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744.25 gmolꢀ1): C 58.10, H 5.48, N 15.06, found: C 58.38, H 5.43, N
14.70.
H37), 5.23 (s, 2H, H30), 6.43 (s, 1H, H6), 6.44 (d, J=8 Hz, 2H, H33,
3
4
H35), 7.03 (d, J=8 Hz, 2H, H32, H36), 7.13 (ddd, J=8 Hz, J=2 Hz,
4
4
4J=1 Hz, 1H, H17), 7.20 (dd, J=2 Hz, J=1 Hz, 1H, H13), 7.22 (s,
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-N-(4-nitrobenzyl)-
quinazolin-4-amine (2a): Erlotinib (200 mg, 0.508 mmol) was
added to an ice-cold solution of tBuOK (63 mg, 1.1 equiv) in dry
DMF (10 mL) and stirred for 10 min at 08C. Then, 4-nitrobenzyl bro-
mide (143 mg, 1.3 equiv) was added and the reaction mixture was
allowed to slowly warm to room temperature and stirred over-
night. After 18 h, the solvent was evaporated, the residue was di-
luted with H2O (10 mL) and extracted with EtOAc (2ꢄ15 mL). The
combined organic phases were dried over MgSO4, filtered and con-
centrated. Purification of the crude product was performed by
flash chromatography on silica gel, eluting with EtOAc/MeOH 20:1,
to give 2a as beige crystals (62 mg, 23%): 1H NMR (500.32 MHz,
[D6]DMSO): d=3.25 (s, 3H, H29), 3.32 (s, 3H, H25), 3.39–3.41 (m,
2H, H27), 3.55–3.57 (m, 2H, H26), 3.70–3.72 (m, 2H, H23), 4.25 (s,
1H, H19), 4.25–4.27 (m, 2H, H22), 5.55 (s, 2H, H30), 6.46 (s, 1H,
H6), 7.26 (s, 1H, H3), 7.28 (ddd, 3J=8 Hz, 4J=2 Hz, 4J=1 Hz, 1H,
H17), 7.31 (ddd, 3J=8 Hz, 4J=1 Hz, 4J=1 Hz, 1H, H15), 7.38 (dd,
J=8 Hz, J=8 Hz, 1H, H16), 7.40 (dd, J=2 Hz, J=1 Hz, 1H, H13),
7.69 (d, J=9 Hz, 2H, H32, H36), 8.15 (d, J=9 Hz, 2H, H33, H35),
8.66 ppm (s, 1H, H8); 13C NMR (125.81 MHz, [D6]DMSO): d=55.4
(C30), 58.7 (C29), 58.8 (C25), 67.7 (C26), 68.5 (C22), 70.0 (C27), 70.4
(C23), 82.3 (C19), 83.1 (C18), 106.1 (C6), 108.6 (C3), 110.6 (C5), 123.7
(C14), 123.9 (C33, C35), 126.5 (C17), 128.4 (C13), 129.3 (C32, C36),
129.3 (C15), 130.7 (C16), 146.8 (C12), 146.9 (C31), 147.0 (C34), 147.1
(C1), 149.5 (C4), 153.2 (C8), 153.9 (C2), 159.7 ppm (C10); MS (ESI)
m/z: 529.40 [M+H]+, 551.35 [M+Na]+; Anal. calcd for
C29H28N4O6·0.1H2O (Mr =530.36 gmolꢀ1): C 65.67, H 5.36, N 10.56,
found: C 65.42, H 5.20, N 10.42.
3
4
4
1H, H3), 7.27 (ddd, J=8 Hz, J=1 Hz, J=1 Hz, 1H, H15), 7.34 (dd,
J=8 Hz, J=8 Hz, 1H, H16), 8.68 ppm (s, 1H, H8); 13C NMR
(125.81 MHz, [D6]DMSO): d=55.4 (C30), 58.7 (C29), 58.7 (C25), 67.7
(C26), 68.5 (C22), 70.0 (C27), 70.4 (C23), 82.0 (C19), 83.2 (C18), 106.4
(C6), 108.5 (C3), 110.8 (C5), 114.2 (C33, C35), 123.4 (C14), 125.1
(C31), 126.7 (C17), 128.6 (C13), 128.9 (C15), 129.4 (C32, C36), 130.5
(C16), 146.9 (C1), 147.2 (C2), 148.1 (C34), 149.4 (C4), 153.3 (C8),
153.7 (C2), 160.1 ppm (C10); MS (ESI) m/z: 499.36 [M+H]+, 521.40
[M+Na]+, 537.38 [M+K]+; Anal. calcd for C29H30N4O4·0.25H2O
(Mr =503.08 gmolꢀ1): C 69.22, H 6.11, N 11.14, found: C 68.95, H
5.93, N 11.05.
(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl (4-(((6,7-bis(2-methox-
yethoxy)quinazolin-4-yl) (3-ethynylphenyl)amino)methyl)phenyl)
carbamate (2c): Compound 2b (80 mg, 0.16 mmol, 1.1 equiv) was
dissolved in dry DMF (6.5 mL) and treated with
9 (47 mg,
0.15 mmol, 1 equiv) and HOBt·H2O (22 mg, 1.1 equiv). The resulting
reaction mixture was stirred overnight at room temperature. After
18 h, it was diluted with brine (30 mL) and extracted with EtOAc
(3ꢄ25 mL). The combined organic phases were dried over MgSO4,
filtered, and concentrated. The crude product was purified by flash
chromatography on silica gel, eluting with hexane/THF 1:2, to give
2c as a pale-yellow powder (83 mg, 77%): 1H NMR (500.32 MHz,
[D6]DMSO): d=3.24 (s, 3H, H29), 3.31 (s, 3H, H25), 3.38–3.40 (m,
2H, H27), 3.53–3.55 (m, 2H, H26), 3.69–3.71 (m, 2H, H23), 3.95 (s,
3H, H48), 4.23 (s, 1H, H19), 4.24–4.26 (m, 2H, H22), 5.26 (s, 2H,
4
4
4
H41), 5.35 (s, 2H, H30), 6.44 (s, 1H, H6), 7.18 (ddd, 3J=8 Hz, J=
4
2 Hz, J=1 Hz, 1H, H17), 7.23 (s, 1H, H3), 7.26–7.36 (m, 8H, H13,
H15, H16, H32, H33, H35, H36, H42), 8.66 (s, 1H, H8), 9.80 ppm (s,
1H, H37); 13C NMR (125.81 MHz, [D6]DMSO): d=34.7 (C48), 55.2
(C30), 55.6 (C41), 58.7 (C29), 58.7 (C25), 67.6 (C26), 68.5 (C22), 70.0
(C27), 70.4 (C23), 82.1 (C19), 83.2 (C18), 106.2 (C6), 108.5 (C3), 110.7
(C5), 118.6 (C33, C35), 123.5 (C14), 126.6 (C17), 128.4 (C13), 128.9
(C32, C36), 129.0 (C15), 129.3 (C42), 130.6 (C16), 132.7 (C31), 133.8
(C46), 138.0 (C34), 146.5 (C44), 147.0 (C1), 147.0 (C12), 149.4 (C4),
153.1 (C38), 153.3 (C8), 153.7 (C2), 160.0 ppm (C10); MS (ESI) m/z:
682.26 [M+H]+; Anal. calcd for C35H35N7O8·0.5 EtOAc·0.5H2O (Mr =
734.76 gmolꢀ1): C 60.48, H 5.49, N 13.34, found: C 60.69, H 5.48, N
13.42.
tert-butyl (4-(((6,7-bis(2-methoxyethoxy)quinazolin-4-yl) (3-ethy-
nylphenyl)amino)methyl)phenyl) carbamate (12): Erlotinib
(500 mg, 1.27 mmol) was added to an ice-cold solution of tBuOK
(185 mg, 1.3 equiv) in dry DMF (25 mL) and stirred for 10 min at
08C. Then, boc-4-aminobenzyl bromide (473 mg, 1.3 equiv) was
added and the reaction mixture was allowed to slowly warm to
ambient temperature and stirred overnight. After 20 h the solvent
was evaporated, the residue was dissolved in EtOAc (50 mL) and
washed with H2O and brine (25 mL each). The organic phase was
dried over Na2SO4, filtered, and concentrated. Purification of the
crude product was performed by flash chromatography on silica
gel, eluting with EtOAc/MeOH 20:1, to give 12 as pale-yellow
powder (263 mg, 35%): 1H NMR (500.32 MHz, [D6]DMSO): d=1.45
(s, 9H), 3.25 (s, 3H), 3.32 (s, 3H), 3.39–3.41 (m, 2H), 3.54–3.56 (m,
2H), 3.70–3.72 (m, 2H), 4.21 (s, 1H), 4.25–4.27 (m, 2H), 5.35 (s, 2H),
6.46 (s, 1H), 7.17 (ddd, 3J=8 Hz, 4J=2 Hz, 4J=1 Hz, 1H), 7.24 (s,
1H), 7.26–7.36 (m, 7H), 8.68 (s, 1H), 9.26 ppm (s, 1H); MS (ESI) m/z:
599.47 [M+H]+.
Stability measurements in aqueous buffer
Compounds 1a, 1b and 2a–c were dissolved in sodium phosphate
buffer (10 mm, pH 7.4, 378C) containing 1% DMSO at a concentra-
tion of 2.5 mm and incubated at 378C. Aliquots were withdrawn at
different time points (0–24 h, every 30 min) and subjected to RP-
HPLC analysis. The HPLC chromatogram peak areas at 420 nm (for
1a, 1b) and 340 nm (for 2a–c) were used to calculate the concen-
tration of the remaining prodrug.
N-(4-aminobenzyl)-N-(3-ethynylphenyl)-6,7-bis(2-methoxyethox-
y)quinazolin-4-amine (2b): A stirred solution of 12 (263 mg,
0.439 mmol) in MeOH (20 mL) was treated with HCl conc. (183 mL,
2.2 mmol). After 20 h, the solvent was evaporated to yield the
product as a pale-yellow foam (240 mg, 96%). To remove the HCl
salt, portions of the product were diluted with NaHCO3 (0.25m)
and extracted with EtOAc. The combined organic phases were
dried over Na2SO4, filtered, and concentrated. Because some erloti-
nib was found in the product, it was purified twice by flash chro-
matography on silica gel (hexane/THF 1:2, followed by CH2Cl2/
MeOH 20:1 + Et3N (0.1%)) to give pure 2b as a beige solid:
1H NMR (500.32 MHz, [D6]DMSO): d=3.24 (s, 3H, H29), 3.32 (s, 3H,
H25), 3.39–3.41 (m, 2H, H27), 3.52–3.54 (m, 2H, H26), 3.70–3.72 (m,
2H, H23), 4.19 (s, 1H, H19), 4.24–4.26 (m, 2H, H22), 4.94 (s, 2H,
E. coli nitroreductase assay
Recombinant E. coli nitroreductase and b-nicotinamide adenine di-
nucleotide (b-NADH, reduced form, dipotassium salt) were pur-
chased from Sigma–Aldrich. The following protocol was applied to
the test compounds: compound stock solution (15 mL, 250 mm) in
DMSO was added to sodium phosphate buffer (410 mL, 10 mm,
pH 7, preheated to 378C) and NADH (75 mL of 1 mm) in sodium
phosphate buffer. The reaction was initiated by addition of E. coli
nitroreductase (1 mL, 2 mgmLꢀ1 in sodium phosphate buffer; final
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ChemMedChem 2016, 11, 1 – 13
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