Novel acetamidothiazole derivatives: Synthesis and in vitro anticancer evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.08.021

A novel series of acetamide derivatives possessing both 2-imino-4-arylthiazoles and morpholine or different piperazines were synthesized and characterized by IR, ¹H NMR, ¹³C NMR, elemental and mass spectral analyses. Twelve compounds were granted NSC codes at National Cancer Institute (NCI), USA for anticancer activity at a single high dose (10^-5 M) in full NCI 60 cell panel. Among the compounds tested, compounds 5a and 6b were found to be the most active candidates of the synthesized series. Assessment of toxicities, druglikeness, and drug score profiles of compounds 5a and 6b are promising. Some of the synthesized compounds showed a good docking score with potential anticancer targets, chosen based on pharmacophore mapping of the established derivatives.

Full text of DOI:10.1016/j.ejmech.2013.08.021

Accepted Manuscript
Novel acetamidothiazole derivatives: Synthesis and in vitro anticancer evaluation
Ahmed R. Ali, Eman R. El-Bendary, Mariam A. Ghaly, Ihsan A. Shehata
PII:
S0223-5234(13)00527-8
10.1016/j.ejmech.2013.08.021
EJMECH 6366
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 April 2013
Revised Date: 9 July 2013
Accepted Date: 12 August 2013
Please cite this article as: A.R. Ali, E.R. El-Bendary, M.A. Ghaly, I.A. Shehata, Novel acetamidothiazole
derivatives: Synthesis and in vitro anticancer evaluation, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.08.021.
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ACCEPTED MANUSCRIPT
The manuscript deals with the synthesis of a novel series of 2-imino-4-
arylthiazoles bearing acetamidomorpholine or acetamidopiperazine
moieties to be evaluated for their anticancer activities.

ACCEPTED MANUSCRIPT
Novel acetamidothiazole derivatives: Synthesis and in
vitro anticancer evaluation
Ahmed R. Ali*, Eman R. El-Bendary, Mariam A. Ghaly, Ihsan A.
Shehata
Department of Medicinal Chemistry, Faculty of Pharmacy,
University of Mansoura, Mansoura 35516, Egypt
* Corresponding author.
E-mail address: ahmed_reda551988@yahoo.com,
ahmed_reda5588@mans.edu.eg.
Abstract:
A novel series of acetamide derivatives possessing both 2-imino-4-
arylthiazoles and morpholine or different piperazines were synthesized
and characterized by IR, 1H NMR, 13C NMR, elemental and mass spectral
analyses. Twelve compounds were granted NSC codes at National Cancer
Institute (NCI), USA for anticancer activity at a single high dose (10-5 M)
in full NCI 60 cell panel. Among the compounds tested, compounds 5a
and 6b were found to be the most active candidates of the synthesized
series. Assessment of toxicities, druglikeness, and drug score profiles of
compounds 5a and 6b are promising. Some of the synthesized
compounds showed a good docking score with potential anticancer
targets, chosen based on pharmacophore mapping of the established
derivatives.
Keywords: aminothiazole, acetamidomorpholine, acetamidopiperazine,
anticancer evaluation.
1.
Introduction
Much attention was given by medicinal chemistry researchers to
thiazole ring system as the basis for development of many drugs with a
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diversity of biological activities. 2-Amino-4-aryl-1,3-thiazoles displayed
a wide range of biological potencies including antimicrobial [1-5],
analgesic [6,7], anticonvulsant [8,9], antioxidant [10], hypolipidemic
[11], anti-HIV-1 [12,13],
adenosine receptor antagonist [14,15],
osteoporosis inhibitor [16] and anti-Alzheimer [17-19] activities.
Antitumor activity of thiazole scaffold was readily established via being
incorporated into a variety of therapeutically active agents like bleomycin
[20], vosaroxin [21], epothilones [22,23] and dasatinib [24-26]. From
literature survey, it was found that aminothiazoles proved to have a broad
spectrum of activity against most of the tested subpanel tumor cell
lines.[27-31] In addition, 2-aminothiazole analogue of the natural
cytotoxic agent, giroline, [32] and cantharidin [33] demonstrated
increased apoptotic activity compared to their parent compounds.
Furthermore, several reports indicated that series of 2-amino-4-
arylthiazoles were synthesized and reported to be active as anticancer
agents.[34-37] Moreover, several acetamide derivatives comprising both
thiazole ring [38-40] or different fused heterocyclic systems [41,42] and
different amines such as morpholine or piperazines were reported to
possess good antitumor activity. Thus, it was proposed to prepare a series
of
2-imino-4-arylthiazoles
bearing
acetamidomorpholine
or
acetamidopiperazine moieties of the general structure (A) to be evaluated
for their antitumor activities.
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2.
Results and discussion
2.1. Chemistry
The synthesis of the starting compounds 3a-c is outlined in Scheme
1.
Scheme 1. Synthesis of the starting compounds 3a-c
2-Amino-4-arylthiazoles 2a-c were prepared utilizing phenacyl
bromide according to a reported procedure [43] which is considered to be
an easy, rapid and purification-free procedure. Thiourea was allowed to
react with phenacyl bromide at room temperature for 2-3 minutes to yield
the corresponding arylthiazole. The formation of 2-amino-4-arylthiazoles
was confirmed by their IR spectra. From literature survey, it was reported
that a variety of aminoheterocyclic systems could yield fused ring
systems containing keto group through reaction with chloroacyl chloride
[44-46] or ethyl chloroacetate [47,48]. Hurd and Hayao [49] reported the
fusion between 2-aminothiazole and 3-bromopropionic acid to produce
5,6-dihydrothiazolo[3,2-a]pyrimidin-7-one hydrobromide in 30% yield.
Recently, it was demonstrated that the reaction between 2-amino-4-
phenylthiazole and chloroacetic acid could be furnished in ethanol
yielding fused imidazothiazole derivative.[50] In the present study, 2-
amino-4-arylthiazoles 2a-c were reacted with chloroacetic acid in glacial
acetic acid in the presence of anhydrous sodium acetate. This was
accomplished via prolonged heating up to 40 h and the products were
obtained in 72-82% yield. The structures of compounds 3a-c were
confirmed on the basis of spectral data. The CH2 protons appeared as a
broad singlet around 3.46-3.87 in the 1H-NMR spectrum.
Mavrova et al. [51] demonstrated non-traditional reaction in
which the condensed thiazole ring underwent cleavage in a basic medium
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and took benefit of this ring opening to prepare many piperazine
derivatives by the reaction between 1,3-thiazolo[3,2-a]benzimidazol-
3(2H)-one and different piperazines. In this study, the new target
compounds 4-7(a-c) were prepared adopting the aforementioned
procedure through the reaction of 3-(un)substituted phenylimidazo[2,1-
b]thiazol-6(5H)-one 3a-c with secondary amines; morpholine or
piperazine derivatives. The synthesis of the new target compounds 4-7(a-
c) is outlined in Scheme 2.
Scheme 2. Synthesis of the title compounds 4-7(a-c)
The structures of the synthesized compounds 4-7(a-c) were
1
13
confirmed by microanalyses and spectral data (IR, H NMR, C NMR
1
and EI-MS) which showed full agreement with their structures. In the H
NMR spectra of these compounds, a singlet peak for CH2-protons
appeared at 4.10-4.23 ppm with a D2O exchangeable peak at 7.47-7.70
ppm for the NH proton. The 13C NMR spectra of these compounds
showed signals for the morpholine and piperazine ring at the expected
regions. In the 1H NMR spectra of compounds 5a-c, a singlet peak for N-
1
CH3 protons appeared at 2.12-2.18 ppm. In the H NMR spectra of
compounds 6a-c, a singlet peak appeared at 1.95-2.17 ppm for -COCH3
13
protons, while C signals of -COCH3 in compound 6a were observed
1
around 22.57 ppm. The H NMR spectra of compounds 7a-c is
characterized by the appearance of signals for phenyl ring attached to
piperazine ring at 6.73-6.79, 6.88-6.93 and 7.17-7.21 ppm. The mass
spectral data of these compounds displayed molecular ion peaks which
confirmed their molecular weights.
2.2. Biological evaluation
2.2.1. In vitro anticancer screening
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The target compounds 4-7(a-c) were submitted to National Cancer
Institute (NCI) [52], Bethesda, Maryland, USA, under the Developmental
Therapeutic Program (DTP) which is designed to screen up to 3,000
compounds per year for potential anticancer activity. The operation of
this screen utilizes 60 different human tumor cell lines, representing
leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast,
prostate, and kidney. Compounds with drug-like properties, based on
computer-aided design, are to be prioritized in the NCI screening service.
Twelve compounds were selected for screening based on their ability to
add diversity to the NCI small molecule compound collection. All
compounds submitted to the NCI 60 cell screen were tested at a single
dose (10-5 M) in the full NCI 60 cell panel. The compounds were added at
a single concentration and the culture was incubated for 48 h. End point
determinations were made with a protein binding dye, Sulforhodamine
B.[53-55]
Results for each compound are reported as a mean graph of the
percent growth of the treated cells when compared to the untreated
control cells. The percentage growth of the target compounds 4-7(a-c)
against the full 60-cell line panel are illustrated in Table 1.
Table 1: Sixty human tumor cell line anticancer screening data at single dose assay (10-5 M
concentration) as percentage cell growth
The mean percentage growth and the growth percentage with the
most sensitive cell lines of all of the tested compounds over the full panel
of cell lines are illustrated in Table 2.
Table 2: Mean percentage growth and screening data of the final compounds with the most
sensitive cell lines represented as percent cell growth
In light of the NCI-60 results, the following considerations could
be made:
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• The tested synthesized compounds exhibited low growth inhibition
against colon cancer cell lines.
• Although the majority of the target compounds 4-7(a-c) were inactive
against melanoma cell lines, compounds like 5c and 6c showed
moderate activity towards UACC-62 cell line with cell promotion
percentage 72.63 and 74.19%, respectively.
• Compounds 6b and 5a were the most active members in the
synthesized series with mean percentage growth values of 89.88 and
91.67%, respectively.
• Compound 6b was found to exhibit moderate potency towards
ovarian OVCAR-4 cancer cell line with cell promotion percentage
77.23%. On the other hand, the remaining tested compounds were
almost inactive against ovarian cancer.
• Anticancer activity of target compounds 4-7(a-c) was found to be
sensitive to the nature of substituent in phenyl ring when compared to
other compounds in the same series. The absence of substituent in
compounds 4-7(a) led to increase in the potency towards CNS SF-295
(when tested), CNS SNB-75, and Renal UO-31 cancer cell lines.
• The introduction of chloro substituent in compounds 4-6(b) led to
more potent compounds towards Leukemia SR and MOLT-4. On the
other hand, reduced potency towards Non-Small Cell Lung HOP-92
cancer cell line was probably associated with chloro substituent in
compounds 4-7(b).
• Methyl group in compounds 4-7(c) has increased the potency towards
Non-Small Cell Lung HOP-92 and Prostate PC-3 cancer cell line than
other compounds in the same series. In addition, when compared to
7c, compound 7a, that has unsubstituted phenyl attached to the
thiazole ring with the phenylpiperazine moiety, showed more
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enhanced potency towards both these two cell lines with cell
promotion percentage of 59.05 and 72.32%, respectively.
We performed COMPARE [56] analyses for compounds 4-7(a-c)
in order to investigate the similarity of their cytotoxicity pattern (mean
graph fingerprints) with those of known anticancer standard agents, NCI
active synthetic compounds and natural extracts, which are present in
public available databases. Such analysis is based on comparing the
patterns of differential growth inhibition for cultured cell lines and can
potentially gain insight into the mechanism of the cytotoxic action. If the
data pattern correlates well with that of compounds belonging to a
standard agent database (Pearson’s correlation coefficient (PCC>0.6), the
compound of interest may have the same mechanism of action.[57,58] On
the other hand, if the activity pattern does not correlate with any standard
agent, it is possible that the compound has a novel mechanism of action.
Standard COMPARE analyses were performed at the GI50 level.
It was established that the majority of the tested compounds
demonstrated considerable to weak correlation levels with rapamycin
(NSC S226080) with PCC values illustrated in Table 3. Such similarity in
COMPARE results could indicate potential resemblance in mechanisms
of action with rapamycin. Rapamycin is reported to be mTOR inhibitor
which is considered to be a key enzyme in regulation of cellular
metabolism, growth, and proliferation.[59-61]
Table 3: PCC values of the tested compounds with rapamycin (NSC S226080)
As shown in Table 3, although compounds 4c, 7b and 7c exhibited
the least correlation with rapamycin, they showed considerable
correlation with other standard anticancer agents. Compound 4c exhibited
a considerable correlation with spirohydantoin mustard (NSC S172112),
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which is a classical bifunctional alkylating agent synthesized in an effort
to develop antitumor agents effective against CNS tumors [62], with PCC
value of 0.522. In addition, compound 7b exhibited a considerable
correlation with hexamethylene bisacetamide (NSC S95580) with PCC
value of 0.472. Hexamethylene bisacetamide interferes with the
morphological and functional differentiation of malignant cells in vitro.
[63] Compound 7c showed a moderate correlation with flavone acetic
acid (NSC S347512) with PCC value of 0.484. Flavone acetic acid
belongs to a class of chemicals having a wide range of biological
properties and is considered to be active in solid cancers.[64]
2.2.2. Total polar surface area and Lipinski’s rule of five
It is well established that more than 80% of the drugs on the market
have an estimated log S value greater than -4. Also, in order to attain a
reasonable probability of being well absorbed, compounds must have log
P value not greater than 5.0. Nearly 40% of drug candidates fail in
clinical trials because of poor ADME [65]. From all the previous
observations, we evaluated the compliance of the designed compounds to
the Lipinski’s rule of five, calculated by Canvas [66] and Osiris [67]
program. Molecules violating more than one of these rules may have
problems with bioavailability. Predictions of ADME properties for the
studied compounds are given in Table 4. The results showed that all the
tested compounds comply with these rules suggesting that the synthesized
compounds would be possible drug candidates.
The total polar surface area (TPSA) using Canvas [66] program
was calculated since it is a key property that has been linked to drug
bioavailability. Thus, passively absorbed molecules with a TPSA > 140
are thought to have low oral bioavailability.[68] Since all the synthesized
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compounds 4-7(a-c) have TPSA value ranging from 37.53 to 53.14, they
theoretically should present good passive oral absorption, but the
differences in their biological activity cannot be attributed to this
property.
Table 4: Solubility, molar refractivity, and calculated Lipinski’s rule of five for target
compounds
2.2.3. Assessment of toxicities, druglikeness, and drug score profiles
Osiris program [67] was used for prediction of the overall toxicity
of the designed derivatives. The prediction process relies on a
substructure search process determining the occurrence frequency of any
fragment (core and constructed fragments) within any of toxicity classes.
All target compounds 4-7(a-c) showed low in silico possible toxicity
risks. Osiris program was also used for calculating the fragment-based
druglikeness by summing up score values of those fragments shredded at
rotatable bonds. The score value of each fragment was estimated based on
their occurrence frequency within the collection of traded drugs and
within the supposedly non drug-like collection of Fluka compounds. A
positive value states that designed molecule contains predominately
fragments which are frequently present in commercial drugs.
The drug score combines druglikeness, cLogP, LogS, molecular
weight and toxicity risks in one handy value that may be used to judge the
compound's overall potential to qualify for a drug. A value of 0.5 or more
makes this compound a promising lead for future development of safe
and efficient drug. Predictions of potential toxicity, druglikeness and drug
score for the studied compounds are given in Table 5. Almost all of the
synthesized compounds possess good values of druglikeness and drug
score. Among all the synthesized compounds, compounds 5a and 6b with
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high values of both druglikeness and drug score showed the lowest mean
percentage growth over 60 cell line panel.
Table 5: Toxicity risks, druglikeness and drug score of the designed compounds
2.2.4. Target fishing
An attempt was made to investigate the potential targets involved
in observed inhibition displayed by the synthesized compounds against
NCI 60 cell panel. In silico target fishing is an emerging technology that
enables the prediction of biological targets of compounds on the basis of
chemical structure by using information from increasingly available
biologically annotated chemical databases.[69] PharmMapper server is
open-accessed web server. PharmMapper automatically finds the best
mapping poses of the query molecule against all the pharmacophore
models using reverse pharmacophore mapping approach.[70]
PharmMapper is available at http://59.78.96.61/pharmmapper.
PharmMapper demonstrated a variety of putative targets that might
exhibit considerable binding affinity to the synthesized compounds.
Seven targets, involved in cancer therapy, are common between the
synthesized compounds. These targets might explain the observed
antiproliferative activity. The fit scores of the synthesized compounds
with the top targets are illustrated in Table 6.
Table 6: Fit score of the synthesized compounds against the top seven enzymes
Compound 7b exhibiting the highest fit score with Vitamin D3
receptor is illustrated in Figure 1. As with the other remaining
compounds, it demonstrated hydrophobic, hydrogen bond acceptor and
hydrogen bond donor centers.
Figure 1: Pharmacophore map with the highest fit score derived from compound 7b
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Targets proposed by PharmMapper are employed in cancer therapy
in a diversity of approaches.[71-89] As a result, we have studied the
potential interaction of the synthesized compounds 4-7(a-c) against these
seven targets which are involved in cancer.
2.2.5. Docking study
The seven potential targets proposed by pharmacophore mapping
approach were used to investigate their interaction with the designed
compounds. The target compounds 4-7(a-c) were comparatively
evaluated in terms of estimated free energy of binding (kcal/mol), and
inhibition constant Ki (uM) to the seven proposed enzymes and the results
are listed in Table 7. Molecular docking simulations were performed for
the synthesized compounds to evaluate their recognition profile at the
binding pocket of the proposed enzymes. The binary complex of the
enzyme coupled with its natural ligand was used as a reference for
docking and modeling in this investigation.
Docking simulations were carried out with the aid of Docking
Server [90], a web-based interface that calculates the geometry and
electronic properties of the ligands at a given pH.
Table 7: Estimated free energy of binding and inhibition constants of the synthesized compounds
with the top seven targets
Compounds 5a and 6b exhibiting the lowest mean percentage
growth over 60 cell line panel showed reasonable docking scores with the
proposed enzymes. The binding mode of compound 7b showing the least
inhibition constant is illustrated in Figure 2. Compound 7b seem to have
hydrogen bond interaction with GLN136, GLY269 and MET270 and
GLU296, polar interaction with HIS295, GLU296 and TYR383, and
hydrophobic interaction with TYR267, MET270 and VAL292 with Ki
equal to 0.093 µM.
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Figure 2: Binding mode of compound 7b
2.2.6. Predicting potential side effects
SePreSA is a server for the prediction of adverse drug reactions.
The server has comprehensive collection of the structural models of
nearly all the well-known serious adverse drug reactions targets and
calculates chemical–protein interactome using the DOCK program. The
server utilizes a 2-directional Z-transformation scoring algorithm, which
computes the relative drug–protein interaction strength based on the
docking-score matrix, thus achieves greater accuracy in prioritizing
targets than simply using dock scoring functions.[91] The server is freely
available at http://SePreSA.Bio-X.cn/.
The drug molecule tends to interact with the protein if Z'-score is
less than -0.5. The protein id, adverse drug reaction and Z'-score for each
compound are recorded in Table 8.
Table 8: Adverse drug reactions and Z'-scores for the designed compounds
From the data obtained, a decrease in the affinity of the synthesized
compounds containing phenylpiperazine moiety 7a-c towards estrogen
and androgen receptors was observed when compared with the remaining
compounds. The absence of substituent in compounds 4-7(a) led to
increase in the affinity towards DNA topoisomerase 2-alpha supposing
possible risk factor for cytotoxicity. In addition, when compared to 7a,
compound 7b showed lower Z'-score with the same receptor.
Cytotoxicity manifested by interaction with DNA topoisomerase 2-alpha
is displayed in the following order: acetylpiperazine series 6a-c >
methylpiperazine series 5a-c
>
morpholine series 4a-c
>
phenylpiperazine series 7a-c. Also, it can be assumed from the previous
findings that the introduction of chloro substituent in compounds 4-6(b)
resulted in increased affinity towards purine nucleoside phosphorylase
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enzyme. The higher affinity of these compounds to the previous enzyme
might suggest potential bone marrow toxicity.
3.
Conclusion
The synthesized compounds 4-7(a-c) were tested at a single dose
of 10 µM at the NCI over 60 cell line panel to screen for their potential
anticancer activity. On the basis of results obtained, it was found that
compounds 5a (NSC 768193) and 6b (NSC 768174) proved to be the
most active members in this study. They showed moderate potency over
certain cancer cell lines. From the obtained results, it is clear that
substituents affect the activity of compounds in different series. The in
silico ADME profiling, toxicity, druglikeness, drug score, and the
docking scores with enzymes derived from pharmacophore similarity
results together with in vitro anticancer activities make 5a and 6b
promising lead compounds for development of more potent anticancer
agents. Further modifications of these compounds in order to improve
their potency are currently under consideration.
4.
4.1. General
2-Amino-4-arylthiazoles 2a-c were prepared following the
Experimental
procedure reported by Dighe [43]. All the reagents and solvents were
obtained from commercial suppliers, and used without purification. TLC
was monitored on Fluka silica gel TLC aluminum cards (0.2 mm
thickness) with fluorescent indicator 254 nm using a mixture of
petroleum ether/ethyl acetate in various proportions.
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Melting points (oC) were recorded using a Fischer-Johns melting
point apparatus and are uncorrected. The IR spectra (KBr) were recorded
on Mattson 5000 FT-IR spectrophotometer (ν in cm-1) in the
1
Microanalytical Unit, Faculty of Science, Mansoura University. H and
13C NMR for compounds 4-7(a) were recorded on Bruker 500 MHz FT
1
NMR spectrometer and H NMR spectra for remaining compounds were
carried out at the National Research Centre using a Varian Gemini 500
MHz FT NMR. Deuteriodimethylsulfoxide (DMSO-d6) is used as a
solvent with the chemical shift being expressed in δ (ppm) and downfield
from tetramethylsilane (TMS) as internal standard.
Electron impact mass spectra (EI-MS), recorded on a Shimadzu
GC/MS QP-2010 Plus mass spectrometer, and elemental analysis (in
accord with the calculated values) were carried out in the Microanalytical
Unit, Faculty of Science, Cairo University. Anticancer evaluation was
performed at National Cancer Institute (NCI), Bethesda, Maryland, USA.
4.2. Synthesis of 3-(un)substituted phenylimidazo[2,1-b]thiazol-6(5H)-
one (3a–c)
A mixture of 2-amino-4-arylthiazoles 2a–c (10 mmol) and chloroacetic
acid (1.89 g, 20 mmol) was dissolved in glacial acetic acid (10 mL) in the
presence of anhydrous sodium acetate (1.64 g, 20 mmol) and refluxed for 40 h.
The reaction mixture was cooled and poured into cold ice water with stirring.
The solid formed was filtered and crystallized from aqueous ethanol.
4.2.1. 3-Phenylimidazo[2,1-b]thiazol-6(5H)-one (3a)
o
Yield: 82%; mp 212-214 C [50]; IR (KBr, ν, cm-1): 3169 (CH
aromatic), 3023, 2987 (CH aliphatic), 1654, 1644 (C=O), 1599, 1584 cm-1
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(C=N); EI-MS (70 eV) m/z (Rel. Int.): 216 (M+, 3.24), 199 (20.06), 176
(100.00), 134 (31.72), 98 (21.04), 77 (11.00).
4.2.2. 3-(4-Chlorophenyl)imidazo[2,1-b]thiazol-6(5H)-one (3b)
o
Yield: 80%; mp 254-256 C; 1H NMR (δ, ppm, DMSO-d6): 3.48 (s, 2H,
CH2), 7.46 (d, 2H, Ar-H), 7.63 (s, 1H, H-thiazole), 7.86 (d, 2H, Ar-H); EI-MS
(70 eV) m/z (Rel. Int.): 252 (M++2, 30.47), 250 (M+, 2.57), 233 (2.36), 210
(100.00), 168 (32.13), 132 (6.05), 111 (11.57); Anal. for C11H7ClN2OS
(250.70) C, H, N.
4.2.3. 3-p-Tolylimidazo[2,1-b]thiazol-6(5H)-one (3c)
Yield: 70%; mp 132-134 oC; 1H NMR (δ, ppm, DMSO-d6): 2.29 (s, 3H, -
CH3), 3.84 (s, 2H, CH2), 7.22 (d, 2H, Ar-H), 7.31 (s, 1H, H-thiazole), 7.62 (d,
2H, Ar-H); EI-MS (70 eV) m/z (Rel. Int.): 230 (M+, 47.37), 198 (100), 176
(41.17); Anal. for C12H10N2OS (230.29) C, H, N.
4.3. Synthesis of 2-(2-imino-4-(un)substituted phenylthiazol-3(2H)-yl)-
1-morpholinoethanone (4a–c)
A mixture of 3-(un)substituted phenylimidazo[2,1-b]thiazol-6(5H)-one
3a-c (10 mmol) and morpholine (1.03 mL, 12 mmol) was dissolved in absolute
ethanol (15 mL) and refluxed for 6 h. The solvent was distilled under reduced
pressure and the obtained product was washed with water and crystallized from
aqueous ethanol to give the corresponding compounds.
4.3.1. 2-(2-Imino-4-phenylthiazol-3(2H)-yl)-1-morpholinoethanone (4a)
o
Yield: 66%; mp 196-198 C; IR (KBr, ν, cm-1): 3453 (NH), 3169
(CH aromatic), 3064, 2986 (CH aliphatic), 1651 (C=O), 1584 cm-1
(C=N); 1H NMR (δ, ppm, DMSO-d6): 3.38 (t, 4H, (CH2)2N-morpholine),
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3.57 (t, 4H, (CH2)2O-morpholine), 4.20 (s, 2H, CH2), 7.33 (t, 2H, Ar-H),
7.43 (t, 1H, Ar-H), 7.58 (s, 1H, H-thiazole), 7.62 (s, 1H, NH), 7.90 (d,
2H, Ar-H); 13C NMR (δ, ppm, DMSO-d6): 40.01 (NCH2CO), 45.16
((CH2)2N-morpholine), 68.32 ((CH2)2O-morpholine), 107.77 (-S-CH-
thiazole), 125.63, 127.69, 128.67 (Ar-CH), 134.33 (quaternary Ar-C),
148.71 (quaternary thiazole-C), 157.96 (-S-C(N)=NH-thiazole), 168.59
(C=O); EI-MS (70 eV) m/z (Rel. Int.): 303 (M+, 0.15), 245 (0.40), 234
(0.11), 218 (30.73), 203 (0.50), 189 (0.39), 176 (100.00), 165 (0.27), 134
(61.83), 119 (1.86), 114 (0.58), 104 (16.27), 77 (21.79); Anal. Calcd for
C15H17N3O2S (303.38) C, H, N.
4.3.2. 2-(4-(4-Chlorophenyl)-2-iminothiazol-3(2H)-yl)-1-
morpholinoethanone (4b)
Yield: 62%; mp 228-230 oC; 1H NMR (δ, ppm, DMSO-d6): 3.32 (t,
4H, (CH2)2N-morpholine), 3.57 (t, 4H, (CH2)2O-morpholine), 4.12 (s, 2H,
CH2), 7.45 (d, 2H, Ar-H), 7.62 (s, 1H, H-thiazole), 7.69 (s, 1H, NH), 7.87
(d, 2H, Ar-H); EI-MS (70 eV) m/z (Rel. Int.): 339 (M++2, 0.99), 337 (M+,
1.88), 279 (0.43), 268 (3.46), 252 (23.82), 237 (2.33), 223 (1.25), 210
(100.00), 199 (0.82), 168 (34.48), 138 (10.82), 114 (3.06), 111 (15.12),
77 (7.21); Anal. Calcd for C15H16ClN3O2S (337.82) C, H, N.
4.3.3. 2-(2-Imino-4-p-tolylthiazol-3(2H)-yl)-1-morpholinoethanone (4c)
Yield: 55%; mp 144-148 oC; 1H NMR (δ, ppm, DMSO-d6): 2.29 (s,
3H, -CH3), 3.50 (t, 4H, (CH2)2N-morpholine), 3.88 (t, 4H, (CH2)2O-
morpholine), 4.11 (s, 2H, CH2),7.19 (d, 2H, Ar-H), 7.34 (s, 1H, H-
thiazole), 7.47 (s, 1H, NH, D2O exchangeable), 7.75 (d, 2H, Ar-H); EI-
MS (70 eV) m/z (Rel. Int.): 317 (M+, 5.54), 259 (33.70), 248 (5.12), 232
(28.20), 217 (52.45), 203 (35.38), 190 (100.00), 179 (90.22), 148 (35.76),
16

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118 (71.22), 114 (8.36), 91 (82.85), 77 (16.87); Anal. Calcd for
C16H19N3O2S (317.41) C, H, N.
4.4. General procedure for the synthesis of compounds 5-7(a-c)
A mixture of 3-(un)substituted phenylimidazo[2,1-b]thiazol-6(5H)-one
3a-c (10 mmol) and piperazine or N-substituted piperazine (12 mmol) was
dissolved in absolute ethanol (10 mL) and refluxed for 6 h. The solvent was
distilled under reduced pressure and the obtained oily product was triturated
with petroleum ether several times in order to remove the excess piperazine.
The obtained solid was crystallized from aqueous ethanol to give the
corresponding compounds.
4.4.1. 2-(2-Imino-4-phenylthiazol-3(2H)-yl)-1-(4-methylpiperazin-1-
yl)ethanone (5a)
o
Yield: 52%; mp 190-192 C; IR (KBr, ν, cm-1): 3434 (NH), 3169
(CH aromatic), 3063, 2985 (CH aliphatic), 1644 (C=O), 1598 cm-1
1
(C=N); H NMR (δ, ppm, DMSO-d6): 2.18 (s, 3H, N-CH3), 2.51 (br s,
4H, (CH2)2N4 piperazine), 3.51 (br s, 4H, (CH2)2N1 piperazine), 4.19 (s,
2H, CH2), 7.32 (t, 2H, Ar-H), 7.42 (t, 1H, Ar-H), 7.57 (s, 1H, H-thiazole),
13
7.63 (s, 1H, NH), 7.90 (d, 2H, Ar-H); C NMR (δ, ppm, DMSO-d6):
39.80 ((CH2)2N1 piperazine), 39.97 (NCH2CO,NCH3), 61.13 ((CH2)2N4
piperazine), 108.00 (-S-CH-thiazole), 125.62, 127.69, 128.67 (Ar-CH),
134.32 (quaternary Ar-C), 148.70 (quaternary thiazole-C), 157.44 (-S-
C(N)=NH-thiazole), 168.60 (C=O); EI-MS (70 eV) m/z (Rel. Int.): 316
(M+, 0.01), 245 (0.01), 234 (0.03), 218 (35.10), 203 (0.55), 189 (0.14),
176 (100.00), 165 (0.04), 141 (0.09), 134 (54.02), 127 (0.08), 104
(12.09), 77 (9.39); Anal. Calcd for C16H20N4OS (316.42) C, H, N.
17

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4.4.2. 2-(4-(4-Chlorophenyl)-2-iminothiazol-3(2H)-yl)-1-(4-
methylpiperazin-1-yl)ethanone (5b)
Yield: 57%; mp 228-230 oC; 1H NMR (δ, ppm, DMSO-d6): 2.12 (s,
3H, N-CH3), 2.46 (br s, 4H, (CH2)2N4 piperazinyl), 3.44 (br s, 4H,
(CH2)2N1 piperazinyl), 4.12 (s, 2H, CH2), 7.45 (d, 2H, Ar-H), 7.63 (s, 1H,
H-thiazole), 7.67 (s, 1H, NH), 7.86 (d, 2H, Ar-H); EI-MS (70 eV) m/z
(Rel. Int.): 352 (M++2, 0.18), 350 (M+, 0.37), 279 (0.17), 268 (2.93), 252
(17.58), 237 (1.12), 223 (0.71), 210 (100.00), 199 (0.35), 168 (35.75),
141 (3.31), 138 (10.59), 127 (1.23), 111 (12.35), 77 (5.85); Anal. Calcd
for C16H19ClN4OS (350.87) C, H, N.
4.4.3. 2-(2-Imino-4-p-tolylthiazol-3(2H)-yl)-1-(4-methylpiperazin-1-
yl)ethanone (5c)
Yield: 51%; mp 142-144 oC; 1H NMR (δ, ppm, DMSO-d6): 2.15 (s,
3H, N-CH3), 2.30 (s, 3H, Ar-CH3), 2.48 (br s, 4H, (CH2)2N4 piperazinyl),
3.47 (br s, 4H, (CH2)2N1 piperazinyl), 4.10 (s, 2H, CH2), 7.18 (d, 2H, Ar-
H), 7.36 (s, 1H, H-thiazole), 7.56 (s, 1H, NH, D2O exchangeable), 7.77
(d, 2H, Ar-H); EI-MS (70 eV) m/z (Rel. Int.): 330 (M+, 0.35), 259 (8.32),
248 (6.25), 232 (24.45), 217 (18.28), 203 (7.23), 190 (83.45), 179
(100.00), 148 (31.26), 141 (1.38), 127 (1.85), 118 (53.36), 91 (46.07), 77
(12.65); Anal. Calcd for C17H22N4OS (330.45) C, H, N.
4.4.4. 1-(4-Acetylpiperazin-1-yl)-2-(2-imino-4-phenylthiazol-3(2H)-
yl)ethanone (6a)
Yield: 33%; mp 180-182 oC; IR (KBr, ν, cm-1): 3433 (NH), 3169
(CH aromatic), 3064, 2986 (CH aliphatic), 1643 (C=O), 1599 cm-1
1
(C=N); H NMR (δ, ppm, DMSO-d6): 2.17 (s, 3H, -COCH3), 3.35 (br s,
8H, piperazine), 4.10 (s, 2H, CH2), 7.32 (t, 2H, Ar-H), 7.43 (t, 1H, Ar-H),
7.59 (s, 1H, H-thiazole), 7.70 (s, 1H, NH), 7.90 (d, 2H, Ar-H); 13C NMR
18

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(δ, ppm, DMSO-d6): 22.57 (-COCH3), 39.76 (NCH2CO), 48.53 (C-
piperazine), 107.80 (-S-CH-thiazole), 125.65, 127.71, 128.72 (Ar-CH),
134.31 (quaternary Ar-C), 148.78 (quaternary thiazole-C), 157.93 (-S-
C(N)=NH-thiazole), 168.60 (2 C=O); EI-MS (70 eV) m/z (Rel. Int.): 344
(M+, 0.44), 245 (0.07), 234 (0.14), 218 (31.73), 203 (1.15), 176 (100.00),
165 (0.88), 155 (0.50), 134 (67.52), 127 (0.18), 104 (17.19), 77 (18.22);
Anal. Calcd for C17H20N4O2S (344.43) C, H, N.
4.4.5. 1-(4-Acetylpiperazin-1-yl)-2-(4-(4-chlorophenyl)-2-iminothiazol-
3(2H)-yl)ethanone (6b)
Yield: 45%; mp 226-228 oC; 1H NMR (δ, ppm, DMSO-d6): 1.95 (s,
3H, -COCH3), 3.32 (br s, 8H, piperazine ring), 4.12 (s, 2H, CH2), 7.45 (d,
2H, Ar-H), 7.63 (s, 1H, H-thiazole), 7.67 (s, 1H, NH), 7.86 (d, 2H, Ar-H);
EI-MS (70 eV) m/z (Rel. Int.): 380 (M++2, 5.17), 378 (M+, 12.98), 279
(8.56), 252 (3.56), 237 (37.29), 223 (3.95), 210 (3.54), 199 (1.45), 169
(2.05), 155 (1.05), 138 (3.05), 127 (7.99), 111 (19.19); Anal. Calcd for
C17H19ClN4O2S (378.88) C, H, N.
4.4.6. 1-(4-Acetylpiperazin-1-yl)-2-(2-imino-4-p-tolylthiazol-3(2H)-
yl)ethanone (6c)
Yield: 40%; mp 140-142 oC; 1H NMR (δ, ppm, DMSO-d6): 1.95 (s,
3H, -COCH3), 2.29 (s, 3H, Ar-CH3), 3.52 (br s, 8H, piperazine ring), 4.12
(s, 2H, CH2), 7.19 (d, 2H, Ar-H), 7.35 (s, 1H, H-thiazole), 7.48 (s, 1H,
NH, D2O exchangeable), 7.73 (d, 2H, Ar-H); EI-MS (70 eV) m/z (Rel.
Int.): 358 (M+, 15.81), 259 (24.62), 232 (19.76), 203 (17.33), 190 (27.66),
169 (20.67), 148 (16.41), 118 (38.30), 91 (50.76), 77 (13.98); Anal. Calcd
for C18H22N4O2S (358.46) C, H, N.
4.4.7. 2-(2-Imino-4-phenylthiazol-3(2H)-yl)-1-(4-phenylpiperazin-1-
yl)ethanone (7a)
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o
Yield: 39%; mp 174-176 C; IR (KBr, ν, cm-1): 3446 (NH), 3179
(CH aromatic), 3069, 2995 (CH aliphatic), 1651 (C=O), 1582 cm-1
1
(C=N); H NMR (δ, ppm, DMSO-d6): 3.17 (t, 4H, (CH2)2N4 piperazine),
3.41 (t, 4H, (CH2)2N1 piperazine), 4.23 (s, 2H, CH2), 6.79 (t, 1H,
piperazine-Ar-H), 6.93 (d, 2H, piperazine-Ar-H), 7.21 (t, 2H, piperazine-
Ar-H), 7.33 (t, 2H, Ar-H), 7.44 (t, 1H, Ar-H), 7.59 (s, 1H, H-thiazole),
13
7.64 (s, 1H, NH), 7.92 (d, 2H, Ar-H); C NMR (δ, ppm, DMSO-d6):
39.99 (NCH2CO), 48.20 ((CH2)2N1 piperazine), 52.50 ((CH2)2N4
piperazine), 107.79 (-S-CH-thiazole), 115.42, 118.82 (piperazine-Ar-C),
125.65, 127.71, 128.69, 128.88 (Ar-CH,piperazine-Ar-C), 134.34
(quaternary Ar-C), 148.73, 148.87 (quaternary thiazole-C,piperazine-Ar-
C), 157.99 (-S-C(N)=NH-thiazole), 168.63 (C=O); EI-MS (70 eV) m/z
(Rel. Int.): 378 (M+, 0.01), 363 (0.05), 336 (0.04), 308 ( 0.05), 288 (0.07),
266 (0.06), 245 (0.03), 234 (0.21), 218 (30.65), 203 (0.89), 189 (0.68),
176 (100.00), 165 (0.66), 161 (1.92), 134 (56.19), 77 (17.34); Anal. Calcd
for C21H22N4OS (378.49) C, H, N.
4.4.8. 2-(4-(4-Chlorophenyl)-2-iminothiazol-3(2H)-yl)-1-(4-
phenylpiperazin-1-yl)ethanone (7b)
Yield: 25%; mp 220-222 oC; 1H NMR (δ, ppm, DMSO-d6): 3.15 (t,
4H, (CH2)2N4 piperazine), 3.47 (t, 4H, (CH2)2N1 piperazine), 4.12 (s, 2H,
CH2), 6.73 (t, 1H, piperazine-Ar-H), 6.88 (d, 2H, piperazine-Ar-H), 7.17
(t, 2H, piperazine-Ar-H), 7.45 (d, 2H, Ar-H), 7.63 (s, 1H, H-thiazole),
7.67 (s, 1H, NH), 7.86 (d, 2H, Ar-H); EI-MS (70 eV) m/z (Rel. Int.): 414
(M++2, 6.88), 412 (M+, 15.95), 397 (1.35), 370 (0.87), 342 (5.01), 322
(0.53), 300 (4.14), 279 (4.23), 268 (4.45), 252 (6.90), 237 (30.01), 223
(8.23), 210 (32.62), 203 (5.11), 199 (6.59), 189 (3.36), 168 (21.60), 161
(28.25), 111 (22.54), 77 (72.85); Anal. Calcd for C21H21ClN4OS (412.94)
C, H, N.
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4.4.9. 2-(2-Imino-4-p-tolylthiazol-3(2H)-yl)-1-(4-phenylpiperazin-1-
yl)ethanone (7c)
Yield: 20%; mp 126-128 oC; 1H NMR (δ, ppm, DMSO-d6): 2.28 (s,
3H, Ar-CH3), 3.15 (t, 4H, (CH2)2N4 piperazine), 3.53 (t, 4H, (CH2)2N1
piperazine), 4.10 (s, 2H, CH2), 6.78 (t, 1H, piperazine-Ar-H), 6.91 (d, 2H,
piperazine-Ar-H), 7.19 (m, 4H, piperazine-Ar-H, thiazole-Ar-H), 7.35 (s,
1H, H-thiazole), 7.48 (s, 1H, NH, D2O exchangeable), 7.78 (d, 2H, Ar-
H); EI-MS (70 eV) m/z (Rel. Int.): 392 (M+, 2.51), 377 (2.30), 350 (4.21),
322 (54.25), 302 (1.52), 280 (30.74), 259 (12.44), 248 (3.02), 232
(12.23), 217 (26.50), 203 (27.12), 190 (53.60), 179 (100.00), 161 (20.12),
148 (20.01), 91 (93.39), 77 (72.45); Anal. Calcd for C22H24N4OS (392.52)
C, H, N.
4.5. In vitro anticancer screening
Twelve of the synthesized compounds including 4-7(a-c) were
subjected to the National Cancer Institute (NCI) in vitro disease-oriented
human cells screening panel assay for in vitro antitumor activity.[52-55]
The human tumor cell lines of the cancer screening panel are grown in
RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-
glutamine. For a typical screening experiment, cells are inoculated into 96
well microtiter plates in 100 µL at plating densities ranging from 5,000 to
40,000 cells/well depending on the doubling time of individual cell lines.
After cell inoculation, the microtiter plates are incubated at 37°C, 5 %
CO2, 95 % air and 100 % relative humidity for 24 h prior to addition of
experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA,
to represent a measurement of the cell population for each cell line at the
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time of drug addition (Tz). Experimental drugs are solubilized in
dimethylsulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug addition,
an aliquot of frozen concentrate is thawed and diluted to twice the desired
final maximum test concentration with complete medium containing 50
µg/mL gentamicin. Additional four, 10-fold or ½ log serial dilutions are
made to provide a total of five drug concentrations plus control. Aliquots
of 100 µL of these different drug dilutions are added to the appropriate
microtiter wells already containing 100 µL of medium, resulting in the
required final drug concentrations.
Following drug addition, the plates are incubated for an additional
48 h at 37°C, 5 % CO2, 95 % air, and 100 % relative humidity. For
adherent cells, the assay is terminated by the addition of cold TCA. Cells
are fixed in situ by the gentle addition of 50 µL of cold 50 % (w/v) TCA
(final concentration, 10 % TCA) and incubated for 60 minutes at 4°C.
The supernatant is discarded, and the plates are washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4
% (w/v) in 1 % acetic acid is added to each well, and plates are incubated
for 10 minutes at room temperature. After staining, unbound dye is
removed by washing five times with 1 % acetic acid and the plates are air
dried. Bound stain is subsequently solubilized with 10 mM trizma base,
and the absorbance is read on an automated plate reader at a wavelength
of 515 nm. For suspension cells, the methodology is the same except that
the assay is terminated by fixing settled cells at the bottom of the wells by
gently adding 50 µL of 80 % TCA (final concentration, 16 % TCA).
Using the seven absorbance measurements [time zero, (Tz), control
growth, (C), and test growth in the presence of drug at the five
concentration levels (Ti)], the percentage growth is calculated at each of
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the drug concentration levels. Percentage growth inhibition is 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
4.6. Assessment of toxicities, druglikeness, and drug score profiles
During the shredding, any molecule was first cut at every rotatable
bond leading to a set of core fragments. These in turn were used to
reconstruct all possible bigger fragments being a substructure of the
original molecule. Afterwards, a substructure search process determined
the occurrence frequency of any fragment (core and constructed
fragments) within all compounds of 3300 traded drugs as well as 15000
commercially available chemicals (Fluka).[67]
4.7. Target fishing
The newly synthesized compounds 4-7(a-c) were uploaded in
Tripos Mol2 format. PharmMapper adopts semi-rigid pharmacophore
mapping protocol. As a result, multiple conformations of the query
molecule were required prior to mapping which could be achieved by
online service provided by the server. PharmMapper found the best
mapping poses of the uploaded molecules against all the targets in
PharmTargetDB and top N potential drug targets (default value is 300) as
well as respective molecule's aligned poses were outputted.[70]
4.8. Docking study
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Docking study was performed with the aid of Docking Server.
Gasteiger partial charges were added to the ligand atoms after energy
minimization using the MMFF94 force field. Non-polar hydrogen atoms
were merged, and rotatable bonds were defined. Essential hydrogen
atoms, Kollman united atom type charges, and solvation parameters were
added with the aid of AutoDock tools to protein model. Affinity (grid)
maps of 20×20×20 A˚ grid points and 0.375 A˚ spacing were generated
using the Autogrid program. Docking simulations were performed using
the Lamarckian genetic algorithm (LGA) and the Solis and Wets local
search method. Initial position, orientation, and torsions of the ligand
molecules were set randomly. Each docking experiment was derived from
10 different runs that were set to terminate after a maximum of 250000
energy evaluations.[90]
4.9. Predicting potential side effects
The key residue ionization state was assigned considering the
most probable one at the physiological pH using Chimera. The
interactome of a drug molecule towards all SADR targets was calculated
using DOCK.
For each adverse drug reaction (ADR) target, residues within a
10 A˚ distance from the ligand were defined as the bioactive pocket of the
protein. The server computes a relative drug-protein interaction score
from a scoring matrix to prioritize SADR targets which might be
affinitive with the user’s compound. The prediction mechanism was
based on the user-oriented interactome, which calculates the Z'-score of
the current molecule from the interactome formed by all molecules
submitted by the user, no matter when and where these previous
molecules are submitted.[91]
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Acknowledgment
We are thankful to the National Cancer Institute (NCI), Bethesda,
Maryland, USA, for performing the anticancer evaluation over the 60-
cancer cell line panel.
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