Synthesis and SAR studies of neuritogenic gentiside derivatives

Article abstract of DOI:10.1248/cpb.c15-00795

Tetradecyl 2,3-dihydroxybenzoate (ABG-001) has been designed and synthesised as a lead compound to treat Alzheimer's disease, based on structure-activity relationships of gentisides. In this paper, the alkyl chain and ester linkage group of ABG-001 were modified. Consequently, several series of novel gentiside derivatives were designed and synthesised, and their neuritogenic activity was evaluated in PC12 cells. Among all the tested compounds, S-dodecyl 2,3-dihydroxybenzothioate (15d, named as ABG-199) was the most potent; the compound induced significant neurite outgrowth at 0.1 μM, which was comparable to that of nerve growth factor at the optimal concentration of 40 ng/mL and ABG-001 at 1 μM. A brief study on the mechanism of action of ABG-199 revealed that extracellular signal-regulated kinase phosphorylation was involved in ABG-199-induced neurite outgrowth in PC12 cells.

Full text of DOI:10.1248/cpb.c15-00795

Vol. 64, No. 2
Chem. Pharm. Bull. 64, 161–170 (2016)
161
Regular Article
Synthesis and SAR Studies of Neuritogenic Gentiside Derivatives
Guangfa Wang, Linglin Bian, Hui Zhang, Yanhui Wang, Lijuan Gao, Kaiyue Sun,
Lan Xiang, and Jianhua Qi*
College of Pharmaceutical Sciences, Zhejiang University; Yu Hang Tang Road 866, Hangzhou 310058, China.
Received October 15, 2015; accepted December 2, 2015
Tetradecyl 2,3-dihydroxybenzoate (ABG-001) has been designed and synthesised as a lead compound
to treat Alzheimer’s disease, based on structure–activity relationships of gentisides. In this paper, the alkyl
chain and ester linkage group of ABG-001 were modified. Consequently, several series of novel gentiside de-
rivatives were designed and synthesised, and their neuritogenic activity was evaluated in PC12 cells. Among
all the tested compounds, S-dodecyl 2,3-dihydroxybenzothioate (15d, named as ABG-199) was the most po-
tent; the compound induced significant neurite outgrowth at 0.1µM, which was comparable to that of nerve
growth factor at the optimal concentration of 40ng/mL and ABG-001 at 1µM. A brief study on the mecha-
nism of action of ABG-199 revealed that extracellular signal-regulated kinase phosphorylation was involved
in ABG-199-induced neurite outgrowth in PC12 cells.
Key words neuritogenic activity; thioester; gentiside derivative
Alzheimer’s disease (AD) is the most prevalent neurodegen- (2) the middle of the alkyl chain (part B) and (3) the linkage
erative disease in aged populations, and the number of cases is atom between the benzene ring and the alkyl chain (part C).
rapidly increasing. As one of the most important neurotrophic
factors, the nerve growth factor (NGF) is essential for neu-
ronal differentiation, growth, survival, function maintenance
Results and Discussion
Chemistry First, hydroxy, 2,3-dihydroxybenzoate, meth-
and prevention of ageing in central and peripheral systems.1,2) oxy, acetate, methane sulphonate and halogen groups were
However, NGF cannot pass through the blood–brain barrier introduced to the end of the ABG-001 alkyl chain to
because of its large molecular size and hydrophilic properties, study effects of the end group in the alkyl chain on the
thereby limiting its use in therapeutic drugs for the treatment neuritogenic activity. Tetradecyl-2,3-dihydroxybenzoate de-
of neurodegenerative diseases such as AD.3) Therefore, small rivatives 3a–e were prepared using tetradecane 1,14-diol
molecular compounds that mimic or enhance the neuritogenic (1a) as a starting material (Chart 1). First, compound 1a
activity of NGF are promising candidates for drug discov- was condensed with 2,3-dihydroxybenzoic acid (2), with
ery. To date, hundreds of neuritogenic substances have been 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
isolated from natural products or synthesised by researchers; (EDC·HCl) as the condenser to afford compounds 3a (35%)
some of which are in clinical or pre-clinical studies for drug and 3b (20%) simultaneously.11,12) Then one hydroxy group of
development.4) The PC12 cell line was cloned from rat pheo- 1a reacted with methyl iodide (MeI), Ac2O, and methylsul-
chromocytoma; this cell line is one of the most important fonyl chloride (MsCl) to afford compounds 1b–d, respectively,
bioassay systems to evaluate the biological activity of neurito- subsequently compounds 1b–d condensed with 2 in the pres-
genic substances in vitro.5–7)
Gentisides are a novel class of neuritogenic phenolic lipids,
ence of EDC·HCl to yield compounds 3c–e, respectively.
The synthetic route of compounds 3f–i is depict-
which we previously isolated from the traditional Chinese ed in Chart 2. Treated 3e with KF in the presence of
8,9)
medicine Gentiana rigescens FRANCH (Fig. 1). These com- 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8,8,8]hexacosane
pounds are potent inducers of neurite outgrowth in PC12 cells. (K222) to afford 3f in low yield (10%). The reason was the
Subsequently, the effects of the alkyl chain length, number nucleophilic ability of phenolic hydroxy was higher than
and position of hydroxy groups on benzene ring, as well as fluoride ion, thereby yielding the intramolecular nucleophilic
the type of linker between the benzene ring and the alkyl products as the main by-products. The same by-product was
chain, on their neuritogenic activity were investigated.10) Re- also afforded when 3e was treated with KCl in the presence of
sults revealed important parts for the neuritogenic activity of K222 to produce a low yield of 3g (13%). Compared with the
gentiside derivatives including the following: (1) at least two fluoride and chloride ions, the nucleophilic ability of the bro-
nearby hydroxy groups on the benzene ring; (2) an ester link- mide and iodide ions was much higher; these ions could react
age group; (3) the alkyl chain length (C10–C22). Among all the with 3e in the presence of K222 to afford 3h (yield: 88%) and
tested compounds, tetradecyl 2,3-dihydroxybenzoate (renamed 3i (yield: 92%), respectively, with high yield.
as ABG-001) was the most important lead compound and ex-
Given that the yield of 3f and g was low and difficult to
hibited significant neuritogenic activity at 1µM (Fig. 1).
separate from mixture of reaction products, synthetic route
In the present structure–activity relationship (SAR) study of was then modified. The phenolic hydroxy groups were protect-
gentisides, we focused our interest mainly on the modification ed. Compound 6 could be synthesised from 413) and condensed
of alkyl chain, as well as the linkage between the benzene with 1d under EDC·HCl to afford compound 7. Subsequently,
ring and the alkyl chain, based on previous results. The modi- 7 reacted with potassium halogen in the presence of K222,
fied parts (Fig. 1) are (1) the end of the alkyl chain (part A), and the p-methoxybenzyl (PMB) group was removed with
*To whom correspondence should be addressed. e-mail: qijianhua@zju.edu.cn
© 2016 The Pharmaceutical Society of Japan

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trifluoroacetic acid (TFA) to afford 3f and g with high yield.14) hydroxybenzothioates (Chart 5) with different alkyl chain
Second, an extra ether or ester group was introduced to the lengths were synthesised. In our previous SAR studies, amide
middle of the alkyl chain of ABG-001 to discover more active and ketone linkages decreased the biological activity; this
compound with better solubility.
effect may be attributed to their improved stability as com-
The ABG-001 derivatives 11a–f were synthesised from the pared with an ester group. In the present work, a thioester was
alkyl-diols 8a–f and the alkyl bromides 9a–f to yield interme- used to replace the ester bond of ABG-001. The alkyl 2,3-di-
diates 10a–f, which were condensed with 2 (Chart 3).
hydroxybenzothioates (15a–e) were prepared from compound
Compounds 14a–f were synthesised from the alkyl-diols 6 which involved of three steps in one pot. First, compound
8a–f and the acyl chlorides 12a–f to yield intermediates 13a– 6, pivaloyl chloride (PivCl), and triethylamine were added
f, which were condensed with 2 (Chart 4).
into dichloromethane at 0°C, warmed up to room temperature,
Third, the oxygen atom between the benzene ring and the and kept stirring until compound 6 disappeared (detected by
alkyl chain was replaced by a sulphur atom; a series of 2,3-di- thin-layer chromatography). Then the corresponding thiol and
4-dimethylaminopyridine were added into the mixture at 0°C,
warmed up to room temperature, and kept stirring for 12h.15)
Finally, TFA (50% in water) was added into the mixture to
remove protecting groups. The solubility of 2,3-dihydroxyben-
zothioates was reduced when alkyl chain length was longer
than 14 carbon atoms. Therefore, 2,3-dihydroxybenzothioates
with alkyl chain lengths between 6 to 14 carbon atoms were
synthesised.
Neuritogenic Activity The neuritogenic activity of the
synthesised gentiside derivatives (3a–i, 11a–f, 14a–f and
15a–e) was evaluated using PC12 cells as compared with the
negative control (0.5% dimethyl sulfoxide (DMSO)) and the
positive NGF control at an optimum concentration of 40ng/
mL, as well as ABG-001 (1µM). Figure 2 shows the maximum
Fig. 1. Chemical Structures of Gentiside A and ABG-001
Reagents and condition: (i) EDC·HCl, DMAP, DCM, rt; (ii) NaH, MeI, DMF, rt; (iii) Ac2O, Et3N, DMAP, DCM, rt; (iv) MsCl, DMAP, pyridine, DCM, rt.
Chart 1. Synthetic Route of Gentiside Derivatives 3a–e
Reagents and condition: (i) KF, K222, CH3CN, 80°C; (ii) KCl, K222, CH3CN, 80°C; (iii) KBr, K222, CH3CN, 80°C; (iv) KI, K222, CH3CN, 80°C; (v) PMBCl, K2CO3,
THF, rt; (vi) 2N NaOH; (vii) EDC·HCl, DMAP, DCM, rt; (viii) (1) KF, K222, CH3CN, 80°C; (2) TFA, DCM, rt; (ix) (1) KCl, K222, CH3CN, 80°C; (2) TFA, DCM, rt.
Chart 2. Synthetic Route of Gentiside Derivatives 3f–i

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Chem. Pharm. Bull.
163
Reagents and condition: (i) NaH, TBAI, DMF, rt; (ii) Compound 2, EDC·HCl, DMAP, DCM, rt.
Chart 3. Synthetic Route of Gentiside Derivatives 11a–f
Reagents and condition: (i) Et3N, DMAP, DCM, rt; (ii) Compound 2, EDC·HCl, DMAP, DCM, rt.
Chart 4. Synthetic Route of Gentiside Derivatives 14a–f
except for the hydroxy group, the introduction of other groups
at the end of the alkyl chain led to minimal changes of bio-
logical activity in this study.
The biological activities of compounds 11a–f and 14a–f are
presented in Figs. 2b and c, respectively. Compounds 11a–f,
with an ether group in different positions of alkyl chain,
did not show significant changes of biological activity, with
a maximum activity of 77, 73, 76, 77, 78 and 75% at 1µM,
respectively, as compared with ABG-001 (78%, 1µM). Com-
pounds 14a–f had an ester group in different positions of the
Reagents and condition: (i) (1) PivCl, Et3N, DCM, rt; (2) alkyl thiol, DMAP, rt;
(3) 50% TFA, rt.
Chart 5. Synthetic Route of Gentiside Derivatives 15a–e
neuritogenic activity of the gentiside derivatives 3a–i, 11a–f, alkyl chain of ABG-001; these compounds showed a large
14a–f and 15a–e at their optimum concentration, respectively. change of biological activity, with a maximum activity of 34,
The biological activity of compounds 3a–i is depicted in 37, 39, 43, 57 and 75% at 1µM, respectively, as compared with
Fig. 2a. Among compounds 3a–i, the 2,3-dihydroxybenzoate ABG-001. With the presence of carboxyl groups close to the
derivatives 3c–e exhibited a slight decrease in the percentage aromatic ring, the decline in the biological activity was more
of neurite outgrowth, with a maximum activity of 75, 73 and obvious.
69%, respectively, at 1µM as compared with ABG-001 (77%,
Compounds 15a–e showed neurite outgrowths of 65%
1µM). The presence of a hydroxy group at the end of the alkyl (15a), 70% (15b), 68% (15c), 77% (15d) and 66% (15e) at
chain (3a, 60%, 3µM) decreased the activity. The introduction 0.1 µM, respectively (Fig. 2d). Among these compounds, S-
of 2,3-dihydroxybenzoate at the end of the alkyl chain (3b, dodecyl 2,3-dihydroxybenzothioate (15d) was the most active
68%, 3µM) also led to decreased activity. When one hydrogen and possessed a 12-carbon alkyl chain. The maximum activity
atom of the terminal methyl was replaced by a halogen atom, of 15d was 77% at 0.1µM, which was comparable to that of
the neuritogenic activity was slightly increased. Compounds ABG-001 (78%) at 1.0µM and NGF at the optimum concentra-
3f–i showed significant neuritogenic activity, with a maximum tion of 40ng/mL (Fig. 2d).
activity of 79, 80, 81 and 82% at 1µM, respectively. Therefore,
Compound 15d showed a dose-dependent increase in the

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Fig. 2. Maximum Neuritogenic Activity of the Gentiside Derivatives 3a–i, 11a–f, 14a–f and 15a–e at Optimum Concentrations in PC12 Cells
Value means are expressed as the average percentage of neurite outgrowth S.D. (n=3). C: control (0.5% DMSO); positive control: NGF (40ng/mL); ABG-001 (1.0µM);
(3a–i, 11a–f, 14a–f and 15a–e: ***p<0.001).
neuritogenic activity, which ranged from 0.03–0.1µM, as
shown in Fig. 3. Compound 15d exhibited neuritogenic ac-
tivity of 50% even at a concentration of 0.03µM and showed
significant neuritogenic activity of 75% at 0.1µM and higher
doses. ABG-001 exhibited neuritogenic activity of 26, 33, 56
and 77% at doses of 0.03, 0.1, 0.3 and 1.0µM, respectively.
Figure 4 shows the morphological changes of PC12 cells after
treatment with 15d at 0.1µM as compared with the solvent
control (0.5% DMSO), positive control (NGF at 40ng/mL) and
ABG-001 at 1µM. Control cells (without adding compounds)
showed few short neurite outgrowths (Fig. 4a). When treated
with 15d at 0.1µM, the cells extended long multipolar neu-
rite outgrowths at 48h after treatment (Fig. 4d), which was
similar to those produced after treatment with ABG-001 at
1.0 µM (Fig. 4c) and NGF at 40ng/mL (Fig. 4b). These results
Fig. 3. Dose-Dependent Response of Neuritogenic Activity of 15d
Compared with ABG-001 at 48h after Treatment
revealed that S-dodecyl 2,3-dihydroxybenzothioate (15d) is
the best neuritogenic compound among all the gentisides and
their derivatives in the present study and in previous studies.
Subsequently, S-dodecyl 2,3-dihydroxybenzothioate (15d) was
C: control (0.5% DMSO); positive control: NGF (40ng/mL), ABG-001 at 0.03,
0.1, 0.3 and 1µM: ***p<0.001; 15d at 0.03, 0.1, 0.3 and 1µM: ***p<0.001.
named as ABG-199.
Subsequently, the key protein in the signalling pathway of kinase (ERKl/2) is a key enzyme during NGF-induced neurite
neuritogenesis was investigated to understand the brief mecha- outgrowth.19) In our previous study, ERKl/2 similarly had
nism of action of neurite outgrowth induced by ABG-199. an important role in ABG-001-induced neurite outgrowth.10)
To date, the cellular mechanism of NGF-induced neuritogen- Therefore, the involvement of ERKl/2 in 15d-induced neurite
esis has been well investigated.16–18) NGF first binds to the outgrowth should be investigated. Results showed that the
transmembrane-specific tyrosine receptor kinase A to induce treatment with 0.1µM of 15d led to a sustained and signifi-
phosphorylation of the specific tyrosine residues located at the cant enhancement of ERKl/2 phosphorylation after 4h (Fig.
intracellular domain, thereby leading to the recruitment and 5). Simultaneously, treatment with 40ng/mL of NGF resulted
activation of a number of kinases. Among these kinases, the in the sustained and strong increase of ERKl/2 phosphoryla-
mitogen-activated protein kinase extracellular signal-regulated tion over 10h. The phosphorylation level induced by 15d was

Vol. 64, No. 2 (2016)
Chem. Pharm. Bull.
165
Fig. 4. Photomicrographs of PC12 Cells under a Phase-Contrast Microscope at 48h after Treatment
(a) Solvent control (0.5% DMSO), (b) NGF (40ng/mL), (c) ABG-001 (1.0µM), and (d) 15d (ABG-199, 0.1µM).
even at a concentration of 0.03µM. Among all the synthesised
gentiside derivatives, the most promising compound was S-
dodecyl 2,3-dihydroxybenzothioate (15d), which was named
as ABG-199. ABG-199 exhibited significant neuritogenic ac-
tivity at 0.1µM, which was comparable to that of NGF at the
optimum concentration of 40ng/mL. The brief study of mech-
anism of action of ABG-199 revealed that ERKl/2 phosphory-
lation was involved in ABG-199-induced neurite outgrowth
in PC12 cells. Intensive studies on neuritogenic activity of
ABG-199 are underway in our laboratory.
Experimental
Fig. 5. Phosphorylation of ERKl/2 in the Presence of the Control (0.5%
Chemistry TLC analyses were done using pre-coated
silica gel plates. The NMR spectra were recorded on a Bruker
AV III-500 spectrometer, the NMR chemical shifts in δ (ppm)
were referenced to the solvent peaks of δC 77.0 and δH 7.26 for
CDCl3, and chemical shifts were given in δ (ppm) and signals
DMSO), 15d (ABG-199, 0.1µM) or NGF (40ng/mL)
The phosphorylation level (%) was analysed with the Image J software and nor-
malised to the control value at different time points (100%).
slightly lower and more delayed than those induced by NGF. were described as singlet (s), doublet (d), triplet (t), double
The data revealed that the significant neuritogenesis induced doublet (dd) and multiplet (m). The high-resolution (HR) elec-
by 15d was dependent on the sustained activation of ERKl/2 trospray ionization-time of flight (ESI-TOF)-MS was recorded
in PC12 cells. The phosphorylation of ERKl/2 was slow using on Agilent 6224A (TOF) LC/MS. All the solvents used were
15d compared with NGF is due to different mechanism of analytical grade.
action between these two substances on neurite outgrowth of
14-Methoxy-1-tetradecanol (1b)
PC12 cells. NGF directly bound to the transmembrane-specific
Tetradecane-1,14-diol (1a, 690mg, 3.00mmol) was dis-
tyrosine receptor kinase A, and resulted in phosphorylation solved in anhydrous N,N-dimethylformamide (DMF) (20mL).
of ERKl/2. However, we found that activation of insulin-like NaH (180mg, 4.5mmol) was added in at 0°C, kept stirring
growth factor 1 receptor (IGF-1R) is involved in ABG-001, for 30min. Warmed up to room temperature for another
an analogue of 15d induced neurite outgrowth of PC12 cells 30min. MeI (511mg, 3.60mmol) was added in at 0°C, kept
in recent study,20) as the chemical structure of 15d is quite stirring overnight. The reaction was stopped by adding 1mL
similar to that of ABG-001. Therefore, we presumed that of methanol, the mixture was washed with 1N HCl solution,
the mechanism of 15d on neurite outgrowth is similar to water, saturated aqueous solution of NaHCO3 and brine, suc-
ABG-001. Furthermore, we need confirm whether ABG-001 cessively. The organic phase was dried over Na2SO4, filtered,
directly or indirectly targets IGF-1R in future study.
and then concentrated. The crude mixture was purified by col-
umn chromatography (petroleum ether–EtOAc 20:1) on silica
1
gel to afford 1b (424mg, 58%) as a colorless powder. H-NMR
Conclusion
Several series of ABG-001 derivatives were prepared via (500MHz, CDCl3) δ: 3.65 (2H, t, J=6.5Hz), 3.36 (2H, t,
convenient synthetic methods. Their structures were con- J=6.5Hz), 3.33 (3H, s), 1.57–1.53 (4H, m), 1.32–1.25 (20H, m).
firmed by spectroscopic data. The alkyl chain of ABG-001
was modified, as well as the ester linkage between benzene
1-Hydroxytetradecyl Acetate (1c)
Tetradecane-1,14-diol (1a, 460mg, 2.00mmol), Et3N
ring and alkyl chain was replaced by a thioester group. Re- (1.01g, 10.0mmol) 4-(dimethylamino)pyridine (DMAP)
sults of SAR studies indicated that property of a group at the (12mg, 0.2mmol) were dissolved in anhydrous CH2Cl2
end of side chain had effects on the biological activity. Intro- (100mL). Cooled down the mixture to 0°C and Ac2O (225mg,
duction of an ether group into the middle position of alkyl 2.20mmol) was added, and kept stirring overnight. The reac-
chain of ABG-001 did not significantly change neuritogenic tion was stopped by adding 1mL of methanol, the mixture
activity. However, the introduction of an ester group into the was washed with 1N HCl solution, saturated aqueous solution
alkyl chain of ABG-001 significantly decreased the neurito- of NaHCO3 and brine, successively. The organic phase was
genic activity of compounds, especially when the ester group dried over Na2SO4, filtered, and then concentrated. The crude
was close to an aromatic ring. When ester linkage between mixture was purified by column chromatography (petroleum
benzene ring and alkyl chain was replaced by a thioester ether–EtOAc 10:1) on silica gel to afford 1c (234mg, 43%) as
1
group, compounds showed significant neuritogenic activity a colorless powder. H-NMR (500MHz, CDCl3) δ: 4.05 (2H, t,

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J=7.0Hz), 3.64 (2H, t, J=6.5Hz), 2.04 (3H, s), 1.63–1.54 (4H, benzoic acid (113mg, 0.73mmol). The pure 3d was obtained
m), 1.34–1.26 (20H, m).
as a colorless powder (72mg, 48%). 1H-NMR (500MHz,
1-Hydroxytetradecyl Methanesulphonate (1d)
CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10 (1H, d,
Tetradecane-1,14-diol (1a, 2.30g, 10.00mmol) was dissolved J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.65 (1H, s), 4.34 (2H, t,
in anhydrous pyridine (20mL), DMAP (122mg, 1.0mmol), J=6.5Hz), 4.05 (2H, t, J=7.0Hz), 2.05 (3H, s), 1.78 (2H, m),
MsCl (0.77mL, 10.00mmol) were added into this mixture at 1.60 (2H, m), 1.45–1.26 (20H, m). 13C-NMR (125MHz, CDCl3)
0°C. Then warmed up to room temperature and keep stirring δ: 171.3, 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.7, 65.7,
for 6h. The reaction was stopped with 2mL methanol, the 64.7, 29.6, 29.5, 29.3, 29.2, 28.6, 28.5, 25.9, 21.0. HR-ESI-MS
mixture was washed with 1N HCl solution, saturated aque- m/z: 431.2372 (Calcd for C23H36O6Na [M+Na]+: 431.2404).
ous solution of NaHCO3 and brine, successively. The organic
phase was dried over Na2SO4, filtered, and then concentrated. (3e)
The crude mixture was purified by column chromatography
14-(Methylsulphonyloxy)tetradecyl 2,3-Dihydroxybenzoate
Compound 1d (920mg, 2.98mmol) was dissolved in anhy-
(CH2Cl2–MeOH 100:1) on silica gel to afford 1d (920mg, drous CH2Cl2 (20mL), DMAP (37mg, 0.3mmol), EDC·HCl
1
30%) as a colorless powder. H-NMR (500MHz, CDCl3) 4.22 (1.14g, 6.0mmol) were added into this mixture. The crude
(2H, t, J=7.0Hz), 3.64 (2H, t, J=6.5Hz), 3.00 (3H, s), 1.75 mixture was purified by column chromatography (petroleum
(2H, m), 1.56 (2H, m), 1.40–1.26 (20H, m).
14-Hydroxytetradecyl 2,3-Dihydroxybenzoate (3a) and a colorless powder. 1H-NMR (500MHz, CDCl3) 10.99 (1H,
1,14-(2,3-Dihydroxybenzoyloxy)tetradecane (3b) s), 7.37 (1H, d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.80 (1H,
ether–EtOAc 5:1) on silica gel to afford 3e (540mg, 41%) as
Tetradecane-1,14-diol (1a, 230mg, 1.00mmol) was dissolved t, J=8.0Hz), 5.64 (1H, s), 4.35 (2H, t, J=6.5Hz), 4.22 (2H,
in anhydrous CH2Cl2 (30mL), DMAP (122mg, 1.0mmol), t, J=6.5Hz), 3.00 (3H, s), 1.79–1.73 (4H, m), 1.44–1.26 (20H,
EDC·HCl (1.92g, 10.0mmol) and 2,3-dihydroxybenzoic acid m). 13C-NMR (125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 120.5,
(1.54g, 10.00mmol) was added into this mixture. Kept stir- 119.7, 119.1, 112.7, 70.2, 65.7, 37.4, 29.6, 29.5, 29.4, 29.2, 29.1,
ring overnight at room temperature, and then the mixture was 29.0, 28.5, 25.9, 25.4. HR-ESI-MS m/z: 467.2081 (Calcd for
concentrated under vacuum. The crude mixture was purified C22H36O7SNa [M+Na]+: 467.2074).
by column chromatography (petroleum ether–EtOAc 30:1)
Methyl 2,3-Bis(4-methoxybenzyloxy)benzoate (5)
on silica gel to afford 3a (101mg, 20%) as a colorless powder
Methyl 2,3-dihydroxybenzoate (4, 1.00g, 5.94mmol) was
and 3b (181mg, 48%) as a colorless powder. 3a: 1H-NMR dissolved in anhydrous tetrahydrofuran (THF) (50mL), K2CO3
(500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), (4.92g, 35.6mmol), tetrabutylammonium iodide (TBAI)
7.10 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.67 (1H, s), (0.22g, 0.6mmol), and PMBCl (2.42mL, 17.82mmol) were
4.34 (2H, t, J=7.0Hz), 3.64 (2H, t, J=6.5Hz), 1.77 (2H, m), added into the solution. The mixture was refluxed for 24h,
1.56 (2H, m), 1.43 (2H, m), 1.35–1.26 (18H, m). 13C-NMR and then filtered. The filtrate was concentrated, and purified
(125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, by column chromatography (petroleum ether–EtOAc 50:1)
112.7, 65.7, 63.1, 32.8, 29.6, 29.5, 29.4, 29.2, 28.5, 25.9, 25.7. to afford 5 (1.70g, 70%) as a colorless powder. 1H-NMR
HR-ESI-MS m/z: 389.2311 (Calcd for C21H34O5Na [M+Na]+: (500MHz, CDCl3) δ: 7.40–7.35 (5H, m), 7.14 (1H, dd, J=8.5,
389.2298). 3b: 1H-NMR (500MHz, CDCl3) δ: 10.99 (2H, 1.5Hz), 7.07 (1H, t, J=8.0Hz), 6.92 (2H, d, J=8.5Hz), 6.85
s), 7.37 (2H, d, J=8.0Hz), 7.10 (2H, d, J=8.0Hz), 6.79 (2H, (2H, d, J=8.5Hz), 5.06 (2H, s), 5.04 (2H, s), 3.87 (3H, s), 3.82
t, J=8.0Hz), 5.65 (2H, s), 4.34 (4H, t, J=7.0Hz), 1.79–1.75 (3H, s), 3.80 (3H, s).
(4H, m), 1.45–1.40 (4H, m), 1.37–1.27 (16H, m). 13C-NMR
2,3-Bis(4-methoxybenzyloxy)benzoic Acid (6)
(125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1,
A solution of 5 (0.94g, 1.83mmol) in dioxane (9mL) and 2N
112.7, 65.7, 29.6, 29.5, 29.2, 28.5, 25.9. HR-ESI-MS m/z: NaOH (4.6mL) was stirred for 24h at room temperature. The
525.2459 (Calcd for C28H38O8Na [M+Na]+: 525.2459).
14-Methoxytetradecyl 2,3-Dihydroxybenzoate (3c)
reaction mixture was concentrated. The residue was stirred
with H2O (5mL) and then acidified to pH 2 with 2N HCl.
Compound 1b (244mg, 1.00mmol) was dissolved in anhy- The colorless powder was filtered, washed with hexane, and
drous CH2Cl2 (20mL), DMAP (12mg, 0.1mmol), EDC·HCl recrystallized from EtOAc–hexane to yield 500mg (69%) of 6
1
(575mg, 3.0mmol) and 2,3-dihydroxybenzoic acid (308mg, as a colorless crystal. H-NMR (500MHz, CDCl3) δ: 7.72 (1H,
2.00mmol) were added into this mixture. Kept stirring over- dd, J=8.0, 2.0Hz), 7.41 (2H, d, J=9.0Hz), 7.27–7.22 (3H, m),
night at room temperature, and then the mixture was concen- 7.18 (1H, t, J=8.0Hz), 6.97–6.95 (2H, m), 6.84–6.82 (2H, m),
trated under vacuum. The crude mixture was purified by col- 5.20 (2H, s), 5.12 (2H, s), 3.85 (3H, s), 3.80 (3H, s). 13C-NMR
umn chromatography (petroleum ether–EtOAc 20:1) on silica (125MHz, CDCl3) δ: 165.2, 160.3, 159.8, 151.3, 147.0, 131.1,
1
gel to afford 3c (181mg, 48%) as a colorless powder. H-NMR 129.6, 127.9, 126.7, 124.9, 124.3, 122.9, 119.0, 114.1, 76.8, 71.3,
(500MHz, CDCl3) δ: 10.99 (1H, s), 7.37 (1H, d, J=8.0Hz), 55.3, 55.2.
7.10 (1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.68 (1H, s),
4.34 (2H, t, J=7.0Hz), 3.36 (2H, t, J=6.5Hz), 3.33 (3H, s), (7)
1.78 (2H, m), 1.59–1.55 (2H, m), 1.43–1.26 (20H, m). 13C-NMR
14-(Methylsulphonyloxy)tetradecyl 2,3-Dihydroxybenzoate
Compound 1d (270mg, 0.88mmol) was dissolved in an-
(125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, hydrous CH2Cl2 (20mL), DMAP (11mg, 0.1mmol) and
112.7, 73.0, 65.7, 58.5, 29.6, 29.5, 29.2, 28.5, 26.1, 25.9. HR- EDC·HCl (503mg, 2.6mmol), and 6 (686mg, 1.75mmol)
ESI-MS m/z: 403.2451 (Calcd for C22H36O5Na [M+Na]+: were added into this mixture. The crude mixture was puri-
403.2455).
fied by column chromatography (petroleum ether–EtOAc 5:1)
14-Acetoxytetradecyl 2,3-Dihydroxybenzoate (3d)
on silica gel to yield 7 (259mg, 43%) as a colorless powder.
The synthesis and purification methods were the same as 1H-NMR (500MHz, CDCl3) δ: 7.37–7.32 (5H, m), 7.12 (1H,
for 3c, condensing 1c (100mg, 0.37mmol) with 2,3-dihydroxy- d, J=8.0Hz), 7.06 (1H, t, J=8.0Hz), 6.91 (2H, m), 6.82 (2H,

Vol. 64, No. 2 (2016)
Chem. Pharm. Bull.
167
m), 5.05 (2H, s), 5.02 (2H, s), 4.26 (2H, t, J=7.0Hz), 4.22 (2H, d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz),
t, J=7.0Hz), 3.83 (3H, s), 3.80 (3H, s), 2.99 (3H, s), 1.77–1.67 5.66 (1H, s), 4.34 (2H, t, J=6.5Hz), 3.19 (2H, t, J=7.0Hz),
(4H, m), 1.41–1.35 (4H, m), 1.31–1.23 (16H, m). 13C-NMR 1.85–1.75 (4H, m), 1.46–1.26 (20H, m). 13C-NMR (125MHz,
(125MHz, CDCl3) δ: 166.5, 159.5, 159.3, 152.8, 148.2, 130.2, CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.6, 119.1, 112.7, 65.7,
129.8, 129.3, 128.7, 127.3, 123.8, 122.6, 117.9, 113.9, 113.6, 33.6, 30.5, 29.6, 29.5, 29.4, 29.2, 28.5, 25.9, 7.3. HR-ESI-MS
77.2, 75.2, 71.1, 70.2, 65.3, 55.3, 55.2, 37.3, 29.6, 29.5, 29.4, m/z: 499.1330 (Calcd for C21H33O4INa [M+Na]+: 499.1316).
29.3, 29.1, 29.0, 28.7, 26.0, 25.4.
2-(Dodecyloxy)ethanol (10a)
14-Fluorotetradecyl 2,3-Dihydroxybenzoate (3f)
Ethanediol (1.1mL, 20.00mmol) was dissolved in 200mL
Compound 7 (10mg, 0.015mmol) was dissolved in 2mL anhydrous DMF, NaH (480mg, 12.0mmol) was added in
anhydrous acetonitrile, KF (1mg, 0.2mmol), K222 (20mg, at 0°C. The mixture was warmed up to room temperature
0.1mmol) were added, the reaction mixture was refluxed for and stirred for 30min. Then 1-bromododecane (2.4mL,
15min. Solvent was evaporated under vacuum to give a crude 10.00mmol), TBAI (369mg, 1.0mmol) were added in at 0°C,
product. The crude mixture was dissolved in 1mL CH2Cl2, kept stirring overnight. The reaction mixture was diluted with
then 1mL TFA was added in with stirring at room tem- 20mL methanol, then washed with 1N HCl solution, water,
perature, kept stirring for 10min. Then the mixture was di- saturated aqueous solution of NaHCO3 and brine, successively.
luted with 10mL distilled water, and subjected to C18 column, The organic phase was dried over Na2SO4, filtered, and then
washed with 2mL distilled water, 2mL EtOH (50%) to yield concentrated. The crude mixture was purified by column
1
3f (4.1mg, 76%) as a colorless powder. H-NMR (500MHz, chromatography (petroleum ether–EtOAc 30:1) on silica
1
CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10 (1H, d, gel to afford 10a as a colorless oil (1.12g, 49%). H-NMR
J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.63 (1H, s), 4.44 (2H, dt, (500MHz, CDCl3) δ: 3.73 (2H, t, J=4.5Hz), 3.53 (2H, t,
J=47.0, 6.5Hz), 4.34 (2H, t, J=6.5Hz), 1.80–1.63 (4H, m), J=4.5Hz), 3.47 (2H, t, J=6.5Hz), 1.59 (2H, m), 1.29–1.26
1.45–1.27 (20H, m). 13C-NMR (125MHz, CDCl3) δ: 170.4, (18H, m), 0.88 (3H, t, J=6.5 Hz).
148.9, 145.9, 120.5, 119.6, 119.1, 112.7, 84.3 (d, J=162.9Hz),
65.7, 30.4, 30.3, 29.6, 29.5, 29.2, 28.5, 25.9, 25.1. HR-ESI-MS
m/z: 391.2265 (Calcd for C21H33O4FNa [M+Na]+: 391.2255).
14-Chlorotetradecyl 2,3-Dihydroxybenzoate (3g)
4-(Decyloxy)butan-1-ol (10b)
The synthesis and purification methods were the same as
for 10a. The pure 10b was obtained as a colorless oil (1.01g,
1
44%). H-NMR (500MHz, CDCl3) δ: 3.64 (2H, t, J=5.5Hz),
The synthesis and purification methods were the same as 3.45 (2H, t, J=5.5Hz), 3.42 (2H, t, J=6.5Hz), 1.70–1.65 (4H,
for 3f, starting from compound 7 (68mg, 0.10mmol), K222 m), 1.56 (2H, m), 1.30–1.25 (14H, m), 0.87 (3H, t, J=7.0 Hz).
(75mg, 0.2mmol) and KCl (37mg, 0.5mmol). The pure 3g
was obtained as a colorless powder (30mg, 79%). H-NMR
6-(Octyloxy)hexan-1-ol (10c)
The synthesis and purification methods were the same as
1
(500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10 for 10a. The pure 10c was obtained as a colorless oil (1.09g,
1
(1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.64 (1H, s), 4.35 47%). H-NMR (500MHz, CDCl3) δ: 3.60 (2H, t, J=6.5Hz),
(2H, t, J=6.5Hz), 3.53 (2H, t, J=6.5Hz), 1.81–1.74 (4H, m), 3.39–3.35 (4H, m), 1.58–1.51 (6H, m), 1.35–1.25 (14H, m), 0.85
1.45–1.41 (4H, m), 1.37–1.27 (16H, m). 13C-NMR (125MHz, (3H, t, J=6.5 Hz).
CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.7, 65.7,
8-(Hexyloxy)octan-1-ol (10d)
45.2, 32.6, 29.6, 29.5, 29.4, 29.2, 28.9, 28.5, 26.9, 25.9. HR-
The synthesis and purification methods were the same as
ESI-MS m/z: 407.1956 (Calcd for C21H33O4ClNa [M+Na]+: for 10a. The pure 10d was obtained as a colorless oil (0.91g,
1
407.1960).
39%). H-NMR (500MHz, CDCl3) δ: 3.63 (2H, t, J=6.5Hz),
3.39 (4H, t, J=6.5Hz), 1.57–1.53 (6H, m), 1.36–1.25 (14H, m),
14-Bromotetradecyl 2,3-Dihydroxybenzoate (3h)
Compound 3e (44mg, 0.10mmol) was dissolved in 2mL 0.88 (3H, t, J=6.5 Hz).
anhydrous acetonitrile, KBr (36mg, 0.3mmol), K222 (38mg,
0.1mmol) were added, the reaction mixture was refluxed for
10-(Butoxy)decan-1-ol (10e)
The synthesis and purification methods were the same as
30min. Solvent was evaporated under vacuum to give the for 10a. The pure 10e was obtained as a colorless oil (1.11g,
1
crude product which was purified by column chromatog- 48%). H-NMR (500MHz, CDCl3) δ: 3.64 (2H, t, J=6.5Hz),
raphy (petroleum ether–EtOAc 5:1) on silica gel to afford 3.41–3.37 (4H, m), 1.58–1.52 (6H, m), 1.40–1.28 (14H, m), 0.92
1
3h (38mg, 88%) as a colorless powder. H-NMR (500MHz, (3H, t, J=7.0 Hz).
CDCl3) δ: 10.99 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10 (1H, d,
J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.64 (1H, s), 4.34 (2H, t,
12-(Ethoxy)dodecan-1-ol (10f)
The synthesis and purification methods were the same as
J=6.5Hz), 3.41 (2H, t, J=6.5Hz), 1.87–1.75 (4H, m), 1.45–1.27 for 10a. The pure 10f was obtained as a colorless oil (0.99g,
1
(20H, m). 13C-NMR (125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 43%). H-NMR (500MHz, CDCl3) δ: 3.63 (2H, t, J=6.5Hz),
120.5, 119.6, 119.1, 112.7, 65.7, 34.0, 32.8, 29.6, 29.5, 29.4, 29.2, 3.46 (2H, m, J=7.0Hz), 3.40 (2H, t, J=7.0Hz), 1.58–1.52 (4H,
28.8, 28.5, 28.2, 25.9. HR-ESI-MS m/z: 451.1457 (Calcd for m), 1.33–1.26 (16H, m), 1.19 (3H, t, J=7.0 Hz).
C21H33O479BrNa [M+Na]+: 451.1454).
14-Iodotetradecyl 2,3-Dihydroxybenzoate (3i)
2-(Dodecyloxy)ethyl 2,3-Dihydroxybenzoate (11a)
The synthesis and purification methods were the same as
Compound 3e (44mg, 0.10mmol) was dissolved in an- for 3a, condensing from 10a (230mg, 1.00mmol) and 2,3-di-
hydrous acetonitrile, KI (33mg, 0.2mmol), K222 (38mg, hydroxybenzoic acid (308mg, 2.00mmol). The pure 11a
1
0.1mmol) were added, the reaction mixture was refluxed for was obtained as a colorless powder (181mg, 49%). H-NMR
30min. Solvent was evaporated to give the crude which was (500MHz, CDCl3) δ: 10.83 (1H, s), 7.41 (1H, d, J=8.0Hz),
purified by column chromatography (hexane–EtOAc 30:1) 7.11 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.64 (1H, s),
on silica gel to afford 3i (44mg, 92%) as a colorless pow- 4.49 (2H, t, J=5.0Hz), 3.76 (2H, t, J=5.0Hz), 3.51 (2H, t,
1
der. H-NMR (500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, J=6.5Hz), 1.58 (2H, m), 1.34–1.25 (18H, m), 0.88 (3H, t,

168
Chem. Pharm. Bull.
Vol. 64, No. 2 (2016)
J=7.0Hz). 13C-NMR (125MHz, CDCl3) δ: 170.2, 148.8, 145.0, (500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10
120.8, 119.8, 119.2, 112.5, 71.6, 68.2, 64.6, 31.9, 29.7, 29.6, (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.66 (1H, s), 4.34
29.4, 29.3, 26.1, 22.7, 14.1. HR-ESI-MS m/z: 389.2291 (Calcd (2H, t, J=6.5Hz), 3.47 (2H, m), 3.40 (2H, t, J=6.5Hz), 1.77
for C21H34O5Na [M+Na]+: 389.2298).
4-(Decyloxy)butyl 2,3-Dihydroxybenzoate (11b)
(2H, m), 1.57 (2H, m), 1.43 (2H, m), 1.34–1.27 (14H, m), 1.20
(3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3) δ: 170.4, 148.9,
The synthesis and purification methods were the same as 145.0, 120.5, 119.6, 119.1, 112.7, 70.8, 66.0, 65.7, 29.8, 29.6,
for 3a, condensing from 10b (230mg, 1.00mmol) and 2,3-di- 29.5, 29.2, 28.5, 26.2, 25.9, 15.2. HR-ESI-MS m/z: 389.2323
hydroxybenzoic acid (308mg, 2.00mmol). The pure 11b (Calcd for C21H34O5Na [M+Na]+: 389.2298).
1
was obtained as a colorless powder (180mg, 47%). H-NMR
2-Hydroxyethyl Dodecanoate (13a)
(500MHz, CDCl3) δ: 10.97 (1H, s), 7.37 (1H, d, J=8.0Hz),
Ethanediol (1.1mL, 20.00mmol), Et3N (5.10g, 50.0mmol)
7.10 (1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.64 (1H, s), and DMAP (122mg, 1.0mmol) were dissolved in 200mL
4.38 (2H, t, J=6.5Hz), 3.47 (2H, t, J=6.0Hz), 3.41 (2H, t, anhydrous CH2Cl2. The C11H23COCl (2.18g, 10.00mmol) was
J=6.5Hz), 1.88 (2H, m), 1.73 (2H, m), 1.56 (2H, m), 1.34–1.23 added dropwise at 0°C. The mixture was warmed up to room
(14H, m), 0.88 (3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3) temperature and kept stirring overnight then washed with 1N
δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.6, 71.1, 70.1, 65.5, HCl solution, water, saturated aqueous solution of NaHCO3
31.9, 29.7, 29.6, 29.5, 29.3, 26.3, 26.2, 25.5, 22.7, 14.1. HR-ESI- and brine, successively. The organic phase was dried over
MS m/z: 389.2311 (Calcd for C21H34O5Na [M+Na]+: 389.2298). Na2SO4, filtered, and then concentrated. The crude mixture
6-(Octyloxy)hexyl 2,3-Dihydroxybenzoate (11c)
was purified by column chromatography (petroleum ether–
The synthesis and purification methods were the same EtOAc 30:1) on silica gel to afford 13a as a colorless oil
1
as for 3a, condensing from 10c (230mg, 1.00mmol) and (1.51g, 62%). H-NMR (500MHz, CDCl3) δ: 4.20 (2H, m),
2,3-dihydroxybenzoic acid (308mg, 2.00mmol). The pure 3.82 (2H, m), 2.34 (2H, t, J=7.5Hz), 1.62 (2H, m), 1.32–1.25
1
11c was obtained as a colorless solid (170mg, 44%). H-NMR (16H, m), 0.87 (3H, t, J=6.5 Hz).
(500MHz, CDCl3) δ: 10.99 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10
(1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.64 (1H, s), 4.35
4-Hydroxybutyl Decanoate (13b)
The synthesis and purification methods were the same as
(2H, t, J=6.5Hz), 3.42–3.38 (4H, m), 1.79 (2H, m), 1.63–1.27 for 13a. The pure 13b was obtained as a colorless oil (1.05g,
1
(18H, m), 0.88 (3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3) 43%). H-NMR (500MHz, CDCl3) δ: 4.10 (2H, t, J=6.5Hz),
δ: 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.7, 71.0, 70.7, 65.6, 3.68 (2H, t, J=6.5Hz), 2.29 (2H, t, J=7.5Hz), 1.72–1.62 (6H,
31.8, 29.8, 29.6, 29.5, 29.3, 28.5, 26.2, 25.9, 25.8, 22.6, 14.1. m), 1.30–1.26 (12H, m), 0.87 (3H, t, J=7.0 Hz).
HR-ESI-MS m/z: 389.2316 (Calcd for C21H34O5Na [M+Na]+:
389.2298).
6-Hydroxyhexyl Octanoate (13c)
The synthesis and purification methods were the same as
8-(Hexyloxy)octyl 2,3-Dihydroxybenzoate (11d)
The synthesis and purification methods were the same as 41%). H-NMR (500MHz, CDCl3) δ: 4.05 (2H, t, J=7.0Hz),
for 3a, condensing from 10d (230mg, 1.00mmol) and 2,3-di- 3.64 (2H, t, J=7.0Hz), 2.28 (2H, t, J=7.5Hz), 1.63–1.55 (6H,
for 13a. The pure 13c was obtained as a colorless oil (1.00g,
1
hydroxybenzoic acid (308mg, 2.00mmol). The pure 11d m), 1.35–1.29 (12H, m), 0.89 (3H, t, J=7.0 Hz).
1
was obtained as a colorless powder (178mg, 48%). H-NMR
8-Hydroxyoctyl Hexanoate (13d)
(500MHz, CDCl3) δ: 10.99 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10
The synthesis and purification methods were the same as
(1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.67 (1H, s), 4.34 for 13a. The pure 13d was obtained as a colorless oil (1.30g,
1
(2H, t, J=6.5Hz), 3.40–3.38 (4H, m), 1.77 (2H, m), 1.59–1.53 53%). H-NMR (500MHz, CDCl3) δ: 4.05 (2H, t, J=7.0Hz),
(4H, m), 1.45–1.27 (14H, m), 0.88 (3H, t, J=7.0Hz). 13C-NMR 3.64 (2H, t, J=7.0Hz), 2.28 (2H, t, J=7.5Hz), 1.63–1.55 (6H,
(125MHz, CDCl3) δ: 170.4, 148.9, 145.3, 120.5, 120.0, 119.1, m), 1.35–1.29 (12H, m), 0.89 (3H, t, J=7.0 Hz).
112.7, 71.0, 70.9, 65.7, 31.7, 29.7, 29.3, 29.2, 28.5, 26.1, 25.9,
22.6, 14.0. HR-ESI-MS m/z: 389.2324 (Calcd for C21H34O5Na
[M+Na]+: 389.2298).
10-Hydroxydecyl Butyrate (13e)
The synthesis and purification methods were the same as
for 13a. The pure 13e was obtained as a colorless oil (1.01g,
1
10-(Butoxy)decyl 2,3-Dihydroxybenzoate (11e)
41%). H-NMR (500MHz, CDCl3) δ: 4.05 (2H, t, J=6.5Hz),
The synthesis and purification methods were the same as 3.63 (2H, t, J=6.5Hz), 2.27 (2H, t, J=7.5Hz), 1.67–1.54 (6H,
for 3a, condensing from 10e (230mg, 1.00mmol) and 2,3-di- m), 1.38–1.24 (12H, m), 0.94 (3H, t, J=7.5 Hz).
hydroxybenzoic acid (308mg, 2.00mmol). The pure 11e
was obtained as a colorless powder (190mg, 52%). H-NMR
12-Hydroxydodecyl Acetate (13f)
The synthesis and purification methods were the same as
1
(500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz), 7.10 for 13a. The pure 13f was obtained as a colorless oil (1.10g,
1
(1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.67 (1H, s), 4.34 45%). H-NMR (500MHz, CDCl3) δ: 4.04 (2H, t, J=6.5Hz),
(2H, t, J=6.5Hz), 3.41–3.78 (4H, m), 1.77 (2H, m), 1.59–1.52 3.63 (2H, m), 2.04 (3H, s), 1.64–1.53 (4H, m), 1.34–1.26 (16H,
(4H, m), 1.45–1.30 (14H, m), 0.92 (3H, t, J=7.5Hz). 13C-NMR m).
(125MHz, CDCl3) δ: 170.4, 148.9, 145.0, 120.5, 119.6, 119.1,
2-(Dodecanoyloxy)ethyl 2,3-Dihydroxybenzoate (14a)
112.7, 70.9, 70.6, 65.7, 31.9, 29.8, 29.5, 29.4, 29.2, 28.5,
The synthesis and purification methods were the same as
26.2, 25.9, 19.4, 13.9. HR-ESI-MS m/z: 389.2293 (Calcd for for 3a, condensing from 13a (230mg, 0.94mmol) and 2,3-di-
C21H34O5Na [M+Na]+: 389.2298).
12-(Ethoxy)dodecyl 2,3-Dihydroxybenzoate (11f)
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14a
was obtained as a colorless powder (190mg, 53%). H-NMR
1
The synthesis and purification methods were the same as (500MHz, CDCl3) δ: 10.75 (1H, s), 7.38 (1H, d, J=8.0Hz),
for 3a, condensing from 10f (230mg, 1.00mmol) and 2,3-di- 7.12 (1H, d, J=8.0Hz), 6.81 (1H, t, J=8.0Hz), 4.55 (2H, m),
hydroxybenzoic acid (308mg, 2.00mmol). The pure 11f 4.43 (2H, m), 2.34 (2H, t, J=7.5Hz), 1.61 (2H, m), 1.29–1.24
1
was obtained as a colorless powder (197mg, 55%). H-NMR (16H, m), 0.88 (3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3)

Vol. 64, No. 2 (2016)
Chem. Pharm. Bull.
169
δ: 173.6, 170.0, 148.9, 145.0, 120.7, 120.0, 119.3, 112.1, 63.2, obtained as a colorless powder (203mg, 57%). 1H-NMR
61.5, 34.1, 31.9, 29.6, 29.4, 29.3, 29.2, 29.1, 24.9, 22.7, 14.1. (500MHz, CDCl3) δ: 11.00 (1H, s), 7.37 (1H, d, J=8.0Hz),
HR-ESI-MS m/z: 403.2089 (Calcd for C21H32O6Na [M+Na]+: 7.10 (1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.78 (1H, s),
403.2091).
4.34 (2H, t, J=6.5Hz), 4.05 (2H, t, J=6.5Hz), 2.05 (3H, s),
4-(Decanoyloxy)butyl 2,3-Dihydroxybenzoate (14b)
1.77 (2H, m), 1.61 (2H, m), 1.45–1.27 (16H, m). 13C-NMR
The synthesis and purification methods were the same as (125MHz, CDCl3) δ: 171.2, 170.4, 148.9, 145.0, 120.5, 119.6,
for 3a, condensing from 13b (230mg, 0.94mmol) and 2,3-di- 119.1, 112.7, 65.7, 64.7, 29.5, 29.2, 28.6, 28.5, 25.9, 21.0. HR-
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14b ESI-MS m/z: 403.2094 (Calcd for C21H32O6Na [M+Na]+:
1
was obtained as a colorless powder (180mg, 50%). H-NMR 403.2091).
(500MHz, CDCl3) δ: 10.93 (1H, s), 7.36 (1H, d, J=8.0Hz),
7.11 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.64 (1H, s),
S-Hexyl 2,3-Dihydroxybenzothioate (15a)
To a solution of compound 6 (79mg, 0.20mmol), triethyl-
4.39 (2H, t, J=6.5Hz), 4.14 (2H, t, J=6.5Hz), 2.30 (2H, t, amine (202mg, 2.0mmol) in dry CH2Cl2 (5mL) at 0°C was
J=7.5Hz), 1.90–1.77 (4H, m), 1.62 (2H, m), 1.32–1.26 (12H, added pivaloyl chloride (24mg, 0.20mmol). The mixture was
m), 0.87 (3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3) δ: warmed to room temperature and was stirred until compound
173.9, 170.3, 148.9, 145.1, 120.5, 119.8, 119.1, 112.4, 65.0, 63.5, 6 disappeared (detected by TLC). Then 1-hexanethiol (47mg,
34.3, 31.8, 29.4, 29.2, 29.1, 25.3, 25.0, 22.6, 14.1. HR-ESI-MS 0.40mmol) and 4-dimethylaminopyridine (2.4mg, 0.02mmol)
m/z: 403.2128 (Calcd for C21H32O6Na [M+Na]+: 403.2091).
6-(Octanoyloxy)hexyl 2,3-Dihydroxybenzoate (14c)
were added into the mixture at 0°C, warmed up to room tem-
perature, and kept stirring for 12h. Finally, trifluoroacetic acid
The synthesis and purification methods were the same as (50% in water, 5mL) was added into the mixture, and kept
for 3a, condensing from 13c (230mg, 0.94mmol) and 2,3-di- stirring for 12h. The mixture was extracted with CH2Cl2, and
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14c washed with water, saturated aqueous solution of NaHCO3
1
was obtained as a colorless powder (199mg, 56%). H-NMR and brine, successively. The organic phase was dried over
(500MHz, CDCl3) δ: 10.97 (1H, s), 7.36 (1H, d, J=8.0Hz), Na2SO4, filtered, and then concentrated. The crude mixture
7.11 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.65 (1H, s), was purified by column chromatography (n-hexane–EtOAc
4.35 (2H, t, J=6.5Hz), 4.08 (2H, t, J=6.5Hz), 2.29 (2H, t, 300:1) on silica gel to afford 15a as a colorless oil (27mg,
1
J=7.0Hz), 1.79 (2H, m), 1.68–1.57 (4H, m), 1.50–1.41 (4H, m), 53%). H-NMR (500MHz, CDCl3) δ: 11.26 (1H, s), 7.42 (1H,
1.30–1.27 (8H, m), 0.87 (3H, t, J=7.0Hz). 13C-NMR (125MHz, d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.81 (1H, t, J=8.0Hz),
CDCl3) δ: 174.0, 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.6, 5.69 (1H, s), 3.07 (2H, t, J=7.0Hz), 1.68 (2H, m), 1.44 (2H, m),
65.5, 64.1, 34.4, 31.7, 29.1, 28.9, 28.5, 28.4, 25.6, 25.0, 22.6, 1.33–1.31 (4H, m), 0.90 (3H, t, J=7.0Hz). 13C-NMR (125MHz,
14.0. HR-ESI-MS m/z: 403.2129 (Calcd for C21H32O6Na CDCl3) δ: 197.9, 146.6, 145.3, 120.0, 119.7, 119.5, 119.4, 31.8,
[M+Na]+: 403.2091).
8-(Hexanoyloxy)octyl 2,3-Dihydroxybenzoate (14d)
The synthesis and purification methods were the same as
for 3a, condensing from 13d (230mg, 0.94mmol) and 2,3-di-
29.3, 29.1, 28.9, 22.6, 14.1. HR-ESI-MS m/z: 255.1063 (Calcd
for C13H19O3S [M+H]+: 255.1049).
S-Octyl 2,3-Dihydroxybenzothioate (15b)
The synthesis and purification methods were the same as
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14d for 15a using 1-octanethiol (29mg, 0.20mmol) as correspond-
1
was obtained as a colorless powder (177mg, 49%). H-NMR ing thiol, the pure 15b was obtained as a colorless oil (30mg,
(500MHz, CDCl3) δ: 10.99 (1H, s), 7.37 (1H, d, J=8.0Hz), 53%). 1H-NMR (500MHz, CDCl3) δ: 11.26 (1H, s), 7.42 (1H, d,
7.10 (1H, d, J=8.0Hz), 6.80 (1H, t, J=8.0Hz), 5.65 (1H, s), J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.81 (1H, t, J=8.0Hz), 5.69
4.34 (2H, t, J=7.0Hz), 4.06 (2H, t, J=7.0Hz), 2.29 (2H, t, (1H, s), 3.07 (2H, t, J=7.0Hz), 1.68 (2H, m), 1.41–1.40 (2H, m),
J=7.5Hz), 1.78 (2H, m), 1.64–1.61 (4H, m), 1.44–1.29 (12H, 1.34–1.28 (8H, m), 0.89 (3H, t, J=7.0Hz). 13C-NMR (125MHz,
m), 0.89 (3H, t, J=7.0Hz). 13C-NMR (125MHz, CDCl3) δ: CDCl3) δ: 197.9, 146.6, 145.3, 120.0, 119.7, 119.5, 119.4, 31.8,
174.0, 170.4, 148.9, 145.0, 120.5, 119.7, 119.1, 112.6, 65.6, 64.3, 29.3, 29.1, 28.9, 22.6, 14.1. HR-ESI-MS m/z: 283.1369 (Calcd
34.4, 31.3, 29.1, 28.6, 28.5, 25.8, 24.7, 22.3, 13.9. HR-ESI-MS for C15H23O3S [M+H]+: 283.1362).
m/z: 403.2118 (Calcd for C21H32O6Na [M+Na]+: 403.2091).
10-(Butyryloxy)decyl 2,3-Dihydroxybenzoate (14e)
S-Decyl 2,3-Dihydroxybenzothioate (15c)
The synthesis and purification methods were the same as
The synthesis and purification methods were the same as for 15a using 1-decanethiol (35mg, 0.20mmol) as correspond-
for 3a, condensing from 13e (230mg, 0.94mmol) and 2,3-di- ing thiol, the pure 15c was obtained as a colorless oil (35mg,
1
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14e 56%). H-NMR (500MHz, CDCl3) δ: 11.26 (1H, s), 7.42 (1H,
1
was obtained as a colorless powder (190mg, 53%). H-NMR d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.81 (1H, t, J=8.0Hz),
(500MHz, CDCl3) δ: 10.98 (1H, s), 7.37 (1H, d, J=8.0Hz), 5.69 (1H, s), 3.07 (2H, t, J=7.5Hz), 1.68 (2H, m), 1.42 (2H,
7.10 (1H, d, J=8.0Hz), 6.79 (1H, t, J=8.0Hz), 5.69 (1H, s), m), 1.34–1.27 (12H, m), 0.88 (3H, t, J=7.0Hz). 13C-NMR
4.34 (2H, t, J=6.5Hz), 4.06 (2H, t, J=6.5Hz), 2.27 (2H, (125MHz, CDCl3) δ: 197.9, 146.6, 145.3, 120.0, 119.7, 119.5,
t, J=7.5Hz), 1.78 (2H, m), 1.67–1.26 (16H, m), 0.95 (3H, t, 119.4, 31.9, 29.5, 29.3, 29.1, 28.9, 22.7, 14.1. HR-ESI-MS m/z:
J=7.5Hz). 13C-NMR (125MHz, CDCl3) δ: 173.8, 170.4, 148.9, 311.1687 (Calcd for C17H27O3S [M+H]+: 311.1675).
145.1, 120.5, 119.7, 119.1, 112.7, 65.7, 64.3, 36.3, 29.4, 29.2,
28.6, 28.5, 25.9, 18.5, 13.7. HR-ESI-MS m/z: 403.2092 (Calcd
for C21H32O6Na [M+Na]+: 403.2091).
S-Dodecyl 2,3-Dihydroxybenzothioate (15d)
The synthesis and purification methods were the same as for
15a using 1-dodecanethiol (40mg, 0.20mmol) as correspond-
12-(Acetoxy)dodecyl 2,3-Dihydroxybenzoate (14f)
ing thiol, the pure 15d was obtained as a colorless oil (39mg,
1
The synthesis and purification methods were the same as 58%). H-NMR (500MHz, CDCl3) δ: 11.23 (1H, s), 7.42 (1H,
for 3a, condensing from 13f (230mg, 0.94mmol) and 2,3-di- d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), 6.81 (1H, t, J=8.0Hz),
hydroxybenzoic acid (308mg, 2.00mmol). The pure 14f was 5.67 (1H, s), 3.07 (2H, t, J=7.5Hz), 1.68 (2H, m), 1.42 (2H,

170
Chem. Pharm. Bull.
Vol. 64, No. 2 (2016)
m), 1.34–1.26 (16H, m), 0.88 (3H, t, J=6.5Hz). 13C-NMR Tyr204) rabbit polyclonal antibody (Cell Signaling Technol-
(125MHz, CDCl3) δ: 197.880, 146.642, 145.300, 120.001, ogy; MA, U.S.A.) for one hour at room temperature. The
119.689, 119.492, 119.350, 31.904, 29.620, 29.557, 29.462, horseradish peroxidase-conjugated secondary antibody (Bei-
29.330, 29.287, 29.104, 28.891, 22.676, 14.098. HR-ESI-MS jing ComWin Biotech Co., Ltd., Beijing, China) was incubated
m/z: 339.1989 (Calcd for C19H31O3S [M+H]+: 339.1988).
S-Tetradecyl 2,3-Dihydroxybenzothioate (15e)
with membrane subsequently. The bands were developed with
enhanced chemiluminescent reaction (Beijing ComWin Bio-
The synthesis and purification methods were the same technology) and analyzed by the software-Image J (National
as for 15a using 1-tetradecanethiol (46mg, 0.20mmol) as Institutes of Health, MD, U.S.A.).
corresponding thiol, the pure 15e was obtained as a color-
less powder (37mg, 50%). 1H-NMR (500MHz, CDCl3) δ:
Acknowledgments This work was financially supported
11.27 (1H, s), 7.42 (1H, d, J=8.0Hz), 7.10 (1H, d, J=8.0Hz), by International Science and Technology Cooperation Pro-
6.81 (1H, t, J=8.0Hz), 5.67 (1H, s), 3.07 (2H, t, J=6.5Hz), gram of China (No. 2014DFG32690), NSFC (81072536 and
1.68 (2H, m), 1.42 (2H, m), 1.34–1.26 (20H, m), 0.88 (3H, t, 81273385). We thank Pharmaceutical Informatics Institute of
J=6.5Hz). 13C-NMR (125MHz, CDCl3) δ: 197.881, 146.643, Zhejiang University for performing NMR spectrometry.
145.299, 120.000, 119.690, 119.490, 119.350, 31.914, 29.643,
29.559, 29.463, 29.346, 29.288, 29.104, 28.883, 22.680, 14.101.
Conflict of Interest The authors declare no conflict of
HR-ESI-MS m/z: 367.2300 (Calcd for C21H35O3S [M+H]+: interest.
367.2301).
Bioassay Biological activity was done using the methods
described in our previous study.6) PC12 cells (20000) were
seeded in each well of a 24-well microplate and incubated
under a humidified atmosphere of 5% CO2 at 37°C. After 24h,
the medium was replaced with 1mL of serum-free Dulbecco’s
modified Eagle’s medium (DMEM) containing a sample or
DMSO (0.5%). NGF (40ng/mL) was used as a positive control.
The morphological changes of the cells were observed under
a microscope. Approximately, 100 cells were watched from a
randomly chosen area. A cell having neurite outgrowth longer
than the diameter of cell body was determined as a positive
cell. Significant differences between each groups were tested
by ANOVA, followed by two-tailed multiple Student–New-
man–Keuls t-tests using SPSS biostatistics software (IBM;
Armonk, NY, U.S.A.). Values of p<0.05 were considered
significant. Independent experiments were repeated for three
times. Each value represents as the mean standard error of
the mean (S.E.M.) of three replicates. ** or *** indicates
significant differences relative to the control at p<0.01 or
p<0.001, respectively.
Western Blot Western blot analysis was performed as
previously described.10) Briefly, 1.5×106 cells were incubated
in 6cm dish with 15d (ABG-199) at a concentration of 0.1µM
for 0, 2, 4, 6, 8 or 10h. The cells were lysed in 150µL lysis
buffer and then sonicated. The supernatant was removed after
centrifugation at 12000rpm for 15min and protein concentra-
tion was determined by Bio-Rad protein assay kit (Bio-Rad
Lab.; CA, U.S.A.). The cells were treated with 40ng/mL NGF
as positive control. A 15µg of protein was loaded into sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) gel and electrophoresised, then protein was transferred
onto the polyvinylidene difluoride (PVDF) membrane. The
membrane was blocked and incubated with anti-phospho-
p44/42 mitogen-activated protein kinase (MAPK) (Thr202/
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