Patent application title: NOVEL COMPOUNDS FOR INHIBITING EEF-2 KINASE ACTIVITY
Alexey G. Ryazanov (Princeton, NJ, US)
The University of Medicine and Dentistry
IPC8 Class: AA61K31519FI
Class name: 1,3-diazines (e.g., pyrimidines, etc.) polycyclo ring system having 1,3-diazine as one of the cyclos tricyclo ring system having 1,3-diazine as one of the cyclos
Publication date: 2012-07-12
Patent application number: 20120178769
The present invention discloses novel compounds for inhibiting eEF2
kinase and methods of use thereof.
7. A compound having a structure selected from the group consisting of ##STR00026## pharmaceutically acceptable salts and prodrugs thereof.
8. A pharmaceutical formulation comprising a compound according to claim 7 in a pharmaceutically acceptable carrier.
9. A method of treating induced cellular apoptosis comprising administering to a patient in need of such treatment a compound according to claim 7.
10. The method of claim 9, wherein the compound has the structure: ##STR00027##
11. The method of claim 9, wherein the induced cellular apoptosis is in the gastrointestinal tract of said patient.
12. The method of claim 11, wherein the induced cellular apoptosis is in the small intestine of said patient.
13. The method of claim 9, wherein the induced cellular apoptosis is radiation or chemotherapy induced.
14. The method of claim 13, wherein the cellular apoptosis is radiation induced.
15. The method of claim 14, wherein the radiation is gamma radiation.
16. The method of claim 9, wherein the induced cellular apoptosis is by increased phosphorylation of eEF2 in said patient.
STATEMENT OF GOVERNMENT SUPPORT
 This invention was made with government support under Grant No. 5R21RR022859-02 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE INVENTION
 The present invention relates to novel compounds for inhibiting eEF2 kinase and methods of use thereof.
BACKGROUND OF THE INVENTION
 The enzyme Elongation Factor 2 Kinase (eEF2-K) belongs to a novel family of protein kinases, with prototypical member being Dictyostelium myosin heavy chain kinase A (MHCK A), which display no homology to conventional eukaryotic protein kinases. This protein kinase is highly specific to Elongation Factor 2 (eEF2) and is responsible for eEF2 phosphorylation. eEF2 promotes ribosomal translocation, the reaction that results in the movement of the ribosome along mRNA during translation. eEF2 was identified among the most prominently phosphorylated proteins in crude tissue and cell lysates. Importantly, it was found that phosphorylation of eEF2 arrests translation, suggesting that this may be a critical mechanism by which the rate of protein synthesis is regulated (Ryazanov, A. G. (1987). Ca2+/calmodulin-dependent phosphorylation of elongation factor 2. FEBS Lett 214, 331-334; Ryazanov, A. G., Shestakova, E. A., and Natapov, P. G. (1988).)
 The activity of this kinase is increased in different types of cancers and may be a valid target for anti-cancer treatment. For example, eEF2-K is reported to be overexpressed in breast cancer cell lines and tumors with little or no activity observed in normal breast tissue. eEF2-L is also activated in rat glioblastoma (Chang et al, (1995) Calmodulin-dependant protein kinases in rat glioblastoma, Cell Growth Diff.). Natural product Rottlerin has been shown to inhibit growth of glioma cell lines by inhibiting eEF2-K. (Parmer et al, (1997) Cell Growth Differ. Vol. 8, 327).
 eEF2-K enzyme has also been shown to have increased activity in human brains of individuals with Alzheimer Disease (Li, X., Alafuzoff, I., Soininen, H., Winblad, B., and Pei, J. J. (2005) Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain. FEBS J. 272, 4211-4220). Although the mechanism and relevance of the enzyme for such purposes are not clear, the eEF2-K activation pathway is believed to play an important role in the pathophysiology of Alzheimer. Thus, eEF2 kinase inhibitors could be used to modulate the pathophysiology of a number of disease states including brain cancer, breast cancer, ischemic heart disease, wherein the eEF2-K pathway may have a role in its etiology.
 It has also been recently discovered that inactivation of a ubiquitous cellular enzyme eEF2 kinase can confer resistance to radiation by suppressing radiation-induced apoptosis. Radiation-induced apoptosis can occur by either of ionizing or non-ionizing type of radiation. Mucosal damage, such as the damage to the intestine is also another major dose-limiting event in radiation therapy and chemotherapy. Aspects of rapid cell turnover, distinct compartmentalization of damage, and known differentiation pathways of crypt cells in the murine and human intestine have been the subject of an ongoing research. Various modalities such as antioxidant therapy and inhibition of serotonin activity at the gastric level, have been suggested and employed in the art to treat such conditions. However, there is still a need to mitigate side effects associated with drug and radiation therapy.
 The present invention provides an alternative approach for achieving this goal. eEF2 kinase inhibitors are proven to be a useful radioprotectors not only against natural radiation but also for patients undergoing radiation therapy. Novel compounds are herein disclosed for use in disorders that have a pathology associated with the eEF2-K activity
BRIEF DESCRIPTION OF THE FIGURES
 FIG. 1 illustrates eEF2 kinase activity assay with purified eEF2 and with different concentration of identified inhibitors.
 FIG. 2 illustrates a reaction scheme for the formation of 1,3 selenazines of the instant invention.
 FIG. 3 illustrates the survival rate of wildtype and eEF2K-KO mice after exposure to gamma radiation.
 FIG. 4 illustrates eEF2K-/- mice have less apoptosis induced by gamma irradiation.
 FIG. 5 illustrates eEF2 kinase activity assay with purified eEF2 and with different concentration of compound PV-02367
 FIG. 6 illustrates in vivo eEF2 kinase activity test with different concentrations of compound PV-2206.
SUMMARY OF THE INVENTION
 The shortcomings of the prior art are now addressed in the present invention. The present invention provides for new and novel compounds directed to eEF2-K enzyme and capable of inhibiting the enzymes activity by at least 10%.
 One aspect of the present invention is directed to the therapeutic use of such compounds in patients suffering from conditions with pathologically abnormal over-expression of eEF2-K. Accordingly the presently disclosed compounds are intended to be used as prophylactically, as a primary mode or alternative mode of treatment in the form of combination therapy in patients suffering from clinically significant over expressed eEF2-K activity.
 In another aspect of the present invention, the novel compounds for the present invention have the structure of Formula I: Het-L-AK, wherein Het is a heteroatom moiety, L is a linking carbon or nitrogen containing chain and AK is a hydrophobic tail.
 In a more preferred embodiment, the compounds of the present invention include but are not limited to the compounds having the following structures:
 The present invention also provides for the enantiomerically enriched variation of these compounds present in a pharmaceutically acceptable composition.
DETAILED DESCRIPTION OF THE INVENTION
 The compounds of the present invention may be optically active and be isolated in either their optically active and enantomerically enriched or racemic forms. Cis and trans geometric isomers of the compounds of the present invention may be isolated as a mixture of isomers or as separated isomeric forms and are intended as a disclosed variation. In the present application, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended to be disclosed.
 The term "substituted," as used herein, means that any one or more hydrogen(s) on the designated atom or ring is replaced with an indicated functional group, provided that the designated atom's or ring atom's normal valency is not exceeded, and that the substitution results in a stable compound. R substitutions presented herein are independent of its definition at every other occurrence.
 As used herein, "alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups containing 1 to 20 carbons, preferably 1 to 18 carbons, more preferably 1 to 16 carbons, in the normal chain, the term encompass methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, and the like. By the term "lower alkyl" it is meant C1-C6 alkyl groups.
 Unless otherwise indicated, the term "alkenyl" as used herein by itself or as part of another group refers to straight or branched chain radicals of 2 to 20 carbons, preferably 2 to 18 carbons, and more preferably 2 to 16 carbons in the normal chain, which include one to six double bonds in the normal chain, such as vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl, 3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like.
 Unless otherwise indicated, the term "alkynyl" as used herein by itself or as part of another group refers to straight or branched chain radicals of 2 to 20 carbons, preferably 2 to 18 carbons and more preferably 2 to 16 carbons in the normal chain, which include one triple bond in the normal chain, such as 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl,3-undecynyl, 4-dodecynyl, and the like.
 Unless otherwise indicated, the term "cycloalkyl" as employed herein alone or as part of another group includes saturated or partially unsaturated (containing 1 or 2 double bonds) cyclic hydrocarbon groups containing 1 to 10 rings, preferably 1 to 3 rings, including monocyclic alkyl, bicyclic alkyl (or bicycloalkyl) and tricyclic alkyl, containing a total of 3 to 20 carbons forming the ring.
 "Halo" or "halogen" as used herein refers to fluoro, chloro, bromo, and iodo; and "haloalkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups, such as for example CF3 or the like.
 Unless otherwise indicated, the term "aryl" as employed herein alone or as part of another group refers to monocyclic and bicyclic aromatic groups containing 6 to 10 carbons in the ring portion (such as phenyl, benzyl, or naphthyl, including 1-naphthyl and 2-naphthyl).
 Unless otherwise indicated, the term "alkoxyl", "aryloxyl" or "aralkoxyl" as employed herein alone or as part of another group includes any of the above alkyl, aralkyl, or aryl groups linked to an oxygen atom.
 Unless specifically specified, as used herein, the term "Het", "aromatic heterocyclic system" or "heteroaryl" is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic aromatic ring which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S and is aromatic in nature.
 The term "cyano" as used herein, refers to a --CN group and are used interchangably.
 The term "nitro" as used herein, refers to an --NO2 group and are used interchangably.
 The term "hydroxy" as used herein, refers to an --OH group and are used interchangably.
 The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in human beings and animals commensurate with a reasonable therapeutic benefit/risk ratio.
 As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making counterpart acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. Moreover the term may refer to counter ions of any moiety that is designated in this disclosure in an ionic form.
 The novel compounds instantly disclosed are also intended to be used in a context of a prodrug. The term "prodrugs" as employed herein includes esters and carbonates of the disclosed compounds formed by reacting one or more hydroxyls of compounds of Formula I -V, with alkyl, alkoxy or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates, and the like.
 The instant invention relates to novel eEF2-K inhibitors compounds, their respective pharmaceutically acceptable salts and analogues thereof. The present invention also relates to pharmaceutically acceptable compositions of said compounds and their use as potential therapeutic means in disorders that have a pathology associated with eEF2-K kinase activity or overexpression. The presently disclosed eEF2-K inhibitors compounds are useful in variety of diagnostic as well as therapeutic applications, prophylactic applications as related to eEF2-K pathway or related disorders.
 In one embodiment, the eEF2-K inhibitor of the present invention is a compound that either binds to or alters the kinase domain of eEF2-K to prevent the enzyme from phosphorylating eEF2. To this end the inhibitor may competitively inhibit the phosphorylative activity of the eEF2K enzyme. Alternatively, the inhibitor may interact with the protein at a site other than the kinase domain, which alters the structure of the enzyme or otherwise causes kinase domain inactivation. To this end, the inhibitor may noncompetitively inhibit eEF2-K phosphorylative activity.
 The preferred embodiment are novel inhibitors of eEF2K that inhibit the activity of the kinase by at least 10, 20, 30, 50 or preferably 80 percent of its normal activity as measured against purified eEF2. These compounds include but are not limited to the compounds having the following structures:
 eEF2 kinase inhibitors also include the compounds having the following structure:
 Based on the ability of each of the above compounds to inhibit eEF2-K phosphorylative activity, the instant invention also encompass structural analogs of any of sphingosine-1-phosphate, L-587, L-207, cetyl pyridinium, PV 2206 or NH-125. As used herein, "analog" or "structural analog" refers to compounds having one or more atoms, functional groups, or substructures replaced or substituted with different atoms, groups, or substructures that are capable of inhibiting the activity of eEF2-K. The desired structural compounds and analogs of the present invention contain a head region and a tail portion, and are collectively represented by Formula I:
Het-L-AK Formula (I)
wherein Het is an optionally substituted aromatic or non-aromatic heterocyclic ring or ring system or an optionally N-substituted guanidine, L is either a linking bridge between Het and AK or a direct bond, a straight or branched alkyl, or alkylene chain, wherein the L may form a 1-8 carbon chain optionally substituted, --NH or --NR1 wherein R1 is a C1-6 alkyl or alkoxyl.
 AK is a straight or branched C14-C18 aliphatic tail which can contain an aromatic ring or at least one alkyene, alkylene group and further be optionally substituted, saturated or unsaturated,. One or more carbons of the aliphatic tail may be substituted with one or more isosteric groups such as one or more aryl or heteroaryl moieties alone or as part of a ring system. Therapeutically valuable analogs having the structure of Formula I, including compounds containing the optional substituents disclosed herein or other known pharmaceutical compound building blocks, may be identified using the screening methods known in the art.
 The foregoing compounds are believed to fit the pharmacophore for inhibition of eEF2-K which is believed to correspond to a C16 alkyl chain with a positive charged head group--either quaternary Nitrogen or basic nitrogen. Thus, they generally contain a 16 carbon aliphatic chain with a positively charged head group that would adapt a configuration capable of interfering with substrate binding domain of the eEF2 kinase. Moreover, the C16 alkyl chain could be replaced by a substituted phenyl ring with appropriate linkers:
 Wherein m, n is a integer from 0 to 10, and preferably m is 4-10 and n is 1-3 and may be linked to the phenyl ring via a hetero atom.
 In at least one embodiment, the novel compounds of the present invention contain a Het moiety, which includes, but is not necessarily limited to or more of the following:
wherein R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 R11 R12 R24, R25, R26, R27, R28, R29, R30 are independently hydrogen (H), hydroxyl (--OH), alkyl, alkylene, alkylyl, halo, haloalkyl, alkoxyl, cycloalkyl, aryl, aryloxyl, substituted variations thereof, carboxyl, nitro, --CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are each independently hydrogen (H), alkyl, cycloalkyl, carboxyl or together with the nitrogen to which they are attached form:
 m, n is a integer from 0 to 10,
 R13, R18 are independently a hydrogen (H), alkyl, haloalkyl, cycloalkyl, aryl including phenyl and benzyl, or substituted variations thereof, --CN, --CH2CN, --CH2CNH2(NOH), OR', S(O)nR', NR' R'' wherein R' and R'' are each independently hydrogen (H), alkyl, cycloalkyl carboxyl or together with the nitrogen to which they are attached form:
 R14, R15, R16, R17 are independently a hydrogen (H), hydroxyl (--OH), alkyl, alkylene, halo, haloalkyl, alkoxyl, cycloalkyl, aryl, carboxyl, alkyl carboxyl, or substituted variations thereof,
 R19, R20 are independently a hydrogen (H), alkyl, aryl, haloalkyl, cycloalkyl, or substituted variations thereof,
 R21, R22, R23 are independently a hydrogen (H), alkyl, haloalkyl, cycloalkyl or substituted variations thereof, --CN, --CH2CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are same as above,
 G represents a C5 to C10 cyclic moiety including cycloalkyl, aryl, aryloxyl, containing a hetero atom and optionally substituted with a halogen,
 X is N, O, or S,
 Y, is O or S,
 V is nitro, --CN, --CH2CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are each independently are same as above,
 Z is independently hydrogen (H), hydroxyl (--OH), alkyl, halo, haloalkyl, alkoxyl, cycloalkyl, aryl, substituted variations thereof, --CN, OR', SR', S(O)nR', --NR' R'' wherein R' and R'' are each independently selected and are same as above, and
 signifies the Het point of attachment to the L or AK.
 In another embodiment, R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 R11 R12 R24, R25, R26, R27, R28, R29, R30 are independently hydrogen (H), hydroxyl (--OH), C1-C16 alkyl, halo, haloalkyl, C1-C8 alkoxyl, C1-C8 cycloalkyl, phenyl, benzyl, naphthyl, carboxyl, --CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are each independently hydrogen (H), C1-C8 alkyl, or together with the nitrogen to which they are attached form:
 m, n is preferably an integer from 0 to 3,
 R13, R18 are independently a hydrogen (H), C1-C8 alkyl, cycloalkyl, phenyl and benzyl, --CN, --CH2CNH2(NOH), OR', S(O)nR', NR' R'' wherein R' and R'' are each independently hydrogen (H), C1-C8 alkyl or together with the nitrogen to which they are attached form:
 R14, R15, R16, R17 are independently a hydrogen (H), hydroxyl (--OH), C1-C8 alkyl, C1-C8 alkoxyl, cycloalkyl, phenyl, benzyl, naphthyl,
 R19, R20 are independently a hydrogen (H), C1-C8 alkyl, phenyl, benzyl,
 R21, R22, R23 are independently a hydrogen (H), C1-C8 alkyl, C1-C8 cycloalkyl or substituted variations thereof, --CN, --CH2CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are same as above,
 G is a C5 to C6 cyclic alkyl or aryl moiety including a nitrogen containing ring,
 X is N, O, or S,
 Y, is O,
 V is nitro, --CN, --CH2CN, OR', S(O)nR', --NR' R'' wherein R' and R'' are each independently are same as above,
 Z is independently hydrogen (H), hydroxyl (--OH), C1-C8 alkyl, C1-C8 alkoxyl, cycloalkyl, phenyl, benzyl, --CN, OR', SR', S(O)nR', --NR' R'' wherein R' and R'' are each independently selected and are same as above, and signifies the point of attachment to Het.
 In a more preferred embodiment the Het is one of following functional groups
##STR00010## ##STR00011## ##STR00012## ##STR00013## ##STR00014##
 Exemplified compounds of Formula I include, but are not limited to, one or more of the following:
wherein the R' and R'' substituents are independently selected and are the same as above. In a more preferred embodiment R' and R'' are H, a straight or branched chain optionally substituted alkyl group, an optionally substituted cycloalkyl.
 Further Examples of compounds of Formula I have the following structures: wherein R is the same as R1 substituent described above for Formula I, and R1, R2 are the same as for Formula I.
 In another embodiments of Formula I, the preferred eEF2-K inhibitor compounds are
 In alternative embodiments, the eEF2K inhibitors of the present invention contains a selenazine compound or an analog thereof. For example, in certain non-limiting embodiments, the eEF2K inhibitor contains an analog of selenazine compounds which are known to inhibit eEF2K phosphorylative activity, namely TS2, TS4, or PS2 which are comprised of the following respective structures:
 To this end, the selenazine analogs of the instant invention contain a 1,3 selenazine core with one or more substituent groups extending therefrom. In certain embodiment, such analogs may be collectively represented by Formula VII:
wherein R1, R2, R3 and R4 are same as above, and in a preferred embodiment are independently selected from H, a straight or branched chain optionally substituted alkyl group, an optionally substituted cycloalkyl group, and an optionally substituted aryl or heteroaryl group, nitro, COOH, --NH-alkyl, --CO--NH-alkyl, --NH-acyl. In a more preferred embodiment R1, R2, R3 and R4 may be selected from lower alkyl, lower alkoxy, nitro, --COOH, --NH-lower alkyl, --CO--NH-lower alkyl, --NH-acyl, and the like. R4 may also include acyl and carboamyl groups.
 One of ordinary skill in the art will appreciate that therapeutically valuable analogs having the structure of Formula VII that are unsubstituted or contain the identified substituents or other pharmaceutical compound building block substituents may be identified using the screening methods discussed herein or with others known in the art.
 In one embodiment, R1 and/or R2 may comprise enone, such as a straight or branched branched chain structure; cycloalkyl structure and/or aryl/heteroaryl groups. Exemplified enones include, but are not limited to the following:
 In one embodiment, R3 is derived from a nitrile. To this end, R3 may comprise an aliphatic, aryl or heteroaryl-substituted nitriles. In alternative embodiment, R3 may be the following structure:
wherein W may be either OMe, NO2, NHR', NHAc, COO2H, CONHR', or the like.
 In even further embodiments, the eEF2K inhibitor is comprised of chalcone, or analogs thereof Rottlerin (IC50 4iM, Cho et al., 2000). In one embodiment, chalcone may be represented by the structure:
 In another embodiment the eEF2-K inhibitory compound have the following structure:
wherein R31 is hydrogen (H), hydroxyl (--OH), C1-C8 alkyl, halo, haloalkyl, C1-C8 alkoxyl, C1-C8 cycloalkyl, phenyl, or benzyl,
 G represents a C5 to C8 cyclic moiety including cycloalkyl, aryl, or halo or hydroxyl substitutes thereof, and
 X and Y, R' and R'' are the same as above.
 In a more preferred embodiment of Formula VII, the compound has the structure:
 eEF2K inhibitory compounds of the present invention may be identified using a high-throughput screening assay, such that the assay discussed in the provisional application U.S. 61/225,875 incorporated herein by reference in its entirety. Specifically, eEF2K can be produced in large quantities by E. coli, or using any other suitable means known in the art.
 Without seeking to limit the possible scope of use of the foregoing compounds, the clinical therapeutic indications envisioned include, but are not limited to, any treatment regimen targeting the inhibition of eEF2K phophorylative activity. Such treatment regimens may include not only the prophylactic measurement, but also as a means to treat side effects associate with post-radiation therapy, breast cancer, Alzheimer's Disease or in disorders wherein induction of Long Term Potentiation of Synaptic Transmissions are deemed clinically useful. The results below also suggest that eEF2K can be an important component of the apoptotic pathway, and its inactivation can confer resistance to radiation. Accordingly, selective inhibitors of eEF2K can be used as drugs to protect tissues from cell death caused by radiation alone or in combination with other like drugs to induce a synergistic effect.
 Compounds of the present invention may be synthesized using methods known in the art or as otherwise specified herein. In one embodiment, the compounds of any of the above Formulas may be used in combination to provide a synergistic clinical alternative. Various methods of manufacturing may be employed to produce the compounds of Formula I-VII. At least in one embodiment compounds of Formula VII are manufactured by reacting a selenamide with an unsaturated ketone. One embodiment of such a reaction is illustrated in FIG. 2. In another embodiment compounds of Formula I, PV-2206 is manufactured by replacing the AK chain of compound L-587 with Fingolimod.
 Unless otherwise specified, a reference to a particular compound of the present invention includes all isomeric faints of the compound, to include all diastereomers, tautomers, enantiomers, racemic and/or other mixtures thereof. Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate (e.g., hydrate), protected forms, and prodrugs thereof. To this end, it may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19, the contents of which are incorporated herein by reference.
 Based on the foregoing, one or more compounds of the present invention, either alone or in combination with another active ingredient, may be synthesized and administered as a therapeutic composition. The compositions of the present invention can be presented for administration to humans and other animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, oil in water and water in oil emulsions containing suitable quantities of the compound, suppositories and in fluid suspensions or solutions. To this end, the pharmaceutical compositions may be formulated to suit a selected route of administration, and may contain ingredients specific to the route of administration. Routes of administration of such pharmaceutical compositions are usually split into five general groups: inhaled, oral, transdermal, parenteral and suppository. In one embodiment, the pharmaceutical compositions of the present invention may be suited for parenteral administration by way of injection such as intravenous, intradermal, intramuscular, intrathecal, or subcutaneous injection. Alternatively, the composition of the present invention may be formulated for oral administration as provided herein or otherwise known in the art. Finally, the compositions of the present invention may be so chosen to prove a synergistice effect, so that the combined use of the compounds would produce a result better than a mere additive effects.
 As used in this specification, the terms "pharmaceutical diluent" and "pharmaceutical carrier," have the same meaning. For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions such as tablets, the compound can be mixed with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methylcellulose and functionally similar materials as pharmaceutical diluents or carriers. Capsules are prepared by mixing the compound with an inert pharmaceutical diluent and filling the mixture into a hard gelatin capsule of appropriate size. Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
 Fluid unit dosage forms or oral administration such as syrups, elixirs, and suspensions can be prepared. The forms can be dissolved in an aqueous vehicle together with sugar or another sweetener, aromatic flavoring agents and preservatives to form a syrup. Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent such as acacia, tragacanth, methylcellulose and the like.
 For parenteral administration fluid unit dosage forms can be prepared utilizing the compound and a sterile vehicle. In preparing solutions the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing. Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle. The composition can be frozen after filling into a vial and the water removed under vacuum. The lyophilized powder can then be scaled in the vial and reconstituted prior to use.
 Dose and duration of therapy will depend on a variety of factors, including (1) the patient's age, body weight, and organ function (M., liver and kidney function) and the capacity of the patient to clear the eEF2K inhibitor; (2) the nature and extent of the disease process to be treated, as well as any existing significant co-morbidity and concomitant medications being taken and (3) drug-related parameters such as the route of administration, the frequency and duration of dosing necessary to effect a cure, the therapeutic index of the drug and the synergistic endpoint. In general, the dose will be chosen to achieve serum levels of 1 ng/ml to 100 ng/ml with the goal of attaining effective concentrations at the target site of approximately 1 ng/ml to 10 μg/ml. Using factors such as this, a therapeutically effective amount may be administered so as to ameliorate the targeted symptoms of and/or treat eEF2K dependent disorders. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art for purposes of achieving desired serum levels between 1 ng/ml to 100 ng/ml, especially in light of the detailed disclosure and examples provided herein.
eEF2K Inhibitor Active Compounds
 The following compounds are three specific inhibitors of eEF2 kinasae: L-207 and L-587:
 All compounds described herein are believed to have radioprotector properties, specifically against radiation induced apoptosis.
eEF2K-KO Mice Resistant to Radiation
 The inventor have found that eEF2 kinase knockout mice are resistant to gamma radiation and do not experience hair loss and hair graying after irradiation. As can be seen in FIG. 3, after exposure to gamma radiation (8 Gy) 50% of wild type mice die within 16 days, however all eEF2 kinase knockout mice survive after irradiation. Moreover, one month after irradiation all surviving wild type mice experienced hair loss and hair graying. However, all eEF2K-KO mice under the same conditions have healthy coats of black hair. Thus, inhibition of eEF2 kinase can protect animals from the damaging effect or radiation and prevent hair loss and hair graying caused by radiation.
 The inventor analyzed long-term survival in EEF2k+/-, EEF2k+/+ and EEF2k-/- mice and found that knockout of eEF2 kinase results in significant increase in mouse life span. Maximal lifespan defined as the average age of the last 10% of surviving mice, was increased by approximately 30% in EEF2k-/- mice and approximately 18% in EEF2k+/- mice in comparison with EEF2k+/+ mice. Since increase in maximal lifespan is observed in both EEF2k-/- and EEF2k+/- mice, the complete elimination of eEF2 kinase is not required for the lifespan extending effect.
 We also investigated the effect of eEF2 kinase inactivation on the sensitivity of gastrointestinal tract to radiation damage. Since cells in the crypts of small intestine are particularly sensitive to radiation (reviewed in Potten, 2004) we investigated radiation induced apoptosis in small intestine in eEF2 kinase knockout mice. Samples of small intestine were obtained from male mice after 24-h of irradiation with 8 Gy. As can be seen in FIG. 4 irradiation results in a dramatic increase in phosphorylation of eEF2. This increase correlated with the increase in the number of apoptotic bodies which can be detected by Hematoxylin staining in the crypts of small intestine (FIG. 4). As can be seen in FIG. 2 and Table 1, the number of apoptotic bodies was significantly lower in eEF2K-/- mice than eEF2K+/+ mice after irradiation.
TABLE-US-00001 TABLE 1 Apoptosis in small intestine after gamma irradiation Apoptotic Dose Genotype bodies/crypt # of mice 0 Gy eEF2K+/+ 0.05 3 8 Gy eEF2K+/+ 3.9 ± 0.5 5 8 Gy eEF2K-/- 1.2 ± 0.6 4
 These results suggest that the inactivation of eEF2K in mice can protect intestinal cells from radiation-induced apoptosis.
 The inventor has analyzed the effect of eEF2K deficiency on apoptosis in cells isolated from eEF2K-/- mice. It was found that cells from eEF2K deficient mice were significantly more resistant to apoptosis induced by radiation, doxorubicin or hydrogen peroxide. Significant cell death was observed in wild type mouse embryonic fibroblasts (MEFs) treated for 24 hours with 1.6 μM doxorubicin. However, much less cell death was observed in eEF2K-deficient cells treated in the same manner. The results of the TUNEL assay suggest that the reduction in cell death in eEF2K-deficient cells is due to decreased apoptosis. To verify that the decreased apoptosis was due to the absence of eEF2K, eEF2K cDNA was introduced into eEF2K- deficient MEFs. Treatment with hydrogen peroxide or doxorubicin, MEFs carrying eEF2K cDNA have more activated caspase 3 than eEF2K-deficient cells from which they were derived, thus confirming that eEF2K enhances apoptosis.
Testing of Radioprotective Properties of eEF2K Inhibitors in Cell Lines and Animals
 Recently, as illustrated in FIG. 1, sphingosine-1-phosphate was demonstrated to inhibit eEF2 kinase in vitro, suggesting a possibility that the radioprotective effect of sphingosine-1-phosphate can be mediated by eEF2 kinase. To this end, the eEF2K inhibitory compounds of the present invention may be used for a radioprotective effect. Accordingly, the present inventors analyzed the effect of various analogs and lead compounds on the radiation induced apoptosis in the intestinal stem cells.
 FIG. 1 illustrates eEF2 kinase activity assay with purified eEF2 and with different concentration of identified inhibitors. Recombinant eEF2 kinase (0.5 μg) was incubated with purified rabbit reticulocyte eEF2 (0.5 μg) in a reaction mixture consisting of 50 mM Hepes-KOH pH 6.6, 10 mM magnesium acetate, 5 mM DTT, 100 μM CaCl2, 0.5 μg calmodulin, 50 μM ATP, and 0.2 μCi [γ-33P]-ATP (3000 Ci/mmol). The reactions were run at 30° C. for 15 minutes, and were terminated by incubation in an ice/water bath and addition of Laemmli sample buffer. Samples were analyzed by SDS-PAGE and autoradiography.
 For the first time the inventor of the present invention developed a cell-based assay of radioprotectors using epithelial cells. It was found that immortalized kidney epithelial cells derived from eEF2 kinase knockout mice are significantly more resistant to radiation-induced apoptosis in comparison with cell lines derived from wild type mice. This assay will be used to investigate radioprotective properties of identified eEF2 kinase inhibitors L-207 and L-587, and V-2206. In addition additional tests on the radioprotective properties of selenazines identified as eEF2 kinase inhibitors previously (Cho et al., 2000) and compounds of Formula VII indicated effective inhibitory affects of the eEF2 in vivo.
 All experiments on gamma irradiation of mice will be performed in collaboration with Dr. Roger Howell (UMDNJ). We will investigate the effect of i.p. injection of various eEF2K inhibitors on the survival of mice after irradiation. In initial experiments we will test five compounds: L-587, L-207, NH-125 and the two most potent selenazine inhibitors of eEF2K, TS4 and PS2. In subsequent experiments we will analyze the radioprotective properties of various analogs and lead compounds developed by Provid. We will first determine the maximum tolerated dose (MTD) of the drug by i.p. injecting each compound at various concentrations and observing mice survival for a period of 30 days. LD90/30 doses will be used in subsequent experiments and will be performed in the following way. Five groups of mice (10 6-8 wk female C57/BL6 mice in each group) will be injected i.p. with the LD90/30 of each compound in 200 μl. Control group will be injected with the same volume of the vehicle. Mice will be irradiated with 8 Gy and 10 Gy of gamma radiation. Mice (whole body) will be acutely irradiated with 137Cs γ-rays using a Mark I irradiator. To keep irradiation time to several minutes (acute), dose rates of ˜2.5 Gy/min will be used. Survival of mice after irradiation for a period of 30 days will be analyzed.
In vivo Inhibition of eEF2 K by PV-2367
 To test in vivo inhibition effect of PV-2367, series concentrations of drugs were used to treat MEFs. After 30 min incubation, cell lysates were collected for western blot. Specific antibodies against phosphorylated eEF2 (cell signal) were used to detect the p-eEF2 levels after treatment. Loading controls were probed with antibodies against GAPDH. FIG. 6 proves the inhibitory effects of the product against eEF2K enzyme and its rate of success in live cells.
 The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the spirit and script of the invention, and all such variations are intended to be included within the scope of the following claims.
Patent applications by Alexey G. Ryazanov, Princeton, NJ US
Patent applications in class Tricyclo ring system having 1,3-diazine as one of the cyclos
Patent applications in all subclasses Tricyclo ring system having 1,3-diazine as one of the cyclos