Sila-Trifluperidol, a Silicon Analogue of the Dopamine (D2) Receptor

Mar 5, 2010 - Antagonist Trifluperidol: Synthesis and Pharmacological ... Trifluperidol (2a) is a dopamine (D2) receptor antagonist of the butyropheno...
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Organometallics 2010, 29, 1652–1660 DOI: 10.1021/om901011t

Sila-Trifluperidol, a Silicon Analogue of the Dopamine (D2) Receptor Antagonist Trifluperidol: Synthesis and Pharmacological Characterization Reinhold Tacke,*,† Binh Nguyen,† Christian Burschka,† W. Peter Lippert,† Alexandra Hamacher,‡ Christian Urban,‡ and Matthias U. Kassack‡ †

Institut f€ ur Anorganische Chemie, Universit€ at W€ urzburg, Am Hubland, D-97074 W€ urzburg, Germany, and ‡ Institut f€ ur Pharmazeutische und Medizinische Chemie, Universit€ at D€ usseldorf, Universit€ atsstrasse 1, D-40225 D€ usseldorf, Germany Received November 23, 2009

Trifluperidol (2a) is a dopamine (D2) receptor antagonist of the butyrophenone type. Carbon/ silicon exchange (sila-substitution) in the 4-position of the piperidine ring of 2a (R3COH f R3SiOH) results in sila-trifluperidol (2b). The silanol 2b was synthesized in a multistep synthesis, starting from triethoxy(vinyl)silane, and was isolated as the hydrochloride 2b 3 HCl. Compound 2b 3 HCl was structurally characterized by single-crystal X-ray diffraction and solution NMR spectroscopy, and the stability of the silanol 2b in aqueous solutions at different pH values was studied by ESI-MS experiments. In addition, the pharmacological profiles of the C/Si analogues trifluperidol (2a) and sila-trifluperidol (2b) and of the related compounds haloperidol (1a) and sila-haloperidol (1b) were studied in functional receptor assays at human dopamine D1 and D2 receptors. Sila-substitution of 1a (f 1b) and 2a (f 2b) affects the pharmacological properties significantly.

Introduction In the late 1950s, butyrophenones (1-phenyl-1-butanones) have been recognized as potent neuroleptic agents and since then have been studied extensively.1-11 The dopamine (D2) receptor antagonists haloperidol (1a) and trifluperidol (2a) are prominent representatives of this class of drugs. Haloperidol is still used clinically in the treatment of schizophrenia, although it may cause severe extrapyramidal side effects (EPSs) including parkinsonism and tardive dyskinesia.12,13 *To whom correspondence should be addressed. Phone: þ49-931-3185250. Fax: þ49-931-888-4609. E-mail: [email protected]. (1) Janssen, P. A. J.; van de Westeringh, C.; Jageneau, A. H. M.; Demoen, P. J. A.; Hermans, B. K. F.; van Daele, G. H. P.; Schellekens, K. H. L.; van der Eycken, C. A. M.; Niemegeers, C. J. E. J. Med. Pharm. 1959, 1, 281–297. (2) Janssen, P. A. J. Arzneim.-Forsch. 1961, 11, 819–824. (3) Gallant, D. M.; Bishop, M. P.; Timmons, E.; Steele, C. A. Curr. Ther. Res. 1963, 5, 463–471. (4) Pratt, J. P.; Bishop, M. P.; Gallant, D. M. Curr. Ther. Res. 1964, 6, 562–571. (5) Janssen, P. A. J. Int. Rev. Neurobiol. 1965, 8, 221–263. (6) Niemegeers, C. J. E.; Janssen, P. A. J. Psychopharmacologia (Berlin) 1965, 8, 263–270. (7) Soudijn, W.; Van Wijngaarden, I.; Allewijn, F. Eur. J. Pharmacol. 1967, 1, 47–57. (8) Janssen, P. A. J.; Allewijn, T. N. Arzneim.-Forsch. 1969, 19, 199– 208. (9) Lewi, P. J.; Heykants, J. J. P.; Janssen, P. A. J. Arzneim.-Forsch. (Drug Res.) 1970, 20, 1701–1705. (10) Givant, Y.; Shani, J.; Goldhaber, G.; Serebrenik, R.; Sulman, F. G. Arch. Int. Pharmacodyn. 1973, 205, 317–327. (11) Honma, T.; Sasajima, K.; Ono, K.; Kitagawa, S.; Inaba, S.; Yamamoto, H. Arzneim.-Forsch. (Drug Res.) 1974, 24, 1248–1256. (12) Levinson, D. F. Clin. Ther. 1991, 13, 326–352. (13) Casey, D. E. Int. Clin. Psychopharmacol. 1995, 10, 105–114. pubs.acs.org/Organometallics

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These side effects (which have been correlated with neurotoxic effects of a pyridinium-type metabolite of 1a; in this context, see refs 14 and 15 and references therein) present a disadvantage in therapy and give motivation for the search of analogues that cause less EPSs. In this context, we have recently synthesized a silicon analogue of haloperidol (1a), sila-haloperidol (1b),14,16,17 and have evaluated the pharmacological properties of the C/Si analogues 1a and 1b.14,17 Whereas in competitive binding assays at human dopamine receptors (hD1-hD5) sila-haloperidol (1b) showed a significantly (5-fold) higher affinity for hD2 receptors than haloperidol (1a), the silicon compound 1b was approximately equipotent to its carbon analogue 1a at all other human dopamine receptors. Furthermore, the subtype selectivity of 1b for hD2 over the other human dopamine receptors was somewhat higher than that of 1a. As already claimed on the basis of theoretical considerations some years ago,17 we have recently also demonstrated that the in vitro metabolic pathway of the C/Si analogues 1a and 1b (phase I and phase II metabolism) is totally different.14,15 A major phase I metabolite of haloperidol (1a), the pyridinium-type metabolite that is claimed to be responsible for the neurotoxic side effects of 1a, is not formed in microsomal incubations (rat (14) Tacke, R.; Popp, F.; M€ uller, B.; Theis, B.; Burschka, C.; Hamacher, A.; Kassack, M. U.; Schepmann, D.; W€ unsch, B.; Jurva, U.; Wellner, E. ChemMedChem 2008, 3, 152–164. (15) Johansson, T.; Weidolf, L.; Popp, F.; Tacke, R.; Jurva, U. Drug Metab. Dispos. 2010, 38, 73–83. (16) Tacke, R.; Heinrich, T. (Amedis Pharmaceuticals Ltd., U.K.) U.K. Patent Appl. GB 2382575A (June 4, 2003). (17) Tacke, R.; Heinrich, T.; Bertermann, R.; Burschka, C.; Hamacher, A.; Kassack, M. U. Organometallics 2004, 23, 4468–4477. r 2010 American Chemical Society

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and human liver microsomes) with sila-haloperidol (1b).15 For sila-haloperidol, three metabolites originating from opening of the piperidine ring were observed, a metabolism that has not been observed for the carbon analogue haloperidol. In addition, one significant phase II metabolite of haloperidol is the glucuronide of the hydroxyl group bound to the piperidine ring, whereas the analogous metabolite was not observed for sila-haloperidol in rat, dog, and human hepatocytes.15

In continuation of these studies, we have now succeeded in synthesizing sila-trifluperidol (2b), a silicon analogue of trifluperidol18 (2a) (which also shows the above-mentioned unfavorable EPSs), and we have studied the inhibitory potencies of the C/Si analogues 2a and 2b at human dopamine D1 and D2 receptors using a functional assay. For reasons of comparison, compounds 1a and 1b were included in these studies. We report here on the synthesis of 2b (isolated as 2b 3 HCl), the crystal structure analyses of 2a 3 HCl and 2b 3 HCl, solution NMR studies of 2a 3 HCl and 2b 3 HCl (solvent, [D6]DMSO), and the pharmacological (18) (a) Wragg, W. R.; Ash, A. S. F.; Creighton, A. M. (May & Baker Ltd., U.K.) U.K. Patent Appl. GB 948,071 (January 29, 1964). (b) Nakatsuka, I.; Kawahara, K.; Kamada, T.; Yoshitake, A. J. Labelled Compd. Radiopharm. 1978, 14, 133–140. (19) Recent original publications dealing with sila-substituted drugs: (a) Daiss, J. O.; Burschka, C.; Mills, J. S.; Montana, J. G.; Showell, G. A.; Fleming, I.; Gaudon, C.; Ivanova, D.; Gronemeyer, H.; Tacke, R. Organometallics 2005, 24, 3192–3199. (b) Daiss, J. O.; Burschka, C.; Mills, J. S.; Montana, J. G.; Showell, G. A.; Warneck, J. B. H.; Tacke, R. Organometallics 2006, 25, 1188–1198. (c) Showell, G. A.; Barnes, M. J.; Daiss, J. O.; Mills, J. S.; Montana, J. G.; Tacke, R.; Warneck, J. B. H. Bioorg. Med. Chem. Lett. 2006, 16, 2555–2558. (d) Ilg, R.; Burschka, C.; Schepmann, D.; W€ unsch, B.; Tacke, R. Organometallics 2006, 25, 5396– 5408. (e) B€ uttner, M. W.; Burschka, C.; Daiss, J. O.; Ivanova, D.; Rochel, N.; Kammerer, S.; Peluso-Iltis, C.; Bindler, A.; Gaudon, C.; Germain, P.; Moras, D.; Gronemeyer, H.; Tacke, R. ChemBioChem 2007, 8, 1688–1699. (f) Warneck, J. B.; Cheng, F. H. M.; Barnes, M. J.; Mills, J. S.; Montana, J. G.; Naylor, R. J.; Ngan, M.-P.; Wai, M.-K.; Daiss, J. O.; Tacke, R.; Rudd, J. A. Toxicol. Appl. Pharmacol. 2008, 232, 369–375. (g) Lippert, W. P.; Burschka, C.; G€ otz, K.; Kaupp, M.; Ivanova, D.; Gaudon, C.; Sato, Y.; Antony, P.; Rochel, N.; Moras, D.; Gronemeyer, H.; Tacke, R. ChemMed Chem 2009, 4, 1143–1152. (h) Tacke, R.; M€uller, V.; B€uttner, M. W.; Lippert, W. P.; Bertermann, R.; Daiss, J. O.; Khanwalkar, H.; Furst, A.; Gaudon, C.; Gronemeyer, H. ChemMedChem 2009, 4, 1797–1802. (20) Reviews: (a) Tacke, R.; Linoh, H. In The Chemistry of Organic Silicon Compounds, Part 2; Patai, S., Rappoport, Z., Eds.; Wiley & Sons: Chichester, 1989; pp 1143-1206. (b) Bains, W.; Tacke, R. Curr. Opin. Drug Discovery Dev. 2003, 6, 526–543. (c) Showell, G. A.; Mills, J. S. Drug Discovery Today 2003, 8, 551–556. (d) Mills, J. S.; Showell, G. A. Expert Opin. Invest. Drugs 2004, 13, 1149–1157. (e) Pooni, P. K.; Showell, G. A. Mini-Rev. Med. Chem. 2006, 6, 1169–1177. (f) Sieburth, S. McN.; Chen, C.-A. Eur. J. Org. Chem. 2006, 311–322. (g) Gately, S.; West, R. Drug Dev. Res. 2007, 68, 156–163. (h) Franz, A. K. Curr. Opin. Drug Discovery Dev. 2007, 10, 654–671.

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characterization of the C/Si pairs 1a/1b and 2a/2b. These studies were performed as part of our systematic investigations on sila-substituted drugs19 (for recent reviews on silicon-based drugs, see ref 20).

Results and Discussion Syntheses. Sila-trifluperidol (2b) was synthesized in a multistep synthesis, starting from triethoxy(vinyl)silane (3), and was isolated as the hydrochloride 2b 3 HCl (Scheme 1). In this synthesis, the use of the 2,4,6-trimethoxyphenyl moiety as a protecting group for silicon played a key role.21 Thus, treatment of 3 with [3-(trifluoromethyl)phenyl]magnesium bromide gave diethoxy[3-(trifluoromethyl)phenyl]vinylsilane (4) (84% yield), which upon reaction with (2,4,6-trimethoxyphenyl)lithium afforded the corresponding (2,4,6trimethoxyphenyl)silane 5 (70% yield). Treatment of 5 with vinylmagnesium chloride gave the divinylsilane 6 (92% yield), which upon reaction with 9-borabicyclo[3.3.1]nonane (9-BBN), followed by sequential treatment with aqueous solutions of sodium hydroxide and hydrogen peroxide, gave the corresponding bis(2-hydroxyethyl)silane 7 (67% yield). Reaction of 7 with methanesulfonyl chloride, in the presence of triethylamine, afforded the corresponding bis[2-(methanesulfonyloxy)ethyl]silane (8), which was pure enough to be used in the following step without further purification. Thus, treatment of 8 with 3-[2-(4-fluorophenyl)-1,3dioxolan-2-yl]propylamine (9) gave the cyclization product, the 4-silapiperidine 10 (37% yield). Deprotection of 10 by treatment with hydrochloric acid finally afforded the title compound 2b, which was isolated as the hydrochloride 2b 3 HCl (74% yield). The identities of 2b 3 HCl, 4-7, and 10 were established by elemental analyses (C, H, N) and NMR studies (1H, 13C, 19F, 29 Si). Compound 2b 3 HCl was additionally characterized by single-crystal X-ray diffraction. Crystal Structure Analyses. The C/Si analogues trifluperidol hydrochloride (2a 3 HCl) and sila-trifluperidol hydrochloride (2b 3 HCl) were structurally characterized by single-crystal X-ray diffraction. The crystal data and the experimental parameters used for these studies are summarized in Table 1; selected bond lengths and angles are given in Table 2. The cations of 2a 3 HCl and 2b 3 HCl are depicted in Figures 1 and 2, respectively. As shown in Figures 1 and 2, the piperidinium ring of 2a 3 HCl and the 4-silapiperidinium ring of 2b 3 HCl adopt a chair conformation, with the exocyclic N-organyl group and the 3-(trifluoromethyl)phenyl group in the equatorial positions. The bond lengths and angles of both compounds are in the expected ranges and therefore do not require any further discussion. Superposition of the structures of the cations of the C/Si analogues 2a 3 HCl and 2b 3 HCl (Figure 3) reveals significant differences in the molecular shape. Due to the longer covalent radius of the silicon atom, the 4-silapiperidinium skeleton of 2b 3 HCl is more “flattened” than the piperidinium skeleton of 2a 3 HCl. As a consequence, the C/Si analogues differ in their relative orientation of the N-organyl group toward the hydroxy and 3-(trifluoromethyl)phenyl groups. Analogous stereochemical features have been observed for the related C/Si analogues 1a 3 HCl and 1b 3 HCl.17 (21) Popp, F.; N€atscher, J. B.; Daiss, J. O.; Burschka, C.; Tacke, R. Organometallics 2007, 26, 6014–6028.

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Tacke et al. Scheme 1

Table 1. Crystal Data and Experimental Parameters for the Crystal Structure Analyses of 2a 3 HCl and 2b 3 HCl empirical formula formula mass, g mol-1 collection T, K λ(Mo KR), A˚ cryst syst space group (No.) a, A˚ b, A˚ c, A˚ β, deg V, A˚3 Z D(calcd), g cm-3 μ, mm-1 F(000) cryst dimens, mm 2θ range, deg index ranges

2a 3 HCl

2b 3 HCl

C22H24ClF4NO2 445.87 100(2) 0.71073 monoclinic C2/c (15) 37.5474(19) 7.0100(4) 15.8691(9) 91.993(3) 4174.3(4) 8 1.419 0.237 1856 0.27  0.15  0.10 4.34-66.92 -57 e h e 58, -7 e k e 10, -24 e l e 22 76 705 8096 0.0439 8096 0 277 1.098 0.0600/3.3470 0.0394 0.1191

C21H24ClF4NO2Si 461.95 193(2) 0.71073 monoclinic P21 (4) 18.074(3) 7.0906(7) 18.197(3) 110.189(17) 2188.8(5) 4 1.402 0.281 960 0.5  0.3  0.2 4.80-58.40 -24 e h e 24, -9 e k e 9, -24 e l e 24 23 053 11 269 0.0383 11 269 326 766 0.991 0.0697/0.0000 0.0444 0.1194 0.07(6) þ0.428/-0.278

no. of collected reflns no. of indep reflns Rint no. of reflns used no. of restraints no. of params Sa weight params a/bb R1c [I > 2σ(I)] wR2d (all data) absolute struct param þ0.638/-0.442 max./min. residual electron density, e A˚-3 P a S = { [w(Fo2 - Fc2)2]/(n - p)}0.5; n = no. of reflections; p = no. of b -1 2 2 2 2 2 w =σ parameters. o ,0)þ 2Fc ]/3. P P (Fo ) þ (aP)Pþ bP, with P=[max(F P c R1= ||Fo|-|Fc||/ |Fo|. d wR2={ [w(Fo2-Fc2)2]/ [w(Fo2)2]}0.5.

NMR Studies. The 1H, 13C, 19F, and 29Si NMR spectra of the C/Si analogues 2a 3 HCl and 2b 3 HCl revealed the

Table 2. Selected Bond Lengths (A˚) and Angles (deg) for the Piperidinium Skeleton of 2a 3 HCl and the 4-Silapiperidinium Skeleton of 2b 3 HCl 2a 3 HCl C-O1 C-C1 C-C8 C-C11 N-C9 N-C10 N-C12 C8-C9 C10-C11 O1-C-C1 O1-C-C8 O1-C-C11 C1-C-C8 C1-C-C11 C8-C-C11 C9-N-C10 C9-N-C12 C10-N-C12 C-C8-C9 C-C11-C10 N-C9-C8 N-C10-C11

2b 3 HCl 1.4292(11) 1.5276(13) 1.5327(14) 1.5370(14) 1.4998(12) 1.5014(13) 1.4988(12) 1.5233(13) 1.5223(13) 111.20(7) 105.29(8) 109.96(8) 111.03(8) 110.20(8) 109.03(7) 109.97(7) 111.08(7) 112.15(7) 111.94(8) 111.89(8) 110.39(8) 110.13(8)

Si-O1 Si-C1 Si-C8 Si-C11 N-C9 N-C10 N-C12 C8-C9 C10-C11 O1-Si-C1 O1-Si-C8 O1-Si-C11 C1-Si-C8 C1-Si-C11 C8-Si-C11 C9-N-C10 C9-N-C12 C10-N-C12 Si-C8-C9 Si-C11-C10 N-C9-C8 N-C10-C11

1.639(2) 1.868(3) 1.866(3) 1.877(3) 1.509(3) 1.505(3) 1.506(3) 1.516(4) 1.522(4) 105.89(12) 113.65(12) 112.30(13) 109.73(12) 112.94(13) 102.50(12) 113.6(2) 112.3(2) 111.8(2) 114.31(17) 110.88(19) 112.3(2) 112.0(2)

existence of two isomers of the respective cations in solution (solvent, [D6]DMSO), with molar ratios of 12:1 (2a 3 HCl) and 2:1 (2b 3 HCl), respectively (Figure 4). These different molar ratios indicate considerable differences in the energies of the respective two isomers of the piperidinium and 4silapiperidinium skeleton. These results are in agreement with those obtained in solution NMR studies of the related C/Si analogues 1a 3 HCl and 1b 3 HCl.17 The existence of the two isomers of 2b 3 HCl was confirmed by 2D 1H,1H EXSY NMR experiments (for details, see Experimental Section). Attempts to determine the structures of the two isomers (2ar/2aβ and 2br/2bβ; Figure 4) by additional NMR experiments (1H,1H NOESY NMR) failed. However, it is

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Figure 1. Molecular structure of the cation in the crystal of 2a 3 HCl.

Figure 2. Molecular structure of the cation in the crystal of 2b 3 HCl.

Figure 3. Superposition of the piperidinium skeleton of 2a 3 HCl (black bonds) and the 4-silapiperidinium skeleton of 2b 3 HCl (gray bonds). The hydrogen atoms are omitted for clarity.

Figure 5. ESI-MS spectra of 10 μM buffered aqueous solutions (pH 7.4) of 2b 3 HCl, showing the signal of the ammonium cation (m/z 426). The spectra were measured 15 min (left) and 24 h (right) after sample preparation at 20 °C (for details, see Experimental Section). Figure 4. The two isomers of the cations of the C/Si analogues 2a 3 HCl (2ar/2aβ) and 2b 3 HCl (2br/2bβ).

reasonable to assume that 2ar and 2br (with the N-organyl group in an equatorial position) are the dominating species in solution. The two isomers (2ar/2aβ; 2br/2bβ) can be interconverted by a process involving nitrogen deprotonation/reprotonation, nitrogen inversion, and ring inversion. ESI-MS Studies. To get information about the stability of 2b toward a potential condensation (disiloxane formation) in aqueous solution, ESI-MS experiments were performed. For this purpose, 10 μM buffered aqueous solutions of 2b 3 HCl at different pH values (pH 1.0, 5.0, 7.4, and 10.0) were analyzed. Some results of these studies are depicted in Figure 5. The mass spectra of the aqueous solutions of 2b 3 HCl at any pH value measured showed only the characteristic peak

for protonated sila-trifluperidol (ammonium cation; m/z 426); that is, no disiloxane formation was observed. This holds true for both freshly prepared samples and solutions that were kept at 20 °C for 24 h. The stability of the silanol in water could also be confirmed for solutions of 2b 3 HCl at higher concentrations (2.5 mM, pH 1 and pH 5; 1 mM, pH 7.4 and pH 10). In any case, no disiloxane formation could be observed. Functional Receptor Studies. The inhibitory potencies of haloperidol (1a), sila-haloperidol (1b), trifluperidol (2a), and sila-trifluperidol (2b) were studied at human dopamine D1 and D2 receptors using a calcium fluorimetric functional assay.22 Table 3 presents the Ki values of 1a, 1b, 2a, and 2b (22) Kassack, M. U.; H€ ofgen, B.; Lehmann, J.; Eckstein, N.; Quillan, J. M.; Sadee, W. J. Biomol. Screening 2002, 7, 233–246.

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Table 3. Inhibitory Potencies of 1a, 1b, 2a, and 2b at Human D1 and D2 Receptors

Table 4. Selectivity of 1a, 1b, 2a, and 2b for Human D1 over Human D2 Receptors

app. Ki [nM]a

ratio of Ki values

receptor subtype

1a

1b

2a

2b

selectivity

1a

1b

2a

2b

hD1 hD2

398 0.36

194 0.12

616 0.024

831 0.24

hD1/hD2

1110

1620

25 700

3460

a Values result from at least two independent experiments carried out in triplicate. Hill slopes were not significantly different from unity.

Table 5. Relative Potency of 1b, 2a, and 2b Compared to Haloperidol (1a) relative Kia receptor subtype

1a

1b

2a

2b

hD1 hD2

1.00 1.00

2.05 3.00

0.65 15.0

0.48 1.50

a Values are expressed as ratio of Ki (1a) over Ki (test compound) for each receptor subtype.

Figure 6. Concentration inhibition curves resulting from inhibition of the agonist (SKF38393, 100 nM)-induced signal using HEK293 cells recombinantly expressing hD1 receptors. Data are means ( SEM of at least two independent experiments performed in triplicate. Slopes are not significantly different from unity.

trifluperidol (2a f 2b) reduced this very high D2 selectivity by around 7-fold, whereas sila-substitution of haloperidol (1a f 1b) had almost no effect on the D2 selectivity (Table 4). Table 5 displays the relative potencies of the four compounds studied, with the potency of haloperidol (1a) set as 1. The remarkably higher potency of 2a compared to 1a found in this study by using a functional readout is in accordance with binding studies from the literature.23,24 The reason for the 10-fold loss of inhibitory potency at D2 receptors upon sila-substitution of trifluperidol (2a f 2b) remains unknown and is opposite of what was found for the sila-substitution of haloperidol (1a f 1b).

Conclusions

Figure 7. Concentration inhibition curves resulting from inhibition of the agonist (quinpirol, 30 nM)-induced signal using HEK293 cells recombinantly expressing hD2 receptors. Data are means ( SEM of at least two independent experiments performed in triplicate. Slopes are not significantly different from unity.

obtained in these studies. Hill slopes were not significantly different from unity, thus assuming a single binding site at all receptors. None of the compounds displayed any agonist activity at D1 or D2 receptors (not shown). The functional data for 1a and 1b were similar to the binding data reported previously.14 Figures 6 and 7 display the inhibition curves of 2a and 2b in comparison to 1a. Whereas sila-substitution of haloperidol (1a f 1b) increased the inhibitory potency by 2-fold (D1) and 3-fold (D2), sila-substitution of trifluperidol (2a f 2b) did not significantly change the Ki value at D1 receptors but surprisingly led to a 10-fold reduction of inhibition at D2 receptors. All compounds studied are more than 1000-fold selective for D2 over D1 receptors (Table 4). However, 2a shows the highest D2 selectivity (25 700-fold). Sila-substitution of

Sila-trifluperidol (2b), a silicon analogue of the dopamine (D2) antagonist trifluperidol (2a), was prepared from triethoxy(vinyl)silane in a multistep synthesis and was isolated as the hydrochloride 2b 3 HCl. In this synthesis, the use of the 2,4,6-trimethoxyphenyl moiety as a protecting group for silicon played a key role. The silanol 2b was found to be very stable in aqueous solution. As shown by ESI-MS studies, no silanol condensation (formation of the corresponding disiloxane) was observed in the pH range 1-10 at 20 °C. Crystal structure analyses of the C/Si analogues 2a 3 HCl and 2b 3 HCl showed that the piperidinium and 4-silapiperidinium ring, respectively, adopt a chair conformation, with the exocyclic N-organyl group and the 3-(trifluoromethyl)phenyl group in equatorial positions. Sila-substitution of 2a 3 HCl (f 2b 3 HCl) resulted in a more “flattened” heterocycle due to the larger covalent radius of silicon compared to carbon. NMR studies revealed the existence of two isomers of the cations of 2a 3 HCl and 2b 3 HCl in solution (solvent, [D6]DMSO), with molar ratios of 12:1 and 2:1, respectively. These different molar ratios indicate considerable differences in the energies of the respective two isomers of the piperidinium and silapiperidinium skeletons. These and other chemical and physiochemical sila-substitution effects (in this context, see also ref 20) may be responsible for the striking biological sila-substitution effects observed for the C/Si analogues trifluperidol (2a) and (23) Burstein, E. S.; Ma, J.; Wong, S.; Gao, Y.; Pham, E.; Knapp, A. E.; Nash, N. R.; Olsson, R.; Davis, R. E.; Hacksell, U.; Weiner, D. M.; Brann, M. R. J. Pharmacol. Exp. Ther. 2005, 315, 1278–1287. (24) Fernandez, J.; Alonso, J. M.; Andres, J. I.; Cid, J. M.; Dı´ az, A.; Iturrino, L.; Gil, P.; Megens, A.; Sipido, V. K.; Trabanco, A. A. J. Med. Chem. 2005, 48, 1709–1712.

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sila-trifluperidol (2b). Functional receptor studies with 2a and 2b at human dopamine D1 and D2 receptors demonstrated that the carbon/silicon switch led to a 10-fold reduction of inhibition at D2 receptors, whereas the inhibitory potency of 2a and 2b at the D1 receptor was not significantly changed; that is, sila-substitution of 2a (f2b) significantly reduced the D2 selectivity by a factor of 7. In this context, it is interesting to note that the sila-substitution of haloperidol 1a (f1b) resulted in an increased inhibitory potency at both D1 (2-fold) and D2 receptors (3-fold); that is, in this case the carbon/silicon switch does not affect D2 selectivity. A satisfactory explanation for this finding is still missing. In conclusion, the studies presented here again demonstrate that the carbon/silicon switch strategy is a powerful tool for drug design. Replacement of a carbon atom by a silicon atom in a known drug changes the geometric and electronic properties and thereby the size, shape, conformational behavior, lipophilicity, and chemical reactivity of the molecule. This can affect the drug-receptor interaction and hence change the pharmacodynamics of the drug. Further, as shown for the C/Si pairs 1a/1b and 2a/2b, carbon/silicon exchange can also significantly change the phase I and phase II metabolism of drugs.

Experimental Section Syntheses. General Procedures. All syntheses were carried out under dry nitrogen. The organic solvents used were dried and purified according to standard procedures and stored under dry nitrogen. A B€ uchi GKR 50 apparatus was used for the bulbto-bulb distillations. Melting points were determined with a B€ uchi melting point B-540 apparatus using samples in sealed glass capillaries. The 1H, 13C, 19F, and 29Si NMR spectra were recorded at 23 °C on a Bruker DRX-300 (1H, 300.1 MHz; 13C, 75.5 MHz; 19F, 282.4 MHz; 29Si, 59.6 MHz), a Bruker Avance 400 (1H, 400.1 MHz; 13C, 100.6 MHz; 19F, 376.5 MHz; 29Si, 79.5 MHz), or a Bruker Avance 500 NMR spectrometer (1H, 500.1 MHz; 13C, 125.8 MHz; 29Si, 99.4 MHz). C6D6, CD2Cl2, or [D6]DMSO were used as the solvent. Chemical shifts (ppm) were determined relative to internal C6HD5 (1H, δ 7.28; C6D6), C6D6 (13C, δ 128.0; C6D6), CHDCl2 (1H, δ 5.32; CD2Cl2), CD2Cl2 (13C, δ 53.8; CD2Cl2), [D5]DMSO (1H, δ 2.49; [D6]DMSO), [D6]DMSO (13C, δ 39.5; [D6]DMSO), external CFCl3 (19F, δ 0; C6D6, CD2Cl2, [D6]DMSO), or external TMS (29Si, δ 0; C6D6, CD2Cl2, [D6]DMSO). Analysis and assignment of the 1H NMR data were supported by 1H,1H gradient-selected COSY, 13C,1H gradient-selected HMQC and gradient-selected HMBC, and 29 Si,1H gradient-selected HMQC (optimized for 2JSiH =7 Hz). Assignment of the 13C NMR data was supported by DEPT 135 and the aforementioned 13C,1H correlation experiments. Preparation of Trifluperidol Hydrochloride (2a 3 HCl). This compound was synthesized according to ref 18. Preparation of 4-Hydroxy-1-[4-oxo-4-(4-fluorophenyl)butyl]4-[3-(trifluoromethyl)phenyl]-4-silapiperidinium Chloride (2b 3 HCl). A solution of 10 (580 mg, 936 μmol) and 2 M hydrochloric acid (950 μL, 1.90 mmol of HCl) in acetone (10 mL) was heated under reflux for 3 h and was then added dropwise at 20 °C with stirring to diethyl ether (70 mL). The resulting mixture was stirred at 20 °C for a further 10 min, and the precipitate was isolated by centrifugation and then further purified by resuspension in diethyl ether (7 mL) and subsequent isolation by centrifugation. This purification procedure was repeated three times, and the resulting product was then dried in vacuo (0.1 mbar, 40 °C, 7 h) to give analytically pure 2b 3 HCl in 74% yield as a colorless solid (320 mg, 694 μmol). The product may be recrystallized from 2-propanol/water (15:10 (v/v)) to afford 2b 3 HCl as a colorless crystalline solid in 49% yield; mp 151-152 °C. 1H NMR

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(500.1 MHz, [D6]DMSO; data for two isomers): δ 1.07-1.13, 1.28-1.43, and 1.57-1.64 (m, 4 H, SiCH2CH2N), 1.98-2.04 and 2.06-2.12 (m, 2 H, NCH2CH2CH2C), 3.13-3.22 (m, 4 H, NCH2CH2CH2C), 3.26-3.32, 3.35-3.43, 3.57-3.62, and 3.68-3.70 (m, 4 H, SiCH2CH2N), 6.94 and 6.98 (s, 1 H, OH), 7.34-7.39 (m, 2 H, H-3/H-5, CC6H4F), 7.64-7.69 (m, 1 H, H-5, SiC6H4(CF3)), 7.79-7.83 (m, 1 H, H-4, SiC6H4(CF3)), 7.96-7.98 (m, 1 H, H-2, SiC6H4(CF3)), 7.99-8.01 (m, 1 H, H6, SiC6H4(CF3)), 8.03-8.09 (m, 2 H, H-2/H-6, CC6H4F), 10.6 and 10.7 (br s, 1 H, NH). 13C NMR (125.8 MHz, [D6]DMSO; data for two isomers, the dominating isomer marked with an asterisk [*], except for those signals that could not be resolved): δ 9.3 and 11.7* (SiCH2CH2N), 18.0* and 18.3 (NCH2CH2CH2C), 35.1 and 35.3* (NCH2CH2CH2C), 50.4 and 51.6* (SiCH2CH2N), 51.8 and 55.8* (NCH2CH2CH2C), 115.7 (d, 2 JCF =21.7 Hz, C-3/C-5, CC6H4F), 124.3 (q, 1JCF =272.7 Hz, CF3), 126.7-126.9 (m, C-4, SiC6H4(CF3)), 128.6 (q, 2JCF=31.2 Hz, C-3, SiC6H4(CF3)), 128.8* and 128.9 (C-5, SiC6H4(CF3)), 129.7-129.9 (m, C-2, SiC6H4(CF3)), 130.86 and 130.90* (d, 3JCF = 9.7 Hz, C-2/C-6, CC6H4F), 133.2 (d, 4JCF = 2.7 Hz, C-1, CC6H4F), 137.0 and 137.4* (C-1, SiC6H4(CF3)), 137.7* and 137.8 (C-6, SiC6H4(CF3)), 165.1 (d, 1JCF = 251.5 Hz, C-4, CC6H4F), 197.33 and 197.34* (CO). 19F NMR (376.5 MHz, [D6]DMSO; data for two isomers, the dominating isomer marked with an asterisk [*]): δ -105.98* and -105.97 (CC6H4F), -61.1* and -61.0 (CF3). 29Si NMR (99.4 MHz, [D6]DMSO; data for two isomers, the dominating isomer marked with an asterisk [*]): δ -11.0* and -10.6. Anal. Calcd for C21H24ClF4NO2Si (461.96): C, 54.60; H, 5.24; N, 3.03. Found: C, 54.3; H, 5.2; N, 3.0. Triethoxy(vinyl)silane (3). This compound was commercially available. Preparation of Diethoxy[3-(trifluoromethyl)phenyl]vinylsilane (4). A solution of [3-(trifluoromethyl)phenyl]magnesium bromide (prepared from 1-bromo-(3-trifluoromethyl)benzene (50.6 g, 225 mmol) and magnesium turnings (5.67 g, 233 mmol) in diethyl ether (275 mL)) was added dropwise at 0 °C within 90 min to a stirred solution of 3 (157 g, 825 mmol) in diethyl ether (400 mL). After the addition was complete, the reaction mixture was allowed to warm to 20 °C and was then stirred at this temperature for a further 20 h. n-Pentane (700 mL) was added, and the resulting suspension was stirred at 20 °C for 1 h. The precipitate was removed by filtration, washed with n-pentane (3  50 mL), and discarded. The filtrate and the wash solutions were combined, the solvents were removed under reduced pressure, and the residue was distilled under reduced pressure to give 92.8 g (488 mmol) of the nonreacted starting material 3. The higher boiling residue was purified by bulb-to-bulb distillation (bp 75-80 °C/0.1 mbar) to give 4 in 84% yield as a colorless liquid (55.0 g, 189 mmol). 1H NMR (500.1 MHz, CD2Cl2): δ 1.25 (δX), 3.84 (δA), and 3.87 (δB) (ABX3 system, 2JAB=10.3 Hz, 3 JAX,BX = 7.0 Hz, 10 H, SiOCHAHBC(HX)3), 5.96 (δA), 6.14 (δB), and 6.22 (δC) (ABC system, 2JAC=4.4 Hz, 3JAB=19.9 Hz, 3 JBC=15.0 Hz, 3 H, SiCHBdCHCHA), 7.51-7.55 (m, 1 H, H-5, SiC6H4(CF3)), 7.68-7.70 (m, 1 H, H-4, SiC6H4(CF3)), 7.84-7.86 (m, 1 H, H-6, SiC6H4(CF3)), 7.90-7.92 (m, 1 H, H-2, SiC6H4(CF3)). 13C NMR (125.8 MHz, CD2Cl2): δ 18.5 (OCH2CH3), 59.3 (OCH2CH3), 124.9 (q, 1JCF = 272.4 Hz, CF3), 127.1 (q, 3JCF = 3.8 Hz, C-4, SiC6H4(CF3)), 128.5 (C-5, SiC6H4(CF3)), 130.2 (q, 2JCF = 31.7 Hz, C-3, SiC6H4(CF3)), 131.4 (q, 3JCF =3.8 Hz, C-2, SiC6H4(CF3)), 131.9 (SiCHdCH2), 135.5 (C-1, SiC6H4(CF3)), 137.7 (SiCHdCH2), 138.4 (q, 5JCF = 1.4 Hz, C-6, SiC6H4(CF3)). 19F NMR (376.5 MHz, CD2Cl2): δ -63.0. 29Si NMR (99.4 MHz, CD2Cl2): δ -34.6. Anal. Calcd for C13H17F3O2Si (290.36): C, 53.78; H, 5.90. Found: C, 53.5; H, 6.0. Preparation of Ethoxy[3-(trifluoromethyl)phenyl](2,4,6-trimethoxyphenyl)vinylsilane (5). A 2.5 M solution of n-butyllithium in hexanes (30 mL, 75 mmol of n-BuLi) was added dropwise at 20 °C within 10 min to a stirred mixture of 1,3,5-

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trimethoxybenzene (10.2 g, 60.6 mmol), 1,2-bis(dimethylamino)ethane (TMEDA; 7.39 g, 63.6 mmol), n-hexane (10 mL), and diethyl ether (30 mL). The resulting suspension was stirred at 20 °C for 16 h and was then added dropwise at 0 °C within 15 min to a stirred solution of 4 (17.6 g, 60.6 mmol) in diethyl ether (60 mL). After the addition was complete, the reaction mixture was allowed to warm to 20 °C and was stirred at this temperature for 2.5 h. The precipitate was removed by filtration, washed with diethyl ether (3  10 mL), and discarded. The filtrate and wash solutions were combined, the solvents were removed under reduced pressure, and the oily residue was purified by bulbto-bulb distillation (bp 155-160 °C/0.06 mbar) to give 5 in 70% yield as a colorless viscous liquid (17.5 g, 42.4 mmol). 1H NMR (500.1 MHz, CD2Cl2): δ 1.21 (δX), 3.76 (δA), and 3.82 (δB) (ABX3 system, 2JAB = 10.2 Hz, 3JAX,BX = 7.0 Hz, 5 H, SiOCHAHBC(HX)3), 3.61 (s, 6 H, o-OCH3, SiC6H2(OCH3)3), 3.83 (s, 3 H, p-OCH3, SiC6H2(OCH3)3), 5.76 (δA), 6.08 (δB), and 6.54 (δC) (ABC system, 2JAB =4.0 Hz, 3JAC =20.5 Hz, 3JBC = 14.7 Hz, 3 H, SiCHCdCHBHA), 6.12 (s, 2 H, H-3/H-5, SiC6H2(OCH3)), 7.44-7.47 (m, 1 H, H-5, SiC6H4(CF3)), 7.59-7.61 (m, 1 H, H-4, SiC6H4(CF3)), 7.75-7.78 (m, 1 H, H-6, SiC6H4(CF3)), 7.82-7.83 (m, 1 H, H-2, SiC6H4(CF3)). 13C NMR (125.8 MHz, CD2Cl2): δ 18.5 (OCH2CH3), 55.4 (o-OCH3, SiC6H2(OCH3)3), 55.6 (p-OCH3, SiC6H2(OCH3)), 59.9 (OCH2CH3), 91.0 (C-3/C5, SiC6H2(OCH3)3), 100.7 (C-1, SiC6H2(OCH3)3), 125.2 (q, 1JCF =272.2 Hz, CF3), 125.6 (q, 3JCF =3.8 Hz, C-4, SiC6H4(CF3)), 127.8 (C-5, SiC6H4(CF3)), 129.4 (q, 2JCF = 31.4 Hz, C-3, SiC6H4(CF3)), 131.0 (q, 3JCF = 3.8 Hz, C-2, SiC6H4(CF3)), 133.2 (SiCHdCH2), 137.4 (SiCHdCH2), 138.0 (q, 5JCF = 1.4 Hz, C-6, SiC6H4(CF3)), 140.1 (C-1, SiC6H4(CF3)), 165.1 (C-4, SiC6H2(OCH3)3), 167.4 (C-2/C-6, SiC6H2(OCH3)3). 19F NMR (376.5 MHz, CD2Cl2): δ -62.7. 29Si NMR (99.4 MHz, CD2Cl2): δ -18.9. Anal. Calcd for C20H23F3O4Si (412.48): C, 58.24; H, 5.62. Found: C, 58.1; H, 5.4. Preparation of [3-(Trifluoromethyl)phenyl](2,4,6-trimethoxyphenyl)divinylsilane (6). A 15 wt % solution of vinylmagnesium chloride (d=0.97 g/mL; 27.0 mL, 45.3 mmol of CH2dCHMgCl) in THF was added dropwise at 20 °C within 10 min to a stirred solution of 5 (16.2 g, 39.3 mmol) in THF (40 mL). After the addition was complete, the reaction mixture was stirred at 20 °C for 1 h, followed by sequential addition of a 0.1 M aqueous solution of potassium carbonate (100 mL) and diethyl ether (80 mL). The aqueous layer was separated and extracted with diethyl ether (2  80 mL), and the combined organic layers were washed with water (1  150 mL) and dried over anhydrous sodium sulfate. The organic solvents were removed under reduced pressure, and the oily residue was purified by bulbto-bulb distillation (150-155 °C/0.06 mbar) to give 6 in 92% yield as a colorless viscous liquid (14.2 g, 36.0 mmol). 1H NMR (500.1 MHz, CD2Cl2): δ 3.60 (s, 6 H, o-OCH3, SiC6H2(OCH3)3), 3.83 (s, 3 H, p-OCH3, SiC6H2(OCH3)3), 5.64 (δC), 6.12 (δB), and 6.61 (δA) (ABC system, 2JBC =3.8 Hz, 3JAB =14.5 Hz, 3JAC = 20.3 Hz, 6 H, SiCHAdCHBHC), 6.12 (s, 2 H, H-3/H-5, SiC6H2(OCH3)3), 7.42-7.46 (m, 1 H, H-5, SiC6H4(CF3)), 7.57-7.60 (m, 1 H, H-4, SiC6H4(CF3)), 7.69-7.71 (m, 1 H, H-6, SiC6H4(CF3)), 7.76-7.77 (m, 1 H, H-2, SiC6H4(CF3)). 13C NMR (125.8 MHz, CD2Cl2): δ 55.4 (o-OCH3, SiC6H2(OCH3)), 55.6 (pOCH3, SiC6H2(OCH3)), 91.2 (C-3/C-5, SiC6H2(OCH3)3), 100.5 (C-1, SiC6H2(OCH3)3), 125.1 (q, 1JCF =272.2 Hz, CF3), 125.4 (q, 3JCF=3.8 Hz, C-4, SiC6H4(CF3)), 127.8 (C-5, SiC6H4(CF3)), 129.5 (q, 2JCF =31.2 Hz, C-3, SiC6H4(CF3)), 131.6 (q, 3 JCF = 3.8 Hz, C-2, SiC6H4(CF3)), 133.6 (SiCHdCH2), 136.6 (SiCHdCH2), 138.7 (q, 5JCF=1.6 Hz, C-6, SiC6H4(CF3)), 139.5 (C-1, SiC6H4(CF3)), 164.9 (C-4, SiC6H2(OCH3)3), 167.1 (C-2/C6, SiC6H2(OCH3)3). 19F NMR (376.5 MHz, CD2Cl2): δ -62.7. 29 Si NMR (99.4 MHz, CD2Cl2): δ -24.8. Anal. Calcd for C20H21F3O3Si (394.47): C, 60.90; H, 5.37. Found: C, 60.7; H, 5.4. Preparation of Bis(2-hydroxyethyl)[3-(trifluoromethyl)phenyl](2,4,6-trimethoxyphenyl)silane (7). A solution of 9-borabicyclo[3.3.1]nonane (16.2 g, 66.4 mmol (based on the 9-BBN dimer))

Tacke et al. and 6 (21.0 g, 53.2 mmol) in THF (260 mL) was stirred at 20 °C for 15 h, followed by the addition of water (55 mL) and a 3 M aqueous sodium hydroxide solution (100 mL). Subsequently, a 30 wt % aqueous hydrogen peroxide solution (100 mL) was added dropwise at 20 °C within 15 min to the stirred reaction mixture, and the resulting mixture was then heated under reflux for 2 h. Upon cooling to 20 °C, a 0.1 M aqueous solution of potassium carbonate (500 mL) and dichloromethane (200 mL) was added. The organic layer was separated, the aqueous layer was extracted with dichloromethane (3  200 mL), and the combined organic phases were dried over anhydrous sodium sulfate. The solvents were removed under reduced pressure, the byproduct cyclooctane-1,5-diol was separated from the crude product by bulb-to-bulb distillation (160 °C/0.02 mbar), and the residue was purified by column chromatography on Al2O3 (neutral, Type 507C, Brockmann I, 150 mesh, 58 A˚; Aldrich cat. no. 199974; deactivated with 6 wt % water) using n-hexane/ dichloromethane/ethanol (20:50:4 (v/v/v)) as the eluent. The relevant fractions were combined, and the solvents were removed under reduced pressure to give 7 in 67% yield as a colorless viscous liquid (15.4 g, 35.8 mmol) that solidified after being left undisturbed at 20 °C for 5 days to give a colorless crystalline solid; mp 50-51 °C. 1H NMR (500.1 MHz, C6D6): δ 1.71-1.88 (m, 4 H, SiCH2CH2OH), 2.39 (s, 2 H, OH), 3.22 (s, 6 H, o-OCH3, SiC6H2(OCH3)3), 3.44 (s, 3 H, p-OCH3, SiC6H2(OCH3)3), 3.88-3.97 (m, 4 H, SiCH2CH2OH), 6.07 (s, 2 H, H-3/ H-5, SiC6H2(OCH3)3), 7.14-7.17 (m, 1 H, H-5, SiC6H4(CF3)), 7.51-7.53 (m, 1 H, H-4, SiC6H4(CF3)), 7.77-7.80 (m, 1 H, H-6, SiC6H4(CF3)), 8.18-8.19 (m, 1 H, H-2, SiC6H4(CF3)). 13C NMR (125.8 MHz, C6D6): δ 20.5 (SiCH2CH2OH), 54.6 (o-OCH3, SiC6H2(OCH3)3), 54.7 (p-OCH3, SiC6H2(OCH3)3), 59.8 (SiCH2CH2OH), 91.2 (C-3/C-5, SiC6H2(OCH3)3), 100.7 (C-1, SiC6H2(OCH3)3), 125.1 (q, 3JCF = 3.8 Hz, C-4, SiC6H4(CF3)), 125.5 (q, 1JCF = 272.4 Hz, CF3), 128.5 (C-5, SiC6H4(CF3)), 129.7 (q, 2JCF = 31.7 Hz, C-3, SiC6H4(CF3)), 130.6 (q, 3JCF =4.0 Hz, C-2, SiC6H4(CF3)), 137.5 (q, 5JCF =1.4 Hz, C-6, SiC6H4(CF3)), 141.7 (C-1, SiC6H4(CF3)), 164.7 (C-4, SiC6H2(OCH3)3), 167.0 (C-2/C-6, SiC6H2(OCH3)3). 19F NMR (282.4 MHz, C6D6): δ -62.4. 29Si NMR (99.4 MHz, C6D6): δ -11.3. Anal. Calcd for C20H25F3O5Si (430.50): C, 55.80; H, 5.85. Found: C, 56.2; H, 6.1. Preparation of 3-[2-(4-Fluorophenyl)-1,3-dioxolan-2-yl]propylamine (9). This compound was synthesized according to ref 25. Preparation of Bis[2-(methanesulfonyloxy)ethyl][3-(trifluoromethyl)phenyl](2,4,6-trimethoxyphenyl)silane (8) and 4-[3-(Trifluoromethyl)phenyl]-1-{3-[2-(4-fluorophenyl)-1,3-dioxolan-2-yl]propyl}-4-(2,4,6-trimethoxyphenyl)-4-silapiperidine (10). Methanesulfonyl chloride (1.57 g, 13.7 mmol) was added dropwise at -25 °C within 10 min to a stirred solution of 7 (2.77 g, 6.43 mmol) and triethylamine (1.89 g, 18.7 mmol) in dichloromethane (60 mL). After the addition was complete, the reaction mixture was stirred for a further 10 min at -25 °C, the mixture was then allowed to warm to 20 °C, and n-pentane (120 mL) was added. The resulting suspension was stirred at 20 °C for 10 min, and the precipitate was removed by filtration, washed with n-pentane (3  15 mL), and discarded. The filtrate and wash solutions were combined, the solvents were removed under reduced pressure, and the residue was dried in vacuo (0.02 mbar, 30 min) to give 8 as a colorless viscous liquid, which was pure enough to be used in the following step without further purification. 1H NMR (500.1 MHz, C6D6): δ 1.74-1.90 (m, 4 H, SiCH2CH2O), 2.36 (s, 6 H, S(O)2CH3), 3.28 (s, 6 H, o-OCH3, SiC6H2(OCH3)3), 3.42 (s, 3 H, p-OCH3, SiC6H2(OCH3)3), 4.37-4.49 (m, 4 H, SiCH2CH2O), 6.04 (s, 2 H, H-3/H-5, SiC6H2(OCH3)3), 7.09-7.12 (m, 1 H, H-5, SiC6H4(CF3)), 7.47-7.49 (m, 1 H, H-4, SiC6H4(CF3)), 7.55-7.57 (m, 1 H, H-6, SiC6H4(CF3)), 7.99-8.00 (m, 1 H, H-2, SiC6H4(CF3)). (25) Ismaiel, A. M.; de Los Angeles, J.; Teitler, M.; Ingher, S.; Glennon, R. A. J. Med. Chem. 1993, 36, 2519–2525.

Article C NMR (125.8 MHz, C6D6): δ 17.9 (SiCH2CH2O), 36.9 (S(O)2CH3), 54.7 (o-OCH3, SiC6H2(OCH3)3), 54.8 (p-OCH3, SiC6H2(OCH3)3), 68.8 (SiCH2CH2O), 91.0 (C-3/C-5, SiC6H2(OCH3)3), 97.4 (C-1, SiC6H2(OCH3)3), 125.1 (q, 1JCF=272.4 Hz, CF3), 125.9 (q, 3JCF = 3.8 Hz, C-4, SiC6H4(CF3)), 128.5 (C-5, SiC6H4(CF3)), 130.2 (q, 2JCF = 32.0 Hz, C-3, SiC6H4(CF3)), 130.3 (q, 3JCF=4.0 Hz, C-2, SiC6H4(CF3)), 137.2 (q, 5JCF=1.2 Hz, C-6, SiC6H4(CF3)), 138.8 (C-1, SiC6H4(CF3)), 165.4 (C-4, SiC6H2(OCH3)3), 167.0 (C-2/C-6, SiC6H2(OCH3)3). 19F NMR (376.5 MHz, C6D6): δ -62.1. 29Si NMR (99.4 MHz, C6D6): δ -13.4. Compound 8 (crude product) was dissolved in acetonitrile (100 mL), triethylamine (2.60 g, 25.7 mmol) and 9 (1.67 g, 7.41 mmol) were added at 20 °C, and the reaction mixture was then heated at 80 °C for 15 h. The solvent and the excess triethylamine were removed under reduced pressure, and dichloromethane (50 mL) and a 0.1 M aqueous solution of potassium carbonate (50 mL) were added to the residue. The organic layer was separated, the aqueous layer was extracted with dichloromethane (2  50 mL), the combined organic phases were dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by column chromatography on Al2O3 (neutral, Type 507C, Brockmann I, 150 mesh, 58 A˚; Aldrich cat. no. 199974; deactivated with 6 wt % water) using n-hexane/ethyl acetate/triethylamine (70:30:1 (v/v/v)) as the eluent. The relevant fractions were combined and the solvents removed by bulb-to-bulb distillation (85 °C, 0.02 mbar, 12 h) to give 10 in 37% yield as a colorless, highly viscous liquid (1.49 g, 2.40 mmol). 1H NMR (500.1 MHz, C6D6): δ 1.67-1.72 and 1.77-1.90 (m, 6 H, SiCH2CH2N, NCH2CH2CH2C), 2.18-2.22 (m, 2 H, NCH2CH2CH2C), 2.44-2.47 (t, 3JHH = 7.1 Hz, 2 H, NCH2CH2CH2C), 2.752.80 and 3.06-3.11 (m, 4 H, SiCH2CH2N), 3.28 (s, 6 H, o-OCH3, SiC6H2(OCH3)3), 3.43-3.45 (m, 5 H, p-OCH3, SiC6H2(OCH3)3, and CH2OC), 3.66-3.68 (m, 2 H, CH2OC), 6.05 (s, 2 H, H-3/H-5, SiC6H2(OCH3)3), 6.91-6.95 (m, 2 H, H-3/H-5, CC6H4F), 7.13-7.16 (m, 1 H, H-5, SiC6H4(CF3)), 7.49-7.53 (m, 3 H, H-4, SiC6H4(CF3), and H-2/H-6, CC6H4F), 7.92-7.94 (m, 1 H, H-6, SiC6H4(CF3)), 8.37-8.39 (m, 1 H, H-2, SiC6H4(CF3)). 13C NMR (125.8 MHz, C6D6): δ 14.6 (SiCH2CH2N), 22.5 (NCH2CH2CH2C), 39.0 (NCH2CH2CH2C), 53.3 (SiCH2CH2N), 54.6 (o-OCH3, SiC6H2(OCH3)3), 54.7 (p-OCH3, SiC6H2(OCH3)3), 58.6 (NCH2CH2CH2C), 64.5 (CH2OC), 91.0 (C-3/C-5, SiC6H2(OCH3)3), 102.0 (C-1, SiC6H2(OCH3)3), 110.5 (CH2OC), 115.0 (d, 2JCF=21.2 Hz, C-3/C-5, CC6H4F), 125.4 (q, 3 JCF = 3.8 Hz, C-4, SiC6H4(CF3)), 125.5 (q, 1JCF = 272.2 Hz, CF3), 128.0 (d, 3JCF = 8.1 Hz, C-2/C-6, CC6H4F), 128.1 (C-5, SiC6H4(CF3)), 129.9 (q, 2JCF=31.2 Hz, C-3, SiC6H4(CF3)), 131.2 (q, 3JCF = 3.8 Hz, C-2, SiC6H4(CF3)), 138.0-138.1 (m, C-6, SiC6H4(CF3)), 139.6 (d, 4JCF=3.0 Hz, C-1, CC6H4F), 141.2 (C-1, SiC6H4(CF3)), 162.9 (d, 1JCF =245.0 Hz, C-4, CC6H4F), 164.5 (C-4, SiC6H2(OCH3)3), 167.0 (C-2/C-6, SiC6H2(OCH3)3). 19F NMR (376.5 MHz, C6D6): δ -115.0 (CC6H4F), -62.0 (CF3). 29 Si NMR (99.4 MHz, C6D6): δ -17.0. Anal. Calcd for C32H37F4NO5Si (619.73): C, 62.02; H, 6.02; N, 2.26. Found: C, 61.8; H, 5.6; N, 2.4. 2D 1H,1H EXSY NMR Studies. 1H, 13C, 19F, and 29Si NMR studies of the C/Si analogues 2a 3 HCl and 2b 3 HCl demonstrated the existence of two isomers of the respective ammonium cations (2a 3 HCl, molar ratio ca. 12:1; 2b 3 HCl, molar ratio ca. 2:1) in solution (solvent, [D6]DMSO). These isomers are configurationally stable on the NMR time scale under the experimental conditions used. To prove the existence of the two isomers, 2D 1 H,1H EXSY NMR experiments with 2b 3 HCl were carried out at 23 °C (solvent, [D6]DMSO). In this study, the site exchange for the SiCH2CH2N and SiCH2CH2N protons of the 4-silapiperidinium skeleton could be observed, showing strong cross-peaks between the respective signals of the two isomers. The mixing time was in the order of the spin-lattice relaxation time T1, calculated by a standard 1D T1-inversion recovery experiment. 13

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ESI-MS Studies. a. Chemicals. Water (HPLC gradient grade) was purchased from Acros (Geel, Belgium). Acetic acid (98%, analytical reagent grade), ammonium hydroxide solution (25%, analytical reagent grade), and ammonium acetate (analytical reagent grade) were purchased from Fluka (Taufkirchen, Germany). b. Sample Preparation. A 10 mM aqueous ammonium acetate buffer was prepared from a 1.0 M stock solution by diluting with water and adjusting the pH with 98% acetic acid (pH 5), 0.25% ammonium hydroxide solution (pH 7.4), or 2.5% ammonium hydroxide solution (pH 10). For measurements at pH 1, 0.1 M hydrochloric acid was used as the solvent. Sample solutions of 2b 3 HCl with a concentration of 10 μM were prepared from a 1.0 mM aqueous stock solution by dilution with 0.1 M hydrochloric acid (pH 1) or with the respective buffer (pH 5, pH 7.4, pH 10) and were analyzed by ESI-MS (i) 15 min and (ii) 24 h after preparation. Sample solutions with concentrations of 2.5 mM (pH 1, pH 5) and 1 mM (pH 7.4, pH 10) of 2b 3 HCl were prepared by dissolving the appropriate amount of 2b 3 HCl in 0.1 M hydrochloric acid (pH 1) or in the respective buffer solution (pH 5, pH 7.4, pH 10). At pH 10, acetonitrile was added as a cosolvent (buffer/acetonitrile, 2:1 (v/v)) for reasons of solubility. The samples were analyzed by ESI-MS (i) 15 min and (ii) 24 h after preparation. c. ESI-MS Analysis. Analysis was performed with a TSQ 7000 tandem mass spectrometer system equipped with an ESI interface (Finnigan MAT, Bremen, Germany) and a syringe pump (Harvard Apparatus, South Natick, MA). Data acquisition and analysis were conducted using Xcalibur Qual Browser Software 1.2/1.3 (Thermo Electron Corp., Dreieich, Germany). A flow rate of 10 μL/min was used. The analysis was performed in the positive ionization mode. The spray capillary voltage was set to 3.2 kV, and the temperature of the heated capillary was 250 °C. Nitrogen served both as sheath (70 psi) and as auxiliary gas (10 L/min). The mass spectrometer was operated in the fullscan mode, m/z 400-1500, with a total scan duration of 1.0 s and a dwell time of 2 ms. Functional Receptor Studies. a. Materials. cDNA for the hD1 dopamine receptor was kindly provided by Dr. D. Grandy (Portland, OR). A pcDNA vector construct for hD2S receptors was a gift from Dr. W. Sadee (San Francisco, CA). All other reagents were purchased from Sigma-Aldrich Chemicals (Taufkirchen, Germany) unless otherwise stated. b. Cell Cultures. HEK293 cells stably expressing dopamine hD1 or hD2S receptors were established as previously described.26,27 Both cell lines were grown in Dulbecco’s modified Eagle medium (DMEM, GlutaMAX; Invitrogen, Karlsruhe, Germany) containing 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). Then 400 μg/mL active G-418 (Calbiochem, San Diego, CA) was added to the medium of the stably transfected cell lines to maintain selection pressure. Cells were incubated at 37 °C in a humidified atmosphere under 5% CO2. Confluent cell lines were subcultured by harvesting with 0.05% trypsin/0.02% EDTA and cultivated in tissue culture flasks (Sarstedt AG & Co., N€ umbrecht, Germany). c. Measurement of Changes in Intracellular [Ca2þ] in HEK293 Cells. Measurement of changes in intracellular [Ca2þ] ([Ca2þ]i) was performed as previously described using a NOVOstar microplate reader with built-in pipettor (BMG LabTech, Offenburg, Germany).26 Culture medium was changed approximately 24 h prior to the assay. A confluent tissue culture flask (175 cm2) of HEK293 cells expressing the respective dopamine receptor was loaded with 0.3 μM Oregon Green 488 BAPTA-1/ AM (Molecular Probes, Eugene, OR) for 45 min at 37 °C and 5% CO2 in FBS-free DMEM containing 0.006% (w/v) Pluronic (26) Kassack, M. U.; H€ ofgen, B.; Decker, M.; Eckstein, N.; Lehmann, J. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2002, 366, 543–550. (27) Kassack, M. U. AAPS PharmSci. 2002, 4, E31.

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F-127. Cells were then harvested with culture medium. After centrifugation (1200 rpm, 4 °C, 4 min), the cell pellet was rinsed with two 800 μL portions of Krebs-HEPES buffer (KHB: 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 4.2 mM NaHCO3, 11.7 mM D-glucose, 1.3 mM CaCl2, 10 mM HEPES, pH 7.4) and then resuspended in 17 mL of the same solution. The cell suspension was evenly plated into a 96-well microplate (Sarstedt AG & Co., N€ umbrecht, Germany). Microplates were kept at 37 °C for 30 min before antagonists, dissolved in KHB containing 0.5% bovine serum albumine (BSA), were added. After a further 30 min incubation at 37 °C, fluorescence intensity was measured at 520 nm (bandwidth 35 nm) for 5 s at 1.0 s intervals to monitor baseline. Control solutions or agonist solutions (hD1: 100 nM SKF38393; hD2: 30 nM quinpirole), dissolved in KHB containing 0.5% BSA, were then injected into separate wells, and fluorescence intensity was monitored at 520 nm (bandwidth 35 nm) for 20 s at 0.4 s intervals. Excitation wavelength was 485 nm (bandwidth 12 nm). d. Data Analysis. The change in [Ca2þ]i is expressed as a change in fluorescence intensity. Data from at least two independent experiments each assayed in triplicates were pooled after normalization, and concentration-inhibition curves were constructed using nonlinear regression curve fit (sigmoidal dose-response equation) by means of Prism software 4.0 from GraphPad (La Jolla, CA). Ki values were calculated

Tacke et al. according to the following equation adapted from Cheng and Prusoff:28

Ki ¼

IC50 L 1þ EC50

where IC50 is the inhibitory concentration 50% of the antagonist, EC50 is the effective concentration 50% of the agonist used (SKF38393 for hD1 and quinpirole for hD2 receptors), and L is the molar concentration of the agonist used. Data are presented as means ( SD (n g 2).

Acknowledgment. Experimental support for the ESIMS studies by W. H€ ummer, Lehrstuhl Lebensmittelchemie, Universit€at W€ urzburg, is gratefully acknowledged. C.U. was supported by the Dr. Hilmer foundation. Supporting Information Available: Crystallographic data for 2a 3 HCl and 2b 3 HCl and inhibitory potencies (pKi values ( SEM) of 1a, 1b, 2a, and 2b. This material is available free of charge via the Internet at http://pubs.acs.org. (28) Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099– 3108.