Ultrasound assisted synthesis of 3-alkynyl substituted 2-chloroquinoxaline derivatives: Their in silico assessment as potential ligands for N-protein of SARS-CoV-2

Article abstract of DOI:10.1016/j.tetlet.2020.152336

In view of recent global pandemic the 3-alkynyl substituted 2-chloroquinoxaline framework has been explored as a potential template for the design of molecules targeting COVID-19. Initial in silico studies of representative compounds to assess their bindi

Full text of DOI:10.1016/j.tetlet.2020.152336

Tetrahedron Letters 61 (2020) 152336
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Ultrasound assisted synthesis of 3-alkynyl substituted
2-chloroquinoxaline derivatives: Their in silico assessment as potential
ligands for N-protein of SARS-CoV-2
Shaik Shahinshavali a, Kazi Amirul Hossain b, Abbaraju Venkata Durga Nagendra Kumar c,
Alugubelli Gopi Reddy d, Deepti Kolli e, Ali Nakhi f, Mandava Venkata Basaveswara Rao a, , Manojit Pal f,
⇑
⇑
a Department of Chemistry, Krishna University, Machilipatnam 521001, Andhra Pradesh, India
b Department of Physical Chemistry, Gdansk University of Technology, Gdan´sk, Poland
c Department of Chemistry, GIS, GITAM Deemed to be University, Visakhapatnam, Andhra Pradesh, INDIA, 530045
d Department of Pharmacy, Sana College of Pharmacy, Kodad 508 206, Telangana, India
e Department of Chemistry, Koneru Lakshmaiah Education Foundation, Green Fields, Vaddeswaram 522502, Andhra Pradesh, India
f Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500046, India
a r t i c l e i n f o
a b s t r a c t
Article history:
In view of recent global pandemic the 3-alkynyl substituted 2-chloroquinoxaline framework has been
explored as a potential template for the design of molecules targeting COVID-19. Initial in silico studies
of representative compounds to assess their binding affinities via docking into the N-terminal RNA-bind-
ing domain (NTD) of N-protein of SARS-CoV-2 prompted further study of these molecules. Thus building
of a small library of molecules based on the said template became essential for this purpose. Accordingly,
a convenient and environmentally safer method has been developed for the rapid synthesis of 3-alkynyl
substituted 2-chloroquinoxaline derivatives under Cu-catalysis assisted by ultrasound. This simple and
straightforward method involved the coupling of 2,3-dichloroquinoxaline with commercially available
terminal alkynes in the presence of CuI, PPh3 and K2CO3 in PEG-400. Further in silico studies revealed
some remarkable observations and established a virtual SAR (Structure Activity Relationship) within
the series. Three compounds appeared as potential agents for further studies.
Received 21 June 2020
Revised 15 July 2020
Accepted 4 August 2020
Available online 27 August 2020
Keywords:
Chloroquinoxaline
Alkyne
Ultrasound
In silico study
COVID-19
Ó 2020 Elsevier Ltd. All rights reserved.
Being the source of a global pandemic COVID-19 (coronavirus
disease 2019) [1], the novel SARS-CoV-2 has already affected the
health and economy of several countries severely. Nearly more
than 450 thousand people have died throughout the world so far
[2] and the number are increasing at a rapid pace. However, there
are no promising vaccines and therapeutic drugs available till date
to curb the spread of the SARS-CoV-2 worldwide. Thus there is an
urgent need to address this global health problem. Multiple studies
are in progress, employing diverse approaches to identify effective
therapeutics to fight against SARS-CoV-2. For example, in vitro
studies have suggested that chloroquine (A, Fig 1), an
immunomodulant drug traditionally used to treat malaria, might
be effective in reducing viral replication in other infections,
including the SARS-associated coronavirus (CoV) and MERS-CoV
[3,4]. Its analogue hydroxychloroquine is also being explored as
an experimental treatment for COVID-19 [5]. Another drug
Favipiravir (T-705, 6-fluoro-3-hydroxypyrazine-2-carboxamide)
(B, Fig. 1), an anti-influenza drug which functions to selectively
inhibit the RNA- dependent RNA polymerase of influenza virus
[6] is being explored for this purpose in Japan. There are several
protease inhibitors that are currently in clinical trials for SARS-
CoV-2 include Indinavir, Saquinavir, Darunavir, ASC09, Ritonavir
and Lopinavir [7].
In 2016 with the goal of finding the potential hit molecules
against human coronavirus (CoV-OC43), Chang et al. conducted a
molecular docking based virtual screening using nucleocapsid
(N)-RNA binding domain as a target protein [8]. The primary func-
tion of this protein is to pack the viral RNA within the viral envel-
ope into a ribonucleoprotein (RNP) complex called the capsid,
which is a fundamental part of viral self-assembly and replication.
Initially, based on docking score eight potential hits were identi-
fied, among which a quinoline derivative (C, Fig. 1) was emerged
as the most potent compound that was supported by the
experimental evidences based on surface plasmon resonance
⇑
Corresponding authors.
E-mail addresses: vbrmandava@yahoo.com (M.V.B. Rao), manojitpal@rediffmail.
com (M. Pal).
https://doi.org/10.1016/j.tetlet.2020.152336
0040-4039/Ó 2020 Elsevier Ltd. All rights reserved.

2
S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
O
N
HN
N
F
N
NH2
OH
N
B
Cl
A
O
H
N
N
N
O
Cl
O
C
Fig. 1. Example of drugs that are being explored against coronavirus.
(SPR) analysis. The crystallographic structure of HCoV-OC43 bound
with the molecule C was deposited. Very recently, a crystallo-
graphic structure of Apo-Nucleocapsid (RNA-binding domain) of
SARS-CoV-2/COVID-19 (PDB: 6M3M) has been deposited by Chen
et al. [9] Nevertheless, discovery and development of small organic
molecule based inhibitor against Nucleocapsid (N) of SARS-CoV-2
is rather uncommon.
Fig. 3. Structural alignment between RNA-binding domain of nucleocapsid protein
of SARS-CoV-2 and HCoV-OC43. Active site residues are shown in stick
representation.
In our effort for the identification of new and potential agents
for COVID-19 we planned to evaluate the library of compounds
based on N-heterocycles. Accordingly, we focused on compounds
based on the template D (Fig 2) at the initial stage. We reasoned
that the template D containing the chloro group and pyrazine ring
of existing experimental agents A and B (Fig. 1) and the alkyne
moiety studied in antiviral research earlier [10,11] would be wor-
thy to explore. Notably, we preferred the bicyclic ring over the
monocyclic one because of the fact that the earlier molecule C tar-
geted towards the human coronavirus nucleocapsid protein was
also a bicyclic compound. Nevertheless, to substantiate our quali-
tative reasoning in favor of choosing the template D we performed
the related docking studies in silico. Firstly, we tried to compare the
N-protein of SARS-CoV-2 with HCoV-OC43 both sequentially and
structurally to find out the common regions between them. While
a 52% sequence identity was observed in the conserve region based
sequence alignment (See Fig. S-6A in ESI) our major interest was to
see secondary structure (which reveals structural pattern) com-
mon in them. Hence we conducted the secondary structure based
sequence alignment (See Fig. S-6B in ESI) using PRALINE web-tool
[12] and performed the structural comparison in PyMol [13]
(Fig. 3). The RMSD was found to be only 0.9 Å whereas the active
site residues were appeared to be conserved. While these data
clearly established the binding site of SARS-CoV-2 nucleocapsid
protein however for further confirmation the computational bind-
ing site prediction was conducted by using fconv program [14]
where the same binding cavity was found with the volume of
279.31 Å3.
To assess the binding affinity of some representative molecules,
e.g. D-1, D-2, D-3 and D-4 (related to the template D) against N
protein of SARS-CoV-2, we performed molecular docking study at
the nucleotide binding (active) site (PDB: 6M3M). The protein
(PDB: 6M3M) as well as all molecules were prepared (e.g. energy
optimization, charge calculation and addition of hydrogen etc.)
using AutoDock tool [15] and all molecules were docked using reli-
able open-source tool AutoDock Vina [16]. The binding energy of
best pose of each molecule is presented in Table 1. Notably, for fur-
ther understanding of the polarity and coulombic electrostatic
potential of the binding site, the hydrophobicity and electrostatic
surface representation was generated using UCSF Chimera [17]
(see the following figures). While a balance between hydrophilic
and hydrophobic residues was evident in case of N-protein and
the binding site was somewhat hydrophobic in nature however
it’s positive charge was essential for nucleotide binding.
Nevertheless, the comparable binding energies of these mole-
cules with the known inhibitor C suggested that compounds based
on framework D might interact with the nucleocapsid (N) of SARS-
CoV-2 too. Indeed, the quinoline ring of molecule D-1 showed
effective pi-pi interactions with residue TYR110 (Fig. 4A). Addition-
ally, it participated in hydrophobic / van der Waals contacts with
residues ALA157, ARG150, THR55, ALA56, ARG89 etc (Fig 4B, see
also Fig. S-1 in ESI).
However, the most effective interactions were observed in case
of molecule D-3 as evident from its binding energy (Table 1). Its
ability to form pi-cation interaction with ARG150, pi-pi interac-
tions with TYR110 and comparatively more hydrophobic interac-
tions with residues ARG108, TYR112, PRO152, SER52, ALA51, and
ARG89 etc (Fig 5, see also Fig S-2 in ESI) could be the reason for this
observation. The molecule D-2 participated in a pi-cation interac-
tion with ARG150 and a pi-pi stacking with TYR110. In addition,
it formed hydrophobic contacts with other residues like ARG108,
TYR112, PRO152, SER52, ALA51 etc (see the Supplementary data).
Similar interactions were observed in case of molecule D-4. Nota-
bly, the reference compound C showed relatively low docking
score perhaps due to the lack of proper aromaticity thereby related
Fig. 2. The 3-alkynyl substituted 2-chloroquinoxaline as a potential template to
target COVID-19.

S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
3
Table 1
Docking of molecules into N-terminal RNA-binding domain (NTD) of N-protein of
SARS-CoV-2.a
Molecules
AutoDock Vina score (Kcal/mol)
À6.4
HO
N
N
D-C1l
D-1
À6.2
N
N
Cl
D-2
D-2
À6.5
À6.1
N
N
Cl
D-3
D-3
N
N
N
N
Cl
D-4
D-4
O
À5.6
H
N
N
O
Cl
O
C
C
Fig. 4. (A) 2D interaction diagram between N-terminal RNA-binding domain (NTD)
of N-protein of SARS-CoV-2 and compound D-1 (where pi-pi interaction shown in
green line), prepared in Maestro visualizer (Schrödinger, LLC). (B) Hydrophobic
surface representation along with 3D interaction diagram (where pi-pi interaction
shown in cyan dashed line).
a
Docking of each individual molecule was performed for 5 times and maximum
difference in score was found to be 0.2.
pi-interactions. Nevertheless, the molecule was involved in
hydrophobic contacts mainly with residues such as ALA157,
ARG150, THR55, ALA56, ARG89, TYR110 etc (see the Supplemen-
tary data).
Over the years the ultrasound-assisted reactions have emerged
as one of the popular approaches in organic synthesis which is evi-
dent from a wide range of applications of these methodologies
both in academia and industrial organizations [25]. The features
and advantages of these reactions include their (i) involvement
as green approaches in organic synthesis [26], (ii) reduction of
waste generation as well as energy requirements [27] and (iii) effi-
ciency and effectiveness for the synthesis of desired products via
employing shorter reaction time and milder conditions at the same
time increasing the product yields [28,29]. The PEG-400 on the
other hand being a high boiling, non-hazardous and polar solvent
has found applications in various reactions. Indeed, due to its easy
recovery from the reaction mixture and recyclability PEG-400 is
considered as an environmentally friendly solvent [30]. As part of
our ongoing effort in the use of ultrasound as an alternative source
of energy and PEG-400 as a greener solvent in various organic reac-
tions we became interested in exploring these reaction conditions
in our current endeavor. Notably, while the CuI/PPh3-catalyzed
Sonogashira coupling of iodoarene with terminal alkynes (i) in
PEG-water under microwave heating or reflux in oil bath [31] (at
120 °C) or (ii) in water [32] (at 120 °C) or (iii) under biphasic con-
ditions [33] (water/organic substrates) have been reported earlier,
a similar coupling of chloroheteroarene with terminal alkynes
under ultrasound is not known.
The alkynyl substituted quinoxaline or related derivatives have
been prepared via Sonogashira type coupling under Pd(0)-Cu catal-
ysis in the presence of Et3N or Et2NH [18–22]. In our effort the syn-
thesis of 3-alkynyl substituted 2-chloroquinoxaline has been
carried out via a selective mono alkynylation of 2,3-dichloro-
quinoxaline (1) earlier [23,24]. The reactions were carried out
using 10%Pd/C-CuI-PPh3 as a catalyst system, Et3N as a base and
EtOH as a solvent and the duration was 2–4 h. However, all these
methodologies involved the use of bi-metallic salts as catalysts
and environmentally harmful alkylamine as a base. Moreover,
the organic solvents used in some of these cases are not environ-
mentally friendly. All these concerns prompted us to explore a
more convenient and environmentally safer method for the syn-
thesis of compounds based on D (Fig. 2) including D-1, D-2, D-3
and D-4 (Table 1). Thus the 3-alkynyl substituted 2-chloroquinox-
aline derivatives (3) were synthesized via a Cu-catalyzed coupling
of 2,3-dichloroquinoxaline (1) with commercially available termi-
nal alkynes (2) under ultrasound irradiation (Scheme 1). The
methodology involved the use of PEG-400 as a solvent under mild
reaction conditions and does not require the use of any co-catalyst.
We now present the details of this study.

4
S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
use of other Cu-catalysts e.g. CuBr or CuCl in the present coupling
reaction but these were found to be less effective (entry 7 and 8,
Table 2). Notably, the CAC coupling did not proceed in the absence
of a catalyst (entry 9, Table 2) indicating key role played by the Cu-
salt in the current alkynylation method. Moreover, though the
reaction proceeded in the absence of ligand PPh3 the product yield
was not particularly high (entry 10, Table 2). The reaction was also
found to be less efficient in terms of product yield when carried out
in the absence of ultrasound (entry 11, Table 2). While the reaction
temperature was maintained at 50 °C during all reactions as men-
tioned above the decrease and increase of temperature was exam-
ined. The reaction did not proceed at lower temperature e.g. at
40 °C and no improvement in product yield was observed at higher
temperature e.g. at 70 °C though the reaction progressed well at
this temperature. Overall, the condition of entry 2 of Table 2 (i.e.
the combination of CuI, PPh3 and K2CO3 in PEG-400 at 50 °C under
ultrasound) appeared to be optimum and was used for the prepa-
ration of analogues of 3a.
A range of terminal alkynes (2) were employed to couple with
the chloro compound (1) under the optimized conditions. The
alkyne may contain a primary, secondary or tertiary hydroxyl
group or an aliphatic chain such as n-butyl, n-pentyl, n-hexyl etc
or an aryl or heteroaryl group. The Cu-catalyzed CAC bond forming
reaction proceeded smoothly in all these cases affording the
desired coupled product in good to acceptable yield (Table 3). It
is worthy to mention that in none of the cases the product yield
was high due to the partial dimerization of the terminal alkyne
used. It is well known that dimerization of terminal alkynes to
the corresponding diyne is often a side reaction under the Sono-
gashira coupling conditions and Cu-salts play a key role in such
oxidative homocoupling (Glaser coupling) of terminal alkynes
[34–36]. Additionally, the formation of bis-alkynylated product in
some cases (particularly in case of 3b, 3c, 3g etc) albeit in trace
quantity perhaps affected the yield of desired monoalkynylated
product. Generally, to avoid the formation of unwanted bis-alkyny-
lated product the use of reactant alkyne (2) was restricted to 1
equivalent. However, a slow evaporation of the corresponding ter-
minal alkyne i.e. 3,3-dimethylbut-1-yne (due to the low boiling
point i.e. 37–38 °C) was observed under the reaction conditions
employed in case of 3b and hence the use of higher quantity of
alkyne (1.5 equiv.) was necessary in this case. Nevertheless, all
the compounds were characterized by using common spectral
(1H and 13C NMR and Mass) data (See ESI).
Fig. 5. (A) 2D interaction diagram between compound D-3 and N-protein of SARS-
CoV-2. (B) Electrostatic surface representation followed by 3D interaction diagram.
Scheme 1. Ultrasound assisted synthesis of 3-alkynyl substituted 2-chloroquinox-
alines (3) under Cu-catalysis.
Based on the results of Table 2 and the earlier reports [31–33] a
proposed reaction mechanism for the Cu-catalyzed coupling of 1
with 2 under ultrasound irradiation is presented in Scheme 2. Ini-
tially, a Cu(I) complex (A) was formed via the interaction of CuI
with the ligand PPh3 under ultrasound irradiation. Indeed, the
complex A was the actual catalytic species that catalyzed the pre-
sent CAC bond forming reaction. On interaction with the terminal
alkyne (2) in the presence of K2CO3 the complex A afforded the
acetylide intermediate E-1 with the generation of KI. Subsequently,
a copper cluster linked with both alkyne as well as heteroarene
moiety (E-2) was formed via the interaction of E-1 with the chloro
compound (1). The Cu-complex E-2 then furnished the desired
mono-alkynylated product 3 along with the regeneration of the
catalyst A (via E-3 in the presence of KI) thereby completing the
catalytic cycle. The results of Table 2 suggested that the present
Cu-catalyzed coupling reaction was accelerated greatly by the
ultrasound irradiation. It is known that the compression of the liq-
uid and then rarefaction (expansion) caused by ultrasound results
in a sudden pressure drop that forms small, oscillating bubbles of
gaseous substances. Subsequently, with each cycle of the applied
ultrasonic energy these bubbles continue to expand till they reach
to an unstable size. At this stage these cavitation bubbles can col-
lide and/or collapse violently which can cause the increase of local
In order to find the optimized reaction conditions for the cou-
pling of 2,3-dichloroquinoxaline (1) with the alkyne i.e. 2-methyl-
but-3-yn-2-ol (2a) was examined under a range of reaction
conditions and the results are summarized in Table 2. The reaction
proceeded well when carried out using 10 mol% CuI as a catalyst,
30 mol% PPh3 as a ligand and K2CO3 as a base in PEG-400 under
ultrasound using a laboratory ultrasonic bath SONOREX SUPER
RK 510H model producing irradiation of 35 kHz (entry 1, Table 2).
However, the desired coupled product 3a was obtained in 54%
yield after 4 h. The increase of CuI loading from 10 mol% to
15 mol% improved the product yield significantly and the reaction
was completed within 1 h (entry 2, Table 2). Encouraged by this
observation we continued our study for possibility of further
improvement in product yield. Thus the quantity of CuI used was
increased further from 15 mol% to 20 mol% but no significant
increase in yield of 3a was observed (entry 3, Table 2). The use of
other solvent e.g. EtOH or n-BuOH (entry 4 and 5, Table 2) in place
of PEG-400 or other base e.g. Et3N (entry 6, Table 2) in place of
K2CO3 did not improve the product yield. The use of pure water
as a solvent was not successful as the partial hydrolysis of 1 was
observed under the conditions employed. We also examined the

S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
5
Table 2
Effect of reaction conditions on coupling of 2,3-dichloroquinoxaline (1) with terminal alkyne (2a).a
HO
N
N
N
N
Cl
Cl
HO
+
Cl
2a
1
3a
Entry
Cu-cat (mol%)
Base
Solvent
Time (h)
Yieldb
1.
2.
3.
4.
5.
6.
7.
8.
CuI (10)
CuI (15)
CuI (20)
CuI (15)
CuI (15)
CuI (15)
CuBr (15)
CuCl (15)
No catalyst
CuI (15)
CuI (15)
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
Et3N
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
PEG-400
PEG-400
PEG-400
EtOH
n-BuOH
PEG-400
PEG-400
PEG-400
PEG-400
PEG-400
PEG-400
4
1
1
1
1
4
4
4
4
1
4
54
73
75
66
62
61
49
37
9.
10.
11.
No reaction
60c
43d
a
All reactions were carried out using the chloro compound 1 (1 equiv.), alkyne 2a (1 equiv.), a Cu-catalyst, PPh3 (30 mol%) and base (2 equiv.) in a solvent (5.0 mL) at 50 °C
under ultrasound irradiation.
b
Isolated yields.
The reaction was performed in absence of PPh3
The reaction was performed in the absence of ultrasound.
c
d
Table 3
Synthesis of 3-alkynyl substituted 2-chloroquinoxaline derivatives (3)a,b (Scheme 1).
OH
OH
N
N
N
N
OH
N
N
N
N
Cl
Cl
Cl
Cl
3c (70%)
3d (74%)
3b (69%)c
3a (73%)
HO
N
N
N
N
N
Cl
N
Cl
N
Cl
N
Cl
3f (70%)
3e (73%)
3h (71%)
3g (67%)
N
N
N
N
N
N
Cl
N
Cl
N
Cl
N
Cl
3i (73%)
3j (77%)
3k (75%)
3l (73%)
aAll reactions were carried out using the chloro compound 1 (1 equiv.), alkyne 2 (1 equiv.), CuI (15 mol%), PPh3 (30 mol%) and K2CO3 (2 equiv.) in PEG-400 (5.0 mL)
at 50 °C under ultrasound irradiation.
bFigure in the bracket represents isolated yield.
c1.5 equivalent of alkyne (3,3-dimethylbut-1-yne) was used in this case.
temperature within the reaction medium. As a result of this the
crossing of energy barrier is facilitated [37] that allows the faster
conversion of reactants to intermediates and subsequently to pro-
duct(s) within short reaction time. The acceleration of various
steps of Scheme 2 by ultrasound in this way explains the rapid for-
mation of product 3 from the chloro compound (1) and alkyne (2).
Having synthesized a small library of molecules (3) based on D
(Fig. 2) we re-focused on assessing their binding affinities via dock-
ing into the N-terminal RNA-binding domain (NTD) of N-protein of
SARS-CoV-2 (PDB: 6M3M). The purpose of this study was to gain
some insights regarding the virtual SAR (Structure-Activity-Rela-
tionship) within this series of compounds. The results are pre-
sented in Table 4 including that of Table 1 for head-to-head
comparison and discussion. It was evident that most of the com-
pounds showed good to acceptable or moderate binding affinities
(>5.0 Kcal/mol) except the compound 3c. The nature and size of

6
S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
CuI
3L
N
N
Cl
Cl
L
Cu
Cl
L
L3CuI
A
N
N
L
1
Cu
L
R
R
L
R
Cl
2
K2CO3
L
KI + KHCO3
E-1
E-2
L
(L = PPh3)
KI
R
N
N
L3CuI
L3CuCl
+
Cl
KCl
E-3
3
Scheme 2. Proposed reaction mechanism for the Cu-catalyzed coupling of 1 with 2 under ultrasound irradiation.
the substituent (other than the chloroquinoxalin moiety) attached
to the alkyne moiety appeared to have played a key role in binding
with the protein in silico (Fig 6). For example, the in silico study of
compound 3c (Fig 7A-B) suggested flipping of the molecule to
adopt a different orientation (aided by the high affinity of its
hydroxyl group towards ARG89) that resulted in losing of several
hydrophobic contacts (Fig 7C) in compared to that of 3k (Fig 7D).
Notably, these hydrophobic contacts contributed significantly
towards overall binding affinity of the most effective compound
3k. Secondly, a steric clash (termed as bad contact, see Fig 7B)
observed between a non-polar H atom of 3c and the residue
THR149 at a distance of 2.37 Å. Nevertheless, generally a bulky
hydroxy cycloalkyl group (3e) or aryl/heteroaryl moiety (3j-l)
was found to be beneficial for binding because of favorable
hydrophobic and/or pi-interactions. Among rest of the molecules
those possessing the hydroxy dimethyl group (3b) and pentyl or
hexyl chain (3g and 3h) were found to be moderately effective.
The affinity was decreased further in case of butyl chain (e.g. 3f)
and tert-butyl group (e.g. 3a). The relatively less number of pi-
interactions in all these cases (only one in each case) could be
the reason (see Fig. S-3A and B in ESI). Notably, a too long alkyl
chain (e.g. 3i) was found to be somewhat less effective due to a
bad steric clash observed between a non-polar H atom of com-
pound 3i and TYR 112 at a distance of 2.46 Å (see Fig. S-3C and
Hydroxy dimethyl,
pentyl or hexyl chain
moderately effective
Hydroxy cycloalkyl or
aryl / heteroaryl moiety
most effective
R
Hydroxy methylene
less effective
N
Butyl chain or tert-butyl
group decreased the affinity
a long alkyl chain decreased
the affinity further
N
Cl
pi-pi interactions
hydrophobic /
van der Waals contacts
pi-cation interaction etc
Hydrophobic contacts
Fig. 6. Summary of in silico binding affinities of compound 3.
D in ESI). While the role of quinoxalin ring has been discussed ear-
lier however the role of its chloro group was not highlighted.
Indeed, the chloro group was prominently involved in hydrophobic
contacts mainly with residues that were in close proximity e.g.
TYR110, ARG108, ARG93 and ALA56 etc (see Fig. S-4 in ESI). Never-
theless, the compound 3e, 3j and 3k appeared as potential agents
for further studies.
In conclusion, the 3-alkynyl substituted 2-chloroquinoxaline
framework has been explored as a potential template for the
design of molecules targeting COVID-19. Initially few representa-
tive molecules were evaluated in silico via assessing their binding
affinities against the N-terminal RNA-binding domain (NTD) of
N-protein of SARS-CoV-2. The encouraging outcome of this study
prompted further evaluation of these molecules. Thus building of
a small library of molecules based on the said template was under-
Table 4
Docking of molecules into N-terminal RNA-binding domain (NTD) of N-protein of
SARS-CoV-2.a
Compound
AutoDock Vina score (Kcal/mol)
3a
3b
3c
À5.5
À5.6
À4.7
À5.0
À6.4
À5.5
À5.6
À5.6
À5.3
À6.2
À6.5
À6.1
À5.6
3d
taken. Accordingly,
a convenient and environmentally safer
3eb
method has been developed based on Cu-catalyzed CAC bond
forming reaction under ultrasound irradiation. The methodology
allowed a rapid synthesis of 3-alkynyl substituted 2-chloroquinox-
aline via the coupling of 2,3-dichloroquinoxaline with commer-
cially available terminal alkynes in the presence of CuI, PPh3 and
K2CO3 in PEG-400. This simple and straightforward method does
not involve the use of bi-metallic salts as catalysts and afforded a
range of target compounds smoothly. All these compounds were
assessed in silico to establish a virtual SAR (Structure Activity Rela-
tionship) within the series. Future study will focus more on valida-
tion of identified compounds in vitro and in vivo. To sum up, the
3f
3g
3h
3i
3jb
3kb
3lb
Compound Cc
a
b
c
See the footnote of Table 1.
3e, 3j, 3k and 3l was D-1, D-2, D-3 and D-4 in Table 1, respectively.
Reference compound (see Table 1).

S. Shahinshavali et al. / Tetrahedron Letters 61 (2020) 152336
7
Fig. 7. (A) The 2D and (B) 3D interaction diagram of compound 3c with N-protein of SARS-CoV-2. (C) The surface representation of docked 3c into the N-protein of SARS-CoV-
2 where no interaction found at the bottom site. (D) The surface representation of docked 3k into the N-protein of SARS-CoV-2 showing hydrophobic contacts at the bottom
site.
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Declaration of Competing Interest
The authors declare that they have no known competing finan-
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Acknowledgement
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Appendix A. Supplementary data
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