C.R. Santos et al. / European Journal of Medicinal Chemistry 44 (2009) 2339–2346
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4.2.2. Preparation of trifluoroacetates 2a–d
152.28; 155.85; 157.62; 170.22; 171.29; m/z (M þ Hþcation) ¼ 430.34
Each compounds 4a–d was dissolved in neat TFA and the reac-
tion was allowed to reach completion (w30 min) at room
temperature. The target trifluoroacetate was precipitated with
anhydrous ethyl ether and isolated after centrifugation at 3300 rpm
and at ꢀ9 ꢁC. Compounds 2a–d were thus isolated as white
hygroscopic solids and their structures confirmed by NMR and MS
(see Supporting information).
(calcd, 430.24).
4.2.5. Synthesis of compounds 7–9
The synthesis and spectroscopic identification of compounds 7
and 8 have been described elsewhere [25,32]. Compound 9 was
prepared in three steps as follows.
4.2.5.1. Preparation of captopril methyl ester (CapMe), 9a. This
compound was prepared as previously reported [33]. Briefly,
captopril (10 mmol) was dissolved in methanol (2 mL) under
magnetic stirring and the solution cooled to 0 ꢁC. Thionyl chloride
4.2.3. Synthesis of compounds 5a–g
Each trifluoroacetates 2a–d (1 eq) was dissolved together with
TEA (1.2 eq) in dry DCM and the solution put under magnetic stir-
ring for 2 h. The solution was then immersed in a water–ice–acetone
bath and the relevant BocAA2OH (1 eq) was then added together
with DMAP (0.1 eq). A suspension of DCCI (1 eq) in dry DCM was
slowly added dropwise, and the reaction allowed to proceed at 0 ꢁC
for 2 h, after which it went on at room temperature for further 48 h.
Procedures onwards were as above described for compound 4.
Compounds of 5 were isolated pure, as whitish ‘‘waxes’’, and their
identity confirmed by NMR and MS (see Supporting information).
(200 mL) was then added dropwise, after which the temperature
was raised to 60 ꢁC and the reaction allowed to evolve at this
temperature for 2 h. The solvent was evaporated and the residue
submitted to column chromatography on silica yielding CapMe as
a colorless oil (yield, 82%) with correct spectral data, as compared to
the literature [33].
4.2.5.2. Synthesis of S-(N-tert-butyloxycarbonylphenylalanyl)captopril
methyl ester (BocPheGlyCapMe), 9b. Captopril methyl ester (9a,
1.8 mmol) was dissolved in dioxane (10 mL), and to this solution
were added Boc-Phe-Gly-OH (1.8 mmol) and DMAP (0.18 mmol),
after which the mixture was cooled to 0 ꢁC. Then, DCCI (1.8 mmol)
was added to the mixture and the reaction allowed to proceed at
room temperature for 72 h. Procedures onwards were as previously
described for compounds 4. The product was isolated as a yellowish
oil with correct spectral data. Methyl N-{3-[S-(N-tert-butyloxy-
carbonylphenylalanylglycyl)]mercapto-2-methyl}propanoylprolinate,
9b: yield, 75%; dH: 1.17 (3H, m); 1.33 (9H, s); 1.95 (3H, m); 2.14
(1H, m); 2.72 (1H, m); 3.00 (4H, m); 3.53 (2H, m); 3.65 (3H, s); 4.05
(2H, m); 4.33 (1H, m); 4.43 (1H, m); 4.91 (1H, br s); 6.58
(1H, t, J ¼ 5.4); 7.19 (5H, m); dC: 16.78; 24.80; 28.21; 29.02; 38.32;
41.17; 46.37; 46.86; 49.04; 52.14; 55.74; 58.63; 80.49; 126.97;
128.67; 129.25; 136.45; 171.65; 172.65; 173.13; 173.49; 196.75; m/z
(M þ Naþ ꢀ Boc) ¼ 458.47 (calcd, 458.17).
4.2.4. Preparation of the target trifluoroacetates 3a–g
The final compounds were obtained as white hygroscopic solids
by acidolysis of their Boc-protected precursors 5a–g, following the
same procedure described above for the preparation of tri-
fluoroacetates 2a–d. Their structures were confirmed by NMR and
EA, as illustrated by the spectral data given below (only for novel
compounds).
4.2.4.1. 9-{[2-(O-Alanylvalyl)hydroxyethoxy]methyl}guanine
fluoroacetic acid salt, 3b. Yield, 95%; dH: 0.83 (6H, m); 1.32 (3H, m);
1.99 (1H, m); 3.66 (3H, m); 4.16 (3H, m); 5.34 (2H, s); 6.57 (2H, br s);
tri-
7.81 (1H, s); 8.10 (3H, br s); 8.55 (1H, d, J ¼ 7.9); 10.7 (1H, s); dC
:
18.04; 19.18; 30.88; 48.76; 58.38; 64.20; 67.84; 72.63; 117.21;
138.49; 152.24; 154.84; 157.61; 170.83; 171.71; m/z
(M þ Hþcation) ¼ 396.72 (calcd, 396.68).
4.2.4.2. 9-{[2-(O-Phenylalanylvalyl)hydroxyethoxy]methyl}guanine
trifluoroacetic acid salt, 3c. Yield, 94%; dH: 0.83 (6H, m); 1.96 (1H,
m); 3.01 (3H, m); 4.17 (5H, m); 5.34 (2H, s); 6.57 (2H, br s); 7.28 (5H,
m); 7.81 (1H, s); 8.17 (3H, br s); 8.67 (1H, m); 10.7 (1H, s); dC: 18.82;
19.55; 30.98; 53.94; 58.33; 64.18; 66.35; 72.64; 117.21; 128.00;
129.34; 130.34; 135.56; 138.47; 152.24; 154.85; 157.58; 169.22;
171.49; m/z (M þ Hþcation) ¼ 472.54 (calcd, 472.77).
4.2.5.3. Preparation of S-(phenylalanyl)captopril methyl ester tri-
fluoroacetic acid salt (PheGlyCapMe, TFA), 9. Compound 9b was
dissolved in 5 mL of 40% trifluoroacetic acid in DCM and the reac-
tion was allowed to evolve at room temperature until it reached
completion (w1 h). Procedures onwards were as described for
compound 2 and the target product, 9, was isolated as a white
hygroscopic solid with correct spectral data. Methyl N-[3-(S-
(phenylalanylglycyl)mercapto-2-methyl]propanoylprolinate
tri-
4.2.4.3. 9-{[2-(O-Glycylalanyl)hydroxyethoxy]methyl}guanine
tri-
fluoroacetic acid salt), 9: yield, 99%; dH: 1.07 (3H, d, J ¼ 6.9 Hz); 1.83
(1H, m); 1.91 (2H, m); 2.16 (1H, m); 2.94 (4H, m); 3.17 (1H, dd,
J ¼ 14.1, 5.1); 3.54 (2H, t, J ¼ 6.6 Hz); 3.59 (3H, s); 4.11 (3H, m); 4.27
fluoroacetic acid salt, 3e. Yield, 99%; dH: 1.23 (1H, m); 3.60 (2H, br
s); 3.69 (2H, t, J ¼ 7.1); 4.29 (2H, m); 4.50 (1H, m); 5.37 (2H, s); 6.72
(2H, s); 7.93 (1H, s); 8.11 (3H, br s); 8.82 (1H, d, J ¼ 8.2); 11.0 (1H, s);
dC: 18.63; 30.88; 58.20; 64.20; 67.33; 72.63; 117.25; 138.50; 152.26;
154.85; 157.66; 167.21; 171.84; m/z (M þ Hþcation) ¼ 354.30 (calcd,
354.48).
(1H, q, J ¼ 4.5); 7.32 (5H, m); 8.24 (3H, br s); 9.28 (1H, t, J ¼ 6.0); dC
:
16.29; 24.38; 28.53; 30.72; 36.71; 37.15; 46.40; 48.69; 51.66; 53.32;
58.17; 127.13; 128.50; 129.46; 134.68; 168.77; 172.19; 173.02;
197.07; m/z (M þ Naþ) ¼ 458.60 (calcd, 458.17).
4.2.4.4. 9-{[2-(O-Glycylphenylalanyl)hydroxyethoxy]methyl}guanine
trifluoroacetic acid salt, 3f. Yield, 78%; dH: 3.67 (4H, m); 4.25 (5H,
m); 5.35 (2H, s); 6.57 (2H, br s); 7.28 (5H, m); 7.82 (1H, s); 8.17 (3H,
br s); 8.63 (1H, m); 10.7 (1H, s); dC: 28.76; 38.23; 57.93; 64.47;
66.32; 72.64; 117.21; 127.15; 128.32; 132.46; 135.56; 138.45;
152.86; 155.36; 158.62; 169.19; 171.21; m/z (M þ Hþcation) ¼ 430.30
(calcd, 430.24).
4.3. General procedure for monitoring of drug release by HPLC-UV
A solution of the compound to be assayed (10ꢀ4 M) in aqueous
buffer (pH 7.4) was kept at a constant temperature of 37.0 ꢄ 0.1 ꢁC;
aliquots were periodically taken and immediately injected (loop of
100
mL) into the HPLC system, using a LichroCart 250-4 Lichrospher
100 RP-8 reverse phase column (250 ꢃ 4 mm, 5
mm); the elution
was isocratic, at varying proportions of CH3CN in aqueous buffer
(sodium hexanesulfonate 10 mM, sodium acetate 2.5 mM, phos-
phoric acid 2.5 mM; pH 6.3) and at a flow rate of 1.0 mL/min, with
UV detection at 240 nm. The hydrolyses of prodrugs in 80% (v/v)
human plasma were also studied by the HPLC-UV method
described above. Plasma was obtained from heparinized blood of
healthy donors, pooled, and frozen at ꢀ70 ꢁC before use. Prodrugs 3
4.2.4.5. 9-{[2-(O-Phenylalanylglycyl)hydroxyethoxy]methyl}guanine
trifluoroacetic acid salt, 3g. Yield, 93%; dH: 3.53 (4H, m); 4.21 (5H,
m); 5.34 (2H, s); 6.57 (2H, br s); 7.29 (5H, m); 7.81 (1H, s); 8.12 (3H,
br s); 8.62 (1H, m); 10.8 (1H, s); dC: 30.98; 40.23; 58.33; 64.27;
66.43; 72.62; 117.21; 128.10; 129.32; 130.32; 135.52; 138.47;