Patent Application
20210317156
The present invention relates to novel polymer linked multimers of guanosine-3′, 5′-cyclic nucleotide monophosphates, including tethered di-, tri- and tetramers and their application in the fields of medicine and pharmacy. The invention also relates to specific monomers as precursors. The invention further relates to the use of such compounds as reagents for signal transduction research and as modulators of cyclic nucleotide-regulated binding proteins and isoenzymes thereof, and as ligands for affinity chromatography, for antibody production or for diagnostic applications e.g. on chip surfaces.
Guanosine-3′,5′-cyclic monophosphate (cGMP) is a purine nucleobase-containing cyclic nucleotide and was discovered as endogenous molecule in 1963. It is well known to act as a second messenger, wherein its intracellular level is altered as a response to (primary) signaling molecules such as toxins, hormones or nitric oxide, which in turn induces diverse cellular processes, such as gene control, chemotaxis, proliferation, differentiation, and apoptosis. Several diseases like retinal degeneration, cardiovascular diseases, asthma or diabetes are associated with unusually high or low levels of cGMP.1 The cGMP signaling cascade therefore has been recognized as a potential pharmacological target and is investigated by numerous academic groups and pharmaceutical companies. Research in this field demands for compounds that effectively modulate different targets of the said cascade. For this purpose, a number of cGMP analogues featuring cell permeability (in contrast to cGMP), enhanced activity and increased resistance to degradation by phosphodiesterases (PDE) have been established.
However, for the use as a drug and/or research tool cGMP analogues need to comply with a complex profile of characteristics that is unique for each biological system, to achieve a maximum effect. While applied compounds are usually selected for their ability to interfere with the main target of a studied mechanism of a disease, condition or disorder, there are always several required characteristics of a compound, that cannot be predicted and demand for testing a large set of analogues. Accordingly, for the increasing number of applications there is growing need for constantly expanding the group of available cGMP analogues with derivatives that feature another combination of characteristics as well as further improved activation potential, target specificity or multi target effects. Also tailor-made modifications such as reporting groups are desired variations for instance for research or diagnostic purposes. One of the targets addressed by cGMP and its analogues is the cGMP dependent protein kinase (PKG) from which three isoforms (Iα, Iß and II) are known. Knowledge on the identity and presence of PKG substrates in different cells, tissues and organisms is restricted. Hence the physiological as well as pathological importance of the cGMP-PKG system is not well understood, which is likely to have reduced the general understanding of cGMP-related phenomena, as well as the development of therapies in diseases and conditions where such substrates are involved. Efficient and reliable PKG activators will make it possible to address this question much more sharply than what can be currently achieved.
Among the PKG isoforms compounds activating PKG Iα have been explored most widely, while far less derivatives targeting Iß and even fewer interfering with PKG II are available. In fact, only a small number of compounds were reported to feature PKG II activation.2 3 For biochemical assays the most potent and predominantly applied one is 8-pCPT-cGMP. A drawback of this compound, however, is associated with the substituent in the 8-position. The 8-pCPT-moiety has been reported to induce additional off target effects besides PKG activation.4 Furthermore it is not suitable for assays involving UV light, as it is decomposed under these conditions. For some applications it is therefore desirable to have strong PKG II agonists that don't feature an 8-pCPT-group.
A desired selective biological effect is always also an issue of the activation potential. The less a compound activates a studied target, the larger amount of substance needs to be applied and thus the higher is the probability of off target effects such as extra cellular bindings.4 In conclusion it is favorable to use agonists with improved activation potential, reasoning a constant need for such compounds.
A class of PKG agonists, which could offer superior activity, are polymer linked multimers (PLM) of cGMP analogues, including di-, tri- and tetramers. This concept comprises the idea of achieving a strong enhancement of activity through addressing multiple binding sites with a single molecule. Prior to the present invention, however, the concept had only been applied once on a dimeric compounds (tri- and tetrameric analogues have not been reported). Therein, just a single type of a polymer linked dimeric cGMP (PLD), or more precisely one homologous series, differing only in the spacer length, has been disclosed. The preparation of said homologues was described as a coupling via the 8-position by reacting 8-thio cGMP with bifunctional PEG vinyl sulfones (VS-PEGn-VS) affording structures of general formula cGMP-8-S—(CH2)2—SO2-PEGn-SO2—(CH2)2—S-8-cGMP (also see
Applicability of the PLM concept has been proposed for further targets,8 but no concrete methods to generate PLMs with superior activating potential for PKG isoforms Iß and II were disclosed and no further coupling strategies or synthetic protocols were reported. Particularly the synthetic access, however, comprises a drawback of the reported PLDs. Applying the published protocol, the desired compound is obtained in poor yields only as a byproduct. This outcome results from a favored addition of vinyl sulfones to the 7-position of cGMP.7 PLDs featuring a different coupling moiety, that supports a more reliable access as well as maintained or preferably increased PKG activation, are therefore needed.
In addition, compounds featuring a similarly increased PKG Iß and/or PKG II activation potential as described for Iα targeting PLDs above, would be valuable tools for numerous research groups. The effects on these isoforms, however, have never been studied in connection with polymer linked di-, tri- or tetramers. Accordingly it was desirable to provide polymer linked multimeric activators of PKG Iß and II, but unknown, whether the concept could be transferred to these isoforms and if, what modifications would be necessary.
The effect of structural manipulations at the nucleobase in context with polymer linked multimers also has not been studied before. This in turn is especially important, though, as established compounds used as biochemical tools often require customized derivatization for specific applications. Among others introduction of a reporter moiety that facilitates a certain assay read out or detection method can be desirable. Coupling of the respective compound to a fluorescent dye, for instance, is a common strategy in this context. This dye in turn allows localizing the intracellular distribution of the compound and its binding proteins in living cells by means of microscopic or spectroscopic techniques. A frequently observed drawback of strategies that involve structural manipulation of an activator, however, is the change of nature of the parent compound. Even minor modifications can result in a significant shift of target affinity and specificity or even loss of activation potential.
In addition, compounds, which simultaneously activate multiple targets such as two or all three PKG isoforms, could be beneficial for some applications and unsolved problems in signal transduction research.
Supported by the results of the only homologoues PLD series reported before (as stated above), however, the enhanced target activation of these compounds appeared to fundamentally depend on an optimum spacer length (between the cGMP units), which is unique for each addressed protein. Therefore, addressing two or more targets, with the same PLD, seemed, if feasible at all, only possible with an intermediate spacer length at which the activation potential for both targets would be significantly decreased.
Accordingly, there is a growing need for activators of the cGMP signaling cascade, tailor-made for individual biochemical applications, featuring superior single or multiple target activation, with or without additional functionalization that for instance facilitates specific assay read outs. To combine all these features within a single monomeric structure can be very difficult to accomplish. On such relatively small compounds each of the multiple modifications necessary can have a significant influence on the original target binding properties.
The situation, however, changes when it comes to multimers, wherein each desired modification can be introduced on a different cGMP unit. Thus, the respective impact on the biochemical activation profile follows different rules. PLMs offer access to a highly potent, so far rarely studied class of compounds. The challenges of transferring their potential to further targets of the cGMP signal transduction cascade (in particular PKG Iß and II) and addressing multiple targets simultaneously, while optionally featuring a reporting group, though, have not been mastered yet.
Therefore the achieved objective of the present invention has been to provide new polymer linked multimeric analogues of guanosine-3′,5′-cyclic monophosphate (PLMs) including di-, tri- and tetrameric derivatives, which are compared to monomeric analogues used so far, improved in terms of PKG Iß and/or PKG II activation potential. Preferably the new PLMs additionally interfere with PKG Iα, wherein it is even more preferred that the activation potential for this isoform exceeds the previously reported one for a polymer linked dimer.5 Another objective of the invention has been, to provide PKG activators, which can be functionalized (e.g. with a reporting group), while essentially maintaining their target affinity. A further objective of the invention has been to provide the new PLMs as pharmaceutically acceptable analogues for treating or diagnosing a disease, condition or disorder associated with dysregulation of a cGMP-effected cellular target, wherein additional targets can be, including, but not limited to, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, a phosphodiesterase (PDE) and a cGMP-gated channel (CNGC). In another aspect, the objective of the invention has been to provide the new PLMs for application as research tool to identify and validate the cGMP-system in cell cultures or tissues or as a diagnostic tool. Embodiments of the invention are directed to new polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG), and related monomeric precursors thereof.
Another achieved objective of the present invention has been to provide related monomeric compounds, which may serve as monomeric precursors of the multimers of the present invention and/or may also show modulating activity. Thus, another objective of the invention has been to provide related monomeric compounds as pharmaceutically acceptable analogues for treating or diagnosing a disease, condition or disorder associated with dysregulation of a cGMP-effected cellular target, wherein additional tar-gets can be, including, but not limited to, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, a phosphodiesterase (PDE) and a cGMP-gated channel (CNGC). In another aspect, the objective of the invention has been to provide the new related monomeric compounds for application as research tool to identify and validate the cGMP-system in cell cultures or tissues or as a diagnostic tool. Embodiments of the invention are directed to new related monomeric compound analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG).
Formula I General constitution of compounds of the invention (branched and linear analogues).
Formula Ib More detailed illustration of Formula I.
Formula II General constitution of compounds of the invention (linear analogues).
Formula IIb More detailed illustration of Formula II.
Formula III General constitution of G units as discrete compounds of the invention or units of compounds according to Formula I or II.
Formula IV and V General constitution of G units according to Formula III, featuring exemplary imidazolinone substitution.
Legend: Guanosine-3′, 5′-cyclic monophosphate-[8-thioethylsulfonyl-(ethyloxy)n-ethylsulfonylethylthio-8]-guanosine-3′, 5′-cyclic monophosphate (polydispers compound arising from synthesis with polydispers bis vinylsulfonyl-PEGn with Mw of 800 g/mol, 1.2 kg/mol, 3.4 kg/mol or 20 kg/mol; or monodispers compound with n=6).
Legend: PKG isozymes Iα (0.2 nM), Iβ (0.15 nM) and II (0.5 nM) were incubated with different concentrations (10 pM to 6 μM) of compounds of the invention and cGMP as reference compound at room temperature for 60 min. The activation values of the compounds are expressed as relative PKG activation compared to cGMP with cGMP set as 1 for each kinase isozyme. The Ka-values of cGMP for half-maximal kinase activation were 28 nM for Iα, 425 nM for Iβ and 208 nM for II. Compound numbers refer to analogues displayed in Table 13.
Legend: RT-PCR on cDNA from mRNA extracted from 661W cell. The 661W cell line expresses the PKG isoforms Iα and II. Heart and muscle tissues were used as positive controls.
Legend: 661W cells were exposed to compounds for 16 hours at different concentrations (1 nM to 10 μM) and percentage of dying cells was evaluated by Ethidium Homodimer assay. Untreated cells are shown as control sample (black bar). Reference compound 8-Br-PET-cGMP is shown as dashed bars. Data are presented as means±SD from at least three biological replicates. Results not including standard deviation refer to single measurements. Asterisks indicate the P value of the unpaired Student's t-test (*P≤0.05, **P≤0.01, ***P≤0.001), statistically assessing significant differences between untreated and treated cells, wherein a p value ≤0.05 was considered significant. Compound numbers refer to analogues displayed in Table 13.
The new polymer linked multimeric cGMP analogues of the invention are compounds having the formula (I) or (II)
wherein:
G units G1 and G2 independently are compounds of formula (III) and G units G3 and G4 independently from G1 and G2 and independently from each other are compounds of formula (III) or absent, wherein in case of formula (II) G4 is always absent if G3 is absent,
and wherein in formula (III)
X, Y and Z are N
R1, R4, R5, R7 and R8 independently can be equal or individual for each G unit (G1, G2, G3 and G4), while
and wherein
linking residues LR1, LR2, LR3 and LR4 independently can replace or covalently bind to any of the particular residues R1, R4 and/or R5 of the G units (G1-4) they connect,
wherein in case they bind to any of the residues R1, R4 and/or R5, an endstanding group of the particular residue (R1, R4 and/or R5), as defined above, is transformed or replaced in the process of establishing the connection and is then further defined as part of the particular linking residue (LR1-4) within the assembled compound,
wherein in case of formula (II) if G4 is absent, LR4 is absent, too, and
wherein in case of formula (II) if G3 and G4 are absent, LR3 and LR4 are absent, too,
and wherein
G1, G2, G3 and G4 can further be salts and/or hydrates
and wherein
G1, G2, G3 and G4 can optionally be isotopically or radioactively labeled, be PEGylated, immobilized or be labeled with a dye or another reporting group,
with the proviso that the compound of formula (I) and/or (II), is not selected from structures depicted in
In a particular embodiment, the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein at least two G units are unequally substituted.
In another particular embodiment, the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein in case of formula (I) G3 and G4 are absent, or in case of formula (II) G3, G4, LR3 and LR4 are absent; and wherein R4 is not H and/or R5 is not NH2.
In a particular variant embodiment, the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein at least one G unit is linked via a position other than R1.
In a further particular embodiment, the precedingly defined compound of formula (I) and/or (II) may be a compound, wherein in case of formula (I) G3 and G4 are absent, or in case of formula (II) G3, G4, LR3 and LR4 are absent.
In still another particular embodiment, any of the precedingly defined compounds of formula (I) and/or (II) may be a compound, wherein all R7 are SH and all R8 are O, or all R7 are O and all R8 are OH.
Any reasonable combination of the before embodiments is possible, too, according to the invention.
Listed below are the definitions of various terms and phrases used to describe the compounds of the present invention. These definitions apply to the terms as they are used throughout the specification.
Halogen refers to F, Cl, Br, and I.
Alkyl refers to an alkyl group, which is a hydrocarbon moiety with 1 to 28, preferably 1 to 20 carbon atoms, with or without (integrated) heteroatoms such as but not limited to O, S, Si, N, Se, B, wherein the point of attachment unless specified otherwise is a carbon atom. Its constitution can be
Linear saturated hydrocarbon moiety—including, but not limited to, methyl, ethyl, propyl, butyl and pentyl or
Linear unsaturated hydrocarbon moiety—containing more preferably 2 to 20 carbon atoms, including, but not limited to, ethylen, propylen, butylen and pentylen or
Branched saturated hydrocarbon moiety—deviating from the general alkyl definition by containing at least 3 carbon atoms and including, but not limited to, isopropyl, sec.-butyl and tert.-butyl or
Branched unsaturated hydrocarbon moiety—deviating from the general alkyl definition by containing at least 3 carbon atoms and including, but not limited to, isopropenyl, isobutenyl, isopentenyl and 4-methyl-3-pentenyl
or
Cyclic saturated hydrocarbon moiety—containing more preferably 3 to 8 ring atoms and including, but not limited to, cyclopentyl, cyclohexyl, cycloheptyl, piperidino, piperazino
or
Cyclic unsaturated hydrocarbon moiety—containing more preferably 3 to 8 ring atoms.
Herein the term saturated means the group has no carbon-carbon double and no carbon-carbon triple bonds. However, in the substituted case of saturated groups one or more carbon-oxygen or carbon-nitrogen double bonds may be present, which may occur as part of keto-enol and imine-enamine tautomerisation respectively. Independent from its constitution, an alkyl group, as defined herein, can be substituted or unsubstituted. Substituents include, but are not limited to, one or more alkyl groups, halogen atoms, haloalkyl groups, (un)substituted aryl groups, (un)substituted heteroaryl groups, amino, oxo, nitro, cyano, azido, hydroxy, mercapto, keto, carboxy, carbamoyl, expoxy, methoxy, ethynyl. In case alkyl, as defined herein, contains a poly ethylene glycol (PEG) moiety, the preferred number of carbon atoms can be exceeded by the number present in the PEG moiety, wherein the PEG moiety can contain a total amount of 1 to 500 ethylene glycol groups (—(CH2CH2O)n— with n=1 to 500).
It has to be noted, that -(EO)n— is used as an abbreviated expression for —(CH2CH2O)n— with n indicating the number of ethylene glycol groups. The number of ethylene glycol groups especially may be n=1-500 or as stated in the particular example.
Aralkyl refers to an alkyl group as described above, that connects to an unsubstituted or substituted aromatic or heteroaromatic hydrocarbon moiety, consisting of one or more aromatic or heteroaromatic rings with 3-8 ring atoms each. Substituents for both the alkyl and aryl part include, but are not limited to, one or more halogen atoms, alkyl or haloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, amino, nitro, cyano, hydroxy, mercapto, carboxy, azido, methoxy, methylthio.
Aryl refers to an aryl group, which is an unsubstituted or substituted aromatic or heteroaromatic hydrocarbon moiety, consisting of one or more aromatic or heteroaromatic rings with 3-8 ring atoms each. Substituents include, but are not limited to, one or more halogen atoms, haloalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted heteroaryl groups, amino, nitro, cyano, hydroxy, mercapto, carboxy, azido, methoxy, methylthio.
Acyl refers to a —C(O)-alkyl group, wherein the alkyl group is as defined above.
Aracyl refers to a —C(O)-aryl group, wherein the aryl group is as defined above.
Carbamoyl refers to a —C(O)—NH2 group, wherein the hydrogens can independently from each other be substituted with an alkyl group, aryl group or aralkyl group, wherein alkyl group, aryl group or aralkyl group are as defined above.
O-acyl refers to an —O—C(O)-alkyl group, wherein the alkyl group is as defined above.
O-alkyl refers to an alkyl group, which is bound through an O-linkage, wherein the alkyl group is as defined above.
O-aracyl refers to a —O—C(O)-aryl group, wherein the aryl group is as defined above.
O-aralkyl refers to an aralkyl group, which is bound through an O-linkage, wherein the aralkyl group is as defined above.
O-aryl refers to an aryl group, which is bound through an O-linkage, wherein the aryl group is as defined above.
O-carbamoyl refers to a carbamoyl group, which is bound through an O-linkage, wherein the carbamoyl group is as defined above.
S-alkyl refers to an alkyl group, which is bound through a S-linkage, wherein the alkyl group is as defined above.
S-aryl refers to an aryl group, which is bound through a S-linkage, wherein the aryl group is as defined above. S-aralkyl refers to an aralkyl group, which is bound through a S-linkage, wherein the aralkyl group is as defined above.
S-aralkyl refers to an aralkyl group, which is bound through an S-linkage, wherein the aralkyl group is as defined above.
Se-alkyl refers to an alkyl group, which is bound through a Se-linkage, wherein the alkyl group is as defined above. Se-aryl refers to an aryl group, which is bound through a Se-linkage, wherein the aryl group is as defined above.
Se-aralkyl refers to an aralkyl group, which is bound through a Se-linkage, wherein the aralkyl group is as defined above. NH-alkyl and N-bisalkyl refer to alkyl groups, which are bound through an N linkage, wherein the alkyl groups are as defined above.
NH-aryl and N-bisaryl refer to aryl groups, which are bound through an N linkage, wherein the aryl groups are as defined above.
NH-carbamoyl refers to a carbamoyl group, which is bound through an N-linkage, wherein the carbamoyl group is as defined above.
Amido-alkyl refers to an alkyl group, which is bound through a NH—C(O)— linkage, wherein the alkyl group is as defined above.
Amido-aryl refers to an aryl group, which is bound through a NH—C(O)— linkage, wherein the aryl group is as defined above.
Amido-aralkyl refers to an aralkyl group, which is bound through a NH—C(O)— linkage, wherein the aralkyl group is as defined above.
Endstanding group refers to a group of a particular residue (R1, R4 and/or R5) which is (sterically) accessible and capable for covalently binding to a particular linking residue (LR1-4). This may be a group at the actual terminal end of the residue (R1, R4 and/or R5) or at any terminal end of any sidechain of the residue (R1, R4 and/or R5), or which is otherwise located in the residue (R1, R4 and/or R5) and sufficiently (sterically) accessible and capable for covalently binding to a particular linking residue (LR1-4). The definition of the term endstanding group, if applicable, is independently also valid for the residues LR5 and/or LRPEG. Further, the term terminus refers to an endstanding group which is actually a terminal end of the concerned residue.
The person skilled in the art is well aware that a particular linking residue (LR1-4) may represent a radical depending on the number of particular G units it binds to. Thus, in compounds of formula (II), the particular linking residue (LR1-4) may be a biradical, or in case it is (intermediary) bound to only one particular G unit it may be a monoradical. Similarly, in case of compounds formula (I), depending on the number of particular G units it binds to, the particular linking residue (LR1) may be a biradical, triradical, or tetraradical, or in case it is (intermediary) bound to only one particular G unit it may be a monoradical.
If an otherwise considered monovalent group is used with the modifier “divalent” as in “divalent alkyl” then this adds a second attachment point. Non limiting examples of divalent alkyl would be —CH2—, —CH2CH2—, —CH2C(CH3)2CH2—.
Whenever side chains or residues are depicted as “floating groups” on a ring system, for example, in the formula:
then these side chains (or residues) may replace any hydrogen atom attached to any of the ring atoms, including depicted, implied, or expressly defined hydrogen, as long as a stable structure is formed. All resulting substitution patterns are thus included. For the given example, this corresponds to
The person skilled in the art understands that many compounds that fall under formula III as defined above have tautomeric forms. It has to 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 saturated six-membered rings, bonds to ring atoms, and the molecular entities attached to such bonds, are termed “axial” or “equatorial” according to whether they are located about the periphery of the ring (“equatorial”), or whether they are orientated above or below the approximate plane of the ring (“axial”). Due to the given stereochemistry of the cyclic phosphate ring, the axial position can only be above the approximate plane of the ring.
In naturally occurring cyclic nucleotide monophosphates (cNMP), both R7 and R8 are oxygen, and the phosphorus double bond is “distributed or dislocated” 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 corresponding compound structures herein are presented as charged compounds with a dislocated double bond at the phosphorus, as long as this is in accordance with valency rules. This style is chosen to account for, depict and disclose all possible “locations” of the phosphorous double bond and distribution of electron density or charge each within a single structure. The dislocated double bond, as used herein, depending on the nature of the particular R7 and R8, however, does not necessarily refer to an equally distributed charge or electron density between R7 and R8.
The term PLM as used herein, stands for polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogue, wherein the term “multimeric” can refer to di-, tri- or tetrameric. To ease understanding of definitions and descriptions the term PLD (polymer linked dimeric cGMP analogue) is used as an equivalent to specifically refer to dimeric compounds.
The term polymer, as used herein, refers to a moiety consisting of at least two (equal) units or monomers.
If R7 and R8 are not equal, the phosphorus atom has four different ligands and becomes chiral resulting in two stereoisomeric forms. To describe the configuration of the chiral phosphorus, the Rp/Sp-nomenclature is used. Therein R/S follows the Cahn-Ingold-Prelog rules while “p” stands for phosphorus.
To give an example: if the equatorial residue R8 is oxygen (while axial R7 is sulphur), the corresponding cyclic guanosine-3′, 5′-monophosphorothioate compound (cGMPS-analogue) is Sp-configurated at phosphorus.
The person skilled in the art knows, that for the use in the field of the medicine especially as part of medicaments certainly only physiologically acceptable salts of the compounds according to the invention may be used.
In an embodiment the invention relates to a compound according to the definition hereinabove, wherein in case of formula (I) G4 is absent, or, wherein in case of formula (II) G4 and LR4 are absent.
In another embodiment the invention relates to a compound according to the definition hereinabove, wherein in case of formula (I) G3 and G4 are absent, or, wherein in case of formula (II) G3, G4, LR3 and LR4 are absent.
In an embodiment the invention relates to a compound according to any definition hereinabove, wherein all R7 are O and all R8 are OH.
According to the invention it is preferred, that linking residues LR1, LR2, LR3 and LR4 are further subdivided as depicted in formula (Ib) and (IIb),
wherein:
coupling functions C1, C1′, C2, C2′, C3, C3′, C4 and C4′ independently from each other can be absent or as defined by structures selected from the group consisting of
while connectivity can be as depicted or reversed as exemplified by
and wherein
in case the coupling function (C1, C1′, C2, C2′, C3, C3′, C4 and/or C4′) does not replace the residue of the G unit (R1, R4 and/or R5 of G1-4) but bind to it, the particular residue (R1, R4 and/or R5) involved in coupling of G units (or G unit with dye(s) or other reporting group(s)) independently from each other is as defined further above, wherein an endstanding group is replaced by or transformed to a coupling function
or
selected from the group depicted hereinafter (wherein if present, Q1 connects to the G unit)
and wherein
the linker (L) is selected from the group consisting of
while
and wherein
spacers (S1, S2, S3 and S4) can be equal or individual within a particular compound, be absent or be —(CH2)n1—(CH2CH2ß)m-(CH2)n2— (with ß=O, S or NH; m=1 to 500, n1=0 to 8, n2=0 to 8, while both n1 and n2 can independently be equal or individual), or —(CH2)n— (with n=1 to 24).
Particularly, in the preferred embodiment of the invention, wherein it is preferred, that linking residues LR1, LR2, LR3 and LR4 are further subdivided as depicted in formula (Ib) and (IIb), containing spacer moieties (S1-4), coupling functions (C1-4, C1-4) and a linker (L, only multimers of structure Ib), coupling functions (C1-4, C1-4) establish covalent bonds between
Coupling functions (C1-4, C1′-4) are generated in a reaction between endstanding groups of the particular precursor parts according to well established methods of the art. Non limiting examples of precursor endstanding groups (of monomeric G units and (commercially available) linkers, dyes, reporting groups and spacers) and the corresponding coupling functions (C1-4, C1′-4), to which they are transformed within the assembled (mono- or multimeric) compound according to the invention, are as depicted in Table 1. Coupling functions (C1-4, C1′-4) can independently further be absent or be equal or individual within a particular mono- or multimeric compound.
A person skilled in the art understands, that synthetic equivalents of the precursor endstanding groups of Table 1, such as but not limited to NHS esters instead of carboxylic acids or triflates instead of halogens can be used as well to generate the particular corresponding coupling function. A person skilled in the art further understands, that endstanding groups of the synthetic precursors (residues R1, R4 and/or R5, linker (L), dye, reporting group and spacer (S1-4)) can be interchanged amongst each other, resulting in reversed connectivity of the coupling function within the mono- or multimeric analogue.
A non limiting example of a multimeric compound according to the invention, illustrating the used and defined variables above is given in
Embodiments of the invention are directed to new polymer linked multimeric guanosine-3′, 5′-cyclic monophosphate (cGMP) analogues that modulate the cGMP-signaling system, preferably having activating properties, and more preferably being activators of cGMP dependent protein kinase (PKG), and related monomeric precursors thereof. The invention is also directed to related monomeric compounds, which may also show modulating activity and/or may serve as monomeric precursors of the multimers.
The idea of addressing more than one binding site of a target protein simultaneously with a single molecule has been reported once before using a polymer linked dimeric cGMP analogue (PLD).5 Therein a homologous series of one PLD (
PKG activation values of the compounds of the invention are expressed as multiple of the cGMP activation with the cGMP activation value set as 1 for each isozyme. It has to be noted, that the applied standard assay conditions only allowed to determine increased activation potencies of up to 140-fold for PKG Iα, 2832-fold for PKG Iß and 408-fold for PKG II, which is due to the employed enzyme concentration in the assays and the phenomenon that the isozymes were activity-titrated in some cases by the highly active compounds of the invention. The actual PKG activation potentials of these particular corns pounds of the invention appear to be significantly higher and are therefore expressed as ≥140-fold for PKG Iα, ≥2832-fold for PKG Iß and ≥408-fold for PKG II.
Very surprisingly, for the analogue of compound 13 lacking the PET moiety (cGMP-8-TMAmd-(PEG pd 2000)-AmdMT-8-cGMP, Table 13, compound 10) the relative enhancement of PKG isoform activity (compared to cGMP) is almost the other way around. Thus it features the so far strongest reported PKG II activation potential (243-fold activity of cGMP) but shows a far less pronounced enhancement on relative PKG Iα and Iß activation (37-fold and 69-fold activity of cGMP). Accordingly, nucleobase manipulation of PLDs and/or variation of the coupling function, which both has not been studied before, surprisingly, overrules the so far proposed target selectivity induced by spacer length.8 5 In particular, variations at Ra and/or R5 (such as the PET moiety) and/or exchange of the sulfonyl coupling function, which overlaps with modifications of the R1 moiety, have a very significant impact on PKG activation. This was especially surprising and not foreseeable, as the monomeric precursors of the two PLD compounds, 8-Br-PET-cGMP and 8-Br-cGMP, differing only in the PET moiety, at least with respect to their PKG Iα and II activatory potential are very alike.2 Yet, as described above, when coupled to a PLD, their PKG activation profile differs significantly. Accordingly, the PKG isoform activation profile of the PLD so far cannot be predicted from the one determined for the corresponding monomers. However, as described above, structural modifications were found to be essential parameters to estimate and manipulate the increasing effects of PLDs on relative PKG isoform activation.
PLDs of the present invention feature a variety of different spacer lengths, as the results described above were also reproduced with homologues PLDs. For instance shorter spacers (19 and 8 ethylene glycol units (-(EO)19— and -(EO)8—), see Table 13, compounds 12, 5, 8, 3) gave similar results, wherein in several cases the PKG enzyme was titrated (
Furthermore, the present invention comprises PLDs containing standard coupling moieties other than amide groups, as the superior activity of new PLDs according to the invention is not limited to this particular type of coupling function. Dimers linked via a triazole group (e.g. compound 23 with ≥2832-fold activity of cGMP for PKG IR, also see
Another structural aspect of PLDs according to the invention concerns the linkage position at which the two cGMP analogues are coupled to each other. The observed activity enhancement of PLDs is not restricted to linkage via the R1 position. It is still present, when linkage is varied along the G unit. Thus, as a non limiting example, PLDs coupled via the PET-moiety (at R4+R5), displayed a similarly increased PKG agonist potential as PET-substituted derivatives tethered via the R1-position (compound 6 and 23,
The nature of the spacer moiety is another motive that affects PLD induced PKG activation. PEG (spacer) units used on the PLD derivatives mentioned so far, can be replaced by or combined with other functionalities such as peptides or alkanes, included in the present invention. Alkanes in particular, however, are restricted in size, as solubility decreases significantly with growing alkyl spacer length. Still alkyl spacers with moderate size (compounds 16, 19 and 21 as non limiting examples of the present invention) are tolerated with respect to maintaining sufficient water solubility. In addition a PKG activation screening performed with compound 16 indicated that such compounds can show an activity increasing effect (PKG II activation approx. 22-fold higher than for cGMP). This effect is less pronounced than for derivatives linked via PEG-spacers but alkyl chain tethered PLDs benefit from increased lipophilicity, which should support better cell permeability. Hence, making them useful tools for biochemical assays and for pharmacological applications.
As stated above, previously established and supported by the work of the inventors linkage to a second cGMP analogue unit appears to be essential to obtain significantly enhanced PKG activation. Moreover, prior to this invention just one type of PLD (
These unexpected findings reveal another new great potential of (mixed) PLDs. As stated above, established effector compounds often need to be derivatized for specific biochemical applications. For instance, introduction of a fluorescent dye, to enable intracellular localization by means of microscopic or spectroscopic techniques, is a very common strategy. In order to obtain representative results, ideally such transformations, meant to facilitate assay read out, should have no impact on the target activation profile. However, these structural manipulations of the original compound frequently do result in a significant shift of target affinity and specificity or even loss of activation potential. This is especially the case, when the particular functionality can only be introduced at a pharmacophoric 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 target compounds, in turn, a change in (or extension of) the target activation profile obviously can also be desirable. Developing a multi target compound, though, sometimes can be just as difficult as producing a target specific one. This is the case, whenever a modification, needed to address one target, inhibits binding to the second. Mixed PLDs as disclosed within the present invention, provide an improved solution to both of these problems. Their advantage springs from the fact, that two cGMP units (instead of one for monomers) contribute to the overall PKG activation profile. As described above, even such modifications, that would give a completely different target affinity (observed for the monomer or the homogeneous PLD), do not erase the enhanced activation characteristics of the parent compound, as long as they are performed at only one cGMP unit. In this respect, the effect of structural manipulation at a single cGMP unit is buffered within mixed PLDs. Thus, mixed PLDs allow a much broader diversity of modifications (at one cGMP unit), while the undesired decrease of PKG activation, caused by these modifications, is much less pronounced if present at all. On the other hand, mixed PLDs also support the design of multi target compounds. Functional groups (e.g. PET-group), intended to address different targets (e.g. different PKG isoform) apparently can be installed at one cGMP unit, giving an extended target activation spectrum of the mixed PLD.
The present invention also comprises the extension of the described concept of polymer linked cGMP analogues from dimers to tri- and tetramers. Therein linkage of the particular G units is accomplished either in a linear or branched fashion (see formula I and II). Compounds 14 and 15 (Table 13) are two non limiting examples of the latter case, featuring particularly strong PKG II activation as predicted from analogues PLD derivatives lacking the PET-moiety (≥416-fold activity of cGMP for compound 14). The increased number of G units within tri- and tetramers results in even more diverse opportunities to combine (different) activator and target independent functionalized G units. While dimeric analogues only allow linkage of an activator G unit to either another one or a target independent functionalized G unit, for tri- and tetramers both can be provided within a single compound. Accordingly, compared to PLDs, they offer a broader scope of applications.
In summary the present invention has established the first activators of PKG Iß and PKG II with PLM structure, which are furthermore significantly improved when compared to state of the art compounds. Among the new PLM are also derivatives, which in addition activate PKG Iα, and mixed PLM, which amongst others are beneficial for functionalization and/or addressing all three PKG isoforms. Nucleobase modifications at R4/R5 and R1 position as a key part of the invention, thereby proved to be powerful modifiers of PKG activation potential. These modifications were shown herein to be able to exceed and overrule the effect of varying spacer lengths, which before was suggested to be the main effector of target selectivity and activity increase (compared to the monomer).5,8 As an exemplary embodiment of the present invention, PLMs coupled via the R1 position, which overlaps with a nucleobase modification at R1, wherein in addition R4 is absent and R5 is NH2 (according to formula III), were found to feature strongly increased PKG II activation potential (compared to the monomer). Prior art PLD compounds5 (see
To test the effect of the new PKG activators of the invention in a cellular system, the 661W cell line was used and increase in cell death after treatment was assessed (for more details see examples section). The 661W cell line is a photoreceptor precursor cell line, which expresses PKG (
Compounds that are selected based on effects on 661W cells are promising tools for research studies on the intracellular signaling pathways mediated by cGMP. Such studies should not be restricted to retinal cells but also include other cell lines sensitive to changes in cGMP levels or activating cGMP targets.10
The present invention as summarized above and defined in the claims, which are particularly incorporated into this description by reference in their entirety, shall be exemplified in the following in more detail by preferred embodiments.
According the invention it is preferred that R1 is selected from group consisting of H, halogen, azido, nitro, alkyl, acyl, aryl, OH, O-alkyl, O-aryl, SH, S-alkyl, S-aryl, S-aralkyl, S(O)-alkyl, S(O)-aryl, S(O)aralkyl, S(O)-benzyl, S(O)2-alkyl, S(O)2-aryl, S(O)2-aralkyl, amino, NH-alkyl, NH-aryl, NH-aralkyl, NR9R10, SiR13R14R15 wherein R9, R10, R13, R14, R15 are alkyl.
According to the invention it is further preferred that R1 is selected from the group consisting of H, CI, Br, I, F, N3, NO2, OH, SH, NH2, CF3, 2-furyl, 3-furyl, 2-bromo-5-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, (4-(7-methoxy)coumarinyl)thio, (2-naphtyl)thio, (2-(1-bromo)naphtyl)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 depicted in Table 2.
According to the invention it is especially preferred that R1 is selected from the group consisting of H, Cl, Br, I, F, N3, NO2, OH, SH, NH2, CF3, 2-furyl, 3-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, (4-(7-methoxy)coumarinyl)thio, (2-naphtyl)thio, (2-(1-bromo)naphtyl)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 depicted in Table 3.
According to the invention it is even more preferred that R1 is selected from the group consisting of H, Cl, Br, SH, 2-furyl, 3-furyl, (2-furyl)thio, (3-(2-methyl)furyl)thio, (3-furyl)thio, 2-thienyl, 3-thienyl, (5-(1-methyl)tetrazolyl)thio, 1,1,2-trifluoro-1-butenthio, (2-(4-phenyl)imidazolyl)thio, (2-benzothiazolyl)thio, (2,6-dichlorophenoxypropyl)thio, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)ethylthio, (4-bromo-2,3-dioxobutyl)thio, [2-[(fluoresceinylthioureido)amino]ethyl]thio, 2,3,5,6-tetrafluorophenylthio, (7-(4-methyl)coumarinyl)thio, (4-(7-methoxy)coumarinyl)thio, (2-naphtyl)thio, (2-(1-bromo)naphtyl)thio, benzimidazolyl-2-thio, benzothiazolylthio, 4-pyridyl, (4-pyridyl)thio, 2-pyridylthio, triphenyliminophosphoranyl or as depicted in Table 4.
In addition to the above or independent to the above it is preferred that according the invention that R4 is selected from group consisting of H, amino, alkyl, aralkyl, nitro, N-oxide or R4 can form together with Y and R5 and the carbon bridging Y and R5 an imidazole ring which can be unsubstituted or substituted with alkyl, aryl or aralkyl, or can form together with Y and R5 and the carbon bridging Y and R5 an imidazolinone as depicted above (structure IV, V, n=1) or an homologous ring (n=2 to 8) which each can be unsubstituted or substituted (not depicted) with alkyl, aryl or aralkyl.
According to the invention it is further preferred that R4 is absent or selected from the group consisting of amino, N-oxide or as depicted in Table 5.
According to the invention it is especially preferred that R4 is absent or selected from the group consisting of amino, N-oxide or as depicted in Table 6.
According to the invention it is even more preferred that R4 is absent or as depicted in Table 7.
In addition to the above or independent to the above it is preferred that according the invention that R5 is selected from the group consisting of H, halogen, NH-carbamoyl-alkyl, NH-carbamoyl-aryl, NH-carbamoyl-aralkyl, amino, NH-alkyl, NH-aryl, NH-aralkyl, NR3OR31 wherein R30 and R31 are alkyl, or can form together with R4, Y and the carbon bridging Y and R5 an imidazole ring which can be unsubstituted or substituted with alkyl, aryl or aralkyl, or can form together with R4, Y and the carbon bridging Y and R5 an imidazolinone ring as depicted above (structure IV, V, n=1) or an homologous ring (n=2 to 8) which each can be unsubstituted or substituted (not depicted) with alkyl, aryl or aralkyl.
According to the invention it is further preferred that R5 is selected from the group consisting of H, NH2, F, Cl, Br, I, methylamino, NH-benzyl, NH-phenyl, NH-4-azidophenyl, NH-phenylethyl, NH-phenylpropyl, 2-aminoethylamino, n-hexylamino, 6-amino-n-hexylamino, 8-amino-3,6-dioxaoctylamino, dimethylamino, 1-piperidino, 1-piperazino or can form together with R4, Y and the carbon bridging Y and R5 a ring system as depicted in Table 5 (entry 2 and 3).
According to the invention it is especially preferred that R5 is selected from the group consisting of H, NH2, F, Cl, Br, I, methylamino, NH-benzyl, NH-phenyl, NH-4-azidophenyl, NH-phenylethyl, NH-phenylpropyl, 2-aminoethylamino, n-hexylamino, 6-amino-n-hexylamino, 8-amino-3,6-dioxaoctylamino, dimethylamino, 1-piperidino, 1-piperazino or can form together with R4, Y and the carbon bridging Y and R5 a ring system as depicted in Table 6 (entry 2 and 3).
According to the invention it is even more preferred that R5 is NH2, or can form together with R4, Y and the carbon bridging Y and R5 a ring system as depicted in Table 7 (entry 2 and 3).
In addition to the above or independent to the above it is preferred that according the invention that R7 is selected from group consisting of OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, SH, S-alkyl, S-aryl, S-aralkyl, borano (BH3), methylborano, dimethylborano, cyanoborano (BH2CN), S-PAP, O-PAP, S-BAP, or O-BAP
According to the invention it is further preferred that R7 is selected from the group consisting of OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy, SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethylthio, phenylthio, benzylthio, 4-acetoxybenzylthio, 4-pivaloyloxybenzylthio, 4-isobutyryloxybenzylthio, 4-octanoyloxybenzylthio, 4-benzoyloxybenzylthio, borano (BH3), methylborano, dimethylborano, cyanoborano (BH2CN).
According to the invention it is further more preferred that R7 is selected from the group consisting of OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy, SH, methylthio, acetoxymethylthio, pivaloyloxymethylthio, methoxymethylthio, propionyloxymethylthio, butyryloxymethylthio, cyanoethylthio, phenylthio, benzylthio, 4-acetoxybenzylthio, 4-pivaloyloxybenzylthio, 4-isobutyryloxybenzylthio, 4-octanoyloxybenzylthio, 4-benzoyloxybenzylthio.
According to the invention it is even more preferred that R7 is OH.
In addition to the above or independent to the above it is preferred that according the invention that R8 is selected from group consisting of OH, O-alkyl, O-aryl, O-aralkyl, O-acyl, O-PAP or O-BAP,
According to the invention it is further preferred that R8 is selected from the group consisting OH, methyloxy, ethyloxy, cyanoethyloxy, acetoxymethyloxy, pivaloyloxymethyloxy, methoxymethyloxy, propionyloxymethyloxy, butyryloxymethyloxy, acetoxyethyloxy, acetoxybutyloxy, acetoxyisobutyloxy, phenyloxy, benzyloxy, 4-acetoxybenzyloxy, 4-pivaloyloxybenzyloxy, 4-isobutyryloxybenzyloxy, 4-octanoyloxybenzyloxy, 4-benzoyloxybenzyloxy, acetyloxy, propionyloxy, benzoyloxy;
According to the invention it is further even more preferred that R8 is OH.
In addition to the above or independent to the above it is preferred that according the invention residues involved in connecting a G unit with another G unit or a dye or another reporting group can be R1, R4 and/or R5, in which case the particular residue is
According to the invention it is further preferred that residues involved in connecting a G unit with another G unit or a dye or another reporting group can be R1, R4 and/or R5, in which case the particular residue is
In addition to the above or independent to the above it is preferred that according the invention that coupling functions (C1-4 and C1′-4) are absent or selected from the group depicted in Table 10.
According to the invention it is further preferred that coupling functions (C1-4 and C1′-4) are absent or selected from the group depicted in Table 11.
In addition to the above or independent to the above it is preferred that according the invention the linker (L) is absent or selected from the group depicted in Table 12.
While n for each sidechain within a particular linker can have an equal or individual value as defined.
In addition to the above or independent to the above it is preferred that in case of formula (I) according to the invention G4 or G4 and G3 are absent
According to the invention it is even more preferred that in case of formula (I) G4 and G3 are absent
Particularly preferred embodiments of the invention based on the above exemplifications, are as defined in anyone of the claims 6, 7, 8 and 9.
Especially preferred according to the invention are the compounds of Table 13, and as defined in claim 10. It has to be noted that in case of doubt the chemical structure as depicted in the formula is the valid one. It further has to be noted, that the compounds of Table 13 are displayed as the free acid. The present invention, however, also comprises salts of these compounds, featuring cations such as but not limited to Na+, Li+, NH4+, Et3NH+ and (i-Pr)2EtNH+.
Monomeric precursor cGMP analogues (G units) for the synthesis of polymer linked multimeric cGMP analogues (PLMs) are compounds of formula (III). As described above, the PKG activation potential is strongly increased, once the monomeric precursor is linked to additional one(s) within a PLM, wherein particularly enhanced PKG isoform activation can be related to a certain extend to structural parameters. Non limiting examples of methods for the transformation of monomeric precursors into exemplary PLMs are given in the examples section. In addition Table 1 gives an overview of exemplary endstanding groups, that can be used for coupling reactions and the corresponding coupling functions within the PLM, to which they are transformed according to established methods of the art.
The invention in one aspect also relates to monomeric compounds of formula (III) and/or monomeric precursors according to formula (III), of any compound of the invention as described herein above, wherein the monomeric compound of formula (III) and/or the monomeric precursor of formula (III) is defined in the context of any said compounds herein above, and preferably wherein the monomeric compound of formula (III) and/or monomeric precursor of formula (III) complies with the following proviso:
and further complies with at least one of the following provisos:
and in addition complies with the proviso that the monomeric compound of formula (III) and/or the monomeric precursor compound of formula (III) is not selected from the group of compounds consisting of
and/or
the monomeric compound of formula (III) and/or the monomeric precursor of the invention is selected from the group depicted in Table 14.
As described above, the compounds according to the present invention may further be labelled, according to well-known labelling techniques. For example, fluorescent dyes may be coupled to the compounds in order to, but not limited to, localize the intracellular distribution of cyclic nucleotide binding proteins in living cells by means of confocal microscopy, for fluorescence correlation spectrometry, for fluorescence energy transfer studies, or for determination of their concentration in living cells.
It should be understood that hydrates of the compounds are also within the scope of the present invention.
Instead of or additional to fluorescent dyes the compounds according to the inventions may be labelled with (radio) nuclides. The person skilled in the art knows many techniques and suitable isotopes that can be used for this.
As described above, the invention also comprises PEGylated forms of the specified compounds, wherein PEGylation is generally known to greatly improve water solubility, pharmacokinetic and biodistribution properties.
The invention further comprises modifications wherein R7 (according to formula III) can be an unsubstituted or substituted thio- or borano function. Both modifications are known in the art to improve resistance towards metabolic degradation.1a, 14
The invention also comprises prodrug forms of the described compounds, wherein the negative charge of the (modified or unmodified) phosphate moiety is masked by a bioactivatable protecting group. It is widely accepted that such structures increase lipophilicity and with that, membrane-permeability and bioavailability resulting in a 10-1000 fold enhanced potency compared to the mother-compound. Such bioactivatable protecting groups can be introduced according to well known techniques of the art and include, but are not limited to acetoxymethyl, propionyloxymethyl, butyryloxymethyl, pivaloyloxymethyl, acetoxyethyl, acetoxybutyl, acetoxyisobutyl. Non limiting examples of corresponding residues R7 and/or R8 according to the invention are acetoxymethyloxy, propionyloxymethyloxy and butyryloxymethyloxy. More labile examples of protecting groups include alkyl or aryl groups as well as substituted alkyl or aryl groups. Non limiting examples for chemically labile protection groups of the R7 and/or R8 position are methyl, ethyl, 2-cyanoethyl, propyl, benzyl, phenyl and polyethylene glycol. These compounds are inactive per se, but extremely membrane-permeable, leading to strongly increased intracellular concentrations. Upon hydrolysis of the ester bond, the biologically active mother compounds are released.
Compounds according to the invention can also feature a photolysable group (also-called “caged”- or photo-activatable protecting group), which can be introduced according to well known techniques of the art. For example, but not limited to, caged groups may be coupled to an R8 oxo-function, leading to compounds with significantly increased lipophilicity and bioavailability. Non limiting examples for caged groups are o-nitro-benzyl, 1-(o-nitrophenyl)-ethylidene, 4,5-dimethoxy-2-nitro-benzyl, 7-dimethylaminocoumarin-4-yl (DMACM-caged), 7-diethylamino-coumarin-4-yl (DEACM-caged) and 6,7-bis(carboxymethoxy)coumarin-4-yl)methyl (BCMCM-caged).
The compounds according to the present invention can also be immobilized to insoluble supports, such as, but not limited to, agarose, dextran, cellulose, starch and other carbohydrate-based polymers, to synthetic polymers such as polacrylamide, polyethyleneimine, polystyrol and similar materials, to apatite, glass, silica, gold, graphene, fullerenes, carboranes, titania, zirconia or alumina, to the surface of a chip suitable for connection with various ligands.
The compounds according to the present invention can also be encapsulated within nanoparticles or liposomes for directed or non-directed delivery and release purposes of the compounds as described in the literature.11
Preferably, the new polymer linked multimeric cGMP analogues of the invention, or the related monomeric compounds of formula (III) of the present invention, respectively, are used for treating or preventing a disease or condition that is associated with low cGMP signaling activity.
Diseases and conditions are preferably treated with polymer linked multimeric cGMP analogues, or the related monomeric compounds of formula (III) of the present invention, respectively, that activate the disease-related unbalanced cGMP-system, and include1c:
It is to be understood herein that the treatment of a pathology, condition or disorder also includes the prevention thereof, even if not explicitly mentioned, unless specifically otherwise indicated.
In another aspect, the invention relates to a method for treating or preventing any of the above pathologies, conditions or disorders by administration of a therapeutically or prophylactically effective amount of an equatorially modified cGMP-analogue of the invention to a subject in need of prophylaxis or therapy.
The compounds according to the present invention, including the related monomeric compounds of formula (III) of the present invention, respectively, can also be used as research tool compound, preferably as research tool compound in regard of a disease or disorder related to an unbalanced cGMP-system, preferably a disease or disorder selected from the group consisting of cancer, cardiovascular disease or disorder, or autoimmune disease or disorder, or neurodegenerative disease or disorder.
The invention is further illustrated by the following figures and examples describing preferred embodiments of the present invention which are, however, not intended to limit the invention in any way.
1. Compound Synthesis
General Experimental Methods
All applied solvents and reagents were available from commercial suppliers. 8-Br-cGMP, 8-Br-PET-cGMP and 4-N3-PET-8-Br-cGMP were available from Biolog Life Science Institute (Bremen, Germany). 8-T-cGMP is established in the literature and was prepared analogously to PET-8-T-cGMP (see examples below). Solvents used were specified as analytical or hplc grade. Dimethyl sulfoxide was stored over activated molecular sieves for at least two weeks before use. Chromatographic operations were performed at ambient temperature. Both reaction progress and purity of isolated products were determined by reversed phase hplc (RP-18, ODS-A-YMC, 120-S-11, 250×4 mm, 1.5 mL/min), wherein UV detection was performed either at 263 nm, an intermediate wavelength suitable to detect most cyclic GMP products and—impurities, or at the λmax of the particular starting material or product. Syntheses were typically performed in a 20-200 μmol scale in 2 mL polypropylene reaction vials with screw cap (reactions requiring inert gas atmosphere and/or degassing were performed in round bottom flasks (typically 10 or 25 mL)). Dissolution of poorly soluble reactants was achieved through sonification or heating (70° C.) prior to addition of reagents. In case dissolution was not elicited by these techniques, which mainly applied to some cGMP analogues carrying a PET-moiety, the suspension was used. Purification of products was accomplished by preparative reversed phase hplc (RP-18, ODS-A-YMC, 12 nm-S-10, 250×16 mm, UV 254 nm). The eluent composition is described in the particular synthetic example and, unless stated otherwise, can be used for analytical purposes as well. Desalting of products was accomplished by repeatedly freeze-drying or by preparative reversed phase hplc (RP-18, ODS-A-YMC, 12 nm-S-10, 250×16 mm, UV 254 nm) according to standard procedures for nucleotides. Solutions were frozen at −70° C. for 15 min prior to evaporation, in case a speedvac concentrator was used to remove the solvent. Products were either isolated as sodium or triethylammonium salt, depending on the applied buffer. Yields refer to the fraction of isolated product featuring the reported purity. They were calculated from UV-absorbance at the λmax, measured on a JASCO V-650 Spectrophotometer (JASCO Germany GmbH, Gross-Umstadt, Germany) according to Lambert-Beers law. Extinction coefficients were estimated from literature known values of structurally related compounds. Mass spectra were obtained with an Esquire LC 6000 spectrometer (Bruker Daltronics, Bremen, Germany) in the ESI-MS mode with 50% water/50% methanol as matrix.
Experimental Procedures for the Preparation of 8-Thio-Modified Guanosine-3′,5′-Cyclic Monophosphate Analogues
General Procedure A:
In a typical experiment the corresponding thiol reactant (8 eq) and NaOH (2 M, 4 eq) were added successively to a solution of the corresponding 8-Br-substituted cGMP analogue (sodium salt, 65 mM, 1 eq) in H2O/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 reach room temperature, neutralized with HCl (1 M) and the solvent was removed through high vacuum evaporation with 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, the residue redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 8-thio-modified cGMP analogue. *In case the residue was not soluble in water, the obtained suspension was washed with MTBE and (if necessary) diluted with MeOH to dissolve remaining precipitate.
General Procedure A2:
In a typical experiment the corresponding thiol(ate) reactant (4.5 eq) was added to a solution of the corresponding 8-Br-substituted cGMP analogue (sodium salt, 65 mM, 1 eq) in H2O/i-PrOH (1:1, v/v). 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 through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with CH2Cl2 (3×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 8-thio-modified cGMP analogue.
General Procedure B:
In a typical experiment a solution of the 8-Br-substituted cGMP analogue (sodium salt, 87 mM, 1 eq) was added portionwise over 2 h to a suspension of the corresponding dithiol (50 mM in water/i-PrOH, 2:3, v/v, 10 eq) 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 through high vacuum evaporation with 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, giving the thiol analogue.
General Procedure C:
In a typical experiment NaOH (2 M, 16 eq) and the corresponding thiol reactant (8 eq) were added successively to a solution of the 8-Br-substituted cGMP analogue (sodium salt, 200 mM, 1 eq) in borate buffer (100 mM, pH 12). 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 reach 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), subjected to preparative reversed phase hplc and desalted.
General Procedure D:
In a typical experiment N,N-diisopropylethylamine (2 eq) and the corresponding bromide (1 eq) were added successively to a solution of the 8-SH-substituted cGMP analogue (sodium or triethylammonium salt, 100 mM, 1 eq) in DMSO. The reaction mixture was stirred until the thiol starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), washed with ethyl acetate (3×), subjected to preparative reversed phase hplc and desalted.
General Procedure E:
For the formation of dimeric cGMP analogues general Procedure D was followed using N,N-diisopropylethylamine (2 eq), the corresponding bis-bromide spacer (0.5 eq) and the 8-SH-substituted cGMP analogue (sodium or triethylammonium salt, 100 mM, 1 eq) in DMSO.
Experimental Procedure for the Transformation of Carboxylic Acid Ester Functionalized Guanosine-3′,5′-Cyclic Monophosphate Analogues into the Corresponding Carboxylic Acid or Amide
General Procedure F:
In a typical experiment NaOH (2 M, 10 eq) was added to a solution of the corresponding ester (80 mM, 1 eq) 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 (1 M) and 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, giving 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 through 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 reversed phase hplc and desalted, giving the carboxylic acid amide analogue.
Experimental Procedures for the Formation of Amide Bonds with 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 successively to a solution of the corresponding acid-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the corresponding amine (1.1 eq)*. The reaction mixture was stirred until the starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with ethyl acetate (5×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the coupled cGMP analogue. *The less valuable reactant was added in slight excess, thus for the reaction with reversed functions the amine-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the acid reactant (1.1 eq) were used.
General Procedure I:
In a typical experiment HOBt (1.1 eq), N,N-diisopropylethylamine (2.2 eq) and EDC (1.1 eq) were added successively to a solution of the corresponding acid-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the corresponding bis-amino spacer (0.5 eq). Workup was performed as described in general procedure H, giving the dimeric cGMP analogue.
General Procedure J:
In a typical experiment N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq) were added successively to a solution of the corresponding carboxylic acid-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the corresponding amine (1.1 eq)*. The reaction mixture was stirred until the starting material was completely consumed or no further reaction progress was observed (usually <10 min). Water (100 μL) was added, stirring was continued for 10 min and the solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), if necessary the pH was adjusted to 6 with NaOH (2 M) or HCl (1 M) and the solution washed with ethyl acetate (5×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the coupled cGMP analogue. *The less valuable reactant was added in slight excess, thus for the reaction with reversed functions the amine-substituted cGMP analogue (100 mM in DMSO, 1 eq) and the acid reactant (1.1 eq) were used.
General Procedure K:
In a typical experiment a solution of the corresponding carboxylic acid-substituted cGMP analogue (100 mM in DMSO, 1 eq) was added portionwise over 40 min to a solution of the bis-amino spacer (400 mM in DMSO, 5 eq), N,N-diisopropyethylamine (2.2 eq) and PyBOP (1.1 eq). More PyBOP (1 eq) was added and the reaction mixture was stirred until the starting material was completely consumed or no further reaction progress was observed (usually <10 min). Workup was performed as described in general procedure J, giving the monomeric cGMP analogue coupling product.
General Procedure L:
General procedure J was followed using the corresponding acid-substituted cGMP analogue (100 mM in DMSO, 1 eq), the bis-amino spacer (0.5 eq), N,N-diisopropylethylamine (2.2 eq) and PyBOP (1.1 eq) to obtain the dimeric cGMP analogue.
General Procedure M:
General procedure J was followed using the corresponding amine-substituted cGMP analogue (33 mM in DMSO, 1 eq), the linker tri-acid (0.3 eq), N,N-diisopropylethylamine (2 eq) and PyBOP (1.3 eq) to obtain the trimeric cGMP analogue.
General Procedure N:
General procedure J was followed using the corresponding amine-substituted cGMP analogue (diisopropylethylammonium salt, 50 mM in DMSO, 1 eq)*, the linker tetra-acid (tetra-diisopropylethylammonium salt, 0.25 eq)*, N,N-diisopropylethylamine (3 eq) and PyBOP (1.3 eq) to obtain the tetrameric cGMP analogue. *To transform the reactants into the diisopropylethylammonium salt they were subjected to N,N-diisopropylethylamine (3 eq per acidic function) in water (0.1-0.3 M) and evaporated to dryness using a speedvac concentrator at high vacuum.
Experimental Procedures for the Preparation of 8-Sulfonyl- and 8-Sulfoxide-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 cGMP analogue (40 mM, 1 eq) in water/MeOH (1:1, v/v). The reaction mixture was stirred until the thio starting material 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, giving the 8-sulfonyl-substituted cGMP analogue.
Derivatives featuring a modified phosphate function, sensitive to oxidation reactions, such as a phosphorothioate, were synthesized starting from the corresponding guanosine, while the (modified) phosphate group was then introduced according to well established methods of the art (e.g. thiophosphorylation protocol12) after oxidation of the 8-thio function.
General Procedure P:
General procedure O was followed, favoring the formation of the 8-sulfoxide-substituted cGMP analogue through shorter reaction time and decreased equivalents of oxidizing agent OXONE® (1.5 eq).
Experimental Procedure for the Generation of 8-Azidoalkylthio-Substituted Guanosine-3′,5′-Cyclic Monophosphate Analogues
General Procedure Q:
In a typical experiment NaN3 (22.5 eq) was added portionwise over 5 h to a solution of 1,2-dibromoalkane (1.5 M, 15 eq) in DMF in an amber flask. The reaction mixture was stirred for 23 h and the 8-SH-substituted cGMP analogue (triethylammonium salt, 1 eq) as well as N,N-diisopropylethylamine (1 eq) were added successively. Stirring was continued until the cGMP analogue starting material was completely consumed or no further reaction progress was observed (usually about 1 h). The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with MTBE (5×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 8-azidoalkylthio-substituted analogue.
Experimental Procedures for the [3+2] Cycloaddition of Azides and Terminal Alkynes on Guanosine-3′,5′-Cyclic Monophosphate Analogues
General Procedure R:
In a typical experiment a solution of the corresponding azide (0.5 M in CH2Cl2, 1.1 eq) was added to the alkyne-substituted cGMP analogue (40 mM in H2O, 1 eq) in an amber flask. Bromotris(triphenylphosphine)copper(I) ([Cu(PPh3)3Br]) (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 (to 1.5 mL) and washed with CH2Cl2 (3×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the triazole addition product.
General Procedure S:
In a typical experiment Cu(PPh3)3Br (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 through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL) and washed with CH2Cl2 (3×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the triazole-containing product.
General Procedure T:
General Procedure S was followed, using Cu(PPh3)3Br (0.05 eq), the corresponding azide-substituted cGMP analogue (23 mM, 1 eq) and the corresponding bis-alkyne (12 mM, 2 eq) in water/N,N-diisopropylethylamine (8:1, v/v). Conditions were chosen to obtain both the monomeric and the dimeric triazole-containing product.
General Procedure U:
General Procedure S was followed, using [Cu(PPh3)3Br] (0.05 eq), the corresponding azide-substituted cGMP analogue (33 mM, 1 eq) and the corresponding bis-alkyne (16 mM, 0.5 eq) in water/N,N-diisopropylethylamine (10:1, v/v) to obtain the dimeric triazole-containing product.
Experimental Procedure for the Transformation of Azido-Substituted Guanosine-3′,5′-Cyclic Monophosphate Analogues into the Corresponding Amines
General Procedure V:
In a typical experiment a solution of the azido-substituted cGMP analogue (2.5 mM in water, 1 eq) in an amber flask was adjusted to pH 10 by addition of 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), subjected to preparative reversed phase hplc and desalted, giving the amine-substituted cGMP analogue.
Experimental Procedures for the Transformation of 8-Azido-Substituted Guanosine-3′,5′-Cyclic Monophosphate Analogues into the Corresponding Iminophosphoranyl Analogue
General Procedure W:
In a typical experiment PPh3 (1.75 eq) and water (100 μL) were added to a solution of 8-azido-substituted cGMP analogue (100 mM, 1 eq) in DMF in an amber flask. The reaction mixture was stirred until the azide starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was suspended in water (1 mL) and washed with toluene (5×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water/MeOH (4:1), subjected to preparative reversed phase hplc and desalted, giving the iminophosphoranyl analogue.
Experimental Procedure for the Suzuki Cross-Coupling of Br-Substituted Guanosine-3′,5′-Cyclic Monophosphate Analogues with Organoboronic Acids
General Procedure X:
In a typical experiment aqueous K2CO3 (2 M, 3 eq) and Pd(dppf)C12 (0.05 eq) were added successively to a solution of the Br-substituted cGMP analogue (52 mM, 1 eq) and the boronic acid (72 mM, 1.4 eq) in EtOH/H2O (1:1, v/v). The reaction mixture was immediately degased 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 through high vacuum evaporation with a speedvac concentrator. The residue was suspended in water and washed with CHCl3 (3×). Methanol was added until dissolution of the precipitate (up to H2O/MeOH=1:1). If an organic phase, containing residual CHCl3, emerged from this composition, it was separated. The aqueous phase was then filtered through a Macherey-Nagel Chromafix C 18 (S) 270 mg cartridge (preconditioned with 10 mL of 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), subjected to preparative reversed phase hplc and desalted, giving the cross-coupling product. * All solvents used, were degassed through sonification under reduced pressure prior to the experiment.
General Procedure X2 (Preparation of Bis Boronic Acid Reagent 4-B(OH)2PhS-PEG5-(CH2)2-4-SPhB(OH)2:
In a typical experiment N,N-diisopropylethylamine (2 eq) was added to a solution of 4-mercaptophenylboronic acid (0.2 M, 1 eq) and Br-(EO)5—(CH2)2—Br (0.5 eq) in DMF. The reaction mixture was stirred until the boronic acid starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in methanol (1 mL) and subjected to preparative reversed phase hplc (62% MeOH) giving 4-B(OH)2PhS-PEG5-(CH2)2-4-SPhB(OH)2 (34% yield).
Experimental Procedure for the Preparation of 1, N2-Functionalized 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 successively to a solution of the corresponding cGMP analogue (50 mM, 1 eq) in DMSO. The reaction mixture was stirred under exclusion of light until the cGMP analogue 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 through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in methanol (0.5 mL) and the pH adjusted to 6-7 with HCl (1 M). In case a precipitate was formed thereby, methanol was added to redissolve it. Otherwise, water was slowly added until all components just remained soluble (max. H2O/MeOH=5:1). The solution was subjected to preparative reversed phase hplc and desalted, giving the 1, N2-etheno-functionalized cGMP analogue.
General Procedure Y2:
In a typical experiment DBU (2 eq) and the corresponding alkyl bromoacetate-reactant (1.1 eq) were added successively to a solution of the corresponding cGMP analogue (100 mM, 1 eq) in DMSO. The reaction mixture was stirred until the cGMP analogue 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 through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in H2O (0.5 mL) and the pH adjusted to 6-7 with HCl (1 M). The solution was subjected to preparative reversed phase hplc and desalted, giving the 1, N2-acyl-functionalized cGMP analogue.
General Procedure Y3:
In a typical experiment N,N-diisopropylethylamine (2 eq) and PyBOP (1.1 eq) were added successively to a solution of the corresponding 1-carboxyalkyl-substituted cGMP analogue (10 mM in DMSO, 1 eq). The reaction mixture was stirred until the cGMP analogue starting material was completely consumed or no further reaction progress was observed. Water (100 μL) was added, stirring was continued for 10 min and the solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in water (1 mL), the pH adjusted to 5-6 with NaOH (2 M) and the solution washed with ethyl acetate (5×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1, N2-acyl-functionalized cGMP analogue.
Experimental Procedures for the Preparation of 1-Substituted 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 successively to a solution of the corresponding cGMP analogue (50-300 mM, 1 eq) in DMSO. The reaction mixture was stirred until the cGMP analogue starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in H2O (0.5 mL) and, in case 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, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1-substituted cGMP analogue.
General Procedure Z2:
In a typical experiment DBU (2 eq) and the corresponding dibromide-reactant (0.5 eq) were added successively to a solution of the corresponding cGMP analogue (15 mM, 1 eq) in DMSO. The reaction mixture was stirred at 90° C. until the cGMP analogue starting material was completely consumed or no further reaction progress was observed. The solvent was removed through high vacuum evaporation with a speedvac concentrator. The residue was dissolved in H2O (0.5 mL), the pH adjusted to 5-7 with HCl (1 M) and the solution was washed with ethyl acetate (4×). The aqueous phase was evaporated under reduced pressure using a rotary evaporator, the residue was redissolved in water, subjected to preparative reversed phase hplc and desalted, giving the 1-substituted dimeric cGMP analogue.
Experimental Procedures for the Preparation of 2′-OR Substituted Guanosine-3′,5′-Cyclic Monophosphate Analogues
General Procedure ZZ:
In a typical experiment 2-azidoacetic anhydride (32 eq, prepared according to Freudenberg13) and 4-dimethylaminopyridine (1 eq) were added successively to the corresponding cGMP analogue (1 eq). The reaction mixture was stirred under exclusion of light until the cGMP analogue starting material was completely consumed or no further reaction progress was observed. The mixture was separated between MTBE and H2O, the organic phase was decanted off and the aqueous phase washed with MTBE (2×). A precipitate was dissolved by adding methanol to the aqueous layer. The solution was subjected to preparative reversed phase hplc and desalted, giving the 2′-O-(2-azidoacetyl)-substituted cGMP analogue.
Synthesis of previously established precursors is described in the following:
Using general procedure C, 8-Br-cGMP was reacted with mercaptoacetic acid to give the title compound.
Yield (Purity): 66% (>99%).
HPLC: (5% MeCN, 100 mM TEAF buffer, pH 6.8).
UV-VIS: λmax=275 nm (pH 7), ε=13700 (est.).
ESI-MS (+): m/z calculated for C12H15N5O9PS ([M+H]+): 436.03, found: 436.
ESI-MS (−): m/z calculated for C12H13N5O9PS ([M−H]−): 434.02, found: 434.
Using general procedure A, 8-Br-cGMP (1 eq) was reacted with sodium methanethiolate (4 eq) to give the title compound.
Yield (Purity): 50% (>99%).
HPLC: (9 MeCN, 20 mM TEAF buffer, pH 6.8).
UV-VIS: λmax=274 nm (pH 7), ε=14000 (est.).
ESI-MS (+): m/z calculated for C11H15N5O7PS ([M+H]+): 392.04, found: 392.
ESI-MS (−): m/z calculated for C11H13N5O7PS ([M−H]−): 390.03, found: 390.
The title compound was synthesized from 8-MPT-cGMP using general procedure F.
Yield (Purity): 56% (>99%).
HPLC: (5 MeCN, 10 mM TEAF buffer, pH 6.8).
UV-VIS: λmax=275 nm (pH 7), ε=13700 (est.).
ESI-MS (+): m/z calculated for C13H17N5O9PS ([M+H]+): 450.05, found: 450.
ESI-MS (−): m/z calculated for C13H15N5O9PS ([M−H]−): 448.33, found: 448.
Using general procedure B, 8-Br-cGMP was reacted with 1,4-benzenedithiol to give the title compound.
Yield (Purity): 41% (>99%).
HPLC: (32% MeOH, 10 mM TEAF buffer, pH 6.8).
UV-VIS: λmax=286 nm (pH 7), ε=21500 (est.).
ESI-MS (+): m/z calculated for C16H17N5O7PS2 ([M+H]+): 486.03, found: 486.
ESI-MS (−): m/z calculated for C16H15N5O7PS2 ([M−H]−): 484.02, found: 484.
The invention is further illustrated by the figures and examples of Table 15 describing preferred embodiments of the present invention which are, however, not intended to limit the invention in any way. Structural examples of novel compounds are depicted in the free acid form. After HPLC workup, compounds are obtained as salts of the applied buffer, but can be transformed to other salt forms or to the free acid by cation exchange according to standard procedures for nucleotides.
Monomeric precursors of the invention and/or momomeric compounds of the invention are further illustrated by the figures and examples of Table 16 describing preferred embodiments of the present invention which are, however, not intended to limit the invention in any way. Structural examples of novel compounds are depicted in the free acid form. After HPLC workup, compounds are obtained as salts of the applied buffer, but can be transformed to other salt forms or to the free acid by cation exchange according to standard procedures for nucleotides.
2. Activation of PKG Isoforms by cGMP Derivatives
Experimental Part
In vitro activation experiments with PKG isozymes Iα, Iß and II were performed with the commercially available luminescence assay ADP-Glo™ Kinase Assay (Cat. #V9101) from Promega Corporation (Madison, Wis., USA) according to the manufacturer's instruction manual (The ADP-Glo™ Kinase Assay Technical Manual #TM313), standardized and conducted by BIAFFIN GmbH & Co KG (Kassel, Germany). Luminescence detection was accomplished with a LUMlstar Optima microplate luminometer from BMG LABTECH GmbH (Ortenberg, Germany). Bovine PKG type Iα was purified from bovine lung. Human PKGIß and PKGII were expressed in Sf9 cells and purified by affinity chromatography.2 Concentrations of enzymes given below refer to the dimeric form. VASPtide (GL Biochem Ltd., Shanghai, China) was used as PKG-selective phosphorylation substrate peptide.2
Assay Conditions:
PKG Iα (0.2 nM), 20 mM Tris (pH 7.4), 10 mM Mg2Cl2, 1 mg/mL BSA, 0.15 mM β-mercaptoethanol, 2.5% DMSO, 130 μM VASPtide, 50 μM ATP, room temperature, 60 min.
PKG Iß (0.15 nM), 20 mM Tris (pH 7.4), 10 mM Mg2Cl2, 1 mg/mL BSA, 2.5% DMSO, 130 μM VASPtide, 50 μM ATP, room temperature, 60 min.
PKG II (0.5 nM), 20 mM Tris (pH 7.4), 10 mM Mg2Cl2, 1 mg/mL BSA, 5 mM β-mercaptoethanol, 2.5% DMSO, 130 μM VASPtide, 50 μM ATP, room temperature, 120 min.
Different concentrations (10 μM to 6 μM) of the compounds of the invention and cGMP as reference compound were incubated with the respective PKG isozyme. To increase assay sensitivity in case of PKG II, cGMP and compounds of the invention were preincubated at room temperature for 30 min. The activation values of the compounds are expressed as relative PKG activation compared to cGMP with cGMP set as 1 for each kinase isozyme. The Ka-values of cGMP for half-maximal kinase activation were 28 nM for Iα, 425 nM for Iß and 208 nM for II.
Results
3. 661W Cell Line: Assessment of Cell Death Using the Ethidium Homodimer Assay
Background
To test the effect of PKG activators, the 661W cell line was used and increase in cell death after treatment was assessed. The 661W cell line is a photoreceptor precursor cell line, immortalized with the SV40 T antigen. As shown in the
The 661W cells were cultured in DMEM with 10% FBS (Fetal Bovine Serum), 2 mM Glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. For the Ethidium Homodimer Assay, cells were plated in a 24 well plate on slides coated with ECM (extracellular matrix) at 20 000 cells/well and left for 24 hours to attach to the slides. The next day the cells were treated with the compounds. Compounds were dissolved in water and then diluted in the medium at concentrations of 1 nM to 10 μM. 16 hours after treatment cells were washed with PBS and fixed in 4% paraformaldehyde. Afterwards slides were dipped into 2 μM Ethidium Homodimer for 2 minutes and nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). Ethidium Homodimer stains nuclei of dying cells. To assess cell death, microphotographs were taken from three different slides for each compound concentration and the total number of cells, as well as the number of dying Ethidium Homodimer positive cells, were counted in each picture. The value for untreated cells was set to 1. To statistically assess significant differences between untreated and treated cells, the unpaired Student's t-test was used and a P value ≤0.05 was considered significant (*≤0.05, **≤0.01, ***≤0.001).
Results
| Number | Date | Country | Kind |
|---|---|---|---|
| 16186700.7 | Aug 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/071859 | 8/31/2017 | WO | 00 |
