Synthesis of indole-tethered [1,3,4]thiadiazolo and [1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one hybrids as anti-pancreatic cancer agents

Article abstract of DOI:10.1016/j.bmcl.2020.127544

New indole-tethered [1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one (8a-j) and [1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one hybrids (9a-e) were synthesized using [4+2] cycloaddition reactions of functionalized 1,3-diazabuta-1,3-dienes with indole-ketenes. All molecul

Full text of DOI:10.1016/j.bmcl.2020.127544

Bioorganic&MedicinalChemistryLetters30(2020)127544
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of indole-tethered [1,3,4]thiadiazolo and [1,3,4]oxadiazolo[3,2-
a]pyrimidin-5-one hybrids as anti-pancreatic cancer agents
⁎
Lalitha Gummidia, Nagaraju Kerrua, Paul Awoladea, Asif Razab, Arun K. Sharmab, ,
⁎
Parvesh Singha,
a School of Chemistry and Physics, University of KwaZulu-Natal, P/Bag X54001, Westville, Durban, South Africa
b Department of Pharmacology, Penn State Cancer Institute, CH72, Penn State College of Medicine, 500 University Drive, Hershey, PA 17033, USA
A R T I C L E I N F O
A B S T R A C T
Keywords:
New indole-tethered [1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one (8a-j) and [1,3,4]oxadiazolo[3,2-a]pyrimidin-5-
one hybrids (9a-e) were synthesized using [4+2] cycloaddition reactions of functionalized 1,3-diazabuta-1,3-
dienes with indole-ketenes. All molecular hybrids were structurally characterized by spectroscopic techniques
(IR, NMR, and HRMS) and screened for their anti-pancreatic cancer activity in vitro. The [1,3,4]oxadiazolo[3,2-
a]pyrimidin-5-one hybrids (9a-e) showed stronger anti-pancreatic cancer activity than the [1,3,4]thiadiazolo
[3,2-a]pyrimidin-5-one hybrids (8a-j) against the PANC-1 cell line. Compound 9d bearing an ortho-chlorophenyl
[4+2] Cycloaddition reaction
Pyrimidinone
Indole
Molecular hybridization
Pancreatic cancer
moiety emerged as the most potent anti-pancreatic cancer agent with an IC50 value of 7.7
0.4 µM, much
superior to the standard drug Gemcitabine (IC50 > 500 µM). The discovery of these [1,3,4]thiadiazolo and
[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one hybrids elicits their potentials as pursuable candidates for pancreatic
cancer chemotherapy.
Pancreatic cancer (PC) persists as one of the seven leading causes of
cancer-related deaths worldwide.1 The mortality rate is estimated at
2.5% and 14.8% per 0.1 million people in developing and developed
countries, respectively.2 This indicates that PC is more frequent and
fatal in developed countries. The death related to PC has increased by
0.3% per year since 1975. In the year 2019 alone in the United States,
approximately 56,770 people were newly diagnosed with PC.3 Many
patients with PC are diagnosed at a metastatic and advanced stage,
missing the chance for curative surgical resection. Although surgical
resection remains the primary curative option for this disease, the
suitability is compromised when the cancer is advanced due to poor
prognosis.4 Moreover, radiotherapy and chemotherapy are also not
remarkably effective for the treatment of metastatic PC.5 Thus, there is
an urgent demand for the development of new, effective and safe
chemotherapeutic agents against PC.
cells,6 while the synthetic derivative monastrol, a promising anticancer
compound as mitotic kinesin inhibitor, can cross the cell membrane.7,8
Functionalized pyrimidinones substituted at C-5 or C-6 positions have
also exhibited diverse biological activities such as antimicrobial, and
EGFR T790M inhibition for lung cancer treatment.9–11 Whereas fused
bicyclic pyrimidinones are promising anti-HIV, antiviral, anti-in-
flammatory and anticancer agents.12–17 The therapeutic value of this
scaffold is also evidenced by the different pyrimidinone-containing
drugs approved by the US Food and Drug Administration (FDA) for PC
chemotherapy such as gemcitabine, capecitabine, folinic acid and
fluorouracil (Fig. 1).18 However, despite the wide availability of drugs
for PC treatment, acquired drug resistance remains a major hurdle for
effective therapy. Besides, the undesirable side effects of existing drugs
due to poor selectivity necessitates the quest for new anti-PC agents.
On the other hand, indole is a prolific scaffold that is both widely
distributed in pharmacologically important natural products and re-
cruited as an organic synthon. The indole nucleus has also been en-
dorsed by the unique ability to mimic peptide derivatives and reversibly
bind to proteins.19 Indole-based synthetics have also found enormous
applications as dyes, agrochemicals, antiproliferative, anti-HIV, anti-
inflammatory and antibacterial agents.20–26 Precisely, in anticancer
drug discovery, indole and its derivatives have attracted considerable
Over the years, medicinal chemistry has provided access to chemical
libraries bearing privileged structures, which have received increased
attention due to their proven therapeutic relevance. Pyrimidinone, the
most important skeleton of diazine heterocycles has attracted great
interest in this regard due to its broad range of pharmacological ap-
plications. For instance, the pyrimidinone-based marine natural alka-
loids batzelladine A and B inhibited the binding of HIV gp-120 to CD4
⁎ Corresponding authors.
E-mail addresses: asharma1@pennstatehealth.psu.edu (A.K. Sharma), singhp4@ukzn.ac.za (P. Singh).
https://doi.org/10.1016/j.bmcl.2020.127544
Received 20 July 2020; Received in revised form 2 September 2020; Accepted 4 September 2020
Availableonline10September2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.

L. Gummidi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127544
with bromoacetic acid following our recently reported method.38 Fi-
nally, the desired pyrimidinone hybrids (8a-j and 9a-e) were synthe-
sized from the [4+2] cycloaddition reaction between 1,3,4-thiadia-
zole/oxadiazole substituted 1,3-diazabutadienes (5a-j and 6a-d) and
indole-ketene (generated in situ from the corresponding indole acids 7a-
b) in the presence of p-toluene sulphonyl chloride and triethylamine in
dry dichloromethane. Different substituents placed on both the thia-
diazole- and oxadiazole-1,3-diazabutadienes did not have any remark-
able effect on the overall yields of desired products.
The structures of synthesized hybrids were fully established by their
spectral data (IR, NMR, and HRMS). For instance, in the 1H NMR
spectrum of compound 8b, the pyrimidinone ring proton resonated as a
characteristic singlet peak at δ 8.30 ppm while the two doublet signals
at δ 7.41 (J = 3.3 Hz) and 6.75 (J = 3.0 Hz) ppm correspond to the CH
protons of indole moiety. Other aromatic ring protons appeared at their
respective aromatic region; the proton peak assignment was further
supported by COSY (details in the experimental section). Furthermore,
the appearance of a carbonyl carbon signal at δ 159.6 ppm in the 13C
NMR spectrum (Fig. 2a) and a strong absorption peak at 1686 cm−1 in
the IR spectrum validated the formation of the pyrimidinone core.
These structural elucidations were further supported by HSQC and
HMBC experiments; the selected HMBC correlations of 8b are shown in
Fig. 2b. High-resolution mass spectrometry (HRMS) showed a mole-
cular ion peak at (M + Na) m/z 444.9732.
Fig. 1. Pyrimidinone-containing drugs.
interest not only as cytotoxins but also as bio-reductively activated
prodrugs which effectively inhibits the growth of human pancreatic
cancer cells.27–30
On this backdrop, this study adopted the molecular hybridization
(MH) strategy to synthesize conjugates of fused-pyrimidinone and in-
dole as potential anti-pancreatic cancer agents. MH has evolved as a
preferred strategy in drug discovery, because of its proficiency to
combine two or more biologically active moieties with a unique or
novel mechanism of action.31 This endorses our research rationale, in
combining the pharmacophores; fused-pyrimidinone and indole into a
single framework using [4+2] cycloaddition reaction. The literature
survey clearly shows that the most effective route to functionalized
pyrimidinones is via the [4+2] cycloaddition reaction of conjugated
1,3-diazabuta-1,3-dienes with appropriate ketene precursors.32,33 As
part of our ongoing interest to find novel bioactive heterocyclic mole-
cules,34,35 herein, we report an efficient route for the synthesis of
functionalized pyrimidinone-indole molecular hybrids (8a-j and 9a-e)
by [4+2] cycloaddition reactions of different functionalized 1,3-diaza-
1,3-butadienes with indole-ketene. Subsequently, the synthesized mo-
lecular hybrids were evaluated in vitro for their anti-pancreatic cancer
activity.
Based on the empirical evidence,39 the [4+2] cycloaddition reac-
tion of 1,3-diazabuta-1,3-dienes (4π component) with indole-ketene
(2π component) proceeds via the nucleophilic addition of N1 in 1,3-
diazabuta-1,3-dienes (5a-f and 6a-d) to the carbonyl group of ketene
(7a-b), which leads to a zwitterionic intermediate (I) (Scheme 2). The
intermediate I then rearranges to dipolar intermediate (II), which after
ring closure and elimination of N,N-dimethyl amine (HN(CH3)2) af-
forded the desired products (8a-j and 9a-e) in good yields.
The potentials of compounds 8a-j and 9a-e as anti-pancreatic cancer
agents were evaluated using the PANC-1 cell line. The cells were treated
with various doses of the test compounds for 48 h, and cell survival was
determined. The results are illustrated in Fig. 3 and Table 1.
Based on the MTS cell viability data, the [1,3,4]oxadiazolo[3,2-a]
pyrimidin-5-ones (9a-e) were stronger cytotoxic agents than the [1,3,4]
thiadiazolo[3,2-a]pyrimidin-5-ones (8a-j) against PANC-1 cell line. The
cytotoxicity of compounds 8a-j and 9a-e was dependent on the type of
substitutions at different positions on the phenyl ring of [1,3,4]thia-
diazolo and [1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one moieties. The
[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one derivatives 9a, 9b, 9c, 9d and
9e showed moderate to potent anticancer activity with IC50 values of
The reaction protocol employed for the preparation of the two series
of indole tethered pyrimidinone molecular hybrids (8a-j and 9a-e) is
depicted in Scheme 1. Firstly, the reaction was carried out between the
appropriate substituted benzoic acids (1a-g) with semicarbazide or
thiosemicarbazide (2a-b) in the presence of phosphorus oxychloride
(POCl3) and aqueous NaOH at 90οC to afford the amine derivatives (3a-
21.1
0.5, 94.9
1.3, 90.5
2.0, 7.7
0.4 and 40.0
1.1 µM,
j
and 4a-d).36 Their subsequent condensation with N,N-di-
respectively (Table 1).
methylformamide dimethyl acetal (DMF-Acetal) at room temperature
resulted in the key precursors 1,3-diazabuta-1,3-dienes (5a-j and 6a-
d).37 Separately the other precursors indoloylglycines (7a-b) were
prepared by a base-promoted condensation of indole or 2-methyl indole
Overall, the ortho-chlorophenyl analogue (9d) emerged as the most
promising anti-pancreatic cancer agent with an IC50 value of
7.7
0.4 µM and was several-folds more potent as compared to the
Scheme 1. Synthesis of pyrimidinones via [4+2] cycloaddition reaction.
2

L. Gummidi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127544
Fig. 2. (a) 13C NMR and (b) HMBC correlation of 8b.
Scheme 2. A plausible mechanism of [4 + 2] cycloaddition reaction for the formation of pyrimidinone hybrids.
Fig. 3. Dose-dependent cell viability inhibitory effects of pyrimidinone compounds 8a-j and 9a-e on PANC-1 cells. The cells were treated with the respective
compounds for 48 h at different doses. Then, cell viability was measured using MTS assay, and the data were analyzed using GraphPad Prism software.
standard drug gemcitabine (IC50 > 500 µM). Whereas compounds 9b
and 9c with para-chloro and bromophenyl units, respectively, showed
had relatively lower potency (IC50 = 40.0
1.1 µM), albeit more
potent than gemcitabine. Surprisingly, no significant activity (IC50
= > 100 µM) was observed for [1,3,4]thiadiazolo[3,2-a]pyrimidin-5-
one hybrids (8a-j) against PANC-1 cell line. These results clearly show
that the presence of 1,3,4-oxadiazole core is crucial for potent
mild activities with IC50 values of 94.9
1.3 and 90.5
2.0 µM,
respectively. The unsubstituted compound 9a also exhibited good cy-
totoxicity (IC50 = 21.1 0.5 µM) while 2-methylindole compound 9e
3

L. Gummidi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127544
Table 1
IC50 values of screened compounds (8a-j and 9a-e) against PANC-1 cancer cell line.
Code
Structure
IC50 (µM)
Code
8i
Structure
IC50 (µM)
8a
> 100
> 100
8b
8c
> 100
> 100
8j
> 100
9a
21.1
0.5
8d
8e
> 100
> 100
9b
9c
94.9
90.5
1.3
2.0
8f
> 100
> 100
9d
9e
7.7
0.4
8g
40.0
1.1
8h
> 100
> 500
Standard drug
Gemcitabine
Fig. 4. SAR analysis of fused pyrimidinone hybrids as potent anti-pancreatic cancer agents.
cytotoxicity against the PANC-1 cell line as compared to 1,3,4-thia-
diazole core. The structure–activity relationship (SAR) analysis of the
test compounds is summarized in Fig. 4.
To sum up, the synthesis of indole linked [1,3,4]thiadiazolo and
[1,3,4]oxadiazolo[3,2-a]pyrimidin-5-one hybrids (8a-j and 9a-e) from
[4+2] cycloaddition reaction has been described. All the novel
4

L. Gummidi, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127544
molecular hybrids were evaluated for their cytotoxicity against the
PANC-1 pancreatic cancer cell line. [1,3,4]oxadiazolo[3,2-a]pyrimidin-
5-one hybrids showed stronger anti-pancreatic cancer activity com-
pared to [1,3,4]thiadiazolo[3,2-a]pyrimidin-5-one hybrids. Compound
9d bearing ortho-chlorophenyl substituent on [1,3,4]oxadiazolo[3,2-a]
pyrimidin-5-one core displayed the most potent anti-pancreatic cancer
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PS thanks to the National Research Foundation (NRF) South Africa
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South Africa for the financial support toward the research efforts (NRF-
TWAS UID: 110887). AKS thanks the Organic Synthesis Shared
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