WO2010056795A1 - Novel synthetic dinucleoside polyphosphate analogs and their use as new therapeutic and/or diagnostic modalities - Google Patents

Abstract

The invention provides novel synthetic dinucleoside polyphosphate analogs and their use as therapeutic compounds and/or diagnostic tools for the treatment and diagnosis of various human diseases and metabolic disorders.

Description

NOVEL SYNTHETIC DINUCLEOSIDE POLYPHOSPHATE

ANALOGS AND THEIR USE AS NEW THERAPEUTIC

AND/OR DIAGNOSTIC MODALITIES

(ATTORNEY DOCKET NO. ZAM-004PC)

BACKGROUND OF THE INVENTION

Field of the invention

The invention relates to synthetic dinucleoside polyphosphate analogs and their use as therapeutic compounds and/or diagnostic tools for the treatment and diagnosis of various human diseases and metabolic disorders.

Summary of the related art

Diadenosine polyphosphates are members of a group of dinucleoside polyphosphates that are widely present in eukaryotic cells and play vital roles in their metabolism [Garrison

et al. 1992, Baxi et al. 1995, McLennan 2000]. Diadenosine polyphosphates have been identified as modulators of cardiovascular and neurotransmitter- like activities in addition to their previously described role in cell proliferation and as signal molecules when cells are undergoing stress [Baxi et al. 1995]. The wide abundance of dinucleoside polyphosphates in almost all types of eukaryotic cells makes their synthetic analogs potentially promising as therapeutic and diagnostic modalities. Several synthetic analogs of diadenosine polyphosphates have been tested successfully as potential therapeutic and diagnostic tools [Holler et al. 1983, Zamecnik et al. 1992, Chan et al. 1997, Elmaleh et al. 1998, Elmaleh

et al. 2006]. Despite the synthesis and characteristics of many diadenosine polyphosphate analogs in the last few decades [Guranowski (1987), Lazewska et al. (1993), Blackburn et

al (1999), Liu et al. (1999), Guranowski et al. (2000), Walkowiak et al. (2002, Blackburn et al. (2002), Guranowski (2003), Guranowski (2004)], only a small number of compounds

has been tested as potential therapeutic or diagnostic compounds. [Holler et al. 1983, Zamecnik et al. 1992, Chan et al. 1997, Elmaleh et al. 1998, Elmaleh et al. 2006, Ratjen F. (2007), Storey S, WaId G. (2008), Kellerman et al. (2008)] US patents and applications # 05306629, 05837861, 06555675, 05681823, 05900407, 06818629, 06864242, 06867199, 06881725, 06977246, 07078391, 07084128, 07101860, 07109181, 07132408, 07223744, 07256183, 07338776, 21031743A1, 22052337A1, 22103158A1, 23027785A1, 23036527A1, 23125299A1, 23207825A1, 24077585A1, 24220133A, 25009778A1, 25085439A1 and WO with numbers 06133375A3, 06133375A2, 02060454A3, 02060454A2, 06016115A3, 06016115A2, 02060454B1, 0216381B1, 0216381BA3, 0216381A2, 08113072A2, 06082397A1, 05097814A3, 05097814A2, 03039473A3, 03039473A2, 0187913C2, 03000056A1, 0187913A3, 0187913A2.

Given the importance of synthesis and screening of the novel dinucleoside polyphosphate analogs as potential therapeutic and diagnostic modalities, based on their important role in the life cycle of eukaryotic cells, the synthesis, testing, and selection of the best candidates as potential therapeutic and diagnostic compounds are needed.

BRIEF SUMMARY OF THE INVENTION

The present invention identifies new synthetic dinucleoside polyphosphate analogs and their use as therapeutic agents and/or diagnostic tools.

In a first aspect, the invention provides compounds having structural formula I:

Wherein X and X' are nucleosides or modified nucleoside groups, deoxynucleoside groups or seconucleoside groups each independently selected from adenosin-5'-yl cytidin-5'-yl, 2,6- diaminopurineribosyl-5'-yl, inosin-5'-yl, guanosin-5'-yl, thymidin-5'-yl, uridin-5'-yl, ethenoadenosin-5'-yl, or other heterocyclic, cyclic or non-cyclic chemical groups attached to a sugar selected from D-ribose, 2-deoxy-D-ribose, 2,3-dideoxy-D-ribose, morpholino-, 2- and/or 3-OMe-ribose, 2- and/or 3-OEt-ribose, 2- and/or 3 -F, -Br, -Cl, -N₃ 2- and/or 3-deoxy- D-ribose, D-arabinose, D-glucose or any other closed ring, open or seco-sugar derivative, or analog thereof, and where X and X' can be the same or different nucleosides or nucleoside analogs. Each A is independently selected from =0, =S, =NH, and other divalent atoms, charged or neutral chemical groups, each B is independently selected from -O , -S^", -NH₃ ⁺, short alkyl, and other charged or neutral chemical groups bonded to the phosphorus atom by a single covalent bond. Each of Y¹, Y², Y³ is separately selected from -O-, -S-, -NH-, -C(O)-CH₂-, -CH₂-CO-, monohalomethylene, dihalomethylene, carboxymethylene, alkylcarboxymethylene, aralkylcarboxymethylene, phosphonomethylene, sulfonomethylene, 1 ,2-ethylene, 1,1 -ethylene, 1,1 -propylene, 1 ,2-propylene, oxy(halo)methylene, halomethyleneoxy, thiomethylene, thiomethyleneoxy, and other linear and other divalent atoms charged or neutral divalent chemical groups serving as a bridge between two adjacent phosphorus atoms, k, 1, m, and n are integral numbers from 0 to 5; where the sum of (k+l+m+n) is ≤ 2 and ≤ 7.

In a second aspect, the invention provides a pharmaceutical formulation comprising a compound according to the invention and a pharmaceutically acceptable diluent, carrier or excipient.

In a third aspect, the invention provides a diagnostic method for detecting atherosclerotic lesions. The method according to this aspect of the invention comprises administering to a patient suspected of having an atherosclerotic lesion a labeled compound according to the invention, allowing the labeled compound to bind to the atherosclerotic lesion and detecting the labeled compound at the site of the atherosclerotic lesion.

In a fourth aspect, the invention provides a therapeutic method for treating a disease or disorder associated with blood disorders. The method according to this aspect of the invention comprises administering to a patient having a blood disorder or a disease associated with a blood disorder a therapeutically effective amount of a compound or pharmaceutical composition according to the invention.

In a fifth aspect, the invention provides a method for modulating an enzyme activity. The method according to this aspect of the invention comprises contacting a cell containing an enzyme with a compound according to the invention. In some preferred embodiments, the cell is in a mammal, such as a human.

In addition to these aspects, the compounds according to the invention are useful as primary phosphoryl energy donors, as agonists or antagonists of specific metabolic signaling pathways, as in toto effector molecules and as precursors of cyclic AMP, cyclic GMP, cyclic CMP, cyclic UMP, cyclic TMP, and other similar cyclic phosphate modifications of altered nucleoside bases of A, C, G, U and T. In addition, as analogs of the bodily compounds (Np_nN) compared with other synthetic therapeutics, the potential products of degradation of the dinucleoside polyphosphate analogs will also be analogs of bodily compounds and as such are expected to have minimal undesirable side effects.

BRIEF DESCRIPTION OF THE DIAGRAMS

Figure 1 shows time course of AdOOpOpCH₂OpOpOAdO hydrolysis catalyzed by narrow-leaved lupin Ap₄A hydrolase monitored by (a) HPLC and (b) chromatography of standard nucleotides.

Figure 2 shows a comparison of binding and modes of reactivity of dinucleotides 3, 4, and 5 by the asymmetrically-acting Ap₄A hydrolases, (a) Major cleavage of 3 to β,γ- methyleneoxy-ATP; (b) minor cleavage of 3 to β,γ-oxymethylene-ATP; (c) frame-shift cleavage of 5 to α,β-oxymethylene-ADP; (d) stability to frame-shift cleavage of 4.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to novel synthetic dinucleoside polyphosphate analogs and their use as therapeutic compounds and/or diagnostic tools for the treatment and diagnosis of various human diseases and metabolic disorders. The references cited herein reflect present knowledge in the art, and are hereby incorporated by reference in their entirety. Any conflicts between the cited references and this specification shall be resolved in favor of the latter.

In a first aspect, the invention provides compounds having structural formula I

Wherein X and X' are nucleosides or modified nucleoside groups, deoxynucleoside groups or seconucleoside groups each independently selected from adenosin-5'-yl cytidin-5'-yl, 2,6- diaminopurineribosyl-5'-yl, inosin-5'-yl, guanosin-5'-yl, thymidin-5'-yl, uridin-5'-yl, ethenoadenosin-5'-yl, or other heterocyclic, cyclic or non-cyclic chemical groups attached to a sugar selected from D-ribose, 2-deoxy-D-ribose, 2,3-dideoxy-D-ribose, morpholino-, 2- and/or 3-OMe-ribose, 2- and/or 3-OEt-ribose, 2- and/or 3 -F, -Br, -Cl, -N₃ 2- and/or 3-deoxy- D-ribose, D-arabinose, D-glucose or any other closed ring, open or seco-sugar derivative, or analog thereof, and where X and X' can be the same or different nucleosides or nucleoside analogs. Each A is independently selected from =0, =S, =NH, and other divalent atoms, charged or neutral chemical groups, each B is independently selected from -O^", -S^", -NH₃ ⁺, short alkyl, and other charged or neutral chemical groups bonded to the phosphorus atom by a single covalent bond. Each of Y¹, Y², Y³ is separately selected from

monohalomethylene, dihalomethylene, carboxymethylene, alkylcarboxymethylene, aralkylcarboxymethylene, phosphonomethylene, sulfonomethylene, 1 ,2-ethylene, 1,1 -ethylene, 1,1 -propylene, 1 ,2-propylene, oxy(halo)methylene, halomethyleneoxy, thiomethylene, thiomethyleneoxy, and other linear and other divalent atoms charged or neutral divalent chemical groups serving as a bridge between two adjacent phosphorus atoms, k, 1, m, and n are integral numbers from 0 to 5; where the sum of (k+l+m+n) is > 2 and < 7.

In some preferred embodiments, the compounds according to the invention have a structure selected from

wherein the two X moieties can be the same or different nucleoside groups or nucleoside analog groups.

In some preferred embodiments, the compounds according to the invention are abbreviated herein as having the structures

AdoOpCH₂OpOCH₂pOAdo (1), AdoOpOCH₂pCH₂OpOAdo (2), AdoOpOpCH₂OpOpOAdo (3), AdoOpCH₂OpOpOCH₂pOAdo (4), AdoOpOCH₂pOpCH₂OpOAdo (5), AdoOpOpOCH₂pCH₂OpOpOAdo (6), NOpOpCH(COOH)pOpON' (37), N0p0pCH(C00CH₂Ph)p0p0N' (38), N0p0pCCl(P0₃H)p0p0N' (39), N0p0pCH₂p0p0N' (40), NOpsOpCH(COOH)pOpsON' (41), NOpsOpCH(COOCH₂Ph)pOpsON' (42), NOpsOpCCl(PO₃H)pOpsON' (43), and NOpsOpCH₂pOpsON' (44) [wherein each N and N' can be the same or different nucleosid- 5'-yl or nucleoside analog moieties, Ado is adenosin-5'-yl, p is a divalent >PO₂ moiety, ps is a divalent >POS moiety, and Ph is a phenyl group].

Alternatively, these compounds may be represented by the structural formulae

The present invention also includes pharmaceutically acceptable salts and prodrugs of compounds of the invention. The term "prodrug" is intended to represent covalently bonded carriers, which are capable of releasing the active ingredient when the prodrug is administered to a mammalian subject, or to a fungal cell. Release of the active ingredient occurs in vivo. Prodrugs can be prepared by techniques known to one skilled in the art. These techniques generally modify appropriate functional groups in a given compound. These modified functional groups however regenerate original functional groups by routine manipulation or in vivo. Prodrugs of compounds of the invention include compounds wherein a hydroxy, amino, carboxylic, phosphate, or a similar group is modified. Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives), carbamates (e.g., N,N-dimethylaminocarbonyl) of hydroxy or amino functional groups, amides (e.g., trifluoroacetylamino, acetylamino, and the like), phosphonamides (e.g. phosphonamides from natural and non-natural α-amino acid esters) and the like. In a second aspect, the invention provides a pharmaceutical formulation comprising a compound according to the invention and a pharmaceutically acceptable diluents, carrier or excipient.

The term "pharmaceutically acceptable carrier" is intended to mean a non-toxic material that is compatible with a biological system in a cell, cell culture, tissue sample or body and that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Thus, compositions according to the invention may contain, in addition to the inhibitor and antifungal agent, diluents, excipients, fillers, salts, buffers, stabilizers, solubilizers, and/or other materials well known in the art. Examples of the preparation of pharmaceutically acceptable formulations are described in, e.g., The Science and Practice of Pharmacy, 20^th Edition, ed. A. R. Gennario, Lippincott, Williams, and Watkins, Baltimore, MD, 2000.

The term "pharmaceutically acceptable salt" is intended to mean a salt that retains the desired biological activity of a compound of the present invention and exhibits minimal or no undesired toxico logical effects. Examples of such salts include, but are not limited to acid addition salts formed with inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like, and salts formed with organic acids, such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, benzenesulfonic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. The compounds can also be in the form of pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula -NR₃ ⁺ + Z^", wherein R is hydrogen, alkyl, or benzyl, and Z is a counter anion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methanesulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamate, mandelate, phenylacetate, and diphenylacetate). As used herein, the term "salt" is also meant to encompass complexes, such as with an alkaline metal or an alkaline earth metal.

The active compounds of a composition of the invention are included in the pharmaceutically acceptable carrier in an amount sufficient to deliver an effective desired amount without causing serious toxic effects to an individual to which the composition is administered.

In a third aspect, the invention provides a diagnostic method for detecting atherosclerotic lesions. The method according to this aspect of the invention comprises administering to a patient suspected of having an atherosclerotic lesion a labeled compound according to the invention, allowing the labeled compound to bind to the atherosclerotic lesion and detecting the labeled compound at the site of the atherosclerotic lesion. The compounds according to the invention provide the possibility of higher specific labeling and detection of atherosclerotic lesions, with earlier recognition of such damage in arteries of the cardiac, cerebral, renal, and other circulatory pathways. In some preferred embodiments, the compounds are labeled either fluorescently or radioactively.

In a fourth aspect, the invention provides a therapeutic method for treating a disease or disorder associated with blood disorders. The method according to this aspect of the invention comprises administering to a patient having a blood disorder or a disease associated with a blood disorder a therapeutically effective amount of a compound or pharmaceutical composition according to the invention.

One of the most common nucleoside polyphosphates, ADP, adenosine diphosphate, plays a primary role in aggregation of blood platelets, at the top of a cascade of sequential reactions culminating in formation of a thrombus (or clot), which occurs within or outside of blood vessel walls and therefore dinucleoside polyphosphates and their analogs can play significant roles as regulators of blood clotting or as therapeutics for treatment of blood related disorders.

The term "effective amount" as employed herein is an amount of a compound of the invention that achieves the effect which is intended with its application. The amount of a compound of the invention which constitutes an "effective amount" will vary depending on the compound, the intended use, the disease state and its severity, the age of the patient to be treated, and the like. The effective amount can be determined routinely by one of ordinary skill in the art.

The term "patient" as employed herein for the purposes of the present invention includes humans and other animals, particularly mammals. Thus, the compounds, compositions and methods of the present invention are applicable to both human therapy and veterinary applications. In a preferred embodiment the patient is a mammal, and in a most preferred embodiment the patient is human.

The terms "treating" or "treatment" as used herein covers the treatment of a disease or disorder in an animal, preferably a mammal, including a human, and includes at least one of: (i) preventing the disease-state from occurring in the animal, in particular, when such animal is predisposed to the disease or disorder but has not yet been diagnosed as having it; (ii) inhibiting the disease or disorder, i.e. arresting its development; and (iii) relieving the disease or disorder, i.e. causing regression of the disease or disorder. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by one of ordinary skill in the art.

Preferred diseases or disorders include, without limitation, hemophilia, thromboses, leukemia, multiple myeloma, malignant melanoma, and other forms of cancer.

Administration of the compounds or formulations according to the invention may be administered by any suitable route, including, without limitation, parenterally, intravenously, or by local implantation or inhalation.

In a fifth aspect, the invention provides a method for modulating an enzyme activity. The method according to this aspect of the invention comprises contacting a cell containing an enzyme with a compound according to the invention. In some preferred embodiments, the cell is in a mammal, such as a human.

As further described above, in some specific embodiments, the invention provides novel diadenosine polyphosphate analogs with oxymethylene bridges replacing one or more interphosphorus oxygens in the polyphosphate chain (compounds 1-6). The invention also provides six other novel compositions, in which the interphosphorus oxygen in polyphosphates of diadenosine polyphosphates are replaced with -CHF-, -CH(COOH)-, - CH(CO₂CH₂Ph)-; -CCl(PO₃H₂)- , -CH₂- chemical groups (Compounds 37-44), as well as novel compounds 43-48 with modifications at internucleoside phosphates and the adenosin- 5'-yl moiety or an analog thereof

In addition to the various aspects of the invention described above, the compounds according to the invention may serve as traffic signals, with a green light opening avenues to rich metabolic areas. They may act as agonists or antagonists of specific metabolic signaling pathways or may substitute for nucleoside di-, tri-, tetra- and higher poly-phosphates, as primary phosphoryl energy donors, in specific signal transduction pathways dictated by the structural features of enzymatic catalysts.

Dinucleoside polyphosphates (Np_nN 's; where N and N' are nucleosides and n = 3-6 phosphate residues) are naturally occurring compounds that may act as signaling molecules. One of the most successful approaches to understanding their biological functions has been the use OfNp_nN' analogs. Here, we present results of studies using novel diadenosine polyphosphate analogs, having an oxymethylene group replacing one or two bridging oxygen(s) in the polyphosphate chain. These have been tested as potential substrates and/or inhibitors of the symmetrically-acting Ap₄A hydrolase (EC 3.6.1.41) from E. coli and of two asymmetrically-acting Ap₄A hydrolases (EC 3.6.1.17) from humans and narrow-leaved lupin.

The following examples are intended to further illustrate certain preferred embodiments of the invention and are not to be construed as limiting the scope of the invention. Enzymes

Homogeneous recombinant asymmetrically-acting human Ap₄A hydrolase (EC 3.6.1.17) [Thorne et al. 1995] was kindly provided by Professor A.G. McLennan and the enzyme from narrow- leaved lupin (Lupinus angustifolius) [Maksel et al. 2001] by Drs. D. Maksel and K. Gayler (University of Melbourne, Australia). Symmetrically acting Ap₄A hydrolase (EC 3.6.1.41) was partially purified from E. coli [Guranowski et al. 1983]. Chemicals

Unlabelled mono- and dinucleotides were from Sigma (St. Louis, MO, USA), and [³H]Ap₄A (740 TBq/mol) was purchased from Moravek Biochemicals (Brea, CA, USA).

Chromatographic systems

Analyses of the hydrolysis OfAp₄A and its analogs were performed on TLC aluminum plates precoated with silica gel containing fluorescent indicator; (Merck Cat. No. 5554) and developed in dioxane/ammonia/water (6:1 :4, by volume).

Enzyme assays

Estimation of the reaction rates and calculation of the K₁ values for the analogs with

the use of radiolabeled Ap₄A were performed as described earlier [Guranowski et al. 2003]. Relative rates of the hydrolysis of dinucleotide substrates and analogs were estimated by the use of HPLC on a reverse-phase column (for details see legend to Fig. 4a) and were based on peak-area analysis. Synthesis of oxymethylene and methyleneoxy analogs of ADP, ATP and ApnA. ADP, ATP and Ap_nA analogs with one -OCH₂- or -CH₂O- group that substitutes for a bridging oxygen in adenosine or diadenosine oligophosphates have not been described previously. The tripolyphosphate analog, -pOCH2pCH2Op-, has been bound to two adenosines, yielding an analog of AP₃A [Walkowiak et al. 2002], but hitherto similar analogs OfAp₄A or Ap₅A have not been made. We have prepared α,β-methyleneoxy-ADP (OpOCH₂pOAdo) (21) and α,β-oxymethylene-ADP (OpCH₂OpOAdo) (24), α,β- methyleneoxy-ATP (pOpOCH₂pOAdo), α,β-oxymethylene-ATP (OpOpCH₂Op)Ado), α,β-oxymethylene-ATP (OpCH₂OpOpOAdo) (18), the unstable α,β-methyleneoxy-ATP (OpOCH₂pOpOAdo) (32), and the six Ap_nA analogs investigated in this study: α,β-bis(methyleneoxy)-Ap₃A (AdoOpCH₂OpOCH₂pOAdo) (1), α,β-bis(oxymethylene)-Ap₃A (AdoOpOCH₂pCH₂OpOAdo) (2), β,β'-methyleneoxy-Ap₄A (AdoOpOpCH₂OpOpOAdo) (3), α,β-bis(methyleneoxy)-Ap₄A (AdoOpCH₂OpOpOCH₂pOAdo) (4), α,β-bis(oxymethylene)-Ap₄A (AdoOpOCH₂pOpCH₂OpOAdo) (5) and β,γ-bis(oxymethylene)-Ap₅A (AdoOpOpOCH₂pCH₂OpOpOAdo) (6). The terminology used supports recognition of the orientation of oxygen- and methylene- components of the oxymethylene bridges in the analogs with respect to their adenosine moieties. Example 1: AdoOpCH₂OpOCH₂pOAdo (1) Monobenzyl phosphonate 8 [Rejman et al. 2006] was esterifϊed with tetrabenzoyladenosine 7 [Rejman et al. 2006] using either 2- chloro-5,5-dimethyl-2-oxido-l,3,2-dioxaphosphinane (NEP)/methoxypyridine-Λ/- oxide/pyridine system [Himmelsbach et al. 1984, Stengele et al. 1990, Rejman et al. 2004] or Mitsunobu conditions (Scheme 1). The DMTr group of phosphonate 9 was removed with acetic acid giving compound 10. Phosphoramidite generated by the reaction of phosphonate 10 with benzyloxybis(diisopropylamino)phosphine [Bannwarth et al. 1987] reacted with a second molecule of 10 to produce the fully protected symmetrical Ap₃A analog 11.

Target compound 1 was obtained by two-step deprotection and DEAE Sephadex column chromatography using a linear gradient of TEAB in water. Benzyl esters were removed by catalytic hydrogenation followed by aqueous ammonia treatment to remove benzoyl protecting groups.

Example 2: AdoOpOCH₂pCH₂OpOAdo (2) Tetrabenzoyladenosine 7 was converted into phosphoramidite 12 by reaction with benzyloxybis(diisopropylamino)phosphine [Bannwarth et al. 1987] (Scheme 2). Phosphoramidite 12 underwent reaction with benzyl bis(hydroxymethane)phosphinate 13 providing fully protected symmetrical Ap₃A analog 14. Final compound 2 was obtained by two-step deprotection and DEAE Sephadex column chromatography using a linear gradient of TEAB in water.

Example 3: AdoOpOpCH₂OpOpOAdo (3) The tributylammonium salt of AMP (15) was reacted with phosphomorpholidate 16 in DMSO. Dibenzyl ester 17 was hydrogenolyzed to give ATP analog 18, purified by DEAE-Sephadex chromatography. Reaction of 18 with AMP morpholidate 19 led to target product 3 after DEAE Sephadex column chromatography, using a linear gradient of TEAB in water.

Example 4: AdoOpCH₂OpOpOCH₂pOAdo (4) Adenosine phosphonate 10 (vs.) was reacted with bis-benzyloxy-(diisopropylamino)phosphine [Bannwarth et al. 1987] with tetrazole catalysis and, after MCPBA oxidation, afforded compound 20 (Scheme 4). Compound 20 was debenzylated by catalytic hydrogenolysis and dimerized using DCC in pyridine. Target compound 4 was obtained pure by DEAE Sephadex column chromatography.

Example 5: AdOOpOCH₂POpCH₂OpOAdO (5) Phosphoramidite 12 was reacted with dibenzyl phosphonate 22 using tetrazole catalysis and, after MCPBA oxidation, afforded compound 23 (Scheme 6). ADP analog 24, obtained by catalytic hydrogenation of 23, was dimerized using DCC in pyridine giving, after DEAE Sephadex column chromatography, target Ap₄A analog 5.

p p p p p Benzyl phosphinate 13, after treatment with bis-benzyloxy-(diisopropylamino)phosphine [Bannwarth et al. 1987] using tetrazole catalysis and MCPBA oxidation, gave compound 25 (Scheme 5). Catalytic hydrogenation of 25 gave bis(hydroxymethylenephosphinic acid) phosphate 26, which underwent condensation with morpholidate 19 to give, after DEAE Sephadex column purification, the target Ap₅A analog 6.

Example 7: pOCH₂pOpOAdo (32) Bis(2-cyanoethyloxy)(diisopropylamino)- -phosphine (27) [Bannwarth and Trzeciak, 1987] was reacted with dibenzyl phosphonate 22 and subsequently with benzyl alcohol. After MCPBA oxidation and catalytic hydrogenation cyanoethyl pyrophosphate analog 30 was obtained (Scheme 7). Pyrophosphate analog 30 underwent standard reaction with adenosine 5'-phosphoromorpholidate 19. After aqueous ammonia deprotection of the cyanoethyl group and DEAE Sephadex column purification, the target ATP analog 32 was obtained.

Example 8: Benzyl bis-(hydroxymethane)-phosphonate (13)

Bis(hydroxymethane)phosphinic acid 33 [Pirat et al. 2002] was reacted with dimethoxytrityl chloride in pyridine to give 34, which was subsequently esterified with benzyl alcohol employing NEP/methoxypyridine-N -oxide/pyridine system [Himmelsbach et al. 1984, Stengele et al. 1990, Rejman et al. 2004]. The benzyl ester 35 obtained was detritylated with

80% aqueous acetic acid to give 13.

Example 9: [Bis(benzyloxy)phosphoryl] methyl hydrogen morpholinophosphonate (16)

Morpholinophosphonic dichloride 36 [Nunez et al. 1987] was treated first with one equivalent of water in pyridine to afford a reactive species that subsequently underwent reaction with dibenzyl hydroxymethanephosphinate 24 to afford the desired reagent 16.

The preparations OfAp_nA and ATP analogs described above employed two main synthetic approaches. Phosphoramidite condensations appeared as the ideal method and gave excellent yields. Phosphoromorpholidate condensation proved to be an alternative method and gave moderate to good yields. While DCC couplings appeared useful, in general they gave lower yields. Using the combination of base-labile benzoyl and hydrogenolytically- removable benzyl groups proved to be compatible with rather unstable polyphosphate products. The structures of all compounds prepared were established by a combination of ¹H and ³¹P NMR and high resolution mass spectroscopy (data not shown). Example 10: Recognition Of Ap_nA oxymethylene analogs by Ap₄A hydrolases

Of various Ap_nA analogs investigated so far as potential substrates and/or inhibitors of specific Ap₄A hydrolases, those with modifications in the polyphosphate chain have been studied most often [Guranowski et al., 1987, Guranowski et al. 1989, McLennan et al. 1989]. Some of them are substrates of the asymmetrically-acting Ap₄A hydrolases from yellow lupin seeds [Guranowski et al. 1987, Guranowski et al. 1989,] and Artemia embryos [McLennan et al. 1989]. AdoOpOpCH₂pOpOAdo and AdoOpCH₂pOpOAdo were hydrolyzed 20- to 50-fold more slowly and AdoOpOpCF₂pOpOAdo, AdoOpOpCHFpOpOAdo, AdoOpOpCHBrpOpOAdo and AdoOpOpCHClpOpOAdo 1.4- to 9-fold more slowly than Ap₄A. As was observed for a series of β,β'-substituted Ap₄A analogs, their efficiencies as substrates of the Ap₄A hydrolase from Artemia increased in direct proportion to increasing electronegativity [McLennan et al. 1989]. Guranowski et al. [Guranowski al. 1989] found that those compounds were not substrates of the symmetrically-acting Ap₄A hydrolase from E. coli but later work by McLennan et al. [McLennan et al. 1989] reported that AdoOpOpCH₂pOpOAdo, AdoOpOpCF₂pOpOAdo and AdoOpOpCHFpOpOAdo underwent slow hydrolysis, using their preparation of bacterial enzyme, with 25-, 50-, and 125-fold reduced rate, respectively, compared to that OfAp₄A hydrolysis. We asked how specific Ap₄A hydrolases might recognize substrate analogs that are non-isosteric (the P-P distance is one atom longer) but are isoelectronic (charge identical) in comparison with natural Ap_nAs. To answer this question, we performed studies on the interaction of the enzymes with aforementioned oxymethylene analogs OfAp_nA. We have found that none of the six new Ap_nA analogs is a substrate of the symmetrically-acting Ap₄A hydrolase, by incubating each of the analogs for up to 16 h with an amount of enzyme sufficient to cleave completely equimolar (0.5 niM) Ap₄A in less than 15 min (analyzing the reaction mixtures in the TLC system that separates each of the analogs tested as potential substrate from possible reaction products). This result is consistent with earlier work [Blackburn et al. 1981, Guranowski et al. 1987, Guranowski et al. 1989, McLennan et al. 1989] that established that the hydrolase from E. coli does not split dinucleoside polyphosphate molecules modified in their ADP-moieties. In addition, none of the oxymethylene analogs investigated inhibited the hydrolysis OfAp₄A catalyzed by the E. coli enzyme. As was shown earlier [Guranowski et al. 1987, Guranowski et al. 1989, McLennan et al. 1989], some methylene or halomethylene analogs OfAp₄A inhibited that bacterial enzyme quite effectively, with K_iS even one order of magnitude lower than the K_m for Ap₄A [Guranowski et al. 1987]. This study thus establishes that the symmetrical Ap₄A hydrolase does not tolerate single (i.e. compound 3) or multiple (i.e. compounds 1, 2, 4-6) atom inserts into the polyphosphate backbone of the six dinucleoside oligophosphate analogs. By contrast, when the same six novel Ap_nA analogs were tested as potential substrates of the asymmetrically-acting Ap₄A hydrolases, compounds 3 and 5 were readily hydrolyzed (Table 1). This was demonstrated both for the human and plant enzymes and the reaction products were clearly identified by comparing them with AMP and the corresponding synthetic oxymethylene analogs of ADP or ATP. In addition to TLC analysis, we also used an HPLC system (see the example of elution profiles in Figs. Ia and Ib) that effectively separated potential substrates from possible products and thus could be used for estimation of relative velocities of the hydrolysis reactions. The asymmetric analog 3 was hydrolyzed by both asymmetric hydrolases to AMP and the β,γ-methyleneoxy-ATP (32) (Fig. 2a), and then the latter, relatively unstable, nucleotide more slowly cleaved spontaneously to give a second AMP.

Table 1. Comparison of the hydrolysis OfAp₄A and its oxymethylene analogs, catalyzed by two asymmetrically-acting Ap₄A hydrolases.

Velocities calculated from time-course of decrease of substrate -peak area as shown on the HPLC profiles exemplified in Fig. Ia. Arrows above substrate formulas indicate sites of cleavage. Compound 3 was degraded by the human hydrolase 6 times faster, and by the lupin enzyme 20 times faster to AMP and OpOCH₂pOpOAdo (large arrow) than to AMP and OpCH₂OpOpOAdO (small arrow).

** **

Profiles shown in Fig. Ia are for reaction mixtures (0.1 ml) containing 50 mM Hepes/KOH (pH 7.6), 0.02 mM dithiothreitol, 5 mM MgCl₂, 0.5 mM substrate, and rate- limiting amounts of the asymmetrically acting Ap₄A hydrolase - incubated at 30⁰C. At time intervals (0, 5, 10, 15 and 20 min), 10 μl aliquots were withdrawn, added to 0.15 ml of 0.1 M KH₂PO₄ (pH 6.0) and the reaction heat-quenched (3 min at 96°C). After centrifugation, samples were filtered and aliquots (0.1 ml) subjected to HPLC on a Discovery C18 column (4.6 x 250 mm, 5 μm; Supelco); flow rate 1 ml min^-1. Gradient elution was performed with 0.1 M KH₂PO₄, pH 6.0, (solvent A); solvent A: methanol (9:1, v/v); (solvent B): 0-9 min, 0% B; 9-15 min, 25% B; 15-17.5 min, 90% B; 17.5-19 min, 100% B; 19-23 min, 100% B and 23-35 min, 0% B. Profiles in Fig. Ib show standards run under identical conditions. An alternative cleavage of analog 3 to α,β-oxymethylene-ATP (18) and AMP was also observed. For the human asymmetric hydrolase this mode of cleavage was approximately six times less frequent than the dominant mode and in case of the lupin enzyme it was over 20 times slower. Such slower cleavage to give 18 could arise either from weaker binding of 3 in the active site of the hydrolase in the reverse orientation (Fig. 2b) or from a reduced rate of cleavage. While the pk_as for the ATP analogs released, 32 and 18 respectively, have not yet been determined, it is reasonable to assume that pK_a4 for 32 is similar to that of ATP {ca. 7.1) while that for 18 will be similar to that of β,γ-methylene-ATP (ca. 8.2. [Guranowski et al. 1994]). The asymmetrical pyrophosphohydrolase from Artemia is known to exhibit a strong dependence on the rate of cleavage on the pK_a of the leaving group (Brønsted coefficient, β_ig 0.5 [McLennan et al. 1989]). A similar β_ig dependence for the human and lupin enzymes studied here would lead to a reduction in rate of about 10-fold for formation of 18 relative to that of 32. Thus, the present kinetic results do not provide any evidence for differential recognition of the alternative orientations on the P-O-C-P bridge for these two enzymes.

The enzymatic hydrolysis of symmetrical analog 5 by both human and plant asymmetric hydrolases yielded only α,β-oxymethylene-ADP (24) and at rates reduced relative to cleavage of 3 (Table 1). This mode of cleavage is a further example of a frame- shift mechanism (Fig. 2c), akin to that shown in the action of the asymmetrical Artemia hydrolase on some α,β-disubstituted analogs OfAp₄A (e.g., ApCHFpOpCHFpA was cleaved at 3% of the rate of AppppA) [McLennan et al. 1989]. It constitutes a symmetrical mode of cleavage of 5 by water attack at Pβ. The failure of these hydrolases to bring about a similar frame-shift symmetrical hydrolysis of 4 is remarkable (Fig. 2d). It appears to indicate that there is specific recognition of the orientation of the P-O-CH₂-P linkage in the β-active site. Taken together, the results of cleavage of compounds 3 and 5 show that the asymmetrically- acting Ap₄A hydrolases can reach the scissile bond either by extending "the frame" relative to Ap₄A, as in the case of compound 3, or by shortening the count, when attacking the Pβ-O- Pβ' bond of compound 5.

As has been established earlier [Guranowski, 2000], the hydrolases do not recognize dinucleoside triphosphates as substrates. Thus it was to be expected that the oxymethylene analogs OfAp₃A, compounds 1 and 2, would not be degraded. The absence of any detectable hydrolysis of compounds 4 and 6 suggests that the enzymes tolerate neither a -CH₂-P_α- sequence, which occurs in 4, nor a -CH₂-P-CH₂- as in 6. Apparently "the frame shift" is unable to accommodate two oxymethylene inserts, as occur in 6.

Finally, we have investigated whether the novel Ap_nA analogs inhibit Ap₄A hydrolysis catalyzed by the asymmetrically-acting Ap₄A hydrolases. Only analogs 3 and 4 acted as competitive inhibitors; with K₁S 2.2 μM (3) and 1.5 μM (4) for the lupin enzyme and 2.1 μM (3) and 2.5 μM (4) for the human counterpart. These K_i values lie in the range of the K_m values for Ap₄A: 2.5 μM for the narrow-leaved lupin [Maksel et al. 2001] and 2 μM for the human enzyme [Guranowski et al. 2003].

The results of binding and cleavage studies on the six Ap_nA analogs described here by the three pyrophosphohydrolases establish the general utility and the limitations of the P-O- CH₂-P bridge as a surrogate for pyrophosphate in nucleotides. First, none of the three enzymes can cleave the P-O bond in the P-O-CH₂-P linkage, which can be attributed to the change in leaving group from an anionic phosphate oxygen to an alcohol. Second, the asymmetric cleaving enzymes accept the P-O-C-P bridge in the position adjacent to the P-O- P cleavage locus in either orientation. Third, hindrance of normal P-O-P cleavage can lead to a frame-shift response, which is facilitated by the extra methylene insertion, but only for one orientation of the P-O-CH₂-P insert. Lastly, the asymmetric hydrolases accept the P-O-CH₂-P inserts as competitive inhibitors while the bacterial symmetrical hydrolases does not. Thus, these novel compounds are tools of specific application for studies on the metabolism of dinucleoside polyphosphates and on Ap₄A-degrading enzymes, and they also merit further attention for the investigation of nucleotide metabolic pathways.

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Claims

What is claimed is:

1. Acompound having structural formula I:

wherein X and X' are nucleosides or modified nucleoside groups, deoxynucleoside groups or seconucleoside groups each independently selected from adenosin-5'-yl cytidin-5'-yl, 2,6- diaminopurineribosyl-5'-yl, inosin-5'-yl, guanosin-5'-yl, thymidin-5'-yl, uridin-5'-yl, ethenoadenosin-5'-yl, or other heterocyclic, cyclic or non-cyclic chemical groups attached to a sugar selected from D-ribose, 2-deoxy-D-ribose, 2,3-dideoxy-D-ribose, morpholino-, 2- and/or 3-OMe-ribose, 2- and/or 3-OEt-ribose, 2- and/or 3 -F, -Br, -Cl, -N₃ 2- and/or 3-deoxy- D-ribose, D-arabinose, D-glucose or any other closed ring, open or seco-sugar derivative, or analog thereof, and where X and X' can be the same or different nucleosides or nucleoside analogs; each A is independently selected from =0, =S, =NH, and other divalent atoms, charged or neutral chemical groups, each B is independently selected from -O^", -S^", -NH₃ ⁺, short alkyl, and other charged or neutral chemical groups bonded to the phosphorus atom by a single covalent bond; each of Y¹, Y², Y³ is independently selected from -O-, -S-, -NH-, -C(O)-CH₂-, -CH₂-CO-, monohalomethylene, dihalomethylene, carboxymethylene, alkylcarboxymethylene, aralkylcarboxymethylene, phosphonomethylene, sulfonomethylene, 1 ,2-ethylene, 1,1 -ethylene, 1,1 -propylene, 1 ,2-propylene, oxy(halo)methylene, halomethyleneoxy, thiomethylene, thiomethyleneoxy, and other linear and other divalent atoms charged or neutral divalent chemical groups serving as a bridge between two adjacent phosphorus atoms, k, 1, m, and n are integral numbers from 0 to 5; where the sum of (k+l+m+n) is > 2 and < 7.

2. The compound according to claim 1 having a structure selected from

wherein each X is independently a purine or pyrimidine nucleoside group or analog thereof.

3. The compound according to claim 1, having a structure selected from AdoOpCH₂OpOCH₂pOAdo (1), AdoOpOCH₂pCH₂OpOAdo (2), AdoOpOpCH₂OpOpOAdo (3), AdoOpCH₂OpOpOCH₂pOAdo (4), AdoOpOCH₂pOpCH₂OpOAdo (5), AdoOpOpOCH₂pCH₂OpOpOAdo (6), NOpOpCH(COOH)pOpON' (37), N0p0pCH(C00CH₂Ph)p0p0N' (38), N0p0pCCl(P0₃H)p0p0N' (39), NOpOpCHφOpON' (40), NOpsOpCH(COOH)pOpsON' (41), N0ps0pCH(C00CH₂Ph)p0ps0N' (42), NOpsOpCC^POs^pOpsON' (43), and N0ps0pCH₂p0ps0N' (44), wherein each N and N' can be the same or different nucleosid-5'-yl or nucleoside analog moieties, Ado is adenosin-5'-yl, p is a divalent >PO₂ ^~ moiety, ps is a divalent >POS^~ moiety, and Ph is a phenyl group.

4. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable diluent, carrier or excipient.

5. A diagnostic method for detecting an atherosclerotic lesion comprising administering to a patient suspected of having an atherosclerotic lesion a labeled compound according to claim 1 , allowing the labeled compound to bind to the atherosclerotic lesion and detecting the labeled compound at the site of the atherosclerotic lesion.

6. The method according to claim 5, wherein the atherosclerotic lesion is in a cardiac, cerebral, pulmonary or renal artery.

7. A method for therapeutically treating a disease or disorder associated with blood disorders comprising administering to a patient having a blood disorder or a disease associated with a blood disorder a therapeutically effective amount of a compound or pharmaceutical composition according to claim 1.

8. The method according to claim 7, wherein the disease or disorder is selected from hemophilia, thromboses, leukemia, multiple myeloma, malignant melanoma, and other forms of cancer.

9. A method for modulating an enzyme activity comprising contacting a cell containing an enzyme with a compound according to claim 1

10. The method according to claim 9, wherein the cell is in a human.