Synthesis and Characterization of all Possible Diastereoisomers of Alvimopan

Number of biological substances and natural products are found to exist as enantiomeric pairs. In this instance, alvimopan 1 as shown in Fig. 1, has three chiral centers thus it will have theoretically eight diastereomers. Out of these, one of the diastereomers is found to be active as opioid antagonist and prescribed for the treatment of postoperative ileus. In order to establish the percentage content of unwanted diastereomers and define the specifications in the drug substance which is unprecedented hitherto therefore we attempted to synthesize and characterize all possible diastereomers. Here in, we present our strategy which has successfully been executed to obtain four practically feasible diastereomers due to anti relationship at 3 and 4 chiral carbon centers. Interestingly, syn pair was not observed thus other four diastereomers are chemically negated.


INTRODUCTION
Number of biological substances and natural products are found to exist as enantiomeric pairs.In this instance, alvimopan 1 as shown in Fig. 1, has three chiral centers thus it will have theoretically eight diastereomers.Out of these, one of the diastereomers is found to be active as opioid antagonist and prescribed for the treatment of postoperative ileus.In order to establish the percentage content of unwanted diastereomers and define the specifications in the drug substance which is unprecedented hitherto therefore we attempted to synthesize and characterize all possible diastereomers.Here in, we present our strategy which has successfully been executed to obtain four practically feasible diastereomers due to anti relationship at 3 and 4 chiral carbon centers.Interestingly, syn pair was not observed thus other four diastereomers are chemically negated.

Alvimopan ([[2(S)-[[4(R)-(3 Hydroxyphenyl
)-3R,4-dimethyl-1-piperidinyl] methyl]-1-oxo-3phenylpropyl]-amino]acetic acid 1 dihydrate is an opioid drug used for the treatment upper and lower gastrointestinal recovery following partial or large and small bowel resection surgery with primary anastomosis. 1The drug is also having the activity towards the treatment of post operative ilieus and chronic idiopathic constipation. 2astereomers were carried out by using Perkin Elmer 2400 FTIR spectrophotometer using KBr pellet.Mass spectra recorded on 4000-Q-trap LC-MS/MS mass spectrometer. 1H and 13 C NMR were recorded in DMSO-d 6 at 400 MHz on a unity INOVA (Varian 400 MHz) FT NMR spectrometer.The chemical shifts are reported in δ(ppm) relative to TMS (δ 0.00).The SOR was recorded at 25 °C at 1.01 concentration in DMSO.

RESULTS AND DISCUSSION
Alvimopan contain total three chiral centers, which leads to form total eight theoretical diastereomers.Two chiral centers are present in the key starting material 2 and the 3 rd one is generated during the preparation of the compound 13.4][5][6] The complete synthesis of alvimopan was disclosed by John A Werner et al., and his co-workers. 7In the reported synthesis no information was available regarding the diastereomers of alvimopan 1.In the synthesis of alvimopan 1, first chiral center was generated during the preparation of the compound 7 by resolution of compound 6 with (+)-diparatoulyl-D-tartaric acid.Unwanted isomer of compound 7 which is 7a can easily be isolated from the filtrate of the compound 7.

Fig. 2. Structures of wanted (7) and unwanted (7a) isomers
Alternately, diastereomer 7a was also synthesized by resolution of 6 with (-)-diparatoulyl-Dtartaric acid.The second chiral center was generated during the preparation of compound 9 by using decalin at higher temperature followed by n-BuLi and dimethyl sulfate.Interestingly, this event was found to be stereoselective (near stereospecific) yielding only anti pairs amounting to two enantiomers 9 and 9a as shown in Figure 3.The 3 rd chiral center was generated during the preparation of the compound 13.Due to anti selectivity during methylation towards preparation of 9 or 9a, it was practically feasible to obtain only four diastereomers (13, 13a, 13b and 13c) as shown in Fig. 4.This Fig. also outlines the retrosynthetic approach to obtain all four diastereomers in sequence.
Eventually, by using 13, 13a, 13b and 13c and following the similar sequence featured in Scheme 1, we were able to prepare all the alvimopan diastereomers (1, 1a, 1b and 1c) as shown in Figure 5.After synthesis, isolation and characterization, we recorded the SOR for alvimopan and its diastereomers and the results are as shown in the Table 1.
In addition to SOR studies we attempted to study the structural features by using Infra-Red spectroscopy.As expected, there were no major changes observed in any of the stretching bands of the functional groups present in the diastereomers as shown in Table 2. H NMR and 13 C NMR for alvimopan and its diastereomers were recorded by using Varian Mercury plus 400 MHz spectrometer.The solvent used for analysis is DMSO-d 6 .The 1 H NMR chemical shifts values were reported on δ scale in ppm relative to TMS 0.00 ppm and 13 C NMR chemical shifts values were reported relative DMSO-d 6 δ=39.50 ppm.Structural elucidation for each and every diastereomer was performed by correlating the δ values of corresponding 1 H NMR and 13 C NMR featured in Table 3 and 4. The number assignment was shown in Figure 6.
indicates that all three diastereomers (1a, 1b and 1c) are distinctly different than alvimopan 1 as shown in Fig. 7. HPLC analytical conditions are as follows: Procedure 1.
Detector: UV The blend chromatogram is shown in Fig. 7.The retention times of the diastereomers (1a, 1b and 1c) and the alvimopan 1 are given in Table 5.In order to establish the relative retention time of the diastereomers 1a, 1b and 1c with respect to alvimopan 1, a blend sample was injected in the HPLC column.The blend sample HPLC chromatogram were compared with alvimopan 1 HPLC chromatogram.This exercise clearly The alvimopan prepared by the reported Scheme 1 and the product contains the diastereomers 1b and 1c are at the level of 0.02 and 1a at the level of 0.12 area percentage.

Synthesis of impurities
All the impurities were prepared synthetically as per the reported sequence of reactions for the preparation of alvimopan shown in Scheme 1.

Synthesis of 1a
Reaction of 12 with benzyl bromide afforded both 13 and 13a in 1:1 ratio as shown Scheme 2. The undesired compound 13a was separated from compound 13 by preferential crystallization in methanol.The impurity 13a was isolated from the mother liquor and purified by column chromatography.Impurity 13a further undergoes the alvimopan reaction sequence (Scheme 2) during the preparation to give rise impurity 1 (1a or αR, 3R, 4R isomer) with same similar process efficiency.

Impurity 1b
During the resolution of compound 6 to obtain compound 7, undesired isomer 7a was also found to be co-precipitated along with compound 7. Thereafter, 7a undergoes transformations involved in the synthesis of alvimopan and eventually afforded impurity 2. The preparation of impurity 2 can alternately be prepared from 6 by resolving it with (-)-diparatoulyl-D-tartaric acid to obtain 7a as isolated product.With this material, by repeating the alvimopan (1) synthetic sequence it was possible to obtain diastereomeric impurity 2

Impurity 1c
During the preparation of compound 13 by reacting the compound 12 with benzyl bromide, both 13 and 13a will form in 1:1 ratios.Similarly the carry forwarded impurity of 12a forms two different isomers of 13b and 13c.Compound 13b undergoes further sequence of reactions and ends up as impurity 2 (S, S, S isomer).Similarly the 13c isomer undergoes the sequence of the reactions and ends up as impurity-3.During the preparation of compound 13b, the obtained mother liquors purified by column chromatography to obtain pure diastereomeric impurity of 13c.This pure compound made to undergo same reaction sequence of alvimopan (1) proceeded to obtain diastereomeric impurity-3 (1c or αR, 3S, 4S isomer).

Structure Elucidation Alvimopan
ESI mass spectrum of alvimopan exhibited protonated molecule peak at m/z 425 (MH) + in positive ion mode, indicating the mass of alvimopan is 424.In 1 H NMR of alvimopan, the signal at 9.10 ppm broad singlet corresponding to phenolic OH proton and another singlet at 5.56-5.58ppm corresponds to amide proton of NH.The complete NMR signals assignment was given in Table 3.In the IR spectrum, a broad band at 3410 cm -1 for OH