Synthesis of [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one

Article abstract of DOI:10.1002/jlcr.3796

Parkinson disease (PD) is a neurodegenerative disorder characterized by the accumulation of α-synuclein into Lewy bodies. 3-Benzylidine-indolin-2-one represents a class of compounds, which are known to inhibit the accumulation of α-synuclein. In this pape

Full text of DOI:10.1002/jlcr.3796

Wen Hongliang (Orcid ID: 0000-0001-8894-5559)
Synthesis of [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one
Jixia Yang, Gongzheng Zhang, Zhaoyang Wang, Zhanxiong Xiao, Hongliang Wen*
School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 102488，China
Correspondence: Hongliang Wen, Beijing Institute of Technology, Fangshan District, Beijing 102488,
China.
Email: wen.hongliang@bit.edu.cn
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the accumulation of α-synuclein
into Lewy bodies. 3-Benzylidine-indolin-2-one represent a class of compounds, which are known to inhibit
the accumulation of α-synuclein. In this paper we report the synthesis of [13C] and [15N] labelled 1-benzyl-
(Z)-3-(benzylidene)indolin-2-one from commercially available [13C2]-chloroacetic acid and [15N]-aniline in
five steps. The product will be used to study its metabolites in human liver microsomes by liquid
chromatography-tandem mass spectrometry.
KEYWORDS: α-synuclein, 3-(benzylidene)indolin-2-one, isotope label, synthesis
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doi: 10.1002/jlcr.3796
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1 | INTRODUCTION
Neurodegenerative diseases are characterized by progressive dysfunction, neuronal death and showing
specific protein inclusion.1,2 For example, Alzheimer’s disease (AD), the most common form of dementia
is diagnosed post-mortem by the appearance of extracellular plaques composed of amyloid beta (Aβ)
peptide and intracellular tangles composed of tau protein aggregated into paired helical filaments
(PHFs).3,4 The diagnosis of neurodegenerative diseases is challenging in the earlier stages. Molecular
imaging by nuclear medicine techniques, and in particular positron emission tomography (PET) molecular
imaging, has considerably evolved providing useful biomarkers for neurodegenerative disease.5 Amyloid
plaques and neurofibrillary tangles in AD and their increased deposition can be detected by various
PET-tracers, for example Pittsburgh Compound B (PiB), a derivative of Thioflavin-T (ThioT), has been
highly useful as a positron emission tomography (PET) probe for selectively quantifying amyloid plaques
in vivo.6-9 Recently a number of 18F and 11C-labeled agents for imaging Aβ are commercially available
permitting large scale clinical use and several tau radiotracers are undergoing evaluation in human imaging
studies.10-13
Parkinson’s disease (PD) is the second most common neurodegenerative disease after Alzheimer’s
disease.14 Its main clinical symptoms including tremors, muscle rigidity, drooping posture and walking
difficulty.15,16 PD affects 1% of people over the age of 65 and approximately 4% of the population aged
over 80 years.17,18 Since PD is becoming a serious concern for many countries,15 early therapy and disease
prevention is essential, which can only be accomplished with early diagnosis. Neuropathological hallmarks
of PD are loss of dopaminergic neurons in the substantia nigra and the formation of intraneuronal protein
inclusions termed Lewy bodies, composed primarily of α-synuclein.19,20 Small molecules that have suitable
pharmaceutical properties as fibrillar α-synuclein ligands and that can be labelled with position emission
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tomography (PET) radionuclides will have great opportunity to serve as PET probes for quantifying
α-synuclein aggregation in the brain.21 But no suitable PET tracer has been reported that can be used to
assess aggregated α-synuclein accumulation in the brain in vivo.
2-Oxindoles are an important class of heterocyclic compounds and possess a larger range of
pharmaceutical activities.22-24 Recently Professor Robert H. Mach synthesized a series of indolinone-diene
analogues and their binding assays for α-syn, Aβ and tau fibrils were conducted.
(Z)-1-(4-(2-Fluoroethoxy)benzyl)-3-((E)-3-(4-nitrophenyl)-allylidene)indolin-2-one showed high affinity
(2 nM) and excellent selectivity for α-syn versus Aβ and tau fibrils and could be used as lead compound to
study PET-based radiotracers for imaging α-syn aggregates which occur in Parkinson’s disease.25
Indolinone derivatives also exhibit high affinity towards receptor tyrosine kinases. SU5416 (Semaxinib)
and SU11248 (Sutent) (Figure 1) have entered preclinical and multi-center clinical studies for
anti-angiogenic
and
antitumor
activity.
Torsten
Kniess
et
al
synthesized
3-[4′-[18F]fluorobenzylidene]indolin-2-one (Figure 2) and studied its biodistribution, clearance and found
two more polar metabolites in arterial blood plasma of Wistar rats.26 However, indolinone metabolites have
not been studies systematically. To study metabolites of indolinone analogues in human liver microsomes
by liquid chromatography-tandem mass spectrometry, we prepared [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-
(benzylidene)indolin-2-one.
FIGURE 1 Structures of tyrosine kinase inhibitors U5416 and SU11248
FIGURE 2 Structure of 3-[4′-[18F]fluorobenzylidene]indolin-2-one
2 | RESULTS AND DISCUSSION
The synthesis of [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one is outlined in Scheme 1.
Treatment of commercially available [13C2]-chloroacetic acid with oxalyl chloride afforded
[13C2]-chloroacetyl chloride (1). Then compound 1 reacted with [15N]-aniline to give [13C2, 15N]-2-chloro-
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N-phenyl-acetamide (2). After cyclization in presence of AlCl3 via Friedel-Crafts reaction, the key
intermediate [13C2, 15N]-1,3-2H-indol-2-one (3) was obtained. Condensation of [13C2, 15N]-1,3-2H-indol-2-
one (3) with benzaldehyde gave (Z)-isomer and (E)-isomer, with separation by column chromatography,
[13C2,
15N]-1,3-2H-(Z)-3-(benzylidene)indolin-2-one
(4)
and
[13C2,
15N]-1,3-
2H-(E)-3-(benzylidene)indolin-2-one (5) were obtained. The configurations of 4 and 5 were assigned by
1
comparison of their H NMR spectrum with unlabeled compounds.27-29 It is reported that Z-isomer has a
higher affinity for α-syn. Finally compound 4 reacted with benzyl bromide to give [13C2, 15N]-1,3-2H-
1-benzyl-(Z)-3-(benzylidene)indolin-2-one (6).
3 | CONCLUSION
The [13C] and [15N] labelled 1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one was prepared in overall
yield 2.9% via a synthetic route involving five steps with [13C2]-chloroacetic acid as starting material. The
product will be useful in experiment to study its metabolites in human liver microsomes by liquid
chromatography-tandem mass spectrometry.
SCHEME 1 Synthesis of [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one
4 | EXPERIMENTAL
[13C2]-Chloroacetic acid (99%) and [15N]-aniline (98%+) were purchased from Cambridge Isotope
Laboratories (Andover, United State). Chemicals were of analytical grade and used without further
purification. Reactions were monitored by thin layer chromatography on pre-coated silica 60 F254
aluminium plates (Merck, Darmstadt, Germany). Spots were visualized by UV light 254 nm. Melting
1
points were determined on XT4A microscopic digital melting-point apparatus and are uncorrected. H
NMR (400 MHz) spectra were recorded on a Bruker Avance 400 NMR spectrometer in CDCl3 (TMS as
internal standard). HRMS Spectra were obtained with Agilent Q-TOF 6520 spectrometer.
4.1 | [13C2]-chloroacetyl chloride (1)
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A solution of [13C2]-chloroacetic acid (250 mg, 2.6 mmol) in dichloromethane (5 mL) was stirred in a
cooling bath under nitrogen gas atmosphere. Oxalyl chloride (0.16 mL, 1.9 mmol) in dry dichloromethane
(2 mL) was added dropwise and the mixture was stirred at 0°C for 1 hour followed by stirring at ambient
temperature for 3 hours. After completion of the reaction, colorless solution of [13C2]-chloroacetyl chloride
(1) was obtained and used to next step with no further purification.
4.2 | [13C2, 15N]-2-chloro-N-phenyl-acetamide (2)
[15N]-Aniline (160 mg, 1.7 mmol) and triethylamine (0.5 mL) were dissolved in dry dichloromethane (2
mL). The resulting solution was cooled to 0°C, then 1 in DCM was added dropwise in 20 minutes. The
reaction mixture was stirred at room temperature for 1 hour. The reaction was quenched by water (30 mL).
The mixture was extracted with DCM (25 mL×4) and washed with saturated ammonium chloride (20
mL×3). The combined organic layer was dried over Na2SO4 and concentrated under vacuum. The crude
product was purified by column chromatography (petroleum ether/EtOAc, 10:1) to give 2 as a white solid
1
(337 mg, 92.6%). m.p. 131.3-133.9 °C. H NMR (400 MHz, CDCl3) δ 8.15 (d, JN-H = 90.4 Hz, 1H),
7.49-7.47 (d, J = 8.0 Hz, 2H), 7.32-7.28 (t, J = 7.4 Hz, 2H), 7.13-7.09 (t, J = 7.2 Hz, 1H), 4.13 (dd, JC-H
=
153.4, JH-H = 3.8 Hz, 2H). HRMS (ESI+) m/z calcd. for 12C613C2H8Cl15NO (M+H) 173.0410, found
173.0407.
4.3 | [13C2, 15N]-1,3-2H-indol-2-one (3)
15
[13C2, N]-2-Chloro-N-phenyl-acetamide (2) (337 mg, 2.0 mmol) was combined with aluminum chloride
(1.2 g, 9.0 mmol) as a mixture of solid. The mixture was then heated in an oil bath at 230°C for 15 minutes.
After cooling to room temperature, diluted hydrochloric acid (50 mL) was added dropwise and the mixture
was extracted with ethyl acetate (30 mL×5). The organic layer was washed with brine, water and dried
over Na2SO4. The solvent was removed under vacuum and the residue was purified by flash column
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chromatography (petroleum ether/EtOAc, 10:1) to give [13C2, 15N]-1,3-2H-indol-2-one (3) (172.5 mg,
66.6%) as a white solid. m.p. 125.5-126.3°C. 1H NMR (400 MHz, CDCl3) δ 8.08 (dd, JN-H = 94.4 Hz, JC-H
= 4.8 Hz, 1H), 7.19-7.13 (m, 2H), 6.97-6.93 (t, J = 7.4 Hz, 1H), 6.82-6.80 (d, J = 8.0 Hz, 1H), 3.47 (dd,
JC-H = 133.4, JH-H = 5.4 Hz, 2H). HRMS (ESI+) m/z calcd. for 12C613C2H715NO (M+H) 137.0643, found
137.0627.
4.4 | [13C2, 15N]-1,3-2H-(Z)-3-(benzylidene)indolin-2-one (4)
Benzaldehyde (0.14 mL, 1.4 mmol) and piperidine (0.1 mL, 1.1 mmol) were added to a solution of [13C2,
15N]-1,3-2H-indol-2-one (3) (172.5 mg, 1.3 mmol) in ethanol. The reaction mixture was refluxed for 1
hour and cooled to room temperature. The obtained suspension was filtered, washed with cold ethanol and
[13C2, 15N]-1,3-2H-(E)-3-(benzylidene)indolin-2-one (5) was obtained. Evaporation of the solvent under
vacuum afforded crude product. Purification of the crude product by flash column chromatography
(petroleum ether/EtOAc, 8:1) gave two isomers.
1
[13C2, 15N]-1,3-2H-(Z)-3-(benzylidene)indolin-2-one (4): 32.2 mg, 11.4%. m.p. 160.5-164.8°C. H NMR
(400 MHz, CDCl3) δ 8.21-8.19 (d, J = 6.8 Hz, 2H), 8.00 (dd, JN-H = 95.2, JC-H = 6.0 Hz, 1H), 7.50-7.37 (m,
4H), 7.18-7.14 (m, 2H), 6.99-6.95 (t, J = 7.4 Hz, 1H), 6.79-6.77 (m, 1H). HRMS (ESI+) m/z calcd. for
12
C
13C2H1115NO (M+H) 225.0956, found 225.0933.
13
15
1
[13C2, N]-1,3-2H-(E)-3-(benzylidene)indolin-2-one (5): 229.4 mg, 80.5%. m.p. 178.5-179.5°C. H NMR
(400 MHz, CDCl3) δ 7.88 (dd, JN-H = 87.2, JC-H = 5.6 Hz, 1H), 7.78-7.75 (m, 1H), 7.60-7.56 (t, J = 9.0 Hz,
3H), 7.42-7.35 (m, 3H), 7.17-7.13 (m, 1H), 6.82-6.79 (t, J = 7.8 Hz, 2H). HRMS (ESI+) m/z calcd. for
12
C
13C2H1115NO (M+H) 225.0956, found 225.0944.
13
4.5 [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one (6)
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A 60% dispersion of sodium hydride (38 mg, 1.6 mmol) was added to a solution of 4 (134 mg, 0.6 mmol)
in DMF (3 mL) at room temperature and stirred for 15 minutes. Then benzyl bromide (290 mg, 1.6 mmol)
was added. After completion of the reaction (1 hour), the solution was quenched by water (20 mL). The
mixture was extracted with methyl tert-butyl ether (20 mL×5) and washed with saturated sodium chloride
(20 mL×3). The organic phase was dried with Na2SO4 and concentrated in vacuo. The yellow oil residual
was purified by flash column chromatography (petroleum ether/EtOAc, 80:1). The yellow solid [13C2,
15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene) indolin-2-one (6) (84.6 mg, 44.8%) was obtained. m.p.
1
131.8-132.5°C. H NMR (400 MHz, CDCl3) δ 8.26-8.24 (m, 2H), 7.54-7.46 (m, 2H), 7.41-7.33 (m, 3H),
7.28-7.15 (m, 5H), 7.13-7.09 (t, J = 7.6 Hz, 1H), 6.98-6.94 (t, J = 7.4 Hz, 1H), 6.66-6.64 (d, J = 7.6 Hz,
12
1H), 4.92-4.91 (d, J = 2.4 Hz, 2H). HRMS (ESI+) m/z calcd. for
found 315.1425.
C
13C2H1715NO (M+H) 315.1426,
20
ACKNOWLEDGEMENTS
The authors thank Beijing Institute of Technology Analysis & Testing Center for spectral data.
CONFLICT OF INTEREST
The authors did not report any conflict of interest.
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O
NH
N
N
N
F
H
H
O
O
N
N
H
H
SU5416 (Semaxinib)
SU11248 (Sutent)
FIGURE 1 Structures of tyrosine kinase inhibitors U5416 and SU11248
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18F
O
N
H
FIGURE 2 Structure of 3-[4′-[18F]fluorobenzylidene]indolin-2-one
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O
Cl
*
*
Cl
a)
OH
Cl
b)
*
*
Cl
*
*
Cl
Cl
+
+
*
*
O
O
O
NH2
N
H
O
1
2
CHO
d)
c)
*
*
*
H
*
O
O
O
+
+
*
*
*
*
*
N
N
N
H
H
3
4
5
e)
Br
*
*
O
O
+
*
*
Z-Isomer
*
*
N
H
N
4
6
SCHEME 1 Synthesis of [13C2, 15N]-1,3-2H-1-benzyl-(Z)-3-(benzylidene)indolin-2-one
a) DCM, DMF, 0°C-rt, 4 hours; b) DCM, triethylamine, 0°C-rt, 1 hour; c) anhydrous alchlor, 230°C, 15 min; d) piperidine,
ethanol, reflux, 1 hour; e) 60% NaH, DMF, rt, 1 hour
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REFERENCES
1. Agarwal S, Yadav A, Chaturvedi RK. Peroxisome proliferator-activated receptors (PPARs) as
therapeutic target in neurodegenerative disorders. Biochem Biophys Res Commun.
2017;483(4):1166-1177.
2. Khanam H, Ali A, Asif M, Shamsuzzaman. Neurodegenerative diseases linked to misfolded proteins and
their therapeutic approaches: A review. Eur J Med Chem. 2016;124:1121-1141.
3. Mohamed T, Shakeri A, Rao PP. Amyloid cascade in Alzheimer's disease: Recent advances in medicinal
chemistry. Eur J Med Chem. 2016;113:258-272.
4. Hroudova J, Singh N, Fisar Z, Ghosh KK. Progress in drug development for Alzheimer's disease: An
overview in relation to mitochondrial energy metabolism. Eur J Med Chem. 2016;121:774-784.
5. Abbasi Gharibkandi N, Hosseinimehr SJ. Radiotracers for imaging of Parkinson's disease. Eur J Med
Chem. 2019;166:75-89.
6. Klunk WE, Engler H, Nordberg A, et al. Imaging brain amyloid in Alzheimer's disease with Pittsburgh
compound-B. Ann Neurol. 2004;55(3):306-319.
7. Vlassenko AG, Benzinger TLS, Morris JC. PET amyloid-beta imaging in preclinical Alzheimer's
disease. Biochim Biophys Acta, Mol Basis Dis. 2012;1822(3):370-379.
8. Maetzler W, Reimold M, Liepelt I, et al. [11C]PIB binding in Parkinson's disease dementia.
Neuroimage. 2008;39(3):1027-1033.
9. Mathis CA, Bacskai BJ, Kajdasz ST, et al. A lipophilic thioflavin-T derivative for positron emission
tomography (PET) imaging of amyloid in brain. Bioorg Med Chem Lett. 2002;12(3):295-298.
18
10. Okamura N, Furumoto S, Harada R, et al. Novel F-labeled arylquinoline derivatives for noninvasive
imaging of tau pathology in Alzheimer disease. J Nucl Med. 2013;54(8):1420-1427.
This article is protected by copyright. All rights reserved.

11. Xia C-F, Arteaga J, Chen G, et al. [18F]T807, a novel tau positron emission tomography imaging agent
for Alzheimer's disease. Alzheimers Dement. 2013;9(6):666-676.
12. Mathis CA, Wang Y, Holt DP, et al. Synthesis and evaluation of 11C-labeled 6-substituted
2-arylbenzothiazoles as amyloid imaging agents. J Med Chem. 2003;46(13):2740-2754.
13. Kotzbauer PT, Tu Z, Mach RH. Current status of the development of PET radiotracers for imaging
alpha synuclein aggregates in Lewy bodies and Lewy neurites. Clin and Transl Imaging.
2017;5(1):3-14.
14. Olanow CW, Stern MB, Sethi K. The scientific and clinical basis for the treatment of Parkinson disease
(2009). Neurology. 2009;72:S1-136.
15. Zhang H, Tong R, Bai L, Shi J, Ouyang L. Emerging targets and new small molecule therapies in
Parkinson's disease treatment. Bioorg Med Chem. 2016;24(7):1419-1430.
16. Bowers S, Truong AP, Ye M, et al. Design and synthesis of highly selective, orally active Polo-like
kinase-2 (Plk-2) inhibitors. Bioorg Med Chem Lett. 2013;23(9):2743-2749.
17. Luo D, Sharma H, Yedlapudi D, et al. Novel multifunctional dopamine D2/D3 receptors agonists with
potential neuroprotection and anti-alpha synuclein protein aggregation properties. Bioorg Med Chem.
2016;24(21):5088-5102.
18. Moore DJ, West AB, Dawson VL, Dawson TM. Molecular pathophysiology of Parkinson's disease.
Annu Rev Neurosci. 2005;28:57-87.
19. George S, Rey NL, Reichenbach N, Steiner JA, Brundin P. alpha-Synuclein: the long distance runner.
Brain pathology. 2013;23(3):350-357.
20. Yu L, Cui J, Padakanti PK, et al. Synthesis and in vitro evaluation of alpha-synuclein ligands. Bioorg
Med Chem. 2012;20(15):4625-4634.
This article is protected by copyright. All rights reserved.

21. Bagchi DP, Yu L, Perlmutter JS, et al. Binding of the radioligand SIL23 to alpha-synuclein fibrils in
Parkinson disease brain tissue establishes feasibility and screening approaches for developing a
Parkinson disease imaging agent. PloS One. 2013;8(2):e55031.
22. Weniger B, Jiang Y, Anton R, et al. Oxindole alkaloids from Neolaugeria resinosa. Phytochemistry.
1993;32(6):1587-1590.
23. Hosoe T, Nozawa K, Kawahara N, et al. Isolation of a new potent cytotoxic pigment along with
indigotin from the pathogenic basidiomycetous fungus Schizophyllum commune. Mycopathologia.
1999;146(1):9-12.
24. Zhu Q, Tang CP, Ke CQ, et al. Constituents of Trigonostemon chinensis. J. Nat. Prod.
2009;73(1):40-44.
25. Chu W, Zhou D, Gaba V, et al. Design, synthesis, and characterization of 3-(benzylidene)indolin-2-one
derivatives as ligands for alpha-synuclein fibrils. J Med Chem. 2015;58(15):6002-6017.
26. Torsten K, Ralf B, Manuela K, Joerg S, Frank W. Synthesis and radiopharmacological investigation of
3-[4'-[18F]fluorobenzylidene]indolin-2-one as possible tyrosine kinase inhibitor. Bioorg Med Chem.
2009;17(22):7732-7742.
27. Sun L, Tran N, Tang F, et al. Synthesis and biological evaluations of 3-substituted Indolin-2-ones: A
Novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine
kinases. J Med Chem. 1998;41(14):2588-2603.
28. Ueda S, Okada T, Nagasawa H. Oxindole synthesis by palladium-catalysed aromatic C-H alkenylation.
Chem Commun. 2010;46(14):2462-2464.
29. Villemin D, Martin B. Potassium fluoride on alumina: Dry synthesis of 3-arylidene-1,3-
dihydro-indol-2-one under microwave irradiation. Synthetic Communications. 2006;28(17):3201-3208.
This article is protected by copyright. All rights reserved.