Synthesis of Four Enantiomers of (1-Amino-3-Hydroxypropane-1,3-Diyl)Diphosphonic Acid as Diphosphonate Analogues of 4-Hydroxyglutamic Acid

All the enantiomers of (1-amino-3-hydroxypropane-1,3-diyl)diphosphonic acid, newly design phosphonate analogues of 4-hydroxyglutamic acids, were obtained. The synthetic strategy involved Abramov reactions of diethyl (R)- and (S)-1-(N-Boc-amino)-3-oxopropylphosphonates with diethyl phosphite, separation of diastereoisomeric [1-(N-Boc-amino)-3-hydroxypropane-1,3-diyl]diphosphonates as O-protected esters, followed by their hydrolysis to the enantiomeric phosphonic acids. The absolute configuration of the enantiomeric phosphonates was established by comparing the 31P NMR chemical shifts of respective (S)-O-methylmandelic acid esters obtained from respective pairs of syn- and anti-[1-(N-Boc-amino)-3-hydroxypropane-1,3-diyl]diphosphonates according to the Spilling rule.


Introduction
As analogues of naturally occurring α-amino acids, α-aminophosphonic acids are pharmacologically significant as they can mimic transition states of several biological processes such as peptide hydrolysis. Owing to the tetrahedral structure of the phosphonic residue, they can act as enzyme inhibitors or antibiotics [1][2][3][4]. Moreover, their activity often depends on the absolute configuration at Cα in α-aminophosphonic acids. Over decades, a vast number of phosphonate analogues of α-amino acids have been synthesized with the intention to study their biological properties ( Figure 1). Among them, analogues of glutamic acid 1, a major excitatory neurotransmitter in the central nervous system, deserve great consideration. For example, 2-amino-4-phosphonobutanic acid (L-AP4) 2 has been obtained as an analogue of glutamic acid and appeared to be a selective agonist for group III glutamate metabotropic receptors (mGluR) [5][6][7][8][9], whereas its α-methylated analogue (MAP4) 3 acts as a competitive antagonist of mGluR [10,11].
In continuation of our research program directed at the syntheses of enantiomerically pure functionalized aminophosphonates, we focus attention on hydroxyglutamic acids, which are widely available in nature, including plants, however this structure is also found as a part of more complex molecules with important biological properties. As expected, the presence of an additional hydroxy group in the glutamic acid framework may have a positive impact on the activity of its analogues. Thus, (2S,4S)-4-hydroxyglutamic acid 4 exhibited potency at mGlu 1a R and mGlu 8a R similar to that of L-glutamic acid [12], and its isomer (2S,4R)-4 demonstrated a significant preference for the NMDA (N-methyl-D-aspartic acid) receptor [13].
Inspired by these observations we considered the synthesis of all four enantiomerically pure diphosphonic acids 5 ( Figure 2). Inspired by these observations we considered the synthesis of all four enantiomerically pure diphosphonic acids 5 ( Figure 2). Our synthetic strategy relied on the formation of the C-P bond by the addition of diethyl phosphite to (R)-and (S)-(1-amino-2-oxoethyl)phosphonates 7, available from the enantiomerically pure N-(1-phenylethyl)-C-(diethoxyphosphoryl)nitrone (S)-10 already described by our research group (Scheme 1) [14].
The aldehyde (R)-7 was subjected to the Abramov reaction with diethyl phosphite in the presence of catalytic amounts of triethylamine to afford a 1:1 mixture of diastereoisomeric diphosphonates (1R,3S)-6 and (1R,3R)-6 (Scheme 3). Attempts to separate the diastereoisomeric mixture of diphosphonates by column (silica gel) and high performance liquid chromatography (HPLC) appeared fruitless as the fractions collected were only enriched for the respective isomers (up to 90%). The ratio of diastereoisomers was established on the basis of 31 P NMR spectra of the crude product. Since two phosphonyl groups are installed in the structure of compound 6, two signals were identified for each of the respective diastereoisomeric diphosphonates (1R,3S)-6 (δ 31 P = 25.26 and 23.58 ppm) and (1R,3R)-6 (δ 31 P = 24.60 and 24.01 ppm). diastereoisomeric mixture of diphosphonates by column (silica gel) and high performance liquid chromatography (HPLC) appeared fruitless as the fractions collected were only enriched for the respective isomers (up to 90%). The ratio of diastereoisomers was established on the basis of 31 P NMR spectra of the crude product. Since two phosphonyl groups are installed in the structure of compound 6, two signals were identified for each of the respective diastereoisomeric diphosphonates (1R,3S)-6 (δ 31 P = 25.26 and 23.58 ppm) and (1R,3R)-6 (δ 31 P = 24.60 and 24.01 ppm).  To complete the full set of stereoisomeric phosphonic acids 5, the aldehyde (S)-7 was used to synthesize diphosphonates (1S,3R)-6 and (1S,3S)-6, which were subsequently O-protected as the respective esters 11 or 12, and then transformed into the final acids (1S,3R)-5 and (1S,3S)-5 by application of an analogous reaction sequence (Scheme 5). diastereoisomeric mixture of diphosphonates by column (silica gel) and high performance liquid chromatography (HPLC) appeared fruitless as the fractions collected were only enriched for the respective isomers (up to 90%). The ratio of diastereoisomers was established on the basis of 31 P NMR spectra of the crude product. Since two phosphonyl groups are installed in the structure of compound 6, two signals were identified for each of the respective diastereoisomeric diphosphonates (1R,3S)-6 (δ 31 P = 25.26 and 23.58 ppm) and (1R,3R)-6 (δ 31 P = 24.60 and 24.01 ppm).   To complete the full set of stereoisomeric phosphonic acids 5, the aldehyde (S)-7 was used to synthesize diphosphonates (1S,3R)-6 and (1S,3S)-6, which were subsequently O-protected as the respective esters 11 or 12, and then transformed into the final acids (1S,3R)-5 and (1S,3S)-5 by application of an analogous reaction sequence (Scheme 5).

General Information
NMR spectra were measured in chloroform-d (CDCl3), benzene-d6 (C6D6), or deuterium oxide (D2O) on a Bruker Avance III (600 MHz). Solvent signals or TMS were used as internal references for 1 H and 13 C chemical shifts (ppm). 31 P signals were referenced through the solvent lock (2H) signal according to the IUPAC recommended secondary referencing method and the manufacturer's protocols (an analogous protocol was used for 13 C NMR spectra recorded in D2O). Coupling constants J are given in Hz. The NMR experiments were conducted at 300K with the following parameters: 1 H NMR spectra were acquired at 600.26 MHz using 30°-pulses (zg30), a spectral width of 12,335.5 Hz, Based on extensive configurational studies of the α-hydroxyphosphonates, Spilling and co-workers concluded that 31 P NMR chemical shifts for the (R)-O-methylmandelic acid esters of (S)-α-hydroxyphosphonates appear in a higher field compared to the signals for the (R)-O-methylmandelates of enantiomeric (R)-alcohols [20]. Accordingly, (S)-Omethylmandelates of (R)-α-hydroxyphosphonates are expected to absorb in a higher field than (S)-O-methylmandelates of (S)-α-hydroxyphosphonates. Indeed, this general rule worked well for our 3-hydroxydiphosphonates 6 ( Figure 3). Thus, the 31 P nucleus at C3 in (