Inverse-Electron-Demand Diels–Alder Reactions for the Synthesis of

Letter pubs.acs.org/OrgLett

Cite This: Org. Lett. XXXX, XXX, XXX−XXX

Inverse-Electron-Demand Diels−Alder Reactions for the Synthesis of Pyridazines on DNA Hailong Li,† Zhen Sun,† Wenting Wu,† Xuan Wang,†,‡ Mingqiang Zhang,† Xiaojie Lu,‡ Wenge Zhong,†,§ and Dongcheng Dai*,† †

Downloaded via UNIV OF TEXAS AT EL PASO on October 27, 2018 at 10:54:01 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Department of Discovery Modalities, Amgen Asia R&D Center, Amgen Research, 4560 Jinke Road, Pudong, Shanghai 201210, P. R. China ‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, 501 Haike Road, Zhang Jiang Hi-Tech Park, Pudong, Shanghai 201203, P. R. China S Supporting Information *

ABSTRACT: The synthesis of pyridazines on DNA has been developed on the basis of inverse-electron-demand Diels−Alder (IEDDA) reactions of 1,2,4,5-tetrazines. The broad substrate scope is explored. Functionalized pyridazine products are selected for subsequent DNA-compatible Suzuki−Miyaura coupling, acylation, and SNAr substitution reactions, demonstrating the feasibility and versatility of IEDDA reactions for DNA-encoded library synthesis.

S

radioactive groups, providing early evidence that IEDDA reactions are DNA compatible.7 However, to the best of our knowledge, the direct usage of IEDDA reactions in building DELs has not been reported. Recently, our laboratories embarked on an effort to develop a DEL technology platform to support small molecule drug discovery and thus decided to explore the potential of IEDDA reactions for DEL synthesis. Pyridazines have been found in biologically active natural products, drugs and drug candidates (Figure 1) and are thought to be a privileged structure in medicinal chemistry.8 One of the most efficient methods for pyridazine synthesis involves IEDDA reactions of 1,2,4,5-tetrazines.9 Here, we report two complementary approaches for the construction of pyridazine scaffolds on DNA via IEDDA reactions of 1,2,4,5tetrazines with alkenes and carbonyl compounds, respectively. We further demonstrate that functionalized pyridazine products can undergo various additional transformations for potential DEL construction. We first investigated the synthesis of pyridazines by the IEDDA reaction between DNA-linked olefin and different 1,2,4,5-tetrazines. To achieve this, the DNA headpiece was

ince its conception by Brenner and Lerner in 1992, DNAencoded library (DEL) technology has increasingly become one of the most powerful hit generation platforms in drug discovery.1 A large number of hits have been identified by DEL technology for various targets,2 and two drug candidates originated from their corresponding initial DEL hits have entered into clinical studies.3 Despite this success, substantial challenges remain in order to fully realize the potential of this technology. One of the most fundamental challenges is the development of new DNA-compatible reactions that will allow more structural diversity and flexibility in DEL design and synthesis. Among the many DNA-compatible reactions developed in recent years,4 the Diels−Alder reaction has emerged as a reliable and useful method in DEL construction. Neri and coworkers first reported the design and synthesis of such DELs and the discovery of carbonic anhydrase IX inhibitors and TNF inhibitors from these libraries.5 Inverse-electron-demand Diels−Alder (IEDDA) reactions are versatile methods and have been extensively employed in the synthesis of heterocyclic natural products and compounds of pharmaceutical interest.6 Indeed, strain-promoted IEDDA reactions of 1,2,4,5-tetrazines have been applied to label DNA by forming (dihydro)pyridazines as linkages between DNA and fluorescent or © XXXX American Chemical Society

Received: September 29, 2018

A

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

furnish pyridazine 12a (Table 2, entry 1).10 With these optimized oxidative aromatization conditions in hand, we explored the olefin substrate scope, and the results are summarized in Table 2. When DNA-linked 10 was subjected to various styrenes, the IEDDA-aromatization reactions proceeded smoothly to produce the pyridazines (Table 2, entries 2−9). The standard reaction conditions were found to be tolerant of many different functional groups such as halo, carboxylic, and amino groups. The regioselectivity of the IEDDA reaction and the structure of product 12b were indirectly established based on a coinjection experiment by LC/MS of 12b from the on-DNA IEDDA reaction and authentic samples 12ba and 12bb from off-DNA syntheses followed by conjugations with DNA headpiece (see the Supporting Information). It is worth pointing out that regiochemical structures of the products are tentatively assigned at this stage. It is believed that at the hit identification and confirmation stage, off-DNA synthesis will help to unequivocally determine the regioselectivity and exact structures. Additional results are also summarized in Table 2. Poor conversion with vinyl naphthalene 11j was observed, likely due to its low solubility in DMSO/water cosolvent (Table 2, entry 10). Heteroaromatic olefins 11k and 11l as well as 1,2-disubstituted alkene 11m were obtained in 74%, 73%, and 63% conversions, respectively (Table 2, entries 11− 13). Interestingly, when tetrazine 10 was treated with acyclic olefin 11n under optimized conditions (200 equiv, 20 °C for 8 h), no desired dihydropyridazine intermediate or 12n was detected. Once the reaction mixture was heated at 50 °C for 8 h, we found that pyridazine 12n was formed in 90% conversion without the need of copper oxidant (Table 2, entry 14). The copper-free IEDDA-oxidative aromatization process was also applied to 11o and 11p. The higher conversion of pyridazine 12o over 12p might be attributed to the carboxyl group on the cyclopentene ring that makes 11o more soluble in aqueous media (Table 2, entries 15 and 16). The major competition reaction for IEDDA reaction of 10 with 11p was thought to be the insertion of water to 10 and formation of hydrazide.11 The IEDDA reaction of 10 with bridged cyclopentene 11q and 2,3-

Figure 1. Pyridazine-containing natural products, drugs, and drug candidates.

covalently conjugated to a commercially available transcyclooctene to form substrate 7. We briefly screened the IEDDA reaction conditions and found that it was optimal to conduct the reaction between 7 and tetrazine 8a (200 equiv) in DMSO/water (1/1) at 20 °C (Table 1, entry 1). Strong oxidants were not required for the oxidative aromatization reaction of dihydropyridazine intermediate to form 9a. In this case, oxidation occurred spontaneously under the ambient atmosphere. As depicted in Table 1, the IEDDA-oxidation reactions of olefin 7 with various tetrazines 8b−f proceeded smoothly to provide pyridazines 9b−f which contained a variety of functional groups such as hydroxyl, methylthio, amino, and halo groups (Table 1, entries 2−6). We next explored the IEDDA reactions of DNA-conjugated tetrazine 10 utilizing more readily available and structurally diverse olefins. The IEDDA reaction of tetrazine 10 with transcyclooctene 11a proceeded efficiently to afford the dihydropyridazine intermediate; however the subsequent oxidative aromatization proved to be difficult without additional oxidants. After a brief optimization of oxidation conditions, we found that the combination of Cu(ClO4)2, bipyridine, and TEMPO effectively promoted the aromatization process to

Table 1. Inverse-Electron-Demand Diels−Alder Reactions between DNA-Linked Alkene 7 and Tetrazine 8a

a

Reaction conditions: unless otherwise noted, all reactions were carried out with 7 (10 nmol) and 8 (2000 nmol, 200 equiv) in DMSO/water (1/ 1) at 20 °C. bRegioselectivity was tentatively assigned. cConversion of pyridazine determined by LCMS. B

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Table 2. Inverse-Electron-Demand Diels−Alder Reactions between DNA-Linked Tetrazine 10 and Alkene 11a

a

Reaction conditions: unless otherwise noted, all reactions were carried out with 10 (10 nmol) and 11 (2000 nmol, 200 equiv) in DMSO/water (1/1) at 20 °C and oxidation in the same reaction tube by Cu(ClO4)2/ bipyridine/TEMPO (1/1/1, 200 nmol, 20 equiv) at 20 °C. b Regioselectivity was tentatively assigned. cConversion of dihydropyridazine or pyridazine determined by LCMS. dIEDDA reactions were carried out at 50 °C with another batch of 11 (2000 nmol, 200 equiv).

(dihydro)pyridazine 12t was identified at 50 °C with prolonged reaction time, and hydrazide was found as the major product, presumably due to lack of strain promotion and/or low solubility in aqueous condition (Table 2, entry 20). Despite the encouraging results of pyridazines synthesis on DNA by the sequential IEDDA-oxidation aromatization reactions, the difficulties encountered in some cases prompted us to find surrogates of alkenes as dienophiles that might avoid the oxidation step. Toward this end, we tested the IEDDA reaction of 10 with 200 equiv of enol ethers 13a and 13b at 50

dihydrofuran 11r worked effectively to give dihydropyridazine adduct 12q and 12r; however, when the oxidation was carried out under the optimal conditions, the desired pyridazine was not identified, and dihydropyridazine intermediates were recovered (Table 2, entries 17 and 18). When 12r was treated with stronger aromatization oxidants, such as DDQ and PhI(OAc)2,12 decomposition of oligo DNA of 12r was observed. For 3,4-dihydropyran 11s, only dihydropyridazine 12s was detected in low conversion even at higher temperatures (Table 2, entry 19). In the case of cyclohexene 11t, no C

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Table 3. Inverse-Electron-Demand Diels−Alder Reactions between DNA-Linked Tetrazine 10 and Alkene Surrogate 13a

a

Reaction conditions: unless otherwise noted, all reactions were carried out with 10 (10 nmol) and 13 (2000 nmol, 200 equiv) in DMSO/water (1/1) at 20 °C in the presence of proline (400 nmol, 200 equiv). bRegioselectivity was tentatively assigned. cConversion of pyridazine determined by LCMS.

°C for 8 h and found that the corresponding pyridazines 14a and 14b were formed without the need of oxidation (Table 3, entries 1 and 2). These initial results together with the work by Boger13 and Wang14 inspired us to explore the IEDDA reaction between 10 and enamines generated in situ from ketones and aldehydes to form pyridazines. After a brief optimization of IEDDA reaction conditions, the substrate scope of ketones and aldehydes was explored. As illustrated in Table 3, proline-promoted IEDDA reactions of tetrazine 10 with acetone 13c and cyclopentanone 13d worked effectively at 20 °C to give 85% of 14b and 87% of 12p, respectively (Table 3, entries 3 and 4). Tetrazine 10 reacted with cyclohexanone 13e, pyranone 13f, piperidinone 13g,h, and thiopyranone 13i to provide the corresponding fused pyridazines efficiently (Table 3, entries 5−9). The conversion of tetrazine 10 with cycloheptanone 13j was low, possibly due to low solubility and/or less effective in proline-promoted enamine formation (Table 3, entry 10).15 The cycloaddition reactions of 10 with aldehydes 13k and 13l proceeded smoothly to furnish the pyridazines on DNA (Table 3, entries 11 and 12). It is worth noting that for some pyridazine products the proline-promoted enamine IEDDA reaction gave superior results compared to those obtained from the corresponding olefins or enol ethers. For example, conversion of 14b was improved from 28% to 85% (Table 3, entry 2 vs entry 3), 12p from 38% to 87% (Table 2, entry 16 vs Table 3

entry 4), and 12t from no detected product formation to 83% (Table 2, entry 20 vs Table 3, entry 5), respectively. With the construction of the pyridazines on DNA by two complementary protocols using the IEDDA reaction, we then conducted additional transformations on the functionalized pyridazines to further confirm the pyridazine structure and investigate the synthetic utility of the pyridazines. The Suzuki− Miyaura cross-coupling reaction between DNA-linked 12c and phenylboronic acid took place efficiently to afford 69% of the desired product 15c (Table 4, entry 1). When 12f was treated with activated tetrazine ester, 85% conversion to the acylated product 15f was observed (Table 4, entry 2). The desired SNAr substitution product 15l was obtained in 80% conversion when 12l was reacted with diethylamine in borate buffer (Table 4, entry 3). In summary, we report herein the synthesis of pyridazines on DNA utilizing IEDDA-aromatization reactions of 1,2,4,5tetrazines with olefins and carbonyl compounds. We demonstrated that both on-DNA processes can be carried out efficiently at low concentrations in aqueous media and have a broad substrate scope, especially for functionalized styrenes and ketones. We further demonstrated that subsequent Suzuki−Miyaura coupling, acylation, and SNAr substitution reactions with the functionalized pyridazines on DNA work effectively, laying a solid foundation for future DEL D

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters

■

Table 4. Post Transformations on DNA-Linked Pyridazine 12

Conversion determined by LCMS.

synthesis. Continued efforts in expanding the IEDDA reactions on DNA and DEL construction will be reported in due course.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03114.

■

REFERENCES

(1) For selected reviews on DNA-encoded library techology, see: (a) Neri, D.; Lerner, R. A. Annu. Rev. Biochem. 2018, 87, 479. (b) Lerner, R. A.; Brenner, S. Angew. Chem., Int. Ed. 2017, 56, 1164. (c) Arico-Muendel, C. C. MedChemComm 2016, 7, 1898. (d) Goodnow, R. A., Jr; Dumelin, C. E.; Keefe, A. D. Nat. Rev. Drug Discovery 2017, 16, 131. (e) Yuen, L. H.; Franzini, R. M. ChemBioChem 2017, 18, 829. (f) Zimmermann, G.; Neri, D. Drug Discovery Today 2016, 21, 1828. (2) For selected reviews on target screening by DNA-encoded libraries: see ref 1c. For selected examples on targets screened by DNA-encoded libraries, see the references with ref 4k. (3) (a) Harris, P. A.; Berger, S. B.; Jeong, J. U.; Nagilla, R.; Bandyopadhyay, D.; Campobasso, N.; Capriotti, C. A.; Cox, J. A.; Dare, L.; Dong, X.; Eidam, P. M.; Finger, J. N.; Hoffman, S. J.; Kang, J.; Kasparcova, V.; King, B. W.; Lehr, R.; Lan, Y.; Leister, L. K.; Lich, J. D.; MacDonald, T. T.; Miller, N. A.; Ouellette, M. T.; Pao, C. S.; Rahman, A.; Reilly, M. A.; Rendina, A. R.; Rivera, E. J.; Schaeffer, M. C.; Sehon, C. A.; Singhaus, R. R.; Sun, H. H.; Swift, B. A.; Totoritis, R. D.; Vossenkämper, A.; Ward, P.; Wisnoski, D. D.; Zhang, D.; Marquis, R. W.; Gough, P. J.; Bertin, J. J. Med. Chem. 2017, 60, 1247. (b) Belyanskaya, S. L.; Ding, Y.; Callahan, J. F.; Lazaar, A. L.; Israel, D. I. ChemBioChem 2017, 18, 837. (4) For selected examples on DNA-compatible reactions, see: (a) Satz, A. L.; Cai, J.; Chen, Y.; Goodnow, R.; Gruber, F.; Kowalczyk, A.; Petersen, A.; Naderi-Oboodi, G.; Orzechowski, L.; Strebel, Q. Bioconjugate Chem. 2015, 26, 1623. (b) Li, Y.; Gabriele, E.; Samain, F.; Favalli, N.; Sladojevich, F.; Scheuermann, J.; Neri, D. ACS Comb. Sci. 2016, 18, 438. (c) Tian, X.; Basarab, G. S.; Selmi, N.; Kogej, T.; Zhang, Y.; Clark, M.; Goodnow, R. A., Jr. MedChemComm 2016, 7, 1316. (d) Thomas, B.; Lu, X.; Birmingham, W. R.; Huang, K.; Both, P.; Juana, E. R. M.; Young, R. J.; Davie, C. P.; Flitsch, S. L. ChemBioChem 2017, 18, 858. (e) Klika Š kopić, M.; Willems, S.; Wagner, B.; Schieven, J.; Krause, N.; Brunschweiger, A. Org. Biomol. Chem. 2017, 15, 8648. (f) Fan, L.; Davie, C. P. ChemBioChem 2017, 18, 843. (g) Lu, X.; Fan, L.; Phelps, C. B.; Davie, C. P.; Donahue, C. P. Bioconjugate Chem. 2017, 28, 1625. (h) Lu, X.; Roberts, S. E.; Franklin, G. J.; Davie, C. P. MedChemComm 2017, 8, 1614. (i) Š kopić, M. K.; Salamon, H.; Bugain, O.; Jung, K.; Gohla, A.; Doetsch, L. J.; Santos, D. d.; Bhat, A.; Wagner, B.; Brunschweiger, A. Chem. Sci. 2017, 8, 3356. (j) Wang, J.; Lundberg, H.; Asai, S.; Martín-Acosta, P.; Chen, J. S.; Brown, S.; Farrell, W.; Dushin, R. G.; O’Donnell, C. J.; Ratnayake, A. S.; Richardson, P.; Liu, Z.; Qin, T.; Blackmond, D. G.; Baran, P. S. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, No. E6404. (k) Wang, X.; Sun, H.; Liu, J.; Dai, D.; Zhang, M.; Zhou, H.; Zhong, W.; Lu, X. Org. Lett. 2018, 20, 4764. (l) Ruff, Y.; Berst, F. MedChemComm 2018, 9, 1188. (m) Kölmel, D. K.; Loach, R. P.; Knauber, T.; Flanagan, M. E. ChemMedChem 2018, 13, 1. (5) DNA-encoded library synthesis and screening based on Diels− Alder reactions: (a) Buller, F.; Mannocci, L.; Zhang, Y.; Dumelin, C. E.; Scheuermann, J.; Neri, D. Bioorg. Med. Chem. Lett. 2008, 18, 5926. (b) Buller, F.; Zhang, Y.; Scheuermann, J.; Schäfer, J.; Bühlmann, P.; Neri, D. Chem. Biol. 2009, 16, 1075. (c) Buller, F.; Steiner, M.; Frey, K.; Mircsof, D.; Scheuermann, J.; Kalisch, M.; Bühlmann, P.; Supuran, C. T.; Neri, D. ACS Chem. Biol. 2011, 6, 336. (6) For selected examples on synthesis of heterocyclic natural products based on IEDDA reactions: (a) Boger, D. L.; Panek, J. S. J. Am. Chem. Soc. 1985, 107, 5745. (b) Boger, D. L.; Coleman, R. S. J. Am. Chem. Soc. 1987, 109, 2717. (c) Boger, D. L.; Zhang, M. J. Am. Chem. Soc. 1991, 113, 4230. (d) Boger, D. L.; Baldino, C. M. J. Am. Chem. Soc. 1993, 115, 11418. (e) Boger, D. L.; Boyce, C. W.; Labroli, M. A.; Sehon, C. A.; Jin, Q. J. Am. Chem. Soc. 1999, 121, 54. (f) Boger, D. L.; Hong, J. J. Am. Chem. Soc. 2001, 123, 8515. (g) Hamasaki, A.; Zimpleman, J. M.; Hwang, I.; Boger, D. L. J. Am. Chem. Soc. 2005, 127, 10767. For selected examples on the synthesis of compounds of pharmaceutical interest based on IEDDA reactions, see: (h) Che, D.; Wegge, T.; Stubbs, M. T.; Seitz, G.; Meier, H.; Methfessel, C. J. Med. Chem. 2001, 44, 47. (i) Gündisch, D.; Kämpchen, T.; Schwarz, S.; Seitz, G.; Siegl, J.; Wegge, T. Bioorg. Med. Chem. 2002, 10, 1. (j) Biros,

a

■

Letter

Experimental procedures and copies of HPLC traces, MS, and NMR spectra (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Xiaojie Lu: 0000-0002-3600-288X Dongcheng Dai: 0000-0001-7962-3718 Present Address §

(W.Z.) Regor Therapeutics, Inc., Shanghai, China.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Jiaxiang Liu and Min Chen from the Shanghai Institute of Materia Medica for LCMS and Dr. David J. St. Jean, Jr. of Amgen for helpful discussions. E

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters S. M.; Moisan, L.; Mann, E.; Carella, A.; Zhai, D.; Reed, J. C.; Rebek, J. Bioorg. Med. Chem. Lett. 2007, 17, 4641. (k) Dalla Via, L.; Gia, O.; Marciani Magno, S.; Braga, A.; González-Gómez, J. C.; PérezMontoto, L. G.; Uriarte, E. Bioorg. Med. Chem. 2010, 18, 5708. (l) Boländer, A.; Kieser, D.; Voss, C.; Bauer, S.; Schön, C.; Burgold, S.; Bittner, T.; Hölzer, J.; Heyny-von Haußen, R.; Mall, G.; Goetschy, V.; Czech, C.; Knust, H.; Berger, R.; Herms, J.; Hilger, I.; Schmidt, B. J. Med. Chem. 2012, 55, 9170. (7) For selected examples on DNA-labeling based on IEDDA reactions, see: (a) Schoch, J.; Wiessler, M.; Jäschke, A. J. Am. Chem. Soc. 2010, 132, 8846. (b) Schoch, J.; Staudt, M.; Samanta, A.; Wiessler, M.; Jäschke, A. Bioconjugate Chem. 2012, 23, 1382. (c) Š ečkutė, J.; Yang, J.; Devaraj, N. K. Nucleic Acids Res. 2013, 41, No. e148. (d) Asare-Okai, P. N.; Agustin, E.; Fabris, D.; Royzen, M. Chem. Commun. 2014, 50, 7844. (e) Rieder, U.; Luedtke, N. W. Angew. Chem., Int. Ed. 2014, 53, 9168. (f) George, J. T.; Srivatsan, S. G. Bioconjugate Chem. 2017, 28, 1529. (8) For selected reviews on pyridazines in medicinal chemistry, see: (a) Wermuth, C. G. MedChemComm 2011, 2, 935. (b) Jaballah, M. Y.; Serya, R. T.; Abouzid, K. Drug Res. (Stuttgart, Ger.) 2017, 67, 138. (9) For selected reviews on pyridazine synthesis based on IEDDA reactions of 1,2,4,5-tetrazines, see: (a) Boger, D. L. Chem. Rev. 1986, 86, 781. (b) Boger, D. L.; Patel, M. Prog. Heterocycl. Chem. 1989, 1, 30. (c) Sauer, J. In Comprehensive Heterocyclic Chemistry II; Pergamon: London, 1996; Vol. 6; p 901. (10) Matsushita, T.; Moriyama, Y.; Nagae, G.; Aburatani, H.; Okamoto, A. Chem. Commun. 2017, 53, 5756. (11) Hoogenboom, R.; Moore, B. C.; Schubert, U. S. J. Org. Chem. 2006, 71, 4903. (12) (a) Selvaraj, R.; Fox, J. M. Tetrahedron Lett. 2014, 55, 4795. (b) Warrener, R.; Harrison, P. Molecules 2001, 6, 353. (13) (a) Hamasaki, A.; Ducray, R.; Boger, D. L. J. Org. Chem. 2006, 71, 185. (b) Boger, D. L.; Schaum, R. P.; Garbaccio, R. M. J. Org. Chem. 1998, 63, 6329. (14) Xie, H.; Zu, L.; Oueis, H. R.; Li, H.; Wang, J.; Wang, W. Org. Lett. 2008, 10, 1923. (15) Castro-Alvarez, A.; Carneros, H.; Costa, A. M.; Vilarrasa, J. Synthesis 2017, 49, 5285.

F

DOI: 10.1021/acs.orglett.8b03114 Org. Lett. XXXX, XXX, XXX−XXX