- Email: [email protected]
Preparation and evaluation of deconstruction analogues of 7-deoxykalafungin as AKT kinase inhibitors
Bioorganic & Medicinal Chemistry Letters 24 (2014) 271–274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Preparation and evaluation of deconstruction analogues of 7-deoxykalafungin as AKT kinase inhibitors Sudha Korwar, Thuy Nguyen, Keith C. Ellis ⇑ Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Biotech 1, 800 East Leigh Street, Richmond, VA 23298, USA
a r t i c l e
i n f o
Article history: Received 5 July 2013 Revised 6 November 2013 Accepted 11 November 2013 Available online 20 November 2013 Keywords: Pyranonaphthoquinone lactone Covalent inhibitor AKT kinase Deconstruction analogues 7-Deoxykalafungin
a b s t r a c t The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify structural features of the natural product scaffold that are essential for potency and selectivity. Using a deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the 3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT. We have also unexpectedly identified a new submicromolar inhibitor of PKA. Ó 2013 Elsevier Ltd. All rights reserved.
Activity-based protein profiling (ABPP) is a chemical proteomics strategy that can be used to selectively identify the activated form of a protein in cells of various phenotypes both in vitro and in vivo.1–3 ABPP is a particularly powerful technique because it moves beyond simply quantifying the overall amount of a protein, active and inactive, in the cellular environment. ABPP detects only the fully activated, functional form of the protein of interest and does not detect forms that are overexpressed but down regulated, inactive because of posttranslational modifications, or not present in the cellular location of interest. ABPP has been used to elucidate the roles of multiple proteins and protein families in diseases including cancer,4,5 inflammation,6,7 rheumatoid arthritis,8,9 and depression.10 ABPP requires activity-based probes (ABPs) that contain three essential elements: (1) the probes must covalently modify a residue in the target protein that is reactive only in the activated form of the protein; (2) the probes must contain a scaffold that binds selectively to the targeted enzyme family or single enzyme; and (3) the probes must contain a tag that can be used for isolation or detection.2 One major source of ABPs has been protein-reactive natural products.11–13 Activity-based probes have been developed from the natural products wortmannin (targeting PI3K),14–16 E-64 (targeting the cathepsins),17 b-lactams (targeting penicillin binding proteins and b-lactamases),18,19 and several others (reviewed in Ref. 6). ⇑ Corresponding author. E-mail address: [email protected] (K.C. Ellis). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.020
AKT is a member of the AGC family of kinases that regulates multiple physiological processes including cell proliferation, metabolism, and survival.20 Hyperactivation of AKT kinase has been implicated in several disease states including cancer,21 type-II diabetes,22 and neurodegenerative diseases.23 To date, no activity-based probes targeting AKT kinase have been reported. The pyranonaphthoquinone (PNQ) lactone natural products lactoquinomycin and frenolicin B were identified as potent and selective inhibitors of AKT kinase in a high-throughput screen (Fig. 1).24 Further work demonstrated that the PNQ lactones inhibit AKT by
Figure 1. Structures of the pyranonaphthoquinone lactone natural products that inhibit AKT kinase.
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covalently alkylating Cys310 on the kinase activation loop.25 Despite the fact that this cysteine is conserved in over half of the enzymes in the AGC kinase family, the PNQ lactones are selective for AKT over these other family members. The preliminary structure-activity relationship (SAR) for the C5 position of the PNQ lactone 7-deoxykalafungin shows that monosubstitution at this position with both small (R = H, Me) and large (R = CH2OBn, 2-thienyl) groups are tolerated but that di-substitution leads to a 10-fold decrease in inhibition.25 This study did not investigate selectivity.25 As the first step in a program to explore the development of the PNQ lactones as an AKT-selective activity-based probe, we sought to understand the structural features of the natural product that impart potency and selectivity to this scaffold. In this communication, we have designed and prepared deconstruction analogues of 7-deoxykalafungin and evaluated their potency against recombinant AKT1 and selectivity for AKT1 over the closely related AGC kinase PKAa. The PNQ lactones must be activated in order to function as covalent inhibitors. Under in vitro and in vivo conditions the quinone is reduced and rearranges to the activated quinone methide (1, Scheme 1), which acts as a Michael acceptor (shown in magenta).26 The nucleophilic Cys310 of AKT then attacks the activated quinone methide 1, resulting in PNQ lactone—AKT adduct 2. The formation of the activated quinone methide 1 by reduction is essential for activity. Analogues missing the lactone ring are inactive as AKT inhibitors up to concentrations of 20 lM.25 The deconstruction analogues that we have designed and prepared are shown in Scheme 2. We designed these analogues to either preserve the latent electrophile by retaining the quinone (analogues 3 and 8) or replace this motif with an already-formed a,b-unsaturated ketone Michael acceptor (shown in magenta, analogues 5–7). We also designed these analogues to systematically remove structural elements from the natural product scaffold that provided steric bulk and conformational rigidity to determine how these elements contributed to both potency and selectivity. Several synthetic approaches to the PNQ lactones have been previously described.27–31 We chose to synthesize 7-deoxykalafungin by a variation of the route of Eid and co-workers32 starting from commercially available 1,4-dimethoxynaphthalene 9 as shown in Scheme 3. Bromination, Heck coupling, Sharpless asymmetric dihydroxylation with concomitant lactone formation, oxaPictet–Spengler reaction, and finally oxidation with CAN afforded the desired 7-deoxykalafungin. Deconstruction analogue 3 was prepared by treatment of intermediate 12 from the 7-deoxykalafungin synthesis with CAN (Scheme 4). Deconstruction analogues 5–7 were prepared by a common route starting from benzoic acid 19 (Scheme 5). EDC coupling afforded the Weinreb amide 20, which was treated with vinylmagnesium bromide in ethyl ether to afford the vinyl ketone 7. Subsequent treatment of 7 with a 2nd generation cross metathesis catalyst (CAS# 253688-91-4) and either 3-methoxyprop-1-ene or
Scheme 2. Deconstruction analysis of 7-deoxykalafungin and the structure of deconstruction analogues 3–8.
methyl pent-4-enoate in refluxing DCM afforded 5 and 6, respectively, both in >20:1 E/Z selectivity. Finally, naphthaquinone 8 was prepared by CAN oxidation of 1,4-dimethoxynaphthalene 9 (not shown). 7-Deoxykalafungin and compounds 3 and 5–8 were tested in the Z’-LYTE assay (Invitrogen) for their ability to inhibit kinase function of AKT1 and PKAa (Table 1). This FRET-based assay measures the ability of a kinase enzyme to phosphorylate a peptide substrate and detects functional inhibitors of kinase activity independent of mechanism of action. Our data confirmed the previous report that 7-deoxykalafungin is a potent inhibitor of AKT1 but
Scheme 1. Activation of 7-deoxykalafungin to form activated quinone methide 1 and AKT adduct 2.
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Table 1 Inhibition of recombinant AKT1 and PKAa by 7-deoxykalafungin and analogues 3-8 Compound
7-Deoxykalafungin 3 5 6 7 8
IC50a (lM) AKT1
PKAa
0.28 2.94 50.0 13.7 12.3 11.0
>100 0.43 >100 >100 >100 n.d. b
a IC50’s were determined using Z’-LYTE assay kits (Invitrogen). 95% confidence intervals can be found in the Supporting information. b Not determined.
Scheme 3. Reagents and conditions: (a) NBS, DCM, 96%. (b) isobutyl but-3-enoate, Pd(tBu3P)2, Cy2NMe, toluene, 81%. (c) K3Fe(CN)6, K2CO3, NaHCO3, (DHQD)2PHAL, CH3SO2NH2, K2OsO42H2O, tBuOH, H2O, 53%, dr >95:5. (d) CH3CHO, BF3OEt2, DCM, 93%, dr >95:5. (e) CAN, ACN, H2O, 72%.
Scheme 4. Reagents and conditions: (a) CAN, ACN, H2O, 65%.
Scheme 5. Reagents and conditions: (a) EDCHCl, NMM, MeON(H)MeHCl, DCM, 84%. (b) vinylmagnesium bromide, Et2O, 55%. (c) 3-methoxyprop-1-ene, cross metathesis catalyst, DCM, 40 °C, 30%, >20:1 E/Z. (d) methyl pent-4-enoate, cross metathesis catalyst, DCM, 40 °C, 41%, >20:1 E/Z.
does not inhibit PKAa. Surprisingly, however, deconstruction analogue 3 showed a reversal of this inhibition pattern. Analogue 3, which presumably forms activated intermediate 3a (Scheme 2), inhibited PKAa at submicromolar concentration but was 10-fold less active toward AKT1 than parent 7-deoxykalafungin. The remaining deconstruction analogues 5–8 were double-digit micro-molar inhibitors of AKT1 and did not inhibit PKAa. The data above, when combined with that previously published,25 gives us insight into the minimum structural elements of the PNQ lactone natural products that are required for potent
Figure 2. PNQ lactone analogue 21.25
and selective inhibition of AKT. Of note is previously reported PNQ lactone analogue 21 (Fig. 2),25 which contains the 3,6-dihydro-2H-pyran ring but lacks a substituent at C5. These data, combined with that reported in this work, demonstrate that the rigidity of 3,6-dihydro-2H-pyran ring is essential for potent and selective inhibition of AKT1. Our data also demonstrates that the activated quinone methide (as seen in 1 and 3a, Schemes 2 and 3) is necessary for submicromolar inhibiton of AKT and that it cannot be replaced with an a,b-unsaturated ketone with similar structural elements (such as 5–7). We have summarized the known data into a pharmacophore model (Fig. 3). The most unexpected result of this study is that deconstruction analogue 3 inhibits PKA at submicromolar concentrations and that it is a better inhibitor of PKA than AKT. PKA also contains a cysteine in the activation loop in a similar position to that of AKT (Cys199 of PKAa vs Cys310 of AKT1). The increased flexibility of deconstruction analogue 3 could be allowing this compound to alkylate PKAa Cys199. Deconstruction analogue 3 represents a new electrophile to explore for the development of an activity-based probe for PKAa. In summary, we have demonstrated that 3,6-dihydro-2H-pyran ring of the PNQ lactones is an essential structural feature for potency and selectivity. With a pharmacophore in hand for inhibition of AKT by the PNQ lactones, we can now begin to develop this scaffold into an AKT-selective activity-based probe. We have also identified a new electrophile and scaffold to explore for the development of an activity-based probe for PKAa.
Figure 3. Pharmacophore model for AKT inhibition by the PNQ lactones.
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Acknowledgments The author thanks Professor Richard A. Glennon, Professor B. Frank Gupton, and Professor John C. Hackett for helpful discussions and Professor Glen Kellogg for assistance in preparing this manuscript. Supplementary data Supplementary data (experimental data for the synthesis and characterization of 7-deoxykalafungin, analogues 3, 5–8, and intermediates 10–20, and assay protocols for inhibition of AKT and PKA) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11.020. References and notes 1. 2. 3. 4.
5. 6.
7.
8. 9.
Liu, Y.; Patricelli, M. P.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 14694. Evans, M. J.; Cravatt, B. F. Chem. Rev. 2006, 106, 3279. Nomura, D. K.; Dix, M. M.; Cravatt, B. F. Nat. Rev. Cancer 2010, 10, 630. Jessani, N.; Humphrey, M.; McDonald, W. H.; Niessen, S.; Masuda, K.; Gangadharan, B.; Yates, J. R.; Mueller, B. M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13756. Chiang, K. P.; Niessen, S.; Saghatelian, A.; Cravatt, B. F. Chem. Biol. 2006, 13, 1041. Ahn, K.; Johnson, D. S.; Mileni, M.; Beidler, D.; Long, J. Z.; McKinney, M. K.; Weerapana, E.; Sadagopan, N.; Liimatta, M.; Smith, S. E.; Lazerwith, S.; Stiff, C.; Kamtekar, S.; Bhattacharya, K.; Zhang, Y.; Swaney, S.; Van Becelaere, K.; Stevens, R. C.; Cravatt, B. F. Chem. Biol. 2009, 16, 411. Johnson, D. S.; Stiff, C.; Lazerwith, S. E.; Kesten, S. R.; Fay, L. K.; Morris, M.; Beidler, D.; Liimatta, M. B.; Smith, S. E.; Dudley, D. T.; Sadagopan, N.; Bhattachar, S. N.; Kesten, S. J.; Nomanbhoy, T. K.; Cravatt, B. F.; Ahn, K. ACS Med. Chem. Lett. 2010, 2, 91. Luo, Y.; Knuckley, B.; Bhatia, M.; Pellechia, P. J.; Thompson, P. R. J. Am. Chem. Soc. 2006, 128, 14468. Slack, J. L.; Causey, C. P.; Luo, Y.; Thompson, P. R. ACS Chem. Biol. 2011, 6, 466.
10. Niphakis, M. J.; Johnson, D. S.; Ballard, T. E.; Stiff, C.; Cravatt, B. F. ACS Chem. Neurosci. 2011, 3, 418. 11. Drahl, C.; Cravatt, B. F.; Sorensen, E. J. Angew. Chem., Int. Ed. 2005, 44, 5788. 12. Böttcher, T.; Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed. 2010, 49, 2680. 13. Krysiak, J.; Breinbauer, R. In Activity-Based Protein Profiling; Sieber, S. A., Ed.; ; Springer: Berlin, Heidelberg, 2012; Vol. 324, p 43. 14. Yuan, H.; Luo, J.; Field, S.; Weissleder, R.; Cantley, L.; Josephson, L. Bioconjugate Chem. 2005, 16, 669. 15. Liu, Y.; Shreder, K. R.; Gai, W.; Corral, S.; Ferris, D. K.; Rosenblum, J. S. Chem. Biol. 2005, 12, 99. 16. Yee, M.-C.; Fas, S. C.; Stohlmeyer, M. M.; Wandless, T. J.; Cimprich, K. A. J. Biol. Chem. 2005, 280, 29053. 17. Greenbaum, D.; Medzihradszky, K. F.; Burlingame, A.; Bogyo, M. Chem. Biol. 2000, 7, 569. 18. Staub, I.; Sieber, S. A. J. Am. Chem. Soc. 2008, 130, 13400. 19. Bottcher, T.; Sieber, S. A. MedChemComm 2012, 3, 408. 20. Liao, Y.; Hung, M.-C. Am. J. Transl. Res. 2010, 2, 19. 21. Qing-Bai, S.; Chandarlapaty, S.; Qing, Y.; Lobo, J.; Haskell, K. M.; Leander, K. R.; DeFeo-Jones, D.; Huber, H. E.; Rosen, N. PLoS One 2008, 3, 1. 22. George, S.; Rochford, J. J.; Wolfrum, C.; Gray, S. L.; Schinner, S.; Wilson, J. C.; Soos, M. A.; Murgatroyd, P. R.; Williams, R. M.; Acerini, C. L.; Dunger, D. B.; Barford, D.; Umpleby, A. M.; Wareham, N. J.; Davies, H. A.; Schafer, A. J.; Stoffel, M.; O’Rahilly, S.; Barroso, I. S. Science 2004, 304, 1325. 23. Griffin, R. J.; Moloney, A.; Kelliher, M.; Johnston, J. A.; Ravid, R.; Dockery, P.; O’Connor, R.; O’Neill, C. J. Neurochem. 2005, 93, 105. 24. Toral-Barza, L.; Zhang, W.-G.; Huang, X.; McDonald, L. A.; Salaski, E. J.; Barbieri, L. R.; Ding, W.-D.; Krishnamurthy, G.; Hu, Y. B.; Lucas, J.; Bernan, V. S.; Cai, P.; Levin, J. I.; Mansour, T. S.; Gibbons, J. J.; Abraham, R. T.; Yu, K. Mol. Cancer Ther. 2007, 6, 3028. 25. Salaski, E. J.; Krishnamurthy, G.; Ding, W.-D.; Yu, K.; Insaf, S. S.; Eid, C.; Shim, J.; Levin, J. I.; Tabei, K.; Toral-Barza, L.; Zhang, W.-G.; McDonald, L. A.; Honores, E.; Hanna, C.; Yamashita, A.; Johnson, B.; Li, Z.; Laakso, L.; Powell, D.; Mansour, T. S. J. Med. Chem. 2009, 52, 2181. 26. Brimble, M. A.; Nairn, M. R. J. Chem. Soc., Perkin Trans. 1 2000, 317. 27. Tatsuta, K.; Akimoto, K.; Annaka, M.; Ohno, Y.; Kinoshita, M. Bull. Chem. Soc. Jpn. 1985, 58, 1699. 28. Kraus, G. A.; Li, J.; Gordon, M. S.; Jensen, J. H. J. Org. Chem. 1995, 60, 1154. 29. Fernandes, R. A.; Brueckner, R. Synlett 2005, 1281. 30. Brimble, M. A. Pure Appl. Chem. 2000, 72, 1635. 31. Donner, C. D. Tetrahedron Lett. 2007, 48, 8888. 32. Eid, C. N.; Shim, J.; Bikker, J.; Lin, M. J. Org. Chem. 2008, 74, 423.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Preparation and evaluation of deconstruction analogues of 7-deoxykalafungin as AKT kinase inhibitors Sudha Korwar, Thuy Nguyen, Keith C. Ellis ⇑ Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Biotech 1, 800 East Leigh Street, Richmond, VA 23298, USA
a r t i c l e
i n f o
Article history: Received 5 July 2013 Revised 6 November 2013 Accepted 11 November 2013 Available online 20 November 2013 Keywords: Pyranonaphthoquinone lactone Covalent inhibitor AKT kinase Deconstruction analogues 7-Deoxykalafungin
a b s t r a c t The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify structural features of the natural product scaffold that are essential for potency and selectivity. Using a deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the 3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT. We have also unexpectedly identified a new submicromolar inhibitor of PKA. Ó 2013 Elsevier Ltd. All rights reserved.
Activity-based protein profiling (ABPP) is a chemical proteomics strategy that can be used to selectively identify the activated form of a protein in cells of various phenotypes both in vitro and in vivo.1–3 ABPP is a particularly powerful technique because it moves beyond simply quantifying the overall amount of a protein, active and inactive, in the cellular environment. ABPP detects only the fully activated, functional form of the protein of interest and does not detect forms that are overexpressed but down regulated, inactive because of posttranslational modifications, or not present in the cellular location of interest. ABPP has been used to elucidate the roles of multiple proteins and protein families in diseases including cancer,4,5 inflammation,6,7 rheumatoid arthritis,8,9 and depression.10 ABPP requires activity-based probes (ABPs) that contain three essential elements: (1) the probes must covalently modify a residue in the target protein that is reactive only in the activated form of the protein; (2) the probes must contain a scaffold that binds selectively to the targeted enzyme family or single enzyme; and (3) the probes must contain a tag that can be used for isolation or detection.2 One major source of ABPs has been protein-reactive natural products.11–13 Activity-based probes have been developed from the natural products wortmannin (targeting PI3K),14–16 E-64 (targeting the cathepsins),17 b-lactams (targeting penicillin binding proteins and b-lactamases),18,19 and several others (reviewed in Ref. 6). ⇑ Corresponding author. E-mail address: [email protected] (K.C. Ellis). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.11.020
AKT is a member of the AGC family of kinases that regulates multiple physiological processes including cell proliferation, metabolism, and survival.20 Hyperactivation of AKT kinase has been implicated in several disease states including cancer,21 type-II diabetes,22 and neurodegenerative diseases.23 To date, no activity-based probes targeting AKT kinase have been reported. The pyranonaphthoquinone (PNQ) lactone natural products lactoquinomycin and frenolicin B were identified as potent and selective inhibitors of AKT kinase in a high-throughput screen (Fig. 1).24 Further work demonstrated that the PNQ lactones inhibit AKT by
Figure 1. Structures of the pyranonaphthoquinone lactone natural products that inhibit AKT kinase.
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covalently alkylating Cys310 on the kinase activation loop.25 Despite the fact that this cysteine is conserved in over half of the enzymes in the AGC kinase family, the PNQ lactones are selective for AKT over these other family members. The preliminary structure-activity relationship (SAR) for the C5 position of the PNQ lactone 7-deoxykalafungin shows that monosubstitution at this position with both small (R = H, Me) and large (R = CH2OBn, 2-thienyl) groups are tolerated but that di-substitution leads to a 10-fold decrease in inhibition.25 This study did not investigate selectivity.25 As the first step in a program to explore the development of the PNQ lactones as an AKT-selective activity-based probe, we sought to understand the structural features of the natural product that impart potency and selectivity to this scaffold. In this communication, we have designed and prepared deconstruction analogues of 7-deoxykalafungin and evaluated their potency against recombinant AKT1 and selectivity for AKT1 over the closely related AGC kinase PKAa. The PNQ lactones must be activated in order to function as covalent inhibitors. Under in vitro and in vivo conditions the quinone is reduced and rearranges to the activated quinone methide (1, Scheme 1), which acts as a Michael acceptor (shown in magenta).26 The nucleophilic Cys310 of AKT then attacks the activated quinone methide 1, resulting in PNQ lactone—AKT adduct 2. The formation of the activated quinone methide 1 by reduction is essential for activity. Analogues missing the lactone ring are inactive as AKT inhibitors up to concentrations of 20 lM.25 The deconstruction analogues that we have designed and prepared are shown in Scheme 2. We designed these analogues to either preserve the latent electrophile by retaining the quinone (analogues 3 and 8) or replace this motif with an already-formed a,b-unsaturated ketone Michael acceptor (shown in magenta, analogues 5–7). We also designed these analogues to systematically remove structural elements from the natural product scaffold that provided steric bulk and conformational rigidity to determine how these elements contributed to both potency and selectivity. Several synthetic approaches to the PNQ lactones have been previously described.27–31 We chose to synthesize 7-deoxykalafungin by a variation of the route of Eid and co-workers32 starting from commercially available 1,4-dimethoxynaphthalene 9 as shown in Scheme 3. Bromination, Heck coupling, Sharpless asymmetric dihydroxylation with concomitant lactone formation, oxaPictet–Spengler reaction, and finally oxidation with CAN afforded the desired 7-deoxykalafungin. Deconstruction analogue 3 was prepared by treatment of intermediate 12 from the 7-deoxykalafungin synthesis with CAN (Scheme 4). Deconstruction analogues 5–7 were prepared by a common route starting from benzoic acid 19 (Scheme 5). EDC coupling afforded the Weinreb amide 20, which was treated with vinylmagnesium bromide in ethyl ether to afford the vinyl ketone 7. Subsequent treatment of 7 with a 2nd generation cross metathesis catalyst (CAS# 253688-91-4) and either 3-methoxyprop-1-ene or
Scheme 2. Deconstruction analysis of 7-deoxykalafungin and the structure of deconstruction analogues 3–8.
methyl pent-4-enoate in refluxing DCM afforded 5 and 6, respectively, both in >20:1 E/Z selectivity. Finally, naphthaquinone 8 was prepared by CAN oxidation of 1,4-dimethoxynaphthalene 9 (not shown). 7-Deoxykalafungin and compounds 3 and 5–8 were tested in the Z’-LYTE assay (Invitrogen) for their ability to inhibit kinase function of AKT1 and PKAa (Table 1). This FRET-based assay measures the ability of a kinase enzyme to phosphorylate a peptide substrate and detects functional inhibitors of kinase activity independent of mechanism of action. Our data confirmed the previous report that 7-deoxykalafungin is a potent inhibitor of AKT1 but
Scheme 1. Activation of 7-deoxykalafungin to form activated quinone methide 1 and AKT adduct 2.
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Table 1 Inhibition of recombinant AKT1 and PKAa by 7-deoxykalafungin and analogues 3-8 Compound
7-Deoxykalafungin 3 5 6 7 8
IC50a (lM) AKT1
PKAa
0.28 2.94 50.0 13.7 12.3 11.0
>100 0.43 >100 >100 >100 n.d. b
a IC50’s were determined using Z’-LYTE assay kits (Invitrogen). 95% confidence intervals can be found in the Supporting information. b Not determined.
Scheme 3. Reagents and conditions: (a) NBS, DCM, 96%. (b) isobutyl but-3-enoate, Pd(tBu3P)2, Cy2NMe, toluene, 81%. (c) K3Fe(CN)6, K2CO3, NaHCO3, (DHQD)2PHAL, CH3SO2NH2, K2OsO42H2O, tBuOH, H2O, 53%, dr >95:5. (d) CH3CHO, BF3OEt2, DCM, 93%, dr >95:5. (e) CAN, ACN, H2O, 72%.
Scheme 4. Reagents and conditions: (a) CAN, ACN, H2O, 65%.
Scheme 5. Reagents and conditions: (a) EDCHCl, NMM, MeON(H)MeHCl, DCM, 84%. (b) vinylmagnesium bromide, Et2O, 55%. (c) 3-methoxyprop-1-ene, cross metathesis catalyst, DCM, 40 °C, 30%, >20:1 E/Z. (d) methyl pent-4-enoate, cross metathesis catalyst, DCM, 40 °C, 41%, >20:1 E/Z.
does not inhibit PKAa. Surprisingly, however, deconstruction analogue 3 showed a reversal of this inhibition pattern. Analogue 3, which presumably forms activated intermediate 3a (Scheme 2), inhibited PKAa at submicromolar concentration but was 10-fold less active toward AKT1 than parent 7-deoxykalafungin. The remaining deconstruction analogues 5–8 were double-digit micro-molar inhibitors of AKT1 and did not inhibit PKAa. The data above, when combined with that previously published,25 gives us insight into the minimum structural elements of the PNQ lactone natural products that are required for potent
Figure 2. PNQ lactone analogue 21.25
and selective inhibition of AKT. Of note is previously reported PNQ lactone analogue 21 (Fig. 2),25 which contains the 3,6-dihydro-2H-pyran ring but lacks a substituent at C5. These data, combined with that reported in this work, demonstrate that the rigidity of 3,6-dihydro-2H-pyran ring is essential for potent and selective inhibition of AKT1. Our data also demonstrates that the activated quinone methide (as seen in 1 and 3a, Schemes 2 and 3) is necessary for submicromolar inhibiton of AKT and that it cannot be replaced with an a,b-unsaturated ketone with similar structural elements (such as 5–7). We have summarized the known data into a pharmacophore model (Fig. 3). The most unexpected result of this study is that deconstruction analogue 3 inhibits PKA at submicromolar concentrations and that it is a better inhibitor of PKA than AKT. PKA also contains a cysteine in the activation loop in a similar position to that of AKT (Cys199 of PKAa vs Cys310 of AKT1). The increased flexibility of deconstruction analogue 3 could be allowing this compound to alkylate PKAa Cys199. Deconstruction analogue 3 represents a new electrophile to explore for the development of an activity-based probe for PKAa. In summary, we have demonstrated that 3,6-dihydro-2H-pyran ring of the PNQ lactones is an essential structural feature for potency and selectivity. With a pharmacophore in hand for inhibition of AKT by the PNQ lactones, we can now begin to develop this scaffold into an AKT-selective activity-based probe. We have also identified a new electrophile and scaffold to explore for the development of an activity-based probe for PKAa.
Figure 3. Pharmacophore model for AKT inhibition by the PNQ lactones.
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Acknowledgments The author thanks Professor Richard A. Glennon, Professor B. Frank Gupton, and Professor John C. Hackett for helpful discussions and Professor Glen Kellogg for assistance in preparing this manuscript. Supplementary data Supplementary data (experimental data for the synthesis and characterization of 7-deoxykalafungin, analogues 3, 5–8, and intermediates 10–20, and assay protocols for inhibition of AKT and PKA) associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.11.020. References and notes 1. 2. 3. 4.
5. 6.
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