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We have recently identified 3-[(1-methyl)-4,5-dihydro-1H-imidazol-2-yl)methyl]pyridine (homoazanicotine, 8) as a novel nicotinic acetylcholinergic (nACh) receptor ligand. In the present investigation, after we determined that 8 binds selectively at nicotinic (K(i) = 7.8 nM) vs muscarinic (K(i)10,000 nM) acetylcholinergic receptors, we examined its structure-affinity relationships for nACh receptor binding. The features investigated included the influence of (i) the composition of connector that separates the two rings, (ii) the N-methyl group, (iii) the ring opening of the imidazoline ring, (iv) the pyridine nitrogen atom, and (v) the aromatization of the imidazoline ring on nACh receptor affinity. As with nicotine, the parent structure seems optimal and most structural changes reduce nACh receptor affinity. Also, as with nicotine analogues, alteration of the spacer group influences affinity in a manner that is somewhat different than that seen with the parent structure.

WB4101 (1)-related compounds 5-10 were synthesized, and their biological profile at alpha(1)-adrenoreceptor (AR) subtypes and 5-HT(1A) serotoninergic receptors was assessed by binding assays in Chinese hamster ovary and HeLa cell membranes expressing the human cloned receptors. Moreover, their receptor selectivity was further determined in functional experiments in isolated rat prostate (alpha(1A)), vas deferens (alpha(1A)), aorta (alpha(1D)), and spleen (alpha(1B)). In functional assays, compound 5 was the most potent at alpha(1D)-ARs with a reversed selectivity profile (alpha(1D)alpha(1A)alpha(1B)) relative to both prototype 1 and phendioxan (2) (alpha(1A)alpha(1D)alpha(1B)), whereas compound 8, bearing a carbonyl moiety at position 1, was the most potent at alpha(1A)-ARs with a selectivity profile similar to that of prototypes. The least potent of the series was the trans isomer 6, suggesting that optimum alpha(1)-AR blocking activity in this series is associated with a cis relationship between the 2-side chain and the 4-phenyl ring rather than a trans relationship as previously observed for the 2-side chain and the 3-phenyl ring in 2 and related compounds. Binding affinity results were not in complete agreement with the selectivity profiles deriving from functional experiments. Although a firm explanation was not available, neutral and negative antagonism and receptor dimerization were considered as two possibilities to account for the difference between binding and functional affinities. Finally, compound 5 was selected for a modeling study in comparison with 1, mephendioxan (3), and open phendioxan (4) to achieve information on the physicochemical interactions that account for its high affinity toward alpha(1d/D)-ARs.

A series of muscarinic agonists, straight chained, branched, cyclic alkyl and aromatic derivatives of the oxime 1 (demox) was designed with the aim of investigating their activity on muscarinic receptor subtypes. Effects on M1 receptor were assessed functionally by a microphysiometer apparatus, while M2, M3, and M4 receptor potency and affinity were studied on isolated preparations of guinea pig heart, ileum, and lung, respectively. The results suggest that the substitution of a hydrogen with a long side-chain or bulky group generally induces a decrease in potency at M1 and M3 subtypes, while a general increase in this parameter is obtained at M2 subtype. Among the agonists 2-18, compound 4 behaves as a full agonist with a preference for M3 subtype. Moreover, compound 12 is inactive at M1 and M4 receptors while it displays a full agonist activity at M2 and M3 subtypes. Since demox displays a variable response on cardiac M2 receptors regulating heart force, an in-depth inquiry of the functional behaviour of this compound was carried out at M2 receptors. In presence of 10(-11) and 10(-10) M demox, the binding of [3H]-NMS was increased by approximately 30% as a consequence of an increase of the association of [3H]-NMS to membranes; this effect was not observed in presence of a higher concentration of [3H]-NMS. Higher concentrations of demox decreased the binding of [3H]-NMS to heart atrial membranes but significantly retarded the dissociation of this radioligand. Our results suggest that demox may interact with orthosteric and allosteric sites of atrial M2 muscarinic receptor.

Pharmacological antagonism of the interaction between MDM2 and/or MDMX with p53 has been pursued as a therapeutic strategy to restore p53 tumour suppressor activity in wild type TP53 tumours with amplification or over-expression of these negative regulators. The majority of small molecule approaches described to date inhibit MDM2 binding to p53, which has E3 ligase activity and through ubiquitination targets p53 for degradation. However, MDMX, although devoid of E3 ligase activity itself, forms heterodimers with MDM2 and augments its activity to induce polyubiquitination of p53. Further complexity in this regulatory network arises from the fact that MDM2 is a target gene of p53 transcription, and that MDM2 can also control the stability of MDMX. Since MDMX is widely reported to confer resistance to established MDM2:p53 antagonists, this study aimed to examine the activity of MDM2:p53 selective inhibitors across a panel of wild-type TP53 cell lines with variable MDM2 and MDMX protein expression, and to contrast this with recently described inhibitors with mixed MDM2:p53 and MDMX:p53 activity. Growth inhibition studies (72h) were performed using XTT assays, but with verification by orthogonal methods (SRB, clonogenic assays). Gene expression was determined at the level of mRNA and protein. The selectivity of all inhibitors was confirmed initially in p53 wild-type and non-functional paired cell lines. As anticipated, MDM2:p53 selective inhibitors (nutlin-3a, MI-773, RG7112) were active in MDM2 amplified cell lines with low MDMX expression (SJSA-1 and T778; GI50 range 0.2 to 1 uM). However, a similar level of activity was also evident in cell lines with low/medium MDM2 in the presence of medium/high MDMX expression (Pre B 697, A375, HCT116, MRK-NU1, and NGP cells: GI50 range 0.2 to 2 uM). Appreciable resistance was only evident in Jeg3 and MCF-7 cells (low MDM2 and high MDMX), averaged compound GI50 values ranging from 9 - 10 uM in Jeg3 and 6 - 14 uM in MCF-7. Whilst a concentration dependent degradation of MDMX was observed in some sensitive cell lines with medium/high MDMX expression, in others no change was evident, suggesting that there are multiple determinants of cellular sensitivity to such inhibitors. Importantly, the mixed MDM2:p53 and MDMX:p53 antagonist RO-5963, displayed a different spectrum of activity, with least growth inhibition evident in SJSA-1 and T778 MDM2 amplified cell lines, and activity being retained in MDMX amplified Jeg3 cells. These studies support the concept that targeting MDMX may provide a different spectrum of antitumour activity to selective MDM2:p53 inhibition. However, MDMX protein levels per se do not constitute a universal resistance factor to selective MDM2:p53 inhibitors and, beyond MDM2 amplification, the identification of additional markers of sensitivity is warranted for this class of agent. Citation Format: Sue Tudhope, Yan Zhao, Anita Wittner, Elaine Wilmore, Annalisa Bertoli, Santosh Adhikari, Suzannah J. Harnor, Fabio Del Bello, Alessandro Piergentili, John Lunec, Ian R. Hardcastle, Roger J. Griffin, Bernard R. Golding, Celine Cano, David R. Newell, Stephen R. Wedge. Profiling inhibitors of MDM2:p53 and MDMX:p53 in relation to MDMX protein levels. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5451. doi:10.1158/1538-7445.AM2014-5451

Analogues of nicotine (1) and azanicotine (3) were prepared with an additional methylene group inserted between the two rings (i.e., homonicotine and homoazanicotine; 6 and 5, respectively). Although 6 (Ki=3110 nM) and 3 (Ki=206 nM) bind at nACh receptors with ≥100-fold lower affinity than nicotine (Ki=2.1 nM), 5 displays high affinity (Ki=7.8 nM). Like nicotine (ED50=12 μg/mouse), both 3 and 5 (ED50=21 and 19 μg/mouse, respectively) produced antinociceptive activity in the tail-flick assay following intrathecal administration. The antinociceptive actions of 3 and 5, unlike those of nicotine, were not antagonized by mecamylamine. Compounds 3 and 5 might represent novel analgesic agents that act via a non-nicotinic mechanism, or via a nicotinic mechanism that is distinct from that mediating the antinociceptive actions of nicotine.

The synthesis and pharmacological testing of two series of novel bis-quinolinium cyclophanes as blockers of the apamin-sensitive Ca(2+)-activated K(+) (SK(Ca)) channel are presented. In these cyclophanes the two 4-aminoquinolinium groups are joined at the ring N atoms (linker L) and at the exocyclic N atoms (linker A). In those cases where A and L contain two or more aromatic rings each, the activity of the compound is not critically dependent upon the nature of the linkers. When A and L each have only one benzene ring, the blocking potency changes dramatically with simple structural variations in the linkers. One of these smaller cyclophanes having A = benzene-1,4-diylbis(methylene) and L = benzene-1, 3-diylbis(methylene) (3j, 6,10-diaza-1,5(1,4)-diquinolina-3(1,3),8(1, 4)-dibenzenacyclodecaphanedium tritrifluoroacetate, UCL 1684) has an IC(50) of 3 nM and is the most potent non-peptidic SK(Ca) channel blocker described to date. Conformational analysis on the smaller cyclophanes using molecular modeling techniques suggests that the differences in the blocking potencies of the compounds may be attributable to their different conformational preferences.

WB 4101-related benzodioxans 3-9 were synthesized, and their biological profiles at alpha(1)-adrenoreceptor subtypes and 5-HT(1A) serotoninergic receptors were assessed by binding assays in CHO and HeLa cells membranes expressing the human cloned receptors. Furthermore, receptor selectivity of selected benzodioxan derivatives was further determined in functional experiments in isolated rat vas deferens (alpha(1A)) and aorta (alpha(1D)) and guinea pig spleen (alpha(1B)), in additional receptor binding assays in rat cortex membranes containing alpha(2)-adrenoreceptors and 5-HT(2) serotoninergic receptors, and in rat striatum membranes containing D(2) dopaminergic receptors. An analysis of the results of receptor binding experiments for benzodioxan-modified derivatives 3-9 showed high affinity and selectivity toward the alpha(1a)-adrenoreceptor subtype for compounds 3-5 and 7 and a reversed selectivity profile for 9, which was a selective alpha(1d) antagonist. Furthermore, the majority of structural modifications performed on the prototype 1 (WB 4101) led to a marked decrease in the affinity for 5-HT(1A) serotoninergic receptors, which may have relevance in the design of selective alpha(1A)-adrenoreceptor antagonists. The exception to these findings was the chromene derivative 8, which exhibited a 5-HT(1A) partial agonist profile.

Radioligand binding assay represents an important technique in pharmacological and pharmaceutical research for assessing the receptor profile of new drugs or of compounds under development. In this study, the pharmacological profile and the receptor specificity of compounds active on dopamine and muscarinic cholinergic receptor subtypes were evaluated using as a receptor source, membrane preparations or frozen sections. Dopamine D1-like receptors were assayed in membrane preparations or frozen sections of rat striatum and kidney with [3H]-SCH 23390 as a ligand. Rat striatum, kidney and atrium were used as a source of dopamine D2-like receptors with [3H]-spiperone as a ligand. The non-selective muscarinic cholinergic receptor antagonist [3H]-N-methyl-scopolamine was used to label muscarinic cholinergic receptors in the rat. Frontal cortex represented the source of M1 receptor subtype, heart the source of M2 receptor subtype, sub maxillary gland the source of M3 receptor subtype and striatum the source of M4 receptor subtype. With the exception of cardiac tissue, no significant differences were noticeable in the affinity of dopaminergic or muscarinic cholinergic compounds tested using membrane particles or 8 microns thick slide-mounted section. In the heart, frozen sections gave lower dissociation constant and inhibition constant values than membranes. The above findings suggest that radioligand binding assay on slide-mounted tissue sections may represent a suitable technique for assessing the receptor profile of drugs under development for the treatment of disorders characterised by dopaminergic or muscarnic cholinergic dysfunction.

To develop ligands that may be useful in exploring muscarinic receptor heterogeneity, we synthesized a series of analogues of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine oxalate and methiodide bearing a modified cationic head. These compounds, when tested on tissues containing the three subtypes M1, M2, and M3, behaved as muscarinic antagonists whose results showed that different substituents on the quaternary and tertiary nitrogen affect affinity and selectivity in different ways. In particular, comparison of the affinities of these ligands with those of the reference compounds points out that compounds bearing an ethyl substituent improve the affinity of the molecule at the three subtypes, while compounds bearing a phenethyl substituent are more selective for the M3 sites.

The observation that all the attempts to characterize imidazoline (I) receptors have been carried out with non-selective or poorly selective ligands prompted us to undertaken research aimed at developing selective ligand(s). In previous work using, as a starting point, cirazoline I, a potent alpha 1-adrenergic receptor agonist that also binds to I receptors, we showed that removal of the cyclopropyl ring (2) retains high affinity for I2 receptors while reducing alpha 1-adrenergic agonist activity. However, it was felt that this residual, albeit modest, alpha 1-adrenergic agonist activity might diminish the usefulness of compound 2, and we now report on our continuing efforts in this field. Starting from compound 2, we first eliminated the alpha 1-agonist component by isosteric replacement and then, by means of conformational restrictions on compound 7, succeeded in discovering tracizoline (9) and benazoline (12). These two new ligands with high affinity (pKi value 8.74 and 9.07, respectively) and unprecedented selectivity with respect to both alpha 2- (I2/alpha 2 7,762 and 18,621) and alpha 1- (I2/alpha 1 2,344 and 2,691) adrenergic receptors, are valuable tools in the study of I receptor structure and function. In addition, the large number of derivatives studied has allowed us to establish congruent qualitative structure-activity relationships and identify some structural elements governing affinity and selectivity.

87 Background: Growing evidence supports the role of α1-ARs in the direct mitogenic effect of catecholamines on prostate cancer (PC) cell growth. The expression of α1D-AR on PC3 prostate cancer cells and the ability of noradrenalin (NA) to stimulate PC3 cell proliferation in a α1D-AR-dependent manner were reported (Quaglia et al., 2005). In addition, TRPV1 expression was also found in prostate cancers (Sanchez et al., 2006). Aim of this study was to investigate the relationship between α1D-AR and TRPV1 receptors and the involvement of TRPV1 in NA-induced proliferation in PC3 cells. Methods: By western blot analysis and confocal microscopy the expression α1D-AR and TRPV1 and localization in PC3 cells were evaluated. PC3 cells were incubated with NA alone or in combination with WS433 and capsazepine (CPZ), α1D-AR and TRPV1 antagonists. Proton release, calcium influx and cell proliferation were assessed in α1D-AR-, TRPV1- or α1D-AR/TRPV1 double-silenced PC3 cells by cytosensor and cytofluorymetric analyses. Finally, lysates from NA-treated PC3 cells alone or in combination with WS433 or CPZ were blotted with anti-phospho ERK, anti-ERK and anti-phospho-(Ser) PKC substrate Abs and Inositol-1,4,5-trisphosphate [3H] radioreceptor assay were performed. Results: α1D-AR and TRPV1 co-localize and are co-immunoprecipitated in PC3 cells. Treatment of PC3 cells with NA strongly stimulated proton release, calcium influx and cell proliferation that were reverted by α1D-AR WS433 and TRPV1 antagonist. NA-induced increase of survival and proliferation was totally abrogated in α1D-AR/TRPV1 silenced cells. In addition, NA stimulates ERK and PKC substrate phosphorylation that was inhibited by WS433 and CPZ. Finally, CPZ treatment inhibited NA-dependent PLC activation, while WS433 had no effect. Conclusions: A functional and structural cross-talk between α1D-AR and TRPV1 receptors control NA-induced proliferation of PC cells. These data strongly suggest the development of new pharmaceutical approaches based on bifunctional antibodies and molecules recognizing both α1D-AR and TRPV1 receptors.

The enantiomers of trans-[2-(2,6-dimethoxyphenoxy)ethyl] [(3-p-tolyl-2,3-dihydro-1,4-benzodioxin-2-yl) methyl]amine (mephendioxan, 2) were synthesized from the chiral trans-3-p-tolyl-2,3-dihydro-1,4-benzodioxin-2-carboxylic acids [(+)-3 and (-)-3] which in turn were obtained through the resolution of the racemic acid with (R)- and (S)-alpha-methylbenzylamine. Comparison of CD spectra of the enantiomers of 2 with that of (2S,3S)-3-methyl-2-phenyl-1,4-benzodioxane allowed the assignment of the 2S,3S configuration to the (-)-enantiomer of 2 and of the 2R,3R configuration to the other enantiomer. The binding profile of the enantiomers of 2 was assessed at alpha 1, alpha 2, D2, and 5-HT1A receptors, in comparison to WB 4101 (1), 5-methylurapidil, and (+)-niguldipine. In addition, the two enantiomers were investigated at native and cloned alpha 1-adrenoreceptor subtypes. (-)-2 was 10-30 times as potent as the (+)-enantiomer at alpha 1-adrenoreceptor subtypes in both functional and binding assays. It was 36-fold selective for the alpha 1A- versus alpha 1B-adrenoreceptor and 60- and 20-fold selective in binding to the alpha 1a-adrenoreceptor relative to alpha 1b and alpha 1d subtypes, respectively. Furthermore, the enantiomer (-)-2 displayed selectivities of 12000-, 2500-, and 250-fold in binding to alpha 1a-adrenoreceptors relative to alpha 2-adrenoreceptors and 5-HT1A and D2 receptors. These results indicate that (-)-2 may be a valuable tool in the characterization of alpha 1-adrenoreceptor subtypes.

Some thio- and the benzoyl-derivatives of deoxamuscarine were synthesized and tested as muscarinic agonists using radioligand binding assays and functional tests. In comparison with deoxamuscarine, used as reference compound, no dimension/distance modification is tolerated for correct lipophilic pocket recognition. The substitution of the ammonium group with a sulphonium group significantly decreased muscarinic potency. The so-called ‘muscarinic sub-site’ accepts relatively bulky functions as long as it is bound to the cyclopentane carrier by an oxygen bridge. Esterification of this moiety increases the M2 subtype selectivity, while etherification heightens that of M3.

Publisher Summary This chapter discusses the discovery of highly selective imidazoline receptor ligands. Phenylethylamines and imidazolines are traditionally thought of as representing the two major classes of α-adrenergic agonists, yet there is evidence to suggest that these two classes of compounds do not interact with α-adrenergic receptors in an identical manner. The chapter discusses the separation procedure of α 1 agonist activity from imidazoline receptor affinity. There is also some discussion about comparative molecular field analysis (CoMFA) as molecular modeling application. The concept of I receptors has gained consensus since the hypothesis of Bousquet that the hypothensive action of clonidine could be related to its imidazoline structure rather than to its interaction with α 2 -adrenergic receptors. Investigations with radiolabeled ligands have shown existence of imidazoline binding sites in several tissues and species including humans. Furthermore, functional studies, though still few in number, have demonstrated that these binding sites may represent functional receptors, and the discovery of agmantine as its natural neurotrasmitter seems to support this view.

Growth plate cartilage is responsible for bone lengthening in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. While earlier studies on growth plate injury repair have identified inflammatory, mesenchymal infiltration, osteogenesis and remodelling repair responses, the molecular mechanisms involved in the bony repair remain unknown. Since the Wnt-β-catenin signalling pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, this study investigated the potential roles of Wnt-β-catenin signalling pathway in the bony repair of injured growth plate in rats. Suggestive of a role of Wnt/β-catenin signalling pathway in the early stage of the repair, our Affymetrix gene microarray analysis with RNA isolated from laser-capture microdissected injury site tissue identified an increase in β-catenin expression and yet a decrease in expression of Wnt signalling inhibitors sFRP1 & 4 at day 4 post-injury vs later time points. Compared to the vehicle control, treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200 mg/kg/day for 8 days, commenced at day 2 post injury) enhanced collagen-2 gene expression (by qRT-PCR), increased proportion of cartilage tissue and decreased level of bone tissue (as measured by histological analysis) at the injury site by day 10 post- injury (n = 8, pb 0.001). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 at 25 mM suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt-β-catenin signalling promotes osteoblast differentiation and suppresses chondrogenic differentiation during bony repair of the injured growth plate, and intervention of this signalling could represent a potential approach in enhancing cartilage regeneration after growth plate injury. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

The synthesis of 2-(4-fluoro-3-hydroxyphenyl)ethylamine (26) and of some N,N-dialkyl derivatives (27-30) starting from 4-fluoro-3-hydroxytoluene and their in vitro binding affinities for dopamine (DA) receptor are reported. The amine 26 can be regarded as a molecular modification of DA in which the para hydroxyl group is replaced by fluorine. The new compounds 26-30 were evaluated for their affinity at D-1 and D-2 DA receptor subtypes by displacement of [3H]SCH 23390 (D-1 selective) and [3H]spiperone (D-2 selective). The amine 26 had about 2-fold less affinity for D-1 and D-2 binding sites than DA. The substitution of the amino group with ethyl, n-propyl, and 2-phenylethyl groups decreased the affinity for D-1 binding sites but greatly enhanced the effectiveness on D-2 binding sites. The N-ethyl- (28) and N-n-propyl-N-(2-phenylethyl)-2-(4-fluoro-3- hydroxyphenyl)ethylamine (30) were the most potent members of the series with high selectivity for D-2 binding sites. A similar effect was observed with isomeric N-n-propyl-N-(2-phenylethyl)-2-(3-fluoro-4-hydroxyphenyl)ethylamine (31) which was approximately 65 times more selective for D-2 sites vs D-1 sites. The introduction of a 2-phenylethyl group on the nitrogen atom induce the highest effect, perhaps as a consequence of an increased liposolubility or of binding to a complementary lipophilic site on the receptor.