A.M. Piloto et al. / Tetrahedron 70 (2014) 650e657
655
that selective cleavage of the photolabile groups was possible by
choosing the most appropriate wavelength, being for the tested
heterocyclic chromophores 350 and 419 nm, and for the o-nitro-
benzyl group 254 nm. Overall, the results showed that the combi-
nations of photolabile protecting groups used in conjugates 6e9 are
wavelength-selective; however, a specific irradiation sequence is
required, meaning that the heterocyclic chromophore should al-
ways be cleaved before the o-nitrobenzyl group.
The possibility of using other classes of photolabile protecting
group, together with other photolysis conditions, and even the use
of two-photon excitation are possibilities under study in our re-
search group.
1344, 1302, 1250, 1216, 1177, 1151, 1117, 1086, 1071, 980, 859, 816,
791, 733, 703, 672. HRMS (EI): calcd for
282.08522; found: 282.08543.
C
12H14N2O6 [Mþ]:
4.2.2. N-(2-Nitrobenzyloxycarbonyl)- -alanine, 2. To a suspension
L
of compound 1 (0.403 g, 1.44ꢁ10ꢀ3 mol) in 1,4-dioxane (4.0 mL) at
0 ꢂC, 1 M aqueous sodium hydroxide (1.44 mL, 1.44ꢁ10ꢀ3 mol) was
added. The solution was stirred at low temperature for 18 h and
acidified to pH 2e3 with 1 M aqueous potassium hydrogensulfate.
The reaction mixture was reduced to half the initial volume in
a
rotary evaporator and extracted with dichloromethane
(3ꢁ10 mL). The organic extracts were combined, dried with anhy-
drous magnesium sulfate and after solvent evaporation, compound
2 was obtained as a colourless oil (0.355 g, 92%). Rf¼0.39 (ethyl
4. Experimental section
4.1. General
acetate). 1H NMR (DMSO-d6, 400 MHz):
d
¼1.17 (d, J 6.8 Hz, 3H,
b-
CH3), 3.50e3.59 (m,1H,
-NH), 7.58 (dt, J 7.6 and 1.2 Hz, 1H, H-4), 7.68 (dd, 1H, J 7.6 and
0.8 Hz, H-6), 7.77 (dt, J 7.2 and 0.8 Hz, 1H, H-5), 8.08 (dd, J 8.4 and
1.2 Hz, 1H, H-3). 13C NMR (DMSO-d6, 100.6 MHz):
¼19.41 ( -CH3),
51.40 ( -CH), 61.76 (CH2), 124.73 (C-3), 128.74 (C-6), 128.86 (C-4),
133.22 (C-1), 134.20 (C-5), 147.12 (C-2), 154.46 (C]O carbamate),
174.33 (C]O acid). IR (film, cmꢀ1):
a-CH), 5.32 (s, 2H, CH2), 6.64 (d, J 6.0 Hz,1H,
a
All melting points were measured on a Stuart SMP3 melting
point apparatus. TLC analyses were carried out on 0.25 mm thick
precoated silica plates (Merck Fertigplatten Kieselgel 60F254) and
spots were visualised under UV light. Chromatography on silica gel
was carried out on Merck Kieselgel (230e240 mesh). IR spectra
were determined on a BOMEM MB 104 spectrophotometer using
KBr discs. UV/vis absorption spectra (200e700 nm) were obtained
using a Shimadzu UV/2501PC spectrophotometer. NMR spectra
were obtained on a Varian Unity Plus Spectrometer at an operating
frequency of 300 MHz for 1H or a Bruker Avance III 400 at an op-
erating frequency of 400 MHz for 1H and 100.6 MHz for 13C using
the solvent peak as internal reference at 25 ꢂC. All chemical shifts
are given in parts per million using dH Me4Si¼0 ppm as reference
and J values are given in hertz. Assignments were made by com-
parison of chemical shifts, peak multiplicities and J values and were
supported by spin decoupling-double resonance and bidimensional
heteronuclear correlation techniques. Mass spectrometry analyses
were performed at the ‘C.A.C.T.I.dUnidad de Espectrometria de
Masas’, at University of Vigo, Spain. Fluorescence spectra were
collected using a FluoroMax-4 spectrofluorometer. Photolysis was
carried out using a Rayonet RPR-100 chamber reactor equipped
with 10 lamps of 254, 300, 350 and 419ꢃ10 nm. HPLC analyses were
d
b
a
n
¼3369, 2987, 1692, 1614, 1576,
1533, 1451, 1343, 1295, 1264, 1215, 1120, 1067, 1020, 982, 936, 896,
860, 802, 738, 704. HRMS (EI): calcd for C11H12N2O6 [Mþ]:
268.06956; found: 268.06986.
4.2.3. N-(2-Nitrobenzyloxycarbonyl)-L-alanine (acridin-9-yl)methyl
ester, 6. To a solution of compound 2 (0.073 g, 2.73ꢁ10ꢀ4 mol) in
dry DMF (3 mL), potassium fluoride (0.047 g, 8.16ꢁ10ꢀ4 mol) and 9-
(bromomethyl)acridine 3 (0.074 g, 2.72ꢁ10ꢀ4 mol) were added. The
reaction mixture was stirred at room temperature for 30 h. Potas-
sium fluoride was removed by filtration, the solvent was removed
by rotary evaporation under reduced pressure and the crude resi-
due was purified by column chromatography using mixtures of
ethyl acetate and light petroleum of increasing polarity as eluent.
Compound 6 was obtained as light brown oil (0.040 g, 32%). Rf¼0.51
(ethyl acetate/light petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
d
¼1.33 (d, J 7.2 Hz, 3H,
b-CH3), 4.30e4.40 (m, 1H, a-CH), 5.45 (s, 2H,
CH2 NB), 5.58 (d, J 7.6 Hz, 1H, NH), 6.08 (d, J 6.8 Hz, 1H, CH2 OAcm),
6.20 (d, J 6.8 Hz, 1H, CH2 OAcm), 7.35e7.45 (m, 1H, H-40), 7.46e7.55
(m, 2H, H-50and H-60), 7.56e7.62 (m, 2H, H-2 and H-7), 7.73e7.79
(m, 2H, H-3 and H-6), 8.043 (dd, J 6.4 and 1.6 Hz, 1H, H-30),
8.20e8.30 (m, 4H, H-1, H-8, H-4 and H-5). 13C NMR (CDCl3,
performed using a Licrospher 100 RP18 (5 mm) column in a JASCO
HPLC system composed by a PU-2080 pump and a UV-2070 de-
tector with ChromNav software. All reagents were used as received.
4.2. Synthesis of compounds 1, 2 and 6e9
100.6 MHz):
d
¼18.14 (
b-CH3), 49.77 (a-CH), 58.57 (CH2 OAcm),
63.37 (CH2 NB),123.74 (C-1 and C-8),124.83 (C-30), 125.16 (C-8a and
C-9a), 126.82 (C-2 and C-7), 128.34 (C-40), 128.41 (C-50), 129.85 (C-4
and C-5), 130.11 (C-3 and C-6), 132.78 (C-10), 133.65 (C-60), 136.49
(C-9), 147.06 (C-20), 148.32 (C-4a and C-10a), 155.04 (C]O carba-
4.2.1. N-(2-Nitrobenzyloxycarbonyl)-
-alanine methyl ester, 1. N,N0-
L
Carbonyldiimidazole (1.078 g, 6.65ꢁ10ꢀ3 mol) was stirred with (2-
nitrophenyl)methanol (1.018 g, 6.65ꢁ10ꢀ3 mol) in dry DMF (4 mL)
at 0 ꢂC for 30 min.
L-Alanine methyl ester hydrochloride (0.928 g,
mate), 172.69 (C]O ester). IR (film, cmꢀ1):
n
¼3420, 3331, 3056,
6.65ꢁ10ꢀ3 mol), previously treated with triethylamine (1.01 mL,
7.32ꢁ10ꢀ3 mol) in dry DMF (2 mL) was added to the reaction
mixture and the mixture was kept stirring for 18 h. The precipitate
was filtered, the solvent was removed by rotary evaporation under
reduced pressure and the crude residue was purified by column
chromatography using ethyl acetate and light petroleum, with
mixtures of increasing polarity as eluent. Compound 1 was ob-
tained as a pink oily solid (0.430 g, 23%). Rf¼0.48 (ethyl acetate/light
2985, 2941, 2878, 1727, 1630, 1611, 1579, 1526, 1452, 1421, 1344,
1305, 1266, 1209, 1170, 1115, 1068, 966, 910, 897, 859, 819, 790, 738,
704, 674, 642. HRMS (EI): calcd for C25H21N3O6 [Mþ]: 459.14313;
found: 459.14292.
4.2.4. N-(2-Nitrobenzyloxycarbonyl)-
3H-naphtho[2,1-b]pyran-1-yl)]methyl ester, 7. To
L
-alanine [(9-methoxy-3-oxo-
solution of
a
compound 2 (0.134 g, 4.99ꢁ10ꢀ4 mol) in dry DMF (3 mL), potassium
fluoride (0.087 g, 1.49ꢁ10ꢀ3 mol) and 1-chloromethyl-9-methoxy-
3-oxo-3H-naphtho[2,1-b]pyran 4 (0.137 g, 4.99ꢁ10ꢀ4 mol) were
added. The reaction mixture was stirred at room temperature for
48 h. Potassium fluoride was removed by filtration, the solvent was
removed by rotary evaporation under reduced pressure and the
crude residue was purified by column chromatography using ethyl
acetate and light petroleum, with mixtures of increasing polarity as
eluent. Compound 7 was obtained as a light yellow solid (0.139 g,
55%); mp 145.0e146.6 ꢂC. Rf¼0.40 (ethyl acetate/light petroleum,
petroleum, 1:1). 1H NMR (CDCl3, 400 MHz):
b
d
¼1.44 (d, J 7.2 Hz, 3H,
-CH3), 3.76 (s, 3H, OCH3), 4.30e4.44 (m, 1H, -CH), 5.49 (broad s,
-NH), 5.53 (s, 2H, CH2), 7.47 (dt, J 7.4 and 1.6 Hz, 1H, H-4), 7.62
a
1H,
a
(dd,1H, J 7.2 and 1.2 Hz, H-6), 7.65 (dt, J 6.8 and 0.8 Hz,1H, H-5), 8.10
(dd, J 8.0 and 0.8 Hz, 1H, H-3). 13C NMR (CDCl3,100.6 MHz):
¼18.54
-CH3), 49.63 ( -CH), 52.49 (OCH3), 63.41 (CH2), 124.92 (C-3),
d
(b
a
128.49 (C-4), 128.56 (C-6), 132.98 (C-1), 133.74 (C-5), 147.22 (C-2),
155.01 (C]O carbamate), 173.32 (C]O ester). IR (KBr 1%, cmꢀ1):
n
¼3345, 3113, 3044, 2991, 2956, 2880, 1729, 1613, 1578, 1526, 1453,