WO2005044181A2 - Protection of tissues and cells from cytotoxic effects of ionizing radiation by abl inhibitors - Google Patents

Protection of tissues and cells from cytotoxic effects of ionizing radiation by abl inhibitors Download PDF

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WO2005044181A2
WO2005044181A2 PCT/US2004/028658 US2004028658W WO2005044181A2 WO 2005044181 A2 WO2005044181 A2 WO 2005044181A2 US 2004028658 W US2004028658 W US 2004028658W WO 2005044181 A2 WO2005044181 A2 WO 2005044181A2
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alkyl
group
alkylene
substituted
methyl
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PCT/US2004/028658
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French (fr)
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WO2005044181A3 (en
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E. Premkumar Reddy
M. V. Ramana Reddy
Stephen C. Cosenza
Kiranmai Gumireddy
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Temple University-Of The Commonwealth System Of Higher Education
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine

Definitions

  • the invention relates to the field of protecting normal cells and tissues from anticipated, planned or inadvertent exposure to ionizing radiation.
  • the invention relates to radioprotective agents administered to an individual prior to or after exposure to ionizing radiation, such as occurs during anticancer radiotherapy.
  • Ionizing radiation has an adverse effect on cells and tissues, primarily through cytotoxic effects. In humans, exposure to ionizing radiation occurs primarily through therapeutic techniques (such as anticancer radiotherapy) or through occupational and environmental exposure.
  • a major source of exposure to ionizing radiation is the administration of therapeutic radiation in the treatment of cancer or other proliferative disorders.
  • Individuals exposed to therapeutic doses of ionizing radiation typically receive between 0.1 and 2 Gy per treatment, and can receive as high as 5 Gy per treatment.
  • Therapeutic radiation is generally applied to a defined area of the individual's body which contains abnormal proliferative tissue, in order to maximize the dose absorbed by the abnormal tissue and minimize the dose absorbed by the nearby normal tissue.
  • normal tissue proximate to the abnormal tissue is also exposed to potentially damaging doses of ionizing radiation throughout the course of treatment.
  • treatments that require exposure of the individual's entire body to the radiation in a procedure called “total body irradiation", or "TBI.”
  • TBI total body irradiation
  • the efficacy of radiotherapeutic techniques in destroying abnormal proliferative cells is therefore balanced by associated cytotoxic effects on nearby normal cells. Because of this, radiotherapy techniques have an inherently narrow therapeutic index which results in the inadequate treatment of most tumors. Even the best radiotherapeutic techniques may result in incomplete tumor reduction, tumor recurrence, increasing tumor burden, and induction of radiation resistant tumors. Numerous methods have been designed to reduce normal tissue damage while still delivering effective therapeutic doses of ionizing radiation.
  • the autologous hematopoietic stem cells are returned to their body. However, if tumor cells have metastasized away from the tumor's primary site, there is a high probability that some tumor cells will contaminate the harvested hematopoietic cell population.
  • the harvested hematopoietic cell population may also contain neoplastic cells if the individual suffers from cancers of the bone marrow such as the various French- American-British (FAB) subtypes of acute myelogenous leukemias (AML), chronic myeloid leukemia (CML), or acute lymphocytic leukemia (ALL).
  • FAB French- American-British
  • the metastasized tumor cells or resident neoplastic cells must be removed or killed prior to reintroducing the stem cells to the individual. If any living tumorigenic or neoplastic cells are re-introduced into the individual, they can lead to a relapse.
  • Prior art methods of removing tumorigenic or neoplastic cells from harvested bone marrow are based on a whole-population tumor cell separation or killing strategy, which typically does not kill or remove all of the contaminating malignant cells. Such methods include leukopheresis of mobilized peripheral blood cells, immunoaffinity-based selection or killing of tumor cells, or the use of cytotoxic or photosensitizing agents to selectively kill tumor cells.
  • the malignant cell burden may still be at 1 to 10 tumor cells for every 100,000 cells present in the initial harvest (Lazarus et al, J. Hematotherapy, 2(4):457-66, 1993).
  • a purging method designed to selectively destroy the malignant cells present in the bone marrow, while preserving the normal hematopoietic stem cells needed for hematopoietic reconstitution in the transplantation subject.
  • Exposure to ionizing radiation can also occur in the occupational setting. Occupational doses of ionizing radiation may be received by persons whose job involves exposure (or potential exposure) to radiation, for example in the nuclear power and nuclear weapons industries. There are currently 104 nuclear power plants licensed for commercial operation in the United States.
  • a radiation count of 0.84 mSv/hour (4000 times the annual limit) was detected in the immediate area.
  • Thirty-nine households (150 people) were evacuated and 200 meter radius around the site was declared Off-limits.
  • the roads within a 3 kilometer radius of the site were closed and residents within 10 kilometer radius of the site were advised to stay indoors.
  • the Tokaimura "criticality event" is ranked as the third most serious accident - behind Three Mile Island and Chernobyl - in the history of the nuclear power industry.
  • Environmental exposure to ionizing radiation may also result from nuclear weapons detonations (either experimental or during wartime), discharges of actinides from nuclear waste storage and processing and reprocessing of nuclear fuel, and from naturally occurring radioactive materials such as radon gas or uranium.
  • radioactive materials such as radon gas or uranium.
  • Radiation exposure from any source can be classified as acute (a single large exposure) or chronic (a series of small low-level, or continuous low-level exposures spread over time).
  • Radiation sickness generally results from an acute exposure of a sufficient dose, and presents with a characteristic set of symptoms that appear in an orderly fashion, including hair loss, weakness, vomiting, diarrhea, skin bums and bleeding from the gastrointestinal tract and mucous membranes. Genetic defects, sterility and cancers (particularly bone marrow cancer) often develop over time. Chronic exposure is usually associated with delayed medical problems such as cancer and premature aging. An acute total body exposure of 125,000 millirem may cause radiation sickness. Localized doses such as are used in radiotherapy may not cause radiation sickness, but may result in the damage or death of exposed normal cells.
  • an acute total body radiation dose of 100,000 - 125,000 millirem (equivalent to 1 Gy) received in less than one week would result in observable physiologic effects such as skin bums or rashes, mucosal and GI bleeding, nausea, diarrhea and/or excessive fatigue.
  • Longer term cytotoxic and genetic effects such as hematopoietic and immunocompetent cell destruction, hair loss (alopecia), gastrointestinal, and oral mucosal sloughing, venoocclusive disease of the liver and chronic vascular hyperplasia of cerebral vessels, cataracts, pneumonites, skin changes, and an increased incidence of cancer may also manifest over time.
  • a sufficiently large acute dose of ionizing radiation for example 500,000 to over 1 million millirem (equivalent to 5 - 10 Gy) may kill an individual immediately. Doses in the hundreds of thousands of millirems may kill within 7 to 21 days from a condition called "acute radiation poisoning." Reportedly, some of the Chernobyl firefighters died of acute radiation poisoning, having received acute doses in the range of 200,000 - 600,000 millirem (equivalent to 2 - 6 Gy). Acute doses below approximately 200,000 millirem do not result in death, but the exposed individual will likely suffer long-term cytotoxic or genetic effects as discussed above. Acute occupational exposures usually occur in nuclear power plant -7-
  • a chronic dose is a low level (i.e., 100 - 5000 millirem) incremental or continuous radiation dose received over time.
  • Examples of chronic doses include a whole body dose of approximately 5000 millirem per year, which is the dose typically received by an adult working at a nuclear power plant.
  • the Atomic Energy Commission recommends that members of the general public should not receive more than 100 millirem per year.
  • Chronic doses may cause long-term cytotoxic and genetic effects, for example manifesting as an increased risk of a radiation-induced cancer developing later in life.
  • Recommended limits for chronic exposure to ionizing radiation are given in Table 2. Table 2:
  • Table 3 sets forth the radiation doses from common sources.
  • Chronic doses of greater tiian 5000 millirem per year may result in long-term cytotoxic or genetic effects similar to those described for persons receiving acute doses. Some adverse cytotoxic or genetic effects may also occur at chronic doses of significantly less than 5000 millirem per year.
  • any dose above zero can increase the risk of radiation-induced cancer (i.e., that there is no threshold).
  • Epidemiological studies have found that the estimated lifetime risk of dying from cancer is greater by about 0.04% per rem of radiation dose to the whole body. While anti-radiation suits or other personal protective equipment (PPE) may be effective at reducing radiation exposure, such specialized PPE is expensive, unwieldy, and generally not available to public.
  • PPE personal protective equipment
  • radioprotective PPE will not protect normal tissue adjacent a tumor from stray radiation exposure during radiotherapy. What is needed, therefore, is a practical way to protect individuals who are scheduled to incur, or are at risk for incurring, exposure to ionizing radiation.
  • therapeutic irradiation it is desirable to enhance protection of normal cells while causing tumor cells to remain vulnerable to the detrimental effects of the radiation.
  • systemic protection from anticipated or inadvertent total body irradiation such as may occur with occupational or environmental exposures, or with certain therapeutic techniques.
  • Pharmaceutical radioprotectants offer a cost-efficient, effective and easily available alternative to radioprotective gear. However, previous attempts at radioprotection of normal cells with pharmaceutical compositions have not been entirely successful.
  • cytokines directed at mobilizing the peripheral blood progenitor cells confer a myeloprotective effect when given prior to radiation (Neta et al, Semin. Radiat. Oncol. 3:16-320, 1996), but do not confer systemic protection.
  • Other chemical radioprotectors administered alone or in combination with biologic response modifiers have shown minor protective effects in mice, but application of these compounds to large mammals was less successful, and it was questioned whether chemical radioprotection was of any value (Maisin, J.R., Bacq and Alexander Award Lecture. "Chemical radioprotection: past, present, and future prospects," Int. J. Radiat. Biol 73:443- 50, 1998).
  • Pharmaceutical radiation sensitizers which are known to preferentially enhance the effects of radiation in cancerous tissues, are clearly unsuited for the general systemic protection of normal tissues from exposure to ionizing radiation.
  • ABL protein Kinase Protein kinases in general are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The phosphorylation serves as a basis for cellular signaling that regulates such fundamental cellular functions such as cellular growth, differentiation and proliferation. Accordingly, disorders associated with abnormal protein kinase activity have included many cancers and other proliferative disorders. Protein kinases are divided into tyrosine kinases and serine-threonine kinases.
  • the protein tyrosine kinases are further classified as receptor tyrosine kinases and non-receptor tyrosine kinases, which are also called cellular tyrosine kinases (CTK's).
  • Receptor tyrosine kinases include epithelial growth factor receptors (EGFR), insulin-like growth factor receptors (IGFR), platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR) and vascular endothelial growth factor receptors (VEGF).
  • Non-receptor tyrosine kinases include Abelson (ABL) tyrosine kinase as well as ten other subfamilies of CTK's.
  • ABL is a tyrosine kinase expressed by the c-abl proto-oncogene.
  • Cloning of the c-abl gene has revealed that it spans at least 230kb, and contains at least 11 exons.
  • Exon la is 19 kb proximal to exon 2.
  • Exon lb which is somewhat smaller than exon la, is more than 200 kb proximal to exon 2.
  • at least two major c-abl messages are transcribed, differing in their 5' regions. See, Shtivelman et al, Cell 47, 277 (1986); Bernards et al, Mol. Cell Biol.
  • exon lb the mRNA is 7.0 kb. If exon la is used, the mRNA is 6.0 kb. Each of exons la and lb are preceded by a transcriptional promoter.
  • the 6-kb c-abl transcript consists of exons la through 11. The 7-kb transcript begins with exon lb, skips the 200 kb distance to exon 2, omits exon la, and joins to exons 2 through 11. Thus, both c-abl messages share a common set of 3' exons, starting from the c-abl exon 2.
  • CML chronic myelogenous leukemia
  • CML the most notable feature of CML is the translocation of the proto-oncogene c-abl from the long arm of chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22, resulting in the formation of bcr-abl hybrid genes.
  • the break occurs near the end of the long arm of chromosome 9 (band 9q34) and in the upper half of chromosome 22 (band 22ql 1).
  • the 9;22 translocation in CML results in the abnormal juxtaposition of abl sequences adjacent to bcr sequences.
  • the c-abl proto-oncogene is expressed in normal cells and plays a critical role in regulating normal hematopoiesis by encoding a protein with tyrosine kinase activity. This activity is augmented in cells carrying bcr-abl hybrid genes.
  • the gene located at the breakpoint on chromosome 22 is called bcr because the break in chromosome 22 in CML occurs in a small 5.8-kilobase (kb) segment (breakpoint cluster region) of the gene on chromosome 22.
  • the fusion of the BCR gene with c-abl leads to an 8.5 kb chimeric mRNA consisting of 5' BCR sequences and 3' abl sequences.
  • the chimeric message is in turn translated into a larger chimeric abl protein (210 kDa) that has increased tyrosine kinase activity
  • a larger chimeric abl protein (210 kDa) that has increased tyrosine kinase activity
  • 210 kDa chimeric abl protein
  • the 210 kDa protein is considerably larger than the normal human protein of 145 kDa, and has a very high tyrosine kinase activity.
  • Oxidants are produced as part of the normal metabolism of all cells but also are an important component of the pathogenesis of many disease processes.
  • Reactive oxygen species ROS
  • ROS reactive oxygen species
  • CNS central nervous system
  • skeletal muscle skeletal muscle
  • Oxygen free radicals also modulate the effects of nitric oxide, thereby contributing to the pathogenesis of vascular disorders, inflammatory diseases and the aging process.
  • Free radicals are molecules with one or more unpaired electrons. Free radicals act as oxidants, rapidly reacting with other molecules, and starting oxidative chain reactions. Free radicals are a normal product of 4181
  • antioxidant compounds have been shown to have cytoprotective properties. Antioxidants are believed to act by scavenging free radicals. Normal cell and organ function is maintained via a balance of oxidant and antioxidant agents. Many antioxidant compounds, e.g., the enzyme superoxide dismutases (SODs) are produced physiologically and balance the naturally occurring free radicals. Several other important antioxidant enzymes are known to exist within cells, including catalase and glutathione peroxidase.
  • extracellular fluids and the extracellular matrix contain only small amounts of these enzymes, other extracellular antioxidants are also known to be present, including radical scavengers and inhibitors of lipid peroxidation, such as ascorbic acid, uric acid, and ⁇ -tocopherol.
  • inhibitors of ABL provide significant and selective systemic protection of normal cells and normal tissues from radiation-induced damage in individuals exposed to ionizing radiation. It is an object of the invention to provide compositions and methods for protecting the normal cells and tissues from the cytotoxic and genetic effects of exposure to ionizing radiation, in individuals who have incurred or are at risk of incurring exposure to ionizing radiation.
  • the exposure to ionizing radiation may occur in controlled doses during the treatment of cancer and other proliferative disorders, or may occur in uncontrolled doses beyond the norm accepted for the population at large during high risk activities or environmental exposures.
  • Radioprotection by Formula I Compounds A method for protecting an individual from cytotoxic side effects of ionizing radiation is provided, comprising administering to said individual an effective amount of at least one compound of formula I: wherein: R is hydrogen, R 2 , or a radical of formula (i) or (ii):
  • X is C orN;
  • R 5 is selected from the group consisting of -H; substituted and unsubstituted aryl, preferably substituted and unsubstituted phenyl; -NH 2 ; -Z-(C ⁇ -C 7 )hydrocarbyl, preferably -Z-(C ⁇ -C 6 )alkyl and -Z-benzyl;
  • n is 1, 2, 3, 4; 5 or 6; preferably 1, 2, 3 or 4; more preferably 1, 2 or 3; most preferably 1 or 2;
  • T is selected from the group consisting of N, C(H) and C(C ⁇ -C 6 alkyl), preferably N and C(H); and T (2) when the designated bond in ' is a single bond, then: p (a) the designated bond in ⁇ is a double bond; (b)R is a radical of formula (ii):
  • R 1 When R 1 is substituted aryl or substituted heteroaryl, it is preferably mono- or di-substituted by substituents independently selected from the group consisting of (C2-C ⁇ )heteroalkyl, preferably (C -C ⁇ o)heteroalkyl; substituted heterocyclyl(C ⁇ -C6)alkyl, preferably substituted non-aromatic monocyclic heterocyclyl(C ⁇ -C6)alkyl, more preferably substituted non-aromatic monocyclic heterocyclyl(C ⁇ -C 4 )alkyl, most preferably mono- or di-substituted by substituents independently selected from the group consisting of -OH, -OCH 3 , and -(C ⁇ -C 6 )alkyl; -0(Ci-C 7 )hydrocarbyl, preferably -0(Ci-C 6 )alkyl, more preferably -0(Ci-C )alkyl; halogen, preferably
  • B is selected from the group consisting of phenyl; pyridyl; pyrazinyl; pyrimidinyl, particularly 2- and 5- pyrimidyl; pyridazinyl; thienyl; furyl; pyrrolyl, particularly 2-pyrrolyl and 1- alkyl-2-pyrrolyl; imidazolyl, particularly 2-imidazolyl; thiazolyl, particularly 2- thiazolyl; oxazolyl, particularly 2-oxazolyl; pyrazolyl, particularly 3- and 5- pyrazolyl; isothiazolyl; 1,2,3-triazolyl; 1,2,4-triazolyl; 1,3,4-triazolyl; tetrazolyl; 1,2,3-thiadiazolyl; 1,2,3-oxadiazolyl; 1,3,4-thiadiazolyl; 1,3,4-oxadiazolyl; indolyl, particularly 2-, 3-,
  • B is selected from the group consisting of phenyl; 2- and 3-indolyl; 2- and 3-pyrrolyl; 2-, 3-benzofuryl; 5-quinolyl, and 2- and 3- benzothienyl.
  • R 3 is substituted heteroaryl, it is preferably mono-, di-, or tri- substituted by substituents independently selected from the group consisting of (C ⁇ -C 7 )hydrocarbyl, preferably (C ⁇ -Ce)alkyl, more preferably (C ⁇ -C4)alkyl, most preferably -CH 3 ; -0(Ci-C 7 )hydrocarbyl, preferably -0(C]-C6)alkyl, more preferably -0(C 1 -C 4 )alkyl; -OH; -(C 1 -C 6 )alkylene-N(C ⁇ -C 6 )alkyl) 2 ; halogen, preferably bromo, chloro and fluoro; -CN; -NH 2 and -N0 2 .
  • R 5 or R ⁇ are substituted aryl, they are preferably mono-, di- or tri- substituted by substituents independently selected from the group consisting of (C ⁇ -C 7 )hydrocarbyl, preferably (C ⁇ -C 6 )alkyl, more preferably (C ⁇ -C )alkyl, most preferably -CH 3 ; -OH; -0(C ⁇ -C 7 )hydrocarbyl, -C0 2 H; CONH 2 ; C0 2 (C ⁇ - Cg)alkyl; halogen, cyano; -N0 2 ; and substituted heterocyclyl, preferably mono- or di-substituted by substituents independently selected from the group consisting of -OH, -OCH 3 , and -(CrGe ⁇ lkyl; -0(Ci-C 7 )hydrocarbyl, halogen, cyano; and -N0 2 ; According to a second embodiment of the invention, there is provided
  • a method for protecting an individual from cytotoxic side effects of ionizing radiation comprising administering to said individual an effective amount of a combination comprising at least one compound of formula I, as defined above, and at least one antioxidant compound.
  • Preferred embodiments of radioprotective compounds of formula I are described as follows.
  • First Embodiment of Formula I Compounds According to a First Embodiment of the radioprotective compounds of formula I: the designated bond in ' is a double bond, R the designated bond in " s a single bond, R is hydrogen, R 2 , or a radical of formula (i):
  • T is selected from the group consisting of N, C(H) and C(Ci- C ⁇ alkyl); or a pharmaceutically acceptable salt of such a compound.
  • the radioprotective compound comprises a compound of formula II:
  • T is selected from the group consisting of N, C(H) and C(C ⁇ - C 6 alkyl);
  • R 1 is selected from the group consisting of -H, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(C ⁇ -C 6 )alkyl, and substituted and unsubstituted heteroaryl(C 1 -C 6 )alkyl; and R is substituted aryl, or substituted or unsubstituted heterocyclyl; or a pharmaceutically acceptable salt of such a compound.
  • the radioprotective compound comprises a compound of formula Ila:
  • R 1 is selected from the group consisting of -(C ⁇ -Ce)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(C 1 -C 6 )alkyl, and substituted and unsubstituted heteroaryl(C 1 -C6)alkyl; or a pharmaceutically acceptable salt of such a compound.
  • Preferred compounds according to formula Ila include: (2- ⁇ 4-[4-amino- 5-(4-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenoxy ⁇ -ethyl)(2-methoxy- ethyl)amine; 1 -(2- ⁇ 4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7- yl]phenyl ⁇ ethyl)piperidin-4-ol; and pharmaceutically acceptable salts thereof.
  • the radioprotective compound comprises a compound of formula lib:
  • R 1 is selected from the group consisting of -(C ⁇ -C6)alkyl, and substituted and unsubstituted aryl; or a pharmaceutically acceptable salt of such a compound.
  • Preferred compounds according to formula Ila include l-(tert-butyl)-3- (4-methylphenyl)pyrazolo[5,4-d]pyrimidine-4-ylamine, and pharmaceutically acceptable salts thereof.
  • the radioprotective compound comprises a compound of formula III:
  • Preferred compounds according to formula III include: ethyl-2- ⁇ 5-[(2- oxo-lH-benzo[d]azolidin-3-ylidene)methyl]-8-quinolyloxy ⁇ -propanoate; 3- ⁇ 2,4-dimethyl-5-[(2-oxo(lH-benzo[d]azolidin-3-ylidene))-methyl]pyrrol-3-yl ⁇ - propanoic acid; 3- ⁇ 4-methyl-2-[(2-oxo(lH-benzo[d]azolidin-3-ylidene))- methyl]pyrrol-3-yl ⁇ propanoic acid; 3-[(3,5-dimethylpyrrol-2-yl)methylene]-lH- benzo[d]azolidin-2-one; 3-[(2-chloro-4-methoxy ⁇ henyl)methylene]- 1 H-benzo- [d]azolidin-2-one; 3-(indol-3-ylmethylene)-5-(2-pipe
  • the radioprotective compound comprises a compound of formula IV:
  • R 3 is substituted heteroaryl, it is preferably mono-, di-, or tri- substituted by substituents independently selected from the group consisting of -(C ⁇ -C 7 )hydrocarbyl, preferably -(C ⁇ -C 6 )alkyl, more preferably -(C ⁇ -C 4 )alkyl, most preferably -CH 3 ; -0(C ⁇ -C 7 )hydrocarbyl, preferably -0(C C 6 )alkyl, more preferably -0(d-C 4 )alkyl; -OH; -(C ⁇ -C 6 )alkylene-N(C ⁇ -C 6 )alkyl) 2 ; halogen, preferably bromo, chloro and fluoro; -CN; -NH 2 and -N0 2 .
  • Preferred compounds according to formula IV include 5-((lZ)-l-cyano- 2-indol-3-ylvinyl)-3-aminopyrazole-4-carbonitrile, and pharmaceutically acceptable salts thereof.
  • the radioprotective compound or combination of compounds is administered before exposure to the ionizing radiation.
  • the radioprotective compound or combination of compounds is administered after exposure to ionizing radiation.
  • Q 1 is phenyl or substituted phenyl, more preferably substituted phenyl.
  • Q 2 is phenyl or substituted phenyl, more preferably substituted phenyl.
  • both Q 1 and Q 2 are phenyl or substituted phenyl, more preferably substituted phenyl.
  • X is selected from the group consisting of (i), (ii) and (iii).
  • X is selected from the group consisting of (i) and (ii).
  • X is (i).
  • the aryl and heteroaryl groups comprising Q 1 and Q 2 are mono-, di- or tri- substituted.
  • the aryl and heteroaryl groups comprising Q 1 and Q 2 are substituted at all substitutable positions.
  • R y are preferably selected from the group consisting of -NH 2 , -N0 2 , N-methylpiperazinyl, and -OR x .
  • R z is selected from the group consisting of -H, -(C ⁇ -C 6 )alkyl,
  • Substituted phenyl R z is preferably mono- di- or tri-substituted, more preferably mono or di substituted, most preferably mono-substituted by substituents independently selected from the group consisting of -R x , -NR X 2 , -N0 2 , -OR x , -CN, -C0 2 R x , halogen, -SR X and S0 2 R x .
  • Substituents on substituted phenyl R z are more preferably independently selected from the group consisting of -(C ⁇ -C6)alkyl, -NH 2 , NH(Cj-C6)alkyl, -N0 2 , -0(C]-C 6 )alkyl, -OH, -CN, -C0 2 (d-C 6 )alkyl, C0 2 H, , halogen, -S(d- C 6 )alkyl, -SH, and S0 2 (d-C 6 )alkyl.
  • Substituents on substituted phenyl R z are most preferably independently selected from the group consisting of methyl, ethyl, -NH 2 , NHCH 3 , -N0 2 , -OCH3, -OH, -CN, -CO2CH3, C0 2 Et, C0 2 H, , halogen, -SCH 3 , -SH, and S0 2 CH 3 .
  • R is more preferably selected from the group consisting of -H, -(d- C 6 )alkyl, and -C(0)(Ci- )alkyl.
  • R is most preferably -H or -(CrC alkyl.
  • R y is preferably selected from -H, -(C ⁇ -C 8 )hydrocarbyl, -0(d- C 8 )hydrocarbyl, substituted phenyl, -NHR X , -(C ⁇ -C 6 )haloalkyl, -(C C3)alkyleneNH 2 , -(C ⁇ -C 3 )alkyleneN(CH 3 ) 2 , -(d-C 3 )alkylene-OR 1 , -(d- C )alkylene-C0 2 R 1 , and ⁇ (C ⁇ -C 4 ) ⁇ erfluoroalkylene-C0 2 R 1 .
  • R y is more preferably selected from -H, -(C ⁇ -C 8 )hydrocarbyl, -0(C ⁇ -
  • R z is preferably selected from the group consisting of-H, -(d-C 6 )alkyl, and phenyl.
  • Preferred compounds according to formula V include, for example: 4- ((U5T)-2- ⁇ [(4-fluorophenyl)methyl]sulfonyl ⁇ vinyl)benzoic acid; 4-((l£)-2- ⁇ [(4- iodophenyl)methyl] sulfonyl ⁇ vinyl)benzoic acid; 4-(( lE)-2- ⁇ [(4-chloropheny 1)- 4181
  • More preferred compounds according to formula V include, for example: 4-((lE)-2- ⁇ [(4-fluorophenyl)methyl]sulfonyl ⁇ vinyl)benzoic acid; 4-((lE)-2- ⁇ [(4-iodo ⁇ henyl)methyl]sulfonyl ⁇ vinyl)benzoic acid; 4-((li?)-2- ⁇ [(4-chloro- phenyl)-methyl]sulfonyl ⁇ vinyl)benzoic acid; l-[5-((l-5)-2- ⁇ [(4-chlorophenyl)- methyl]-sulfonyl ⁇ vinyl)-2-fluorophenyl]-2-(dimethylamino)ethan-l-one; (1-5)- 2-(2,4-difluorophenyl)-l- ⁇ [(4-bromophenyl)methyl]sulfonyl ⁇ ethene; (lE)-2-(
  • a pharmaceutical composition comprising administering to the individual an effective amount of at least one compound of formula I, and an effective amount of at least one antioxidant compound.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, at least one compound according to formula I as defined above, and at least one compound according to formula V as defined above.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier, at least one compound according to formula I as defined above, and at least one antioxidant compound.
  • a method of treating an individual with a proliferative disorder comprising: (a) administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound; and (b) administering an effective amount of therapeutic ionizing radiation.
  • the proliferative disorder is cancer.
  • a method of safely increasing the dosage of therapeutic ionizing radiation used in the treatment of cancer or other proliferative disorders comprising administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to 4181
  • the radioprotective compound or combination of compounds is administered prior to administration of the therapeutic ionizing radiation.
  • the radioprotective compound or combination of compounds induces a temporary radioresistant phenotype in the normal tissue of the individual.
  • a method for treating an individual who has incurred, or is at risk for incurring, remediable radiation damage from exposure to ionizing radiation comprising administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound
  • the compound or compounds may be administered before or after incurring remediable radiation damage from exposure to ionizing radiation.
  • a method is provided of reducing the number of malignant cells in bone marrow of an individual, comprising: (1) removing a portion of the individual's bone marrow; (2) administering to the removed bone marrow an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound; and (3) irradiating the bone marrow with an effective amount of ionizing radiation.
  • the bone marrow is reimplanted into the individual.
  • the individual receives therapeutic ionizing radiation prior to reimplantation of the bone marrow.
  • the individual receives therapeutic ionizing radiation prior to reimplantation of the bone marrow, and is administered a radioprotective compound or combination of compounds as defined above prior to receiving the therapeutic ionizing radiation.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof is used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof, and a compound of formula V are used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof, and an antioxidant compound are used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation.
  • the compounds are for administration before exposure to ionizing radiation.
  • the compounds are for administration after exposure to ionizing radiation.
  • the formula I compounds are for administration before or after administration of therapeutic ionizing radiation, for treatment of a proliferative disorder.
  • the compounds are for treating an individual who has incurred or is at risk of incurring remediable radiation damage from exposure to ionizing radiation.
  • the compounds are used for the preparation of a medicament for treating bone marrow prior to irradiating the bone marrow with an effective amount of ionizing radiation.
  • the compounds are used for the preparation of a medicament for safely increasing the dosage of therapeutic ionizing radiation used in the treatment of cancer or other proliferative disorders.
  • the term "individual” includes human beings and non-human animals and, as used herein, refers to an organism which is scheduled to incur, is at risk of incurring, or has incurred, exposure to ionizing radiation.
  • ionizing radiation is radiation of sufficient energy that, when absorbed by cells and tissues, induces formation of reactive oxygen species and DNA damage. This type of radiation includes X-Rays, gamma rays, and particle bombardment (e.g., neutron beam, electron beam, protons, mesons and others), and is used for medical testing and treatment, scientific purposes, industrial testing, manufacturing and sterilization, weapons and weapons development, and many other uses.
  • the Sv is the Gy dosage multiplied by a factor that includes tissue damage done.
  • penetrating ionizing radiation e.g., gamma and beta radiation
  • an effective amount of ionizing radiation is meant an amount of ionizing radiation effective in killing, or in reducing the proliferation, of abnormally proliferating cells in an individual.
  • effective amount of ionizing radiation means an amount of ionizing radiation effective in killing, or in reducing the proliferation, of malignant cells in a bone marrow sample removed from an individual.
  • acute exposure to ionizing radiation or "acute dose of ionizing radiation” is meant a dose of ionizing radiation absorbed by an individual in less than 24 hours. The acute dose may be localized, as in radiotherapy techniques, or may be absorbed by the individual's entire body.
  • Acute doses are typically above 10,000 millirem (0.1 Gy), but may be lower.
  • chronic exposure to ionizing radiation or “chronic dose of ionizing radiation” is meant a dose of ionizing radiation absorbed by an individual over a period greater than 24 hours. The dose may be intermittent or continuous, and may be localized or absorbed by the individual's entire body. Chronic doses are typically less than 10,000 millirem (0.1 Gy), but may be higher.
  • at risk of incurring exposure to ionizing radiation is meant that an individual may intentionally, e.g., by scheduled radiotherapy sessions, or inadvertently be exposed to ionizing radiation in the future. Inadvertent exposure includes accidental or unplanned environmental or occupational exposure.
  • small molecule in meant a monomeric organic compound having a molecular weight of less than about 1000.
  • a “radioprotective ⁇ , ⁇ -unsaturated (aryl or heteroaryl) sulfone, sulfonamide or carboxamide” is meant a compound of the formula V:
  • Q 1 — X— CH CH— Q 2 V wherein, Q 1 and Q 2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; preferably substituted or unsubstituted phenyl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below: 4181
  • antioxidant is meant a pharmaceutically acceptable chemical compound that prevents or slows the breakdown of another substance by oxygen.
  • antioxidants useful in the methods of the present invention are small molecule organic compounds.
  • effective amount in the context of an amount of radioprotective compound is meant an amount, alone, or in combination with either another radioprotective compound or an antioxidant compound, which is effective to reduce or eliminate the toxicity associated with radiation in normal cells of the individual.
  • an effective amount of a radioprotective compound means an amount of the compound effective to reduce or eliminate the toxicity associated with radiation in bone marrow removed from an individual.
  • alkyl by itself or as part of another substituent means, unless otherwise stated, a straight, branched or cyclic chain saturated hydrocarbon radical, including di- and multi-radicals, having the number of carbon atoms designated in an expression such as (C x -C y )alkyl.
  • (C x -C y )alkyl wherein x ⁇ y, represents an alkyl chain containing a minimum of x carbon atoms and a maximum of y carbon atoms.
  • Examples include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl and cyclopropyhnethyl.
  • Preferred is (C ⁇ -C 3 )alkyl, particularly ethyl, methyl and isopropyl.
  • cycloalkyl refers to alkyl groups that cyclic, i.e., that contain at least one cyclic structure. Examples include cyclohexyl, cyclopentyl, norbomyl, adamantyl and cyclopropylmethyl.
  • alkylene refers to a divalent alkyl radical having the number of carbon atoms designated (i.e.
  • (Ci-C ⁇ ) means ⁇ CH 2 -; -CH 2 CH 2 -; -CH 2 CH 2 CH 2 -; -CH 2 CH 2 CH 2 CH 2 -; -CH 2 CH 2 CH 2 CH 2 -; and -CH2CH 2 CH2CH2CH 2 CH 2 -, and also includes branched divalent structures such as, for example, -CH 2 CH(CH 3 )CH 2 CH 2 - and -CH(CH 3 )CH(CH 3 )-, and divalant cyclic structures such as, for example 1,3-cyclopentyl.
  • arylene by itself or as part of another substituent means, unless otherwise stated, a divalent aryl radical.
  • divalent phenyl radicals or "phenylene” groups, particularly 1,4-divalent phenyl radicals.
  • heteroarylene by itself or as part of another substituent means, unless otherwise stated, a divalent heteroaryl radical.
  • Preferred are five- or six-membered monocyclic heteroarylene.
  • heteroarylene moieties comprising divalent heteroaryl rings selected from the group consisting of pyridine, piperazine, pyrimidine, pyrazine, furan, thiophene, pyrrole, thiazole, imidazole and oxazole, such as, for example 2,5-divalent pyrrole, thiophene, furan, thiazole, oxazole, and imidazole.
  • alkoxy employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-pro ⁇ oxy, 2- ⁇ ro ⁇ oxy (isopropoxy) and the higher homologs and isomers. Preferred are (d- C6)alkoxy, particularly ethoxy and methoxy.
  • the carbon chains in the alkyl and alkoxy groups which may occur in the compounds of the invention may be cyclic, straight or branched, with straight chain being preferred.
  • hydrocarbyl refers to any moiety comprising only hydrogen and carbon atoms.
  • heteroalkyl by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain radical consisting of the stated number of carbon atoms and one, two or three heteroatoms selected from the group consisting of O, N, and S, and wherem the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized.
  • halo or halogen by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl means an alkyl group as defined above, wherein at least one hydrogen atom is substituted by a halogen atom.
  • haloalkyl groups are substituted by bromine, chlorine or fluorine, more preferably chlorine or fluorine, most preferably fluorine. Examples include: 3-bromopropyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, 1-chloroethyl and 2- chloropropyl.
  • aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (4n + 2) delocalized ⁇ (pi) electrons).
  • aromatic is intended to include not only ring systems containing only carbon ring atoms but also systems containing one or more non- carbon atoms as ring atoms. Systems containing one or more non-carbon atoms may be known as “heteroaryl” or “heteroaromatic” systems. The term “aromatic” thus is deemed to include “aryl” and “heteroaryl” ring systems.
  • aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl; anthracyl; and naphthyl which may be substituted or unsubstituted. The aforementioned listing of aryl moieties is intended to be representative, not limiting.
  • heterocycle or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, monocyclic or polycyclic heterocyclic ring system which consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom which affords a stable structure.
  • Heterocyclyl groups are inclusive of monocyclic and polycyclic heteroaryl groups and monocyclic and polycyclic groups that are not aromatic, such as saturated and partially saturated and monocyclic and polycyclic partially saturated monocyclic and polycyclic groups.
  • the term "heteroaryl” or “heteroaromatic” refers to a heterocycle having aromatic character, and includes both monocyclic heteroaryl groups and polycyclic heteroaryl groups.
  • a polycyclic heteroaryl group may include one or more rings which are partially, saturated.
  • Examples of monocyclic heteroaryl groups include: Pyridyl; pyrazinyl; pyrimidinyl, particularly 2- and 5-pyrimidyl; pyridazinyl; thienyl; furyl; pyrrolyl, particularly 2-pyrrolyl and l-alkyl-2-pyrrolyl; imidazolyl, particularly 2-imidazolyl; thiazolyl, particularly 2-thiazolyl; oxazolyl, particularly 2- oxazolyl; pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl; and 1,3,4-oxadiazolyl.
  • monocyclic heterocycles that are not aromatic include saturated monocyclic groups such as: Aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, 1,4-dioxane, 1,3-dioxane, sulfolane, tetrahydrofuran, thiophane, piperazine, morpholine, thiomorpholine, tetrahydropyran, homopiperazine, homopiperidine, 1,3-dioxepane, hexamethyleneoxide and piperidine; and partially saturated monocyclic groups such as: 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, 2,3-dihydrofuran, 2,5-dihydrofuran , 2,3-dihydropyran, 1,2-dihydro
  • polycyclic heteroaryl groups include: Indolyl, particularly 3-, 4-, 5-, 6- and 7-indolyl, quinolyl, isoquinolyl, particularly 1- and 5- isoquinolyl, cinnolinyl, quinoxalinyl, particularly 2- and 5-quinoxalinyl, quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, benzofuryl, particularly 3-, 4-, 1,5-naphthyridinyl, 5-, 6- and 7-benzofuryl, 1,2- benzisoxazolyl, benzothienyl, particularly 3-, 4-, 5-, 6-, and 7-benzothienyl, benzoxazolyl, benzthiazolyl, particularly 2-benzothiazolyl and 5-benzothiazolyl, purinyl, benzimidazolyl, particularly 2-benzimidazolyl, benztriazolyl,
  • non-aromatic polycyclic heterocycles include: pyrrolizidinyl and quinolizidinyl.
  • the aforementioned listing of non-aromatic heterocyclic moieties and heteroaryl moieties is intended to be representative, not limiting.
  • Preferred heteroaryl groups are 2-, 3- and 4-pyridyl; pyrazinyl; 2- and 5- pyrimidinyl; 3-pyridazinyl; 2- and 3-thienyl; 2- and 3-furyl; pyrrolyl; particularly N-methylpyrrol-2-yl; 2-imidazolyl; 2-thiazolyl; 2-oxazolyl; pyrazolyl; particularly 3- and 5-pyrazolyl; isothiazolyl; 1,2,3-triazolyl; 1,2,4- 4181
  • heteroaryl groups are 2, 3- and 4-pyridyl; 2- and 3- thienyl; 2- and 3-furyl; 2-pyrrolyl; 2-imidazolyl; 2-thiazolyl; 2-oxazolyl; 2- and 3-indolyl; 2-, and 3-benzofuryl; 3-(l,2-benzisoxazolyl); 2-, and 3 -benzothienyl; 2-benzoxazolyl; 1- and 2-benzimidazolyl, 2-, 3- and 4-quinolyl; and 2- and 5- benzthiazolyl.
  • Most preferred heteroaryl groups are 2- and 3-indolyl; 2- and 3- ⁇ yrrolyl, 2-, and 3-benzofuryl; and 2-, and 3 -benzothienyl.
  • substituted means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group.
  • substituted refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted.
  • the substituents are independently selected, and substitution may be at any chemically accessible position.
  • geometric when used in the name of a compound is an abbreviation of the term “geminal” designates that two substituents are bonded to the same atom.
  • a gem-difluoro(C ⁇ -C 6 )alkyl group includes 2,2- difluoropropyl, 1,1-difTuoroethyl and difluoromethyl.
  • Some of the radioprotective small molecule inhibitors of ABL kinase and all of the ⁇ , ⁇ -unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides are characterized by isomerism resulting from the presence of a double bond. This isomerism is commonly referred to as cis-trans isomerism, but the more comprehensive naming convention employs E and Z designations.
  • the indolones of formula III contain a double bond and are prepared as either the E- or Z-isomer or as a mixture of both isomers.
  • the proportions of an isomeric mixture may be an equilibrium mixture influenced by differential steric strain of the two isomers. Alternately, a mixture of isomers may be enriched in one isomer by selected reaction conditions or by a purification method.
  • radioprotective ABL inhibitor Unless otherwise indicated, both configurations and mixtures thereof are included in the scope of "radioprotective ABL inhibitor” and in the scope of " ⁇ , ⁇ -unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides.”
  • Some of the radioprotective small molecule inhibitors of ABL kinase and some of the ⁇ , ⁇ -unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides may be characterized by isomerism resulting from the presence of a chiral center. The isomers resulting from the presence of a chiral center 181
  • Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light.
  • Single enantiomers are designated according to the Cahn-Ingold-Prelog system. See March, Advanced Organic Chemistry, 4 th Ed., (1992), p. 109. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S).
  • the Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.
  • FIG. 1 shows quantitatively the inhibition of ABL kinase activity by compounds 1 ( ⁇ ), 2 ( ⁇ ), 3 (V), 4 (0), 5 (•) and 6 ( ⁇ ) plotted as percent of solvent treated control.
  • FIG. 2 shows quantitatively the inhibition of ABL kinase activity by compounds 7 ( ⁇ ), 8 ( ⁇ ), 9 (V), 10 (0), 11 (•) and 12 (o), plotted as percent of solvent treated control.
  • FIG. 3 shows quantitatively the inhibition of ABL kinase activity by compounds 13 ( ⁇ ), 14 ( ⁇ ), 15 (repeated three times as V, •, and o), and 16 (0), plotted as percent of solvent treated control.
  • FIG. 4 shows quantitatively the inhibition of ABL kinase activity by compounds 17 ( ⁇ ), 18 ( ⁇ ), and 19 (V) plotted as percent of solvent treated control.
  • FIG. 5 shows quantitatively the inhibition of ABL kinase activity by compounds 20 ( ⁇ ), 21 ( ⁇ ), and 22 (V) plotted as percent of solvent treated control.
  • small molecule inhibitors of ABL activity are also capable of protecting cells, tissues and individuals from the cytotoxic effects of ionizing radiation.
  • the radioprotective compounds protect normal cells and tissues from the effects of acute and chronic exposure to ionizing radiation.
  • compositions comprising a small molecule inhibitor of ABL activity, in combination with either an antioxidant compound or an ⁇ , ⁇ - unsaturated (aryl or heteroaryl) sulfone, sulfonamide or carboxamide, are capable of protecting cells, tissues and individuals from the cytotoxic effects of ionizing radiation.
  • Preferred antioxidant compounds useful in combination small molecule inhibitors of ABL activity include, for example, carotenoids, catechins, isoflavones, flavanones, flavanols, flavanoid chalcones, vitamin E compounds, (3-aminopropyl)[2-(phosphonothio)ethyl]amine, ascorbic acid, cysteine, glutathione, probucol, ⁇ -mercaptoethanol dithiothreitol, pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, ubiquinone, and porphyrin compounds such as those disclosed in EP 1,045,851, the entire contents of which is incorporated herein by reference.
  • Preferred carotenoids include ⁇ -carotene, ⁇ - -44-
  • catechins include gallic acid, propyl gallate, (+)-catechin, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)- epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-catechin gallate (CG), and (-)-gallocatechin gallate.
  • Preferred isoflavones include genistein and daidzein.
  • Preferred flavanols include hesperitin, hesperidin, and quercetin, kaempferol, myricetin.
  • Preferred flavanoid chalcones include xanthohumol and isoxanthohumol.
  • Preferred vitamin E compounds include tocopherols and tocotrienols.
  • Antioxidant compounds more preferably include, for example, ⁇ - carotene, ⁇ -carotene, lutein, lycopene, gallic acid, propyl gallate, (+)-catechin, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-catechin gallate (CG), (-)-gallocatechin gallate, genistein, hesperitin, hesperidin, quercetin, kaempferol, myricetin, xanthohumol, isoxanthohumol, tocopherols, tocotrienol, (3-amin
  • a metal selected from the group consisting of iron, copper, cobalt, nickel and zinc.
  • Such disorders include cancerous and non-cancer proliferative disorders.
  • the present compounds and pharmaceutical compositions are believed effective in protecting normal cells during therapeutic irradiation of a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, lung, colorectal, brain (i.e., glioma) and renal.
  • the compounds and compositions are also effective in protecting normal cells during therapeutic irradiation of leukemic cells.
  • the compounds are also believed useful in protecting normal cells during therapeutic irradiation of abnormal tissues in non-cancer proliferative disorders, including but not limited to the following: hemangiomatosis in newborn, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Paget's Disease of the bone, fibrocystic disease of the breast, Peronies and Duputren's fibrosis, restenosis and cirrhosis.
  • the radioprotective ABL inhibitors of formula I useful in the method of the invention may be prepared by organic synthesis using techniques that are known in the art or readily adapted from techniques known in the art.
  • An activated acid such as, for example, acid chloride intermediate 1 is first reacted with malononitrile, to form an enol intermediate, preferably in a suitable inert solvent in the presence of a suitable base.
  • an inert solvent is meant a solvent that does not react or degrade under the reaction conditions to a degree that would interfere with the desired reaction.
  • Suitable inert solvents for the enol preparation include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, for example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or N-methylpyrrolidinone (NMP).
  • alkyl alcohols such as methanol, ethanol, and isopropanol
  • esters such as methyl acetate or ethyl acetate
  • halogenated solvents such as methylene chloride, chloroform or carbon tetrachloride
  • Suitable bases include bases capable of removing a proton from malononitrile such as, for example, sodium hydride or potassium hydride.
  • the enol synthesis reaction is conveniently carried out at a temperature in the range from about 0° C to about 50° C, preferably in the range from about 25° C to about 30° C, and conveniently at about room temperature.
  • the enol intermediate produced by the above reaction is isolated from the reaction 181
  • the enol intermediate is then alkylated to yield intermediate 2, using a suitable alkylating reagent, preferably dimethyl sulfate, in the presence of a suitable base, preferably in a suitable inert solvent.
  • a suitable alkylating reagent preferably dimethyl sulfate
  • Suitable bases for the alkylation reaction include those described above in the enol synthesis reaction.
  • Suitable solvents for the alkylation reaction include aqueous solvents such as, for example mixtures of THF and water or mixtures of dioxane and water.
  • the alkylation reaction is conveniently carried out at a temperature in the range from about 25° C.
  • Intermediate 2 is cyclized to intermediate 3 by reaction with a suitable hydrazine derivative, or a salt thereof, preferably in the presence of a suitable inert solvent and in the presence of a suitable base.
  • suitable hydrazine derivatives include alkyl hydrazines such as tert-butyl hydrazine, preferably added to the reaction as an acid salt such as an HC1 salt.
  • Suitable inert solvents include those described above in the enol synthesis reaction.
  • the solvent is an alkyl alcohol, for example ethanol.
  • Suitable bases for use in the cyclization reaction include bases described for use in the enol synthesis described above.
  • an amine base is employed, such as triethylamine.
  • the cyclization reaction is conveniently carried out at a temperature in the range from about 30° C. to about 100° C, preferably in the range from about 50° C to about 80° C, most conveniently at the reflux temperature of the reaction mixture.
  • the pyrazole 3 may be isolated from the reaction mixture, for example by removing the volatiles and subjecting the residue obtained thereby to chromatographic separation.
  • C. Pyrazole 3 is reacted with formamide to generate the product pyrazolopyrimidine of formula Ila.
  • the reaction is preferably performed at an elevated temperature from about 50° C. to about 200° C, preferably in the range from about 100° C to about 200° C, most conveniently at the reflux temperature of the reaction mixture. 181
  • haloalkyl ketone 4 is reacted with aryl amine 5, preferably in the presence of a suitable inert solvent and in the presence of a suitable base, to generate intermediate 6.
  • suitable solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as DMSO, DMF or NMP.
  • the solvent is an alkyl alcohol such as, for example, ethanol.
  • Suitable bases for the reaction include organic amine bases such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or l,8-diazabicyclo[5.4.0]undecane; alkali or 4181
  • alkaline earth metal carbonates or hydroxides for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide
  • alkali metal or alkaline earth metal amides for example sodium amide or sodium bis(trimethylsilyl)amide
  • reagents that are immobilized on a solid phase support such as solid phase amine bases, e.g., diisopropylethylamine bound to polystyrene.
  • the reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably in the range from about 50° C to about 100° C, and conveniently at the reflux temperature of the reaction mixture.
  • Suitable solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as DMSO, DMF, or NMP.
  • alkyl alcohols such as methanol, ethanol, and isopropanol
  • esters such as methyl acetate or ethyl acetate
  • halogenated solvents such as methylene chloride, chloroform or carbon tetrachloride
  • ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethyl
  • Suitable bases include bases capable of removing a proton from malononitrile such as, for example, sodium metal, lithium metal, lithium naphthalide, sodium hydride or potassium hydride.
  • the reaction is conveniently carried out at a temperature in the range from about 0° C. to about 100° C, preferably at the reflux temperature of the reaction mixture.
  • C. Pyrrole intermediate 7 is reacted with a phosgene equivalent such as triethyl orthoformate, with or without additional solvent.
  • the reaction is conveniently carried out at an elevated temperature, preferably in the range of from about 100° C. to about 200° C, preferably at the reflux temperature of the reaction mixture.
  • An intermediate addition product is isolated by concentrating the reaction mixture under vacuum and removing the precipitated product by filtration. The intermediate addition product is taken up in a suitable inert 18
  • Suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol.
  • the reaction is conveniently carried out at an elevated temperature, conveniently in the range of from about 100° C to about 200° C, preferably from about 100° C to about 150° C.
  • the product amino pyrrolopyrimidine may be isolated by cooling the reaction mixture and removing the solid precipitated produce by filtration.
  • 2-fluoronitrobenzene analog 8 is reacted with a malonate ester, preferably in the presence of a suitable inert solvent, and in the presence of a suitable base.
  • suitable inert solvents include, for example, ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, for example, DMSO, DMF, or NMP.
  • Suitable bases include bases capable of removing a proton from a malonate ester such as, for example, sodium metal, lithium metal, lithium naphthalide, sodium hydride or potassium hydride.
  • the reaction is conveniently carried out at a temperature in the range from about 25° C to about 150° C, preferably at about 100 C.
  • the intermediate 2-phenyl malonate 9 is subjected to hydrolytic conditions, such as, for example an aqueous strong acid such as aqueous hydrochloric acid, to produce the phenyl acetic acid intermediate 10.
  • the hydrolytic reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably at about 100° C.
  • the intermediate phenyl acetic acid 10 is sub ected to reduction, preferably in the presence of a suitable inert solvent, to yield intermediate substituted indolone 11.
  • Suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; and aromatic solvents such as toluene.
  • the reaction is conveniently carried out at a temperature in the range of from about 0° C to about 100° C, preferably about room temperature.
  • the reduction conditions are preferably catalytic hydrogenation conditions.
  • the reduction is preferably performed in the presence of a hydrogen source, such as, for example pressurized hydrogen gas, in the presence of a transition metal catalyst such as for example tin, iron, platinum, palladium or zinc.
  • a hydrogen source such as, for example pressurized hydrogen gas
  • a transition metal catalyst such as for example tin, iron, platinum, palladium or zinc.
  • Intermediate indolone 11 is reacted with aryl aldehyde intermediate 12, preferably in the presence of a suitable inert solvent.
  • suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or t-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, DMSO, DMF, or NMP.
  • the reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably at the reflux temperature of the reaction mixture.
  • Aldehyde intermediate 13 is reacted with aryl or heteroaryl acetonitrile 14, preferably in the presence of a suitable inert solvent.
  • suitable inert solvents include for example alkyl alcohols such as methanol, ethanol, and isopropanol; esters such as ethyl acetate; halogenated solvents such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran or 1,4-dioxan; aromatic solvents such as toluene; and polar aprotic solvents such as DMF, NMP or DMSO. Alcohols, particularly ethanol and isopropanol are preferred.
  • the reaction is carried out at a temperature in the range from about 25° C. to about 150° C, preferably in the range from about 25° C to about 100° C. Most preferably, the reaction is carried out at the reflux temperature of the reaction mixture.
  • the ⁇ , ⁇ -unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides useful in the claimed method may be prepared by organic synthesis using techniques that are known in the art or readily adapted from techniques known in the art. The following general synthesis methods are representative of methods whereby the compounds may be prepared.
  • a mercaptan 15 is slowly added to a solution of sodium hydroxide (8 g, 0.2 mol) in methanol (200 mL). Then, chloroacetic acid (0.1 mol) is added portiomvise and the reaction mixture may be refluxed for 2-3 hours, then cooled to ambient temperature. The cooled reaction mixture is poured onto crushed ice and neutralized with dilute hydrochloric acid (200 mL). The resulting thioacetic acid 16 (0.1 mol) may be oxidized to the corresponding sulfonyl acetic acid 17b by use of any reagent capable of oxidizing a sulf ⁇ de to a sulfone.
  • the thioacetic acid 16 may alternately be oxidized to the sulfinyl acetic acid 17a by treatment with any reagent capable of selectively oxidizing a sulf ⁇ de to a sulfoxide.
  • Suitable oxidizing reagents for both oxidation reactions include peroxides such as hydrogen peroxide, peracids such as meta-chloroperoxybenzoic acid (MCPBA) or persulfates such as OXONE® (potassium peroxymonosulfate).
  • MCPBA meta-chloroperoxybenzoic acid
  • OXONE® potential peroxymonosulfate
  • the reaction is preferably carried out in the presence of a suitable solvent.
  • Suitable solvents include, for example, water, acetic acid or non-polar solvents such as dichloromethane (DCM).
  • Reaction to selectively form the sulfinyl acetic acid 17a is preferably performed at low temperature, more preferably from about -10 to about 20°C.
  • a reaction to form the sulfinyl acetic acid 17a is preferably monitored so as to terminate the reaction prior to appreciable oxidation to the sulfonyl acetic acid 17b.
  • the reaction mixture may be poured onto crushed ice.
  • a solid precipitate may be collected by filtration and recrystallized from hot water to yield the purified sulfinyl acetic acid 17a.
  • Reaction to form the sulfonyl acetic acid 17 b may be performed at higher temperature, for example, from about 30 to about 100°C with 30% hydrogen peroxide (0.12 mol) in glacial acetic acid (25 mL) by refluxing for 1-2 hours.
  • the reaction mixture may be cooled to ambient temperature and poured onto crushed ice. A solid precipitate may be collected by filtration and recrystallized from hot water to yield the purified sulfonyl acetic acid 17b.
  • the ⁇ , ⁇ -unsaturated sulfone 19b may be prepared by mixing the sulfonyl acetic acid 17b (0.001 mol), an aromatic aldehyde 18 (0.001 mol) and benzylamine (1 mL) in glacial acetic acid (15 mL) and heating the mixture at reflux temperature for 2-3 hours.
  • the ⁇ , ⁇ -unsaturated sulfoxide 19a may be prepared by mixing the sulfinyl acetic acid 17a (0.001 mol), an aromatic aldehyde 18 (0.001 mol) and benzylamine (1 mL) in glacial acetic acid (15 mL) and heating the mixture at reflux temperature for 2-3 hours.
  • the reaction mixture may be cooled to ambient temperature and treated with dry ether (50 mL). Any precipitated product may be collected by filtration. The filtrate may be diluted with more ether and washed successively with a saturated solution of sodium bicarbonate (20 mL), sodium bisulfite (20 mL), dilute hydrochloric acid (20 mL) and finally with water (35 mL). Evaporation of the dried ether layer yields a solid compound of formula V in many cases. However, in some cases a syrupy material separates and may be solidified on treatment with 2-propanol. The purity of the product may be checked by TLC (silica gel, hexane/ethyl acetate 3:1). -55-
  • an aromatic mercaptan may be converted to the corresponding sodium thiolate 21.
  • an alkyl alcohol is added an aryl acetylene 20.
  • the reaction is preferably performed at elevated temperature, more preferably at the reflux temperature of the reaction mixture.
  • the reaction mixture may be poured onto water ice.
  • the crude product may be collected by filtration and purified, preferably by recrystallization from a suitable solvent, to yield a pure (2)- , ⁇ -unsaturated (aryl or heteroaryl)sulfide 22.
  • Preferable recrystallization solvents include water miscible alcohols and aqueous mixtures of water-miscible alcohols.
  • the (Z)- ⁇ , ⁇ -unsaturated (aryl or heteroaryl)sulfide 22 may be oxidized to the corresponding sulfone by use of any reagent capable of oxidizing a sulfide to a sulfone.
  • the ( )- , ⁇ -unsaturated (aryl or heteroaryl)sulfide 22 may be oxidized to the corresponding sulfoxide by use of any reagent capable of oxidizing a sulfide to a sulfoxide.
  • Suitable reagents and conditions for oxidation to a sulfone or sulfoxide are the same as the conditions for preparation of sulfonyl acetic acid 17b and sulfinyl acetic acid 17a.
  • unsaturated (aryl or heteroaryl)sulfone or sulfoxide may be ascertained by thin layer chromatography and geometrical configuration may be assigned by analysis of infrared and nuclear magnetic resonance spectral data.
  • Sulfonamides of formula V containing an (£)-double bond may be prepared according to the method of Reddy et al, WO 02/067865, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 9. 1. Na 2 S0 3 C
  • the sodium sulfoacetate intermediate is then reacted with a chlorinating agent, preferably PCI 5 , to form the methyl (or ethyl) ⁇ -chlorosulfonylacetate intermediate 24.
  • a chlorinating agent preferably PCI 5
  • Reaction of intermediate 24 with the aromatic amine 25 yields the arylaminosulfonylacetate intermediate 26.
  • the latter reaction is conducted in a nonprotic solvent in the presence of a base.
  • the same compound may serve as both the nonprotic solvent and the base.
  • dual-function solvents include, for example, pyridine, substituted pyridines, trimethylamine and triethylamine.
  • arylaminosulfonylacetate 26 is then converted to the corresponding arylaminosulfonylacetic acid compound 27 by any base capable of hydrolyzing the ester function of 26 to an acid.
  • bases include, for example, KOH and NaOH.
  • the arylaminosulfonylacetic acid compound is condensed with arylaldehyde 18 in the presence of a basic catalyst via a Knoevenagel reaction and decarboxylation of an intermediate.
  • Basic catalysts include, for example, pyridine and benzylamine. The reaction yields the desired N-(aryl)-2-arylethenesulfonamide of formula V.
  • Carboxamides of Formula V (£)- ⁇ , ⁇ -unsaturated (aryl or heteroaryl) carboxamides of formula II may be prepared according to the method of Reddy et al, US provisional patent application 60/406,766, filed August 29. 2002. and PCT Patent Application WO 04037751. filed August 28. 2003 and published May 6. 2004. the entire disclosures of which are incorporated herein by reference. The method is described in Scheme 10.
  • an intermediate (E)- or ( ⁇ -aromatic acryloylhalide 34 may be prepared from the corresponding aromatic acrylic acid 33.
  • the aromatic acrylic acid is reacted with a halogenating agent such as for example, thionyl chloride or phosphorous pentachloride to form the intermediate carboxylic acid halide 34.
  • a halogenating agent such as for example, thionyl chloride or phosphorous pentachloride to form the intermediate carboxylic acid halide 34.
  • a solution of an aromatic amine 35 (10 mmol) in pyridine (75 mL) is reacted with the (E) or (Z)-aromatic acryloyl halide 34 (10 mmol) for 4 to 6 hours at 80° C.
  • therapeutic ionizing radiation may be administered to an individual on any schedule and in any dose consistent with the prescribed course of treatment.
  • the radioprotective compound is administered prior to the therapeutic ionizing radiation.
  • the course of treatment differs from individual to individual, and those of ordinary skill in the art can readily determine the appropriate dose and schedule of therapeutic radiation in a given clinical situation.
  • radioprotective compound shall mean administration of a radioprotective compound according to formula I alone, or in combination with either a radioprotective compound of formula V, or an antioxidant compound.
  • the formula V compound or antioxidant compound may be administered simultaneously with the formula I compound, or may be administered separately.
  • the compounds may be administered by the same or by different routes.
  • the administration times are preferably optimized to obtain the radioprotective benefit of the combination based on the pharmacokinetic profiles of the compounds administered.
  • the formula I compound and (i) the formula V compound or (ii) the antioxidant compound are administered simultaneously, the administration may be by the same or by different routes.
  • simultaneous administration is done by administering the compounds as part of the same pharmaceutical composition.
  • the radioprotective compound should be administered far enough in advance of the therapeutic radiation such that the compound is able to reach the normal cells of the individual in sufficient concentration to exert a radioprotective effect on the normal cells.
  • the radioprotective compound may be administered as much as about 24 hours, preferably no more than about 18 hours, prior to administration of the radiation.
  • the radioprotective compound is administered at least about 6-12 hours before administration of the therapeutic radiation. Most preferably, the radioprotective compound is administered once at about 18 hours and again at about 6 hours before the radiation exposure.
  • One or more radioprotective compounds may be administered simultaneously, or different radioprotective compounds may be administered at different times during the treatment.
  • radioprotective compounds may be administered either simultaneously or at different times during the treatment.
  • an about 24 hour period separates administration of a radioprotective compound and the therapeutic radiation.
  • the administration of a radioprotective compound and the therapeutic radiation is separated by about 6 to 18 hours. This strategy will yield significant reduction in radiation-induced side effects without affecting the anticancer activity of the therapeutic radiation.
  • therapeutic radiation at a dose of 0.1 Gy may be given daily for five consecutive days, with a two-day rest, for a total period of 6-8 weeks.
  • One or more compounds according to the invention may be administered to the individual 18 hours previous to each round of radiation. It should be pointed out, however, that more aggressive treatment schedules, i.e., delivery of a higher dosage, is contemplated according to the present invention due to the protection of the normal cells afforded by the radioprotective compounds.
  • the radioprotective effect of the radioprotective compound increases the therapeutic index of the therapeutic radiation, and may permit the physician to safely increase the dosage of therapeutic radiation above presently recommended levels without risking increased damage to the surrounding normal cells and tissues.
  • the radioprotective compounds of the invention are further useful in protecting normal bone marrow cells from radiological treatments designed to destroy hematological neoplastic cells or tumor cells which have metastasized into the bone marrow.
  • Such cells include, for example, myeloid leukemia cells.
  • the appearance of these cells in the bone marrow and elsewhere in the body is associated with various disease conditions, such as the French-American-British (FAB) subtypes of acute myelogenous leukemias (AML), chronic myeloid leukemia (CML), and acute lymphocytic leukemia (ALL).
  • FAB French-American-British subtypes of acute myelogenous leukemias
  • CML chronic myeloid leukemia
  • ALL acute lymphocytic leukemia
  • CML in particular, is characterized by abnormal proliferation of immature granulocytes (e.g., neutrophils, eosinophils, and basophils) in the blood, bone marrow, spleen, liver, and other tissues and accumulation of granulocytic precursors in these tissues.
  • immature granulocytes e.g., neutrophils, eosinophils, and basophils
  • the individual who presents with such symptoms will typically have more than 20,000 white blood cells per microliter of blood, and the count may exceed 400,000.
  • Virtually all CML patients will develop "blast crisis", the terminal stage of the disease during which immature blast cells rapidly proliferate, leading to death.
  • Other individuals suffer from metastatic tumors, and require treatment with total body irradiation (TBI).
  • TBI total body irradiation
  • TBI will also kill the individual's hematopoietic cells
  • a portion of the individual's bone marrow is removed prior to irradiation for subsequent reimplantation.
  • metastatic tumor cells are likely present in the bone marrow, and reimplantation often results in a relapse of the cancer within a short time.
  • Individuals presenting with neoplastic diseases of the bone marrow or metastatic tumors may be treated by removing a portion of the bone marrow (also called “harvesting"), purging the harvested bone marrow of malignant stem cells, and reimplanting the purged bone marrow.
  • the individual is simultaneously treated with radiation or some other anti-cancer therapy.
  • the invention provides a method of reducing the number of malignant cells in bone marrow, comprising the steps of removing a portion of the individual's bone marrow, administering an effective amount of at least one radioprotective compound of formula I and irradiating the treated bone marrow with a sufficient dose of ionizing radiation such that neoplastic or tumor cells in the bone marrow are killed.
  • malignant cell means any uncontrollably proliferating cell, such a tumor cell or neoplastic cell.
  • the radioprotective compound protects the normal hematopoietic cells present in the bone marrow from the deleterious effects of the ionizing radiation.
  • each radioprotective compound is administered in a concentration from about 0.25 to about 100 micromolar; more preferably, from about 1.0 to about 50 micromolar; in particular from about 2.0 to about 25 181
  • the radioprotective compound may be added directly to the harvested bone marrow, but are preferably dissolved in an organic solvent such as dimethylsulfoxide (DMSO). Pharmaceutical formulations of radioprotective compounds such as are described in more detail below may also be used.
  • DMSO dimethylsulfoxide
  • the radioprotective compound is added to the harvested bone marrow about 20 hours prior to radiation exposure, preferably no more than about 24 hours prior to radiation exposure. In one embodiment, the radioprotective compound is administered to the harvested bone marrow at least about 6 hours before radiation exposure.
  • One or more radioprotective compounds of formula I may be administered simultaneously, or different radioprotective compounds may be administered at different times. Other dosage regimens may also be used. If the individual is to be treated with ionizing radiation prior to reimplantation of the purged bone marrow, the individual may be treated with one or more radioprotective compounds of formula I prior to receiving the ionizing radiation dose, as described above.
  • An individual may also be exposed to ionizing radiation from occupational or environmental sources, as discussed in the background section.
  • the source of the radiation is not as important as the type (i.e., acute or chronic) and dose level absorbed by the individual. It is understood that the following discussion encompasses ionizing radiation exposures from both occupational and environmental sources. Individuals suffering from effects of acute or chronic exposure to ionizing radiation that are not immediately fatal are said to have remediable radiation damage. Such remediable radiation damage can be reduced or eliminated by the compounds and methods of the present invention.
  • An acute dose of ionizing radiation which may cause remediable radiation damage includes a localized or whole body dose, for example, between about 10,000 millirem (0.1 Gy) and about 1,000,000 millirem (10 Gy) in 24 hours or less, preferably between about 25,000 millirem (0.25 Gy) and about 200,000 (2 Gy) in 24 hours or less, and more preferably between about 100,000 millirem (1 Gy) and about 150,000 millirem (1.5 Gy) in 24 hours or less.
  • a chronic dose of ionizing radiation which may cause remediable radiation damage includes a whole body dose of about 100 millirem (.001 Gy) to about 10,000 millirem (0.1 Gy), preferably a dose between about 1000 millirem (.01 Gy) and about 5000 millirem (.05 Gy) over a period greater than 24 hours, or a localized dose of 15,000 millirem (0.15 Gy) to 50,000 millirem (0.5 Gy) over a period greater than 24 hours.
  • the invention therefore provides a method for treating individuals who have incurred remediable radiation damage from acute or chronic exposure to ionizing radiation, comprising reducing or eliminating the cytotoxic effects of radiation exposure on normal cells and tissues by administering an effective amount of at least one radioprotective compound of formula I.
  • the compound is preferably administered in as short a time as possible following radiation exposure, for example between 0 - 6 hours following exposure.
  • Remediable radiation damage may take the form of cytotoxic and genotoxic (i.e., adverse genetic) effects in the individual.
  • a method of reducing or eliminating the cytotoxic and genotoxic effects of radiation exposure on normal cells and tissues comprising administering an effective amount of at least one radioprotective compound prior to acute or chronic radiation exposure.
  • the compound may be administered, for example about 24 hours prior to radiation exposure, preferably no more than about 18 hours prior to radiation exposure.
  • the compound is administered at least about 6 hours before radiation exposure. Most preferably, the compound is administered at about 18 and again at about 6 hours before the radiation exposure.
  • One or more radioprotective compounds of formula I may be administered simultaneously, or different compounds may be administered at different times.
  • the administration times are preferably optimized to obtain the radioprotective benefit of the combination based on the pharmacokinetic profile of the compounds administered.
  • the administration may be by the same or by different routes.
  • simultaneous administration of more than one compound of formula I is done by administering the compounds as part of the same pharmaceutical composition.
  • the radioprotective compound may be administered multiple times. For example, if fire or rescue personnel must enter contaminated areas multiple times, the radioprotective compound may be administered prior to each exposure.
  • an about 24-hour period separates administration of radioprotective compound and the radiation exposure. More preferably, the administration of a radioprotective compound and the radiation exposure is separated by about 6 to 18 hours. It is also contemplated that a worker in a nuclear power plant may be administered an effective amount of radioprotective compound prior to beginning each shift, to reduce or eliminate the effects of exposure to ionizing radiation. If an individual anticipates chronic exposure to ionizing radiation, the radioprotective compound may be administered periodically throughout the duration of anticipated exposure. For example, a nuclear power plant worker or a soldier operating in a forward area contaminated with radioactive fallout may be given a radioprotective compound of formula I every 24 hours, preferably every 6-18 hours, in order to mitigate the effects of radiation damage. Likewise, the radioprotective compound may be periodically administered to civilians living in areas contaminated by radioactive fallout until the area is decontaminated or the civilians are removed to a safer environment. 4181
  • administered means the act of making the radioprotective compounds of formula I (alone, or in combination with a compound of formula V or an antioxidant) available to the individual such that a pharmacological effect of radioprotection is realized.
  • This pharmacological effect may manifest as the absence of expected physiologic or clinical symptoms at a certain level of radiation exposure.
  • One skilled in the art may readily determine the presence or absence of radiation-induced effects, by well- known laboratory and clinical methods.
  • the radioprotective compound may thus be administered by any route which is sufficient to bring about the desired radioprotective effect in the patient.
  • Routes of administration include, for example enteral (e.g., oral, rectal, intranasal, etc.) and parenteral administration.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal, intravesical (e.g., into the bladder), intradermal, topical or subcutaneous administration.
  • parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal, intravesical (e.g., into the bladder), intradermal, topical or subcutaneous administration.
  • a depot of a radioprotective compounds of formula I may be administered to the patient more than 24 hours before the administration of radiation.
  • at least a portion of the compound is retained in the depot and not released until an about 6-18 hour window prior to the radiation exposure.
  • the radioprotective compound may be administered in the form of a pharmaceutical composition comprising one or more compounds of formula I in combination with a pharmaceutically acceptable carrier.
  • the active compound in such formulations may comprise from 0.1 to 99.99 weight percent.
  • pharmaceutically acceptable carrier is meant any carrier, diluent or excipient, which is compatible with the other ingredients of the formulation and is not deleterious to the individual. It is within the skill in the art to formulate appropriate pharmaceutical compositions with radioprotective compounds.
  • the radioprotective compound may be formulated into pharmaceutical compositions according to standard practices in the field of pharmaceutical preparations. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, PA.
  • Suitable pharmaceutical compositions include, for example, tablets, capsules, solutions (especially parenteral solutions), troches, suppositories, or suspensions.
  • the radioprotective compound may be mixed with a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose (glucose) and related sugar solutions, cyclodextrans or a glycol such as propylene glycol or polyethylene glycol.
  • Solutions for parenteral administration preferably contain a pharmaceutically acceptable, water-soluble salt of the radioprotective compound. Stabilizing agents, antioxidizing agents and preservatives may also be added.
  • Suitable antioxidizing agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • the radioprotective compound may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, or other suitable oral dosage forms.
  • the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
  • the specific dose and schedule of radioprotective compound to obtain the radioprotective benefit will, of course, be determined by the particular circumstances of the individual patient including, the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease, and the route of administration, and the specific toxicity of the radiation.
  • a daily dosage of from about 0.01 to about 150 mg/kg/day may be utilized, more preferably from about 0.05 to about 50 mg/kg/day.
  • the dose may be given over multiple administrations, for example, two administrations of 3.5 mg/kg. Higher or lower doses are also contemplated.
  • the radioprotective compounds may take the form of pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable.
  • Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid.
  • organic acids may be selected from the group consisting of aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, beta-hydroxybutyric, galactaric
  • Suitable pharmaceutically acceptable base addition salts include metallic salts made from calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,iV- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding radioprotective compound by reacting, for example, the appropriate acid or base with the free acid or free base of the compound.
  • the compositions useful in the method of the present invention may also be formulated so as to provide slow or controlled-release of the active ingredient therein.
  • a controlled-release preparation is a composition capable of releasing the active ingredient at the required rate to maintain constant 181
  • Such dosage forms may provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than other non-controlled formulations.
  • U.S. Patent No. 5,674,533 discloses controlled-release compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive.
  • U.S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances.
  • U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers.
  • U.S. Patent No. 5,073,543 discloses controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle.
  • U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. The patents cited above are incorporated herein by reference.
  • Biodegradable microparticles may be used in controlled-release formulations useful in the method of this invention.
  • U.S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient.
  • U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions. These patents are incorporated herein by reference.
  • the controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • the controlled-release component can swell and form porous openings large enough to release the active ingredient after administration to a patient.
  • controlled-release component in the context of the present invention is defined herein as a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres, that facilitate the controlled-release of the radioprotective compound of formula I in a pharmaceutical composition.
  • the controlled-release component may be biodegradable, induced by exposure to the aqueous environment, pH, temperature, or enzymes in the body.
  • sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature.
  • This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.
  • a patient preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.
  • the practice of the invention is illustrated by the following non- limiting examples. Examples
  • Example 1 Synthesis of 5-((lZ)-l-cyano-2-indol-3-yIvinyl)-3- amin ⁇ pyrazole-4-carbonitriIe.
  • the title compound of Example 1, a tyrphostin compound of formula IV, is synthesized according to Scheme 12.
  • Indole-3-carboxaldehyde la [487-89-8] Sigma Aldrich, 12,944-5)(0.29 g, 2 mmol)
  • 3-amino-4-cyano-5-cyanomethyl-2-pyrazole lb (0.29 g, 2 mmol)
  • ⁇ -alanine (20 mg) are heated at reflux in ethanol (30 mL) for four hours.
  • reaction mixture is then cooled to ambient temperature and filtered to yield the crude product, lc, which is further purified by preparative HPLC.
  • Example 2 Synthesis of l-(tert-butyl)-3-(4-methyIphenyI)pyrazolo[5,4- d]pyrimidine-4-ylamine.
  • the title compound of Example 2, a pyrazolopyrimidine compound of formula lib, is synthesized according to Scheme 13.
  • Example 3 Synthesis of (2- ⁇ 4-[4-amino-5-(4-methoxyphenyl)pyrroIo[2,3- d]pyrimidin-7-yl]phenoxy ⁇ ethyI)(2-methoxyethyl)amine.
  • the title compound of Example 3, a pyrrolopyrimidine compound of formula Ila is synthesized according to Scheme 14.
  • Petroleum ether is added and the purified product, (tert-butoxy)-N-(2-methoxyethyl)-N-[2- (4- ⁇ [2-(4-methoxyphenyl)-2-oxoethyl]amino ⁇ phenoxy)ethyl]carboxamide, 3f crystallizes and is collected by filtration.
  • reaction mixture is stirred overnight. A precipitate forms and is collected by filtration. The collected material is redissolved in 8% ammonia in ethanol (150 mL). The reaction mixture is heated in a pressure vessel to 130° C for four hours. After cooling to ambient temperature, the precipitated product is collected by filtration. This BOC-protected intermediate is stirred overnight at ambient temperature in DCM (100 mL) with trifluoroacetic acid (3 mL).
  • Example 4 Synthesis of 4- ⁇ 4-[(2-oxo-lH-benzo[d]azolidin-3- ylidene)methyl]phenyl ⁇ piperazinecarbaldehyde.
  • the title compound of Example 4 an indoline-2-one compound of formula III is synthesized according to Scheme 15.
  • reaction mixture is then poured into ice-cold sodium hydroxide (IN) and stirred at ambient temperature for one hour.
  • the organic layer is separated and the aqueous layer is extracted with ethyl acetate.
  • the combined organic fraction is washed with brine, dried (sodium sulfate), and concentrated under vacuum.
  • the residue is purified by column chromatography to yield 4-(l-formylpiperazin-4- yPbenzaldehyde, 4b.
  • Example 5 Inhibition of ABL Tyrosine Kinase by Compounds of Formula I: Method A. The inhibition of ABL activity by compounds of formula I is demonstrated as follows. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged, Panvera, CA) is incubated with various concentrations of a compound of formula I for 30 minutes in kinase buffer (50 mM HEPES (N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl 2 , pH 7.5) at room temperature in a total volume of 15 ⁇ l.
  • kinase buffer 50 mM HEPES (N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM
  • kinase reactions are performed by adding cold ATP and ⁇ -32P-ATP in the presence of 6 ⁇ g of ABL kinase substrate (recombinant murine Crk) for 20 minutes at 30°C.
  • the reactions are stopped by the addition of 20 ⁇ l of 2x SDS sample buffer.
  • the samples are boiled and resolved on a 10 % SDS-polyacrylamide gel. The gel is fixed, and exposed to x-ray film. Quantitation of Crk phosphorylation is determined using a phosphoimager system (Fuji).
  • Example 6 Inhibition of ABL Tyrosine Kinase by Compounds of Formula I: Method B - Quantitation using filter assay. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged, Panvera, CA) is incubated with various concentrations of compounds of formula I for 30 minutes in kinase buffer (50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl 2 , pH 7.5) at room temperature in a total volume of 15 ⁇ l.
  • kinase buffer 50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl 2 , pH 7.5
  • kinase reactions are performed by adding cold ATP and ⁇ -32P-ATP in the presence of Crk (6 ⁇ g) for 20 minutes at 30° C. After incubation, 10 ⁇ l aliquots are spotted onto 2 cm x 2 cm P81 phosphocellulose paper. The paper is air dried and then washed 3x with 0.75% phosphoric acid, and fixed with acetone for 5 minutes. The wet filters are then placed into scintillation vials containing scintillation fluid (Ecolume) and counted for 32P using a scintillation counter. The counts per minute (CPM) of each treated sample are compared to the amount of radioactivity resulting from control reactions in the presence of DMSO. The reactions are performed in triplicates, and the average CPM +/- SD for each reaction is plotted as percent of solvent treated control.
  • CPM counts per minute
  • Example 7 Inhibition of ABL Tyrosine Kinase by a Composition Comprising at Least One Compound of Formula I and at Least One Compound of Formula V: Abi-1 protein (Panvera) is incubated with different concentrations of a mixture of a compound of formula I and a compound of formula V such as, for example, 4-((lE)-2- ⁇ [(4-chlorophenyl)methyl]suhOnyl ⁇ vinyl)benzoic acid (Compound 23 in Table 4) in a 15 ⁇ l reaction mixture (50 mM HEPES, 10 mM MgC12, 1 M EDTA, 2 mM DTT and 0.01% NP-40, pH 7.5) for 30 min at room temperature.
  • a compound of formula V such as, for example, 4-((lE)-2- ⁇ [(4-chlorophenyl)methyl]suhOnyl ⁇ vinyl)benzoic acid (Compound 23 in Table 4) in a 15 ⁇ l
  • Kinase reactions are initiated by the addition of 2 ⁇ l of 1 mM ATP (lOO ⁇ M concentration), 2 ⁇ l of ⁇ 32pATP (40 ⁇ ci final concentration) and 6 ⁇ g of recombinant GST-Crk (Upstate). Reactions are performed for 20 min at 30°C and are stopped by the addition of 20 ⁇ l of 2XSDS-PAGE buffer, boiled and subjected to SDS-PAGE using a 12% polyacrylamide gel. Following electrophoresis, the gel is dried and exposed to X-ray film for 3-10 min.
  • Example 8 Inhibition of ABL Tyrosine Kinase by Compounds according to Formula V: Method B - Quantitation using filter assay. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged,
  • Panvera, CA was incubated with various concentrations of each compound of Formula V listed in Table 5 (Compounds 1-22) for 30 minutes in kinase buffer (50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl 2 , pH 7.5) at room temperature in a total volume of 15 ⁇ l.
  • kinase buffer 50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl 2 , pH 7.5
  • kinase reactions were performed by adding cold ATP and ⁇ - 32P-ATP in the presence of Crk (6 ⁇ g) for 20 minutes at 30° C. After incubation, 10 ⁇ l aliquots were spotted onto 2 cm x 2 cm P81 phosphocellulose paper.
  • Example 9 Radioprotective Effect of Compounds of Formula I, and Compositions Comprising Compounds of Formula I on Cultured Normal Cells: The radioprotective effect of: (a) compounds of formula I; (b) combinations of at least one compound of formula I and at least one compound of formula V; and (c) combinations of at least one compound of formula I and at least one antioxidant compound on cultured normal cells is evaluated as follows.
  • HFL-1 cells which are normal diploid lung fibroblasts, are plated into 24 well dishes at a cell density of 3000 cells per 10 mm 2 in DMEM completed with 10% fetal bovine serum and antibiotics.
  • a compound of formula I (or a composition comprising a compound of formula I) is added to the cells 24 hours later at concentrations in a range from about 0.25 to about 2.0 micromolar, using DMSO as a solvent.
  • Control cells are treated with DMSO alone.
  • the cells are exposed to the test compound (or composition) or DMSO for 24 hrs.
  • the cells are then irradiated with either 10 Gy or 15 Gy of ionizing radiation (IR) using a J.L. Shepherd Mark I, Model 30-1 Irradiator equipped with 137 cesium as a source. After irradiation, the medium on the test and control cells is removed and replaced with fresh growth medium without the test compounds or DMSO.
  • the irradiated cells are incubated for 96 hours and duplicate wells are trypsinized and replated onto 100 mm 2 tissue culture dishes.
  • the replated cells are grown under normal conditions with one change of fresh medium for 3 weeks.
  • the number of colonies from each 100 mm 2 culture dish which represents the number of surviving cells, is determined by staining the dishes as described below.
  • the cells are stained with a 1:10 diluted Modified Giemsa staining solution (Sigma) for 20 minutes. The stain is removed, and the plates are washed with tap water. The plates are air-dried, the number of colonies from each plate is counted and the average from duplicate plates is determined.
  • Example 10 Treatment of bcr-abl Transformed Leukemic Cells by a Composition Comprising at Least One Compound of Formula I and at Least One Compound of Formula V: K562 cells, a cell line isolated from a 35 year old patient with chronic myelogenous leukemia (CML), transformed due to the Philadelphia chromosome translocation resulting in the expression of bcr-abl kinase is used as the target cell line.
  • CML chronic myelogenous leukemia
  • K562 cells are plated at a cell density of 1.0 x 10 s cells/mL in 12 well dishes and treated with a constant concentration (a concentration that reduces the growth of K562 cells by no more than 20% based upon dose response assays treating with the compound of formula I alone) of a compound of formula I, and a series of concentrations of a compound of formula V. Following an incubation period of 96 hours at 37°C under 5% C0 2 , the number of viable cells remaining in each well is determined by counting using a hemacytometer and trypan blue staining. The total number of viable cells remaining is calculated and plotted as the percent of vehicle treated control cells.
  • Example 11 Effect of Exposure to Ionizing Radiation on Normal and Malignant Hematopoietic Progenitor Cell Growth After Pretreatment with Compounds of Formula I: The effect of ionizing radiation on normal and malignant hematopoietic progenitor cells which are pretreated with a compound of formula I is determined by assessing cloning efficiency and development of the pretreated cells after irradiation.
  • human bone marrow cells (BMC) or peripheral blood cells (PB) are obtained from normal healthy, or acute or chronic myelogenous leukemia (AML, CML), volunteers by Ficoll- Hypaque density gradient centrifugation, and are partially enriched for hematopoietic progenitor cells by positively selecting CD34 + cells with immunomagnetic beads (Dynal A.S., Oslo, Norway).
  • the CD34 + cells are suspended in supplemented alpha medium and incubated with mouse anti- HPCA-I antibody in 1:20 dilution, 45 minutes, at 4° C with gentle inverting of tubes.
  • CD34 + cells are washed x 3 in supplemented alpha medium, and then incubated with beads coated with the Fc fragment of goat anti-mouse IgGi (75 ⁇ l of immunobeads/107 CD34 + cells). After 45 minutes of incubation (4° C), cells adherent to the beads are positively selected using a magnetic particle concentrator as directed by the manufacturer. 2 x 10 CD34 + cells are incubated in 5 mL polypropylene tubes (Fisher Scientific, Pittsburgh, PA) in a total volume of 0.4 mL of Iscove's modified Dulbecco's medium (IMDM) containing 2% human AB serum and 10 mM Hepes buffer.
  • IMDM Iscove's modified Dulbecco's medium
  • a compound of formula I is added to the cells; in four different concentrations (0.25 ⁇ M, 0.5 ⁇ M, 1.0 ⁇ M and 2.0 ⁇ M) is added separately to the cells.
  • Control cells receive DMSO alone. The cells are incubated for 20-24 hours and irradiated with 5 Gy or 10 Gy of ionizing radiation. Immediately after irradiation, the medium is removed and replaced with fresh medium without the test compound or DMSO. Twenty-four hours after irradiation, the treatment and control cells are prepared for plating in plasma clot or methylcellulose cultures. Cells (1 x 10 4 CD34 + cells per dish) are not washed before plating.
  • Example 12 Bone Marrow Purging with Ionizing Radiation After Pretreatment with Compounds of Formula I.
  • Bone marrow is harvested from the iliac bones of an individual under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the individual will be able to receive about 4 x 10 8 to about 8 x 10 8 processed marrow cells per kg of body weight. Thus, about 750 to 1000 mL of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 mL of medium.
  • TC-199 Gibco, Grand Island, New York
  • the aspirated marrow is filtered through three progressively finer meshes to obtain a cell suspension devoid of cellular aggregates, debris and bone particles.
  • the filtered marrow is then processed further into an automated cell separator (e.g., Cobe 2991 Cell Processor) which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets).
  • the buffy coat preparation is then placed in a transfer pack for further processing and storage. It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation.
  • the purging procedure is carried out as follows.
  • Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 10 7 /mL in TC-199 containing about 20% autologous plasma.
  • a compound of formula I for example, at a concentration of from 0.25 ⁇ M to 2.0 ⁇ M is added to the transfer packs containing the cell suspension and incubated in a 37° C waterbath for 20- 24 hours with gentle shaking. The transfer packs are then exposed to 5-10 Gy ionizing radiation.
  • Recombinant human hematopoietic growth factors e.g., rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of hematopoietic neoplasms and thereby increase their sensitivity to ionizing radiation.
  • the cells may then either be frozen in liquid nitrogen or washed once at

Abstract

Pre-treatment with one or more compounds of formula (I), as described herein, protects normal cells from the toxic side effects of ionizing radiation. Administration of one or more radioprotective compounds of formula (I) to a patient prior to anticancer radiotherapy reduces the cytotoxic side effects of the radiation on normal cells. The radioprotective effect of one or more compounds of formula (I) allows for the safe increase the dosage of anticancer radiation. Amelioration of toxicity following inadvertent radiation exposure may also be mitigated with administration of one or more compounds of formula (I).

Description

Protection of Tissues and Cells from Cytotoxic Effects of Ionizing Radiation by ABL Inhibitors
Cross-Reference to Related Application This application claims the benefit of copending U.S. Provisional
Application Serial No. 60/501,748, filed September 9, 2003, the entire disclosure of which is incorporated herein by reference. Field of the Invention The invention relates to the field of protecting normal cells and tissues from anticipated, planned or inadvertent exposure to ionizing radiation. In particular, the invention relates to radioprotective agents administered to an individual prior to or after exposure to ionizing radiation, such as occurs during anticancer radiotherapy. Background of the Invention Ionizing radiation has an adverse effect on cells and tissues, primarily through cytotoxic effects. In humans, exposure to ionizing radiation occurs primarily through therapeutic techniques (such as anticancer radiotherapy) or through occupational and environmental exposure.
Sources and Effects of Ionizing Radiation A major source of exposure to ionizing radiation is the administration of therapeutic radiation in the treatment of cancer or other proliferative disorders. Individuals exposed to therapeutic doses of ionizing radiation typically receive between 0.1 and 2 Gy per treatment, and can receive as high as 5 Gy per treatment. Depending on the course of treatment prescribed by the treating physician, multiple doses may be received by an individual over the course of several weeks to several months. Therapeutic radiation is generally applied to a defined area of the individual's body which contains abnormal proliferative tissue, in order to maximize the dose absorbed by the abnormal tissue and minimize the dose absorbed by the nearby normal tissue. However, it is difficult (if not impossible) to selectively administer therapeutic ionizing radiation to the abnormal tissue. Thus, normal tissue proximate to the abnormal tissue is also exposed to potentially damaging doses of ionizing radiation throughout the course of treatment. There are also some treatments that require exposure of the individual's entire body to the radiation, in a procedure called "total body irradiation", or "TBI." The efficacy of radiotherapeutic techniques in destroying abnormal proliferative cells is therefore balanced by associated cytotoxic effects on nearby normal cells. Because of this, radiotherapy techniques have an inherently narrow therapeutic index which results in the inadequate treatment of most tumors. Even the best radiotherapeutic techniques may result in incomplete tumor reduction, tumor recurrence, increasing tumor burden, and induction of radiation resistant tumors. Numerous methods have been designed to reduce normal tissue damage while still delivering effective therapeutic doses of ionizing radiation. These techniques include brachytherapy, fractionated and hyperfractionated dosing, complicated dose scheduling and delivery systems, and high voltage therapy with a linear accelerator. However, such techniques only attempt to strike a balance between the therapeutic and undesirable effects of the radiation, and full efficacy has not been achieved. For example, one treatment for individuals with metastatic tumors involves harvesting their hematopoietic stem cells and then treating the individual with high doses of ionizing radiation. This treatment is designed to destroy the individual's tumor cells, but has the side effect of also destroying their normal hematopoietic cells. Thus, a portion of the individual's bone marrow (containing the hematopoietic stem cells) is removed prior to radiation therapy. Once the individual has been treated, the autologous hematopoietic stem cells are returned to their body. However, if tumor cells have metastasized away from the tumor's primary site, there is a high probability that some tumor cells will contaminate the harvested hematopoietic cell population. The harvested hematopoietic cell population may also contain neoplastic cells if the individual suffers from cancers of the bone marrow such as the various French- American-British (FAB) subtypes of acute myelogenous leukemias (AML), chronic myeloid leukemia (CML), or acute lymphocytic leukemia (ALL). Thus, the metastasized tumor cells or resident neoplastic cells must be removed or killed prior to reintroducing the stem cells to the individual. If any living tumorigenic or neoplastic cells are re-introduced into the individual, they can lead to a relapse. Prior art methods of removing tumorigenic or neoplastic cells from harvested bone marrow are based on a whole-population tumor cell separation or killing strategy, which typically does not kill or remove all of the contaminating malignant cells. Such methods include leukopheresis of mobilized peripheral blood cells, immunoaffinity-based selection or killing of tumor cells, or the use of cytotoxic or photosensitizing agents to selectively kill tumor cells. In the best case, the malignant cell burden may still be at 1 to 10 tumor cells for every 100,000 cells present in the initial harvest (Lazarus et al, J. Hematotherapy, 2(4):457-66, 1993). Thus, there is needed a purging method designed to selectively destroy the malignant cells present in the bone marrow, while preserving the normal hematopoietic stem cells needed for hematopoietic reconstitution in the transplantation subject. Exposure to ionizing radiation can also occur in the occupational setting. Occupational doses of ionizing radiation may be received by persons whose job involves exposure (or potential exposure) to radiation, for example in the nuclear power and nuclear weapons industries. There are currently 104 nuclear power plants licensed for commercial operation in the United States. Internationally, a total of 430 nuclear power plants are operating in 32 countries. All personnel employed in these nuclear power plants may be exposed to ionizing radiation in the course of their assigned duties. Incidents such as the March 28, 1979 accident at Three Mile Island nuclear power plant, which released radioactive material into the reactor containment building and surrounding environment, illustrate the potential for harmful exposure. Even in the absence of catastrophic events, workers in the nuclear power industry are subject to higher levels of radiation than the general public. Military personnel stationed on vessels powered by nuclear reactors, or soldiers required to operate in areas contaminated by radioactive fallout, risk similar exposure to ionizing radiation. Occupational exposure may also occur in rescue and emergency personnel called in to deal with catastrophic events involving a nuclear reactor or radioactive material. For example, the men who fought the April 26, 1986 reactor fire at the Chernobyl nuclear power plant suffered radiation exposure, and many died from the radiation effects. In August 2000, navy and civilian rescue personnel risked exposure to radiation when attempting to rescue the crew of the downed Russian nuclear-powered submarine Kursk. Salvage crews may still face radiation exposure if the submarine's reactor plant was damaged. Other sources of occupational exposure may be from machine parts, plastics, and solvents left over from the manufacture of radioactive medical products, smoke alarms, emergency signs, and other consumer goods. Occupational exposure may also occur in persons who serve on nuclear powered vessels, particularly those who tend the nuclear reactors, in military personnel operating in areas contaminated by nuclear weapons fallout, and in emergency personnel who deal with nuclear accidents. Humans and other animals (such as livestock) may also be exposed to ionizing radiation from the environment. The primary source of exposure to significant amounts of environmental radiation is from nuclear power plant accidents, such as those at Three Mile Island, Chernobyl and Tokaimura. A 1982 study by Sandia National Laboratories estimated that a "worst-case" nuclear accident could result in a death toll of more than 100,000 and long-term radioactive contamination of large areas of land. For example, the estimated number of deaths from the Chernobyl accident is from 8,000 to 300,000, and in the Ukraine alone, over 4.6 million hectares of land was contaminated with varying levels of radiation. Fallout was detected as far away as Ireland, northern Scandinavia, and coastal Alaska in the first weeks after the accident. 135,000 people were evacuated from a 30-mile radius "dead zone" around the Chernobyl plant, an area that is still not fit for human habitation. Approximately 1.2 million people continue to live in areas of low-level radiation outside the "dead-zone." Other nuclear power plant accidents have released significant amounts of radiation into the environment. The Three Mile Island accident was discussed above. In Japan, a cracked pipe leaked 51 tons of coolant water from the Tsuruga 2 nuclear plant in July of 1999. A more serious accident occurred on September 30, 1999 at a uranium reprocessing facility in Tokaimura, Japan, where 69 people received significant radiation exposure. The accident occurred when workers inadvertently started a self-sustaining nuclear chain reaction, causing a release of radiation into the atmosphere. A radiation count of 0.84 mSv/hour (4000 times the annual limit) was detected in the immediate area. Thirty-nine households (150 people) were evacuated and 200 meter radius around the site was declared Off-limits. The roads within a 3 kilometer radius of the site were closed and residents within 10 kilometer radius of the site were advised to stay indoors. The Tokaimura "criticality event" is ranked as the third most serious accident - behind Three Mile Island and Chernobyl - in the history of the nuclear power industry. Environmental exposure to ionizing radiation may also result from nuclear weapons detonations (either experimental or during wartime), discharges of actinides from nuclear waste storage and processing and reprocessing of nuclear fuel, and from naturally occurring radioactive materials such as radon gas or uranium. There is also increasing concern that the use of ordnance containing depleted uranium results in low-level radioactive contamination of combat areas. Radiation exposure from any source can be classified as acute (a single large exposure) or chronic (a series of small low-level, or continuous low-level exposures spread over time). Radiation sickness generally results from an acute exposure of a sufficient dose, and presents with a characteristic set of symptoms that appear in an orderly fashion, including hair loss, weakness, vomiting, diarrhea, skin bums and bleeding from the gastrointestinal tract and mucous membranes. Genetic defects, sterility and cancers (particularly bone marrow cancer) often develop over time. Chronic exposure is usually associated with delayed medical problems such as cancer and premature aging. An acute total body exposure of 125,000 millirem may cause radiation sickness. Localized doses such as are used in radiotherapy may not cause radiation sickness, but may result in the damage or death of exposed normal cells. For example, an acute total body radiation dose of 100,000 - 125,000 millirem (equivalent to 1 Gy) received in less than one week would result in observable physiologic effects such as skin bums or rashes, mucosal and GI bleeding, nausea, diarrhea and/or excessive fatigue. Longer term cytotoxic and genetic effects such as hematopoietic and immunocompetent cell destruction, hair loss (alopecia), gastrointestinal, and oral mucosal sloughing, venoocclusive disease of the liver and chronic vascular hyperplasia of cerebral vessels, cataracts, pneumonites, skin changes, and an increased incidence of cancer may also manifest over time. Acute doses of less than 10,000 millirem (equivalent to 0.1 Gy) typically will not result in immediately observable biologic or physiologic effects, although long term cytotoxic or genetic effects may occur. A sufficiently large acute dose of ionizing radiation, for example 500,000 to over 1 million millirem (equivalent to 5 - 10 Gy) may kill an individual immediately. Doses in the hundreds of thousands of millirems may kill within 7 to 21 days from a condition called "acute radiation poisoning." Reportedly, some of the Chernobyl firefighters died of acute radiation poisoning, having received acute doses in the range of 200,000 - 600,000 millirem (equivalent to 2 - 6 Gy). Acute doses below approximately 200,000 millirem do not result in death, but the exposed individual will likely suffer long-term cytotoxic or genetic effects as discussed above. Acute occupational exposures usually occur in nuclear power plant -7-
workers exposed to accidental releases of radiation, or in fire and rescue personnel who respond to catastrophic events involving nuclear reactors or other sources of radioactive material. Suggested limits for acute occupational exposures in emergency operations were developed by the Brookhaven National Laboratories, and are given in Table 1. For radiation doses listed in column 1 of Table 1, 100,000 millirem equals one sievert (Sv). For penetrating radiation such as gamma radiation, one Sv equals approximately one Gray (Gy). Thus, the dosage in Gy can be estimated as 1 Gy for every 100,000 millirem.
Table 1:
Figure imgf000008_0001
A chronic dose is a low level (i.e., 100 - 5000 millirem) incremental or continuous radiation dose received over time. Examples of chronic doses include a whole body dose of approximately 5000 millirem per year, which is the dose typically received by an adult working at a nuclear power plant. By contrast, the Atomic Energy Commission recommends that members of the general public should not receive more than 100 millirem per year. Chronic doses may cause long-term cytotoxic and genetic effects, for example manifesting as an increased risk of a radiation-induced cancer developing later in life. Recommended limits for chronic exposure to ionizing radiation are given in Table 2. Table 2:
Figure imgf000008_0002
By way of comparison, Table 3 sets forth the radiation doses from common sources.
Table 3:
Figure imgf000009_0001
Chronic doses of greater tiian 5000 millirem per year (0.05 Gy per year) may result in long-term cytotoxic or genetic effects similar to those described for persons receiving acute doses. Some adverse cytotoxic or genetic effects may also occur at chronic doses of significantly less than 5000 millirem per year. For radiation protection purposes, it is assumed that any dose above zero can increase the risk of radiation-induced cancer (i.e., that there is no threshold). Epidemiological studies have found that the estimated lifetime risk of dying from cancer is greater by about 0.04% per rem of radiation dose to the whole body. While anti-radiation suits or other personal protective equipment (PPE) may be effective at reducing radiation exposure, such specialized PPE is expensive, unwieldy, and generally not available to public. Moreover, radioprotective PPE will not protect normal tissue adjacent a tumor from stray radiation exposure during radiotherapy. What is needed, therefore, is a practical way to protect individuals who are scheduled to incur, or are at risk for incurring, exposure to ionizing radiation. In the context of therapeutic irradiation, it is desirable to enhance protection of normal cells while causing tumor cells to remain vulnerable to the detrimental effects of the radiation. Furthermore, it is desirable to provide systemic protection from anticipated or inadvertent total body irradiation, such as may occur with occupational or environmental exposures, or with certain therapeutic techniques. Pharmaceutical radioprotectants offer a cost-efficient, effective and easily available alternative to radioprotective gear. However, previous attempts at radioprotection of normal cells with pharmaceutical compositions have not been entirely successful. For example, cytokines directed at mobilizing the peripheral blood progenitor cells confer a myeloprotective effect when given prior to radiation (Neta et al, Semin. Radiat. Oncol. 6:306-320, 1996), but do not confer systemic protection. Other chemical radioprotectors administered alone or in combination with biologic response modifiers have shown minor protective effects in mice, but application of these compounds to large mammals was less successful, and it was questioned whether chemical radioprotection was of any value (Maisin, J.R., Bacq and Alexander Award Lecture. "Chemical radioprotection: past, present, and future prospects," Int. J. Radiat. Biol 73:443- 50, 1998). Pharmaceutical radiation sensitizers, which are known to preferentially enhance the effects of radiation in cancerous tissues, are clearly unsuited for the general systemic protection of normal tissues from exposure to ionizing radiation.
ABL protein Kinase Protein kinases in general are enzymes that catalyze the phosphorylation of hydroxy groups on tyrosine, serine and threonine residues of proteins. The phosphorylation serves as a basis for cellular signaling that regulates such fundamental cellular functions such as cellular growth, differentiation and proliferation. Accordingly, disorders associated with abnormal protein kinase activity have included many cancers and other proliferative disorders. Protein kinases are divided into tyrosine kinases and serine-threonine kinases. The protein tyrosine kinases are further classified as receptor tyrosine kinases and non-receptor tyrosine kinases, which are also called cellular tyrosine kinases (CTK's). Receptor tyrosine kinases include epithelial growth factor receptors (EGFR), insulin-like growth factor receptors (IGFR), platelet-derived growth factor receptors (PDGFR), fibroblast growth factor receptors (FGFR) and vascular endothelial growth factor receptors (VEGF). Non-receptor tyrosine kinases include Abelson (ABL) tyrosine kinase as well as ten other subfamilies of CTK's. ABL is a tyrosine kinase expressed by the c-abl proto-oncogene.
Cloning of the c-abl gene has revealed that it spans at least 230kb, and contains at least 11 exons. Two alternative first exons exist, namely exon la and exon lb, which are spliced to the common splice acceptor site, exon 2. Exon la is 19 kb proximal to exon 2. Exon lb, which is somewhat smaller than exon la, is more than 200 kb proximal to exon 2. As a result of this configuration, at least two major c-abl messages are transcribed, differing in their 5' regions. See, Shtivelman et al, Cell 47, 277 (1986); Bernards et al, Mol. Cell Biol. 7, 3231 (1987); and Fainstein et al, Oncogene 4, 1477-1481 (1989), the entire disclosures of which are incorporated herein by reference. If exon lb is used, the mRNA is 7.0 kb. If exon la is used, the mRNA is 6.0 kb. Each of exons la and lb are preceded by a transcriptional promoter. The 6-kb c-abl transcript consists of exons la through 11. The 7-kb transcript begins with exon lb, skips the 200 kb distance to exon 2, omits exon la, and joins to exons 2 through 11. Thus, both c-abl messages share a common set of 3' exons, starting from the c-abl exon 2. Consequently, the messages code for two proteins that share most of their amino acid sequence, except for the N- termini. Since the coding begins with the first exon, exonic selection will determine the protein product. Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease caused by malignant transformation of stem cells as a result of fusion of the c-abl gene to the bcr gene. See, Calabretta et al, US patent 5,652,222, the entire disclosure of which is incorporated herein by reference. At the molecular level, the most notable feature of CML is the translocation of the proto-oncogene c-abl from the long arm of chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22, resulting in the formation of bcr-abl hybrid genes. The break occurs near the end of the long arm of chromosome 9 (band 9q34) and in the upper half of chromosome 22 (band 22ql 1). The 9;22 translocation in CML results in the abnormal juxtaposition of abl sequences adjacent to bcr sequences. The c-abl proto-oncogene is expressed in normal cells and plays a critical role in regulating normal hematopoiesis by encoding a protein with tyrosine kinase activity. This activity is augmented in cells carrying bcr-abl hybrid genes. The gene located at the breakpoint on chromosome 22 is called bcr because the break in chromosome 22 in CML occurs in a small 5.8-kilobase (kb) segment (breakpoint cluster region) of the gene on chromosome 22. The fusion of the BCR gene with c-abl leads to an 8.5 kb chimeric mRNA consisting of 5' BCR sequences and 3' abl sequences. The chimeric message is in turn translated into a larger chimeric abl protein (210 kDa) that has increased tyrosine kinase activity See, Konopka et al, Cell 37, 1035 (1984); Kloetzer et al, Virology 140, 230 (1985); Konopka et al, Proc. Natl Acad. Sci. U.S.A. 82, 1810 (1985), the entire disclosures of which are incorporated herein by reference. The 210 kDa protein is considerably larger than the normal human protein of 145 kDa, and has a very high tyrosine kinase activity. Recently, the drug {4-[(4-methylpiperazinyl)methyl]phenyl}-N-{4- methyl-3-[(4-(3-pyridyl)-pyrimidin-2-yl)amino]phenyl}carboxamide has been shown to selectively inhibit ABL activity and to be useful in the treatment of chronic myelogenous leukemia (CML). See Fabbro et al, Current Opinion in Drug Discovery & Development, 2002, Vol. 5, No. 5, page 701-712, the entire disclosure of which is incorporated herein by reference. Zimmerman et al. discloses a structure activity relationship (SAR) of a group of 2- anilinopyrimidine compounds (including {4-[(4- methylpiperazmyl)methyl]phenyl}-N-{4-methyl-3-[(4-(3-pyridyl)-pyrimidin-2- yl)amino]phenyl}carboxamide) for ABL inhibition. See, Zimmerman; Bioorg. & Med. Chem. Lett., Vol. 7, No. 2, pages 187-192, 1997, the entire disclosure of which is incorporated herein by reference. Several experimental compounds have also been shown to selectively inhibit ABL activity, e.g., Warmuth et al. Blood, Vol. 101, No. 2, January 15, 2003) (disclosing pyrrolopyrimidine compounds); Wisniewski et al., Cancer Research, Vol. 62, pages 4244-4255, August 1, 2002 (disclosing pyridopyrimidine compounds); Boschelli et al, US Patent 6,521,618 (disclosing 4-anilino-3-quinolinecarbonitriles); Gibson et al, WO 96/33980 (disclosing quinazoline compounds that demonstrate an SAR parallel to the 4-anilino-3- quinolinecarbonitriles against receptor tyrosine kinases); Kaur et al, Anticancer Drugs, 1994, Vol. 5, pages 213-222 (disclosing tyrphostin compounds); and Battastini et al, WO 97/46551 (disclosing indoline-2-ones). The entire disclosures of the above references are incorporated herein by reference.
Antioxidant Compounds Oxidants are produced as part of the normal metabolism of all cells but also are an important component of the pathogenesis of many disease processes. Reactive oxygen species (ROS), for example, contribute to the pathogenesis of diseases of the lung, the central nervous system (CNS) and skeletal muscle.
Oxygen free radicals also modulate the effects of nitric oxide, thereby contributing to the pathogenesis of vascular disorders, inflammatory diseases and the aging process. Free radicals are molecules with one or more unpaired electrons. Free radicals act as oxidants, rapidly reacting with other molecules, and starting oxidative chain reactions. Free radicals are a normal product of 4181
-13-
metabolis . However, ionizing radiation can significantly increase the number of free radicals in the body. Certain antioxidant compounds have been shown to have cytoprotective properties. Antioxidants are believed to act by scavenging free radicals. Normal cell and organ function is maintained via a balance of oxidant and antioxidant agents. Many antioxidant compounds, e.g., the enzyme superoxide dismutases (SODs) are produced physiologically and balance the naturally occurring free radicals. Several other important antioxidant enzymes are known to exist within cells, including catalase and glutathione peroxidase. While extracellular fluids and the extracellular matrix contain only small amounts of these enzymes, other extracellular antioxidants are also known to be present, including radical scavengers and inhibitors of lipid peroxidation, such as ascorbic acid, uric acid, and α-tocopherol.
Summary of the Invention We have now found that inhibitors of ABL, in particular small molecule inhibitors, provide significant and selective systemic protection of normal cells and normal tissues from radiation-induced damage in individuals exposed to ionizing radiation. It is an object of the invention to provide compositions and methods for protecting the normal cells and tissues from the cytotoxic and genetic effects of exposure to ionizing radiation, in individuals who have incurred or are at risk of incurring exposure to ionizing radiation. The exposure to ionizing radiation may occur in controlled doses during the treatment of cancer and other proliferative disorders, or may occur in uncontrolled doses beyond the norm accepted for the population at large during high risk activities or environmental exposures.
Radioprotection By Formula I Compounds A method for protecting an individual from cytotoxic side effects of ionizing radiation is provided, comprising administering to said individual an effective amount of at least one compound of formula I:
Figure imgf000015_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000015_0002
" ' indicates that the designated bond is either a single bond or a double bond; K indjca es that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(C]-C6)alkyl, substituted and unsubstituted heteroaryl(Cι-C6)alkyl, and (CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(Cι-C6alkyl); preferably from N, C(=0) and C(H); R2 is substituted aryl or substituted or unsubstituted heterocyclyl, preferably substituted or unsubstituted heteroaryl; p is 1, 2 or 3; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι- C6)alkyl; B is aryl, preferably phenyl; or heteroaryl; each R4 is independently selected from the group consisting of -(CrC7)hydrocarbyl; halogen; -OH; -NH2; -NH(Cι-C6)alkyl -N(Cι-C6alkyl)2; -N(C2-C6 heteroalkyl)2; -N02; -CN; -S02(CrC6)alkyl
Figure imgf000015_0003
-S(C1-C6)alkyl; -0(C,-C6)alkyl; -C(=0)NH2
Figure imgf000016_0001
C6)alkyl; -S02NH2; -S02NH(Cι-C6)alkyl;
Figure imgf000016_0002
-S02- (non-aromatic heterocycle); -(C!-C6)alkylene-NH2, preferably -(C2- C4)alkylene-NH2; - (Cι-C6)alkylene-OH, preferably -(C2-C4)alkylene- OH; -(CrC6)alkylene-C02H, preferably -(C2-C4)alkylene-C02H;
-NHC(=0)(C1-C6)alkyl; -NHSO^d-Ce^lkyl; -C(=NH)NH2; -Z(C C6)alkylene-NH2; -Z(Cι-C6)alkylene-NH(Cι-C6)alkyl; -Z(Cι-
C6)alkylene-N(Cι-C6alkyl)2; -Z(C1-C6)alkylene-C02(Cι-C6)alkyl; -Z(Cι- C6)alkylene-C02H; -Z(Ci-C6)alkylene-C02(C1-C3)alkyl; -Z(C,- C6)alkylene-C(=0)NH2; -Z(Ci-C6)alkylene-C(=0)NH(Ci-C6)alkyl;
-Z(C1-C6)alkylene-C(=0)N(Cι-C6alkyl)2; and substituted heterocyclyl, preferably substituted non-aromatic heterocyclyl, more preferably substituted monocyclic non-aromatic heterocyclyl, most preferably mono-, di- or tri-substituted by substituents independently selected from the group consisting of -(Cι-C6)alkyl, -C(=0)C1-C6)alkyl, and
-C(=0)H; X is C orN; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, preferably substituted and unsubstituted phenyl; -NH2; -Z-(Cι-C7)hydrocarbyl, preferably -Z-(Cι-C6)alkyl and -Z-benzyl;
-Z-(Cι-C6)alkylene-heterocyclyl, preferably containing non-aromatic heterocyclic groups; and -Z-(CH(Rb))nC(=0)R ; n is 1, 2, 3, 4; 5 or 6; preferably 1, 2, 3 or 4; more preferably 1, 2 or 3; most preferably 1 or 2; R6 is selected from the group consisting of -H; -CN; substituted and unsubstituted aryl, preferably substituted and unsubstituted phenyl; -Z-(Cι-C7)hydrocarbyl, preferably -Z-(C]-C6)alkyl and -Z-benzyl; -Z- (Cι-C6)alkylene-heterocyclyl, preferably containing non-aromatic heterocyclic groups, more preferably containing monocyclic non- aromatic heterocyclic groups; and -Z-(CH(Rb))πC(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Ci-C6)alkyl, -NH2, -NH(CrC6)alkyl,
Figure imgf000017_0001
and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of -H and -(C1-C6)alkyl; provided: (1) when the designated bond in :T ' ; isC a double bond, then: (a) the designated bond in ^ is a single bond; (b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000017_0002
(c) T is selected from the group consisting of N, C(H) and C(Cι-C6alkyl), preferably N and C(H); and T (2) when the designated bond in ' is a single bond, then: p (a) the designated bond in ^ is a double bond; (b)R is a radical of formula (ii):
Figure imgf000017_0003
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound. , Λ O044181
-17-
When R1 is substituted aryl or substituted heteroaryl, it is preferably mono- or di-substituted by substituents independently selected from the group consisting of (C2-Cι )heteroalkyl, preferably (C -Cιo)heteroalkyl; substituted heterocyclyl(Cι-C6)alkyl, preferably substituted non-aromatic monocyclic heterocyclyl(Cι-C6)alkyl, more preferably substituted non-aromatic monocyclic heterocyclyl(Cι-C4)alkyl, most preferably mono- or di-substituted by substituents independently selected from the group consisting of -OH, -OCH3, and -(Cι-C6)alkyl; -0(Ci-C7)hydrocarbyl, preferably -0(Ci-C6)alkyl, more preferably -0(Ci-C )alkyl; halogen, preferably bromo, chloro and fluoro; cyano; and -N02; When R2 is substituted aryl or substituted heterocyclyl, it is preferably mono- or di-substituted by substituents independently selected from the group consisting of -(Cι-C )hydrocarbyl, preferably -(Cι-Ce)alkyl, more preferably -(Cι-C )alkyl, most preferably -CH3; -0(Cι-C7)hydrocarbyl, preferably -0(Cr Cδ)alkyl, more preferably -0(C]-C )alkyl; halogen, preferably bromo, chloro and fluoro; and -N02. According to a preferred sub-embodiment, B is selected from the group consisting of phenyl; pyridyl; pyrazinyl; pyrimidinyl, particularly 2- and 5- pyrimidyl; pyridazinyl; thienyl; furyl; pyrrolyl, particularly 2-pyrrolyl and 1- alkyl-2-pyrrolyl; imidazolyl, particularly 2-imidazolyl; thiazolyl, particularly 2- thiazolyl; oxazolyl, particularly 2-oxazolyl; pyrazolyl, particularly 3- and 5- pyrazolyl; isothiazolyl; 1,2,3-triazolyl; 1,2,4-triazolyl; 1,3,4-triazolyl; tetrazolyl; 1,2,3-thiadiazolyl; 1,2,3-oxadiazolyl; 1,3,4-thiadiazolyl; 1,3,4-oxadiazolyl; indolyl, particularly 2-, 3-, 4-, 5-, 6- and 7-indolyl; quinolyl; isoquinolyl, particularly 1- and 5-isoquinolyl; cinnolinyl; quinoxalinyl, particularly 2- and 5- quinoxalinyl; quinazolinyl; phthalazinyl; 1,8-naphthyridinyl; 1,5-naphthyridinyl, particularly l,5-naphthyridin-3-yl and l,5-naphthyridin-4-yl; 1,4- benzodioxanyl; coumarin; benzofuryl, particularly 2-, 3-, 4-, 5-, 6- and 7- benzofuryl; 1 ,2-benzisoxazolyl; benzothienyl, particularly 3-, 4-, 5-, 6-, and 7- benzothienyl; benzoxazolyl; benzthiazolyl, particularly 2-benzothiazolyl and 5- benzothiazolyl; purinyl; benzimidazofyl, particularly 2-benzimidazolyl; benztriazolyl; thioxanthinyl; carbazolyl; carbolinyl; and acridinyl, particularly 6- acridinyl. Preferably, B is selected from the group consisting of phenyl; 2, 3- and
4-pyridyl; 2- and 3-thienyl; 2- and 3-furyl; 2-pyrrolyl; 2-imidazolyl; 2-thiazolyl; 2-oxazolyl; 2- and 3-indolyl; 2-, and 3-benzofuryl; 3-(l,2-benzisoxazolyl); 2- and 3-benzothienyl; 2-benzoxazolyl; 1- and 2-benzimidazolyl, 2-, 3-, 4-, 5-and
8-quinolyl; and 2- and 5-benzthiazolyl. More preferably, B is selected from the group consisting of phenyl; 2- and 3-indolyl; 2- and 3-pyrrolyl; 2-, 3-benzofuryl; 5-quinolyl, and 2- and 3- benzothienyl. When R3 is substituted heteroaryl, it is preferably mono-, di-, or tri- substituted by substituents independently selected from the group consisting of (Cι-C7)hydrocarbyl, preferably (Cι-Ce)alkyl, more preferably (Cι-C4)alkyl, most preferably -CH3; -0(Ci-C7)hydrocarbyl, preferably -0(C]-C6)alkyl, more preferably -0(C1-C4)alkyl; -OH; -(C1-C6)alkylene-N(Cι-C6)alkyl)2; halogen, preferably bromo, chloro and fluoro; -CN; -NH2 and -N02. When R5 or Rδ are substituted aryl, they are preferably mono-, di- or tri- substituted by substituents independently selected from the group consisting of (Cι-C7)hydrocarbyl, preferably (Cι-C6)alkyl, more preferably (Cι-C )alkyl, most preferably -CH3; -OH; -0(Cι-C7)hydrocarbyl, -C02H; CONH2; C02(Cι- Cg)alkyl; halogen, cyano; -N02; and substituted heterocyclyl, preferably mono- or di-substituted by substituents independently selected from the group consisting of -OH, -OCH3, and -(CrGe^lkyl; -0(Ci-C7)hydrocarbyl, halogen, cyano; and -N02; According to a second embodiment of the invention, there is provided a method for protecting an individual from cytotoxic side effects of ionizing radiation, comprising administering to said individual an effective amount of a combination comprising at least one compound of formula I, as defined above, and at least one radioprotective α,β-unsaturated aryl or heteroaryl sulfone, sulfoxide, sulfonamide or carboxamide. According to a third embodiment of the invention, there is provided a method for protecting an individual from cytotoxic side effects of ionizing radiation, comprising administering to said individual an effective amount of a combination comprising at least one compound of formula I, as defined above, and at least one antioxidant compound. Preferred embodiments of radioprotective compounds of formula I are described as follows. First Embodiment of Formula I Compounds According to a First Embodiment of the radioprotective compounds of formula I: the designated bond in ' is a double bond, R the designated bond in " s a single bond, R is hydrogen, R2, or a radical of formula (i):
Figure imgf000020_0001
T is selected from the group consisting of N, C(H) and C(Ci- Cβalkyl); or a pharmaceutically acceptable salt of such a compound.
According to one preferred sub-embodiment, the radioprotective compound comprises a compound of formula II:
Figure imgf000020_0002
wherein: T is selected from the group consisting of N, C(H) and C(Cι- C6alkyl); R1 is selected from the group consisting of -H,
Figure imgf000020_0003
substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Cι-C6)alkyl, and substituted and unsubstituted heteroaryl(C1-C6)alkyl; and R is substituted aryl, or substituted or unsubstituted heterocyclyl; or a pharmaceutically acceptable salt of such a compound. According to one preferred sub-embodiment of the compounds of formula II the radioprotective compound comprises a compound of formula Ila:
Figure imgf000021_0001
wherein: R1 is selected from the group consisting of -(Cι-Ce)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(C1-C6)alkyl, and substituted and unsubstituted heteroaryl(C1-C6)alkyl; or a pharmaceutically acceptable salt of such a compound. Preferred compounds according to formula Ila include: (2-{4-[4-amino- 5-(4-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]phenoxy}-ethyl)(2-methoxy- ethyl)amine; 1 -(2- {4-[4-amino-5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7- yl]phenyl}ethyl)piperidin-4-ol; and pharmaceutically acceptable salts thereof.
According to another preferred sub-embodiment of the compounds of formula II, the radioprotective compound comprises a compound of formula lib:
Figure imgf000021_0002
wherem: R1 is selected from the group consisting of -(Cι-C6)alkyl, and substituted and unsubstituted aryl; or a pharmaceutically acceptable salt of such a compound. Preferred compounds according to formula Ila include l-(tert-butyl)-3- (4-methylphenyl)pyrazolo[5,4-d]pyrimidine-4-ylamine, and pharmaceutically acceptable salts thereof.
Second Embodiment of Formula I Compounds According to a Second Embodiment of the radioprotective compounds of formula I: the designated bond in — T ' ; is, a single bond; and X is C. According to one preferred sub-embodiment, the radioprotective compound comprises a compound of formula III:
Figure imgf000022_0001
wherein: R1 is -H or (CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; ww, indicates a single bond in either the Z- or E- conformation, preferably in the E- conformation; B is aryl, preferably phenyl; or heteroaryl; each R4 is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(Cι-C6)alkyl, -N(Cι-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(Ci-C6)alkyl, -SO(Cι-C6)alkyl, -S(d-C6)alkyl, -0(d-C6)alkyl, -C(=0)NH2, -C(=0)NH(C,-C6)alkyl, ' -C(=0)N(C,-C6alkyl)2, -C02H, -C02(d- C6)alkyl, -S02NH2, -S02NH(Ci-C6)alkyl, -S02N(d-C6alkyl)2, -S02- (non-aromatic heterocycle), -(d-C6)alkylene-NH2, -(Cι-C6)alkylene- OH, -(Ci-C6)alkylene-C02H -NHC(=0)(C1-C6)alkyl, -(CrC6)alkylene- C02H, -NHS02(Cι-C6)alkyl, -C(=NH)NH2, -Z(Cι-C6)alkylene-NH2, 4181
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-Z(Cι-C6)alkylene-NH(Cι-C6)alkyl, -Z(d-C6)alkylene-N(Cι-C6alkyl)2, -Z(C,-C6)alkylene-C02(d-C6)alkyl, -Z(d-C6)alkylene-C02H, -Z(Cι- C6)alkylene-C(=0)NH2, -Z(C1-C6)alkylene-C(=0)NH(d-C6)alkyl, and -Z(Ci-C6)alkylene-C(=0)N(Ci-C6alkyl)2; p is 1, 2 or 3; R and R are independently selected from the group consisting of -H; -CN; substituted and unsubstituted aryl, preferably substituted and unsubstituted phenyl; -Z-(Cι-C7)hydrocarbyl, preferably -Z-(Cr Cδ)alkyl or -Z-benzyl; -Z-(C)-C6)alkylene-heterocyclyl, preferably containing non-aromatic heterocyclic groups; and -Z-(CH(Rb))nC(=0)R ; n is 1, 2, 3, 4; 5 or 6; preferably 1, 2, 3 or 4; more preferably 1, 2 or 3; most preferably 1 or 2; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Ci-C6)alkyl, -NH2, -NH(d-C6)alkyl, -N((d-C6)alkyl)2, and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group may be present as a carboxyl group, an amide or a carboxylic ester; and each Rb is -H or (Cι-C6)alkyl; or a pharmaceutically acceptable salt of such a compound. Preferred compounds according to formula III include: ethyl-2-{5-[(2- oxo-lH-benzo[d]azolidin-3-ylidene)methyl]-8-quinolyloxy}-propanoate; 3- {2,4-dimethyl-5-[(2-oxo(lH-benzo[d]azolidin-3-ylidene))-methyl]pyrrol-3-yl}- propanoic acid; 3-{4-methyl-2-[(2-oxo(lH-benzo[d]azolidin-3-ylidene))- methyl]pyrrol-3-yl}propanoic acid; 3-[(3,5-dimethylpyrrol-2-yl)methylene]-lH- benzo[d]azolidin-2-one; 3-[(2-chloro-4-methoxyρhenyl)methylene]- 1 H-benzo- [d]azolidin-2-one; 3-(indol-3-ylmethylene)-5-(2-piperidylacetyl)- 1 H-benzo[d]- azolidin-2-one; N-{3-[(5-methoxyindol-3-yl)methylene]-2-oxo(lH-benzo[3,4- d]azolidin-5-yl)}-3-piperidylpropanamide; 3-{4-methyl-5-[(2-oxo-6-phenyl(lH- benzo [d]azolidin-3 -y lidene))methyl]pyrrol-3 -yl }propanoic acid; 3 -(5- { [6-(3 - -23-
methoxyphenyl)-2-oxo(lH-benzo[d]azolidin-3-ylidene)]methyl}-4-methyl- pyrrol-3-yl)propanoic acid; 2-oxo-3-(pyrrolo[2,3-b]pyridin-3-ylmethylene)-lH- benzo[d]-azolidine-5-carbonitrile; 3-{[4-(4-carbonylpiperazinyl)phenyl]methyl- ene}-lH-benzo[d]azolidin-2-one; and pharmaceutically acceptable salts thereof.
Third Embodiment of Formula I Compounds According to a Third Embodiment of the radioprotective compounds of formula I: the designated bond in :T is a double bond; and R is a radical of formula (ii):
Figure imgf000024_0001
wherein: R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι- C6)alkyl. According to one preferred sub-embodiment, the radioprotective compound comprises a compound of formula IV:
Figure imgf000024_0002
wherein: T is C(H) orN, preferably C(H); and R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι- Cβ)alkyl; or a pharmaceutical salt of such a compound. When R3 is substituted heteroaryl, it is preferably mono-, di-, or tri- substituted by substituents independently selected from the group consisting of -(Cι-C7)hydrocarbyl, preferably -(Cι-C6)alkyl, more preferably -(Cι-C4)alkyl, most preferably -CH3; -0(Cι-C7)hydrocarbyl, preferably -0(C C6)alkyl, more preferably -0(d-C4)alkyl; -OH; -(Cι-C6)alkylene-N(Cι-C6)alkyl)2; halogen, preferably bromo, chloro and fluoro; -CN; -NH2 and -N02. Preferred compounds according to formula IV include 5-((lZ)-l-cyano- 2-indol-3-ylvinyl)-3-aminopyrazole-4-carbonitrile, and pharmaceutically acceptable salts thereof. According to some embodiments of the invention, the radioprotective compound or combination of compounds is administered before exposure to the ionizing radiation. According to some embodiments of the invention, the radioprotective compound or combination of compounds is administered after exposure to ionizing radiation. According to one embodiment of the invention, a method for protecting an individual from cytotoxic side effects of ionizing radiation is provided comprising administering to the individual an effective amount of at least one compound of formula I, and an effective amount of at least one compound of formula V: Q1 — X— CH=CH— Q2 V wherein: Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; preferably substituted or unsubstituted phenyl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000025_0001
(i) (ϋ)
Figure imgf000025_0002
(iii) (iv) wherein: n is one or zero, preferably one; and Rx is -H, -(CrC8)hydrocarbyl or -C(=0)(d-C8)hydrocarbyl; or a pharmaceutically acceptable salt thereof. According to some preferred embodiments of compounds according to formula V, Q1 is phenyl or substituted phenyl, more preferably substituted phenyl. According to other preferred embodiments of compounds according to formula V, Q2 is phenyl or substituted phenyl, more preferably substituted phenyl. According to still other preferred embodiments of compounds according to formula V, both Q1 and Q2 are phenyl or substituted phenyl, more preferably substituted phenyl. According to one sub-embodiment of the compounds according to formula V, X is selected from the group consisting of (i), (ii) and (iii). According to another sub-embodiment of the compounds according to formula V, X is selected from the group consisting of (i) and (ii). According to yet another sub-embodiment of the compounds according to formula V, X is (i). According to some embodiments of compounds according to formula V, the aryl and heteroaryl groups comprising Q1 and Q2 are mono-, di- or tri- substituted. According to other embodiments of compounds according to formula V, the aryl and heteroaryl groups comprising Q1 and Q2 are substituted at all substitutable positions. Substituents for substituted aryl and heteroaryl groups comprising Q1 and Q2 are preferably independently selected from the group consisting of halogen, -Rx -NRX 2, -NHC(=0)Ry, -NHS02Ry, -NH(Ci-C )alkylene-C02Rx, -C02Rx, -C(=0)NHRx, -N02, -CN, -ORx, phosphonato, dimethylamino(C2-C6 alkoxy), -NHC(=NH)NHRX, -(d-C6)haloalkyl, -(Cι-C6)haloalkoxy, -(C=0)(Cι- C4)alkylene-NRX 2 and -NH-CH(Rz)-C02Rx, -(C=0)CH(Rz)-NRx 2; wherein: Ry is selected from -H, -{d-Cj hydrocarbyl, -0(C,- C8)hydrocarbyl, substituted phenyl, substituted heterocyclyl(Cι-C3)alkyl, heteroaryl(Cι-C3)alkyl, -(C2-Cιo)heteroalkyl, -(d-C6)haloalkyl, -NHC(RZ)NHRX, -NHRX, -(Ci-C3)alkyleneNH2, -(d- C3)alkyleneN(CH3)2, -(Cι-C3)ρerfluoroalkyleneN(CH3)2, -(Ci- C3)alkyleneN+(C1-C3)3, -(Cι-C3)alkylene-N+(CH2CH2OH)3, -(d- C3)alkylene-OR1, -(d-C4)alkylene-C02R1, -(d-C4)alkylene- C(=0)halogen, and -(Cι-C4)perfluoroalkylene-C02RI . Substituents on substituted phenyl Ry are preferably selected from the group consisting of -NH2, -N02, N-methylpiperazinyl, and -ORx. Rz is selected from the group consisting of -H, -(Cι-C6)alkyl,
-(CH2)3-ΝH-C(ΝH2)(=ΝH), -CH2C(=0)NH2, -CH2COOH, -CH2SH, -(CH2)2C(=0)-NH2, -(CH2)2COOH, -CH2-(2-imidazolyl), -{CH2)4-NH2, -(CH2)2-S-CH3, phenyl, substituted phenyl, -CH2-phenyl, -CH2-OH, -CH(OH)- CH3, -CH2-(3-indolyl), and -CH2-(4-hydroxyphenyl). Substituted phenyl Rz is preferably mono- di- or tri-substituted, more preferably mono or di substituted, most preferably mono-substituted by substituents independently selected from the group consisting of -Rx, -NRX 2, -N02, -ORx, -CN, -C02Rx, halogen, -SRX and S02Rx. Substituents on substituted phenyl Rz are more preferably independently selected from the group consisting of -(Cι-C6)alkyl, -NH2, NH(Cj-C6)alkyl, -N02, -0(C]-C6)alkyl, -OH, -CN, -C02(d-C6)alkyl, C02H, , halogen, -S(d- C6)alkyl, -SH, and S02(d-C6)alkyl. Substituents on substituted phenyl Rz are most preferably independently selected from the group consisting of methyl, ethyl, -NH2, NHCH3, -N02, -OCH3, -OH, -CN, -CO2CH3, C02Et, C02H, , halogen, -SCH3, -SH, and S02CH3. Substituents on substituted heterocyclyl(Cι-C6)alkyl Ry are preferably selected from -(C C7)hyrocarbyl, preferably -(d-C6)alkyl; -C(=0)d-C6)alkyl, preferably -C(=0)Cι-C3)alkyl, most preferably acetyl; and -(d- C6)perfluoroalky 1, preferably -(C 1 -C3)perfluoroalkyl, most preferably -CF3. Substituents for substituted aryl and heteroaryl groups comprising Q1 and Q2 are more preferably independently selected from the group consisting of halogen, -Rx -NRX 2, -NHC(=0)Ry, -NHS02Ry, -NH(d-C4)alkylene-C02Rx,
-C02Rx, -C(=0)NHRx, -N02, -CN, -ORx, phosphonato, dimethylamino(C2-C6 alkoxy), -NHC(=NH)NHRX, -(CrC6)haloalkyl and -(d-C6)haloalkoxy. Substituents for substituted aryl and heteroaryl groups comprising Q1 and Q2 are most preferably independently selected from the group consisting of halogen, -NRX 2, -NHC(=0)Ry, -NHS02Ry, -NH(Ci-C4)alkylene-C02Rx,
-Cθ2(Cι-C8)hydπ)carbyl, -C(=0)NHRx, -N02, -CN, -OH, -OCH3 and phosphonato. Rx is preferably selected from the group consisting of-H, -(Cι-C(5)alkyl, benzyl, -C(=0)(d-C6)alkyl and -C(=0)benzyl. R is more preferably selected from the group consisting of -H, -(d- C6)alkyl, and -C(0)(Ci- )alkyl. R is most preferably -H or -(CrC alkyl. Ry is preferably selected from -H, -(Cι-C8)hydrocarbyl, -0(d- C8)hydrocarbyl, substituted phenyl, -NHRX, -(Cι-C6)haloalkyl, -(C C3)alkyleneNH2, -(Cι-C3)alkyleneN(CH3)2, -(d-C3)alkylene-OR1, -(d- C )alkylene-C02R1, and ~(Cι-C4)ρerfluoroalkylene-C02R1. Ry is more preferably selected from -H, -(Cι-C8)hydrocarbyl, -0(Cι-
C8)hydrocarbyl, substituted phenyl, -(Cι-C6)perfluoroalkyl, -NHRX, -(Ci- C3)alkyleneNH2, -(Cι-C3)alkyleneN(CH3)2, -(Ci-C3)alkylene-0(d-
C8)hydrocarbyl, -(Cι-C4)alkylene-Cθ2(Cι-C8)hydrocarbyl and -(Cj- C4)perfluoroalkylene-C02(Ci-C8)hydrocarbyl. Rz is preferably selected from the group consisting of-H, -(d-C6)alkyl, and phenyl. Preferred compounds according to formula V include, for example: 4- ((U5T)-2-{[(4-fluorophenyl)methyl]sulfonyl}vinyl)benzoic acid; 4-((l£)-2-{[(4- iodophenyl)methyl] sulfonyl} vinyl)benzoic acid; 4-(( lE)-2- { [(4-chloropheny 1)- 4181
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methyljsulfonyl} vinyl)benzoic acid; 1 -[5-(( 1 E)-2- { [(4-chlorophenyl)methyl]- sulfonyl}vinyl)-2-fluorophenyl]-2-(dimethylammo)ethan-l-one; (\E)-2-(2,4- difluorophenyl)- 1 - { [(4-bromophenyl)methyl] sulfonyl} ethene; ( lE)-2-(3 -amino- 4-fluorophenyl)- 1 - { [(4-chlorophenyl)methyl]sulfonyl} ethene; 2-(5-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)-acetic acid; (R)-2-(5- (((£)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)- propanoic acid; (S)-2-(5-(((-5)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2- methoxyphenylamino)-propanoic acid; racemic-2-(5-(((£)-2,4,6-trimethoxy- sryrylsulfonyl)methyl)-2-methoxyphenyl-amino)propanoic acid; (R)-2-(5-(((E)- 2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)-2-phenylacetic acid; (■S)-2-(5-(((£)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenyl- amino)-2-phenylacetic acid; racemic-2-(5-(((£)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-metlιoxyphenyl-amino)-2-phenylacetic acid; N-(3 -(((-5)- 2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenyl)-4-(4-methyl- piperazin- 1 -yl)benzamide; 2-((^)-2-(4-methoxybenzylsulfonyl)vinyl)- 1,3,5- trimethoxybenzene; l-(((£)-4-chlorostyrylsulfonyl)methyl)-2-chloro-4-fluoro- benzene; 4-((^)-27(4-chlorobenzylsulfonyl)vinyl)- 1 -fluoro-2-nitro-benzene; 4- ((E)-2-(3 -amino-4-methoxybenzylsulfonyl)vinyl)benzoic acid; 5-(((E)-2,4- difluorostyrylsulfonyl)methyl)-2-methoxybenzenamine; l-bromo-4-(((£)-per- fluorostyrylsulfonyl)methyl)benzene; l-(((is)-2,3,5,6-tefrafluorostyryl-sulfonyι)- methyl)-4-bromobenzene; l-(((^)-2,4,5-trifluorostyrylsulfonyl)-methyl)-4- bromobenzene; l-(((Jδ)-2,4,6-trifluorostyrylsulfonyl)methyl)-4-bromobenzene; l-(((^)-2,3,6-trifluorostyrylsulfonyl)methyl)-4-bromobenzene; 5-(((E)-2,4- difluorostyrylsulfonyl)methyl)-2-bromobenzenamine; 4-(((i^-2,4-difluorostyryl- sulfonyl)methyl)benzoic acid; 5-(((Jδ)-2,4,6-trimethoxystyryl-sulfonyl)methyl)- 2-bromobenzenamine; 4-((F)-2-(4-bromobenzylsulfonyl)- vinyl)- 1 -fluoro-2- nitrobenzene; 4-((-5)-2-(4-chloro-2-nitrobenzylsulfonyl)vinyl)- 1 -fluoro-2-nitro- benzene; 5-((-5)-2-(4-bromobenzylsulfonyl)vinyl)-2-fluoro-benzenamine; 5- ((£)-2-(4-iodobenzylsulfonyl)vinyl)-2-fluorobenzenamine; 5-(((E)-2,4,6-tή- methoxystyrylsulfonyl)methyl)-2-methoxybenzonitrile; 4-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)benzoic acid; 3 -((£)-2-(4-bromobenzyl- sulfonyl)vinyl)-2,6-difluorophenol; and salts thereof. More preferred compounds according to formula V include, for example: 4-((lE)-2- { [(4-fluorophenyl)methyl]sulfonyl}vinyl)benzoic acid; 4-((lE)-2- {[(4-iodoρhenyl)methyl]sulfonyl}vinyl)benzoic acid; 4-((li?)-2-{[(4-chloro- phenyl)-methyl]sulfonyl}vinyl)benzoic acid; l-[5-((l-5)-2-{[(4-chlorophenyl)- methyl]-sulfonyl}vinyl)-2-fluorophenyl]-2-(dimethylamino)ethan-l-one; (1-5)- 2-(2,4-difluorophenyl)-l-{[(4-bromophenyl)methyl]sulfonyl}ethene; (lE)-2-(3- amino-4-fluorophenyl)- 1 - { [(4-chlorophenyl)methyl] sulfonyl} ethene; 1 -(((£)-4- chlorostyrylsulfonyl)methyl)-2-chloro-4-fluorobenzene;l-(((-5 )-2,4,5-trifluoro- styrylsulfonyl)-methyl)-4-bromobenzene; N-(3-(((£)-2,4,6-trimethoxystyryl- sulfonyl)methy l)-2-methoxyρhenyl)-4-(4-methy 1-piperazin- 1 -yl)benzamide; 4- ((£)-2-(3-ammo-4-methoxybenzylsulfonyl)vinyl)benzoic acid; 5-(((E)-2,4- difluorostyrylsulfonyl)methyl)-2-methoxybenzenamine; l-(((-5 -2,4,6-trifluoro- styrylsulfonyl)methyl)-4-bromobenzene; l-(((^)-2,3,6-trifluorostyrylsulfonyl)- methyl)-4-bromobenzene; 5-(((^)-2,4,6-trimethoxystyryl-sulfonyl)methyl)-2- bromobenzenamine; 5-((^)-2-(4-iodobenzylsulfonyl)vinyl)-2-fluoro- benzenamine; 5-(((£)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxy- benzonitrile; 4-(((^)-2,4,6-trimethoxystyrylsulfonyl)methyl)benzoic acid; 3- ((-5)-2-(4-bromobenzyl-sulfonyl)vinyl)-2,6-difluorophenol; (R)-2-(5-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)-propanoic acid; (iS)- 2-(5-(((-5)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)- propanoic acid; racemic-2-(5-(((-5)-2,4,6-trimetlιoxy-styrylsulfonyl)methyl)-2- methoxyphenyl-amino)propanoic acid; (R)-2-(5-(((^)-2,4,6-trimethoxy- styrylsulfonyl)methyl)-2-methoxyphenylamino)-2-phenylacetic acid; (S)-2-(5- (((£)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenyl-amino)-2- phenylacetic acid; racemic-2-(5-(((-5)-2,4,6-trimethoxystyryl-sulfonyl)methyl)- 2-methoxyphenyl-amino)-2-phenylacetic acid; 5-((2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxy-N-methylbenzenamine and pharmaceutically acceptable salts thereof. According to another embodiment of the invention, a method for protecting an individual from cytotoxic side effects of ionizing radiation is 4181
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provided comprising administering to the individual an effective amount of at least one compound of formula I, and an effective amount of at least one antioxidant compound. According to another embodiment of the invention, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable carrier, at least one compound according to formula I as defined above, and at least one compound according to formula V as defined above. According to another embodiment of the invention, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable carrier, at least one compound according to formula I as defined above, and at least one antioxidant compound. According to another embodiment of the invention, a method of treating an individual with a proliferative disorder is provided, comprising: (a) administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound; and (b) administering an effective amount of therapeutic ionizing radiation. According to one sub-embodiment of the method of the invention for treating a proliferative disorder, the proliferative disorder is cancer.
According to another embodiment of the invention, a method of safely increasing the dosage of therapeutic ionizing radiation used in the treatment of cancer or other proliferative disorders is provided, comprising administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to 4181
-31- formula V as defined above, or at least one antioxidant compound.
According to one sub-embodiment of the above method of safely increasing dosages of therapeutic ionizing radiation, the radioprotective compound or combination of compounds is administered prior to administration of the therapeutic ionizing radiation. According to a further sub-embodiment of the above method of safely increasing dosages of therapeutic ionizing radiation, the radioprotective compound or combination of compounds induces a temporary radioresistant phenotype in the normal tissue of the individual.
According to another embodiment of the invention, a method is provided for treating an individual who has incurred, or is at risk for incurring, remediable radiation damage from exposure to ionizing radiation, comprising administering to the individual an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound The compound or compounds may be administered before or after incurring remediable radiation damage from exposure to ionizing radiation.
According to another embodiment of the invention, a method is provided of reducing the number of malignant cells in bone marrow of an individual, comprising: (1) removing a portion of the individual's bone marrow; (2) administering to the removed bone marrow an effective amount of either: (i) at least one radioprotective compound according to formula I; or (ii) at least one radioprotective compound according to formula I, and an effective amount of either at least one compound according to formula V as defined above, or at least one antioxidant compound; and (3) irradiating the bone marrow with an effective amount of ionizing radiation. The bone marrow is reimplanted into the individual. According to another sub-embodiment of the bone marrow treatment method, the individual receives therapeutic ionizing radiation prior to reimplantation of the bone marrow. According to another sub-embodiment of the above method of bone marrow treatment, the individual receives therapeutic ionizing radiation prior to reimplantation of the bone marrow, and is administered a radioprotective compound or combination of compounds as defined above prior to receiving the therapeutic ionizing radiation. According to another embodiment of the invention, a compound of formula I, or a pharmaceutically acceptable salt thereof, is used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation. According to another aspect of the invention, a compound of formula I, or a pharmaceutically acceptable salt thereof, and a compound of formula V, are used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation. According to another aspect of the invention, a compound of formula I, or a pharmaceutically acceptable salt thereof, and an antioxidant compound are used in the manufacture of a medicament for protecting an individual from cytotoxic side effects of ionizing radiation. According to one embodiment, the compounds are for administration before exposure to ionizing radiation. According to another embodiment, the compounds are for administration after exposure to ionizing radiation. According to another embodiment, the formula I compounds are for administration before or after administration of therapeutic ionizing radiation, for treatment of a proliferative disorder. According to yet another embodiment of the invention, the compounds, are for treating an individual who has incurred or is at risk of incurring remediable radiation damage from exposure to ionizing radiation. According to another embodiment of the invention, the compounds are used for the preparation of a medicament for treating bone marrow prior to irradiating the bone marrow with an effective amount of ionizing radiation. According to another embodiment of the invention, the compounds are used for the preparation of a medicament for safely increasing the dosage of therapeutic ionizing radiation used in the treatment of cancer or other proliferative disorders.
Definitions The term "individual" includes human beings and non-human animals and, as used herein, refers to an organism which is scheduled to incur, is at risk of incurring, or has incurred, exposure to ionizing radiation. As used herein, "ionizing radiation" is radiation of sufficient energy that, when absorbed by cells and tissues, induces formation of reactive oxygen species and DNA damage. This type of radiation includes X-Rays, gamma rays, and particle bombardment (e.g., neutron beam, electron beam, protons, mesons and others), and is used for medical testing and treatment, scientific purposes, industrial testing, manufacturing and sterilization, weapons and weapons development, and many other uses. Radiation is typically measured in units of absorbed dose, such as the rad or gray (Gy), wherein 1 rad = 0.01 Gy, or in units of dose equivalence, such as the rem or sievert (Sv), wherein 1 rem = 0.01 Sv. The Sv is the Gy dosage multiplied by a factor that includes tissue damage done. For example, penetrating ionizing radiation (e.g., gamma and beta radiation) have a factor of about 1, so 1 Sv = ~1 Gy. Alpha rays have a factor of 20, so 1 Gy of alpha radiation = 20 Sv. By "effective amount of ionizing radiation" is meant an amount of ionizing radiation effective in killing, or in reducing the proliferation, of abnormally proliferating cells in an individual. As used with respect to bone marrow purging, "effective amount of ionizing radiation" means an amount of ionizing radiation effective in killing, or in reducing the proliferation, of malignant cells in a bone marrow sample removed from an individual. By "acute exposure to ionizing radiation" or "acute dose of ionizing radiation" is meant a dose of ionizing radiation absorbed by an individual in less than 24 hours. The acute dose may be localized, as in radiotherapy techniques, or may be absorbed by the individual's entire body. Acute doses are typically above 10,000 millirem (0.1 Gy), but may be lower. By "chronic exposure to ionizing radiation" or "chronic dose of ionizing radiation" is meant a dose of ionizing radiation absorbed by an individual over a period greater than 24 hours. The dose may be intermittent or continuous, and may be localized or absorbed by the individual's entire body. Chronic doses are typically less than 10,000 millirem (0.1 Gy), but may be higher. By "at risk of incurring exposure to ionizing radiation" is meant that an individual may intentionally, e.g., by scheduled radiotherapy sessions, or inadvertently be exposed to ionizing radiation in the future. Inadvertent exposure includes accidental or unplanned environmental or occupational exposure. By "small molecule" in meant a monomeric organic compound having a molecular weight of less than about 1000. By a "radioprotective α,β-unsaturated (aryl or heteroaryl) sulfone, sulfonamide or carboxamide" is meant a compound of the formula V:
Q1 — X— CH=CH— Q2 V wherein, Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; preferably substituted or unsubstituted phenyl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below: 4181
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Figure imgf000036_0001
(0 (ϋ)
Figure imgf000036_0002
(iii) (iv) wherein n is one or zero; and Rx is -H, -(Cι-C8)hydrocarbyl or -C(=0)(Ci-C8)hydrocarbyl; or a pharmaceutically acceptable salt thereof. By the term "antioxidant" is meant a pharmaceutically acceptable chemical compound that prevents or slows the breakdown of another substance by oxygen. Preferably, antioxidants useful in the methods of the present invention are small molecule organic compounds. By the expression "effective amount," in the context of an amount of radioprotective compound is meant an amount, alone, or in combination with either another radioprotective compound or an antioxidant compound, which is effective to reduce or eliminate the toxicity associated with radiation in normal cells of the individual. As used with respect to bone marrow purging, "effective amount" of a radioprotective compound means an amount of the compound effective to reduce or eliminate the toxicity associated with radiation in bone marrow removed from an individual. The term "alkyl", by itself or as part of another substituent means, unless otherwise stated, a straight, branched or cyclic chain saturated hydrocarbon radical, including di- and multi-radicals, having the number of carbon atoms designated in an expression such as (Cx-Cy)alkyl. The expression (Cx-Cy)alkyl wherein x < y, represents an alkyl chain containing a minimum of x carbon atoms and a maximum of y carbon atoms. Examples include: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, cyclohexyl and cyclopropyhnethyl. Preferred is (Cι-C3)alkyl, particularly ethyl, methyl and isopropyl. The term "cycloalkyl" refers to alkyl groups that cyclic, i.e., that contain at least one cyclic structure. Examples include cyclohexyl, cyclopentyl, norbomyl, adamantyl and cyclopropylmethyl. Preferred is (C3-C12)cycloalkyl, particularly cyclopentyl, norbomyl, and adamantyl. The term "alkylene" refers to a divalent alkyl radical having the number of carbon atoms designated (i.e. (Ci-Cβ) means ~CH2-; -CH2CH2-; -CH2CH2CH2-; -CH2CH2CH2CH2-; -CH2CH2CH2CH2CH2-; and -CH2CH2CH2CH2CH2CH2-, and also includes branched divalent structures such as, for example, -CH2CH(CH3)CH2CH2- and -CH(CH3)CH(CH3)-, and divalant cyclic structures such as, for example 1,3-cyclopentyl. The term "arylene", by itself or as part of another substituent means, unless otherwise stated, a divalent aryl radical. Preferred are divalent phenyl radicals, or "phenylene" groups, particularly 1,4-divalent phenyl radicals. The term "heteroarylene", by itself or as part of another substituent means, unless otherwise stated, a divalent heteroaryl radical. Preferred are five- or six-membered monocyclic heteroarylene. More preferred are heteroarylene moieties comprising divalent heteroaryl rings selected from the group consisting of pyridine, piperazine, pyrimidine, pyrazine, furan, thiophene, pyrrole, thiazole, imidazole and oxazole, such as, for example 2,5-divalent pyrrole, thiophene, furan, thiazole, oxazole, and imidazole. The term "alkoxy" employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-proρoxy, 2-ρroρoxy (isopropoxy) and the higher homologs and isomers. Preferred are (d- C6)alkoxy, particularly ethoxy and methoxy. The carbon chains in the alkyl and alkoxy groups which may occur in the compounds of the invention may be cyclic, straight or branched, with straight chain being preferred. The term "hydrocarbyl" refers to any moiety comprising only hydrogen and carbon atoms. The term includes, for example, alkyl, alkenyl, alkynyl, aryl and benzyl groups. Preferred are (d-C7)hydrocarbyl. More preferred are (Ci- Cδ)alkyl and (C3-Ci2)cycloalkyl. The term "heteroalkyl" by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain radical consisting of the stated number of carbon atoms and one, two or three heteroatoms selected from the group consisting of O, N, and S, and wherem the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quatemized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkyl group and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O- CH2-CH2-CH3, -CH2-CH2CH2-OH, -CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=0)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or-CH2-CH2-S-S-CH3. The terms "halo" or "halogen" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term "haloalkyl" means an alkyl group as defined above, wherein at least one hydrogen atom is substituted by a halogen atom. Preferably, haloalkyl groups are substituted by bromine, chlorine or fluorine, more preferably chlorine or fluorine, most preferably fluorine. Examples include: 3-bromopropyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, 1-chloroethyl and 2- chloropropyl. The term "aromatic" refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (4n + 2) delocalized π (pi) electrons). The term "aromatic" is intended to include not only ring systems containing only carbon ring atoms but also systems containing one or more non- carbon atoms as ring atoms. Systems containing one or more non-carbon atoms may be known as "heteroaryl" or "heteroaromatic" systems. The term "aromatic" thus is deemed to include "aryl" and "heteroaryl" ring systems. The term "aryl" employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl; anthracyl; and naphthyl which may be substituted or unsubstituted. The aforementioned listing of aryl moieties is intended to be representative, not limiting. The term "heterocycle" or "heterocyclyl" or "heterocyclic" by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, monocyclic or polycyclic heterocyclic ring system which consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quatemized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom which affords a stable structure. Heterocyclyl groups are inclusive of monocyclic and polycyclic heteroaryl groups and monocyclic and polycyclic groups that are not aromatic, such as saturated and partially saturated and monocyclic and polycyclic partially saturated monocyclic and polycyclic groups. The term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character, and includes both monocyclic heteroaryl groups and polycyclic heteroaryl groups. A polycyclic heteroaryl group may include one or more rings which are partially, saturated. Examples of monocyclic heteroaryl groups include: Pyridyl; pyrazinyl; pyrimidinyl, particularly 2- and 5-pyrimidyl; pyridazinyl; thienyl; furyl; pyrrolyl, particularly 2-pyrrolyl and l-alkyl-2-pyrrolyl; imidazolyl, particularly 2-imidazolyl; thiazolyl, particularly 2-thiazolyl; oxazolyl, particularly 2- oxazolyl; pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl; and 1,3,4-oxadiazolyl. Examples of monocyclic heterocycles that are not aromatic include saturated monocyclic groups such as: Aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, 1,4-dioxane, 1,3-dioxane, sulfolane, tetrahydrofuran, thiophane, piperazine, morpholine, thiomorpholine, tetrahydropyran, homopiperazine, homopiperidine, 1,3-dioxepane, hexamethyleneoxide and piperidine; and partially saturated monocyclic groups such as: 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, 2,3-dihydrofuran, 2,5-dihydrofuran , 2,3-dihydropyran, 1,2-dihydrothiazole, 1,2- dihydrooxazole, 1,2-dihydro imidazole and 4,7-dihydro-l,3-dioxepin. Examples of polycyclic heteroaryl groups include: Indolyl, particularly 3-, 4-, 5-, 6- and 7-indolyl, quinolyl, isoquinolyl, particularly 1- and 5- isoquinolyl, cinnolinyl, quinoxalinyl, particularly 2- and 5-quinoxalinyl, quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, benzofuryl, particularly 3-, 4-, 1,5-naphthyridinyl, 5-, 6- and 7-benzofuryl, 1,2- benzisoxazolyl, benzothienyl, particularly 3-, 4-, 5-, 6-, and 7-benzothienyl, benzoxazolyl, benzthiazolyl, particularly 2-benzothiazolyl and 5-benzothiazolyl, purinyl, benzimidazolyl, particularly 2-benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, tetrahydroquinolyl; 1,2,3,4-tetrahydroisoquinolyl; dihydrocoumarinyl; 2,3- dihydrobenzofuryl; 2,3-dihydrobenzothienyl, N-methyl-2-indolinyl; and indolinyl. Examples of non-aromatic polycyclic heterocycles include: pyrrolizidinyl and quinolizidinyl. The aforementioned listing of non-aromatic heterocyclic moieties and heteroaryl moieties is intended to be representative, not limiting. Preferred heteroaryl groups are 2-, 3- and 4-pyridyl; pyrazinyl; 2- and 5- pyrimidinyl; 3-pyridazinyl; 2- and 3-thienyl; 2- and 3-furyl; pyrrolyl; particularly N-methylpyrrol-2-yl; 2-imidazolyl; 2-thiazolyl; 2-oxazolyl; pyrazolyl; particularly 3- and 5-pyrazolyl; isothiazolyl; 1,2,3-triazolyl; 1,2,4- 4181
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triazolyl; 1,3,4-triazolyl; tetrazolyl, 1,2,3-thiadiazolyl; 1,2,3-oxadiazolyl; 1,3,4- thiadiazolyl and 1,3,4-oxadiazolyl; indolyl, particularly 2-, 3-, 4-, 5-, 6- and 7- indolyl; cinnolinyl; quinoxalinyl, particularly 2- and 5-quinoxalinyl; quinazolinyl, particularly 2-, 5-, 6-, 7- and 8-quinazolinyl; phthalazinyl; 1,8- naphthyridinyl; 1,5-naphthyridinyl, particularly l,5-naphthyridin-3-yl and 1,5- naphthyridin-4-yl; 1,4-benzodioxanyl; coumarinyl; benzofuryl, particularly 2-, 3- 5-, 6- and 7-benzofuryl; 1,2-benzisoxazolyl; benzothienyl, particularly 2-, 3-, 4-, 5-, 6-, and 7-benzothienyl; benzoxazolyl; benzthiazolyl, particularly 2- benzothiazolyl and 5-benzothiazolyl; purinyl; benzimidazolyl, particularly 2- benzimidazolyl; benztriazolyl; thioxanthinyl; carbazolyl; carbolinyl; and acridinyl, particularly 6-acridinyl. More preferred heteroaryl groups are 2, 3- and 4-pyridyl; 2- and 3- thienyl; 2- and 3-furyl; 2-pyrrolyl; 2-imidazolyl; 2-thiazolyl; 2-oxazolyl; 2- and 3-indolyl; 2-, and 3-benzofuryl; 3-(l,2-benzisoxazolyl); 2-, and 3 -benzothienyl; 2-benzoxazolyl; 1- and 2-benzimidazolyl, 2-, 3- and 4-quinolyl; and 2- and 5- benzthiazolyl. Most preferred heteroaryl groups are 2- and 3-indolyl; 2- and 3-ρyrrolyl, 2-, and 3-benzofuryl; and 2-, and 3 -benzothienyl. The term "substituted" means that an atom or group of atoms has replaced hydrogen as the substituent attached to another group. For aryl and heteroaryl groups, the term "substituted" refers to any level of substitution, namely mono-, di-, tri-, tetra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position. The term "gem" when used in the name of a compound is an abbreviation of the term "geminal" designates that two substituents are bonded to the same atom. For example, a gem-difluoro(Cι-C6)alkyl group includes 2,2- difluoropropyl, 1,1-difTuoroethyl and difluoromethyl. Some of the radioprotective small molecule inhibitors of ABL kinase and all of the α,β-unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides are characterized by isomerism resulting from the presence of a double bond. This isomerism is commonly referred to as cis-trans isomerism, but the more comprehensive naming convention employs E and Z designations. The compounds are named according to the Cahn-Ingold-Prelog system, the IUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc., New York, NY, 4th ed., 1992, p. 127-138. Using this system of nomenclature, the four groups about a double bond are prioritized according to a series of rules. Then, that isomer with the two higher ranking groups on the same side of the double bond is designated Z (for the German word "zusammen", meaning together). The other isomer, in which the two higher-ranking groups are on opposite sides of the double bond, is designated E (for the German word "entgegen", which means "opposite"). Thus if the four groups on a carbon-carbon double bond are ranked with A being the lowest rank and D being highest, A > B > C > D, the isomers would be named as in Scheme 1.
Figure imgf000042_0001
Z configuration Scheme 1 E connSuration In particular the indolones of formula III contain a double bond and are prepared as either the E- or Z-isomer or as a mixture of both isomers. The proportions of an isomeric mixture may be an equilibrium mixture influenced by differential steric strain of the two isomers. Alternately, a mixture of isomers may be enriched in one isomer by selected reaction conditions or by a purification method. Unless otherwise indicated, both configurations and mixtures thereof are included in the scope of "radioprotective ABL inhibitor" and in the scope of "α,β-unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides." Some of the radioprotective small molecule inhibitors of ABL kinase and some of the α,β-unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides may be characterized by isomerism resulting from the presence of a chiral center. The isomers resulting from the presence of a chiral center 181
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comprise a pair of nonsuperimposable isomers that are called "enantiomers." Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. See March, Advanced Organic Chemistry, 4th Ed., (1992), p. 109. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated (R) and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated (S). In the example below, the Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.
Figure imgf000043_0001
(if) configuration (S) configuration Scheme 2 Unless otherwise indicated, both absolute configurations and mixtures thereof are included in the scope of "radioprotective ABL inhibitor" and in the scope of "α,β-unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides." Nomenclature employed herein for providing systematic names for compounds useful in the claimed method is derived using the Nomenclator® facility within the computer program package, ChemDraw®. When compounds of the invention characterized by E-/Z- isomerism or by isomerism resulting from the presence of a chiral center, are named herein, the absence of E- or Z-, or R- or S- designation in the name is intended to mean that the named compound includes all possible isomeric forms and all mixtures thereof. Brief Description of the Figures FIG. 1 shows quantitatively the inhibition of ABL kinase activity by compounds 1 (■), 2 (Δ), 3 (V), 4 (0), 5 (•) and 6 (α) plotted as percent of solvent treated control. 4181
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FIG. 2 shows quantitatively the inhibition of ABL kinase activity by compounds 7 (■), 8 (Δ), 9 (V), 10 (0), 11 (•) and 12 (o), plotted as percent of solvent treated control. FIG. 3 shows quantitatively the inhibition of ABL kinase activity by compounds 13 (■), 14 (Δ), 15 (repeated three times as V, •, and o), and 16 (0), plotted as percent of solvent treated control. FIG. 4 shows quantitatively the inhibition of ABL kinase activity by compounds 17 (■), 18 (Δ), and 19 (V) plotted as percent of solvent treated control. FIG. 5 shows quantitatively the inhibition of ABL kinase activity by compounds 20 (■), 21 (Δ), and 22 (V) plotted as percent of solvent treated control.
Detailed Description of the Invention It has now been found that small molecule inhibitors of ABL activity are also capable of protecting cells, tissues and individuals from the cytotoxic effects of ionizing radiation. The radioprotective compounds protect normal cells and tissues from the effects of acute and chronic exposure to ionizing radiation. Further, compositions comprising a small molecule inhibitor of ABL activity, in combination with either an antioxidant compound or an α,β- unsaturated (aryl or heteroaryl) sulfone, sulfonamide or carboxamide, are capable of protecting cells, tissues and individuals from the cytotoxic effects of ionizing radiation. Preferred antioxidant compounds useful in combination small molecule inhibitors of ABL activity include, for example, carotenoids, catechins, isoflavones, flavanones, flavanols, flavanoid chalcones, vitamin E compounds, (3-aminopropyl)[2-(phosphonothio)ethyl]amine, ascorbic acid, cysteine, glutathione, probucol, β-mercaptoethanol dithiothreitol, pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, ubiquinone, and porphyrin compounds such as those disclosed in EP 1,045,851, the entire contents of which is incorporated herein by reference. Preferred carotenoids include β-carotene, α- -44-
carotene, lutein. lycopene. Preferred catechins include gallic acid, propyl gallate, (+)-catechin, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)- epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-catechin gallate (CG), and (-)-gallocatechin gallate. Preferred isoflavones include genistein and daidzein. Preferred flavanols include hesperitin, hesperidin, and quercetin, kaempferol, myricetin. Preferred flavanoid chalcones include xanthohumol and isoxanthohumol. Preferred vitamin E compounds include tocopherols and tocotrienols. Antioxidant compounds more preferably include, for example, β- carotene, α-carotene, lutein, lycopene, gallic acid, propyl gallate, (+)-catechin, (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-catechin gallate (CG), (-)-gallocatechin gallate, genistein, hesperitin, hesperidin, quercetin, kaempferol, myricetin, xanthohumol, isoxanthohumol, tocopherols, tocotrienol, (3-aminopropyl)[2- (phosphonothio)ethyl]amine, ascorbic acid, cysteine, glutathione, probucol, β- mercaptoethanol dithiothreitol, pyrrolidine dithiocarbamate, N-acetyl-L- cysteine, ubiquinone, meso-tetrakis-(N-alkylpyridinium-2-yl)porphyrins, meso- tetrakis-(N-alkylpyridinium-3-yl)porphyrins, and salts of such compounds. The meso-tetrakis-(N-alkylpyridinium-2-yl)porphyrins, meso-tetrakis- (N-alkylpyridinium-3-yl)ρorphyrins are disclosed in EP 1,045,851 as having the formulae VI and VII:
Figure imgf000045_0001
wherein Alkyl is preferably (Cι-C8) alkyl, more preferably (Cι-C4)alkyl; and each M is independently selected from the group consisting of-H, -N02, -CN, -CH=CH2 and -CHO; and the compound is complexed with a metal selected from the group consisting of iron, copper, cobalt, nickel and zinc. The precise mechanism of action of the radioprotective compounds disclosed herein is unknown. However, based on experimental models, and without wishing to be bound by any theory, the compounds are believed to inhibit the tyrosine kinase activity of the ABL protein. Individuals may be exposed to ionizing radiation when undergoing therapeutic irradiation for the treatment of proliferative disorders. Such disorders include cancerous and non-cancer proliferative disorders. For example, the present compounds and pharmaceutical compositions are believed effective in protecting normal cells during therapeutic irradiation of a broad range of tumor types, including but not limited to the following: breast, prostate, ovarian, lung, colorectal, brain (i.e., glioma) and renal. The compounds and compositions are also effective in protecting normal cells during therapeutic irradiation of leukemic cells. The compounds are also believed useful in protecting normal cells during therapeutic irradiation of abnormal tissues in non-cancer proliferative disorders, including but not limited to the following: hemangiomatosis in newborn, secondary progressive multiple sclerosis, chronic progressive myelodegenerative disease, neurofibromatosis, ganglioneuromatosis, keloid formation, Paget's Disease of the bone, fibrocystic disease of the breast, Peronies and Duputren's fibrosis, restenosis and cirrhosis. The radioprotective ABL inhibitors of formula I useful in the method of the invention may be prepared by organic synthesis using techniques that are known in the art or readily adapted from techniques known in the art. The following general synthesis methods are representative of methods whereby the compounds useful in the claimed method may be prepared. Svnthesis of Formula Ila Compounds The compounds of formula Ila comprise pyrazolopyrimidines which may be prepared according to the method of Bishop et al, J. Am. Chem. Soc, 1999, 121, pp. 627-631 and Dow et al, US Patent 5,593,997, the entire disclosures of which are incorporated herein by reference. The method is described in Scheme 3:
Figure imgf000047_0001
formamide
Figure imgf000047_0002
Scheme 3
Figure imgf000047_0003
A. An activated acid such as, for example, acid chloride intermediate 1 is first reacted with malononitrile, to form an enol intermediate, preferably in a suitable inert solvent in the presence of a suitable base. By an "inert solvent" is meant a solvent that does not react or degrade under the reaction conditions to a degree that would interfere with the desired reaction. Suitable inert solvents for the enol preparation include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, for example, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), or N-methylpyrrolidinone (NMP). Suitable bases include bases capable of removing a proton from malononitrile such as, for example, sodium hydride or potassium hydride. The enol synthesis reaction is conveniently carried out at a temperature in the range from about 0° C to about 50° C, preferably in the range from about 25° C to about 30° C, and conveniently at about room temperature. The enol intermediate produced by the above reaction is isolated from the reaction 181
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mixture, for example by neutralization of the reaction mixture and extraction of the reaction mixture with a suitable water immiscible solvent. The enol intermediate is then alkylated to yield intermediate 2, using a suitable alkylating reagent, preferably dimethyl sulfate, in the presence of a suitable base, preferably in a suitable inert solvent. Suitable bases for the alkylation reaction include those described above in the enol synthesis reaction. Suitable solvents for the alkylation reaction include aqueous solvents such as, for example mixtures of THF and water or mixtures of dioxane and water. The alkylation reaction is conveniently carried out at a temperature in the range from about 25° C. to about 150° C, preferably in the range from about 50° C to about 80° C. B. Intermediate 2 is cyclized to intermediate 3 by reaction with a suitable hydrazine derivative, or a salt thereof, preferably in the presence of a suitable inert solvent and in the presence of a suitable base. Suitable hydrazine derivatives include alkyl hydrazines such as tert-butyl hydrazine, preferably added to the reaction as an acid salt such as an HC1 salt. Suitable inert solvents include those described above in the enol synthesis reaction. Preferably, the solvent is an alkyl alcohol, for example ethanol. Suitable bases for use in the cyclization reaction include bases described for use in the enol synthesis described above. Preferably an amine base is employed, such as triethylamine.
The cyclization reaction is conveniently carried out at a temperature in the range from about 30° C. to about 100° C, preferably in the range from about 50° C to about 80° C, most conveniently at the reflux temperature of the reaction mixture. The pyrazole 3 may be isolated from the reaction mixture, for example by removing the volatiles and subjecting the residue obtained thereby to chromatographic separation. C. Pyrazole 3 is reacted with formamide to generate the product pyrazolopyrimidine of formula Ila. The reaction is preferably performed at an elevated temperature from about 50° C. to about 200° C, preferably in the range from about 100° C to about 200° C, most conveniently at the reflux temperature of the reaction mixture. 181
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Synthesis of Formula lib Compounds The compounds of formula lib comprise pyrrolopyrimidines which may be prepared according to the method of Missbach et al, US Patent 5,869,485, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 4:
Figure imgf000049_0001
Scheme 4 A. According to Scheme 4, haloalkyl ketone 4 is reacted with aryl amine 5, preferably in the presence of a suitable inert solvent and in the presence of a suitable base, to generate intermediate 6. Suitable solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as DMSO, DMF or NMP. Preferably, the solvent is an alkyl alcohol such as, for example, ethanol. Suitable bases for the reaction include organic amine bases such as pyridine, 2,6-lutidine, collidine, 4-dimethylaminopyridine, triethylamine, morpholine, N-methylmorpholine or l,8-diazabicyclo[5.4.0]undecane; alkali or 4181
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alkaline earth metal carbonates or hydroxides, for example sodium carbonate, potassium carbonate, calcium carbonate, sodium hydroxide or potassium hydroxide; alkali metal or alkaline earth metal amides, for example sodium amide or sodium bis(trimethylsilyl)amide; and reagents that are immobilized on a solid phase support, such as solid phase amine bases, e.g., diisopropylethylamine bound to polystyrene. The reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably in the range from about 50° C to about 100° C, and conveniently at the reflux temperature of the reaction mixture. B. Intermediate 6 is reacted with malononitrile, preferably in the presence of suitable inert solvent and in the presence of a suitable base to generate pyrrole intermediate 7. Suitable solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as DMSO, DMF, or NMP. Suitable bases include bases capable of removing a proton from malononitrile such as, for example, sodium metal, lithium metal, lithium naphthalide, sodium hydride or potassium hydride. The reaction is conveniently carried out at a temperature in the range from about 0° C. to about 100° C, preferably at the reflux temperature of the reaction mixture. C. Pyrrole intermediate 7 is reacted with a phosgene equivalent such as triethyl orthoformate, with or without additional solvent. The reaction is conveniently carried out at an elevated temperature, preferably in the range of from about 100° C. to about 200° C, preferably at the reflux temperature of the reaction mixture. An intermediate addition product is isolated by concentrating the reaction mixture under vacuum and removing the precipitated product by filtration. The intermediate addition product is taken up in a suitable inert 18
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solvent and the resulting solution is saturated with ammonia and reacted, preferably at elevated temperature in a pressure reactor. Suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol. The reaction is conveniently carried out at an elevated temperature, conveniently in the range of from about 100° C to about 200° C, preferably from about 100° C to about 150° C. The product amino pyrrolopyrimidine may be isolated by cooling the reaction mixture and removing the solid precipitated produce by filtration. Synthesis of Formula III Compounds The compounds of formula III comprise indolones which may be prepared according to the method of Battistini et al, US Patent 5,905,149, the entire disclosure of which is incorporated herein by reference. See also, Corbett et al, US Patent 6,307,056, and Sun et al, J. Med. Chem. 1998, 41, pp. 2588- 2603, the entire disclosures of which are incorporated herein by reference. The method is described in Scheme 5:
Figure imgf000051_0001
A. According to Scheme 5, 2-fluoronitrobenzene analog 8 is reacted with a malonate ester, preferably in the presence of a suitable inert solvent, and in the presence of a suitable base. Suitable inert solvents include, for example, ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, for example, DMSO, DMF, or NMP. Suitable bases include bases capable of removing a proton from a malonate ester such as, for example, sodium metal, lithium metal, lithium naphthalide, sodium hydride or potassium hydride. The reaction is conveniently carried out at a temperature in the range from about 25° C to about 150° C, preferably at about 100 C. B. The intermediate 2-phenyl malonate 9 is subjected to hydrolytic conditions, such as, for example an aqueous strong acid such as aqueous hydrochloric acid, to produce the phenyl acetic acid intermediate 10. The hydrolytic reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably at about 100° C. C. The intermediate phenyl acetic acid 10 is sub ected to reduction, preferably in the presence of a suitable inert solvent, to yield intermediate substituted indolone 11. Suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; ethers such as tetrahydrofuran, 1,4-dioxane or tert-butylethylether; and aromatic solvents such as toluene. The reaction is conveniently carried out at a temperature in the range of from about 0° C to about 100° C, preferably about room temperature. The reduction conditions are preferably catalytic hydrogenation conditions. The reduction is preferably performed in the presence of a hydrogen source, such as, for example pressurized hydrogen gas, in the presence of a transition metal catalyst such as for example tin, iron, platinum, palladium or zinc. D. Intermediate indolone 11 is reacted with aryl aldehyde intermediate 12, preferably in the presence of a suitable inert solvent. Suitable inert solvents include, for example alkyl alcohols, such as methanol, ethanol, and isopropanol; esters such as methyl acetate or ethyl acetate; halogenated solvents, such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran, 1,4-dioxane or t-butylethylether; aromatic solvents such as toluene; and polar aprotic solvents such as, DMSO, DMF, or NMP. The reaction is conveniently carried out at a temperature in the range from about 50° C to about 150° C, preferably at the reflux temperature of the reaction mixture. -52-
Synthesis of Formula IV Compounds The compounds of formula IV comprise tyrphostins which may be prepared according to the method of Hirth et al, US Patent 5,700,822, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 6:
Figure imgf000053_0001
Scheme 6 Aldehyde intermediate 13 is reacted with aryl or heteroaryl acetonitrile 14, preferably in the presence of a suitable inert solvent. Suitable inert solvents include for example alkyl alcohols such as methanol, ethanol, and isopropanol; esters such as ethyl acetate; halogenated solvents such as methylene chloride, chloroform or carbon tetrachloride; ethers such as tetrahydrofuran or 1,4-dioxan; aromatic solvents such as toluene; and polar aprotic solvents such as DMF, NMP or DMSO. Alcohols, particularly ethanol and isopropanol are preferred. The reaction is carried out at a temperature in the range from about 25° C. to about 150° C, preferably in the range from about 25° C to about 100° C. Most preferably, the reaction is carried out at the reflux temperature of the reaction mixture. The α,β-unsaturated (aryl or heteroaryl) sulfones, sulfonamides and carboxamides useful in the claimed method may be prepared by organic synthesis using techniques that are known in the art or readily adapted from techniques known in the art. The following general synthesis methods are representative of methods whereby the compounds may be prepared.
Synthesis of α.β-Unsaturated (Aryl or Heteroaryl) Sulfones and Sulfoxides of Formula V A. Synthesis of α,β-unsaturated (aryl or heteroaryl) sulfones and sulfoxides of formula V containing an (iζ)-double bond, may be accomplished 181
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according to the method of Reddy et al, US Patent 6,359,013, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 7:
Figure imgf000054_0001
According to Scheme 7, a mercaptan 15 is slowly added to a solution of sodium hydroxide (8 g, 0.2 mol) in methanol (200 mL). Then, chloroacetic acid (0.1 mol) is added portiomvise and the reaction mixture may be refluxed for 2-3 hours, then cooled to ambient temperature. The cooled reaction mixture is poured onto crushed ice and neutralized with dilute hydrochloric acid (200 mL). The resulting thioacetic acid 16 (0.1 mol) may be oxidized to the corresponding sulfonyl acetic acid 17b by use of any reagent capable of oxidizing a sulfϊde to a sulfone. The thioacetic acid 16 may alternately be oxidized to the sulfinyl acetic acid 17a by treatment with any reagent capable of selectively oxidizing a sulfϊde to a sulfoxide. Suitable oxidizing reagents for both oxidation reactions include peroxides such as hydrogen peroxide, peracids such as meta-chloroperoxybenzoic acid (MCPBA) or persulfates such as OXONE® (potassium peroxymonosulfate). The reaction is preferably carried out in the presence of a suitable solvent. Suitable solvents include, for example, water, acetic acid or non-polar solvents such as dichloromethane (DCM). Reaction to selectively form the sulfinyl acetic acid 17a is preferably performed at low temperature, more preferably from about -10 to about 20°C. A reaction to form the sulfinyl acetic acid 17a is preferably monitored so as to terminate the reaction prior to appreciable oxidation to the sulfonyl acetic acid 17b. When the reaction is complete, the reaction mixture may be poured onto crushed ice. A solid precipitate may be collected by filtration and recrystallized from hot water to yield the purified sulfinyl acetic acid 17a. Reaction to form the sulfonyl acetic acid 17 b may be performed at higher temperature, for example, from about 30 to about 100°C with 30% hydrogen peroxide (0.12 mol) in glacial acetic acid (25 mL) by refluxing for 1-2 hours. When the reaction is complete, the reaction mixture may be cooled to ambient temperature and poured onto crushed ice. A solid precipitate may be collected by filtration and recrystallized from hot water to yield the purified sulfonyl acetic acid 17b. The α,β-unsaturated sulfone 19b may be prepared by mixing the sulfonyl acetic acid 17b (0.001 mol), an aromatic aldehyde 18 (0.001 mol) and benzylamine (1 mL) in glacial acetic acid (15 mL) and heating the mixture at reflux temperature for 2-3 hours. Similarly, the α,β-unsaturated sulfoxide 19a may be prepared by mixing the sulfinyl acetic acid 17a (0.001 mol), an aromatic aldehyde 18 (0.001 mol) and benzylamine (1 mL) in glacial acetic acid (15 mL) and heating the mixture at reflux temperature for 2-3 hours. When the reaction is complete, (the reaction forming sulfone 19b or (the reaction forming sulfoxide 19a) the reaction mixture may be cooled to ambient temperature and treated with dry ether (50 mL). Any precipitated product may be collected by filtration. The filtrate may be diluted with more ether and washed successively with a saturated solution of sodium bicarbonate (20 mL), sodium bisulfite (20 mL), dilute hydrochloric acid (20 mL) and finally with water (35 mL). Evaporation of the dried ether layer yields a solid compound of formula V in many cases. However, in some cases a syrupy material separates and may be solidified on treatment with 2-propanol. The purity of the product may be checked by TLC (silica gel, hexane/ethyl acetate 3:1). -55-
B. Synthesis of α,β-unsaturated (aryl or heteroaryl) sulfones and sulfoxides of formula V containing a (Z)-double bond, may be accomplished according to the method of Reddy et al, US Patent 6,359,013, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 8:
Figure imgf000056_0001
(Z)-Sulfone Compound of formula V ( )-Sulfoxide Compound of formula V Scheme 8
A. According to Scheme 8, an aromatic mercaptan may be converted to the corresponding sodium thiolate 21. To the thiolate, in an alkyl alcohol is added an aryl acetylene 20. The reaction is preferably performed at elevated temperature, more preferably at the reflux temperature of the reaction mixture. When the reaction is complete, the reaction mixture may be poured onto water ice. The crude product may be collected by filtration and purified, preferably by recrystallization from a suitable solvent, to yield a pure (2)- ,β-unsaturated (aryl or heteroaryl)sulfide 22. Preferable recrystallization solvents include water miscible alcohols and aqueous mixtures of water-miscible alcohols.
B. The (Z)-α,β-unsaturated (aryl or heteroaryl)sulfide 22 may be oxidized to the corresponding sulfone by use of any reagent capable of oxidizing a sulfide to a sulfone. Likewise, the ( )- ,β-unsaturated (aryl or heteroaryl)sulfide 22 may be oxidized to the corresponding sulfoxide by use of any reagent capable of oxidizing a sulfide to a sulfoxide. Suitable reagents and conditions for oxidation to a sulfone or sulfoxide are the same as the conditions for preparation of sulfonyl acetic acid 17b and sulfinyl acetic acid 17a. The purity of the (Z)-α,β- 181
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unsaturated (aryl or heteroaryl)sulfone or sulfoxide may be ascertained by thin layer chromatography and geometrical configuration may be assigned by analysis of infrared and nuclear magnetic resonance spectral data. Synthesis of α,β-Unsaτurated (Aryl or Heteroaryl) Sulfonamides of Formula V A. Sulfonamides of formula V containing an (£)-double bond, may be prepared according to the method of Reddy et al, WO 02/067865, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 9. 1. Na2S03 C| Br' NCOOR' S02 OOR • + Qr NH 2. PC13 23 24 25 \
Figure imgf000057_0001
Scheme 9 According to Scheme 9, a methyl (or ethyl) β-chlorosulfonylacetate intermediate 24 is prepared from methyl (or ethyl) bromoacetate (R' = methyl or ethyl). To do this, methyl (or ethyl) bromoacetate is reacted with sodium sulfate to form the sodium sulfoacetate intermediate Na2OS0 CH2C02R'. Potassium sulfate may be used as a substitute for sodium sulfate. The sodium sulfoacetate intermediate is then reacted with a chlorinating agent, preferably PCI5, to form the methyl (or ethyl) β-chlorosulfonylacetate intermediate 24. Reaction of intermediate 24 with the aromatic amine 25 yields the arylaminosulfonylacetate intermediate 26. The latter reaction is conducted in a nonprotic solvent in the presence of a base. The same compound may serve as both the nonprotic solvent and the base. Such dual-function solvents include, for example, pyridine, substituted pyridines, trimethylamine and triethylamine. The -57-
arylaminosulfonylacetate 26 is then converted to the corresponding arylaminosulfonylacetic acid compound 27 by any base capable of hydrolyzing the ester function of 26 to an acid. Such bases include, for example, KOH and NaOH. In the final step, the arylaminosulfonylacetic acid compound is condensed with arylaldehyde 18 in the presence of a basic catalyst via a Knoevenagel reaction and decarboxylation of an intermediate. Basic catalysts include, for example, pyridine and benzylamine. The reaction yields the desired N-(aryl)-2-arylethenesulfonamide of formula V.
Synthesis of (i?)-α,β-Unsaturated (aryl or heteroaryl") Carboxamides of Formula V (£)-α,β-unsaturated (aryl or heteroaryl) carboxamides of formula II may be prepared according to the method of Reddy et al, US provisional patent application 60/406,766, filed August 29. 2002. and PCT Patent Application WO 04037751. filed August 28. 2003 and published May 6. 2004. the entire disclosures of which are incorporated herein by reference. The method is described in Scheme 10.
Figure imgf000058_0001
Scheme 10
A: Synthesis of an Alkyl -2(N-(Aryl or heteroarypaminocarbonyDacetate 26: According to Scheme 10, to a solution of an aromatic amine 30 (10 mmol) and TEA (10 mmol) in dichloromethane (DCM) (50 mL) at room temperature is slowly added a solution of an alkyl malonyl chloride (10 mmol) in DCM. The reaction is stirred for 1 hour. The reaction mixture is filtered and solvent is removed under reduced pressure to yield an oily material. The crude 4181
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product is purified by column chromatography to yield an alkyl-2-(N-(aryl or heteroaryl)aminocarbonyl)-acetate 31
B: Synthesis of 3-fAryl or heteroaryPamino-3-oxoρropanoic acid 32: The alkyl-2-(Ν-(aryl or heteroaryl)aminocarbonyl)-acetate 31 is refluxed for 2.5 hours in a solution of sodium hydroxide (9.0 g) in water (90 mL) and ethanol (90 mL). The reaction mixture is subsequently cooled and acidified with HC1 to precipitate the crude product acid. The crude 3-(aryl or heteroaryl)amino-3-oxopropanoic acid 32 is removed by filtration and recrystallized from hot water. C: Condensation of a 3-(aryl or heteroaryPamuio-3-oxopropanoic acid 27 with an aromatic aldehyde 18: A solution of the (aryl or heteroaryl)amino-3-oxoproρanoic acid 32 (10 mmol), an aromatic aldehyde 18 (10 mmol) and benzylamine (0.4 mL) is refluxed for 3 hours in glacial acetic acid (10 mL). The solution is then cooled. Cold ether (50 mL) is added. The organic layer is separated and washed with a saturated solution of sodium bicarbonate (30 mL), sodium bisulfite (30 mL) and dilute hydrochloric acid (30 mL). The ether solution is then dried over anhydrous sodium sulfate and evaporated under reduced pressure to yield the corresponding N-(aryl or heteroaryl)-3-(aryl or heteroaryl)-2-propenamide of formula V (JF-isomer).
Synthesis of (E) or (Z ,β-Unsaturated (Aryl or Heteroaryl) Carboxamides of Formula V (E)- ox ( )-α,β-unsaturated (aryl or heteroaryl) carboxamides of formula V may be prepared according to the method of Reddy et al, US provisional patent application 60/406,766, the entire disclosure of which is incorporated herein by reference. The method is described in Scheme 11. -59-
Figure imgf000060_0001
Condensation of (E) or (Z)- Aromatic acryloyl chlorides with aromatic amines: According to Scheme 11, an intermediate (E)- or (^-aromatic acryloylhalide 34 may be prepared from the corresponding aromatic acrylic acid 33. To do this, the aromatic acrylic acid is reacted with a halogenating agent such as for example, thionyl chloride or phosphorous pentachloride to form the intermediate carboxylic acid halide 34. A solution of an aromatic amine 35 (10 mmol) in pyridine (75 mL) is reacted with the (E) or (Z)-aromatic acryloyl halide 34 (10 mmol) for 4 to 6 hours at 80° C. The reaction mixture is cooled and poured into ice water (IL) and concentrated hydrochloric acid (100 mL) is added. The precipitated product is separated by filtration and crystallized to yield a pure E or Z-N-(aryl or heteroaryl)-3-(aryl or heteroaryl)-2-propenamide of formula V. Administration of ABL inhibitors Administration in Association with Therapeutic Ionizing Radiation According to the present invention, therapeutic ionizing radiation may be administered to an individual on any schedule and in any dose consistent with the prescribed course of treatment. The radioprotective compound is administered prior to the therapeutic ionizing radiation. The course of treatment differs from individual to individual, and those of ordinary skill in the art can readily determine the appropriate dose and schedule of therapeutic radiation in a given clinical situation. Hereinafter, reference to administration of radioprotective. compound shall mean administration of a radioprotective compound according to formula I alone, or in combination with either a radioprotective compound of formula V, or an antioxidant compound. The formula V compound or antioxidant compound may be administered simultaneously with the formula I compound, or may be administered separately. The compounds may be administered by the same or by different routes. Where the formula I compound and (i) the formula V compound or (ii) the antioxidant compound are administered at different times, the administration times are preferably optimized to obtain the radioprotective benefit of the combination based on the pharmacokinetic profiles of the compounds administered. Where the formula I compound and (i) the formula V compound or (ii) the antioxidant compound are administered simultaneously, the administration may be by the same or by different routes. Preferably, simultaneous administration is done by administering the compounds as part of the same pharmaceutical composition. The radioprotective compound should be administered far enough in advance of the therapeutic radiation such that the compound is able to reach the normal cells of the individual in sufficient concentration to exert a radioprotective effect on the normal cells. The radioprotective compound may be administered as much as about 24 hours, preferably no more than about 18 hours, prior to administration of the radiation. In one embodiment, the radioprotective compound is administered at least about 6-12 hours before administration of the therapeutic radiation. Most preferably, the radioprotective compound is administered once at about 18 hours and again at about 6 hours before the radiation exposure. One or more radioprotective compounds may be administered simultaneously, or different radioprotective compounds may be administered at different times during the treatment. Where the therapeutic radiation is administered in serial fashion, it is preferable to intercalate administration of one or more radioprotective compounds within the schedule of radiation treatments. As above, different radioprotective compounds may be administered either simultaneously or at different times during the treatment. Preferably, an about 24 hour period separates administration of a radioprotective compound and the therapeutic radiation. More preferably, the administration of a radioprotective compound and the therapeutic radiation is separated by about 6 to 18 hours. This strategy will yield significant reduction in radiation-induced side effects without affecting the anticancer activity of the therapeutic radiation. For example, therapeutic radiation at a dose of 0.1 Gy may be given daily for five consecutive days, with a two-day rest, for a total period of 6-8 weeks. One or more compounds according to the invention may be administered to the individual 18 hours previous to each round of radiation. It should be pointed out, however, that more aggressive treatment schedules, i.e., delivery of a higher dosage, is contemplated according to the present invention due to the protection of the normal cells afforded by the radioprotective compounds. Thus, the radioprotective effect of the radioprotective compound increases the therapeutic index of the therapeutic radiation, and may permit the physician to safely increase the dosage of therapeutic radiation above presently recommended levels without risking increased damage to the surrounding normal cells and tissues.
Administration in Bone Marrow Treatment The radioprotective compounds of the invention are further useful in protecting normal bone marrow cells from radiological treatments designed to destroy hematological neoplastic cells or tumor cells which have metastasized into the bone marrow. Such cells include, for example, myeloid leukemia cells. The appearance of these cells in the bone marrow and elsewhere in the body is associated with various disease conditions, such as the French-American-British (FAB) subtypes of acute myelogenous leukemias (AML), chronic myeloid leukemia (CML), and acute lymphocytic leukemia (ALL). CML, in particular, is characterized by abnormal proliferation of immature granulocytes (e.g., neutrophils, eosinophils, and basophils) in the blood, bone marrow, spleen, liver, and other tissues and accumulation of granulocytic precursors in these tissues. The individual who presents with such symptoms will typically have more than 20,000 white blood cells per microliter of blood, and the count may exceed 400,000. Virtually all CML patients will develop "blast crisis", the terminal stage of the disease during which immature blast cells rapidly proliferate, leading to death. Other individuals suffer from metastatic tumors, and require treatment with total body irradiation (TBI). Because TBI will also kill the individual's hematopoietic cells, a portion of the individual's bone marrow is removed prior to irradiation for subsequent reimplantation. However, metastatic tumor cells are likely present in the bone marrow, and reimplantation often results in a relapse of the cancer within a short time. Individuals presenting with neoplastic diseases of the bone marrow or metastatic tumors may be treated by removing a portion of the bone marrow (also called "harvesting"), purging the harvested bone marrow of malignant stem cells, and reimplanting the purged bone marrow. Preferably, the individual is simultaneously treated with radiation or some other anti-cancer therapy. Thus, the invention provides a method of reducing the number of malignant cells in bone marrow, comprising the steps of removing a portion of the individual's bone marrow, administering an effective amount of at least one radioprotective compound of formula I and irradiating the treated bone marrow with a sufficient dose of ionizing radiation such that neoplastic or tumor cells in the bone marrow are killed. As used herein, "malignant cell" means any uncontrollably proliferating cell, such a tumor cell or neoplastic cell. The radioprotective compound protects the normal hematopoietic cells present in the bone marrow from the deleterious effects of the ionizing radiation. The number of malignant cells in the bone marrow is significantly reduced prior to reimplantation, thus minimizing the occurrence of a relapse. Preferably, each radioprotective compound is administered in a concentration from about 0.25 to about 100 micromolar; more preferably, from about 1.0 to about 50 micromolar; in particular from about 2.0 to about 25 181
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micromolar. Particularly preferred concentrations are 0.5, 1.0 and 2.5 micromolar and 5, 10 and 20 micromolar. Higher or lower concentrations may also be used. The radioprotective compound may be added directly to the harvested bone marrow, but are preferably dissolved in an organic solvent such as dimethylsulfoxide (DMSO). Pharmaceutical formulations of radioprotective compounds such as are described in more detail below may also be used. Preferably, the radioprotective compound is added to the harvested bone marrow about 20 hours prior to radiation exposure, preferably no more than about 24 hours prior to radiation exposure. In one embodiment, the radioprotective compound is administered to the harvested bone marrow at least about 6 hours before radiation exposure. One or more radioprotective compounds of formula I may be administered simultaneously, or different radioprotective compounds may be administered at different times. Other dosage regimens may also be used. If the individual is to be treated with ionizing radiation prior to reimplantation of the purged bone marrow, the individual may be treated with one or more radioprotective compounds of formula I prior to receiving the ionizing radiation dose, as described above.
Administration in Treatments Related to Non-therapeutic Exposure to Ionizing Radiation An individual may also be exposed to ionizing radiation from occupational or environmental sources, as discussed in the background section. For purposes of the invention, the source of the radiation is not as important as the type (i.e., acute or chronic) and dose level absorbed by the individual. It is understood that the following discussion encompasses ionizing radiation exposures from both occupational and environmental sources. Individuals suffering from effects of acute or chronic exposure to ionizing radiation that are not immediately fatal are said to have remediable radiation damage. Such remediable radiation damage can be reduced or eliminated by the compounds and methods of the present invention. An acute dose of ionizing radiation which may cause remediable radiation damage includes a localized or whole body dose, for example, between about 10,000 millirem (0.1 Gy) and about 1,000,000 millirem (10 Gy) in 24 hours or less, preferably between about 25,000 millirem (0.25 Gy) and about 200,000 (2 Gy) in 24 hours or less, and more preferably between about 100,000 millirem (1 Gy) and about 150,000 millirem (1.5 Gy) in 24 hours or less. A chronic dose of ionizing radiation which may cause remediable radiation damage includes a whole body dose of about 100 millirem (.001 Gy) to about 10,000 millirem (0.1 Gy), preferably a dose between about 1000 millirem (.01 Gy) and about 5000 millirem (.05 Gy) over a period greater than 24 hours, or a localized dose of 15,000 millirem (0.15 Gy) to 50,000 millirem (0.5 Gy) over a period greater than 24 hours. The invention therefore provides a method for treating individuals who have incurred remediable radiation damage from acute or chronic exposure to ionizing radiation, comprising reducing or eliminating the cytotoxic effects of radiation exposure on normal cells and tissues by administering an effective amount of at least one radioprotective compound of formula I. The compound is preferably administered in as short a time as possible following radiation exposure, for example between 0 - 6 hours following exposure. Remediable radiation damage may take the form of cytotoxic and genotoxic (i.e., adverse genetic) effects in the individual. In another embodiment, there is therefore provided a method of reducing or eliminating the cytotoxic and genotoxic effects of radiation exposure on normal cells and tissues, comprising administering an effective amount of at least one radioprotective compound prior to acute or chronic radiation exposure. The compound may be administered, for example about 24 hours prior to radiation exposure, preferably no more than about 18 hours prior to radiation exposure. In one embodiment, the compound is administered at least about 6 hours before radiation exposure. Most preferably, the compound is administered at about 18 and again at about 6 hours before the radiation exposure. One or more radioprotective compounds of formula I may be administered simultaneously, or different compounds may be administered at different times. For administration of more than one radioprotective compound of formula I at different times, the administration times are preferably optimized to obtain the radioprotective benefit of the combination based on the pharmacokinetic profile of the compounds administered. For administration of more than one radioprotective compound of formula I simultaneously, the administration may be by the same or by different routes. Preferably, simultaneous administration of more than one compound of formula I is done by administering the compounds as part of the same pharmaceutical composition. When multiple acute exposures are anticipated, the radioprotective compound may be administered multiple times. For example, if fire or rescue personnel must enter contaminated areas multiple times, the radioprotective compound may be administered prior to each exposure. Preferably, an about 24-hour period separates administration of radioprotective compound and the radiation exposure. More preferably, the administration of a radioprotective compound and the radiation exposure is separated by about 6 to 18 hours. It is also contemplated that a worker in a nuclear power plant may be administered an effective amount of radioprotective compound prior to beginning each shift, to reduce or eliminate the effects of exposure to ionizing radiation. If an individual anticipates chronic exposure to ionizing radiation, the radioprotective compound may be administered periodically throughout the duration of anticipated exposure. For example, a nuclear power plant worker or a soldier operating in a forward area contaminated with radioactive fallout may be given a radioprotective compound of formula I every 24 hours, preferably every 6-18 hours, in order to mitigate the effects of radiation damage. Likewise, the radioprotective compound may be periodically administered to civilians living in areas contaminated by radioactive fallout until the area is decontaminated or the civilians are removed to a safer environment. 4181
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Routes of Administration As used herein, "administered" means the act of making the radioprotective compounds of formula I (alone, or in combination with a compound of formula V or an antioxidant) available to the individual such that a pharmacological effect of radioprotection is realized. This pharmacological effect may manifest as the absence of expected physiologic or clinical symptoms at a certain level of radiation exposure. One skilled in the art may readily determine the presence or absence of radiation-induced effects, by well- known laboratory and clinical methods. The radioprotective compound may thus be administered by any route which is sufficient to bring about the desired radioprotective effect in the patient. Routes of administration include, for example enteral (e.g., oral, rectal, intranasal, etc.) and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intravaginal, intravesical (e.g., into the bladder), intradermal, topical or subcutaneous administration. Also contemplated within the scope of the invention is the instillation of drug in the body of the patient in a controlled formulation, with systemic or local release of the drug to occur at a later time. For example, a depot of a radioprotective compounds of formula I may be administered to the patient more than 24 hours before the administration of radiation. Preferably, at least a portion of the compound is retained in the depot and not released until an about 6-18 hour window prior to the radiation exposure.
Compositions for Administration The radioprotective compound may be administered in the form of a pharmaceutical composition comprising one or more compounds of formula I in combination with a pharmaceutically acceptable carrier. The active compound in such formulations may comprise from 0.1 to 99.99 weight percent. By "pharmaceutically acceptable carrier" is meant any carrier, diluent or excipient, which is compatible with the other ingredients of the formulation and is not deleterious to the individual. It is within the skill in the art to formulate appropriate pharmaceutical compositions with radioprotective compounds. For example, the radioprotective compound may be formulated into pharmaceutical compositions according to standard practices in the field of pharmaceutical preparations. See Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, PA. Suitable pharmaceutical compositions include, for example, tablets, capsules, solutions (especially parenteral solutions), troches, suppositories, or suspensions. For parenteral administration, the radioprotective compound may be mixed with a suitable carrier or diluent such as water, an oil, saline solution, aqueous dextrose (glucose) and related sugar solutions, cyclodextrans or a glycol such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a pharmaceutically acceptable, water-soluble salt of the radioprotective compound. Stabilizing agents, antioxidizing agents and preservatives may also be added. Suitable antioxidizing agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. For oral administration, the radioprotective compound may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, or other suitable oral dosage forms. For example, the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods. The specific dose and schedule of radioprotective compound to obtain the radioprotective benefit will, of course, be determined by the particular circumstances of the individual patient including, the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease, and the route of administration, and the specific toxicity of the radiation. For example, a daily dosage of from about 0.01 to about 150 mg/kg/day may be utilized, more preferably from about 0.05 to about 50 mg/kg/day. Particularly preferred are doses from about 1.0 to about 10.0 mg/kg/day, for example, a dose of about 7.0 mg/kg/day. The dose may be given over multiple administrations, for example, two administrations of 3.5 mg/kg. Higher or lower doses are also contemplated. The radioprotective compounds may take the form of pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from the group consisting of aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, beta-hydroxybutyric, galactaric and galacturonic acid. Suitable pharmaceutically acceptable base addition salts include metallic salts made from calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,iV- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding radioprotective compound by reacting, for example, the appropriate acid or base with the free acid or free base of the compound. The compositions useful in the method of the present invention may also be formulated so as to provide slow or controlled-release of the active ingredient therein. In general, a controlled-release preparation is a composition capable of releasing the active ingredient at the required rate to maintain constant 181
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pharmacological activity for a desirable period of time. Such dosage forms may provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than other non-controlled formulations. For example, U.S. Patent No. 5,674,533 discloses controlled-release compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive. U.S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances. U.S. Patent No. 5, 591,767 discloses a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-inflammatory agent with potent analgesic properties. U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers. U.S. Patent No. 5,073,543 discloses controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle. U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. The patents cited above are incorporated herein by reference. Biodegradable microparticles may be used in controlled-release formulations useful in the method of this invention. For example, U.S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient. U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions. These patents are incorporated herein by reference. The controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. Various mechanisms of drug release exist. For example, in one embodiment, the controlled-release component can swell and form porous openings large enough to release the active ingredient after administration to a patient. The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres, that facilitate the controlled-release of the radioprotective compound of formula I in a pharmaceutical composition. In another embodiment, the controlled-release component may be biodegradable, induced by exposure to the aqueous environment, pH, temperature, or enzymes in the body. In another embodiment, sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature. This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient. The practice of the invention is illustrated by the following non- limiting examples. Examples
Example 1. Synthesis of 5-((lZ)-l-cyano-2-indol-3-yIvinyl)-3- aminøpyrazole-4-carbonitriIe. The title compound of Example 1, a tyrphostin compound of formula IV, is synthesized according to Scheme 12. Indole-3-carboxaldehyde la [487-89-8] (Sigma Aldrich, 12,944-5)(0.29 g, 2 mmol), 3-amino-4-cyano-5-cyanomethyl-2-pyrazole lb (0.29 g, 2 mmol) and β-alanine (20 mg) are heated at reflux in ethanol (30 mL) for four hours.
The reaction mixture is then cooled to ambient temperature and filtered to yield the crude product, lc, which is further purified by preparative HPLC.
Figure imgf000071_0001
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Example 2. Synthesis of l-(tert-butyl)-3-(4-methyIphenyI)pyrazolo[5,4- d]pyrimidine-4-ylamine. The title compound of Example 2, a pyrazolopyrimidine compound of formula lib, is synthesized according to Scheme 13.
Figure imgf000072_0001
A. [Methoxy(4-methylphenyl)methylene]methane-l,l-dicarbonitrile, 2b. Sodium hydride (10 mmol, 0.4 g, 60% in mineral oil) is washed with pentane (10 mL) and suspended in dry THF (40 mL) and stirred at ambient temperature under argon. Malononitrile [109-77-3] (5 mmol, 0.33 g) is added dropwise followed by the dropwise addition of 4-methylbenzoyl chloride, 2a [874-60-2]. The resulting mixture is stirred at ambient temperature for 0.5 hours. When the reaction is complete, the reaction mixture diluted with water and extracted with ethyl acetate. The extract is dried (sodium sulfate), filtered, and concentrated under vacuum. The residue is redissolved in dioxane/water (6/1). Sodium bicarbonate (25 mmol, 2.1 g) is added. Dimethyl sulfate is added dropwise over five minutes and the reaction mixture is warmed to 80° C for one hour. The reaction mixture is then concentrated under vacuum and the residue is purified by preparative HPLC to yield [methoxy(4- methylphenyl)methylene]methane- 1 , 1 -dicarbonitrile, 2b. B. 2-Amino-l-(tert-butyl)-4-(4-methylphenyl)pyrrole-3-carbonitrile, 2c. [Methoxy(4-methylphenyl)methylene]methane- 1 , 1 -dicarbonitrile, 2b (4 181
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mmol, 0.8 g) is dissolved in ethanol (40 mL). tert-Butyl hydrazine hydrochloride salt [7400-27-3] (Sigma Aldrich, 19,497-2)(4 mmol, 0.5 g) is added. The reaction mixture is stirred at reflux for one hour. The reaction mixture is then cooled to ambient temperature, concentrated under vacuum and the residue is purified by preparative HPLC to yield 2-amino-l-(tert-butyl)-4-(4- methylphenyl)pyrrole-3-carbonitrile, 2c.
C. 1 -(tert-Butyl)-3-(4-methylphenyl)pyrazolo[5,4-d]pyrimidine-4-ylamine, 2d. 2- Amino- 1 -(tert-butyl)-4-(4-methylphenyl)pyrrole-3-carbonitrile, 2c (3 mmol, 0.76 g) is dissolved in formamide (2.0 mL) and warmed to 180° C for 12 hours. The reaction mixture is added dropwise to rapidly stirred ice- water (50 mL). The product, l-(tert-butyl)-3-(4-methylphenyl)pyrazolo[5,4-d]pyrimidine- 4-ylamine, precipitates and is removed by filtration.
Example 3. Synthesis of (2-{4-[4-amino-5-(4-methoxyphenyl)pyrroIo[2,3- d]pyrimidin-7-yl]phenoxy}ethyI)(2-methoxyethyl)amine. The title compound of Example 3, a pyrrolopyrimidine compound of formula Ila is synthesized according to Scheme 14.
Figure imgf000074_0001
A. (2-Methoxyethyl)[2-(4-nitrophenoxy)ethyl] amine, 3b. 4-nitrophenol [93951-79-2] (10 g, 144 mmol), 2-chloroethyl mesylate [3570-58-9] (0.2 mol) and 4-DMAP (catalytic) are stirred at reflux in acetonitrile (100 mL) overnight. Volatiles are removed and the residue is purified by preparative HPLC. The intermediate chloroethoxy compound is stirred at reflux in neat methoxyethyl amine [109-85-3] (100 mL) overnight. The volatiles are removed under vacuum. The residue is purified by preparative HPLC to yield (2-methoxyethyl)[2-(4-nitrophenoxy)ethyl]amine, 3b.
B. (tert-Butoxy)-N-(2-methoxyethyl)-N-[2-(4-nitrophenoxy)ethyl]- carboxamide, 3c. Intermediate 3b (10 g, 42 mmol) and di-tert-butyldicarbonate [24424- 99-5] (9.2 g, 42 mmol) are stirred together in dry THF at ambient temperature overnight. Volatiles are removed under vacuum and the residue is purified by preparative HPLC to generate the intermediate (tert-butoxy)-N-(2- methoxyethyl)-N-[2-(4-nitrophenoxy)ethyl]-carboxamide, 3c. C. N-[2-(4-Aminophenoxy)ethyl](tert-butoxy)-N-(2-methoxyethyl)- carboxamide, 3d. Intermediate 3c (lOg, 29 mmol) and 10% palladium on carbon (500 mg) are taken up in ethanol (250 mL) and shaken under hydrogen (3 atm.) until the stoichiometric amount of hydrogen is taken up. The reaction mixture is then filtered through a pad of diatomaceous earth. The filtrate is concentrated under vacuum and the crude product N-[2-(4-aminophenoxy)ethyl](tert-butoxy)-N-(2- methoxyethyl)-carboxamide, 3d is used for the next step without further purification. D. (tert-Butoxy)-N-(2-methoxyethyl)-N-[2-(4-{[2-(4-methoxyphenyl)-2- oxoethyl]amino}phenoxy)ethyl]carboxamide, 3f. 2-Bromo-4'-methoxyacetophenone [2632-13-5] 3e [(Acros AC15190) (5.7 g, 25 mmol), intermediate 3d (8g, 26 mmol), and powdered sodium carbonate (25 g) are stirred at reflux for three hours in ethanol (150 mL). The mixture is then cooled to ambient temperature. Water is added and the product precipitates and is collected by filtration. The crude product is taken up in methylene chloride, washed with water, dried over magnesium sulfate and filtered. The filtrate is concentrated to about two thirds volume. Petroleum ether is added and the purified product, (tert-butoxy)-N-(2-methoxyethyl)-N-[2- (4- { [2-(4-methoxyphenyl)-2-oxoethyl]amino}phenoxy)ethyl]carboxamide, 3f crystallizes and is collected by filtration.
E. N-(2-{4-[2-Amino-3-cyano-4-(4-methoxyphenyl)pyrrolyl]phenoxy}- ethyl)(tert-butoxy)-N-(2-methoxyethyl)carboxamide, 3g. Sodium (600 mg, 26 mmol) is dissolved in ethanol (60 mL). Malononitrile (1.5 g, 23 mmol) is added dropwise and the resulting mixture is stirred at ambient temperature for fifteen minutes. Intermediate 3f (20 mmol) is added dropwise and the resulting mixture is heated to reflux for one hour. When the reaction is complete, water is added to the mixture while still at reflux until the clear mixture becomes cloudy. The mixture is then allowed to slowly cool to ambient temperature. The product N-(2-{4-[2-amino-3-cyano-4-(4- methoxyphenyl)pyrrolyl]phenoxy}-ethyl)(tert-butoxy)-N-(2-methoxyethyl)- carboxamide, 3g crystallizes out and is collected by filtration.
F. (2-{4-[4-Amino-5-(4-methoxyphenyl)pyrrolo[2,3-d]pyrimidin-7-yl]- phenoxy}ethyl)(2-methoxyethyl)amine; Compound of Example 3. Intermediate 3g (7.6 g, 15 mmol) is heated to reflux for 2.5 hours in triethyl orthoformate (140 mL) with acetic anhydride (0.5 mL). When the reaction is complete the reaction mixture is concentrated under vacuum, and the residue is crystallized from diethyl ether/petroleum ether. This intermediate is redissolved in ethanol (50 mL) and treated with ammonia in ethanol (8%, 100 mL). The reaction mixture is stirred overnight. A precipitate forms and is collected by filtration. The collected material is redissolved in 8% ammonia in ethanol (150 mL). The reaction mixture is heated in a pressure vessel to 130° C for four hours. After cooling to ambient temperature, the precipitated product is collected by filtration. This BOC-protected intermediate is stirred overnight at ambient temperature in DCM (100 mL) with trifluoroacetic acid (3 mL). The volatiles are removed under vacuum and the residue is purified by preparative HPLC to yield the compound of example 3, (2-{4-[4-amino-5-(4- methoxyphenyl)-pyrrolo[2,3-d]pyrimidin-7-yl]-phenoxy}etiιyl)(2-methoxy- ethyl)amine.
Example 4. Synthesis of 4-{4-[(2-oxo-lH-benzo[d]azolidin-3- ylidene)methyl]phenyl}piperazinecarbaldehyde. The title compound of Example 4, an indoline-2-one compound of formula III is synthesized according to Scheme 15.
Figure imgf000077_0001
A. 4-(l-Formylpiperazin-4-vPbenzaldehyde.4b. POCl3 (30 mL, 0.3 mol) is added dropwise to a solution of DMF (30 mL, 0.3 mol) in anhydrous 1,2-dichloroathane at 0° C in an ice- water bath. The reaction mixture is allowed to come to ambient temperature over 30 minutes. The reaction mixture is then cooled to 0° C in an ice- water bath and 1 -phenyl piperazine 4a [92-54-6] (16.0 g, 0.1 mol) is added portionwise over 15 minutes. The resulting mixture is warmed to 50° C for one hour. The reaction mixture is then poured into ice-cold sodium hydroxide (IN) and stirred at ambient temperature for one hour. The organic layer is separated and the aqueous layer is extracted with ethyl acetate. The combined organic fraction is washed with brine, dried (sodium sulfate), and concentrated under vacuum. The residue is purified by column chromatography to yield 4-(l-formylpiperazin-4- yPbenzaldehyde, 4b.
B . 4- (4- [(2-Oxo- 1 H-benzo [di azolidin-3 -ylidene')methyllphenyl> piperazine- carbaldehvde. 4d. Oxindole 4c [59-48-3] (Aldrich, O-980-8) (135 mg, 1 mmol), 4-(l- formylpiperazin-4-yl)benzaldehyde, 4b (1 mmol) and piperidine (3 drops) are warmed to 90° C in ethanol (2 mL) for five hours. The mixture is then cooled to ambient temperature and the resulting precipitate collected by filtration. The collected material is triturated with ether hexane and dried to yield 4d, 4-{4-[(2- oxo-lH-benzo[d]azolidin-3-ylidene)methyl]phenyl}piperazinecarbaldehyde.
Example 5: Inhibition of ABL Tyrosine Kinase by Compounds of Formula I: Method A. The inhibition of ABL activity by compounds of formula I is demonstrated as follows. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged, Panvera, CA) is incubated with various concentrations of a compound of formula I for 30 minutes in kinase buffer (50 mM HEPES (N-2- hydroxyethylpiperazine-N-2-ethanesulfonic acid), 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl2, pH 7.5) at room temperature in a total volume of 15 μl. Following the incubation period, kinase reactions are performed by adding cold ATP and γ-32P-ATP in the presence of 6 μg of ABL kinase substrate (recombinant murine Crk) for 20 minutes at 30°C. The reactions are stopped by the addition of 20 μl of 2x SDS sample buffer. The samples are boiled and resolved on a 10 % SDS-polyacrylamide gel. The gel is fixed, and exposed to x-ray film. Quantitation of Crk phosphorylation is determined using a phosphoimager system (Fuji).
Example 6: Inhibition of ABL Tyrosine Kinase by Compounds of Formula I: Method B - Quantitation using filter assay. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged, Panvera, CA) is incubated with various concentrations of compounds of formula I for 30 minutes in kinase buffer (50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl2, pH 7.5) at room temperature in a total volume of 15 μl. Following the incubation period, kinase reactions are performed by adding cold ATP and γ-32P-ATP in the presence of Crk (6 μg) for 20 minutes at 30° C. After incubation, 10 μl aliquots are spotted onto 2 cm x 2 cm P81 phosphocellulose paper. The paper is air dried and then washed 3x with 0.75% phosphoric acid, and fixed with acetone for 5 minutes. The wet filters are then placed into scintillation vials containing scintillation fluid (Ecolume) and counted for 32P using a scintillation counter. The counts per minute (CPM) of each treated sample are compared to the amount of radioactivity resulting from control reactions in the presence of DMSO. The reactions are performed in triplicates, and the average CPM +/- SD for each reaction is plotted as percent of solvent treated control.
Example 7: Inhibition of ABL Tyrosine Kinase by a Composition Comprising at Least One Compound of Formula I and at Least One Compound of Formula V: Abi-1 protein (Panvera) is incubated with different concentrations of a mixture of a compound of formula I and a compound of formula V such as, for example, 4-((lE)-2-{[(4-chlorophenyl)methyl]suhOnyl}vinyl)benzoic acid (Compound 23 in Table 4) in a 15 μl reaction mixture (50 mM HEPES, 10 mM MgC12, 1 M EDTA, 2 mM DTT and 0.01% NP-40, pH 7.5) for 30 min at room temperature. Kinase reactions are initiated by the addition of 2 μl of 1 mM ATP (lOOμM concentration), 2 μl of γ32pATP (40 μci final concentration) and 6 μg of recombinant GST-Crk (Upstate). Reactions are performed for 20 min at 30°C and are stopped by the addition of 20 μl of 2XSDS-PAGE buffer, boiled and subjected to SDS-PAGE using a 12% polyacrylamide gel. Following electrophoresis, the gel is dried and exposed to X-ray film for 3-10 min.
Example 8: Inhibition of ABL Tyrosine Kinase by Compounds according to Formula V: Method B - Quantitation using filter assay. Purified (5-10 ng) recombinant human c-abl (Abll, Histidine tagged,
Panvera, CA) was incubated with various concentrations of each compound of Formula V listed in Table 5 (Compounds 1-22) for 30 minutes in kinase buffer (50 mM HEPES, 0.1% NP-40, 300 mM, 2.0 mM DTT, 1.0 mM EDTA, 10 mM MgCl2, pH 7.5) at room temperature in a total volume of 15 μl. Following the incubation period, kinase reactions were performed by adding cold ATP and γ- 32P-ATP in the presence of Crk (6 μg) for 20 minutes at 30° C. After incubation, 10 μl aliquots were spotted onto 2 cm x 2 cm P81 phosphocellulose paper. The paper was air dried and then washed 3x with 0.75% phosphoric acid, and fixed with acetone for 5 minutes. The wet filters were then placed into scintillation vials containing scintillation fluid (Ecolume) and counted for 32P using a scintillation counter. The counts per minute (CPM) of each treated sample were compared to the amount of radioactivity resulting from control reactions in the presence of DMSO. The reactions were performed in triplicates, and the average CPM +/- SD for each were plotted as percent of solvent treated control. The ICso's of compounds 1-24 are listed in Table 4. The dose response curves for compounds 1-6, 7-12, 13-16, 17-19, and 20-22 are provided in Figures 1, 2, 3, 4 and 5, respectively. Table 5
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
* indicates IC50 > lOμM; ** indicates lμM> IC50> 10μM; *** indicates IC50 < lμM
Example 9: Radioprotective Effect of Compounds of Formula I, and Compositions Comprising Compounds of Formula I on Cultured Normal Cells: The radioprotective effect of: (a) compounds of formula I; (b) combinations of at least one compound of formula I and at least one compound of formula V; and (c) combinations of at least one compound of formula I and at least one antioxidant compound on cultured normal cells is evaluated as follows. HFL-1 cells, which are normal diploid lung fibroblasts, are plated into 24 well dishes at a cell density of 3000 cells per 10 mm2 in DMEM completed with 10% fetal bovine serum and antibiotics. A compound of formula I (or a composition comprising a compound of formula I) is added to the cells 24 hours later at concentrations in a range from about 0.25 to about 2.0 micromolar, using DMSO as a solvent. Control cells are treated with DMSO alone. The cells are exposed to the test compound (or composition) or DMSO for 24 hrs. The cells are then irradiated with either 10 Gy or 15 Gy of ionizing radiation (IR) using a J.L. Shepherd Mark I, Model 30-1 Irradiator equipped with 137cesium as a source. After irradiation, the medium on the test and control cells is removed and replaced with fresh growth medium without the test compounds or DMSO. The irradiated cells are incubated for 96 hours and duplicate wells are trypsinized and replated onto 100 mm2 tissue culture dishes. The replated cells are grown under normal conditions with one change of fresh medium for 3 weeks. The number of colonies from each 100 mm2 culture dish, which represents the number of surviving cells, is determined by staining the dishes as described below. To visualize and count the colonies derived from the clonal outgrowth of individual radioprotected cells, the medium is removed and the plates are washed one time with ambient temperature phosphate buffered saline. The cells are stained with a 1:10 diluted Modified Giemsa staining solution (Sigma) for 20 minutes. The stain is removed, and the plates are washed with tap water. The plates are air-dried, the number of colonies from each plate is counted and the average from duplicate plates is determined.
Example 10: Treatment of bcr-abl Transformed Leukemic Cells by a Composition Comprising at Least One Compound of Formula I and at Least One Compound of Formula V: K562 cells, a cell line isolated from a 35 year old patient with chronic myelogenous leukemia (CML), transformed due to the Philadelphia chromosome translocation resulting in the expression of bcr-abl kinase is used as the target cell line. K562 cells are plated at a cell density of 1.0 x 10s cells/mL in 12 well dishes and treated with a constant concentration (a concentration that reduces the growth of K562 cells by no more than 20% based upon dose response assays treating with the compound of formula I alone) of a compound of formula I, and a series of concentrations of a compound of formula V. Following an incubation period of 96 hours at 37°C under 5% C02, the number of viable cells remaining in each well is determined by counting using a hemacytometer and trypan blue staining. The total number of viable cells remaining is calculated and plotted as the percent of vehicle treated control cells.
Example 11: Effect of Exposure to Ionizing Radiation on Normal and Malignant Hematopoietic Progenitor Cell Growth After Pretreatment with Compounds of Formula I: The effect of ionizing radiation on normal and malignant hematopoietic progenitor cells which are pretreated with a compound of formula I is determined by assessing cloning efficiency and development of the pretreated cells after irradiation. To obtain hematopoietic progenitor cells, human bone marrow cells (BMC) or peripheral blood cells (PB) are obtained from normal healthy, or acute or chronic myelogenous leukemia (AML, CML), volunteers by Ficoll- Hypaque density gradient centrifugation, and are partially enriched for hematopoietic progenitor cells by positively selecting CD34+ cells with immunomagnetic beads (Dynal A.S., Oslo, Norway). The CD34+ cells are suspended in supplemented alpha medium and incubated with mouse anti- HPCA-I antibody in 1:20 dilution, 45 minutes, at 4° C with gentle inverting of tubes. Cells are washed x 3 in supplemented alpha medium, and then incubated with beads coated with the Fc fragment of goat anti-mouse IgGi (75 μl of immunobeads/107 CD34+ cells). After 45 minutes of incubation (4° C), cells adherent to the beads are positively selected using a magnetic particle concentrator as directed by the manufacturer. 2 x 10 CD34+ cells are incubated in 5 mL polypropylene tubes (Fisher Scientific, Pittsburgh, PA) in a total volume of 0.4 mL of Iscove's modified Dulbecco's medium (IMDM) containing 2% human AB serum and 10 mM Hepes buffer. A compound of formula I is added to the cells; in four different concentrations (0.25 μM, 0.5 μM, 1.0 μM and 2.0 μM) is added separately to the cells. Control cells receive DMSO alone. The cells are incubated for 20-24 hours and irradiated with 5 Gy or 10 Gy of ionizing radiation. Immediately after irradiation, the medium is removed and replaced with fresh medium without the test compound or DMSO. Twenty-four hours after irradiation, the treatment and control cells are prepared for plating in plasma clot or methylcellulose cultures. Cells (1 x 104 CD34+ cells per dish) are not washed before plating. Assessment of the cloning efficiency and development of the treated hematopoietic progenitor cells are carried out essentially as reported in Gewirtz et al, Science 242, 1303-1306 (1988), the disclosure of which is incorporated herein by reference.
Example 12: Bone Marrow Purging with Ionizing Radiation After Pretreatment with Compounds of Formula I. Bone marrow is harvested from the iliac bones of an individual under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the individual will be able to receive about 4 x 108 to about 8 x 108 processed marrow cells per kg of body weight. Thus, about 750 to 1000 mL of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 mL of medium. The aspirated marrow is filtered through three progressively finer meshes to obtain a cell suspension devoid of cellular aggregates, debris and bone particles. The filtered marrow is then processed further into an automated cell separator (e.g., Cobe 2991 Cell Processor) which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets). The buffy coat preparation is then placed in a transfer pack for further processing and storage. It may be stored until purging in liquid nitrogen using standard procedures. Alternatively, purging can be carried out immediately, then the purged marrow may be stored frozen in liquid nitrogen until it is ready for transplantation. The purging procedure is carried out as follows. Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 107/mL in TC-199 containing about 20% autologous plasma. A compound of formula I, for example, at a concentration of from 0.25 μM to 2.0 μM is added to the transfer packs containing the cell suspension and incubated in a 37° C waterbath for 20- 24 hours with gentle shaking. The transfer packs are then exposed to 5-10 Gy ionizing radiation. Recombinant human hematopoietic growth factors, e.g., rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of hematopoietic neoplasms and thereby increase their sensitivity to ionizing radiation. The cells may then either be frozen in liquid nitrogen or washed once at
4° C in TC-199 containing about 20% autologous plasma. Washed cells are then infused into the individual. Care must be taken to work under sterile conditions wherever possible and to maintain scrupulous aseptic techniques at all times. All references discussed herein are incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.

Claims

CLAIMS What is claimed is:
1. A method for protecting an individual from cytotoxic side effects of ionizing radiation, comprising administering to said individual an effective amount of at least one compound of formula I:
Figure imgf000091_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000091_0002
: ' indicates that the designated bond is either a single bond or a double bond; "■^indicates that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Cι-C6)alkyl, substituted and unsubstituted heteroaryl(Cι-C6)alkyl, and -(CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and CCd-Cβalkyl); p is 1, 2 or 3; R2 is substituted aryl or substituted or unsubstituted heterocyclyl; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(C C6)alkyl; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(d-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(Cι-C6)alkyl, -N(Cι-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(C1-C6)alkyl, -SO(C1-C6)alkyl, -S(Cι-C6)alkyl, -0(d-C6)alkyl, -C(=0)NH2, -C(=0)NH(d-C6)alkyl, -C(=0)N(d-C6alkyl)2, -C02H, -C02(Cι- C6)alkyl, -S02NH2, -S02NH(d-C6)alkyl, -S02N(Cι-C6alkyl)2, -S02-
(non-aromatic heterocycle), -(Cι-C6)alkylene-NH2, -(Cι-C6)alkylene- OH, -(Cι-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(Cι- C6)alkyl, -C(=NH)NH2, -Z(Cι-C6)alkylene-NH2, -Z(d-C6)alkylene- NH(Cι-C6)alkyl, -Z(Cι-C6)alkylene-N(Cι-C6alkyl)2, -Z(C,-C6)alkylene- C02(Ci-C6)alkyl, -Z(d-C6)alkylene-C02H, -Z(Cι-C6)alkylene-
C(=00)NH2, -Z(C1-C6)alkylene-C(=0)NH(C1-C6)alkyl, -Z(d- C6)alkylene-C(=0)N(C]-C6alkyl)2, and substituted heterocyclyl; X is selected from the group consisting of C and N; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-Cέ)alkylene- heterocyclyl, and -Z-(CH(Rb))„C(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; -CN; substituted and unsubstituted aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-C,5)alkylene- heterocyclyl, and -Z-(CH(Rb))„C(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Cι-C6)alkyl, -NH2, -NH(d-C6)alkyl, -N((Cι-C6)alkyl)2, and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (C,-C6)alkyl; provided: (1) when the designated bond in :T ' i«s a double bond, then: (a) the designated bond in •" is a single bond, ( b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000093_0001
(c) T is selected from the group consisting of N, C(H) and C(C)-C6alkyl); and (2) when the designated bond in ' is a single bond, then: (a) the designated bond in ^ is a double bond; (b) R is a radical of formula (ii):
Figure imgf000093_0002
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound.
2. The method of claim 1 wherein the designated bond in ' is a double bond, the designated bond in •"< is a single bond, R is hydrogen, R2, or a radical of formula (i):
Figure imgf000093_0003
T is selected from the group consisting of N, C(H) and C(d- Cβalkyl).
3. The method of claim 2 wherein the administered compound is a compound of formula II:
Figure imgf000094_0001
wherein: T is selected from the group consisting of N, C(H) and C(Cι- dalkyl); R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Cι-C6)alkyl, and substituted and unsubstituted heteroaryl(Cι-C6)alkyl; and R2 is substituted aryl, or substituted or unsubstituted heterocyclyl; or a pharmaceutically acceptable salt of such a compound.
4. The method of claim 3 wherein the administered compound is a compound of formula Ila:
Figure imgf000094_0002
wherein: R1 is selected from the group consisting of -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Cι-C6)alkyl, and substituted and unsubstituted heteroaryl(Cι-C6)alkyl; or a pharmaceutically acceptable salt of such a compound.
5. The method of claim 4 wherein the administered compound is selected from the group consisting of: (2-{4-[4-amino-5-(4-methoxyphenyl)pyrrolo[2,3- d]pyrimidin-7-yl]-phenoxy} -ethyl)(2-methoxy-ethyl)amine; 1 -(2- {4-[4-amino- 5-(3-methoxyphenyl)pyrrolo[2,3-d]pyrimdin-7-yl]-ρhenyl}ethyl)piperidine-4-ol; and pharmaceutically acceptable salts thereof.
6. The method of claim 3 wherein the administered compound is a compound of formula lib:
Figure imgf000095_0001
wherein: R1 is selected from the group consisting of -(Cι-Cfi)alkyl, and substituted and unsubstituted aryl; or a pharmaceutically acceptable salt of such a compound.
7. The method of claim 6 wherein the administered compound is l-(tert- butyl)-3-(4-methylphenyl)pyrazolo[5,4-d]pyrimidine-4-ylamine, or a pharmaceutically acceptable salt thereof.
8. The method of claim 1 wherein T the designated bond in ■ is a single bond;, and X is C.
9. The method of claim 8 wherein the administered compound is a compound of formula III:
Figure imgf000096_0001
wherein: R1 is -H or (CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; ' ww indicates a single bond in either a Z- or E- conformation; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(Cι-C6)alkyl, -N Ci-dalky j, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(Ci-C6)alkyl, -SO(C1-C6)alkyl, -S(d-C6)alkyl, -0(d-C6)alkyl, -C(=0)NH2, -C(=0)NH(d-C6)alkyl, -C(=0)N(d-C6alkyl)2, -C02H, -C02(Cι- C6)alkyl, -S02NH2, -S02NH(C1-C6)alkyl, -S02N(Ci-C6alkyl)2, -S02- (non-aromatic heterocycle), -(Ci-C6)alkylene-NH2, -(Cι-C6)alkylene- OH, -(Ci-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(C C6)alkyl, -C(=NH)NH2, -Z(d-C6)alkylene-NH2, -Z(Cι-C6)alkylene- NH(Cι-C6)alkyl, -Z(d-C6)alkylene-N(Cι-C6alkyl)2, -Z(Cι-C6)alkylene- C02(Ci-C6)alkyl, -Z(Ci-C6)alkylene-C02H, -Z(d-C6)alkylene- C(=00)NH2, -Z(Ci-C6)alkylene-C(=0)NH(Ci-C6)alkyl, -Z(Cr C6)alkylene-C(=0)N(Cι-C6alkyl)2, and substituted heterocyclyl; p is 1, 2 or 3; R5 and R6 are independently selected from the group consisting of -H, aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-C6)alkylene-heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Ci-C6)alkyl, -NH2, -NH(d-C6)alkyl, -N((Cι-C6)alkyl)2, and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group may be present as a carboxyl group, an amide or a carboxylic ester; and each Rb is -H or (CrC6)alkyl; or a pharmaceutically acceptable salt of such a compound.
10. The method of claim 9 wherein the administered compound is selected from the group consisting of: ethyl-2-{5-[(2-oxo-lH-benzo[d]azolidin-3- ylidene)methyl]-8-quinolyloxy}-propanoate; 3-{2,4-dimethyl-5-[(2-oxo(lH- benzo[d]azolidin-3-ylidene))-methyl]pyrrol-3-yl}propanoic acid; 3-{4-methyl- 2-[(2-oxo( 1 H-benzo [d]azolidin-3 -ylidene))methy l]-pyrrol-3 -yl} propanoic acid; 3 - [(3 , 5 -dimethylpyrrol-2-yl)methylene]- 1 H-benzo [d]azolidin-2-one; 3 - [(2- chloro-4-methoxyphenyl)methylene]-lH-benzo[d]azolidin-2-one; 3-(indol-3- ylmethylene)-5-(2-piperidylacetyl)-lH-benzo[d]-azolidin-2-one; N-{3-[(5- methoxyindol-3-yl)methylene]-2-oxo(lH-benzo[3,4-d]azolidin-5-yl)}-3- piperidylpropanamide; 3-{4-methyl-5-[(2-oxo-6-phenyl(lH-benzo[d]azolidin-3- ylidene))methyl]pyrrol-3-yl}propanoic acid; 3-(5-{[6-(3-methoxyphenyl)-2- oxo( 1 H-benzo [d] azolidin-3 -ylidene)]methyl} -4-methylpyrrol-3 -y l)ρropanoic acid; 2-oxo-3-(pyrrolo[2,3-b]pyridm-3-ylmethylene)-lH-benzo[d]-azolidine-5- carbonitrile; 3-{[4-(4-carbonylpiperazinyl)phenyl]methylene}-lH-benzo[d]- azolidin-2-one; and pharmaceutically acceptable salts thereof.
11. The method of claim 1 wherein the designated bond in ' is a double bond and R is a radical of formula (i):
Figure imgf000097_0001
wherein: R3 is substituted heteroaryl, -C(=0)ΝH2 or -C(=0)NH(d- C6)alkyl.
12. The method of claim 11 wherein the administered compound is a compound of formula IV:
Figure imgf000098_0001
wherein: T is C(H) orN; and R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι C6)alkyl; or a pharmaceutical salt of such a compound.
13. The method of claim 12 wherein the administered compound is 5-((lZ)- l-cyano-2-indol-3-ylvinyl)-3-aminopyrazole-4-carbonitrile, or a pharmaceutically acceptable salt thereof.
14. The method of claim 1, wherein the radioprotective compound is administered before exposure to the ionizing radiation.
15. The method of claim 1, wherein the radioprotective compound is administered after exposure to ionizing radiation.
16. The method of claim 1, further comprising administering to said individual an effective amount of at least one compound of formula V:
Q1 — X— CH=CH— Q2 V wherein, Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000099_0001
(iϋ) (iv) wherein, n is one or zero; and Rx is -H, -(Cι-C8)hydrocarbyl or -C(=0)(d-C8)hydrocarbyl; or a pharmaceutically acceptable salt thereof.
17. The method of claim 16, wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000099_0002
18. The method of claim 17, wherein Q1 is phenyl or substituted phenyl.
19. The method of claim 17, wherein Q2 is phenyl or substituted phenyl.
20. The method of claim 19, wherein the compound of formula V is selected from the group consisting of: 4-((l-5)-2-{[(4-fluorophenyl)methyl]- sulfonyl}vinyl)benzoic acid; 4-((1.5)-2-{[(4-iodophenyl)methyl]sulfbnyl}- viny benzoic acid; 4-(( 1 E)-2- { [(4-chlorophenyl)-methyl] sulfonyl } viny l)benzoic acid; l-[5-((l£)-2-{[(4-chlorophenyl)methyl]-sulfonyl}vinyl)-2-fluorophenyl]- 2-(dimethylamino)ethan- 1 -one; ( l-5T)-2-(2,4-difluorophenyl)- 1 - { [(4-bromo- phenyl)methyl] sulfonyl}ethene; ( E)-2-(3 -amino-4-fluorophenyl)- 1 - { [(4- chlorophenyl)methyl]sulfonyl}ethene; 2-(5-(((-5)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxyphenylamino)-acetic acid; (JR)-2-(5-(((£)_2 >4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)propanoic acid; (S)- 2-(5-(((^)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)- propanoic acid; racemic-2-(5-(((£)-2,4,6-trimethoxy-styrylsulfonyl)methyl)-2- methoxyphenyl-amino)propanoic acid; (R)-2-(5-(((-B)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxyphenylamino)-2-phenylacetic acid; (S)-2-(5-(((-5)- 2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenyl-amino)-2- phenylacetic acid; racemic-2-(5-(((JE)-2,4,6-trimethoxystyryl-sulfonyl)methyl)- 2-methoxyphenyl-amino)-2-phenylacetic acid; N-(3-(((.5)-2,4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxyphenyl)-4-(4-methyl-piperazin-l- yl)benzamide; 2-(( )-2-(4-methoxybenzylsulfonyl)vinyl)-l,3,5-trimethoxy- benzene; 1 -(((£)-4-chlorostyrylsulfonyl)methy l)-2-chloro-4-fluorobenzene; 4- ((-5)-2-(4-chlorobenzylsulfonyl)vinyl)-l-fluoro-2-nitrobenzene; 4-((E)-2-(3- amino-4-methoxybenzylsulfonyl)vinyl)benzoic acid; 5-(((£)-2,4-difluorostyryl- sulfonyl)methyl)-2-methoxybenzenamine; l-bromo-4-(((£)-perfluorostyryl- sulfonyl)methyl)benzene; l-(((Jδ)-2,3,5,6-tetrafluorostyryl-sulfonyl)methyl)-4- bromobenzene; l-(((.5)-2,4,5-trifluorostyrylsulfonyl)-methyl)-4-bromobenzene; 1 -(((-5)-2,4,6-trifluorostyrylsulfonyl)methyl)-4-bromobenzene; 1 -(((E)-2,3,6- trifluorostyrylsulfonyl)methyl)-4-bromobenzene; 5-(((-5)-2,4-difluorostyryl- sulfonyl)methyl)-2-bromobenzenamine; 4-(((-^-2,4-difluorostyrylsulfonyl)- methyl)benzoic acid; 5-(((£T)-2,4,6-trimethoxystyryl-sulfonyl)methyl)-2-bromo- benzenamine; 4-((£)-2-(4-bromobenzylsulfonyl)-vinyl)- 1 -fluoro-2- nitrobenzene; 4-((£T)-2-(4-chloro-2-nitrobenzylsulfonyl)vinyl)- 1 -fluoro-2- nitrobenzene; 5-((^)-2-(4-bromobenzylsulfonyl)vinyl)-2-fluoro-benzenamine; 5- ((-5)-2-(4-iodobenzylsulfonyl)vinyl)-2-fluorobenzenamine; 5-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxybenzonitrile; 4-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)benzoic acid; 3 -((£)-2-(4-bromobenzyl- sulfonyl)vinyl)-2,6-difluorophenol; 5-((2,4,6-trimethoxystyrylsulfonyl)methyl)- 2-methoxy-N-methylbenzenamine and pharmaceutically acceptable salts thereof.
21. The method of claim 1, further comprising administering to said individual an effective amount of at least one antioxidant compound.
22. The method of claim 21 wherein the antioxidant compound is selected from the group consisting of carotenoids, catechins, isoflavones, flavanones, flavanols, flavanoid chalcones, vitamin E compounds, (3-aminopropyl)[2- (phosphonothio)ethyl]amine, ascorbic acid, cysteine, glutathione, probucol, β- mercaptoethanol dithiothreitol, pyrrolidine dithiocarbamate, N-acetyl-L- cysteine, ubiquinone, meso-tetrakis-(N-alkylpyridinium-2-yl)porphyrins, meso- tetrakis-(N-alkylpyridinium-3-yl)po hyrins, and salts thereof.
23. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, (A) at least one compound according to formula I:
Figure imgf000101_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000101_0002
■ ■ indicates that the designated bond is either a single bond or a double bond; - ■R ^ indicates that the designated bond is either a single bond or a double bond RR11 iiss sseelleecctteedd from the group consisting of -H, -(Ci-Cejalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(d-C6)alkyl, substituted and unsubstituted heteroaryl(Cι-C6)alkyl, and -(CH2)m-C(=0)R ; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(Cι-C6alkyl); p is 1, 2 or 3; R2 is substituted aryl or substituted or unsubstituted heterocyclyl; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι- C6)alkyl; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(d-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(Cι-C6)alkyl, -N d-dalkyPz, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(d-C6)alkyl, -SO(Cι-C6)alkyl, -S(Cι-C6)alkyl, -0(Cι-C6)alkyl, -C(=0)NH2,
Figure imgf000102_0001
C6)alkyl, -S02NH2, -S02NH(d-C6)alkyl, -S02N(C C6alkyl)2, -S02- (non-aromatic heterocycle), -(Cι-C6)alkylene-NH2, -(Ci-Ce)alkylene- OH, -(Ci-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(C C6)alkyl, -C(=NH)NH2, -Z(C,-C6)alkylene-NH2, -Z(d-C6)alkylene- NH(d-C6)alkyl, -Z(Cι-C6)alkylene-N(Cι-C6alkyl)2, -Z(d-C6)alkylene-
C02(Ci-C6)alkyl, -Z(CrC6)alkylene-C02H, ' -Z(d-C6)alkylene- C(=00)NH2, -Z(C,-C6)alkylene-C(=0)NH(d-C6)alkyl, -Z(C,- C6)alkylene-C(=0)N(Cι-C6alkyl)2, and substituted heterocyclyl; X is selected from the group consisting of C and N; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(C]-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; substituted and unsubstituted aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(C]-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Ci-C6)alkyl, -NH2, -NH(C,-C6)alkyl, -N((d-C6)alkyl)2, and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (d-C6)alkyl; provided: T (1) when the designated bond in ' is a double bond, then: (a) the designated bond in r\ js a singje bond, (b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000103_0001
(c) T is selected from the group consisting of N, C(H) and C(d-C6alkyl); and (2) when the designated bond in ' is a single bond, then: (a) the designated bond in "^ is a double bond; (b) R is a radical of formula (ii):
Figure imgf000103_0002
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound; and (B) either (1) , at least one compound according to formula V: Q1 χ__CH= CH_Q2 V wherein, Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000104_0001
(D (ϋ)
Figure imgf000104_0002
(iii) (iv) wherein, q is one or zero; and Rx is-H, -(Cι-C8)hydrocarbyl or
Figure imgf000104_0003
or a pharmaceutically acceptable salt of such a compound; or (2) at least one antioxidant compound.
24. The pharmaceutical composition of claim 21, wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000104_0004
25. The composition of claim 24, wherein part B comprises a compound of formula V.
26. The composition of claim 25, wherein the compound of formula II is selected from the group consisting of: 4-((l£)-2-{[(4-fluorophenyl)- methyl] sulfonyl} vinypbenzoic acid; 4-((lE)-2- { [(4-iodophenyl)methyl]- sulfonyl}vinyl)benzoic acid; 4-((l£ -2-{[(4-chlorophenyl)methyl]sulfonyl}- vinyl)benzoic acid; l-[5-((1.5)-2-{[(4-chlorophenyl)methyl]-sulfonyl}vinyl)-2- fluorophenyl]-2-(dimethylamino)ethan- 1 -one; ( lii)-2-(2,4-difluorophenyl)- 1 - { [(4-bromophenyl)methyl] sulfonyl} ethene; (l£)-2-(3-amino-4-fluorophenyl)- 1 - { [(4-chlorophenyl)methyl]sulfonyl} ethene; 2-(5-(((-5)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxyphenylamino)acetic acid; (R)-2-(5-(((E)-2,4,6- trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)propanoic acid; (S)- 2-(5-(((-^)-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)- propanoic acid; racemic-2-(5-(((-5)-2,4,6-trimethoxy-styrylsulfonyl)methyl)-2- methoxyphenyl-amino)propanoic acid; (R)-2-(5-(((-5)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxyphenylamino)-2-phenylacetic acid; (S)-2-(5-(((E)- 2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxyphenylamino)-2-phenylacetic acid; racemic-2-(5-(((£)-2,4,6-trimethoxystyryl-sulfonyl)methyl)-2-methoxy- phenylamino)-2-phenylacetic acid; N-(3-(((-5)-2,4,6-trimethoxystyryl- sulfonyl)methyl)-2-methoxyphenyl)-4-(4-methyl-piperazin-l-yl)benzamide; 2- ((.5)-2-(4-methoxybenzylsulfonyl)vinyl)-l,3,5-trimethoxybenzene; l-(((E)-4- chlorostyrylsulfonyl)methyl)-2-chloro-4-fluorobenzene; 4-((iT)-2-(4-chloro- benzylsulfonyl)vinyl)-l-fluoro-2-nitrobenzene; 4-((-5)-2-(3-amino-4-methoxy- benzylsulfonyl)vinyl)benzoic acid; 5-(((^)-2,4-difluorostyrylsulfonyl)methyl)-2- methoxybenzenamine; l-bromo-4-(((-5)-perfluorostyrylsulfonyl)methyl)- benzene; l-(((£)-2,3,5,6-tetrafluorostyryl-sulfonyl)methyl)-4-bromobenzene; 1- (((JB)-2,4,5-trifluorostyrylsulfonyl)-methyl)-4-bromobenzene; l-(((E)-2,4,6- trifluorostyrylsulfonyl)methyl)-4-bromobenzene; 1 -(((E)-2, ,6-trifluorostyryl- sulfonyl)methyl)-4-bromobenzene; 5-(((J5)-2,4-difluorostyrylsulfonyl)methyl)- 2-bromobenzenamine; 4-(((£)-2,4-difluorostyrylsulfonyl)methyl)benzoic acid; 5-(((-5)-2,4,6-trimethoxystyryl-sulfonyl)methyl)-2-bromobenzenamine; 4-((E)- 2-(4-bromobenzylsulfonyl)-vinyl)- 1 -fluoro-2-nitrobenzene; 4-((£)-2-(4-chloro- 2-nitrobenzylsulfonyl)vinyl)- 1 -fluoro-2-nitrobenzene; 5-((£)-2-(4-bromobenzyl- sulfonyl)vinyl)-2-fluorobenzenamine; 5-((JB)-2-(4-iodobenzylsulfonyl)vinyl)-2- fluorobenzenamine"; 5-((( )-2,4,6-trimethoxystyrylsulfonyl)methyl)-2-methoxy- benzonitrile; 4-(((-5)-2,4,6-trimetlioxystyrylsulfonyl)methyl)benzoic acid; 3- ((-5)-2-(4-bromobenzylsulfonyl)vinyl)-2,6-difluorophenol; 5-((2,4,6-trimethoxy- styrylsulfonyl)methyl)-2-methoxy-N-methylbenzenamine and pharmaceutically acceptable salts thereof.
27. The composition of claim 20, wherein part B comprises an antioxidant compound.
28. The composition of claim 27 wherein the antioxidant compound is selected from the group consisting of carotenoids, catechins, isoflavones, flavanones, flavanols, flavanoid chalcones, vitamin E compounds, (3- aminopropyl)[2-(phosphonothio)ethyl]amine, ascorbic acid, cysteine, glutathione, probucol, β-mercaptoethanol dithiothreitol, pyrrolidine dithiocarbamate, N-acetyl-L-cysteine, ubiquinone, meso-tetrakis-(N- alkylpyridinium-2-yl)porphyrins, meso-tetrakis-(N-alkylpyridinium-3- yl)porphyrins, and salts thereof.
29. A method of treating an individual fot a proliferative disorder, comprising: (a) administering to the individual an effective amount of at least one radioprotective compound of formula I:
Figure imgf000106_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000107_0001
• ' indicates that the designated bond is either a single bond or a double bond; K indicates that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Cι-C6)alkyl, substituted and unsubstituted heteroaryl(Cι -C6)alkyl, and -(CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(Cι-C6alkyl); p is 1, 2 or 3; R2 is substituted aryl or substituted or unsubstituted heterocyclyl; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(C C6)alkyl; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(Cι-C6)alkyl,
-N(Cι-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(d-C6)alkyl, -SO(C,-C6)alkyl, -S(d-C6)alkyl, -0(d-C6)alkyl, -C(=0)NH2, -C(=0)NH(d-C6)alkyl, -C(=0)N(C,-C6alkyl)2, -C02H, -C02(d- C6)alkyl, -S02NH2, -S02NH(C1-C6)alkyl, -S02N(d-C6alkyl)2, -S02- (non-aromatic heterocycle), -(Cι-Cβ)alkylene-NH2, -(Cι-Cδ)alkylene-
OH, -(C,-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(d- C6)alkyl, -C(=NH)NH2, -Z(C,-C6)alkylene-NH2, -Z(Cι-C6)alkylene- NH(d-C6)alkyl, -Z(C1-C6)alkylene-N(Cι-C6alkyl)2, -Z(d-C6)alkylene- C02(Ci-C6)alkyl, -Z(Ci-C6)alkylene-C02H, -Z(d-C6)alkylene- C(=00)NH2, -Z(d-C6)alkylene-C(=0)NH(Ci-C6)alkyl, -Z(C,- C6)alkylene-C(=0)N(Cι-C6alkyl)2, and substituted heterocyclyl; X is selected from the group consisting of C and N; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(d-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; substituted and unsubstituted aryl, -Z-(C1-C7)hydrocarbyl, -Z-(d-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0 , -N(Rb)C(=0 and -N(Rb)-; each Ra is independently selected from the group consisting of
-OH, -0(Cι-C6)alkyl, -NH2, -NH(Cι-C6)alkyl, -N((Cι-C6)alkyl)2, and an N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (Cι-C6)alkyl; provided: (1) when the designated bond in ' is a double bond, then: (a) the designated bond in ^ is a single bond, ( b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000108_0001
(c) T is selected from the group consisting of N, C(H) and C(d-C6alkyl); and (2) when the designated bond in ' is a single bond, then: p (a) the designated bond in ^ is a double bond; (b) R is a radical of formula (ii):
Figure imgf000109_0001
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound; and (b) administering an effective amount of therapeutic ionizing radiation.
30. The method of claim 29 wherein the proliferative disorder is cancer.
31. The method of claim 29, further comprising administering an effective amount of either: (A) at least one compound of formula V: q1 X— CH=CH— Q2 V wherein: Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000110_0001
(iii) (iv) wherein: q is one or zero; and Rx is -H, -(Ci-C8)hydrocarbyl or -C(=0)(d-C8)hydrocarbyl; or a pharmaceutically acceptable salt of such a compound; or (B) at least one antioxidant compound, prior to administering an effective amount of therapeutic ionizing radiation.
32. The method of claim 31 , wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000110_0002
33. A method of safely increasing the dosage of therapeutic ionizing radiation used in the treatment of cancer or other proliferative disorders, comprising administering to an individual an effective amount of at least one radioprotective compound according to formula I:
Figure imgf000111_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000111_0002
' indicates that the designated bond is either a single bond or a double bond; K in(}icates that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted aryl(Ci-C6)alkyl, substituted and unsubstituted heteroaryl(C1-C6)alkyl, and -(CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(C,-C6alkyl); p is 1, 2 or 3; R2 is substituted aryl or substituted or unsubstituted heterocyclyl; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(C C6)alkyl; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(d-C6)alkyl, -N(d-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(Ci-C6)alkyl, -SO(d-C6)alkyl, -S(d-C6)alkyl, -0(Ci-C6)alkyl, -C(=0)NH2, -C(=0)NH(Cι-C6)alkyl, -C(=0)N(C1-C6alkyl)2, -C02H, -C02(d- C6)alkyl, -S02NH2, -S02NH(d-C6)alkyl, -S02N(d-C6alkyl)2, -S02- (non-aromatic heterocycle), -(Cι-C6)alkylene-NH2, -(Cι-C6)alkylene- OH, -(Cι-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(d- C6)alkyl, -C(=NH)NH2, -Z(d-C6)alkylene-NH2, -Z(d-C6)alkylene- NH(d-C6)alkyl, -Z(Cι-C6)alkylene-N(d-C6alkyl)2, -Z(d-C6)alkylene-
C02(Ci-C6)alkyl, -Z(Ci-C6)alkylene-C02H, -Z(d-C6)alkylene- C(=00)NH2, -Z(Ci-C6)alkylene-C(=0)NH(Ci-C6)alkyl, -Z(Cι- C6)alkylene-C(=0)N(Cι-C6alkyl)2, and substituted heterocyclyl; X is selected from the group consisting of C andN; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; substituted and unsubstituted aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; each Z is independently selected from the group consisting of -O-, -S-, -C(=0>, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(Ci-C6)alkyl, -NH2, -NH(d-C6)alkyl, -N((d-C6)alkyl)2, and an
N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (Cι-C6)alkyl; provided: (1) when the designated bond in ;T ■ ; isC a double bond, then: p (a) the designated bond in ^ is a single bond, (b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000113_0001
(c) T is selected from the group consisting of N, C(H) and C(d-C6alkyl); and (2) when the designated bond in :T ' isC a single bond, then: (a) the designated bond in r\ js a UOUDle bond; (b) R is a radical of formula (ii):
Figure imgf000113_0002
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound; prior to administration of the therapeutic ionizing radiation, which radioprotective compound induces a temporary radioresistant phenotype in the normal tissue of the individual.
34. The method of claim 33, further comprising administering an effective amount of either: (A) at least one compound of formula V: Q1 X— CH=CH— Q2 V wherein, Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000114_0001
(ii)
Figure imgf000114_0002
(iii) (iv) wherein, q is one or zero; and R is -H, -(d-C8)hydrocarbyl or -C(=0)(Cι-C8)hydrocarbyl; or a pharmaceutically acceptable salt of such a compound; or (B) at least one antioxidant compound, prior to administering an effective amount of therapeutic ionizing radiation.
35. The method of claim 34, wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000114_0003
36. A method for treating an individual who has incurred or is at risk for incurring remediable radiation damage from exposure to ionizing radiation, comprising administering an effective amount of at least one radioprotective compound according to formula I:
Figure imgf000115_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000115_0002
' indicates that the designated bond is either a single bond or a double bond; p r\ indicates that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-Cβ)alkyl, substituted and unsubstituted aryl, substimted and unsubstituted heteroaryl, substimted and unsubstituted aryl(C1-C6)alkyl, substimted and unsubstituted heteroaryl(Cι-C6)alkyl, and -(CH2)m-C(=0)R ; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(d-C6alkyl); p is 1, 2 or 3; R2 is substituted aryl or substimted or unsubstituted heterocyclyl; R3 is substimted heteroaryl, -C(=0)NH2 or -C(=0)NH(Cι- C6)alkyl; B is aryl or heteroaryl; each R4 is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(d-C6)alkyl, -N(Cι-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(d-C6)alkyl, -SO(Cι-C6)alkyl, -S(Cι-C6)alkyl, -0(Cι-C6)alkyl, -C(=0)NH2, -C(=0)NH(d-C6)alkyl, -C(=0)N(d-C6alkyl)2, -C02H, -C02(Cr C6)alkyl, -S02NH2, -S02NH(d-C6)alkyl, -S02N(d-C6alkyl)2, -S02- (non-aromatic heterocycle), -(d-C6)alkylene-NH2, -(Cι-C6)alkylene- OH, -(Ci-C6)alkyleneC02H, -NHC(=0)(d-C6)alkyl, -NHS02(d- C6)alkyl, -C(=NH)NH2, -Z(Cι-C6)alkylene-NH2, -Z(d-C6)alkylene- NH(Cι-C6)alkyl, -Z(d-C6)alkylene-N(Cι-C6alkyl)2, -Z(C,-C6)alkylene-
C02(Ci-C6)alkyl, -Z(Ci-C6)alkylene-C02H, -Z(d-C6)alkylene- C(=00)NH2, -Z(d-C6)alkylene-C(=0)NH(Ci-C )alkyl, -Z(d- C6)alkylene-C(=0)N(Cι-C6alkyl)2, and substituted heterocyclyl; X is selected from the group consisting of C and N; R5 is selected from the group consisting of -H; substituted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; substimted and unsubstituted aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(d-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb>; each Ra is independently selected from the group consisting of -OH, -0(C,-C6)alkyl, -NH2, -NH(Cι-C6)alkyl, -N((Cι-C6)alkyl)2, and an
N-terminally linked peptidyl residue containing from! 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -Cθ2Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (Cι-C6)alkyl; provided: (1) when the designated bond in ' is a double bond, then: (a) the designated bond in •"< is a single bond, (b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000117_0001
(c) T is selected from the group consisting of N, C(H) and C(d-C6alkyl); and (2) when the designated bond in ' is a single bond, then: (a) the designated bond in r\ is a double bond; (b) R is a radical of formula (ii):
Figure imgf000117_0002
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound; prior to or after the individual incurs remediable radiation damage from exposure to ionizing radiation.
37. The method of claim 36, further comprising administering an effective amount of either: (A) a compound of formula V: Q1 X— CH=CH— Q2 V wherem, Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substimted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000118_0001
(i) (ϋ)
O O - N— C ii— - - §-(CH2)n-S II- i- Rx (iii) (iv) wherein: q is one or zero; and Rx is -H, -(d-C8)hydrocarbyl or-C(=0)(Ci-C8)hydrocarbyl; or a pharmaceutically acceptable salt of such a compound; or (B) at least one antioxidant compound, prior to or after the individual incurs remediable radiation damage from exposure to ionizing radiation.
38. The method of claim 37, wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000118_0002
39. The method of claim 37 wherein the radioprotective compound is administered before incurring remediable radiation damage from exposure to ionizing radiation.
40. The method of claim 37, wherein the radioprotective compound is administered after incurring remediable radiation damage from exposure to ionizing radiation
41. A method of reducing the number of malignant cells in bone marrow of an individual, comprising: (1) removing a portion of the individual's bone marrow; (2) administering to the removed bone marrow an effective amount of at least one radioprotective compound according to formula I:
Figure imgf000119_0001
wherein: R is hydrogen, R2, or a radical of formula (i) or (ii):
Figure imgf000119_0002
T ' indicates that the designated bond is either a single bond or a double bond; K in(}ica es that the designated bond is either a single bond or a double bond; R1 is selected from the group consisting of -H, -(Cι-C6)alkyl, substituted and unsubstituted aryl, substimted and unsubstituted heteroaryl, substimted and unsubstituted aryl(Cι-C6)alkyl, substimted and unsubstituted heteroaryl(d-C6)alkyl, and -(CH2)m-C(=0)Ra; m is 1, 2, 3, or 4; T is selected from the group consisting of N, C(H) C(=0) and C(d-C6alkyl); p is 1, 2 or 3; R2 is substituted aryl or substimted or unsubstituted heterocyclyl; R3 is substituted heteroaryl, -C(=0)NH2 or -C(=0)NH(C,- C6)alkyl; B is aryl or heteroaryl; each R is independently selected from the group consisting of -(Cι-C7)hydrocarbyl, halogen, -OH, -NH2, -NH(C,-C6)alkyl, -N(Cι-C6alkyl)2, -N(C2-C6heteroalkyl)2, -N02, -CN, -S02(d-C6)alkyl, -SO(Cι-C6)alkyl, -S(d-C6)alkyl, -0(d-C6)alkyl, -C(=0)NH2, -C(=0)NH(C,-C6)alkyl, -C(=0)N(d-C6alkyl)2, -C02H, -C02(d-
C6)alkyl, -S02NH2, -S02NH(C,-C6)alkyl, -S02N(d-C6alkyl)2, -S02- (non-aromatic heterocycle), -(Cι-C6)alkylene-NH2, -(Cι-Ce)alkylene- OH, -(Ci-C6)alkyleneC02H, -NHC(=0)(Cι-C6)alkyl, -NHS02(d- C6)alkyl, -C(=NH)NH2, -Z(d-C6)alkylene-NH2, -Z(d-C6)alkylene- NH(d-C6)alkyl, -Z(Cι-C6)alkylene-N(d-C6alkyl)2, -Z(d-C6)alkylene-
C02(Ci-C6)alkyl, -Z(Ci-C6)alkylene-C02H, -Z(d-C6)alkylene- C(=00)NH2, -Z(Ci-C6)alkylene-C(=0)NH(Ci-C6)alkyl, -Z(d- C6)alkylene-C(=0)N(d-C6alkyl)2, and substimted heterocyclyl; X is selected from the group consisting of C andN; R5 is selected from the group consisting of -H; substimted and unsubstituted aryl, -NH2, -Z-(Cι-C7)hydrocarbyl, -Z-(Cι-Ce)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; n is 1, 2, 3, 4, 5 or 6; R6 is selected from the group consisting of -H; substimted and unsubstituted aryl, -Z-(Cι-C7)hydrocarbyl, -Z-(d-C6)alkylene- heterocyclyl, and -Z-(CH(Rb))nC(=0)Ra; each Z is independently selected from the group consisting of -0-, -S-, -C(=0)-, -N(Rb)C(=0)- and -N(Rb)-; each Ra is independently selected from the group consisting of -OH, -0(CrC6)alkyl, -NH2, -NH(Cι-C6)alkyl, -N((C1-C6)alkyl)2, and an
N-terminally linked peptidyl residue containing from 1 to 3 amino acids in which the terminal carboxyl group of the peptidyl residue is present as a functional group selected from the group consisting of -C02Rb and -C(=0)NRb 2; and each Rb is independently selected from the group consisting of-H and (Cι-C6)alkyl; provided: (1) when the designated bond in ' is a double bond, then: p (a) the designated bond in ^ is a single bond, (b) R is hydrogen, R2, or a radical of formula (i):
Figure imgf000121_0001
(c) T is selected from the group consisting of N, C(H) and C(d-C6alkyl); and (2) when the designated bond in ' is a single bond, then: p (a) the designated bond in "^ is a double bond; (b) R is a radical of formula (ii):
Figure imgf000121_0002
(c) T is C(=0); or a pharmaceutically acceptable salt of such a compound; to the bone marrow; and (3) irradiating the bone marrow with an effective amount of ionizing radiation.
42. The method of claim 34, further comprising administering to the removed bone marrow an effective amount of either: (A) at least one compound of formula V: Q -X— CH=CH— 0 V wherein: Q1 and Q2 are, same or different, are substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and X is selected from the group consisting of (i), (ii), (iii) and (iv) below:
Figure imgf000122_0001
(0 (ϋ)
O O - $ N— C H— $- - I-(CH2)„-S ll- I- Rx (iii) (iv) wherein: q is one or zero; and Rx is -H, -(Cι-C8)hydrocarbyl or
Figure imgf000122_0002
or a pharmaceutically acceptable salt of such a compound; or (B) at least one antioxidant compound, prior to irradiating the bone marrow with an effective amount of ionizing radiation.
43. The method of claim 42, wherein X is selected from the group consisting of (i), (ii), and (iii) below:
Figure imgf000122_0003
44. The method of claim 42, further comprising reimplanting the bone marrow into the individual.
45. The method of claim 44, wherein the individual receives therapeutic ionizing radiation prior to reimplantation of the bone marrow.
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