# Synthesis and Structure of Novel Phenothiazine Derivatives, and Compound Prioritization via In Silico Target Search and Screening for Cytotoxic and Cholinesterase Modulatory Activities in Liver Cancer Cells and In Vivo in Zebrafish

## TL;DR

This study identifies new phenothiazine compounds that show promise as anticancer agents and cholinesterase modulators in liver cancer cells and zebrafish.

## Contribution

The paper introduces novel phenothiazine derivatives with enhanced cytotoxicity and low in vivo toxicity, identified through a combination of in silico, in vitro, and in vivo methods.

## Key findings

- Several novel phenothiazine derivatives showed higher cytotoxicity than the basic PTZ core in liver cancer cell lines.
- Compounds 1, 3, PCP, and TFP interacted with acetylcholinesterase similarly to Huprin W, suggesting cholinesterase modulation.
- Compounds 8 and 10 exhibited significant cytotoxic and cholinesterase modulatory effects with low toxicity in zebrafish.

## Abstract

Phenothiazines (PTZ) are antipsychotics known to modulate
a variety
of neurotransmitter activities that include dopaminergic and cholinergic
signaling and have been identified as potential anticancer agents
in vitro. However, it is important to also test whether a highly cytotoxic,
repurposed, or novel PTZ has low toxicity and neuromodulatory activity
in vivo using vertebrate model organisms, such as zebrafish. In this
study, we synthesized novel phenothiazines and screened them in vitro
in liver cancer and in vivo in zebrafish embryos/larvae. The syntheses
of several intermediate PTZ 10-yl acyl chlorides were followed by
elemental analysis and determination of 1H NMR and 13C NMR mass (ESI+) spectra of a large number of
novel PTZ 10-carboxamides. Cytotoxicities of 28 PTZ derivatives (1–28) screened against Hep3B and SkHep1 liver cancer
cell lines revealed five intermediate and five novel leads along with
trifluoperazine (TFP), prochlorperazine (PCP), and perphenazine, which
are relatively more cytotoxic than the basic PTZ core. Overall, the
derivatives were more cytotoxic to Hep3B than SkHep1 cells. Moreover,
in silico target screening identified cholinesterases as some of the
commonest targets of the screened phenothiazines. Interestingly, molecular
docking studies with acetylcholinesterase (AChE) and butyrylcholinesterase
proteins showed that the most cytotoxic compounds 1, 3, PCP, and TFP behaved similar to Huprin W in their amino
acid interactions with the AChE protein. The highly cytotoxic intermediate
PTZ derivative 1 exhibited a relatively lower toxicity
profile than those of 2 and 3 during the
zebrafish development. It also modulated in vivo the cholinesterase
activity in a dose-dependent manner while significantly increasing
the total cholinesterase activity and/or ACHE mRNA
levels, independent of the liver cancer cell type. Our screen also
identified novel phenothiazines, i.e., 8 and 10, with significant cytotoxic and cholinesterase modulatory effects
in liver cancer cells; yet both compounds had low levels of toxicity
in zebrafish. Moreover, they modulated the cholinesterase activity
or expression of ACHE in a cancer cell line-specific
manner, and compound 10 significantly inhibited the cholinesterase
activity in zebrafish. Accordingly, using a successful combination
of in silico, in vitro, and in vivo approaches, we identified several
lead anticancer and cholinesterase modulatory PTZ derivatives for
future research.