- Email: [email protected]
Scalable syntheses of the BET bromodomain inhibitor JQ1
Tetrahedron Letters 56 (2015) 3454–3457
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Scalable syntheses of the BET bromodomain inhibitor JQ1 Shameem Sultana Syeda, Sudhakar Jakkaraj, Gunda I. Georg ⇑ Department of Medicinal Chemistry and Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, United States
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 8 February 2015 Accepted 13 February 2015
Keywords: Bromodomains BET inhibitors Triazolothienodiazepine (+)-JQ1 Male contraceptive Thionation One-pot method
a b s t r a c t We have developed methods involving the use of alternate, safer reagents for the scalable syntheses of the potent BET bromodomain inhibitor JQ1. A one-pot three step method, involving the conversion of a benzodiazepine to a thioamide using Lawesson’s reagent, followed by amidrazone formation and installation of the triazole moiety furnished JQ1. This method provides good yields and a facile purification process. For the synthesis of enantiomerically enriched (+)-JQ1, the highly toxic reagent diethyl chlorophosphate, used in a previous synthesis, was replaced with the safer reagent diphenyl chlorophosphate in the three-step one-pot triazole formation without effecting yields and enantiomeric purity of (+)-JQ1. Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction The bromodomain (BRD) proteins are an important class of histone reader proteins that recognize acetylated lysine residues (KAc) on histone tails and direct transcription complexes to turn on genes.1 The bromodomain motif is short with approximately 110 amino acids that are conserved in several human genes.2 The human genome encodes 61 BRDs, which are present in 46 diverse proteins.3 Crystal structures of BRDs from more than 20 different proteins demonstrated that bromodomains share a conserved deep hydrophobic pocket formed by a left-handed four-helical bundle (aA, aB, aC, aZ) and loop regions of various lengths (ZA and BC),3,4 which constitute an attractive pocket for the development of selective protein–protein interaction inhibitors. Among the eight BRD families, the BRD and BET proteins have been found to be tractable for drug discovery. Chemical inhibition of BET proteins exerts a broad spectrum of desirable biological effects such as anticancer, anti-inflammatory, and male contraceptive properties.1,5 Triazolothienodiazepine (+)-JQ1 is one of the first selective inhibitors with nanomolar affinity for BET proteins (BRD2, BRD3, BRD4 and BRDT).6 The discovery of this potent, selective, and permeable inhibitor for BETs has stimulated research activity in diverse therapeutic areas, particularly in oncology, inflammation, and viral infection. Two other BET inhibitors I-BET7627,8 and OTX0155 (Fig. 1) that share the JQ1 scaffold are in clinical trials for the treatment of NUT midline carcinoma (a rare cancer caused by
BRD4-NUT fusions), acute myeloid leukemia, Burkitt’s lymphoma, and multiple myeloma.6,9,10 (+)-JQ1 also served as a probe to validate testis-specific BRDT as a promising reversible male contraceptive agent.11 As a part of our collaborative efforts to discover non-hormonal male contraceptive agents, we required a significant quantity of (+)-JQ1 for further evaluations. To meet the demand of (+)-JQ1, we developed a scalable and safer route to JQ1 based on reported methods. The reported synthesis that is shown in Scheme 1 provides racemic JQ1, although the synthesis employs the L-amino acid (Fmoc-Asp-(OtBu)-OH) (2). Racemization was observed at several different stages of the synthesis, including the peptide coupling, the aminoketone cyclization, and the thionation.6 Scheme 2 details the synthesis of enantiomerically enriched (+)-JQ1 with 90% optical purity. While both methods provide short and efficient methods for the synthesis of JQ1, we were concerned about the large scale use of excess P2S5 for the conversion of amide 5 to
⇑ Corresponding author. Tel.: +1 612 626 6320; fax: +1 612 626 6318. E-mail address: [email protected] (G.I. Georg).
Figure 1. Structures of BET inhibitors.
http://dx.doi.org/10.1016/j.tetlet.2015.02.062 0040-4039/Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457
3455
Scheme 1. Filippakopoulos et al. method for the synthesis of (±)-JQ1.6
Scheme 2. Filippakopoulos et al. method for the synthesis of enantiomerically enriched (+)-JQ1.6
thioamide 6 (Scheme 1) and the use of diethyl chlorophosphate for the conversion of intermediate 5 to (+)-JQ1 (Scheme 2). Excess P2S5 is problematic during work-up because of H2S gas evolution. This gas is challenging to trap on a large scale, and possesses a noxious, sulfur-related odor. In addition, the reagent diethyl chlorophosphate used in the conversion of amide 5 to (+)-JQ1 (Scheme 2) is classified as possessing acute oral, dermal, and inhalation toxicities. Results and discussion In our efforts to circumvent H2S associated issues for thioamide formation, we replaced P2S5 (4 equiv) with Lawesson’s reagent (0.5 equiv) for thionation. Initially, 1 mmol of amide 5, prepared by methods shown in Scheme 1, was treated with Lawesson’s reagent in refluxing toluene to afford thioamide 6 in 75% yield (Scheme 3). Although thionation proceeded efficiently on a 1 mmol scale, it provided only a moderate yield (50%) on a larger scale. Because of the presence of poorly soluble byproducts, purification of this reaction was difficult and required multiple column
Scheme 3. Thionation of amide 5 with Lawesson’s reagent.
purifications to obtain pure thioamide 6. While we were pursuing the reaction with Lawesson’s reagent Zhang et al. reported12 a similar JQ1 synthesis using Lawesson’s reagent for amide thionation. They observed moderate yields, analogous to our results. Next we aimed at optimizing a scalable strategy for the synthesis of JQ1. To achieve this, we designed a one-pot strategy13 for the conversion of amide 5 to JQ1. Our one-pot strategy (Scheme 4) started with the treatment of amide 5 with Lawesson’s reagent in THF at 80 °C for 2 h (monitored by TLC), followed by the addition of excess hydrazine hydrate (10 equiv) at 0 °C. The reaction mixture was stirred for 30 min (monitored by TLC) to yield amidrazone 7, which was used for the next step directly after aqueous work-up. Amidrazone 7 was heated to 110 °C for 2 h in a mixture of
Scheme 4. Synthesis of (±)-JQ1 via a one-pot method.
3456
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457
trimethyl orthoacetate and toluene (2:3) to yield the target compound (±)-JQ1 in 60% yield over three steps. We found that the newly designed one-pot strategy (thionation and amidrazone formation) greatly minimized sulfur related concerns (the strong, unpleasant odor of sulfur byproducts). The purification process was also facile and the reaction proceeded with a slightly improved overall yield of 60% compared to the reactions in Scheme 1 (55% overall yield). Using this efficient method we have synthesized 30 g of (±)-JQ1. The reaction was carried out in four batches to minimize sulfur-related odors but can in principle be performed on a larger scale. A related approach for the synthesis of bromodomain inhibitors involving Lawessons’s reagent was disclosed in a recent patent.13 Our next goal was to prepare enantiomerically enriched JQ1, according to the procedure that has been reported by Filippakopoulos et al. (Scheme 2).6 A concern about the reported method is the use of highly toxic diethyl chlorophosphate for the installation of the triazole ring from amide 5 on a larger scale. We thus investigated alternate conditions to replace the highly toxic diethyl chlorophosphate and found that the less toxic diphenyl chlorophosphate (corrosive) was equally reactive to activate the amide functionality and led to the formation of (+)-JQ1 in excellent yields without effecting enantiomeric purity. To the best of our knowledge, there are no previous examples in the literature for the use of diphenyl chlorophosphate for a triazole synthesis. Following the reported method (Scheme 2), benzodiazepine 5 was prepared and then treated with KOtBu and diphenyl chlorophosphate to form phosphorylimidate. 8 (Scheme 5), which was not isolated and subsequently reacted with acetylhydrazide at room temperature for 1 h followed by heating at 90 °C to furnish enantiomerically enriched (+)-JQ1 in 82% yield. The enantiopurity of (+)-JQ1 was determined by chiral HPLC. A 91:9 ratio of the two enantiomers was observed, which is the same as was previously reported for the synthesis of enantioenriched (+)-JQ1 (Scheme 1).6 In conclusion, we have developed methods involving the use of alternate, safer reagents for the synthesis of racemic and enantioenriched JQ1.14 We examined the conversion of amide 5 to thioamide 6 using Lawesson’s reagent and were able to develop a one-pot method for the three-step conversion of amide 5 to racemic JQ1. We found that this method significantly improved the purification process and provided JQ1 in good yields. For the synthesis of enantiomerically enriched (+)-JQ1 we replaced the highly toxic diethyl chlorophosphate reagent used in the literature procedure with the safer reagent diphenyl chlorophosphate in a three-step one-pot synthesis without affecting the yield and the
Scheme 5. Synthesis of enantiomerically enriched (+)-JQ1 with diphenyl chlorophosphate.
enantiomeric purity of (+)-JQ1. These methods should be equally useful to access other BET inhibitors such as I-BET762, OTX015, and related analogues with the same or similar scaffolds. Acknowledgments Financial support for this project is gratefully acknowledged from the NIH/NICHD: 1U01HD076542 and HHSN275201300017C. The determination of the enantiomeric purity of (+)-JQ1 was carried out by Shanghai ChemPartner Co., Ltd China.
References and notes 1. Filippakopoulos, P.; Knapp, S. Nat. Rev. Drug Discovery 2014, 13, 337–356. 2. Haynes, S. R.; Dollard, C.; Winston, F.; Beck, S.; Trowsdale, J.; Dawid, I. B. Nucleic Acids Res. 1992, 20, 2603. 3. Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.-P.; BarsyteLovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.; Gingras, A.-C.; Arrowsmith, C. H.; Knapp, S. Cell 2012, 149, 214–231. 4. Jacobson, R. H.; Ladurner, A. G.; King, D. S.; Tjian, R. Science 2000, 288, 1422– 1425. 5. Arkin, M. R.; Tang, Y.; Wells, J. A. Chem. Biol. 2014, 21, 1102–1114. 6. Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith William, B.; Fedorov, O.; Morse Elizabeth, M.; Keates, T.; Hickman Tyler, T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown Michael, R.; Wang, Y.; Christie Amanda, L.; West, N.; Cameron Michael, J.; Schwartz, B.; Heightman Tom, D.; La Thangue, N.; French Christopher, A.; Wiest, O.; Kung Andrew, L.; Knapp, S.; Bradner James, E. Nature 2010, 468, 1067–1073. The thionation reaction was carried out on 0.5 mmol scale. 7. Mirguet, O.; Gosmini, R.; Toum, J.; Clement, C. A.; Barnathan, M.; Brusq, J.-M.; Mordaunt, J. E.; Grimes, R. M.; Crowe, M.; Pineau, O.; Ajakane, M.; Daugan, A.; Jeffrey, P.; Cutler, L.; Haynes, A. C.; Smithers, N. N.; Chung, C.-W.; Bamborough, P.; Uings, I. J.; Lewis, A.; Witherington, J.; Parr, N.; Prinjha, R. K.; Nicodeme, E. J. Med. Chem. 2013, 56, 7501–7515. 8. Chung, C.-W.; Coste, H.; White, J. H.; Mirguet, O.; Wilde, J.; Gosmini, R. L.; Delves, C.; Magny, S. M.; Woodward, R.; Hughes, S. A.; Boursier, E. V.; Flynn, H.; Bouillot, A. M.; Bamborough, P.; Brusq, J.-M. G.; Gellibert, F. J.; Jones, E. J.; Riou, A. M.; Homes, P.; Martin, S. L.; Uings, I. J.; Toum, J.; Clement, C. A.; Boullay, A.B.; Grimley, R. L.; Blandel, F. M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.; Nicodeme, E. J. Med. Chem. 2011, 54, 3827–3838. 9. Dawson, M. A.; Prinjha, R. K.; Dittman, A.; Giotopoulos, G.; Bantscheff, M.; Chan, W.-I.; Robson, S. C.; Chung, C.-W.; Hopf, C.; Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.; Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet, O.; Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.; Lee, K.; Huntly, B. J. P.; Kouzarides, T. Nature 2011, 478, 529–533. 10. Delmore Jake, E.; Issa Ghayas, C.; Lemieux Madeleine, E.; Rahl Peter, B.; Shi, J.; Jacobs Hannah, M.; Kastritis, E.; Gilpatrick, T.; Paranal Ronald, M.; Qi, J.; Chesi, M.; Schinzel Anna, C.; McKeown Michael, R.; Heffernan Timothy, P.; Vakoc Christopher, R.; Bergsagel, P. L.; Ghobrial Irene, M.; Richardson Paul, G.; Young Richard, A.; Hahn William, C.; Anderson Kenneth, C.; Kung Andrew, L.; Bradner James, E.; Mitsiades Constantine, S. Cell 2011, 146, 904–917. 11. Matzuk, M. M.; McKeown, M. R.; Filippakopoulos, P.; Li, Q.; Ma, L.; Agno, J. E.; Lemieux, M. E.; Picaud, S.; Yu, R. N.; Qi, J.; Knapp, S.; Bradner, J. E. Cell 2012, 150, 673–684. 12. Zhang, C.-J.; Tan, C. Y. J.; Ge, J.; Na, Z.; Chen, G. Y. J.; Uttamchandani, M.; Sun, H.; Yao, S. Q. Angew. Chem. 2013, 52, 14060–14064. The reaction scale for the thionation was not provided. 13. Albrecht, B. K.; Audia, J. E.; Cote, A.; Gehling, V. S.; Harmange, J.-C.; Hewitt, M. C.; Naveschuk, C. G.; Taylor, A. M.; Vaswani, R. G. WO 2012075456A1. 14. Experimental procedures: tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2thioxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (6). In a 25 mL round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (0.42 g, 1.00 mmol) in toluene (10 mL). To this solution Lawesson’s reagent (0.21 g, 0.52 mmol) was added and the resulting suspension was heated to reflux. The reaction mixture was allowed to stir at reflux for 2 h at which point TLC indicated the consumption of the starting material. The solvent was removed under reduced pressure. The resulting residue was purified by column chromatography (silica gel, 0–10% ethyl acetate in hexanes) to afford thioamide 6 (0.32 g, 75%) as a yellow solid. Spectroscopic data for compound 6 were consistent with those described in the literature.12 (±)-JQ1: One-pot method. In a 500 mL round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (9.50 g, 22.72 mmol) in THF (150 mL). To this solution Lawesson’s reagent (4.59 g, 11.36 mmol) was added and the resulted suspension was at reflux for 2 h at which point TLC indicated the consumption of the starting material. The reaction mixture was cooled to 0 °C and hydrazine hydrate solution (50–60%) (14.50 mL, 226.73 mmol) was added dropwise to the reaction mixture over 10 min and continued to stir at 0 °C for 30 min. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3 50 mL). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, and concentrated in vacuo to furnish amidrazone 7, which was used for the next step without purification. The crude
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457 amidrazone 7 (10.50 g, 24.30 mmol) was dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (200 mL) and stirred at 110 °C for 1 h until the reaction was complete (monitored by TLC). The reaction mixture was concentrated in vacuo and the resulting residue was purified by column chromatography (silica gel, 0–100% ethyl acetate in hexanes) to furnish (±)-JQ1 (6.22 g, 60%). The spectral data of compound 3 matched with those described in the literature.6 (+)-JQ1: In a 500 mL two neck round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (3.40 g, 8.13 mmol) in THF (60 mL) and cooled to 78 °C. While stirring at 78 °C, potassium tert-butoxide (1.0 M solution in THF, 8.95 mL, 8.94 mmol, 1.10 equiv) was added. The reaction mixture was warmed to 10 °C and stirred for 30 min. The reaction mixture was cooled to 78 °C and diphenyl chlorophosphate (2.02 mL, 9.76 mmol) was added to the reaction mixture. The resulting mixture was warmed to 10 °C and stirred at this temperature for 45 min. Acetylhydrazide (0.90, 12.20 mmol, 1.50 equiv)
3457
was added to the reaction mixture. The reaction mixture was stirred at room temperature. After 1 h, 1-butanol (70 mL) was added to the reaction mixture, which was heated to 90 °C. After 1 h, all solvents were removed under reduce pressure. The residue was dissolved in CH2Cl2 and washed with saturated aq. NaHCO3, brine, dried over anhydrous Na2SO4 and concentrated in vacuo to obtain crude (+)-JQ1, which was purified by flash column chromatography on silica gel (0–100% ethyl acetate in hexanes) to afford (+)-JQ1 (3.04 g, 82%) as a white solid and with a 91:9 er (determined with Supercritical Fluid Chromatography (SFC) using the CHIRALPAK AS-H column, CO2–EtOH (3:1), 210 nm, tR (R-enantiomer) 1.62 min, tR (S-enantiomer) 3.51 min). This product was further purified by preparative SFC using a CHIRALPAK AS-H column to obtain (+)-JQ1 (S-enantiomer) with >99% ee. The spectral data of (+)-JQ1 matched with those described in the literature6 with the exception of the 22 12 optical rotation. [a]22 ( )D +41 (c 0.50, CHCl3), (Lit. [a]D +55 (c 0.50, CHCl3); JQ1: >99% ee, [a]22 39 (c 0.55, CHCl3), (Lit. [a]22 55 (c 0.50, CHCl3).6 D D
Contents lists available at ScienceDirect
Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet
Scalable syntheses of the BET bromodomain inhibitor JQ1 Shameem Sultana Syeda, Sudhakar Jakkaraj, Gunda I. Georg ⇑ Department of Medicinal Chemistry and Institute for Therapeutics Discovery and Development, College of Pharmacy, University of Minnesota, Minneapolis, MN 55414, United States
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 8 February 2015 Accepted 13 February 2015
Keywords: Bromodomains BET inhibitors Triazolothienodiazepine (+)-JQ1 Male contraceptive Thionation One-pot method
a b s t r a c t We have developed methods involving the use of alternate, safer reagents for the scalable syntheses of the potent BET bromodomain inhibitor JQ1. A one-pot three step method, involving the conversion of a benzodiazepine to a thioamide using Lawesson’s reagent, followed by amidrazone formation and installation of the triazole moiety furnished JQ1. This method provides good yields and a facile purification process. For the synthesis of enantiomerically enriched (+)-JQ1, the highly toxic reagent diethyl chlorophosphate, used in a previous synthesis, was replaced with the safer reagent diphenyl chlorophosphate in the three-step one-pot triazole formation without effecting yields and enantiomeric purity of (+)-JQ1. Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction The bromodomain (BRD) proteins are an important class of histone reader proteins that recognize acetylated lysine residues (KAc) on histone tails and direct transcription complexes to turn on genes.1 The bromodomain motif is short with approximately 110 amino acids that are conserved in several human genes.2 The human genome encodes 61 BRDs, which are present in 46 diverse proteins.3 Crystal structures of BRDs from more than 20 different proteins demonstrated that bromodomains share a conserved deep hydrophobic pocket formed by a left-handed four-helical bundle (aA, aB, aC, aZ) and loop regions of various lengths (ZA and BC),3,4 which constitute an attractive pocket for the development of selective protein–protein interaction inhibitors. Among the eight BRD families, the BRD and BET proteins have been found to be tractable for drug discovery. Chemical inhibition of BET proteins exerts a broad spectrum of desirable biological effects such as anticancer, anti-inflammatory, and male contraceptive properties.1,5 Triazolothienodiazepine (+)-JQ1 is one of the first selective inhibitors with nanomolar affinity for BET proteins (BRD2, BRD3, BRD4 and BRDT).6 The discovery of this potent, selective, and permeable inhibitor for BETs has stimulated research activity in diverse therapeutic areas, particularly in oncology, inflammation, and viral infection. Two other BET inhibitors I-BET7627,8 and OTX0155 (Fig. 1) that share the JQ1 scaffold are in clinical trials for the treatment of NUT midline carcinoma (a rare cancer caused by
BRD4-NUT fusions), acute myeloid leukemia, Burkitt’s lymphoma, and multiple myeloma.6,9,10 (+)-JQ1 also served as a probe to validate testis-specific BRDT as a promising reversible male contraceptive agent.11 As a part of our collaborative efforts to discover non-hormonal male contraceptive agents, we required a significant quantity of (+)-JQ1 for further evaluations. To meet the demand of (+)-JQ1, we developed a scalable and safer route to JQ1 based on reported methods. The reported synthesis that is shown in Scheme 1 provides racemic JQ1, although the synthesis employs the L-amino acid (Fmoc-Asp-(OtBu)-OH) (2). Racemization was observed at several different stages of the synthesis, including the peptide coupling, the aminoketone cyclization, and the thionation.6 Scheme 2 details the synthesis of enantiomerically enriched (+)-JQ1 with 90% optical purity. While both methods provide short and efficient methods for the synthesis of JQ1, we were concerned about the large scale use of excess P2S5 for the conversion of amide 5 to
⇑ Corresponding author. Tel.: +1 612 626 6320; fax: +1 612 626 6318. E-mail address: [email protected] (G.I. Georg).
Figure 1. Structures of BET inhibitors.
http://dx.doi.org/10.1016/j.tetlet.2015.02.062 0040-4039/Ó 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457
3455
Scheme 1. Filippakopoulos et al. method for the synthesis of (±)-JQ1.6
Scheme 2. Filippakopoulos et al. method for the synthesis of enantiomerically enriched (+)-JQ1.6
thioamide 6 (Scheme 1) and the use of diethyl chlorophosphate for the conversion of intermediate 5 to (+)-JQ1 (Scheme 2). Excess P2S5 is problematic during work-up because of H2S gas evolution. This gas is challenging to trap on a large scale, and possesses a noxious, sulfur-related odor. In addition, the reagent diethyl chlorophosphate used in the conversion of amide 5 to (+)-JQ1 (Scheme 2) is classified as possessing acute oral, dermal, and inhalation toxicities. Results and discussion In our efforts to circumvent H2S associated issues for thioamide formation, we replaced P2S5 (4 equiv) with Lawesson’s reagent (0.5 equiv) for thionation. Initially, 1 mmol of amide 5, prepared by methods shown in Scheme 1, was treated with Lawesson’s reagent in refluxing toluene to afford thioamide 6 in 75% yield (Scheme 3). Although thionation proceeded efficiently on a 1 mmol scale, it provided only a moderate yield (50%) on a larger scale. Because of the presence of poorly soluble byproducts, purification of this reaction was difficult and required multiple column
Scheme 3. Thionation of amide 5 with Lawesson’s reagent.
purifications to obtain pure thioamide 6. While we were pursuing the reaction with Lawesson’s reagent Zhang et al. reported12 a similar JQ1 synthesis using Lawesson’s reagent for amide thionation. They observed moderate yields, analogous to our results. Next we aimed at optimizing a scalable strategy for the synthesis of JQ1. To achieve this, we designed a one-pot strategy13 for the conversion of amide 5 to JQ1. Our one-pot strategy (Scheme 4) started with the treatment of amide 5 with Lawesson’s reagent in THF at 80 °C for 2 h (monitored by TLC), followed by the addition of excess hydrazine hydrate (10 equiv) at 0 °C. The reaction mixture was stirred for 30 min (monitored by TLC) to yield amidrazone 7, which was used for the next step directly after aqueous work-up. Amidrazone 7 was heated to 110 °C for 2 h in a mixture of
Scheme 4. Synthesis of (±)-JQ1 via a one-pot method.
3456
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457
trimethyl orthoacetate and toluene (2:3) to yield the target compound (±)-JQ1 in 60% yield over three steps. We found that the newly designed one-pot strategy (thionation and amidrazone formation) greatly minimized sulfur related concerns (the strong, unpleasant odor of sulfur byproducts). The purification process was also facile and the reaction proceeded with a slightly improved overall yield of 60% compared to the reactions in Scheme 1 (55% overall yield). Using this efficient method we have synthesized 30 g of (±)-JQ1. The reaction was carried out in four batches to minimize sulfur-related odors but can in principle be performed on a larger scale. A related approach for the synthesis of bromodomain inhibitors involving Lawessons’s reagent was disclosed in a recent patent.13 Our next goal was to prepare enantiomerically enriched JQ1, according to the procedure that has been reported by Filippakopoulos et al. (Scheme 2).6 A concern about the reported method is the use of highly toxic diethyl chlorophosphate for the installation of the triazole ring from amide 5 on a larger scale. We thus investigated alternate conditions to replace the highly toxic diethyl chlorophosphate and found that the less toxic diphenyl chlorophosphate (corrosive) was equally reactive to activate the amide functionality and led to the formation of (+)-JQ1 in excellent yields without effecting enantiomeric purity. To the best of our knowledge, there are no previous examples in the literature for the use of diphenyl chlorophosphate for a triazole synthesis. Following the reported method (Scheme 2), benzodiazepine 5 was prepared and then treated with KOtBu and diphenyl chlorophosphate to form phosphorylimidate. 8 (Scheme 5), which was not isolated and subsequently reacted with acetylhydrazide at room temperature for 1 h followed by heating at 90 °C to furnish enantiomerically enriched (+)-JQ1 in 82% yield. The enantiopurity of (+)-JQ1 was determined by chiral HPLC. A 91:9 ratio of the two enantiomers was observed, which is the same as was previously reported for the synthesis of enantioenriched (+)-JQ1 (Scheme 1).6 In conclusion, we have developed methods involving the use of alternate, safer reagents for the synthesis of racemic and enantioenriched JQ1.14 We examined the conversion of amide 5 to thioamide 6 using Lawesson’s reagent and were able to develop a one-pot method for the three-step conversion of amide 5 to racemic JQ1. We found that this method significantly improved the purification process and provided JQ1 in good yields. For the synthesis of enantiomerically enriched (+)-JQ1 we replaced the highly toxic diethyl chlorophosphate reagent used in the literature procedure with the safer reagent diphenyl chlorophosphate in a three-step one-pot synthesis without affecting the yield and the
Scheme 5. Synthesis of enantiomerically enriched (+)-JQ1 with diphenyl chlorophosphate.
enantiomeric purity of (+)-JQ1. These methods should be equally useful to access other BET inhibitors such as I-BET762, OTX015, and related analogues with the same or similar scaffolds. Acknowledgments Financial support for this project is gratefully acknowledged from the NIH/NICHD: 1U01HD076542 and HHSN275201300017C. The determination of the enantiomeric purity of (+)-JQ1 was carried out by Shanghai ChemPartner Co., Ltd China.
References and notes 1. Filippakopoulos, P.; Knapp, S. Nat. Rev. Drug Discovery 2014, 13, 337–356. 2. Haynes, S. R.; Dollard, C.; Winston, F.; Beck, S.; Trowsdale, J.; Dawid, I. B. Nucleic Acids Res. 1992, 20, 2603. 3. Filippakopoulos, P.; Picaud, S.; Mangos, M.; Keates, T.; Lambert, J.-P.; BarsyteLovejoy, D.; Felletar, I.; Volkmer, R.; Muller, S.; Pawson, T.; Gingras, A.-C.; Arrowsmith, C. H.; Knapp, S. Cell 2012, 149, 214–231. 4. Jacobson, R. H.; Ladurner, A. G.; King, D. S.; Tjian, R. Science 2000, 288, 1422– 1425. 5. Arkin, M. R.; Tang, Y.; Wells, J. A. Chem. Biol. 2014, 21, 1102–1114. 6. Filippakopoulos, P.; Qi, J.; Picaud, S.; Shen, Y.; Smith William, B.; Fedorov, O.; Morse Elizabeth, M.; Keates, T.; Hickman Tyler, T.; Felletar, I.; Philpott, M.; Munro, S.; McKeown Michael, R.; Wang, Y.; Christie Amanda, L.; West, N.; Cameron Michael, J.; Schwartz, B.; Heightman Tom, D.; La Thangue, N.; French Christopher, A.; Wiest, O.; Kung Andrew, L.; Knapp, S.; Bradner James, E. Nature 2010, 468, 1067–1073. The thionation reaction was carried out on 0.5 mmol scale. 7. Mirguet, O.; Gosmini, R.; Toum, J.; Clement, C. A.; Barnathan, M.; Brusq, J.-M.; Mordaunt, J. E.; Grimes, R. M.; Crowe, M.; Pineau, O.; Ajakane, M.; Daugan, A.; Jeffrey, P.; Cutler, L.; Haynes, A. C.; Smithers, N. N.; Chung, C.-W.; Bamborough, P.; Uings, I. J.; Lewis, A.; Witherington, J.; Parr, N.; Prinjha, R. K.; Nicodeme, E. J. Med. Chem. 2013, 56, 7501–7515. 8. Chung, C.-W.; Coste, H.; White, J. H.; Mirguet, O.; Wilde, J.; Gosmini, R. L.; Delves, C.; Magny, S. M.; Woodward, R.; Hughes, S. A.; Boursier, E. V.; Flynn, H.; Bouillot, A. M.; Bamborough, P.; Brusq, J.-M. G.; Gellibert, F. J.; Jones, E. J.; Riou, A. M.; Homes, P.; Martin, S. L.; Uings, I. J.; Toum, J.; Clement, C. A.; Boullay, A.B.; Grimley, R. L.; Blandel, F. M.; Prinjha, R. K.; Lee, K.; Kirilovsky, J.; Nicodeme, E. J. Med. Chem. 2011, 54, 3827–3838. 9. Dawson, M. A.; Prinjha, R. K.; Dittman, A.; Giotopoulos, G.; Bantscheff, M.; Chan, W.-I.; Robson, S. C.; Chung, C.-W.; Hopf, C.; Savitski, M. M.; Huthmacher, C.; Gudgin, E.; Lugo, D.; Beinke, S.; Chapman, T. D.; Roberts, E. J.; Soden, P. E.; Auger, K. R.; Mirguet, O.; Doehner, K.; Delwel, R.; Burnett, A. K.; Jeffrey, P.; Drewes, G.; Lee, K.; Huntly, B. J. P.; Kouzarides, T. Nature 2011, 478, 529–533. 10. Delmore Jake, E.; Issa Ghayas, C.; Lemieux Madeleine, E.; Rahl Peter, B.; Shi, J.; Jacobs Hannah, M.; Kastritis, E.; Gilpatrick, T.; Paranal Ronald, M.; Qi, J.; Chesi, M.; Schinzel Anna, C.; McKeown Michael, R.; Heffernan Timothy, P.; Vakoc Christopher, R.; Bergsagel, P. L.; Ghobrial Irene, M.; Richardson Paul, G.; Young Richard, A.; Hahn William, C.; Anderson Kenneth, C.; Kung Andrew, L.; Bradner James, E.; Mitsiades Constantine, S. Cell 2011, 146, 904–917. 11. Matzuk, M. M.; McKeown, M. R.; Filippakopoulos, P.; Li, Q.; Ma, L.; Agno, J. E.; Lemieux, M. E.; Picaud, S.; Yu, R. N.; Qi, J.; Knapp, S.; Bradner, J. E. Cell 2012, 150, 673–684. 12. Zhang, C.-J.; Tan, C. Y. J.; Ge, J.; Na, Z.; Chen, G. Y. J.; Uttamchandani, M.; Sun, H.; Yao, S. Q. Angew. Chem. 2013, 52, 14060–14064. The reaction scale for the thionation was not provided. 13. Albrecht, B. K.; Audia, J. E.; Cote, A.; Gehling, V. S.; Harmange, J.-C.; Hewitt, M. C.; Naveschuk, C. G.; Taylor, A. M.; Vaswani, R. G. WO 2012075456A1. 14. Experimental procedures: tert-Butyl 2-(5-(4-chlorophenyl)-6,7-dimethyl-2thioxo-2,3-dihydro-1H-thieno[2,3-e][1,4]diazepin-3-yl)acetate (6). In a 25 mL round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (0.42 g, 1.00 mmol) in toluene (10 mL). To this solution Lawesson’s reagent (0.21 g, 0.52 mmol) was added and the resulting suspension was heated to reflux. The reaction mixture was allowed to stir at reflux for 2 h at which point TLC indicated the consumption of the starting material. The solvent was removed under reduced pressure. The resulting residue was purified by column chromatography (silica gel, 0–10% ethyl acetate in hexanes) to afford thioamide 6 (0.32 g, 75%) as a yellow solid. Spectroscopic data for compound 6 were consistent with those described in the literature.12 (±)-JQ1: One-pot method. In a 500 mL round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (9.50 g, 22.72 mmol) in THF (150 mL). To this solution Lawesson’s reagent (4.59 g, 11.36 mmol) was added and the resulted suspension was at reflux for 2 h at which point TLC indicated the consumption of the starting material. The reaction mixture was cooled to 0 °C and hydrazine hydrate solution (50–60%) (14.50 mL, 226.73 mmol) was added dropwise to the reaction mixture over 10 min and continued to stir at 0 °C for 30 min. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3 50 mL). The combined organic layers were washed with brine (80 mL), dried over Na2SO4, and concentrated in vacuo to furnish amidrazone 7, which was used for the next step without purification. The crude
S. S. Syeda et al. / Tetrahedron Letters 56 (2015) 3454–3457 amidrazone 7 (10.50 g, 24.30 mmol) was dissolved in a 2:3 mixture of trimethyl orthoacetate and toluene (200 mL) and stirred at 110 °C for 1 h until the reaction was complete (monitored by TLC). The reaction mixture was concentrated in vacuo and the resulting residue was purified by column chromatography (silica gel, 0–100% ethyl acetate in hexanes) to furnish (±)-JQ1 (6.22 g, 60%). The spectral data of compound 3 matched with those described in the literature.6 (+)-JQ1: In a 500 mL two neck round-bottomed flask equipped with a magnetic stir bar was dissolved amide 5 (3.40 g, 8.13 mmol) in THF (60 mL) and cooled to 78 °C. While stirring at 78 °C, potassium tert-butoxide (1.0 M solution in THF, 8.95 mL, 8.94 mmol, 1.10 equiv) was added. The reaction mixture was warmed to 10 °C and stirred for 30 min. The reaction mixture was cooled to 78 °C and diphenyl chlorophosphate (2.02 mL, 9.76 mmol) was added to the reaction mixture. The resulting mixture was warmed to 10 °C and stirred at this temperature for 45 min. Acetylhydrazide (0.90, 12.20 mmol, 1.50 equiv)
3457
was added to the reaction mixture. The reaction mixture was stirred at room temperature. After 1 h, 1-butanol (70 mL) was added to the reaction mixture, which was heated to 90 °C. After 1 h, all solvents were removed under reduce pressure. The residue was dissolved in CH2Cl2 and washed with saturated aq. NaHCO3, brine, dried over anhydrous Na2SO4 and concentrated in vacuo to obtain crude (+)-JQ1, which was purified by flash column chromatography on silica gel (0–100% ethyl acetate in hexanes) to afford (+)-JQ1 (3.04 g, 82%) as a white solid and with a 91:9 er (determined with Supercritical Fluid Chromatography (SFC) using the CHIRALPAK AS-H column, CO2–EtOH (3:1), 210 nm, tR (R-enantiomer) 1.62 min, tR (S-enantiomer) 3.51 min). This product was further purified by preparative SFC using a CHIRALPAK AS-H column to obtain (+)-JQ1 (S-enantiomer) with >99% ee. The spectral data of (+)-JQ1 matched with those described in the literature6 with the exception of the 22 12 optical rotation. [a]22 ( )D +41 (c 0.50, CHCl3), (Lit. [a]D +55 (c 0.50, CHCl3); JQ1: >99% ee, [a]22 39 (c 0.55, CHCl3), (Lit. [a]22 55 (c 0.50, CHCl3).6 D D