Microwave-assisted rapid synthesis of spirooxindole-pyrrolizidine analogues and their activity as anti-amyloidogenic agents

Article abstract of DOI:10.1016/j.bioorg.2021.105128

A library of Sox-pyrrolizidines was rapidly prepared by microwave-assisted, one-pot, three-component, 1,3-dipolar cycloaddition of azomethine ylides from l-proline and isatin, with various β-nitrostyrenes. Nitro-Sox compounds, 4b, 4d and 4e inhibit HEWL amyloid fibril formation by ThT studies with percentages of fluorescence intensity of 55.4, 42.9 and 40.3%, respectively. Further studies with MTT assay, Raman spectroscopy, TEM and molecular docking supported these promising candidates for activity against amyloid misfolding, a phenomenon leading to Alzheimer's disease pathology.

Full text of DOI:10.1016/j.bioorg.2021.105128

Bioorganic Chemistry 114 (2021) 105128
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Microwave-assisted rapid synthesis of spirooxindole-pyrrolizidine
analogues and their activity as anti-amyloidogenic agents
Nilamuni H. de Silva, Suneela Pyreddy, Ewan W. Blanch, Helmut M. Hügel*,
Subashani Maniam*
School of Science, STEM College, RMIT University, 124 La Trobe Street, Melbourne, VIC 3001, Australia
A R T I C L E I N F O
A B S T R A C T
Keywords:
A library of Sox-pyrrolizidines was rapidly prepared by microwave-assisted, one-pot, three-component, 1,3-
dipolar cycloaddition of azomethine ylides from L-proline and isatin, with various β-nitrostyrenes. Nitro-Sox
compounds, 4b, 4d and 4e inhibit HEWL amyloid fibril formation by ThT studies with percentages of fluores-
cence intensity of 55.4, 42.9 and 40.3%, respectively. Further studies with MTT assay, Raman spectroscopy, TEM
and molecular docking supported these promising candidates for activity against amyloid misfolding, a phe-
nomenon leading to Alzheimer’s disease pathology.
Spicocyclics
Alzheimer’s disease
Neuroprotection
Amyloid beta
Protein misfolding
Fluorescence inhibition
1. Introduction
shown promising activity as anti-amyloidogenic agents [10–14]. How-
ever, high molecular weight, multiple-step synthesis, and poor
The appearance of amyloid plaques in the human brain is considered
as the late stage decisive indicator of Alzheimer’s disease (AD) [1,2].
Plaques are comprised primarily of amyloid-beta (Aβ) peptides that are
formed by the sequential cleavage of the amyloid precursor proteins
(APP). These peptides can aggregate and form soluble oligomers that
could self-assemble into fibrils which subsequently deposit as plaques
[3,4]. These different aggregated states of Aβ peptides associate with
progressive neurodegeneration.
bioavailability has challenged the translation of these natural products
into therapeutic agents [15,16]. The naturally occurring spirocyclic
compounds with a quaternary carbon center (spiroatom) that connects
two ring systems, have shown increasing importance in drug discovery
[17–19]. The activity of spirocyclics, especially spirooxindole (Sox)
compounds as anti-cancer [20], anaesthetic [21] and anti-viral [22]
agents has been reported.
However, there is limited research on both natural and synthetic
spirocyclics as anti-amyloidogenic agents with lack of primary Thio-
flavin T (ThT) screening and majority of the compounds targeting to-
wards AchE or β-secretase 1 (BACE1) inhibition and neuroprotection
against Aβ-induced toxicity. Ethanolic extracts (10 µM) of Uncaria
rhynchophylla could reduce the formation of Aβ1-42 by 40.7% and
destabilize pre-formed Aβ fibrils by 17.7% at the same concentrate [11].
Two main alkaloids of this extract were identified as rhynchophylline
(RIN) and isorhynchophylline (IRN) (Fig. 1) and were studied as neu-
roprotective agents against Aβ induced toxicity [23–26].
The food and drug administration (FDA) has approved acetylcho-
linesterase (AchE) inhibitors such as donepezil, galantamine and riva-
stigmine, which are symptom suppressors for early cases of AD. The
other class of therapeutics, N-methyl-D-aspartate (NMDA) receptor an-
tagonists such as memantine are used as treatment agents [5]. These
classes of drugs target the disruptions caused by the Aβ-induced toxicity,
therefore they are only able to suppress symptoms but cannot prevent
the occurrence or the progression of AD. Additionally, these agents are
not effective for the broad spectrum of patients, are low in bioavail-
ability and cause major side effects such as diarrhea, dizziness, hallu-
cinations, agitation, insomnia and urinary incontinence [6–8].
Therefore, inhibition of Aβ generation, prevention of Aβ fibril formation,
or removal of pre-formed Aβ fibrils would be attractive strategies for AD
treatment [9].
Kwon et al. isolated six new Sox alkaloids from Aspergillus duricaulis
and identified 16-oxo-paraherquamide B as an Aβ inhibitor reducing
aggregation by 30% (100 µM) and decreasing Aβ-induced toxicity [13].
Liu et al. published the anti-Aβ aggregation activities of spi-
ropyrrolizidine oxindole alkaloids from plant extracts of Isatis indigotica
Fortune and then performed ThT fluorescence assays for the isolated
Several natural products including polyphenols and alkaloids have
* Corresponding authors.
E-mail addresses: hmhugel100@gmail.com (H.M. Hügel), subashani.maniam@rmit.edu.au (S. Maniam).
https://doi.org/10.1016/j.bioorg.2021.105128
Received 10 May 2021; Received in revised form 16 June 2021; Accepted 23 June 2021
Available online 29 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
compounds. Among the 4 pairs of enantiomers, isatindigotindolin a and
b (Fig. 1) yielded excellent percentage inhibitions of 85.8% and 73.6%,
respectively, at 20 µM [12].
Sox-based drug design as anti-cancer agents with analogues capable of
better hydrogen bonding and hydrophobic interactions with various
biological receptors [33,34]. Aromatic groups provide a handle to
introduce various functional groups, however it introduces high mo-
lecular weight, poor solubility and high lipophilicity. Thus, aromatic
groups should be maintained in moderation and kept below three
[35–38]. Electron withdrawing groups, such as nitro substituent creates
electron deficient sites within the structure that would enable interac-
tion with nucleophilic binding sites in biological systems such as amino
acids [39]. Combination of these various substituents has allowed us to
design Sox-compounds for Aβ misfolding studies.
There is only one literature on Aβ aggregation inhibition using syn-
thetic Sox compounds. Marques and co-workers synthesized
a
spiroketal-oxindole via a rhodium(I)-catalyzed addition of dioxolane to
N-1,2,3-triazol-isatin derivative. The reaction was performed in toluene
at 120 ◦C for 18 hrs, giving a yield of 79%. The product was subjected to
ThT fluorescence assay resulting in a low percentage inhibition of 8.3%
[27]. Sox compounds incorporating nitro groups, nitro-Soxs, have also
shown an increasing importance in drug design [28,29]. The synthesis
between β-nitrostyrenes and Amy including isatin and sarcosine was
recently reported [30]. The regioselective reaction was performed under
reflux using methanol for 4.5–7 hrs and the products were obtained after
several purification steps. The authors followed the same reaction pro-
cedures to obtain pyrrolizidine Sox compounds by substituting sarcosine
with proline [31], however, the regiochemistry of the products were
opposite to the ones they previously obtained. Subsequently, Rajesh
et al. optimized the reaction conditions and work-up of this reaction
[29] to obtain the similar regiochemistry. Previously it was reported that
for pyrrolidine Sox, microwave-assisted synthesis was performed in
Here, we present a rapid synthesis to access a variety of pyrrolizidine
nitro-Soxs using microwave irradiation and, quantify and evaluate their
anti-amyloidogenic properties using ThT fluorescence assays, in vitro cell
toxicity studies, TEM imaging, Raman spectroscopy and molecular
docking. The synthesis of the Soxs involve a 1,3-dipolar cycloaddition
between azomethine ylide (Amy), synthesized in situ using isatin and L-
proline, with β-nitrostyrenes prepared using the nitro aldol reaction
(Henry reaction, ESI, Table S1.1). We were able to conduct the cyclo-
addition reaction in 10 min using microwave irradiation and the prod-
ucts were isolated in good yields without chromatographic purification.
A model reaction using isatin, L-proline and 1-methoxy-4-(2-nitrovinyl)
benzene was performed to optimize the microwave reaction conditions.
When ethanol was used as the solvent, regardless of the different
time/temperature combinations, the maximum product yield was 44%
(Table 1, entry 1). Water was also utilized (Table 1, entries 11–14);
however, no reaction was detected. Upon using methanol as the solvent,
◦
water at 100 C in 10 min and the products were separated by chro-
matographic techniques [32].
1.1. Rational design
Our objective is to identify/promote the selection of readily available
bioinspired synthetic pyrrolizidine nitro-Sox with optimal molecular
properties to effectively prevent AD. Despite several natural/synthetic
compounds being identified as potential therapeutic agents against Aβ
misfolding, the structure–activity relationship of Aβ misfolding has not
been methodically explored. This could be attributed to the gaps in the
understanding of molecular mechanism of Aβ pathology. An example of
Sox alkaloids, spirotryprostatin A/B has been a molecular platform for
◦
a good yield of 72% was observed at 65 C (Table 1, entry 9). These
reaction conditions (methanol, 65 ◦C, 10 min) were selected to prepare a
library of functionalized Sox compounds, with various substituted
β-nitrostyrenes.
Fig. 1. Examples of Sox natural products with anti-AD activity.
2

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
Table 1
Optimization of reaction conditions.a
Entry
Solvent
Time/min
Temperature/oC
Yieldb/%
1
ethanol
ethanol
ethanol
ethanol
methanol
methanol
methanol
methanol
methanol
methanol
water
5
80
44
22
18
26
35
19
18
33
72
45
NP
NP
NP
NP
2
5
90
3
10
10
5
80
4
90
5
60
6
5
65
7
5
80
8
10
10
10
5
60
9
65
10
11
12
13
14
80
100
110
100
110
water
5
water
10
10
water
a
Molar ratio of isatin: L-proline: 1-methoxy-4-(2-nitrovinyl)benzene is 1:1:1 in the respective solvents (2 mL), microwave irradiation (200 W, 20 psi) at
given temperature/time combinations.
b
isolated yield of Sox. NP = No product.
2. Results and discussion
cycloaddition reaction of the two nucleophilic carbons of Amy with the
electrophilic β-carbon of the β-nitrostyrene. This addition produces
–
2.1. Chemical synthesis
three new C C bonds with four contiguous stereo centres including a
quaternary carbon, which results in the spirocyclic structure. Therefore,
two regioisomers are theoretically possible. High regio- and stereo-
selectivity depends on the nature of the amino acid and the dipolar-
ophile associated in the 1,3-dipolar cycloaddition reaction [41]. Two
possible regioisomers are a result of two possible transition states, TS1
and TS2. TS2 is more sterically hindered/strained compared to TS1 and
this results in unfavourable and less stable product, which is not
observed experimentally. Thus, this is consistent with the obtained
product of only one regioisomer from TS1. Irrespective of having 4 stereo
centres, the reaction favours the aromatic group attached to the pyrro-
lizidine moiety being cis to the carbonyl group of the oxindole and trans
to the nitro group. This formation is favoured to minimize steric hin-
drance between the aryl group attached to the pyrrolizidine moiety and
The nitro-Sox products 4a-4h were obtained in good yields without
any further purification and were fully characterized (Fig. 3 and ESI,
S1). Using the 1H NMR spectrum of 4b as an example, the most
deshielded aliphatic proton appeared as a doublet of doublets at 6.22
ppm. Based on the molecular structure, only one regioisomer possibility
will give a doublet of doublets multiplicity. In the 2D COSY NMR
spectrum, the resonance of this proton displayed cross-peaks with the
pyrrolizidine protons’ resonances, confirming that the nitro group is
adjacent to the pyrrolizidine moiety, giving a single isomer and this
regioisomer agrees with previous literature [29].
Here we propose the first step of the reaction mechanism involves a
condensation reaction to form an Amy (Scheme 1). This is followed by a
Scheme 1. Proposed mechanism for the synthesis of nitro-Sox compounds.
3

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
the benzo group of the oxindole moiety.
mediated fluorescence responses of the nitro-Soxs were reported at day 6
to determine the fluorescence intensities of each analyte with respect to
the non-inhibited HEWL solution (Fig. 4). The fluorescence intensity is
directly indicative of the inhibition properties, thus the lower the fluo-
rescent emission intensity, the higher the inhibition of Aβ fibril capa-
bility of the compound.
The structure explicated from NMR spectroscopy and proposed by
the mechanism was confirmed by single crystal X-ray studies (Fig. 2,
CCDC number: 2081840) [42].
Brain permeation is at the epicenter of dementia drug effectiveness
and Lipinski’s [43] insights, inspired advances into the concept of brain
estimation and prediction of drug-likeness, preventing and reducing
drug attrition rates. The high number of oxygenated functional groups
typical in many natural and synthetic compounds is reflected in their
high polarity (topological polar surface area (TPSA), required to be 90
Å2 or less) that limits/prevents their blood brain barrier (BBB) perme-
ability [44] and optimal brain exposure. These compounds were
designed to adhere to Lipinski’s rule of five for orally active drug. The
TPSA values for the synthesized compounds are in the range of 78.16 to
107.62 Å2, which could be further improved with future derivatives
(Table 2).
The acquired ThT data showed that EGCG exhibited the highest anti-
amyloidogenic activity with the lowest relative fluorescence intensity of
25.0%. EGCG is a polyphenol with 8 hydroxy substituents within its
structure, which could readily undergo oxidation in the ThT assay me-
dium [49,50]. Irrespective of the promising activities EGCG exhibits in
vitro, it is easily metabolism in the human digestive tract [51–54]. In
contrast, the enantiomeric RIN and IRN alkaloids have previously been
reported to have better bioavailability in the brain [55]. No ThT studies
have been reported for these alkaloids and the present study revealed
that RIN and IRN exhibit promising activity against HEWL fibril for-
mation with percentage fluorescence intensities of 37.6% and 30.4%,
respectively. However, practical challenges such as cumbersome natural
product extraction and separation, lengthy total synthesis and difficulty
in mass production had limited the translation of RIN and IRN into
therapeutics [56].
2.2. Thioflavin T spectrometric assay
To evaluate the anti-amyloidogenic properties of these nitro-Sox
compounds, in vitro screening of the compounds was done against Aβ
fibril formation using ThT fluorescence assay [45] in triplicates. Hen egg
white lysozyme (HEWL) was used as a model protein [46] which under
The percentage fluorescence intensities of the Soxs ranged from
40.3% (4e) to 97.5% (4a) (Fig. 4). Compound 4a with one methoxy
group at the para position of the adjoining aryl group, had the highest
percentage fluorescence intensity, and thus the lowest activity. Com-
pounds 4b, 4c, 4d and 4e are isomers of dimethoxy functional groups.
However, their activities were very different from each other; 4e with
methoxy substituents attached at the 3 and 4 positions had the lowest
fluorescence intensity of 40.3% which corresponds to the highest
inhibitory activity. In contrast, 4c having methoxy substituents attached
at the 2 and 5 positions had the highest relative fluorescence intensity of
90.3% hence displayed the poorest inhibition activity. Also, 4f, with a
hydroxy group and, 4g and 4h, which are halogen containing nitro-Sox
compounds, did not exhibit satisfactory relative fibril inhibition values.
This wide range of fluorescence variability suggests that their molecular
interactions with HEWL are not an exclusive property of the single Sox
◦
mild denaturing conditions (pH 2, incubation temperature 70 C) un-
dergoes misfolding and forms fibrils. When ThT interacts with Aβ fibrils,
a characteristic elevation of fluorescence intensity of ThT is observed
which is used to quantify the formation of amyloid fibrils [47,48]. In
addition to these nitro-Sox inhibitors, green tea polyphenol, epi-
gallocatechingallate (EGCG), which has been extensively studied for its
anti-AD activities and two Uncaria rhynchophylla alkaloids RIN and IRN,
which have similar Sox scaffolds, were also subjected to the assay as
reference compounds. For a timescale ranging from day 1 (24 hrs) – day
12 (288 hrs), the presence of HEWL fibrils were recorded with respect to
the fluorescence intensity of an uninhibited protein solution (control).
The fibril growth followed a sigmoidal pattern (ESI, Fig. S3.1) and
reached maximum fibril formation on day 6 (144 hrs), therefore ThT
Fig. 2. Single crystal X-ray ORTEP diagram of 4b.
4

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
NO2
O
O
O2N
in situ
N
N
[2+3]
cycloaddition
OH
+
O
+
MW
NH
O
N
O
R
N
N
H
H
R
MW
H
[1]
[2]
[3]
[4a-h]
NO2
NO2
OCH3
N
NO2
O
NO2
O
OCH3
OCH3
N
N
N
O
O
OCH3
OCH3
N
H
N
N
H
N
H
H3CO
H
OCH3
[4a, 72%]
[4b, 66%]
[4c, 39%]
[4d, 50%]
NO2
NO2
NO2
NO2
N
N
N
N
OCH3
OH
OCH3
OCH3
O
O
O
O
Cl
Br
N
N
N
N
H
H
H
H
[4e, 78%]
[4f, 68%]
[4h, 87%]
[4g, 62%]
Fig. 3. Synthesis of nitro-Sox compounds 4a-4h with their percentage product yields. Molar ratio of isatin: L-proline: substituted β-nitrostyrenes is 1:1:1 (1.0 mmol),
methanol (2 mL), microwave irradiation (200 W, 20 psi), 65 ◦C for 10 min.
Table 2
Molecular parameters comparison of 4a-4h based on Lipinski’s rule of five and TPSA.
Entry
No. of H-bond
No. of H-bond acceptors
Molecular mass/Da
Lipophilicity /cLogP
No. of rotatable bonds
Rule
TPSA/Å2 [<90
donors[<5]
[<10]
[<500 Da]
[<5]
[<10]
violations
Å2]
4a
1
1
1
1
1
2
1
1
8
1
1
7
8
8
8
8
8
6
6
11
6
6
379.42
409.44
409.44
409.44
409.44
395.42
383.83
428.29
458.38
384.48
384.48
3.13
2.65
2.65
2.62
2.72
2.42
3.75
3.89
2.25
2.83
2.83
3
4
4
4
4
3
2
2
4
5
5
0
0
0
0
0
0
0
0
2
0
0
87.39
96.63
96.63
96.63
96.63
107.62
78.16
78.16
197.36
67.88
67.88
4b
4c
4d
4e
4f
4g
4h
EGCG
RIN
IRN
The predicted TPSA of compounds were obtained using Molinspiration Cheminformatics software.[40]
scaffold but varies with the substituents attached to the adjoining aro-
matic rings.
compounds 4b, 4d, 4e and RIN showed cell viabilities of 73.9 ± 1.0%,
54.11 ± 6.2%, 54.06 ± 3.9% and 77.02 ± 1.2%, respectively. This data
indicates that the Aβ-induced toxicity of the HEWL did not significantly
reduce in the presence of 4d and 4e, while toxicity reduction is observed
in the presence of 4b and the reference compound RIN by ~18 and
~22%, respectively.
Next, the effect of the synthesized compounds on mature fibrils (ESI,
S4) was also examined. Compounds were introduced to the mature fi-
brils and ThT fluorescence intensity was monitored over time. None of
the compounds exhibited promising mature fibril destabilization.
2.3. MTT assay
2.4. TEM imaging
To evaluate whether the nitro-Sox compounds provide neuro-
protection activities against Aβ induced toxicity and to understand
whether they display toxicity to cells, we performed MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays of
Human Embryonic Kidney (HEK) cells against Aβ fibrils-induced toxicity
(ESI, S5). Treatment of cells with HEWL fibrils in the absence of the
compounds caused reduction in cell viability by 55.2 ± 7.0% compared
with the control. The samples with HEWL fibrils along with the
In parallel to the ThT studies, microscopic analysis was carried out
using Transmission Electron Microscopy (TEM) to visually analyze the
nature of the protein morphology during HEWL aggregation (Fig. 5 and
ESI, Fig. S6.1). The TEM images obtained show a significant morpho-
logical difference between the native HEWL on day 0 and uninhibited
HEWL at maximum fibrillation (as monitored by ThT studies) on day 6.
Native HEWL on day 0 was visualized as disordered aggregates
(Fig. 5A). In contrast, for the uninhibited HEWL fibrils on day 7, a very
5

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
Fig. 4. Percentage ThT fluorescence intensity of selected compounds at 144 hrs. HEWL (4 × 105 monomer units) were dissolved in 900 µL of phosphate buffer
(0.695 mM in the final solution of 1000 µL) and inhibitor compounds dissolved in 100 µL PBS (0.695 mM in the final solution of 1000 µL) containing DMSO (10% in
the final mixture of 1000 µL) were introduced and immediately incubated in 70 ◦C at pH 2. Fluorescence measurements were taken daily between day 0 (t = 0 hrs)
and day 7 (t = 168 hrs). ThT induced fluorescence intensities reached a maximum on day 6 hence the intensities measured at 485 nm on day 6 (144 hrs) were used to
calculate percentage fluorescence intensities of each analyte.
Fig. 5. TEM images of native HEWL on day 0 (A), HEWL acidified to pH 2 and incubated for 7 days at 70 ◦C without (B) and with (C) Sox inhibitor 4e. HEWL (4 ×
105 monomer units) were dissolved in 900 µL of phosphate buffer (0.695 mM in the final solution of 1000 µL) and inhibitor compounds dissolved in 100 µL PBS
(0.695 mM in the final solution of 1000 µL) containing DMSO (10% in the final mixture of 1000 µL) were introduced and immediately incubated in 70 ◦C at pH 2. 10
µL aliquots of each HEWL sample were taken on day 0 and day 7 for TEM imaging.
complex and dense network of linear and rigid fibrils of a few micro-
meter lengths was evident (Fig. 5B), which further confirmed the for-
mation of HEWL fibrils under the denaturation conditions introduced.
For the HEWL incubated with inhibitor compound 4e, the fibrils
appeared shorter and more flexible (Fig. 5C) compared to those of the
uninhibited lysozyme. Similar morphologies were recorded for com-
pounds 4b and 4d (ESI, Fig. S6.1). These observations suggest that the
nitro-Sox inhibitor directs the HEWL fibrillation towards an alternate
6

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
aggregation morphology which is less ordered.
The peak corresponding to tryptophan which shifted from 1358 to 1336
cmꢀ 1 for the uninhibited HEWL from day 0 to day 7, disappeared over
time for the HEWL incubated with 4e. This indicates that the microen-
vironments surrounding tryptophan in the two cases are significantly
different hence this observation supports the suggestion that 4e influ-
ence HEWL to form alternate aggregates as seen in the TEM analysis.
2.5. Raman spectroscopic studies
From the primary screening of the synthesized compounds, the three
nitro-Sox compounds having the most promising anti-amyloidogenic
activities, 4b, 4d and 4e were subjected to Raman spectroscopic
studies to understand the effect of these inhibitor molecules on the
conformational transitions of the HEWL protein secondary structure
2.6. Molecular docking studies
during fibrillation (Fig. 6 and ESI, S7). In previous studies,
α-helical
Additionally, molecular docking tools were used to predict the
orientation of the selected nitro-Sox compounds 4b, 4d, and 4e within
the constraints of the protein binding pockets. These studies helped to
understand the types and the strength of interactions between the in-
hibitor compounds and HEWL amino acids. Binding interactions of the
nitro-Sox compounds with amino acid residues 57–107 are significant as
they are in the highly amyloidogenic regions of the lysozyme [61]. The
docking studies revealed that these compounds fit well within the sur-
face pocket of HEWL (PDB ID – 6LYZ) (Fig. 7 and ESI, S8). Nitro-Sox 4e
had the highest binding affinity of ꢀ 8.2 kcal/mol while 4b and 4d ob-
tained docking scores of ꢀ 6.1 and ꢀ 7.5 kcal/mol, respectively. Two of
the reference compounds EGCG and RIN were also subjected to molec-
ular docking studies to predict the binding affinities of the two com-
pounds. EGCG had the highest binding affinity of ꢀ 8.8 kcal/mol while
RIN had a predicted affinity of ꢀ 6.8 kcal/mol. Two of the synthesized
inhibitor compounds, 4d and 4e had better affinities than the reference
alkaloid.
domains of proteins contribute to amide I and III bands at ~1645–1655
cmꢀ 1 and ~1260–1340 cmꢀ 1, respectively, while those of β-sheets are
present at ~1665–1680 cmꢀ 1 and ~1230–1245 cmꢀ 1, respectively
[57,58]. However, for native HEWL in this study, the Raman spectrum
on day 0 (Fig. 6, black trace) contained prominent bands in the amide I
and amide III regions at 1670 cmꢀ 1 and 1236 cmꢀ 1, respectively, even
before the protein was introduced to denaturing conditions. These
amide band positions are assigned to β-sheet structure rather than
α
-helix [58].
These unusual peak positions for native HEWL could be attributed to
the presence of DMSO. When the HEWL underwent fibrillation and
reached a maximum fibril formation (Fig. 6, grey trace), the Raman peak
position and shape in the amide I region did not change. The preser-
vation of the sharp peak shape is indicative of the existence of β-sheet
structure stabilized with H-bonds. As for the amide III region, the peak at
1236 cmꢀ 1 on day 0 did not change its position but broadened in shape.
Additionally, an intense peak at 1358 cmꢀ 1 appeared on day 0, dis-
played a downward shift to 1336 cmꢀ 1 on day 7 with a decrease in in-
tensity. This peak is assigned to the tryptophan residue and its intensity
is a marker of hydrophobicity around the amino acid, and consequently
of changes in protein structure, upon undergoing denaturation [59,60].
For the HEWL incubated with inhibitor compound 4e, the prominent
peak in the amide I region at 1670 cmꢀ 1 on day 0 did not change in peak
position over time but dramatically lost the sharp profile on day 7
(Fig. 6, red trace). This peak broadening suggests that with 4e, the
HEWL does not form highly ordered classical β-sheets but directs the
fibrillation towards an alternate aggregate state which is less ordered.
Following the promising binding affinities identified by the inhibitor
molecules, post-docking binding interaction studies were performed to
identify the binding sites as well as the types of binding interactions
between the inhibitor molecules and HEWL (Fig. 7 and ESI, Fig. S8.1).
Both nitro-Sox compounds, 4d and 4e show interactions with the amino
acid residues via the nitro substituent of the pyrrolizidine moiety,
further confirming the effectiveness of incorporating a nitro group into
the Sox structures. All three compounds displayed binding interactions
with amino acid residues, especially between 57 and 107 which indicate
that these compounds potentially affect the protein aggregation by
interacting with amino acid residues within the highly amyloidogenic
Fig. 6. Comparison of Raman spectra in the region of 800–2000 cmꢀ 1 of native HEWL on day 0 (black trace), and HEWL acidified to pH 2 and incubated at 70 ◦
C
without (grey trace) and with (red trace) the presence of Sox inhibitor compound 4e on day 7. All three analytes contain 10% DMSO. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
7

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
Fig. 7. Surface binding diagram of 4e (A). Post-docking 2D interaction representation for 4b (B), 4d (C), 4e (D), EGCG (E) and RIN (F) with 6LYZ. Interacting HEWL
amino acids are animated as bubbles.
region of HEWL, confirming the observation from the ThT fluorescence
assay studies.
an alternate form which is less ordered than β-sheets. The Soxs do not
significantly display cell toxicity, moreover 4b protects HEK cells
against Aβ-induced toxicity. We believe that nitro-Soxs are promising
bioactive lead molecules for activity against amyloid misfolding and AD
prevention. Further extension of the substrate scope and biophysical
analysis are currently in progress in our lab.
3. Conclusion
In conclusion, a rapid synthesis of nitro-Sox compounds using mi-
crowave irradiation are presented. The compounds have excellent
binding affinities towards highly amyloidogenic areas of HEWL, and
thus inhibit aggregation of HEWL fibrils and direct fibrillation towards
8

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
4. Experimental
δ177.8, 160.2, 158.9, 143.4, 130.0, 128.7, 127.9, 125.0, 121.2, 113.7,
109.9, 105.5, 98.8, 92.6, 74.7, 63.6, 55.7, 55.5, 51.2, 42.9, 27.9, 25.5;
MS (ESI): m/z 410 [M + H]+. HRMS (ESI) calculated for C22H23N3O5 [M
+ H]+ 410.1716; obtained: 410.1762.
4.1. Chemical synthesis
4.1.1. General experimental details
2′-(2,5-dimethoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro
[indoline-3,3′-pyrrolizin]-2-one (4c)
The starting materials and reagents were purchased from Sigma
Aldrich, Merck, AK Scientific, Alfa Aeser and were used without further
purification. Reactions were monitored by TLC analysis using Merck
pre-coated thin Al plates coated with silica gel and F254 indicator and
visualized by the aid of UV light at a wavelength of 254 nm. Melting
points were determined on a digital electrothermal melting point
apparatus (Stuart MP 10) and are uncorrected. Ramp rates were set at
20 ◦C per minute to plateau and 2 ◦C per minute to melt. Nuclear
Magnetic Resonance spectra (1H, and 13C) were recorded on a 300 MHz
(Bruker) NMR spectrometer, using TMS as an internal reference. The
chemical shifts values are expressed in parts per million (ppm) relative
to DMSO and coupling constants (J) in Hz. Splitting patterns of multi-
plicities are designated as s, singlet; d, doublet; t, triplet; q, quartet; dd,
doublet of doublets; m, multiplets. High-resolution mass spectrometric
data were obtained by using a LC-TOF mass spectrometer (150 eV). Low-
resolution mass spectrometric data were obtained on a Bruker GC-TOF
mass spectrometer.
The NMR and mass spectra are consistent with the reported data
from Rajesh et al. 2011 [29]. White solid, Yield 39%, Melting point:
217–219 ◦C, 1H NMR (300 MHz, DMSO d6): δ10.34 (s, 1H), 7.62 (d, J =
7.5 Hz, 1H), 7.20 (t, J = 7.5 Hz, 1H), 7.01–6.96 (m, 2H), 6.72–6.69 (m,
3H), 6.64 (d, J = 7.5 Hz, 1H), 6.24 (dd, J = 10.5 Hz, 1H), 5.16 (d, J =
10.5 Hz, 1H), 4.70–4.61 (m Hz, 1H), 3.62 (s, 3H), 3.47 (s, 3H),
3.27–3.24 (m, 1H), 2.66–2.61 (m, 1H), 2.08–1.89 (m, 2H), 1.70–1.61
(m, 1H), 1.40–1.25 (m, 1H); MS (ESI): m/z 410 [M + H] + . MS (ESI)
calculated for C22H23N3O5 [M + H]+ 410.2; obtained: 410.2.
2′-(2,6-dimethoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro
[indoline-3,3′-pyrrolizin]-2-one (4d)
1
◦
White solid, yield 48%, melting point: 197–199 C, H NMR (300
MHz, DMSO d6): δ10.09 (s, 1H), 7.55 (d, J = 7.5 Hz, 1H), 7.13–7.01 (m,
2H), 6.95 (t, J = 7.5 Hz, 1H), 6.65–6.56 (m, 2H), 6.45–6.42 (m, 2H),
5.23 (d, J = 9.9 Hz, 1H), 4.76 (q, J = 8.1,8.1,8.4 Hz, 1H), 3.56 (s, 6H),
3.18 (q, J = 8.7,6.3,8.7 Hz, 1H), 2.69 (t, J = 6.9,7.2 Hz, 1H), 2.01–1.71
(m, 3H), 1.33–1.30 (m, 1H); 13C NMR (75 MHz, DMSO d6); δ 178.2,
146.4, 145.4, 142.2, 139.2, 126.2, 125.3, 123.9, 122.5, 121.25, 114.5,
110.5, 110.4, 91.4, 75.2, 64.1, 55.5, 53.1, 51.2, 27.9, 25.7; MS (ESI): m/
z 410 [M + H]+. HRMS (ESI) calculated for C22H23N3O5 [M + H]+
410.1716; obtained: 410.1757.
4.1.2. General procedure for the synthesis of substituted β-nitrostyrenes,
3a-3h
β-Nitrostyrenes were synthesized using known procedures [62].
Substituted aldehyde (1.0 mmol), nitro methane (1.5 mmol) and
ammonium acetate (0.8 mmol) were reacted using a Discover® CEM
microwave synthesizer (200 W, 20 psi, 30 s ramp time) at 90 ◦C for 45
min. Sealed microwave vessels (10 mL) were used in microwave-assisted
organic synthesis (MAOS). After completion of the reaction, the reaction
mixture was cooled down and the formed crystals were filtered, washed
with methanol and recrystallized using methanol to give the β-nitro-
styrene derivatives 3a-3h.
2′-(3,4-dimethoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro
[indoline-3,3′-pyrrolizin]-2-one (4e)
The NMR and mass spectra are consistent with the reported data
from Rajesh et al. 2011 [29]. White solid, Yield 78%, Melting point:
173–175 ◦C, 1H NMR (300 MHz, DMSO d6): δ10.25 (s, 1H), 7.88 (d, J =
7.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.76–6.60
(m, 4H), 6.37 (dd, J = 10.5 Hz, 1H), 4.65–4.56 (m, 1H), 4.47 (d, J =
10.5 Hz, 1H), 3.64 (s, 3H), 3.48 (s, 3H), 2.65–2.60 (m, 1H), 2.10–1.71
(m, 4H); MS (ESI): m/z 410 [M + H]+. MS (ESI) calculated for
4.1.3. General procedure for the synthesis of nitro-aromatic spirooxindoles,
4a-4h
C
22H23N3O5 [M + H]+ 410.2; obtained: 410.2.
A mixture of isatin (1.0 mmol), L-proline (1.0 mmol) and aryl
β-nitrostyrene (1.0 mmol) dissolved in 2 mL of methanol, were irradi-
ated under microwave irradiation using a Discover® CEM microwave
synthesizer (200 W, 20 psi, 30 s ramp time) at 65 ◦C for 10 min. Sealed
microwave vessels (10 mL) were used in MAOS. After completion of the
reaction, the crude product formed was filtered and washed with
methanol. The obtained product was dried overnight under vacuum and
washed with diethyl ether and filtered to obtain the nitro-aromatic
spirooxindoles 4a-4h.
2′-(4-hydroxy-3-methoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahy-
drospiro[indoline-3,3′-pyrrolizin]-2-one (4f)
1
◦
White solid, yield 68%, melting point: 119–121 C, H NMR (300
MHz, DMSO d6): δ 10.24 (s, 1H), 8.90 (s, 1H), 7.85 (d, J = 7.5 Hz, 1H),
7.24 (t, J = 7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.6 (d, J = 7.5 Hz, 1H),
6.58–6.52 (m, 3H), 6.32 (dd, J = 10.5 Hz, 1H), 4.64–4.56 (m Hz, 1H),
4.43 (d, J = 10.5 Hz, 1H), 3.49 (s, 3H), 3.28–3.26 (m, 1H), 2.62 (t, J =
6.9 Hz, 1H), 2.05–1.90 (m, 2H), 1.69–1.60 (m, 1H), 1.42–1.32 (m, 1H);
13C NMR (75 MHz, CDCl3); δ 178.2, 146.4, 145.4, 142.2, 139.2, 126.2,
125.3, 123.9, 122.5, 121.25, 114.5, 110.5, 110.4, 91.4, 75.2, 64.1, 55.5,
53.1, 51.2, 27.9, 25.7; MS (ESI): m/z 396 [M + H]+. HRMS (ESI)
calculated for C21H21N3O5 [M + H]+ 396.1560; obtained: 396.1601
2′-(4-chlorophenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indo-
line-3,3′-pyrrolizin]-2-one (4g)
2′-(4-methoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’
hexahydrospiro
[indoline-3,3′-pyrrolizin]-2-one (4a)
The NMR and mass spectra are consistent with the reported data
from Rajesh et al. 2011 [29]. White solid, yield 31%., Melting point:
1
◦
221–223 C, H NMR (300 MHz, DMSO d6): δ 10.26 (s, 1H), 8.90 (s,
1H), 7.85 (d, J = 7.5 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz,
1H), 6.66 (d, J = 7.5 Hz, 1H), 6.59–6.52 (m, 3H), 6.32 (dd, J = 10.5 Hz,
1H), 4.64–4.56 (m, 1H), 4.44 (d, J = 10.5 Hz, 1H), 3.49 (s, 3H),
3.42–3.35 (m, 2H), 2.65–2.60 (m, 1H), 2.05–1.90 (m, 2H), 1.69–1.63
(m, 1H), 1.40–1.33 (m, 1H); MS (ESI): m/z 380 [M + H]+. MS (ESI)
calculated for C21H21N3O4 [M + H]+ 380.2; obtained: 380.2.
2′-(2,4-dimethoxyphenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro
[indoline-3,3′-pyrrolizin]-2-one (4b)
The NMR and mass spectra are consistent with the reported data
from Rajesh et al. 2011 [29]. White solid, Yield 62%, Melting point:
126–128 ◦C, 1H NMR (300 MHz, DMSO d6): δ10.30 (s, 1H), 7.89 (d, J =
7.5 Hz, 1H), 7.25–7.16 (m, 5H), 7.05 (t = 7.5 Hz, 1H), 6.66 (d, J = 7.5
Hz, 1H), 6.41 (dd, J = 10.5 Hz, 1H), 4.65–4.5 (m, 2H), 2.63–2.59 (m,
1H), 2.06–1.32 (m, 4H); MS (ESI): m/z 384 [M + H]+. MS (ESI) calcu-
lated for C20H18ClN3O3 [M + H]+ 384.1; obtained: 384.1.
2′-(4-bromophenyl)-1′-nitro-1′,2′,5′,6′,7′,7a’-hexahydrospiro[indo-
line-3,3′-pyrrolizin]-2-one (4h)
1
◦
White solid, yield 65%, melting point: 216–218 C, H NMR (300
MHz, DMSO d6): δ10.27 (s,1H), 7.62 (d, J = 7.5 Hz, 2H), 7.30 (d, J =
8.7 Hz), 7.20 (t, J = 7.8 Hz, 1H), 7.00 (t, J = 7.8 Hz, 1H), 6.63 (d, J = 7.5
Hz, 1H), 6.42 (dd, J = 8.7 Hz, 1H), 6.30–6.29 (m, 1H), 6.22 (dd, J =
10.5 Hz, 1H), 5.11 (d, J = 10.5 Hz, 1H), 4.67–4.59 (m, 1H), 3.66 (s, 3H),
3.50 (s, 3H), 3.26–3.21 (m, 1H), 2.65–2.61 (m, 1H), 2.04–1.97 (m, 2H),
1.69–1.61 (m, 1H), 1.37–1.33 (m, 1H); 13C NMR (75 MHz, DMSO d6):
The NMR and mass spectra are consistent with the reported data
from Rajesh et al. 2011 [29]. White solid, yield 87%, melting point:
208–210 ◦C, 1H NMR (300 MHz, DMSO d6): δ10.31 (s, 1H), 7.89 (d, J =
7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 2H), 7.24 (t, J = 7.5 Hz, 1H), 7.11–7.09
(m, 2H), 7.05 (t, J = 7.5 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 6.41 (dd, J =
10.5 Hz, 1H), 4.65–4.54 (m, 2H), 2.60–2.56 (m, 1H), 2.62–1.39 (m, 4H);
9

N.H. de Silva et al.
Bioorganic Chemistry 114 (2021) 105128
MS (ESI): m/z 428 [M + H]+. MS (ESI) calculated for C20H18BrN3O3 [M
+ H]+ 428.06; obtained: 428.06.
org/10.1016/j.bioorg.2021.105128.
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Declaration of Competing Interest
The authors declare that they have no known competing financial
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AD: Alzheimer’s disease
Kappa CCD diffractometer, ϕ and
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Aβ: amyloid-beta
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APP: amyloid precursor proteins
FDA: food and drug administration
AchE: acetylcholinesterase
NMDA: N-methyl-D-aspartate
Sox: spirooxindole
ThT: thioflavin T
BACE1: β-secretase 1
RIN: rhynchophylline
IRN: isorhynchophylline
Amy: azomethine ylid
NMR: Nuclear magnetic resonance
COSY: homonuclear correlation spectroscopy
CCDC: Cambridge Crystallographic Data Centre
TPSA: topological polar surface area
BBB: blood brain barrier
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HEWL: hen egg white lysozyme
EGCG: epigallocatechingallate
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
HEK: human Embryonic Kidney
TEM: Transmission Electron Microscopy
DMSO: dimethylsulphoxide
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fibrils: localisation and implications, J. Struct. Biol. 149 (2005) 30–37.
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PDB: protein data bank
11