COMMUNICATION DOI: 10.1002/asia.201402241
Facile Synthesis of the CERT Inhibitor HPA-12 and Some Novel Derivatives Essa M. Saied,[a, b] Stephanie Diederich,[a] and Christoph Arenz*[a]
Abstract: HPA-12 is an inhibitor of CERT-mediated non-vesicular transport of ceramide from the ER membranes to the Golgi apparatus. The inhibitor effectively blocks the synthesis of the membrane lipid sphingomyelin and may represent a novel drug prototype. Previous syntheses relied on non-commercial catalysts or specialized chemistries. Here we present a straightforward and effective method to synthesize HPA-12 from commercially available protected lserinol in four steps. Some new analogues were synthesized and evaluated for their CERT-binding activity.
stereochemistry of the most active HPA-12 diasteromer was revised from R,R to R,S.[8a, 14] Previous methods induce stereochemical orientation at both chiral carbon atoms with high ratios of the desired diastereomers and allow further structural variation. However, these syntheses also include specialized chemistries and rely on catalysts that are not commercially available. The recently published (and probably most practical) synthesis by Berkes et al.,[8a] for example, allows a five-step synthesis of HPA-12 from oxoamino acids using crystallization-induced asymmetric transformation. However, the oxoamino acids are not commercially available and the alkyl or aryl substituents are pre-set in the synthetic route very early, thus making the synthesis of many precursors necessary. Hence, there is still a need to develop an easy, practical, and versatile approach for the synthesis of HPA-12 and its derivatives. The initial structure–activity-relationship (SAR) revealed that inhibitory potency is at maximum for acyl chain lengths of 12 and 13.[9] The hydroxy groups and the stereochemical orientation at both chiral centers are key features for full inhibitory potency.[9, 10] By contrast, the role of the phenyl ring or other potential groups at this position have not been fully elucidated yet. With respect to the aforementioned and our interest in the synthesis of sphingolipid-related bioactive molecules, we planned to develop a more facile, convenient, and versatile synthesis of HPA-12 and its derivatives that relies on readily available starting materials and methods that are well established in many laboratories. Furthermore, we aimed at synthesizing a set of novel derivatives of the active diasteromer (R,S) HPA-12 in order to gain further insight into the SAR for this very interesting compound. l-Serine is the usual starting material for sphingosine synthesis (Scheme 1 A). After formation of Garners aldehyde 2,[11] the second chiral center is formed upon nucleophilic attack of the aldehyde by either an alkyne lithium that is subsequently reduced to an E-olefin[11] or, alternatively, by treatment with vinylmagnesium bromide and subsequent olefin metathesis.[12] The main difference between sphingosine and the HPA core structure, however, is only an additional methylene between the two stereocenters. We thus decided to just shift the position for a nucleophilic attack by one methylene group. Towards this end, we planned to reduce the protected serine to the corresponding serinol and to substitute the newly formed hydroxy group by a nitrile. Nitriles have been reported to be readily attacked by C-nucleophiles like Grignard reagents to yield the respective ketones.[13] The latter then can be stereoselectively reduced, as
Sphingolipids are ubiquitous components of mammalian plasma membranes and play important roles in cell regulation and human disease.[1] Sphingolipid biosynthesis is located at the cytosolic and luminal sides of the endoplasmatic reticulum and Golgi apparatus and requires distinct cellular trafficking to make the intermediates available at the subsequent sites of conversion. The ceramide transportation protein CERT mediates the non-vesicular transport of ceramide from the ER to Golgi and has been shown to be essential for sphingomyelin biosynthesis.[2] The ceramide analogue HPA-12 has been reported to potently inhibit CERT-mediated ceramide transport.[3] HPA-12-mediated blockage of sphingomyelin biosynthesis has been shown to interfere with hepatitis C infections,[4] to inhibit the growth of Chlamydia trachomatis,[5] and to sensitize cancer cells to chemotherapeutic treatment[6] or irradiation.[7] Therefore, a great deal of attention has been devoted to studies of the biological processes regulated by HPA-12. However, HPA-12 is not commercially available, which significantly hampers basic research on the role of CERT and HPA-12 in health and disease. Recently, several novel syntheses of HPA-12 have been reported.[8] In the course of these investigations, the
[a] E. M. Saied, S. Diederich, Prof. Dr. C. Arenz Institute for Chemistry Humboldt Universitt zu Berlin Brook-Taylor-Str. 2, 12489 Berlin (Germany) Fax: (+ 49) 30-2093 6947 E-mail: [email protected] [b] E. M. Saied Chemistry Department Faculty of Science Suez Canal University Isia (Egypt) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402241.
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backbone, the limitation to organolithiums and the incompatibility of Grignard reagents as nucleophiles were dissatisfying, since this would cause limitations in the structural diversity of prospective HPA analogues. We therefore decided to adapt the synthetic route in a way that would allow the use of Grignard nucleophiles as well. Towards this end, the nitrile 4 was reduced to the correspondScheme 1. A) Most common synthetic routes to sphingosine starting from Garners aldehyde 2. B) Retrosyning aldehyde 7 using DIBAL-H. thetic analysis of HPA-12 with l-serine as a starting material. The subsequent reaction of aldehyde 7 with phenylmagnesium bromide in THF at 78 8C afforded the expected aminoshown before in the HPA synthesis by Ueno et al.,[14] followed by deprotection and N-acylation to give the desired diol in a good yield (72 %). Unfortunately, the undesired diHPA-12 (Scheme 1 B). asteromer 8 a of the HPA backbone was obtained with The previously described and commercially available proa high excess (8 a:6 a; anti:syn 9:1). Oxidation of the alcohol tected serinol 3[11] was readily prepared from l-serine in 8 a using standard Swerns conditions, however, yielded the intermediate 5 a, which in turn was reduced using l-Selecfour steps on a multigram scale (overall yield of 79 % over tride (see also Table S1, Supporting Information) in order to four steps, see the Supporting Information for details). The obtain the correct stereochemistry. This procedure now enasubsequent conversion into the corresponding nitrile 4 was bled us to conveniently synthesize both, the R,S and the R,R achieved after in situ activation of the hydroxy group with backbone of HPA-12 stereoisomers, respectively. 1-(p-toulenesulfonyl) imidazole (TsIm) in a moderate yield Having the protected intermediates 6 a and 8 a in hand, (58 %) or, alternatively, through mesylation and subsequent we tried to deprotect both the hydroxy and the amino group conversion to the nitrile with much better efficiency (90 % in one step, by using TFA in dichloromethane or 4 m HCl in yield over two steps) (Scheme 2). When we tried to react 1,4-dioxane, which are our standard procedures for sphingothe thus acquired nitrile 4 with Grignard reagents, we did sine synthesis. After N-acylation with dodecanoyl chloride not observe significant conversion, although different condiin the presence of Hnigs base, however, we noticed that instead of the desired HPA diasteromers, we obtained the respective dehydration products 11 a and 12 a (Scheme 3). Surprisingly, both products represented the pure diastereomers, thereby suggesting that protonation of the secondary hydroxy group triggered an intramolecular SN2 reaction, which Scheme 2. Reagents and conditions: a) Tos-imidazole, KCN, DMF, 95 8C, 16 h; b) MsCl, Pyr, 40 8C, 1 h, then was obviously kinetically faKCN, DMF, 90 8C, 16 h; c) RLi (2.0 equiv), toluene, 78 8C, 1 h; d) l-Selectride, THF, 78 8C, 1 h; e) DIBALvored over a possible SN1 reacH, CH2Cl2, 40 8C, 1 h; f) PhMgBr, THF, 78 8C, 1 h; h) DMSO, (COCl)2, DIPEA; i) 4 m HCl, MeOH reflux, tion. Notably, to the best of our 2 h, then C11H23COCl, DIPEA, CH2Cl2. knowledge, the R,S isomer has tions (excess reagent, temperature, etc.) were tried. By contrast, a more activated nucleophile, phenyl lithium, yielded the desired ketone 5 a in an acceptable yield (74 %). The ketone 5 a was then stereoselectively reduced with l-Selectride to the desired syn-HPA backbone 6 a as the predominant stereoisomer in 91 % yield (syn:anti; 11:1). Several other reducing agents were screened for diastereoselective reduction, but gave lower yields of the desired compound (see also Table S1 in the Supporting Information). Although Scheme 3. Reagents and conditions: a) TFA in CH2Cl2, 0 8C, 1 h or 4 m we succeeded in exploring a very facile route to the HPA-12 HCl in 1,4-dioxane, 0 8C, 1 h; b) C11H23COCl, DIPEA, 0 8C to rt, 30 min.
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the synthesis of many new derivatives that will result in a more detailed SAR and hopefully more potent HPA-12 analogues very soon.
not been described before. Since aromatic ethanolamines often possess bioactivity,[15] we kept both compounds for prospective investigations of their potential impact on sphingolipid metabolism. In order to obtain the desired products, we treated the protected intermediates 6 a or 8 a with 4 m HCl in aqueous methanol under reflux followed by N-acylation under standard conditions to furnish both isomers 9 a and 10 a in good yields (83 % and 81 % over the two steps, respectively). Accordingly, to test the versatility of this method and to gain some information about the importance of the phenyl ring in HPA-12, two other R,S-configured derivatives 9 b and 9 c were prepared by reacting the nitrile 4 with nBuLi or tBuLi, respectively. To assess the CERT-binding activity of all compounds synthesized, we established an in vitro CERT binding assay, similar to a method described recently.[16] Towards this end, CERT was expressed and purified from E. coli cells as described previously.[2b] Briefly, an NBD derivative of ceramide[17] was pulled down together with the His-tagged CERT using Ni-NTA magnetic beads, and the bound fraction was quantified afterwards by its fluorescence intensity. Addition of the respective competitors reduced the amount of NBDCer-C16 pulled down together with CERT (Table 1). As ex-
Acknowledgements This work was supported by the Humboldt Universitt zu Berlin. E.M.S. is grateful for a Yousef Jameel Fellowship. The authors thank Kentaro Hanada, Tokyo, for the CERT expression plasmid.
Keywords: binding assays · chiral pool · inhibitors · lipid trafficking · sphingolipids
[1] T. Kolter, K. Sandhoff, Angew. Chem. Int. Ed. 1999, 38, 1532 – 1568; Angew. Chem. 1999, 111, 1632 – 1670. [2] a) K. Hanada, Mol. Cell. Biochem. 2006, 286, 23 – 31; b) K. Hanada, K. Kumagai, S. Yasuda, Y. Miura, M. Kawano, M. Fukasawa, M. Nishijima, Nature 2003, 426, 803 – 809. [3] S. Yasuda, H. Kitagawa, M. Ueno, H. Ishitani, M. Fukasawa, M. Nishijima, S. Kobayashi, K. Hanada, J. Biol. Chem. 2001, 276, 43994 – 44002. [4] H. Sakamoto, K. Okamoto, M. Aoki, H. Kato, A. Katsume, A. Ohta, T. Tsukuda, N. Shimma, Y. Aoki, M. Arisawa, M. Kohara, M. Sudoh, Nat. Chem. Biol. 2005, 1, 333 – 337. [5] C. A. Elwell, S. Jiang, J. H. Kim, A. Lee, T. Wittmann, K. Hanada, P. Melancon, J. N. Engel, Plos Pathog. 2011, 7, e1002198. [6] C. Swanton, M. Marani, O. Pardo, P. H. Warne, G. Kelly, E. Sahai, F. Elustondo, J. Chang, J. Temple, A. A. Ahmed, J. D. Brenton, J. Downward, B. Nicke, Cancer Cell 2007, 11, 498 – 512. [7] A. Charruyer, S. M. Bell, M. Kawano, S. Douangpanya, T. Y. Yen, B. A. Macher, K. Kumagai, K. Hanada, W. M. Holleran, Y. Uchida, J. Biol. Chem. 2008, 283, 16682 – 16692. ˇ urisˇ, T. Wiesenganger, D. Moravcˇkova, P. Baran, J. Kozˇsˇek, [8] a) A. D A. Dach, D. Berkesˇ, Org. Lett. 2011, 13, 1642 – 1645;b) J. R. Snider, J. T. Entrekin, T. S. Snowden, D. Dolliver, Synthesis 2013, 1899 – 1903; c) S. Kobayashi, R. Matsubara, H. Kitagawa, Org. Lett. 2002, 4, 143 – 145; d) S. Kobayashi, R. Matsubara, Y. Nakamura, H. Kitagawa, M. Sugiura, J. Am. Chem. Soc. 2003, 125, 2507 – 2515. [9] Y. Nakamura, R. Matsubara, H. Kitagawa, S. Kobayashi, K. Kumagai, S. Yasuda, K. Hanada, J. Med. Chem. 2003, 46, 3688 – 3695. [10] N. Kudo, K. Kumagai, R. Matsubara, S. Kobayashi, K. Hanada, S. Wakatsuki, R. Kato, J. Mol. Biol. 2010, 396, 245 – 251. [11] P. Garner, J. M. Park, J. Org. Chem. 1987, 52, 2361 – 2364. [12] I. Ojima, E. S. Vidal, J. Org. Chem. 1998, 63, 7999 – 8003. [13] R. L. Jones, M. Gordon, D. E. Pearson, J. Org. Chem. 1972, 37, 3369. [14] M. Ueno, Y. Y. Huang, A. Yamano, S. Kobayashi, Org. Lett. 2013, 15, 2869 – 2871. [15] a) K. P. Bhabak, C. Arenz, Bioorg. Med. Chem. 2012, 20, 6162 – 6170; b) C. Bedia, D. Canals, X. Matabosch, Y. Harrak, J. Casas, A. Llebaria, A. Delgado, G. Fabrias, Chem. Phys. Lipids 2008, 156, 33 – 40. [16] S. Combemale, C. Santos, F. Rodriguez, V. Garcia, C. Galaup, C. Frongia, V. Lobjois, T. Levade, C. Baudoin-Dehoux, S. Ballereau, Y. Genisson, RSC Adv. 2013, 3, 18970 – 18984. [17] K. P. Bhabak, D. Proksch, S. Redmer, C. Arenz, Bioorg. Med. Chem. 2012, 20, 6154 – 6161. Received: March 16, 2014 Published online: && &&, 0000
Table 1. Competitor assay versus NBD-ceramide-C16 (1.54 mm) bound to CERT. Compound
Concentration
Ceramide bound [%][a]
9a 10 a 9b 9c 11 a 12 a
1 mm (5 mm) 5 mm 5 mm 5 mm 5 mm 5 mm
18 3 (1 1) 87 17 51 7 96 6 93 5 99 11
[a] Including standard deviations.
pected, and in agreement with the recently revised stereochemistry of HPA-12,[8a, 14] the R,S-diastereomer 9 a was found to be more active than the R,R diasteromer 10 a. The n-butyl derivative 9 b, but not the tert-butyl derivative 9 c, showed significant binding to CERT, thus suggesting that the SAR at this position may be further investigated in the future. As expected and in full agreement with the observation that intact hydroxy groups are essential for binding to CERT, the anhydro-derivatives 11 a and 12 a were inactive as well. In conclusion, we developed a highly efficient, versatile, and straightforward four-step approach for the synthesis of the potent CERT inhibitor HPA-12 and its derivatives starting from a commercially available l-serinol synthon. The key steps in the developed route are the nucleophilic attack of nitrile 4 with organolithium reagents and the subsequent selective syn-reduction of the formed ketones to secondary hydroxyl groups. We are convinced that the ease and cost effectiveness of this method will stimulate research on the CERT-dependent pathways and ultimately enable
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COMMUNICATION Enzyme Inhibitors Essa M. Saied, Stephanie Diederich, Christoph Arenz* &&&&—&&&& Facile Synthesis of the CERT Inhibitor HPA-12 and Some Novel Derivatives
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As easy as it gets: The potent CERT inhibitor HPA-12 was synthesized from protected serinol in only four steps. No chiral catalysts or expensive protecting groups were needed. The synthetic route allows variation in one stereo-
4
genic center (green) and attachment of alkyl and aryl modifications (red). Preliminary CERT-binding assay data suggest the synthesis of further derivatives for structural optimization.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
Facile Synthesis of the CERT Inhibitor HPA-12 and Some Novel Derivatives Essa M. Saied,[a, b] Stephanie Diederich,[a] and Christoph Arenz*[a]
Abstract: HPA-12 is an inhibitor of CERT-mediated non-vesicular transport of ceramide from the ER membranes to the Golgi apparatus. The inhibitor effectively blocks the synthesis of the membrane lipid sphingomyelin and may represent a novel drug prototype. Previous syntheses relied on non-commercial catalysts or specialized chemistries. Here we present a straightforward and effective method to synthesize HPA-12 from commercially available protected lserinol in four steps. Some new analogues were synthesized and evaluated for their CERT-binding activity.
stereochemistry of the most active HPA-12 diasteromer was revised from R,R to R,S.[8a, 14] Previous methods induce stereochemical orientation at both chiral carbon atoms with high ratios of the desired diastereomers and allow further structural variation. However, these syntheses also include specialized chemistries and rely on catalysts that are not commercially available. The recently published (and probably most practical) synthesis by Berkes et al.,[8a] for example, allows a five-step synthesis of HPA-12 from oxoamino acids using crystallization-induced asymmetric transformation. However, the oxoamino acids are not commercially available and the alkyl or aryl substituents are pre-set in the synthetic route very early, thus making the synthesis of many precursors necessary. Hence, there is still a need to develop an easy, practical, and versatile approach for the synthesis of HPA-12 and its derivatives. The initial structure–activity-relationship (SAR) revealed that inhibitory potency is at maximum for acyl chain lengths of 12 and 13.[9] The hydroxy groups and the stereochemical orientation at both chiral centers are key features for full inhibitory potency.[9, 10] By contrast, the role of the phenyl ring or other potential groups at this position have not been fully elucidated yet. With respect to the aforementioned and our interest in the synthesis of sphingolipid-related bioactive molecules, we planned to develop a more facile, convenient, and versatile synthesis of HPA-12 and its derivatives that relies on readily available starting materials and methods that are well established in many laboratories. Furthermore, we aimed at synthesizing a set of novel derivatives of the active diasteromer (R,S) HPA-12 in order to gain further insight into the SAR for this very interesting compound. l-Serine is the usual starting material for sphingosine synthesis (Scheme 1 A). After formation of Garners aldehyde 2,[11] the second chiral center is formed upon nucleophilic attack of the aldehyde by either an alkyne lithium that is subsequently reduced to an E-olefin[11] or, alternatively, by treatment with vinylmagnesium bromide and subsequent olefin metathesis.[12] The main difference between sphingosine and the HPA core structure, however, is only an additional methylene between the two stereocenters. We thus decided to just shift the position for a nucleophilic attack by one methylene group. Towards this end, we planned to reduce the protected serine to the corresponding serinol and to substitute the newly formed hydroxy group by a nitrile. Nitriles have been reported to be readily attacked by C-nucleophiles like Grignard reagents to yield the respective ketones.[13] The latter then can be stereoselectively reduced, as
Sphingolipids are ubiquitous components of mammalian plasma membranes and play important roles in cell regulation and human disease.[1] Sphingolipid biosynthesis is located at the cytosolic and luminal sides of the endoplasmatic reticulum and Golgi apparatus and requires distinct cellular trafficking to make the intermediates available at the subsequent sites of conversion. The ceramide transportation protein CERT mediates the non-vesicular transport of ceramide from the ER to Golgi and has been shown to be essential for sphingomyelin biosynthesis.[2] The ceramide analogue HPA-12 has been reported to potently inhibit CERT-mediated ceramide transport.[3] HPA-12-mediated blockage of sphingomyelin biosynthesis has been shown to interfere with hepatitis C infections,[4] to inhibit the growth of Chlamydia trachomatis,[5] and to sensitize cancer cells to chemotherapeutic treatment[6] or irradiation.[7] Therefore, a great deal of attention has been devoted to studies of the biological processes regulated by HPA-12. However, HPA-12 is not commercially available, which significantly hampers basic research on the role of CERT and HPA-12 in health and disease. Recently, several novel syntheses of HPA-12 have been reported.[8] In the course of these investigations, the
[a] E. M. Saied, S. Diederich, Prof. Dr. C. Arenz Institute for Chemistry Humboldt Universitt zu Berlin Brook-Taylor-Str. 2, 12489 Berlin (Germany) Fax: (+ 49) 30-2093 6947 E-mail: [email protected] [b] E. M. Saied Chemistry Department Faculty of Science Suez Canal University Isia (Egypt) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402241.
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These are not the final page numbers! ÞÞ
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2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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www.chemasianj.org
Christoph Arenz et al.
backbone, the limitation to organolithiums and the incompatibility of Grignard reagents as nucleophiles were dissatisfying, since this would cause limitations in the structural diversity of prospective HPA analogues. We therefore decided to adapt the synthetic route in a way that would allow the use of Grignard nucleophiles as well. Towards this end, the nitrile 4 was reduced to the correspondScheme 1. A) Most common synthetic routes to sphingosine starting from Garners aldehyde 2. B) Retrosyning aldehyde 7 using DIBAL-H. thetic analysis of HPA-12 with l-serine as a starting material. The subsequent reaction of aldehyde 7 with phenylmagnesium bromide in THF at 78 8C afforded the expected aminoshown before in the HPA synthesis by Ueno et al.,[14] followed by deprotection and N-acylation to give the desired diol in a good yield (72 %). Unfortunately, the undesired diHPA-12 (Scheme 1 B). asteromer 8 a of the HPA backbone was obtained with The previously described and commercially available proa high excess (8 a:6 a; anti:syn 9:1). Oxidation of the alcohol tected serinol 3[11] was readily prepared from l-serine in 8 a using standard Swerns conditions, however, yielded the intermediate 5 a, which in turn was reduced using l-Selecfour steps on a multigram scale (overall yield of 79 % over tride (see also Table S1, Supporting Information) in order to four steps, see the Supporting Information for details). The obtain the correct stereochemistry. This procedure now enasubsequent conversion into the corresponding nitrile 4 was bled us to conveniently synthesize both, the R,S and the R,R achieved after in situ activation of the hydroxy group with backbone of HPA-12 stereoisomers, respectively. 1-(p-toulenesulfonyl) imidazole (TsIm) in a moderate yield Having the protected intermediates 6 a and 8 a in hand, (58 %) or, alternatively, through mesylation and subsequent we tried to deprotect both the hydroxy and the amino group conversion to the nitrile with much better efficiency (90 % in one step, by using TFA in dichloromethane or 4 m HCl in yield over two steps) (Scheme 2). When we tried to react 1,4-dioxane, which are our standard procedures for sphingothe thus acquired nitrile 4 with Grignard reagents, we did sine synthesis. After N-acylation with dodecanoyl chloride not observe significant conversion, although different condiin the presence of Hnigs base, however, we noticed that instead of the desired HPA diasteromers, we obtained the respective dehydration products 11 a and 12 a (Scheme 3). Surprisingly, both products represented the pure diastereomers, thereby suggesting that protonation of the secondary hydroxy group triggered an intramolecular SN2 reaction, which Scheme 2. Reagents and conditions: a) Tos-imidazole, KCN, DMF, 95 8C, 16 h; b) MsCl, Pyr, 40 8C, 1 h, then was obviously kinetically faKCN, DMF, 90 8C, 16 h; c) RLi (2.0 equiv), toluene, 78 8C, 1 h; d) l-Selectride, THF, 78 8C, 1 h; e) DIBALvored over a possible SN1 reacH, CH2Cl2, 40 8C, 1 h; f) PhMgBr, THF, 78 8C, 1 h; h) DMSO, (COCl)2, DIPEA; i) 4 m HCl, MeOH reflux, tion. Notably, to the best of our 2 h, then C11H23COCl, DIPEA, CH2Cl2. knowledge, the R,S isomer has tions (excess reagent, temperature, etc.) were tried. By contrast, a more activated nucleophile, phenyl lithium, yielded the desired ketone 5 a in an acceptable yield (74 %). The ketone 5 a was then stereoselectively reduced with l-Selectride to the desired syn-HPA backbone 6 a as the predominant stereoisomer in 91 % yield (syn:anti; 11:1). Several other reducing agents were screened for diastereoselective reduction, but gave lower yields of the desired compound (see also Table S1 in the Supporting Information). Although Scheme 3. Reagents and conditions: a) TFA in CH2Cl2, 0 8C, 1 h or 4 m we succeeded in exploring a very facile route to the HPA-12 HCl in 1,4-dioxane, 0 8C, 1 h; b) C11H23COCl, DIPEA, 0 8C to rt, 30 min.
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ÝÝ These are not the final page numbers!
www.chemasianj.org
the synthesis of many new derivatives that will result in a more detailed SAR and hopefully more potent HPA-12 analogues very soon.
not been described before. Since aromatic ethanolamines often possess bioactivity,[15] we kept both compounds for prospective investigations of their potential impact on sphingolipid metabolism. In order to obtain the desired products, we treated the protected intermediates 6 a or 8 a with 4 m HCl in aqueous methanol under reflux followed by N-acylation under standard conditions to furnish both isomers 9 a and 10 a in good yields (83 % and 81 % over the two steps, respectively). Accordingly, to test the versatility of this method and to gain some information about the importance of the phenyl ring in HPA-12, two other R,S-configured derivatives 9 b and 9 c were prepared by reacting the nitrile 4 with nBuLi or tBuLi, respectively. To assess the CERT-binding activity of all compounds synthesized, we established an in vitro CERT binding assay, similar to a method described recently.[16] Towards this end, CERT was expressed and purified from E. coli cells as described previously.[2b] Briefly, an NBD derivative of ceramide[17] was pulled down together with the His-tagged CERT using Ni-NTA magnetic beads, and the bound fraction was quantified afterwards by its fluorescence intensity. Addition of the respective competitors reduced the amount of NBDCer-C16 pulled down together with CERT (Table 1). As ex-
Acknowledgements This work was supported by the Humboldt Universitt zu Berlin. E.M.S. is grateful for a Yousef Jameel Fellowship. The authors thank Kentaro Hanada, Tokyo, for the CERT expression plasmid.
Keywords: binding assays · chiral pool · inhibitors · lipid trafficking · sphingolipids
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Table 1. Competitor assay versus NBD-ceramide-C16 (1.54 mm) bound to CERT. Compound
Concentration
Ceramide bound [%][a]
9a 10 a 9b 9c 11 a 12 a
1 mm (5 mm) 5 mm 5 mm 5 mm 5 mm 5 mm
18 3 (1 1) 87 17 51 7 96 6 93 5 99 11
[a] Including standard deviations.
pected, and in agreement with the recently revised stereochemistry of HPA-12,[8a, 14] the R,S-diastereomer 9 a was found to be more active than the R,R diasteromer 10 a. The n-butyl derivative 9 b, but not the tert-butyl derivative 9 c, showed significant binding to CERT, thus suggesting that the SAR at this position may be further investigated in the future. As expected and in full agreement with the observation that intact hydroxy groups are essential for binding to CERT, the anhydro-derivatives 11 a and 12 a were inactive as well. In conclusion, we developed a highly efficient, versatile, and straightforward four-step approach for the synthesis of the potent CERT inhibitor HPA-12 and its derivatives starting from a commercially available l-serinol synthon. The key steps in the developed route are the nucleophilic attack of nitrile 4 with organolithium reagents and the subsequent selective syn-reduction of the formed ketones to secondary hydroxyl groups. We are convinced that the ease and cost effectiveness of this method will stimulate research on the CERT-dependent pathways and ultimately enable
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Christoph Arenz et al.
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COMMUNICATION Enzyme Inhibitors Essa M. Saied, Stephanie Diederich, Christoph Arenz* &&&&—&&&& Facile Synthesis of the CERT Inhibitor HPA-12 and Some Novel Derivatives
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As easy as it gets: The potent CERT inhibitor HPA-12 was synthesized from protected serinol in only four steps. No chiral catalysts or expensive protecting groups were needed. The synthetic route allows variation in one stereo-
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genic center (green) and attachment of alkyl and aryl modifications (red). Preliminary CERT-binding assay data suggest the synthesis of further derivatives for structural optimization.
2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!
