Synthesis and antibacterial activity of 4-(4-methylpent-3-enyl)cyclohex-4- ene-1,2-dicarboxamide

Article abstract of DOI:10.14233/ajchem.2013.14431

In this paper, 4-(4-methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide was synthesized by β-myrcene through Diels-Alder reaction and ammonolysis reaction. The synthetic compound was proved to be the target product with the help of NMR, FT-IR and GC-MS. Di

Full text of DOI:10.14233/ajchem.2013.14431

Asian Journal of Chemistry; Vol. 25, No. 12 (2013), 6718-6720
http://dx.doi.org/10.14233/ajchem.2013.14431
Synthesis and Antibacterial Activity of 4-(4-Methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide
1
1
1
3
4
JIANWEI LIAN , JIANLING XIA1,2,*, MEI WANG , XIAODONG TANG , LISHA GAO and YAFENG YANG
1Institute of Chemical Industry of Forestry Products, CAF; Key Lab. of Biomass Energy and Material, Jiangsu Province; National Engineering
Lab. for Biomass Chemical Utilization; Key and Lab. on Forest Chemical Engineering, SFA, Nanjing 210042, P.R. China
2Institute of Forest New Technology, CAF, Beijing 100091, P.R. China
3Quality, Safety and Environmental Department, Petrochina Hebei Marketing Company, Shijiazhuang 050000, Hebei Province, P.R. China
4Special Operation Division, The Third Engineering Company of CPP, Zhengzhou 451450, Henan Province, P.R. China
*Corresponding author: E-mail: xiajianling@126.com
(Received: 9 August 2012;
Accepted: 27 May 2013)
AJC-13556
In this paper, 4-(4-methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide was synthesized by β-myrcene through Diels-Alder reaction
and ammonolysis reaction. The synthetic compound was proved to be the target product with the help of NMR, FT-IR and GC-MS. Diels-
Alder reaction has been optimized under the following conditions: 150 ºC, reacting for 3 h, no catalyst and the production rate of 4-(4-
methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide is 88.12 %, the selectivity is 97.49 %. Then get down to ammonolysis reaction
under 0.4 MPa ammonia pressure for 18 h with methanol as the solvent and the production rate turned up to 88.7 %. At last, the test of the
antibacterial activity of the target product showed optimistic to Staphylococcus aureus but passimistic to Escherichia coli.
Key Words: β-Myrcene, Diels-Alder reaction, Ammonolysis reaction, Antibacterial activity.
INTRODUCTION
EXPERIMENTAL
In China, rosin resources are very rich, especially in
Guangdong, Guangxi and Yunnan provinces1 and the content
of β-pinene in rosin can reach to 30.3 %. β-Myrcene can
be obtained by β-pinene through thermal isomerization in
industry, whose purity is 80 %. β-Myrcene is also refered as
geraniolene and the chemical name is 7-methyl-3-methylene-
1,6-octadiene and there are three unsaturated carbon-carbon
double bonds, which are easy to generate chemical reactions,
such as Diels-Alder reaction, oxidizing reaction and reduction
reaction. Goldblatt and Palkin2, Yin et al.3-6 and Luo et al.7
have investigated the Diels-Alder reaction of β-myrcene,
however, none of them had investigated the further application
of the Diels-Alder product in industry.
β-Myrcene, (Jiangxi Kaiyuan spices Co. Ltd., China), NH3
(Nanjing special gas Co. Ltd., China), Dimethyl maleate (AR),
CH3OH (AR), CaCl2 (AR), HCl (AR).
500 MHz NMR (Bruker, Germany), 7890N/5975N GC-
MS (Agilent, USA), 7890 GC (Agilent, USA), IS10 FT-IR
(Nicolet, USA), WRS-1B Melting PointApparatus (Shanghai
exact science instrument Co. Ltd., China), BS110S electronic
balance (Bejing Saiduoli Inc., China), SHZ-C vacuum pump
(Yingyu Electronic Instrument Factory, China), temperature
control system, JJ-1 electric mixer (Guohua Inc., China), HH-
4 digital electronic constant temperature water-bath (Aorui
Equipment Factory, China).
Antibacterial activity: The antibacterial activity of
chemicals was estimated by a disc paper method. The test
chemicals were dissolved in DMSO to 25 mg/mL. As test
species, Staphylococcus aureus (gram-positive bacteria) and
Escherichia coli (Gram-negative bacteria) were cultivated in
beef extract-peptone for 1 week. After cultivation, a small
amount of (1 scratch ca. 2 scratch) fresh bacteria from the
culture medium were added into the culture solution. The
culture solution was diluted 10 fold to a concentration of
(5.0~10.0) × 106 (CFU) mL-1. 1 mL of the bacterial solution
Diamide compounds have significant applications in
chemical industry, which are widely used as chemical
materials, agricultural materials, pharmaceuticals, fuel and
intermediates of pigment productions. Synthesis of diamide
compounds have been investigated for years8-11 and in this
paper, we firstly synthesized 4-(4-methylpent-3-enyl)cyclohex-
4-ene-1,2-dicarboxamide by β-myrcene through Diels-Alder
reaction and ammonolysis reaction and the antibacterial
activity of the product was studied as well.

Vol. 25, No. 12 (2013)
Synthesis and Antibacterial Activity of 4-(4-Methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide 6719
1
described above was then evenly coated on 90 mm plate of
beef extract peptone medium. Six millimeter diameter sterile
filter paper was dipped in the solution of antibacterial test
chemicals for 10 min and then the paper was taken out and
placed on the plate. The inhibition zones were measured by
calipers in millimeters at the end of a 24 h incubation period.
All experiments were conducted in triplicate to ensure repro-
ducibility at a given concentration.
1252(w), 1190(w), 1114(w), 787(w); H NMR (500 MHz,
DMSO-d6) δ/ppm: 7.18, 6.65 (m, 4H, 2*NH2), 5.37 (d, J = 4.1
Hz, 1H, C8-H), 5.09-5.06 (m, 1H, C4-H), 2.53-2.41 (m, 2H,
C10-H and C11-H), 2.19-1.92 (m, 8H, C9-H2, C12-H2, C5-H2 and
C6-H2), 1.64 (2s, 6H, C1-H3 and C2-H3); 13C NMR (125 MHz,
DMSO-d6) δ/ppm: 180.5 (C13 and C14), 137.4(C7), 132.5(C3),
125.1(C4), 121.2(C8), 49.5(C10), 44.2(C11), 38.4(C6), 30.4(C12),
28.3(C5), 26.8(C9), 25.8(C2), 17.8(C1); GC-MS (m/z): 250.1
[M]+, 233.1 [M-NH3]+, 205.1 [M-HCONH2]+. The reaction eqn.
(2) shows as follows:
Diameter of inhibition zone (mm) = A1-A0
(1)
where A0 is the diameter of the control (blank, ethanol and
sterile water without an antibacterial test chemical) and A1 is
the diameter in the presence of test chemical.
O
O
9
1
NH3 CH3OH
CaCl2
8
13 NH
2
2
O
O
10
11
5
Synthesis of dimethyl 4-(4-methylpent-3-enyl)cyclohex
-4-ene-1,2-dicarboxylate: 72 g dimethyl maleate was added
into 250 mL flask which was connected with water bath, stir
device, thermograph, dropping funnel and reflux condenser.
Turning on the stirring device and adding β-myrcene drop-
wisely, then heat up to 150 ºC for 3 h. When reaction finishes,
draw out fraction of 205 ºC/1.05 kPa through vacuum distilla-
tion method and the left is 4-(4-methylpent-3-enyl)cyclohex-
4-ene-1,2-dicarboxylate, which is light yellow liquid and its
weight is 119.7 g. The yield is 88.1 %. IR (ν/cm-1): 2951[s,
-CH3, νas], 2920[s, -CH2, νas], 2852[s, -CH2, νs], 1736[s, ν(C=O)],
1672[w, ν(C=C)], 1436[m, -C-CH3, δas], 1376[m, -C-CH3, δs],
1354(m), 1279(m), 1203[s, ν(C-O-C)], 1170(s), 1105(w, C-O-
C, νs), 1074(w), 1024(m), 926(w), 848(w), 792(w); 1H NMR
(500 MHz, CD3OD) δ/ppm: 5.37 (s, 1H, C8-H), 5.08 (t, J = 6.8
Hz, 1H, C4-H), 3.65 (2s, 6H, C15-H3 and C16-H3), 3.08-3.00 (m,
2H, C10-H and C11-H), 2.51-2.27 (m, 4H, C9-H2 and C12-H2),
2.08-1.96 (m, 4H, C5-H2 and C6-H2), 1.67 (2s, 6H, C1-H3 and
C2-H3); 13C NMR (125 MHz, CD3OD) δ/ppm: 175.3 (C13 and
C14), 137.2 (C7), 132.3 (C3), 125.1 (C4), 120.0 (C8), 52.2 (C15
and C16), 49.5 (C10), 41.4 (C11), 38.5 (C6), 29.7 (C12), 27.3 (C5),
26.8 (C9), 25.9 (C2), 17.8 (C1); GC-MS (m/z): 280.1 [M-e]+,
248.1 [M-CH3OH-e]+, 220.1 [M-CH3COOH-e]+. The reaction
eqn. (1) shows as follows:
3
(2)
NH
14
7
2
12
4
6
O
O
2
3
RESULTS AND DISCUSSION
Effects on different conditions towards Diels-Alder
reaction between β-myrcene and dimethyl maleate: Effects
on different conditions towards productivity and selectivity
of Diels-Alder reaction between β-myrcene and dimethyl
maleate are given in Table-1. Entry 10-14 indicate that with
the extension of the reaction time, the productivity rises at
first and then fluctuates in a level, which indicates Diels-Alder
reaction between β-myrcene and dimethyl maleate is reversible
and it will get to balance in a certain extent and the producti-
vities are different under different temperatures (entrys 9, 12).
The contrast of data in the table (entry 1, 4, 5, 8, 12, 21) shows
that within the same reaction time, the productivity continues
rising with the increase of reaction temperature until the
temperature rises up to 150 ºC. The data of Table-1 (entry 1-3,
TABLE-1
EFFECTS OF DIFFERENT FACTORS ON THE DIELS-ALDER
REACTION OF β-MYRCENE AND DIMETHYL MALEATE
Temp
(ºC)
Time
(h)
Yielda
(%)
Selectivityb
(%)
Entry
Catalyst
O
O
O
9
1
2
75
3
3
5
3
3
1
2
3
4
1
2
3
4
5
1
2
3
4
1
2
3
-
12.08
81.86
80.52
18.25
41.45
72.18
81.05
80.79
84.19
78.26
84.61
88.12
86.19
86.98
86.22
88.70
85.99
87.01
87.35
88.72
84.54
93.43
94.91
94.96
95.19
95.11
93.10
94.51
93.58
95.52
94.82
96.13
97.49
94.35
94.89
95.74
96.50
94.38
94.89
95.31
93.95
93.68
15
16
1
8
O
O
13
14
O
O
10
11
75
LiClO4
5
(1)
3
150 ºC
7
3
75
LiClO4
2
12
4
6
O
4
95
-
1
2
5
110
135
135
135
135
150
150
150
150
150
150
150
150
150
160
160
160
-
6
-
Synthesis of 4-(4-methylpent-3-enyl)cyclohex-4-ene-1,
2-dicarboxamide: Add 11.8 g of 4-(4-methylpent-3-enyl)-
cyclohex-4-ene-1, 2-dicarboxylate into 150 mL stainless steel
reaction kettle, then add 9.3 g anhydrous CaCl2 and 60 mL
anhydrous CH3OH. Put them into ice water bath airtightly and
turn on the magnetic stir device, at the same time, enter NH3
till the pressure of stainless steel reaction kettle ups to 0.4
MPa. When the temperature downs to 8-10 ºC, stop adding of
NH3, warm up to 80 ºC slowly and keep it for 18 h. When
reaction finishes, adjust pH to 5-6 with diluted HCl and wash
the product till there is no Cl–, then after filtering and vacuum
drying, 4-(4-methylpent-3-enyl)cyclohex-4-ene-1,2-dicarbo-
xamide is obtained, which is tan solid. The yield is 88.7 %.
m.p.: 209-211 ºC; IR (ν/cm-1): 3417(w), 3330[m, -NH2, νas],
3202[m, -NH2, νs], 2967[m, -CH3, νas], 2919[m, -CH2, νas],
2849[m, -CH2, νs], 1711(w), 1657(s, peak of primary amide),
1610(m, peak of primary amide), 1371[m, -C-CH3, δs],
7
-
8
-
9
-
10
11
12
13
14
15
16
17
18
19
20
21
-
-
-
-
-
LiClO4
LiClO4
LiClO4
LiClO4
-
-
-
aMass fraction of product in the reaction system whose retention time
is 19.0-21.0 min; bMass fraction of product retention time at 20.38 min
in the yield

6720 Lian et al.
Asian J. Chem.
10-18) shows that the catalyst LiClO4 can shorten the reaction
time and reduce the temperature to the same balance, but at
the same time reduce the product selectivity and increases the
difficulty of the product post-processing, which complicates
the experiment operation and increase the cost.
The test shows big rate at the beginning of the reaction and
the productivity will not increase when it gets to balance after
reacting for 18 h. Therefore, the best reaction time is 18 h.
Antibacterial activity of the 4-(4-methylpent-3-enyl)-
cyclohex-4-ene-1,2-dicarboxamide: Table-3 shows that
4-(4-methylpent-3-enyl)cyclohex-4-ene-1,2-dicarboxamide
has antibacterial effect to Staphylococcus aureus but not to
Escherichia coli, which indicates the antibacterial activity of
4-(4-methylpent-3-enyl)cyclohex-4-ene-1, 2-dicarboxamide is
specific.
The contrast of data in Table-1 (entry1-21) shows that the
balance forms under 150 ºC after reacting for 3 h has both
high productivity and good selectivity. Considering the cost
of catalyst and post-processing of the product, the reaction
conditions are made as follows: no catalyst, reaction tempe-
rature of 150 ºC and reaction time of 3 h.
TABLE-3
ANTIBACTERIAL ACTIVITIES OF THE 4-(4-METHYLPENT
-3-ENYL)CYCLOHEX-4-ENE-1,2-DICARBOXAMIDE
Effects on productivity of ammonolysis reaction of
dimethyl 4-(4-methylpent-3-enyl)cyclohex-4-ene-1,2-
dicarboxylate: Effects on productivity of ammonolysis
reaction of dimethyl 4-(4-methylpent-3-enyl)cyclohex-4-ene-
1,2-dicarboxylate are given in Table-2. Data from entry 1-4
indicates that the demand of solvent in this reaction is strict,
because when acetone, formamide and DMF are used as
solvents, there is none of 4-(4-methylpent-3-enyl)cyclohex-
4-ene-1, 2-dicarboxamide. The reason may be that the reaction
needs large concentration of NH3 and the hydrogen bond
between H-N or O-H from -OH in methanol and NH3 increases
the solubility of NH3 in the reaction system which promotes
the reaction to a great extent.
Bacterias
Diameter of inhibition zone (mm)
Escherichia coli
Staphylococcus aureus
0.40
2.66
Conclusion
The optimized β-myrcene and dimethyl maleate Diels-
Alder reaction condition is 150 ºC, react for 3 h and no catalyst.
Productivity of dimethyl 4-(4-methylpent-3-enyl)cyclohex-4-
ene-1,2-dicarboxylate is 88.12 % and the selectivity is 97.49 %.
Target compound 4-(4-methylpent-3-enyl)cyclohex-4-
ene-1,2-dicarboxamide was synthesized by β-myrcene through
Diels-Alder reaction and ammonolysis reaction. The optimized
ammonolysis reaction condition is methanol as solvent, reacting
under 0.4 MPa ammonia pressure for 18 h and the yield is
88.7 %. The antibacterial activity of 4-(4-methylpent-3-enyl)-
cyclohex-4-ene-1,2-dicarboxamide have been studied, which
shows an efficient antibacterial effect towards Staphylococcus
aureus.
TABLE-2
OPTIMIZATION OF DIFFERENT FACTORS ON THE AMMONO-
LYSIS REACTION OF DIMETHYL 4-(4-METHYLPENT-3-
ENYL)CYCLOHEX-4-ENE-1,2-DICARBOXYLATE
Temp
(ºC)
Pressure
(MPa)
Time
(h)
Yielda
(%)
Entry
Solvent
1
2
Acetone
Formamide
DMF
80
80
80
80
60
70
90
80
80
80
80
80
80
80
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.2
0.3
0.4
0.4
0.4
0.4
0.4
24
24
24
24
24
24
24
24
24
24
6
-
-
3
-
ACKNOWLEDGEMENTS
4
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
Methanol
88.9
40.7
67.4
86.5
49.6
75.8
88.7
74.8
85.6
88.7
88.7
5
This work was supported by the Chinese Academy of
Forestry and Public welfare forestry research projects
(201104031).
6
7
8
9
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10
11
12
13
14
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Data of entry 2 show that the reaction temperature should
be 80 ºC. With the rise of temperature, 4-(4-methylpent-
3-enyl)cyclohex-4-ene-1,2-dicarboxamide will turn to 5-(4-
methylpent-3-enyl)-3a,4,7,7a-tetrahydroisobenzofuran-1,3-
dione (structure can be proved through GC-MS method). The
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the cost of NH3, 0.4 MPa is chosen to be the reaction pressure.
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