Letter pubs.acs.org/acscombsci
Expedient Synthesis of N1-Substituted Triazole Peptidomimetics Steven B. Coffey,*,† Gary Aspnes,†,‡ and Allyn T. Londregan† †
Pfizer Worldwide Medicinal Chemistry, Eastern Point Road, Groton, Connecticut 06340, United States Pfizer Worldwide Medicinal Chemistry, 610 Main Street, Cambridge, Massachusetts 02139, United States
‡
S Supporting Information *
ABSTRACT: A general procedure for the rapid diversification of peptide scaffolds is described. A one-pot click reaction between a peptide-alkyne and a series of in situ generated aryl/ alkyl azides affords novel N1-substituted triazole peptidomimetics. This transformation is of broad scope, operates under mild conditions, and is parallel chemical synthesis compatible. KEYWORDS: triazole peptidomimetics, rapid diversification, one-pot click reaction, mild conditions, parallel synthesis
T
stitued-1,2,3-triazoles were well tolerated in our initial SAR studies and prompted our efforts in developing a parallel chemistry protocol. In the design of our peptidomimetics, diversity would originate from varied aryl, heteroaryl and aliphatic azide reaction partners. From the onset, however, we were concerned about the safety of isolating and utilizing organic azides. Additionally, the scarce commercial availability of such monomers presented a significant limitation. As a solution to this problem, a number of research groups have developed onepot procedures for preparing 1,2,3-triazoles directly from aryl halides and terminal alkynes in the presence of sodium azide.21−29 The method developed by Andersen et al.29 was of particular interest. As shown in Scheme 1, we envisioned utilizing a one-pot procedure where the requisite azide would be generated in situ from the corresponding aryl/heteroaryl/alkyl halide (4) and sodium azide. This species would subsequently undergo a click reaction directly with the appropriate peptide-alkyne (5) to afford the desired peptidomimetic (6). Conveniently, in the
he development of peptides into orally bioavailable medicines is of significant interest to the pharmaceutical industry. Peptides often exhibit compelling biological activities but are impractical as oral drug candidates because of their relatively poor in vivo pharmacokinetic properties. Rapid proteolytic cleavage and low cellular membrane permeability1 are among the most significant challenges to achieving sufficient systemic exposure. The synthesis and development of peptidomimetics, where peptides are derivatized to increase their binding affinity or improve their physicochemical properties, is one approach to this issue. This strategy usually incorporates non-natural amino acids or other moieties into a peptide scaffold to help improve its drug-like properties.2−4 A robust method for the late-stage modification of complex molecules, including peptides, is the copper-catalyzed azide− alkyne cycloaddition (CuAAC) or click reaction.5 Pioneered independently by the Meldal6 and Sharpless7 laboratories, this copper-catalyzed variant of Huisgen’s (3 + 2) cycloaddition affords 1,2,3-triazoles in high yields and regioselectivity from azide and alkyne reaction partners. The click reaction has been used extensively in the field of chemical biology,8−12 where carrier systems are linked to parent molecules via 1,2,3triazoles, often in the presence of complex biomolecules.8 The synthesis of 1,2,3-triazoles via the click reaction is well documented and has been used for specific protein,13−16 and peptide6,17−20 modifications. However, little has been shown regarding CuAAC solution phase chemistry with heterocycles in a one-pot format in the context of peptidomimetics. Owing to the mild and substrate-tolerant nature of the click reaction, we surmised that it would be an effective means to rapidly synthesize a series of peptidomimetics of interest to us. Specifically, we wished to diversify the N-termini of privileged peptide scaffolds with various aryl, heteroaryl, and aliphatic substituents. We reasoned that a 1,4-disubstituted-1,2,3-triazole could serve to replace the N-terminal amide bond and eliminate a charged terminal amine, both modifications that could influence permeability, while providing a useful point of diversity in developing our SAR. Conveniently, N1-sub© 2015 American Chemical Society
Scheme 1. General Protocol Design
Received: September 23, 2015 Published: November 12, 2015 706
DOI: 10.1021/acscombsci.5b00150 ACS Comb. Sci. 2015, 17, 706−709
Letter
ACS Combinatorial Science case of aryl and heteroaryl halide substrates, the copper(I) iodide would catalyze both the in situ azidonation and the final click reaction. This protocol would be particularly useful in parallel format where the handling/isolation of hazardous organic azide intermediates is undesirable. Unique to this work is the significant expansion of the reaction substrate scope beyond simple aryl-halides and aryl/alkyl-alkynes. To our knowledge, no examples of such a protocol have been described in the context of sensitive peptide-based substrates. As exemplified in Scheme 2, four privileged tripeptide alkyne substrates (5a−d) were synthesized via HBTU mediated30
Table 1. Substrate Scope for the One-Pot Click Reaction with Various Halides and Peptide Alkynesa,b
Scheme 2. Synthesis of Peptide Alkyne Substratesa
a
7 (0.22 mmol), 8a−d (0.22 mmol), iPr2NEt (0.66 mmol), HBTU (0.22 mmol), 4 mL of DMF, 20 °C, 16 h. Peptide = −LeuValPhe-OBn (in 8a and 5a), LeuValTyr-OMe (in 8b and 5b), −PheAlaPro-OBn (in 8c and 5c), −AlaAlaPro-OBn (in 8d and 5d).
amide couplings between 2,2-dimethylbut-3-ynoic acid (7) and 8a−d. With alkyne 5a in hand, a one-pot azide/click reaction with 3-bromopyridine (Table 1, entry 1) was examined. We utilized conditions conducive to parallel format, where stock solutions in both DMSO (peptide-alkyne 5a, trans-N1,N2dimethylcyclohexane-1,2-diamine, CuI) and water (sodium ascorbate, sodium azide) were first prepared. In a 2-dram vial containing the halide (4), each of the stock solutions were added in the sequence presented above. The reaction was then purged with nitrogen, capped, and heated to 70 °C. Gratifyingly, after just 5.5 h, the reaction was complete with the desired product (6a) the major component by LCMS analysis. The reaction was quenched with ice cold water and extracted with ethyl acetate directly in reaction vial. The organic layer was separated and then concentrated to afford a crude residue, which was purified by silica gel chromatography to afford 6a in 70% yield. To assess the compatibility of these conditions with varied reaction partners, the above protocol was repeated using 5a and each of the halides shown in Table 1. In general, all reactions examined proceeded in excellent to modest yields after silica gel chromatography. Numerous heteroaryl bromides were effective reaction partners, with 3-bromo-6-methoxypyridine (entry 2) affording 80% isolated yield of 6b. Pleasingly, benzyl and phenethyl bromides were excellent substrates for the in situ azide formation as well, and afforded good yields of product (entries 7 and 8). To diversify the reaction scope beyond commercially available halides, various alcohols were converted to the corresponding mesylates and tosylates and effectively utilized (entries 9−11). Common protecting groups (entry 4 and 11) were also well tolerated under the reaction conditions. Of particular note is the chemoselective reaction of 5-bromoindazole (entry 5), which suggests that unprotected and potentially reactive functionality is compatible with this copper(I)-based protocol. With confidence in the substrate scope and reproducibility of this protocol, we initiated a full combinatorial library31 with the remaining three peptide-alkynes. A ChemGlass Optichem heater block was employed with a 24-well plate. Several duplicate experiments with alkyne 5a (entries 3,7) were
Peptide = −LeuValPhe-OBn (in 5a), LeuValTyr-OMe (in 5b), −PheAlaPro-OBn (in 5c), −AlaAlaPro-OBn (in 5d). bReaction conditions: 4 (0.05 mmol), sodium azide (0.067 mmol), 5a−d (0.05 mmol), sodium ascorbate (0.005 mmol), CuI (0.005 mmol), ligand: trans-N1,N2-dimethylcyclohexane-1,2-diamine (0.005 mmol), 1.5 mL of DMSO/water 5:1, 70 °C, 5.5 h. cIsolated yield after RP-HPLC purification. dIsolated yield after silica gel chromatography. a
included to compare this full production mode with the individual test reactions. In general, all reactions examined proceeded acceptably and were purified by RP-HPLC purification. Isolated yields were comparable to those obtained using silica gel chromatography in the initial screens with 707
DOI: 10.1021/acscombsci.5b00150 ACS Comb. Sci. 2015, 17, 706−709
Letter
ACS Combinatorial Science
(8) Hong, V. H.; Presolski, S. I.; Ma, C.; Finn, M. G. Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem., Int. Ed. 2009, 48, 9879−9883. (9) Zhang, L.; Chen, X.; Xue, P.; Sun, H. H.; Williams, I. D.; Sharpless, K. B.; Fokin, V. V.; Jia, G. Ruthenium-Catalyzed Cycloaddition of Alkynes and Organic Azides. J. Am. Chem. Soc. 2005, 127, 15998−15999. (10) Katritzky, A. R.; Zhang, Y.; Singh, S. K. 1,2,3-Triazole Formation Under Mild Conditions via 1,3-Dipolar Cycloaddition of Acetylenes with Azides. Heterocycles 2003, 60, 1225−1239. (11) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click Chemistry In Situ: Acetylcholinesterase as a Reaction Vessel for the Selective Assembly of a Femtomolar Inhibitor from an Array of Building Blocks. Angew. Chem., Int. Ed. 2002, 41, 1053−1057. (12) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew. Chem., Int. Ed. 2001, 40, 2004−2021. (13) Ballet, S.; Betti, C.; Novoa, A.; Tomboly, S.; Nielsen, C.; Helms, H.; Lesniak, A.; Kleczkowska, P.; Chung, N.; Lipkowski, A.; Brodin, B.; Tourwe, D.; Schiller, P. In Vitro Membrane Permeation Studies and In Vivo Antinociception of Glycosylated Dmt1-DALDA Analogues. ACS Med. Chem. Lett. 2014, 5, 352−357. (14) Lau, Y.; de Andrade, P.; McKenzie, G.; Venkitaraman, S.; Spring, D. Linear Aliphatic Dialkynes as Alternative Linkers for Double-Click Stapling of p53-Derived Peptides. ChemBioChem 2014, 15, 2680−2683. (15) Zhang, J.; Kemmink, J.; Rijkers, D.; Liskamp, R. Synthesis of 1,5Triazole Bridged Vancomycin CDE-ring Bicyclic Mimics using RuAAC Macro Cyclization. Chem. Commun. 2013, 49, 4498−4500. (16) Jacobsen, O.; Maekawa, H.; Ge, N.; Gorbitz, C.; Rongved, P.; Ottersen, O.; Amiry-Moghaddam, M.; Klaveness, J. Stapling of a 310Helix with Click Chemistry. J. Org. Chem. 2011, 76, 1228−1238. (17) Dirksen, A.; Madsen, M.; Iacono, G.; Matin, M.; Bacica, M.; Stankovic, N.; Callans, S.; Bhat, B. Parallel Synthesis and Screening of Peptide Conjugates. Bioconjugate Chem. 2014, 25, 1052−1060. (18) Kotha, S.; Goyal, D.; Bitra, A.; Thota, N.; Kruger, T.; Anand, R. Diversity Oriented Approach to Triazole Based Peptidomimetics as Mammalian Sterile 20 Kinase Inhibitors. RSC Adv. 2013, 3, 24447− 24454. (19) Valverde, I. E.; Mindt, T. L. 1,2,3-Triazoles as Amide-Bond Surrogates in Peptidomimetics. Chimia 2013, 67, 262−267. (20) Pedersen, D. S.; Abell, A. 1,2,3-Triazoles in Peptidomimetic Chemistry. Eur. J. Org. Chem. 2011, 13, 2399−2411. (21) Gehringer, M.; Forster, M.; Laufer, S. A. Solution-Phase Parallel Synthesis of Ruxolitinib-Derived Janus Kinase Inhibitors via CopperCatalyzed Azide-Alkyne Cycloadditions. ACS Comb. Sci. 2015, 17, 5− 10. (22) Dangroo, N. A.; Dar, A. A.; Dar, B. A. An Efficient Protocol for Domino One Pot Synthesis of 1,2,3-Triazoles from Natural Organic Acids and Phenols. Tetrahedron Lett. 2014, 55, 6729−6733. (23) Echemendia, R.; Concepcion, O.; Morales, F.; Paixao, M. W. The Cu1-Catalyzed Alkyne-Azide Cycloaddition as Direct Conjugation/Cyclization Method of Peptides to Steroids. Tetrahedron 2014, 70, 3297−3305. (24) Pehere, A. D.; Abell, A. D. New β−Strand Templates Constrained by Huisgen Cycloaddition. Org. Lett. 2012, 14, 1330− 1333. (25) Kolarovic, A.; Schnurch, M.; Mihovilovic, M. D. Tandem Catalysis: From Alkynoic Acids and Aryl Iodides to 1,2,3-Triazoles in One-pot. J. Org. Chem. 2011, 76, 2613−2618. (26) Latyshev, G. V.; Baranov, M. S.; Kazantsev, A. V.; Averin, A. D.; Lukashev, N. V.; Beletskaya, I. P. Copper-Catalyzed [1,3]-Dipolar Cycloaddition for the Synthesis of Macrocycles Containing Acyclic, Aromatic and Steroidal Moieties. Synthesis 2009, 15, 2605−2615. (27) Kumar, R.; Maulik, P. R.; Misra, A. K. Significant Rate Accelerated Synthesis of Glycosyl Azides and Glycosyl 1,2,3-Triazole Conjugates. Glycoconjugate J. 2008, 25, 595−602.
alkyne 5a. Overall, this methodology was highly reproducible in parallel format and tolerant of diverse reaction partners. In all examples attempted, the desired product was observed as the major component of the reaction mixture. In conclusion, a one pot solution phase method was used to synthesize varied N1-substitued triazole peptidomimetics via a Cu(I) catalyzed tandem azide addition−cycloaddition reaction. This one-pot, two-step approach reduced the potential hazard of isolating azide intermediates and proceeded in good yields with highly varied reaction partners. The mild nature of this reaction was compatible with sensitive peptide functionality. While N-terminal modification was exemplified, one could easily envision extending this methodology to side chain or Cterminal modification through incorporation of alternative alkynyl monomers. This general procedure should find continued use in the field of peptidomimetic synthesis.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acscombsci.5b00150. Experimental procedures and characterization for all new compounds (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail: steven.b.coffey@pfizer.com. Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Dr. Vincent Mascitti, Dr. David Piotrowski, and Mr. Jason Ramsay of Pfizer, Inc., for their assistance in this research.
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REFERENCES
(1) Craik, D. J.; Fairlie, D. P.; Liras, S.; Price, D. The Future of Peptide-Based Drugs. Chem. Biol. Drug Des. 2013, 81, 136−147. (2) Boutureira, O.; Bernardes, G. J. L. Advances in Chemical Protein Modification. Chem. Rev. 2015, 115, 2174−2195. (3) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings. Adv. Drug Delivery Rev. 2012, 64, 4. (4) Chan, A. O.; Ho, C.; Chong, H.; Leung, Y.; Huang, J.; Wong, M.; Che, C. Modification of N-Terminal α-Amino Groups of Peptides and Proteins Using Ketenes. J. Am. Chem. Soc. 2012, 134, 2589−2598. (5) Meldal, M.; Tornøe, C. Cu-Catalyzed Azide-Alkyne Cycloaddition. Chem. Rev. 2008, 108, 2952−3015. (6) Tornøe, C.; Christensen, C.; Meldal, M. Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)-Catalyzed 1,3Dipolar Cycloadditions of Terminal Alkynes to Azides. J. Org. Chem. 2002, 67, 3057−3062. (7) Rostovtsev, V.; Green, L.; Fokin, V.; Sharpless, K. B. A Stepwise Huisgen Cycloaddition Process: Copper (I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem., Int. Ed. 2002, 41, 2596−2599. 708
DOI: 10.1021/acscombsci.5b00150 ACS Comb. Sci. 2015, 17, 706−709
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ACS Combinatorial Science (28) Kocalka, P.; Andersen, N. K.; Jensen, F.; Nielsen, P. Synthesis of 5-(1,2,3-Triazol-4-yl)-2′-Deoxyuridines by a Click Chemistry Approach: Stacking of Triazoles in the Major Groove Gives Increased Nucleic acid Duplex Stability. ChemBioChem 2007, 8, 2106−2116. (29) Andersen, J.; Bolvig, S.; Liang, X. Efficient One-pot Synthesis of 1-Aryl 1,2,3-Triazoles from Aryl Halides and Terminal Alkynes in the Presence of Sodium Azide. Synlett 2005, 19, 2941−2947. (30) General procedure for the amidation of the peptides: In an 8 mL vial was dissolved c-terminal protected peptide (0.22 mmol), 2,2dimethylbut-3-ynoic acid (0.22 mmol), iPr2NEt (0.66 mmol), and HBTU (0.22 mmol) into 4 mL of DMF. The reaction was stirred at ambient temperature for 16 h. The reaction was concentrated, dissolved into ethyl acetate, and washed with sat. NaHCO3, brine, and the organic layer was dried over Na2SO4, filtered, and concentrated to a colorless oil. TLC in 7:3 ethyl acetate/heptane shows single spot Rf = 0.6. The crude material was purified using a 5 g silica precolumn and a 12 g silica analytical column with a 0−50% ethyl acetate/heptane gradient. (31) General library procedure: The following stock solutions were prepared: DMSO, alkyne (0.3 mmol), N1,N2-dimethylcyclohexane1,2-diamine (6.4 mg, 0.03 mmol) into 1.2 mL of DMSO; aqueous, sodium ascorbate (6 mg, 0.03 mmol), sodium azide (20.8 mg, 0.319 mmol) dissolved into 0.27 mL of water. CuI (5.8 mg, 0.03 mmol) was dissolved in 0.09 mL of DMSO. To each vial containing the halide (0.050 mmol, 1 equiv), was added 200 uL of an alkyne stock solution, 45 μL of the aqueous stock solution and 15 μL of the CuI stock solution giving a 5:1 DMSO/water mixture. The reactions were purged and filled with nitrogen 3× and stirred at 70 °C (5.5 h); then, they were stirred at ambient temperature for 14 h. One mL of water was added, and the reactions were extracted with 3 mL of ethyl acetate. The organic layer was transferred to a clean vial and concentrated using a Genevac. The resulting crude oils were dissolved into 1 mL DMSO and purified by HPLC (mass triggered), Atlantis dC18 5 um, 4.6 × 50 mm column, acetonitrile/water/0.05% TFA, 5 min run time, 2 mL/min., starting gradient was 95/5 (ACN/water).
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DOI: 10.1021/acscombsci.5b00150 ACS Comb. Sci. 2015, 17, 706−709
