Diclofenac-Based Hydrazones and Spirothiazolidinones: Synthesis, Characterization, and Antimicrobial Properties

Article abstract of DOI:10.1002/ardp.201700010

We report here the synthesis, structural characterization, and biological evaluation of novel diclofenac-based hydrazone (4a–f) and spirothiazolidinone (5a–f, 6a–f) derivatives designed as potential antimicrobial agents. The compounds were evaluated in vi

Full text of DOI:10.1002/ardp.201700010

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2017, 350, e1700010
Archiv der Pharmazie
Full Paper
Diclofenac-Based Hydrazones and Spirothiazolidinones: Synthesis,
Characterization, and Antimicrobial Properties
1
1, Lieve Naesens2, Emel Mataracı-Kara3, Mebble Nassozi1,
€
€
€
ꢀ
AysSe Kocabalkanlı , GokcSe Cihan-Ustundag
1
€
and Gultaze CS apan
1
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Istanbul University, Istanbul, Turkey
Department of Microbiology and Immunology, Rega Institute for Medical Research, KU Leuven, Leuven,
Belgium
2
3
Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Istanbul University, Istanbul, Turkey
We report here the synthesis, structural characterization, and biological evaluation of novel
diclofenac-based hydrazone (4a–f) and spirothiazolidinone (5a–f, 6a–f) derivatives designed as
potential antimicrobial agents. The compounds were evaluated in vitro for their antiviral activity
against a wide spectrum of DNA and RNA viruses. They were further screened in vitro against
different strains of bacteria and fungi. The hydrazone derivatives, 4a and 4c–f, were found to be
active against herpesviruses (HSV-1, HSV-2, and HSV-1 TKꢀ), vaccinia virus, and Coxsackie B4 virus,
with EC50 values between 6.6 mg/mL and 14.7 mg/mL, and the selectivity index values were greater
than 10 for 4a and 4f. The newly synthesized compounds (4–6) were inactive against the bacterial
and the fungal strains tested, at levels below 2500, 1250, or 625 mg/mL. Interestingly, the key
intermediate 3 with a free hydrazide moiety displayed antifungal properties against Candida albicans
and C. parapsilosis at MIC values of 4.88 mg/mL and 78.12 mg/mL, respectively.
Keywords: Antimicrobial activity / Cytotoxic activity / Rational drug design / Structure elucidation /
Synthesis
Received: January 10, 2017; Revised: March 7, 2017; Accepted: March 7, 2017
DOI 10.1002/ardp.201700010
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
postoperative pain, dysmenorrhea, and various ocular
conditions [1].
Introduction
Diclofenac is a proven and widely used non-steroidal anti-
inflammatory drug (NSAID) of the phenylacetic acid class
(Fig. 1). As with most NSAIDs, diclofenac exerts anti-
inflammatory, analgesic, and antipyretic actions via inhibition
of cyclooxygenases (COXs). Diclofenac was first synthesized by
Alfred Sallmann and Rudolf Pfister in 1973 and since then the
drug has been in clinical use for the treatment of rheumatoid
arthritis, osteoarthritis, ankylosing spondylitis, gout attacks,
Besides its use in pathological processes related to
inflammation, diclofenac and its chemical derivatives have
been thoroughly investigated for a wide range of pharmaco-
logical effects [2–9]. Several scientific communications report
the broad-spectrum antibacterial activity of diclofenac in vitro
and in vivo against isolates of various gram-positive and gram-
negative bacteria that were either sensitive or resistant to
antibiotics [10–13]. The drug was reported to be highly
bactericidal and exerts its action by inhibiting the DNA
synthesis of bacteria [11, 13]. Likewise, several small molecules
bearing the 2-[(2,6-dichlorophenyl)amino]benzyl unit were
shown to possess antibacterial potential [14, 15].
€
€
€
ꢀ
Correspondence: Dr. GokcSe Cihan-Ustundag, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, Istanbul University,
Istanbul 34116, Turkey.
Antiviral activity of diclofenac was first studied by Gordon
et al. in 1998. These researchers investigated the direct
pharmacological effects of topical NSAIDs on adenoviral
replication in vitro and only diclofenac appeared to have an
E-mail: gokcechn@istanbul.edu.tr
Fax: þ90-212-4400252
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Cl
Cl
(OCH , OH, NHOH)
R
3
O
NH
NH
NH2
(OH)
Cl
N
Cl
OH
O
O
(b)
(a)
(H, Alkyl, Aryl)
R
H
(Cl)
N
H
N
Ar
Ar :
N
OH
S
N
O
S
O
OH
CH3
(c)
Figure 1. Chemical structures of (a) the NSAID diclofenac, (b) the neuraminidase inhibitor diclofenac derivatives, and (c) the
influenza A virus fusion inhibitor spirothiazolidinone compounds.
inhibitory effect against different adenovirus serotypes [16].
In a recent patent, the inventors claimed that diclofenac or its
pharmaceutically acceptable salt inhibit the activity of herpes
viruses, including herpes simplex virus type 1 (HSV-1), herpes
simplex virus type 2 (HSV-2), and varicella-zoster virus. The
drug was found to reduce the lesion size, lesion number, and
viral titer when topically administered to herpesvirus lesions
in animals [17]. In a more recent study, some NSAIDs,
antioxidants, and peroxisome proliferator-activated receptor
gamma agonists were tested for their ability to interfere
with rotavirus ECwt (wild type) infectivity in ICR mice. The
work has provided evidence that the treatment of rotavirus-
infected mice with diclofenac led to reduced infection of
villus cells [18]. Further, Zhang et al. synthesized novel
compounds (Fig. 1) against influenza A/H3N2 viruses were
described in three reports [20–22]. The lead compounds were
proven to act as influenza A virus fusion inhibitors by
preventing the conformational change of the influenza virus
hemagglutinin and inhibiting the hemagglutinin-mediated
membrane fusion at low pH.
Encouraged by the above data, it was anticipated that a
combinationofdiclofenacandspirothiazolidinonestructuresin
one molecule could be interesting to potentially achieve
antimicrobial compounds. Inthis study, wereportthesynthesis,
structural identification, and in vitro antiviral, antibacterial,
and antifungal activities of 2-{2-[(2,6-dichlorophenyl)amino]-
phenyl}-N-[2,8-(non)substituted-3-oxo-1-thia-4-azaspiro[4.5]-
dec-4-yl]acetamides (5a–f, 6a–f), together with their precursors
2-{2-[(2,6-dichlorophenyl)amino]phenyl}-N0-[4-(non)substitute
dcyclohexylidene]acetohydrazides (4a-f) and 2-{2-[(2,6-dichlor
ophenyl)amino]phenyl}acetohydrazide (3).
diclofenac derivatives bearing
a substituted pyrrolidine
residue and evaluated the compounds for their ability to
inhibit influenza neuraminidase (NA) which is the drug target
of current influenza blockers (Fig. 1). Diclofenac analogs were
reported to display promising influenza NA inhibitory activity,
although in all cases, the activity was lower than that of
oseltamivir [19].
Results and discussion
Chemistry
During the last few years, our research group has focused
on the synthesis and biological evaluation of compounds
containing N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide
(spirothiazolidinone) backbone. The potent activity and
structure–activity relationships of various spirothiazolidinone
The synthetic pathways for the preparation of the target
products are illustrated in Scheme 1. The key intermediate 3
and hydrazone derivative 4a were previously reported
elsewhere [23, 24]. The structures of the compounds were
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established by microanalysis, IR, 1H-NMR, 13C-NMR (APT), 2D-
NMR (HSQC, HMBC), and electrospray ionization mass
spectrometry (ESI-MS). The X-ray crystal structure of com-
pound 4d was further determined and reported in a prior
paper [25].
multiplets. 13C-NMR resonances of compounds 4a–f were
further observed as duplicated signals (see Experimental
section for details). All these molecules were predicted to exist
as mixtures of rotamers due to the restricted rotation of two
phenyl rings. The stereoisomeric content and the purity of
hydrazones were verified by X-ray diffraction of compound
4d [25]. The X-ray study has revealed that the compound exists
as two crystallographically independent molecules in which
the dihedral angles between the benzene rings are 70.1° and
63.8°, and the crystal structure is stabilized by intramolecular
N–H ꢁ ꢁ ꢁ O and C–H ꢁ ꢁ ꢁ O hydrogen bonding interactions (for
X-ray structures, see the Supporting Information). The
isomeric composition was calculated from the integral values
of duplicate NH, phenyl H3, and CH2CO peaks, and the isomers
were found in the ratios of 33–40% and 60–67%.
The IR spectrum of the intermediate hydrazide (3) exhibited
N–H and C O bands in the 3325 cmꢀ1 and 1637 cmꢀ1 regions
–
–
which shifted to 3211–3228 cmꢀ1 and 1645–1662 cmꢀ1
,
respectively in compounds 4a–f. The absence of the NH2
resonance of compound 3 at d 4.31 ppm together with the
new signals assigned to the cycohexylidene residue at about d
0.83–3.10 ppm in the 1H-NMR spectra of 4a–f supported the
structure of the new adducts. The 1H-NMR spectra of 4a–f
displayed two sets of signals for most of the protons.
Arylamine N–H and carboxamide N–H protons were observed
as two separate singlets. CH2CO protons resonated as a singlet
and two doublets (resulting from geminal interactions) with
coupling constants in the range of 13.6–14.0 Hz. Aromatic
proton resonances appeared as duplicated signals or distorted
The spectra of cyclization products (5, 6) provided evidence
–
for ring closure. New bands assigned to the lactam C O
–
group of the spirothiazolidinone ring appeared in the
1693–1728 cmꢀ1 region. The spirothiazolidinone C2–H2
Cl
Cl
Cl
ii
i
iii
NH
NH
NH
O
Cl
Cl
O
Cl
O
O
OH
ONa
CH3
2
1
Cl
Cl
Cl
v
iv
NH
NH
NH
R
Cl
O
N
Cl
O
Cl
O
N
R
HN
HN
HN
NH2
S
O
4a–f
3
5a–f
vi
Cl
NH
4–6a: R =H
R
4–6b : R=CH3
4–6c : R=C2H5
4–6d : R=C3H7
Cl
O
N
HN
4–6e : R=C(CH3)3
4–6f : R=4-C6H4(OH)
S
O
6a–f
CH3
Scheme 1. Synthesis of compounds 4–6. Reagents and conditions: (i) H2O, conc. HCl; (ii) CH3OH, conc. H2SO4, reflux, 3 h; (iii) H2NNH2.
H2O, EtOH, reflux, 6 h; (iv) 4-(non)substituted cyclohexanone, abs. EtOH, reflux, 5–6 h; (v) mercaptoacetic acid, dry benzene, reflux,
5–6 h; (vi) 2-mercaptopropionic acid, dry benzene, reflux, 5–6 h.
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protons of 5a–f resonated at about d 3.56–3.62 ppm as singlets
and C2–H protons of 6a–f resonated at about d 3.86–3.92 ppm
as quartets. The remaining spirodecane protons resonated at
about d 0.73–2.20 ppm together with the alkyl substituents on
the spiroheterocycle. 13C-NMR (APT) and 2D-NMR (HSQC,
HMBC) experiments allowed unambiguous assignment of
the proton and carbon chemical shifts. The absence of upfield
aureus ATCC 29213, S. epidermidis ATCC 12228, Enterococcus
faecalis ATCC 29212, Candida albicans ATCC 10231, C.
parapsilosis ATCC 22019, C. tropicalis ATCC 750. Compounds
were assayed using twofold dilutions starting at 2500 mg/mL.
The lowest concentration of compound giving complete
inhibition of visible growth was referred as the MIC. The
tested compounds showed no inhibition against the micro-
organisms at concentrations below 2500, 1250, or 625 mg/mL,
except for compound 3. The key intermediate 2-{2-[(2,6-
dichlorophenyl)-amino]phenyl}acetohydrazide (3) was syn-
thesized and reported by different research groups, but to our
knowledge, its antimicrobial potential has not been investi-
gated before. The compound exhibited antifungal activity
against C. albicans and C. parapsilosis, showing complete
inhibition at MIC values of 4.88 mg/mL and 78.12 mg/mL,
respectively.
–
C N carbon resonances of 4a–f (d 157.18–163.30 ppm) and
–
observation of new shifts in the d 71.13–73.00 ppm region
assigned to the C5 carbon of the spirothiazolidinone system in
the 13C-NMR spectra of 5–6 confirmed the formation of
expected spirothiazolidinones. Cross peaks observed between
the spirothiazolidinone 2-CH3, C2–H protons and the lactam
–
C O in the HMBC spectrum of 5b and 6e enabled definite
–
–
assignment of the lactam C O (d 168.31–171.01 ppm) and the
–
CONH (d 171.43–171.62 ppm) carbons.
ESI-MS was used to verify the molecular weights of
compounds 4a, 4b, 4e, 4f, 5a–d, and 6c–f. Deprotonated
[M–H]ꢀ or protonated [MþH]þ ions observed in the ESI-MS
confirmed the calculated molecular weight of the compounds.
Conclusion
A series of hydrazone (4a–f) and spirothiazolidinone (5a–f and
6a–f) derivatives bearing diclofenac residue were prepared
and their activities against various viral, bacterial, and fungal
strains were evaluated. Of the 19 compounds tested, five
hydrazone derivatives, 4a and 4c–f, were found to be active
against herpes viruses (HSV-1, HSV-2, and HSV-1 TKꢀ), vaccinia
virus, and Coxsackie B4 virus with EC50 values between 6.6
mg/mL and 14.7 mg/mL. The highest inhibitory effect was
displayed by 4f bearing a 4-hydroxyphenyl substitution.
Cytotoxicity was further examined in all of the cell cultures
studied and two of the active compounds, 4a and 4f, exhibited
the highest selectivity indices (>10). The preliminary antiviral
screening results showed that the free hydrazone moiety was
crucial for biological activity since the transformation to a
spirothiazolidinone ring system led to compounds with no
inhibitory activity.
The newly synthesized compounds (4–6) showed no activity
against any of the bacterial or fungal strains tested, at levels
below 2500, 1250, or 625 mg/mL. Interestingly, compound 3
with a free hydrazide moiety displayed antifungal properties
against C. albicans and C. parapsilosis at MIC values of 4.88
mg/mL and 78.12 mg/mL, respectively. The inhibitory effect of
compound 3 on fungal growth makes this system attractive
for further structural optimization.
Biological activity
Compounds 3, 4a–f, 5a–f, and 6a–f were evaluated against a
variety of DNA and RNA viruses in cell culture, that is, herpes
simplex virus type-1 (HSV-1) and type-2 (HSV-2), an acyclovir-
resistant thymidine kinase-deficient (TKꢀ) form of HSV-1,
vaccinia virus, vesicular stomatitis virus, parainfluenza-3 virus,
reovirus, Sindbis virus, Coxsackie B4 virus, Punta Toro virus,
respiratory syncytial virus, feline corona virus, feline herpes
virus, influenza A, and influenza B virus. Hydrazone derivatives
4a,4e,and4fweremoderatelyinhibitorytoherpesviruses(HSV-
1, HSV-2, andHSV-1TKꢀ)andvacciniavirusinHELcells(Table1).
TheantiviralEC50 valueswere in the range of 6.6–14mg/mL. The
most prominent inhibitory effect was demonstrated by
compound 4f which bears a 4-hydroxyphenyl group at 4-
position of the cyclohexylidene residue. The values for the
selectivity index (ratio of cytotoxic to antivirally effective
concentration) were found to be greater than 10 for some test
results (Table 1). Furthermore, compounds 4c and 4d inhibited
the replication of Coxsackie B4 virus in Vero cells with EC50
values of 6.7 mg/mL and 14.7 mg/mL, respectively. Although a
straightforward structure–activity relationship could not be
derived, the antiviral screening results showed that the
hydrazone moiety had an essential role in antiviral activity,
since their cyclic spirothiazolidinone analogs (5a–f, 6a–f) did
not produce any antiviral effect at the tested concentrations.
Compounds 3, 4b, 5a–f, and 6a–f did not display activity
against any of the other DNA or RNA viruses tested. On the
other hand, this antiviral testing allowed to determine the
cytotoxicity of the test compounds in diverse mammalian cell
lines. The cytotoxicity test results are given in Table 2.
Antimicrobial activity of compounds 4a–f, 5a–f, and 6a–f,
along with the precursor hydrazide (3), was further studied in
vitro against the following strains: Pseudomonas aeruginosa
ATCC 27853, Escherichia coli ATCC 25922, Klebsiella pneumo-
niae ATCC 4352, P. mirabilis ATCC 14153, Staphylococcus
Experimental
Chemistry
General
Melting points were determined in open capillary tubes with
a Buechi B-540 melting point apparatus and are uncorrected.
Microanalyses were performed on a Thermo Finnigan Flash
EA 1112 elemental analyzer. IR spectra were recorded in
KBr discs (ymax in cmꢀ1) on a Perkin-Elmer 1600 FTIR and
Shimadzu IRAffinity-1 FTIR spectrophotometer. 1H-NMR
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Table 1. Antiviral activity in HEL and Vero cell cultures infected with diverse DNA and RNA viruses.
Antiviral assays in HELa) cells
Antiviral assays in Verob) cells
c)
Antiviral EC50 value (mg/mL)
Antiviral EC50 value (mg/mL)
Herpes
Herpes
simplex
virus-1
(KOS)
Herpes
simplex
virus-1
TKꢀ KOS
ACVr
Para-
influenza-
3
simplex
virus-2
(G)
Vesicular
stomatitis
virus
Punta
Toro
Vaccinia
virus
MCCd)
Reovirus-
Sindbis
virus
Coxsackie
MCC
Compound
(mg/mL)
virus
1
B4 virus
virus
(mg/mL)
3
>31
8.5
>31
9.6
>31
13.6
>31
10.3
>31
>31
>6.2
>6.2
>6.2
>6.2
>6.2
31
4a
>100
ꢂ100
>100
>100
>100
>100
>100
100
(ꢂ11.7)
>40
(ꢂ10.4)
>40
(ꢂ7.4)
>40
(ꢂ9.7)
>40
4b
4c
>40
>42
>40
>42
>8.1
>42
>8.1
>42
>8.1
>42
>8.1
6.7
>8.1
>42
40
>42
>42
>42
>42
>42
(>6.3)
14.7
4d
4e
4f
>43
>43
>43
>43
>43
>100
>100
>43
ꢂ100
ꢂ100
>43
>100
>100
>43
>100
>100
>100
>43
>100
>100
>43
>100
>100
>43
100
100
(>2.9)
>100
11.3
(ꢂ8.8)
7.5
10.2
(ꢂ9.8)
6.6
12.0
(ꢂ8.3)
9.2
14.0
(ꢂ7.1)
8.9
>100
(ꢂ13.3)
>100
>9.6
>9.9
>10
(ꢂ15.2)
>100
>9.6
>9.9
>10
(ꢂ10.9)
>100
>9.6
>9.9
>10
(ꢂ11.2)
>100
>9.6
>9.9
>10
5a
>100
>9.6
>9.9
>10
100
ꢂ9.6
ꢂ9.9
ꢂ10
ꢂ4
>100
>9.6
>9.9
>10
>100
>9.6
>9.9
>10
>100
>9.6
>9.9
>10
>100
>9.6
>9.9
>10
20
48
5b
>9.6
>9.9
>10
5c
49
5d
51
5e
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
>100
>100
>100
>10
20
5f
4
20
6a
>100
>10
ꢂ10
10
20
6b
>10
>10
10
6c
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
6d
>2
>2
>2
>2
>2
>2
>2
>2
>2
>2
6e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
>100
>100
>100
>100
>100
>100
>100
>100
4.0
>100
>100
58
>100
>100
10
20
6f
4
ꢂ4
>100
DS-
10.000e)
Ribavirinf)
Brivudinf)
Ganciclovirf)
146
>250
>250
>250
126
>250
0.06
0.08
250
250
4
10
>250
>100
>250
>250
0.09
>100
Values shown are the mean of three independent tests. In parenthesis, the selectivity index (i.e., ratio of MCC to antiviral EC50
)
is given.
Bold values indicate significant activities.
a)
HEL: human embryonic lung fibroblast cells.
Vero: African green monkey kidney cells.
b)
c) EC50: 50% effective concentration, producing 50% inhibition of virus-induced cytopathic effect, as determined by microscopy.
d) MCC: minimum inhibitory concentration, or compound concentration causing minimal changes in cell morphology, as assessed
by microscopy.
e)
DS-10.000: dextran sulfate with MW 10.000.
For these compounds, data are expressed in micromolar.
f)
(DMSO-d6), 13C-NMR (APT) (DMSO-d6), and heteronuclear
correlation 1H-13C (HSQC, HMBC) (DMSO-d6) spectra were run
on a VarianUNITYINOVA (500 MHz) instrument. Chemical shifts
are reported as d (ppm) relative to TMS as internal standard
and coupling constants (J) are given in hertz (Hz). MS (ESIþ/ꢀ)
were determined on a Finnigan LCQ Advantage Max mass
spectrometer (br.: broad, cyc.: cyclohexylidene, ar.: aromatic,
sp.: spirodecane).
The NMR spectra as well as the InChI codes of the investigated
compounds together with some biological activity data are
provided as Supporting Information.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}acetic acid (1)
Diclofenac sodium (0.01 mol) was dissolved in 25 mL of water
with heating. Concentrated HCl was added dropwise with
stirring until the precipitation was completed. The white
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Table 2. Cytotoxic activity in diverse mammalian cell linesa)
MCCb) (mg/mL)
CC50 (mg/mL)
c)
Compound
HEL
HeLa
Vero
MDCK
CRFK
3
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
>31
ꢂ100
>40
>42
>43
ꢂ100
ꢂ100
100
ꢂ9.6
ꢂ9.9
ꢂ10
ꢂ4
ꢂ31
20
ꢂ40
ꢂ42
ꢂ43
20
31
100
40
>42
>43
100
100
20
48
49
51
20
ꢂ6.2
ꢂ20
8.1
>31
>100
3.7
8.4
3.0
5.4
ꢂ8.6
20
>100
62.7
9.8
>48
>49
5.4
20
20
>100
>48
>49
51
>100
ꢂ9.6
ꢂ49
10
0.8
4
>100
9.6
1.9
5f
4
4
20
6a
6b
6c
6d
>100
>10
ꢂ10
10
100
20
4
4
20
20
>100
>10
ꢂ10
ꢂ2
>100
1.4
13
>10
>10
10
2.1
8.9
6e
4
20
0.8
6f
4
ND
ND
>100
ꢂ4
ꢂ4
0.4
ND
>100
ND
ND
1.9
ND
ND
>100
DS-10.000d)
Ribavirine)
Ganciclovire)
Brivudine)
>100
>250
ND
ND
>100
>250
ND
ND
>250
ND
ND, not done.
a)HEL: human embryonic lung fibroblast cells; HeLa: human cervix carcinoma cells; Vero: African green monkey kidney cells;
MDCK: Madin–Darby canine kidney cells; CRFK: Crandell–Rees feline kidney cells.
b)MCC: minimum inhibitory concentration, or compound concentration causing minimal changes in cell morphology, as assessed
by microscopy.
c)CC50: 50% cytotoxic concentration, assessed by the spectroscopic MTS cell viability assay.
d)DS-10.000: dextran sulfate with MW 10.000.
e)For these compounds, data are expressed in micromolar.
precipitate was filtered, washed with water and used without
further purification (CAS number: 15307-86-5).
(DMSO-d6/500 MHz): d 3.51, 3.87 (2H, 2s, CH2CO), 4.31 (2H, s,
NH2), 6.29 (1H, d, J ¼ 7.8 Hz, H3), 6.83 (1H, t, J ¼ 7.3 Hz, H5),
7.03 (1H, td, J ¼ 7.8, 1.5 Hz, H4), 7.10–7.18 (2H, m, H6, H40), 7.62
(2H, d, J ¼ 8.3 Hz, H30, H50), 8.44, 8.52 (1H, 2s, NH), 9.26, 9.47
(1H, 2s, CONH) (CAS number: 133239-50-6).
Methyl 2-{2-[(2,6-dichlorophenyl)amino]phenyl}acetate
(2)
A solution of 1 (0.01 mol) in methanol (20 mL) was refluxed
with a few drops of concentrated H2SO4 for 3 hours. The
obtained solid was separated, washed with NaHCO3 solution
(5%), and used without further purification (CAS number:
15307-78-5).
General procedure for the synthesis of 2-{2-[(2,6-
dichlorophenyl)amino]phenyl}-N0-[4-(non)-
substitutedcyclohexylidene]acetohydrazides (4a–f)
A mixture of 3 (0.005 mol) and an appropriate cyclohexanone
(0.007 mol) was refluxed in 15 mL abs. ethanol for 5–6 h. The
precipitate obtained was purified by recrystallization from
ethanol.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}acetohydrazide
(3) [23]
A mixture of 2 (0.01 mol), ethanol (25 mL), and H2NNH2.
H2O (98%, 0.08 mol) was heated under reflux for 6 hours.
After the completion of reaction, the reaction mixture
was cooled, diluted with water, and allowed to stand
overnight. The resulting white solid was filtered off and
recrystallized from methanol. White powder (89.5%); mp
N0-(Cyclohexylidene)-2-{2-[(2,6-dichlorophenyl)amino]-
phenyl}acetohydrazide (4a) [24]
White powder (74.3%); mp: 202–205°C; IR (KBr): ymax 3215
(N–H), 1662 (C O); 1H-NMR (DMSO-d6/500 MHz): d 1.54–1.62
–
–
(6H, m, CH2-cyc.), 2.22–2.26 (2H, m, CH2-cyc.), 2.39–2.44 (2H, m,
CH2-cyc.), 3.73, 3.99 (2H, 2s, CH2CO), 6.24, 6.28 (1H, 2d,
154–155°C; IR (KBr): ymax 3325 (N–H), 1637 (C O); 1H-NMR
–
–
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Archiv der Pharmazie
J ¼ 7.8 Hz, H3), 6.81, 6.84 (1H, 2t, J ¼ 7.3 Hz, H5), 7.01–7.06
(1H, m, H4), 7.13–7.28 (2H, m, H6,H40), 7.51, 7.52 (2H, 2d,
J ¼ 8.3 Hz, H30,H50), 7.83, 8.30 (1H, 2s, NH), 10.55, 10.68 (1H, 2s,
CONH); 13C-NMR (APT) (DMSO-d6/125 MHz): d 25.78, 26.30,
26.41, 27.42, 27.58, 28.12, 35.66, 35.92 (CH2-cyc.), 36.45, 38.43
(CH2-CO), 116.00, 116.65 (C3), 121.03, 121.42 (C5), 124.94,
125.93 (C1), 125.72 (C40), 127.82, 128.03 (C4), 129.83, 129.95
(C30,C50), 130.23, 130.24, 130.95, 130.97 (C20,C60), 131.15,
131.33 (C6), 137.69, 137.85 (C10), 143.69, 143.85 (C2), 157.91,
129.83, 129.96 (C30,C50), 130.19, 130.20, 131.00, 131.01 (C20,
C60), 131.10, 131.27 (C6), 137.69, 137.84 (C10), 143.69, 143.85
(C2), 158.07, 163.07 (CN), 168.65, 173.79 (CO). Anal. calcd.
for C22H25Cl2N3O (418.36): C, 63.16; H, 5.98; N, 10.04. Found:
C, 62.68; H, 5.85; N, 9.55.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N0-(4-
propylcyclohexylidene)acetohydrazide (4d) [25]
White needles (66.7%); mp: 172.5–173°C; IR (KBr): ymax 3226
162.95 (C N), 168.76, 173.82 (C O). MS (ESIþ) m/z (%): 390.00
(N–H), 1651 (C O); 1H-NMR (DMSO-d6/500 MHz): d 0.91–0.95
–
–
–
–
–
–
([MH]þ, 100), 392.00 ([MHþ2]þ, 72.34), 394.00 ([MHþ4]þ,
9.32). Anal. calcd. for C20H21Cl2N3O (390.31): C, 61.55; H, 5.42;
N, 10.77. Found: C, 61.88; H, 5.76; N, 10.54 (CAS number:
145262-77-7).
(3H, m, 4-CH2CH2CH3-cyc.), 1.04–1.16 (2H, m, CH2-cyc.), 1.22–
1.29, 1.32–1.40 (4H, 2m, 4-CH2CH2CH3-cyc.), 1.57–1.62 (1H, m,
CH-cyc.), 1.88–2.01 (3H, m, CH2-cyc.), 2.21–2.29 (1H, m, CH2-
cyc.), 2.38–2.42 (1H, m, CH2-cyc.), 2.95–3.03 (1H, m, CH2-cyc.),
3.76 and 4.03, 4.07 (2H, s, 2d, J ¼ 13.6 Hz, CH2CO), 6.30, 6.36
(1H, 2d, J ¼ 7.8 Hz, H3), 6.88, 6.92 (1H, 2t, J ¼ 7.3 Hz, H5), 7.08–
7.12 (1H, m, H4), 7.21–7.32 (2H, m, H40,H6), 7.58, 7.59 (2H, 2d,
J ¼ 7.8 Hz, H30,H50), 7.87, 8.35 (1H, 2s, NH), 10.60, 10.62 (1H, 2s,
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N0-
(4-methylcyclohexylidene)acetohydrazide (4b)
White powder (71.8%); mp: 201–202°C; IR (KBr): ymax 3216
1
(N–H), 1660 (C O); H-NMR (DMSO-d /500 MHz): d 0.84, 0.87
CONH); 13C-NMR (APT) (DMSO-d6/125 MHz):
d 14.89 (4-
–
–
6
(3H, 2d, J ¼ 6.8 Hz, 4-CH3-cyc.), 0.96–1.08 (2H, m, CH2-cyc.),
1.60–1.65 (1H, m, CH-cyc.), 1.75–1.92 (3H, m, CH2-cyc.), 2.13–
2.19 (1H, m, CH2-cyc.), 2.26–2.30 (1H, br. d, J ¼ 13.6 Hz, CH2-
cyc.), 2.88–2.92 (1H, m, CH2 cyc.), 3.66 and 3.91, 3.97 (2H, s, 2d,
J ¼ 14.0 Hz, CH2CO), 6.19, 6.26 (1H, 2d, J ¼ 8.3 Hz, H3), 6.80,
6.83 (1H, 2t, J ¼ 7.6 Hz, H5), 6.99–7.04 (1H, m, H4), 7.12–7.23
(2H, m, H6,H40), 7.48, 7.49 (2H, 2d, J ¼ 8.3 Hz, H30, H50), 7.81,
8.28 (1H, 2s, NH-disappeared on D2O exchange), 10.53, 10.55
(1H, 2s, CONH-disappeared on D2O exchange); 13C-NMR
(HSQC) (DMSO-d6/125 MHz): d 21.95 (4-CH3-cyc.), 26.51, 27.09
(CH2-cyc.), 31.69, 31.74 (CH-cyc.), 34.26, 34.31, 34.86, 35.13,
35.52 (CH2-cyc.), 36.43, 38.59 (CH2-CO), 115.89, 116.82 (C3),
121.06, 121.39 (C5), 124.91, 126.37 (C1), 125.80, 125.89 (C40),
127.90, 127.96 (C4), 129.87, 130.19 (C30/C50), 130.99 (C20,C60),
131.19, 131.99 (C6), 137.68, 137.83 (C10), 143.67, 143.84 (C2),
CH2CH2CH3-cyc.), 20.30, 20.33 (4-CH2CH2CH3-cyc.), 31.88,
32.32, 32.38, 33.43, 33.52, 34.85, 35.12 (CH2-cyc./4-
CH2CH2CH3-cyc.), 36.24, 36.35 (CH-cyc.), 36.44, 38.50 (CH2-
CO), 115.99, 116.72 (C3), 121.01, 121.33 (C5), 124.94, 125.92
(C1), 125.70 (C40), 127.81, 128.05 (C4), 129.82, 129.94 (C30,C50),
130.17, 130.19, 130.97, 130.99 (C20,C60), 131.11, 131.28 (C6),
137.69, 137.85 (C10), 143.69, 143.85 (C2), 158.05, 163.06 (CN),
–
168.68, 173.79 (C O). Anal. calcd. for C23H27Cl2N3O (432.39):
–
C, 63.89; H, 6.25; N, 9.72. Found: C, 63.43; H, 6.37; N, 9.61 (CAS
number: 1236490-02-0).
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N0-(4-tert-
butylcyclohexylidene)acetohydrazide (4e)
Whiteflakes(76.0%);mp:210.5–211°C;IR(KBr):ymax 3211(N–H),
1
–
1645 (C O); H-NMR (DMSO-d /500MHz): d 0.83, 0.85 (9H, 2s,
–
6
–
–
157.76, 162.75 (C N), 168.65, 173.78 (C O). MS (ESIþ) m/z
4-C(CH3)3-cyc.), 1.00–1.18 (2H, m, CH2-cyc.), 1.23–1.32 (1H, m,
CH-cyc.), 1.73–1.89 (3H, m, CH2-cyc.), 2.12–2.21 (1H, m, CH2-cyc.),
2.36 (1H, d, J ¼ 13.6Hz, CH2-cyc.), 2.97, 3.05 (1H, 2d, J ¼ 13.6Hz,
CH2-cyc.), 3.68 and3.94, 4.00(2H, s, 2d, J ¼ 13.6Hz, CH2CO), 6.22,
6.29 (1H, 2d, J ¼ 8.3 Hz, H3), 6.80, 6.84 (1H, 2t, J ¼ 7.3 Hz, H5),
7.01–7.05 (1H, m, H4), 7.13–7.25 (2H, m, H40,H6), 7.50, 7.51 (2H,
2d, J ¼ 7.8 Hz, H30,H50), 7.79, 8.28 (1H, 2s, NH), 10.52 (1H, s,
CONH); 13C-NMR (APT) (DMSO-d6/125MHz): d 27.04, 27.64
(CH2-cyc.),28.03,28.06(4-C(CH3)3-cyc.),32.84,32.86,35.31,35.56
(CH2-cyc./4-C(CH3)3-cyc.), 36.43, 38.51 (CH2-CO), 47.06, 47.14
(CH-cyc.), 115.86, 116.71 (C3), 120.85, 121.02 (C5), 124.82, 125.91
(C1), 125.78 (C40), 127.91, 128.09 (C4), 129.83, 129.96 (C30,C50),
130.17, 131.10(C20,C60),131.28 (C6), 137.65, 137.84 (C10),143.67,
–
–
(%): 404.43 ([MH]þ, 100), 406.46 ([MHþ2]þ, 60.9), 408.44
([MHþ4]þ, 10.0). Anal. calcd. for C21H23Cl2N3O (404.33): C,
62.32; H, 5.63; N, 10.39. Found: C, 62.83; H, 6.12; N, 10.67.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N0-
(4-ethylcyclohexylidene)acetohydrazide (4c)
White powder (76.1%); mp: 177.5–178.5°C; IR (KBr): ymax
3228 (N–H), 1660 (C O); 1H-NMR (DMSO-d6/500 MHz): d
–
–
0.84, 0.87 (3H, 2d, J ¼ 7.3 Hz, 4-CH2CH3-cyc.), 0.96–1.08
(2H, m, CH2-cyc.), 1.19–1.26 (2H, m, 4-CH2CH3-cyc.), 1.40–
1.44 (1H, m, CH-cyc.), 1.81–1.92 (3H, m, CH2-cyc.), 2.15–2.19
(1H, m, CH2-cyc.), 2.31–2.34 (1H, m, CH2-cyc.), 2.88–2.96
(1H, m, CH2-cyc.), 3.69 and 3.95, 3.99 (2H, s, 2d, J ¼ 13.7 Hz,
CH2CO), 6.23, 6.28 (1H, 2d, J ¼ 7.8 Hz, H3), 6.81, 6.84 (1H, 2t,
J ¼ 7.3 Hz, H5), 7.00–7.05 (1H, m, H4), 7.13–7.25 (2H, m, H40,
H6), 7.50, 7.51 (2H, 2d, J ¼ 8.3 Hz, H30, H50), 7.81–8.28 (1H, 2s,
NH), 10.53, 10.55 (1H, 2s, CONH); 13C-NMR (APT) (DMSO-d6/
125 MHz): d 12.15, 12.18 (4-CH2CH3-cyc.), 28.86, 31.98, 33.13,
34.83, 35.09 (CH2-cyc./4-CH2CH3-cyc.), 36.43, 38.50 (CH2-CO),
38.29, 38.32 (CH-cyc.), 115.98, 116.71 (C3), 121.05, 121.33
(C5), 124.91, 125.91 (C1), 125.72 (C40), 127.87, 128.05 (C4),
–
143.84 (C2), 158.05, 163.30 (CN), 168.62, 173.74 (C O). MS (ESIþ)
–
m/z (%): 446.35 ([MH]þ, 100), 448.32 ([MHþ2]þ, 70.3), 450.34
([MHþ4]þ, 12.6).Anal. calcd. forC24H29Cl2N3O(446.41):C,64.57;
H, 6.55; N, 9.41. Found: C, 64.70; H, 6.69; N, 9.48.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N0-[4-(4-hydroxy
phenyl)cyclohexylidene]acetohydrazide (4f)
Beige powder (52.5%); mp: 150–151°C; IR (KBr): ymax 3624,
1
–
3219 (O–H, N–H), 1651 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
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Archiv der Pharmazie
1.40–1.62 (2H, m, CH2-cyc.), 1.86–2.04 (3H, m, CH2-cyc.), 2.31–
2.43 (2H, m, CH2-cyc.), 2.68–2.76 (1H, m, CH-cyc.), 3.06, 3.10
(1H, br. 2d, J ¼ 14.1 Hz, CH2-cyc.), 3.71 and 3.98, 4.02 (2H, s, 2d,
J ¼ 13.6 Hz, CH2CO), 6.24, 6.29 (1H, 2d, J ¼ 7.8 Hz, H3), 6.66 (2H,
d, J ¼ 8.0 Hz, 4-ar.(H)-cyc.), 6.82, 6.85 (1H, 2t, J ¼ 7.3 Hz, H5),
6.98–7.07 (3H, m, H4,4-ar.(H)-cyc.), 7.13–7.24 (2H, m, H40,H6),
7.51, 7.53 (2H, 2d, J ¼ 7.8 Hz, H30,H50), 7.80, 8.29 (1H, 2s, NH-
disappeared on D2O exchange), 9.11 (1H, s, OH-disappeared
on D2O exchange), 10.58, 10.61 (1H, 2s, CONH-disappeared on
D2O exchange); 13C-NMR (APT) (DMSO-d6/125 MHz): d 33.82,
34.95, 35.48, 35.77 (CH2-cyc.), 36.46, 38.51 (CH2-CO), 42.32,
42.50 (CH-cyc.), 115.75, 115.79 (4-ar.(CH)-cyc.), 115.95, 116.71
(C3), 121.00, 121.34 (C5), 124.88, 125.89 (C1), 125.74 (C40),
125.94, 126.32, 128.04, 128.24 (C4/4-ar.(CH)-cyc.), 129.85,
129.97 (C30,C50), 130.21, 131.06 (C20,C60), 131.13, 131.31
(C6), 136.65 (4-ar.(C)-cyc.), 137.68, 137.84 (C10), 143.69,
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(8-methyl-3-
oxo-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (5b)
White needles (97.0%); mp: 277–278.5°C; IR (KBr): ymax 3350,
1
–
3227 (N–H); 1715, 1681 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
0.81 (3H, br. s, 8-CH3-sp.), 1.09 (3H, br. s, CH-sp., CH2-sp.), 1.51–
1.72 (6H, m, CH2-sp.), 3.58 (2H, s, SCH2-sp.), 3.70 (2H, s, CH2CO),
6.28 (1H, d, J ¼ 8.3 Hz, H3), 6.87 (1H, t, J ¼ 7.3 Hz, H5), 7.05 (1H,
t, J ¼ 7.3 Hz, H4), 7.17 (1H, t, J ¼ 7.8 Hz, H40), 7.28 (1H, d,
J ¼ 7.3 Hz, H6), 7.43 (1H, s, NH-disappeared on D2O exchange),
7.51 (2H, d, J ¼ 7.8 Hz, H30,H50), 10.40 (1H, s, CONH-disap-
peared on D2O exchange); 13C-NMR (HMBC) (DMSO-d6/
125 MHz): d 22.43 (8-CH3-sp.), 28.52 (C2-sp.), 31.24, 31.58,
37.23 (CH/CH2-sp.), 37.80 (CH2CO), 72.69 (C5-sp.), 116.76 (C3),
121.58 (C5), 125.17 (C1), 126.19 (C40), 128.20 (C4), 129.87 (C30,
C50), 130.62 (C20,C60) 131.24 (C6), 137.80 (C10), 143.47 (C2),
–
168.34 (C O-sp.), 171.53 (CONH). MS (ESIꢀ) m/z (%): 476.16
–
143.85 (C2), 156.28 (4-ar.(C)-cyc.), 157.18, 162.15 (C N),
([MꢀH]ꢀ, 100), 478.13 ([MꢀHþ2]ꢀ, 66.7), 480.03 ([M–Hþ4]ꢀ,
15.8). Anal. calcd. for C23H25Cl2N3O2S (478.44): C, 57.74; H,
5.27; N, 8.78. Found: C, 57.35; H, 5.33; N, 8.73.
–
–
þ
–
168.71, 173.83 (C O). MS (ESIþ) m/z (%): 482.10 ([MH] ,
–
100), 484.00 ([MHþ2]þ, 60.11), 486.00 ([MHþ4]þ, 13.15). Anal.
calcd. for C26H25Cl2N3O2 (482.40): C, 64.73; H, 5.22; N, 8.71.
Found: C, 64.16; H, 5.39; N, 8.26.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(8-ethyl-3-
oxo-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (5c)
White powder (66.3%); mp: 268–269°C; IR (KBr): ymax 3362,
1
–
General procedure for the synthesis of 2-{2-[(2,6-
3227 (N–H); 1727, 1667 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
dichlorophenyl)amino]phenyl}-N-[2,8-(non)substituted-3-
oxo-1-thia-4-azaspiro[4.5]dec-4-yl]acetamides (5a–f, 6a–f)
A mixture of 4a–f (0.0025 mol) and mercaptoacetic acid or 2-
mercaptopropionic acid (0.01 mol) was refluxed in 20 mL dry
benzene for 5–6 h using a Dean-Stark water separator. Excess
benzene was evaporated in vacuo. The resulting residue was
triturated with saturated NaHCO3 solution until CO2 evolu-
tion ceased and was allowed to stand overnight or in some
cases refrigerated until solidification. The solid thus obtained
was washed with water, dried, and recrystallized from
ethanol or ethanol/chloroform.
0.79 (3H, t, J ¼ 7.3 Hz, 8-CH2CH3-sp.), 0.83–0.88 (1H, m, CH-sp),
1.03–1.06 (2H, m, CH2-sp), 1.13 (2H, quin, J ¼ 7.3 Hz, 8-CH2CH3-
sp.), 1.65–1.73 (6H, m, CH2-sp.), 3.58 (2H, s, SCH2-sp.), 3.70
(2H, s, CH2CO), 6.27 (1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H, td, J ¼ 7.3,
1.0 Hz, H5), 7.05 (1H, td, J ¼ 7.8, 1.5 Hz, H4), 7.17 (1H, t,
J ¼ 8.3 Hz, H40), 7.27 (1H, dd, J ¼ 7.8, 1.5 Hz, H6), 7.41 (1H, s,
NH), 7.50 (2H, d, J ¼ 8.3 Hz, H30,H50), 10.39 (1H, s, CONH); 13C-
NMR (HSQC) (DMSO-d6/125 MHz): d 12.00 (8-CH2CH3-sp.),
28.52 (C2-sp.), 29.33, 29.55 (8-CH2CH3-sp., CH/CH2-sp.), 37.20
(CH/CH2-sp.), 37.83 (CH2CO), 72.98 (C5-sp.), 116.76 (C3), 121.59
(C5), 125.17 (C1), 126.19 (C40), 128.20 (C4), 129.87 (C30,C50),
130.60 (C20,C60), 131.30 (C6), 137.80 (C10), 143.47 (C2), 168.34
(CO-sp.), 171.53 (CONH). MS (ESIþ) m/z (%): 492.00 ([MH]þ,
100), 494.00 ([MHþ2]þ, 73.53), 496.00 ([MHþ4]þ, 19.77). Anal.
calcd. for C24H27Cl2N3O2S (492.46): C, 58.53; H, 5.53; N, 8.53.
Found: C, 58.57; H, 5.59; N, 8.37.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(3-oxo-1-thia-
4-azaspiro[4.5]dec-4-yl)acetamide (5a)
White powder (54.0%); mp: 252–253°C; IR (KBr): ymax 3348,
1
–
3227 (N–H), 1724, 1668 (C O); H-NMR (DMSO-d /500 MHz):
–
6
d 0.87 (1H, br. q, J ¼ 13.2 Hz, CH2-sp.), 1.37 (2H, br. d,
J ¼ 11.7 Hz, CH2-sp.), 1.49 (1H, br. d, J ¼ 13.1 Hz, CH2-sp.),
1.63–1.72 (6H, m, CH2-sp.), 3.57 (2H, s, SCH2-sp.), 3.71 (2H, s,
CH2CO), 6.28 (1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H, t, J ¼ 7.3 Hz,
H5), 7.05 (1H, td, J ¼ 7.5, 1.4 Hz, H4), 7.16 (1H, t, J ¼ 7.8 Hz,
H40), 7.28 (1H, dd, J ¼ 7.3, 1.5 Hz, H6), 7.44 (1H, s, NH), 7.51
(2H, d, J ¼ 8.2 Hz, H30,H50), 10.40 (1H, s, CONH); 13C-NMR
(APT) (DMSO-d6/125 MHz): d 23.53, 24.61, 24.73 (CH2-sp.),
28.54 (C2-sp.), 37.52 (CH2-sp.), 37.83 (CH2CO), 72.85
(C5-sp.), 116.77 (C3), 121.58 (C5), 125.20 (C1), 126.18
(C40), 128.19 (C4), 129.87 (C30,C50), 130.63 (C20,C60), 131.26
(C6), 137.82 (C10), 143.51 (C2), 168.31 (CO-sp.), 171.59
(CONH). MS (ESIꢀ) m/z (%): 462.28 ([M–H]ꢀ, 100), 464.26
([M–Hþ2]ꢀ, 67.2), 466.28 ([M–Hþ4]ꢀ, 8.5). Anal. calcd. for
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(3-oxo-8-
propyl-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (5d)
White needles (71.5%); mp: 240–241.5°C; IR (KBr): ymax
3356, 3225 (N–H), 1725, 1674 (C O); 1H-NMR (DMSO-d6/
–
–
500 MHz): d 0.81 (3H, t, J ¼ 7.3 Hz, 8-CH2CH2CH3-sp.), 0.88–
1.11 (5H, m, 8-CH2CH2CH3-sp., CH/CH2-sp), 1.22 (2H, q,
J ¼ 7.3 Hz, 8-CH2CH2CH3-sp.), 1.63–1.72 (6H, m, CH2-sp), 3.58
(2H, s, SCH2-sp.), 3.70 (2H, s, CH2CO), 6.27 (1H, d, J ¼ 8.3 Hz,
H3), 6.87 (1H, td, J ¼ 7.3, 1.0 Hz, H5), 7.05 (1H, td, J ¼ 7.8,
1.5 Hz, H4), 7.17 (1H, t, J ¼ 8.3 Hz, H40), 7.28 (1H, dd, J ¼ 7.8,
1.5, H6), 7.40 (1H, s, NH), 7.51 (2H, d, J ¼ 8.3, H30,H50), 10.39
(1H, s, CONH); 13C-NMR (APT) (DMSO-d6/125 MHz): d 14.79
(8-CH2CH2CH3-sp.), 20.13 (8-CH2CH2CH3-sp.), 28.53 (C2-sp.),
29.87, 30.05, 37.23, 38.94 (8-CH2CH2CH3-sp./CH2-sp.), 35.74
(CH-sp.), 37.83 (CH2CO), 73.00 (C5-sp.), 116.76 (C3), 121.60
C
22H23Cl2N3O2S (464.41): C, 56.90; H, 4.99; N, 9.05. Found: C,
56.42; H, 4.98; N, 8.82.
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(C5), 125.17 (C1), 126.22 (C40), 128.25 (C4), 129.88 (C30,C50),
130.62 (C20,C60), 131.33 (C6), 137.80 (C10), 143.48 (C2),
168.35 (CO-sp.), 171.51 (CONH). MS (ESIþ) m/z (%): 506.00
([MH]þ, 100), 508.00 ([MHþ2]þ, 64.88), 510.00 ([MHþ4]þ,
14.96). Anal. calcd. for C25H29Cl2N3O2S (506.49): C, 59.28; H,
5.77; N, 8.30. Found: C, 59.56; H, 5.86; N, 8.15.
7.05 (1H, td, J ¼ 7.3, 1.5 Hz, H4), 7.17 (1H, t, J ¼ 8.3 Hz, H40),
7.29 (1H, d, J ¼ 7.3 Hz, H6), 7.41 (1H, s, NH), 7.50 (2H, d,
J ¼ 7.8 Hz, H30,H50), 10.44 (1H, s, CONH); 13C-NMR (APT)
(DMSO-d6/125 MHz): d 20.37 (2-CH3-sp.), 23.39, 23.82, 24.60,
24.72, 24.61 (CH2-sp.), 37.39 (C2-sp.), 37.82 (CH2CO), 71.51 (C5-
sp.), 116.74 (C3), 121.48 (C5), 125.15 (C1), 126.15 (C40), 128.17
(C4), 129.88 (C30,C50), 130.65 (C20,C60), 131.25 (C6), 137.84
0
–
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(3-oxo-8-tert-
(C1 ), 143.51 (C2), 170.95 (C O-sp.), 171.51 (CONH). Anal.
–
butyl-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (5e)
calcd. for C23H25Cl2N3O2S (478.44): C, 57.74; H, 5.27; N, 8.78.
Found: C, 57.69; H, 5.22; N, 8.73.
White powder (55.0%); mp: 272.5–274°C; IR (KBr): ymax 3370,
1
–
3250 (N–H), 1711, 1665 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
0.74–0.83 (10H, m, CH2/CH-sp., 8-C(CH3)3-sp.), 1.14–1.16
(2H, m, CH/CH2-sp.), 1.64–1.77 (6H, m, CH2-sp.), 3.56 (2H, s,
SCH2-sp.), 3.70 (2H, s, CH2CO), 6.28 (1H, d, J ¼ 7.8 Hz, H3), 6.87
(1H, t, J ¼ 7.8 Hz, H5), 7.05 (1H, t, J ¼ 8.3 Hz, H4), 7.17 (1H, t,
J ¼ 8.3 Hz, H40), 7.28 (1H, dd, J ¼ 7.8, 1.5 Hz, H6), 7.39 (1H, s,
NH), 7.50 (2H, d, J ¼ 8.3 Hz, H30,H50), 10.38 (1H, s, CONH); 13C-
NMR (APT) (DMSO-d6/125 MHz): d 24.30 (CH2-sp.), 27.97 (8-
C(CH3)3-sp.), 28.52 (C2-sp.), 32.58, 37.23, 37.55 (8-C(CH3)3-sp./
CH2-sp.), 37.84 (CH2CO), 46.45 (CH-sp.), 72.88 (C5-sp.), 116.77
(C3), 121.63 (C5), 125.18 (C1), 126.22 (C40), 128.25 (C4), 129.88
(C30,C50), 130.61, 130.62 (C20,C60), 131.41 (C6), 137.80 (C10),
143.48 (C2), 168.35 (CO-sp.), 171.50 (CONH). Anal. calcd. for
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(2,8-dimethyl-
3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (6b)
White powder (77.3%); mp: 222–222.5°C; IR (KBr): ymax 3352,
1
–
3243 (N–H), 1718, 1678 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
0.81 (3H, d, J ¼ 5.1 Hz, 8-CH3-sp.), 1.03–1.11 (3H, m, CH/CH2-
sp.), 1.38 (3H, d, J ¼ 7.3 Hz, 2-CH3-sp.), 1.60–1.69 (6H, m, CH2-
sp.), 3.70 (2H, s, CH2CO), 3.87 (1H, q, J ¼ 7.3 Hz, SCH-sp.), 6.27
(1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H, t, J ¼ 7.3 Hz, H5), 7.05 (1H, t,
J ¼ 7.8 Hz, H4), 7.17 (1H, t, J ¼ 8.3 Hz, H40), 7.28 (1H, d,
J ¼ 7.8 Hz, H6), 7.40 (1H, s, NH), 7.51 (2H, d, J ¼ 7.8 Hz, H30,H50),
10.45 (1H, s, CONH); 13C-NMR (APT) (DMSO-d6/125 MHz): d
20.41 (2-CH3-sp.), 22.46 (8-CH3-sp.), 31.24 (CH(C8)-sp.), 31.77,
31.90, 32.23, 32.35 (CH2-sp.), 37.40 (C2-sp.), 37.82 (CH2CO),
71.36 (C5-sp.), 116.73 (C3), 121.48 (C5), 125.15 (C1), 126.23
(C40), 128.28 (C4), 129.88 (C30,C50), 130.69 (C20,C60), 131.25
(C6), 137.83 (C10), 143.48 (C2), 170.99 (CO-sp.), 171.49 (CONH).
Anal. calcd. for C24H27Cl2N3O2S (492.46): C, 58.53; H, 5.53; N,
8.53. Found: C, 58.41; H, 5.65; N, 8.34.
C
26H31Cl2N3O2S (520.51): C, 59.99; H, 6.00; N, 8.07. Found: C,
59.48; H, 5.95; N, 7.96.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-[8-(4-
hydroxyphenyl)-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl]-
acetamide (5f)
Beige powder (62.8%); mp: 280–283°C; IR (KBr): ymax 3350,
3238 (O–H, N–H), 1707, 1667 (C O); 1H-NMR (DMSO-d6/
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(8-ethyl-2-
methyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide
(6c)
–
–
500 MHz): d 1.54–1.56 (2H, m, CH2-sp.), 1.73–1.85 (6H, m, CH2-
sp.), 2.20 (1H, t, J ¼ 11.1 Hz, CH-sp.), 3.62 (2H, s, SCH2-sp.), 3.73
(2H, s, CH2CO), 6.29 (1H, d, J ¼ 7.8 Hz, H3), 6.66 (2H, d,
J ¼ 8.3 Hz, 8-ar.(H)-sp.), 6.89 (1H, td, J ¼ 7.3, 1.0 Hz, H5), 6.94
(2H, d, J ¼ 8.3 Hz, 8-ar.(H)-sp.), 7.06 (1H, td, J ¼ 7.8, 1.5 Hz, H4),
7.17 (1H, t, J ¼ 7.8 Hz, H40), 7.30 (1H, dd, J ¼ 7.6, 1.5 Hz, H6),
7.44 (1H, s, NH), 7.51 (2H, d, J ¼ 8.3 Hz, H30,H50), 9.10 (1H, s,
OH), 10.44 (1H, s, CONH); 13C-NMR (APT) (DMSO-d6/125 MHz):
d 28.60 (C2-sp.), 31.43, 37.61, 37.73 (CH2-sp.), 37.85 (CH2CO),
41.46 (CH-sp.), 72.46 (C5-sp.), 115.79 (8-ar.(CH)-cyc.), 116.84
(C3), 121.65 (C5), 125.21 (C1), 126.20 (C40), 128.06, 128.34 (C4/
8-ar.(CH)-cyc.), 129.89 (C30,C50), 130.63, 130.64 (C20,C60) 131.37
(C6), 136.63 (8-ar.(C)-cyc.), 137.89 (C10), 143.53 (C2), 156.28 (8-
White powder (54.5%); mp: 197–197.5°C; IR (KBr): ymax 3363,
1
–
3233 (N–H), 1726, 1671 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
0.79 (3H, t, J ¼ 7.3 Hz, 8-CH2CH3-sp.), 0.81–0.86 (1H, m, CH-sp),
1.02–1.22 (4H, m, CH2-sp, 8-CH2CH3-sp.), 1.38 (3H, d, J ¼ 6.8 Hz,
2-CH3-sp.), 1.64–1.70 (6H, m, CH2-sp.), 3.69 (2H, s, CH2CO), 3.86
(1H, q, J ¼ 6.8 Hz, SCH-sp.), 6.27 (1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H,
t, J ¼ 7.3 Hz, H5), 7.04 (1H, t, J ¼ 8.3 Hz, H4), 7.17 (1H, t,
J ¼ 8.3 Hz, H40), 7.27 (1H, d, J ¼ 7.3 Hz, H6), 7.42 (1H, s, NH),
7.51 (2H, d, J ¼ 7.8 Hz, H30,H50), 10.46 (1H, s, CONH); 13C-NMR
(HSQC) (DMSO-d6/125 MHz): d 11.99 (8-CH2CH3-sp.), 20.41 (2-
CH3-sp.), 29.36 (8-CH2CH3-sp.), 29.40, 29.86, 37.31 (CH(C8)/
CH2-sp.), (37.43 (C2-sp.), 37.80 (CH2CO), 71.68 (C5-sp.), 116.69
(C3), 121.51 (C5), 125.29 (C1), 126.18 (C40), 128.12 (C4), 129.87
(C30,C0), 130.66 (C20,C60), 131.27 (C6), 137.86 (C10), 143.50 (C2),
170.89 (CO-sp.), 171.52 (CONH). MS (ESIꢀ) m/z (%): 504.20
([M–H]ꢀ, 100), 506.20 ([MꢀHþ2]ꢀ, 75.39), 508.20 ([MꢀHþ4]ꢀ,
14.64). Anal. calcd. for C25H29Cl2N3O2S (506.49): C, 59.28; H,
5.77; N, 8.30. Found: C, 58.77; H, 5.54; N, 7.87.
–
–
ar.(C)-cyc.), 168.38 (C O-sp.), 171.62 (CONH). Anal. calcd. for
28H27Cl2N3O3S (556.50): C, 60.43; H, 4.89; N, 7.55. Found: C,
C
59.99; H, 4.99; N, 7.54.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(2-methyl-3-
oxo-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (6a)
White needles (56.7%); mp: 240–241.5°C; IR (KBr): ymax 3362,
3233 (N–H), 1722, 1672 (C O); H-NMR (DMSO-d /500 MHz): d
1
–
–
6
0.88 (1H, br. q, J ¼ 12.7 Hz, CH2-sp.), 1.31–1.44 (5H, m, CH2-sp.,
2-CH3-sp.), 1.49 (1H, br. d, J ¼ 12.6 Hz, CH2-sp.), 1.64–1.70
(6H, m, CH2-sp.), 3.70 (2H, s, CH2CO), 3.87 (1H, q, J ¼ 7.1 Hz,
SCH-sp.), 6.28 (1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H, t, J ¼ 7.3 Hz, H5),
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(2-methyl-3-
oxo-8-propyl-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide (6d)
White powder (68.7%); mp: 218–219°C; IR (KBr): ymax 3356,
1
–
3245 (N–H), 1728, 1668 (C O); H-NMR (DMSO-d /500 MHz): d
–
6
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Archiv der Pharmazie
0.82 (3H, t, J ¼ 7.3 Hz, 8-CH2CH2CH3-sp.), 0.84–1.11 (5H, m, 8-
CH2CH2CH3, CH/CH2-sp), 1.23 (2H, q, J ¼ 7.3 Hz, 8-CH2CH2CH3-
sp.), 1.38 (3H, d, J ¼ 7.3 Hz, 2-CH3-sp.), 1.64–1.70 (6H, m, CH2-
sp.), 3.69 (2H, s, CH2CO), 3.87 (1H, q, J ¼ 7.3 Hz, SCH-sp.), 6.27
(1H, d, J ¼ 7.3 Hz, H3), 6.88 (1H, td, J ¼ 7.3, 1.0 Hz, H5), 7.04 (1H,
td, J ¼ 7.8, 1.5 Hz, H4), 7.17 (1H, t, J ¼ 8.3 Hz, H40), 7.28 (1H, dd,
J ¼ 7.8, 1.5 Hz, H6), 7.38 (1H, s, NH), 7.51 (2H, d, J ¼ 8.3 Hz,
H3b0,H50), 10.43 (1H, s, CONH); 13C-NMR (APT) (DMSO-d6/
125 MHz): d 14.79 (8-CH2CH2CH3-sp.), 20.13 (8-CH2CH2CH3-
sp.), 20.47 (2-CH3-sp.), 29.73, 30.12 (8-CH2CH2CH3-sp./CH2-sp.),
35.71 (CH(C8)-sp.), 37.37(8-CH2CH2CH3-sp./CH2-sp.), 37.40 (C2-
sp.), 37.81 (CH2CO), 38.96 (8-CH2CH2CH3-sp./CH2-sp.), 71.66
(C5-sp.), 116.72 (C3), 121.55 (C5), 125.13 (C1), 126.26 (C40),
128.18 (C4), 129.88 (C30,C50), 130.69 (C20,C60), 131.32 (C6),
137.82 (C10), 143.78 (C2), 170.99 (CO-sp.), 171.44 (CONH). MS
(ESI–) m/z (%): 518.40 ([M–H]ꢀ, 100), 520.30 ([M–Hþ2]ꢀ,
78.03), 522.20 ([MꢀHþ4]ꢀ, 6.87). Anal. calcd. for
D2O exchange); 13C-NMR (APT) (DMSO-d6/125 MHz): 20.39 (2-
CH3-sp.), 31.24, 31.78 (CH2-sp.), 37.45 (C2-sp.), 37.69 (CH2-sp.),
37.83 (CH2CO), 41.43 (CH(C8)-sp.), 71.13 (C5-sp.), 115.79 (8-ar.
(CH)-cyc.), 116.78 (C3), 121.60 (C5), 125.17 (C1), 126.23 (C40),
127.89, 128.25 (C4/8-ar.(CH)-cyc.), 129.88 (C30,C50), 130.69,
130.71 (C20,C60), 131.35 (C6), 136.64 (8-ar.(C)-cyc.), 137.86
0
–
(C1 ), 143.53 (C2), 156.27 (8-ar.(C)-cyc.), 171.01 (C O-sp.),
–
171.54 (CONH). MS (ESIþ) m/z (%): 570.1 ([MH]þ, 100), 572.2
([MHþ2]þ, 57.2), 574.2 ([MHþ4]þ, 15.6). Anal. calcd. for
C
29H29Cl2N3O3S (570.53): C, 61.05; H, 5.12; N, 7.37. Found: C,
60.67; H, 5.23; N, 7.31.
Biological assays
Antiviral activity assays
The compounds were evaluated for antiviral activity in cell
culture by using cytopathic effect (CPE) reduction assays with
a broad and diverse panel of DNA and RNA viruses [26, 27].
Human cervix carcinoma HeLa cells were used to study
vesicular stomatitis virus, Coxsackie B4 virus, and respiratory
syncytial virus. African green monkey kidney Vero cells
were used to evaluate para-influenza virus type 3, reovirus
type 1, Sindbis virus, Coxsackie B4 virus, and Punta Toro virus.
Viruses evaluated on human embryonic lung fibroblast
cells were herpes simplex virus types 1 and 2, vaccinia virus,
and vesicular stomatitis virus. Madin–Darby canine kidney
(MDCK) cells were used for antiviral evaluation against
influenza A and B viruses. Finally, feline herpes virus and
feline canine virus were grown in Crandell–Rees feline kidney
cells [20].
To perform the antiviral assays, the viruses were added to
subconfluent cultures of the cells in 96-well plates, and at the
same time, the test compounds were added in serial dilutions.
Appropriate reference compounds were included, that is, the
viral entry inhibitor dextran sulfate (MW 5000); the broad
antiviral agent ribavirin; the antiherpetic agents ganciclovir
and brivudin and the HIV inhibitor azidothymidine. After 3–6
days, incubation at 37°C (or 35°C in the case of influenza
virus), the compounds’ inhibitory effect on virus-induced
cytopathic effect as well as their cytotoxicity was monitored
by light microscopy or by performing the MTS cell viability
assay (CellTiter 961 AQueous One Solution Cell Proliferation
Assay from Promega). Antiviral activity was expressed as the
50% effective concentration (EC50), whereas cytotoxicity was
defined as the minimal cytotoxic concentration (based on
microscopy) or CC50 (50% cytotoxic concentration, assessed by
the MTS assay).
C
26H31Cl2N3O2S (520.51): C, 59.99; H, 6.00; N, 8.07. Found:
C, 59.60; H, 6.53; N, 8.00.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-(2-methyl-3-
oxo-8-tert-butyl-1-thia-4-azaspiro[4.5]dec-4-yl)acetamide
(6e)
Whitepowder(51.6%);mp:196–197°C;IR(KBr):ymax 3370,3258
1
–
(N–H), 1724, 1672 (C O); H-NMR (DMSO-d /500 MHz): d 0.73–
–
6
0.83 (10H, m, CH/CH2-sp., 8-C(CH3)3-sp.), 1.10–1.19 (2H, m, CH/
CH2-sp.), 1.39 (3H, d, J ¼ 7.3 Hz, 2-CH3-sp.), 1.65–1.74 (6H, m,
CH2-sp.), 3.70 (2H, s, CH2CO), 3.86 (1H, q, J ¼ 7.1 Hz, SCH-sp.),
6.28 (1H, d, J ¼ 7.8 Hz, H3), 6.87 (1H, t, J ¼ 7.3 Hz, H5), 7.05 (1H, t,
J ¼ 8.3 Hz, H4), 7.16 (1H, t, J ¼ 7.8 Hz, H40), 7.29 (1H, dd, J ¼ 7.3,
1.5 Hz,H6),7.37(1H,s,NH),7.50(2H,d,J ¼ 7.8 Hz,H30,H50),10.45
(1H, s, CONH); 13C-NMR (HMBC)(DMSO-d6/125 MHz):d 20.39 (2-
CH3-sp.), 24.18, 24.61 (CH2-sp.), 27.94 (8-C(CH3)3-sp.), 32.56 (8-C
(CH3)3-sp.), 37.41 (C2-sp.), 37.55 (CH2-sp.), 37.82 (CH2CO), 46.38
(CH(C8)-sp.), 71.55 (C5-sp.), 116.73 (C3), 121.56(C5), 125.15 (C1),
126.23 (C40), 128.18 (C4), 129.86 (C30,C50), 130.65 (C20,C60),
131.39 (C6), 137.81 (C10), 143.47 (C2), 170.98 (CO-sp.), 171.43
(CONH). MS (ESIꢀ) m/z (%): 532.30 ([MꢀH]ꢀ, 100), 534.30
([MꢀHþ2]ꢀ, 63.45), 536.30 ([M–Hþ4]ꢀ, 6.87). Anal. calcd. for
C27H33Cl2N3O2S (534.54): C, 60.67; H, 6.22; N, 7.86. Found: C,
60.34; H, 6.27; N, 7.85.
2-{2-[(2,6-Dichlorophenyl)amino]phenyl}-N-[8-(4-
hydroxyphenyl)-2-methyl-3-oxo-1-thia-4-azaspiro[4.5]-
dec-4-yl]acetamide (6f)
Beige crystals (60.0%); mp: 217–219°C; IR (KBr): ymax 3445,
1
–
3377, 3262 (O–H, N–H), 1693, 1651 (C O); H-NMR (DMSO-d /
–
6
500 MHz): d 1.41 (3H, d, J ¼ 7.3 Hz, 2-CH3-sp.), 1.49–1.60 (2H, m,
CH2-sp.), 1.73–1.99 (6H, m, CH2-sp.), 2.18 (1H, t, J ¼ 11.7 Hz, CH-
sp.), 3.35 (br. s, 6H, OH with DMSO-d6), 3.73 (2H, s, CH2CO),
3.92 (1H, q, J ¼ 7.3 Hz, SCH-sp.), 6.29 (1H, d, J ¼ 7.8 Hz, H3), 6.65
(2H, d, J ¼ 8.3 Hz, 8-ar(H)-sp.), 6.89 (1H, td, J ¼ 7.3, 1.0 Hz, H5),
6.94 (2H, d, J ¼ 8.3 Hz, 8-ar(H)-sp.), 7.07 (1H, td, J ¼ 7.5, 1.3 Hz,
H4), 7.17 (1H, t, J ¼ 7.8 Hz, H40), 7.31 (1H, dd, J ¼ 7.5, 1.5 Hz,
H6), 7.40 (1H, s, NH-disappeared on D2O exchange), 7.51 (2H,
d, J ¼ 8.3 Hz, H30,H50), 10.50 (1H, s, CONH-disappeared on
Antimicrobial activity assays
Antimicrobial activity against S. aureus ATCC 29213, S.
epidermidis ATCC 12228, E. coli ATCC 25922, K. pneumoniae
ATCC 4352, P. aeruginosa ATCC 27853, P. mirabilis ATCC 14153,
E. faecalis ATCC29212, C. albicans ATCC 10231, C. parapsilosis
ATCC 22019, and C. tropicalis ATCC 750 was determined by the
microbroth dilutions technique using the Clinical Laboratory
Standards Institute (CLSI) recommendations [28, 29]. Mueller–
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Archiv der Pharmazie
Hinton broth for bacteria and RPMI-1640 medium for yeast
strains were used as the test medium. Serial twofold dilutions
ranging from 2500 mg/mL to 1.22mg/mL were prepared in
medium. The inoculum was prepared using a 4–6-hour broth
cultureofeachbacteriatypeand24-hourcultureofyeaststrains
adjusted to a turbidity equivalent to 0.5 McFarland standard,
diluted in broth media to give a final concentration of
5 ꢃ 105 cfu/mL for bacteria and 5 ꢃ 103 cfu/mL for yeast in the
test tray. The trays were covered and placed into plastic bags to
prevent evaporation. The trays containing Mueller–Hinton
broth were incubated at 35°C for 18–20hours while the trays
containing RPMI-1640 medium were incubated at 35°C for 46–
50 hours. The MIC was defined as the lowest concentration of
compound giving complete inhibition of visible growth. As
control, antimicrobial effects of the solvents were investigated
against test microorganisms. According to values of the
controls, the results were evaluated. Amikacin and fluconazole
were used as reference antibiotics for bacteria and yeast,
respectively. The MIC values of the amikacin and fluconazole
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