Design, Synthesis of Mannich Bases Derivatives of Thiosemicarbazide and their Evaluation for Anticancer Activity using Potato Disk Bioassay Method

Mannich bases and thiosemicarbazide individually are known to show verity of pharmacological activities such as anti-inflammatory, anticancer, antifilarial, antibacterial, antifungal, anticonvulsant, anthelmintic, antitubercular, analgesic, anti-HIV, antimalarial, antipsychotic, antiviral and so forth. As novel attempt in present work in first step synthesis of verity of mannich bases is done using structurally different types of aldehyde, ketones and amines. The condensation of synthesized mannich bases is done with thiosemicarbazide to form a novel class of compounds called mannich bases of thiosemicarbazide. Use of bioassay is one of the ways of carrying out preliminary investigation of activity. A. tumefaciens induced potato disc tumor assay has been used to investigate anticancer activity of synthesized compounds. The compounds B2, B4, B25, B26, B28, B29 and B30 have shown same or better inhibitory activity compared to Gemcitabine used as standard. Original Research Article Patil and Pishawikar; JPRI, 34(27B): 53-62, 2022; Article no.JPRI .83427 54


INTRODUCTION
There are number of life-threatening diseases prevalent globally and slowly cancer has become a big threat to human beings. Newer types of cancer are coming into picture. Through various reports on cancer cases it is estimated that 19.3 million new cancer cases (18.1 million excluding nonmelanoma skin cancer) and almost 10.0 million cancer deaths (9.9 million excluding nonmelanoma skin cancer) occurred globally in 2020 [1].
In India, category wise i.e. the total number of new cases in males will increased from 0.589 million in 2011 to 0.934 million by the year 2026. In females the new cases of cancer increased from 0.603 to 0.935 million [1][2].
Cancer has become second largest disease after cardiovascular disorders responsible for maximum mortality with around 0.3 million deaths per year in India [1][2]. This has led to the continuous increase in demand for new antitumor drugs with different mechanism of action [3][4][5][6][7][8].
As per the CPCAC guidelines 3 Rs likewise replacement, reduction and refinement have led restricted use of animal in research. There is increase emphasis on replacement of animal methods by non-animal methods. The refinement of experimental method emphasizes on reducing the pain and suffering of animals used [2][3]. Most convenient and inexpensive alternative to animal studies are plant tumor assay methods that can be used for screening new anticancer drugs [9][10].
Bioassay methods offer special advantages in establishing the biological activities like antitumor, antibacterial, antioxidant and phytotoxic properties and are becoming a preliminary step in drug discovery.
For preliminary investigation of anticancer activity Potato disc assay has become most useful method. The bioassay is based on inhibition of tumour caused by Agrobacterium tumefaciens in potato disc. The mechanism of initiating tumour in plant tissues, is similar to tumour generated by carcinogenic agents in humans which validates the use of assay in screening new anti-cancer drugs. In fact, Kempf, et al., have confirmed that Bartonella henselae, a bacterium causing tumour in human shows a similar pathogenic strategy as shown by plant pathogen A.
tumefaciens. The similarities include the use of common toxins, secretion system, adhesion mechanism, invasion and regulation. The antitumor potato disc assay is considered to be sensitive for screening chemical compounds having different modes of action for interfering with cell cycle whereby they can show anticancer activity [9][10][11].
Mannich base and thiosemicarbazide individually are known to show different types of pharmacological activities as well as act as important pharmacophores or lead structures used in synthesis of various potential agents with high medicinal value [12,4].
The aim of present work was to combine two pharmacologically active entities in synthesizing thiosemicarbazide derivatives of mannich bases as novelty with expectation that each derivative would show good anticancer activity.

Synthesis of Mannich Bases of Thiosemicarbazide
Step One Synthesis of mannich bases is done using aldehyde, ketone and amines having aliphatic, aromatic, cyclic and heterocyclic nature in proportion 1.00 molecular equivalent of carbonyl compound (ketone), 1.05-1.10 molecular equivalent of amine in the form of hydrochloride salt and 1.5-2.0 molecular equivalence of aldehyde. The reaction conditions had to be optimized individually and the time of reaction varied from 45 minutes to 12-14 hr.

Step Two
The synthesized mannich bases were condensed with thiosemicarbazide in one is to one proportion to get mannich bases of thiosemicarbazide.

Identification and Characterization of Synthesized Compounds
All the synthesized compounds were identified and characterized by following methods:

Scheme 1. Scheme for Synthesis
 Physicochemical characterization  Qualitative chemical Analysis  Thin layer chromatography  Infra-red spectroscopy  UV-Visible spectroscopy  Nuclear magnetic resonance spectroscopy Results are mentioned in Table. 9

Potato disk bioassay
The inhibition of A. tumefaciens-induced tumors (or Crown Gall) in potato disc tissue is an assay based on antimitotic activity and can detect a broad range of known and novel antitumor effects.

Procedure
Fresh Russet potatoes were collected from a local grocery store. Sterilized in laboratory using a 20% bleaching solution. The potato discs of dimension 1 cm x 1 cm x 0.5 cm were cut. Five discs were placed in 1.5% agar media in petri plates and allowed to submerged up to 2/3. The experiment was performed in three groups; Test group (Solutions of different synthesized compounds), control group (DMSO with Sterile water) and Standard group (Gemcitabine). 10 ml of solution of each synthesized mannich bases of thiosemicarbazide having concentrations of 100 PPM and 10PPM was prepared using DMSO in disposable culture tubes. Further inoculums were prepared as follows: 1) 1.5 ml sterile water, 2.5 ml 48 hrs incubated bacterial culture and 5 ml sample were added in DMSO.
2) Controls were prepared by replacing extract with only DMSO with Sterile water.
3) Same procedure was followed for standard anticancer drug Gemcitabine.
From different groups, 0.05 ml sample were added on five potato discs in respective labeled Petri plates (test with respective compounds, standard and control). Plates were covered, tape the lids using cello tape (to minimize moisture loss), and incubate under dry conditions at room temperature for 7 -12 days. After 7 -12 days incubation, the potatoes discs were analyzed using a colony counter magnifying glass after staining with Lugol's Solution and experiment was repeated in triplicate. The tumors lack of starch will turn orange to black in the presence of the stain while the potato discs will turn dark blue. Potato discs inoculated with the control solutions should average 10-30 tumors. Finally calculation of the percentage inhibition of crown gall tumors was done using following formula: Average number of tumors in control

DISCUSSION
Compounds like B2 and B4 in which all components used for synthesis of mannich base are aromatic in nature have shown comparable activity to standard.
The compound with nitro substituted aromatic ketone like B25 have shown good activity.
Compound having combination of Aromatic -Aldehyde, Aliphatic-Ketone and Heterocyclic: Amine like compound B26 has shown highest activity.

CONCLUSION
From bioassay results it can be concluded that thiosemicarbazide derivatives of mannich base as new chemical entity have shown promising results. By carrying out further In-vitro and in-vivo screening studies confirmation of the potential of developed mannich bases of thiosemicarbazide as anticancer drugs as well as new chemical entity needs to be done.