Design, synthesis and biological evaluation of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives as CDK inhibitors

Figures & data

Figure 1. Discovery of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives as CDK inhibitors.

Scheme 1. Synthesis of 2-((4-sulfamoylphenyl)amino) substituted derivatives. Reagents and conditions: (a) arylamine, Xantphos, Pd₂ (db)₃, Cs₂CO₃, DMF, 110 °C, microwave irradiation.

Scheme 2. Synthesis of 2-((4-sulfamoylphenyl)amino) substituted derivatives. Reagents and conditions: (a) K₂CO₃, DCM,RT; (b) Pd/C, Methanol; (c) 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide, Xantphos, Pd₂ (db)₃, Cs₂CO₃, DMF, 110 °C, microwave irradiation; (d) 2 M hydrochloride in Methanol.

Scheme 3. Preparation of 2-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidine derivatives. Reagents and conditions: (a) 2-methoxyphenylamine, DIPEA, acetonitrile, 0 °C; (b) cyclopentylamine, acetonitrile;(c) 4-chloro-2-(methylthio)pyrimidine-5-carbaldehyde, DIPEA, acetonitrile, MW, 80 °C; (d) Cs₂CO₃, MW, 120 °C, 30 min, acetonitrile; (e) m-CPBA, DCM; (f) N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)formamide or N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)formamide, Cs₂CO₃, DMSO, 85 °C

Table 1. Anti-proliferative activity of 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives in pancreatic cancer MIA PaCa-2 cell culture.

Cpd.	R1	R2	X	IC50 (µM) in MIA PaCa-2^a	CDK9 inhibitory rate (%) @ 50nM^b
1			CH	4.98 ± 2.68	98.6
2a			CH	13.00 ± 0.37	100.3
2b			CH	14.35 ± 0.21	98.7
2c			CH	18.80 ± 5.54	99.1
2d			CH	6.23 ± 1.25	91.8
2e			CH	10.50 ± 0.74	89.3
2f			CH	18.90 ± 2.121	96.5
2g			CH	0.52 ± 0.67	97.8
2h^c		-SO2-R2 not exist	CH	45.05 ± 16.64	93.2
2i			N	>100	27.4
2j			N	69.13 ± 15.51	25.8
5b			CH	0.94 ± 0.25	98.2
5c			CH	7.80 ± 1.04	99.1
5d			CH	2.85 ± 0.52	80.6
5e			CH	4.35 ± 1.61	81.8
10a			CH	40.76 ± 3.26	−3.1
10b			CH	80.76 ± 15.99	54.4
Ribociclib				16.53 ± 7.00	16.9
GCTB				1.04 ± 0.27

^aCell viability was determined after 72 h drug exposure to cells. Compounds 2b, 2c, and 5c precipitated in cell culture medium during incubation. ^bTest compounds were screened against on CDK9/cyclinT1 by Lance Ultra Assay with ATP concentration at Km. ^ccompound 2h was previously reported²³.

Table 2. Anti-proliferative activity of active 2-((4-sulfamoylphenyl)amino)-pyrrolo[2,3-d]pyrimidine derivatives in different PDAC cell lines.

Cpd.	IC50 (µM)^a
	AsPC-1	BxPC-3	PANC-1
1	2.598 ± 0.997	0.409 ± 0.060	3.679 ± 2.499
2g	1.403 ± 0.034	0.226 ± 0.065	11.022 ± 4.967
5b	1.479 ± 0.090	0.249 ± 0.099	5.814 ± 1.384
GCTB	0.956 ± 0.140	0.388 ± 0.119	5.789 ± 4.206

^aCell viability was determined after 72 h drug exposure to cells.

Figure 2. Compound 2g induced slightly cell cycle arrest and late apoptosis of MIA PaCa-2 cells. The cells were incubated without or with 2g at indicated concentrations for 24 h. The cell cycle distribution (Upper lane) or apoptosis (Lower lane) was detected using propidium iodide (PI) staining or both PI and annexin V-FITC staining, respectively, and analysed by flow cytometry assays. Graphs showing the results of a representative experiment, and the data was presented as mean ± standard deviation.

Figure 3. The anti-proliferative activity of new synthetic compounds is mainly mediated by CDK9. (A) 2g inhibited the phosphorylation of Rb and RNA polymerase II, induced apoptosis through down-regulation of Mcl-1 and c-Myc protein. MIA PaCa-2 cells were incubated with or without 2g at indicated concentration for 6 h. (B) CDK9 was dose-dependently knockdown by its specific siRNA after 48 h treatment in MIA PaCa-2 cells. (C) Downregulation of CDK9 using siCDK9 (50 nM) decreased the sensitivity of MIA PaCa-2 cell to compound 2g (0.2 µM). The cells were incubated with 2g for 72 h. NS no significance; ***p < 0.001; ****p < 0.0001. Whole-cell lysates were subjected to immunoblotting. A representative protein band of three independent experiments is shown. (D) Representative illustration of the binding of synthetic compound to CDK9/cyclin T. Small molecules 1 (left column), 2g (middle column), and 2i (right column) were shown in stick representation with carbons colored yellow.

Table 3. IC₅₀ (nM) values of anti-proliferative compounds against CDK activity.

Cpd.	IC50 (nM)^a
	CDK1	CDK2	CDK4	CDK7	CDK9
1	515.8	91.6	89	>10^4	5.6
2d	ND^b	7984.4	148	>10^4	7.7
2g	54	21	12	>10^4	5.0
5b	333	147	20	4967	3.2
5c	550	227	35	>10^4	6.3
5d	906	2338.3	170	>10^4	20
5e	439	235.9	52	>10^4	13
Ribociclib	ND	ND	7.1	ND	2695
Staurosporine	2.5	0.9	28	70	15

^aIC₅₀ values of test compounds were determined with a serial of 3-fold diluted concentrations from top concentration of 10 μM and ATP concentration at Km in Mobility shift assay by single experiment.

^bND represents not determined.

Figure 4. Oral PK study of compounds 2g and 5 b after oral administration to rats (n = 3) at dose of 30 mg/kg.

Figure 5. In vivo efficacy evaluation in AsPC-1 cell-derived xenograft mice model (n = 6).

Figure 6. Representative H&E-stained tissue section (scale bar, 50 µM for all) from CDX mice treated with vehicle, GCTB or 2g alone, or combination of GCTB and 2g for 3 weeks.

Table 4. Drug concentrations in tumour tissue and plasma sample after 30 mg/kg single dosing of 2g to tumour-bearing mice (n = 3).

Time after dosing (h)	Drug concentration (µM)
	Tumour	Plasma
1.0	3.13 ± 0.64	7.46 ± 1.95
4.0	0.48 ± 0.34	0.53 ± 0.41

Shi X, Quan Y, Wang Y, Wang Y, Li Y. Design, synthesis, and biological evaluation of 2,6,7-substituted pyrrolo[2,3-d]pyrimidines as cyclin dependent kinase inhibitor in pancreatic cancer cells. Bioorg Med Chem Lett. 2021;33:127725.

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