US20210317156A1 - New polymer linked multimers of guanosine-3', 5'-cyclic monophosphates - Google Patents

New polymer linked multimers of guanosine-3', 5'-cyclic monophosphates Download PDF

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US20210317156A1
US20210317156A1 US16/329,031 US201716329031A US2021317156A1 US 20210317156 A1 US20210317156 A1 US 20210317156A1 US 201716329031 A US201716329031 A US 201716329031A US 2021317156 A1 US2021317156 A1 US 2021317156A1
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cyclic monophosphate
guanosine
phenyl
thio
cyclic
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Hans-Gottfried Genieser
Frank Schwede
Andreas Rentsch
Valeria Marigo
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Calcimedica Inc
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Mireca Medicines GmbH
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Definitions

  • the present invention relates to novel polymer linked multimers of guanosine-3′, 5′-cyclic nucleotide monophosphates, including tethered di-, tri- and tetramers and their application in the fields of medicine and pharmacy.
  • the invention also relates to specific monomers as precursors.
  • the invention further relates to the use of such compounds as reagents for signal transduction research and as modulators of cyclic nucleotide-regulated binding proteins and isoenzymes thereof, and as ligands for affinity chromatography, for antibody production or for diagnostic applications e.g. on chip surfaces.
  • cGMP Guanosine-3′,5′-cyclic monophosphate
  • the cGMP signaling cascade therefore has been recognized as a potential pharmacological target and is investigated by numerous academic groups and pharmaceutical companies. Research in this field demands for compounds that effectively modulate different targets of the said cascade. For this purpose, a number of cGMP analogues featuring cell permeability (in contrast to cGMP), enhanced activity and increased resistance to degradation by phosphodiesterases (PDE) have been established.
  • cell permeability in contrast to cGMP
  • PDE phosphodiesterases
  • cGMP analogues need to comply with a complex profile of characteristics that is unique for each biological system, to achieve a maximum effect. While applied compounds are usually selected for their ability to interfere with the main target of a studied mechanism of a disease, condition or disorder, there are always several required characteristics of a compound, that cannot be predicted and demand for testing a large set of analogues. Accordingly, for the increasing number of applications there is growing need for constantly expanding the group of available cGMP analogues with derivatives that feature another combination of characteristics as well as further improved activation potential, target specificity or multi target effects. Also tailor-made modifications such as reporting groups are desired variations for instance for research or diagnostic purposes.
  • cGMP dependent protein kinase PKG
  • I ⁇ , Iß and II three isoforms
  • a desired selective biological effect is always also an issue of the activation potential.
  • PLM polymer linked multimers
  • This concept comprises the idea of achieving a strong enhancement of activity through addressing multiple binding sites with a single molecule.
  • PMD polymer linked dimeric cGMP
  • the achieved objective of the present invention has been to provide new polymer linked multimeric analogues of guanosine-3′,5′-cyclic monophosphate (PLMs) including di-, tri- and tetrameric derivatives, which are compared to monomeric analogues used so far, improved in terms of PKG Iß and/or PKG II activation potential.
  • PLMs guanosine-3′,5′-cyclic monophosphate
  • the new PLMs additionally interfere with PKG I ⁇ , wherein it is even more preferred that the activation potential for this isoform exceeds the previously reported one for a polymer linked dimer.
  • Another objective of the invention has been, to provide PKG activators, which can be functionalized (e.g. with a reporting group), while essentially maintaining their target affinity.
  • a further objective of the invention has been to provide the new PLMs as pharmaceutically acceptable analogues for treating or diagnosing a disease, condition or disorder associated with dysregulation of a cGMP-effected cellular target, wherein additional targets can be, including, but not limited to, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, a phosphodiesterase (PDE) and a cGMP-gated channel (CNGC).
  • HCNGC hyperpolarization-activated cyclic nucleotide-gated
  • PDE phosphodiesterase
  • CNGC cGMP-gated channel
  • the objective of the invention has been to provide the new PLMs for application as research tool to identify and validate the cGMP-system in cell cultures or tissues or as a diagnostic tool.
  • Embodiments of the invention are directed to new polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG), and related monomeric precursors thereof.
  • cGMP polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate
  • PKG cGMP dependent protein kinase
  • Another achieved objective of the present invention has been to provide related monomeric compounds, which may serve as monomeric precursors of the multimers of the present invention and/or may also show modulating activity.
  • another objective of the invention has been to provide related monomeric compounds as pharmaceutically acceptable analogues for treating or diagnosing a disease, condition or disorder associated with dysregulation of a cGMP-effected cellular target, wherein additional tar-gets can be, including, but not limited to, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, a phosphodiesterase (PDE) and a cGMP-gated channel (CNGC).
  • HCN hyperpolarization-activated cyclic nucleotide-gated
  • PDE phosphodiesterase
  • CNGC cGMP-gated channel
  • the objective of the invention has been to provide the new related monomeric compounds for application as research tool to identify and validate the cGMP-system in cell cultures or tissues or as a diagnostic tool.
  • Embodiments of the invention are directed to new related monomeric compound analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG).
  • FIG. 1 Previously reported polymer linked dimeric cGMP 5
  • FIG. 2 Example of a trimeric compound according to the invention, illustrating the used variables.
  • FIGS. 3 to 5 In vitro activation of PKG isoforms by polymer linked cGMP derivatives featuring different spacer lengths with and without PET-modification ( FIG. 3 ), varied linking position ( FIG. 4 ) and unequal cGMP (analogue) units with and without unequal linking positions ( FIG. 5 ).
  • PKG isozymes I ⁇ (0.2 nM), I ⁇ (0.15 nM) and II (0.5 nM) were incubated with different concentrations (10 pM to 6 ⁇ M) of compounds of the invention and cGMP as reference compound at room temperature for 60 min.
  • the activation values of the compounds are expressed as relative PKG activation compared to cGMP with cGMP set as 1 for each kinase isozyme.
  • the K a -values of cGMP for half-maximal kinase activation were 28 nM for I ⁇ , 425 nM for I ⁇ and 208 nM for II.
  • Compound numbers refer to analogues displayed in Table 13.
  • FIG. 6 Expression of PKG isoforms in 661W cells.
  • RT-PCR on cDNA from mRNA extracted from 661W cell The 661W cell line expresses the PKG isoforms I ⁇ and II. Heart and muscle tissues were used as positive controls.
  • FIG. 7 Increased cell death in the 661W cell line after treatment with different polymer linked dimeric cGMP analogues.
  • the new polymer linked multimeric cGMP analogues of the invention are compounds having the formula (I) or (II)
  • G units G 1 and G 2 independently are compounds of formula (III) and G units G 3 and G 4 independently from G 1 and G 2 and independently from each other are compounds of formula (III) or absent, wherein in case of formula (II) G 4 is always absent if G 3 is absent,
  • R 1 , R 4 , R 5 , R 7 and R 8 independently can be equal or individual for each G unit (G 1 , G 2 , G 3 and G 4 ), while
  • linking residues LR 1 , LR 2 , LR 3 and LR 4 independently can replace or covalently bind to any of the particular residues R 1 , R 4 and/or R 5 of the G units (G 1-4 ) they connect,
  • an endstanding group of the particular residue (R 1 , R 4 and/or R 5 ), as defined above, is transformed or replaced in the process of establishing the connection and is then further defined as part of the particular linking residue (LR 1-4 ) within the assembled compound,
  • G 1 , G 2 , G 3 and G 4 can further be salts and/or hydrates
  • G 1 , G 2 , G 3 and G 4 can optionally be isotopically or radioactively labeled, be PEGylated, immobilized or be labeled with a dye or another reporting group,
  • the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein at least two G units are unequally substituted.
  • the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein in case of formula (I) G3 and G4 are absent, or in case of formula (II) G 3 , G 4 , LR 3 and LR 4 are absent; and wherein R 4 is not H and/or R 5 is not NH 2 .
  • the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein at least one G unit is linked via a position other than R 1 .
  • the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein in case of formula (I) G 3 and G 4 are absent, or in case of formula (II) G 3 , G 4 , LR 3 and LR 4 are absent.
  • any of the precedingly defined compounds of formula (I) and/or (II) may be a compound, wherein all R 7 are SH and all R 8 are O, or all R 7 are O and all R 8 are OH.
  • Halogen refers to F, Cl, Br, and I.
  • Alkyl refers to an alkyl group, which is a hydrocarbon moiety with 1 to 28, preferably 1 to 20 carbon atoms, with or without (integrated) heteroatoms such as but not limited to O, S, Si, N, Se, B, wherein the point of attachment unless specified otherwise is a carbon atom. Its constitution can be
  • Linear saturated hydrocarbon moiety including, but not limited to, methyl, ethyl, propyl, butyl and pentyl or
  • Linear unsaturated hydrocarbon moiety containing more preferably 2 to 20 carbon atoms, including, but not limited to, ethylen, propylen, butylen and pentylen or
  • Branched saturated hydrocarbon moiety deviceiating from the general alkyl definition by containing at least 3 carbon atoms and including, but not limited to, isopropyl, sec.-butyl and tert.-butyl or
  • Branched unsaturated hydrocarbon moiety deviceiating from the general alkyl definition by containing at least 3 carbon atoms and including, but not limited to, isopropenyl, isobutenyl, isopentenyl and 4-methyl-3-pentenyl
  • Cyclic saturated hydrocarbon moiety containing more preferably 3 to 8 ring atoms and including, but not limited to, cyclopentyl, cyclohexyl, cycloheptyl, piperidino, piperazino
  • Cyclic unsaturated hydrocarbon moiety containing more preferably 3 to 8 ring atoms.
  • saturated means the group has no carbon-carbon double and no carbon-carbon triple bonds.
  • one or more carbon-oxygen or carbon-nitrogen double bonds may be present, which may occur as part of keto-enol and imine-enamine tautomerisation respectively.
  • an alkyl group as defined herein, can be substituted or unsubstituted.
  • Substituents include, but are not limited to, one or more alkyl groups, halogen atoms, haloalkyl groups, (un)substituted aryl groups, (un)substituted heteroaryl groups, amino, oxo, nitro, cyano, azido, hydroxy, mercapto, keto, carboxy, carbamoyl, expoxy, methoxy, ethynyl.
  • alkyl contains a poly ethylene glycol (PEG) moiety
  • PEG poly ethylene glycol
  • -(EO) n — is used as an abbreviated expression for —(CH 2 CH 2 O) n — with n indicating the number of ethylene glycol groups.
  • Aralkyl refers to an alkyl group as described above, that connects to an unsubstituted or substituted aromatic or heteroaromatic hydrocarbon moiety, consisting of one or more aromatic or heteroaromatic rings with 3-8 ring atoms each.
  • Substituents for both the alkyl and aryl part include, but are not limited to, one or more halogen atoms, alkyl or haloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, amino, nitro, cyano, hydroxy, mercapto, carboxy, azido, methoxy, methylthio.
  • Aryl refers to an aryl group, which is an unsubstituted or substituted aromatic or heteroaromatic hydrocarbon moiety, consisting of one or more aromatic or heteroaromatic rings with 3-8 ring atoms each.
  • Substituents include, but are not limited to, one or more halogen atoms, haloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, amino, nitro, cyano, hydroxy, mercapto, carboxy, azido, methoxy, methylthio.
  • Acyl refers to a —C(O)-alkyl group, wherein the alkyl group is as defined above.
  • Aracyl refers to a —C(O)-aryl group, wherein the aryl group is as defined above.
  • Carbamoyl refers to a —C(O)—NH 2 group, wherein the hydrogens can independently from each other be substituted with an alkyl group, aryl group or aralkyl group, wherein alkyl group, aryl group or aralkyl group are as defined above.
  • O-acyl refers to an —O—C(O)-alkyl group, wherein the alkyl group is as defined above.
  • O-alkyl refers to an alkyl group, which is bound through an O-linkage, wherein the alkyl group is as defined above.
  • O-aracyl refers to a —O—C(O)-aryl group, wherein the aryl group is as defined above.
  • O-aralkyl refers to an aralkyl group, which is bound through an O-linkage, wherein the aralkyl group is as defined above.
  • O-aryl refers to an aryl group, which is bound through an O-linkage, wherein the aryl group is as defined above.
  • O-carbamoyl refers to a carbamoyl group, which is bound through an O-linkage, wherein the carbamoyl group is as defined above.
  • S-alkyl refers to an alkyl group, which is bound through a S-linkage, wherein the alkyl group is as defined above.
  • S-aryl refers to an aryl group, which is bound through a S-linkage, wherein the aryl group is as defined above.
  • S-aralkyl refers to an aralkyl group, which is bound through a S-linkage, wherein the aralkyl group is as defined above.
  • S-aralkyl refers to an aralkyl group, which is bound through an S-linkage, wherein the aralkyl group is as defined above.
  • Se-alkyl refers to an alkyl group, which is bound through a Se-linkage, wherein the alkyl group is as defined above.
  • Se-aryl refers to an aryl group, which is bound through a Se-linkage, wherein the aryl group is as defined above.
  • Se-aralkyl refers to an aralkyl group, which is bound through a Se-linkage, wherein the aralkyl group is as defined above.
  • NH-alkyl and N-bisalkyl refer to alkyl groups, which are bound through an N linkage, wherein the alkyl groups are as defined above.
  • NH-aryl and N-bisaryl refer to aryl groups, which are bound through an N linkage, wherein the aryl groups are as defined above.
  • NH-carbamoyl refers to a carbamoyl group, which is bound through an N-linkage, wherein the carbamoyl group is as defined above.
  • Amido-alkyl refers to an alkyl group, which is bound through a NH—C(O)— linkage, wherein the alkyl group is as defined above.
  • Amido-aryl refers to an aryl group, which is bound through a NH—C(O)— linkage, wherein the aryl group is as defined above.
  • Amido-aralkyl refers to an aralkyl group, which is bound through a NH—C(O)— linkage, wherein the aralkyl group is as defined above.
  • Endstanding group refers to a group of a particular residue (R 1 , R 4 and/or R 5 ) which is (sterically) accessible and capable for covalently binding to a particular linking residue (LR 1-4 ). This may be a group at the actual terminal end of the residue (R 1 , R 4 and/or R 5 ) or at any terminal end of any sidechain of the residue (R 1 , R 4 and/or R 5 ), or which is otherwise located in the residue (R 1 , R 4 and/or R 5 ) and sufficiently (sterically) accessible and capable for covalently binding to a particular linking residue (LR 1-4 ).
  • the definition of the term endstanding group if applicable, is independently also valid for the residues LR 5 and/or LR PEG . Further, the term terminus refers to an endstanding group which is actually a terminal end of the concerned residue.
  • a particular linking residue (LR 1-4 ) may represent a radical depending on the number of particular G units it binds to.
  • the particular linking residue (LR 1-4 ) may be a biradical, or in case it is (intermediary) bound to only one particular G unit it may be a monoradical.
  • the particular linking residue (LR 1 ) may be a biradical, triradical, or tetraradical, or in case it is (intermediary) bound to only one particular G unit it may be a monoradical.
  • divalent alkyl any organic radical that is used with the modifier “divalent” as in “divalent alkyl” then this adds a second attachment point.
  • divalent alkyl would be —CH 2 —, —CH 2 CH 2 —, —CH 2 C(CH 3 ) 2 CH 2 —.
  • bonds to ring atoms, and the molecular entities attached to such bonds are termed “axial” or “equatorial” according to whether they are located about the periphery of the ring (“equatorial”), or whether they are orientated above or below the approximate plane of the ring (“axial”). Due to the given stereochemistry of the cyclic phosphate ring, the axial position can only be above the approximate plane of the ring.
  • both R7 and R8 are oxygen, and the phosphorus double bond is “distributed or dislocated” between both atoms.
  • the compound In water at physiological pH, the compound has a negative charge between both oxygens, and a corresponding cation, such as H+ or Na+.
  • the corresponding compound structures herein are presented as charged compounds with a dislocated double bond at the phosphorus, as long as this is in accordance with valency rules. This style is chosen to account for, depict and disclose all possible “locations” of the phosphorous double bond and distribution of electron density or charge each within a single structure.
  • the dislocated double bond as used herein, depending on the nature of the particular R7 and R8, however, does not necessarily refer to an equally distributed charge or electron density between R7 and R8.
  • PLM polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogue, wherein the term “multimeric” can refer to di-, tri- or tetrameric.
  • PLD polymer linked dimeric cGMP analogue
  • polymer refers to a moiety consisting of at least two (equal) units or monomers.
  • the phosphorus atom has four different ligands and becomes chiral resulting in two stereoisomeric forms.
  • Rp/Sp-nomenclature is used. Therein R/S follows the Cahn-Ingold-Prelog rules while “p” stands for phosphorus.
  • the invention relates to a compound according to the definition hereinabove, wherein in case of formula (I) G 4 is absent, or, wherein in case of formula (II) G 4 and LR 4 are absent.
  • the invention relates to a compound according to the definition hereinabove, wherein in case of formula (I) G 3 and G 4 are absent, or, wherein in case of formula (II) G 3 , G 4 , LR 3 and LR 4 are absent.
  • the invention relates to a compound according to any definition hereinabove, wherein all R 7 are O and all R 8 are OH.
  • linking residues LR 1 , LR 2 , LR 3 and LR 4 are further subdivided as depicted in formula (Ib) and (IIb),
  • coupling functions C 1 , C 1′ , C 2 , C 2′ , C 3 , C 3′ , C 4 and C 4′ independently from each other can be absent or as defined by structures selected from the group consisting of
  • the linker (L) is selected from the group consisting of
  • linking residues LR 1 , LR 2 , LR 3 and LR 4 are further subdivided as depicted in formula (Ib) and (IIb), containing spacer moieties (S 1-4 ), coupling functions (C 1-4 , C 1-4 ) and a linker (L, only multimers of structure Ib), coupling functions (C 1-4 , C 1-4 ) establish covalent bonds between
  • Coupling functions (C 1-4 , C 1′-4 ) are generated in a reaction between endstanding groups of the particular precursor parts according to well established methods of the art.
  • Non limiting examples of precursor endstanding groups (of monomeric G units and (commercially available) linkers, dyes, reporting groups and spacers) and the corresponding coupling functions (C 1-4 , C 1′-4 ), to which they are transformed within the assembled (mono- or multimeric) compound according to the invention, are as depicted in Table 1.
  • Coupling functions (C 1-4 , C 1′-4 ) can independently further be absent or be equal or individual within a particular mono- or multimeric compound.
  • FIG. 2 A non limiting example of a multimeric compound according to the invention, illustrating the used and defined variables above is given in FIG. 2 .
  • Embodiments of the invention are directed to new polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG), and related monomeric precursors thereof.
  • the invention is also directed to related monomeric compounds, which may also show modulating activity and/or may serve as monomeric precursors of the multimers.
  • PLD polymer linked dimeric cGMP analogue
  • the PKG Iß activation potential determined for this new PLD is the strongest so far observed 1735-fold activity of cGMP) while PKG II is rather poorly activated (2.5-fold activity of cGMP).
  • this compound not only causes increased PKG I ⁇ activation but the effect is also more than 4-fold greater ( ⁇ 140-fold potential of cGMP) as determined for the best PLD agonist (PEG-spacer of 282 Da) described before.
  • PKG activation values of the compounds of the invention are expressed as multiple of the cGMP activation with the cGMP activation value set as 1 for each isozyme. It has to be noted, that the applied standard assay conditions only allowed to determine increased activation potencies of up to 140-fold for PKG I ⁇ , 2832-fold for PKG Iß and 408-fold for PKG II, which is due to the employed enzyme concentration in the assays and the phenomenon that the isozymes were activity-titrated in some cases by the highly active compounds of the invention.
  • the actual PKG activation potentials of these particular corns pounds of the invention appear to be significantly higher and are therefore expressed as ⁇ 140-fold for PKG I ⁇ , ⁇ 2832-fold for PKG Iß and ⁇ 408-fold for PKG II.
  • PLDs of the present invention feature a variety of different spacer lengths, as the results described above were also reproduced with homologues PLDs. For instance shorter spacers (19 and 8 ethylene glycol units (-(EO) 19 — and -(EO) 8 —), see Table 13, compounds 12, 5, 8, 3) gave similar results, wherein in several cases the PKG enzyme was titrated ( FIG. 3 ). As mentioned above, a titrated enzyme corresponds to a value beyond the measurement limit, indicating a very strong activation potential. The substantial major effect of nucleobase manipulation, in particular R 4 /R 5 substitution, can again be observed by comparing for instance compounds 3 and 5 ( FIG. 3 ). Therein compound 3 is identical to 5 but lacks the PET moiety.
  • the present invention comprises PLDs containing standard coupling moieties other than amide groups, as the superior activity of new PLDs according to the invention is not limited to this particular type of coupling function.
  • Dimers linked via a triazole group e.g. compound 23 with ⁇ 2832-fold activity of cGMP for PKG IR, also see FIG. 4
  • featuring no additional coupling function besides the thio ether group directly connected to the nucleobase e.g. compound 1 with 231-fold activity of cGMP for PKG II
  • two random non limiting examples of the present invention gave comparable results.
  • PLDs Another structural aspect of PLDs according to the invention concerns the linkage position at which the two cGMP analogues are coupled to each other.
  • the observed activity enhancement of PLDs is not restricted to linkage via the R 1 position. It is still present, when linkage is varied along the G unit.
  • PLDs coupled via the PET-moiety displayed a similarly increased PKG agonist potential as PET-substituted derivatives tethered via the R 1 -position (compound 6 and 23, FIG. 4 ).
  • PLDs of the invention comprise a variety of possible linking positions as defined further above.
  • spacer moiety is another motive that affects PLD induced PKG activation.
  • PEG (spacer) units used on the PLD derivatives mentioned so far can be replaced by or combined with other functionalities such as peptides or alkanes, included in the present invention.
  • Alkanes in particular, however, are restricted in size, as solubility decreases significantly with growing alkyl spacer length.
  • Still alkyl spacers with moderate size are tolerated with respect to maintaining sufficient water solubility.
  • a PKG activation screening performed with compound 16 indicated that such compounds can show an activity increasing effect (PKG II activation approx. 22-fold higher than for cGMP).
  • compound 18 is a strong activator of PKG 11 (381-fold activity of cGMP) while 6 shows virtually no increased effect on this isoform (1.3-fold activity of cGMP). Even though 6 or this type of G unit respectively therefore appears to be unable to contribute to PKG II activation, the mixed PLD hybrid 22 still is a very strong activator of PKG II (194-fold activity of cGMP).
  • the second G unit can even be a significantly less effective activator of PKG (observed for the respective homogenous PLD) while the superior PKG activation of the first G unit (again observed for the respective homogenous PLD) is substantially preserved within the mixed PLD hybrid.
  • mixed PLDs allow a much broader diversity of modifications (at one cGMP unit), while the undesired decrease of PKG activation, caused by these modifications, is much less pronounced if present at all.
  • mixed PLDs also support the design of multi target compounds. Functional groups (e.g. PET-group), intended to address different targets (e.g. different PKG isoform) apparently can be installed at one cGMP unit, giving an extended target activation spectrum of the mixed PLD.
  • the present invention also comprises the extension of the described concept of polymer linked cGMP analogues from dimers to tri- and tetramers. Therein linkage of the particular G units is accomplished either in a linear or branched fashion (see formula I and II).
  • Compounds 14 and 15 (Table 13) are two non limiting examples of the latter case, featuring particularly strong PKG II activation as predicted from analogues PLD derivatives lacking the PET-moiety ( ⁇ 416-fold activity of cGMP for compound 14).
  • the increased number of G units within tri- and tetramers results in even more diverse opportunities to combine (different) activator and target independent functionalized G units.
  • the present invention has established the first activators of PKG Iß and PKG II with PLM structure, which are furthermore significantly improved when compared to state of the art compounds.
  • PLM are also derivatives, which in addition activate PKG I ⁇ , and mixed PLM, which amongst others are beneficial for functionalization and/or addressing all three PKG isoforms.
  • Nucleobase modifications at R 4 /R 5 and R 1 position as a key part of the invention, thereby proved to be powerful modifiers of PKG activation potential. These modifications were shown herein to be able to exceed and overrule the effect of varying spacer lengths, which before was suggested to be the main effector of target selectivity and activity increase (compared to the monomer).
  • PLMs coupled via the R 1 position which overlaps with a nucleobase modification at R 1 , wherein in addition R 4 is absent and R 5 is NH 2 (according to formula III), were found to feature strongly increased PKG II activation potential (compared to the monomer).
  • Prior art PLD compounds 5 (see FIG. 1 ) also fall under the scope of this general structural paradigm, however, only by coincidence, and are expressively disclaimed from the present invention. Their appearance in the art was connected to a different question, wherein a different target (PKG I ⁇ and CNGC) was addressed and the crucial role of a different modifier (spacer length instead of nucleobase modification) was concluded.
  • the new PLM compounds of the present invention furthermore differ in and benefit from improved synthetic coupling strategies.
  • Prior art synthetic protocol for PLDs involved coupling of a thiol-group in the 8-(R 1 )position with a bifunctional PEG vinylsulfone. 5
  • the reported conditions as published later and in accordance with our own experience, however, favour addition at the 7-(R 2 ) instead of the 8-(R 1 )position.
  • various more robust, regioselective and higher yielding methods were developed for the present invention, involving for instance peptide (amid)- and click chemistry.
  • the 661W cell line was used and increase in cell death after treatment was assessed (for more details see examples section).
  • the 661W cell line is a photoreceptor precursor cell line, which expresses PKG ( FIG. 6 ). This makes them a suitable model for examining PKG activity using cell death as readout since increased PKG activity was previously associated with increased cell death.
  • 9 Results were compared to untreated cells and to incubation with 8-Br-PET-cGMP as reference.
  • 8-Br-PET-cGMP is a well established commercially available PKG activator, which has been applied in various cellular systems and is furthermore a synthetic precursor of some of the exemplary PLDs of the invention.
  • the most potent PLDs of the invention display a 5-6 fold increase in cell death when compared to untreated cells and 3-4 fold increase in cell death when compared to the reference 8-Br-PET-cGMP.
  • R 1 is selected from group consisting of H, halogen, azido, nitro, alkyl, acyl, aryl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, S-aralkyl, S(O)-alkyl, S(O)-aryl, S(O)aralkyl, S(O)-benzyl, S(O) 2 -alkyl, S(O) 2 -aryl, S(O) 2 -aralkyl, amino, NH-alkyl, NH-aryl, NH-aralkyl, NR9R10, SiR13R14R15 wherein R9, R10, R13, R14, R15 are alkyl.
  • R 1 is selected from the group consisting of H, CI, Br, I, F, N 3 , NO 2 , OH, SH, NH 2 , CF 3 , 2-furyl, 3-furyl, 2-bromo-5-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-
  • R 1 is selected from the group consisting of H, Cl, Br, I, F, N 3 , NO 2 , OH, SH, NH 2 , CF 3 , 2-furyl, 3-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioure
  • Q S, S(O), S(O) 2 , NH.
  • R 1 is selected from the group consisting of H, Cl, Br, SH, 2-furyl, 3-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetra
  • Q S.
  • R 4 is absent or selected from the group consisting of amino, N-oxide or as depicted in Table 5.
  • X 2 can be H, Ph, 2-naphtyl, 9-phenanthryl, 1-pyrenyl, 2,3-dihydro-1,4-benzodioxin-6-yl, dibenzo[b,d]furan-2-yl, 2,3-dihydro-1-benzofuran-5-yl, 1-benzothien-5-yl, 1-benzofuran-5-yl, cycopropyl, 1-adamantyl, C(Ph) 3 , 2-thienyl, 3-chloro-2-thienyl, 3-thienyl, 1,3-thiazol-2-yl, 2- pyridinyl, 5-chloro-2-thienyl, 1-benzofuran-2-yl, X 3 , X 4 and X 5 can independently be H, OH, NH, CH 3 , Cl, Br, F, CN, N 3 , CF 3 , OCF 3 , NO 2 , C(O)OH, C(
  • n 1-6.
  • R 4 is absent or selected from the group consisting of amino, N-oxide or as depicted in Table 6.
  • X 2 can be H, Ph, 2-naphtyl, 9-phenanthryl, 1-pyrenyl, 2,3-dihydro-1,4-benzodioxin-6-yl, dibenzo[b,d]furan-2-yl, 2,3-dihydro-1-benofuran-5-yl, 1-benzothien-5-yl, 1-benzofuran-5-yl, cycopropyl, 1-adamantyl, C(Ph) 3 , 2-thienyl, 3-chloro-2-thienyl, 3-thienyl, 1,3-thiazol-2-yl, 2- pyridinyl, 1-benzofuran-2-yl; X 3 , X 4 and X 5 can independently be H, OH, NH, CH 3 , Cl, Br, F, CN, N 3 , CF 3 , OCF 3 , NO 2 , C(O)OH, C(O)OCH 3 , OCH 3
  • n 1-6.
  • R 4 is absent or as depicted in Table 7.
  • X 2 can be H, Ph, 2-naphtyl, 9-phenanthryl, 1-pyrenyl, 2,3-dihydro-1,4-benzodioxin-6-yl, dibenzo[b,d]furan-2-yl, 2,3-dihydro-1-benofuran-5-yl, 1-benzothien-5-yl, 1-benzofuran-5-yl, cycopropyl, 1-adamantyl, C(Ph) 3 , 2-thienyl, 3-chloro-2-thienyl, 3-thienyl, 1,3-tiazol-2-yl, 2- pyridinyl, 1-benzofuran-2-yl; X 3 , X 4 and X 5 can independently be H, OH, NH, CH 3 , Cl, Br, F, CN, N 3 , CF 3 , OCF 3 , NO 2 , C(O)OH, C(O)OCH 3 , OCH
  • R 5 is selected from the group consisting of H, NH 2 , F, Cl, Br, I, methylamino, NH-benzyl, NH-phenyl, NH-4-azidophenyl, NH-phenylethyl, NH-phenylpropyl, 2-aminoethylamino, n-hexylamino, 6-amino-n-hexylamino, 8-amino-3,6-dioxaoctylamino, dimethylamino, 1-piperidino, 1-piperazino or can form together with R 4 , Y and the carbon bridging Y and R 5 a ring system as depicted in Table 5 (entry 2 and 3).
  • R 5 is selected from the group consisting of H, NH 2 , F, Cl, Br, I, methylamino, NH-benzyl, NH-phenyl, NH-4-azidophenyl, NH-phenylethyl, NH-phenylpropyl, 2-aminoethylamino, n-hexylamino, 6-amino-n-hexylamino, 8-amino-3,6-dioxaoctylamino, dimethylamino, 1-piperidino, 1-piperazino or can form together with R 4 , Y and the carbon bridging Y and R 5 a ring system as depicted in Table 6 (entry 2 and 3).
  • R 5 is NH 2 , or can form together with R 4 , Y and the carbon bridging Y and R 5 a ring system as depicted in Table 7 (entry 2 and 3).
  • R 7 is selected from group consisting of OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, SH, S-alkyl, S-aryl, S-aralkyl, borano (BH 3 ), methylborano, dimethylborano, cyanoborano (BH 2 CN), S-PAP, O-PAP, S-BAP, or O-BAP
  • R 7 is selected from the group consisting of OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy, SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethyl
  • R 7 is selected from the group consisting of OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy, SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethyloxy, SH, methylthio
  • R7 is OH
  • R8 is selected from group consisting of OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, O-PAP or O-BAP,
  • R8 is selected from the group consisting OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy;
  • R8 is OH
  • residues involved in connecting a G unit with another G unit or a dye or another reporting group can be R 1 , R 4 and/or R 5 , in which case the particular residue is
  • Residues R1, R4 and R5 involved in connecting a G unit with another G unit or a dye or another reporting group (if present Q 1 connects to the G unit).
  • n 0-6;
  • m 0-6;
  • Q1 absent, S, NH, O;
  • Q2 NH, S, O, C(O), CH 2 , OC(O), NC(O);
  • Q1 absent, S, NH, O;
  • Q1 absent, S, NH, O;
  • Q2 NH, S, O, C(O), CH 2 , OC(O), NC(O);
  • residues involved in connecting a G unit with another G unit or a dye or another reporting group can be R 1 , R 4 and/or R 5 , in which case the particular residue is
  • R 1 , R 4 and R 5 involved in connecting a G unit with another G unit or a dye or another reporting group (if present Q 1 connects to the G unit)
  • Q1 S;
  • Q2 NH, S, O, CH 2 , NC(O);
  • coupling functions are absent or selected from the group depicted in Table 10.
  • coupling functions are absent or selected from the group depicted in Table 11.
  • linker (L) is absent or selected from the group depicted in Table 12.
  • n for each sidechain within a particular linker can have an equal or individual value as defined.
  • Especially preferred according to the invention are the compounds of Table 13, and as defined in claim 10 . It has to be noted that in case of doubt the chemical structure as depicted in the formula is the valid one. It further has to be noted, that the compounds of Table 13 are displayed as the free acid.
  • the present invention also comprises salts of these compounds, featuring cations such as but not limited to Na + , Li + , NH 4 + , Et 3 NH + and (i-Pr) 2 EtNH + .
  • Monomeric precursor cGMP analogues (G units) for the synthesis of polymer linked multimeric cGMP analogues (PLMs) are compounds of formula (III).
  • the PKG activation potential is strongly increased, once the monomeric precursor is linked to additional one(s) within a PLM, wherein particularly enhanced PKG isoform activation can be related to a certain extend to structural parameters.
  • Non limiting examples of methods for the transformation of monomeric precursors into exemplary PLMs are given in the examples section.
  • Table 1 gives an overview of exemplary endstanding groups, that can be used for coupling reactions and the corresponding coupling functions within the PLM, to which they are transformed according to established methods of the art.
  • the invention in one aspect also relates to monomeric compounds of formula (III) and/or monomeric precursors according to formula (III), of any compound of the invention as described herein above, wherein the monomeric compound of formula (III) and/or the monomeric precursor of formula (III) is defined in the context of any said compounds herein above, and preferably wherein the monomeric compound of formula (III) and/or monomeric precursor of formula (III) complies with the following proviso:
  • the monomeric compound of formula (III) and/or the monomeric precursor compound of formula (III) is not selected from the group of compounds consisting of
  • the monomeric compound of formula (III) and/or the monomeric precursor of the invention is selected from the group depicted in Table 14.
  • the compounds according to the present invention may further be labelled, according to well-known labelling techniques.
  • fluorescent dyes may be coupled to the compounds in order to, but not limited to, localize the intracellular distribution of cyclic nucleotide binding proteins in living cells by means of confocal microscopy, for fluorescence correlation spectrometry, for fluorescence energy transfer studies, or for determination of their concentration in living cells.
  • the compounds according to the inventions may be labelled with (radio) nuclides.
  • the person skilled in the art knows many techniques and suitable isotopes that can be used for this.
  • the invention also comprises PEGylated forms of the specified compounds, wherein PEGylation is generally known to greatly improve water solubility, pharmacokinetic and biodistribution properties.
  • the invention further comprises modifications wherein R 7 (according to formula III) can be an unsubstituted or substituted thio- or borano function. Both modifications are known in the art to improve resistance towards metabolic degradation. 1a, 14
  • the invention also comprises prodrug forms of the described compounds, wherein the negative charge of the (modified or unmodified) phosphate moiety is masked by a bioactivatable protecting group. It is widely accepted that such structures increase lipophilicity and with that, membrane-permeability and bioavailability resulting in a 10-1000 fold enhanced potency compared to the mother-compound.
  • bioactivatable protecting groups can be introduced according to well known techniques of the art and include, but are not limited to acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, acetoxyethyl, acetoxybutyl, acetoxyisobutyl.
  • Non limiting examples of corresponding residues R7 and/or R8 according to the invention are acetoxymethyloxy, propionyloxymethyloxy and butyryloxymethyloxy. More labile examples of protecting groups include alkyl or aryl groups as well as substituted alkyl or aryl groups.
  • Non limiting examples for chemically labile protection groups of the R7 and/or R8 position are methyl, ethyl, 2-cyanoethyl, propyl, benzyl, phenyl and polyethylene glycol. These compounds are inactive per se, but extremely membrane-permeable, leading to strongly increased intracellular concentrations. Upon hydrolysis of the ester bond, the biologically active mother compounds are released.
  • Compounds according to the invention can also feature a photolysable group (also-called “caged”- or photo-activatable protecting group), which can be introduced according to well known techniques of the art.
  • a photolysable group also-called “caged”- or photo-activatable protecting group
  • caged groups may be coupled to an R8 oxo-function, leading to compounds with significantly increased lipophilicity and bioavailability.
  • Non limiting examples for caged groups are o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylaminocoumarin-4-yl (DMACM-caged), 7-diethylamino-coumarin-4-yl (DEACM-caged) and 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM-caged).
  • DMACM-caged 7-dimethylaminocoumarin-4-yl
  • DECM-caged 7-diethylamino-coumarin-4-yl
  • BCMCM-caged 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl
  • the compounds according to the present invention can also be immobilized to insoluble supports, such as, but not limited to, agarose, dextran, cellulose, starch and other carbohydrate-based polymers, to synthetic polymers such as polacrylamide, polyethyleneimine, polystyrol and similar materials, to apatite, glass, silica, gold, graphene, fullerenes, carboranes, titania, zirconia or alumina, to the surface of a chip suitable for connection with various ligands.
  • insoluble supports such as, but not limited to, agarose, dextran, cellulose, starch and other carbohydrate-based polymers, to synthetic polymers such as polacrylamide, polyethyleneimine, polystyrol and similar materials, to apatite, glass, silica, gold, graphene, fullerenes, carboranes, titania, zirconia or alumina, to the surface of a chip suitable for connection with various ligands
  • the compounds according to the present invention can also be encapsulated within nanoparticles or liposomes for directed or non-directed delivery and release purposes of the compounds as described in the literature. 11
  • the new polymer linked multimeric cGMP analogues of the invention, or the related monomeric compounds of formula (III) of the present invention, respectively, are used for treating or preventing a disease or condition that is associated with low cGMP signaling activity.
  • Diseases and conditions are preferably treated with polymer linked multimeric cGMP analogues, or the related monomeric compounds of formula (III) of the present invention, respectively, that activate the disease-related unbalanced cGMP-system, and include 1c :
  • the invention relates to a method for treating or preventing any of the above pathologies, conditions or disorders by administration of a therapeutically or prophylactically effective amount of an equatorially modified cGMP-analogue of the invention to a subject in need of prophylaxis or therapy.
  • the compounds according to the present invention can also be used as research tool compound, preferably as research tool compound in regard of a disease or disorder related to an unbalanced cGMP-system, preferably a disease or disorder selected from the group consisting of cancer, cardiovascular disease or disorder, or autoimmune disease or disorder, or neurodegenerative disease or disorder.
  • a disease or disorder related to an unbalanced cGMP-system preferably a disease or disorder selected from the group consisting of cancer, cardiovascular disease or disorder, or autoimmune disease or disorder, or neurodegenerative disease or disorder.
  • 8-Br-cGMP, 8-Br-PET-cGMP and 4-N 3 -PET-8-Br-cGMP were available from Biolog Life Science Institute (Bremen, Germany). 8-T-cGMP is established in the literature and was prepared analogously to PET-8-T-cGMP (see examples below). Solvents used were specified as analytical or hplc grade. Dimethyl sulfoxide was stored over activated molecular sieves for at least two weeks before use. Chromatographic operations were performed at ambient temperature.
  • reaction progress and purity of isolated products were determined by reversed phase hplc (RP-18, ODS-A-YMC, 120-S-11, 250 ⁇ 4 mm, 1.5 mL/min), wherein UV detection was performed either at 263 nm, an intermediate wavelength suitable to detect most cyclic GMP products and—impurities, or at the ⁇ max of the particular starting material or product.
  • Syntheses were typically performed in a 20-200 ⁇ mol scale in 2 mL polypropylene reaction vials with screw cap (reactions requiring inert gas atmosphere and/or degassing were performed in round bottom flasks (typically 10 or 25 mL)).
  • Dissolution of poorly soluble reactants was achieved through sonification or heating (70° C.) prior to addition of reagents. In case dissolution was not elicited by these techniques, which mainly applied to some cGMP analogues carrying a PET-moiety, the suspension was used. Purification of products was accomplished by preparative reversed phase hplc (RP-18, ODS-A-YMC, 12 nm-S-10, 250 ⁇ 16 mm, UV 254 nm). The eluent composition is described in the particular synthetic example and, unless stated otherwise, can be used for analytical purposes as well.
  • Desalting of products was accomplished by repeatedly freeze-drying or by preparative reversed phase hplc (RP-18, ODS-A-YMC, 12 nm-S-10, 250 ⁇ 16 mm, UV 254 nm) according to standard procedures for nucleotides. Solutions were frozen at ⁇ 70° C. for 15 min prior to evaporation, in case a speedvac concentrator was used to remove the solvent. Products were either isolated as sodium or triethylammonium salt, depending on the applied buffer. Yields refer to the fraction of isolated product featuring the reported purity.
  • aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 8-thio-modified cGMP analogue.
  • N,N-diisopropylethylamine (2 eq) and the corresponding bromide (1 eq) were added successively to a solution of the 8-SH-substituted cGMP analogue (sodium or triethylammonium salt, 100 mM, 1 eq) in DMSO.
  • the reaction mixture was stirred until the thiol starting material was completely consumed or no further reaction progress was observed.
  • the solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), washed with ethyl acetate (3 ⁇ ), subjected to preparative reversed phase hplc and desalted.
  • the aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the coupled cGMP analogue.
  • the less valuable reactant was added in slight excess, thus for the reaction with reversed functions the amine-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the acid reactant (1.1 eq) were used.
  • N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq) were added successively to a solution of the corresponding carboxylic acid-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the corresponding amine (1.1 eq)*.
  • the reaction mixture was stirred until the starting material was completely consumed or no further reaction progress was observed (usually ⁇ 10 min).
  • Water (100 ⁇ L) was added, stirring was continued for 10 min and the solvent was removed through high vacuum evaporation with a speedvac concentrator.
  • Derivatives featuring a modified phosphate function, sensitive to oxidation reactions, such as a phosphorothioate, were synthesized starting from the corresponding guanosine, while the (modified) phosphate group was then introduced according to well established methods of the art (e.g. thiophosphorylation protocol 12 ) after oxidation of the 8-thio function.
  • aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water/MeOH (4:1), subjected to preparative reversed phase hplc and desalted, giving the iminophosphoranyl analogue.
  • N,N-diisopropylethylamine (2 eq) was added to a solution of 4-mercaptophenylboronic acid (0.2 M, 1 eq) and Br-(EO) 5 —(CH 2 ) 2 —Br (0.5 eq) in DMF.
  • the reaction mixture was stirred until the boronic acid starting material was completely consumed or no further reaction progress was observed.
  • the solvent was removed through high vacuum evaporation with a speedvac concentrator.
  • N,N-diisopropylethylamine (2 eq) and PyBOP (1.1 eq) were added successively to a solution of the corresponding 1-carboxyalkyl-substituted cGMP analogue (10 mM in DMSO, 1 eq).
  • the reaction mixture was stirred until the cGMP analogue starting material was completely consumed or no further reaction progress was observed.
  • Water (100 ⁇ L) was added, stirring was continued for 10 min and the solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), the pH adjusted to 5-6 with NaOH (2 M) and the solution washed with ethyl acetate (5 ⁇ ).
  • the aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1, N 2 -acyl-functionalized cGMP analogue.
  • aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1-substituted cGMP analogue.
  • aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1-substituted dimeric cGMP analogue.
  • HPLC (5% MeCN, 100 mM TEAF buffer, pH 6.8).
  • HPLC (9 MeCN, 20 mM TEAF buffer, pH 6.8).
  • the title compound was synthesized from 8-MPT-cGMP using general procedure F.
  • HPLC (5 MeCN, 10 mM TEAF buffer, pH 6.8).
  • HPLC (32% MeOH, 10 mM TEAF buffer, pH 6.8).
  • Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetra-[(8- methylamidoethylthio)guanosine-3′,5′-cyclic monophosphate] (EG-N,N,N′,N′-tetra(8- MAmdET-cGMP))
  • EGTA Ethylene glycol-bis(2- aminoethylether)-N,N,N′,N′-tetraacetic acid
  • PET-cGMP- 8-T-(CH 2 ) 12 -T-8-cGMP-PET PET-8-T-cGMP was reacted with 1,12-dibromdodecane to give the title compound. Yield (Purity): 24% (>99%). HPLC: (36% MeCN, 20 mM TEAF buffer, pH 6.8).
  • the reaction mixture was stirred until no further reaction progress was observed ( ⁇ 10% remaining starting material).
  • the solvent was removed through high vacuum evaporation with a speedvac concentrator.
  • the residue was dissolved in MeCN/water (8:1, v/v), washed with petroleum ether (3 ⁇ ) and the aqueous phase evaporated to dryness using a rotary evaporator.
  • the crude product was dissolved in DMF (115 mM). 4,4′-Thiobisbenzenthiol (0.5 eq) and N,N-diisopropylethylamine (2.2 eq) were added successively.
  • the reactin mixture was stirred until the starting material was completely consumed.
  • the solvent was removed through high vacuum evaporation with a speedvac concentrator.
  • Monomeric precursors of the invention and/or momomeric compounds of the invention are further illustrated by the figures and examples of Table 16 describing preferred embodiments of the present invention which are, however, not intended to limit the invention in any way. Structural examples of novel compounds are depicted in the free acid form. After HPLC workup, compounds are obtained as salts of the applied buffer, but can be transformed to other salt forms or to the free acid by cation exchange according to standard procedures for nucleotides.
  • 32 8-(4-Boronatephenylthio)-guanosine-3′,5′-cyclic monophosphate (8-pB(OH) 2 PT- cGMP) Using general procedure A, 8-Br-cGMP was reacted with 4-mercaptophenylboronic acid to give the title compound. Yield (Purity): 71% (>99%).
  • 66 8-(3-Boronatephenyl)amidoethylthio-guanosine-3′,5′-cyclic monophosphate (8- mB(OH) 2 PAmdET-cGMP) Using general procedure H, 8-CET-cGMP was reacted with 3-aminophenylboronic acid to give the title compound.
  • 8-Methylpropionylthioguanosine-3′,5′-cyclic monophosphate 8-MPT-cGMP
  • 8-T-cGMP was reacted with methyl 3-bromopropionate and equivalents were increased stepwise (methyl 3-bromopropionate up to 9 eq, N,N- diisopropylethylamine up to 8 eq) to improve the yield of the title compound.
  • HPLC (15% MeOH, 10 mM TEAF buffer, pH 6.8).
  • 8-Methoxyethylamidoethylthio-guanosine-3′,5′-cyclic monophosphate (8- MeOEAmdEt-cGMP) Using general procedure H, 8-CET-cGMP was reacted with 2-methoxyethylamine to give the title compound. Yield (Purity): 65% (>99%).
  • 8-(4-Thiophenyl-4′′-thiophenylthio)guanosine-3′,5′-cyclic monophosphate (8-pTP- pTPT-cGMP) Using general procedure B, 8-Br-cGMP was reacted with 4,4′-thiobisbenzenethiol to give the title compound.
  • 8-Carboxymethylthio- ⁇ -phenyl-1,N 2 -ethenoguanosine-3′,5′-cyclic monophosphate (8-CMT-PET-cGMP) Using general procedure C, 8-Br-PET-cGMP was reacted with mercaptoacetic acid to give the title compound. Yield (Purity): 68% (>99%).
  • 8-Ethylthio- ⁇ -phenyl-1,N2-ethenoguanosine-3′,5′-cyclic monophosphate (8-ET- PET-cGMP) Using general procedure A, 8-Br-PET-cGMP (1 eq) was reacted with ethanethiol (12 eq) in a tube with screw cap at 70° C.
  • 8-Bromo-(4-methoxy- ⁇ -phenyl-1,N2-etheno)guanosine-3′,5′-cyclic monophosphate (8-Br-pMeO-PET-cGMP) Using general procedure Y, 8-Br-cGMP was reacted with 2-bromo-4′-methoxyacetophenone to give the title compound.
  • 153 ⁇ -(4-Aminophenyl)-1,N 2 -etheno-8-bromoguanosine-3′,5′-cyclic monophosphate pNH 2 -PET-8-Br-cGMP
  • the title compound was synthesized from 4-N 3 -PET-8-Br-cGMP using general procedure V. Yield (Purity): 10% (>98%).
  • 8-Bromo-(9-phenanthrenyl-1,N 2 -etheno)guanosine-3′,5′-cyclic monophosphate (8-Br-(9-Phethr)ET-cGMP) Using general procedure Y, 8-Br-cGMP was reacted with 9-(2-bromoacetyl)phenantrene to give the title compound.
  • 159 1,N 2 -Etheno-8-(2-phenylethyl)thioguanosine-3′,5′-cyclic monophosphate (ET-8- PhEtT-cGMP) Using general procedure A, 8-Br-Et-cGMP (B 177) was reacted with 2-phenyethanethiol to give the title compound.
  • PKGIß and PKGII were expressed in Sf9 cells and purified by affinity chromatography. 2 Concentrations of enzymes given below refer to the dimeric form. VASPtide (GL Biochem Ltd., Shanghai, China) was used as PKG-selective phosphorylation substrate peptide. 2
  • FIGS. 3 to 5 show that all tested PLMs produce significantly higher relative PKG activation for at least 2 of the 3 PKG isozymes compared to the reference compound cGMP. Furthermore, it has to be noted, that the applied standard assay conditions only allowed to determine increased activation potencies of up to 140-fold for PKG I ⁇ , 2832-fold for PKG Iß and 416-fold for PKG II, which is due to the employed enzyme concentration in the assays and the phenomenon that the isozymes were activity-titrated in some cases by the highly active compounds of the invention.
  • the 661W cell line was used and increase in cell death after treatment was assessed.
  • the 661W cell line is a photoreceptor precursor cell line, immortalized with the SV40 T antigen.
  • the 661W cells express PKG. This makes them a suitable model for examining PKG activity using cell death as readout since increased PKG activity was previously associated with increased cell deaths. Because of potentially complex outcomes from the activation of different PKG isoforms this analysis is interpreted as a proof of principle on the use of these compounds in PKG-expressing cells or tissues.
  • the 661W cells were cultured in DMEM with 10% FBS (Fetal Bovine Serum), 2 mM Glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin.
  • FBS Fetal Bovine Serum
  • ECM extracellular matrix
  • Ethidium Homodimer stains nuclei of dying cells.
  • microphotographs were taken from three different slides for each compound concentration and the total number of cells, as well as the number of dying Ethidium Homodimer positive cells, were counted in each picture. The value for untreated cells was set to 1. To statistically assess significant differences between untreated and treated cells, the unpaired Student's t-test was used and a P value ⁇ 0.05 was considered significant (* ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001).
  • FIG. 7 shows percentage of cells undergoing cell death after treatment with non limiting exemplary polymer linked dimeric cGMP analogues of the invention (12 compounds).
  • Six of the tested compounds led to significantly increased cell death at one or more concentrations when compared to untreated cells.
  • the most potent compounds of the invention display a 5-6 fold increase in cell death when compared to untreated cells and 3-4 fold increase in cell death when compared to the reference 8-Br-PET-cGMP.

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