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Design and synthesis of paracaseolide A analogues as selective protein tyrosine phosphatase 1B inhibitors† Jian-Peng Yin,‡a Chun-Lan Tang,‡b Li-Xin Gao,b Wei-Ping Ma,b Jing-Ya Li,b Ying Li,*a Jia Li*b and Fa-Jun Nan*b
Received 27th January 2014, Accepted 24th March 2014
A series of structurally related analogues of the natural product paracaseolide A were synthesized and
DOI: 10.1039/c4ob00214h
identified as potent PTP1B inhibitors. Among these analogues, compound 10 in particular showed improved PTP1B enzyme inhibitory activity, high selectivity for PTP1B over TC-PTP, and improved cellular
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effects.
Introduction As the global incidence of type 2 diabetes and obesity continues to grow, there is a clear need for a new therapeutic agent to treat these diseases. Protein tyrosine phosphatase 1B (PTP1B) has been shown to play an essential role in signal transduction for both insulin and leptin pathways.1,2 Gene knockout studies have also demonstrated that PTP1B-deficient mice display increased insulin sensitivity and are resistant to diet-induced obesity.3,4 Thus, PTP1B is considered to be one of the best validated biological targets for the treatment of type 2 diabetes. Several structurally diverse small-molecule inhibitors of PTP1B have previously been developed;5–12 many of these were designed to bind to the PTP1B active site.5 Achieving high selectivity for PTP1B over other closely associated PTPs (LAR, SHP-1, SHP-2 and TC-PTP), however proved to be a major challenge. The catalytic domains share a high degree of primary and tertiary structural similarity and have similar active sites.1,13 Oral bioavailability has been another challenge in the development of potent and selective PTP1B inhibitors, as the majority of the active site-directed PTP1B inhibitors exhibit limited cell permeability.14 Therefore, there is an urgent need to develop novel potential drug scaffolds targeting PTP1B.
a State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China. E-mail: [email protected] b The National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: 1H and 13C NMR data for all new compounds and experimental data for compounds. See DOI: 10.1039/ c4ob00214h ‡ These authors contributed equally.
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Paracaseolide A, which was isolated from the stem bark of the Chinese mangrove plant Sonneratia paracaseolaris by Guo and co-workers,15 is characterized by a tetracyclic nucleus (tetraquinane oxa-cage bislactone) bearing two linear alkyl chains at C-2 and C-7. By virtue of its unique scaffold and biological activity, paracaseolide A is an attractive target for chemists. The first total synthesis of paracaseolide A was accomplished by Vassilikogiannakis and co-workers via a biosynthetic [4 + 2] dimerization of 12,16 as proposed by Guo et al. Recently, Kraus and co-workers also accessed paracaseolide A in 8 steps from a known symmetric bis-lactone compound.17 In the initial bioassay, paracaseolide A showed significant inhibitory activity against dual specificity phosphatase CDC25B with an IC50 value of 6.44 μM.15 The structure–activity relationship of this novel skeleton has not yet been reported for CDC25B or other phosphatases. We recently found that synthetic paracaseolide A – which is equipotent against CDC25B compared to the natural sample – displays potent inhibitory activity against PTP1B and T-cell protein tyrosine phosphatase (TC-PTP) in our PTP panel assays, with IC50 values of 1.50 μM and 6.01 μM respectively. Modest selectivity (T/P = 4) was observed between the two highly associated PTPs. Herein, we describe the synthesis of a series of structurally related analogues of paracaseolide A. Of the synthesized compounds, 9 and 10 were identified as the most potent inhibitors of PTP1B, with sub-micro molar IC50s and high selectivity for inhibition of PTP1B over TC-PTP (17-fold selectivity) (Table 1).
Results and discussion The synthesis of diverse paracaseolide A analogues was generally achieved by means of an intermolecular Diels–Alder [4 + 2]
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Paper Table 1
Organic & Biomolecular Chemistry Inhibitory activity for other phosphatases compared with PTP1B
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IC50 ± SD (μM)
6 7 8 9 10 13 14 15 a b
PTP1Ba
TC-PTP
LAR
SHP-1
SHP-2
CDC25B
3.25 ± 0.85 4.27 ± 0.68 NAb 0.27 ± 0.02 0.27 ± 0.03 1.50 ± 0.33 6.00 ± 0.30 20.4 ± 2.47
13.16 ± 0.51 31.79 ± 6.15 NA 2.55 ± 0.27 4.73 ± 1.28 6.01 ± 0.33 9.38 ± 0.35 19.79 ± 1.36
NA NA NA NA NA NA NA NA
NA NA NA 3.86 ± 0.32 4.41 ± 0.58 NA NA NA
NA NA NA 3.13 ± 0.58 1.71 ± 0.38 NA NA NA
2.34 ± 0.17 3.93 ± 0.17 NA 0.22 ± 0.02 0.20 ± 0.02 2.34 ± 0.00 8.33 ± 1.32 17.0 ± 1.68
Selected test compounds were screened for TC-PTP, LAR, SHP-1, SHP-2 inhibitory activity. IC50 values were means ± SD of three replications. NA: no activity (inhibitory rate less than 50% at dose of 20 μg mL−1).
dimerization of monomer 5,18 which, in turn, was prepared by a three-step sequence from commercially available n-tetradecanal 1 and (S)-ethyl-lactate 3, as outlined in Scheme 1. n-Tetradecanal was transformed into α,β-unsaturated ester 2 in 87% yield using a Wittig reaction.19 THP protection of (S)-ethyllactate 3 followed by reduction with DIBAL at −78 °C gave aldehyde 4 in 98% yield.20 Addition of the tetrahydropyranyl ether 4 to the dienolate derived from 2, followed by acetal hydrolysis, lactonization using p-toluenesulfonic acid in methanol, and then dehydration (MsCl, Et3N, CH2Cl2), provided 5 as a mixture of geometrical isomers (Z : E 8 : 1); the Z isomer could be fully transformed into the E isomer 5 in the presence of iodine under direct sunlight.21 With compound E-5 in hand, the key Diels–Alder [4 + 2] dimerization reaction was optimized for the solvent, Lewis acid, temperature, and reaction time. Ultimately, running the reaction in toluene–water (2 : 1) with BHT at 170 °C for 20 hours yielded the dimerization product 6 in 68% yield (Scheme 2).18 When the reaction conditions were modified in toluene with BHT at 210 °C for 20 h, the dimerization product 8 was unexpectedly obtained as the major product in 50% yield. Selective saponification of 6, and subsequent oxidation with Dess–Martin periodinane gave compound 9 in 68% yield. Divergently, α,β-unsaturated lactone 6 was also transformed into 7 using NaOMe in methanol in 83% yield. Analogue 10 was obtained from 7 by selective lactone hydrolysis and oxidation. The stereochemistry of compounds 6–10 was assigned using HMBC and HMQC.
Scheme 1 Reagents and conditions: (a) Ph3PvCHCOOEt, CH2Cl2, R.T., 10 h, 87%; (b) (i) DHP, PPTS, CH2Cl2, R.T., 9 h; (ii) DIBAL, −78 °C, 40 min; then MeOH, H2O, R.T., 1 h, 98%, over 2 steps; (c) (i) LDA, HMPA, THF, −78 °C, 1 h; then 4; (ii) TsOH·H2O, MeOH, R.T., 8 h; then NEt3; (iii) MsCl, NEt3, CH2Cl2, 0 °C, 2.5 h, 57%, over 3 steps; (d) I2, CH2Cl2, hν, 8 h, 85%.
3442 | Org. Biomol. Chem., 2014, 12, 3441–3445
Scheme 2 Reagents and conditions: (a) BHT (cat.), toluene–H2O (2 : 1), sealed tube, 170 °C, 20 h, 68%; (b) NaOMe, MeOH, 50 °C, 6 h, 83%; (c) (i) t-BuOH/1 M KOH aq. (1 : 1), R.T., 2.5 h, (ii) Dess–Martin periodinane, CH2Cl2, R.T., 6 h, 65%, over 2 steps; (d) BHT (cat.), toluene, sealed tube, 210 °C, 20 h, 50%.
Following the work of Vassilikogiannakis and co-workers,16 paracaseolide A (13) can be obtained from 12 by an intermolecular Diels–Alder [4 + 2] dimerization; 12 is accessible from butenolide 5 in two steps.22 Selective ozonolysis of 13 gave aldehyde 14 and subsequent reduction with sodium cyanoborohydride gave alcohol 15 in 80% yield in two steps (Scheme 3). Synthesized analogues 6–10, 14, 15 and synthetic natural product paracaseolide A (13) were tested in PTP1B inhibition assays, as shown in Table 1. Paracaseolide A, 6 and 7 exhibited similar activities with IC50 values of approximately 2–4 μM. This suggests that the tetrahydrofuran ring does not obviously affect activity as it relates to PTP1B inhibition. Compound 9, which has one lactone ring opened and a free carboxylic acid group, displayed a more potent inhibitory activity, with an IC50 of 0.27 μM. This is a 5-fold improvement compared to paracaseolide A. Our results, together with recent reports that stearic acid is an inhibitor of PTP1B, imply that aliphatic carboxylic acids contribute to PTP1B inhibitory activity.23 Compound 10, which differs from 9, only in the arrangement of the conjugated double bond from α,β- to β,γ-position, exhibited potent activity, with an IC50 value of 0.27 μM. These results
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Scheme 3 Reagents and conditions: (a) NEt3, CH2Cl2; then TBSOTf, R.T., 4 h, 91%; (b) m-CPBA, CH2Cl2, −78 °C, 30 min, 92%; (c) O3, CH2Cl2, −78 °C, 0.5 min; then S(CH3)2, 97%; (d) THF–AcOH (10 : 1), NaBH3CN, 0 °C, 0.5 h; then R.T. 0.5 h, 83%.
indicate that the α,β-unsaturated carbonyl in 9 may not serve as a Michael receptor to covalently modify the catalytic residue. A kinetic study was performed in order to identify the inhibitory mechanism of compound 9 (see Fig. 1 in ESI†). In enzyme kinetics assays, 9 demonstrated a time-independent inhibition of PTP1B. The results indicate that 9 is a fastbinding inhibitor of PTP1B. We further determined the inhibition modality of 9, which inhibits PTP1B with characteristics typical of a competitive inhibitor, as indicated by increased Km values and unchanged Vmax values when the inhibitor concentration is increased. A Lineweaver–Burk plot confirmed that 9 is a competitive inhibitor of PTP1B. Together, these results suggest that 9 binds to the catalytic pocket of PTP1B and behaves as a competitor to the substrate. Natural product paracaseolide A has two long alkyl chains, which make the compound highly lipophilic (clog P ≈ 11). In order to decrease the lipophilicity, compounds 14 and 15 were synthesized through ozonolysis of one alkyl chain and subsequent reduction. To our disappointment, the activity of both compounds was significantly decreased, indicating that the hydrophobic interaction between the long alkyl chains and protein surface contribute to the remarkable binding affinity. This conclusion was further supported by compound 8, the other dimerization product, which bears a dramatically different skeleton and showed no activity against PTPs. The selectivity of these novel PTP1B inhibitors against TC-PTP, SHP-1, SHP-2, LAR, and CDC25B are outlined in Table 1. All synthetic compounds including paracaseolide A selectively inhibit PTP1B, CDC25B and TC-PTP over LAR, SHP-1, and SHP-2. Analogues 9 and 10, were not only the most potent PTP1B inhibitors with IC50 values of 0.27 ± 0.02 µM and 0.27 ± 0.03 µM, respectively, but also showed high selectivity (9- to 17-fold) for PTP1B over TC-PTP. TC-PTP has the highest homology to PTP1B, with 72% sequence identity in the catalytic domain.14 These compounds exhibited significant improvement in both potency and selectivity compared to the natural product paracaseolide A. In order to further understand the activity and the selectivity of compound 10, molecular docking analysis was carried
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Paper
out using LibDock available with Discovery Studio 2.1. The X-ray crystal structure of PTP1B (PDB-ID: 1NNY) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). The preferred coordination modes of compound 10 with PTP1B are presented in Fig. 1. Fig. 1a depicts the binding of the fused ring of compound 10 in the active site. Fig. 1b shows the binding interactions of 10 with PTP1B. The carbonyl of the –COOH moiety may interact with Asn111 via a hydrogen bonding interaction, while the ketone is hydrogen bonded to Ser216 and Ala217. Comparison with compound 13 suggests that the opening of lactone ring and the free carboxylic acid group in 10 may contribute significantly to its improved inhibitory activity. It has been proposed that dual-binding inhibitors, which occupy both the active site and second phosphotyrosine ( pTyr) binding site adjacent to the catalytic site of PTP1B would provide both increased binding affinity and the opportunity for improved selectivity over highly homologous phosphatase TC-PTP.6,10,14 The second pTyr binding site adjacent to the catalytic site is lined by Arg24, Asp48, Val49, Ile219, Arg254, Met258, Gly259 and Gln262.14 Fig. 1 shows that compound 10 extends the full span of active site and second pTyr binding site of PTP1B, the long alkyl chain in 10 binds in the
Fig. 1 Molecular docking analysis of compound 10, showing proposed binding modes with PTP1B. Ligands are shown in stick representation with carbon atoms in cyan. Hydrogen bonds are displayed as green dashed lines. (a) Binding pose of compound 10 in the protein surface of PTP1B. (b) Interaction of compound 10 with catalytic residues of PTP1B.
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Notes and references
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Fig. 2 Effects of 9, 10 and paracaseolide A (13) on insulin signaling. CHO/hIR cells were treated with compounds or PC for 1 h, followed by insulin (10 nM). The expression and phosphorylation levels of IR were detected using IRβ and PY-20 antibodies, respectively. PC ( positive control): sodium orthovanadate (250 μM); NC (negative control): DMSO.
second pTyr binding site by hydrophobic interaction with Ile219, Met258 and Gly259. Thus, the activity against PTP1B of compound 10 and its good selectivity over TC-PCP can be well explained. PTP1B acts as a negative regulator of insulin signaling via dephosphorylation of the insulin receptor (IR) and insulin receptor substrate proteins (IRS).24 Increased expression and activity of PTP1B has been found in diabetic and obese humans and mice, while PTP1B knockout mice exhibit improved insulin sensitivity and resistance to diet-induced obesity.4 To explore the cellular effect of PTP1B inhibitors, 9, 10 and 13, the phosphorylation level of IR was detected in CHO/hIR cells after incubating with the compounds (5 to 20 μM) for 1 h. As shown in Fig. 2, compound 10 obviously improved p-IR compared to the negative control (NC) in a dose-dependent manner. This result indicates that the analogues of paracaseolide A hold promise as novel PTP1B inhibitors to counter insulin resistance.
Conclusion In conclusion, a series of paracaseolide A analogues were synthesized and evaluated for their inhibitory activity for various PTPs (PTP1B, TC-PTP, LAR, SHP-1, SHP-2 and CDC25B). Most of these analogues exhibit similar or greater activity against PTP1B than the natural product. Compounds 9 and 10, in particular, exhibit potent inhibitory activity and modest to high selectivity over TC-PTP. The improved cellular activity of these novel inhibitors also correlated with an improvement in enzymatic inhibitory activity, although the membrane permeability of these highly lipophilic compounds requires further optimization. These compounds provide a new starting point for further development of paracaseolide A-inspired PTP1B inhibitors, the work on which is currently underway in our laboratory.
Acknowledgements This work was supported by the National Program on Key Basic Research Project (2012CB524906), the National Natural Science Foundation of China (81125023), and Shanghai Commission of Science and Technology (13DZ2290300).
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1 J. N. Andersen, O. H. Mortensen, G. H. Peters, P. G. Drake, L. F. Iversen, O. H. Olsen, P. G. Jansen, H. S. Andersen, N. K. Tonks and N. P. Moller, Mol. Cell. Biol., 2001, 21, 7117–7136. 2 S.-C. Yip, S. Saha and J. Chernoff, Trends Biochem. Sci., 2010, 35, 442–449. 3 M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. HimmsHagen, C.-C. Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay and B. P. Kennedy, Science, 1999, 283, 1544–1548. 4 L. D. Klaman, O. Boss, O. D. Peroni, J. K. Kim, J. L. Martino, J. M. Zabolotny, N. Moghal, M. Lubkin, Y.-B. Kim, A. H. Sharpe, A. Stricker-Krongrad, G. I. Shulman, B. G. Neel and B. B. Kahn, Mol. Cell. Biol., 2000, 20, 5479–5489. 5 T. R. Burke, H. K. Kole and P. P. Roller, Biochem. Biophys. Res. Commun., 1994, 204, 129–134. 6 Y. A. Puius, Y. Zhao, M. Sullivan, D. S. Lawrence, S. C. Almo and Z. Y. Zhang, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 13420–13425. 7 G. Scapin, S. B. Patel, J. W. Becker, Q. Wang, C. Desponts, D. Waddleton, K. Skorey, W. Cromlish, C. Bayly, M. Therien, J. Y. Gauthier, C. S. Li, C. K. Lau, C. Ramachandran, B. P. Kennedy and E. Asante-Appiah, Biochemistry, 2003, 42, 11451–11459. 8 B. G. Szczepankiewicz, G. Liu, P. J. Hajduk, C. Abad-Zapatero, Z. Pei, Z. Xin, T. H. Lubben, J. M. Trevillyan, M. A. Stashko, S. J. Ballaron, H. Liang, F. Huang, C. W. Hutchins, S. W. Fesik and M. R. Jirousek, J. Am. Chem. Soc., 2003, 125, 4087–4096. 9 A. P. Combs, W. Zhu, M. L. Crawley, B. Glass, P. Polam, R. B. Sparks, D. Modi, A. Takvorian, E. McLaughlin, E. W. Yue, Z. Wasserman, M. Bower, M. Wei, M. Rupar, P. J. Ala, B. M. Reid, D. Ellis, L. Gonneville, T. Emm, N. Taylor, S. Yeleswaram, Y. Li, R. Wynn, T. C. Burn, G. Hollis, P. C. C. Liu and B. Metcalf, J. Med. Chem., 2006, 49, 3774–3789. 10 S. Zhang and Z. Zhang, Drug Discovery Today, 2007, 12, 373–381. 11 Y. Wei, Y.-T. Chen, L. Shi, L.-X. Gao, S. Liu, Y.-M. Cui, W. Zhang, Q. Shen, J. Li and F.-J. Nan, MedChemComm, 2011, 2, 1104–1109. 12 Y.-T. Chen, C.-L. Tang, W.-P. Ma, L.-X. Gao, Y. Wei, W. Zhang, Q. Shen, J.-Y. Li, J. Li and F.-J. Nan, Eur. J. Med. Chem., 2013, 69, 399–412. 13 T. Tiganis and A. M. Bennett, Biochem. J., 2007, 402, 1–15. 14 A. P. Combs, J. Med. Chem., 2010, 53, 2333–2344. 15 X.-L. Chen, H.-L. Liu, J. Li, G.-R. Xin and Y.-W. Guo, Org. Lett., 2011, 13, 5032–5035. 16 D. Noutsias and G. Vassilikogiannakis, Org. Lett., 2012, 14, 3565–3567. 17 T. Guney and G. A. Kraus, Org. Lett., 2013, 15, 613–615.
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22 S. K. Bagal, R. M. Adlington, R. A. B. Brown and J. E. Baldwin, Tetrahedron Lett., 2005, 46, 4633– 4637. 23 A. Tsuchiya, T. Kanno and T. Nishizaki, Cell. Physiol. Biochem., 2013, 32, 1451–1459. 24 K. A. Kenner, E. Anyanwu, J. M. Olefsky and J. Kusari, J. Biol. Chem., 1996, 271, 19810–19816.
Published on 25 March 2014. Downloaded by Colorado State University on 22/08/2014 11:27:08.
18 D. C. Rideout and R. Breslow, J. Am. Chem. Soc., 1980, 102, 7816–7817. 19 T. Xu, C.-c. Li and Z. Yang, Org. Lett., 2011, 13, 2630–2633. 20 Y. Ito, Y. Kobayashi, T. Kawabata, M. Takase and S. Terashima, Tetrahedron, 1989, 45, 5767–5790. 21 D. J. Hart, W.-L. Wu and A. P. Kozikowski, J. Am. Chem. Soc., 1995, 117, 9369–9370.
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Design and synthesis of paracaseolide A analogues as selective protein tyrosine phosphatase 1B inhibitors† Jian-Peng Yin,‡a Chun-Lan Tang,‡b Li-Xin Gao,b Wei-Ping Ma,b Jing-Ya Li,b Ying Li,*a Jia Li*b and Fa-Jun Nan*b
Received 27th January 2014, Accepted 24th March 2014
A series of structurally related analogues of the natural product paracaseolide A were synthesized and
DOI: 10.1039/c4ob00214h
identified as potent PTP1B inhibitors. Among these analogues, compound 10 in particular showed improved PTP1B enzyme inhibitory activity, high selectivity for PTP1B over TC-PTP, and improved cellular
www.rsc.org/obc
effects.
Introduction As the global incidence of type 2 diabetes and obesity continues to grow, there is a clear need for a new therapeutic agent to treat these diseases. Protein tyrosine phosphatase 1B (PTP1B) has been shown to play an essential role in signal transduction for both insulin and leptin pathways.1,2 Gene knockout studies have also demonstrated that PTP1B-deficient mice display increased insulin sensitivity and are resistant to diet-induced obesity.3,4 Thus, PTP1B is considered to be one of the best validated biological targets for the treatment of type 2 diabetes. Several structurally diverse small-molecule inhibitors of PTP1B have previously been developed;5–12 many of these were designed to bind to the PTP1B active site.5 Achieving high selectivity for PTP1B over other closely associated PTPs (LAR, SHP-1, SHP-2 and TC-PTP), however proved to be a major challenge. The catalytic domains share a high degree of primary and tertiary structural similarity and have similar active sites.1,13 Oral bioavailability has been another challenge in the development of potent and selective PTP1B inhibitors, as the majority of the active site-directed PTP1B inhibitors exhibit limited cell permeability.14 Therefore, there is an urgent need to develop novel potential drug scaffolds targeting PTP1B.
a State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P.R. China. E-mail: [email protected] b The National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China. E-mail: [email protected], [email protected] † Electronic supplementary information (ESI) available: 1H and 13C NMR data for all new compounds and experimental data for compounds. See DOI: 10.1039/ c4ob00214h ‡ These authors contributed equally.
This journal is © The Royal Society of Chemistry 2014
Paracaseolide A, which was isolated from the stem bark of the Chinese mangrove plant Sonneratia paracaseolaris by Guo and co-workers,15 is characterized by a tetracyclic nucleus (tetraquinane oxa-cage bislactone) bearing two linear alkyl chains at C-2 and C-7. By virtue of its unique scaffold and biological activity, paracaseolide A is an attractive target for chemists. The first total synthesis of paracaseolide A was accomplished by Vassilikogiannakis and co-workers via a biosynthetic [4 + 2] dimerization of 12,16 as proposed by Guo et al. Recently, Kraus and co-workers also accessed paracaseolide A in 8 steps from a known symmetric bis-lactone compound.17 In the initial bioassay, paracaseolide A showed significant inhibitory activity against dual specificity phosphatase CDC25B with an IC50 value of 6.44 μM.15 The structure–activity relationship of this novel skeleton has not yet been reported for CDC25B or other phosphatases. We recently found that synthetic paracaseolide A – which is equipotent against CDC25B compared to the natural sample – displays potent inhibitory activity against PTP1B and T-cell protein tyrosine phosphatase (TC-PTP) in our PTP panel assays, with IC50 values of 1.50 μM and 6.01 μM respectively. Modest selectivity (T/P = 4) was observed between the two highly associated PTPs. Herein, we describe the synthesis of a series of structurally related analogues of paracaseolide A. Of the synthesized compounds, 9 and 10 were identified as the most potent inhibitors of PTP1B, with sub-micro molar IC50s and high selectivity for inhibition of PTP1B over TC-PTP (17-fold selectivity) (Table 1).
Results and discussion The synthesis of diverse paracaseolide A analogues was generally achieved by means of an intermolecular Diels–Alder [4 + 2]
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Paper Table 1
Organic & Biomolecular Chemistry Inhibitory activity for other phosphatases compared with PTP1B
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IC50 ± SD (μM)
6 7 8 9 10 13 14 15 a b
PTP1Ba
TC-PTP
LAR
SHP-1
SHP-2
CDC25B
3.25 ± 0.85 4.27 ± 0.68 NAb 0.27 ± 0.02 0.27 ± 0.03 1.50 ± 0.33 6.00 ± 0.30 20.4 ± 2.47
13.16 ± 0.51 31.79 ± 6.15 NA 2.55 ± 0.27 4.73 ± 1.28 6.01 ± 0.33 9.38 ± 0.35 19.79 ± 1.36
NA NA NA NA NA NA NA NA
NA NA NA 3.86 ± 0.32 4.41 ± 0.58 NA NA NA
NA NA NA 3.13 ± 0.58 1.71 ± 0.38 NA NA NA
2.34 ± 0.17 3.93 ± 0.17 NA 0.22 ± 0.02 0.20 ± 0.02 2.34 ± 0.00 8.33 ± 1.32 17.0 ± 1.68
Selected test compounds were screened for TC-PTP, LAR, SHP-1, SHP-2 inhibitory activity. IC50 values were means ± SD of three replications. NA: no activity (inhibitory rate less than 50% at dose of 20 μg mL−1).
dimerization of monomer 5,18 which, in turn, was prepared by a three-step sequence from commercially available n-tetradecanal 1 and (S)-ethyl-lactate 3, as outlined in Scheme 1. n-Tetradecanal was transformed into α,β-unsaturated ester 2 in 87% yield using a Wittig reaction.19 THP protection of (S)-ethyllactate 3 followed by reduction with DIBAL at −78 °C gave aldehyde 4 in 98% yield.20 Addition of the tetrahydropyranyl ether 4 to the dienolate derived from 2, followed by acetal hydrolysis, lactonization using p-toluenesulfonic acid in methanol, and then dehydration (MsCl, Et3N, CH2Cl2), provided 5 as a mixture of geometrical isomers (Z : E 8 : 1); the Z isomer could be fully transformed into the E isomer 5 in the presence of iodine under direct sunlight.21 With compound E-5 in hand, the key Diels–Alder [4 + 2] dimerization reaction was optimized for the solvent, Lewis acid, temperature, and reaction time. Ultimately, running the reaction in toluene–water (2 : 1) with BHT at 170 °C for 20 hours yielded the dimerization product 6 in 68% yield (Scheme 2).18 When the reaction conditions were modified in toluene with BHT at 210 °C for 20 h, the dimerization product 8 was unexpectedly obtained as the major product in 50% yield. Selective saponification of 6, and subsequent oxidation with Dess–Martin periodinane gave compound 9 in 68% yield. Divergently, α,β-unsaturated lactone 6 was also transformed into 7 using NaOMe in methanol in 83% yield. Analogue 10 was obtained from 7 by selective lactone hydrolysis and oxidation. The stereochemistry of compounds 6–10 was assigned using HMBC and HMQC.
Scheme 1 Reagents and conditions: (a) Ph3PvCHCOOEt, CH2Cl2, R.T., 10 h, 87%; (b) (i) DHP, PPTS, CH2Cl2, R.T., 9 h; (ii) DIBAL, −78 °C, 40 min; then MeOH, H2O, R.T., 1 h, 98%, over 2 steps; (c) (i) LDA, HMPA, THF, −78 °C, 1 h; then 4; (ii) TsOH·H2O, MeOH, R.T., 8 h; then NEt3; (iii) MsCl, NEt3, CH2Cl2, 0 °C, 2.5 h, 57%, over 3 steps; (d) I2, CH2Cl2, hν, 8 h, 85%.
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Scheme 2 Reagents and conditions: (a) BHT (cat.), toluene–H2O (2 : 1), sealed tube, 170 °C, 20 h, 68%; (b) NaOMe, MeOH, 50 °C, 6 h, 83%; (c) (i) t-BuOH/1 M KOH aq. (1 : 1), R.T., 2.5 h, (ii) Dess–Martin periodinane, CH2Cl2, R.T., 6 h, 65%, over 2 steps; (d) BHT (cat.), toluene, sealed tube, 210 °C, 20 h, 50%.
Following the work of Vassilikogiannakis and co-workers,16 paracaseolide A (13) can be obtained from 12 by an intermolecular Diels–Alder [4 + 2] dimerization; 12 is accessible from butenolide 5 in two steps.22 Selective ozonolysis of 13 gave aldehyde 14 and subsequent reduction with sodium cyanoborohydride gave alcohol 15 in 80% yield in two steps (Scheme 3). Synthesized analogues 6–10, 14, 15 and synthetic natural product paracaseolide A (13) were tested in PTP1B inhibition assays, as shown in Table 1. Paracaseolide A, 6 and 7 exhibited similar activities with IC50 values of approximately 2–4 μM. This suggests that the tetrahydrofuran ring does not obviously affect activity as it relates to PTP1B inhibition. Compound 9, which has one lactone ring opened and a free carboxylic acid group, displayed a more potent inhibitory activity, with an IC50 of 0.27 μM. This is a 5-fold improvement compared to paracaseolide A. Our results, together with recent reports that stearic acid is an inhibitor of PTP1B, imply that aliphatic carboxylic acids contribute to PTP1B inhibitory activity.23 Compound 10, which differs from 9, only in the arrangement of the conjugated double bond from α,β- to β,γ-position, exhibited potent activity, with an IC50 value of 0.27 μM. These results
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Scheme 3 Reagents and conditions: (a) NEt3, CH2Cl2; then TBSOTf, R.T., 4 h, 91%; (b) m-CPBA, CH2Cl2, −78 °C, 30 min, 92%; (c) O3, CH2Cl2, −78 °C, 0.5 min; then S(CH3)2, 97%; (d) THF–AcOH (10 : 1), NaBH3CN, 0 °C, 0.5 h; then R.T. 0.5 h, 83%.
indicate that the α,β-unsaturated carbonyl in 9 may not serve as a Michael receptor to covalently modify the catalytic residue. A kinetic study was performed in order to identify the inhibitory mechanism of compound 9 (see Fig. 1 in ESI†). In enzyme kinetics assays, 9 demonstrated a time-independent inhibition of PTP1B. The results indicate that 9 is a fastbinding inhibitor of PTP1B. We further determined the inhibition modality of 9, which inhibits PTP1B with characteristics typical of a competitive inhibitor, as indicated by increased Km values and unchanged Vmax values when the inhibitor concentration is increased. A Lineweaver–Burk plot confirmed that 9 is a competitive inhibitor of PTP1B. Together, these results suggest that 9 binds to the catalytic pocket of PTP1B and behaves as a competitor to the substrate. Natural product paracaseolide A has two long alkyl chains, which make the compound highly lipophilic (clog P ≈ 11). In order to decrease the lipophilicity, compounds 14 and 15 were synthesized through ozonolysis of one alkyl chain and subsequent reduction. To our disappointment, the activity of both compounds was significantly decreased, indicating that the hydrophobic interaction between the long alkyl chains and protein surface contribute to the remarkable binding affinity. This conclusion was further supported by compound 8, the other dimerization product, which bears a dramatically different skeleton and showed no activity against PTPs. The selectivity of these novel PTP1B inhibitors against TC-PTP, SHP-1, SHP-2, LAR, and CDC25B are outlined in Table 1. All synthetic compounds including paracaseolide A selectively inhibit PTP1B, CDC25B and TC-PTP over LAR, SHP-1, and SHP-2. Analogues 9 and 10, were not only the most potent PTP1B inhibitors with IC50 values of 0.27 ± 0.02 µM and 0.27 ± 0.03 µM, respectively, but also showed high selectivity (9- to 17-fold) for PTP1B over TC-PTP. TC-PTP has the highest homology to PTP1B, with 72% sequence identity in the catalytic domain.14 These compounds exhibited significant improvement in both potency and selectivity compared to the natural product paracaseolide A. In order to further understand the activity and the selectivity of compound 10, molecular docking analysis was carried
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out using LibDock available with Discovery Studio 2.1. The X-ray crystal structure of PTP1B (PDB-ID: 1NNY) was retrieved from the RCSB Protein Data Bank (http://www.rcsb.org). The preferred coordination modes of compound 10 with PTP1B are presented in Fig. 1. Fig. 1a depicts the binding of the fused ring of compound 10 in the active site. Fig. 1b shows the binding interactions of 10 with PTP1B. The carbonyl of the –COOH moiety may interact with Asn111 via a hydrogen bonding interaction, while the ketone is hydrogen bonded to Ser216 and Ala217. Comparison with compound 13 suggests that the opening of lactone ring and the free carboxylic acid group in 10 may contribute significantly to its improved inhibitory activity. It has been proposed that dual-binding inhibitors, which occupy both the active site and second phosphotyrosine ( pTyr) binding site adjacent to the catalytic site of PTP1B would provide both increased binding affinity and the opportunity for improved selectivity over highly homologous phosphatase TC-PTP.6,10,14 The second pTyr binding site adjacent to the catalytic site is lined by Arg24, Asp48, Val49, Ile219, Arg254, Met258, Gly259 and Gln262.14 Fig. 1 shows that compound 10 extends the full span of active site and second pTyr binding site of PTP1B, the long alkyl chain in 10 binds in the
Fig. 1 Molecular docking analysis of compound 10, showing proposed binding modes with PTP1B. Ligands are shown in stick representation with carbon atoms in cyan. Hydrogen bonds are displayed as green dashed lines. (a) Binding pose of compound 10 in the protein surface of PTP1B. (b) Interaction of compound 10 with catalytic residues of PTP1B.
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Notes and references
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Fig. 2 Effects of 9, 10 and paracaseolide A (13) on insulin signaling. CHO/hIR cells were treated with compounds or PC for 1 h, followed by insulin (10 nM). The expression and phosphorylation levels of IR were detected using IRβ and PY-20 antibodies, respectively. PC ( positive control): sodium orthovanadate (250 μM); NC (negative control): DMSO.
second pTyr binding site by hydrophobic interaction with Ile219, Met258 and Gly259. Thus, the activity against PTP1B of compound 10 and its good selectivity over TC-PCP can be well explained. PTP1B acts as a negative regulator of insulin signaling via dephosphorylation of the insulin receptor (IR) and insulin receptor substrate proteins (IRS).24 Increased expression and activity of PTP1B has been found in diabetic and obese humans and mice, while PTP1B knockout mice exhibit improved insulin sensitivity and resistance to diet-induced obesity.4 To explore the cellular effect of PTP1B inhibitors, 9, 10 and 13, the phosphorylation level of IR was detected in CHO/hIR cells after incubating with the compounds (5 to 20 μM) for 1 h. As shown in Fig. 2, compound 10 obviously improved p-IR compared to the negative control (NC) in a dose-dependent manner. This result indicates that the analogues of paracaseolide A hold promise as novel PTP1B inhibitors to counter insulin resistance.
Conclusion In conclusion, a series of paracaseolide A analogues were synthesized and evaluated for their inhibitory activity for various PTPs (PTP1B, TC-PTP, LAR, SHP-1, SHP-2 and CDC25B). Most of these analogues exhibit similar or greater activity against PTP1B than the natural product. Compounds 9 and 10, in particular, exhibit potent inhibitory activity and modest to high selectivity over TC-PTP. The improved cellular activity of these novel inhibitors also correlated with an improvement in enzymatic inhibitory activity, although the membrane permeability of these highly lipophilic compounds requires further optimization. These compounds provide a new starting point for further development of paracaseolide A-inspired PTP1B inhibitors, the work on which is currently underway in our laboratory.
Acknowledgements This work was supported by the National Program on Key Basic Research Project (2012CB524906), the National Natural Science Foundation of China (81125023), and Shanghai Commission of Science and Technology (13DZ2290300).
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1 J. N. Andersen, O. H. Mortensen, G. H. Peters, P. G. Drake, L. F. Iversen, O. H. Olsen, P. G. Jansen, H. S. Andersen, N. K. Tonks and N. P. Moller, Mol. Cell. Biol., 2001, 21, 7117–7136. 2 S.-C. Yip, S. Saha and J. Chernoff, Trends Biochem. Sci., 2010, 35, 442–449. 3 M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. HimmsHagen, C.-C. Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay and B. P. Kennedy, Science, 1999, 283, 1544–1548. 4 L. D. Klaman, O. Boss, O. D. Peroni, J. K. Kim, J. L. Martino, J. M. Zabolotny, N. Moghal, M. Lubkin, Y.-B. Kim, A. H. Sharpe, A. Stricker-Krongrad, G. I. Shulman, B. G. Neel and B. B. Kahn, Mol. Cell. Biol., 2000, 20, 5479–5489. 5 T. R. Burke, H. K. Kole and P. P. Roller, Biochem. Biophys. Res. Commun., 1994, 204, 129–134. 6 Y. A. Puius, Y. Zhao, M. Sullivan, D. S. Lawrence, S. C. Almo and Z. Y. Zhang, Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 13420–13425. 7 G. Scapin, S. B. Patel, J. W. Becker, Q. Wang, C. Desponts, D. Waddleton, K. Skorey, W. Cromlish, C. Bayly, M. Therien, J. Y. Gauthier, C. S. Li, C. K. Lau, C. Ramachandran, B. P. Kennedy and E. Asante-Appiah, Biochemistry, 2003, 42, 11451–11459. 8 B. G. Szczepankiewicz, G. Liu, P. J. Hajduk, C. Abad-Zapatero, Z. Pei, Z. Xin, T. H. Lubben, J. M. Trevillyan, M. A. Stashko, S. J. Ballaron, H. Liang, F. Huang, C. W. Hutchins, S. W. Fesik and M. R. Jirousek, J. Am. Chem. Soc., 2003, 125, 4087–4096. 9 A. P. Combs, W. Zhu, M. L. Crawley, B. Glass, P. Polam, R. B. Sparks, D. Modi, A. Takvorian, E. McLaughlin, E. W. Yue, Z. Wasserman, M. Bower, M. Wei, M. Rupar, P. J. Ala, B. M. Reid, D. Ellis, L. Gonneville, T. Emm, N. Taylor, S. Yeleswaram, Y. Li, R. Wynn, T. C. Burn, G. Hollis, P. C. C. Liu and B. Metcalf, J. Med. Chem., 2006, 49, 3774–3789. 10 S. Zhang and Z. Zhang, Drug Discovery Today, 2007, 12, 373–381. 11 Y. Wei, Y.-T. Chen, L. Shi, L.-X. Gao, S. Liu, Y.-M. Cui, W. Zhang, Q. Shen, J. Li and F.-J. Nan, MedChemComm, 2011, 2, 1104–1109. 12 Y.-T. Chen, C.-L. Tang, W.-P. Ma, L.-X. Gao, Y. Wei, W. Zhang, Q. Shen, J.-Y. Li, J. Li and F.-J. Nan, Eur. J. Med. Chem., 2013, 69, 399–412. 13 T. Tiganis and A. M. Bennett, Biochem. J., 2007, 402, 1–15. 14 A. P. Combs, J. Med. Chem., 2010, 53, 2333–2344. 15 X.-L. Chen, H.-L. Liu, J. Li, G.-R. Xin and Y.-W. Guo, Org. Lett., 2011, 13, 5032–5035. 16 D. Noutsias and G. Vassilikogiannakis, Org. Lett., 2012, 14, 3565–3567. 17 T. Guney and G. A. Kraus, Org. Lett., 2013, 15, 613–615.
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22 S. K. Bagal, R. M. Adlington, R. A. B. Brown and J. E. Baldwin, Tetrahedron Lett., 2005, 46, 4633– 4637. 23 A. Tsuchiya, T. Kanno and T. Nishizaki, Cell. Physiol. Biochem., 2013, 32, 1451–1459. 24 K. A. Kenner, E. Anyanwu, J. M. Olefsky and J. Kusari, J. Biol. Chem., 1996, 271, 19810–19816.
Published on 25 March 2014. Downloaded by Colorado State University on 22/08/2014 11:27:08.
18 D. C. Rideout and R. Breslow, J. Am. Chem. Soc., 1980, 102, 7816–7817. 19 T. Xu, C.-c. Li and Z. Yang, Org. Lett., 2011, 13, 2630–2633. 20 Y. Ito, Y. Kobayashi, T. Kawabata, M. Takase and S. Terashima, Tetrahedron, 1989, 45, 5767–5790. 21 D. J. Hart, W.-L. Wu and A. P. Kozikowski, J. Am. Chem. Soc., 1995, 117, 9369–9370.
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