Neuritogenic effect of epolactaene derivatives on human neuroblastoma cells which lack high-affinity nerve growth factor receptors

Full text of DOI:10.1021/jm960719a

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© Copyright 1997 by the American Chemical Society
Volume 40, Number 4
February 14, 1997
Communications to the Editor
We have screened microbial products to discover
Neu r itogen ic Effect of Ep ola cta en e
neuritogenic compounds which induced neurite out-
growth in the human neuroblastoma cell line SH-SY5Y
that lacks significant TRK family mRNAs.17,18 During
the screening, we isolated a novel, non-protein neu-
rotrophic compound, epolactaene (Figure 1), produced
by Penicillium sp. 1689-P, having an epoxide-containing
γ-lactam ring in the molecule.19 However, only small
quantities of epolactaene could be isolated from the
fungus; furthermore, the compound was unstable under
light because of a labile triene group in the side chain
and an epoxide fused to a γ-lactam ring. In the present
paper, we prepared a number of epolactaene derivatives,
3-substituted 3-pyrrolin-2-ones possessing a double bond
in the γ-lactam ring, to find compounds with more
effective neuritogenic activity. In addition, we described
the structure-activity relationship of 3-substituted
3-pyrrolin-2-ones on the neuritogenic effect in SH-SY5Y
cells. MT-5, 3-acetyl-4,5-dimethyl-5-octadecyloxy-3-pyr-
rolin-2-one, the most effective derivative for neuritogenic
activity in SH-SY5Y cells, also arrested cell cycle
progression at G0/G1 like epolactaene.
Der iva tives on Hu m a n Neu r obla stom a
Cells Wh ich La ck High -Affin ity Ner ve
Gr ow th F a ctor Recep tor s
Hideaki Kakeya, Chizuko Onozawa, Masakazu Sato,†
Koshi Arai,† and Hiroyuki Osada*
Antibiotics Laboratory, The Institute of Physical and
Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi,
Saitama 351-01, J apan, and Medicinal Research
Laboratories, Taisho Pharmaceutical Co., Ltd.,
Yoshino-cho 1-403, Ohmiya-shi, Saitama 330, J apan
Received October 14, 1996
Neurotrophic factors such as nerve growth factor
(NGF) are involved in survival, growth, and differentia-
tion in normal and neoplastic nerve cells.1,2 At least
three receptors bearing tyrosine kinase activity for
neurotrophic factors have beeen cloned;3-5 TRK-A en-
codes a high-affinity receptor for NGF and also binds
neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5).6-8
TRK-B encodes a receptor for brain-derived neu-
rotrophic factor (BDNF) and also binds NT-3 and NT-
4/5, and TRK-C encodes a receptor for NT-3.9-11 Despite
high sequence homology among the receptors, the
developmental and physiological functions of each neu-
rotrophic factor appear to be different, suggesting that
each factor has a unique role.
In addition, it has been reported that most benign
neuroblastomas express high levels of TRK-A, while
malignant neuroblastomas, especially those with N-myc
amplification, express little or no detectable TRK-A
mRNA.12-16 NGF is expected to induce the differentia-
tion of benign neuroblastomas to neurons, but NGF has
no effect on malignant neuroblastomas lacking NGF
receptors. Therefore, compounds which induce the
differentiation of neuroblastomas might be good candi-
dates for pharmaceutical agents for various neurode-
generative diseases.
Ch em istr y. We focused on the γ-lactam ring of
epolactaene in developing derivatives that have more
simple structure and potent activity. The intermediates
were synthesized essentially according to the procedure
of Howard et al.,20 in which R,â-diketones, including
aliphatic diketones, react smoothly in aqueous solution
pH 7-10 at room temperature with a variety of aceta-
mides possessing a strong electron-withdrawing group
in the R-position to give 3-substituted 5-hydroxy-3-
pyrrolin-2-ones. For example, as shown in Scheme 1,
R,â-diketone (1) reacted with acetoacetamide (2) to give
3 and the following treatment with alcohols under acidic
conditions gave 3-acetyl-4,5-dimethyl-5-(octadecyloxy)-
3-pyrrolin-2-one (MT-5) in a moderate yield. N-Alky-
lpyrrolinones were also synthesized by a similar proce-
dure.
Resu lts a n d Discu ssion . Neurite outgrowth activ-
ity was measured as follows. SH-SY5Y cells were plated
at a density of 3 × 103 cells per 100 µL per well on a
96-well plate which was precoated with collagen and
incubated for 24 h at 37 °C in 5% CO2 in Dulbecco’s
modified Eagle medium (DMEM) containing 10% fetal
* Corresponding author: Tel: +81-(48)-467-9541. FAX: +81-(48)-
462-4669. E-mail: antibiot@postman.riken.go.jp.
† Taisho Pharmaceutical Co., Ltd.
S0022-2623(96)00719-4 CCC: $14.00 © 1997 American Chemical Society

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392 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Communications to the Editor
Ta ble 1. Neuritogenic Effect by
3-Acetyl-4,5-dimethyl-3-pyrrolin-2-ones in SH-SY5Y Cells
F igu r e 1. Structure of epoplactaene.
Sch em e 1. Synthesis of
3-Acetyl-4,5-dimethyl-5-(octadecyloxy)-3-pyrrolin-2-one
(MT-5)
entry compound
R1
R2
activitya
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
MT-1
MT-9
H
H
H
H
H
H
H
H
H
CH3
C2H5
n-C3H7
n-C4H9
n-C8H17
n-C12H25
n-C16H33
n-C18H37
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
C2H5
CH3
C2H5
-
-
MT-10
MT-15
MT-16
MT-21
MT-20
MT-19
MT-17
MT-3
MT-2
MT-8
MT-4
MT-6
-
-
-
++b
c
+
d
+
e
H
+
CH3
C2H5
n-C3H7
CH(CH3)2
n-C6H13
-
-
-
-
f
+
-
MT-7
MT-5
n-C14H29
n-C18H37
calf serum (FCS). After addition of test samples, the
cells were incubated for 2 days at 37 °C in 5% CO2.
Neurite formation was measured as described by Guzwits
and Cunningham.21 In brief, cells exhibiting at least
one clearly defined neurite equal to or longer than a
diameter of a cell body were scored positive.
+++g
-
MT-51
MT-52
MT-53
MT-29
MT-54
MT-22
MT-24
MT-11
MT-13
MT-12
MT-14
CH2CH2NO2
CH2CH2CN
CH2Ph
CH2CH2Ph
CH2CH2OCH3
CH2CH2CO2CH3
CH2CH2CH2ONO2
CH3
-
-
-
-
-
A significant portion of epolactaene-treated cells
exhibited a bipolar morphology, in which two neurites
extend from opposite sides of the cell body in a dose-
dependent manner.19 A series of 3-substituted 3-pyr-
rolin-2-ones were tested for their ability to induce
neurite outgrowth in SH-SY5Y cells (Tables 1 and 2).
In the case of 3-acetyl-4,5-dimethyl-5-hydroxy-N-alkyl-
3-pyrrolin-2-ones, the compounds with a straight long
chain alkyl group, e.g., MT-21, MT-20, MT-19, and MT-
17, showed the moderate neuritogenic activity, and MT-
21 with an octyl group was most effective within this
group (Table 1). In the case of 3-acetyl-4,5-dimethyl-
5-alkoxy-3-pyrrolin-2-ones, MT-5, which possessed an
octadecyloxy group at the 5-position, induced neurite
outgrowth most effectively among compounds with a
straight chain alkoxy group. MT-5 caused character-
istic changes in the morphology of SH-SY5Y cells
(Figure 2a). The control cells cultured without drugs
extended quite a few neurites (an average of <5%
neurite-bearing cells/total cells). When cells were treated
with MT-5 at concentrations of 7.4-59.4 µM, many
neurites were extended from cell bodies in a dose-
dependent manner (an average of 66.6% neurite-bearing
cells/total cells at a dose of 59.4 µM). Neurite formation
in SH-Y5Y cells was also induced by 1 mM of dibutyryl
cAMP (an average of 62% neurite-bearing cells/total
cells) as shown in Figure 2b. Other compounds with
an electron-withdrawing moiety on the alkoxy group,
e.g., a nitro ethoxy group or a cyano ethoxy group, were
completely inactive even at higher concentrations.
Next, we investigated the effect of the subsituted
group at the 3-position of 3-pyrrolin-2-ones as shown
in Table 2. Interestingly, the compounds with a cyano,
a carbamoyl, a methoxycarbonyl, or a benzyl group were
completely inactive in contrast to the compounds with
an acetyl group at the 3-position.
-
-
CH3
C2H5
C2H5
-
-
-
a
Plus sign indicates a significant difference in the percentage
of cells exhibiting a bipolar morphology at 48 h relative to 0.1%
MeOH- or 0.1% DMSO-treated control cells. One plus, two plus,
and three plus signs indicate that the percentage of neurite-
bearing cells/total cells are 5-20%, 20-40%, and >40%, respec-
tively. A minus sign indicates no significant difference from
control. The compounds which were inactive on the neurite
outgrowth were tested at concentrations which were not toxic. At
least two independent experiments with triplicate samples were
performed for each treatment. 17.8 µM. c 29.5 µM. 25.4 µM.
b
d
e 118.8 µM. f 98.8 µM. 59.4 µM.
g
induced them to differentiate. Thus, the effect of SH-
SY5Y cells on the cell cycle progression was investigated
by a flow cytometry. SH-SY5Y cells were plated at a
density of 1 × 105 cells per 500 µL per well on a 24-well
plate precoated with collagen. After incubation with
various concentrations of MT-5 or MT-21 for 2 days at
37 °C, the cells were collected and stained with the 50
µg/mL of propidium iodide solution containing 0.1%
sodium citrate and 0.2% Nonidet P-40. DNA histograms
were obtained by using a flow cytometer equipped with
an argon-ion laser at 488 nm (Figure 3). In the control
cells, cells with 2C DNA (G0/G1 phase) and 4C DNA
(G2/M phase) and cells between 2C and 4C (S phase)
were observed. In the cells treated with MT-5 or MT-
21, the fraction of S-phase cells decreased while those
in G0/G1 phase increased. The ratios of G1-phase cells
to S-phase cells are 2.9, 7.0, and 7.1 in the cells treated
with 0.1% MeOH, MT-5, and MT-21, respectively,
suggesting that MT-5 (59.4 µM) and MT-21 (8.9 µM)
significantly arrested the cell cycle at G0/G1 phase.
Epolactaene also had the same effect in SH-SY5Y cells.
Taxol which did not induce the neurite outgrowth
caused accumulation of the M phase cells with 4C DNA
Addition of 3-substituted 3-pyrrolin-2-ones such as
MT-5 and MT-21 stopped cells from proliferating and

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Communications to the Editor
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4 393
Ta ble 2. Neuritogenic Effect by 3-Substituted
4,5-Dimethyl-3-pyrrolin-2-ones in SH-SY5Y Cells
entry
compound
R1
R3
activitya
16
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
MT-5
n-C18H37
H
CH3
n-C3H7
n-C18H37
CH2CH2CN
CH2CH2Ph
H
C(O)CH3
CN
CN
CN
CN
+++b
-
MT-32
MT-33
MT-47
MT-38
MT-35
MT-41
MT-40
MT-42
MT-48
MT-43
MT-45
MT-57
MT-58
MT-59
MT-85
-
-
-
CN
CN
-
-
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
C(O)NH2
CO2CH3
CO2CH3
CO2CH3
C(O)Ph
-
CH3
-
n-C3H7
n-C18H37
CH2CH2Ph
H
CH3
n-C18H37
CH3
-
-
-
-
-
-
-
a
Plus sign indicates a significant difference in the percentage
of cells exhibiting a bipolar morphology at 48 h relative to 0.1%
MeOH- or 0.1% DMSO-treated control cells. Three plus indicates
that the percentage of neurite-bearing cells/total cells is >40%. A
minus sign indicates no significant difference from control. The
compounds which were inactive on the neurite outgrowth were
tested at concentrations which were not toxic. At least two
independent experiments with triplicate samples were performed
F igu r e 2. Neuritogenic effect by MT-5 in SH-SY5Y cells. (a)
Photomicrographs of SH-SY5Y cells viewed 48 h after the
following treatments: (A) 0.1% MeOH; (B) 59.4 µM MT-5. (b)
Neuritogenic effect: The number of neurite-bearing cells were
counted as described in the text. Bu2cAMP indicates dibutyryl
cyclic AMP. All experiments were performed in triplicate.
Values are means ( SD. *p < 0.01, **p < 0.001 relative to
0.1% MeOH-treated control cells.
b
for each treatment. 59.4 µM.
contents. The IC50s of inhibitory activity on SH-SY5Y
cell growth were >238 µM (MT-5) and >35.6 µM (MT-
21) as determined by viable staining with the dye
MTT.22
Our studies on the 3-substituted 3-pyrrolin-2-ones,
epolactaene analogs, reveal the importance of several
chemical groups in the structure for biological activity
in SH-SY5Y cells. MT-1, MT-38, MT-43, and MT-59
are completely inactive, but MT-5 and MT-21 are more
effective in the neuritogenic activity in SH-SY5Y cells
as shown in Tables 1 and 2. The acetyl group at the
3-position is more important, as is one straight long
chain alkyl group connected to the γ-lactam ring at
least. These groups may be involved in the primary
recognition of the target for activity. In compounds
which showed moderate neuritogenic activity, the po-
tency of neuritogenic effect in SH-SY5Y cells is MT-5
> MT-21 > MT19, MT-20, but that of neuritogenic
effect in rat pheochromocytoma PC12 cells is MT-19,
MT-20 > MT-5, MT-21 (data not shown). MT-21
revealed greater cytotoxicity than its neuritogenic effect
in PC12 cells, inducing apoptotic cell death (data not
shown). PC12 cells express high levels of TRK-A, while
SH-SY5Y cells express little or no detectable TRK-A
mRNA. The mode of action of these MT compounds
might be due to the different signal transduction in
PC12 cells and SH-SY5Y cells.
F igu r e 3. Flow cytometry analyses of propidium iodide-
stained SH-SY5Y cells. Cells were cultured with (A) 0.1%
MeOH, (B) 59.4 µM MT-5, (C) 8.9 µM MT-21, and (D) 1 µM
Taxol. After treatment, the cells were analyzed as described
in the text. Results are representative of three independent
experiments. Values are means ( SD. *p < 0.01, **p < 0.001
relative to 0.1% MeOH-treated control cells.
Recently, it has been reported that lactacystin inhib-
ited the cell cycle progression at G0/G1 phase and
induced neurite outgrowth in mouse neuroblastoma cell
line Neuro 2A, through the inhibition of proteasome
activity.23-26 Lactacystin does not act on the nerve
growth factor signaling pathway, at least as manifested
in PC12 cells, but like MT-21, it is toxic to PC12 cells.25
In addition, specific interactions between protease and
protease inhibitors have been suggested to play a role
in the regulation of neurite outgrowth.27,28 However,
MT-5, MT-19, MT-20, and MT-21 did not inhibit the

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394 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 4
Communications to the Editor
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activity of some proteases, papain, trypsin, cathepsin
B, and R-chymotrypsin in vitro, even at much higher
concentrations than the effective dose on neurite out-
growth in cultured cells (data not shown). Defining the
target molecule for these compounds and clarifying the
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a
Con clu sion . A series of 3-substituted 3-pyrrolin-2-
ones were designed as epolactaene, a novel non-protein
neurotrophic factor, related analogs. The following
relationships between the structures and neuritogenic
activity in SH-SY5Y cells were noted: an epoxide ring
fused to the γ-lactam ring in epolactaene is not always
necessary for the biological activity, and at least one
straight long chain alkyl group and a carbonyl group
at the 3-position are required for the biological activity.
The observed structure-activity relationships suggested
that epolactaene and related 3-pyrrolin-2-ones analogs
may act via acylation of one or more relevant target
molecule(s) in the cell. We obtained several promising
compounds, MT-5, MT-19, MT-20, and MT-21 on the
based on the ability to selectively induce neurite out-
growth in different cultured cells. As most neuroblas-
toma cell lines are derived from patients with advanced
stage disease and have poor clinical prognosis, these
results demonstrated a promising class of potential
chemotherapeutics which can induce the differentiation
of neuroblastoma tumors.
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Ack n ow led gm en t. We would like to thank Drs. H.
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gift of SH-SY5Y cells. We are also grateful to Dr. Phillip
C. C. Liu (The University of Michigan Medical School)
for the preparation of the manuscript. This work was
supported in part by Special Grant for Promotion of
Research from The Institute of Physical and Chemical
Research (RIKEN).
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pages). Ordering information is given on any current mast-
head page.
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