Synthesis of novel spin-labeled podophyllotoxin derivatives as potential antineoplastic agents: Part XXV

Abstract

A series of novel spin-labeled 4β-[(4-substituted)-1,2,3-triazol-1-yl]podophyllotoxin derivatives (17a–h) were firstly designed and synthesized with significant regioselectivity by employing Cu(I) catalyzed click approach, and evaluated for cytotoxicity against four human tumor cell lines (A-549, DU145, KB, and KBvin). Among them, compound 17h displayed the highest cytotoxic activity against the tumor cell lines tested. Significantly, compound 17h showed superior cytotoxic activity compared with etoposide (IC₅₀ 6.30 to>10 μM), a clinically available anticancer drug. Significant activity toward the drug resistant KBvin cell line revealed promising future for compound 17h as a new generation of epipodophyllotoxin-derived antitumor clinical trial candidate.

Introduction

Podophyllotoxin (1, PPT), a naturally occurring aryltetralin lignan, has been used as a lead compound in the development of new potent anticancer drugs (Garcia and Azambuja, 2004). Its powerful cytotoxic properties have been attributed by its binding to tubulin during mitosis and thus inhibiting microtubule assembly (Jardine, 1980). The extensive chemical modifications of 1 led to two anticancer drugs, etoposide (2) and teniposide (3), as well as etopophos (4), a water-soluble prodrug of 2 (Stahelin and von Wartburg, 1989; Canetta et al., 1982; Joel 1996). These compounds are currently used as drugs, alone or in association, in clinical cancer chemotherapy against small cell lung cancer, acute leukemia, lymphoma, testicular carcinoma, and Kaposi’s sarcoma. Notably, these two structural modifications also led to a change in the mechanism of action. While PPT acts as antimicrotubule agent, 2 and 3 function as topoisomerase II (topo II) inhibitors (Canela et al., 2000; Imbert, 1998). However, the therapeutic use of 2 and 3 is often hindered by problems such as acquired drug-resistance and poor water solubility (Liu et al., 2008). To get more potent analogs and to overcome drug-resistance, recently some nonsugar substituted analogs, particularly N-linked congeners, exhibit superior pharmacological properties to etoposide, and consequently, several newer-generation clinical candidates, including NPF (5), GL-331 (6), and TOP-53 (7), have emerged through C-4 modification as alternatives to overcome the drawbacks of etoposide (Cragg and Newman, 2004; Lee, 1999; Liu et al., 1989). These successful examples imply that C-4 substitution plays an important role in the activity profiles of 1-analogs, and that optimization of this compound class through rational C-4 modification is quite feasible. Both a composite pharmacophore model and comparative molecular field analysis also further demonstrated that the C-4 molecular area could accommodate considerable structural diversity (Cho et al., 1996).

Recently, the applications of click chemistry are increasingly interest in all aspects of drug discovery, ranging from initial lead identification through combinatorial chemistry and target-templated in situ chemistry, to proteonmics and DNA research, using bioconjugation reaction. The copper(I)-catalyzed 1,2,3-trizole formation from azides and terminal acetylenes is a particularly powerful linking reaction, in addition to be passive linkers, 1,2,3-triazole ring is a widespread functional group in drugs (Kolb and Sharpless, 2003). Accordingly, it is intriguing to attach 1,2,3-triazoles to podophyllotoxin parent nucleus and has generated various potent aniline, phenol, thiophenol, and carbohydrate-based 1,2,3-triazole derivatives, some of which exhibited significant antitumor activity (Bhat et al., 2008; Reddy et al., 2008a, b; Chen et al., 2011, 2012). Additionally, a recent docking studies revealed that 1,2,3-triazole derivatives with various substituents in triazolemoiety showed better binding ability to topoisomerase II enzyme than etoposide (Reddy et al., 2011). From this standpoint, logic-based design utilizing click chemistry could be advantageous.

In our previous studies, we have introduced a stable nitroxyl radical into different positions in the PPT skeleton and proved that the resulting analogs can exhibit significant antitumor activity against several mouse transplantable tumors with remarkably decreased toxicity (Jin et al., 2006; Liu and Tian, 2005; Tian et al., 1997, 2002). Especially, GP-11 (8) is a typical example which has promise to be a new antitumor drug, GP-11 has been found which could increased the mitotic index and resulted in G2/M phase, and to a lesser extent, S arrest (Wang et al., 1993).

Inspired by the growing impact of click chemistry on drug discovery as well as our previous studies, we introduced the nitroxyl radical moiety into the molecule of podophyllotoxin at its C-4 via 1,2,3-triazol spacer as a part of our drug discovery program. Herein, a series of novel spin-labeled 4β-[(4-substituted)-1,2,3-triazol-1-yl] podophyllotoxin derivatives (17a–h) were firstly designed, synthesized, and evaluated for their in vitro cytotoxic activity against four tumor cell lines (A-549, DU145, KB, and KBvin) (Fig. 1).

Fig. 1.

Structures of podophyllotoxin derivatives

Results and discussion

Chemistry

As illustrated in Scheme 1, the starting materials, N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl-oxycarbonyl) amino acids 14a–h, for the preparation of the target compounds 17a–h were synthesized according to our previous procedure (Zhang et al., 2010; Liu et al., 2012; Hankovszky et al., 1979). Briefly, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl 10 was prepared by catalytic oxidation of 4-hydroxy-2,2,6,6-tetramethylpiperidine 9 with sodium tungstate-hydrogen peroxide-EDTA in yield 85 %. Following, the reaction of compound 10 with N,N′-carbonyldiimidazol proceeds to give N-(1-oxyl-2,2,6,6-tetramethyl piperidinyloxycarbonyl)-imidazole 11 by the modified method. Compound 11, without further purification, was reacted with p-toluenesulfonic acid monohydrate to give its higher reactive tosylate 12. Compound 12 is instantaneously converted into alkoxycarbonyl azide 13 when dissolved in an aqueous solution of sodium azide. Compounds 14a–h were obtained in good yield by reaction of 13 with free amino acids in presence of MgO. As illustrated in Scheme 2, 4β-Azido podophyllotoxin 15 was obtained in excellent yield by treating podophyllotoxin 1 with hydrazoic acid (HN₃) in the presence of BF₃·OEt₂ (Tian et al., 1997). Compound 15 was allowed to react with propargyl alcohol in the presence of CuSO₄·5H₂O, sodium ascorbate in t-butyl alcohol, and water (1:2) at room temperature to selectively get 4β-[4-(4-methylol)-1,2,3-triazol-1-yl] podophyllotoxin 16. Compound 16 was then condensed with the appropriate nitroxide free radical 14a–h in the presence of 1,3-diisopropylcarbodiimide (DIPC), 4-dimethylaminopyridine (DMAP) to provide the target compounds 17a–h in moderate yields. Synthesized target compounds 17a–h were characterized by melting point, ESR, IR, and HRMS spectral analyses.

Scheme 1.

Synthesis of N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl) amino acids 14a–h

Reagents and conditions: (i) Na₂WO₄/H₂O₂/EDTA; (ii) N,N′-carbonyldimidazole/THF; (iii) p-toluenesulfonic acid monohydrate; (iv) NaN₃/water, stir; (v) amino acids/MgO, stir, 24 h.

Scheme 2.

Synthesis of target compounds 3a–f

Reagents and conditions: (i) HN₃/BF₃·Et₂O, CH₂Cl₂; (ii) Propargyl alcohol, CuSO₄,Sodium ascorbate, H₂O: t-BuOH=2:1; (iii) DIPC/DMAP, dry CH₂Cl₂

Biology

Target compounds 17a–h were evaluated for in vitro cytotoxic activity against four different tumor cell lines, KB (nasopharyngeal), A-549 (lung), DU-145 (prostate), and KBvin (an MDR KB subline), using a sulforhodamine B colorimetric assay with triplicate experiments (Skehan et al., 1990). Etoposide was used as reference compound. The screening results are shown in Table 1. Remarkably, compound 17h exhibited significant inhibitory activities against A549, DU-145, and KB with the IC₅₀ value in the range of 5.49–5.6.30 μg/mL. Also, compound 17h displayed more potent cytotoxic activity against KBvin than etoposide (IC₅₀>10 μg/mL). Surprisingly, all other compounds only displayed weak cytotoxicity against the four human tumor cell lines. However, the reason is not clear to our current knowledge and has to be subject of further investigations.

Table 1.

In vitro cytotoxic assay against four tumor cell lines

Entry	IC50 (μg/mL)
	A549	DU-145	KB	KBvin
17a	>10	>10	>10	>10
17b	>10	>10	>10	>10
17c	>10	>10	>10	>10
17d	>10	>10	>10	>10
17e	>10	>10	>10	>10
17f	>10	>10	>10	>10
17g	>10	>10	>10	>10
17h	5.91 ± 0.53	5.49 ± 0.42	5.71 ± 0.11	6.30 ± 0.41
Etoposide	1.51 ± 0.25	1.19 ± 0.12	2.28 ± 0.19	>10

Experimental

General

Melting points were determined in Kofler apparatus and were uncorrected. IR spectra were measured on a Nicolet 5DX-FT-IR spectrometer on neat samples placed between KBr plates. Mass spectra were recorded on a Bruker Daltonics APEXII49e spectrometer with ESI source as ionization. The electron spin resonance (ESR) spectra were obtained from 10⁻³ M ethanol solution using a Bruker ER200D-SRC spectrometer. ¹H NMR spectra were recorded at 400 MHz on a Bruker AM-400 spectrometer using TMS as reference (Bruker Company, USA). The N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-amino acids 14a–h for the experiments were prepared by following a modified previous procedure as shown in Scheme 1 (Zhang et al., 2010; Liu et al., 2012; Hankovszky et al., 1979).

Synthesis of 4β-azido podophyllotoxin (15)

To a solution of Podophyllotoxin 1 (10 g, 24 mmol) in appropriate, anhydrous dichloromethane was added hydrazoic acid (25 mL, C = 1.04 M, benzene) with stirring. BF₃·Et₂O (4.5 mL) was slowly drop into the mixture so as to keep temperature below −10 °C. Through TLC analysis, when most of the starting material had been consumed, pyridine (4.5 mL) and distilled water (80 mL) were added. The separated organic phase was washed successively by distilled water, 5 % hydrochloric acid, distilled water, 2 % sodium bicarbonate, distilled water, then dried over anhydrous sodium sulfate, and evaporated on rotary evaporator. The residue was recrystallized in acetone–methanol to afford a white solid. Yield 75 %. mp: 110–112 °C; IR(KBr)ν/cm⁻¹: 3430 (NH₂); 1 776 (Lactone); 1588, 1550, 1484 (aromatic C=C); 935 (OCH₂O), ¹H NMR(CDCl₃) δ: 6.84 (s, 1H, H-5), 6.47 (s, 1H, H-8), 6.36 (s, 2H, H-2′, 6′), 5.97 (s, 2H, OCH₂O), 4.58 (d, J = 5.1 Hz, 1H, H-1), 4.32 (m, 2H, H-11), 4.25 (d, J = 4.0 Hz, H-4), 3.80 (s, 3H, 4′-OCH₃), 3.75 (s, 6H, 3′,5′-OCH₃), 3.32 (q, 1H, H-2), 2.92–2.63 (m, 1H, H-3), 1.83 (d, 2H, 4-NH₂).

Synthesis of 4β-[4-(4-methylol)-1,2,3-triazol-1-yl] podophyllotoxin (16)

A mixture of propargyl alcohol (30.6 mg, 0.55 mmol), CuSO₄·5H₂O (138 mg, 0.55 mmol), and sodium ascorbate (181 mg, 0.92 mmol) in t-butyl alcohol and water (1:2, 25 mL) was added 4β-azido podophyllotoxin 15 (200 mg, 0.46 mmol). The reaction mixture was stirred for 6 h at room temperature. After completion, the reaction mixture was diluted with 50 mL of water and extracted with chloroform (3 × 30 mL). The combined extracts were washed with saturated salt water, dried over anhydrous sodium sulfate, and evaporated on rotary evaporator. The residue was chromatographed on a silica gel column and eluted with ethyl acetate–petroleum ether to afford a white powder 80 mg, yield 35 %. mp: 160–162 °C; IR (KBr) cm⁻¹: 3384, 2926, 1763, 1611, 1516, 1483, 1232, 1109, 1033, 997, 928; ¹H NMR (CDCl₃) δ: 7.25 (s, 1H), 6.66 (s, 1H), 6.62 (s, 1H), 6.32 (s, 2H), 6.06 (d, J = 4.89 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 4.76 (d, J = 4.77 Hz, 1H), 4.42–4.39 (t, J = 6.79 Hz,1H), 3.77 (s, 6H), 3.74 (s, 2H), 3.28–3.17 (m, 2H), 3.09–3.05 (dd, J = 8.57 Hz, 4.88 Hz, 1H); ESI–MS: 482 [M +1].

General procedure of synthesis of 17a–h

Compound 16 (0.4 mmol) and appropriate nitroxide free radical 14a–h (0.48 mmol) were dissolved in appropriate anhydrous dichloromethane at room temperature, and to this solution, 1,3-diisopropylcarbodiimide (DIPC 0.19 mL, 1.2 mmol) and 4-dimethylaminopyridine (DMAP 97.6 mg, 0.8 mmol) were added at 0 °C and then warm to room temperature and left overnight. The product was washed with 0.1 M HCl (3 × 20 mL) and then washed with saturated salt water; the organic layer was dried over anhydrous sodium sulfate and evaporated on rotary evaporator. The residue was chromatographed on a silica gel column and eluted with ethyl acetate–petroleum ether to afford 17a–h.

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-glycinemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17a)

Yield: 55 %; red powder; mp: 126–129 °C; IR (cm⁻¹) 3370, 2973, 2939, 1778, 1713, 1420, 1361 (N–O^·), 934; ESR: An = 16.04G, g₀ = 2.0061; HRMS (ESI) 773. 2866 for [M + Na]⁺ (calcd 773.2879 for C₃₇H₄₄O₁₂N₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-alaninemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17b)

Yield: 62 %; red powder; mp: 128–131 °C; IR (cm⁻¹) 3343, 2975, 2939, 1779, 1711, 1422, 1360 (N–O^·), 934; ESR: An = 15.90G, g₀ = 2.0060; HRMS (ESI) 787. 3063 for [M + Na]⁺ (calcd 787.3035 for C₃₈H₄₆O₁₂N₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-prolinemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17c)

Yield: 60 %; red powder; mp: 142–146 °C; IR (cm⁻¹) 3343 2973, 2939, 1780, 1702, 1419, 1365 (N–O^·), 929; ESR: An = 16.12G, g₀ = 2.0060; HRMS (ESI) 813. 3173 for [M + Na]⁺ (calcd 813.3192 for C₄₀H₄₈O₁₂N₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-phenylalaninemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17d)

Yield: 56 %; red powder; mp: 127–131 °C; IR (cm⁻¹) 3343, 2972, 2936, 1779, 1715, 1420, 1388 (N–O^·), 933; ESR: An = 16.12G, g₀ = 2.0061; HRMS (ESI) 863. 3356 for [M + Na]⁺ (calcd 863.3348 for C₄₄H₅₀O₁₂N₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-methioninemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17e)

Yield: 55 %; red powder; mp: 115–119 °C; IR (cm⁻¹) 3334, 2972, 2936, 1779, 1712, 1421, 1360 (N–O^·), 935; ESR: An = 16.01G, g₀ = 2.0060; HRMS (ESI) 847. 3081 for [M + Na]⁺ (calcd 847.3069 for C₄₀H₅₀O₁₂SN₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-isoleucinemethyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17f)

Yield: 55 %; red powder; mp: 121–126 °C; IR (cm⁻¹) 3364, 2969, 2936, 1780, 1714, 1420, 1360 (N–O^·), 934; ESR: An = 16.02G, g₀ = 2.0060; HRMS (ESI) 829. 3525 for [M + Na]⁺ (calcd 829.3035 for C₄₁H₅₂O₁₂N₅).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-tryptophanmetheyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17g)

Yield: 39 %; red powder; mp: 148–154 °C; IR (cm⁻¹) 3354, 2971, 2936, 1777, 1714, 1507, 1422, 1361 (N–O^·), 932; ESR: An = 16.28G, g₀ = 2.0061; HRMS (ESI) 902.3442 for [M + Na]⁺ (calcd 902.3457 for C₄₆H₅₁O₁₂N₆).

4β-[{4-[N-(1-Oxyl-2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl)-L-valine methyl]}-1,2,3-triazol-1-yl] podophyllotoxin (17h)

Yield: 45 %; red powder; mp: 122–125 °C; IR (cm⁻¹) 3352, 2969, 2935, 1780, 1713, 1420, 1361 (N–O^·), 934; ESR: An = 16.04G, g₀ = 2.0061; HRMS (ESI) 793. 3543 for [M + H]⁺ (calcd 793.3529 for C₄₀H₅₀O₁₂N₅).

Conclusion

In conclusion, a series of novel 4β-[(N-substituted)-1,2,3-triazol-1-yl] podophyllotoxin derivatives were synthesized by employing click chemistry protocol, and their structural information was extensively characterized by using IR, ESR, and HRMS. Also, their cytotoxic activity was evaluated against four tumor cell lines (A549, DU-145, KB, and KBvin) in vitro by using an SRB-assay. Especially, compound 17h displayed more potent cytotoxic activity against KBvin than etoposide. The cytotoxic results indicated that compound 17h may be a good lead for further structural modification.