Mahesh Kumar
Gundluru
a,
Alan
Pourpak
b,
Xiaoli
Cui
b,
Stephan W.
Morris
bc and
Thomas R.
Webb
*a
aChemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, U.S.A. E-mail: thomas.webb@stjude.org; Fax: +901 595 5715; Tel: +901 595 3928
bPathology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, U.S.A
cOncology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, Tennessee 38105, U.S.A
First published on 21st July 2011
A novel and simplified synthetic scaffold based on pladienolide was designed using a consensus pharmacophore hypothesis. An initial target was synthesized and evaluated to examine the role of the 3-hydroxy group and the methyl groups present at positions 10, 16, 20, 22 in 1, on biological activity. We report the first totally synthetic analog of this macrolide that shows biological activity. Our novel synthetic strategy enables the rapid synthesis of other new analogs of pladienolide in order to develop selective anticancer lead compounds.
Fig. 1 Natural and synthetic analogs of pladienolide and FR901464. |
To date, only three approaches have been reported for the synthesis of these unique macrolides,3,15,16 including a total synthesis of pladienolide B and D.3 However, access to multi-gram quantities of totally synthetic pladienolides for in vivo studies remains a significant challenge, due to the synthetic complexity inherent to this class of compounds. Fortunately our pharmacophore design has allowed us to target simplified analogs of pladienolides that possess the potential to be potent and more drug-like than either FR901464 or pladienolide. As part of our ongoing efforts to extend our successful pladienolide–FR901464 consensus pharmacophore-based design approach to simplified synthetic analogs of pladienolides, we now report the synthesis and biological evaluation of our first synthetic pladienolide analogs 5 and E-26, as the starting point in our development of active simplified analogs of this natural product. The design of this scaffold is based on our published consensus-based pharmacophore (see Fig. 2) that indicates that the C2 through C5 carbon atoms (highlighted in green) do not overlap with atoms in FR901464 analogs and are therefore not a likely component of the active pharmacophore.13,14 In particular the C3 atom bearing the hydroxyl group does not appear to have an important interaction in this model, therefore this functionality is excluded from our design for the basic framework molecule 5 (see Scheme 1). However, inspection of the overlay in Fig. 1 shows that the C6 OH group can readily overlay with the amide NH of FR901464 analogs, indicating that this feature could be a critical hydrogen-bond donor in the pharmacophore. In addition, from this overlay we assume that the methyl groups present at 10, 12, 16, 20, 22 positions of 1 are responsible for locking the free rotational bonds of the molecule. So, our preliminary hypothesis is that the side-chain chiral methyl centers may have limited importance in the activity of pladienolide. Our previous work that led to the development of the active analogs 6, 7, and 8, has already shown the critical importance of the carbonyloxy group (at the C7 position in pladienolide B) in the pharmacophore.13,14
Scheme 1 The retrosynthetic analysis of simplified pladienolide analog scaffold 5. BT = COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundBenzothiazole; PG = protecting group |
Fig. 2 A 3D representation of an example of an overlay of the presumed key interaction groups in a low energy conformation of 3-deoxypladienolide (shown in yellow, some atoms are not shown) and compound 6, showing that the epoxy group and the carbonyloxy groups are the same distance in both molecules. In the pladienolide model the C2 through C5 carbons are highlighted in green. This alignment represents the best S value (S = 167.18) for molecules matching the hypothetical pharmacophore, and the second best overall of 69 alignments from 500 iterations. The alignment was prepared using the Molecular Operating System (MOE 2007.09, Chemical Computing Group, Inc.) using the Flexible Alignment function with both molecules, following a conformational minimization using MOE default settings.14 |
Our approach to the synthesis of 5 began with a novel stereoselective approach to the C1–C9 fragment as shown in Scheme 2. The Mukaiyama aldol reaction18 between the 2,2-disubstituted silylenolether 13 (obtained by a regioselective silylation of COMPOUND LINKS
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Download mol file of compound2-methylcyclohexanone)19 and COMPOUND LINKS
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Download mol file of compound3-trimethylsilylpropynal provided the corresponding racemic aldol product (syn:anti 10:90) from which the pure anti isomer (±)-14 was isolated in 60% overall yield.20 It is worth noting that BF3·OEt2 afforded a cleaner reaction with better anti stereoselectivity among the Lewis acids that we explored: i.e. 1 eq. TiCl4 (syn:anti 23:77, 60% yield), 10 mol% TiCl4 (syn:anti 25:75, 40% yield) and 1 eq. SnCl4 (syn:anti 10:90, 32% yield).21 It can be noted that we explored conditions that could be expected to provide syn stereoselectivity by application of the Nicholas reaction with 13 (without success) using the dicobalt hexacarbonyl complex of 3-trimethylsilylpropynal20 and other conditions. The next step involved the inversion of C7 stereocenter by means of a Mitsunobu inversion-saponification protocol. Accordingly, the major anti aldol adduct (±)-14 was converted into its syn isomer (±)-15 in 40% yield using COMPOUND LINKS
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Download mol file of compound4-nitrobenzoic acid under Mitsunobu conditions.22
As anticipated, the acetylenic TMS group was also cleaved during the hydrolysis of the ester group. Baeyer–Villiger oxidation of (±)-15 then provided lactone (±)-16 in 70% yield as a single stereo- and regioisomer, as expected.23 The structure assignment, including the relative stereochemistry at the C6–C7 position, was confirmed by X-ray crystallographic analysis of lactone (±)-16 (see Scheme 2).24Methanolysis of this lactone in the presence of Et3N, followed by controlled partial hydrogenation using Lindlar catalyst, gave diol (±)-17 in 97% yield.25 Installation of the acetonide diol protecting group and hydrolysis of the resulting methylester produced the acetonide acid (±)-12, in quantitative yield.
The synthesis of the C1–C14 unit was accomplished as shown in Scheme 3 giving aldehyde 18 in 4 steps, as previously reported.26 The Brown allylation of aldehyde 18 provided the enantioenriched (85% ee) alcohol 19 in 89% isolated yield as shown in Scheme 3.27,28 The acid (±)-12 was then coupled with 19, using the Yamaguchi protocol, to furnish 20a and 20b as an inseparable mixture of diastereomers (1:1) in 88% yield.29 The ring-closing metathesis (RCM) of this diastereomeric pair, with the 2nd generation Hoveyda–Grubbs catalyst, smoothly afforded the macrolides 21a and 21b in 49% combined yield.30 As shown in Scheme 3, an interesting diastereoselection occurred during the RCM reaction, resulting in a 3:2 mixture of the desired 21a and the undesired 21b diastereomers respectively.31,32 This diastereomeric mixture was separated by standard flash chromatography. The absolute configuration at the C7 carbinol center was determined by converting 21a into (R)- and (S)-MTP derivatives followed by a modified Mosher's ester protocol (see Supporting Information).33,34 The desired major diastereomer 21a was then further converted into the corresponding acetyl-aldehyde derivative 10. The deprotection of the acetonide then chemoselective acetylation of the C7 hydroxyl group followed by PMB-deprotection and finally Dess–Martin oxidation, furnished aldehyde 10 in excellent yields.
Scheme 3 Synthesis of the C1–C14 unit. Reagents and conditions: a) (–)-Ipc2BOMe, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundAllylmagnesium bromide, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compounddiethyl ether, 89%; b) 19, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound2,4,6-trichloro benzoylchloride, Et3N, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMAP, 88%, 1:1 diastereomers; c) 2nd generation Hoveyda-Grubbs catalyst, 49%, 3:2 diastereomers; d) i. PPTS, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundMeOH, 80 °C, 66%; ii. Et3N, Ac2O, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMAP, quant; e) i. DDQ, CH2Cl2, 70%; ii. Dess–Martin periodinane, CH2Cl2, 95%. |
Next, the preparation of the C15–C22 side-chain unit was undertaken, as shown in Scheme 4. Commercially available COMPOUND LINKS
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Download mol file of compound4-penten-1-ol was protected as the benzothiazole, followed by cross-metathesis with COMPOUND LINKS
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Download mol file of compound(S)-penten-2-ol smoothly afforded the desired olefin 24 as a 96:4 mixture of E:Z isomers respectively. Following the selective oxidation of 24, the resulting sulfone was subjected to Shi asymmetric epoxidation conditions to afford a 5:1 mixture of the β:α epoxides, from which the desired β-epoxide was isolated in 59% isolated yield. Silylation of the free hydroxy moiety furnished the side chain fragment 25.
Scheme 4 The synthesis of C15–C22 unit, fragment coupling and the synthesis of simplified pladienolide analogs E-26 and 5. Reagents and conditions: a) i. COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound2-mercaptobenzothiazole, TPP, DIAD, 90%; ii. COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound(S)-4-penten-2-ol, 2nd generation Grubbs catalyst, 60%; b) i. (NH3)6Mo7(H2O)4, H2O2, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundEtOH, 72%; ii. Shi epoxidation catalyst, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundoxone, K2CO3, 59%; iii. TBSOTf, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compound2,6-lutidine, 86% for compound 25; Ethyl vinyl ether, PPTS, CH2Cl2, 85% for compound2727; c) 10, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundNaHMDS, THF, E:Z 72:28, 54% for compound 26; mixture of stereoisomers, 38% for compound2828; d) SFC; e) 2828, PPTS, MeOH, E:Z 72:28, 25%. (The reaction sequence with ethoxyethyl protecting group are presented in italics). |
The final steps in the synthesis of the simplified pladienolide analog 5 entailed the coupling of sulfone 25 with aldehyde 10. This reaction provided compound 26 as a 72:28 mixture of E:Z isomers in 54% yield, which was quantitatively separated by Supercritical Fluid Chromatography (SFC) using an OD-H column to isolate major stereoisomer E-26. Unfortunately desilylation of compound E-26 under a variety of conditions produced inseparable complex mixtures. In order to circumvent this problem the alternate Julia coupling precursor 27 was prepared and used to successfully complete the synthesis of 5. Thus, employing a similar reaction sequence, compound 28 was prepared in 38% yield as an inseparable mixture of stereoisomers. Desilylation of 28 with PPTS/COMPOUND LINKS
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Download mol file of compoundMeOH provided the target analog 5 as a 72:28 inseparable mixture of E:Z isomers in 25% yield. This inseparable mixture of cis and trans isomers (5) was submitted for cytotoxicity assay along with compound E-26 and with our previous FR active analogs 6 and 7 as positive controls.
Fig. 3 Modulation of MDM2 mRNA splicing. SK-MEL-2 melanoma cells were treated with vehicle (COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDMSO) or the designated concentrations of compounds E-26, 22, or 7 for 6 h. Following RNA extraction and reverse transcription, cDNA was amplified by PCR using primers for MDM2 and the intronless gene ubiquitin. Due to the presence of endogenous, transcriptionally inactive, mutant p53 in the SK-MEL-2 cell line, basal levels of MDM2 are low-to-undetectable in the untreated cells.35,36 The asterisk (*) denotes the size of properly spliced MDM2; arrows indicate splice variants of MDM2. |
Cell line | Cancer type | IC50 (μM) | |||
---|---|---|---|---|---|
E-26 | 5 | 6 | 7 | ||
SK-MEL-2 | Melanoma | 10.71 ± 0.77 | >20 | 0.39 ± 0.11 | 0.70 ± 0.10 |
JeKo-1 | Mantle cell lymphoma | 11.60 ± 0.27 | >20 | 0.11 ± 0.01 | 0.29 ± 0.02 |
MOLT-4 | Acute lymphoblastic leukemia | 15.46 ± 0.76 | ND | 0.18 ± 0.01 | 0.66 ± 0.07 |
PC-3 | Prostate | 18.03 ± 0.66 | >20 | 2.02 ± 0.39 | 4.16 ± 0.68 |
MCF7 | Breast | 19.15 ± 0.55 | ND | 0.72 ± 0.02 | 1.69 ± 0.48 |
NCI-H226 | Non-small cell lung cancer | >20 | ND | >10 | >10 |
Footnotes |
† CCDC reference numbers 805182. For crystallographic data in CIF or other electronic format see DOI: 10.1039/c1md00040c |
‡ Author roles: M.K.G. performed all of the chemistry content of this work, collaborated on the design of targets with T.R.W., and wrote the initial draft manuscript including all supporting information; T.R.W. directed the project, designed the initial synthetic targets, directed the chemistry and drafted the final manuscript; A.P. and X.C. performed all of the biology studies described herein, with supervision from S.W.M. |
This journal is © The Royal Society of Chemistry 2011 |