Microwave assisted synthesis and anti-influenza virus activity of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide derivatives

Article abstract of DOI:10.1016/j.bmc.2012.09.064

A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl) carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4. 5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC₅₀: 1.4 μM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.

Full text of DOI:10.1016/j.bmc.2012.09.064

Bioorganic & Medicinal Chemistry 20 (2012) 7155–7159
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Microwave assisted synthesis and anti-influenza virus activity
of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide
derivatives
Füsun Göktasß a, , Evelien Vanderlinden b, Lieve Naesens b, Nesrin Cesur a, Zafer Cesur a
⇑
a Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 34116 Beyazıt, Istanbul, Turkey
b Rega Institute for Medical Research, University of Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2012
Revised 25 September 2012
Accepted 26 September 2012
Available online 12 October 2012
A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthe-
sis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety.
The structures of the compounds were confirmed by spectral and elemental analysis. All compounds
were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK
cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-
3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor
[antiviral EC50: 1.4 lM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis
assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational
change of the influenza virus hemagglutinin at low pH.
Keywords:
Adamantane
Spirothiazolidinone
Microwave
Ó 2012 Elsevier Ltd. All rights reserved.
Antiviral
Influenza virus
Hemagglutinin
1. Introduction
of some neurological conditions, type 2 diabetes or acne vulgaris.4
Furthermore, incorporation of an adamantyl moiety into a pharma-
Influenza viruses are important respiratory pathogens causing
significant morbidity and mortality and considerable economic
losses during the epidemic periods. Vaccination represents the pri-
mary protection but, due to the antigenic variability, the vaccine
requires annual updating. Vaccine development is retarded when
a new influenza virus, such as the 2009 pandemic variant, ap-
pears.1 The influenza virions carry a lipid envelope in which three
viral surface proteins are embedded: the hemagglutinin (HA), the
neuraminidase (NA) and the M2 proton channel protein.2 The lat-
ter two are validated antiviral targets: M2 channel activity is
blocked by the adamantane compounds amantadine and rimanta-
dine, whereas the NA is inhibited by zanamivir and oseltamivir.3
Besides its application in anti-influenza virus therapeutics, the
adamantyl moiety represents a versatile pharmacophore, since
adamantane derivatives have also been explored for the treatment
cologically active molecule often improves the therapeutic profile
of the parent agent.5
The anti-influenza virus action of the M2 blockers amantadine
and rimantadine is situated during the viral entry process. After cel-
lular uptake of the influenza virus by receptor-mediated endocyto-
sis, the low pH inside the endosomes activates the M2 proton
channel. Transport of protons into the virion interior leads to
uncoating, that is, release of the viral ribonucleoprotein from the
M1 capsid protein. In addition, the low endosomal pH provokes an
irreversible change in the conformation of the HA protein, leading
to fusion of the viral and endosomal membranes. Inhibition of M2
channel function is an explored strategy.6,7 Although amantadine
is effective in both prevention and prophylaxis of influenza A virus
infections, its clinical use is limited by its neurological side effects
and low barrier for resistance. Its analogue rimantadine has been
considered a better antiviral drug because of its enhanced activity
and fewer side effects.8 Recently gained insight in the protein struc-
ture of M2 and the precise M2 binding mode of amantadine and
rimantadine9,10 may lead to more potent M2 inhibitors which retain
activity against virus mutants carrying amantadine- or rimanta-
dine-associated resistance mutations in the M2 protein.11
Abbreviations: HA, hemagglutinin; NA, neuraminidase; MDCK, madin darby
canine kidney cells; EC50, 50% effective concentration; RBC, red blood cells; DMSO,
dimethyl sulfoxide; CPE, cytopathic effect; PFU, plaque forming unit; MTS, 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo-
lium; MCC, minimal cytotoxic concentration; Mp, melting point; adm, adamantane;
spd, spiro[4.5]decane.
In comparison, the development of small molecule inhibitors of
the HA-mediated fusion process has proven to be relatively
⇑
Corresponding author. Tel.: +90 212 4555700/13460; fax: +90 212 4400252.
E-mail address: fusung@istanbul.edu.tr (F. Göktasß).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.064

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F. Göktaßs et al. / Bioorg. Med. Chem. 20 (2012) 7155–7159
A mixture of 1-adamantanecarbohydrazide, the appropriate
cyclic ketone and sulfanylacetic acid or 2-sulfanylpropanoic acid
in toluene, was heated under microwave irradiating conditions at
120 °C and 400 W for 10 min. After solvent evaporation, the resi-
due was made alkaline and allowed to solidify.
O
S
O
N
NH
N
The IR spectra of 2a–f and 3a–f showed common characteristic
absorption bands at 3468–3265 cmÀ1 (NH), 1683–1658 cmÀ1
(NH–C@O) and 1720–1697 cmÀ1 (C@O), which provided evidence
for the cycloaddition reaction between azomethine intermediates
and sulfanylacetic or 2-sulfanylpropanoic acids. In the 1H
NMR spectra of 2a–f and 3a–f, CONH proton was observed at
9.57–9.25 ppm as a singlet. The resonance of the CH2 protons 2a–f
at position 2 of the spiro system was observed at 3.57–3.49 ppm
as a singlet. Additional support was obtained from the spectra of
3a–f, which showed the same resonance with almost the same
chemical shift values (3.84–3.78 ppm) as a quartet. CH3 protons of
3a–f resonated at 1.41–1.37 ppm as a doublet. Adamantil protons
were observed as expected, together with the remaining protons
of the spiro system in the range of C6–10. Synthesis of 3a–f was per-
formed using racemic 2-sulfanylpropanoic acid. On the other hand,
in spirocyclic compounds, two rings meet at a single atom. This
means that the two rings are orthogonal about the tetrahedral atom.
Even symmetrically looking compounds are unexpectedly chiral.15
Thus, our target compounds structurally are stereoisomers. Enan-
tiomeric separation was not performed. The detailed spectral data
of the compounds (2a–f and 3a–f) are given in the Section 4.
S
N
Figure 1. Structure of 6-methyl-N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-
4-yl) imidazo[2,1-b][1,3]thiazole-5-carboxamide (4c).
difficult. Most reported fusion inhibitors suffer from having sub-
type-dependent antiviral activity and a low barrier for resistance
selection.12 We recently identified a new class of N-(1-thia-4-aza-
spiro[4.5]decan-4-yl)carboxamide derivatives with potent activity
against influenza A/H3N2 viruses.13 We demonstrated that the
lead compound [6-methyl-N-(2,8-dimethyl-3-oxo-1-thia-4-aza-
spiro[4.5]dec-4-yl)imidazo[2,1-b][1,3]thiazole-5-carboxamide; en-
coded 4c in Ref. 13] (Fig. 1) inhibits the HA-mediated membrane
fusion at low pH, due to its specific binding interaction with a
hydrophobic pocket within the HA protein. We now report on re-
lated analogues carrying an 1-adamantyl substituent. This creates
the possibility to combine two pharmacological activities (i.e.,
M2 and HA inhibition) into one single agent.
2. Results and discussion
2.1. Chemistry
2.2. Biological activity
The test compounds were evaluated for anti-influenza virus
activity by a cytopathic effect (CPE) reduction assay.13 MDCK cells
were infected with one of three influenza virus strains: A/PR/8/34
(A/H1N1); A/X-31 (A/H3N2) or B/HK/5/72. After 72 h incubation at
35 °C, microscopy followed by the MTS cell viability test were per-
formed, to determine the following parameters for antiviral and
cytotoxic activity: EC50, or compound concentration producing
50% inhibition of virus-induced cytopathic effect; MCC, or
minimum cytotoxic concentration (defined as the compound
Microwave (MW) irradiation has been recently introduced to
perform diverse chemical reactions in a short time, and to produce
compounds in high yields. In the green-chemistry area, the objec-
tive is to perform organic reactions solvent-free under dry media
conditions, thereby reducing the use of toxic solvents, and prevent-
ing unnecessary pollution. Thus, MW reaction methods are conve-
nient, clean and economical.14
Table 1
Anti-influenza virus activity and cytotoxicity in MDCK cell cultures
c
Compound
Cytotoxicity (lM)
EC50
(lM) against influenza virus
MCCb
A/H1N1 (A/PR/8/34)
CPE
A/H3N2 (A/X-31)
CPE
B (B/HK/5/72)
CPE
a
CC50
MTS
MTS
MTS
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
2f
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
42
>100
>100
>100
>100
>100
>100
>100
>20
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
53
>100
>100
1.6
>100
51
>100
29
>100
>100
>100
>100
>100
>100
1.4
21
1.7
44
23
34
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>20
20
>100
>100
>100
>100
>100
>100
3f
4c [13]
>100
>100
>100
>100
6.8
9.0
>100
>100
Zanamivir
Ribavirin
Amantadine
Rimantadine
>100
>100
>500
218
>100
>20
>500
500
0.44
8.9
58
0.48
8.4
75
12
53
0.072
0.094
>500
>500
0.063
0.14
>500
>500
8.9
224
45
8.5
208
40
2.7
2.5
ND: Not done
MDCK cells: Madin Darby canine kidney cells.
a
50% Cytotoxic concentration, as determined by measuring the cell viability with the colorimetric MTS assay.
Minimum cytotoxic concentration, causing a microscopically detectable alteration in cell morphology.
50% Effective concentration, or concentration producing 50% inhibition of virus replication,as determined by microscopical scoring of the cytopathic effect (CPE) or by the
b
c
MTS cell viability assay.

F. Göktaßs et al. / Bioorg. Med. Chem. 20 (2012) 7155–7159
7157
O
R
O
Compound
R
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
R1
H
H
CH3
CH3
C2H5
C2H5
C3H7
C3H7
C(CH3)3
C(CH3)3
C6H5
C6H5
R
O
1
O
2a
3a
2b
3b
2c
3c
2d
3d
2e
3e
2f
N
S
N
C
H
O
N
NH2
OH
H
R
SH
R1
(2a-f) (3a-f)
CH3
3f
Figure 2. Strategy for the synthesis of compounds 2a–f and 3a–f.
concentration causing minimal changes in cell morphology), and
CC50 (the compound concentration causing 50% reduction in cell
viability based on the MTS assay).
which an inhibitory effect on the conformational change of HA
was clearly demonstrated,13 3b strongly inhibits influenza-virus
induced hemolysis at low pH. Compound 3b represents a hybrid
molecule combining the adamantyl pharmacophore for inhibition
of the influenza virus M2 protein, and the spirothiazolidinone
backbone, which interferes with HA-mediated fusion. This creates
the possibility to combine two antiviral modes of action into one
single pharmacological agent.
As shown in Table 1, compound 3b was the most potent ana-
logue among the series, with an antiviral EC50 value of 1.5
against the A/H3N2 virus. This compound produced no cytotoxic
effects at 100 M (the highest concentration tested), yielding a
lM
l
selectivity index (or ratio of cytotoxic to antiviral concentration)
of >67. Since 3b carries the N-(1-thia-4-azaspiro[4.5]decan-4-
yl)carboxamide backbone that is characteristic for our series of
influenza virus fusion inhibitors,13 we further evaluated the activ-
ity of 3b in a hemolysis inhibition assay. The A/H3N2 virus was
preincubated with test compound prior to its addition to red blood
cells. After lowering the pH, the degree of hemolysis was deter-
mined by spectrophotometry. In the absence of compound, low
pH-induced refolding of HA resulted in perforation of the RBC
membrane and hemolysis. Compound 3b produced a dose-depen-
dent inhibition of hemolysis (data not shown), with an IC50 of
4. Experimental protocols
4.1. Chemistry
Commercially available chemicals were obtained from Merck
and Aldrich. Melting points (Mp) were determined on a Büchi
530 capillary melting point apparatus in open capillaries and were
uncorrected. Compound purity was measured by thin-layer chro-
motography (TLC) on Silica gel 60 F254. Infrared (IR) spectra were
recorded on a Pelkin Elmer 1600 FT infrared spectrophotometer
3.7
our previously identified lead compound 4c,13 which was included
as a reference (IC50 of 2.6 M).
Structure–activity analysis on this new series of 1-adamantyl
substituted
lM. This value was in the same range as that obtained with
in potassium bromide pellets (t
in cmÀ1). 1H nuclear magnetic res-
l
onance (NMR) spectra were recorded on a Varian UNITY INOVA
500 MHz NMR spectrophotometer and Varian Mercury 400 MHz
High Performance Digital FT-NMR Spectrometer using tetrameth-
ylsilane as internal standard. Microwave reactions were carried
out using a Milestone Start S microwave apparatus. Elementary
analyses were performed on a Thermo Finnigan Flash EA 1112
Series elemental analyzer.
N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamides
(Fig. 2) revealed which substituents are required for the antiviral
activity. Overall, these data were in nice agreement with the struc-
ture–activity relationship that we previously obtained for 4c and
analogues.13 An important new insight is that the anti-influenza
virus activity is unchanged when the aromatic ring system in 4c
is replaced by a non-aromatic and bulky adamantyl moiety. With
regard to the spiro moiety, substitution at position 2 and 8 is
clearly essential. The methyl substituent at position 2 (R) is obvi-
ously required, since the analogues 2c and 2d lacking this group
were less active than their 2-methylated counterparts 3c and 3d.
Regarding position 8 of the spiro ring, a smaller alkyl group (i.e.,
methyl in 3b, ethyl in 3c or n-propyl in 3d) is preferable over a lar-
ger alkyl (i.e., t-butyl; 3e) or phenyl (3f) substituent. Compounds
lacking a substituent at this position 8 (3a) were devoid of any
anti-influenza virus activity. In analogy to our findings with 4c,
the novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide deriv-
atives had activity against influenza A/H3N2 but not A/H1N1 or B
viruses.
4.2. General synthesis of 2a–f and 3a–f
A one-pot three component mixture of 1-adamantane carbo-
hydrazide (0.005 mol), the appropriate cyclic ketone (0.005 mol)
and sulfanylacetic acid or 2-sulfanylpropanoic acid in 12 mL of tol-
uene was added to a glass tube with a magnetic stirring bar. The
mixture was heated under microwave irradiating conditions at
120 °C and 400 W for 10 min. After cooling, completion of the reac-
tions was checked by TLC, and the compounds were transferred to
a glass flask. Excess toluene was evaporated in vacuo. The residue
was triturated with saturated NaHCO3 until CO2 effervescence
ceased and was allowed to stand overnight. The solid thus obtained
was filtered, washed with H2O, and crystallized from the C2H5OH–
H2O mixture.
3. Conclusion
4.2.1. N-(3-Oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-
carboxamide (2a)
Among this novel series of 1-adamantyl substituted spiro-
thiazolidinones, compound 3b emerged as a potent and selective
inhibitor of influenza A/H3N2 virus. Similar to its congener 4c for
Yield: 87.35%. Mp 245–7 °C. IR: 3280 (NH), 1705, 1683 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.96–1.98 (25H, m, adm-H and spd.

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F. Göktaßs et al. / Bioorg. Med. Chem. 20 (2012) 7155–7159
C6–10-H), 3.50 (2H, s, spd. C2-H), 9.45 (1H, s, CONH). Anal. calcd for
4.2.9. N-(8-Ethyl-2-methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (3c)
C
19H28N2O2SÁH2O (366.51): C, 62.26; H, 8.25; N, 7.64. Found: C,
63.03; H, 7.55; N, 8.13.
Yield: 89.23%. Mp 209–10 °C. IR: 3468 (NH), 1703, 1668 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.82 (3H, t, J = 7.5 Hz, –CH2CH3),
1.00–1.19 (5H, m, adm-H, spd. C6–10-H and CH2CH3), 1.37 (3H, d,
J = 6.8 Hz, C2-CH3), 1.64–1.97 (21H, m, adm-H and spd. C6–10-H),
3.78 (1H, q, J = 6.8 Hz, spd. C2-H), 9.50 (1H, s, CONH). Anal. calcd
for C22H34N2O2S (408.59): C, 66.14; H, 9.41; N, 7.04. Found: C,
66.12; H, 8.83; N, 7.01.
4.2.2. N-(8-Methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide(2b)
Yield: 93.37%. Mp 229–31 °C. IR: 3265 (NH), 1705, 1681 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.84 (3H, d, J = 6.3 Hz, ÀCH3), 1.09–
1.97 (24H, m, adm-H and spd. C6–10-H), 3.50 (2H, s, spd. C2-H),
9.45 (1H, s, CONH). Anal. calcd for C20H30N2O2SÁ0.5H2O (371.53):
C, 64.65; H, 8.41; N, 7.54. Found: C, 64.49; H, 8.09; N, 8.23.
4.2.10. N-(2-Methyl-3-oxo-8-propyl-1-thia-4-azaspiro[4.5]dec-
4-yl)adamantane-1-carboxamide (3d)
Yield: 88.11%. Mp 198–9 °C. IR: 3462 (NH), 1701, 1662 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.83 (3H, t, J = 7.3 Hz, –CH2CH2CH3),
1.10–1.13 (4H, m, –CH2CH2CH3 and adm-H), 1.26 (2H, q,
J = 7.8 Hz, –CH2CH2CH3), 1.37 (3H, d, J = 6.8 Hz, spd. C2-CH3),
1.66–1.97 (22H, m, adm-H and spd. C6–10-H), 3.78 (1H, q,
J = 6.8 Hz, spd. C2-H), 9.50 (1H, s, CONH). Anal. calcd for
C23H36N2O2SÁH2O (422.62): C, 65.36; H, 9.06; N, 6.63. Found: C,
65.58; H, 8.97; N, 6.93.
4.2.3. N-(8-Ethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (2c)
Yield: 88.82%. Mp 195–7 °C. IR: 3448 (NH), 1701, 1660 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.83 (3H, t, J = 7.3 Hz, –CH2CH3),
1.10–1.17 (2H, m, –CH2CH3), 1.66–1.97 (24H, m, adm-H and spd.
C6–10-H), 3.50 (2H, s, spd. C2-H), 9.45 (1H, s, CONH). Anal. calcd
for C21H32N2O2SÁ0.5C2H5OH (399.58): C, 63.11; H, 8.82; N, 7.01.
Found: C, 62.85; H, 8.45; N, 7.65.
4.2.11. N-(2-Methyl-3-oxo-8-tert-butyl-1-thia-4-
4.2.4. N-(3-Oxo-8-propyl-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide(2d)
Yield: 93.33%. Mp 188–90 °C. IR: 3446 (NH), 1701, 1658 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.84 (3H, t, J = 7.3 Hz, –CH2CH2CH3),
1.08–1.13 (4H, m, –CH2CH2CH3 and adm-H), 1.26 (2H, q,
J = 7.3 Hz, –CH2CH2CH3), 1.64–1.97 (22H, m, adm-H and spd.
azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide (3e)
Yield: 76.07%. Mp 256–7 °C. IR: 3273 (NH), 1720, 1670 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.81 (9H, s, –C(CH3)3), 1.12–1.22
(2H, m, adm-H and spd. C6-10-H), 1.37 (3H, d, J = 6.8 Hz, spd.
C2-CH3), 1.64–1.97 (22H, m, adm-H and spd. C6-10-H), 3.78 (1H,
q, J = 6.8 Hz, spd. C2-H), 9.50 (1H, s, CONH). Anal. calcd for
C6-10-H), 3.49 (2H, s, spd. C2-H), 9.45 (1H, s, CONH). Anal. calcd
C
24H38N2O2SÁH2O (436.64): C, 66.01; H, 9.23; N, 6.42. Found: C,
for C22H34N2O2SÁH2O (408.59): C, 64.06; H, 8.88; N, 6.85. Found:
66.10; H, 8.91; N, 6.75.
C, 64.53; H, 8.75; N, 7.13.
4.2.12. N-(2-Methyl-3-oxo-8-phenyl-1-thia-4-azaspiro[4.5]dec-
4-yl)adamantane-1-carboxamide (3f)
4.2.5. N-(3-Oxo-8-tert-butyl-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (2e)
Yield: 84.01%. Mp 146–8 °C. IR: 3462 (NH), 1697, 1670 (C@O).
1H NMR (DMSO-d6) d (ppm): 1.41 (3H, d, J = 6.8 Hz, spd. C2-CH3),
Yield: 83.66%. Mp 230–2 °C. IR: 3446 (NH), 1701, 1670 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.81 (9H, s, –C(CH3)3), 1.16–1.97
(24H, m, adm-H and spd. C6–10-H), 3.49 (2H, s, spd. C2-H), 9.45
(1H, s, CONH). Anal. calcd for C23H36N2O2SÁH2O (422.62): C,
65.36; H, 8.58; N, 6.92. Found: C, 64.99; H, 8.63; N: 7.05.
1.60–1.99 (24H, m, adm-H and spd.
C6-10-H), 3.84 (1H, q,
J = 6.8 Hz, spd. C2-H), 7.15–7.29 (5H, m, phenyl-H), 9.57 (1H, s,
CONH). Anal. calcd for C26H34N2O2SÁ1.5 H2O (465.64): C, 67.05;
H, 8.01; N, 6.01. Found: C, 66.98; H, 7.53; N, 6.27.
4.3. Antiviral assay
4.2.6. N-(3-Oxo-8-phenyl-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (2f)
The multicycle CPE reduction assay for antiviral evaluation of
the test compounds can be found elsewhere.13 MDCK cells, seeded
into 96-well plates, were exposed to serial dilutions of the com-
pounds, together with the influenza virus (multiplicity of infection:
0.0004 PFU per cell). After three days incubation at 35 °C, micros-
copy was performed for visual scoring of the CPE and cytotoxic
effects. The data were confirmed by the spectrophotometric MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium] cell viability assay.
Yield: 78.30%. Mp 252–4 °C. IR: 3292 (NH), 1697, 1681 (C@O).
1H NMR (DMSO-d6) d (ppm): 1.63–2.00 (24H, m, adm-H and spd.
C
6–10-H), 3.57 (2H, s, spd. C2-H), 7.18–7.31 (5H, m, phenyl-H),
9.57 (1H, s, CONH). Anal. calcd for C25H32N2O2SÁH2O (422.61): C,
67.83; H, 7.74; N, 6.33. Found: C, 68.28; H, 7.48; N, 6.77.
4.2.7. N-(2-Methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (3a)
Yield: 77.34%. Mp 241–3 °C. IR: 3304 (NH), 1708, 1670 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.98–1.98 (25H, m, adm-H and spd.
4.4. Hemolysis inhibition assay
C
6–10-H), 1.38 (3H, d, J = 6.8 Hz, spd. C2-CH3), 3.79 (1H, q,
J = 6.8 Hz, spd. C2-H), 9.50 (1H, s, CONH). Anal. calcd for
20H30N2O2S (362.52): C, 66.26; H, 8.34; N, 7.73. Found: C, 66.02;
The detailed methodology has already been published.13 An
allantois stock of influenza virus A/H3N2 (strain A/X-31) was pre-
incubated with serial dilutions of the test compounds, and incu-
bated at 37 °C for 1 h. Chicken red blood cells (RBC) were added
and, after 10 min incubation at 37 °C, the RBC (with adsorbed
virus) were collected by centrifugation. The RBC pellets were
resuspended in buffer containing the corresponding compound
concentrations, and the pH was lowered to 5.0. After 25 min incu-
bation at 37 °C, the samples were neutralized, and intact chicken
RBC were removed by centrifugation. The supernatant was trans-
ferred to a 96-well plate, and the optical density was measured
at 540 nm. Background values were derived from mock-infected
C
H, 7.84; N, 7.91.
4.2.8. N-(2,8-Dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-
yl)adamantane-1-carboxamide (3b)
Yield: 93.61%. Mp 255–6 °C. IR: 3273 (NH), 1708, 1668 (C@O).
1H NMR (DMSO-d6) d (ppm): 0.85 (3H, d, J = 6.0 Hz, spd. C8-CH3),
1.08–1.98 (27H, m, adm-H, spd. C2-CH3 and spd. C6–10-H), 3.81
(1H, q, J = 6.8 Hz, spd. C2-H), 9.25 (1H, s, CONH). Anal. calcd for
C
21H32N2O2S (376.55): C, 66.98; H, 8.57; N, 7.44. Found: C, 66.87;
H, 8.82; N, 7.91.

F. Göktaßs et al. / Bioorg. Med. Chem. 20 (2012) 7155–7159
7159
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of the compounds was expressed as the IC50, relative to the value
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Acknowledgments
7. Duque, M. D.; Ma, C.; Torres, E.; Wang, J.; Naesens, L.; Jiminez, J. J.; Camps, P.;
Luque, F. J.; DeGrado, W. F.; Lamb, R. A.; Pinto, L. H.; Vázquez, S. J. Med. Chem.
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This work was supported by Istanbul University Scientific
Research Project (Project Number: BYP-2906 and BYP-12603), the
Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO No.
9.0188.07) and the Geconcerteerde Onderzoeksacties (GOA/10/
014). E.V. is a recipient of a PhD grant from the KULeuven. We
appreciate the dedicated assistance from Leentje Persoons and
Wim van Dam.
8. De Clercq, E. Nat. Rev. Drug Disc. 2006, 5, 1015.
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