Reprofiling of pyrimidine-based DAPK1/CSF1R dual inhibitors: identification of 2,5-diamino-4-pyrimidinol derivatives as novel potential anticancer lead compounds

Figures & data

Figure 1. Reported pyrimidine-based anticancer molecules and design rationale for anticancer 2,5-diamino-4-pyrimidinol derivatives.

Table 1. General structure and specific examples of the compounds being evaluated for their antiproliferative effects in this report.

Compound	R^1	R^2	X	Y	Z
6a	OMe	3,5-Dimethoxy	CH	CH	Morpholino
6b	OMe	3-MeO	CH	CH	Morpholino
6c	OMe	3-CF3	CH	CH	Morpholino
6d	OMe	3,5-Dimethoxy	CH	N	Morpholino
6e	OMe	3,5-Dimethoxy	CH	N	Piperidino
6f	OMe	3,5-Dimethoxy	CH	CH	Fluoro
6g	CF3	3,5-Dimethoxy	CH	N	Morpholino
6h	CF3	H	N	N	Morpholino

Table 2. GI₅₀ values (μM) exhibited by selected compounds over M-NFS-60 murine myeloblastic leukaemia cell line.

Compound	M-NFS-60 cell line	CSF1R	DAPK1
	% Inhibition^a (GI50 in μM^b)	Inhibition (%)^a	Inhibition (%)^a
6a	77.87 ± 0.46 (5.81 ± 1.03)	95.34 ± 1.28	65.95 ± 2.21^c
6b	92.31 ± 0.8 (4.10 ± 1.08)	91.85 ± 1.31	75.06 ± 1.26
6c	52.57 ± 0.61^c (10.2 ± 1.15)	74.70 ± 1.28	69.70 ± 1.19^c
6d	88.81 ± 1.38 (4.96 ± 1.11)	77.94 ± 0.52	66.22 ± 0.26
6e	99.18 (1.97 ± 1.40)	75.99 ± 0.49	67.28 ± 2.95
6f	ND	35.20	65.00
6g	ND	ND	17.16 ± 2.10
6h	ND	ND	7.12 ± 2.98

^a % Inhibition ± SD value at 10 μM dose.

^b GI₅₀±SD value data were generated from the dose-response curve plotted from a 10-dose experiment starting at 10 μM performed in duplicate.

^c Measured at 20 μM.

ND: Not determined.

Table 3. % Inhibition of kinase reactions of a panel of kinases by 10 μM doses of compound 6e.

Kinase	% Inhibition	Kinase	% Inhibition
AXL	0.44	FLT3	14.52
c-Kit	3.63	JAK3	−16.66
c-MER	−5.95	KDR/VEGFR2	−16.61
c-MET	−16.40	PDGFRa	−5.94
CDK2/cyclin A	−7.09	PDGFRb	−1.76
EGFR	−9.78	RET	−3.48
FLT1/VEGFR1	−5.79	TYRO3/SKY	−10.84

The significance of bold values represent the highest values.

Figure 2. Measured % growth inhibition of various hematological cancers triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 3. Measured % growth inhibition of various NSCLC cells triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 4. Measured % growth inhibition of various colon cancers triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 5. Measured % growth inhibition of various brain cancer cell lines triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 6. Measured % growth inhibition of various melanoma cell lines triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 7. Measured % growth inhibition of various ovarian cancer cell lines triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 8. Measured % growth inhibition of various renal cancer cells triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 9. Measured % growth inhibition of various prostate cancer cell lines triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 10. Measured % growth inhibition of various breast cancer cells triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Figure 11. Average % values of measured growth inhibition of nine cancer diseases triggered by 10 µM doses of compounds 6a–h and hit compound 4. Values are the averages of duplicate assays.

Table 4. In vitro measured effective permeability (P_e) of compound 6e as well as verapamil and ranitidine using PAMPA-GIT.

Compound	Pe (10^−6 cm/s)±SD
6e	19.0 ± 1.1^a
Verapamil^b	83.8 ± 2.9
Ranitidine^c	0.26 ± 0.02

^a A 12.5 μM concentration was used and the results are the means of three independent experiments ± standard deviation.

^b Verapamil (50 μM) was used as the positive control agent for the PAMPA assay.

^c Ranitidine (50 μM) was used as the negative control agent for the PAMPA assay.

Figure 12. Calculated binding modes and interactions of compound 6e: (A) Compound 6e docked within kinase binding site of CSF1R (PDB code: 3KRJ); (B) Predicted interactions of compound 6e within the binding pocket of CSF1R (PDB code: 3KRJ); (C) Compound 6e docked within substrate-binding site of DAPK1 (PDB code: 4TXC); (D) Predicted interactions of compound 6e within the substrate binding pocket of DAPK1 (PDB code: 4TXC).

Scheme 1. Synthesis of compounds 6g–h. Reagents and conditions: (a) 6-morpholinopyridin-3-amine, pyridine, THF, rt, 12 h; (b) H₂, 10% Pd/C, MeOH, rt, 12 h; (c) appropriate acyl chloride, DIPEA, DCM, −78 °C to rt, 2 h.