R. Patle et al.
Bioorganic & Medicinal Chemistry Letters 42 (2021) 128062
activate PKM2 and act as potent anticancer agents.8 The much-studied
compounds are micheolide, Ml-265, DASA-58, SAICAR, and its deriva-
tives.9–12 Some promising PKM2 activators having appreciable biolog-
ical potential are shown in Fig. 1. Covalent modifiers like micheliode
onsets covalent modification of target leading to anticancer effects.
However, its applicability has been largely impeded owing to associated
safety concerns.13 Several studies have demonstrated significant syner-
gistic effects of PKM2 modulators in combination with other wide range
of established oncology drugs.14 It is imperative to mention that the
development of kinase activators will be highly useful as it will smartly
bypass the functional consequences associated with kinase inhibitors.15
On the contrary, activator binding may stabilize the binding-competent
state. This may increase the target affinity and often pushes the dynamic
range to lower target concentrations.16
The idea to synthesize boronic acid-based molecules stemmed from
our recent findings where nitrile substituted phenylaminothiazoles
(PAT) were found to act as B-cell lymphoma-2 (Bcl-2) inhibitors.32 The
molecules were found to pan assay interference compounds (PAINS) free
and had adequate ADMET properties to guide the apoptosis in cancerous
cells. Considering PAT moiety (blue highlighted) insignia of anticancer
activity, we started tinkering with nitrile moiety isosteric to carbonyl
moiety (yellow highlighted) (Fig. 3).
Since we were inclined to target PKM2, we found that 1,3 dicarbonyl
moiety (yellow highlighted) existing in curcumin directly modulates
PKM2.33 Upon analyzing the structure, it was further found that Knoe-
venagel condensation of carbonyl substituted phenylboronic acid with
active methylene of 1,3 dicarbonyl moiety yields cinnamate, which is
also a known PKM2 modulator.34 We chose ethyl ester owing to the
toxicity of methyl esters. Further, as explained in the introduction, the
boronic acid moiety involved in the modulation of kinases was incor-
porated. Moreover, it may be metabolized to a phenolic moiety (6c-OH)
by simultaneous generation of non-toxic boric acid.26
Thus, there is a need to develop novel PKM2 activators as anticancer
agents. It has been demonstrated that boronic acid-based molecules are
electrophilic and potent antiproliferative agents.17–21 For instance,
bortezomib is chiefly employed in multiple myelocytic leukemia, and
targets the serine/threonine amino acids of proteasome through its
electrophilic boronic acid core (Fig. 2).22 Boron-containing functional
groups like diazaborines, boronic acids, esters, and benzoxaboroles have
been successfully incorporated into therapeutics. Aryl boronic acids also
provide a viable biomimetic of phenolic group having poor bioavail-
ability profiles.23
Further, to synthesize the molecules, phenyl isothiocyanates (2)
were prepared from aniline (1) derivatives and carbon disulfide via
dithiocarbamate salt intermediate (Scheme 1).35 Tetramethylguanidine
adducts (3) were synthesized by the treatment of isothiocyanate de-
rivatives (2) (1 eq.) with 1,1,3,3 tetramethylguanidine (1 eq.) at room
temperature.36
Boronic acid functional group improves the water solubility of potent
anticancer combretastatin CA-4. The moiety also increases the lip-
ophilicity and affinity of a drug towards the hydrophobic region of the
receptor. It can improve the stability of a drug towards metabolic
degradation.24 At the cell surface, an intense cluster of polysaccharides
is present that forms glycocalyx. The boronic acids can readily form
boronate esters with glycocalyx that may enhance drug selectivity to-
wards tumor cells.25 The widespread utility of boronic acids stems from
its vacant p-orbital of boron. Boron has chameleonic behavior that
switches between electrophilic and nucleophilic states under physio-
logical conditions.26
Tetramethylguanidine adducts (3) underwent cyclization in the
presence of 4-chloroacetoacetate that led to thiazole-based (4) core
structure having a side-chain of 1,3 dicarbonyl moiety.37 Desired com-
pounds (6a-6d) were synthesized via the Knoevenagel condensation
method (Fig. 4).38 (For detailed procedure of synthesis, please refer to
the supplementary information).
PKM2 dimeric form is a hallmark of cancer, while the tetrameric
(active) form is incharge of pyruvate generation. The PKM2 enzyme
activity was ascertained by lactate dehydrogenase (LDH)-coupled
enzyme assay.39 The results indicated that 6a-6d are acting as an acti-
vator with AC50 of 7.15 µM, 6.11 µM, 5.69 µM, and 8.17 µM, respec-
tively. Fig. 5 is showing % relative activity of PKM2 following treatment
with 6a-6d.
Here, we designed and synthesized four novel compounds based on
boronic acids centered around the thiazole core, which is generally
considered as master keys in medicinal chemistry.27–30 We further
describe the enzyme assay of the molecules synthesized, their inhibitory
effects on various cancer cell lines, including triple-negative breast
cancer and mesenchymal stem cells, along with computational studies to
elucidate the mechanism of action of our molecules. This was followed
by the development of a formulation to target colon cancer reported to
have the highest expression of PKM2. Considering the poor aqueous
solubility of 6c, a contemporary nanoparticle-based formulation was
developed. Recently in vitro metabolite profiling of 6c was successfully
done and published.31 We speculated that thiolated chitosan-based
formulation may enhance mucoadhesion to colon cells and can have a
marked impact on gastric retention. Our results strongly support that
rationally designed boronic acid-based PKM2 activators can orchestrate
multifaceted cancer cell elimination responses. Thus, targeting PKM2
with activators may represent a novel anticancer strategy. Modelling
studies suggested that boronic acid scaffold could position itself within
proximity of PKM2 dimers which could enhance the binding kinetics.
To further ascertain the potential of 6a-6d, we screened them against
different cell lines (Table 1). Here, we used a wide range of cancer cell
lines, including drug-resistant MDA-MB and drug-sensitive cell lines like
MCF-7, Bcl-2 Jurkat, and Colo-201.40,41 All the cell lines and normal
fibroblast cells were also treated with varying concentrations of 6a-6d
and assayed for the cell viability by MTT (3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyl tetrazolium) assay.42 It is pertinent to mention that all the
cell lines are known to have sufficient PKM2 expression. Cell growth was
inhibited in all treated cancer cell lines (IC50, 80 nM ꢀ 58.7 μM) in a
concentration-dependent manner (Table 1). It may be noted that the
PKM2 activation at a particular concentration leads to the formation of
tetramer and has reverted the cancerous phenotype leading to
cytotoxicity.
The MCF-7 cell line was found to be more sensitive to 6c, with an
IC50 of 0.08 μM. The activities showed (IC50) in Table 1 established that
6c showed promise against almost all cell lines at various
concentrations.
Fig. 1. Reported PKM2 activators (ML-265, DASA-58, Micheliolide).
2