Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Aug 4.
Published in final edited form as: Adv Exp Med Biol. 2009;611:207–208. doi: 10.1007/978-0-387-73657-0_95

Stereoselective Synthesis of (3S)- and (3R)-3-Hydroxy-3-(2,6-Dimethyl-4-Hydroxyphenyl)Propanoic Acid and its Incorporation into a Cyclic Enkephalin Analogue

Grazyna Weltrowska 1, Carole Lemieux 1, Nga N Chung 1, Brian C Wilkes 1, Peter W Schiller 1
PMCID: PMC2720549  NIHMSID: NIHMS128231  PMID: 19400162

Introduction

2’,6’-dimethyl substitution of the Tyr1 residue of opioid agonist peptides and deletion of the N-terminal amino group have been shown to represent a general structural modification to convert opioid peptide agonists into antagonists [1]. This conversion required the syntheses of opioid peptide analogues containing 3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid (Dhp) in place of Tyr1. The cyclic enkephalin analogue Dhp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 is a potent µ opioid antagonist and a less potent δ and κ antagonist [2]. An analogue of this peptide with β-methylated Dhp, (3S)-3-methyl-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(3S)-Mdp], in place of Dhp showed increased antagonist activity at all three receptors, whereas an analogue containing β-isopropyl-substituted Dhp [(3R)-Idp] turned out to be a mixed κ agonist/µ antagonist [3]. To further investigate the SAR at the β position of Dhp in the cyclic analogue, we examined the effect of substituting β-hydroxylated Dhp on the opioid activity profile. This required the development of stereoselective syntheses of (3S)- and (3R)-3-hydroxy-3-(2,6-dimethyl-4-hydroxyphenyl)propanoic acid [(3S)-and (3R)-Hdp].

Results and Discussion

(3S)- and (3R)-Hdp were synthesized using Evans chemistry [4] (Scheme 1). Aldol reaction between silyl-protected aldehyde 2 and the dibutylboron enolate derived from chloroacetyloxazolidinone 3 or 4 afforded the desired intermediate 5 or 6 in 70% and 75% yield, respectively. The diastereoselectivity ratio for 5 (78:22) and for 6 (82:18) was determined by 1H NMR spectroscopy. Similar to this moderate diastereomeric excess, limited stereoselectivity had also been observed upon introduction of alkyl substituents in the β-position of Dhp [2]. However, the diastereomers were easily separated by silica gel chromatography, yielding the pure diastereomer 5 or 6. Dechlorination of 5 or 6 was accomplished by treatment with zinc dust and glacial acetic acid to afford 7 or 8 in 50% yield in both cases. Silylation of the free hydroxyl group in 80% yield for 9 and 85% yield for 10 was followed by removal of the chiral auxiliary in standard manner [5] to give 11 or 12. Structures were confirmed by 1H NMR spectroscopy and EI mass spectrometry.

Scheme 1.

Scheme 1

Open in a new tab

(i) TBDMSCl, imidazole, DMF, rt., 95%; (ii) Cl-CH 2-COXc, TEA, Bu 2BOTF, CH2Cl2, −78°C to rt., 1,5h, phosphate buffer pH7, 30% H2O2, ether, 0°C, 1h; (iii) Zn, AcOH, THF, rt., 15min, 50%; (iv) TBDMSCl, imidazole, DMF, 80%; (v) LiOH, 30% H2O2, THF/H2O, 0°C to rt., 90%.

The target peptides (3S)- and (3R)-Hdp-c[D-Cys-Gly-Phe(pNO2)-D-Cys)]NH2 were prepared by a combination of manual solid-phase and solution techniques. The linear precursor peptide of H-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 was assembled on a p-methylbenzhydrylamine resin, using Boc-protection of the α-amino function and Acm protection of the Cys side chain. After cleavage from the resin by HF/anisole treatment, disulfide bond formation was carried out in MeOH/H2O using iodine as oxidation agent and the peptide was purified by preparative reversed-phase HPLC. (3S)- or (3R)-Hdp(TBDMS)2 (11,12) was attached to the N-terminal amino group of H-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 using HBTU as coupling agent. After removal of the silyl protection by 90% TFA/anisole treatment, the final peptide products were purified by reversed-phase HPLC, and their purity and structural identity were established by TLC, analytical HPLC and FAB-MS.

(3S)- and (3R)-Hdp-c[D-Cys-Gly-Phe(pNO2)-D-Cys-NH2 both showed neither agonist nor antagonist activity in the guinea pig ileum and mouse vas deferens assays at concentrations up to 10 µM. In the opioid receptor binding assays, both the (3S)-Hdp1- and the(3R)-Hdp1 -analogues showed no µ and κ receptor binding affinity at concentrations up to 10 µM and very weak δ receptor binding affinity with Ki values of 7.65 and 26.3 µM, respectively. This result indicates that introduction of the hydrophilic hydroxyl group at the β-position of Dhp is not tolerated at all three receptors.

Acknowledgments

This work was supported by grants from the CIHR and the NIH.

References

  • 1.Lu Y, Nguyen TM-D, Weltrowska G, Berezowska I, Wilkes BC, Lemieux C, Chung NN, Schiller PW. J. Med. Chem. 2001;44:3048–3053. doi: 10.1021/jm0101186. [DOI] [PubMed] [Google Scholar]
  • 2.Weltrowska G, Lu Y, Lemieux C, Chung NN, Schiller PW. Bioorg. Med. Chem. Lett. 2004;14:4731–4733. doi: 10.1016/j.bmcl.2004.06.077. [DOI] [PubMed] [Google Scholar]
  • 3.Schiller PW, Weltrowska G, Berezowska I, Lemieux C, Chung NN, Wilkes BC. In: Blondelle SE, editor. Understanding Biology Using Peptides (Proceedings of the 19th American Peptide Symposium); American Peptide Society; San Diego. 2005. pp. 31–35. [Google Scholar]
  • 4.Evans DA, Fitch DM, Smith TS, Cee VJ. J. Am. Chem. Soc. 2000;122:10033–10046. [Google Scholar]
  • 5.Evans DA, Britton DC, Ellman JA. Tetrahedron Lett. 1987;28:6141–6144. [Google Scholar]

RESOURCES