A Multi-Gram-Scale Stereoselective
`Synthesis of Z-Endoxifen
`Lech-Gustav Milroya*, Bartjan Koning,b
`Daphne S. v. Scheppingena, Nynke G. L. Jagerc, Jos H. Beijnenc,d, Jan Koekb, Luc Brunsvelda*
`a Laboratory of Chemical Biology and Institute for Complex Molecular Systems (ICMS), Department of
`Biomedical Engineering, Technische Universiteit Eindhoven, Den Dolech 2, 5612 AZ, Eindhoven, The
`Netherlands
`b Syncom B.V., Kadijk 3, 9747 AT Groningen, The Netherlands
`c Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute - Antoni van Leeuwenhoek and
`MC Slotervaart, Louwesweg 6, 1066 EC, Amsterdam, The Netherlands
`d Faculty of Science, Division of Pharmacoepidemiology and Clinical Pharmacology, Department of
`Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands
`OH
`- 5 steps
`- 18% overall yield
`- 470 mmol scale synthesis delivered 37 g
`- >97% purity, >99/1 Z/E
`- Two columns chromatographies
`- Two triturations
`- No RP-HPLC!
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`Leave this area blank for abstract info.
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`O
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`NH
`Me
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`O
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`Me
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`HO
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`OH
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`Z-endoxifen
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`Experimental Section
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`General
`
`Reagents and solvents for the initial and optimized synthesis
`Column chromatography was carried out using either silica gel (40-63 μm, ScreeningDevices b.v.) or
`neutral alumina (activated, neutral, Brockmann Activity I, Sigma-Aldrich). Dihydroxybenzophenone,
`trimethylacetyl chloride, propiophenone, zinc dust, titanium tetrachloride were all purchased from
`Sigma-Aldrich and used without further purification. Sodium hydroxide was purchased from Merck.
`Ethyl acetate, dichloromethane, hexanes, and methanol were all purchased from Biosolve B.V. For
`the synthesis of alcohol 4: 2-(methylamino)ethanol
`(Sigma-Aldrich), ethyl chloroformate,
`trimethylamine (all Sigma-Aldrich), dichloromethane. THF (both Biosolve B.V.). All deuterated
`solvents for NMR analysis were purchased from Cambridge Isotope Laboratories Inc. and used
`without further treatment. Compounds 3, 5, 6 & Z-endoxifen were synthetized under reduced light
`under the fumehood and stored in the dark at -30 °C. H2O refers to Millipore grade distilled water.
`
`Reagents and solvents for the scaled-up synthesis
`All reagents and solvents were obtained from commercial sources (Sigma Aldrich, Acros, Fluorochem
`and Combi-Blocks) and were used without further purification unless otherwise specified. All deuterated
`solvents for NMR analysis were purchased from Acros. Reverse-phase liquid chromatography-mass
`spectrometry (LC-MS) analysis of Z-endoxifen reported in Figure S1 was performed on an Applied
`Biosystems Single Quadrupole Electrospray Ionization Mass Spectrometer API-150EX in positive
`mode using a Jupiter C4-column 150 x 2.0 mm. Eluent conditions (CH3CN/H2O/1% formic acid): 0-
`2 min, isocratic, 5 % CH3CN; 2-10 min, linear gradient, 5 – 70 %; 10-12 min, isocratic, 70 %; 12-15
`min, linear gradient, 70 – 5 %. CH3CN refers to HPLC grade acetonitrile purchased from Biosolve
`B.V. The formic acid used was ULC-MS grade, 99% purchased from Biosolve B.V. H2O refers to
`MilliQ Ultrapure water for UHPLC and LC-MS. LC-UV-MS analysis of Z-endoxifen reported in
`Figure S16 was performed using a Shimadzu LC-MS with Phenomenex Luna 5u C18(2) 100A (100 x
`4.6 mm) column with PDA Detection. 1H-NMR measurements made on Z-endoxifen during the initial
`synthesis work (Figures S2-S4) were performed on a 400 MHz NMR (Varian Mercury). Proton
`chemical shifts in 1D 1H-NMR spectra are reported in ppm and calibrated against either the
`tetramethylsilane or residual CHCl3 (s, 7.26 ppm), d5-DMSO (quintet, JHD = 1.9 Hz), as the internal
`standard. Carbon chemical shifts in the 1D 13C-NMR spectra are reported in ppm and calibrated
`against the CDCl3 (77.16 ppm, t, JCD = 32 Hz) or d6-DMSO signals (39.52 ppm, septet, JCD = 21
`Hz). 1H-NMR, 13C-NMR and 19F NMR measurements made during the optimized synthesis of Z-
`endoxifen (Figures S6-S15) were recorded on a Varian VNMRS: 7.05 Tesla magnet from Oxford
`Instruments, indirect detection probe 300 MHz 1H{15N-31P}, Direct drive console including PFG module
`and a Varian MP300: 7.05 Tesla magnet from Oxford Instruments, 4 nuclei autoswitchable probe
`1H/19F/{15N-31P}, Mercury plus console. The splitting patterns are designated as: s, singlet; d, doublet;
`t, triplet; q, quartet; br s, broad singlet; m, multiplet. The purity-determination by qNMR with 3,5-
`dinitrobenzoic acid as reference were calculated by comparing the combined integrals of the aromatic
`signals of 3,5-dinitrobenzoic acid and the combined integral of the signals at δ 4.08, 3.25 and 0.83 ppm of
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`Z-Endoxifen. For the purity-determination by qNMR with maleic acid the integral of the signal at δ
`6.16 ppm was compared with the integral of the combined signals at δ 4.05 and 0.83 ppm.
`
`Determination of E/Z ratios of endoxifen by HPLC analysis described in Figures S5
`Measurement were performed according to the methods described in Teunissen et al., 2011.1
`
`Reagents and chemicals
`Acetonitrile and methanol were obtained from Biosolve Ltd. (Amsterdam, the Netherlands).
`Ammonium formate was purchased from Acros Organics (Geel, Belgium). Formic acid and
`LiChrosolv water for HPLC were purchased from Merck (Darmstadt, Germany).
`
`Instrumentation & gradient conditions
`An Agilent HPLC system was used consisting of an 1100 series binary pump, column oven, on-line
`degasser and autosampler (Agilent Technologies, Palo Alto, CA, USA). Mobile phase A was
`prepared by adjusting a 4.0 mM ammonium formate solution to pH 3.5 with a 98% formic acid
`solution. Mobile phase B consisted of 100% acetonitrile. Mobile phases A and B were pumped
`through a Kinetex C18 100 Å column (150 x 2.1 mm I.D., 2.6 μm; Phenomenex) at a flow rate of 0.4
`mL/min using a gradient as shown in Table 2. The analytical column was protected by a
`KrudKatcher inline filter (Phenomenex, Torrance, CA, USA). The separation was performed at 60˚C.
`Volumes of 15 μL were injected using the autosampler thermostatted at 7 ˚C. The column was
`equilibrated for 3 minutes before the next injection, leading to a total run time of 10 minutes. The
`autosampler needle was rinsed with acetonitrile before and after each injection. During the first and
`last 1.0 minute the eluate was directed to waste using a divert valve to prevent the introduction of
`endogenous compounds into the mass spectrometer. The HPLC gradient conditions used to separate
`Z- and E-isomers of endoxifen using the above conditions are as follows: under a constant flow rate
`0.40 mL/min, mobile phase A = 4.0 mM ammonium formate buffer pH 3.5, mobile phase B =
`acetonitrile; t = 0.00 min, A = 70%, B = 30%; t = 6.00 min, 47.5/52.5; t = 6.01 min, 20/80; t = 7.00
`min, 20/80; t = 7.01 min, 70/30; t = 10.00 min, 70/30.
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`Chemical synthesis of Z-endoxifen
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`Me
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`OH
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`O
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`NH
`Me
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`Initial synthesis
`The initial synthesis of Z-endoxifen was performed according to the synthetic route depicted in
`Figure 2 of the main manuscript and the synthetic protocols described in previous work published by
`Gauthier & Labrie et al.2 and Fauq et al.3 The crude material was purified by silica gel column
`chromatography (MeOH/CH2Cl2) to afford Z-endoxifen (Figure S1) as a 3.6/1 E/Z mixture determined
`by 1H-NMR (Figures S2 & S3) and HPLC (Figure S5, left panel).
`
`Optimized synthesis
`For the optimized synthesis, the crude material was instead purified by column chromatography
`using neutral alumina (MeOH/CH2Cl2) to afford Z-endoxifen as a 96/4 Z/E mixture determined by
`1H-NMR (Figure S4) and HPLC (Figure S5, right panel)
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`Figure S1. LC-MS analysis of Z-endoxifen after purification by silica gel column chromatography
`(MeOH/CH2Cl2). Expected 374.21, observed 374.25 [M+H]+.
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`Figure S2. 1D 1H NMR (d6-DMSO, 400 MHz) of Z-endoxifen after purification by silica gel column
`chromatography (MeOH/CH2Cl2). Z/E ratio = 3.6/1 (see Figure S5)
`
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`Comparison of experimental and literature 1D 1H NMR shift values for Z-endoxifen
`
`Literature [S. M. Ali, et al. Bioorg. Med. Chem. Lett., 2010, 20, 2665]:4 1H-NMR (400 MHz, d6-
`DMSO): δ 0.85 (t, J = 7.24 Hz, 3H), 2.29 (s, 3H), 2.42 (q, J = 7.2 Hz, 2 H), 2.74 (t, J = 5.59 Hz, 3H), 3.86
`(t, J = 5.56 Hz, 2H), 6.58 (d, J = 8.78 Hz, 2H), 6.71 (d, J = 8.56 Hz, 2H), 6.75 (d, J = 8.68 Hz, 2H), 6.98
`(d, J = 8.2 Hz, 2H), 7.08–7.13 (m, 3H), 7.15–7.19 (m, 2H), 9.38 (br s, 1H)
`
`This publication (only values for major isomer, Z-isomer are reported) [internal code: DS_010_1stp]:
`1H-NMR (400 MHz, d6-DMSO): δ 0.84 (t, J = 7.2 Hz, 3H), 2.31 (s, 3H), 2.40 (q, J = 7.2 Hz, 2 H), 2.78
`(t, J = 5.6 Hz, 3H), 3.87 (t, J = 5.6 Hz, 2H), 6.56 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.8 Hz, 2H), 6.75 (d, J =
`8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 7.07–7.11 (m, 3H), 7.15–7.19 (m, 2H), 9.40 (br s, 1H).
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`9.39 ppm
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`OH
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`Me
`NH
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`O
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`Me
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`(Z)
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`Me
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`(E)
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`Me
`NH
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`O
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`OH
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`9.14 ppm
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`Figure S3. A portion of the 1D 1H NMR spectrum (d6-DMSO, 400 MHz) of Z-endoxifen shown in
`Figure 2 after aqueous work-up but before (crude, top) and then after purification (bottom) by silica gel
`chromatography. Z/E ratio = 3.6/1 (see Figure S5)
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`Figure S4. 1D 1H NMR (d6-DMSO, 400 MHz) of Z-endoxifen after purification using neutral alumina.
`Z/E ratio = 96/4 (see Figure S5)
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`9.42 ppm
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`OH
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`Me
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`(Z)
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`Me
`NH
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`O
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`Figure S5. Comparison of LC-UV data for Z-endoxifen analyzed after purification on silica gel
`chromatography (left) – Z/E ratio = 3.6/1 – and neutral alumina (right) – Z/E ratio = 96/4.
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`Large-scale synthesis of Z-endoxifen
`
`
`i. PivCl, Et3N
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`OH
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`ii, LiOH
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`HO
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`O
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`2
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`O
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`Me
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`OPiv
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`Zn, TiCl4
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`O
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`1
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`OPiv
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`HO
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`Me
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`(E)
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`OPiv
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`OH
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`Me
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`(E)
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`LiOH
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`Me
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`(Z)
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` DIAD
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`PPh3,
`e
`OH
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`NM
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`CF3
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`6
`>95:5 E/Z
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`O
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`NMe
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`O
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`Z-endoxifen
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`O
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`Me
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`NH
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`CF3
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`
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`3
`>95:5 E/Z
`
`OH
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`7
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`O
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`Scheme S1. Overview of large-scale synthetic route to Z-endoxifen.(see Figure 3 of main manuscript)
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`4-(4-hydroxybenzoyl)phenyl pivalate 2
`Bis(4-hydroxyphenyl)methanone 1 (100 g, 0.47 mol) was dissolved in 1 L of THF and cooled to 0
`°C. Triethylamine (188.7 g, 1.87 mol) was added followed by the dropwise addition of PivCl (141.7
`g, 1.17 mol). More THF (250 mL) was added and the mixture was stirred overnight. The mixture was
`quenched with 500 mL of water and extracted with ethyl acetate (2x). The combined organic layers
`were washed with 1N HCl (500 mL) and brine and dried over Na2SO4. Concentration afforded 194.3
`g of the crude bis protected benzophenone. The material was dissolved in 750 mL of THF and 50 mL
`of methanol. LiOH.H2O (21.4 g, 0.5 mol) was added and the mixture was stirred for 1 hour. More
`LiOH.H2O (5 g, 0.12 mol) was added and the mixture was stirred for an additional 30 min. The
`mixture was concentrated. At this point a second equal batch was run and combined with the first
`one. The combined batches were coated on silica and purified by means of column chromatography
`(silica; CH2Cl2/EtOAc 20:1→ CH2Cl2/THF 20:1) affording 4-(4-hydroxybenzoyl)phenyl pivalate as
`a white solid (140 g, 47%). 1H-NMR (300 MHz, CDCl3): δ 1.38 (s, 9H) 6.41 (br s, 1H), 6.90 (d, J = 9
`Hz, 2H), 7.18 (d, J = 8,7 Hz, 2H), 7.75 (d, J = 8,7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H).
`
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`Figure S6. 1D 1H NMR spectrum of pure 2 (CDCl3, 300 MHz).
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`2,2,2-trifluoro-N-(2-hydroxyethyl)-N-methylacetamide 7
`Ethyl trifluoroacetate (92.3 g, 0.65 mol) was slowly added to 2-(methylamino)ethanol (50 g, 0.67 mol) at
`10 °C. The mixture was stirred for 3 hours at rt and CH2Cl2 was added. The mixture was washed with 1N
`HCl and brine and dried over Na2SO4. Evaporation of the solvent afforded 2,2,2-trifluoro-N-(2-
`hydroxyethyl)-N-methylacetamide as colorless oil (84 g, 75%, 2.7:1 mixture of rotamers). 1H-NMR (300
`MHz, CDCl3): δ 2.28 (br s, 1H), 3.10 (apparent s, 3H – minor rotamer), 3.22 (m, 3H – major rotamer),
`3.56-3.62 (m, 2H) 3.81-3.86 (m, 2 H); 13C-NMR (75 MHz, CDCl3): δ 35.3, 36.2 (q, J = 3.8 Hz), 51.3 (q, J
`= 3.0 Hz), 51.9, 59.3, 59.8, 116.44 (q, J = 285 Hz), 116.41 (q, J = 285 Hz), 157,40 (q, J = 35.3 Hz),
`157,55 (q, J = 35.3 Hz); 19F-NMR (282 MHz) -69.9 (major rotamer), -68.2 (minor rotamer).
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`Figure S6. 1D 1H NMR spectrum of pure 7 (CDCl3, 300 MHz).
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`Figure S7. 1D 13C NMR spectrum of pure 7 (CDCl3, 75 MHz).
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`Figure S8. 1D 19F NMR spectrum of pure 7 (CDCl3, 282 MHz).
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`(E)-4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl pivalate 3
`To a suspension of zinc dust (153.4 g, 2360 mmol) in anhydrous THF (2 L) was added TiCl4 (223.8
`g, 1180 mmol) dropwise at 0-10 °C. The mixture was refluxed for 2 h and then cooled to 40 °C. A
`mixture of 4-(4-hydroxybenzoyl)phenyl pivalate (88 g, 295 mmol) and propiophenone (123.5 g, 922
`mmol) in anhydrous THF (4 L) was added at once and the mixture was refluxed for 5 h. Upon
`completion, the reaction mixture was cooled to 0 °C and quenched with 10% K2CO3 (4 L). Celite
`was added and the mixture was stirred for 30 min before the organic layer was sucked from the
`mixture and filtered over Celite. The aqueous/celite mixture was stirred up with EtOAc and the
`organic layer was sucked from the mixture and filtered over Celite as before. This was repeated three
`times. The combined organic extracts were successively washed with 10% K2CO3 and brine, dried
`over MgSO4, filtered, and the filtrate concentrated under reduced pressure to afford a crude (E)-4-(1-
`(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl pivalate (234 g). After thorough evaporation of the
`solvents the crude material was triturated in 200 mL of methanol (4x) affording pure (E)-4-(1-(4-
`hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl pivalate as a white solid. The filtrate was concentrated
`and the obtained material was triturated in 200 mL of methanol (3x). Extra material was obtained and
`this was combined with the first crop. The material was dried in vacuo at 50 °C overnight to afford
`65 g of pure and dry (E)-4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl pivalate (55%). 1H-
`NMR (300 MHz, CDCl3): δ 0.91 (t, J = 7.2 Hz, 3H), 1.37 (s, 9H), 2.46 (q, J = 7.2 Hz, 2H), 4,50 (s, 1H),
`6.47 (d, J = 8.4 Hz, 2H), 6.72 (d, J = 8,7 Hz, 2H), 7.03-7.25 (m, 9H).
`
`Figure S9. 1D 1H NMR spectrum of pure 3 (CDCl3, 300 MHz).
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`(E)-4-(2-phenyl-1-(4-(2-(2,2,2-trifluoro-N-methylacetamido)ethoxy)phenyl)but-1-en-1-yl)phenyl
`pivalate 6
`(E)-4-(1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl)phenyl pivalate (49.5 g, 123.8 mmol) was
`dissolved in 2 L of THF and PPh3 (66.8 g, 255 mmol) was added. The mixture was cooled to 10°C
`and a mixture of DIAD (50.5 g, 250 mmol) and 2,2,2-trifluoro-N-(2-hydroxyethyl)-N-methylacetamide
`(42.8 g, 250 mmol) in 500 mL of THF was added drop wise over 5 hours. The mixture was stirred for an
`additional 18 hours. Since the reaction was not complete more PPh3 (39.3 g, 150 mmol) was added to the
`mixture. Also a mixture of DIAD (30.3 g, 150 mmol) and 2,2,2-trifluoro-N-(2-hydroxyethyl)-N-
`methylacetamide (25.7 g, 150 mmol) in 300 mL of THF was added drop wise over 5 hours. The mixture
`was stirred overnight and subsequently concentrated in vacuo. Note that some stereorandomization of 6 is
`detected on aqueous workup, but not if the crude is purified by neutral alumina column chromatography
`(heptanes/EtOAc 10:1) immediately after evaporation of the reaction solvents under reduced pressure
`affording
`(E)-4-(2-phenyl-1-(4-(2-(2,2,2-trifluoro-N-methylacetamido)ethoxy)phenyl)but-1-en-1-
`yl)phenyl pivalate as a white solid (56.8 g, 83%, 2.6:1 mixture of rotamers).1H-NMR (300 MHz, CDCl3):
`δ 0.91 (t, J = 6 Hz, 3H), 1.36 (s, 9H), 2.47 (q, J = 6 Hz, 2H), 3.11 (s, 3H – minor rotamer), 3.23 (s, 3H –
`major rotamer), 3.72-3.77 (m, 2H – mixture of rotamers), 3.99-4.07 (m, 2H – mixture of rotamers), 6.52
`(d, J = 9 Hz, 2H), 6.77 (d, J = 9 Hz, 2H), 7.03-7.24 (m, 9H); 13C-NMR (75 MHz, CDCl3): δ 13.53, 27.14,
`29.05, 36.05, 37.07, 39.10, 48.67, 49.50, 65.43, 65.98, 113.22, 116.35 (q, J = 286 Hz), 116.45 (q, J = 286
`Hz), 121.12, 126.14, 127.91, 129.62, 130.38, 132.06, 135.93, 136.17, 137.16, 140.99, 141.98, 142.20,
`149.72, 155.88, 156.13, 157,12 (q, J = 35.3 Hz), 157,14 (q, J = 36.0 Hz); 19F-NMR (282 MHz) -69.9
`(major rotamer), -68.3 (minor rotamer).
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`Figure S10. 1D 1H NMR spectrum of pure 6 (CDCl3, 300 MHz).
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`Figure S11. 1D 13C NMR spectrum of pure 6 (CDCl3, 75 MHz).
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`Figure S12. 1D 19F NMR spectrum of pure 6 (CDCl3, 282 MHz).
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`Z-endoxifen
`(E)-4-(2-phenyl-1-(4-(2-(2,2,2-trifluoro-N-methylacetamido)ethoxy)phenyl)but-1-en-1-yl)phenyl pivalate
`(56.8 g, 103 mmol) was dissolved in 1 L of THF and 150 mL of methanol. The mixture was cooled to 0
`°C and LiOH.H2O (21.6 g, 0.51 mol) was added portion wise. The mixture was stirred for 3 hours at rt.
`NH4Cl-sat (500 mL) was added and the mixture was extracted with EtOAc (2x). The combined organic
`layers were washed with NaHCO3-sat and brine, dried over Na2SO4 and concentrated affording a crude
`material (42 g). From a previous batch, 6.8 g of crude Z-endoxifen was added and the combined solids
`were triturated twice with 200 mL Et2O affording pure Z-endoxifen as a white solid (37 g, 83% -
`recalculated from total amount of starting materials). 1H-NMR (300 MHz, CDCl3): δ 0.91 (t, J = 7.2 Hz,
`3H), 2.48 (q, J = 7.5 Hz, 2H), 2.50 (s, 3H), 2.93 (t, J = 5.1 Hz, 2H), 3.96 (t, J = 5.4 Hz, 2H), 6.49 (d, J =
`8.7 Hz, 2H), 6.71-6.75 (m, 4H), 7.02 (d, J = 8.7 Hz, 2H), 7.08-7.18 (m, 5H).
`
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`Figure S13. 1D 1H NMR spectrum of pure Z-endoxifen (CDCl3, 300 MHz).
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`qNMR-experiments
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`Figure S14. qNMR spectrum of maleic acid (11.31 mg) and Z-endoxifen (13.66 mg) in 2 mL of DMSO-
`d6 (300 MHz) – purity 98.3%.
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`Figure S15. qNMR spectrum of 3,5-dinitrobenzoic acid (20.49 mg) and Z-endoxifen (14.35 mg) in 2 mL
`of DMSO-d6 (300 MHz) – purity 97.8%.
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`Figure S16. LC-MS-UV analysis of Z-endoxifen.
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`3.0
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`7
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`10
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`Prikersty Pos Segeacall Event |
`Pobrity Meg Seren] -Event?
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`Accord Exhibit 1025
`Page 20 of 21
`PGR2023-00043
`
`Accord Exhibit 1025
`Page 20 of 21
`PGR2023-00043
`
`

`

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`References
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`1. Teunissen, S. F.; Jager, N. G. L.; Rosing, H.; Schinkel, A. H.; Schellens, J. H. M.; Beijnen, J. H. J.
`Chromatogr. B 2011, 879, 1677–1685.
`2. Gauthier, S.; Mailhot, J.; Labrie, F. J. Org. Chem. 1996, 61, 3890–3893.
`3. Fauq, A. H.; Maharvi, G. M.; Sinha, D. Bioorg. Med. Chem. Lett. 2010, 20, 3036–3038.
`4. Ali, S. M.; Ahmad, A.; Shahabuddin, S.; Ahmad, M. U.; Sheikh, S.; Ahmad, I. Bioorg. Med. Chem. Lett.
`2010, 20, 2665–2667.
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`Accord Exhibit 1025
`Page 21 of 21
`PGR2023-00043
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