WO2012032513A1 - Boranophosphate derivatives for the treatment of osteoarthritis - Google Patents

Boranophosphate derivatives for the treatment of osteoarthritis Download PDF

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WO2012032513A1
WO2012032513A1 PCT/IL2011/000713 IL2011000713W WO2012032513A1 WO 2012032513 A1 WO2012032513 A1 WO 2012032513A1 IL 2011000713 W IL2011000713 W IL 2011000713W WO 2012032513 A1 WO2012032513 A1 WO 2012032513A1
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hydrocarbyl
independently
pharmaceutical composition
methyl
alkyl
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PCT/IL2011/000713
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French (fr)
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Bilha Fischer
Jean Sevigny
Shay Eliahu
Joanna Lecka
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Bar-Ilan University
Universite Laval
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    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06Pyrimidine radicals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00Drugs for skeletal disorders
    • A61P19/02Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16Purine radicals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

  • the present invention relates to pharmaceutical compositions and methods for treatment and management of osteoarthritis.
  • Nucleoside triphosphate diphosphohydrolase-1 , -2, -3 and -8 (NTPDasel , -2, -3 and -8; EC 3.6.1 .5) and nucleotide pyrophosphatase phosphodiesterase- 1 and 3 (NPP 1 and NPP3 ; EC 3.1 .3.1 , EC 3.6.1.9) are the dominant ectonucleotidases that terminate nucleotide signaling through the hydrolysis of nucleotide agonists of the P2X and P2Y receptors (Kukulski et al, 2005; Nahum et al, 2006; Shirley et al, 2009).
  • NTPDasel , -2, -3 and -8 are plasma membrane-bound with an extracellular active site, which catalyze the hydrolysis of the terminal phosphate of nucleoside triphosphates, e.g., ATP and UTP, and diphosphates, e.g., ADP and UDP, at different rates.
  • nucleoside triphosphates e.g., ATP and UTP
  • diphosphates e.g., ADP and UDP
  • NTPDasel (CD39/ATPDase/ectoapyrase/ecto-ADPase) hydrolyzes ATP and ADP equally well (Sevigny et al , 1997), while NTPDase2 (ecto-ATPase/CD39Ll ) is a preferential triphosphonucleosidase (Heine et al, 1999). Both NTPDase3 (CD39L3/HB6) and NTPDase8 are functional intermediates between NTPDasel and NTPDase2 (Kukulski et al , 2005). NTPDase4-7, are mainly associated with intracellular organelles and are therefore not expected to significantly affect P2 receptor activation.
  • AMP NTPDase activity
  • CD73 ecto-5 '-nucleotidase
  • NPP family members are conserved eukaryotic enzymes which, as for NTPDases, exist as membrane glycoproteins with an extracellular active site.
  • Three members of this family, in particular, NPP1 -3, are capable of hydrolyzing phosphodiester and pyrophosphate bonds found in a variety of endogenous nucleotides and their derivatives, e.g., nucleotide triphosphates (NTPs), nucleotide diphosphates (NDPs), dinucleotides, oligonucleotides, nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) and uracil diphosphate (UDP) sugars (Bollen et al.
  • NPP 1 can hydrolyze both phosphodiester, e.g., cAMP, and pyrophosphate (PPi), e.g., ATP, bonds (Belli et al , 1993), and in the latter case hydrolysis could be performed between phosphate-a and phosphate-/? (Stefan et al , 2005).
  • PPi pyrophosphate
  • NPP l and NPP3 are closely related, with -50% identity, and share 39% and 41 % identity, respectively, with NPP2 (Deissler et al , 1 995).
  • NPP2 has a much lower capacity to hydrolyzc nucleotides than NPP l and NPP3 , and therefore may not play an important role in the regulation of P2 receptor activation.
  • NPP l is a membrane protein consisting of 925 amino acids organized into six main domains including an N-terminus cytoplasmic tail, a transmembrane domain, an extracellular region, a phosphodiesterase domain and a nuclease domain (Stefan et al , 2005).
  • the catalytic site of NPP l is located in the extracellular phosphodiesterase domain.
  • Gijsbers et al. (2001 ) proposed a structural model and a catalytic reaction mechanism for mouse NPPs based on secondary structure similarities to known crystal lographic structures of alkaline phosphatase, independent phosphoglycerate mutase and arylsulfatase.
  • a substrate e.g., ATP
  • one of its negatively charged oxygens can partially coordinate both binding site di-valent metal ions, thereby bringing the phosphate group into close proximity with the nucleophilic oxygen of Thr238, which can then hydrolyze the ATP molecule to generate a protein- nucleoside mono-phosphate adduct and release PPi.
  • AM P can be easily released from the protein- AMP adduct through hydrolysis occurring by an active site water molecule.
  • Zalatan et al. (2006) determined the structure of the bacterial NPP Xanthomonas axonopodis pv.
  • NPP l is expressed in different tissues, especially in bone (osteoblasts) and cartilage (chondrocytes), and has a role in regulating skeletal remodeling and calcification.
  • NPP l affects skeletal remodeling and calcification by regulating processes such as bone mineralization and soft tissue calcification.
  • NPP l The primary role of NPP l is to regulate extracellular PPi levels thereby contributing to the balance between the extracellular levels of phosphate (Pi) and PPi that is a key factor in mineralization process ( Stefan et al , 2005).
  • phosphate Pi
  • PPi phosphate
  • PPi PPi
  • TNAP tissue-nonspecific alkaline phosphatase
  • ANK progressive ankylosis protein
  • ATPases specific for ATP
  • pyrophosphatases specific for PPi
  • US 7,368,439 discloses diribo-, di-2'-deoxyribo, and ribo-2'-deoxyribo-nucleoside boranophosphate derivatives that can be useful for prevention or treatment of diseases or disorders modulated by P2Y receptors such as type 2 diabetes, cystic fibrosis and cancer.
  • WO 2009/066298 discloses non-hydrolyzable adenosine and uridine polyphosphate derivatives, said to be useful for prevention or treatment of diseases modulated by P2Y- receptors such as type 2 diabetes.
  • the present invention provides a pharmaceutical composition for treatment of osteoarthritis comprising a pharmaceutically acceptable carrier and either a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II:
  • X and X' each independently is an adenine residue of the formula la, linked through the 9 -position:
  • Ri is H, halogen, -O-hydrocarbyl, -S -hydro carbyl, -NR 4 R 5 , heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 2 , -OR 4 , -SI 4 , -NR4R5 or heteroaryl, wherein R 4 and R 5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
  • R 2 and R 3 each independently is H or hydrocarbyl
  • X and X' each independently is an uracil residue of the formula lb, linked through the 1 -position:
  • R 6 is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -N RXRQ, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 2 , -OR$, -SR 8 , -NR 8 R-9 or heteroaryl, wherein R 8 and Rt> each independently is H or hydrocarbyl, or R 8 and R9 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
  • R 7 is O or S
  • Y and Y' each independently is H, -OH or -NH 2 ;
  • Zi , Z 2 , Z 3 , Z 4 and Z s each independently is -O " , -S " or -BH 3 " , provided that at least one of Zi to Z 5 in the general formula I is -BH 3 " , and at least one of Z) to Z 3 in the general formula II is -BH 3 " ;
  • W i , W 2 , W 3 and W 4 each independently is -0-, -NH- or -C(RioRn )-, wherein R ! 0 and Ri i each independently is H or halogen, provided that at least one of W i to W 4 in the general formula I is not -0-, and at least one of W
  • n and n' each independently is 0 or 1 ;
  • n 3, 4 or 5;
  • the present invention provides a dinucleoside boranophosphatc derivative of the general formula I or a nucleoside boranophosphatc derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for use in treatment of osteoarthritis.
  • the present invention relates to use of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for the preparation of a pharmaceutical composition for treatment of osteoarthritis.
  • the present invention relates to a method for treatment of osteoarthritis in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • the present invention relates to a diadenosine boranophosphatc derivative of the general formula II I :
  • Ad is an adenine residue of the formula la, linked through the 9-position:
  • Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR 4 R 5 , heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N0 2 , -OR 4 , -SR 4 , -NR R5 or heteroaryl, wherein R 4 and R 5 each independently is H or hydrocarbyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
  • R and R each independently is H or hydrocarbyl ;
  • Y and Y' each independently is H, -OH or -NH 2 ;
  • W] , W 2 , W 3 and W 4 each independently is -0-, -NH- or -C(RioRn)-, wherein Rio and Ri i each independently is H or halogen, provided that two of W
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a diadcnosine boranophosphate derivative of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, and a pharmaceutically acceptable carrier.
  • Fig. 1 shows proposed structures for nucleotide-BPj Mg 2+ complexes leading to products 1 and 2 (upper left side) and products 3 and 4 (upper right side), wherein Im represents imidazolyl; and Nuc represents 2'-deoxy-adenosyl.
  • FIG. 2 shows the effects of analogues 1 -4 on NTPDase and eclo-5 '-nuclcotidase activity.
  • Either ATP (for NTPDases) or AMP (for ecto-5 '-nucleotidase) was used as a substrate in the presence of compound 1 (panel A), 2 (panel B), 3 (panel C), or 4 (panel D). Both substrate and analogues 1-4 were used at 100 ⁇ .
  • the 100% activity was set with the nucleotide substrate alone: 1270 ⁇ 35, 928 ⁇ 55, 202 ⁇ 37, 129 ⁇ 1 1 , and 357 ⁇ 10 nmol of Pi min " ' (mg protein "1 ) for NTPDasel , -2, -3 and -8, and ecto-5 '-nucleotidase, respectively. Data are presented as the mean ⁇ SD of 3 experiments carried out in triplicate.
  • Hgs. 3A-3D show that analogues 1 -4 inhibit NPP activities.
  • Substrates and analogues 1-4 were studied at a concentration of 100 ⁇ . In the control (ctrl), the substrate only was tested and was set to 100% of activity. The percentage of residual activity is presented at the top of each bar. Data are presented as the mean ⁇ SD of 3-6 experiments carried out in triplicate. [0019] Fig.
  • analogues 1-4 inhibit NPP activity at the surface of HTB-85 and HT29 cells.
  • Substrate, pnp-TMP, and analogues 1-4 were used at the concentration of 100 ⁇ . In the control, the substrate only was tested and was set to 100% activity. The percentage of residual activity is presented at the top of each bar. Data are presented as the mean ⁇ SD of 3 experiments performed in triplicate.
  • Figs. 5A-5B show relative concentration-response plots for analogues 1 -4 via the P2Yi i receptor (5A) and the P2Y
  • Data were obtained from 1321 N 1 cells stably expressing the P2Yu GFP receptor (5A) or P2Yi GFP receptor (5B), triggering the ligand-induced change in [Ca 2+ ]j.
  • Cells were pre-incubated with 2 ⁇ fura-2 AM for 30 min, and the change in fluorescence (AF 3 4o/F 3ii o) was monitored.
  • Fig. 6 shows that analogues 22-24 are poor substrates of human NTPDases. Bars represent the mean of one experiment performed in triplicate. The relative activity was calculated using ATP hydrolysis as 100% (white bar), which was, in nmoles Pi-min " ' -mg protein " 1 , 467 for NTPDasel ; 512 for NTPDase2; 496 for NTPDase3 ; and 192 for NTPDase8.
  • Fig. 7 shows hydrolysis of pnp-TMP and analogues 22-24 by human NPPs. Bars represent the mean of one experiment performed in triplicate. The relative activity was calculated using pnp-TMP hydrolysis as 100% (white bar), which was, in nmoles pnp- ⁇ ⁇ -min ' ' -mg protein " 1 , 24 for NPP 1 ; and 53 for NPP3.
  • Fig. 8 shows the effect of analogues 22-24 on human NTPDase activity. Bars represent the mean of one experiment performed in triplicate. ATP (substrate) and analogues 22-24 were all used at the concentration of 100 ⁇ . The ATPase activity of each NTPDase is indicated in Fig. 6.
  • Fig. 9 shows that analogues 22-24 are potent inhibitors of human NPP 1. Bars represent the mean of one experiment performed in triplicate. Pnp-TMP (substrate) and analogues 22-24 were all used at the concentration of 100 ⁇ . The activity with pnp-TMP of both NPPs is indicated in Fig. 7.
  • Nucleotide pyrophosphatase phosphodiesterase 1 -3 (NPP 1 -3) have a nucleotide pyrophosphatase activity and metabolize nucleotide triphosphate (NTP) directly to nucleotide monophosphate (NMP) and pyrophosphate (PPi) (Stefan et al. , 2006).
  • NPPs ectonucleotidases
  • NPP specific inhibitors which do not affect other ectonucleotidases such as NTPDases and 5'- ectonucleotidase and do not trigger nor interfere with P2 receptor activation, would be extremely valuable.
  • potent and selective NPP inhibitors could be used as therapeutic agents for the treatment of osteoarthritis (Tenenbaum et al , 1 981 ) and chondrocalcinosis (Johnson and Terkeltaub, 2005).
  • Nucleotide scaffolds suffer from inherent limitations as therapeutic agents as they interact with numerous proteins (Nahum et al , 2006) and are metabolically unstable (Sellers et al , 2001 ). Therefore, in the study described herein, a dinucleoside polyphosphate scaffold, which offers better stability and selectivity than nucleotides (Nahum et al , 2006), was selected for the development of NPP inhibitors, and four diadenosine polyphosphate derivatives herein identified by the Arabic numbers 1-4 in bold, more particularly, two diadenosine pentaphosphate derivatives identified as analogues 1 and 2, and two diadenosine tetraphosphate derivatives identified as analogues 3 and 4, were synthesized, taking into consideration the following points: (i) In order to prevent any activity of those derivatives toward the P2Yi receptor, the adenine ring was conserved without a mcthylthio substitution at the C-2 position, known to enhance potency toward the
  • Analogue 1 is also identified by the name diadenosine 5', 5"- P ' ,P 5 ,o; , jS-methylene-5,e-methylene-pentaphosphate-7-borano; analogue 2 is also identified by the name di-2'-deoxyadenosine 5',5"-P ' ,P 5 , k; ⁇ /3-methylene-5, 6-methylene penta- phosphate-y-borano; analogue 3 is also identified by the name (..adenosine 5',5"-? ?
  • ⁇ - methylene-7,5-methylene-tetraphosphate and analogue 4 is also identified by the name di- 2'-deoxyadenosine 5',5"-P ' ,P 5 ,o;,j3-methylene-7,5-methylene-tetraphosphate.
  • Analogues 1-4 were evaluated for their protein selectivity as either agonists of P2Y I , 2,I I receptors or substrates for the major ectonucleotidases; and their inhibitory activity and NPP subtype selectivity were evaluated by comparison of their effects on the other main ectonucleotidases, in the presence of pnp-TMP, Ap 5 A or ATP as substrates.
  • these analogues were evaluated as inhibitors of cell surface NPP activity in two cancer cell lines. As described hereinafter, based on the various experiments conducted, a most selective NPP inhibitor was identi fied, and important structure-activity relationships for such inhibitors was established.
  • analogues 1-4 strongly inhibited the metabolism of both synthetic (pnp-TMP) and natural substrates (Ap 5 A and ATP) by
  • NPP 1 NPP 1 . Additionally, analogues 1 and 4 inhibited the hydrolysis of pnp-TMP, Ap 5 A, and
  • analogues 1 -4 were not hydrolyzed by NTPDases and did not affect hydrolysis of ATP by NTPDase l and -8.
  • NTPDase2 and -3 activities were reduced by __30% by these analogues.
  • analogues 1 and 2 exhibited no inhibitory effect toward ecto-5'-nucleotidase.
  • dinucleotides having either a penta- or tctraphosphate linker such as analogues 1 and 2, and 3 and 4, respectively, do not, or barely, affect NTPDase activity, and indeed, such dinucleotides are recognized neither as substrates nor as inhibitors by these enzymes.
  • analogues 1 and 2 were not recognized by ecto-5'-nucleotidase may indicate that an Ap 5 A scaffold is especially suitable for designing NPP-selective inhibitors.
  • Analogues 1 and 2 having a pentaphosphate linker inhibited Ap 5 A hydrolysis by NPP 1 better than analogues 3 and 4 bearing a tetraphosphate chain. Yet, analogue 1 inhibited the hydrolysis of ATP by NPP 1 better than analogue 2, implying that 1 competes with ATP because it has a 2'-OH group, i.e., recognition of ATP by NPP 1 probably involves the 2'-OH group. This requirement is not important for a NPP 1 inhibitor directed against Ap 5 A hydrolysis, possibly since recognition of Ap 5 A does not involve a 2'-OH group.
  • N PP3 -mcdiated hydrolysis of ATP was sensitive to inhibition by the dinucleotide analogues 1 -4. Apparently, the patterns of recognition of Ap 5 A and ATP by NPP3 are different than those for NPP l , and therefore, NPP3 was not affected by analogues 1-4 as much as NPP l .
  • NPP2 nucleotidase activity for both the membrane-bound forms and the secreted forms was highly affected by analogues 1-4. It is noteworthy that in addition to its nucleotidase activity, NPP2 prefers Iysophospholipids as substrates. Since the hydrolysis of Iysophospholipids and nucleotides is performed by the same catalytic site (Gij sbers et al. , 2003 ; Koh et al. , 2003), it may be speculated that analogues 1-4 might also inhibit the hydrolysis of Iysophospholipids by NPP2, and potentially also by NPP4-7.
  • [0035 j As for a /3-methylene-ADP, a known ecto-5 '-nucleotidase inhibitor (Bar and Simonson, 1 75), the methylene groups between , ⁇ and ⁇ , ⁇ phosphates conferred strong inhibitory activity to analogues 3 and 4 toward ecto-5 '-nucleotidase. In contrast, compounds 1 and 2 had no effect on ecto-5 '-nucleotidase activity, further emphasizing the specificity of the latter analogues as NPP inhibitors.
  • P ⁇ -P bridging oxygen atoms in the aforesaid compound with methylene groups, yielding analogue 1, a decreased activity toward the P2Y ] receptor was observed.
  • Analogue 3 was ⁇ 60-fold less potent than ATP, while Ap 4 A itself had a potency similar to that of ATP (Shaver et al , 2005), indicating that replacing the bridging oxygen atom with a methylene group reduces P2Yi agonist potency.
  • Analogue 2 was >200-fold less potent than ATP, indicating the importance of the 2'- hydroxyl group for molecular recognition by the P2Y ) receptor.
  • Ap 4 A may be considered as an agonist of P2Yn , which is normally activated by ATP derivatives (Communi et al , 2001 ; Patel et al , 2001 ).
  • Ap 4 A derivatives 3 and 4 were poor P2Yi i receptor agonists or completely inactive, probably due to the replacement of the bridging oxygen atoms in the polyphosphate chain with methylene groups, as observed for the P2Yi receptor.
  • the most potent P2Yn agonist among analogues 1-4 was analogue 1.
  • Analogue 21 is also identified by the name 2-MeS-adenosine-5'-0-(a-borano- triphosphate); analogue 22 is also identified by the name 3,7-CH 2 -2-MeS-adenosine-5'- triphosphate; analogue 23 is also identified by the name adenosine-/3,7-CH 2 -5'-0-(o;- borano-triphosphate); and analogue 24 is also identified by the name 2-MeS-adenosine- i8,y-CH 2 -5'-0-(a-borano-triphosphate).
  • analogues 22 and 24 (B isomer) proved potent P2Y i receptor agonists, probably due to improved interactions of 2-MeS- adenine moiety (vs. adenine) with the P2Y i receptor binding-pocket (Mohamady and Jakeman, 2005), analogues 23 (A and B isomers) and 24 (A isomer) were practically inactive at this receptor. As further shown, analogues 22-24 were hardly degraded by all known sub-types of NTPDase, and are not inhibitors of NTPDase.
  • analogues 22-24 were specific inhibitors of NPP 1 and were not hydrolyzed by NTPDases or NPPs, indicating that analogue 23 could be useful as a specific inhibitor of NPP 1 .
  • analogues 1 -4 arc moderate but effective inhibitors of NPP 1 activity in either cell extracts or intact cells.
  • Analogues 1 and 4 strongly blocked the activity of both NPP 1 and -3.
  • analogue 2 did not significantly block NPP3 activity, had no activity on NTPDasel , -2, -3, and -8, as well as ecto-5' -nucleotidase, and virtually no activity toward the P2Yi , P2Y 2 and P2Yn receptors, and is therefore the most specific inhibitor of NPP 1 among the analogues tested, and can be useful in treatment or management of osteoarthritis.
  • analogues 23A/23B were practically inactive at P2Y i -R and P2Y 4 /6-Rs; were chemically stable being hydrolyzed under conditions mimicking gastric juice pH (pH 1 .4 and 37°C) with hal f-lives of 14.1 and 47.1 h, respectively, as compared to ATP which was hydrolyzed with half-life of 3.6 h; and completely resisted hydrolysis by alkaline phosphatase for 30 min at 37°C.
  • analogues 23A/23B were hardly degraded by all plasma-bounded NTPDase sub-types, and were not inhibitors and only weakly bound to NTPDase.
  • Analogue 23A was a specific and potent inhibitor (AT, 500 nM) of NPP 1 and was not hydrolyzed by NTPDases or NPPs. Therefore, analogue 23A could be useful as a specific inhibitor of NPP 1 .
  • the present invention thus provides, in one aspect, a pharmaceutical composition for treatment of osteoarthritis comprising cither a dinuclcoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, in which at least one but preferably two of the bridging-oxygens in the dinucleoside boranophosphate derivative, more preferably the , ⁇ - and ⁇ 5,e-bridging- oxygens, and at least one of the bridging-oxygens in the nucleoside boranophosphate derivative, each is replaced with a group selected from -NH- or -C(RioRn)-, wherein Rio and Ri i each independently is H or halogen.
  • halogen includes fluoro, chloro, bromo, and iodo, and is preferably fluoro or chloro.
  • hydrocarbyl in any of the definitions of the different radicals R] to R , Rs and Rg refers to a radical containing only carbon and hydrogen atoms that may be saturated or unsaturated, linear or branched, cyclic or acyclic, or aromatic, and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and aryl.
  • alkyl typically means a straight or branched hydrocarbon radical having 1 -8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl. n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n- hcptyl. n-octyl, and the like.
  • Preferred are (C ] -C fi )alkyl groups, more preferably (C i - C 4 )alkyl groups, most preferably methyl and ethyl.
  • alkenyl and alkynyl typically mean straight or branched hydrocarbon radicals having 2-8 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, propenyl, 3-buten- l -yl, 2- ethenylbutyl, 3-octen-l -yl, and the like, and propynyl, 2-butyn-l -yl, 3-pentyn- l -yl, and the like.
  • Preferred are (C 2 -C 6 )alkenyl and (C 2 -C 6 )alkynyl, more preferably (C 2 -C 4 )alkenyl and (C:-C4)alkynyl.
  • Each one of the alkyl, alkenyl and alkynyl may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH , - N OT, -CN, -SCN, aryl, or heteroaryl, and/or interrupted by one or more heteroatoms selected from nitrogen, oxygen or sul fur.
  • halogen e.g., F, CI or Br
  • cycloalkyl as used herein means a mono- or bicyclic saturated hydrocarbyl group having 3- 10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1 ]octyl, bicyclo[2.2.1 ]heptyl, and the like, which may be substituted, e.g., with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N0 2 , -CN, -SCN, (C,-C 8 )alkyl, -0-(C, - C g )alkyl, -S-(C ,-C 8 )alkyl, -NH 2 , -NH-(C,-C 8 )alkyl, or -N
  • cycloalkenyl as used herein means a mono- or bicyclic unsaturated hydrocarbyl group having 3- 1 0 carbon atoms and 1 double bond, and include cyclopropenyl, cyclobutenyl, cyclopentcnyl, cyclohcxenyl, cycloheptenyl, cyclooctenyl, cyclononcnyl, cyclodecenyl, hexahydropentalenyl, octahydronaphtalenyl, bicycle[4.2.0]oct-2-enyl, and the like.
  • aryl denotes an aromatic carbocyclic group having 6- 14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl. Preferred are (C6-Cio)aryl, more preferably phenyl.
  • the aryl radical may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N0 2 , -CN, -SCN, (C , -C 8 )alkyl, -0-(C , -C 8 )alkyl, -S-(C , -C 8 )alkyl, -NH 2 , -NH- (C
  • halogen e.g., F, CI or Br
  • heteroaryl refers to a radical derived from a mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1 or 2, heteroatoms selected from N, O or S.
  • heteroaryl is a monocyclic ring, it is preferably a radical of a 5-6- membered ring such as, but not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1 ,2,3-triazinyl, 1 ,3,4-triazinyl, and 1 ,3,5-triazinyl.
  • Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobcnzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[ l ,2- «]pyridyl, bcnzimidazolyl, bcnzthiazolyl, benzoxazolyl, pyrido[ l ,2-a]pyrimidinyl and 1 ,3-benzodioxinyl.
  • the heteroaryl may be substituted. It is to be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings.
  • heterocyclic ring denotes a mono- or poly-cyclic non-aromatic ring of 4- 12 atoms containing at least one carbon atom and one to three, preferably 1 -2 heteroatoms selected from N, O or S, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond.
  • heterocyclic rings may optionally be substituted at any carbon atom as well as at a second nitrogen atom of the ring, if present, with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N0 2 , -CN, -SCN, (C,-C 8 )alkyl, -0-(C, -C 8 )alkyl, -S-(C, -C 8 )alkyl, -NH 2 , -NH-(C,- C 8 )alkyl, or -N-((C) -C 8 )alkyl) 2 .
  • halogen e.g., F, CI or Br
  • Non-limiting examples of radicals -NR4R 5 and -NRsR.9 include amino, dimethylamino, diethylamino, ethylmethylamino, phenylmethyl-amino, pyrrolidino, piperidino, tetrahydropyridino, piperazino, ethylpiperazino, hydroxyethyl piperazino, morpholino, thiomorpholino, thiazolino, and the like.
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein (i) both n and n' arc 1 , two of Wi to W 4 are -0-, and the other two of Wi to W 4 each independently is -C(RioRi i)-; (ii) n is 0 and n' is 1 , one of W 2 to W 4 is -0-, and the other two of W 2 to W 4 each independently is -C(R
  • the compound of the general formula I is a dinucleoside penta(borano)phosphate derivative wherein n and n' are 1.
  • These derivatives may have (i) a sole borano group at position (or a'), namely, Z ⁇ (or Z 5 ) is -BH 3 " , and Z 2 , Z 3 , Z 4 and Z 5 (or Z ⁇ , Z 2 , Z 3 and Z 4 ) are -O " ; at position ⁇ (or ⁇ '), namely, Z 2 (or Z 4 ) is -BH 3 " , and Zi , Z 3 , Z 5 and Z 4 (or Zi , Z 2 , Z 3 and Z 4 ) are -O " ; or at position ⁇ , namely, Z 3 is -BH 3 ⁇ , and Z
  • the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein Z 3 is -BH 3 " , Z, , Z 2 , Z 4 and Z5 are -O " , W 2 and W 3 are -0-, and W
  • the compound of the general formula I is a dinucleoside tetra(borano)phosphate derivative wherein n is 0 and n' is 1 .
  • These derivatives may have (i) a sole borano group at position a (or a'), namely, Z ⁇ (or Z 5 ) is - BH " , and Z 3 , Z and Z 5 (or Z ⁇ , Z 3 and Z 4 ) are -O " ; or at position ⁇ (or ⁇ '), namely, Z 3 (or Z 4 ) is -BH 3 ⁇ , and Zi , Z 4 and Z 5 (or Z ⁇ , Z 3 and Z 5 ) are -O " ; (ii) two borano groups at positions ⁇ , ⁇ (or ⁇ , ⁇ '), namely, Z ⁇ and Z 3 (or Z 4 and Z 5 ) are -BH 3 " , and Z 4 and Z5 (or Z ⁇ and Z 3 ) are O
  • the compound of the general formula 1 is a dinucleoside tri(borano)phosphate derivative wherein n and n' are 0.
  • These derivatives may have (i) a sole borano group at position (or ⁇ '), namely, Z ⁇ (or Z 5 ) is BH 3 " , and Z 3 and Z 5 (or Z ⁇ and Z ) are O " ; or at position ⁇ , namely, Z 3 is BH 3 " , and Zi and Z 5 are O " ; (ii) two borano groups at positions ⁇ , ⁇ (or ⁇ , ⁇ ), namely, Zi and Z 3 (or Z 3 and Z 5 ) are BH 3 " , and Z 5 (or Z
  • the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein Y and Y' each independently is H or -OH.
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la.
  • X and X' are an adenine residue, wherein Ri each independently is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl , -NR4R5, heteroaryl, or hydrocarbyl; R 4 and R 5 each independently is H or hydrocarbyl, or R 4 and R 5 together with the nitrogen atom to which they are attached fonn a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hcteroatoms selected from N, O or S; and R2 and R 3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C
  • R ] each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl;
  • R4 and R 5 each independently is H or hydrocarbyl;
  • R 2 and R 3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C
  • Ri each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R 5 each independently is H or hydrocarbyl; and R 2 and R 3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
  • the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la, wherein R i each independently is I I, -O- hydrocarbyl, -S-hydrocarbyl, -NR 4 R 5 , or hydrocarbyl; R 4 and R5 each independently is H or hydrocarbyl; R 2 and R 3 are H; Y and Y' each independently is H or -OH; n and n' are 1 ; m is 5; Z 3 is -BH 3 " ; Zi , Z 2 , Z 4 and Z 5 are -O " ; W 2 and W are -0-; and Wi and W 4 each independently is -C(R
  • each independently is H, -O- methyl or -S-methyl
  • R 2 and R 3 are H
  • Y and Y' each independently is H or -OH
  • n and n' are 1
  • m is 5
  • Z 3 is -BH 3 "
  • , Z 2 , Z4 and Zs are -O "
  • W 2 and W 3 arc -0-
  • and W 4 each independently is -CH 2 -, -CC1 2 - or -CF 2 -, preferably -CH 2 -.
  • the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la, wherein R
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an uracil residue of the formula lb.
  • X and X' arc an uracil residue, wherein R 6 each independently is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, - NR 8 R 9 , heteroaryl, or hydrocarbyl; Rg and R y each independently is H or hydrocarbyl, or Rg and R 9 together with the nitrogen atom to which they are attached form a 5- or 6- membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R 7 is O, wherein said hydrocarbyl each independently is (Ci -C 8 )alkyl, preferably (Ci -C4)alkyl, more preferably methyl or ethyl, (C -C 8 )alkenyl, preferably (C 2 -C 4 )alkenyl, (C 2 -Cg)alkynyl, preferably (C 2 -C 4 )alkyn
  • R 6 each independently is H, -O-hydrocarbyl, - S-hydrocarbyl, -NR g R 9 , or hydrocarbyl;
  • R 8 and R9 each independently is H or hydrocarbyl; and
  • R 7 is O, wherein said hydrocarbyl each independently is (Ci -C 4 )alkyl, preferably methyl or ethyl, (C 2 -C 4 )alkenyl, (C 2 -C 4 )alkynyl, or (C 6 -Ci 0 )aryl, preferably phenyl.
  • R f each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NRgRy, or hydrocarbyl ;
  • R 8 and Ry each independently is I I or hydrocarbyl; and
  • R7 is O, wherein said hydrocarbyl each independently is methyl or ethyl.
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • the active agent is a compound of the general formula 11, or a diastereomer or mixture of diastereoisomers thereof, wherein (i) n is 1 , one of W ] and W 2 is -0-, and another one of W , and W 2 is -C(R ioRn )-; or (ii) n is 0, and W 2 is - C(R,oRn)-.
  • the compound of the general formula II is a nucleoside tri(borano)phosphate derivative wherein n is 1 .
  • These derivatives may have (i) a sole borano group at position a, namely, Z ⁇ is -BH 3 " , and Z 2 and Z 3 are -O " ; at position ⁇ , namely, Z 2 is -BH 3 ⁇ , and Z ⁇ and Z 3 are -O " ; or at position ⁇ , namely, Z 3 is -BH 3 ⁇ , and Z ⁇ and Z 2 are -O " ; (ii) two borano groups at positions , ⁇ , namely, Z] and Z 2 are -BH 3 ⁇ , and Z 3 is - O " ; at positions ⁇ , ⁇ , namely, Z ⁇ and Z 3 are -BH 3 " , and Z 2 is -O " ; or at positions , ⁇ , namely, Z 2 and Z 3 are -BH " , and Z
  • the active agent is a compound of the general formula II, or a diastereomer or mixture of diastereoisomers thereof, wherein Z ⁇ is -BH 3 " , Z 2 and Z 3 are -O " , W , is -0-, and W 2 is -C(R
  • the compound of the general formula II is a nucleoside di(borano)phosphate derivative wherein n is 0.
  • These derivatives may have (i) a sole borano group at position a, namely, Zj is -BH 3 " , and Z 3 is -O " ; or at position ⁇ , namely, Z 3 is -BH 3 " , and Z ⁇ is -O " ; or (ii) two borano groups at positions ⁇ , ⁇ , namely, Z ⁇ and Z 3 are -BH 3 " .
  • the active agent is a compound of the general formula I I , or a diastereomer or mixture of diastereoisomers thereof, wherein Y is H or -OH.
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la.
  • X is an adenine residue, wherein R
  • R 4 and R 5 each independently is H or hydrocarbyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R 2 and R 3 each independently is H or hydrocarbyl, wherein said hydro carbyl each independently is (Ci-C 8 )alkyl, preferably (d-C 4 )alkyl, more preferably methyl or ethyl, (C 2 -C 8 )alkenyl, preferably (C 2 -C 4 )alkenyl, (C 2 -C 8 )alkynyl, preferably (C 2 -C 4 )alkynyl, or (C 6 -C l 4 )aryl, preferably (C6-C i () )aryl, more preferably phenyl; and said heteroaryl is a 5-6- membcrcd monocycl
  • R] is H, -O-hydrocarbyl, -S-hydrocarbyl, - NR4R5, or hydrocarbyl;
  • R 4 and R 5 each independently is H or hydrocarbyl;
  • R 2 and R 3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (Ci -C4)alkyl, preferably methyl or ethyl, (C 2 -C 4 )alkenyl, (C 2 -C 4 )alkynyl, or (C 6 -Cio)aryl, preferably phenyl.
  • Ri is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R 4 and R 5 each independently is H or hydrocarbyl; and R 2 and R 3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
  • the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein R ⁇ is H, -O-hydrocarbyl, -S-hydrocarbyl, - NR4R5, or hydrocarbyl; R 4 and R 5 each independently is H or hydrocarbyl; R 2 and R 3 are H ; Y is H or -OH; n is 1 ; m is 4; Z, is -BH 3 ⁇ ; Z 2 and Z 3 are -O " ; W, is -0-; and W 2 is - C(R ioR
  • is H, -O-methyl or -S-methyl
  • R 2 and R 3 are H
  • Y is H or -OH
  • n is 1
  • m is 4
  • Z is -BH 3 "
  • Z 2 and Z 3 are -O "
  • W is -0-; and W 2 is -CH 2 -, - CC1 2 - or -CF 2 -, preferably -CH 2 -.
  • the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein Ri is H; R 2 and R 3 are H ; Y is -OH ; n is 1 ; m is 4; 7 ⁇ is -BH 3 " ; Z 2 and Z 3 are -O " ; W, is -0-; and W 2 is -CH 2 - ( analogues 23).
  • Both isomers 23A and 23B can be used, i.e., the isomers having a retention time (Rt) of 7.64 min or 9.67 min, respectively, when separated from a mixture of diastereoisomers using a semi-preparative reverse-phase Gemini 5u column (C- 1 8 1 1 OA, 250x 1 0 mm, 5 micron), and isocratic elution [ 100 mM triethylammonium acetate, pH 7: MeOH, 89: 1 1 ] with flow rate of 5 ml/min.
  • Rt retention time
  • the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein Ri is S-methyl; R 2 and R 3 are H; Y is -OH; n is 1 ; m is 4; Z] is -BH 3 " ; Z 2 and Z 3 are -O " ; Wi is -0-; and W 2 is -CH 2 - (analogues 24).
  • the preferred isomer in this case is 24A, i.e., the isomer having a retention time (Rt) of 5.29 min when separated from a mixture of diastereoisomers using a semi-preparative reverse- phase Gemini 5u column (C- 1 8 1 1 0A, 250 x 10 mm, 5 micron), and isocratic elution [ 100 mM triethylammonium acetate, pH 7 : MeOH, 75 :25 J with flow rate of 5 ml/min.
  • Rt retention time
  • the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an uracil residue of the formula lb.
  • X is an uracil residue, wherein R 6 is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR 8 R 9 , heteroaryl, or hydrocarbyl; R 8 and Rq each independently is H or hydrocarbyl, or R 8 and RQ together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S ; and R 7 is O, wherein said hydrocarbyl each independently is (Ci -C 8 )alkyl, preferably (Ci- C )alkyl, more preferably methyl or ethyl, (C 2 -C 8 )alkenyl, preferably (C 2 -C 4 )alkenyl, (C 2 - Cs)alkynyl, preferably (C 2 -C 4 )alkynyl, or (C 6
  • R 6 is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR 8 R9, or hydrocarbyl;
  • R 8 and RQ each independently is H or hydrocarbyl;
  • R 7 is O, wherein said hydrocarbyl each independently is (Ci -C 4 )alkyl, preferably methyl or ethyl, (C 2 -C )alkenyl, (C 2 -C 4 )alkynyl, or (C 6 -C io)aryl, preferably phenyl.
  • Re is H, -O- hydrocarbyl, -S-hydrocarbyl, -NR 8 R 9 , or hydrocarbyl; R 8 and R9 each independently is H or hydrocarbyl; and R 7 is O, wherein said hydrocarbyl each independently is methyl or ethyl.
  • the compounds of the general formula I or II may be synthesized according to any technology or procedure known in the art. Procedures for the synthesis of compounds of the general formula I are described in detail, e.g., in US 7,368,439 and in the Examples section hereinafter. Procedures for the preparation of compounds of the general formula II may are described, inter alia, in WO 2009/066298. [0072] Both the compounds of the general formula I and the compounds of the general formula II may have one or more asymmetric centers, e.g., in the Pa, and may accordingly exist as pairs of diastereoisomers.
  • the separation and characterization of the different diastereoisomers may be accomplished using any technology known in the art, e.g., using HPLC.
  • treatment of osteoarthritis could be carried out by administration of all such isomers and mixtures thereof.
  • the compounds of the general formula I are in the form of pharmaceutically acceptable salts.
  • the cation B is an inorganic cation of an alkali metal, e.g., lithium, sodium or potassium, or an alkaline earth metal, e.g., calcium or magnesium.
  • the cation B is ammonium ( ⁇ 4 ' ) or is an organic cation derived from an amine of the formula wherein each one of the Rs independently is selected from H, C
  • N, S and O such as pyrrolydine
  • the cation B is a cationic lipid or a mixture of cationic lipids.
  • Cationic lipids are often mixed with neutral lipids prior to use as delivery agents.
  • Neutral lipids include, but are not limited to, lecithins; phosphatidylethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; phosphatidylglycerol
  • Neutral lipids also include cholesterol and other 3 ⁇ hydroxy-sterols.
  • Examples of cationic lipid compounds include, without being limited to, Lipofectin* ( Li fe Technologies, Burlington, Ontario) ( 1 : 1 (w/w) formulation of the cationic lipid N-[ l -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine 1 M (Life Technologies, Burlington, Ontario) (3 : 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent),
  • compositions provided by the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19 th Ed., 1995.
  • the compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the general formula I or II, into association with a liquid carrier, a finely divided solid earner, or both, and then, i f necessary, shaping the product into the desired formulation.
  • the compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients.
  • the pharmaceutical composition of the present invention is formulated as nanoparticles.
  • compositions can be formulated for any suitable route of administration, but they are preferably formulated for parenteral, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, subcutaneous, transdermal or topical, or for oral administration.
  • parenteral e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, subcutaneous, transdermal or topical, or for oral administration.
  • the dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.
  • the pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleagenous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent.
  • Acceptable vehicles and solvents include, without limiting, water, Ringer's solution and isotonic sodium chloride solution.
  • compositions of the invention when formulated for administration route other than parenteral administration, may be in a form suitable for oral use, e.g., as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets.
  • excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, e.g., magnesium stearate, stearic acid, or talc.
  • inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate
  • granulating and disintegrating agents e.g., corn starch or alginic acid
  • binding agents e.g., starch, gelatin or acacia
  • lubricating agents e.g., magnesium ste
  • the tablets may be either uncoated or coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostcarate or glyceryl distearate may be employed. They may also be coated using the techniques described in the US Patent Nos. 4,256, 108, 4, 166,452 and 4,265,874 to form osmotic therapeutic tablets for control release.
  • the pharmaceutical composition of the invention may also be in the form of oil-in-water emulsion.
  • compositions according to the invention when formulated for inhalation, may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like.
  • the pharmaceutical compositions of the invention may be formulated for controlled release of the active agent.
  • Such compositions may be formulated as controlled- release matrix, e.g., as controlled-release matrix tablets in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo).
  • compositions comprise the active agent formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters.
  • Another contemplated formulation is depot systems, based on biodegradable polymers, wherein as the polymer degrades, the active agent is slowly released.
  • the most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules.
  • Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLC).
  • the present invention provides a dinucleoside boranophosphatc derivative of the general formula 1 or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for use in treatment of osteoarthritis.
  • the present invention relates to use of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for the preparation of a pharmaceutical composition for treatment o f osteoarthritis.
  • the present invention relates to a method for treatment of osteoarthritis in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof.
  • Osteoarthritis also known as degenerative arthritis or degenerative joint disease, is the most common form of arthritis and refers to a group of mechanical abnormalities involving degradation of joints, including articular cartilage and subchondral bone.
  • Symptoms may include joint pain, tenderness, stiffness, locking, and sometimes an effusion, i.e., an accumulation of excess fluid in or around the knee joint.
  • an effusion i.e., an accumulation of excess fluid in or around the knee joint.
  • a variety of causes including hereditary, developmental, metabolic and mechanical may initiate processes leading to loss of cartilage. When bone surfaces become less well protected by cartilage, bone may be exposed and damaged. As a result of decreased movement secondary to pain, regional muscles may atrophy, and ligaments may become more lax.
  • Osteoarthritis can be either primary or secondary in case there is an identifiable underlying cause, although the resulting pathology is the same.
  • Primary osteoarthritis is a chronic degenerative disorder related to but not caused by aging. As a person ages, the water content of the cartilage decreases as a result of a reduced proteoglycan content, thus causing the cartilage to be less resilient. Without the protective effects of the proteoglycans, the collagen fibers of the cartilage can become susceptible to degradation and thus exacerbate the degeneration. Inflammation of the surrounding joint capsule can also occur, though often mild compared to that which occurs in rheumatoid arthritis.
  • Secondary osteoarthritis is caused by other factors such as congenital disorders of joints; diabetes; inflammatory diseases, e.g., Perthes' disease and Lyme disease, and all chronic forms of arthritis, e.g., costochondritis, gout and rheumatoid arthritis; injury to joints as a result of an accident or orthodontic operations; septic arthritis, i.e., infection of a joint; ligamentous deterioration; Marfan syndrom, obesity; alkaptonuria; and hemochromatosis and Wilson's disease.
  • CPPD calcium pyrophosphate dehydrate
  • the CPPD crystals deposition is lead by excess of extracellular PPi resulting from over expression of NPP 1 . Controlled blocking of NPP 1 could thus control the concentration of extracellular PPi and consequently decrease symptoms of CPPD. Crystal formation in both articular cartilage and synovial fluid, as well as low-grade inflammation, a consequence of crystal deposition, should be monitored during therapy.
  • the only available treatment for osteoarthritis is symptomatic and does not deal with the causes underlying the disease.
  • the compounds of the general formulas I and II are useful in treatment or management of osteoarthritis.
  • treatment as used herein with respect to osteoarthritis refers to blocking of NPP 1 over expression and consequently extracellular PPi concentration, thus reducing CPPD crystals deposition and attenuating, i .e., limiting or reducing, the various symptoms of the disease as defined above.
  • the term “management” as used herein with respect to osteoarthritis refers to a continuous treatment of the disease during which NPP 1 over expression is constantly controlled so as to maintain balanced levels of extracellular PPi thus constantly reducing CPPD crystals deposition.
  • therapeutically effective amount as used herein refers to the quantity of the compound of the general formula I or II as defined above, or a diastereomer or mixture of diastereomers thereof, that is useful to treat or manage osteoarthritis.
  • the present invention relates to a diadenosine pcnta(" ⁇ - borano)phosphatc derivative of the general formula III as defined above, i.e., a particular embodiment of the compound defined by the general formula I above, in which a borano group replaces a non-bridging oxygen atom at position ⁇ and two of the bridging- oxygens, preferably the ⁇ , ⁇ - and ⁇ 5,e-bridging-oxygens, each is replaced with a group selected from -NH- or -C(Rio n)-, wherein Rio and Rn each independently is H or halogen.
  • the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III, wherein W 2 and W 3 are -0-, and W , and W 4 each independently is -NH- or -C(Ri oR u )-, preferably -CH 2 -, -CC3 ⁇ 4- or -CF 2 -.
  • the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III, wherein Y and Y' each independently is H or -OH.
  • the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each independently is an adenine residue of the formula la.
  • Ad each independently is an adenine residue, wherein R
  • each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl
  • R 4 and Rj each independently is H or hydrocarbyl
  • R 2 and R 3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C] -C 4 )alkyl, preferably methyl or ethyl, (C 2 -C 4 )alkenyl, (C 2 -C 4 )alkynyl, or (C 6 -Cio)aryl, preferably phenyl.
  • each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl;
  • R 4 and R 5 each independently is H or hydrocarbyl; and
  • R 2 and R 3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
  • the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each is an adenine residue of the formula la, wherein R
  • Ri each independently is I I, -O-methyl or -S-mcthyl
  • R 2 and R 3 arc H
  • Y and Y' each independently is H or -OH
  • W 2 and W 3 are -0-
  • Wj and W 4 each independently is -CH 2 -, -CC1 2 - or -CF 2 -, preferably -CH 2 -.
  • the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each is an adenine residue of the formula la, wherein Ri is H; R 2 and R 3 are H; Y and Y' each independently is -OH or H; W 2 and W 3 are -0-; and Wi and W 4 are -CH 2 - (analogues 1 and 2, respectively).
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a diadenosine boranophosphate derivative of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof and a pharmaceutically acceptable carrier.
  • BPj boranophosphate
  • [Ca 2+ ]j intracellular Ca 2+ concentration
  • CDI carbodiimidazole
  • MF N,N-dimethylformamide
  • E-NPP ecto-nucleotide pyrophosphatase/phosphodiesterase
  • E-NTPDasc ecto-nucleoside triphosphate diphosphohydrolase
  • ESI electron spray ionization
  • FBS fetal bovine serum
  • HRMS- MALDI high resolution mass spectrometry matrix-assisted laser desorption/ionization
  • MPLC medium pressure liquid chromatography
  • pnp-TMP thymidine 5 '- monophosphate p-nitrophenyl ester
  • P2R P2 receptor
  • RT room temperature
  • triethylammonium acetate.
  • Nucleotides were characterized also by 1 P NMR in D 2 0, using 85% H 3 P0 4 as an external reference on Bruker AC-200 and DMX-600 spectrometers. High resolution mass spectra were recorded on an AutoSpec-E FIS ION VG mass spectrometer by chemical ionization. Nucleotides were analyzed using electron spray ionization (ESI) on a Q-TOF micro-instrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a column of Sephadex DEAE-A25, swollen in 1 M NaHC0 3 at 3°C for 24 h. The resin was washed with deionized water before use.
  • ESI electron spray ionization
  • LC separation was monitored by UV detection at 280 nm.
  • Final purification of the nucleotides and separation of the diastereomeric pairs were achieved on an HPLC (Merck-Hitachi) system using a semi- preparative reverse-phase column (Gemini 5u C- 1 8 1 1 OA 250x 10 mm; 5 micron; Phenomenex, Torrance, USA).
  • the purity of the dinucleotides was evaluated on an analytical reverse-phase HPLC column system (Gemini 5u C-18 1 1 OA, 1 50 x 3.60 mm; 5 micron; Phenomenex) in two-solvent systems with either solvent systems I and It or solvent system III.
  • Solvent system I was: (A) 100 mM TEAA, pH 7, (B) MeOH; solvent system II : (A) 100 mM TEAA, pH 7, (B) CH 3 CN; solvent system III: (A) 0.01 M KH 2 P0 3 , pH - 3.5, (B) CH 3 CN.
  • solvent system III (A) 0.01 M KH 2 P0 3 , pH - 3.5, (B) CH 3 CN.
  • the details of the solvent system gradients used for the separation of each product are provided below.
  • the products, obtained as triethylammonium salts, were generally 3 ⁇ 45% pure. All reactants in moisture-sensitive reactions were dried overnight in a vacuum oven.
  • Compound 1 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 75:25 A:B over 15 min at a flow rate of 3 ml/min. Retention time: 12.88 min.
  • Purity data obtained on an analytical column retention time: 1.81 min (99.97% purity) using solvent system II with a gradient from 80:20 to 70:30 A:B over 10 min at a flow rate of 1 ml/min.
  • Compound 2 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 70:30 A:B over 20 min at a flow rate of 5 ml/min. Retention time: 15.35 min.
  • Compound 4 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 75:25 A:B over 20 min at a flow rate of 5 ml/min. Retention time: 16.26 min.
  • HTB-85 and HT29 cell lines were grown in 10 cm-plates and were then transferred into 1 cm-plates and incubated at 37°C in a-MEM medium and Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12), respectively, in the presence of 10% FBS. After reaching full confluence, cells were used in intact cell assays (see below).
  • DMEM/F-12 Dulbecco's modified Eagle's medium/F-12 nutrient mixture
  • Ectonucleotidases were produced by transiently transfecting COS-7 cells in 10- cm plates using Lipofectamine (Invitrogen, Burlington, ON, Canada), as previously described (Kukulski et ai , 2005). Briefly, 80-90% confluent cells were incubated for 5 h at 37°C in Dulbecco's modified Eagle's medium, nutriment mix F- 12 (DMEM/F- 12) in the absence of FBS with 6 ⁇ g of plasmid DNA and 23 ⁇ of Lipofectamine reagent. The reaction was stopped by the addition of an equal volume of DMEM/F- 12 containing 20% FBS and the cells were harvested 33-72 h later. The conditioned medium of NPP2- transfected cells was also collected.
  • Lipofectamine Invitrogen, Burlington, ON, Canada
  • Green fluorescent protein (GFP) constructs of human P2Y ] and P2Yn receptors were expressed in 132 1 N 1 astrocytoma cells, which lack endogenous expression of both P2X and P2Y receptors.
  • the respective cDNA of the given receptor gene was cloned into the pEGFPN l vector, and after transfection using the FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany), cells were selected with 0.5 mg/ml G3 1 8 (Merck Chemicals, Darmstadt, Germany) and grown in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C and 5% C0 2 .
  • the functional expression of the receptor was confirmed by GFP fluorescence and the change in intracellular Ca 2 ' concentration ([Ca 2+ ]j) upon incubation with the respective standard receptor agonists.
  • transfected cells were washed three times with Tris-saline buffer at 4°C, collected by scraping in harvesting buffer (95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 35 mM Tris at pH 7.5), and washed twice again by 300 g centrifugation for 10 min at 3°C. Cells were re-suspended in the harvesting buffer supplemented with 10 mg/ml of aprotinin, and sonicated.
  • harvesting buffer 95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 35 mM Tris at pH 7.5
  • NPP2 The secreted form of NPP2 was prepared from the conditioned media of transfected cells, which were frozen and stored at -80°C until tested for activity. Protein concentration was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as standard (Bradford, 1976). Enzymatic activity assays
  • NTPDases and eto-5 '-nucleotidase Activity was measured as previously described (Kukulski et al , 2005) in 0.2 ml of incubation medium Tris-Ringer buffer (in mM, 120 NaCl, 5 KC1, 2.5 CaCl 2 , 1.2 MgS0 3 , 25 NaHC0 3 , 5 glucose, 80 Tris, pH 7.3) at 37°C with or without analogues 1-4 (final concentration 100 ⁇ ), and with or without 100 ⁇ ATP (for NTPDases) or 100 ⁇ AMP (for ecto-5 '-nucleotidase) as a substrate.
  • Tris-Ringer buffer in mM, 120 NaCl, 5 KC1, 2.5 CaCl 2 , 1.2 MgS0 3 , 25 NaHC0 3 , 5 glucose, 80 Tris, pH 7.3
  • Tris-Ringer buffer in mM, 120 NaCl, 5 KC1, 2.5 CaCl
  • the analogues were added without ATP or AMP when tested as potential substrate, and with ATP or AMP when tested for their effect on nucleotide hydrolysis.
  • Either NTPDase or ecto-5 '-nucleotidase protein extracts were added to the incubation mixture and pre- incubated at 37°C for 3 min. The reaction was initiated by the addition of a substrate (ATP, AMP or one of analogues 1-4) and stopped after 15 min with 50 ⁇ of malachite green reagent.
  • the released inorganic phosphate (Pj) was measured at 630 nm as previously described (Baykov et al. , 1988).
  • NPPs Evaluation of the effect of compounds 1-4 on human NPP 1 , -2 and -3 activity was carried out either with pnp-TMP, ATP or Ap 5 A as a substrate (Belli and Goding, 1994). The reactions were carried out at 37°C in 0.2 ml of the following incubation mixture: in mM, 1 CaCl 2 , 1 30 NaCl, 5 KC1 and 50 Tris, pH 8.5, with or without compounds 1-4 and/or substrates. Substrates and compounds 1-4 were all used at a final concentration of 100 ⁇ .
  • the reaction was initiated by addition of the substrate.
  • pnp-TMP hydrolysis the production of p-nitrophenol was measured at 405 nm, 15 min after the initiation of the reaction.
  • Ap 5 A and ATP the reaction was stopped after 30 min by transferring an aliquot of 0.1 ml from the reaction mixture to 0.125 ml ice-cold 1 M perchloric acid. The samples were centrifuged for 5 min at 13,000 x g.
  • Activity assays with intact HTB-85 and HT29 cell lines were carried out in 0.25 ml of the incubation mixture containing 1 35 mM NaCl in 24-well plates. Reaction was initiated by the addition of pnp-TMP to obtain a final concentration of 100 ⁇ . After 20 min, 0.2 ml of the reaction mixture was transferred to a 96-well plate and the production of p-nitrophenol was measured at 3 10 nm.
  • [001 13 ] 1 321 N 1 astrocytoma cells were transfected with the respective plasmid for P2YR-GFP expression, i.e., pEGFPN l expression vector plasmids encoding the cDNA for human P2Yi or P2Yn receptors (Ecke el al. , 2006), and the P2Y 2 receptor (Ginsburg- Shmuel el al. , 201 0; Tulapurkar el al. , 2004), respectively.
  • HEPES-buffered saline in mM, 135 NaCl, 5.3 C1, 1 .8 CaCl 2 , 1 MgCl 2 , 25 glucose, 20 HEPES/Tris, pH 7.3
  • Dinucleoside polyphosphates are conventionally prepared via the activation of the 5'-terminal phosphate of a nucleotide, thus forming a phosphoryl donor (P-donor), followed by reaction with a non-activated nucleoside 5'-phosphate, or phosphonatc analogue (phosphoryl acceptor; P-acceptor).
  • P-donor phosphoryl donor
  • P-acceptor phosphonatc analogue
  • a common method for activation of phosphates/phosphonates uses CDI to form phosphoroimidazolides. The latter may be generated in situ or isolated prior to the reaction with the corresponding nucleotides (Zatorski el al , 1995).
  • 3-5,e-dimethylene-pentaphosphonate, 2) were prepared as described in the Experimental from the ⁇ , -methylene-ADP building blocks, 9, and a,
  • ⁇ ,/3-Methylene-ADP derivatives were synthesized as previously described (Davisson et al , 1987).
  • adenosine analogue 6 which is 2' and 3'-OH protected, was activated with tosyl chloride to form the activated nucleoside 7, which was then coupled with a tris(tetra-/7-butylammonium)methylene diphosphonate salt to form analogue 8, followed by removal of the protecting group, which provided product 9.
  • the related scaffold 13 was prepared from 2'-deoxyadenosine. A selective tosylation at the 5'-OH position of 11 was earned out at 0°C to form product 12, which was then coupled with a tris(tetra-n-butylammonium) methyl enediphosphonate salt to yield product 1 (Liang et al. , 2008).
  • Nucleotides 9 and 13 were activated with CDI in situ to form P-donors 10 and 14, respectively, which were then treated with BP,, 15, as a P-acceptor.
  • MgCl 2 was added as an activator to overcome the low nuclcophilicity of BP, as a P-acceptor (Hoard and Ott, 1 965).
  • Compounds 1 and 2 were obtained in 10% and 21 % overall yields, respectively, after LC separation.
  • the activated forms o ⁇ -methylene-ADP-Im, 10, and a,j8-methylene-2'-dcoxy- ADP-Im, 14, become P-donors, whereas ⁇ ,/3-methylene-ADP, 9, and a /3-methylcne-2'- deoxy-ADP, 13, rather than BP,, function as P-acceptors (Fig 1 , upper right side, path c). Furthermore, since the phosphonate is assumed to be a better nucleophile than BP,, 15, byproducts 3/4 are obtained and not adcnosine-a ⁇ -CHi-T-borano-triphosphate, 16.
  • Example 2 The effect of analogues 1-4 on ectonucleotidase activity and on
  • NTPDase2 and -3 were modestly inhibited ( 10-30%) by these analogues. While analogues 3 and 4 inhibited ecto-5 '-nucleotidase activity by 90 and 80%, respectively, analogues 1 and 2 did not affect the latter enzymatic activity.
  • NPP l When using ATP as the substrate, NPP l was inhibited by -70-80% in the presence of analogues 2 and 3, and by more than 90% by analogues 1 and 4 ( Figs. 3C-3D).
  • the presence of analogues 1 -4 reduced the hydrolysis of pnp-TMP by NPP3 by -30% (Fig. 3A), and the hydrolysis of Ap 5 A by - 1 0-60% (Fig. 3C).
  • the inhibition of NPP3 activity using ATP as the substrate was more pronounced (-90%) in the presence of analogues 1 and 4, and around 65% with analogues 2 and 3 (Fig. 3D).
  • Dinuclcotide analogues 1 -4 (each at 1 00 ⁇ ) were incubated with the indicated ectonucleotidases.
  • the activity with 1 00 ⁇ ATP (for NTPDases) or AMP (for the ecto-5 '-nucleotidase) was set as 1 00%: 1270 ⁇ 35; 928 ⁇ 55; 2()2 ⁇ 37; 1 29 ⁇ 1 1 ; and 357 ⁇ 1 0 nmol Pi min " 1 (mg protein " ' ) for NTPDase l , -2, -3, and -8, and ecto-5 '- nucleotidase, respectively.
  • pnp-TMP substrate and analogues 1-4 were used in the concentration range of 2.5x 1 0 "5 to l x l 0 " M .
  • the pnp-TMP concentration was 5x l 0 '5 M and the concentrations of the inhibitors were in the range of 5x 1 0 "7 to 1 1 0° M. All experiments were performed three times in triplicate.
  • NPP 1 is critical in regulating mineralization by generating inorganic pyrophosphate, a potent inhibitor of hydroxyapatite crystal growth.
  • NPP3 is associated with carcinogenesis.
  • Human osteoblastic SaOS-2 cells (HTB-85) are used to investigate the activity of NPP 1 (Vaingankar et al. , 2004), as well as HT29, a human colon cancer cell line (Baricault et al , 1 995).
  • HTB-85 and HT29 catabolized pnp- TMP, indicating the presence o f NPPs at their surface.
  • the enzymes obtained from cell extract NPP activity exhibited by both cell lines was blocked by -90% by analogues 1 and 2, and by about 80% by analogues 3 and 4, as shown in Fig. 4.
  • Example 4 The activity of analogues 1-4 on the P2Y], P2Y 2 and P2Yn receptors
  • GFP constructs of human P2Yi and P2Y, , receptors were expressed in 1 21 N l astrocytoma cells, which lack endogenous expression of both P2X and P2Y receptors.
  • the cells were then incubated with various concentrations of analogues 1-4, and the Ca " response to each one of the analogues was compared with that due to ATP, as shown in Figs. 5A-5B.
  • analogues 2-4 were weak agonists of the P2Y i receptor.
  • the 2'-deoxy analogues 2 and 4 exhibited comparably weak activities with EC 5 o values of SO ⁇ for the P2Yj receptor. No clear plateau was reached up to 100 ⁇ for analogue 2.
  • Analogue 3 was found to be a very weak agonist of the P2Y ] , receptor, with an HC 5 o 3 ⁇ 40 ⁇ , whose maximal response corresponded only to 1 5% of that of ATP.
  • the 2'-deoxy analogues 2 and 4 were both inactive at concentrations ⁇ >0 ⁇ .
  • Analogues 1 -4 were completely inactive toward the P2Y 2 receptor at concentrations ⁇ 5 ⁇ .
  • Table 3 EC50 values for [Ca 2+ ]i elevation by analogues 1-4, mediated by the P2Yi , 2 , n receptors
  • a ATP was selected as the common reference agonist at both P2Y i and P2Y n receptors, although ADP is the preferred endogenous P2Y
  • Example 7 The effect of analogues 22-24 on human ectonuclcotidase activity, NPP l and NPP3 activity, and on the P2Y,, P2Y 2 and P2Y 4 and P2Y 6 receptors
  • Ectonucleotidases were produced by transiently transfecting 293T cells using Lipofcctamine (Invitrogen, Burlington, ON, Canada), and protein fractions were prepared as described in Experimental. [00134] NTPDase activity was measured as previously described (Kukulski et ⁇ , , 2005) in 0.2 ml of Tris-Ringer buffer (in raM 120 NaCl, 5 C1, 2.5 CaCl 2 , 1 .2 MgS0 4 , 25 NaHC0 3 , 5 glucose, 80 Tris, pH 7.4) at 37°C with or without analogues 21 -24.
  • NTPDase protein extracts were added to the incubation mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by the addition of the substrate (ATP, ADP or one o f the analogues) to a final concentration of 1 00 ⁇ and stopped after 20 min with 50 ⁇ of malachite green reagent. The released inorganic phosphate (Pj) was measured at 630 nm according to Baykov et al. ( 1988). The activity obtained with protein extracts from untransfected cells was subtracted from the activity obtained with extracts from NTPDase transfected cells. The activity with this control protein extract did not exceed 5% of the activity of any NTPDase extract.
  • the substrate ATP, ADP or one o f the analogues
  • pnp-TMP For pnp-TMP, the production of p-nitrophenol was measured at 410 nm, 20 min after the initiation of the reaction. When one of the analogues was used as a substrate, the reaction was stopped after 20 min by transferring a 0. 1 ml aliquot from the reaction mixture to 0.1 25 ml ice-cold 1 M perchloric acid. These samples were centrifuged for 5 min at 13,000 g. Supematants were neutralized with 1 M KOH (4°C) and centrifuged for 5 min at 13,000 g. An aliquot of 20 ⁇ was separated by reverse-phase HPLC to evaluate the decrease in the analogue level. Protein extracts from non-transfected cells did not show any NPP activity.
  • the substrates and their products were separated on a SUPELCOSILTM LC- 1 8-T column ( 1 5 cm x 4.6 mm, 3 ⁇ Supelco, Bcllefonte, Pennsylvania, USA) with a mobile phase composed of 25 mM TBA, 5 mM EDTA, 100 mM ⁇ 3 ⁇ 4 ⁇ 0 4 / ⁇ 2 ⁇ 0 4 , pH 7.0 and 2% (v/v) methanol at a flow rate of 1 ml/min.
  • SUPELCOSILTM LC- 1 8-T column 1 5 cm x 4.6 mm, 3 ⁇ Supelco, Bcllefonte, Pennsylvania, USA
  • a mobile phase composed of 25 mM TBA, 5 mM EDTA, 100 mM ⁇ 3 ⁇ 4 ⁇ 0 4 / ⁇ 2 ⁇ 0 4 , pH 7.0 and 2% (v/v) methanol at a flow rate of 1 ml/min.
  • analogues 22-24 were almost not hydrolyzed by NTPDases.
  • Analogue 22 was the most efficiently hydrolyzed analogue by NTPDases, and was hydrolyzed by human NTPDasc l , 3 and 8 at about 7-8% of the rate of ATP and by human NTPDase2, at about 2% of the rate o f ATP.
  • NPP 1 hydrolyzed 22 at 20% the rate of pnp-TMP hydrolysis
  • human N PP3 hydrolyzed 22 and 24A at ⁇ 0% of the rate of pnp-TMP hydrolysis.
  • the other analogues were hydrolyzed by these ectonucleotidases at less than 5% of the rate of pnp- TMP hydrolysis.
  • analogues 22-24 did not affect signi ficantly the activity o f NTPDases.
  • a weak inhibition of ATP hydrolysis by human NTPDasel 1 7% was observed when equal concentrations of the substrate ATP and 23 were used, and similar levels of inhibition of human NTPDase3 activity were obtained with 22 ( 1 7%), 24A ( 16%) and 24B ( 1 8%)), as shown in Fig. 8.
  • Similar inhibition profiles were obtained for analogues 22-24 with ADP as a substrate (data not shown).
  • Fig. 9 shows that all analogues 22-24 inhibited the hydrolysis of pnp-TMP by human NPP 1 .
  • the molecule with the weakest inhibitory properties was 22 that blocked 66% of NPP 1 activity and the most potent inhibitor was 23 that inhibited 93% of the hydrolysis of pnp-TMP.
  • the activity of human NPP3 was more modestly inhibited (between 1 5-23%) by the analogues 22-24.
  • analogues 22-24 were further examined at the G protein-coupled P2Y Rs, P2Y
  • analogues 22 and 24B were agonists of the P2Y , R with FiC 5 o's of 0.08 and 1 7.2 ⁇ , respectively, as compared to 0.004 ⁇ for 2-MeSADP, and were virtually ineffective agonists of P2Y 2 R, P2Y 4 R and P2Y 6 R.
  • Analogues 23A, 23B and 24A had insignificant activities at all the P2YRs tested.
  • HT-29 a cultured human colon cancer cell line, to study the effect of femiented milks on colon cancer cell growth and di fferentiation.
  • the hydrolysis of lysophospholipids and nucleotides by autotaxin (NPP2) involves a single catalytic site.
  • Patcl Barnes, A., Camacho, J ., Paterson, C, Boughtflower, R., Cousens, D., Marshall, P., Activity of diadenosine polyphosphates at P2Y receptors stably expressed in 1 321 N 1 cells. Eur. J. Pharmacol , 2001 , 430, 203-210
  • Rucckcr B., Almeida, M.E., Libermann, T.A., Zerbini, L.F., Wink, M.R., Sarkis, J.J. P., Biochemical characterization of ecto-nucleotide pyrophosphatase/ phosphodiesterase (E-NPP, E.C. 3.1 .4.1 ) from rat heart left ventricle. Mol. Cell. Biochem. , 2007, 306, 247- 254
  • Tcrkcltaub R., Physiologic and pathologic functions o f the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP 1 in calcification. Purinergic Signal, 2006, 2, 371 -377

Abstract

The present invention provides pharmaceutical compositions and methods for treatment and management of osteoarthritis using certain dinucleotide boranophosphate derivatives or nucleoside boranophosphate derivatives. The invention further provides particular diadenosine penta(ϒ-borano)phosphate derivative such as diadenosine 5',5''-P1,P5,α¸β-methylene-δ,ε- methylene-pentaphosphate-ϒ-borano and di-2'-deoxyadenosine 5',5''-P1,P5,α¸β-methylene-δ,ε-methylene pentaphosphate-ϒ-borano, and pharmaceutical compositions thereof.

Description

BORANOPHOSPHATE DERIVATIVES FOR THE TREATMENT OF OSTEOARTHRITIS
TECHNICAL FIELD
[0001 ] The present invention relates to pharmaceutical compositions and methods for treatment and management of osteoarthritis. BACKGROUND ART
[0002] Nucleoside triphosphate diphosphohydrolase-1 , -2, -3 and -8 (NTPDasel , -2, -3 and -8; EC 3.6.1 .5) and nucleotide pyrophosphatase phosphodiesterase- 1 and 3 (NPP 1 and NPP3 ; EC 3.1 .3.1 , EC 3.6.1.9) are the dominant ectonucleotidases that terminate nucleotide signaling through the hydrolysis of nucleotide agonists of the P2X and P2Y receptors (Kukulski et al, 2005; Nahum et al, 2006; Shirley et al, 2009).
[0003] NTPDasel , -2, -3 and -8 are plasma membrane-bound with an extracellular active site, which catalyze the hydrolysis of the terminal phosphate of nucleoside triphosphates, e.g., ATP and UTP, and diphosphates, e.g., ADP and UDP, at different rates. NTPDasel (CD39/ATPDase/ectoapyrase/ecto-ADPase) hydrolyzes ATP and ADP equally well (Sevigny et al , 1997), while NTPDase2 (ecto-ATPase/CD39Ll ) is a preferential triphosphonucleosidase (Heine et al, 1999). Both NTPDase3 (CD39L3/HB6) and NTPDase8 are functional intermediates between NTPDasel and NTPDase2 (Kukulski et al , 2005). NTPDase4-7, are mainly associated with intracellular organelles and are therefore not expected to significantly affect P2 receptor activation. The product of NTPDase activity, AMP, is further hydrolyzed by ecto-5 '-nucleotidase (CD73) giving adenosine, which is the natural ligand of P I receptors (Colgan et al., 2006; Resta et al., 1993).
[0004] The NPP family members are conserved eukaryotic enzymes which, as for NTPDases, exist as membrane glycoproteins with an extracellular active site. Three members of this family, in particular, NPP1 -3, are capable of hydrolyzing phosphodiester and pyrophosphate bonds found in a variety of endogenous nucleotides and their derivatives, e.g., nucleotide triphosphates (NTPs), nucleotide diphosphates (NDPs), dinucleotides, oligonucleotides, nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) and uracil diphosphate (UDP) sugars (Bollen et al. , 2000), and thus control purinergic signaling. NPP 1 can hydrolyze both phosphodiester, e.g., cAMP, and pyrophosphate (PPi), e.g., ATP, bonds (Belli et al , 1993), and in the latter case hydrolysis could be performed between phosphate-a and phosphate-/? (Stefan et al , 2005). NPP l and NPP3 are closely related, with -50% identity, and share 39% and 41 % identity, respectively, with NPP2 (Deissler et al , 1 995). NPP2 has a much lower capacity to hydrolyzc nucleotides than NPP l and NPP3 , and therefore may not play an important role in the regulation of P2 receptor activation.
[0005] NPP l is a membrane protein consisting of 925 amino acids organized into six main domains including an N-terminus cytoplasmic tail, a transmembrane domain, an extracellular region, a phosphodiesterase domain and a nuclease domain (Stefan et al , 2005). The catalytic site of NPP l is located in the extracellular phosphodiesterase domain. Gijsbers et al. (2001 ) proposed a structural model and a catalytic reaction mechanism for mouse NPPs based on secondary structure similarities to known crystal lographic structures of alkaline phosphatase, independent phosphoglycerate mutase and arylsulfatase. According to this mechanism, when a substrate, e.g., ATP, approaches the active site, one of its negatively charged oxygens can partially coordinate both binding site di-valent metal ions, thereby bringing the phosphate group into close proximity with the nucleophilic oxygen of Thr238, which can then hydrolyze the ATP molecule to generate a protein- nucleoside mono-phosphate adduct and release PPi. At the second step of the catalysis, AM P can be easily released from the protein- AMP adduct through hydrolysis occurring by an active site water molecule. In a later study, Zalatan et al. (2006) determined the structure of the bacterial NPP Xanthomonas axonopodis pv. citri (Xac) in the apo form (PDB code 2GSN), in complex with vanadate (PDB code 2GSO) and in complex with AMP (PDB codes 2GSU and 2RH6). These structures contributed to the understanding of the NPP active site and its catalytic reaction.
[0006] NPP l is expressed in different tissues, especially in bone (osteoblasts) and cartilage (chondrocytes), and has a role in regulating skeletal remodeling and calcification. Studies have shown that deficiency in bone NPP l in mice leads to hyper calcification (Okawa et al , 1998), whereas over expression is linked with reduced bone calci fication (Johnson et al. , 1999). NPP l affects skeletal remodeling and calcification by regulating processes such as bone mineralization and soft tissue calcification. The primary role of NPP l is to regulate extracellular PPi levels thereby contributing to the balance between the extracellular levels of phosphate (Pi) and PPi that is a key factor in mineralization process ( Stefan et al , 2005). Examples of other proteins that contribute to the mineralization process through the regulation of Pi and PPi levels include the tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes PPi into Pi, the progressive ankylosis protein (ANK) that is responsible for the intracellular-to-extracellular channeling of PPi, and possibly also ATPases (specific for ATP) and pyrophosphatases (specific for PPi) (Terkeltaub et al , 2006).
|00()7] Under normal physiological conditions, extracellular PPi levels are balanced leading to a normal mineralization process whereby crystals of hydroxyapatite Ca l 0(PO4)6(OH)2 are generated in their expected locations, such as bone and cartilage, through the matrix vesicles (Stefan et al , 2005). However, under pathological conditions, deficiencies in NPP l and ANK lead to low levels of extracellular PPi and consequently to ectopic calcification. In contrast, over expression of NPP l elevates extracellular PPi levels and thus leads to the deposition of calcium pyrophosphate dihydrate crystals (¾(Ρ2θ7)·2Η2θ, a condition known as the calcium pyrophosphate dehydrate (CPPD) disease. Crystal deposition often occurs in the articular cartilage and is therefore termed chondrocalcinosis. This phenomenon often occurs in aging chondrocytes and accompanies age-related osteoarthritis, a degenerative joint disease (Stefan et al , 2005). These CPPD crystals can be detected in the synovial fluid causing stiffness and severe pain and eventually leading to cartilage damage (Bjelle and Sundstrom, 1975; Nalbant et al. , 2003). PPi not only initiates but also regulates mineralization by suppressing hydroxyapatite crystal deposition from amorphous calcium phosphate (Ali, 1992; Anderson, 1988).
[0008| There is currently a lack of speci fic NPP inhibitors, and therefore, the therapeutic potential of NPP inhibition for the treatment of health disorders such as chondrocalcinosis (Johnson and Terkeltaub, 2005) remains virtually unexplored. Indeed, NPP inhibitors have scarcely been reported. Thus, suramin was reported to reduce the hydrolysis of / Nph-5 '- TMP by NPP by -36% at 250 μΜ (Ruecker et al , 2007). It is noteworthy that suramin and its derivatives antagonize most P2 receptors and also efficiently inhibit NTPDases and cannot therefore be considered as specific NPP inhibitors (Munkonda et al , 2007). Recently, [3-(/-butyldimethylsilyloxy)-phenyl]-l ,3 ,3-oxadiazole-2 (3H)-thione was reported as an NPP l inhibitor (^=100 μΜ) (Khan et al , 2009). Likewise, biscoumarin derivatives were identified as pure non-competitive inhibitors of snake venom and human NPP l enzymes, with K and IC50 values as low as 50 and 164 μΜ, respectively, for human NPP l (Choudhary et al , 2006).
[0009] US 7,368,439 discloses diribo-, di-2'-deoxyribo, and ribo-2'-deoxyribo-nucleoside boranophosphate derivatives that can be useful for prevention or treatment of diseases or disorders modulated by P2Y receptors such as type 2 diabetes, cystic fibrosis and cancer. WO 2009/066298 discloses non-hydrolyzable adenosine and uridine polyphosphate derivatives, said to be useful for prevention or treatment of diseases modulated by P2Y- receptors such as type 2 diabetes. Both publications, based on studies conducted in the laboratories of the present inventors, are herewith incorporated by reference in their entirety as i f fully described herein.
SUMMARY OF INVENTION
[0010] In one aspect, the present invention provides a pharmaceutical composition for treatment of osteoarthritis comprising a pharmaceutically acceptable carrier and either a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II:
Figure imgf000005_0001
or a diastereomer or mixture of diastereoisomers thereof,
wherein
X and X' each independently is an adenine residue of the formula la, linked through the 9 -position:
Figure imgf000005_0002
wherein
Ri is H, halogen, -O-hydrocarbyl, -S -hydro carbyl, -NR4R5, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N02, -OR4, -SI 4, -NR4R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
R2 and R3 each independently is H or hydrocarbyl;
or X and X' each independently is an uracil residue of the formula lb, linked through the 1 -position:
Figure imgf000006_0001
wherein
R6 is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -N RXRQ, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N02, -OR$, -SR8, -NR8R-9 or heteroaryl, wherein R8 and Rt> each independently is H or hydrocarbyl, or R8 and R9 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
R7 is O or S;
Y and Y' each independently is H, -OH or -NH2;
Zi , Z2, Z3, Z4 and Zs each independently is -O", -S" or -BH3 ", provided that at least one of Zi to Z5 in the general formula I is -BH3 ", and at least one of Z) to Z3 in the general formula II is -BH3 ";
W i , W2, W3 and W4 each independently is -0-, -NH- or -C(RioRn )-, wherein R! 0 and Ri i each independently is H or halogen, provided that at least one of W i to W4 in the general formula I is not -0-, and at least one of W| to W2 in the general formula II is not - 0-;
n and n' each independently is 0 or 1 ;
m is 3, 4 or 5; and
B+ represents a pharmaceutically acceptable cation. [001 1 ] In another aspect, the present invention provides a dinucleoside boranophosphatc derivative of the general formula I or a nucleoside boranophosphatc derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for use in treatment of osteoarthritis.
[0012] In a further aspect, the present invention relates to use of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for the preparation of a pharmaceutical composition for treatment of osteoarthritis.
[0013] In still a further aspect, the present invention relates to a method for treatment of osteoarthritis in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof.
[0014] In yet another aspect, the present invention relates to a diadenosine boranophosphatc derivative of the general formula II I :
Figure imgf000007_0001
or a diastereomer or mixture of diastereoisomers thereof,
wherein
Ad is an adenine residue of the formula la, linked through the 9-position:
Figure imgf000007_0002
wherein
Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N02, -OR4, -SR4, -NR R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
R and R each independently is H or hydrocarbyl ;
Y and Y' each independently is H, -OH or -NH2;
W] , W2, W3 and W4 each independently is -0-, -NH- or -C(RioRn)-, wherein Rio and Ri i each independently is H or halogen, provided that two of W | to W4 are not -0-; and
represents a pharmaceutically acceptable cation.
[0015| In still another aspect, the present invention provides a pharmaceutical composition comprising a diadcnosine boranophosphate derivative of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF DRAWINGS
[0016] Fig. 1 shows proposed structures for nucleotide-BPj Mg2+ complexes leading to products 1 and 2 (upper left side) and products 3 and 4 (upper right side), wherein Im represents imidazolyl; and Nuc represents 2'-deoxy-adenosyl.
10017) Fig. 2 shows the effects of analogues 1 -4 on NTPDase and eclo-5 '-nuclcotidase activity. Either ATP (for NTPDases) or AMP (for ecto-5 '-nucleotidase) was used as a substrate in the presence of compound 1 (panel A), 2 (panel B), 3 (panel C), or 4 (panel D). Both substrate and analogues 1-4 were used at 100 μΜ. The 100% activity was set with the nucleotide substrate alone: 1270±35, 928±55, 202±37, 129±1 1 , and 357±10 nmol of Pi min"' (mg protein"1) for NTPDasel , -2, -3 and -8, and ecto-5 '-nucleotidase, respectively. Data are presented as the mean ± SD of 3 experiments carried out in triplicate.
[ 0018] Hgs. 3A-3D show that analogues 1 -4 inhibit NPP activities. The activity of human N PP 1 and NPP3 with either pnp-TMP (3A), Ap5A (3C) or ATP (3D) as the substrate, as well as the activity of human NPP2 (membrane-bound form) with pnp-TMP (3B) as the substrate, is shown. Substrates and analogues 1-4 were studied at a concentration of 100 μΜ. In the control (ctrl), the substrate only was tested and was set to 100% of activity. The percentage of residual activity is presented at the top of each bar. Data are presented as the mean ± SD of 3-6 experiments carried out in triplicate. [0019] Fig. 4 shows that analogues 1-4 inhibit NPP activity at the surface of HTB-85 and HT29 cells. Substrate, pnp-TMP, and analogues 1-4 were used at the concentration of 100 μΜ. In the control, the substrate only was tested and was set to 100% activity. The percentage of residual activity is presented at the top of each bar. Data are presented as the mean ± SD of 3 experiments performed in triplicate.
[0020] Figs. 5A-5B show relative concentration-response plots for analogues 1 -4 via the P2Yi i receptor (5A) and the P2Y| receptor (5B). Data were obtained from 1321 N 1 cells stably expressing the P2Yu GFP receptor (5A) or P2Yi GFP receptor (5B), triggering the ligand-induced change in [Ca2+]j. Cells were pre-incubated with 2 μΜ fura-2 AM for 30 min, and the change in fluorescence (AF34o/F3iio) was monitored.
[0021 ] Fig. 6 shows that analogues 22-24 are poor substrates of human NTPDases. Bars represent the mean of one experiment performed in triplicate. The relative activity was calculated using ATP hydrolysis as 100% (white bar), which was, in nmoles Pi-min"' -mg protein" 1 , 467 for NTPDasel ; 512 for NTPDase2; 496 for NTPDase3 ; and 192 for NTPDase8.
[0022] Fig. 7 shows hydrolysis of pnp-TMP and analogues 22-24 by human NPPs. Bars represent the mean of one experiment performed in triplicate. The relative activity was calculated using pnp-TMP hydrolysis as 100% (white bar), which was, in nmoles pnp- ΤΜ Ρ-min' ' -mg protein" 1 , 24 for NPP 1 ; and 53 for NPP3.
[0023| Fig. 8 shows the effect of analogues 22-24 on human NTPDase activity. Bars represent the mean of one experiment performed in triplicate. ATP (substrate) and analogues 22-24 were all used at the concentration of 100 μΜ. The ATPase activity of each NTPDase is indicated in Fig. 6.
[0024] Fig. 9 shows that analogues 22-24 are potent inhibitors of human NPP 1. Bars represent the mean of one experiment performed in triplicate. Pnp-TMP (substrate) and analogues 22-24 were all used at the concentration of 100 μΜ. The activity with pnp-TMP of both NPPs is indicated in Fig. 7.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Nucleotide pyrophosphatase phosphodiesterase 1 -3 (NPP 1 -3) have a nucleotide pyrophosphatase activity and metabolize nucleotide triphosphate (NTP) directly to nucleotide monophosphate (NMP) and pyrophosphate (PPi) (Stefan et al. , 2006). These enzymes can be discriminated from other ectonucleotidases, such as NTPDases, by their ability to hydrolyze the diadenosine-5',5"-polyphosphate analogues, ApnA, to AMP and adenosine nucleoside 5'-(«-l ) phosphate (Rotlian el al , 2002). The regulation of the dinucleotide levels by NPPs may, in fact, be one of the dominant functions exerted by these enzymes (Stefan et al , 2006).
[0026] The field of NPP enzymology is still in its infancy. Therefore, NPP specific inhibitors, which do not affect other ectonucleotidases such as NTPDases and 5'- ectonucleotidase and do not trigger nor interfere with P2 receptor activation, would be extremely valuable. Furthermore, potent and selective NPP inhibitors could be used as therapeutic agents for the treatment of osteoarthritis (Tenenbaum et al , 1 981 ) and chondrocalcinosis (Johnson and Terkeltaub, 2005).
[0027] Nucleotide scaffolds suffer from inherent limitations as therapeutic agents as they interact with numerous proteins (Nahum et al , 2006) and are metabolically unstable (Sellers et al , 2001 ). Therefore, in the study described herein, a dinucleoside polyphosphate scaffold, which offers better stability and selectivity than nucleotides (Nahum et al , 2006), was selected for the development of NPP inhibitors, and four diadenosine polyphosphate derivatives herein identified by the Arabic numbers 1-4 in bold, more particularly, two diadenosine pentaphosphate derivatives identified as analogues 1 and 2, and two diadenosine tetraphosphate derivatives identified as analogues 3 and 4, were synthesized, taking into consideration the following points: (i) In order to prevent any activity of those derivatives toward the P2Yi receptor, the adenine ring was conserved without a mcthylthio substitution at the C-2 position, known to enhance potency toward the P2Y | receptor (Fliahu et al , 2009); (ii) Since N PP 1 hydrolyzes the ?α-?β or Pfi-Pf phosphodiester bond in ApnAs (Nahum et al , 2006), the oxygen atoms bridging I P/3 and Ρδ£ in analogue 1, and the oxygen atoms bridging Ρα-Ρβ and Ργ-Ρ^ in analogue 3, were substituted with methylene groups to obtain a hydrolysis-resistant scaffold; (iii) Since substitution of the non-bridging oxygen atom on the central phosphate of Ap3A with a BH3 group conferred resistance to hydrolysis by NPP 1 and NPP3 (Nahum et al , 2006), a central boranophosphate moiety was introduced in analogue 1 ; and (iv) Since 2'- deoxyadenosine-5 '-(a-thio)triphosphate was shown to be a potent inhibitor of NPPs (Wojcik et al , 2007), the 2'-deoxy analogues of both analogues 1 and 3, herein identified analogues 2 and 4, respectively, were synthesized as well.
[0028] The full chemical structures of analogues 1-4 are depicted in Appendix A and in Schemes 1 -2 hereinafter. Analogue 1 is also identified by the name diadenosine 5', 5"- P ' ,P5,o;,jS-methylene-5,e-methylene-pentaphosphate-7-borano; analogue 2 is also identified by the name di-2'-deoxyadenosine 5',5"-P ' ,P5 , k;</3-methylene-5, 6-methylene penta- phosphate-y-borano; analogue 3 is also identified by the name (..adenosine 5',5"-? ? αβ- methylene-7,5-methylene-tetraphosphate; and analogue 4 is also identified by the name di- 2'-deoxyadenosine 5',5"-P ' ,P5,o;,j3-methylene-7,5-methylene-tetraphosphate.
[0029] Analogues 1-4 were evaluated for their protein selectivity as either agonists of P2Y I ,2,I I receptors or substrates for the major ectonucleotidases; and their inhibitory activity and NPP subtype selectivity were evaluated by comparison of their effects on the other main ectonucleotidases, in the presence of pnp-TMP, Ap5A or ATP as substrates. In addition, these analogues were evaluated as inhibitors of cell surface NPP activity in two cancer cell lines. As described hereinafter, based on the various experiments conducted, a most selective NPP inhibitor was identi fied, and important structure-activity relationships for such inhibitors was established.
[0030] As shown in the Examples section hereinafter, analogues 1-4 strongly inhibited the metabolism of both synthetic (pnp-TMP) and natural substrates (Ap5A and ATP) by
NPP 1 . Additionally, analogues 1 and 4 inhibited the hydrolysis of pnp-TMP, Ap5A, and
ATP by NPP3, by 30, 50, and >90%, respectively. The kinetic parameters of the inhibition with pnp-TMP as substrate indicated that these analogues are NPP 1 inhibitors.
Interestingly, analogues 1 -4 were not hydrolyzed by NTPDases and did not affect hydrolysis of ATP by NTPDase l and -8. Likewise, NTPDase2 and -3 activities were reduced by __30% by these analogues. In addition, analogues 1 and 2 exhibited no inhibitory effect toward ecto-5'-nucleotidase.
[003 1 ] In view of these findings it seems that dinucleotides having either a penta- or tctraphosphate linker, such as analogues 1 and 2, and 3 and 4, respectively, do not, or barely, affect NTPDase activity, and indeed, such dinucleotides are recognized neither as substrates nor as inhibitors by these enzymes. Furthermore, the fact that analogues 1 and 2 were not recognized by ecto-5'-nucleotidase may indicate that an Ap5A scaffold is especially suitable for designing NPP-selective inhibitors.
[0032] Analogues 1 and 2 having a pentaphosphate linker inhibited Ap5A hydrolysis by NPP 1 better than analogues 3 and 4 bearing a tetraphosphate chain. Yet, analogue 1 inhibited the hydrolysis of ATP by NPP 1 better than analogue 2, implying that 1 competes with ATP because it has a 2'-OH group, i.e., recognition of ATP by NPP 1 probably involves the 2'-OH group. This requirement is not important for a NPP 1 inhibitor directed against Ap5A hydrolysis, possibly since recognition of Ap5A does not involve a 2'-OH group.
[ 00331 N PP3 -mcdiated hydrolysis of ATP was sensitive to inhibition by the dinucleotide analogues 1 -4. Apparently, the patterns of recognition of Ap5A and ATP by NPP3 are different than those for NPP l , and therefore, NPP3 was not affected by analogues 1-4 as much as NPP l .
[0034] Finally, NPP2 nucleotidase activity for both the membrane-bound forms and the secreted forms (data not shown) was highly affected by analogues 1-4. It is noteworthy that in addition to its nucleotidase activity, NPP2 prefers Iysophospholipids as substrates. Since the hydrolysis of Iysophospholipids and nucleotides is performed by the same catalytic site (Gij sbers et al. , 2003 ; Koh et al. , 2003), it may be speculated that analogues 1-4 might also inhibit the hydrolysis of Iysophospholipids by NPP2, and potentially also by NPP4-7.
[0035 j As for a /3-methylene-ADP, a known ecto-5 '-nucleotidase inhibitor (Bar and Simonson, 1 75), the methylene groups between ,β and γ,δ phosphates conferred strong inhibitory activity to analogues 3 and 4 toward ecto-5 '-nucleotidase. In contrast, compounds 1 and 2 had no effect on ecto-5 '-nucleotidase activity, further emphasizing the specificity of the latter analogues as NPP inhibitors.
[0036] Adenine nucleotide analogues with a methylene group substituting for a bridging oxygen atom and with the replacement of a nonbridging oxygen in ?a by a BH3 group, were shown to be weak agonists of P2Y | 4 6 receptors (Eliahu et al. , 2009). Likewise, replacing the Pa-P 3 bridging-oxygen in the potent P2Yi receptor agonist 2-MeS-ADP with a dihalomethylene group such as CC12 and CF2 resulted in reductions in potency of 390- and 1200-fold (Eliahu et al. , 2010). In view of these findings, in the present study, both P„- ?g and Pa-Pc bridging oxygen atoms were replaced with methylene groups to prevent activity of the dinucleotide analogues toward the P2Y receptors.
[0037] As previously shown, PJ-borano P ' ,P5-5 -diadenosine-pentaphosphate is a highly potent P2Yi receptor agonist with EC50=63 nM vs. 1 00 nM for 2-MeS-ADP (Nahum el al. , 2006). Upon replacing both
Figure imgf000012_0001
and P§-P( bridging oxygen atoms in the aforesaid compound with methylene groups, yielding analogue 1, a decreased activity toward the P2Y ] receptor was observed. The boranophosphate modification in analogue 1, which in case of Ap„A resulted in significant activity via the P2Yi receptor, increased the potency of analogue 1 toward the P2Yi receptor as compared to analogue 3, possibly indicating an improved binding to P2Y i receptor due to the presence of the relatively lipophilic borane moiety (Shaw et al , 2000). Analogue 3 was ~60-fold less potent than ATP, while Ap4A itself had a potency similar to that of ATP (Shaver et al , 2005), indicating that replacing the bridging oxygen atom with a methylene group reduces P2Yi agonist potency. Analogue 2 was >200-fold less potent than ATP, indicating the importance of the 2'- hydroxyl group for molecular recognition by the P2Y ) receptor.
[0038| Among natural diadenosine polyphosphates, only Ap4A may be considered as an agonist of P2Yn , which is normally activated by ATP derivatives (Communi et al , 2001 ; Patel et al , 2001 ). As found in the present study, Ap4A derivatives 3 and 4 were poor P2Yi i receptor agonists or completely inactive, probably due to the replacement of the bridging oxygen atoms in the polyphosphate chain with methylene groups, as observed for the P2Yi receptor. The most potent P2Yn agonist among analogues 1-4 was analogue 1. Although a boranophosphate modi fication increased the potency of analogue 1 as a P2Y n agonist as compared to the other analogues tested, it had a lower potency than ATP, with EC5o= 1 3 μΜ vs. 3.3 μΜ for ATP. The 2'-deoxy-related ApsA scaffold, 2, was inactive up to 50 μΜ, indicating the essential role of the 2'-hydroxyl group for activity at the P2Yn receptor, as noted above for the P2Y | receptor.
[0039] In a further set of experiments, various ATP analogues described in WO 2009/066298, herein identified by the Arabic numbers 21-24 in bold, more particularly, two ATP analogues having methylthio substitution at the C-2 position of the adenine, in which either the non-bridging oxygen atom at Pa: is replaced by a borano group or the P/S- Ργ bridging oxygen atom is replaced by methylene group (analogues 21 and 22, respectively), and two ATP analogues without or with methylthio substitution at the C-2 position of the adenine, having both borano group at the Pa and methylene group at position P/5-Ργ (analogues 23 and 24, respectively) were synthesized and characterized as potential NPP inhibitors. The synthesis of these analogues was carried out following the exact procedure described in WO 2009/066298. Analogues 23 and 24 were obtained as diastereomeric pairs that were separated on an HPLC column as described in the Examples.
[0040] The full chemical structures of analogues 21 -24 are depicted in Appendix A. Analogue 21 is also identified by the name 2-MeS-adenosine-5'-0-(a-borano- triphosphate); analogue 22 is also identified by the name 3,7-CH2-2-MeS-adenosine-5'- triphosphate; analogue 23 is also identified by the name adenosine-/3,7-CH2-5'-0-(o;- borano-triphosphate); and analogue 24 is also identified by the name 2-MeS-adenosine- i8,y-CH2-5'-0-(a-borano-triphosphate). The synthesis of those analogues was earned out following the exact procedure described in WO 2009/066298, and their chemical stability, enzymatic stability to alkaline phosphatase, and stability at human blood serum were evaluated as further described in the aforesaid publication.
[ 0041 ] As shown in the Examples hereinafter, while analogues 22 and 24 (B isomer) proved potent P2Y i receptor agonists, probably due to improved interactions of 2-MeS- adenine moiety (vs. adenine) with the P2Y i receptor binding-pocket (Mohamady and Jakeman, 2005), analogues 23 (A and B isomers) and 24 (A isomer) were practically inactive at this receptor. As further shown, analogues 22-24 were hardly degraded by all known sub-types of NTPDase, and are not inhibitors of NTPDase. This is a significantly beneficial feature of these analogues, as the local concentration of P2YR agonists in the vicinity of P2YRs is not reduced by binding to neighboring proteins of the NTPDase f amily. Previously we found that A- and B-isomers of analogue 21 were hydrolyzed by NTPDasc l at 14% and 59% the rate of ATP, respectively (Padyukova et al , 1 999). Here, the addition of a j3,7-bridging methylene to the scaffold of 21 significantly improved the resistance to enzymatic hydrolysis with negligible hydrolysis (0.9% of ATP) for both 24 (A and B isomers). Most importantly, as shown in this study, analogues 22-24, more specifically analogue 23, were specific inhibitors of NPP 1 and were not hydrolyzed by NTPDases or NPPs, indicating that analogue 23 could be useful as a specific inhibitor of NPP 1 .
[0042] In summary, the study described herein shows that analogues 1 -4 arc moderate but effective inhibitors of NPP 1 activity in either cell extracts or intact cells. Analogues 1 and 4 strongly blocked the activity of both NPP 1 and -3. Yet, analogue 1, found here to be an effective NPP 1 inhibitor, exhibited a low activity toward the P2Yi and P2Yn receptors. On the other hand, analogue 2 did not significantly block NPP3 activity, had no activity on NTPDasel , -2, -3, and -8, as well as ecto-5' -nucleotidase, and virtually no activity toward the P2Yi , P2Y2 and P2Yn receptors, and is therefore the most specific inhibitor of NPP 1 among the analogues tested, and can be useful in treatment or management of osteoarthritis.
[0043] As further shown, analogues 23A/23B were practically inactive at P2Y i -R and P2Y4/6-Rs; were chemically stable being hydrolyzed under conditions mimicking gastric juice pH (pH 1 .4 and 37°C) with hal f-lives of 14.1 and 47.1 h, respectively, as compared to ATP which was hydrolyzed with half-life of 3.6 h; and completely resisted hydrolysis by alkaline phosphatase for 30 min at 37°C. In addition, analogues 23A/23B were hardly degraded by all plasma-bounded NTPDase sub-types, and were not inhibitors and only weakly bound to NTPDase. Analogue 23A was a specific and potent inhibitor (AT, 500 nM) of NPP 1 and was not hydrolyzed by NTPDases or NPPs. Therefore, analogue 23A could be useful as a specific inhibitor of NPP 1 .
[0044] The present invention thus provides, in one aspect, a pharmaceutical composition for treatment of osteoarthritis comprising cither a dinuclcoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, in which at least one but preferably two of the bridging-oxygens in the dinucleoside boranophosphate derivative, more preferably the ,β- and <5,e-bridging- oxygens, and at least one of the bridging-oxygens in the nucleoside boranophosphate derivative, each is replaced with a group selected from -NH- or -C(RioRn)-, wherein Rio and Ri i each independently is H or halogen.
[0045| As used herein, the term "halogen" includes fluoro, chloro, bromo, and iodo, and is preferably fluoro or chloro.
[0()46| The term "hydrocarbyl" in any of the definitions of the different radicals R] to R , Rs and Rg refers to a radical containing only carbon and hydrogen atoms that may be saturated or unsaturated, linear or branched, cyclic or acyclic, or aromatic, and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and aryl.
[0047] The term "alkyl" as used herein typically means a straight or branched hydrocarbon radical having 1 -8 carbon atoms and includes, e.g., methyl, ethyl, n-propyl, isopropyl. n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 2,2-dimethylpropyl, n-hexyl, n- hcptyl. n-octyl, and the like. Preferred are (C ] -Cfi)alkyl groups, more preferably (C i - C4)alkyl groups, most preferably methyl and ethyl. The terms "alkenyl" and "alkynyl" typically mean straight or branched hydrocarbon radicals having 2-8 carbon atoms and 1 double or triple bond, respectively, and include ethenyl, propenyl, 3-buten- l -yl, 2- ethenylbutyl, 3-octen-l -yl, and the like, and propynyl, 2-butyn-l -yl, 3-pentyn- l -yl, and the like. Preferred are (C2-C6)alkenyl and (C2-C6)alkynyl, more preferably (C2-C4)alkenyl and (C:-C4)alkynyl. Each one of the alkyl, alkenyl and alkynyl may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH , - N OT, -CN, -SCN, aryl, or heteroaryl, and/or interrupted by one or more heteroatoms selected from nitrogen, oxygen or sul fur. [0048] The term "cycloalkyl" as used herein means a mono- or bicyclic saturated hydrocarbyl group having 3- 10 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, adamantyl, bicyclo[3.2.1 ]octyl, bicyclo[2.2.1 ]heptyl, and the like, which may be substituted, e.g., with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N02, -CN, -SCN, (C,-C8)alkyl, -0-(C, - Cg)alkyl, -S-(C ,-C8)alkyl, -NH2, -NH-(C,-C8)alkyl, or -N-((C,-C8)alkyl)2. The term "cycloalkenyl" as used herein means a mono- or bicyclic unsaturated hydrocarbyl group having 3- 1 0 carbon atoms and 1 double bond, and include cyclopropenyl, cyclobutenyl, cyclopentcnyl, cyclohcxenyl, cycloheptenyl, cyclooctenyl, cyclononcnyl, cyclodecenyl, hexahydropentalenyl, octahydronaphtalenyl, bicycle[4.2.0]oct-2-enyl, and the like.
[0049 ] The term "aryl" as used herein denotes an aromatic carbocyclic group having 6- 14 carbon atoms consisting of a single ring or multiple rings either condensed or linked by a covalent bond such as, but not limited to, phenyl, naphthyl, phenanthryl, and biphenyl. Preferred are (C6-Cio)aryl, more preferably phenyl. The aryl radical may optionally be substituted by one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N02, -CN, -SCN, (C , -C8)alkyl, -0-(C , -C8)alkyl, -S-(C , -C8)alkyl, -NH2, -NH- (C | -C8)alkyl, or -N-((C, -C8)alkyl)2.
[ 0050 J The term "heteroaryl" refers to a radical derived from a mono- or poly-cyclic heteroaromatic ring containing one to three, preferably 1 or 2, heteroatoms selected from N, O or S. When the heteroaryl is a monocyclic ring, it is preferably a radical of a 5-6- membered ring such as, but not limited to, pyrrolyl, furyl, thienyl, thiazinyl, pyrazolyl, pyrazinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, 1 ,2,3-triazinyl, 1 ,3,4-triazinyl, and 1 ,3,5-triazinyl. Polycyclic heteroaryl radicals are preferably composed of two rings such as, but not limited to, benzofuryl, isobcnzofuryl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, imidazo[ l ,2-«]pyridyl, bcnzimidazolyl, bcnzthiazolyl, benzoxazolyl, pyrido[ l ,2-a]pyrimidinyl and 1 ,3-benzodioxinyl. The heteroaryl may be substituted. It is to be understood that when a polycyclic heteroaryl is substituted, the substitution may be in any of the carbocyclic and/or heterocyclic rings.
[0051] In the groups -NP R5 and -NR8RQ, P and R5, and R8 and R9, respectively, each independently is H or hydrocarbyl as defined above, or form together with the nitrogen atom to which they are attached a saturated or unsaturated heterocyclic ring optionally containing 1 or 2 further heteroatoms selected from N, O or S. The term "heterocyclic ring" denotes a mono- or poly-cyclic non-aromatic ring of 4- 12 atoms containing at least one carbon atom and one to three, preferably 1 -2 heteroatoms selected from N, O or S, which may be saturated or unsaturated, i.e., containing at least one unsaturated bond. Preferred are 5- or 6-membered heterocyclic rings. The heterocyclic ring may optionally be substituted at any carbon atom as well as at a second nitrogen atom of the ring, if present, with one or more groups each independently selected from halogen, e.g., F, CI or Br, -OH, -N02, -CN, -SCN, (C,-C8)alkyl, -0-(C, -C8)alkyl, -S-(C, -C8)alkyl, -NH2, -NH-(C,- C8)alkyl, or -N-((C) -C8)alkyl)2. Non-limiting examples of radicals -NR4R5 and -NRsR.9 include amino, dimethylamino, diethylamino, ethylmethylamino, phenylmethyl-amino, pyrrolidino, piperidino, tetrahydropyridino, piperazino, ethylpiperazino, hydroxyethyl piperazino, morpholino, thiomorpholino, thiazolino, and the like.
[0052] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof.
[0053] In particular embodiments, the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein (i) both n and n' arc 1 , two of Wi to W4 are -0-, and the other two of Wi to W4 each independently is -C(RioRi i)-; (ii) n is 0 and n' is 1 , one of W2 to W4 is -0-, and the other two of W2 to W4 each independently is -C(R|oRn)-; or (iii) both n and n' are 0, and W2 and W3 each independently is -C(RioRi 1 )-.
[0054] In certain particular embodiments, the compound of the general formula I is a dinucleoside penta(borano)phosphate derivative wherein n and n' are 1. These derivatives may have (i) a sole borano group at position (or a'), namely, Z\ (or Z5) is -BH3 ", and Z2, Z3, Z4 and Z5 (or Z\ , Z2, Z3 and Z4) are -O"; at position β (or β'), namely, Z2 (or Z4) is -BH3 " , and Zi , Z3, Z5 and Z4 (or Zi , Z2, Z3 and Z4) are -O"; or at position γ, namely, Z3 is -BH3 ~, and Z| , Z2, Z4 and Z5 are -O"; (ii) two borano groups at positions α,β (or οί,β'), namely,
Figure imgf000017_0001
and Z2 (or Z4 and Z5) are -BH3\ and Z3, Z4 and Z5 (or Z| , Z2 and Z3) arc -O"; at positions (¾γ (or ct,y), namely, Z| and Z3 (or Z3 and Z5) are -BH3 ", and Z2, Z4 and Z5 (or Z] , Z and Z4) are -O"; at positions α,δ (or οί,δ'), namely, Zi and Z4 (or Z2 and Z5) are -BH3 ", and Z2, Z3 and Z5 (or Z\ , Z3 and Z4) are -O"; at positions a,e (or d,e'), namely, Z\ and Z5 are -BH3 ", and Z2, Z3 and Z4 are -O"; at positions β,γ (or β',γ), namely, Z2 and Z3 (or Z3 and Z ) are - BH3 ", and Zj , Z and Z5 (or Zi, Z2 and Z5) are -O"; or at positions β,δ (or β',δ'), namely, Z2 and Z4 are -BH3 ", and Z\ , Z3 and Z5 are -O"; (iii) three borano groups at positions α, ,γ (θΓ οί,β',Ύ), namely, Z| , Z2, and Z3 (or Z3, Z4 and Z5) are -BH3 ", and Z4 and Z5 (or Z| and Z2) are -O"; at positions α,β,δ (or οί,β',δ'), namely, Z\ , Z2 and Z4 (or Z2, Z4 and Z5) are -BH3 ~, and Z5 and Z3 (or Z| and Z3) are -O"; at positions ,β,€ (or ά,β',β'), namely, Zi , Z2 and Z5 (or
Figure imgf000018_0001
, Z4 and Z5) are -BH3 ", and Z3 and Z4 (or Z2 and Z3) are -O"; at positions α,γ,δ (or α',γ,δ'), namely, Z] , Z3 and Z4 (or Z2, Z3 and Z5) are -BH3 ", and Z2 and Z5 (or Z\ and Z4) are -O"; at positions ,j,e (or , '), namely, Z\ , Z3 and Z5 are -BH3 ", and Z2 and Z4 are -O"; at positions β,-γ,ε (or ',γ,ε'), namely, Z2, Z3 and Z5 (or Z\ , Z3 and Z4) are -BH3 ", and Z\ and Z4 (or Z2 and Z5) are -O"; or at positions ,γ,δ (or β',γ,δ'), namely, Z2, Z3 and Z4 are -BH3 ", and Z\ and Z5 are -O"; (iv) four borano groups at positions α,β,Ύ,δ (or α', ',γ,δ'), namely, Z\ , Z2, Z3 and Z4 (or Z2, Z3, Z4 and Z5) are -BH3 ", and Z5 (or Z\) is -O"; at positions α,β,γ,ε (or οί,β',Ύ,€'), namely, Z] , Z2, Z3 and Z5 (or Z\ , Z3, Z4 and Z5) are -BH3 ", and Z4 (or Z2) is -O"; or at positions α,β,δ,β (or ά,β',δ',β'), namely, Z\ , Z2, Z4 and Z5 are -BH3 ", and Z3 is -O"; or (v) five borano groups at positions α,β,Ύ,δ,ο, namely, Z\ , Z2, Z3, Z4 and Z5 are -BH3 ". In specific such embodiments, the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein Z3 is -BH3 ", Z, , Z2, Z4 and Z5 are -O", W2 and W3 are -0-, and W| and W4 each independently is -C(R|0Ri 1 )-.
[0055] In certain particular embodiments, the compound of the general formula I is a dinucleoside tetra(borano)phosphate derivative wherein n is 0 and n' is 1 . These derivatives may have (i) a sole borano group at position a (or a'), namely, Z\ (or Z5) is - BH ", and Z3, Z and Z5 (or Z\ , Z3 and Z4) are -O"; or at position β (or β'), namely, Z3 (or Z4) is -BH3 ~, and Zi , Z4 and Z5 (or Z\ , Z3 and Z5) are -O"; (ii) two borano groups at positions α,β (or οΐ,β'), namely, Z\ and Z3 (or Z4 and Z5) are -BH3 ", and Z4 and Z5 (or Z\ and Z3) are O"; at positions α,γ (θΓ c ,γ1), namely, Z\ and Z (or Z3 and Z5) are -BH3 ", and Z3 and Z5 (or Z| and Z4) are O"; at positions α,δ (or α',δ'), namely, Z\ and Z5 are -BH3 ", and Z3 and Z4 are O"; or at positions ,γ (or /3',Y), namely, Z3 and Z4 are BH3 ", and Z\ and Z5 are O"; (iii) three borano groups at positions α,β, (οτ οΐ,β,' ), namely, Z\ , Z3 and Z4 (or Z3, Z4 and Z5) are BH3 ", and Z (or Z\) is O"; or at positions ,β,δ (or οΐ,β',δ'), namely, Z\ , Z3 and Z¾ (or Z| , Z4 and Z5) are BH ", and Z4 (or Z3) is O"; or (iv) four borano groups at positions α,β,γ,δ, namely, Z | , Z3, Z4 and Z5 are BH3 ".
[0056] In certain particular embodiments, the compound of the general formula 1 is a dinucleoside tri(borano)phosphate derivative wherein n and n' are 0. These derivatives may have (i) a sole borano group at position (or α'), namely, Z\ (or Z5) is BH3 ", and Z3 and Z5 (or Z\ and Z ) are O"; or at position β, namely, Z3 is BH3 ", and Zi and Z5 are O"; (ii) two borano groups at positions α,β (or οΐ,β), namely, Zi and Z3 (or Z3 and Z5) are BH3 ", and Z5 (or Z| ) is O"; or at positions α,γ (or α',γ), namely, Zi and Z5 are BH3 ", and Z3 is O"; or (iii) three borano groups at positions α,β,-γ (ογ οΐ,β',1), namely, Z| , Z3 and Z5 are BH3 " .
[0057] In particular embodiments, the active agent is a compound of the general formula I, or a diastereomer or mixture of diastereoisomers thereof, wherein Y and Y' each independently is H or -OH.
| 0058] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la. In particular such embodiments, X and X' are an adenine residue, wherein Ri each independently is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl , -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached fonn a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hcteroatoms selected from N, O or S; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C | -C )alkyl, preferably (C | - C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- C8)alkynyl, preferably (C2-C4)alkynyl, or (C6-C i4)aryl, preferably (C6-C i o)aryI, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S. In more particular such embodiments, R ] each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C | -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-C |0)aryl, preferably phenyl. In most particular such embodiments, Ri each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
[0059] In specific such embodiments, the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la, wherein R i each independently is I I, -O- hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; R2 and R3 are H; Y and Y' each independently is H or -OH; n and n' are 1 ; m is 5; Z3 is -BH3 "; Zi , Z2, Z4 and Z5 are -O"; W2 and W are -0-; and Wi and W4 each independently is -C(R|0R n )-, wherein said hydrocarbyl each independently is methyl or ethyl . More speci fic such embodiments are those wherein R | each independently is H, -O- methyl or -S-methyl; R2 and R3 are H; Y and Y' each independently is H or -OH; n and n' are 1 ; m is 5; Z3 is -BH3 "; Z| , Z2, Z4 and Zs are -O"; W2 and W3 arc -0-; and W | and W4 each independently is -CH2-, -CC12- or -CF2-, preferably -CH2-. In particular speci fic such embodiments, the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an adenine residue of the formula la, wherein R| is H; R2 and R3 are H; Y and Y' each independently is -OH or H; n and n' are 1 ; m is 5; Z3 is -BH3 "; Zi , Z2, Z4 and Z5 are -O"; W2 and W3 are -0-; and W | and W4 each independently is -CH2- (analogues 1 and 2, respectively).
[0060] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula I as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X and X' each is an uracil residue of the formula lb. In particular such embodiments, X and X' arc an uracil residue, wherein R6 each independently is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, - NR8R9, heteroaryl, or hydrocarbyl; Rg and Ry each independently is H or hydrocarbyl, or Rg and R9 together with the nitrogen atom to which they are attached form a 5- or 6- membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R7 is O, wherein said hydrocarbyl each independently is (Ci -C8)alkyl, preferably (Ci -C4)alkyl, more preferably methyl or ethyl, (C -C8)alkenyl, preferably (C2-C4)alkenyl, (C2-Cg)alkynyl, preferably (C2-C4)alkynyl, or (C(,-C |.i)aryl, preferably (C6-C |0)aryl, more preferably phenyl; and said heteroaryl is a 5-6- membered monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S. In more particular such embodiments, R6 each independently is H, -O-hydrocarbyl, - S-hydrocarbyl, -NRgR9, or hydrocarbyl; R8 and R9 each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is (Ci -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-Ci0)aryl, preferably phenyl. In most particular such embodiments, Rf, each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NRgRy, or hydrocarbyl ; R8 and Ry each independently is I I or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is methyl or ethyl. [0061] In other embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof.
[0062] In particular embodiments, the active agent is a compound of the general formula 11, or a diastereomer or mixture of diastereoisomers thereof, wherein (i) n is 1 , one of W ] and W2 is -0-, and another one of W , and W2 is -C(R ioRn )-; or (ii) n is 0, and W2 is - C(R,oRn)-.
[0063] In certain particular embodiments, the compound of the general formula II is a nucleoside tri(borano)phosphate derivative wherein n is 1 . These derivatives may have (i) a sole borano group at position a, namely, Z\ is -BH3 ", and Z2 and Z3 are -O"; at position β, namely, Z2 is -BH3 ~, and Z\ and Z3 are -O"; or at position γ, namely, Z3 is -BH3 ~, and Z\ and Z2 are -O"; (ii) two borano groups at positions ,β, namely, Z] and Z2 are -BH3 ~, and Z3 is - O"; at positions α,γ, namely, Z\ and Z3 are -BH3 ", and Z2 is -O"; or at positions ,γ, namely, Z2 and Z3 are -BH ", and Z\ is -O"; or (iii) three borano groups at positions α, ,γ, namely, Zi , Z and Z3 are -BH3 ".
[0064] In specific such embodiments, the active agent is a compound of the general formula II, or a diastereomer or mixture of diastereoisomers thereof, wherein Z\ is -BH3 ", Z2 and Z3 are -O", W , is -0-, and W2 is -C(R |0Ri i )-.
[0065] In certain particular embodiments, the compound of the general formula II is a nucleoside di(borano)phosphate derivative wherein n is 0. These derivatives may have (i) a sole borano group at position a, namely, Zj is -BH3 ", and Z3 is -O"; or at position β, namely, Z3 is -BH3 ", and Z\ is -O"; or (ii) two borano groups at positions α,β, namely, Z\ and Z3 are -BH3 ".
[0066] In particular embodiments, the active agent is a compound of the general formula I I , or a diastereomer or mixture of diastereoisomers thereof, wherein Y is H or -OH.
[0067] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la. In particular such embodiments, X is an adenine residue, wherein R| is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R.s each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydro carbyl each independently is (Ci-C8)alkyl, preferably (d-C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2-C8)alkynyl, preferably (C2-C4)alkynyl, or (C6-C l 4)aryl, preferably (C6-C i())aryl, more preferably phenyl; and said heteroaryl is a 5-6- membcrcd monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S. In more particular such embodiments, R] is H, -O-hydrocarbyl, -S-hydrocarbyl, - NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (Ci -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-Cio)aryl, preferably phenyl. In most particular such embodiments, Ri is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
[0068] In specific such embodiments, the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein R\ is H, -O-hydrocarbyl, -S-hydrocarbyl, - NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; R2 and R3 are H ; Y is H or -OH; n is 1 ; m is 4; Z, is -BH3 ~; Z2 and Z3 are -O"; W, is -0-; and W2 is - C(R ioR | | )-, wherein said hydrocarbyl each independently is methyl or ethyl. More specific such embodiments are those wherein R | is H, -O-methyl or -S-methyl; R2 and R3 are H; Y is H or -OH; n is 1 ; m is 4; Z, is -BH3 "; Z2 and Z3 are -O"; W, is -0-; and W2 is -CH2-, - CC12- or -CF2-, preferably -CH2-. In one particular such embodiments, the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein Ri is H; R2 and R3 are H ; Y is -OH ; n is 1 ; m is 4; 7Λ is -BH3 "; Z2 and Z3 are -O"; W, is -0-; and W2 is -CH2- ( analogues 23). Both isomers 23A and 23B can be used, i.e., the isomers having a retention time (Rt) of 7.64 min or 9.67 min, respectively, when separated from a mixture of diastereoisomers using a semi-preparative reverse-phase Gemini 5u column (C- 1 8 1 1 OA, 250x 1 0 mm, 5 micron), and isocratic elution [ 100 mM triethylammonium acetate, pH 7: MeOH, 89: 1 1 ] with flow rate of 5 ml/min.
[0069| In another particular such embodiments, the active agent comprised within the pharmaceutical composition of the invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an adenine residue of the formula la, wherein Ri is S-methyl; R2 and R3 are H; Y is -OH; n is 1 ; m is 4; Z] is -BH3 "; Z2 and Z3 are -O"; Wi is -0-; and W2 is -CH2- (analogues 24). The preferred isomer in this case is 24A, i.e., the isomer having a retention time (Rt) of 5.29 min when separated from a mixture of diastereoisomers using a semi-preparative reverse- phase Gemini 5u column (C- 1 8 1 1 0A, 250x 10 mm, 5 micron), and isocratic elution [ 100 mM triethylammonium acetate, pH 7 : MeOH, 75 :25 J with flow rate of 5 ml/min.
[ 0070] In certain embodiments, the active agent comprised within the pharmaceutical composition of the present invention is a compound of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein X is an uracil residue of the formula lb. In particular such embodiments, X is an uracil residue, wherein R6 is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR8R9, heteroaryl, or hydrocarbyl; R8 and Rq each independently is H or hydrocarbyl, or R8 and RQ together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S ; and R7 is O, wherein said hydrocarbyl each independently is (Ci -C8)alkyl, preferably (Ci- C )alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- Cs)alkynyl, preferably (C2-C4)alkynyl, or (C6-Ci )aryl, preferably (C6-Ci0)aryl, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic hetero aromatic ring containing 1 -2 heteroatoms selected from N, O or S. In more particular such embodiments, R6 is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR8R9, or hydrocarbyl; R8 and RQ each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is (Ci -C4)alkyl, preferably methyl or ethyl, (C2-C )alkenyl, (C2-C4)alkynyl, or (C6-C io)aryl, preferably phenyl. In most particular such embodiments, Re is H, -O- hydrocarbyl, -S-hydrocarbyl, -NR8R9, or hydrocarbyl; R8 and R9 each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is methyl or ethyl.
[ 0071 J The compounds of the general formula I or II may be synthesized according to any technology or procedure known in the art. Procedures for the synthesis of compounds of the general formula I are described in detail, e.g., in US 7,368,439 and in the Examples section hereinafter. Procedures for the preparation of compounds of the general formula II may are described, inter alia, in WO 2009/066298. [0072] Both the compounds of the general formula I and the compounds of the general formula II may have one or more asymmetric centers, e.g., in the Pa, and may accordingly exist as pairs of diastereoisomers. In cases a pair of diastereoisomers exists, the separation and characterization of the different diastereoisomers may be accomplished using any technology known in the art, e.g., using HPLC. According to the present invention, treatment of osteoarthritis could be carried out by administration of all such isomers and mixtures thereof.
10073] The compounds of the general formula I are in the form of pharmaceutically acceptable salts.
[0074] In certain embodiments, the cation B is an inorganic cation of an alkali metal, e.g., lithium, sodium or potassium, or an alkaline earth metal, e.g., calcium or magnesium. In other embodiments, the cation B is ammonium (ΝΗ4 ' ) or is an organic cation derived from an amine of the formula wherein each one of the Rs independently is selected from H, C|-C22, preferably Ci-C6 alkyl, such as methyl, ethyl, propyl, isopropyl, butyl, and the like, phenyl, or heteroaryl such as pyridyl, imidazolyl, pyrimidinyl, and the like, or two of the Rs together with the nitrogen atom to which they are attached form a 3-7 membered ring optionally containing a further heteroatom selected from N, S and O, such as pyrrolydine, piperidine and morpholine.
[0075] In further embodiments, the cation B is a cationic lipid or a mixture of cationic lipids. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents. Neutral lipids include, but are not limited to, lecithins; phosphatidylethanolamine; diacyl phosphatidylethanolamines such as dioleoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyloleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine; phosphatidylcholine; diacyl phosphatidylcholines such as dioleoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, palmitoyloleoyl phosphatidylcholine and distearoyl phosphatidylcholine; phosphatidylglycerol; diacyl phosphatidylglycerols such as dioleoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol and distearoyl phosphatidylglycerol; phosphatidylserine; diacyl phosphatidylserines such as dioleoyl- or dipalmitoyl phosphatidylserine; and diphosphatidylglycerols; fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3β hydroxy-sterols. [0076] Examples of cationic lipid compounds include, without being limited to, Lipofectin* ( Li fe Technologies, Burlington, Ontario) ( 1 : 1 (w/w) formulation of the cationic lipid N-[ l -(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoylphosphatidyl-ethanolamine); Lipofectamine 1 M (Life Technologies, Burlington, Ontario) (3 : 1 (w/w) formulation of polycationic lipid 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl- 1 -propanamin-iumtrifluoroacetate and dioleoylphosphatidyl-ethanolamine), Lipofectamine Plus (Life Technologies, Burlington, Ontario) (Lipofectamine and Plus reagent), Lipofectamine 2000 (Life Technologies, Burlington, Ontario) (Cationic lipid), Effectene (Qiagen, Mississauga, Ontario) (Non liposomal lipid formulation), Metafectene (Biontex, Munich, Gennany) (Polycationic lipid), Eu-fectins (Promega Biosciences, San Luis Obispo, Calif.) (ethanolic cationic lipids numbers 1 through 12: C52H i06N6O4-4CF3CO2H, C88H I 78N804S2 '4CF3C02H, C40H84NO3PCF3CO2H, C5oH ,o3N703-4CF3C02H, C55H, 16N802 '6CF3C02I-I,
C49H io2N603-4CF3C02H, C44H89N503-2CF3C02H, CuoHzoeNuC- Sz SCFsCOzH, C ,62H330N22O9- 13CF3CO2H, C43H88N402-2CF3C02H, C43H88N403-2CF3C02H,
C4 | H7 NOKP); Cytofectene (Bio-Rad, Hercules, Cali f.) (mixture of a cationic lipid and a neutral lipid), GencPORTER* (Gene Therapy Systems, San Diego, Calif.) (formulation of a neutral lipid (Dope) and a cationic lipid) and FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, Ind.) (Multi-component lipid based non-liposomal reagent).
[0077] The pharmaceutical compositions provided by the present invention may be prepared by conventional techniques, e.g., as described in Remington: The Science and Practice of Pharmacy, 19th Ed., 1995. The compositions can be prepared, e.g., by uniformly and intimately bringing the active agent, i.e., the compound of the general formula I or II, into association with a liquid carrier, a finely divided solid earner, or both, and then, i f necessary, shaping the product into the desired formulation. The compositions may be in liquid, solid or semisolid form and may further include pharmaceutically acceptable fillers, carriers, diluents or adjuvants, and other inert ingredients and excipients. In one embodiment, the pharmaceutical composition of the present invention is formulated as nanoparticles.
[0078] The compositions can be formulated for any suitable route of administration, but they are preferably formulated for parenteral, e.g., intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, subcutaneous, transdermal or topical, or for oral administration. The dosage will depend on the state of the patient, and will be determined as deemed appropriate by the practitioner.
[0079] The pharmaceutical composition of the invention may be in the form of a sterile injectable aqueous or oleagenous suspension, which may be formulated according to the known art using suitable dispersing, wetting or suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be employed include, without limiting, water, Ringer's solution and isotonic sodium chloride solution.
[0080] The pharmaceutical compositions of the invention, when formulated for administration route other than parenteral administration, may be in a form suitable for oral use, e.g., as tablets, troches, lozenges, aqueous, or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binding agents, e.g., starch, gelatin or acacia; and lubricating agents, e.g., magnesium stearate, stearic acid, or talc. The tablets may be either uncoated or coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostcarate or glyceryl distearate may be employed. They may also be coated using the techniques described in the US Patent Nos. 4,256, 108, 4, 166,452 and 4,265,874 to form osmotic therapeutic tablets for control release. The pharmaceutical composition of the invention may also be in the form of oil-in-water emulsion.
[0081] Pharmaceutical compositions according to the invention, when formulated for inhalation, may be administered utilizing any suitable device known in the art, such as metered dose inhalers, liquid nebulizers, dry powder inhalers, sprayers, thermal vaporizers, electrohydrodynamic aerosolizers, and the like. [0082] The pharmaceutical compositions of the invention may be formulated for controlled release of the active agent. Such compositions may be formulated as controlled- release matrix, e.g., as controlled-release matrix tablets in which the release of a soluble active agent is controlled by having the active diffuse through a gel formed after the swelling of a hydrophilic polymer brought into contact with dissolving liquid (in vitro) or gastro-intestinal fluid (in vivo). Many polymers have been described as capable of forming such gel, e.g., derivatives of cellulose, in particular the cellulose ethers such as hydroxypropyl cellulose, hydroxymethyl cellulose, methylcellulose or methyl hydroxypropyl cellulose, and among the different commercial grades of these ethers are those showing fairly high viscosity. In other configurations, the compositions comprise the active agent formulated for controlled release in microencapsulated dosage form, in which small droplets of the active agent are surrounded by a coating or a membrane to form particles in the range of a few micrometers to a few millimeters.
[0083] Another contemplated formulation is depot systems, based on biodegradable polymers, wherein as the polymer degrades, the active agent is slowly released. The most common class of biodegradable polymers is the hydrolytically labile polyesters prepared from lactic acid, glycolic acid, or combinations of these two molecules. Polymers prepared from these individual monomers include poly (D,L-lactide) (PLA), poly (glycolide) (PGA), and the copolymer poly (D,L-lactide-co-glycolide) (PLC).
[0084] In another aspect, the present invention provides a dinucleoside boranophosphatc derivative of the general formula 1 or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for use in treatment of osteoarthritis.
[0085] In a further aspect, the present invention relates to use of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof, for the preparation of a pharmaceutical composition for treatment o f osteoarthritis.
[ 0086] In still a further aspect, the present invention relates to a method for treatment of osteoarthritis in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined above, or a diastereomer or mixture of diastereoisomers thereof. [0087] Osteoarthritis, also known as degenerative arthritis or degenerative joint disease, is the most common form of arthritis and refers to a group of mechanical abnormalities involving degradation of joints, including articular cartilage and subchondral bone. Symptoms may include joint pain, tenderness, stiffness, locking, and sometimes an effusion, i.e., an accumulation of excess fluid in or around the knee joint. A variety of causes including hereditary, developmental, metabolic and mechanical may initiate processes leading to loss of cartilage. When bone surfaces become less well protected by cartilage, bone may be exposed and damaged. As a result of decreased movement secondary to pain, regional muscles may atrophy, and ligaments may become more lax.
[0088] Osteoarthritis can be either primary or secondary in case there is an identifiable underlying cause, although the resulting pathology is the same. Primary osteoarthritis is a chronic degenerative disorder related to but not caused by aging. As a person ages, the water content of the cartilage decreases as a result of a reduced proteoglycan content, thus causing the cartilage to be less resilient. Without the protective effects of the proteoglycans, the collagen fibers of the cartilage can become susceptible to degradation and thus exacerbate the degeneration. Inflammation of the surrounding joint capsule can also occur, though often mild compared to that which occurs in rheumatoid arthritis. This can happen as breakdown products from the cartilage are released into the synovial space, and the cells lining the joint attempt to remove them. New bone outgrowths, called "spurs" or osteophytes, can form on the margins of the joints, possibly in an attempt to improve the congruence of the articular cartilage surfaces. These bone changes, together with the inflammation, can be both painful and debilitating. Secondary osteoarthritis is caused by other factors such as congenital disorders of joints; diabetes; inflammatory diseases, e.g., Perthes' disease and Lyme disease, and all chronic forms of arthritis, e.g., costochondritis, gout and rheumatoid arthritis; injury to joints as a result of an accident or orthodontic operations; septic arthritis, i.e., infection of a joint; ligamentous deterioration; Marfan syndrom, obesity; alkaptonuria; and hemochromatosis and Wilson's disease.
[0089] One of the conditions frequently coexisting and associated with osteoarthritis is known as the calcium pyrophosphate dehydrate (CPPD) disease, i.e., the deposition of calcium pyrophosphate dehydrate crystals in the synovial fluid, which causes sti ffness and severe pain, and eventually leads to cartilage damage. The CPPD crystals deposition is lead by excess of extracellular PPi resulting from over expression of NPP 1 . Controlled blocking of NPP 1 could thus control the concentration of extracellular PPi and consequently decrease symptoms of CPPD. Crystal formation in both articular cartilage and synovial fluid, as well as low-grade inflammation, a consequence of crystal deposition, should be monitored during therapy.
[0090] The only available treatment for osteoarthritis is symptomatic and does not deal with the causes underlying the disease. The compounds of the general formulas I and II are useful in treatment or management of osteoarthritis. The term "treatment" as used herein with respect to osteoarthritis refers to blocking of NPP 1 over expression and consequently extracellular PPi concentration, thus reducing CPPD crystals deposition and attenuating, i .e., limiting or reducing, the various symptoms of the disease as defined above. The term "management" as used herein with respect to osteoarthritis refers to a continuous treatment of the disease during which NPP 1 over expression is constantly controlled so as to maintain balanced levels of extracellular PPi thus constantly reducing CPPD crystals deposition. The term "therapeutically effective amount" as used herein refers to the quantity of the compound of the general formula I or II as defined above, or a diastereomer or mixture of diastereomers thereof, that is useful to treat or manage osteoarthritis.
1 091 ] In yet another aspect, the present invention relates to a diadenosine pcnta(" <- borano)phosphatc derivative of the general formula III as defined above, i.e., a particular embodiment of the compound defined by the general formula I above, in which a borano group replaces a non-bridging oxygen atom at position Ργ and two of the bridging- oxygens, preferably the α,β- and <5,e-bridging-oxygens, each is replaced with a group selected from -NH- or -C(Rio n)-, wherein Rio and Rn each independently is H or halogen.
[0092] In certain embodiments, the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III, wherein W2 and W3 are -0-, and W , and W4 each independently is -NH- or -C(Ri oR u )-, preferably -CH2-, -CC¾- or -CF2-.
[0093] In certain embodiments, the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III, wherein Y and Y' each independently is H or -OH.
[0094] In certain embodiments, the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each independently is an adenine residue of the formula la. In particular such embodiments, Ad each independently is an adenine residue, wherein R| each independently is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C]-C8)alkyl, preferably (Cp C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- C8)alkynyl, preferably (C2-C4)alkynyl, or (C6-C|4)aryl, preferably (C6-Cio)aryl, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S. In more particular such embodiments, R| each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and Rj each independently is H or hydrocarbyl; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C] -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-Cio)aryl, preferably phenyl. In most particular such embodiments, R| each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
[0095] In specific such embodiments, the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each is an adenine residue of the formula la, wherein R| each independently is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; R2 and R3 are H; Y and Y' each independently is H or -OH; W2 and W3 are -0-; and Wi and W4 each independently is -C(R|oRn)-, wherein said hydrocarbyl each independently is methyl or ethyl. More specific such embodiments are those wherein Ri each independently is I I, -O-methyl or -S-mcthyl; R2 and R3 arc H; Y and Y' each independently is H or -OH; W2 and W3 are -0-; and Wj and W4 each independently is -CH2-, -CC12- or -CF2-, preferably -CH2-. In particular specific such embodiments, the diadenosine penta(borano)phosphate derivative of the invention is a compound of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof, wherein Ad each is an adenine residue of the formula la, wherein Ri is H; R2 and R3 are H; Y and Y' each independently is -OH or H; W2 and W3 are -0-; and Wi and W4 are -CH2- (analogues 1 and 2, respectively). [0096] In still another aspect, the present invention provides a pharmaceutical composition comprising a diadenosine boranophosphate derivative of the general formula III as defined above, or a diastereomer or mixture of diastereoisomers thereof and a pharmaceutically acceptable carrier.
[0097] The invention will now be illustrated by the following non-limiting Examples.
EXAMPLES
Abbreviations
[0098] BPj, boranophosphate; [Ca2+]j, intracellular Ca2+ concentration; CDI, carbodiimidazole; MF, N,N-dimethylformamide; E-NPP, ecto-nucleotide pyrophosphatase/phosphodiesterase; E-NTPDasc, ecto-nucleoside triphosphate diphosphohydrolase; ESI, electron spray ionization; FBS, fetal bovine serum; HRMS- MALDI , high resolution mass spectrometry matrix-assisted laser desorption/ionization; MPLC, medium pressure liquid chromatography; pnp-TMP, thymidine 5 '- monophosphate p-nitrophenyl ester; P2R, P2 receptor; RT, room temperature; ΊΈΑΑ, triethylammonium acetate.
Experimental General
[0099] All commercial reagents were used without further purification, unless otherwise noted. All air- and moisture-sensitive reactions were carried out in flame-dried, argon- flushed, two-neck flasks sealed with rubber septa, and the reagents were introduced with a syringe. Progress of reactions was monitored by TLC using pre-coated Merck silica gel plates (60F-253). Reactants and products were visualized using UV light (Isco, UA-5). Compounds were characterized by NMR using Bruker AC-200, DPX-300 or DMX-600 spectrometers. Ή NMR spectra were measured at 200, 300 or 600 MHz. Nucleotides were characterized also by 1 P NMR in D20, using 85% H3P04 as an external reference on Bruker AC-200 and DMX-600 spectrometers. High resolution mass spectra were recorded on an AutoSpec-E FIS ION VG mass spectrometer by chemical ionization. Nucleotides were analyzed using electron spray ionization (ESI) on a Q-TOF micro-instrument (Waters, UK). Primary purification of the nucleotides was achieved on a LC (Isco UA-6) system using a column of Sephadex DEAE-A25, swollen in 1 M NaHC03 at 3°C for 24 h. The resin was washed with deionized water before use. LC separation was monitored by UV detection at 280 nm. Final purification of the nucleotides and separation of the diastereomeric pairs were achieved on an HPLC (Merck-Hitachi) system using a semi- preparative reverse-phase column (Gemini 5u C- 1 8 1 1 OA 250x 10 mm; 5 micron; Phenomenex, Torrance, USA). The purity of the dinucleotides was evaluated on an analytical reverse-phase HPLC column system (Gemini 5u C-18 1 1 OA, 1 50 x 3.60 mm; 5 micron; Phenomenex) in two-solvent systems with either solvent systems I and It or solvent system III. Solvent system I was: (A) 100 mM TEAA, pH 7, (B) MeOH; solvent system II : (A) 100 mM TEAA, pH 7, (B) CH3CN; solvent system III: (A) 0.01 M KH2P03, pH - 3.5, (B) CH3CN. The details of the solvent system gradients used for the separation of each product are provided below. The products, obtained as triethylammonium salts, were generally ¾5% pure. All reactants in moisture-sensitive reactions were dried overnight in a vacuum oven. 2',3'-0-Methoxymethylidene adenosine, 6 (Nahum et al , 2002), α, 3-methylene-ADP, 9, and 2'-dcoxy-o lS-methyleme-ADP, 13 (Davisson et al , 1 987) were prepared as previously described. α,/3-methylene-ADP and 2'-deoxy-ct!,j3- mcthylcne-ADP were separated using a MPLC system (Biotage; Kungsgatan, Uppsala, Sweden) using a RP-C 1 8 ( 12+M) column and the following gradient scheme: 3 column volumes of 100:0 (A:B ratio) (A: 100 mM TEAA; B : MeOH), 9 column volumes of a gradient from 1 00:0 to 60:30 A:B, followed by 5 column volumes of 60:30 A:B, at a flow rate of 1 2 ml/min. Typical procedure for the preparation of diadenosine-5 ',5 "-(boranated) poly- phosphonate derivatives 1-4
[00100] As depicted in Schemes 1 and 2, respectively, ¾/3-methylene-ADP(Bu3NH)+ 2, 9, and 2'-deoxy-a,lS-methylene-ADP (Bu3NH)+ 2, 13, were prepared by applying the corresponding ADP analogues through a column of activated Dowex 50WX-8 (200 mesh, H+ form). The eluate was collected in an ice-cooled flask containing tributylamine (2 eq) and EtOH. The resulting solution was freeze-dried to yield 9 and 13 as viscous oil. Bis(tributylammonium) o j3-methylene-ADP salt ( 100 mg, 0.16 mmol) was dissolved in dry DM F (2 ml), and CDI ( 1 80 mg, 1 . 1 1 mmol, 5 eq) was added. The resulting solution was stirred at RT for 1 2 h. BP,(Bu3NlT )2 , 15, ( 1 30 mg, 0.27 mmol , 1 .7 eq) in dry DMF ( 1 .5 ml), and MgCl2 ( 1 20 mg, 1 .28 mmol, 8 cquiv) were added. The resulting solution was stirred at RT for 23 h. The semisolid obtained after evaporation of the solvent was chromatographed at RT on a Sephadex DEAE-A25 column, which was swelled in 1 M NaHC0 prior to column preparation. The separation was monitored by UV detection (λ- 280 nm). A buffer gradient of water (1 1) to 0.7 M NH4HCO3 (1 1) was applied. The relevant fractions were pooled and freeze-dried to yield a white solid. Final purification was achieved on a semipreparative CI 8 HPLC column. Compound 1 was obtained in 10% yield (16 mg). Compound 3 was obtained in 20% yield (29 mg) (Pankiewicz et al, 1997). The spectral data for 3 are consistent with literature (Pankiewicz et al, 1997). Compound 2 was obtained in 21% yield (15 mg). Compound 4 was obtained in 28% yield (20 mg) after LC separation.
Diadeiiosine 5', 5"-P', P5, a, -methylene-h,t-methylene-pentaphosphate-y-borano, 1, - purification and characterization
[00101] Compound 1 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 75:25 A:B over 15 min at a flow rate of 3 ml/min. Retention time: 12.88 min. Ή NMR (D20; 300 MHz): δ 8.31 (s; H-8; 2H), 8.08 (s; H-2; 2H), 5.99 (d; J = 6.00 Hz; H-l'; 2H), (Η2' signals are hidden by the water signal at 3.78 ppm), 3.53 (m; H-3'; 2H), 3.29 (m; H-3', 2H), 3.13 (m; H-5'; 3H), 2.31 (t;J = 20.71 Hz; CH2; 3H), 0.50 (m; BH3; 3H) ppm.3,P NMR (D20; 81 MHz): 78.38 (m; P BH3; IP), 20.55 (d, J= 7.93 Hz; P„; 2P), 10.77 (m; P^; 2P) ppm. HRMS MALD1 (negative) m/z C22H27D5N10O20P5: calculated 916.0796 found 916.0791 [MD5]. TLC (NH4OH:H20: isopropanol 2:8:11), Rj = 0.17. Purity data obtained on an analytical column: retention time: 1.81 min (99.97% purity) using solvent system II with a gradient from 80:20 to 70:30 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 8.00 min (98.31% purity) using solvent system III with a gradient from 100:0 to 70:30 A:B over 10 min at a flow rate of 1 ml/min.
Di-2'-deoxy adenosine 5', 5"-P' , P5, αβ-methylene-h ,e-methylene pentaphosphate-y- borano, 2, - purification and characterization
[00102] Compound 2 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 70:30 A:B over 20 min at a flow rate of 5 ml/min. Retention time: 15.35 min. Ή NMR (D20; 300 MHz): <58.33 (s; H-8; 1H), 8.33 (s; H-8; 1H), 8.06 (s; H-2; 1H), 8.05 (s; H-2; 1H), 6.32 (t; J= 6.90 Hz; H-l'; 2H), (Η2' and H3' signals are hidden by the water signal at 3.78 ppm), 3.19 (m; H-3', 2H), 3.07 (m; H-5'; 311), 2.38 (t; J = 20.70 Hz; CH2; 3H), 0.50 (m; BH3; 3H) ppm.31P NMR (D20; 81 MHz): 76.00 (m; P BH3, IP), 17.82 (s; ?a; 2P), 9.13 (d; J= 32.08 Hz; ?β; 2P) ppm. MS ESI m/z: 899 (M" Na+). TLC (NH4OH:H20:isopropanol 2:8:11), R = 0.12. Purity data obtained on an analytical column: retention time: 2.18 min (93.1% purity) using solvent system 1 with a gradient from 70:30 to 50:50 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 1.37 min (99.5% purity) using solvent system III with a gradient from 85: 15 to 50:50 A:B over 10 min at a flow rate of 1 ml/min.
Di-2'-deoxy adenosine 5', 5"-P' , P5, at -methylene-%h-methylene-tetraphosphate, 4, - purification and characterization
[00103] Compound 4 was purified by HPLC on a semi-preparative reverse-phase column, using solvent system I, with a gradient from 95:5 to 75:25 A:B over 20 min at a flow rate of 5 ml/min. Retention time: 16.26 min. Ή NMR (D20; 300 MHz): δ 8.33 (s; H-8; 2H), 8.06 (s; H-2; 2H), 6.32 (t; J = 6.30 Hz; I -1'; 2H), (Η2' and H3' signals are hidden by the water signal at 3.78 ppm), 3.18 (m; H-3\ 2H), 3.06 (m; H-5'; 3H), 2.31 (t; J= 2.31 Hz; CH2; 3H) ppm.3,P NMR (D20; 81 MHz): 18.02 (s; P„; 2P), 8.15 (s; Ρβ; 2P) ppm. HRMS MALDI (negative) m/z C22H31N10O15P3: calculated 799.0920 found 799.0915 [M3"]. TLC (NH4OH:H20:isopropanol 2:8:11), R/= 0.22. Purity data obtained on an analytical column: retention time: 3.39 min (100% purity) using solvent system I with a gradient from 80:20 to 60:30 A:B over 10 min at a flow rate of 1 ml/min. Retention time: 1.51 min (98.5% purity) using solvent system III with a gradient from 85:15 to 50:50 A:B over 10 min at a flow rate of 1 ml/min. Plasmids
[00104] The plasmids used in this study, except for human NPP2, have all been described in published reports: human NTPDasel (GenBank accession No. U87967) (Kaczmarek et al., 1996); human NTPDase2 (NM_203368) (Knowles and Chiang, 2003); human NTPDase3 (AF033830) (Smith and Kirley, 1998); human NTPDase8 (AY330313) (Fausther el ai, 2007); human NPP1 (NMJ)06208) (Belli and Coding, 1994); and human NPP3 (NM_005021) (Jin-Hua et ai, 1997). The unpublished human NPP2 (XXU 13871, submitted to NCBI by JA Malone) was subcloned here in the expression vector pcDNA3.1.
Cell culture and transfection
[00105] HTB-85 and HT29 cell lines were grown in 10 cm-plates and were then transferred into 1 cm-plates and incubated at 37°C in a-MEM medium and Dulbecco's modified Eagle's medium/F-12 nutrient mixture (DMEM/F-12), respectively, in the presence of 10% FBS. After reaching full confluence, cells were used in intact cell assays (see below).
[00106] Ectonucleotidases were produced by transiently transfecting COS-7 cells in 10- cm plates using Lipofectamine (Invitrogen, Burlington, ON, Canada), as previously described (Kukulski et ai , 2005). Briefly, 80-90% confluent cells were incubated for 5 h at 37°C in Dulbecco's modified Eagle's medium, nutriment mix F- 12 (DMEM/F- 12) in the absence of FBS with 6 μg of plasmid DNA and 23 μΐ of Lipofectamine reagent. The reaction was stopped by the addition of an equal volume of DMEM/F- 12 containing 20% FBS and the cells were harvested 33-72 h later. The conditioned medium of NPP2- transfected cells was also collected.
100107] Green fluorescent protein (GFP) constructs of human P2Y ] and P2Yn receptors were expressed in 132 1 N 1 astrocytoma cells, which lack endogenous expression of both P2X and P2Y receptors. The respective cDNA of the given receptor gene was cloned into the pEGFPN l vector, and after transfection using the FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals, Mannheim, Germany), cells were selected with 0.5 mg/ml G3 1 8 (Merck Chemicals, Darmstadt, Germany) and grown in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 U/ml streptomycin at 37°C and 5% C02. The functional expression of the receptor was confirmed by GFP fluorescence and the change in intracellular Ca2 ' concentration ([Ca2+]j) upon incubation with the respective standard receptor agonists.
Preparation of protein fractions
J 00108 ] For the preparation of protein extracts enriched in membrane proteins, transfected cells were washed three times with Tris-saline buffer at 4°C, collected by scraping in harvesting buffer (95 mM NaCl, 0.1 mM phenylmethylsulphonyl fluoride (PMSF) and 35 mM Tris at pH 7.5), and washed twice again by 300 g centrifugation for 10 min at 3°C. Cells were re-suspended in the harvesting buffer supplemented with 10 mg/ml of aprotinin, and sonicated. Nucleus and cellular debris were discarded by centrifugation at 300 g for 10 min at 3°C and the supernatant (crude protein extract) was aliquoted and stored at -80°C until used for activity assays. The secreted form of NPP2 was prepared from the conditioned media of transfected cells, which were frozen and stored at -80°C until tested for activity. Protein concentration was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as standard (Bradford, 1976). Enzymatic activity assays
[00109] NTPDases and eto-5 '-nucleotidase. Activity was measured as previously described (Kukulski et al , 2005) in 0.2 ml of incubation medium Tris-Ringer buffer (in mM, 120 NaCl, 5 KC1, 2.5 CaCl2, 1.2 MgS03, 25 NaHC03, 5 glucose, 80 Tris, pH 7.3) at 37°C with or without analogues 1-4 (final concentration 100 μΜ), and with or without 100 μΜ ATP (for NTPDases) or 100 μΜ AMP (for ecto-5 '-nucleotidase) as a substrate. The analogues were added without ATP or AMP when tested as potential substrate, and with ATP or AMP when tested for their effect on nucleotide hydrolysis. Either NTPDase or ecto-5 '-nucleotidase protein extracts were added to the incubation mixture and pre- incubated at 37°C for 3 min. The reaction was initiated by the addition of a substrate (ATP, AMP or one of analogues 1-4) and stopped after 15 min with 50 μΐ of malachite green reagent. The released inorganic phosphate (Pj) was measured at 630 nm as previously described (Baykov et al. , 1988).
[001 10] NPPs. Evaluation of the effect of compounds 1-4 on human NPP 1 , -2 and -3 activity was carried out either with pnp-TMP, ATP or Ap5A as a substrate (Belli and Goding, 1994). The reactions were carried out at 37°C in 0.2 ml of the following incubation mixture: in mM, 1 CaCl2, 1 30 NaCl, 5 KC1 and 50 Tris, pH 8.5, with or without compounds 1-4 and/or substrates. Substrates and compounds 1-4 were all used at a final concentration of 100 μΜ. Recombinant human NPP 1 , -2 or -3 cell lysates, as well as soluble proteins containing the secreted form of NPP2, were added to the incubation mixture and preincubatcd at 37°C for 3 min. The reaction was initiated by addition of the substrate. For pnp-TMP hydrolysis, the production of p-nitrophenol was measured at 405 nm, 15 min after the initiation of the reaction. For Ap5A and ATP, the reaction was stopped after 30 min by transferring an aliquot of 0.1 ml from the reaction mixture to 0.125 ml ice-cold 1 M perchloric acid. The samples were centrifuged for 5 min at 13,000 x g. Supernatants were neutralized with 1 M KOH (3°C) and centrifuged for 5 min at 1 3 ,000 x g. An aliquot of 20 μΐ was separated by reverse-phase HPLC to evaluate the nucleotide content of each reaction sample (see below). The type of inhibition, IC5o and K{, were calculated by plotting the data of three independent experiments using pnp-TMP as a substrate according to Dixon and Cornish-Bowden kinetics.
[00111] Separation and quantification of nucleotides and dinucleotides by HPLC. An aliquot of 20 μΐ of the reaction products (described above) was used for nucleotide analysis by HPLC using a 15 cm x 3.6 mm, 3 μιτι SUPELCOSIL™ LC- 18-T column (Supelco, Bcllcfonte, PA). ATP, Ap5A, analogues 1 -4 and their hydrolysis products were separated with a mobile phase made of 25 mM TBA, 5 mM EDTA, 100 mM H2PO3/K2HPO3, pH 7.0 and 2% methanol (v/v), at a flow rate of 1 ml/min for 30 min. Separated nucleotides were detected by UV absorption at 260 and 253 nm, identified and quantified by the comparison of the retention time with the appropriate standards.
[00112] Activity assays with intact HTB-85 and HT29 cell lines. For intact cells, activity assays at the cell surface were carried out in 0.25 ml of the incubation mixture containing 1 35 mM NaCl in 24-well plates. Reaction was initiated by the addition of pnp-TMP to obtain a final concentration of 100 μΜ. After 20 min, 0.2 ml of the reaction mixture was transferred to a 96-well plate and the production of p-nitrophenol was measured at 3 10 nm.
Calcium measurements
[001 13 ] 1 321 N 1 astrocytoma cells were transfected with the respective plasmid for P2YR-GFP expression, i.e., pEGFPN l expression vector plasmids encoding the cDNA for human P2Yi or P2Yn receptors (Ecke el al. , 2006), and the P2Y2 receptor (Ginsburg- Shmuel el al. , 201 0; Tulapurkar el al. , 2004), respectively. Cells plated on cover slips (22- mm diameter) and grown to approximately 80% density were incubated with 2 μΜ fura 2/AM and 0.02% pluronic acid in HEPES-buffered saline (in mM, 135 NaCl, 5.3 C1, 1 .8 CaCl2, 1 MgCl2, 25 glucose, 20 HEPES/Tris, pH 7.3) for 30 min at 37°C. Cells were supervised ( 1 ml/min, 37°C) with different concentrations of compounds 1-4 and the change in [Ca2+]j was monitored from the respective emission intensity at 510 nm after alternating between 330 nm and 380 nm as excitation wavelengths (Ubl el al , 1998). Concentration-response data were analyzed with the SigmaPlot software (SPSS Inc., Chicago, IL, USA) using the ratio of the fluorescence intensities with 330 nm and 380 nm excitation wavelengths (Ecke el al , 2006; Ecke et al , 2008). Example 1. Synthesis of dinucleoside polyphosphonate analogues 1-4
[001 14] Dinucleoside polyphosphates are conventionally prepared via the activation of the 5'-terminal phosphate of a nucleotide, thus forming a phosphoryl donor (P-donor), followed by reaction with a non-activated nucleoside 5'-phosphate, or phosphonatc analogue (phosphoryl acceptor; P-acceptor). A common method for activation of phosphates/phosphonates uses CDI to form phosphoroimidazolides. The latter may be generated in situ or isolated prior to the reaction with the corresponding nucleotides (Zatorski el al , 1995). We had previously synthesized diadenosine (T-borano)penta- phosphate, 5, using an inorganic boranophosphate salt (BPj) (Nahum and Fischer, 2004) as a P-acceptor and two nucleoside phosphoroimidazolides (NDP-Im) as P-donors (Nahum et a!.. 2006). Here, wc used the same synthetic method to prepare the corresponding phosphonatc analogues, 1 and 2.
[00115] As depicted in Schemes 1 and 2, respectively, diadenosine o iS-5,e-dimethylene- pentaphosphonate, 1 , and di-2'-deoxyadenosine (¾|3-5,e-dimethylene-pentaphosphonate, 2, were prepared as described in the Experimental from the α, -methylene-ADP building blocks, 9, and a,|3-methylene-2'-deoxy-ADP, 13, respectively. α,/3-Methylene-ADP derivatives were synthesized as previously described (Davisson et al , 1987). As specifically shown in Scheme 1 , adenosine analogue 6, which is 2' and 3'-OH protected, was activated with tosyl chloride to form the activated nucleoside 7, which was then coupled with a tris(tetra-/7-butylammonium)methylene diphosphonate salt to form analogue 8, followed by removal of the protecting group, which provided product 9. As shown in Scheme 2, the related scaffold 13 was prepared from 2'-deoxyadenosine. A selective tosylation at the 5'-OH position of 11 was earned out at 0°C to form product 12, which was then coupled with a tris(tetra-n-butylammonium) methyl enediphosphonate salt to yield product 1 (Liang et al. , 2008).
[001 16] Nucleotides 9 and 13 were activated with CDI in situ to form P-donors 10 and 14, respectively, which were then treated with BP,, 15, as a P-acceptor. MgCl2 was added as an activator to overcome the low nuclcophilicity of BP, as a P-acceptor (Hoard and Ott, 1 965). Compounds 1 and 2 were obtained in 10% and 21 % overall yields, respectively, after LC separation.
[00117] Dinucleoside poly(borano)phosphonate 1 and 2 are probably formed due to a preorganization of a P-acceptor (BPj) and two P-donors (nucleoside phosphorimidazolides) coordinated with one Mg2+ ion, as shown in Fig. 1. Specifically, the Mg2 ion probably stabilized the folded structure, which involved two molecules of (¾ 3-methylene-ADP- imidazolide (Im) or o j3-methylene-2'-deoxy-ADP-rrn and one BPi ion. This structure provided the correct orientation and proximity for a nucleophilic attack of both nucleoside phosphoroimidazolide reactants by BPj in an octahedral complex (Fig. 1, upper left side, paths a and b). Although we expected to obtain analogues 1 and 2 as exclusive products, by-products 3 and 4 were also isolated with 20% and 28% yields, respectively. The formation of 3 and 4 products is driven by a,j3-mcthylcnc-ADP, 9, and o j3-methylenc-2'- deoxy-ADP, 13, which remained in the reaction mixture due to incomplete reaction with CDI. Thus, the activated forms o^-methylene-ADP-Im, 10, and a,j8-methylene-2'-dcoxy- ADP-Im, 14, become P-donors, whereas α,/3-methylene-ADP, 9, and a /3-methylcne-2'- deoxy-ADP, 13, rather than BP,, function as P-acceptors (Fig 1 , upper right side, path c). Furthermore, since the phosphonate is assumed to be a better nucleophile than BP,, 15, byproducts 3/4 are obtained and not adcnosine-a^-CHi-T-borano-triphosphate, 16.
[001 18] The identity and purity of compounds 1-4 were established by Ή and i P NMR, ES I or MALDI negative mass spectrometry, and HPLC in two solvent systems. 3 1 P NMR spectra of compounds 1 and 2 showed a typical Ργ signal as a multiplet at about 80 ppm in addition to two phosphonate signals at 20 and 10 ppm. Ή NMR spectra showed borane hydrogen atoms as a very broad signal at -0.3 ppm, and a typical triplet at 2.3 ppm of the bridging methylene group.
Example 2. The effect of analogues 1-4 on ectonucleotidase activity and on
recombinant ectonucleotidases
[001 19] The experiments were carried out with protein extracts of COS-7 cells transfected with an expression vector encoding one of each ectonucleotidase or, alternatively, with intact cell lines exhibiting NPP activity. In addition, medium from NPP2-transfected cells was assayed for activity to test the secreted form of this enzyme. The protein extracts and media of non-transfectcd COS-7 cells exhibited a negligible level of NTP ase and NPP activity, thus allowing the analysis of each ectonucleotidase in its native membrane-bound form (Levesque et al. , 2007).
[00120] As shown in Tabic 1 , none of the analogues 1 -4 was metabolized by human NTPDases or ecto-5 ' -nucleotidase, and all of them were modestly hydrolyzed by NPPs. While analogues 1 and 3 were hydrolyzed by NPP 1 and -3 at -7.5- 13% of the level observed for ATP hydrolysis, analogues 2 and 4 were more resistant to hydrolysis. As shown in Fig. 2, the hydrolysis of ATP by NTPDasel and -8 was not affected by any o f the analogues 1 -4 when used at the same concentration as the substrate ( 100 μΜ). NTPDase2 and -3 were modestly inhibited ( 10-30%) by these analogues. While analogues 3 and 4 inhibited ecto-5 '-nucleotidase activity by 90 and 80%, respectively, analogues 1 and 2 did not affect the latter enzymatic activity.
[0012 1 1 The effect of analogues 1 -4 on NPP activities was tested using synthetic (pnp- TMP) and natural substrates (Ap5A and ATP). The level of hydrolysis of pnp-TM P by N P P 1 was decreased by over 90% by all compounds tested, as shown in Fig. 3A. In the presence of analogues 1-4, the hydrolysis of pnp-TMP by NPP2 was similarly blocked at -95%, as shown in Fig. 3B, and at 60-70% by the secreted form of NPP2 (data not shown). The activity of NPP l with Ap5A as the substrate was reduced by 60-80% by analogues 1 , 2 and 4, and by 20% by analogue 3. When using ATP as the substrate, NPP l was inhibited by -70-80% in the presence of analogues 2 and 3, and by more than 90% by analogues 1 and 4 ( Figs. 3C-3D). The presence of analogues 1 -4 reduced the hydrolysis of pnp-TMP by NPP3 by -30% (Fig. 3A), and the hydrolysis of Ap5A by - 1 0-60% (Fig. 3C). The inhibition of NPP3 activity using ATP as the substrate was more pronounced (-90%) in the presence of analogues 1 and 4, and around 65% with analogues 2 and 3 (Fig. 3D).
Table 1 : Hydrolysis of analogues 1-4 by human ectonucleotidases
Figure imgf000040_0001
Dinuclcotide analogues 1 -4 (each at 1 00 μΜ) were incubated with the indicated ectonucleotidases. The activity with 1 00 μΜ ATP (for NTPDases) or AMP (for the ecto-5 '-nucleotidase) was set as 1 00%: 1270±35; 928±55; 2()2±37; 1 29± 1 1 ; and 357± 1 0 nmol Pi min" 1 (mg protein"' ) for NTPDase l , -2, -3, and -8, and ecto-5 '- nucleotidase, respectively. 100% of the activity with 1 00 μΜ Ap5A as a substrate was 71 ±5 and 98±9 nmol of nucleotide min" 1 (mg protein" 1 ) for NPP l and NPP3 , respectively (n=3). ND = no hydrolysis detected.
| ()() 122] As analogues 1-4 significantly reduced the activity of human NPP l , we have tested IC50 values as well as some kinetic parameters {K and K,') of the inhibition with pnp-TMP as the substrate. IC50 values were similar for all tested analogues, on the order of 1 0-60 μΜ, while inhibition constants (K ) were in the range of 10-50 μΜ, always smaller than (enzyme-substrate)-inhibitor dissociation constants (Κ,) that were in the range of 30- 1 50 μΜ. The lowest K value was observed for analogue 2, as shown in Table 2. Kinetic analysis of K, and K using the Dixon and Comish-Bowden methods showed a mixed-type, predominantly competitive, inhibition of NPP l by all tested analogues (data not shown). Table 2: IC50, Kt and K analysis of the inhibition of NPP 1 by analogues 1-4
Figure imgf000041_0001
For the determination of K and K , pnp-TMP substrate and analogues 1-4 were used in the concentration range of 2.5x 1 0"5 to l x l 0" M . For the determination of IC >, the pnp-TMP concentration was 5x l 0'5 M and the concentrations of the inhibitors were in the range of 5x 1 0"7 to 1 1 0° M. All experiments were performed three times in triplicate.
Example 3. The effect of analogues 1-4 on NPP activity at the surface of two cell lines (00123] NPP 1 is critical in regulating mineralization by generating inorganic pyrophosphate, a potent inhibitor of hydroxyapatite crystal growth. On the other hand, NPP3 is associated with carcinogenesis. Human osteoblastic SaOS-2 cells (HTB-85) are used to investigate the activity of NPP 1 (Vaingankar et al. , 2004), as well as HT29, a human colon cancer cell line (Baricault et al , 1 995). HTB-85 and HT29 catabolized pnp- TMP, indicating the presence o f NPPs at their surface. As for the enzymes obtained from cell extract, NPP activity exhibited by both cell lines was blocked by -90% by analogues 1 and 2, and by about 80% by analogues 3 and 4, as shown in Fig. 4.
Example 4. The activity of analogues 1-4 on the P2Y], P2Y2 and P2Yn receptors
[00124] GFP constructs of human P2Yi and P2Y, , receptors were expressed in 1 21 N l astrocytoma cells, which lack endogenous expression of both P2X and P2Y receptors. The cells were then incubated with various concentrations of analogues 1-4, and the Ca" response to each one of the analogues was compared with that due to ATP, as shown in Figs. 5A-5B.
| ()0125] As shown in Table 3, analogues 2-4 were weak agonists of the P2Y i receptor. Analogue 1 was 2-fold less potent than the standard agonist ATP (EC50=0. 15 μΜ) and 30- fold more potent than the Ap4A derivative 3 (EC50=9 μΜ). The 2'-deoxy analogues 2 and 4 exhibited comparably weak activities with EC5o values of SO μΜ for the P2Yj receptor. No clear plateau was reached up to 100 μΜ for analogue 2.
[00126] Analogue 1 also exhibited the highest P2Yn receptor agonist potency (EC5o=13 μΜ) among the diadenosine polyphosphate analogues. The maximal response reached -80% of that obtained with the standard agonist ATP (EC50=3.3 μΜ), but with a 4- fold lower potency. Analogue 3 was found to be a very weak agonist of the P2Y ] , receptor, with an HC5o ¾0 μΜ, whose maximal response corresponded only to 1 5% of that of ATP. The 2'-deoxy analogues 2 and 4 were both inactive at concentrations ≤>0 μΜ. Analogues 1 -4 were completely inactive toward the P2Y2 receptor at concentrations≤5 μΜ. Table 3: EC50 values for [Ca2+]i elevation by analogues 1-4, mediated by the P2Yi ,2, n receptors
Figure imgf000042_0001
a ATP was selected as the common reference agonist at both P2Y i and P2Y n receptors, although ADP is the preferred endogenous P2Y | receptor agonist.
Example 5. Adenosinc-/3,Y-CIl2-5'-0-(a-borano-triphosphate), 23, separation and characterization
[00127] The separation of the diastereomeric pair of 23, obtained as described in WO 2009/066298, was accomplished using a semi-preparative reverse-phase Gemini 5u column (C- 1 8 1 10A, 250x 10.00 mm, 5 micron) and isocratic elution using Solvent System I, by appl ying (A) 100 mM TEAA, pH 7 to (13) MeOH, at a flow rate o f 5 ml/min, at 89: 1 1 A:B at a flow rate of 5 ml/min, followed by a final separation of the two diastereoisomers using an analytical Gemini 5u column (C- 1 8 1 1 OA, 150x4.60 mm) by applying Solvent System I with a gradient from 90: 10 to 70:30 A:B over 20 min at a flow rate of 1 ml/min. Fractions containing the same isomer [Rt = 6.33 min (isomer A), 7.73 min (isomer B)] were collected and freeze-dried. The excess buffer was removed by repeated freeze-drying cycles with the solid residue dissolved each time in deionized water. Diastereoisomers 23A and 23B were obtained in 36% overall yield after LC separation. Adenosine-&,y-CH2-5'-0-( .-borano-triphosphate), 23 A, characterization
|00128) Retention time on a semi-preparative column: 7.64 min. Ή NMR (D20, 600 MHz): δ 8.59 (s, 11-8, HI), 8.25 (s, lf-2, HI), 6.14 (d,J=4.8 Hz, Η-Γ, 111), (Η2' signal is hidden by the water signal at 4.78 ppm), 4.60 (m, H-3', 1H), 4.39 (m, H-4', 1H), 4.27 (m, H-5', 1H), 4.14 (m, H-5", 1H), 2.25 (t, J= 20.4 Hz, CH2, 2H), 0.37 (m, BH3, 3H) ppm.31P NMR (D20, 600 MHz): δ 82.81 (m, P„-BH3), 13.92 (s, Ργ), 11.22 (br s, Ρβ) ppm. MS-ESI m/z: 502 (M"). TLC (NH4OH:H20:isopropanol 2:8:11), Rf = 0.23. Purity data obtained on an analytic column: Retention time: 3.55 min (100% purity) using Solvent System I with a gradient from 90:10 to 70:30 A:B over 10 min at a flow rate of 1 ml/min). Retention time: 2.53 min (95.5% purity) using Solvent System II, a gradient from 90:10 to 80:20 of (A) 0.01 M KH2P04, pH = 4.5 to (B) McOI I over 10 min at a How rate of 1 ml/min).
Adenosine- ,"Y-CH2-5'-0-(Qi-borano-triphosphate), 23 B, characterization
|()0129] Retention time on a semi-preparative column: 9.67 min. Ή NMR (D20, 300 MHz): δ 8.56 (s, H-8, 1H), 8.24 (s, H-2, 1H), 6.14 (d,J= 5.1 Hz, H-l\ 1H), (H2* signal is hidden by the water signal at 4.78 ppm), 4.52 (m, H-3', 1H), 4.39 (m, H-4', 1H), 4.23 (m, H-5', 1H), 4.17 (m, H-5", 1H), 2.30 (t,J= 20.10 Hz, CH2, 2H), 0.40 (m, BH3, 3H) ppm.31P NMR (D20, 600 MHz): δ 82.50 (m, P„-BH3), 14.10 (s, P7), 11.03 (br s, P^) ppm. MS-ESI m/z: 502 (M"). TLC (NH4OH:H20:isopropanol 2:8:11), R/= 0.23. Purity data obtained on an analytic column: Retention time: 4.09 min (92.6% purity) using Solvent System I with a gradient from 90:10 to 70:30 A:B over 10 min at a flow rate of 1 ml/min). Retention time: 3.66 min (95.5% purity) using Solvent System II with a gradient from 95:10 to 80:20 A:B over 10 min at a flow rate of 1 ml/min).
Example 6.2-MeS-adenosine-|3,7-CH2-5'-0-(a-borano-triphosphate), 24, separation and characterization
[00130] The separation of the diastereomcne pair of 24, obtained as described in WO 2009/066298, was accomplished using a semi-preparative reverse-phase Gemini 5u column (C-18 110A, 250x10.00 mm, 5 micron), and isocratic elution by applying Solvent System I (sec Example 5) at 75:25 A:B at a flow rate of 5 ml/min. Final separation of the two diastercoisomers was achieved using an analytical Gemini 5u column (C-18 11 OA, 150x4.6 mm) and Solvent System I with a gradient from 82:18 to 74:26 A:B over 20 min at a flow rate of 1 ml/min. Fractions containing the same isomer [Rt = 9.79 min (isomer A), 11.53 min (isomer B)] were collected and freeze-dried. The excess buffer was removed by repeated freeze-drying cycles with the solid residue dissolved each time in deionized water. Diastereoisomers 24A and 24B were obtained in 28% overall yield after LC separation.
2-\l eS-aile nasi ην-β,γ-αΐ 2-5 '-O-(a-boi ano-triphosphate), 24 A, characterization.
[ 00131 ) Retention time on a semi-preparative column: 5.29 min. Ή NMR (D20, 600 MHz): <5 8.30 (s, H-8, 1 H), 6. 12 (d, J = 4.98 Hz, H- l ', 1 H), (Η2' signal is hidden by the water signal at 4.78 ppm), 4.50 (m, H-3', 1 H), 4.25 (m, H-4', 1 H), 4.14 (m, H-5', 1 H), 4.05 (m, H-5", 1 H), 2.95 (s, CH3, 3H), 2.17 (t, J = 20.10 Hz, CH2, 2H), 0.42 (m, BH3, 3H) ppm. 3 ,P NMR (D20, 600 MHz): δ 83.60 (m, Pa-BH3), 14.61 (s, PT), 10.26 (br s, P^) ppm. MS- ES m/z: 548 (M"). TLC (NH4OH :H20:isopropanol 2: 8 : 1 1 ), Rf = 0.44. Purity data obtained on an analytic column: Retention time: 4.24 min (94.3% purity) using Solvent System I with a gradient from 80:20 to 60:40 A :B over 10 min at a flow rate of 1 ml/min). Retention time: 2.99 min (99.5% purity) using Solvent System II (see Example 5) with a gradient from 75 :25 to 65:35 A :B over 1 0 min at a How rate of 1 ml/min). 2-MeS-adenosine^,y-CH2-5'-0~(oi-boraiio-triphosphate), 24B, characterization
[00132] Retention time on a semi-preparative column: 5.57 min. Ή NMR (D2O, 600 MHz): <5 8.29 (s, H-8, 1 H), 6.99 ( m, H- 1 ', 1 H), (Η2' signal is hidden by the water signal at 4.78 ppm). 4.47 (m, H-3', 1 H), 4.27 (m, H-4', 1 H), 4. 1 5 (m, H-5', 1 H), 4.08 (m, H-5", 1 H), 2.49 (s, CH3, 3 H) 2. 1 8 (t, J = 19.20 Hz, CH2, 2H), 0.32 (m, BH3, 3H) ppm. 3 IP NMR (D20, 600 MHz): δ 84.13 (m, Pa-BH3), 14.85 (s, P7), 1 0.04 (br s, P^) ppm. MS-ESI m/z: 548 (M"). TLC (NH4OH:H20:isopropanol 2:8 : 1 1 ), R = 0.44. Retention time: 2.12 min (94% purity) using a gradient of (A) 1 00 mM TEAA, pH 7 to (B) CH3CN from 70:30 to 40:60 A :B over 1 0 min at a flow rate of 1 ml/min). Retention time: 1.38 min (100% purity) using Solvent System II with a gradient from 50:50 to 40:60 A:B over 10 min at a flow rate of 1 ml/min).
Example 7. The effect of analogues 22-24 on human ectonuclcotidase activity, NPP l and NPP3 activity, and on the P2Y,, P2Y2 and P2Y4 and P2Y6 receptors
(00133] Ectonucleotidases were produced by transiently transfecting 293T cells using Lipofcctamine (Invitrogen, Burlington, ON, Canada), and protein fractions were prepared as described in Experimental. [00134] NTPDase activity was measured as previously described (Kukulski et αί, , 2005) in 0.2 ml of Tris-Ringer buffer (in raM 120 NaCl, 5 C1, 2.5 CaCl2, 1 .2 MgS04, 25 NaHC03, 5 glucose, 80 Tris, pH 7.4) at 37°C with or without analogues 21 -24. NTPDase protein extracts were added to the incubation mixture and pre-incubated at 37°C for 3 min. The reaction was initiated by the addition of the substrate (ATP, ADP or one o f the analogues) to a final concentration of 1 00 μΜ and stopped after 20 min with 50 μΐ of malachite green reagent. The released inorganic phosphate (Pj) was measured at 630 nm according to Baykov et al. ( 1988). The activity obtained with protein extracts from untransfected cells was subtracted from the activity obtained with extracts from NTPDase transfected cells. The activity with this control protein extract did not exceed 5% of the activity of any NTPDase extract.
[00135] Evaluation of the inhibition of human NPP l and NPP3 activities by each one of the analogues was carried out with pnp-TMP as the substrate, as previously described previously. Reactions were carried out at 37°C in 0.2 ml of the following incubation mixture, in iiiM 1 CaCl2, 140 NaCl, 5 Cl, and 50 Tris, pH 8.5, with or without an analogue ( 1 00 μΜ). Human NPP l or NPP3 extract was added to the incubation mixture and pre-incubated at 37°C for 3 min. Reaction was initiated by the addition o f the indicated substrate (pnp-TMP or an analogue) at the final concentration of 100 μΜ. For pnp-TMP, the production of p-nitrophenol was measured at 410 nm, 20 min after the initiation of the reaction. When one of the analogues was used as a substrate, the reaction was stopped after 20 min by transferring a 0. 1 ml aliquot from the reaction mixture to 0.1 25 ml ice-cold 1 M perchloric acid. These samples were centrifuged for 5 min at 13,000 g. Supematants were neutralized with 1 M KOH (4°C) and centrifuged for 5 min at 13,000 g. An aliquot of 20 μΐ was separated by reverse-phase HPLC to evaluate the decrease in the analogue level. Protein extracts from non-transfected cells did not show any NPP activity. After NPP l and NPP3 enzymatic reactions, the substrates and their products were separated on a SUPELCOSIL™ LC- 1 8-T column ( 1 5 cm x 4.6 mm, 3 μιη Supelco, Bcllefonte, Pennsylvania, USA) with a mobile phase composed of 25 mM TBA, 5 mM EDTA, 100 mM Ι<¾Ρ042ΗΡ04, pH 7.0 and 2% (v/v) methanol at a flow rate of 1 ml/min.
[00136] As shown in Fig. 6, in comparison to the physiological substrate ATP, analogues 22-24 were almost not hydrolyzed by NTPDases. Analogue 22 was the most efficiently hydrolyzed analogue by NTPDases, and was hydrolyzed by human NTPDasc l , 3 and 8 at about 7-8% of the rate of ATP and by human NTPDase2, at about 2% of the rate o f ATP. As shown in Fig. 7, NPP 1 hydrolyzed 22 at 20% the rate of pnp-TMP hydrolysis, while human N PP3 hydrolyzed 22 and 24A, at \ 0% of the rate of pnp-TMP hydrolysis. The other analogues were hydrolyzed by these ectonucleotidases at less than 5% of the rate of pnp- TMP hydrolysis.
[00137] In general, analogues 22-24 did not affect signi ficantly the activity o f NTPDases. A weak inhibition of ATP hydrolysis by human NTPDasel ( 1 7%) was observed when equal concentrations of the substrate ATP and 23 were used, and similar levels of inhibition of human NTPDase3 activity were obtained with 22 ( 1 7%), 24A ( 16%) and 24B ( 1 8%)), as shown in Fig. 8. Similar inhibition profiles were obtained for analogues 22-24 with ADP as a substrate (data not shown).
[00138] Fig. 9 shows that all analogues 22-24 inhibited the hydrolysis of pnp-TMP by human NPP 1 . The molecule with the weakest inhibitory properties was 22 that blocked 66% of NPP 1 activity and the most potent inhibitor was 23 that inhibited 93% of the hydrolysis of pnp-TMP. The activity of human NPP3 was more modestly inhibited (between 1 5-23%) by the analogues 22-24.
1001391 The activities of analogues 22-24 were further examined at the G protein-coupled P2Y Rs, P2Y | , P2Y:, P2Y4 and P2Y6, expressed in human 1321 N 1 astrocytoma cells. As shown in Table 4, analogues 22 and 24B were agonists of the P2Y , R with FiC5o's of 0.08 and 1 7.2 μΜ, respectively, as compared to 0.004 μΜ for 2-MeSADP, and were virtually ineffective agonists of P2Y2R, P2Y4R and P2Y6R. Analogues 23A, 23B and 24A had insignificant activities at all the P2YRs tested.
Table 4: Activity of analogues 22, 23 and 24 at P2Y i/2/4/6R
Figure imgf000046_0001
* sr = slight response at 1 00 μΜ; nr = no response Example 8. In vivo models for evaluating NPP1 inhibitors activity
[00140) Several animal models to study crystal deposition diseases have been reported. Unfortunately, there are no such models that are totally satisfactory for mimicking osteoarthritis symptoms in humans, but may be useful for studying osteoarthritis aspects like CPPD decrease in deposition.
[00141 ] First promising animal model comes from the use of guinea pigs (Bendele et al , 1989). These animals spontaneously develop extensive calcification of the meniscus. The animals start calcification from birth under gene control, but it is speculated that direct intervention with the drug wil l slow the progression of the chronic crippling arthritic condition. The guinea pig model is currently being used to ascertain changes in calci fying meniscus and articular cartilage degeneration in the presence of both salt forms of phosphocitrate (Sallis and Cheung, 2003).
[00142] Mouse phenotypes express pathologic calcification in soft tissues and articular cartilage, allowing insights into the extra- and intracellular events associated with osteoarthritis (Masuda and Hirose, 2002). Using a murine progressive ankylosis model of ank/ank mice, rug et al. ( 1993) have reported a therapeutic effect of phosphocitrate to decrease calcification before the disease is fully established. Histology and clinical function tests (e.g., ability to cling to cage wire) all confirmed an impressive improvement in the disease progression. This model itself has limitations for interpreting the role of crystals in osteoarthritis because it more closely resembles reactive arthritis rather than the degenerative disease.
[00143] Using a different experimental model (the rat air pouch model), new studies conclude that increasing levels of low density lipoprotein might be beneficial in CPPD crystal-induced inflammation (Kumagai ct !. , 2001 ). In associated calcific studies in the rat with phosphocitrate, the response to a new and even more potent formulation of this compound recently was reported (Demadis et al., 2001 ). This model allowed long-term studies as a calcium-sodium salt of phosphocitrate (as opposed to the neutralized tctrasodium form) is several times stronger in blocking calcification of a chemically induced calcific plaque in rats.
[00144] All publications mentioned in this Example are herewith incorporated by reference in their entirety as if fully described herein. APPENDIX A
Figure imgf000048_0001
Figure imgf000048_0003
Figure imgf000048_0002
Figure imgf000048_0004
Scheme 1 : Synthesis of compounds 1 and 3
Figure imgf000049_0001
10
Figure imgf000049_0002
Figure imgf000049_0003
B P i 1 5
Reaction conditions
a) CH1CI2, DMAP, TsCl, RT, 12 h, 67%; b) Tetra-(n-butylammonium)methyl enediphosphonate, dry DMF, RT , 38 h, 63%; c) 1) 18% HCl, pH 2.3, RT, 3 h; and 2) 23% NH4OH, pH 9, RT, 35 min, 63%; d) CDI, DMF, RT, 12 h; e) BPi; 15, MgCl2, RT, 23 h, 1 and 3 were obtained in 1 0 and 20% yields, respectively. Scheme 2 : Synthesis of compounds 2 and 4
Figure imgf000050_0001
14
Figure imgf000050_0002
Figure imgf000050_0003
BPi 15 Ad
Reaction conditions
a) Pyridine, DMAP, TsCl, 0°C, 3 h, 62%; b) Tetra-(n-butylammonium)methyl enediphosphonate, dry DMF, RT , 38 h, 62%; c) CD1, DMF, RT, 12 h; d) BP,, 15, MgCl2, RT, 23 h, 2 and 4 were obtained in 21 and 28% overall yield of step c and d, respectively. R EFERENCES
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Claims

CLA IMS
1 . A pharmaceutical composition for treatment of osteoarthritis comprising a pharmaceutically acceptable earner and either a dinucleoside boranophosphate derivative of the general formula 1 or a nucleoside boranophosphate derivative of the general formula II :
Figure imgf000058_0001
or a diastereomer or mixture of diastereoisomers thereof,
wherein
X and X ' each independently is an adenine residue of the formula la, l inked through the 9-position:
Figure imgf000058_0002
wherein
Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -NO,, -OR4, -SR4, -N R4R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
R2 and R3 each independently is H or hydrocarbyl; or X and X' each independently is an uracil residue of the formula lb, linked through the 1 -position:
Figure imgf000059_0001
wherein
R-6 is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR8 9, heteroaryl, or hydrocarbyl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -N02, -OR8, -SR8, -NR8R9 or heteroaryl, wherein R8 and R9 each independently is H or hydrocarbyl, or R8 and R9 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hcteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl; and
R7 is O or S;
Y and Y' each independently is H, -OH or -NH2;
Zi , Z2, Z3, Z4 and Z5 each independently is -O", -S" or -BH3\ provided that at least one of Zi to Z5 in the general formula I is -BH3 ", and at least one of Zi to Z3 in the general formula II is -BH3 ";
VV | , W2, W3 and W each independently is -0-, -NH- or -C(R i R n )-, wherein R )() and R 1 1 each independently is H or halogen, provided that at least one o f W i to W4 in the general onnula I is not -0-, and at least one of W i to W2 in the general formula 11 is not - 0-;
n and n' each independently is 0 or 1 ;
m is 3, 4 or 5 ; and
B+ represents a pharmaceutically acceptable cation.
2. The pharmaceutical composition of claim 1 , comprising a dinuclcoside boranophosphatc derivative of the general formula I. or a diastcreomer or mixture of diastercoisomers thereof.
3. The phamiaceiitical composition of claim 1, comprising a nucleoside boranophosphate derivative of the general formula II, or a diastereomer or mixture of diastereoisomers thereof.
4. The pharmaceutical composition of claim 2, wherein:
(i) n and n' are 1, two of W| to W4 are -0-, and the other two of W) to W4 each independently is -C(Rio i i)-;
(ii) n is 0 and n' is 1, one of W2 to W4 is -0-, and the other two of W2 to W4 each independently is -C(RioR| i)-; or
(iii) n and n' are 0, and W2 and W3 each independently is -C(RmRi i)-.
5. The pharmaceutical composition of claim 4, wherein n and n' are 1, and:
(i) Zi (or Zs) is -BH3 ", and Z2, Z3, Z4 and Z<¾ (or Z\, Z2, Z3 and Z4) are -O"; Z2 (or Z4) is -BH3 ", and Z\, Z , Z5 and Z4 (or Z\, Z , Z3 and Z4) are -O"; or Z3 is -BH3 ~ . and Zi, Z2, Z4 and Z5 are -O";
(ii) Zi and Z2 (or Z4 and Z5) are -BH3 ~, and Z3, Z4 and Z5 (or Z\, Z2 and Z3) are -O" ; Z\ and Z3 (or Z3 and Z5) are -BH3 ~, and Z2, Z4 and Z5 (or Z\, Z2 and Z4) are - O"; Z| and Z4 (or Z2 and Z5) arc -BH3 ~, and Z2, Z3 and Z5 (or Z], Z and Z ) are -O"; Z\ and Z5 are -BH3 ", and Z2, Z3 and Z are -O"; Z2 and Z3 (or Z3 and Z4) are -BH3 ~, and Z\, Z4 and Z5 (or Z\, Z2 and Z5) are -O"; or Z2 and Z4 are -BH3 ", and Z\, Z3 and Z$ are -O";
(iii) Z], Z2, and Z3 (or Z3, Z4 and Z5) are -BH3 ~, and Z4 and Z5 (or Z\ and Z2) are - O"; Z|, Z2 and Z (or Z2, Z and Z5) arc -BH ", and Z5 and Z3 (or Z( and Z3) are -O"; Z|, Z and Z5 (or Z\, Z and Z5) are -BH3 ", and Z3 and Z4 (or Z2 and Z3) are -O ; Zi, Z3 and Z4 (or Z2, Z3 and Z5) arc -BH ", and Z2 and Z5 (or Z\ and Z4) are -O"; Z\, Z3 and Z5 arc -BH3 ~, and Z2 and Z4 are -O"; Z2, Z3 and Z5 (or Zi, Z3 and Z ) are -BH3 ", and Z| and Z4 (or Z2 and Z5) are -O"; or Z2, Z3 and Z4 are -BH3 ", and Z\ and Z5 are -O";
(iv) Zi, Z2, Z3 and Z4 (or Z2, Z3, Z4 and Z5) are -BH3 ", and Z5 (or Zi) is -O"; Zj, Z2, Z3 and Z5 (or Z], Z3, Z4 and Z5) are -BH3 ", and Z4 (or Z2) is -O"; or Z,, Z2, Z and Z5 are -BH3 ~, and Z3 is -O"; or
(v) Z), Z2, Z , Z and Z5 are -BH ".
6. The pharmaceutical composition of claim 4, wherein n is 0 and n' is 1, and: (i) Zi (or Z5) is -BH3 ~, and Z3, Z4 and Z5 (or Zi, Z3 and Z4) are -O"; or Z3 (or Z4) is -BH ~, and Z], Z4 and Zs (or Z,, Z3 and Z5) are -O";
(ii) Z| and Z3 (or Z4 and Z5) are -BH3 ~, and Z4 and Z5 (or Z| and Z3) are O"; Z, and Z4 (or Z3 and Z5) are -BH3 ", and Z3 and Z5 (or Zi and Z4) are O"; Z| and Z5 are -BH3 ", and Z3 and Z4 are O"; or Z3 and Z4 are BH3 ~, and Zi and Z5 are O"; or
(iii) Z|, Z and Z4 (or Z3, Z4 and Z5) are BH3 ", and Z5 (or Z|) is O"; Zi, Z3 and Z5 (or Z|, Z4 and Z5) are BH3 ", and Z4 (or Z3) is O"; or
(iv) Z|, Z , Z4 and Z5 are BH3 ".
7. The pharmaceutical composition of claim 4, wherein n and n' are 0, and:
(i) Z| (or Zs) is BH3 ", and Z3 and Zs (or Z| and Z3) are O"; or Z3 is BH ", and Z|
Figure imgf000061_0001
(ii) Z] and Z3 (or Z3 and Z5) are BH3 ", and Z5 (or
Figure imgf000061_0002
) is O"; or Z| and Z5 are BH3 ", and Z3 is O"; or
(iii) Z|, Z3 and Z5 are BH3 ~
8. The pharmaceutical composition of claim 5, wherein Z3 is -BH3 ", Zi, Z2, Z4 and Z5 arc -O", W2 and W3 are -0-, and W] and W4 each independently is -C(Ri0Ri 1)-.
9. The phannaceutical composition of claim 2, wherein Y and Y' each independently is H or -OH.
10. The phannaceutical composition of any one of claims 2 or 4 to 9, wherein X and X' are an adenine residue, wherein R] each independently is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl, -NR R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl. or R4 and Rs together with the nitrogen atom to which they are attached form a 5- or 6-mcmbered saturated or unsaturated heterocyclic ring optionally containing 1-2 further hctcroatoms selected from N, O or S; and R2 and R each independently is H or hydrocarbyl,
wherein said hydrocarbyl each independently is (C]-C8)alkyl, preferably (Ci- C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- C8)alkynyl, preferably (C2-C4)alkynyl, or (C6-Ci )aryl, preferably (C6-Ci0)aryl, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1-2 heteroatoms selected from N, O or S.
1 1 . The pharmaceutical composition of claim 1 0, wherein R , each independently is H, - O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R3 each independently is H or hydrocarbyl ; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C i -C4)alkyl, preferably methyl or ethyl, (C2- C4)alkenyl, (C2-C4)alkynyl, or (C6-C io)aryl, preferably phenyl.
12. The pharmaceutical composition of claim 1 1 , wherein R\ each independently is H, - O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
1 3. The pharmaceutical composition of any one of claims 2 or 4 to 9, wherein X and X' arc an uracil residue, wherein R() each independently is H, halogen, -O-hydrocarbyl, -S- hydrocarbyl, -NR8RQ, heteroaryl, or hydrocarbyl ; R8 and R9 each independently is H or hydrocarbyl, or R8 and R9 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hctcroatoms selected from N, O or S ; and R7 is O,
wherein said hydrocarbyl each independently is (C| -C8)alkyl, preferably (Cp C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- Cs)alkynyl, preferably (C2-C4)alkynyl, or (C6-C i )aryl, preferably (C6-C i0)aryl, more preferably phenyl ; and said heteroaryl is a 5-6-membcrcd monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S.
14. The pharmaceutical composition of claim 13 , wherein R6 each independently is H, - O-hydrocarbyl, -S-hydrocarbyl, -NR8R9, or hydrocarbyl; R8 and R9 each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is (Q- C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-C |0)aryl, preferabl y phenyl .
1 5. The pharmaceutical composition of claim 14, wherein R6 each independently is H, - O-hydrocarbyl, -S-hydrocarbyl, -NR8R or hydrocarbyl; RS and R9 each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is methyl or ethyl.
16. The pharmaceutical composition of claim 10, wherein R] each independently is H, - O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl ; R4 and R5 each independently is H or hydrocarbyl; R2 and R3 are H ; Y and Y ' each independently is H or -OH; n and n' arc 1 ; m is 5; Z3 is -BH3 ~; Z| , Z2, Z4 and Z5 are -O"; W2 and W3 are -0-; and W , and W4 each independentl y is -C(R ioR | 1 )-, wherein said hydrocarbyl each independently is methyl or ethyl .
17. The pharmaceutical composition of claim 16, wherein Ri each independently is H, - O-methyl or -S-methyl; R2 and R3 are H; Y and Y' each independently is H or -OH; n and n' are 1 ; m is 5; Z3 is -BH3 "; \ , Z2, Z4 and Z5 are -O"; W2 and W3 are -0-; and W| and W4 each independently is -CH2-, -CC12- or -CF2-, preferably -CH2-.
1 8. The pharmaceutical composition of claim 3, wherein:
(i) n is 1 , one of W | and W2 is -0-, and another one o f W | and W2 is -C(R i oRn )-; or
(ii) n is 0, and W2 is -C(R l 0Rn )-.
1 9. The pharmaceutical composition of claim 1 8, wherein n is 1 , and:
(i) Zi is -BH3 ", and Z2 and Z3 are -O"; Z2 is -BH3 ", and Zi and Z3 are -O"; or Z3 is - BH3 ~, and Zi and Z2 are -O";
(ii) Z] and Z2 arc -BH3 ", and Z3 is -O"; Z( and Z3 are -BH3\ and Z2 is -O"; or Z2 and Z3 arc -BH3 ", and Zi is -O"; or
(iii) Zi . Z2 and Z3 arc -BH3\
20. The pharmaceutical composition of claim 18, wherein n is 0, and:
(i) Zi is -BH3 ", and Z3 is -O"; or Z3 is -BH3 ", and Zi is -O"; or
(ii) Zi and Z3 are -BH3 ".
21 . The pharmaceutical composition of claim 19, wherein Z| is -BH3 ~, Z2 and Z3 are -O" , W , is -0-, and W2 is -C(R , 0R n )-.
22. The pharmaceutical composition of claim 3, wherein Y is H or -OH .
23. The pharmaceutical composition of any one of claims 3 or 1 8 to 22, wherein X is an adenine residue, wherein Ri is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hcteroatoms selected from N, O or S; and R2 and R3 each independently is H or hydrocarbyl,
wherein said hydrocarbyl each independently is (Ci-C8)alkyl, preferably (Q - C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2- Cs)alkynyl, preferably (CVC4)alkynyl, or (C6-C,4)aryl, preferably (C6-C io)aryl, more preferably phenyl ; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1 -2 hcteroatoms selected from N, O or S.
24. The pharmaceutical composition of claim 23 , wherein Ri is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (Ci-C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (CVC io)aryl, preferably phenyl.
25. The pharmaceutical composition of claim 24, wherein R i is H, -O-hydrocarbyl, -S- hydrocarbyl, -N R4R5, or hydrocarbyl; R4 and Rs each independently is H or hydrocarbyl ; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
26. The pharmaceutical composition of any one of claims 3 or 1 8 to 22, wherein X is an uracil residue, wherein R6 is H, halogen, -O-hydrocarbyl, -S -hydrocarbyl, -NR8R<5, heteroaryl, or hydrocarbyl; Rs and R9 each independently is H or hydrocarbyl, or R8 and Ry together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S; and R7 is O,
wherein said hydrocarbyl each independently is (C| -C8)alkyl, preferably (C | -
C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C )alkenyl, (C2- C8)alkynyl, preferably (C2-C4)alkynyl, or (C6-Ci4)aryl, preferably (C6-C i0)aryl, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S.
27. The pharmaceutical composition o f claim 26, wherein R6 is H , -O-hydrocarbyl, -S- hydrocarbyl, -N RxRy, or hydrocarbyl ; RK and \ each independently is H or hydrocarbyl ; and R7 is O, wherein said hydrocarbyl each independently is (C] -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C4)alkynyl, or (C6-Ci0)aryl, preferably phenyl.
28. The pharmaceutical composition of claim 27, wherein R6 is H, -O-hydrocarbyl, -S- hydrocarbyl, -NRgRg, or hydrocarbyl; R8 and R9 each independently is H or hydrocarbyl; and R7 is O, wherein said hydrocarbyl each independently is methyl or ethyl.
29. The pharmaceutical composition of claim 23 , wherein R| is H, -O-hydrocarbyl, -S- hydrocarbyl, -NR4R5, or hydrocarbyl ; R4 and R5 each independently is H or hydrocarbyl ; R2 and R3 are H ; Y is H or -OH; n is 1 ; m is 4; Z , is -BH3 "; Z2 and Z arc -O"; W, is -0-; and W2 is -C(R ioR n )-, wherein said hydrocarbyl each independently is methyl or ethyl .
30. The pharmaceutical composition of claim 29, wherein Ri is H, -O-methyl or -S- methyl; R2 and R3 are H; Y is H or -OH; n is 1 ; m is 4; Zi is -BH3 "; Z2 and Z3 are -O"; Wi is -0-; and W2 is -CH2-, -CC12- or -CF2-, preferably -CH2-.
3 1 . The phannaceutical composition of claim 1 , wherein B is a cation o f an alkali metal. N i l.) ' , an organic cation of the formula R4N ' wherein each one of the Rs independently is H or C i -C22, preferably C | -C6, alkyl, a cationic lipid or a mixture of cationic lipids.
32. The phannaceutical composition of any one of claims 1 to 3 1 , for intravenous, intraarterial, intramuscular, intraperitoneal, intrathecal, subcutaneous, transdermal, topical or oral administration.
33. A dinucleoside boranophosphatc derivative o f the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined in claim 1 , or a diastercomer or mixture of diastereoisomers thereof, for use in treatment of osteoarthritis.
34. Use of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined in claim 1 . or a diastereomer or mixture of diastereoisomers thereof, for the preparation of a phannaceutical composition for treatment of osteoarthritis.
35. A method for treatment of osteoarthritis in an individual in need thereof, comprising administering to said individual a therapeutical ly effective amount of a dinucleoside boranophosphate derivative of the general formula I or a nucleoside boranophosphate derivative of the general formula II as defined in claim 1 , or a diastereomer or mixture of diastereoisomers thereof.
36. A diadenosine boranophosphate derivative of the general formula III:
Figure imgf000066_0001
or a diastereomer or mixture of diastereoisomers thereof,
wherein
Ad is an adenine residue of the fonnula la, linked through the 9-position:
wh
Figure imgf000066_0002
R i is H, halogen, -O-hydrocarbyl , -S-hydrocarbyl, -NR4R5, hetcroaryl , or hydrocai'byl optionally substituted by one or more groups each independently selected from halogen, -CN, -SCN, -NC)2, -OR4, -SR4, -NR4R5 or heteroaryl, wherein R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a saturated or unsaturated heterocyclic ring optionally containing 1 -2 further heteroatoms selected from N, O or S, wherein the additional nitrogen is optionally substituted by alkyl ; and
R2 and R3 each independently is H or hydrocarbyl;
Y and Y ' each independently is H, -OH or -NH2;
W i , W2, W3 and W4 each independently is -0-, -NH- or -C(Ri0Ri i)-, wherein io and R11 each independently is H or halogen, provided that two of W| to W4 are not -0-; and
B+ represents a pharmaceutically acceptable cation.
37. The diadenosine boranophosphate derivative of claim 36, wherein W2 and W3 are - 0-, and W, and W4 each independently is -NH- or -C(RioRn)-, preferably -CH2-, -CC12- or
-CF2-.
38. The diadenosine boranophosphate derivative of claim 36, wherein Y and Y' each independently is H or -OH.
39. The diadenosine boranophosphate derivative of any one of claims 36 to 38, wherein R i each independently is H, halogen, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, heteroaryl, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl, or R4 and R5 together with the nitrogen atom to which they are attached form a 5- or 6-membered saturated or unsaturated heterocyclic ring optionally containing 1 -2 further hctcroatoms sel ected from
N, O or S; and R2 and R3 each independently is 11 or hydrocarbyl,
wherein said hydrocarbyl each independently is (C | -C8)alkyl, preferably (Cj -
C4)alkyl, more preferably methyl or ethyl, (C2-C8)alkenyl, preferably (C2-C4)alkenyl, (C2-
Cg)alkynyl, preferably (C2-C4)alkynyl, or (C6-Ci4)aryl, preferably (C6-Ci0)aryl, more preferably phenyl; and said heteroaryl is a 5-6-membered monocyclic heteroaromatic ring containing 1 -2 heteroatoms selected from N, O or S.
40. The diadenosine boranophosphate derivative of claim 39, wherein R | each independently is H, -O-hydrocarbyl. -S-hydrocarbyl, -NR4R5, or hydrocarbyl ; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 each independently is H or hydrocarbyl, wherein said hydrocarbyl each independently is (C| -C4)alkyl, preferably methyl or ethyl, (C2-C4)alkenyl, (C2-C )alkynyl, or (C6-Ci0)aryl, preferably phenyl.
41 . The diadenosine boranophosphate derivative of claim 40, wherein Ri each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR4R5, or hydrocarbyl ; R4 and R5 each independently is H or hydrocarbyl; and R2 and R3 are H, wherein said hydrocarbyl each independently is methyl or ethyl.
42. The diadenosine boranophosphate derivative of claim 39, wherein
Figure imgf000067_0001
each independently is H, -O-hydrocarbyl, -S-hydrocarbyl, -NR R5, or hydrocarbyl; R4 and R5 each independently is H or hydrocarbyl ; R2 and R3 are H, Y and Y' each independently is H or -OH; W2 and W3 are -0-; and Wi and W each independently is -C(RioRi i)-, wherein said hydrocarbyl each independently is methyl or ethyl.
43. The diadenosinc boranophosphate derivative of claim 42, wherein Ri each independently is H, -O-methyl or -S-methyl; R2 and R3 are H; Y and Y' each independently is H or -OH; W2 and W3 are -0-; and Wi and W4 each independently is -CH2-, -CC12- or - CIV, preferably -CH2-.
44. The diadenosinc boranophosphate derivative of claim 36, wherein B is a cation of an alkali metal, NH4 an organic cation of the formula R4N+ wherein each one of the Rs independently is H or C i -C22, preferably C i -C , alkyl, a cationic lipid or a mixture of cationic lipids.
45. A pharmaceutical composition comprising a diadenosine boranophosphate derivative according to any one of claims 36 to 44 and a pharmaceutically acceptable carrier.
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