Novel indole-thiazolidinone conjugates: Design, synthesis and whole-cell phenotypic evaluation as a novel class of antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2018.10.008

In connection with our research program on the development of novel anti-tubercular candidates, herein we report the design and synthesis of two different sets of indole-thiazolidinone conjugates (8a,b; 11a-d) and (14a-k; 15a-h). The target compounds were evaluated for their in vitro antibacterial and antifungal activities against selected human pathogens viz. Staphylococcus aureus (Gram positiveve), Pseudomonas aeruginosa, Escherichia coli (Gram negative), Mycobacterium tuberculosis (Acid-fast bacteria), Aspergillus fumigates and Candida albicans (fungi). Moreover, eukaryotic cell-toxicity was tested via an integrated ex vivo drug screening model in order to evaluate the selective therapeutic index (SI) towards antimicrobial activity when microbes are growing inside primary immune cells. Also, the cytotoxicity towards a panel of cancer cell lines and human lung fibroblast normal cell line, WI-38 cells, was explored to assure their safety. Compound 15b emerged as a hit in this study with potent broad spectrum antibacterial (MIC: 0.39–0.98 μg/mL) and antifungal (MIC: 0.49–0.98 μg/mL) activities, in addition to its ability to kill mycobacteria M. aurum inside an infected macrophage model with good therapeutic window. Moreover, compound 15b displayed promising activity towards resistant bacteria strains MRSA and VRE with MIC values equal 3.90 and 7.81 μg/mL, respectively. These results suggest compound 15b as a new therapeutic lead with good selectivity for further optimization and development.

Full text of DOI:10.1016/j.ejmech.2018.10.008

Accepted Manuscript
Novel indole-thiazolidinone conjugates: Design, synthesis and whole-cell phenotypic
evaluation as a novel class of antimicrobial agents
Mahmoud F. Abo-Ashour, Wagdy M. Eldehna, Riham F. George, Marwa M. Abdel-
Aziz, Mahmoud M. Elaasser, Nagwa M. Abdel Gawad, Antima Gupta, Sanjib Bhakta,
Sahar M. Abou-Seri
PII:
S0223-5234(18)30870-5
10.1016/j.ejmech.2018.10.008
EJMECH 10794
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 March 2018
Revised Date: 20 September 2018
Accepted Date: 2 October 2018
Please cite this article as: M.F. Abo-Ashour, W.M. Eldehna, R.F. George, M.M. Abdel-Aziz, M.M.
Elaasser, N.M. Abdel Gawad, A. Gupta, S. Bhakta, S.M. Abou-Seri, Novel indole-thiazolidinone
conjugates: Design, synthesis and whole-cell phenotypic evaluation as a novel class of
antimicrobial agents, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.10.008.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel indole-thiazolidinone conjugates: Design, synthesis and whole-cell
phenotypic evaluation as a novel class of antimicrobial agents
Mahmoud F. Abo-Ashoura, Wagdy M. Eldehnab,*, Riham F. Georgec, Marwa M. Abdel-Azizd,
Mahmoud M. Elaasserd, Nagwa M. Abdel Gawadc, Antima Guptae, Sanjib Bhaktae, Sahar M. Abou-
Seric
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo 11829, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
Kafrelsheikh, Egypt
cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt
dThe Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt
eMycobacteria Research Laboratory, Department of Biological Sciences, Institute of Structural
and Molecular Biology, Birkbeck, University of London, Malet Street, London, WC1E 7HX,
United Kingdom
ABSTRACT
In connection with our research program on the development of novel anti-tubercular candidates,
herein we report the design and synthesis of two different sets of indole-thiazolidinone
conjugates (8a,b; 11a-d) and (14a-k; 15a-h). The target compounds were evaluated for their in
vitro antibacterial and antifungal activities against selected human pathogens viz. Staphylococcus
aureus (Gram positiveve), Pseudomonas aeruginosa, Escherichia coli (Gram negative),
Mycobacterium tuberculosis (Acid-fast bacteria), Aspergillus fumigates and Candida albicans
(fungi). Moreover, eukaryotic cell-toxicity was tested via an integrated ex vivo drug screening
model in order to evaluate the selective therapeutic index (SI) towards antimicrobial activity
when microbes are growing inside primary immune cells. Also, the cytotoxicity towards a panel
of cancer cell lines and human lung fibroblast normal cell line, WI-38 cells, was explored to
assure their safety. Compound 15b emerged as a hit in this study with potent broad spectrum
antibacterial (MIC: 0.39-0.98 µg/ml) and antifungal (MIC: 0.49-0.98 µg/ml) activities, in
addition to its ability to kill mycobacteria M. aurum inside an infected macrophage model with
good therapeutic window. Moreover, compound 15b displayed promising activity towards
resistant bacteria strains MRSA and VRE with MIC values equal 3.90 and 7.81 µg/mL,
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respectively. These results suggest compound 15b as a new therapeutic lead with good
selectivity for further optimization and development.
__________
Keywords: Antimicrobial resistance (AMR); Anti-tubercular agents; Indole and Isatin;
Macrophage Infection Model; MRSA and VRE; Thiazolidinone.
* Corresponding authors. wagdy2000@gmail.com (W.M. Eldehna).
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1. Introduction
With the regular rise of antimicrobial resistance (AMR) to our best available last stage of
antibiotics, our advance medical system is rapidly reaching towards apocalypse by having no
available treatment and millions of death along with a serious threat of a return to the pre-
antibiotic era. If there is no action taken place then there is an estimate of drug-resistant
infections that will kill 10 million people a year by 2050 [1]. It is truly difficult to treat an ever-
increasing range of infections caused by superbugs (resistant bacteria, parasites, viruses and
fungi) due to serious lack of effective antibiotics.
Although antibacterial resistance is a widely recognized public health threat, but the burden
of drug-resistant fungal infections are also an emerging issue [2]. Other than ESKAPE and
MRSA, Tuberculosis (TB) is still having continuous global impact on the health worldwide, as
nearly one-third of the world`s population is directly and indirectly infected with TB [3]. In
2016, Asia occupied the first rank in new TB cases with about 45% of the total new cases
followed by Africa with 25% of the new cases [4]. Nevertheless, multi-drug resistant (MDR) TB
and extremely drug resistant (XDR) TB are rapidly emerged, increasing the urgency to develop
new antiTB agents based on innovative scaffolds, thus lacking the cross resistance with current
therapeutics. Continuous efforts in all different directions to develop broad spectrum as well as
specific and targeted drugs against different microbes, are required. A recent strategy to design
new agents based on hybridization of different pharmacophores binding diverse biomolecular
targets to gain an effective therapy against drug resistant infectious diseases [5].
As per recent report, only eight out of the 51 new biologicals in clinical development are new
class of drugs that could add value to the current therapies while most of others are only short-
term solutions [6]. During the last two decades, thiazolidinone ring has emerged as novel
important core in many chemotherapeutic agents special when conjugated with substituted
aminothiazoles derivatives. For example, the aminothiazole derivative I was found to exhibit
promising antibacterial and antifungal activities [7]. Moreover, ethyl 2-amino-4-methylthiazole-
5-carboxylate derivative II revealed potent activity against the virulent species of M.
tuberculosis H37Rv [8]. Therefore, our research team was motivated to synthesize some
thiazolidinone/2-amino-4-methylthiazole hybrids bearing 5-acetyl/5-ethyl carboxylate
functionalities as potential anti-tubercular agents [9]. Among the synthesized hybrids, compound
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III exhibited good antitubercular effect (MIC=1.56 µg/mL) with broad spectrum antibacterial
and antifungal activities [9]. It possessed an excellent safety profile with high selectivity toward
M. tubercolosis over normal human lung cells (selectivity index= 52.11) and considered as good
lead for further optimization. Literature survey revealed that, the antimicrobial spectrum and
potency of thiazolylamino-thiazoledinone derivatives can be manipulated by changing type or
nature of arylidene substituent at position 5 of thiazoledinone ring. It was reported that the
incorporation of indolyl moiety as in N'-[(1H-indol-3-yl)methylene]-isonicotinohydrazide
derivative (IV) or 2-oxindolyl moiety as in (1,3,4-thiadiazol-2-yl)imino)indolin-2-one derivative
(V) showed excellent to good anti-tubercular activity [10, 11]. Thus, in continuation of our team
work to obtain an optimized scaffold with potent anti-tubercular, antibacterial and antifungal
activities [12-15], two series of thiazolylamino-thiazolidinone hybrids were designed in which
the 5-pyridylidene moiety in the hybrid III was replaced by N-(un)substituted 5-[(1H-indol-3-
yl)methylene] moiety in compounds 8a,b and 11a-d or by (un)substituted-2-oxindole in series
14a-k and 15a-h to study the effect of such structure modifications on activity (Figure 1). As a
part of the urgent focus on the discovery of novel lead molecules to fuel the pipeline of antibiotic
design, discovery and development in order to tackle the alarming emergence of antimicrobial
resistance, we aim to explore the potential of few novel chemical entities with their promising
antimicrobial activities and broad-spectrum mode of antibiotic action. The target compounds
8a,b, 11a-d, 14a-k and 15a-h will be evaluated for their in vitro anti-tubercular, antibacterial and
antifungal activities against selected human pathogens viz. M. tuberculosis, Staphylococcus
aureus, Pseudomonas aeruginosa, Escherichia coli, Aspergillus fumigates and Candida
albicans. Furthermore, an integrated ex vivo drug screening model will be used to evaluate
certain selected conjugates for their intracellular inhibition and selectivity towards
Mycobacterium aurum in mouse macrophages RAW 264.7. Moreover, the cytotoxicity of the
most active conjugate towards a panel of cancer cell lines and human lung fibroblast normal cell
line, WI-38 cells, will be explored to assure its safety.
Figure 1
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2. Results and Discussion
2.1. Chemistry
The synthetic pathways employed to prepare the new target conjugates are depicted in
Schemes 1-3. α-Chloro-β-dicarbonyl derivatives 2a,b were obtained via chlorination of acetyl
acetone 1a or ethyl acetoacetate 1b, using sulfuryl chloride in dry toluene. Subsequent
heterocyclization of compounds 2a,b with thiourea furnished the corresponding 2-
aminothiazoles 3a,b. The latter were acetylated with chloroacetyl chloride in dry DMF to furnish
compounds 4a,b, which were cyclized with ammonium thiocyanate in absolute ethanol to give
the key intermediates 5a,b in 75 and 70% yield (Scheme 1).
Scheme 1
In Scheme 2, the synthesis of the N-substituted-indole-3-carbaldehyde derivatives 10a,b was
accomplished via formylation of 1H-indole 6 with phosphorus oxychloride and DMF to afford
1H-indole-3-carbaldehyde 7, which subsequently alkylated with bromomethane 9a or benzyl
bromide 9b in DMF in the presence of sodium hydride. The first series of the target conjugates
8a,b and 11a-d were obtained in good yields (75-83%) through Knoevenagel condensation of
intermediates 5a,b with 1H-indole-3-carbaldehyde 7 or N-substituted-indole-3-carbaldehydes
10a, b in glacial acetic acid in the presence of anhydrous sodium acetate.
Scheme 2
Finally, alkylation of isatins 12a,c with bromomethane 9a or benzyl bromide 9b was carried
out via heating in DMF in the presence of potassium carbonate to furnish N-alkylated isatins
13a-d. The target compounds (14a-k and 15a-h) were obtained through condensation of the
intermediates 5a,b with the appropriate isatin derivative (12a-f and 13a-d) in glacial acetic acid
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in the presence of anhydrous sodium acetate under reflux temperature with 64-75% yields
(Scheme 3).
Scheme 3
Postulated structures of the newly prepared conjugates 8a,b, 11a-d, 14a-k and 15a-h were in
full agreement with their spectral and elemental analyses data. IR spectra of the target
compounds showed the absorption bands due to the (NH) and (C=O) groups in the regions 3117-
1
3395 and 1651-1740 cm-1, respectively. The H NMR spectra of all new compounds showed
D2O-exchangeable singlet signals corresponding to thiazolidinone NH proton at range δ = 11.92-
1
13.21 ppm, additionally, H NMR spectra of compounds 8a,b displayed another D2O-
exchangeable singlet signals attributable for indole NH at δ 12.11 and 12.13 ppm, respectively,
whereas the oxindole NH protons of compounds 14a-k appeared at range δ 10.86-11.42 ppm.
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Moreover, the H NMR spectra of the prepared conjugates revealed the presence of singlet
signals around δ 2.60 ppm attributed to the CH3 group at position 4 of the thaizole ring . The
acetyl protons (CH3C=O) of conjugates 8a, 11a,b, 14a-f and 15a-d appeared as singlet signals
around δ 2.49 ppm, while the ethyl ester protons (CH3CH2COO-) of conjugates 8b, 11c,d, 14g-k
and 15e-h displayed as triplet and quartet signals around δ 1.24 and 4.21 ppm, respectively.
1
Furthermore, H NMR spectra of compounds 8a,b and 11a-d confirmed the presence of the
singlet signal of the olefinic protons (CH=N) around δ 8.0 ppm. The N-methyl substituted
derivatives 11a,c and 15a,b,e,f showed single signals at the range δ= 3.94-3.23 ppm attributed to
N-CH3, while those with N-benzyl substituent 11b,d and 15c,d,g,h revealed singlet signals at δ
range 5.01-5.60 ppm due to the benzylic (-CH2) protons.
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Moreover, C NMR spectra of conjugates 8a,b, 11a-d, 14a-k and 15a-h displayed signals
resonating in the range δ 18.1-19.1 ppm attributable for the carbon of the thiazole CH3, while the
carbons of the acetyl groups (-CH3C=O) of conjugates 8a, 11a,b, 14a-f and 15a-d appeared as
two signals around δ 30.6 and 190.8 ppm, respectively, and the carbons of the ethyl ester groups
(CH3CH2COO-) in conjugates 8b, 11c,d, 14g-k and 15e-h appeared as three signals around δ
14.6, 61.0 and 168.0 ppm, respectively. Also, signals attributable to (N-CH3) carbon, as
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compounds 11a,c, and benzylic carbon (-CH2), as compounds 11b,d, appeared around δ 33.9 and
50.2 ppm, respectively.
2.2. Biological Evaluation
2.2.1. Antimicrobial activity
Anti-mycobacterial, antibacterial and antifungal activities for the target conjugates (8a,b,
11a-d, 14a-k and 15a-h) and reference drugs were in vitro evaluated at the Regional Center for
Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The results are
displayed in Tables 1-3.
2.2.1.1. Anti-tubercular Activity; M. tuberculosis (RCMB 010126)
Target conjugates (8a,b, 11a-d, 14a-k and 15a-h) were examined for their anti-tubercular
activity against M. tuberculosis (RCMB 010126) using the Microplate Alamar Blue Assay
(MABA) [16]. Isoniazid was used as reference drug. The results of such in vitro anti-
mycobacterial screening are summarized in Table 1, as percent inhibition and minimum
inhibitory concentration (MIC). As indicated in Table 1, compounds 14i, 14k, 15a, 15b, 15d,
15e, 15g and 15h (MIC = 0.39 µg/mL) exhibited the highest potency against M. tuberculosis
(RCMB 010126), with 2-fold increased activity than the reference drug isoniazid (MIC = 0.78
µg/mL). Also, compounds 14c, 14g and 15f (MIC = 0.78 µg/mL), displayed potent activity
which is equipotent to isoniazid.
Table 1
2.2.1.1.1. Structure activity relationship (SAR)
From the obtained anti-tubercular results, the following structure activity relationship can be
concluded: the 1H-indole containing compounds 8a,b exhibited moderate antitubercular activity.
The 5-acetyl derivatives 8a revealed better activity than its ester analog 8b (MIC= 3.12 and 6.24
µg/ml, respectively), this activity was increased by two folds upon N-benzylation of the indole as
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observed in compound 11b. On the other hand, N-methylation (11c) or N-benzylation (11d) on
the indole ring of ethyl carboxylate derivative 8b abolished the activity. Direct attachment of 5-
(un)substituted 2-oxindole to the lead scaffold in 14a-k resulted in compounds with moderate to
potent activity. Generally the ethyl carboxylate derivatives 14g-k (MIC range = 0.39-1.56 µg/ml)
revealed higher activity than their acetyl counterparts 14a-e (MIC range = 0.78-12.48 µg/ml).
The highest activity in this series was associated with the 5-chloro substituent on the 2-oxindole
ring. N-methylation or N-benzylation of the 2-oxindole in acetyl derivatives 15a-d enhanced the
activity by 4 and 2 folds, respectively, as compared to the N-unsubstituted analogs, 14a and 14c.
However, applying the same structure modification in ethyl carboxylate derivatives 15e-h
resulted in 2 fold increase in activity of the unsubstituted 2-oxindole 14g only.
2.2.1.2. Antibacterial Activity
All the prepared conjugates (8a,b, 11a-d, 14a-k and 15a-h) were evaluated for their potential
antimicrobial activity against one Gram-positive bacteria: S. aureus (RCMB 010028, Sa), and
two Gram-negative bacteria: P. aeruginosa (RCMB010043, Pa) and E. coli (RCMB 010052,
Ec). The results are displayed in Table 2, as inhibition zone (I.Z) and minimum inhibitory
concentration (MIC). From the obtained results, it can be observed that compounds 11b, 14c,
14e, 14g, 14i, 14k and 15a-h displayed broad-spectrum antibacterial activity against the tested
Gram positive and Gram negative bacteria as compared with ciprofloxacin.
Regarding Gram positive bacteria, compounds 14c, 14g, 14i, 14k, 15a, 15b, 15d and 15h
(MIC = 0.98 µg/mL) were two times more active than ciprofloxacin. On the other hand
compounds 11b, 14e, 15c, and 15e-g were equipotent to the ciprofloxacin. As for the Gram
negative bacteria P. aeruginosa, compounds 14c, 14i, 15a, 15b and 15d (MIC = 0.98 µg/mL)
were four times more potent than ciprofloxacin, while compounds 14e, 14g, 14k and 15e-h (MIC
= 1.95 µg/mL) were two times more active than ciprofloxacin, whereas compounds 14a, 14h, 14j
and 15c were equipotent to the ciprofloxacin. Regarding Gram negative bacteria E. coli, the
compounds 14c, 15a, 15b and 15d (MIC = 0.49 µg/mL) exhibited the highest potency, they were
four times more potent than the reference drug ciprofloxacin (MIC = 1.95 µg/mL). Moreover,
compounds 14a, 14i, 14k, 15c and 15f-h (MIC = 0.98 µg/mL) were two times more potent than
ciprofloxacin. While compounds 11a, 11b, 14e, 14g, 14h, 14j and 15e were equipotent to the
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ciprofloxacin. Thus, 2-oxindole based compounds exhibited better broad spectrum antibacterial
activity than indole congeners.
Table 2
2.2.1.3. Antifungal activity
Target conjugates and Amphotericin B, a reference antifungal drug, were evaluated for their
antifungal activity, by I.Z. technique and MIC, towards A. fumigates and C. albicans, Table 3.
Aspergillus and Candida spp. account for the majority of documented fungal infections.
Clinically, candidiasis and aspergillosis account for between 80% and 90% of systemic fungal
infections in immunocompromised patients. Data in Table 3 revealed that conjugates 14c, 14i,
15a, 15b, 15d and 15g (MIC = 0.49 µg/mL) exhibited the highest potency against C. albicans
organism being two times more active than the reference drug Amphotericin B (MIC = 0.98
µg/mL). While conjugates 14a, 14g, 14k, 15c and 15e were equipotent to the Amphotericin B.
On the other hand, compounds 14c, 14g, 15a, 15b, 15d and 15g revealed two fold increase in
activity than Amphotericin B, with MIC = 0.98 µg/mL against A. fumigatus organism. Moreover,
conjugates 14a, 14e, 14h, 14i, 14k, 15c, 15e and 15h were equipotent to Amphotericin B.
Table 3
2.2.1.4. Whole-cell phenotypic evaluation and anti-tubercular selectivity
Compounds 14g, 14i, 14k, 15a, 15b, 15e, 15g, 15h which displayed the most potent anti-
tubercular activity were predicted to have acceptable ADME parameters (Supplementary
Materials), therefor they were selected for further investigation of their anti-TB activity against
fast growing non-pathogenic M. aurum in an ex vivo drug screening model using SPOTi method
(Figure 2) [17]. M. aurum is a good drug screening surrogate proved to be close towards
pathogenic M. tuberculosis because of its similar cell wall components and intracellular
therapeutic targets. An integrated ex vivo drug screening model was used to provide additional
information about intracellular inhibition inside host’s primary immune cells and to evaluate the
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selected conjugates towards their permeability to macrophages RAW 264.7, their stability inside
macrophages over the time of incubation, cytotoxicity against RAW 264.7 and their selective
killing towards M. aurum [18]. Intracellular growth inhibition of M. aurum was observed in the
presence and absence of selected conjugates. All the selected conjugates were found to be
permeable through eukaryotic cells RAW 264.7 and stable over the time of incubation.
Figure 2
Table 4 displays the results of the anti-tubercular screening, cytotoxicity and the selectivity
index (SI) towards intracellular killing of mycobacterium. As shown, in Table 4, compound 15b
produced an effective killing of intracellular M. aurum at 25 µg/mL, while compounds 15a and
15h killed mycobacteria at 50 µg/mL (Table 4). Also, compounds 14g displayed inhibitory
activity at higher MIC value (100 µg/mL). On the other hand, the other tested compounds are
showing inhibition towards intracellular bacilli at higher concentrations. These results, also,
suggested that N-methylation of the 2-oxindole in acetyl derivatives enhanced the intracellular
inhibition.
SI, the ratio between growth inhibition concentration values (GIC50) in macrophages and
MIC values in bacteria, indicates the safety and druggability of the target conjugates. The tested
conjugates showed moderate cytotoxic activity towards macrophage RAW 264.7 cells, with
GIC50 values ranging from 62.5 to 500 µg/mL (Table 4). However, the most potent compound
15b exhibited the highest selectivity among the tested conjugates (SI = 5).
Table 4
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2.2.1.5. Antibacterial Activity against MRSA and VRE
Compounds 15a and 15b, which produced an effective killing of intracellular M. aurum,
were evaluated for their potential antibacterial activity against two resistant bacteria; methicillin-
resistant Staphylococcus aureus (MRSA ATCC 33592) and vancomycin- resistant Enterococcus
faecium (ATCC 700221), using Vancomycin and Ciprofloxacin as reference drugs, respectively.
The results were displayed in Table 5 as minimum inhibitory concentration (MIC in µg/mL).
Table 5
As shown in Table 5, conjugate 15b displayed potent activity towards both MRSA and VRE
(MIC = 3.90 and 7.81 µg/mL, respectively) with two-fold decreased activity than the reference
drugs Vancomycin (MIC = 1.95 µg/mL) and Ciprofloxacin (MIC = 3.90 µg/mL). Whereas,
compound 15a showed modest activity with MIC values equal 15.63 and 62.50 towards MRSA
and VRE strains, respectively.
2.2.2. In Vitro Cytotoxicity
2.2.2.1. In vitro cytotoxic activity against human normal WI-38 cells
Compounds 15a, 15b and 15h were evaluated for their ability to induce cytotoxic effect
against human normal lung fibroblast cell line (WI-38 cells), to investigate their safety [19]. The
results were expressed as half maximal inhibitory concentration (IC50), then selectivity index was
calculated (Table 6). In particular, compounds 15b and 15h showed non-significant cytotoxic
action with IC50 values of 19.5 and 52.1 µg/mL, respectively, (S.I. = 50 and 133, respectively).
Table 6
2.2.2.2. NCI, USA Cytotoxicity assay towards 60 cancer cell lines
Compounds 15b and 15h were screened by the National Cancer Institute (NCI)
Developmental Therapeutic Program (www.dtp.nci.nih.gov) for their cytotoxic activity in vitro.
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The cytotoxicity assays were performed in accordance with the protocol of the Drug Evaluation
Branch, NCI, Bethesda [20-22]. Compounds 15b and 15h were examined at one dose primary
cytotoxicity assay towards a panel of approximately 60 cancer cell lines (concentration 10-5 M).
A 48 hrs drug exposure protocol was used and sulforhodamine B (SRB) protein assay was
applied to estimate the cell viability and growth [23]. The data represented as mean growth
percentage over 60 cell lines (Table 6). The tested compounds 15b and 15h showed non-
significant cytotoxic action with mean growth = 101.5 % and 101.2, respectively. Therefore
these results ruled out nonspecific binding of compounds 15b and 15h to the basic cell
macromolecules like proteins, plasma membrane, carbohydrates and also biocompatibility to the
eukaryotic cells.
3. Conclusion
In summary, conjugating thiazolylamino-thiazolidinone with either indole 8a,b and 11a-d or
2-oxindole 14a-k and 15a-h resulted in the discovery of promising scaffold with good
antimicrobial activities. It was concluded that compound 15b revealed the best activity profile as
a broad spectrum agent with antimicrobial activity against Gram positive and Gram negative
bacteria (MIC: 0.39-0.98 µg/ml) as well as antifungal activity (MIC: 0.49-0.98 µg/ml).
Furthermore, it has in vitro and ex vivo anti-tubercular activity. An integrated ex vivo drug
screening model depicted the intracellular inhibition of mycobacterium against selected
conjugates, their permeability through primary immune cells and stability inside mouse
macrophages over the time of incubation. Compound 15b produced an effective killing of
intracellular M. aurum at 25 µg/mL, with weak cytotoxic activity towards macrophage RAW
264.7 cells (GIC50 = 125 µg/mL). Furthermore, compound 15b displayed promising activity
towards both MRSA and VRE (MIC = 3.90 and 7.81 µg/mL, respectively) with two-fold
decreased activity than the reference drugs Vancomycin (MIC = 1.95 µg/mL) and Ciprofloxacin
(MIC = 3.90 µg/mL). Also, safety of compound 15b was approved via investigating its
cytotoxicity towards cancer cell lines and human lung fibroblast normal cell line, WI-38 cells.
The results suggest some potential molecules with greater SI values to be developed further for
new therapeutic leads. Noteworthy, the broad-spectrum antimicrobial activities of 15b with MIC
spanning from 0.49 to 0.98 µg/mL indicate the possible endogenous target for compound 15b
could be a fundamental mechanism of bacterial cells (i.e. RNA, DNA, protein synthesis and/or
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cellular respiration) which must be common in various cell-types. So, further mechanistic studies
will be in progress in our laboratories and will be reported upon in the near future.
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR 8400S
spectrophotometer. Mass spectral data are given by GCMS-QP1000 EX and Helwett Packard
5988 spectrometers at 70 e.V. NMR Spectra were recorded on a Varian Mercury NMR
1
13
spectrometer or Bruker spectrophotometer. H spectrum was run at 400 MHz and C spectrum
was run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d6) and deuterated trifluoroacetic
acid (TFA-d1). Chemical shifts are expressed in values (ppm) using the solvent peak as internal
standard. All coupling constant (J) values are given in hertz. The abbreviations used are as
follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional
Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Analytical thin
layer chromatography (TLC) on silica gel plates containing UV indicator was employed
routinely to follow the course of reactions and to check the purity of products.
4.1.2. Synthesis of compounds 2a,b, 3a,b and 4a,b
Compounds 2a,b, 3a,b and 4a,b were prepared according to the literature procedure [24, 25].
4.1.3. Synthesis of 2-[(4-Methylthiazol-2-yl)amino]thiazol-4(5H)-one derivatives 5a,b
To a solution of 2-chloro-N-(4-methylthiazol-2-yl)acetamide derivatives 4a,b (5 mmol) in
absolute ethanol (15 mL), ammonium thiocyanate (0.76 g, 10 mmol) was added. This mixture
was heated under reflux for 6 hrs. The formed precipitate was filtered off while hot, and washed
with ethanol and petroleum ether, and then recrystallized from dioxane to obtain the targeted
compounds 5a,b [9, 26].
4.1.3.1. 2-[(5-Acetyl-4-methylthiazol-2-yl)imino)thiazolidin-4-one (5a).
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Yellow powder (yield: 0.87 g, 75 %), m.p. 220-221 °C; IR (KBr, ν cm-1): 3117 (NH) and 1740,
1717 (2 C=O); 1H NMR (DMSO-d6, 400 MHz)
4.02 (s, 2H, CH2), 12.27 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100 MHz)
ppm: 19.0 (CH3), 30.6 (CH3CO), 35.7 (CH2), 132.1, 155.9, 166.8, 170.9, 174.6 (C=O amide),
δ ppm: 2.49 (s, 3H, CH3CO), 2.60 (s, 3H, CH3),
δ
190.9 (C=O acetyl); MS, m/z [%]: 255 [M+, 19.94], 71 [100]; Anal. Calcd. for C9H9N3O2S2
(255.31): C, 42.34; H, 3.55; N, 16.46; Found C, 41.98; H, 3.71; N, 16.70.
4.1.3.2. Ethyl-4-methyl-2-[(4-oxothiazolidin-2-ylidene)amino]thiazole-5-carboxylate (5b).
Yellow powder (yield: 1.0 g, 70 %), m.p. 210-211 °C; IR (KBr, ν cm-1): 3128 (NH), 1708, 1697
1
(2 C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.24 (t, 3H, CH3-CH2, J = 7.20 Hz), 2.57 (s,
3H, CH3), 4.02 (s, 2H, CH2), 4.21 (q, 2H, CH3-CH2, J = 7.20 Hz), 12.24 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz)
ppm: 14.6 (CH3CH2), 17.8 (CH3), 35.7
δ
(CH2), 61.3 (CH3CH2), 117.9, 158.0, 162.1, 166.7, 171.1 (C=O ester), 174.5 (C=O amide); MS,
m/z [%]: 285 [M+, 67.30], 71 [100]; Anal. Calcd. for C10H11N3O3S2 (285.34): C, 42.09; H, 3.89;
N, 14.73; found C, 41.87; H, 4.02; N, 14.98.
4.1.4. Synthesis of 1H-indole-3-carbaldehyde 7
Compound 7 was prepared according to the literature procedures [27].
4.1.5. General procedure for synthesis of N-substituted-1H-indole-3-carbaldehydes 10a,b.
To a stirred suspension of NaH (60 percent in mineral oil, 1 gm, 2.5 mmol) and 1H-indole-3-
carbaldehyde 7 (3.0 g, 2 mmol) in DMF, bromomethane 9a or benzyl bromide 9b (2 mmol) was
added drop wise at 00C. The reaction mixture was stirred for 24 hrs at room temperature after
complete addition of alkyl halide. The reaction mixture was poured into ice water, the formed
precipitate was filtered off, washed with water and petroleum ether then recrystallized from
ethanol to obtain compounds 10a,b [28].
4.1.6. General procedure for synthesis of target compounds 8a,b and 11a-d
To a well-stirred hot solution of the key intermediates 5a,b (3 mmol) and sodium acetate
(0.49 g, 6 mmol) in acetic acid (15 mL), the appropriate 1H-indole-3-carbaldehyde derivative 7,
10a or 10b (4.5 mmol) was added. The reaction mixture was heated under reflux for 3 hrs. The
14

ACCEPTED MANUSCRIPT
formed solid was filtered off while hot, washed with hot ethanol, and crystallized from DMF to
furnish the target compounds 8a, b and 11a-d, respectively.
4.1.6.1. 5-[(1H-Indol-3-yl)methylene]-2-((5-acetyl-4-methylthiazol-2-yl)imino)thiazolidin-4-one
(8a).
Yellow powder (yield: 0.51 g, 45%), m.p. >300 °C; IR (KBr, ν cm-1): 3244, 3190 (2 NH) and
1701, 1651 (2 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.48 (s, 3H, CH3C=O), 2.68 (s, 3H,
CH3), 7.17-7.26 (m, 2H, H-5, H-6 of indolyl), 7.50 (d, 1H, H-7 of indolyl, J=8.0 Hz), 7.79 (s,
1H, C-2 of indol), 7.89 (d, 1H, H-4 of indolyl, J=8.0), 8.01 (s, 1H, -CH=), 12.11 (s, 1H, NH
indol exchanged with D2O of indolyl), 12.62 (s, 1H, NH exchanged.D2O.); 13C.NMR (DMSO-d6,
100 MHz)
δ ppm: 19.0 (CH3), 30.6 (CH3C=O), 111.1, 112.8, 112.9, 117.1, 118.9, 121.6, 123.2,
123.6, 125.9, 127.3, 129.6, 136.1, 136.7, 156.3 (aromatic carbons), 167.3 (C=O amide), 190.8
(C=OCH3); Anal. Calcd. for C18H14N4O2S2 (382.46): C, 56.53; H, 3.69; N, 14.65; found C,
56.38; H, 3.78; N, 14.92.
4.1.6.2.
Ethyl
2-{[-5-((1H-indol-3-yl)methylene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (8b).
Yellow powder (yield: 0.49 g, 40%), m.p. 257-260°C; IR (KBr, ν cm-1): 3309 (NH) and 1701,
1678 (2C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.26 (t, 3H, CH3-CH2-, J = 6.80 Hz), 2.66
(s, 3H, CH3), 4.24 (q, 2H, CH3-CH2-, J=6.80 Hz), 7.24 (t, 1H, H-6 of indolyl, J=7.32), 7.32 (t,
1H, H-5 of indolyl, J=7.6Hz), 7.50 (d, 1H, H-7 of indolyl, J=8.0), 7.79 (s, 1H, C-2 of indolyl),
7.88 (d, 1H, H-4 of indolyl, J=8.0), 8.0 (s, 1H, -CH=), 12.13 (s, 1H, NH of indolyl exchanged
with D2O), 12.63 (s, 1H, NH exchanged with D2O); 13C NMR (DMSO-d6, 100 MHZ)
δ ppm :
14.6 (CH3CH2), 18.0 (CH3), 61.3 (CH2CH3), 111.1, 112.9, 117.1, 118.9, 121.6, 123.6, 125.8,
127.3, 129.6, 136.7, 158.3, 159.2, 162.1 (aromatic carbons), 167.3 (C=O ester), 170.6 (C=O
amide); Anal. Calcd. for C19H16N4O3S2 (412.48): C, 55.33; H, 3.91; N, 13.58; found C, 55.49; H,
3.98; N, 13.67
4.1.6.3.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-((1-methyl-1H-indol-3-
yl)methylene)thiazolidin-4-one (11a).
Yellow powder (yield: 0.62 g, 52%), m.p. 295-297 °C ; IR (KBr, ν cm-1) 3395 (NH) and 1701,
1659 (2C=O); 1H NMR (DMSO-d6, 400 MHz)
δ
ppm: 2.48 (s, 3H, CH3C=O), 2.62 (s, 3H, CH3),
15

ACCEPTED MANUSCRIPT
3.95 (s, 3H, N-CH3 of indolyl), 7.21-7.33 (m, 2H, H-5, H-6 of indolyl), 7.56 (d, 1H, H-7 of indol,
J = 8.0), 7.82 (s, 1H, C-2 of indol), 7.91 (d, 1H, H-4 of indolyl, J = 8.0 Hz), 7.99 (s, 1H, -CH=),
12.59 (s, 1H, NH exchanged with D2O); Anal. Calcd. for C19H16N4O2S2 (396.48): C, 57.56; H,
4.07; N, 14.13; found C, 57.17; H, 4.05; N, 14.25.
4.1.6.4.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-((1-benzyl-1H-indol-3-
yl)methylene)thiazolidin-4-one (11b).
Yellow powder (yield: 0.55 g, 39%), m.p. 297-300°C; IR (KBr, ν cm-1): 3150 (NH) and 1701,
1
1658 (2 C=O); H NMR (DMSO-d6)
δ ppm: 2.48 (s, 3H, CH3C=O), 2.68 (s, 3H, CH3), 5.60 (s,
2H, CH2-Ar of indol), 7.20-7.36 (m, 7H, H-5, H-6 of indolyl and 5H of phenyl ring), 7.59 (d, 1H,
H-7 of indolyl, J = 7.20), 7.91-7.94 (m, 2H, C-2, H-4 of indol), 8.00 (s, 1H, -CH=), 12.62 (s, 1H,
NH exchanged with D2O); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 19.1 (CH3), 30.6 (CH3C=O),
50.2 (CH2), 111.8, 117.8, 119.3, 122.0, 123.8, 125.2, 127.9, 128.3, 129.1, 132.0, 136.7, 137.3,
156.3, 159.3, 167.3 (aromatic carbons), 170.4 (C=O amide), 190.8 (C=OCH3); Anal. Calcd. for
C25H20N4O2S2 (472.58): C, 63.54; H, 4.27; N, 11.86; found C, 63.50; H, 4.52; N, 11.42
4.1.6.5. Ethyl 4-methyl-2-{[-5-((1-methyl-1H-indol-3-yl)methylene)-4-oxothiazolidin-2-
ylidene]amino}thiazole-5-carboxylate (11c).
Yellow powder (yield: 0.51 g, 40%), m.p. 280-282 °C ; IR (KBr, ν cm-1): 3120 (NH), 1701, 1678
1
(2C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.27 (t, 3H, CH3-CH2-, J = 7.20 Hz), 2.69 (s,
3H, CH3) , 3.94 (s, 3H, N-CH3 of indolyl), 4.24 (q, 2H, CH3-CH2-, J=7.2 Hz), 7.23 (t, 1H, H-6
of indol, J=7.60 Hz), 7.31 (t, 1H, H-5 of indolyl, J=7.60 Hz), 7.56 (d, 1H, H-7 of indolyl, J=8.0),
7.81 (s, 1H, C-2 of indolyl), 7.90 (d, 1H, H-4 of indol, J=8.0), 7.98 (s, 1H, -CH=), 12.58 (s, 1H,
NH exchanged with D2O); 13C NMR (DMSO-d6, 100 MHz)
δ ppm: 14.64 (CH3CH2), 18.1
(CH3), 33.9 (N-CH3), 61.3 (CH3CH2), 110.0, 111.3, 117.2, 119.0, 121.9, 123.6, 125.3, 127.7,
133.0, 136.8, 137.3, 158.5, 159.1, 162.1 (aromatic carbons), 167.30 (C=O ester), 170.5 (C=O
amide); MS m/z [%]: 426 [M+, 17.15], 187 [100]; Anal. Calcd. for C20H18N4O3S2 (426.5): C,
56.32; H, 4.25; N, 13.14; found C, 56.08; H, 4.41; N, 13.29.
4.1.6.6. Ethyl 2-{[-5-((1-benzyl-1H-indol-3-yl)methylene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (11d).
16

ACCEPTED MANUSCRIPT
Yellow powder (yield: 0.6 g, 40%), m.p. 260-263 °C; IR (KBr, ν cm-1): 3150 (NH) and 1701,
1678 (2C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.28 (t, 3H, CH3-CH2-, J = 7.20 Hz), 2.66
(s, 3H, CH3), 4.24 (q, 2H, CH3-CH2-, J=6.80 Hz), 5.60 (s, 2H, CH2-Ar of indol), 7.20-7.35 (m,
7H, H-5, H-6 of indolyl and 5H of phenyl ring), 7.59 (d, 1H, H-7, J=8.80 Hz), 7.91-7.96 (m, 2H,
13
H-2, H-4 of indol), 8.00 (s, 1H, -CH=), 12.62 (s, 1H, NH exchanged with D2O), C NMR
(DMSO-d6, 100 MHz)
δ ppm: 14.63 (CH3CH2), 18.0 (CH3), 50.2 (CH2Ph), 61.3 (CH3CH2),
110.8, 111.7, 117.2, 117.8, 119.2, 122.0, 123.8, 125.1, 127.7, 127.9, 128.3, 129.20, 132.0, 136.7,
137.4, 158.4, 159.20, 162.0 (aromatic carbons), 167.2 (C=O ester), 170.5 (C=O amide); MS m/z
[%]: 502 [M+, 44.70], 263 [100]; Anal. Calcd. for C26H22N4O3S2 (502.61) : C, 62.13; H, 4.41; N,
11.15; found C, 62.01; H, 4.38; N, 11.35.
4.1.7. General procedure for synthesis of N-substituted isatins 13a-d.
To a mixture of isatins 12a,c (3 mmol) and anhydrous potassium carbonate (1.24 g, 9 mmol)
in dry DMF (15 mL), bromomethane 9a or benzyl bromide 9b (6 mmol) was added. The mixture
was stirred at room temperature for 24 hrs. The reaction mixture was poured into ice water. The
formed precipitate was filtered off and washed with water and petroleum ether and recrystallized
from ethanol to afford compounds 13a-d [29, 30].
4.1.8. General procedure for synthesis of target compounds 14a-k and 15a-h.
To a well-stirred hot solution of 2-(thiazol-2-ylimino)thiazolidin-4-one derivatives 5a,b
(0.003 mol) and sodium acetate (0.49 gm, 6 mmol) in acetic acid (15 mL), the appropriate isatins
12a-f or 13a, d (4 mmol) were added. The reaction mixture was heated under reflux for 3 hrs.
The formed precipitate was filtered off while hot, washed with hot ethanol and crystallized from
DMF to furnish the target compounds 14a-k and 15a-h, respectively.
4.1.8.1. 2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-2-oxoindolin-3-ylidene)thiazolidin-4-one
(14a).
Red powder (yield: 0.57 g, 50%), m.p. > 300°C; IR (KBr, ν cm-1): 3178, 3128 (2NH) and 1716,
1693, 1663 (3 C=O); 1H NMR (DMSO-d6, 300 MHz)
δ ppm: 2.49 (s, 3H, CH3C=O), 2.64 (s, 3H,
CH3), 6.93 (d, 1H, H-7 of isatin, J = 7.80 Hz), 7.06 (t, 1H , H-5 of isatin, J = 7.80 Hz), 7.37 (t,
17

ACCEPTED MANUSCRIPT
1H, H-6 of isatin, J = 7.80 Hz), 8.84 (br s, 1H, H-4 of isatin , 11.10 (s, 1H, NH of isatin
exchanged with D2O), 13.05 (s, 1H, NH exchanged with D2O); MS m/z [%]: 384 [M+, 42.78],
175 [100]; Anal. Calcd. for C17H12N4O3S2 (384.43): C, 53.11; H, 3.15; N, 14.57; found C, 53.28;
H, 3.27; N, 14.81
4.1.8.2.
2-[(5-Acetyl-4-methylthiazol-2-yl)amino]-5-(-5-fluoro-2-oxoindolin-3-
ylidene)thiazolidin-4-one (14b).
Red powder (yield 0.54 g, 45%), m.p. >300°C; IR (KBr, ν cm-1): 3159, 3116 (2NH) and 1716,
1701, 1654 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δppm: 2.47 (s, 3H, CH3C=O), 2.61 (s, 3H,
CH3), 6.87-6.90 (m, 1H, H-6 of isatin), 7.19 (t, 1H , H-7 of isatin, J = 8.80 Hz), 8.60 (s, 1H, H-4
of isatin), 11.11 (s, 1H, NH of isatin exchanged with D2O), 13.07 (s, 1H, NH exchanged with
D2O); 13C NMR (TFA-d1, 100 MHz)
δ ppm: 13.11, 27.71, 116.66, 116.94, 120.05, 121.84,
122.09, 127.35, 128.77, 133.45, 139.14, 146.90, 159.42, 165.89, 168.87, 171.17, 193.50; MS m/z
[%]: 402 [M+, 93.21], 193 [100]; Anal. Calcd. for C17H11FN4O3S2 (402.42): C, 50.74; H, 2.76; N,
13.92; found C, 50.98; H, 2.67; N, 14.25.
4.1.8.3.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-5-chloro-2-oxoindolin-3-
ylidene)thiazolidin-4-one (14c).
Red powder (yield 0.54 g, 43%), m.p. > 300 °C; IR (KBr, ν cm-1): 3120, 3105 (2NH) and 1701,
1680, 1666 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.47 (s, 3H, CH3C=O), 2.61 (s, 3H,
CH3), 6.89 (d, 1H, H-6 of isatin, J = 8.40 Hz), 7.37 (d, 1H, H-7 of isatin, J = 7.60 Hz), 8.81 (s,
1H, H-4 of isatin), 11.25 (s, 1H, NH of isatin exchanged with D2O), 13.13 (s, 1H, NH exchanged
with D2O); 13C NMR (TFA-d1, 100 MHz)
δ ppm: 18.97, 33.58, 120.98, 123.21, 124.33, 126.57,
133.35, 134.71, 137.55, 143.89, 147.58, 152.79, 171.74, 174.76, 175.17, 177.04, 199.38; MS m/z
[%]: 420 [M++2, 1.21], 418 [M+, 4.68], 96 [100]; Anal. Calcd. for C17H11ClN4O3S2 (418.87): C,
48.75; H, 2.65; N, 13.38; found C, 48.91; H, 2.78; N, 13.52
4.1.8.4.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-5-bromo-2-oxoindolin-3-
ylidene)thiazolidin-4-one (14d).
Red powder (yield 0.52 g, 38%), m.p. > 300 °C; IR (KBr, ν cm-1): 3178, 3124 (2 NH) and 1693,
1670, 1647 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.47 (s, 3H, CH3C=O), 2.59 (s, 3H,
18

ACCEPTED MANUSCRIPT
CH3), 6.86 (d, 1H, H-6 of isatin, J = 7.60 Hz), 7.49 (br s, 1H, H-7 of isatin), 8.98 (s, 1H, H-4 of
isatin), 11.18 (s, 1H, NH of isatin exchanged with D2O), 13.04 (s, 1H, NH exchanged with D2O);
MS m/z [%]: 464 [M++2, 63.88], 462 [M+, 57.48], 265 [100]; Anal. Calcd. for C17H11BrN4O3S2
(463.32): C, 44.07; H, 2.39; N, 12.09; found C, 44.23; H, 2.06; N, 12.43......................................
4.1.8.5.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-5-methoxy-2-oxoindolin-3-
ylidene)thiazolidin-4-one (14e).
Reddish black powder (yield 0.63 g, 50%), m.p. > 300 °C; IR (KBr, ν cm-1): 3152, 3113 (2 NH)
1
and 1720, 1693, 1670 (3 C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.48 (s, 3H, CH3C=O),
2.61 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 6.78 (d, 1H, H-6 of methoxy isatin, J = 8.40 Hz), 6.93 (d,
1H, H-7 of methoxy isatin, J = 8.40 Hz), 8.48 (s, 1H, H-4 of methoxy isatin), 10.86 (s, 1H, NH
of methoxy isatin exchanged with D2O), 12.97 (s, 1H, NH exchanged with D2O); Anal. Calcd.
for C18H14N4O4S2 (414.45): C, 52.16; H, 3.40; N, 13.52; found C, 52.44; H, 3.53; N, 13.79.
4.1.8.6.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-2-oxo-5-(trifluoromethoxy)indolin-3-
ylidene)thiazolidin-4-one (14f).
Red powder (yield 0.71 g, 51%), m.p. > 300 °C; IR (KBr, ν cm-1): 3150, 3124, (2 NH) and 1720,
1708, 1651 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.48 (s, 3H, CH3C=O), 2.65 (s, 3H,
CH3), 6.99 (d, 1H, H-6 of isatin, J = 8.80 Hz), 7.33 (d, 1H, H-7 of isatin, J = 8.80 Hz), 8.84 (s,
1H, H-4 of isatin), 11.35 (s, 1H, NH of isatin exchanged with D2O), 13.16 (s, 1H, NH exchanged
13
with D2O); C NMR (TFA-d1, 100 MHz)
δ ppm: 19.06, 33.68, 118.35, 119.31, 124.44, 125.99,
129.06, 130.84, 133.77, 134.86, 137.93, 147.30, 152.15, 152.88, 171.87, 174.88, 177.05, 199.45;
MS m/z [%]:468 [M+, 47.97], 259 [100]; Anal. Calcd. for C18H11F3N4O4S2 (468.43): C, 46.15; H,
2.37; N, 11.96; found C, 46.28; H, 2.29; N, 12.21.
4.1.8.7.
Ethyl
4-methyl-2-{[-4-oxo-5-(-2-oxoindolin-3-ylidene)thiazolidin-2-
ylidene]amino}thiazole-5-carboxylate (14g).
Red powder (yield 0.72 g, 52%), m.p. > 300 °C; IR (KBr, ν cm-1): 3174, 3140 (2 NH) and 1720,
1708, 1693 (3 C=O); 1H NMR (DMSO-d6, 300 MHz)
δ ppm: 1.28 (t, 3H, CH3-CH2-, J = 6.8 Hz),
2.62 (s, 3H, CH3), 4.24 (q, 2H, CH3-CH2-, J = 6.3 Hz), 6.91 (d, 1H, H-7 of isatin, J = 7.80), 7.04
(t, 1H ,H-5 of isatin, J = 7.80 Hz), 7.36 (t, 1H, H-6 of isatin, J=7.50 Hz), 8.81 (br s, 1H, H-4 of
isatin), 11.13 (s, 1H, NH of isatin exchanged with D2O), 13.03 (s, 1H, NH exchanged with D2O);
19

ACCEPTED MANUSCRIPT
13C NMR (TFA-d1, 100 MHz)
δ ppm: 12.01, 12.04, 64.66, 112.81, 113.32, 115.62, 116.13,
119.433, 124.69, 125.49, 130.07, 133.25, 135.79, 142.99, 147.45, 166.37, 168.72, 170.93; MS
m/z [%]: 414 [M+, 11.55], 175 [100]; Anal. Calcd. for C18H14N4O4S2 (414.45): C, 52.16; H, 3.40;
N, 13.52; found C, 52.40; H, 3.68; N, 13.74.
4.1.8.8. Ethyl 2-{[-5-(-5-fluoro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (14h).
Red powder (yield 0.65 g, 50%), m.p. >300 °C; IR (KBr, ν cm-1): 3120, 3159 (2 NH) and 1720,
1
1716, 1693 (3 C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.28 (t, 3H, CH3-CH2-, J = 6.40
Hz), 2.61 (s, 3H, CH3), 4.24 (q, 2H, CH3-CH2-, J = 6.40 Hz), 6.87-6.91 (m, 1H, H-6 of fluoro
isatin), 7.20 (br s, 1H, H-7 of flouroisatin), 8.60 (br s, 1H, H-4 of fluoroisatin), 11.14 (s, 1H, NH
of isatin exchanged with D2O), 13.03 (s, 1H, NH exchanged with D2O); MS m/z [%]:432 [M+,
100]; Anal. Calcd. for C18H13FN4O4S2 (432.44): C, 49.99; H, 3.03; N, 12.96; found C, 50.15; H,
3.11; N, 13.23.
4.1.8.9. Ethyl 2-{[-5-(-5-chloro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (14i).
Red powder (yield 0.54 g, 40%), m.p. >300 °C; IR (KBr, ν cm-1): 3155, 3109 (2 NH) and 1708,
1685, 1670 (3 C=O); 1H NMR (DMSO-d6, 300 MHz)
δ ppm: 1.29 (br s, 3H, CH3-CH2-), 2.55 (s,
3H, CH3), 4.24 (br s, 2H, CH3-CH2-), 6.92 (br s, 1H, H-6 of chloroisatin), 7.33 (br s, 1H, H-7 of
chloroisatin), 9.03 (br s, 1H, H-4 of chloroisatin), 11.07 (s, 1H, NH of chloroisatin exchanged
with D2O), 11.94 (s, 1H, NH exchanged with D2O); 13C NMR (TFA-d1, 100 MHz)
δ ppm: 17.96,
24.39, 70.57, 118.55, 125.62, 126.21, 133.69, 135.23, 136.38, 137.26, 140.75, 147.11, 153.44,
168.23, 171.74, 174.54, 175.25, 176.20; MS m/z [%]: 450 [M++2, 8.29], 448 [M+, 23.70], 209
[100]; Anal. Calcd. for C18H13ClN4O4S2 (448.9): C, 48.16; H, 2.92; N, 12.48; found C, 48.02; H,
3.08; N, 12.65.
4.1.8.10. Ethyl 2-{[-5-(-5-bromo-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (14j).
Red powder (yield 1.0 g, 70%), m.p. > 300 °C; IR (KBr, ν cm-1): 3155, 3101 (2 NH) and 1708,
1685, 1670 (3 C=O); 1H NMR (DMSO-d6, 300 MHz)
δ ppm: 1.29 (br s, 3H, CH3-CH2-), 2.55 (s,
20

ACCEPTED MANUSCRIPT
3H, CH3), 4.24 (br s, 2H, CH3-CH2-), 6.92 (br s, 1H, 4,H-6 of bromoisatin), 7.34 (br s, 1H ,H-7
of bromoisatin), 9.02 (s, 1H, H-4 of bromoisatin), 11.07 (s, 1H, NH of bromoisatin exchanged
with D2O), 11.92 (s, 1H, NH exchanged with D2O); Anal. Calcd. for C18H13BrN4O4S2 (493.35):
C, 43.82; H, 2.66; N, 11.36; found C, 43.58; H, 2.72; N, 11.52.
4.1.8.11. Ethyl 2-{[-5-(-5-methoxy-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (14k).
Reddish black powder (yield 0.8 g, 60%), m.p. > 300 °C; IR (KBr, ν cm-1): 3155, 3132 (2 NH)
and 1708, 1701, 1693 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.27 (t, 3H, CH3-CH2-, J
= 6.4 Hz), 2.59 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 4.22 (q, 2H, CH3-CH2-, J=6.8 Hz), 6.78 (d,
1H, H-6 of methoxy isatin, J=8.40 Hz), 6.92 (d, 1H, H-7 of methoxy isatin, J= 8.0 Hz), 8.48 (s,
1H,H-4of methoxy isatin), 11.42 (s, 1H, NH of methoxy isatin exchanged with D2O), 13.18 (s,
1H, NH exchanged with D2O); 13C NMR (TFA-d1, 100 MHz)
δ ppm: 17.71, 24.39, 61.87, 70.34,
118.48, 121.88, 125.22, 125.92, 127.88, 132.57, 138.44, 143.82, 153.17, 160.92, 168.23, 171.77,
174.38, 175.33, 176.36; MS m/z [%]: 444 [M+, 35.58], 72 [100]; Anal. Calcd. for C19H16N4O5S2
(444.48): C, 51.34; H, 3.63; N, 12.61; found C, 51.58; H, 3.71; N, 12.49.
4.1.8.12.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-1-methyl-2-oxoindolin-3-
ylidene)thiazolidin-4-one (15a).
Red powder (yield 0.65 g, 55%), m.p. >300 °C; IR (KBr, ν cm-1): 3113 (NH) and 1689, 1662,
1658 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.47 (s, 3H, CH3C=O), 2.61 (s, 3H, CH3),
3.22 (s, 3H, N-CH3), 7.04-7.12 (m, 2H, H-5, H-7 of isatin), 7.43 (t, 1H, H-6 of isatin, J = 7.6
Hz), 8.85 (s, 1H, H-4 of isatin), 12.98 (s, 1H, NH exchanged with D2O); MS m/z [%]: 398 [M+,
17.55], 71 [100]; Anal. Calcd. for C18H14N4O3S2 (398.46): C, 54.26; H, 3.54; N, 14.06; found C,
54.18; H, 3.60; N, 14.23.
4.1.8.13.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-5-chloro-1-methyl-2-oxoindolin-3-
ylidene)thiazolidin-4-one (15b).
Red powder (yield 0.78 g, 60%), m.p. >300 °C; IR (KBr, ν cm-1): 3136 (NH) and 1716, 1689 (3
C=O); 1H NMR (DMSO-d6, 400 MHz)
3H, N-CH3), 7.13 (d, 1H, H-6 of isatin, J = 8.4 Hz), 7.51 (d, 1H, H-7 of isatin, J = 8.8 Hz), 8.90
δ ppm: 2.48 (s, 3H, CH3C=O), 2.64 (s, 3H, CH3), 3.23 (s,
(s, 1H, H-4 of isatin), 13.04 (s, 1H, NH , exchanged with D2O); 13C NMR (TFA-d1, 100 MHz)
δ
21

ACCEPTED MANUSCRIPT
ppm: 13.07, 25.85, 27.87, 110.40, 119.99, 127.50, 128.12, 129.56, 130.89, 131.41, 134.71,
143.64, 147.22, 165.86, 167.57, 168.96, 171.25, 193.70; MS m/z [%]: 434 [M++2, 29.18], 432
[M+, 65.73], 223 [100]; Anal. Calcd. for C18H13ClN4O3S2 (432.9): C, 49.94; H, 3.03; N, 12.94;
found C, 49.87; H, 3.14; N, 13.20.
4.1.8.14.. 2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-1-benzyl-2-oxoindolin-3-
ylidene)thiazolidin-4-one (15c).
Red powder (yield 0.92 g, 65%), m.p. 285-287°C; IR (KBr, ν cm-1): 3109 (NH) and 1708, 1693,
1651 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.48 (s, 3H, CH3C=O), 2.65 (s, 3H, CH3),
5.02 (s, CH2-Ar), 7.04 (d, 1H, H-7 of isatin, J = 8.0 Hz), 7.10 (t, 1H, H-5 of isatin, J = 8 Hz),
7.21-7.37 (m, 6H, H-6 of isatin, Ar-H of benzyl ring), 8.88 (br s, 1H, H-4 of isatin), 13.08 (s, 1H,
13
NH exchanged with D2O); C NMR (TFA-d1, 100 MHz)
δ ppm: 19.06, 33.83, 50.50, 116.75,
121.11, 122.15, 124.46, 125.32, 130.88, 132.36, 134.17, 134.75, 136.18, 139.04, 139.14, 141.51,
150.85, 153.14, 172.26, 174.09, 177.62, 199.73; MS m/z [%]: 474 [M+, 19.65], 265 [100]; Anal.
Calcd. for C24H18N4O3S2 (474.55): C, 60.74; H, 3.82; N, 11.81; found C, 60.89; H, 3.89; N,
12.07.
4.1.8.15.
2-[(5-Acetyl-4-methylthiazol-2-yl)imino]-5-(-1-benzyl-5-chloro-2-oxoindolin-3-
ylidene)thiazolidin-4-one (15d).
Red powder (yield 1.07 g, 70%), m.p. > 300°C; IR (KBr, ν cm-1): 3109 (NH) and 1693, 1665,
1655 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 2.48 (s, 3H, CH3C=O), 2.63 (s, 3H, CH3),
5.02 (s, CH2-Ar), 7.04 (d, 1H, H-6 of chloroisatin, J = 8.8 Hz), 7.26-7.34 (m, 5H, Ar-H of benzyl
ring), 7.43 (d, 1H, H-7 of chloroisatin, J = 8.00 Hz), 8.90 (s, 1H, H-4 of chloroisatin), 13.04 (s,
1H, NH exchanged with D2O); MS m/z [%]: 511 [M++2, 1.52], 509 [M+, 5.85], 466 [100]; Anal.
Calcd. for C24H17ClN4O3S2 (509.0): C, 56.63; H, 3.37; N, 11.01; found C, 56.48; H, 3.31; N,
11.28.
4.1.8.16.
Ethyl
4-methyl-2-{[-5-(-1-methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene]amino}thiazole-5-carboxylate (15e).
Red powder (yield 0.83 g, 65%), m.p. > 300 °C; IR (KBr, ν cm-1): 3109 (NH) and 1720, 1710,
1655 (3 C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.27 (t, 3H, CH3-CH2-, J = 7.2 Hz), 2.62
(s, 3H, CH3) , 3.22 (s, 3H, N-CH3), 4.24 (q, 2H, CH3-CH2-, J=7.20 Hz), 7.07-7.13 (m, 2H, H-5,
22

ACCEPTED MANUSCRIPT
H-7 of isatin), 7.43 (t, 1H, H-6 of isatin, J = 7.8 Hz), 8.84 (br s, 1H, H-4 of isatin), 13.05 (s, 1H,
13
NH exchanged with D2O); C NMR (TFA-d1, 100 MHz)
δ ppm: 12.20, 25.82, 64.74, 65.73,
110.00, 112.91, 113.41, 119.20, 125.00, 125.56, 130.12, 133.12, 135.62, 145.43, 147.55, 166.51,
168.04, 168.84, 171.02; MS m/z [%]: 428 [M+, 33.42], 189 [100]; Anal. Calcd. for C19H16N4O4S2
(428.48): C, 53.26; H, 3.76; N, 13.08; found C, 53.40; H, 3.88; N, 13.29.
4.1.8.17.
Ethyl
2-{[-5-(-5-chloro-1-methyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene]amino}-4-methylthiazole-5-carboxylate (15f).
Red powder (yield 0.73 g, 53%), m.p. > 300 °C; IR (KBr, ν cm-1): 3117 (NH) and 1708, 1701,
1693 (3C=O); 1H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.28 (t, 3H, CH3-CH2-, J = 6.8 Hz), 2.64
(s, 3H, CH3), 3.23 (s, 3H, N-CH3 of chloroisatin), 4.25 (q, 2H, CH3-CH2-, J = 7.2 Hz), 7.13 (d,
1H, H-6 of chloroisatin, J = 8.4 Hz), 7.51 (d, H1, H-7 of chloroisatin, J = 8.4 Hz), 8.88 (s, 1H,
H-4 of chloroisatin), 13.05 (s, 1H, NH exchanged with D2O); MS m/z [%]: 464 [M++2, 25.08],
462 [M+, 55.88], 223 [100]. Anal. Calcd. for C19H15ClN4O4S2 (462.92): C, 49.30; H, 3.27; N,
12.10; found C, 49.64; H, 3.08; N, 12.43.
4.1.8.18. Ethyl 2-{[-5-(-1-benzyl-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-ylidene]amino}-4-
methylthiazole-5-carboxylate (15g).
Red powder (yield 0.6 g, 40%), m.p. 260-261°C; IR (KBr, ν cm-1): 3186 (NH) and 1716, 1693,
1
1670 (3 C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.27 (br s, 3H, CH3-CH2-), 2.61 (s, 3H,
CH3) , 4.23 (q, 2H, CH3-CH2-, J = 6.4 Hz) , 5.00 (s, 2H, CH2-Ar) , 7.01 (d, 1H, H-7 of isatin, J =
8.0 Hz) , 7.08 (t, 1H, H-5 of isatin, J = 8.0 Hz), 7.23-7.37 (m, 6H, H-4 of isatin, 5H of benzyl
ring), 8.86 (br s, 1H, H-4 of isatin), 13.05 (s, 1H, NH exchanged with D2O); MS m/z [%]: 504
[M+, 1.76], 164 [100]; Anal. Calcd. for C25H20N4O4S2 (504.58): C, 59.51; H, 4.00; N, 11.10
found C, 59.32; H, 4.13; N, 11.43.
4.1.8.19.
Ethyl
2-{[-5-(-1-benzyl-5-chloro-2-oxoindolin-3-ylidene)-4-oxothiazolidin-2-
ylidene]amino}-4-methylthiazole-5-carboxylate (15h).
Red powder (yield 1.0 g, 67%), m.p. > 300 °C; IR (KBr, ν cm-1): 3109 (NH) and 1693, 1666,
1
1655 (3 C=O); H NMR (DMSO-d6, 400 MHz)
δ ppm: 1.27 (br s, 3H, CH3-CH2-), 2.61 (s, 3H,
CH3), 4.23 (q, 2H, CH3-CH2- , J=6.8 Hz), 5.01 (s, 2H, CH2-Ar), 7.05 (d, 1H, H-6 of chloroisatin
J = 8.2 Hz), 7.24-7.34 (m, 5H, of benzyl ring), 7.42 (d, 1H, H-7 of chloroisatin J = 8.2), 8.89 (s,
23

ACCEPTED MANUSCRIPT
1H, H-4 of chloroisatin), 13.21 (s, 1H, NH exchanged with D2O); 13C NMR (TFA-d1, 100 MHz)
δ
ppm: 12.17, 24.39, 44.67, 64.73, 110.00, 111.61, 112.89, 120.25, 126.41, 127.76, 128.33,
128.86, 129.62, 130.86, 131.38, 132.84, 134.57, 143.05, 147.64, 166.05, 167.84, 168.75, 170.48;
Anal. Calcd. for C25H19ClN4O4S2 (539.02): C, 55.71; H, 3.55; N, 10.39; found C, 56.02; H,
3.67; N, 10.18.
4.2. Biological Evaluation
4.2.1. Anti-tubercular Activity
M. tuberculosis (RCMB 010126) strain was obtained from the culture collection of the Regional
Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Isoniazid
was used as reference drug. Anti-tubercular activity of the newly synthesized molecules (8a,b,
11a-d, 14a-k and 15a-h) was evaluated using the Microplate Alamar Blue Assay (MABA) as
reported earlier [12, 16]. For ex vivo SPOTi assay, M. aurum [Optical Density (OD)600≈1] were
first checked for quality control using cold Ziehl–Neelsen (ZN) staining (also called ‘acid fast
staining’; TB-colour staining kit, BDH/Merck) according to the manufacturer’s protocol. Wells
with DMSO or H2O were used as negative controls and isoniazid as a positive control.
4.2.2. Intracellular assay using a mycobacteria-infected macrophage model
5
For the intracellular killing assay, RAW 264.7 macrophages (5×10 cells per well) were
◦
infected with M. aurum at 1:10 MOI (multiplicity of infectivity) for 1 h at 37 C in a 24-well
plate. The culture was washed with RPMI-1640 thrice and incubated with different
concentration of inhibitors (0, 12.5, 25, 50, 100 and 200 µg/mL) in RPMI-1640 complete
medium. Inhibitors were incubated along with infected macrophages for 48 h at 37°C CO2
incubator. Macrophages were then washed twice with RPMI-1640 and lysed in 500 µL of
distilled water at room temperature for 10 min. The lysed cells were centrifuged and
suspended in 50 µL of distilled water. Then, 5 µL of suspended cells was spotted onto wells
°
of a 24-well plate containing MB7H10 OADC agar and incubated at 37 C for 4 days to
determine intracellular survival and ex vivo MIC.
4.2.3. Antimicrobial activity
24

ACCEPTED MANUSCRIPT
All tested strains were provided from culture collection of the RCMB. Antibacterial and
antifungal activities were expressed as the diameter of inhibition zones; agar well diffusion
method was used as reported earlier [13, 14]. While, MIC was performed by a serial dilution
technique described by Irobiet al. [31]. Amphotericin B and ciprofloxacin were used as
antifungal and antibacterial positive control; respectively. The results reported are means of
at least three separate experiments.
4.2.4. In vitro cytotoxic activity by NCI-USA.
The anticancer assays were performed in accordance with the protocol of the Drug
Evaluation Branch, NCI, Bethesda [20]. A 48 h drug exposure protocol was used and
sulforhodamine B (SRB) protein assay [23] was applied to estimate the cell viability and
growth, as reported earlier [32]. The human tumor cell lines of the cancer screening panel
were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-
glutamine. After cell inoculation, the microtiter plates were incubated at 37 oC, 5% CO2, 95%
air and 100% relative humidity for 24 h prior to addition of experimental drugs, then two
plates of each cell line were fixed in situ with trichloroacetic acid, to represent a
measurement of the cell population for each cell line at the time of drug addition (Tz). Aliquot
of 100 µl of the drug dilution was added to the appropriate microtiter wells already
containing 100 µl of medium, resulting in the required final drug concentration (10 µM).
Triplicate wells were prepared for each individual dose. Following drug addition, the plates
o
were incubated for an additional 48 h at 37 C, 5% CO2, 95% air, and 100% relative
humidity. For adherent cells, the assay was terminated by the addition of cold TCA. Cells
were fixed in situ by the gentle addition of 50 µl of cold 50% (w/v) TCA (final
o
concentration, 10% TCA) and incubated for 60 min at 4 C. The supernatant was discarded,
then the plates were washed 5 times with tap water and air dried. Sulforhodamine B (SRB)
solution (100 µl) was added to each well, and plates were incubated for 10 min at room
temperature. After staining, unbound dye was removed by washing five times with 1% acetic
acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM
trizma base, and the absorbance was read on an automated plate reader at a wavelength of
515 nm. For suspension cells, the methodology was the same except that the assay was
terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of 80%
25

ACCEPTED MANUSCRIPT
TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero
(Tz), control growth (C), and test growth in the presence of drug (Ti)], the percentage growth
was calculated at the drug concentration level. Percentage growth inhibition was calculated
as:
[(Ti – Tz) / (C – Tz)] x 100 for concentrations for which Ti ≥ Tz
[(Ti – Tz) / Tz] x 100 for concentrations for which Ti < Tz
4.2.5. In vitro cytotoxic activity WI-38 cells (human lung fibroblast normal cell line)
WI-38 cells (normal breast cells), were obtained from American Type Culture Collection.
The cells were propagated in DMEM supplemented with 10% heat-inactivated FBS (Hyclone),
10 ug/ml of insulin (Sigma), and 1% penicillin-streptomycin. All of the other chemicals and
reagents were from Sigma, or Invitrogen. Cytotoxicity was determined using MTT assay [19] as
reported earlier [33]. The 50% inhibitory concentration (IC50) was estimated from graphic plots
of the dose response curve for each conc. using Graphpad Prism software (San Diego, CA.
USA). The data presented are the mean of at least three separate experiments.
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28