Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer.

In this article we describe the preclinical characterization of 5-(3-chlorophenylamino) benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first orally available small molecule inhibitor of protein CK2 in clinical trials for cancer. CX-4945 was optimized as an ATP-competitive inhibitor of the CK2 holoenzyme (Ki = 0.38 nM). Iterative synthesis and screening of analogs, guided by molecular modeling, led to the discovery of orally available CX-4945. CK2 promotes signaling in the Akt pathway and CX-4945 suppresses the phosphorylation of Akt as well as other key downstream mediators of the pathway such as p21. CX-4945 induced apoptosis and caused cell cycle arrest in cancer cells in vitro. CX-4945 exhibited a dose-dependent antitumor activity in a xenograft model of PC3 prostate cancer model and was well tolerated. In vivo time-dependent reduction in the phosphorylation of the biomarker p21 at T145 was observed by immunohistochemistry. Inhibition of the newly validated CK2 target by CX-4945 represents a fresh therapeutic strategy for cancer.

Pharmacological characterization of a novel nonpeptide antagonist of the human gonadotropin-releasing hormone receptor, NBI-42902.

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K(i) = 0.56 nm). Tritiated NBI-42902 binds with high affinity (K(d) = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca(2+) flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

Design, synthesis, in vitro, and in vivo characterization of phenylpiperazines and pyridinylpiperazines as potent and selective antagonists of the melanocortin-4 receptor.

Benzylamine and pyridinemethylamine derivatives were synthesized and characterized as potent and selective antagonists of the melanocortin-4 receptor (MC4R). These compounds were also profiled in rodents for their pharmacokinetic properties. Two compounds with diversified profiles in chemical structure, pharmacological activities, and pharmacokinetics, 10 and 12b, showed efficacy in an established murine cachexia model. For example, 12b had a K(i) value of 3.4 nM at MC4R, was more than 200-fold selective over MC3R, and had a good pharmacokinetic profile in mice, including high brain penetration. Moreover, 12b was able to stimulate food intake in the tumor-bearing mice and reverse their lean body mass loss. Our results provided further evidence that a potent and selective MC4R antagonist with appropriate pharmacokinetic properties might potentially be useful for the treatment of cancer cachexia.

Rational design, synthesis, and structure-activity relationships of aryltriazoles as novel corticotropin-releasing factor-1 receptor antagonists.

Following the discovery of the very high binding affinity of 4-anilinopyrimidines against corticotropin-releasing factor receptor-1 (CRF(1)) (e.g., 1, K(i) = 2 nM), a new series of triazoles bearing different groups has been synthesized and evaluated. The compounds were prepared by cyclizations of N-acyl-S-methylisothioureas with alkylhydrazines or by cyclizations with hydrazine followed by alkylation. While members of this series showed potent binding affinity against CRF(1) receptor, there were important differences between the different regio- (7 and 12) and stereoisomeric aryltriazoles where the R(1) or R(2) side chain in 7 has an asymmetric center. In terms of overall potency, aryltriazole analogues such as 7r bearing an N-(alpha-branched benzyl)-N-propylamino side chain were the most potent, followed by analogues such as 7a, with an N-bis(cyclopropyl)methyl-N-propylamino side chain, and analogues such as 7m, with an N-(alpha-branched aliphatic)-N-propylamino side chain. While the N-propyl group was crucial for high potency, we hypothesized that the terminal methyl mimicked the 5-methyl of pyrazolo[1,5-a]pyrimidines 3 and 4. Correlation of the low-energy conformers of compounds of type 3 and 7 generated by computational analyses was very good. The size and shape of the N-alkyl group dramatically changed the potency of the triazoles, which is in contrast to the SAR seen for bicyclic CRF(1) antagonists. In general, the S-enantiomer was much more potent than the corresponding R-isomer. Furthermore, to a limited extent in the aryltriazole series the substituent on the 5-phenyl ring changed the potency up to 9-fold. (S)-1-Methyl-3-[N-(4-fluorophenylpentyl)-N-propyl]amino-5-(2-methoxy-4-dichlorophenyl)-1H-[1,2,4]triazole [(S)-7r] showed very potent binding affinity (K(i) = 2.7 nM) to CRF(1) receptors with an IC(50) of 49 nM in a cAMP inhibition assay.

Potent, orally active corticotropin-releasing factor receptor-1 antagonists containing a tricyclic pyrrolopyridine or pyrazolopyridine core.

Two new classes of tricyclic-based corticotropin-releasing factor (CRF(1)) receptor-1 antagonists were designed by constraining known 1H-pyrrolo[2,3-b]pyridine and 1H-pyrazolo[3,4-b]pyridine ligands. Pyrrole- and pyrazole-based molecules 19g and 22a, respectively, were discovered that potently bind the recombinant CRF(1) receptor (K(i) = 3.5, 2.9 nM) and inhibit adrenocorticotropic hormone (ACTH) release from rat pituitary cell culture (IC(50) = 14, 6.8 nM). These compounds show good oral bioavailabity (F = 24%, 7.0%) and serum half-lives in rats (t(1/2) = 6.3, 12 h) and penetrate the rat brain ([brain]/[plasma] = 0.27, 0.52) but tend toward large volumes of distribution (V(D) = 38, 44 L kg(-1)) and rapid clearances (CL = 70, 43 mL min(-1) kg(-1)). When given orally, both the pyrazole and the pyrrole leads dose-dependently inhibit stress-induced ACTH release in vivo. ACTH reductions of 84-86% were observed for 30 mg kg(-1) doses.

Design and synthesis of tricyclic imidazo[4,5-b]pyridin-2-ones as corticotropin-releasing factor-1 antagonists.

The synthesis and SAR studies of tricyclic imidazo[4,5-b]pyridin-2-ones as human corticotropin-releasing factor receptor (CRF(1)) antagonists are discussed herein. Compound 16g was identified as a functional antagonist that inhibited CRF-stimulated cyclic adenosine monophosphate production and CRF-induced adrenocorticotrophic hormone release. Pharmacokinetics studies in rats showed that 16g was orally bioavailable, had good brain penetration, and had a moderate half-life. In our effort to identify CRF(1) antagonists with improved pharmacokinetics properties, 16g exhibited a favorably lower volume of distribution.

Identification of 1-arylmethyl-3- (2-aminoethyl)-5-aryluracil as novel gonadotropin-releasing hormone receptor antagonists.

Based on SAR from bicyclic GnRH antagonists such as 6-aminomethyl-7-arylpyrrolo[1,2-a]pyrimid-4-ones (1) and 2-aryl-3-aminomethylimidazolo[1,2-a]pyrimid-5-ones (2a,b), a series of novel uracil compounds (4) were derived as the GnRH antagonists. Their syntheses and initial SAR are discussed herein. This is the first time that monocycle-based GnRH receptor antagonists are reported.

Design of 2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylaminopyrazolo[1,5-a]pyrimidine (NBI 30775/R121919) and structure--activity relationships of a series of potent and orally active corticotropin-releasing factor receptor antagonists.

We have previously shown that 3-phenylpyrazolo[1,5-a]pyrimidines exemplified by 8 were potent antagonists of the human corticotropin-releasing factor-1 receptor. A series of 3-pyridylpyrazolo[1,5-a]pyrimidines 15, 25-30, 34, and 35 containing a weakly basic pyridine ring at the 3-position of the bicyclic nucleus was designed to reduce lipophilicity from the initial leads such as 7. Here, we showed that these 3-pyridyl compounds exhibited potent antagonists at the human CRF(1) receptor. Moreover, the hydrophilic and weakly basic pyridine moiety increased the water solubility of some analogues. Compound 26 h exhibited good binding affinity at the human CRF(1) receptor with a K(i) value of 3.5 nM. As a functional antagonist, it dose-dependently inhibited CRF-stimulated cAMP production in cells expressing the CRF(1) receptor (IC(50) = 50 nM), and CRF-stimulated ACTH release from cultured rat pituitary cells (IC(50) = 20 nM). 26 h had a log P value of 4.9 and water solubility of greater than 10 mg/mL. Pharmacokinetic studies in rats showed that 26 h was orally bioavailable and able to penetrate into the brain. 26 h has been demonstrated in vivo efficacy in animal behavioral models that measure anxiolytic activity. These results suggest that analogues from this series were potent CRF(1) receptor antagonists with proper physicochemical properties and good pharmacokinetic profiles. 26 h was developed into a clinical compound and exhibited efficacy in patients with major depression.

3-(2-aminoalkyl)-1-(2,6-difluorobenzyl)-5- (2-fluoro-3-methoxyphenyl)-6-methyl-uracils as orally bioavailable antagonists of the human gonadotropin releasing hormone receptor.

Uracils possessing N-3 side chains derived from various amino alcohols were designed and synthesized as potent human gonadotropin releasing hormone receptor antagonists. The compounds herein presented displayed superior metabolic stability than their predecessor molecules. Selected compounds from this series featured good oral bioavailability in mice and cynomolgus monkeys.

Synthesis and structure-activity relationships of 1-arylmethyl-5-aryl-6-methyluracils as potent gonadotropin-releasing hormone receptor antagonists.

Based on the SAR from bicyclic gonadotropin-releasing hormone (GnRH) antagonists such as 6-aminomethyl-7-aryl-pyrrolo[1,2-a]pyrimid-4-ones (5) and 2-aryl-3-aminomethyl-imidazolo[1,2-a]pyrimid-5-ones (6a,b), a series of novel uracil compounds (8) were derived as GnRH antagonists. The synthesis and SAR studies of 6-methyluracils as human GnRH receptor antagonists are discussed herein. Introduction of a small methyl substituent at the beta-position of the N3 side-chain improved the GnRH binding potency by 5-10-fold. Introduction of a methyl group of (R)-configuration at the alpha-carbon of the N-3 side-chain gave a modest improvement in binding affinity over the unsubstituted ethylene analogues. This modification enabled us to make uracil compounds without the labile 2-pyridylethyl motif on the basic nitrogen while still maintained excellent potency against the hGnRH receptor.

Discovery and SAR of 5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxylic acid (CX-4945), the first clinical stage inhibitor of protein kinase CK2 for the treatment of cancer.

Herein we chronicle the discovery of CX-4945 (25n), a first-in-class, orally bioavailable ATP-competitive inhibitor of protein kinase CK2 in clinical trials for cancer. CK2 has long been considered a prime cancer drug target because of the roles of deregulated and overexpressed CK2 in cancer-promoting prosurvival and antiapoptotic pathways. These biological properties as well as the suitability of CK2's small ATP binding site for the design of selective inhibitors, led us to fashion novel therapeutic agents for cancer. The optimization leading to 25n (K(i) = 0.38 nM) was guided by molecular modeling, suggesting a strong binding of 25n resulting from a combination of hydrophobic interactions, an ionic bridge with Lys68, and hydrogen bonding with the hinge region. 25n was found to be highly selective, orally bioavailable across species (20-51%) and efficacious in xenograft models. The discovery of 25n will allow the therapeutic targeting of CK2 in humans for the first time.

A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolites.

An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8 min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation.

In vivo pharmacological characterization of indiplon, a novel pyrazolopyrimidine sedative-hypnotic.

Indiplon (NBI 34060; N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-alpha]pyrimidin-7-yl]phenyl]acetamide), a novel pyrazolopyrimidine and high-affinity allosteric potentiator of GABA(A) receptor function, was profiled for its effects in rodents after oral administration. In mice, indiplon inhibited locomotor activity (ED(50) = 2.7 mg/kg p.o.) at doses lower than the nonbenzodiazepine hypnotics zolpidem (ED(50) = 6.1 mg/kg p.o.) and zaleplon (ED(50) = 24.6 mg/kg p.o.), a sedative effect that was reversed by the benzodiazepine site antagonist flumazenil. Indiplon inhibited retention in the mouse passive avoidance paradigm over a dose range and with a temporal profile that coincided with its sedative activity. Indiplon, zolpidem, and zaleplon were equally effective in inhibiting locomotor activity in the rat and produced dose-related deficits on the rotarod. In a rat vigilance paradigm, indiplon, zolpidem, and zaleplon produced performance deficits over a dose range consistent with their sedative effects, although indiplon alone showed no significant increase in response latency. Indiplon produced a small deficit in the delayed nonmatch to sample paradigm at a dose where sedative effects became apparent. Indiplon was active in the rat Vogel test of anxiety, but it showed only a sedative profile in the mouse open field test. The pharmacokinetic profile of indiplon in both rat and mouse was consistent with its pharmacodynamic properties and indicated a rapid T(max), short t(1/2), and excellent blood-brain barrier penetration. Therefore, indiplon has the in vivo profile of an efficacious sedative-hypnotic, in agreement with its in vitro receptor pharmacology as a high-affinity allosteric potentiator of GABA(A) receptor function, with selectivity for alpha1 subunit-containing GABA(A) receptors.