Bioorganic & Medicinal Chemistry Letters 24 (2014) 4482–4485
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and analgesic activity of alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin Saoussen Hammami a, Zine Mighri a, Colette T. Dooley b, Adel Nefzi b,⇑ a b
Faculty of Sciences of Monastir, Tunisia Torrey Pines Institute for Molecular Studies, Port Saint Lucie, FL 34987, USA
a r t i c l e
i n f o
Article history: Received 24 April 2014 Revised 29 July 2014 Accepted 31 July 2014 Available online 8 August 2014 Keywords: Peptidomimetics Oligoheterocyclic peptidomimetics Solid-phase synthesis Combinatorial chemistry Opioid receptors
a b s t r a c t We report the design and the parallel solid phase synthesis of linear and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The described peptidomimetics represent different unique scaffolds that distribute in the space the peptidyl side chains of amino acids essential for biological activity and mimic the bioactive conformation of the Leu-enkephalin peptide. All the compounds were screened in competitive radioligand binding assays to determine their affinities for l-(MOR), and j-(KOR) opioid receptors. A reduced analog of Leu-enkephalin TPI1879-26 with activity Ki = 60 nM for the mu receptor was identified. Ó 2014 Elsevier Ltd. All rights reserved.
Biologically active peptides are recognized to have significant therapeutic potential but serious limitations, especially for oral dosing.1,2 These limitations have generated an intensive search for peptidomimetics.1–6 The alteration of peptides to peptidomimetics has included peptide side chain manipulation, amino acid extensions, deletions, substitutions and most recently backbone modification.3,4 The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor/ acceptor binding and for signal transduction. Continuing with our efforts toward the generation of acyclic and heterocyclic compounds from modified peptides,7–24 we report the synthesis and opioid activity of linear and oligoheterocyclic peptidomimetic analogs of the endogenous pentapeptide Leu-enkephalin. Following exhaustive reduction of the amide bonds and using different bifunctional reagents, the polyamide backbone has been transformed to different oligoheterocyclic peptidomimetics. The insertion of heterocycles provides additional binding sites, which render them of particular interest to synthetic and medicinal chemists alike. The parallel synthesis of all linear and oligoheterocyclic peptidomimetics were generated from predesigned reduced ⇑ Corresponding author. Tel./fax: +1 772 345 4739. E-mail address: [email protected] (A. Nefzi). http://dx.doi.org/10.1016/j.bmcl.2014.07.090 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
peptides, in which we alternate two secondary amides and tertiary amides (used as spacer). The tertiary amide incorporated can be derived from N-alkylated amino acids or proline. Following exhaustive reduction of the amide bonds, the generated pre-designed separated pairs of secondary amines were treated with bifunctional reagents such as carbonyldiimidazole, thiocarbonyldiimidazole, cyanogen bromide or oxalyldiimidazole to generate the corresponding oligoheterocycles. Chiral polyamines are generated in our laboratory following exhaustive reduction of resin-bound peptides. As was reported in numerous publications, typical reaction conditions consist of the treatment of resin-bound peptides with the complex BH3-THF.10,13–15 The generated resin-bound complexed borane-amines are disproportioned by transamination following overnight treatment of the solid-support complex with neat piperidine at 65 °C. As was reported earlier by our group and others, the reduction of polyamides with borane is free of racemization.13–15,25 We previously reported the identification of potent and highly selective chiral tri-amine and tetra-amine l opioid receptors ligands following screening of a polyamine library. Forty individual hepta-amines were identified and served as starting ‘hits’ for further SAR studies. The individual compounds showed IC50 values ranging from 14 to 345 nM.33 As might be anticipated by the known studies of mu opiate antagonists, the identified active hepta-amines possessed aromatic rings derived from phenylalanine and tyrosine amino acid side chains. Following SAR studies, a truncation
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analog, reduced and permethylated YYF-NH2, was found to bind with high affinity to mu receptors in rat brains (0.5 nM) as a selective l antagonist in the guinea pig ileum bioassay and a pA2 of 6 nM was found in the GTPcS assay.33 Our approach toward the parallel solid-phase synthesis of alkylated, reduced and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin and their opioid activities against Mu and Kappa receptors is outlined in Scheme 1. Starting from p-methyl benzhydrylamine resin (MBHA), Boc-Leu-OH, was coupled using standard coupling conditions (DIC and HOBt in DMF). The subsequent couplings of sarcosine (N-methyl glycine), N-methyl phenylalanine and N-methyl tyrosine were performed using standard coupling deprotection cycles in the presence of DIC, HOBt as activating agents and 55% trifluoroacetic acid in DCM for Boc deprotection. Following deprotection of the N-terminal protected amino acid tyrosine and exhaustive reduction of the amide groups (5 amide bonds), of the desired compound containing two secondary amines was obtained (Scheme 1). The treatment of resin bound chiral polyamines with thiocarbonyldiimidazole or oxalyldiimidazole followed by HF cleavage provided the corresponding heterocyclic peptidomimetics TPI1853-7 and TPI1853-1, respectively. Some of the bis-diketopiperazine Leu-enkephalin analog on the resin was further reduced to provide, after cleavage from the resin, the desired bis-piperazine Leu-enkephalin analog TPI1853-3. Also, a batch of the resin-bound reduced polyamides was cleaved from the resin leading to the corresponding polyamine Leu-encephalin analog TPI1853-11. Following extraction and lyophilization, the identity of all the linear and oligoheterocyclic peptidomimetics were confirmed by LC–MS. All the compounds were purified using preparative RP-HPLC and tested for their analgesic activities against the MOR and KOR receptors. Moderate activities were observed for all compounds with Kis ranging from 1.50 lM to 15.20 lM for the mu receptor and Kis ranging from 1.11 to 90.40 lM for the Kappa receptor.
O BocHN
O N
N H
O
H N
N
O
H N
N H
O
HO
Another set of oligoheterocyclic peptidomimetics were prepared in which we changed the distribution of the pairs of secondary and tertiary amides (Scheme 2). Thus, different from the approach used in Scheme 1, we incorporated glycine instead of sarcosine for the third amino acid coupling while the N terminal Boc protecting group was kept. The exhaustive reduction of polyamides led to different separated pairs of secondary amine. The reduction of carbamate tertbutyloxycarbonyl on the N-methyl-terminal group of tyrosine led to the corresponding N,N-dimethyl amine. As before, the treatment of the resin bound polyamines with thiocarbonyl diimidazole, cyanogen bromide or diketopiperazine followed by reduction of the oxamide groups and cleavage of the solid support provided the—bis-cyclic thioureas, bis cyclic guanidines and bis cyclic piperazine Leu-enkephalin analogs, respectively. Following purification of all compounds and their screening against l-(MOR), and j-(KOR) opioid receptors, all compounds showed moderate mixed activities with Kis ranging from 0.74 to 1.15 lM for the mu receptor and Kis ranging from 2.30 to 11.30 lM for the Kappa receptor. Similarly, by shortening the peptide chain by eliminating the two sarcosines in Scheme 1 and using proline instead, we performed the synthesis of the analogs described in Scheme 3. The exhaustive reduction of the polyamides led to one pair of secondary amines and three tertiary amines. The treatment of the resin bound polyamines with thiocarbonyldiimidazole and oxalyldiimidazole followed by HF cleavage of the solid support provided the corresponding cyclic thiourea TPI1879-23 and diketopiperazine TPI1879-21 analogs. Two extra sets of resins were used and cleaved for the synthesis of the corresponding polyamides TPI-1879-27 and polyamines TPI-1879-26. The compounds were purified and tested. While all compounds showed moderate activity against KOR receptors with Kis ranging from 1.28 to 10.59 lM, the polyamine analog TPI-1879-26 was
1) TFA, DCM
HO
2) BH 3-THF
H N
N
N H
HF
1) (COIm)2 2) HF
H N
N
N H
O 1) CSIm2 2) HF
HO
N
N H
H N
N
NH2
O
TPI1853-11 Molecular Weight: 526.80 μ= Ki: 1.50 μM κ= Ki: 1.11μM
HO
1) (COIm)2 2) BH3 -THF 3) HF
N
N
O
O
N
N
N NH
O O
TPI1853-1 Molecular Weight: 634.81 μ = Ki: 15.20 μ M κ= Ki: 94.40 μM HO
HO
N
N
N
N
S
N
N S
N
N
N
NH
TPI1853-3 Molecular Weight: 578.87 μ = Ki: 2.65 μ M κ= Ki: 3.12 μM
TPI1853-7 Molecular Weight: 610.92 μ= Ki: 7.11 μM κ= Ki: 3.42 μM Scheme 1.
N NH
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S N
N O BocHN
H N
N H
O
H N
N
O
O
O
HO
N
N
HO N H
1) CS(Im)2 2) HF
HN N
N
N H
H N
N
N H
HO
1) CNBr 2) HF
N H
S
TPI1853-22 Molecular Weight: 610.92 μ= Ki: 0.74 μM κ= Ki: 2.30 μ M
BH 3 -THF
H N
N
N
N
N
N
HO
NH
HN TPI1853-23 Molecular Weight: 576.82 μ= Ki:1.08 μM κ= Ki: 11.30 μM
1) (COIm)2 2) BH3 -THF 3) HF
N
N
N
N
N
NH
HO TPI1853-17 Molecular Weight: 578.87 μ= Ki: 1.15 μM κ= Ki: 3.43 μ M Scheme 2.
O
BocHN
O
N
HO
H N
N O
HF N H
O
H N
N
NH 2
O TPI1879-27 Molecular Weight: 565.70 μ= Ki: 2.14 μM κ = Ki: 2.03 μ M
N N
O
N
HO
BH3 -THF
HO
O
HN
O
H N
N
1) CS(Im)2 2) HF N H
S N
N
HO
N
NH
N 1) (COIm)2 2) HF
HF
TPI1879-23 Molecular Weight: 565.86 μ= Ki: 0.43 μM κ = Ki: 3.32 μ M O
N
N
HO
N H
NH 2
N
HO
N
N
N
O NH
N TPI1879-26
TPI1879-21
Molecular Weight: 523.80 μ= Ki: 0.06 μM κ= Ki: 1.28 μM
Molecular Weight: 577.80 μ= Ki: 0.25 μM κ= Ki: 10.59 μ M Scheme 3.
identified to be highly active against mu receptor with Ki = 60 nM. The corresponding cyclic thiourea and cyclic guanidine showed good activity as well against the mu receptor with Ki = 0.43 lM and Ki = 0.25 lM, respectively (Scheme 4).
By using 5-aminopentanoic acid instead of the two sarcosines in Scheme 1, an extra set of analogs were made in which we eliminated one amide bond while maintaining the length of the peptide chain. Tyrosine was used for the N-terminal instead of N-Me
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S. Hammami et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4482–4485
HO
HO O
H N
H 2N
N
O
N H
O
H N
HF
H 2N
NH2
N H
O
O
TPI1879-37
1) CS(Im)2 2) HF
Molecular Weight: 553.69 μ= Ki: 38.86 μM κ = Ki: 22.5 μ M
2) HF
HO
OH H N
N
O
O
1) BH 3-THF
H 2N
O
H N
HN N
N H
NH 2
S N
N
S
N
NH
TPI1879-33 Molecular Weight: 581.88
TPI1879-36 Molecular Weight: 497.76
μ= Ki: 10.95 μM κ = Ki: 9.48 μ M
μ= Ki: 1.79 μM κ= Ki: 1.62 μ M Scheme 4.
tyrosine. Following Boc deprotection and reduction of the amide bonds, the resin bound polyamines were treated with thiocarbonyldiimidazole to yield the desired bi-cyclic thiourea. Parallel to the cyclic thiourea analog, we also prepared the corresponding polyamide TPI1879-37 and polyamine TPI1879-36 analogs. Low lM activities were obtained for all compounds in mu and Kappa receptors. The parallel synthesis of all compounds was performed using the ‘tea-bag’ methodology.32 Binding assays were conducted using [3H]-DAMGO and [3H]-U69,593 as radioligands for MOR, and KOR, respectively, as was reported in numerous publications.26–31 In conclusion, we performed the parallel solid synthesis of different alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The nature of the chemical diversity of the prepared structures, as well as the large number of compounds making up each class of structures described in this paper, is expected to greatly increase the probability of identifying novel linear and oligoheterocyclic peptidomimetics with desirable biological properties to aid in the study of the mechanisms of analgesic efficacy, addiction and tolerance, and may lead to effective new forms of analgesics or alternative treatments for drug abuse. Acknowledgments ‘This work was funded in part through the Florida Drug Discovery Acceleration Program by the State of Florida, Department of Health’. References and notes 1. 2. 3. 4.
Hruby, V. J.; Cai, M. Annu. Rev. Pharmacol. Toxicol. 2013, 53, 557. Avan, I.; Dennis Hall, C.; Katritzky, Alan R. Chem. Soc. Rev. 2014, 43, 3575. Vagner, J.; Qu, H.; Hruby, V. J. Curr. Opin. Chem. Biol. 2008, 12, 292. Kharb, R.; Rana, M.; Sharma, P. C.; Yar, M. S. J. Chem. Pharm. Res. 2011, 3, 173.
5. Cerminara, I.; Chiummiento, L.; Funicello, M.; Ambra Guarnaccio, A.; Lupattelli, P. Pharmaceuticals 2012, 5, 297. 6. Giri, A. K.; Hruby, V. J. Expert Opin. Invest. Drugs 2014, 23, 227. 7. Nefzi, A.; Ostresh, J. M.; Houghten, R. A. Biopolym. (Pept. Sci.) 2001, 60, 212. 8. Nefzi, A.; Dooley, C.; Ostresh, J. M.; Houghten, R. A. Bioorg. Med. Chem. Lett. 1998, 8, 2273. 9. Nefzi, A.; Ong, N. A.; Houghten, R. A. Tetrahedron Lett. 2000, 5441. 10. Nefzi, A.; Giulianotti, M. A.; Houghten, R. A. Tetrahedron Lett. 2000, 41, 2283. 11. Alvarez-Gutierrez, J. M.; Nefzi, A.; Houghten, R. A. Tetrahedron Lett. 2000, 41, 851. 12. Alvarez-Gutierrez, J. M.; Nefzi, A.; Houghten, R. A. Tetrahedron Lett. 2000, 41, 609. 13. Nefzi, A.; Giulianotti, M.; Houghten, R. A. Tetrahedron Lett. 1999, 40, 8539. 14. Nefzi, A.; Giulianotti, M.; Houghten, R. A. Tetrahedron 2000, 56, 3319. 15. Houghten, R. A.; Pinilla, C.; Appel, J. R.; Blondelle, S. E.; Dooley, C. T.; Eichler, J.; Nefzi, A.; Ostresh, J. M. J. Med. Chem. 1999, 42, 3743. 16. Nefzi, A.; Ostresh, J. M.; Giulianotti, M.; Houghten, R. A. J. Comb. Chem. 1999, 1, 195. 17. Nefzi, A.; Ostresh, J. M.; Houghten, R. A. Solid-Phase Synthesis—A Practical Guide In Kates, S. A., Albericio, F., Eds.; Marcel Dekker: New York, 2000; pp 617–647. 18. Nefzi, A.; Ostresh, J. M.; Yu, Y.; Houghten, R. A. J. Org. Chem. 2004, 69, 3603. 19. Murru, S.; Dooley, C. T.; Nefzi, A. Tetrahedron Lett. 2013, 54, 7062. 20. Michaels, H. A.; Velosa, D. C.; Nefzi, A. ACS Comb. Sci. 2014, 16, 1. 21. Arutyunyan, S.; Nefzi, A. J. Comb. Chem. 2010, 12, 315. 22. Nefzi, A.; Appel, J.; Arutyunyan, S.; Houghten, R. A. Bioorg. Med. Chem. Lett. 2009, 19, 5169. 23. Nefzi, A.; Santos, R. T. Tetrahedron Lett. 2006, 47, 3819. 24. Nefzi, A.; Santos, R. T. J. Org. Chem. 2005, 70, 9622. 25. Manku, S.; Laplante, C.; Kopac, D.; Chan, T.; Hall, D. G. J. Org. Chem. 2001, 66, 874. 26. Dooley, C. T.; Chung, N. N.; Wilkes, B. C.; Schiller, P. W.; Bidlack, J. M.; Pasternak, G. W.; Houghten, R. A. Science 1994, 266, 2019. 27. Dooley, C. T.; Spaeth, C. G.; Craymer, K.; Adapa, I. D.; Brandt, S. R.; Houghten, R. A.; Toll, L. J. Pharmacol. Exp. Ther. 1997, 283, 735. 28. Dooley, C. T.; Houghten, R. A. Analgesia 1995, 1, 400. 29. Dooley, C. T.; Houghten, R. A. Biopolym. (Pept. Sci.) 2000, 51, 379. 30. Dooley, C. T.; Houghten, R. A. Biopolymers 1999, 51, 379. 31. Dooley, C. T.; Ny, P.; Bidlack, J. M.; Houghten, R. A. J. Biol. Chem. 1998, 273, 18848. 32. Houghten, R. A. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 5131. 33. Nefzi, A.; Ostresh, J. M.; Appel, J. R.; Bidlack, J.; Dooley, C. T.; Houghten, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 4331.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and analgesic activity of alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin Saoussen Hammami a, Zine Mighri a, Colette T. Dooley b, Adel Nefzi b,⇑ a b
Faculty of Sciences of Monastir, Tunisia Torrey Pines Institute for Molecular Studies, Port Saint Lucie, FL 34987, USA
a r t i c l e
i n f o
Article history: Received 24 April 2014 Revised 29 July 2014 Accepted 31 July 2014 Available online 8 August 2014 Keywords: Peptidomimetics Oligoheterocyclic peptidomimetics Solid-phase synthesis Combinatorial chemistry Opioid receptors
a b s t r a c t We report the design and the parallel solid phase synthesis of linear and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The described peptidomimetics represent different unique scaffolds that distribute in the space the peptidyl side chains of amino acids essential for biological activity and mimic the bioactive conformation of the Leu-enkephalin peptide. All the compounds were screened in competitive radioligand binding assays to determine their affinities for l-(MOR), and j-(KOR) opioid receptors. A reduced analog of Leu-enkephalin TPI1879-26 with activity Ki = 60 nM for the mu receptor was identified. Ó 2014 Elsevier Ltd. All rights reserved.
Biologically active peptides are recognized to have significant therapeutic potential but serious limitations, especially for oral dosing.1,2 These limitations have generated an intensive search for peptidomimetics.1–6 The alteration of peptides to peptidomimetics has included peptide side chain manipulation, amino acid extensions, deletions, substitutions and most recently backbone modification.3,4 The side chain groups of amino acid residues in polypeptide hormones, neurotransmitters, growth factors, substrates, antigens, and other bioactive peptides have been demonstrated to be extremely important pharmacophores for receptor/ acceptor binding and for signal transduction. Continuing with our efforts toward the generation of acyclic and heterocyclic compounds from modified peptides,7–24 we report the synthesis and opioid activity of linear and oligoheterocyclic peptidomimetic analogs of the endogenous pentapeptide Leu-enkephalin. Following exhaustive reduction of the amide bonds and using different bifunctional reagents, the polyamide backbone has been transformed to different oligoheterocyclic peptidomimetics. The insertion of heterocycles provides additional binding sites, which render them of particular interest to synthetic and medicinal chemists alike. The parallel synthesis of all linear and oligoheterocyclic peptidomimetics were generated from predesigned reduced ⇑ Corresponding author. Tel./fax: +1 772 345 4739. E-mail address: [email protected] (A. Nefzi). http://dx.doi.org/10.1016/j.bmcl.2014.07.090 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
peptides, in which we alternate two secondary amides and tertiary amides (used as spacer). The tertiary amide incorporated can be derived from N-alkylated amino acids or proline. Following exhaustive reduction of the amide bonds, the generated pre-designed separated pairs of secondary amines were treated with bifunctional reagents such as carbonyldiimidazole, thiocarbonyldiimidazole, cyanogen bromide or oxalyldiimidazole to generate the corresponding oligoheterocycles. Chiral polyamines are generated in our laboratory following exhaustive reduction of resin-bound peptides. As was reported in numerous publications, typical reaction conditions consist of the treatment of resin-bound peptides with the complex BH3-THF.10,13–15 The generated resin-bound complexed borane-amines are disproportioned by transamination following overnight treatment of the solid-support complex with neat piperidine at 65 °C. As was reported earlier by our group and others, the reduction of polyamides with borane is free of racemization.13–15,25 We previously reported the identification of potent and highly selective chiral tri-amine and tetra-amine l opioid receptors ligands following screening of a polyamine library. Forty individual hepta-amines were identified and served as starting ‘hits’ for further SAR studies. The individual compounds showed IC50 values ranging from 14 to 345 nM.33 As might be anticipated by the known studies of mu opiate antagonists, the identified active hepta-amines possessed aromatic rings derived from phenylalanine and tyrosine amino acid side chains. Following SAR studies, a truncation
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analog, reduced and permethylated YYF-NH2, was found to bind with high affinity to mu receptors in rat brains (0.5 nM) as a selective l antagonist in the guinea pig ileum bioassay and a pA2 of 6 nM was found in the GTPcS assay.33 Our approach toward the parallel solid-phase synthesis of alkylated, reduced and oligoheterocyclic peptidomimetic analogs of Leu-enkephalin and their opioid activities against Mu and Kappa receptors is outlined in Scheme 1. Starting from p-methyl benzhydrylamine resin (MBHA), Boc-Leu-OH, was coupled using standard coupling conditions (DIC and HOBt in DMF). The subsequent couplings of sarcosine (N-methyl glycine), N-methyl phenylalanine and N-methyl tyrosine were performed using standard coupling deprotection cycles in the presence of DIC, HOBt as activating agents and 55% trifluoroacetic acid in DCM for Boc deprotection. Following deprotection of the N-terminal protected amino acid tyrosine and exhaustive reduction of the amide groups (5 amide bonds), of the desired compound containing two secondary amines was obtained (Scheme 1). The treatment of resin bound chiral polyamines with thiocarbonyldiimidazole or oxalyldiimidazole followed by HF cleavage provided the corresponding heterocyclic peptidomimetics TPI1853-7 and TPI1853-1, respectively. Some of the bis-diketopiperazine Leu-enkephalin analog on the resin was further reduced to provide, after cleavage from the resin, the desired bis-piperazine Leu-enkephalin analog TPI1853-3. Also, a batch of the resin-bound reduced polyamides was cleaved from the resin leading to the corresponding polyamine Leu-encephalin analog TPI1853-11. Following extraction and lyophilization, the identity of all the linear and oligoheterocyclic peptidomimetics were confirmed by LC–MS. All the compounds were purified using preparative RP-HPLC and tested for their analgesic activities against the MOR and KOR receptors. Moderate activities were observed for all compounds with Kis ranging from 1.50 lM to 15.20 lM for the mu receptor and Kis ranging from 1.11 to 90.40 lM for the Kappa receptor.
O BocHN
O N
N H
O
H N
N
O
H N
N H
O
HO
Another set of oligoheterocyclic peptidomimetics were prepared in which we changed the distribution of the pairs of secondary and tertiary amides (Scheme 2). Thus, different from the approach used in Scheme 1, we incorporated glycine instead of sarcosine for the third amino acid coupling while the N terminal Boc protecting group was kept. The exhaustive reduction of polyamides led to different separated pairs of secondary amine. The reduction of carbamate tertbutyloxycarbonyl on the N-methyl-terminal group of tyrosine led to the corresponding N,N-dimethyl amine. As before, the treatment of the resin bound polyamines with thiocarbonyl diimidazole, cyanogen bromide or diketopiperazine followed by reduction of the oxamide groups and cleavage of the solid support provided the—bis-cyclic thioureas, bis cyclic guanidines and bis cyclic piperazine Leu-enkephalin analogs, respectively. Following purification of all compounds and their screening against l-(MOR), and j-(KOR) opioid receptors, all compounds showed moderate mixed activities with Kis ranging from 0.74 to 1.15 lM for the mu receptor and Kis ranging from 2.30 to 11.30 lM for the Kappa receptor. Similarly, by shortening the peptide chain by eliminating the two sarcosines in Scheme 1 and using proline instead, we performed the synthesis of the analogs described in Scheme 3. The exhaustive reduction of the polyamides led to one pair of secondary amines and three tertiary amines. The treatment of the resin bound polyamines with thiocarbonyldiimidazole and oxalyldiimidazole followed by HF cleavage of the solid support provided the corresponding cyclic thiourea TPI1879-23 and diketopiperazine TPI1879-21 analogs. Two extra sets of resins were used and cleaved for the synthesis of the corresponding polyamides TPI-1879-27 and polyamines TPI-1879-26. The compounds were purified and tested. While all compounds showed moderate activity against KOR receptors with Kis ranging from 1.28 to 10.59 lM, the polyamine analog TPI-1879-26 was
1) TFA, DCM
HO
2) BH 3-THF
H N
N
N H
HF
1) (COIm)2 2) HF
H N
N
N H
O 1) CSIm2 2) HF
HO
N
N H
H N
N
NH2
O
TPI1853-11 Molecular Weight: 526.80 μ= Ki: 1.50 μM κ= Ki: 1.11μM
HO
1) (COIm)2 2) BH3 -THF 3) HF
N
N
O
O
N
N
N NH
O O
TPI1853-1 Molecular Weight: 634.81 μ = Ki: 15.20 μ M κ= Ki: 94.40 μM HO
HO
N
N
N
N
S
N
N S
N
N
N
NH
TPI1853-3 Molecular Weight: 578.87 μ = Ki: 2.65 μ M κ= Ki: 3.12 μM
TPI1853-7 Molecular Weight: 610.92 μ= Ki: 7.11 μM κ= Ki: 3.42 μM Scheme 1.
N NH
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S N
N O BocHN
H N
N H
O
H N
N
O
O
O
HO
N
N
HO N H
1) CS(Im)2 2) HF
HN N
N
N H
H N
N
N H
HO
1) CNBr 2) HF
N H
S
TPI1853-22 Molecular Weight: 610.92 μ= Ki: 0.74 μM κ= Ki: 2.30 μ M
BH 3 -THF
H N
N
N
N
N
N
HO
NH
HN TPI1853-23 Molecular Weight: 576.82 μ= Ki:1.08 μM κ= Ki: 11.30 μM
1) (COIm)2 2) BH3 -THF 3) HF
N
N
N
N
N
NH
HO TPI1853-17 Molecular Weight: 578.87 μ= Ki: 1.15 μM κ= Ki: 3.43 μ M Scheme 2.
O
BocHN
O
N
HO
H N
N O
HF N H
O
H N
N
NH 2
O TPI1879-27 Molecular Weight: 565.70 μ= Ki: 2.14 μM κ = Ki: 2.03 μ M
N N
O
N
HO
BH3 -THF
HO
O
HN
O
H N
N
1) CS(Im)2 2) HF N H
S N
N
HO
N
NH
N 1) (COIm)2 2) HF
HF
TPI1879-23 Molecular Weight: 565.86 μ= Ki: 0.43 μM κ = Ki: 3.32 μ M O
N
N
HO
N H
NH 2
N
HO
N
N
N
O NH
N TPI1879-26
TPI1879-21
Molecular Weight: 523.80 μ= Ki: 0.06 μM κ= Ki: 1.28 μM
Molecular Weight: 577.80 μ= Ki: 0.25 μM κ= Ki: 10.59 μ M Scheme 3.
identified to be highly active against mu receptor with Ki = 60 nM. The corresponding cyclic thiourea and cyclic guanidine showed good activity as well against the mu receptor with Ki = 0.43 lM and Ki = 0.25 lM, respectively (Scheme 4).
By using 5-aminopentanoic acid instead of the two sarcosines in Scheme 1, an extra set of analogs were made in which we eliminated one amide bond while maintaining the length of the peptide chain. Tyrosine was used for the N-terminal instead of N-Me
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S. Hammami et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4482–4485
HO
HO O
H N
H 2N
N
O
N H
O
H N
HF
H 2N
NH2
N H
O
O
TPI1879-37
1) CS(Im)2 2) HF
Molecular Weight: 553.69 μ= Ki: 38.86 μM κ = Ki: 22.5 μ M
2) HF
HO
OH H N
N
O
O
1) BH 3-THF
H 2N
O
H N
HN N
N H
NH 2
S N
N
S
N
NH
TPI1879-33 Molecular Weight: 581.88
TPI1879-36 Molecular Weight: 497.76
μ= Ki: 10.95 μM κ = Ki: 9.48 μ M
μ= Ki: 1.79 μM κ= Ki: 1.62 μ M Scheme 4.
tyrosine. Following Boc deprotection and reduction of the amide bonds, the resin bound polyamines were treated with thiocarbonyldiimidazole to yield the desired bi-cyclic thiourea. Parallel to the cyclic thiourea analog, we also prepared the corresponding polyamide TPI1879-37 and polyamine TPI1879-36 analogs. Low lM activities were obtained for all compounds in mu and Kappa receptors. The parallel synthesis of all compounds was performed using the ‘tea-bag’ methodology.32 Binding assays were conducted using [3H]-DAMGO and [3H]-U69,593 as radioligands for MOR, and KOR, respectively, as was reported in numerous publications.26–31 In conclusion, we performed the parallel solid synthesis of different alkylated, reduced and constrained oligoheterocyclic peptidomimetic analogs of Leu-enkephalin. The nature of the chemical diversity of the prepared structures, as well as the large number of compounds making up each class of structures described in this paper, is expected to greatly increase the probability of identifying novel linear and oligoheterocyclic peptidomimetics with desirable biological properties to aid in the study of the mechanisms of analgesic efficacy, addiction and tolerance, and may lead to effective new forms of analgesics or alternative treatments for drug abuse. Acknowledgments ‘This work was funded in part through the Florida Drug Discovery Acceleration Program by the State of Florida, Department of Health’. References and notes 1. 2. 3. 4.
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