Synthesis and anti-coronavirus activity of a series of 1-thia-4-azaspiro[4.5]decan-3-one derivatives

Article abstract of DOI:10.1002/ardp.201800330

A series of 1-thia-4-azaspiro[4.5]decan-3-ones bearing an amide group at C-4 and various substitutions at C-2 and C-8 were synthesized and evaluated against human coronavirus and influenza virus. Compounds 7m, 7n, 8k, 8l, 8m, 8n, and 8p were found to inhibit human coronavirus 229E replication. The most active compound was N-(2-methyl-8-tert-butyl-3-oxo-1-thia-4-azaspiro[4.5]decan-4-yl)-3-phenylpropanamide (8n), with an EC₅₀ value of 5.5 μM, comparable to the known coronavirus inhibitor, (Z)-N-[3-[4-(4-bromophenyl)-4-hydroxypiperidin-1-yl]-3-oxo-1-phenylprop-1-en-2-yl]benzamide (K22). Compound 8n and structural analogs were devoid of anti-influenza virus activity, although their scaffold is shared with a previously discovered class of H3 hemagglutinin-specific influenza virus fusion inhibitors. These findings point to the 1-thia-4-azaspiro[4.5]decan-3-one scaffold as a versatile chemical structure with high relevance for antiviral drug development.

Full text of DOI:10.1002/ardp.201800330

Received: 15 November 2018
DOI: 10.1002/ardp.201800330
Revised: 25 March 2019
Accepted: 31 March 2019
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F U L L P A P E R
Synthesis and anti‐coronavirus activity of a series of
1‐thia‐4‐azaspiro[4.5]decan‐3‐one derivatives
Çağla Begüm Apaydın1
Zafer Cesur1
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Nesrin Cesur1
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Annelies Stevaert2
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Lieve Naesens2
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1Faculty of Pharmacy, Department of
Pharmaceutical Chemistry , Istanbul
University, Istanbul, Turkey
Abstract
A series of 1‐thia‐4‐azaspiro[4.5]decan‐3‐ones bearing an amide group at C‐4 and
various substitutions at C‐2 and C‐8 were synthesized and evaluated against human
coronavirus and influenza virus. Compounds 7m, 7n, 8k, 8l, 8m, 8n, and 8p were
found to inhibit human coronavirus 229E replication. The most active compound was
N‐(2‐methyl‐8‐tert‐butyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐phenylpropana-
mide (8n), with an EC50 value of 5.5 µM, comparable to the known coronavirus
inhibitor, (Z)‐N‐[3‐[4‐(4‐bromophenyl)‐4‐hydroxypiperidin‐1‐yl]‐3‐oxo‐1‐phenylprop‐
1‐en‐2‐yl]benzamide (K22). Compound 8n and structural analogs were devoid of
anti‐influenza virus activity, although their scaffold is shared with a previously
discovered class of H3 hemagglutinin‐specific influenza virus fusion inhibitors. These
findings point to the 1‐thia‐4‐azaspiro[4.5]decan‐3‐one scaffold as a versatile
chemical structure with high relevance for antiviral drug development.
2Laboratory of Virology and Chemotherapy,
Rega Institute, KU Leuven, Leuven, Belgium
Correspondence
Dr. Çağla Begüm Apaydın, Faculty of
Pharmacy, Department of Pharmaceutical
Chemistry, Istanbul University, 34126
Istanbul, Fatih, Turkey.
Email: caglabegum@gmail.com
Funding information
Istanbul University, Grant/Award Number:
TDK‐2016‐20602
K E Y W O R D S
antimicrobial activity, cycloaddition, structure elucidation, synthesis
Middle East Respiratory Syndrome virus (MERS‐CoV) appeared[6]
which, to date, caused 2249 cases with a CFR of 36%.[8] The high
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1
INTRODUCTION
Coronaviruses (CoVs) are a large family of enveloped RNA viruses
with positive‐sense single‐stranded RNA genome, affecting a wide
range of animal species. Some widely spread human coronaviruses
(HCoVs, i.e., strains 229E, OC43, NL63, and HKU1) are the etiologic
agents in 15–30% of common cold disease,[1] a relatively benign yet
highly contagious respiratory illness that can possibly be complicated
by virus‐induced asthma or exacerbations of chronic obstructive
pulmonary disease.[2] These HCoVs can also cause high‐morbidity
lower respiratory tract conditions, such as pneumonia, bronchiolitis,
and croup, especially in immunocompromised patients, the elderly
and young children.[3–5] Besides, two highly pathogenic CoVs
originating from animal reservoirs have had a major medical and
socio‐economic impact. In 2003, the newly discovered SARS‐CoV[6]
responsible for the severe acute respiratory syndrome, suddenly
emerged in South‐East Asia, after which it caused 8096 infections
worldwide with a case‐fatality (CFR) rate of 9.6% and a total societal
cost estimated at 40 billion USD.[7] In 2012, the thus far unknown
virulence of SARS‐ and MERS‐CoVs, combined with their sudden
emergence and clear pandemic potential, has boosted the develop-
ment of preventive (i.e., vaccines) or therapeutic (i.e., antiviral drugs)
interventions to address serious CoV infections in humans. Thus far,
except for quarantine measures, no effective intervention exists.
In our laboratory, we initiated
a small‐molecule screening
program in HCoV‐infected cells, aimed at identifying new chemical
entities and CoV targets with relevance for antiviral drug develop-
ment. In the present report, we describe the identification of a new
class of CoV inhibitors carrying a 1‐thia‐4‐azaspiro[4.5]decan‐3‐one
scaffold (Scheme 1). A series of structural analogs were chemically
synthesized to delineate the structure‐activity relationship. Intrigu-
ingly, the same scaffold is present in a class of H3 hemagglutinin‐
specific influenza virus fusion inhibitors that was discovered by our
team some time ago.[9] This points to the 1‐thia‐4‐azaspiro[4.5]‐
decan‐3‐one scaffold as a versatile chemical structure with high
relevance for antiviral drug development.
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Arch. Pharm. Chem. Life Sci. 2019;e1800330.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800330

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APAYDIN ET AL.
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Compound
R
H
R1
H
n
1
2
2
2
2
2
3
1
2
2
2
2
2
2
Compound
R
H
R1
H
n
1
2
2
2
2
2
2
2
1
2
2
2
2
2
2
2
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
8e
8f
3-CH3
4-CH3
4-C(CH3)3
4-CF3
4-C6H5
H
H
H
H
H
4-CH3
4-C2H5
4-C3H7
4-C(CH3)3
4-CF3
4-C6H5
H
H
H
H
H
H
H
H
7g
7h
7i
H
8g
8h
8i
H
H
CH3
CH3
CH3
CH3
H
H
CH3
CH3
7j
3-CH3
4-CH3
8j
H
7k
7l
8k
8l
4-CH3 CH3
4-C2H5 CH3
4-C3H7 CH3
4-C(CH3)3 CH3
4-C(CH3)3 CH3
7m
7n
4-CF3
CH3
CH3
8m
8n
8o
8p
4-C6H5
SCHEME 1 Synthetic route and
chemical structures for compound series 7
and 8
4-CF3
CH3
4-C6H5 CH3
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2
RESULTS AND DISCUSSION
Chemistry
[4.5]dec‐4‐yl/[4.6]undec‐4‐yl)‐2‐phenoxyacetamide (7) or N‐(substituted
3‐oxo‐1‐thia‐4‐azaspiro[4.4]non‐4‐yl/[4.5]decan‐4‐yl)‐3‐phenylpropana-
mides (8). The first evidence for the formation of 7 and 8 was the
observation of a new band between 1728–1697 cm−1 in their IR spectra
due to 3‐carbonyl group. The C=O stretching band of the carboxamide
moiety was also observed at 1660–1693 cm−1. Bands between 3468–
3167 cm−1 region confirmed the presence of NH stretching bands. In the
1H‐NMR spectra of 7, CONH protons were observed at δ 9.91–10.59
ppm as singlets. C2–methylene protons of 7a–g and 8a–h resonated at δ
3.52–3.64 ppm as singlets, while C2‐methine protons of 7h–n and 8i–p
gave quartets at δ 3.80–3.93 ppm. The 13C‐NMR spectra of 7a–m and 8j
showed spirothiazolidinone C2 carbon (δ 27.76–38.08 ppm) and
spirothiazolidinone C=O carbon (δ 167.59–170.77 ppm) resonances as
another evidence for the formation of a ring system. Molecular weights
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2.1
Hydrazides (3, 4) were obtained by stepwise reactions, firstly by
esterification of acids in methanol in the presence of H2SO4 to give
esters (1, 2), and secondly hydrazinolysis of 1, 2 to give 3, 4.[10] 3, 4 were
reacted with cyclic ketones to afford corresponding derivatives 5, 6 that
are commercially available or recorded earlier, except 5a and 6a.[11,12]
Spectral and analytical data of 5a and 6a are given under “General
procedure for the synthesis of N'‐(cycloalkylidene)‐2‐phenoxyacetohy-
drazides (5) or N'‐(cycloalkylidene)‐3‐phenylpropanehydrazides (6)”.
Cyclocondensation of 5 or 6 with sulphanylacetic acid or 2‐sulphanyl-
propanoic acid led to N‐(substituted 3‐oxo‐1‐thia‐4‐azaspiro[4.4]non‐4‐yl/

APAYDIN ET AL.
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(for the group coded as 7) were verified by taking mass spectra to get
another proof. ESI method was applied in positive or negative modes and
[M+H]+ or [M‒H]− ions formed confirmed the molecular weights.
can be classified in either Group 7, carrying a phenoxyethyl side
chain, or Group 8, with a phenethyl side chain. Our previously
discovered H3 hemagglutinin‐specific influenza virus fusion inhibi-
tors[9] have the same scaffold but carry an imidazo[2,1‐b]thiazole or
o‐hydroxyphenyl side chain. The current series of molecules show no
activity against influenza viruses at concentrations up to 100 µM
(data not shown), probably phenoxymethyl and phenethyl side
chains are not suitable for binding to influenza virus hemagglutinin.
Seven compounds, i.e. 7m, 7n, 8k, 8l, 8m, 8n, and 8p, were found to
inhibit human coronavirus 229E with low cytotoxicity (Table 1). The
compounds showing activity all carry a methyl substituent at the C‐2
position of the azaspiro[4.5]decane (R1), whereas the unmethylated
analogs (i.e., 7e, 7f, 8c, 8d, 8e, 8f, 8h) were devoid of antiviral activity. The
same observation was previously made for the structurally related
influenza virus fusion inhibitors.[9] In addition, for compound series 8, the
anti‐coronavirus activity clearly depended on the bulkiness of the C‐8
substituent of the azaspiro[4.5]decane (R). No activity was seen for the
unsubstituted compound 8j (R = H). Intermediate activity was observed
for 8k and 8l (EC50 = 28 and 18 µM), which carry a 4‐methyl and 4‐ethyl‐
group, respectively. A further increase in activity was seen for 8m, 8n,
and 8p (EC50 values 5.5–8.1 µM), carrying, respectively, a 4‐propyl, 4‐tert‐
butyl and 4‐phenyl group. An exception here is 8o (R = 4‐CF3), which did
not show antiviral activity at concentrations up to 100 µM. Its direct
analog belonging to Group 7 (7m) however, was moderately active
(EC50 = 31 µM), and so was 7n (EC50 = 12 µM), which carries the same
substituent (4‐phenyl) as 8p. Molecules 7k and 7l, which are the
counterparts of 8k and 8n, were not active.
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2.2
Biological activity
The compounds were evaluated for antiviral activity against human
coronavirus 229E in HEL 299 fibroblast cells, and against influenza
A/H1N1, A/H3N2, and B viruses in MDCK cells. The test compounds
TABLE 1 Activity in human coronavirus 229E‐infected HELa cells
b
Compounds
7a
Antiviral activity (EC50
)
Cytotoxicity (MCCc)
>100
>100
>100
≥100
>100
>100
>100
>100
>100
>100
>100
>100
>100
100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
8a
31
1
1
12
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
28
>100
>100
>100
>100
>100
>100
>100
100
8b
8c
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3
CONCLUSION
8d
8e
We report the chemical synthesis of a compound series with 4‐
carboxamido‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]nonan/[4.5]decan/[4.6]undecan
structure. Upon evaluation in virus‐infected cell culture assays, seven
compounds proved to be able to inhibit human coronavirus 229E virus
replication, with the most potent analog having an EC50 value of 5.5 µM.
The anti‐coronavirus activity was found to be strongly dependent on the
substituents at the C‐2 and C‐8 positions of the azaspiro[4.5]decane
scaffold. While a series of closely related analogs was previously
demonstrated to target influenza virus fusion by interfering with
hemagglutinin refolding at low pH, the present molecules have no
activity against human influenza A/H1N1, A/H3N2, and B viruses.
Ongoing mechanistic investigations will reveal the biochemical basis for
their inhibitory effect on coronavirus.
8f
8g
8h
8i
>100
>100
>100
≥100
100
8j
8k
8l
0
5
18
8m
8n
8o
8p
K22
8.1 2.2
5.5 0.7
>100
≥100
>100
50
6.1 0.2
3.3 1.0
50
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4
EXPERIMENTAL
Chemistry
Data represent the averages SEM of 3–4 independent tests.
aHEL: human embryonic lung fibroblast cells.
bEC50: compound concentration (µM) producing 50% inhibition of virus
replication, as determined by microscopic scoring of the cytopathic effect
(CPE).
cMCC: minimum cytotoxic concentration (µM), that is, compound
concentration producing minimal changes in cell morphology, as
estimated by microscopy.
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4.1
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4.1.1
General
Melting points were determined in open capillary tubes with a Büchi
B‐540 melting point apparatus and are uncorrected. IR spectra were

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APAYDIN ET AL.
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recorded in KBr disks on a Shimadzu IR Affinity‐1 FTIR spectro-
photometer. 1H and 13C‐NMR spectra were recorded on Varian Unity
Inova (500 MHz) and Varian Mercury (400 MHz) spectrophotometers. All
chemical shifts were reported as δ (ppm) values and spin‐spin couplings
(J) were expressed in Hz. Microanalyses were performed on a Thermo
Finnigan Flash EA 1112 elemental analyzer. Electrospray ionization (ESI)/
MS were determined on Agilent 6460 TripQuad LC‐MS/MS and Waters
2695 Alliance Micromass ZQ LC/MS spectrometers.
([M+Na]+, 40), 315 ([M+H]+, 100). Anal. calcd. for C15H17 F3N2O2
(314.30): C: 57.32, H: 5.45, N: 8.91. Found: C: 57.19, H: 5.40, N: 8.94.
N'‐[4‐(Trifluoromethyl)cyclohexylidene]‐3‐
phenylpropanehydrazide (6a)
Yield: 69%. mp: 117–120°C; IR (KBr) ʋ (cm−1): 3234, 3176 (N‐H),
1668 (C=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.28–1.47; 1.86–2.00;
2.23–2.41; 2.59–2.62 (9H, m, cyclohexane C2‐6‐H), 2.79–2.88 (4H, m,
CH2), 7.15–7.29 (5H, m, phenyl C2‐6‐H), 10.17, 10.25 (1H, 2s, NH).
Anal. calcd. for C16H19F3N2O (312.33): C: 61.53, H: 6.13, N: 8.97.
Found: C: 61.60, H: 6.21, N: 9.12.
The original spectra of the investigated compounds are provided
as Supporting Information. The InChI codes of the investigated
compounds together with some biological activity data are also
provided as Supporting Information.
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4.1.5
General procedure for the synthesis of
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4.1.2
Synthesis of methyl phenoxyacetate (1) or
N‐(substituted 3‐oxo‐1‐thia‐4‐azaspiro[4.4]non‐4‐yl/
[4.5]dec‐4‐yl/[4.6]undec‐4‐yl)‐2‐phenoxyacetamides
(7) and N‐(substituted 3‐oxo‐1‐thia‐4‐azaspiro[4.4]‐
non‐4‐yl/[4.5]decan‐4‐yl)‐3‐phenylpropanamides (8)
methyl 3‐phenylpropanoate (2)
A solution of phenoxyacetic acid (0.01 mol) or 3‐phenylpropionic acid
(0.01 mol) and sulfuric acid (98%) (0.1 ml) in 50 ml of methanol was
heated for 24 hr. The solution was neutralized with 0.01 mol of
sodium bicarbonate solution, the mixture was extracted with diethyl
ether and, after distillation of solvent, the ester was used without
further purification.[10,13]
A mixture of 5 or 6 (0.005 mol) and sulfanylacetic acid or 2‐
sulfanylpropanoic acid (1.5 ml) in 30 ml of dried toluene was refluxed
for 4–10 hr, using a Dean Stark water separator. Toluene was
evaporated in vacuo. The residue was neutralized with saturated
sodium bicarbonate and was allowed to solidify. The crude product
was filtered and recrystallized from ethanol or ethanol/water
mixture.
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4.1.3
Synthesis of 2‐phenoxyacetohydrazide (3) or
3‐phenylpropane‐hydrazide (4)
A mixture of 1 or 2 (0.01 mol), ethanol (50 ml), and hydrazine (0.0125
mol) was refluxed for 1 hr. The solid was filtered off, washed with
cold water and used without recrystallization.[10,13]
N‐(3‐Oxo‐1‐thia‐4‐azaspiro[4.4]non‐4‐yl)‐2‐
phenoxyacetamide (7a)
Yield: 65%. mp: 113–117°C; IR (KBr) ʋ (cm−1): 3383, 3238 (N‐H),
1722 (C=O), 1687 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 1.52–
1.62 (4H, m, spirononane C7,8‐H), 1.76, 1.95–2.07 (2H, 2H, dt, J = 13
Hz, 6 Hz, m, spirononane C6,9‐H), 3.64 (2H, s, spirononane C2‐H), 4.68
(2H, s, OCH2), 6.95–6.99 (3H, m, phenyl C2,4,6‐H), 7.27–7.32 (2H, m,
phenyl C3,5‐H), 10.36 (1H, s, NH). 13C‐NMR (proton decoupled)
(DMSO‐d6/125 MHz): 22.94 (spirononane C7,8), 29.17 (spirononane
C2), 38.42 (spirononane C6,9), 66.49 (OCH2), 76.15 (spirononane C5),
115.23, 121.81, 129.89 (phenyl C2‐6), 158.01 (phenyl C1), 167.87
(NHCO), 168.13 (CO). (ESI+) MS m/z (%): 329 ([M+Na]+, 50), 307
([M+H]+, 100), 233 (90). Anal. calcd. for C15H18N2O3S (306.38): C:
58.80, H: 5.92, N: 9.14. Found: C: 58.28, H: 6.43, N: 9.18.
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4.1.4
General procedure for the synthesis of
N'‐(cycloalkylidene)‐2‐phenoxyacetohydrazides (5) or
N'‐(cycloalkylidene)‐3‐phenylpropanehydrazides (6)
A solution of hydrazide (3 or 4) and appropriate ketone (0.01 mol) in
20 ml of ethanol (96%) was refluxed for 1–3 hr and then the solution
was allowed to cool. The solid thus formed was filtered off, dried and
directly used in the next step.
N'‐[4‐(Trifluoromethyl)cyclohexylidene]‐2‐
phenoxyacetohydrazide (5a)
Yield: 80%. mp: 140–142°C; IR (KBr) ʋ (cm−1): 3331, 3184, 3082
(N‐H), 1683 (C=O). 1H‐NMR (dimethyl sulfoxide [DMSO]‐d6/400
MHz): 1.27–1.53 (2H, m, cyclohexane C2,6‐ax‐H), 1.86–2.10, 2.23–
2.46, 2.51–2.72 (6H, 3m, C2,6‐eq‐H, C3,5‐H), 2.89, 3.05 (1H, 2d, J = 16
N‐(7‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7b)
Yield: 91%. mp: 78–80°C; IR (KBr) ʋ (cm−1): 3487, 3224 (O‐H/N‐H),
1714 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.62
(1H, qd, J = 13 Hz, 3 Hz, C8‐ax‐H), 0.83 (3H, d, J = 6 Hz, spirodecane
C7‐CH3), 1.21–1.74 (8H, m, C6‐H, C7‐H, C8‐eq‐H, C9‐H and C10‐H),
3.56 (2H, s, spirodecane C2‐H), 4.69 (2H, s, OCH2), 6.95–7.00 (3H, m,
phenyl C2,4,6‐H), 7.27–7.32 (2H, m, phenyl C3,5‐H), 10.29 (1 H, s, NH).
13C‐NMR (proton decoupled) (DMSO‐d6/125 MHz): 22.45 (spirode-
cane C7‐CH3), 22.83 (spirodecane C9), 28.35 (spirodecane C2), 29.92
(spirodecane C7), 33.06 (spirodecane C8), 36.73 (spirodecane C10),
45.39 (spirodecane C6), 66.41 (OCH2), 72.62 (spirodecane C5),
Hz, C4‐H), 4.59, 4.95 (2H, 2s, OCH2), 6.90–7.00 (3H, m, phenyl C2,4,6
‐
H), 7.20–7.40 (2H, m, phenyl C3,5‐H), 10.45, 10.62 (1H, 2s, NH). 13C‐
NMR (proton decoupled) (100 MHz) (DMSO‐d6): 23.56, 24.01, 24.79,
25.03, 32.34, 32.45 (cyclohexane C2,3,5,6), 38.87–40.12 (C‐CF3 with
(CD3)2SO), 64.70, 65.91 (OCH2), 114.35, 114.50, 114.69, 120.59,
2
121.01, 121.20, 129.33, 129.43 (phenyl C2‐6), 124.98 (q, JC‐F = 277
Hz, CF3), 153.97, 157.70, 157.87, 158.18 (phenyl C1), 160.90, 163.97
(cyclohexane C1), 166.69, 169.29 (CO). (ESI+) MS m/z (%): 337

APAYDIN ET AL.
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115.29, 121.80, 129.91 (phenyl C2‐6), 158.00 (phenyl C1), 167.84
(NHCO), 168.06 (CO). (ESI+) MS m/z (%): 357 ([M+Na]+, 50), 335
([M+H]+, 100), 261 (40). Anal. calcd. for C17H22N2O3S.0.5H2O
(343.43): C: 59.40, H: 6.70, N: 8.16. Found: C: 59.31, H: 6.36, N: 8.03.
N‐(8‐Phenyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7f)
Yield: 93%. mp: 165–169°C; IR (KBr) ʋ (cm−1): 3460, 3253 (N‐H), 1724
(C=O), 1680 (NHC = O). 1H‐NMR (DMSO‐d6/400 MHz): 1.55–1.70 (2H,
m, spirodecane C7,9‐ax‐H), 1.74–1.94 (6H, m, spirodecane C7,9‐eq‐H and
C6,10‐H), 2.35 (1H, tt, J = 13 Hz, 3 Hz, spirodecane C8‐H), 3.61 (2H, s,
spirodecane C2‐H), 4.72 (2H, s, OCH2), 6.96–7.07 (3H, m, phenoxy
C2,4,6‐H), 7.14–7.22 (3H, m, phenyl C8‐H and phenoxy C3,5‐H), 7.25–7.37
(4H, m, phenyl‐H), 10.36 (1H, s, NH). 13C‐NMR (proton decoupled)
(DMSO‐d6/125 MHz): 28.34, 30.88 (spirodecane C6,7,9,10), 37.30
(spirodecane C2), 41.93 (spirodecane C8), 66.52 (OCH2), 72.17
(spirodecane C5), 115.17, 115.35, 121.84, 129.91, 129.96 (phenoxy
C2‐6), 126.59, 127.00, 128.85, (phenyl C2‐6), 146.24 (phenyl C1), 158.05
(phenoxy C1), 168.00 (NHCO), 168.14 (CO). (ESI−) MS m/z (%): 395
([M‒H]‒, 100), 321 (12). Anal. calcd. for C22H24N2O3S (396.50):
C: 66.64, H: 6.10, N: 7.07. Found: C: 66.55, H: 5.99, N: 7.05.
N‐(8‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7c)
Yield: 98%. mp: 122–124°C; IR (KBr) ʋ (cm−1): 3446, 3221 (N‐H),
1726 (C=O), 1681 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.83
(3H, d, J = 6 Hz, CH3), 1.03–1.22 (3H, m, spirodecane C7‐ax‐H, C8‐H
and C9‐ax‐H), 1.63 (2H, d, J = 13 Hz, C7,9‐eq‐H), 1.66–1.75 (2H, m,
C6,10‐H), 3.57 (2H, s, spirodecane C2‐H), 4.68 (2H, s, OCH2), 6.95–
7.00 (3H, m, phenyl C2,4,6‐H), 7.27–7.32 (2H, m, phenyl C3,5‐H), 10.33
(1H, s, NH). 13C‐NMR (proton decoupled) (DMSO‐d6/100 MHz):
21.76 (C8‐CH3), 27.76 (spirodecane C2), 30.38 (spirodecane C8),
31.28, 36.54 (spirodecane C6,7,9,10), 65.95 (OCH2), 71.99 (spirode-
cane C5), 114.77, 121.27, 129.38 (phenyl C2‐6), 157.56 (phenyl C1),
167.45 (NHCO), 167.59 (CO). (ESI−) MS m/z (%): 333 ([M‒H]−, 100),
259 (32). Anal. calcd. for C17H22N2O3S (334.43): C: 61.05, H: 6.63, N:
8.38. Found: C: 60.86, H: 6.56, N: 8.32.
N‐(3‐Oxo‐1‐thia‐4‐azaspiro[4.6]undec‐4‐yl)‐2‐
phenoxyacetamide (7g)
Yield: 60%. mp: 115–119°C; IR (KBr) ʋ (cm−1): 3462, 3236 (N‐H),
1726 (C=O), 1681 (NHC = O). 1H‐NMR (DMSO‐d6/500 MHz): 1.32–
1.43 (4H, m, C8,9‐H), 1.43–1.68 (4H, m, C7,10‐H), 1.87 (2H, dd, J = 14.5
Hz, 8 Hz, C6/11‐H), 1.99 (2H, broad dd, J = 14.5 Hz, 8H, C6/11‐H), 3.57
(2H, s, C2‐H), 4.70 (2H, s, OCH2), 6.96–7.01 (3H, m, phenyl C2,4,6‐H),
7.29–7.33 (2H, m, phenyl C3,5‐H), 10.33 (1H, s, NH). 13C‐NMR
(proton decoupled) (DMSO‐d6/125 MHz): 22.62, 28.53 (spirounde-
cane C6‐11), 28.60 (spiroundecane C2), 66.43 (OCH2), 74.79 (spir-
oundecane C5), 115.22, 121.79, 129.90 (phenyl C2‐6), 158.06 (phenyl
C1), 167.42 (NHCO), 168.07 (CO). (ESI+) MS m/z (%): 357 ([M+Na]+,
100), 335 ([M+H]+, 80), 261 (90). Anal. calcd. for C17H22N2O3S
(334.43): C: 61.05, H: 6.63, N: 8.38. Found: C: 61.15, H: 6.59, N: 8.72.
N‐(8‐tert‐Butyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7d)
Yield: 79%. mp: 166–170°C; IR (KBr) ʋ (cm−1): 3466, 3234 (N‐H), 1724
(C=O), 1681 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.70–0.90 (1H,
m, C8‐H), 0.81 (9H, s, tert‐butyl CH3), 1.07–1.23 (2H, m, spirodecane
C7,9‐ax‐H), 1.54–1.71 (6H, m, C7,9‐eq‐H and C6,10‐H), 3.55 (2H, s,
spirodecane C2‐H), 4.68 (2H, s, OCH2), 6.95–7.00 (3H, m, phenyl
C2,4,6‐H), 7.27–7.32 (2H, m, phenyl C3,5‐H), 10.30 (1H, s, NH). 13C‐NMR
(proton decoupled) (DMSO‐d6/125 MHz): 24.11 (spirodecane C7‐9),
27.74 (tert‐butyl CH3), 28.27 (spirodecane C2), 32.34 (spirodecane C8‐
C), 37.35 (spirodecane C6,10), 46.13 (spirodecane C8), 66.47 (OCH2),
72.64 (spirodecane C5), 115.32, 121.81, 129.89 (phenyl C2‐6), 158.01
(phenyl C1), 167.95 (NHCO), 168.02 (CO). (ESI−) MS m/z (%): 375
([M‒H]−, 100), 301 (22). Anal. calcd. for C20H28N2O3S (376.51): C:
63.80, H: 7.50, N: 7.44. Found: C: 63.86, H: 8.00, N: 7.41.
N‐(2‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]non‐4‐yl)‐2‐
phenoxyacetamide (7h)
Yield: 80%. mp: 121–125°C; IR (KBr) ʋ (cm−1): 3448, 3230 (N‐H),
1728 (C=O), 1685 (NH=O). 1H‐NMR (DMSO‐d6/400 MHz): 1.41 (3H,
d, J = 7 Hz, CH3), 1.49–1.62 (4H, m, spirononane C7,8‐H), 1.72, 1.80
(1H, 1H, dt, J = 13 Hz, 6 Hz, C6,9‐H), 1.95–2.15 (2H, m, C6,9‐H), 3.92
(1H, q, J = 7 Hz, spirononane C2‐H), 4.68 (2H, s, OCH2), 6.95–6.99
(3H, m, phenyl C2,4,6‐H), 7.28–7.32 (2H, m, phenyl C3,5‐H), 10.39 (1H,
s, NH). 13C‐NMR (proton decoupled) (DMSO‐d6/125 MHz): 19.62
(CH3), 23.03, 23.10, 38.80 (spirononane C6‐9), 38.08 (spirononane
C2), 66.50 (OCH2), 74.68 (spirononane C5), 115.23, 121.81, 129.89
(phenyl C2‐6), 158.02 (phenyl C1), 168.10 (NHCO), 170.57 (CO).
(ESI−) MS m/z (%): 319 ([M‒H]−, 100), 245 (8). Anal. calcd. for
C16H20N2O3S (320.40): C: 59.98, H: 6.29, N: 8.74. Found: C: 59.88,
H: 6.21, N: 8.56.
N‐[3‐Oxo‐8‐(trifluoromethyl)‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl]‐2‐
phenoxyacetamide (7e)
Yield: 100%. mp: 131–135°C; IR (KBr) ʋ (cm−1): 3468, 3167 (O‐H/N‐
H), 1697 (C=O), 1660 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz):
1.36–1.52 (2H, m, spirodecane C6,10‐ax‐H), 1.69–1.95 (6H, m, C6,10‐eq
‐
H and C7,9‐H), 2.10‐2.25 (1H, m, C8‐ax‐H), 3.63 (2H, s, spirodecane C2‐
H), 4.69 (2H, s, OCH2), 6.97–7.03 (3H, m, phenyl C2,4,6‐H), 7.31–7.35
(2H, m, phenyl C3,5‐H), 10.38 (1H, s, NH). 13C‐NMR (APT) (DMSO‐d6/
125 MHz): 22.06, 35.25 (spirodecane C6,7,9,10), 28.23 (spirodecane
2
C2), 39.00 (q, JC‐F = 26 Hz, C8), 66.46 (OCH2), 71.47 (spirodecane
1
C5), 115.31, 121.79, 129.94 (phenyl C2‐6), 124.80, 128.13 (q, JC‐F
=
N‐(2‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7i)
Yield: 94%. mp: 134–136°C; IR (KBr) ʋ (cm−1): 3444, 3230 (N‐H),
1728 (C=O), 1687 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.92
(1H, broad q, J = 13 Hz, spirodecane C8‐ax‐H), 1.39 (3H, d, J = 7 Hz,
277 Hz, CF3), 158.04 (phenyl C1), 167.93 (NHCO), 168.19 (CO).
(ESI−) MS m/z (%): 387 ([M‒H]−, 100), 313 (20). Anal. calcd. for
C17H19F3N2O3S.2H2O (424.43): C: 48.11, H: 5.46, N: 6.60. Found: C:
48.39, H: 5.13, N: 6.79.

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CH3), 1.24–1.45 (2H, m, spirodecane C7,9‐ax‐H), 1.50 (1H, broad d, J =
13 Hz, spirodecane C8‐eq‐H), 1.56–1.78 (6H, m, C6,10‐H, C7,9‐eq‐H),
3.87 (1H, q, J = 7 Hz, spirodecane C2‐H), 4.69 (2H, s, OCH2), 6.96–
7.02 (3H, m, phenyl C2,4,6‐H), 7.29–7.34 (2H, m, phenyl C3,5‐H), 10.37
(1H, s, NH). 13C‐NMR (proton decoupled) (DMSO‐d6/100 MHz):
19.58 (CH3), 22.59, 23.03, 23.80, 36.57, and 36.86 (spirodecane
C6‐10), 37.75 (spirodecane C2), 65.90 (OCH2), 70.73 (spirodecane C5),
114.72, 121.23, 129.35 (phenyl C2‐6), 157.50 (phenyl C1), 167.52
(NHCO), 169.97 (CO). (ESI+) MS m/z (%): 357 ([M+Na]+, 100), 335
([M+H]+, 85), 247 (75). Anal. calcd. for C17H22N2O3S (334.43):
C: 61.05, H: 6.63, N: 8.38. Found: C: 61.24, H: 6.46, N: 8.50.
spirodecane C7,9‐ax‐H), 1.38 (3H, d, J = 7 Hz, spirodecane CH3), 1.54–1.79
(6H, m, spirodecane C7,9‐eq‐H, C6,10‐H), 3.85 (H, q, J = 7 Hz, spirodecane
C2‐H), 4.68 (2H, s, OCH2), 6.94–7.00 (3H, m, phenyl C2,4,6‐H), 7.27–7.31
(2H, m, phenyl C3,5‐H), 10.34 (1H, s, NH). 13C‐NMR (proton decoupled)
(DMSO‐d6/100 MHz): 19.50 (spirodecane C2‐CH3), 23.38, 23.82, 36.90,
and 37.60 (spirodecane C6,7,9,10), 27.16 (tert‐butyl CH3), 31.77 (tert‐butyl
C), 36.60 (spirodecane C2), 45.50 (spirodecane C8), 65.90 (OCH2), 70.73
(spirodecane C5), 114.61, 114.76, 121.24, 129.33 (phenyl C2‐6), 157.47,
157.50 (phenyl C1), 166.50, 167.43 (NHCO), 170.04 (CO). (ESI+) MS
m/z (%): 413 ([M+Na]+, 75), 391 ([M+H]+, 100), 303 (40). Anal. calcd. for
C21H30N2O3S (390.53): C: 64.58, H: 7.74, N: 7.17. Found: C: 64.04, H:
7.75, N: 7.27.
N‐(2,7‐Dimethyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7j)
N‐[2‐Methyl‐3‐oxo‐8‐(trifluoromethyl)‐1‐thia‐4‐azaspiro[4.5]‐
dec‐4‐yl]‐2‐phenoxyacetamide (7m)
Yield: 92%. mp: 132–138°C; IR (KBr) ʋ (cm−1): 3383, 3251 (N‐H),
1712 (C=O), 1687 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.62
(1H, qd, J = 13 Hz, 3 Hz, spirodecane C8‐ax‐H), 0.82 (3H, d, J = 7 Hz,
C7‐CH3), 1.29–1.73 (8H, m, spirodecane C6‐H, C7‐H, C8‐eq‐H, C9‐H
and C10‐H), 1.39 (3H, d, J = 7 Hz, spirodecane C2‐CH3), 3.86 (1H, q,
J = 7 Hz, spirodecane C2‐H), 4.68 (2H, s, OCH2), 6.95–7.00 (3H, m,
phenyl C2,4,6‐H), 7.27–7.32 (2H, m, phenyl C3,5‐H), 10.33 (1H, s, NH).
13C‐NMR (proton decoupled) (DMSO‐d6/125 MHz): 19.90 (spirode-
cane C2‐CH3), 22.54 (spirodecane C7‐CH3), 23.12 (spirodecane C9),
29.78 (spirodecane C7), 37.16 (spirodecane C2), 33.06 (spirodecane
C8), 36.78 (spirodecane C10), 46.22 (spirodecane C6), 66.41 (OCH2),
71.24 (spirodecane C5), 115.28, 121.79, 129.91 (phenyl C2‐6), 158.00
(phenyl C1), 168.03 (NHCO), 170.46 (CO). (ESI+) MS m/z (%): 371
([M+Na]+, 40), 349 ([M+H]+, 100), 261 (38). Anal. calcd. for
Yield: 100%. mp: 78–81°C; IR (KBr) ʋ (cm−1): 3444, 3196 (O‐H/N‐H),
1716 (C=O), 1685 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 1.29–
1.53 (2H, m, spirodecane C6,10‐ax‐H), 1.39 (3H, d,
J = 7 Hz,
spirodecane C2‐CH3), 1.66–1.99 (6H, m, spirodecane C6,10‐eq‐H and
C7,9‐H), 2.08–2.25 (1H, m, C8‐H), 3.92 (1H, q, J = 7 Hz, spirodecane
C2‐H), 4.66 (2H, s, OCH2), 6.94–7.00 (3H, m, phenyl C2,4,6‐H), 7.28–
7.32 (2H, m, phenyl C3,5‐H), 10.40 (1H, s, NH). 13C‐NMR (proton
decoupled) (DMSO‐d6/100 MHz): 19.51 (CH3), 21.43, 21.86, 34.90,
35.71 (spirodecane C6,7,9,10), 36.73 (spirodecane C2), 39.49 (q,2JC‐F
=
26 Hz, spirodecane C8), 65.99 (OCH2), 69.67 (spirodecane C5),
114.83, 121.32, 129.47 (phenyl C2‐6), 127.65 (q,1JC‐F = 277 Hz, CF3),
157.57 (phenyl C1), 167.71 (NHCO), 170.08 (CO). (ESI+) MS m/z (%):
425 ([M+Na]+, 32), 403 ([M+H]+, 100), 315 (72). Anal. calcd. for
C18H24N2O3S (348.45): C: 62.04, H: 6.94, N: 8.04. Found: C: 62.26,
C18H21F3N2O3S.0.5H2O (411.43): C: 52.49, H: 5.34, N: 6.80. Found:
H: 6.92, N: 8.56.
C: 52.34, H: 5.52, N: 6.75.
N‐(2,8‐Dimethyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐phe-
noxyacetamide (7k)
N‐(2‐Methyl‐8‐phenyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7n)
Yield: 100%. mp: 147–152°C; IR (KBr) ʋ (cm−1): 3390, 3259 (N‐H), 1726
(C=O), 1685 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.82 (3H, d, J =
6 Hz, spirodecane C8‐CH3), 0.96–1.25 (3H, m, spirodecane C7,9‐ax‐H and
C8‐H), 1.38 (3H, d, J = 7 Hz, spirodecane C2‐CH3), 1.56–1.85 (6H, m,
spirodecane C6,10‐H, C7,9‐eq‐H), 3.86 (1H, q, J = 7 Hz, spirodecane C2‐H),
4.68 (2H, s, OCH2), 6.93–6.99 (3H, m, phenyl C2,4,6‐H), 7.26–7.32 (2H, m,
phenyl C3,5‐H), 10.31 (1H, s, NH). 13C‐NMR (proton decoupled; DMSO‐
d6/100 MHz): 19.65 (spirodecane C2‐CH3), 21.81 (spirodecane C8‐CH3),
30.38 (spirodecane C8), 31.13, 31.62, 37.53 (spirodecane C6,7,9,10), 36.65
(spirodecane C2), 65.96 (OCH2), 70.65 (spirodecane C5), 114.69, 114.78,
121.20, 121.29, 129.42 (phenyl C2‐6), 157.58 (phenyl C1), 166.69, 167.59
(NHCO), 170.11 (CO). (ESI−) MS m/z (%): 347 ([M‒H]−, 100), 259 (25).
Anal. calcd. for C18H24N2O3S (348.45): C: 62.04, H: 6.94, N: 8.04. Found:
C: 61.61, H: 6.89, N: 8.15.
Yield: 100%. mp: 78–80°C; IR (KBr) ʋ (cm−1): 3334, 3197 (N‐H), 1712
(C=O), 1676 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 1.43 (3H, d, J =
8 Hz, spirodecane C2‐CH3), 1.48–1.71 (2H, m, spirodecane C7,9‐ax‐H),
1.73–2.03 (6H, m, spirodecane C7,9‐eq‐H, C6,10‐H), 2.35 (1H, broad t, J =
13 Hz, spirodecane C8‐H), 3.93 (1H, q, J = 8 Hz, spirodecane C2‐H), 4.73
(2H, s, OCH2), 6.95–7.06 (3H, m, phenoxy C2,4,6‐H), 7.14–7.21 (3H, m,
phenoxy C3,5‐H and C8‐phenyl‐H), 7.25–7.37 (4H, m, phenyl‐H), 10.44
(1H, s, NH). 13C‐NMR (proton decoupled) (DMSO‐d6/100 MHz): 18.46
(spirodecane C2‐CH3), 30.12, 30.68, 36.85, 37.71 (spirodecane C6,7,9,10),
36.69 (spirodecane C2), 41.36 (spirodecane C8), 65.96 (OCH2), 70.26
(spirodecane C5), 114.79, 121.28, 129.40 (phenoxy C2‐6), 126.03,
128.30 (phenyl C2‐6), 145.72 (phenyl C1), 157.52 (phenoxy C1), 167.57
(NHCO), 170.07 (CO). (ESI−) MS m/z (%): 409 ([M‒H]−, 100), 321 (10).
Anal. calcd. for C23H26N2O3S.C2H5OH (456.59): C: 65.76, H: 7.06, N:
6.14. Found: C: 65.48, H: 6.79, N: 6.50.
N‐(2‐Methyl‐8‐tert‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]dec‐4‐yl)‐2‐
phenoxyacetamide (7l)
Yield: 100%. mp: 155–158°C; IR (KBr) ʋ (cm−1): 3444, 3251 (N‐H), 1722
(C=O), 1693 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.70–0.89 (1H, m,
spirodecane C8‐H), 0.80 (9H, s, tert‐butyl CH3), 1.04–1.24 (2H, m,
N‐(2‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]nonan‐4‐yl)‐3‐
phenylpropanamide (8a)
Yield: 89%. mp: 190–193°C; IR (KBr) ʋ (cm−1): 3221, 3196 (N‐H), 1716
(C=O), 1670 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.04–1.82 (8H,

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m, spirononane C6‐9‐H), 2.49–2.53 (m, CH2, and DMSO‐d6), 2.86 (2H, t,
J = 7 Hz, CH2), 3.61 (2H, s, spirononane C2‐H), 7.18–7.29 (5H, m,
phenyl C2‐6‐H), 9.97 (1H, s, NH). Anal. calcd. for C16H20N2O2S
(304.40): C: 63.13, H: 6.62, N: 9.20. Found: C: 63.08, H: 6.71, N: 9.23.
C2‐H), 7.17–7.29 (5H, m, phenyl‐CH), 9.91 (1H, s, NH). Anal. calcd. for
C21H30N2O2S. 0.5C2H5OH (397.54): C: 66.46, H: 8.37, N: 7.05.
Found: C: 66.84, H: 8.14, N: 7.04.
N‐[3‐Oxo‐(8‐trifluoromethyl)‐1‐thia‐4‐azaspiro[4.5]decan‐4‐
yl]‐3‐phenylpropanamide (8g)
N‐(3‐Oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8b)
Yield: 94%. mp: 94–100°C; IR (KBr) ʋ (cm−1): 3392, 3186 (N‐H), 1697
(C=O), 1670 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.39–2.09
(9H, m, spirodecane C6‐10), 2.49–2.53 (m, CH2 and DMSO‐d6), 2.88
(2H, t, J = 7 Hz, CH2), 3.59 (2H, s, spirodecane C2‐H), 7.16–7.31 (5H,
m, phenyl‐CH), 10.00 (1H, s, NH). Anal. calcd. for C18H21F3N2O2S.
0.5H2O (395.43): C: 54.68, H: 5.82, N: 7.08. Found: C: 54.71, H: 5.97,
N: 7.12.
Yield: 100%. mp: 98–99°C; IR (KBr) ʋ (cm−1): 3354, 3167 (N‐H), 1703
(C=O), 1670 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.88–1.62
(10H, m, spirodecane C6‐10), 2.49–2.52 (m, CH2, and DMSO‐d6), 2.86
(2H, t, J = 7 Hz, CH2), 3.52 (2H, s, spirodecane C2‐H), 7.16–7.29 (5H,
m, phenyl C2‐6‐H), 9.91 (1H, s, NH). Anal. calcd. for C17H22N2O2S.
0.5C2H5OH (321.43): C: 63.31, H: 7.38, N: 8.20. Found: C: 62.78,
H:6.81, N: 8.59.
N‐(8‐Phenyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8h)
N‐(8‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8c)
Yield: 100%. mp: 194–196°C; IR (KBr) ʋ (cm−1): 3300, 3223 (N‐H),
1726 (C=O), 1681 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.04–
2.31 (9H, m, spirodecane C6‐10), 2.49–2.56 (m, CH2 and DMSO‐d6),
2.90 (2H, t, J = 7 Hz, CH2), 3.58 (2H, s, spirodecane C2‐H), 7.19–7.34
(10H, m, phenyl‐CH), 10.00 (1H, s, NH). Anal. calcd. for C23H26N2O2S
(394.52): C: 70.02, H: 6.64, N: 7.10. Found: C: 69.99, H: 6.58, N: 7.19.
Yield: 100%. mp: 102–105°C; IR (KBr) ʋ (cm−1): 3408, 3232 (N‐H),
1705 (C=O), 1674 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.81–
1.60 (9H, m, spirodecane C6‐10), 0.84 (3H, d, J = 7 Hz, CH3), 2.49–2.51
(m, CH2, and DMSO‐d6), 2.86 (2H, t, J = 7 Hz, CH2), 3.53 (2H, s,
spirodecane C2‐H), 7.16–7.28 (5H, m, phenyl C2‐6‐H), 9.91 (1H, s,
NH). Anal. calcd. for C18H24N2O2S. 0.5C2H5OH (355.46): C: 64.61, H:
7.79, N: 7.73. Found: C: 64.13, H: 7.35, N: 8.03.
N‐(2‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]nonan‐4‐yl)‐3‐
phenylpropanamide (8i)
N‐(8‐Ethyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8d)
Yield: 90%. mp: 169–171°C; IR (KBr) ʋ (cm−1): 3380, 3190 (N‐H),
1716 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.04–
1.88 (8H, m, spirononane C6‐9‐H), 1.40 (3H, d, J = 7 Hz, CH3), 2.49–
2.53 (m, CH2, and DMSO‐d6), 2.86 (2H, t, J = 7 Hz, CH2), 3.90 (1H, q,
J = 7 Hz, spirononane C2‐H), 7.18–7.29 (5H, m, phenyl‐CH), 10.02
(1H, s, NH). Anal. calcd. for C17H22N2O2S (318.43): C: 64.12, H: 6.96,
N: 8.80. Found: C: 64.16, H: 6.80, N: 8.87.
Yield: 97%. mp: 145–147°C; IR (KBr) ʋ (cm−1): 3396, 3230 (N‐H),
1705 (C=O), 1674 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz):
0.83–1.62 (9H, m, spirodecane C6‐10), 0.84 (3H, t, J = 7 Hz, CH3),
1.15 (2H, qu, J = 7 Hz, spirodecane C8‐CH2), 2.49–2.52 (m, CH2, and
DMSO‐d6), 2.86 (2H, t, J = 7 Hz, CH2), 3.52 (2H, s, spirodecane
C2‐H), 7.15–7.28 (5H, m, phenyl C2‐6‐H), 9.91 (1H, s, NH). Anal.
calcd. for C19H26N2O2S. 0.5C2H5OH (369.48): C: 65.01, H:7.51,
N: 7.58. Found: C: 65.16, H:7.52, N: 7.68.
N‐(2‐Methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8j)
Yield: 100%. mp: 141–145°C; IR (KBr) ʋ (cm−1): 3390, 3290 (N‐H),
1716 (C=O), 1681 (NHC=O). 1H‐NMR (DMSO‐d6/400 MHz): 0.86–
1.59 (10H, m, spirodecane C6‐10), 1.35 (3H, d, J = 7 Hz, C2‐CH3), 2.46–
2.54 (m, CH2, and DMSO‐d6), 2.85 (2H, t, J = 7 Hz, CH2), 3.80 (1H, q,
J = 7 Hz, spirodecane C2‐H), 7.14–7.28 (5H, m, phenyl C2‐6‐H), 9.95
(1H, s, NH). 13C‐NMR (DEPT) (DMSO‐d6/400 MHz): 19.68 (CH3),
22.59, 23.40, 23.74, 30.66, 34.67 (spirodecane C2‐6 and 2CH2), 36.62
(spirodecane C2), 70.55 (spirodecane C5), 125.99, 128.18, 128.35
N‐(3‐Oxo‐8‐propyl‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8e)
Yield: 100%. mp: 102–107°C; IR (KBr) ʋ (cm−1): 3383, 3230 (N‐H),
1705 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.86
(3H, t, J = 7 Hz, CH3), 0.99–1.61 (9H, m, spirodecane C6‐10), 1.05 (2H,
t, J = 7 Hz, spirodecane C8‐CH2), 1.23–1.31 (2H, m, spirodecane C8‐
CH2), 2.49–2.52 (m, CH2, and DMSO‐d6), 2.87 (2H, t, J = 7 Hz, CH2),
3.52 (2H, s, spirodecane C2‐H), 7.17–7.29 (5H, m, phenyl C2‐6‐H),
9.91 (1H, s, NH). Anal. calcd. for C20H28N2O2S. 0.5C2H5OH (383.51):
C: 65.76, H: 8.15, N: 7.30. Found: C: 65.58, H: 7.86, N: 7.29.
(phenyl
C2‐6), 140.52 (phenyl C1), 170.04 (amide CO), 170.77
(spirodecane CO). Anal. calcd. for C18H24N2O2S (332.46): C: 65.03,
H: 7.28, N: 8.43. Found: C: 64.77, H: 7.11, N: 8.49.
N‐(8‐tert‐Butyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8f)
Yield: 100%. mp: 154–155°C; IR (KBr) ʋ (cm−1): 3385, 3253 (N‐H),
1701 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.72–
1.64 (9H, m, spirodecane C6‐10), 0.82 (9H, s, CH3), 2.49–2.52 (m, CH2,
and DMSO‐d6), 2.87 (2H, t, J = 7 Hz, CH2), 3.52 (2H, s, spirodecane
N‐(2,8‐Dimethyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8k)
Yield: 100%. mp: 68–71°C; IR (KBr) ʋ (cm−1): 3421, 3261 (N‐H), 1701
(C=O), 1670 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.79–1.60 (9H,
m, spirodecane C6‐10), 0.84 (3H, d, J = 7 Hz, CH3), 1.37 (3H, d, J = 7 Hz,
C2‐CH3), 2.49–2.53 (m, CH2, and DMSO‐d6), 2.87 (2H, t, J = 7 Hz, CH2),

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3.83 (1H, q, J = 7 Hz, spirodecane C2‐H), 7.16–7.29 (5H, m, phenyl C2‐6
‐
(1H, s, NH). Anal. calcd. for C24H28N2O2S (408.55): C: 70.55, H: 6.91,
N: 6.86. Found: C: 70.57, H: 6.79, N: 6.94.
H), 9.96 (1H, s, NH). Anal. calcd. for C19H26N2O2S. 0.5H2O (346.48):
C: 64.22, H: 7.88, N: 7.88. Found: C: 63.98, H: 7.55, N: 8.04.
|
4.2
Antiviral activity assays
N‐(8‐Ethyl‐2‐methyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐3‐
phenylpropanamide (8l)
Cytopathic effect (CPE) reduction assays were performed to
determine antiviral activity against human coronavirus 229E and
influenza A/H1N1, A/H3N2, and B virus. The HCoV‐229E stock was
obtained from ATCC (VR‐740™) and expanded in human embryonic
lung (HEL) 299 fibroblast cells (ATCC CCL‐137™). For the CPE
reduction assay, HEL cells were seeded in 96‐well plates and grown
for 6 days until they reached confluence.
Yield: 94%. mp: 144–146°C; IR (KBr) ʋ (cm−1): 3310, 3186 (N‐H),
1720 (C=O), 1674 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.81–
1.63 (9H, m, spirodecane C6‐10), 0.84 (3H, t, J = 7 Hz, CH3), 1.16 (2H,
qu, J = 7 Hz, spirodecane C8‐CH2), 1.37 (3H, d, J = 7 Hz, C2‐CH3),
2.49–2.51 (m, CH2, and DMSO‐d6), 2.87 (2H, t, J = 7 Hz, CH2), 3.83
(1H, q, J = 7 Hz, spirodecane C2‐H), 7.17–7.29 (5 H, m, phenyl C2‐6‐H),
9.97 (1H, s, NH). Anal. calcd. for C20H28N2O2S (360.51): C: 66.63,
H: 7.83, N: 7.77. Found: C: 66.68, H: 7.55, N: 7.90.
The influenza virus strain A/HK/7/87 (A/H3N2) was a kind gift
from J. Neyts (Leuven, Belgium), whereas the clinical isolates A/Ned/
378/05 (A/H1N1) and B/Ned/537/05 were generously provided by
R. Fouchier (Rotterdam, The Netherlands). Virus stocks were
prepared in 10‐day‐old embryonated hen eggs; the clinical isolates
first underwent one passage in eggs. The influenza CPE reduction
assays were performed in Madin‐Darby canine kidney (MDCK) cells
(a kind gift from M. Matrosovich, Marburg, Germany) which were
seeded 1 day before infection at 7500 cells per well in 96‐well plates.
The cells were infected with the respective viruses at a multi-
plicity of infection of 100 (HCoV‐229E) or 50 (influenza virus) “50%
cell culture infective doses” (CCID50) per well in the presence of
serial dilutions of the test compounds. After five (HCoV‐229E) or
three (influenza virus) days incubation at 35°C, virus‐induced CPE
and compound cytotoxicity (in uninfected cells) were determined by
microscopic scoring. Antiviral activity was expressed as EC50 or
concentration showing 50% effectivity; cytotoxicity was expressed as
MCC, that is, minimum cytotoxic concentration producing minimal
changes in cell morphology. For HCoV‐229E, K22 [(Z)‐N‐[3‐[4‐(4‐
bromophenyl)‐4‐hydroxypiperidin‐1‐yl]‐3‐oxo‐1‐phenylprop‐1‐en‐2‐
N‐(2‐Methyl‐3‐oxo‐8‐propyl‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐
3‐phenylpropanamide (8m)
Yield: 93%. mp: 157–159°C; IR (KBr) ʋ (cm−1): 3388, 3188 (N‐H),
1724 (C=O), 1670 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.86
(3H, t, J = 7 Hz, CH3), 0.99–1.61 (9H, m, spirodecane C6‐10), 1.27 (2H,
m, spirodecane C8‐CH2), 1.37 (3H, d, J = 7 Hz, C2‐CH3), 2.49–2.52 (m,
CH2, and DMSO‐d6), 2.86 (2H, t, J = 7 Hz, CH2), 3.82 (1H, q, J = 7 Hz,
spd C2‐H), 7.17–7.29 (5 H, m, phenyl C2‐6‐H), 9.96 (1 H, s, NH). Anal.
calcd. for C21H30N2O2S (374.54): C: 67.34, H: 8.07, N: 7.48. Found:
C: 67.21, H: 7.76, N: 7.57.
N‐(2‐Methyl‐8‐tert‐butyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐
yl)‐3‐phenylpropanamide (8n)
Yield: 100%. mp: 122–123°C; IR (KBr) ʋ (cm−1): 3385, 3125 (N‐H),
1701 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 0.69–
1.62 (9H, m, spirodecane C6‐10), 0.82 (9H, s, CH3), 1.37 (3H, d, J = 7
Hz, C2‐CH3), 2.49–2.52 (m, CH2 and DMSO‐d6), 2.87 (2H, t, J = 7 Hz,
CH2), 3.82 (1H, q, J = 7 Hz, spirodecane C2‐H), 7.17–7.29 (5H, m,
phenyl C2‐6‐H), 9.96 (1H, s, NH). Anal. calcd. for C22H32N2O2S.H2O
(406.56): C: 64.93, H: 8.36, N: 6.88. Found: C: 64.73, H: 8.16, N: 6.98.
yl]benzamide; purchased from ChemDiv][14] was included as
a
reference compound. The following reference compounds were
included for influenza virus: ribavirin (Virazole from ICN Pharma-
ceuticals, Costa, CA), rimantadine and amantadine (both from Sigma‐
Aldrich, Belgium). Compounds showing anti‐HCoV activity were
evaluated in 3–4 independent experiments.
N‐[2‐Methyl‐3‐oxo‐(8‐trifluoromethyl)‐1‐thia‐4‐azaspiro[4.5]‐
decan‐4‐yl]‐3‐phenylpropanamide (8o)
Yield: 95%. mp: 126–130°C; IR (KBr) ʋ (cm−1): 3390, 3196 (N‐H),
1697 (C=O), 1660 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.27–
2.09 (9H, m, spirodecane C6‐10), 1.39 (3H, d, J = 7 Hz, C2‐CH3), 2.47–
2.53 (m, CH2 and DMSO‐d6), 2.88 (2H, t, J = 7 Hz, CH2), 3.89 (1H, q,
J = 7 Hz, spirodecane C2‐H), 7.15–7.31 (5H, m, phenyl C2‐6‐H), 10.06
(1H, s, NH). Anal. calcd. for C19H23F3N2O2S (418.45): C: 54.53,
H: 6.02, N: 6.69. Found: C: 54.61, H: 6.11, N: 6.91.
ACKNOWLEDGMENTS
This work was supported in part by a research fund from Istanbul
University (Project number TDK‐2016‐20602). A.S. and L.N. acknowl-
edge dedicated technical assistance from Leentje Persoons and
Benjamin Van Loy.
N‐(2‐Methyl‐8‐phenyl‐3‐oxo‐1‐thia‐4‐azaspiro[4.5]decan‐4‐yl)‐
3‐phenylpropanamide (8p)
CONFLICT OF INTERESTS
Yield: 96%. mp: 206–207°C; IR (KBr) ʋ (cm−1): 3350, 3182 (N‐H),
1720 (C=O), 1668 (NHC=O). 1H‐NMR (DMSO‐d6/500 MHz): 1.04–
2.31 (9H, m, spirodecane C6‐10), 1.41 (3H, d, J = 7 Hz, C2‐CH3), 2.49–
2.56 (m, CH2, and DMSO‐d6), 2.90 (2 H, t, J = 7 Hz, CH2), 3.89 (1H, q,
J = 7 Hz, spirodecane C2‐H), 7.19–7.32 (10H, m, phenyl‐CH), 10.05
The authors declared that they have no conflict of interests.
ORCID
Çağla Begüm Apaydın
http://orcid.org/0000-0001-6703-9389

APAYDIN ET AL.
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