Molecular Pharmaceutics
ARTICLE
Germany) with suitable visualization. Purifications by flash chroma-
tography were performed using Combiflash Companion with
Boc-4-amino-phenylalanine (11). Compound 11 was
synthesized from Boc-4-nitro-L-phenylalanine (9) (Aldrich, St.
Louis, MO, USA) (1.15 g, 3.71 mmol) using the same method as
for 10 to give a pink solid (1 g, 96%). 1H NMR (CDCl3): δ 1.41
(s, 9H), 3.00 (d, 2H), 4.44 (s, 1H), 5.13 (s, 1H), 6.21 (s, 2H),
6.62 (d, 2H), 6.96 (d, 2H).
1
RediSep Columns (Teledyne ISCO, Lincoln, USA). H and 13C
nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Avance 500 spectrometer (Bruker Biospin, Fallanden,
Switzerland) operating at (500.13 and 125.75) MHz, respectively,
using tetramethylsilane (TMS) or deuterated solvent as an internal
standard. Furthermore, the products were characterized by mass
spectrometry with a Finnigan LCQ quadrupole ion trap mass
spectrometer (Finnigan MAT, San Jose, CA) equipped with an
electrospray ionization source, and the purity was determined by
elemental analysis (C, H, N) with a ThermoQuest CE Instruments
EA 1110-CHNS-O elemental analyzer (CE Instruments, Milan,
Italy).
Valproic Acid Chloride (5). 2-Propylpentanoic acid (Acros
Organics, Waltham, MA, USA) (1 g, 6.93 mmol) was dissolved in
DCM (30 mL), and the mixture was cooled in an ice-water bath.
Thionyl chloride (Merck, Darmstadt, Germany) (0.554 mL, 7.63
mmol) was then added slowly. The mixture was refluxed until
chloride formation was complete (4 h). Solvents were evapo-
rated using a rotary evaporator and vacuum pump, and the crude
product was used as such without further purification.
m-Valpromidoyl-Boc-phenylalanine (12). The cooled mix-
ture of 10 (0.9 g, 3.21 mmol) in 1 M NaOH (5 mL) and DCM
(20 mL) was stirred vigorously, and 5 in DCM (0.87 g, 5.35
mmol) was added dropwise to the solution. The reaction was
stirred overnight in room temperature. The aqueous layer was
separated and extracted with ethyl acetate (3 ꢁ 5 mL). The
combined organic layers were washed first with a saturated
aqueous NaHCO3 and then with H2O. The separated organic
phase was dried over Na2SO4 and evaporated to dryness under
reduced pressure. Purified with flash chromatography (MeOH/
DCM = 1:99 to 1:1) to give an off-white solid of 0.79 g (61%) of
1
the title compound. H NMR (CDCl3): δ 0.88 (t, 6H), 1.32
(m, 4H), 1.36 (s, 9H), 1.65 (m, 2H), 1.63 (m, 2H), 2.59 (m, 1H),
3.14 (d, 2H), 4.16 (t, 1H), 7.00 (d, 1H), 7.03 (s, 1H), 7.18
(d, 1H), 7.37 (t, 1H).
p-Valpromidoyl-Boc-phenylalanine (13). Compound was
synthesized starting from 11 (1 g, 3.57 mmol) and 5 (0.78 g, 4.82
mmol) with the similar method as 12, yielding an off-white solid
of 0.79 g (55%). Flash chromatography (MeOH/DCM = 3:97 to
m-Valproylphenylalanine (1). DL-m-Tyrosine (6) (Fluka, St.
Louis, MO, USA) (1 g, 5.52 mmol) was dissolved in trifluor-
oacetic acid (TFA) (30 mL) in an ice-water bath, and the mixture
was stirred for 0.5 h. 5 (1.08 g, 6.62 mmol) was added slowly to
the mixture. The reaction was brought to room temperature and
stirred overnight. Solvents were evaporated using a rotary
evaporator and vacuum pump. The crude product was purified
with a column chromatography on silica gel (MeOH/DCM =
1:9) to give product as a TFA salt. The crude product was
dissolved in acetonitrile (ACN; 20 mL), and dry HCl gas was
introduced to the solution for 0.5 h. Excess HCl was flushed with
N2, and ACN was evaporated with a rotary evaporator and
1
1:1). H NMR (CDCl3): δ 0.89 (t, 6H), 1.35 (m, 4H), 1.40
(s, 9H), 1.46; 1.68 (m, 4H), 2.27 (m, 1H), 3.12 (d, 2H), 4.59
(d, 1H), 5.02 (d, 1H) 7.08 (d, 2H), 7.53 (d, 2H), 7.70 (s, 1H).
m-Valpromidoylphenylalanine (3). Compound 12 was dis-
solved in ACN (30 mL), and HCl gas was introduced to the
mixture for 0.5 h. The excess of the HCl was flushed with N2, and
the ACN was evaporated with rotary evaporator and vacuum
pump to give the crude product. The product was recrystallized
from water/ethanol (pH = 1ꢀ4) to give a white solid of 0.55 g
(83%). 1H NMR (DMSO): δ 0.86 (t, 6H), 1.24 (m, 4H), 1.30
(m, 4H), 1.53 (m, 2H), 2.43 (m, 1H), 3.04 (dd, 2H), 3.88 (t, 1H),
6.96 (d, 1H), 7.21 (t, 1H), 7.53 (d, 1H), 7.56 (s, 1H), 9.95 (s, 1H).
13C NMR (DMSO): δ 14.0, 20.2, 34.9, 36.3, 46.1, 53.9, 117.9,
120.2, 124.0, 128.7, 136.2, 139.5, 170.2, 174.3 MS: calcd. for
C17H26N2O3 306.4 [M]+, found 307.28 [M + 1]+. Anal.
(C17H26N2O3 HCl 1/2EtOH 2/5ACN) calcd (%): C, 59.06;
1
vacuum pump to give a white solid 0.32 g (30%). H NMR
(DMSO): δ 0.93 (t, 6H), 1.38 (m, 4H), 1.52 (m, 2H), 1.63 (m,
2H), 2.59 (m, 1H), 3.14 (d, 2H), 4.16 (t, 1H), 7.00 (d, 1H), 7.03
(s, 1H), 7.18 (d, 1H), 7.37 (t, 1H). 13C NMR (DMSO): δ 13.9,
22.5, 34.1, 35.3, 44.4, 53.0, 120.6, 122.6, 127.0, 129.6, 136.9,
150.4, 170.2, 174.1. MS: calcd. for C17H26NO4 307.38 [M]+,
found 308.18 [M + 1]+. Anal. (C17H25NO4 HCl 6/5H2O
3
3
3
3
3
3
ACN) calcd (%): C, 56.14; H, 7.79; N, 6.89; found: C, 55.8;
H, 7.8; N, 6.72.
H, 8.23; N, 8.79; found: C, 59.26; H, 8.59; N, 9.14.
p-Valpromidoylphenylalanine (4). Compound 4 was
synthesized from 13 with a similar method as 3 to yield a white
solid 0.48 g (81%). 1H NMR (DMSO): δ 0.86 (t, 6H), 1.23 (m,
4H), 1.30 (m, 2H), 1.52 (m, 2H), 2.43 (m. 1H), 3.07 (d, 2H),
4.08 (t, 1H), 7.17(d, 2H), 7.58 (d, 2H), 8.43 (s, 3H), 10.01
(s, 1H). 13C NMR (DMSO): 14.0, 20.2, 34.9, 36.3, 46.0, 54.0,
117.9, 120.2, 124.0, 128.7, 136.2, 139.5, 170.2, 174.3. MS: calcd.
for C17H26N2O3 306.4 [M]+, found 307.19 [M + 1]+. Anal.
(C17H26N2O3 HCl 7/10H2O 3/10ACN) calcd (%): C, 57.48;
p-Valproylphenylalanine (2). Compound 2 was synthesized
from L-tyrosine(7) (Sigma, St. Louis, MO, USA) (1 g, 5.52mmol)
and 5 (1.08 g, 6.62 mmol) with the same procedures as 1 to give a
white solid (0.43 g, 23%). 1H NMR (CDCl3): δ 0.93 (t, 6H), 1.38
(m, 4H), 1.50 (m, 2H), 1.67 (m, 2H), 2.54 (t, 1H), 3.26 (d, 2H),
4.23 (t, 1H), 6.94 (d, 2H), 7.35 (d, 2H). 13C NMR (MeOD): δ
14.3, 21.7, 35.8, 36.6, 46.5, 55.1, 123.4, 131.7, 133.4, 151.9, 171.1,
176.9. MS: calcd. for C17H26NO4 307.38 [M]+, found 308.18
[M +1]+. Anal. (C17H25NO4 7/5HCl 2/5ACN) calcd (%): C,
3
3
3
H, 8.03; N, 8.76; found: C, 57.32; H, 8.33; N, 8.96.
3
3
57.03; H, 7.42; N, 5.23; found: C, 57.08; H, 7.41; N, 5.32.
Boc-3-amino-phenylalanine (10). Pd (10% on activated
charcoal) (0.12 g) was added carefully to Boc-3-nitro-L-pheny-
lalanine (8) (Aldrich, St. Louis, MO, USA) (1 g, 3.22 mmol) in
MeOH (30 mL). The reaction was stirred in H2-atmosphere
until complete. The mixture was filtered through Celite, and the
solvent was evaporated by a rotary evaporator and vacuum pump.
A pink solid formed in vacuum to give 0.9 g (100%) of the title
Analytical Procedure. The amount of the prodrugs 1ꢀ4 in
analytical and biological samples was analyzed by the Agilent
1100 HPLC system (Agilent Technologies, Waldbronn, Karls-
ruhe, Germany) that consisted of a binary pump G1312A, a
vacuum degasser G1379A, an automated injector system auto-
sampler Hewlett-Packard 1050, an UV detector Hewlett-Packard
1050 variable wavelength detector, and an analyst software
Agilent ChemStation for LC Systems Rev. A.10.02. An reverse-
phase high performance liquid chromatography (RP-HPLC)
C-8 column (Agilent Technologies, Little Falls Wilmington,
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compound. H NMR (CDCl3): δ 1.41 (s, 9H), 3.02 (d, 2H),
4.48 (dd, 1H), 6.63 (m, 3H), 7.08 (t, 1H).
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dx.doi.org/10.1021/mp2001878 |Mol. Pharmaceutics 2011, 8, 1857–1866