Electronic Supplementary Material 1

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1 Electronic Supplementary Material 1 Molecular clefts of Rebek revisited: potential application as drug carriers for the antiviral acyclovir Fernanda M. F. Roleira a*, Elisiário J. Tavares da Silva a, José A. C. Pereira b, Francesco rtuso c, Stefano Alcaro c, Madalena M. M. Pinto d a CC-IBILI and Grupo de Química Farmacêutica, Faculdade de Farmácia, Universidade de Coimbra, Azinhaga de Santa Comba, Coimbra, Portugal b ICBAS Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira n º 228, Porto, Portugal REQUIMTE/CEQUP Centro de Química da Universidade do Porto, Rua D. Manuel II, Apartado 55142, Porto, Portugal c Laboratorio di Chimica Farmaceutica, Dipartimento di Scienze della Salute, Università Magna Græcia di Catanzaro, Viale Europa, 88100, Catanzaro, Italy d Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP) and Laboratório de Química rgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira nº 228, Porto, Portugal *Corresponding author: Fernanda M. F. Roleira; froleira@ff.uc.pt; Phone: ; Fax: Contents: Synthetic strategy scheme, experimental conditions for the synthesis, spectroscopic data and equilibrium constants calculation using MR data, 1 MR titration data for protons (a) and (b) Journal of Inclusion Phenomena and Macrocyclic Chemistry 1

2 C 2 C 2 C 2 C 2 C 2 i ii iii CCl iv v vi, vii C 2 C 2 C viii a b ix C 2 C Scheme 1 Synthetic strategy followed for the preparation of receptors 5 and 9, according to Rebek procedures. 1,3 i) sublimation at 180 o C (1 cmg); ii) 4 conc., 4-dimethylaminopyridine, 110 o C; iii) SCl 2, reflux; iv) anhydrous pyridine, 4-dimethylaminopyridine, CCl 3, 90 o C, 2; v) anhydrous pyridine, 4-dimethylaminopyridine, anhydrous C 2Cl 2, reflux, 2; vi) LiB 4, anhydrous tetrahydrofuran, 0 o C; vii) trifluoroacetic acid, triethylsilane, anhydrous C 2Cl 2; viii) phenylchloroformate, diisopropylethylamine, anhydrous C 2Cl 2, reflux; ix) guanidine hydrochloride, 60% a, diisopropylethylamine, anhydrous tetrahydrofuran, 90 o C, 2. 2

3 Experimental conditions for de synthesis and association studies: Compounds 5 and 9 (Scheme 1) were prepared according to previous descriptions. 1,3 Acyclovir 10 was purchased from Wellcome Laboratories. Reagents were purchased from Sigma-Aldrich and solvents were purchased from Merck. 1 MR and 13 C MR spectra of individual compounds were obtained with a Brucker AMX 200 or 300 spectrometer using 5 mm o.d. MR tubes. δ is expressed in ppm, relatively to Me4Si. Coupling constants (J) are expressed in z. Mass spectra (electronic impact) were obtained with a mass spectrometer itachi Perkin-Elmer, RMU-6M. 1 MR and 13 C MR spectra in titrations were obtained with a Brucker AMX 300 spectrometer using 5 mm o.d. MR tubes. Measurements were carried out at 298 K in DMS-d6 relatively to Me4Si; δ is expressed in ppm or z. PLC chromatograms were obtained with a Jasco model liquid chromatographic system, a Jasco 875 variable wavelength UV photometric detector and a Varian 4270 integrator. Standard PLC conditions were 65:35 Me/2 containing 0.1% of Et3 for the association studies of 5 and 10 and 75:25 Me/2 containing 0.1% of Et3 for the association studies of 9 and 10 with a reversed phase RP-8 (Lichrospher) column (Merck) with 10 mm. The UV detector was set at 254 nm. Melting points and spectroscopic data for compounds 2 to 9: Mp, IV and 1 MR data are in accordance with those published in the literature. In addition, new 13 C MR data were acquired. Compound 2: Mp: o C (lit o C). IR (KBr) νmax cm -1 : , 1795, 1760, 1700, 1460, 1285, 1220, 1185, 1130, 1090, MR (200 Mz, pyridine-d5) δ: (12, m), 2.02 (1, dt, J=13.3, 2.1, 2.1), 2.92 (2, dd, J=14.2, 1.9). 13 C MR (50.3 Mz, pyridine-d5) δ: 25.4 (two carbons), 30.4, 40.4 (two carbons), 42.0, 42.1, 44.5 (two carbons), (two carbons), Compound 3: Mp: > 300 o C (lit. 1 > 300 o C). IR (KBr) νmax cm -1 : 3140, 3070, 2970, 1730, 1705, 1455, 1380, 1310, 1220, MR (300 Mz, DMS-d6) δ: 1.07 (6, s), 1.01 (3, s), 1.18 (2, d, J=13.5), 1.37 (1, d, J=13.2), 1.87 (1, d, J=12.9), 2.35 (2, d, J=13.5), (1, s), (1, s). 13 C MR (75.47 Mz, DMS-d6) δ: 24.5 (two carbons), 30.7, 39.3 (two carbons), 41.0, 42.8 (two carbons), 43.2, (two carbons), Compound 4: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 3200, 3100, 2980, 1780, 1720, 1690, 1460, 1385, 1205, 925, 895, MR (200 Mz, CDCl3) δ: (12, m), 2.02 (1, dt, J=13.5, 2.2, 2.2), 2.75 (2, dd, J=15.2, 1.9), 7.70 (1, s). 13 C MR (50.3 Mz, CDCl3) δ: 24.1 (two carbons), 30.2, 40.0 (two carbons), 44.1, 44.3 (two carbons), 52.3, (two carbons), MS: ([M+2] +. ), ([M+1] +. ), ([M] +. ). 3

4 Compound 5: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 3380, 3200, 3090, 2960, 1720, 1690, 1545, 1450, 1385, 1360, MR (300 Mz, DMS-d6) δ: 1.13 (6, s), 1.22 (3, s), 1.26 (2, d, J=14.4), 1.41 (1, d, J=12.9), 1.90 (1, d, J=12.6), 2.72 (2, d, J=13.8), (7, m), 9.40 (1, s), (1, s). 13 C MR (75.47 Mz, DMS-d6) δ: 24.6 (two carbons), 30.6, 39.4 (two carbons), 42.4, 43.0 (two carbons), 43.6, 117.8, 122.2, 124.6, 126.1, 127.2, 127.4, 127.5, 130.0, 133.2, 136.5, 173.2, (two carbons). MS: ([M] +. ). Compound 6: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 3230, 3095, 2965, 2930,1730, 1685, 1500, 1460, 1380, 1240, 1205, 1175, MR (300 Mz, CDCl3) δ: 1.07 (3, s), 1.11 (2, d, J=14.3), 1.23 (6, s), 1.31 (1, d, J=13.3), 1.93 (1, d, J=13.3), 2.65 (2, dd, J=14.4, 1.5), 3.05 (2, t, J=7.2), 4.28 (2, t, J=7.2), (8, m, being 1, s at 7.64). 13 C MR (75.47 Mz, CDCl3) δ: 24.3 (two carbons), 30.4, 34.7, 40.1 (two carbons), 42.1, 43.8 (two carbons), 44.4, 65.3, 125.4, 126.0, (two carbons), 127.5, 127.6, 128.0, 132.2, 133.5, 135.0, 175.1, (two carbons). Compound 7: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 3200, 2920, 1720, 1660, 1455, 1160, MR (300 Mz, CDCl3) δ: 0.85 (3, s), 0.99 (2, d, J=13.8), 1.05 (3, s), 1.12 (3, s), 1.17 (1, m), 1.60 (1, d, J=12.8), 2.42 (1, d, J=13.9), 2.59 (1, d, J=13.9), (2, m), 3.09 (2, t, J=6.9), (2, m), 5.34 (1, s), (7, m). 13 C MR (75.47 Mz, CDCl3) δ: 25.0, 28.7, 30.4, 31.1, 34.8, 38.4, 42.3, 45.1, 45.2, 46.0, 52.9, 65.0, 125.4, 126.0, 127.5, 127.6, 128.0, 132.3, 133.5, 135.6, 175.5, Compound 8: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 2970, 1785, 1725, 1660, 1490, 1455, 1280, 1200, MR (300 Mz, CDCl3) δ: 1.02 (3, s), 1.06 (3, s), 1.14 (2, d, J=14.1), 1.23 (3, s), 1.32 (1, dd, J=13.0, 2.5), 1.82 (1, d, J=12.9), 2.57 (1, d, J=14.2), 2.70 (1, d, J=14.0), 3.03 (2, t, J=7.0), 3.39 (1, dd, J=12.3, 1.8), 3.79 (1, dd, J=12.4, 2.5), (2, m), (12, m). 13 C MR (75.47 Mz, CDCl3) δ: 25.8, 28.9, 30.6, 31.4, 34.7, 41.4, 42.1, 44.9, 45.7, 45.8, 58.6, 65.3, (two carbons), 125.2, 125.7, 126.0, 127.3, (two carbons), 127.5, 127.7, (two carbons), 132.2, 133.4, 135.4, 150.7, 152.4, 174.4,

5 Compound 9: Mp: o C (lit o C). IR (KBr) νmax cm -1 : 3400, 3200, 1720, 1615, 1590, 1515, 1460, 1185, MR (300 Mz, CDCl3) δ: (13, m), 2.42 (1, d, J=14.4), 2.57 (1, d, J=13.9), 3.00 (2, t, J=6.9), 3.15 (1, d, J=14.7), 3.95 (1, d, J=14.5), (2, m), 5.24 (1, s), 5.86 (1, s), (7, m). 1 MR (300 Mz, DMS-d6) δ: (12, m), 1.74 (1, d, J=12.6), 2.24 (1, d, J=14.4), 2.36 (1, d, J=13.5), 3.01 (2, t, J=7.0), 3.05 (1, d, J=14.4), 3.70 (1, d, J=13.8), (1, m), (1, m), 7.13 (1, s), 7.17 (1, s), (7, m). 13 C MR (75.47 Mz, CDCl3) δ: 26.0, 29.1, 30.0, 30.9, 34.9, 38.3, 42.0, 43.2, 46.0, 47.4, 54.7, 65.5, , 127.4, (two carbons), 127.6, 127.9, 132.2, 133.5, 135.3, 155.6, 165.6, 172.1, C MR (75.47 Mz, DMS-d6) δ: 25.8, 28.6, 29.5, 30.3, 34.0, 37.7, 41.5, 41.7, 45.1, 46.3, 54.0, 64.9, , 127.1, 127.3, 127.4, 127.5, 127.7, 131.8, 133.0, 135.3, 154.8, 165.4, 170.7, MS: ([M] +. ). 5

6 Equilibrium constants calculation using MR data: The equilibrium constant for the complex formed between the antiviral drug (A) and the synthetic receptor (R), described by the chemical equation: A + R AR where K1:1 = [AR]/[A][R] were calculated by least-squares regression of a parametric non-linear function over the experimental fast equilibrium chemical shift data. The chemical shift function describes the variation of the observed chemical shift of R nuclei, δ, as: δ = ([R] δr + [AR] δar)/[r]t (1) where δr and δar are the chemical shifts of species R and AR, respectively and [R]t = [R] + [AR]. With the substitutions Δ = δ - δr, ΔR = δr - δr = 0 and ΔAR = δar - δr, equation (1) can be rewritten as: Δ = [AR] ΔAR/[R]t (2) By doing the convenient substitutions in (2), it is possible to express Δ as a function of two known quantities, [R]t and [A]t = [A] + [AR], and two unknown parameters, K1:1 and ΔAR. A FRTRA program, a straightforward application of subroutine E04FDF from AG Library (umerical Algorithms Group), was used for the minimization of the function: S = Ʃ (Δcalc Δobs) 2 Where Δcalc is the calculated chemical shift (equation (2)) and Δobs is the observed chemical shift, by adjusting the values attributed to K1:1 and ΔAR. 6

7 Table 1. 1 MR titration data for proton (a) [Acyclovir 10] [Receptor 9] Δobs. 1) Δcalc. 2) Δobs.- Δcalc x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Concentrations are in M and chemical shifts in z. 1) bserved chemical shift variation of proton (a) of 9 after addition of several equivalents of 10. 2) Calculated chemical shift variation of proton (a) of 9 after addition of several equivalents of 10. 7

8 Table 2. 1 MR titration data for proton (b) [Acyclovir 10] [Receptor 9] Δobs. 1) Δcalc. 2) Δobs.- Δcalc x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x Concentrations are in M and chemical shifts in z. 1) bserved chemical shift variation of proton (b) of 9 after addition of several equivalents of 10. 2) Calculated chemical shift variation of proton (b) of 9 after addition of several equivalents of 10. 8