Structure-activity relationships of neuritogenic gentiside derivatives

Full text of DOI:10.1002/cmdc.201100348

MED
DOI: 10.1002/cmdc.201100348
Structure–Activity Relationships of Neuritogenic Gentiside Derivatives
Yan Luo,[a] Kaiyue Sun,[a] Lin Li,[b] Lijuan Gao,[a] Guangfa Wang,[a] Yuan Qu,[a] Lan Xiang,[c] Ling Chen,*[b]
Yongzhou Hu,[a] and Jianhua Qi*[a]
Neurotrophic factors are target-derived peptides that play an
important role in the development and survival of responsive
neuronal populations. Nerve growth factor (NGF), one of the
most important neurotrophic factors, is essential for neuronal
differentiation, growth, survival, function maintenance, and
prevention of aging in the central and peripheral systems.[1,2]
However, because of its large molecular size and hydrophilic
properties, NGF cannot pass through the blood–brain barrier,
Table 1. Structures of gentiside derivatives 1a–y, 2a–d and 3a–b.
limiting its use as a therapeutic agent. Therefore, synthetic
low-molecular weight compounds that possess equivalent or
better neuritogenic activity compared with NGF are promising
agents for the treatment of neurodegenerative diseases, such
as Alzheimer’s disease.[3] The PC12 cell line, cloned from rat
pheochromocytoma, is widely used as a model system to eval-
uate the biological activity of neuritogenic substances.[4–7]
Compd
R1
R2
R3
R4
R5
X
R
Recently, screening for neuritogenic substances from tradi-
tional Chinese medicine resulted in the isolation of 11 novel
alkyl benzoates (gentisides A–K) from Gentiana rigescens
(Franch.).[8,9] These compounds are structurally different from
one another through varying alkyl chain lengths and the pres-
ence or absence of an isobutyl or isopropyl group at the end
of the alkyl chain. The structure–activity relationships within
gentisides reveal that the alkyl chain length is important for ac-
tivity, but structural diversity at the end of the alkyl chain is
not. Gentisides D, E, and F have similar alkyl chain lengths. In
spite of the different structures at the end of the alkyl chain,
these compounds exhibit similar neuritogenic activities at the
optimum concentration of 3 mm. Gentiside E, which has a
straight alkyl chain of 18 carbon atoms, exhibits higher activity
(74% at 3 mm) than gentiside K, which has 24 carbon atoms in
the alkyl chain (48% at 30 mm).[9]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
OH
H
OCH3
OH
OCH3
OH
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
OH
OH
OH
H
H
H
H
OH
OH
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
C2H5
C5H11
C8H17
C10H21
C12H25
C14H29
C16H33
C18H37
C20H41
C22H45
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C14H29
C12H25
C12H25
C13H25
C13H25
H
H
H
H
OH
OH
H
H
OH
H
H
H
H
H
OH
H
H
H
H
OH
OH
OH
OH
OCH3
OCH3
OH
OH
OH
OH
OH
OCH3
OH
OCH3
OH
O
O
O
O
1s
1t
To study the structure–activity relationships and discover
lead compounds for drug development, a series of gentiside
derivatives were designed and synthesized. First, 2,3-dihydrox-
ybenzoates 1a–j (Table 1) with different alkyl chain lengths
were synthesized to find the optimum length of the alkyl
chain. Second, tetradecylbenzoates 1k–u (Table 1) were pre-
1u
1v
1w
1x
1y
2a
2b
2c
2d
O
NH
NH
NH
NH
CH2
CH2
CH2
CH2
H
OCH3
H
H
OCH3
OH
H
H
[a] Y. Luo, K. Sun, L. Gao, Dr. G. Wang, Y. Qu, Prof. Dr. Y. Hu, Prof. Dr. J. Qi
College of Pharmaceutical Sciences, Zhejiang University
Yu Hang Tang Road 388, Hangzhou 310058, PR China
E-mail: qijianhua@zju.edu.cn
pared to study the importance of the number and position of
the hydroxy groups on the benzene ring. Third, to find the
best linkage group between the benzene ring and alkyl chain,
tetradecyl-benzamides 1v–y, phenylketones 2a–d, 2,3-dihy-
droxyphenyl tetradecanoate (3 a) and 3-(tetradecyloxy)ben-
zene-1,2-diol (3 b) were synthesized. Full experimental details
can be found in the Supporting Information.
[b] L. Li, Prof. Dr. L. Chen
Department of Physiology, Nanjing Medical University
Hanzhong Road 140, Nanjing 210029, PR China
E-mail: lingchen@njmu.edu.cn
[c] Dr. L. Xiang
College of Biosystems Engineering & Food Sciences, Zhejiang University
Yu Hang Tang Road 388, Hangzhou 310058, PR China
Ethyl-2,3-dihydroxybenzoate 1a and pentyl-2,3-dihydroxy-
benzoate 1b were prepared by reacting 2,3-dihydroxybenzoic
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100348.
1986
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ChemMedChem 2011, 6, 1986 – 1989

acid with the corresponding alcohol in the presence of a cata-
lytic amount of concentrated sulfuric acid. Compounds 1c–k,
1m–n, 1p and 1r were synthesized by esterification of substi-
tuted benzoic acid with the corresponding alcohol in dry tetra-
hydrofuran (THF) using N,N’-dicyclohexylcarbodiimide (DCC).[10]
2,3-Dihydroxyphenyl tetradecanoate (3 a) was prepared from
pyrogallic acid and tetradecanoic acid also using DCC as a cou-
pling agent. Compounds 1l, 1s, and 1t were obtained using
1-(3-dimethylaminopropyl)-3-eth-
dent response of the NGF-mimicking activity of each com-
pound was tested. The concentration that induced the highest
percentage of neurite outgrowth (maximum activity) of PC12
cells is reported. Figure 1 shows the maximum neuritogenic ac-
tivity of gentiside analogues 1a–y, 2a–d and 3a–b.
Figure 1a shows the maximum neuritogenic activity of com-
pounds 1a–j. Compounds 1a and 1b, which have short alkyl
chain lengths (C2 and C5), exhibited 38% (10 mm) and 37%
ylcarbodiimide
(EDC) as an activating reagent.
Tetradecyl 3,5-dihydroxyben-
hydrochloride
zoate (1o) and tetradecyl 2,3,4-
trihydroxybenzoate (1q) were
prepared by a Mitsunobu reac-
tion using diethyl azodicarboxy-
late (DEAD) as the condenser.[11]
Methylation of compound 1 f
gave tetradecyl 3-hydroxy-2-me-
thoxybenzoate (1u).[12]
Benzamides 1v, 1w and 1x
were synthesized by amidation
of 2,3-dihydroxybenzoic acid,
3,4-dihydroxybenzoic acid or
3,4,5-trihydroxybenzoic acid with
tetradecan-1-amine in the pres-
ence of DCC and 1-hydroxyben-
zotriazole hydrate (HOBt).[13] 3-
Hydroxy-2-methoxy-N-tetradecyl-
benzamide (1y) was prepared
via a methylation reaction ac-
cording to the procedure report-
ed for 1u. The 3,4-dimethoxy-
phenyl ketone 2a was prepared
using veratrole through a Frie-
del–Crafts reaction. 2,3-Dime-
thoxyphenyl ketone 2c was ob-
tained by a Grignard reaction.[14]
Deprotection of the methoxy-
phenyl ketones 2a and 2c with
boron tribromide yielded hy-
droxyphenyl ketones 2b and 2d,
respectively.
3-(Tetradecyloxy)-
benzene-1,2-diol (3b) was pre-
pared by a Mitsunobu reaction
between pyrogallol and 1-tetra-
decanol.[15] All compounds were
purified by silica gel open
column chromatography.
The neuritogenic activities of
the synthesized gentiside deriva-
tives were evaluated in PC12
cells and compared with a nega-
tive control (0.5% DMSO) and a
positive control (NGF at the opti-
Figure 1. Maximum NGF-mimicking activity of gentiside derivatives 1a–y, 2a–d, and 3a–b at the optimum con-
centration. Values represent the mean neurite outgrowth (%) Æ SD (n=3). C: control (0.5% DMSO); NGF: positive
mum
concentration
of
40 ngmLÀ1). The dose-depen- control (40 ngmLÀ1). 1a–y, 2a–d, 3a–b: *** p <0.001.
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(3 mm) neuritogenic activity, respectively. Compounds 1c–g,
with alkyl chain lengths of 8 to 16 carbon atoms, showed max-
imum activities of 71%, 80%, 82%, 87%, and 80%, respectively
at 1 mm. Compounds 1h–j, with alkyl chain lengths of 18 to 22
carbon atoms exhibited maximum activities of 81% (3 mm),
78% (10 mm), and 70% (30 mm), respectively. These results
reveal that alkyl chain length had significant effects on neurito-
genic activity. Tetradecyl benzoate (1 f) exhibited the highest
neuritogenic activity at 1 mm. Therefore, a straight chain of 14
carbon atoms was considered to be the optimum structure for
biological activity.
After determination of the best alkyl chain length, the syn-
thetic strategy was targeted to find the optimum number and
position of hydroxy groups on the benzene ring. Tetradecyl
monohydroxybenzoate (1k), tetradecyl 2,6-dihydroxybenzoate
(1l), tetradecyl 2,4-dihydroxybenzoate (1m), tetradec-
yl 2,5-dihydroxybenzoate (1n), and tetradecyl 3,5-di-
hydroxybenzoate (1o) exhibited maximum activities
of 32% (3 mm), 28% (3 mm), 38% (1 mm), 40% (1 mm),
and 43% (1 mm), respectively (Figure 1b). Tetradecyl
3,4-dihydroxybenzoate (1p), tetradecyl 2,3,4-trihy-
droxybenzoate (1q) and tetradecyl 3,4,5-trihydroxy-
benzoate (1r) showed maximum activities of 83%
(3 mm), 69% (1 mm) and 80% (1 mm), respectively (Fig-
ure 1b). These results reveal that at least two ortho-
hydroxy groups on the benzene ring are necessary
for significant neuritogenic activity. Furthermore, the
maximum activity of 29% (10 mm) for 1s, 33%
(10 mm) for 1t, and 44% (1 mm) for 1u indicate that
the presence of hydroxy groups is important for neu-
ritogenic activity (Figure 1b).
Figure 2. Dose-dependent response of the NGF-mimicking activity of ABG-
001 (1 f) 48 h after treatment. C: control (0.5% DMSO); NGF: positive control
(40 ngmLÀ1). 1 f at 0.03 mm, ** p <0.01; 0.1, 0.3, 1 mm: *** p <0.001.
Finally, suitable groups linking the benzene ring
and the alkyl chain were investigated. Compounds
Figure 3. Photomicrographs of PC12 cells under a phase-contrast microscope 48 h after
treatment: a) solvent control (0.5% DMSO); b) ABG-001 (1 f) (1 mm); c) NGF (40 ngmLÀ1);
d) ABG-001 (1 f) (0.03 mm).
1v–y, 2a–d and 3a–b induced maximum activities of
51% (0.1 mm), 57% (0.3 mm), 48% (0.3 mm), 42%
(0.2 mm), 42% (4 mm), 41% (0.3 mm), 36% (2 mm), 46%
(0.3 mm), 38% (3 mm), and 43% (0.3 mm), respectively (Fig-
ure 1c). These results indicate that an ester linkage, rather than
a ketone, amido, ether, or modified ester linkage, is optimal for
neuritogenic activity.
comparison with the solvent control (0.5% DMSO), and the
positive control (NGF ). Control cells (without any test com-
pound) showed very few short neurite outgrowth (Figure 3a).
When treated with ABG-001 (1 f) at 1 mm, the cells extended
long multipolar neurite outgrowths 48 h after treatment (Fig-
ure 3b), which were similar to those produced following treat-
ment with NGF at 40 ngmLÀ1 (Figure 3c). At a dose even as
low as 0.03 mm, ABG-001 (1 f) induced significant neurite out-
growth (Figure 3d). However, 2,3-dihydroxybenzoic acid only
or tetradecan-1-ol did not give rise to obvious neurite out-
growth in PC12 cells (data not shown). These results reveal
that both the hydrophobic alkyl chain and the 2,3-dihydroxy-
benzoic acid moiety are necessary for neuritogenic activity.
Subsequently, the signaling pathway of ABG-001 (1 f)-in-
duced neuritogenesis was investigated. The cellular mecha-
nism of NGF-induced neuritogenesis has been well investigat-
ed.[16-18] NGF first binds to the transmembrane-specific receptor,
tyrosine receptor kinase A (TrkA) to induce phosphorylation of
the specific tyrosine residues located at the intracellular
domain, leading to recruitment and activation of a number of
kinases. Among them, the mitogen-activated protein (MAP)
Taking the above results and structural features into consid-
eration, tetradecyl 2,3-dihydroxybenzoate (1 f), tetradecyl 3,4-
dihydroxybenzoate (1p), and tetradecyl 3,4,5-trihydroxyben-
zoate (1r) were selected as lead compounds and renamed
ABG-001, ABG-002, and ABG-003, respectively. ABG-001 (1 f) ex-
hibited the highest neuritogenic activity against PC12 cells of
all synthesized compounds. Thus, this compound was further
evaluated.
ABG-001 (1 f) showed dose-dependent activity when evalu-
ated in the concentration range of 0.03 to 1 mm (Figure 2). The
maximum neuritogenic activity of ABG-001 (1 f) at the best
concentration of 1 mm was comparable to that seen for NGF at
40 ngmLÀ1, the best concentration for this protein. Significant
neuritogenic activity (p <0.01) of ABG-001 (1 f) was observed
even at 0.03 mm (Figure 2). The solvent control also induced
some neurite outgrowth. Figure 3 shows the morphological
changes of PC12 cells after treatment with ABG-001 (1 f) in
1988
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kinase extracellular signal-regulated kinase (ERK) is a key
enzyme during NGF-induced neurite outgrowth. To investigate
the cellular events of significant neurite outgrowth induced by
ABG-001 (1 f), specific inhibitor tests were performed to find
the possible target kinases for ABG-001-induced neuritogene-
sis. Therefore, the TrkA-specific inhibitor K252a[19] and the ERK-
specific inhibitor U0126[20] were applied to examine whether
TrkA and ERK kinases are necessary for neuritogenesis induced
by ABG-001 (1 f). The results show that the percentage of neu-
rite outgrowth induced by ABG-001 (1 f) did not significantly
decrease when treated with K252a at various concentrations
(data not shown). However, U0126 treatment led to a reduc-
tion of neuritogenesis induced by ABG-001 (1 f) from 89% to
9%. To confirm these results, phosphorylation of TrkA and ERK
in PC12 cells was examined using Western blot analysis after
treatment with ABG-001 (1 f) at 1 mm. Sustained and strongly
enhanced ERK phosphorylation was observed over 28 h when
PC12 cells were treated with NGF at 40 ngmLÀ1 (Figure 4).
However, ABG-001 (1 f) treatment resulted in sustained and
significantly enhanced ERK phosphorylation 16 h after treat-
ment (Figure 4). At 1 mm, ABG-001 (1 f) did not activate TrkA
(data not shown). Phosphorylation levels induced by ABG-001
(1 f) were slightly lower and more delayed than those induced
by NGF. These results reveal that significant neuritogenesis in-
duced by ABG-001 (1 f) was dependent on the sustained acti-
vation of ERK in PC12 cells.
groups on the benzene ring, and the type of linker between
the benzene ring and alkyl chain had distinct effects on the
neuritogenic activities of these derivatives. ABG-001 (1 f), ABG-
002 (1p), and ABG-003 (1r), with 14 carbons, two or three
close hydroxy groups on the benzene ring, and an ester link-
age, exhibited significant activities at 1 or 3 mm. Furthermore,
ABG-001 (1 f) induced significant neurite outgrowth via activa-
tion of the ERK signaling pathway. This compound was exam-
ined using specific-inhibitor experiments and Western blot
analysis. Intensive studies on these neuritogenic compounds
are currently underway in our lab.
Experimental Section
The experimental details of compound syntheses, characterization
data, biological evaluation, and mechanism studies are available in
the Supporting Information.
Acknowledgements
This work was financially supported by the NSFC (30873152 and
81072536), the Qianjiang Talent Program of Zhejiang Province
(2010R10052) and the Natural Science Foundation of Zhejiang
Province, China (Y2110105 to L.X.).
Keywords: ABG-001
·
biological activity
·
gentiside
In conclusion, a series of gentiside derivatives were prepared
through effective and convenient synthetic methods. Most of
these derivatives are new compounds. Their structures were
confirmed by NMR (13C NMR for new compounds only), IR, and
high-resolution ESI-TOF-MS (Supporting Information). The
length of the alkyl chain, number and position of hydroxy
derivatives · PC12 cells · structure–activity relationships
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Figure 4. Phosphorylation of ERK in the presence of control, ABG-001 (1 mm)
and NGF (40 ngmLÀ1). The phosphorylation levels (%) were measured using
a luminescent image analyzer after Western blotting and normalized to the
^
control value at 0 min (100%) for phospho-ERK. = control; &= 1 f (1 mm);
Received: July 16, 2011
Published online on August 10, 2011
~= NGF (40 ngmLÀ1).
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1989