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The synthesis and biological evaluation of a series of new derivatives of 2-substituted 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), are reported. Chemical and pharmacological studies on compounds 6 have been pursued with the aim of expanding the SAR data and validating the previously proposed model of interaction of this class of compounds with the κ-opioid receptor. The synthesis of the previously described compounds 6 has been reinvestigated in order to obtain a more direct synthetic procedure. To study the relationship between the stereochemistry and the receptor binding affinity, compounds 6e and 6k were selected on the basis of their evident structural resemblance to tifluadom. Since a different specificity of action could be expected for the enantiomers of 6e and 6k, owing to the results shown by (S)- and (R)-tifluadom, their racemic mixtures have been resolved by means of liquid chromatography with chiral stationary phases (CSP), and the absolute configuration of the enantiomers has been studied by circular dichroism (CD) and ¹H NMR techniques. Moreover, some new 2-[(acylamino)ethyl]-1,4-benzodiazepine derivatives, 6a−d,f,g,j, have been synthesized, while the whole series (6a−o) has been tested for its potential affinity toward human cloned κ-opioid receptor. The most impressive result obtained from the binding studies lies in the fact that this series of 2-[2-(acylamino)ethyl]-1,4-benzodiazepine derivatives binds the human cloned κ-opioid receptor subtype very tightly. Indeed, almost all the ligands within this class show subnanomolar Ki values, and the least potent compound 6o shows, in any case, an affinity in the nanomolar range. A comparison of the affinities obtained in human cloned κ-receptor with the correspondent one obtained in native guinea pig κ-receptor suggests that the human cloned κ-receptor is less effective in discriminating the substitution pattern than the native guinea pig κ-receptor. Furthermore, the results obtained are discussed with respect to the interaction with the homology model of the human κ-opioid receptor, built on the recently solved crystal structure of rhodopsin. Finally, the potential antinociceptive and antiamnesic properties of compounds 6e and 6i have been investigated by means of the hot-plate and passive avoidance test in mice, respectively.

We have recently reported the potent hypocalcemic effects of side-chain lactam-bridged analogues of human calcitonin (hCT) (Kapurniotu, A.; et al. Eur. J. Biochem. 1999, 265, 606−618). To extend these studies, we have now synthesized a new series of (Asp¹⁷, Lys²¹) and (Asp¹⁷, Orn²¹) side-chain bridged salmon calcitonin (sCT) and hCT analogues. The affinities of these analogues for the human calcitonin receptor, hCTRI1-, and for rat-brain membrane receptors were assayed in competitive binding assays, and agonist potencies at the hCTRI1- receptors were assessed, using a cAMP-responsive gene-reporter assay. The bridged sCT analogues had activities similar to sCT itself. In contrast, an (Asp¹⁷, Orn²¹) side-chain bridged hCT analogue, cyclo(17−21)-[Nle⁸, Phe¹², Asp¹⁷, Orn,²¹ Tyr²²)-hCT, was 80 and 450 times more active than hCT in the hCTRI1- and rat-brain receptor binding assays, respectively, and was 90 times more potent than hCT and 16 times more potent than sCT in initiating receptor signaling. An uncyclized, isosteric analogue of this peptide was also more potent than hCT, demonstrating that the cyclization constraint and these single-residue substitutions enhance the activities of hCT in an additive fashion. This study demonstrates that the potency-enhancing effects of lactam-bridge constraints at hCT residues 17−21 are not transferable to sCT. We also show that, in comparison to the hCT analogues, sCT and its analogues are less potent agonists than expected from their hCTRI1- affinities. This suggests that it may be possible to preserve the efficient signal transduction of hCT while introducing additional receptor affinity-enhancing elements from sCT into our potent lactam-bridged hCT analogue, thereby creating new super-potent, hCT-based agonists.

A double-blind, double-dummy clinical trial was conducted in which the efficacy of cyclandelate 1600mg daily was compared with that of flunarizine 10mg daily in 40 patients (25 men and 15 women) with dementia of cerebrovascular origin. Parameters were assessed before treatment, and after 45 and 90 days of therapy. At 90 days, significant improvements were observed in patients given cyclandelate in measurements of P100latency in the left eye, neurological impairment, dementia scores, ischaemia scores, Gottfries mental deterioration scale, Hamilton depression scores, short term visual memory, long term memory, Bender- Gestalt test and Koh’s blocks test. In flunarizine recipients, improvements were observed in neurological impairment, ischaemia scores, Gottfries scale and Hamilton depression scores. Patients treated with cyclandelate showed significantly greater ameliorations in symptoms as assessed by the ischaemia scale, evoked visual potential, visual memory and Koh’s block test compared with those given flunarizine. However, in none of the parameters was flunarizine superior to cyclandelate. Les auteurs ont conduit une étude clinique en double-insu avec double-placebo afin de comparer l’efficacité du cyclandélate (1600 mg/jour) à celle de la flunarizine (10 mgljour) chez 40 patients (25 hommes et 15 femmes) atteints de démence consécutive à une atteinte vasculaire cérébrale. Les paramètres ont été évalués avant le traitement puis 45 et 90 jours après son début. A 90 jours, les patients traités par la cyclandélate ont présenté des améliorations significatives de la latence P100de l’oeil gauche, de la détérioration neurologique, des scores de démence, du score d’ischémie, du score de l’échelle de détérioration mentale de Gottfries, du score de l’échelle de la dépression de Hamilton, de la mémoire visuelle à court terme, de la mémoire à long terme, du test de Bender-Gestalt et du test des cubes de Koh. Chez les patients traités par la flunarizine, on a observé des améliorations de la détérioration neurologique, du score d’ischémie, de l’échelle de Gottfries et du score de l’échelle de la dépression de Hamilton. Les patients traités par le cyclandélate ont présenté des améliorations symptomatologiques significativement supérieures à ceux sous flunarizine, comme le montrent les évaluations de l’échelle d’ischémie, des potentiels évoqués visuels, de la mémoire visuelle et des cubes de Koh. En revanche, la flunarizine n’a été supérieure au cyclandélate sur aucun des paramètres étudiés. E’stata condotta una sperimentazione a doppio cieco, a doppio placebo, per confrontare l’efficacia di 1600 mg al giorno di ciclandelato con quella di 10 mg al giorno diflunarizina in 40 pazienti (25 uomini e 15 donne) con demenza di origine cerebrovascolare. Furono determinati i parametri relativi: prima del trattamento, dopo 45 e dopo 90 giorni di terapia. Al novantesimo giorno, nei pazienti trattati con ciclandelato, si osservarono significativi miglioramenti délie determinazioni nell’occhio sinistro con latenza P100, della compromissione neurologica, dei gradi di demenza e di ischemia secondo la Scala di Gottfries, di quelli della depressione secondo Hamilton, della memoria visiva a breve e a lungo termine, del test Bender-Gestalt e di quello dei blocchi di Koh. Nei pazienti trattati con flunarizina si osservarono miglioramenti delle turbe neurologiche, dei gradi di ischemia, dei gradi délia depressione secondo la Scala di Gottfries e dei gradi di depressione secondo Hamilton. Nei pazienti trattati con ciclandelato si evidenziarono maggiori miglioramenti dei sintomi, valutati secondo la scala délia ischemia, dei potenziali visivi indotti (PEV) e dei test dei blocchi di Koh, più significativi rispetto a quelli dati dalla flunarizina. Perd in nessuno dei parametri la flunarizina è risultata superiore al ciclandelato. Er werd een vergelijkend, dubbelblind klinisch onderzoek uitgevoerd naar de effectiviteit van resp. 1600 mg cyclandelaat en 10 mg flunarizine per dag. Men selecteerde daarvoor 40 proefpersonen (25 mannen en 15 vrouwen) met dementie van cerebrovasculaire oorsprong. Voor en na 45 en 90 dagen behandeling werden de parameters geëvalueerd. Na 90 dagen werden significante verbeteringen vastgesteld in de testresultaten bij patiënten die met cyclandelaat behandeld waren: de P100-latentietijd van het linkeroog, de neurologische vermindering, de dementiescores, de ischemiescores, de “mental deterioration scale” van Gottfries, de Hamilton-depressiescores, het korte termijn visuele geheugen, het lange termijn geheugen, de Bender-Gestalttest en Kohs blokkentest verbeterden. Bij de flunarizine patiënten werden verbeteringen waargenomen op het vlak van de neurologische vermindering, de ischemiescores, de schaal van Gottfries en de Hamilton-depressiescores. De proefpersonen die cyclandelaat toegediend kregen, vertoonden significant grotere verbeteringen van de Symptomen die gemeten werden op de ischemieschaal, de evoked visual potential, het visuele geheugen en Kohs blokkentest dan de patiënten uit de flunarizinegroep. Flunarizine bleek voor geen enkele parameter betere resultaten op te leveren dan cyclandelaat.

The synthesis and biological evaluation of a series of 2-substitued 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a κ-opioid agonist and a cholecystokinin-A (CCK-A) antagonist, are reported. The radioligand binding models used in these studies were [¹²⁵I](BH)-CCK-8 in rat pancreas (CCK-A), [³H](MeNLE^28,31)-CCK-8 in guinea pig cerebral cortex (CCK-B), and [³H]U-69593 (κ1), [³H]DAMGO (μ), and [³H]DADLE (δ) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for κ-opioid receptors. In particular, the 2-thienyl derivative 7a (X = H) with a Ki = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig κ (κ1)-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure−affinity relationships in this class of receptors.

Clearance studies in rats using kappa opioid agonists have demonstrated that agonists that can cross the blood-brain barrier are more potent water diuretics than agonists which have limited access to the brain. The mechanism of kappa agonist-induced water diuresis is unclear but may involve inhibition of vasopressin secretion and/or an adrenomedullary factor. In the present study the effect of an alpha-2 adrenoceptor antagonist (yohimbine, 10 micrograms/kg.min i.v.) on kappa agonist-induced water diuresis was evaluated in conscious chronically instrumented rats. BRL 53117 (1-[(3,4-dichlorophenyl)acetyl]-2-[(3-hydroxy-1-pyrrolidinyl) methyl]4,4-dimethyl piperidine), a kappa agonist that can cross the blood-brain barrier, caused a dose-dependent (1-100 micrograms/kg) water diuresis which was attenuated by yohimbine. The effective dose to cause a free water clearnace of zero for BRL 53117 was 13 +/- 5 micrograms/kg in vehicle-treated rats and 37 +/- 12 micrograms/kg in yohimbine-treated rats. BRL 52974 (5-[(3,4-dichlorophenyl)acetyl]4-(1-pyrrolidinylmethyl)-4,5,6,7-te trahydro- 1H-imidazo[4,5-c]pyridine), a compound with limited ability to cross the blood-brain barrier, also caused a dose-dependent water diuresis, albeit at higher doses (30-3000 micrograms/kg), and thus a higher effective dose to cause a free water clearance of zero (129 +/- 61 micrograms/kg); however, the effect was abolished by yohimbine. The data suggest that kappa agonists cause a water diuresis by both a central mechanism involving inhibition of vasopressin secretion and a peripheral mechanism involving stimulation of renal alpha-2 receptors.

Data suggest that kappa opioid agonist-induced water diuresis involves inhibition of vasopressin (AVP) secretion; however, it is not clear whether this action involves kappa receptors in the neurohypophysis or receptors behind the blood-brain barrier (BBB). We have investigated the site of action using three selective kappa agonists, BRL 52656 (S(-)-2-(1-pyrrolidinylmethyl)-1-(4-trifluoromethylphenyl) acetyl piperidine hydrochloride), BRL 53114 ((-)-1-(4-trifluoromethylphenyl) acetyl-2-(1-pyrrolidinymethyl)3,3- dimethyl piperidine hydrochloride) and BRL 52974 (4-(1-pyrrolidinylmethyl)5-(3,4-dichlorophenyl)acetyl-4,5,6,7-t etrahydroimidazo [4,5-c] pyridine), with varying abilities to cross the BBB. Chemical and functional assays indicate that BRL 52974 has limited ability to cross the BBB, whereas BRL 53114 and BRL 52656 can freely penetrate. BRL 52974 was significantly less potent than BRL 52656 and BRL 53114 in causing a water diuresis in conscious rats. The ED10S (i.v. doses to cause a positive free water clearance of 10 microliters/min.100 g) for BRL 52974, BRL 52656 and BRL 53114 were 181, 9 and 3.4 mg/kg, respectively. Furthermore, in dogs BRL 52656 and BRL 53114 but not BRL 52974 (30 micrograms/kg i.v.) were able to cause a significant water diuresis. The data demonstrate that opiate receptors behind the BBB are primarily involved in kappa agonist-induced water diuresis and possibly inhibition of AVP secretion.

The roles of various types of opioid receptors in opioid-induced local inhibition of gastrointestinal transit were studied in rats 5 min after a charcoal meal, injecting i.p. relatively selective agonists and antagonists. The proposed mu agonists, morphine and [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO), and the nonselective delta agonist, [D-Ala2,D-Leu5]enkephalin (DADLE), produced a dose-related inhibition of gastrointestinal transit at the peak time and the i.p. doses producing a 50% reduction of the control values (A50) were 0.015, 0.006 and 0.023 mg/kg, respectively, for morphine, DAMGO and DADLE. The effect of the other nonselective delta agonist, [D-Ser2,L-Leu5]enkephalyl-Thr (DSLET), was not linearly related with the dose. The proposed selective delta agonist, [D-Pen2,D-Pen5]enkephalin (DPDPE), and the selective kappa agonist, U-69,593, up to 2 and 15 mg/kg i.p., respectively, did not delay gastrointestinal transit. Naloxone (0.05 mg/kg i.p.) injected 1 min before each agonist produced a significant parallel shift to the right of the dose-response curves for morphine and DAMGO, but only partly antagonized the effects of DADLE and DSLET. The selective delta antagonist ICI 174,864 (1 mg/kg i.p., injected 1 min before each agonist) shifted the dose-response curve of DADLE, but not of the mu agonists, slightly, but significantly to the right, and had an inconsistent effect on DSLET. Naloxone prevented DADLE's effect on the gut with a nonlinear dose-response curve and much higher doses of naloxone were required to prevent fully DADLE-induced effects than to antagonize doses of morphine equiactive to DADLE on the gut.(ABSTRACT TRUNCATED AT 250 WORDS)