Chiral 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimine analogues as novel potent neonicotinoids: Synthesis, insecticidal evaluation and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2010.09.047

A new series of 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimines (4a-4x) were designed and synthesized as novel chiral neonicotinoid analogues. The single-crystal structure of 4n was further determined by X-ray diffraction, and its S configuration was confirmed. Preliminary bioassay showed that compound 4e, 4k, 4u, 4v exhibited excellent insecticidal activities at 100 mg/L, while 4k had >90% mortality at 10 mg/L, which suggested it could be used as a lead for future development. Modeling the inhibitor-nAChR complexes by molecular docking studies explained the structure-activity relationships observed in vitro, and revealed an intriguing molecular binding mode at the active site of nAChR, which raised the possibility that these analogues may arbitrate their insecticidal activity through a mechanism other than imidacloprid.

Full text of DOI:10.1016/j.ejmech.2010.09.047

European Journal of Medicinal Chemistry 46 (2011) 11e20
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chiral 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimine analogues as
novel potent neonicotinoids: Synthesis, insecticidal evaluation and molecular
docking studies
,
a
a
a
b
a
Chuanwen Sun a , Jun Zhu , Haifeng Wang , Jia Jin , Jiahua Xing , Dingrong Yang
*
a College of Life and Environment Sciences, Shanghai Normal University, No.100 Guilin Road, XuHui district Shanghai, Shanghai 200234, China
b Bioassay Department, Branch of National Pesticide R&D South Center, Hangzhou 310023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimines (4ae4x) were designed and
synthesized as novel chiral neonicotinoid analogues. The single-crystal structure of 4n was further
determined by X-ray diffraction, and its S configuration was confirmed. Preliminary bioassay showed that
compound 4e, 4k, 4u, 4v exhibited excellent insecticidal activities at 100 mg/L, while 4k had >90%
mortality at 10 mg/L, which suggested it could be used as a lead for future development. Modeling the
inhibitor-nAChR complexes by molecular docking studies explained the structureeactivity relationships
observed in vitro, and revealed an intriguing molecular binding mode at the active site of nAChR, which
raised the possibility that these analogues may arbitrate their insecticidal activity through a mechanism
other than imidacloprid.
Received 11 February 2010
Received in revised form
18 August 2010
Accepted 19 September 2010
Available online 25 September 2010
Keywords:
Neonicotinoid
Chirality
1,3,5-Hexahydrotriazine
Insecticidal activity
Molecular docking
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistance to NNs have been observed in a range of species after
frequent field applications during the past decades [18e20]. Espe-
Neonicotinoid insecticides [1] (NNs) have recently received
considerable interests from both agricultural chemistry and
medicinal fields, due to their broad spectrum of biological activities
and high selectivity for crop protection and public health. Since
imidacloprid [2] (IMI) was introduced to the market, many new
generation NNs, such as thiacloprid [3] (THIA), thiamethoxam [4]
and dinotefuran [5] are now on the market with their own prom-
inence (Fig. 1) and they account for one-fifth of the global insecti-
cide market. These NNs have agonistic effects on the nicotinic
acetylcholine receptor (nAChR) [6] and confer high selectivity
[7e9] and much lower toxicity [10,11] against mammals, birds,
aquatic life than insects, due to the differential binding affinities
with the nAChR of their neurosystem [12].
cially, recently collected strains of the whitefly exhibited much
stronger resistance to imidacloprid and thiamethoxam [21,22].
Hence, research in the design and screening for novel insecticidal
lead compounds with less resistance is a high priority.
Since structure modification of the existing NNS is one of the
most effective resistance-management tactics, we developed a new
design strategy to innovate a novel serious of neonicotinoid
analogues. As well known, electron-withdrawing groups such as
nitro play important roles in the major classes of NNs insecticides,
and the corresponding NNs showed excellent insecticidal activities
[22,23]. In addition, the hydroheterocyles and amino acid esters
have also been proved to be useful sections in the scaffold of active
insecticides, which may be involved in the recognition and inter-
actions with the insect target(s) [24e26]. Based on these reports,
a new series of chiral neonicotinoid analogues (4ae4x) were
designed and synthesized by introducing the 1,3,5-hexahydro-
triazine pharmacophore with various ethoxycarbonyl-alkyl
substituents at 5-position into the scaffolds of imidacloprid and
thiamethoxam, and their insecticidal activities against Aphis med-
icagini were evaluated. To obtain the precise three-dimensional
structural information, compound 4n was further crystallized and
its single-crystal structure was determined by X-ray diffraction. The
However, a well-recognized potential problem facing all insec-
ticides is the insects’ acquisition of resistance [13,14]. Although the
neonicotinoids have proven relatively resilient to the development
of resistance [15e17], significant increases in resistance and cross-
* Corresponding author. Fax: þ86 021 64321926.
E-mail address: willin@shnu.edu.cn (C. Sun).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.047

12
C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
Fig. 1. Structures of commercial available neonicotinoid insecticides, 4I and 4II are the target compounds based on imidacloprid and thiamethoxam, respectively.
binding activities of these analogues into the nAChR model were
also studied by molecular docking investigations, which prompted
some useful information for future design of new NNs pesticides.
3. Result and discussion
3.1. Single-crystal structure of compound 4n
To further obtain precise three-dimensional structural infor-
mation, the single-crystal structure of chiral compound 4n was
investigated by X-ray diffraction analysis (CCDC Number: 751235),
with its S configuration confirmed. As indicated in Fig. 2, the
molecules with chiral carbon atom (C10) were symmetrical to the
center of unit cell and adopted the single S configuration, which
was also supported by the value of abs_structure_Flack (ꢁ0.01).
Due to the electron-withdrawing force of nitro, the distance of N
(3)-C(7) (1.337 Å), N(2)-C(2) (1.341 Å) were remarkably shorter
than standard CeN single bond (1.47 Å) but close to C]N (1.33 Å).
The crystal data also showed that the dihedral angles made by the
triazine ring with the pyridine ring are approximate 65.99ꢀ and the
hexahydrotriazine ring of 4n was in a chair form conformation,
which makes this molecule more stable.
2. Chemistry
The route adapted for the synthesis of the target compounds
(4ae4x) is summarized in Scheme 1. Amino acid ethyl ester
hydrochlorides 2ae2l were prepared via acylation of various amino
acids 1 with dichloro sulfoxide [27], followed by alcoholysis at 70 ꢀC
with >80% yield. These amino acids included Gly,
acid, -aminopropanoic acid, -(Ala, Leu, Ile, Val, Met, Phe, Asp,
Glu), and -4-Chlorophenylglycine. Correspondingly, 5-substituted
g-aminobutanoic
b
L-a
D
1,3,5-hexahydrotriazine-2-N-nitroimines 3ae3l were synthesized
by Manich reaction with 50e60% yield [28], in the presence of Et3N.
The three components of this Manich reaction were nitroguanidine,
37% formaldehyde aqueous solution, and an excess of amino acid
ethyl ester hydrochlorides 2ae2l in the ratio of 1: 3: 1.5. The target
compounds 4ae4x were then prepared by reaction of 3ae3l,
2-chloro-5-chloromethly thiazole (Cl-Thy-CH2Cl) or 6-chloro-
3-chloromethly pyridine (Cl-Pyr-CH2Cl) and K2CO3 with catalysis
amount of CsCl at 60 ꢀC [29,30], which promoted the conversion
and selectivity. Aprotic solvent must be selected in this reaction,
since the chloro atom on the thiazolemethyl and pyridinemethyl
group may be apt to react with proton solvent. The best choice of
aprotic solvent herein is acetonitrile, because of its high polarity
and low boiling point.
3.2. Evaluation of insecticidal activities
As depicted in Table 1, most of our designed compounds
exhibited good insecticidal activities against A. medicagini and had
>60% mortality at 100 mg/L. Among all these analogues, 4k affor-
ded the best in vitro activity, and had >90% mortality at 10 mg/L,
which is comparable to that of imidacloprid and thiamethoxam.
When different substituents were introduced to the 5-position of
Scheme 1. Synthesis of 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimines (4ae4x). Reagents and conditions: (a) (1) SOCl2, 0 ꢀC; (2) EtOH, 70 ꢀC; (b) 2ae2l, HCHO, Et3N,
EtOH, 60 ꢀC; (c) 2-chloro-5-chloromethyl-thiazole and 2-chloro-5-chloromethyl-pyridine, K2CO3, CsCl, CH3CN, 60 ꢀC.

C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
13
the hexahydrotriazine ring, their insecticidal activities decreased in
the order 4u, 4v (n ¼ 2) > 4q, 4r (n ¼ 1) >4a, 4x (n ¼ 0), respec-
tively, which indicated that the parameter n was strongly related to
their insecticidal potency. However, in most cases, the substitution
of the 6-chloro-3-pyridylmethyl (Cl-Pyr-CH2) or 2-chloro-5-thia-
zolylmethyl (Cl-Thy-CH2) had no distinct influence on their insec-
ticide activities.
As for the substituent R, compounds (4e, 4f) with a long and
flexible alkyl group at
a-position exhibited excellent inhibitory
activities at 10 mg/L, whereas the corresponding compounds
substituted with benzyl (4m, 4n) showed much lower inhibitory
efficiency, which is possibly due, in part, to the relatively rigid
structure of this substituent. In addition, the introduction of
electron-withdrawing ester groups (4o) as R resulted in higher
insecticidal potency than the corresponding analogues with elec-
tron-donating groups (4a). Compared with 4b, compound 4p also
displayed increasing tendency in biological activity. Hence, the
observations herein suggest that compound 4k could be used as
a lead for further developing new neonicotinoid type products,
and the insecticidal potency of our designed analogues depends
significantly on the length and flexibility of the substituted ethyl
ester groups on the 5-position of the hexahydrotrizine ring.
Fig. 2. Molecular structure of compound 4n (Number CCDC 751235), with its single S
configuration confirmed by X-ray diffraction.
3.3. Molecular docking study
To validate observed structureeactivity relationships, molecular
docking studies of selected analogues were performed. Since the
amino acids forming the active pockets are both structurally and
functionally consistent in the diverse nAChRs and AchBPs, the
homomeric ls-AChBP (PDB ID: 2zju) was used as a structural
surrogate of the insect nAChR. Among all the modeled molecules,
compound 4k attained the highest score (data not shown) and
fitted the best in the interfacial binding pocket between the two
adjacent subunits. As illustrated in Fig. 3, seven hydrogen bonds
were found between compound 4k and the active site residues,
versus five for compound 4e, 4u and six for 4v, which was in good
agreement with their high insecticidal efficiency observed in vitro.
The binding conformation of 4k exhibited two hydrogen bonds via
its nitryl O21 and O22 with the side chain HeN of Gln55 and
phenolic hydroxy of Tyr164 (Fig. 3b), respectively. Its ethox-
ycarbonyl interacts with the phenolic hydroxy O of Tyr192 and its
thiazole N hydrogen bonds the Tyr185 side chain. Moreover, 4k also
showed the important additional H bonding interactions with
Met114 at the interface of two adjacent subunits. These distinctions
likely account for the high inhibitory potency of inhibitor 4k.
As indicated in the ligand superimposition in Fig. 4, most other
active compounds also exhibited significant hydrogen bonding
interactions with the receptor, and shared a quite similar binding
mode with 4k (colored in magenta). However, the backbones of 4j
and 4m were not nicely nestled in the subunit interfacial binding
pocket of this receptor (data not shown). Consistent with this
simulation, they had little biological activity in vitro. In summary,
the structural similarity to interactions of neonicotinoids with
other nAChRs, calculated binding affinities, and similar rank order
of complementarity scores suggest that our predicted binding
modes accurately capture the ligandereceptor interactions.
Meanwhile, the different binding mode observed herein has raised
the possibility that some analogues may arbitrate their insecticidal
activity through a mechanism other than imidacloprid.
Table 1
Insecticidal activities of 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimines
(4ae4x) against Aphis medicagini
Compd
n
R
Aryl
Concn (mg/L)a
500 100
10
4a
4b
4c
4d
4e
4f
4g
4h
4i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
2
2
0
0
e
e
CH3
CH3
(CH3)2CH
(CH3)2CH
(CH3)2CHCH2
(CH3)2CHCH2
Thy-Cl þþþþ
Pyr-Cl þþþ
Thy-Cl þþþþ
Pyr-Cl þþþþ
þþ
þþ
þ
þþ
þþ
þ
þþþ
þþ
Thy-Cl þþþþþþ þþþþþþ þþþþ
Pyr-Cl þþþþþþ þþþþ
þþþ
þ
CH3CH2(CH3)CH Thy-Cl þþþþ
þþ
CH3CH2(CH3)CH Pyr-Cl þþþþ
þþþþ
þþ
þþ
þ
CH3SCH2CH2
CH3SCH2CH2
4-CleC6H4
4-CleC6H4
C6H5CH2
Thy-Cl þþþþþ
Pyr-Cl þþþ
4j
4k
4l
þþ
ntb
Thy-Cl þþþþþþ þþþþþþ þþþþþ
Pyr-Cl þþþ
Thy-Cl þþþ
Pyr-Cl þþþ
þþ
þ
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
4x
e
þþ
nt
C6H5CH2
þþ
þ
CH2COOCH2CH3 Thy-Cl þþþþþ
CH2COOCH2CH3 Pyr-Cl þþþþþ
þþþþ
þþþ
þþþ
þþ
þþ
þþþ
þþ
þþ
H
H
Thy-Cl þþþþþþ þþþ
Pyr-Cl þþþþþþ þþþþ
CH2COOCH2CH3 Thy-Cl þþþþþ
þþþ
CH2COOCH2CH3 Pyr-Cl þþþþþþ þþþ
H
H
H
H
Thy-Cl þþþþþþ þþþþþþ þþþþ
Pyr-Cl þþþþþþ þþþþþþ þþþþ
Thy-Cl þþþþ
Pyr-Cl þþþþ
þþþ
þþþ
þþ
þþ
4. Conclusion
Imidacloprid
Thiamethoxam
Thy-Cl þþþþþþ þþþþþþ þþþþþþ
Pyr-Cl þþþþþþ þþþþþþ þþþþþþ
e
This paper describes the design and synthesis of a new series of
chiral 1,5-disubstituted 1,3,5-hexahydrotriazine-2-N-nitroimines
(4ae4x) analogues as potent NNs. Most of these compounds
a
Rating system for the mortality percentage: þþþþþþ, 100%; þþþþþ, ꢂ90%;
þþþþ, ꢂ80%; þþþ, ꢂ70%; þþ, ꢂ60%; þ, ꢂ50%; ꢁ, <50%.
b
nt ¼ not tested.

14
C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
Fig. 3. View of the binding mode of compound 4k in the binding site of ls-AChBP structural surrogate of the insect nAChR, suggested by molecular docking studies. (a) 4k is bound
into the interfacial binding pocket between the (þ)-face and (ꢁ)-face of two adjacent subunits. (b) The binding interactions between 4k and the active site residues. For clarity, only
two subunits (A and B) of the five identical subunits are extracted and shown from the homomeric AChBP pentamer, and they are colored cyan and purple, respectively. Key H-
bonds are indicated by magenta dotted lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
exhibited good insecticidal activity against A. medicagini at 100 mg/
L, while analogue 4k afforded the best in vitro activity, and had
>90% mortality at 10 mg/L. The single-crystal structure of 4n was
further determined by X-ray diffraction, with its single S configu-
ration confirmed. In addition, to further explore the structural
features important for better activity, molecular docking studies
were also performed to model the ligand-nAChR complexes and
analyze their binding interactions. The simulation demonstrated
new binding modes of 4k and other active analogues at the active
site of nAChR, which is in good agreement with their high insec-
ticidal activities observed in vitro. Further pharmacological studies
on these compounds are ongoing. Hence, this study has gained
some insight into the molecular mechanism of this new series of
neonicotinoids, which may provide some useful information, and
facilitate future receptor structure-guided design of novel insecti-
cides with less resistance.
5. Experimental section
5.1. Chemistry reagents and instruments
All reagentsobtained fromcommercial sources wereof analytical
reagent grade and used without further purification, except for
acetonitrile, which was dried by refluxing in the presence of CaH2
anddistilled priortouse. All
a-amino acids are L types, exceptfor D-p-
Chlorophenylglycine. Melting points were measured using a XT4A
Fig. 4. Superimposition of active compounds docked into nAChR. The backbone of the most potent 4k is shown in magenta, while the protein is represented as line. The atoms of
other molecules are colored as follows: O, red; N, blue; C, grey; S, yellow; Cl, light green. (For interpretation of the references to colour in this figure legend, the reader is referred to
the web version of this article.)

C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
15
microscopic melting point apparatus. IR spectra were obtained on
Nicolet5DX FT-IR spectrophotometer in the region
O) 1642 (C]N), 1367 (NO2), 1189 (CeOeC), 1101 (CeN). Anal. calcd
a
for C11H21N5O4: C45.98, H 7.37, N 24.38; found C 46.07, H 7.43, N
20
4000e400 cmꢁ1 using KBr discs. 1H NMR spectrum (CDCl3) was
recorded on a Bruker AVANCE-400 MHz with TMS as an internal
standard. Coupling constants (J values) are in Hertz. Elemental
analysis was performed with a PerkineElmer 2400 instrument.
Mass spectra (MS-ESI) were recorded on a TOF-LCT mass spec-
trometer. The single-crystal structure of compound 4n was deter-
mined on a Bruker SMART 1000 CCD diffractometer.
24.31. ESI-MS (M þ H) m/z: 288.3, [
a
]
¼ ꢁ16.70(c ¼ 0.1 g/L in
D
acetone).
5.3.4. 5-(2-Methyl-1-ethoxycarbonylbutyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3d)
White crystals, yield 60.8%, m.p.167e169 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.00 (2H, s, 2NH), 4.60e4.57 (2H, d, J ¼ 13.2 Hz, triazine-
2H), 4.50e4.47 (2H, d, J ¼ 13.2 Hz, triazine-2H), 4.19e4.10 (2H, m,
OCH2), 3.27e3.25 (1H, d, J ¼ 6.8 Hz, CHC]O), 1.44e1.41 (2H, t,
J ¼ 7.2 Hz CH2CH3), 1.28e1.25 (3H, t, J ¼ 7.2 Hz, OCH2CH3), 1.20e1.17
(1H,m, CH), 0.94e0.91 (6H, t, J ¼ 6.8 Hz, 2CH3). IR(potassium
5.2. General procedure for the synthesis of compounds 2ae2l
(exemplified by 2a)
A mixture of alanine (3.56 g, 0.04 mol) in ethanol (30 mL), with
thionyl chloride (3.6 mL) dropwise added at ꢁ5 ꢀC, was refluxed in
ethanol at 78 ꢀC under stirring within 1.5 h. Subsequently, the
solution was distilled in vacuum to afford Alanine ethyl ester
hydrochloride 2a in >80% yield. The physical constants of 2ae2l are
identical with the corresponding data in reference [24].
bromide, cmꢁ1
) n: 3424 (NeH), 2969 (CH3), 1721 (C]O) 1618 (C]
N), 1377 (NO2), 1181 (CeOeC), 1110 (CeN). Anal. calcd for
C11H21N5O4: C45.98, H 7.37, N 24.38; found C 46.04, H 7.33, N 24.42.
20
ESI-MS (M þ H) m/z: 288.5, [
a
]
¼ þ8.95(c ¼ 0.1 g/L in acetone).
D
5.3.5. 5-(3-Methylthioyl-1-ethoxycarbonylpropyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3e)
5.3. General method for preparation of compounds 3ae3l
(exemplified by 3a)
White crystals, yield 52.4%, m.p. 83e85 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.17 (2H, s, 2NH), 4.59e4.48(4H, m, triazine-4H), 4.21e4.11
(2H, m, OCH2), 3.76e3.73(1H, t, J ¼ 6.8 Hz, CHC]O), 2.56e2.52 (2H,
m, SCH2), 2.10(3H,d, J ¼ 6.8 Hz, SCH3), 2.07e2.05(2H, m, J ¼ 6.4 Hz,
CH2),1.29e1.25(3H,ddd, J ¼ 1.2 Hz,1.2 Hz, 0.8 Hz, CH3). IR (potassium
A mixture of 37% formaldehyde aqueous (3.65 g, 0.045 mol),
triethylamine (3.15 g, 0.03 mol) and Alanine ethyl ester hydrochlo-
ride 2a (3.44 g, 0.0225 mol) in ethanol were added into to a solution
of nitroguanidine (1.58 g, 0.015 mol) in 20 mL ethanol dropwise,
with stirring at 60 ꢀC for 6 h. When the mixture was cooled to room
temperature, the crude product was deposited fromthe solution and
removed by filtration. Compound 3a was recrystallized using the
solvent mixture of EtOHeH2O to afford the pure product.
bromide, cmꢁ1
) n: 3423(NeH), 2976(CH3), 1740(C]O), 1644(C]N),
1371(NO2), 1190(CeOeC), 1087 (CeN), 712(CeS). Anal. calcd for
C10H19N5O4S: C39.33, H 6.27, N 22.94; found C 39.37, H 6.35, N 22.89.
20
ESI-MS (M þ H) m/z: 306.3, [
a]
¼ ꢁ21.93(c ¼ 0.1 g/L in acetone).
D
5.3.6. 5-(1-(4-chlorophenyl)-1-ethyoxycarbonylmethyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3f)
5.3.1. 5-(1-ethoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-
White crystals, yield 58.5%, m.p.211e213 ꢀC. 1H NMR(
d, ppm,
nitroimine (3a)
CDCl3): 8.85 (2H, s, 2NH), 7.46e7.38 (4H, m, phenyl), 4.64 (1H, s,
CHC]O), 4.55e4.52 (2H, d, J ¼ 13.6 Hz, triazine-2H), 4.40e4.37
(2H, d, J ¼ 13.6 Hz, triazine-2H), 4.25e4.14 (2H, m, OCH2), 1.27e1.31
White crystals, yield 55.3%, m.p.129e131 ꢀC 1H NMR(
d, ppm,
CDCl3): 9.04 (2H, s, 2NH), 4.65e4.62 (2H, m, J ¼ 1.6 Hz, triazine-2H),
4.53e4.49 (2H, m, J ¼ 6.4 Hz, triazine-2H), 4.26e4.19 (2H, m, OCH2),
3.75e3.70(1H, m, CH), 1.48 (3H, d, J ¼ 7.2 Hz, CH3CH), 1.28e1.32
(3H, t, J ¼ 7.2 Hz, CH3); IR(potassium bromide, cmꢁ1
) n: 3438 (NeH),
3082(phenyl), 2973 (CH3), 1727(C]O) 1647 (C]N), 1342 (NO2),
1215 (CeOeC), 1112 (CeN). Anal. calcd for C13H16N5O4Cl: C 47.40, H
(3H, t, J ¼ 7.2 Hz, CH2CH3). IR (potassium bromide, cmꢁ1
) n: 3351
(NeH), 2973, 2936 (CH3), 1739 (C]O), 1586 (C]N), 1376 (NO2),
1187 (CeOeC), 1110 (CeN). Anal. calcd for C8H15N5O4:C 39.18,H 6.17,
4.83, N 19.74; found C 47.46, H 4.91, N 19.67. ESI-MS (M þ H) m/z:
20
355.4, [
a
]
¼ þ24.33(c ¼ 0.1 g/L in acetone).
D
N 28.56; found C 39.26, H 6.11,N 28.52. ESI-MS (M þ H) m/z: 245.4,
20
[
a]
¼ ꢁ12.08(c ¼ 0.1 g/L in acetone).
5.3.7. 5-(2-Phenyl-1-ethyoxycarbonylethyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3g)
D
5.3.2. 5-(2-Methyl-1-ethoxycarbonylpropyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3b)
White crystals, yield 62.6%, m.p.140e142 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.13 (2H, s, 2NH), 7.31e7.18 (5H, m, phenyl), 4.61 (4H, m,
triazine-4H),4.06e3.97 (2H, m, OCH2), 3.85e3.81 (1H, t, J ¼ 7.2 Hz,
CHC]O), 3.15e3.10 (2H, m, ph-CH2), 1.09e1.06 (3H, t, J ¼ 7.2 Hz,
White crystals, yield 58.3%, m.p.169e171 ꢀC 1H NMR(
d, ppm,
CDCl3): 9.06 (2H, s, 2NH), 4.61e4.58 (2H, m, J ¼ 1.2 Hz, triazine-2H),
4.50e4.46 (2H, m, J ¼ 1.6 Hz, triazine-2H), 4.10e4.18 (2H, m, OCH2),
3.12 (1H, J ¼ 7.8 Hz d, CHC]O), 2.15e2.06 (1H, m, CH), 1.25e1.28
(3H, t, J ¼ 6.8 Hz, CH3CH2), 1.01e1.02 (3H, d, J ¼ 6.4 Hz, CHCH3),
CH3); IR(potassium bromide, cmꢁ1
) n: 3438 (NeH), 3082 (phenyl),
2973 (CH3), 1727 (C]O) 1647 (C]N), 1342 (NO2), 1215 (CeOeC),
1112 (CeN). Anal. calcd for C14H19N5O4: C 52.33, H 5.96, N 21.79;
0.93e0.95 (3H, d, J ¼ 6.4 Hz, CHCH3). IR(potassium bromide, cmꢁ1
)
found C 52.40, H 5.91, N 21.85. ESI-MS (M þ H) m/z: 322.3,
20
n
: 3443 (NeH), 2926 (CH3), 1742 (C]O) 1642 (C]N), 1366(NO2),
[a
]
¼ ꢁ10.52(c ¼ 0.1 g/L in acetone).
D
1189(CeOeC), 1101 (CeN). Anal. calcd for C10H19N5O4: C43.95, H
7.01 N 25.63; found C 43.89, H 7.10, N 25.57. ESI-MS (M þ H) m/z:
5.3.8. 5-(1,2-diethoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-
20
274.3, [
a
]
¼ þ10.47 (c ¼ 0.1 g/L in acetone).
nitroimine (3h)
D
White crystals, yield 53.8%, m.p.119e121 ꢀC 1H NMR(
d, ppm,
5.3.3. 5-(3-Methyl-1-ethoxycarbonylbutyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3c)
CDCl3): 9.02(2H, s, 2NH), 4.58(4H, d, J ¼ 2 Hz, triazine-4H), 4.26e4.08
(4H, m, 2OCH2), 4.06e4.02 (1H, t, J ¼ 7.2 Hz, CHC]O), 2.95e2.89 (1H,
m, CH2C]O), 2.83e2.77 (1H, m, CH2C]O),1.30e1.28(3H, t, J ¼ 6.4 Hz,
White crystals, yield 63.6%, m.p.153e155 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.01 (2H, s, 2NH), 4.61e4.58 (4H, s, triazine-4H), 4.20e4.11
(2H, m, OCH2), 3.61e3.57 (1H, dd, J ¼ 6 Hz, J ¼ 6.8 Hz, CHC]O),
1.73e1.65 (2H, m, CH2), 1.45e1.41 (1H,t, J ¼ 7.2 Hz CH), 1.29e1.26
(3H, t, J ¼ 7.2 Hz, OCH2CH3), 0.96e0.93 (6H, t, J ¼ 6 Hz, 2CH3). IR
CH3),1.27e1.25 (3H, t, J¼ 6.4Hz, CH3).IR(potassiumbromide, cmꢁ1
)n:
3350 (NeH), 2983,2936 (CH3), 1721 (C]O) 1603 (C]N), 1372 (NO2),
1240 (CeOeC), 1109 (CeN). Anal. calcd for C11H19N5O6: C 41.64, H
6.04, N 22.07; found C 41.72, H 5.98, N 22.16. ESI-MS (M þ H) m/z:
20
(potassium bromide, cmꢁ1
)
n
: 3454 (NeH), 2961 (CH3), 1742 (C]
318.3, [
a
]
¼ ꢁ15.12(c ¼ 0.1 g/L in acetone).
D

16
C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
5.3.9. 5-(2-ethoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-
CH3),1.26e1.23 (3H, t, J ¼ 7.2 Hz, CH3). IR (potassium bromide, cmꢁ1
)n:
nitroimine (3i)
3282 (NeH), 2995 (thiazole), 1732 (C]O) 1584 (C]N), 1395 (NO2),
1242 (CeOeC), 1100 (CeN). Anal. calcd for C12H17ClN6O4S: C 38.25, H
White crystals, yield 64.7%, m.p.164e166 ꢀC 1H NMR(
d, ppm,
CDCl3): 9.08 (2H, s, 2NH), 4.45e4.44 (4H, d, J ¼ 2 Hz, triazine-4H),
4.20e4.15 (2H, m, OCH2), 3.11e3.08 (2H, t, J ¼ 6.4 Hz, NCH2),
2.56e2.59 (2H, t, J ¼ 6.8 Hz, CH2C]O), 1.27e1.31 (3H,t,J ¼ 7.2 Hz,
4.55, N 22.30; found C 38.21, H 4.62, N 22.36. ESI-MS (M þ H) m/z:
20
377.1, [
a
]
¼ þ28.08(c ¼ 0.1 g/L in acetone).
D
CH3). IR(potassium bromide, cmꢁ1
) n: 3398 (NeH), 2975 (CH3),1730
5.4.2. 1-(6-Chloro-3-pyridinemethyl)-5-(1-ethoxycarbonylethyl)-
(C]O) 1650 (C]N), 1381 (NO2), 1210 (CeOeC), 1089 (CeN). Anal.
calcd for C8H15N5O4: C39.18, H 6.17, N 28.56; found C 39.14, H 6.24,
N 28.60. ESI-MS (M þ H) m/z: 246.5.
1,3,5-hexahydrotriazine-2-N-nitroimine (4b)
White crystals, yield 38.2%, m.p.126e128 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.64 (1H,s, NH), 8.33e8.32 (1H, d, J ¼ 2.4 Hz, pyridine-H),
7.85e7.82 (1H,dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.38e7.36 (1H, d,
J ¼ 4.4 Hz, pyridine-H), 4.70e4.66 (1H, d, J ¼ 15.6 Hz, CH2-pyridine),
4.56e4.46 (4H, m, triazine-4H), 4.38e4.35 (1H, d, J ¼ 12.8 Hz, CH2-
pyridine), 4.22e4.04 (2H, m, OCH2), 3.55e3.50 (2H, s, CHC]O),
1.33e1.31 (3H, d, J ¼ 6.8 Hz, CH3), 1.28e1.24 (3H, t, J ¼ 7.2 Hz, CH3).
5.3.10. 5-(1,3-Diethoxycarbonyl-2-propyl)-1,3,5-
hexahydrotriazine-2-N-nitroimine (3j)
White crystals, yield 46.7%, m.p. 101e103 ꢀC 1H NMR(
d, ppm,
CDCl3): 8.99 (2H, s, 2NH), 4.59e4.50 (4H, m, triazine-4H), 4.21e4.11
(4H, m, 2OCH2), 3.64e3.61 (1H, t, J ¼ 7.2 Hz, CHC]O), 2.48e2.33
(2H, m, CH2C]O), 2.19e2.07 (2H, m, CHCH2), 1.30e1.26 (6H, ddd,
IR (potassium bromide, cmꢁ1
) n: 3132 (NeH), 2986 (pyridine), 1739
(C]O) 1584 (C]N), 1395 (NO2), 1248 (CeOeC), 1111 (CeN),. Anal.
J ¼ 1.2 Hz,1.2 Hz,1.2 Hz, CH3). IR (potassium bromide, cmꢁ1
) n: 3352
calcd for C14H19ClN6O4: C 45.35, H 5.16, N 22.67; found C 45.29, H
(NeH), 2984 (CH3), 1722 (C]O) 1612 (C]N), 1375 (NO2), 1231
(CeOeC), 1108 (CeN). Anal. calcd for C12H21N5O6: C 43.50, H 6.39, N
20
5.20, N 22.74. ESI-MS (M þ H) m/z: 371.2, [
a
]
¼ þ22.33 (c ¼ 0.1 g/L
D
in acetone).
21.14; found C 43.43, H 6.42, N 21.18. ESI-MS (M þ H) m/z: 332.4,
20
[
a]
¼ ꢁ2.82 (c ¼ 0.1 g/L in acetone).
D
5.4.3. 1-(2-Chloro-5-thiazomethyl)-5-(2-methyl-1-
ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-nitroimine(4c)
5.3.11. 5-(3-ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-
White crystals, yield 35.2%, m.p.96e98 ꢀC. 1H NMR(
d, ppm,
nitroimine (3k)
CDCl3): 9.52 (1H, s, NH), 7.46 (1H, s, thiazole-H), 4.76e4.72 (1H, d,
J ¼ 16 Hz, CH2-thiazole), 4.65 (1H, d, J ¼ 2 Hz, CH2-thiazole),
4.18e4.03 (2H, m, OCH2), 3.00e2.97 (1H, d, J ¼ 10 Hz, CHC]O),
2.02e1.96 (1H, m, CH), 1.27e1.23 (3H, t, J ¼ 7.2 Hz, CH3), 0.99e0.97
(3H, d, J ¼ 6.8 Hz, CH3), 0.92e0.91 (3H, d, J ¼ 6.4 Hz, CH3). IR
White crystals, yield 70.6%, m.p.131e133 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.06 (2H, s, 2NH), 4.42e4.41 (4H, d, J ¼ 2 Hz, triazine-4H),
4.17e4.12 (2H, m, OCH2), 2.82e2.78 (2H, t, J ¼ 7.2 Hz, NCH2),
2.43e2.39 (3H, t, J ¼ 7.2 Hz, CH2C]O), 1.92e1.85 (2H,m, CH2),
1.29e1.26 (3H, t, J ¼ 7.2 Hz, CH2CH3). IR (potassium bromide, cmꢁ1
) n:
(potassium bromide, cmꢁ1
) n: 3128 (NeH), 2926 (thiazole), 1736
3398 (NeH), 2975(CH3), 1730(C]O) 1650(C]N), 1381(NO2), 1210
(CeOeC), 1089 (CeN). Anal. calcd for C9H17N5O4: C 41.69, H 6.61, N
27.01; found C 41.75, H 6.68, N 27.14, ESI-MS (M þ H) m/z: 260.5.
(C]O), 1642 (C]N), 1395 (NO2), 1194 (CeOeC), 1106 (CeN). Anal.
calcd for C14H21ClN6O4S: C 41.53, H 5.23 N 20.76; found C 41.60, H
20
5.14, N 20.81. ESI-MS (M þ H) m/z: 405.3 [
a
]
¼ þ27.83(c ¼ 0.1 g/L
D
in acetone).
5.3.12. 5-(1-ethoxycarbonylmethyl)-1,3,5-hexahydrotriazine-2-N-
nitroimine (3l)
5.4.4. 1-(6-Chloro-3-pyridinemethyl)-5-(2-methyl-1-
Yellow crystals, yield 60.6%, m.p.185e187 ꢀC. 1H NMR (
d, ppm,
ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4d)
CDCl3): 9.15 (2H, s, 2NH), 4.74 (4H, s, 2CH2), 4.29e4.22 (2H, m,
CH2CH3), 3.61 (2H, s, NCH2C]O), 1.33e1.30 (3H, t, J ¼ 7.2 Hz,
White crystals, yield 37.5%, m.p.126e128 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.64 (1H, s, NH), 8.33 (1H, d, J ¼ 8 Hz, pyridine-H), 7.81e7.79
(1H, d, J ¼ 8 Hz, pyridine-H), 7.36e7.34 (1H, d, J ¼ 8 Hz, pyridine-H),
4.80e4.77 (1H, d, J ¼ 15.2 Hz, CH2-pyridine), 4.66e4.63 (1H, d,
J ¼ 12.4 Hz, CH2-pyridine), 4.47e4.30 (4H, m, triazine-4H), 4.18e4.04
(2H, m, OCH2), 2.98e2.96 (1H, d, J ¼ 10 Hz, CHC]O), 1.98e1.94 (1H,
m, CH),1.28e1.24 (3H, m, CH3), 0.97e0.95 (3H, d, J ¼ 6.8 Hz, CHCH3),
CH2CH3). IR (potassium bromide, cmꢁ1
) n: 3462 (NeH), 2977(CH3),
1743 (C]O) 1604 (C]N), 1371 (NO2), 1223 (CeOeC), 1108 (CeN)
cmꢁ1. Anal. calcd for C7H13N5O4: C36.36, H 5.67, N 30.29; found C
36.30, H 5.72, N 30.31. ESI-MS m/z: 232.1.
5.4. General procedure for the synthesis of analogues 4ae4x
(exemplified by 4a)
0.91e0.90 (3H, d, J ¼ 6.4 Hz, CHCH3). IR (potassium bromide, cmꢁ1
) n:
3307 (NeH), 2963 (pyridine), 1714 (C]O), 1587 (C]N), 1397 (NO2),
1186 (CeOeC), 1118 (CeN). Anal. calcd for C16H23ClN6O4: C 48.18, H
The solution of compound 3a (1.22 g, 0.005 mol) in dry aceto-
nitrile (20 mL) was mixed with K2CO3 (0.7 g, 0.005 mol) and a spot
of CsCl, and was heated to 55 ꢀC. The solution of 2-chloro-5-
chloromethyl thiazole (2.86 g, 0.017 mol) in 15 mL dry acetonitrile
was slowly added to this mixture. After stirring under 55 ꢀC for 6 h,
the mixture was filtrated and poured into water (10 mL), and
extracted with ethyl acetate (3 ꢃ 20 mL). Then it was washed with
saturation salt water and dried with anhydrous MgSO4. After
removing the solvent under vacuum, the residue was purified by
silica gel chromatography using ethyl acetate and petroleum ether
in the ratio of 1:1 as the flush to afford 4a.
5.81, N 21.07; found C 48.25, H 5.90, N 21.14. ESI-MS (M þ H) m/z:
20
399.7, [
a
]
¼ þ6.69 (c ¼ 0.1 g/L in acetone).
D
5.4.5. 1-(2-Chloro-5-thiazomethyl)-5-(3-methyl-1-
ethoxycarbonylbutyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4e)
White crystals, yield 46.2%, m.p.130e132 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.54 (1H, s, NH), 7.47 (1H, s, thiazole-H), 4.73e4.69 (1H, d,
J ¼ 15.6 Hz, CH2-thiazole), 4.61e4.57 (1H, m, CH2-thiazole),
4.55e4.47 (4H, m, triazine-4H), 4.21e4.03 (2H, m, OCH2), 3.47e3.43
(1H, m, CHC]O), 1.60e1.52 (2H, m, CH2), 1.37e1.35 (1H, m,
CH),1.28e1.24 (3H, t, J ¼ 7.2 Hz, OCH2CH3), 0.91e0.90 (3H, d,
J ¼ 2 Hz, CHCH3), 0.89e0.88 (3H, d, J ¼ 1.6 Hz, CHCH3). IR (potas-
5.4.1. 1-(2-Chloro-5-thiazolmethyl)-5-(1-ethoxycarbonylethyl)-
sium bromide, cmꢁ1
) n: 3307 (NeH), 2963 (thiazole), 1714 (C]O),
1,3,5-hexahydrotriazine-2-N-nitroimine (4a)
White crystals, yield 40.2%, m.p.129e131 ꢀC. 1H NMR(
d, ppm,
1587 (C]N), 1397 (NO2), 1186 (CeOeC), 1118 (CeN). Anal. calcd for
C15H23ClN6O4S: C 43.01, H 5.53, N 20.06; found C 42.97, H 5.59, N
CDCl3): 9.47 (1H,s, NH), 7.45 (1H, s, thiazole-H), 4.73e4.70 (2H, d,
J ¼ 15.2 Hz, CH2-thiazole), 4.61e4.46 (4H, m, triazine-4H), 4.21e4.03
(2H, m, OCH2), 3.49e3.44 (2H, s, CHC]O),1.32e1.29(3H, d,J ¼ 11.2 Hz,
20
20.10. ESI-MS (M þ H) m/z: 419.2, [
a]
¼ ꢁ4.68(c ¼ 0.1 g/L in
D
acetone).

C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
17
5.4.6. 1-(6-Chloro-3-pyridinemethyl)-5-(3-methyl-1-
7.82e7.79 (1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d,
J ¼ 8 Hz, pyridine-H), 4.77e4.73 (1H, d, J ¼ 15.2 Hz, CH2-pyridine),
4.63e4.60 (1H, m, CH2-pyridine), 4.51e4.36 (4H, m, triazine-4H),
4.22e4.03 (2H, m, OCH2), 3.66e3.63 (1H, t, J ¼ 7.2 Hz, CHC]O),
2.53e2.46 (2H, m, SCH2), 2.08 (3H, s, SCH3), 2.02e1.91 (2H, m, CH2),
1.28e1.25 (3H, t, J ¼ 7.2 Hz, OCH2CH3). IR (potassium bromide,
ethoxycarbonylbutyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4f)
White crystals, yield 50.2%, m.p.104e106 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.67 (1H, s, NH), 8.33 (1H, d, J ¼ 2 Hz, pyridine-H), 7.83e7.80
(1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d, J ¼ 8 Hz,
pyridine-H), 4.77e4.73 (1H, d, J ¼ 15.6 Hz, CH2-pyridine), 4.64e4.60
(1H, dd, J ¼ 2.4 Hz, 2.4 Hz, CH2-pyridine), 4.47e4.29 (4H, m,
triazine-4H), 4.16e4.07 (2H, m, OCH2), 3.48e3.45 (1H,m, CHC]O),
1.64e1.51 (2H, m, CH2), 1.49e1.42 (1H, m, CH), 1.28e1.24 (3H,t,
J ¼ 7.2 Hz, OCH2CH3), 0.90e0.89 (3H, d, J ¼ 2.4 Hz, CHCH3),
cmꢁ1
) n: 3394 (NeH),2980 (pyridine),1734 (C]O),1544 (C]N),1398
(NO2), 1193 (CeOeC), 1094 (CeN). Anal. calcd for C16H23ClN6O4S: C
44.60, H 5.38, N 19.50; found C 44.53, H 5.43, N 19.55. ESI-MS
20
(M þ H) m/z: 431.2, [
a]
¼ ꢁ3.73 (c ¼ 0.1 g/L in acetone).
D
0.89e0.88 (3H, d, J ¼ 2 Hz, CHCH3). IR (potassium bromide, cmꢁ1
) n:
3259 (NeH), 2961 (pyridine), 1742 (C]O), 1587 (C]N), 1397 (NO2),
1186 (CeOeC), 1105 (CeN). Anal. calcd for C17H25ClN6O4: C 49.45, H
5.4.11. 1-(2-Chloro-5-thiazomethyl)-5-(1-(4-chlorophenyl)-1-
ethyoxycarbonylmethyl)-1,3,5-hexahydrotriazine-2-N-nitroimine
(4k)
6.10, N 20.36; found C 49.52, H 6.14, N 27.35. ESI-MS (M þ H) m/z:
20
413.4, [
a
]
¼ ꢁ5.97 (c ¼ 0.1 g/L in acetone).
White crystals, yield 44.7%, m.p.226e228 ꢀC. 1H NMR(
d, ppm,
D
CDCl3): 9.41 (1H, d, J ¼ 0.8 sHz, NH), 7.37 (1H, s, thiazole-H), 7.35
(1H, s, phenyl-H), 7.30 (1H, s, phenyl-H), 7.24e7.22 (2H, d,
J ¼ 8.8 Hz, phenyl-H), 4.64e4.60 (1H, d, J ¼ 15.6 Hz, CH2-thiazole),
4.48e4.46 (1H, d, J ¼ 16 Hz, CH2-thiazole), 4.41e4.27 (4H, m,
triazine-4H), 4.09e4.05 (2H, m, OCH2), 1.59 (1H, t, J ¼ 6.8 Hz, CHC]
5.4.7. 1-(2-Chloro-5-thiazomethyl)-5-(2-methyl-1-
ethoxycarbonylbutyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4g)
White crystals, yield 45.3%, m.p.135e137 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.52 (1H, d, J ¼ 0.8 Hz, NH), 7.46 (1H, s, thiazole-H),
4.74e4.70 (1H, d, J ¼ 15.6 Hz, CH2-thiazole), 4.63e4.59 (1H, dd,
J ¼ 2 Hz, 2.4 Hz, CH2-thiazole), 4.53e4.49 (2H, m, triazine-2H),
4.43e4.39 (2H, m, triazine-2H), 4.19e4.04 (2H, m, OCH2), 3.13e3.11
(1H, d, J ¼ 9.6 Hz, CHC]O), 1.61e1.55 (2H, m, CH2), 1.27e1.24 (3H, t,
J ¼ 6.8 Hz, OCH2CH3),1.17e1.13 (1H, t, J ¼ 7.2 Hz, CH), 0.91e0.87 (6H,
O), 1.15e1.11 (3H, t, J ¼ 7.2 Hz, OCH3). IR (potassium bromide, cmꢁ1
)
n
: 3254 (NeH), 2965 (thiazole), 1740 (C]O), 1590 (C]N), 1399
(NO2), 1190 (CeOeC), 1053 (CeN). Anal. calcd for C18H19Cl2N6O4S: C
43.14, H 3.83, N 17.75; found C 43.09, H 3.89, N 17.81. ESI-MS
20
(M þ H) m/z: 473.3 [
a
]
¼ ꢁ12.75 (c ¼ 0.1 g/L in acetone).
D
m, 2CH3). IR (potassium bromide, cmꢁ1
) n: 3291 (NeH), 2972
(thiazole), 1733 (C]O), 1585 (C]N), 1395 (NO2), 1195 (CeOeC),
1046 (CeN). Anal. calcd for C15H23ClN6O4S: C 43.01, H 5.53, N 20.06;
5.4.12. 1-(6-Chloro-3-pyridinemethyl)-5-(1-(4-chlorophenyl)-1-
ethyoxycarbonylmethyl)-1,3,5-hexahydrotriazine-2-N-nitroimine
(4l)
found C 42.95, H 5.58, N 20.17. ESI-MS (M þ H) m/z: 419.1
20
[
a]
¼ þ11.53 (c ¼ 0.1 g/L in acetone).
White crystals, yield 41.5%, m.p.160e162 ꢀC. 1H NMR (
d, ppm,
D
CDCl3): 9.56 (1H, s, NH), 8.23 (1H, d, J ¼ 2.4 Hz, pyridine-H), 7.81e7.78
(1H, dd, J ¼ 2 sHz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d, J ¼ 7.6 sHz,
pyridine-H), 7.33 (1H, s, phenyl-H), 7.28 (1H, s, phenyl-H), 7.24e7.22
(2H, d, J ¼ 8.4 Hz, phenyl-H), 4.60e4.56 (1H, d, J ¼ 15.2 Hz, CH2-
pyridine), 4.51e4.48 (1H, dd, J ¼ 0.4 Hz, 0.4 Hz, CH2-pyridine),
4.45e4.23 (4H, m, triazine-4H), 4.12e4.01 (2H, m, OCH2),1.27 (1H, s,
CHC]O), 1.14e1.11 (3H, t, J ¼ 7.2 Hz, CH3). IR (potassium bromide,
5.4.8. 1-(6-Chloro-3-pyridinemethyl)-5-(2-methyl-1-
ethoxycarbonylbutyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4h)
White crystals, yield 43.9%, m.p.141e143 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.66 (1H, s, NH), 8.33e8.32 (1H, d, J ¼ 6.4 Hz, pyridine-H),
7.82e7.79 (1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d,
J ¼ 8 Hz, pyridine-H), 4.81e4.77 (1H, d, J ¼ 15.6 Hz, CH2-pyridine),
4.64e4.60 (1H, dd, J ¼ 2.4 Hz, 2.4 Hz, CH2-pyridine), 4.47e4.29
(4H, m, triazine-4H), 4.19e4.06 (2H, m, OCH2), 3.12e3.09 (1H, d,
J ¼ 9.6 Hz, CHC]O), 1.59 (3H, s, CHCH2), 1.28e1.25 (3H, t,
J ¼ 7.2 Hz, OCH2CH3), 0.89e0.85 (6H, m, 2CH3). IR (potassium
cmꢁ1
)n:3342(NeH), 2985(pyridine),1727(C]O),1545(C]N),1397
(NO2), 1193 (CeOeC), 1112 (CeN). Anal. calcd for C20H21Cl2N6O4: C
48.83, H 4.31, N 17.98; found C 48.78, H 4.38, N 18.02. ESI-MS (M þ H)
20
m/z: 467.3 [
a
]
¼ ꢁ22.50 (c ¼ 0.1 g/L in acetone).
D
bromide, cmꢁ1
) n: 3288 (NeH), 2965 (pyridine), 1732 (C]O), 1585
(C]N), 1396 (NO2), 1189 (CeOeC), 1106 (CeN). Anal. calcd for
5.4.13. 1-(2-Chloro-5-thiazomethyl)-5-(2-pheny-1-
ethyoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-nitroimine
(4m)
C17H25ClN6O4: C 49.45, H 6.10 1, N 20.36; found C 49.40, H 6.18, N
20
20.40 ESI-MS (M þ H) m/z: 413.5, [
a
]
¼ ꢁ6.48 (c ¼ 0.1 g/L in
D
acetone).
White crystals, yield 52.8%, m.p.106e108 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.50 (1H, d, J ¼ 0.8 Hz, NH), 7.46 (1H, s, thiazole-H), 7.30e7.25
(3H, m, phenyl-H), 7.11e7.09 (2H, m, phenyl-H), 4.63e4.59 (2H, m,
CH2-thiazole), 4.56e4.51 (4H, m, triazine-4H), 4.07e3.90 (2H, m,
OCH2), 3.69e3.65 (1H, m, CHC]O), 3.06e2.94 (2H, m, CH2Ph),
5.4.9. 1-(2-Chloro-5-thiazomethyl)-5-(3-meththioyl-1-
ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4i)
White crystals, yield 35.8%, m.p.89e91 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.55 (1H, d, J ¼ 0.8 Hz, NH), 7.47 (1H, s, thiazole-H),
4.72e4.68 (1H, d, J ¼ 16 Hz, CH2-thiazole), 4.62e4.57 (1H, m, CH2-
thiazole), 4.53e4.45 (4H, m, triazine-4H), 4.21e4.04 (2H, m, OCH2),
3.65e3.62 (1H, t, J ¼ 6.8 Hz, CHC]O), 2.57e2.45 (2H, m, SCH2), 2.09
(3H, s, CH3), 2.02e1.95 (2H, m, CH2), 1.28e1.25 (3H, t, J ¼ 7.2 Hz,
1.09e1.05 (3H, t, J ¼ 7.2 Hz, OCH3). IR (potassium bromide, cmꢁ1
) n:
3289 (NeH), 2962 (thiazole), 1729 (C]O), 1585 (C]N), 1396 (NO2),
1189 (CeOeC),1049 (CeN). Anal. calcd for C18H21ClN6O4S: C 47.73, H
4.67, N 18.56; found C 47.80, H 4.72, N 18.52. ESI-MS (M þ H) m/z:
20
453.6, [
a
]
¼ þ13.67 (c ¼ 0.1 g/L in acetone).
D
OCH2CH3). IR (potassium bromide, cmꢁ1
) n: 3395 (NeH), 3008
(thiazole), 1744 (C]O), 1589 (C]N), 1396 (NO2), 1197 (CeOeC),
1108 (CeN). Anal. calcd for C14H21ClN6O4S2: C 38.48, H 4.84, N
5.4.14. 1-(6-Chloro-3-pyridinemethyl)-5-(2-pheny-1-
ethyoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4n)
19.23; found C 38.39, H 4.89, N 19.31. ESI-MS (M þ H) m/z: 437.6,
White crystals, yield 50.5%, m.p.131e133 ꢀC. 1H NMR (
d, ppm,
20
[
a]
¼ þ8.48 (c ¼ 0.1 g/L in acetone).
CDCl3): 9.64 (1H, s, NH), 8.32 (1H, d, J ¼ 2 Hz, pyridine-H), 7.82e7.79
(1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.36e7.34 (1H, d, J ¼ 7.6 Hz,
pyridine-H), 7.30e7.25 (3H, m, phenyl-H), 7.12e7.10 (2H, t, J ¼ 2 Hz,
phenyl-H), 4.65e4.60 (2H, m, CH2-pyridine), 4.54e4.38 (4H, m,
triazine-4H), 4.09e3.92 (2H, m, OCH2), 3.71e3.67 (1H, m, CHC ¼ O),
3.06e2.92 (2H, m, CH2Ph), 1.11e1.08 (3H, t, J ¼ 7.2 Hz, CH3). IR
D
5.4.10. 1-(6-Chloro-3-pyridinemethyl)-5-(2-methanthioyl-1-
ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4j)
White crystals, yield 33.6%, m.p.68e70 ꢀC. 1H NMR (
d, ppm,
CDCl3): 9.68 (1H, s, NH), 8.43e8.33 (1H, d, J ¼ 2.4 Hz, pyridine-H),

18
C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
(potassium bromide, cmꢁ1
O),1545 (C]N),1392 (NO2),1185 (CeOeC),1122 (CeN),. Anal. calcdfor
C20H23ClN6O4: C 53.75, H 5.19, N 18.81; found C 53.81, H 5.23, N 18.78.
ESI-MS (M þ H) m/z:447.4, [
)
n
: 3300 (NeH), 2982 (pyridine),1715 (C]
(4H, m, 2OCH2), 3.51e4.48 (1H, m, NCH), 2.45e2.23 (2H, m, O]
CCH2), 2.11e1.97 (2H, m, CH2), 1.29e1.24 (6H, m, 2CH3). IR (potas-
sium bromide, cmꢁ1
) n: 3450 (NeH), 2930 (thiazole), 1725 (C]O),
20
a
]
¼ þ16.58 (c ¼ 0.1 g/L in acetone).
1586 (C]N), 1390 (NO2), 1185 (CeOeC), 1095 (CeN). Anal. calcd for
D
C16H23ClN6O6S:C 41.51, H5.01, N 18.15; foundC 41.48, H5.06, N 18.12.
20
5.4.15. 1-(2-Chloro-5-thiazomethyl)-5-(1,2-diethoxycarbonylethyl)-
ESI-MS (M þ H) m/z:463.2, [
a]
¼ ꢁ57.08(c ¼ 0.1 g/L in acetone).
D
1,3,5-hexahydrotriazine-2-N-nitroimine (4o)
White crystals, yield 34.8%, m.p.100e102 ꢀC. 1H NMR (
d, ppm,
5.4.20. 1-(6-Chloro-3-pyridinemethyl)-5-(1,3-diethoxycarbonyl-2-
CDCl3): 9.53 (1H, s, NH), 7.48 (1H, s, thiazole-H), 4.77e4.74 (1H, d,
J ¼ 15.6 Hz, CH2-thiazole), 4.60e4.56 (1H, dd, J ¼ 2 Hz, 3 Hz, CH2-
thiazole), 4.54e4.46 (4H, m, triazine-4H), 4.30e4.01 (4H, m,
2OCH2), 3.97e3.93 (1H, t, J ¼ 7.2 Hz, CHC ¼ O), 2.83e2.67 (2H, m,
CH2C]O), 1.28e1.27 (3H, m, CH3), 1.26e1.24 (3H, m, CH3). IR
propyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4t)
White crystals, yield 38.6%, m.p.101e103 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.67 (1H, s, NH), 8.34 (1H, d, J ¼ 2 Hz, pyridine-H), 7.82e7.79
(1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d, J ¼ 8 Hz,
pyridine-H), 4.75e4.71 (1H, d, J ¼ 11.6 Hz, CH2-pyridine), 4.61e4.57
(1H, dd, J ¼ 2 Hz, 2.4 Hz, CH2-pyridine), 4.54e4.35 (4H, m, triazine-
4H), 4.22e4.03 (4H, m, 2OCH2), 3.53e3.50 (1H, t, J ¼ 7.2 Hz, NCH),
2.41e2.28 (2H, m, CH2C]O), 2.07e1.98 (2H, m, CH2),1.28e1.25 (6H,
(potassium bromide, cmꢁ1
) n: 3423 (NeH), 2986 (thiazole), 1735
(C]O), 1584 (C]N), 1373 (NO2), 1155 (CeOeC), 1056 (CeN). Anal.
calcd for C15H21ClN6O6S: C 40.14, H 4.72, N 18.72; found C 40.20, H
20
4.65, N 18.75. ESI-MS (M þ H) m/z: 449.5, [
a
]
¼ þ11.92 (c ¼ 0.1 g/L
m, 2CH3). IR (potassium bromide, cmꢁ1
) n: 3450 (NeH), 2922
(pyridine), 1738 (C]O), 1586 (C]N), 1388 (NO2), 1182 (CeOeC),
D
in acetone).
1113 (CeN). Anal. calcd for C18H25ClN6O6: C 47.32, H 5.52, N 18.39;
20
5.4.16. 1-(6-Chloro-3-pyridinemethyl)-5-(1,2-
diethoxycarbonylethyl)-1,3,5-hexahydrotriazine-2-N-nitroimine
found C 47.29, H 5.55, N 18.35. ESI-MS (M þ H) m/z: [
a]
¼ þ4.22
D
(c ¼ 0.1 g/L in acetone).
(4p)
White crystals, yield 38.6%, m.p.109e111 ꢀC. 1H NMR(
d, ppm,
5.4.21. 1-(2-Chloro-5-thiazomethyl)-5-(3-ethoxycarbonylpropyl)-
CDCl3): 9.67 (1H, s, NH), 8.34 (1H, d, J ¼ 2.4 Hz, pyridine-H),
7.81e7.79 (1H, dd, J ¼ 2.4 Hz, 2.4 Hz, pyridine-H), 7.37e7.35 (1H, d,
J ¼ 8 Hz, pyridine-H), 4.83e4.79 (1H, d, J ¼ 11.6 Hz, CH2-pyridine),
4.61 (1H, d, J ¼ 2 Hz, CH2-pyridine), 4.58e4.38 (4H, d, J ¼ 0.8 Hz,
triazine-4H), 4.25e4.03 (4H, m, 2OCH2), 3.97e3.93 (1H, t, J ¼ 7.2 Hz,
NCH), 2.83e2.66 (2H, m, CH2C]O), 1.29e1.27 (3H, m, CH3),
1,3,5-hexahydrotriazine-2-N-nitroimine (4u)
White crystals, yield 56.4%, m.p. 127e129 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.49 (1H, s, NH), 7.47 (1H, s, thiazole-H), 4.63 (2H, s, CH2-
thiazole), 4.38e4.35 (4H, m, triazine-4H), 4.16e4.10 (2H, m, OCH2),
2.60e2.56 (2H, t, J ¼ 7.2 Hz, NCH2), 2.35e2.31 (2H, t, J ¼ 7.2 Hz,
CH2C]O), 1.77e1.73 (2H, m, CH2), 1.28e1.24 (3H, t, J ¼ 7.2 Hz,
1.26e1.25 (3H, m, CH3). IR (potassium bromide, cmꢁ1
)
n: 3424
OCH2CH3). IR (potassium bromide, cmꢁ1
) n: 3423 (NeH), 2969
(NeH), 2989 (pyridine), 1734 (C]O), 1584 (C]N), 1386 (NO2), 1178
(CeOeC), 1113 (CeN). Anal. calcd for C17H23ClN6O6: C 46.11, H 5.23,
(thiazole), 1720 (C]O), 1588 (C]N), 1375 (NO2), 1189 (CeOeC),
1055 (CeN). Anal. calcd for C13H19ClN6O4S: C 39.95, H 4.90, N 21.50;
found C 40.00, H 4.86, N 21.52. ESI-MS (M þ H) m/z:391.2.
N 18.98; found C 46.13, H 5.22, N 19.02. ESI-MS (M þ H) m/z: 443.6
20
[
a]
¼ þ13.37 (c ¼ 0.1 g/L in acetone)
D
5.4.22. 1-(6-Chloro-3-pyridinemethyl)-5-(3-
5.4.17. 1-(2-Chloro-5-thiazomethyl)-5-(2-ethoxycarbonylethyl)-
ethoxycarbonylpropyl)-1,3,5-hexahydrotriazine-2-N-nitroimine
1,3,5-hexahydrotriazine-2-N-nitroimine (4q)
(4v)
White crystals, yield 60.8%, m.p.134e136 ꢀC. 1H NMR (
d, ppm,
White crystals, yield 58.7%, m.p.70e72 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.54 (1H, d, J ¼ 0.8 Hz, NH), 7.48 (1H, s, thiazole-H), 4.64 (2H,
m, CH2-thiazole), 4.39(4H, d, J ¼ 2 Hz, triazine-4H), 4.19e4.13 (2H, m,
OCH2), 2.88e2.85 (2H, t, J ¼ 6.4 Hz, NCH2), 2.46e2.43 (2H, t, CH2C]
O), 1.03e1.26 (3H, t, J ¼ 7.2 Hz, OCH2CH3). IR (potassium bromide,
CDCl3): 9.61 (1H, s, NH), 8.33 (1H, d, J ¼ 2.4 Hz, pyridine-H),
7.81e7.79 (1H, m, 1.2 Hz, pyridine-H), 7.36e7.34 (1H, d, J ¼ 8 Hz,
pyridine-H), 4.62 (2H, s, CH2-pyridine), 4.39 (2H, d, J ¼ 0.8 Hz,
triazine-2H), 4.28 (2H, s, triazine-2H), 4.14e4.09 (2H, m, OCH2),
2.64e2.60 (2H, t, J ¼ 7.2 Hz, NCH2), 2.33e2.29 (2H, t, J ¼ 7.2 Hz,
CH2C]O), 1.75e1.68 (2H, m, CH2), 1.27e1.23 (3H, m, CH3). IR
cmꢁ1
) n: 3270(NeH), 2988 (thiazole),1716 (C]O),1585 (C]N),1396
(NO2), 1197 (CeOeC), 1047 (CeN). Anal. calcd for C12H17ClN6O4S: C
38.25, H 4.55, N 22.30;foundC 38.28, H 4.70, N 22.26. ESI-MS(M þ H)
(potassium bromide, cmꢁ1
) n: 3473 (NeH), 3083 (pyridine), 1720
20
m/z: 377.1, [
a]
¼ þ3.29 (c ¼ 0.1 g/L in acetone).
(C]O), 1544 (C]N), 1383 (NO2), 1189 (CeOeC),1121 (CeN). Anal.
calcd for C15H21ClN6O4: C 46.82, H 5.50, N 21.84; found C 46.83, H
5.53, N 21.88. ESI-MS (M þ H) m/z: 385.4.
D
5.4.18. 1-(6-Chloro-3-pyridinemethyl)-5-(2-ethoxycarbonylethyl)-
1,3,5-hexahydrotriazine-2-N-nitroimine (4r)
White crystals, yield 62.7%, m.p.157e159 ꢀC. 1H NMR(
d, ppm,
5.4.23. 1-(6-Chloro-3-thiazolmethyl)-5-(1-ethoxycarbonylmethyl)-
CDCl3): 9.59 (1H, s, NH), 8.34 (1H, d, J ¼ 2 Hz, pyridine-H), 7.83e7.81
(1H, d, J ¼ 8 Hz, pyridine-H), 4.64 (2H, s, CH2-pyridine), 4.41 (2H, s,
triazine-2H), 4.31 (2H, s, triazine-2H), 4.18e4.12 (2H, m, OCH2),
2.92e2.89 (2H, t, J ¼ 6.4 Hz, NCH2), 2.43e2.40 (2H, t, J ¼ 6.4 Hz,
CH2C]O), 1.28e1.25 (3H, t, J ¼ 6.8 Hz, CH3). IR (potassium bromide,
1,3,5-hexahydrotriazine-2-N-nitroimine (4w)
White crystals, yield 46.7%, m.p.175e177 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.51 (1H,s, NH), 7.44 (1H,s, thiazole-H), 4.61 (2H,s, CH2-
thiazole), 4.48e4.49 (4H, d, J ¼ 5.2 Hz, triazine-4H), 4.21e4.15 (2H,
m, OCH2), 3.32 (2H, s, CHC ¼ O) 1.29e1.26 (3H, t, J ¼ 7.2 Hz, CH3). IR
cmꢁ1
)
n
: 3300 (NeH), 2978 (pyridine), 1730 (C]O), 1542 (C]N),
(potassium bromide, cmꢁ1
) n: 3288 (NeH), 3000 (thiazole), 1730
1396 (NO2),1189(CeOeC),1126 (CeN). Anal. calcd for C14H19ClN6O4:
(C]O) 1587 (C]N), 1398 (NO2), 1224 (CeOeC), 1105 (CeN). Anal.
calcd for C11H15ClN6O4S: C 36.42, H 4.17, N 23.16; found C 36.40, H
4.23, N 23.19. ESI-MS m/z: 363.8.
C 45.35, H 5.16, N 22.67; found C 45.33, H 5.20, N 22.72. ESI-MS
20
(M þ H) m/z: 371.2, [
a
]
¼ ꢁ5.08 (c ¼ 0.1 g/L in acetone).
D
5.4.19. 1-(2-Chloro-5-thiazomethyl)-5-(1,3-diethoxycarbonyl-2-
propyl)-1,3,5-hexahydrotriazine-2-N-nitroimine (4s)
5.4.24. 1-(6-Chloro-3-pyridinemethyl)-5-(1-
ethoxycarbonylmethyl)- 1,3,5-hexahydrotriazine-2-N-nitroimine
White crystals, yield 42.5%, m.p.103e105 ꢀC. 1H NMR(
d,
(4x)
ppm,CDCl3): 9.53 (1H, s, NH), 7.47 (1H, s, thiazole-H), 4.68e4.60 (2H,
t, J ¼ 16 Hz, thiazole-CH2), 4.56e4.47 (4H, m, triazine-4H), 4.20e4.03
White crystals, yield 51.7%, m.p.170e172 ꢀC. 1H NMR(
d, ppm,
CDCl3): 9.66 (1H,s, NeH), 8.33e8.32 (1H, d, J ¼ 2.4 Hz, pyridine-H),

C. Sun et al. / European Journal of Medicinal Chemistry 46 (2011) 11e20
19
7.83e7.81 (1H, dd, J ¼ 2 Hz, 2.4 Hz, pyridine-H), 7.38e7.37 (1H, d,
J ¼ 4.4 Hz, pyridine-H), 4.62 (2H,s, pyridine-CH2), 4.52 (2H, s,
triazine-2H), 4.43 (2H, s, triazine-2H), 4.20e4.14 (2H, m, OCH2),
3.38 (2H, s, CH2C]O), 1.29e1.25 (3H, t, J ¼ 7.2 Hz, CH3). IR(potas-
subunits A and B, which are adjacent, were extracted from the
homopentamer for docking, and the interfacial binding pocket
between the (þ)-face and (ꢁ)-face of these two subunits was used
for modeling. The protein was prepared for docking by addition of
polar hydrogens, and subjected to a side chain conformational
search. The putative active binding site was characterized by
selecting all residues within a 12 Å radius of the original binding
substrate in the X-ray structure. All co-crystallized molecules were
deleted, and each ligand was iteratively minimized and assigned
the GasteigereHückel charges.
sium bromide, cmꢁ1
) n: 3278 (NeH), 2994 (thiazole), 1731 (C]O),
1586 (C]N), 1391 (NO2), 1215 (CeOeC), 1111 (CeN). Anal. calcd for
C13H17ClN6O4: C 43.77, H 4.80, N 23.56; found C 43.75, H 4.84, N
23.52. ESI-MS m/z: 357.8.
5.5. Crystal growth and X-ray data for crystal structure of 4n
Colorless crystal of compound 4n with approximate dimensions
of 0.16 mm ꢃ 0.12 mm ꢃ 0.10 mm, which is suitable for single-
crystal X-ray diffraction, was obtained by slowly evaporating
a solution of 4n in mixed with acetone and petroleum at 25 ꢀC. The
crystal was mounted on a glass fiber for data collection on a Bruker
Smart Apex CCD diffractometer equipped with a graphite-mono-
5.7.2. Molecular modeling and results analysing
All selected compounds were flexibly docked and investigated
for their binding activities into protein nAChR. The AMBER force
field was used to calculate a three-dimensional grid of interaction
energies for the target nAChR by the AutoGrid component of the
program, and these grids were precomputed to store the electro-
static and van der Waals values. Default values were used for all
docking parameters with 15 independent docking runs for each
ligand. Intermolecular energy, torsional free energy and intermo-
lecular hydrogen bonds were included to evaluate their binding
free energy. The docking conformations were subjected to cluster
analysis using a root mean square deviation (RMSD) tolerance of
0.5 Å. The top hits of each cluster were examined and the confor-
mations with the lowest binding energy were chosen for further
comparison and analysis. Accelrys DS visualizer 2.5[Accelrys Inc.,
San Diego, CA (2009)] was used for molecular modeling to deter-
mine the binding orientations of these analogues and their inter-
actions with the active site.
chromatic Mo K
a
radiation (
q
l
¼ 0.71073 Å). The
4-u scan mode in
the range of 1.54 ꢄ
ꢄ 26.00ꢀ and 2.29 ꢄ
q
ꢄ 25.50ꢀ at 298 K was
used. A total of 6826 reflections were collected with 5544 unique
ones (Rint ¼ 0.0546), of which 4717 with I > 2
s (I) were considered
as observed and used in the subsequent refinements. The structure
was refined by full-matrix least-squares method on F2 with
anisotropic thermal parameters for all nonhydrogen atoms.
Crystal data for 4n: space group P21/c with a ¼ 8.877(2) Å,
b ¼ 9.176(2) Å, c ¼ 13.661(2) Å, V ¼ 1068.2(4)Å3, Dc ¼ 1.386 g/cm3, F
(000) ¼ 466,
m
¼ 0.219 mmꢁ1, Z ¼ 24,.The final refinement gave
S ¼ 1.017, R ¼ 0.0504 and w ¼ 1/[
s
2(Fo2) þ (0.0996P)2]þ0.000P,
where P ¼ (Fo2 þ 2F2c)/3. The crystal was analyzed with SHELXTL-
9727 software package and the structural plots were drawn with
Otrep. Crystallographic data (excluding structure factors) for the
structure in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication nos.
CCDC 751235. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, (fax:
þ44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
Acknowledgments
This work was supported by Innovation Program of Shanghai
Municipal Education Commission (09YZ157 and ssd08013) and
Leading Academic Discipline Project of Shanghai Normal University
(DZL808). We are also grateful for the support from Key scientific
and technological project of Shanghai Science and Technology
Commission (09391912100).
5.6. Insecticidal activity assay
The insecticidal activities of compounds 4ae4x were measured
against A. medicagini according to the standard test [31] with a slight
modification. The test analogues were dissolved in DMF and serially
diluted with water containing Triton X-80 (0.1 mg/L) to get the
required concentrations. The insects were reared at 25ꢀ (ꢅ1) ꢀC, and
groups of 10 were transferred to glass Petridishes and sprayed with
the aforementioned solutions using a Potter sprayer. Assessments
weremadeafter72 hbythenumberandsizeofliveinsectsrelativeto
that in the negative control, and evaluations are based on
a percentage scale of 0e100, in which 100 total kill and 0 no activity.
The mortality rates were subjected to probit analysis. The reference
compounds were imidacloprid and thiamethoxam, while water
containing Triton X-80 (0.1 mg/L) was used as a negative control. All
experiments were carried out in three replicates according to
statistical requirements, and the results were shown in Table 1.
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