Design, synthesis and biological evaluation of novel 2-methylpyrimidine-4- ylamine derivatives as inhibitors of Escherichia coli pyruvate dehydrogenase complex E1

Article abstract of DOI:10.1016/j.bmc.2012.01.019

As potential inhibitors of Escherichia coli pyruvate dehydrogenase complex E1 (PDHc E1), a series of novel 2-methylpyrimidine-4-ylamine derivatives were designed based on the structure of the active site of PDHc E1 and synthesized using 'click chemistry'.

Full text of DOI:10.1016/j.bmc.2012.01.019

Bioorganic & Medicinal Chemistry 20 (2012) 1665–1670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of novel 2-methylpyrimidine-
4-ylamine derivatives as inhibitors of Escherichia coli pyruvate
dehydrogenase complex E1
Junbo He , Lingling Feng , Jing Li, Ruijuan Tao, Fang Wang, Xun Liao, Qiushuang Sun, Qingwu Long,
⇑
⇑
Yanliang Ren, Jian Wan , Hongwu He
Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Department of Chemistry, Central China Normal University, Wuhan 430079, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 21 January 2012
As potential inhibitors of Escherichia coli pyruvate dehydrogenase complex E1 (PDHc E1), a series of novel
2-methylpyrimidine-4-ylamine derivatives were designed based on the structure of the active site of
PDHc E1 and synthesized using ‘click chemistry’. Their inhibitory activity in vitro against PDHc E1 and
fungicidal activity were examined. Some of these compounds such as 3g, 3l, 3n, 3o, and 5b demonstrated
to be effective inhibitors of PDHc E1 from E. coli and exhibited antifungal activity. SAR analysis indicated
that both, the inhibitory potency against E. coli PDHc E1 and the antifungal activity of title compounds,
could be increased greatly by optimizing substituent groups in the compounds. The structures of substi-
tuent group in 5-position on the 1,2,3-triazole and 4-position on the benzene ring in title compounds
were found to play a pivotal role in both above-mentioned biological activities. Amongst all the com-
pounds, compound 5b with iodine in the 5-position of 1,2,3-triazole and with nitryl group in the 4-posi-
tion of benzene ring acted as the best inhibitor against PDHc E1 from E. coli. It was also found to be the
most effective compound with higher antifungal activity against Rhizoctonia solani and Botrytis cinerea at
Keywords:
2-Methylpyrimidine-4-ylamine
PDHc E1 inhibitor
Antifungal activity
Click chemistry
the dosage of 100
ther optimization.
l
g mLꢀ1. Therefore, in this study, compound 5b was used as a lead compound for fur-
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
E1 from plants and showed potential utility as herbicide.13 These
results encouraged further exploration and research of new inhib-
Pyruvate dehydrogenase complex (PDHc) plays a pivotal role in
cellular metabolism catalyzing the oxidative decarboxylation of
pyruvate and the subsequent acetylation of coenzyme A (CoA) to
acetyl-CoA.1–4 The complex is comprised of three different en-
zymes components and a number of cofactors. Pyruvate dehydro-
genase complex E1 component (PDHc E1, EC 1.2.4.1) is the initial
member of PDHc, which catalyzes the first step of the multistep
process, using thiamine diphosphate (ThDP) (Fig. 1) and Mg2+ as
cofactors.5–7 Therefore, it has been studied clearly that the ThDP
plays an important role in the enzyme reaction and the catalysis
mechanism.8
A systematic study for the rational design for the potential use
of a novel effective inhibitor of PDHc E1 as herbicide has been
previously carried out in our laboratory for several years.9–12 Some
of 1-(substituted phenoxyacetoxy)alkylphosphonates (such as
HW02) were successfully found to be effective inhibitors of PDHc
itors of PDHc E1 possessing bactericidal or fungicidal activity based
on the structure of PDHc E1 in the microorganism.
Due to the important role of fungicide in modern agriculture, it
is essential to develop efficient fungicide with novel structures or
modes of action to overcome fungicide resistance. In order to find
a novel fungicide or antifungal activity compound based on the de-
sign of inhibitor of PDHc E1, Escherichia coli PDHc E1 was selected
as the target pattern of microorganism. Considering the impor-
tance of ThDP in the metabolic process of pyruvate, this research
also focuses on designing novel ThDP analogs as inhibitors of PDHc
E1 in E. coli. Through literature review, it was noticed that some
ThDP analogs (Fig. 2) have been chemically synthesized and were
reported as effective inhibitors of E. coli PDHc E1.14–17 Although
these analogs of ThDP showed potent inhibition against E. coli
PDHc E1, they exhibit no potential utility as fungicide due to their
complex structure with highly charged pyrophosphate. These com-
plex structures are unsuitable for good bioavailability.18 Moreover,
there was no report about their bactericidal or fungicidal activity.
Aiming at the above-mentioned problems, both the pyrophos-
phate and the thiazolium ring moiety in ThDP were replaced by
substituted benzene ring and 1,2,3-triazole ring, respectively. It
⇑
Corresponding authors. Tel./fax: +86 (0)27 67867960.
E-mail addresses: jianwan@mail.ccnu.edu.cn (J. Wan), he1208@mail.ccnu.edu.cn
(H. He).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.01.019

1666
J. He et al. / Bioorg. Med. Chem. 20 (2012) 1665–1670
R1
R1
NH2
N
H
CO2
Me
R2
Me
R2
N
N
N
S
N
Me
Me
HO
k3
R2
HO
Me
S
S
R1
CH3COCO2
H3C
CO2
k2'
LThDP
H
4-aminopyrimidine
Me
Me
R2
NH
N
N
S
N
R2
R1
S
HN
1,4-iminopyrimidine
R1
Yield
-H1'
Me
thiazolium
k5
Me
R2
Me
R2
N
S
-H4'
k-4/k4
N
Me
Me
HO
CH3CHO
S
HO
NH2
H
H
enamine
HEThDP
N
S
N
R1=4-amino-2-methyl-5-pyrimidyl
R2
β -hydroxyethyldiphosphate
Me
2
H3C
N
H
R =
4-aminopyrimidinium
Figure 1. Mechanism of pyruvate dehydrogenase complex (E1) component.
2. Chemistry
NH2
N
NH2
CH3
CH3
S
3-
3-
OP2O6
OP2O6
N
N
N
N
Triazole-based pyrophosphate analogs of ThDP had been re-
ported by Leeper and co-workers,20 followed their method, the
synthetic route employed to obtain the title compounds 3a–3j is
depicted in Scheme 1. The 10 terminal alkynes 1a–1j were synthe-
sized from the 3-bromopropyne with corresponding substituted
phenol in refluxing acetone with K2CO3 as base. The 5-azido-
methyl-2-methylpyrimidine-4-ylamine 2 was prepared readily
from thiamine hydrochloride according to the literature method
as described.20 The Cu-catalyzed 1,3-dipolar cycloaddition reac-
tion21 was introduced to assemble the final target compounds
3a–3j. A combination of copper(II) sulfate/sodium ascorbate was
utilized in situ to prepare the copper(I) species, and a ‘click chem-
istry’ was achieved in 12–24 h at room temperature.
S
H3C
H3C
N
O
tetrahydro-ThDP
ThTDP
NH2
NH2
CH3
CH3
S
3-
6
3-
OP2O
OP2O6
N
N
N
N
S
HO
H3C
N
H3C
N
S
CH3
P(O2)OCH3
PLThDP
ThTTDP
Figure 2. Structures of ThDP analogs.
General synthetic procedure for compounds 5a and 5b is shown
in Scheme 2. A one-pot, two-stage sequence was used to synthe-
size 5-iodotirazoles 5a and 5b. The two terminal alkynes were
treated with N-iodomorpholine22 catalyzed with copper(I) iodide
(CuI) within 60 min, the corresponding 1-iodoalkynes (4a and
4b) could be purified by filtration through a pad of neutral alumina
in excellent yield. The 1-iodoalkynes and azide 2 were catalyzed by
copper(I) iodide-triethylamine (TEA) in THF, and gave 5-iodotriaz-
oles 5a and 5b in excellent yield.
The chemical structures of all the target compounds (Table 1)
were confirmed by recording their 1H NMR, 13C NMR, mass spectra
and elementary analysis. The 1H NMR spectrum of all the com-
pounds showed a singlet for three (CH3) protons at d (2.30–2.33),
another two singlets for two (OCH2 and CH2) protons at d
(5.10–5.47 and 5.35–5.52, respectively), and a singlet for two
(NH2) protons at d (5.59–5.60 for CDCl3 and 5.59–5.98 for DMSO-
d6, respectively). Interestingly, it must be noted that one proton
(pyrimidine-H) disappeared for compounds 5a and 5b, but on the
has been verified that some hit compounds with aminopyrimidine,
triazole and benzene ring moiety could be effective in occupying
the ThDP-binding pocket of PDHc E1 and also may be bound in a
pocket of PDHc-E1 by using structure-based molecular docking
methods.19 Therefore, a series of novel 2-methylpyrimidine-4-yla-
mine derivatives 3 and 5 as potential inhibitors of E. coli PDHc E1
were designed based on the structure of ThDP and properties of
the active site of PDHc E1. The design rationality of title com-
pounds 3 and 5 was further verified by later experiments.
In this paper, we reported the synthesis of 12 novel 1,2,3-tria-
zole containing 2-methylpyrimidine-4-ylamine derivatives 3 and
5 using click chemistry. All these compounds were tested for their
inhibition against PDHc E1 from Escherichia coli. The title com-
pounds, including some previously reported analogous com-
pounds, were further evaluated on the basis of their practicality,
antifungal properties and structure–activity relationships.
OH
a
R2
R2
O
R1
1a-1j
R1
N
NH2
NH
N
2
NH2
N
b
c
R2
N
N3
N
N
N
N
N
OH
O
S
H3C
H3C
N
H3C
R1 = H
2
3a-3j
Scheme 1. Reagents and conditions: (a) 3-Bromopropyne, K2CO3, acetone, reflux, 5 h; (b) NaN3, Na2SO3, H2O, 60–65 °C, 6 h; (c) 1a–1j, sodium ascorbate, CuSO4ꢁ5H2O,
t-BuOH/H2O (2:1), rt, 12–24 h.

J. He et al. / Bioorg. Med. Chem. 20 (2012) 1665–1670
1667
R1
R1
N
NH2
N
O
OH
O
b
R2
a
c
N
N
N
R2
R2
R2
O
H3C
R = H, NO2
4a, 4b
5a, 5b
R1 = I
Scheme 2. Reagents and conditions: (a) 3-Bromopropyne, K2CO3, acetone, reflux, 5 h; (b) N-iodomorpholine, CuI, THF, rt, 1 h; (c) 2, CuI, THF, Et3N, rt, 6 h.
itory activity. However, the inhibitory activity was highly depen-
dent upon both, the structure and position of substituent R2 on
the benzene ring. Comparing the data in Table 1, the compounds
3 with 2-Cl, 4-NO2 and 4-NO2 as R2 on the benzene ring showed
a great promotive effect to the inhibitory activity followed by
2,4-2NO2, such as 3n and 3l with 2-Cl, 4-NO2 and 4-NO2 as R2
Table 1
Structures and IC50 values against PDHc E1 inhibitory activities of derivatives 3a–3o
and 5a–5b
R1
NH2
N
N
N
O
H3C
N
R2
had IC50 values of 6.88 and 8.80
tively. However, compound 3h with 4-CH2CO2Me as R2 showed the
lowest inhibitory activity (IC50 = 101.77 M). It showed that the
lM against E. coli PDHc E1, respec-
N
R1
R2
IC50 (lM)
a
Compounds
l
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
5a
5b
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
I
4-OCH3
2-NO2
4-Cl
81.62
36.29
26.44
32.73
28.99
43.32
11.35
101.77
18.74
26.20
28.75
8.80
55.15
6.88
15.10
19.56
5.33
more bulky substituent at 4-position of benzene ring was not
favorable for the PDHc E1 inhibitory activity. We found that 4-
NO2 could significantly enhance the inhibitory activity against
the PDHc-E1. In conclusion, all the compounds with 4-NO2 in the
benzene ring displayed much higher inhibitory potency than other
compounds. The relationship between the inhibitory activity and
the functional groups in title compounds could be reasonable ex-
plained by jointly using molecular docking and MD simulation.
For instance, the recent result analyses of representative com-
pound 3n with 2-Cl-4-NO2 as R2 has revealed that the aminopy-
rimidine ring could interact with the active site of PDHc-E1 by
3-CF3
2-Cl-5-CH3
2-Cl-4-F
2,4-2NO2
4-CH2CO2Me
2,4-2Cl
3-CH3-4-Cl
4-CO2Et
4-NO2
H
2-Cl-4-NO2
4-COOH
H
strong
p–p and hydrogen bonds, and the triazole could form two
hydrogen bond with the residues Glu522 and His640, respectively.
Moreover, the nitryl group on benzene ring presented a special
case as it not only formed three hydrogen bonds with Gly231,
Asn260 and Lys392, but also coordinated with the Mg2+ in the ac-
tive site. Furthermore, it appears that the benzene group appears
I
4-NO2
a
The values were estimated statistically by origin 7.0 software using a personal
computer.
formed
p–p
stacking with the residue His106.19 Using the afore-
mentioned results, a powerful inhibitor can be designed by focus-
ing on enhancing its direct interactions with the key residues pres-
ent in the active site of PDHc-E1.
basis of the above-mentioned data, it can be conclude that the
structures of compounds 5a and 5b have been identified correctly.
13C NMR spectra of all the compounds were taken in DMSO-d6 and
the obtained signal further verified the proposed structures. All the
compounds showed a signal at d (25.2–25.4), which is due to the
presence of methyl carbon (2-methylpyrimidine). Two methylene
carbons displayed signals at 46.7–48.0 and 60.3–63.6, respectively.
The mass spectrum of all the compounds showed molecular ion
peak at m/z = [M+1], which is in agreement with the molecular
formula. The spectral values for all the compounds and C, H, N
analyses are given in the experimental part.
We have noticed that the introduction of halogens sometimes
can lead to an improved activity profile.23 Owing to the larger
van der Waals radius (with 2.15 Å) and total size (with 4.29 Å3)
of the C–I bond compared with that of C–H bond (with 1.20 Å
and 2.29 Å3, respectively),23 the introduction of iodine would be
better to satisfy the steric requirement at the receptor site. So,
the H and 4-NO2 as R2 were kept constant, while further optimiza-
tion of the design was achieved by the introduction of iodine in the
5-position of 1,2,3-triazole of compounds 3. Compounds 5a and 5b
were synthesized to investigate the effect of introduction of iodine
on the inhibitory activity of PDHc E1. As shown in Table 1, com-
3. Results and discussion
pounds 5a and 5b with I as R1 (IC50 = 19.56
respectively) showed higher enzyme inhibitory activity than that
of the corresponding compounds 3m and 3l with
as R1
(IC50 = 55.15 M and 8.80 M, respectively). Based on the results,
lM and 5.33 lM,
3.1. In vitro inhibition of E. coli PDHc E1
H
All the synthesized compounds 3a–3j and 5a–5b, including pre-
viously reported analogous compounds 3k–3o,19 were evaluated
for their inhibitory activity against PDHc E1 from Escherichia coli.
The IC50 values are summarized in Table 1. It was observed that
l
l
it can be concluded that the 5-position of 1,2,3-triazole plays an
important role in the PDHc E1 inhibitory activity. Additionally,
the inhibitory activity could be further enhanced by introducing I
in the 5-position of 1,2,3-triazole of compounds. Once again, it
was also found that 4-NO2 could significantly enhance the inhibi-
tory activity of PDHc-E1, irrespective of H or I as R1. The com-
pounds with 4-NO2 in the benzene ring displayed much higher
inhibitory potency than other compounds. Furthermore, the
compound 5b with iodine as R1 in the 5-position of 1,2,3-triazole
and with nitryl group as R2 in the 4-position of benzene ring was
found to be the best inhibitor against PDHc E1 from E. coli.
many compounds exhibited good inhibitory activity (IC50 <30 lM).
A simple structure–activity relationship analysis showed that the
PDHc E1 inhibitory potency closely related to the substituent
group R2 on the benzene ring.
It was noticed that the compounds with electron-donating
substituent as R2 on the benzene ring had very weak inhibitory
activity against PDHc E1 from Escherichia coli, while the presence
of electron-withdrawing groups as R2 slightly improved the inhib-

1668
J. He et al. / Bioorg. Med. Chem. 20 (2012) 1665–1670
Table 2
The antifungal activities of some compounds 3 and 5a–5b at 100 l
g mLꢀ1
Compounds
R1
R2
Inhibition rate (%)
G. zeaea
R. solania
B. cinereaa
A. solania
A. alternate Keisslera
C. orbicularea
3c
3g
3n
3o
3m
5a
3l
5b
H
H
H
H
H
I
4-Cl
51
48
0
21
0
59
0
68
98
23
0
31
0
73
54
44
67
53
88
37
84
96
44
0
52
0
50
42
57
42
40
45
43
35
97
2,4-2NO2
2-Cl-4-NO2
4-COOH
H
H
4-NO2
4-NO2
20
33
25
57
24
66
100
36
57
43
23
33
59
100
0
96
24
97
100
H
I
Difenoconazol
a
G. zeae, Gibberella zeae; R. solani, Rhizoctonia solani; B. cinerea, Botrytis cinerea; A. solani, Alternaria solani; A. alternate Keissler, Alternaria alternate Keissler; C. orbiculare,
Colletotrichum orbicul.
3.2. Fungicidal activity
pounds 5b will be as used a lead compound for further
optimization.
In order to examine the practicality of title compounds, some of
the compounds with higher enzyme inhibitory activity were cho-
sen from groups 3 and 5 to further evaluate their antifungal activ-
ity against G. zeae, R. solani, B. cinerea, A. solani, A. alternate Keissler,
and C. orbiculare. The results in Table 2 showed that most of the
tested compounds 3 did not display obvious fungicidal activity, ex-
cept compounds 3c and 3o, which showed fair fungicidal activity
against B. cinerea. It should be noted that the compounds 3m and
3l had weak fungicidal activity (<60% inhibitory rate) against all
5. Experimental
5.1. Chemistry
Melting points (mp) were measured on an electrothermal melt-
ing point apparatus and were uncorrected. 1H NMR and 13C NMR
spectra were recorded at 400 MHz or 600 MHz, in CDCl3 or
DMSO-d6 solution on a Varian Mercury-Plus 400 or 600 spectrom-
eter and chemical shifts were recorded in parts per million (ppm)
with TMS as the internal reference. Mass spectra (MS) were
obtained on a QTRAP LC/MS/MS system (API2000; Applied Biosys-
tems, Foster City, CA, USA), and signals were given in m/z. Elemen-
tal analysis (EA) was measured on a Vario ELIII CHNSO elemental
analyzer. Unless otherwise noted, reagents were purchased from
commercial suppliers and used without further purification. Inter-
mediate 2 was synthesized according to the existing methods.20
types of fungi at 100 l
g mLꢀ1. However the corresponding com-
pounds 5a and 5b with I as R1 in the 5-position of the 1,2,3-triazole
ring exhibited significantly better fungicidal activity than that of
compounds 3m and 3l. Compounds 5a and 5b exhibited good to
excellent fungicidal activity against B. cinerea and R. solani, espe-
cially compounds 5b also displayed obvious fungicidal activity
against G. zeae and A. solani at the dosage of 100 l
g mLꢀ1. The test
results indicated that the fungicidal activity could be significantly
enhanced by introducing iodine in the 5-position of the 1,2,3-tria-
zole ring.
5.2. General procedure for preparation of compounds 3a–3j
4. Conclusion
A solution of 3-bromopyropyne (0.71 g, 6 mmol), corresponding
substituted phenol (5 mmol) and K2CO3 (1.38 g, 10 mmol) in ace-
tone (20 mL) was heated under reflux until the reaction was com-
plete based on TLC monitoring. Then the solvent was removed
under reduced pressure. The residue was dissolved in water and
the aqueous layer was extracted twice with dichloromethane.
The combined organic phases were dried with MgSO4 and evapo-
rated at reduced pressure to obtain product, which was used
directly for the next step reaction without further purification.
To a stirred solution of 5-azidomethyl-2-methylpyrimidine-4-
ylamine 2 (1 mmol, 1.0 equiv) and substituted (prop-2-yn-1-
yloxy)benzene 1a–1j (1.1 mmol, 1.1 equiv) in tert-butanol/water
(9 mL, 2:1) were added sodium ascorbate (99 mg, 0.5 mmol) and
CuSO4ꢁ5H2O (12.5 mg, 0.05 mmol). The reaction mixture was stir-
red at room temperature for 12–24 h. It was poured into cold water
(50 mL), and the precipitate was collected by filtration and dried in
the atmospheric pressure. Recrystallization with appropriate sol-
vent afforded the desired solid compounds 3a–3j.
Based on the structure of active site of PDHc E1, where is occu-
pied by ThDP, a series of 2-methylpyrimidine-4-ylamine deriva-
tives as the analogs of ThDP were designed as E. coli PDHc E1
inhibitors and synthesized using click chemistry. SAR analyses
indicated that the inhibitory potency against E. coli PDHc E1 and
antifungal activity of title compounds could be increased greatly
by optimizing substituent groups in the compounds. All the com-
pounds with 4-NO2 in the benzene ring displayed much higher
inhibitory potency against E. coli PDHc E1 than other compounds.
Moreover, introduction of iodine into the 5-position of 1,2,3-tria-
zole of title compounds could further increased the inhibitory
activity of E. coli PDHc E1. However, the obvious antifungal activity
of title compound could be achieved by a reasonable combination
of both, the substituted benzene ring and substituted 1,2,3-triazole
ring moieties in the parent compound. The results of the activity
evaluation showed that the 5-position of the 1,2,3-triazole played
a pivotal role in both E. coli PDHc E1 inhibitory activity and
fungicidal activity. Therefore, compound 5b with I as R1 in the
5-position of 1,2,3-triazole and with 4-NO2 as R2 in the benzene
ring, acted as the best inhibitor against PDHc E1 from E. coli and
was found to be the most effective compound with higher
antifungal activity against R. solani and B. cinerea at the dosage of
5.2.1. 5-((4-((4-Methoxyphenoxy)methyl)-1H-1,2,3-triazol-1-
yl)methyl)-2-methylpyrimidin-4-amine (3a)
White solid, yield: 87%, mp 115–117 °C; 1H NMR (CDCl3,
600 MHz): d 2.30 (s, 3H, CH3), 3.77 (s, 3H, CH3), 5.13 (s, 2H,
OCH2), 5.36 (s, 2H, CH2), 5.60 (s, 2H, NH2), 6.82–6.83 (d, 2H,
J = 9.0 Hz, Ar-H), 6.89–6.90 (d, 2H, J = 8.4 Hz, Ar-H), 7.61 (s, 1H, tri-
azole CH), 8.19 (d, 1H, pyrimidine CH). 13C NMR (DMSO-d6,
100 MHz): d 25.2, 46.8, 55.4, 61.6, 108.3, 114.6, 115.7, 124.5,
100 l
g mLꢀ1. The above-mentioned results of SAR analyses will
be useful for further research on designing more potent E. coli
PDHc E1 inhibitors with higher antifungal activity, while com-

J. He et al. / Bioorg. Med. Chem. 20 (2012) 1665–1670
1669
143.1, 151.1, 152.1, 153.6, 161.5, 167.1. ESI-MS m/z: 327 (M+1)+.
5.2.7. 5-((4-((2,4-Dinitrophenoxy)methyl)-1H-1,2,3-triazol-1-
Anal. Calcd For C16H18N6O2: C, 58.88; H, 5.56; N, 25.75. Found: C,
58.95; H, 5.86; N, 25.45.
yl)methyl)-2-methylpyrimidin-4-amine (3g)
Yellow solid, yield: 50%, mp 210–211 °C; 1H NMR (DMSO-d6,
400 MHz): d 2.30 (s, 3H, CH3), 5.47 (s, 2H, OCH2), 5.52 (s, 2H,
CH2), 6.98 (s, 2H, NH2), 7.80–7.83 (d, 1H, J = 14.4 Hz, Ar-H), 8.27
(s, 1H, pyrimidine CH), 8.51–8.54 (m, 1H, Ar-H), 8.75–8.75 (d, 1H,
J = 4.2 Hz, Ar-H). 13C NMR (DMSO-d6, 150 MHz): d 25.2, 46.9,
63.6, 108.5, 116.2, 121.2, 125.3, 129.2, 138.7, 139.9, 141.0, 155.2,
155.9, 161.6, 167.3. ESI-MS m/z: 387 (M+1)+. Anal. Calcd For
5.2.2. 2-Methyl-5-((4-((2-nitrophenoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)pyrimidin-4-amine (3b)
Yellow solid, yield: 90%, mp 184–185 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 5.34 (s, 2H, OCH2), 5.46 (s, 2H,
CH2), 6.95 (s, 2H, NH2), 7.13–7.15 (t, 1H, J = 7.2 Hz, 7.8 Hz, Ar-H),
7.57–7.58 (d, 1H, J = 8.4 Hz, Ar-H), 7.65–7.68 (d, 1H, J = 7.8 Hz,
Ar-H), 7.85–7.87 (d, 1H, J = 7.8 Hz, Ar-H), 8.01 (s, 1H, triazole CH),
8.23 (s, 1H, pyrimidine CH). 13C NMR (DMSO-d6, 150 MHz): d
25.2, 46.8, 62.5, 115.1, 116.0, 120.7, 121.1, 121.5, 124.3, 124.7,
125.4, 133.8, 134.7, 139.7, 141.8, 150.6, 161.5. ESI-MS m/z: 342
(M+1)+. Anal. Calcd For C15H15N7O3: C, 52.78; H, 4.43; N, 28.73.
Found: C, 52.64; H, 4.36; N, 28.31.
C15H14N8O5: C, 46.63; H, 3.65; N, 29.01. Found: C, 46.45; H, 3.86;
N, 28.81.
5.2.8. Methyl 2-(4-((1-((4-amino-2-methylpyrimidin-5-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)acetate (3h)
White solid, yield: 85%, mp 142–143 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.30 (s, 3H, CH3), 3.59 (s, 5H, CH2 + CH3), 5.10 (s, 2H,
OCH2), 5.44 (s, 2H, CH2), 6.93 (s, 2H, NH2), 6.96–6.97 (d, 2H,
J = 8.4 Hz, Ar-H), 7.16–7.18 (d, 2H, J = 8.4 Hz, Ar-H), 8.01 (s, 1H, tri-
azole CH), 8.20 (s, 1H, pyrimidine CH). 13C NMR (DMSO-d6,
150 MHz): d 25.2, 39.9, 46.7, 51.6, 61.0, 108.2, 114.4, 114.6,
124.5, 126.6, 130.9, 142.8, 156.9, 161.5, 167.0, 171.8. ESI-MS m/z:
369 (M+1)+. Anal. Calcd For C19H22N6O3: C, 58.69; H, 5.47; N,
22.81. Found: C, 58.77; H, 5.48; N, 22.67.
5.2.3. 5-((4-((4-Chlorophenoxy)methyl)-1H-1,2,3-triazol-1-
yl)methyl)-2-methylpyrimidin-4-amine (3c)
White solid, yield: 78%, mp 168–170 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 5.13 (s, 2H, OCH2), 5.45 (s, 2H,
CH2), 6.95 (s, 2H, NH2), 7.05–7.06 (d, 2H, J = 9.0 Hz, Ar-H), 7.32–
7.34 (d, 2H, J = 9.6 Hz, Ar-H), 8.01 (s, 1H, triazole CH), 8.21 (s, 1H,
pyrimidine CH). 13C NMR (DMSO-d6, 150 MHz): d 25.3, 46.9, 61.3,
109.1, 116.5, 124.7, 129.3, 142.6, 156.2, 156.9, 161.5, 167.7. ESI-
MS m/z: 331 (M+1)+. Anal. Calcd For C15H15ClN6O: C, 54.47; H,
4.57; N, 25.41. Found: C, 54.74; H, 4.43; N, 25.17.
5.2.9. 5-((4-((2,4-Dichlorophenoxy)methyl)-1H-1,2,3-triazol-1-
yl)methyl)-2-methylpyrimidin-4-amine (3i)
Yellow solid, yield: 70%, mp 175–177 °C; 1H NMR (DMSO-d6,
600 MHz): d2.31 (s, 3H, CH3), 5.24 (s, 2H, OCH2), 5.46 (s, 2H,
CH2), 6.96 (s, 2H, NH2), 7.38 (s, 2H, Ar-H), 7.57 (s, 1H, Ar-H), 8.01
(s, 1H, triazole CH), 8.25 (s, 1H, pyrimidine CH). 13C NMR (DMSO-
d6, 100 MHz): d 25.2, 46.8, 62.3, 108.2, 115.6, 122.5, 124.8, 128.0,
129.3, 142.0, 152.5, 156.3, 161.5, 167.1. ESI-MS m/z: 365 (M+1)+.
Anal. Calcd For C15H14Cl2N6O: C, 49.33; H, 3.86; N, 23.01. Found:
C, 49.40; H, 4.20; N, 22.89.
5.2.4. 2-Methyl-5-((4-((3-(trifluoromethyl)phenoxy)methyl)-
1H-1,2,3-triazol-1-yl)methyl)pyrimidin-4-amine (3d)
White solid, yield: 89%, mp 160–161 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 5.23 (s, 2H, OCH2), 5.46 (s, 2H,
CH2), 6.96 (s, 2H, NH2), 7.30–7.31 (d, 1H, J = 7.8 Hz, Ar-H), 7.34–
7.36 (t, 2H, J = 8.4 Hz, 12.0 Hz, Ar-H), 7.52–7.53 (d, 1H, J = 7.8 Hz,
Ar-H), 8.02 (s, 1H, triazole CH), 8.24 (s, 1H, pyrimidine CH). 13C
NMR (DMSO-d6, 150 MHz): d 25.2, 46.8, 61.4, 108.2, 111.3, 117.4,
118.9, 123.1, 124.8, 124.9, 130.1, 130.3, 130.5, 130.7, 142.3,
156.4, 158.3, 161.6, 167.2. ESI-MS m/z: 365 (M+1)+. Anal. Calcd
For C16H15F3N6O: C, 52.75; H, 4.15; N, 23.07. Found: C, 52.46; H,
4.35; N, 22.94.
5.2.10. 5-((4-((4-Chloro-3-methylphenoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)-2-methylpyrimidin-4-amine (3j)
Yellow solid, yield: 74%, mp 142–143 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.28 (s, 3H, CH3), 2.31 (s, 3H, CH3), 5.11 (s, 2H,
OCH2), 5.45 (s, 2H, CH2), 6.95 (s, 2H, NH2), 6.89 (s, 1H, Ar-H),
7.03–7.04 (d, 1H, J = 3.6 Hz, Ar-H), 7.28–7.31 (d, 1H, J = 14.4 Hz,
Ar-H), 8.00 (s, 1H, triazole CH), 8.20 (s, 1H, pyrimidine CH). 13C
NMR (DMSO-d6, 100 MHz): d 19.7, 25.2, 46.7, 61.2, 113.8, 117.4,
124.6, 124.9, 129.4, 136.5, 139.9, 140.7, 142.5, 150.2, 156.7,
161.5. ESI-MS m/z: 345 (M+1)+. Anal. Calcd For C16H17ClN6O: C,
55.73; H, 4.97; N, 24.37. Found: C, 55.63; H, 4.86; N, 24.39.
5.2.5. 5-((4-((2-Chloro-5-methylphenoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)-2-methylpyrimidin-4-amine (3e)
White solid, yield: 80%, mp 184–185 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.30 (s, 3H, CH3), 2.31 (s, 3H, CH3), 5.20–5.21 (d, 2H,
J = 5.4 Hz, OCH2), 5.46–5.47 (d, 2H, J = 6.0 Hz, CH2), 6.79 (s, 1H,
Ar-H), 6.96 (s, 2H, NH2), 7.17–7.18 (d, 1H, J = 5.4 Hz, Ar-H), 7.27–
7.29 (d, 1H, J = 7.2 Hz, Ar-H), 8.03 (s, 1H, triazole CH), 8.25 (s, 1H,
pyrimidine CH). 13C NMR (DMSO-d6, 150 MHz): d 20.9, 25.2, 46.8,
61.9, 108.3, 115.0, 118.3, 122.3, 124.8, 129.5, 138.1, 142.4, 153.0,
156.3, 161.5, 167.4. ESI-MS m/z: 345 (M+1)+. Anal. Calcd For
5.3. One-pot, two-stage procedure for preparation of
compounds 5a and 5b
Terminal alkynes19 (1.5 mmol) was dissolved in THF (5 mL) and
treated with N-iodomorpholine (0.56 g, 1.65 mmol) followed by
CuI (0.014 g, 0.075 mmol). The reaction mixture was stirred at
room temperature for 60 min, after which time a fine white precip-
itate had formed. The suspension was poured onto a pad of neutral
alumina (20 mL) and the solution was collected under vacuum. The
pad was then washed three times with THF (5 mL). This solution
was charged with 5-azidomethyl-2-methylpyrimidine-4-ylamine
2 (0.25 g, 1.5 mmol), followed by TEA (0.30 g, 3 mmol) and finally
CuI (0.014 g, 0.075 mmol). The reaction mixture was stirred at
room temperature for 12 h. The volatile components were re-
moved by evaporation, and the resulting residue was suspended
in water and diethyl ether. A precipitate formed upon vigorous
stirring and was isolated by filtration, give compounds 5a and 5b
as a fine yellow powder.
C
16H17ClN6O: C, 55.73; H, 4.97; N, 24.37. Found: C, 55.41; H,
4.99; N, 24.44.
5.2.6. 5-((4-((2-Chloro-4-fluorophenoxy)methyl)-1H-1,2,3-
triazol-1-yl)methyl)-2-methylpyrimidin-4-amine (3f)
White solid, yield: 78%, mp 169–171 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 5.21 (s, 2H, OCH2), 5.46 (s, 2H,
CH2), 6.95 (s, 2H, NH2), 7.18–7.21 (t, 1H, J = 6.6 Hz, 7.8 Hz, Ar-H),
7.35–7.38 (q, 1H, J = 4.8 Hz, Ar-H), 7.43–7.44 (d, 1H, J = 6.0 Hz,
Ar-H), 8.01 (s, 1H, triazole CH), 8.23 (s, 1H, pyrimidine CH). 13C
NMR (DMSO-d6, 150 MHz): d 25.2, 46.8, 62.6, 114.5, 114.6, 115.3,
117.1, 117.3, 122.1, 122.2, 124.8, 142.2, 150.2, 155.1, 156.2,
156.7, 161.5, 167.3. ESI-MS m/z: 349 (M+1)+. Anal. Calcd For
C15H14ClFN6O: C, 51.66; H, 4.05; N, 24.10. Found: C, 51.18; H,
4.04; N, 24.02.

1670
J. He et al. / Bioorg. Med. Chem. 20 (2012) 1665–1670
5.3.1. 5-((5-Iodo-4-(phenoxymethyl)-1H-1,2,3-triazol-1-
yl)methyl)-2-methylpyrimidin-4-amine (5a)
A. alternate Keissler, and C. orbiculare and their relative inhibitory
ratio (%) had been determined by using the mycelium growth rate
method.26 A set amount of each sample was dissolved in acetone to
which a drop of emulsifier, Tween 80, was added. The solution was
then diluted in water until it reached the concentrations required.
The final concentration of the compounds in the medium was
Yellow solid, yield: 75%, mp 169–171 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.32 (s, 3H, CH3), 5.06 (s, 2H, CH2), 5.45 (s, 2H, CH2),
6.97 (s, 2H, NH2), 7.05 (s, 2H, Ar-H), 7.31 (s, 2H, Ar-H), 7.69 (s,
1H, Ar-H). 13C NMR (DMSO-d6, 150 MHz): d 25.27, 48.01, 61.37,
86.15, 107.49, 114.80, 121.16, 129.58, 147.38, 155.09, 158.16,
161.41, 166.86. ESI-MS m/z: 423 (M+1)+. Anal. Calcd For
tested at 100 l
g mLꢀ1. The solutions (1.5 mL) were mixed rapidly
with thawed potato glucose agar culture medium (9 cm) under
50 °C. The mixtures were poured into Petri dished. After the dishes
were cooled, the solidified plates were incubated with 5 mm myce-
lium disk, inverted, and incubated at 28 °C for 48 h. The mixed
medium without sample was used as the blank control. Three rep-
licates of each test were carried out. The mycelial elongation ra-
dium (mm) of fungi settlements was measured after 48 h of
culture. The growth inhibition rates were calculated with the fol-
lowing equation: I = [(C ꢀ T)/C] ꢂ 100%. Here, I is the growth inhi-
bition rate (%), C is average diameter of mycelia in the blank
control, T is the average diameter of mycelia in the presence of
those compounds. The inhibition ratio of those compounds at the
C15H15IN6O: C, 42.67; H, 3.58; N, 19.90. Found: C, 42.89; H, 3.59;
N, 19.95.
5.3.2. 5-((5-Iodo-4-((4-nitrophenoxy)methyl)-1H-1,2,3-triazol-
1-yl)methyl)-2-methylpyrimidin-4-amine (5b)
Yellow solid, yield: 74%, mp 215–217 °C; 1H NMR (DMSO-d6,
600 MHz): d 2.31 (s, 3H, CH3), 5.25 (s, 2H, OCH2), 5.47 (s, 2H,
CH2), 6.92 (s, 2H, NH2), 7.28–7.29 (d, 2H, J = 7.8 Hz, Ar-H), 8.23–
8.27 (d, 2H, J = 8.7 Hz, Ar-H). 13C NMR (DMSO-d6, 150 MHz): d
25.4, 48.0, 62.3, 86.6, 107.8, 115.3, 124.5, 125.9, 141.2, 146.5,
158.3, 161.3, 163.2. ESI-MS m/z: 468 (M+1)+. Anal. Calcd For
C15H14IN7O3: C, 38.56; H, 3.02; N, 20.99. Found: C, 38.20; H, 3.00;
dose of 100 l
g mLꢀ1 was summarized in Table 2.
N, 21.35.
Acknowledgments
5.4. Evaluation of inhibitory activity of PDHc E1
This work was supported by the National Basic Research Pro-
gram of China (No. 2010CB126100), the Natural Science Founda-
tion of China (Nos. 20772042, 21172090), the research was also
supported in part by the PCSIRT (No. IRT0953).
The expressing plasmid pMal-C2X-PDHc-E1 was transformed
into E. coli stain TB1 and inoculated in Luria–Bertani (LB) broth
containing 2% glucose and 30 mg/ml ampicillin at 37 °C until
reaching a cell density to A600 of 0.6–0.8. Then cells were induced
with a final concentration of 0.5 mM IPTG for 7 h at 25 °C before
harvesting. Purification of the fusion protein was carried out using
a MBP affinity column attached to an AKTA purifier 10 (UPC-F920,
GE Healthcare Life Sciences). The concentrations of purified pro-
teins were determined by the method of Bradford24 using bovine
serum albumin (Tiangen) as standard. The final purify (>95%) of
the sample was verified by SDS–PAGE and then the purified protein
was stored in 50 % (v/v) glycerol at ꢀ20 °C.
References and notes
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The inhibitory activities of synthesized compounds were mea-
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modified methods of Nemeria,15 and measured by monitoring
the reduction of 2,6-dichlorophenolindophenol (2,6-DCIP) at
600 nm using a microplate reader (BioTek Synergy2, USA). The to-
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tal volume of 100
7.2, 2.0 mM sodium pyruvate as substrate, 0.8 mM 2,6-DCIP,
7.1 M enzyme and different concentration of inhibitors. The reac-
tion mixtures were incubated for 3 min at 37 °C, then added differ-
ent concentration of ThDP (ranging from 0 to 200 M) to initial
lL reaction mixture contained 50 mM K3PO4, pH
l
l
reaction. To determine the inhibitor concentration of synthesized
compounds at 50% inhibition (IC50), initial rate data taken at satu-
rating substrate, fixed effectors, and systematically varied inhibitor
concentrations were fit to Hill equation, V = V0 ꢀ (V0 ꢀ V )/((IC50
/
1
I)n + 1),25 Where V, V0, and V are the velocity, maximum velocity
1
(at I = 0), and the limiting velocity (at I saturating); n is the Hill
coefficient associated with the inhibitor; and IC50 is the inhibition
concentration of synthesized compounds at 50% inhibition. All ki-
netic data were fit to the growth/sigmoidal model from origin 7.0
software. One unit of activity is defined as the amount of 2,6-DCIP
22. Koyama, M.; Ohtani, N.; Kai, F.; Moriguchi, I.; Inouye, S. J. Med. Chem. 1987, 30,
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23. Jeschke, P. Pest Manag. Sci. 2010, 66, 10.
reduced (lmol/min/mg of PDHc-E1).
24. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
25. Hines, J. K.; Kruesel, C. E.; Fromm, H. J.; Honzatko, R. B. J. Biol. Chem. 2007, 282,
24697.
5.5. Antifungal activities assay
26. Chen, N. C. Bioassay of Pesticides; Beijing Agricultural University Press: Beijing,
China, 1991. pp 161–162.
The fungicidal activities of a part of compounds 3 and 5a–5b
were tested in vitro against G. zeae, R. solani, B. cinerea, A. solani,