Journal of Medicinal Chemistry
Article
Compound 7 could not be readily separated, and the reaction mixture
was used as-is for the next reaction. LRMS (ESI+): m/z = 749.3 [M +
H]+.
−3.3 (c = 0.12, CH3OH). 1H NMR (500 MHz, DMSO-d6): δ 8.49 (s,
1H, CHtriazole), 8.33 (s, 2H, SO2NH2), 6.32 (d, J = 9.1 Hz, 1H, H1′),
5.61 (t, J = 9.4 Hz, 1H, H2′), 5.57 (s, 2H, CH2), 5.52 (t, J = 9.4 Hz,
1H, H3′), 5.16 (t, J = 9.7 Hz, 1H, H4′), 4.35 (m, 1H, H5′), 4.10 (m,
2H, H6′), 2.57 (s, 3H, NCOCH3), 2.02 (s, 3H, COCH3), 1.99 (s, 3H,
COCH3), 1.96 (s, 3H, COCH3), 1.73 (s, 3H, COCH3). 13C NMR
(125 MHz, DMSO-d6): δ 171.5 (NCOCH3), 170.5 (OCOCH3), 170.0
(OCOCH3), 169.8 (OCOCH3), 168.8 (OCOCH3), 166.7 (C5), 161.5
(C2), 143.1 (Ctriazole), 123.4 (CHtriazole), 84.4 (C1′), 73.8 (C5′), 72.5
(C3′), 70.7 (C2′), 68.0 (C4′), 62.2 (C6′), 43.9 (NCH2), 22.4
(NCOCH3), 21.0 (OCOCH3), 20.8 (OCOCH3), 20.7 (OCOCH3),
20.2 (OCOCH3). LRMS (ESI+): m/z = 634.6 [M + H]+. HRMS
(ESI+): m/z calcd for C21H27N7O12S2Na1 [M + Na+], 656.1051;
found, 656.1052.
5-(N-[1-{β-D-Glucopyranosyl}-1H-1,2,3-triazol-4-yl]methyl-N-
[1-phenyl-1H-1,2,3-triazol-4-yl]methyl)amino-1,3,4-thiadia-
zole-2-sulfonamide (3). Title compound 3 was synthesized from the
reaction mixture comprising the per-O-acetylated precursor 7 (0.063 g,
0.084 mmol, 1 equiv) upon treatment with 8% HCl in MeOH (5 mL).
The reaction mixture was sonicated for 1 min, the colorless solution
turned cloudy, and a suspension was formed. The reaction was allowed
to stir for 3 days and was monitored by normal-phase silica gel TLC
using EtOAc as eluent and RP-18 silica gel using 10% ACN/H2O as
eluent. The solvent was evaporated under reduced pressure. Column
chromatography using RP-18 silica gel with 10% ACN/H2O (25 mL)
followed by 40% ACN/H2O (100 mL) gave title compound 3 (0.015
g, 31%) as a hygroscopic off-white solid. Rf = 0.43 (ACN/H2O 4:6).
5-(1-[β-D -Glucopyranosyl]-1H-1,2,3-triazol-4-yl)-
methylamino-1,3,4-thiadiazole-2-sulfonamide (9). Title com-
pound 9 was synthesized from per-O-acetylated precursor 12 (0.205 g,
0.324 mmol, 1 equiv) upon treatment with 8% HCl in MeOH (8 mL).
The reaction mixture was sonicated for 1 min, the colorless solution
turned cloudy, and a suspension was formed. The reaction was allowed
to stir for 5 days and was monitored by normal-phase silica gel TLC
using EtOAc as eluent and RP-18 silica gel using 10% ACN/H2O as
eluent. The solvent was evaporated under reduced pressure. Column
chromatography using RP-18 silica gel with 10% ACN/H2O gave title
compound 9 (0.100 g, 73%) as a hygroscopic off-white solid. Rf = 0.92
1
[α]2D5 +1.77 (c = 0.1, CH3OH). H NMR (500 MHz, DMSO-d6): δ
8.62 (s, 1H, CHtriazole‑Ar), 8.33 (s, 1H, CHtriazole‑Glc), 7.92 (s, 2H,
SO2NH2), 7.84 (d, J = 7.5 Hz, 2H, H2′′,6′′), 7.61 (t, J = 7.5 Hz, 2H,
H3′′,5′′), 7.52 (t, J = 7.5 Hz, 1H, H4′′), 5.56 (d, J = 9.3 Hz, 1H, H1′),
4.70 (s, 2H, NCH2‑triazole‑Ar), 4.68 (s, 2H, NCH2‑triazole‑Glc), 3.78 (t, J =
9.1 Hz, 1H, H2′), 3.72 (m, 1H, H6′), 3.47 (m, 2H, H5′, H6′), 3.40 (t, J =
8.9 Hz, 1H, H3′), 3.26 (t, J = 8.9 Hz, 1H, H4′). 13C NMR (125 MHz,
DMSO-d6): δ 172.6 (C5), 154.5 (C2), 142.6 (Ctriazole‑Ar), 142.2
(Ctriazole‑Glc), 137.0 (C1′′), 130.3 (C3′′,5′′), 129.2 (C4′′), 123.9
(CHtriazole‑Glc), 123.1 (CHtriazole‑Ar), 120.8 (C2′′,6′′), 88.0 (C1′), 80.4
(C5′), 77.3 (C3′), 72.6 (C2′), 70.0 (C4′), 61.2 (C6′), 43.0
(NCH2triazole‑Ar), 42.6 (NCH2triazole‑Glc). LRMS (ESI+): m/z = 581.18
[M+1]+. HRMS (ESI): m/z calcd for C20H24N10O7S2Na1 [M + Na+],
603.1163; found, 603.1178.
(ACN/H2O 9:1). [α]3D2 −0.14 (c = 0.12, CH3OH). H NMR (500
1
MHz, D2O): δ 8.94 (s, 1H, NH), 8.25 (s, 1H, CHtriazole), 8.10 (s, 2H,
SO2NH2), 5.54 (d, J = 9.3 Hz, 1H, H1′), 4.62 (s, 2H, NCH2), 3.76 (t, J
= 9.1 Hz, 1H, H2′), 3.70 (d, J = 10.2 Hz, 1H, H6′), 3.45 (m, 2H, H5′,
H6′), 3.38 (t, J = 9.1 Hz, 1H, H3′), 3.23 (t, J = 9.1 Hz, 1H, H4′). 13C
NMR (125 MHz, DMSO-d6): δ 171.1 (C5), 158.7 (C2), 143.8
(Ctriazole), 122.9 (CHtriazole), 87.9 (C1′), 80.4 (C5′), 77.5 (C3′), 72.6
(C2′), 70.0 (C4′), 61.2 (C6′), 40.1 (NCH2). LRMS (ESI+): m/z =
424.29 [M + H]+. HRMS (ESI+): m/z calcd for C11H17N7O7S2Na1 [M
+ Na+], 446.0523; found, 446.0523.
5-N-[(1-Phenyl-1H-1,2,3-triazol-4-yl)methyl]acetamido-
1,3,4-thiadiazole-2-sulfonamide (11). Title compound 11 was
synthesized from alkyne 10 (2.67 g, 10.26 mmol, 1 equiv) and
phenylazide (1.27 g, 10.66 mmol, 1 equiv) according to general
procedure 1 in 8 h. Purification of crude product by flash
chromatography (15% MeOH/CH2Cl2) gave the product, triazole
11 (2.98 g, 77%), as a pale yellow solid. Rf = 0.62 (EtOAc/hexane 4:1).
Protein X-ray Crystallography. The hCA II protein was
expressed, purified, and crystallized as reported previously.26 Briefly,
the protein was expressed in Escherichia coli and purified using ion
exchange and gel filtration chromatography; it was then concentrated
to 14 mg/mL and crystallized in 2.9 M ammonium sulfate with 0.1 M
Tris buffer, pH 8.5, at 20 °C. Crystal soaking with compound 3 lasted
for 8 days and was done by sprinkling the compound directly into the
crystallization drop. The crystals were subsequently cryo-cooled in
liquid nitrogen before obtaining data from the Australian Synchrotron
MX-2 beamline. The data were indexed using XDS27 and scaled using
SCALA.28 Molecular replacement was done using Phaser29 using the
structure of hCA II in complex with the saccharin ligand (PDB code
4cq026) as the initial starting model. The model was manually rebuilt
using Coot30 and refined using Phenix.31 Density was found for
compound 3, and the initial structure and cif dictionary were generated
using the eLBOW program within Phenix.32
CA Inhibition Assay. An Applied Photophysics stopped-flow
instrument was used for assaying the CA-catalyzed CO2 hydration
activity.33 IC50 values were obtained from dose−response curves
working at seven different concentrations of test compound by fitting
squares methods; values represent the mean of at least three different
determinations, as described by us previously.34 The inhibition
constants (Ki) were then derived by using the Cheng−Prusoff
equation as follows: Ki = IC50/(1 + [S]/Km), where [S] represents the
CO2 concentration at which the measurement was carried out, and Km,
the concentration of substrate at which the enzyme activity is at half
maximum. All enzymes used were recombinant, produced in E. coli as
reported earlier.35,36 The concentration of enzymes used in the assay
were as follows: hCA I, 10.4 nM; hCA II, 8.3 nM.
1
mp 233−235 °C (dec.). H NMR (500 MHz, DMSO-d6): δ 8.88 (s,
1H, Htriazole), 8.32 (s, 2H, SO2NH2), 7.89 (d, J = 7.8 Hz, 2H, H2′,6′),
7.59 (t, J = 7.8 Hz, 2H, H3′,5′), 7.49 (t, J = 7.8 Hz, 1H, H4′), 5.65 (s,
2H, NCH2), 2.67 (s, 3H, COCH3). 13C NMR (125 MHz, DMSO-d6):
δ 171.7 (C5), 166.7 (CO), 161.5 (C2), 143.5 (Ctriazole), 136.9 (C1′),
130.3 (C3′,5′), 129.2 (C4′), 122.4 (CHtriazole), 120.6 (C2′,6′), 44.0
(NCH2), 22.7 (COCH3). LRMS (ESI+): m/z = 380.6 [M + H]+.
HRMS (ESI+): m/z calcd for C13H13N7O3S2Na1 [M + Na+], 402.0413;
found, 402.0413.
5-(1-Phenyl-1H-1,2,3-triazol-4-yl)methylamino-1,3,4-thiadia-
zole-2-sulfonamide (8). To a suspension of compound 11 (0.770 g,
2.029 mmol, 1 equiv) in MeOH (20 mL) was added methanolic
sodium methoxide (25% w/v, 0.700 mL, 3.06 mmol, 1.5 equiv), and
the reaction was stirred at room temperature for 4 h. On completion,
the reaction was neutralized with HCl (1.0 M), and the resulting
suspension was filtered to obtain title compound 8 (0.605 g, 88%) as a
1
solid. Rf = 0.74 (EtOAc). mp 265−267 °C (dec.). H NMR (500
MHz, DMSO-d6): δ 8.84 (br s, 1H, NH), 8.80 (s, 1H, CHtriazole), 8.10
(br s, 2H, SO2NH2), 7.90 (d, J = 7.5 Hz, 2H, H2′,6′), 7.61 (t, J = 7.5
Hz, 2H, H3′,5′), 7.50 (t, J = 7.5 Hz, 1H, H4′), 4.70 (s, 2H, CH2). 13C
NMR (125 MHz, DMSO-d6): δ 171.2 (C5), 158.8 (C2), 145.0
(Ctriazole), 137.1 (C1′), 130.4 (C3′,5′), 129.2 (C4′), 122.1 (CHtriazole),
120.5 (C2′,6′), 40.2 (CH2). LRMS (ESI+): m/z = 338.8 [M + H]+.
HRMS (ESI+): m/z calcd for C11H11N7O2S2Na1 [M + Na+], 360.0308;
found, 360.0313.
5-N-([1-{2′,3′,4′,6′-Tetra-O-acetyl-β-D-glucopyranosyl}-1H-
1,2,3-triazol-4-yl]methyl)acetamido-1,3,4-thiadiazole-2-sulfo-
namide (12). Title compound 12 was synthesized from alkyne 10
(0.520 g, 1.998 mmol, 1 equiv) and β-D-glycopyranosyl azide (0.756 g,
2.025 mmol, 1 equiv) according to general procedure 1 in 8 h.
Purification of the crude product by flash chromatography (EtOAc/
hexane 1:1) gave the product, triazole 12 (0.90 g, 71%), as a white
solid. Rf = 0.45 (EtOAc/hexane 4:1). mp 129−131 °C (dec.). [α]D32
Coordinates. The atomic coordinates and structure factors of hCA
II/3 complex have been deposited in the RCSB Protein Data Bank
with the access number 4RN4.
F
J. Med. Chem. XXXX, XXX, XXX−XXX