Novel equatorially modified polymer-linked multimers of 3',5'-cyclic guanosine monophosphate

Abstract

The present invention provides novel equatorially modified cGMP analogues for the inhibition of cell death pathways activated during retinal degeneration processes. [Solution] Embodiments of the present invention relate to novel equatorially modified polymer-linked multimers of guanosine-3',5'-cyclic monophosphate (cGMP) analogs that inhibit the cGMP signaling pathway. The present invention also relates to related monomeric compounds that may serve as monomeric precursors to the multimers and / or may also exhibit inhibitory activity themselves and / or may affect the inhibitory activity of related multimers. The present invention further relates to the use of the compounds as reagents for signal transduction studies and as modulators of cyclic nucleotide-regulated binding proteins and their isoenzymes, as well as ligands for affinity chromatography, for antibody production, or for diagnostic applications, e.g., on chip surfaces.

Description

[Technical field]

[0001] summary Embodiments of the present invention relate to novel equatorially modified polymer-linked multimers of 3',5'-cyclic guanosine monophosphate (cGMP) analogs that inhibit the cGMP signaling pathway. The present invention also relates to related monomeric compounds that serve as monomeric precursors to the multimers and / or that may also exhibit inhibitory activity themselves and / or affect the inhibitory activity of related multimers.

[0002] FIELD OF THEINVENTION The present invention also relates to novel equatorially modified polymer-linked multimers (including tethered dimers, trimers and tetramers) of 3',5'-cyclic nucleotide guanosine monophosphate and their uses in the medical and pharmaceutical fields. The present invention also relates to unique precursor monomers. The present invention further relates to the use of said compounds as reagents for signal transduction studies and as modulators of cyclic nucleotide-regulated binding proteins and their isoenzymes, as well as ligands for affinity chromatography, for antibody production or for diagnostic applications, e.g. on chip surfaces. [Background technology]

[0003] 2. Background of the Invention 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are purine nucleobase-containing cyclic nucleotides that were discovered as endogenous molecules in 1957 and 1963, respectively. They act as second messengers in numerous cellular processes, such as genetic control, chemotaxis, proliferation, differentiation, and programmed cell death. Several diseases are associated with abnormally high or low levels of cGMP and / or cAMP. 1The synthesis of sulfur-modified Rp-guanosine-3',5'-monophosphorothioate (Rp-cGMPS) and Rp-8-Cl-cGMPS, in which a sulfur atom is introduced at the equatorial exocyclic position of the 3',5'-cyclic phosphate, has been described, and their inhibitory effects on cGMP-dependent protein kinase (PKG) as a member of the cellular cGMP system have been reported. However, Rp-cGMPS in particular showed a lack of specificity for PKG over cAMP-dependent protein kinase (PKA), which is inhibited at similar concentrations. Furthermore, the poor membrane permeability of hydrophilic analogs such as Rp-cGMPS and Rp-8-Cl-cGMPS is the main limitation for biological experiments and prevents the wider application of these analogs. In the last decades, several Rp-cGMPS analogs with partially improved membrane permeability and biological activity have been developed, e.g., Rp-8-Br-cGMPS. 2 and Rp-8-Br-PET-cGMPS 2b、3 were developed. These analogs were now at least potent enough to allow a broader testing of cyclic nucleotide-based inhibitors of the cGMP system in biological environments. However, the analogs are still not optimal and may have to be applied in higher μmolar to millimolar ranges to exert their biological effects. Especially in cells with an upregulated cGMP system, either by pharmacological agents or pathological situations, analogs such as Rp-8-Br-PET-cGMPS showed suboptimal potency or even partial agonistic properties. 4 .

[0004] Knowledge of the identity and presence of PKG substrates in various cells, tissues and organs is limited.Therefore, the physiological and pathological importance of cGMP-PKG system is not fully understood, which may limit the general understanding of cGMP-related phenomena as well as the development of treatments for diseases and conditions in which said substrates are involved.If more effective and reliable cGMP analogue-based inhibitors can be developed, they are expected to address cGMP system-related problems more sharply than they can be achieved at present. Retinal dystrophies (RD) are neurodegenerative diseases that result in severe damage to the eye, progressively reducing visual function. These diseases affect rod and / or cone photoreceptors, the sensory neurons of the retina responsible for converting light stimuli into electrochemical signals that allow us to see. The primary degeneration can affect either the rods or the cones, but often progresses to complete blindness. RD is genetically heterogeneous, associated with over 250 different genes with different functions and expression patterns in the retina. This large number of target genes hampers the development of alternative cellular approaches for gene therapy and broad mutation-dependent neuroprotective therapies that target common cell death mechanisms. Published studies have provided some information on the intracellular mechanisms underlying the degenerative process and identified several factors that play key roles in photoreceptor cell death. These studies have mostly been derived from cell and animal models that display genetic mutations homologous to RD found in patient cohorts. One such approach uses primary photoreceptors differentiated from retinal stem cells. 5 , respectively, have demonstrated a suitable model for characterizing retinal cell death pathways when induced by or derived from the RD model, and have demonstrated utility for testing small molecules with neuroprotective activity. 6 Another approach involves retinal explants from RD models, which can also be used to study cell death mechanisms and experimental treatments. 4 .

[0005] Genes mutated in RD usually involve photoreceptor-specific functions. cGMP plays a direct role in the phototransduction cascade that occurs within photoreceptor cells when they are struck by light. In many cases, RD mutations lead to the excessive accumulation of cGMP in photoreceptors, for example in situations where genes for enzymes involved in cGMP metabolism in photoreceptors are affected. 7 Importantly, however, photoreceptor accumulation of cGMP can also be seen in situations where the mutated gene is not directly related to cGMP metabolism. 7, thus identifying the cGMP system as a possible target for mutation-dependent treatment approaches. For genes directly involved in cGMP metabolism, this is the case for mutations in phosphodiesterase 6, the photoreceptor enzyme that hydrolyzes cGMP to 5'-GMP (its subunits are encoded by the genes PDE6B, PDE6A, and PDE6G in rod photoreceptors and by the genes PDE6C and PDE6H in cone photoreceptors). The Pde6b gene is mutated in the retinal degeneration 1 (rd1) mouse model of retinitis pigmentosa (RP), which has been well studied in many laboratories. In a postulated sequence of events, the accumulation of cGMP in photoreceptors of PDE6B-mutated retinas occurs as a direct consequence of the actual genetic defect and thus can be seen as an early and mechanistically fundamental component of degeneration. In a next step, the cGMP increase can be expected to have at least one of the following four targets: 1) PKGs, which, when activated by cGMP, phosphorylate specific proteins; 2) PKGs, which, when activated by cGMP, phosphorylate Na; + and Ca 2+ ) cyclic nucleotide-gated channels (CNGCs) that allow cGMP-regulated influx of cyclic nucleotides (CGMPs), 3) PDEs, and 4) hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. The first two cGMP targets are directly related to photoreceptor degeneration, 4、8 , cGMP-targeted PDEs and HCN channels are further involved in the degenerative process. Due to their direct association with early degenerative events, PKG and CNGC can be considered as disease drivers, even though the downstream mechanisms are still not fully understood. 4、8 There may be other cGMP targets.

[0006] Previously, it has been shown that certain equatorially modified cGMP-derived PKG inhibitors, e.g., Rp-8-Br-cGMPS, Rp-8-Br-PET-cGMPS, offer some protection of rd1 and rd2 mutant photoreceptors in both in vitro (rd1 and rd2) and in vivo (rd1) model systems of analysis. 4 The rd2 model harbors mutations apparently unrelated to the cGMP system. 4、7However, these current state-of-the-art equatorially modified cGMP analogues must be applied in high extracellular doses to exert their inhibitory effect on the pathologically imbalanced cGMP system, thus implying the risk of extracellular or intracellular side effects. The inventors have now realised that a class of compounds that promise to show improved inhibitory efficacy compared to the state-of-the-art compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS are equatorially modified (inhibitory) polymer-linked multimeric cGMP (PLM) analogues, which have not been previously synthesized or studied. Secondly, chemically related activating polymer-linked dimeric cGMP (PLD) analogues lacking the equatorial modification have been reported to induce a marked increase in activation of either PKGIα or CNGC, depending on the spacer length, when compared to monomeric cGMP. 10 However, one drawback of this single related report was the low-yielding synthetic related strategy that did not provide effective access. 10、12 . Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, there is no approved prophylactic or therapeutic method available for RD, and there is a need for the development of new and improved compounds that can directly interfere with cell death pathways and prevent photoreceptor death. Therefore, the object of the present invention is to provide novel equatorially modified cGMP analogs for the inhibition of cell death pathways activated during retinal degeneration processes. Preferably, to demonstrate their effectiveness for the potential treatment of photoreceptor cell death, the novel equatorially modified cGMP analogs should be effective inhibitors of cell death in primary photoreceptors differentiated from retinal stem cells and in RD-related cells or tissues. A further object of the present invention is to provide novel equatorially modified cGMP analogs as research tools for identifying and validating the cGMP system in other cell systems of neuronal origin and in general cell culture or tissue systems. In a further object, the novel equatorially modified cGMP analogs should be more effective than Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS in inhibiting the cGMP system in cell culture and tissue systems. Another object of the present invention is to provide novel equatorially modified cGMP analogs for affinity chromatography, for antibody production, for diagnostic applications, or as additives for organ or tissue transplant storage solutions. A further object of the present invention is to provide novel equatorially modified cGMP analogs for the pharmacological inhibition of disease-related imbalanced cGMP even outside the retina and retinal photoreceptors within a medical environment. Another object of the present invention is to establish novel equatorially modified (inhibitory) polymer-linked multimeric cGMP analogs, while comparing them with the single related report of potentiated PLD compounds. 10 To this end, a more robust and regioselective synthetic method with improved yields is applied to give efficient access. [Means for solving the problem]

[0008] Summary of the Invention Thus, the object of the present invention is solved by providing novel equatorially modified (inhibitory) polymer-linked multimeric cGMP analogs, while also applying a more robust and regioselective synthetic method with improved yields to give efficient access to said novel equatorially modified (inhibitory) polymer-linked multimeric cGMP analogs. In a further aspect, the object of the present invention is achieved by novel pharma- cetically acceptable equatorially modified polymer-linked multimeric cGMP (PLM) analogs or their related monomeric precursors, which have improved properties compared to the state-of-the-art compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS for treating or diagnosing pathologies, conditions or disorders associated with dysregulation of cGMP-acting cellular targets, preferably at least one of, for example but not limited to, cGMP-dependent protein kinase (PKG), hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, phosphodiesterases (PDEs) and cGMP-gated channels (CNGCs). In a further aspect, the object of the present invention is achieved by novel equatorially modified polymer-linked multimeric cGMP analogs or their related monomeric precursors, which are utilized as research tools suitable for interfering with the cGMP system in cell cultures or tissues or as diagnostic tools. Preferably, the cGMP analog is a chemically conjugated multimer of equatorial modified guanosine-3',5'-cyclic nucleotide monophosphate, including tethered dimers, trimers and tetramers of formula (I) or formula (II) or a monomeric precursor cGMP analog of formula (III).

[0009] BRIEF DESCRIPTION OF THE FIGURES AND FORMULA Formula I shows the general structure of the compounds of the invention (branched and linear analogs). Formula Ib shows a more detailed illustration of Formula I. Formula II shows the general structure of the compounds of the invention (linear analogs). Formula IIb shows a more detailed illustration of Formula II. Formula III shows the general structure of a G unit as an individual compound of the invention or a unit of a compound of formula I or II. Formulas IV and V show the general structure of the G units of formula III featuring typical imidazolinone substitutions. [Brief description of the drawings]

[0010] [Figure 1] Examples of trimer compounds of the present invention are given below to illustrate the variables used. [Diagram 2] Protective effect of exemplary compounds of the invention against cell death in primary rod-like cells (compared to known compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS). Description: Primary rod-like cells from rd1 mutant mice undergo spontaneous cell death 11 days after differentiation. Rod-like cells were exposed to compounds on day 10 of culture and analyzed after 24 hours. A.: Test compound at 0.1 μM concentration. B.: Test compound at 1 μM concentration. Percentage of dead cells was assessed by ethidium homodimer assay. Untreated cells are shown as control samples (black bars). Reference compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS are shown as dashed bars. Data are shown as mean ± SD from at least three biological replicates. [Diagram 3] The culture paradigm for rd1 explant experiments is shown. Description: Animals aged 5 days after birth (PN5) were killed by decapitation and retinas with attached retinal pigment epithelium were dissected as previously described9. After flattening the retinas on the membrane of a commercially available 6-well culture insert, 1.5 ml of custom-made culture medium was added to each well. These explants were then maintained in culture for 2 days without any treatment, after which the test analogs of the invention were added at the desired concentration and the medium was changed at PN7 ("7" in the figure). The same concentration of the analogs of the invention was then changed to fresh medium at PN9 ("9" in the figure) and the cultures were maintained until PN11 ("11" in the figure). At this point the experiment was terminated by a fixation procedure. This paradigm is therefore referred to as PN5+2+4. Controls, i.e. rd1 explants without any treatment, used the same paradigm. Healthy animals (wild type, wt) can be used for comparison. The lighter parts of the horizontal bars represent the untreated initial period, while the darker parts indicate the actual treatment time. [Figure 4]Protective effect of exemplary compounds of the invention against cell death in retinal explants (compared to known compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS). Description: Effect of selected analogs of the invention on photoreceptor cell death in rd1 explants at given concentrations. After cell death was assessed by so-called TUNEL staining on fixed and sectioned material, the number of dead cells was counted and analyzed and compared to the number of dead cells in untreated rd1 explants. To allow a more direct comparison between the various analogs and concentrations, the ratio of treated / untreated specimens was calculated. The leftmost bar represents the untreated explant as is, which has a ratio of 1.0 since there is no effect. And the next bar is for 50 μM Rp-8-Br-PET-cGMPS, with an effect ratio of about 0.78. This means that this treatment reduced photoreceptor cell death by more than 20%. The rest of the treatments can be interpreted in a similar manner. Bars represent standard deviation, the number of tests was 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention The present invention relates to novel equatorially modified polymer-linked multimeric cGMP (PLM) analogs and related monomer precursors, in which the term "equatorial modification" refers to the modification of the equatorial exocyclic position of the 3',5'-cyclic monophosphate (R 8 The present invention has utility as improved pharmaceutical agents and research tools. The concept of potentially achieving increased activity by simultaneously addressing more than one binding site on a target protein with a single molecule has previously been reported using activated polymer-linked dimeric cGMP analogs (PLDs) without equatorial modifications. 10In that report, a series of PLDs differing only in the length of the PEG spacer were synthesized and tested for their ability to activate cGMP-dependent protein kinase Iα (PKG Iα) and cyclic nucleotide-gated ion channels (CNG channels). These results suggested that PLDs possess enhanced activation potential compared to monomeric cGMP, but this enhancement is essentially determined by the optimal spacer length (between cGMP units) specific to each target protein. Thus, as the deviation from this optimal spacer length increases, the effect is reported to decrease and eventually disappear.

[0012] Based on this work, it has been proposed that the inhibitory potency of known antagonistic cGMP analogs featuring phosphorothioate groups with the Rp configuration can also be improved through linkage via a polymer spacer to the second unit of the analog. 11 However, this was not pursued in practice. The proposal did not include any recommendation or evaluation of suitable positions within the molecule for the attachment of a linking spacer. It was therefore unclear at which positions a spacer would be tolerated to achieve an increased inhibitory effect, especially when no increased inhibitory effect was obtained with respect to the other PKG isoforms Iβ and II. Furthermore, apart from one synthesis that had already been carried out, there was no synthetic protocol for the preparation of the multimeric antagonistic compounds, nor a description of the nature of the coupling functionalities or functionalization of the precursors suitable for effecting the linking to further units and / or spacers. In said synthesis (of the activator analogue), cGMP bearing a thiol group at the 8-position was reacted with a bifunctional PEG vinylsulfone to give a dimer linked at the 8-position. 10-11 No yields were provided for this method. However, as later published 12 , and in accordance with our own experience, the reported conditions favor addition at the 7-position rather than the 8-position. It must therefore be concluded that the desired dimer linked at the 8-position was only obtained in poor yield using this strategy. Thus, a more robust and regioselective method with improved yields was needed to provide effective access to inhibitory analogues of the compound for further exploration.

[0013] As mentioned above, the potential of PLD has so far only been investigated for a single homologue series of activator analogs and their effects on PKG isoform Iα and CNG channels. In this context, other targets of the cGMP signaling cascade, such as PKG Iβ and II, were not studied, and therefore it was not known what modifications would be required to address these targets. In complex cell systems such as primary rod-like cells used here as a model for retinal cell death pathways, the cGMP system is dysregulated. Thus, overactivation of more than one cGMP target can cause cell death. It was unclear which cGMP target needed to be addressed to achieve a protective effect. For example, if inhibition of multiple targets was required, it was unclear whether PLD or PLM analogs would be generally more suitable. Previous studies had only focused on spacer length as a modifier to improve and optimize the activity of PLD compounds, and the results suggested fairly selective target affinity depending on the spacer length. Thus, addressing more than one target with the same PLD, even if feasible, seemed possible only at intermediate spacer lengths where the activation potential of both targets would be significantly reduced. The effects of nucleobase modifications (eg substitutions), variations in the coupling moiety or linkage position, as well as the combination of two different cGMP units within the PLM on activation potential have not been addressed before. The most essential factors related to the concept of inhibiting the cGMP signaling pathway with multimeric cGMP analogs have been unclear.

[0014] When we set out to discover and establish the first compound of this kind, we first sought to identify the synthetically more easily accessible activator analogues (R 8 Starting with 8-(R =O, see formula III), we investigated their ability to activate PKG isoforms Iα, Iβ and II. 1To replace the poor coupling strategies of the prior art, various more robust, regioselective and higher yielding methods involving, for example, peptide (amide) and click chemistry were developed. The new PLDs were significantly more active than those reported in the art. 10 They found that all applied novel coupling methods gave very similar results, while also essentially maintaining their improved activation potential over a fairly wide variation in spacer length. Surprisingly, it was further found that the nucleobase manipulation of PLD and / or the variation of the coupling functionalities, both of which have not been studied before, invalidated previously proposed targets selectively induced by spacer length. In particular, R 4 and / or R 5 Variations in (e.g., β-phenyl-1, N 2 The -etheno (PET) moiety; see Formula III) induced ultrastrong PKG Iα activation even at spacer lengths where elevated activation had not previously been observed, an effect that was much more potent than any of the spacer length-related effects. 1 The replacement of the sulfonyl coupling functional group with the modification of the moiety also significantly enhanced PKG activation. Another structural aspect of the novel PLDs relates to the linkage position that couples the two cGMP analogs to each other. The observed activity enhancement of the PLDs is therefore due to the 1 Linkage was not limited by position. Activity enhancement was present even when linkage was varied along the G unit. Thus, as a non-limiting example, 4 +R 5 The PLD connected by R 1 Position-tethered PET-substituted derivatives showed increased PKG agonist potential as well. Surprisingly, mixed (heterogeneous) PLDs featuring two unequal G units with different binding affinities (e.g., containing one PET-cGMP unit and one lacking the PET moiety) conferred PKG (isoform) activation profiles that to a large extent resembled the properties of both G units in their corresponding cognate PLDs. Furthermore, mixed PLDs containing mixed linkage positions (e.g., 4 +R 5-PET-cGMP analog unit linked with a PET moiety and R 1The first G unit lacking the PET moiety linked at position 1 behaved similarly. These results imply that, although linkage to a second cGMP (analog) is required to obtain a highly enhanced PKG activity, the second G unit does not necessarily have to be of the same kind. As mentioned, the superior PKG activation of the first G unit (again observed for each of the cognate PLDs) is substantially preserved in the mixed PLD hybrids, although the second G unit may even be a very less potent activator of PKG (as observed for each of the cognate PLDs). These unexpected findings reveal another new and great potential of (mixed) PLDs. Established effector compounds often need to be derivatized for specific biochemical applications. For example, the introduction of fluorescent dyes is a very common strategy to allow intracellular localization using microscopy or spectroscopic techniques. To obtain representative results, the transformation, which is ideally meant to facilitate assay interpretation, should not affect the target activation profile. However, these structural manipulations of the original compounds often lead to significant shifts in target affinity and specificity, and even to loss of activation potential. This is especially the case when a specific moiety can only be introduced at the pharmacophore group or when it inhibits or weakens binding to the target protein due to steric hindrance. For applications that benefit from the use of multiple targeting compounds, a change in the target activation profile (or its extent) may also be clearly desirable. However, the development of multi-targeting compounds may be just as difficult as the production of target-specific compounds. This is difficult whenever the modifications required to address one target inhibit binding to a second target. The mixed PLDs disclosed within the scope of the present invention provide an improved solution to both of these problems. Their advantage stems from the fact that two cGMP units (not the units for the monomers) contribute to the overall PKG activation profile. As mentioned above, even those modifications that would give completely different target affinities (observed for the monomers or for the cognate PLDs) do not erase the improved activation properties of the parent compound, as long as they are only performed on one cGMP unit.In this respect, the effect of structural manipulation at a single cGMP unit is buffered in the mixed PLD. Thus, the mixed PLD allows a much wider variety of modifications (at one cGMP unit), while the undesirable decrease in PKG activation caused by these modifications is hardly noticeable, if any. On the other hand, the mixed PLD also supports the design of multi-target compounds. Functional groups (e.g. PET groups) that are intended to address different targets (e.g. different PKG isoforms) can be introduced explicitly at one cGMP unit, giving the mixed PLD a broad target activation spectrum.

[0015] The concept of polymer-linked cGMP analogues has been extended from dimers to trimers and tetramers. In that respect, the linkage of specific cGMP units is achieved either in a linear or branched manner (see formulae I and II). The increase in the number of cGMP units in trimers and tetramers provides more diverse opportunities for combining (different) activators and target-dependent functionalized cGMP units. The tested analogues also provided significantly improved PKG activation similar to the dimeric analogues. For a more detailed description of the properties of multimeric activated cGMP analogues, please refer to the co-pending European patent application with application number 16186700.7. Thus, experiments with related activator analogs of the present invention have revealed many beneficial new properties of multimeric cGMP analogs. The present invention clearly identifies positions R1, R4, and R5 as important modifiers of PKG activation potential. These positions tolerate fairly wide variation both when used as linking positions and when used to attach substituents.

[0016] This new knowledge was then adapted and tested for the inhibitory equatorially modified PLM analogs of the present invention. Given the uncertainties mentioned above, it was not expected that the corresponding dimeric analogs compounds 1 and 2 (Table 13) would both show significantly improved potency in preventing cell death of primary rod-like cells, a biological system known to have an activated cGMP-signaling system, compared to the state-of-the-art compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS (Figure 2). This result is particularly interesting as it is the first demonstration of an inhibitory effect due to a PLD analog and represents a proof of concept in support of the present invention. Moreover, for the corresponding stimulatory analogs, R 4 and / or R 5 It was found that derivatives bearing substituents at the R position, e.g. PET groups (as present in compound 2), confer PKG affinity very similar to analogs linked at these positions. This strongly suggests that equatorially modified PLM analogs featuring said alternative linking positions will show similar protective effects. Compounds 1 and 2 differ only with respect to the PET moiety. This moiety similarly has a very strong effect on the PKG affinity profile as experienced by the corresponding activators. It must therefore be concluded that at least for this test system, a fairly extensive modification of the PKG affinity profile and thus of the substituents is tolerated. Moreover, if both compounds give good results separately, it is speculated that, according to the educated inventors, mixed equatorially modified PLDs, characterized in that one cGMP unit has a PET substituent and one does not, will very likely constitute similarly potent inhibitors in accordance with the above findings, despite their different PKG affinity profiles. It also seems very likely that this concept can be applied to trimeric and tetrameric equatorially modified PLMs as well. To further improve the protective effect, one focus will be on the R 1 , R 4 and / or R 5 The aim of the study was to find better substituents at positions 1 and 2 of the activator complex. These modifiers had a strong effect on the target affinity as judged by the corresponding activators. Therefore, a number of analogues featuring said modifications were synthesized and tested at the monomer precursor stage to select promising candidates.

[0017] Surprisingly, all tested compounds of the present invention showed significantly improved potency in preventing cell death of primary rod-like cells compared to the state-of-the-art compounds Rp-8-Br-cGMPS and p-8-Br-PET-cGMPS (Figure 2). The majority were much more effective than the dimeric compounds 1 and 2, making them promising candidates for conversion into PLM analogues. Non-limiting examples of this substance β-1 include N 2 -acetyl-8-bromoguanosine-3',5'-cyclic monophosphorothioate (Rp isomer, compound 21, Table 14) was assembled into the corresponding dimer 3 (Table 13), which showed further improved protective effect in primary rod-like cells. Another non-limiting example of this substance is 8-bromo-(3-thiophene-yl-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphorothioate (Rp isomer, compound 23, Table 14) was assembled into the corresponding dimer 20 (Table 13), which showed further improved protective effect in primary rod-like cells. It was further unexpected that all the tested monomeric and multimeric compounds of the present invention produced significantly improved cell viability compared to Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS in retinal explants from rd1 mice, a recognized animal model for RP that has pathologically high cGMP levels and an imbalanced cGMP system in photoreceptor cells (Figure 4). Particularly surprisingly, not only the monomeric and multimeric compounds of the invention having higher lipophilicity than Rp-8-Br-PET-cGMPS, but also those compounds having lower lipophilicity (Table 17) showed improved protection against cell death in primary photoreceptor cells and explants derived from rd1 mice compared to Rp-8-Br-cGMPS.

[0018] The novel equatorially modified cGMP analogs are compounds of formula (I) or (II):

[0019] [ka]

[0020] During the ceremony, G unit G 1 and G 2 are independently compounds of formula (III) and each G unit G 3 and G 4 is G 1 and G 2 and independently of each other, a compound of formula (III) or absence, where in the case of formula (II), G 3 If does not exist, then G 4 is always non-existent,

[0021] [ka] (III)

[0022] In the formula (III), X, Y and Z are N; R 1 , R 4 , R 5 , and R 8 is the unit of each G (G 1 , G 2 , G 3 and G 4 ) may be independently equal or individual; On top of that, R 1 are independently H, halogen, azido, cyano, acyl, aracyl, nitro, alkyl, aryl, aralkyl, amido-alkyl, amido-aryl, amido-aralkyl, amido-O-alkyl, amido-O-aryl, amido-O-aralkyl, OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, O-aracyl, SH, S-alkyl, S-aryl, S-aralkyl, S-acyl, S-aracyl, S(O)-alkyl, S(O)-aryl, S(O)-aralkyl, S(O)-acyl, S(O)-aracyl, S(O) 2 -Alkyl, S(O) 2 -aryl, S(O) 2 -Aralkyl, S(O) 2 -Acyl, S(O) 2-aracil, SeH, Se-alkyl, Se-aryl, Se-aralkyl, NR9R10, carbamoylR11R12, NH-carbamoylR11R12, O-carbamoylR11R12, SiR13R14R15, where R9, R10, R11, R12, R13, R14, R15 can be independently H, alkyl, aryl, aralkyl; R 2 is non-existent; R 3 is OH; R 4 may independently be absent, H, amino, alkyl, aralkyl, nitro, N-oxide, or Y and R 5 And Y and R 5 together with the carbons bridging Y and R may form an imidazole ring, which may be unsubstituted or substituted with alkyl, aryl or aralkyl, or Y and R may 5 And Y and R 5 together with the carbons bridging them may form the following imidazolinones (structures IV, V, n=1) or homologous rings (n=2-8) which may be unsubstituted or substituted with alkyl, aryl or aralkyl (not shown);

[0023] [ka]

[0024] R 5 are independently H, halogen, azido, cyano, acyl, aracyl, nitro, alkyl, aryl, aralkyl, amido-alkyl, amido-aryl, amido-aralkyl, amido-O-alkyl, amido-O-aryl, amido-O-aralkyl, OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, O-aracyl, SH, S-alkyl, S-aryl, S-aralkyl, S-acyl, S-aracyl, S(O)-alkyl, S(O)-aryl, S(O)-aralkyl, S(O)-acyl, S(O)-aracyl, S(O) 2 -Alkyl, S(O) 2 -aryl, S(O) 2 -Aralkyl, S(O) 2-Acyl, S(O) 2 -aracyl, SeH, Se-alkyl, Se-aryl, Se-aralkyl, NR30R31, carbamoylR32R33, NH-carbamoylR32R33, O-carbamoylR32R33, SiR34R35R36, where R30, R31, R32, R33, R34, R35, R36 may each independently be H, alkyl, aryl, aralkyl, or R 4 , Y and Y and R 5 together with the carbons bridging R to form an imidazole ring, which may be unsubstituted or substituted with alkyl, aryl or aralkyl, or R 4 , Y and Y and R 5 together with the carbons bridging them may form an imidazolinone ring (structures IV, V, n=1) or homologous ring (n=2-8) as above, each of which may be unsubstituted or substituted with alkyl, aryl or aralkyl (not shown); R 6 is OH; R 7 is O; and R 8 is SH, S-alkyl, S-aryl, S-aralkyl, SeH, Se-alkyl, Se-aryl or Se-aralkyl, borano (BH 3 ), methylborano, dimethylborano, cyanoborano (BH 2 CN), S-PAP, Se-PAP, S-BAP or Se-BAP; where PAP is a photoactivatable protecting group, non-limiting examples of which are optionally PAP = o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylamino-coumarin-4-yl (DMACM caged), 7-diethylamino-coumarin-4-yl (DEACM caged) and 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM caged); and BAP is a bioactivatable protecting group, non-limiting examples of which are optionally BAP=methyl, acetoxymethyl, pivaloyloxymethyl, methoxymethyl, propionyloxymethyl, butyryloxymethyl, cyanoethyl, phenyl, benzyl, 4-acetoxybenzyl, 4-pivaloyloxybenzyl, 4-isobutyryloxybenzyl, 4-octanoyloxybenzyl, 4-benzoyloxybenzyl; and, Linking residue LR 1 , L.R. 2 , L.R. 3 and L.R. 4 are independently determined by the number of G units to which they are attached (G 1-4 ) specific residue R 1 , R 4 and / or R 5 or may be covalently attached to either Here, they are residues R 1 , R 4 and / or R 5 When the specific residue (R 1 , R 4 and / or R 5 The end-standing groups of the aryl groups are converted or replaced during the process of establishing the bond to form specific linking residues (LR 1-4 ), at the same time L-R 1 is (a) a trivalent or tetravalent branched hydrocarbon moiety or (b) a divalent hydrocarbon moiety, each of which may or may not incorporate heteroatoms such as, for example and without limitation, O, N, S, Si, Se, B, the backbone of which preferably contains 1 to 28 carbon atoms and may be saturated or unsaturated, substituted or unsubstituted; at the same time each attachment point may independently be a substituted or unsubstituted carbon or heteroatom; When polyethylene glycol (PEG) moieties are incorporated according to the definition, the preferred number of carbon atoms may exceed the number present in the PEG moieties, and all PEG moieties taken together Divalent Linking Residue (LR 1 ) is 1 to 500 ethylene glycol groups (-(CH 2 CH 2 O) n -, n = 1 to 500), or Divalent Linking Residue (LR 1 ) in the case of 1 to 750 ethylene glycol groups (-(CH 2 CH 2 O) n -, n = 1 to 750) or Tetravalent Linking Residues (LR 1 ) is 1 to 1000 ethylene glycol groups (-(CH 2 CH 2 O) n -, n = 1 to 1000), And if replaced, Substituents may optionally 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; and / or the substituents may further be linked together to form a ring system having 1 to 4 rings, which may be saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic, with or without incorporated heteroatoms; L-R 2 , L.R. 3 and L.R. 4 is, for example and without limitation, a divalent hydrocarbon moiety with or without incorporated heteroatoms (which may be heteroatoms O, N, S, Si, Se, B), the backbone of which preferably contains 1 to 28 carbon atoms and may be saturated or unsaturated, substituted or unsubstituted; at the same time each attachment point may independently be a substituted or unsubstituted carbon or heteroatom; and When polyethylene glycol (PEG) moieties are incorporated according to the definition, the preferred number of carbon atoms may exceed the number present in the PEG moieties, with all PEG moieties combined having a total of 1 to 500 ethylene glycol groups (-(CH 2 CH 2 O) n -, n = 1 to 500), And if replaced, Substituents may optionally 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, epoxy, methoxy, ethynyl; and / or the substituents may further be linked together to form a ring system having 1 to 4 rings, which may be saturated or unsaturated, substituted or unsubstituted, aliphatic or aromatic, with or without incorporated heteroatoms; Here, in the case of formula (II), G 4 If non-existent, then LR 4 is also non-existent, and In the case of formula (II), G 3 and G 4 If non-existent, then LR 3 and L.R. 4 is also non-existent, and G 1 , G 2 , G 3 and G 4 may further be a salt and / or a hydrate, At the same time, optionally, non-limiting examples of suitable salts of a particular phosphate moiety are lithium, sodium, potassium, calcium, magnesium, zinc or ammonium, and trialkylammonium, dialkylammonium, alkylammonium, such as triethylammonium, trimethylammonium, diethylammonium and octylammonium; and G 1 , G 2 , G 3 and G 4may be optionally isotopically or radiolabeled, PEGylated, immobilized or labeled with a dye or other reporting group; Where: The reporting group and / or dye may be (a) Each G unit (G 1 , G 2 , G 3 and / or G 4 ) independently for a specific residue R 1 , R 4 and / or R 5 A linking residue (LR) covalently attached to or replacing either 5 ) through G 1 , G 2 , G 3 and / or G 4 is coupled to At the same time, L-R 5 ,LR 2 may be as defined by or (b) In the case of formula (I), G 3 and / or G 4 You can replace Where: Examples of optionally suitable dyes include, but are not limited to, fluorescent dyes such as fluorescein, anthraniloyl, N-methylanthraniloyl, dansyl, or nitro-benzofurazanyl (NBD) series, rhodamine-based dyes such as Texas Red or TAMRA, cyanine dyes such as Cy TM 3. Cy TM 5. Cy TM 7. EVOblue TM 10. EVOblue TM 30. EVOblue TM 90. EVOblue TM 100 (EVOblue TM -Family), BODIPY TM - Family, Alexa Fluor TM -family, DY-family, such as DY-547P1, DY-647P1, coumarins, acridines, oxazones, phenalenones, fluorescent proteins, such as GFP, BFP and YFP, and near-infrared and far-infrared dyes; and Reporting groups optionally include, but are not limited to, quantum dots, biotin, and tyrosyl methyl ester; and PEGylation can be performed using single or multiple LR PEG The attachment of the LR group is independent. PEG ,LR 2 where (i) each G unit (G 1 , G 2 , G 3 and / or G 4 ) independently for a specific residue R 1 , R 4 and / or R 5 By covalently bonding to or substituting either 2 Only one end of the 1 , G 2 , G 3 and / or G 4 ), and (ii) LR 2 provided that the other terminus of is either an alkyl group or a reactive group that allows for conjugation reactions and / or hydrogen bonding; At the same time, optionally, non-limiting examples of reactive groups include -NH 2 , -SH, -OH, -COOH, -N 3 , -NHS-ester, halogen group, epoxide, ethynyl, allyl, and (iii) L.R. PEG is the ethylene glycol moiety (-(CH 2 CH 2 O) n -, n=2~500) is included.

[0025] chemistry definition Listed below are definitions of various terms and phrases used to describe the compounds of this invention. These definitions apply to the terms as used throughout this specification. Halogen refers to F, Cl, Br, and I. Alkyl refers to an alkyl group, which is a hydrocarbon moiety having 1-28, preferably 1-20 carbon atoms, with or without (incorporated) heteroatoms, such as, for example and without limitation, O, S, Si, N, Se, B, and unless otherwise specified, the point of attachment is a carbon atom. The structure can be: Straight chain saturated hydrocarbon moieties - such as, but not limited to, methyl, ethyl, propyl, butyl, and pentyl. or Linear unsaturated hydrocarbon moieties - more preferably containing 2 to 20 carbon atoms, such as, but not limited to, ethylene, propylene, butylene, and pentylene. or Branched saturated hydrocarbon moieties - deviate from the general alkyl definition by containing at least 3 carbon atoms, such as, but not limited to, isopropyl, sec.-butyl, and tert.-butyl. or Branched unsaturated hydrocarbon moieties - deviate from the general alkyl definition by containing at least three carbon atoms, such as, but not limited to, isopropenyl, isobutenyl, isopentenyl, and 4-methyl-3-pentenyl. or Cyclic saturated hydrocarbon moieties - more preferably containing 3 to 8 ring atoms, such as, but not limited to, cyclopentyl, cyclohexyl, cycloheptyl, piperidino, piperazino, etc. or Cyclic unsaturated hydrocarbon moieties - more preferably containing from 3 to 8 ring atoms.

[0026] As used herein, the term saturated means that the group has no carbon-carbon double bonds and no carbon-carbon triple bonds. However, when a saturated group is substituted, one or more carbon-oxygen or carbon-nitrogen double bonds may be present, which may exist as part of keto-enol and imine-enamine tautomerism, respectively. Regardless of its structure, an alkyl group as defined herein may 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, epoxy, methoxy, ethynyl. When an alkyl as defined herein includes a polyethylene glycol (PEG) moiety, the preferred number of carbon atoms may exceed that present in the PEG moiety, with the PEG moiety being a total of 1 to 500 ethylene glycol groups (-(CH 2 CH 2 O) n -, n = 1 to 500). -(CH 2 CH 2 O) n -(EO) as an abbreviation for - (where n represents the number of ethylene glycol groups) n It should be noted that the use of - The number of ethylene glycol groups can be, in particular, n=1 to 500 or as stated in the specific examples.

[0027] Aralkyl refers to an alkyl group as described above attached to an unsubstituted or substituted aromatic or heteroaromatic hydrocarbon moiety consisting of one or more aromatic or heteroaromatic rings, each having from 3 to 8 ring atoms. Substituents on both the alkyl and aryl moieties 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, and 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, each having 3 to 8 ring atoms. 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, and methylthio.

[0028] Acyl refers to a -C(O)-alkyl group, where alkyl is as defined above. Aracyl refers to the group -C(O)-aryl, where aryl is as defined above. Carbamoyl is -C(O)-NH 2 “R” refers to a group in which the hydrogens may be replaced independently of one another by an alkyl group, an aryl group, or an aralkyl group, where the alkyl group, the aryl group, or the aralkyl group are as defined above. O-Acyl refers to an -OC(O)-alkyl group, where the alkyl group is as defined above. O-alkyl refers to an alkyl group attached through an O linkage, where the alkyl group is as defined above. O-Aracyl refers to the group -OC(O)-aryl, where aryl is as defined above. O-aralkyl refers to an aralkyl group attached through an O linkage, where the aralkyl group is as defined above. O-aryl refers to an aryl group attached through an O linkage, where the aryl group is as defined above. O-carbamoyl refers to a carbamoyl group attached through an O-linkage, where the carbamoyl group is as defined above.

[0029] S-alkyl refers to an alkyl group attached through an S linkage, where the alkyl group is as defined above. S-aryl refers to an aryl group attached through an S linkage, where the aryl group is as defined above. S-aralkyl refers to an aralkyl group attached through an S linkage, where the aralkyl group is as defined above. S-aralkyl refers to an aralkyl group attached through an S linkage, where aralkyl groups are as defined above. Se-alkyl refers to an alkyl group attached through a Se linkage, where the alkyl group is as defined above. Se-aryl refers to an aryl group attached through a Se linkage, where the aryl group is as defined above. Se-aralkyl refers to an aralkyl group attached through a Se linkage, where the aralkyl group is as defined above. NH-alkyl and N-bisalkyl refer to an alkyl group attached through a N linkage, where the alkyl group is as defined above. NH-aryl and N-bisaryl refer to an aryl group attached through an N linkage, where the aryl group is as defined above. NH-carbamoyl refers to a carbamoyl group attached through an N-linkage, where the carbamoyl group is as defined above. Amido-alkyl refers to an alkyl group attached through an NH-C(O) linkage, where the alkyl group is as defined above. Amido-aryl refers to an aryl group attached through an NH-C(O) linkage, where the aryl group is as defined above. Amido-aralkyl refers to an aralkyl group attached through an NH-C(O) linkage, where aralkyl groups are as defined above.

[0030] The end standing group is a (sterically) available and specific linking residue (LR 1-4 ) can be covalently bonded to a specific residue (R 1 , R 4 and / or R 5 ) group. This refers to the residue (R 1 , R 4 and / or R 5) or at the actual end of the residue (R 1 , R 4 and / or R 5 ) at either end of the side chain of any of the residues (R 1 , R 4 and / or R 5 ) are located differently within the LR 1-4 The definition of the term end-standing group includes, where applicable, residues LR 5 and / or L.R. PEG Furthermore, the term terminal refers to an end-standing group that is actually at the end of the relevant residue. Those skilled in the art will recognize that certain linking residues (LR 1-4 It is well known that the linking residue (LR) may represent a radical depending on the specific number of G units to which it is attached. Thus, in the compound of formula (II), the specific linking residue (LR 1-4 ) may be a biradical, or, if it is only (intermediately) attached to one particular G unit, it may be a monoradical. Similarly, in the case of compounds of formula (I), depending on the particular number of G units to which it is attached, a particular linking residue (LR 1 ) can be a biradical, triradical, or tetraradical, or if it is only (intermediate) attached to one particular G unit, it can be a monoradical.

[0031] When another contemplated monovalent group is used with the modifier "divalent", as in "divalent alkyl", this provides a second point of attachment. A non-limiting example of a divalent alkyl is -CH 2 -, -CH 2 CH 2 -, -CH 2 C(CH 3 ) 2 CH 2 -It becomes. Ring systems, such as those of the formula:

[0032] [ka]

[0033] Whenever side chains or residues are depicted as "floating groups" in, these side chains (or residues) may replace any hydrogen atom attached to any of the ring atoms, including hydrogens depicted, implied, or explicitly defined, so long as a stable structure is formed. Thus, all resulting substitution patterns are included. For the given example, this amounts to:

[0034] [ka]

[0035] Those skilled in the art will understand that many compounds that fall under formula III as defined above have tautomeric forms. It should be noted that according to this specification, all tautomeric forms fall under formula III if at least one of the tautomers falls under formula III as defined above. In the chair form of a saturated six-membered ring, the bonds to the ring atoms and the molecular entities attached to the bonds are called "axial" or "equatorial" depending on whether they are located on the periphery of the ring ("equatorial") or whether they are positioned above or below the approximate plane of the ring ("axial"). For a given stereochemistry of the cyclic phosphate ring, axial positions can only be in the approximate plane of the ring. In naturally occurring cyclic nucleotide monophosphates (cNMPs), R7 and R8 are both oxygens, and the phosphorus double bond is "distributed or rearranged" between both atoms. In water at physiological pH, the compound has a negative charge between both oxygens, and a corresponding cation such as H+ or Na+. The compounds of the present invention have different functional groups, e.g., the equatorial (R8) oxygen replaced by sulfur, while the axial (R7) oxygen can also be optionally replaced. Regardless of the nature of the newly introduced R7 and / or R8, the corresponding compound structures herein are represented as charged compounds with a rearranged double bond at phosphorus, so long as it follows the valence rules. This style is chosen to consider, depict, and disclose all possible "positions" of the double bond of each phosphorus within a single structure and the distribution of electron density or each charge. However, the rearranged double bond as used herein, depending on the nature of the specific R7 and R8, does not necessarily refer to an equal distribution of charge or electron density between R7 and R8. When R7 and R8 are not equal, the phosphorus atom becomes chiral with four different ligands, resulting in stereoisomeric forms. To describe the chiral phosphorus configuration, the Rp / Sp nomenclature is used, in which R / S follows the Cahn-Ingold-Prelog rule and "p" is an abbreviation for phosphorus. To give an example: Equatorial residue R 8 In the case of sulfur (on the other hand, axial R 7 is oxygen), the corresponding cyclic guanosine-3',5'-monophosphorothioate compound (cGMPS analogue) has the Rp configuration at phosphorus and the equatorial residue R 8 When is a borano group, the corresponding cyclic guanosine-3',5'-monoboranophosphate compound (cGMPB analogue) has the Sp configuration at phosphorus. The skilled artisan knows that for use in the medical field, in particular as part of a medicament, only physiologically acceptable salts of the compounds according to the invention may be used.

[0036] Further details of the structure In one embodiment, the present invention relates to a compound represented by the formula (I) in the above definition, 4 is absent or, in the case of formula (II), G4 and L.R. 4 is absent. In another embodiment, the present invention relates to a compound represented by the formula (I) in the above definition, 3 and G 4 is absent or, in the case of formula (II), G 3 , G 4 , L.R. 3 and L.R. 4 is absent. In a further embodiment, the present invention relates to a compound according to the above definition, wherein in formula (I), G 2 , G 3 , G 4 and L.R. 1 is absent or, in the case of formula (II), G 2 , G 3 , G 4 , L.R. 2 , L.R. 3 and L.R. 4 is absent. In this case, said embodiment corresponds to compounds which are precursors of the multimers of the invention. In one embodiment, the present invention provides a method for the preparation of a compound comprising: 8 is SH. According to the present invention, a linking residue LR is provided as shown in the following formulas (Ib) and (IIb). 1 , L.R. 2 , L.R. 3 and L.R. 4 is preferably further subdivided.

[0037] [ka]

[0038] During the ceremony, Coupling Functional Group C 1 , C 1’ , C 2 , C 2’ , C 3 , C 3’ , C 4 and C 4’may be defined by a structure that is, independently of each other, absent or selected from the group consisting of:

[0039] [ka]

[0040] At the same time, connectivity can be depicted or inverted as illustrated by the following: G 1 -OC(O)-NH-S 2 vs. G 1 -NH-C(O)-OS 2 Where: Coupling functional group (C 1 , C 1’ , C 2 , C 2’ , C 3 , C 3’ , C 4 and / or C. 4’ ) is a residue of G unit (G 1-4 R 1 , R 4 and / or R 5 ), a particular residue (R 1 , R 4 and / or R 5 ) are, independently of each other, As further defined above, the end standing group is replaced or converted into a coupling functional group. or The following groups (if present, Q1 is attached to the G unit):

[0041] [ka]

[0042] [ka]

[0043] is selected from and The linker (L) is the following linker

[0044] [ka] TIFF0007672368000011.tif249162 TIFF0007672368000012.tif251157 TIFF0007672368000013.tif33167

[0045] is selected from the group consisting of at the same time n for each side chain in a particular linker in the above list may have equal or distinct values ​​as defined; and All chiral, diastereomeric, racemic, and all geometric isomeric forms of the linkers (L) listed above are included herein even if not explicitly depicted; and Cationic linkers (L), such as ammonium derivatives, are salts containing chloride, bromide, iodide, phosphate, carbonate, sulfate, acetate or any other physiologically acceptable counterion; and Spacer (S 1 , S 2 , S 3 and S 4 ) are equal or individual within a particular compound and are absent or -(CH 2 ) n1 -(CH 2 CH 2 β) m -(CH 2 ) n2 -(β=O, S or NH; m=1-500, n1=0-8, n2=0-8, and both n1 and n2 can be independently equal or individual) or -(CH 2 ) n - (n=1 to 24).

[0046] In particular, the linking residues LR 1 , L.R. 2 , L.R. 3 and L.R. 4 is further subdivided as shown in formula (Ib) and (IIb), and the spacer portion (S 1-4 ), coupling functional group (C 1-4 , C 1’-4’ In a preferred embodiment of the present invention, it is preferred that the aryl group contains a coupling functional group (C 1-4 , C 1’-4’ )teeth, residue R 1 , R 4 and / or R 5 or by replacing either one of the spacer and G units (G 1-4 ) (compare with the structure of formula III) and / or Between the spacer and the linker (L), dye or another reporting group and / or (If no specific spacer is present) Residue R 1 , R 4 and / or R 5 By combining with or replacing any of the G units (G 1-4 ) and a dye or another reporting group and / or (When the specified spacer is absent and / or the G unit is replaced by a dye or other reporting group) between the linker (L) and the dye or other reporting group or the G unit (G 1-4 , residue R 1 , R 4 and / or R 5 by combining with or replacing any of Establish a covalent bond.

[0047] Coupling functional group (C 1-4 , C 1’-4’) are generated by reaction between end-standing groups of specific precursor moieties according to methods well established in the art. The precursor end-standing groups (of monomeric G units and (commercially available) linkers, dyes, reporting groups and spacers) and the corresponding coupling functionalities (C) to which they are converted in the (monomeric or multimeric) compounds constructed according to the present invention. 1-4 , C 1’-4’ Non-limiting examples of coupling functional groups (C 1-4 , C 1’-4’ ) may further independently be absent or equal or distinct within a particular monomeric or multimeric compound.

[0048] Table 1 End standing groups and corresponding coupling functional groups (C 1-4 , C 1’-4’ ) TIFF0007672368000014.tif208146 TIFF0007672368000015.tif197146 TIFF0007672368000016.tif83144

[0049] One of skill in the art will appreciate that the precursor end-standing groups of Table 1, such as, but not limited to, NHS esters in place of carboxylic acids or triflates in place of halogens, can be used similarly to generate specific corresponding coupling functionalities. 1 , R 4 and / or R 5 , linker (L), dye, reporting group and spacer (S 1-4 It is further understood that the )) can be interchanged with one another to provide inverted connectivity of the coupling functionalities within the monomer or multimer analogs. Non-limiting examples of multimeric compounds of the present invention illustrating the variables defined above are provided in FIG.

[0050] Preferred compounds of the present invention According to the present invention, R1 is H, halogen, azide, 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, preferably R9, R10, R13, R14, R15 being alkyl. According to the present invention, R1 is H, Cl, Br, I, F, N 3 , NO 2 , O.H., S.H., and N.H. 2 , C.F. 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-butenethio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, More preferably, it is selected from the group consisting of (4-(7-methoxy)coumarinyl)thio, (2-naphthyl)thio, (2-(1-bromo)naphthyl)thio, benzimidazolyl-2-thiobenzothiazolylthio, 4-pyridyl, (4-pyridyl)thio, 2-pyridylthio, 5-amino-3-oxopentylamino, 8-amino-3,6-dioxaoctylamino, 19-amino-4,7,10,13,16-pentaoxanonadecylamino, 17-amino-9-aza-heptadecylamino, 4-(N-methylanthranoyl)aminobutylamino, dimethylamino, diethylamino, 4-morpholino, 1-piperidino, 1-piperazino, triphenyliminophosphoranyl or as shown in Table 2 below.

[0051] Table 2 Residue R 1 . TIFF0007672368000017.tif177145

[0052] According to the present invention, R1 is H, Cl, Br, I, F, N 3 , NO 2 , O.H., S.H., and N.H. 2 , C.F. 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-butenethio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, (4-(7-methoxy)coumarinyl)thio, (2-naphthyl)thio, Particularly preferred are selected from the group consisting of (2-(1-bromo)naphthyl)thio, benzimidazolyl-2-thio, benzothiazolylthio, 4-pyridyl, (4-pyridyl)thio, 2-pyridylthio, 5-amino-3-oxopentylamino, 8-amino-3,6-dioxaoctylamino, 19-amino-4,7,10,13,16-pentaoxanonadecylamino, 17-amino-9-aza-heptadecylamino, 4-(N-methylanthranoyl)aminobutylamino, dimethylamino, diethylamino, 4-morpholino, 1-piperidino, 1-piperazino, triphenyliminophosphoranyl or as shown in Table 3 below.

[0053] Table 3 Residue R 1 . TIFF0007672368000018.tif145150

[0054] According to the present invention, R1 is 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-butenethio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2, More preferably, the aryl group is selected from the group consisting of 3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, (4-(7-methoxy)coumarinyl)thio, (2-naphthyl)thio, (2-(1-bromo)naphthyl)thio, benzimidazolyl-2-thio, benzothiazolylthio, 4-pyridyl, (4-pyridyl)thio, 2-pyridylthio, triphenyliminophosphoranyl, or as shown in Table 4 below.

[0055] Table 4 Residue R 1 . TIFF0007672368000019.tif109144

[0056] According to the present invention, in addition to or independently of the above, R4 is selected from the group consisting of H, amino, alkyl, aralkyl, nitro, N-oxide, or R4 is selected from the group consisting of Y and R 5 And Y and R 5 Together with the carbons bridging Y and R may form an imidazole ring, which may be unsubstituted or substituted with alkyl, aryl or aralkyl, or Y and R may form an imidazole ring, which may be unsubstituted or substituted with alkyl, aryl or aralkyl. 5 And Y and R 5 together with the carbons bridging them, can form the imidazolinones (structures IV, V, n=1) or homologous rings (n=2-8) described above, each of which can be unsubstituted or substituted with alkyl, aryl or aralkyl (not shown). It is further preferred according to the present invention that R4 is absent or selected from the group consisting of amino, N-oxide or as shown in Table 5 below. Table 5 Group R 4 . TIFF0007672368000020.tif203147 TIFF0007672368000021.tif93142

[0057] According to the present invention, it is particularly preferred that R4 is absent or selected from the group consisting of amino, N-oxide or as shown in Table 6 below.

[0058] Table 6 Residue R 4 . TIFF0007672368000022.tif182145 TIFF0007672368000023.tif93141

[0059] It is further preferred according to the present invention that R4 is absent or as shown in Table 7 below.

[0060] Table 7 Residue R 4 . TIFF0007672368000024.tif187145 TIFF0007672368000025.tif62131

[0061] Additionally or independently of the above, according to the present invention, R5 is selected from the group consisting of H, halogen, azide, acyl, aracyl, nitro, alkyl, aryl, aralkyl, amido-alkyl, amido-aryl, amido-aralkyl, amido-O-alkyl, amido-O-aryl, amido-O-aralkyl, NH-carbamoyl-alkyl, NH-carbamoyl-aryl, NH-carbamoyl-aralkyl, OH, O-alkyl, O-aryl, O-aralkyl, SH, S-alkyl, S-aryl, S-aralkyl, amino, NH-alkyl, NH-aryl, NH-aralkyl, NR30R31, SiR34R35R36, where R30, R31, R34, R35, R36 are alkyl, or R 4 , Y and Y and R 5 together with the carbons bridging R form an imidazole ring which may be unsubstituted or substituted with alkyl, aryl or aralkyl, or 4 , Y and Y and R 5 together with the carbons bridging them, it is preferred to form the imidazolinone ring (structures IV, V, n=1) or homologous ring (n=2-8) as described above, each of which may be unsubstituted or substituted with alkyl, aryl or aralkyl (not shown).

[0062] According to the present invention, R5 is H, NH 2, F, Cl, Br, I, nitro, methyl, ethyl, n-propyl, n-hexyl, 6-amino-n-hexyl, trifluoromethyl, phenyl, 4-N,N-dimethylaminophenyl, benzyl, 4-azidobenzyl, amido-n-butyl, amidoisobutyl, amido(6-amino-n-hexyl), OH, methyloxy, n-hexyloxy, phenyloxy, benzyloxy, SH, methylthio, ethylthio, 6-amino-n-hexylthio, phenylthio, 4-azidophenyl nylthio, benzylthio, 4-azidobenzylthio, 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, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl or R 4 , Y and Y and R 5 can be formed together with the carbons bridging the ring systems described in Table 5 (entries 2 and 3).

[0063] According to the present invention, R5 is H, NH 2 , F, Cl, Br, I, nitro, SH, methylthio, ethylthio, 6-amino-n-hexylthio, phenylthio, 4-azidophenylthio, benzylthio, 4-azidobenzylthio, 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 R 4 , Y and Y and R 5 can be formed together with the carbons bridging the ring systems described in Table 6 (entries 2 and 3). According to the present invention, R5 is NH 2 or R 4 , Y and Y and R 5and the carbons bridging them can form the ring systems described in Table 7 (entries 2 and 3).

[0064] Additionally or independently of the above, the present invention provides a method for the preparation of a cyclic alkyl group in which R8 is selected from the group consisting of SH, S-alkyl, S-aryl, S-aralkyl, borano (BH 3 ), methylborano, dimethylborano, cyanoborano (BH 2 CN), S-PAP, Se-PAP, S-BAP or Se-BAP, Here, PAP is a photoactivatable protecting group, PAP = o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylamino-coumarin-4-yl (DMACM caged), 7-diethylamino-coumarin-4-yl (DEACM caged) and 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM caged). and wherein BAP is a bioactivatable protecting group, BAP=methyl, acetoxymethyl, pivaloyloxymethyl, methoxymethyl, propionyloxymethyl, butyryloxymethyl, cyanoethyl, phenyl, benzyl, 4-acetoxybenzyl, 4-pivaloyloxybenzyl, 4-isobutyryloxybenzyl, 4-octanoyloxybenzyl, 4-benzoyloxybenzyl.

[0065] According to the present invention, R8 is selected from the group consisting of SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethylthio, phenylthio, benzylthio, 4-acetoxybenzylthio, 4-pivaloyloxybenzylthio, 4-isobutyryloxybenzylthio, 4-octanoyloxybenzylthio, 4-benzoyloxybenzylthio, borano(BH 3 ), methylborano, dimethylborano, cyanoborano (BH 2 More preferably, it is selected from the group consisting of: According to the invention, it is particularly preferred that R8 is selected from the group consisting of SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethylthio, phenylthio, benzylthio, 4-acetoxybenzylthio, 4-pivaloyloxybenzylthio, 4-isobutyryloxybenzylthio, 4-octanoyloxybenzylthio, 4-benzoyloxybenzylthio. It is further preferred according to the invention that R8 is SH.

[0066] Additionally or independently of the above, according to the present invention, the residue involved in linking a G unit to another G unit or to a dye or other reporting group can be R 1 , R 4 and / or R 5 where the particular residue is As defined for the preferred embodiment above, where the end standing group is replaced or converted to a coupling functionality or It is preferably selected from the group shown in Table 8 below (Q1, if present, is linked to the G unit).

[0067] Table 8 Residues R involved in linking a G unit to another G unit or to a dye or other reporting group 1 , R 4 and R 5 (If present, Q 1 binds to G units) TIFF0007672368000026.tif78135

[0068] According to the present invention, the residues involved in linking a G unit to another G unit or to a dye or other reporting group are R 1 , R 4 and / or R 5 where the particular residue is As defined for the preferred embodiment, the end standing group is replaced or converted to a coupling functional group. or More preferably, it is selected from the group shown in Table 9 below (Q1, if present, is linked to the G unit).

[0069] Table 9. Residues R involved in linking a G unit to another G unit or to a dye or other reporting group 1 , R 4 and R 5 (If present, Q 1 binds to G units) TIFF0007672368000027.tif135166

[0070] Additionally or independently of the above, according to the present invention, a coupling functional group (C 1-4 and C 1’-4’ ) is absent or selected from the group shown in Table 10 below.

[0071] Table 10 Coupling functional groups (C 1-4 and C 1’-4’ ). TIFF0007672368000028.tif78149

[0072] According to the present invention, the coupling functional group (C 1-4 and C 1’-4’ It is even more preferred that ) is absent or selected from the group shown in Table 11 below.

[0073] Table 11 Coupling functional groups (C 1-4 and C 1’-4’ ). TIFF0007672368000029.tif67147

[0074] Additionally or independently of the above, it is preferred according to the present invention that the linker (L) is absent or selected from the group shown in Table 12 below.

[0075] Table 12 Linkers (L). TIFF0007672368000030.tif213147

[0076] At the same time, n for each side chain in a particular linker can have equal or distinct values ​​as defined. In addition or independently of the above, according to the present invention, in the case of formula (I), G 4 Or G 4 and G 3 is non-existent or or in the case of formula (II), G 4 and L.R. 4 Or G 4 , L.R. 4 , G 3 and L.R. 3 is preferably absent. According to the present invention, in the case of formula (I), G 4 and G 3 is non-existent or in the case of formula (II), G 4 , L.R. 4 , G 3 and L.R. 3 It is even more preferred that is absent. Particularly preferred embodiments of the invention based on the above examples are as defined in any one of claims 5, 6, 7 and 8. According to the present invention, the compounds of Table 13 and the compounds according to claim 9 are particularly preferred. In case of doubt, it should be noted that the chemical structures shown in the formulas are the ones that are valid. It should further be noted that the compounds of Table 13 are shown as free acids. However, the present invention is not limited to Na + , Li + , N.H. 4 + , Et 3 NH + and (i-Pr) 2 EtNH + Also included are salts of these compounds with cations such as:

[0077] Table 13. Structures of novel equatorially modified polymer-linked multimeric cGMP compounds of the present invention TIFF0007672368000031.tif227170 TIFF0007672368000032.tif223170 TIFF0007672368000033.tif224170 TIFF0007672368000034.tif240170

[0078] As used herein, the term equatorial modification refers to a group R 8 It should be noted that this refers to modification of the R 8 For the non-limiting example of the invention where is SH and represents a phosphorothioate group, the resulting configuration is Rp. Care should be taken not to confuse this situation with the mirrored case (shown below), which is not part of the invention, where there is also a sulfur modification at the equatorial position, but the resulting configuration is Sp.

[0079] [ka]

[0080] Structures 1, 2, 4-13 and 15-19 from the priority application contained this obviously editorially incorrect structural element. In this respect, they have been corrected so that the skilled person can undoubtedly derive it directly from the priority application. Editorially incorrect structural elements are easily produced by mistake when the lower ribose part is inverted for optical or symmetric reasons during the drawing of the structure. However, the inversion would result in the projection of a chiral phosphorus center whose Rp configuration has been erroneously converted to Sp. Such editorially incorrectly described structural elements in the Sp configuration are obvious errors for the skilled person, since general formula III does not allow this situation and supports that all the monomer precursors applied in the synthesis of the illustrated specific examples of the present invention (Table 13) were in the Rp configuration. The skilled person will undoubtedly know that this configuration cannot be inverted under the reaction conditions used, and therefore all G units in the construction multimers of Table 13 must be in the Rp configuration.

[0081] The monomeric equatorially modified precursor cGMP analogs (G units) for the synthesis of equatorially modified polymer-linked multimeric cGMP analogs (PLMs) are compounds of formula (III). As mentioned above, the potency of blocking cell death in primary rod-like cells and retinal explants from rd1 mice is strongly increased when the monomeric precursor is linked to additional precursors in the PLM. Non-limiting examples of new robust and regioselective methods for the conversion of monomeric precursors to typical equatorially modified PLMs are provided in the Examples section. Furthermore, Table 1 provides an overview of typical end-standing groups that can be used in the coupling reaction and the corresponding coupling functionalities in the PLMs that are converted according to established methods in the art.

[0082] The present invention in one aspect also relates to a monomeric compound of formula (III) and / or a monomeric precursor of formula (III) of any of the compounds of the present invention described above, the monomeric compound of formula (III) and / or the monomeric precursor of formula (III) being defined in the context of any of said compounds hereinabove, and preferably the monomeric compound of formula (III) and / or the monomeric precursor of formula (III) being represented by the following formula: R 8 is not a substituted or unsubstituted borano functional group and furthermore, the monomer compound of formula (III) and / or the monomer precursor compound of formula (III) are not selected from the group of compounds consisting of the following compounds:

[0083] [ka]

[0084] In a preferred embodiment of the present invention, the monomeric compound of formula (III) and / or the monomeric precursor according to formula (III) of any of the above compounds of the present invention is R 8 is not a substituted or unsubstituted borano functional group, and R 4 is not H, but R 5 NH 2 Is it or R 5 NH 2 , H, 4-methoxytrityl, R 4 and R 5 but

[0085] [ka]

[0086] Isn't it? or R 4 and R 5 but

[0087] [ka]

[0088] and And R 1 is -NH-, -S-, -S(O)- or -S(O) 2 -bridge or carbon-carbon bond attachment, -SH, NH 2 , -S(CH 2 ) n NH 2 , -S(CH 2 ) n OH, -NH(CH 2 ) n NH 2 and -NH(CH 2 ) n Selected from the above group excluding OH (n=1-100) or R 1 is as described immediately above, except for monopara-substituted phenylthio and monopara-substituted phenylamino. The following conditions are met.

[0089] In another preferred embodiment of the present invention, the monomeric compound of formula (III) and / or the monomeric precursor according to formula (III) of any of the above compounds of the present invention is R 8 is not a substituted or unsubstituted borano functional group, and R 4 is not H, but R 4 +R 5 but

[0090] [ka]

[0091] Isn't it? or R 4 is H and R 5 NH 2 Not and (a) R 1 Isn't it H? Or (b) R 5 Is not H or 4-methoxytrityl? or R 4 and R 5 but

[0092] [ka]

[0093] and R 1 is -NH-, -S-, -S(O)- or -S(O) 2 -bridge or carbon-carbon bond attachment, -SH, NH 2 , -S(CH 2 ) n NH 2 , -S(CH 2 ) n OH, -NH(CH 2 ) n NH 2 and -NH(CH 2 ) n Selected from the above group excluding OH (n=1-100) or R 1 is as described immediately above, except for monopara-substituted phenylthio and monopara-substituted phenylamino. The following conditions are met.

[0094] In a further preferred embodiment of the present invention of any of the compounds of the present invention according to formula (III) and / or monomer precursors according to formula (III), the monomer compounds of formula (III) and / or monomer precursors of the present invention are selected from the group shown in Table 14 below.

[0095] Table 14. Structures of novel monomer precursor compounds of the present invention. TIFF0007672368000041.tif250163 TIFF0007672368000042.tif246170 TIFF0007672368000043.tif240170 TIFF0007672368000044.tif251158 TIFF0007672368000045.tif249154 TIFF0007672368000046.tif249157 TIFF0007672368000047.tif47163

[0096] As mentioned above, the compounds of the present invention can be further labeled according to well-known labeling techniques.For example, but not limited to, fluorescent dyes can be attached to the compounds for fluorescence correlation spectroscopy, for fluorescence energy transfer studies, or for measuring their concentration in living cells, to localize the intracellular distribution of cyclic nucleotide binding proteins in living cells using confocal or other microscopes. Of course, hydrates of the compounds are also within the scope of the present invention. Instead of or in addition to fluorescent dyes, the compounds of the invention can be labelled with (radio)nuclides. The skilled person is aware of the numerous techniques and suitable isotopes which can be used for this purpose. As noted above, the present invention also includes PEGylated forms of certain compounds, and PEGylation is generally known to greatly improve aqueous solubility, pharmacokinetic and biodistribution properties.

[0097] The present invention further includes prodrug forms of the described compounds, in which the negative charge of the equatorial modified phosphate moiety is masked with a bioactivatable protecting group. It is widely accepted that this structure enhances lipophilicity, and thus membrane permeability and bioavailability, resulting in a 10-1000-fold increase in potency compared to the parent compound. The bioactivatable protecting group can be introduced according to techniques well known in the art, and includes, but is not limited to, acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, acetoxyethyl, acetoxybutyl, and acetoxyisobutyl. Non-limiting examples of the corresponding residue R8 of the present invention are acetoxymethylthio, propionyloxymethylthio, and butyryloxymethylthio. More labile examples of protecting groups include alkyl or aryl groups as well as substituted alkyl or aryl groups. Non-limiting examples of chemically labile protecting groups at the R8 position are methyl, ethyl, 2-cyanoethyl, propyl, benzyl, phenyl, and polyethylene glycol. Although these compounds are inactive by themselves, they are extremely membrane permeable, leading to very high intracellular concentrations. Hydrolysis of the ester bond liberates the biologically active parent compound.

[0098] The compounds of the present invention may also be equipped with photolysable groups (also called "caged" or photoactivatable protecting groups), which can be introduced according to techniques well known in the art. For example, but not limited to, caged groups can be attached to the R8 thio functional group to provide compounds with significantly increased lipophilicity and bioavailability. Non-limiting examples of caged groups are o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylamino-coumarin-4-yl (DMACM caged), 7-diethylamino-coumarin-4-yl (DEACM caged), and 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM caged).

[0099] The compounds of the present invention can also be immobilized on insoluble supports, including but not limited to agarose, dextran, cellulose, starch and other carbohydrate-based polymers, synthetic polymers such as polyacrylamide, polyethyleneimine, polystyrene and similar materials, apatite, glass, silica, gold, graphene, fullerene, carborane, titania, zirconia or alumina, chip surfaces suitable for binding with various ligands, and the like. As described in the literature, the compounds of the invention can also be encapsulated within nanoparticles or liposomes for directed or undirected delivery and release of the compounds. 13 .

[0100] Furthermore, the compounds of the present invention are suitable for use as research tool compounds, preferably for diseases or disorders, preferably selected from the group consisting of retinal diseases or disorders or neurological or neurodegenerative diseases or disorders. The term "research tool" or "research tool compound" as used herein defines any experimental use in laboratory and preclinical research of a compound, and specifically excludes any use in humans and preventive and / or medical treatment. In particular, said term relates to any experimental use of a compound in laboratory and preclinical research that does not apply to humans, but is used to study a disease or disorder in laboratory and preclinical settings, preferably a disease or disorder selected from the group consisting of a retinal disease or disorder or a neurological or neurodegenerative disease or disorder. The compounds of the present invention are suitable for use in the treatment of a disease or disorder, preferably a disease or disorder selected from the group consisting of a retinal disease or disorder or a neurological or neurodegenerative disease or disorder.

[0101] In this specification, the treatment of a pathology, condition or disorder is to be understood as also embracing its prevention, even if not expressly mentioned, unless specifically specified to the contrary. The equatorially modified cGMP analogs of the present invention are preferably used to treat or prevent retinal diseases or conditions. The equatorially modified cGMP analogs of the present invention are preferably used to treat retinal diseases and conditions by suppressing the disease-related imbalanced cGMP system, including rare genetic diseases of the retina, such as retinitis pigmentosa, Stargardt's disease, fundus flaviparis, juvenile Best's disease, adult vitelliform foveomacular dystroph (adult vitelliform degeneration), familial drusen (North Carolina macular dystrophy), Bietti crystalline dystrophy, progressive cone dystrophy, Alport syndrome, benign familial spotted retinopathy, Leber's congenital amaurosis, congenital monochromatism, and hereditary macular dystrophies. Additionally, these equatorial modified cGMP analogs of the present invention can be used to treat several metabolic and neurodegenerative diseases, secondary pigmentary retinal degenerations occurring in various syndromes and other ocular diseases, including retinitis pigmentosa and deafness associated with Usher, Waardenburg, Alstrom, Alport, Refsum, and other systemic conditions (all with their own systemic manifestations), short stature, renal insufficiency, and polydactyly are some symptoms of Bardet-Biedl or Laurence-Moon syndromes when accompanied by pigmentary retinopathy, and mucopolysaccharidoses can be associated with retinitis pigmentosa (e.g., Hurler, Scheie's, and Sanfilippo's syndromes), as well as the mitochondrial disorder Kearns-Sayre syndrome. In addition to the above, these also include: Friedreich's ataxia, mucopolysaccharidoses, myotonic dystrophy, Batten syndrome, Bassen-Kornzweig syndrome, homocystinuria, oxalosis, ocular and retinal trauma, glaucoma with retinal pigment epithelium changes, end-stage chloroquine retinopathy, end-stage thioridazine retinopathy, end-stage syphilitic neuroretinitis, and cancer-associated retinopathies. These equatorial modified cGMP analogs of the present invention can also be used to treat other common diseases of the retina, such as diabetic retinopathy, age-related macular degeneration, macular hole / Pucker, eye tumors such as retinoblastoma, retinal detachment, and river blindness / onchocerciasis.

[0102] Furthermore, the equatorially modified cGMP analogs of the present invention can be used to treat completely different conditions associated with disease-related imbalanced cGMP systems, such as neurological or neurodegenerative disorders, stroke, anosmia, inflammatory and neuropathic pain, axonal regeneration and repair after spinal cord injury. The equatorially modified cGMP analogs of the present invention can also be used to treat cardiovascular disease, hypertension, acute shock and cancer. This also includes certain parasitic diseases such as malaria, sleeping sickness (African trypanosomiasis), and Chagas disease, where parasite survival is highly dependent on an active cGMP system. In another aspect, the present invention relates to a method for treating or preventing the above-mentioned pathologies, conditions or disorders by administering to a subject in need of such prevention or treatment a therapeutically or prophylactically effective amount of an equatorial modified cGMP analog of the present invention. The present invention is further illustrated by the following examples which describe preferred embodiments of the invention and are not intended to limit the invention in any way. EXAMPLES

[0103] Working Example 1. Compound Synthesis General Experimental Method All applied solvents and reagents were available from commercial suppliers. Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS were available from Biolog Life Science Institute (Bremen, Germany). Solvents used were designated as analytical or hplc grade. Dimethylsulfoxide was stored over activated molecular sieves for at least 2 weeks before use. Chromatographic runs were performed at ambient temperature. Reaction progress as well as purity of isolated products were determined by reverse hplc (RP-18, ODS-A-YMC, 120-S-11, 250 × 4 mm, 1.5 mL / min) with UV detection at 263 nm, a mid-wavelength suitable for the detection of most cyclic GMP products and impurities, or at λ 200 nm for specific starting materials or products. maxThe synthesis was typically carried out on a 20-200 μmol scale in 2 mL polypropylene reaction vials with screw caps (reactions requiring an inert gas atmosphere and / or degassing were carried out in round bottom flasks (typically 10 or 25 mL)). Dissolution of poorly soluble reagents was achieved by sonication or heating (70 °C) prior to the addition of the reagents. Suspensions were used when dissolution did not occur with these techniques, which are mainly applied to some cGMP analogues bearing a PET moiety. Purification of the products was achieved by preparative reversed phase hplc (RP-18, ODS-A-YMC, 12 nm-S-10, 250 × 16 mm, UV 254 nm). The composition of the eluent is given in the individual synthesis examples and can be used for analytical purposes as well, unless otherwise stated. Desalting of the products was achieved by repeated lyophilization 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. When removing the solvent using a speedvac concentrator, the solution was frozen at -70 °C for 15 min before evaporation. Products were isolated as sodium or triethylammonium salts, depending on the applied buffer. Yields represent the percentage of isolated product with the reported purity. They were measured at λ = 0.01, 0.01, 0.01 and 0.02, respectively, on a JASCO V-650 spectrophotometer (JASCO Germany GmbH, Gross-Umstadt, Germany). max The extinction coefficients were estimated from known values ​​in the literature for structurally related compounds. Mass spectra were obtained on an Esquire LC 6000 spectrometer (Bruker Daltronics, Bremen, Germany) in ESI-MS mode with 50% water / 50% methanol as matrix.

[0104] Experimental Procedure for the Preparation of 8-Thio-Substituted Equatorially Modified Guanosine-3',5'-Cyclic Monophosphate Analogues General Procedure A: In a typical experiment, the corresponding thiol reactant (8 eq) and NaOH (2M, 4 eq) were dissolved in H 2The resulting mixture was then added sequentially to a solution of the corresponding 8-Br-substituted equatorial modified cGMP analog (sodium salt, 65 mM, 1 eq) in O / i-PrOH (1:1, v / v). The reaction mixture was heated to 90° C. and stirred until the bromide starting material was completely consumed or no further reaction progress was observed. The solution was then allowed to warm to room temperature, neutralized with HCl (1 M), and the solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with MTBE (3×). * The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reversed phase HPLC and desalted to give the 8-thio-substituted equatorial modified cGMP analogue. * If the residue was not soluble in water, the resulting suspension was washed with MTBE and diluted (if necessary) with MeOH to dissolve any remaining precipitate. General Procedure A2: In a typical experiment, H 2 To a solution of the corresponding 8-Br-substituted equatorial modified cGMP analog (sodium salt, 65 mM, 1 eq) in O / i-PrOH (1:1, v / v) was added the corresponding thiol(ate) reactant (4.5 eq). The reaction mixture was stirred at room temperature until the bromide starting material was completely consumed or no further reaction progress was observed. The solution was then adjusted to pH 6 with NaOH (10%) and the solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL) and purified by HCl. 2 Cl 2 The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reversed phase HPLC and desalted to give the 8-thio-substituted equatorially modified cGMP analogue.

[0105] General Procedure B: In a typical experiment, a solution of 8-Br-substituted equatorial modified cGMP analogue (sodium salt, 87 mM, 1 eq) was added portionwise over 2 h to a suspension of the corresponding dithiol (50 mM, 10 eq in water / i-PrOH, 2:3, v / v) and NaOH (2 M, 5 eq). The reaction mixture was heated to 90° C. and stirred until the bromide starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was suspended in water (1 mL), neutralized with HCl (1 M) and filtered. The crude product solution was subjected to preparative reversed phase HPLC and desalted to give the thiol analogue. General Procedure C: In a typical experiment, to a solution of 8-Br-substituted cGMP analog (sodium salt, 200 mM, 1 eq) in borate buffer (100 mM, pH 12) was added NaOH (2 M, 16 eq) and the corresponding thiol reactant (8 eq) sequentially. The reaction mixture was heated to 90° C. and stirred until the bromide starting material was completely consumed or no further reaction progress was observed. The solution was then allowed to warm to room temperature and neutralized with HCl (1 M). The solvent was removed under reduced pressure using a rotary evaporator. The residue was dissolved in water (1 mL) and subjected to preparative reversed phase HPLC and desalted.

[0106] General Procedure D: In a typical experiment, N,N-diisopropylethylamine (2 eq) and the corresponding bromide (1 eq) were added sequentially to a solution of 8-SH-substituted equatorial modified 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 via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL), washed with ethyl acetate (3x), subjected to preparative reversed phase hplc and desalted. General Procedure E: For the formation of dimeric equatorially modified cGMP analogs, general procedure D was followed using N,N-diisopropylethylamine (2 eq), the corresponding bisbromide spacer (0.5 eq) and the 8-SH-substituted equatorially modified cGMP analog (sodium or triethylammonium salt, 100 mM, 1 eq) in DMSO.

[0107] Experimental procedures for the conversion of carboxylate-functionalized equatorially modified guanosine-3',5'-cyclic monophosphate analogues to the corresponding carboxylic acids or amides General Procedure F: In a typical experiment, NaOH (2M, 10eq) was added to a solution of the corresponding ester (80mM, 1eq) in water / MeOH (1:1, v / v). The reaction mixture was stirred until the ester starting material was completely consumed or no further reaction progress was observed. The solution was then neutralized with HCl (1M) and the solvent was removed under reduced pressure using a rotary evaporator. The residue was dissolved in water (1mL) and subjected to preparative reverse phase hplc and desalted to give the carboxylic acid analogue. General Procedure G: In a typical experiment, the corresponding ester (1 eq) was dissolved in excess methanolic ammonia (4.2 M, 200 eq). The reaction mixture was stirred until the starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), neutralized with HCl (1 M) and filtered through a syringe filter. The crude product was subjected to preparative reverse phase hplc and desalted to give the carboxylic acid amide analogue.

[0108] Experimental procedure for the formation of amide bonds with equatorially modified guanosine-3',5'-cyclic monophosphate analogues General Procedure H: In a typical experiment, HOBt (1.1 eq), N,N-diisopropylethylamine (2.2 eq), and EDC (1.1 eq) were added sequentially to a solution of the corresponding acid-substituted equatorially modified cGMP analog (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. The solvent was removed via high vacuum evaporation using a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with ethyl acetate (5x). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc, and desalted to give the coupled equatorial modified cGMP analog. *Less effective reactants were used in slight excess. Thus, for reactions with functional group inversion, amine-substituted equatorially modified cGMP analogs (100 mM in DMSO, 1 eq) and acid reactants (1.1 eq) were used.

[0109] General Procedure I: In a typical experiment, HOBt (1.1 eq), N,N-diisopropylethylamine (2.2 eq) and EDC (1.1 eq) were added sequentially to a solution of the corresponding acid-substituted equatorially modified cGMP analog (100 mM in DMSO, 1 eq) and the corresponding bisamino spacer (0.5 eq). Work-up was carried out as described in General Procedure H to give the dimeric equatorially modified cGMP analog. General Procedure J: In a typical experiment, N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq) were added sequentially to a solution of the corresponding carboxylic acid-substituted equatorially modified cGMP analog (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 μL) was added, stirring was continued for 10 min, and the solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL), the pH was adjusted to 6 with NaOH (2 M) or HCl (1 M) if necessary, and the solution was 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 to give the coupled equatorial modified cGMP analog. *Less efficient reactants were used in slight excess, thus for reactions with functional group inversion, amine-substituted equatorially modified cGMP analogs (100 mM in DMSO, 1 eq) and acid reactants (1.1 eq) were used.

[0110] General Procedure K: In a typical experiment, a solution of the corresponding carboxylic acid substituted equatorially modified cGMP analog (100 mM in DMSO, 1 eq) was added in portions over 40 min to a solution of bisamino spacer (400 mM in DMSO, 5 eq), N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq). Additional PyBOP (1 eq) was added and the reaction mixture was stirred until either the starting material was completely consumed or no further reaction progress was observed (usually <10 min). Workup was carried out as described in General Procedure J to give the monomeric equatorially modified cGMP analog coupling product. General Procedure L: General procedure J was followed using the corresponding acid-substituted equatorially modified cGMP analog (100 mM in DMSO, 1 eq), bisamino spacer (0.5 eq), N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq) to obtain the dimeric equatorially modified cGMP analog. General Procedure M: The trimeric equatorially modified cGMP analog was obtained according to the general procedure using the corresponding amine-substituted equatorially modified cGMP analog (33 mM in DMSO, 1 eq), linker triacid (0.3 eq), N,N-diisopropylethylamine (2 eq) and PyBOP (1.3 eq). General Procedure N: Prepare the corresponding amine-substituted equatorially modified cGMP analogue (diisopropylethylammonium salt, 50 mM in DMSO, 1 eq) according to general procedure J. * , linker tetraacid (tetradiisopropylethylammonium salt, 0.25eq) * , N,N-diisopropylethylamine (3 eq) and PyBOP (1.3 eq) were used to obtain the tetrameric equatorial modified cGMP analogue. *To convert the reactants to their diisopropylethylammonium salts, the reactants were subjected to N,N-diisopropylethylamine (3 eq per acidic functional group) in water (0.1-0.3 M) and evaporated to dryness using a speedvac concentrator under high vacuum.

[0111] Experimental Procedure for the Preparation of 8-Sulfonyl and 8-Sulfoxide Substituted Equatorial Modified Guanosine-3',5'-Cyclic Monophosphate Analogues General Procedure O: In a typical experiment, a solution of OXONE® (180 mM, 5 eq) in NaOAc buffer (2 M, pH 4.2) was added dropwise to a solution of the corresponding 8-thio-substituted guanosine analog (40 mM, 1 eq) in water / MeOH (1:1, v / v). The reaction mixture was stirred until the thiogenerator was completely consumed or no further reaction progress was observed. The solution was then neutralized with NaOH (2 M) and filtered through a syringe filter. The solvent was removed under reduced pressure using a rotary evaporator. The residue was dissolved in water (1 mL), subjected to preparative reversed-phase HPLC, and desalted to give the 8-sulfonyl-substituted guanosine analog. The thiophosphorylation was then carried out according to established thiophosphorylation protocols. 2b Conversion to the corresponding equatorially modified cGMP analogues was carried out according to General Procedure P: General procedure O was followed, but with shorter reaction times and fewer equivalents of oxidizing agent OXONE® (1.5 eq) to favor the formation of the 8-sulfoxide substituted equatorially modified cGMP analog.

[0112] Experimental procedure for the generation of 8-azidoalkylthio substituted equatorially modified guanosine-3',5'-cyclic monophosphate analogues General Procedure Q: In a typical experiment, NaN was added to a solution of 1,2-dibromoalkane in DMF (1.5 M, 15 eq) in an amber flask over a period of 5 h. 3(22.5 eq) was added in portions. The reaction mixture was stirred for 23 h and the 8-SH-substituted equatorially modified cGMP analog (triethylammonium salt, 1 eq) was added sequentially as well as N,N-diisopropylethylamine (1 eq). Stirring was continued until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed (usually about 1 h). The solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with MTBE (5x). The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reverse phase hplc and desalted to give the 8-azidoalkylthio substituted analog.

[0113] Experimental Procedure for the [3+2] Cycloaddition of Azides and Terminal Alkynes to Equatorially Modified Guanosine-3',5'-Cyclic Monophosphate Analogues General Procedure R: In a typical experiment, an alkyne-substituted equatorial modified cGMP analog (H 2 A solution of the corresponding azide (40 mM, 1 eq in CH2HO) 2 Cl 2 Bromotris(triphenylphosphine)copper(I) ([Cu(PPh 3 ) 3 [Br] (0.05 eq) was added and the reaction mixture was stirred until the alkyne starting material was completely consumed or no further reaction progress was observed. The mixture was diluted with water (up to 1.5 mL) and diluted with CH 2 Cl 2 The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reverse phase HPLC and desalted to give the triazole-containing product.

[0114] General Procedure S: In a typical experiment, [Cu(PPh 3 ) 3[Br] (0.05 eq) was added to a solution of the corresponding azide (13 mM, 1 eq) and the corresponding alkyne (13 mM, 1 eq) in water / N,N-diisopropylethylamine (7:1, v / v) in an amber flask. The reaction mixture was stirred at 65 °C until the starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation using a speedvac concentrator. The residue was dissolved in water (1 mL) and diluted with CH 2 Cl 2 The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reverse phase HPLC and desalted to give the triazole-containing product. General procedure T: [Cu(PPh 3 ) 3 General procedure S was followed using [Br] (0.05 eq), the corresponding azide-substituted equatorial modified cGMP analog (23 mM, 1 eq) and the corresponding bis-alkyne (12 mM, 2 eq). Conditions were selected to afford monomeric and dimeric triazole-containing products. General Procedure U: [Cu(PPh 3 ) 3 The dimeric triazole-containing product was obtained following general procedure S using [Br] (0.05 eq), the corresponding azide-substituted equatorial modified cGMP analog (33 mM, 1 eq) and the corresponding bisalkyne (16 mM, 0.5 eq).

[0115] Experimental Procedure for the Conversion of Azide-Substituted Equatorially Modified Guanosine-3',5'-Cyclic Monophosphate Analogues to the Corresponding Amines General Procedure V: In a typical experiment, a solution of azide-substituted equatorially modified cGMP analog (2.5 mM in water, 1 eq) was adjusted to pH 10 in an amber flask by adding triethylamine and cooled to 10° C. DL-dithiothreitol (5 eq) was added and the reaction mixture was stirred until the azide starting material was completely consumed or no further reaction progress was observed (usually <20 min). The mixture was evaporated to dryness under reduced pressure using a rotary evaporator. The residue was dissolved in water (1 mL) and subjected to preparative reverse phase HPLC and desalted to give the amine-substituted equatorially modified cGMP analog.

[0116] Experimental Procedure for Suzuki Cross-Coupling of Br-Substituted Equatorially Modified Guanosine-3',5'-Cyclic Monophosphate Analogues with Organoboronic Acids General Procedure W: In a typical experiment, EtOH / H 2 A solution of Br-substituted equatorially modified cGMP analogue (52 mM, 1 eq) and boronic acid (72 mM, 1.4 eq) in HO (1:1, v / v) was added to K 2 CO 3 Aqueous solution (2M, 3eq) and Pd(dppf)Cl 2 (0.05eq) was added sequentially. The reaction mixture was degassed by immediately applying three cycles of freeze-pump-thaw technique and stirred at 90°C under argon until the bromide starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation with a speedvac concentrator. The residue was suspended in water and CHCl 3 (3x) until the precipitate was dissolved (H 2 Methanol was added to the composition until the mixture was 1:1 (O / MeOH). 3Once the organic phase containing was visible, it was separated. The aqueous phase was then filtered through a Macherey-Nagel Chromafix C 18 (S) 270 mg cartridge (preconditioned with 10 mL MeOH, 50% MeOH and 30% MeOH, respectively) and rinsed with 30% MeOH (6 mL). The solvent was removed under reduced pressure using a rotary evaporator. The residue was dissolved in water (1 mL) and subjected to preparative reversed phase hplc and desalted to give the cross-coupled product. * All solvents used were degassed by sonication under reduced pressure prior to the experiments.

[0117] General Procedure X (Bisboronic Acid Reagent 4-B(OH) 2 PhS-(EO) 5 -(CH 2 ) 2 -4-SPhB(OH) 2 Preparation of: In a typical experiment, 4-mercaptophenylboronic acid (0.2 M, 1 eq) and Br-(EO) in DMF were 5 -(CH 2 ) 2 To a solution of 4-Br (0.5 eq) was added N,N-diisopropylethylamine (2 eq). 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 via high vacuum evaporation using a speedvac concentrator. The residue was dissolved in methanol (1 mL) and subjected to preparative reverse phase hplc (62% MeOH) to give 4-B(OH). 2 PhS-(EO) 5 -(CH 2 ) 2 -4-SPhB(OH) 2 was obtained (yield 34%).

[0118] 1,N 2 -Experimental Procedure for the Preparation of Functionalized Equatorially Modified Guanosine-3',5'-Cyclic Monophosphate Analogues General Procedure Y: In a typical experiment, DBU (7 eq) and the corresponding 2-bromo-aceto reactant (3.5 eq) were added sequentially to a solution of the corresponding equatorially modified cGMP analog in DMSO (50 mM, 1 eq). The reaction mixture was stirred under the exclusion of light until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation with a speedvac concentrator. The residue was dissolved in methanol (0.5 mL) and the pH was adjusted to 6-7 with HCl (1 M). If a precipitate thereby formed, methanol was added to redissolve it. Otherwise, water was slowly added (maximum H) until all components were just dissolved. 2 The solution was subjected to preparative reversed-phase HPLC, desalted and diluted to 1N 2 -etheno-functionalized equatorial modified cGMP analogues were obtained.

[0119] General procedure Y2: In a typical experiment, DBU (2 eq) and the corresponding alkyl bromoacetate reactant (1.1 eq) were added sequentially to a solution of the corresponding equatorially modified cGMP analog in DMSO (100 mM, 1 eq). The reaction mixture was stirred until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was purified by H 2 The solution was dissolved in HO (0.5 mL) and the pH was adjusted to 6-7 with HCl (1 M). The solution was subjected to preparative reversed-phase HPLC, desalted and purified to 1N 2 -acyl-functionalized equatorial modified cGMP analogues were obtained. General procedure Y3: In a typical experiment, N,N-diisopropylethylamine (2 eq) and PyBOP (1.1 eq) were added sequentially to a solution of the corresponding 1-carboxyalkyl-substituted equatorially modified cGMP analog (10 mM in DMSO, 1 eq). The reaction mixture was stirred until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed. Water (100 μL) was added, stirring was continued for 10 min, and the solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was dissolved in water (1 mL), the pH was adjusted to 5-6 with NaOH (2 M), and the solution was 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 to give 1,N 2 -acyl-functionalized equatorial modified cGMP analogues were obtained.

[0120] Experimental procedure for the preparation of 1-substituted equatorially modified guanosine-3',5'-cyclic monophosphate analogues General Procedure Z: In a typical experiment, DBU (4 eq) and the corresponding bromide (or iodide) reactant (4 eq) were added sequentially to a solution of the corresponding equatorially modified cGMP analog in DMSO (50-300 mM, 1 eq). The reaction mixture was stirred until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was purified by H 2 The residue was dissolved in 20O (0.5 mL) and, if the resulting solution was not neutral, the pH was adjusted to 7 with HCl (1 M). The solution was washed with ethyl acetate (4×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator and the residue was redissolved in water and subjected to preparative reversed phase HPLC and desalted to give the 1-substituted equatorial modified cGMP analog.

[0121] General procedure Z2: In a typical experiment, DBU (2 eq) and the corresponding dibromide reactant (0.5 eq) were added sequentially to a solution of the corresponding equatorially modified cGMP analog in DMSO (15 mM, 1 eq). The reaction mixture was stirred at 90° C. until the equatorially modified cGMP analog starting material was completely consumed or no further reaction progress was observed. The solvent was removed via high vacuum evaporation in a speedvac concentrator. The residue was purified by H 2 The mixture was dissolved in HO (0.5 mL), the pH was adjusted to 5-7 with HCl (1 M), and the solution was washed with ethyl acetate (4x). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, and the residue was redissolved in water and subjected to preparative reversed-phase HPLC and desalted to obtain the 1-substituted dimeric equatorial modified cGMP analog.

[0122] The present invention is further illustrated by the figures and examples (Table 15) which describe preferred embodiments of the present invention, but are not intended to limit the present invention in any way. The structural examples of the novel compounds are depicted in the free acid form. After HPLC workup, the compounds are obtained as salts in the application buffer, but can be converted to other salt forms or to the free acid by cation exchange following standard procedures for nucleotides.

[0123] Table 15. Examples of novel equatorially modified polymer-linked multimeric cGMP compounds of the present invention TIFF0007672368000048.tif224170 TIFF0007672368000049.tif198170 TIFF0007672368000050.tif223170 TIFF0007672368000051.tif229170 TIFF0007672368000052.tif225170 TIFF0007672368000053.tif236170 TIFF0007672368000054.tif233170 TIFF0007672368000055.tif167170

[0124] The monomer precursors of the present invention are illustrated by the following figures and examples in Table 16, which describe preferred embodiments of the present invention. The following further describes the structures and / or monomeric compounds of the present invention, but are not intended to limit the present invention in any way. The structural examples of the novel compounds are depicted in the form of the free acid. After HPLC workup, the compounds are obtained as salts in the applied buffer, but can be converted to other salt forms or to the free acid by cation exchange according to standard procedures for nucleotides.

[0125] Table 16. Examples of monomer precursors and / or monomer compounds of the present invention TIFF0007672368000056.tif203146 TIFF0007672368000057.tif177142 TIFF0007672368000058.tif187143 TIFF0007672368000059.tif171139 TIFF0007672368000060.tif177142 TIFF0007672368000061.tif192146 TIFF0007672368000062.tif187143 TIFF0007672368000063.tif192142 TIFF0007672368000064.tif234145 TIFF0007672368000065.tif208142 TIFF0007672368000066.tif208145 TIFF0007672368000067.tif223150 TIFF0007672368000068.tif218146 TIFF0007672368000069.tif218144

[0126] 2. Determination of Lipophilicity A commonly accepted indicator of the predicted ability of a given analogue to cross cellular lipid bilayers by passive diffusion is the octanol / water partition coefficient, Log P, but determination of this data is rather difficult for polar structures such as cyclic nucleotides, and therefore lipophilicity information is often only obtained by fragment analysis and corresponding calculations. An established HPLC method based on retention data on RP-18 reversed-phase silica during gradient elution was used to determine lipophilicity. Instead of log P, this method uses a descriptor log k' which also ranks analytes according to their lipophilicity on a logarithmic scale. g Since charged molecules such as cyclic nucleotides have little retention on reversed phase, ion-pair chromatography with the lipophilic triethylammonium cation is used. Unmodified cGMP itself (log k' g The β-aspartate phosphate moiety (0.77) is considered not membrane permeable by passive diffusion, meaning that only analogs with significant hydrophobic modifications and substitutions, respectively, countering the negative charge at the phosphate moiety, can be used for extracellular application. Previous analysis of cyclic nucleotide analogs widely used in our laboratory has shown that they diffuse appreciably into cells with log k's of at least 1.2. g This corresponds well with the work of Werner et al. (2011). 14 . The corresponding analytical results of 12 novel equatorially modified cGMP analogs with Rp phosphorothioates are shown below in Table 17. For comparison and control, three established structures within this series (compounds A-C) were reanalyzed.

[0127] Table 17. Log k' of exemplary compounds of the present invention g values ​​(compound numbers refer to structures listed in Tables 13 and 14). TIFF0007672368000070.tif130150 * The values ​​for the dimeric analogues are not directly comparable to those for the monomeric analogues.

[0128] All monomeric analogs are log k' g had a value >1.2 and thus were sufficiently lipophilic to cross the cell membrane. Most of the monomeric analogs have similar or even higher lipophilicity than Rp-8-Br-PET-cGMPS. g All monomeric analogs with a value >2.831 are expected to have improved membrane permeation in biological systems and therefore improved properties compared to Rp-8-Br-PET-cGMPS. Table 17 also shows lipophilicity data for two cGMPS dimers (compounds 1 and 2). However, because these structures have two negative charges at physiological pH, the corresponding values ​​are log k' obtained for the monomeric analogs that have only a single negative charge. g Not directly comparable to the value.

[0129] 3. Primary Rod-like Cells: Assessment of Cell Death Using the Ethidium Homodimer Assay background Primary photoreceptors derived from retinal stem cells after in vitro differentiation have been demonstrated to be a suitable in vitro system for studying mechanisms of cell death associated with retinal degeneration and cGMP imbalance, as well as for screening compounds with neuroprotective activity. 5~6 Therefore, data obtained by screening drugs in this in vitro system can be used for further research studies on retinal explants and in vivo in eyes of animal models of disease.

[0130] Experimental Part Primary rod-like cells were obtained by isolating stem cells from the ciliary epithelium of mouse eyes. 15Cells were cultured in DMEM / F12 containing FGF (20ng / ml), heparin (2μg / ml), N2 (1x), glucose (0.6%), HEPES (5μM) and 1% penicillin / streptomycin until they formed neurospheres. Single neurospheres were selected and plated on ECM-coated glass slides in the same medium as before, but with reduced FGF concentration (10ng / ml), to induce attachment. After 4 days, the medium was changed to DMEM / F12 containing N2, glucose, HEPES and penicillin / streptomycin suspended in 1% FBS to allow differentiation into rod-like photoreceptors. Compound treatments began 10 days after neurosphere plating. This time point was chosen because cells from rd1 mutant eyes showed a peak in cell death and activated cell death pathways similar to those in the in vivo retina. 5b Compounds were dissolved in water and then diluted in differentiation medium at concentrations ranging from 1 nM to 100 μM. After 24 h of treatment, cells were washed with PBS and fixed in 4% PFA. Slides were then immersed in 2 μM ethidium homodimer for 2 min, and nuclei were stained with DAPI. Ethidium homodimer stains the nuclei of dead cells. To assess cell death, microscopic photographs were taken from three different slides for each compound concentration, and the total number of cells, as well as the number of ethidium homodimer-positive dead cells, were counted in each photograph. To statistically evaluate the significant differences between untreated and treated cells, an unpaired Student's t-test was used, with a p-value ≤ 0.05 considered significant ( * ≦0.05, ** ≦0.01, *** ≦0.001).

[0131] result Figure 2 shows the protective effect of exemplary compounds of the invention. All test compounds of the invention resulted in significantly improved survival of primary rod-like cells at both test compound concentrations of 0.1 μM (Figure 2a) and 1 μM (Figure 2b) compared to untreated cells (black bars) and compared to the reference compounds Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS (dashed bars). The strongest precedents of the exemplary compounds of the invention showed a 4.7- to 9-fold better reduction in cell death compared to known compounds.

[0132] 4. Retinal Explants: Determining Photoreceptor Cell Death background In addition to using cell lines that degenerate retinal photoreceptors or photoreceptor-like cells to evaluate the properties of various cyclic nucleotide analogs, a serum-free organotypic explant culture system can be used in which retinas from young animals are explanted in culture and maintained for up to three weeks. 4、9 This explant system allows the assessment of photoreceptor survival in an in vivo-like histological context. It keeps most of the cytoarchitecture intact, but without the risk of degeneration or dilution, for example by body fluids, of any treatment compound, which would otherwise be dangerous in vivo. The rd1 mouse is a very well-studied model for RD and its degenerative characteristics, including the early onset and rapid progression of photoreceptor cell death. rd1 degeneration can be easily achieved in the time frame of explant culture. This offers the benefit of easy pharmacological intervention to look for neuroprotective potential, which has been exploited repeatedly. 4、8 These studies have made it possible, among other things, to outline several disease steps.

[0133] Experimental Part The effect of various compounds of formula (I), (II) and their monomeric precursor, formula (III), on the degeneration of retinal photoreceptors from a mouse model suffering from inherited retinal degeneration was investigated using the retinal explant system described above. In this experiment, retinas are dissected from young animals, usually on postnatal day 5 (PN5), and cultured for several days in serum-free medium (see also FIG. 3 for the rd1 culture paradigm), with medium changes usually every other day. To observe the effect of the various analogs on the degeneration, the retinas are fixed (preserved) at the end of the experiment, after which they are prepared for histological and other analyses, in particular the so-called TUNEL staining, which allows the quantification of photoreceptor cell death.

[0134] result Figure 4 shows the results of a series of tests with analogs of the invention, with the effect on photoreceptor cell death expressed as a ratio of treated vs. untreated (see figure legend). The leftmost bar represents untreated rd1 explants, while the other bars show selected analogs of the invention used at concentrations of either 50 μM, 10 μM, or 1 μM. The effects of these analogs are compared to a concentration-matched previously available analog, Rp-8-Br-PET-cGMPS. Note that at all concentrations of Rp-8-Br-cGMPS and Rp-8-Br-PET-cGMPS, one or more of the analogs of the invention works better.

[0135] List of acronyms TIFF0007672368000071.tif208156 TIFF0007672368000072.tif73157

[0136] literature 1.Schlossmann, J.; Schinner, E., cGMP becomes a drug target.Naunyn Schmiedebergs Arch Pharmacol 2012, 385 (3), 243-52. 2.(a) Kawada, T.; Toyosato, A.; Islam, MO; Yoshida, Y.; Imai, S., cGMP-kinase mediates cGMP- and cAMP-induced Ca2+ desensitization of skinned rat artery.Eur J Pharmacol 1997, 323 (1), 75-82; (b) Genieser, H.-G.; Walter, U.; Butt, E.Derivatives of cyclic guanosine-3',5'-monophosphorothioate.USPatent 5,625,056 Apr.29, 1997. 3.Butt, E.; Pohler, D.; Genieser, H.G.; Huggins, J.P.; Bucher, B., Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS.Br J Pharmacol 1995, 116 (8), 3110-6. 4.Paquet-Durand, F.; Hauck, S.M.; van Veen, T.; Ueffing, M.; Ekstrom, P., PKG activity causes photoreceptor cell death in two retinitis pigmentosa models.J Neurochem 2009, 108 (3), 796-810. 5.(a) Mussolino, C.; Sanges, D.; Marrocco, E.; Bonetti, C.; Di Vicino, U.; Marigo, V.; Auricchio, A.; Meroni, G.; Surace, E.M., Zinc-finger-based transcriptional repression of rhodopsin in a model of dominant retinitis pigmentosa.EMBO Mol Med 2011, 3 (3), 118-128; (b) Sanges, D.; Comitato, A.; Tammaro, R.; Marigo, V., Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors.Proc Natl Acad Sci USA 2006, 103 (46), 17366-17371. 6.Comitato, A.; Sanges, D.; Rossi, A.; Humphries, M.M.; Marigo, V., Activation of Bax in Three Models of retinitis pigmentosa.Invest Ophthalmol Vis Sci 2014, 55 (6), 3555-3562. 7.Arango-Gonzalez, B.; Trifunovic, D.; Sahaboglu, A.; Kranz, K.; Michalakis, S.; Farinelli, P.; Koch, S.; Koch, F.; Cottet, S.; Janssen-Bienhold, U.; Dedek, K.; Biel, M.; Zrenner, E.; Euler, T.; Ekstrom, P.; Ueffing, M.; Paquet-Durand, F., Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration.PloS One 2014, 9 (11), e112142-e112142. 8.Paquet-Durand, F.; Beck, S.; Michalakis, S.; Goldmann, T.; Huber, G.; Muhlfriedel, R.; Trifunovic, D.; Fischer, M.D.; Fahl, E.; Duetsch, G.; Becirovic, E.; Wolfrum, U.; van Veen, T.; Biel, M.; Tanimoto, N.; Seeliger, M.W., A key role for cyclic nucleotide gated (CNG) channels in cGMP-related retinitis pigmentosa.Hum Mol Genet 2011, 20 (5), 941-7. 9.Caffe, A.R.; Ahuja, P.; Holmqvist, B.; Azadi, S.; Forsell, J.; Holmqvist, I.; Soderpalm, A.K.; van Veen, T., Mouse retina explants after long-term culture in serum free medium.J Chem Neuroanat 2001, 22 (4), 263-73. 10.Kramer, R.H.; Karpen, J.W., Spanning Binding Sites on Allosteric Proteins with Polymer-linked Ligand Dimers.Nature 1998, 395, 710 - 713. 11.Kramer, R.H.; Karpen, J.W.Multimeric Tethered Ligands and Their Use in Receptor-Ligand Interaction.WO 99 / 25384 1999. 12.Strassmaier, T.; Karpen, J., Novel N7- and N1-substituted cGMP Derivatives Are Potent Activators of Cyclic Nucleotide-Gated Channels.J.Med.Chem.2007, 50, 4186-4194. 13.(a) Bala, I.; Hariharan, S.; Kumar, M.N., PLGA nanoparticles in drug delivery: the state of the art.Crit Rev Ther Drug Carrier Syst 2004, 21 (5), 387-422; (b) Basu, S.C.; Basu, M., Liposome Methods and Protocols.Humana Press: 2002; (c) Gregoriadis, G., Liposome Technology.Informa Healthcare: 2006; (d) Paquet-Durand, F.; Gaillard, P.J.; Maringo, V.; Ekstrom, P.; Genieser, H.-G.; Rentsch, A.Targeted liposomal delivery of cGMP analogues.PCT / EP2016 / 055659. 14.Werner, K.; Schwede, F.; Genieser, H.G.; Geiger, J.; Butt, E., Quantification of cAMP and cGMP analogs in intact cells: pitfalls in enzyme immunoassays for cyclic nucleotides.Naunyn Schmiedebergs Arch Pharmacol 2011, 384 (2), 169-76. 15.Giordano, F.; De Marzo, A.; Vetrini, F.; Marigo, V., Fibroblast growth factor and epidermal growth factor differently affect differentiation of murine retinal stem cells in vitro.Mol Vis 2007, 13, 1842-50.

Claims

1. The following formula (III) 【Chemistry 1】 (III) (In formula (III), X, Y and Z are N; R 1 is H, halogen, azido, SH, S-alkyl or S-acyl; R 2 is non-existent; R 3 is OH; R 4 Y and R 5 And Y and R 5 together with the carbons bridging the two groups form an imidazole ring substituted with a monosubstituted aryl or aralkyl; R 6 is OH; R 7 is O; and R 8 is SH, S-alkyl, S-aryl, S-aralkyl, S-PAP, S-BAP, Wherein PAP is a photoactivatable protecting group, PAP = o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylamino-coumarin-4-yl (DMACM caged), 7-diethylamino-coumarin-4-yl (DEACM caged) or 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM caged); and BAP is a bioactivatable protecting group, where BAP=methyl, acetoxymethyl, pivaloyloxymethyl, methoxymethyl, propionyloxymethyl, butyryloxymethyl, cyanoethyl, phenyl, benzyl, 4-acetoxybenzyl, 4-pivaloyloxybenzyl, 4-isobutyryloxybenzyl, 4-octanoyloxybenzyl or 4-benzoyloxybenzyl. A monomeric compound having the formula: The monomeric compound may be in the form of a salt and / or a hydrate, At the same time, suitable salts of a particular phosphate moiety are lithium, sodium, potassium, calcium, magnesium, zinc, ammonium, trialkylammonium, dialkylammonium or alkylammonium; however, 8-Bromo-(4-methyl-β-phenyl-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphorothioate, Rp-isomer; and β-(4-azidophenyl)-1,N 2 -Etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer; The monomer compounds described above, excluding

2. R 1 H, Cl, Br, I, F, N 3 or SH or R 1 is shown below as residue entry 1, Residue entry 1: 【Chemistry 3】 (In the formula, m=0-3； Q=S; X 1 is H or i-Pr); and / or R 4 But Y and R 5 And Y and R 5 together with the bridging carbons to form an imidazole ring, shown below as residue entry 1, Residue entry 1: 【Chemistry 11】 (In the formula, X 1 is H; X 3 are OH, NH, CH 3 , Cl, Br, F, CN, N 3 , C.F. 3 , OCF 3 , NO 2 , C(O)OH, C(O)OCH 3 , O.C.H. 3 , S.C.H. 3 , N(CH 3 ) 2 , S(O) 2 CH 3 or C(O)NH 2 and X 4 and X 5 is H); and / or R8 is SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethylthio, phenylthio, benzylthio, 4-acetoxybenzylthio, 4-pivaloyloxybenzylthio, 4-isobutyryloxybenzylthio, 4-octanoyloxybenzylthio or 4-benzoyloxybenzylthio; The monomeric compound according to claim 1.

3. R 1 H, Cl, Br, I, F, N 3 or SH or R 1 is shown below as residue entry 1, Residue entry 1: 【Chemistry 13】 (In the formula, m=0-3； Q=S; X 1 is H or i-Pr); and / or R 4 But Y and R 5 And Y and R 5 together with the bridging carbons to form an imidazole ring, shown below as residue entry 1, Residue entry 1: 【Chemistry 18】 (In the formula, X 1 is H; X 3 are OH, NH, CH 3 , Cl, Br, F, N 3 , C.F. 3 , OCF 3 , NO 2 , O.C.H. 3 , S.C.H. 3 or N(CH 3 ) 2 and X 4 and X 5 is H); and / or R 8 is SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, 4-pivaloyloxybenzylthio or 4-isobutyryloxybenzylthio; The monomer compound according to any one of claims 1 to 2.

4. R 1 is H, Cl or Br, or R 1 is shown below as residue entry 1, Residue entry 1: 【Chemistry 20】 (In the formula, m=0-3； Q=S; X 1 is H or i-Pr); and / or R 4 But Y and R 5 And Y and R 5 together with the bridging carbons to form an imidazole ring, shown below as residue entry 1, Residue entry 1: 【Chemistry 25】 (In the formula, X 1 is H; X 3 are OH, NH, CH 3 , Cl, Br, OCH 3 , S.C.H. 3 or N(CH 3 ) 2 and X 4 and X 5 is H); and / or R 8 is SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio or butyryloxymethylthio; The monomer compound according to any one of claims 1 to 3.

5. R 8 is SH. The monomer compound according to any one of claims 1 to 4.

6. A composition for use in the treatment of a disease or disorder comprising a monomeric compound according to any one of claims 1 to 5.

7. The monomer compound according to any one of claims 1 to 5 is contained. a) Retinitis pigmentosa or another genetic disorder of the retina; b) Secondary pigmentary retinal degeneration as a result of a metabolic or neurodegenerative disease, syndrome or eye disease; c) Diseases of the retina including diabetic retinopathy, age-related macular degeneration, macular hole / pucker, eye tumors, retinoblastoma, retinal detachment and river blindness; d) neurological or neurodegenerative disorders, stroke, anosmia, inflammatory and neuropathic pain, axonal regeneration and repair after spinal cord injury; e) Parasitic diseases, f) Cardiovascular disease, hypertension, cancer, or acute shock The composition for use in treating at least one of the following:

Images