4882
L. A. Ho et al. / Bioorg. Med. Chem. Lett. 26 (2016) 4879–4883
water system (Millipore, Australia). All reagents were purchased from Sigma-
Aldrich, Fluka, Merck, or Boron Molecular and used without further
purification, unless otherwise stated.
Thin layer chromatography (TLC) was performed on Merck silica gel 60 F254
pre-coated aluminium sheets. Visualisation of developed plates was achieved
(570 mg, 1.07 mmol) was dissolved in anhydrous CH2Cl2 (15 mL) and the
mixture was cooled to 0 °C in an ice bath, under an atmosphere of argon gas. To
this cooled mixture was added freshly prepared Ag2O (588 mg, 2.54 mmol), KI
(294 mg, 1.77 mmol), and TsCl (305 mg, 1.60 mmol). The resulting mixture
was left to stir at 0 °C for 30 min, then at reflux for 18 h. After this time, the
Ag2O was filtered off through a pad of celite eluted with 1:12 MeOH/CH2Cl2.
The filtrate was then concentrated under reduced pressure. The resulting crude
material was subjected to silica gel column chromatography (40:60 acetone/
CH2Cl2 ? 5:95 MeOH/CH2Cl2 ? 20:80 MeOH/CH2Cl2) to give compound 4 as a
colourless oil (551 mg, 75% yield, Rf = 0.55 in 1:10 MeOH/CH2Cl2). 1H NMR
(399.85 MHz, CD3OD): d = 7.76 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 7.40–7.29 (m,
4H, 4 Â ArH), 7.27–7.18 (m, 3H, 3 Â ArH), 7.14–7.03 (m, 5H, 5 Â ArH), 6.76 (app
d, J = 8.9 Hz, 2H, 2 Â ArH), 6.54 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 4.10–4.06 (m,
2H, OCH2), 3.93 (t, J = 5.6 Hz, 2H), 3.61–3.42 (m, 12H), 2.82 (t, J = 5.6 Hz, 2H),
2.69 (t, J = 5.6 Hz, 2H), 2.43 (q, J = 7.4 Hz, 2H, CH2CH3), 2.40 (s, 3H, CH3), 2.34 (s,
3H, NCH3), 0.89 (t, J = 7.4 Hz, 3H, CH2CH3); 13C NMR (100.54 MHz, CD3OD):
d = 158.0, 146.4, 145.0, 143.6, 142.6, 139.8, 136.9, 134.4, 132.9 (ArCH), 131.0
(ArCH), 130.8 (ArCH), 130.4 (ArCH), 129.2 (ArCH), 129.0 (ArCH), 128.9 (ArCH),
127.7 (ArCH), 127.2 (ArCH), 114.5 (ArCH), 71.5 (CH2), 71.4 (CH2), 71.3 (CH2),
70.9 (CH2), 69.6 (CH2), 69.5 (CH2), 66.3 (CH2), 57.8 (CH2), 57.2 (CH2), 43.5 (CH3),
29.9 (CH2), 21.7 (CH3), 13.9 (CH3); HRMS (ESI): calcd C40H50NO7S [M+H]+
688.3308, found 688.3320.
through the use of
a 254 nm or 365 nm UV lamp or staining with
phosphomolybdic acid stain solution. Column chromatography was
performed using silica gel 60 (0.063–0.200 nm) as supplied by Merck, unless
otherwise stated. HPLC was conducted using an Agilent 1200 with
photodiode array detector (PDA). Separation was achieved using
a
a
250 Â 10 mm i.d., 5
lm, Apollo C18 reversed phase column (Grace-Division)
with a 33 mm  7 mm guard column of the same material.
1H and 13C NMR spectra were acquired in the specified deuterated solvent
using either a Bruker AV600 (600.13 MHz for 1H and 150.9 MHz for 13C), a
Bruker AV500 (500.13 MHz for 1H and 125.8 MHz for 13C), or a Varian Gemini-
400 (399.85 MHz for 1H and 100.5 MHz for 13C) spectrometer at 25 °C.
Chemical shifts are reported in parts per million downfield from
tetramethylsilane using the residual solvent resonance as internal
standard.20 Data are reported as follows: chemical shift, multiplicity
(app = apparent, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad, sept = septet), coupling constant, integration, and assignment.
High resolution mass spectra (HRMS) were acquired on
a Waters liquid
chromatograph premier (LCT) mass spectrometer using electrospray ionisation
(ESI).
Synthesis of (E)-S-(1-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)-3-methyl-6,9,12-
trioxa-3-azatetradecan-14-yl)ethanethioate
(5):
Compound
4
(98 mg,
Synthesis of N-desmethyl tamoxifen (1): Tamoxifen (2.12 g, 5.71 mmol) was
dissolved in anhydrous CH2Cl2 (60 mL) and the mixture was cooled to 0 °C in
an ice bath, under an atmosphere of argon gas. To this cooled mixture was
added 1-chloroethyl chloroformate (0.68 mL, 0.90 g, 6.30 mmol) in one
portion. After 15 min stirring at 0 °C, the resulting mixture was heated at
reflux for 22 h. After this time, the solvent was removed under reduced
pressure to obtain a yellow oil. The crude mixture was then dissolved in
methanol, refluxed for a further 3 h, and then concentrated under reduced
pressure. The resulting crude material was subjected to silica gel flash column
chromatography (CH2Cl2 ? 10:90 MeOH/CH2Cl2) to give compound 1 as a
colourless solid (2.05 g, 91% yield, Rf = 0.46 in 1:10 MeOH/CH2Cl2). 1H NMR
(399.8 MHz, d6-DMSO): d = 7.38–7.30 (m, 2H, 2 Â ArH), 7.26–7.05 (m, 8H,
8 Â ArH), 6.76–6.69 (m, 2H, 2 Â ArH), 6.66–6.58 (m, 2H, 2 Â ArH), 4.06 (t,
J = 5.2 Hz, 2H), 3.16 (t, J = 5.2 Hz, 2H), 2.32 (q, J = 7.2 Hz, 2H, CH2CH3), 0.80 (t,
J = 7.2 Hz, 3H, CH2CH3); 13C NMR (125.77 MHz, CD3OD): d = 157.3, 144.9, 143.6,
143.0, 139.7, 137.9, 133.0 (ArCH), 130.8, 130.4 (ArCH), 129.2 (ArCH), 128.9
(ArCH), 127.7 (ArCH), 127.2 (ArCH), 114.6 (ArCH), 64.2 (CH2), 49.5 (CH2), 33.8
(CH3), 29.9 (CH2), 13.8 (CH3); HRMS (ESI): calcd Chemical Formula: C25H28NO
[M+HÀCl]+ 358.2171, found 358.2162.
0.14 mmol) was dissolved in anhydrous THF (2 mL), under an atmosphere of
argon gas. To this mixture was added KSAc (81 mg, 0.71 mmol). The resulting
mixture was heated at reflux for 16 h. After this time, the residue was diluted
with EtOAc and decoloursing charcoal was added. The decoloursing charcoal
was filtered off through a pad of celite eluted with 1:12 MeOH/CH2Cl2 and the
filtrate was then concentrated under reduced pressure. The resulting crude
material was purified via silica gel flash column chromatography (40:60
acetone/CH2Cl2 ? 5:95 MeOH/CH2Cl2) to give thioacetate 5 as an oil (38 mg,
46% yield, Rf = 0.71 in 1:12 MeOH/CH2Cl2). 1H NMR (399.85 MHz, CD3OD):
d = 7.36–7.28 (m, 2H, 2 Â ArH), 7.27–7.19 (m, 3H, 3 Â ArH), 7.19–7.04 (m, 5H,
5 Â ArH), 6.75 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 6.59 (app d, J = 8.9 Hz, 2H,
2 Â ArH), 3.93 (t, J = 5.9 Hz, 2H), 3.69–3.46 (m, 12H), 3.06 (t, J = 6.5 Hz, 2H),
2.80 (t, J = 5.9 Hz, 2H), 2.68 (t, J = 5.9 Hz, 2H), 2.44 (q, J = 7.4 Hz, 2H), 2.35 (s, 3H,
CH3), 2.30 (s, 3H, CH3), 0.91 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (100.54 MHz,
CD3OD): d = 195.4, 156.7, 143.8, 142.4, 141.3, 138.3, 135.5, 131.8 (ArCH), 129.7
(ArCH), 129.5 (ArCH), 128.1 (ArCH), 127.9 (ArCH), 126.5 (ArCH), 126.0 (ArCH),
113.4 (ArCH), 70.6 (CH2), 70.5 (CH2), 70.4 (CH2), 70.3 (CH2), 69.8 (CH2), 69.2
(CH2), 65.7 (CH2), 57.1 (CH2), 56.5 (CH2), 43.4 (CH3), 30.6 (CH3), 29.0 (CH2), 28.9
(CH2), 13.6 (CH3); HRMS (ESI): calcd C35H46NO5S [M+H]+ 592.3097, found
592.3085.
Synthesis of 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl 4-methylbenzenesul-
fonate (2): Tetraethylene glycol (2 mL, 2.25 g, 11.58 mmol) was dissolved in
anhydrous CH2Cl2 (25 mL) and the mixture was cooled to 0 °C in an ice bath,
under an atmosphere of argon gas. To this cooled mixture was added freshly
prepared Ag2O (4.03 g, 17.39 mmol), KI (0.77 g, 4.64 mmol), and TsCl (2.21 g,
11.59). The resulting mixture was left to stir at 0 °C for 30 min. After this time,
the Ag2O was filtered off through a pad of celite eluted with 1:12 MeOH/
CH2Cl2. The filtrate was then concentrated under reduced pressure. The
resulting crude material was subjected to silica gel column chromatography
(CH2Cl2 ? 1:4 acetone/CH2Cl2) to give compound 2 as a colourless oil (1.78 g,
44% yield, Rf = 0.58 in 1:8 acetone/CH2Cl2). 1H NMR (399.85 MHz, CDCl3):
d = 7.78 (app d, J = 8.4 Hz, 2H, 2 Â ArH), 7.34 (app d, J = 8.4 Hz, 2H, 2 Â ArH),
4.16–4.13 (m, 2H, CH2OTs), 3.70–3.56 (m, 14H, 7 Â OCH2), 2.93 (s, 1H, OH),
2.43 (s, 3H, CH3); 13C NMR (100.54 MHz, CDCl3): d = 144.8 (ArC), 132.8 (ArC),
129.8 (ArCH), 127.8 (ArCH), 72.4 (OCH2), 70.6 (OCH2), 70.5 (OCH2), 70.3 (OCH2),
70.2 (OCH2), 69.2 (OCH2), 68.5(OCH2), 61.5 (OCH2), 21.5 (CH3); HRMS (ESI):
calcd C15H25O7S [M+H]+ 349.1321, found 349.1323.
Synthesis of (E)-1-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)-3-methyl-6,9,12-tri-
oxa-3-azatetradecan-14-ol (3): N-Desmethyl tamoxifen (2.05 g, 5.20 mmol)
and glycol 2 (2.72 g, 7.80 mmol) were dissolved in anhydrous DMF (60 mL),
under an atmosphere of argon gas. To this stirring mixture was added K2CO3
(7.19 g, 5.21 mmol) in one portion. The resulting mixture was left to stir at
85 °C for 24 h. After this time, DMF was evaporated under reduced pressure
and the crude mixture was diluted by the addition of ethyl acetate. The excess
K2CO3 was filtered off through a pad of celite eluted with 1:12 MeOH/CH2Cl2
and the filtrate was then concentrated under reduced pressure. The resulting
crude material was purified via silica gel flash column chromatography (30:70
acetone/CH2Cl2 ? 5:95 MeOH/CH2Cl2 ? 20:80 MeOH/CH2Cl2) to give com-
pound 3 as an oil (1.54 mg, 55% yield, Rf = 0.25 in 1:12 MeOH/CH2Cl2). 1H NMR
(500.13 MHz, CD3OD): d = 7.35–7.31 (m, 2H, 2 Â ArH), 7.27–7.21 (m, 3H,
3 Â ArH), 7.18–7.09 (m, 5H, 5 Â ArH), 6.75 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 6.53
(app d, J = 8.9 Hz, 2H, 2 Â ArH), 3.95 (t, J = 5.9 Hz, 2H, OCH2), 3.70–3.55 (m,
14H), 3.40 (s, 1H, OH), 2.79 (t, J = 5.9 Hz, 2H), 2.67 (t, J = 5.9 Hz, 2H), 2.44 (q,
J = 7.4 Hz, 2H, CH2CH3), 2.34 (s, 3H, NCH3), 0.93 (t, J = 7.4 Hz, 3H, CH2CH3); 13C
NMR (125.77 MHz, CD3OD): d = 156.8, 143.9, 142.5, 141.4, 138.3, 131.9 (ArCH),
129.8 (ArCH), 129.5 (ArCH), 128.2 (ArCH), 127.9 (ArCH), 126.6 (ArCH), 126.1
(ArCH), 113.5 (ArCH), 73.0, 72.7 (CH2), 70.7 (CH2), 70.6 (CH2), 70.4, 69.3 (CH2),
65.7 (CH2), 61.8 (CH2), 61.7 (CH2), 57.1 (CH2), 56.6 (CH2), 43.4 (CH3), 29.1 (CH2),
13.7 (CH3); HRMS (ESI): calcd C33H44NO5 [M+H]+ 534.3219, found 534.3233. IR
Synthesis of (E)-1-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)-3-methyl-6,9,12-tri-
oxa-3-azatetradecane-14-thiol (6): Thioacetate
dissolved in degassed EtOH (400 L), under an atmosphere of argon gas. To this
mixture was added a solution of NaOH in degassed H2O (200 L, 7 M). The
5 (50 mg, 0.084 mmol) was
l
l
resulting mixture was heated at reflux for 2 h. After this time, the reaction
mixture was diluted with dichloromethane (4 mL). The layers were separated
and the organic layer was washed with brine (1 mL). The organic layer was
then filtered through a small plug of silica eluted with 5:95 MeOH/CH2Cl2 and
concentrated to give thiol 6 as an oil (33 mg, 71% yield, 0.72 in 1:12 MeOH/
CH2Cl2). 1H NMR (600.13 MHz, CDCl3): d = 7.36–7.32 (m, 2H, 2 Â ArH), 7.28–
7.22 (m, 3H, 3 Â ArH), 7.19–7.15 (m, 2H, 2 Â ArH), 7.13–7.10 (m, 3H, 3 Â ArH),
6.76 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 6.53 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 3.97–
3.93 (m, 2H, OCH2), 3.72–3.56 (m, 12H), 2.86 (t, J = 6.72 Hz, 2H), 2.83–2.78 (m,
2H), 2.72–2.66 (m, 2H), 2.45 (q, J = 7.42 Hz, 2H, CH2CH3), 2.36 (s, 3H, NCH3),
0.92 (T, J = 7.42 Hz, 3H, CH2CH3); 13C NMR (100.54 MHz, CDCl3): d = 156.8,
143.9, 142.5, 141.4, 138.3, 135.6, 131.9 (ArCH), 129.8 (ArCH), 129.5 (ArCH),
128.1 (ArCH), 127.9 (ArCH), 126.6 (ArCH), 126.0 (ArCH), 113.4 (ArCH), 77.4,
70.7 (CH2), 70.6 (CH2), 70.5 (CH2), 70.4 (CH2), 69.5 (CH2), 65.9 (CH2), 57.2 (CH2),
56.7 (CH2), 43.5 (CH3), 30.1 (CH2), 29.1 (CH2), 13.7 (CH3); HRMS (ESI): calcd
C
33H44NO4S [M+H]+ 550.2991, found 550.3018.
Conjugation of (E)-1-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)-3-methyl-6,9,12-
trioxa-3-azatetradecane-14-thiol with BODIPY fluorophore (7): Commercially
available BODIPYÒFL N-(2-aminoethyl)maleimide (Life Technology, 5 mg) was
dissolved in dry DMSO (2 Â 0.25 mL) and transferred to a flask containing thiol
6 (10 mg). The resulting mixture was left to stir at room temperature under an
atmosphere of argon gas, overnight, in the dark. After this time, the reaction
mixture was diluted with methanol, separated into two batches and then
freeze-dried using a lyophilizer to give a crude orange solid. Each batch of dry
lyophilised sample was then dissolved in methanol (1 mL) and separated by
HPLC. The column was eluted at 4 mL/min with an isocratic mobile phase
consisting of 65% acetonitrile: 35% of 0.1% TFA/water over 40 min. UV
absorbance was measured at wavelengths of 280, 488, and 508 nm. Fractions
were collected every minute for 40 min. A sample of 7 eluted between 15 and
20 min. The fractions containing the appropriate product were then concen-
trated under reduced pressure to remove acetonitrile and then freeze-dried
using a lyophilizer to give 7 as an orange solid (3 mg). 1H NMR (600.13 MHz,
CD3OD): d = 7.41 (s, 1H, C@CH), 7.37–7.33 (m, 2H, 2 Â ArH), 7.29–7.25 (m, 1H,
1 Â ArH), 7.22–7.20 (m, 2H, 2 Â ArH), 7.17–7.07 (m, 5H, 5 Â ArH), 6.99 (app d,
J = 4.14 Hz, 1H, C@CH), 6.83 (app d, J = 8.9 Hz, 2H, 2 Â ArH), 6.65 (app d,
J = 8.9 Hz, 2H, 2 Â ArH), 6.30 (app d, J = 4.14 Hz, 1H, C@CH), 6.20 (s, 1H, C@CH),
4.24–4.22 (m, 2H), 3.84–3.80 (m, 2H), 3.70–3.55 (m, 12H), 3.05–2.99 (m, 2H),
2.97 (s, 3H, CH3), 2.86–2.80 (m, 2H), 2.55–2.52 (m, 2H), 2.49 (s, 3H, CH3), 2.44
(neat):
1443, 1349 cmÀ1
Synthesis of (E)-1-(4-(1,2-diphenylbut-1-en-1-yl)phenoxy)-3-methyl-6,9,12-tri-
oxa-3-azatetradecan-14-yl 4-methylbenzenesulfonate (4): Compound
m = 3415 (OH), 2870 (AOACH2), 1605 (AC@CA), 1573, 1507, 1461,
.
3