Solid-Phase Methodology for Synthesis of O-Alkylated Aromatic Oligoamide Inhibitors of α-Helix-Mediated Protein–Protein Interactions

Rapid access to rigid rods: A method is described for the synthesis of 3-O-alkylated aromatic oligobenzamide foldamers that could be used for assembly of libraries of α-helix mimetic inhibitors of protein-protein interactions (see scheme; Fmoc=9-fluorenylmethoxycarbonyl).

Micromass GCT Premier, using electron impact ionisation (EI) or a Bruker Daltonics micrOTOF, using electron spray ionisation (ES). LC-MS experiments were run on a Bruker Daltronics esquire TM series spectrometer, samples ionised by electrospray and analysed by a quadrupole ion trap mass spectrometer. All experiments were run through a C18 column on an acetonitrile/water gradient (typically 0-100% acetonitrile over 3 minutes). Intermediates 3a, 3e, 3h, 4a and 4e have previously been described. [1][2][3] Assignment of the compounds is as follows; naming proceeds from N to C terminus where each hydroxyl amino benzoic acid (HABA) residue is assigned a number with respect to its position on the chain. Side-chain assignment follows a peptide nomenclature pattern in which the carbon attached to the alkoxy oxygen is assigned as Ca. Examples of oligomers are given below however monomer intermediates follow the same assignment.

Procedure A (RBr Alkylation)
To a stirred solution of methyl-3-hydroxy-4-nitrobenzoate 2 (1 eq.) and potassium carbonate (3 eq.) in dimethylformamide (20 mL / g) is added RBr (1.2 eq.) and the resulting mixture stirred at 50 C overnight under a nitrogen atmosphere. The resultant red liquid is allowed to cool and poured into water (40 mL / g) and extracted with ethyl acetate. The combined organic fractions are thoroughly washed with water and further washed by brine, dried (magnesium sulfate), filtered and evaporated to dryness.

Procedure B (Mitsunobu)
A stirred solution containing methyl-3-hydroxy-4-nitrobenzoate 2 (1 eq.), ROH (1.1 eq.) and triphenylphosphine (1.5 eq.) in anhydrous tetrahydrofuran (30 mL / g) is cooled to 0°C. Diisopropyl azodicarboxylate (1.5 eq.) is added and the resulting solution allowed to warm to room temperature and left stirring overnight under a nitrogen atmosphere. Organic solvents are removed under reduced pressure and the product is purified via column chromatography.

Procedure C (Tin Reduction)
To a stirred solution containing either i) nitro/ester or ii) nitro/acid (1 eq.) in ethyl acetate (20 mL / g) tin(II) chloride dihydrate (6 eq.) is added and the resulting mixture stirred at 50 C overnight, under a nitrogen atmosphere (with a calcium chloride drying tube attached). On completion, the reaction Hγ mixture is allowed to cool and poured over ice. The pH is made slightly basic (~pH 8) by addition of a saturated sodium bicarbonate solution and the resulting basic mixture is allowed to stir for an hour.
The aqueous mixture is extracted with ethyl acetate and the combined organic fractions washed thoroughly with brine, dried (magnesium sulfate), filtered and evaporated to dryness

Procedure D (Hydrogenation)
A solution containing either i) nitro/ester or ii) nitro/acid (1 eq.) in methanol (20 mL / g) and palladium on carbon (10 wt%) is evacuated and flushed with nitrogen (3 times) and left under vacuum. Hydrogen is drawn into and the flask and the reaction is left stirring at room temperature overnight. On completion, the reaction mixture is filtered through a celite pad and evaporated to dryness.

Procedure E(Cobalt assisted reduction)
To a solution containing either i) nitro/ester or ii) nitro/acid (1 eq.) in methanol, cobalt (II) chloride hexahydrate (3 eq.) is added and allowed to dissolve. Sodium borohydride (6 eq.) is added slowly and allowed to stir for 30 minutes. A further portion of cobalt (II) chloride hexahydrate (3 eq.) and sodium borohydride (6 eq.) is added as above. The solution is filtered through a celite pad which is washed with dichloromethane and the filtrate thoroughly washed with 1M hydrochloric acid to remove the metal. The organic solvents are washed with brine, dried (magnesium sulfate), filtered and evaporated to dryness.

Procedure F (NaOH Saponification)
To a solution containing either i) amine/ester or ii) nitro/ester (1 eq.) in a 1:1 mixture of methanol: tetrahydrofuran (25 mL / g), a 10% sodium hydroxide solution (5 mL / g) is added and the resulting mixture is allowed to stir at RT overnight. Addition of further portions of the hydroxide solution may be necessary. The organic solvents are removed under reduced pressure and water is added to dissolve the solid. The resulting solution is extracted with dichloromethane (unreacted starting material) and the aqueous layer acidified via the addition of hydrochloric acid (conc) to pH 4. The resulting precipitate is extracted into dichloromethane and the combined organic extracts are washed with water and further washed with brine, dried (magnesium sulfate), filtered and evaporated to dryness.

Procedure G (LiOH Saponification)
To a solution containing either i) amine/ester or ii) nitro/ester (1 eq.) in a 1:1 mixture of tetrahydrofuran / water (25 mL / g), a saturated lithium hydroxide solution (1 eq.) is added and the resulting mixture is allowed to stir at RT overnight. The organic solvent is removed under reduced pressure and an additional amount of water is added. The resulting solution is extracted with dichloromethane (unreacted starting material) and the aqueous layer acidified via the addition of 1M potassium bisulfate solution to pH 4. The resulting precipitate is extracted into dichloromethane and the combined organic extracts are washed with water and further washed with brine, dried (magnesium sulfate), filtered and evaporated to dryness.
This solution was filtered and the organic solvents removed under reduced pressure and the solid was crystallised (chloroform/ methanol) to yield the title compound (
Work up yielded the title compound ( LC-MS analysis of this reaction indicated reasonable coupling of the central indole monomer to give the target trimer, however it was not possible to purify and isolate this oligomer. ESI-MS found m/z conformation displaying an alternative arrangement of side-chains however, has a relative potential energy of +3.2 kJ mol -1 demonstrating a variety of rotamers are accessible. Using a crystal structure of gp41 (PDB ID: 1AIK) we took a series of superpositions from our hexamer using different combinations of side chains (eg side chains 1,2 + 3 or 5,4 + 3) and the extended helix using different combinations of residues (e.g. i, i + 3 and i + 7 or i, i + 4 and i + 8) and at varying positions on the helix (e.g. towards the N or C terminus). From the relatively small set we sampled in comparison to the available combinations, we achieved RMSD (Root Mean Square Deviation) values ranging from 0.421-0.788 when superimposing 3 atom pairs consisting of the oxygen of the alkoxy group and the α carbon of the amino acids.
The superposition of the lowest energy conformation of the hexamer using the alkoxy oxygen from rings 2, 3, and 4 with residues at i, i + 3 and i + 7 positions respectively (residues Thr569, Leu566 and Gln562) is shown in Figure S1a. This demonstrates side chains from rings 1, 2, 3, 4 and 5 are orientated in a very similar fashion to residues at the i -4, i, i + 3, i + 7 and i + 10 positions.
Demonstrated by molecular modelling studies and crystal structures ( Fig. 3c-e), the possibility of side chains having different arrangements is thermodynamically viable. With this in mind, the Ar-CO bond on ring 5 has been rotated and Figure S1 b-d shows side chain 6 is found to occupy the same space as residues in the i + 14, i + 15, and i + 16 positions. These studies thus show the prospect of the longer oligoamides to mimic extended a-helices. supplementary crystallographic data for this structure. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Crystal Data II Trimer O2N-[O-iPr (3-HABA)]-[O-iPr (3-HABA)]-[O-iPr (3-HABA)]CO 2 Me:
Prismatic crystals were obtained by the slow evaporation of a solution of the compound in chloroform/ cyclohexane. A crystal of size 0.14 x 0.03 x 0.01 mm was used for data collection; ? range = 3.03≤ θ ≤ 22.50°, Crystals belong to Monoclinic; Space group P 21/c; Formula = C 31.5 H 35.5 Cl 1.5 N 3 O 9 ; Formula weight = 653.30; a = 19.020(4) Å, b = 6.9807 (14)  diffractometer equipped with an Apex II CCD detector and using graphite monochromated Mo-Ka radiation from a FR591 rotating anode generator. The structure was solved by direct methods using SHELXLS-97 and refined using SHELXL-97. The compound crystallises in the monoclinic space group P21/c with one molecule and half of a CHCl 3 in the asymmetric unit. Within the unit cell, each full CHCl 3 molecule is disordered across two symmetry generated positions. All non-hydrogen atoms were refined anisotropically. Hydrogen atoms were placed in calculated positions and refined using a riding model. All Uiso(H) values were constrained to be 1.2 times (1.5 for methyl) Ueq of the parent atom. C-Cl bond lengths were restrained to be chemically reasonable. Crystals were very small and did not diffract to high angles hence the data was cut at 2θ = 45º. The data collected was weak leading to high residual R-factors. CCDC 906042 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Crystal Data III Trimer O2N-[O-iPr (3-HABA)]-[O-iPr (3-HABA)]-[O-iPr (3-HABA)]CO 2 Me:
Prismatic crystals were obtained by the slow evaporation of a solution of the compound in chloroform/ cyclohexane. A crystal of size 0.21 x 0.15 x 0.11 mm was used for data collection; ? range = 2.42≤ θ ≤ 28.48°, Crystals belong to Triclinic; Space group P-1; Formula = C 32 H 38 N 3 O 9.5 ; Formula weight = 616.65; a = 6.4288 (6)  diffractometer equipped with an Apex II CCD detector and using graphite monochromated Mo-Ka radiation from a FR591 rotating anode generator. The structure was solved by direct methods using SHELXLS-97 and refined using SHELXL-97. Compound crystallises in the triclinic space group P-1 with one molecule and half of an EtOH in the asymmetric unit. The half EtOH is disordered over two positions in the asymmetric unit and there is further symmetry-imposed disorder across the inversion centre. All non-hydrogen atoms were refined anisotropically.