Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-based compounds for inhibiting α-synuclein in transgenic C. elegans model of Synucleinopathy

Article abstract of DOI:10.1016/j.bmc.2020.115640

Parkinson's disease (PD), a calamitous neurodegenerative disorder with no cure till date, is closely allied with the misfolding and aggregation of α-Synuclein (α -Syn). Inhibition of α-Syn aggregation is one of the optimistic approaches for the treatment for PD. Here, we carried out hypothesis-driven studies towards synthesising a series of pyrazolo-pyridine carboxylate containing compounds (7a–7m) targeted at reducing deleterious α-Syn aggregation. The target compounds were synthesized through multi-step organic synthesis reactions. From docking studies, compounds 7b, 7g and 7i displayed better interaction with the key residues of α-Syn with values: ?6.8, ?8.9 and ?7.2 Kcal/mol, respectively. In vivo transgenic C. elegans model of Synucleinopathy was used to evaluate the ability of the designed and synthesized compounds to inhibit α-Syn aggregation. These lead compounds 7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-Syn with respect to the control. Further, the strategy of employing pyrazolo-pyridine-based compounds worked with success and these scaffolds could be further modified and validated for betterment of endpoints associated with PD.

Full text of DOI:10.1016/j.bmc.2020.115640

Journal Pre-proofs
Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-
based compounds for inhibiting α-synuclein in Transgenic C. elegans Model
of Synucleinopathy
Mudasir Maqbool, Roshani Rajvansh, Kottapalli Srividya, Nasimul Hoda
PII:
DOI:
Reference:
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https://doi.org/10.1016/j.bmc.2020.115640
BMC 115640
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Bioorganic & Medicinal Chemistry
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30 June 2020
4 July 2020
Please cite this article as: Maqbool, M., Rajvansh, R., Srividya, K., Hoda, N., Deciphering the robustness of
pyrazolo-pyridine carboxylate core structure-based compounds for inhibiting α-synuclein in Transgenic C.
elegans Model of Synucleinopathy, Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
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Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-based
compounds for inhibiting α-synuclein in Transgenic C. elegans Model of
Synucleinopathy
Mudasir Maqbool1, Roshani Rajvansh2, Kottapalli Srividya2, Nasimul Hoda1*
1Drug Design and Synthesis Lab., Department of Chemistry, Jamia Millia Islamia, Jamia
Nagar, New Delhi-110025, India.
2Laboratory of Functional Genomics and Molecular Toxicology, CSIR-Central Drug
Research Institute, (CSIR-CDRI), Jankipuram Extension, Sitapur Road, Lucknow-226031,
India.
Corresponding author
Nasimul Hoda, PhD
Professor
Department of Chemistry,
Jamia Millia Islamia,
Jamia Nagar, New Delhi-110025, India.
Email: nhoda@jmi.ac.in

Abstract
Parkinson’s disease (PD), a calamitous neurodegenerative disorder with no cure till date, is
closely allied with the misfolding and aggregation of α-Synuclein (α -Syn). Inhibition of α-
Syn aggregation is one of the optimistic approaches for the treatment for PD. Here, we
carried out hypothesis-driven studies towards synthesising a series of pyrazolo-pyridine
carboxylate containing compounds (7a-7m) targeted at reducing deleterious α-synuclein
aggregation. The target compounds were synthesized through multi-step organic synthesis
reactions. From docking studies, compounds 7b, 7g and 7i displayed better interaction with
the key residues of α-Syn with values: -6.8, -8.9 and -7.2 Kcal/mol, respectively. In vivo
transgenic C. elegans model of Synucleinopathy was used to evaluate the ability of the
designed and synthesized compounds to inhibit α-Syn aggregation. These lead compounds
7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-Syn with respect to the control.
Further, the strategy of employing pyrazolo-pyridine-based compounds worked with success
and these scaffolds could be further modified and validated for betterment of endpoints
associated with PD.
Keywords: Parkinson’s disease, synucleinopathy, medicinal chemistry, drug development,
pyrazolo-pyridine, α-synuclein, C. elegans.

Graphical abstract

1. Introduction
Parkinson’s disease (PD) is the second-largest death causing neurodegenerative disorder
after Alzheimer’s disease (AD) and most common Synucleinopathy, still lacking a complete
cure. PD includes aggregation of α-synuclein (α-Syn), a 140-residue protein, known for
Lewy bodies and Lewy neurites, and the massive loss of midbrain dopamine neurons in the
substantia nigra pars compacta resulting in bradykinesia, tremor, and postural instability.1–3
Aggregates of α-Syn can spread the disease through self-amplification, propagation and
cell-to-cell transmission.4–6 The process initiates from misfolding of the naturally unfolded
monomers, giving birth to oligomers and ultimately the formation of fibrils, via
intermolecular interactions that are primarily hydrophobic and/or H-bonding.7 α-Syn
oligomerization and amyloid fibrillation are believed to be due to mutations of A30P, E46K
or A53T or the overexpression of wild-type (wt) α-Syn. Protofibrils or oligomers produced
during the initial phases of α-synuclein fibrillization process are the major cytotoxic species
responsible for neuronal cell death, however the mature fibrils, such as Lewy bodies are less
toxic for the cells. Therefore, the inhibition of protein oligomerization is an optimistic
approach for the therapeutics of neurodegenerative diseases. In vitro treatment by silica
nanoparticles (SiNPs) have been found to eliminate the formation of toxic oligomeric
intermediates in amyloid fibrillogenesis.8 Secretagogin, a secreted calcium sensor has been
found to prevent α-Syn fibrillation.9
Majority of the therapies intend to increase the dopamine level in the brain. An approach
of inhibiting α-Syn aggregation has also been suggested as a hopeful treatment for PD.10
However, the etiology and pathomechanism of PD is very multifaceted and not copiously
understood. Drugs available in the market (Figure 1) are only able to minimize the
symptoms. Consequently, there is a dire need for novel methods of treatment and prevention
of PD.
Figure 1. Structures of some available anti-PD drugs.

Pyrazolopyridine compounds like etazolate are famous for the non-sedative
anxiolytic-like properties.11 Etazolate is reported to modulate GABA (A) receptors through
12,13
the inhibition of adenosine receptors
and proved to be a capable agent for AD
therapy.14,15 Recently, A series of pyrazolo[1,5-a]pyridine-3-carboxamide derivatives was
reported as a new antituberculosis agents with slightest cytotoxicity against Vero cells.16
One more similar type of series of dual-target phosphodiesterase 9 and histone deacetylase
inhibitors was reported as safe and efficient pharmacological tool compounds for evaluating
the insinuations of these two drug targets in Alzheimer’s disease.17
Here we designed and synthesized a series of pyrazolo-pyridine-carboxylate
comprising compounds as persuasive α-Syn disaggregating agents for the treatment of PD.
The motivation for this work was the known heterocyclic systems having very similar size,
shape and intermolecular force of attraction necessary for α-synuclein disruption and
strongly and selectively protecting diverse cell types from α-synuclein toxicity (Figure 2).18
Dihydropyrazolo pyrimidine-carboxylic acid based compounds not only significantly
inhibits the misfolding and aggregation of α-synuclein and protects neuroblastoma cells
from α-synuclein toxicity.19 The present work is in continuation to our previously reported
optimistic results of the pyrazolo-pyridine moiety for inhibiting 81.65% of self-induced
amyloid-β aggregation.20 Pyrrolopyridine and pyrazolopyridine containing compounds are
positive allosteric modulators (PAM) of the M1 receptor. These compounds may be useful
for the treatment of diseases mediated by the M1 receptor such as Alzheimer’s disease,
cognitive impairment, schizophrenia, pain, and sleep disorders.21
Figure 2. Figure explaining the rationale for using pyrazolo-pyridine-carboxylate-based
compounds for the inhibition of α-syn aggregation.
To evaluate the effect of these compounds on PD, we used transgenic C. elegans
model. The transparent anatomy of the nematode made the study of aggregation of the

neural protein α-Syn convenient.22 The easy availability of C. elegans for us and its 60-80%
of genetic homology to humans made us to choose this model of PD for the screening of our
synthesized molecules.23 To obtain a better understanding of possible interactions with
biological targets, molecular docking studies were also performed.
2.
Results and discussion
2.1. Synthesis of the target compounds (7a–7m) from differently substituted
phenylhydrazines.
The target molecules were synthesized via multiple-step reactions as depicted in
Scheme 1. Differently substituted aromatic amines (1a–1d) were converted into substituted
phenylhydrazines (2a–2d). The substituted phenylhydrazines were coupled with ethyl
(ethoxymethylene)cyanoacetate in ethanol to obtain phenyl-pyrazole amino carboxylates
(3a–3d). In the next step the phenyl-pyrazole amino carboxylate was decarboxylated to get
4a–4d. The phenyl-pyrazole carboxylates (4a-4d) were coupled with diethyl 2-
◦
(propoxymethylene)malonate in ethanol and under reflux at 80 C for 24 h to attain 5a–5d
which were further treated with phosphorus oxychloride to get chloro-phenyl-pyrazolo-
pyridine-carboxylate (6a–6d) as reported in the literature.24,25 The chloro-compounds (6a–
6d) were treated with different nucleophiles in presence of 1,4-dioxane and K2CO3 under
reflux to obtain the target molecules (7a–7m) as per our previously reported work.26,27
Column chromatography technique was used for the purification of all the molecules and
1H-NMR, 13C-NMR, ESI MS and elemental analysis for characterisation purposes.

Scheme 1: Synthesis of the target compounds (7a–7m)
◦
Reagents and conditions: (i) HCl, H2O, NaOH, DCM (ii) EtOH, 12 h, reflux 80 C (iii)
H3PO4, reflux 170 ◦C, 52 h (iv) EtOH, 12 h, reflux 80 ◦C (v) POCl3, 12 h, reflux 110 ◦C (vi)
R2, 1,4-dioxane, 24 h, reflux 110 ◦C.
Compound Substrate
R2
2-(piperazin-1-yl)nicotinonitrile
7-chloro-4-(piperazin-1-yl)quinoline
5-methyl-7-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidine
2-(piperazin-1-yl)nicotinonitrile
2-(piperazin-1-yl)nicotinonitrile
2-(piperazin-1-yl)nicotinonitrile
1-methylpiperazine
6a
6a
6a
6b
6c
6d
6a
6a
6a
6a
6a
6a
6a
7a
7b
7c
7d
7e
7f
7g
7h
7i
morpholine
piperidine
N-(4-(piperazin-1-ylsulfonyl)phenyl)acetamide
4-(piperazin-1-yl)quinolin-2(1H)-one
quinolin-8-ol
7j
7k
7l
7-(piperazin-1-yl)-5-(trifluoromethyl)-[1,2,4]triazolo[1,5-
a]pyrimidine
7m

2.2.Pyrazolo-pyridine carboxylate-based compounds decrease the α-Syn content in
transgenic C. elegans
Synucleinopathy is the characteristic feature of PD associated with progressive loss
of structure and function of dopamine producing neurons. We carried out hypothesis driven
studies towards synthesising a series of pyrazolo-pyridine containing compounds (7a-7m)
targeted at reducing deleterious α-Syn aggregation moieties. We tested a 1% working
concentration of the test compounds in transgenic model of C. elegans NL5901 (expressing
human α-Syn). Screening of these compounds revealed that, there is significant decrease in
α-Syn expression levels upon treatment with these compounds (Figure 3).
Figure 3. Effect of 13 test compounds (7a–7m) on α-Synuclein aggregation: α- Synuclein
aggregation was measured in transgenic strain of C. elegans, NL5901[unc-54p::alpha
synuclein::YFP + unc-119(+)] All images were taken on the same exposure time and gain
settings .Code for the compounds are as follows - (a) 7a. (b) 7b. (c) 7c. (d) 7d. (e) 7e. (f) 7f.
(g) 7g. (h) 7h. (i) 7i. (j) 7j. (k) 7k. (l) 7l. (m) 7m.
Experiments were carried out by two independent investigators and the average percent
inhibition for each compound was calculated. It was observed that compounds 7a–7m led to
inhibition of α-Syn aggregation by 33.15%, 39.25%, 31.88%, 27.32%, 26.06%, 13.52%,
52.96%, 30.21%, 47.66%, 17.71%, 14.66%, 24.45%, and 14.10% respectively, with respect
to that of control group (Figure 4). We conclude that three compounds, 7b, 7g and 7i,
leading to marked decrease in aggregation (more than 30% inhibition) make promising
candidates for further mechanistic studies. These lead compounds displayed 1.7, 2.4 and
1.5-fold inhibition of α-Syn with respect to the control. Further, the strategy of employing

pyrazolo-pyridine containing compounds worked with success and these scaffolds could be
further modified and validated for betterment of endpoints associated with PD.
Figure 4. Graphical representation of transgenic strain of C. elegans [NL5901unc
54p::alpha synuclein::YFP + unc-119(+)].(a) Analysis of α-Syn aggregation upon
treatment with compounds 7a–7m with respect to control. (b) Mean percentage inhibition of
α-Syn by the test compounds with respect to control. Analysis was done using unpaired
Students T-test using *p<0.05,**p<0.01,***p<0.0001,ns-Non significant.
2.3.Design of pyrazolo-pyridine-carboxylate based α-Syn inhibitors
The molecular mechanism for the inhibition of α-synuclein by our synthesized
compounds is unknown, however, the docking studies predict the most possible way of the
interactions between the ligands and the protein. The amino acid residues of α-Syn like
Tyr125, Tyr133, and Tyr136 present in the hydrophilic tail of the C-terminus of α-Syn play
a crucial role in fibrilization process.28,29 The C-terminal region also has an essential part in
the interaction of protofibrils required for maturation of fibrils.30 Autoinhibition of α-Syn by
the interaction of C-terminus region with the NAC region has been found to prevent
fibrillation under normal conditions.31,32 Previously, the binding of ceftriaxone to bovine
serum albumin (BSA) has been reported to increase the polarity and hydrophilicity of the C-
terminal of the protein, thereby, increasing the compactness of monomeric α-Syn and
inhibited its fibril formation.31,33 Consequently, binding of a putative ligand to the C-
terminal region should affect the aggregation of α-Syn. Even though the protein topology
reveals it in a micelle bound state, the unfolded hydrophilic tail (Asp98-Ala140) can be well
suited as a docking target.34
Choice for selecting pyrazolo-pyridine-carboxylate core moiety was due to its bicyclic
aromatic system and an aliphatic ester facilitating non-covalent interactions like π-π

stacking interaction and H-bonding with the essential residues of the target protein,
respectively. AutoDock Vina version 1.5.6 was used for flexible docking and redocking.
During docking process, the simulation boxes were prepared large enough to cover the
interaction residues of the protein and the ligand.35 The docking results provided the
approximate binding affinity
(-ΔG in Kcal/mol) between the ligands 7a-7m and α-Syn (PDB ID 1XQ8) mentioned in
Table 1. It was found that the alkyl or aralkyl groups with varying hydrophobicity and
electronic properties decided the binding affinity of the ligands to the drug target. The
Binding free energy of three best compounds 7b, 7g and 7i with α-Syn was found to be -6.8,
-8.9 and -7.2 Kcal/mol, respectively, suggesting a very high binding affinity between the
ligands and the enzyme. PyMOL viewer was used for the analysis of the docked structures.
From the interpretation of the docking results as shown in Figure 5, we came to know that,
the binding interactions of the compounds 7b, 7g and 7i display a strong H-bond between
the carbonyl oxygen of the carboxylate group and one of the hydrogens of Gly132. In
addition, π-π stacking interactions was observed between the aromatic system attached to
pyrazolo pyridine and Tyr136. These theoretic results were also validated with in vivo α-Syn
inhibition assay and showed full agreement; hence such molecules could be used as lead to
develop potent α-Syn inhibitors.
Table 1: The calculated free energies of selected derivatives in molecular docking using
Auto Dock Vina.35

Bolded values represent the most active compounds 7b, 7g and 7i.
Compound name
Binding affinity (kcal/mol)
7a
7b
7c
7d
7e
7f
-6.2
-6.8
-6.2
-6.1
-6.1
-5.9
-8.9
-5.6
-7.2
-6.1
-6.6
-5.9
-6.3
7g
7h
7i
(a)
Figure 5.
7j
Binding
7k
7l
7m
interactions of compound 7b with α-Syn (PDB ID 1XQ8). (b) Interaction between α-Syn and

compound 7g. (c) compound 7i showing H-bonding and π-π interactions with the key
residues of α-Syn. (d) showing ligand binding site and position of tyrosine triad in α-Syn.
Tyr is shown in magentas, Glu in cyan, Gly in red, Pro in green, Met in yellow and Ser in
blue colours. H-bonding and π-π interactions are shown with yellow and red coloured
dotted lines respectively.
3. Conclusion
Our hypothesis for designing and synthesizing a series of pyrazolo-pyridine containing
compounds (7a-7m) as α-Syn aggregation inhibitors proved to be positive by means of
theoretical and in vivo studies. Compounds 7b, 7g and 7i showed computation binding
affinity with α-Syn as -6.8, -8.9 and -7.2 kcal/mol, respectively. The test compounds were
examined for their α-Syn inhibiting effect on transgenic model of C. elegans NL5901
(expressing human α-Syn). The computationally verified lead compounds evidenced to be
good in α-Syn aggregation inhibition in C. elegans. It was observed that compounds 7b, 7g
and 7i led to inhibition of α-Syn aggregation by 39.25%, 52.96% and 47.66%, respectively
which implies 1.7, 2.4 and 1.5-fold with respect to the control. Overall, the approach of
engaging pyrazolo-pyridine-based compounds for α-Syn inhibition worked with success and
these scaffolds could be further modified and corroborated for furtherance of endpoints
associated with PD.

4. Experimental
4.1.Chemistry
All reagents and solvents were commercially available and used as purchased without
further purification. Melting points (mp) were determined on a Stuart Scientific SMP1
apparatus and are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Bruker 400 MHz FT spectrometer. Proton chemical shifts are reported in ppm
(TMS, δ 0.00) or with the solvent reference relative to TMS employed as the internal
standard (CDCl3, δ 7.26; DMSO-d6 δ 2.54) and multiplicities of NMR signals are designated
as s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), br (broad), m
(multiplet, for unresolved lines). LCMS of the compounds were carried out on applied
biosystem (absciex 2000 triple quad). Column chromatography was performed on columns
packed with alumina from Merck (70−230 mesh). Aluminum oxide thin-layer
chromatography (TLC) cards from Merck (aluminum oxide-precoated aluminum cards with
fluorescent indicator detectable at 254 nm) were used for TLC. Developed plates were
visualized by a Spectroline ENF 260C/FE UV apparatus. Organic solutions were dried over
anhydrous sodium sulfate. Evaporation of the solvents was carried out on a chi rotavapor
R-100 e uipped with a chi -100 vacuum controller. Elemental analyses were obtained
in an Elementar Vario analyser. Elemental analyses of the compounds were found to be
within ±0.4% of the theoretical values. The purity of tested compounds was >95%.
4.1.1. General procedure for the synthesis of substituted phenylhydrazine (2a-2d)
2.7 g (24.29 mmol) of substituted phenylamine was suspended in 10 mL of
37 % aqueous HCl at 0 °C and was carefully treated with 1.7 g (2.75 mmol, 1.05 equivalent)
of sodium nitrite dissolved in 20 mL of ice-cold water. The solution was stirred for 1 h at 0
°C and 30 min at rt. Then, 27 g (0.12 mol, 5 equivalent) of tin (II) chloride in 30 mL HCl
were dropped carefully into the cooled solution (0 °C) and stirred for 6 h. The reaction was
neutralized and basified with aqueous NaOH (pH 14). The aqueous phase was extracted
three times with dichloromethane. The combined organic phase was washed with brine,
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The product obtained was
used for the next reaction step without purification.
4.1.2. General procedure for the synthesis of phenyl-pyrazole amino carboxylate (3a–
3d)25
2.5 g (23.1 mol) of substituted phenylhydrazine in 20 mL of ethanol was stirred under
reflux conditions for
2
h, followed by the dropwise addition of ethyl

(ethoxymethylene)cyanoacetate (3 g, 17.73 mol) dissolved in ethanol. The reaction mixture
was refluxed for 24 h. On the completion of the reaction, the reaction mixture was poured
into ice-cold water. The light-yellow coloured precipitate obtained was washed with cold
water, dried and produced for further reactions without purification.
4.1.3. General procedure for the synthesis of phenyl-pyrazole carboxylate
(4a–4d)25
A mixture of compounds 3a–d, 1 g (4.3 mol) and 15 mL of 85% phosphoric acid was
◦
refluxed at 170 C for 36 h. The reaction mixture was poured into ice water and neutralised
to pH 7 with aqueous NaOH solution. The organic phase was extracted with
dichloromethane (3 x 50 mL), dried over anhydrous Na2SO4, concentrated in vacuo to
obtain compounds 4a–4d as brownish oily liquid.
4.1.4. General procedure for the synthesis of phenyl-pyrazol-amino methylene
malonate (5a–5d)25
2.3 g (14.4 mol) of compounds 4a–4d were dissolved in 20 mL of ethanol followed by
the dropwise addition of 3.1 g (14.3 mol) of diethyl 2-(propoxymethylene)malonate in
◦
ethanol. The reaction mixture was allowed to stir and reflux at 80 C for 24 h. The reaction
mixture was poured into 50 mL of ice-cold water. The precipitate obtained was washed with
cold water, dried and produced for further reactions without purification.
4.1.5. General procedure for the synthesis of chloro-phenyl-pyrazolo-pyridine-
carboxylate (6a–6d)24,25
2.87 g (8.71 mol) of compounds 5a–5d were treated with 30 mL of phosphorus
oxychloride under reflux conditions for 12 h. The reaction was carefully quenched with
crushed ice and the mixture was neutralised to pH 7 by aqueous NaOH solution. The
organic part was extracted with dichloromethane (3 x 50 mL). The crude chloro-compounds
were concentrated under vacuum and purified using column chromatography (ethylacetate :
hexane = 3:7). The pure compounds were solid and off white in colour.
4.1.6. General procedure for the synthesis of final compounds (7a–7m)26,27
To a solution of compound R2 (0.7 mol) in 20 mL 1,4-dioxane, K2CO3 (0.9 mol) was
added at room temperature with regular stirring. After half an hour, the chloro-compound
(6a–6d) dissolved in 1,4-dioxane was added dropwise to the stirring reaction mixture. The
◦
reaction mixture was allowed to reflux at 110 C over night. After the completion of the

reaction, the excessive solvent was removed under vacuum and the crude compound (7a–
7m) was purified using column chromatography (ethylacetate : hexane = 6:4).
4.1.6.1.Synthesis of ethyl 4-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (7a)
◦
1
Dark yellow solid, yield=61% (Rf =0.4 in pure ethylacetate); m.p. 240–242 C.; H
NMR (300 MHz, CDCl3) δ 8.83 (s, 1H), 8.40 (d, J = 4.8 Hz, 1H), 8.30 (s, 1H), 8.12 (d, J =
8.1 Hz, 2H), 7.84 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 7.8 Hz, 2H), 7.27 (m, 1H), 6.84 (dd, J =
7.5, 4.8 Hz, 1H), 4.40 (dd, J = 14.1, 7.2 Hz, 2H), 4.01 (d, J = 4.8 Hz, 2H), 3.82 (t, J = 5.7
13
Hz, 2H), 3.71 (d, J = 13.5 Hz, 2H), 3.66 (d, J = 3.0 Hz, 2H), 1.42 (t, J = 6.9 Hz, 3H). C
NMR (75 MHz, CDCl3) δ 166.89, 160.31, 153.73, 152.55, 151.93, 143.96, 138.90, 134.73,
129.08, 126.75, 122.33, 114.60, 109.35, 108.56, 94.98, 90.75, 70.05, 66.06, 62.39, 61.09,
51.54, 47.65, 14.36. IR νmax (%T)/cm-1 2221 (C≡N), 1689 (C=O ester), 1363 (N=N), 1278
(C-N),; LCMS: (ESI, m/z): [M+H]+ calcd. for C25H23N7O2 453.1; found 454.1.; Anal.
Calcd. for C25H23N7O2; C, 66.21; H, 5.11; N, 21.62; O, 7.06%; found C, 66.43; H, 5.33; N,
21.84; O, 7.28%.
4.1.6.2.Synthesis of ethyl 4-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)-1-phenyl-1H-
pyrazolo[3,4-b]pyridine-5-carboxylate (7b)
◦
1
Yellow solid, yield=76% (Rf =0.4 in pure ethylacetate); m.p. 210–212 C.; H NMR
(300 MHz, CDCl3) δ 8.89 (s, 1H), 8.79 (d, J = 4.8 Hz, 1H), 8.31 (s, 1H), 8.15 (d, J = 8.7 Hz,
2H), 8.03 (d, J = 9.0 Hz, 1H), 7.52 (dd, J = 15.0, 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 1H), 6.95
(d, J = 5.1 Hz, 1H), 4.41 (dd, J = 14.1, 6.9 Hz, 2H), 3.91 (s, 4H), 3.52 (s, 4H), 2.11 (s, 2H),
1.43 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 175.35, 166.72, 156.95, 153.91,
152.81, 151.38, 149.39, 138.92, 135.58, 134.56, 129.12, 128.26, 126.74, 124.99, 122.19,
121.68, 109.72, 109.21, 108.96, 61.08, 51.95, 29.68, 20.99, 14.40. IR νmax (%T)/cm-1 1713
(C=O ester), 1574 (C=C), 1363 (N=N), 1230 (C-N), 741 (C-Cl); LCMS: (ESI, m/z):
[M+H]+ calcd. for C28H25ClN6O2 512.1; found 513.0.; Anal. Calcd. for C28H25ClN6O2; C,
65.56; H, 4.91; Cl, 6.91; N, 16.38; O, 6.24%; found C, 65.78; H, 5.13; Cl, 7.13; N, 16.50; O,
6.36%.
4.1.6.3.Synthesis of ethyl 4-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)piperazin-
1-yl)-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7c)
◦
Cream coloured solid, yield=71% (Rf =0.4 in pure ethylacetate); m.p. 226–228 C.;
1H NMR (300 MHz, CDCl3) δ 8.94 (s, 1H), 8.37 (s, 1H), 8.32 (s, 1H), 8.16 (d, J = 8.7 Hz,

1H), 7.54 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.27 (s, 1H), 6.26 (s, 1H), 4.41 (dd, J
= 14.4, 7.2 Hz, 2H), 4.17 – 4.07 (m, 4H), 3.95 – 3.85 (m, 4H), 2.64 (s, 3H), 1.43 (t, J = 7.2
Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.59, 165.21, 154.43, 154.01, 152.86, 150.03,
138.95, 134.57, 129.17, 126.83, 122.16, 112.48, 109.71, 108.86, 94.73, 61.12, 51.51, 47.78,
29.69, 25.32, 14.44. IR νmax (%T)/cm-1 2921 (C-H stretching, alkyl), 1695 (C=O ester),
1332 (N=N), 1235 (C-N); LCMS: (ESI, m/z): [M+H]+ calcd. for C25H25N9O2 483.5; found
484.1.; Anal. Calcd. for C25H25N9O2; C, 62.10; H, 5.21; N, 26.07; O, 6.62%; found C,
62.32; H, 5.43; N, 26.29; O, 6.84%;
4.1.6.4.Synthesis
of
ethyl
4-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-1-(3-
(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7d)
◦
Light brown solid, yield=68% (Rf =0.4 in pure ethylacetate); m.p. 236–238 C.; 1H
NMR (300 MHz, CDCl3) δ 8.85 (s, 1H), 8.60 (s, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.40 (dd, J =
4.5, 1.2 Hz, 1H), 8.30 (s, 1H), 7.84 (dd, J = 7.8, 1.5 Hz, 1H), 7.68 – 7.54 (m, 2H), 6.84 (dd,
J = 7.5, 4.8 Hz, 1H), 4.41 (q, J = 6.9 Hz, 2H), 4.03 (s, 4H), 3.82 (s, 4H), 1.43 (t, J = 6.9 Hz,
3H). 13C NMR (75 MHz, CDCl3) δ 166.73, 160.32, 153.86, 153.01, 152.42, 151.94, 143.91,
139.65, 135.28, 131.74, 129.56, 124.43, 122.77, 118.40, 117.89, 114.64, 109.62, 109.05,
95.03, 61.15, 51.57, 47.63, 29.68, 14.37. IR νmax (%T)/cm-1 2220 (C≡N), 1718 (C=O
ester), 1374 (N=N), 1229 (C-N), 1114 (C-F); LCMS: (ESI, m/z): [M+H]+ calcd. for
C26H22F3N7O2 521.4; found 522.2; Anal. Calcd. for C26H22F3N7O2 C, 59.88; H, 4.25; F,
10.93; N, 18.80; O, 6.14%; found C, 60.1; H, 4.47; F, 11.15; N, 19.02; O, 6.63%.
4.1.6.5.Synthesis of ethyl 4-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-1-(2-fluorophenyl)-
1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7e)
Yellow sticky, yield=59% (Rf =0.4 in pure ethylacetate); m.p. 198–200 ◦C.; 1H NMR
(300 MHz, CDCl3) δ 8.81 (s, 1H), 8.40 (dd, J = 4.5, 1.5 Hz, 1H), 8.34 (s, 1H), 7.84 (dd, J =
7.6, 1.5 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.49 – 7.41 (m, 1H), 7.36 – 7.26 (m, 2H), 6.84
(dd, J = 7.5, 4.8 Hz, 1H), 4.39 (dd, J = 14.4, 7.2 Hz, 2H), 4.06 (t, J = 3.9 Hz, 4H), 3.84 (t, J
= 2.4 Hz, 4H), 1.40 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.78, 160.28,
154.16, 153.63, 152.56, 151.88, 143.87, 135.49, 130.29, 128.50, 124.58, 117.85, 117.09,
116.83, 114.56, 108.55, 108.33, 94.96, 60.98, 51.49, 47.60, 29.60, 14.32. IR νmax (%T)/cm-
1 2220 (C≡N), 1700 (C=O ester), 1380 (N=N), 1290 (C-N), 1036 (C-F); LCMS: (ESI, m/z):
[M+H]+ calcd. for C25H22FN7O2 471.4; found 472.0; Anal. Calcd. for C25H22FN7O2 C,
63.69; H, 4.70; F, 4.03; N, 20.80; O, 6.79%; found C, 63.91; H, 4.92; F, 4.25; N, 21.02; O,
7.01%.

4.1.6.6.Synthesis
of
ethyl
4-(4-(3-cyanopyridin-2-yl)piperazin-1-yl)-1-(3,4-
difluorophenyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7f)
◦
White solid, yield=79% (Rf =0.4 in pure ethylacetate); m.p. 241–243 C.; 1H NMR
(300 MHz, CDCl3) δ 8.83 (d, J = 0.9 Hz, 1H), 8.48 – 8.35 (m, 1H), 8.27 (s, 1H), 8.04 (d, J =
10.2 Hz, 1H), 7.84 (dd, J = 8.7, 1.2 Hz, 1H), 7.47 – 7.04 (m, 2H), 6.85 (dd, J = 7.8, 4.8 Hz,
1H), 4.51 (q, J = 6.9 Hz, 2H), 4.12 (t, J = 4.2 Hz, 4H), 3.90 (t, J = 5.4 Hz, 4H), 1.42 (t, J =
7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ 166.73, 160.34, 153.82, 152.78, 152.45, 151.95,
143.91, 135.61, 135.01, 117.89, 117.49, 117.25, 114.67, 111.40, 111.11, 109.49, 108.99,
95.07, 84.18, 61.15, 51.57, 47.64, 14.37. IR νmax (%T)/cm-1 2202 (C≡N), 1700 (C=O
ester), 1386 (N=N), 1235 (C-N), 1036 (C-F); LCMS: (ESI, m/z): [M+H]+ calcd. for
C25H21F2N7O2 489.4; found 490.1; Anal. Calcd. for C25H21F2N7O2 C, 61.34; H, 4.32; F,
7.76; N, 20.03; O, 6.54 %; found C, 61.56; H, 4.54; F, 7.98; N, 20.25; O, 6.76 %.
4.1.6.7.Synthesis of ethyl 4-(4-methylpiperazin-1-yl)-1-phenyl-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate (7g)
◦
Light pink solid, yield=73% (Rf =0.4 in pure ethylacetate); m.p. 238–240 C.; 1H
NMR (300 MHz, CDCl3) δ 8.79 (s, 1H), 8.23 (s, 1H), 8.14 (d, J = 7.8 Hz, 2H), 7.52 (t, J =
7.5 Hz, 2H), 7.31 (dd, J = 15.0, 7.8 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 3.66 (d, J = 3.9 Hz,
13
4H), 2.67 (d, J = 3.9 Hz, 4H), 2.40 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). C NMR (75 MHz,
CDCl3) δ 167.10, 153.78, 152.82, 152.58, 139.09, 134.71, 129.06, 126.55, 122.12, 109.50,
108.81, 60.91, 54.78, 52.02, 46.10, 14.36. IR νmax (%T)/cm-1 1706 (C=O ester), 1380
(N=N), 1229 (C-N),; LCMS: (ESI, m/z): [M+H]+ calcd. for C20H23N5O2 365.1; found 365.9;
Anal. Calcd. for C20H23N5O2 C, 65.73; H, 6.34; N, 19.16; O, 8.76%; found C, 65.95; H,
6.56; N, 19.38; O, 8.98%.
4.1.6.8.Synthesis of ethyl 4-morpholino-1-phenyl-1H-pyrazolo[3,4-b]pyridine-5-
carboxylate (7h)
◦
Light yellow solid, yield=68% (Rf =0.4 in pure ethylacetate); m.p. 220–222 C.; 1H
NMR (300 MHz, CDCl3) δ 8.84 (s, 1H), 8.24 (s, 1H), 8.15 (d, J = 8.4 Hz, 2H), 7.52 (t, J =
7.5 Hz, 2H), 7.34 (t, J = 7.5 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 4.02 (t, J = 4.5 Hz, 4H), 3.68
13
(t, J = 4.5 Hz, 4H), 1.40 (t, J = 7.2 Hz, 3H). C NMR (75 MHz, CDCl3) δ 174.31, 166.85,
153.88, 152.83, 139.02, 134.59, 129.08, 126.63, 122.11, 109.49, 108.79, 66.64, 60.95,
52.55, 14.35. IR νmax (%T)/cm-1 1700 (C=O ester), 1380 (N=N), 1229 (C-N), 1102 (C-O-C
stretch); LCMS: (ESI, m/z): [M+H]+ calcd. for C19H20N4O3 352.3; found 353.0; Anal.

Calcd. for C19H20N4O3 C, 64.76; H, 5.72; N, 15.90; O, 13.62%; found C, 64.76; H, 5.72; N,
15.90; O, 13.62%.
4.1.6.9.Synthesis of ethyl 1-phenyl-4-(piperidin-1-yl)-1H-pyrazolo[3,4-b]pyridine -5-
carboxylate (7i)
◦
Yellow solid, yield=66% (Rf =0.4 in pure ethylacetate); m.p. 225–227 C.; 1H NMR
(300 MHz, CDCl3) δ 8.85 (s, 1H), 8.60 (s, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.40 (d, J = 3.3 Hz,
1H), 8.30 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.68 – 7.50 (m, 2H), 6.84 (dd, J = 7.5, 4.8 Hz,
1H), 4.41 (q, J = 6.9 Hz, 2H), 4.03 (s, 4H), 3.82 (s, 4H), 1.43 (t, J = 6.9 Hz, 3H). 13C NMR
(75 MHz, CDCl3) δ 166.73, 160.32, 153.86, 151.94, 143.91, 135.28, 129.56, 124.43, 114.64,
109.62, 95.03, 61.15, 51.57, 47.63, 29.68, 14.37. IR νmax (%T)/cm-1 1706 (C=O ester),
1340 (N=N), 1229 (C-N),; LCMS: (ESI, m/z): [M+H]+ calcd. for C20H22N4O2 350.4; found
351.0; Anal. Calcd. For C20H22N4O2 C, 68.55; H, 6.33; N, 15.99; O, 9.13%; found C, 68.77;
H, 6.55; N, 16.21; O, 9.35%.
4.1.6.10.
Synthesis of ethyl 4-(4-(4-acetamidophenylsulfonyl)piperazin-1-yl)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7j)
◦
1
Yellow solid, yield=70% (Rf =0.4 in pure ethylacetate); m.p. 218–220 C.; H NMR
(300 MHz, DMSO) δ 10.21 (s, 1H), 8.42 (s, 1H), 8.38 (s, 1H), 7.95 (d, J = 8.1 Hz, 2H), 7.67
(d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 7.17 (t, J = 7.2 Hz,
1H), 4.05 (q, J = 6.6 Hz, 2H), 3.45 (s, 4H), 2.92 (s, 4H), 1.90 (s, 3H), 1.03 (t, J = 6.8 Hz,
3H). 13C NMR (75 MHz, DMSO) δ 169.59, 166.76, 153.29, 152.66, 152.38, 144.20, 136.31,
129.56, 129.36, 128.57, 126.86, 124.19, 121.85, 119.21, 109.35, 109.13, 61.19, 51.30,
46.19, 24.63, 14.42. IR νmax (%T)/cm-1 3326 (N-H stretch), 1706 (C=O ester), 1568 (C=O
amide), 945 (N-S), 848 (S-O stretch), LCMS: (ESI, m/z): [M+H]+ calcd. for C27H28N6O5S
548.6; found 549.0; Anal. Calcd. For C27H28N6O5S C, 59.11; H, 5.14; N, 15.32; O, 14.58; S,
5.84%; found C, 59.33; H, 5.36; N, 15.54; O, 14.80; S, 6.06%.
4.1.6.11.
Synthesis of ethyl 4-(4-(2-oxo-1,2-dihydroquinolin-4-yl)piperazin-1-yl)-1-
phenyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (7k)
◦
1
Light yellow solid, yield=61% (Rf =0.4 in pure ethylacetate); m.p. 233–235 C.; H
NMR (300 MHz, DMSO) δ 11.20 (s, 1H), 8.43 (d, J = 6.0 Hz, 2H), 7.97 (d, J = 8.1 Hz, 2H),
7.62 (s, 1H), 7.40 – 7.23 (m, 3H), 7.13 (dd, J = 16.2, 7.5 Hz, 2H), 6.98 (t, J = 7.5 Hz, 1H),
5.73 (s, 1H), 4.13 (q, J = 6.9 Hz, 2H), 3.61 (s, 4H), 3.11 (s, 4H), 1.13 (t, J = 6.9 Hz, 3H). 13C
NMR (75 MHz, CDCl3) δ 175.35, 166.72, 156.95, 153.91, 152.73, 151.38, 149.39, 138.92,

135.58, 134.56, 129.12, 128.26, 126.74, 124.99, 122.19, 121.68, 109.72, 109.21, 108.96,
61.08, 51.95, 29.68, 20.99, 14.40. IR νmax (%T)/cm-1 1706 (C=O ester), 1640 (C=O amide),
1368 (N=N), 1290 (C-N),; LCMS: (ESI, m/z): [M+H]+ calcd. for C28H26N6O3 494.2; found
495.0; Anal. Calcd. For C28H26N6O3 C, 68.00; H, 5.30; N, 16.99; O, 9.71%; found C, 68.22;
H, 5.52; N, 17.21; O, 9.93%
4.1.6.12.
Synthesis of ethyl 1-phenyl-4-(quinolin-8-yloxy)-1H-pyrazolo[3,4-
b]pyridine-5-carboxylate (7l)
◦
Brown solid, yield=59% (Rf =0.4 in pure ethylacetate); m.p. 217–219 C.; 1H NMR
(300 MHz, CDCl3) δ 9.19 (s, 1H), 8.88 (dd, J = 4.2, 1.8 Hz, 2H), 8.26 (d, J = 8.1 Hz, 1H),
8.12 (d, J = 8.7 Hz, 1H), 7.81 (d, J = 8.1 Hz, 1H), 7.62 – 7.25 (m, 5H), 6.72 (s, 1H), 4.35
(dd, J = 14.1, 6.9 Hz, 2H), 1.41 (d, J = 7.8 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ 164.65, 153.85, 153.60, 152.22, 150.88, 140.77, 138.83, 136.11, 132.98,
129.92, 129.08, 126.67, 126.55, 125.44, 122.28, 121.69, 118.55, 110.81, 108.91, 61.16,
29.69, 14.11. IR νmax (%T)/cm-1 1725 (C=O ester), 1374 (N=N), 1247 (C-N), 1169 (C-O-C
stretch); LCMS: (ESI, m/z): [M+H]+ calcd. for C24H18N4O3 410.4; found 411.0; Anal.
Calcd. For C24H18N4O3 C, 70.23; H, 4.42; N, 13.65; O, 11.69%; found C, 70.43; H, 4.22; N,
13.85; O, 11.72%.
4.1.6.13.
Synthesis of ethyl 1-phenyl-4-(4-(5-(trifluoromethyl)-[1,2,4]triazolo[1,5-
a]pyrimidin-7-yl)piperazin-1-yl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate
(7m)
◦
1
Cream yellow, yield=63% Rf =0.4 in pure ethylacetate); m.p. 182–184 C.; H NMR
(300 MHz, CDCl3) δ 8.93 (s, 1H), 8.47 (s, 1H), 8.28 (s, 1H), 8.16 (d, J = 8.4 Hz, 2H), 7.53
(t, J = 7.8 Hz, 2H), 7.3–7.27 (m, 1H), 6.63 (s, 1H), 4.42 (q, J = 7.2, Hz, 2H), 4.34 (d, J = 3.3
Hz, 4H), 3.92 (d, J = 3.0 Hz, 4H), 1.44 (t, J = 7.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
166.40, 156.78, 155.54, 153.97, 152.66, 152.04, 150.84, 138.86, 134.46, 129.13, 126.83,
122.11, 109.61, 108.73, 89.82, 72.77, 61.13, 51.31, 48.11, 29.69, 14.39. LCMS: (ESI, m/z):
[M+H]+ calcd. for C25H22F3N9O2; 537.1; found 538.1; Anal. Calcd. For C25H22F3N9O2 C,
55.86; H, 4.13; F, 10.60; N, 23.45; O, 5.95%; found C, 70.43; H, 4.22; N, 13.85; O, 11.49%.
4.2. In vivo evaluation of the synthesized compounds
4.2.1. Culture and maintenance of C. elegans
The culture and maintenance of the worms was done as per the previously described
methods.36 Briefly, worms were cultured using nematode growth medium (NGM) agar

plates, seeded with E. coli-OP50 bacteria using standard techniques at 22 °C. For the
preparation of NGM, 50 mM NaCl (Merck), 2.5 gL-1 peptone (Sigma), 17 gL-1 Agar (Hi-
media) were added to 975 mL double distilled water and autoclaved for 30 to 40 minutes at
15 lb/inch.37 The media was allowed to cool to 50-60 °C and 5 µgmL-1 cholesterol solution
(Sigma) prepared in ethanol, 1 mM calcium chloride (Sigma), 1 mM Magnesium Sulphate
(Sigma) and 25 mM potassium dihydrogen phosphate (SRL) additives were added. The
transgenic strain which was used in this study was NL5901 (Punc-54:: α- synuclein::
YFP+unc-119; expressing human alpha α-Syn protein with YFP expression in the muscles).
This strain was obtained from the Caenorhabditis Genetics Centre (University of Minnesota,
USA).
4.2.2. Obtaining synchronous nematode population by embryo isolation
Alkaline sodium hypochlorite bleaching method was adopted to synchronize the
worms for the isolation of embryos from various strains of C. elegans. This method includes
the washing of worms with M-9 buffer and their treatment with axenizing solution (2 mL of
sodium hypochlorite and 5 mL of 1 M sodium hydroxide solution) followed by intermittent
vortexing. The worms were washed again (twice) with M-9 buffer to obtain the worms in
their synchronized embryo stage.37
4.2.3. Treatment of Worms with Test Compounds
In this study, test compounds were initially dissolved in DMSO and ethanol (1:1) to
prepare a stock of 10%. Working concentration of 1% of compound in DMSO and ethanol
(1:1) was then prepared in OP50. The OP50-test compound mixtures were seeded onto 6-
well NGM plates. Age synchronized worms were grown on the plates till early adult stage,
for further studies.
4.2.4. Assay for analysis of α-Syn aggregation
The α-Syn aggregation assay was performed as reported earlier.38 Briefly, the worms
were grown on control media and the media was treated with 1% of the test compounds
(7a–7m) for 48 h. The worms were washed with M-9 buffer in order to eliminate the
adhering bacteria followed by immobilization with 100 mM sodium azide (Sigma, cat no.
71289). The worms were then mounted onto glass slides and sealed with a cover slip.
Images of the worms were captured by Carl Zeiss fluorescence microscope, which were
further quantified by measuring fluorescence intensity using image J Software (Image J,
National Institutes of Health, Bethesda, MD). At least 10 worms were analyzed for each
group, and data was subjected to statistical evaluation followed by plotting on graphs.

4.3. Docking protocol
Crystal structure of α-Syn (PDB ID 1XQ8) in pdb format was downloaded from Protein
Data Bank (www.rcsb.org).39 AutoDock Vina version 1.5.6 was used to perform docking
with the ligand molecules. PyMOL visualization tool was used to visualize the molecular
interactions. Weak non-covalent interactions like π-π stacking interaction and H-bonding
were observed between the key residues of α-Syn and the ligand molecules lying within a
range of 2.5 Å. In addition, binding free energy was calculated on the basis of which the
designed molecules were screened.
Acknowledgement: The authors are highly thankful to Dr Aamir Nazir, Toxicology &
Experimental Medicine CSIR-Central Drug Research Institute, Lucknow for allowing the
biological screening of the compounds in his research lab. MM is thankful to The Council
of Scientific and Industrial Research (CSIR), Government of India for financial assistance in
the form of senior research fellowship (SRF).

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Highlights
 A series of pyrazolo-pyridine carboxylate hybrids (7a-7m) were designed and
synthesized.
 Docking was used to understand the binding of the ligands with the protein.
 In vivo transgenic C. elegans model of Synucleinopathy was used to evaluate the
ability of the test compounds to inhibit α-synuclein aggregation.
 Compounds 7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-synuclein
with respect to the control.