Synthesis of pyrophosphate analogues and their cytotoxic activities

Abstract

Six pyrophosphate analogues are prepared from zerumbone, murrayafoline A, acridone, and 4-hydroxycoumarin via 1,3-dipolar cycloaddition reactions. Their in vitro cytotoxic activity is evaluated against HepG2, LU-1, and HeLa cancer cell lines. Among them, diisopropyl ((ethoxy((4-((1-methoxy-3-methyl-9H-carbazol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6a) and diisopropyl ((ethoxy((4-(((3-methyl-9H-carbazol-1-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6b) are found to show activity against HepG2, LU-1, and HeLa cancer cell lines, with IC₅₀ values ranging from 7.31 to 17.88 μM.

Introduction

The pyrophosphate group plays an important role in the biochemistry of living organisms. ¹ A number of pyrophosphate analogues have been used as drugs for the treatment of osteoporosis, ² for example, alendronate, risedronate, ibandronate, and zoledronate, and as human farnesyl pyrophosphate synthase inhibitors.^3,4

Pyrophosphates and their analogues often have a high negative charge and subsequent poor bioavailability. ⁵ Because of their high charge at physiological pH, pyrophosphates and their analogues can have difficulty diffusing across cell membranes. ⁶ To remedy this, pyrophosphonates have been considered as replacements for pyrophosphates so enabling a problematic molecule to overcome a biological obstacle. ⁷ In addition, several studies have shown that the potencies of pyrophosphate analogues can be increased by administering them as prodrugs in which negative charges on the phosphate moieties are masked.^7,8 Pyrophosphonate prodrugs such as tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate ⁸ and tetrakis-pivaloyloxymethyl 2-(3,4-dibromophenyl)ethylidene1,1-bisphosphonate ⁹ have been found to potently inhibit the in vitro growth of a variety of tumor cell lines (Figure 1). A number of pyrophosphonate prodrugs, such as SRP3042, ¹⁰ also have the ability to accelerate the degradation of HMG-CoA reductase, which is a rate-limiting enzyme in the cholesterol biosynthetic pathway, and reduce serum cholesterol level (Figure 1).

Figure 1.

Example of pyrophosphate analogues.

Specifically, pyrophosphonate analogues of nucleosides such as AOPCP and AB680 (Figure 1) express strong anti-cancer activity through inhibition of the enzyme CD73^11–13 or ecto-5’-nucleotidase, an enzyme that is able to catabolize 5’-adenosine monophosphate (AMP) into adenosine at the extracellular level. The overexpression of CD73 has been linked to many diseases including breast cancer, bladder cancer, ovarian cancer, and melanoma. ¹⁴ Therefore, the CD73 enzyme is a potential target for cancer prevention and treatment.

In our previous report, a series of 1,2,3-triazoles containing a bisphosphonate unit (P-C-P-C) was prepared and their CD73 inhibitory and cytotoxic activities were documented. ⁵ In addition, the non-adenosine bisphosphonates (Figure 1) were reported to inhibit the growth of human cancer cell lines. These findings encouraged us to investigate the cytotoxicity of new non-adenosine pyrophosphate analogues. Therefore, in this paper, several novel pyrophosphonate analogues of murrayafoline A, azazerumbone, acridone, and 4-hydroxycoumarin are synthesized and evaluated for their cytotoxic activity against human carcinoma cell lines: HepG2, LU-1, and HeLa.

Results and discussion

The synthesis was initiated with acridone, 4-hydroxycoumarin, and two bioactive natural compounds, murrayafoline A and zerumbone, which can be easily isolated as the major active ingredients from the rhizomes of Glycosmis stenocarpa ¹⁵ and Zingiber zerumbet, ¹⁶ respectively. Compounds 4a–d were obtained from murrayafoline A and zerumbone using the procedures of Cuong et al. ¹⁵ and Chinh et al. ¹⁷ The Huisgen 1,3-dipolar cycloaddition was used as the key reaction for the preparation of the desired final products 6a–f.

First, the key intermediate diisopropyl {[(azidomethyl)(ethoxy)phosphoryl]methyl}phosphonate (3) was synthesized from a commercially available diisopropyl methyl phosphonate in three steps and a 36% overall yield (Scheme 1) as described by Ghoteimi and co-workers. ⁵

Scheme 1.

Synthesis of the key intermediate 3.

In the next step, the terminal alkynes 5a–f were prepared from natural products and were the starting compounds for the synthesis of the drugs. Murrayafoline A and zerumbone are the two promising anti-cancer agents. They can be isolated in large scale from Vietnamese traditional medicinal plants (G. stenocarpa and Z. zerumbet). Acridone and 4-hydroxycoumarin are the two starting compounds for the synthesis of the drugs: acridone acetic acid and warfarin. The synthesis of 5a–f was reported in our previous study, see Scheme 2. ¹⁸

Scheme 2.

Synthesis of terminal alkynes 5a–f.

In the final step, a 1,3-dipolar cycloaddition reaction was used to couple the intermediate diisopropyl {[(azidomethyl)(ethoxy)phosphoryl]methyl}phosphonate (3) with the appropriate terminal alkyne 5a–f in the presence of CuSO₄/sodium ascorbate in DMF to afford pyrophosphate analogues 6a–f in 46%–79%. In this step, EDTA solution 1% was used to remove Cu(II) ions (Scheme 3). In cases of 6a and 6b, the ¹H NMR also indicated that they contained a small amount of 5a (11%) and 5b (13%); these intermediates were difficult to separate from 6a and 6b by column chromatography. The spectral data of the pyrophosphate analogues 6a–f were in excellent agreement with their structures.

Scheme 3.

Synthesis of pyrophosphate analogues 6a–f.

All the synthesized compounds 6a–f were screened for their cytotoxic activity against HepG2, LU-1, and HeLa cancer cell lines (Table 1), according to the methods described by Likhitwitayawuid et al. ¹⁹ and Skehan et al. ²⁰ The IC₅₀ values were compared with those of ellipticine as a positive control.

Table 1.

Cytotoxic activity of compounds 6a–f against cancer cell lines.

Compound	IC50 (µM)
	HepG2	LU-1	HeLa
6a	15.18	14.6	7.31
6b	8.84	17.88	17.87
6c	>20	>20	>20
6d	>20	>20	>20
6e	>20	>20	>20
6f	>20	>20	>20
Ellipticine	0.35	0.30	0.16

As shown in Table 1, pyrophosphonates 6a–f showed weak or no activity against the tested cell lines. The two compounds containing murrayafoline A, 6a and 6b, displayed moderate cytotoxic activity against HepG2, LU-1, and HeLa cells with IC₅₀ values ranging from 7.31 to 17.88 μM. However, these results did not demonstrate the role of the bisphosphonate structure in 6a–f in impacting cytotoxic activity in comparison to those of their precursors 5a–f. Therefore, the presence of an adenosine moiety may be required for the cytotoxic activity of these pyrophosphonates. In addition, the bulky isopropyl group in the pyrophosphate analogue units may also cause a reduction of their cytotoxic activity compared with those of unprotected pyrophosphate analogues.

The reference substance, ellipticine, exhibited cytotoxic activity against human cancer cells, including HepG2 (ATCC-HB-8065), LU-1 (ATCC-HTB-57), and HeLa (ATCC-CCL-2) cells with IC₅₀ value of 0.35, 0.30, and 0.16 μM, respectively. The values shown for these compounds are the average of three determinations.

Conclusion

In this study, a five-step procedure was used to synthesize six pyrophosphonates 6a–f. Their structures were elucidated by ¹H NMR, ¹³C NMR, and ³¹P NMR spectroscopy and HRMS. The products were tested for cytotoxic activity against HepG2, LU-1, and HeLa cancer cell lines and the role of the bisphosphonate moiety in their structures is discussed.

Experimental

General

¹H NMR and ¹³C NMR spectra were recorded at ambient temperature on a Bruker Avance 500 MHz spectrometer in CDCl₃. Chemical shifts δ are quoted in parts per million (ppm) referenced to the residual solvent peak (CDCl₃ at 7.26 and 77 ppm) relative to TMS. Mass spectra were recorded using an Agilent LC/MSD Trap SL. Thin-layer chromatography was performed on precoated Silica Gel 60 F254 aluminum sheets (Merck), and products were visualized under a UV lamp at 254 nm. Column chromatography was carried out on Silica Gel (40–230 mesh). The terminal alkynes 5a–f were prepared by the procedures described in the literature. ¹⁸

Synthesis of alkynes 5a–f

Synthesis of intermediates: 1-methoxy-3-methyl-9-(prop-2-yn-1-yl)-9H-carbazole (5a, 90%), 3-methyl-9H-1-(prop-2-yn-1-yloxy)carbazole (5b, 75%), (3E,7E,11E)-3,7,10,10-tetramethyl-1-(prop-2-yn-1-yl)-1-azacyclododeca-3,7,11-trien-2-one (5c, 73%), N-propargylazazerumbone oxide (5d, 77%), 10-(prop-2-yn-1-yl)acridin-9(10H)-one (5e, 85%), and 4-(prop-2-yn-1-yloxy)-2H-chromen-2-one (5f, 77%) and their properties were given in our previous report. ¹⁸

General procedure for the synthesis of compounds 6a–f

To a solution of the intermediate 3 (1 equiv.) in DMF (0.8 mL/mmol) were added the appropriate terminal alkyne 5a–f (1.1 equiv.), CuSO₄ (0.03 equiv.), and sodium ascorbate (0.1 equiv.). The resulting mixture was stirred at 70 °C until TLC showed the disappearance of the starting material. Water and EtOAc were added and the aqueous layer was extracted with EtOAc (3 × 20 mL). The combined EtOAc layer was next extracted with EDTA 1% and dried over anhydrous sodium sulfate, then filtered and evaporated under reduced pressure. The crude products were purified by column chromatography on silica gel eluting with CH₂Cl₂/acetone.

Diisopropyl ((ethoxy((4-((1-methoxy-3-methyl-9H-carbazol-9-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6a):

Compound 6a (300 mg, 46%) was obtained as a white solid from intermediate 3 (328 mg, 1 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 4:1). ¹H NMR (500 MHz, CDCl₃): δ 7.96 (d, J = 7.8 Hz, 1H, H-Ar), 7.52 (d, J = 8.3 Hz, 1H, H-Ar), 7.46 (s, 1H, H-triazole), 7.41 (s, 1H, H-Ar), 7.40-7.34 (m, 1H, H-Ar), 7.20-7.13 (m, 1H, H-Ar), 6.74 (s, 1H, H-Ar), 5.92 (d, J = 1.0 Hz, 2H, N-CH₂), 4.98-4.51 (m, 4H, N-CH₂-P, CH(CH₃)₂), 4.06-3.91 (m, 2H, O-CH₂-CH₃), 3.96 (s, 3H, O-CH₃), 2.49 (s, 3H, CH₃), 2.36-2.26 (m, 2H, P-CH₂-P), 1.38-1.08 (m, 15H, CH₃). ¹³C NMR (125 MHz, dimethyl sulfoxide (DMSO)): δ 146.87 (C-Ar), 146.80 (C=CH), 141.12, 129.88 (C-Ar), 128.80 (C=CH), 126.10, 125.48, 123.80, 120.50, 119.57, 113.18, 109.80, 109.41 (C-Ar), 72.51 (d, J = 6.6 Hz, CH(CH₃)₂), 72.14 (d, J = 6.6 Hz, CH(CH₃)₂), 62.90 (d, J = 6.8 Hz, O-CH₂-CH₃), 56.05 (O-CH₃), 48.75 (d, J = 101.9 Hz, N-CH₂-P), 41.29 (N-CH₂), 27.87 (dd, J = 135.6, 86.1 Hz, P-CH₂-P), 24.22 (ddd, J = 2.9, 8.3, 7.5 Hz, CH₃), 22.01 (CH₃), 16.50 (d, J = 6.1 Hz, O-CH2-CH₃). ³¹P NMR (202 MHz, CDCl₃): δ 34.97, 15.70. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₇H₃₉N₄O₆P₂: 577.2267; found: 577.2330.

Diisopropyl ((ethoxy((4-(((3-methyl-9H-carbazol-1-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6b):

Compound 6b (349 mg, 54%) was obtained as a white solid from intermediate 3 (328 mg, 1 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 3:1). ¹H NMR (500 MHz, CDCl₃): δ 8.87 (s, 1H, NH), 8.04 (s, 1H, H-triazole), 7.99 (d, J = 7.8 Hz, 1H, H-Ar), 7.50 (s, 1H, H-Ar), 7.43 (d, J = 8.1 Hz, 1H, H-Ar), 7.39-7.33 (m, 1H, H-Ar), 7.22-7.13 (m, 1H, H-Ar), 6.85 (s, 1H, H-Ar), 5.36 (s, 2H, O-CH₂), 5.17-4.96 (m, 2H, N-CH₂-P), 4.93-4.70 (m, 2H, CH(CH₃)₂), 4.31-4.05 (m, 2H, O-CH₂-CH₃), 2.51 (s, 3H, CH₃), 2.47-2.30 (m, 2H, P-CH_2-P), 1.44-1.22 (m, 15H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 144.81 (C-Ar), 144.04 (C=CH), 139.86, 129.29, 128.63, 125.66 (C-Ar), 124.84 (C=CH), 124.73, 123.47, 120.47, 119.13, 113.60, 111.15, 109.88 (C-Ar), 72.57 (d, J = 6.6 Hz, CH(CH₃)₂), 72.06 (d, J = 6.7 Hz, CH(CH₃)₂), 62.91 (d, J = 6.7 Hz, O-CH₂-CH₃), 55.65 (O-CH₂), 48.65 (d, J = 101.3 Hz, N-CH₂-P), 27.73 (dd, J = 135.9, 86.3 Hz, P-CH_2-P), 24.20 (d, J = 3.5 Hz, CH₃), 24.07 (d, J = 4.0 Hz, CH₃), 23.99 (d, J = 5.4 Hz, CH₃), 21.95 (CH₃), 16.46 (d, J = 6.0 Hz, O-CH2-CH₃). ³¹P NMR (202 MHz, CDCl₃): δ 35.19, 15.67. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₃₇N₄O₆P₂: 563.2110; found: 563.2175.

Diisopropyl ((ethoxy((4-(((3E,7Z,11E)-5,5,8,12-tetramethyl-2-oxoazacyclododeca-3,7,11-trien-1-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6c):

Compound 6c (279 mg, 47%) was obtained as a white oil from intermediate 3 (328 mg, 1 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 3:1). ¹H NMR (500 MHz, CDCl₃): δ 7.82 (s, 1H, H-triazole), 6.35 (d, 1H, J = 15.9 Hz, H-4), 5.83 (d, J = 15.9 Hz, 1H, H-3), 5.02-4.85 (m, 4H, H-7, H-11, N-CH₂-P), 4.84-4.72 (m, 2H, H-17), 4.71-4.61 (m, 2H, CH(CH₃)₂), 4.22-4.08 (m, 2H, O-CH₂-CH₃), 2.90 (d, J = 37.2 Hz, 2H, P-CH₂-P), 2.55-2.33 (m, 1H, H-9), 2.29-1.94 (m, 5H, H-6, H-9, H-10), 1.86 (s, 3H, H-13), 1.56 (s, 3H, H-14), 1.31 (ddd, J = 24.3, 12.2, 6.5 Hz, 15H, CH₃), 1.07 (s, 6H, H-15, H-16). ¹³C NMR (125 MHz, CDCl₃): δ 167.12 (C-2), 149.02 (C-4), 145.03 (C=CH), 134.40 (C-8), 134.00 (C-12), 132.72 (C-11), 125.24 (C-7), 125.11 (C=CH), 122.98 (C-3), 72.29 (d, J = 6.3 Hz, CH(CH₃)₂), 71.95 (d, J = 6.6 Hz, CH(CH₃)₂), 62.70 (d, J = 6.7 Hz, O-CH₂-CH₃), 48.57 (d, J = 102.1 Hz, N-CH₂-P), 40.36 (C-9), 39.58 (C-17), 38.82 (C-6), 36.64 (C-5), 27.77 (dd, J = 135.6, 86.0 Hz, P-CH₂-P), 25.60 (C-10), 24.17 (dd, J = 6.5, 3.7 Hz, CH₃), 24.00 (dd, J = 8.9, 5.3 Hz, CH₃), 16.47 (d, J = 5.9 Hz, O-CH2-CH₃), 15.57 (C-14), 15.31 (C-13). ³¹P NMR (202 MHz, CDCl₃): δ 35.18, 15.90. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₄₉N₄O₆P₂: 599.3049; found: 599.3111.

Diisopropyl ((ethoxy((4-(((4E,8E)-1,5,10,10-tetramethyl-7-oxo-13-oxa-6-azabicyclo[10.1.0]trideca-4,8-dien-6-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6d):

Compound 6d (300 mg, 49%) was obtained as a white oil from intermediate 3 (328 mg, 1 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 3:1). ¹H NMR (500 MHz, CDCl₃): δ 7.83 (d, J = 6.8 Hz, 1H, H-triazole), 6.61 (d, J = 15.7 Hz, 1H, H-4), 5.88 (d, J = 15.8 Hz, 1H, H-3), 5.20-4.70 (m, 6H, H-11, H-17b, N-CH₂-P, CH(CH₃)₂), 4.55 (d, J = 15.0 Hz, 1H, H-17a), 4.30-4.03 (m, 2H, O-CH₂-CH₃), 2.50-2.24 (m, 4H, H-7, H-10b, P-CH₂-P), 2.23-2.12 (m, 2H, H-9b, H-10a), 1.95 (s, 3H, H-13), 1.86 (dd, J = 15.4, 6.7 Hz, 1H, H-6b), 1.52 (d, J = 15.4 Hz, 1H, H-6a), 1.37-1.26 (m, 15H, CH₃), 1.25 (s, 3H, H-14), 1.21 (s, 3H, H-16), 1.18-1.12 (m, 1H, H-9a), 1.03 (s, 3H, H-15). ¹³C NMR (125 MHz, CDCl₃): δ 166.16 (C-2), 150.37 (C-4), 144.65 (C=CH), 134.92 (C-12), 131.48 (C-11), 125.23 (C=CH), 120.81 (C-3), 72.30 (d, J = 6.5 Hz, CH(CH₃)₂), 71.93 (d, J = 6.5 Hz, CH(CH₃)₂), 62.66 (d, J = 6.2 Hz, O-CH₂-CH₃), 48.58 (dd, J = 101.9, 14.1 Hz, N-CH₂-P), 62.60 (C-7), 60.50 (C-8), 40.09 (C-17), 38.44 (C-9), 38.15 (C-6), 36.77 (C-5), 30.99 (C-15), 28.90-26.76 (m, P-CH₂-P), 26.63 (C-16), 24.67 (C-10), 24.16 (dd, J = 7.9, 3.7 Hz, CH₃), 23.99 (dd, J = 9.8, 5.1 Hz, CH₃), 16.57 (C-13), 16.47 (d, J = 5.3 Hz, O-CH2-CH₃), 15.80 (C-14). ³¹P NMR (202 MHz, CDCl₃): δ 35.12, 15.83. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₈H₄₉N₄O₇P₂: 615.2998; found: 615.3060.

Diisopropyl ((ethoxy((4-((9-oxoacridin-10(9H)-yl)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6e):

Compound 6e (364 mg, 65%) was obtained as a white oil from intermediate 3 (289 mg, 0.88 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 1:1). ¹H NMR (500 MHz, CDCl₃): δ 8.52 (d, J = 1.5 Hz, 1H, H-Ar), 8.50 (d, J = 1.5 Hz, 1H, H-Ar), 7.67 (d, J = 1.6 Hz, 1H, H-Ar), 7.66 (s, 1H, H-triazole), 7.64 (d, J = 1.6 Hz, 1H, H-Ar), 7.56 (d, J = 8.6 Hz, 2H, H-Ar), 7.25 (dd, J = 13.6, 6.2 Hz, 2H, H-Ar), 5.65 (s, 2H, N-CH₂), 4.93 (dd, J = 9.8, 2.2 Hz, 2H, N-CH₂-P), 4.75-4.53 (m, 2H, CH(CH₃)₂), 4.05 (m, 2H, O-CH₂-CH₃), 2.45-2.06 (m, 2H, P-CH₂-P), 1.34-1.06 (m, 15H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 178.12 (C=O), 143.86 (C=CH), 142.08, 134.33, 128.13 (C-Ar), 123.79 (C=CH), 122.79, 121.91, 114.84 (C-Ar), 72.45 (d, J = 6.5 Hz, CH(CH₃)₂), 71.92 (d, J = 6.6 Hz, CH(CH₃)₂), 62.75 (d, J = 6.7 Hz, O-CH₂-CH₃), 48.67 (d, J = 101.4 Hz, N-CH₂-P), 43.18 (CH₂), 27.76 (dd, J = 135.2, 86.6 Hz, P-CH₂-P), 23.98 (ddd, J = 3.3, 10.6, 7.7 Hz CH₃), 16.37 (d, J = 6.0 Hz, O-CH2-CH₃). ³¹P NMR (202 MHz, CDCl₃): δ 34.73, 15.52. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₃₆N₄O₆P₂: 561.1954; found: 561.2009.

Diisopropyl ((ethoxy((4-(((2-oxo-2H-chromen-4-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)phosphoryl)methyl)phosphonate (6f):

Compound 6f (414 mg, 79%) was obtained as a white oil from intermediate 3 (328 mg, 1 mmol) using the general procedure (24 h). Purification was performed on silica gel (CH₂Cl₂/acetone 1:1). ¹H NMR (500 MHz, CDCl₃): δ 8.15 (s, 1H, H-triazole), 7.80 (dd, J = 8.0, 1.5 Hz, 1H, H-Ar), 7.56 (ddd, J = 8.7, 7.3, 1.6 Hz, 1H, H-Ar), 7.39-7.30 (m, 1H, H-Ar), 7.30-7.23 (m, 1H, H-Ar), 5.91 (s, 1H, CH), 5.38 (s, 2H, O-CH₂), 5.23-5.05 (m, 2H, N-CH₂-P), 4.87 (ddt, J = 12.4, 7.8, 6.2 Hz, 1H, CH(CH₃)₂), 4.79 (ddt, J = 12.4, 7.9, 6.2 Hz, 1H, CH(CH₃)₂), 4.45-4.08 (m, 2H, O-CH₂-CH₃), 2.87-2.34 (m, 2H, P-CH_2-P), 1.37 (tdd, J = 18.8, 12.7, 6.3 Hz, 15H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 165.04 (C=CH), 162.65 (C=O), 153.44 (C-Ar), 141.92 (C=CH triazole), 132.63 (C-Ar), 125.36 (C=CH triazole), 124.00, 123.24, 116.83, 115.52 (C-Ar), 91.33 (C=CH), 72.54 (d, J = 6.5 Hz, CH(CH₃)₂), 71.99 (d, J = 6.6 Hz, CH(CH₃)₂), 62.91 (d, J = 6.7 Hz, O-CH₂-CH₃), 62.73 (O-CH₂), 48.74 (d, J = 101.3 Hz, N-CH₂-P), 27.89 (dd, J = 135.7, 86.4 Hz, P-CH_2-P), 24.19 (d, J = 3.6 Hz, CH₃), 24.01 (d, J = 5.3 Hz, CH₃), 23.92 (d, J = 5.2 Hz, CH₃), 16.46 (d, J = 6.0 Hz, O-CH2-CH₃). ³¹P NMR (202 MHz, CDCl₃): δ 34.90, 15.57. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₃₃N₃O₈P₂: 528.1586; found: 528.1655.

Cytotoxic evaluation

Stock solutions of the pyrophosphate analogues 6a–f were prepared in DMSO at a concentration of 4 mM, and were diluted 200-fold to obtain solutions with the highest concentration (20 μM) tested on 96-well plates. These solutions were serially diluted for bioassay. Determination of IC₅₀ values was carried out using three human cancer cell lines: HepG2, LU-1, and HeLa. Ellipticine was used as a positive control. The IC₅₀ values were determined from dose-dependent curves plotted from five different concentration regimens (0–20 μM). At each regimen, mean of triplicate experiments was used for a point in the curve.

Supplemental Material

sj-pdf-1-chl-10.1177_17475198211043439 – Supplemental material for Synthesis of pyrophosphate analogues and their cytotoxic activities

Supplemental material, sj-pdf-1-chl-10.1177_17475198211043439 for Synthesis of pyrophosphate analogues and their cytotoxic activities by Van-Tai Nguyen, Minh-Quan Pham, Thi-Ha Vu, Thi-Hong-Ha Tran, Duy-Tien Doan, Dinh-Luyen Nguyen, Phong Le and Van-Chinh Luu in Journal of Chemical Research