SYNTHESIS OF THE AB RING SYSTEM OF CLIFEDNAMIDE

Moritz Sinast, Inga Loke, Guillaume Bentzinger, Julia Holz, Aruna Raja, Aman Bhasin, Florenz Sasse, Andreas Köhn, Rainer Schobert, and Sabine Laschat Institut für Organische Chemie, Universität Stuttgart, 70569 Stuttgart, Germany Abteilung Chemische Biologie, Helmholtz-Zentrum für Infektionsforschung, 38124 Braunschweig, Germany Institut für Theoretisch Chemie, Universität Stuttgart, 70569 Stuttgart, Germany Lehrstuhl für Organische Chemie, Universität Bayreuth, 95447 Bayreuth, Germany


Synthesis of the AB ring system of clifednamide utilizing Claisen rearrangement and Diels-Alder reaction as key steps
Inga Loke, Guillaume Bentzinger, Julia Holz, Aruna Raja, Aman Bhasin, Florenz Sasse, Andreas Köhn,* Rainer Schobert and Sabine Laschat* Electronic Supplementary Information [(2S)-(8,8-dimethyl-6,10-dioxaspiro[4.5]dec-2-yl)methyl]triphenylphosphonium iodide (8a).In a microwave reactor S5 (10 mg, 0.03 mmol), 1 mL of CH 3 CN, drops of NaHCO 3 and PPh 3 (11 mg, 0.04 mmol) were added.The mixture was heated at 130°C with a power of 300 W for the given time (Table S1).Then the solvent was evaporated and the crude product dissolved in 0.2 mL of CH 2 Cl 2 and 10 mL of Et 2 O.The solid was filtered and the filtrate concentrated Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2015 under reduced pressure to give a light brown solid.No yield in 8a could be calculated because of the amount of PPh 3 remaining in the product.

6) Hydrolysis of derivative 11a
Reaction of 11a with aqueous KOH in THF proceeded quantitatively, however, isomerization of the double bond occurred giving the bicyclic system S7 rather than hydrolysis of the ester.

7) Deuteration experiments of derivative 20b
Tert-butyldiphenylsilyl ether 20b was deprotonated with LDA and the reaction was quenched with D 2 O. Chromatographic purification gave a pure fraction suitable for 1 H NMR investigation, where the signals of the olefinic protons H-2 and H-3 which remain unaffected by the deuteration were set to 1 as reference (Fig. S1a).formed by coupling between H and D (Fig. S1b).Thus, integration of signals for the deuteration 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 ppm 10.87 3.24 2.01 1.82 2.17 at position 5 gave a percentage of 13% (20b), of 25% monodeuterated and of 28% double deuterated derivative.The degree of deuteration is 87%.

8) Quantum-chemical investigation of the selective formation of 11a
As pointed out by Boeckman et al. 10 in a study on a similar compound, the conformation of the diastereomeric centres of 11a can be understood by inspection of the different transition states leading from E,E-ketotriene ester 12a to the final product.Here, we carried out a detailed quantum-chemical investigation of the different transition states, which underpins the formation of the intended diastereomer.
Our study is based on density functional theory (DFT).We employed the PBE and PBE0 density functionals 34 together with the def2-SVP and def2-TZVPP basis sets. 35The D3 dispersion correction was used throughout. 36The impact of solvation effects has been tested by applying the continuum solvation approach COSMO. 37Computations were carried out with the TURBOMOLE V6.6 suite of programs, 38 using the multipole-accelerated RIJ technique 39 and appropriate auxiliary basis functions. 40mpound 11a carries a bulky TBDPS group and both isomers resulting from the different stereochemistry at the C-1' centre have to be considered.The large configuration space of the TBDPS residue complicates the computations and we therefore decided to first investigate a simpler derivative, where TBDPS was replaced by methyl.Nonetheless, we also carried out computations on the full compound to check the effect of the much bulkier TBDPS residue.
The computations focus on the two transition states leading from triene 12a to the compound 11a and 11a' (Scheme 6 of the article).For the initial computations we introduced the simplified predicted difference in activation energy increases significantly to more than 30 kJ mol -1 .This is in line with the experimental observation of only a single diastereomer (except to the two possible configurations at C-1').

9) Investigation of biological activities
Presumably the biosynthetic formation of secondary metabolites such as clifednamide is of advantage for the producing organisms.No biological activities have been reported from clifednamides so far, but since other polycyclic tetramate macrolactams were shown to be active against eukaryotes, we suppose that also clifednamides are biologically active.Since the biosynthesis of such a complex structure has evolved during evolution we also assume that simpler precursors at least may have a slight advantage for the producer.With the different precursors and bicyclic ester 11a in hand we started a study on their biological acitivity.The inhibition of proliferation of L-929 mouse fibroblasts and the human KB-3-1 cervix carcinoma cell line was investigated in a standard MTT assay. 41The results in Table S4 show that the possible biosynthetic precursors of the clifednamides indeed have antiproliferative activities in micromolar range with triene 12a showing the highest potency (IC 50 = 6 M) against L-929 mouse fibroblasts (entry 6).Overall, the cytotoxicity increased from the more distant precursor 28 in the order 14a, 13a to triene 12a.The inhibitory activity of the AB-ring system 11a against L-929 mouse fibroblasts is poorer than that of triene 12a (IC 50 = 17 M and 6 M, resp.) while the cytotoxicity of 11a and 12a against the KB-3-1 cell line is in a similar range (IC 50 = 9 M and 11 M, resp.).a IC 50 values are calculated as mean ± S.D. of two assays in parallel.b,c Two diastereomers of alcohol 32 which could be separated but not assigned unambiguously.

Fig. S1 1 H
Fig. S1 1 H NMR spectrum (500 MHz, CDCl 3 ) of starting silyl ether 20b after deuteration (a) and detail of the spectrum showing the resolved region of protons H-5 and H-6 (b).

Fig. S2
Fig. S2 Representations of the transition state structures leading to compound P1 (upper row, front and side view) and to compound P2 (lower row, front and side view).In the side view pictures, the hydrogens at atoms C-5 and C-6 have been marked in green to highlight the different conformation.

Table S3
Computed reaction energies (RE) and activation energies (AE, all energies in kJ mol -1 ) for the reaction of 12 to 11a or 11a'.Only one isomer concerning the stereocentre C-1' has been a Based on PBE/def2-SVP structures and zero-point vibrational energies.b As previous column but with solvation energies included (COSMO,  = 2.5).