Synthesis of ginkgolic acid analogues and evaluation of their molluscicidal activity

Article abstract of DOI:10.3390/molecules16054059

Based on the molluscicidal activity of ginkgolic acids (GAs) isolated from Ginkgo biloba L, a series of Z/E isomers of GA analogues were prepared and evaluated for their molluscicidal activities against the host snail Oncomelania hupensis. The results and analysis of the structure-activity relationship revealed that the E-isomers showed better molluscicidal activities than their Z-isomers. Molluscicidal activities decreased with the shortening of the alkenyl chain lengths.

Full text of DOI:10.3390/molecules16054059

Molecules 2011, 16, 4059-4069; doi:10.3390/molecules16054059
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Ginkgolic Acid Analogues and Evaluation of Their
Molluscicidal Activity
Peng Zhang 1, Jiahu Pan 1,Wanxing Duan 2, Xuedong Li 2, Yao Zhang 1, Yibiao Zhou 3,
Qingwu Jiang 3, Zuohua Mao 2,* and Peizhong Yu 1,*
1
School of Pharmacy, Fudan University, Shanghai 201203, China
2
Shanghai Medical College, Fudan University, Shanghai 200032, China
3
School of Public Health, Fudan University, Shanghai 200032, China
* Author to whom correspondence should be addressed; E-Mails: zhmao@shmu.edu.cn (Z.M.);
pzyu@fudan.edu.cn (P.Y.); Tel.: +86 21 54237197 (Z.M.); E-Mail: Tel.: +86 21 51980103 (P.Y.).
Received: 16 March 2011; in revised form: 23 April 2011 / Accepted: 9 May 2011 /
Published: 17 May 2011
Abstract: Based on the molluscicidal activity of ginkgolic acids (GAs) isolated from
Ginkgo biloba L, a series of Z/E isomers of GA analogues were prepared and evaluated for
their molluscicidal activities against the host snail Oncomelania hupensis. The results and
analysis of the structure-activity relationship revealed that the E-isomers showed better
molluscicidal activities than their Z-isomers. Molluscicidal activities decreased with the
shortening of the alkenyl chain lengths.
Keywords: ginkgolic acid analogues; synthesis; molluscicidal activity; snail; schistosomiasis
1. Introduction
Schistosomiasis ranks as the second most prevalent parasitic disease in tropical and subtropical
regions of the World after malaria. It is estimated that more than 200 million people are infected and
600 million at risk of infection in approximately 74 countries [1]. China is currently facing a new
challenge because this disease has re-emerged in some regions due to an increase in habitats suitable
for the snails, these including increased flood areas and large water resource development projects [2].
The snail Oncomelania hupensis is the main host responsible for the transmission of this disease
in China.

Molecules 2011, 16
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The use of molluscicides is still considered the most important means to control the transmission of
schistosoma parasites by host snails. Although the molluscicide niclosamide (Nic) is the only
commercially available molluscicide recommended by the WHO [3], its use is hampered by its high
cost and toxicity to fish. This factor has stimulated new avenues of research to identify novel
molluscidal agents from natural sources [4-8] and then to improve the molluscicidal properties of
known natural compounds, leading to the synthesis of new chemicals analogues with increased
activities [9-12].
We have previously described the molluscicidal activities of ginkgolic acids (GAs), a group of
6-alkylsalicylic acids, extracted from the sarcotesta and leaves of Ginkgo biloba [13,14]. Five GA
monomers were identified and all differed in their chain lengths (13~17 carbon atoms). The alkyl chain
was also found to contain different numbers (0~2) of carbon double bonds. The order of molluscicidal
activity for these five monomers were C13:0 ~C15:1 > C15:0 > C17:1 > C17:2. The LD50 values after 24 and
48 hours were 20.79 mg·L−1 and 5.06 mg·L−1 for C13:0, 27.28 mg·L−1 and 3.27 mg·L−1 for C15:1
,
respectively [14]. Besides the GAs found in the sarcotesta and leaves of Ginkgo biloba, additional
6-alkylsalicylic acid derivatives have also been found in other plant species, e.g. anacardic acids
isolated from the cashew (Anacardium occidentale). They have similar structures to GA C15:1 but have
a different number (1~3) of double bonds in the alkyl chain. Kubo and colleagues found these
compounds to have molluscicidal activities against the South American snail Biomphalaria glabratus
with LD50 values of 1 mg·L−1 for C15:1, 0.6 mg·L−1 for C15:2, 0.3 mg·L−1 for C15:3, respectively, proving
that the double bond is related to their activity [15].
All of the known GAs and anacardic acids are derivatives of salicylic acid each having a long
carbon side chain substituted at the 6-position. It is suggested that the alkyl group side chain plays an
important role in their molluscicidal activities. The major differences in these compounds are the
carbon side chain lengths and the number of unsaturated bonds present. No naturally occuring GAs or
anacardic acids having side chain length less than 13 carbons in length have been identified.
In order to understand the structure-activity relationship for these compounds and improve their
molluscicidal activity, in this study a series of Z/E isomers of GA analogues with different chain
lengths (5~13 carbon atoms) and phenyl rings based on one added inside double bond have been
synthesized from 6-methylsalicylic acid. Their molluscicidal effects against the host snail
Oncomelania hupensis were evaluated, and the structure-activity relationships were explored.
2. Results and Discussion
2.1. Synthesis of the compounds
The unhydrolyzed mixtures of Z and E isomers 5a–5g were synthesized from ethyl 6-
methylsalicylate according to the reaction sequence delineated in Scheme 1 [16]. These were separated
by column chromatography on silica gel. Target compounds 8a–8g, 9a–9g were obtained by ester
hydrolysis and extracted from acidic solution with chloroform. Based on the spin–spin coupling
constants of the double bond protons, the isomer with smaller coupling constant (J = 11~12 Hz) was
assigned to the Z-configuration, and the larger coupling constant (J = 15~16 Hz) was assigned to the
E configuration.

Molecules 2011, 16
Scheme 1. Synthesis of GA analogues from ethyl 6-methylsalicylate.
4061
OH
O
AcO
O
AcO
O
AcO
O
OEt
a
c
OEt
b
OEt
OEt
PPh3
Br
Me
Me
CH2Br
1
2
3
4
Z
E
Z/E
6a 29.2%
6b 25.7%
6c 20.5%
37/63
33/67
35/65
32/68
36/54
40/60
50/50
7a 50.4%
7b 52.3%
7c 37.3%
AcO
O
AcO
O
AcO
O
d
OEt
e
OEt
OEt
6d 16.4% 7d 34.4%
7e 30.2%
7f 27.7%
7g 35.8%
R
6e 21.2%
6f 18.7%
6g 35.8%
R
R
5a-5g
6a-6g
7a-7g
a. R = CH2CH2CH3
b. R = CH2(CH2)3CH3
c. R = CH2(CH2)5CH3
d. R = CH2(CH2)7CH3
e. R = CH2(CH2)8CH3
f. R = CH2(CH2)9CH3
g. R = Phenyl
OH
O
OH
O
f
OH
OH
R
R
8a-8g
9a-9g
Reagents and conditions: (a) (CH3CO)2O, H2SO4, 70 °C, 15min, 95%; (b) CCl4, NBS, AIBN, 90%;
(c) Ph3P, 0 to 130 °C, CHCl3, (CH3CH2)2O, 98%; (d) R-CHO, Et3N, DMSO, 125 °C, 21~26 h,
46.4~80%; (e) separated by silica gel column chromatography with gradient mixtures of n-hexane-
acetone (30:1); (f) EtOH, NaOH, reflux, 5~8 h, 83~96%.
2.2. Assay for molluscicidal activity
The results of the molluscicidal assays and the representative LD10, LD50, and LD90 values with the
confidence limit are shown in Table 1. The above results revealed that all of the E-isomers of the
synthesized GA analogues had better molluscicidal activities than their Z-isomers. According to the
LD50 values 9f(E) and 9d(E) showed the strongest activities among all of the analogues tested. Indeed,
9f and 9d were found to have greater activity than that of GA C13:0, the most active ingredient among
the natural GAs, proving that the molluscicidal activity was improved by the simple addition of an E
double bond in the alkyl chain. Molluscicidal activities were also found to decrease with the shortening
of the alkyl chain lengths in the following sequence: f > d >c > b > a. The respective LD50 values for
these compounds were 22.3 μM, 23.6 μM, 27.5 μM, 54.2 μM, and 56.1 μM, respectively. The
molluscicidal activity was dramatically decreased when the number of carbon atoms was less than 7.
The results of the LD10 and LD90 data also showed a similar pattern to that of the LD50. This pattern of
molluscicidal activity were similar to the rules revealed by de Villiers for the varying length acyl chain
of one or disubstituted amides [17].

Molecules 2011, 16
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Table 1. Molluscicidal activity (μM) of the Z/E isomers of GA analogues against snail
O.hupensis under laboratory conditions and 48 h exposure.
Compound
LD10 (C.L.)a
8 (Z)
LD50 (C.L.)a
8 (Z)
107.2
LD90 (C.L.)a
8 (Z)
786.6
9 (E)
14.6
9 (E)
9 (E)
115.9
a
b
c
d
e
f
27.2
(9.3–44.1)
33.6
56.1
(7.6–57.8)
22
(32.4–133.8) (16.7–122.6) (322.6–1213.8) (35.1–643.1)
73.1
54.2
243.6
(134.9–1215)
98.2
214.5
(78.3–462.8)
74.3
(7.5–75.6)
12.5
(6.9–35.3)
6.5
(48.4–130.4) (21.4–132.2)
50.9
(6.5–68.7)
43.7
27.5
(14.8–87.1)
23.6
(4.8–19.6)
18.4
(0.8–9.1)
10.9
(25.5–150.6)
135.7
(34.2–95.0)
68.3
(1.7–27.7)
11.1
(1.6–17.3)
7.7
(22.9–89.2)
56.9
(10.5–26.9)
31.4
(14.2–199.3)
362.9
(63.4–801.6)
88.7
(3.0–17.0)
13.3
(0.3–15.0)
5.5
(24.9–63.6)
47.2
(21.1–80.3)
22.4
(83.4–595.5)
338.7
(46.7–121.1)
50.9
(0.3–26.6)
14.6
(1.5–6.2)
11.4
(31.4–57.7)
64.4
(4.3–25.3)
37.2
(82.2–364.6)
283.9
(23.3–62.8)
122.0
g
(3.3–25.9)
(3.6–18.8)
(40.1–127.7) (23.9–57.0)
(138.6–2001)
(74.6–366.6)
GA C13:0
*
1.6
29.2
(4.2–60.8)
1.5
64.6
(0.8–8.6)
0.7
(37.7–180.2)
3.4
Niclosamide
(0.3–0.9)
(1.1–2.1)
(2.3–7.9)
The above data were the average from 4 dependent experiments; * GA C13:0 was isolated from the
sarcotesta and leave of Ginkgo biloba L.; a Confidence limit.
3. Experimental
3.1. General
Melting points were determined on an X4 micro-melting point apparatus, and are uncorrected. IR
1
spectra (KBr) were obtained on Avatar 360 ESP FT-IR spectrometer (v in cm–1). H-NMR spectra
were recorded in acetone-d6 or CDCl3 on a Varian Mercury Plus 400 NMR spectrometer or a Bruker
Avance III 400 MHz NMR spectrometer. The chemical shifts (δ) were given in ppm and the coupling
constants (J) in Hz. MS spectra were measured with a Thermo-Finnigan model LCQ Classic
LC/MS/MS ion trap mass spectrometer and Agilent 1100 chromatography system coupled with an
Agilent G1946D mass detector. High-resolution electrospray ionization mass spectra (HRESIMS)
were measured on a Bruker Daltonics micrOTOF-QII mass spectrometer. Column chromatography
(CC) was performed with silica gel 200–300 mesh (Qingdao Haiyang Chemical Co., Ltd.) and C18
reversed-phase silica gel 15–35 μm (Unicorn). Different aldehydes were purchased from Shanghai
Jingchun Reagent Co., Ltd. Other reagents were purchased from commercial suppliers and used as
received, unless otherwise noted.

Molecules 2011, 16
4063
3.2. Synthesis
3.2.1. Ethyl 6-methylsalicylate (1)
Crude 1 was synthesized according to Hamada as a brown oil [18]. The pure colorless crystals of
1
ethyl 6-methylsahcylate were obtained by steam distillation; Mp: 43–44 °C; H-NMR (CDCl3,
400 MHz) δ1.43 (t, J = 7.0 Hz, 3H), 2.55 (s, 3H), 4.43 (q, J = 7.0 Hz, 2H), 6.70 (d, J = 7.4 Hz, 1H),
6.83 (d, J = 8.2 Hz, 1H), 7.26 (dd, J = 8.2, 7.4 Hz, 1H), 11.38 (s, 1H); ESIMS (positive) m/z: 181.1
([M+H]+).
3.2.2. Ethyl O-acetyl-6-methylsalicylate (2)
A mixture of acetic anhydride (18 mL) and 6-methylsalicylate (7.2 g, 40 mmol), was stirred at room
temperature until the solid dissolved completely. Sulfuric acid (18 drops) was then added dropwise.
After 10 min, the mixture was placed into an oil bath of 70 °C for 15 min and then left to cool to room
temperature. The reaction mixture was then poured into ice-cold water (50 mL), extracted three times
with chloroform (50 mL), and the organic layer was washed with saturated sodium bicarbonate
solution several times until no bubble emerges. The sample was then dried over Na2SO4 to give the
desired product 2 as a yellow oil (8.46 g, 95%) [19]. 1H-NMR (CDCl3, 400 MHz) δ 1.37 (t, J = 7.1 Hz,
3H), 2.04 (s, 3H), 2.41 (s, 3H), 4.37 (q, J = 7.1 Hz, 2H), 6.96 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 7.5 Hz,
1H), 7.32 (dd, J = 8.3, 7.5 Hz, 1H).
3.2.3. Ethyl O-acetyl-6-bromomethylsalicylate (3)
To a stirred solution of compound 2 (8.34 g, 37.6 mmol) dissolved in carbon tetrachloride (60 mL)
were added a mixture of N-bromosuccinimide (7.44 g, 41.8 mmol) and azobisisobutyronitrile (246 mg,
0.15 mmol). This solution was then heated at reflux for 3 h. After cooling, the reaction mixture was
filtered and the filtrate was condensed under reduced pressure to give the crude product
Chromatographic purification of the crude material (silica gel, petroleum ether-EtOAc = 15:1) gave the
bromide 3 (10.24 g, 90%) as a white powder. This power was directly used for the next reaction
without further identification.
3.2.4. 3-Acetoxy-2-ethoxycarbonylbenzyltriphenylphosphonium bromide (4)
A mixture of compound 3 (10.2 g, 34 mmol) and triphenylphosphine (8.91 g, 34 mmol) was added
to a magnetically stirred round bottomed flask at 0 °C, and then slowly heated to 130 °C. After all the
power melted together, the reaction mixture was cooled to 70 °C. A small amount of chloroform was
added and refluxed until all the solid had dissolved. Anhydrous ether (30 mL) was then added and the
reaction mixture was filtered and to give the phosphonium salt as a slightly yellow powder (18.7 g,
98%).
3.2.5. General procedure for preparation of ethyl O-acetyl-6-(1-alkenyl)salicylates 5a–5g
The phosphonium salt 4 (563 mg, 1 mmol) was dissolved in DMSO (5 mL). Triethylamine
(0.170 mL, 1.2 mmol) and different aldehydes was added. The reaction mixture was heated to 125 °C

Molecules 2011, 16
4064
for 21–26 h, then allowed to cool to ambient temperature, poured onto ice-cold water (25 mL), and
extracted three times with ether (each 50 mL). The organic layer was washed with water (50 mL) and
saturated brine (50 mL), and dried over Na2SO4. The solution was then filtered, and concentrated
under reduced pressure to give the crude product as a mixture of geometric isomers.
3.2.6. Separation of Z and E isomers 6a–6g and 7a–7g of ethyl O-acetyl-6-(1-alkenyl)salicylates
Chromatographic separation of the crude material (silica gel, n-hexane and acetone, about 30:1)
gave the Z (6a–6g) and E (7a–7g) isomers as yellow oils with a ratio of 37/63 to 50/50 (Z/E) (Scheme 1).
3.2.7. General procedure for preparation of 6-(1-alkenyl)salicylic acids
Triple molecular quantities of 1N NaOH and the ester 6a–6g and 7a–7g, in EtOH was heated at
reflux for 5–8 h, respectively. After removal of the solvent, the residue was acidified to pH 2 with 10%
HCl, diluted with water, and extracted three times with dichloromethane. The organic layer was
washed with water and saturated brine, dried over Na2SO4, and concentrated in vacuo to give the crude
products. These were purified by chromatography (C18, MeOH-H2O = 17:3) to obtain the salicylic
acids (Z-isomer 8a–8g and E-isomer 9a–9g) as yellowish or white powders.
3.2.8. (Z)-6-(1-Pentenyl)salicylic acid (8a)
(Z)-6-(1-Pentenyl)salicylic acid (8a, 49.1 mg, 94%) was obtained as a yellowish powder from the
hydrolysis of 6a (70 mg, 0.25 mmol) by 1N NaOH (0.75 mL, 0.75 mmol) in EtOH (1.54 mL) for 5 h.
Mp 85–86 °C; IR (KBr, cm−1) ν 3425, 3027, 2956, 2870, 1646, 1602, 1486, 1448, 1301, 1237, 1164;
1H-NMR (acetone-d6, 400 MHz) δ 0.85 (t, J = 7.4 Hz, 3H), 1.40 (m, 2H), 2.08 (m, 2H), 5.63 (dt,
J = 11.4, 7.4 Hz, 1H), 6.76 (d, J = 7.4 Hz, 1H), 6.85 (d, J = 11.4 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 7.43
(dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 205.0854. Calcd: 205.0865 for C12H13O3 ([M-H]−).
3.2.9. (E)-6-(1-Pentenyl)salicylic acid (9a)
(E)-6-(1-Pentenyl)salicylic acid (9a, 85.4 mg, 88%) was obtained as a yellowish powder from the
hydrolysis of 7a (130 mg, 0.47 mmol) by 1N NaOH (1.41 mL, 1.41 mmol) in EtOH (2.86 mL) for 6 h.
1
Mp 96–98 °C, IR (KBr, cm−1) ν 3414, 3052, 2958, 2930, 2873, 1652, 1602, 1445, 1245, 1170; H-
NMR (acetone-d6, 400 MHz) δ 0.95 (t, J = 7.2 Hz, 3H), 1.47 (m, 2H), 2.19 (m, 2H), 6.04 (dt, J = 15.7,
7.0 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 15.7 Hz, 1H), 7.38 (dd,
J = 8.2, 7.8 Hz, 1H); HRESIMS found: 205.0807. Calcd: 205.0865 for C12H13O3 ([M-H]−).
3.2.10. (Z)-6-(1-Heptenyl)salicylic acid (8b)
(Z)-6-(1-Heptenyl)salicylic acid (8b, 46.5 mg, 93%) was obtained as a yellowish powder from the
hydrolysis of 6b (65 mg, 0.21 mmol) by 1N NaOH (0.63 mL, 0.63 mmol) in EtOH (1.43 mL) for 5 h.
Mp 55–56 °C, IR (KBr, cm−1) ν 3423, 3058, 2959, 2925, 2844, 1652, 1599, 1455, 1384, 1113;
1H-NMR (acetone-d6, 400 MHz) δ 0.83 (t, J = 7.0 Hz, 3H), 1.23 (m, 4H), 1.40 (m, 2H), 2.10 (m, 2H),
5.63 (dt, J = 11.7, 7.4 Hz, 1H), 6.76 (d, J = 7.4 Hz, 1H), 6.81 (d, J = 11.7 Hz, 1H), 6.87 (d, J = 8.2 Hz,
1H), 7.43 (dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 233.1166. Calcd: 233.1178 for C14H17O3 ([M-H]−).

Molecules 2011, 16
4065
3.2.11. (E)-6-(1-Heptenyl)salicylic acid (9b)
(E)-6-(1-Heptenyl)salicylic acid (9b, 108.4 mg, 88%) was obtained as a yellowish powder from the
hydrolysis of 7b (160 mg, 0.53 mmol) by 1N NaOH (1.59 mL, 1.59 mmol) in EtOH (3.52 mL) for 6 h.
Mp 70–72 °C, IR (KBr, cm−1) ν 3430, 3035, 2957, 2925, 2851, 1655, 1601, 1446, 1243, 1219, 1171,
1064; 1H-NMR (acetone-d6, 400 MHz) δ 0.87 (m, 3H), 1.35 (m, 4H), 1.49 (m, 2H), 2.21 (m, 2H), 6.04
(dt, J = 15.7, 6.7 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 7.4 Hz, 1H), 7.11 (d, J = 15.7 Hz, 1H),
7.37 (J = 8.2, 7.4 Hz, 1H); HRESIMS found: 257.1142. Calcd: 257.1154 for C14H18NaO3 ([M+Na]+).
3.2.12. (Z)-6-(1-Nonenyl)salicylic acid (8c)
(Z)-6-(1-Nonenyl)salicylic acid (8c, 44.5 mg, 94%) was obtained as a yellowish powder from the
hydrolysis of 6c (60 mg, 0.18 mmol) by 1N NaOH (0.54 mL, 0.54 mmol) in EtOH (1.32 mL) for 5 h.
Mp 40–42 °C; IR (KBr, cm–1) ν 3421, 3029, 2956, 2924, 2854, 1651, 1601, 1449, 1384, 1298, 1188;
1H-NMR (acetone-d6, 400 MHz) δ 0.84 (t, J = 7.0 Hz, 3H), 1.22 (m, 8H), 1.40 (m, 2H), 2.11 (m, 2H),
5.62 (dt, J = 11.4, 7.4 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 11.4 Hz, 1H), 6.87 (d, J = 8.2 Hz,
1H), 7.42 (dd, J = 8.2, 7.8 Hz, 1H); HRESIMS found: 261.1433. Calcd: 261.1491 for C16H21O3
([M-H]−).
3.2.13. (E)-6-(1-Nonenyl)salicylic acid (9c)
(E)-6-(1-Nonenyl)salicylic acid (9c, 79 mg, 87%) was obtained as a yellowish powder from the
hydrolysis of 7c (115 mg, 0.35 mmol) by 1N NaOH (1.05 mL, 1.05 mmol) in EtOH (2.53 mL) for 7 h.
Mp 75–77 °C, IR (KBr, cm−1) ν 3416, 3058, 2959, 2925, 2850, 1640, 1601, 1440, 1300, 1215, 1171,
707; 1H-NMR (acetone-d6, 400 MHz) δ 0.88 (t, J = 7.0 Hz, 3H), 1.22 (m, 8H), 1.49 (m, 2H), 2.22 (m,
2H), 6.05 (dt, J = 15.7, 6.7 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 7.4 Hz, 1H), 7.13 (d,
J = 15.7 Hz, 1H), 7.37 (dd, J = 8.6, 7.4 Hz, 1H); HRESIMS found: 261.1486. Calcd: 261.1491 for
C16H21O3 ([M-H]−).
3.2.14. (Z)-6-(1-Undecenyl)salicylic acid (8d)
(Z)-6-(1-Undecenyl)salicylic acid (8d, 37.2 mg, 92%) was obtained as a yellowish powder from the
hydrolysis of 6d (50 mg, 0.14 mmol) by 1N NaOH (0.42 mL, 0.42 mmol) in EtOH (1.10 mL) for 5 h.
Mp 47–49 °C; IR (KBr, cm−1) ν 3418, 3046, 2956, 2924, 2853, 1650, 1634, 1586, 1455, 1384, 1206,
1114, 1063; 1H-NMR (CDCl3, 400 MHz) δ 0.89 (t, J = 6.8 Hz, 3H), 1.24 (m, 12H), 1.41 (m, 2H), 2.09
(m, 2H), 5.82 (dt, J = 11.2, 7.6 Hz, 1H), 6.75 (d, J = 7.6 Hz, 1H), 6.79 (d, J = 11.2 Hz, 1H), 6.96 (d,
J = 8.4 Hz, 1H), 7.25 (dd, J = 8.4, 7.6 Hz, 1H); HRESIMS found: 289.1835. Calcd: 289.1804 for
C18H25O3 ([M-H]−).
3.2.15. (E)-6-(1-Undecenyl)salicylic acid (9d)
(E)-6-(1-Undecenyl)salicylic acid (9d, 73.9 mg, 83%) was obtained as a yellowish powder from the
hydrolysis of 7d (110 mg, 0.31 mmol) by 1N NaOH (0.93 mL, 0.93 mmol) in EtOH (2.42 mL) for 7 h.
Mp 79–81 °C; IR (KBr, cm−1) ν 3444, 3058, 2960, 2922, 2849, 1642, 1600, 1440, 1213, 1171;

Molecules 2011, 16
4066
1H-NMR (acetone-d6, 400 MHz) δ 0.87 (t, J = 6.7 Hz, 3H), 1.29 (m, 12H), 1.48 (m, 2H), 2.22 (m, 2H),
6.04 (dt, J = 15.7, 6.7 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 7.12 (d, J = 15.7 Hz,
1H), 7.37 (dd, J = 8.2, 7.8 Hz, 1H); HRESIMS found: 289.1787. Calcd: 289.1804 for C18H25O3
([M-H]−).
3.2.16. (Z)-6-(1-Dodecenyl)salicylic acid (8e)
(Z)-6-(1-Dodecenyl)salicylic acid (8e, 54.8 mg, 96%) was obtained as a white powder from the
hydrolysis of 6e (70 mg, 0.19 mmol) by 1N NaOH (0.57 mL, 0.57 mmol) in EtOH (1.54 mL) for 5 h.
Mp 52–54 °C; IR (KBr, cm−1) ν 3386, 3058, 2956 ,2917, 2850, 1728, 1659, 1600, 1450, 1383, 1288,
1
1189; H-NMR (acetone-d6, 400 MHz) δ 0.85 (t, J = 7.0 Hz, 3H), 1.25 (m, 14H), 1.36 (m, 2H), 2.10
(m, 2H), 5.61 (dt, J = 11.4, 7.4 Hz, 1H), 6.75 (d, J = 7.4 Hz, 1H), 6.81 (d, J = 11.4 Hz, 1H), 6.86 (d,
J = 8.2 Hz, 1H), 7.41 (dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 303.1927. Calcd: 303.1960 for
C19H27O3 ([M-H]−).
3.2.17. (E)-6-(1-Dodecenyl)salicylic acid (9e)
(E)-6-(1-Dodecenyl)salicylic acid (9e, 75 mg, 88%) was obtained as a white powder from the
hydrolysis of 7e (105 mg, 0.28 mmol) by 1N NaOH (0.84 mL, 0.84 mmol) in EtOH (2.31 mL) for 8 h.
Mp 82–84 °C; IR (KBr, cm−1) ν 3446, 3058, 2959, 2922, 2850, 1641, 1601, 1469, 1443, 1302, 1216,
1
1170; H-NMR (acetone-d6, 400 MHz) δ 0.87 (t, J = 7.0 Hz, 3H), 1.28 (m, 14H), 1.48 (m, 2H), 2.22
(m, 2H), 6.04 (dt, J = 15.7, 6.7 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 7.4 Hz, 1H), 7.12 (d,
J = 15.7 Hz, 1H), 7.37 (dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 303.1966. Calcd: 303.1960 for
C19H27O3 ([M-H]−).
3.2.18. (Z)-6-(1-Tridecenyl)salicylic acid (8f)
(Z)-6-(1-Tridecenyl)salicylic acid (8f, 47.3 mg, 89%) was obtained as a white powder from the
hydrolysis of 6f (65 mg, 0.17 mmol) by 1N NaOH (0.51 mL, 0.51 mmol) in EtOH (1.43 mL) for 5 h.
Mp 55–57 °C; IR (KBr, cm−1) ν 3431, 3055, 2957, 2921, 2850, 1731, 1656, 1600, 1448, 1384, 1300,
1
1189; H-NMR (acetone-d6, 400 MHz) δ 0.87 (t, J = 6.7 Hz, 3H), 1.25 (m, 16H), 1.37 (m, 2H), 2.11
(m, 2H), 5.62 (dt, J = 11.3, 7.4 Hz, 1H), 6.76 (d, J = 7.4 Hz, 1H), 6.83 (d, J = 11.3 Hz, 1H), 6.87 (d,
J = 8.2 Hz, 1H), 7.4 (dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 317.2152. Calcd: 317.2117 for
C20H29O3 ([M-H]−).
3.2.19. (E)-6-(1-Tridecenyl)salicylic acid (9f)
(E)-6-(1-Tridecenyl)salicylic acid (9f, 73.8 mg, 90%) was obtained as a white powder from the
hydrolysis of 7f (100 mg, 0.26 mmol) by 1N NaOH (0.78 mL, 0.78 mmol) in EtOH (2.2 mL) for 8 h.
Mp 90–92 °C; IR (KBr, cm−1) ν 3425, 3063, 2961, 2920, 2848, 1642, 1601, 1467, 1439, 1304, 1214,
1
1171; H-NMR (acetone-d6, 400 MHz) δ 0.87 (t, J = 7.0 Hz, 3H), 1.29 (m, 16H), 1.49 (m, 2H), 2.22
(m, 2H), 6.05 (dt, J = 15.7, 7.0 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 7.4 Hz,1H), 7.11 (d,
J = 15.7 Hz, 1H), 7.37 (dd, J = 8.2, 7.4 Hz, 1H); HRESIMS found: 317.2114. Calcd: 317.2117 for
C20H29O3 ([M-H]−).

Molecules 2011, 16
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3.2.20. (Z)-6-Phenylethenyl salicylic acid (8g)
(Z)-6-Phenylethylenyl salicylic acid (8g, 72 mg, 93%) was obtained as a yellowish powder from the
hydrolysis of 6g (100 mg, 0.32 mmol) by 1N NaOH (0.96 mL, 0.96 mmol) in EtOH (2.2 mL) for 5 h.
Mp 84–86 °C; IR (KBr, cm−1) ν 3422, 3059, 3025, 2948, 2924, 2852, 1733, 1652, 1599, 1448, 1398,
1
1297, 1205, 1171; H-NMR (acetone-d6, 400 MHz) δ 6.57 (d, J = 12.1 Hz, 1H), 6.62 (d, J = 7.4 Hz,
1H), 6.85 (d, J = 8.6 Hz, 1H), 7.05–7.18 (m, 6H), 7.23 (t, J = 7.8 Hz, 1H); HRESIMS found: 263.0677.
Calcd: 263.0684 for C15H12NaO3 ([M+Na]+).
3.2.21. (E)-6-Phenylethenyl salicylic acid (9g)
(E)-6-Phenylethylenyl salicylic acid (9g, 70.8 mg, 92%) was obtained as a yellowish powder from
the hydrolysis of 7g (100 mg, 0.32 mmol) by 1N NaOH (0.96 mL, 0.96 mmol) in EtOH (2.2 mL) for
5 h. Mp 130–132 °C; IR (KBr, cm−1) ν 3443, 3061, 3025, 2959, 2925, 2851, 1674, 1652, 1600, 1449,
1
1384, 1205, 1171; H-NMR (acetone-d6, 400 MHz) δ 6.88 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 16.0 Hz,
1H), 7.20 (d, J = 7.8 Hz, 1H), 7.27 (m, 1H), 7.34–7.43 (m, 3H), 7.59 (d, J = 7.8 Hz, 2H), 8.16 (d,
J = 16.0 Hz, 1H); HRESIMS found: 239.0736. Calcd: 239.0786 for C15H11O3 ([M-H]−).
3.3. Molluscicidal activity tests
The molluscicidal test was carried out in accordance with WHO guidelines for laboratory
molluscicidal tests [20]. The Oncomelania hupensis snails were collected from the epidemic area of
Schistosomiasis in Anhui Province, China. Healthy adult snails were used in all molluscicidal tests.
Each compound was dissolved into a small amount of alcohol (less than 0.1% final concentration) and
then diluted with dechlorinated water. Niclosamide (Nic) was used as a positive control and
dechlorinated water as the control. The molluscicidal test was carried out using the immersion method.
Snails (20 snails in each group) were placed into beakers containing each test compound at different
concentrations for a period of 48 h at 22 ± 2 °C. Each beaker was covered with nylon netting to
prevent the snails from escaping. Snail mortality was carefully checked and all tests were carried out in
duplicate. The LD10, LD50 and LD90 values were determined using Probit analysis [21] of the mortality
data.
4. Conclusions
Synthetic GA analogues represent a new class of chemical entities with promising molluscicidal
activities. In this study, a series of Z/E isomers were prepared and evaluated for their molluscicidal
activities against the host snail O. hupensis. The results and analysis of the structural–activity
relationship revealed that the E-isomers were better molluscicidal agents than their respective Z-
isomers. Activity was found to decrease regularly with the shortening of the alkyl chain lengths. New
lead compounds have been identified that warrant further study.
Conflict of Interest
The authors declare no conflict of interest.

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Acknowledgements
This work was supported by the National Natural Science Funds of China (30872203). We are
grateful to Yuliang He for cultivating the snails in the laboratory.
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