Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death

Article abstract of DOI:10.1016/j.bmcl.2017.08.005

Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds.

Full text of DOI:10.1016/j.bmcl.2017.08.005

Accepted Manuscript
Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and
their effects on neuronal cell death
Kyoji Furuta, Yu Kawai, Yosuke Mizuno, Yurika Hattori, Hiroko Koyama,
Yoko Hirata
PII:
S0960-894X(17)30793-X
http://dx.doi.org/10.1016/j.bmcl.2017.08.005
BMCL 25199
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 June 2017
31 July 2017
3 August 2017
Please cite this article as: Furuta, K., Kawai, Y., Mizuno, Y., Hattori, Y., Koyama, H., Hirata, Y., Synthesis of 3-
[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.005
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Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and
their effects on neuronal cell death
Kyoji Furutaa,b,c,*, Yu Kawaic, Yosuke Mizunoc, Yurika Hattoric, Hiroko Koyamaa,c, Yoko Hirataa,b
a Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan
b Field of Biological Molecular Sciences, United Graduate School of Drug Discovery and
Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
c Regeneration and Advanced Medical Sciences, Graduated School of Medicine, Gifu University,
1-1 Yanagido, Gifu 501-1194, Japan
Keywords:
Oxindole derivative
SAR
Suppression of neuronal cell death
Oxidative stress
* Corresponding author. E-mail address: furuta@gifu-u.ac.jp

ABSTRACT
Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the
following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with
(4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with
[(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the
oxindole skeleton by anion-based methods provided additional substituted derivatives for
structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative
stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of
2–4 significantly suppressed the neuronal cell death. No significant change occurred in the
activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core
was also irrelevant for the neuroprotective effects of these compounds.

As part of our studies on the design and synthesis of small molecules with neuroprotective
activities,1–9 we succeeded in elaborating a novel oxindole 1 (Figure 1) and its analogs that
significantly suppressed the oxidative stress-induced death of neuronal cells.10 Structure–activity
relationship (SAR) studies clarified that substitutions with less polar functional groups on the
benzene or lactam ring of the oxindole skeleton positively affect the potency, while polar groups
intensely reduced the activity. In addition, the stereochemistry at C-3 of the oxindole core was
not a crucial factor for the activity of the compounds. However, the effects by modifications at
the methylene part, marked by the arrow in Figure 1, have not yet been investigated. This series
of compounds comprise two structural blocks of the oxindole skeleton and
(dimethylamino)phenyl group. The methylene part that connects the two blocks is assumed to
play an important role in regulating the spatial arrangement between them. Therefore, we
planned the synthesis and validation of the neuroprotective activity of the homologs of 1 and
related derivatives.
Figure 1. Structure of neuroprotective oxindole derivative 1.
Structures of the designed 3-[(4-dimethylamino)phenyl]alkyl-2-oxindole derivatives with various
substituents on the oxindole core are illustrated in Figure 2. The alkyl chain length was in the
range of 2–4 (n = 2–4) by considering the water solubility of the compounds. We also appended
some analogs with modified or deformed structures for further SAR studies as depicted in
Figure 3. Most of the compounds were synthesized by either one of the following two pathways:
(1) a sequence of Knoevenagel condensation–hydrogenation (the upper part in Scheme 1),11–14 or
(2) alkylation of oxindole dianion with halides (Scheme 2).15 Further alkylation at C-3 and/or
N-1 was accomplished by the procedure described in the lower part of Scheme 1. The syntheses
of the commercially unavailable starting materials are described as schemes in the
Supplementary material.
1

Figure 2. Compounds 2–14
Compd
n
2
3
4
3
3
3
3
3
3
3
3
3
3
Y1
Y2
H
Y3
2
3
H
H
H
H
H
4
H
H
H
5
H
Me
Me
Me
Me
Me
H
H
6
F
H
7
HO
H
8
MeO
H
9
MeOCH2CH2O
H
H
10
11
12
13
14
6-F3C
H
H
Me
MeO
MeO
H
Me
Me
Me
CH2CONMe2
CH2COOH
4-Methoxyphenyl
Figure 3. Compounds 15–23.
Knoevenagel
condensation
of
unsubstituted
or
substituted
oxindole
and
(4-dimethylamino)cinnamaldehyde afforded the intermediates A as E/Z mixtures.16 The
following palladium-mediated hydrogenation of A gave compounds 3 and 10 in good yields.
Compounds 15, 16 and 18 were similarly prepared by this two-step method using the
corresponding aldehyde units in the total yields of 45%, 59% and 89%, respectively. The
2

C-3-alkylated derivatives 5–9 were obtained by a consecutive five-step method; (1) Knoevenagel
condensation giving A; (2) N-Boc protection of A; (3) hydrogenation of A to the saturated
compound B; (4) methylation of the amide enolate generated from B; and (5) deprotection of the
N-Boc group. We also applied this method for the synthesis of 17 using the appropriate aldehyde
unit (five steps, 14% yield). Furthermore, the conversions of 8 to 12 and 13 were easily
accomplished by N-alkylation with halides via amidate formation. For the synthesis of 11 and 14,
minor modifications were made. Thus, the direct methylation of A (Y1 = H) via its amidate
formation, followed by hydrogenation gave 11 (two steps, 60%), while Knoevenagel
condensation of 1-(4-methoxyphenyl)-2-oxindole with (4-dimethylamino)cinnamaldehyde and
subsequent hydrogenation produced a saturated intermediate, which in turn was methylated at
C-3 via the amide enolate to produce 14. The total yield of 14 based on the starting oxindole was
63%.
Scheme 1. Synthetic route based on Knoevenagel reaction.
Reagents and conditions: (i) piperidine (0.6 equiv), methanol, reflux, 4–22 h; (ii) H2, 10% Pd-C,
THF, r.t., 6–24 h; (iii) Boc2O (1.5 equiv), DMAP (1.5 equiv), CH3CN, r.t., 15–30 min; (iv) NaH
(1.2 equiv), DMF, 0 °C, 15 min, then CH3I (1.2 equiv), 0 °C to r.t., 20–60 min; (v) trifluoroacetic
acid (7 equiv), CH2Cl2, r.t., 0.5–1 h; (vi) for 12, NaH (1.1 equiv), DMF, r.t., 15 min, then
ClCH2CONMe2 (1 equiv), r.t., 1 h; (vii) for 13, NaH (1.1 equiv), DMF, r.t., 20 min, then
ClCH2COOt-Bu (1 equiv), r.t., 30 min; (viii) for 13, trifluoroacetic acid/CH2Cl2 (1/1, v/v), r.t., 3
h.
3

Compounds 2, 4, 21 and 22 were synthesized by the oxindole dianion-based method (Scheme 2)
according to the reported procedure.15 Namely, the treatment of oxindole with two equivalents of
BuLi produces the dianion that preferentially reacts with an alkyl halide at the C-3 position. This
method enabled the synthesis of 2 and 4, each in a single step. In a similar manner, we obtained
21 through the reaction with N-(5-iodopentyl)-N-methylaniline. Furthermore, the dianion reacted
with N,N-bis(2-iodoethyl)aniline to form the cyclic derivative 22. The stepwise dual-alkylation
mechanism via an intermolecular anion-exchange reaction between the monoalkylated amide
anion intermediates is proposed for this type of substrate.15
Scheme 2. Synthetic route based on alkylation of oxindole dianion.
Reagents and conditions: (a) BuLi (2 equiv), TMEDA (2 equiv), THF, –78 °C, 1 h; (b)
4-(Me2N)C6H4(CH2)nI (1.2 equiv), –78 °C to r.t., 2 h; (c) PhN(Me)(CH2)5I (2 equiv), –78 to
–20 °C, 30 min; (d) PhN(CH2CH2I)2 (2 equiv), –78 to 0 °C, 3 h.
The dianion-based method was also applicable for the synthesis of 19 (Scheme 3). The initial
attempt
to
alkylate
the
dianion
generated
from
25
with
1-[(4-dimethylamino)phenoxy]-2-iodoethane, by which 19 should be obtainable in a single step,
was unsuccessful. However, we found that the dianion could react with
1-iodo-2-(2-tetrahydro-2H-pyranoxy)ethane to afford 26. After protection of the acidic N-H in
26 with a benzyl group and deprotection of the THP ether, the resulting alcohol 28 was coupled
with 4-nitrophenol under Mitsunobu conditions to produce 29. Subsequent conversion of the
nitro group to dimethylamino and deprotection of the N-benzyl group provided the desired
4

product 19. The total yield of 19 by this scheme was 5% (yields are not optimized). The
synthesis of 23 was also accomplished by an analogous procedure (Scheme 4): The dianion of
31 reacted with a long-chain alkyl halide to produce 32, which was successfully converted to the
desired compound 23 by the usual method. The amide-type compound 20 was synthesized by
N-acylation of triptamine followed by oxidation of the indole ring as depicted in Scheme 5.17
Scheme 3. Synthesis of 19.
Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), THF, –78 °C, 30 min, then MeI
(1.1 equiv), –78 °C, 2 h, 65%; (ii) BuLi (2 equiv), TMEDA (2 equiv), THF, –78 °C, 30 min, then
ICH2CH2OTHP (2 equiv), –78 °C, 2 h, 37%; (iii) NaH (1 equiv), CH2Cl2, 0 °C, 10 min, then
benzyl bromide (1.1 equiv), 0 °C, 3 h, 73%, (iv) TsOH·H2O, MeOH, r.t., 30 min; (v)
4-nitrophenol (1.5 equiv), PPh3 (2 equiv), diisopropyl azodicarboxylate (2 equiv), benzene, r.t., 1
h, 63% (2 steps); (vi) H2, 10% Pd-C, THF, r.t., 16 h; (vii) (CH2O)n (10 equiv), NaBH3CN (5
equiv), AcOH, 0 °C to r.t., 5 h, 87% (2 steps); (viii) Na (7.6 equiv), liq NH3/THF (2/1, v/v),
–40 °C to r.t., 5 min, 50%.
Scheme 4. Synthesis of 23.
Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), –78 °C, 45 min, then
1-iodo-7-(2-tetrahydro-2H-pyranoxy)heptane (1.1 equiv), –78 °C to r.t., 2 h, 35%; (ii)
TsOH·H2O, MeOH, r.t., 40 min; (iii) I2 (2 equiv), PPh3 (2.5 equiv), imidazole (2.5 equiv),
benzene, r.t., 3 h, 75% (2 steps); (iv) Me2NH·HCl (2 equiv), K2CO3 (4 equiv), MeCN, r.t., 2.5 h,
89%.
5

Scheme 5. Synthesis of 20.
Reagents and conditions: (i) 4-(dimethylamino)benzoyl chloride (1 equiv), Et3N (2 equiv),
CH2Cl2, r.t., 16 h, 95%; (ii) DMSO/3 M HCl (1/9, v/v), 60 °C, 21 h, 50%.
All the oxindole derivatives synthesized in this study are racemates. In a preceding study, we
found that the stereochemistry at C-3 of compound 1 was not a crucial factor for the
neuroprotective activity.10 In order to verify whether this is also the case for the homologs with
an elongated carbon chain, we separated, as a representative example, the racemate 8 into two
enantiomers 8a and 8b by HPLC using a chiral column (Daicel Chiralcel OD-H).10 The isomer
8a has a shorter retention time during the HPLC compared to 8b. The absolute configurations of
both enantiomers were not assigned.
Evaluation of the effect of the compounds on the oxidative stress-induced neuronal cell death
was conducted in a similar manner as in the previous report.8,10,18 Briefly, HT22 cells were treated
with each compound at 25 µM and 50 µM with 10 mM glutamate. After incubation for 24 h, the
extent of cell death was determined by measuring lactate dehydrogenase (LDH), a marker of
injured cells, released into the culture medium using a cytotoxicity detection kit (LDH assay).
All compounds by themselves exerted no cytotoxic effects on HT22 cells up to 100 µM (data not
6

shown). Ascorbic acid, a well-known antioxidant, prevented the glutamate-induced cell death by
ca. 50% at 25 µM in this assay system.
The assay results are shown in Figure 4. Overall, elongation of the carbon chain of 1 did not
appreciably affect the activity. Thus, compounds 2, 3 and 4, simple homologs of 1, suppressed
the neuronal cell death induced by oxidative stress. Although the activity of 2 decreased at 25
µM, compound 18, an analog of 2 with the same carbon-chain number but has methyl
substituents on the chain, exhibited an activity comparable to 3 and 4. These results indicated
that considerable fluctuation in the relative spatial arrangement of the two aromatic skeletons
was permissible for interaction with a target. Among the derivatives, compounds 3, 6 and 7
exhibited significantly increased activities. The low-dose effects of 3, 6 and 7, together with that
for 1, are summarized in Figure 5. Evidently, these two-carbon elongated homologs exhibited
increased activities compared to 1. For compound 7, however, auxiliary involvement of the
antioxidant effect of the phenolic moiety is not ruled out.19,20 The estimated IC50 values for 1 and 3
were 7.1 µM and 5.5 µM, respectively. Of interest, the observed LDH levels were somewhat
lower than that of the control in some cases (compounds 5, 6, 7, 11, 15 and 16, for example).
This means that the compounds concerned suppressed the cell death by over 100%. Under the
experimental conditions employed, a small portion of HT22 cells dies to release LDH during
incubation even in the absence of glutamate. That level of LDH was set at 100% as the control in
our experiments. When HT22 cells were incubated with the compounds in the absence of
glutamate, the LDH levels similarly decreased less than the control (data not shown). Therefore,
we assume that the compounds also prevented such cell death together with the
glutamate-mediated death, although further validation is needed. The approximate IC50 values
corrected by subtracting this overlapping activity for 6 and 7 were 1.2 µM and 1.9 µM,
respectively.
Figure 4. Effects of 1–23 on glutamate-induced cell death of HT22 cells.
HT22 cells were incubated with each compound (open bar, 0; dot, 25 µM; check, 50 µM) and
glutamate (10 mM) for 24 h, and cell death was determined by measuring LDH released into the
culture medium. The LDH level of the control cultures (c) was set at 100%.
7

Figure 5. Low-dose effects of 1, 3, 6, 7, 8a and 8b on glutamate-induced cell death of HT22
cells.
HT22 cells were incubated with each compound (open bar, 0; right-down oblique, 1.0 µM;
right-up oblique, 2.5 µM; lattice, 5.0 µM; lateral stripes, 10 µM; dot, 25 µM) and glutamate (10
mM) for 24 h, and cell death was determined by measuring LDH released into the culture
medium. The LDH level of the control cultures (c) was set at 100%.
Furthermore, the activity was only slightly affected by substituents introduced on the oxindole
skeleton (Figure 4). Consequently, the amide N–H and benzylic C–H were unnecessary to exert
the effect.10 Changes in the position and alkyl substituents of the amino moiety on the side-chain
aromatic ring was also permissible (compounds 15–17). Of special interest is the retained
activity of 17 possessing a large phenyl group at the amino moiety. Therefore, a considerably
broad hydrophobic region around the side-chain aromatic ring is assumed to exist as the
compounds' pharamcophore. In sharp contrast, compound 13 bearing a polar carboxylic acid
group completely lacked any activity, suggesting a tendency to avoid polar interactions around
the oxindole skeleton. This tendency coincided very closely with those obtained for compound 1
as previously observed.10
Modifications by inserting a heteroatom into the carbon-chain also showed contrasting results.
While the ether type compound 19 exhibited a comparable activity to that of the parent analog 8,
8

the amide derivative 20 was completely inactive. Further studies are needed to elucidate what
types of factors contribute to the reduction in activity of 20. Another intriguing observation is
that compound 21, whose nitrogen atom was translocated from the terminal end to the inside
position of the side chain, exerted a potent suppressive effect. We designed 21 to have a nitrogen
atom at the spatial position overlapping with that of 1. The analogous inside-nitrogen type
compound 22 with a shortened and cyclized chain resulted in a loss of activity. Therefore, the
appropriate positioning of the nitrogen atom is supposed to be necessary to exert the effect. In
addition, the total loss in activity of the aliphatic analog 23 indicated the indispensable
requirement for the aminophenyl group located in the side chain. Finally, assay results for the
enantiomers of 8 are shown in Figure 5. Both 8a and 8b showed comparable activities,
demonstrating that the stereochemistry at C-3 of the oxindole core was irrelevant for the
neuroprotective effects of the compounds.10
In conclusion, we found that elongation of the carbon chain of compound 1 modestly enhanced
the effect on the suppression of neuronal cell death induced by oxidative stress. The concise SAR
studies proved that further modifications of the oxindole skeleton with less-polar substituents
were feasible without decreasing the activities. These findings provide insights into the adequate
design of compounds with desirable drug-like properties.21 Additional pharmacological features
of our oxindole derivatives, including the effects on the induction of neurotrophic factors, will be
described in a separate paper. Although preliminary, we observed that 8 but not 5 induced glial
cell line-derived neurotrophic factor (GDNF). This finding suggests that some of the oxindole
derivatives may have distinct mechanisms of actions in their neuroprotective activity. More
detailed biochemical analysis to elucidate the molecular mechanisms and in vivo studies using
Parkinson's disease model mice are currently in progress.
Acknowledgment
This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for
the Promotion of Science.
9

A. Supplementary material
Supplementary data associated with this article can be found, in the online version at http:
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12

Figure Legends
Figure 1. Structure of neuroprotective oxindole derivative 1.
Figure 2. Compounds 2–14.
Figure 3. Compounds 15–23.
Figure 4. Effects of 1–23 on glutamate-induced cell death of HT22 cells. HT22 cells were
incubated with each compound (open bar, 0; dot, 25 µM; check, 50 µM) and glutamate (10 mM)
for 24 h, and cell death was determined by measuring LDH released into the culture medium.
The LDH level of the control cultures (c) was set at 100%.
Figure 5. Low-dose effects of 1, 3, 6, 7, 8a and 8b on glutamate-induced cell death of HT22
cells. HT22 cells were incubated with each compound (open bar, 0; right-down oblique, 1.0 µM;
right-up oblique, 2.5 µM; lattice, 5.0 µM; lateral stripes, 10 µM; dot, 25 µM) and glutamate (10
mM) for 24 h, and cell death was determined by measuring LDH released into the culture
medium. The LDH level of the control cultures (c) was set at 100%.
Scheme 1. Synthetic route based on Knoevenagel reaction. Reagents and conditions: (i)
piperidine (0.6 equiv), methanol, reflux, 4–22 h; (ii) H2, 10% Pd-C, THF, r.t., 6–24 h; (iii) Boc2O
(1.5 equiv), DMAP (1.5 equiv), CH3CN, r.t., 15–30 min; (iv) NaH (1.2 equiv), DMF, 0 °C, 15
min, then CH3I (1.2 equiv), 0 °C to r.t., 20–60 min; (v) trifluoroacetic acid (7 equiv), CH2Cl2, r.t.,
0.5–1 h; (vi) for 12, NaH (1.1 equiv), DMF, r.t., 15 min, then ClCH2CONMe2 (1 equiv), r.t., 1 h;
13

(vii) for 13, NaH (1.1 equiv), DMF, r.t., 20 min, then ClCH2COOt-Bu (1 equiv), r.t., 30 min;
(viii) for 13, trifluoroacetic acid/CH2Cl2 (1/1, v/v), r.t., 3 h.
Scheme 2. Synthetic route based on alkylation of oxindole dianion. Reagents and conditions: (a)
BuLi (2 equiv), TMEDA (2 equiv), THF, –78 °C, 1 h; (b) 4-(Me2N)C6H4(CH2)nI (1.2 equiv),
–78 °C to r.t., 2 h; (c) PhN(Me)(CH2)5I (2 equiv), –78 to –20 °C, 30 min; (d) PhN(CH2CH2I)2 (2
equiv), –78 to 0 °C, 3 h.
Scheme 3. Synthesis of 19. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), THF,
–78 °C, 30 min, then MeI (1.1 equiv), –78 °C, 2 h, 65%; (ii) BuLi (2 equiv), TMEDA (2 equiv),
THF, –78 °C, 30 min, then ICH2CH2OTHP (2 equiv), –78 °C, 2 h, 37%; (iii) NaH (1 equiv),
CH2Cl2, 0 °C, 10 min, then benzyl bromide (1.1 equiv), 0 °C, 3 h, 73%, (iv) TsOH·H2O, MeOH,
r.t., 30 min; (v) 4-nitrophenol (1.5 equiv), PPh3 (2 equiv), diisopropyl azodicarboxylate (2 equiv),
benzene, r.t., 1 h, 63% (2 steps); (vi) H2, 10% Pd-C, THF, r.t., 16 h; (vii) (CH2O)n (10 equiv),
NaBH3CN (5 equiv), AcOH, 0 °C to r.t., 5 h, 87% (2 steps); (viii) Na (7.6 equiv), liq NH3/THF
(2/1, v/v), –40 °C to r.t., 5 min, 50%.
Scheme 4. Synthesis of 23. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv),
–78 °C, 45 min, then 1-iodo-7-(2-tetrahydro-2H-pyranoxy)heptane (1.1 equiv), –78 °C to r.t., 2 h,
35%; (ii) TsOH·H2O, MeOH, r.t., 40 min; (iii) I2 (2 equiv), PPh3 (2.5 equiv), imidazole (2.5
equiv), benzene, r.t., 3 h, 75% (2 steps); (iv) Me2NH·HCl (2 equiv), K2CO3 (4 equiv), MeCN, r.t.,
2.5 h, 89%.
Scheme 5. Synthesis of 20. Reagents and conditions: (i) 4-(dimethylamino)benzoyl chloride (1
equiv), Et3N (2 equiv), CH2Cl2, r.t., 16 h, 95%; (ii) DMSO/3 M HCl (1/9, v/v), 60 °C, 21 h, 50%.
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Graphical Abstract
Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on
neuronal cell death
Kyoji Furuta, Yu Kawai, Yosuke Mizuno, Yurika Hattori, Hiroko Koyama, Yoko Hirata
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