• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Bioorg Med Chem Lett. Author manuscript; available in PMC May 1, 2013.
Published in final edited form as:
PMCID: PMC3331937
NIHMSID: NIHMS364681

Analogues of the RSK inhibitor SL0101: Optimization of in vitro biological stability

Abstract

The Ser/Thr protein kinase, RSK, is important in the etiology of tumor progression including invasion and motility. The natural product kaempferol-3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside), called SL0101, is a highly specific RSK inhibitor. Acylation of the rhamnose moiety is necessary for high affinity binding and selectivity. However, the acetyl groups can be cleaved by esterases, which accounts for the poor in vitro biological stability of SL0101. To address this problem a series of analogues containing acetyl group replacements were synthesized and their in vitro stability evaluated. Monosubstituted carbamate analogues of SL0101 showed improved in vitro biological stability while maintaining specificity for RSK. These results should facilitate the development of RSK inibitors derived from SL0101 as anticancer agents.

Keywords: SL0101, RSK inhibitor, RSK-specific, Breast cancer, Protein kinase

The members of the p90 ribosomal S6 kinase (RSK) family of Ser/Thr protein kinases have been shown to play a role in a number of different cancers as key drivers of proliferation and metastasis.1-8 These discoveries have been enabled in part by our report of the identification and isolation of the RSK inhibitor SL0101 (1, Figure 1).9 SL0101 is a flavonoid glycoside (kaempferol 3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside)) isolated from Forsteronia refracta, a variety of dogbane found in the South American rainforest. SL0101 is highly specific for RSK, inhibiting RSK1/2 but not unrelated kinases nor the closely related kinases MSK1 and p70S6K1.2,9,10 SL0101 inhibits the proliferation of breast and prostate cancer lines but not their normal counterparts even though it inhibits RSK activity in all the lines.1,5,9 Thus it appears that some cancer cells have become addicted to RSK, which suggests that RSK may be a potential new target for cancer therapeutics. SL0101, owing to its exquisite specificity, is a compelling lead compound from which to begin the process of identifying drug-like RSK inhibitors.

Figure 1
The RSK inhibitor SL0101 and two previously reported analogues.13

We and others have reported the total synthesis and biological evaluation of SL0101 and a number of analogues, with the ultimate goal of developing an anticancer drug that targets RSK.11-15 These analogues have provided key information about the SAR of both the aglycone and carbohydrate portions of the natural product. In the course of this work we discovered that the 3″ and 4″ acetyl groups of the carbohydrate are critical for potency and specificity for RSK.13 TriOH-SL0101 (2), lacking these acetyl groups, is 12-fold less potent for inhibition of RSK in vitro and does not inhibit the growth of cancer cell lines, likely due to poor membrane permeability.13 These results indicate that SL0101 is not a suitable candidate for in vivo evaluation, as hydrolysis of the acetates by esterases would generate a less potent inhibitor.

An analogue that replaces these acetates with ethyl ethers (3) inhibits RSK with potency roughly equivalent to SL0101.13 We previously determined that the specificity of SL0101 and its analogues for RSK could be evaluated by their preferential ability to inhibit the growth of the human breast cancer line, MCF7, compared to the normal human breast line, MCF-10A.12 Unexpectedly, we observed that the ethyl ether analogue 3 inhibited both lines to a similar extent, which indicates that it has a decreased specificity for RSK.13 These results demonstrate that the acetates are a key modulator of specificity and thus a more carefully considered approach is necessary to identify suitable replacements. Accordingly, we have focused our efforts on identifying analogues bearing replacements for the acetates that confer greater biological stability without decreasing potency or specificity for RSK. Herein we present our approach, which has led to the identification of SL0101 analogues that are both specific for RSK and more biologically stable in vitro than the parent compound.

The only structural difference between the diethyl analogue 3 and the diacetyl parent compound is the replacement of two methylenes with two carbonyl groups. It is surprising that such a seemingly small structural feature can regulate specificity for RSK. To recover this specificity, in the design of new analogues we sought to better mimic the acetates and particularly the acetate carbonyls, sterically and electronically, in a way that would confer a greater resistance to metabolism by esterases. In one approach we investigated the dependence of potency and specificity on the relative position of the carbonyl group. To this end we prepared an analogue 9 in which the acetates are replaced by alkoxyacetones (Scheme 1), moving the carbonyl group one carbon further from the carbohydrate ring and replacing the labile ester bond with an ether bond. The desired functionality could be installed at a late stage in the synthesis of the analogue. Alkylation of known diol 613 with propargyl bromide provided bis-alkyne 7, which after mercury-catalyzed hydration provided bis-ketone 8. Removal of the benzyl ether protecting groups by hydrogenolysis using Pearlman's catalyst provided the completed analogue 9.

Scheme 1
Synthesis of a bis-ketone analogue of SL0101. Reagents and conditions: (a) NaH, propargyl bromide, THF, 0 °C to rt, 66%; (b) Hg(OAc)2, PPTS, water, acetone, rt, 62%; (c) H2, Pd(OH)2/C, MeOH, EtOAc, 50%. Yields are unoptimized.

In a second approach we retained the acetate carbonyl in the correct position but in a more biologically stable form in a series of analogues in which we replaced the acetates with bioisosteric mono- or disubstituted carbamates. Late-stage installation of the carbamate was desirable for maximum synthetic efficiency. Thus, carbamoylation of diol 6 with the appropriate isocyanate or diaklycarbamoyl chloride followed by hydrogenolysis of the benzyl ethers provided mono- or dialkylated carbamates 10-15 (Scheme 2).

Scheme 2
General scheme for the preparation of carbamate analogues of SL0101. Reagents and conditions: (a) R1NCO, Et3N, DMF, 45 °C, 44-66%; (b) R2R3NCOCl, NaH, DMF, 0 °C to rt, 26-69%; (c) H2, Pd(OH)2/C, MeOH, EtOAc, rt, 46-94%. Yields are unoptimized. ...

The ability of all new analogues to inhibit RSK activity was determined in an in vitro kinase assay and compared with the parent compound 1 (Table 1). The ketone analogue 9 was 2-fold more potent than 1 at inhibiting RSK2. Analogues 11, 13, and 14 were as potent as SL0101, and analogues 10, 12, and 15 were slightly (2- to 3-fold) less potent. Overall, we found that the ability of an analogue to inhibit RSK was not greatly influenced by the structure of the acetate replacement, which is consistent with previous observations.

Table 1
Potency of analogues in in vitro kinase and MCF7 cell-based assays. IC50 is concentration needed for 50% inhibition; the 95% CI is shown in parentheses; n=3 in triplicate; * p <0.05.PS; partially soluble.

We also determined the ability of all new analogues to inhibit MCF7 cell proliferation (Table 1). The ketone analogue 9 was again the most potent of the new analogues. The three monosubstituted carbamates, analogues 10-12, were similarly potent to the parent compound, with a trend toward improved potency with increasing lipophilicity of the carbamate substituent, presumably due to improved membrane permeability. In the disubstituted carbamate series, the dimethyl analogue 13 and 1-pyrrolidinyl carbamate analogue 14 exhibited poor solubility in cell culture media and therefore their ability to inhibit cell growth was not determined. The morpholino bis-carbamate 15 showed improved solubility but was unable to inhibit cell proliferation despite its ability to inhibit RSK in the in vitro kinase assay, most likely due to poor membrane permeability.

Analogues that inhibited MCF7 cell proliferation were evaluated along with 1 for their stability in a MCF7 cell-based assay. The inhibitor was added when the cells were plated and proliferation analyzed at various time points to determine the persistence of the inhibitory effect. SL0101 (1) was able to inhibit MCF7 proliferation for 48 h (Fig. 2). However, at longer time points the cells began to proliferate indicating that SL0101 was no longer effective, which we hypothesize is due to degradation of the inhibitor by esterases to the inactive triol 2. Treatment of cells with either the bis-ketone analogue 9 or the ethyl carbamate analogue 10 did not result in sustained growth inhibition, indicating poor in vitro stability of these analogues. As the 3″ and 4″ substituents of analogue 9 are non-hydrolyzable, its poor stability was initially surprising. However, MCF7 cells express aldo-keto reductases (AKRs), well known to be Phase I metabolizing enzymes for a variety of drugs bearing carbonyl groups.16,17 Thus an alternative metabolic pathway is available to analogue 9 whereby one or both ketones could be reduced by AKRs to secondary alcohols, leading either directly to a less potent RSK inhibitor or indirectly as the secondary alcohols could be further metabolized by conjugation.17

Figure 2
In vitro determination of analogue stability. The inhibitor (100μM) was added to MCF7 cells at time 0 and percentage of growth determined for the indicated time points. (n=3 in quadruplicate, # p ≤ 0.05 at 48 h when compared to vehicle ...

Encouragingly, the more lipophilic monosubstitued carbamate analogues 11 and 12 demonstrated improved in vitro stability, as cells treated with these compounds did not proliferate over the full time course (Fig. 2). We further examined the stability of analogues 11 and 12 by determining whether cyclin D1 levels were inhibited (Fig. 3). Previously, we found that SL0101 inhibits proliferation in breast cancer cell lines by inducing a cell cycle block in G1, which is due to RSK regulation of cyclin D1 levels.1,18 In agreement with the MCF7 stability results we observed that SL0101 decreased the levels of cyclin D1 at 48 h compared to the control, but that cyclin D1levels began to increase at later time points, indicating degradation of the inhibitor. However, cyclin D1 levels remained low in cells treated with 11 or 12, indicating persistent inhibition of RSK and therefore improved biological stability of the carbamate anlogues over the parent compound. Taken together, these results indicate that analogues 11 and 12 have improved stability over SL0101 (1).

Figure 3
Persistence of RSK inhibition. MCF7 cells were treated with SL0101 or the more stable analogues 11 and 12 (100 μM). At the indicated time in hours (h) the cells were lysed and the lysates immunoblotted. Each analogue was analyzed on a single membrane ...

We then investigated whether our strategy of reintroducing the carbonyl group improved the specificity of 11 and 12 relative to the diethyl analogue 3.13 We have previously shown that the specificity of an analogue for RSK can be evaluated by determining its antiproliferative activity in both MCF-10A and MCF7 cells, with the most specific analogues showing no inhibition of MCF-10A but substantial inhibition of MCF7 proliferation, due to the differential dependence of the growth of these cell lines on RSK.9,12 We have also previously shown that while SL0101 does not inhibit the growth of MCF-10A cells up to a concentration of 100 μM, the diethyl analogue 3 significantly inhibits the growth of MCF-10A cells, indicating reduced specificity for RSK.13 We found that analogues 11 and 12, like SL0101, significantly inhibited the growth of MCF7 cells but did not significantly inhibit the growth MCF-10A cells (Figure 4). These results suggest that analogues 11 and 12, like SL0101, specifically inhibit RSK.2,9,10 The only significant differences in biological activity between the two compounds are slightly improved potencies for 11 versus 12 in both the kinase and MCF7 cell proliferation assays. As these small differences are unlikely to be physiologically important, either carbamate modification should render an analogue suitable for in vivo evaluation.

Figure 4
Inhibition of growth of MCF-10A vs. MCF7 cells by SL0101 and select analogues. The inhibitor concentration was 50 μM. (n=3 in quadruplicate, * p ≤ 0.05 when compared to vehicle)

In summary, the C3″ and C4″ acetates on the carbohydrate moiety of SL0101 are required for both potent and specific inhibition of RSK but we predict that they would be metabolized rapidly by esterases in vivo, a fact which is supported by the poor biological stability of the natural product in vitro. Thus, SL0101 is not suitable for in vivo evaluation and analogues with improved stability are needed. The number of suitable replacements for these acetates that would confer greater biological stability is surprisingly limited as a simple change from acetyl to ethyl leads to a reduction in specificity for RSK. As a solution to this problem, bioisosteric replacement of the acetates by carbamates provided analogues that are more biologically stable than SL0101 in vitro and are as specific as SL0101 for RSK. These modifications along with others aimed at further improving the stability and potency of SL0101 analogues are currently being investigated in our laboratory with the goal of identifying a RSK inhibitor that could be advanced to preclinical testing.

Supplementary Material

01

Acknowledgments

This work was supported by the Department of Defense #W81XWH-11-1-0068 to MKH and GM084386 to DAL.

Footnotes

Supplementary Material: Experimental procedures and compound characterization for all new compounds can be found online at

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will under go copy editing, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References and notes

1. Eisinger-Mathason TS, Andrade J, Lannigan DA. Steroids. 2010;75:191. [PMC free article] [PubMed]
2. Doehn U, Hauge C, Frank SR, Jensen CJ, Duda K, Nielsen JV, Cohen MS, Johansen JV, Winther BR, Lund LR, Winther O, Taunton J, Hansem SH, Frodin M. Mol Cell. 2009;35:511. [PMC free article] [PubMed]
3. Smolen GA, Zhang J, Zubrowski MJ, Edelman EJ, Luo B, Yu M, Ng LW, Scherber CM, Schott BJ, Ramaswamy S, Irimia D, Root DE, Haber DA. Genes Dev. 2010;24:2654. [PMC free article] [PubMed]
4. Cho YY, Yao K, Kim HG, Kang BS, Zheng D, Bode AM, Dong Z. Cancer Res. 2007;67:8104. [PMC free article] [PubMed]
5. Clark DE, Errington TM, Smith JA, Frierson HF, Jr, Weber MJ, Lannigan DA. Cancer Res. 2005;65:3108. [PubMed]
6. Kang S, Dong S, Gu TL, Guo A, Cohen MS, Lonial S, Khoury HJ, Fabbro D, Gilliland DG, Bergsagel PL, Taunton J, Polakiewicz RD, Chen J. Cancer Cell. 2007;12:201. [PMC free article] [PubMed]
7. Kang S, Elf S, Lythgoe K, Hitosugi T, Taunton J, Zhou W, Xiong L, Wang D, Muller S, Fan S, Sun SY, Marcus AI, Gu TL, Polakiewicz RD, Chen ZG, Khuri FR, Shin DM, Chen J. Clin Invest. 2010;120:1165. [PMC free article] [PubMed]
8. Kang S, Elf S, Dong S, Hitosugi T, Lythgoe K, Guo A, Ruan H, Lonial S, Khoury HJ, Williams IR, Lee DH, Roesel JL, Karsenty G, Hanauer A, Taunton J, Boggon TJ, Gu TL, Chen J. J Mol Cell Biol. 2009;29:2105. [PMC free article] [PubMed]
9. Smith JA, Poteet-Smith CE, Xu Y, Errington TM, Hecht SM, Lannigan DA. Cancer Res. 2005;65:1027. [PubMed]
10. Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H, Klevernic I, Arthur JS, Alessi DR, Cohen P. Biochem J. 2007;408:297. [PMC free article] [PubMed]
11. Maloney DJ, Hecht SM. Org Lett. 2005;7:1097. [PubMed]
12. Smith JA, Maloney DJ, Clark DE, Xu Y, Hecht SM, Lannigan DA. Bioorg Med Chem. 2006;14:6034. [PubMed]
13. Smith JA, Maloney DJ, Hecht SM, Lannigan DA. Bioorg Med Chem. 2007;15:5018. [PubMed]
14. Shan M, O'Doherty GA. Org Lett. 2006;8:5149. [PMC free article] [PubMed]
15. Shan M, O'Doherty GA. Org Lett. 2010;12:2986. [PMC free article] [PubMed]
16. Ruiz FX, Porté S, Gallego O, Moro A, Ardèvol A, Del Río-Espínola A, Rovira C, Farrés J, Parés X. Biochem J. 2011;440:335. [PubMed]
17. Jin Y, Penning TM. Annu Rev Pharmacol Toxicol. 2007;47:263. [PubMed]
18. Eisinger-Mathason TS, Andrade J, Groehler AL, Clark DE, Muratore-Schroeder TL, Pasic L, Smith JA, Shabanowitz J, Hunt DF, Macara IG, Lannigan DA. Mol Cell. 2008;31:722. [PMC free article] [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links