WO2012032513A1 - Dérivés de boranophosphate destinés au traitement de l'arthrose - Google Patents

Dérivés de boranophosphate destinés au traitement de l'arthrose 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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Bilha Fischer
Jean Sevigny
Shay Eliahu
Joanna Lecka
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Bar-Ilan University
Universite Laval
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A61K31/7072—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid having two oxo groups directly attached to the pyrimidine ring, e.g. uridine, uridylic acid, thymidine, zidovudine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H23/00—Compounds 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

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Abstract

La présente invention concerne des compositions pharmaceutiques et des procédés de traitement et de gestion de l'arthrose au moyen de certains dérivés de boranophosphate dinucléotide ou de boranophosphate nucléoside. L'invention concerne, en outre, un dérivé particulier de diadénosine penta(γ-borano)phosphate tel qu'une diadénosine 5',5''-P1,P5,α¸β-méthylène-δ,ε- méthylène-pentaphosphate-γ-borano et di-2'-désoxyadénosine 5',5''-P1,P5,α¸β-méthylène-δ,ε-méthylène pentaphosphate-γ-borano, et des compositions pharmaceutiques de ceux-ci.
PCT/IL2011/000713 2010-09-07 2011-09-07 Dérivés de boranophosphate destinés au traitement de l'arthrose WO2012032513A1 (fr)

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US11633416B1 (en) 2020-03-06 2023-04-25 Arcus Biosciences, Inc. Oral formulations of CD73 compounds
US11780849B2 (en) 2020-05-04 2023-10-10 Volastra Therapeutics, Inc. Imino sulfanone inhibitors of ENPP1
US12091412B2 (en) 2020-06-16 2024-09-17 Volastra Therapeutics, Inc. Heterocyclic inhibitors of ENPP1

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