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C.B. Albinana et al. / European Journal of Medicinal Chemistry 121 (2016) 100e109
106
12.0 Hz, 1H), 3.26e3.08 (m, 1H), 2.76 (dd, J ¼ 17.7, 5.1 Hz, 1H),
2.26e2.08 (m, 1H), 2.04 (s, 3H), 1.58 (s, 2H), 1.55e1.44 (m, 4H), 1.29
(t, J ¼ 7.1 Hz, 3H), 0.90 (td, J ¼ 7.4, 3.8 Hz, 6H). 13C NMR (75 MHz,
7.4 Hz, 6H). 13C NMR (151 MHz, D2O, t-BuOH as reference)
d 175.9,
134.2, 133.5 (d, J ¼ 165.8 Hz), 85.0, 76.8 (d, J ¼ 18.9 Hz), 53.7, 50.6 (d,
J ¼ 13.7 Hz), 30.0 (d, J ¼ 11.2 Hz), 26.2, 25.9, 23.0, 9.3, 9.1. All spectral
properties matched literature values [28]. HR-ESI-MS calculated for
CDCl3)
d 171.1, 166.4, 137.8, 129.5, 81.7, 77.6, 77.2, 76.7, 75.0, 60.8,
58.9, 49.3, 33.7, 26.2, 25.7, 23.6, 14.2, 9.6, 9.3. HR-ESI-MS calculated
C
13H25O5N2NaP (M þ Na)þ 343.1393, found 343.1395.
for C16H29O4N2 (M þ H)þ 313.2122, found 313.2122.
4.1.7. Ethyl (3R,4R,5S)-4-acetamido-5-[(tert-butoxycarbonyl)-
amino]-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylate (7)
4.1.4. Ethyl (3R,4R,5S)-4-acetamido-5-[N2,N3-bis(tert-
butoxycarbonyl)guanidino]-3-(1-ethylpropoxy)-1-cyclohexene-1-
carboxylate (4)
To a solution of oseltamivir free base 3 (0.38 g, 1.21 mmol) in
DCM (3 mL) was added triethylamine (0.61 g, 6.08 mmol) followed
by addition of di-tert-butyl dicarbonate (0.53 g, 2.42 mmol) and
then the reaction mixture was stirred for 4 h at room temperature.
The mixture was diluted with water (10 mL) and then extracted
with DCM (3 ꢂ 10 mL). The combined organic phase was washed
with brine, dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by flash column chromatog-
raphy (eluent DCM to DCM/MeOH, 20:1) to afford the protected
amine (0.49 g, 98% yield) as a white solid. 1H NMR (300 MHz, CDCl3)
Mercury(II) chloride (0.11 g, 0.406 mmol) was added portion-
wise to a solution of oseltamivir base 3 (0.10 g, 0.320 mmol),
N,N0ꢀdi-(tert-butoxycarbonyl)thiourea (0.11 g, 0.400 mmol) and
triethylamine (0.11 mL, 0.800 mmol) in DMF (15 mL) at 0 ꢁC. The
reaction mixture was stirred for 24 h at room temperature. The
mixture was diluted with EtOAc (10 mL), filtered through a pad of
Celite and concentrated under reduced pressure. The residue was
diluted with water (30 mL) and extracted with EtOAc (3 ꢂ 10 mL).
The combined organic phase was washed with water, brine, dried
over Na2SO4, filtered and evaporated. The residue was purified by
flash column chromatography (eluent Hexanes/EtOAc, 2:1) to
afford the Boc-protected guanidine derivative 4 (0.16 g, 89%). TLC
d
6.78 (s, 1H), 5.80 (d, J ¼ 8.2 Hz, 1H), 5.11 (d, J ¼ 9.0 Hz, 1H), 4.20 (q,
J ¼ 7.1 Hz, 2H), 4.13e4.00 (m, 1H), 3.97 (s, 1H), 3.79 (dd, J ¼ 9.7,
5.1 Hz, 1H), 3.52e3.28 (m, 1H), 2.74 (dd, J ¼ 18.1, 4.9 Hz, 1H), 2.29
(dd, J ¼ 17.7, 9.6 Hz, 1H), 1.98 (s, 3H), 1.51 (dd, J ¼ 5.5, 4.1 Hz, 4H),
(Hexanes/EtOAc 1:1) Rf ¼ 0.4. 1H NMR (300 MHz, CDCl3)
d
11.33 (s,
1.42 (s, 9H), 1.28 (t, J ¼ 7.1 Hz, 3H), 0.88 (dd, J ¼ 13.6, 7.3 Hz, 6H). 13
C
1H), 8.57 (d, J ¼ 8.1 Hz, 1H), 6.76 (s, 1H), 6.16 (d, J ¼ 8.9 Hz, 1H),
4.43e4.23 (m, 1H), 4.20e4.01 (m, 3H), 3.96 (d, J ¼ 7.7 Hz, 1H),
3.36e3.21 (m, 1H), 2.72 (dd, J ¼ 17.6, 5.3 Hz, 1H), 2.42e2.23 (m, 1H),
1.85 (s, 3H), 1.55e1.49 (m, 22H), 1.22 (dd, J ¼ 12.6, 5.5 Hz, 3H), 0.82
NMR (75 MHz, CDCl3) d 170.9, 166.1, 156.4, 137.7, 129.5, 82.3, 79.8,
76.0, 61.1, 54.5, 49.2, 31.1, 28.5, 26.3, 25.8, 23.5, 14.3, 9.6, 9.4. HR-ESI-
MS calculated for C21H37O6N2 (M þ H)þ 413.2646, found 413.2648.
(dt, J ¼ 10.3, 7.4 Hz, 6H). 13C NMR (101 MHz, CDCl3)
d
170.3, 166.0,
4.1.8. (3R,4R,5S)-4-Acetamido-5-[(tert-butoxycarbonyl)-amino]-3-
(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (8)
163.3, 157.0, 152.7, 138.1, 128.7, 83.6, 82.8, 79.6, 76.3, 61.1, 54.5, 48.2,
30.6, 28.41, 28.2, 26.2, 25.9, 23.4,14.3, 9.7, 9.4. HR-ESI-MS calculated
for C27H47O8N4 (M þ H)þ 555.3388, found 555.3389.
An aqueous solution of NaOH (0.5 M; 4.35 mmol; 8.7 mL) was
added dropwise to a stirred solution of the ester 7 (0.9 g,
2.18 mmol) in 1,4-dioxane (8.7 mL). The reaction mixture was
stirred for 24 h at room temperature. The pH was adjusted to
neutral by addition of Amberlite IR 120 hydrogen form. Amberlite
was removed and the filtrate was concentrated under reduced
pressure. The residue was purified by flash column chromatog-
raphy (eluent DCM/MeOH gradient 10:1 to 10:3) to furnish the free
acid 8 (1.28 g, 95% yield) as a white solid. 1H NMR (300 MHz, CDCl3)
4.1.5. (3R,4R,5S)-4-Acetamido-5-[N2,N3-bis(tert-butoxycarbonyl)
guanidino]-3-(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid (5)
An aqueous solution of NaOH (0.5 M; 0.55 mL) was added to a
solution of ester 4 (0.15 g, 0.27 mmol) in 1,4-dioxane (0.55 mL). The
reaction mixture was stirred overnight at room temperature. The
pH was adjusted to neutral by addition of Amberlite IR 120
hydrogen form. Amberlite was removed and the liquid phase was
concentrated under reduced pressure. The residue was purified by
flash column chromatography (Toluene/EtOAc/AcOH 1:1:0.5%) to
afford the free acid 5 (0.07 g, 48%). TLC (Toluene/EtOAc/AcOH
d
6.81 (s, 1H), 6.69 (s, 1H), 5.72 (d, J ¼ 9.3 Hz, 1H), 4.02 (d, J ¼ 6.8 Hz,
2H), 3.75 (dd, J ¼ 13.1, 8.4 Hz, 1H), 3.39e3.26 (m, 1H), 2.70 (dd,
J ¼ 17.5, 5.0 Hz, 1H), 2.25 (dd, J ¼ 17.7, 11.1 Hz, 1H), 1.99 (s, 3H), 1.49
(dd, J ¼ 14.0, 6.7 Hz, 4H), 1.41 (s, 9H), 0.86 (dd, J ¼ 16.5, 7.4 Hz, 6H).
1:1:0.5%) Rf ¼ 0.2. 1H NMR (300 MHz, CDCl3)
d
8.64 (d, J ¼ 8.1 Hz,
13C NMR (75 MHz, CDCl3)
d 171.5, 169.1, 156.9, 139.2, 129.2, 82.3,
1H), 6.82 (s,1H), 6.23 (d, J ¼ 8.9 Hz,1H), 4.39 (dd, J ¼ 8.3, 6.0 Hz,1H),
4.26e4.16 (m, 2H), 4.16e4.08 (m, 1H), 4.06e3.98 (m, 1H), 3.41e3.29
(m, 1H), 2.78 (dd, J ¼ 17.6, 5.3 Hz, 1H), 2.47e2.32 (m, 1H), 1.91 (s,
3H), 1.61e1.41 (m, 22H), 1.27 (dt, J ¼ 10.9, 7.1 Hz, 4H), 0.89 (dt,
79.8, 76.2, 55.2, 49.6, 31.0, 28.5, 26.3, 25.7, 23.4, 9.8, 9.2. HR-ESI-MS
calculated for C19H33O6N2 (M þ H)þ 385.2333, found 385.2335.
4.1.9. (3R,4R,5S)-4-Acetamido-5-[(tert-butoxycarbonyl)-amino]-3-
(1-ethylpropoxy)-1-Bromocyclohexene (9)
J ¼ 10.3, 7.4 Hz, 6H). 13C NMR (75 MHz, CD3OD)
d 173.6, 169.3, 164.4,
157.7, 153.7, 138.7, 130.4, 84.6, 84.0, 80.5, 76.6, 54.6, 50.0, 31.4, 28.5,
28.2, 27.3, 26.9, 22.7, 9.9, 9.7. HR-ESI-MS calculated for C25H43O8N4
(M þ H)þ 527.3075, found 527.3076.
S-(1-Oxido-2-pyridyl)-N,N,N0N0-tetramethylthiuronium hexa-
fluorophosphate (0.22 g, 0.592 mmol) was added to a solution of
the acid 8 (0.21 g, 0.538 mmol), triethylamine (0.22 mL, 1.61 mmol)
and 4-(dimethylamino)pyridine (0.007 g, 0.053 mmol) in dry THF
(4 mL). The reaction was stirred in the dark for 40 min at room
temperature. The solvent was removed by evaporation under
reduced pressure. The remaining green oil was dissolved in DCM
(2 mL) and bromotrichloromethane (2 mL). The formed solution
was irradiated (refluxed) with a flood lamp for 90 min. The mixture
was concentrated and purified by flash column chromatography
(eluent Toluene/EtOAc gradient 2:1 to 1:1) to afford the vinyl bro-
mide (0.15 g, 66% yield). TLC (Toluene/EtOAc 1:1) Rf ¼ 0.45. 1H NMR
4.1.6. (3R,4R,5S)-4-Acetamido-5-amino-3-(1-ethylpropoxy)-1-
cyclohexene-1-phosphonate (6)
Trimethylsilyl bromide (0.41 mL, 3.18 mmol) and 2,6-lutidine
(0.44 mL, 3.81 mmol) were added to a solution of the Boc-
protected phosphonic acid 13 (0.14 g, 0.318 mmol) in DCM
(10 mL) and the reaction was allowed to stir for 9 h at room tem-
perature. The solvent was evaporated and TFA (50% in water, 10 mL)
was added. Then the reaction was allowed to stir for 1 h and the
solvent was evaporated under reduced pressure. The residue was
(300 MHz, CDCl3)
d
6.07 (s, 1H), 5.52 (d, J ¼ 9.4 Hz, 1H), 5.35 (d,
purified by preparative HPLC. 1H NMR (400 MHz, D2O)
d
6.31 (t,
J ¼ 9.0 Hz, 1H), 4.09 (dd, J ¼ 9.1, 6.8 Hz, 1H), 3.88 (dd, J ¼ 7.9, 5.5 Hz,
1H), 3.83 (s, 1H), 3.39e3.25 (m, 1H), 2.68 (m, J ¼ 26.0, 18.0, 6.8 Hz,
2H), 1.99 (s, 3H), 1.59 (s, 1H), 1.49 (dd, J ¼ 7.3, 6.0 Hz, 3H), 1.42 (s,
J ¼ 17.6 Hz, 1H), 4.25 (s, 1H), 4.07 (t, J ¼ 10.2 Hz, 1H), 3.62e3.47 (m,
2H), 2.95e2.74 (m, 1H), 2.52 (d, J ¼ 12.1 Hz, 1H), 2.09 (s, 3H),
1.67e1.52 (m, 3H), 1.48 (dd, J ¼ 14.1, 7.0 Hz, 1H), 0.88 (dt, J ¼ 17.1,
9H), 0.88 (t,
J
¼
7.4 Hz, 6H). HR-ESI-MS calculated for