Synthesis, in vitro Antimicrobial, and Cytotoxic Activities of New 1,3,4-Oxadiazin-5(6H)-one Derivatives from Dehydroabietic Acid

Article abstract of DOI:10.1002/jccs.201700358

A series of new 1,3,4-oxadiazin-5(6H)-one derivatives (6a–n) of dehydroabietic acid were designed and synthesized as potential antimicrobial and antitumor agents. Their structures were characterized by IR, ¹H NMR, ¹³C NMR, MS, and elemental analyses. All the title compounds were evaluated for their antimicrobial activity against four bacterial and three fungal strains using the serial dilution method. Among them, compound 6e showed the highest antibacterial activity against Bacillus subtilis with a minimum inhibitory concentration (MIC) value of 1.9 μg/mL. In addition, the in vitro cytotoxic activities of the title compounds were also assayed against three human carcinoma cell lines (MCF-7, SMMC-7721, and HeLa) through the MTT colorimetric method. As a result, compounds 6b, 6g, 6k, and 6m exhibited significant inhibition against at least one cell line with IC₅₀ values below 10 μM. Compound 6m was especially found to be the most potent derivative with IC₅₀ values of 2.26 ± 0.23, 0.97 ± 0.11, and 1.89 ± 0.31 μM against MCF-7, SMMC-7721, and HeLa cells, respectively, comparable to positive control etoposide.

Full text of DOI:10.1002/jccs.201700358

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
Article
Synthesis, in vitro Antimicrobial, and Cytotoxic Activities of New 1,3,4-Oxadiazin-5(6H)-
one Derivatives from Dehydroabietic Acid
†
†
*
Xiao-Yan Jin, Kang-Ping Zhang, Hao Chen, Ting-Ting Miao, Shi-Fa Wang and Wen Gu
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-forest Biomass, Jiangsu Key Lab of
Biomass-based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing Forestry University,
Nanjing 210037, P. R. China
(Received: October 12, 2017; Accepted: December 23, 2017; DOI: 10.1002/jccs.201700358)
A series of new 1,3,4-oxadiazin-5(6H)-one derivatives (6a–n) of dehydroabietic acid were designed
and synthesized as potential antimicrobial and antitumor agents. Their structures were character-
1
13
ized by IR, H NMR, C NMR, MS, and elemental analyses. All the title compounds were evalu-
ated for their antimicrobial activity against four bacterial and three fungal strains using the serial
dilution method. Among them, compound 6e showed the highest antibacterial activity against
Bacillus subtilis with a minimum inhibitory concentration (MIC) value of 1.9 μg/mL. In addition,
the in vitro cytotoxic activities of the title compounds were also assayed against three human carci-
noma cell lines (MCF-7, SMMC-7721, and HeLa) through the MTT colorimetric method. As a
result, compounds 6b, 6g, 6k, and 6m exhibited significant inhibition against at least one cell line
with IC50 values below 10 μM. Compound 6m was especially found to be the most potent deriva-
tive with IC50 values of 2.26 ꢀ 0.23, 0.97 ꢀ 0.11, and 1.89 ꢀ 0.31 μM against MCF-7, SMMC-
7
721, and HeLa cells, respectively, comparable to positive control etoposide.
Keywords: Dehydroabietic acid; 1,3,4-Oxadiazin-5(6H)-one; Synthesis; Antimicrobial activity;
Cytotoxic activity.
INTRODUCTION
derivatives have drawn much attention for their broad
spectrum of biological activities, including antimicro-
bial, antiprotozoal, antitumor, antiviral, anti-aging,
gastroprotective, antifeedant, and BK-channel opening
As one of the leading causes of mortality all over
the world, cancer poses a tremendous threat to human’s
1
health. In spite of the significant advances in cancer
6–13
therapy in recent years, there still exists an urgent need
for the discovery of new anticancer agents. In addition,
the rapid development of multidrug-resistant microbial
pathogens has made the treatment of infectious diseases
an escalating problem, which exerts a continuous need
activities.
These results suggest that DAA is a prom-
ising starting material for the discovery of new antimi-
crobial or anticancer agents.
Nitrogen-containing heterocyclic compounds have
drawn the interest of chemists for their extensive biolog-
ical activities, which can afford extremely versatile
building blocks for the manufacture of bioactive com-
pounds in the pharmaceutical drug design and agro-
2
for new antibiotics with better efficacy. In recent years,
there has been increased interest in the search of new
antimicrobial and anticancer drugs from natural prod-
ucts. Many clinically used antibiotics, such as penicillin,
14–16
chemical industry.
The 1,3,4-oxadiazines represent
3
tetracycline, cephalosporin, etc, and antitumor drugs,
an important type of six-membered heterocycles with
two adjacent nitrogen atoms. Many 1,3,4-oxadiazine
derivatives exhibit a great diversity of biological effects,
such as antibacterial, antitumor, antileishmanial, miti-
cidal, insecticidal, and monoamine oxidase (MAO)
such as vinblastine, doxorubicin, and paclitaxel, were
discovered from natural products or natural product-
derived compounds. Dehydroabietic acid (DAA, 1) is
a natural-occurring diterpene resin acid obtained from
Pinus rosin or commercial disproportionated rosin. Its
4
,5
17–21
inhibitory
activities.
Some
1,3,4-oxadiazine
*Corresponding author. Email: njguwen@163.com
†The two authors contributed equally to this work.
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1

Article
Jin et al.
derivatives, e.g., indoxacarb, have been developed as a
potent voltage-dependent sodium channel blocker and
have been widely used as a commercially successful
20,22
insecticide.
These findings suggest that the introduc-
tion of the 1,3,4-oxadiazine motif into the scaffold of
DAA may probably produce new derivatives with
improved antimicrobial and anticancer properties. In
continuation of our research on the bioactive deriva-
2
3–25
tives of resin acid,
a series of new 1,3,4-oxadiazin-5
Scheme 2. Synthetic route for compounds 6n from
i. Reagents and conditions: (a) Fe pow-
(
6H)-one derivatives of DAA have been designed and
6
ꢁ
synthesized, and their in vitro antimicrobial and cyto-
toxic activities are also presented.
der, HCl, EtOH, H O, 85 C, 2 h.
2
compounds (6a–n) were purified by silica gel column
chromatography and recrystallization.
RESULTS AND DISCUSSION
Chemistry
The structures of 6a–n were characterized on the
The synthetic routes for the target compounds 6a–
p were depicted in Schemes 1 and 2. Briefly, DAA (1)
was converted to dehydroabietate chloride (2) through a
common chloridization reaction. Then, diazotization of
different substituted anilines (3a–m) followed by reduc-
1
13
basis of MS, IR, H NMR, C NMR spectra, and ele-
mental analyses. For example, the ESIMS of compound
6
b displayed a peak at m/z 445.3, corresponding to the
+
quasimolecular ions [M + H] , which confirmed the
molecular formula (C H N O ) in combination with
29
36
2
2
tion with SnCl afforded the corresponding substituted
2
the data of the elemental analysis. The absorption
phenylhydrazine hydrochlorides (4a–m), which were fur-
ther reacted with the dehydroabietate chloride 2 to give
−
1
bands at 1693 and 1648 cm in the IR spectrum of 6b
suggested the presence of C═O and C═N groups. In the
0
the substituted N -phenylcarbohydrazides (5a–m). Sub-
1
H NMR spectrum of 6b, the isopropyl group could be
sequently, the title compounds (6a–m) were obtained in
moderate to good yields by treating compounds 5a–m
with chloroacetyl chloride and K CO to form 1,3,4-
oxadiazin-5(6H)-one rings. In addition, the nitro group
of compound 6i could be reduced by Fe powder to
afford the p-aminophenyl derivative 6n. All the title
confirmed by a doublet at δ 1.23 ppm corresponding to
six methyl protons (H-19 and H-20) together with a
multiplet at δ 2.83 ppm due to the vicinal methine pro-
ton (H-18). Three singlets at δ 1.25, 1.31, and 2.36 ppm
could be assigned to methyl protons at C-17, C-16, and
2
3
0
0
4
-Me, respectively. Two doublets, each containing one
proton, at δ 4.54 and 4.59 ppm were due to the methy-
0
lene protons at C-1 on the 1,3,4-oxadiazin-5(6H)-one
ring. One double doublet at δ 7.01 (1H, J = 8.2, 1.7 Hz)
and two doublets at δ 6.90 (1H, J = 1.1 Hz) and δ 7.18
(1H, J = 8.2 Hz) could be attributed to three aromatic
protons at C-12, C-14, and C-11, respectively. Protons
on the p-methylphenyl ring appeared as two doublets at
0
0
00
00
00
13
δ 7.20 (H-3 , H-5 ) and δ 7.55 (H-2 , H-6 ). In the
C
NMR spectrum of 6b, there were 26 peaks appearing in
the range from δ 17.2 to 168.3 ppm, corresponding to
29 carbon atoms. The presence of 1,3,4-oxadiazin-5
(6H)-one ring could especially be confirmed by three
Scheme 1. Synthetic pathway to target compounds
6
a–m from dehydroabietic acid. Reagents
and conditions: (a) SOCl
2
, benzene, reflux
ꢁ
for 3 h; (b) NaNO
2
, HCl, 0~5 C, 1 h;
ꢁ
(
c) SnCl
2
, HCl, 0~5 C, 1–2 h; (d) differ-
0
signals at δ 67.4, 158.8, and 168.3 ppm due to C-1 , C-
ent substituted phenylhydrazine hydro-
0
ꢁ
15, and C-2 , respectively. The assignments of the sig-
chloride 4, Et
3
N, cyclohexane, 0 C for
1
13
nals in the H and C NMR spectra of 6b were in good
1
h and 3 h at rt.; (e) chloroacetyl chlo-
ride, 2-butanone, K CO , reflux for 3 h.
accordance with its structure.
2
3
2
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J. Chin. Chem. Soc. 2018

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
New Oxadiazine Derivatives of Dehydroabietic Acid
Antimicrobial activity
15.6 μg/mL. Specifically, compound 6e exerted the high-
est inhibitory effect against B. subtilis, with an MIC
value of 1.9 μg/mL, close to that of the positive control
amikacin (0.9 μg/mL). This compound also showed
strong inhibition against E. coli and S. aureus (MIC:
3.9 and 7.8 μg/mL, respectively), indicating its antibac-
terial potential against both Gram-positive and Gram-
negative strains. In addition, compound 6j exhibited
considerable inhibitory activity against two Gram-
negative bacteria, with an MIC value of 7.8 μg/mL. On
the other hand, the rest of the title compounds only
showed weak (MIC: 31.2 μg/mL) or no inhibition (MIC
>50 μg/mL) against the four tested bacteria. Concern-
ing antifungal activity, most of the title compounds
showed mild or no inhibitory activity against the three
tested fungi. Specifically, compounds 6d, 6e, 6i, and 6j
displayed weak antifungal activity (MIC: 31.2 μg/mL)
against C. albicans and/or C. tropicalis, while all the
compounds were inactive to P. citrinum.
The in vitro antimicrobial activities of all the title
compounds were evaluated using a modified twofold
serial dilution method to obtain the minimum inhibitory
26
concentrations (MICs) for different microorganisms.
The strains used in the study were two Gram-positive
bacteria (Staphylococcus aureus CGMCC 1.1361,
Bacillus subtilis CGMCC 1.1162), two Gram-negative
bacteria (Escherichia coli CGMCC 1.1571, Pseudomo-
nas fluorescens CGMCC 1.1828), and three fungi
(
Candida albicans CGMCC 2.2086, Candida tropicalis
CGMCC 2.3967, and Penicillium citrinum CGMCC
.825). All strains were bought from the China General
3
Microbiological Culture Collection Center (CGMCC).
Amikacin sulfate and ketoconazole were coassayed
as a positive control for tested bacteria and fungi,
respectively.
The in vitro antibacterial and antifungal data of
the synthesized compounds are shown in Table 1. From
the results, most of the title compounds showed inhibi-
tory activity against both Gram-positive and Gram-
negative bacteria with MIC values <50 μg/mL. Among
them, compounds 6d, 6e, 6g, 6j, 6l, and 6n displayed
considerable antibacterial activity against at least one
bacterial strain, with MIC values ranging from 1.9 to
The antimicrobial results indicated that this series
of derivatives possessed considerably superior antibacter-
ial properties compared to their antifungal properties.
The antibacterial activities of these compounds were sub-
stantially influenced by the substituents on the benzene
rings. In general, derivatives with electron-withdrawing
Table 1. The antimicrobial activity of the compounds (1, 6a–n) against four test bacteria and three test fungi
MIC (μg/mL)
Gram-negative bacteria
Gram-positive bacteria
Fungi
Compd.
E. coli
P. fluorescens
S. aureus
B. subtilis
C. albicans
C. tropicalis
P. citrinum
1
> 50
> 50
31.2
31.2
31.2
3.9
> 50
31.2
31.2
31.2
7.8
> 50
31.2
31.2
15.6
1.9
> 50
> 50
> 50
> 50
> 50
15.6
31.2
> 50
> 50
> 50
7.8
> 50
> 50
> 50
31.2
0.9
> 50
> 50
31.2
> 50
15.6
7.8
31.2
> 50
> 50
31.2
15.6
> 50
31.2
> 50
31.2
0.9
> 50
31.2
> 50
31.2
15.6
1.9
31.2
15.6
31.2
31.2
15.6
31.2
15.6
> 50
15.6
0.9
> 50
> 50
> 50
> 50
31.2
31.2
> 50
> 50
> 50
31.2
31.2
> 50
> 50
> 50
> 50
−
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
31.2
> 50
> 50
> 50
> 50
−
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
−
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a
b
c
d
e
f
g
h
i
j
k
l
m
n
Amikacin
Ketoconazole
−
−
−
−
3.9
3.9
7.8
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Article
Jin et al.
substituents, such as ─F, ─Cl and ─CN groups,
displayed stronger antibacterial activities than those with
electron-donating ones (─Me, ─Et and ─OMe). Among
them, compounds 6e and 6j with p-Cl and p-CN groups
showed stronger activities against tested bacteria than
derivatives with ─F, ─Br, ─CF , and ─NO groups, indi-
cating that these two substituents were the most beneficial
to the antibacterial activity of target derivatives. In addi-
tion, replacement of the p-Cl group (6e) with the o-Cl
group (6l) significantly reduced the antibacterial activity.
Similar results could also be observed for ─Me or ─OMe
groups. These phenomena suggested that the position of
the substituents would also influence the antibacterial
activity. For the same substituent, para substitution was
generally more favorable than ortho substitution.
Table 2. The cytotoxicity of the compounds (1, 6a–n) against
MCF-7, SMMC-7721, and HeLa cells
IC50 (μM)
Compd.
MCF-7
> 50
SMMC-7721
HeLa
> 50
18.79 ꢀ 2.58
12.71 ꢀ 1.98
18.03 ꢀ 1.34
27.03 ꢀ 2.43
22.31 ꢀ 1.75
> 50
1
> 50
21.32 ꢀ 1.89
8.16 ꢀ 0.73
12.38 ꢀ 2.11
24.71 ꢀ 2.66
20.14 ꢀ 2.05
> 50
5.91 ꢀ 0.20
37.76 ꢀ 3.69
> 50
3
2
6a
6b
6c
6d
6e
6f
27.43 ꢀ 2.31
17.39 ꢀ 3.07
18.87 ꢀ 2.16
17.21 ꢀ 1.33
33.72 ꢀ 3.79
43.78 ꢀ 3.20
7.21 ꢀ 0.37
> 50
6g
6h
7.39 ꢀ 0.18
> 50
6
6
6
6
6
6
i
j
k
l
m
n
> 50
> 50
> 50
> 50
> 50
6.53 ꢀ 0.86
19.78 ꢀ 2.48
2.26 ꢀ 0.23
14.75 ꢀ 1.95
0.77 ꢀ 0.12
3.03 ꢀ 0.42
13.21 ꢀ 2.02
0.97 ꢀ 0.11
12.37 ꢀ 1.34
0.62 ꢀ 0.16
3.78 ꢀ 0.28
11.26 ꢀ 1.59
1.89 ꢀ 0.31
18.72 ꢀ 1.13
0.83 ꢀ 0.22
Cytotoxic activity
All the title compounds were also evaluated for
their in vitro cytotoxic activity against the human
breast cancer cell line (MCF-7), hepatocarcinoma cell
line (SMMC-7721), and cervical carcinoma cell line
Etoposide
2
7
(
HeLa) through the MTT assay method. The antican-
activity of the title compounds. For example, deriva-
cer drug etoposide was coassayed as the positive con-
trol. The results of the tested compounds expressed as
IC50 values (concentration required to inhibit tumor cell
proliferation by 50%) are presented in Table 2.
tives with electron-donating substituents (─Me, ─Et,
─OMe and ─NH ) exhibited considerably stronger
2
cytotoxic activity than those derivatives with strong
electron-withdrawing ones (─NO , ─CN and ─CF ),
2
3
As illustrated in Table 2, the target compounds
exhibited varying degrees of cytotoxicity against three
cancer cell lines. It was found that compounds 6b, 6g,
which suggested that, contrary to antibacterial activity,
the electron-donating substituents could significantly
increase the cytotoxic activity of these derivatives.
Among these electron-donating substituents, the ─OMe
group was shown to be the most beneficial to the cyto-
toxicity. Derivatives with halogen substituents (─F,
─Cl, and ─Br) exhibited moderate antiproliferative
activities, and their cytotoxic activities decreased in the
order of F > Cl > Br. In addition, the cytotoxic activi-
ties of compounds 6k–m with ortho-substituents were
generally more potent than their analogs containing
para-substituents, indicating that the positions of the
substituents on the benzene rings could affect the inter-
action with their potential targets in cancer cells, there-
fore considerably influencing the antiproliferative
potency of these derivatives.
6k, and 6m showed significant cytotoxic activities against
at least one cancer cell line (IC50 < 10 μM). Among
them, compound 6m exhibited the most potent cytotoxic
activity against MCF-7, SMMC-7721, and HeLa cells
with IC50 values of 2.26 ꢀ 0.23, 0.97 ꢀ 0.11, and
1
.89 ꢀ 0.31 μM, respectively, nearly equipotent to the
positive control etoposide. In addition, compound 6a,
c–e, 6l, and 6n displayed moderate inhibitory activities
to three cancer cell lines. On the other hand, compounds
f and 6h only showed weak inhibition, while 6i and 6j
6
6
were inactive to all three cell lines (IC50 > 50 μM).
From these results, it could be observed that the
cytotoxic activity of most title compounds with 1,3,4-
oxadiazin-5(6H)-one moieties were stronger than the
starting material DDA, indicating that the introduction
of this heterocyclic moiety could enhance the cytotoxic-
ity. Moreover, the substituents on the benzene ring
could also exert a significant effect on the cytotoxic
CONCLUSIONS
In summary, a series of new 1,3,4-oxadiazin-5
(6H)-one derivatives of DAA was synthesized and
4
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JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
New Oxadiazine Derivatives of Dehydroabietic Acid
evaluated for their in vitro antimicrobial and cytotoxic
activities. As a result, some title compounds exhibited
promising antimicrobial and/or cytotoxic activities.
Among them, compound 6e possessed prominent anti-
bacterial properties against four tested bacteria, while
compound 6m exhibited potent cytotoxic activity
against three cancer cell lines. The activities of the title
compounds were significantly influenced by the proper-
ties and positions of the substituents on the benzene
ring. Further studies will be carried out to explore the
in-depth structure–activity relationships (SAR) and the
mechanisms of action of these compounds.
The solid product was filtered, washed with 35% HCl
thrice, and dried in a vacuum desiccator containing
anhydrous CaCl . The product could be used in subse-
2
quent reactions without further purification.
General procedure for the preparation of acylhydrazine
derivatives (5a–5m)
To a solution of DAA (30 g, 0.1 mol) in 100 mL
of benzene, 10.9 mL of SOCl (17.85 g, 0.15 mol) was
2
added slowly. The mixture was then refluxed for 3 h.
After cooling, the solvent and excess SOCl2 were
removed in vacuo to yield dehydroabietate chloride (2)
as a yellow oily product, which was used in the next
step without further purification.
EXPERIMENTAL
Chemistry
To a solution of dehydroabietate chloride (2,
0
.638 g, 2 mmol) in 10 mL of cyclohexane, different
Melting points were measured on an XT-4 appara-
tus (Taike Corp., Beijing, China) and were uncorrected.
IR spectra were measured on a Nexus 870 FT-IR spec-
trometer, and the absorption bands were expressed in
substituted phenylhydrazine hydrochloride (3 mmol)
ꢁ
and triethylamine (0.606 g, 6 mmol) at 0 C were
ꢁ
added. The mixture was stirred for 1 h at 0 C and then
for 3 h at room temperature. At the end of reaction, the
mixture was filtered to remove the precipitate. The fil-
trate was washed with water and brine, dried over
anhydrous Na SO , and concentrated in vacuo. The
−1
cm . The ESI-MS spectra were recorded on a Mariner
1
13
System 5304 mass spectrometer. H NMR and
C
NMR spectra were accomplished in CDCl on Bruker
3
2
4
AV-300, AV-500, and DRX-600 NMR spectrometers
using TMS as the internal standard. Elemental analyses
were carried out using the Elementar Vario El cube ele-
mental analyzer. Reactions and the resulted products
were monitored by TLC, which was carried out on silica
gel IB-F flexible sheets obtained from Mallinckrodt
Baker Inc., Germany and visualized in UV light
residue was purified by recrystallization in MeOH to
afford compound 5a–5 m as yellow resin-like solids.
General procedure for the synthesis of 1,3,4-oxadiazin-5
(6H)-one derivatives (6a–6m)
Chloracetyl chloride (40 μL, 0.5 mmol) was added
to a solution of compound 5 (0.26 mmol) in 10 mL of
anhydrous 2-butanone. The mixture was refluxed at
(254 nm). Silica gel (300–400 mesh) for column chroma-
ꢁ
tography was purchased from Qingdao Marine Chemi-
cal Factory, China. DAA (98%) was bought from
Yijing Industrial Co., Ltd. (Shanghai, China). The
reagents (chemicals), all being of A.R. grade, were pur-
chased from Shanghai Chemical Reagent Company
85 C for 1 h. After cooling, anhydrous K CO3
2
(0.415 g, 3 mmol) was added, and the mixture was
refluxed for another 3 h. At the end of the reaction, the
solvent was removed in vacuo, and the residue was dis-
solved in 20 mL of ethyl acetate. The solution was
washed with water and brine, dried over anhydrous
Na SO , and concentrated in vacuo. The residue was
(Shanghai, China).
2
4
General procedure for the preparation of
phenylhydrazine hydrochlorides (4a–4m)
then purified by column chromatography on silica gel
and eluted with petroleum ether-acetone (200:1~10:1,
v/v) to afford compounds 6a–6m.
A solution of NaNO (0.73 g, 10.5 mmol) in 2 mL
2
of H O was added dropwise to a solution of substituted
2
aniline (3, 10 mmol) in 10 mL of 20% HCl while cool-
2-Dehydroabietyl-4-phenyl-4H-1,3,4-oxadiazin-5(6H)-
one (6a)
ing with an ice water bath. The reaction mixture was
ꢁ
ꢁ
stirred at 0~5 C for 1 h to give a clear solution. Then,
Yellow solid; yield: 56%; mp 130~133 C; IR
−
1
a solution of SnCl (20 mmol) in 6 mL of 35% HCl at
(KBr, cm ): 2954, 2925, 2867, 1694, 1646, 1595, 1493,
2
ꢁ
1
0
~5 C was added dropwise to this solution. The mix-
1374, 1309, 1274, 1052, 817, 758, 685; H NMR
ture was then stirred at room temperature for 1–2 h.
(600 MHz, CDCl ): δ 1.23 (d, J = 7.0 Hz, 6H), 1.25
3
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5

Article
Jin et al.
13
(s, 3H), 1.32 (s, 3H), 1.51 (dt, J = 12.8, 3.6 Hz, 1H),
(d, J = 8.5 Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H);
C
1
2
.60~1.90 (m, 6H), 2.20 (dd, J = 12.5, 2.0 Hz, 1H),
.33 (brd, J = 13.2 Hz, 1H), 2.83 (m, 1H), 2.87~2.92
NMR (100 MHz, CDCl ): δ 15.2, 17.2, 18.5, 20.9, 24.0,
3
25.3, 28.0, 29.9, 33.5, 36.7, 37.4, 37.9, 41.1, 46.4, 67.3,
122.2, 124.0, 124.1, 129.8, 126.9, 146.2, 134.5, 138.7,
(m, 2H), 4.56 (d, J = 15.1 Hz, 1H), 4.61 (d,
+
J = 15.1 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H), 7.02 (dd,
J = 8.1, 1.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 7.25 (t,
J = 7.4 Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.70 (d,
146.0, 146.5, 158.8, 168.3; MS (ESI): m/z [M + H] :
459.3; Anal. Calc. for C H N O : C 78.56; H 8.35; N
30
38
2
2
6.11; found: C 78.61; H 8.39; N 6.18.
13
J = 7.7 Hz, 2H); C NMR (100 MHz, CDCl ): δ 17.2,
3
1
4
1
8.5, 20.9, 24.1, 25.3, 29.8, 33.5, 36.7, 37.4, 37.9, 41.1,
6.3, 67.4, 120.1, 123.9, 124.1, 125.2, 126.9, 128.6,
2
−
-Dehydroabietyl-4-(4-fluorophenyl)-4H-1,3,4-oxadiazin
5(6H)-one (6d)
Yellow gum; yield: 44%; IR (KBr, cm ): 2949,
34.5, 138.6, 146.0, 146.4, 158.8, 168.3; MS (ESI): m/z
−1
+
[
M + H] : 431.3; Anal. Calc. for C H N O : C 78.10;
2
8
34
2
2
2
1
927, 2862, 1695, 1649, 1604, 1504, 1460, 1375, 1285,
229, 1207, 1180, 1099, 1059, 969, 833, 723, 694, 633;
H 7.96; N 6.51; found: C 78.16; H 7.91; N 6.58.
1
H NMR (600 MHz, CDCl ): δ 1.23 (d, J = 7.0 Hz,
3
2-Dehydroabietyl-4-(p-tolyl)-4H-1,3,4-oxadiazin-5(6H)-
6
H), 1.25 (s, 3H), 1.31 (s, 3H), 1.50 (dt, J = 12.5,
.0 Hz, 1H), 1.60~1.90 (m, 6H), 2.20 (dd, J = 12.5,
.1 Hz, 1H), 2.34 (brd, J = 12.8 Hz, 1H), 2.83 (m, 1H),
.87~2.92 (m, 2H), 4.55 (d, J = 15.2 Hz, 1H), 4.60 (d,
one (6b)
2
2
2
−1
Yellow gum; yield: 49%; IR (KBr, cm ): 2953,
927, 2863, 1727, 1693, 1648, 1506, 1460, 1374, 1300,
274, 1210, 1172, 1116, 1063, 967, 819, 711, 634; H
2
1
1
J = 15.2 Hz, 1H), 6.90 (brs, 1H), 7.02 (dd, J = 8.1,
NMR (600 MHz, CDCl ): δ 1.23 (d, J = 6.9 Hz, 6H),
3
1
1
.3 Hz, 1H), 7.09 (t, J = 8.7 Hz, 2H), 7.18 (d, J = 8.2,
H), 7.67 (dd, J = 9.1, 4.9 Hz, 2H); C NMR
1.25 (s, 3H), 1.31 (s, 3H), 1.50 (dt, J = 12.8, 3.8 Hz,
1H), 1.60~1.89 (m, 6H), 2.20 (dd, J = 12.5, 2.1 Hz,
1H), 2.33 (brd, J = 13.1 Hz, 1H), 2.36 (s, 3H), 2.83 (m,
1H), 2.87~2.92 (m, 2H), 4.54 (d, J = 15.1 Hz, 1H), 4.59
13
(100 MHz, CDCl ): δ 17.2, 18.5, 20.8, 24.0, 25.3, 29.8,
3
3
3.5, 36.7, 37.5, 37.9, 41.1, 46.3, 67.4, 115.4 (d,
J = 8.9 Hz), 116.2 (d, J = 22.8 Hz), 123.9, 124.1, 126.9,
34.5, 138.8 (d, J = 2.3 Hz), 146.0, 146.5, 158.7, 160.1
(
(
(
d, J = 15.1 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H), 7.01
dd, J = 8.2, 1.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.20
d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H);
1
+
(d, J = 243.9 Hz), 168.4; MS (ESI): m/z [M + H] :
13
C
4
49.3; Anal. Calc. for C H FN O : C 74.97; H 7.42;
28
33
2
2
NMR (100 MHz, CDCl ): δ 17.2, 18.4, 20.8, 21.0, 24.0,
3
N 6.25; found: C 74.90; H 7.36; N 6.32.
2
1
1
5.2, 29.8, 33.5, 36.6, 37.4, 37.9, 41.1, 46.3, 67.3, 121.8,
23.8, 124.1, 126.9, 129.5, 134.5, 137.4, 138.7, 146.0,
+
46.4, 158.9, 168.2; MS (ESI): m/z [M + H] : 445.3;
4-(4-Chlorophenyl)-2-dehydroabietyl-4H-1,3,4-oxadiazin
−5(6H)-one (6e)
Anal. Calc. for C H N O : C 78.34; H 8.16; N 6.30;
found: C 78.39; H 8.10; N 6.24.
2
9
36
2
2
−1
Yellow gum; yield: 45%; IR (KBr, cm ): 2949,
2
927, 2854, 1696, 1490, 1461, 1369, 1282, 1209, 1179,
1
2-Dehydroabietyl-4-(4-ethylphenyl)-4H-1,3,4-oxadiazin-5
1137, 1089, 1059, 1012, 968, 826, 753, 708, 631; H
(
6H)-one (6c)
NMR (600 MHz, CDCl ): δ 1.23 (d, J = 7.0 Hz, 6H),
3
−1
Yellow gum; yield: 37%; IR (KBr, cm ): 2958,
928, 2866, 1734, 1693, 1650, 1507, 1460, 1375, 1309,
1.25 (s, 3H), 1.32 (s, 3H), 1.50 (dt, J = 12.7, 3.5 Hz,
1H), 1.59~1.90 (m, 6H), 2.19 (dd, J = 12.6, 2.1 Hz,
1H), 2.34 (brd, J = 12.5 Hz, 1H), 2.83 (m, 1H),
2.87~2.92 (m, 2H), 4.55 (d, J = 15.2 Hz, 1H), 4.60 (d,
J = 15.2 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H), 7.02 (dd,
J = 8.2, 1.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.36 (d,
2
1
8
288, 1283, 1209, 1179, 1118, 1076, 1057, 968, 899,
1
29, 706; H NMR (600 MHz, CDCl ): δ 1.23 (d,
3
J = 6.9 Hz, 6H), 1.24 (t, J = 7.6 Hz, 3H), 1.25 (s, 3H),
.31 (s, 3H), 1.50 (dt, J = 12.7, 3.8 Hz, 1H), 1.60~1.90
m, 6H), 2.20 (dd, J = 12.4, 2.1 Hz, 1H), 2.33 (brd,
J = 13.2 Hz, 1H), 2.66 (q, J = 7.6 Hz, 2H), 2.83 (m,
H), 2.87~2.92 (m, 2H), 4.55 (d, J = 15.1 Hz, 1H), 4.59
1
(
13
J = 8.9 Hz, 2H), 7.69 (d, J = 8.9 Hz, 2H); C NMR
(100 MHz, CDCl ): δ 17.2, 18.5, 20.9, 24.0, 25.3, 29.8,
3
1
(
(
33.5, 36.5, 37.3, 38.0, 41.1, 46.3, 67.4, 120.6, 123.9,
124.1, 126.9, 129.3, 130.4, 134.5, 138.5, 146.0, 146.4,
d, J = 15.1 Hz, 1H), 6.90 (d, J = 1.2 Hz, 1H), 7.01
dd, J = 8.2, 1.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.23
+
158.6, 168.2; MS (ESI): m/z [M + H] : 465.2; Anal.
6
www.jccs.wiley-vch.de
© 2018 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Chin. Chem. Soc. 2018

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
New Oxadiazine Derivatives of Dehydroabietic Acid
Calc. for C H ClN O : C 72.32; H 7.15; N 6.02;
found: C 72.37; H 7.09; N 6.09.
1349, 1324, 1312, 1284, 1213, 1165, 1125, 1068, 1015,
28
33
2
2
1
968, 843, 822; H NMR (600 MHz, CDCl ): δ 1.23 (d,
3
J = 6.9 Hz, 6H), 1.26 (s, 3H), 1.34 (s, 3H), 1.51 (dt,
J = 13.0, 3.4 Hz, 1H), 1.60~1.89 (m, 6H), 2.21 (dd,
J = 12.5, 2.1 Hz, 1H), 2.34 (brd, J = 12.6 Hz, 1H), 2.83
4
−
-(4-Bromophenyl)-2-dehydroabietyl-4H-1,3,4-oxadiazin
5(6H)-one (6f)
Yellow gum; yield: 47%; IR (KBr, cm ): 2955,
−1
(m, 1H), 2.87~2.92 (m, 2H), 4.57 (d, J = 15.3 Hz, 1H),
2926, 2861, 1696, 1649, 1488, 1460, 1369, 1282, 1210,
4
7
7
.62 (d, J = 15.3 Hz, 1H), 6.91 (d, J = 1.2 Hz, 1H),
1
179, 1110, 1074, 1010, 969, 877, 823, 783, 719, 621;
.02 (dd, J = 8.2, 1.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H),
1
H NMR (600 MHz, CDCl ): δ 1.23 (d, J = 6.9 Hz,
13
3
.66 (d, J = 8.6, 2H), 7.92 (d, J = 8.5, 2H); C NMR
6
H), 1.25 (s, 3H), 1.32 (s, 3H), 1.50 (dt, J = 13.0,
.5 Hz, 1H), 1.59~1.90 (m, 6H), 2.19 (dd, J = 12.5,
.0 Hz, 1H), 2.33 (brd, J = 12.5 Hz, 1H), 2.83 (m, 1H),
.87~2.92 (m, 2H), 4.54 (d, J = 15.2 Hz, 1H), 4.60 (d,
(
100 MHz, CDCl ): δ 17.2, 18.4, 20.8, 24.0, 25.2, 29.7,
3
3
2
2
3
1
1
1
3.5, 36.6, 37.4, 37.9, 41.2, 46.3, 67.3, 123.9, 124.1,
22.5 (q, J = 271.2 Hz, CF ), 126.6 (q, J = 3.8 Hz),
3
26.9, 127.1 (q, J = 4.1 Hz), 131.1 (q, J = 32.5 Hz),
J = 15.2 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H), 7.02 (dd,
J = 8.2, 1.6 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.51 (d,
34.3, 141.2, 146.0, 146.3, 158.8, 168.3; MS (ESI): m/z
+
[M + H] : 499.3; Anal. Calc. for C H F N O : C
29
33
3
2
2
1
3
J = 8.9 Hz, 2H), 7.64 (d, J = 8.9 Hz, 2H); C NMR
100 MHz, CDCl ): δ 17.2, 18.5, 20.8, 24.0, 25.3, 29.8,
6
9.86; H 6.67; N 5.62; found: C 69.82; H 6.70; N 5.68.
(
3
3
1
1
3.5, 36.7, 37.4, 37.9, 41.1, 46.2, 67.4, 116.9, 121.7,
24.0, 124.1, 126.9, 131.6, 134.5, 138.3, 146.0, 146.4,
58.8, 168.3; MS (ESI): m/z [M + H] : 509.2; Anal.
2
-Dehydroabietyl-4-(4-nitrophenyl)-4H-1,3,4-oxadiazin-5
(
6H)-one (6i)
+
ꢁ
Yellow solid; yield: 58%; m.p. 120~124 C; IR
Calc. for C H BrN O : C 66.01; H 6.53; N 5.50;
found: C 66.07; H 6.58; N 5.43.
−1
28
33
2
2
(KBr, cm ): 2952, 2924, 2852, 1704, 1650, 1591, 1520,
1
493, 1458, 1335, 1297, 1110, 1056, 852, 823, 745, 688;
1
H NMR (600 MHz, CDCl ): δ 1.23 (d, J = 6.9 Hz,
3
2
-Dehydroabietyl-4-(4-methoxyphenyl)-4H-1,3,4-
6
H), 1.27 (s, 3H), 1.36 (s, 3H), 1.52 (dt, J = 12.5,
oxadiazin-5(6H)-one (6g)
−1
3.7 Hz, 1H), 1.60~1.90 (m, 6H), 2.21 (dd, J = 12.5,
Yellow gum; yield: 53%; IR (KBr, cm ): 3444,
949, 2929, 2836, 1799, 1730, 1679, 1598, 1514, 1461,
389, 1331, 1248, 1214, 1176, 1121, 1078, 1029,
2
2
.1 Hz, 1H), 2.35 (brd, J = 12.1 Hz, 1H), 2.84 (m, 1H),
.87~2.92 (m, 2H), 4.59 (d, J = 15.4 Hz, 1H), 4.64 (d,
2
1
9
1
J = 15.5 Hz, 1H), 6.91 (d, J = 1.0 Hz, 1H), 7.03 (dd,
J = 8.2, 1.6 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 8.06 (d,
72, 876, 821; H NMR (600 MHz, CDCl ): δ 1.22 (d,
3
J = 6.9 Hz, 6H), 1.26 (s, 3H), 1.37 (s, 3H), 1.49 (dd,
J = 12.7, 4.0 Hz, 1H), 1.60~1.88 (m, 6H), 2.20 (dd,
J = 12.5, 2.2 Hz, 1H), 2.34 (brd, J = 13.4 Hz, 1H), 2.82
13
J = 9.3 Hz, 2H), 8.27 (dd, J = 9.3 Hz, 2H); C NMR
100 MHz, CDCl ): δ 17.2, 18.5, 20.9, 24.0, 25.3, 29.8,
(
3
3
1
1
3.5, 36.6, 37.4, 37.9, 41.1, 46.3, 67.5, 115.2, 124.0,
24.1, 124.5, 127.0, 134.5, 140.9, 142.6, 146.0, 146.4,
(m, 1H), 2.87~2.92 (m, 2H), 3.89 (s, 3H), 4.56 (d,
J = 15.8 Hz, 1H), 4.65 (d, J = 15.8 Hz, 1H), 6.91 (d,
J = 2.0 Hz, 1H), 6.94 (dd, J = 8.7, 2.2 Hz, 2H), 7.02
+
58.7, 168.4; MS (ESI): m/z [M + H] : 476.3; Anal.
Calc. for C H N O : C 70.71; H 6.99; N 8.84; found:
C 70.78; H 6.96; N 8.80.
28
33
3
4
(dd, J = 8.2, 2.1 Hz, 1H), 7.18 (d, J = 8.3, 1H), 7.56 (d,
1
3
J = 8.7, 2.1, 2H); C NMR (100 MHz, CDCl ): δ
3
1
4
1
7.2, 18.5, 20.8, 24.0, 25.2, 29.7, 33.5, 36.6, 37.4, 37.9,
1.1, 46.3, 55.7, 67.4, 115.2, 122.1, 124.0, 124.1, 126.9,
4
-(2-Dehydroabietyl-5-oxo-5,6-dihydro-4H-1,3,4-
oxadiazin-4-yl)benzonitrile (6j)
32.6, 134.5, 146.0, 146.4, 158.8, 161.3, 168.3; MS
−1
+
Yellow gum; yield: 41%; IR (KBr, cm ): 2948,
(ESI): m/z [M + H] : 461.3; Anal. Calc. for
2
1
1
927, 2853, 2223, 1703, 1646, 1601, 1494, 1458, 1368,
343, 1163, 835, 819; H NMR (600 MHz, CDCl ): δ
.23 (s, J = 6.9 Hz, 6H), 1.26 (s, 3H), 1.34 (s, 3H), 1.51
C H N O : C 75.62; H 7.88; N 6.08; found: C 75.57;
H 7.84; N 6.12.
2
9
36
2
3
1
3
2
-Dehydroabietyl-4-(4-(trifluoromethyl)phenyl)-4H-1,3,4-
(dt, J = 12.6, 2.9 Hz, 1H), 1.60~1.90 (m, 6H), 2.20 (dd,
J = 12.5, 2.1 Hz, 1H), 2.35 (brd, J = 12.3 Hz, 1H), 2.83
(m, 1H), 2.87~2.92 (m, 2H), 4.57 (d, J = 15.4 Hz, 1H),
4.62 (d, J = 15.4 Hz, 1H), 6.90 (d, J = 1.1 Hz, 1H),
oxadiazin-5(6H)-one (6h)
−1
Yellow gum; yield: 52%; IR (KBr, cm ): 2958,
931, 2867, 1702, 1648, 1613, 1514, 1496, 1461, 1365,
2
J. Chin. Chem. Soc. 2018
© 2018 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.jccs.wiley-vch.de
7

Article
Jin et al.
+
7
7
.02 (dd, J = 8.2, 1.6 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H),
MS (ESI): m/z [M + H] : 465.2; Anal. Calc. for
13
.69 (d, J = 8.9 Hz, 2H), 7.99 (d, J = 8.9 Hz, 2H);
C
C H ClN O : C 72.32; H 7.15; N 6.02; found: C
28
33
2
2
NMR (100 MHz, CDCl ): δ 17.2, 18.5, 20.9, 24.0, 25.3,
72.34; H 7.19; N 5.98.
3
2
1
1
4
9
9.8, 33.5, 36.7, 37.4, 37.9, 41.1, 46.3, 67.4, 118.6,
20.1, 123.9, 124.1, 125.2, 126.9, 128.6, 134.5, 138.6,
46.0, 146.4, 158.8, 168.3; MS (ESI): m/z [M + H] :
2
-Dehydroabietyl-4-(2-methoxyphenyl)-4H-1,3,4-
oxadiazin-5(6H)-one (6m)
Yellow solid; yield: 59%; m.p. 123~126 C; IR
+
ꢁ
56.3; Anal. Calc. for C H N O : C 76.45; H 7.30; N
2
9
33
3
2
−
1
(KBr, cm ): 3311, 2955, 2929, 2867, 1690, 1597, 1498,
.22; found: C 76.51; H 7.23; N 9.20.
1
7
1
463, 1380, 1274, 1170, 1112, 1074, 1024, 977, 821,
1
97, 751, 635, 610; H NMR (600 MHz, CDCl ): δ
3
2-Dehydroabietyl-4-(o-tolyl)-4H-1,3,4-oxadiazin-5(6H)-
.22 (d, J = 7.0 Hz, 6H), 1.23 (s, 6H), 1.49 (m, 1H),
one (6k)
Yellow solid; yield: 44%; m.p. 134~136 C; IR
ꢁ
1.60~1.90 (m, 6H), 2.19 (dd, J = 12.7, 2.1 Hz, 1H),
2.31 (brd, J = 13.2 Hz, 1H), 2.83 (m, 1H), 2.87~2.92
−
1
(KBr, cm ): 2953, 2928, 2868, 1695, 1639, 1495, 1459,
(m, 2H), 3.87 (s, 3H, OCH ), 4.57 (d, J = 15.2 Hz, 1H),
3
1
7
385, 1283, 1219, 1176, 1131, 1057, 971, 879, 822, 761,
1
4.62 (d, J = 15.2 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H),
6
7
17, 628; H NMR (600 MHz, CDCl ): δ 1.22 (d,
3
.96 (m, 2H), 7.02 (m, 2H), 7.19 (d, J = 8.1 Hz, 1H),
J = 7.0 Hz, 6H), 1.23 (s, 6H), 1.49 (dt, J = 13.0,
13
.58 (d, J = 8.2 Hz, 1H); C NMR (100 MHz,
3
2
2
1
1
1
1
3
1
1
.8 Hz, 1H), 1.60~1.89 (m, 6H), 2.19 (dd, J = 12.5,
.2 Hz, 1H), 2.25 (s, 3H), 2.32 (brd, J = 13.1 Hz, 1H),
.83 (m, 1H), 2.87~2.92 (m, 2H), 4.59 (d, J = 15.1 Hz,
H), 4.64 (d, J = 15.1 Hz, 1H), 6.89 (d, J = 1.7 Hz,
H), 7.01 (dd, J = 8.2, 2.0 Hz, 1H), 7.17 (d, J = 8.1 Hz,
CDCl ): δ 17.2, 18.5, 20.8, 24.0, 25.3, 29.7, 33.5, 36.6,
3
3
1
1
7.4, 37.9, 41.1, 46.3, 55.8, 67.5, 112.8, 121.5, 123.9,
24.1, 126.9, 128.1, 130.7, 140.8, 134.5, 146.0, 146.5,
+
50.3, 158.7, 168.3; MS (ESI): m/z [M + H] : 461.3;
1
3
Anal. Calc. for C H N O : C 75.62; H 7.88; N 6.08;
29 36 2 3
found: C 75.57; H 7.83; N 6.13.
H), 7.28 (m, 4H); C NMR (100 MHz, CDCl ): δ
3
7.2, 17.6, 18.4, 20.8, 24.0, 25.3, 29.8, 33.5, 36.7, 37.5,
7.8, 41.1, 46.3, 67.3, 123.8, 124.1, 124.5, 126.3, 126.9,
27.6, 131.3, 133.9, 134.5, 138.1, 146.0, 146.5, 158.8,
General procedure for the synthesis of 1,3,4-oxadiazin-5
(
6H)-one derivatives (6n)
+
68.3; MS (ESI): m/z [M + H] : 445.3; Anal. Calc. for
Reduced Fe powder (0.112 g, 2 mmol) and
C H N O : C 78.34; H 8.16; N 6.30; found: C 78.39;
H 8.20; N 6.27.
2
9
36
2
2
0
.6 mL of concentrated HCl was added to a solution of
compound 6i (0.12 g, 0.26 mmol) in 15 mL of EtOH
and 1.5 mL of water. The mixture was refluxed for 2 h
at 85 C and monitored by TLC. After cooling, the
mixture was poured into 20 mL of ice-cold water,
which was extracted with CH Cl (3 × 20 mL). The
organic layer was combined; washed with water, satu-
rated NaHCO , and brine; dried over anhydrous
Na SO ; and concentrated in vacuo. The residue was
4
−
-(2-Chlorophenyl)-2-dehydroabietyl-4H-1,3,4-oxadiazin
5(6H)-one (6l)
Yellow solid; yield: 54%; m.p. 166~168 C; IR
ꢁ
ꢁ
2
2
−
1
(KBr, cm ): 2953, 2928, 2867, 1702, 1639, 1582, 1481,
1
8
447, 1386, 1287, 1219, 1176, 1137, 1071, 1031,
3
1
21, 763, 751, 736, 722, 621; H NMR (600 MHz,
2
4
CDCl ): δ 1.22 (d, J = 8.6 Hz, 6H), 1.23 (s, 6H), 1.49
3
purified by column chromatography on silica gel, elut-
ing with petroleum ether-acetone (5:1, v/v) to afford
compound 6n as a yellow gum.
(
dt, J = 12.6, 3.8 Hz, 1H), 1.60~1.87 (m, 6H), 2.19 (dd,
J = 12.5, 2.2 Hz, 1H), 2.32 (brd, J = 13.1 Hz, 1H), 2.83
m, 1H), 2.87~2.92 (m, 2H), 4.61 (d, J = 15.2 Hz, 1H),
(
4
7
7
1
2
1
4
1
.66 (d, J = 15.1 Hz, 1H), 6.90 (d, J = 2.0 Hz, 1H),
.01 (dd, J = 8.2, 2.0 Hz, 1H), 7.17 (d, J = 8.1 Hz, 1H),
.33 (dt, J = 7.4, 2.0 Hz, 1H), 7.37 (dt, J = 7.5, 1.8 Hz,
H), 7.41 (dd, J = 7.4, 2.0 Hz, 1H), 7.50 (dd, J = 7.4,
4-(4-Aminophenyl)-2-dehydroabietyl-4H-1,3,4-oxadiazin
−5(6H)-one (6n)
−1
Yellow gum; yield: 91%; IR (KBr, cm ): 3431,
3368, 2949, 2925, 2857, 1731, 1682, 1629, 1511, 1460,
1382, 1283, 1210, 1184, 1125, 1084, 1057, 1021,
13
.0 Hz, 1H); C NMR (100 MHz, CDCl ): δ 17.2,
3
1
8.5, 20.9, 24.1, 25.3, 29.8, 33.5, 36.6, 37.3, 38.0, 41.1,
6.3, 67.5, 123.2, 123.8, 124.1, 126.9, 127.3, 130.1,
30.5, 131.1, 134.5, 137.2, 146.0, 146.4, 158.7, 168.2;
969, 884, 825, 750, 712, 685; H NMR (600 MHz,
CDCl ): δ 1.24 (d, J = 7.0 Hz, 6H), 1.27 (s, 3H), 1.34
3
(s, 3H), 1.49 (dt, J = 12.6, 3.6 Hz, 1H), 1.60~1.86 (m,
8
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J. Chin. Chem. Soc. 2018

JOURNAL OF THE CHINESE
CHEMICAL SOCIETY
New Oxadiazine Derivatives of Dehydroabietic Acid
2
7
6
H), 2.18 (dd, J = 12.5, 2.2 Hz, 1H), 2.32 (brd,
(HeLa) via the MTT colorimetric method. Briefly, dif-
ferent tumor cells were grown in a DMEM medium
supplemented with 10% fetal bovine serum, penicillin
(100 U/mL), and streptomycin (50 μg/mL). Cells were
harvested at the log phase of growth and seeded in 96-
J = 12.9 Hz, 1H), 2.83 (m, 1H), 2.87~2.92 (m, 2H),
4
5
J = 1.6 Hz, 1H), 7.00 (dd, J = 8.1, 1.6 Hz, 1H), 7.17 (d,
J = 8.2 Hz, 1H), 7.40 (dd, J = 8.8, 2.1 Hz, 2H);
.53 (d, J = 15.1 Hz, 1H), 4.58 (d, J = 15.1 Hz, 1H),
.30 (s, 2H), 6.69 (dt, J = 8.8, 2.1 Hz, 2H), 6.89 (d,
13
5
C
well plates (100 μL/well at a density of 2 × 10 cells/
mL). After 24 h incubation at 37 C and 5% CO to
ꢁ
NMR (100 MHz, CDCl ): δ 17.2, 18.5, 20.8, 24.0, 25.2,
3
2
2
1
1
9.7, 33.5, 36.6, 37.4, 37.9, 41.1, 46.3, 67.5, 112.2,
22.3, 124.0, 124.1, 126.9, 128.9, 134.5, 143.8, 146.0,
46.4, 158.7, 168.3; MS (ESI): m/z [M + H] : 446.3;
allow cell attachment, cultures were exposed to various
concentrations of the isolated compounds for 48 h.
Finally, MTT solution (2.5 mg/mL in PBS) was added
(40 μL/well). Plates were further incubated for 4 h at
+
Anal. Calc. for C H N O : C 75.47 H 7.92; N 9.43;
found: C 75.51; H 7.86; N 9.38.
2
8
35
3
2
ꢁ
37 C, and the formazan crystals formed were dissolved
by adding 150 μL/well of DMSO. Absorption at
570 nm was measured with an ELISA plate reader. The
results were expressed as IC50 values with standard
deviations, which were defined as the concentration at
which 50% survival of cells could be discerned. Etopo-
side was coassayed as positive control.
Antimicrobial assay
All the target compounds were screened for their
antibacterial and antifungal activity. Seven strains were
used as test microorganisms, including four bacteria:
Escherichia coli (CGMCC 1.1571), Pseudomonas fluor-
escens (CGMCC 1.1828), Staphylococcus aureus
(
1
2
CGMCC 1.1361), and Bacillus subtilis (CGMCC
.1162) and three fungi: Candida albicans (CGMCC
.2086), Candida tropicalis (CGMCC 2.3967), and Pen-
ACKNOWLEDGMENTS
This work was supported by the National Natural
Science Foundation of China (31770616), the Natural
Science Foundation of Jiangsu Province (BK201516),
the Natural Science Foundation for Colleges and Uni-
versities in Jiangsu Province (17KJA220002), Top-
notch Academic Programs Project of Jiangsu Higher
Education Institutions (PPZY2015C221), and a project
funded by the Priority Academic Program Develop-
ment of Jiangsu Higher Education Institutions (PAPD).
The authors are grateful to the Advanced Analysis &
Testing Center of Nanjing Forestry University for the
measurements of NMR data.
icillium citrinum (CGMCC 3.825). The test microbes
were obtained from the China General Microbiological
Culture Collection Center (CGMCC), China. The anti-
microbial activity was assessed in terms of MICs by a
modified microdilution method. Compounds were dis-
solved in DMSO, and serial double dilutions of each
compound (75 μL) were prepared in 96-well micro
trays. The same amount of test microorganisms in Mar-
2
6
5
tin’s (for bacteria) or PD (for fungi) broth (~ 10
colony-forming unit (CFU)/mL) was added to each well
to give a final volume of 150 μL. After incubation at 37
ꢁ
ꢁ
C for 24 h (for bacteria) or 28 C for 48 h (for fungi),
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negative control. All assays were performed in
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J. Chin. Chem. Soc. 2018