key: cord-1002381-6fn7mxlw authors: Lüttenberg, Sebastian; Sondermann, Frank; Scherkenbeck, Jürgen title: Anthelmintic PF1022A: stepwise solid-phase synthesis of a cyclodepsipeptide containing N-methyl amino acids date: 2012-02-25 journal: Tetrahedron DOI: 10.1016/j.tet.2011.12.026 sha: c56a60abb43d8f0ac75a55e3f07eefa8355acef4 doc_id: 1002381 cord_uid: 6fn7mxlw Cyclodepsipeptides of the enniation-, PF1022-, and verticilide-family represent a diverse class of highly interesting natural products with respect to their manifold biological activities. However, until now no stepwise solid-phase synthesis has been accomplished due to the difficult combination of N-methyl amino acids and hydroxycarboxylic acids. We report here the first stepwise solid-phase synthesis of the anthelmintic cyclooctadepsipeptide PF1022A based on an Fmoc/THP-ether protecting group strategy on Wang-resin. The standard conditions of our synthesis allow an unproblematic adaption to an automated peptide synthesizer. Depsipeptides are defined as heterodetic peptides with at least one ester bond. While most depsipeptides contain only few, usually irregular ester groups, depsipeptides with alternating ester bonds are less common. Examples (Fig. 1 ) of these highly symmetric natural products comprise PF1022A (1), verticilide (3), bassianolide (4), valinomycin (5) , and the enniatins (6e9). 1 These regular cyclodepsipeptides share several characteristic structural features, among those the occurrence of (R)-hydroxycarboxylic acids and Nmethyl amino acids. Natural depsipeptides, in particular cyclodepsipeptides, are of increasing interest for pharmaceutical research because of their wide range of biological activities (Fig. 1 ). 2 For instance, valinomycin (5) a well known potassium-selective ionophore was recently reported to be the most potent agent against severe acute respiratory-syndrome coronavirus (SARS-CoV). 3 Verticilide (3), a cyclooctadepsipeptide isolated from a Verticilium sp. shows a strong and selective inhibiting activity on ryanodine binding in insects. 4 Bassianolide (4), another cyclooctadepsipeptide, and the enniatins (6e9), a large family of cyclohexadepsipeptides, display diverse biological activities, including antibiotic, antifungal, insecticidal, antiproliferative, and cell migration inhibitory activities. 5 The 24-membered cyclooctadepsipeptide PF1022A (1), a metabolite of Mycelia sterilia (Rosselinia sp.), originally isolated from leaves of Camellia japonica has been established as a resistance-breaking anthelmintic with low toxicity in animals ( Fig. 1) . 6, 7 Other cyclodepsipeptides act by selective ion transport through cellular membranes. Despite the high potential of cyclodepsipeptides for drug discovery, only very limited screening and structureeactivity data have been reported until now, probably due to the difficult access to these compounds. The combination of amino acids and bulky hydroxycarboxylic acids represents a formidable challenge for a solid-phase synthesis, which is on the other hand a prerequisite for the preparation of larger compound collections needed for biological screening. The stepwise assembly of such compounds is expected to be particularly difficult when N-methyl amino acids are involved. 8 Due to the increased steric hindrance couplings need more time with the consequence that dioxomorpholine formation and epimerization can become the predominant side-reactions. 9e11 The difficulties, resulting from the stepwise assemblage of N-methyl amino acids have been impressively demonstrated in several cyclosporine syntheses. 12, 13 Although advanced reagents, such as BMTB and DFET have been developed and applied for the coupling of sterically hindered N-methyl amino acids, high-yielding ester bond assemblage on solid support remains a synthetic problem. 14, 15 Besides standard reagents, such as DIC/DMAP alsotriphosgene and hexafluoroacetonides of a-hydroxycarboxylic acids have been employed for the construction of depsipeptides. 16e18 Abbreviations: ACN, acetonitrile; Boc, tert-butyloxycarbonyl; BOPCl, N,N 0 -bis(2oxo-3-oxazolidinyl)phosphinic chloride; DCM, dichloromethane; DEAD, diethylazodicarboxylate; DHP, 3,4-dihydro-2H-pyrane; DIC, N,N 0 -diisopropylcarbodiimide; DIEA, diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; EDCI, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide; Fmoc, 9-Fluorenyl-methoxycarbonyl; HATU, N,N,N 0 ,N 0 -tetramethyl-O-(7-azabenzo-triazol-1-yl)uroniumhexa-fluorophosphate; HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxy-benzotriazole; MeOH, methanol; TEA, triethylamine; TFA, trifluoro acetic acid; THF, tetrahydrofuran; THP, tetrahydropyranyl; TPP, triphenylphosphane; p-TsOH, para-toluenesulfonic acid. Usually, the ester bonds are either assembled beforehand in solution and only the peptide bonds are formed on the resin or the hydroxycarboxylic acids are introduced without any protection. 19, 20 Only a few procedures have been published for stepwise solidphase depsipeptide syntheses, the most impressive one comprises a twenty-four step total synthesis of the cyclododecadepsipeptide valinomycine (5), including the on-resin formation of six ester bonds. 21e23 However, no stepwise solid-phase syntheses of depsipeptides containing N-methyl amino acids have been accomplished until now. In this paper we wish to report a solid-phase total synthesis of the cyclooctadepsipeptide PF1022A in which all esterand amide-bonds are formed on the support. In principle, the most frequently used Fmoc-and Boc-protecting group strategies should be applicable also for the solid-phase synthesis of depsipeptides. However, both of them have their own problems when ester bonds are involved. The basic conditions needed for the removal of the Fmoc-group may cause a partial ester cleavage, diketopiperazine formation and enhanced racemization of the chiral hydroxy acids. On the other hand, Fmoc-protecting schemes are well established for standard supports, such as the Wang-resin and can be easily adapted on a peptide synthesizer. Diketopiperazine formation is drastically reduced or even suppressed under the strong acidic cleavage conditions used for the cleavage of Boc protecting groups. However, activated ester bonds as those from PhLac for instance, may be affected, too. Additionally, the resins compatible with Boc-protecting, mainly the Kaiseroxime and the PAM-resin are significantly more expensive and have lower loadings compared to Wang-resin. Silyl ethers and acetyl groups have been employed in solidphase syntheses as protecting groups for the OH-function of hydroxy acids. 24 While acetyl groups are cleaved under strong basic conditions, bearing again the risk of racemization, silyl ethers appear generally unsuited for the coupling of N-methyl amino acids and hydroxy acids due to the sterical shielding of the bulky silyl residue. For these reasons we chose the THP-protecting group, which is cleaved under mild acidic conditions, compatible even with the Boc-protecting group. 25 Fmoc protected N-methyl leucine and THP protected hydroxy acids were prepared according to standard literature procedures (Scheme 1). 26e30 First we used the Kaiser-oxime resin, which we had already applied successfully in the course of a didepsipeptide segment synthesis of PF1022A (1) and emodepside (2) . However, in the considerably longer stepwise synthesis of PF1022A, deletion-and failure-sequences accumulated, accompanied by premature cleavages from the resin, which resulted in an inseparable mixture of products after six or seven couplings. In a second attempt we chose the Wang-resin, being well aware of the potential problems arising from the basic cleavage conditions of the Fmoc-group. Since the strongly solvating DMF has been shown to be an unfavorable solvent in critical couplings with respect to yield, diketopiperazine formation and degree of racemization we used the less solvating THF, in which all reagents were soluble and the Wang-resin still showed good swelling properties. 14, 31 After each coupling step a small portion of the resin was cleaved and the product analyzed by HPLC-MS. The results of our optimization experiments are summarized in Table 1 . As expected, the coupling-yields were strongly dependent on the residue used as the first building block, on the coupling reagents and conditions. THP-PhLac 18, which was used as the anchoring-residue due to its lower tendency to form dioxomorpholines compared to THP-Lac 15, could be coupled to the resin in over 90% yield with the reagent mixture DIC/DMAP/HOBt (entry 1b, Table 1 ) in two coupling cycles. Without HOBt the reactivity of the reagent system was distinctly reduced and even after three couplings the yield was only 62%. Remarkably, EDCI failed completely and even HATU, which has been used quite frequently as reagent for ester formation gave only a moderate coupling yield in the anchoring step. 32, 33 In the first chain-extension reaction an ester bond between the anchored PhLac and Fmoc-NMe-Leu had to be established. Best yields (93%) were obtained again with DIC/ DMAP/HOBt after two coupling cycles. The next step, removal of the Fmoc-group from the didepsipeptide segment 20, is in particular prone to the formation of dioxomorpholines. Extensive studies in the literature demonstrate the critical role of the first and second amino acid as well as the coupling conditions. Sterically demanding amino acids (Val, Leu, Phe) in the first position and N-methyl amino acids in the second position, which exactly complies with the PF1022A situation, generally facilitate piperazinedione formation. 34e36 Even after careful optimizations, we were not able to reduce the dioxomorpholine formation for this step to a content lower than 10e15% in the cleavage solution. Noteworthy, in all following Fmoc-group removals dixomorpholine formation was found between only 1% and 5% at maximum. Several methods were tested for the coupling of THP-Lac 15 to resin-bound NMe-Leu (20/21), including THP-Lac acid chloride (entry 3e, Table 1 and Scheme 2). While this method gave only low yields largely due to insufficient coupling, epimerization, and resincleavages, good results for the amide bond formation were obtained with HATU and the DIC/DMAP/HOBt reagent. Since the carbodiimides are known for their risk of racemization, HATU was used for all subsequent amide forming couplings. The reaction between resin-bound Lac and NMeLeu (21/22) turned out to be more difficult than the previous ester formations between the Wang-resin and PhLac (18/19) on the one side and PhLac/NMeLeu (19/20) on the other side (Scheme 2). Thus, in addition to DIC, further procedures frequently used for the formation of difficult ester bonds, such as the highly reactive Fmoc-NMe-Leu acid chloride, the Yamaguchi dichlorobenzoate active ester (entry 4d, Table 1 ) and the Mitsunobo (entry 4e, Table 1 ) coupling with (S)-lactic acid were studied. 37 Step ( and the Yamaguchi method afforded insufficient yields while the Mitsunobo reaction gave somewhat higher yields. However, the DIC/DMAP/HOBt reagent system remained superior also for this coupling step. Standard conditions were used for the removal of the Fmocprotecting group (THF, 25% piperidine, 30 min). The THPprotecting groups were removed quantitatively with diluted p-TsOH (5 mg/mL in DCM/MeOH 97:3, 2Â1 h) according to the procedure published by Kuisle. 23 Due to the symmetry of PF1022A the remaining four chain-elongation reactions are simple repeats of the first couplings. Thus, no more optimizations were conducted. The yield of the linear octadepsipeptide 25 after removal of the N-terminal Fmoc-group and cleavage from the resin (50% TFA in DCM, 1.5 h, quantitative) was 16%, which corresponds to an average yield of 89% for each of the sixteen steps on solid support (Scheme 2). The macrocyclization reaction, performed under high-dilution conditions in DCM with BOPCl as coupling reagent, afforded PF1022A in almost quantitative yield, regardless of whether the purified or the crude precursor 25 was employed. We found it very convenient to work with the crude octadepsipeptide because this avoided a costly and time-consuming preparative HPLC purification. Furthermore, PF1022A can be easily separated from noncyclized by-products by flash-chromatography with silica gel due to its considerably higher lipophilicity. The unusual high macrocyclization yield can be attributed to a cis-amide bond between a leucine and lactic acid, which brings the N-and C-terminus in close proximity and thus facilitates the cylization reaction. 39 PF1022A (1) obtained by synthesis was completely identical with respect to biological and spectroscopical data with a natural reference sample. We have developed a methodology for the stepwise coupling of N-methyl amino acids and hydroxycarboxylic acids on solid support, which allows the preparation of the anthelmintic cyclooctadepsipeptide PF1022A (1) in an overall yield in a range of 13e16% (89% average yield for each step). The synthesis comprises seventeen steps, sixteen of which are performed on solid support followed by a solution macrocyclization. Albeit there is still room for improvements in particular in the amide-bond forming steps, our standard conditions allow an easy adaption to an automated synthesizer as a prerequisite for the preparation of cyclodepsipeptide screening-libraries. The starting material (D)-phenyllactic acid, and was either purchased or prepared by standard literature procedures. 28 All reactions except the saponification were performed in dried solvents. Dichloromethane and the reagents DIEA and piperidine were refluxed for 1 h over calcium hydride and distilled. THF was refluxed for several hours over LiAlH 4 and then distilled and toluene was dried by filtration through basic alumina. The instrumentation used was as follows: 1 H NMR and 13 C NMR spectra were recorded at 25 C 4.3.6. General procedure for the cleavage from the resin. The resin (500 mg) was suspended in a solution (10 mL) consisting of TFA/ DCM 1:1 and agitated for 1.5 h at room temperature. The resin was filtered of and washed with DCM (10Â, 10 mL each). The combined DCM rinses were washed with brine, dried with Na 2 SO 4 , and concentrated in vacuo. Usually, the crude product was immediately used for the macrocyclization reaction without further purification. Cyclooctadepsipeptide PF1022A (1). BOPCl (62.0 mg, 0.24 mmol) was added to a solution of the crude linear octadepsipeptide 25 ppm; 13 Combinatorial Chemistry Generous financial support by Bayer Animal Health GmbH is gratefully acknowledged.