Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones

Article abstract of DOI:10.1016/j.bmc.2019.115130

The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of supe

Full text of DOI:10.1016/j.bmc.2019.115130

Journal Pre-proofs
Superior inhibition of influenza virus hemagglutinin-mediated fusion by in-
dole-substituted spirothiazolidinones
Gökçe Cihan-Üstündağ, Muhammet Zopun, Evelien Vanderlinden, Elif
Ozkirimli, Leentje Persoons, Gültaze Çapan, Lieve Naesens
PII:
S0968-0896(19)31025-9
https://doi.org/10.1016/j.bmc.2019.115130
BMC 115130
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
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22 June 2019
12 September 2019
18 September 2019
Please cite this article as: G. Cihan-Üstündağ, M. Zopun, E. Vanderlinden, E. Ozkirimli, L. Persoons, G. Çapan,
L. Naesens, Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted
spirothiazolidinones, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115130
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Superior inhibition of influenza virus
hemagglutinin-mediated fusion by indole-
substituted spirothiazolidinones
Leave this area blank for abstract info.
Gökçe Cihan-Üstündağa, Muhammet Zopuna, Evelien Vanderlindenb, Elif Ozkirimlic, Leentje Persoonsb,
Gültaze Çapana and Lieve Naesensb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, Istanbul 34116, Turkey
bRega Institute for Medical Research, KU Leuven, Department of Microbiology, Immunology and Transplantation, B-3000 Leuven,
Belgium
cChemical Engineering Department, Bogazici University, Istanbul 34342 Turkey

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-
substituted spirothiazolidinones
Gökçe Cihan-Üstündağa, Muhammet Zopuna, Evelien Vanderlindenb, Elif Ozkirimlic, Leentje Persoonsb,
Gültaze Çapana and Lieve Naesensb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Istanbul University, Istanbul 34116, Turkey
bRega Institute for Medical Research, KU Leuven, Department of Microbiology, Immunology and Transplantation, B-3000 Leuven, Belgium
cChemical Engineering Department, Bogazici University, Istanbul 34342 Turkey
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by
endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented
by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors
with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC50
value against influenza A/H3N2 virus of 1 nM and selectivity index of almost 2000. Resistance
data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA
binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to
achieve superior inhibitors of HA-mediated fusion.
Available online
Keywords:
Indole
Spirothiazolidinone
Influenza virus
Hemagglutinin
Fusion
2009 Elsevier Ltd. All rights reserved.
Antiviral
Inhibitor
In silico
———
 Corresponding author. Tel.: +32-16-322098; e-mail: lieve.naesens@kuleuven.be

1. Introduction
Small molecule inhibitors of HA refolding have been known
for more than two decades, with diverse scaffolds reported in the
literature (reviewed in:15). More recently, several broadly
neutralizing anti-HA antibodies (bnAbs) were found to interfere
with HA-mediated fusion21, renewing also the relevance of small
molecules to prevent this event. For example, one small molecule
was rationally designed to optimally fit in the HA binding pocket
of a group 1-specific bnAb.22 Thus far, arbidol (Fig. 1) is the only
influenza virus fusion inhibitor that is commercially available in
Russia (generic name: umifenovir) and China; it is under clinical
evaluation in the USA.23 Besides its inhibitory effect on HA-
mediated fusion, arbidol exhibits a more general membrane-
perturbing effect, explaining its broad activity against several
viruses distinct from influenza virus.24 A recent cocrystallo-
graphic study revealed the precise binding mode of arbidol within
the stem region of H3 and H7 HAs.16 The arbidol binding pocket
in H3 HA (Fig. 2) partially overlaps with that of tert-butyl
hydroquinone (TBHQ; Fig. 1), a group 2-specific inhibitor.17 For
other fusion inhibitors, the binding mode is less well defined
although evidence was provided by virus resistance, HA
interaction or in silico docking studies. Except for arbidol, all
reported influenza virus fusion inhibitors display narrow activity
against specific HA subtypes, which appears related to the size,
accessibility or charge of their HA binding pocket. In this context,
the “TBHQ pocket” appears particularly important, since it is the
binding site for TBHQ and arbidol in H3 HA, and proposedly also
for some aniline-based inhibitors with HA group 1 (H1 and H5)
specificity.25
Seasonal influenza is a major public health problem caused by
globally circulating influenza
A and B viruses. Serious
complications are common in fragile persons such as infants,
elderly or chronically ill individuals.1 Besides, novel influenza A
viruses can emerge from zoonotic reservoirs and cause severe
pandemics such as occurred in 2009 (pH1N1) and at three
occasions in the 20th century, namely in 1918 (Spanish influenza,
H1N1), 1957 (H2N2) and 1968 (H3N2).2 Two avian subtypes that
are causing severe infections in humans, i.e. H5N1 and H7N9, are
feared for their potential to evolve into a human-transmissible
virus. The annual vaccination against seasonal influenza is only
partially effective and prone to antigenic mismatch.3 In the past
years, vaccine effectiveness was particularly low for the H3N2
subtype4, which also tends to cause more severe disease compared
to pH1N1.5
The influenza A virus subtype is determined by two antigens
embedded in the viral envelope: hemagglutinin (HA) and
neuraminidase (NA). The clinical benefit of NA inhibitors
(oseltamivir and zanamivir) was recently reviewed6, yet is
threatened by potential drug resistance as was globally seen for
oseltamivir in the period 2007-2009.7 Widespread resistance is
particularly problematic for the M2 channel blockers (amantadine
and rimantadine). Diverse antivirals are in the (pre)clinical
pipeline8,9 and two polymerase inhibitors, favipiravir10 and
baloxavir marboxil11, were recently approved in a few countries.
A strategy preventing HA-mediated entry into host cells
appears attractive to shut-off the viral and inflammatory cascades,
yet is complicated by pronounced HA sequence variation. The
eighteen HA subtypes are classified in two groups. The above-
mentioned H1, H2 and H5 HAs fall in group 1 whereas the H3 and
H7 HAs belong to group 2. The entry process starts with virus
attachment, which requires recognition of sialylated cell surface
glycans by the receptor binding domain in the globular head of
HA.12 After endocytic virus uptake followed by gradual endosome
acidification from pH ~6 to pH ~5, the low pH induces drastic
conformational rearrangements in HA. As
a result, the
hydrophobic fusion peptide leaves its buried position in the HA
stem to provoke fusion of the viral and endosomal membranes.13
The fusion pore allows migration of the viral genome segments
into the cytoplasm, followed by their nuclear import to initiate
transcription and replication.14,15
(A)
Figure 2. Reported HA binding mode for arbidol and TBHQ. (A) Binding
site of arbidol in complex with trimeric H3 HA16, relative to location of the
fusion peptide (of which the N-terminus is marked by a green sphere). The
three HA protomers are shown in lavender, blue and beige ribbons. Each of the
three arbidol molecules (red vdW spheres) binds at the interface between two
protomers. (B) Binding mode of arbidol (in red) and TBHQ (in white) in
reported crystal structures16,17, showing their interactions with monomer 1
(HA1 in violet and HA2 in blue) and monomer 2 (HA1 in orange and HA2 in
pink). The Figure presents the H3 HA protein and arbidol ligand in PDB entry
5T6N, onto which the binding pose of TBHQ (from PDB entry 3EYM) was
superimposed.
OH
N
O
O
HO
Br
N
S
OH
TBHQ
Arbidol
(B)
N
We previously identified a class of influenza virus fusion
inhibitors with N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide
(spirothiazolidinone) scaffold and strong activity against H3 HA.18
The lead compound, B1, is shown in Fig. 1. Combined A/H3N2
resistance, biochemical and in silico docking experiments
indicated that the spirothiazolidinone moiety binds to H3 HA in
the same pocket as TBHQ.17 In addition, the imidazo[2,1-
b]thiazole part of B1 was predicted to form favorable hydrophobic
interactions. Subsequent SAR analyses showed that this moiety
could be replaced by 2-hydroxyphenyl (B2)18, 1-adamantyl (B3)19
or 5-chloro-2-hydroxyphenyl (B4)20 (Fig. 1), although antiviral
efficacy and selectivity were not drastically changed. In the
N
H
S
Ar
N
Ar :
N
B1
O
S
Cl
O
OH
OH
B3
B4
B2
Figure 1. Chemical structures of influenza virus fusion inhibitors relevant
for this study. (A) Arbidol and tert-butylhydroquinone (TBHQ) which have
been cocrystallized with H3 HA16,17; (B) H3 HA-specific spirothiazolidinone
compounds discovered by our group and for which a few variations in the aryl
(Ar) moiety were explored as indicated18-20
.

present study, we report the chemical synthesis and influenza virus
inhibition for a new and particularly potent series of analogues
carrying an indole function. This core was found to offer an
impressive 4000-fold gain in antiviral activity compared to the
original lead B1. The best analogue, 5f, displays nanomolar
activity against influenza A/H3N2 virus and classifies as the most
potent influenza virus fusion inhibitor ever described. Resistance
data with mutant H3 HA proteins were rationalized by in silico
docking, showing partial binding similarities between 5f and
arbidol or TBHQ. We propose that 5f owes its superior anti-
A/H3N2 activity to optimal interactions with H3 HA, combined
with a capability for endosomal accumulation.
together with the alkyl substituents on the spiroheterocycle. The
spirodecane C2-H2 protons of 4a-j resonated at about  3.55-3.71
ppm as singlets (except for the spectrum of compound 4i in which
C2-H2 protons were observed as separate doublets due to the
geminal interaction) and C2-H protons of 5a-j resonated at about
 3.84-3.99 ppm as quartets. 13C-NMR (APT) experiments and
two-dimensional HSQC and HMBC experiments allowed
complete assignment of proton and carbon signals. Resonances at
 162.19-163.58 ppm and  168.33-171.09 ppm regions were
assigned to the indolecarboxamide carbonyl and spirocyclic
lactam carbonyl groups, respectively. This assignment was
substantiated by the cross peaks between the spirodecane C2-H, 2-
CH3 protons and lactam C=O carbon and the cross peak between
the indole-3-CH3 protons and indolecarboxamide C=O carbon in
the HMBC spectrum of compound 5e.
2. Results and discussion
Deprotonated [M-H]- or protonated [M+H]+ ions observed in
the ESI-MS confirmed the molecular weight of the compounds.
Additional [(M-H)+2]- or [(M+H)+2]+ isotope peaks
approximately one-third the intensity of the molecular ion peak
were further observed because of the 37Cl isotope.
2.1. Chemical synthesis and structural characterization
The synthetic pathways for spirothiazolidinones 4a-j and 5a-j
are shown in Scheme 1. The diazonium salt, obtained from 4-
chloroaniline and NaNO2 plus HCl, was reacted with ethyl 2-ethyl-
3-oxobutanoate to achieve compound 1 according to the Japp-
Klingemann reaction. The Fischer indole synthesis was carried out
in acidic media to cyclize 1 into ethyl 5-chloro-3-methyl-1H-
indole-2-carboxylate (2). Subsequent exposure of 2 to an excess of
hydrazine hydrate afforded compound 3.26 The target spirocyclic
compounds (4a-j, 5a-j) were synthesized by treatment of the key
intermediate 3 with an appropriate cyclic ketone and mercapto
acids in a one-pot reaction.27
150
100
5b
100
50
50
0
0
1
1
1
1
.
0
1
1
1
1
0
0
0
0
0
0
.
0
1
0
0
.
0
1
0
.
0
150
100
50
100
50
0
5d
Cl
O
O
Cl
iv
Cl
O
iii
i
N
C2H5
N
C2H5
N
O
ii
H
NH2
H
0
1
0
1
1
1
0
0
0
.
0
0
.
0
1
0
0
0
0
0
1
1
1
.
0
.
0
0
0
0
R2
R3
R4
O
Cl
O
Cl
O
Cl
v
R1
150
100
50
N
H
HN
N
N
H
HN NH2
100
50
0
5e
N
HN
N
S
R
S
H
O
O
4b-j
4a
vi
R2
R3
R4
O
Cl
O
0
Cl
R1
1
1
1
1
.
0
0
0
0
0
0
1
N
H
HN
N
.
0
0
0
.
0
N
S
HN N
.
R
0
S
H
O
O
5a
5b-j
150
100
50
5f
100
50
0
Scheme 1. Synthesis of 4a-j and 5a-j. Reagents and conditions: (i) 7%
NaNO2, EtOH, conc. HCl, 0 oC; (ii) ethyl 2-ethyl-3-oxo-butanoate, KOH,
o
EtOH, 0 C; (iii) conc. HCl, reflux, 4h; (iv) H2NNH2.H2O, EtOH, reflux, 6h;
(v) cyclopentanone/4-(non)substituted cyclohexanone, mercaptoacetic acid,
dry toluene, reflux, 5–6 h; (vi) cyclopentanone/4-(non)substituted
cyclohexanone, 2-mercaptopropionic acid, dry toluene, reflux, 5-6 h.
Compounds: 4b, 5b: R,R1,R2,R3,R4=H; 4c, 5c: R=CH3; R1,R2,R3,R4=H; 4d, 5d:
R1=CH3; R,R2,R3,R4=H; 4e, 5e: R2=CH3; R,R1,R3,R4=H; 4f, 5f: R2=C2H5;
R,R1,R3,R4=H; 4g, 5g: R2=C3H7; R,R1,R3,R4=H; 4h, 5h: R2=C(CH3)3;
R,R1,R3,R4=H; 4i, 5i: R1,R3,R4=CH3; R,R2=H; 4j,5j: R2=C6H5; R,R1,R3,R4=H).
0
1
1
1
1
.
0
0
0
0
0
0
0
1
.
0
.
0
0
.
0
150
100
50
Ribavirin
100
50
0
0
The new compounds were characterized by combustion
analysis, IR, 1H-NMR, 13C-NMR (APT), 2D-NMR (HSQC,
HMBC) and electrospray ionization mass spectrometry (ESI-MS).
The shifts observed in the amide carbonyl bands (1645-1685 cm-
1) when compared to that of 3 (1606 cm-1) and the presence of new
lactam carbonyl absorptions (1692-1707 cm-1) characteristic for 4-
thiazolidinones in the IR spectra of 4, 5 proved the aimed
cyclization. The NH2 resonance of the intermediate hydrazide (3)
at  4.53 ppm disappeared in the 1H-NMR spectra of 4 and 5 as the
group participated in ring formation and new resonances assigned
to the spirodecane system appeared in the  0.75-2.55 ppm region
1
0
0
0
1
1
Concentration (µM)
Figure 3. Dose-response curves. The graphs, generated with Graphpad
Prism, show the dose-dependent inhibition of virus-induced CPE (O; left axis)
versus cytotoxicity (●; right axis), both determined by MTS cell viability assay.
Data points are the mean ± SEM (N=4).

Table 1. Antiviral activity and selectivity in MDCKa cells infected with influenza A/H3N2 virus.
Compound
R
R1
R2
R3
R4
EC50b (µM) against influenza A/H3N2 virus
Cytotoxicity (µM)
MCCc
CC50
d
CPE
MTS
Analogues lacking the 2-methyl in the spiro ring
4a
4b
4c
4d
4e
4f
4g
4h
4i
-
H
Me
H
H
H
H
H
H
H
-
H
H
Me
H
H
H
H
Me
H
-
H
H
H
Me
Et
Pr
t-Bu
H
-
H
H
H
H
H
H
H
Me
H
-
H
H
H
H
H
H
H
Me
H
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
≥20
≥100
≥100
≥4
0.8
0.8
>100
5.0
>100
>100
>100
>100
>100
94
1.3
2.1
>100
2.4
4j
Ph
Analogues carrying the 2-methyl in the spiro ring
5a
5b
5c
5d
5e
5f
5g
5h
5i
-
H
Me
H
H
H
H
H
H
H
-
-
H
H
Me
H
H
H
H
Me
H
-
-
H
H
-
H
H
H
H
H
H
H
Me
H
-
-
H
H
H
H
H
H
H
Me
H
-
>100
0.085 ± 0.041
>100
0.031 ± 0.023
0.063 ± 0.027
0.0012 ± 0.0008
>100
>100
0.063 ± 0.022 [92]
>100
>100
≥4
≥20
≥4
0.87
3.0
0.8
≥4
20
1.0
≥100
>100
5.8
88
H
0.023 ± 0.019 [1435]
0.045 ± 0.014 [62]
0.00077 ± 0.00023 [1948]
>100
33
Me
Et
Pr
t-Bu
H
2.8
1.5
1.9
0.9
85
>100
>100
>100
5.0 ± 2.3
>100
>100
>100
5j
B1
Ph
-
2.3
>100
3.2 ± 2.3 [>31]
Ribavirin
Amantadine
8.4 ± 1.6
3.8 ± 0.2
4.6 ± 3.2
1.2 ± 0.2
≥100
>200
>100
>200
a Madin-Darby canine kidney cells.
b Antiviral EC50 (mean ± SEM; N = 4): compound concentration producing 50% inhibition of virus-induced CPE, as determined by microscopy or by the MTS cell
viability assay. The value in square brackets represents the selectivity index or ratio of CC50 to EC50 based on the MTS assay.
c Minimum compound concentration that causes a microscopically detectable alteration of normal cell morphology.
d 50% cytotoxic concentration based on the cell viability MTS assay.
than the corresponding imidazo[2,1-b]thiazole analogue (B1; EC50:
4 µM).18
2.2 SAR analysis for inhibition of influenza virus replication
Several compounds were devoid of cytotoxicity at 100 µM (the
The newly synthesized compounds were evaluated in Madin-
highest concentration tested) (Table 1). For most other molecules,
Darby canine kidney (MDCK) cells infected with influenza
there was consensus between the cytotoxicity values obtained by
A/H1N1, A/H3N2 or B virus. Antiviral activity was estimated from
the microscopic (MCC) or MTS (CC50) assay. There was no
protection against virus-induced cytopathic effect (CPE) using
correlation between antiviral and cytotoxic activity. For 5b, 5d, 5e
microscopic scoring and MTS-based cell viability assay; both
and 5f, the superior activity and selectivity are evident from the
assays yielded very similar antiviral EC50 values. These methods
dose-response curves shown in Fig. 3.
were also used to monitor compound cytotoxicity. Table 1 shows
the clearly delineated SAR for inhibition of influenza A/H3N2
virus. There was no effect on influenza A/H1N1 or B virus (data
2.3. Inhibitory effect on HA-mediated membrane fusion
not shown). The four compounds showing strong activity, i.e. 5b,
5d, 5e and 5f, all carry a methyl substituent at position 2- of the
spirocyclic ring system; their counterparts lacking this moiety, i.e.
4b, 4d, 4e and 4f, were inactive (Table 1). The most active analogue
5f, with an 8-ethyl function, had an antiviral EC50 value as low as 1
nM and an impressive selectivity index (ratio of CC50 to EC50; both
based on the MTS assay) of almost 2000. Its activity was 27-, 54-
and 74-fold superior compared to the analogues bearing,
respectively, a 7-methyl (5d), 8-methyl (5e) or no substituent (5b)
on the spiro ring. Moving the methyl group to position 6- (5c) was
deleterious. Also, a larger substituent at position 8- was not
tolerated, i.e. in the compounds carrying a propyl (5g), tert-butyl
(5h) or phenyl (5j). Compound 5a bearing a spiro-fused
cyclopentane ring (instead of cyclohexane) was also devoid of
antiviral activity. The SAR of the indole-spirothiazolidinones
nicely agrees with what we previously reported for the imidazo[2,1-
b]thiazole; 2-hydroxyphenyl18; 1-adamantyl19; and 5-chloro-2-
hydroxy-phenyl20 series. Still, the indole moiety provided an
impressive gain in anti-influenza virus activity since indole
compound 5f (antiviral EC50: 1 nM) was 4000-fold more potent
We next evaluated the compounds in the polykaryon assay, in
which cell-cell fusion is monitored when influenza virus HA-
expressing cells are briefly exposed to a buffer at pH 5. In cells
transfected with wild-type (WT) H3 HA, very comparable EC50
values were noted for the four indole derivatives tested, i.e. 5b, 5d,
5e and 5f, and their imidazo[2,1-b]thiazole analogue B1 (Table 2).
As evident from the dose-response curves in Fig. 4A, the
spirothiazolidinone compounds had stronger inhibitory effect on
polykaryon formation than TBHQ (~10-fold less active) and arbidol
(~40-fold less active). We also tested activity against two mutant
forms of H3 HA, which were previously identified when we
passaged A/H3N2 virus in cell culture under selective pressure of
B1.18 All five spirothiazolidinone molecules were highly impacted
by HA mutations E572K and D1122N (Table 2 and photographs in
Fig. 4B). [Note: throughout the text, suffix 1 corresponds to
residues from HA1 and suffix 2 to residues from HA2]. Resistance
of the E572K mutant is explained by our model showing
involvement of E572 in compound binding (see below). The E572K
mutant HA was cross-resistant to the structurally distinct inhibitor

effect against the E572K and D1122N-mutant forms of H3 HA (representative
photographs; original magnification 200x).
arbidol, but showed unchanged sensitivity to TBHQ (Fig. 4B). The
other substituted residue, D1122, lies in the vicinity of the HA
fusion peptide with which this aspartic acid residue is forming
hydrogen bonds.28 Mutation D1122N was shown29 to increase the
fusion pH from 5.2 for WT-HA to 5.6 for the mutant (Table 2). This
means that HA refolding occurs at higher pH, rendering the mutated
protein insensitive to diverse fusion inhibitors, including
TBHQ.17,30
One striking observation was the marked potency shift between
the HA-polykaryon assay and antiviral assay in MDCK cells. For
indole compound 5f, the difference between both assays was ~900-
fold (= ratio between its EC50 value against A/H3N2 virus in Table
1 and EC50 value against WT-HA in Table 2). This shift is not
explained by differences in the H3 HA protein sequences of
A/HK/7/87 and A/X-31, used in the antiviral and polykaryon
assays, respectively, since 5f exhibited similar potency against both
virus strains (EC50 value in the CPE reduction assay: 7-fold lower
for A/HK/7/87 compared to A/X-31; data not shown). The
pronounced shift in EC50 value between the antiviral assay and
polykaryon assay was also seen with the other three indole
compounds 5b, 5d and 5e. In contrast, imidazo[2,1-b]thiazole
compound B1 displayed very similar EC50 values in both assays.
We hypothesize that the basic indole moiety in compound 5f is
protonated at low pH, leading to a positively charged indolium
cation that may get trapped in the endosomes. A similar indolium
ion trapping phenomenon is observed in pH-sensitive cyanine
dyes.31 This plausibly results in high intra-endosomal compound
concentrations for 5f and amplification of its inhibitory effect on
Table 2. Inhibitory effect in the polykaryon assay with WT or
mutanta forms of H3 HA.
Compound
EC50b (µM)
HA-WT
pH: 5.2c
HA-E572K
pH: 5.2c
HA-D1122N
pH: 5.6c
5b
5d
5e
5f
B1
TBHQ
Arbidol
1.3 ± 0.2
1.4 ± 0.2
2.1 ± 0.8
0.88 ± 0.15
1.1 ± 0.2
9.9 ± 0.4
40 ± 11
>25
>25
≥13
>25
>25
>25
≥21
>25
>25
9.0 ± 2.6
12 ± 3
>200
>400
>200
a The two mutations in the HA2 subunit (E572K and D1122N) were previously entering virus. For comparison, the imidazo[2,1-b]thiazole ring
identified in influenza A/H3N2 viruses selected for resistance to imidazo[2,1-
system in compound B1 does not possess a basic nitrogen.
b]thiazole analogue B1.18 The mutations were introduced in an HA-expression
plasmid to perform the polykaryon assay.
b EC50 (mean ± SEM; N = 3): compound concentration at which the number of
polykaryons was 50% relative to the number observed in the no compound 2.4 Molecular docking studies
control.
c Fusion pH (values reported in18): pH at which the number of polykaryons was
50% relative to the number seen at pH 4.9.
In the TBHQ- (PDB ID: 3EYM) and arbidol- (PDB ID: 5T6N)
bound crystal structures with H3 HA, the two ligands bind to the
trimeric HA stem in an overlapping hydrophobic cavity situated at
the interface between two protomers (Fig. 2A), with three ligand
molecules bound per HA trimer. The inhibitors act as a glue to
prevent the conformational rearrangement of HA required for
membrane fusion. As noted previously16, the overall structural
arrangement is quite similar in the two cocrystal structures and H3
HA apo form. Comparison of the arbidol and TBHQ cocrystal
structures shows that the binding site residues also assume similar
conformations, except for the R542 to N602 loop from monomer 1,
which moves by about 1.5 Å RMSD, and E572 and K582 sidechains.
These residues move away from the pocket to accommodate the
larger arbidol molecule. In addition, the sidechain of K3071 from
monomer 1 points towards the amine group of arbidol while
pointing away from the pocket in the TBHQ-bound structure.
(A)
5b
100
5d
5e
5f
50
B1
Arbidol
TBHQ
0
0.1
1
10
100
µM
WT
E572K
D1122N
(B)
The tert-butyl moiety of TBHQ (Fig 2B, ligand shown in white)
makes hydrophobic interactions with residues I291, L982 and A1012
of protomer 1, while the hydroquinone part pointed toward the
solvent is surrounded by the charged residues R542 and E572 of
protomer 1 and E972 of protomer 2. Arbidol (Fig. 2B, ligand shown
in red) occupies the same hydrophobic pocket through its
thiophenyl group, while its substituted indole ring occupies the
pocket lined by E572 and K582 (protomer 1) and E972 (protomer 2).
The indole makes hydrogen bonding and π-cation interactions with
these residues as well as the aromatic residues W922 and F2941
(both in protomer 1).
25 µM 5f
25 µM B1
200 µM arbidol
400 µM TBHQ
No compound
5f was docked into the two H3 HA structures (PDB ID: 3EYM
and 5T6N) using an induced fit protocol that allows movement for
ligand atoms and protein sidechain atoms surrounding the ligand.
As expected given the resemblance between the two starting
structures, the docked poses of 5f were similar with comparable
Glide Gscores of around -8 kcal/mol. In both cases the azaspiro
group inserts into the hydrophobic pocket and the indole group is
oriented towards the pocket mouth, which is lined by a network of
ionizable residues. In the prediction (Fig. 5) starting from the
arbidol-bound structure (PDB ID: 5T6N), the azaspiro group of 5f
Figure 4. Inhibition in the HA polykaryon assay. (A) Dose-response curves
for inhibition of wild-type (WT) H3 HA. Data points are the mean of three
independent experiments. (B) The spirothiazolidinone compounds have no

engages the hydrophobic pocket with its cyclohexyl ring occupying (see above and Table 1) related to formation of hydrophobic
the same space as the thiophenyl group of arbidol and the interactions with the target protein32, namely (i) the absolute
thiazolidine ring of 5f reaching out to interact with F2941 of requirement for the thiazolidine methyl substituent at position 2 of
monomer 1 similar to the ester moiety of arbidol. Fig. 5A shows a the spirocyclic ring system that interacts with L982 and L1022 in the
comparison of arbidol (red) and 5f (magenta) binding. The indole hydrophobic pocket of protomer 1, and (ii) the favorable small alkyl
groups of 5f and arbidol both point towards the solvent and make substituent at position 8 of the cyclohexane ring, which makes
electrostatic interactions with the charged residues lining the hydrophobic contacts with P2931 and F2941 of protomer 1 (Fig.
pocket, such as E572 of monomer 1, which interacts with the 5C). Hydrophobic interactions with L982 and L1022 were shown to
positively charged indole nitrogen. When the TBHQ-bound be critical for the binding of TBHQ17 and the cyclohexyl group of
structure was used, a similar binding pose was predicted for the N-cyclohexyltaurine33. Arbidol has also been reported to make CH-
spirolactam group of 5f, while the indole plane was slightly rotated π interactions with F2941 of monomer 1.16
to position its positively charged nitrogen toward E972 of monomer
Finally, the structural models offer an explanation for resistance
2 and its carbonyl substituent towards R542 and K582 of monomer
of the E572K-mutant H3 HA protein, as demonstrated in the
1. In both poses, an electrostatic network of interactions surrounded
polykaryon experiments. Substitution of E572 by a lysine would
the indole moiety and it was stabilized by the presence of the
introduce an extra positive charge very close to R542 and K582 of
positively charged indole nitrogen, suggesting that these
the aforementioned electrostatic network (Fig. 5D). Because these
consistently observed interactions are valid.
residues are solvent exposed, mutation E572K is tolerated by the
protein and does not change the fusion pH. However, the mutant
HA is resistant to arbidol and the spirothiazolidinones 5f and B1.
A
B
The resistance factor proved to be higher for 5f than B1, since the
ratio in EC50 values for mutant versus WT was >28 for 5f and 8 for
B1 (Table 2). While both compounds have a carboxamide moiety,
5f (alike arbidol) has an indole nitrogen that makes direct hydrogen
bonding interactions with E572 of monomer 1, which would be lost
upon substitution by lysine. Mutation E572K does not cause
resistance to TBHQ. In the H3 HA trimer complexed with TBHQ,
E572 adopts different orientations in the three ligand binding
pockets17, suggesting that the presence of TBHQ does not stabilize
the local interactions engaged by E572. Our biological data with
E572K mutant HA nicely support the binding mode of our fusion
inhibitors and of arbidol. This was not the case for resistance
mutations detected in a previous study on arbidol, since these were
located at regions remote from the arbidol binding pocket identified
by X-ray studies.16,34
D
C
3. Conclusion
We report a class of indole-substituted spirothiazolidinone
inhibitors of HA-mediated fusion with unprecedented nanomolar
activity against influenza A/H3N2 virus. The pronounced potency
shift between the HA polykaryon versus antiviral assay indicates
that the indole substituent in these inhibitors might lead to
endosomal accumulation, a favorable characteristic that has not
been recognized with fusion inhibitors before. Our inhibitors
proved inactive against E572K-mutant HA protein, consistent with
in silico predictions of their HA binding mode. While having an
overlapping binding pocket with the known fusion inhibitors
TBHQ and arbidol, the indole-substituted spirothiazolidinones
have far higher inhibitory activity with obvious relevance for
antiviral drug development.
Figure 5. Predicted HA binding mode of 5f and B1. The molecules were
docked in the arbidol-H3 HA cocrystal structure (PDB entry: 5T6N) between
monomer 1 (chain C and D) and monomer 2 (chain E and F). (A, B) Overlay of
5f (magenta) versus arbidol (red; panel A) or B1 (green; panel B). The protein
residues are colored according to HA chain (monomer 1: HA1 in violet and HA2
in blue; monomer 2: HA1 in orange and HA2 in pink). (C) Interaction network
between 5f and surrounding residues; H-bonds are shown as dashed lines. (D)
Rationale for the E572K resistance mutation. E572 forms H-bonds with the
carboxamide and indole moiety of 5f.
The same docking exercise with B1 into the arbidol-bound
structure (Fig. 5B, green) predicted that the spiro moiety is again
buried within the hydrophobic pocket while the imidazo[2,1-
b]thiazole group faces the pocket mouth. Since this group lacks a
positively charged nitrogen, it does not make interactions with E572
of monomer 1. However, the electrostatic network that surrounds
the indole of arbidol and 5f is also present around B1. The
electrostatic bridge that forms between E972 of monomer 2 and
K582 of monomer 1 was previously recognized to enhance the
stability of the HA trimer.17 The network of interactions formed by
these ionizable residues is stabilized by the carboxamide group
present in both 5f and B1 (Fig. 5B, magenta and green). This may
explain why, in the polykaryon experiments (see above), the two
compounds have comparable activity towards H3 HA-mediated
fusion. The docking results further clarify two aspects of the SAR
4. Experimental section
4.1. Chemistry
4.1.1. General methods
Melting points were determined in open capillary tubes with a
Buchi B-540 melting point apparatus and are uncorrected.
Microanalyses were performed on a Thermo Finnigan Flash EA
1112 elemental analyzer. IR spectra were recorded in KBr discs
(υmax in cm-1) on a Shimadzu IRAffinity-1 FTIR spectrophotometer.
1H-NMR (DMSO-d6), 13CNMR (APT) (DMSO-d6) and
heteronuclear correlation 1H-13C (HSQC, HMBC) (DMSO-d6)

spectra were run on VarianUNITYINOVA (500 MHz) instrument.
White needles (38.3%); mp 287-288 °C; IR(KBr): υmax 3244,
Chemical shifts are reported as  (ppm) relative to TMS as internal 3159 (N-H), 1707, 1654 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
standard and coupling constants (J) are given in hertz (Hz). MS 1.00-1.12 (1H, m, CH2-sp.), 1.42-1.50 (2H, m, CH2-sp.), 1.58 (1H,
(ESI+/-) were determined on a Finnigan LCQ Advantage Max mass d*, J=13.1, CH2-sp.), 1.75 (2H, d*, J=13.5, CH2-sp.), 1.80-1.90
spectrometer (*: broad/distorted, ind.:indole, ar.:aromatic, sp.: (4H, s*, CH2-sp.), 2.48-2.52 (5H, m, 3-CH3-ind., DMSO-d6), 3.64
spirodecane).
(2H, s, C2-H2-sp.), 7.24 (1H, dd, J=8.5, 1.9, H6-ind.), 7.43 (1H, d,
J=8.3, H7-ind.), 7.70 (1H, d, J=1.9, H4-ind.), 10.10 (1H, s, CONH),
11.50 (1H, s, NH); 13C-NMR (APT) (DMSO-d6/125 MHz):  10.39,
10.46 (3-CH3-ind.), 23.61, 24.78, 28.62, 28.71, 37.82 (CH2-sp.),
72.91 (C5-sp.), 114.41, 114.51 (C7-ind.), 115.54 (C3-ind.), 119.93,
119.97 (C4-ind.), 124.67 (C5-ind.), 124.97, 125.08 (C6-ind.),
127.86 (C2-ind.), 129.53 (C3a-ind.), 134.92 (C7a-ind.), 162.25
(CO-NH), 168.39 (CO-sp.). MS (ESI-) m/z (%): 376.1 ([M-H]-,
100), 378.1 ([(M-H)+2]-, 37.7). Anal. Calcd for C18H20ClN3O2S
(377.88): C, 57.21; H, 5.33; N, 11.12. Found: C, 57.23; H, 5.20; N,
11.21.
4.1.2. Ethyl 2-[2-(4-chlorophenyl)hydrazinylidene]butanoate (1)
To a solution of 4-chloroaniline (0.02 mol) in ethanol (10 mL),
water (10 mL) and conc. HCl (6mL), 7% aqueous NaNO2 solution
(30mL) was added dropwise at 0 C with stirring. The resulting
°
solution of diazonium salt was poured into a cooled (0 °C) mixture
of ethyl 2-ethyl-3-oxobutanoate (0.02 mol), ethanol (10 mL), water
(10 mL), and KOH (5.4 g) while stirring. The resulting mixture was
refrigerated overnight. The red oily solid residue thus obtained was
separated, washed with water, and used without further purification.
4.1.8.
5-Chloro-3-methyl-N-(6-methyl-3-oxo-1-thia-4-azaspiro-
4.1.3. Ethyl 5-chloro-3-methyl-1H-indole-2-carboxylate (2)
[4.5]decan-4-yl)-1H-indole-2-carboxamide (4c)
A solution of 1 (0.02 mol) in conc. HCl (20 mL) was heated
under reflux on water bath (70-80 C) for 4 h. The crude product
°
Beige crystals (51.3%); mp 270-272 °C; IR(KBr): υmax 3327,
3240 (N-H), 1697, 1670 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
0.98 (3H, d, J=5.4, 6-CH3-sp.), 1.15-1.23 (2H, m, CH/CH2-sp.),
1.33-1.42 (1H, m, CH/CH2-sp.), 1.56 (1H, d, J=6.3, CH/CH2-sp.),
1.64 (1H, d, J=6.3, CH/CH2-sp.), 1.72 (1H, d, J=13.6, CH/CH2-sp.),
1.93-2.01 (3H, m, CH/CH2-sp.), 2.49-2.50 (5H, m, 3-CH3-ind.,
DMSO-d6), 3.57 (2H, s, C2-H2-sp.), 7.24 (1H, dd, J=8.5, 1.9, H6-
ind.), 7.44 (1H, d, J=8.3, H7-ind.), 7.70 (1H,d, J=1.9, H4-ind.),
10.05, 10.08 (1H, 2s, CONH), 11.50, 11.52 (1H, 2s, NH); 13C-NMR
(APT) (DMSO-d6/125 MHz):  10.40, 10.46 (3-CH3-ind.), 16.90
(6-CH3-sp.), 23.50, 25.39, 29.35, 32.08, 39.15 (CH2-sp.), 39.32
(C6-sp.), 77.08 (C5-sp.), 114.41, 114.50 (C7-ind.), 115.79 (C3-
ind.), 119.93, 119.97 (C4-ind.), 124.68 (C5-ind.), 124.99, 125.10
(C6-ind.), 127.86 (C2-ind.), 129.52 (C3a-ind.), 134.89 (C7a-ind.),
162.51, 163.58 (CO-NH), 168.48, 169.41 (CO-sp.). MS (ESI+) m/z
(%): 391.9 ([M+H]+, 100), 394.0 ([(M+H)+2]+, 32.2). Anal. Calcd
for C19H22ClN3O2S (391.91): C, 58.23; H, 5.66; N, 10.72. Found:
C, 58.70; H, 5.48; N, 10.66.
was filtered off, washed with water until tested neutral to litmus and
used without further purification.
4.1.4. 5-Chloro-3-methyl-1H-indole-2-carbohydrazide (3)26
A mixture of 2 (0.02 mol), ethanol (20 mL) and H2NNH2.H2O
(98%, 8 mL) was heated under reflux on a water bath (70-80 °C) for
6 h. The resulting yellowish-brown solid was filtered off and
recrystallized from ethanol. Mp 271-272 °C; IR(KBr): υmax 3296
(N-H), 1606 (C=O); 1H-NMR (DMSO-d6/500MHz):  2,44 (3H, s,
3-CH3), 4,53 (2H, s, NH2), 7,18 (1H, dd, J=8,8 ;2,0 Hz, H6), 7,38
(1H, d, J=8,8 Hz, H7), 7,63 (1H, d, J=2,4 Hz, H4), 9,19 (1H, s,
CONH), 11,32 (1H, s, NH).
4.1.5. General procedure for the synthesis of 5-chloro-3-methyl-N-
(2,6,7,8,9-(non)substituted-3-oxo-1-thia-4-azaspiro-
[4.4]nonan/decan-4-yl)-1H-indole-2-carboxamides (4a-4j, 5a-5j)
A
mixture of
3
(0.0025 mol), an appropriate
cyclohexanone/cyclopentanone (0.003 mol) and mercaptoacetic
4.1.9.
5-Chloro-3-methyl-N-(7-methyl-3-oxo-1-thia-4-azaspiro-
acid or 2-mercaptopropionic acid (0.01 mol) was refluxed in 20 mL
[4.5]decan-4-yl)-1H-indole-2-carboxamide (4d)
o
dry toluene for 5–6 h (150-200 C) using a Dean-Stark water
White crystals (45.0%); mp 285-286 °C; IR(KBr): υmax 3340 (N-
H), 1701, 1670 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.76-0.82
(1H, m, CH/CH2-sp.), 0.91 (3H, d*, J=4.8, 7-CH3-sp.), 1.40-1.88
(8H, m, CH/CH2-sp.), 2.48-2.52 (5H, m, 3-CH3-ind., DMSO-d6),
3.64 (2H, s, C2-H2-sp.), 7.20 (1H, dd, J=8.7, 1.9, H6-ind.), 7.43
(1H, d, J=8.3, H7-ind.), 7.70 (1H,d, J=1.9, H4-ind.), 10.10 (1H, s,
CONH), 11.40 (1H, s, NH); 13C-NMR (APT) (DMSO-d6/125
MHz):  10.38, 10.45 (3-CH3-ind.), 22.75 (7-CH3-sp.), 23.15, 28.70
(CH2-sp.), 30.29 (C7-sp.), 33.47, 37.19, 46.00 (CH2-sp.), 72.90
(C5-sp.), 114.42, 114.51 (C7-ind.), 115.64 (C3-ind.), 119.92,
119.97 (C4-ind.), 124.68 (C5-ind.), 124.99, 125.11 (C6-ind.),
127.81 (C2-ind.), 129.53 (C3a-ind.), 134.92 (C7a-ind.), 162.24
(CO-NH), 168.34 (CO-sp.). Anal. Calcd for C19H22ClN3O2S
(391.91): C, 58.23; H, 5.66; N, 10.72. Found: C, 57.96; H, 5.75; N,
10.75.
separator. Excess toluene was evaporated in vacuo. The resulting
residue was triturated with saturated NaHCO3 solution until CO2
evolution ceased and was allowed to stand overnight or in some
cases refrigerated until solidification. The solid thus obtained was
washed with water, dried, and recrystallized from ethanol or
ethanol–water.
4.1.6. 5-Chloro-3-methyl-N-(3-oxo-1-thia-4-azaspiro[4.4]nonan-
4-yl)-1H-indole-2-carboxamide (4a)
White powder (55.0%); mp 269-272 °C; IR(KBr): υmax 3304 (N-
H), 1697, 1651 (C=O); 1H-NMR (DMSO-d6/500MHz):  1.65-1.73
(4H, m, CH2-sp.), 1.89-1.93 (2H, m, CH2-sp.), 2.20 (2H, s*, CH2-
sp.), 2.49-2.51 (6H, m, 3-CH3-ind., DMSO-d6), 3.71 (2H, s, C2-H2-
sp.), 7.23 (1H, dd, J=8.7, 1.9, H6-ind.), 7.43 (1H, d, J=8.7, H7-ind.),
7.70 (1H, d, J=1.9, H4-ind.), 10.15 (1H, s, CONH), 11.49 (1H, s,
NH); 13C-NMR (APT) (DMSO-d6/125 MHz):  10.36, 10.43 (3-
CH3-ind.), 23.36, 29.41, 29.51, 29.61, 38.92 (CH2-sp.), 76.45 (C5-
sp.), 114.43, 114.53 (C7-ind.), 115.41 (C3-ind.), 119.95, 119.99
(C4-ind.), 124.69 (C5-ind.), 125.00, 125.12 (C6-ind.), 127.82 (C2-
ind.), 129.52 (C3a-ind.), 135.00 (C7a-ind.), 162.28 (CO-NH),
168.36 (CO-sp.). MS (ESI-) m/z (%): 362.2 ([M-H]-, 100), 364.2
([(M-H)+2]-, 29.9). Anal. Calcd for C17H18ClN3O2S (363.86): C,
56.12; H, 4.99; N, 11.55. Found: C, 56.25; H, 5.03; N, 11.33.
4.1.10. 5-Chloro-3-methyl-N-(8-methyl-3-oxo-1-thia-4-azaspiro-
[4.5]decan-4-yl)-1H-indole-2-carboxamide (4e)
White needles (54.1%); mp 264-266 °C; IR(KBr): υmax
1
3248,3161 (N-H), 1701, 1654 (C=O); H-NMR (DMSO-d6/500
MHz):  0.87, 0.92 (3H, 2d, J=6.3, 8-CH3-sp.), 1.10-1.40 (3H, m,
CH/CH2-sp.), 1.52-1.93 (6H, m, CH/CH2-sp.), 2.44-2.50 (5H, m, 3-
CH3-ind., DMSO-d6), 3.64, 3.65 (2H, 2s, C2-H2-sp.), 7.23 (1H, dd,
J=8.7, 1.9, H6-ind.), 7.43 (1H, d, J=8.7, H7-ind.), 7.70 (1H,d, J=1.9,
H4-ind.), 10.13, 10.21 (1H, 2s, CONH), 11.51, 11.57 (1H, 2s, NH);
13C-NMR (APT) (DMSO-d6/125 MHz):  10.43 (3-CH3-ind.),
4.1.7. 5-Chloro-3-methyl-N-(3-oxo-1-thia-4-azaspiro[4.5]decan-
4-yl)-1H-indole-2-carboxamide (4b)

22.53 (8-CH3-sp.), 28.55, 28.62, 28.99 (CH2-sp.), 31.32 (C8-sp.), 4.1.14.
5-Chloro-3-methyl-N-(7,7,9-trimethyl-3-oxo-1-thia-4-
32.08, 37.55 (CH2-sp.), 72.73, 72.95 (C5-sp.), 114.45 (C7-ind.), azaspiro[4.5]decan-4-yl)-1H-indole-2-carboxamide (4i)
115.05, 115.46 (C3-ind.), 119.97 (C4-ind.), 124.65 (C5-ind.),
White powder (42.9%); mp 288-290 °C; IR(KBr): υmax 3321 (N-
125.01 (C6-ind.), 127.88 (C2-ind.), 129.52 (C3a-ind.), 134.91,
H), 1701, 1668 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.77 (1H,
134.96 (C7a-ind.), 162.25, 162.41 (CONH), 168.39, 168.43 (CO-
t, J=12.7, CH/CH2-sp.), 0.92 (6H, s*, 7-(CH3)2-sp.), 1.09 (3H, s*,
sp.). MS (ESI-) m/z (%): 390.2 ([M-H]-, 100), 392.2 ([(M-H)+2]-,
9-CH3-sp.), 1.23-1.37 (2H, m, CH/CH2-sp.), 1.74-1.89 (4H, m,
37.9). Anal. Calcd for C19H22ClN3O2S (391.91): C, 58.23; H, 5.66;
CH/CH2-sp.), 2.48-2.51 (5H, m, 3-CH3-ind., DMSO-d6), 3.55 (1H,
N, 10.72. Found: C, 58.24; H, 5.35; N, 10.78.
d, J=16.1, C2-H2-sp.), 3.68 (1H, d, J=15.7, C2-H2-sp.), 7.24 (1H,
4.1.11. 5-Chloro-N-(8-ethyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4- dd, J=8.4, 2.0, H6-ind.), 7.44 (1H, d, J=8.3, H7-ind.), 7.69 (1H, d,
yl)-3-methyl-1H-indole-2-carboxamide (4f)
J=2.0, H4-ind.), 10.06 (1H, s, CONH), 11.49 (1H, s, NH); 13C-
NMR (APT) (DMSO-d6/125 MHz):  10.35, 10.42 (3-CH3-ind.),
22.55, 26.30, 27.83 (7-(CH3)2, 9-CH3-sp.), 28.82, 32.53 (CH2-sp.),
34.57 (C9-sp.), 45.98, 47.52 (CH2-sp., C7-sp.), 72.21 (C5-sp.),
114.42, 114.51 (C7-ind.), 115.67 (C3-ind.), 119.92, 119.96 (C4-
ind.), 124.69 (C5-ind.), 125.00, 125.11 (C6-ind.), 127.84 (C2-ind.),
129.52 (C3a-ind.), 134.92 (C7a-ind.), 162.33 (CONH), 168.33
(CO-sp.). Anal. Calcd for C21H26ClN3O2S (419.97): C, 60.06; H,
6.24; N, 10.01. Found: C, 59.73; H, 6.06; N, 10.06.
White needles (40.2%); mp 264-265 °C; IR(KBr): υmax 3323 (N-
H), 1701, 1685 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.84 (3H,
t, J=7.3, 8-CH2CH3-sp.), 1.02-1.26 (5H, m, CH/CH2-sp., 8-
CH2CH3-sp.), 1.60-1.96 (6H, m, CH/CH2-sp.), 2.48-2.52 (5H, m, 3-
CH3-ind., DMSO-d6), 3.64, 3.65 (2H, 2s, C2-H2-sp.), 7.23 (1H, dd,
J=8.8, 2.4, H6-ind.), 7.40 (1H, d, J=8.3, H7-ind.), 7.70 (1H,d, J=1.9,
H4-ind.), 10.14, 10.18 (1H, 2s, CONH), 11.50, 11.56 (1H, 2s, NH);
13C-NMR (APT) (DMSO-d6/125 MHz):  10.39, 10.46 (3-CH3-
ind.), 12.05 (8-CH2CH3-sp.), 28.62, 28.71, 29.44, 29.67, 37.53 4.1.15. 5-Chloro-3-methyl-N-(8-phenyl-3-oxo-1-thia-4-azaspiro-
(CH2-sp., 8-CH2CH3-sp.), 37.89 (C8-sp.), 73.03 (C5-sp.), 114.40, [4.5]decan-4-yl)-1H-indole-2-carboxamide (4j)
114.49 (C7-ind.), 115.47 (C3-ind.), 119.94, 119.98 (C4-ind.),
White crystals (61.8%); mp 161-163 °C; IR(KBr): υmax 3325,
124.66 (C5-ind.), 124.94, 125.06 (C6-ind.), 127.90 (C2-ind.),
3194 (N-H), 1705, 1653 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
129.54 (C3a-ind.), 134.92 (C7a-ind.), 162.24 (CONH), 168.40
1.06 (1H, t, J=7.0, CH2-sp.), 1.70 (2H, q, J=12.6, CH2-sp.), 1.90
(CO-sp.). Anal. Calcd for C20H24ClN3O2S (405.94): C, 59.17; H,
(2H, d*, J=12.2, CH2-sp.), 1.99 (2H, d*, J=11.2, CH2-sp.), 2.15 (1H,
5.96; N, 10.35. Found: C, 59.23; H, 6.09; N, 10.49.
s*, CH2-sp.), 2.49-2.52 (5H, m, 3-CH3-ind., DMSO-d6), 2.54 (1H,
4.1.12. 5-Chloro-3-methyl-N-(8-propyl-3-oxo-1-thia-4-azaspiro- s, CH-sp.), 3.69 (2H, s, C2-H2-sp.), 7.19 (1H, tt, J=7.3, 1.5, 8-
[4.5]decan-4-yl)-1H-indole-2-carboxamide (4g)
C6H5(H4)-sp.), 7.23 (2H, dd, J=7.5, 1.5, 8-C6H5(H2,H6)-sp.), 7.25
(1H, dd, J=8.8, 2.0, H6-ind.), 7.29 (2H, t, J=7.3, 8-C6H5(H3,H5)-
sp.), 7.46 (1H, d, J=8.8, H7-ind.), 7.72 (1H, d, J=2.4, H4-ind.),
10.18 (1H, s, CONH), 11.51 (1H, s, NH); 13C-NMR (APT) (DMSO-
d6/125 MHz):  10.46, 10.53 (3-CH3-ind.), 28.69, 31.21, 37.77
(CH2-sp.), 42.24 (C8-sp.), 56.74 (CH2-sp.), 72.47 (C5-sp.), 114.42,
114.52 (C7-ind.), 115.65 (C3-ind.), 119.97, 120.00 (C4-ind.),
124.70 (C5-ind.), 125.00, 125.12 (C6-ind.), 127.21, 127.34 (8-
ar.(CH)-sp.), 127.85 (C2-ind.), 129.01, 129.14 (8-ar.(CH)-sp.),
129.57 (C3a-ind.), 134.94 (C7a-ind.), 146.55 (8-ar.(C)-sp.), 162.29
(CONH), 168.46 (CO-sp.). MS (ESI+) m/z (%): 453.9 ([M+H]+,
100), 455.9 ([(M+H)+2]+, 36.6). Anal. Calcd for C24H24ClN3O2S
(453.98) C, 63.49; H, 5.33; N, 9.26. Found: C, 63.41; H, 5.22; N,
9.06.
White powder (41.3%); mp 246-247 °C; IR(KBr): υmax 3316 (N-
H), 1701, 1645 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.84 (3H,
t, J=7.3, 8-CH2CH2CH3-sp.), 1.14-1.31 (7H, m, CH/CH2-sp., 8-
CH2CH2CH3-sp.), 1.74-1.89 (6H, m, CH/CH2-sp.), 2.48-2.51 (5H,
m, 3-CH3-ind., DMSO-d6), 3.63 (2H, s, C2-H2-sp.), 7.23 (1H, dd,
J=8.7, 1.9, H6-ind.), 7.43 (1H, d, J=8.8, H7-ind.), 7.70 (1H,d, J=2.0,
H4-ind.), 10.11 (1H, s, CONH), 11.48 (1H, s, NH); 13C-NMR
(APT) (DMSO-d6/125 MHz):  10.39, 10.46 (3-CH3-ind.), 14.85
(8-CH2CH2CH3-sp.), 20.18 (8-CH2CH2CH3-sp.), 28.63, 29.74,
30.06 (CH2-sp., 8-CH2CH2CH3-sp.), 35.83 (C8-sp.), 37.51, 39.07
(CH2-sp.), 73.04 (C5-sp.), 114.40, 114.50 (C7-ind.), 115.54 (C3-
ind.), 119.93, 119.97 (C4-ind.), 124.67 (C5-ind.), 124.94, 125.07
(C6-ind.), 127.87 (C2-ind.), 129.54 (C3a-ind.), 134.91 (C7a-ind.),
162.42 (CONH), 168.40 (CO-sp.). MS (ESI+) m/z (%): 420.0 4.1.16. 5-Chloro-3-methyl-N-(2-methyl-3-oxo-1-thia-4-azaspiro-
([M+H]+, 100), 421.9 ([(M+H)+2]+, 36.9). Anal. Calcd for [4.4]nonan-4-yl)-1H-indole-2-carboxamide (5a)
C21H26ClN3O2S (419.97) C, 60.06; H, 6.24; N, 10.01. Found: C,
White powder (60.2%); mp 282-284 °C; IR(KBr): υmax 3307 (N-
59.81; H, 6.02; N, 10.14.
H), 1694, 1651 (C=O); 1H-NMR (DMSO-d6/500 MHz):  1.47 (3H,
4.1.13. N-(8-tert-butyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-5- d, J=7.3, 2-CH3-sp.), 1.64-1.72 (4H, m, CH2-sp.), 1.83-1.95 (2H, m,
chloro-3-methyl-1H-indole-2-carboxamide (4h)
CH2-sp.), 2.18-2.30 (2H, m, CH2-sp.), 2.49-2.51 (6H, m, 3-CH3-
ind., DMSO-d6), 3.99 (1H, q, J=6.8, C2-H-sp.), 7.24 (1H, dd, J=8.8,
2.0, H6-ind.), 7.43 (1H, d, J=9.2, H7-ind.), 7.70 (1H,d, J=2.0, H4-
ind.), 10.20 (1H, s, CONH), 11.50 (1H, s, NH); 13C-NMR (APT)
(DMSO-d6/125 MHz):  10.35, 10.43 (3-CH3-ind.), 20.09 (2-CH3-
sp.), 23.47, 23.51 (CH2-sp.), 38.35, 38.49 (C2-sp.), 39.32 (CH2-sp.),
75.01 (C5-sp.), 114.43, 114.53 (C7-ind.), 115.37 (C3-ind.), 119.96,
120.00 (C4-ind.), 124.69 (C5-ind.), 125.00, 125.12 (C6-ind.),
127.82 (C2-ind.), 129.53 (C3a-ind.), 134.97 (C7a-ind.), 162.27
(CO-NH), 171.07 (CO-sp.). MS (ESI-) m/z (%): 376.3 ([M-H]-,
100), 378.2 ([(M-H)+2]-, 37.2). Anal. Calcd for C18H20ClN3O2S
(377.89): C, 57.21; H, 5.33; N, 11.12. Found: C, 57.37; H, 5.24; N,
11.19.
Beige crystals (48.1%); mp 252-254 °C; IR(KBr): υmax 3327,
3179 (N-H), 1692, 1670 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
0.84 (9H, s, 8-C(CH3)3-sp.), 0.95-0.98 (1H, m, CH/CH2-sp.), 1.23-
1.28 (2H, m, CH/CH2-sp.), 1.78 (2H, d, J=12.6, CH/CH2-sp.), 1.92
(4H, s*, CH/CH2-sp.), 2.49-2.51 (5H, m, 3-CH3-ind., DMSO-d6),
3.63 (2H, s, C2-H2-sp.), 7.24 (1H, dd, J=8.6, 2.0, H6-ind.), 7.43
(1H, d, J=8.8, H7-ind.), 7.70 (1H, d, J=2.0, H4-ind.), 10.12 (1H, s,
CONH), 11.47 (1H, s, NH); 13C-NMR (APT) (DMSO-d6/125
MHz):  10.41, 10.48 (3-CH3-ind.), 24.45 (CH2-sp.), 28.02 (8-
C(CH3)3-sp.), 28.61 (CH2-sp.), 32.66 (8-C(CH3)3-sp.), 37.82 (CH2-
sp.), 46.48 (C8-sp.), 72.92 (C5-sp.), 114.40, 114.50 (C7-ind.),
115.55 (C3-ind.), 119.92, 119.97 (C4-ind.), 124.67 (C5-ind.),
124.95, 125.07 (C6-ind.), 127.85 (C2-ind.), 129.54 (C3a-ind.), 4.1.17. 5-Chloro-3-methyl-N-(2-methyl-3-oxo-1-thia-4-azaspiro-
134.91 (C7a-ind.), 162.21 (CONH), 168.41 (CO-sp.). Anal. Calcd [4.5]decan-4-yl)-1H-indole-2-carboxamide (5b)
for C22H28ClN3O2S (433.99) C, 60.88; H, 6.50; N, 9.68. Found: C,
White needles (40.1%); mp 281-283 °C; IR(KBr): υmax 3311 (N-
60.99; H, 6.42; N, 9.67.
H), 1693, 1654 (C=O); 1H-NMR (DMSO-d6/500 MHz):  1.03-1.09

(1H, m, CH2-sp.), 1.30-1.60 (3H, m, CH2-sp.), 1.45 (3H, d, J=6.8, sp.), 31,29, 31.93, 32.38, 37.51 (CH/CH2-sp.), 38.54 (C2-sp.),
2-CH3-sp.), 1.70-2.00 (6H, m, CH2-sp.), 2.48-2.50 (5H, m, 3-CH3- 71.39 (C5-sp.), 114.95 (C7-ind.), 115.45 (C3-ind.), 119.97 (C4-
ind., DMSO-d6), 3.95 (1H, q, J=7.3, C2-H-sp.), 7.24 (1H, dd, J=8.7, ind.), 124.66 (C6-ind.), 125.01 (C5-ind.), 127.90 (C2-ind.), 129.54
1.9, H6-ind.), 7.43 (1H, d, J=8.8, H7-ind.), 7.70 (1H, d, J=1.9, H4- (C3a-ind.), 134.91 (C7a-ind.), 162.23 (CONH), 171.06 (C=O).
ind.), 10.10 (1H, s, CONH), 11.50 (1H, s, NH); 13C-NMR (HSQC) Anal. Calcd for C20H24ClN3O2S (405.94): C, 59.17; H, 5.96; N,
(DMSO-d6/125 MHz):  10.43 (3-CH3-ind.), 20.77 (2-CH3-sp.), 10.35. Found: C, 59.06; H, 6.21; N, 10.32.
23.46, 23.88, 24.74 (CH2-sp.), 37.49, 37.92 (CH2-sp., C2-sp.),
4.1.21.
5-Chloro-N-(8-ethyl-2-methyl-3-oxo-1-thia-4-azaspiro-
38.83 (CH2-sp.), 71.53 (C5-sp.), 114.45 (C7-ind.), 115.46 (C3-
ind.), 119.97 (C4-ind.), 124.65 (C5-ind.), 125.01 (C6-ind.), 127.88
(C2-ind.), 129.50 (C3a-ind.), 134.90 (C7a-ind.), 162.23 (CO-NH),
170.99 (CO-sp.). Anal. Calcd for C19H22ClN3O2S (391.91): C,
58.23; H, 5.66; N, 10.72. Found: C, 57.78; H, 5.47; N, 10.64.
[4.5]decan-4-yl)-3-methyl-1H-indole-2-carboxamide (5f)
White crystals (52.1%); mp 258-261 °C; IR(KBr): υmax 3317,
3265 (N-H), 1701, 1653 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
0.85 (3H, t, J=7.3, 8-CH2CH3-sp.), 1.00-1.30 (5H, m, CH/CH2-sp.,
8-CH2CH3-sp.), 1.45 (3H, d, J=6.8, 2-CH3-sp.), 1.70-1.90 (6H, m,
CH/CH2-sp.), 2.48-2.52 (5H, m, 3-CH3-ind., DMSO-d6), 3.93 (1H,
q, J=6.8, C2-H-sp.), 7.32 (1H, dd, J=8.7, 1.9, H6-ind.), 7.43 (1H, d,
4.1.18. N-(2,6-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-
5-chloro-3-methyl-1H-indole-2-carboxamide (5c)
White flakes (55.7%); mp 261-264 °C; IR(KBr): υmax 3217, 3134 J=8.6, H7-ind.), 7.70 (1H,d, J=1.9, H4-ind.), 10.18, 10.22 (1H, 2s,
(N-H), 1701, 1645 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.98 CONH), 11.50, 11.56 (1H, 2s, NH); 13C-NMR (APT) (DMSO-
(3H, d*, J=5.9, 6-CH3-sp.), 1.14-1.35 (3H, m, CH/CH2-sp.), 1.44, d6/125 MHz):  10.39, 10.45 (CH3-ind.), 12.03 (8-CH2CH3-sp.),
1.45 (3H, 2d, J=6.8, 7.3, 2-CH3-sp.), 1.54-1.58 (1H, m, CH/CH2- 20.65 (2-CH3-sp.), 28.45, 29.50, 29.95 (CH2-sp., 8-CH2CH3-sp.),
sp.), 1.63 (1H, d*, J=8.3, CH/CH2-sp.), 1.72 (1H, d*, J=13.6, 37.45, 37.60, 37.85 (C2-sp., C8-sp.), 71.68 (C5-sp.), 114.40, 114.49
CH/CH2-sp.), 1.88-2.00 (3H, m, CH/CH2-sp.), 2.49-2.51 (5H, m, 3- (C7-ind.), 115.43 (C3-ind.), 119.94, 119.98 (C4-ind.), 124.66 (C6-
CH3-ind., DMSO-d6), 3.84, 3.94 (1H, 2q, J=6.8, C2-H-sp.), 7.24 ind.), 124.94, 125.05 (C5-ind.), 127.92 (C2-ind.), 129.55 (C3a-
(1H, dd, J=8.5, 2.0, H6-ind.), 7.43, 7.44 (1H, 2d, J=8.3, H7-ind.), ind.), 134.92 (C7a-ind.), 162.22 (CONH), 171.04 (C=O). MS (ESI-
7.70 (1H, d, J=2.0, H4-ind.), 10.08 (1H, s*, CONH), 11.51 (1H, s, ) m/z (%): 418.2 ([M-H]-, 100), 420.2 ([(M-H)+2]-, 37.4). Anal.
NH); 13C-NMR (APT) (DMSO-d6/125 MHz):  10.39, 10.46 (3- Calcd for C21H26ClN3O2S (419.97) C, 60.06; H, 6.24; N, 10.01.
CH3-ind.), 16.95 (6-CH3-sp.), 23.46, 25.41, 29.71, 32.24 (CH2-sp.), Found: C, 60.12; H, 6.18; N, 9.96.
38.11, 38.31 (C2-sp.), 39.26 (C6-sp.), 75.58, 75.98 (C5-sp.),
4.1.22. 5-Chloro-3-methyl-N-(2-methyl-8-propyl-3-oxo-1-thia-4-
114.40, 114.48 (C7-ind.), 115.71 (C3-ind.), 119.94, 119.97 (C4-
azaspiro[4.5]decan-4-yl)-1H-indole-2-carboxamide (5g)
ind.), 124.67 (C5-ind.), 124.97, 125.09 (C6-ind.), 127.89 (C2-ind.),
129.53 (C3a-ind.), 134.88 (C7a-ind.), 162.48 (CO-NH), 170.97,
171.09 (CO-sp.). Anal. Calcd for C20H24ClN3O2S (405.94): C,
59.17; H, 5.96; N, 10.35. Found: C, 59.21; H, 5.68; N, 10.11.
White crystals (42.3%); mp 173-176 °C; IR(KBr): υmax 3329,
3254 (N-H), 1709, 1648 (C=O); 1H-NMR (DMSO-d6/500 MHz): 
0.84 (3H, t, J=7.3, 8-CH2CH2CH3-sp.), 1.14-1.31 (7H, m, CH/CH2-
sp., 8-CH2CH2CH3-sp.), 1.46 (3H, d, J=7.3, 2-CH3-sp.), 1.75-1.91
(6H, m, CH/CH2-sp.), 2.49-2.52 (5H, m, 3-CH3-ind., DMSO-d6),
3.92 (1H, q, J=6.8, C2-H-sp.), 7.23 (1H, dd, J=8.8, 2.0, H6-ind.),
4.1.19. N-(2,7-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-
5-chloro-3-methyl-1H-indole-2-carboxamide (5d)
White crystals (46.0%); mp 214-216 °C; IR(KBr): υmax 3408, 7.42, 7.43 (1H, 2d, J=8.8, H7-ind.), 7.70 (1H,d, J=2.0, H4-ind.),
1
3304 (O-H/N-H), 1697, 1654 (C=O); H-NMR (DMSO-d6/500 10.16, 10.23 (1H, 2s, CONH), 11.48, 11.57 (1H, 2s, NH); 13C-NMR
MHz):  0.75-0.83 (1H, m, CH/CH2-sp.), 0.91 (3H, d, J=5.8, 7- (APT) (DMSO-d6/125 MHz):  10.39, 10.46 (3-CH3-ind.), 14.85
CH3-sp.), 1.06 (3H, t, J=7.3, CH3CH2OH), 1.48 (3H, d, J=7.3, 2- (8-CH2CH2CH3-sp.), 20.19 (8-CH2CH2CH3-sp.), 20.63 (2-CH3-
CH3-sp.), 1.50-2.00 (8H, m, CH/CH2-sp.), 2.48-2.52 (5H, m, 3- sp.), 29.86, 29.92, 30.34 (CH2-sp., 8-CH2CH2CH3-sp), 35.81 (C8-
CH3-ind., DMSO-d6), 3.43 (2H, m, CH3CH2OH), 3.92-3.97 (1H, m, sp.), 37.44, 37.59 (C2-sp.), 39.09 (CH2-sp., 8-CH2CH2CH3-sp),
C2-H-sp.), 4.34 (1H, t, J=6.8, CH3CH2OH), 7.25 (1H, dd, J=8.7, 71.58, 71.68 (C5-sp.), 114.38, 114.49 (C7-ind.), 115.50 (C3-ind.),
1.9, H6-ind.), 7.44 (1H, d, J=8.7, H7-ind.), 7.72 (1H,d, J=1.9, H4- 119.92, 119.97 (C4-ind.), 124.67 (C5-ind.), 124.94, 125.06 (C6-
ind.), 10.50 (1H, s, CONH), 11.50 (1H, s, NH); 13C-NMR (HSQC) ind.), 127.88 (C2-ind.), 129.55 (C3a-ind.), 134.91, 134.98 (C7a-
(DMSO-d6/125 MHz):  10.44 (3-CH3-ind.), 19.27 (CH3CH2OH), ind.), 162.20, 162.45 (CONH), 171.04 (CO-sp.). Anal. Calcd for
20.65 (2-CH3-sp.), 22.70 (7-CH3-sp.), 23.42 (CH2-sp.), 30.17 (C7- C22H28ClN3O2S (433.99) C, 60.88; H, 6.50; N, 9.68. Found: C,
sp.), 30.60, 33.47 (CH2-sp.), 38.30 (C2-sp.), 38.83, 46.00 (CH2-sp.), 60.58; H, 6.33; N, 9.61.
56.74 (CH3CH2OH), 71.54 (C5-sp.), 114.47 (C7-ind.), 115.59 (C3-
4.1.23.
N-(2-methyl-8-tert-butyl-3-oxo-1-thia-4-azaspiro-
ind.), 119.98 (C4-ind.), 124.66 (C5-ind.), 125.04 (C6-ind.), 127.84
(C2-ind.), 129.54 (C3a-ind.), 134.90 (C7a-ind.), 162.22 (CO-NH),
170.91 (CO-sp.). MS (ESI-) m/z (%): 404.1 ([M-H]-, 100), 406.1
([(M-H)+2]-, 28.4). Anal. Calcd for C20H24ClN3O2S.CH3CH2OH
(405.94+46.07): C, 58.41; H, 6.63; N, 9.29. Found: C, 58.20; H,
6.54; N, 9.26.
[4.5]decan-4-yl)-5-chloro-3-methyl-1H-indole-2-carboxamide
(5h)
White powder (53.5%); mp 226-228 °C; IR(KBr): υmax 3308 (N-
H), 1698, 1658 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.84 (9H,
s, 8-C(CH3)3-sp.), 0.96 (1H, t*, J=12.2, CH/CH2-sp.), 1.17-1.32
(2H, m, CH/CH2-sp.), 1.46 (3H, d, J=6.8, 2-CH3-sp.), 1.78 (2H, d,
J=12.2, CH/CH2-sp.), 1.89-1.95 (4H, m, CH/CH2-sp.), 2.49-2.53
(5H, m, 3-CH3-ind., DMSO-d6), 3.93 (1H, q, J=6.8, C2-H-sp.), 7.23
4.1.20. N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-
5-chloro-3-methyl-1H-indole-2-carboxamide (5e)
White crystals (75.3%); mp 235-238 °C; IR(KBr): υmax 3321, (1H, dd, J=8.8, 2.0, H6-ind.), 7.43 (1H, d, J=8.8, H7-ind.), 7.70 (1H,
3246 (N-H), 1701, 1678 (C=O); 1H-NMR (DMSO-d6/500 MHz):  d, J=2.0, H4-ind.), 10.16 (1H, s, CONH), 11.47 (1H, s, NH); 13C-
0.88, 0.94 (3H, 2d, J=6.8, 8-CH3-sp.), 1.12-1.38 (4H, m, CH/CH2- NMR (APT) (DMSO-d6/125 MHz):  10.40, 10.46 (3-CH3-ind.),
sp.), 1.48 (3H, d, J=7.3, 2-CH3-sp.), 1.60-2.10 (5H, m, CH/CH2- 20.04 (2-CH3-sp.), 24.30, 24.71 (CH2-sp.), 28.01 (8-C(CH3)3-sp.),
sp.), 2.48-2.52 (5H, m, 3-CH3-ind., DMSO-d6), 3.96 (1H, q, J=7.3, 32.67 (8-C(CH3)3-sp.), 37.45, 37.60 (C2-sp.), 46.42 (C8-sp.), 71.58
C2-H-sp.), 7.24 (1H, dd, J=8.7, 1.9, H6-ind.), 7.44 (1H, d, J=8.7, (C5-sp.), 114.39, 114.49 (C7-ind.), 115.50 (C3-ind.), 119.93,
H7-ind.), 7.70 (1H,d, J=1.9, H4-ind.), 10.18, 10.26 (1H, 2s, 119.97 (C4-ind.), 124.66 (C5-ind.), 124.96, 125.05 (C6-ind.),
CONH), 11.52, 11.58 (1H, 2s, NH); 13C-NMR (HMBC) (DMSO- 127.87 (C2-ind.), 129.55 (C3a-ind.), 134.91 (C7a-ind.), 162.19
d6/125 MHz):  10.43 (CH3-ind.), 20.56 (2-CH3-sp.), 22.54 (8-CH3- (CONH), 171.06 (CO-sp.). MS (ESI-) m/z (%): 446.4 ([M-H]-,

100), 448.3 ([(M-H)+2]-, 33.0). Anal. Calcd for C23H30ClN3O2S giving minimal alterations in cell morphology). Next, the
(448.02) C, 61.66; H, 6.75; N, 9.38. Found: C, 61.88; H, 6.84; N, colorimetric MTS cell viability assay (CellTiter 96® AQueous One
9.44.
Solution Cell Proliferation Assay from Promega) was done, the
results of which were expressed as antiviral EC50 and 50% cytotoxic
concentration (CC50).35
4.1.24. 5-Chloro-3-methyl-N-(2,7,7,9-tetramethyl-3-oxo-1-thia-4-
azaspiro[4.5]decan-4-yl)-1H-indole-2-carboxamide (5i)
The polykaryon assay (see18 for all details) was performed with
pCAGEN plasmids expressing wild-type or mutant (E572K and
D1122N) forms of H3 HA (cloned from strain A/X-31). HeLa cells
seeded into 12-well plates were transfected with HA plasmid using
Fugene 6® reagent (Promega). Two days later, the HA was activated
by brief exposure to TPCK-treated trypsin. After two rinses with
PBS containing Ca2+ en Mg2+ (PBS-CM), the cells were pre-
incubated with compound during 15 min. Next, the cells were
exposed to acidic buffer (PBS-CM adapted to pH 5) containing
compound, and incubated for exactly 15 min at 37 °C. The wells
were rinsed with PBS-CM, after which medium with 10% fetal calf
serum was added. After 3 h incubation, the cells were fixated with
ethanol and stained with Giemsa. Microscopy at 200x
magnification was used to count the number of polykaryons
(containing five or more nuclei) in four randomly selected
microscopic fields.
White flakes (47.7%); mp 263-265 °C; IR(KBr): υmax 3312 (N-
H), 1699, 1660 (C=O); 1H-NMR (DMSO-d6/500 MHz):  0.77 (1H,
t*, J=12.8, CH/CH2-sp.), 0.92 (6H, s, 7-(CH3)2-sp.), 1.09 (3H, d,
J=10.7, 9-CH3-sp.), 1.23-1.37 (2H, m, CH/CH2-sp.), 1.46, 1.47 (3H,
2d, J=7.3, 2-CH3-sp.), 1.70-1.89 (4H, m, CH/CH2-sp.), 2.49-2.51
(5H, m, 3-CH3-ind., DMSO-d6), 3.89, 3.94 (1H, 2q, J=6.8, C2-H-
sp.), 7.24 (1H, dd, J=8.7, 2.0, H6-ind.), 7.44 (1H, d, J=8.8, H7-ind.),
7.70 (1H, d, J=2.0, H4-ind.), 10.10, 10.11 (1H, 2s, CONH), 11.48,
11.50 (1H, 2s, NH); 13C-NMR (APT) (DMSO-d6/125 MHz): 
10.35, 10.40 (3-CH3-ind.), 18.89 (2-CH3-sp.), 22.51, 22.68, 25.98,
26.53, 27.80, 27.90 (7,7,9-CH3-sp.), 29.88, 32.56 (CH2-sp.), 34.54,
34.65 (C9-sp.), 37.12, 37.26 (C2-sp.), 45.93, 47.50 (CH2-sp., C7-
sp.), 70.73 (C5-sp.), 114.42, 114.50 (C7-ind.), 115.65 (C3-ind.),
119.92, 119.96 (C4-ind.), 124.67 (C5-ind.), 124.98, 125.10 (C6-
ind.), 127.84 (C2-ind.), 129.52 (C3a-ind.), 134.92 (C7a-ind.),
162.34 (CONH), 170.91 (CO-sp.). MS (ESI+) m/z (%): 434.0
([M+H]+, 100), 435.9 ([(M+H)+2]+, 34.7). Anal. Calcd for
C22H28ClN3O2S (433.99): C, 60.88; H, 6.50; N, 9.68. Found: C,
60.63; H, 6.36; N, 9.58.
4.3. Molecular docking studies
Unless stated otherwise, all calculations were done using the
Schrödinger Suite (version 2015-4, Schrödinger, LLC, New York,
NY, 2015).
4.1.25. 5-Chloro-3-methyl-N-(2-methyl-8-phenyl-3-oxo-1-thia-4-
azaspiro[4.5]decan-4-yl)-1H-indole-2-carboxamide (5j)
White flakes (67.9%); mp 162-164 °C; IR(KBr): υmax 3323, 3188
(N-H), 1693, 1653 (C=O); 1H-NMR (DMSO-d6/500 MHz):  1.06
(2H, t, J=7.0, CH2-sp.), 1.49 (3H, d, J=6.8, 2-CH3-sp.), 1.63-2.25
(6H, m, CH2-sp.), 2.49-2.51 (5H, m, 3-CH3-ind., DMSO-d6), 2.55
(1H, s, CH-sp.), 3.99 (1H, q, J=6.8, C2-H-sp.), 7.18 (1H, tt, J=7.5,
1.5, 8-C6H5(H4)-sp.), 7.22 (2H, dd, J=7.4, 1.5, 8-C6H5(H2,H6)-sp.),
7.25 (1H, dd, J=8.8, 2.0, H6-ind.), 7.30 (2H, t, J=7.3, 8-
C6H5(H3,H5)-sp.), 7.46 (1H, d, J=9.2, H7-ind.), 7.72 (1H, d, J=2.0,
H4-ind.), 10.23 (1H, s, CONH), 11.52 (1H, s, NH); 13C-NMR
(APT) (DMSO-d6/125 MHz):  10.46, 10.53 (3-CH3-ind.), 20.58
(2-CH3-sp.), 31.00, 31.56 (CH2-sp.), 37.55, 37.69 (C2-sp.), 37.75
(CH2-sp.), 42.26 (C8-sp.), 56.75 (CH2-sp.), 71.12 (C5-sp.), 114.41,
114.51 (C7-ind.), 115.62 (C3-ind.), 119.97, 120.00 (C4-ind.),
124.71 (C5-ind.), 125.00, 125.12 (C6-ind.), 126.74, 127.05 (8-
ar.(CH)-sp.), 127.87 (C2-ind.), 129.01, 129.14 (8-ar.(CH)-sp.),
129.58 (C3a-ind.), 134.95 (C7a-ind.), 146.55 (8-ar.(C)-sp.), 162.28
(CONH), 171.09 (CO-sp.). MS (ESI-) m/z (%): 466.6 ([M-H]-,
100), 468.5 ([(M-H)+2]-, 34.6). Anal. Calcd for C25H26ClN3O2S
(468.01) C, 64.16; H, 5.60; N, 8.98. Found: C, 64.39; H, 5.75; N,
8.59.
For ligand preparation, the selected ligand was drawn with the
2D Sketcher in Maestro and the LigPrep tool (version 2.5) was used
to generate the 3D conformers. Tautomers and ionization state of
the ligands were determined at neutral pH using Epik.36,37
Stereoisomers were generated with one low-energy ring
conformation for each stereoisomer.
The cocrystal structures of H3 HA with TBHQ (PDB ID: 3EYM;
H3 HA sequence A/Aichi/2/1968) and arbidol (PDB ID: 5T6N; H3
HA sequence A/Hong Kong/1/1968) were retrieved and the
proteins were prepared by removing the ligands from the respective
hydrophobic pockets and applying the Protein Preparation
Wizard.38 Coordinates for the missing residues and missing atoms
were filled. In both PDB entries, only the last four residues are
missing in the coordinate sets and the residues in the ligand binding
site are all resolved. Prior to docking, the protein structures were
energy minimized. The Glide grid, defined by taking the centroid
of the cocrystallized ligands as the box center, included residues
R542, L552, E572 of monomer 1 (chain D in PDB 5T6N) and Y942,
E972 and L992 of monomer 2 (chain F in PDB 5T6N).
The ligand was docked into the binding site between monomer
1 (chain C and D in 5T6N) and monomer 2 (chain E and F)
according to the Induced Fit Docking (IFD) protocol, which is
4.2. Biological methods
Inhibitory activity against influenza virus was determined in a based on combining Prime energy and GlideScore39-41 and in which
CPE reduction assay, described in full detail elsewhere.18 Briefly, side chains in the binding site are allowed to reorient.40 The poses
semiconfluent cultures of Madin-Darby canine kidney (MDCK) were ranked based on Glide Gscore values and the top ranking
cells (kindly donated by M. Matrosovich, Marburg, Germany) in poses in which the indole group of the spirothiazolidinones is
96-well plates were infected with influenza A/H1N1 (strain positioned similar to that of the arbidol ligand were selected for
A/Ned/378/05; a kind gift from R. Fouchier, Rotterdam, the visual analysis of the protein – compound interactions.
Netherlands); A/H3N2 (A/HK/7/87; from ATCC); or influenza B
virus (B/Ned/537/05; also from R. Fouchier) at a multiplicity of
infection of 0.0004 plaque-forming units per cell. At the same time,
the compounds were added at serial dilutions. After four days
Acknowledgments
incubation at 35 °C, microscopy was performed to score the
This work was supported in part by the Research Fund of
compounds’ inhibitory effect on viral CPE, expressed as 50%
Istanbul University (Project Number T-20867). E.O. would like to
effective concentration (EC50), and cytotoxicity, expressed as the
thank Prof. Amedeo Caflisch for hosting her during her sabbatical
minimal cytotoxic concentration (MCC; compound concentration

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at University of Zürich. L.N. acknowledges dedicated help from
Ria Van Berwaer and Nathalie Van Winkel.
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Graphical abstract

Declarations of interest
None.