Construction of C-glycosides of heterocycles containing the pyrimidin-2-amine or the 1 H -pyrazolo[3,4- b ]pyridine moiety and their biological evaluation for anticancer activities

Abstract

A series of novel C-glycosides of heterocyclic derivatives containing a pyrimidin-2-amine or a 1H-pyrazolo[3,4-b]pyridine moiety were synthesized using condensation reactions of the substituted puerarin with guanidine or 3-amino-5-hydroxypyrazole in methyl alcohol. Their chemical structures were characterized by Fourier-transform infrared spectroscopy, nuclear magnetic resonance, and high-resolution mass spectrometry. In addition, their biological activity has been demonstrated by in vitro evaluation against the human leukemia cells K562 and human prostate cancer cells PC-3 by MTT-based assays, using the commercially available standard drug of cis-platin as a positive control. The results also demonstrated that most of the compounds showed considerable cytotoxicity to these two cell lines of K562 and PC-3, and indicated that novel C-glycosides of heterocyclic derivatives may be potential leads for further biological screenings and may generate drug-like molecules.

Introduction

Glycosides have received increasing interest in medicinal chemistry as carbohydrate biomimetics.^1–3 As compared to O-glycosidic linkage in glycosides the C-glycosidic linkage offers stability to glycosidic bonds toward acidic or enzymatic hydrolysis and therefore these compounds are comparatively more stable. C-glycosides of synthetic and natural origins have been reported to possess a vast array of biological activities, such as antitumor, ⁴ antibiotic, ⁵ or SGLT2 inhibitor activity. ⁶ Among these compounds, C-glycosides of heterocycles are essential and reliable platforms for the development of many anticancer and antitumor drugs ⁷ and have been regarded as good glycosyl donors, in addition to their biological activities such as inhibition of enzyme activity. ⁸

In Chinese herbal medicine, flavonoids constitute the largest and most important group of polyphenolic compounds, which possess various beneficial biological properties, including anti-inflammatory, ⁹ antiviral, ¹⁰ and antitumor activities. ¹¹ Puerarin, in which a β-D-glucopyranose is directly linked through a carbon–carbon bond to the A-ring of daidzein[7-hydroxy-3-(4-hydroxyphenyl)chromone], was isolated from Pueraria lobota, ¹² one of the most popular Chinese herbal medicines. It has beneficial effects on cardiovascular, neurological, and hyperglycemic disorders.^13,14 It could suppress the phosphorylation and degradation of NF-kB ¹⁵ and inhibit mRNA and protein expression of C-reactive protein in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells, suggesting anti-inflammatory activity. ¹⁶ In addition, previous research has found that the puerarin nanosuspensions suppressed the HT-29 proliferation, increased the early apoptosis, and enhanced the in vivo anticancer effectiveness toward human colon cancer HT-29 cells. ¹⁷ Subsequently, a finding suggested that a puerarin nanosuspension effectively inhibited the growth of HepG2 cells in vitro. ¹⁸ Recently, puerarin has been reported to significantly inhibit LPS-induced MCF-7 and MDA-MB-231 cell migration, invasion, and adhesion, ¹⁹ and suppress the enzymatic activity of cyclooxygenase-2 (COX-2) in A549 cells and its expression in tumor tissues with pulmonary metastasis. ²⁰ In the reported experiments, the efforts were centered mostly on the substitutions on the aromatic rings (either phenolic hydroxyl or glucosyl group) of puerarin.^21–24 It was reported that the chromone fragment can generate a 1,3-diketone equivalent in the presence of alkali, which readily reacts with amidines^25,26 and hydrazine ²⁷ to form the corresponding pyrimidines and pyrazoles. We report herein a facile construction of novel C-glycosides of heterocycles containing the pyrimidin-2-amine or the 1H-pyrazolo[3,4-b]pyridine 3 fragments via condensation reactions of the substituted puerarin with guanidine or 3-amino-5-hydroxypyrazole. In particular, their biological activities against human lung cancer cells A549 and human prostate cancer cells PC-3 have been evaluated.

Results and discussion

Initially, the reaction of puerarin (1a) with guanidine (2a) was investigated under various reaction conditions, as shown in Table 1. We conducted the reaction using different bases in alcohol. As a result, no products were observed when the base is triethylamine (NEt₃) or potassium carbonate (K₂CO₃) (Table 1, entries 1 and 2). Sodium methylate (MeONa) was found to be the most effective of the four bases examined for the preparation of 3a (Table 1, entry 4). Afterwards, different solvents were screened (Table 1, entries 4–9) and we were pleased to find that methyl alcohol (MeOH) gave satisfactory results (Table 1, entry 5). Then the effect of the molar ratios (1a:2a:base) on the yield was evaluated (Table 1, entries 10–14). We found that the yield of 3a was high for the cyclocondensation reaction when the molar ratio was 1:3:3. Thus, the optimal reaction conditions we established were puerarin (1a, 1 mmol), guanidine (2a, 3 mmol), and MeONa (3 mmol) in 20 mL of MeOH under reflux until complete disappearance of 1a.

Table 1.

Screening of reaction conditions ^a .

Entry	Solvent b	Base	Molar ratios (1a:2a:base)	Yield c (%)
1	EtOH	NEt3	1:1:2	nr d
2	EtOH	K2CO3	1:1:2	nr d
3	EtOH	NaOH	1:1:2	25
4	EtOH	MeONa	1:1:2	41
5	MeOH	MeONa	1:1:2	55
6	THF	MeONa	1:1:2	36
7	MeCN	MeONa	1:1:2	32
8	DMSO	MeONa	1:1:2	40
9	DMF	MeONa	1:1:2	44
10	MeOH	MeONa	1:1:3	62
11	MeOH	MeONa	1:1:4	58
12	MeOH	MeONa	1:2:3	66
13	MeOH	MeONa	1:3:3	72
14	MeOH	MeONa	1:4:3	68

THF: tetrahydrofuran; DMSO: dimethyl sulfoxide; DMF: dimethylformamide.

a

All reactions were carried out on a 1 mmol scale of 1a in the indicated solvent (20 mL) until complete disappearance of 1a.

b

Reactions at reflux in EtOH, MeOH, THF, and MeCN at 90 °C in DMF and DMSO.

c

Isolated yield after chromatography.

d

No reaction.

The optimized conditions were applied to the cyclocondensation of the substituted puerarin (1a–c) with guanidine or 3-amino-5-hydroxypyrazole. 4,5-Diphenylpyrimidin-2-amine (3a–c) and 3-hydroxy-5,6-diphenyl-1H-pyrazolo[3,4-b]pyridine (3d–f) were produced in good yields (38%–75%; Table 2). Yields were slightly lower when the hydroxyl group was present on ring A of puerarin (Table 2, entries 1 and 3). It seems plausible that phenolate ions generated under basic conditions from the hydroxyl group present in puerarin might have a greater electron-donating ability than the corresponding ethyloxy puerarin, thereby affecting the mechanism. The hydroxyl group on ring B had a limited effect on the efficiency of the condensation reactions. All of the products were characterized by Fourier-transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS).

Table 2.

Synthesis of C-glycosides of heterocycles 3.

Entry	Substrate	R1	R2	Product	Yield a (%)
1	1a	H	H	3a	38
2	1b	Et	H	3b	67
3	1c	Et	Et	3c	70

a

Isolated yield after chromatographic purification.

Entry	Substrate	R1	R2	Product	Yield a (%)
4	1a	H	H	3d	41
5	1b	Et	H	3e	72
6	1c	Et	Et	3f	75

a

Isolated yield after chromatographic purification.

To explain the formation of 3, a plausible reaction mechanism of puerarin (1a) with guanidine (2a) or 3-amino-5-hydroxypyrazole (2b) is shown in Scheme 1. It was reported that 3-phenyl-4H-chromene may undergo ring opening reaction when refluxing in the presence of alkali to form an α,β-unsaturated ketone intermediate 4 (Scheme 1). ²⁸ Subsequent attack of the primary amine group of the guanidine (2a) at the β-carbon atom of 4, followed by a ring-closure reaction between the secondary amine and the carbonyl carbon atom, leads to product 3a. Similarly, C4 of 3-amino-5-hydroxypyrazole (2b) attacks the β-carbon atom of 4, followed by ring closure between the primary amine and the carbonyl carbon giving 3d.

Scheme 1.

The proposed mechanism of cyclocondensation.

Subsequently, to further demonstrate the potential activities of these synthesized C-glycosides of heterocycles 3, their activity was evaluated in vitro against human leukemia cells K562 and human prostate cancer cells PC-3 by the MTT-based assay using the commercially available standard drug cis-platin as a positive control, and their IC₅₀ concentrations are depicted in Table 3. The results demonstrated that most of the compounds 3 showed considerable cytotoxicity to these two cell lines K562 and PC-3. Although a general structure–activity relationship of C-glycosides of heterocycles 3 to anticancer effect could not be summarized just from these in vitro inhibitory activity data, the following points were noteworthy: the compounds 3b, 3c, 3e, and 3f showed IC₅₀ ranging from 9.2 to 31.5 µM in vitro inhibitory activity against human leukemia cells K562 and showed equipotent or more potent activity than 1a and the positive control of cis-platin. Compounds 3a, 3b, and 3f had comparable in vitro inhibitory activity against human prostate cancer cells PC-3 with 1a and the positive control of cis-platin. The results indicated that C-glycosides of heterocycles 3 may be useful leads for further biological screenings.

Table 3.

In vitro antitumor activities of target compounds.

Compounds	K562 IC50 (µM) a	PC-3 IC50 (µM)
3a	52.4	16.7
3b	25.3	15.4
3c	11.5	71.8
3d	40.3	51.2
3e	9.2	73.4
3f	31.5	27.5
1a	27.8	32.5
cis-platin	28.7	25.8

a

The IC₅₀ concentration represents the concentration which results in a 50% decrease in cell growth after 2 days of incubation. The given values are mean values of three parallel experiments.

Experimental

The ¹H and ¹³C NMR spectra were recorded on Bruker Avance DMX 300 MHz NMR spectrometers in dimethyl sulfoxide (DMSO)-d₆ using tetramethylsilane (TMS) as the internal standard. Chemical shifts were reported as δ values (ppm). High-resolution mass spectrometry electrospray ionization (HRMS-ESI) spectra were obtained on a Micro^™ Q-TOF mass spectrometer. The infrared (IR) spectra were recorded on a PerkinElmer One FTIR spectrograph using KBr pellets. Melting points (m.p.) were uncorrected and recorded on an Electrothermal IA9100 digital melting point apparatus.

Reagents were purchased from commercial sources and used as received unless mentioned otherwise. Reactions were monitored by thin-layer chromatography (TLC) using silica gel GF₂₅₄ plates. Column chromatography was performed on silica gel (300–400 mesh).

Synthesis of 7-alkoxy-3-(4-alkoxyphenyl)-8-C-β-D-glucopyranosyl-4H-chromen-4-one (1b–c)

The mixture of puerarin 1a (1 mmol), bromoethane (2 mmol), and NaOH (2 mmol) was refluxed in MeOH (20 mL) for 3 h. ²⁴ The reaction mixture was concentrated to 5 mL using a rotary evaporator. The residue was poured into ice water (20 mL) and adjusted to neutrality with the solution of 5% HCl. A white precipitate appeared and was filtered after 15 min. Then the precipitate was dissolved in MeOH and dried over MgSO₄, then concentrated under reduced pressure, and purified by column chromatography on a silica gel column, using chloroform–methanol (18:1) to give the corresponding pure products 1b and 1c.

Synthesis of the ethylated products of puerarin (3a–c)

The mixture of puerarin or its derivatives (1, 1 mmol), guanidine (2a, 3 mmol), and MeONa (3 mmol) was heated to reflux. All reactions were monitored by TLC, which showed the disappearance of 1 that was indicative of the reaction being complete. After MeOH was removed under reduced pressure, the residue was diluted by water and adjusted to neutrality with the solution of 5% HCl, extracted by n-butyl alcohol six times, and dried over anhydrous sodium sulfate. Then concentration under reduced pressure gave a crude product, which was purified by column chromatography on a silica gel column using chloroform–methanol (5:1–8:1) to give the corresponding pure products 3a–c.

4-(2,4-Dihydroxy-3-C-β-D-glucopyranosylphenyl)-5-(4-hydroxyphenyl)pyrimidin-2-amine (3a): Yellow solid; yield 38%; m.p. 175.1–176.6 °C; IR (KBr), (ν cm⁻¹): 3350, 1609, 1539, 1572, 1400, 1289, 1209, 1117, 910, 819, 753. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 8.12 (s, 1H), 7.01 (d, J = 8.0 Hz, 2H), 6.78 (d, J = 8.0 Hz, 2H), 6.70 (d, J = 8.6 Hz, 1H), 5.96 (d, J = 8.8 Hz, 1H), 4.71 (d, J = 9.7 Hz, 1H), 4.06–4.0 (m, 1H), 3.20 (d, J = 12.1 Hz, 3H). ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 161.6, 161.0, 159.8, 159.0, 157.9, 156.3, 131.1, 130.0, 128.5, 120.4, 115.7, 112.5, 110.5, 106.1, 81.3, 78.9, 74.0, 70.6, 70.2, 61.1. HRMS (ESI) calcd for C₂₂H₂₄N₃O₈ [M + H]⁺ 458.1562, found 458.1548.

4-(2-Hydroxy-3-C-β-D-glucopyranosyl-4-ethyoxyphenyl)-5-(4-hydroxyphenyl)pyrimidin-2-amine (3b): Yellow solid; yield 51%; m.p.: 186.6–187.4 °C; IR (KBr), (ν cm⁻¹): 3331, 2935, 1601, 1557, 1411, 1289, 1154, 945, 807, 734. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 10.30 (s, 1H), 9.61 (s, 1H), 8.15 (s, 1H), 7.01–6.77 (m, 5H), 6.16 (d, J = 8.6 Hz, 1H), 4.72 (s, 1H), 4.02–3.40 (m, 8H), 1.26 (m, 3H); ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 162.1, 161.6, 160.5, 160.2, 158.0, 157.1, 131.6, 130.5, 128.8, 120.7, 116.2, 115.2, 106.6, 103.0, 82.0, 79.8, 74.2, 71.2, 70.9, 63.5, 62.2, 15.1. HRMS (ESI) calcd for C₂₄H₂₈N₃O₈ [M + H]⁺ 486.1864, found 486.1849.

4-(2-Hydroxy-3-C-β-D-glucopyranosyl-4-ethyoxyphenyl)-5-(4-ethyoxyphenyl)pyrimidin-2-amine (3c): Yellow solid; yield 70%; m.p.: 187.7–188.4 °C; IR (KBr), (ν cm⁻¹): 3349, 1613, 1543, 1582, 1395, 1291, 1214, 1109, 908, 823, 748. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 8.17 (s, 1H), 7.13 (d, J = 8.0 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 8.9 Hz, 1H), 6.15 (d, J = 8.7 Hz, 1H), 4.73 (s, 1H), 4.03–3.40 (m, 8H), 1.27 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 161.8, 160.6, 159.9, 158.0, 131.7, 130.5, 120.6, 115.2, 113.3, 103.3, 82.0, 79.7, 74.0, 71.2, 70.8, 64.1, 63.4, 62.1, 15.1. HRMS (ESI) calcd for C₂₆H₃₂N₃O₈ [M + H]⁺ 514.2175, found 514.2158.

Synthesis of 3-hydroxy-5,6-diphenyl-1H-pyrazolo[3,4-b]pyridines (3d–f)

The mixture of puerarin or its derivatives (1, 1 mmol), 3-amino-5-hydroxypyrazole (2b, 3 mmol), and MeONa (3 mmol) was heated to reflux. All reactions were monitored by TLC, which showed the disappearance of 1 that was indicative of the reaction being complete. After MeOH was removed under reduced pressure, the residue was diluted by water and adjusted to neutrality with the solution of 5% HCl, extracted by n-butyl alcohol six times, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a crude product, which was purified by column chromatography on a silica gel column using chloroform–methanol (10:1–13:1) to give the corresponding pure products 3d–f.

3-Hydroxy-5-(4-hydroxyphenyl)-6-(2,4-dihydroxy-3-C-β-D-glucopyranosyl)-1H-pyrazolo[3,4-b]pyridine (3d): Red solid; yield 41%; m.p.: 165.1–167.3 °C; IR (KBr), (ν cm⁻¹): 3297, 2982, 1675, 1621, 1523, 1431, 1393, 1227, 1118, 989, 841, 803, 701. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 7.99 (s, 1H), 7.01 (d, J = 7.8 Hz, 2H), 6.73 (d, J = 7.8 Hz, 2H), 6.61 (d, J = 8.4 Hz, 1H), 6.03 (d, J = 8.6 Hz, 1H), 4.75 (d, J = 9.5 Hz, 1H), 3.88–3.39 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 157.8, 157.7, 156.6, 156.2, 155.5, 150.4, 133.7, 132.1, 131.5, 130.7, 128.0, 115.8, 115.5, 112.9, 106.7, 103.9, 81.6, 79.2, 74.9, 71.7, 70.4, 61.3, 56.0. HRMS (ESI) calcd for C₂₄H₂₄N₃O₉ [M + H]⁺ 498.1518, found 498.1504.

3-Hydroxy-5-(4-hydroxyphenyl)-6-(2-hydroxy-3-C-β-D-glucopyranosyl-4-ethyoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (3e): Red solid; yield 56%; m.p.: 172.7–173.8 °C; IR (KBr), (ν cm⁻¹): 3062, 2841, 1623, 1155, 1439, 1341, 1285, 1212, 1163, 1081, 1032, 961, 833, 789, 699. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 12.09 (s, 1H), 11.00 (s, 1H), 9.51 (s, 1H), 8.03 (s, 1H), 7.04 (d, J = 7.7 Hz, 2H), 6.74 (m, 3H), 6.24 (d, J = 8.5 Hz, 1H), 4.76 (d, J = 9.5 Hz, 1H), 3.91–3.42 (m, 8H), 1.28 (m, 3H); ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 158.7, 156.9, 156.6, 156.1, 154.9, 150.4, 133.0, 131.7, 130.7, 128.9, 115.7, 114.7, 104.1, 103.3, 81.6, 79.3, 74.6, 71.5, 70.6, 64.1, 61.6, 15.1. HRMS (ESI) calcd for C₂₆H₂₉N₃O₉ [M + H]⁺ 526.1826, found 526.1808.

4-Hydroxy-5-(4-ethyoxyphenyl)-6-(2-hydroxy-3-C-β-D-glucopyranosyl-4-ethyoxyphenyl)-1H-pyrazolo[3,4-b]pyridine (3f): Red solid; yield 75%; m.p.: 166.4–167.3 °C; IR (KBr), (ν cm⁻¹): 3155, 2841, 1623, 1489, 1454, 1419, 1353, 1238, 1431, 1356, 1234, 1147, 1103, 1024, 948, 831. ¹H NMR (300 MHz, DMSO-d₆), δ (ppm): 8.00 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.76 (d, J = 8.5 Hz, 1H), 6.26 (d, J = 8.5 Hz, 1H), 4.77 (d, J = 9.5 Hz, 5H), 4.03–3.38 (m, 10H), 1.27 (m, 6H); ¹³C NMR (75 MHz, DMSO-d₆), δ (ppm): 158.7, 157.7, 156.6, 156.2, 154.9, 150.7, 133.2, 132.6, 131.5, 130.7, 128.8, 121.6, 114.7, 104.0, 103.4, 81.6, 79.2, 74.7, 71.6, 70.5, 64.1, 63.3, 61.5, 15.1. HRMS (ESI) calcd for C₂₈H₃₂N₃O₉ [M + H]⁺ 554.2142, found 554.2126.

Bioassay of the antitumor activities ²⁹

Cell culture

The leukemic cells K562 and human prostate cancer cells PC-3 were incubated with an RPMI-1640 medium. ²⁹ The number of tumor cells is 5 × 10³ cells per hole in a 96-well plate and the tumor cells were incubated for 24 h at 37 °C, and the air in the incubator is humid air with 5% CO₂.

Antitumor activity evaluation

DMSO solutions of compounds 1a and 3a–f were prepared for reserve. When the cells were incubated for 24 h, the solution of each compound was added to each pore of the 96-well plates, and the concentrations of each compound were 5, 10, 20, 40, and 80 μmol L⁻¹, respectively. After incubating for 48 h, 10 μL of 5 mg mL⁻¹ MTT solution was added to each hole. The tumor cells were incubated for 4 h at 37 °C and the air in the incubator is humid air with 5% CO₂. 150 μL DMSO was added to each hole and the solution was clarified after shaking for 10 min. The absorbance of each pore sample was measured by a microplate reader. Inhibition rates were calculated using the following formula

$$\mathrm{IR}=\left(1-\frac{{\mathrm{OD}}_{\mathrm{Evaluated}}}{{\mathrm{OD}}_{\mathrm{Contrast}}}\right)\times 100\%$$

The IC₅₀ values were calculated by prohibiting regression using SPSS Statistics17.0 software.