Discovery of a Novel, Highly Potent, and Selective Thieno[3,2-d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent.

In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.

p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) inhibits amyloid aggregation of mutant p53 in cancer cells.

p53 mutants can form amyloid-like structures that accumulate in cells. p53 reactivation with induction of massive apoptosis-1 (PRIMA-1) and its primary active metabolite, 2-methylene-3-quinuclidinone (MQ), can restore unfolded p53 mutants to a native conformation that induces apoptosis and activates several p53 target genes. However, whether PRIMA-1 can clear p53 aggregates is unclear. In this study, we investigated whether PRIMA-1 can restore aggregated mutant p53 to a native form. We observed that the p53 mutant protein is more sensitive to both PRIMA-1 and MQ aggregation inhibition than WT p53. The results of anti-amyloid oligomer antibody assays revealed that PRIMA-1 reverses mutant p53 aggregate accumulation in cancer cells. Size-exclusion chromatography of the lysates from mutant p53-containing breast cancer and ovarian cell lines confirmed that PRIMA-1 substantially decreases p53 aggregates. We also show that MDA-MB-231 cell lysates can "seed" aggregation of the central core domain of recombinant WT p53, corroborating the prion-like behavior of mutant p53. We also noted that this aggregation effect was inhibited by MQ and PRIMA-1. This study provides the first demonstration that PRIMA-1 can rescue amyloid-state p53 mutants, a strategy that could be further explored as a cancer treatment.

Inhibition of the glutaredoxin and thioredoxin systems and ribonucleotide reductase by mutant p53-targeting compound APR-246.

The tumor suppressor p53 is commonly inactivated in human tumors, allowing evasion of p53-dependent apoptosis and tumor progression. The small molecule APR-246 (PRIMA-1Met) can reactive mutant p53 in tumor cells and trigger cell death by apoptosis. The thioredoxin (Trx) and glutaredoxin (Grx) systems are important as antioxidants for maintaining cellular redox balance and providing electrons for thiol-dependent reactions like those catalyzed by ribonucleotide reductase and peroxiredoxins (Prxs). We show here that the Michael acceptor methylene quinuclidinone (MQ), the active form of APR-246, is a potent direct inhibitor of Trx1 and Grx1 by reacting with sulfhydryl groups in the enzymes. The inhibition of Trx1 and Grx1 by APR-246/MQ is reversible and the inhibitory efficiency is dependent on the presence of glutathione. APR-246/MQ also inhibits Trxs in mutant p53-expressing Saos-2 His-273 cells, showing modification of Trx1 and mitochondrial Trx2. Inhibition of the Trx and Grx systems leads to insufficient reducing power to deoxyribonucleotide production for DNA replication and repair and peroxiredoxin for removal of ROS. We also demonstrate that APR-246 and MQ inhibit ribonucleotide reductase (RNR) in vitro and in living cells. Our results suggest that APR-246 induces tumor cell death through both reactivations of mutant p53 and inhibition of cellular thiol-dependent redox systems, providing a novel strategy for cancer therapy.

PRIMA-1Met suppresses colorectal cancer independent of p53 by targeting MEK.

PRIMA-1Met is the methylated PRIMA-1 (p53 reactivation and induction of massive apoptosis) and could restore tumor suppressor function of mutant p53 and induce p53 dependent apoptosis in cancer cells harboring mutant p53. However, p53 independent activity of PRIMA-1Met remains elusive. Here we reported that PRIMA-1Met attenuated colorectal cancer cell growth irrespective of p53 status. Kinase profiling revealed that mitogen-activated or extracellular signal-related protein kinase (MEK) might be a potential target of PRIMA-1Met. Pull-down binding and ATP competitive assay showed that PRIMA-1Met directly bound MEK in vitro and in cells. Furthermore, the direct binding sites of PRIMA-1Met were explored by using a computational docking model. Treatment of colorectal cancer cells with PRIMA-1Met inhibited p53-independent phosphorylation of MEK, which in turn impaired anchorage-independent cell growth in vitro. Moreover, PRIMA-1Met suppressed colorectal cancer growth in xenograft mouse model by inhibiting MEK1 activity.Taken together, our findings demonstrate a novel p53-independent activity of PRIMA-1Met to inhibit MEK and suppress colorectal cancer growth.

Drug in adhesive patch of palonosetron: Effect of pressure sensitive adhesive on drug skin permeation and in vitro-in vivo correlation.

Palonosetron (PAL) is recommended for the prevention of chemotherapy-induced nausea and vomiting. The aim of this study was to develop a long-acting PAL transdermal patch to improve patient compliance. We were particularly concerned about the effect of pressure sensitive adhesives (PSAs) on PAL skin permeability. Formulation factors including PSAs, backing films and drug loadings were investigated in the in vitro skin permeation study using rabbit skin. Fourier transform infrared spectrometer study and thermal analysis were conducted to investigate the drug-PSA interaction and thermodynamic activity of PSAs, respectively. The results indicated that high drug skin permeation amount was obtained in PSA DURO-TAK(®)87-2516, which had low interaction potential with PAL and high thermodynamic activity. The optimized patch was composed of PAL of 8 %, DURO-TAK(®)87-2516 as PSA, CoTran™ 9700 as backing film and Scotchpak™ 9744 as release liner. The in vitro skin permeation amount of the optimized patch was 734.0±55.8µg/cm(2) during 3-day administration. The absolute bioavailability of the optimized patch was 43 % in rabbit and a good in vitro-in vivo correlation coefficient was obtained (R(2)=0.989). These results indicated the feasibility of PAL transdermal patch in the prevention of chemotherapy-induced nausea and vomiting.

PRIMA-1Met induces apoptosis in Waldenström's Macroglobulinemia cells independent of p53.

PRIMA-1Met has shown promising preclinical activity in various cancer types. However, its effect on Waldenström's Macroglobulinemia (WM) cells as well as its exact mechanism of action is still elusive. In this study, we evaluated the anti- tumor activity of PRIMA-1Met alone and in combination with dexamethasone or bortezomib in WM cell lines and primary samples. Treatment of WM cells with PRIMA-1Met resulted in induction of apoptosis, inhibition of migration and suppression of colony formation. Upon PRIMA-1Met treatment, p73 was upregulated and Bcl-xL was down-regulated while no significant change in expression of p53 was observed. Furthermore, siRNA knockdown of p53 in WM cell line did not influence the PRIMA-1Met-induced apoptotic response whereas silencing of p73 inhibited latter response in WM cells. Importantly, combined treatment of BCWM-1 cells with PRIMA-1Met and dexamethasone or bortezomib induced synergistic reduction in cell survival. Our study provides insights into the mechanisms of anti-WM activity of PRIMA-1Met and supports further clinical evaluation of PRIMA-1Met as a potential novel therapeutic intervention in WM.

In silico discovery of potential VEGFR-2 inhibitors from natural derivatives for anti-angiogenesis therapy.

Angiogenesis is the growth of new capillaries from existing blood vessels that supply oxygen and nutrients and provide gateways for immune surveillance. Abnormal vessel growth in term of excessive angiogenesis is a hallmark of cancer, inflammatory and eye diseases. VEGFR-2 (vascular endothelial growth factor receptor 2) dominating the process of angiogenesis has led to approval of therapeutic inhibitors and is becoming a promising target for anti-angiogenic drugs. Notwithstanding these successes, the clinical use of current VEGFR-2 blockers is more challenging than anticipated. Taking axitinib as a reference drug, in our study we found three potent VEGFR-2 inhibitors (ZINC08254217, ZINC08254138, and ZINC03838680) from natural derivatives. Each of the three inhibitors acquired a better grid score than axitinib (-62.11) when docked to VEGFR-2. Molecular dynamics simulations demonstrated that ZINC08254217- and ZINC08254138-VEGFR-2 complexes were more stable than axitinib. Similar to bind free energy for axitinib (-54.68 kcal/mol), such for ZINC03838680, ZINC08254217, and ZINC08254138 was -49.37, -43.32, and -32.73 kcal/mol respectively. These results suggested these three compounds could be candidate drugs against angiogenesis, with comparable VEGFR-2 binding affinity of axitinib. Hence findings in our study are able to provide valuable information on discovery of effective anti-angiogenesis therapy.

Prolonged inhibition of 5-HT3 receptors by palonosetron results from surface receptor inhibition rather than inducing receptor internalization.

BACKGROUND AND PURPOSE: The 5-HT3 receptor antagonist palonosetron is an important treatment for emesis and nausea during cancer therapy. Its clinical efficacy may result from its unique binding and clearance characteristics and receptor down-regulation mechanisms. We investigated the mechanisms by which palonosetron exerts its long-term inhibition of 5-HT3 receptors for a better understanding of its clinical efficacy. EXPERIMENTAL APPROACH: Cell surface receptors (recombinantly expressed 5HT3A or 5HT3AB in COS-7 cells) were monitored using [³H]granisetron binding and ELISA after exposure to palonosetron. Receptor endocytosis was investigated using immunofluorescence microscopy. KEY RESULTS: Chronic exposure to palonosetron reduced the number of available cell surface [³H]granisetron binding sites. This down-regulation was not sensitive to either low temperature or pharmacological inhibitors of endocytosis (dynasore or nystatin) suggesting that internalization did not play a role. This was corroborated by our observation that there was no change in cell surface 5-HT3 receptor levels or increase in endocytic rate. Palonosetron exhibited slow dissociation from the receptor over many hours, with a significant proportion of binding sites being occupied for at least 4 days. Furthermore, our observations suggest that chronic receptor down-regulation involved interactions with an allosteric binding site. CONCLUSIONS AND IMPLICATIONS: Palonosetron acts as a pseudo-irreversible antagonist causing prolonged inhibition of 5-HT3 receptors due to its very slow dissociation. In addition, an irreversible binding mode persists for at least 4 days. Allosteric receptor interactions appear to play a role in this phenomenon.

Loss of muscarinic and purinergic receptors in urinary bladder of rats with hydrochloric acid-induced cystitis.

OBJECTIVES: To clarify the basic mechanism involved in the pathophysiology of cystitis by characterizing the urodynamic parameters, pharmacologically relevant (muscarinic and purinergic) receptors, and the in vivo release of adenosine triphosphate (ATP) in the bladder of hydrochloric acid (HCl)-treated rats. METHODS: The muscarinic and purinergic receptors in rat tissue were measured by radioreceptor assays using (N-methyl-³H) scopolamine methyl chloride ([³H]NMS) and αß-methylene-ATP (2,8-³H) tetrasodium salt ([³H]αß-MeATP), respectively. The urodynamic parameters and ATP levels were measured using a cystometric method and the luciferin-luciferase assay, respectively. RESULTS: In the HCl-treated rats, the micturition interval and micturition volume were significantly (48% and 55%, respectively, P <.05) decreased and the number of micturitions was significantly (3.2-fold, P <.05) increased compared with those of the control rats. The maximal number of binding sites for [³H]NMS and [³H]αß-MeATP was significantly (55% and 72%, respectively, P <.001) decreased in the bladder of HCl-treated rats, suggesting downregulation of both muscarinic and purinergic receptors. In the HCl-treated rats, the inhibition constant, K(i), values for oxybutynin, solifenacin, and darifenacin were significantly (1.3-1.4-fold, P <.05) increased, but those for tolterodine and AF-DX116 were unchanged. Similarly, the inhibition constant for A-317491, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, and MRS2273 was significantly (5.5, 11, and 7.6-fold, respectively, P <.001) increased. Furthermore, the in vivo release of ATP was significantly (P <.05) enhanced in the HCl-treated rat bladder. CONCLUSIONS: Both muscarinic and purinergic mechanisms might be, at least in part, associated with the urinary dysfunction due to cystitis.

Palonosetron: a second generation 5-hydroxytryptamine 3 receptor antagonist.

BACKGROUND: Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles and patient characteristics (female gender, younger age, low alcohol consumption, history of motion sickness) are the major risk factors for CINV. OBJECTIVE: This review provides a detailed description of palonosetron, a second generation 5-HT3 receptor antagonist. METHODS: The chemistry and pharmacology of palonosetron are described, as well as the initial and recent clinical trials. RESULTS/CONCLUSION: Palonosetron has a longer half-life and a higher binding affinity than the first generation 5-HT3 receptor antagonists. Palonosetron has been approved for the prevention of acute CINV in patients receiving either moderately or highly emetogenic chemotherapy and for the prevention of delayed CINV in patients receiving moderately emetogenic chemotherapy. In recent studies, compared to the first generation 5-HT3 receptor antagonists, palonosetron in combination with dexamethasone demonstrated better control of delayed CINV in patients receiving highly emetogenic chemotherapy. There were no clinically relevant adverse reactions reported in the palonosetron clinical trials that were different from the common reactions reported for the 5-HT3 receptor antagonist class. Due to its efficacy in controlling both acute and delayed CINV, palonosetron may be very effective in the clinical setting of multiple-day chemotherapy and bone marrow transplantation.

Protection from DNBS-induced colitis by the tachykinin NK(1) receptor antagonist SR140333 in rats.

Inflammation is known to be associated with changes in tachykinin expression both in human and animal models: substance P and NK(1) receptor expression are increased in patients with inflammatory bowel disease, and similar changes are reported in experimental models of inflammation. We investigated the effect of the tachykinin NK(1) receptor antagonist SR140333 (10 mg/kg orally) on 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in rats. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-alpha tissue levels on day 6 after induction of colitis. An enzyme immunoassay technique was used to measure colonic substance P levels. DNBS administration impaired body weight gain and markedly increased all inflammatory parameters as well as colonic tissue levels of substance P. Treatment with SR140333 significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity and TNF-alpha tissue levels. Colonic tissue levels of substance P were also reduced by SR140333, although this effect did not reach statistical significance. In conclusion, treatment with SR140333 protects from DNBS-induced colitis in rats. These results suggest a role for NK(1) receptors and substance P in the development of intestinal inflammation and indicate tachykinin receptors as a potential pharmacological target in the treatment of inflammatory bowel disease.

The selective nicotinic acetylcholine receptor alpha7 agonist JN403 is active in animal models of cognition, sensory gating, epilepsy and pain.

Several lines of evidence suggest that the nicotinic acetylcholine receptor alpha7 (nAChR alpha7) is involved in central nervous system disorders like schizophrenia and Alzheimer's disease as well as in inflammatory disorders like sepsis and pancreatitis. The present article describes the in vivo effects of JN403, a compound recently characterized to be a potent and selective partial nAChR alpha7 agonist. JN403 rapidly penetrates into the brain after i.v. and after p.o. administration in mice and rats. In the social recognition test in mice JN403 facilitates learning/memory performance over a broad dose range. JN403 shows anxiolytic-like properties in the social exploration model in rats and the effects are retained after a 6h pre-treatment period and after subchronic administration. The effect on sensory inhibition was investigated in DBA/2 mice, a strain with reduced sensory inhibition under standard experimental conditions. Systemic administration of JN403 restores sensory gating in DBA/2 mice, both in anaesthetized and awake animals. Furthermore, JN403 shows anticonvulsant potential in the audiogenic seizure paradigm in DBA/2 mice. In the two models of permanent pain tested, JN403 produces a significant reversal of mechanical hyperalgesia. The onset was fast and the duration lasted for about 6h. Altogether, the present set of data suggests that nAChR alpha7 agonists, like JN403 may be beneficial for improving learning/memory performance, restoring sensory gating deficits, and alleviating pain, epileptic seizures and conditions of anxiety.

Structural elucidation of in vivo metabolites of penehyclidine in rats by the method of liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry and isotope ion cluster.

The structural elucidation of metabolites of penehyclidine in rats, a novel anti-cholinergic drug, by the method of liquid chromatography-electrospray ionization mass spectrometry, gas chromatography-mass spectrometry with electron impact ion source and stable isotope ion cluster was described. Identification and elucidation of the phase I and phase II metabolites were performed by comparing the daughter ion pairs of stable isotope cluster, changes of the protonated molecular masses, full scan MS(n) spectra and retention times with those of the parent drug, penehyclidine and penehyclidine deuterium-labeled. Penehyclidine was easily biotransformed by the pathways of oxidative, hydroxylated, methoxylated and phase II conjugated reactions to form several metabolites that retained the some features of the parent molecules. Twelve metabolites (penehyclidine monoxide, hydroxypenehyclidine, penehyclidine dioxide, hydroxypenehyclidine monoxide, dihydroxypenehyclidine, dihydroxypenehyclidine (position isomer), dihydroxypenehyclidine monoxide, trihydroxypenehyclidine, methoxypenehyclidine dioxide, dimethoxypenehyclidine, trihydroxymethoxypenehyclidine and glucuronide conjugated hydroxypenehyclidine) were identified. The results from electrospray ion and electron impact ion data with the stable isotope cluster showed the qualitative differences in the mass spectral patterns, suggesting that these technologies should be used in parallel to ensure comprehensive metabolites detection and characterization. The described method has wide applicability to rapidly screen and provide structural information of metabolites.

Zinc uptake is mediated by M1 muscarinic acetylcholine receptors in differentiated SK-SH-SY5Y cells.

Alzheimer's disease (AD), the most prevalent form of dementia, is characterized by several major morphological hallmarks such as senile plaques, neurofibrillary tangles and a loss of cholinergic basal forebrain neurons. Apart from cholinergic markers like choline acetyltransferase and acetylcholinesterase, there have been reports on changes in muscarinic acetylcholine receptors (mAChR) as well as on influences of zinc metabolism in the disease. As recent studies gave hints about a possible link between mAChRs and zinc uptake, the human neuroblastoma cell line SK-SH-SY5Y was used to evaluate the role of M1-mAChR on zinc uptake. Zinc levels were semi-quantitatively detected by using the zinc-specific fluorophor Zn-AF2-DA. In the presence of 1 microM extracellular zinc, M1-mAChR stimulation with talsaclidine increased intracellular zinc levels as did stimulation of PKC by phorbol esters. Furthermore, the effect of extracellular zinc on the expression of the zinc finger protein PNUTS (protein phosphatase 1 nuclear targeting subunit 10) was investigated and revealed an upregulation of PNUTS expression in the presence of 1 microM extracellular zinc by 294% when compared to incubation in zinc free medium. In summary, this report demonstrates that intracellular zinc uptake in SK-SH-SY5Y cells is controlled by M1-mAChR mediated signalling pathways and that zinc may act as a cofactor for transcriptional regulation of zinc finger genes such as PNUTS.

Comparison of the functional blockade of rat substance P (NK1) receptors by GR205171, RP67580, SR140333 and NKP-608.

Extensive screening of compound libraries was undertaken to identify compounds with high affinity for the rat NK(1) receptor based on inhibition of [(125)I]-substance P binding. RP67580, SR140333, NKP-608 and GR205171 were selected as compounds of interest, with cloned rat NK(1) receptor binding K(i) values of 0.15-1.9 nM. Despite their high binding affinity, NKP-608 and GR205171 exhibited only a moderate functional antagonism of substance P-induced inositol-1-phosphate accumulation and acidification rate at 1 microM using cloned or native rat NK(1) receptors in vitro. The ability of the compounds to penetrate the CNS was determined by inhibition of NK(1) agonist-induced behaviours in gerbils and rats. GR205171 and NKP-608 potently inhibited GR73632-induced foot drumming in gerbils (ID(50) 0.04 and 0.2 mg/kg i.v., respectively). In contrast, RP67580 and SR140333 were poorly brain penetrant in gerbils (no inhibition at 10 mg/kg i.v.) and were not examined further in vivo. In rats, only high doses of GR205171 (10 or 30 mg/kg s.c.) inhibited NK(1) agonist-induced sniffing and hypertension, whilst NKP-608 (1 or 10 mg/kg i.p.) was without effect. GR205171 (3-30 mg/kg s.c.) caused only partial inhibition of separation-induced vocalisations in rat pups, a response that is known to be NK(1) receptor mediated in other species. These observations demonstrate the shortcomings of currently available NK(1) receptor antagonists for rat psychopharmacology assays.

Improving the nicotinic pharmacophore with a series of (Isoxazole)methylene-1-azacyclic compounds: synthesis, structure-activity relationship, and molecular modeling.

A series of (isoxazole)methylene-1-azacyclic compounds was prepared. The compounds were tested for affinity to central nicotinic acetylcholine receptors (nAChRs) and central muscarinic receptors. The compounds covered a broad range of affinities for the nAChRs (IC(50) = 0.32 to >1000 nM), with selectivities for the nAChRs over the muscarinic receptors in the range of 3-183. The high-affinity compound (Z)-26 (3-(4-methyl-5-isoxazolyl)methylene-1-azabicyclo[2.2. 2]octane, IC(50) = 3.2 nM) having only one energy minimum was used as the reference structure in a computational study. This ligand has enabled definition of an important distance parameter, and the existence of this parameter was supported by showing that other potent nicotinic ligands (for example, nicotine and epibatidine) fit the model.

The interaction of the enantiomers of aceclidine with subtypes of the muscarinic receptor.

The pharmacological activity of the enantiomers of aceclidine was investigated in Chinese hamster ovary cells transfected with the M1 through M5 subtypes of the muscarinic receptor and also in the rat heart and parotid gland that express primarily M2 and M3 receptors, respectively. When measured by stimulation of phosphoinositide hydrolysis in Chinese hamster ovary cells transfected with the M1, M3 and M5 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 2- to 4-fold greater than that of R-(-)-aceclidine, whereas the maximal response of the R-(-)-isomer was only 44 to 64% that of the S-(+)-isomer. When measured by inhibition of forskolin-stimulated cyclic AMP accumulation in Chinese hamster ovary cells transfected with the M2 and M4 muscarinic subtypes, the potency of S-(+)-aceclidine was approximately 3.5-fold greater than that of R-(-)-aceclidine. In cells transfected with the M2 muscarinic receptor, the maximal responses of the enantiomers were the same, whereas the maximal response of R-(-)-aceclidine was 86% that of S-(+)-aceclidine in cells transfected with the M4 muscarinic subtype. The activities of the enantiomers of aceclidine at native M2 and M3 muscarinic receptors coupled to inhibition of adenylyl cyclase activity in the heart and stimulation of phosphoinositide hydrolysis in the parotid gland, respectively, were similar to those observed in Chinese hamster ovary cells transfected with the corresponding receptor subtypes. We devised a simple quantitative method for using our data in Chinese hamster ovary cells to predict the relative potencies of agonists in a more sensitive assay in which the agonists produce a full maximum response. By using this method, we were able to predict the relative potencies of the enantiomers for eliciting contractions in the guinea pig ileum, an M3 muscarinic response, from their activity in Chinese hamster ovary cells transfected with the M3 muscarinic subtype. Our method of analysis should have application in a variety of studies in which transfected cells are used to determine the pharmacological activity of agonists.

In vivo autoradiographic and dissection evaluation of isomers of 125I-labeled 1-azabicyclo[2.2.2] oct-3-yl-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP).

(R,S)-[125I]IQNB has been used extensively in in vivo studies in rats and has been of utility in demonstrating the in vivo subtype selectivity of nonradioactive ligands in competition studies. Radiolabeled Z- and E-(-,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z- and E-[-,-]-[125I]IQNP) are analogs of (R,S)-[125I]IQNB. Preliminary rat brain regional dissection studies have indicated that Z- and E-(-,-)-[125I]IQNP, in general, are distributed similarly to (R,S)-[125I]IQNB. An important observation is that Z-(-,-)-[125I]IQNP binds to the muscarinic receptors in those brain regions enriched in the m2 subtype with approximately a two- to fivefold higher percent dose/g compared to (R,S)-[125I]IQNB. These observations are confirmed here by in vivo autoradiographic comparison of the time-courses of (R,S)-[125I]IQNB, Z-(-,-)-[125I]IQNP, and E-(-,-)-[125I]IQNP. Thus, in vivo competition studies against Z-(-,-)-[125I]IQNP would provide a potentially more sensitive and accurate probe for demonstrating the in vivo m2 selectivity of the nonradioactive ligands. In addition, Z-(-,-)-[123I]IQNP would potentially be useful for SPECT imaging of muscarinic receptor loss in neurodegenerative diseases.

Resolution and in vitro and initial in vivo evaluation of isomers of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate: a high-affinity ligand for the muscarinic receptor.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 1), is a highly selective ligand for the muscarinic acetylcholinergic receptor (mAChR). There are eight stereoisomers in the racemic mixture. The optical isomers of alpha-hydroxy-alpha-phenyl-alpha-(1-propyn-3-yl)acetic acid were resolved as the alpha-methylbenzylamine salts, and the optical isomers of 3-quinuclidinol were resolved as the tartrate salts. The E and Z isomers were prepared by varying the reaction conditions for the stannylation of the triple bond followed by purification utilizing flash column chromatography. In vitro binding assay of the four stereoisomers containing the (R)-(-)-3-quinuclidinyl ester demonstrated that each isomer of 1 bound to mAChR with high affinity. In addition, (E)-(-)-(-)-IQNP demonstrated the highest receptor subtype specificity between the m1 molecular subtype (KD, nM, 0.383 +/- 0.102) and the m2 molecular subtype (29.6 +/- 9.70). In vivo biodistribution studies demonstrated that iodine-125-labeled (E)-(-)-(+)-1 cleared rapidly from the brain and heart. In contrast, iodine-125-labeled (E)-(-)-(-)-, (Z)-(-)-(-)-, and (Z)-(-)-(+)-1 have high uptake and retention in mAChR rich areas of the brain. It was also observed that (E)-(-)-(-)-IQNP demonstrated an apparent subtype selectivity in vivo with retention in M1 (m1, m4) mAChR areas of the rain. In addition, (Z)-(-)-(-)-IQNP also demonstrated significant uptake in tissues containing the M2 (m2) mAChR subtype. These results demonstrate that the iodine-123-labeled analogues of the (E)-(-)-(-)- and (Z)-(-)-(-)-IQNP isomers are attractive candidates for single-photon emission-computed tomographic imaging of cerebral and cardiac mAChR receptor densities.

Synthesis and biodistribution of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate. A new ligand for the potential imaging of muscarinic receptors by single photon emission computed tomography.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 3), an analogue of QNB in which a phenyl ring has been replaced with an iodopropenyl substituent, was prepared and evaluated in vitro and in vivo for m-AChR selectivity and specificity. High specific activity [125]IQNP ([125I]-3) was synthesized in greater than 60% yield utilizing an electrophilic iododestannylation reaction with hydrogen peroxide for the oxidation of iodide. In in vitro receptor binding studies, 3 demonstrated high affinity for M1 (Ki = 0.78 nM), M2 (Ki = 1.06 nM), and M3 (Ki = 0.27 nM) subtypes. In vivo biodistribution studies in female rats [125I]-3 demonstrated high uptake in areas rich in muscarinic receptors such as the brain (cortex and striatum) and the heart. Blocking studies were performed with a series of receptor specific agents and demonstrated that the uptake of [125I]-3 was selective and specific for cerebral muscarinic receptor rich areas and that the binding to m-AChR is reversible. The high-yield preparation and specificity and selectivity of high specific activity [125I]IQNP for muscarinic receptors suggest that this is an attractive new agent for potential imaging of cerebral receptors using single photon tomographic imaging (SPECT).