Discovery of potent and selective inhibitors of calmodulin-dependent kinase II (CaMKII).

We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/ß). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat F = 73%) and IV pharmacological studies.

Discovery of Dihydrobenzoxazepinone (GS-6615) Late Sodium Current Inhibitor (Late INai), a Phase II Agent with Demonstrated Preclinical Anti-Ischemic and Antiarrhythmic Properties.

Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

The novel late Na+ current inhibitor, GS-6615 (eleclazine) and its anti-arrhythmic effects in rabbit isolated heart preparations.

BACKGROUND AND PURPOSE: Enhanced late Na+ current (late INa ) in the myocardium is pro-arrhythmic. Inhibition of this current is a promising strategy to stabilize ventricular repolarization and suppress arrhythmias. Here, we describe GS-6615, a selective inhibitor of late INa , already in clinical development for the treatment of long QT syndrome 3 (LQT3). EXPERIMENTAL APPROACH: The effects of GS-6615 to inhibit late INa , versus other ion currents to shorten the ventricular action potential duration (APD), monophasic APD (MAPD) and QT interval, and decrease to the incidence of ventricular arrhythmias was determined in rabbit cardiac preparations. To mimic the electrical phenotype of LQT3, late INa was increased using the sea anemone toxin (ATX-II). KEY RESULTS: GS-6615 inhibited ATX-II enhanced late INa in ventricular myocytes (IC50  = 0.7 µM), shortened the ATX-II induced prolongation of APD, MAPD, QT interval, and decreased spatiotemporal dispersion of repolarization and ventricular arrhythmias. Inhibition by GS-6615 of ATX-II enhanced late INa was strongly correlated with shortening of myocyte APD and isolated heart MAPD (R2  = 0.94 and 0.98 respectively). In contrast to flecainide, GS-6615 had the minimal effects on peak INa . GS-6615 did not decrease the maximal upstroke velocity of the action potential (Vmax) nor widen QRS intervals. CONCLUSIONS AND IMPLICATIONS: GS-6615 was a selective inhibitor of late INa , stabilizes the ventricular repolarization and suppresses arrhythmias in a model of LQT3. The concentrations at which the electrophysiological effects of GS-6615 were observed are comparable to plasma levels associated with QTc shortening in patients with LQT3, indicating that these effects are clinically relevant.

Discovery of triazolopyridinone GS-462808, a late sodium current inhibitor (Late INai) of the cardiac Nav1.5 channel with improved efficacy and potency relative to ranolazine.

Previously we disclosed the discovery of potent Late INa current inhibitor 2 (GS-458967, IC50 of 333nM) that has a good separation of late versus peak Nav1.5 current, but did not have a favorable CNS safety window due to high brain penetration (3-fold higher partitioning into brain vs plasma) coupled with potent inhibition of brain sodium channel isoforms (Nav1.1, 1.2, 1.3). We increased the polar surface area from 50 to 84Å(2) by adding a carbonyl to the core and an oxadiazole ring resulting in 3 GS-462808 that had lower brain penetration and serendipitously lower activity at the brain isoforms. Compound 3 has an improved CNS window (>20 rat and dog) relative to 2, and improved anti-ischemic potency relative to ranolazine. The development of 3 was not pursued due to liver lesions in 7day rat toxicology studies.

Discovery of triazolopyridine GS-458967, a late sodium current inhibitor (Late INai) of the cardiac NaV 1.5 channel with improved efficacy and potency relative to ranolazine.

We started with a medium throughput screen of heterocyclic compounds without basic amine groups to avoid hERG and ß-blocker activity and identified [1,2,4]triazolo[4,3-a]pyridine as an early lead. Optimization of substituents for Late INa current inhibition and lack of Peak INa inhibition led to the discovery of 4h (GS-458967) with improved anti-arrhythmic activity relative to ranolazine. Unfortunately, 4h demonstrated use dependent block across the sodium isoforms including the central and peripheral nervous system isoforms that is consistent with its low therapeutic index (approximately 5-fold in rat, 3-fold in dog). Compound 4h represents our initial foray into a 2nd generation Late INa inhibitor program and is an important proof-of-concept compound. We will provide additional reports on addressing the CNS challenge in a follow-up communication.

A1 adenosine receptor antagonists, agonists, and allosteric enhancers.

Intense efforts of many pharmaceutical companies and academicians in the A(1) adenosine receptor (AR) field have led to the discovery of clinical candidates that are antagonists, agonists, and allosteric enhancers. The A(1)AR antagonists currently in clinical development are KW3902, BG9928, and SLV320. All three have high affinity for the human (h) A(1)AR subtype (hA(1) K (i) < 10 nM), > 200-fold selectivity over the hA(2A) subtype, and demonstrate renal protective effects in multiple animal models of disease and pharmacologic effects in human subjects. In the A(1)AR agonist area, clinical candidates have been discovered for the following conditions: atrial arrhythmias (tecadenoson, selodenoson and PJ-875); Type II diabetes and insulin sensitizing agents (GR79236, ARA, RPR-749, and CVT-3619); and angina (BAY 68-4986). The challenges associated with the development of any A(1)AR agonist are to obtain tissue-specific effects but avoid off-target tissue side effects and A(1)AR desensitization leading to tachyphylaxis. For the IV antiarrhythmic agents that act as ventricular rate control agents, a selective response can be accomplished by careful IV dosing paradigms. The treatment of type II diabetes using A(1)AR agonists in the clinic has met with limited success due to cardiovascular side effects and a well-defined desensitization of full agonists in human trials (GR79236, ARA, and RPR 749). However, new partial A(1)AR agonists are in development, including CVT-3619 hA(1) AR K(i) = 55nM, hA(2A:hA2B:hA(3))1,000:20, CV Therapeutics), which have the potential to provide enhanced insulin sensitivity without cardiovascular side effects and tachyphylaxis. The nonnucleosidic A(1)AR agonist BAY 68-4986 (capadenoson) represents a novel approach to angina wherein both animal studies and early human studies are promising. T-62 is an A(1)AR allosteric enhancer that is currently being evaluated in clinical trials as a potential treatment for neuropathic pain. The challenges associated with developing A(1)AR antagonists, agonists, or allosteric enhancers for therapeutic intervention are now well defined in humans. Significant progress has been made in identifying A(1)AR antagonists for the treatment of edema associated with congestive heart failure (CHF), A(1)AR agonists for the treatment of atrial arrhythmias, type II diabetes and angina, and A(1)AR allosteric enhancers for the treatment of neuropathic pain.

A1 adenosine receptor agonists and their potential therapeutic applications.

BACKGROUND: The challenges in developing any A(1) adenosine receptor (A(1)-AdoR) agonist involve having the desired effect on target tissue while avoiding side effects due to activation of A(1)-AdoR on other tissues. A(1)-AdoR de-sensitization leading to tachyphylaxis is also another challenge. OBJECTIVES: The major goal of this review is twofold: to highlight the structure affinity relationships (SAR) of A(1)-AdoR agonists, starting with initial lead compounds that were the genesis for second-generation compounds with high selectivity, affinity, and partial agonism; and to give an overview of the A(1)-AdoR agonists under development for various indications. RESULTS: Intense efforts by many pharmaceutical companies and academicians in the A(1)-AdoR agonist field have led to the discovery of clinical candidates for the following conditions: atrial arrhythmias - Tecadenoson, Selodenoson and PJ-875; type 2 diabetes (T2D) and insulin-sensitizing agents - GR79236, ARA, and CVT-3619; pain management - SDZ WAG 994, GW493838; and angina - BAY-68-4986. For the i.v. antiarrhythmic agents that act as ventricular rate control agents, a selective response can be accomplished by careful dosing paradigms. The treatment of T2D using A(1)-AdoR agonists has been met by limited success due to cardiovascular side effects and well-defined desensitization of full agonists in both animal models and human trials (GR79236 and ARA). However, new partial A(1)-AdoR agonists are in development, including CVT-3619 (hA(1)-AdoR K(i) = 55 nm, selectivity A(2A) > 200; A(2B) > 1000; A(3) > 20, CV Therapeutics), that have the potential to provide enhanced insulin sensitivity without cardiovascular side effects or tachyphylaxis. The A(1)-AdoR agonists GW493838 and GR792363 are under evaluation for pain management. The non-nucleosidic A(1)-AdoR agonist, BAY-68-4986 (Capadenoson), represents a unique approach to angina wherein both animal studies and early human studies are promising. CONCLUSION: The challenges associated with developing an A(1)-AdoR agonist for therapeutic intervention are now well defined in humans. Significant progress has been made in identifying agents for the treatment of atrial arrhythmias, T2D, and angina.

Discovery of a novel A2B adenosine receptor antagonist as a clinical candidate for chronic inflammatory airway diseases.

Recently, we have reported a series of new 1,3-symmetrically (R 1 = R 3) substituted xanthines ( 3 and 4) which have high affinity and selectivity for the human adenosine A 2B receptors (hA(2B)-AdoR). Unfortunately, this class of compounds had poor pharmacokinetic properties. This prompted us to investigate the effect of differential alkyl substitution at the N-1 and N-3 positions ( N 1-R not equal to N 3-R) on A(2B)-AdoR affinity and selectivity; we had the dual objectives of enhancing affinity and selectivity for the A(2B)-AdoR, as well as improving oral bioavailability. This effort has led to the discovery of compound 62, that displayed high affinity and selectivity for the hA(2B)-AdoR (K(i) = 22 nM). In addition, compound 62 showed high functional potency in inhibiting the accumulation of cyclic adenosine monophosphate induced by 5'- N-ethylcarboxamidoadenosine in HEK-A(2B)-AdoR and NIH3T3 cells with K(B) values of 6 and 2 nM, respectively. In a single ascending-dose phase I clinical study, compound 62 had no serious adverse events and was well tolerated.

Selective, high affinity A(2B) adenosine receptor antagonists: N-1 monosubstituted 8-(pyrazol-4-yl)xanthines.

A series of N-1 monosubstituted 8-pyrazolyl xanthines have been synthesized and evaluated for their affinity for the adenosine receptors (AdoRs). We have discovered two compounds 18 (CVT-7124) and 28 (CVT-6694) that display good affinity for the A(2B) AdoR (K(i)=6 nM and 7 nM, respectively) and greater selectivity for the human A(1), A(2A), and A(3) AdoRs (>1000-, >830-, and >1500-fold; >850-, >700-, and >1280-fold, respectively). CVT-6694 has been shown to block the release of interleukin-6 and monocyte chemotactic protein-1 from bronchial smooth muscle cells (BSMC), a process believed to be promoted by activation of A(2B) AdoR.

N6-Cycloalkyl-2-substituted adenosine derivatives as selective, high affinity adenosine A1 receptor agonists.

A series of new selective, high affinity A(1)-AdoR agonists is reported. Compound 23 that incorporated a carboxylic acid functionality in the 4-position of the pyrazole ring displayed K(iL) value of 1 nM for the A(1)-AdoR and >5000-fold selectivity over the A(3) and A(2A)-AdoRs. In addition, compound 19 that incorporated a carboxamide functionality in the 4-position of the pyrazole ring displayed subnanomolar affinity for the A(1)-AdoR (K(iL)=0.6 nM) and >600-fold selectivity over the A(3) and A(2A)-AdoRs.

Novel 1,3-disubstituted 8-(1-benzyl-1H-pyrazol-4-yl) xanthines: high affinity and selective A2B adenosine receptor antagonists.

Adenosine has been suggested to induce bronchial hyperresponsiveness in asthmatics, which is believed to be an A(2B) adenosine receptor (AdoR) mediated pathway. We hypothesize that a selective, high-affinity A(2B) AdoR antagonist may provide therapeutic benefit in the treatment of asthma. In an attempt to identify a high-affinity, selective antagonist for the A(2B) AdoR, we synthesized 8-(C-4-pyrazolyl) xanthines. Compound 22, 8-(1H-pyrazol-4-yl)-1,3-dipropyl xanthine, is a N-1 unsubstituted pyrazole derivative that has favorable binding affinity (K(i) = 9 nM) for the A(2B) AdoR, but it is only 2-fold selective versus the A(1) AdoR. Introduction of a benzyl group at the N-1-pyrazole position of 22 resulted in 19, which had moderate selectivity. The initial focus of the SAR study was on the preparation of substituted benzyl derivatives of 19 because the corresponding phenyl, phenethyl, and phenpropyl derivatives showed a decrease in A(2B) AdoR affinity and selectivity relative to 19. The preferred substitution on the phenyl ring of 19 contains an electron-withdrawing group, specifically F or CF(3) at the m-position, as in 33 and 36 respectively, increases the selectivity while retaining the affinity for the A(2B) AdoR. Exploring disubstitutions on the phenyl ring of derivatives 33 and36 led to the 2-chloro-5-trifluoromethylphenyl derivative 50, which retained the A(2B) AdoR affinity but enhanced the selectivity relative to 36. After optimization of the substitution on the 8-pyrazole xanthine, 1,3-disubstitution of the xanthine core was explored with methyl, ethyl, butyl, and isobutyl groups. In comparison to the corresponding dipropyl analogues, the smaller 1,3-dialkyl groups (methyl and ethyl) increased the A(2B) AdoR binding selectivity of the xanthine derivatives while retaining the affinity. However, the larger 1,3-dialkyl groups (isobutyl and butyl) resulted in a decrease in both A(2B) AdoR affinity and selectivity. This final SAR optimization led to the discovery of 1,3-dimethyl derivative 60, 8-(1-(3-(trifluoromethyl) benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl xanthine, a high-affinity (K(i) = 1 nM) A(2B) AdoR antagonist with high selectivity (990-, 690-, and 1,000-) for the human A(1), A(2A,) and A(3) AdoRs.

Novel 1,3-dipropyl-8-(1-heteroarylmethyl-1H-pyrazol-4-yl)-xanthine derivatives as high affinity and selective A2B adenosine receptor antagonists.

A series of new 1,3-dipropyl-8-(1-heteroarylmethyl-1H-pyrazol-4-yl)-xanthine derivatives as A(2B)-AdoR antagonists have been synthesized and evaluated for their binding affinities for the A(2B), A(1), A(2A), and A(3)-AdoRs. 8-(1-((3-phenyl-1,2,4-oxadiazol-5-yl)methyl)-1H-pyrazol-4-yl)-1,3-dipropyl-1H-purine-2,6(3H,7H)-dione (4) displayed high affinity (K(i)=1 nM) and selectivity for the A(2B)-AdoR versus A(1), A(2A), and A(3)-AdoRs (A(1)/A(2B), A(2A)/A(2B), and A(3)/A(2B) selectivity ratios of 370, 1100, and 480, respectively). The synthesis and SAR of this novel class of compounds are presented herein.

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability.

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the 'Western Portion' of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC50=380 nM rat mitochondria) with favorable PK properties (F=93%, t(1/2)=13.6h, dog).

2-Pyrazolyl-N(6)-substituted adenosine derivatives as high affinity and selective adenosine A(3) receptor agonists.

We describe the synthesis of new high affinity and selective A(3)-adenosine receptor (A(3)-AdoR) agonists. Introduction of a methyl group at the N(6)-position of the A(2A)-AdoR selective 2-pyrazolyl-adenosine analogues (Figure 2) brought about a substantial increase in the A(3)-AdoR binding affinity and selectivity. While the N(6)-desmethyl analogues 3a and 4 were inactive at the A(3)-AdoR (K(i) > 10 microM), the corresponding N(6)-methyl analogues 5 and 22 showed good binding affinity at the A(3)-AdoR (K(i) = 73 and 97 nM, respectively). Replacement of the carboxamide group in 5 with different heteroaryl groups resulted in analogues with high affinities and selectivity for the A(3)-AdoR. (2R,3S,4R)-tetrahydro-2-(hydroxymethyl)-5-(6-(methylamino)-2-(4-(pyridin-2-yl)-1H-pyrazol-1-yl)-9H-purin-9-yl)furan-3,4-diol (15, K(i) = 2 nM) displayed high selectivity for the A(3)-AdoR versus A(1)- and A(2A)-AdoRs (selectivity ratios of 1900 and >2000, respectively).

Structure-affinity relationships of 5'-aromatic ethers and 5'-aromatic sulfides as partial A1 adenosine agonists, potential supraventricular anti-arrhythmic agents.

Atrial fibrillation (AF) is the most commonly encountered sustained clinical arrhythmia with an estimated 2.3 million cases in the US (2001). A(1) adenosine receptor agonists can slow the electrical impulse propagation through the atrioventricular (AV) node (i.e., negative dromotropic effect) resulting in prolongation of the stimulus-to-His bundle (S-H) interval to potentially reduce ventricular rate. Compounds that are full agonists of the A(1) adenosine receptor can cause high grade AV block. Therefore, it is envisioned that a compound that is a partial agonist of the A(1) adenosine receptor could avoid this deleterious effect. 5(') Phenyl sulfides (e.g., 17, EC(50)=1.26 microM) and phenyl ethers (e.g., 28, EC(50)=0.2 microM) are partial agonists with respect to their AV nodal effects in guinea pig isolated hearts. Additional affinity, GTPgammaS binding data suggesting partial activity of the A(1) adenosine receptor, and PK results for 5(') modified adenosine derivatives are shown.

Novel inhibitors of fatty acid oxidation as potential metabolic modulators.

We describe the synthesis of novel inhibitors of fatty acid oxidation as potential metabolic modulators for the treatment of stable angina. Replacement of the 2H-benzo[d]1,3-dioxolene ring system in our initial lead 3 with different benzthiazoles, benzoxazoles and introducing small alkyl substituents into the piperazine ring resulted in analogues with enhanced inhibitory activity against 1-(14)[C]-palmitoyl-CoA oxidation in isolated rat heart mitochondria (6, IC(50)=70 nM; 25, IC(50)=23 nM).

New fatty acid oxidation inhibitors with increased potency lacking adverse metabolic and electrophysiological properties.

New inhibitors of palmitoylCoA oxidation were synthesized based on a structurally novel lead, CVT-3501 (1). Investigation of structure-activity relationships was conducted with respect to potency of inhibition of cardiac mitochondrial palmitoylCoA oxidation and metabolic stability. Potent and metabolically stable analogues 33, 42, and 43 were evaluated in vitro for cytochrome P450 inhibition and potentially adverse electrophysiological effects. Compound 33 was also found to have favorable pharmacokinetic properties in rat.

Structure-affinity relationships of the affinity of 2-pyrazolyl adenosine analogues for the adenosine A2A receptor.

The structure-affinity relationships of two novel 2-substituted adenosine series containing a substituted pyrazole attached at the N-1 or C-4 position for the adenosine (ADO) A2A receptor are described. Compounds in the 2-(N-1-pyrazolyl) adenosine series IV provided the highest affinity for the ADO A2A receptor as compared to the 2-(C-4-pyrazolyl) series V. The main structural differences between the two series point to the N-1 nitrogen of series IV imparting more favorable binding interactions with the receptor than those of series V.