Novel 4H-1,3,4-oxadiazin-5(6H)-ones with hydrophobic and long alkyl chains: Design, synthesis, and bioactive diversity on inhibition of monoamine oxidase, chitin biosynthesis and tumor cell

Article abstract of DOI:10.1016/j.ejmech.2008.10.015

A new series of nitrogen-containing heterocycles 4H-1,3,4-oxadiazin-5(6H)-ones derivatives with hydrophobic and long chains were designed and synthesized by direct cyclization reaction of N′-alkylation substituted aroylhydrazines with chloroacetyl chloride. The preliminary assays showed that some of the compounds displayed moderate to good inhibitory activities toward monoamine oxidase (MAO) at the concentration of 10^-5-10^-3 M, and antitumor activities against human lung cancer A-549 and human prostate cancer PC-3 cell lines at μM level, which might provide new scaffold for anticancer agents. Furthermore, compounds 5i and 5m exhibited significant inhibitory activity on chitin biosynthesis, which might represent a novel class of highly potential inhibitors of chitin synthesis. Crown Copyright

Full text of DOI:10.1016/j.ejmech.2008.10.015

European Journal of Medicinal Chemistry 44 (2009) 2113–2121
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 4H-1,3,4-oxadiazin-5(6H)-ones with hydrophobic and long alkyl chains:
Design, synthesis, and bioactive diversity on inhibition of monoamine oxidase,
chitin biosynthesis and tumor cell
,
b
b
b,
a
Shao-Yong Ke a, Xu-Hong Qian a , Feng-Yi Liu , Ni Wang , Qing Yang
*
**
, Zhong Li
a Shanghai Key Laboratory of Chemical Biology, Institute of Pesticides and Pharmaceuticals, School of Pharmacy, East China University of Science and Technology,
Shanghai 200237, China
b Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, 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 nitrogen-containing heterocycles 4H-1,3,4-oxadiazin-5(6H)-ones derivatives with
hydrophobic and long chains were designed and synthesized by direct cyclization reaction of N0-alkyl-
ation substituted aroylhydrazines with chloroacetyl chloride. The preliminary assays showed that some
of the compounds displayed moderate to good inhibitory activities toward monoamine oxidase (MAO) at
the concentration of 10ꢀ5–10ꢀ3 M, and antitumor activities against human lung cancer A-549 and human
Received 25 September 2008
Received in revised form
13 October 2008
Accepted 17 October 2008
Available online 1 November 2008
prostate cancer PC-3 cell lines at
mM level, which might provide new scaffold for anticancer agents.
Furthermore, compounds 5i and 5m exhibited significant inhibitory activity on chitin biosynthesis,
which might represent a novel class of highly potential inhibitors of chitin synthesis.
Crown Copyright Ó 2008 Published by Elsevier Masson SAS. All rights reserved.
Keywords:
1,3,4-Oxadiazin-5(6H)-ones
Heterocycle
Bioactive diversity
1. Introduction
structural variants of 4H-1,3,4-oxidiazine-5(6H)-ones derivatives
and their related intermediates.
Azaheterocyclic compounds are of synthetic interest and display
extensive biological activities, which constitute an important class
of natural and unnatural products and are extremely versatile
building blocks for the manufacture of bioactive compounds in
pharmaceutical drug design and agrochemical industry [1–6]. Thus,
developing new nitrogen-containing heterocycles derivatives as
pharmaceuticals is still an important area of interest in the life
science. The interest in six-membered heterocycles with two
adjacent nitrogen atoms stems from the occurrence of saturated
and partially saturated pyridazine in biologically active compounds
[7,8]. Many pyridazine derivatives are used in various applications
as pharmaceuticals and agrochemicals, especially those with oxa-
diazines heterocycles have a diversity of biological effects such as
cardiovascular, antibacterial, antimicrobial, plant-growth regu-
lating, miticidal and nematocidal, acricidal, and insecticidal activi-
ties and monoamine oxidase (MAO) inhibition [9–19]. The
promising bioactive diversity of this class of heteroaryl compounds
urge us to synthesize and biologically evaluate a series of novel
In 2000, Gellman and co-workers [20,21] at University of
Wisconsin developed novel rigid amphiphiles for membrane
protein manipulation. It is known that membrane proteins
represent a large proportion of the proteins produced by living
organisms and perform many crucial functions including trans-
port, catalysis, photosynthesis, respiration and signal trans-
duction. Their research results showed that the hydrophobic and
long substituents in the molecules A and B (Fig. 1) were very
important moieties for the binding with proteins. Whereafter, the
researchers at FMC Corporation [22,23] developed
a High-
Throughput Screening (HTS) assay against the insect ecdysone
receptor trying to identify new, unique scaffolds at a highly
researched target site. From their results, many simple and unique
scaffolds are of great interest such as isoxazolidine-3,5-dione
derivative C and N0,N0-dibutyl-N-phenylthiourea D (Fig. 1). Both of
them possess two hydrophobic and long alkyl chains, which might
play a key role in regulating the octanol/water partition coeffi-
cient, improving chemical behavior and enhancing activities
of the molecules. Recently, during the course of drug discovery,
the researchers at Abbott Laboratories [24] have synthesized
a series of 4,4-dialkyl-1-hydroxy-3-oxo-3,4-dihydronaphthalene-
* Corresponding author.
** Corresponding author.
3-yl benzothiadiazine derivatives
E (Fig. 1), and evaluated
their activities as Hepatitis C NS5B polymerase inhibitors. The
E-mail addresses: xhqian@ecust.edu.cn (X.-H. Qian), qingyang@dlut.edu.cn
(Q. Yang).
results indicated that the representative gem-dibutyl series of
0223-5234/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.10.015

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S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
O
O
O
O
O
N
O
N
R
N
N
NH
S
N
O
N
O
R = H, Me
A
B
D
C
O
O
H
N
S
O
O
OH
N
S
S
R2
O
O
OH
N
N
H
N
H
O
O
R1
R1
E
F
Fig. 1. Representative structures containing hydrophobic alkyl chains.
compounds F showed the best overall in vitro activity and
improved pharmacokinetic behavior, these compounds with
hydrophobic and long alkyl chains also displayed interesting
bioactivity.
scope of such kind of compounds, the easy derivatives 5j–m were
obtained conveniently in a different way by replacing the linear
alkyl chains at 4-position of heterocycles with the different
substituents such as 2-CNCH2CH2 and n-BuOCOCH2CH2 groups
(Method B).
Inspired by these reports, we developed an idea to introduce
hydrophobic and long alkyl chains into the important bioactive
nitrogen-containing oxadiazinone heterocycles. Considering that
the incorporation of hydrophobic and long alkyl chains into the
heterocyclic skeleton may provide better interaction with the cell’s
microstructure and the active site of the enzymes, which lead to the
promotion of the biological activity of the compounds and extend
activity profiles, we utilized 1,3,4-oxadiazinone scaffold as proto-
type molecule and speculated that it might be able to improve on
the properties of compounds by extending the active system.
Therefore, a series of novel 4H-1,3,4-oxadiazin-5(6H)-one deriva-
tives were designed and synthesized as shown in Scheme 1, and
their antitumor activities, MAO inhibition activities and chitin
synthesis inhibition were also evaluated. The availability of a reli-
able diversity measurement makes possible a comparison of data
bases, so the aim of the present study is to evaluate the bioactive
diversity and get an insight into the effect of the conjugates of 4H-
1,3,4-oxadiazine-5(6H)-ones with hydrophobic and long alkyl
chains on the biological activities.
The various synthesized benzoates were treated with hydrazine
hydrate in ethanol to afford the corresponding hydrazides 2. It is
well known that alkylation of a hydrazide usually occurs at
different positions depending on different reaction conditions.
Generally, in neutral medium, the terminal N0-atom is alkylated
first, nevertheless in alkaline medium, the position of alkylation is
distinctly determined by the nature of the solvent, namely, aprotic
solvents such as benzene etc. are helpful to N-substitution, and in
protic solvents like ethanol, N0-substitution plays a dominant role
[25,26]. The condensation reaction (Method A in Scheme 2)
between hydrazides 2 and nonan-5-one followed by a selective
reduction of C]N double bond which was efficiently performed
with a suspension of NaBH4 in THF led exclusively to the key
intermediates N0-alkylation products 3a–i. The other four impor-
tant alkylation products 4a–d were obtained under mild reaction
conditions by classical Michael addition of appropriate hydrazides
2 to
a,b-unsaturated systems such as acrylonitrile and n-butyl
acrylate (Method B in Scheme 2). In the above-described experi-
mental conditions, all the alkylation reactions reached completion
with a very high yield. The following cyclizations of the interme-
diates N0-(nonan-5-yl)aroylhydrazides 3a–i, N0-(2-cyanoehtyl)ar-
oylhydrazides and N0-(butoxycarbonylethyl)hydrazides 4a–d were
brought about with chloroaectyl chloride in CHCl3 and following in
the presence of potassium carbonate for about 0.5–2 h to afford the
target compounds 5,6-dihydro-4H-1,3,4-oxadiazin-5-ones deriva-
tives 5a–m in excellent yields.
2. Results and discussion
2.1. Synthesis of substituted 1,3,4-oxadiazin-5(6H)-one derivatives
The synthesis and reaction conditions of the novel 4H-1,3,4-
oxadiazin-5(6H)-one derivatives containing n-butyl chains 5a–i
are outlined in Scheme 2. Furthermore, in order to broaden the
Aromatic (hetero)cycle
X
X
R
O
N
N
N
N
Hydrophobic chain
R1
R2
O
O
Oxadiazinone Bioactive Scaffold
1,3,4-oxadiazinone
heterocycle
Scheme 1. Design strategy of new 1,3,4-oxadiazinone derivatives.

S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
2115
O
H
N
CONHNH2
COOH
N
H
a and b
c and d
A
R1
R1
R1
X
X
X
1
2
3a-i
f and g
e
B
O
5a-i: R2 = ( Bu)2CH-
n
H
R1
O
N
f and g
O
2
N
H
R'
5j-l
: R = CNCH2CH2-
R1
X
N N
n
5m: R2 = BuOCOCH2CH2-
R2
X
5a-m
4a-d
Scheme 2. Reagents and conditions: a. EtOH, Conc. H2SO4; b. 5 equiv NH2NH2$H2O, reflux for 8 h; c. 1.1 equiv nonan-5-one, EtOH, reflux for 6–8 h; d. 2 equiv NaBH4, THF, r.t. to
40 ꢁC for 15 h; e. EtOH, acrylonitrile or n-butyl acrylate, at 60 ꢁC for 30–48 h; f. 1.2 equiv ClCH2COCl, CHCl3, reflux for 1 h; g. 5 equiv K2CO3, EtOH, reflux for 0.5–2 h.
2.2. Spectroscopy
activity and cytotoxicity assays. The in vitro inhibition activities
against MAO of the synthesized compounds and the related inter-
mediates were investigated by kynuramine fluorimetric assay
method [27,28]. Enzymatic assays revealed that some of the tested
compounds were weak to moderate MAO inhibitors at low
concentrations (0.08 mmol/L). The inhibition activity of some
active compound 5 and the intermediates 3 and 4 against MAO are
shown in Tables 1 and 2, respectively.
Structures of target compounds 5a–m were confirmed by their
1H and 13C NMR spectra and high-resolution electron impact
mass spectra (HR-EIMS). Their 1H NMR spectra showed distinc-
tive signals of methylene between oxygen and carbonyl in
1,3,4-oxadiazin-5(6H)-one ring, which presented a singlet at about
4.68–4.79 ppm. The multiplets at 4.57–4.70 ppm in the 1H NMR
spectra of compounds 5a–i were assigned to the methine proton
next to N atom in heterocycles as shown in the representative
spectra (Fig. 2). For compounds 5a–i, the signals that appeared in
their 1H NMR spectra in the ranges 0.88–0.89 ppm, 1.19–1.39 ppm,
1.48–1.59 ppm and 1.72–1.92 ppm were attributed to the aliphatic
protons of the dibutyl attached to the heterocycles. In particular, the
two methylenes linked to the methine presents distinctly different
signals at 1.48–1.59 ppm and 1.72–1.92 ppm, respectively, which
may be related to the phenomena of magnetically nonequivalent
protons due to the obstruction or restriction of the C–C bond free
rotation by the long aliphatic chains. The 13C NMR spectra of
compounds 5a–m indicated signals at 158.79–161.41 ppm as well as
at 147.37–148.88 ppm, corresponding to carbonyl carbon and C]N
carbon in the oxadiazinone heterocycle, respectively.
As shown in Table 1, compounds 5b, 5d, 5g, 5j take on
moderate inhibitory activity against MAO at the concentration
of 0.08 mmol/L. Particularly, the compound 5b bearing trime-
thoxyphenyl moiety exhibits better activity and the IC50 value
is up to 0.41 mmol/L, which further indicates that trimethox-
yphenyl moiety is an important active unit due to its naturally
derived characterization [29–32]. For comparison, the inter-
mediates 3 and 4 have been tested for the inhibition activity.
On the basis of the results in Table 2, the intermediate 4a
exhibits better inhibitory activity against MAO compared with
the other intermediates 3b–i, the IC50 is up to 0.24 mmol/L.
Furthermore, from Tables 1 and 2, it can be figured that the
cyclization products 5b, 5d, 5g have better activities than the
corresponding intermediates 3b, 3d, 3g, respectively, which
demonstrates that the 4H-1,3,4-oxadiazin-5(6H)-one derivatives
containing hydrophobic and long chains may be developed as
potential lead compounds for optimization of novel MAO
inhibitors that are useful not only in the treatment of neuro-
degenerative diseases (MAOI-B), but also for affective disorders
(MAOI-A).
2.3. Biological activity evaluation
2.3.1. MAO inhibitory activity
The prepared compounds were submitted to the Chinese
National Center for Drug Screening for in vitro MAO inhibitory
Fig. 2. Representative 1H NMR spectral analysis of compound 5e.

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S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
Table 1
Table 3
In vitro inhibition activity of some active 4H-1,3,4-oxadiazin-5(6H)-one derivatives 5
Cytotoxicities of some target compounds 5 against cell lines of A-549 and PC-3.
against MAO by kynuramine fluorimetric assay.
Entry Compound Substituents
no.
Growth-inhibitory
Entry Compound Substituents
no.
Inhibitions against
MAO (%) (mmol/L)
a
properties IC50
(m mol/L)
R1
R2
A-549b
PC-3c
R1
R2
0.008 0.08
0.8
IC50
a
1
2
3
4
5b
5d
5g
5j
3,4,5-Trimethoxy Nonan-5-yl
3,4-Tetramethine Nonan-5-yl
p-Chloro
p-Ethyl
9.91
>100
24.79
14.42
42.45
NDd
1
2
3
4
5b
5d
5g
5j
3,4,5-Trimethoxy Nonan-5-yl
3,4-Tetramethine Nonan-5-yl
p-Chloro
p-Ethyl
14.04 23.54 61.36 0.41
12.84 20.32 54.59 0.60
11.15 28.18 58.79 0.42
Nonan-5-yl
Nonan-5-yl
2-Cyanoethyl >100
>100
2-Cyanoethyl 9.73 25.49 59.68 0.43
a
b
c
IC50 – 50% Inhibition concentration.
Human lung cancer cell line.
Human prostate cancer cell line.
ND – Not determination.
a
IC50 – 50% inhibition concentration.
d
2.3.2. Antitumor activity evaluation
The 4H-1,3,4-oxadiazin-5(6H)-one heterocyclic derivatives
5
2.3.3. Chitin synthesis inhibitory activity
were further evaluated for their in vitro cytotoxicity effects against
human lung cancer cell A-549 and human prostate cancer cell PC-3
using sulforhodamine B (SRB) dye-staining assay method [33]. Some
of the results are summarized in Table 3. The IC50 value represents
The chitin synthesis inhibition activities of the synthesized
compounds were estimated using yeast Saccharomyces cerevisiae
cell extracts by a non-radioactive chitin synthase assay according to
a modified procedure described by Lecuro et al. [34]. Fig. 3 indicates
the enzymatic activities in the presence of compounds 5a–m.
The inhibitory activities against chitin synthesis were confirmed
by control with nikkomycin Z (NZ) and diflubenzuron (DFB) to
compare the potency of our compounds 5a–m. As shown in Fig. 3,
all the synthesized 4H-1,3,4-oxadiazin-5(6H)-one derivatives 5a–m
displayed moderate to good inhibition activities on chitin synthesis
the drug concentration (mM) requires to inhibit cell growth by 50%.
In general, some of the new derivatives showed medium cyto-
toxic activity against A-549 and PC-3 cell lines. Among them, 4H-
1,3,4-oxadiazin-5(6H)-one containing trimethoxyphenyl moiety 5b
represents most potential growth inhibitory activity against A-549
and PC-3 cell lines with IC50 values of 9.91 and 14.42 mM, respec-
tively. Also, the cytotoxicities of compound 5d exhibited selectivity
for a special human prostate cancer cell type, the inhibition against
PC-3 cell was obviously higher than A-549 cell. The results of
preliminary antitumor assay indicated the heterocyclic molecule
containing trimethoxyphenyl group might be an active scaffold,
which further confirmed the compound 5b should be a potential
lead molecule for discovery of N-heterocyclic derivatives as
potential drugs.
at 250
exhibited significant inhibitory effect on enzyme activity compared
with NZ up to 98% and 95% at 250 M, respectively. However,
mM concentration, especially compounds 5i and 5m
m
compound DFB produced almost no inhibition of chitin synthesis in
this system. Compound 5i bearing alkoxy-substituted pyridine ring
exhibited obvious chitin synthesis inhibition, which might be due
to the hydrophobic and long alkyl chains, e.g. dibutyl side chains
increasing the lipophilicity of molecule, and the ether group in the
structure possesses hydrophilicity as well, the double effects
resulted in a better lipid–water partition coefficients of molecule 5i.
Additionally, compound 5m containing n-BuOCOCH2CH2 moiety
also showed a strong inhibition effect on chitin synthesis, however,
the presence of cyanoethyl group attached to oxadiazinone
heterocycle (5j and 5k) decreased the inhibitory activity relative to
the other analogues. Comparison of compounds 5e and 5j, the
aromatic rings at C-2 of oxadiazinone scaffold are the same, but the
different alkyl-substituents attached to the nitrogen atom of oxa-
diazinone heterocycle lead to the obviously different inhibition
activity, which further reveals that the hydrophobic and long alkyl
chains are superior to the cyanoethyl substituent.
Table 2
In vitro inhibition activity of some active intermediates 3 and 4 against MAO by
kynuramine fluorimetric assay.
In addition, we introduced various electron-withdrawing
groups (such as chlorine atoms) and electron-donating substitu-
ents (Me, MeO, Et, etc.) into the aromatic ring for exploring the
influence of structural changes on activity. As described in Fig. 3,
the different substituents at the periphery of the molecules 5a–i led
to the obviously different inhibition activities. Among all the
compounds containing electron-withdrawing groups, the
compound containing o-chloro-substituent 5f was the comparably
highest potent, which led to 86% inhibition. Nevertheless, position
changes of the chloro-substituent within the same ring
(compounds 5f, 5g and 5h) dramatically changed the inhibition
activity, suggesting that the presence of a group in the ortho-
position of benzene ring attached to oxadiazinone heterocycle
Entry Compound Substituents
no.
Inhibitions against
MAO (%) (mmol/L)
R1
R2
0.01 0.1
1
IC50
a
X
1
2
3
4
5
6
7
8
3b
3c
3d
3f
3g
3h
3i
C
C
C
C
C
C
N
C
3,4,5-Trimethoxy Nonan-5-yl
3,4-Dioxomethene Nonan-5-yl
3,4-Tetramethine Nonan-5-yl
o-Chloro
p-Chloro
2,4-Dichloro
o-Ethoxy
p-Ethyl
4.67 8.16 35.53 2.30
b
0.00 13.77 62.82
0.00 8.47 33.15
6.40 5.13 14.21
7.52 2.71 28.56 2.61
9.33 3.52 10.51
0.00 6.09 21.70
–
–
–
Nonan-5-yl
Nonan-5-yl
Nonan-5-yl
Nonan-5-yl
–
–
4a
2-Cyanoethyl 2.80 36.98 71.18 0.24
a
IC50 – 50% Inhibition concentration.
–: Not determination.
b

S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
2117
R1
X
O
N
O
N
R2
120
100
80
60
40
20
0
Substituents
Compd.
250 µM
Control
X
R1
R2
No.
n
5a
5b
5c
5d
5e
5f
3,5-Me2
C
C
C
C
C
C
C
C
N
C
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
( Bu)2CH-
n
3,4,5-(MeO)3
3,4-OCH2O
3,4-(CH)4
p-Et
o-Cl
p-Cl
n
n
n
n
n
5g
n
5h
2,4-Cl2
n
5i
5j
2-EtO
( Bu)2CH-
CNCH2CH2-
CNCH2CH2-
CNCH2CH2-
p-Et
5k
C
C
C
H
a
5l
p-F
m-Me
NZ DFB 5a 5b
5c
5d 5e
5f
5g 5h
5i
5j
5k 5m
n
5m
BuOCOCH2CH2-
Compd. No.
a
- Not determination.
Fig. 3. The structures and relative activity relationships of 4H-1,3,4-oxadiazin-5(6H)-one derivatives 5a–m, and the chitin synthase activities were assayed in a total membrane
fraction from Saccharomyces cerevisiae.
could introduce important steric and electronic effects. On the
other hand, para-ethyl group bound to benzene rings in compound
5e was better for inhibitory activity than the other alkyl- or alkoxy-
substituted analogues. The drastic decrease in potency observed for
compounds 5b, 5c, and 5d can be explained by the reducing
hydrophilicity resulting from the decreasing of ether group
attached to the benzene ring. In comparison with the structures
and activities of compounds 5b, 5c and 5d, we can find the presence
of trimethoxyphenyl moiety (5b) in the molecules is more favor-
able for inhibition activities, which may have a better lipid–water
partition coefficient.
Furthermore, since compounds 5i and 5m displayed significant
inhibition against chitin synthesis compared with NZ, five serial
dilutions of them were further tested for enzyme activity. As indi-
cated in Fig. 4, the inhibitory effects on chitin synthesis of target
compounds showed obvious concentration-dependent manner.
tested compounds exhibited moderate to good inhibition activi-
ties against MAO at the dosage of 0.08 mmol Lꢀ1, and several
compounds showed good antitumor activities against A-549 and
PC-3 cell lines. Meanwhile, compounds 5i and 5m exhibited better
inhibitory activity on chitin synthesis at 250 mM level. The present
work demonstrated that the integration of 1,3,4-oxadiazin-5-ones
ring with hydrophobic and long alkyl chains showed an important
biological effect and exhibited broad activity profiles, further
modification of these structures might result in new compounds
with high lipophilicity and variously potent activities. The
preliminary structure–activity relationship established the
importance of the alkoxy-substituted pyridine ring and highly
lipophilic moieties.
4. Experimental section
4.1. Instrumentation and chemicals
3. Conclusion
All melting points (m.p.) were obtained using a Bu¨chi Melting
Point B540, and are uncorrected. 1H and 13C NMR spectra were
recorded on a Brucker AM-400 (400 MHz) spectrometer with CDCl3
as the solvent and TMS as the internal standard. Chemical shifts are
In summary, the design, synthetic approach, analytical, spec-
troscopic and biological data of 2-substituted-aryl-5,6-dihydro-4-
alkyl-4H-1,3,4-oxadiazin-5-one derivatives with hydrophobic and
long alkyl chains (5a–m) have been presented here. Some of the
reported in
d
(parts per million) values. Coupling constants nJ are
reported in Hz. High-resolution electron mass (HR-EIMS) spectra
were recorded under electron impact (70 eV) condition using
a MicroMass GCT CA 055 instrument. Analytical thin-layer chro-
matography (TLC) was carried out on precoated plates (silica gel 60
F254), and spots were visualized with ultraviolet (UV) light. All
chemicals or reagents were purchased from standard commercial
supplies. Anhydrous CHCl3 and THF were prepared by standard
methods. All other solvents and reagents were analytical reagent
and used directly without purification, except for chloroacetyl
chloride, which were distilled before use.
100
NZ
90
5i
80
5m
70
60
50
40
30
20
10
0
4.2. General synthetic procedure for N0-(nonan-5-yl)-
aroylhydrazides 3a–i
Synthesis of the intermediates aroylhydrazones: the equal
amounts of appropriate acylhydrazide (0.01 mol), nonan-5-one
(1.42 g, 0.01 mol) were refluxed in absolute ethanol (15 mL) for
several hours, which was monitored by TLC. Then the solution was
concentrated, and the condensation product hydrazone separated
out on cooling and was recrystallized from aqueous ethanol.
1
1.2
1.4
1.6
1.8
LogC
2
2.2
2.4
2.6
Fig. 4. Semi-logarithmic plot of the inhibitory activity of compounds NZ, 5i and 5m
against chitin synthesis at concentrations of 15.63, 31.25, 62.5, 125 and 250
expressed as residual activity of the enzyme.
mM,

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S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
The reduction of intermediates aroylhydrazones: sodium
(m, 4H, Ph-H), 3.07–3.13 (m, 1H, CH), 1.49–1.63 (m, 4H, CH2), 1.39–
borohydride (0.76 g, 0.02 mol) was suspended in anhydrous THF
(30 mL) under nitrogen, the above step product hydrazone
(0.01 mol) was added batch to the stirred suspension solution
under ice bath. The mixture was stirred at low temperature for
about 0.5 h. After this, the mixture was allowed to react at 35–
40 ꢁC for about 10–15 h. The excess sodium borohydride was
decomposed with 1 N hydrochloric acid under ice bath, the
mixture was extracted with dichloromethane. The organic layer
was washed with water and then brine, dried over anhydrous
sodium sulfate, and concentrated in vacuo to obtain crude prod-
ucts. The residue was purified by silica gel column chromatog-
raphy to give the pure hydrazide, the eluent was petroleum ether/
AcOEt (v/v, 6:1 / 4:1). Their physico-chemical properties and the
spectra data are as follows.
1.45 (m, 8H, CH2), 0.93 (t, J ¼ 7 Hz, 6H, CH3); 13C NMR (100 MHz,
CDCl3):
d
¼ 165.76, 133.23, 131.64, 131.07, 130.31, 130.29, 127.09,
60.55, 31.96, 27.74, 22.94, 13.99; MS: m/z ¼ 296 (Mþ), 239, 139, 111.
4.2.7. 4-Chloro-N0-(nonan-5-yl)benzohydrazide (3g)
This compound was obtained as white solid following the above
method, yield 86%, m.p. 99.8–102.2 ꢁC; 1H NMR (CDCl3):
d
¼ 7.42–
7.82 (q, 4H, Ar-H), 4.69 (s, 1H, NH), 3.09–3.12 (m, 1H, CH), 1.34–1.59
(m, 12H, CH2), 0.92 (t, J ¼ 7 Hz, 6H, CH3); 13C NMR (100 MHz,
CDCl3):
d
¼ 166.10, 138.83, 129.75, 129.27, 129.03, 128.84, 126.93,
61.74, 31.04, 27.55, 22.77, 13.91; MS: m/z ¼ 296 (Mþ), 239, 156,
139, 111.
4.2.8. 2,4-Dichloro-N0-(nonan-5-yl)benzohydrazide (3h)
This compound was obtained as white solid following the
4.2.1. 3,5-Dimethyl-N0-(nonan-5-yl)benzohydrazide (3a)
above method, yield 84%, m.p. 92.6–93.4 ꢁC; 1H NMR (CDCl3):
This compound was obtained as pale yellow liquid following the
d
¼ 7.43–7.62 (m, 2H, Ph-H), 7.31–7.35 (m,1H, Ph-H), 2.92–2.97 (m,
above method, yield 82%; 1H NMR (CDCl3):
d
¼ 7.36 (s, 2H, Ph-H),
1H, CH), 1.25–1.47 (m, 12H, CH2), 0.92 (t, J ¼ 6.8 Hz, 6H, CH3); 13C
7.15 (s,1H, Ph-H), 4.63 (s,1H, NH), 2.89–2.94 (m,1H, CH), 2.37 (s, 6H,
NMR (100 MHz, CDCl3):
d
¼ 164.75, 137.18, 131.77, 131.42, 130.14,
Ph-CH3), 1.25–1.54 (m, 12H, CH2), 0.92 (t, J ¼ 6.8 Hz, 6H, CH3); 13C
127.59, 60.33, 32.17, 27.75, 22.98, 14.04; MS: m/z ¼ 330 (Mþ), 273,
NMR (100 MHz, CDCl3):
d
¼ 167.59, 138.39, 133.36, 132.92, 124.82,
190, 173, 142, 111, 84.
124.58, 60.38, 32.25, 27.86, 22.99, 21.22, 14.05; MS: m/z ¼ 290 (Mþ),
233, 190, 150, 133, 105.
4.2.9. 2-Ethoxy-N0-(nonan-5-yl)nicotinohydrazide (3i)
This compound was obtained as white powder following the
4.2.2. 3,4,5-Trimethoxy-N0-(nonan-5-yl)benzohydrazide (3b)
This compound was obtained as white powder following the
above method, yield 88%, m.p. 60.2–61.9 ꢁC; 1H NMR (CDCl3):
above method, yield 75%, m.p. 55.6–56.2 ꢁC; 1H NMR (CDCl3):
d
¼ 9.31 (s, 1H, N–H), 8.51 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.2 Hz, 1H, Py-H),
8.26 (dd, 3J ¼ 7.6 Hz, 4J ¼ 1.2 Hz, 1H, Py-H), 7.06 (dd, 3J ¼ 5 Hz,
4J ¼ 2.4 Hz, 1H, Py-H), 4.57 (q, J ¼ 7 Hz, 2H, OCH2CH3), 2.87–2.91
(m, 1H, CH), 1.26–1.51 (m, 15H, aliphatic CH2 and OCH2CH3), 0.93
(t, J ¼ 7 Hz, 6H, aliphatic CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 6.98 (s, 2H, Ph-H), 3.89 (s, 6H, OCH3), 3.88 (s, 3H, OCH3), 2.89–
2.91 (m, 1H, CH), 1.32–1.45 (m, 12H, CH2), 0.91 (t, J ¼ 6 Hz, 6H, CH3);
13C NMR (100 MHz, CDCl3):
d
¼ 167.01, 153.31, 141.19, 128.33,
104.25, 60.90, 60.39, 56.31, 32.38, 27.88, 22.99, 14.05; MS: m/
z ¼ 352 (Mþ), 295, 211, 195.
d
¼ 162.93, 160.27, 149.60, 141.23, 117.65, 114.55, 62.97, 60.45,
32.35, 27.86, 23.02, 14.64, 14.03; MS: m/z ¼ 307 (Mþ), 250, 167, 150,
122, 94.
4.2.3. N0-(Nonan-5-yl)benzo[d][1,3]dioxole-5-carbohydrazide (3c)
This compound was obtained as white solid following the above
4.3. General synthetic procedure for aroylhydrazides 4a–d
method, yield 78%, m.p. 35.4–36.2 ꢁC; 1H NMR (CDCl3):
d
¼ 7.23–7.29
(m, 1H, Ph-H), 6.77–6.88 (m, 2H, Ph-H), 6.03 (s, 2H, OCH2O), 5.95 (s,
1H, N–H), 4.59 (s,1H, N–H), 2.85–2.90 (m,1H, CH),1.26–1.51 (m,12H,
CH2), 0.92 (t, J ¼ 7 Hz, 6H, CH3); 13C NMR (100 MHz, CDCl3):
A mixture of appropriate acylhydrazide (10 mmol) and acrylo-
nitrile or n-butyl acrylate (12.5 mmol) in EtOH (20 mL) was
refluxed for 30–48 h, which was monitored by TLC. The solvent was
removed in vacuo. The resulting crop was chromatographed,
eluting with petroleum ether/AcOEt (v/v, 4:1 / 3:1). Their phys-
ico-chemical properties and the spectra data are as follows.
d
¼ 166.69, 150.57, 148.04, 127.11, 121.47, 120.46, 108.15, 101.71, 60.32,
32.33, 27.83, 23.00, 14.04; MS: m/z ¼ 306 (Mþ), 249, 165, 149, 121,
84, 65.
4.2.4. N0-(Nonan-5-yl)-2-naphthohydrazide (3d)
4.3.1. N0-(2-Cyanoethyl)-4-ethylbenzohydrazide (4a)
This compound was obtained as white solid following the above
This compound was obtained as white powder following the
method, yield 84%, m.p. 58.4–60.8 ꢁC; 1H NMR (CDCl3):
d
¼ 8.29 (s,
above method, yield 81%, m.p. 103.8–104.9 ꢁC; 1H NMR (400 MHz,
1H, Ar-H), 7.79–7.93 (m, 4H, Ar-H), 7.53–7.60 (m, 2H, Ar-H), 2.94–
CDCl3):
d
¼ 8.14 (bs, 1H, N–H), 7.71 (d, J ¼ 8 Hz, 2H, Ph-H), 7.26 (d,
2.99 (m, 1H, CH), 1.35–1.57 (m, 12H, CH2), 0.93 (t, J ¼ 7 Hz, 6H, CH3);
J ¼ 8 Hz, 2H, Ph-H), 4.93 (bs, 1H, N–H), 3.24 (t, J ¼ 6.2 Hz, 2H,
CH2CN), 2.70 (q, J ¼ 7.2 Hz, 2H, CH2-Ph), 2.59 (t, J ¼ 6.4 Hz, 2H, N–
CH2), 1.24 (t, J ¼ 7.8 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3):
13C NMR (100 MHz, CDCl3):
128.92, 128.62, 127.78, 127.44, 126.85, 123.24, 60.43, 32.39, 27.88,
23.02, 14.07.
d
¼ 167.33, 134.86, 132.62, 130.22,
d
¼ 168.25, 149.02, 129.50, 128.24, 127.14, 118.89, 47.46, 28.82, 17.46,
15.26; MS: m/z ¼ 217 (Mþ), 177, 164, 149, 133, 105, 79.
4.2.5. 4-Ethyl-N0-(nonan-5-yl)benzohydrazide (3e)
This compound was obtained as yellow liquid following the
4.3.2. N0-(2-Cyanoethyl)-benzohydrazide (4b)
above method, yield 81%; 1H NMR (CDCl3):
d
¼ 7.68 (d, J ¼ 8.4 Hz,
This compound was obtained as white powder following the
2H, Ar-H), 7.27 (d, J ¼ 8.4 Hz, 2H, Ar-H), 4.66 (s, 1H, NH), 2.89–2.94
(m, 1H, CH), 2.70 (q, 2H, Ar-CH2), 1.35–1.46 (m, 12H, CH2), 1.26 (t,
J ¼ 7.6 Hz, 6H, Ar-CH2CH3), 0.92 (t, J ¼ 7 Hz, 6H, CH3); 13C NMR
above method, yield 76%, m.p. 120.4–121.2 ꢁC; 1H NMR (400 MHz,
CDCl3):
d
¼ 7.80 (s, 1H, N–H), 7.45–7.78 (m, 5H, Ph-H), 4.25 (bs, 1H,
N–H), 3.28 (t, J ¼ 6.4 Hz, 2H, CH2CN), 2.61 (t, J ¼ 6.4 Hz, 2H, N–CH2);
(100 MHz, CDCl3):
d
¼ 167.25, 148.45, 130.39, 128.14, 126.92, 60.35,
13C NMR (100 MHz, CDCl3):
126.98, 118.68, 47.51, 17.56.
d
¼ 156.17, 132.32, 132.11, 128.84,
32.33, 28.79, 27.85, 23.00, 15.26, 14.03; MS: m/z ¼ 290 (Mþ), 233,
133, 105, 79, 57.
4.3.3. N0-(2-Cyanoethyl)-4-fluorobenzohydrazide (4c)
4.2.6. 2-Chloro-N0-(nonan-5-yl)benzohydrazide (3f)
This compound was obtained as white solid following the above
This compound was obtained as white crystal following the above
method, yield 70%, m.p. 93.4–95.7 ꢁC; 1H NMR (400 MHz, CDCl3):
method, yield 82%, m.p. 36.2–37.5 ꢁC; 1H NMR (CDCl3):
d
¼ 7.35–7.72
d
¼ 7.91 (bs, 1H, N–H), 7.81 (q, J ¼ 8.8 Hz, 2H, Ph-H), 7.14 (t,

S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
2119
J ¼ 8.6 Hz, 2H, Ph-H), 5.06 (bs, 1H, N–H), 3.26 (t, J ¼ 6.2 Hz, 2H,
CH2CN), 2.61 (t, J ¼ 6.4 Hz, 2H, N–CH2); 13C NMR (100 MHz, CDCl3):
m/z ¼ 346 (Mþ), 289, 233, 220, 206, 175, 149, 121; EI-HRMS: calcd
for C19H26N2O4 (Mþ), 346.1893; found, 346.1893.
d
¼ 167.27, 132.32, 129.49, 129.40, 128.83, 126.98, 116.07, 115.85,
47.44, 17.62; MS: m/z ¼ 207 (Mþ), 167, 154, 123, 95.
4.4.4. 2-(Naphthalen-2-yl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5d)
4.3.4. N0-(Butoxycarbonylethyl)-3-methylbenzohydrazide (4d)
This compound was obtained as white solid following the above
This compound was obtained as pale yellow liquid following the
method, yield 78%, m.p. 70.6–72.3 ꢁC; 1H NMR (400 MHz, CDCl3):
above method, yield 83%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.78 (s,1H,
d
¼ 8.32 (s, 1H, Ar-H), 8.01 (d, J ¼ 8.8 Hz, 1H, Ar-H), 7.92 (d, J ¼ 8 Hz,
N–H), 7.59 (s, 1H, Ph-H), 7.32–7.54 (m, 3H, Ph-H), 4.11 (t, J ¼ 6.6 Hz,
2H, OCH2), 3.26 (t, J ¼ 6.4 Hz, 2H, NHCH2), 2.57 (t, J ¼ 6.4 Hz, 2H,
CH2CO), 2.40 (s, 3H, Ph-CH3), 1.58–1.65 (m, 2H, COOCH2CH2), 1.33–
1.43 (m, 2H, CH3CH2), 0.93 (t, J ¼ 7.4 Hz, 3H, CH3CH2); 13C NMR
1H, Ar-H), 7.86 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.55 (m, 2H, Ar-H), 4.79 (s,
2H, OCH2), 4.63–4.70 (m, 1H, CH), 1.82–1.92 (m, 2H, CH2), 1.57–1.63
(m, 2H, CH2),1.26–1.38 (m, 8H, CH2), 0.89 (t, J ¼ 6.6 Hz, 6H, CH3); 13
C
NMR (100 MHz, CDCl3):
d
¼ 159.11, 148.50, 134.35, 132.75, 128.79,
(100 MHz, CDCl3):
d
¼ 172.54, 167.54, 138.60, 132.65, 128.56, 127.63,
128.10, 127.75, 127.37, 126.63, 126.61, 123.43, 64.92, 54.81, 32.33,
28.39, 22.52, 14.02; MS: m/z ¼ 352 (Mþ), 295, 239, 226, 212, 181,
155, 127; EI-HRMS: calcd for C22H28N2O2 (Mþ), 352.2151; found,
352.2150.
123.78, 64.61, 47.61, 33.53, 30.60, 21.32, 19.11, 13.68; MS: m/z ¼ 278
(Mþ), 205, 163, 144, 91, 77, 65.
4.4. General synthetic procedure for 4H-1,3,4-oxadiazin-5(6H)-one
derivatives 5a–m
4.4.5. 2-(4-Ethylphenyl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5e)
This compound was obtained as yellow oil following the above
To a solution of N0-alkylaroylhydrazides (2 mmol) in anhydrous
CHCl3 (25 mL) was added chloroacetyl chloride (0.23 g, 2 mmol)
dropwise at room temperature, and the stirred mixture was
refluxed for 30 min. After cooling and evaporation of the solvent,
the resulting crop was taken up into ethanol (20 mL) and charged
with an excess of anhydrous K2CO3 (1.38 g, 10 mmol). The mixture
was stirred to reflux for 0.5–2 h and then filtered and evaporated
in vacuo. On completion of the reaction, as ascertained by TLC
analysis, the residue was chromatographed with petroleum ether/
AcOEt (v/v, 5:1 / 3:1) and recrystallized from EtOH to give
substituted 1,3,4-oxadiazin-5(6H)-one derivatives.
method, yield 76%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.80 (d, J ¼ 8 Hz,
2H, Ph-H), 7.25 (d, J ¼ 8 Hz, 2H, Ph-H), 4.70 (s, 2H, OCH2), 4.58–4.66
(m, 1H, CH), 2.70 (q, 2H, Ph-CH2), 1.76–1.86 (m, 2H, CH2), 1.50–1.57
(m, 2H, CH2), 1.22–1.36 (m, 11H, CH2 and Ph-CH2CH3), 0.88 (t,
J ¼ 6.8 Hz, 6H, CH3); 13C NMR (100 MHz, CDCl3):
¼ 159.07, 148.67,
d
147.40, 127.90, 127.48, 126.64, 64.81, 54.66, 32.29, 28.83, 28.35,
22.52, 15.40, 14.01; MS: m/z ¼ 330 (Mþ), 273, 217, 204, 190, 159, 133;
EI-HRMS: calcd for C20H30N2O2 (Mþ), 330.2307; found, 330.2307.
4.4.6. 2-(2-Chlorophenyl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5f)
This compound was obtained as yellow oil following the above
4.4.1. 2-(3,5-Dimethylphenyl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5a)
method, yield 65%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.60 (d,
J ¼ 7.6 Hz, 1H, Ph-H), 7.47 (d, J ¼ 8 Hz, 1H, Ph-H), 7.39 (t, J ¼ 7.6 Hz,
1H, Ph-H), 7.32 (t, J ¼ 7.6 Hz, 1H, Ph-H), 4.73 (s, 2H, OCH2), 4.59–
4.66 (m, 1H, CH), 1.74–1.83 (m, 2H, CH2), 1.49–1.57 (m, 2H, CH2),
1.23–1.39 (m, 8H, CH2), 0.89 (t, J ¼ 6.8 Hz, 6H, CH3); 13C NMR
This compound was obtained as yellow oil following the above
method, yield 76%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.49 (s, 2H, Ph-
H), 7.09 (s, 1H, Ph-H), 4.69 (s, 2H, OCH2), 4.59–4.66 (m, 1H, CH), 2.37
(s, 6H, Ph-CH3), 1.78–1.87 (m, 2H, CH2), 1.51–1.59 (m, 2H, CH2), 1.19–
1.37 (m, 8H, CH2), 0.88 (t, J ¼ 6.8 Hz, 6H, CH3); 13C NMR (100 MHz,
(100 MHz, CDCl3):
d
¼ 159.08, 148.47, 133.45, 131.49, 130.98, 130.72,
129.62, 126.66, 64.95, 54.75, 32.26, 28.36, 22.51, 14.03; MS: m/
z ¼ 336 (Mþ), 279, 223, 139, 111; EI-HRMS: calcd for C18H25ClN2O2
(Mþ), 336.1605; found, 336.1605.
CDCl3):
d
¼ 159.12, 148.81, 137.99, 132.45, 129.87, 129.25, 124.83,
124.32, 64.80, 54.70, 32.29, 28.38, 22.51, 21.27,14.02; MS: m/z ¼ 330
(Mþ), 273, 217, 204, 190, 159, 133, 105; EI-HRMS: calcd for
C20H30N2O2 (Mþ), 330.2307; found, 330.2307.
4.4.7. 2-(4-Chlorophenyl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5g)
4.4.2. 2-(3,4,5-Trimethoxyphenyl)-4-(nonan-5-yl)-4H-1,3,4-
oxadiazin-5(6H)-one (5b)
This compound was obtained as white solid following the above
method, yield 76%, m.p. 45.1–46.0 ꢁC; 1H NMR (400 MHz, CDCl3):
This compound was obtained as white solid following the above
d
¼ 7.81 (d, J ¼ 8.4 Hz, 2H, Ph-H), 7.39 (d, J ¼ 8.4 Hz, 2H, Ph-H), 4.72
method, yield 81%, m.p. 79.2–80.8 ꢁC; 1H NMR (400 MHz, CDCl3):
(s, 2H, OCH2), 4.59–4.66 (m, 1H, CH), 1.74–1.83 (m, 2H, CH2), 1.51–
d
¼ 7.13 (s, 2H, Ph-H), 4.71 (s, 2H, OCH2), 4.59–4.66 (m, 1H, CH), 3.91
1.56 (m, 2H, CH2), 1.23–1.32 (m, 8H, CH2), 0.88 (t, J ¼ 7 Hz, 6H, CH3);
(s, 6H, OCH3), 3.89 (s, 3H, OCH3), 1.75–1.85 (m, 2H, CH2), 1.51–1.59
13C NMR (100 MHz, CDCl3):
d
¼ 158.79, 147.48, 136.84, 128.63,
(m, 2H, CH2),1.23–1.35 (m, 8H, CH2), 0.88 (t, J ¼ 6.8 Hz, 6H, CH3); 13
C
128.53, 127.81, 64.83, 54.77, 32.27, 28.33, 22.49, 13.99; MS: m/
z ¼ 336 (Mþ), 279, 223, 139, 111; EI-HRMS: calcd for C18H25ClN2O2
(Mþ), 336.1605; found, 336.1605.
NMR (100 MHz, CDCl3):
d
¼ 158.96, 153.11, 148.23, 140.53, 125.25,
103.94, 64.89, 60.94, 56.20, 54.73, 32.33, 28.33, 22.51, 14.00; MS: m/
z ¼ 392 (Mþ), 335, 279, 266, 252, 195, 111; EI-HRMS: calcd for
C21H32N2O5 (Mþ), 392.2311; found, 392.2311.
4.4.8. 2-(2,4-Dichlorophenyl)-4-(nonan-5-yl)-4H-1,3,4-oxadiazin-
5(6H)-one (5h)
4.4.3. 2-(Benzo[d][1,3]dioxol-5-yl)-4-(nonan-5-yl)-4H-1,3,4-
oxadiazin-5(6H)-one (5c)
This compound was obtained as yellow oil following the above
method, yield 74%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.56 (d,
This compound was obtained as yellow oil following the above
J ¼ 8.4 Hz, 1H, Ph-H), 7.49 (d, J ¼ 2 Hz, 1H, Ph-H), 7.31 (dd,
3J ¼ 8.4 Hz, 4J ¼ 2 Hz, 1H, Ph-H), 4.73 (s, 2H, OCH2), 4.59–4.66 (m,
1H, CH), 1.72–1.81 (m, 2H, CH2), 1.48–1.56 (m, 2H, CH2), 1.21–1.37
(m, 8H, CH2), 0.89 (t, J ¼ 7.2 Hz, 6H, CH3); 13C NMR (100 MHz,
method, yield 72%; 1H NMR (400 MHz, CDCl3):
d
¼ 7.40 (dd,
3J ¼ 8.4 Hz, 4J ¼ 1.6 Hz,1H, Ph-H), 7.36 (d, 4J ¼ 1.6 Hz,1H, Ph-H), 6.83
(d, J ¼ 8.0 Hz, 1H, Ph-H), 6.03 (s, 2H, OCH2O), 4.68 (s, 2H, OCH2),
4.57–4.64 (m, 1H, CH), 1.74–1.83 (m, 2H, CH2), 1.49–1.57 (m, 2H,
CH2), 1.19–1.38 (m, 8H, CH2), 0.88 (t, J ¼ 7 Hz, 6H, CH3); 13C NMR
CDCl3):
d
¼ 158.88, 147.37, 136.96, 134.21, 131.67, 130.71, 127.99,
127.07, 64.96, 54.82, 32.27, 28.34, 22.49, 14.02; MS: m/z ¼ 370 (Mþ),
313, 257, 245, 230, 199, 173, 145; EI-HRMS: calcd for C18H24Cl2N2O2
(Mþ), 370.1215; found, 370.1215.
(100 MHz, CDCl3):
d
¼ 159.00, 149.86, 148.33, 147.79, 124.05, 121.36,
108.00, 106.86, 101.54, 64.86, 54.65, 32.27, 28.34, 22.51, 14.01; MS:

2120
S.-Y. Ke et al. / European Journal of Medicinal Chemistry 44 (2009) 2113–2121
4.4.9. 2-(2-Ethoxylpyridin-3-yl)-4-(nonan-5-yl)-4H-1,3,4-
oxadiazin-5(6H)-one (5i)
kynuramine as a substrate according to the reported method
[27,28] with some modifications. The in vitro cytotoxicity assays
was evaluated against human lung cancer A-549 and prostate
cancer PC-3 cell lines. Growth inhibitory effect on the cell lines
(A-549 and PC-3) was measured by the sulforhodamine B (SRB)
dye-staining assay [33]. The chitin synthesis activity from yeast
S. cerevisiae was estimated using a non-radioactive chitin syn-
thase assay according to a modified procedure described by
Lecuro et al. [34].
This compound was obtained as yellow oil following the above
method, yield 68%; 1H NMR (400 MHz, CDCl3):
d
¼ 8.23 (dd,
3J ¼ 4.8 Hz, 4J ¼ 2 Hz, 1H, Py-H), 7.86 (dd, 3J ¼ 7.6 Hz, 4J ¼ 2 Hz, 1H,
Py-H), 6.93 (q, 3J ¼ 4.8 Hz, 4J ¼ 3 Hz,1H, Py-H), 4.68 (s, 2H, OCH2CO),
4.58–4.65 (m, 1H, CH), 4.45 (q, J ¼ 7.2 Hz, 2H, OCH2CH3), 1.74–1.84
(m, 2H, CH2), 1.49–1.56 (m, 2H, CH2), 1.43 (t, J ¼ 7.2 Hz, 3H,
OCH2CH3), 1.23–1.38 (m, 8H, CH2), 0.89 (t, J ¼ 7 Hz, 6H, CH3); 13C
NMR (100 MHz, CDCl3):
d
¼ 161.41, 159.30, 148.88, 147.91, 139.10,
116.07, 114.06, 64.95, 62.41, 54.68, 32.21, 28.38, 22.51, 14.66, 14.04;
MS: m/z ¼ 347 (Mþ), 290, 234, 221, 207, 176, 150, 121, 93; EI-HRMS:
calcd for C19H29N3O3 (Mþ), 347.2209; found, 347.2209.
Acknowledgements
This work was financiallysupported by the NationalKey Project for
Basic Research (2003CB114400), National 863 High-Tech Research
Award (2006AA10A201) and the National Natural Science Foundation
of China (20536010) are highlyappreciated. The authorsalsothank for
the partial support from Shanghai Leading Academic Discipline
Project (B507) and Shanghai Foundation of Science and Technology
(073919107).
4.4.10. 2-(4-Ethylphenyl)-4-(cyanoethyl)-4H-1,3,4-oxadiazin-
5(6H)-one (5j)
This compound was obtained as white solid following the above
method, yield 88%, m.p. 107.2–109.1 ꢁC; 1H NMR (400 MHz, CDCl3):
d
¼ 7.78 (d, J ¼ 8 Hz, 2H, Ph-H), 7.25 (d, J ¼ 8 Hz, 2H, Ph-H), 4.75 (s,
2H, OCH2CO), 4.10 (t, J ¼ 6.8 Hz, 2H, NCH2), 2.82 (t, J ¼ 6.8 Hz, 2H,
CH2CN), 2.70 (q, 2H, Ph-CH2), 1.26 (t, J ¼ 7.6 Hz, 3H, CH3); 13C NMR
Appendix. Suplementary information
(100 MHz, CDCl3):
d
¼ 159.12, 149.66, 148.05, 128.04, 126.76, 126.46,
117.21, 64.83, 42.85, 28.84, 16.34, 15.29; MS: m/z ¼ 257 (Mþ), 217,
159, 132, 116, 103, 77; EI-HRMS: calcd for C14H15N3O2 (Mþ),
257.1164; found, 257.1164.
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.ejmech.2008.10.015.
References
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