Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole derivatives

Article abstract of DOI:10.1007/s00706-019-02457-9

Abstract: A series of indole-based spirothiazolidinones have been designed, synthesized and evaluated, in vitro, for their antitubercular, antiviral, antibacterial, and antifungal activities. The structures of the new compounds were established by IR,

Full text of DOI:10.1007/s00706-019-02457-9

Monatshefte für Chemie - Chemical Monthly (2019) 150:1533–1544
https://doi.org/10.1007/s00706-019-02457-9
ORIGINAL PAPER
Design, synthesis, antitubercular and antiviral properties of new
spirocyclic indole derivatives
Gökçe Cihan‑Üstündağ1 · Lieve Naesens2 · Dilek Şatana3 · Gonca Erköse‑Genç3 · Emel Mataracı‑Kara4 ·
Gültaze Çapan1
Received: 12 March 2019 / Accepted: 12 June 2019 / Published online: 17 July 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
A series of indole-based spirothiazolidinones have been designed, synthesized and evaluated, in vitro, for their antitubercular,
antiviral, antibacterial, and antifungal activities. The structures of the new compounds were established by IR, 1H NMR,
13C NMR (proton decoupled, APT, and DEPT), electrospray ionization mass spectrometry, and microanalysis. Compounds
bearing a phenyl substituent at position 8 of the spiro ring, exhibited signifcant antitubercular activity against Mycobacte-
rium tuberculosis H37Rv ATCC 27294 at concentrations of 3.9 and 7.8 µM. Still, some of the tested compounds displayed
activity on mycobacteria with MIC values of 16 and 31 µM. Four of the indole-spirothiazolidinone derivatives were found
to be moderately active against Punta Toro virus, yellow fever virus or Sindbis virus in Vero cells. The antiviral EC50 val-
ues were in the range of 1.9–12 µM and the selectivity index (ratio of cytotoxic to antivirally efective concentration) was
above 10 in some cases. The most potent efect was seen with the compound that is methylated at positions 2 and 8 of the
spirothiazolidinone system.
Graphic abstract
Keywords Heterocycles · Spirothiazolidinone · Antitubercular activity · Antiviral activity · Cytotoxicity · Drug research
Introduction
Tuberculosis (TB) is a highly infectious disease caused
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-019-02457-9) contains
supplementary material, which is available to authorized users.
by the bacillus Mycobacterium tuberculosis. For the past
5 years, it has been the leading cause of mortality from a
3
* Gökçe Cihan-Üstündağ
Department of Microbiology and Clinical Microbiology,
gokcechn@istanbul.edu.tr
Istanbul Faculty of Medicine, Istanbul University,
Istanbul 34116, Turkey
1
Department of Pharmaceutical Chemistry, Faculty
4
Department of Pharmaceutical Microbiology, Faculty
of Pharmacy, Istanbul University, Istanbul 34116, Turkey
of Pharmacy, Istanbul University, 34116 Istanbul, Turkey
2
Department of Microbiology, Immunology
and Transplantation, Rega Institute for Medical Research,
KU Leuven, 3000 Louvain, Belgium
Vol.:(0123456789)
1 3

1534
G. Cihan-Üstündağ et al.
single infectious disease, ranking above HIV/AIDS [1].
Major problems associated with the currently available TB
treatment include long treatment duration, inadequate com-
pliance, concurrent HIV infection, and increasing incidence
of multidrug-resistant (MDR) and extensively drug-resistant
(XDR) tuberculosis [2–5]. This emergence of difcult to
treat strains necessitates the discovery and development of
novel antitubercular drugs. After this domain has been inac-
tive for several decades, two new drugs became available,
i.e., the mitochondrial ATP synthase inhibitor bedaquiline
and mycolic acid biosynthesis inhibitor delamanid, which
received accelerated approval for the treatment of MDR
tuberculosis in 2012 and 2014, respectively [6]. Besides,
diverse novel drug candidates are in preclinical or clinical
development [6–8].
tenfold lower than those that produced cytotoxicity in mam-
malian cell lines.
Furthermore, several spirothiazolidinone compounds syn-
thesized in our laboratory were found to be efcient inhibi-
tors of membrane fusion mediated by infuenza virus hemag-
glutinin (HA) [18–20]. As demonstrated in Fig. 1d, these
compounds have a similar backbone structure, consisting of
an aromatic/polycyclic ring linked to a non-aromatic spiro
system via an amide bridge. Some analogues displayed low
micromolar activity against the infuenza A/H3N2 subtype
with a favorable selectivity index.
Based on these insights and our objective to optimize
the antimicrobial activity of indolyl thiazolidinones and
spirothiazolidinones, we here report the chemical synthe-
sis, structural characterization and in vitro antitubercular,
antiviral, antibacterial, and antifungal evaluation of new
5-chloro-3-phenyl-N-(2,7,8,9-substituted/nonsubstituted-
3-oxo-1-thia-4-azaspiro[4.4]nonan/[4.5]decan-4-yl)-1H-in-
dole-2-carboxamides 4a–4i, 5a–5h (Fig. 1e).
Indole-2-carboxamides incorporating an alicyclic sys-
tem (Fig. 1a) have been extensively studied by diferent
research groups [9–14]. This type of compounds was found
to be highly active against both drug-susceptible and drug-
resistant strains of Mycobacterium tuberculosis by acting on
the MmpL3 transporter protein. In previous investigations,
we have identifed the indole-spirothiazolidinone system
(Fig. 1b) as a promising scafold against the Mycobacterium
tuberculosis H37Rv strain [15, 16]. Some of these analogues
exhibited in vitro antitubercular activity with GI (growth
inhibition) values of 91–95% at a MIC (minimum inhibitory
concentration) value of 6.25 µg/cm3. Recently, we reported
on the synthesis of novel 5-fluoro-3-phenyl-1H-indole
derivatives containing a 4-thiazolidinone nucleus (instead
of spirothiazolidinone system) [17]. Two molecules (Fig. 1c)
displayed notable antitubercular activity at concentrations
Results and discussion
Chemistry and structural characterization
The synthetic pathways for the preparation of the spirothia-
zolidinones 4a–4i and 5a–5h are illustrated in Scheme 1.
Thus, the diazonium salt, formed by the reaction of 4-chloro-
aniline with NaNO2 and HCl, was reacted with ethyl 2-ben-
zyl-3-oxobutanoate to obtain compound 1 [21] according
(a)
(b)
(c)
(e)
(d)
Fig. 1 Structures of a, b, c indole-2-carboxamide-based antitubercular agents and d the spirothiazolidinone compounds acting as infuenza A
virus fusion inhibitors
1 3

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole…
1535
Scheme 1
(i) 7% NaNO2, EtOH, conc. HCl, 0 oC; (ii) ethyl 2-benzyl-3-oxo-butanoate, KOH, EtOH, 0 oC; (iii) conc. HCl, reflux, 4 h;
.
(iv) H2NNH2 H2O, EtOH, reflux, 6 h; (v) cyclopentanone/3,4,5-(non)substituted cyclohexanone, mercaptoacetic acid,
dry toluene, reflux, 5–6 h; (vi) cyclopentanone/3,4,5-(non)substituted cyclohexanone, 2-mercaptopropionic acid, dry
toluene, reflux, 5–6 h. 4b, 5b: R,R1,R2,R3=H; 4c, 5c: vR=CH3;R1,R2,R3,=H; 4d, 5d: R1=CH3;R,R2,R3=H;
1
4e, 5e: R1=C2H5;R,R2,R3=H; 4f, 5f: R1=C3H7;R,R2,R3=H; 4g, 5g: R,R2,R3=CH3;R1=H; 4h,5h:R =C6H5;R,R2,R3=H;
1
2
3
4i:
R =C(CH3)3;R,R ,R =H
to the Japp–Klingemann reaction. The Fischer indole syn-
thesis was carried out in acidic medium to cyclize 1 into
ethyl 5-chloro-3-phenyl-1H-indole-2-carboxylate 2 [22].
Subsequent exposure of 2 to an excess of hydrazine hydrate
aforded compound 3 [23]. The target spirocyclic com-
pounds 4a–4i, 5a–5h were synthesized by treatment of the
key intermediate 3 with appropriate cyclic ketones and mer-
capto acids in a one-pot reaction [15].
together with the absence of the NH2 resonance of the inter-
1
mediate hydrazide 3 in the H NMR spectra of 4 and 5,
provided further evidence for the formation of new adducts.
The S–CH2 (4a–4i) and S–CH (5a–5h) protons of the newly
formed spiroalkane residue resonated at about 3.50-3.65 and
3.87-3.93 ppm, respectively. The S–CH2 protons of 4a–4i
appeared as singlets except for the methylene hydrogens of
compound 4g which were observed as separate doublets with
large coupling constants (J = 16.1 Hz) due to the geminal
coupling resulting from the chiral centers of the spirothiazo-
lidinone ring. The 1H NMR spectra of 5a–5h displayed the
thiazolidinone S–CH protons as quartets or broad/distorted
singlets and doublets. Assignment of the indole protons was
achieved on the basis of the values and coupling constants
reported for the 2,3,5-trisubstituted indole ring [15, 16,
24, 25]. The carbon resonances were assigned by chemical
shifts and comparison with previously reported 13C NMR
data for compounds having a similar backbone structure [15,
19, 26]. CH3, CH2, CH, and C signals were assigned by
The structures of the new compounds were characterized
1
by IR, H NMR, 13C NMR (proton decoupled, APT, and
DEPT), electrospray ionization mass spectrometry (ESI-
MS), and combustion analysis. The IR spectra of 4a–4i and
5a–5h exhibited the ring and the exocyclic C=O bands in
the 1692–1713 cm−1 and 1651–1670 cm−1, respectively.
The shifts observed in the amide bands when compared to
that of 3 (1636 cm−1) and the presence of additional lac-
tam bands provided defnite proof for the aimed cycliza-
tion. Observation of NH signals assigned to the indole NH
(δ = 12.11–12.18 ppm) and amide NH (9.97–0.21 ppm)
1 3

1536
G. Cihan-Üstündağ et al.
Table 1 Antitubercular activity against M. tuberculosis H37Rv
4f, and 4i) displayed some activity on mycobacteria with
MIC values of 16 and 31 µM.
ATCC 27294
R1
R2
R3
MICa/µM
Looking at the chemical structures of the active com-
pounds, it can be observed that the presence of a methyl
group at position 2 of the spirocyclic system (series 5) led to
a signifcant reduction in antitubercular activity. Introduction
of a bulky aromatic substituent (C6H5) at position 8 of the
ring, as in 4h and 5h, enhanced the antitubercular activity.
Compound
R
4a
–
H
CH3
H
H
H
CH3
H
H
–
H
CH3
H
H
H
CH3
H
–
–
H
H
–
H
H
H
H
H
CH3
H
H
–
H
H
H
H
H
CH3
H
–
–
H
H
H
H
H
CH3
H
H
–
H
H
H
H
H
CH3
H
–
1000
16
16
31
250
31
500
3.9
16
4b
4c
4d
CH3
C2H5
C3H7
H
C6H5
C(CH3)3
–
4e
4f
Antiviral activity
4g
4h
Compounds 4a–4i and 5a–5h were evaluated against a vari-
ety of DNA and RNA viruses in cell culture, namely: herpes
simplex virus type-1 (HSV-1) and type-2 (HSV-2), an acy-
clovir-resistant thymidine kinase-defcient (TK−) mutant of
HSV-1, vaccinia virus, human adenovirus-2, human corona-
virus, vesicular stomatitis virus, Coxsackie B4 virus, respira-
tory syncytial virus, parainfuenza-3 virus, reovirus, Sindbis
virus, Punta Toro virus, yellow fever virus, and infuenza A
and infuenza B virus. The cytopathic efect reduction assays
revealed that compounds 4b, 4c, 5b, and 5d were moderately
active against Punta Toro virus, yellow fever virus or Sind-
bis virus in Vero cells (Table 2). The antiviral EC50 values
were in the range of 1.9–12 µM and the selectivity index
(SI: ratio of cytotoxic to antivirally efective concentration)
was above 10 in some cases (see values between brackets in
Table 2). The most potent efect was seen with compound
5d that is methylated at positions 2 and 8 of the spirothiazo-
lidinone system. Of note, no antiviral activity was obtained
for the analogues carrying a spiro-fused cyclopentane ring
instead of cyclohexane (i.e., 4a and 5a) or a bulkier group
than methyl on the cyclohexane residue. Introduction of a
methyl group at position 2 of the ring system (e.g., compare
compounds 4b and 5b) seemed to have a slightly positive
efect on antiviral activity.
4i
5a
250
250
500
5b
H
H
5c
5d
CH3
C2H5
C3H7
H
C6H5
–
1000
1000
1000
1000
7.8
5e
5f
5g
5h
Rifampicin
<0.1 µg/cm3
The experiment was performed twice and the same results were
obtained
aMIC, the actual minimum inhibitory concentration required to
inhibit the growth of 100% of organisms
APT and DEPT experiments. Observation of the typical C5
(δ=70.59–76.40 ppm) and C=O (167.64–170.57 ppm) reso-
nances of the spirothiazolidinone structure in the 13C NMR
spectra of 4 and 5, verifed the formation of the expected
spiro ring system.
Deprotonated [M − H]− or protonated [M + H]+ ions
observed in the ESI-MS confrmed 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 resulting from the 37Cl isotope further
confrmed the new structures.
The test compounds did not exhibit activity against any
of the other DNA- or RNA-viruses tested. Nevertheless,
this broad antiviral testing allowed to determine the com-
pounds’ cytotoxic activity in diferent mammalian cell lines
(Table 3). In general, the compounds endowed with antiviral
activity (4b, 4c, 5b, and 5d) tended to be less cytotoxic than
the inactive derivatives.
Antitubercular activity
The antitubercular activity of compounds 4a–4i and
5a–5h was tested in vitro against M. tuberculosis H37Rv
ATCC 27294 using the microdilution method. The lowest
concentration of compound that inhibited 100% of myco-
bacterial growth in the culture was defned as the MIC.
Rifampicin was used as the reference drug. Compounds
were assayed using twofold dilutions starting at 1000 µM.
As shown in Table 1, compounds 4 h and 5h, bearing a phe-
nyl substituent at position 8 of the spiro ring, exhibited the
highest anti-TB activity at concentrations of 3.9 and 7.8 µM,
respectively. Most of the compounds in series 4 (4b, 4c, 4d,
Antibacterial and antifungal activity
The broad antibacterial and antifungal activity of compounds
4a–4i and 5a–5 h was further assessed using Pseudomonas
aeruginosa ATCC 27853, Escherichia coli ATCC 25922,
Klebsiella pneumoniae ATCC 4352, Proteus mirabilis
ATCC 14153, Staphylococcus aureus ATCC 29213, Staphy-
lococcus epidermidis ATCC 12228, Enterococcus faecalis
ATCC 29212, Candida albicans ATCC 10231, Candida
parapsilosis ATCC 22019, and Candida tropicalis ATCC
1 3

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole…
1537
Table 2 Antiviral activity in Veroa cell cultures infected with diverse RNA viruses
Compound
R
R1
R2
R3
Antiviral assays in Vero cells
Antiviral ECb50 value/µM
MCCc/µM
Parain-
fuenza-3
virus
Reovirus-1 Sindbis
virus
Cox-
Punta Toro Yellow
sackie B4 virus
virus
Fever virus
4a
–
H
CH3
H
H
H
CH3
H
H
–
H
CH3
H
H
H
CH3
H
–
–
–
–
H
H
–
–
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
111
>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
11 [9]
12 [8]
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4.0
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
5.6 [>18]
>100
1.9 [≥11]
>100
>100
>100
>100
15
>100
100
100
20
4b
H
H
H
H
H
H
H
H
H
H
4c
4d
CH3
C2H5
C3H7
H
C6H5
C(CH3)3
–
4e
20
20
20
20
4f
4g
CH3 CH3 >100
4h
H
H
–
H
H
H
H
H
H
H
–
H
H
H
H
H
>100
>100
>100
>100
>100
>100
>100
>100
4i
20
5a
>100
>100
>100
≥20
20
20
>100
20
5b
H
H
5c
5d
CH3
C2H5
C3H7
H
C6H5
–
8.9 [≥2] >100
5e
>100
>100
>100
>100
0.8
8.9
0.4
>100
>100
>100
>100
34
>250
>100
5f
5g
CH3 CH3 >100
H
–
–
–
5h
H
–
–
>100
>100
111
DS-10,000d
Ribavirin
>100
>250
>100
–
–
111
1.7
>250
0.8
Mycophenolic acid
–
0.4
1.5
Values shown are the mean of two independent tests. In square brackets, the selectivity index (i.e., ratio of MCC to antiviral EC50) is given
aVero: African green monkey kidney cells
bEC50: 50% efective concentration, producing 50% inhibition of virus-induced cytopathic efect, as determined by microscopy
cMCC: minimal cytotoxic concentration, or compound concentration causing minimal changes in cell morphology, as assessed by microscopy
dDS-10,000: dextran sulfate with MW 10,000; for this compound, data are expressed in µg per cm3
750. The compounds were assayed using twofold dilutions
from 2500 to 156 µM. Neither of the test compounds pro-
duced antimicrobial activity below this concentration range.
and 7.8 µM, respectively. Compounds 4b, 4c, 5b, and 5d
exhibited inhibitory efect on the replication of Punta Toro
virus, yellow fever virus or Sindbis virus in Vero cells. The
antiviral EC50 values were in the range of 1.9–12 µM and the
selectivity index (SI: ratio of cytotoxic to antivirally efec-
tive concentration) was above 10 in some cases. The most
potent efect was seen with compound 5d that is methylated
at positions 2 and 8 of the spirothiazolidinone system. Nei-
ther of the indole-spirothiazolidinone compounds showed
activity against any of the bacterial or fungal strains tested,
at concentrations below 2500–156 µM.
Conclusion
In the search for efective antimicrobial agents, we achieved
the synthesis of novel spirothiazolidinone derivatives 4a–4i
5a–5h with the 5-chloro-3-phenyl-1H-indole scaffold.
Structures of the new compounds were characterized and
1
confrmed by spectrometric methods (IR, H NMR, 13C
NMR, and ESI-MS) and elemental analysis. Compounds
4a–4i and 5a–5h were evaluated for in vitro antitubercu-
lar, antiviral, antibacterial, and antifungal activity against
various viral, bacterial, and fungal strains. Compounds 4h
and 5h, bearing a bulky phenyl group at position 8 of the
spiro ring, displayed appreciable anti-TB activity against M.
tuberculosis H37Rv ATCC 27294 with MIC values of 3.9
Experimental
All purchased solvents and chemicals were of analytical
grade and used as received. Melting points were determined
in open capillary tubes with a Buchi B-540 melting point
apparatus. Microanalyses were performed on a Thermo
1 3

1538
G. Cihan-Üstündağ et al.
30 cm3 aqueous NaNO2 solution (7%) 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-ben-
zyl-3-oxobutanoate (0.02 mol), 10 cm3 ethanol, 10 cm3
water, and 5.4 g KOH while stirring. The resulting mixture
was refrigerated overnight. The red oily solid residue thus
obtained was separated, washed with water, and used with-
out further purifcation. Yield: 72.4%; m.p.: 90–93 °C (Ref.
[21] 87–93 °C).
Table 3 Cytotoxic activity in diverse mammalian cell linesa
Compound
MCCb/µM
CCc50/µM
MDCK
HEL
HeLa
Vero
MDCK
4a
>100
100
100
20
20
20
20
20
20
>100
100
>100
20
20
20
≥100
20
ND
ND
>100
>250
>100
100
100
20
20
20
20
20
20
>100
≥100
>100
20
20
20
100
20
>100
≥250
ND
ND
>100
100
100
20
20
20
20
20
20
>100
>100
>100
≥20
20
20
>100
20
>100
>250
ND
ND
>100
≥20
≥20
4
4
4
4
4
4
≥100
100
>100
≥20
4
4
100
4
ND
>100
ND
ND
>100
63
64
2.3
9.9
2.4
20
2.3
2.3
>100
>100
>100
>100
18
2.3
>100
2.3
ND
>100
ND
ND
4b
4c
4d
4e
4f
Ethyl 5‑chloro‑3‑phenyl‑1H‑indole‑2‑carboxylate (2) [22] A
solution of 1 (0.02 mol) in 20 cm3 conc. HCl was heated
under refux on a water bath (70–80 °C) for 4 h. The crude
product was fltered of, washed with water until tested neu-
tral to litmus and used without further purifcation. Yield:
67.8%; m.p.: 160–162 °C (Ref. [22] 158–160 °C).
4g
4h
4i
5a
5b
5c
5d
5‑Chloro‑3‑phenyl ‑1H‑indole‑2‑carbohydrazide (3)
[23] A mixture of 2 (0.02 mol), 20 cm3 ethanol, and 8 cm3
H2NNH2·H2O (98%) was heated under refux on a water bath
(70–80 °C) for 6 h. The resulting brown solid was fltered
of and recrystallized from ethanol. Yield: 53.7%; m.p.:
227–230 °C (Ref. [23] 234–236 °C).
5e
5f
5g
5h
DS-10,000d
Ribavirin
Ganciclovir
Brivudin
General procedure for the synthesis
of 5‑chloro‑3‑phenyl‑N‑(2,7,8,9‑substituted/
nonsubstituted‑3‑oxo‑1‑thia‑4‑azaspiro[4.4]nonan/
[4.5]decan‑4‑yl)‑1H‑indole‑2‑carboxamides 4a–4i,
5a–5h
ND not done
aHEL human embryonic lung fbroblast cells, HeLa human cervix
carcinoma cells, Vero African green monkey kidney cells, MDCK
Madin–Darby canine kidney cells
bMCC minimal cytotoxic concentration, or compound concentration
causing minimal changes in cell morphology, as assessed by micros-
copy
A mixture of 3 (0.0025 mol), an appropriate cyclohex-
anone/cyclopentanone (0.003 mol), and mercaptoacetic
acid or 2-mercaptopropionic acid (0.01 mol) in 20 cm3
dry toluene was heated to refux with a heating mantle for
5–6 h using a Dean–Stark water separator. Excess tolu-
ene was evaporated in vacuo. The resulting residue was
treated with saturated NaHCO3 solution until CO2 evo-
lution ceased and was allowed to stand overnight or in
some cases refrigerated until solidifcation. The solid thus
obtained was washed with water, dried, and recrystallized
from ethanol or ethyl acetate.
cCC50 50% cytotoxic concentration, assessed by the spectroscopic
MTS cell viability assay
dDS-10,000 dextran sulfate with MW 10,000; for this compound, data
are expressed in µg per cm3
Finnigan Flash EA 1112 elemental analyzer and their results
were found to be in good agreement ( 0.3%) with the cal-
culated values. IR spectra were recorded in KBr discs (̄ꢀ /
cm−1) on a Shimadzu IRAfnity-1 FTIR spectrophotometer.
1H NMR (DMSO-d6), 13C NMR (APT, DMSO-d6) were run
on VarianUNITYINOVA (500 MHz) instrument. 13C NMR
(DEPT, DMSO-d6) were run Bruker ARX (300 MHz).
Chemical shifts are reported as δ (ppm) relative to TMS
as internal standard and coupling constants (J) are given
in hertz (Hz). MS (ESI ) were determined on a Finnigan
LCQ Advantage Max mass spectrometer (*: broad/distorted,
ind.:indole, ar.:aromatic, sp.: spirodecane).
5‑Chloro‑N‑(3‑oxo‑1‑thia‑4‑azaspiro[4.4]nonan‑4‑yl)‑3‑phe‑
nyl‑1H‑indole‑2‑carboxamide (4a, C22H20ClN3O2S) White
crystals; yield: 87.7%; m.p.: 297–300 °C; Rf=0.46 (EtOAc/
CHCl3 1:1); IR (KBr): ̄ꢀ =3277 (N–H), 1709, 1651 (C=O)
cm−1; 1H NMR (DMSO-d6, 500 MHz): δ=1.58–1.69 (4H,
m, CH2-sp.), 1.72–1.74 (2H, m, CH2-sp.), 1.94 (2H, s*,
CH2-sp.), 3.65 (2H, s, S–CH2-sp.), 7.29 (1H, dd, J = 8.5,
2.0 Hz, H6-ind.), 7.37 (1H, tt, J=7.3, 1.5 Hz, 3-C6H5(H4)-
ind.), 7.45 (2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-ind.), 7.51–
7.54 (4H, m, H4, H7, 3-C6H5(H2,H6)-ind.), 10.10 (1H, s,
CONH), 12.13 (1H, s, NH) ppm; 13C NMR (APT, DMSO-
d6, 125 MHz): δ = 22.40, 29.05, 37.91 (CH2-sp.), 76.40
Ethyl 2‑[2‑(4‑chlorophenyl)hydrazinylidene]‑3‑phenylpro‑
panoate (1) [21] To a solution of 4-chloroaniline (0.02 mol)
in 10 cm3 ethanol, 10 cm3 water, and 6 cm3 conc. HCl,
1 3

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole…
1539
(C5-sp.), 114.62, 114.67 (CH-ar.), 118.64 (C-ar.), 119.32,
119.41 (CH-ar.), 124.69 (CH-ar.), 125.50 (C-ar.), 127.75
(CH-ar.), 127.81 (C-ar.), 128.03 (C-ar.), 128.79 (CH-ar.),
130.44 (CH-ar.), 133.27 (C-ar.), 134.65 (C-ar.), 162.08 (CO–
NH), 167.87 (CO-sp.) ppm; MS (ESI-): m/z (%) = 424.5
([M−H]−, 100), 426.3 ([(M−H)+2]−, 36.5).
CH2-sp.), 3.57, 3.58 (2H, 2 s, S–CH2-sp.), 7.29, 7.30 (1H,
2dd, J=8.5, 2.4 Hz, H6-ind.), 7.36 (1H, tt, J=8.0, 1.5 Hz,
3-C6H5(H4)-ind.), 7.46 (2H, t, J=7.8 Hz, 3-C6H5(H3,H5)-
ind.), 7.51-7.55 (4H, m, H4, H7, 3-C6H5(H2,H6)-ind.),
10.05, 10.06 (1H, 2 s, CONH), 12.11, 12.12 (1H, 2 s, NH)
ppm; 13C NMR (proton decoupled and DEPT, DMSO-
d6, 75 MHz): δ = 22.23 (8-CH3-sp.), 28.19 (CH2-sp.),
30.70 (C8-sp.), 31.73, 36.76 (CH2-sp.), 72.39 (C5-sp.),
114.50 (CH-ar.), 118.43 (C-ar.), 119.25 (CH-ar.), 124.52
(CH-ar.), 125.38 (C-ar.), 127.26 (CH-ar.), 127.73 (C-ar.),
128.06 (C-ar.), 128.76 (CH-ar.), 130.37 (CH-ar.), 133.26
(C-ar.), 134.53 (C-ar.), 162.00 (CO–NH), 167.69 (CO-sp.)
ppm; MS (ESI-): m/z (%) = 452.6 ([M − H]−, 100), 454.4
([(M−H)+2]−, 34.3).
5‑Chloro‑N‑(3‑oxo‑1‑thia‑4‑azaspiro[4.5]decan‑4‑yl)‑3‑phe‑
nyl‑1H‑indole‑2‑carboxamide (4b, C23H22ClN3O2S) White
crystals; yield: 76.9%; m.p.: 229–232 °C; Rf=0.48 (EtOAc/
CHCl3 1:1); IR (KBr): ̄ꢀ =3279 (N–H), 1709, 1651 (C=O)
cm−1; 1H NMR (DMSO-d6, 500 MHz): δ=1.01–1.11 (2H,
m, CH2-sp.), 1.35–1.42 (2H, m, CH2-sp.), 1.56 (1H, d*,
J = 13.2 Hz, CH2-sp.), 1.64–1.72 (5H, m, CH2-sp.), 3.58
(2H, s, S–CH2-sp.), 7.30 (1H, dd, J=8.8, 2.4 Hz, H6-ind.),
7.37 (1H, tt, J=7.3, 1.5 Hz, 3-C6H5(H4)-ind.), 7.46 (2H, t,
J=7.6 Hz, 3-C6H5(H3,H5)-ind.), 7.51–7.56 (4H, m, H4, H7,
3-C6H5(H2,H6)-ind.), 10.09 (1H, s, CONH), 12.14 (1H, s,
NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz): δ=23.33,
24.31, 28.29, 37.10 (CH2-sp.), 72.63 (C5-sp.), 114.56,
114.64 (CH-ar.), 118.50 (C-ar.), 119.31, 119.39 (CH-ar.),
124.54, 124.66 (CH-ar.), 125.47 (C-ar.), 127.41 (CH-ar.),
127.82 (C-ar.), 128.23 (C-ar.), 128.90 (CH-ar.), 130.51 (CH-
ar.), 133.38 (C-ar.), 134.62 (C-ar.), 162.14 (CO–NH), 167.73
(CO-sp.) ppm.
5‑Chloro‑N‑(8‑ethyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (4e,
C25H26ClN3O2S) White flakes; yield: 88.2%; m.p.: 236–
237 °C; Rf= 0.53 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3283
(N–H), 1713, 1651 (C=OC=O) cm−1; 1H NMR (DMSO-d6,
500 MHz): δ=0.89 (3H, t, J=7.3 Hz, 8-CH2CH3-sp.), 1.04–
1.35 (5H, m, CH/CH2-sp., 8-CH2CH3-sp.), 1.62–1.96 (6H,
m, CH/CH2-sp.), 3.58, 3.59 (2H, 2 s, S–CH2-sp.), 7.30 (1H,
dd, J=8.8, 2.0 Hz, H6-ind.), 7.36 (1H, tt, J=7.3, 1.5 Hz,
3-C6H5(H4)-ind.), 7.45 (2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-
ind.), 7.51-7.56 (4H, m, H4, H7, 3-C6H5(H2,H6)-ind.),
5‑Chloro‑N‑(7‑methyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (4c,
C24H24ClN3O2S) White powder; yield: 83.4%; m.p.: 268–
270 °C; Rf= 0.52 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3231
10.09, 10.10 (1H, 2 s, CONH), 12.13 (1H, s, NH) ppm; 13
C
NMR (APT, DMSO-d6, 125 MHz): δ=11.94 (8-CH2CH3-
sp.), 28.29, 28.35, 29.22, 29.41, 36.84 (CH2-sp., 8-CH2CH3-
sp.), 37.45 (C8-sp.), 72.75 (C5-sp.), 114.55, 114.63 (CH-
ar.), 118.48 (C-ar.), 119.29, 119.38 (CH-ar.), 124.64
(CH-ar.), 125.47 (C-ar.), 127.40 (CH-ar.), 127.81 (C-ar.),
128.24 (C-ar.), 128.87 (CH-ar.), 130.48 (CH-ar.), 133.38
(C-ar.), 134.60 (C-ar.), 162.11 (CO–NH), 167.78 (CO-sp.)
ppm; MS (ESI−): m/z (%) = 466.8 ([M-H]−, 100), 468.5
([(M-H)+2]−, 30.9).
1
(N–H), 1711, 1651 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ=0.76–0.85 (1H, m, CH/CH2-sp.), 0.87 (3H,
d, J=6.4 Hz, 7-CH3-sp.), 1.24–1.70 (8H, m, CH/CH2-sp.),
3.58 (2H, s, S–CH2-sp.), 7.29 (1H, dd, J = 8.8, 2.0 Hz,
H6-ind.), 7.37 (1H, tt, J = 7.3, 1.5 Hz, 3-C6H5(H4)-ind.),
7.45 (2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-ind.), 7.49 (1H, d,
J=2.0 Hz, H4-ind.), 7.52-7.54 (3H, m, H7, 3-C6H5(H2,H6)-
ind.), 10.06 (1H, s, CONH), 12.14 (1H, s, NH) ppm; 13C
NMR (APT, DMSO-d6, 125 MHz): δ=22.42 (7-CH3-sp.),
22.88, 28.36 (CH2-sp.), 29.97 (C7-sp.), 32.98, 36.54, 45.20
(CH2-sp.), 72.59 (C5-sp.), 114.56, 114.63 (CH-ar.), 118.50
(C-ar.), 119.28, 119.37 (CH-ar.), 124.54 (CH-ar.), 125.45
(C-ar.), 127.42 (CH-ar.), 127.85 (C-ar.), 128.25 (C-ar.),
128.91 (CH-ar.), 130.51 (CH-ar.), 133.43 (C-ar.), 134.62
(C-ar.), 162.18 (CO–NH), 167.66 (CO-sp.) ppm.
5‑Chloro‑3‑phenyl‑N‑(8‑propyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
d e c a n ‑ 4 ‑ y l ) ‑ 1 H‑ i n d o l e ‑ 2 ‑ c a r b o x a m i d e ( 4 f ,
C26H28ClN3O2S) Beige powder; yield: 75.7%; m.p.: 233-
236 °C; Rf =0.55 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ =3291,
3217 (N–H), 1707, 1661 (C=O) cm−1; 1H NMR (DMSO-
d6, 500 MHz): δ=0.88 (3H, t, J=7.3 Hz, 8-CH2CH2CH3-
sp.), 1.05–1.34 (7H, m, CH/CH2-sp., 8-CH2CH2CH3-sp.),
1.60–1.82 (6H, m, CH/CH2-sp.), 3.58 (2H, s, S–CH2-
sp.), 7.29 (1H, dd, J= 8.8, 2.0 Hz, H6-ind.), 7.35 (1H, tt,
J=7.3, 1.5 Hz, 3-C6H5(H4)-ind.), 7.45 (2H, t, J=7.8 Hz,
3-C6H5(H3,H5)-ind.), 7.51 (1H, d, J = 2.0 Hz, H4-ind.),
7.53–7.56 (3H, m, H7, 3-C6H5(H2,H6)-ind.), 10.10 (1H, s,
CONH), 12.13 (1H, s, NH) ppm; 13C NMR (APT, DMSO-
d6, 125 MHz): δ=14.58 (8-CH2CH2CH3-sp.), 19.99, 28.30,
28.36, 29.77 (CH2-sp., 8-CH2CH2CH3-sp.), 35.25 (C8-sp.),
36.85, 38.76 (CH2-sp., 8-CH2CH2CH3-sp.), 72.74 (C5-sp.),
5‑Chloro‑N‑(8‑methyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (4d,
C24H24ClN3O2S) White powder; yield: 91.2%; m.p.: 292–
293 °C; Rf= 0.51 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3285
1
(N–H), 1709, 1651 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ=0.89, 0.95 (3H, 2d, J=6.4 Hz, 8-CH3-sp.),
1.08–1.32 (3H, m, CH/CH2-sp.), 1.64–1.75 (6H, m, CH/
1 3

1540
G. Cihan-Üstündağ et al.
114.55, 114.61 (CH-ar.), 118.47 (C-ar.), 119.30, 119.38
(CH-ar.), 124.54 (CH-ar.), 125.47 (C-ar.), 127.30 (CH-ar.),
127.67 (C-ar.), 128.28 (C-ar.), 128.84 (CH-ar.), 130.51 (CH-
ar.), 133.42 (C-ar.), 134.61 (C-ar.), 162.12 (CO–NH), 167.77
(CO-sp.) ppm.
500 MHz): δ=0.84 (9H, s, 8-C(CH3)3-sp.), 0.90-0.96 (1H,
m, CH/CH2-sp.), 1.12–1.22 (2H, m, CH/CH2-sp.), 1.64-1.72
(6H, m, CH/CH2-sp.), 3.58 (2H, s, S–CH2-sp.), 7.30 (1H,
dd, J=8.8, 2.0 Hz, H6-ind.), 7.36 (1H, tt, J=7.3, 1.5 Hz,
3-C6H5(H4)-ind.), 7.45 (2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-
ind.), 7.50 (1H, d, J =2.0 Hz, H4-ind.), 7.52-7.56 (3H, m,
H7, 3-C6H5(H2,H6)-ind.), 10.13 (1H, s, CONH), 12.14
(1H, s, NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz):
δ=24.14 (CH2-sp.), 27.80 (8-C(CH3)3-sp.), 28.28 (CH2-sp.),
32.39 (8-C(CH3)3-sp.), 37.11 (CH2-sp.), 45.96 (C8-sp.),
72.57 (C5-sp.), 114.60 (CH-ar.), 118.42 (C-ar.), 119.27,
119.35 (CH-ar.), 124.64 (CH-ar.), 125.45 (C-ar.), 127.40
(CH-ar.), 127.84 (C-ar.), 128.38 (C-ar.), 128.90 (CH-ar.),
130.51 (CH-ar.), 133.44 (C-ar.), 134.58 (C-ar.), 162.13 (CO–
NH), 167.79 (CO-sp.) ppm; MS (ESI-): m/z (%)=494.5 ([M-
H]−, 100), 496.3 ([(M-H)+2]−, 37.6).
5‑Chloro‑N‑(7,7,9‑trimethyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (4g,
C26H28ClN3O2S) White crystals; yield: 85.1%; m.p.: 264-
266.5 °C; Rf =0.56 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ =3277,
1
3219 (N–H), 1705, 1695, 1665 (C=O) cm−1; H NMR
(DMSO-d6, 500 MHz): δ = 0.76 (1H, t, J = 12.7 Hz, CH/
CH2-sp.), 0.90 (6H, s*, 7-(CH3)2-sp.), 1.04 (3H, s*, 9-CH3-
sp.), 1.20-1.34 (2H, m, CH/CH2-sp.), 1.58-1.84 (4H, m, CH/
CH2-sp.), 3.50 (1H, d, J=16.1 Hz, S–CH2-sp.), 3.61 (1H,
d, J = 16.1 Hz, S–CH2-sp.), 7.29 (1H, dd, J = 8.6, 2.0 Hz,
H6-ind.), 7.37 (1H, t*, J=7.3 Hz, 3-C6H5(H4)-ind.), 7.44–
7.48 (3H, m, H4, 3-C6H5(H3,H5)-ind.), 7.51–7.54 (3H,
m, H7, 3-C6H5(H2,H6)-ind.), 9.97 (1H, s, CONH), 12.13
(1H, s, NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz):
δ=22.24, 25.97, 27.64 (7-(CH3)2, 9-CH3-sp.), 28.50, 32.24
(CH2-sp.), 34.09 (C9-sp.), 45.23, 46.80, 47.31 (CH2-sp.,
C7-sp.), 71.87 (C5-sp.), 114.56, 114.64 (CH-ar.), 118.50
(C-ar.), 119.28, 119.38 (CH-ar.), 124.58 (CH-ar.), 125.45
(C-ar.), 127.44 (CH-ar.), 127.96 (C-ar.), 128.18 (C-ar.),
128.99 (CH-ar.), 130.54 (CH-ar.), 133.47 (C-ar.), 134.62
(C-ar.), 162.40 (CO–NH), 167.64 (CO-sp.) ppm.
5‑Chloro‑N‑(2‑methyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.4]‑
nonan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5a,
C23H22ClN3O2S) Beige powder; yield: 85.4%; m.p.: 270–
272 °C; Rf= 0.60 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3283
1
(N–H), 1709, 1651 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ = 1.42 (3H, d, J = 6.8 Hz, 2-CH3-sp.), 1.59-
1.77 (6H, m, CH2-sp.), 1.91-2.08 (2H, m, CH2-sp.), 3.93
(1H, q, J=6.8 Hz, S–CH-sp.), 7.29 (1H, dd, J=8.8, 2.0 Hz,
H6-ind.), 7.36 (1H, tt, J = 7.8, 1.5 Hz, 3-C6H5(H4)-ind.),
7.44 (2H, t, J = 7.8 Hz, 3-C6H5(H3,H5)-ind.), 7.50-7.55
(4H, m, H4, H7, 3-C6H5(H2,H6)-ind.), 10.14 (1H, s,
CONH), 12.13 (1H, s, NH) ppm; 13C NMR (APT, DMSO-
d6, 125 MHz): δ = 19.81 (2-CH3-sp.), 22.47 (CH2-sp.),
37.93, 38.06 (C2-sp.), 38.25, 38.37 (CH2-sp.), 74.96 (C5-
sp.), 114.60, 114.66 (CH-ar.), 118.64 (C-ar.), 119.33, 119.41
(CH-ar.), 124.63, 124.74 (CH-ar.), 125.49 (C-ar.), 127.48
(CH-ar.), 127.84 (C-ar.), 128.02 (C-ar.), 128.81 (CH-ar.),
130.48 (CH-ar.), 133.27 (C-ar.), 134.65 (C-ar.), 162.05 (CO–
NH), 170.57 (CO-sp.) ppm.
5‑Chloro‑3‑phenyl‑N‑(8‑phenyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
d e c a n ‑ 4 ‑ y l ) ‑ 1 H‑ i n d o l e ‑ 2 ‑ c a r b o x a m i d e ( 4 h ,
C29H26ClN3O2S) Beige powder; yield: 72.8%; m.p.: 285–
287 °C; Rf =0.58 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ =3277,
3237 (N–H), 1707, 1663 (C=O) cm−1; 1H NMR (DMSO-
d6, 500 MHz): δ=1.60–1.91 (8H, m, CH2-sp.), 2.47–2.49
(1H, m, CH-sp.), 3.64 (2H, s, S–CH2-sp.), 7.21 (1H, tt*,
J=7.3, 1.5 Hz, 8-C6H5(H4)-sp.), 7.25 (2H, d*, J=7.3 Hz,
8-C6H5-sp.), 7.29–7.34 (3H, m, H6-ind., 8-C6H5-sp.), 7.41
(1H, tt*, J=7.8, 1.5 Hz, 3-C6H5(H4)-ind.), 7.50–7.61 (6H,
m, H4, H7, 3-C6H5(H2,H3,H5,H6)-ind.), 10.18 (1H, s,
CONH), 12.17 (1H, s, NH) ppm; 13C NMR (APT, DMSO-
d6, 125 MHz): δ=28.38, 30.92, 37.12 (CH2-sp.), 41.95 (C8-
sp.), 72.10 (C5-sp.), 114.57, 114.64 (CH-ar.), 118.60 (C-ar.),
119.34, 119.42 (CH-ar.), 124.62 (CH-ar.), 125.51 (C-ar.),
126.98 (8-ar.(CH)-sp.), 127.09 (CH-ar.), 127.73 (8-ar.(CH)-
sp.), 127.88 (C-ar.), 128.28 (C-ar.), 128.95 (CH-ar.), 130.57
(CH-ar.), 133.45 (C-ar.), 134.63 (C-ar.), 146.26 (8-ar.(C)-
sp.), 162.18 (CO–NH), 167.84 (CO-sp.) ppm.
5‑Chloro‑N‑(2‑methyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5b,
C24H24ClN3O2S) White crystals; yield: 90.7%; m.p.: 245–
248 °C; Rf =0.62 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ =3285
1
(N–H), 1711, 1651 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ=1.02–1.11 (2H, m, CH2-sp.), 1.31–1.38 (2H,
m, CH2-sp.), 1.41 (3H, d, J=6.8 Hz, 2-CH3-sp.), 1.56 (1H,
d, J=12.7 Hz, CH2-sp.), 1.62–1.75 (5H, m, CH2-sp.), 3.93
(1H, q, J=6.4 Hz, S–CH-sp.), 7.30 (1H, dd, J=8.8, 2.0 Hz,
H6-ind.), 7.37 (1H, t, J = 7.3 Hz, 3-C6H5(H4)-ind.), 7.47
(2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-ind.), 7.50-7.55 (4H, m,
H4, H7, 3-C6H5(H2,H6)-ind.), 10.12 (1H, s, CONH), 12.14
(1H, s, NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz):
δ=20.01 (2-CH3-sp.), 23.59, 24.31 (CH2-sp.), 37.12 (C2-
sp.), 38.02 (CH2-sp.), 71.29 (C5-sp.), 114.56, 114.63
(CH-ar.), 118.50 (C-ar.), 119.31, 119.39 (CH-ar.), 124.63
N‑ ( 8 ‑ ter t‑ B u t y l ‑ 3 ‑ o x o ‑ 1 ‑ t h i a ‑ 4 ‑ a z a s p i r o [ 4 . 5 ] ‑
decan‑4‑yl)‑5‑chloro‑3‑phenyl‑1H‑indole‑2‑carboxamide
(4i, C27H30ClN3O2S) White crystals; yield: 79.3%; m.p.: 230-
232 °C; Rf= 0.57 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3285
1
(N–H), 1702, 1662 (C=O) cm−1; H NMR (DMSO-d6,
1 3

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole…
1541
(CH-ar.), 125.46 (C-ar.), 127.42 (CH-ar.), 127.84 (C-ar.),
128.23 (C-ar.), 128.90 (CH-ar.), 130.54 (CH-ar.), 133.37
(C-ar.), 134.62 (C-ar.), 162.11 (CO–NH), 170.36 (CO-sp.)
ppm.
198–200 °C; Rf= 0.64 (EtOAc/CHCl3 1:1); IR (KBr):
̄ꢀ =3383 (N–H), 1711, 1653 (C=O) cm−1; 1H NMR (DMSO-
d6, 500 MHz): δ=0.89 (3H, t, J=7.3 Hz, 8-CH2CH3-sp.),
1.02–1.24 (5H, m, CH/CH2-sp., 8-CH2CH3-sp.), 1.41 (3H, d,
J=6.8 Hz, 2-CH3-sp.), 1.62-1.88 (6H, m, CH/CH2-sp.), 3.87
(1H, q*, J=6.3 Hz, S–CH-sp.), 7.30 (1H, dd, J=8.8, 2.0 Hz,
H6-ind.), 7.37 (1H, tt, J = 7.8, 1.5 Hz, 3-C6H5(H4)-ind.),
7.46 (2H, t, J=7.8 Hz, 3-C6H5(H3,H5)-ind.), 7.51 (1H, d,
J=2.0 Hz, H4-ind.), 7.52–7.56 (3H, m, H7, 3-C6H5(H2,H6)-
ind.), 10.13 (1H, s, CONH), 12.13 (1H, s, NH) ppm; 13C
NMR (APT, DMSO-d6, 125 MHz): δ=11.92 (8-CH2CH3-
sp.), 20.35 (2-CH3-sp.), 29.23, 29.67, 36.98 (CH2-sp.,
8-CH2CH3-sp.), 37.14, 37.27 (C2-sp.), 37.42 (C8-sp.), 37.85
(CH2-sp., 8-CH2CH3-sp.), 71.42, 71.71 (C5-sp.), 114.55,
114.61 (CH-ar.), 118.50 (C-ar.), 119.32, 119.39 (CH-ar.),
124.56, 124.64 (CH-ar.), 125.46 (C-ar.), 127.39 (CH-ar.),
127.85 (C-ar.), 128.24 (C-ar.), 128.88 (CH-ar.), 130.51 (CH-
ar.), 133.39 (C-ar.), 134.60 (C-ar.), 162.07 (CO–NH), 170.41
(CO-sp.) ppm.
5‑Chloro‑N‑(2,7‑dimethyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5c,
C25H26ClN3O2S) White crystals; yield: 86.0%; m.p.: 262–
264 °C; Rf= 0.65 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3235
1
(N–H), 1694, 1661 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ = 0.75–0.80 (1H, m, CH/CH2-sp.), 0.87
(3H, d, J = 6.0 Hz, 7-CH3-sp.), 1.23–1.68 (8H, m, CH/
CH2-sp.), 1.41 (3H, d, J = 6.3 Hz, 2-CH3-sp.), 3.88 (1H,
s*, S–CH-sp.), 7.29 (1H, dd, J = 8.6, 2.0 Hz, H6-ind.),
7.37 (1H, tt, J= 7.3, 1.5 Hz, 3-C6H5(H4)-ind.), 7.45–7.49
(3H, m, H4, 3-C6H5(H3,H5)-ind.), 7.52-7.54 (3H, m, H7,
3-C6H5(H2,H6)-ind.), 10.07 (1H, s, CONH), 12.12 (1H, s,
NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz): δ=20.01,
20.03 (2-CH3-sp.), 22.38, 22.48 (7-CH3-sp.), 22.72, 23.12
(CH2-sp.), 29.86, 30.25 (C7-sp.), 32.98, 36.66 (CH2-sp.),
37.27, 37.38 (C2-sp.), 37.71, 45.41, 46.10 (CH2-sp.), 71.22,
71.26 (C5-sp.), 114.55, 114.63 (CH-ar.), 118.52 (C-ar.),
119.29, 119.39 (CH-ar.), 124.56, 124.65 (CH-ar.), 125.45
(C-ar.), 127.42 (CH-ar.), 127.88 (C-ar.), 128.25 (C-ar.),
128.90 (CH-ar.), 130.55 (CH-ar.), 133.43 (C-ar.), 134.62
(C-ar.), 162.14 (CO–NH), 170.30 (CO-sp.) ppm; MS (ESI-):
m/z (%)=466.7 ([M-H]−, 100), 468.5 ([(M-H)+2]−, 33.4).
5‑Chloro‑N‑(2‑methyl‑8‑propyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5f,
C27H30ClN3O2S) Beige crystals; yield: 89.1%; m.p.:
194–197 °C; Rf= 0.67 (EtOAc/CHCl3 1:1); IR (KBr):
̄ꢀ =3287, 3184 (N–H), 1713, 1692, 1653 (C=O) cm−1; 1H
NMR (DMSO-d6, 500 MHz): δ = 0.88 (3H, t, J = 7.3 Hz,
8-CH2CH2CH3-sp.), 1.00–1.34 (7H, m, CH/CH2-sp.,
8-CH2CH2CH3-sp.), 1.42 (3H, d, J = 6.8 Hz, 2-CH3-sp.),
1.60–1.84 (6H, m, CH/CH2-sp.), 3.89 (1H, d*, J=5.8 Hz,
S–CH-sp.), 7.30 (1H, dd, J = 8.8, 2.0 Hz, H6-ind.), 7.36
(1H, tt, J = 7.3, 1.5 Hz, 3-C6H5(H4)-ind.), 7.45 (2H, t,
J = 7.3 Hz, 3-C6H5(H3,H5)-ind.), 7.50 (1H, d, J = 2.0 Hz,
H4-ind.), 7.52-7.56 (3H, m, H7, 3-C6H5(H2,H6)-ind.), 10.13
(1H, s, CONH), 12.13 (1H, s, NH) ppm; 13C NMR (APT,
DMSO-d6, 125 MHz): δ=14.57 (8-CH2CH2CH3-sp.), 20.00
(CH2-sp., 8-CH2CH2CH3-sp.), 20.31 (2-CH3-sp.), 29.62,
30.05 (CH2-sp., 8-CH2CH2CH3-sp.), 35.26 (C8-sp.), 37.00
(CH2-sp., 8-CH2CH2CH3-sp.), 37.16, 37.29 (C2-sp.), 37.86,
38.79 (CH2-sp., 8-CH2CH2CH3-sp.), 71.39, 71.75 (C5-sp.),
114.53, 114.60 (CH-ar.), 118.50 (C-ar.), 119.32, 119.39
(CH-ar.), 124.55, 124.64 (CH-ar.), 125.47, 125.52 (C-ar.),
127.36 (CH-ar.), 127.66 (C-ar.), 128.27 (C-ar.), 128.84
(CH-ar.), 130.55 (CH-ar.), 133.32, 133.43 (C-ar.), 134.61,
134.66 (C-ar.), 162.08 (CO–NH), 170.37, 170.41 (CO-sp.)
ppm; MS (ESI-): m/z (%) = 494.7 ([M − H]−, 100), 496.4
([(M−H)+2]−, 34.2).
5‑Chloro‑N‑(2,8‑dimethyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5d,
C25H26ClN3O2S) White crystals; yield: 95.1%; m.p.: 223-
225.5 °C; Rf=0.65 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ =3337,
3262 (N–H), 1694, 1670 (C=O) cm−1; 1H NMR (DMSO-d6,
500 MHz): δ=0.89, 0.96 (3H, 2d, J=6.4 Hz, 8-CH3-sp.),
1.03-1.36 (3H, m, CH/CH2-sp.), 1.41 (3H, d, J = 6.8 Hz,
2-CH3-sp.), 1.50-1.74 (6H, m, CH/CH2-sp.), 3.87 (1H, q*,
J=6.8 Hz, S–CH-sp.), 7.30, 7.31 (1H, 2dd, J=8.8, 2.0 Hz,
H6-ind.), 7.37 (1H, tt, J = 7.3, 1.5 Hz, 3-C6H5(H4)-ind.),
7.46 (2H, t, J=7.3 Hz, 3-C6H5(H3,H5)-ind.), 7.50 (1H, d,
J=2.0 Hz, H4-ind.), 7.52–7.58 (3H, m, H7, 3-C6H5(H2,H6)-
ind.), 10.10 (1H, s, CONH), 12.13 (1H, s, NH) ppm; 13C
NMR (proton decoupled and DEPT, DMSO-d6, 75 MHz):
δ = 20.24 (2-CH3-sp.), 22.25 (8-CH3-sp.), 30.71 (C8-
sp.), 31.60, 32.02, 36.91 (CH2-sp.), 37.10 (C2-sp.), 37.78
(CH2-sp.), 71.05 (C5-sp.), 114.50 (CH-ar.), 118.43 (C-ar.),
119.26 (CH-ar.), 124.52 (CH-ar.), 125.38 (C-ar.), 127.27
(CH-ar.), 127.75 (C-ar.), 128.08 (C-ar.), 128.76 (CH-ar.),
130.41 (CH-ar.), 133.27 (C-ar.), 134.53 (C-ar.), 161.98 (CO–
NH), 170.33 (CO-sp.) ppm.
5‑Chloro‑N‑(2,7,7,9‑tetramethyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5g,
C27H30ClN3O2S) White crystals; yield: 75.5%; m.p.: 266-
268 °C; Rf= 0.68 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3287
5‑Chloro‑N‑(8‑ethyl‑2‑methyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5e,
C26H28ClN3O2S) White needles; yield: 87.2%; m.p.:
1
(N–H), 1705, 1655 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ=0.76 (1H, t, J=12.7 Hz, CH/CH2-sp.), 0.90
1 3

1542
G. Cihan-Üstündağ et al.
(6H, s*, 7-(CH3)2-sp.), 1.03 (3H, d, J=6.4 Hz, 9–CH3-sp.),
1.24–1.42 (2H, m, CH/CH2-sp.), 1.38 (3H, d, J = 6.8 Hz,
2-CH3-sp.), 1.62–1.80 (4H, m, CH/CH2-sp.), 3.87 (1H, s*,
S–CH-sp.), 7.29, 7.30 (1H, 2dd, J =8.8, 2.0 Hz, H6-ind.),
7.38 (1H, t*, J = 7.3 Hz, 3-C6H5(H4)-ind.), 7.45–7.48
(3H, m, H4, 3-C6H5(H3,H5)-ind.), 7.50–7.54 (3H, m, H7,
3-C6H5(H2,H6)-ind.), 10.02 (1H, s, CONH), 12.13, 12.17
(1H, 2 s, NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz):
δ=20.05 (2-CH3-sp.), 22.23, 25.68, 26.21, 27.69 (7-(CH3)2,
9-CH3-sp.), 32.27 (CH2-sp.), 36.84, 36.96 (C9-sp.), 37.46,
37.57 (C2-sp.), 45.22, 46.80, 47.29 (CH2-sp., C7-sp.),
70.59 (C5-sp.), 114.56, 114.63 (CH-ar.), 118.52 (C-ar.),
119.29, 119.38 (CH-ar.), 124.58, 124.67 (CH-ar.), 125.44
(C-ar.), 127.44 (CH-ar.), 127.98 (C-ar.), 128.18, 128.24
(C-ar.), 128.97 (CH-ar.), 130.54 (CH-ar.), 133.46, 133.48
(C-ar.), 134.61 (C-ar.), 162.44 (CO–NH), 170.24 (CO-sp.)
ppm; MS (ESI+): m/z (%)=497.1 ([M+H]+, 31.2), 426.3
([(M+H)+2]+, 10.7).
Middlebrook 7H9 broth medium (Becton and Dickinson,
USA) was used and the medium was adjusted to pH 7.0 at
25 °C. Each bottle was controlled for sterility before it was
used. Resazurin purchased from Sigma-Aldrich (St Louis,
USA) was dissolved in sterile distilled water to a fnal con-
centration of 0.02% and sterilized by fltration, then stored
at 4 °C until use. Rifampicin purchased from Becton–Dick-
inson (BD, USA) was dissolved in sterile distilled water to a
fnal concentration of 1 μg/cm3 (critical concentration). The
synthesized compounds were dissolved in 100% dimethyl
sulfoxide according to CLSI methods [27, 28]. Stock solu-
tions were obtained by 40-fold dilution in DMSO followed
by sterile fltration. From here, working stocks at 4000 μM
were obtained by diluting 1/10 in MB7H9 medium. The fnal
concentrations were 1000 μM to 0.49 μM for the synthesized
compounds. For rifampicin, the critical concentration (1 μg/
cm3) was used [27, 28].
Inoculum suspensions of mycobacteria were prepared
according to the CLSI guidelines as described previously.
The isolates were subcultured on Löwenstein Jensen medium
and incubated at 37 °C for 20–25 days. A few colonies from
freshly grown M. tuberculosis were suspended in Middle-
brook 7H9 broth medium to obtain 1.0 McFarland turbidity,
then diluted ten times with the same medium.
5‑Chloro‑N‑(2‑methyl‑8‑phenyl‑3‑oxo‑1‑thia‑4‑azaspiro[4.5]‑
decan‑4‑yl)‑3‑phenyl‑1H‑indole‑2‑carboxamide (5h,
C30H28ClN3O2S) Beige powder; yield: 82.3%; m.p.: 213–
215 °C; Rf= 0.69 (EtOAc/CHCl3 1:1); IR (KBr): ̄ꢀ = 3289
1
(N–H), 1705, 1663 (C=O) cm−1; H NMR (DMSO-d6,
500 MHz): δ=1.45 (3H, d, J=6.8 Hz, 2-CH3-sp.), 1.58-2.00
(8H, m, CH2-sp.), 2.45-2.51 (2H, m, CH-sp., DMSO-d6),
3.92 (1H, d*, J=5.6 Hz, S–CH-sp.), 7.20 (1H, tt*, J=7.3,
1.5 Hz, 8-C6H5(H4)-sp.), 7.25 (2H, d*, J=6.8 Hz, 8-C6H5-
sp.), 7.30-7.34 (3H, m, H6-ind., 8-C6H5-sp.), 7.42 (1H, tt*,
J=7.3, 1.5 Hz, 3-C6H5(H4)-ind.), 7.51-7.61 (6H, m, H4, H7,
3-C6H5(H2,H3,H5,H6)-ind.), 10.21 (1H, s, CONH), 12.18
(1H, s, NH) ppm; 13C NMR (APT, DMSO-d6, 125 MHz):
δ=20.03 (2-CH3-sp.), 30.76, 31.24 (CH2-sp.), 37.38 (C2-
sp.), 38.10 (CH2-sp.), 41.95 (C8-sp.), 70.78 (C5-sp.),
114.57, 114.63 (CH-ar.), 118.61 (C-ar.), 119.34, 119.43
(CH-ar.), 124.59, 124.69 (CH-ar.), 125.49 (C-ar.), 126.97
(8-ar.(CH)-sp.), 127.08 (CH-ar.), 127.43 (8-ar.(CH)-sp.),
127.91 (C-ar.), 128.27 (C-ar.), 128.95 (CH-ar.), 130.63
(CH-ar.), 133.45 (C-ar.), 134.63 (C-ar.), 146.28 (8-ar.(C)-
sp.), 162.14 (CO–NH), 170.47 (CO-sp.) ppm; MS (ESI-):
m/z (%)=528.8 ([M-H]−, 100), 530.8 ([(M-H)+2]−, 33.8).
The broth microdilution test was performed in sterile
96-well U-shaped microdilution plates (LP Italiano SPA,
Milano, Italy). Rows A–F contained 100 mm3 of the com-
pound dilutions, whereas rows G (positive control) and H
(negative control) contained 100 mm3 medium. One hundred
mm3 of the corresponding inoculum was added to all wells
except for row H. The microplates were incubated at 35 °C
for about 7–10 days, when mycobacterial growth was clearly
visible as a white sediment in the positive control. Microbial
growth was confrmed by Ehrlich–Ziehl–Neelsen acid-fast
stain. Resazurin solution (30 mm3) was added to each well
and the plates were incubated for one additional day. At that
time, the frst purple colored well in which no growth was
visible, was defned as the compounds’ MIC value (Table 1).
Antiviral activity assays
Stock solutions of the test and reference compounds were
prepared in 100% DMSO at 5–25 mM. During incubation
with the cells, the highest test concentration was 100 µM (or
250 µM for ribavirin). The antiviral reference compounds
were: ganciclovir, brivudin, zanamivir, amantadine, ribavi-
rin, dextran sulfate-10,000, and mycophenolic acid. Antivi-
ral evaluation was carried out with a broad panel of viruses
using cytopathic efect (CPE) reduction assays. Human
infuenza A (H1N1 and H3N2) and B viruses were exam-
ined on Madin–Darby canine kidney (MDCK) cells. Res-
piratory syncytial virus, vesicular stomatitis virus and Cox-
sackie B4 virus were evaluated on human cervix carcinoma
Antitubercular activity assays
The microdilution method was performed according to a
standard protocol from the Clinical and Laboratory Stand-
ard Institute (CLSI) [27, 28]. The resazurin microtitre assay
(REMA) has been developed as a colorimetric and standard
method for drug susceptibility testing. The minimum inhibi-
tory concentrations (MICs) were determined according to
color changes at the end of incubation [29–31]. The strain
used, i.e., Mycobacterium tuberculosis H37Rv ATCC 27294
is susceptible to all common antimycobacterial drugs.
1 3

Design, synthesis, antitubercular and antiviral properties of new spirocyclic indole…
1543
HeLa cells. African Green Monkey Vero cells were used for
parainfuenza-3 virus, reovirus-1, Sindbis virus, Coxsackie
B4 virus, Punta Toro virus and yellow fever virus. Human
embryonic lung (HEL) fbroblast cells were infected with
herpes simplex virus types 1 and 2, vaccinia virus, human
adenovirus-2, and human coronavirus 229E.
References
1. World Health Organization (WHO) (2017) Global Tuberculosis
Report. http://www.who.int/tb/publications/global_report/en/.
Accessed 10 June 2018
2. Streicher EM, Müller B, Chihota V, Mlambo C, Tait M, Pillay M,
Trollip A, Hoek KGP, Sirgel FA, Pittius NCG, Helden PD, Victor
TC, Warren RM (2012) Infect Gene Evol 12:686
Semiconfluent cell cultures in 96-well plates were
infected with virus at a multiplicity of infection of 100
CCID50 (50% cell culture infective dose) per well. Together
with the virus, fourfold dilutions of the test or reference
compounds were added. The plates were incubated at 37 °C
(or 35 °C for infuenza- and coronavirus) until far advanced
CPE was visible, i.e., during 3–6 days or 10 days in the
case of adenovirus-2. Then, microscopy was performed to
score the CPE and calculate the 50% antivirally efective
concentration (EC50). Microscopy was also done to assess
cytotoxicity, expressed as the compound concentration caus-
ing minimal changes in cell morphology (minimal cytotoxic
concentration; MCC).
3. Zumla AI, Gillespie SH, Hoelscher M, Philips PP, Cole ST,
Abubakar I, McHugh TD, Schito M, Maeurer M, Nunn AJ (2014)
Lancet Infect Dis 14:327
4. Zignol M, Dean AS, Falzon D, Gemert W, Wright A, Deun A,
Portaels F, Laszlo A, Espinal MA, Pablos-Méndez A, Bloom A,
Aziz MA, Weyer K, Jaramillo E, Nunn P, Floyd K, Raviglione MC
(2016) N Engl J Med 375:1081
5. Ankale P, Nair G, Uppe A, Mathew A, Shah R (2017) Eur Respir
J 50:PA2727
6. Wallis RS, Maeurer M, Mwaba P, Chakaya J, Rustomjee R,
Migliori GB, Marais B, Schito M, Churchyard G, Swaminathan
S, Hoelscher M, Zumla A (2016) Lancet Infect Dis 16:e34
7. Poce G, Cocozza M, Consalvi S, Biava M (2014) Eur J Med Chem
86:335
8. Chetty S, Ramesh M, Singh-Pillay A, Soliman MES (2017)
Bioorg Med Chem Lett 27:370
9. Onajole OK, Pieroni M, Tipparaju SK, Lun S, Stec J, Chen G,
Gunosewoyo H, Guo H, Ammerman NC, Bishai WR, Kozikowski
AP (2013) J Med Chem 56:4093
Antibacterial and antifungal activity assays
10. Lun S, Guo H, Onajole OK, Pieroni M, Gunosewoyo H, Chen
G, Tipparaju SK, Ammerman NC, Kozikowski AP, Bishai WR
(2013) Nat Commun 4:2907
Antimicrobial activity against Pseudomonas aeruginosa
ATCC 27853, Escherichia coli ATCC 25922, Klebsiella
pneumoniae ATCC 4352, Proteus mirabilis ATCC 14153,
Staphylococcus aureus ATCC 29213, Staphylococcus epi-
dermidis ATCC 12228, Enterococcus faecalis ATCC 29212,
Candida albicans ATCC 10231, Candida parapsilosis
ATCC 22019, and Candida tropicalis ATCC 750 was deter-
mined by the microbroth dilutions technique using CLSI
recommendations [32, 33]. Serial twofold dilutions ranging
from 2500 µM to 1.22 µM were prepared in the test medium,
i.e., Mueller–Hinton broth for bacteria and RPMI-1640
medium for yeast strains. The inoculum was prepared using
a 4–6 h broth culture of each bacteria type and 24 h cul-
ture of yeast strains adjusted to a turbidity equivalent to 0.5
McFarland standard, diluted in broth media to give a fnal
concentration in the test tray of 5×105 cfu/cm3 for bacteria
and 5×103 cfu/cm3 for yeast. The trays were covered and
placed into plastic bags to prevent evaporation. The bacteria
trays were incubated at 35 °C for 18–20 h while the yeast-
containing trays were incubated at 35 °C for 46–50 h. The
MIC was defned as the lowest concentration of compound
giving complete inhibition of visible growth. Amikacin and
fuconazole were used as reference antibiotics for bacteria
and yeast, respectively; their MIC values were within the
accuracy range of the CLSI guidelines [34].
11. Kondreddi RR, Jiricek J, Rao SP, Lakshminarayana SB, Camacho
LR, Rao R, Herve M, Bifani P, Ma NL, Kuhen K, Goh A, Chatter-
jee AK, Dick T, Diagana TT, Manjunatha UH, Smith PW (2013)
J Med Chem 56:8849
12. Rao SP, Lakshminarayana SB, Kondreddi RR, Herve M, Camacho
LR, Bifani P, Kalapala SK, Jiricek J, Ma NL, Tan BH, Ng SH,
Nanjundappa M, Ravindran S, Seah PG, Thayalan P, Lim SH, Lee
BH, Goh A, Barnes WS, Chen Z, Gagaring K, Chatterjee AK,
Pethe K, Kuhen K, Walker J, Feng G, Babu S, Zhang L, Blasco
F, Beer D, Weaver M, Dartois V, Glynne R, Dick T, Smith PW,
Diagana TT, Manjunatha UH (2013) Sci Transl Med 5:214ra168
13. Stec J, Onajole OK, Lun S, Guo H, Merenbloom B, Vistoli G,
Bishai WR, Kozikowski AP (2016) J Med Chem 59:6232
14. Franz ND, Belardinelli JM, Kaminski MA, Dunn LC, Moura
VCN, Blaha MA, Truong DD, Li W, Jackson M (2017) Bioorg
Med Chem 25:3746
15. Cihan-Üstündağ G, Çapan G (2012) Mol Divers 16:525
16. Güzel Ö, Terzioğlu N, Çapan G, Salman A (2006) Arkivoc xii:98
17. Cihan-Üstündağ G, Şatana D, Özhan G, Çapan G (2015) J Enzyme
Inhib Med Chem 31:369
18. Vanderlinden E, Göktaş F, Cesur Z, Froeyen M, Reed ML, Russell
CJ, Cesur N, Naesens L (2010) J Virol 84:4277
19. Göktaş F, Vanderlinden E, Naesens L, Cesur N, Cesur Z (2012)
Bioorg Med Chem 20:7155
20. Göktaş F, Vanderlinden E, Naesens L, Cesur Z, Cesur N, Taş P
(2015) Phosphorus. Sulfur Silicon Relat Elem 190:1075
21. Yamamoto H, Inaba S, Okamoto T, Hirohashi T, Ishizumi K,
Yamamoto M, Maruyama I, Mori K, Kobayashi T (1969) Tran-
quilizer benzodiazepine derivatives. South African Patent ZA
6803041 A, Jan 18, 1969; (1969) Chem Abstr 71:124519
22. Abdelrahman MH, Aboraia AS, Youssif BGM, Elsadek BEM
(2017) Chem Biol Drug Des 90:64
Acknowledgements This work was supported in part by the Research
Fund of Istanbul University (Project Number BYP-57695). LN
acknowledges dedicated assistance from Leentje Persoons and her
team members.
23. Hiremath SP, Mruthyunjayaswamy BHM, Purohit MG (1978)
Indian J Chem 16B:789
1 3

1544
G. Cihan-Üstündağ et al.
24. Ergenç N, Günay NS, Demirdamar R (1998) Eur J Med Chem
33:143
30. Martin A, Panaiotov S, Portaels F, Hoffner S, Palomino JC,
Angeby K (2008) J Antimicrob Chemother 62:56
25. Kline T, Andersen NH, Harwood EA, Bowman J, Malanda A,
Endsley S, Erwin AL, Doyle M, Fong S, Harris AL, Mendelsohn
B, Mdluli K, Raetz CRH, Stover CK, Witte PR, Yabannavar A,
Zhu S (2002) J Med Chem 45:3112
31. Coban AY, Akbal AU, Uzun M, Durupınar B (2015) Mem Inst
Oswaldo Cruz 110:649
32. CLSI document (1997) Approved standard-2nd edition M27-A2.
Clinical and Laboratory Standards Institute, Wayne
26. Kocabalkanlı A, Cihan-Üstündağ G, Naesens L, Mataracı-Kara
E, Nassozi M, Çapan G (2017) Arch Pharm Chem Life Sci 350:1
27. Amsterdam D (2005) In: Lorian V (ed) Antibiotics in laboratory
medicine, 5th edn. Williams and Wilkins, Philadelphia
28. Clinical and Laboratory Standards Institute (2011) Susceptibility
testing of mycobacteria, nocardiae and other aerobic actinomy-
cetes. Approved Standard Second Edition M24-A2 Clinical and
Laboratory Standards Institute, Wayne
33. CLSI document (2006) Approved standard-7th edition M7-A7.
Clinical and Laboratory Standards Institute, Wayne
34. CLSI document (2010) 7th informational supplement M100-S20.
Clinical and Laboratory Standards Institute, Wayne
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
29. Martin A, Portaels F, Palomino JC (2007) J Antimicrob Chem-
other 59:175
1 3