Preparation and antiplasmodial activity of 3',4'-dihydro-1'H-spiro(indoline-3,2'-quinolin)-2-ones

Article abstract of DOI:10.1111/cbdd.13598

A series of 3',4'-dihydro-1'H-spiro(indoline-3,2'-quinolin)-2-ones were prepared by the inverse-electron-demand aza-Diels–Alder reaction (Povarov reaction) of imines derived from isatin and substituted anilines, and the electron-rich alkenes trans-isoeuge

Full text of DOI:10.1111/cbdd.13598

DR AMANDA LOUISE ROUSSEAU (Orcid ID : 0000-0002-9339-1797)
Article type : Research Article
Preparation and antiplasmodial activity of 3',4'-dihydro-1'H-spiro(indoline-3,2'-quinolin)-2-ones
Synthesis of antiplasmodial spiroindolones
Bakolise Mathebula,1 Kamogelo Rosinah Butsi,1,3 Robyn Lynne van Zyl,2,3 Natasha Colleen Jansen van Vuuren,2,3
Heinrich Carl Hoppe,4 Joseph Philip Michael,1 Charles Bernard de Koning1 and Amanda Louise Rousseau1,3
*
1Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Private Bag 3, PO
WITS, 2050, South Africa;
2Pharmacology Division, Department of Pharmacy and Pharmacology; Faculty of Health Sciences,
University of the Witwatersrand, 7 York Road, Parktown 2193, South Africa.
3WITS Research Institute for Malaria (WRIM), Faculty of Health Sciences, University of the Witwatersrand,
7 York Road, Parktown 2193, South Africa.
4Department of Biochemistry and Microbiology, Rhodes University, Grahamstown 6140, South Africa.
*Corresponding author: Amanda Rousseau, Molecular Sciences Institute, School of Chemistry, University of
the Witwatersrand, Private Bag 3, PO WITS, 2050, South Africa; email: Amanda.Rousseau@wits.ac.za, Tel:
+27117176706, Fax: +27117176749
ACKNOWLEDGMENTS
The authors wish to thank Ms Amy Naicker for her assistance in aspects of this project. Research reported in
this publication was supported by the South African Medical Research Council under a Self-Initiated Research
Grant. BM and KRB thank the National Research Foundation (South Africa) for funding.
ABSTRACT
A series of 3',4'-dihydro-1'H-spiro(indoline-3,2'-quinolin)-2-ones were prepared by the inverse electron
demand aza Diels-Alder reaction (Povarov reaction) of imines derived from isatin and substituted anilines, and
the electron rich alkenes trans-isoeugenol and 3,4-dihydro-2H-pyran. These compounds were assessed for in
vitro antiplasmodial activity against drug-sensitive and drug-resistant forms of the P. falciparum parasite.
Three compounds derived from 3,4-dihydro-2H-pyran and four compounds derived from trans-isoeugenol
showed antiplasmodial activity in the low micromolar range against the drug-resistant FCR-3 strain (1.52 – 4.20
µM). Only compounds derived from trans-isoeugenol showed antiplasmodial activity against the drug-
sensitive 3D7 strain (1.31 - 1.80 µM).
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13598
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KEY WORDS
Spiroindolone, Povarov reaction, antiplasmodial activity
1
INTRODUCTION
Malaria is a disease caused by parasitic protozoa of the genus Plasmodium. An estimated 435 000 malaria
deaths were reported in 2017, with 92% of these occurring in sub-Saharan Africa and due to P. falciparum
infection (WHO, 2018). Although there has been a 48% decline in the number of malaria fatalities globally over
the last 15 years, the rate of decline has decreased significantly since 2014 owing to a number of factors,
including insecticide resistance and increasing reports of drug resistant strains of the parasite. There is thus
still a need for the identification of new antimalarial agents with novel modes of action.
Cipargamin (also known as NITD609 or KAE609 1, Figure 1) is a spiroindolone targeting a parasitic P-type
cation-transporter ATPase4 and is the first antimalarial drug candidate with a novel mechanism of action
identified in the last 25 years (Rottmann et al., 2010). Significantly, cipargamin displays potent activity against
both sexual and asexual stages of the parasite, and thus shows promise as a transmission blocking agent,
capable of preventing the spread of malaria to healthy individuals (Upton et al., 2015; van Pelt-Koops et al.,
2012). We became interested in spiroindolones owing to their potent antimalarial properties and noted that
they can be prepared by means of an inverse electron demand aza Diels-Alder reaction (Povarov reaction)
from relatively simple precursors.
Of interest to us is the use of isatin 3 in the Povarov reaction, which affords spiroindolones such as 2 (Scheme
1). Aromatic imines derived from isatin have successfully been used in the Povarov reaction with
vinylbenzenes (Kouznetsov, Bello Forero, & Amado Torres, 2008; Shi, Xing, Zhu, Tan, & Tu, 2013), vinylindoles
(Zhang et al., 2014), 3,4-dihydro-2H-pyran (Ramesh, Elamparuthi, & Raghunathan, 2008), acetylene
dicarboxylates (Karmakar, Kayal, Bhattacharya, & Maiti, 2014) β-enamino esters (Gao, Sun, & Yan, 2014) and
α,β-unsaturated N,N-dimethylhydrazones (Bianchini, Ribelles, Becerra, Ramos, & Menendez, 2016) to afford
spiroindolones. Herein we describe the synthesis and biological evaluation of a series of substituted
spiroindolones 2 prepared by Povarov reaction of ketimines with electron rich alkenes. To the best of our
knowledge, these compounds have not previously been assessed for antiplasmodial activity in vitro.
2
METHODS AND MATERIALS
2.1 Chemistry
All reagents were purchased from Sigma-Aldrich (South Africa) and used as received. Dichloromethane used
for reactions was distilled from calcium hydride under an inert atmosphere prior to use. Absolute ethanol was
obtained from commercial sources without any further purification. Solvents for column chromatography
(ethyl acetate and hexane) were purified by distillation prior to use.
Thin Layer Chromatography (TLC) was carried out using Merck aluminium foil F254 backed plates coated with
silica gel 60. Column chromatography was performed on Macherey-Nagel silica gel 60 particle size 0.063 –
0.200 mm, while Merck silica gel (particle size 0.035 – 0.070 mm) was employed for flash chromatography.
1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopic data were recorded on a Bruker 300 or 500 MHz
spectrometer using specified deuterated solvents. For those compounds soluble in deuterated chloroform
(CDCl3), chemical shifts were referenced against the internal standard tetramethylsilane (TMS) which occurs at
zero parts per million. Chemical shifts are recorded in ppm, while coupling constants are recorded in Hertz.
High Resolution Mass Spectra (HRMS) were obtained on a SYNAPT G2 HDMS mass spectrometer (ESI).
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2.1.1 General procedure for the synthesis of aryl-imines (4a-n)
Isatin (3.00 g, 20.4 mmol) and the appropriately substituted aniline (1.0 eq, 20.4 mmol) were dissolved in
absolute ethanol (120 ml) and treated with glacial acetic acid (0.8 ml). The resulting heterogeneous mixture
was stirred at reflux (100°C) under a nitrogen atmosphere for 5-15 hours. The reaction was then cooled and
concentrated by removal of the solvent to allow the product to precipitate out of solution. The mixture was
filtered and the filter cake was further purified by recrystallization from ethanol to afford the desired imine.
Imines 4a-n were prepared using this procedure. A representative example is given below.
3-(p-Tolylimino)indolin-2-one (Kouznetsov et al., 2008) (4a) was isolated as a mixture of E:Z isomers in a ratio
of 3:1. 1H NMR (300 MHz, DMSO-d6) E Isomer δ 10.97 (1H, s, NH), 7.38 – 7.22 (3H, m, ArH), 6.93 – 6.85 (3H, m,
ArH), 6.72 (1H, td, J = 7.7, 1.0 Hz, ArH), 6.49 (1H, dd, J = 7.8, 1.2 Hz, ArH), 2.35 (3H, s, CH3); Z isomer δ 10.85
(1H, s, NH), 7.57 (1H, dd, J = 7.5, 1.2 Hz, ArH), 7.43 (1H, td, J = 7.7, 1.3 Hz, ArH), 7.11 (2H, d, J = 8.0 Hz, ArH),
7.05 (1H, td, J = 7.6, 0.9 Hz, ArH), 6.99 – 6.92 (2H, m, ArH), 6.91 – 6.81 (1H, m, ArH, signal overlapping with ArH
of E-isomer), 2.30 (3H, s, CH3); 13C NMR (75 MHz, DMSO-d6) E Isomer δ 163.5, 154.7, 147.8, 146.9, 134.2,
134.1, 129.9, 125.2, 121.6, 117.4, 115.7, 111.4, 20.5; Z Isomer δ 158.4, 152.5, 146.1, 145.4, 133.9, 133.9,
128.7, 122.5, 122.2, 119.6, 115.7, 110.6, 20.5; HRMS (m/z), calculated for C15H13N2O: 237.1028, found (M +
H)+: 237.1022.
2.1.2 General procedure for the synthesis of 2',3',4',4a',6',10b'-hexahydrospiro[indoline-3,5'-pyrano[3,2-
c)quinolin]-2-ones (2a-h)
Aryl imines 4a-h (1.8 mmol) were each dissolved in anhydrous dichloromethane (30 ml) and treated with
.
BF3 OEt2 (0.9 mmol) under an inert atmosphere and allowed to stir for fifteen minutes. 3,4-Dihydro-2H-pyran
(2.7 mmol) was then added dropwise, and the reaction progress monitored by TLC. Typically, after 72 hours,
the reaction mixture was treated with saturated aqueous NaHCO3 solution (20 ml) and extracted with ethyl
acetate (3 × 20 ml). The organic layer was dried (Na2SO4), concentrated under vacuum, and the resulting
residue purified by either recrystallization (hexane/EtOAc) or silica gel column chromatography (30-50%
EtOAc/hexane). The following products were prepared by this method:
9'-Methyl-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2a) (Ramesh et al.,
2008)
1H NMR (300 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.18 (td, J = 7.5, 1.5 Hz, 1H), 7.07 (d, J = 2.2 Hz, 1H), 6.91 – 6.80
(m, 4H), 6.52 (d, J = 8.1 Hz, 1H), 6.19 (s, 1H), 5.07 (d, J = 5.2 Hz, 1H), 3.55 – 3.48 (m, 1H), 3.47 – 3.36 (m, 1H),
2.21 (s, 3H), 2.01 – 1.94 (dd, J = 13.4, 4.3 Hz, 1H), 1.82 – 1.73 (m, 1H), 1.54 – 1.32 (m, 3H); 13C NMR (126 MHz,
DMSO-d6) δ 182.7, 147.2, 145.7, 140.2, 134.0, 133.8, 132.4, 130.3, 128.8, 126.7, 122.7, 119.5, 114.7, 74.7, 68.6,
+
66.0, 42.3, 30.0, 25.6, 25.4; HRMS (m/z), calculated for C20H21N2O2 : 321.1598, found (M + H)+: 321.1603.
7'-Fluoro-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2b)
1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.21 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 7.4 Hz,
1H), 6.99 – 6.90 (m, 2H), 6.88 (d, J = 7.8 Hz, 1H), 6.78 (td, J = 7.9, 5.0 Hz, 1H), 5.29 (d, J = 5.0 Hz, 1H), 4.40 (s,
1H), 3.67 – 3.61 (m, 1H), 3.60 – 3.53 (m, 1H), 2.15 – 2.03 (m, 2H), 1.67 – 1.53 (m, 3H); 13C NMR (126 MHz,
DMSO-d6) δ 178.9, 151.1 (d, JC-F = 240.0 Hz), 139.2, 134.0, 131.8 (d, JC-F = 12.3 Hz), 129.4, 124.2, 123.1 (d, JC-F
=
3.2 Hz), 123.0, 121.5 (d, JC-F = 2.5 Hz), 117.9 (d, JC-F = 7.2 Hz), 114.0 (d, JC-F = 17.9 Hz), 110.4, 69.7, 64.2, 62.0,
37.8, 25.3, 20.7; HRMS (m/z), calculated for C19H18N2O2F+: 325.1347, found (M + H)+: 325.1364.
7'-Chloro-9'-methyl-2',3',4',4a',6',10b'-hexahydro-spiro[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2c)
1H NMR (500 MHz, DMSO-d6) δ 10.32 (s, 1H), 7.24 – 7.16 (m, 1H), 7.13 – 7.03 (m, 2H), 6.85 (d, J = 4.3 Hz, 2H),
6.83 (d, J = 7.6 Hz, 1H), 6.05 (s, 1H), 5.13 (d, J = 5.2 Hz, 1H), 3.62 – 3.48 (m, 1H), 3.37 (td, J = 11.1, 3.2 Hz, 1H),
2.24 (s, 3H), 2.04 – 1.97 (m, 1H), 1.83 – 1.76 (m, 1H), 1.56 – 1.41 (m, 2H), 1.35 – 1.25 (m, 1H); 13C NMR (126
MHz, DMSO-d6) δ 177.0, 140.7, 138.0, 134.9, 128.7, 128.5, 126.2, 126.1, 123.2, 121.3, 119.8, 117.3, 109.5,
69.1, 63.3, 60.7, 36.9, 24.7, 20.1, 19.9; HRMS (m/z), calculated for C20H20N2O2Cl+: 355.1208, found (M + H)+:
355.1207.
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8'-Chloro-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2d) (Ramesh et al.,
2008)
1H NMR (500 MHz, DMSO-d6) δ 10.34 (s, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.25 – 7.22 (m, 1H), 7.21 – 7.17 (m, 1H),
6.91 – 6.85 (m, 2H), 6.84 (d, J = 7.7 Hz, 1H), 6.73 (t, J = 7.7 Hz, 1H), 6.26 (s, 1H), 5.16 (d, J = 5.2 Hz, 1H), 3.59 –
3.49 (m, 1H), 3.37 (td, J = 11.2, 3.3 Hz, 1H), 2.06 – 1.93 (m, 1H), 1.86 – 1.77 (m, 1H), 1.56 – 1.44 (m, 2H), 1.37 –
1.23 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ 176.9, 140.7, 140.4, 134.9, 128.6, 128.3, 125.9, 123.2, 121.3,
119.8, 117.3, 117.0, 109.5, 69.0, 63.2, 60.7, 36.8, 24.7, 20.1; HRMS (m/z), calculated for C19H18N2O2Cl+:
341.1051, found (M + H)+: 341.1066.
9'-Chloro-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2e) (Ramesh et al.,
2008)
1H NMR (500 MHz, DMSO-d6) δ 10.40 (s, 1H), 7.27 – 7.14 (m, 2H), 7.06 (dd, J = 8.6, 1.7 Hz, 1H), 6.95 (d, J = 7.3
Hz, 1H), 6.92 – 6.81 (m, 2H), 6.66 (s, 1H), 6.63 (d, J = 8.6 Hz, 1H), 5.08 (d, J = 5.0 Hz, 1H), 3.55 (d, J = 11.0 Hz,
1H), 3.44 – 3.41 (m, 1H), 2.03 – 1.91 (m, 1H), 1.89 – 1.80 (m, 1H), 1.58 – 1.40 (m, 2H), 1.37 – 1.28 (m, 1H); 13
C
NMR (126 MHz, DMSO-d6) δ 177.0, 143.1, 140.4, 134.4, 128.6, 127.8, 126.4, 123.4, 121.5, 119.9, 119.2, 115.5,
109.4, 68.8, 64.8, 62.9, 36.6, 24.4, 20.1; HRMS (m/z), calculated for C19H18N2O2Cl+: 341.1051, found (M + H)+:
341.1053.
9'-Bromo-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2f) (Ramesh et al.,
2008)
1H NMR (500 MHz, DMSO-d6) δ 10.42 (s, 1H), 7.25 – 7.19 (m, 2H), 6.95 (d, J = 7.4 Hz, 1H), 6.93 – 6.88 (m, 1H),
6.85 (d, J = 7.7 Hz, 1H), 6.75 (s, 1H), 6.68 (dd, J = 8.2, 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 5.05 (d, J = 5.1 Hz, 1H),
3.60 – 3.49 (m, 1H), 3.45 – 3.39 (m, 1H), 2.00 – 1.91 (m, 1H), 1.86 (dt, J = 10.0, 4.8 Hz, 1H), 1.63 – 1.38 (m, 2H),
1.36 – 1.26 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ 177.2, 145.7, 140.6, 134.3, 132.5, 129.1, 128.9, 123.5,
121.7, 116.5, 116.2, 112.9, 109.6, 68.9, 64.9, 63.0, 36.9, 24.5, 20.25.
7',9'-Dimethyl-2',3',4',4a',6',10b'-hexahydrospiro-[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2g)
1H NMR (500 MHz, DMSO-d6) δ 10.29 (s, 1H), 7.16 (td, J = 7.1, 2.4 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 6.85 – 6.78
(m, 3H), 6.75 (d, J = 2.0 Hz, 1H), 5.48 (s, 1H), 5.11 (d, J = 5.3 Hz, 1H), 3.56 – 3.44 (m, 1H), 3.41 – 3.33 (m, 1H),
2.19 (s, 3H), 2.06 – 1.94 (m, 4H), 1.73 (dt, J = 10.2, 4.8 Hz, 1H), 1.56 – 1.29 (m, 3H); 13C NMR (126 MHz, DMSO-
d6) δ 177.6, 140.5, 140.0, 135.4, 130.1, 128.3, 124.8, 124.66, 123.4, 121.9, 121.2, 117.4, 109.4, 69.5, 63.6, 60.4,
+
36.9, 24.9, 20.3, 20.1, 17.5; HRMS (m/z), calculated for C21H23N2O2 : 335.1754, found (M + H)+: 335.1761.
2',3',4',4a',6',10b'-Hexahydrospiro[indoline-3,5'-pyrano[3,2-c]quinolin]-2-one (2h) (Ramesh et al., 2008)
1H NMR (500 MHz, DMSO-d6) δ 10.36 (d, J = 3.2 Hz, 1H), 7.34 – 7.14 (m, 2H), 7.07 – 6.80 (m, 4H), 6.72 – 6.53
(m, 2H), 6.40 (d, J = 8.7 Hz, 1H), 5.11 (d, J = 5.1 Hz, 1H), 3.51 (d, J = 11.0 Hz, 1H), 3.41 (td, J = 10.7, 2.9 Hz, 1H),
2.01 – 1.77 (m, 2H), 1.60 – 1.21 (m, 3H); 13C NMR (126 MHz, DMSO-d6) δ 176.3, 143.1, 139.4, 133.8, 127.5,
126.9, 125.9, 122.4, 120.3, 116.3, 115.5, 112.9, 108.4, 68.2, 62.1, 59.7, 35.9, 23.6, 19.1; HRMS (m/z), calculated
+
for C19H19N2O2 : 307.1441, found (M + H)+: 307.1448.
2.1.3 General procedure for the synthesis of 4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-
spiro[indoline-3,2'-quinolin]-2-ones (2i-2p)
Aryl imines 4a-n (1.8 mmol) were each dissolved in anhydrous dichloromethane (30 ml) and treated with
.
BF3 OEt2 (1.8 mmol) under an inert atmosphere and allowed to stir for fifteen minutes. Trans-isoeugenol (1.5
eq, 2.7 mmol) was then added, and the reaction progress monitored by TLC. Typically, after 72 hours, the
reaction mixture was treated with saturated aqueous NaHCO3 solution (20 ml) and extracted with ethyl
acetate (3 × 20 ml). The organic layer was dried (Na2SO4), concentrated under vacuum, and the resulting
residue purified by either recrystallization (hexane/EtOAc) or silica gel column chromatography (30-50%
EtOAc/hexane). The following products were prepared by this method:
4'-(4-Hydroxy-3-methoxyphenyl)-3',6'-dimethyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2i)
(Kouznetsov et al., 2008)
1H NMR (500 MHz, CDCl3) δ 10.50 (s, 1H), 8.82 (s, 1H), 7.20 (t, J = 7.6 Hz, 1H), 6.95 (d, J = 7.3 Hz, 1H), 6.90 (t, J =
7.5 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.75 – 6.65 (m, 3H), 6.56 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 8.0 Hz, 1H), 6.32 (s,
1H), 6.22 (s, 1H), 3.75 (d, J = 11.8 Hz, 1H), 3.70 (s, 3H), 2.32 (dq, J = 13.1, 6.7 Hz, 1H), 2.02 (s, 3H), 0.31 (d, J =
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6.5 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 177.7, 147.4, 144.9, 141.5, 141.2, 134.6, 132.0, 129.4, 128.3, 127.4,
124.5, 123.8 (2C), 121.8, 121.4, 115.5, 114.2, 113.0, 109.3, 64.8, 55.5, 46.5, 39.7, 20.2, 13.1; HRMS (m/z),
+
calculated for C25H25N2O3 : 401.1860, found (M + H)+: 401.1865.
8'-Fluoro-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2j)
1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.84 (s, 1H), 7.33 – 7.26 (m, 1H), 7.23 (td, J = 7.7, 1.2 Hz, 1H),
7.05 (dd, J = 7.5, 1.2 Hz, 1H), 6.95 (td, J = 7.5, 1.0 Hz, 1H), 6.86 (dd, J = 7.8, 1.0 Hz, 1H), 6.76 – 6.70 (m, 3H), 6.59
(t, J = 7.8 Hz, 2H), 6.26 (s, 1H), 3.85 (d, J = 12.1 Hz, 1H), 3.71 (s, 3H), 2.32 (dq, J = 13.1, 6.6 Hz, 1H), 0.31 (d, J =
6.6 Hz, 3H); 13C NMR (126 MHz, DMSO) δ 177.0, 151.1, 149.2, 140.7, 134.8, 132.7, 132.6, 128.6, 123.3, 122.6,
121.4, 120.6, 120.5, 115.9, 115.9, 113.7, 113.5, 109.5, 69.0, 62.9, 61.0, 36.9, 24.7, 20.2.
8'-Chloro-4'-(4-hydroxy-3-methoxyphenyl)-3',6'-dimethyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-
one (2k)
1H NMR (500 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.94 (s, 1H), 7.27 (t, J = 7.0 Hz, 1H), 7.07 – 6.94 (m, 3H), 6.91 (d, J
= 7.4 Hz, 1H), 6.79 (d, J = 7.4 Hz, 1H), 6.75 (s, 1H), 6.62 (s, 1H), 6.36 (s, 1H), 6.21 (s, 1H), 3.85 (d, J = 11.8 Hz,
1H), 3.76 (s, 3H), 2.44 – 2.26 (m, 1H), 2.09 (s, 3H), 0.37 (d, J = 5.7 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ
177.6, 147.6, 145.2, 142.0, 137.5, 133.9, 131.9, 128.7, 128.5, 127.6, 126.0, 125.3, 123.7, 121.8 (2C), 117.2,
+
115.7, 115.2, 110.6, 109.4, 64.1, 55.7, 46.4, 19.8, 13.0; HRMS (m/z), calculated for C25H24ClN2O3 : 435.1470,
found (M + H)+: 435.1485.
7'-Chloro-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2l)
1H NMR (500 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.74 (s, 1H), 7.22 (t, J = 7.3 Hz, 1H), 6.98 (t, J = 7.7 Hz, 1H), 6.93 –
6.82 (m, 2H), 6.77 (d, J = 7.1 Hz, 1H), 6.71 – 6.65 (m, 3H), 6.62 (t, J = 7.4 Hz, 2H), 6.48 (d, J = 7.7 Hz, 1H), 3.75 (d,
J = 10.2 Hz, 1H), 3.67 (s, 3H), 2.37 – 2.26 (m, 1H) 0.52 (d, J = 6.2 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 176.6,
147.3, 147.1, 144.6, 141.7, 135.9, 134.3, 130.8, 128.7, 128.1, 123.0, 122.9, 121.9, 120.9, 118.8, 115.3, 114.1,
+
113.5, 109.8, 63.8, 55.7, 46.5, 44.7, 13.4; HRMS (m/z), calculated for C24H22ClN2O3 : 421.1313, found (M + H)+:
421.1323.
6'-Chloro-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2m)
1H NMR (500 MHz, DMSO-d6) δ 10.57 (s, 1H), 8.89 (d, J = 4.1 Hz, 1H), 7.23 (t, J = 7.1 Hz, 1H), 7.06 – 7.02 (m,
2H), 6.96 (d, J = 6.9 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.76 (d, J = 7.7 Hz, 1H), 6.74 – 6.69 (m, 2H), 6.59 (d, J = 8.1
Hz, 1H), 6.56 – 6.43 (m, 2H), 3.82 (d, J = 11.9 Hz, 1H), 3.72 (s, 3H), 2.32 (dq, J = 13.6, 6.4 Hz, 1H), 0.31 (d, J = 6.6
Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 177.5, 147.7, 145.2, 145.0, 141.5, 133.6, 131.7, 131.2, 130.9, 128.7,
124.0, 122.8, 122.2, 121.5, 115.7, 115.6, 113.0, 112.6, 109.6, 63.9, 55.6, 45.8, 40.1, 13.0; HRMS (m/z),
+
calculated for C24H22ClN2O3 : 421.1313, found (M + H)+: 421.1331.
6'-Bromo-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2n) (Kouznetsov et al., 2008)
1H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.81 (s, 1H), 7.21 (td, J = 7.6, 1.7 Hz, 1H), 6.96 (d, J = 7.4 Hz, 1H),
6.92 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.78 – 6.67 (m, 3H), 6.57 (d, J = 8.0 Hz, 1H), 6.46 (d, J = 8.1 Hz,
1H), 6.33 (s, 1H), 6.23 (s, 1H), 3.76 (d, J = 11.6 Hz, 1H), 3.72 (s, 3H), 2.33 (dd, J = 11.9, 6.5 Hz, 1H), 0.32 (d, J =
6.4 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 178.3, 148.0, 145.5, 142.1, 141.8, 135.1, 132.6, 130.0, 128.9,
128.0, 125.1, 124.4 (2C), 122.4, 122.0, 116.1, 114.8, 113.7, 109.9, 64.6, 56.1, 47.1, 40.6, 13.7.
4'-(4-Hydroxy-3-methoxyphenyl)-3',6',8'-trimethyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one (2o)
1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.81 (s, 1H), 7.19 (t, J = 7.3 Hz, 1H), 6.95 – 6.86 (m, 2H), 6.84 (d, J
= 7.6 Hz, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 6.63 (s, 1H), 6.53 (d, J = 7.5 Hz, 1H), 6.19 (s, 1H), 5.56 (s, 1H),
3.75 (d, J = 11.9 Hz, 1H), 3.69 (s, 3H), 2.38 – 2.20 (m, 1H), 2.00 (s, 3H), 1.98 (s, 3H), 0.30 (d, J = 6.2 Hz, 3H); 13C
NMR (126 MHz, DMSO-d6) δ 178.1, 147.5, 144.9, 141.8, 139.3, 134.9, 132.5, 129.0, 128.2, 127.4, 124.1, 123.8,
123.7, 121.8, 121.5, 121.4, 115.5, 113.2, 109.2, 64.9, 55.6, 46.6, 40.1, 20.2, 17.6, 13.2; HRMS (m/z), calculated
+
for C26H27N2O3 : 415.2016, found (M + H)+: 415.2036.
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4'-(4-Hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2p)
(Kouznetsov et al., 2008)
1H NMR (500 MHz, DMSO-d6) δ 10.54 (s, 1H), 8.83 (s, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 6.95 –
6.84 (m, 3H), 6.73 (d, J = 7.4 Hz, 1H), 6.70 (s, 1H), 6.58 (d, J = 7.3 Hz, 1H), 6.52 (d, J = 7.5 Hz, 1H), 6.50 – 6.41 (m,
3H), 3.79 (d, J = 11.8 Hz, 1H), 3.70 (s, 3H), 2.41 – 2.28 (m, 1H), 0.32 (d, J = 5.6 Hz, 3H); 13C NMR (126 MHz,
DMSO-d6) δ 177.8, 147.6, 145.1, 143.6, 141.6, 134.4, 132.2, 129.2, 128.5, 126.8, 123.9, 123.8, 122.0, 121.6,
+
116.3, 115.5, 114.0, 113.1, 109.5, 64.0, 55.6, 46.4, 40.1, 13.2; HRMS (m/z), calculated for C24H23N2O3 :
387.1703, found (M + H)+: 387.1716.
4'-(4-Hydroxy-3-methoxyphenyl)-3'-methyl-8'-(trifluoromethyl)-3',4'-dihydro-1'H-spiro[indoline-3,2'-
quinolin]-2-one (2q)
1H NMR (500 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.84 (s, 1H), 7.33 – 7.26 (m, 1H), 7.23 (td, J = 7.7, 1.2 Hz, 1H),
7.05 (dd, J = 7.5, 1.2 Hz, 1H), 6.95 (td, J = 7.5, 1.0 Hz, 1H), 6.86 (dd, J = 7.8, 1.0 Hz, 1H), 6.76 – 6.70 (m, 3H), 6.59
(t, J = 7.8 Hz, 2H), 6.26 (s, 1H), 3.85 (d, J = 12.1 Hz, 1H), 3.71 (s, 3H), 2.32 (dq, J = 13.1, 6.6 Hz, 1H), 0.31 (d, J =
6.6 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 177.7, 147.8, 145.3, 142.1, 141.1, 133.6, 133.3, 131.6, 128.6,
128.1, 126.4, 124.9 (q, JC-F = 271.0 Hz), 124.7, (q, JC-F = 6.1 Hz), 123.9 (2C), 121.9, 115.7, 115.2, 111.2, (q, JC-F
=
+
28.4 Hz), 109.4, 64.9, 55.7, 45.8, 38.7, 12.9; HRMS (m/z), calculated for C25H22F3N2O3 : 455.1577, found (M +
H)+: 455.1583.
6'-Chloro-4'-(4-hydroxy-3-methoxyphenyl)-3',8'-dimethyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-
one (2r)
1H NMR (500 MHz, DMSO-d6) δ 10.52 (s, 1H), 8.91 (s, 1H), 7.21 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 7.1 Hz, 1H), 6.92
(t, J = 7.3 Hz, 1H), 6.90 – 6.82 (m, 2H), 6.74 (d, J = 7.9 Hz, 1H), 6.70 (s, 1H), 6.56 (s, 1H), 6.29 (s, 1H), 5.99 (s, 1H),
3.80 (d, J = 11.9 Hz, 1H), 3.71 (s, 3H), 2.36 – 2.18 (m, 1H), 2.02 (s, 3H), 0.30 (d, J = 5.9 Hz, 3H); 13C NMR (126
MHz, DMSO-d6) δ 177.9, 147.7, 145.2, 141.8, 140.7, 133.8, 132.2, 128.5, 127.5, 126.2, 125.4, 123.9, 123.6,
121.9 (2C), 119.0, 115.7, 113.0, 109.4, 64.9, 55.7, 46.2, 39.1, 17.5, 13.0; HRMS (m/z), calculated for
+
C25H24ClN2O3 : 435.1470, found (M + H)+: 435.1476.
4'-(4-Hydroxy-3-methoxyphenyl)-3'-methyl-7'-(trifluoromethyl)-3',4'-dihydro-1'H-spiro[indoline-3,2'-
quinolin]-2-one (2s)
1H NMR (500 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.73 (s, 1H), 7.21 (td, J = 7.7, 0.9 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H),
6.95 – 6.84 (m, 3H), 6.80 (d, J = 7.5 Hz, 2H), 6.64 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 1.6 Hz, 1H), 6.40 (dd, J = 8.1, 1.8
Hz, 1H), 5.75 (s, 1H), 3.95 (d, J = 10.1 Hz, 1H), 3.64 (s, 3H), 2.26 (dq, J = 13.3, 6.6 Hz, 1H), 0.50 (d, J = 6.7 Hz, 3H);
13C NMR (126 MHz, DMSO-d6) δ 176.6, 146.9, 146.7, 144.9, 141.8, 136.2, 130.7, 128.8, 128.0 (q, JC-F = 29.2 Hz),
127.3, 124.6 (q, JC-F = 274.6 Hz), 122.92, 122.89, 122.0, 121.3, 119.7, 116.5 (q, JC-F = 6.3 Hz), 115.2, 113.7, 109.9,
+
63.3, 55.8, 45.1, 40.1, 13.6; HRMS (m/z), calculated for C25H22F3N2O3 : 455.1577, found (M + H)+: 455.1572.
4'-(4-Hydroxy-3-methoxyphenyl)-3'-methyl-6'-(trifluoromethyl)-3',4'-dihydro-1'H-spiro[indoline-3,2'-
quinolin]-2-one (2t)
1H NMR (500 MHz, DMSO-d6) δ 10.63 (s, 1H), 8.90 (s, 1H), 7.27 (s, 1H), 7.25 – 7.20 (m, 2H), 7.10 (d, J = 7.5 Hz,
1H), 6.97 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.7 Hz, 1H), 6.79 – 6.72 (m, 2H), 6.70 (s, 1H), 6.66 (d, J = 8.5 Hz, 1H),
6.61 (d, J = 8.0 Hz, 1H), 3.88 (d, J = 12.0 Hz, 1H), 3.71 (s, 3H), 2.33 (dq, J = 13.5, 6.7 Hz, 1H), 0.32 (d, J = 6.5 Hz,
3H); 13C NMR (126 MHz, DMSO-d6) δ 177.5, 147.7, 146.9, 145.3, 141.6, 132.9, 131.6, 128.8, 126.0 (q, JC-F = 5.2
Hz), 125.2 (q, JC-F = 270.0 Hz), 124.2, 124.00 (q, JC-F = 3.4 Hz), 123.4, 122.3, 122.0, 115.8 (q, JC-F = 5.0 Hz), 115.5,
+
113.4 (2C), 109.6, 63.9, 55.7, 45.6, 38.7, 13.0; HRMS (m/z), calculated for C25H22F3N2O3 : 455.1577, found (M +
H)+: 455.1587.
7'-Bromo-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2u)
1H NMR (500 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.76 (s, 1H), 7.21 (t, J = 7.5 Hz, 1H), 6.92 – 6.84 (m, 3H), 6.82 (d, J
= 7.9 Hz, 1H), 6.74 (d, J = 7.5 Hz, 1H), 6.68 – 6.62 (m, 4H), 6.45 (d, J = 8.0 Hz, 1H), 3.69 (d, J = 10.0 Hz, 1H), 3.66
(s, 3H), 2.34 – 2.26 (m, 1H), 0.53 (d, J = 6.3 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 176.6, 147.5, 147.0, 144.6,
141.7, 135.8, 130.7, 128.7, 128.5, 125.1, 124.4, 123.0, 122.3, 121.9, 121.3, 115.3, 114.8, 114.1, 109.8, 63.8,
+
55.7, 48.4, 45.2, 13.5; HRMS (m/z), calculated for C24H2279BrN2O3 : 465.0808, found (M + H)+: 465.0814
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8'-Chloro-4'-(4-hydroxy-3-methoxyphenyl)-3'-methyl-3',4'-dihydro-1'H-spiro[indoline-3,2'-quinolin]-2-one
(2v)
1H NMR (500 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.85 (s, 1H), 7.23 (t, J = 7.7 Hz, 1H), 7.09 (d, J = 6.9 Hz, 1H), 6.99
(d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 7.7 Hz, 1H), 6.74 (d, J = 8.0 Hz, 1H), 6.70 (s, 1H), 6.57 (d, J =
8.0 Hz, 1H), 6.51 – 6.44 (m, 2H), 6.35 (s, 1H), 3.84 (d, J = 11.8 Hz, 1H), 3.70 (s, 3H), 2.33 (dq, J = 13.6, 7.1, 6.5 Hz,
1H), 0.32 (d, J = 6.6 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 177.6, 147.7, 145.2, 142.0, 139.8, 133.6, 131.9,
128.6, 128.2, 127.2, 126.0, 123.8, 121.9, 121.6, 117.1, 116.4, 115.6, 113.1, 109.4, 64.1, 55.6, 46.2, 39.9, 13.0;
+
HRMS (m/z), calculated for C24H22ClN2O3 : 421.1313, found (M + H)+: 421.1325.
2.1.4 General procedure for the one-pot synthesis of spiroindolones
A mixture of isatin (1 mmol), the appropriately substituted aniline (1.0 eq, 1 mmol) and either 3,4-dihydro-2H-
pyran or isoeugenol (1.0 eq, 1 mmol) were dissolved in acetonitrile (10 ml) and treated with InCl3 (0.2 eq, 0.2
mmol). The resulting heterogeneous mixture was stirred at room temperature under a nitrogen atmosphere
for 72 hours. The reaction was quenched with water (10 ml) and extracted with ethyl acetate (3 × 20 ml). The
organic layer was dried (Na2SO4), concentrated under vacuum, and the resulting residue purified by silica gel
column chromatography (30-50% EtOAc/hexane).
2.2 In vitro antiplasmodial assays
3
2.2.1 H-Hypoxanthine incorporation assay against FCR-3 strain. The chloroquine-resistant Gambian FCR-3
strain was cultured in vitro according to the method described by Jensen and Trager (Trager & Jensen, 1976)
and van Zyl et al (Van Zyl, Seatlholo, & Viljoen, 2010) under an optimal atmosphere of 5% CO2, 5% O2, 90% N2
in human erythrocytes taken from volunteers of various blood groups and Rh factors. For experimental
purposes the cultures were synchronized with 5% D-sorbitol when the parasites were in the ring stage
(Lambros & Vanderberg, 1979). The antimalarial activity of the various compounds was determined using the
tritiated hypoxanthine incorporation assay (Desjardins, Canfield, Haynes, & Chulay, 1979). The parasite
suspension, consisting of predominately the ring stage, was adjusted to a 0.5% parasitaemia and 1%
haematocrit in hypoxanthine-free RPMI-1640 culture medium with 10% human plasma and exposed to 6 to 7
concentrations of each compound for a single cycle of parasite growth. All assays were carried out using
3
untreated parasites and uninfected red blood cells as controls. Labelled H-hypoxanthine was added after 24 h
and the parasitic 3H-DNA harvested after a further 24 h incubation period at 37°C in a humidified environment.
The concentration that inhibited 50% of parasite growth (IC50 value) was determined from the sigmoidal log
dose response curve generated by the Enzfitter and GraphPad Prism software.
2.2.2 pLDH assay against 3D7 strain. Three-fold serial dilutions of the test compounds were incubated with
3D7 strain P. falciparum parasites (2% parasitaemia, 1% haematocrit) in 96-well plates containing RPMI 1640
medium supplemented with 25 mM HEPES, 0.5% (w/v) Albumax II, 22 mM glucose, 0.65 mM hypoxanthine,
0.05 mg/mL gentamicin and 1% (v/v) human erythrocytes. Incubations were initiated when parasites were
predominantly in the trophozoite stage and continued for 48 h at 37°C in sealed containers filled with an
atmosphere of 5% CO2, 5% O2, 90% N2. Subsequently, a colourimetric assay for parasite lactate dehydrogenase
(pLDH) activity in individual wells was carried out (Makler et al., 1993). Twenty µL of culture was removed from
the individual wells and transferred to a second 96-well plate containing 125 μL per well pLDH assay reagent
(44 mM Tris buffer, pH 9, containing 0.18 M L-lactic acid, 0.13 mM acetylpyridine adenine dinucleotide, 0.39
mM nitrotetrazolium blue chloride, 0.048 mM phenazine ethosulfate and 0.16% (v/v) Triton X-100) and
incubated at ambient temperature for 10 – 30 minutes, after which Abs620 was measured in a Spectramax M3
plate reader. Absorbance values were used to calculate percentage parasite viability relative to control wells
containing untreated parasite cultures. IC50 values for individual compounds were calculated from plots of %
viability vs. log[compound] by non-linear regression using GraphPad Prism.
3
RESULTS AND DISCUSSION
The Povarov reaction of ketimines derived from isatin typically requires the use an acid catalyst owing to the
low reactivity of the ketimine. Lewis acids, such as indium trichloride (Bianchini et al., 2016; Ramesh et al.,
2008), antimony pentachloride (Karmakar et al., 2014) and boron trifluoride (Kouznetsov et al., 2008), p-
toluenesulfonic acid (Gao et al., 2014), as well as chiral phosphoric acids (Shi et al., 2013; Zhang et al., 2014)
have been reported to catalyse this reaction. We wanted to assess the ability of a range of catalysts to
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facilitate the Povarov reaction of a ketimine derived from isatin. To this end, ketimine 4a was prepared for a
model study by reaction of isatin 3 with p-toluidine in absolute ethanol (Scheme 2). The imine was then
treated with 3,4-dihydro-2H-pyran in the presence of an acid catalyst as described in Table 1, to afford
spiroindolone 2a as a single regioisomer.
In each case, the cis product was isolated from the reaction mixture as the major product, with only traces of
the trans product identified. Raghunathan and co-workers reported the use of InCl3 in this reaction affording
the desired product as a mixture of isomers in a yield of 66% (77:23 cis:trans) (Ramesh et al., 2008). In our
hands, only the cis product was isolated in a yield of 42% under these conditions (entry 1 of Table 1).
Increasing the temperature of the reaction or catalyst loading did not significantly improve the yield of the
reaction (entry 2 and 3 of Table 1), with starting material recovered in each case (approximately 35%), even
when the reaction was run for extended periods of time. By comparison, CuOTf, Montmorillonite K10 and
Montmorillonite KSF afforded the product in comparatively lower yields (entry 4, 5 and 6 of Table 1). For
ZnCl2, a change in solvent resulted in improved yields of product (entry 10 and 11 of Table 1), while in other
cases this had little effect (eg. for Montmorillonite K10, entry 7 and 8 of Table 1). Increasing the temperature
of the reactions catalysed by ZnCl2 and Montmorillonite K10, or the use of microwave (MW) irradiation,
resulted in a slight decrease in the yield of product isolated (compare entries 7, 8 and 9, and 11, 12 and 13 of
Table 1), while only starting material was recovered for the reactions with p-TsOH and Ti(OiPr)4 as catalyst
.
(entry 14, 15 and 16 of Table 1). The use of catalytic BF3 OEt2 afforded a reasonable yield of the product (entry
.
17 of Table 1), with a higher yield of product obtained when 0.5 mole equivalents of BF3 OEt2 was used (entry
18 of Table 1). Increasing this further resulted in a decrease in product yield (entry 19 of Table 1). We
.
therefore chose to employ BF3 OEt2 as catalyst in the synthesis of a range of spiroindolones derived from 3,4-
dihydro-2H-pyran.
A series of ketimines 4b-h were then prepared by reaction of isatin 3 with substituted anilines, followed by
.
Povarov reaction with 3,4-dihydro-2H-pyran in the presence of BF3 OEt2 to afford spiroindolones 2b-h in
moderate yields (Table 2), with starting material recovered in most cases. Once again, the cis product was
isolated as the major product from each reaction.
Other electron rich alkenes were tested in the Povarov reaction, including trans-isoeugenol 5 and ethyl vinyl
.
ether. While spiroindolone products derived from trans-isoeugenol were formed using BF3 OEt2 as catalyst, we
were unable to isolate any of the desired products when ethyl vinyl ether was used as the electron rich alkene.
As shown in Scheme 3, when imine 4a derived from isatin 3 and p-toluidine was reacted with trans-isoeugenol
5 using the optimised conditions described above for the Povarov reaction with 3,4-dihydro-2H-pyran, the
.
desired trans product 2i was isolated in a yield of 32%. Increasing the amount of BF3 OEt2 used in the reaction
to one mole equivalent resulted in the product being isolated in a yield comparable to that reported previously
(Kouznetsov et al., 2008).
.
Ketimines 4b-n were then reacted with trans-isoeugenol in the presence of 1 mole equivalent of BF3 OEt2 to
afford spiroindolones 2j-v, in moderate yields (Table 3). With the use of trans-isoeugenol as the substrate, the
trans product was isolated from the reaction mixture as the major product in each case.
We then tested the one-pot reaction of isatin 3, p-toluidine and 3,4-dihydro-2H-pyran to form the product 2a
in a single step (entry 1 of Table 4). In this case, we found that the use of InCl3 was best to facilitate the one-
pot preparation of spiroindolones 2, as reported by Raghunathan (Ramesh et al., 2008). The one-pot reaction
resulted in a higher overall yield of product 2a, and therefore we repeated the one-pot reaction on selected
substrates that were particularly low yielding in the two step process (Table 4). With the exception of 2v
(entry 5 of Table 4), the yields of isolated products improved significantly, suggesting that the one-pot method
is superior to the two step approach for these compounds.
Compounds prepared were then assessed for antiplasmodial activity in a whole cell P. falciparum screen
against a drug-sensitive strain (3D7) and a drug-resistant strain (FCR-3) of the parasite. Those compounds that
were found to inhibit parasite growth by 50% or more at a concentration of 5 µM (for the FCR-3 strain) or by
75% or more at a concentration of 20 µM (for the 3D7 strain) were then assessed further and IC50 values
obtained (Table 5).
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As can be seen from the results in Table 5, a wider range of spiroindolones showed moderate antiplasmodial
activity against the drug resistant FCR-3 strain, than against the drug sensitive 3D7 strain. Compounds derived
from an aniline bearing a substituent in the 3-position did not show any antiplasmodial activity. Only
compounds derived from isoeugenol and an aniline bearing an electron withdrawing group in either the 2- or
the 4-position of the aromatic ring showed activity against the 3D7 strain (2j, 2n, 2r, entry 5, 7 and 8 of Table
5). Two of these compounds also showed activity against the FCR-3 strain (2n and 2r, entry 7 and 8 of Table 5),
along with an additional two compounds derived from isoeugenol (2i and 2k, entry 4 and 6 of Table 5).
Furthermore, three compounds derived from 3,4-dihydro-2H-pyran showed moderate activity against the FCR-
3 strain (2a, 2f and 2g, entry 1, 2 and 3 of Table 5). Significantly, all the compounds displaying activity against
the FCR-3 strain were derived from an aniline bearing either a methyl substituent or a halogen at the 4-
position of the aromatic ring, irrespective of whether isoeugenol or 3,4-dihydro-2H-pyran was used as the
diene in the Povarov reaction.
4
CONCLUSIONS
We have prepared a series of 3',4'-dihydro-1'H-spiro(indoline-3,2'-quinolin)-2-ones by the Povarov reaction of
electron rich alkenes and ketimines derived from isatin and assessed them for antiplasmodial activity in vitro.
Eight of the compounds prepared displayed antiplasmodial activity in the low micromolar range against either
the 3D7 or the FCR-3 strain. Interestingly, compounds derived from 3,4-dihydro-2H-pyran did not show
activity against the 3D7 strain but did show activity in the low micromolar range against the FCR-3 strain.
Compounds derived from isoeugenol displayed moderate antiplasmodial activity against both drug-resistant
and drug-sensitive strains of the parasite. Although the compounds were less potent than Chloroquine, the
structure-activity relationships observed from this study have been utilised in the design of a second
generation of analogues currently under investigation in our laboratories.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable
request.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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FIGURES AND SCHEMES:
FIGURE 1 Cipargamin, 1 and spiroindolones 2 prepared by Povarov reaction.
SCHEME 1 Reaction of isatin 3 in Povarov reaction to afford spiroindolones 2.
SCHEME 2 Reagents and conditions: (i) p-toluidine, abs. EtOH, gl. AcOH, reflux, 15h, 96 %; (ii) 3,4-dihydro-2H-pyran,
conditions as described in Table 1.
.
.
.
SCHEME 3 Reagents and conditions: (i) BF3 OEt2; CH2Cl2, rt, 32% (0.5 eq BF3 OEt2); 45% (1.0 eq. BF3 OEt2).
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TABLES:
TABLE 1 Screening of catalysts for Povarov reaction of ketimine 4a with 3,4-dihydro-2H-pyran
Catalyst
Solvent Temp Time (h)
Yield of 2a
42 %
47 %
48 %
38 %
34 %
28 %
42 %
38 %
28 %
No reaction
36 %
22 %
1
2
3
4
5
6
7
8
9
InCl3 (20 mol %)
InCl3 (20 mol %)
InCl3 (50 mol %)
CuOTf (20 mol %)
Montmorillonite K10 (0.2 mass eq.) CH2Cl2
Montmorillonite KSF (0.2 mass eq.) CH2Cl2
Montmorillonite K10 (1 mass eq.)
Montmorillonite K10 (1 mass eq.)
Montmorillonite K10 (1 mass eq.)
CH3CN
CH3CN
CH3CN
CH2Cl2
rt†
72
24
72
72
72
72
72
72
3
40°C
rt
rt
rt
rt
CH2Cl2
CH3CN
CH3CN
CH2Cl2
CH3CN
CH3CN
CH3CN
CH2Cl2
CH2Cl2
CH3CN
CH2Cl2
CH2Cl2
CH2Cl2
rt
reflux
65°C‡
rt
10 ZnCl2 (20 mol %)
11 ZnCl2 (20 mol %)
12 ZnCl2 (20 mol %)
13 ZnCl2 (20 mol %)
14 p-TsOH (5 mol %)
15 Ti(OiPr)4 (10 mol %)
16 Ti(OiPr)4 (10 mol %)
72
72
72
3
72
72
24
72
72
72
rt
reflux
65°C‡
rt
rt
reflux
rt
rt
rt
30 %
No reaction
No reaction
No reaction
45%
63%
15%
.
17 BF3.OEt2 (20 mol %)
18 BF3.OEt2 (50 mol %)
19 BF3 OEt2 (1 mol eq.)
†rt = room temperature; ‡reactions conducted under MW irradiation (150W)
TABLE 2 Spiroindolones 2 prepared by Povarov reaction of imines 4 with 3,4-dihydro-2H-pyran
Entry
R
Yield % Yield %
RSM %†
1
2
3
4
5
6
7
8
4-Me
2-F
2-Cl, 4-Me 4c 35
3-Cl
4-Cl
4-Br
4a 96
4b 73
2a 63
2b 68
2c 22
2d 49
2e 54
2f 60
2g 62
2h 45
15
11
48
26
18
22
14
33
4d 84
4e 65
4f 82
2,4-diMe 4g 75
4h 58
H
†RSM % = % starting material recovered
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TABLE 3 Spiroindolones 2 prepared by Povarov reaction of imines 4 with trans-isoeugenol
Entry
1
2
3
4
5
6
7
R
4-Me
2-F
Yield %
4a 96
4b 73
Yield %
RSM %†
2i
45
34
62
49
41
24
29
9
25
68
18
32
22
30
66
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
2t
2u
2v
25
28
36
52
48
65
54
24
64
42
56
45
30
2-Cl, 4-Me 4c 35
3-Cl
4-Cl
4-Br
4d 84
4e 65
4f 82
2,4-diMe 4g 75
H
2-CF3
2-Me, 4-Cl 4j
3-CF3
4-CF3
3-Br
8
9
4h 58
4i
46
82
10
11
12
13
14
4k 67
4l 58
4m 72
4n 80
2-Cl
†RSM % = % starting material recovered
TABLE 4 Spiroindolones 2 prepared by one-pot reaction†
Entry Compound Yield %
RSM %‡
1
2
3
4
5
2a
2c
2i
2k
2v
72
45
53
52
31
12
21
26
38
42
†InCl3 (20 mol %), CH3CN, rt, 72 hr; ‡RSM % = % starting material recovered
TABLE 5 In vitro antiplasmodial activity of spiroindolones 2
Entry Compound
IC50 3D7
IC50 FCR-3
strain (µM)† strain (µM) ‡
1
2
3
4
5
6
7
8
9
2a
2f
2g
2i
Not active
Not active
Not active
Not active
1.48 ± 1.50
Not active
1.80 ± 1.86
1.31 ± 1.18
0.0127
3.75 ± 0.39
3.73 ± 1.57
3.21 ± 1.27
2.28 ± 1.73
Not active
4.20 ± 1.29
1.67 ± 0.71
1.52 ± 0.84
0.110 ±
2j
2k
2n
2r
CQ§
± 0.0030¶
0.002
Data are presented as the mean ± SD of three experiments; †IC50 values determined by pLDH assay; ‡IC50 values determined
¶
by 3H-hypoxanthine incorporation assay; CQ = chloroquine; similar IC50 value obtained for CQ using H-hypoxanthine
§
3
incorporation assay
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This article is protected by copyright. All rights reserved.