Study of the anti(myco)bacterial and antitumor activities of prolinate and N-amidocarbothiolprolinate derivatives based on fused tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione, bearing an indole ring

Article abstract of DOI:10.1007/s00706-018-2286-8

Abstract: A series of prolinate and N-amidocarbothiolprolinate derivatives based on a fused pyrrole-3,4-dione core, bearing indole ring systems, are prepared from the corresponding amino acid and an aldehyde via thermal 1,3-dipolar cycloaddition of azomet

Full text of DOI:10.1007/s00706-018-2286-8

Monatshefte für Chemie - Chemical Monthly
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ORIGINAL PAPER
Study of the anti(myco)bacterial and antitumor activities of prolinate
and N-amidocarbothiolprolinate derivatives based on fused
tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-dione, bearing an indole
ring
Samet Poyraz1 • Necmiye Canacankatan2 • Samet Belveren Derya Yetkin3 • Kezban Kibar3 •
1 •
4 •
5
6
•
3 •
1
¨
•
¨
Mahmut Ulger Jos e´ M. Sansano Nefise Dilek Ozcelik S¸ . Necat Yılmaz H. Ali D o¨ nda s¸
Received: 6 July 2018 / Accepted: 19 August 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
A series of prolinate and N-amidocarbothiolprolinate derivatives based on a fused pyrrole-3,4-dione core, bearing indole
ring systems, are prepared from the corresponding amino acid and an aldehyde via thermal 1,3-dipolar cycloaddition of
azomethine ylides and condensation with benzoylisothiocyanate. Products are fully characterized by NMR, FT-IR, MS,
and an X-ray crystal structure. The prepared compounds are screened for their antibacterial activity against a range of
Gram-positive and Gram-negative bacteria and their antimycobacterial activity against M. tuberculosis H37Rv strain. In
addition, two selected target compounds are evaluated for cytotoxicity, apoptosis, and anti-inflammatory effects on MCF-7
(
breast carcinoma) cell lines. The incorporation of indole ring and –C(O)NHC(S)– moiety resulted to be beneficial since
the biological point of view.
Graphical abstract
Keywords Amino acids Á Cycloadditions Á Heterocycles Á MCF-7 cells Á Apoptosis Á Anti(myco)bacterial
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00706-018-2286-8) contains
supplementary material, which is available to authorized
users.
5
H. Ali D o¨ nda s¸
yakdas25@mersin.edu.tr
´mica Org a´ nica, Instituto de S ´ı ntesis
Departamento de Quı
nica y Centro de Innovaci o´ n en Qu ´ı mica Avanzada
(
03080 Alicante, Spain
&
Orga
´
ORFEO-CINQA), Universidad de Alicante, Apdo. 99,
1
2
3
4
Department of Chemistry, Faculty of Pharmacy, Mersin
University, 33169 Mersin, Turkey
6
Department of Physics, Faculty of Science, Aksaray
University, Aksaray, Turkey
Department of Biochemistry, Faculty of Pharmacy, Mersin
University, 33169 Mersin, Turkey
Department of Histology and Embryology, Faculty of
Medicine, Mersin University, 33169 Mersin, Turkey
Department of Pharmaceutical Microbiology, Faculty of
Pharmacy, Mersin University, 33169 Mersin, Turkey
123

S. Poyraz et al.
Introduction
Results and discussion
Extensive studies to synthesize compounds possessing –
C(O)NHC(S)– moiety [1–4] and their metal complexes
Selected novel pyrrolidines 6a–6d and aroylaminocarbo-N-
thioylpyrrolidines 7a–7d were prepared. Prolinates 6 were
obtained from the corresponding imines 4, derived from
tryptophan methyl or ethyl ester and aldehydes, via thermal
sequence imine-azomethine ylide-1,3-dipolar cycloaddi-
tion reaction using N-alkyl/aryl-maleimides 5 as dipo-
larophiles in refluxing toluene for 36–48 h (see
‘‘Experimental part’’). The prepared pyrrolidines were then
condensed with benzoylisothiocyanate in dry acetonitrile at
reflux for 24–36 h affording the desired functionalized N-
amidocarbothiolpyrrolidines 7a–7d (Scheme 1), in good to
excellent yields (73–90%). These reactions were performed
in a larger scale (25 mmol) obtaining the same chemical
yields. The relative configuration was unambiguously
[
5–7] have been focused on the preparation of variety of
products with very attractive biological activity, for
example, antitumoural agent [8] including a P53 activator
tenovin-1 [9] (e.g, 1, Fig. 1), as nucleotide binding inhi-
bitor (NBI) [10], microsomal epoxide hydrolase inhibitors
[
11], DNA-topoisomerase inhibitor activity [12], and some
potentially important antifungal and antimicrobial activi-
ties [13–15]. Some pyrrolidine rings have been found to be
bioactive in a range of biological properties [16] including
antibacterial activity, virus inhibitors [17], such as HIV
integrase inhibitors ZINC04885876 [18] (e.g., 2, Fig. 1). In
addition, the presence of an indole unit is known to play an
important role in medicinal chemistry increasing the anti-
cancer [19, 20], antimicrobial, anti-oxidant [21–23], anti-
tubercular, anti-inflammatory activity, for example indo-
methacin (e.g., 3, Fig. 1), and so forth [24]. So, the
compounds bearing all these three pharmacophore moieties
1
13
determined by H NMR, C NMR, FT- IR, and MS and
also by X-ray diffraction analysis of the crystalline struc-
ture 6a (see Supporting Information).
Antibacterial activity
[
–C(O)NHC(S)–, prolinate and indole heterocycle] are
very attractive candidates to be tested in biological assays.
We have previously reported the synthesis and some
biological evaluation of pyrrolidine, aroylaminocarbo-N-
thioylpyrrolidine [7, 25], their several metal complexes [7],
thiohydantoin [26], and pyrrolidine thiazole derivatives
Antibacterial activity was investigated against Staphylo-
coccus aureus (ATCC 25925), Bacillus subtilis (ATCC
6633), Aeromonas hydrophila (ATCC 95080), Escherichia
coli (ATCC 25923), and Acinetobacter baumanii (ATCC
02026) which provided by the Refik Saydam Hıfzısıhha
Institute. Ampicillin was used as a reference drug. The
minimum inhibitory concentrations (MIC) values were
determined by broth microdilution method in duplicate as
recommended by the Clinical Laboratory Standards Insti-
tute [28]. Stock solutions were prepared by dissolving the
compounds in DMSO and then diluting in Mueller–Hinton
broth and Triptic soy broth and the test medium were
[
27]. As a continuation of our work, and according to our
experience, in this article we have prepared some new
selected tetrahydropyrrolo[3,4-c]pyrrole-1,3(2H,3aH)-
dione structures incorporating –C(O)NHC(S)– moiety,
prolinate and indole ring system in order to get potent
anti(myco)bacterial agents and evaluate their cytotoxicity,
apoptosis, and anti-inflammatory effects on MCF-7 cell
lines.
prepared at concentrations of 500, 250, 125, 62.5, 31.25,
3
15.62, 7.8, 3.9, and 1.9 lg/cm . Effects of DMSO were
controlled by inoculated broth supplement at the same
solutions. The observed data on the inhibited growth of
bacteria at (MIC) values are given in Table 1.
Fig. 1 Representative examples of biologically important aroyl thiourea/pyrrolidine and indole scaffolds
1
23

Study of the anti(myco)bacterial and antitumor activities of prolinate and…
Scheme 1
123

S. Poyraz et al.
The tested compounds 6a–6d and 7a–7d inhibited the
growth of bacteria at (MIC) values in the range of
reference drug (Table 1). The compounds 7a, 7b, and 7d
showed moderate activity with the MIC values of
3
3
1
5.62–250 lg/cm . The control, ampicillin, showed activ-
7.81–15.62 lg/cm , whereas the compounds 6a–6d
showed the lowest activity with the MIC values of
ity against the tested bacteria with a range of 0.9–125 lg/
cm . The compounds 7a, 7b, 7d were found to show the
3
3
62.5–125 lg/cm .
highest activity against Aeromonas hydrophila (ATCC
3
It is also important to note the incorporation of
–C(O)NHC(S)– moiety resulted to be very beneficial in
most cases.
9
5080) in MIC values of 15.62 lg/cm , and the compounds
a, 6b, 6d and 7b, 7d showed better activity than ampi-
6
cillin against Acinetobacter baumannii (ATCC 02026),
whereas the tested compounds 6a–6d and 7a–7d showed
the lowest activity to other bacteria with the MIC values of
Biological evaluation on MCF-7 (breast
carcinoma) cell lines
3
6
2.5-250 lg/cm as indicated in Table 1.
Two selected target compounds 6a and 7a were chosen to
test their cytotoxicity and their effects on apoptosis and
inflammation on the human breast cancer MCF-7 cells.
Anti-tuberculosis (TB) activity
Anti-TB activity of the compounds was investigated
according to literature method [29, 30] utilizing Microplate
Alamar Blue assay against M. tuberculosis H37Rv which
provided by the Refik Saydam National Public Health
Agency, National Tuberculosis Reference Laboratory,
Ankara, Turkey.
Cytotoxicity
Cell proliferation experiments were carried out using the
xCELLigence RTCA DP instrument, in a humidified
incubator at 37 °C and 5% CO according to the instruc-
2
The compounds 6a–6d and 7a–7d were screened and
3
tions of the supplier (Roche Applied Science and ACEA
Biosciences). Cell suspension was seeded into the wells of
E-plates (30,000 cells/well) [31]. Impedance value of each
well was automatically monitored by the xCELLigence
system or duration of 24 h and was expressed as a cell
index (CI) value. At the beginning of the log phase, 7a and
6a were added into the wells in 10, 20, 50, 100 lM con-
centrations. 0.01% DMSO was used as a vehicle group.
Cells were grown with complete medium was used as
control while 0.01% DMSO was used as a vehicle group.
Three replicates of each group were used, and the experi-
ment was ended at 100th h. All data have been recorded by
the supplied RTCA software (version 1.2.1).
measured by means of MIC values (lg/cm ). Ethambutol
(
EMB, Sigma E4630) and isoniazid (INH, Sigma I3377)
were used as the standard reference drugs and the results
with MIC values are shown in Table 1.
The tested compounds 6a–6d, 7a–7d exhibited anti-tu-
3
berculosis activity in the range of 3.90–125 lg/cm sug-
gesting that better MIC values when compared their
antibacterial activity. Especially the compound 7c, possess
Cl on the phenyl ring and –CO Et moiety in the pyrro-
2
–
lidine ring, exhibited the highest activity against M.
3
tuberculosis H37Rv strain (3.90 lg/cm ) even better
activity than known reference drugs ethambutol and mod-
erate activity when compared to isoniazid as known
3
Table 1 The MIC values (lg/cm ) of the target compounds against the bacteria and M. tuberculosis H37Rv strain
Staphylococcus Escherichia coli
aureus (ATCC 25925) (ATCC 25923)
Acinetobacter
baumannii (ATCC
Bacillus subtilis
(ATCC 6633)
Aeromonas
hydrophila (ATCC
95080)
M.
tuberculosis
H37Rv
0
2026)
6
6
6
6
7
7
7
7
a
b
c
125
62.5
62.5
250
31.25
31.25
125
125
125
250
125
125
125
125
125
0.9
31.25
31.25
250
62.5
125
62.5
250
62.5
125
d
a
b
c
62.5
62.5
62.5
250
62.5
62.5
62.5
250
31.25
62.5
31.25
15.62
15.62
250
7.81
7.81
3.90
15.62
31.25
125
d
62.5
62.5
15.62
31.25
125
15.62
31.25
Ampicillin 31.25
Isoniazid
0.2 and 1
5 and 10
Ethambutol
1
23

Study of the anti(myco)bacterial and antitumor activities of prolinate and…
Fig. 2 Red: control, green: DMSO group, navy blue:7a at 10 lM, pink: 7a at 20 lM, turquoise: 7a at 50 lM, purple: 7a at 100 lM (color figure online)
Fig. 3 Red: control, green: DMSO group, navy blue: 6a at 10 lM, pink: 6a at 20 lM, turquoise: 6a at 50 lM, purple: 6a at 100 lM (color figure online)
Treatment of the cells with 7a at 10 and 20 lM caused a
significant increase of cell index, whereas at 50 and
which has no treatment. For the experimental groups, they
were treated either with 0.01% DMSO (DMSO group) or
30 lM 7a (7a group) or 100 lM 6a (6a group) in complete
medium (n = 2). After 24 and 48 h of treatment, cells were
1
00 lM caused a significant drop in the cell index (Fig. 2)
and IC50 value of 7a was calculated as 30 lM. In addition,
treatment of the cells with 6a at 10, 20, and 50 lM caused
a significant increase of cell index whereas at 100 lM
caused a significant inhibition (50%) in the cell index
3
harvested and re-suspended in 100 mm of D-PBS for the
biochemical investigations.
(
Fig. 3).
Apoptosis
Cell culture
Caspase-8 and -9 enzyme activities were evaluated as
apoptotic markers [32]. For the determination of protein,
MCF-7 cells were homogenized in lysis buffer by 10 min
of centrifugation at 14,0009g at 4 °C. Protein levels were
evaluated with respect to Lowry assay [33] in cytosolic
extract. Caspase-8, caspase-9, and COX-2 enzyme activi-
ties were analyzed in supernatants. Caspase-8 and -9
enzyme activities were evaluated using Caspase-8 Colori-
metric Assay Kit (BioVision Research Product, Mountain
View, CA, USA) and Caspase-9 Colorimetric Assay Kit
MCF-7 cells (Cat. No: 00092502 S¸ ap Enstit u¨ s u¨ Ankara,
Turkey) were grown on six-well plate petri dishes con-
taining RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS), 1% L-glutamine, 1% penicillin–
streptomycin, and amphotericin-B as monolayer culture
until they reached 50–60% confluence.
Biochemical analysis
(BioVision Research Product, Mountain View, CA, USA),
In this study, four groups were included. MCF-7 cells were
respectively. All enzymatic activities were evaluated
with respect to the manufacturer’s instructions.
incubated with complete medium (n = 2) as control group
123

S. Poyraz et al.
Fig. 4 Effect of compounds 7a and 6a on caspase 8 and on caspase 9
on MCF-7 cell line. **p \ 0.05 vs. DMSO 48 h
Fig. 5 Effect of compounds 7a and 6a on COX2 on MCF-7 cell line.
p \ 0.05 vs. control 24 h
#
Spectrophotometric methods were based on finding out
chromophore pNA after splitting from the labeled sub-
strate. Caspase-8 and -9 enzyme activities were quantified
by free pNA cleavage from IETD-pNA and LEHD-pNA,
respectively.
MCF-7 cell lines is an important evidence for the preven-
tion of inflammation in tumor development.
Statistical analysis
There was no statistically significant difference between
the groups in caspase-8 enzyme activity which is the ini-
tiator caspase of death receptor induced (extrinsic) pathway
of apoptosis (Fig. 4). These results can be considered that
the evaluated compounds (6a, 7a) have no effects on this
extrinsic pathway. Caspase-9 which is the apical caspase of
the mitochondrial pathway and is recruited to in response
to signals originating from inside the cell. There was a
statistically significant increase in the 6a 48 h group as
compared with the DMSO 48 h group, but in caspase
activity. Caspase-9 was also increased in 7a 48 h group
compared with the DMSO 48 h group, but it was not sig-
nificant. 6a increased apoptosis by altering caspase-9
enzyme activity, suggesting that it has the potential effect
on the mitochondrial (intrinsic) pathway (Fig. 4).
Descriptive statistics (mean ± standard deviation) were
calculated in each group for all parameters. For the com-
parison of groups, one-way ANOVA (ANalysis of VAri-
ance) with post hoc Tukey was used. P \ 0.05 was
estimated to be statistically significant.
Atomic arrangement-activity studies
Although the mode of action of these molecules with the
biological targets is unknown at the moment, it is worth to
furthering this work by modifying such these substrates on
the aspect of structural diversity and subsequently testing
of other biological activities. Structure–activity relation-
ship (SAR) is very risky due to small amount of structured
tested. However, a reasonable prediction can be attempted
after analyzing the minimum energy for compounds 7b
(this work) and 8 [27] (Fig. 6). The rigid core fused
bicyclic entity in 7b is very important to get the free
accessible sulfur atom able to interact as nucleophile with
the natural target. However, in the absence of this rigid
skeleton in compound 8, the sulfur atom is flanked by two
phenyl groups avoiding a favorable approach to the living
media. Two more details need to be pointed. The presence
of aromatic rings can attract another p-cloud favoring
specific approaches in both examples and the NH bond of
the indole would promote another important hydrogen
bond with a nucleophilic moiety of the biological target. In
fact, a hydrogen bond can be formed between this indole-
NH and the ester group in both molecules 7b and 8
Inflammation
COX2 levels were measured to assess inflammation in
MCF-7 cells. COX2 enzyme activity was assayed with
respect to the manufacturer’s guidance (Shang-
hai Sunred Biological Technology Co., Ltd. Assay kit).
The results were stated in U/mg protein.
COX2 levels decreased statistically significant in 6a
4 h group compared with the control 24 h groups (Fig. 5).
2
COX1 and COX2 mediate the tissue repair process in the
alimentary tract and take part in roles in tumor develop-
ment at these sites [34]. COX2 is found over expressed in
many cancer types [35]. Reduction of COX2 level of 6a in
(Fig. 6).
1
23

Study of the anti(myco)bacterial and antitumor activities of prolinate and…
7b
8
Fig. 6 MM2 minimal energy calculations of compounds 7b and 8 (calculations were run with ChemBio Draw program using the MM2 package
at a basic level)
Conclusion
anti-inflammatory effects on MCF-7 cell lines. In addi-
tion, the tested compounds 6a and 7a reflect moderate
activity against some of the used tumor cells which can
be consider as potential bioactive to possible anti-car-
cinogenic properties on MCF-7 cell lines. It is also
important to note that the incorporation of indole ring and
–C(O)NHC(S)– moiety resulted to be crucial for the
increment of activity since the biological point of view,
so they can consider promising candidates for antitumor
agents. As tentative proposal, the sulfur atom can play a
crucial role in the effective interaction with the living
target in order to get the experimentally observed inhi-
bition of the cell/bacteria.
We have described synthesis of four novel 6a–6d, 7a–7d
pyrrolidine and aminocarbothiol pyrrolidine derivatives
based on pyrrole-3,4-dione, bearing indole ring system in
good yields. The prepared compounds were screened for
their anti(myco)bacterial activity against a range of bac-
teria and M. tuberculosis H37Rv strain. The screened
compounds showed moderate antibacterial activity against
various bacteria strains, whereas showed better anti-TB
activity against M. tuberculosis H37Rv strain. In general,
better bactericide activity was observed than those pre-
viously reported by our group. The obtained results dis-
played that these target compounds can be considered as
potential anti(myco)bacterial activity agent, especially
anti-tuberculosis agent (e.g., 7a, 7b, and 7c). The com-
pound 7c exhibited higher activity against M. tuberculosis
Experimental
All chemicals were purchased from Merck or Sigma-
Aldrich and used without further purification. Melting
points were determined on a Stuart SMP3 hot stage appa-
ratus. The structurally most important peaks of the IR
spectra (Perkin-Elmer FT-IR using horizontal ATR) are
3
H37Rv strain in 3.90 lg/cm values and shows even
better activity than known reference drugs ethambutol and
moderate activity when compared to isoniazid as known
reference drugs. Moreover, two selected target compounds
-
1
6
a and 7a were evaluated for cytotoxicity, apoptosis and
listed and wave numbers are given in cm . Nuclear
123

S. Poyraz et al.
magnetic resonance spectra were determined on a Bruker
Biospin GmbH Ultra Shield Plus 400 MHz spectrometer.
Chemical shifts are reported in parts per million (d)
downfield from tetramethylsilane as internal standard.
Spectra were determined in deuterated chloroform or
(1S,3R,3aS,6aR)-Ethyl 1-[(1H-indol-2-yl)methyl]-3-(4-meth-
oxyphenyl)-4,6-dioxo-5-phenyloctahydropyrrolo[3,4-c]pyr-
role-1-carboxylate (6b, C31
Et O: hexane as colorless prisms. Yield 87%; m.p.: 268-
270 °C (decomp.); R
): d = 11.01 (bs, 1H, NH), 7.60–6.89 (m, 14H, Ar–H),
5.11 (dd, 1H, J = 9.38 Hz, 4.8 Hz, 5-H), 4.13–4.04 (m, 2H,
CH CH ), 3.88 (dd,1H, J = 9.34 Hz, 7.68 Hz, 4-H), 3.77
(dd, 1H, J = 7.56 Hz, 1,44 Hz, 3-H), 3.74 (s, 3H, OCH ),
H N O ) Crystallized from
29 3 5
2
1
= 0.24; H NMR (400 MHz, DMSO-
f
DMSO-d as stated. The following abbreviations are used:
6
d
6
s = singlet, d = doublet, t = triplet, q = quartet, m = mul-
tiplet and br s = broad singlet. Flash column chromatog-
raphy was performed using silica gel 60. Mass spectra were
recorded on an Agilent LC/MSD, Agilent 6460 Triple
Quad LC/MS/MS mass spectrometer. The X-ray crystal
structure data were collected on Bruker APEX-II CCD
model X-ray diffractometer. Flash column chromatography
was performed using silica gel 60 (230–400 mesh). High-
resolution mass spectra (HRMS) were measured in a Fin-
nigan MAT 95S using ESI.
2
3
3
3.44 (d, 1H, J = 14.56 Hz, 6-H), 3.34 (d, 1H,
J = 14.50 Hz, 6-H’), 2.44 (d, 1H, J = 4.68 Hz, N–H), 1.25
3
1
(t, 3H, J = 7.12 Hz, CH
CH ) ppm; C NMR (100 MHz,
3
2
DMSO-d ): d = 175.4 (C=O), 174.2 (C=O), 171.3 (C=O),
6
158.7, 136.9, 132.2, 130.8, 128.8 (2 C), 128.5 (2 C), 128.2,
127.7, 126.7 (2 C), 124.3, 121.0, 120.2, 118.5, 118.1,
113.3, 111.5, 108.0, 70.4, 60.4, 59.5, 55.0, 54.1, 49.1, 30.2,
-
1
1
3.9 ppm; IR: mꢀ = 3356, 3067, 2995, 1772, 1708 cm
;
?
General procedure for the synthesis of bicyclic
pyrrolidines 6
MS (ESI): m/z = 524.3 ([M ? H] , 100).
1S,3R,3aS,6aR)-Ethyl 1-[(1H-indol-2-yl)methyl]-3-(4-chloro-
phenyl)-5-methyl-4,6-dioxooctahydropyrrolo[3,4-c]pyrrole-
-carboxylate (6c, C H ClN O ) Crystallized from Et O:
(
The pyrrolidine derivatives 6a–6d prepared by adaption of
the literature procedures [4, 7, 15, 27]. Thus, aldehydes
1
2
5
24
3
4
2
hexane as colorless prisms. Yield 76%; m.p.: 213–215 °C
3
1.1 mmol) in 10 cm were added to L-tryptophan methyl/
(
1
decomp.); R = 0.20; H NMR (400 MHz, CDCl3):
(
3
f
ethyl esters (1 mmol) in 20 cm dry DCM to form corre-
sponding imines 4. Then, N-maleimides (1 mmol) in dry
toluene were added to imine solution (1 mmol). The
reaction mixture was stirred under reflux for 36–48 h. After
completion of the reaction by monitoring TLC, the reaction
mixture was evaporated and crystallized from ether-hex-
d = 8.25 (bs, 1H, NH), 7.56 (d, 1H, J = 7.88 Hz, Ar–H),
7.25–6.96 (m, 8H, Ar–H), 4.85 (dd, 1H, J = 9.1 Hz,
3
.60 Hz, 5-H), 4.33–4.17 (m, 2H, CH CH ), 3.61 (d, 1H,
3
2
J = 14.68 Hz, 6-H), 3.55 (dd, 1H, J = 9.0 Hz, 7.66 Hz, 4-
H), 3.44 (d, 1H, J = 7.48 Hz, 3-H), 3.24 (d, 1H,
J = 14.64 Hz, 6-H’), 2.79 (s, 3H, NCH ), 2.50 (d, 1H,
3
ane. R values for each compounds 6a–6d were calculated
f
J = 3.16 Hz, N–H), 1.34 (t, 3H, J = 7.16 Hz, CH CH )
3
2
in Et O: hexane (2:1).
2
13
ppm; C NMR (100 MHz, CDCl ): d = 175.7 (C=O),
3
(
1S,3R,3aS,6aR)-Methyl 1-[(1H-indol-2-yl)methyl]-3-(4-chloro-
174.7 (C=O), 171.6 (C=O), 137.8, 136.4, 135.9, 130.0,
128.6 (3C), 126.1, 123.2, 122.4, 119.9, 118.2, 111.5, 109.2,
71.1, 61.7, 60.6, 54.0, 49.5, 24.9, 21.4, 14.1 ppm; IR:
mꢀ = 3324, 2959, 1777, 1725, 1698, 1430 1282, 1198, 1091,
phenyl)-5-ethyl-4,6-dioxooctahydropyrrolo[3,4-c]pyrrole-1-
carboxylate (6a, C H ClN O ) Crystallized from Et O:
hexane as colorless prisms. Yield 58%; m.p.: 237-239 °C;
R = 0.20; H NMR (400 MHz, DMSO-d ): d = 11.00 (br
s, 1H, NH), 7.57–6.95 (m, 9H, Ar–H), 5.02 (dd, 1H,
25
24
3
4
2
1
-1
?
1012,785, 740 cm ; MS (ESI): m/z = 466.2 ([M ? H] ,
?
100), 468.2 ([M ? H] , 35); HRMS (ES): C25
f
6
H24ClN
O
3
4
?
calc. [M ? H] 465.1455, found 465.1449.
J = 4.68 Hz, 9.4 Hz, 5-H), 3.72 (dd, 1H, J = 8.38 Hz,
7
.52 Hz, 4-H), 3.68 (s, 3H, OCH ), 3.60 (dd, 1H,
3
(
1S,3R,3aS,6aR)-Ethyl 1-[(1H-indol-2-yl)methyl]-3-(4-meth-
oxyphenyl)-5-methyl-4,6-dioxooctahydropyrrolo[3,4-c]pyr-
role-1-carboxylate (6d, C H N O ) Crystallized from
J = 7.46 Hz, 1.38 Hz, 3-H), 3.43 (d, 1H, J = 14.6 Hz, 6-
H), 3.30 (d, 1H, J = 14.56 Hz, 6-H’), 3.25-3.13 (m, 2H,
CH CH ), 2.44 (d, 1H, J = 3.52 Hz, N–H), 0.91 (t, 3H,
2
6 27 3 5
2
3
Et O: hexane as colorless prisms. Yield 75%; m.p.: 185–
2
1
3
J = 7.16 Hz, CH CH ) ppm; C NMR (100 MHz, DMSO-
2
3
1
1
87 °C; R = 0.16; H NMR (400 MHz, DMSO-d6):
f
d6): d = 175.8 (C=O), 174.6 (C=O), 171.6 (C=O), 138.2,
d = 8.19 (s, 1H, NH), 7.59 (d, 1H, J = 7.88 Hz, Ar–H),
7
1
1
3
35.9, 131.8, 129.2 (2 C), 127.8 (2 C), 127.6, 124.4, 121.0,
18.6, 118.0, 111.5, 107.9, 70.2, 58.8, 53.4, 51.5, 48.5,
2.9, 30.3, 12.7 ppm; IR mꢀ = 3339, 2981, 2944, 2840, 1774
.25–6.80 (m, 8H, Ar–H), 4.85 (dd, 1H, J = 9.06 Hz,
4
.76 Hz, 5-H), 4.33–4.16 (m, 2H,CH CH ), 3.75 (s, 3H,
2
3
OCH ), 3.59 (d, 1H, J = 14.56 Hz, 6-H), 3.53 (dd, 1H,
3
-
1
(
C=O), 1739 (C=O), 1683 (C=O), 744 cm ; MS (ESI): m/
J = 9.00 Hz, 7.52 Hz, 4-H), 3.45 (d, 1H, J = 7.44 Hz, 3-
?
?
z = 466.3 ([M ? H] , 100), 468.3 ([M ? H] , 35); HRMS
H), 3.26 (d, 1H, J = 14.64 Hz, 6-H’), 2.80 (s, 3H, NCH3),
?
(
ES): C H ClN O calc. [M ? H] 465.1455, found
25 24 3 4
2
.55 (d, 1H, J = 4.52 Hz, N–H), 1.32 (t, 3H, J = 7.16 Hz,
1
4
65.1449.
3
CH CH ) ppm;
3
C
NMR (100 MHz, DMSO-d6):
2
d = 175.9 (C=O), 175.1 (C=O), 171.7 (C=O), 159.5, 136.9,
1
23

Study of the anti(myco)bacterial and antitumor activities of prolinate and…
1
1
4
1
1
29.8, 128.3 (2 C), 127.9, 123.2, 122.2, 119.8, 118.3,
13.8, (2 C), 111.3, 109.5, 71.0, 61.6, 61.0, 55.2, 54.3,
9.8, 30.8, 24.9, 14.1 ppm; IR: mꢀ = 3414, 3342, 2981,
8.22–6.51 (m, 40H, Ar–H, NH major and minor), 5.57 (d,
1H, J = 11.00 Hz, 5-H minor), 5.53 (d, 1H, J = 11.32 Hz,
5-H major), 4.98 (d, 1H, J = 15.4 Hz, 6-H minor), 4.48 (d,
1H, J = 15.24 Hz, 6- H’ minor), 4.43–4.18 (m, 4H, CH2-
-
1
?
780, 1727 cm ; MS (ESI): m/z = 462.3 ([M ? H] ,
00).
CH major and minor), 3.94 (d, 1H, J = 14.96 Hz, 6-H
3
major), 3.91–3.83 (m, 2H, 4-H major and minor) 3.86 (d,
General procedure for the synthesis of bicyclic
1H, J = 14.96 Hz, 6- H’ major), 3.74 (s, 3H, OCH minor),
3
benzoylaminocarbo-N-thiol-pyrrolidines 7
3.73 (s, 3H, OCH major), 2.82 (dd, 1H, J = 10.44 Hz,
3
10.32 Hz, 3-H major), 2.55 (dd, 1H, J = 10.9 Hz, 9.04 Hz,
Bicyclic benzoylaminocarbo-N-thioylpyrrolidines 7a–7d
were prepared by adaption of literature procedures
3-H minor), 1.43 (t, 3H, J = 6.96 Hz, CH CH major), 1.30
2
3
1
3
(t, 3H, J = 7.12 Hz, CH CH minor) ppm; C NMR
2 3
[
(
4, 7, 15, 27]. Thus, to a stirred solution of pyrrolidine
3
1.2 mmol) in 25 cm dry acetonitrile was added a solution
(100 MHz, CDCl ): d = 187.0 (C = S major), 173.0
3
(C = S minor), 172.9 (C=O major), 172.6 (C=O major),
172.3 (C=O minor), 171.9 (C=O minor), 169.6 (C=O
major), 167.8 (C=O minor), 164.0 (C=O minor), 159.1
(C=O major), 135.9 (2 C major), 133.7 (C major), 133.7 (C
minor), 133.1 (C minor), 132.9 (C major), 132.7 (C major),
131.7 (C major), 131.0 (C minor), 130.9 (C major), 130.2
(2 C minor), 129.2 (2 C minor), 129.0 (4 C major), 128.9 (3
C minor), 128,6 (4 C major), 128,5 (2 C minor), 127.8 (C
minor), 127.7 (C minor), 127.6 (2 C minor), 127.5 (C
minor), 126.7 (C minor), 125.9 (5 C major), 125.7 (C
minor), 124.3 (C major), 122.8 (C major), 122.5 (C minor),
3
of benzoyl isothiocyanate (1.22 mmol) in 15 cm of dry
acetonitrile dropwise. The resulting mixture was stirred at
reflux for appropriate time (24–36 h) monitoring by TLC.
After completion of the reaction, the solvent was removed
and purified by flash chromatography. Rf values for com-
pounds 7a–7d were calculated in Et O: hexane (2:1).
2
(
1S,3R)-Methyl
1-[(1H-indol-3-yl)methyl]-2-(benzoylcar-
bamothioyl)-3-(4-chlorophenyl)-5-ethyl-4,6-dioxooctahy-
dropyrrolo[3,4-c]pyrrole-1-carboxylate
(7a, C H ClN O S) Reaction time 24 h. The product
33 29 4 5
crystallized from Et O:hexane as pale-yellow prisms as a1:3
1
20.6 (C major), 120.4 (C minor), 118,1 (C major), 117.9
C minor), 113.9 (C minor), 113.7 (C major), 111.9 (C
major), 111.8 (C minor), 108.9 (C major), 108.5 (C minor),
6.1 (minor), 69.1 (C major), 68.6 (C minor), 62,1 (C
major), 55.4 (C major), 55.2 (2 C minor), 54.3 (C minor),
2.8 (C major), 49.5 (C minor), 48.2 (C major), 33.0 (C
major), 29.7 (C minor), 28.2 (C minor), 14.0 (2 C major)
2
(
rotamer mixture. Yield 73%; m.p.: 145–147 °C; R = 0.25;
f
1
H NMR (400 MHz, CDCl ): d = 8.55 (br s, 1H, NH, major
3
7
rotamer), 8.46 (br s, 1H, NH minor rotamer), 8.19–7.00 (m,
3
0H, Ar–H, NH major and minor), 5.63 (d, 1H,
5
J = 11.52 Hz, 5-H minor), 5.43 (d, 1H, J = 11.2 Hz, 5-H
major), 4.81 (d, 2H, J = 15.44 Hz NCH CH major), 4.42(d,
2
3
-1
ppm; IR: mꢀ = 3440, 2990, 1731, 1711, 1245, 1213 cm
MS (ESI): m/z = 687.3 ([M-H] , 100).
;
2
H, J = 15.04 Hz NCH CH minor), 3.96 (s, 3H, OCH
2 3 3
?
major), 3.90 (s, 3H, OCH minor), 3.20–2.99 (m, 4H, 6-H, 6-
3
H’ major and minor), 2.77- 2.44 (m, 2H, 3-H major and
(1S,3R,3aS,6aR)-Ethyl 1-[(1H-indol-3-yl)methyl]-2-(benzoyl-
carbamothioyl)-3-(4-chlorophenyl)-5-methyl-4,6-dioxooc-
tahydropyrrolo[3,4-c]pyrrole-1-carboxylate
minor), 1.41–1.19 (m, 5H, 4-H major and minor, CH CH
2
3
1
3
minor), 0.68 (t, 3H, J = 7.2 Hz, CH CH major) ppm;
2
C
3
NMR (100 MHz, CDCl ): d = 187.3 (C = S), 178.8 (C=O),
(7c, C H ClN O S) Reaction time 24 h. The product
3
33 29
4 5
1
1
1
6
73.8 (C=O), 173.1 (C=O), 170.2 (C=O), 135.9, 135.0,
33.9, 133.6, 133.3, 133.1, 129.2 (2 C), 128.8, 128.7 (2 C),
27.8, 127.6 (2 C), 124.3, 122.9, 121.7, 120.7, 117.9, 69.0,
8.3, 53.1, 49.4, 47.8, 34.0, 33.9, 12.5, 12.3 ppm; IR:
crystallized from Et O: hexane as pale-yellow prisms as a
2
1:1 rotamer mixture. Yield 78%; m.p.: 128–130 °C; R
f-
1
= 0.22; H NMR (400 MHz, CDCl ): d = 8.61 (br s, 1H,
3
NH, minor rotamer), 8.55 (br s, 1H, NH major rotamer),
8.20 (s, 1H, NH minor), 8.18 (s, 1H, NH major), 7.79–7.02
(m, 28H, Ar–H, major and minor), 5.60 (d, 1H, J = 11.40,
5-H minor), 5.40 (d, 1H, J = 11.08, 5-H major), 4.59–4.20
(m, 8H, CH CH , 4-H major and minor), 3.94 (d, 1H,
mꢀ = 3374, 2981, 2946, 2925, 1782 (C=O), 1740 (C=O), 1702
-
C=O), 1228, 710 cm ; MS (ESI): m/z = 627.3 ([M-H ],
1
?
(
?
1
00), 629.3 ([M-H ], 45); HRMS (ES): C H ClN O S
33 29 4 5
?
calc. [M ? H] 628.1547, found 628.1546.
2
3
J = 15.52, 6-H major), 3.87 (d, 1H, J = 14.36, 6-H’
(
1S,3R,3aS,6aR)-Ethyl 1-[(1H-indol-2-yl)methyl]-2-(benzoyl-
minor), 3.73–3.68 (m, 2H, 3-H major and minor), 2.57 (s,
carbamothioyl)-3-(4-methoxyphenyl)-4,6-dioxo-5-phenyloc-
tahydropyrrolo[3,4-c]pyrrole-1-carboxylate
(7b, C H N O S) Reaction time 24 h. The product
39 34 4 6
crystallized from Et O: hexane as pale-yellow prisms as a
3
H, NCH minor), 2.45 (s, 3H, NCH major), 1.48–1.33
3 3
1
3
(
(
(
m, 6H, CH CH major and minor) ppm; C NMR
2 3
100 MHz, CDCl ): d = 187.2 (C = S major), 179.7
C = S minor), 174.1 (C=O minor), 173.8 (C=O major),
3
2
1
:1 rotamer mixture. Yield 90%; m.p.: 170–172 °C; R
f-
173.3 (C=O major), 173.0 (C=O minor), 172.1 (C=O
1
=
0.20; H NMR (400 MHz, CDCl ): d = 8.56 (br s, 1H,
3
minor), 169.6 (C=O major), 169.4 (C=O minor), 164.0
NH major rotamer), 8.49 (br s, 1H, NH minor rotamer),
123

S. Poyraz et al.
(
C=O major), 135.9 (C major), 135.8 (C minor), 135.4 (C
major), 134.9 (C minor), 134.6 (C minor), 133.9 (C major),
33.7 (C major), 133.3 (C minor), 133.1 (C major), 133.0
C minor), 132.3 (C minor), 132.1 (C major), 131.9. (C
14.0 (3 C major) ppm; IR: mꢀ = 3346, 2980, 1734, 1706,
1246, 1210 cm ; MS (ESI): m/z = 625.3 ([M-H] , 100).
-
1
?
1
Acknowledgements We would like to thank Mersin University,
Research Foundation (Project No: BAP 2015-AP2-1342) for financial
support.
(
major), 129.23 (2 C minor), 129.0 (C minor), 128.7 (2 C
major), 128.6 (C major), 128.5 (C minor), 128.3 (C major),
1
28.0 (C minor), 127.8 (C minor), 127.6 (C major), 127.4
2 C minor), 126.2 (2 C major), 126.0 (C minor), 124.4 (C
major), 122.8 (C major), 122.4 (C minor), 120.6 (C major),
20.3 (C minor), 117.9 (C minor), 117.8 (C major), 111.9
C major), 111.8 (C minor), 108.7 (C major), 108.1 (C
minor), 76.1 (C major), 68.8 (C minor), 68.3 (C minor),
2.5 (C major), 54.1 (C major), 52.4 (C minor), 49.5 (C
minor), 48.0 (C major), 36.7 (C minor), 32.9 (C major),
7.70 (C minor), 24.7 (C major), 24.5 (2 C minor), 14.0 (2
C major) ppm; IR: mꢀ = 3364, 3055, 2975, 2937, 1785,
(
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-
1
1
707, 1599, 1563, 1489, 1377, 1232, 1090, 745, 711 cm ;
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(
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8
1
.20–7.04 (m, 30H, Ar–H, NH major and minor), 4.92 (d,
1
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3
(
s, 3H, OCH minor), 2.81 (s, 3H, NCH minor), 2.45 (s,
3 3
3
H, NCH3 major), 1.47 (t, 3H, J = 7.12 Hz, CH CH
2
3
1
3
major), 1.33 (t, 3H, J = 7.12 Hz, CH CH minor) ppm;
2
C
3
NMR (100 MHz, CDCl ): d = 186.9 (C = S minor), 174.1
3
1
(
C = S major), 173.9 (C=O minor), 173.5 (C=O major),
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123