Journal of Medicinal Chemistry
ARTICLE
fraction was washed with a chilled brine solution (100 mL) to afford 3
as a colorless liquid (55 g, 39.6%). Purity by GC, 96.72%. H NMR
(CDCl3, 400 MHz) δ 2.1 (s, 3H, -COCH3), 3.50 (t, 2H, J = 3.96 Hz,
-CH2Br), 4.4 (t, 2H, J = 6.16 Hz, -CH2O); IR (nujol) cm-1 1753
(CdO).
vigorously for 10 min at pH 10.75 and at 25 °C. The organic layer was
separated, washed with brine solution (250 mL), filtered, and the solvent
was evaporated completely under vacuum to get very viscous oil at
40 °C, which on cooling to -15 to -10 °C for 4 h solidified to the
colorless desired solid product 6 (5.1 g, 85%), mp 71.2 °C. Chromato-
graphic purity (HPLC): 98.15%. MS (ESIþ) m/z = 382.1 (M þ H)þ.
UV max (methanol) 276 nm (48 mM-1 cm-1). IR (KBr) cm-1 3304
(N-H), 1750 (CdO), 1723 (CdO)cm-1; 1H NMR (CDCl3, 400 MHz)
δ 2.02 (s, 3H, -COCH3), 3.83 (s, 2H, -Ph-CH2-CO-), 4.28-4.37
(2 m, 4H, -O-CH2-CH2-O), 6.55 (d, 1H, J = 8.0 Hz, Ar-H), 6.85
(bs, 1H, -NH), 6.94 (m, 2H, Ar-H), 7.11 (t, 1H, J = 7.5, Ar-H), 7.23
(d, 1H, J = 7.5, Ar-H), 7.33 and 7.35 (2s, 2H, Ar-H); 13C NMR
(CDCl3, 100 MHz) δ 20.88 (-CH3), 38.53 (2C, -CH2-Ph), 67.03
(C, -OCH2CH2O-), 62.14 (2C, -OCH2CH2 O-),118.46-142.85
(10C, Ar),170.93 (-OCOCH3), 172.30 (ArCH2CO-).
GC Analysis. a. Analysis Method for Promoities 1 and 2. GC
analysis was performed using a Perkin-Elmer GC model Clarus 500,
column DB-1, 30 m ꢀ 0.53 mm, 1.5 μm, by maintaining the follow-
ing chromatographic parameters: oven temperature, 75 °C; ramp rate,
10 °C/min up to 200 °C; final oven temperature, 200 °C for 10 min;
injection temperature, 150 °C; detector temperature, 250 °C; flow
(carrier), 5.0 mL/min; injection volume, 0.2 μL; split, 1:20.
b. Analysis Method for Promoity 3. A Perkin-Elmer GC, model
Clarus 500, column DB-5, 30 m ꢀ 0.53 mm, 5 μm, was used. Chro-
matographic parameters were as follows: detector, FID; carrier gas flow
(N2), 3.5 mL/min; initial oven temperature, 50 °C; initial time, 6.0 min;
rate 1, 15 °C/min, up to 110 °C for 6 min; rate 2, 35 °C/min up to
250 °C for 10 min; injection temperature, 125 °C; detector temperature,
270 °C; flow (carrier), 5.0 mL/min; injection volume, 0.2 μL; split, 1:10.
HPLC Analysis. In-process analysis and determination of chroma-
tographic purity and partition coefficients of prodrugs 4, 5, 6 were
performed by HPLC instrument (Waters alliance), pump 2695, and UV
detector 2487 with the following chromatographic parameters: wave-
length 254 nm; column, YMC-Pack C8, 100 mm ꢀ 4.6 mm, 3 μm;
injection volume, 20 μL; run time, 20 min. Separation was performed as
isocratic elution. Mode of operation was isocratic. Mobile phase was as
follows. Solution A: mix of 2.0 mL of glacial acetic acid in 1000 mL of
water (pH 3.0). Solution B: acetonitrile (filtered and degassed). Mobile
phase mix ratio was 30 volumes of solution A and 70 volumes of solution
B. Flow rate was 0.8 mL/min at 25 °C.
Metabolic Stability Analysis. Analysis was performed by using
an HPLC instrument (Waters alliance), pump 2695, and PDA detector
2996 with the following chromatographic parameters: wavelength,
275 nm; column, Inertsil C18, 3V, 250 mm ꢀ 4.6 mm, 5 μm; injection
volume, 20 μL; run time, 20 min. Mode of operation was isocratic.
Solution A was as follows: mix of 2.0 mL of glacial acetic acid in 1000 mL
of water (pH 3.0), Solution B was as follows: acetonitrile (filtered and
degassed). Mobile phase was a mixture of 30 volumes of solution A and
70 volumes of solution B. Flow rate was 1.0 mL/min at 25 °C.
Aqueous Solubility. Diclofenac sodium and prodrug solubility
was determined in various pH buffers at pH 1.0, pH 3.0, pH 5.2, pH 7.4,
and pH 9.0 at 25 °C. An excess compound was added to buffer solutions,
and the suspension was shaken for 5 h at 25 °C on mechanical shaker at
350 rpm. The solution was filtered through a Millipore filter (0.22 μm)
and analyzed quantitatively by HPLC.
1
Pivoloxymethyl 2-[(2,6-Dichlorophenyl)Amino]Benzene
Acetate (4). A solution of 1a (10 g, 31.4 mmol) in DMAc (45 mL)
was treated with sodium carbonate (0.33 g, 3.1 mmol) at -5 °C for
10 min under a N2 atmosphere. The mixture was cooled to -15 °C,
followed by addition of iodomethyl pivalate 1 (7.6 g, 31.4 mmol). The
mixture was stirred vigorously at -15 to -13 °C for 60 min under N2
atmosphere. The reaction progress was monitored by HPLC. The
reaction mixture was added under vigorous stirring to a mixture of ethyl
acetate (120 mL), water (400 mL), and sodium thiosulfate (3.2 g). The
organic layer was separated after 10 min of stirring at 25 °C. The organic
layer was washed with brine solution (2 ꢀ 200 mL), filtered, and the
solvent was removed in vacuum. Isopropyl ether (50 mL) was added.
The solid product was filtered, washed with isopropyl ether (50 mL),
and dried under vacuum at 45 °C for 8 h to afford 4 as a white crystalline
powder (9.8 g, 83.8%), mp 95.2 °C. Chromatographic purity (HPLC):
99.50%. MS (ESIþ) m/z = 410 (M þ H)þ. UV max (methanol):
275 nm (26 mM-1 cm-1). IR (KBr) cm-1 3358 (N-H), 1750 (CdO);
1H NMR (CDCl3, 400 MHz) δ 1.15 [s, 9H, -C(CH3)3 ], 3.86 (s, 2H,
-CO-CH2-Ar), 5.80 (s, 2H, -O-CH2-O-), 6.55 (d, 1H, J =
8.0 Hz, 7Ar-H), 6.71 (bs, 1H, -NH), 6.94-7.01 (m, 2H, Ar-H), 7.11
(td, 1H, J = 8.0 Hz and J = 1.4 Hz, Ar-H), 7.22 (dd, 1H, J = 7.5 Hz and
J = 1.2 Hz, Ar-H), 7.33 (s, 1H, Ar-H), 7.35 (s, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) δ 26.95 [(3C, (CH3)3)], 38.38-38.89 (2C, CH2),
79.84 (1C, OCH2O), 118.7-142.8 (12C, ArC), 171.16 (1C, CdO),
177.23 (1C, CdO).
1-[(1-Methylethoxy)carbonyloxy]methyl 2-[(2,6-Dichlo-
rophenyl)amino]phenyl Acetate (5). A solution of 1a (5 g,
15.7 mmol) in DMAc (22.5 mL) was treated with micronized sodium
carbonate (0.25 g, 2.35 mmol) at 30 °C for 10 min under N2 atmo-
sphere. The mixture was cooled to -10 °C, and iodomethylisopropyl
carbonate 2 (3.76 g, 15.4 mmol) was added. The reaction mixture was
stirred at -10 °C for 45 min under N2 atmosphere. The reaction
progress was monitored by HPLC. Reaction mixture was added under
stirring to mixture of ethyl acetate (60 mL), water (200 mL), and sodium
thiosulfate (2 g). The mass was stirred vigorously for 10 min at 25 °C.
The organic layer was separated and washed with brine solution
(2 ꢀ 125 mL). The organic layer was treated with activated carbon
(1 g) for 10 min and filtered, and the solvent was evaporated completely
under high vacuum followed by cooling of the sample at -15 to -10 °C
to afford a yellowish solid product 5 (5.6 g, 86.4%). Chromatographic
purity (HPLC), 99.58%. Mp 52.5 °C. MS (ESIþ) m/z = 412 (M þ H)þ.
UV max (methanol) 274.2 nm (45 mM-1 cm-1). IR (KBr) cm-1 3306
(N-H), 1756 (CdO), 1735 (CdO); 1H NMR (DMSO, 400 MHz) δ
1.22 (d, 6H, J= 6.3 Hz, -CH(CH3)2), 3.90 (s, 2H, -Ph-CH2-CO-),
4.77 (sep, 1H, J = 6.3 Hz, -O-CH(CH3)2), 5.73 (s, 2H, -O-
CH2-O-), 6.21 (d, 1H, J = 8.0 Hz, Ar-H), 6.82 (t, 1H, J = 7.4 Hz,
Ar-H), 7.02 (bs, 1H, -NH), 7.06 (t, 1H, J = 7.7 Hz, Ar-H), 7.18-7.24
(m, 2H, Ar-H), 7.53 (s, 1H, Ar-H), 7.55 (s, 1H, Ar-H); 13C NMR
(CDCl3, 100 MHz) δ 21.77 (2C, (CH3)2), 38.28 (C, CH2Ar), 73.34 (C,
-CH(CH3)2), 82.33 (C, -OCH2O-), 118.73-142.83 (10C, Ar),
153.41 (C, -OCOO-), 170.96 (C, -OCOCH2-).
Partition Coefficient. The partition coefficient of diclofenac
sodium and prodrugs was determined by the HPLC method. Before
a partition coefficient, 1-octanol and buffer solution were mutually
saturated for 24 h by stirring vigorously on a mechanical shaker at
25 °C, and the phases were allowed to separate for 24 h at 25 °C. A stock
solution of test compound was prepared at 25 °C, filtered through
0.22 μm membrane filter, and the percentage of test compound was
determined by HPLC. Three tests were carried out in duplicate with
various volume ratios of octanol (stock solution) to presaturated buffer,
2-Acetoxyethyl 2-[(2,6-Dichlorophenyl)amino]phenyl
Acetate (6). A solution of 1a (5 g, 15.7 mmol) was treated with
sodium carbonate (0.33 g, 3.14 mmol) in DMAc (20 mL) at 30 °C for
10 min under N2 atmosphere. 2-Acetoxyethyl bromide 3 (3.28 g,
19.64 mmol) was added at 35 °C, and the mixture was stirred at 40 °C for
60 min, 55 °C for 4 h under N2 atmosphere. The reaction mixture
was added under stirring to the mixture of ethyl acetate (60 mL), water
(200 mL), and sodium thiosulfate (2 g), and the mixture was stirred
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dx.doi.org/10.1021/jm101095e |J. Med. Chem. 2011, 54, 1202–1210