Methyl (S)-3-((1r*,3R*)-3-((1H-indol-3-yl)methyl)cyclobutane-1-carboxamido)-2-(phenylsulfonamido)propanoate

Abstract

The cyclobutane ring has attractive properties as a scaffold in medicinal chemistry but is underused, potentially due to the limited methods available for synthesising highly functionalised cyclobutanes. This short note describes the synthesis of the 1,3-cis disubstituted cyclobutane, methyl (S)-3-((1r*,3R*)-3-((1H-indol-3-yl)methyl)cyclobutane-1-carboxamido)-2-(phenylsulfonamido)propanoate using a one-pot thermal stepwise cycloaddition reaction and its characterisation by NMR spectroscopy and HRMS.

1. Introduction

The cyclobutane ring has attractive properties as a scaffold in medicinal chemistry, such as structural rigidity replacing alkane chains, reduced planarity compared to aromatic rings and improved metabolic stability or solubility [1,2]. However, there are limited methods available for synthesising highly functionalised cyclobutanes.

The integrin family of cell surface adhesion receptors are a popular target for drug discovery [3,4]. The subfamily of integrins which bind to the Arg-Gly-Asp (RGD) tripeptide sequence in extracellular matrix proteins have been extensively investigated with aims to develop anti-thrombotic [5], anti-fibrotic [6,7,8], and anti-angiogenic [9] agents and molecules targeting other processes in cancer progression [10,11,12]. Most molecules targeting RGD-binding integrins have been designed to mimic the RGD tripeptide sequence [3,13]. Elarofiban was developed from the KQAGD sequence in fibrinogen [14]; however, its pharmacophore is essentially identical to a RGD mimetic. Snake venom disintegrins have proven to be a useful source of biologically active peptide sequences targeting integrins [15,16]. Disintegrin CC8 is a heterodimer containing both RGD and WGD tripeptide sequences; the presence of WGD significantly enhances its anti-integrin activity [17]. As part of a larger project developing cyclobutane-based tripeptide mimetics as potential integrin antagonists [18,19], we aimed to synthesise an analogue containing a W-mimetic sidechain (Figure 1).

2. Results

The synthetic route to methyl (S)-3-((1r*,3R*)-3-((1H-indol-3-yl)methyl)cyclobutane-1-carboxamido)-2-(phenylsulfonamido)propanoate 1 is shown in Scheme 1. The WG-mimetic skeleton in compound 1 was obtained by a telescoped 4-step reaction sequence starting with protected aldehyde 2. Reaction with diethylamine forms an enamine which adds to methyl acrylate in a thermal stepwise [2 + 2] cycloaddition to yield an intermediate cyclobutane [20]. The intermediate is quaternised with methyl iodide then base-mediated elimination generates the cyclobutene which is easily isolated by flash column chromatography. Cyclobutene 3 was obtained in moderate (40%) yield accompanied by aldehyde-containing side-products which may have resulted from the conjugated cyclobutene being unstable to reaction with environmental water leading to ring opening. Therefore, it was identified by ¹H NMR (characteristic cyclobutene alkene proton at 6.87 ppm) then immediately hydrogenated to give cyclobutane 4. The stereochemistry of 4 was assigned by comparison with other cyclobutanes—the H-1 proton adjacent to the ester appears as a triple triplet at 2.99 ppm with two large couplings of 8.6 and 9.6 Hz (see Supplementary Materials for spectrum) which is characteristic of 1,3-cis substituents. The H-1 proton in 1.3-trans-disubstituted cyclobutanes appears slightly downfield (~3.05 ppm) as a narrower and generally more complex multiplet [18].

To allow coupling with the D-sidechain mimetic 6, the methyl ester of 4 was hydrolysed under basic conditions. The Boc-protecting group was unstable to the hydrolysis conditions and the reaction yielded fully deprotected carboxylic acid 5. Successful coupling was indicated by the presence of characteristic signals in the ¹H NMR arising from the functionalised propionic acid sidechain. A broad triplet at 5.88 ppm integrating as one hydrogen and coupling to the two beta hydrogens is consistent with the amide proton in compound 1 and the adjacent broad doublet at 5.78 ppm is consistent with the sulfonamide proton coupling to the one alpha hydrogen. These two signals are consistent with other compounds with 3-amino-2-arylsulfonylamino-propionic acid-based sidechains synthesised in related work [18,19]. Amide and ester carbonyls (175.9 and 170.1 ppm) were also observed in the ¹³C NMR and the expected molecular ion ([M + H]⁺, 470) by mass spectrometry. The indole signals in aromatic region of the spectrum of 1 are similar to those of the starting acid 5 suggesting no undesired functionalisation of this ring has occurred. The yield of 1 has not been optimised.

3. Discussion

The synthesis of substituted cyclobutane 1 extends information available on the substrate scope of [2 + 2] stepwise synthesis of cyclobutenes and cyclobutanes. The intermediate cyclobutene 3 appears less stable than molecules with longer arginine mimetic sidechains in the allylic position synthesised previously [18] but is substantially more stable than cyclobutenes with sp² hybridised carbon attached directly to this position [21]. The full W-mimetic sidechain is introduced at the start of the synthesis allowing a short synthesis of a relatively complex disubstituted cyclobutane. Similar cyclobutanes with a range of heterocyclic substituents may find wider applications in medicinal chemistry.

4. Materials and Methods

4.1. General

Chemical reagents and anhydrous solvents were obtained from Sigma-Aldrich (Gillingham, Dorset, UK) and used without further purification. All other solvents were supplied by VWR (Lutterworth, Leicestershire, UK). Unless otherwise stated, reactions were carried out in anhydrous solvent and were not air sensitive. Petroleum ether (PE) refers to the fraction boiling between 60 and 80 °C. Flash chromatography was carried out on silica gel (Merck 9385 Kieselgel 60 (230–400 ASTM) (VWR) or Davisil 60A, 40–63 μm (Fisher Scientific (Loughborough, Leicestershire, UK))). Analytical TLC was carried out on 0.25 mm thick aluminium plates precoated with Merck Kieselgel F₂₅₄ silica gel (VWR) and visualised by UV and aqueous alkaline potassium permanganate solution. Preparative TLC was carried out on Analtech silica plates with UV245 indicator (Sigma-Aldrich, Gillingham, Dorset, UK). NMR spectra were recorded on a Bruker DPX400 spectrometer (Coventry, UK). Low resolution mass spectra were recorded on a Micromass Quattro Ultima spectrometer (Waters, Milford, MA, USA). High resolution mass spectra were recorded on a LTQ Orbitrap XL spectrometer (Thermofisher, Altrincham, Cheshire, UK) at the EPSRC National Mass Spectrometry Service Facility, Swansea University, UK. The synthesis of aldehyde 2 has been described by Roman et al. [22]. (S)-3-Amino-2-benzenesulfonylamino-propionic acid methyl ester 6 was synthesised as previously described [18].

4.2. Synthetic Methods

4.2.1. Tert-butyl 3-((3-(methoxycarbonyl)cyclobut-2-enyl)methyl)-1H-indole-1-carboxylate 3

To a stirred solution of tert-butyl 3-(3-oxopropyl)-1H-indole-1-carboxylate (888 mg, 3.25 mmol) in acetonitrile (18 mL), diethylamine (0.673 mL, 476 mg, 6.51 mmol) and K₂CO₃ (898 mg, 6.51 mmol) were added and the reaction mixture was stirred at room temperature for 2.15 h. Methyl acrylate (0.741 mL, 560 mg, 6.51 mmol) was added and the reaction mixture was stirred for a further 45 h. The mixture was filtered through Celite and the solvent was removed under reduced pressure. The residue was redissolved in acetonitrile (21 mL), methyl iodide (1.01 mL, 2.31 g, 16.25 mmol) was added and the resulting solution was stirred at room temperature for 2 h. The solution was then concentrated in vacuo, the residue was redissolved in chloroform (21 mL), DBU (0.486 mL, 495 mg, 3.25 mmol) was added and the solution heated to 80 °C for 21 h. The reaction mixture was then concentrated in vacuo and purified by flash column chromatography (EtOAc:PE 1:9 → 2:5) to yield tert-butyl 3-((3-(methoxycarbonyl)cyclobut-2-enyl)methyl)-1H-indole-1-carboxylate (447 mg, 40%) as a yellow oil. Rf 0.71 (EtOAc:PE 3:7). δH (400 MHz, CDCl₃) 8.13 (1H, brd, J = 7.6 Hz), 7.51 (1H, d, J = 7.6 Hz), 7.36–7.40 (1H, m), 7.32 (1H, dt, J = 5.1, 8.6 Hz), 7.24 (1H, dt, J = 1.0, 8.1 Hz), 6.87 (1H, d, J = 1.0 Hz, H-2), 3.74 (3H, s, OCH₃), 3.12 (1H, dddt, J = 1.0, 1.5, 4.5, 8.1 Hz, H-3), 2.91 (1H, dd, J = 4.2, 13.6 Hz, H-4), 2.88 (2H, brd, J = 8.1 Hz, CH₂Indole), 2.42 (1H, dd, J = 1.5, 13.6 Hz, H-4), 1.67 (9H, s, (CH₃)₃). Due to concerns over stability, the product was used in the next step without further characterisation.

4.2.2. Tert-butyl 3-(((1s*,3r*)-3-(methoxycarbonyl)cyclobutyl)methyl)-1H-indole-1-carboxylate 4

To a stirred solution of tert-butyl 3-((3-(methoxycarbonyl)cyclobut-2-enyl)methyl)-1H-indole-1-carboxylate (229 mg, 0.672 mmol) in EtOAc (20 mL), 10% Pd/C (20 mg) was added and the reaction mixture was degassed × 3 and stirred under 1 atm H₂ for 22 h. The reaction mixture was filtered through 1 cm SiO₂ and concentrated in vacuo and the residue resubjected to the reaction conditions a further 2 times (72 h total reaction time, 3 batches of catalyst) to yield the title compound (230 mg, 100%) as a yellow oil. Rf 0.71 (EtOAc:PE 3:7). δH (400 MHz, CDCl₃) 8.10 (1H, br), 7.50 (1H, d, J = 7.1 Hz), 7.26–2.32 (2H, m), 7.22 (1H, dt, J = 1.0, 8.6 Hz), 3.68 (3H, s, OCH₃), 2.99 (1H, tt, J = 8.6, 9.6 Hz, H-1), 2.77 (2H, d, J = 7.6 Hz, CH₂Indole), 2.63 (1H, tdd, J = 7.6, 8.1, 9.1 Hz, H-3), 2.36 (2H, dddd, J = 2.5, 8.1, 8.6, 9.1 Hz, H-2,4), 2.03 (2H, dddd, J = 2.5, 8.1, 8.6, 9.1 Hz, H-2,4), 1.66 (9H, s, C(CH₃)₃); δC (100 MHz, CDCl₃) 175.7 (C), 149.8 (C), 135.4 (C), 130.7 (C), 124.2 (CH), 122.4 (CH), 122.3 (CH), 120.4 (C), 119.0 (CH), 115.2 (CH), 83.4 (C), 51.6 (CH₃), 34.1 (CH), 32.0 (CH₂), 31.7 (CH₂), 31.3 (CH), 28.3 (CH₃); m/z (ES+) 361 ([M + NH₄]⁺, 100%); HRMS Found 361.2125 [M + NH₄]⁺, C₂₀H₂₉N₂O₄ req. 361.2122.

4.2.3. (1r*,3s*)-3-((1H-indol-3-yl)methyl)cyclobutanecarboxylic Acid 5

To a stirred solution of tert-butyl 3-(((1s*,3r*)-3-(methoxycarbonyl)cyclobutyl)methyl)-1H-indole-1-carboxylate (46 mg, 0.134 mmol) in THF (5 mL) and methanol (0.5 mL), 1 M aqueous NaOH solution (0.67 mL, 0.670 mmol) was added and the reaction mixture was heated to reflux for 30 h, at which point a further 1 M NaOH solution (0.268 mL) was added and the reaction continued for a further 16 h. The reaction mixture was diluted with 5% aqueous HCl (30 mL), extracted with EtOAc (3 × 7 mL) and the combined organic layers were dried (MgSO₄) and concentrated in vacuo to yield the title compound (24 mg, 78%) as a pale brown oil; Rf 0.11 (EtOAc:PE, 3:7); δH (400 MHz, CDCl₃) 7.94 (1H, brs), 7.59 (1H, d, J = 8.1 Hz), 7.35 (1H, d, J = 8.1 Hz), 7.19 (1H, dt, J = 1.0, 8.1 Hz), 7.14 (1H, dt, J = 1.0, 8.1 Hz), 6.95 (1H, d, J = 2.5 Hz), 3.02 (1H, qn, J = 8.6 Hz, H-1), 2.86 (2H, d, J = 7.1 Hz, CH₂Indole), 2.66 (1H, ttt, J = 7.1, 7.6, 9.6 Hz, H-3), 2.38 (2H, ddt, J = 2.5, 7.6, 8.6 Hz, H-2,4), 2.07 (2H, dtd, J = 2.5, 8.6, 9.6 Hz, H-2,4); δC (100 MHz, CDCl₃) 181.2 (C), 136.3 (C), 127.6 (C), 121.9 (CH), 121.3 (CH), 119.2 (CH), 118.9 (CH), 114.4 (C), 111.1 (CH), 34.0 (CH), 32.3 (CH₂), 32.1 (CH), 31.6 (CH₂); m/z (ES+) 230 ([M + H]⁺, 100%). Found 228.1034 [M − H]⁻, C₁₄H₁₄O₂N req. 228.1030.

4.2.4. Methyl (S)-3-((1r*,3R*)-3-((1H-indol-3-yl)methyl)cyclobutane-1-carboxamido)-2-(phenylsulfonamido)propanoate 1

To a stirred solution of (1r*,3s*)-3-((1H-indol-3-yl)methyl)cyclobutanecarboxylic acid (59 mg, 0.258 mmol) in DMF (6. mL), (S)-3-amino-2-benzenesulfonylamino-propionic acid methyl ester 6 (73 mg, 0.283 mmol), EDCI.HCl (148 mg, 0.773 mmol), HOBt (104 mg, 0.773 mmol) and DIPEA (0.224 mL, 167 mg, 1.29 mmol) were added. The reaction mixture was stirred at room temperature for 21 h then poured into water (50 mL) and extracted with EtOAc (4 × 5 mL). The combined organic layers were dried (MgSO₄), filtered, concentrated in vacuo and purified by PTLC (DCM:MeOH, 95:5) to yield the title compound (17 mg, 14%); Rf 0.07 (DCM:MeOH, 95:5); δH (400 MHz, CDCl₃) 8.05 (1H, brs, indole), 7.83 (2H, d, J = 7.1 Hz, ArH-o), 7.55–7.58 (2H, m, ArH-p + indole), 7.49 (2H, t, J = 7.1 Hz, ArH-m), 7.34 (1H, d, J = 8.1 Hz, indole), 7.18 (1H, dt, J = 1.0, 7.1 Hz, indole), 7.10 (1H, dt, J = 1.0, 7.6 Hz, indole), 6.95 (1H, d, J = 2.5 Hz, indole), 5.88 (1H, brt, J = 6.1 Hz, NHCH₂), 5.78 (1H, d, J = 7.6 Hz, NHSO₂Ph), 3.98 (1H, dt, J = 4.6, 6.6, 7.6 Hz, CHNHSO₂Ph), 3.59 (1H, ddd, J = 4.6, 6.1, 14.1 Hz CHH’NH), 3.55 (3H, s, OCH₃), 3.51 (1H, ddd, J = 6.1, 6.6, 14.1 Hz, CHH’NH), 2.83 (2H, d, J = 7.6 Hz, indole-CH₂), 2.78 (1H, tt, J = 8.6, 9.6 Hz, H-1), 2.59 (qd, J = 7.6, 9.1 Hz, H-3), 2.23–2.30 (2H, m, H-2,4), 1.93–2.02 (2H, m, H-2,4); δC (100 MHz, CDCl₃) 175.9 (C), 170.1 (C), 139.3 (C), 136.3 (C), 133.1 (CH), 129.2 (CH), 127.6 (C), 127.2 (CH), 121.8 (CH), 121.5 (CH), 119.1 (CH), 118.9 (CH), 114.4 (C), 111.1 (CH), 55.8 (CH₃), 53.1 (CH), 41.7 (CH₂), 35.8 (CH), 31.9 (CH₂), 31.8 (CH), 31.5 (CH₂), 31.4 (CH₂); m/z (ES+) 470 ([M + H]⁺, 100%) Found 470.1738; C₂₃H₂₈O₅N₃S req. 470.1744.