Pyrrolidine analogs of arylceramide HPA-12 †

: Conformational constraint is the usual way to modify the properties of bioactive molecules. In some cases, such modiﬁcation improves their activity as well as their affinity for their biological target. At the beginning of the millennium, the ( R,R )-HPA-12 was found to be the first antagonist of ceramide transporter CERT, which has been identified as a key factor for the ER-to-Golgi trafficking of ceramides. Ten years later, we have revised the stereochemistry of the most active HPA-12 diastereomer to ( R , S )-diastereomer and developed synthesis of more potent alkyl substituted HPA-12 analogs. In this contribution, we would like to describe a straightforward approach to the enantiomerically pure constraint pyrrolidine analogs of both ( R,S )-HPA-12 and ( R,R )-HPA-12 starting from 3-substituted pyroglutamic acids easily accessible via our methodology. The results of the binding assays to the CERT protein will be discussed.


Introduction
Sphingolipids are membrane lipids that play important roles in the regulation of cell functions such as cell growth, differentiation, apoptosis, and homeostasis.Alterations in their metabolism have been associated with several pathologies.The regulation of these functions depends on a fine dynamic balance between some species whose relative intracellular concentrations are decisive for the cell´s fate.Ceramides are important signal molecules that control important cell vital functions such as division, apoptosis, or autophagy.They are formed in the endoplasmic reticulum and are the basis of glycosphingolipids and phospholipids whose synthesis takes place in the Golgi apparatus.Transport of ceramide from ER to GA provides CERT protein [1].Aryl substituted ceramide analog -HPA-12 was discovered as the first specific inhibitor of intracellular transport of ceramides by CERT [2].In this way, it is possible to regulate the ratio of sphingolipids and ceramides in the cell, which contributes to the proper functioning of the cell.The original structure of the HPA-12 was revised in 2011 by our research group [3] and finally confirmed by X-ray analysis [4].In the last five years, several new synthetic methodologies of HPA-12 and analogs were published [5].In our group, the substituted alkyl analogs of HPA-12 -structurally closer to the natural ligand of CERT protein -ceramide itself were prepared with m-hexyl HPA-12 analogs being the most effective ligand [6].As part of our ongoing research focused on this field, we were particularly interested in the design and synthesis of constraint HPA-12 analogs starting from enantiomerically pure 3-aroyl pyroglutamic acids.

Methods
Scheme 1 illustrates the planned synthesis of the desired HPA-12 analogs (1).The design of the synthesis was based on the easy accessibility of enantiomerically pure -oxo--aminobutanoic acids (2) via own CIAT methodology [7,8].The subsequent N-chloroacetylation followed by intramolecular cyclization represents a convenient and economical synthesis of substituted enantiomerically pure 3-aroyl-5-oxo prolines (4).The important goal of their further elaboration was the stereoselective reduction of the carbonyl group.The main product in all tested conditions was the (R)-5 stereoisomer.The desired (S)-5 diastereoisomer was prepared by Mitsunobu inversion.The subsequent synthetic steps including standard functional groups manipulation, Sonogashira coupling, and several reduction steps followed our previous synthetic strategy published for alkyl analogs of HPA-12.

Scheme 1. Retrosynthetic analysis of the HPA-12 constrained analogs
The starting substituted oxoamino acids (2) were prepared using the aza-Michael addition of (R)phenylethylamine to aroylacrylic acids in tandem with crystallization-induced asymmetric transformation (CIAT) [7].This convenient multigram methodology was developed in our laboratory, and its main advantage is the simple product isolation by filtration.The prepared oxo amino acids (2) were subsequently transformed into aroyl pyroglutamic acids (4) in two steps using N-chloroacetylation in non-basic condition with propylene oxide as an HCl scavenger [9].The second step was highly stereoselective intramolecular cyclization using aqueous sodium hydroxide.The cyclisation provided almost pure diastereomer in high yield.The product is thermodynamically more stable trans-diastereomer with high stereoselectivity (d.r.97:3).The practically pure diastereomer was obtained via simple crystallization from methanol.

Scheme 3. Stereoselective cyclisation
The key transformation in our synthetic pathway to the targeted analogs of HPA-12 was the stereoselective reduction of carbonyl function.Despite testing a wide variety of methods for reducing a carbonyl group, we failed to prepare diastereomer with the correct (S)-5 configuration.We decided to prepare him via Mitsunobu reaction by inversion of the major (R)-5 diastereoisomer.The carboxyl function was protected by esterification with methyl iodide.The product can be purified by simple crystallization.

Figure 1. X-ray of the product of Mitsunobu inversion
The high affinity of m-hexyl-substituted HPA-12 for CERT protein led us to prepare also m-hexylsubstituted conformationally restricted pyrrolidine analogs of HPA-12.For this purpose, Sonogashira reaction was also used.Both prepared diastereomers with protected carboxyl function were applied as the starting materials for this reaction.Both reactions were carried out with high conversion.

Scheme 5. Sonogashira coupling
Final elaboration of polysubstituted oxoprolines to the targeted pyrrolidine analogs of HPA-12 was consistent with our previously published strategy. 6For the preparation of the hexyl, substituent was used as a simple method of catalytic reduction of the unsaturated bond using palladium on carbon with hydrogen.Subsequent reduction by lithium aluminum hydride reduced the lactam and ester group to the desired prolinol derivative.The last step was catalytic N-debenzylation in the presence of two equivalents of acetic acid.The products were finally purified chromatographically.The final conformationally limited pyrrolidine analogs of HPA-12 were obtained by chemoselective N-acylation.

Results and discussion
This study describes conformationally restricted HPA-12 analogs with incorporated pyrrolidine scaffold as biologically active inhibitors of the CERT protein.The objectives of the work consisted of the preparation of aroyl-substituted oxoproline, reduction of the carbonyl group, preparation of the target pyrrolidine analogs of HPA-12 and determination of their chemical and physical characteristics.Research has been made to find out most effective synthetic methods for the preparation of optically pure secondary alcohols from the substituted ketones.Effective and simple reduction with sodium borohydride and crystallization resulted in the preparation of only one pure diastereomer with d.r.greater than 99: 1 in a yield of 60%.The opposite isomer was finally prepared using the Mitsunobu inversion.The inverse configuration at the newly-formed stereogenic center was confirmed by basic hydrolysis.Also, the ability of this product to well crystallize helped us by X-ray analysis to detect relative configurations of the resulting stereogenic center and thus to define the absolute configuration of the molecule.Further functional groups manipulation led to the synthesis of the final pyrrolidine analogs of HPA-12.Finally, both diastereomers of the target molecule were prepared and characterized by optical rotation measurement and HRMS spectra.Further optimization of the last synthetic steps is desirable, but the analogs have been prepared in sufficient quantity.Target pyrrolidine analogs of HPA-12 were sent for testing their affinity to the CERT protein using previously developed FRET methodology [10].Preliminary results obtained from the Evotec International GmbH laboratory Toulouse, France show EC50 for CERT comparable with those for HPA-12.

Conclusions
Constrained analogs of HPA-12 were successfully prepared in high diastereomeric purity and in good yields.Final constrained analogs of HPA-12 were sent for testing their affinity to the CERT protein.
Preliminary results France show EC50 for CERT comparable with those for HPA-12.