Synthesis of dipeptide mimics based on amino phosphinate backbones and cyclic derivatives

Dialkyl phosphinates are valuable peptide mimics for metallopeptidase targets. Despite their large pharmaceutical potential, the synthesis of many phosphinates remains challenging. An additional drawback for many applications is the high polarity of the phosphinate group. Herein we describe the synthesis of stereoisomerically pure GPI ( 1 ), a phosphinate with high binding affinity for the cancer specific zinc peptidase PSMA (prostate specific membrane antigen). In addition, analogous cyclic phosphinate esters 13 , 16 and 23 are reported that might be useful as less polar ligands for metallo peptidase binding. The key step to these new 1,2-oxaphosphorinan-2-ones is an intramolecular cyclization of an intermediate H -phosphinate. The cyclizations work with modest diastereoselectivities of ~2:1 in favor of the trans arrangement of substituents at 2-and 4-position of the 1,2-oxaphosphorinan-2-one scaffolds.


Introduction
2] An altered level of expression is often associated with malignant neoplasy.A range of metallopeptidases have therefore been used as tumor markers and are valuable targets for the development of targeted cancer therapeutics and imaging reagents.][5] The general structural requirements for small molecules in this context are a peptidomimetic backbone for recognition of the peptidase and a zinc binding moiety (Scheme 1).Suitable zinc binding groups are thiols, carboxylates, hydroxamates, phosphonates and phosphinates.8][9][10] Scheme 1. Tetrahedral intermediate upon peptide hydrolysis in an active site of a zinc peptidase and a phosphinate analogue.In addition, the PSMA-specific ligand GPI (1) is shown.
Herein we describe the synthesis of stereoisomerically pure GPI (1), a phosphinate with high binding affinity to the cancer specific zinc peptidase PSMA (prostate specific membrane antigen).In addition, analogous cyclic phosphinates are reported that might be useful as less polar precursors for metallo peptidase binding.

Results and Discussion
GPI (1), a mimic of the native dipeptide NAAG (N-acetylaspartylglutamic acid), is a high affinity binder for the tumor marker PSMA and has been used for prostate cancer targeting (Scheme 2). 17- 191] However, the stereoselective synthesis of GPI proved to be difficult and only low diastereoselectivities have been realized so far. 22For a comparative study of stereoisomeric imaging reagents based on GPI as a targeting ligand, we needed both the (S,S) and the corresponding (S,R) isomer.Following a known protocol, we started our synthesis with enantiomerically pure Cbz-(S)-vinylglycine (3), which is readily available by a two-step synthesis from Cbz-(S)-methionine (2). 23In parallel, dibenzyl glutarate (5)  was prepared from benzyl acrylate (4) via a Baylis-Hilman type reaction with (n-Bu)3P. 24Addition of ammonium hypophosphite gave phosphinic acid 6 as a racemate.Following a protocol of Vitharana et al., 25 stepwise crystallization with first yohimbine and second (S)-methylbenzylamine gave (S)-6 and (R)-6 in 31 and 28% yield, respectively over two steps.Both enantiomers of 6 were then treated with vinylglycine (3) and BSA to the target compounds (S,S)-7 and (S,R)-7.Both diastereomers were obtained with a de > 95 according to 1 H-NMR, proving indirectly the high enantiomeric purity of intermediate phosphinic acids (S)-6 and (R)-6 after crystallization.Scheme 2. Synthesis of (S)-vinylglycine (3) and isomerically pure (S,S)-7 and (S,R)-7.BSA = Bis(trimethylsilyl)acetamide.
The high polarity of the phosphinic acid group in peptidase inhibitors like GPI (1) is an inherent problem not only for the bioavailability of the compounds but also for workup of protected derivatives like 7. The affinity of phosphinates for silica often leads to significant loss of yield upon column chromatography.To decrease the polarity of the target compounds, we planned to introduce the dialkyl phosphinate part of the peptidase binders as a cyclic phosphinate ester (1,2-oxaphosphorinan-2-one), which upon hydrolysis would then release the phosphinic acid and an alcohol group.Our first attempt started with the conversion of allyl alcohol 8 to homoallyl alcohol 10 in a two-step procedure involving bromination and a subsequent In-mediated Barbier reaction of bromide 9 with formaldehyde (Scheme 3).The resulting homoallyl alcohol 10 was converted to the corresponding phosphinic acid ester 12.In our hands, the mixed-anhydride of phosphinic acid 11 which was prepared with pivaloyl chloride gave the best yields for this esterification.Ester 12 was then treated with BSA to give the cyclic phosphinate 13 upon intramolecular cyclization in reasonable 46% yield for the two-step sequence.[28][29] Scheme 3. Synthesis of the cyclic phosphinate 13 via intramolecular cyclization of ester 12. BSA = Bis(trimethylsilyl)acetamide; PivCl = pivaloyl chloride; only one enantiomer of racemic compound trans-13 is shown.
The intramolecular cyclization of ester 12 to cyclic phosphinate 13 proceeds with a moderate diastereoselectivity of 2:1 in favor of trans-13.The preferred formation of trans-13 may be rationalized with the mechanistic proposal depicted in Scheme 4.

Scheme 4.
Mechanistic proposal for the intramolecular cyclization of ester 12 to the cyclic phosphinate trans-13.Only one enantiomer of racemic compound trans-13 is shown.
Using the same approach, we have treated homoallyl alcohol 10 with phosphinic acid 14, which is readily available from (S)-vinylglycine (3).Ester 15 was again formed via the mixed anhydride of 14 with PivCl.Subsequent intramolecular cyclization gave the target cyclic phosphinate 16, which is a dipeptide mimic.Along with the methyl ester 16, we isolated a significant amount of the corresponding acid 17.Because we have used enantiomerically pure phosphonic acid 14 as a starting material, we expected the formation of all four possible stereoisomers in 16.To our surprise, we observed only two diastereoisomers in the 31 P-NMR spectra of 16 (Scheme 5, A).However, this seems to be the consequence of a coincidental signal overlap of each two stereoisomers.In the corresponding acid derivative 17, which was formed as a deprotected side product, all four isomers are observable in the 31  One of the reasons for synthesizing cyclic phosphinates like 13 and 16 was their decreased polarity compared to phosphinic acid analogues.In addition, we hoped that the intramolecular alkylation of the H-phosphinate intermediate might give better diastereoselectivities then acyclic variants.To introduce a chiral auxiliary in the vicinity of the newly formed stereogenic centers, we have prepared the chiral ester 20 by reaction of (-)-borneol (19) with acid 18 (Scheme 6).
Deprotection with TBAF gave alcohol 21, which was coupled to the mixed anhydride of phosphinic acid 14 to give ester 22. Intramolecular cyclization gave cyclic phosphinate 23 as a mixture of four stereoisomers.Finally, hydrolysis with aqueous HCl to 24 was performed to decrease the number of possible stereoisomers and a 2:1-mixture of two diastereoisomers was observed in the 13 C-NMR-spectra.Alternatively, the alkylation of H-phosphinate 14 with αmethylene-γ-butyrolactone (25) gave also dipeptide mimic 24, but with no diastereoselectivity at all.Scheme 6. Auxiliary synthesis of dipeptide mimic 24 via intramolecular cyclization of ester 22 and an alternative approach from α-methylene-γ-butyrolactone (25).Only one major stereoisomer for 23 and 24 is shown.

Conclusions
We have described the synthesis of isomerically pure (S,S)-and (S,R)-7 via fractional crystallization of the intermediate H-phosphinate rac-6.The target compounds 7 are protected GPI (1) derivatives and thus ligands for the prostate cancer specific peptidase PSMA.To reduce the polarity of the targeted phosphinic acids, we synthesized cyclic phosphinate esters.The key step to these new 1,2-oxaphosphorinan-2-ones is an intramolecular cyclization of an intermediate Hphosphinate.The cyclizations work with modest diastereoselectivities of ~2:1 in favor of the trans arrangement of substituents at 2-and 4-position of the 1,2-oxaphosphorinan-2-one. The resulting cyclic phosphinates are peptide mimetics of relatively low polarity compared to the corresponding acyclic phosphinic acids.

Scheme 5 .
Scheme 5. Synthesis of dipeptide mimic 16 via intramolecular cyclization of ester 15.Only one out of four stereoisomers for 16 and 17 is shown.A: stretch of the 31 P-NMR spectra of 16; B: stretch of the 31 P-NMR spectra of 17.