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1 Electronic Supplementary Information Selective Steroid Recognition by a Partially Bridged Resorcin[4]arene Cavitand Martina Cacciarini, a,b Vladimir A. Azov, a Paul Seiler, a Hermann Künzer c and François Diederich* a a Laboratorium für Organische Chemie, ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Fax: (+41) ; diederich@org.chem.ethz.ch b Dipartimento di Chimica Organica U. Schiff, Università di Firenze, Polo Scientifico, I Sesto Fiorentino (FI), Italy; martina.cacciarini@unifi.it c Schering AG, Corporate Research, Research Centre Europe, Medicinal Chemistry, D Berlin, Germany 1

2 I. Synthesis of receptor 1 ESI Synthesis of octol A (a): To an ice-cooled stirred solution of resorcinol (16.5 g, 0.15 mol) in EtOH (60 ml) and conc. HCl (20 ml), dodecanal (32.3 ml, 0.15 mol) was added dropwise over 1 h. A white precipitate formed. The mixture was gradually warmed to 20 C, then heated to 90 C for 17 h, which led to the dissolution of the precipitate. After cooling to 20 C, the orange gummy suspension was transferred into water (600 ml) and the resulting precipitate collected by vacuum filtration, then washed with 600 ml of water to neutrality and dried under vacuum at 80 C. The resulting yellowish solid was recrystallised from MeOH and dried under high vacuum over P 2 O 5. Yield 31.6 g ( mol, 76%) of octol A as a slightly yellow powder. Mp ºC (from MeOH); δ H (300 MHz, (CD 3 ) 2 CO) 8.45 (8 H, s, OH), 7.55 (4 H, s, meta to OH), 6.24 (4 H, s, ortho to OH), 4.30 (4 H, t, J 8.0 Hz, CHCH 2 ), 2.25 (8 H, m, CHCH 2 ), (72 H, m, CHCH 2 (CH 2 ) 9 CH 3 ), 0.89 (12 H, t, J 6.6 Hz, CH 3 ); δ C (75 MHz, (CD 3 ) 2 CO) , , , , 33.69, 32.05, 29.88, 29.83, 29.51, 22.72, Synthesis of cavitand B (b): Octol A (2.8 g, 2.53 mmol) was suspended in Me 2 SO (80 ml) under N 2, then 2,3- dichloroquinoxaline (2.1 g, mmol) and K 2 CO 3 (2.44 g, mmol) were added. The orange suspension became dark red. The mixture was stirred at 20 C for 1 h and heated at 2

3 60 C overnight, then it was cooled to 20 C, transferred into 400 ml of water and the resulting pink precipitate collected by vacuum filtration and washed with water (2x200 ml). The crude solid was dissolved in CH 2 Cl 2 (100 ml) and the organic solution washed with water (3x20 ml) and sat. aq. NaCl solution and dried over Na 2 SO 4. Filtration and evaporation of the solvent gave a yellow solid that was purified by flash chromatography (SiO 2 ; CH 2 Cl 2 :EtOAc 100:0 98:2) to yield 2.2 g of cavitand B (1.37 mmol, 54%) as a yellow solid. Mp ºC; R f 0.70 (SiO 2 ; CH 2 Cl 2 :EtOAc 2%); δ H (300 MHz, CDCl 3 ) 8.15 (4 H, s, Ar-H bottom), (8 H, AA BB system, Qx-H), (AA BB system, Qx-H), 7.21 (H, s, 4Ar-H top), 5.56 (4 H, t, J 7.5 Hz, CH), (8 H, m, CHCH 2 ), (72 H, m, CHCH 2 (CH 2 ) 9 CH 3 ), (12 H, m, CH 3 ); δ C (75 MHz, CDCl 3 ) , , , , , , , , 34.36, 32.46, 32.03, 29.81, 29.50, 28.05, 22.81, Synthesis of 1 (c): Cavitand B (1.185 g, mmol) was dissolved in dry THF (15 ml) and the solution added to a round-bottom flask containing DMF (100 ml) heated to 80 C, then CsF (2.24 g, 14.7 mmol) and finally a freshly prepared solution (0.5 M, 4.4 ml) of catechol in DMF were added. The mixture became darker while heating to 80 C for 1.5 h, then it was concentrated to half the volume and poured into 1 L of sat. aq. NaCl solution. A green solid formed, and the mixture was subjected for 30 min to magnetic stirring in order to improve the size and the quality of the precipitate which was then filtered and dried under vacuum over P 2 O 5 overnight. Purification by flash chromatography (SiO 2 ; CH 2 Cl 2 :EtOAc 98:2 92:8) gave 1 (500 mg, mmol, 50%). Colorless crystals of compound 1 were obtained by slow evaporation of an acetone solution. White solid. Mp >300 ºC (from Me 2 CO, dec.); R f 0.20 (SiO 2 ; CH 2 Cl 2 :EtOAc 92:8); δ H (300 MHz, (CD 3 ) 2 CO) 9.01 (4 H, s, OH), (4 H, 3

4 AA BB system, Qx-H), 7.72 (4 H, s, Ar-H bottom), (4 H, AA BB system, Qx- H), 7.18 (4 H, s, Ar-H top), 5.51 (2 H, t, J 8.4 Hz, CH below Qx), 4.42 (2 H, t, J 8.1 Hz, CH below OH s), (8 H, m, CHCH 2 ), (72 H, m, CHCH 2 (CH 2 ) 9 CH 3 ), (12 H, m, CH 3 ); δ H (300 MHz, CDCl 3 ) 7.67 (4 H, br s), 7.42 (4 H, br s), 7.26 (4 H, s), 7.23 (4 H, s), 7.18 (4 H, s), 5.45 (2 H, t, J 8.0 Hz), 4.34 (2 H, t, J 7.5 Hz), (8 H, m, CHCH 2 ), (72 H, m, CHCH 2 (CH 2 ) 9 CH 3 ), (12 H, m, CH 3 ); δ C + DEPT(90) (75 MHz, CDCl 3 ) , , , , (CH), , (CH), (CH), (CH), (CH), 33.78, 32.50, 32.05, 29.95, 29.90, 29.85, 29.52, 28.17, 22.81, (CH 3 ); m/z (MALDI HRMS) (C 88 H 116 N 4 O 8 + (MH + ) requires ), (C 88 H 116 N 4 O 8 Na + (MNa + ) requires ); found: C, 77.28; H, 8.65; N, 4.11; C 88 H 116 N 4 O 8 0.5H 2 O requires: C, 77.27; H, 8.69; N, II. Binding Studies ESI Determination of the association constants K a and the dimerisation constants K dim 1 H NMR titrations and dilution experiments were performed at 298 K on a Varian Gemini 300 Spectrometer (300 MHz) and on a Bruker 500 MHz Spectrometer in CDCl 3 (stored over 4 Å molecular sieves and filtered over basic Al 2 O 3 before use) purchased from Merck. Host 1 was dissolved in CDCl 3 to give mm stock solution. This solution was used first to dissolve steroids 2a-p (5-15 mm) to give an NMR sample with the initial high concentration of a steroid guest. The aliquots of the stock solution were successively added by a syringe (12-14 additions) to the NMR tube, so that the host concentration remained constant and the guest concentration gradually decreased during the titration. 1 H NMR spectra were recorded after each addition and the change in chemical shift of the HO-protons of receptor 1 as a function of the guest concentration was analyzed with purpose-written software package provided by Prof. C. A. Hunter (binding isotherms were created using the NMRTit HG, HH 4

5 and GG programs on an Apple Macintosh computer). In few cases, the HO-protons signal of 1 split into two signals of equal intensity upon addition of the chiral, non-racemic steroid guest. The K a values for both signals was then evaluated and the average value reported. Uncertainty in K a is one standard deviation from duplicate or triplicate runs. Dilution experiments were carried out with a solution of 1 (30 mm) or with a solution of the steroids (5-15 mm depending on the steroid solubility), which was progressively diluted with CDCl 3. The change in chemical shift of the HO-protons for receptor 1 and the HO- protons or the C(18)-Me protons for the steroids was recorded after each of CDCl 3 additions. Dilution curves and K dim values were obtained using the NMRDil Dimer program on an Apple Macintosh computer. Determination of the binding stoichiometry by Job plot analysis The 1:1 binding stoichiometry of the 1 2a-p complexes was confirmed as follows. Two stock solutions were prepared; solution A: 5 mm of 1 in CDCl 3. Solution B: 5 mm of 2 in CDCl 3. The two solutions were combined to give a series of samples of identical total concentration but containing different mole fractions of the two components. 1 H NMR spectra were recorded at 500 MHz for each sample and then used to draw the Job plots (e.g. see Fig. 7 ESI) using HO- and Me-resonances. 5

6 Figure 1 ESI. Dilution curve of receptor 1 at 298 K in CDCl 3, using the HO-signal. Figure 2a ESI. Dilution curve of 2n at 298 K in CDCl 3, using the CH 3 on C(18). 6

7 Figure 2b ESI. Dilution curve of 2n at 298 K in CDCl 3, using the HO-signal on C(17). Figure 3 ESI. Dilution curve of steroid 2m at 298 K in CDCl 3, using the HO-signal on C(17). 7

8 Figure 4 ESI. 1 H NMR titration of the 1 2g complex at 298 K in CDCl 3 Figure 5a ESI. 1 H NMR titration of the 1 2n complex at 298 K in CDCl 3 (first set of HOsignals) 8

9 Figure 5b ESI. 1 H NMR titration of the 1 2n complex at 298 K in CDCl 3 (second set of HO-signals) Figure 6 ESI. Job plot to prove the 1:1 binding stoichiometry of the 1 2g complex (298 K, CDCl 3 ) Job Plot with 2g 0,016 0,014 0,012 complexg 0,01 0,008 0,006 0,004 0, ,2 0,4 0,6 0,8 1 molar fraction of 1 9

10 Figure 7 ESI. Job plot to prove the 1:1 binding stoichiometry of the 1 2n complex (298 K, CDCl 3 ) 0,02 Job Plot with 2n 0,018 0,016 0,014 comple 0,012 0,01 0,008 0,006 0,004 0, ,2 0,4 0,6 0,8 1 molar fraction of 1 10

11 III. Crystal structure data for 1 ESI. Crystal data at 220(2) K for (C 88 H 116 N 4 O 8 ) 2(C 3 H 6 O) [M r = ], triclinic, space group P 1 (no.2), Dc = g cm -3, Z = 2, a = (2), b = (2), c = (4) Å, α = (1), β = (1), γ = (1) o, V = (11) Å 3. Bruker-Nonius Kappa-CCD diffractometer, MoKα radiation, λ = Å, µ = mm -1. A colorless crystal of 1 (linear dimensions ca x 0.28 x 0.26 mm) was obtained by slow evaporation of an acetone solution. Number of measured and unique reflections are and 11913, respectively (R int = 0.025). The structure was solved by direct methods (SIR97) [1] and refined by full-matrix least-squares analysis (SHELXL-97) [2], using an isotropic extinction correction, and w = 1/[σ 2 (Fo 2 ) + (0.1172P) P], where P = (Fo 2 + 2Fc 2 )/3. One of the two C 3 H 6 O solvents exhibits static and dynamic disorder, which could not be resolved. All heavy atoms of 1 were refined anisotropically, H-atoms isotropically, whereby H-positions are based on stereochemical considerations. Final R(F) = 0.082, wr(f 2 ) = for 959 parameters and 8241 reflections with I > 2σ(I) and 2.82 < θ < o (corresponding R-values based on all reflections are and respectively). Crystallographic data (excluding structure factors) for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as deposition No. CCDC Copies of the data can be obtained, free of charge, on application to the CCDC, 12 union Road, Cambridge CB2 1EZ UK (fax:+44(1223) ; e- mail:deposit@ccdc.cam.ac.uk). [1] A. Altomare, M. Burla, M. Camalli, G. Cascarano, C. Giacovazzo, A. Guagliardi, A. G. G. Moliterni, G. Polidori and R. Spagna, J. Appl. Crystallogr., 1999, 32,

12 [2] G. M. Sheldrick, SHELXL-97 Program for the Refinement of Crystal Structures. University of Göttingen, Germany,