Journal of Medicinal Chemistry
Article
1H), 7.69 (d, J = 8.8 Hz, 1H), 7.53 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 9.8
Hz, 1H), 7.38 (d, J = 8.1 Hz, 2H), 6.98 (dd, J = 8.9, 2.6 Hz, 1H), 6.85
(d, J = 2.6 Hz, 1H), 6.83 (dd, J = 9.8, 2.0 Hz, 1H), 6.31 (d, J = 2.0 Hz,
1H), 6.05−5.89 (m, 1H), 5.13 (s, 2H), 4.59−4.41 (m, 1H), 4.12−3.95
(m, 1H), 3.79−3.63 (m, 1H), 1.93−1.79 (m, 2H), 1.79−1.71 (m, 1H),
1.70−1.59 (m, 1H), 1.51 (s, 9H), 1.46 (s, 9H), 1.36 (s, 9H). 13C NMR
(151 MHz, chloroform-d) δ 186.35, 163.08, 162.62, 161.11, 154.83,
149.82, 145.66, 145.59, 137.95, 134.72, 134.26, 131.60, 131.44, 128.48,
128.17, 121.36, 114.35, 106.76, 101.12, 84.33, 79.98, 79.61, 70.56,
53.99, 43.96, 29.05, 28.45, 28.03, 24.60. ESI-HRMS (MH+) calcd for
C40H51N6O10 775.3667, found 775.3684. Boc-groups were cleaved
using a 1:1 TFA/DCM mixture (3 mL) at rt for 3 h. The solvents were
evaporated under reduced pressure, and the resulting solid was
dissolved in a minimal amount of methanol (0.5 mL). The methanolic
solution was poured in ether (10 mL), and the resorufin conjugate was
collected as an orange solid as TFA salt. It was washed with ether
several times and dried under vacuum, yielding 44 mg (88%) of pure
3H), 2.41 (s, 3H), 2.01 (s, 3H), 1.74−1.63 (m, 1H), 1.60−1.43 (m,
−
3H), 1.38 (s, 6H). ESI-HRMS (M−) calcd for C26H35N4O7S2
1
579.1953, found 579.1940. According to H NMR spectra, 95% pure.
Bzls-D-Arg(Pbf)-Gly-OH (9). Carboxylic acid 8 (150 mg, 0.26 mmol)
and NHS (38.6 mg, 0.33 mmol) were dissolved in 1,2-dimethoxy-
ethane (3 mL), and DCC (68 mg, 0.33 mmol) was added as solid at 0
°C. The resulting solution was stirred for 3 h at 0 °C, followed by 1 h
at room temperature. The urea was filtered off and solvent was
evaporated under vacuum, obtaining the succinimide Bzls-Arg(Pbf)-
OSuc as a foam which was used in the next step without purification.
The solution of succinimide in dioxane (10 mL) was added to a
solution of glycine (60 mg, 0.8 mmol) and NaHCO3 (67.2 mg, 0.8
mmol) in water (6 mL) at 10 °C. The suspension was stirred
vigorously for 1 h at 5−10 °C and then left overnight in the fridge.
Most of the dioxane was evaporated under vacuum, and the resulting
water solution was filtered, the filtrate was washed with ethyl acetate,
and the water phase was collected and then acidified with acetic acid to
pH 4. The water phase was again extracted with ethyl acetate, and the
extracts were washed with brine then dried over MgSO4. The solvent
was evaporated, and the residue was purified by SiO2 column
chromatography using 8% methanol in CH2Cl2 with 1% acetic acid,
yielding the dipeptide 9 (31 mg, 19%) as a white foam. 1H NMR (600
MHz, methanol-d4) δ 7.44−7.37 (m, 2H), 7.37−7.27 (m, 3H), 4.40−
4.24 (m, 2H), 3.99−3.82 (m, 3H), 3.28−3.07 (m, 2H), 2.99 (s, 2H),
2.58 (s, 3H), 2.52 (s, 3H), 2.08 (s, 3H), 1.77−1.67 (m, 1H), 1.66−
1.52 (m, 3H), 1.44 (s, 6H). 13C NMR (151 MHz, methanol-d4) δ
173.13, 171.53, 158.50, 156.79, 138.05, 132.89, 132.16, 130.74, 129.63,
128.11, 128.01, 124.68, 117.07, 86.29, 58.84, 56.37, 53.43, 42.54,
40.55, 30.19, 27.31, 18.22, 17.04, 11.11. ESI-HRMS (M−) calcd for
1
product 5. H NMR (600 MHz, methanol-d4) δ 7.79 (d, J = 8.9 Hz,
1H), 7.67 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 9.8 Hz, 1H), 7.49 (d, J = 8.4
Hz, 2H), 7.14 (dd, J = 8.9, 2.6 Hz, 1H), 7.11 (d, J = 2.6 Hz, 1H), 6.85
(dd, J = 9.8, 2.1 Hz, 1H), 6.33 (d, J = 2.1 Hz, 1H), 5.24 (s, 2H), 4.04
(t, J = 6.5 Hz, 1H), 3.25 (t, J = 7.0 Hz, 2H), 2.10−1.88 (m, 2H), 1.83−
1.66 (m, 2H). 13C NMR (151 MHz, methanol-d4) δ 187.01, 166.79,
163.34, 157.28, 150.63, 145.79, 144.98, 137.66, 135.16, 133.20, 132.51,
131.49, 128.63, 128.33, 128.33, 119.83, 114.63, 105.36, 100.84, 70.17,
53.33, 48.17, 48.03, 40.40, 28.48, 24.07. ESI-HRMS (MH+) calcd for
C25H27N6O4 475.2094, found 475.2082. According to 1H NMR
spectra and HPLC, 99% pure.
Boc-D-Phe-Pro-OMe (6). To a solution of Boc-D-Phe-OH (250 mg,
0.95 mmol) and TBTU (369 mg, 1.15 mmol) in DMF (10 mL) was
added DIEA (296 mg, 400 μL, 2.3 mmol), and the solution was stirred
for 15 min at rt. H-Pro-OMe·HCl (190 mg, 1.15 mmol) was dissolved
in DMF (2 mL), mixed with DIEA (148 mg, 200 μL, 1.15 mmol), and
added to the reaction mixture. After stirring overnight at rt, the solvent
was evaporated and to the residue was added water and ethyl acetate.
The extracts were dried over MgSO4, the solvent was evaporated, and
the residue was chromatographed on silica with 10% ethyl acetate in
DCM as eluent. The dipeptide 6 (260 mg, 73%) was obtained as white
foam. 1H NMR (600 MHz, chloroform-d) δ 7.32−7.13 (m, 5H), 5.37
(d, J = 8.6 Hz, 1H), 4.62 (td, J = 9.1, 5.4 Hz, 1H), 4.28 (dd, J = 8.4, 3.9
Hz, 1H), 3.71 (s, 3H), 3.57−3.47 (m, 1H), 3.05 (dd, J = 12.9, 5.5 Hz,
1H), 2.91 (dd, J = 12.8, 9.5 Hz, 1H), 2.68−2.56 (m, 1H), 1.95−1.88
(m, 1H), 1.88−1.78 (m, 2H), 1.53−1.46 (m, 1H), 1.43 (s, 9H).
Boc-D-Phe-Pro-OH (7). To the solution of methyl ester 6 (260 mg,
0.7 mmol) in THF (2 mL) was added water (10 mL), and the
emulsion was cooled to 0 °C. A solution of NaOH (0.5 N, 20 mL) was
added dropwise, and the reaction mixture was stirred for 3 h at 0 °C.
The base was neutralized with acetic acid, and the product was
extracted with ethyl acetate. The extracts were dried over MgSO4, the
solvent was evaporated under reduced pressure, and the residue was
purified by column chromatography on silica using 5% methanol in
DCM with 1% acetic acid. The product 7 (200 mg, 79%) was obtained
as white crystals. 1H NMR (600 MHz, chloroform-d) δ 7.32−7.15 (m,
5H), 5.35 (d, J = 8.5 Hz, 1H), 4.64 (td, J = 9.3, 5.6 Hz, 1H), 4.36 (d, J
= 5.9 Hz, 1H), 3.62−3.50 (m, 1H), 3.07 (dd, J = 12.8, 5.5 Hz, 1H),
2.94 (dd, J = 12.8, 10.0 Hz, 1H), 2.53 (td, J = 9.4, 6.5 Hz, 1H), 2.32−
2.19 (m, 1H), 1.88−1.76 (m, 1H), 1.65−1.51 (m, 2H), 1.43 (s, 9H).
Bzls-D-Arg(Pbf)-OH (8). To a suspension of H-D-Arg(Pbf)-OH (400
mg, 0.94 mmol) in water (0.95 mL) was added dropwise 1 M solution
of NaOH (0.95 mL) at 0 °C. Phenylmethanesulfonyl chloride (200
mg, 1.05 mmol) was added portionwise to the reaction mixture at 0
°C, followed by triethylamine (111 mg, 153 μL, 1.1 mmol) and
acetone (2 mL). Stirring was continued at 0 °C for 1 h then at rt
overnight. The solution was acidified with 10% KHSO4 and extracted
5 times with ethyl acetate. The extracts were washed with brine and
concentrated under reduced pressure. The residue was purified by
column chromatography using 7.5% methanol in DCM with 1% of
−
1
C28H38N5O8S2 636.2167, found 636.2186. According to H NMR
spectra, 99% pure.
Enzyme Kinetic Studies. Michaelis−Menten kinetics were
measured for the substrate 1a using UV−vis and for the substrate 1
using both fluorescence and UV−vis. Thrombin solution (200 μL, 1
nM) which also contained Tris buffer (50 mM, pH 8.3) and NaCl
(130 mM) was placed in a cuvette. A 20× solution of substrate (10
μL) in water was added to the enzyme (substrate final concentration
1−20 μM), and the enzymatic reaction was monitored over time. For
substrate 1a the absorption increase at 405 nm (ε = 9600 M−1 cm−1)
was measured and for 1, the absorption increase at 570 nm (ε = 53000
M−1 cm−1) or emission increase at 583 nm. The reaction rate ν was
calculated for each substrate concentration [s]. The data were fit to the
following equation: ν = νmax × [s]/(KM + [s]).
Specificity of Substrate 1 for Thrombin. The activity of a series
of enzymes was assayed in the presence of substrate 1. The following
enzymes were used: myoglobin horse heart (17 kDa), cytochrome C
horse heart (12.4 kDa), trypsin (23.3 kDa), BSA (66.5 kDa), factor Xa
(43 kDa), and human thrombin (36 kDa). A 96-well microtiter plate
was charged with the enzymes solutions (350 μL, 100 pM), also
containing Tris buffer (50 mM, pH 8.3) and NaCl (130 mM) and the
emission increase at 583 nm started to be measured immediately after
substrate 1 addition (5 μM). The initial reaction rates were
determined.
Thrombin Detection Assay. A series of thrombin solutions with
concentrations in low range (0.5−10 pM) and high range (5−100
pM) were prepared and loaded onto a 96-well microtiter plate. The
thrombin solution contained also Tris buffer (50 mM, pH 8.3), NaCl
(130 mM), and BSA (0.1 mg/mL). The emission increase
measurement started immediately after substrate (5 μM) addition.
For the calibration curve, the initial reaction rates were plotted versus
thrombin concentration. Each measurement was carried out in
triplicate.
Dabigatran Quantification in Human Plasma. Human plasma
was spiked with dabigatran at different concentrations (30−300 ng/
mL). The thrombin solution (100 pM) with Tris buffer (50 mM, pH
8.3), NaCl (130 mM), urea (500 mM), aprotinin (150 mU/mL),
polybrene (100 ng/mL), and BSA (0.1 mg/mL) was loaded onto a 96-
well microtiter plate (350 μL per well). Plasma spiked with dabigatran
(14 μL) was added to thrombin solution and incubated for 10 min.
The substrate 1 (5 μM) was added, and the emission increase at 583
1
acetic acid, yielding 150 mg (27%) of 8 as white powder. H NMR
(600 MHz, chloroform-d) δ 7.35−7.26 (m, 5H), 4.20 (q, J = 13.8 Hz,
2H), 3.30−3.27 (m, 1H), 3.12−3.01 (m, 2H), 2.88 (s, 2H), 2.48 (s,
F
J. Med. Chem. XXXX, XXX, XXX−XXX