Synthesis of imidazole-containing analogues of farnesyl pyrophosphate and evaluation of their biological activity on protein farnesyltransferase

Abstract

With the aim of creating new bisubstrate inhibitors of protein farnesyltransferase (FTase), new carboxylic farnesyl pyrophosphate analogues have been designed and synthesized. The original structures are built around three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring. All the compounds were evaluated for their ability to inhibit FTase and compared with the corresponding derivatives lacking the imidazole ring, synthesized for that purpose. These new compounds are not bisubstrate inhibitors probably because the imidazole ring is not in the right position to interact with the zinc atom. However these derivatives display FPP competitive inhibition with a good activity in the carboxylic farnesyl pyrophosphate analogues series.

Keywords::

• farnesyltransferase inhibitors
• imidazole
• isoprenyl
• inhibitors

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Erratum

Introduction

The protein farnesyltransferase (FTase) appears to be an important target in anticancer therapy [1] and, recently, in antiviral and antiparasitic fields [2]. FTase catalyzes the farnesylation of many proteins (Scheme 1) and this post-translational modification is a critical step for several proteins involved in the intracellular signal transduction and the cell proliferation. Indeed, the hydrophobic farnesyl group is necessary for membrane targeting and also for protein-protein interactions of these proteins making the farnesylation process a crucial step [1, 3].

Scheme 1.  Protein farnesylation.

FTase is a heterodimeric zinc metalloenzyme [4] which transfers the farnesyl group, a 15-carbon isoprenoid lipid unit, from the farnesyl pyrophosphate (FPP) to the cysteine of the C-terminal CaaX motif (C: cysteine, a: aliphatic amino acid, X: serine or methionine or alanine or glutamine) [3]. All the data concerning FTase catalytic reaction suggest that its mechanism is associative with an “exploded” transition state. [5]

Numerous FTase inhibitors (FTIs) have been developed and reported [6]. Some of them are currently in preclinical or clinical trials and have shown efficacy as anticancer agents alone or in combination with cytotoxic compounds [7]. Most of FTIs are FPP or CaaX competitive inhibitors [8]. However, bisubstrate compounds, i.e. able to bind to both FPP and CAAX sites, are expected to have a high affinity and specificity for FTase [9]. Therefore, we focused our interest on the design and the synthesis of potential bisubstrate FTIs and herein our approach and our first results are described.

On the course of our research of original new FTIs, based on the structures of already described FTIs, we designed potential bisubstrate compounds (Figure 1). Our first series of analogues 1 where a 1,4-diacidic moiety was connected to the peptidic chain through an imidazole ring, was unable to bind to the FFP binding site [10]. Therefore to accomplish this binding, we designed a new series 2 composed by three elements: a prenyl moiety, a 1,4-diacid motif and an imidazole ring.

Figure 1.  Potential bisubstrate analogues.

We chose a 1,4-diacid motif to mimic the pyrophosphate of the FPP because this acidic moiety is present in several potent natural FPP competitive inhibitors like chaetomellic acid A (Figure 2) [11]. Concerning the imidazole ring, it is also a common moiety of potent FTIs [12]. By its basic nitrogen, it realizes a strong interaction with the zinc atom in the FTase catalytic binding site. To evaluate the importance of a free NH in these structures, we synthesized and evaluated the structures with a free or a methyl amine.

Figure 2.  Chaetomellic acid A.

Experimental protocols

Commercial compounds were used without any further purification. Tetrahydrofuran (THF) was freshly distilled from sodium/benzophenone. Pyridine was stored over KOH and other solvents over 3å molecular sieves. Column chromatography was performed with silica gel 60 (35-70 μm). Preparative TLC (PLC) was performed on SDS TLC with silica gel 60.

NMR spectra (¹H and ¹³C) were recorded on a Brucker Avance 300 (300 MHz), DRX400 (400 MHz) and Avance 500 (500 MHz). Chemical shifts are given in ppm relative to CDCl₃ (¹H: 7.27 ppm; ¹³C: 77.14 ppm), CD₃OD (¹H: 3.34 ppm; ¹³C: 49.9 ppm) or (CH₃)₄Si, as an internal standard. Splitting patterns are designed as: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad and combinations thereof. IR spectra were recorded on a Perkin-Elmer Spectrum BX. Mass spectra were recorded on Thermofinningan Automass with a quadripole detection (IE) and on Thermoquest AQA Navigator with a TOF detection (ESI-HRMS).

Chemistry

General procedure A: Alkylation at C-2 position of the imidazole ring

To a stirred solution of compound 3a-b in anhydrous THF under argon at -78°C was added n-butyllithium (1.6 M in hexane, 1 equiv). After 40-50 min under argon at −78°C, compound 4a-c (1.5 equiv.) was added dropwise. The solution was stirred 20-150 min under argon at −78°C then it was slowly allowed to warm to room temperature. The reaction mixture was quenched with water and concentrated under reduced pressure. The mixture was diluted with EtOAc washed three times with brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to afford compounds 5a-d.

3-Methyl-1-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-but-2-en-1-ol (5a):

Prepared according to general procedure A on SEM-1H-imidazole 3a [34] (0.29 g, 1.5 mmol) in anhydrous THF (3 mL) with n-butyllithium (1.0 mL, 1.6 mmol), 50 min, and 3-methylcrotonaldehyde 4a (210 μL, 2.2 mmol), 20 min. After column chromatography (CH₂Cl₂/MeOH : 96/4) 5a was isolated as a yellow wax (0.28 g, 68% yield); IR (CH₂Cl₂) ν : 3400; 3133; 3111; 1679; 1248 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 0 (s, 9H); 0.90 (t, 2H, J = 8.0 Hz); 1.80 (bs, 3H); 1.82 (bs, 3H); 3.49 (t, 2H, J = 8.0 Hz); 5.31 (s, 2H); 5.55 (d, 1H, J = 9.0 Hz); 5.61 (dd, 1H, J = 9.0 Hz and J’ = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: -1.4 (3C); 17.9; 18.3; 26.0; 63.9; 66.3; 75.1; 120.5; 127.0; 124.4; 137.0; 149.9; MS (IE, MeOH) m/z 282 [M]^+..

3-Methyl-1-(1-methyl-1H-imidazol-2-yl)-but-2-en-1-ol (5b):

Prepared according general procedure A on N-methyl-1H-imidazole 3b (1.96 g, 23.9 mmol) in anhydrous THF (34 mL) with n-butyllithium (18.0 mL, 28.8 mmol), 45 min, and 3-methylcrotonaldehyde 4a (3.4 mL, 33.5 mmol), 30 min. The crude product was purified by precipitation in ether to afford 5b as a white powder (2.7 g, 68% yield); IR (CH₂Cl₂) ν: 3073; 2964; 2897; 2825; 1676; 1282 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 1.76 (bs, 6H); 3.65 (s, 3H); 5.45 (d, 1H, J = 9.0 Hz); 5.56 (bd, 1H, J = 9.0 Hz); 6.77 (bs, 1H); 6.85 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 18.1; 28.7; 32.8; 63.9; 122.6; 123.9; 126.5; 136.7; 149.2; HRMS (ESI, MeOH) calculated for C₉H₁₅N₂O [M+H]⁺: 167.1184; found 167.1147.

3,7-Dimethyl-1-(1-methyl-1H-imidazole-2-yl)-octa-2,6-dien-1-ol (5c):

Prepared according general procedure A on N-methyl-1H-imidazole 3b (0.35 mL, 4.4 mmol) in anhydrous THF (3.4 mL) with n-butyllithium (3.3 mL, 5.3 mmol), 45 min, and geranial 4b [34] (1.07 g, 7.02 mmol), 2h30. After column chromatography (CH₂Cl₂/MeOH : 96/4) 5c was isolated as a light yellow oil (1.0 g, 100% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.58 (bs, 3H); 1.66 (bs, 3H); 1.78 (d, 3H, J = 1.0 Hz); 2.09 (m, 4H); 3.63 (s, 3H); 5.06 (m, 1H); 5.49 (m, 2H); 6.79 (d, 1H, J = 1.0 Hz); 6.90 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.6; 17.7; 25.7; 26.3; 32.8; 39.6; 64.1; 121.8; 123.8; 123.9; 126.7; 131.8; 140.5; 149.4; HRMS (ESI, MeOH) calculated for C₁₄H₂₃N₂O [M+H]⁺: 235.1810; found: 235.1799.

3,7,11-Trimethyl-1-(1-methyl-1H-imidazol-2-yl)-dodeca-2, 6, 10-trien-1-ol (5d):

Prepared according general procedure A on N-methyl-1H-imidazole 3b (0.4 mL, 5.0 mmol) in anhydrous THF (4 mL) with n-butyllithium (3.75 mL, 6.0 mmol), 40 min, and farnesal 4c [36] (1.61 g, 7.3 mmol), 2 h. After column chromatography (CH₂Cl₂/MeOH : 95/5) 5d was isolated as a light yellow oil (1.45 g, 96% yield); IR (CH₂Cl₂) ν: 3110; 2960; 2913; 2857; 1668; 1279 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 1.58 (bs, 3H); 1.60 (bs, 3H); 1.68 (bs, 6H); 1.80 (bs, 3H); 1.91-2.19 (m, 8H); 3.63 (s, 3H); 5.08 (m, 2H); 5.48 (d, 1H, J = 9.0 Hz); 5.55 (bd, 1H, J = 9.0 Hz); 6.81 (bs, 1H); 6.92 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 16.0; 16.7; 17.7; 25.7; 26.3; 26.7; 32.8; 39.6; 39.7; 64.1; 121.8; 123.6; 123.7; 124.3; 126.8; 131.4; 135.5; 140.8; 149.2; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₁₉H₃₁N₂O [M+H]^+: 303.2436; found: 303.2443.

General procedure B. Oxidation of the secondary alcohol

Anhydrous THF was added to manganese dioxide (IV) and the alcohols 5a-d under argon at 0°C. After stirring 2 h at 0°C, the reaction mixture was filtered over Celite^® and concentrated under reduced pressure.

3-Methyl-1-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-but-2-en-1-one (6a):

Prepared according to general procedure B on 5a (152 mg, 0.54 mmol) in anhydrous THF (12 mL) with MnO₂ (0.52 g, 6.0 mmol). 6a was obtained without purification as a yellow wax (151 mg, 100% yield); IR (CH₂Cl₂) ν: 2952; 1656; 1609; 1247 cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ: 0 (s, 9H); 1.03 (t, 2H, J = 8.5 Hz); 2.02 (s, 3H); 2.27 (s, 3H); 3.58 (t, 2H, J = 8.5 Hz); 5.85 (s, 2H); 7.18 (bs, 1H); 7.27 (bs, 1H); 7.29 (s, 1H); ¹³C NMR (62.5 MHz, CDCl₃) δ: -1.5 (3C); 17.8; 21.0; 28.2; 66.8; 76.5; 121.2; 124.1; 129.0; 144.4; 158.4; 181.8; HRMS (ESI, EtOAc) calculated for C₁₄H₂₄N₂O₂SiNa [M+Na]⁺: 303.1505; found: 303.1518.

3-Methyl-1-(1-methyl-1H-imidazol-2-yl)-but-2-en-1-one (6b):

Prepared according to general procedure B on 5b (0.95 g, 5.7 mmol) in anhydrous THF (40 mL) with MnO₂ (5.9 g, 67.8 mmol). The crude product was purified by column chromatography on silica gel (CH₂Cl₂/EtOAc : 9/1) to afford 6b as a white solid (0.84 g, 90% yield); IR (CH₂Cl₂) ν: 2939; 1651; 1609 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ : 2.01 (bs, 3H); 2.26 (bs, 3H); 4.04 (s, 3H); 6.98 (bs, 1H); 7.11 (bs, 1H); 7.28 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 21.1; 28.3; 36.5; 121.2; 126.7; 128.6; 144.7; 158.1; 182.0; HRMS (ESI, MeOH) calculated for C₉H₁₃N₂O [M+H]⁺: 165.1028; found: 165.1005.

3,7-Dimethyl-1-(1-methyl-1H-imidazole-2-yl)-octa-2,6-dien-1-one (6c):

Prepared according to general procedure B on 5c (1.0 g, 4.3 mmol) in THF (26 mL) with MnO₂ (5.63 g, 64.7 mmol). The crude product was purified by column chromatography on silica gel (Heptane/EtOAc : 8/2) to afford 6c as a light yellow oil (0.8 g, 80% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.62 (bs, 3H); 1.68 (bs, 3H); 2.27 (m, 7H); 4.04 (s, 3H); 5.12 (m, 1H); 6.99 (bs, 1H); 7.13 (bs, 1H); 7.30 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ : 17.7; 19.7; 25.7; 26.5; 36.4; 42.0; 120.7; 123.2; 126.6; 128.5; 132.4; 144.7; 161.6; 182.2; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₁₄H₂₁N₂O [M+H]⁺: 233.1654; found : 233.1650.

3,7,11-Trimethyl-1-(1-methyl-1H-imidazol-2-yl)-dodeca-2,6,10-trien-1-one (6d):

Prepared according to general procedure B on 5d (1.27 g, 4.2 mmol) in THF (28 mL) with MnO₂ (5.58 g, 64 mmol). The crude product was purified by column chromatography on silica gel (Heptane/EtOAc : 8/2) to afford 6d as a light yellow oil (1.16 g, 92% yield); IR (CH₂Cl₂) ν: 2960; 2913; 2863; 1656; 1609 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 1.59 (bs, 3H); 1.62 (bs, 3H); 1.67 (bs, 3H); 2.02 (m, 4H); 2.28 (bs, 7H); 4.05 (s, 3H); 5.11 (m, 2H); 6.99 (bs, 1H); 7.13 (bs, 1H); 7.32 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 16.5; 17.7; 19.7; 25.7; 26.4; 26.7; 36.4; 39.7; 42.0; 120.7; 123.1; 124.3; 126.6; 128.5; 131.4; 136.1; 144.7; 161.6; 182.2; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₁₉H₂₈N₂ONa [M+Na]⁺: 323.2099; found: 323.2108.

General procedure C. Reformatsky reactions

Anhydrous THF was added to a bromocarboxylic ester, α,β-insaturated ketones 6a-d and HCl-activated zinc under argon at room temperature. After 5 h in an ultrasound bath (temperature around 40°C), the reaction mixture was filtered over Celite^® and concentrated. The crude product was dissolved in ether and washed with 10% HCl, twice with saturated NaHCO₃ and once with brine (a) or dissolved in EtOAc and washed with KOH 1M (c-d). The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to afford compounds 8, 10a-d.

2-(2-Methyl-propenyl)-5-oxo-2-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-tetrahydro-furan-3-carboxylic acid methyl ester (8):

Prepared according to general procedure C on 6a (0.7 g, 2.5 mmol) in anhydrous THF (5 mL) with 2-bromosuccinic acid dimethyl ester 7 [37] (1.13 g, 5.0 mmol), and HCl-activated zinc (0.33 g, 5.0 mmol). After column chromatography on silica gel (CH₂Cl₂/EtOAc : 96/4) 8 was isolated as a colorless oil (0.62 g, 63% yield); IR (CH₂Cl₂) ν: 2950; 1789; 1737; 1248; 1094; 1080 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 0 (s, 9H); 0.92 (m, 2H); 1.34 (bs, 3H); 1.80 (bs, 6H); 2.83 (dd, 1H, J = 8.5 Hz and J’ = 17.5 Hz); 3.14 (dd, 1H, J = 10.0 Hz et J’ = 17.5 Hz); 3.52 (m, 3H); 3.68 (s, 3H); 5.29 (s, 2H); 5.64 (s, 1H); 6.94 (d, 1H, J = 1.0 Hz); 7.07 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: -1.4 (3C); 18.1; 18.4; 27.5; 32.4; 51.6; 52.3; 66.8; 76.0; 84.7; 121.1; 124.3; 127.6; 139.8; 144.1; 170.1; 173.8; HRMS (ESI, CH₃CN) calculated for C₁₉H₃₀N₂O₅SiNa [M+Na]⁺: 417.1822; found: 417.1810.

3-Hydroxy-5-methyl-3-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-hex-4-enic acid tert-butyl ester (10a):

Prepared according to general procedure C on 6a (1.95 g, 7.0 mmol) in anhydrous THF (14.2 mL) with bromoacetic acid tert-butyl ester 9 (3.1 mL, 21.4 mmol), and HCl-activated zinc (1.44 g, 22.0 mmol). After column chromatography on silica gel (CH₂Cl₂/EtOAc : 9/1) 10a was isolated as a yellow oil (1.4 g, 51% yield); IR (CH₂Cl₂) ν: 3418; 2952; 2916; 1702; 1697; 1367; 1352; 1153 cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ: 0 (s, 9H); 0.91 (t, 2H, J = 8.5 Hz); 1.27 (bs, 3H); 1.46 (s, 9H); 1.67 (bs, 3H); 2.70 (d, 1H, J = 16.5 Hz); 3.26 (d, 1H, J = 16.5 Hz); 3.48 (t, 2H, J = 8.5 Hz); 5.38 (d, 1H, J = 10.0 Hz); 5.43 (d, 1H, J = 10.0 Hz); 5.49 (s, 1H); 6.89 (bs, 1H); 7.04 (bs, 1H); ¹³C NMR (62.5 MHz, CDCl₃) δ: -1.3 (3C); 17.4; 18,2; 27.1; 28.1 (3C); 44.0; 66.3; 72.4; 76.0; 81.8; 119.8; 126.6; 128.7; 135.2; 150.0; 173.4; HRMS (ESI, MeOH) calculated for C₂₀H₃₆N₂O₄SiNa [M+Na]⁺: 419.2342; found: 419.2347.

3-Hydroxy-5-methyl-3-(1-methyl-1H-imidazol-2-yl)-hex-4-enoic acid tert-butyl ester (10b):

Prepared according to general procedure C on 6b (786 mg, 4.8 mmol) in anhydrous THF (10 mL) with bromoacetic acid tert-butyl ester 9 (2.1 mL, 14.6 mmol), and HCl-activated zinc (0.97 mg, 14.9 mmol). After column chromatography on silica gel (Heptane/EtOAc : 7/3) 10b was isolated as a yellow oil (1.0 g, 74 % yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.21 (bs, 3H); 1.46 (s, 9H); 1.69 (bs, 3H); 2.69 (d, 1H, J = 16.0 Hz); 3.25 (d, 1H, J = 16.0 Hz); 3.66 (s, 3H); 5.38 (s, 1H); 5.46 (s, 1H); 6.78 (bs, 1H); 6.83 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 17.2; 27.3; 28.3 (3C); 34.3; 44.1; 72.6; 81.9; 122.6; 126.1; 128.3; 134.9; 149.9; 173.7; HRMS (ESI, MeOH) calculated for C₁₅H₂₄N₂O₃Na [M+Na]⁺: 303.1685; found: 303.1691.

3-Hydroxy-5,9-dimethyl-3-(1-methyl-1H-imidazol-2-yl)-deca-4,8-dienoic acid tert-butyl ester (10c):

Prepared according to general procedure C on 6c (764 mg, 3.3 mmol) in anhydrous THF (6.6 mL) with bromoacetic acid tert-butyl ester 9 (1.5 mL, 10.3 mmol), and HCl-activated zinc (0.65 mg, 10.0 mmol). After column chromatography on silica gel (CH₂Cl₂/EtOAc : 95/5) 10c was isolated as a yellow oil (0.91 g, 79 % yield); ¹H NMR (300 MHz, CDCl₃): 1.19 (d, 3H, J = 1.1 Hz); 1.48 (s, 9H); 1.58 (bs, 3H); 1.67 (bs, 3H); 2.01 (m, 4H); 2.68 (d, 1H, J = 16.5 Hz); 3.26 (d, 1H, J = 16.5 Hz); 3.66 (s, 3H); 5.05 (m, 1H); 5.42 (s, 1H); 5.47 (bs, 1H); 6.78 (d, 1H, J = 1.0 Hz); 6.83 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 15.2; 17.7; 25.7; 26.5; 28.1 (3C); 34,0; 40.8; 43.7; 72.3; 81.7; 122.4; 123.8; 125.8; 128.3; 131.8; 138.1; 149.7; 173.5; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₂₀H₃₃N₂O₃ [M+H]⁺: 349.2491; found: 349.2502.

3-Hydroxy-5,9,13-trimethyl-3-(1-methyl-1H-imidazol-2-yl)-tetradeca-4,8,12-trienoic acid tert-butyl ester (10d):

Prepared according to general procedure C on 6d (1.0 g, 3.3 mmol) in anhydrous THF (6.8 mL) with bromoacetic acid tert-butyl ester 9 (1.5 mL, 10.3 mmol), and HCl-activated zinc (0.65 mg, 10.0 mmol). After column chromatography on silica gel (CH₂Cl₂/EtOAc : 95/5) 10d was isolated as a pale yellow oil (1.0 g, 74 % yield); IR (CH₂Cl₂) ν: 3110; 2975; 2927; 1698; 1367; 1349; 1152 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 1.21 (bs, 3H); 1.48 (s, 9H); 1.58 (bs, 3H); 1.60 (bs, 3H); 1.68 (bs, 3H); 1.92-2.12 (m, 8H); 2.69 (d, 1H, J = 16.5 Hz); 3.27 (d, 1H, J = 16.5 Hz); 3.67 (s, 3H) 5.08 (m, 2H); 5.41 (bs, 1H); 5.49 (bs, 1H); 6.78 (bs, 1H); 6.84 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 15.2; 16.0; 17.7; 25.7; 26.5; 26.7; 28.1 (3C); 34.1; 39.7; 40.8; 43.6; 72.3; 81.7; 122.3; 123.6; 124.,3; 125.8; 128.2; 131.5; 135.5; 138.1; 149.7; 173.5; HRMS (ESI, MeOH) calculated for C₂₅H₄₁N₂O₃ [M+ H]⁺: 417.3117; found: 417.3102.

General procedure D. Dehydration

To a stirred solution of hydroxyesters 10a-d in pyridine under argon at 0°C POCl₃ was added dropwise. After stirring 14 hours at room temperature, the reaction mixture was diluted with ether and quenched carefully with water. The solution was basified with KOH 10M then extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to afford compounds 11a-d.

5-Methyl-3[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-hexa-2,4-dienoaic acid tert-butyl ester (11a):

Prepared according general procedure D on 10a (1.03 g, 2.6 mmol) in pyridine (6.5 mL) with POCl₃ (1.2 mL, 12.9 mmol). After column chromatography (Heptane/EtOAc : 6/4) 11a was isolated as a yellow oil (737 mg, 75% yield); IR (CH₂Cl₂) ν: 2953; 1704; 1627; 1248; 1147 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 0 (s, 9H); 0.88 (t, 2H, J = 8.0 Hz); 1.33 (s, 9H); 1.36 (bs, 3H); 1.85 (bs, 3H); 3.47 (t, 2H, J = 8.0 Hz); 5.09 (s, 2H); 6.00 (d, 1H, J = 1.0 Hz); 6.04 (bs, 1H); 7.09 (bs, 1H); 7.13 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: -1,1 (3C); 18.2; 19.4; 28.3 (3C); 28.6; 67.0; 75.8; 80.7; 119.4; 124.9; 125.6; 129.2; 141.6; 144.3; 144.7; 165.0; HRMS (ESI, EtOAc) calculated for C₂₀H₃₅N₂O₃Si [M+H]⁺: 379.2417; found: 379.2422.

5-Methyl-3-(1-methyl-1H-imidazol-2-yl)-hexa-2,4-dienoic acid tert-butyl ester (11b):

Prepared according to procedure D on 10b (0.82 g, 3 mmol) in pyridine (7.5 mL) with POCl₃ (1.4 mL, 15.2 mmol). After column chromatography (Heptane/EtOAc : 1/1) 11b was isolated as a yellow oil (646 mg, 82% yield); IR (CH₂Cl₂) ν: 2976; 2933; 1698; 1145 cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ: 1.20 (bs, 3H); 1.85 (bs, 3H); 1.33 (s, 9H); 3.47 (s, 3H); 6.03 (s, 2H); 6.91 (bs, 1H); 7.09 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 18.5; 28.2 (3C); 28.5; 32.9; 80.4; 120.4; 125.4; 125.9; 128.6; 141.6; 144.2; 144.7; 165.0; HRMS (ESI, EtOAc) calculated for C₁₅H₂₃N₂O₂ [M+H]⁺: 263.1760; found: 263.1733.

5,9-Dimethyl-3-(1-methyl-1H-imidazol-2-yl)-deca-2,4,8-trienoic acid tert-butyl ester (11c):

Prepared according to procedure D on 10c (0.79 g, 2.3 mmol) in pyridine (5.7 mL) with POCl₃ (1.06 mL, 11.4 mmol). After column chromatography (Heptane/EtOAc : 6/4) 11c was isolated as a yellow oil (613 mg, 82% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.20 (d, 3H, J = 1.0 Hz); 1.32 (s, 9H); 1.59 (bs, 3H); 1.68 (bs, 3H); 2.11 (m, 4H); 3.45 (s, 3H); 5.06 (m, 1H); 6.00 (bs, 1H); 6.04 (bs, 1H); 6.90 (d, 1H, J = 1.0 Hz); 7.08 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.7; 17.7; 25.7; 26.5; 28.0 (3C); 32.7; 42.0; 80.3; 120.2; 123.4; 124.9; 126.2; 128.6; 132.1; 141.5; 144.8; 147.4; 164.9; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₂₀H₃₁N₂O₂ [M+H]⁺: 331.2386; found: 331.2386.

5,9,13-Trimethyl-3-(1-methyl-1 H-imidazol-2-yl)-tetradeca-2,4,8,12-tetraenoic acid tert-butyl ester (11d):

Prepared according to procedure D on 10d (2.16 g, 5.2 mmol) in pyridine (16 mL) with POCl₃ (2.41 mL, 25.9 mmol). After column chromatography (CH₂Cl₂/EtOAc : 9/1) 11d was isolated as a yellow oil (1.77 g, 85% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.17 (bs, 3H); 1.29 (s, 9H); 1.57 (bs, 6H); 1.65 (bs, 3H); 1.90-2.02 (m, 4H); 2.09 (bs, 4H); 3.42 (s, 3H); 5.05 (m, 2H); 5.92 (bs, 1H); 6.01 (bs, 1H); 6.87 (d, 1H, J = 1.0 Hz); 7.04 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.4; 17.1; 18.0; 26.0; 26.8; 27.0; 28.3 (3C); 33.0; 40.0; 42.3; 80.6; 120.5; 123.6; 124.6; 125.2; 126.4; 128.8; 131.7; 136.1; 141.8; 145.0; 147.8; 165.2; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₂₅H₃₉N₂O₂ [M+ H]⁺: 399.3012; found: 399.3024.

General procedure E. Reduction of the conjugated double bond

Anhydrous methanol was added to the conjugated alkenes 11a-d and magnesium turnings under argon at room temperature. After controlling the temperature with an ice bath at the beginning, the reaction mixture was stirred for 3 h and quenched with 3N HCl. The solution was basified with 3M KOH, the formed gel was filtered and the filtrate was extracted with EtOAc. The combined organic layers were dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to afford compounds 13a-d.

5-Methyl-3[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-2-yl]-hex-4-enoic acid tert-butyl ester (13a):

Prepared according to general procedure E on 11a (327 mg, 0.87 mmol) in anhydrous methanol (7 mL) with Mg (0.32 g, 13.3 mmol). After column chromatography (Heptane/EtOAc : 6/4) 13a was isolated as a yellow oil (205 mg, 62% yield); ¹H NMR (300 MHz, CDCl₃) δ: 0 (s, 9H); 0.89 (dd, 2H, J = 7.5 Hz and J’ = 9.0 Hz); 1.38 (s, 9H); 1.69 (bs, 3H); 1.77 (bs, 3H); 2.59 (dd, 1H, J = 7.5 Hz and J’ = 15.0 Hz); 2.92 (dd, 1H, J = 8.0 Hz and J’ = 15.0 Hz); 3.45 (dd, 2H, J = 7.5 Hz and J’ = 9.0 Hz); 4.23 (m, 1H); 5.23 (bs, 1H); 5.24 (s, 2H); 6.88 (bs, 1H); 6.95 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: -1.1 (3C); 18.2; 18.6; 26.1; 28.4 (3C); 33.6; 40.9; 66.3; 75.1; 80.6; 119.8; 125.0; 127.8; 133.4; 150.4; 171.7; HRMS (ESI, MeOH) calculated for C₂₀H₃₆N₂O₃SiNa [M+Na]⁺: 403.2393; found: 403.2388.

5-Methyl-3-(1-methyl-1H-imidazol-2-yl)-hex-4-enoic acid tert-butyl ester (13b):

Prepared according to general procedure E on 11b (434 mg, 1.66 mmol) in anhydrous methanol (9.6 mL) with Mg (0.51 g, 21.2 mmol). After column chromatography (ether) 13b was isolated as a yellow oil (352 mg, 80% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.36 (s, 9H); 1.68 (bs, 3H); 1.75 (bs, 3H); 2.56 (dd, 1H, J = 7.5 Hz and J’ = 16.0 Hz); 2.92 (dd, 1H, J = 8.0 Hz and J’ = 16.0 Hz); 3.54 (s, 3H); 4.07 (ddd, 1H, J = 7.5 Hz, J’ = 8.0 Hz and J’’ = 10.0 Hz); 5.13 (bd, 1H, J = 10.0 Hz); 6.21 (d, 1H, J = 1.0 Hz); 6.88 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 18.5; 26;9; 28.2 (3C); 32.7; 33.4; 40.4; 80.5; 120.7; 124.5; 127.2; 133.1; 149.7; 171.7; HRMS (ESI, EtOAc) calculated for C₁₅H₂₄N₂O₂Na [M+Na]⁺ : 287.1735; found: 287.1719.

5,9-Dimethyl-3-(1-methyl-1H-imidazol-2-yl)-deca-4,8-dienoic acid tert-butyl ester (13c):

Prepared according to general procedure E on 11c (488 mg, 1.48 mmol) in anhydrous methanol (12.5 mL) with Mg (0.73 g, 30.4 mmol). After column chromatography (CH₂Cl₂/EtOAc : 8/2) 13c was isolated as a yellow oil (347 mg, 71% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.37 (s, 9H); 1.55 (bs, 3H); 1.64 (bs, 3H); 1.76 (d, 3H, J = 1.5 Hz); 2.01 (m, 4H); 2.57 (dd, 1H, J = 7.0 Hz and J’ = 16.0 Hz); 2.97 (dd, 1H, J = 8.0 Hz and J’ = 16.0 Hz); 3.54 (s, 3H); 4.09 (m, 1H); 5.02 (m, 1H); 5.13 (bd, 1H, J = 10.0 Hz); 6.73 (d, 1H, J = 1.0 Hz); 6.89 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.4; 17.7; 25.7; 26.5; 28.0 (3C); 32.4; 33.1; 39.5; 40.0; 80.2; 120.5; 123.2; 123.9; 127.0; 131.6; 136.5; 149.5; 171.5; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₂₀H₃₃N₂O₂ [M+H]⁺ : 333.2542; found: 333.2511.

5,9,13-Trimethyl-3-(1-methyl-1 H-imidazol-2-yl)-tetradeca-4,8,12-trienoic acid tert-butyl ester (13d):

Prepared according to general procedure E on 11d (1.4 g, 3.5 mmol) in anhydrous methanol (28 mL) with Mg (1.77 g, 74 mmol). After column chromatography (CH₂Cl₂/EtOAc : 8/2) 13d was isolated a yellow oil (1.0 g, 71% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.34 (s, 9H); 1.53 (bs, 3H); 1.55 (bs, 3H); 1.63 (bs, 3H); 1.73 (bs, 3H); 1.86-2.11 (m, 8H); 2.54 (dd, 1H, J = 7.0 Hz and J’ = 16.0 Hz); 2.95 (dd, 1H, J = 8.0 Hz and J’ = 16.0 Hz); 3.51 (s, 3H); 4.06 (m, 1H); 5.02 (m, 2H); 5.11 (d, 1H, J = 10.0 Hz); 6.68 (d, 1H, J = 1.0 Hz); 6.85 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.3; 16.7; 18.0; 26.0; 26.7; 27.0; 28.3 (3C); 33.7; 33.3; 39.8; 39.9; 40.2; 80.5; 120.7; 123.9; 124.4; 124.6; 127.2; 131.5; 135.5; 136.8; 149.7; 171.7; HRMS (ESI, MeOH) calculated for C₂₅H₄₁N₂O₂ [M+H]⁺: 401.3268; found: 401.3169.

General procedure F. Succinic moiety building

n-Butyllithium (1.6 M in hexane) was added to a cooled (−78°C) solution of diisopropyl amine in anhydrous THF (1.6 mL) under argon at -78°C. After 35 min, a solution of the saturated esters 13a-d in anhydrous THF was added dropwise under argon at -78°C. After 35 min at -78°C, bromoacetic acid tert-butyl ester 9 was added dropwise under argon. The solution was stirred for 4 h at -78°C and was slowly allowed to warm to room temperature. The reaction mixture was quenched with water, poured in EtOAc and washed three times with brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to afford compounds 15a-d.

2-{3-Methyl-1-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-yl]-but-2-enyl} succinic acid di-tert-butyl ester (15a):

Prepared according to general procedure F on 13a (303 mg, 0.8 mmol) in anhydrous THF (1 mL) with in situ prepared LDA (n-Butyl lithium (595 μL) and diisopropyl amine (134 μL), 0.95 mmol) in anhydrous THF (1.6 mL) and 10 (140 μL, 0.95 mmol). After column chromatography (Heptane/ EtOAc : 8/2) 15a was isolated as a pale yellow oil (0.23 g, 72% yield with 81% conversion). We evaluated the diastereomeric rate by HPLC on Symmetry^® column 250x4.6mm with H₂O/MeOH 20/80, flow: 1 mL/min (t_r= 24.94 min for the diastereoisomer 15aA, 47% and 29.57 min for the diastereoisomer 15aB, 53 %); ¹H NMR (300 MHz, CDCl₃) δ: Compound 15aA: 0 (s, 9H); 0.90 (t, 2H, J = 8.0 Hz); 1.38 (s, 9H); 1.40 (s, 9H); 1.69 (bs, 6H); 2.25 (dd, 1H, J = 4.0 Hz and J’ = 11.0 Hz); 2.56 (m, 1H); 3.32 (m, 1H); 3.44 (t, 2H, J = 8.0 Hz); 4.00 (m, 1H); 5.21 (s, 2H); 5.37 (d, 1H, J = 10.0 Hz); 6.86 (bs, 1H); 6.98 (bs, 1H); Compound 15aB: 0 (s, 9H); 0.88 (m, 2H); 1.28 (s, 9H); 1.41 (s, 9H); 1.69 (bs, 3H); 1.71 (bs, 3H); 2.56 (m, 2H); 3.19 (m, 1H); 3.43 (t, 2H, J = 8.5 Hz); 4.12 (t, 1H, J= 10.5 Hz); 5.12 (d, 1H, J = 11.0 Hz); 5.35 (d, 1H, J = 10.5 Hz); 5.37 (d, 1H, J = 11.0 Hz); 6.83 (bs, 1H); 6.94 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: Compound 15aA: -0.3 (3C); 18.1; 18.6; 26.2; 28.2 (3C); 28.4 (3C); 36.6; 39.0; 47.1; 66.3; 75.1; 80.6; 80.7; 119.9; 123.7; 128.1; 133.9; 148.9; 171.4; 173,3; Compound 15aB: -0.8 (3C); 18.1; 18.8; 26,3; 28.1 (3C); 28.4 (3C); 35.6; 37.9; 47.3; 66.2; 75.1; 80.7; 80.8; 119.5; 122.6; 127.8; 135.2; 149.5; 171.5; 173.3; HRMS (ESI, MeOH) calculated for C₂₆H₄₇N₂O₅Si [M+H]⁺: 495.3254; found: 495.3253.

2-[3-Methyl-1-(1-methyl-1H-imidazol-2-yl)-but-2-enyl]-succinic acid di-tert-butyl ester (15b):

Prepared according to general procedure F on 13b (327 mg, 0.86 mmol) in anhydrous THF (1.2 mL) with in situ prepared LDA (n-Butyl lithium (700 μL) and diisopropyl amine (155 μL), 1.1 mmol) in anhydrous THF (1.9 mL) and 9 (160 μL, 1.1 mmol). After column chromatography (Heptane/ EtOAc : 4/6) 15b was isolated as a pale yellow oil (223 mg, 76% yield, 91% conversion); ¹H NMR (300 MHz, CDCl₃) δ : 1.29 (s, 9H); 1.42 (s, 9H); 1.70 (bs, 3H); 1.72 (bs, 3H); 2.57 (m, 2H); 3.20 (td, 1H, J = 5.0 Hz and J’ = 9.0 Hz); 3.60 (s, 3H); 4.01 (dd, 1H, J = 9.0 Hz and J’ = 10.5 Hz); 5.30 (bd, 1H, J = 10.5 Hz); 6.71 (d, 1H, J = 1.0 Hz); 6.91 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 18.4; 25.9; 27.7 (3C); 28.1 (3C); 32.6; 35.3; 37.6; 46.6; 80.5 (2C); 120.1; 122.3; 127.2; 134.7; 148.7; 171.2; 173.0; HRMS (ESI, EtOAc) calculated for C₂₁H₃₅ N₂O₄ [M+H]⁺: 379.2597; found: 379.2610.

2-[3,7-Dimethyl-1-(1-methyl-1H-imidazole-2-yl)-octa-2,6- dienyl]-succinic acid di-tert-butyl ester (15c):

Prepared according to general procedure F on 13c (230 mg, 0.69 mmol) in anhydrous THF (1.0 mL) with in situ prepared LDA (n-Butyl lithium (600 μL) and diisopropyl amine (125 μL), 0.96 mmol) in anhydrous THF (1.5 mL) and 9 (130 μL, 0.89 mmol). After column chromatography (Heptane/ EtOAc : 6/4) 15c was isolated as a pale yellow oil (128 mg, 42% yield); ¹H NMR (300 MHz, CDCl₃) δ: 1.29 (s, 9H); 1.43 (s, 9H); 1.57 (bs, 3H); 1.65 (bs, 3H); 1.69 (d, 3H, J = 1.0 Hz); 2.05 (m, 4H); 2.52 (dd, 1H, J = 9.5 Hz and J’ = 16.0 Hz); 2.65 (dd, 1H, J = 4.0 Hz and J’ = 16.0 Hz); 3.22 (td, 1H, J = 4.0 Hz and J’ = 9.5 Hz); 3.59 (s, 3H); 4.01 (dd, 1H, J = 9.5 Hz and J’ = 10.5 Hz); 5.03 (m, 1H); 5.28 (bd, 1H, J = 10.5 Hz); 6.70 (d, 1H, J = 1.0 Hz); 6.91 (d, 1H, J = 1.0 Hz); ¹³C NMR (75 MHz, CDCl₃) δ: 16.7; 17.7; 25.7; 26.3; 27.8 (3C); 29.1 (3C); 32.6; 35.4; 37.5; 39.7; 46.6; 80.5 (2C); 120.1; 122.6; 123.9; 127.2; 131.8; 138.1; 148.8; 171.3; 173.0; HRMS (ESI, MeOH) calculated for C₂₆H₄₃N₂O₄ [M+H]⁺: 447.3223; found: 447.3235.

2-[3,7,1-Trimethyl-1-(1-methyl-1H-imidazol-2-yl)-dodeca-2,6,10-trienyl]-succinic acid di-tert-butyl ester (15d):

Prepared according to general procedure F on 13d (200 mg, 0.5 mmol) in anhydrous THF (1.2 mL) with in situ prepared LDA (n-Butyl lithium (410 μL) and diisopropyl amine (91 μL), 0.65 mmol) in anhydrous THF (1.5 mL) and 9 (95 μL, 0.65 mmol). After column chromatography (Heptane/ EtOAc : 8/2) 15d was isolated as a pale yellow oil (124 mg, 48% yield); ¹H NMR (300 MHz, CDCl₃) δ : 1.29 (s, 9H); 1.43 (s, 9H); 1.57 (bs, 3H); 1.59 (bs, 3H); 1.67 (bs, 3H); 1.70 (bs, 3H); 1.90-2.13 (m, 8H); 2.53 (dd, 1H, J = 9.0 Hz and J’ = 16.0 Hz); 2.66 (dd, 1H, J = 4.0 Hz and J’ = 16.0 Hz); 3.18-3.31 (m, 1H); 3.60 (s, 3H); 4.05 (dd, 1H, J = 9.5 Hz and J’ = 10.5 Hz); 5.05 (m, 2H); 5.31 (d, 1H, J = 10.5 Hz); 6.71 (bs, 1H); 6.92 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃) δ: 16.4; 17.1; 18.0; 26.0; 26.7; 27.1; 28.1 (3C); 28;4 (3C); 32.4; 35.7; 37.7; 40.0; 46.9; 80.8 (2C); 120.4; 122.7; 124.1; 124.7; 127.4; 131.6; 135.8; 138.7; 149.2; 171.6; 173.3; HRMS (ESI, MeOH/CH₂Cl₂) calculated for C₃₁H₅₁N₂O₄ ([M+H]⁺: 515.3849; found: 515.3834.

General procedure G. Deprotection of SEM-compounds

TFA (3.6 mL) was added drop by drop to a stirred solution of ester (0.24 mmol) in dichloromethane (1.8 mL) at room temperature. After 5 hours, the solvent was evaporated. The crude product was precipitated to afford the expected acid 12a, 14a or 2a.

3-(1H-imidazol-2-yl)-5-methyl-hexa-2,4-dienoic acid (12a):

General procedure G on 11a (91 mg, 0.24 mmol) afforded 12a as a white powder after precipitation with ether in methanol (27 mg, 36% yield); IR (CH₂Cl₂) ν: 3148; 3121; 2916; 1557; 1346 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ: 1.73 (bs, 3H); 1.95 (bs, 3H); 6.08 (bs, 1H); 6.11 (bs, 1H); 7.40 (bs, 2H); ¹³C NMR (75 MHz, CD₃OD) δ: 20.5; 27.3; 122.5; 123.1 (2C); 130.1; 134.2; 145.3; 145.4; 171.9; HRMS (ESI, MeOH) calculated for C₁₀H₁₂N₂O₂Na (M+Na)⁺: 215.0796; found: 215.0802.

3-(1H-imidazol-2-yl)-5-methyl-hex-4-enoic acid (14a):

General procedure G on 13a (108 mg, 0.28 mmol) afforded 14a as a white powder after precipitation with acetone (41 mg, 49% yield); IR (CH₂Cl₂) ν: 3147; 2978; 2916; 1666; 1180; 1137 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ: 1.57 (bs, 3H); 1.60 (bs, 3H); 2.63 (dd, 1H, J = 6.5 Hz and J’ = 17.0 Hz); 2.75 (dd, 1H, J = 7.7 Hz and J’ = 17.0 Hz); 4.25 (m, 1H); 5.11 (d, 1H, J = 9.5 Hz); 7.23 (bs, 2H); ¹³C NMR (75 MHz, CD₃OD) δ: 19.0; 26.7; 35.3; 39.7; 120.8 (2C); 121.6; 141.0; 151.6; 174.5; HRMS (ESI, MeOH) calculated for C₁₀H₁₅N₂O₂ [M+H]⁺: 195.1134, found: 195.1149.

2-[1-(1H-imidazol-2-yl)-3-methyl-but-2-enyl]-succinic acid (2a):

General procedure G on 15a (57 mg, 0.11 mmol) afforded 2a as a pale yellow oil (44 mg, 100% yield). HPLC on a Symmetry^® column 150 × 4.6 mm, elution with a gradient 100% H₂O, 1% TFA to 100% CH₃CN, 1% TFA; t_r= 10.1 min for the diastereoisomer 2aA and 10.7 min for the diastereoisomer 2aB; IR (CH₂Cl₂) ν: 3154; 2976; 2915; 1666; 1190 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ: diastereoisomer 2aA: 1.50 (bs, 3H); 1.66 (bs, 3H); 2.25 (dd, 1H, J = 6.5 Hz and J’ = 17.0 Hz); 2.56 (dd, 1H, J = 8.0 Hz and J’ = 17.0 Hz); 3.12 (m, 1H); 4.16 (dd, 1H, J= 6.0 Hz and J’ = 10.0 Hz); 5.19 (d, 1H, J = 10.0 Hz); 7.22 (bs, 1H); 7.23 (bs, 1H); diastereoisomer 2aB: 1.55 (bs, 3H); 1.60 (bs, 3H); 2.46 (dd, 1H, J = 5.0 Hz and J’ = 17.0 Hz); 2.56 (dd, 1H, J = 7.0 Hz and J’ = 17.0 Hz); 3.12 (m, 1H); 4.26 (t, 1H, J= 10.0 Hz); 5.06 (d, 1H, J = 10.0 Hz); 7.22 (bs, 2H); ¹³C NMR (75 MHz, CD₃OD) δ: diastereoisomer 2aA: 19.2; 26.9; 36.1; 39.9; 47.3; 118.4; 121.0 (2C); 142.3; 150.6; 175.6 (2C); diastereoisomer 2aB : 19.1; 26.7; 35.4; 40.1; 47.1; 120.5; 120.8 (2C); 143.5; 150.9; 175.6; 176.3; HRMS (ESI, MeOH) calculated for C₁₂H₁₇N₂O₄ [M+H]⁺: 253.1144; found: 253.1167.

General procedure H. Deprotection of the N-methyl-isoprenyl-compounds

Formic acid (5 mL) was added to ester (0.25 mmol) at room temperature. After 19 hours, the solvent was evaporated to afford the corresponding acid 12b, 14b or 2b.

5-Methyl-3-(1-methyl-1H-imidazol-2-yl)-hexa-2,4-dienoic acid (12b):

General procedure H on 11b (66 mg, 0.25 mmol) afforded 12b as a pale orange oil (52 mg, 100% yield). IR (CH₂Cl₂) ν: 3396; 2970; 2920; 2852; 1698; 1600; 1265 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ: 1.25 (bs, 3H); 1.90 (bs, 3H); 3.60 (s, 3H); 6.13 (bs, 1H); 6.39 (bs, 1H); 7.40 (bs, 1H); 7.46 (bs, 1H); 8.20 (s, 1H); ¹³C NMR (75 MHz, CD₃OD) δ: 18.6; 28.3; 34.6; 122.3; 124.0; 124.1; 132.9; 133.3; 145.5 (2C); 169.4; HRMS (ESI, MeOH) calculated for C₁₁H₁₄N₂O₂Na [M+Na]⁺: 229.0953; found: 229.0924.

5-Methyl-3-(1-methyl-1H-imidazol-2-yl)-hex-4-enoic acid (14b):

General procedure H on 13b (60 mg, 0.23 mmol) afforded 14b as a pale yellow powder (47 mg, 100% yield); IR (CH₂Cl₂) ν: 3375; 2960; 2915; 2857; 1713; 1274 cm⁻¹; ¹H NMR (400 MHz, CD₃OD) δ: 1.56 (bs, 3H); 1.58 (bs, 3H); 2.56 (dd, 1H, J = 6.0 Hz and J’ = 16.5 Hz); 2.74 (dd, 1H, J = 9.0 Hz and J’ = 16.5 Hz); 3.66 (s, 3H); 4.25 (m, 1H); 5.01 (bd, 1H, J = 9.5 Hz); 7.16 (bs, 2H); 8.04 (s, 1H); ¹³C NMR (75 MHz, CD₃OD) δ: 17.0; 24.5; 32.2; 33.5; 38.3; 119.1; 119.9; 122.6; 137.5; 149.3; 165.3; 173.5; HRMS (ESI, MeOH) calculated for C₁₁H₁₆N₂O₂Na [M+Na]⁺: 231.1109; found: 231.1078.

2-[3-Methyl-1-(1-methyl-1H-imidazol-2-yl)-but-2-enyl]-succinic acid (2b):

General procedure H on 15b (88.5 mg, 0.23 mmol) afforded 2b as a pale yellow oil (54 mg, 87% yield); IR (CH₂Cl₂) ν: 3376; 1572; 1567; 1557; 1385 cm⁻¹; ¹H NMR (300 MHz, CD₃OD) δ: 1.29 (bs, 3H); 1.30 (bs, 3H); 2.29 (d, 2H, J = 5.5 Hz); 2.84 (td, 1H, J = 5.5 Hz and J’ = 10.5 Hz); 3.43 (s, 3H); 4.05 (t, 1H, J = 10.5 Hz); 4.77 (d, 1H, J = 10.5 Hz); 6.96 (s, 2H); 7.64 (s, 1H); ¹³C NMR (75 MHz, CD₃OD) δ: 19.5; 26.7; 35.4; 36.1; 38.2; 46.6; 120.3; 120.4; 125.0; 142.5; 150.8; 165.8; 175.7; 176.4; HRMS (ESI, MeOH) calculated for C₁₃H₁₉N₂O₄ [M+H]⁺: 267.1345; found: 267.1318.

General procedure I. Deprotection of the N-methyl-geranyl and N-methyl-farnesyl compounds

The ester in toluene (3 mL) with silica gel (35-70 μm) was refluxed with vigorous agitation for 14 hours. After cooling, the reaction mixture was filtered over Celite^®, washed with CH₂Cl₂/MeOH 8/2 and concentrated. The crude product was purified by preparative TLC to afford the corresponding acid 12c-d, 14c-d or 2c-d.

5,9-Dimethyl-3-(1-methyl-1H-imidazol-2-yl)-deca-2,4,8-trienoic acid (12c):

Prepared according general procedure I on 11c (36 mg, 0.11 mmol) with SiO₂ (550 mg). After preparative TLC (CH₂Cl₂/MeOH : 9/1) 12c was isolated as an orange oil (18 mg, 60% yield); IR (CH₂Cl₂) ν: 3127; 2960; 2925; 2857; 1591; 1373 cm⁻¹; ¹H NMR (500 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 0.96 (bs, 3H); 1.29 (bs, 3H); 1.38 (bs, 3H); 1.81 (m, 4H); 3.17 (s, 3H); 4.81 (m, 1H); 5.76 (bs, 1H); 6.05 (bs, 1H); 6.66 (bs, 1H); 6.85 (bs, 1H); ¹³C NMR (125 MHz, CDCl₃/Pyridine-d₅ 1/2) δ: 16.7; 17.8; 25.7; 26.8; 33.0; 41.8; 121.2; 124.0; 125.4; 127.7; 128.1; 131.8; 146.1; 149.0; HRMS (ESI, MeOH) calculated for C₁₆H₂₁N₂O₂ [M-H]⁻: 273.1603; found: 273.1619.

5,9-Dimethyl-3-(1-methyl-1H-imidazol-2-yl)-deca-4,8-dienoic acid (14c):

Prepared according general procedure I on 13c (38 mg, 0.11 mmol) with SiO₂ (575 mg). After preparative TLC (CH₂Cl₂/MeOH : 9/1) 14c was isolated as a colorless oil (18 mg, 58% yield); IR (CH₂Cl₂) ν: 3392; 2960; 2915; 2857; 1574; 1392 cm⁻¹; ¹H NMR (500 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 1.52 (bs, 3H); 1.62 (bs, 3H); 1.79 (s, 3H); 1.95-2.09 (m, 4H); 2.85 (dd, 1H, J = 6.0 Hz and J’ = 16.0 Hz); 3.32 (dd, 1H, J = 8.0 Hz and J’ = 16.0 Hz); 3.50 (s, 3H); 4.30 (m, 1H); 5.03 (m, 1H); 5.27 (bd, 1H, J = 9.5 Hz); 6.76 (d, 1H, J = 1.0 Hz); 6.98 (d, 1H, J = 1.0 Hz); ¹³C NMR (125 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 16.3; 17.6; 25.6; 26.5; 32.2; 33.1; 39.5 (2C); 120.8; 124.1; 124.6; 126.7; 131.3; 136.5; 150.0; 174.4; HRMS (ESI, MeOH) calculated for C₁₆H₂₃N₂O₂ [M-H]⁻: 275.1760; found: 275.1743.

2-[3,7-Dimethyl-1-(1-methyl-1H-imidazole-2-yl)-octa-2,6-dienyl]-succinic acid (2c):

Prepared according general procedure I on 15c (55 mg, 0.12 mmol) with SiO₂ (620 mg). After preparative TLC (CH₂Cl₂/MeOH : 85/15) 2c was isolated as a colorless oil (13.5 mg, 34% yield); IR (CH₂Cl₂) ν: 3396; 2970; 2927; 2852; 1715; 1614; 1395; 1264 cm⁻¹; ¹H NMR (300 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 1.53 (bs, 3H); 1.64 (bs, 3H); 1.79 (bs, 3H); 2.15-2.02 (m, 4H); 3.10 (m, 2H); 3.62 (s, 3H); 3.88 (m, 1H); 4.54 (m, 1H); 5.06 (m, 1H); 5.55 (bd, 1H, J = 10.5 Hz); 6.82 (bs, 1H); 7.04 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 16.5; 17.7; 25.6; 26.4; 32.8; 34.3; 37.0; 39.7; 46.0; 120.8; 122.4; 124.1; 125.3; 131.4; 138.6; 149.5; 174.6; 175.4; HRMS (ESI, MeOH) calculated for C₁₈H₂₇N₂O₄ [M+H]⁺: 335.1971; found: 335.1940.

5,9,13-Trimethyl-3-(1-methyl-1H-imidazol-2-yl)-tetradeca-2,4,8,12-tetraenoic acid (12d):

Prepared according general procedure I on 11d (32.5 mg, 0.08 mmol) with SiO₂ (400 mg). After preparative TLC (CH₂Cl₂/MeOH : 9/1) 12d was isolated as a white powder (16.5 mg, 59% yield); IR (CH₂Cl₂) ν: 3353; 2960; 2922; 2846; 1693; 1253 cm⁻¹; ¹H NMR (500 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 1.29 (bs, 3H); 1.60 (bs, 3H); 1.61 (bs, 3H); 1.67 (bs, 3H); 1.97-2.05 (m, 2H); 2.05-2.18 (m, 6H); 3.50 (s, 3H); 5.14 (m, 2H); 6.12 (bs, 1H); 6.36 (bs, 1H); 7.00 (bs, 1H); 7.17 (bs, 1H); ¹³C NMR (125 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 16.0; 16.8; 17.7; 25.7; 26.5; 26.8; 33.1; 39.8; 41.8; 121.1; 123.7; 124.5; 125.3; 126.2; 127.9; 131.1; 135.7; 144.6; 144.8; 146.8; 167.6; HRMS (ESI, MeOH) calculated for C₂₁H₃₁N₂O₂ [M+H]⁺: 343.2386; found: 343.2372.

5,9,13-Trimethyl-3-(1-methyl-1 H-imidazol-2-yl)-tetradeca-4,8,12-trienoic acid (14d):

Prepared according general procedure I on 13d (50 mg, 0.125 mmol) with SiO₂ (680 mg). After preparative TLC (CH₂Cl₂/MeOH : 9/1) 14d was isolated as a colorless oil (29.0 mg, 67% yield); IR (CH₂Cl₂) ν: 3390; 2965; 2916; 2857; 1714; 1383 cm⁻¹; ¹H NMR (300 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 1.53 (bs, 3H); 1.55 (bs, 3H); 1.63 (bs, 3H); 1.78 (bs, 3H); 1.90-2.11 (m, 8H); 2.86 (dd, 1H, J = 5.5 Hz and J’ = 16.0 Hz); 3.42 (dd, 1H, J = 8.5 Hz and J’ = 16.0 Hz); 3.55 (s, 3H); 4.33 (m, 1H); 5.08 (m, 2H); 5.34 (d, 1H, J = 9.5 Hz); 6.82 (bs, 1H); 7.01 (bs, 1H); ¹³C NMR (75 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 16.2; 16.5; 17.8; 25.9; 26.6; 26,9; 32.9; 33.1; 39.7; 39.9 (2C); 121.1; 124.0; 124.1; 124.7; 125.5; 131.2; 135.3; 137.3; 150.0; 174.3; HRMS (ESI, MeOH) calculated for C₂₁H₃₁N₂O₂ (M-H)⁻: 343.2386; found: 343.2380.

2-[3,7,1-Trimethyl-1-(1-methyl-1H-imidazol-2-yl)-dodeca-2,6,10-trienyl]-succinic acid (2d):

Prepared according general procedure I on 15d (41 mg, 0.08 mmol) with SiO₂ (420 mg). After preparative TLC (EtOAc/MEK/HCO₂H/H₂O : 5/5/0.3/0.3 ) 2d was isolated as a colorless oil (14.5 mg, mixture of 2 diastereoisomer couples, 45% yield); IR (CH₂Cl₂) ν : 3428; 2922; 2852; 1722; 1712; 1384; ¹H NMR (300 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 2dA: 1.50 (bs, 3H); 1.53 (bs, 3H); 1.61 (bs, 3H); 1.76 (bs, 3H); 1.88-2.08 (m, 8H); 2.74 (dd, 1H, J = 7.0 Hz and J’ = 17.0 Hz); 3.16 (dd, 1H, J = 7.0 Hz and J’ = 17.0 Hz); 3.60 (s, 3H); 3.75 (m, 1H); 4.36 (dd, 1H, J = 4.5 Hz and J’ = 10.5 Hz); 5.05 (m, 2H); 5.43 (d, 1H, J = 10.5 Hz); 6.96 (bs, 1H); 7.04 (bs, 1H); 2dB: 1.54 (bs, 3H); 1.55 (bs, 3H); 1.63 (bs, 3H); 1.77 (bs, 3H); 1.90-2.16 (m, 8H); 3.19 (m, 2H); 3.82 (s, 3H); 4.15 (m, 1H); 4.81 (m, 1H); 5.09 (m, 2H); 5.76 (d, 1H, J = 10.5 Hz); 7.16 (bs, 2H); ¹³C NMR (75 MHz, CDCl₃/Pyridine-d₅ 1/1) δ: 2dA: 16.2; 16.9; 17.9; 25.9; 26.7; 27.0; 33.5; 35.2; 37.4; 39.8; 39.9; 45.7; 119.8; 122.1; 123.9; 124.1; 124.7; 131.1; 135.8; 141.3; 149.2; 174.6; 174.9; 2dB: 16.2; 16.8; 17.7; 25.7; 25.8; 26.9; 33.9; 34.2; 35.1; 39.7; 39.8; 45.3; 120.4; 121.1; 121.6; 123.6; 124.7; 131.1; 135.5; 141.1; 150.3; 174.7; 175.8; HRMS (ESI, MeOH) calculated for C₂₃H₃₃O₄N₂ [M-H]⁻: 401.2440; found : 401.2444.

Synthesis of (2E,4E,8E)-ethyl 5,9,13-trimethyltetradeca-2,4,8,12-tetraenoate (21):

Triethyl phosphonoacetate 20 (674 μL, 3.4 mmol, 1.5 equiv.) was added to a cooled (0°C) suspension of NaH (60% in oil, 155 mg, 3.9 mmol, 1.7 equiv.) in anhydrous THF (28 mL) under argon. After 10 min at 0°C and 30 min at room temperature, a solution of farnesal 4c (504 mg, 2.3 mmol) in anhydrous THF (4.5 mL) was added. The reaction was stopped after stirring 4h30 at room temperature by addition of HCl 10%. After standard work-up, the residue was purified by column chromatography on silica gel (Heptane/CH₂Cl₂ : 7/3) to afford 21 (482 mg, 74% yield) as a yellow oil. ¹H NMR (300 MHz, CDCl₃) δ: 1.28 (t, 3H, J = 7.0 Hz); 1.59 and 1.67 (bs, 6H); 1.59 (bs, 3H); 1.89 (bs, 3H); 1.93-2.11 (m, 4H); 2.15 (m, 4H); 4.19 (q, 2H, J = 7.0 Hz); 5.07 (m, 2H); 5.77 (d, 1H, J = 15.0 Hz); 5.98 (d, 1H, J = 11.5 Hz); 7.57 (dd, 1H, J = 11.5 Hz and J’ = 15.0 Hz). ¹³C NMR (75 MHz, CDCl₃) δ: 14,7; 16,3; 17,7; 18.0; 26.0; 26.5; 27.0; 40.0; 40.6; 60.4; 119.1; 123.6; 124.5; 131.7; 136.2; 141.3; 150.0; 168.0. HRMS (ESI, MeOH-CH₂Cl₂) calculated for C₁₉H₃₀O₂Na [M+Na]⁺: 313.2144; found : 313.2122.

Synthesis of (4E,8E)-ethyl 5,9,13-trimethyltetradeca-4,8,12-trienoate (24):

Ethyl acetate (0.8 mL, 8.2 mmol, 2.05 equiv.) was added to a cooled (-110°C) suspension of CuI (3.2 g, 16.8 mmol, 4.2 equiv.) in THF (30 mL) under argon. After few minutes, a LDA solution (0.82 M in THF, 8.2 mmol, 2.05 equiv.) previously prepared at -78°C, was added dropwise. After stirring 2h at -110°C, farnesyl bromide 23 (1.08 mL, 4 mmol) was added and the reaction mixture was stirred 2 h at -110°C. The reaction was quenched by saturated ammonium chloride. After standard work-up with heptane, the residue was purified by column chromatography on silica gel (Heptane/EtOAc : 97/3 then Heptane/CH₂Cl₂, 3/7) to afford 24 (642 mg, 69% yield) as a yellowish oil. ¹H NMR (300 MHz, CDCl₃) δ: 1.25 (t, 3H, J = 7.1 Hz); 1.59 and 1.68 (bs, 6H); 1.59 (bs, 3H); 1.62 (bs, 3H); 1.92-2.12 (m, 8H); 2.31 (m, 4H); 4.12 (q, 2H, J = 7.0 Hz); 5.10 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ: 14.6; 16.3; 18.0; 26.0; 23.9; 26.9; 27.1; 34.9; 40.0; 60.5; 122.7; 124.4; 124.7; 131.5; 135.3; 136.8; 173.7. HRMS (ESI, MeOH-CH₂Cl₂) calculated for C₁₉H₃₂O₂Na [M+Na]⁺: 315.2300; found: 315.2299.

Synthesis of (E)-diethyl 2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienylidene) succinate (26):

A solution of diethyl 2-(diethoxyphosphoryl)succinate 25 (500 mg, 1.6 mmol, 1.5 equiv.) in anhydrous THF (0.7 mL) was added to a cooled (0°C) suspension of NaH (60% in oil, 155 mg, 3.9 mmol, 1.7 equiv.) in anhydrous THF (0.7 mL) under argon. After 10 min at 0°C and 30 min at room temperature, a solution of farnesal 4c (234 mg, 1.1 mmol) in anhydrous THF (2.2 mL) was added. The reaction was stopped after stirring 4h30 at room temperature by addition of HCl 10%. After standard work-up, the residue was purified by column chromatography on silica gel (Heptane/CH₂Cl₂ : 4/6) to afford 26 (271 mg, 66% yield) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ: 1.24 (t, 3H, J = 7.0 Hz); 1.28 (t, 3H, J = 7.0 Hz); 1.59 (bs, 3H); 1.59 and 1.66 (bs, 6H); 1.91 (bs, 3H); 1.96 (m, 2H); 2.04 (m, 2H); 2.17 (m, 4H); 3.42 (s, 2H); 4.13 (q, 2H, J = 7.0 Hz); 4.21 (q, 2H, J = 7.0 Hz); 5.08 (m, 2H); 6.06 (d, 1H, J = 12.0 Hz); 7.64 (d, 1H, J = 12.0 Hz). ¹³C NMR (75 MHz, CDCl₃) δ: 14.5; 14.6; 16.4; 17.9; 18.0; 26.0; 26.7; 27.0; 32.8; 40.0; 41.1; 61.0; 61.1; 120.3; 122.2; 123.6; 124.6; 131.7; 136.2; 137.2; 151.0 168.1; 171.4. HRMS (MALDI-TOF, CH₂Cl₂) calculated for C₂₃H₃₇O₄ [M+H]⁺: 377.2691; found : 377.2701.

Synthesis of diethyl 2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)succinate (28):

Prepared according to general procedure E on 26 (60 mg, 0.16 mmol) in anhydrous methanol (1.3 mL) with Mg (67 mg, 2.8 mmol). After preparative TLC (Heptane/EtOAc : 9/1) 28 was isolated as a colorless oil (21 mg, 38% yield) as a mixture of ethyl-methyl and dimethyl esters (13 and 25% respectively); ¹H NMR (500 MHz, CDCl₃) δ: 1.26 (t, 3H, J = 7.0 Hz); 1.60 (bs, 18H); 1.69 (bs, 18H); 1.95-2.10 (m, 24H); 2.27 (dt, 3H, J= 8.0 and 14.5 Hz); 2.37 (dt, 3H, J= 6.5 and J’ = 14.5 Hz); 2.45 (m, 3H); 2.66-2.73 (m, 3H); 2.81-2.92 (m, 3H); 3.67 (s, 9H); 3.70 (s, 6H); 4.16 (q, 2H, J = 7.0 Hz); 5.09 (m, 9H). ¹³C NMR (75 MHz, CDCl₃) δ: 14.5; 16.3; 16.4; 16.5; 18.0; 26.0; 26.9; 27.1; 30.4; 35.2; 35.3; 40.1; 41.8; 41.9; 52.0; 52.2; 52.4; 60.9; 120.3; 120.4; 124.3; 124.7; 131.6; 135.6; 138.8; 138.9; 172.9; 173.0; 174.9; 175.4. HRMS (ESI, MeOH-CH₂Cl₂) calculated for C₂₁H₃₄O₄Na [M+Na]⁺: 373.2355; found : 373.2335 for the dimethyl ester.

General procedure J. Saponification of the farnesyl pyrophosphate analogues

A solution of sodium hydroxide 2M (1.25 mmol, 1.6 equiv.) was added to the ester in ethanol (3 mL) and the reaction mixture was heated at 70°C for 14 hours. After cooling, the reaction mixture was acidified with HCl 1M and extracted twice with ethyl acetate. The organic layers were pooled, washed with brine, dried and concentrated. The crude product was purified by reverse phase (C18) column chromatography (H₂O/CH₃CN : 80/20 to 0/100) to afford the corresponding acid 16, 17, 18 and 19.

(2E,4E,8E)-5,9,13-trimethyltetradeca-2,4,8,12-tetraenoic acid (16) :

Prepared according general procedure J on 21 (230 mg, 0.79 mmol). Compound 16 (86.9 mg, 42% yield) was obtained after purification as a colorless oil; ¹H NMR (300 MHz, CD₃OD) δ: 1.62, 1.64 and 1.69 (bs, 9H); 1.93 (bs, 3H); 1.97-2.14 (m, 4H); 2.22 (m, 4H); 5.12 (m, 2H); 5.78 (bd, 1H, J = 15.0 Hz); 6.07 (d, 1H, J = 11.5 Hz); 7.62 (dd, 1H, J = 11.5 Hz and J’ = 15.0 Hz). ¹³C NMR (75 MHz, CD₃OD) δ: 17.0; 18.2; 18.6; 26.8; 28.1; 28.6; 41.7; 42.1; 120.9; 125.4; 125.5; 126.2; 133.0; 137.7; 143.5; 151.9; 172.0. HRMS (MALDI-TOF, CH₃OH-CH₂Cl₂) calculated for C₁₇H₂₆O₂Na [M+Na]⁺: 285.1830; found : 285.1839.

(4E,8E)-5,9,13-trimethyltetradeca-4,8,12-trienoic acid (17) :

Prepared according general procedure J on 24 (253 mg, 0.87 mmol). Compound 17 (38.9 mg, 17% yield) was obtained after purification as a colorless oil; ¹H NMR (300 MHz, CDCl₃) δ: 1.60 (bs, 6H); 1.69 (bs, 3H); 1.69 (bs, 3H); 1.93-2.14 (m, 8H); 2.28-2.44 (m, 4H); 5.11 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ: 16.4; 18.0; 26.0; 26.3; 26.9; 27.1; 34.6; 40.0; 40.1; 122.3; 124.4; 124.7; 131.6; 135.5; 137.4; 180.0. HRMS (ESI, CH₃OH) calculated for C₁₇H₂₇O₂ [M-H]⁻: 263.2011; found : 263.1971.

(E)-2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienylidene)succinic acid (18) :

Prepared according general procedure J on 26 (52 mg, 0.14 mmol). Compound 18 (29.6 mg, 67% yield) was obtained after purification as a white amorphous solid; ¹H NMR (300 MHz, CDCl₃) δ: 1.60 (bs, 3H); 1.61 (bs, 3H); 1.69 (bs, 3H); 1.95 (bs, 3H); 1.97-2.09 (m, 4H); 2.13-2.27 (m, 4H); 3.47 (s, 2H); 5.09 (m, 2H); 6.10 (bd, 1H, J = 12.0 Hz); 7.78 (d, 1H, J = 12.0 Hz). ¹³C NMR (75 MHz, CDCl₃) δ: 16.4; 17.0; 18.1; 26.0; 26.7; 27.0; 33.3; 40.0; 41.2; 120.2; 120.6; 123.5; 124.6; 131.7; 136.4; 139.9; 153.4; 173.9; 178.0. HRMS (ESI, CH₃OH) calculated for C₁₉H₂₇O₄ [M-H]⁻: 319.1909; found : 319.1895.

2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trienyl)succinic acid (19) :

Prepared according general procedure J on 28 (20 mg, 0.06 mmol). Compound 19 (4.6 mg, 25% yield) was obtained after purification as a colorless oil; ¹H NMR (300 MHz, CDCl₃) δ: 1.63 (bs, 6H); 1.66 (bs, 3H); 1.70 (bs, 3H); 1.98-2.17 (m, 8H); 2.31-2.41 (m, 2H); 2.46 (dd, 1H, J = 5.0 Hz and J’ = 16.5 Hz); 2.61 (dd, 1H, J = 9.0 Hz and J’ = 16.5 Hz); 2.75-2.82 (m, 1H); 5.10-5.20 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ: 17.0; 17.2; 18.6; 27.7; 28.4; 28.7; 32.0; 37.5; 41.7; 41.8; 44.3; 123.0; 126.2; 126.4; 133.0; 137.0; 139.9; 178.1; 180.1. HRMS (ESI, CH₃OH-CH₂Cl₂) calculated for C₁₉H₂₉O₄ [M-H]⁻: 321.2066; found : 321.2045.

Biological assays

Yeast FTase assay

Assays were realized on 96-well plates, prepared with Biomek NKMC and Biomek 3000 from Beckman Coulter and read on Wallac Victor fluorimeter from Perkin-Elmer. Per well 20 μL of farnesyl pyrophosphate (10 μM) was added to 180 μL of a solution containing 2 μL of varied concentrations of 11-15a-d, 2a-d and 16-19 (dissolved in DMSO) and 178 μL of a solution composed by 0.1 mL of partially purified recombinant yeast FTase (2.2 mg/mL) and 7.0 mL of Dansyl-GCVLS peptide (in the following buffer: 5.8 mM DTT, 6 mM MgCl₂, 12 μM ZnCl₂ and 0.09% (w/v) CHAPS, 53 mM Tris/HCl, pH 7.5). Then the fluorescence development was recorded for 15 min (0.7 s per well, 20 repeats) at 30 °C with an excitation filter at 340 nm and an emission filter at 486 nm. Each measurement was realized twice as duplicate or triplicate.

Production of recombinant human FTase

To obtain recombinant heterodimeric human FTase in Escherichia Coli, the two subunits were expressed as a translationally coupled operon under transcriptional control by the bacteriophage T7 in the plasmid pET-DUET-1 (Novagen). Translational coupling was achieved by placing the alpha subunit coding sequence upstream of the ß subunit coding sequence. A (His)₆-tag was fused to the C-terminal part of the alpha subunit, allowing affinity purification of the heterodimer from E. Coli extracts.

The transformed strain BL21 RIL(DE3) was grown at 37°C in 14 liters of LB-rich medium with 50 μg.mL⁻¹ ampicillin and induced at a cell density of 0.6 A₆₀₀ with 1 mM isopropyl-ß-D-thiogalactopyranoside. After 12-15 h, the cells were collected by centrifugation. A fraction (22.5 g) was diluted in 50 mL buffer A (25 mM Tris,HCl pH 7.4, 0.5 M NaCl) plus protease inhibitors and lysed with Dyno mill (0.2 mm) 25 times for 30 s at 4 °C, 4500 rpm. The lysate solution and rinsing (60 mL) were pooled and centrifuged at 10000 rpm (JA 25 50) for 20 min. The supernatant was loaded on a His crude column, (5 mL, Amersham) and eluted stepwise with buffer A and buffer B (25 mM Tris,HCl pH 7.4, 0.5 M NaCl, 0.5M imidazole): 0-4%B, 4-25%B, 25-100%B. The most active fractions (~80% pure) eluted at 25% buffer B were pooled, concentrated by ultrafiltration (YM30 filter) and stored in buffer A with 5% glycerol at 4°C.

Human FTase assay

Assays were realized on 96-well plates, as described for yeast FTase but Octyl-D-glucopyranoside (0.18%) was used instead of CHAPS and the solution contains 5 μL of partially purified human FTase (1.5 mg/mL) in 1 mL peptide solution.

The kinetic experiments have been realized under the same conditions either with FPP as varied substrate with constant concentration of Dns-GCVLS of 2 μM or with Dns-GCVLS as varied substrate with constant concentration of FPP of 5 μM. Non linear regression were made by KaleidaGraph 4.03 software.

Assay for in vitro inhibition of P. falciparum growth

The chloroquine-resistant strain FcB1/Colombia of Plasmodium falciparum was maintained in vitro on human erythrocytes in RPMI 1640 medium supplemented by 8% (v/v) heat-inactivated human serum, at 37°C, under an atmosphere of 3% CO₂, 6% O₂, 91% N₂ [38]. In vitro drug susceptibility assays were performed using a modification of the semi-automated microdilution technique of Desjardins et al. [39]. Drugs were prepared in DMSO at a 10 mM concentration. Compounds were serially diluted two-fold with 100 μL culture medium in 96-well plates. Asynchronous parasite cultures (100 μL, 1% parasitaemia and 1% final hematocrite) were then added to each well and incubated for 24 h at 37°C prior to the addition of 0.5 μCi of [³H]-hypoxanthine (GE Healthcare, France, 1 to 5 Ci·mmol/mL) per well. After a further incubation of 24 h, plates were frozen and thawed. Cell lysates were then collected onto glass-fiber filters and counted in a liquid scintillation spectrometer. The growth inhibition for each drug concentration was determined by comparison of the radioactivity incorporated in the treated culture with that in the control culture maintained on the same plate. The concentration causing 50% growth inhibition (IC₅₀) was obtained from the drug concentration-response curve and the results were expressed as the mean values ± standard deviations determined from several independent experiments.

Results and Discussion

Chemistry

A convergent synthesis consisting on the addition of the succinic moiety to the imidazole ring branched with the prenyl group has first been explored (Scheme 2). The synthetic pathway was undertaken for compound 2a with one isoprenyl unit (n = 0) and a free amine. Our starting material was the N-SEM-imidazole 3a, easily obtained by classical protection of the N-1 imidazole with SEM-Cl.[13]

Scheme 2.  Convergent synthesis pathway. a) nBuLi, THF, −78°C, 50 min then addition of 4a, −78°C, 20 min to RT 1h (68%); b) MnO₂, THF, 0°C, 2h (100%); c) 7, THF, Zn, ultrasounds, 40°C, 4h30 (63%).

The deprotonation with nBuLi of the N-1 protected imidazole ring 3a [14] followed by the addition on the 3-methylcrotonaldehyde 4a led to the allylic alcohol 5a which was easily oxidized by MnO₂ [15]. The direct addition of the carbanion on the methyl ester of 3-methylcrotonic acid was considered but the high reactivity of the formed ketone 6a to nucleophilic addition led to a mediocre yield (26%). To introduce the succinic moiety two reaction types were applied. In the classic Horner-Wadsworth-Emmons procedure, the diethyl phosphonosuccinic acid diethyl ester [16] didn’t react with the α,β-insaturated ketone 6a. Whereas the Reformatsky reaction between the 2-bromo-succinic acid dimethyl ester 7 and the α,β-insaturated ketone 6a in presence of activated zinc in an ultrasound bath [17] led to the lactone 8. This lactone resulted from the attack of the alcoholate formed during the Reformatsky reaction [18]. Our efforts to avoid the formation of the lactone 8 or to open it were unsuccessful.

Therefore, we decided to build up the succinic motif from two acetic units (Scheme 3). The first unit was introduced by a Reformatsky reaction between the ketones 6a-d and the tert-butyl bromoacetate 9. Then, POCl₃ in pyridine [19] dehydrated efficiently the tertiary hydroxyesters 10a-d to the corresponding conjugated Z-alkenes 11a-d which were regioselectively reduced by magnesium in dry methanol [20] in good yields. Finally, the second acetic unit was introduced by alkylation at the α-position of the saturated esters 13a-d using LDA and the tert-butyl bromoacetate 9.

Scheme 3.  Synthesis of farnesyl acids. a) NaH and 20, THF, 0°C, 10 min then 30 min RT, then addition of 4c, RT, 4h30 (74%); b) Mg, MeOH, RT, 4h (38%); c) CuI, LDA, THF, 2h, −110°C, then addition of 23, −110°C, 2h (69%); d) NaH and 25, THF, 0°C, 10 min then RT, 40 min, then addition of 4c, RT, 2h15 (66%); e) LDA, THF, −78 °C, 35 min then addition of 27, −78°C; f) NaOH 2M, EtOH, 70 °C, 15h (77-100%).

The deprotection step of 11a-d, 13a-d and 15a-d appeared more complex than expected. Indeed, the final double bond of the prenyl group presented a higher reactivity in the case of the geranyl (n = 1) and farnesyl (n = 2) moieties. TFA [21] and formic acid [22] provided the expected deprotected compounds 12a-b, 14a-b and 2a-b from the compounds a (R = SEM, R’ = tBu, n = 0) and b (R = CH₃, R’ = tBu, n = 0) respectively. However, in the case of the compounds c (R = CH₃, R’ = tBu, n = 1) and d (R = CH₃, R’ = tBu, n = 2), only silica in refluxing toluene [23] provided the desired compounds 12c-d, 14c-d and 2c-d. In the TFA and formic acid conditions, we observed the addition of the carboxylate group of the acidic reagent on the terminal double bond of compounds c and d [24].

To study the influence of the imidazole ring on the activity of compounds 2d, 12d and 14d, we also synthesized the corresponding FPP analogues 16, 17, 18 and 19 according the synthetic pathway described in Scheme 4. Compound 21 was obtained by a Horner-Wadsworth-Emmons reaction with triethylphosphonoacetate 20 on the aldehyde 4c. Reduction of the double bond with magnesium in dry methanol did not afford the desired compound 24 in sufficient yield. This derivative was finally obtained by a SN₂ reaction of the anion of ethyl acetate 22 on farnesyl bromide 23 [26]. Contrary to compounds 13, it was impossible to obtain compound 28 by alkylation of 24 with bromo acetate. Therefore we synthesized compound 26 by a Horner-Wadsworth-Emmons reaction with tetraethylphosphonosuccinate 25 [10] and reduced the conjugated double bond by magnesium in dry methanol. As observed with compound 21, the yield was moderate and we obtained a mixture of transesterified compounds 28. It is likely that the presence of the imidazole ring in compounds 12 favored the reduction of this double bond by additional conjugation. Compounds 21, 24, 26 and 28 were saponified in warm sodium hydroxide in ethanol to afford the corresponding acids 16, 17, 18 and 19 in good to excellent yields.

Scheme 4.  Second synthetic pathway. a) nBuLi, THF, −78°C, 45 min then addition of 4a-c, −78°C, 20 min to 2h30 to RT 1h (68-100%); b) MnO₂, THF, 0°C, 2h (80-100%); c) 9, THF, Zn, ultrasounds, 40°C, 5h (51-79%); d) POCl₃, Pyridine, 0°C to RT 14h (75-85%); e) for a (R = SEM, n = 0) TFA, CH₂Cl₂, RT, 5h, for b (R = CH₃, n = 0) HCO₂H, RT, 19h and for c (R = CH₃, n = 1) and d (R = CH₃, n = 2) SiO₂, toluene, reflux, 14h; f) Mg, MeOH, RT, 3h (62-80%); g) LDA, THF, −78°C, 35 min then addition of 9, −78°C, 4h (42-72%).

Biological Evaluation

Inhibitory activity of compounds 11d, 12a-d, 13d, 14a-d, 15d and 2a-d was evaluated in a fluorescence-based assay [27] against yeast and human recombinant FTases [28, 29]. Results are summarized in Tables 1 and 2. The inhibition rate was measured at 10⁻³M for the a and b series (Table 1) and IC₅₀ was estimated for compounds which presented measurable activities in the c and d series and compared with the chaetomellic acid A activity measured under our conditions (Table 2).

Table 1.  Activity on yeast FTase of a and b compounds (n = 0).

Compounds	R	Inhibition at 10^−3M	Compounds	R	Inhibition at 10^−3M
12a	H	8%	12b	Me	8%
14a	H	21%	14b	Me	25%
2a	H	Inactive	2b	Me	Inactive

Table 2.  Activity on recombinant yeast and human FTase of c and d compounds (R = CH₃ and FPP analogues).

Compounds	n	IC50yeast FTase	IC50human FTase
Chaetomellic acid^a		0.34 ± 0.03 μM	0.175 ± 0.01 μM
11d	2	600 ± 100 μM	300 ± 60 μM
12c	1	inactive	ND^b
12d	2	130 ± 15 μM	175 ± 15 μM
13d	2	inactive	inactive
14c	1	inactive	ND
14d	2	700 ± μM	500 ± 40 μM
15d	2	ND	700 ± 65 μM
2c	1	850 ± 50 μM	590 ± 45 μM
2d	2	28 ± 3 μM	12 ± 0.8 μM
16	2	40 ± 5 μM	130 ± 15 μM
17	2	42 ± 7 μM	170 ± 20 μM
18	2	16 ± 1.2 μM	6.5 ± 1.0 μM
19	2	2.5 ± 0.5 μM	5.4 ± 0.9 μM

a: IC₅₀ measured under our conditions.

b: not determined.

The very similar results obtained in both a and b series (Table 1) indicate that the presence of a hydrogen or a methyl on the N-1 position of the imidazole ring does not change the interaction with FTase. Therefore, only the N-methylimidazole derivatives of the c and d series were studied. As expected, the farnesyl compounds (series d) are much more active than their geranyl analogues (series c). Indeed, they are able to realize more interactions with the hydrophobic pocket. Concerning the acidic part, the succinic moiety (2d) shows its superiority to mimic the FPP pyrophosphate compared to a monoacid unit (12d, 14d). Generally, the presence of the free carboxylic acid (12d, 14d and 2d) led to a better inhibition. Concerning the ester analogues (11d, 13d and 15d) it is surprising that the unsaturated monoester 11d is the most active compound whereas the corresponding acid 12d is less active than the succinic analogue 2d. However, the ester activities are weak and it would not be meaningful to draw conclusions from these results. The activity of compounds 16-19 also shows that a diacid is more active than a monoacid but here the presence of the double bond has little influence on the activity even if the more active compound is the succinic acid 19. Finally, these results show that the presence of the imidazole ring unexpectedly does not enhance the activity.

In order to explain this result, kinetic studies were performed with compound 2d to precise the binding mode of our imidazole containing compounds.

As shown in Figure 3, compound 2d is competitive to FPP (K_i^app = 10.5 μM) like chaetomellic acid A (K_i^app = 0.19 μM measured in our assay conditions) and uncompetitive to the peptidic substrate DnsGCVLS (K_i’^app = 160 μM). Therefore, our imidazole containing compounds are unable to bind to the peptide binding site. The imidazole ring apparently can not interact efficiently with the zinc atom as was initially planned. This explains the similar activity we observed for the succinic analogues 2d and 19.

Figure 3.  Kinetic experiments on compound 2d. a) double reciprocal with DnsGCVLS as the varied substrate and fixed concentration of FPP. Concentrations of 2d were: 200μM (▪), 100μM (⦿), 50μM (

), 25μM (⧫), 0μM (•); b) double reciprocal with FPP as the varied substrate and fixed concentrations of DnsGCVLS. Concentrations of 2d were: 20μM (▪), 10μM (⧫), 5 μM (

), 0μM (•); c) K_i^,app measured from competition with DnsGCVLS; d) K_i^,app measured from competition with DnsGCVLS; d) K_i,^app measured from competition with FPP.

Two reasons can be put forward: firstly, the distance between the farnesyl moiety and the imidazole ring is too short to allow zinc binding or/and the imidazole ring might not be well oriented to achieve this interaction with the zinc atom. Thus, to improve the activity of compound 2d, the imidazole ring could be attached further from the farnesyl group or/and functionalized on another position.

Though less active than chaetomellic acid A, we have synthesized new succinic analogues of FPP with IC₅₀ and K_i in the micromolar range. When compared to the other dicarboxylic analogues of FPP described in the literature, these compounds are among the most active ones [30–33].

All our derivatives of the d series were evaluated for their ability to block the in vitro growth of the intraerythrocytic form of Plasmodium falciparum. Only compound 15d displayed activity against P. falciparum (IC₅₀ = 30 ± 2 μM). It is generally observed that the esters are more active than the corresponding acids in cellular assays, because of differences in cell penetration. These results are in good agreement with the enzymatic assays where the succinyl derivative displayed the best activity.

Conclusion

We have synthesized twelve new compounds (type 2) formed by an imidazole ring, a prenyl group and an acidic moiety. Some of them revealed encouraging inhibitory activities in the micromolar range against human or yeast FTase as well as against the growth of Plasmodium falciparum. Four analogues devoid of the imidazole ring have also been synthesized and evaluated showing a similar activity on FTase. These derivatives are not able to bind to both substrate binding sites but they are competitive FPP inhibitors with a rather good activity in this field. The structural difference with Chaetomellic acid A, relies on the absence of a double bond between the two carboxyl groups and a methyl on this double bond. It would be worth investigating other modifications on these analogues to find new more potent FPP inhibitors.

In our course of designing bisubstrate inhibitors, we have synthesized two series of compounds. The first series of compounds (type 1) were only CaaX inhibitors and the series reported herein are only FPP inhibitors. Therefore a combination of both series would likely lead to the expected bisubstrate inhibitors. This is currently under investigation as well as modifications of the connecting position on the imidazole ring and of the length between the imidazole moiety and the FPP analogue part of the molecule.

Acknowledgements

The authors thank J.-B. Créchet for the generous gift of the E. Coli strain expressing the yeast FTase, Dr. J. Ouazzani and P. Lopez for the production and purification of recombinant yeast and human FTases and Pr P. Grellier for evaluation of P.falciparum growth inhibition and Research Minister and CNRS for financial support.

Declaration of interest: The authors report no conflicts of interest.