Z. Cheng, et al.
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
4.9. Synthesis of 2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]phenazin-1-
yl 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)
butanoate, (compound 7)
NMR (100 MHz, CDCl3, 303 K) δ: 190.9, 187.6, 172.7, 161.3, 158.0,
156.4, 152.3, 143.4, 143.1, 142.6, 139.0, 138.7, 133.3, 131.0, 129.5,
127.3, 126.9, 126.2, 123.7, 122.9, 120.0, 119.3, 117.6, 111.0, 101.1,
92.0, 68.6, 57.5, 47.5, 38.3, 28.8, 27.5, 21.5, 14.4, 12.7, 12.3. EI/HRMS
(m/z) [M + H]+: 661.2646. Cald. for [C38H36N4O4]+: 661.2657.
N,N′-Dicyclohexylcarbodiimide (31 mg, 0.15 mmol) and trimethyl
lock quinone (70 mg, 0.2 mmol) were dissolved in 3 mL of CH2Cl2 at
0 °C under stirring until precipitation (20 min). Separately, 4-(di-
methylamino)pyridine (10 mg, 0.1 mmol) and 2,2-dimethyl-1,2-dihy-
drobenzo[a]furo[2,3-c]phenazin-1-ol (4), (63 mg, 0.2 mmol) were dis-
solved in 5 mL of CH2Cl2 under stirring for 20 min, then added dropwise
for 10 min to DCC solution at 0 °C. The mixture was heated to 25 °C and
keep under stirring for 24 h. The mixture was extracted with CH2Cl2
and water and dried over Na2SO4. Solvent was evaporated under re-
duced pressure and the crude was purified under sílica gel 60 and n-
4.12. Synthesis of (1-(2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]
phenazin-1-yl)-1H-1,2,3-triazol-4-yl)methanol
Compound 10 was prepared following procedure described above.12
Compound 10 was obtained as a yellow solid (122 mg, 0.36 mmol, 70%
yield); m.p. 291–292 °C. 1H NMR (400 MHz, DMSO-d6, 303 K) δ: 9.34
(d, J = 7.5 Hz, 1H), 8.30 (dd, J = 3.4 and 6.5 Hz, 1H), 8.16 (d,
J = 6.5 Hz, 1H), 8.03–7.95 (m, 3H), 7.89 (s, 1H), 7.86 (dd, J = 3.4 and
6.5 Hz, 2H), 6.63 (s, 1H), 5.72 (s, 1H), 4.41 (d, J = 5.6 Hz, 2H), 1.73 (s,
3H), 1.14 (s, 3H). 13C NMR (100 MHz, DMSO-d6, 303 K) δ: 160.4,
148.5, 142.6, 142.4, 141.2, 140.2, 133.0, 131.5, 131.3, 130.7, 130.2,
129.8, 128.9, 126.4, 124.6, 123.6, 123.6, 110.2, 93.5, 68.1, 55.6, 27.6,
21.6. HRMS (ES+) calculated for C23H19N5O2 [M+H]+: 398.1613;
found: 398.1612.
hexane/EtOAc 25:1 to provide compound (7) as
a yellow solid
(66.8 mg, 0.12 mmol, 60% yield); m.p. 205–207 °C. 1H NMR (400 MHz,
CDCl3, 303 K) δ: 9.37 (d, J = 8.0 Hz, 1H), 8.30–8.28 (m, 1H), 8.08 (d,
J = 8.0 Hz, 1H), 8.03–8.01 (m, 1H), 7.86–7.74 (m, 4H), 6.65 (s, 1H),
3.64 (d, J = 16.0 Hz, 1H), 2.47 (d, J = 16.0 Hz, 1H), 1.58 (s, 3H),
1.54–1.52 (m, 9H), 1.39 (s, 3H), 1.28 (s, 3H), 0.97 (s, 3H). 13C NMR
(100 MHz, CDCl3, 303 K) δ: 190.6, 185.7, 172.0, 160.1, 151.6, 142.4,
142.1, 142.0, 140.6, 140.3, 138.3, 137.3, 133.1, 130.1, 130.0, 129.8,
129.5, 128.4, 128.3, 126.3, 124.2, 122.7, 110.4, 78.3, 48.3, 38.5, 28.4,
28.3, 25.9, 21.0, 13.7, 12.0, 11.0. EI/HRMS (m/z) [M+H]+: 549.2383.
Cald. for [C34H33N2O5]+: 549.2384.
4.13. Synthesis of 2-(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)-5,5-
dimethyl-4,5-dihydrofuro[3′,2′:3,4]naphtho[1,2-d]oxazol-2-yl)phenol
Compound 11 was prepared following procedure described above.12
Compound (11) was obtained as a white solid (69 mg, 0.18 mmol, 40%
yield); m.p. 297–298 °C. 1H NMR (400 MHz, DMSO-d6, 303 K) δ: 10.99
(s, 1H), 8.55 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.3 Hz, 1H), 7.92 (s, 1H),
7.86–7.80 (m, 2H), 7.69 (t, J = 7.3 Hz, 1H), 7.45 (t, J = 7.3 Hz, 1H),
7.11 (d, J = 8.3 Hz, 1H), 7.02 (t, J = 7.3 Hz, 1H), 6.67 (s, 1H), 5.11 (t,
4.10. Synthesis of (1-(2,2-dimethyl-1,2-dihydrobenzo[a]furo[2,3-c]
phenazin-1-yl)-1H-1,2,3-triazol-4-yl)methyl 3-methyl-3-(2,4,5-trimethyl-
3,6-dioxocyclohexa-1,4-dien-1-yl)butanoate
Compound 8 was prepared according to the classical methodology
described by Sharpless, Fokin and co-workers with minor modifica-
tion.12 Alkyne (253 mg, 0.87 mmol), CuSO4·5H2O (12 mg, 0.05 mmol)
and sodium ascorbate (25 mg, 0.13 mmol) were added to a solution of
azido-phenazine (5) (200 mg, 0.58 mmol) in 10 mL of CH2Cl2:H2O
(1:1). The mixture was stirred at room temperature for 24 h and was
monitored by TLC. The organic phase was extracted with CH2Cl2, dried
over sodium sulfate and concentrated under reduced pressure. The
obtained residue was purified by column chromatography on silica gel,
using a gradient mixture of hexane/ethyl acetate with increasing po-
larity as an eluent. Compound (8) was obtained as a yellow solid
(284 mg, 0.45 mmol, 77% yield); m.p. 286–287 °C. 1H NMR (400 MHz,
CDCl3, 303 K) δ: 9.45 (d, J = 8.0 Hz, 1H), 8.30–8.28 (m, 1H), 8.20 (d,
J = 8.0 Hz, 1H), 8.03–8.00 (m, 1H), 7.92 (t, J = 7.0 Hz, 1H), 7.77–7.75
(m, 2H), 7.12 (s, 1H), 6.66 (s, 1H), 5.00 (d, J = 13.0 Hz, 1H), 4.96 (d,
J = 13.0 Hz, 2H), 2.90 (d, J = 16.3 Hz, 1H), 2.76 (d, J = 16.3 Hz, 1H),
1.97 (s, 3H), 1.86 (s, 3H), 1.80 (s, 3H), 1.77 (s, 3H), 1.26 (s, 3H), 1.23
(s, 3H), 1.14 (s, 3H). 13C NMR (100 MHz, CDCl3, 303 K) δ: 190.9, 187.5,
172.6, 160.5, 152.4, 143.1, 142.9, 142.6, 141.7, 141.1, 140.7, 138.9,
138.5, 133.5, 130.4, 130.2, 130.2, 129.9, 129.1, 129.0, 126.4, 124.2,
123.4, 123.1, 108.7, 92.9, 69.0, 57.6, 47.4, 38.2, 28.7, 27.8, 21.7, 14.3,
J = 5.7 Hz, 1H), 4.48 (d, J = 5,7 Hz, 2H), 1.72 (s, 3H), 1.13 (s, 3H). 13
C
NMR (100 MHz, DMSO-d6, 303 K) δ: 160.5, 157.4, 156.1, 148.9, 143.7,
133.8, 130.5, 129.7, 127.6, 126.8, 126.6, 123.7, 123.6, 123.1, 119.2,
120.7, 117.8, 111.5, 103.1, 92.5, 67.5, 55.6, 27.5, 21.6. EI/HRMS (m/z)
[M+H]+: 429.1563. Cald. for [C24H20N4O4]+: 429.1557.
5. Procedures, additional spectroscopy and biological assays. In
vitro spectral characterization
Spectroscopic data were recorded on
a Synergy HT spectro-
photometer (Biotek). Compounds were dissolved at the indicated con-
centrations and spectra were recorded at r.t. Spectra are represented as
means from at least two independent experiments with n = 3.
6. Enzyme activation assay
hNQO1 was purchased from Sigma-Aldrich. Solutions of 50 μM of
compounds 7, 8 and 9 were prepared in a buffer solution (pH 7.4)
containing 25 mM Tris, BSA, Tween-20, 5 μM FAD and 0.1 mM NADH.
1 μM of hNQO1 was prepared using the same buffer solution and was
pre-incubated at 37 °C. 10 μL of each compound was added to 10 μL of
hNQO1 solution and the experiments were performed in a 384-well
plate at 37 °C. The samples were excited at 405 nm and the fluorescence
was collected at 490 nm. Fluorescence measurements were collected
every 30 s for 30 min.
12.7, 12.2. HRMS (ES+) calculated for
630.2710; found: 630.2711.
C
37H35N5O5 [M+H]+
:
4.11. Synthesis of (1-(2-(2-hydroxyphenyl)-5,5-dimethyl-4,5-dihydrofuro
[3′,2′:3,4]naphtho[1,2-d]oxazol-4-yl)-1H-1,2,3-triazol-4-yl)methyl-3-
methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butanoate
7. Enzyme kinetics
Compound 9 was prepared following procedure described above.12
Compound (9) was obtained as a yellow oil (184 mg, 0.5 mmol, 80%
yield). 1H NMR (400 MHz, CDCl3, 303 K) δ: 11.27 (s, 1H), 8.50 (d,
J = 8.3 Hz, 1H), 8.18 (d, J = 8.3 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.80
(t, J = 7.2 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H),
7.07 (s, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.48 (s, 1H), 5.02 (s, 2H), 2.93 (d,
J = 16.3 Hz, 1H), 2.82 (d, J = 16.3 Hz, 1H), 2.01 (s, 3H), 1.87 (s, 3H),
1.79 (s, 3H), 1.76 (s, 3H), 1.27 (s, 3H), 1.23 (s, 3H), 1.21 (s, 3H). 13C
Solutions of different concentrations of compound 7 (5 μM, 7.5 μM,
10 μM, 20 μM, 30 μM and 40 μM) were prepared in a buffer solution (pH
7.4) containing 25 mM Tris, BSA, Tween-20, 5 μM FAD and 0.1 mM
NADH. 1 μM of hNQO1 was prepared using the same buffer solution
and pre-incubated at 37 °C. Each assay was performed by adding 10 μL
of compound 7 to 10 μL of hNQO1 solution in a 384-well plate at 37 °C.
The assay was excited at 405 nm and the fluorescence was collected at
490 nm. Fluorescence measurements were collected every 30 s for the
7