C.-N. Chen et al. / Bioorg. Med. Chem. 17 (2009) 3011–3017
3015
of 3-amino-5-benzylthio-1,2,4-triazole. The resulting solution was
heated at reflux for 5 days. On cooling to room temperature the so-
lid which had separated was collected by filtration, washed with
cold ethanol and dissolved in 100 mL of water. The resulting yel-
low solution was acidified with concentrated HCl to precipitate a
solid. The solid was collected by filtration and dried to yield 6.4 g
(94%) of the desired product as a white solid, mp 202–209 °C
(decomposition) [lit.,16 199–210 °C (decomposition)]. 1H NMR
spectra were in agreement with the assigned structure. 1H NMR
(400 MHz, DMSO): d 4.38 (s, 2H), 7.26–7.42 (m, 6H), 12.92 (br s,
2H).
100 mL of methylene chloride, 50 mL of water cooled to ꢁ1 °C.
During the course of the addition the temperature of the reaction
mixture was maintained below 5 °C. When the reaction was com-
plete (TLC analysis) the organic layer was separated and the aque-
ous layer was extracted twice with methylene chloride. The
combined organic phases were dried (MgSO4) and evaporated at
reduced pressure to obtain crude product and it was used straight
without further purification.
4.6. Preparation of N-(2,6-difluorophenyl)-5-methoxy-1,2,4-
triazolo[1,5-a]pyrimidine-2-sulfonamide (Y6610)
4.2. Preparation of 2-benzylthio-5,7-dichloro-1,2,4-
triazolo[1,5-a]pyrimidine (3)
The starting 2,6-difluoroaniline (7.8 g, 60 mmol) was dissolved
in 100 mL of pyridine and the above suspension in 100 mL dichlo-
romethane was added. The reaction mixture was stirred at room
temperature for 10 h. The pyridine was removed by evaporation
at reduced pressure, and the residue was treated with 200 mL of
0.5 N NaOH and 100 mL dichloromethane. After stirring to dissolve
all soluble material the aqueous layer was separated and acidified
with 3 N HCl. The precipitate which separated upon acidification
was collected by filtration and dried to give 5.2 g (38% overall yield
from 5) of the desired product as a yellow solid. Mp 230–231 °C; 1H
NMR (400 MHz, DMSO): d 4.05 (s, 3H), 7.05 (d, 1H, J = 7.2 Hz), 7.12
(d, 2H, J = 8.0 Hz), 7.41 (t, 1H, J = 8.0 Hz), 9.24 (d, 1H, J = 7.2 Hz),
10.75 (br s, 1H). ESI-MS: m/z (%) 342 ([M+1]+), 340 ([Mꢁ1]+); Anal.
Calcd for C12H9F2N5O3S: C, 42.23; H, 2.66; N, 20.52. Found: C,
42.12; H, 2.57; N, 20.36.
A suspension of 11.0 g (0.04 mol) of 2-benzylthio-5,7-dihy-
droxy-1,2,4-triazolo[1,5-a]pyrimidine and 57.0 mL (0.6 mol) of
phosphorous oxychloride was heated at reflux for 5 h. The result-
ing solution was concentrated by evaporation under reduced pres-
sure. The residue was partitioned between cold water and
methylene chloride, and the organic phase was separated and
dried (MgSO4). The organic phase was filtered with silica and con-
centrated by evaporation under reduced pressure to yield the de-
sired product as 8.7 g (70%) of yellow solid. mp 102–104 °C
[lit.,16 97–100 °C]. 1H NMR (400 MHz, CDCl3): d 4.52 (s, 2H), 7.13
(s, 1H), 7.25–7.30 (m, 3H), 7.47 (d, 2H, J = 7.2 Hz).
4.3. Preparation of 2-benzylthio-5-chloro-1,2,4-triazolo[1,5-
a]pyrimidine (4)
4.7. X-ray diffraction analysis
A zinc–copper couple was prepared by stirring 1.0 g of copper
sulfate in 20 mL of water with 15.0 g of zinc dust for 2 h. The cou-
ple was collected by filtration, washed with acetone, and dried
Colorless blocks of Y6610 (0.40 mm ꢀ 0.04 mm ꢀ 0.02 mm)
were counted on a quartz fiber with protection oil. Cell dimensions
and intensities were measured at 298 K on a Bruker SMART CCD
area detector diffractometer with graphite monochromated Mo
overnight under vacuum at 100 °C. To
a solution of 33.0 g
(106 mmol) of 2-benzylthio-5,7-dichloro-1,2,4-trizolo[1,5-
K
a
radiation (k = 0.71073 Å); hmax = 25.99; 14,210 measured reflec-
tions; 2723 independent reflections (Rint = 0.1417) of which 1251
had I > 2 (I). Data were corrected for Lorentz and polarization ef-
a]pyrimidine in 12.5 mL (213 mmol) of acetic acid 50 mL of meth-
anol and 300 mL of fresh distilled tetrahydrofuran was added
20.5 g of Zn–Cu couple. When the reaction was complete (TLC anal-
ysis) the mixture was filtered through Celite and the filtrate was
concentrated by evaporation at reduced pressure. The residue
was triturated with methanol to separated a solid. The solid was
collected by filtration to yield the desired product as 26.6 g (91%)
of orange solid, mp 123–125 °C [lit.,16 125–127 °C]. 1H NMR
(400 MHz, CDCl3): d 4.50 (s, 2H), 7.05 (d, 1H, J = 7.2 Hz), 7.25–
7.32 (m, 3H), 7.45 (d, 2H, J = 7.2 Hz), 8.61 (d, 1H, J = 7.2 Hz).
r
fects and for absorption (Tmin = 0.8959; Tmax = 0.9944). The struc-
ture was solved by direct methods using SHELXS-2001;17 all other
calculations were performed with Bruker SAINT System and Bruker
SMART programs.18 Full-matrix least-squares refinement based on
F2 using the weight of 1/[
values of R = 0.0506,
r
2(F2o) + (0.0849P)2 + 0.0555P] gave final
R = 0.1152, and GOF(F) = 0.897. Hydrogen
x
atoms were observed and refined with a fixed value of their isotro-
pic displacement parameter.
4.4. Preparation of 2-benzylthio-5-methoxy-1,2,4-triazolo[1,5-
4.8. Methods of computational simulations
a]pyrimidine (5)
The X-ray crystal structures of A. thaliana AHAS in complex with
herbicide molecules have been reported.19–22 To save computa-
tional time we constructed a dimmer model of A. thaliana AHAS
for further study since the active site is formed at the interface be-
tween two monomers. The initial monomer coordinates were cho-
sen from the X-ray crystal structure of A. thaliana AHAS in complex
with Chlorsulfuron (PDB code: 1YHZ).22 The crystal structure of the
dimmer AHAS from yeast (PDB code: 1NOH) was used as the tem-
plate. After the herbicide molecule was removed, flumetsulam and
Y6610 were docked into the active site of AHAS with FlexX. The
residues within 6.5 Å around the ligand were defined as the active
site and the final models were selected on the basis of total score.
The MD simulations were performed using the Sander module of
the AMBER 8 program. The partial atomic charges of ligands and
cofactors ThDP and flavin adenine dinucleotide (FAD) were calcu-
lated using the RESP protocol after electrostatic potential calcula-
tions at the Hartree-Fock (HF) level with 6-31G* basis set using
the GAUSSIAN 03 program. Parm99 force field was used to establish
A mixture of 6.0 g (22 mmol) of 2-benzylthio-5-chloro-1,2-4-
triazolo[1,5-a]pyrimidine in 25 mL of methanol was treated with
5.0 g (23.8 mmol) of a 25% solution of sodium methoxide in meth-
anol. When the reaction was complete (TLC analysis) the reaction
mixture was diluted with 100 mL of water and neutralized with
3 N HCL (aq). The solid which separated was collected by filtration,
washed with water and dried to afford 4.5 g (76%) of the desired
product as a white solid, mp 124–126 °C [lit.,16 126–128 °C]. 1H
NMR (400 MHz, CDCl3): d 4.08 (s, 3H), 4.52 (s, 2H), 6.49 (d, 1H,
J = 7.2 Hz), 7.26–7.34 (m, 3H), 7.46 (d, 2H, J = 7.2 Hz), 8.40 (d, 1H,
J = 7.2 Hz).
4.5. Preparation of 5-methoxy-1,2,4-triazolo[1,5-a]pyrimidine-
2-sulfonyl chloride (6)
Chlorine was bubbled into a suspension of 10.9 g (40 mmol) of
2-benzylthio-5-methoxy-1,2,4-triazolo[1,5-a]
pyrimidine
in
Chen, Chao-Nan
