Novel Glu-based pyrazolo[3,4-d]pyrimidine analogues: design, synthesis and biological evaluation as DHFR and TS dual inhibitors

Figures & data

Figure 1. Structures of some antifolates and design strategy of novel Glu-based pyrazolo[3,4-d]pyrimidine analogues.

Scheme 1. Synthesis of target compounds 6a–l. (i) R₂-NHNH₂, EtOH, reflux, 6 h; (ii) R₃COOH, POCl₃, reflux, 2h; (iii) ClCH₂COCl, THF, reflux, 1 h; (iv) 5, DMF, K₂CO₃, NaI, 80 °C, 1–2 h.

Scheme 2. Synthesis of acid derivatives 7a,b. (i) aq. NaOH, 25 °C, 2h.

Scheme 3. Synthesis of substituted 1,4-dihydropyrimidine derivatives 9a–c. (i) HCl, EtOH, reflux, 6 h; (ii) 5, DMF, K₂CO₃, NaI, RT, 8 h.

Figure 2. MGI% for compounds 6a–l and 9a–c towards a full panel of 59 cell lines.

Figure 3. GI% of the most active derivative 6i against NCI-59 cancer cells.

Table 1. NCI-59 cancer cell five-dose screening results of compound 6i (NSC: 838397).

Subpanel/cancer cell lines		Compound 6i
	GI50 (µM)	TGI (µM)	LC50 (µM)
Leukaemia
HL-60(TB)	>100	>100	>100
CCRF-CEM	14.5	>100	>100
MOLT-4	>100	>100	>100
K-562	>100	>100	>100
SR	71.0	>100	>100
RPMI-8226	22.7	>100	>100
Non-small cell lung cancer
EKVX	10.4	27.3	71.7
A549/ATCC	9.81	57.3	>100
HOP-62	5.11	19.8	57.5
HOP-92	6.01	23.7	66.2
NCI-H226	4.54	27.8	>100
NCI-H23	7.64	26.7	82.1
NCI-H322M	21.3	66.6	>100
NCI-H460	13.7	34.4	86.9
NCI-H522	17.1	38.2	85.0
Colon cancer
COLO 205	14.0	36.7	96.0
HCC-2998	35.1	>100	>100
HCT-116	9.85	>100	>100
SW-620	8.98	44.4	>100
HCT-15	26.0	>100	>100
KM12	16.1	>100	>100
HT29	18.4	71.7	>100
CNS cancer
SF-268	7.01	32.8	>100
SF-295	14.2	31.9	71.6
SF-539	14.8	33.0	73.6
SNB-19	12.1	27.3	61.3
SNB-75	14.9	40.9	>100
U251	13.2	>100	>100
Melanoma
M14	>100	>100	>100
LOX IMVI	5.3	20.6	58.4
MALME-3M	15.0	29.9	59.4
SK-MEL-2	27.6	89.1	>100
MDA-MB-435	12.2	34.3	96.1
SK-MEL-5	19.4	43.1	95.8
SK-MEL-28	10.1	22.5	50.2
UACC-62	13.3	32.1	77.8
UACC-257	31.5	>100	>100
Ovarian cancer
OVCAR-3	11.7	61.3	>100
IGROV1	23.9	74.2	>100
OVCAR-5	20.2	56.3	>100
OVCAR-4	13.4	32.1	76.7
SK-OV-3	21.8	67.3	>100
OVCAR-8	8.17	>100	>100
Renal cancer
A498	24.9	48.1	93.0
786-0	8.66	>100	>100
CAKI-1	10.4	22.1	47.0
ACHN	15.1	34.3	78.3
SN12C	29.2	>100	>100
RXF 393	15.9	50.9	>100
UO-31	18.1	55.9	>100
TK-10	31.3	86.2	>100
Prostate cancer
DU-145	7.47	28.8	>100
PC-3	5.79	19.4	47.7
Breast cancer
MDA-MB-231/ATCC	11.5	26.8	62.5
MCF7	3.93	16.5	59.4
BT-549	4.54	>100	>100
HS 578 T	12.7	45.7	>100
MDA-MB-468	13.5	51.3	>100
T-47D	45.6	>100	>100

Table 2. MG-MID values and selectivity index for compound 6i towards the herein examined subpanels.

Subpanel cancer cell line	Compound
	6i
	MG-MID	Selectivity index
Leukaemia	68.03	0.30
Colon cancer	18.34	1.11
Non-small cell lung cancer	10.62	1.92
Melanoma	16.8	1.21
CNS cancer	12.70	1.61
Renal cancer	19.19	1.06
Ovarian cancer	16.52	1.23
Breast cancer	15.29	1.33
Prostate cancer	6.63	3.08
Full panel MG-MID	20.45

Figure 4. preliminary SAR study of target compounds.

Figure 5. Flow cytometric analyses: cell cycle distribution analysis using PI staining method in which MCF-7 cells were treated by 6i (10 µM) and DMSO (negative control; NC) for 48 h; (A) 6i, (B) DMSO, (C) cells percentage in different phases (G0-G1, S, G2/M); apoptotic induction using annexin-V-FITC/PI staining method in which cancer cells were treated by 6i (10 µM) and DMSO (negative control; NC) for 24 h; (D) 6i, (E) DMSO, (F) histogram for induction of apoptosis.

Figure 6. Binding modes to the active sites of DHFR and TS enzymes: (A) 3D representation of 6i displaying an overlay with the crystal structure of MTX in the active site of DHFR; (B) 3D representation of 6i demonstrating an overlay with the crystal structure of PMX in the active site of TS; (C) 2D diagram of 6i in DHFR active site; (D) 2D diagram of 6i in TS active site.

Table 3. Growth inhibitory activity of compound 6i against MCF-7, PC-3, OVCAR-3, and HSF cells.

Comp.	IC50 (µM)^a				Selectivity index
	MCF-7	PC-3	OVCAR-3	HSF	MCF-7	PC-3	OVCAR-3
6i MTX	4.40 ± 0.26	12.42 ± 0.62	11.52 ± 0.58	48.52 ± 2.43	11.02	3.91	4.21
	0.58 ± 0.03	2.72 ± 0.14	6.31 ± 0.32	13.68 ± 0.69	23.71	5.03	2.16

^aValues are the mean ± SD from triplicates.

Table 4. DHFR and TS inhibitory activity of compound 6i and reference drugs.

	IC50 (µM)
	DHFR	TS
6i	2.41 ± 0.13	8.88 ± 0.46
MTX	0.11 ± 0.01	–
5-FU	–	3.60 ± 0.19

Note: ± represents the standard deviation from triplicates.

Table 5. Lipinski’s rule of five calculated parameters for the most active anti-cancer compound 6i.

Parameter							Pfizer rule
Log P^a	TPSA^b	MW^c	nHBD^d	nHBA^e	nRB^f	Log S^g
3.32	163.51	666.14	2	13	16	−5.07	Accepted

^aCalculated lipophilicity.

^bTotal polar surface area.

^cMolecular weight.

^dHydrogen bond donors.

^eHydrogen bond acceptors.

^fRotatable bonds.

^gLog of the aqueous solubility.

Table 6. Predicted ADMET properties for the most active anti-cancer compound 6i.

SAscore	HIA%	F20%	PPB	CYP1A2 CYP2C19 CYP2D6 inhibition	CL	Carcinogenicity	Acute toxicity rule
3.14	≥90%	≥20%	91.72%	Non-inhibitor	2.47	Noncarcinogenic	Non-toxic