Synthesis, anticancer evaluation, molecular docking and ADME study of novel pyrido[4',3':3,4]pyrazolo[1,5-a]pyrimidines as potential tropomyosin receptor kinase A (TrKA) inhibitors

Abstract

The starting compound 3-amino-1,7-dihydro-4H-pyrazolo[4,3-c]pyridine-4,6(5H)-dione (1) is reacted with each of diketone and β-ketoester, forming pyridopyrazolo[1,5-a]pyrimidines 4a,b and 14a,b, respectively. The compounds 4 and 14 reacted with each of aromatic aldehyde and arenediazonium salt to give the respective arylidenes and arylhydrazo derivatives, respectively. The structure of the new products was established using spectroscopic techniques. The cytotoxic activity of selected targets was tested in vitro against three cancer cell lines MCF7, HepG2 and HCT116. The data obtained from enzymatic assays of TrKA indicated that compounds 7b and 16c have the strongest inhibitory effects on TrKA with IC₅₀ = 0.064 ± 0.0037 μg/ml and IC₅₀ = 0.047 ± 0.0027 μg/ml, respectively, compared to the standard drug Larotrectinib with IC₅₀ = 0.034 ± 0.0021 μg/ml for the HepG2 cancer cell line. In cell cycle analysis, compounds 7b, 15b, 16a and 16c caused the greatest arrest in cell cycle at the G2/M phase. In addition, compound 15b has a higher apoptosis-inducing effect (36.72%) than compounds 7b (34.70%), 16a (21.14) and 16c (26.54%). Compounds 7b, 16a and 16c were shown fit tightly into the active site of the TrKA kinase crystal structure (PDB: 5H3Q). Also, ADME study was performed on some selected potent anticancer compounds described in this study.

Keywords: ADME studies; Anticancer activity; Apoptotic activity; Molecular docking; Pyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]pyrimidines; TrKA enzyme.

Fig. 1

The three types of tropomyosin receptor kinases (Trks)

Fig. 2

Larotrectinib and entrectinib as type-I multi-target kinase inhibitors

Fig. 3

Some pyrazolo[1,5-a]pyrimidines such as I–III with a standard drug dinaciclib as anticancer agents

Fig. 4

Our target compounds as anticancer agents and TrKA inhibitors compared to larotrectinib

Scheme 1

Synthesis of pyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]pyrimidines 4a,b

Scheme 2

Synthesis of compounds 7a–t

Scheme 3

Synthetic route of arylhydrazonopyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]pyrimidine diones

Scheme 4

Synthesis of arylhydrazono derivatives 11a–d

Scheme 5

Synthetic route for 4-hydroxy-2-substituted pyrido[4ʹ,3ʹ:3,4]pyrazolo[1,5-a]-pyrimidine-8,10-diones 14a,b

Scheme 6

Synthesis of arylmethylene derivatives 15a–f

Scheme 7

Synthesis of arylhydrazono derivatives 16a–h

Fig. 5

IC₅₀ values of 1

Fig. 6

IC₅₀ values of 4a

Fig. 7

IC₅₀ values of 4b

Fig. 8

IC₅₀ values of 7a

Fig. 9

IC₅₀ values of 7b

Fig. 10

IC₅₀ values of 7c

Fig. 11

IC₅₀ values of 7k

Fig. 12

IC₅₀ values of 7l

Fig. 13

IC₅₀ values of 9a

Fig. 14

IC₅₀ values of 9b

Fig. 15

IC₅₀ values of 9c

Fig. 16

IC₅₀ values of 14a

Fig. 17

IC₅₀ values of 15b

Fig. 18

IC₅₀ values of 16a

Fig. 19

IC₅₀ values of 16b

Fig. 20

IC₅₀ values of 16c

Fig. 21

Structure activity relationship (SAR) of some synthesized compounds

Fig. 22

Enzyme inhibition of tested compounds

Fig. 23

Cell cycle analysis of compounds 7b, 15b, 16a and 16c. Compounds 7b, 15b, 16a and 16c increased the ratio of cells at phases in the G2/M by about 4, 4, 2.5 and 3 times, respectively, and HepG2 cells were arrested in the cell cycle at G2/M phase by compounds 7b and 15b

Fig. 24

Cell cycle of control HepG-2

Fig. 25

Cell cycle of compound 7b

Fig. 26

Cell cycle of compound 15b

Fig. 27

Cell cycle of compound 16a

Fig. 28

Cell cycle of compound 16c

Fig. 29

Apoptosis and necrosis of tested compounds 7b, 15b, 16a, 16c with control

Fig. 30

Interaction of 5H3Q with the active site in 2D and 3D

Fig. 31

Interaction of larotrectinib with the active site of 5H3Q in 2D and 3D

Fig. 32

Interaction of 7b with the active site of 5H3Q in 2D and 3D

Fig. 33

Interaction of 16a with the active site of 5H3Q in 2D and 3D

Fig. 34

Interaction of 16c with the active site of 5H3Q in 2D and 3D

Fig. 35

Boiled-egg depicts gastrointestinal absorption and brain penetration of compounds 4a, 7a–c, 9c, 15b and 16a–c