Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4655
δ 7.58-7.32 (bd, 4H, Ar), 4.41 (m, 1H, R-CH:Glu), 3.96-3.74 (m,
3H, R-CH:Glu, CH2:Gly), 2.81-2.62 (m, 2H, γ-CH2:Glu), 2.54-
2.39 (m, 2H, γ-CH2:Glu), 2.20-1.94 (m, 4H, β-CH2:Glu). 13C
NMR (75 MHz, CD3OD/CF3COOD) δ 173.9, 173.8, 172.9,
171.8, 167.4 (C(dO), 138.4, 131.8, 123.6, 119.2 (CAr), 54.2, 53.6
(R-C:Glu), 42.3 (CH2:Gly), 32.8, 30.6, 28.7, 27.5 (β-C:Glu, γ-C:
Glu). ESI HRMS m/z 503.0754 (M þ H)þ; C18H23BrN4O8 þ Hþ
requires 503.0778. HPLC purity (detected at 254 nm) = 99.4%,
Reverse phase HPLC was run on Varian Microsorb column (C18, 5
μm, 4.6 mm ꢀ 250 mm) using two solvent systems with 0.5 mL/min
flow rate and detected at 254 nm. Solvent system 1: 0.04 M TEAB
(triethylammonium bicarbonate) in water/70% acetonitrile in
water=1/1, tR=5.31 min, purity=99.4%. Solvent system 2: 0.04
M TEAB in water/70% acetonitrile in water=20-100% B linear,
tR=13.42 min, purity=98.99%.
polymerization product and then diluted with distilled water.
The acidic impurities were removed by passing the diluted
distillate through Amberlite-400 resin (carbonate form, pre-
pared by stirring 450 mL of chloride form of resin with an
aqueous solution containing 40 g of Na2CO3). The methyl
glyoxal solution thus obtained after filtering off the resin was
standardized by the method of Friedemann.29 Enzyme assays
were performed (30 °C, 0.05 M phosphate buffer (pH 6.6)) using
a thermostatted Beckman DU 7400 spectrophotometer. A fresh
GSH solution was prepared, on the day of the assays, using
distilled, deionized water. For each assay, the cell contained a
total volume of 3.0 mL, which was no more than 6.0 mM with
respect to methylglyoxal and 1.3 mM with respect to GSH.
Sufficient amounts of glyoxalase I, in the presence of 0.1%
bovine serum albumin (Sigma) as a stabilizing agent, were
employed to give an easily measurable initial rate, which was
followed by an increase in absorption at 240 nm. Stock inhibitor
solutions were prepared in the distilled water and adjusted, when
necessary, to pH 6.6. Methylglyoxal, GSH, and buffer (and
inhibitor) were added to a cuvette and allowed to stand for 6 min
in the thermostatted compartment of the spectrophotometer to
allow complete hemimercaptal equilibration. Hemimercaptal
substrate concentrations were calculated from the concentra-
tions of GSH and methylglyoxal added, using a value of 3.1 ꢀ
10-3 M for the dissociation constant of the hemimercaptal at pH
6.6. Data were analyzed using an Enzyme kinetics module of
Sigmaplot 9.0 from Systat Software Inc.
γ-Glutamyltranspeptidase Assay. The stability of glyoxalase I
inhibitors toward γ-GT mediated degradation was determined
by incubating 100 μL of 10 mM solutions of these inhibitors
(dissolved in 200 mM of 2-amino-2-methyl-1,3-propanediol
buffer at pH 8.5) with 10 μL of 0.54 mg/100 μL equine kidney
γ-glutamyltranspeptidase in the above buffer in presence of
20 μL of acceptor dipeptide gly gly (40 mM in the above buffer).
At selected time intervals, a compound from each tube was
spotted on silica gel TLC plate during its incubation at 37 °C.
Thin layer chromatography of these samples was performed
on silica gel TLC plates and compared with the authentic
predicted degradation products (solvent system: (6:2.5:1.5)
butanol/acetic acid/water). Visual detection of TLC spots was
performed under UV and also by iodine and fluorescamine
staining solution.
TFA NH2-γ-Gla[-Glu(CON(OH)-p-bromophenyl)Gly-OH]-
3
OH (26). A mixture of 36 (100 mg, 0.14 mmol) and TFA-
CH2Cl2 (1:1 v/v, 6 mL) was stirred at room temperature for 4 h.
After concentration, the residue was triturated with ether and
EtOAc to get a white residue. Recrystallization by first dissol-
ving in 0.1 N HCl at rt and then cooling in ice/salt gave
analytically pure 7 (66 mg, 78% yield) as a feathery solid. Rf
0.55 (butanol/acetic acid/H2O, 12:5:3); [R]D -19.2 (c 0.5, 1 N
HCl). 1H NMR (300 MHz, CD3OD/CF3COOD) δ 7.59 (d, J=
9.0, 2H, Ar), 7.49 (d, J=8.7 Hz, 2H, Ar), 4.60 (q, J=4.8, 8.4 Hz,
1H, R-CH:Glu), 4.48 (q, J=4.8, 7.8 Hz, 1H, R-CH:Dap), 4.17
(m, 2H, CH2:Gly), 3.68-3.62, 3.41-3.34 (2m, 2H, β-CH2:
Dap), 3.24-2.98 (m, 2H, β-CH2:Glu). 13C NMR (75 MHz,
CD3OD/CF3COOD) δ 174.5, 172.6, 170.5, 159.2, 157.1
(C(dO)), 140.6 (CArNOH), 131.5 (CAr: ortho to Br), 121.1
(CArBr), 117.7 (CAr: ortho to NOH), 52.4 (R-C:Glu), 49.1 (R-C:
Dap), 44.3 (β-C:Dap), 42.6 (CH2:Gly), 31.5 (β-C:Glu). ESI-
HRMS m/z 504.0740 (M þ H)þ; C17H22BrN5O8 þ Hþ requires
504.0730. Reverse phase HPLC was run on Varian Microsorb
column (C18, 5 μm, 4.6 mm ꢀ 250 mm) using two solvent
systems with 0.5 mL/min flow rate and detected at 254 nm.
Solvent system 1: 0.04
bicarbonate) in water/70% acetonitrile in water = 1/1, tR
M TEAB (triethylammonium
=
7.00 min, purity=96.06%. Solvent system 2: 0.04 M TEAB in
water/70% acetonitrile in water=20-100% B linear, tR=14.67
min, purity=95.90%.
TFA NH2-γ-Glu[-Dab(N-(p-bromobenzoyl)-N0-hydroxyl)-Gly-
3
OH]-OH (39). Protected tripeptide 68 (100 mg, 0.14 mmol) was
treated with a mixture of TFA and CH2Cl2 (1:1 v/v, 6 mL) at room
temperature for 4 h. The reaction mixture was then concentrated
and the residue obtained was triturated with ether and EtOAc to
afford a white solid. Recrystallization of the TFA salt from 0.1 N
HCl aqueous gave analytically pure 8 (60 mg, 70% yield). Rf 0.60
(butanol/acetic acid/H2O, 12:5:3); [R]D -12.6 (c 0.44, 1 N HCl).
1H NMR (300 MHz, CD3OD/CF3COOD) δ 7.59 (d, J=9.0, 2H,
Ar), 7.49 (d, J=8.7 Hz, 2H, Ar), 4.43 (m, 1H, R-CH:Glu), 4.17-
3.89 (m, 3H, R-CH:Dab, CH2:Gly), 3.44 (m, 2H, γ-CH2:Dab),
2.55-1.89 (m, 6H, β-CH2:Dab, β-CH2:Glu, γ-CH2:Glu). 13C
NMR (75 MHz, CD3OD/CF3COOD) δ 173.5, 173.1, 172.6,
170.3, 167.2 (C(dO)), 139.7, 130.8, 123.7, 119.4 (CAr), 54.1, 52.4
(R-C:Glu, R-C:Dab), 46.4 (γ-C:Dab), 42.4 (CH2:Gly), 31.9 (γ-C:
Glu), 28.7, 23.2 (β-C:Glu, β-C:Dab). ESI-HRMS m/z 503.0751
(M þ H)þ; C18H23BrN4O8 þ Hþ requires 503.0772. Reverse
phase HPLC was run on Varian Microsorb column (C18, 5 μm,
4.6 mm ꢀ 250 mm) using two solvent systems with 0.5 mL/min
flow rate and detected at 254 nm. Solvent system 1: 0.04 M TEAB
(triethylammonium bicarbonate) in water/70% acetonitrile in
water=1/1, tR=5.45 min, purity=96.65%. Solvent system 2:
0.04 M TEAB in water/70% acetonitrile in water=20 - 100% B
linear, tR=6.86 min, purity=95.07%.
Acknowledgment. This work was supported financially by
a grant from the Center for Drug Design, Academic Health
Center, University of Minnesota. We thank Jay Brownell for
his guidance while performing the glyoxalase I enzyme ki-
netics assays.
Supporting Information Available: Experimental details for
the synthesis of all new compounds and thin-layer chromato-
grams for the γ-glutamyltranspeptidase assay. This material is
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tors were tested for their ability to inhibit yeast glyoxalase I in an
enzyme kinetic assay. Yeast glyoxalase I and the substrate
methylglyoxal were purchased from Sigma Chemical Co. The
commercial 40% methylglyoxal solution was distilled to remove