Synthesis of new 7-amino-3,4-dihydroquinolin-2(1H)-one-peptide derivatives and their carbonic anhydrase enzyme inhibition, antioxidant, and cytotoxic activities

Article abstract of DOI:10.1002/ardp.202100122

Six new monopeptides, seven new dipeptides, and two deprotected monopeptide dihydroquinolinone conjugates were prepared by the benzothiazole-mediated method and their structures were confirmed by nuclear magnetic resonance, mass, infrared spectroscopy, and elemental analysis methods. The human carbonic anhydrase (hCA) I and hCA II enzyme inhibition activities of the compounds were determined using the stopped-flow instrument. The synthesized peptide–dihydroquinolinone conjugates 2, 3, 6, 10, 13, and 15 showed inhibition against the hCA II enzyme in the range of 15.7–65.7 μM. However, none of the compounds showed inhibition of hCA I at a concentration of 100 μM. The antioxidant activities of the compounds were also examined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method at concentrations of 12.5–125 μg/ml, but when compared with the standard antioxidant compounds α-tocopherol and butylated hydroxyanisole (BHA), weak antioxidant activities were detected. The cytotoxic effects of four compounds against the A549 and BEAS-2B cell lines were also investigated. Among the compounds studied, compound 7 was found to be most effective, with the IC₅₀ values on the A549 cells for 48 and 72 h being 26.87 and 9.979 μg/ml, respectively, and the IC₅₀ values on the BEAS-2B cells being >100 μg/ml. None of the tested compounds showed antimicrobial activity in the concentration range (800–1.56 μg/ml) studied.

Full text of DOI:10.1002/ardp.202100122

Received: 26 March 2021
Revised: 30 June 2021
Accepted: 1 July 2021
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DOI: 10.1002/ardp.202100122
F U L L P A P E R
Synthesis of new 7‐amino‐3,4‐dihydroquinolin‐2(1H)‐one‐
peptide derivatives and their carbonic anhydrase enzyme
inhibition, antioxidant, and cytotoxic activities
Hasan Küçükbay1
| Zeynep Gönül1 | Fatümetüzzehra Küçükbay2
|
Zehra Tekin2,3 | Andrea Angeli4 | Gianluca Bartolucci4 | Claudiu T. Supuran4
Eray Tatlıcı5 | Elif Apohan5 | Özfer Yeşilada5
|
1Department of Chemistry, Faculty of Arts
and Sciences, İnönü University, Malatya,
Abstract
Turkey
Six new monopeptides, seven new dipeptides, and two deprotected monopeptide
dihydroquinolinone conjugates were prepared by the benzothiazole‐mediated
method and their structures were confirmed by nuclear magnetic resonance,
mass, infrared spectroscopy, and elemental analysis methods. The human carbonic
anhydrase (hCA) I and hCA II enzyme inhibition activities of the compounds were
2Department of Basic Pharmaceutical
Sciences, Faculty of Pharmacy, İnönü
University, Malatya, Turkey
3Department of Basic Pharmaceutical
Sciences, Faculty of Pharmacy, Adıyaman
University, Adıyaman, Turkey
4Dipartimento Neurofarba, Sezione Di
Scienze Farmaceutiche e Nutraceutiche e
Laboratorio Di Chimica Bioinorganica,
Università Degli Studi Di Firenze, Florence,
Italy
determined
using
the
stopped‐flow
instrument.
The
synthesized
peptide–dihydroquinolinone conjugates 2, 3, 6, 10, 13, and 15 showed inhibition
against the hCA II enzyme in the range of 15.7–65.7 µM. However, none of the
compounds showed inhibition of hCA I at a concentration of 100 µM. The anti-
oxidant activities of the compounds were also examined using the DPPH (2,2‐
diphenyl‐1‐picrylhydrazyl) radical scavenging method at concentrations of
12.5–125 µg/ml, but when compared with the standard antioxidant compounds α‐
tocopherol and butylated hydroxyanisole (BHA), weak antioxidant activities were
detected. The cytotoxic effects of four compounds against the A549 and BEAS‐2B
cell lines were also investigated. Among the compounds studied, compound 7 was
found to be most effective, with the IC50 values on the A549 cells for 48 and 72 h
being 26.87 and 9.979 µg/ml, respectively, and the IC50 values on the BEAS‐2B cells
being >100 µg/ml. None of the tested compounds showed antimicrobial activity in
the concentration range (800–1.56 µg/ml) studied.
5Department of Biology, Faculty of Science,
İnönü University, Malatya, Turkey
Correspondence
Hasan Küçükbay, Department of Chemistry,
Faculty of Arts and Sciences, İnönü University,
44280 Malatya, Turkey.
Email: hasan.kucukbay@inonu.edu.tr
Claudiu T. Supuran, Dipartimento Neurofarba,
Sezione Di Scienze Farmaceutiche e
Nutraceutiche e Laboratorio Di Chimica
Bioinorganica, Università Degli Studi Di
Firenze, Sesto Fiorentino, 50019 Florence,
Italy.
Email: claudiu.supuran@unifi.it
K E Y W O R D S
antioxidant, carbonic anhydrase, cytotoxicity, peptide, peptide–dihydroquinolin‐2‐one
conjugates, quinolones
© 2021 Deutsche Pharmazeutische Gesellschaft
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Arch. Pharm. 2021;e2100122.
wileyonlinelibrary.com/journal/ardp
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https://doi.org/10.1002/ardp.202100122

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1
INTRODUCTION
human carbonic anhydrase I (hCA I) and II (hCA II) enzymes. The
cytotoxic activities of some mono‐ and dipeptide–dihydroquinolin‐
2‐one conjugates were also tested.
The first drug containing quinoline structure and possibly derived
from natural sources, quinine was isolated from cinchona tree
bark in 1820.[1] The effect of chloroquine, a quinoline derivative,
against the COVID‐19 virus has recently led to an increase in
interest in the synthesis of quinoline derivatives. Possible impacts
of quinoline derivatives such as chloroquine and hydroxy-
chloroquine on the COVID‐19 virus are reported to act by af-
fecting the viral receptor S protein to which the virus binds.[2]
Quinoline derivatives have a wide variety of biological activities
such as antimalarial,[3–5] antifungal,[6–9] antitumor,[9–13] anti-
tubercular,[13,14] HIV replication inhibitor,[15] antibacterial,[16,17]
antiparasitic,[18] antiviral,[19] and carbonic anhydrase enzyme in-
hibition[20–22] properties according to the substituents they con-
tain. For example, the antibiotic properties of quinolines were first
determined in the 1960s. Although they were initially active in a
narrow spectrum, they were transformed into broad‐spectrum
antibiotic‐effective compounds through structural modifica-
tions.[23] Heterocyclic compounds are generally compounds with
various physiological properties and are often found in the
structure of many natural compounds and drugs. For a long time,
we have been working on the synthesis and investigation of the
biological properties of compounds containing benzimida-
zole,[24,25] benzothiazole,[26] furan,[27] coumarin,[28] quinolone,[28]
quinine,[29] morpholine,[30] piperidine,[30] pyridine,[31] and isa-
tine.[32] Carbonic anhydrase is an enzyme found in red blood cells,
gastric mucosa, pancreatic cells, and renal tubules that catalyzes
the interconversion of carbon dioxide (CO2) and carbonic acid
(H2CO3). Carbonic anhydrase plays an important role in respira-
tion by affecting the transport of CO2 in the blood.[33] Therefore,
the synthesis of compounds that inhibit the carbonic anhydrase
enzyme is an important research topic. The zinc ion, which is in the
active center of carbonic anhydrase enzymes, plays a significant
role in the catalytic reaction. Therefore, the preparation of new
compounds with properties that can bind with the zinc ion in the
active center and that has excellent inhibition‐effectiveness is an
extremely important research area. The existence of a possible
relationship between the carbonic anhydrase enzyme and cancer
has added extra importance to these studies. Related to the most
commonly used artificial antioxidant substances BHT (2,6‐di‐tert‐
butyl‐4‐methylphenol), BHA (2‐tert‐butyl‐4‐methoxyphenol) and
propyl gallate (propyl 3,4,5‐trihydroxybenzoate) studies have in-
creased the suspicions of carcinogens about these compounds.
The emergence of new and biocompatible antioxidant substances
has led to an increase in the search. Within the framework of
these searches, it was aimed to investigate their antioxidant
properties by preparing compounds containing a series of new
peptide–dihydroquinolinone structures, inspired by peptides,
which are natural antioxidants.[34–36] In this study, we aimed to
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The mono‐ and dipeptide–quinolone derivatives reported in this
study were prepared in good yields from the reaction of 7‐amino‐3,4‐
dihydroquinolin‐2(1H)‐one with benzotriazole‐activated N‐protected
peptide under microwave heating conditions. Removal of the Boc
protecting group for compounds I,[21]II,[21] and 11 was done in a
mixture of trifluoroacetic acid (TFA) and dichloromethane (1:1) with
stirring at room temperature for 2 h according to the literature
method.[37]
The synthesis scheme of the N‐protected mono‐ and
dipeptide–quinolin‐2‐one derivatives is summarized in Scheme 1.
The structures of the newly synthesized compounds were elu-
cidated using spectroscopic methods and elemental analysis mea-
surements. In the nuclear magnetic resonance (NMR) spectra of the
compounds, the carbamate NH peak in the dihydroquinolinone ring
was generally shifted to the downfield in the range of 0.24–0.34 ppm
compared with the carbamate NH peak in the starting compound
(9.84 ppm). In compound 3 containing the amino acid L‐tryptophan,
the NH resonance at position 1 of the indole was observed at
10.58 ppm lower than the carbamate NH peaks. The observation of
carbamate NH resonances at 10.20 and 10.18 ppm and amide NH
resonances at 10.15 and 9.94 ppm in compound
4 containing
L‐glutamic acid with two dihydroquinolinone rings confirms the
structure. The amino resonance observed at 4.97 ppm in the starting
compound 7‐amino‐3,4‐dihydroquinolin‐2(1H)‐one was observed in
peptide–dihydroquinoline conjugates at around 10 ppm slightly
lower
than
in
carbamate
NH
resonances.
In
peptide–dihydroquinoline conjugates (1–13), the shift of this peak to
the downfield by about 5 ppm confirms that the amino group is
acylated. While the aliphatic methylene peaks in the quinoline ring of
the dihydroquinolinone derivatives resonated in the range of
2.82–2.85 ppm adjacent to the carbonyl group, the other methylene
resonated in the range of 2.44–2.47 ppm. The aliphatic methylene
carbons in the quinoline ring of the dihydroquinolinone derivatives
resonated between 31.1–34.1 ppm and 24.1–23.1 ppm, respectively.
The singlet peaks of the tert‐butoxy group of around 1.3 ppm dis-
appeared in compounds 14 and 15 in which the Boc protecting group
was removed. Similarly, the quaternary carbon peak at around
78 ppm and the carbamate carbonyl peak at around 156 ppm seen in
the 13C NMR spectra of compounds I and 11 disappeared in com-
pounds 14 and 15. After removing the Boc protecting group, the free
amino group formed was converted into trifluoroacetate salt by
taking a proton from TFA. This was confirmed by a broad singlet
peak corresponding to three protons at 8.3 ppm in the proton NMR
spectra of compounds 14 and 15 and their element analysis results.
prepare
mono‐
and
dipeptide
derivatives
containing
dihydroquinolin‐2‐one and to investigate their antioxidant capa-
cities, antimicrobial properties, and inhibition properties against

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SCHEME 1 Synthesis pathways of the new dihydroquinolinone–peptide conjugates. Conditions and reagents: (i) room temperature (rt), 2 h
in tetrahydrofuran (THF); (ii) rt, 12 h in H2CCl2; (iii) 70°C, 1 h in THF; and (iv) rt, 3 h
In compounds 14 and 15, carbon peaks belonging to the tri-
fluoroacetate group were seen as quartet in the carbon‐13 spectra at
around 160 and 116 ppm. All other protons and carbon peaks were
present in the spectra, confirming the proposed structures.
living organisms.[38] As many heterocyclic compounds exhibit CA
inhibitor properties,[39,40] we synthesized novel type mono‐ and
dipeptide–dihydroquinolinone conjugates to explore their possible
carbonic anhydrase enzyme inhibition capacities against human
carbonic anhydrase hCA I, II, IX, and XII. To investigate the inhibitory
capacity of all novel mono‐ and dipeptide–dihydroquinolin‐2‐one
conjugates (1–15) prepared, two human CA isoforms (hCA I and
hCA) were evaluated using a stopped‐flow CO2 hydrase assay. The
inhibition results of the compounds are presented in Table 1 to-
gether with the result of acetazolamide (AAZ) used as the standard
inhibitor. When the results in Table 1 are analyzed, the following
structure–activity relationships can be obtained. While none of the
newly synthesized peptide–dihydroquinolinone conjugates showed
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2.2
Biology
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2.2.1
Carbonic anhydrase inhibition
Among the biological activities, human carbonic anhydrase (hCA; EC
4.2.1.1) inhibition has been the subject of several investigations since
the discovery of the biological importance of this enzyme in several

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TABLE 1 Inhibition data of hCA I and hCA II, with compounds 1–15 and the standard sulfonamide inhibitor acetazolamide (AAZ) by a
stopped‐flow CO2 hydrase assay
Ki (µM)a
Compound no.
1
hCA I
hCA II
>100
>100
2
3
>100
>100
60.8
53.9
4
>100
>100
5
6
>100
>100
>100
32.5
7
8
9
>100
>100
>100
>100
>100
24.4
10
>100
15.7

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TABLE 1 (Continued)
Ki (µM)a
Compound no.
11
hCA I
hCA II
>100
>100
12
13
>100
>100
>100
23.8
14
>100
>100
15
>100
0.25
65.7
AAZ
0.012
aMean from three different assays, by a stopped‐flow technique (errors were in the range of 5%–10% of the reported values).
an inhibition effect against the hCA I enzyme at concentrations of
100 µM, compounds 2, 3, 6, 9, 10, 13, and 15 showed an inhibition
effect against the hCA II enzyme with Ki values in the range of
15.7–65.7 µM. According to the hCA II results, it is understood that
the amino acids methionine, phenylalanine, tryptophan, and iso-
leucine contribute to the hCA II enzyme inhibition. When the
structures of the compounds (2, 3, 6, 9, 10, 13, and 15) showing
inhibitory effects on hCA II were examined, dipeptide–
dihydroquinolin‐2‐one derivatives (6, 9, 10, and 13) were found to be
more effective than monopeptide–dihydroquinolin‐2‐one derivatives
(2, 3, and 15). When the Ca I and CA II enzyme inhibition results of
the deprotected compounds (14 and 15) were compared with their
initial states (I[] and 11), no change was observed for CA I enzyme,
whereas an increase in CA II enzyme inhibition of compound 15 was
observed. When the structures of the compounds showing the best
CA II inhibition are examined, it is seen that the most effective
compounds with Ki 15.7 and 23.8 µM (compounds 10 and 13,
respectively) are dipeptide–dihydroquinolin‐2‐one conjugates con-
taining the amino acids methionine and phenylalanine. The type of
protecting group in these compounds also did not make much of a
difference to their inhibition properties.
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2.2.2
Antioxidant testing
2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging activity
The antioxidant activity of the compounds was determined using
the widely used DPPH method.[41] The antioxidant activity re-
sults of mono‐ and dipeptide–dihydroquinolin‐2‐one compounds
are shown in Table 2. From Table 2, it is clear that the newly
synthesized mono‐ and dipeptide–dihydroquinolin‐2‐one con-
jugates do not show significant antioxidant activity. Although
peptide–dihydroquinolinone conjugates generally do not show
significant antioxidant activity, those that show antioxidant

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TABLE 2 Antioxidant activities of the synthesized mono‐ and dipeptide–quinolinone conjugates
DPPH free radical scavenging activity (%)
Compound no.
12.5 µg/ml
25 µg/ml
37.5 µg/ml
62.5 µg/ml
125 µg/ml
1
2.52
1.89
1.93
3.31
3.38
2
3
2.25
2.20
3.02
1.89
3.65
1.89
3.72
2.83
4.86
4.72
4
3.50
4.93
8.42
11.23
19.45
5
6
2.93
3.50
3.34
3.92
4.81
4.64
7.43
9.22
10.85
13.63
7
8
9
4.33
4.83
5.23
4.62
5.25
8.74
7.84
7.54
11.25
11.91
11.61
17.91
16.74
20.62
28.40
10
3.85
4.74
5.24
8.92
12.43

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TABLE 2 (Continued)
DPPH free radical scavenging activity (%)
Compound no.
11
12.5 µg/ml
25 µg/ml
37.5 µg/ml
62.5 µg/ml
125 µg/ml
2.81
2.93
2.94
3.25
3.83
12
13
2.74
2.20
3.13
1.57
3.04
1.57
3.42
1.57
3.85
1.89
14
15
9.75
8.80
10.13
10.01
11.47
12.19
13.77
13.56
18.41
17.15
BHA
61.1
62.9
63.0
63.4
67.5
68.4
71.0
72.8
72.4
74.0
α‐Tocopherol
Abbreviations: BHA, butylated hydroxyanisole; DPPH, 2,2‐diphenyl‐1‐picrylhydrazyl.
activity at a concentration of 125 µg/ml by about one‐third
compared with standard antioxidants are compounds 4, 8, 9, 14,
and 15. When the structures of the compounds showing relative
antioxidant activity are examined, it can be said that L‐aspartic
acid and methionine amino acids may be responsible for anti-
oxidant activity.
more cytotoxic effects on A549 cells compared with BEAS‐2B cells.
Compound 7 showed more cytotoxicity on A549 cells after 48 and 72 h
incubation. While the IC50 values of compound 7 on A549 cells for 48
and 72 h were 26.87 and 9.98 µg/ml, respectively, IC50 values on
BEAS‐2B cells were >100 µg/ml. Moreover, the IC50 values of compound
15 on A549 and BEAS‐2B cells for 72 h were found to be 13.94 and
60.95 µg/ml, respectively. When compounds 11 and 15 are compared, it
can be seen that protection group removal did not have a positive effect
on the cytotoxic effect at 24 and 48 h incubation times, but increased
cytotoxicity at 72 h. On the contrary, when compounds 11 and 15 were
compared in terms of their effects on healthy cells, it was observed that
removal of the protecting group did not contribute positively to cyto-
toxicity. The tested compounds (7, 10, 11, and 15) showed better cyto-
toxicity against A549 cell lines than the standard drug cisplatin during
the first 24 ‐h incubation period and less cytotoxicity in healthy cells
(BEAS‐2B) at 48 and 72 h incubation times.
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2.2.3
Cytotoxic activity
The cytotoxicity of some newly synthesized peptide–dihydroquinolinone
conjugates on A549 (lung cancer cells) and BEAS‐2B (healthy human lung
bronchial epithelium cells) cells was determined, and the IC50 values of
the compounds are presented in Table 3 with the standard drug cisplatin.
The compounds tested were generally more effective on the A549 than
on BEAS‐2B cells. As shown in Table 3, compounds 7, 11, and 15 had

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TABLE 3 IC50 (µg/ml) values of some peptide–dihydroquinolinone conjugates on A549 and BEAS‐2B cells
A549
Time (h)
BEAS‐2B
Compound no.
7
24
48
72
24
48
72
1.74
26.87
9.98
1.93
>100
>100
10
1.90
>100
>100
1.96
>100
>100
11
15
1.60
1.80
29.89
40.96
>100
13.94
1.75
1.94
>100
48.52
>100
60.95
Cisplatin
4.44
2.72
2.56
3.17
2.60
2.53
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2.2.4
Antimicrobial activity
showed antimicrobial activity in the concentration range
(800–1.56 µg/ml) studied.
The antimicrobial activity of 16 new peptide–dihydroquinolinone
conjugates against bacteria and yeasts was tested using microdilu-
tion and disc diffusion methods. However, these newly synthesized
compounds 1–15 had no antimicrobial activity against any of the
microorganisms used according to these methods.
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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
|
3
CONCLUSIONS
All solvents and reagents were purchased from Sigma‐Aldrich,
Acros, Novabiochem, Merck, Isolab, and Alkomed. All reactions
were synthesized under normal atmospheric conditions. 1H NMR
(400 MHz) and 13C NMR (100 MHz) spectra (see the Supporting
Information) were recorded using a Bruker Advance III 400 or
300 MHz spectrometer in dimethyl sulfoxide (DMSO)‐d6. The as-
signment of exchangeable protons (OH and NH) was confirmed by
the addition of D2O. Positive‐ion electrospray ionization (ESI)
mass spectra were recorded on a double‐focusing Finnigan MAT
95 instrument with BE geometry. Infrared spectra were recorded
with the ATR equipment in the range 4000–650 cm−1 on a Per-
kinElmer Spectrum one FTIR spectrophotometer. Micro‐analyses
were performed using a LECO CHNS‐932 elemental analyzer.
In this study, 15 new peptide–dihydroquinolinone conjugates were
synthesized under benzotriazole‐mediated mild reaction conditions
and their structures were determined using spectrometric and ana-
lytical methods. Among them, compounds 2, 3, 6, 10, 13, and 15
showed an inhibition effect against hCA II in the range of
15.7–65.7 µM. None of the compounds showed inhibition against the
hCA I enzyme at a concentration of 100 µM. The antioxidant prop-
erties of the synthesized compounds were examined using the DPPH
method in the concentration range of 12.5–125 µg/ml, but the
compounds were found to have weak antioxidant activity. Com-
pound 7, the methionine–dihydroquinolinone derivative with the Cbz
protecting group, was found to be most effective among the two
benzyloxycarbonyls, a tert‐butoxycarbonyl protecting group and de-
protected peptide–dihydroquinolinone conjugates tested for cyto-
toxic activity. The IC50 values of compound 7 on A549 cells for 48
and 72 h were 26.87 and 9.979 µg/ml, respectively, and IC50 values
on BEAS‐2B cells were >100 µg/ml. None of the tested compounds
Melting points were recorded in
a capillary tube using an
Electrothermal‐9200 melting point apparatus, and are un-
corrected. All dihydroquinolinone–peptide derivatives synthesized
by microwave heating were obtained in a microwave synthesis
instrument manufactured by Milestone Start S. All initial

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N‐protected mono‐ and dipeptides used in this study were pre-
pared according to literature methods.[42,43]
(d, 1H, NH, J = 8 Hz), 7.70 (d, 1H, Ar–H, J = 8 Hz), 7.36–6.97 (m, 1H,
Ar–H), 5.01–4.94 (m, 1H, CH2O), 5.07–4.84 (m, 2H, Ar–H), 4.50–4.44
(s, H, NHCH), 3.13–3.12 and 3.03–3.01 (2 m, 2H, CH2‐diasterotopic),
2.82 (t, 2H, COCH2CH2, J = 8 Hz), 2.43 (t, 2H, COCH2CH2, J = 8 Hz).
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information.
13C NMR (DMSO‐d6, 400 MHz)
δ 171.0 (CH2CONH), 170.8
(NHCOCH), 156.6 (COOCH2Ph), 138.9, 138.3, 137.4, 136.5, 128.8,
128.2, 128.1, 127.7, 124.4, 121.4, 119.1, 119.0, 118.7, 113.6, 111.8,
110.3, 107.0 (Ar–C), 65.8 (OCH2Ph), 56.5 (CHNH), 31.1 (CH2‐indole),
28.3 (COCH2CH2), 24.8 (COCH2CH2). Elemental analysis:
|
4.1.2
General procedure for the synthesis of
quinolinone–peptide conjugates 1–13
A mixture of the appropriate N‐protected aminoacylbenzotriazole
and equivalent amounts of 7‐amino‐3,4‐dihydroquinolin‐2(1H)‐one
was subjected to microwave irradiation (100 W, 70°C) for 1 h in
anhydrous tetrahydrofuran. After the completion of the reaction, all
volatiles were removed by a rotavapor and the resulting crude
product was crystallized from methanol.
C28H26N4O4 required C, 69.70; H, 5.43; N, 11.61; found C, 69. 43; H,
5.41; N, 11.37. HRMS m/z for C28H26N4O4 [M+Na]+ calcd. 506.1,
found 505.2; [M+K]+ calcd. 521.2, found 523.2.
Benzyl {(S)‐1,5‐dioxo‐1,5‐bis[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐7‐
yl)amino]pentan‐2‐yl}carbamate (4)
Beige solid (87%); m.p. 211–212°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.20 and 10.18 (2 s, 2H, NHlactam), 10.15 and 9.94 (2 s, 2H, NHamide),
7.94 (d, 1H, NH, J = 4 Hz), 7.65 (m, 4H, Ar–H), 7.49 (s, 1H, Ar–H), 7.40
(m, 2H, Ar–H), 7.39–7.33 (m, 2H, Ar–H), 7.32–7.25 (s, 1H, Ar–H),
7.22–7.04 (s, 1H, Ar–H), 5.06 (s, 2H, OCH2Ph), 4.34–4.03 (m, 1H,
CHNH), 2.80 (t, 4H, COCH2CH2, J = 8 Hz), 2.50–2.29 (m, 6H,
COCH2CH2 and COCH2CH2CH), 2.10–2.05 and 2.04–1.92 (2 m, 2H,
COCH2CH2CH). 13C NMR (DMSO‐d6, 400 MHz) δ 170.9 and 170.8
(COCH2), 170.2 and 170.0 (CONH), 156.4 (COOCH2Ph), 148.3,
138.9, 138.8, 138.7, 138.3, 137.4, 128.8, 128.3, 128.1, 119.0, 118.6,
113.2, 108.4, 106.8, 106.6, 101.3 (Ar–C), 65.9 (CH2Ph), 55.4 (CHNH),
33.2 (COCH2CH2), 31.7 (COCH2CH2), 31.1 (CHCH2), 28.0
(COCH2CH2), 24.7 (COCH2CH2), 24.5 (COCH2CH2CH). Elemental
analysis: C31H31N5O6 required C, 65.37; H, 5.49; N, 12.30; found C,
65. 12; H, 5.46; N, 11.97. HRMS m/z for C31H31N5O6 [M+H]+ calcd.
570.2, found 570.2; [M+Na]+ calcd. 592.2, found 592.1; [M−H]− calcd.
568.2, found 568.2; [M+Cl]− calcd. 604.2, found 604.1.
Benzyl {(R)‐3‐methyl‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐7‐
yl)amino]butan‐2‐yl}carbamate (1)
White solid (78%); m.p. 225–226°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.13 (s, 1H, NHlactam), 10.01 (s, 1H, NH), 7.49 (d, 1H, NH, J = 8 Hz),
7.38–7.06 (m, 7H, Ar–H), 5.05 (s, 2H, CH2Ph), 3.99 (t, 1H, CHNH,
J = 8 Hz), 2.81 (t, 2H, COCH2CH2, J = 8 Hz), 2.42 (t, 2H, COCH2CH2,
J = 8 Hz), 2.01–1.97 (m, 1H, CH(CH3)2), 0.90 (d, 6H, CH3, J = 8 Hz).
13C NMR (DMSO‐d6, 400 MHz)
δ 171.7 (NHCOCH2), 171.0
(NH–C═O), 156.7 (COOCH2Ph), 138.9, 138.2, 137.5, 128.8, 128.3,
128.2, 128.1, 119.0, 113.3, 106.7 (Ar–C), 65.9 (OCH2Ph), 61.4
(CHNH), 31.1 (COCH2CH2), 30.7 (COCH2CH2), 24.7 (CH(CH3)2), 19.6
and 18.9 (CH3)2CH). Elemental analysis: C22H25N3O4 required C,
66.82; H, 6.37; N, 10.63; found C, 66.72; H, 6.35; N, 10.59. HRMS m/z
for C22H25N3O4 [M+H]+ calcd. 396.2, found 396.2; [M+Na]+ calcd.
418.2, found 418.1; [M+Cl]− calcd. 430.2, found 430.1.
Benzyl {(2R,3R)‐3‐methyl‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]pentan‐2‐yl}carbamate (2)
Benzyl [2‐oxo‐2‐({2‐oxo‐2‐[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐7‐
yl)amino]ethyl}amino)ethyl]carbamate (5)
Beige solid (91%); m.p. 142–143°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.13 (s, 1H, NHlactam), 10.03 (s, 1H, NH), 7.52 (d, 1H, NH, J = 12 Hz),
7.37–7.06 (m, 8H, Ar–H), 5.04 (s, 2H, CH2NH), 4.04–4.00 (m, 1H,
NHCH), 2.81 (t, 2H, COCH2CH2, J = 8 Hz), 2.42 (t, 2H, COCH2CH2,
J = 8 Hz), 1.78–1.76 (m, 1H, CHCH3), 1.51–1.49 and 1.20–1.16 (2 m,
2H, CH3CH2), 0.85–0.81 (m, 6H, CH3CH2 + CH3). 13C NMR (DMSO‐
d6, 400 MHz) δ 171.0 (COCH2), 170.8 (COCH), 156.6 (COOCH2Ph),
138.9, 138.2, 137.5, 128.8, 128.4, 128.2, 120.8, 199.0, 113.4, 106.8
(Ar–C), 65.8 (OCH2Ph), 60.2 (CHNH), 36.7 (COCH2CH2), 31.1
(COCH2CH2), 25.0 (CHCH3), 24.8 (CH3CH2), 15.7 (CH2CH3), 11.2
(CH3CH). Elemental analysis: C23H27N3O4 required C, 67.46; H, 6.65;
N, 10.26; found C, 67. 02; H, 6.51; N, 9.97. HRMS m/z for
White solid (74%); m.p. 235–236°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.14 (s, 1H, NHlactam), 9.85 (s, 1H, NH), 8.22 (t, 1H, NH, J = 6 Hz),
7.56 (t, 1H, NH, J = 6 Hz), 7.38–7.07 (m, 8 H, Ar–H), 5.06 (s, 2H,
CH2Ph), 3.89 (d, 2H, CH2NH, J = 6 Hz), 3.66 (d, 2H, CH2NH, J = 6 Hz),
2.81 (t, 2H, COCH2CH2, J = 8 Hz), 2.42 (t, 2H, COCH2CH2, J = 8 Hz).
13C NMR (DMSO‐d6, 400 MHz)
δ 170.7 (COCH2CH2), 170.0
(COCH2), 167.8 (COCH2), 157.8 (COOCH2Ph), 138.9, 138.2, 137.4,
128.8, 128.3, 128.2, 118.9, 113.3, 106.7 (Ar–C), 66.0 (CH2Ph), 44.0
(CHNH), 43.0 (CHNH), 31.1 (OCH2CH2), 24.7 (COCHCH2). Elemental
analysis: C21H22N4O5 required C, 61.45; H, 5.40; N, 13.65; found C,
61. 42; H, 5.38; N, 13.59.
C
23H27N3O4 [M+H]+ calcd. 410.2, found 410.1; [M+Na]+ calcd. 432.2,
found 432.1; [M+Cl]− calcd. 444.2, found 444.1.
Benzyl [(S)‐1‐oxo‐1‐({(R)‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]‐3‐phenylpropan‐2‐yl}amino)propan‐
2‐yl]carbamate (6)
White solid (62%); m.p. 178–179°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.18 (s, 1H, NHlactam), 10.00 (s, 1H, NH), 8.13 (d, 1H, NH, J = 8 Hz),
7.41–7.13 (m, 13 H, Ar–H + NH), 5.08 (d, 2H, CH2Ph), 4.71 (t, 1H,
Benzyl {(S)‐3‐(1H‐indol‐3‐yl)‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]propan‐2‐yl}carbamate (3)
Yellow solid (89%); m.p. 119–120°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.58 (s, 1H, NHindole), 10.15 (s, 1H, NHlactam), 10.13 (s, 1H, NH), 7.70

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CHNH, J = 6 Hz), 4.10 (t, 1H, CHNH, J = 6 Hz), 3.12–3.00 and
2.97–2.94 (2 m, 2H, CH2Ph), 2.86 (t, 2H, COCH2CH2, J = 8 Hz), 2.47
(t, 2H, COCH2CH2, J = 8 Hz), 1.20 (d, 3H, CH3, J = 8 Hz). 13C NMR
7.57–7.09 (m, 9H, Ar–H + NH), 5.04 (s, 2H, CH2Ph), 4.47–4.46 (m, 1H,
CHNH), 4.17–4.12 (m, 1H, CHNH), 2.81 (t, 2H, COCH2, J = 8 Hz),
2.44–2.06 (m, 6H, COCH2CH2 + CH2SCH3), 2.03 (s, 6H, CH3),
1.91–1.80 (m, 4H, CH2CH2S). 13C NMR (DMSO‐d6, 400 MHz) δ 172.1
(COCH2), 170.8 (COCH), 170.3 (COCH), 156.5 (COOCH2Ph), 138.9,
138.2, 137.4, 128.8, 128.3, 128.2, 119.1, 113.4, 108.4, 106.8 (Ar–C),
65.9 (CH2Ph), 54.3 (CHNH), 53.2 (CHNH), 32.4 (COCH2CH2), 32.1
(COCHCH2), 31.7 (CHCH2CH2), 31.1 (CHCH2CH2), 30.1 (SCH3), 24.8
(SCH3), 15.2 (CH2CH2S), 15.1 (CH2CH2S). Elemental analysis:
(DMSO‐d6, 400 MHz)
δ 172.8 (COCH2), 170.7 (COCH), 170.1
(COCH), 156.1 (COOCH2Ph), 138.9, 138.1, 137.9, 137.4, 129.7,
128.8, 128.5, 128.3, 128.2, 128.1, 126.9, 119.2, 113.5, 108.9 (Ar–C),
65.9 (CH2Ph), 54.9 (CHNH), 50.6 (CHNH), 38.1 (CHCH2Ph), 31.0
(COCH2CH2), 24.7 (COCH2CH2), 18.5 (CHCH3). Elemental analysis:
C
29H30N4O5 required C, 67.69; H, 5.88; N, 10.89; found C, 67.54; H,
5.83; N, 10.62. HRMS m/z for C29H30N4O5 [M+H]+ calcd. 515.2,
C27H34N4O5S2 required C, 58.04; H, 6.13; N, 10.03; S, 11.48; found
found 515.2; [M+Cl]− calcd. 549.2, found 549.1.
C, 57. 92; H, 6.01; N, 9.93; S, 11.39. HRMS m/z for C27H34N4O5S2 [M
+H]+ calcd. 559.2, found 559.2; [M+Cl]− calcd. 553.2, found 553.1.
Benzyl [(S)‐4‐(methylthio)‐1‐oxo‐1‐({2‐oxo‐2‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]ethyl}amino)butan‐2‐yl]‐
Benzyl [(S)‐4‐(methylthio)‐1‐oxo‐1‐({(S)‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]‐3‐phenylpropan‐2‐yl}amino)butan‐2‐
yl]carbamate (10)
carbamate (7)
Beige solid (75%); m.p. 158–159°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.13 (s, 1H, NHlactam), 9.82 (s, 1H, NH), 8.28 (t, 1H, NH, J = 6 Hz), 7.92
(bs, 1H, Ar–H), 7.62 (d, 1H, NH, J = 8 Hz), 7.38–7.31 (m, 5H, Ar–H), 7.26 (s,
1H, Ar–H), 7.08 (s, 2H, Ar–H), 5.04 (s, 2H, CH2Ph), 4.17–4.13 (m, 1H,
CHNH), 3.91–3.85 (m, 2H, CH2NH), 2.81 (t, 2H, COCH2CH2, J = 8 Hz),
2.44–2.40 (m, 4H, COCH2CH2), 2.05 (s, 3H, CH3), 1.96–1.92 and
1.86–1.81 (2 m, 2H, CH2CH2S). 13C NMR (DMSO‐d6, 400 MHz) δ 170.4
(NHCOCH2), 170.3 (COCH), 167.8 (COCH), 156.4 (COOCH2Ph), 148.2,
139.0, 137.3, 128.8, 128.2, 118.8, 113.2, 108.3, 106.4, 101.4 (Ar–C), 63.4
(CH2Ph), 54.3 (CHNH), 43.0 (CHNH), 31.0 (CHCH2), 30.1 (COCH2CH2),
24.8 (COCHCH2), 15.6 (CH2CH2), 15.1 (CH3). Elemental analysis:
Beige solid (67%); m.p. 216–217°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.17 (s, 1H, NHlactam), 10.04 (s, 1H, NH), 8.19 (d, 1H, NH, J = 8 Hz),
7.56 (d, 1H, NH, J = 8 Hz), 7.42–7.14 (m, 13 H, Ar–H), 5.09 (d, 2H,
CH2Ph, J = 4 Hz), 4.73–4.72 (m, 1H, CHNH), 4.17–4.11 (m, 1H,
CHNH), 3.12–3.09 and 2.99–2.94 (2 m, 2H, CH2Ph), 2.86 (t, 2H,
COCH2CH2, J = 8 Hz), 2.49–2.41 (m, 4H, COCH2CH2, and SCH2),
2.06 (s, 3H, CH3), 1.87–1.78 (m, 2H, SCH2CH2). 13C NMR (DMSO‐d6,
400 MHz) δ 171.8 (NHCOCH2), 170.7 (COCH), 170.1 (COCH), 156.4
(COOCH2Ph), 139.0, 138.2, 137.8, 137.4, 129.7, 128.8, 128.5, 128.3,
128.2, 126.9, 119.2, 113.4, 106.9 (Ar–C), 66.0 (CH2Ph), 55.0 (CHNH),
54.4 (CHNH), 38.1 (CHCH2), 32.2 (COCH2CH2), 31.0 (COCH2CH2),
30.0 (SCH2), 24.8 (SCH3), 15.1 (SCH2CH2). Elemental analysis:
C24H28N4O5S required C, 59.49; H, 5.82; N, 11.56; S, 6.62; found C, 59.
32; H, 5.81; N, 11.47; S, 6.51. HRMS m/z for C24H28N4O5S [M+H]+ calcd.
485.2, found 485.1; [M+Cl]− calcd. 519.2, found 519.1.
C31H34N4O5S required C, 64.79; H, 5.96; N, 9.75; S, 5.58; found C,
64.72; H, 5.91; N, 9.67; S, 5.51. HRMS m/z for C31H34N4O5S [M+H]+
calcd. 575.2, found 575.2; [M+Na]+ calcd. 597.2, found 597.2; [M
+Cl]− calcd. 609.2, found 609.2.
Benzyl [(S)‐4‐(methylthio)‐1‐oxo‐1‐({(S)‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]propan‐2‐yl}amino)butan‐2‐yl]‐
carbamate (8)
Beige solid (72%); m.p. 202–203°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.17 (s, 1H, NHlactam), 9.97 (s, 1H NH), 8.21 (d, 1H, N–H, J = 8 Hz),
7.63–7.11 (m, 9H, Ar–H + NH), 5.09 (s, 2H, CH2Ph), 4.47–4.42 (m, 1H,
CHNH), 4.21–4.17 (m, 1H, CHNH), 2.85 (t, 2H, COCH2CH2, J = 8 Hz),
2.46 (t, 2H, COCH2CH2, J = 8 Hz), 2.09 (s, 3H, CH3), 1.94–1.85 (2 m,
2H, CHCH2CH2), 1.36–1.33 (m, 3H, CHCH3). 13C NMR (DMSO‐d6,
400 MHz) δ 171.3 (COCH2), 170.7 (COCH), 156.4 (COOCH2Ph),
138.9, 138.4, 137.4, 128.8, 128.2, 118.9, 113.2, 106.6 (Ar–C), 65.9
(CH2Ph), 54.7 (CHNH), 54.2 (CHNH), 49.4 (CH2CH2), 31.3
(COCH2CH2), 30.1 (COCHCH2), 24.7 (SCH3), 18.5 (CH2CH2S), 15.1
(CHCH3). Elemental analysis: C25H30N4O5S required C, 60.22; H, 6.07;
N, 11.24; S, 6.43; found C, 59. 97; H, 6.06; N, 11.22; S, 6.23. HRMS m/z
for C25H30N4O5S [M+H]+ calcd. 499.2, found 499.1; [M+Na]+ calcd.
521.2, found 521.1; [M+Cl]− calcd. 533.2, found 533.1.
tert‐Butyl (S)‐{3‐methyl‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐
7‐yl)amino]butan‐2‐yl}carbamate (11)
White solid (84%); m.p. 217–218°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.12 (s, 1H, NHlactam), 9.92 (s, 1H, NH), 7.24 (s, 1H, Ar–H), 7.23–7.06
(m, 2H, Ar–H), 6.85 (d, 1H, NH, J = 12 Hz), 3.92–3.88 (m, 1H, CHNH),
2.80 (t, 2H, COCH2CH2, J = 8 Hz), 2.40 (t, 2H, COCH2CH2, J = 8 Hz),
1.99–1.92 (m, 1H, CH(CH3)2), 1.39 (s, 9H, (CH3)3C), 0.89 (d, 6H,
(CH3)2CH). 13C NMR (DMSO‐d6, 400 MHz) δ 171.0 (NHCOCH2),
170.7 (CHCONH), 155.9 (COOCH2Ph), 138.9, 138.2, 128.2, 118.9,
113.3, 106.7 (Ar–C), 78.4 (CO(CH3)3), 60.9 (CHNH), 31.1
(COCH2CH2), 30.8 (COCH2CH2), 28.7 [(CH3)2CHCH], 24.8 [CH
(CH3)3], 19.6 and 19.0 [(CH3)2CH]. Elemental analysis: C19H27N3O4
required C, 63.14; H, 7.53; N, 11.63; found C, 62. 72; H, 7.47;
N, 11.52.
Benzyl [(S)‐4‐(methylthio)‐1‐({(S)‐4‐(methylthio)‐1‐oxo‐1‐[(2‐oxo‐
1,2,3,4‐tetrahydroquinolin‐7‐yl)amino]butan‐2‐yl}amino)‐1‐
oxobutan‐2‐yl]carbamate (9)
Beige solid (72%); m.p. 202–203°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.13 (s, 1H, NHlactam), 9.98 (s, 1H, NH), 8.19 (d, 1H, NH, J = 8 Hz),
tert‐Butyl {(2R,3R)‐3‐methyl‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]pentan‐2‐yl}carbamate (12)
White solid (83%); m.p. 199–200°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.12 (s, 1H, NHlactam), 9.93 (s, 1H, NH), 7.24 (s, 1H, Ar–H), 7.24–7.06
(m, 2H, Ar–H), 6.89 (d, 1H, NH, J = 8 Hz), 3.94 (t, 1H, CHCHNH,

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J = 8 Hz), 2.80 (t, 2H, COCH2CH2, J = 8 Hz), 2.42 (t, 2H, COCH2CH2,
J = 8 Hz), 1.74–1.45 and 1.47–1.45 (m, 2H, CH3CH2CH), 1.39 (s, 9H,
(CH3)3C), 1.16–1.13 (m, 1H, CH3CH2CH), 0.85–0.81 (m, 6H,
(CH3)2CH). 13C NMR (DMSO‐d6, 400 MHz) δ 171.1 (NHCOCH2),
170.7 (CHNHCO), 155.9 (COOC(CH3)3), 138.9, 138.2, 128.2, 118.9,
113.3, 106.7 (Ar–C), 78.5 (CO(CH3)3), 59.8 (CHNH), 36.8
(COCH2CH2), 31.1 (COCH2CH2), 28.7 (CHCH2CH3), 25.0 (C(CH3)3),
24.8 (CHCH2CH3), 15.8 (CH3), 11.2 (CH3). Elemental analysis:
(S)‐3‐Methyl‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐7‐yl)‐
amino]butan‐2‐aminium 2,2,2‐trifluoroacetate (15)
Cream solid (72%); m.p. 81–82°C; 1H NMR (400 MHz, DMSO) δ
10.49 (s, 1H, NHlactam), 10.20 (s, 1H, NH), 8.31 (s, 3H, NH3+),
7.23–7.16 (m, 2H, Ar–H), 6.73 (s, 1H, Ar–H), 3.80–3.73 (m, 1H,
NHCH), 2.84 (t, 2H, COCH2CH2, J = 8 Hz), 2.44 (t, 2H, COCH2CH2,
J = 78 Hz (t, J = 78 Hz), 2.20–2.12 (m, 1H, [(CH3)2CH]), 0.98 (d, 6H,
[(CH3)2CH, J = 4.0 Hz). 13C NMR (101 MHz, DMSO)
δ 170.8
2
C
20H29N3O4 required C, 63.98; H, 7.79; N, 11.19; found C, 62.72; H,
(NHCOCH2), 167.2 (COCH), 159.4 (F3CCO, q, J(C–F) = 30 Hz), 139.7,
1
7.77; N, 11.07. HRMS m/z for C20H29N3O4 [M+Na]+ calcd. 398.2,
found 398.1; [M−H]− calcd. 374.2, found 374.1; [M+Cl]− calcd. 410.2,
found 410.1.
134.6, 129.2, 121.3, 117.7 (q, CF3CO, J(C–F) = 256.7 Hz), 115.0,
108.3 (Ar–C), 58.3 (CHNH), 30.8 (COCH2CH2), 30.4 (COCH2CH2),
24.7 [CHCH(CH3)2], 18.8 and 18.2 [CHCH(CH3)2]. Elemental analysis:
C14H16F3N3O4 required C, 48.12; H, 4.64; N, 12.10; found C, 48.02
tert‐Butyl [(R)‐1‐({(R)‐4‐(methylthio)‐1‐oxo‐1‐[(2‐oxo‐1,2,3,4‐
tetrahydroquinolin‐7‐yl)amino]butan‐2‐yl}amino)‐1‐oxo‐3‐
phenylpropan‐2‐yl]carbamate (13)
12; H, 4.63; N, 11.98.
White solid (80%); m.p. 197–198°C; 1H NMR (400 MHz, DMSO‐d6) δ
10.19 (s, 1H, NHlactam), 10.06 (s, 1H, NH), 8.21 (d, 1H, NH, J = 8 Hz),
7.36–7.02 (m, 8H, Ar–H + NH), 4.59–5.56 (m, 1H, CHNH), 4.30–4.24 (m,
1H, CHNH), 3.07–3.03 (m, 1H, CH2Ph), 2.88–2.85 (m, 2H,
COCH2CH2 + CH2Ph), 2.50–2.46 (m, 4H, COCH2CH2 + CHCH2CH2S),
2.12 (s, 5H,CH3), 2.04–1.95 (m, 2H, CHCH2CH2S), 1.36 (s, 9H, OC(CH3)3).
13C NMR (DMSO‐d6, 400 MHz) δ 172.3 (NHCOCH2), 170.8 (COCH),
170.2 (COCH), 155.8 (COOC(CH3)3), 138.9, 138.6, 138.2, 129.7, 128.5,
128.2, 126.6, 119.2, 113.5, 106.9 (Ar–C), 78.6 (OCH(CH3)3), 56.2 (CHNH),
53.2 (CHNH), 32.6 (COCH2CH2), 31.1 (CHCH2), 30.9 (CHCH2CH2S), 29.9
(COCH2CH2), 28.6 (SCH3), 24.8 (CHCH2CH2S), 15.1 (OCH(CH3)3). Ele-
mental analysis: C28H36N4O5S required C, 62.20; H, 6.71; N, 10.36; S,
5.93; found C, 62. 12; H, 6.70; N, 10.27; S, 5.83.
4.2
Biological assays
|
|
4.2.1
CA Inhibition
An Applied Photophysics Stopped‐Flow instrument was used for
assaying the CA‐catalyzed CO2 hydration activity using the
method of Khalifah.[44] Phenol red (at a concentration of 0.2 mM)
has been used as an indicator, working at the absorbance max-
imum of 557 nm, with 20 mM HEPES (pH 7.5) as buffer, and
20 mM Na2SO4 (for maintaining constant ionic strength), following
the initial rates of the CA‐catalyzed CO2 hydration reaction for a
period of 10–100 s. The CO2 concentrations ranged from 1.7 to
17 mM for the determination of the kinetic parameters and in-
hibition constants. For each inhibitor, at least six traces of the
initial 5–10% of the reaction have been used to determine
the initial velocity. The uncatalyzed rates were determined in the
same manner and subtracted from the total observed rates.
Stock solutions of inhibitor (0.1 mM) were prepared in
distilled–deionized water, and dilutions up to 0.01 nM were per-
formed thereafter with the assay buffer. Inhibitor and enzyme
solutions were pre‐incubated together for 15 min at room tem-
perature before the assay, to allow for the formation of the E–I
complex. The inhibition constants were obtained by nonlinear
least‐squares methods using PRISM (www.graphpad.com), and
nonlinear least‐squares methods, values representing the mean of
at least three different determinations, as described earlier by
us.[45–47]
|
4.1.3
General procedure to remove the Boc
protecting group
An appropriate Boc protected compound (0.15 mmol) was dissolved
in a TFA/dichloromethane mixture (1.5/1.5 ml) and stirred for 2 h at
room temperature according to the literature method.[37] The reac-
tion mixture was concentrated at reduced pressure, and the excess
of diethyl ether was added to afford the desired cream powder. This
powder was filtered and air‐dried.
(R)‐1‐Oxo‐1‐[(2‐oxo‐1,2,3,4‐tetrahydroquinolin‐7‐yl)amino]propan‐
2‐aminium 2,2,2‐trifluoroacetate (14)
Cream solid (86%); m.p. 239–240°C; 1H NMR (400 MHz, DMSO) δ
10.46 (s, 1H, NHlactam), 10.22 (s, 1H, NH), 8.29 (s, 3H, NH3+), 7.23 (s,
1H, Ar–H), 7.12 (s, 1H, Ar–H), 4.06–3.97 (m, 1H, CH3CH), 2.82 (t, 2H,
COCH2CH2, J = 8 Hz), 2.43 (t, 2H, COCH2CH2, J = 7.5 Hz (t,
J = 78 Hz), 1.44 (d, 3H, CH3, J = 7.0 Hz). 13C NMR (101 MHz, DMSO)
δ 170.8 (NHCOCH2), 168.5 (COCH), 158.8 (F3CCO, q, 2J(C–F) = 30 Hz),
139.1, 137.6, 128.5, 119.7, 117.6 (q, CF3CO, 1J(C–F) = 296 Hz), 113.4,
106.9 (Ar–C), 49.4 (CHCH3), 31.0 (COCH2CH2), 24.8 (COCH2CH2),
17.6 (CH3). Elemental analysis: C14H16F3N3O4 required C, 48.12; H,
4.64; N, 12.10; found C, 48.02 12; H, 4.63; N, 11.98.
|
4.2.2
Antioxidant testing
DPPH radical scavenging activity
Antioxidant activities of the compounds were determined based on
the ability of the antioxidants to act as radical scavengers toward the
stable free radical, 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH). As detailed
by Yang et al.,[41] 1 ml of antioxidant solution (solubilized in ethanol)
was added to 3 ml of a 0.1 mM ethanolic solution of DPPH. After

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|
30 min at ambient temperature in darkness, absorbance readings
were taken at 517 nm. Inhibition (%) was calculated using the fol-
lowing equation:
Sabouraud dextrose agar were used for bacteria and yeasts, re-
spectively. The suspensions of the microbial cultures at 0.5
McFarland were spread on agar plates using cotton swabs. Then,
the filter paper discs containing 30 μl compounds were placed on
agar surfaces and these cultures were incubated for 24 and 48 h at
37°C for bacteria and yeasts, respectively. The diameter of the
inhibition of growth zones shows the antimicrobial activity of the
compounds.[49,50]
[1 − (As − Ao)/Ab] × 100,
where As is the absorbance reading for samples containing the an-
tioxidant, Ao is the absorbance of the antioxidant in pure methanol,
and Ab corresponds to the absorbance of the DPPH solution.
ACKNOWLEDGMENT
|
4.2.3
Cytotoxic activity
This study was financially supported by the İnönü University
Research Fund (FYL‐2018‐909).
Stock solutions of the peptide–dihydroquinolinone conjugates
were prepared in DMSO and then, further dilutions were made
with a fresh culture medium. The concentration of DMSO in the
final culture medium was <0.1%. A549 and BEAS‐2B cell lines
were plated in 96‐well plates (5 × 103 cells/well) for 24 h. After the
cells attached to the surface of the plate, the tested
peptide–dihydroquinolinone conjugates were added to the wells
to obtain the final concentrations in the range of 0–100 µg/ml.
Then, the cells were incubated at 37°C in a humidified incubator
with 5% CO2 for 24, 48, and 72 h. Their cytotoxic effects against
the cells were determined using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐
2,5‐diphenyl tetrazolium bromide (MTT) cell proliferation assay
according to the methodology previously described by our
group.[48] Twelve wells were used for every concentration, and
IC50 values (µg/ml) were defined as the compound concentrations
reducing absorbance to 50% of the control values.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Hasan Küçükbay
https://orcid.org/0000-0002-7180-9486
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