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WB4101-Related Compounds: New, Subtype-Selective r1-Adrenoreceptor Antagonists (or Inverse Agonists?) Marco Pallavicini,† Roberta Budriesi,‡ Laura Fumagalli,† Pierfranco Ioan,‡ Alberto Chiarini,‡ Cristiano Bolchi,† Maria Paola Ugenti,‡ Simona Colleoni,§ Marco Gobbi,§ and Ermanno Valoti*,† Istituto di Chimica Farmaceutica e Tossicologica, UniVersita` degli Studi di Milano, Viale Abruzzi 42, I-20131 Milano, Italy, Dipartimento di Scienze Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, I-40126 Bologna, Italy, and Istituto di Ricerche Farmacologiche ‘Mario Negri’, Via Eritrea 62, I-20157 Milano, Italy ReceiVed March 28, 2006
Our previous structure-affinity relationship study had considered the enantiomers of the naphthodioxane, tetrahydronaphthodioxane, and 2-methoxy-1-naphthoxy analogues (compounds 1, 3, and 2, respectively) of 2-(2,6-dimethoxyphenoxyethylaminomethyl)-1,4-benzodioxane, the well-known R1-adrenoceptor (R1-AR) antagonist WB4101, showing that such modifications significantly modulate the affinity and selectivity profile for R1-AR subtypes and 5-HT1A receptor. Here, we extend investigations to antagonist activity enclosing new enantiomeric pairs, namely those of the methoxytetrahydronaphthoxy and methoxybiphenyloxy WB4101 analogues (4 and 5-7, respectively) and of a double-modified WB4101 derivative (8) resulting from hybridization between 2 and 3. We found that (S)-2 is a very potent (pA2 10.68) and moderately selective R1D-AR antagonist and the hybrid (S)-8 is a potent (pA2 7.98) and highly selective R1A-AR antagonist. Both of these compounds and (S)-WB4101 seem to act as inverse agonists in a vascular model. The results, which generally validate the logic we followed in designing these eight compounds, are acceptably rationalized by comparative SAR analysis of binding and functional affinities. Introduction The tissue distribution and physiological role of the three R1adrenoreceptor (R1-AR) subtypes (R1A, R1B, and R1D) in humans have been intensively investigated in recent years as well as involvement of their overstimulation in the etiology of pathological disorders, in particular of hypertension and lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH).1,2 The latest evidence indicates that the R1A subtype and, with aging, the R1B subtype would be the principal mediators of blood pressure control, while the R1A and R1D subtypes, predominating in prostatic stroma and bladder detrusor muscle, respectively, mediate LUTS. Owing to these effects, R1-AR blockers were initially developed for the treatment of hypertension and, successively, of symptomatic BPH. Unfortunately, few ligands recognize only one R1-AR subtype and such a lack of selectivity limits their administration in vivo and their therapeutic use. Although the relative importance of the various adrenergic receptor subtypes is still controversial, selectivity for the R1A and/or R1D subtypes over the R1B subtype appears to be important for clinical “uroselectivity”, that is, high effectiveness in relieving LUTS with low incidence of cardiovascular and hypotensive adverse effects. Our research group has long been involved in designing new R1-AR ligands structurally related to 2-(2,6-dimethoxyphenoxy)ethylaminomethyl-1,4-benzodioxane (WB4101), a potent R1 antagonist displaying a slight selectivity for R1A- and, to a minor extent, for R1D-ARs with respect to R1B-AR and 5-HT1A serotoninergic receptor.3-8 Recently, we have demonstrated that it is possible to significantly modulate the affinity and selectivity * Corresponding author. Tel. +39-2-50317553; Fax: +39-2-50317565; e-mail:
[email protected]. † Universita ` degli Studi di Milano. ‡ Universita ` di Bologna. § Istituto di Ricerche Farmacologiche ‘Mario Negri’.
profile of (S)-WB4101, the more potent enantiomer, simply fusing a cyclohexane or an additional benzene ring with its benzodioxane or phenoxy moiety.6 Successively, by a wide number of ortho monosubstituted phenoxy analogues, we have proved that removal of one or both o-methoxy substituents adversely affects the affinity for the three R1-AR subtypes, but not for the 5-HT1A receptor, for which (S)-WB4101 analogues unsubstituted or suitably o-monosubstituted at the phenoxy moiety exhibit very high and significantly specific affinity.7 Consistent with such indications, the affinity data for a series of new ortho disubstituted analogues we have lately studied show that o-disubstitution and the presence of at least one o-methoxyl play a crucial role for the interaction of the phenoxy moiety with the R1-AR and, especially, with the R1a subtype.8 On these bases, we designed new WB4101 analogues 4-8 (Chart 1) in order to further investigate the consequences of different patterns of substitution, in particular with saturated or unsaturated carbocycles, at the phenoxy and benzodioxane moieties. The hypothesis was that such modifications, as those previously carried out and resulting in compounds 1-3, would remarkably affect the affinity and selectivity profile and, hopefully, the R1 activity spectrum. The ultimate aim was to obtain potent and selective R1A and/or R1D antagonists. Here, we describe the synthesis of the S and R enantiomers of 4-8 and the biological profile of such novel compounds and of closely related congeners 1-3 (Chart 1) in binding and functional experiments at R1-AR subtypes and the 5-HT1A receptor. Furthermore, the nature of inverse agonist of WB4101 enantiomers and of the most potent and/or selective derivatives in the present series was studied and semiquantitatively determined by means of a vascular model. Finally, the SAFIRa and SAR data were comparatively analyzed. a
Abbreviations: SAFIR, structure affinity relationship.
10.1021/jm060358r CCC: $33.50 © 2006 American Chemical Society Published on Web 11/03/2006
Subtype SelectiVe R-Adrenoreceptor Antagonists
Chart 1
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 24 7141
Scheme 2. Synthesis of Compound 9a
a Reagents and conditions: (a) AcCl, Py, DCM; (b) AlCl , dichloroben3 zene; (c) benzyl bromide, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (d) m-CPBA, EtOAc; (e) 2.5 N NaOH, MeOH; (f) MeI, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (g) H2, Pd/C, EtOAc; (h) ethyl chloroacetate, NaH, DMSO; (i) LiAlH4, THF; (j) mesyl chloride, Et3N, DCM.
Scheme 1. Synthesis of Compounds (S)-4-(S)-8.
Scheme 3. Synthesis of Compound 10a
a Reagents and conditions: (a) AcCl, Py, DCM; (b) AlCl , dichloroben3 zene; (c) benzyl bromide, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (d) m-CPBA, EtOAc; (e) 2.5 N NaOH, MeOH; (f) MeI, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (g) H2, Pd/C, acetone; (h) 1,2dibromoethane, tetrabutylammonium bromide, 2.5 N NaOH, DCM.
Chemistry Of compounds 1-8, which are required for the current study, 1-3 have been previously reported in both enantiomeric forms,6 while 4-8 are new. The enantiomeric pairs of the latter were prepared by the same strategy as the former: enantiopure 2-aminomethyl- or 2-mesyloxymethyl-substituted benzodioxane or tetrahydronaphthodioxane, whose syntheses we have already described,6,9,10 was reacted with 2-aryloxyethyl mesylate 9 or bromide 10 or with 2-aryloxyethylamines 11-13, as exemplified in Scheme 1 for the S isomers. Mesylate 9 was prepared as outlined in Scheme 2. Esterification of 5,6,7,8-tetrahydro-1-naphthol with acetyl chloride, followed by Fries rearrangement, afforded 15, which was benzylated and converted to 1-benzyloxy-5,6,7,8-tetrahydro-2naphthol (17) by oxidation to acetate and ester hydrolysis. This was O-methylated and the resultant diether 18 debenzylated, yielding 2-methoxy-5,6,7,8-tetrahydro-1-naphthol (19), whose sodium salt was reacted with ethyl chloroacetate to obtain tetrahydronaphthoxyacetate 20. Successive reduction of the ester to alcohol 21 and final mesylation of the latter led to 9.
Bromide 10 was synthesized from 2-phenylphenol by the reaction sequence illustrated in Scheme 3: (a) esterification with acetyl chloride, (b) Fries rearrangement, (c) O-benzylation of 2-phenyl-6-acetylphenol (23), (d) oxidation to acetate and ester hydrolysis, (e) O-methylation of 2-benzyloxy-3-phenylphenol (25), (f) debenzylation, and (g) reaction with dibromoethane to give 10. The syntheses of m- and p-phenyl-substituted o-methoxyphenoxyethylamines, 11 and 12, respectively, are shown in Scheme 4. They were both prepared from 2-acetyl-4-phenylphenol (29), which in turn was obtained from 4-phenylphenol by acetylation and subsequent Fries rearrangement. Phenol 29 was O-methylated, transformed by oxidation to the acetate and by ester hydrolysis into 2-methoxy-5-phenylphenol (31), and etherified with dibromoethane. The resultant 2-bromoethyl ether 32 was converted into azide 33 and finally reduced to amine 11. Alternatively, phenol 29 was O-benzylated, oxidized to the acetate, and hydrolyzed to 2-benzyloxy-5-phenylphenol 35, which was O-methylated and debenzylated to give 37, the isomer of 31 with exchanged methoxyl and hydroxyl positions. Like the latter, 37 was etherified with dibromoethane, converted into the azide, and finally reduced, yielding amine 12. Nucleophilic displacement of bromide by azide ion and successive reduction with hydrazine were also accomplished to synthesize amine 13 from 1-(2-bromoethoxy)-2-methoxynaphthalene, whose preparation has been previously reported.6 Biology Binding Experiments. As previously reported for the enantiomers of WB4101 and of compounds 1-3,6 the pharmacologi-
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PallaVicini et al.
Scheme 4. Synthesis of Compounds 11 and 12a
a Reagents and conditions: (a) AcCl, Py, DCM; (b) AlCl , dichloroben3 zene; (c) MeI, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (d) m-CPBA, EtOAc; (e) 2.5 N NaOH, MeOH; (f) 1,2-dibromoethane, KOH, DMSO; (g) NaN3, DMF; (h) hydrazine, PdO, MeOH; (i) benzyl bromide, tetrabutylammonium bromide, 2.5 N NaOH, DCM; (j) H2, Pd/C, MeOH; (k) 1,2-dibromoethane, tetrabutylammonium bromide, 2.5 N NaOH, DCM.
cal profile of the enantiomeric pairs of 4-8 was initially evaluated by measuring affinities for cloned human R1a-, R1b-, and R1d-AR subtypes and for native 5-HT1A serotoninergic receptor from rat hippocampus. [3H]prazosin was used to label cloned human R1-ARs expressed in Chinese hamster ovary (CHO) cells, while [3H]-8-OH-DPAT was the radioligand used in the binding assays at 5-HT1A receptor, carried out in membrane preparations from rat hippocampus. The affinity constants, expressed as pKi, of (S)- and (R)-WB4101 and of the enantiomers of 1-8 for these four receptors, are listed in Table 1. Functional Assays. R1-AR subtypes and R2-AR blocking activity of both enantiomers of WB4101 and of 2 and of the S isomers of compounds 1 and 3-8 was determined on different rat tissues. In particular, R1-AR subtypes blocking activity was assessed by antagonism of (-)-noradrenaline (NA)-induced contraction of vas deferens prostatic portion (R1A)11 or thoracic
aorta (R1D)12 and by antagonism to (-)-phenylephrine-induced contraction of spleen (R1B),12 while R2-adrenoreceptor blocking activity was determined by antagonism to clonidine-inhibited twitch responses of the field-stimulated prostatic portion of vas deferens. Furthermore, considering the demonstrated R1A-AR involvement in maintaining prostate smooth muscle tone and the consequent therapeutic potential of agents reducing the latter for LUTS, the antagonist affinity was also evaluated in rat prostate for the lead compound, its enantiomers, and (S)-8, which is the most R1A-AR selective compound in the series.13 All the antagonist affinities, expressed as pA2, of WB4101 and 2 enantiomers and of the S forms of 1 and 3-8 are listed in Table 2 together with those of WB4101 racemate, some of which have been previously reported. For WB4101, its enantiomers, (S)-2, (R)-2, (S)-5, and (S)-8, inverse agonism was assessed by preincubating calcium-depleted guinea pig thoracic aorta with different concentrations of such antagonists and then evaluating the corresponding diminutions of successively Ca2+-induced increase in the resting tone (IRT) of that tissue with respect to the IRT consequent on the same Ca2+administration, but in the absence of antagonist.14 The results are shown by the histogram represented in Figure 1. Discussion SARs. As previously reported,6 binding tests of (S)-1 and (S)-3 at the three cloned human R1-AR subtypes and 5-HT1A receptor had indicated that both the naphthodioxane and tetrahydronaphthodioxane derivatives have almost equally (about 2 orders of magnitude) decreased R1a, R1b, and 5-HT1A affinities with respect to (S)-WB4101, whereas, for R1d, the loss of affinity is near 3 orders of magnitude. Such a trend slightly improves the R1a/R1b and R1a/5-HT1A selectivities of (S)-WB4101 and produces, what is noteworthy, a moderate R1a/R1d selectivity (R1a/R1d affinity ratio of 12.3 and 13.5, respectively), of which the lead compound is completely devoid (R1a/R1d affinity ratio of 1.3), so that (S)-1 and (S)-3 have still good R1a affinity (pKi ) 7.47 and 7.60, respectively) and higher specificity for R1aAR than (S)-WB4101. Analogous enlargement had been accomplished at the phenoxy moiety of (S)-WB4101 by addition of a fused benzene ring, leading to (S)-2, which shows a significantly higher R1a affinity (pKi ) 8.80) than (S)-1 and (S)3, with only slightly decreased R1a specificity with respect to the latter [affinity ratios R1a/R1b of 10, R1a/R1d of 4, and R1a/
Table 1. Affinity Constants, Expressed as pKi (-log Ki, M), of WB4101 Enantiomers and of Compounds 1-8 for Cloned Human R1-Adrenoceptor Subtypes and 5-HT1A Receptora affinity ratiosb
pKi ((σEM) S-WB4101 R-WB4101 (S)-1 (R)-1 (S)-2 (R)-2 (S)-3 (R)-3 (S)-4 (R)-4 (S)-5 (R)-5 (S)-6 (R)-6 (S)-7 (R)-7 (S)-8 (R)-8 a
R1a
R1b
R1d
5-HT1A
R1a/R1b
R1a/R1d
R1a/5-HT1A
9.39 ( 0.06 7.95 ( 0.04 7.47 ( 0.05 6.99 ( 0.08 8.80 ( 0.09 8.34 ( 0.05 7.60 ( 0.15 7.00 ( 0.07 8.55 ( 0.15 7.94 ( 0.05 8.92 ( 0.12 8.03 ( 0.07 8.25 ( 0.14 7.79 ( 0.03 7.06 ( 0.03 6.63 ( 0.03 7.18 ( 0.01 6.49 ( 0.11
8.24 ( 0.04 7.14 ( 0.06 6.05 ( 0.04 6.37 ( 0.04 7.80 ( 0.07 7.07 ( 0.07 6.24 ( 0.03 6.40 ( 0.05 7.72 ( 0.07 7.31 ( 0.03 7.75 ( 0.05 6.82 ( 0.06 7.96 ( 0.14 7.21 ( 0.07 6.73 ( 0.12 6.26 ( 0.15 6.43 ( 0.04 6.26 ( 0.10
9.29 ( 0.11 7.98 ( 0.08 6.38 ( 0.04 6.53 ( 0.04 8.18 ( 0.13 7.65 ( 0.05 6.47 ( 0.06 6.69 ( 0.04 7.84 ( 0.08 7.80 ( 0.04 8.47 ( 0.09 7.54 ( 0.07 7.82 ( 0.07 7.27 ( 0.05 6.67 ( 0.07 6.44 ( 0.06 6.83 ( 0.03 6.02 ( 0.11
8.61 ( 0.04 7.39 ( 0.03 6.46 ( 0.04 6.00 ( 0.04 7.95 ( 0.06 7.17 ( 0.04 6.44 ( 0.03 6.30 ( 0.03 8.07 ( 0.08 6.88 ( 0.04 8.16 ( 0.07 7.11 ( 0.06 7.28 ( 0.04 6.44 ( 0.04 7.13 ( 0.02 6.06 ( 0.06 5.77 ( 0.02 5.44 ( 0.06
14.1 6.5 26.3 4.2 10.0 18.6 22.9 4.0 6.8 4.3 14.8 16.2 1.9 3.8 2.1 2.3 5.6 1.7
1.3 0.9 12.3 2.9 4.2 4.9 13.5 2.0 5.1 1.3 2.8 3.1 2.7 3.3 2.5 1.5 2.2 3.0
6.0 3.6 10.2 9.8 7.1 14.8 14.5 5.0 3.0 11.5 5.8 8.3 9.3 22.4 0.9 3.7 25.7 11.2
Data of WB4101 and 1-3 are reported in ref 6. b Antilog of ∆pKi.
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Table 2. Antagonist Affinities, Expressed as pA2, of WB4101, Its Enantiomers, and Compounds 1-8 (S Isomers and, for 2, Also the R Isomer) at R1and R2-Adrenoceptors on Isolated Rat Tissues, Namely, Prostatic Vas Deferens (R1A and R2), Prostate (R1A), Spleen (R1B), and Thoracic Aorta (R1D) pA2a R1A compd
vas deferens
prostate
WB4101 S-WB4101 R-WB4101 (S)-1 (S)-2 (R)-2 (S)-3 (S)-4 (S)-5 (S)-6 (S)-7 (S)-8
9.36 ( 0.04 9.98 ( 0.01 7.79 ( 0.03 5.46 ( 0.02 8.96 ( 0.05 7.23 ( 0.07
