Optimization of biguanide derivatives as selective antitumor agents blocking adaptive stress responses in the tumor microenvironment

Abstract

Adaptive cellular responses resulting from multiple microenvironmental stresses, such as hypoxia and nutrient deprivation, are potential novel drug targets for cancer treatment. Accordingly, we focused on developing anticancer agents targeting the tumor microenvironment (TME). In this study, to search for selective antitumor agents blocking adaptive responses in the TME, thirteen new compounds, designed and synthesized on the basis of the arylmethylbiguanide scaffold of phenformin, were used in structure activity relationship studies of inhibition of hypoxia inducible factor (HIF)-1 and unfolded protein response (UPR) activation and of selective cytotoxicity under glucose-deprived stress conditions, using HT29 cells. We conducted luciferase reporter assays using stable cell lines expressing either an HIF-1-responsive reporter gene or a glucose-regulated protein 78 promoter-reporter gene, which were induced by hypoxia and glucose deprivation stress, respectively, to screen for TME-targeting antitumor drugs. The guanidine analog (compound 2), obtained by bioisosteric replacement of the biguanide group, had activities comparable with those of phenformin (compound 1). Introduction of various substituents on the phenyl ring significantly affected the activities. In particular, the o-methylphenyl analog compound 7 and the o-chlorophenyl analog compound 12 showed considerably more potent inhibitory effects on HIF-1 and UPR activation than did phenformin, and excellent selective cytotoxicity under glucose deprivation. These compounds, therefore, represent an improvement over phenformin. They also suppressed HIF-1- and UPR-related protein expression and secretion of vascular endothelial growth factor-A. Moreover, these compounds exhibited significant antiangiogenic effects in the chick chorioallantoic membrane assay. Our structural development studies of biguanide derivatives provided promising candidates for a novel anticancer agent targeting the TME for selective cancer therapy, to be subjected to further in vivo study.

Keywords:

• HIF-1
• UPR
• antiangiogenesis
• hypoxia
• glucose deprivation

Supplementary material

Materials and methods

Proton (¹H) and carbon-13 (¹³C) nuclear magnetic resonance (NMR) spectra were recorded using a JEOL JNM-EX400, JNM-AL400, or JNM-ECA-500 spectrometer (JEOL Ltd, Tokyo, Japan) at 400 or 500 MHz (¹H NMR) and 100 or 125 MHz (¹³C NMR) in deuterated chloroform (CDCl₃) (Cambridge Isotope Laboratories, Tewksbury, MA, USA), methanol (CD₃OD) (Wako Pure Chemical Industries, Ltd), and dimethyl sulfoxide DMSO-d₆ (Cambridge Isotope Laboratories). Chemical shifts of ¹H-NMR are referenced to tetramethylsilane (TMS). Chemical shifts of ¹³C-NMR were referenced to CDCl₃ (77.0 ppm), CD₃OD (49.0), and DMSO-d₆ (39.7), unless otherwise specified. Electron impact (EI) mass spectra and fast atom bombardment (FAB) mass (m-nitrobenzyl alcohol was used as the matrix) spectra were measured on a JEOL JMS-SX102A mass spectrometer (JEOL Ltd). Direct analysis in real time (DART^®) mass spectra were measured on a JEOL JMS-T100TD DART mass spectrometer (JEOL Ltd). Elementary analysis was performed on a Micro Corder JM10 (J-SCIENCE LAB Co, Ltd, Kyoto, Japan). Thin-layer chromatography was carried out on silica gel (Art 7749 Kieselgel 60 PF₂₅₄, Merck KGaA, Darmstadt, Germany) with visualization of components by ultraviolet (UV) light (254 nm). Column chromatography was carried out on silica gel (AP-300S; Taiko-Shoji, Nagoya, Japan). Unless otherwise noted, all chemicals were obtained from commercial sources and used without further purification.

Syntheses of phenformin (1) and intermediate compounds

2-phenethylbiguanide hydrochloride (compound 1: phenformin)

Following the procedure described in the literature,¹ phenethylamine (compound 15) (254.5 mg, 2.1 mmol) was added to a solution of dicyandiamide (176.6 mg, 2.1 mmol) in 3.7 mL of dry CH₃CN, and TMSCl (228.1 mg, 2.3 mmol) was slowly added dropwise to the mixture. The mixture was stirred and irradiated with adjustable power in the range of 0–400 W, at 2.45 GHz, for 15 minutes at 150°C, using microwave reactor (Biotage^® Initiator 2.0, Biotage AB, Uppsala, Sweden). After the mixture was cooled down to approximately 50°C, isopropyl alcohol (iPrOH) (0.49 mL, 6.3 mmol) was added slowly, and the mixture was further stirred and irradiated at 125°C for 1 minute. The precipitation of the biguanide hydrochloride salt was washed with CH₃CN twice to afford compound 1² (307.3 mg, 73%). The analytical sample was obtained by recrystallization from iPrOH as a white powder. mp 175°C–177°C (lit 175°C–178°C); ¹H NMR (400 MHz, DMSO-d₆): δ=2.77 (t, J=7.0 Hz, 2H), 3.33 (q, J=7.0 Hz, 2H), 7.23–7.43 (m, 12H); FABMS: m/z calcd for [M-Cl]⁺: 206, found 206.

S-methylisothiourea hydroiodide (compound 27)

A solution of thiourea (compound 26) (5.0 g, 65.7 mmol) and MeI (4.74 mL, 72.3 mmol) in EtOH (10 mL) was refluxed for 2 hours. After this, the resulting mixture was cooled to 0°C, and then, Et₂O was added to the mixture. The precipitate was filtered and washed with Et₂O and dried to give compound 27³ (13.3 g, 93%) as a pale brown solid. ¹H NMR (400 MHz, DMSO-d₆): δ=2.49 (s, 3H), 8.79 (s, 4H); ¹³C NMR (100 MHz, DMSO-d₆): δ=13.4, 171.1.

N,N′-bis(tert-butoxycarbonyl)-S-methylisothiourea (compound 28)

To a solution of compound 27 (5.0 g, 22.9 mmol) in a mixture of H₂O (50 mL) and 1,4-dioxane (50 mL) were added 1 M NaOH aqueous solution (23 mL, 23.0 mmol) and (Boc)₂O (12.5 g, 57.4 mmol). The reaction mixture was vigorously stirred for 9 hours at room temperature. The precipitate was filtered off and washed with a small amount of H₂O. The filtrate was concentrated under reduced pressure to approximately half the volume, and the solid was separated by filtration. The solid was combined and suspended in H₂O (150 mL) at approximately 50°C and filtered. Then it was dried under reduced pressure at room temperature to give compound 28³ (5.3 g, 79%) as a white powder. ¹H NMR (400 MHz, CDCl₃): δ=1.52 (s, 9H), 1.53 (s, 9H), 2.40 (s, 3H), 11.60 (s, 1H); EIMS: m/z calcd for [M]⁺: 290, found 290.

N,N′-Bis(tert-butoxycarbonyl)-N″-2-phenethylguanidine (compound 29)

To a solution of compound 28 (1.16 g, 4.0 mmol) in CH₂Cl₂ (8 mL) was added phenethylamine (compound 15) (969 mg, 8.0 mmol), and the reaction mixture was stirred at room temperature for 14 hours. Then the solvent was evaporated under reduced pressure, and the residue was dissolved in EtOAc (50 mL). The solution was washed with 10% citric acid aqueous solution (5×20 mL), saturated NaHCO₃ aqueous solution (3×10 mL), and H₂O (3×10 mL). The organic phase was dried over anhydrous MgSO₄ and concentrated in vacuo to give compound 29 (1.25 g, 86%) as a white powder. The analytical sample was obtained by recrystallization from n-hexane and EtOAc as a colorless powder. mp 128°C–129°C; ¹H NMR (400 MHz, CDCl₃): δ=1.48 (s, 9H), 1.50 (s, 9H), 2.87 (t, J=7.2 Hz, 2H), 3.67 (q, J=7.2 Hz, 2H), 7.21–7.28 (m, 5H), 8.36 (s, 1H), 11.46 (s, 1H); ¹³C NMR (100 MHz, DMSO-d₆): δ=28.0, 28.3, 35.3, 42.2, 79.2, 83.0, 126.5, 128.5, 128.8, 138.5, 153.2, 156.1, 163.6; DARTMS: m/z calcd for [M + H]⁺: 364, found 364. Anal Calcd for C₁₉H₂₉N₃O₄: C, 62.79; H, 8.04; N, 11.56. Found: C, 62.84; H, 8.10; N, 11.58.

2-phenethylguanidine hydrochloride (compound 2)

According to the procedure described in the literature,² a solution of compound 29 (500 mg, 1.38 mmol) in 4 M HCl-1,4-dioxane (4 mL) was stirred at room temperature for 15 hours. The solvent was removed under reduced pressure. The residue was reevaporated from Et₂O three times. After EtOAc was added, the precipitate was filtered and washed with EtOAc and dried to give compound 2⁴ (271 mg, 99%). The analytical sample was obtained by recrystallization from EtOAc and EtOH as a colorless powder. mp 133°C–134°C (lit 135°C–138°C); ¹H NMR (400 MHz, CD₃OD): δ=2.88 (t, J=7.2 Hz, 2H), 3.46 (t, J=7.2 Hz, 2H), 7.20–7.36 (m, 5H); ¹³C NMR (100 MHz, CD₃OD): δ=36.7, 44.5, 128.6, 130.5, 130.6, 140.1, 159.4. DARTMS: m/z calcd for [M-Cl]⁺:164, found 164.

1-(2-phenethyl)urea (compound 3)

According to the literature procedure,⁵ a mixture of ethyl carbamate (1.06 g, 11.9 mmol), Al₂O₃ (1.06 g, 10.4 mmol), and phenethylamine (compound 15) (2.88 g, 23.8 mmol) in toluene (7 mL) was refluxed for 48 hours. The reaction mixture was filtered on Celite^® (Wako Pure Chemical Industries, Ltd) and evaporated in vacuo. The residue was purified by chromatography (MeOH/CHCl₃ =1:40) to give compound 3⁶ (603.3 mg, 31%). The analytical sample was obtained by recrystallization from H₂O as a colorless powder. mp 110°C–111°C (lit 110°C–111°C); ¹H NMR (400 MHz, CDCl₃): δ=2.80 (t, J=6.8 Hz, 2H), 3.40 (q, J=6.8 Hz, 2H), 4.49 (s, 2H), 4.91 (s, 1H), 7.21–7.29 (m, 5H); ¹³C NMR (100 MHz, CDCl₃): δ=36.2, 41.7, 126.4, 128.6, 128.8, 139.0, 158.9; EIMS: m/z calcd for [M⁺]: 164, found 164.

1-(2-phenethyl)thiourea (compound 4)

According to the literature procedure,⁷ a mixture of phenethylamine hydrochloride (compound 30) (2.0 g, 12.7 mmol) and ammonium isothiocyanate (1.01 g, 13.4 mmol) in bromobenzene (3.5 mL) was refluxed under a nitrogen atmosphere for 1.5 hours and then cooled to 0°C. The precipitate was washed with H₂O, filtered, then washed with n-hexane and dried to give compound 4⁸ (965.6 mg, 42%). The analytical sample was obtained by recrystallization from EtOH as a colorless powder. mp 136°C–137°C (lit 137°C). ¹H NMR (500 MHz, DMSO-d₆): δ=2.79 (br s, 2H), 3.57 (br s, 2H), 7.19–7.32 (m, 5H), 6.99, 7.56, 7.70 (br s, total 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ=34.9, 45.2, 126.0, 128.2, 128.7, 139.3, 183.2; EIMS: m/z calcd for [M]⁺: 180, found 180.

1,4-bis(2-tert-butoxycarbonylaminoethyl)benzene (compound 32)

According to the literature procedure,⁹ to a stirred solution of 1,4-phenylenediacetonitrile (compound 31) (4.69 g, 30 mmol) in dry methanol (200 mL) at 0°C, (Boc)₂O (26.2 g, 120 mmol) and NiCl₂ · 6H₂O (713 mg, 10 mol%) were added. NaBH₄ (15.9 g, 280 mmol) was then added in small portions over 2 hours. The resulting reaction mixture was allowed to warm to room temperature and stirred for 19 hours. Then diethylentriamine (6.5 mL, 60 mmol) was added, and the mixture was allowed to stir for another 30 minutes. After evaporation of solvent, the purple residue was dissolved in ethyl acetate and washed with a NaHCO₃ saturated solution (2×150 mL) and brine (50 mL). The organic layer was dried over anhydrous MgSO₄, filtered, and evaporated to give the crude product, which was purified by silica gel column chromatography (CHCl₃/MeOH =10:1) to afford compound 32 (7.02 g, 64%). The analytical sample was obtained by recrystallization from CH₂Cl₂ and n-hexane as pale yellow needles. mp 154°C–155°C; ¹H NMR (400 MHz, CDCl₃): δ=1.44 (s, 18H), 2.77 (t, J=7.0 Hz, 4H), 3.36 (br, 4H), 4.55 (s, 2H), 7.13 (br, 4H); ¹³C NMR (125 MHz, CDCl₃): δ=28.4, 35.8, 41.7, 79.2, 129.0, 137.0, 155.8; DARTMS: m/z calcd for [M + H]⁺: 365, found 365; Anal Calcd for C₂₀H₃₂N₂O₄: C, 65.91; H, 8.85; N, 7.69. Found: C, 65.67; H, 8.57; N, 7.95.

1,4-bis(2-aminoethyl)benzene dihydrochloride (compound 33)

A mixture of compound 32 (3.98 g, 10.9 mmol) and 5% HCl/MeOH (70 mL) was stirred for 13 hours. The solvent was removed in vacuo to give compound 33¹⁰ (2.61 g, quant) as a white solid. ¹H NMR (400 MHz, CD₃OD): δ=2.97 (t, J=7.7 Hz, 4H), 3.15 (t, J=7.7 Hz, 4H), 7.28 (br, 4H).

1-(2-tert-butoxycarbonylaminoethyl)-4-(2-aminoethyl)benzene (compound 34)

According to the procedure described in the literature,¹¹ to the solution of compound 33 (1.0 g, 4.2 mmol) in MeOH (1.5 mL) at 0°C, 0.9 mL of 34% NaOH aqueous solution (5.9 mmol) was added carefully, then stirred for 1 hour at room temperature. The solution of (Boc)₂O (1.29 g, 5.9 mmol) in MeOH (2 mL) was added at 0°C for 30 minutes, and the mixture was stirred for 66 hours at room temperature. The mixture was concentrated in vacuo, and 10% citric acid aqueous solution (5 mL) was added to the residue. Bis-Boc amine compound 32 was removed by extraction with diethyl ether (8×30 mL) (884.5 mg, 58% recovery). The aqueous layer was neutralized with NaOH (1 g) and extracted with CHCl₃ (3×15 mL). The combined organic layer was washed with 10 mL of brine, dried over anhydrous MgSO₄, and concentrated in vacuo to yield compound 34¹² (383.8 mg, 34%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ=1.44 (s, 9H), 2.71–2.77 (m, 4H), 2.96 (t, J=6.8 Hz, 2H), 3.36 (br, 2H), 4.56 (s, 1H), 7.13 (br, 4H).

1-(2-acetylaminoethyl)-4-(2-tert-butoxycarbonylaminoethyl)benzene (compound 35)

According to the literature procedure,¹³ triethylamine (0.73 mL) was added to a stirred solution of compound 34 (383.8 mg, 1.45 mmol) and N,N-dimethyl-4-aminopyridine (DMAP) (18 mg, 0.15 mmol) in anhydrous CH₂Cl₂ (1.2 mL) at 0°C. Then the mixture was added dropwise to acetic anhydride (0.2 mL, 2.2 mmol) and stirred for 4 hours at room temperature. The mixture was washed with H₂O (20 mL), saturated citric acid solution (20 mL), saturated NaHCO₃ solution (2×20 mL), and brine (10 mL) and dried over anhydrous MgSO₄. After filtration, the solvent was evaporated under reduced pressure to give the residue, which was purified by silica gel column chromatography (ethyl acetate/n-hexane =3:1) to afford compound 35 (268.3 mg, 60%) as a white solid. mp 117°C–118°C; ¹H NMR (400 MHz, CDCl₃): δ=1.44 (s, 9H), 1.95 (s, 3H), 2.79 (m, 4H), 3.36 (br, 2H), 3.50 (q, J=6.8 Hz, 2H), 4.55 (s, 1H), 5.47 (s, 1H), 7.14 (br, 4H); ¹³C NMR (125 MHz, CDCl₃): δ=23.3, 28.4, 35.2, 35.8, 40.6, 41.8, 79.2, 128.9, 129.0, 136.9, 137.2, 155.8, 170.0; DARTMS: m/z calcd for [M + H]⁺: 307, found 307; Anal Calcd for C₁₇H₂₆N₂O₃: C, 66.64; H, 8.55; N, 9.14. Found: C, 66.45; H, 8.55; N, 9.03.

1-(2-acetylaminoethyl)-4-(2-aminoethyl)benzene (compound 22)

A solution of compound 35 (263.3 mg, 0.86 mmol) in 5% HCl/MeOH (2.5 mL) was stirred for 22 hours at room temperature. The solvent was removed in vacuo to give compound 22¹⁴ hydrochloride as a white solid; then it was dissolved in saturated NaHCO₃ aqueous solution (5 mL) and extracted with CHCl₃ (with 30 mL, 20 mL, and 20 mL). The extracts were washed with sat. brine (10 mL), dried over anhydrous MgSO₄, and concentrated in vacuo to give compound 22 (165.9 mg, 94%) as a yellow oil; ¹H NMR (500 MHz, CDCl₃): δ=1.94 (s, 3H), 2.72 (t, J=7.0 Hz, 2H), 2.79 (t, J=7.0 Hz, 2H), 2.95 (t, J=7.0 Hz, 2H), 3.49 (q, J=7.0 Hz, 2H), 5.74 (s, 1H), 7.11–7.16 (m, 4H).

Figure S1 Synthesis of compounds 2–4 and 22.

Notes: Bolded numbers signify an individual compound. Reagent and conditions: (A) (a) MeI, EtOH, reflux; (b) (Boc)₂O, H₂O/1,4-dioxane, NaOH aq, rt; (c) phenethylamine (15), CH₂Cl₂, rt; (d) 4 M HCl-1,4-dioxane, rt; (B) (e) Al₂O₃, toluene, reflux; (C) (f) bromobenzene, reflux under N₂; and (D) (g) 1) (Boc)₂O, NiCl₂ · 6H₂O, NaBH₄, dry MeOH, 0°C to rt; 2) diethylenetriamine, rt; (h) 5% HCl/MeOH, rt; (i) Boc₂O, NaOH aq, MeOH, 0°C to rt; (j) Ac₂O, NEt₃, DMAP, dry CH₂Cl₂, 0°C to rt; (k) 1) 5% HCl/MeOH, rt; 2) sat. NaHCO₃ aq.
Abbreviations: Boc, tert-butoxycarbonyl; aq, aqueous solution; rt, room temperature; sat., saturated.

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Acknowledgments

This work was supported in part by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research [B], number 24390029, to HN) and a Research Grant for Clinical Study Promotion (2013) from Gifu University. We thank Ms Emi Inaba, Ms Masako Hayashi, and Ms Miharu Hotta for their technical assistance.

Disclosure

The authors report no conflicts of interest in this work.