yl)pent-4-yn-1-yl)-3-(methoxymethoxy)benzoate (alkynol) 4. n-butyllithium (152 µL, 1.1 M in hexane, ... Alkynol 4 (23.7 mg, 0.045 mmol) was dissolved in EtOAc.
Supporting Information
Designed Spiroketal Protein Modulation Marcel Scheepstra, Sebastian A. Andrei, M. Yagiz Unver, Anna K. H. Hirsch, Seppe Leysen, Christian Ottmann, Luc Brunsveld,* and Lech-Gustav Milroy* anie_201612504_sm_miscellaneous_information.pdf
Cheminformatic analysis of spiroketals. A structure search was made of the SciFinder (substructure) and Reaxys (substructure → on all atoms → product → align results with query) databases for spiroketals, and more specifically [6,6]- & [6,5]-benzannulated spiroketals, and compared against the total no. of substances bearing the generic benzodiazepine structure. The search results are summarized below.
These data suggest that more examples of spiroketals have been reported in the Reaxys database (Total no. 30,561) than benzodiazepines (26,558), while Scifinder returned significantly more examples of both compound classes (95,498 vs. 117,933) compared to the Reaxys database, as evidence of the general abundance of both in the literature. The chromane isomer of the [6,6]monobenzannulated spiroketals is the most abundant sub-type of monobenzannulated spiroketal (Reaxys, 522; SciFinder, 1095) of which >80% are represented by [6,6]-bisbenzannulated, thus highlighting the abundance of this specific spiroketal subset.
Polarization assay. His-RXRα-LBD (1 µM), labeled co-activator peptide (D22, TIF2, Pro22) (0.1 µM), and the ligand at the indicated concentration in the assay buffer: 10 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT, 0.1% v/v bovine serum albumin were incubated for 60 minutes at 4 °C and protected from light. Fluorescent polarization signals (mP) were measured with a Tecan Infinite F500 microplate reader. Experiments were performed in triplicate. The labeled D22 peptide (Thermo Fisher, catalog number PV4386) contains a C-terminal carboxylate and is labelled directly at the N-terminus with fluorescein (FAM, ex 495 nm, em 519 nm). The labeled TIF2 & Pro22 peptides were both custom-made by GenScript and contain C-terminal amidation and an Nterminal FITC (ex 495 nm, em 519 nm) label attached via an aminohexanoic (Ahx) linker.
D22 LG100268 D22 peptide peptide LG100268 D22 peptide peptide (±)-1 D22 Spiroketal 1
Polarization / mP
160
EC50 %eff 302 ± 15 nM 100 425 ± 79 nM 49.6 Sequence D22 peptide: LPYEGSLLLKLLRAPVEEV
140
LG100268 (±)-1
120
100
80
60 -8,5
-8,0
-7,5
-7,0
-6,5
-6,0
-5,5
-5,0
-4,5
Concentration ligand / Log(M)
TIF2 TIF2peptide peptideLG100268 LG100268 TIF2peptide peptideSpiroketal (±)-1 TIF2 1
220 200
Polarization / mP
180
EC50 %eff LG100268 478 ± 18 nM 100 (±)-1 465 ± 58 nM 28.4 Sequence TIF2 peptide: KHKILHRLLQDSS
160 140 120 100 80 60 40 -8,5
-8,0
-7,5
-7,0
-6,5
-6,0
-5,5
Concentration ligand / Log(M)
-5,0
-4,5
M2H peptide Pro22 peptideLG100268 LG100268 Pro22 peptideSpiroketal (±)-1 M2H peptide 1
240
Polarization / mP
220 200
LG100268 (±)-1
180 160 140
EC50 %eff 250 ± 7 nM 100 426 ± 12 nM 58.7 Sequence Pro22 peptide: LTARHPLLMRLLLSPS
120 100 -8,5
-8,0
-7,5
-7,0
-6,5
-6,0
-5,5
-5,0
-4,5
Concentration ligand / Log(M)
Figure S1 | Stability over 24 hours of the enantiomers. Polarization assay RXR (1 μM) with Pro22 peptide (0.1 μM). LG100268, (±)-1, 1-ent1, 1-ent2 at indicated concentrations. Measurements over a time course of 24 hours.
Table S1 | EC50 values and efficacies. for LG100268, (±)-1, 1-ent1 and 1-ent2 over a time course of 24 hours. 1 hour
2 hours
4 hours
24 hours
EC50 nM
% Eff
EC50 nM
% Eff
EC50 nM
% Eff
EC50 nM
% Eff
LG100268
231 (13)
100
259 (19)
100
247 (19)
100
264 (18)
100
(±)-1
957 (15)
65
1021 (163)
66
1130 (165)
68
1614 (266)
77
1-ent1
275 (18)
69
379 (20)
71
369 (30)
77
508 (81)
81
1-ent2
>2500
-
>2500
-
>2500
-
>2500
-
Co-crystallization of the RXR ligand binding domain. The histidine-tagged LBD of human RXRα (in a pET15b vector) was expressed in Escherichia coli BL21(DE3). The precise amino acid sequence of this construct can be found in Scheepstra et al., Angew. Chem. Int. Ed. 2014, 53, 6443–6448.10 this Cells were grown at 37 °C in LB medium supplemented with 100 μg mL-1 ampicillin until OD600 reached about 0.7. Protein expression was induced through addition of isopropyl-b-dthiogalactoside (IPTG) to a final concentration of 0.1 mM. After an additional incubation for 15 h at 15°C, cell cultures were harvested by centrifugation at 8,000 ´ g for 20 min. The cell pellet of 2 liters of culture expressing RXR LBD was resuspended in 50 mL buffer A (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM imidazole) supplemented with a protease inhibitor (PMSF) and DNAse I. The suspension was then lysed by sonication and centrifuged at 35,000 g and 4 °C for 45 min. The supernatant was loaded onto a 5 ml Ni2+-affinity column, preequilibrated with buffer A. The column was washed with 10 volumes of buffer A and 10 volumes of buffer A supplemented with 50 mM imidazole. Bound proteins were eluted with buffer A containing 200 mM imidazole. The fractions containing RXRα LBD were pooled, concentrated and desalted to buffer B (10 mM TrisHCl pH 7.5, 150 mM NaCl, 5 mM DTT). To remove the histidine-tag the protein was incubated for 16 h at 4°C with thrombin (1 unit/mg RXR). The protein was passed through a Ni2+ column and a superdex gel filtration column. The protein was concentrated and stored at -80°C until further use. Before crystallization the protein was mixed with a 1.5-fold molar excess of compound 1 and a 3-fold excess of TIF2 NR2 cofactor peptide (686-KHKILHRLLQDSS-698). The complex was incubated for 1h at 4 °C. Initial screening was performed using JCSG+ (Qiagen), NR LBD screen (molecular dimensionsTM) and PACT (Quigen®) at 4 °C using sitting drop vapor diffusion. 200 nL reservoir solution was automatically mixed with 200 nL protein solution using a pipetting robot (Mosquito® Crystal) in a 96-well plate. The drops were equilibrated against 75 µL of reservoir solution. After two days crystals had appeared in 6 conditions. Reproduction of the crystals were performed in 15well plates using the hanging drop vapor diffusion method. Drops with a size of 2 - 3 µL using different reservoir to protein ratio were manually mixed and equilibrated against reservoirs with a volume of 500 μL. Optimal crystals were grown in two days in 3 µL drops with protein solution to reservoir ratio of 1:2 with: 1 M MIB buffer (Malonic acid, Imidazole, Boric acid), pH 8 25% (w/v) PEG 1500. The crystals were cryo-cooled in liquid nitrogen using sucrose as cryo-protectant for Xray data collections. X-ray diffraction data was then collected using an in-house Rigaku Micromax-003 sealed tube X-ray source and a Dectris Pilatus 200K detector at 100K. The data was indexed and integrated using iMosflm and scaled and merged using Aimless. 2 Phasing was done by molecular replacement using Phaser3 and 4OC7 as a starting model, followed by iterative rounds of refinement and manual
model building using Phenix.Refine4 and COOT5 respectively. Model validation was performed using MolProbity prior to PDB submission (PDB code: 5LYQ). Table S2 Crystallographic statistics for Spiroketal (±)-1 Spiroketal (±)-1 Data collection Resolution (Å) a
42.58-2.17 (2.24-2.17)
Space group
P43212
Cell parameters (Å)
a=b=64.63, c=113.21
CC1/2
(%)a,b,c
99.9 (87.8)
Rmerge a, c
0.092 (0.803)
Average I/σ(I) a,c
22.3 (2.1)
Completeness (%) a, c
99.9 (99)
Redundancy a, c
10.2 (5.8)
Refinement Number of protein/solvent/ligand atoms
1870/165/30
Rwork/Rfree (%)
21.3/25.4
No. of reflections
13159
Ramachandran favored (%)
98.7
Ramachandran allowed (%)
1.3
Ramachandran outliers (%)
0
R.m.s. deviations from ideal values bond lengths 0.003 / 0.56 (Å) / bond angles (°) Average protein/solvent/ligand B-factor (Å2)
30.59/39.15/30.59
a
number in parentheses is for the highest resolution shell
b
CC1/2 = Pearson's intra-dataset correlation coefficient, as described by Karplus and Diederichs. 6
c
Values as reported by Aimless version 0.5.27. 2
Figure S2: Screenshots taken from COOT4 during refinement of the spiroketal containing crystal structure. The 2Fo-Fc density map is displayed in blue at 1 σ, the Fo-Fc map is displayed in red (negative values) and green (positive values) at 2.5 σ. Left: The R-enantiomer is modeled in the density, showing good overlap in the 2FoFc map and only minor peaks in the Fo-Fc map. Right: The S-enantiomer shows a less than optimal fit into the 2Fo-Fc map and large deviations in the Fo-Fc map.
Both the S- and R-enantiomer of 1 were modeled into the observed ligand density, and by visual inspection the R-enantiomer shows a much better fit into the electron density. This is supported by an electron density fit analysis, which yields a correlation coefficient of 0.63 for the Renantiomer compared to 0.43 for the S-enantiomer (+1 means perfect fit, -1 means perfect mismatch). Additionally, a geometry analysis yields a z-score of 1.12 for the R-enantiomer and 2.84 for the S-enantiomer. The geometry z-score is the number of standard deviations that the current conformation of the molecule deviates from the bond angle/length and plane restraints that have been defined for the ligand. These restraints are generated from the MOGUL 7 crystallographic database.
Modeling and docking. The X-ray crystal structure of the complex of RXR (PDB code: 1MVC)
with full agonist BMS649 was used for our modeling studies summarized in figure 1b. The energy of the system was minimized using the MAB force field as implemented in the computer program MOLOC.8 All types of interactions (H-bonding, lipophilic and repulsive interactions) between spiroketal products – both R-1 and S-1 – and protein were analyzed in MOLOC. Designed compounds were subsequently docked into the active site of RXR by using the FlexX docking module in the LeadIT suite.9 During the docking, the binding site in the protein was restricted to 6.5 Å around the co-crystallized ligand BMS649, and the 30 top scored FlexX solutions were retained, subsequently post-scored with SEESAR.10 The best scored pose for R1 is shown in Figure 1 of the main manuscript. A similar strategy was used for the modeling studies summarized below in figure S3, except this time starting from our co-crystal structure of R-1 bound to hRXRα/TIF2 (PDB code: 5LYQ). The best scored pose for R-1 and S-1 are shown, superimposed with the pose fitted in the crystal structure. a
b
c
Figure S3 | Top ranked poses for R-1 (a) and S-1 (b) docking into the space occupied by R-1 in the hRXRα–R-1 co-crystal structure reported in this work (PDB code: 5LYQ) superimposed with the hRXRα–R-1 co-crystal structure (c).
Luciferase assays with U2OS cells. The mammalian two-hybrid (M2H) assay conditions used for this manuscript are the same as those described in the supporting information of Scheepstra et al., Angew. Chem. Int. Ed. 2014, 53, 6443–6448.10
Chiral column chromatography. Chiral column chromatography was performed on a Chiralpak IA3 column with a particle size of 3 μm, dimensions 2.1 mm x 100 mm, amylose immobilized on silica gel. Eluting with hexane/2-propanol (95:5 v/v) with 0.1% formic acid at a flowrate of 0.15 mL/min. Semi-preparative column chromatography was performed on a ChiralCel®OD cellulose column, 0.46x25 cm with hexane/2-propanol (98:2 v/v) with 0.1% formic acid and a flowrate of 1 mL/min. The UV signal was recorded with a PDA SPD-M10Avp between 200-350 nm. a
b
c
Figure S4 | Results of chiral column chromatography. a) (±)-1 crude mixture. b) 1-ent1 after chiral column chromatography. c) 1-ent2 after chiral column chromatography. Chiral column chromatography was performed on a Chiralpak IA-3 column with a particle size of 3 μm, dimensions 2.1 mm x 100 mm, amylose immobilized on silica gel. Eluting with hexane/2-propanol (95:5 v/v) with 0.1% formic acid
a
b
LCMS trace of 1-ent1, (m/z): [M+H] + calcd
LCMS trace of 1-ent2, (m/z): [M+H]+ calcd 407.22,
407.22, found 407.25.
found 407.25.
Figure S5 | LC-MS traces after chiral column chromatogrpy of the separated enantiomers. a) 1-ent1 and b) 1-ent2
Cahn–Ingold–Prelog priority for spiroketal (±)-1
Figure S6 | The Cahn-Ingold-Prelog ordering for spiroketal (±)-1. The priority was determined in accordance with the CIP priority rules. Numbers of the corresponding carbons are depicted, C 0 and O0 correspond to “ghost” carbons or oxygens. Synthetic procedures General considerations synthetic procedures. Water was purified using a Millipore purification train. All the reagents are commercially available and used without purification. All the NMR data were recorded on a Varian Gemini 200 or 400 MHz NMR or a Bruker Cryomagnet for NMR spectroscopy 400 MHz (400 or 200 MHz for 1H-NMR and 100 or 50 MHz for 13C-NMR). Proton experiments are reported in parts per million (ppm) downfield of TMS and were relative to the residual chloroform (7.26 ppm). All 13C spectra were reported in ppm relative to residual chloroform (77 ppm) Analytical LC-MS was performed on a C4, Jupiter SuC4300A, 150x2.00 mm column with a gradient 5% to 100% acetonitrile in H2O in 15 min. Silica column chromatography was performed manually using silica with particle size 60 – 200 µm. Preparative HPLC was performed on a Gemini S4 110A 150x21.20 mm column using H2O with 0.1% Formic Acid (F.A.) and acetonitrile with 0.1% F.A. Purity and exact mass of the final compound was determined using a High Resolution LC-MS system consisting of a Waters ACQUITY UPLC I-Class system coupled to a Xevo G2 Quadrupole Time of Flight (Q-tof). The system was comprised of a Binary Solvent Manager and a Sample Manager with Fixed-Loop (SM-FL). compounds were separated (0.3 mL min -1) by the column (Polaris C18A reverse phase column 2.0 x 100 mm, Agilent) using a 15% to 75% acetonitrile
gradient in water supplemented with 0.1% v/v formic acid before analysis in positive mode in the mass spectrometer. Compound LG100268 was commercially obtained from Sigma Aldrich and used without further purification.
methyl 4-(allyloxy)benzoate 9. methyl 4-hydroxy-benzoate 8 (6.0 g, 39.4 mmol) was dissolved in acetone (44 mL). To this solution were added K2CO3 (16.3 g, 118 mmol) and 3-bromoprop-1-ene (3.5 mL, 40.2 mmol). The reaction was stirred at 65 °C for 20 h. The solvent was evaporated and the residue separated between CH2Cl2 and H2O. The aqueous layer was extracted twice with CH2Cl2. The combined organic layers were washed with brine, dried over Na 2SO4, filtered and concentrated in vacuo. The product was purified via column chromatography, eluting with CH 2Cl2 to obtain methyl 4-(allyloxy)benzoate as colorless oil in quantitative yield. 7.56 g, 39.3 mmol. Silica gel TLC Rf = 0.53 (CH2Cl2). GCMS (EI) expected m/z: 192, most abundant peaks observed: 192, 161, 133; 1H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.8 Hz, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.09 – 6.00 (m, 1H), 5.42 (d, J = 17.3 Hz, 1H), 5.31 (d, J = 10.6 Hz, 1H), 4.58 (d, J = 5.1 Hz, 2H), 3.88 (s, 3H); 13C NMR (50 MHz, CDCl3) δ 166.92, 162.42, 132.67, 131.67, 122.82, 118.22, 114.41, 68.95, 51.97. methyl 3-allyl-4-hydroxybenzoate 10. methyl 4-(allyloxy)benzoate (1.1 g, 5.9 mmol) was dissolved in chlorobenzene (2.0 mL) in a microwave reaction vessel equipped with stirring bar. After the vessel was sealed, the sample was irradiated for 240 min at 245 °C. The reaction mixture was subsequently cooled to room temperature and the solvent was evaporated to yield methyl 3-allyl-4hydroxybenzoate as off white solid, 1.1 g, 5.9 mmol. The material was used without further purification. Silica gel TLC Rf = 0.18 (CH2Cl2); GCMS (EI) expected m/z: 192, most abundant peaks observed: 192, 161. methyl 3-allyl-4-(methoxymethoxy)benzoate 11. methyl 3-allyl-4-hydroxybenzoate (1.1 g, 5.9 mmol) was dissolved in CH2Cl2 (11.5 mL). The solution was cooled to 0 °C and iPr2NEt (1.9 mL, 10.63 mmol) was added. Finally chloromethyl methyl ether (0.63 mL, 8.3 mmol) was added and the mixture was stirred at 0 °C for 30 min. The reaction was then allowed to warm to room temperature and was stirred until all the starting material was consumed. After 4 hours the reaction mixture was separated between CH2Cl2 and H2O. The aqueous layer was extracted twice with CH2Cl2. The
combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified via column chromatography eluting with CH 2Cl2 to yield methyl 3-allyl-4(methoxymethoxy)benzoate as colorless oil in 80% yield over two steps. 1.1 g, 4.74 mmol. Silica gel TLC Rf = 0.46 (CH2Cl2); GCMS (EI) expected m/z: 236, most abundant peaks observed: 236, 191; 1
H NMR (200 MHz, CDCl3) δ 7.92 – 7.83 (m, 2H), 7.09 (d, J = 8.2 Hz, 1H), 6.10 – 5.88 (m, 1H),
5.26 (s, 2H), 5.12 – 5.00 (m, 2H), 3.88 (s, 3H), 3.48 (s, 3H), 3.42 (d, J = 6.5 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 167.07, 158.72, 136.29, 131.70, 129.73, 129.22, 123.43, 116.07, 113.04, 94.12, 56.40, 52.02, 34.46. methyl-3-(3-hydroxypropyl)-4-(methoxymethoxy)
benzoate
12.
methyl
3-allyl-4-
(methoxymethoxy)benzoate (330.76 mg, 1.4 mmol) was dissolved in THF (15 mL) and cooled to 0 °C. Borane dimethyl sulfide complex (266 μL, 2.8 mmol) was added drop wise and the reaction was stirred for 5 h at 0 °C. The reaction mixture was allowed to warm to room temperature and was stirred overnight. The excess of borane complex was quenched with MeOH (1.5 mL). NaOH (1 M, 1.55 mL) was added carefully and finally H2O2 (35% 1.7 mL) was added drop wise. The reaction was stirred for 24 h. The reaction mixture was separated between H2O (15 mL) and Et 2O (15 mL), the aqueous layer was extracted with Et 2O (twice 15 mL). The combined organic layers were dried over MgSO4, filtered and evaporated. The product was purified via column chromatography eluting with 40 % v/v EtOAC in heptane to obtain methyl 3-(3-hydroxypropyl)-4-(methoxymethoxy)benzoate as colorless oil, 190 mg, 0.75 mmol 54%. Silica gel TLC Rf = 0.19 (Hexane/EtOAc 40% v/v); LC-MS (ESI): calc. for C 13H18O5 [M+H]: 255.28, observed 254.83, LC, Rt=5.10 min; 1HNMR (400 MHz, CDCl3): δ (ppm) 7.90 – 7.81 (m, 2H), 7.09 (d, J = 9.2 Hz, 1H), 5.26 (s, 2H), 3.88 (s, 3H), 3.87 (s, 1H), 3.66 (t, J = 6.3 Hz, 2H), 3.49 (s, 3H), 2.77 (t, J = 14.4 Hz, 2H), 1.89 (dd, J = 8.5, 6.4 Hz, 2H); 13
C NMR (50 MHz, CDCl3) δ 167.16, 158.97, 131.81, 130.70, 129.55, 123.46, 113.13, 94.34, 62.20,
56.48, 52.07, 32.77, 26.35. methyl
4-(methoxymethoxy)-3-(3-oxopropyl)benzoate
3.
Methyl
3-(3-hydroxypropyl)-4-
(methoxymethoxy) benzoate (127.5 mg, 0.50 mmol) was dissolved in CH 2Cl2 (1.1 mL). Trichloroisocyanuric acid (117.8 mg, 0.5 mmol) was added and the mixture was cooled to 0 °C. (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (0.8 mg, 5,0 µmol) was added and the mixture was stirred at 0 °C for 2 minutes. The mixture was then allowed to warm to room temperature and stirred for 20 minutes, passed through celite® eluting with CH2Cl2. The organic phase was washed with sat. aq. Na2CO3, 1 M HCl, dried with Na2SO4, filtered and evaporated. The product was purified via column chomatography eluting with 35% v/v EtOAc in heptane to yield methyl 4(methoxymethoxy)-3-(3-oxopropyl)benzoate as a white solid. 114 mg, 0.45 mmol, 90%. Silica gel TLC Rf = 0.30 (Heptane/EtOAc 35% v/v); GC-MS (EI): calc. for: C 13H16O5: 252.26, observed most abundant peaks: 252, 190, 176; 1H NMR (400 MHz, CDCl3): δ 9.84 (t, J = 1.4 Hz, 1H), 7.91 - 7.84
(m, 2H), 7.10 (d, J = 8.5 Hz, 1H), 5.26 (s, 2H), 3.88 (s, 3H), 3.48 (s, 3H), 3.00 (t, J = 7.5 Hz, 2H), 2.76 (dd, J = 15.8, 8.5 Hz, 2H); 13C NMR (50 MHz, CDCl3) δ 201.85, 166.90, 158.87, 131.61, 130.04, 129.23, 123.52, 113.14, 94.22, 56.49, 52.07, 43.78, 23.39.
5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol 15. 2,5-dichloro-2,5-dimethylhexane 13 (4.0 g, 21.84 mmol) and phenol 14 (2.1 g, 21.84 mmol) were dissolved in dry CH2Cl2 (24 mL) in an oven dried flask. To this solution AlCl3 (988 mg, 0.33 mmol) was added. The reaction was stirred at room temperature for 2 hours and then at 40 °C for 1.5 hours. The reaction mixture was cooled to 0 °C and 2 M HCl (24 mL) was poured into the mixture. The aqueous layers was extracted with Et 2O three times. The combined organic layers were washed with brine, dried over Na 2SO4, filtered and evaporated. The product was purified via column chromatography eluting with 7% EtOAc in pentane to yield 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol as white solid. 3.2 g, 15.7 mmol, 72%. Silica gel TLC Rf = 0.37 (pentane/EtOAc 7% v/v); LC-MS (ESI): calc. for C 14H20O [M+H]: 205.32, observed 205.25, LC, Rt=8.20 min; 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 2.8 Hz, 1H), 6.64 (dd, J = 8.5, 2.8 Hz, 1H), 4.68 (s, 1H), 1.67 (s, 4H), 1.26 (s, 6H), 1.25 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 153.11, 146.71, 137.45, 127.88, 113.22, 112.80, 35.28, 35.21, 34.51, 33.80, 32.14, 31.93. 3-iodo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol 16. To a solution of 5,5,8,8-tetramethyl5,6,7,8-tetrahydronaphthalen-2-ol 15 (3.18 g, 15.56 mmol) in MeOH (37.5 mL) were added sodium iodide (2.3 g, 15.56 mmol) and sodium hydroxide (627 mg, 15.56 mmol). The mixture was cooled to 0 °C and sodium hypochlorite (5%, 21.53 mL, 15.56 mmol) was added dropwise. The reaction was stirred for 2 hours at 0 °C and then quenched with 10% w/v Na 2S2O3. The pH was adjusted to 6 with 1 M HCl and the aqueous layer was extracted with Et 2O three times. The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified via column chromatography, eluting with 7% v/v EtOAc in heptane to yield 3-iodo-5,5,8,8tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol as white solid. 4.21 g, 12.76 mmol, 82%.Silica gel TLC Rf = 0.21 (heptane/EtOAc 7% v/v); 1H NMR (400 MHz, CDCl3) δ 7.54 (s, 1H), 6.94 (s, 1H),
5.06 (s, 1H), 1.66 (s, 4H), 1.26 (s, 6H), 1.25 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 152.49, 147.96, 140.03, 136.27, 112.82, 83.19, 77.16, 35.10, 34.96, 34.51, 33.86, 32.10, 31.83. 6-iodo-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene 17. To a solution of 3iodo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol 16 (3.97 g, 12.02 mmol) in CH2Cl2 (23 mL) was added diisopropylethylamine (3.8 mL, 21.64 mmol) and chloro(methoxy)methane (1.2 mL, 16.83 mmol) carefully. The cooling bath was removed after 30 min and the reaction was stirred at room temperature for 5 hours. The reaction mixture was separated between CH2Cl2 and H2O. The aqeous layer was extracted twice with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified via column chromatography, eluting with 7% EtOAc in heptane to obtain 6-iodo-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2,3,4tetra hydronaphthalene. 4.1 g, 10.97 mmol, 91%. Silica gel TLC Rf = 0.48 (heptane/EtOAc 7% v/v); GC-MS (EI): calc. for C 16H23IO2: 374.26, observed most abundant peaks: 374, 359, 329; 1H NMR (400 MHz, CDCl3) δ 7.65 (s, 1H), 6.99 (s, 1H), 5.20 (s, 2H), 3.53 (s, 3H), 1.65 (d, J = 1.6 Hz, 4H), 1.26 (s, 6H), 1.25 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 154.05, 147.00, 141.22, 137.52, 113.30, 95.51, 84.66, 56.58, 35.06, 35.04, 34.68, 33.92, 31.97, 31.84 4-(3-(methoxymethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahy-dronaphthalen-2-yl)-2-methylbut-3-yn-2ol 18. An oven dried schlenk tube was charged with 6-iodo-7-(methoxymethoxy)-1,1,4,4-tetramethyl1,2,3,4-tetrahydronaphthalene 17 (904 mg, 2.4 mmol), PdCl2(PPh3)2 (214 mg, 0.31 mmol) and CuI (60.1 mg, 0.32 mmol). The flask was evacuated and backfilled wit argon three times, then NEt 3 (35 mL) and 2-methylbut-3-yn-2-ol (290 µL, 2.9 mmol) were added under a positive argon flow. The reaction was stirred at 80 °C for 20 hours. The reaction mixture was allowed to cool to room temperature and was passed through celite, eluting with EtOAc and concentrated under reduced pressure. The product was purified via column chromatography eluting with heptane/EtOAc 80/20 v/v to yield a colorless oil, 68.7 mg, 87%. Silica gel TLC Rf = 0.22 (20% EtOAc/heptane); GCMS (EI) expected m/z: 330.2, observed [M+]: 330; 1HNMR (400 MHz, CDCl3): δ (ppm) 7.30 (s, 1H), 6.97 (s, 1H), 5.20 (s, 2H), 3.53 (s, 3H), 1.64 (s, 4H). 1.62 (s, 6H), 1.242 (s, 6H), 1.237 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 155.41, 147.26, 139.04, 131.71, 114.16, 111.28, 96.81, 95.78, 78.94, 65.81, 56.41, 35.07, 35.02, 34.73, 33.82, 31.93, 31.79, 31.67. 6-ethynyl-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2,
3,4
–tetrahydronaphthalene
2.
4-(3-
(methoxymethoxy) -5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-methylbut-3-yn-2-ol 18 (221.44 mg, 0.67 mmol) was disolved in dry toluene (17.6 mL). NaOH (137 mg, 3.43 mmol) was added and the reaction was refluxed at 120 °C for 3.5 hour. The reaction mixture was allowed to cool to room temperature and was quenched with sat. aq. NH4Cl, the mixture was diluted with H2O and extracted twice with Et 2O. The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified via column chromatography eluting with 17% v/v
EtOAc
in
heptane
to
yield
6-ethynyl-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2,3,4-
tetrahydronaphthalene as a white solid. 164 mg, 0.6 mmol, 90%. Silica gel TLC Rf = 0.30 (heptane/EtOAc 7% v/v); GC-MS (EI): calc. for: C 18H24O2: 272,38, observed most abundant peaks: 272, 257, 227, 225; 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 7.04 (s, 1H), 5.23 (s, 2H), 3.53 (s, 3H), 3.22 (s, 1H), 1.66 (s, 4H), 1.26 (s, 6H), 1.25 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 156.12, 147.98, 138.89, 132.41, 113.35, 110.22, 95.47, 80.76, 79.90, 56.39, 35.03, 34.86, 33.83, 31.94, 31.80. methyl4-(3-hydroxy-5-(3-(methoxymethoxy)-5,5,8,8-tetra
methyl-5,6,7,8-tetrahydronaphthalen-2-
yl)pent-4-yn-1-yl)-3-(methoxymethoxy)benzoate (alkynol) 4. n-butyllithium (152 µL, 1.1 M in hexane, 0.17 mmol) was added dropwise to a stirred solution of 6-ethynyl-7-(methoxymethoxy)-1,1,4,4tetramethyl-1,2,3,4-tetrahydronaphthalene 2 (52.1 mg, 0.19 mmol) in THF (600 µL) at -78 °C. The mixture was stirred at -78 °C for 40 minutes. methyl 4-(methoxymethoxy)-3-(3-oxopropyl)benzoate 3 (38,6 mg, 153 mmol) in THF (300 µL) was added dropwise and the reaction was stirred for 60 minutes at -78 °C. The reaction mixture was then allowed to warm to room temperature and was stirred for 2.5 hour. The reaction was then quenched with H2O and extracted with EtOAc three times. The combined organic layers were washed with brine and evaporated. The product was purified via column chromatography, eluting with 30 % EtOAc in heptane, to yield a pale yellow oil. 47 mg, 58%. Silica gel TLC Rf = 0.25 (heptane/EtOAc 30% v/v); LC-MS (ESI): calc. for C 31H40O7 [M+Na]: 547.63, observed 547.50, LC, Rt=9.02 min; 1H NMR (200 MHz, CDCl3) δ 7.94 – 7.83 (m, 1H), 7.33 (s, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.01 (s, 1H), 5.26 (s, 2H), 5.21 (s, 2H), 4.64 (t, J = 6.4 Hz, 1H), 3.87 (s, 3H), 3.51 (s, 3H), 3.48 (s, 3H), 2.98 – 2.87 (m, 2H), 2.24 – 2.04 (m, 2H), 1.65 (s, 4H), 1.25 (s, 6H), 1.24 (s, 6H); 13C NMR (50 MHz, CDCl3) δ 167.06, 159.04, 155.64, 147.54, 138.94, 131.86, 130.51, 129.67, 123.48, 113.68, 113.17, 110.92, 95.59, 94.28, 92.77, 82.08, 62.76, 56.46, 56.37, 52.01, 37.93, 35.07, 34.81, 33.84, 31.95, 31.81, 26.28. methyl 4-(3-hydroxy-5-(3-(methoxymethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl) pentyl)-3-(methoxymethoxy)benzoate 5. Alkynol 4 (23.7 mg, 0.045 mmol) was dissolved in EtOAc (0.6 mL). KHCO3 (16.8 mg, 0.17 mmol) and 10 % palladium on carbon (18 mg, 0.17 mmol) were added and the reaction was stirred at room temperature under an atmosphere of hydrogen. After 2 hours the mixture was diluted with EtOAc and passed through celite, to obtain the title compound as colorless oil. 21.7 mg, 0.041 mmol, 91%. Silica gel TLC Rf = 0.21 (heptane/EtOAc 30% v/v); LCMS (ESI): calc. for C 31H44O7 [M+Na]: 551.67, observed 551.42, LC, Rt=9.15 min; 1H NMR (200 MHz, CDCl3) δ 7.91 – 7.79 (m, 2H), 7.08 – 6.92 (m, 3H), 5.25 (s, 2H), 5.17 (s, 2H), 3.88 (s, 3H), 3.68 – 3.56 (m, 1H), 3.49 (s, 3H), 3.47 (s, 3H), 2.82 – 2.62 (m, 4H), 1.85 – 1.69 (m, 4H), 1.65 (s, 4H), 1.26 (s, 6H), 1.23 (s, 6H); 13C NMR (50 MHz, cdcl3) δ 167.10, 158.90, 153.22, 144.00, 138.38, 131.79, 131.26, 129.43, 128.44, 128.09, 123.50, 113.13, 111.80, 95.05, 94.28, 70.71, 56.44, 56.25, 52.02, 38.17, 37.52, 35.30, 34.44, 33.80, 32.05, 31.99, 26.52, 26.37.
methyl
3-(methoxymethoxy)-4-(5-(3-(methoxymethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalen-2-yl)-3-oxopentyl)benzoate (ketone) 6. Compound 5 (21.7 mg, 0.041 mmol) was dissolved in CH2Cl2 (2.4 mL). Dess–Martin periodinane (38.4 mg, 0.091 mmol) was added and the reaction was stirred for 18 hours at room temperature. The reaction was quenched with saturated aq. KHCO3 (5 mL) and 10 % Na2S2O3 (5 mL) solution. The aqueous layer was extracted with CH2Cl2 three times. The combined organic layers were washed with brine, dried over Na 2SO4, filtered and evaporated to yield the title compound. 18.5 mg, 0.035 mmol, 86%. Silica gel TLC Rf = 0.41 (heptane/EtOAc 30% v/v); LC-MS (ESI): calc. for C 31H42O7 [M+Na]: 549.65, observed: 549.42; LC, rt 9.50 min; 1H NMR (200 MHz, CDCl3) δ 7.92 – 7.80 (m, 2H), 7.12 – 6.93 (m, 3H), 5.25 (s, 2H), 5.15 (s, 2H), 3.88 (s, 3H), 3.47 (s, 6H), 3.01 – 2.65 (m, 8H), 1.65 (s, 4H), 1.25 (s, 6H), 1.23 (s, 6H); 13
C NMR (50 MHz, CDCl3) δ 210.04, 167.00, 158.92, 153.29, 144.35, 138.25, 131.59, 130.07, 129.77,
128.05, 127.43, 123.47, 113.11, 111.72, 94.83, 94.17, 56.44, 56.20, 52.05, 43.36, 42.63, 35.29, 34.47, 33.82, 32.05, 31.96, 25.11, 24.82. Spiroketal 7. To a solution of ketone 6 (18.5 mg, 35 µmol) in CH2Cl2 (350 µL) was added trimethylsilyl bromide (46 µL, 351 µmol) at -30 °C. The reaction was stirred for 1 hour at -30 °C and 1 hour at 0 °C. the reaction was then quenched with H2O and extracted with EtOAc three times. The combined organic layers were washed with brine, dried over Na2SO4, filtered and evaporated. The product was purified via column chromatography eluting with 17% EtOAc in heptane. to yield the title compound as a white solid, 9,5 mg, 23 µmol, 64%. Silica gel TLC Rf = 0.41 (heptane/EtOAc 17% v/v); LC-MS (ESI): calc. for C 27H32O4 [M+H]: 421.24, observed 421.25, LC, Rt=10.10 min. Individual 1H and 13C resonance peaks were assigned by comparison with data for (±)-1. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H, Ar), 7.76 (dd, J = 8.6, 2.3 Hz, 1H, Ar), 7.02 (s, 1H, Ar), 6.78 (d, J = 8.5 Hz, 1H, Ar), 6.64 (s, 1H, Ar), 3.87 (s, 3H, CO2CH3), 3.34 (ddd, J = 13.2, 10.4, 4.8, 1H, 4 or 4’Hax), 3.22 (ddd, J = 15.2, 10.8, 4.4, 1H, 4 or 4’-Hax), 2.80 (ddd, J = 13.2, 4.8, 2.0, 1H, 4 or 4’-Heq), 2.71 (ddd, J = 12.8, 4.8, 2.0, 1H, 4 or 4’-Heq), 2.20 – 2.27 (m, 2H, 3 and 3’-Heq), 1.93 – 2.01 (m, 2H, 3 and 3’-Hax), 1.63 (s, 4H, H10/H11), 1.26 (s, 3H, H13/H14), 1.26 (s, 3H, H13/H14), 1.21 (s, 3H, H13/H14), 1.17 (s, 3H, H13/H14); 13C NMR (126 MHz, CDCl3) δ 167.2 (CO2CH3), 156.6 (Ar, ipso), 149.9 (Ar, ipso), 144.7 (Ar, ipso), 137.7 (Ar, ipso), 131.3 (Ar), 129.1 (Ar), 126.9 (Ar), 122.7 (Ar, ipso), 122.3 (Ar, ipso), 119.4 (Ar, ipso), 117.5 (Ar), 114.2 (Ar), 97.0 (q, 2), 52.0 (CO2CH3), 35.4 (C10/C11), 35.3 (C10/C11), 34.3 (C9/C12, q), 33.8 (C9/C12, q), 32.3 (C13/C14), 32.2 (C13/C14), 32.1 (C13/C14), 32.0 (C13/C14), 31.5 (C3/C3’), 31.4 (C3/C3’), 21.0 (C4/C4’), 20.7 (C4/C4’). Spiroketal (±)-1. Spiroketal 7 (9 mg, 21.4 μmol) was dissolved in 1,4-dioxane/MeOH (14:5 v/v, 0.5 mL). NaOH (4 M, 50 μL) was added and the reaction was stirred at 40 °C. After 4 h the solvent was evaporated and the residue was redissoved in H2O. The aqueous solution was acidified to pH 4 and extracted three times with CH2Cl2, the combined organic layers were washed with brine, dried over
Na2SO4, filtered and evaporated to obtain compound 1 in quantitative yield. The racemate (±)-1 was further purified by RP-HPLC or by chiral HPLC prior to biochemical evaluation. Individual 1H and 13C resonance peaks were assigned by comparing 1D 1H & 13C NMR spectra with 2D COSY, HSQC and HMBC spectra of the sample. LC-MS (ESI): calc. for C26H30O4 [M+H]: 407.22 observed 407.25, LC, Rt=8.98 min; 1H NMR (400 MHz, CDCl3) δ 7.92 (s, 1H, Ar), 7.84 (d, J = 8.6 Hz, 1H, Ar), 7.03 (s, 1H, Ar), 6.81 (d, J = 8.6 Hz, 1H, Ar), 6.65 (s, 1H, Ar), 3.33 (ddd, J = 17.3, 12.8, 5.8 Hz, 1H, 4 or 4’Hax), 3.22 (ddd, J = 16.1, 12.9, 5.7 Hz, 1H, 4 or 4’-Hax), 2.80 (ddd, J = 16.5, 5.8, 2.8 Hz, 1H, 4 or 4’Heq), 2.70 (ddd, J = 16.1, 5.8, 2.5 Hz, 1H, 4 or 4’-Heq), 2.19 – 2.27 (m, 2H, 3 and 3’-Heq), 1.91 – 2.01 (m, 2H, 3 and 3’-Hax), 1.64 (s, 4H H10/H11), 1.27 (s, 3H, H13/H14), 1.26 (s, 3H, H13/H14), 1.22 (s, 3H, H13/H14), 1.17 (s, 3H, H13/H14); 13C NMR (101 MHz, CDCl3) δ 171.6 (CO2H), 157.3 (Ar), 149.7 (Ar), 144.6 (Ar), 137.6 (Ar), 131.9 (Ar), 129.8 (Ar), 126.8 (Ar), 122.3 (Ar), 121.6 (Ar), 119.3 (Ar), 117.5 (Ar), 114.0 (Ar), 97.0 (q, 2), 35.2 (C10/C11), 35.1 (C10/C11), 34.2 (C9/C12, q), 33.7 (C9/C12, q), 32.2 (C13/C14), 32.1 (C13/C14), 31.9 (C13/C14), 31.8 (C13/C14), 31.3 (C3/C3’), 31.2 (C3/C3’), 20.9 (C4/C4’), 20.6 (C4/C4’); HRMS (m/z): [M + H] + calcd 407.2222, found 407.2224.
1H & 13 NMR spectra for methyl 4-(allyloxy)benzoate 9
1H & 13 NMR spectra for methyl 3-allyl-4-(methoxymethoxy)benzoate 11
1H & 13 NMR spectra for methyl-3-(3-hydroxypropyl)-4-(methoxymethoxy) benzoate 12
1H & 13 NMR spectra for methyl 4-(methoxymethoxy)-3-(3-oxopropyl)benzoate 3
1H & 13 NMR spectra for 5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol 15
1H & 13 NMR spectra for 3-iodo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-ol 16
1H
&
13
NMR
tetrahydronaphthalene 17
spectra
for
6-iodo-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2,3,4-
1H & 13 NMR spectra for 4-(3-(methoxymethoxy)-5,5,8,8-tetramethyl-5,6,7,8-tetrahy-dronaphthalen2-yl)-2-methylbut-3-yn-2-ol 18
1H & 13 NMR spectra for 6-ethynyl-7-(methoxymethoxy)-1,1,4,4-tetramethyl-1,2, tetrahydronaphthalene 2
3,4 –
1H & 13C NMR spectra for methyl4-(3-hydroxy-5-(3-(methoxymethoxy)-5,5,8,8-tetra methyl-5,6,7,8tetrahydronaphthalen-2-yl)pent-4-yn-1-yl)-3-(methoxymethoxy)benzoate (alkynol) 4
1H NMR spectrum for methyl 4-(3-hydroxy-5-(3-(methoxymethoxy)-5,5,8,8-tetramethyl-5,6,7,8tetrahydronaphthalen-2-yl) pentyl)-3-(methoxymethoxy)benzoate 5
1H & 13 NMR spectra for methyl 3-(methoxymethoxy)-4-(5-(3-(methoxymethoxy)-5,5,8,8tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-3-oxopentyl)benzoate (ketone) 6
1H & 13 NMR spectra for spiroketal 7
1H & 13 NMR spectra for spiroketal (±)-1
References: [1] Evans PR, Murshudov GN. How good are my data and what is the resolution? Acta Crystallogr Section D: Biol Crystallogr; 69, 1204–1214 (2013). [2] McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007). [3] Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C. & Zwart, P. H. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Section D, Biol Crystallogr 66, 213–21 (2010). [4] Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-32, (2004). [5] Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012). [6] Bruno I. J. et al. Retrieval of crystallographically-derived molecular geometry information. J. Chem. Inf. Comput. Sci. 44, 2133–2144 (2004). [7] Gerber, P. R. & Müller, K. MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry. J. Comput. Aided. Mol. Des. 9, 251–268 (1995). [8] BioSolveIT GmbH, Sankt Augustin. http://www.biosolveit.de, LeadIT, version 2. 1. 3. [9] BioSolveIT GmbH, Sankt Augustin. http://www.biosolveit.de, SeeSar, version 5.3. [10] Scheepstra, M. et al. A natural-product switch for a dynamic protein interface. Angew. Chem. Int. Ed. 53, 6443–6448 (2014).
