Z. Wang et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
(100 MHz, DMSO‑d6, ppm) δ: 166.32, 157.58, 156.75, 148.74, 143.21,
140.62, 140.23, 139.19, 136.48, 135.35, 130.54, 126.96, 122.19 (q,
J = 275.93 Hz), 120.82, 118.80, 117.40, 114.10, 99.99, 53.21. ESI-MS:
m/z 450.3 (M + 1).C19H11ClF3N5O3 (449.05).
(monitored by TLC). The solvent was removed under reduced pressure
and the residue was taken up in 40 mL of water. The mixture was ex-
tracted with ethyl acetate (3 × 40 mL). Then the combined organic
layers were washed with 1 N hydrochloric acid solution (1 × 30 mL)
and saturated sodium chloride solution (2 × 30 mL). The organic phase
was dried over anhydrous MgSO4, filtered, and purified by flash column
chromatography. The product was recrystallized from ethyl acetate/
hexane to afford the target compound 9. White solid, yield: 11.0%, mp:
147–149 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ: 7.93 (d, J = 7.2 Hz,
1H), 7.79–7.72 (m, 1H), 7.60 (dd, J = 2.3, 1.2 Hz, 1H), 7.56–7.51 (m,
1H), 6.70 (d, J = 7.4 Hz, 1H), 5.66 (s, 2H), 5.24 (s, 2H), 4.77 (t,
J = 6.2 Hz, 1H), 3.19 (s, 3H), 1.21 (d, J = 6.2 Hz, 6H). 13C NMR
(100 MHz, DMSO‑d6, ppm) δ: 157.61, 156.50, 153.60, 153.05, 144.83,
140.57, 137.99, 135.39, 130.70 (q, J = 32.12 Hz), 127.01, 122.11 (q,
J = 278.15 Hz), 121.61, 118.83, 117.40, 114.11, 100.81, 72.65, 70.14,
43.89, 28.01, 21.81. ESI-MS: m/z 559.4 (M + 18), 564.4 (M + 23).
4.1.7.7. 2-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)
pyridin-1(2H)-yl)-N-(pyridin-3-yl)acetamide (8g). White solid, yield:
43.6%, mp: 132–134 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ: 10.34
(s, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.72–7.64 (m, 1H), 7.58–7.38 (m, 4H),
7.09 (t, J = 8.9 Hz, 2H), 6.62 (d, J = 7.2 Hz, 1H), 4.79 (s, 2H). 13C NMR
(100 MHz, DMSO‑d6, ppm) δ: 166.33, 157.58, 156.75, 148.74, 143.21,
140.62, 139.19, 136.47, 135.35, 130.70 (q, J = 31.92 Hz), 126.96,
124.9, 122.19 (q, J = 275.83 Hz), 121.61, 118.80, 117.40, 114.10,
99.99, 53.20. ESI-MS: m/z 449.4 (M + 1). C20H12ClF3N4O3 (448.06).
4.1.7.8. 2-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)
pyridin-1(2H)-yl)-N-(pyridin-4-yl)acetamide (8h). White solid, yield:
62.1%, mp: 157–159 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ: 10.49
(s, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.82–7.68 (m, 1H), 7.62–7.40 (m, 6H),
6.69 (d, J = 7.2 Hz, 1H), 4.87 (s, 2H). 13C NMR (100 MHz, DMSO‑d6,
ppm) δ: 165.27, 157.59, 156.76, 140.2, 139.27, 138.32, 135.37,
132.15, 130.64 (q, J = 31.82 Hz), 126.96, 122.2 (q, J = 275.83 Hz),
121.48, 118.77, 117.42, 115.68, 114.08, 99.91, 53.45. ESI-MS: m/z
449.4 (M + 1). C20H12ClF3N4O3 (448.06).
C22H19ClF3N5O6 (541.1).
4.1.9. Synthesis
of
(3-((3-(3-chloro-5-cyanophenoxy)-2-oxo-4-
(trifluoromethyl)pyridin-1(2H)-yl)methyl)-4-methyl-5-oxo-4,5-dihydro-
1H-1,2,4-triazol-1-yl)methyl dihydrogen phosphate (10)
Doravirine (0.5 g, 1.17 mmol) and KI (0.39 g, 2.35 mmol) were
dissolved in redistilled DMF (20 mL), followed by addition of NaH
(0.061 g, 1.53 mmol) in an ice bath. The reaction mixture was stirred at
room temperature for 30 min under a nitrogen atmosphere. Then, di-
tert-butyl(chloromethyl)phosphate (1.518 g, 5.87 mmol) was added and
subsequently the temperature was increased to 60 °C for 16 h (mon-
itored by TLC). The solvent was removed under reduced pressure and
the residue was taken up in 40 mL of water. The mixture was extracted
with ethyl acetate (3 × 40 mL). Then the combined organic layers were
washed with 1 N hydrochloric acid solution (1 × 30 mL) and saturated
sodium chloride solution (2 × 30 mL). The organic phase was dried
over anhydrous MgSO4, filtered, and concentrated to give the crude
product di-tert-butyl ((3-((3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(tri-
fluoromethyl)pyridin-1(2H)-yl)methyl)-4-methyl-5-oxo-4,5-dihydro-
1H-1,2,4-triazol-1-yl)methyl) phosphate (17), which was used directly
in the next step without further purification. A solution of 17 in acetone
(5 mL) and water (5 mL) was stirred at room temperature for 8 h
(monitored by TLC). The solvent was removed under reduced pressure
and subsequently recrystallized with DMF/water to afford target com-
pound 10. White solid, yield: 4.0%, mp: 172–174 °C. 1H NMR
(400 MHz, DMSO‑d6, ppm) δ: 7.94 (d, J = 7.3 Hz, 1H), 7.42 (d,
J = 1.6 Hz, 1H), 7.33–7.17 (m, 1H), 7.13 (t, J = 2.0 Hz, 1H), 6.68 (d,
J = 7.3 Hz, 1H), 5.65 (s, 2H), 5.26 (s, 2H), 3.20 (s, 3H). 13C NMR
(100 MHz, DMSO‑d6, ppm) δ: 157.67, 156.52, 155.50, 143.74, 140.57,
137.80, 135.39, 130.57 (q, J = 32.02), 126.99, 122.13 (q, J = 276.03),
121.63, 118.90, 117.38, 114.12 , 100.70 , 74.50 (d, J = 7.27 Hz),
43.83, 27.19. ESI-MS: m/z 553.5 (M + 18), 558.4 (M + 23).
4.1.7.9. 2-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)
pyridin-1(2H)-yl)-N-(1H-pyrazol-4-yl)acetamide (8i). White solid, yield:
64.8%, mp: 224–226 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ: 12.38
(s, 1H), 10.85 (s, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.78–7.73 (m, 1H), 7.63
(d, J = 10.9 Hz, 1H), 7.53 (dd, J = 2.3, 1.3 Hz, 1H), 7.50 (d,
J = 4.2 Hz, 1H), 6.66 (d, J = 7.2 Hz, 1H), 6.45 (s, 1H), 4.86 (s, 2H).
13C NMR (100 MHz, DMSO‑d6, ppm) δ: 164.19, 157.64, 156.73, 147.17,
140.16, 139.34, 135.36, 130.71 (q, J = 31.66 Hz), 129.23, 126.92,
122.21 (q, J = 276.03 Hz), 121.61, 118.75, 117.42, 114.09, 99.75,
96.30, 52.76. ESI-MS: m/z 438.4 (M + 1) C18H11ClF3N5O3 (437.05).
4.1.7.10. 2-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)
pyridin-1(2H)-yl)-N-(1H-1,2,4-triazol-5-yl)acetamide (8j). White solid,
yield: 77.4%, mp: 209–211 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ:
12.38 (s, 1H), 10.85 (s, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.78–7.73 (m,
1H), 7.63 (d, J = 10.9 Hz, 1H), 7.53 (dd, J = 2.3, 1.3 Hz, 1H), 7.50 (d,
J = 4.2 Hz, 1H), 6.66 (d, J = 7.2 Hz, 1H), 6.45 (s, 1H), 4.86 (s, 2H). 13
C
NMR (100 MHz, DMSO‑d6, ppm) δ: 164.19, 157.64, 156.73, 147.17,
140.16, 139.34, 135.36, 130.71 (q, J = 31.66 Hz), 129.23, 126.92,
122.21 (q, J = 276.03 Hz), 121.61, 118.75, 117.42, 114.09, 99.75,
96.30, 52.76. ESI-MS: m/z 438.4 (M + 1). C18H11ClF3N5O3 (437.05).
4.1.7.11. 2-(3-(3-chloro-5-cyanophenoxy)-2-oxo-4-(trifluoromethyl)
pyridin-1(2H)-yl)-N-(1H-pyrazol-5-yl)acetamide (8k). White solid, yield:
52.3%, mp: 295–296 °C. 1H NMR (400 MHz, DMSO‑d6, ppm) δ: 13.38
(s, 1H), 11.93 (s, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.81–7.54 (m, 2H), 7.47
(d, J = 17.6 Hz, 2H), 6.62 (d, J = 7.1 Hz, 1H), 4.85 (s, 2H). 13C NMR
(100 MHz, DMSO‑d6, ppm) δ: 164.19, 157.65, 156.74, 147.16, 140.17,
139.33, 135.36, 130.55 (q, J = 31.92 Hz), 129.23, 126.91, 122.22 (q,
J = 275.93 Hz), 121.60, 118.75, 117.41, 114.10, 99.74, 52.76. ESI-MS:
m/z 439.4 (M + 1). C17H10ClF3N6O3 (438.05).
C18H14ClF3N5O7P (535.03).
4.2. In vitro assay of anti-HIV activities
Antiviral activity and cytotoxicity of the synthesized compounds
were evaluated against HIV-1 wt strain (IIIB), HIV-1 double mutant
strain (RES056) and HIV-2 strain (ROD) in MT-4 cell cultures utilizing
the
bromide
(MTT) method as described previously25,26. Stock solutions (10 × final
concentration) of test compounds were added in 25 μL volumes to two
series of triplicate wells in order to allow simultaneous evaluation of the
effects on mock- and HIV-infected cells. Using a Biomek 3000 robot
(Beckman Instruments, Fullerton, CA) to dilute the test compounds to 5-
fold serially (final 200 μL volume per well) in flat-bottomed 96-well
microtiter trays, including untreated control HIV-1 and mock-infected
cell samples for each sample. HIV-1 wt strain (IIIB), HIV-1 double
mutant strain (RES056) or HIV-2 strain (ROD) stock (50 μL at 100–300
CCID50) (50% cell culture infectious dose) or culture medium was
4.1.8. Synthesis
of
(3-((3-(3-chloro-5-cyanophenoxy)-2-oxo-4-
(trifluoromethyl)pyridin-1(2H)-yl)methyl)-4-methyl-5-oxo-4,5-dihydro-
1H-1,2,4-triazol-1-yl)methyl isopropyl carbonate (9)
Doravirine (0.5 g, 1.17 mmol) and KI (0.39 g, 2.35 mmol) were
dissolved in redistilled DMF (20 mL), followed by addition of NaH
(0.061 g, 1.53 mmol) in an ice bath. The reaction mixture was stirred at
room temperature for 30 min under a nitrogen atmosphere. Then
chloromethyl isopropyl carbonate (0.896 g, 5.87 mmol) was added, and
subsequently the temperature was increased to 60 °C for 16 h
8