Ethyl cinnamate derivatives as promising high-efficient acaricides against Psoroptes cuniculi: Synthesis, bioactivity and structure-activity relationship

Article abstract of DOI:10.1248/cpb.c14-00765

This paper reported the synthesis, structure-activity relationship (SAR) and acaricidal activity in vitro against Psoroptes cuniculi, a mange mite, of 25 ethyl cinnamate derivatives. All target compounds were synthesized and elucidated by means of MS, ¹H- and ¹³C-NMR analysis. The results showed that 24 out of 25 tested compounds at 1.0 mg/mL demonstrated acaricidal activity in varying degrees. Among them, 6, 15, 26, 27 and 30 showed significant activity with median lethal concentration values (LC₅₀) of 89.3, 119.0, 39.2, 29.8 and 41.2 μg/mL, respectively, which were 2.1- to 8.3-fold the activity of ivermectin (LC₅₀=247.4 μg/mL), a standard drug in the treatment of Psoroptes cuniculi. Compared with ivermectin, with a median lethal time value (LT₅₀) of 8.9 h, 27 and 30 showed smaller LTM₅₀ values of 7.9 and 1.3 h, respectively, whereas 6, 15 and 26 showed slightly larger LT50 values of 10.6, 11.0 and 10.4 h at 4.5 μmol/mL. SARs showed that the presence of o-NO₂ or m-NO₂ on the benzene ring significantly improved the activity, whereas the introduction of a hydroxy, methoxy, acetoxy, methylenedioxy, bromo or chloro group reduced the activity. (E)-Cinnamates were more effective than their (Z)-isomer. Nevertheless, the carbon-carbon double bond in the acrylic ester moiety was proven not to be essential to improve the activity of cinnamic acid esters. Thus, the results strongly indicate that cinnamate derivatives, especially their dihydro derivatives, should be promising candidates or lead compounds for the development of novel acaricides for the effective control of animal or human acariasis.

Full text of DOI:10.1248/cpb.c14-00765

Vol. 63, No. 4
Chem. Pharm. Bull. 63, 255–262 (2015)
255
Regular Article
Ethyl Cinnamate Derivatives as Promising High-Efficient Acaricides
against Psoroptes cuniculi: Synthesis, Bioactivity and Structure–Activity
Relationship
a,#
a,#
b
a
a
a
Bingyu Zhang, Chao Lv, Weibo Li, Zhiming Cui, Dongdong Chen, Fangjun Cao,
,
b
,a
Fang Miao,* and Le Zhou*
a
b
College of Science, Northwest A&F University; Yangling, Shaanxi 712100, China: and College of Life Science,
Northwest A&F University; Yangling, Shaanxi 712100, China.
Received November 7, 2014; accepted January 28, 2015; advance publication released online February 11, 2015
This paper reported the synthesis, structure–activity relationship (SAR) and acaricidal activity in vitro
against Psoroptes cuniculi, a mange mite, of 25 ethyl cinnamate derivatives. All target compounds were syn-
1
13
thesized and elucidated by means of MS, H- and C-NMR analysis. The results showed that 24 out of 25
tested compounds at 1.0mg/mL demonstrated acaricidal activity in varying degrees. Among them, 6, 15, 26,
2
7 and 30 showed significant activity with median lethal concentration values (LC ) of 89.3, 119.0, 39.2, 29.8
50
and 41.2µg/mL, respectively, which were 2.1- to 8.3-fold the activity of ivermectin (LC ꢀ247.4 µg/mL), a
5
0
standard drug in the treatment of Psoroptes cuniculi. Compared with ivermectin, with a median lethal time
value (LT ) of 8.9h, 27 and 30 showed smaller LT values of 7.9 and 1.3h, respectively, whereas 6, 15 and
5
0
50
2
6 showed slightly larger LT50 values of 10.6, 11.0 and 10.4h at 4.5µmol/mL. SARs showed that the presence
of o-NO or m-NO on the benzene ring significantly improved the activity, whereas the introduction of a hy-
2
2
droxy, methoxy, acetoxy, methylenedioxy, bromo or chloro group reduced the activity. (E)-Cinnamates were
more effective than their (Z)-isomer. Nevertheless, the carbon–carbon double bond in the acrylic ester moiety
was proven not to be essential to improve the activity of cinnamic acid esters. Thus, the results strongly indi-
cate that cinnamate derivatives, especially their dihydro derivatives, should be promising candidates or lead
compounds for the development of novel acaricides for the effective control of animal or human acariasis.
Key words cinnamic acid ester; ethyl 3-phenylpropionate; acaricidal activity; acaricide; Psoroptes cuniculi
1
3)
Acariasis is a skin disease caused by mites, an ectoparasite been proven to have a variety of pharmacological activities,
14,15)
16–18)
18)
which widely occurs in animals and human. Psoroptes cu- such as anticancer,
antimicrobial,
anti-Mycobactrium tuberculosis,
and can be parasitic in sheep, horse, rabbit, goat, cattle and antiviral, anti-human immunodeficiency virus (HIV),
antioxidative,
15,19–21)
22–24)
niculi is an animal ear mite living in the ear canals of animal anti-inflammatory,
2
5)
26–28)
1)
29)
30)
31)
buffalo. Psoroptic acariasis is a highly contagious disease, antidiabetic, anticholesterolemic, analgesic, hepatopro-
immunoprotective, inducing neural progenitor
tion, the formation of crusts and scabs, anorexia and reduction cell proliferation and anxiolytic activity. Especially, what
32,33)
34)
which causes intense pruritus, serous exudations, inflamma- tective,
35)
36)
2)
of weight gain, or even death of animals. Therefore, the in- interests us is that cinnamic acid derivatives also have sig-
37)
38)
fection of this mite species may severely reduce the productiv- nificant antiparasitic activities on plasmodia, Leishmania
3)
39)
ity and the quality of animal products.
and nematode. Furthermore, the acaricidal activity of ethyl
Traditionally, organophosphates, organochlorine, pyre- cinnamate and trans-cinnamaldehyde as an analogue of
thrins, ivermectin and abamectin have been used as effec- cinnamic acid were reported as well. Thus, cinnamic acid
tive drugs for treatment and control of animal acariasis. How- derivatives are often used as promising starting compounds
ever, the chemical control could increase resistance of target for the development of new, highly effective drugs. Neverthe-
4
0)
41)
4)
5)
6
)
7,8)
species to acaricides, toxicity and environmental hazards.
less, until now no systematic research on acaricidal activity of
These problems have made researchers’ efforts to discover cinnamic acid esters and their structure–activity relationship
new effective acaricides derived from natural products due to (SAR) were reported.
37–41)
their easy degradation in the environment, less or not remain
Our interest in the excellent antiparasitic activities
and
of cinnamic acid derivatives prompted us
to explore their acaricidal activity and extend their pharmaco-
11,12)
in livestock, not being prone to resistance and relative safety the low toxicity
9)
for humans, animals and environment.
Cinnamic acid and its ester derivatives are widely distrib- logic activities. This investigation presented the preparation
uted in plants including cereals, legumes, oilseeds, fruits, veg- of a series of cinnamic acid ester derivatives and evaluation
10)
etables and tea or coffee beverages. Due to their common of their acaricidal activity against P. cuniculi as well as the
11,12)
occurrence in plants and their low toxicity,
cinnamic acid discussion of their preliminary SAR.
derivatives have attracted much attention of many pharma-
cologists. In the past decades, cinnamic acid derivatives in- Results and Discussion
cluding natural, semi-synthetic and synthetic compounds had
Chemistry Compound 6 was obtained by esterifica-
tion reaction of commercially available trans-cinnamic acid
with ethanol using thionyl chloride as a catalyst in 95%
#
These authors contributed equally to this work.
*
To whom correspondence should be addressed. e-mail: miaofangmf@163.com;
zhoulechem@nwsuaf.edu.cn
© 2015 The Pharmaceutical Society of Japan

2
56
Chem. Pharm. Bull.
Vol. 63, No. 4 (2015)
a. i) DMF, POCl , 0–100°C; ii) NaOAc, 100°C, 11% yield; b. Paraformaldehyde, conc. HCl, r.t., 80% yield; c. DMSO, NaHCO , 90°C, 48% yield; d. Bromine, AcOH,
3
3
9
2%; e. DMSO, NaHCO , 90°C, 37% yield; f. SOCl , EtOH, r.t., 95%; g. Ph P=CHCO Et. EtOH or toluene, 42–92%; h. NaBH , CuCl, MeOH, 97%; i. 2-(Bis(2-(tert-
3 2 3 2 4
butyl)phenoxy)phosphoryl)acetate, KOH, THF, 73%; j. (CH ) SO , K CO , acetone, 53%; k. (Ac) O/Et N, 94–99%.
3
2
4
2
3
2
3
Chart 1. Synthesis of Ethyl Cinnamate Derivatives
42)
yield. Compounds 7–9, 11, 12, 16–28 were synthesized 22, other tested compounds showed the activity at various
by Wittig reaction of ethyl triphenylphosphanylideneacetate degrees at 1.0mg/mL. Among them, 6, 11, 15, 26, 27 and 30
[
(C H ) P=CHCO Et] and aromatic aldehyde in ethanol or displayed the highest activity with the mite mortality of 100%,
6 5 3 2
43)
toluene. Compounds 10 and 13–15 were obtained by typical absence of significant difference from that of ivermectin
methyl-etherification or acetylation reaction of the correspond- (98.3%) (p>0.05) and the others showed low to moderate ac-
ing hydroxyl-substituted trans-cinnamic acid esters (7–9). tivity (6.7–62.5%). For the higher active compounds 6, 11, 15,
Compound 29 was synthesized by Horner–Wadsworth–Em- 26, 27 and 30, further tests were conducted at lower concen-
mons (HWE) reaction of benzaldehyde with ethyl 2-(bis(2- trations. The results showed that at 0.5 or 0.25mg/mL, these
4
4)
(tert-butyl)phenoxy)phosphoryl)acetate in 73% yield. Com- compounds were significantly more active than ivermectin
pound 30 was prepared by reduction of 1 with NaBH in the (p<0.05) with the exception of 11 (Table 1).
4
45)
presence of CuCl in 97% yield. Aromatic aldehyde 1 was
Acaricidal Toxicity The excellent activity of 6, 15, 26, 27
obtained from sesamol by the reaction of Vilsmeier–Haack and 30 in Table 1 encouraged us to further determine their
4
6)
formylation in 11% yield. 1,3-Benzodioxole reacted with acaricidal toxicity on P. cuniculi in order to get insight into
paraformaldehyde in a concentrated HCl solution to provide their acaricidal potency. The assay method was the same as
4
7)
2
,
and followed by treatment with bromine in glacial ace- that described above. Ivermectin was used as a reference drug
4
8)
tic acid to yield 4 in 92% yield. Compounds 2 and 4 were control. The activities caused by the treatment with various
oxidized by dimethylsulphoxide oxidation in the presence of concentrations of the compounds for 24h and caused by the
49)
NaHCO to yield intermediates 3 and 5, respectively. (Chart treatment with the same concentration (4.5µmol/mL) of the
3
1).
compounds for various times were shown in Figs. 1A and
Compounds 6–30 were identified by electrospray ionization B, respectively. Toxicity regression equations for concentra-
1
13
(
ESI)-MS, H- and C-NMR spectra. In positive or negative tion–effect and time–effect of the compounds and their cor-
ESI-MS spectra, 6–30 showed their corresponding molecular responding median lethal concentration values (LC ) and
5
0
ion peaks, quasi-molecular or pseudo-molecular ion peaks median lethal time values (LT ) were listed in Tables 2 and
5
0
+
+
−
[
M+H] , [M+Na] or [M] . The NMR data were agreement 3, respectively.
with the corresponding literature data.
Figure 1A clearly showed that the activity of all the tested
Pharmacology Acaricidal Activity in Vitro Compounds compounds including the positive drug ivermectin increased
–30 were screened for the acaricidal activity in vitro against with increase of their respective test concentrations in a cer-
6
5
0–52)
P. cuniculi according to our previously reported method.
tain range of concentration. Statistical analysis further showed
Ivermectin, a standard acaricidal drug, was used as a refer- that at the post-treatment 24h, each of the compounds had
ence control. The results listed in Table 1 showed that except a significant linear correlation between the mortality rate

Vol. 63, No. 4 (2015)
Chem. Pharm. Bull.
257
Table 1. The Substitution Patterns and Acaricidal Activity of the Synthesized Compounds against P. cuniculi
a)
ArCH=CHCO Et
Mortality % (mean± S.D.)
2
Compound
E/Z
Ar
1.0mg/mL
0.5mg/mL
0.25mg/mL
c)
6
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
Z
C H –
100.0± 0.0 a
26.7± 5.2 f
22.5± 5.0 f
25.0± 5.8 f
62.5± 9.6 b
100.0± 0.0 a
55.0± 5.5 c
61.7± 4.1 b
52.5± 9.6 c
100.0± 0.0 a
13.3± 5.2g
13.3± 5.2g
13.3± 5.2 gh
8.3± 4.1 gh
6.7± 5.2 hi
21.7± 4.1 f
1.7± 4.1 ij
36.7± 5.2 e
55.0± 5.5 c
50.0± 8.2 c
100.0± 0.0 a
100.0± 0.0 a
50.0± 0.0 c
43.3± 5.2 d
100± 0.0 a
98.3± 4.1 a
0.0± 0.0 j
100.0± 0.0 a
95.0± 5.5 a
ND
6
5
c)
b)
7
2-OH-C H –
ND
6
5
c)
8
3-OH-C H –
ND
ND
ND
6
5
c)
9
4-OH-C H –
ND
6
5
c)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2-OCH –C H –
ND
ND
3
6
5
3-OCH –C H –
35.0± 5.8 c
ND
ND
3
6
5
c)
4-OCH –C H –
ND
3
6
5
2-OAc–C H –
ND
ND
6
5
3-OAc–C H –
ND
ND
6
5
4-OAc–C H –
100.0± 0.0 a
ND
73.3± 5.2 b
ND
6
5
2-OH–3-OCH –C H –
3
6
5
c)
c)
4-OH–3-OCH –C H –
ND
ND
3
6
5
3,4-(OCH ) –C H –
ND
ND
3
2
6
5
2,4,5-(OCH ) –C H –
ND
ND
3
3
6
5
c)
3,4,5-(OCH ) –C H –
ND
ND
3
3
6
5
3,4-OCH O–C H –
ND
ND
2
6
5
2-OH–4,5-OCH O–C H –
ND
ND
2
6
5
2-Br–4,5-OCH O–C H –
ND
ND
2
6
5
4-Cl–C H –
ND
ND
6
5
4-Br–C H –
ND
ND
6
5
2-NO –C H –
100.0± 0.0 a
100.0± 0.0 a
ND
100.0± 0.0 a
96.7± 5.2 a
ND
2
6
5
3-NO –C H –
2
6
5
4-NO –C H –
2
6
5
c)
c)
C H –
ND
ND
6
5
100.0± 0.0 a
75.0± 5.8 b
0.0± 0.0 d
100.0± 0.0 a
45.0± 10.0 c
0.0± 0.0 d
Ivermectin
Control
a) The differences between data with the different lowercases within a column are significant (p<0.05). b) ND denotes no determination. c) Natural compounds.
Fig. 1. Effects of Tested Concentrations (A) and Treatment Times (B) of the Compounds on the Acaricidal Activity against P. cuniculi
Table 2. Toxicity Regression Equations for Concentration–Effect of the Compounds and Their LC50 Values (24h)
LC50
a)
2
b)
c)
Compound
Regression equation
R
95% CI
RA
Linear scope (µg/mL)
µg/mL
mmol/L
6
y=3.8792x−2.5675
y=2.3377x+0.1483
y=3.2104x−0.1148
y=2.1307x+1.8600
y=7.6979x−7.4278
y=1.3165x+1.8491
0.9849
0.9679
0.9906
0.9550
0.9536
0.9804
89.3
119.0
39.2
0.51
0.51
0.18
0.13
0.23
0.28
86.8–91.9
109.4–129.3
37.8–40.7
2.8
2.1
6.3
8.3
6.0
1.0
60–250
60–360
16–120
16–120
30–70
1
2
2
3
5
6
7
0
29.8
24.8–35.8
41.2
39.9–42.5
Ivermectin
247.4
197.9–310.2
50–1600
a) y: Probability of average mortality; x: lg[C(µg/mL)]. b) 95% Confidence interval. c) Relative activity=LC50 (µg/mL) of ivermectin/LC50 (µg/mL) of the tested compound.

2
58
Chem. Pharm. Bull.
Vol. 63, No. 4 (2015)
Linear range (h)
Table 3. Toxicity Regression Equations for Time–Effect of the Compounds at 4.5µmol/mL and Their LT50 Values
a)
2
b)
c)
Compound
Regression equation
R
LT50 (h)
95% CI
RA
6
y=16.534x−11.969
y=8.5501x−3.9043
y=9.7377x−4.9128
y=11.965x−5.7690
y=10.376x−14.739
y=5.5047x−0.2254
0.9022
0.9912
0.9489
0.9277
0.9903
0.9840
10.6
11.0
10.4
7.9
10.5–10.7
10.9–11.1
10.2–10.7
7.8–8.1
0.84
0.81
0.86
1.13
6.85
1.00
10–14
8–19
8–16
7–11
1–2
1
2
2
3
5
6
7
0
1.3
1.3–1.3
Ivermectin
8.9
8.8–9.0
8–17
a) y: Probability of average mortality. For 6, 15, 26 and 27, x=lg[t(h)]; for 30, x=lg[t(min)]. b) 95% Confidence interval. c) Relative activity=LT50 of ivermectin/LT50 of
the tested compound.
probabilities and lg[concentration (µg/mL)] values in differ- of the carbon–carbon double bond in the acrylic ester moiety
2
ent concentration ranges (R >0.95) (Table 2). As expected, was not essential to improve the activity of cinnamic acid
6
, 15, 26, 27 and 30 displayed the smaller LC50 values of esters, which was evidenced by the fact that 6 (LC =89.3 µg/
50
29.8–119.0 µg/mL than ivermectin (LC =247.4 µg/mL) and mL, LT =10.6h) was less active than its dihydro derivative
5
0
50
their relative activities (RA) reached up to 2.8-, 2.1-, 6.3-, 30 (LC =41.2 µg/mL, LT =1.3h). Although the similar case
5
0
50
8
.3- and 6.0-fold the activity of ivermectin (Table 2). The was also found for HIV-1 integrase activity of cinnamic acid
53)
results above were agreement with that observed in the activ- esters, the double bond was thought to be crucial for other
ity screening experiment (Table 1). Among these compounds, bioactivities such as leishmanicidal and cytotoxic,
3
8)
anti-
2
2,24)
16)
27 showed the highest activity with a LC50 value of 29.8µg/ tuberculosis (TB)
and anti-candida albicans biofilm. The
mL followed by 26 and 30 (LC =39.2, 41.2µg/mL). In addi- leishmanicidal and cytotoxic action of cinnamic acid esters
5
0
38)
tion, comparison of the change trend of the various curves in had been explained as a Michael addition mechanism, in
Fig. 1A showed that various compounds had different activity which cinnamic acid esters as one type of α,β-unsaturated
sensitivity to the change of test concentration with the order of carbonyl compounds were considered to have a conjugated
3
0>27≈26>6>15>ivermectin.
addition reaction with nucleophilic groups in biomolecules of
Figure 1B showed that the activity of each compound at the natural receptors. This mechanism was also reported for
4
.5µmol/mL exhibited treatment-time-dependent effects in a other α,β-unsaturated carbonyl compounds such as lactones,
5
4–56)
certain time range. Linear regress analysis showed that each of chalcones and coumarins.
Obviously, the present results
the compounds showed a significant linear correlation between strongly suggest that the acaricidal action of cinnamic acid
the probability values of mite mortality and lg[treatment time esters might not be Michael addition mechanism and different
2
(
h or min)] values in a specific time range (R >0.90) (Table 3). action mechanisms may be responsible for the different bioac-
Compared with ivermectin with a LT50 value of 8.9h, 27 and tivity of cinnamic acid esters.
0 (LT =7.9, 1.3h) showed the higher activity whereas 6, 15 Furthermore, the conjecture above was also supported by
and 26 (LT =10.6, 11.0, 10.4h) showed the slightly lower ac- the fact that the effects of substituents on the benzene on the
3
5
0
5
0
tivity. Especially, the relative activity of 30 attained 6.85-fold acaricidal activity of cinnamic acid esters and other bioactivi-
of that of ivermectin. On the other hand, the change trend of ties are different. The present research showed that the intro-
the various curves in Fig. 1B presented that the activity sus- duction of a hydroxyl, methoxyl or acetoxyl on the benzene
ceptibilities of the various compounds to the treatment time ring did not improve the acaricidal activity. But in the leish-
3
8)
were as the following order: 30>27>26≈6>15>ivermectin.
manicidal activity and cytotoxicity of cinnamic acid esters,
57)
58)
SAR The present research showed that almost all the cin- chalcones and coumarins, the presence of hydroxyl or
namate derivatives have the acaricidal activity at a certain de- methoxyl groups led to enhancement of the activities.
gree (Table 1). Comparison of the activity and structure of the
From the point of view of median lethal mass concentra-
various compounds revealed that a substituent on the benzene tion values (µg/mL), compounds 6, 15, 26, 27 and 30 should
ring and its substitution site can significantly influence the theoretically have a great advantage in practice application
activity of ethyl cinnamate. Compared with an unsubstituted over ivermectin, due to their lower LC50 values (µg/mL) than
compound 6 (LC =89.3 µg/mL), the presence of o-NO or ivermectin (Table 2). However, as far as SAR analysis is con-
5
0
2
m-NO (26 and 27) led to a significant improvement of the cerned, researchers prefer to compare median lethal molar
2
activity (LC =39.2, 29.8 µg/mL) whereas p-NO derivative concentration values (mol/L) of various compounds. Though
5
0
2
(28) showed the lower activity (Table 1). Unlike the situation 6 and 15 had the different median lethal mass concentration
of nitro group, the introduction of a hydroxyl, methoxyl or values (µg/mL), lower than that of ivermectin, they had ap-
acetoxyl to any site on the benzene ring (7–15) led to reduce proximately the same median lethal molar concentration val-
the activity. Meanwhile, the activities of methoxyl-substituted ues (mol/L), larger than that ivermectin (Table 2). Therefore,
compounds (18–20) reduced with increasing the number for single molecule, 6 and 15 had the same acaricidal activity,
of substituted methoxy groups. In addition, the presence of indicating that the presence of 4-acetyl hardly influenced the
methylenedioxy, bromo or chloro groups at some sites also led activity (Fig. 2).
to reduction of the activity, such as 21–25.
As is shown in Table 3, at the same molar concentration
Compound 6 showed the higher activity than its configura- (4.5 µmol/mL), 6, 15, 26, 27 and 30 showed lower or slightly
tional isomer 29 (Table 1), indicating that the E configuration higher LT50 values (1.3–11.0h) than ivermectin (LT =8.9h).
50
of cinnamic acid esters is more beneficial for the activity than However, it was worth mentioning that the molecular weight
the corresponding Z configuration. Nevertheless, the presence of ivermectin (MW=875) is much larger than that of 6, 15,

Vol. 63, No. 4 (2015)
Chem. Pharm. Bull.
259
hai, China. Other chemicals used in the present study were
purchased from J&K Chemical Ltd., Beijing, China and used
without further purification.
Apparatus Melting points (mp) were determined on an
1
XT-4 micro-melting point apparatus and uncorrected. H- and
13
C-NMR spectra were recorded on a Bruker AVANCE III op-
erating at 500 or 400MHz, respectively and using tetramethyl
silane (TMS) as an internal standard. ESI-MS was measured
on a Trace mass spectrometer.
Synthesis of Compound 6 According to the literature’s
42)
method, compound 6 was obtained by the reaction of cin-
namic acid (1.48g, 10mmol) with absolute ethanol (15mL)
at 0°C to room temperature by dropwise addition of thionyl
chloride (3.57g, 30mmol).
(
E)-Ethyl Cinnamate (6): Colorless liquid in 95% yield. The
Fig. 2. The Structure of Ivermectin (22,23-Dihydroavermectin B Con-
sisting of B1a and B1b)
1
13
1
data of H- and C-NMR were consistent with those in the
literature. Positive ESI-MS m/z: 177 [M+H] .
Synthesis of Compounds 7–9, 11, 12 and 16–28 Ethyl
59)
+
2
6, 27 and 30 (MW=176–234). At the test concentration of triphenylphosphanylideneacetate ((C H ) P=CHCO Et) (4.2g,
6 5 3 2
4.5µmol/mL, the mass concentration of ivermectin (3.94mg/ 12mmol) reacted with aromatic aldehyde (10mmol) in 50mL
mL) was 3.6–5.0 times of that of 6, 15, 26 and 27 (0.8–1.1mg/ ethanol at reflux for 1–4h (compds. 7, 11, 12, 16–28) or 50mL
mL). Based on the results that at the same test molar concen- toluene at reflux for 0.5h (compds. 8 and 9) to yield the de-
43)
tration, 6, 15, 26, 27 and 30 possessed lower or slightly higher sired compounds according to the reported method with
LT50 values (1.3–11.0) than ivermectin (LT =8.9h), it was slight modification. After removal of the solvent, the resulting
50
deduced that 6, 15, 26 and 27 probably have much lower LT50 residue was subjected to a short silica gel column chroma-
values than ivermectin if the same test mass concentration tography (ϕ40mm×L 40mm) using petroleum ether–ethyl
was used, which may be evidenced by the results in Fig. 1A acetate as eluent to remove polar triphenylphosphine oxide.
to some degree.
For the preparation of compounds 8, 9, 19, 20, 22, 23, 26–28,
The present research strongly suggests that further research the obtained crude products were directly recrystallized in
should be necessary on the acaricidal mechanism of cinnamic petroleum ether–ethyl acetate (8, 9, 19, 20, 23, 26–28) or pe-
acid esters as well as more diverse structural modification troleum ether–ethanol (22); for the purification of the target
including cinnamamides, 3-aryl fatty acid esters or amides compounds 7, 11, 12, 16–18, 21, 24 and 25, the obtained crude
and even 3-aryl aliphatic ketones. At present, these works are products were re-chromatographed over silica gel (ϕ26mm×L
partly underway in our lab.
140mm or ϕ40mm×L 200mm) using petroleum ether–ethyl
acetate (7, 16, 17, 21) or petroleum ether–ethyl ether (11, 12,
18, 24, 25) as eluent.
Conclusion
In conclusion, the present study reported the synthesis of
a series of ethyl cinnamate derivatives and the acaricidal yield, mp 80–81°C (lit. 83–86°C). The data of H- and
activity in vitro against Psoroptes cuniculi, a mange mite.
Furthermore, the preliminary SAR was discussed. Almost tive ESI-MS m/z: 193 [M+H] .
all the compounds were found to have the activity in varying
degrees at 1.0mg/mL for post-treatment 24h and of which 5 tal in 64% yield, mp 65–66°C (lit. 67.7–68.7°C). H-NMR
(E)-Ethyl 2-Hydroxycinnamate (7): White crystal in 73%
6
0)
1
13
61)
C-NMR were consistent with those in the literature. Posi-
+
(E)-Ethyl 3-Hydroxycinnamate (8): White lamellar crys-
62)
1
showed much lower LC50 values and slightly lower or higher (500MHz, CDCl ) δ: 7.64 (1H, d, J=16.0Hz), 7.24 (1H, t,
3
LT50 values than a standard drug ivermectin. SAR showed that J=8.1Hz), 7.06–7.07 (2H, m), 6.91–6.93 (1H, m), 6.83 (1H,
the presence of o-NO or m-NO on the benzene ring led to brs, OH), 6.40 (1H, d, J=16.0Hz), 4.28 (2H, q, J=7.1Hz), 1.34
2
2
1
3
significant improvement of the activity. In contrast, a hydroxy, (3H, t, J=7.1Hz). C-NMR (125MHz, CDCl ) δ: 167.9, 156.5,
methoxy, acetoxy, methylenedioxy, bromo or chloro group 145.2, 135.7, 130.1, 120.6, 118.1, 117.8, 114.7, 61.0, 14.3. Posi-
led to activity reduction. The E-isomer was found to be more tive ESI-MS m/z: 193 [M+H] .
beneficial for improving the activity than the corresponding
Z-isomer. Nevertheless, the carbon–carbon double bond in the in 78% yield, mp 74–75°C (lit. 73°C). The data of H- and
acrylic ester moiety was proven not to be essential to improve
the activity of cinnamic acid esters. Thus, the present study tive ESI-MS m/z: 193 [M+H] .
strongly suggests that cinnamic acid esters and their dihydro
3
+
(E)-Ethyl 4-Hydroxycinnamate (9): White rod-like crystal
63)
1
13
64)
C-NMR were consistent with those in the literature. Posi-
+
65)
(E)-Ethyl 3-Methoxycinnamate (11): Yellow oil in 78%
1
derivatives are promising candidates or lead compounds for yield. The data of H-NMR were consistent with those in
the development of novel drugs for the effective control of the literature.
6
6) 13
C-NMR (125MHz, CDCl ) δ: 166.9, 159.9,
3
animal or human acariasis.
144.5, 135.9, 129.9, 120.8, 118.6, 116.1, 112.9, 60.5, 55.3, 14.3.
Positive ESI-MS m/z: 207 [M+H] .
(E)-Ethyl 4-Methoxycinnamate (12): Faint yellow powder in
+
Experimental
6
7)
1
Materials Ivermectin (≥91% 22,23-dihydroavermectin 62% yield, mp 45–46°C (lit. 48–50°C). The data of H- and
13
68)
B1 consisting of 95% avermectin B1a and 5% avermectin B1b)
C-NMR were consistent with those in the literature. Posi-
was purchased from Sigma-Aldrich Trading Co., Ltd., Shang- tive ESI-MS m/z: 207 [M+H] .
+

2
60
Chem. Pharm. Bull.
Vol. 63, No. 4 (2015)
6
7)
1
(
E)-Ethyl 2-Hydroxy-3-methoxycinnamate (16): White 64% yield, mp 135–136°C (lit. 138–140°C). The data of H-
6
9)
1
13
81)
crystal in 92% yield, mp 59–60°C (lit. 68–69°C). H-NMR and C-NMR were agreement with those in the literature.
−
(
400MHz, CDCl ) δ: 7.94 (1H, d, J=16.2Hz), 7.08 (1H, dd, Negative ESI-MS m/z: 221 [M] .
3
J=6.9, 2.4Hz), 6.81–6.87 (2H, m), 6.60 (1H, d, J=16.2Hz),
Synthesis of Compound 10 According to a typical me-
.20 (1H, s, –OH), 4.26 (2H, q, J=7.1Hz), 3.90 (3H, s), 1.34 thoxylation method of phenols, compound 7 (0.58g, 3.0mmol)
3H, t, J=7.1Hz). C-NMR (100MHz, CDCl ) δ: 167.5, 146.8, reacted with dimethyl sulfate (0.38g, 3.0mmol) in 20mL ace-
45.4, 139.5, 120.93, 120.89, 119.6, 119.3, 111.7, 60.3, 56.2, tone in the presence of potassium carbonate (0.5g, 3.6mmol)
6
(
1
1
13
3
−
4.4. Negative ESI-MS m/z: 221 [M−H] .
to yield compound 10.
(E)-Ethyl 4-Hydroxy-3-methoxycinnamate (17): White pow-
(E)-Ethyl 2-Methoxycinnamate (10): Faint yellow oil in
der in 55% yield, mp 42–43°C (lit. 57.3–58.0°C). The data 53% yield (lit. mp 33–34°C). The data of H-NMR were
of H- and C-NMR were consistent with those in the litera- agreement with those in the literature.
CDCl ) δ: 167.6, 158.4, 140.0, 131.4, 128.9, 123.5, 120.7, 118.8,
E)-Ethyl 3,4-Dimethoxylcinnamate (18): Faint yellow 111.1, 60.4, 55.5, 14.4. Positive ESI-MS m/z: 207 [M+H] .
Synthesis of Compounds 13–15 General Procedure Ac-
of H- and C-NMR were consistent with those in the litera- cording to a typical acetylation method of phenols, the solu-
70)
65)
1
1
13
66) 13
C-NMR (125MHz,
71)
−
ture. Negative ESI-MS m/z: 221 [M−H] .
(
3
+
7
2)
powder in 81% yield, mp 52–53°C (lit. 49–51°C). The data
1
13
7
3)
+
ture. Positive ESI-MS m/z: 237 [M+H] .
(
tion of compounds 7–9 (0.4g, 2.1mmol) and acetic anhydride
E)-Ethyl 2,4,5-Trimethoxylcinnamate (19): Faint yellow (0.6g, 6.2mmol) in 20mL triethylamine was stirred for 1h at
74)
1
crystal in 60% yield, mp 63–64°C (lit. 68–69°C). H-NMR room temperature to provide the desired compounds (13–15).
400MHz, CDCl ) δ: 7.97 (1H, d, J=16.0Hz), 7.01 (1H, s), (E)-Ethyl 2-Acetoxycinnamate (13): Faint yellow oil in 99%
(
6
3
1
13
.50 (1H, s), 6.37 (1H, d, J=16.0Hz), 4.28 (2H, q, J=7.1Hz), yield. The data of H- and C-NMR were consistent with
82)
+
3
.93 (3H, s), 3.88 (3H, s), 3.86 (3H, s), 1.34 (3H, t, J=7.1Hz). those in the literature. Positive ESI-MS m/z: 235 [M+H] .
(E)-Ethyl 3-Acetoxycinnamate (14) : Faint yellow oil
39.0, 115.3, 114.4, 110.2, 96.3, 59.7, 55.9, 55.8, 55.5, 13.9. Posi- in 94% yield. H-NMR (500MHz, CDCl ) δ: 7.65 (1H, d,
13
83)
C-NMR (100MHz, CDCl ) δ: 167.3, 153.3, 151.5, 142.7,
3
1
1
3
+
tive ESI-MS m/z: 267 [M+H] .
(
J=16.0Hz), 7.37–7.41 (2H, m), 7.25–7.26 (1H, m), 7.10–7.12
E)-Ethyl 3,4,5-Trimethoxylcinnamate (20): White needle (1H, m), 6.42 (1H, d, J=16.0Hz), 4.26 (2H, q, J=7.1Hz), 2.31
7
5
)
1
3
crystal in 42% yield, mp 66–67°C (lit. 68–69.5°C). The (3H, s), 1.33 (3H, t, J=7.1Hz). C-NMR (125MHz, CDCl3)
1
75)
data of H-NMR were consistent with those in the literature.
δ: 169.3, 166.7, 151.1, 143.4, 136.1, 129.9, 125.6, 123.4, 120.9,
C-NMR (100MHz, CDCl ) δ: 167.0, 153.4, 144.6, 140.0, 119.4, 60.6, 21.1, 14.3. Positive ESI-MS m/z: 235 [M+H] .
13
+
3
1
2
30.0, 117.5, 105.2, 61.0, 60.5, 56.1, 14.3. Positive ESI-MS m/z:
(E)-Ethyl 4-Acetoxycinnamate (15): White powder in 98%
yield, mp 37–38°C (lit. mp 40–42°C). The data of H- and
+
84)
1
67 [M+H] .
13
84)
(E)-Ethyl 3,4-Methylenedioxycinnamate (21): White crystal
C-NMR were consistent with those in the literature. Posi-
76)
1
+
in 78% yield, mp 59–60°C (lit. 68–70°C). The data of H- tive ESI-MS m/z: 235 [M+H] .
13
76)
and C-NMR were consistent with those in the literature.
Positive ESI-MS m/z: 221 [M+H] .
(
Synthesis of Compound 29 According to the literature
method, compound 29 was obtained by HWE reaction of
E)-Ethyl 2-Hydroxy-4,5-methylenedioxycinnamate (22): ethyl 2-(bis(2-(tert-butyl)phenoxy)phosphoryl)acetate (0.476g,
1.1mmol) with benzaldehyde (0.106g, 1mmol) 15mL anhy-
49–150°C). The data of H- and C-NMR were agreement drous tetrahydrofuran in the presence of KOH (0.09g, 1mmol)
Positive ESI-MS m/z: 237 at 0°C for 2h.
(Z)-Ethyl Cinnamate (29): Colorless liquid in 73% yield.
E)-Ethyl 2-Bromo-4,5-methylenedioxycinnamate (23): The H-NMR data were consistence with those in the litera-
+
44)
43)
Bright yellow crystal in 76% yield, mp 149–150°C (lit.
1
13
1
43)
with those in the literature.
M+H] .
(
Faint yellow needle crystal in 58% yield, mp 113–114°C (lit.
mp 78–80°C). The data of H- and C-NMR were consis-
+
[
1
7
7)
59)
ture.
Synthesis of Compound 30 According to the literature
tent with those in the literature. Positive ESI-MS m/z: 299 method, compound 30 was prepared by reduction of com-
1
13
7
7)
45)
+
[
M+H] .
(
pound 6 (0.88g, 5mmol) with borohydride (0.19g, 5mmol) in
E)-Ethyl 4-Chlorocinnamate (24): Yellow liquid in 83% 20mL methanol in the presence of cuprous chloride (0.29g,
6
8)
1
13
yield. The data of H- and C-NMR were agreement with 3mmol).
those in the literature. Positive ESI-MS m/z: 211 [M+H] .
(
yield. The data of H- and C-NMR were agreement with those in the literature.
those in the literature. Positive ESI-MS m/z: 279 [M+Na] .
(
6
8)
+
Ethyl 3-Phenylpropionate (30): Colorless liquid in 95%
1
13
E)-Ethyl 4-Bromocinnamate (25): Yellow liquid in 87% yield. The data of H- and C-NMR were agreement with
1
13
85)
78)
+
Pharmacology In Vitro Acaricidal Activity Assay In
E)-Ethyl 2-Nitrocinnamate (26): Faint yellow crystal in vitro acaricidal activity of 6–30 was performed according to
2% yield, mp 38–39°C (lit. 42°C). The data of H-NMR our previously reported method.
79)
1
50–52)
5
Ivermectin, a standard
6
6) 13
were agreement with those in the literature.
C-NMR acaricidal drug, was used as a reference control. All the tested
(
125MHz, CDCl ) δ: 165.8, 148.4, 139.8, 133.5, 130.7, 130.3, compounds were dissolved in a mixed solvent of dimethyl
3
1
29.1, 124.9, 123.4, 60.9, 14.3. Negative ESI-MS m/z: 221 sulfoxide (DMSO), Tween-80 and normal saline (1:1:8, v/v/v)
−
[
M] .
to prepare the test solution with the concentration of 1, 0.5
(
E)-Ethyl 3-Nitrocinnamate (27): Faint yellow needle crys- or 0.25mg/mL. Psoroptes cuniculi adult mites of both sexes
8
0)
1
tal in 57% yield, mp 73–74°C (lit. 74–75°C). The data of H- isolated from naturally infected rabbits were used as the tested
and C-NMR were agreement with those in the literature.
Negative ESI-MS m/z: 221 [M] .
13
80)
objects. The scabs and the cerumen, collected from the infect-
ed ears, were observed by means of a stereoscopic microscope
−
(E)-Ethyl 4-Nitrocinnamate (28): Yellow rod-like crystal in to isolate adult mites of both sexes. Mites were placed in 24-

Vol. 63, No. 4 (2015)
Chem. Pharm. Bull.
261
well flat-bottomed cell culture plates (10 adult mites per each tional Natural Science Foundation of China (NSFC 31172365
well) and followed by addition of 0.6mL of the tested solution and 31101469).
into each well. Each 20 mites in 2 wells were set as one test
and three replicates were made for each concentration. The
Conflict of Interest The authors declare no conflict of
same solution without the tested compound was used as an interest.
untreated control. Ivermectin in the same solvent represented
the treated control.
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