Patent Grant
10016432
The present disclosure relates to a novel family of adenosine kinase inhibitors, and more particularly to the prevention of epilepsy and its progression using the novel adenosine kinase inhibitors.
Endogenous adenosine (i.e., naturally occurring adenosine) acts on G protein-coupled receptors (adenosine receptors, ARs) in the central nervous system to suppress seizures and pain, and to blunt the effects of ischemia (a restriction in blood supply to tissues). In addition, adenosine has AR-independent epigenetic effects based on interactions with the transmethylation pathway. There is a dynamic equilibrium between extracellular adenosine levels and its intracellular content that is mediated by either equilibrative (ENTs) or concentrative (CNTs) transporters of nucleosides. Within the brain the concentration of adenosine is largely under the control of metabolic clearance through astrocytic adenosine kinase (AdK), which converts adenosine to 5′-AMP. By inhibiting AdK, the adenosine concentration can be exogenously raised.
It may therefore be possible to target the AR-independent effects of adenosine while avoiding excessive AR activation, by administering brain-penetrant human (h) AdK inhibitors.
The present disclosure is directed to adenosine kinase inhibitors having the formula
where J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—; R1 is C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl; R2 and R3 are independently C1-C6 alkyl; R4 is hydrogen, or C1-C6 alkyl; and R5 and R6 are independently C6-C12 aryl, C3-C8 cycloalkyl, or C3-C8 heteroaryl, that is optionally further substituted.
In another aspect, the disclosure is directed to pharmaceutical compositions that include an adenosine kinase inhibitor having the formula
where J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—; R1 is C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl; R2 and R3 are independently C1-C6 alkyl; R4 is hydrogen, or C1-C6 alkyl; and R5 and R6 are independently C6-C12 aryl, C3-C8 cycloalkyl, or C3-C8 heteroaryl, that is optionally further substituted.
In yet another aspect, the disclosure is directed to a method of preventing epilepsy and its progression in a patient that includes administering an effective amount of an adenosine kinase inhibitor having the formula:
where J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—; R1 is C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl; R2 and R3 are independently C1-C6 alkyl; R4 is hydrogen, or C1-C6 alkyl; and R5 and R6 are independently C6-C12 aryl, C3-C8 cycloalkyl, or C3-C8 heteroaryl, that is optionally further substituted.
A raised level of adenosine in the brain counteracts seizures by activating the neuroprotective A1AR and attenuates epilepsy progression by decreasing S-adenosyl methionine dependent DNA methylation as an epigenetic mechanism of action. Increased DNA methylation is a pathological hallmark of chronic epilepsy and associated with disease progression and maintenance of the epileptic state. Efficient transmethylation reactions, for example in the liver, require the removal of adenosine by AdK, and this effect has been demonstrated to occur in the brain, as well. Unlike adenosine itself, synthetic A1AR agonists, which are also proposed for seizure treatment, would not inhibit DNA methylation. Overexpression of AdK in the brain is both a result of astroglial activation and a contributing factor to epileptic seizures.
Without wishing to be bound by theory, it is believed that an inhibitor of AdK might provide an advantage in seizure control, because it would combine a pharmacological mechanism (increased A1AR activation) with epigenetic mechanisms (decreased DNA methylation) and might preferentially act on pathologically increased AdK as opposed to normal baseline levels of the enzyme. The advantages of ‘adenosine augmentation therapies’ for epilepsy and its associated comorbidities have already been discussed.
Several classes of AdK inhibitors have already been introduced and explored for the treatment of seizures and pain (see
A common approach in medicinal chemistry to enhance the activity or selectivity of flexible biologically active, small molecules is to introduce a conformational constraint to achieve a desired conformation for interacting with a target biopolymer, i.e. here an enzyme. This may lower the energy barrier of the binding process and can eliminate undesired interactions with other molecular targets that prefer a different conformation of the ligand. One means of sterically constraining the ribose ring of nucleoside derivatives, as already applied to antiviral agents and to receptor ligands, is to incorporate a bicyclic ribose substitute in a conformation that is preferred when the molecule is bound to the protein target. The methanocarba([3.1.0]bicyclohexane) ring system is applied to hold the ribose-like ring in either a North (N) or a South (S) conformation. The X-ray structure of human AdK shows a bound nucleoside inhibitor 1c containing a ribose in the (S) conformation, which is similar to the ribose conformation preferred by other nucleoside kinases. The present disclosure is directed to selected sterically constraining nucleoside inhibitors of human AdK using methanocarba rings.
In one aspect, the disclosure is directed to adenosine kinase inhibitors having the formula:
where R1 is —NH2, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl. The substituents R2 and R3 are independently C1-C6 alkyl. The substituent R4 is hydrogen, or C1-C6 alkyl.
The R5 and R6 moieties are independently selected from C6-C12 aryl, C3-C8 cycloalkyl, and C3-C8 heteroaryl. The R5 and R6 moieties are optionally and independently further substituted.
Where R5 or R6 is an aryl substituent, the aryl substituent is optionally further substituted one or more times by a halogen, a C1-C6 haloalkyl, a C1-C6 hydroxyalkyl, a C1-C6 alkoxy, a sulfonyloxy, carboxyalkyl, a nitro, a C1-C6 sulfonyloxyalkyl, or arylcarbonyl, and any combination thereof.
Where R5 or R6 is a cycloalkyl, the cycloalkyl substituent is optionally further substituted one or more times by a halogen, a C1-C6 haloalkyl, a C1-C6 hydroxyalkyl, a C1-C6 alkoxy, a sulfonyloxy, carboxyalkyl, a C1-C6 sulfonyloxyalkyl, or arylcarbonyl, and any combination thereof.
Where R5 or R6 is a heteroaryl substituent, the heteroaryl substituent is optionally further substituted one or more times by a halogen, a C1-C6 haloalkyl, an amino, a nitro, a C1-C6 alkyl, a C1-C6 hydroxyalkyl, a C1-C6 alkoxy, aryl, hydroxyl, carboxyl, sulfonyloxy, carboxyalkyl, a C1-C6 sulfonyloxyalkyl, a sulfonamide, a C1-C6 alkylcarbonyl, an arylcarbonyl, and any combination thereof.
In one aspect of the invention, the R5 and R6 moieties are independently each an aryl that is optionally further substituted by halogen, C1-C6 alkyl, C1-C6 perfluoroalkyl, hydroxy, C1-C6 alkoxy, or NO2. In another aspect of the invention, the R5 and R6 moieties are each phenyl or phenyl substituted one or more times by fluorine.
Either J and K, considered in combination, or K and L, considered in combination, are —CH2— and form a three-membered cyclopropane ring fused to the cyclopentane ring of the formula. Where J and K, in combination, are —CH2— the resulting compound has the formula:
Similarly, where K, and L, in combination, are —CH2— the resulting compound has the formula:
Where the substituents R1-R6 are as defined above.
In an alternative aspect, the disclosure is directed to adenosine kinase inhibitors wherein R1 is methyl or hydroxymethyl, R2 and R3 are independently hydrogen or methyl, R4 is hydrogen, and each of R5 and R6 is phenyl or substituted phenyl.
The adenosine kinase inhibitors of the present disclosure may alternatively be selected from compounds having the following formulae:
In some aspects, the disclosed adenosine kinase inhibitors may be incorporated into a pharmaceutical composition. That is, the disclosed inhibitors may be combined with one or more additional substances to facilitate the administration of the adenosine kinase inhibitors to a subject. For example, the adenosine kinase inhibitors may be present in a formulation that further includes one or more pharmaceutically acceptable carriers, excipients, preservatives, and/or diluents.
Pharmaceutically acceptable excipients may be selected to confer a therapeutic enhancement on the selected adenosine kinase inhibitor in its final dosage form, such as by facilitating drug absorption, reducing viscosity, or enhancing solubility, among other effects. Alternatively or in addition, excipients may possess utility for the manufacturing process, such as to aid in the handling of the adenosine kinase inhibitor for example by facilitating powder flowability or non-stick properties, aiding in vitro stability such as by preventing or minimizing denaturation or aggregation over an expected shelf life. The selection of appropriate excipients may also depend upon the route of administration and the dosage form.
The composition may be administered to a subject using any medically acceptable and effective technique. For example, the adenosine kinase inhibitor may be administered intravenously, orally, transdermally, subcutaneously, mucosally, intramuscularly, intranasally, intrapulmonary, parenterally, intrarectally or topically, without limitation. The disclosed pharmaceutical composition may be formulated to be fully compatible with a selected means of administering.
The adenosine kinase inhibitors of the present disclosure may be particularly useful when formulated so as to readily, or at least more readily, cross the blood-brain barrier. For example, the adenosine kinase inhibitor may be conjugated to one or more small molecules that confer brain-penetrant properties onto the molecule, such as one or more selected brain-penetrant peptides. Alternatively, or in addition, the adenosine kinase inhibitor may be administered in combination with one or more additional substances to facilitate passage of the inhibitor across the blood-brain barrier.
Where the adenosine kinase inhibitors of the present disclosure exhibit limited blood-brain barrier permeability, the adenosine kinase inhibitors may be administered directly to the patient via an by intracranial injection either acutely, or chronically via an infusion pump or other delivery device.
In yet another alternative aspect of the disclosure, administration of a selected adenosine kinase inhibitor may be considered during the course of a standard surgical resection of an epileptogenic focus, in order to prevent or moderate secondary postsurgical epileptogenesis. That is, the inhibitors of the disclosure may be administered during the course of another procedure during which the brain may be accessible. In such cases, administration of the adenosine kinase inhibitors may be performed within the context of a planned surgery.
Where the adenosine kinase inhibitor is being administered to the brain during a procedure, the inhibitor may be delivered by placing a drug-delivery substance in contact with the desired brain tissue, for example into a resectional cavity. Any appropriate drug-delivery substance may be used, for example such as a polymer that is a synthetic polymer, natural or artificial silk, and the like configured to release the selected adenosine kinase inhibitor over a desired time frame, such as days or even weeks. Administration of the adenosine kinase inhibitors via contact with a drug-delivery substance may permit the delivery of the inhibitor to a specific target area in the brain.
The present disclosure may also be directed to a method of preventing epilepsy and/or preventing its progression. The method includes administering an effective amount of the adenosine kinase inhibitors of the present disclosure to a subject in need thereof. An effective amount is that amount sufficient to produce a measurable decrease in the progression of epilepsy, or symptoms associated with epilepsy, in the subject. The method may include a method of preventing the recurrence of epilepsy in a patient by administering an adenosine kinase inhibitor of the present disclosure. Alternatively or in addition, the method may include inhibiting or suppressing epileptic seizures in a patient.
The present disclosure may additionally include methods of treating a disorder in a patient that may be characterized by pathological overexpression of adenosine kinase, such as for example Parkinson's disease, Alzheimer's disease, or cancer. Such methods may include administering an effective amount of the adenosine kinase inhibitors of the present disclosure to a subject sufficient to produce a measurable decrease in the symptoms associated with the disorder.
Discussion
The application of AdK inhibitors to CNS disorders such as epilepsy and chronic pain experienced a hiatus during the past decade, due to possible side effects of the known inhibitors. Critical gaps to be addressed are isoform selectivity (cytoplasmic versus nuclear AdK), cell-type selectivity (neuron versus astrocyte), and trans-membrane transport. Thus, there is a need for a new class of inhibitors with enhanced selectivities, such as nucleosides that have a major distinction from the known riboside inhibitors, such as 1a and 2.
An alternative ribose-like ring system, e.g. two isomeric methanocarba pseudoribose substitutions, has been utilized in nucleoside derivatives to demonstrate that inhibition of AdK is compatible with these major structural changes. By virtue of the bridged bicycloaliphatic ring, a rigid (S) 3′-exo or (N) 2′-exo envelope conformation is permanently locked in these nucleoside analogues. The most effective inhibitors, (S)-methanocarba derivatives 34, 38a, 38b and 38c and (N)-methanocarba derivative 55 were approximately two-fold less potent than the reference compound 2. The apparent discrepancy for the IC50 of 2 in our study of 48 nM, compared to its previously reported sub-nM IC50, is because of the use of a different assay system. The previous assay used was based on a radioactive assay using adenosine as a substrate, and ours is a commercial spectrophotometric assay based on inosine as substrate.
The presence of a p-F substitution in either the 5-phenyl 38b or 4-phenylamino 38c ring in the (S)-methanocarba series maintained the inhibitory potency of analogue 38a. We synthesized and compared potencies of two pairs of corresponding (S) and (N)-methanocarba 4′-CH2OH derivatives. We conclude that both conformations maintain potency at hAdK and can provide a path to novel nucleoside inhibitors. 5-Iodo analogues 32 (S) and 57 (N) displayed comparable potency, and the pair of more potent 4-phenylamino-5-phenyl analogues 34 (S) and 55 (N) also displayed similar IC50 values. This conclusion was consistent with X-ray structures of known inhibitors bound to hAdK32, and with molecular docking and MD analyses of several of the present derivatives.
A similar attempt to constrain analogues of 2′-deoxynucleoside inhibitors of adenosine deaminase (ADA) using the [3.1.0]bicyclohexane ring system failed. Although the enzyme preferred the (N) methanocarba nucleoside over the (S), the relative rate of deamination was ˜100-fold lower than adenosine, which suggested a possible role of the 4′-oxygen atom of native ribose in an anomeric effect to assist hydrolysis by ADA. In (S)-methanocarba nucleosides, the syn-conformation of the pseudoglycosidic bond is thought to be more stable than the anti-conformation, but in the X-ray structure of hAdK complexes, the nucleoside anti-conformation is present. The analysis of MD simulation starting from the X-ray complex suggests that the anti conformation suits at best the electrostatic potential distribution of the enzyme active site, featuring a highly hydrophobic cavity that hosts the purine core next to a region full of charged residues that anchors the ribose ring.
As far as the conformational preference of the enzyme is concerned, the active site of AdK appears to be highly flexible, as both (S)-methanocarba (C3′-exo, P=198°) compound 34 and its (N)-methanocarba (C2′-exo, P=342°) analogue 55 are equipotent inhibitors. It has to be noted that the synthesized locked conformers deviate by ±36° from the (N) and (S) conformations of the natural ribose counterparts. Interestingly, the near equipotency of (N) and (S) methanocarba isomers did not apply to conformationally locked analogues of 1a lacking the phenyl moieties. (S)-Conformers 30 and 49 were weak inhibitors of hAdK, while the inhibitory potencies of 1a and its (N)-methanocarba counterpart 57 were comparable. A possible explanation is provided by the modeling results: the bulky N6,C7-diphenyl substituents do not fit into the hAdK closed form and, as emerged from MD simulations, induced the opening of the small lid domain. The enzyme open conformation is expected to exhibit a higher plasticity with respect to closed conformation to which smaller 1a mimics are predicted to bind. As a consequence, this enzyme conformation could accommodate the pseudo-sugar ring locked in both the (S) and (N) conformations. Thus, hAdK might indeed prefer the (N)-conformer as inhibitor in the closed/catalytic-phase of the enzyme.
Thus, we have discovered novel high potency inhibitors of hAdK, which can now be evaluated in vivo, for example in effects on DNA methylation, in comparison to riboside inhibitors such as 1a. Although nucleosides often have low entry into the CNS, the slightly greater hydrophobicity of the (S)-methanocarba derivatives (c Log P of 49 is −0.11; tPSA is 120 Å) compared to ribosides (c Log P of the tetrahydrofuryl equivalent of 49 is −1.15; tPSA is 130 Å) might be beneficial for crossing the blood brain barrier. Furthermore, there remains the possibility that the specificity for AdK is enhanced in these (S)-methanocarba derivatives, which is crucial for avoiding possible side effects already noted for known AdK inhibitors. Although we have shown the feasibility of using this ribose conformational constraint in AdK inhibitors, there remains room for structural optimization to improve the inhibitory potency, considering that some of the known inhibitors achieve sub-nM affinity.
The interaction of these (S)-methanocarba analogues with nucleoside transporters that are relevant to adenosine derivatives, such as ENT1 and CNT2, remains to be characterized. The ability to serve as substrate or inhibitor of nucleoside transporters could affect the biodistribution or availability of the compounds in vivo. An analogue of the potent ENT1 inhibitor S-(4-nitrobenzyl)-thioinosine containing the opposite ring twist conformation ((N) methanocarba) was shown to inhibit ENT1, and other fixed conformations of 2′-deoxynucleosides were evaluated. (S)-Methanocarba 2′-deoxyadenosine inhibited both ENTs and CNTs, although less potently than 2′-deoxyadenosine.
The bioavailability and the in vivo activity of these inhibitors remain to be determined. Other nucleoside derivatives were protective in seizure models when administered peripherally. For example, the Br analogue of carbocyclic nucleoside 2 was found to be orally active in vivo in models of pain and inflammation. 5′-Deoxynucleoside analogues of 1a were noted to be more potent in vivo than 5′-amino analogues, possibly because of enhanced passage across the blood-brain barrier by virtue of being less polar. Among the new (S)-methanocarba AdK inhibitors, 5′-deoxy analogue 38a and especially the fluoro analogues 38b and 38c appear to be the least polar, based on their tendency to dissolve in organic solvents.
It is possible that transient treatment with AdK inhibitors would have long-lasting therapeutic benefits for treatment of CNS disorders, not only by raising the basal level of AR activation but also through epigenetic reprogramming. A high level of adenosine in the brain drives the enzymatic equilibrium in the presence of S-adenosylhomocysteine (SAH) hydrolase in the direction of increased SAH formation. SAH in turn inhibits DNA methyltransferases through product inhibition. Because the epigenetic effects related to changes in the DNA methylation status would persist, it might be possible to reduce the duration of drug administration, thus avoiding toxicities associated with prolonged, chronic dosing. A transient dosing regimen might also avoid possible side effects, such as liver toxicity. Compound 38a was submitted to the Psychoactive Drug Screening Program (PDSP) for screening at 45 receptors, channels and transporters. It was found to inhibit radioligand binding at the human 5HT7 (serotonin) receptor with a Ki value of 0.71 μM and did not substantially inhibit binding at any of the other off-target sites examined (<50% inhibition at 10 μM). Also, 38a was found to be inactive (10 μM) as agonist or antagonist at human P2Y1, P2Y2, P2Y4, and P2Y11Rs (calcium transients) expressed in 1321N1 astrocytoma cells and protease-activated receptor (PAR)1 expressed in mouse KOLF cells. Nevertheless, the offtarget interactions of the present set of compounds would have to be examined more extensively.
In conclusion, the development of novel AdK inhibitors, by virtue of their ability to raise the level of endogenous adenosine, particularly in disease states, remains of interest for the potential treatment of seizures and neurodegenerative and inflammatory conditions. We demonstrated that the class of constrained bicyclic ribonucleoside analogues retain inhibitory activity at this enzyme and are amenable to structural modification to enhance the potency. The SAR for ring-constrained analogues roughly parallels the SAR determined previously for ribosides, but analogues with aryl groups at the 7-deaza and N6 positions of adenine are greatly favored, with (S)≈(N). We determined that the (S) conformation permits a range of substitutions, but amino derivatives 45 and 49 were much less potent than expected from the ribose equivalents. However, a difference between the (N) and (S)-methanocarba series is that the methanocarba equivalent of reference riboside 1a is more potent in the (N) than in the (S) series. We have identified compounds 34, 38a, 38b, 38c, and 55 as hAdK inhibitors with IC50 values of ˜100 nM. The successful docking of selected members of this series in the enzyme structure suggests that a structure-based design approach for further enhancement is possible. Although we have not yet explored all of the potential off-target effects and adenosine-receptor related side effects of this structural class, it is possible that the novel non-ribose ring system will provide a cleaner pharmacological profile. The potent AdK inhibitors in this study are now ready for further tests in animal models of epilepsy and its development.
Synthesis of Adenosine Kinase Inhibitors
The intermediate 24 containing the (S)-methanocarba ring with 1′-amino functionalization was required as an intermediate for the target compounds (see the reaction scheme of
Using the (S)-methanocarba intermediate 24 as a precursor, analogues of known AdK nucleoside inhibitors were prepared. The 7-deazaadenine core was constructed by reacting 24 with symmetrical dichloropyrimidine bearing an acetaldehyde moiety (25), which on iodination using NBS followed by the removal of protecting groups in aqueous trifluoroacetic acid resulted in 28. Similarly, the aminolysis of 27 and deprotection rendered 30, the (S)-methanocarba analogue of 1a, in moderate yield.
Substitution of chlorine on the 6-position of 7-deazapurine using aniline and sodium acetate achieved a complete reaction, but the use of a stronger base i.e. potassium tert-butoxide at lower temperature produced N6-phenyl derivatives 31a and 31b in increased yields. A Suzuki reaction involving arylboronic acids and 31a,b, followed by the removal of a silyl protecting group gave 35a-c, which were subjected to Barton-McCombie deoxygenation to yield 5′-deoxy compounds 37a-c. Removal of the protecting groups from 31, 33 and 37a-c gave 32, 34 and 38a-c, respectively, in low to moderate yields. Compounds 38b and 38c differ from 38a only in the presence of a fluorine atom in the p-position of either the 4-phenyl-amino or 5-phenyl ring, respectively.
In an attempt to synthesize 5′-azido compounds (see
To arrive at 49 from 39, a Curtius rearrangement strategy was adapted, and to realize this, the 4′-hydroxylmethyl group in 39 was converted to the corresponding carboxylic acid 46 employing a TEMPO-BAIB oxidation in aqueous acetonitrile. Initial efforts to prepare the Boc-protected analog of urethane 48 from 46 using DPPA and tert-butanol failed, but instead, after deprotection of the isopropylidine group, resulted in dimer 47. However, a similar reaction with benzyl alcohol formed Cbz-protected compound 48, which on deprotection using 33% HBr in acetic acid provided the desired compound 49, as a minor product, and 50 as the major product. Unfortunately, an attempt to convert 50 to the desired compound 49 using NIS in DMF at elevated temperature did not materialize.
The synthesis of several corresponding (N)-methanocarba analogues was performed as shown in
Biochemical Evaluation of Adenosine Kinase Inhibitors
The nucleoside derivatives were tested for inhibitory activity at hAdK using a commercial, nonisotopic assay for the measurement of hAdK kinetic activity based on continuous monitoring of enzymatic activity at 340 nm (appearance of reduced nicotinamide-adenine dinucleotide from a coupled enzymatic reaction). The hAdK-catalyzed phosphorylation of inosine to inosine 5′-monophosphate is coupled to the inosine 5′-monophosphate dehydrogenase-dependent oxidation of the product. Thus, the assay was performed in the presence of dithiothreitol, oxidized nicotinamide-adenine dinucleotide (NAD, 2.5 mM) and ATP (2.75 mM) as cofactors and inosine (2.5 mM) as substrate. hAdK activity was continuously measured via absorption at 340 nm for four hours where measurements were made at five min intervals; however, for graphing and analysis purposes we focused on the first 40 min of activity, which is mostly linear, for determining slope activity as percent of AdK activity without the addition of any inhibitor (See
abased on slope of the linear portion (0-40 min interval) of the activity plot of hAdK, compared to control with DMSO vehicle.
Because the 5′-OH derivative 30 was a poor inhibitor of hAdK, we hypothesized that it might serve as a substrate for hAdK, unlike the corresponding riboside 1a, which is not primarily a substrate. The Km of adenosine as substrate of hAdK is 700 nM, and the nucleoside analogue concentrations that we used in inhibition experiments far exceeded that concentration. By comparison in another kinase system, (S)-methanocarbathymidine, but not (N)-methanocarbathymidine, is a good substrate for herpes simplex virus type 1 thymidine kinase. Moreover, (S)-methanocarbathymidine is not a substrate for human cytosolic thymidine kinase isoenzyme 1. Thus, it was important to probe the substrate qualities of compound 30, (S)-methanocarbaadenosine derivative, at hAdK. Therefore, we performed three different assays under similar conditions: first, with substrate in the presence of 1 μM 30; second, without substrate in the presence of 1 μM 30; and third as a control, without 30. After 4 h incubation, the resulting mixtures were studied using LC-MS in negative and positive mode (Supporting information), which could identify and qualitatively measure the 7 levels of nucleosides and their phosphorylated products. In the first and last cases, we detected the final enzymatic product, i.e. xanthosine-5′-phosphate (MW 364+1). However in the first two cases, we found no evidence from the LC-MS analysis that 30 serves as a substrate of hAdK. There was a prominent signal (MW 402+1) from unchanged compound 30, and no detectable or prominent signal of the corresponding 5′-phosphate (MW 482±1).
Molecular Modeling
The selection of novel AdK inhibitors was informed using molecular modeling studies. To date, three X-ray structures of the hAdK in complex with both nucleoside and nonnucleoside inhibitors have been solved, which revealed at least two different enzyme conformations. A closed form was observed for the complexes of hAdK with 1c (PDB ID: 2I6A) and 7-ethynyl-7-deazaadenosine (PDB ID: 4O1L), but the enzyme adopted an open conformation in the complex with a bulkier alkynylpyrimidine inhibitor (PDB ID: 2I6B). Given the structural similarity of our compounds to 1c, we used the corresponding co-crystallized structure as macromolecular starting point of our analysis.
MD Simulation of the X-Ray Complex.
In a first instance, we subjected the experimentally determined complex (PDB ID: 2I6A) to 30 ns of all atom Molecular Dynamics (MD) simulation, in order to identify the residues mostly involved in the interaction and to explore in detail the conformational space of the inhibitor. In the starting structure, 1c bound with the glycosidic bond (χ) in the anti conformation (χ=−134.7°) and the sugar moiety in the C1′-exo conformation (P=125.3°). The analysis of the trajectory revealed that the anti conformation was retained throughout the simulation, while the pseudo-sugar ring explored different conformational states. The anti conformation of the glycosidic bond seemed to be compatible with the charge distribution of the residues surrounding the enzyme active site. Indeed, the inhibitor established persistent H-bond interactions with negatively charged residues through the C2′ and C3′ hydroxyl groups and a stable π-π stacking interaction with Phe170 through the purine core. Concerning the ribose ring conformations, the starting C1′-exo conformation featuring a bidentate H-bond interaction between the C2′ and C3′ hydroxyl groups and the sidechain of Asp18 was the most favorable in terms of ligand-protein interaction energy (IE) during the simulation. However, after approximately 12 ns of MD simulations, the H-bond network was lost, and the pseudo-sugar ring adopted a C2′-endo (S) conformation (P=156.7°, Figure S2ii) with the C2′ and C3′ hydroxyl groups interacting with the Asp18 sidechain and water molecules, respectively. At the end of the simulation, the ring adopted a less favorable—in terms of IE—C3′-endo (N) conformation (P=36.4°, Figure S2iii) that was accompanied by a rotameric switch of the Asp18 sidechain to establish a bidentate H-bond interaction with Arg132 while still interacting with the C3′ hydroxyl group (data not shown). To assess whether this conformation was persistent, we restarted the simulation for another 30 ns in which the inhibitor remained in the (N) conformation. These results prompted us to consider nucleoside inhibitors constrained by the methanocarba ring system in the (N) as well as the (S) conformation. We therefore analyzed from a molecular point of view the comparison between two selected isomers, namely 34 and 55.
Docking of Selected Methanocarba-Nucleoside Derivatives.
The compounds were docked into the closed form of hAdK by using the Induced Fit Docking (IFD) procedure, because a preliminary attempt to dock the structures into the rigid enzyme in the closed form failed. In both docking poses (see
MD Simulations of Methanocarba-Nucleoside hAdK Complexes.
The docking poses described above were subjected to 30 ns of all-atom MD simulation, and the corresponding IE profiles are reported in
hAdK Conformation During MD Simulations.
As mentioned above, initial attempts to dock the methanocarba ligands in the closed conformation of the enzyme failed. We therefore resorted to an IFD protocol to obtain initial docking poses that we subsequently subjected to MD simulation. The analysis of the trajectories revealed that after a few ns of simulation, in both cases the opening of the small lid domain occurred. Superimposition of MD average protein structures with the enzyme in the closed (PDB ID: 2I6A) and open (PDB ID: 2I6B) conformation, revealed that while the enzyme remained in the closed conformation for the 1c-hAdK complex, in the complexes with 34 and 55 the enzyme approached an open conformation. In particular, in the MD average structures the small lid domain was rotated outward by about 24° with respect to the large domain for both the 34-hAdK and 55-hAdK complexes (comparison with the enzyme starting structure obtained after IFD), while the rotation angle was approximately 9° for the 1c-hAdK complex. The preference for an open enzyme conformation as well as the rotation angle of the small lid domain during MD simulations of nucleoside inhibitors agreed with modeling studies reported by other authors.
Epigenetic Activity in ADK-L Cells.
As a secondary screening tool we engineered BHK cell lines that stably overexpress either the nuclear isoform ADK-L or the cytoplasmic isoform ADK-S in an ADK-null background. We show that overexpression of ADK-L results in >50% increase in baseline DNA methylation (see
Next, we tested whether known ADK inhibitors, as well as compound 55 and compound 38A differ in the specificity of ADK-L vs. ADK-S.
ADK-L cells were treated for 24 hours with vehicle or the ADK inhibitors 5-iodotubercidin (5-ITU, 26 nM), ABT-702 (1.7 nM), A-134974 (60 μM), compound 55 (26 nM), or compound 38a (26 nM). The in vitro IC50 for each conventional ADK inhibitor was selected as the treatment dose. Compounds 55 and 38a were used at equimolar concentrations to 5-ITU. Purified genomic DNA (100 ng) was run on a 5-methylcytosine (5 mC) ELISA. Data shown in
No Off-Target Activity in Adk-Null Cells.
To test for nonspecific activities of ADK inhibitors on DNA methylation, we tested our set of inhibitors on ADK-null cells, which are hypomethylated and can be used to identify treatments that increase DNA methylation (whereas ADK-L cells display a hypermethylated ceiling effect). The results are shown in
As shown in
Epigenetic activity in the brain. Swiss Webster mice received 3 i.p. doses of vehicle, 5-ITU (3.1 mg/kg) or compound 38a (3.1 mg/kg) once every 12 hours. Tissue was harvested 1 hour after the 3rd dose.
As shown in
As shown in
Side effects were indexed by mouse activity in an open field for 20 minutes following a single i.p. injection of vehicle, 5-ITU or compound 38a. As shown in
When injected systemically into mice, compound 38A (3.1 mg/kg i.p. tid) reduced DNMT activity and DNA methylation in the brain (
Summary and Safety Considerations.
As determined in our primary assay compound 38A has an IC50 value for ADK<100 nM which is superior to 5-ITU. As determined in our secondary assay compound 38A reduces DNA methylation specifically in ADK-L cells without any off-target effects in ADK-Null or ADK-S cells. As deter-mined in our tertiary assay a systemic dose of compound 38A (3.1 mg/kg, i.p. tid) is bioactive in the brain and reduces DNA methylation in the hippocampus.
In addition, the following arguments support general safety of the proposed therapeutic methods:
The following series of paragraphs, some or all of which may be alphanumerically designated for clarity and efficiency, describe additional aspects and features of the adenosine kinase inhibitors of the present disclosure. Each of these paragraphs can be combined with one or more other paragraphs, and/or with disclosure from elsewhere in this application, including the Appendices, in any suitable manner. Some of the paragraphs below expressly refer to and further limit other paragraphs, providing without limitation examples of some of the suitable combinations.
A1. An adenosine kinase inhibitor having the formula:
wherein J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—;
R1 is —NH2, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl;
R2 and R3 are independently C1-C6 alkyl;
R4 is hydrogen, or C1-C6 alkyl; and
R5 and R6 are independently aryl that is optionally further substituted by C1-C6 alkyl, hydroxy, C1-C6 alkoxy, or NO2.
A2. The adenosine kinase inhibitor of paragraph A1, having the formula:
A3. The adenosine kinase inhibitor of paragraph A1, having the formula:
A4. The adenosine kinase inhibitor of paragraph A1, wherein R1 is methyl or hydroxymethyl, R2 and R3 are independently hydrogen or methyl; R4 is hydrogen; and each of R5 and R6 is phenyl or substituted phenyl.
A5. The adenosine kinase inhibitor of paragraph A1, wherein each of R5 and R6 is phenyl or phenyl substituted by fluorine.
A6. The adenosine kinase inhibitor of paragraph A1, having the formula:
A7. The adenosine kinase inhibitor of paragraph A1, having the formula:
A8. The adenosine kinase inhibitor of paragraph A1, having the formula:
A9. The adenosine kinase inhibitor of paragraph A1, having the formula:
A10. The adenosine kinase inhibitor of paragraph A1, having the formula:
B1. A pharmaceutical composition, comprising an adenosine kinase inhibitor having the formula:
wherein J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—;
R1 is —NH2, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl;
R2 and R3 are independently C1-C6 alkyl;
R4 is hydrogen, or C1-C6 alkyl; and
R5 and R6 are independently aryl that is optionally further substituted by C1-C6 alkyl, hydroxy, C1-C6 alkoxy, or NO2.
B2. The pharmaceutical composition of paragraph B1, further comprising one or more pharmaceutically acceptable carriers, excipients, preservatives, and/or diluents.
C1. A method of inhibiting seizures in a patient, comprising: administering an effective amount of an adenosine kinase inhibitor having the formula
wherein J and K, considered in combination, are —CH2—, or K and L, considered in combination, are —CH2—;
R1 is —NH2, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 hydroxyalkyl;
R2 and R3 are independently C1-C6 alkyl;
R4 is hydrogen, or C1-C6 alkyl; and
R5 and R6 are independently aryl that is optionally further substituted by C1-C6 alkyl, hydroxy, C1-C6 alkoxy, or NO2.
C2. The method of paragraph C1, wherein administering the adenosine kinase inhibitor includes administering the adenosine kinase inhibitor intravenously, orally, transdermally, subcutaneously, mucosally, intramuscularly, intranasally, intrapulmonary, parenterally, intrarectally or topically.
Although the present invention has been shown and described with reference to the foregoing operational principles and preferred embodiments, it will be apparent to those skilled in the art that various changes in form and detail may be made without departing from the spirit and scope of the invention. The present invention is intended to embrace all such alternatives, modifications and variances that fall within the scope of the appended claims.
It is believed that the disclosure set forth above encompasses multiple distinct embodiments with independent utility. While each of these embodiments has been disclosed in its preferred form, the specific aspects as disclosed and illustrated herein are not to be considered in a limiting sense as numerous variations are possible. The subject matter of the inventions includes all novel and non-obvious combinations and subcombinations of the various elements, features, functions and/or properties disclosed herein. Similarly, where the claims recite “a” or “a first” element or the equivalent thereof, such claims should be understood to include incorporation of one or more such elements, neither requiring nor excluding two or more such elements.
Various combinations and subcombinations of features, functions, elements, and/or properties may be claimed through presentation of new claims in a related application. Such new claims, whether they are directed to a different invention or directed to the same invention, whether different, broader, narrower or equal in scope to the original claims, are also regarded as included within the subject matter of the present disclosure.
This application is based upon and claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 62/342,854, filed May 27, 2016, which is incorporated herein by reference in its entirety for all purposes.
One or more inventions disclosed herein was made with Government support under NIDDK Intramural Research Program, Contract No. ZIA DK031117. The Government has certain rights in said invention or inventions.
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| Number | Date | Country | |
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| 20170340640 A1 | Nov 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62342854 | May 2016 | US |
