Molecular editing of kinase-targeting Resorcylic Acid Lactones (RAL): Fluoroenone RAL

Article abstract of DOI:10.1002/cmdc.201000047

(Chemical Equation Presented) Molecular editing: The resorcylic acid lactones (RAL) are known small-molecule irreversible inhibitors of select kinases, and represent a unique pharmacophore with potential for further development in kinase research. The bas

Full text of DOI:10.1002/cmdc.201000047

MED
DOI: 10.1002/cmdc.201000047
Molecular Editing of Kinase-Targeting Resorcylic Acid Lactones (RAL):
Fluoroenone RAL
Rajamalleswaramma Jogireddy, Sofia Barluenga, and Nicolas Winssinger*[a]
Kinases have become one of the most intensely pursued
target class for therapeutic intervention, particularly for appli-
cations in oncology.[1] Most small-molecule inhibitors are het-
erocycles reminiscent of the adenosine scaffold, which target
the nucleotide binding site of the kinase. Resorcylic acid lac-
tones (RAL) bearing a suitably positioned cis-enone, such as
hypothemycin,[2,3] LL-Z1640-2,[4,5] L-783277,[6] radicicol A[7,8]
(Figure 1), have been shown to irreversibly inhibit select kinas-
enone may accentuate the Michael acceptor properties while
constituting a fairly neutral steric permutation. This “molecular
editing”[13] may provide further benefits from a synthetic per-
spective, as well as favorably impede the propensity of these
cis-enones to isomerize to the thermodynamically more favora-
ble and biologically less active trans-enone. As shown in
Figure 2, the synthesis could capitalize on our previously estab-
lished strategy,[7,10,14] namely, disconnection of the ester and
benzylic position. However, the difference of reactivi-
ty between a fluoroalkene and an alkene could be
harnessed to introduce the diol moiety by dihydrox-
ylation chemistry.
The synthesis of fluoroenones 1 and 2 began with
the straightforward conversion of ester 5 into fluo-
roenal 7 via aldehyde 6 relying on a Horner–Wads-
worth–Emmons reaction with triethyl 2-fluoro-2-
phosphonoacetate to install the desired E-fluoro-
enoal (Scheme 1). Reaction of aldehyde 7 with alkyl
lithium 8 afforded the desired alcohol as a diastero-
meric mixture (diastereoisomeric ratio (dr), 1:1),
which was reduced to the cis-alkene using Lindlar’s
catalyst (H2, Pd/CaCO3) to afford compound 9. As
the hydroxy group will ultimately be oxidized to the
ketone, the lack of selectivity is inconsequential.
Figure 1. Irreversible inhibition of kinases bearing a suitably positioned cysteine residue
in the nucleotide binding site (most prominently: VEGFRs, MEKs, FLT3, GAK, MKNKs,
PDGFRs, TAK1, KIT) by cis-enone resorcylic acid lactones.
es and represent a unique pharmacophore that has been
shown to be effective in vivo.[9] Through a bioinformatic analy-
sis of the kinome, Santi and co-workers identified 46 kinases
that could potentially be targeted by this family of com-
pounds.[2] Our efforts to expand the diversity of this natural
pharmacophore has led to the identification of several tolerat-
ed modifications, such as substitution of the benzylic carbon
by an oxygen which offers simpler synthetic access.[7,10,11]
Figure 2. Key disconnections for the synthesis of 5-fluoro-cis-enone resorcy-
lides.
Based on the fact that these compounds react with the cys-
teine residue in the kinase nucleotide binding site through a
Michael addition, modulation of the electronic properties of
the enone may further enhance the inhibitory properties of
this pharmacophore. Previously, we have shown that substitu-
tion at the b-position of the enone with a methyl group abol-
ishes activity.[11] These results were concurrently confirmed by
researchers from Eisai.[12]
Benzoyl protection of alcohol 9 thus afforded 10 wherein the
terminal OPMB group was converted to an iodide 11 (DDQ; I2,
PPh3, imidazole). Attempts to alkylate a phenol using 11 led to
rapid elimination of the iodide to give a conjugated triene
(product not shown). To circumvent this elimination, which
was thought to be facilitated by the presence of the alkene,
compound 10 was dihydroxylated (dr, 2.5:1 in favor of the de-
sired isomer 12) and converted to acetonide 13. Conversion of
the terminal protected PMB ether into an iodide afforded the
key intermediate 14.
Fluoroenones 1 and 2 were synthesized from key intermedi-
ate 14 via the route shown in Scheme 2. Following previously
established chemistry, fragment 14 was coupled to the aromat-
ic moiety 15 bearing a benzylic selenide by alkylation followed
by oxidation/elimination of the selenide to afford 17. Con-
To further pursue modulation of the Michael acceptor, we
reasoned that a fluorine substituent at the a-position of the
[a] Dr. R. Jogireddy, Dr. S. Barluenga, Prof. N. Winssinger
Institut de Science et Ingꢀnierie Supramolꢀculaires (ISIS-UMR 7006)
Universitꢀ de Strasbourg - CNRS
8 allꢀe Gaspard Monge, 67000 Strasbourg (France)
Fax: (+33)36-885-5112
E-mail: winssinger@unistra.fr
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000047.
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ChemMedChem 2010, 5, 670 – 673

bound sulfonic acid, respective-
ly, afforded the desired fluoro-
enone resorcylic acids 1 (from
19) and 2 (from 20). We and
others had previously noted the
sensitivity of the cis-enone
system of LL-Z1640-2 and relat-
ed analogues to acid conditions
that could lead to isomeriza-
tion.[10,15,16] As anticipated based
on the higher thermodynamic
stability of E-fluoroalkene, ana-
logues 1 and 2 proved to be
more resistant to epimerization,
and no trace of isomerization
was observed even with pro-
longed reaction times. Interest-
ingly, the different diastereoiso-
mers originating from the non-
Scheme 1. Synthesis of key intermediate 14 from ester 5. Reagents and conditions: a) TBDPSCl (1.1 equiv), imid-
azole (2.0 equiv), CH2Cl2, 238C, 13 h, 90%; b) Dibal-H (1.1 equiv), PhMe, ꢀ788C, 1 h, 85%; c) 1. (EtO)2POCHFCO2Et,
nBuLi, THF, 08C, 1 h; 2. 6 (1.0 equiv), 0!238C, 12 h, 83%; d) Dibal-H (2.5 equiv), CH2Cl2, 0!238C, 85%; e) (COCl)2
(1.5 equiv), DMSO (2.45 equiv), Et3N (4.0 equiv), CH2Cl2, 1 h, 98%; f) 1. PMBO(CH2)2CCH (2.1 equiv), nBuLi
(2.0 equiv), THF, ꢀ788C, 1 h; 2. 7 (1.0 equiv), ꢀ788C, 1 h, 62%; g) H2, Pd/CaCO3, MeOH, 238C, 25 min, 97%; h) BzCl
(1.5 equiv), pyridine (2.5 equiv), CH2Cl2, 0!238C, 14 h, 91%; i) DDQ (1.2 equiv), CH2Cl2/H2O, 238C, 3 h, 92%; j) PPh3
(1.8 equiv), imidazole (3.0 equiv), I2, CH2Cl2, 238C, 1.5 h, 90%; k) OSO4 (1%), NMO·H2O (2.0 equiv), THF, 238C, 15 h,
78%; l) 2-methoxy propene (1.5 equiv), PPTS (0.1 equiv), CH2Cl2, 238C, 1 h, 93%. Abbreviations: Bz, benzoyl; Dibal-
H, diisobutylaluminium hydride; DDQ, 2,3-dichloro-5,6-dicyanobenzoquinone; NMO, N-methylmorpholine N-oxide;
PPTS, pyridinium p-tolenesulfonate; TBDPS, tert-butyldiphenylsilyl.
selective
dihydroxylation
proved to have significant dif-
ference in the kinetics of depro-
tection, which offered a conven-
ient means to separate them.
To gage the indiscriminate re-
activity of these enone resorcyl-
ic acids with thiols, fluoroenone
2 and the parent analogue lacking the fluoride were treated
with one equivalent of DTT (2 mm) as a prototypical thiol nu-
cleophile in phosphate-buffered saline (pH 7.4) containing 1%
DMSO. Interestingly, a clear difference in reactivity was ob-
served with the fluoroenone 2 reacting slower. With fluoroe-
nones 1 and 2 in hand, we tested their efficacy against VEGF-
R2, which was frequently found to be the most inhibited
kinase in our profile of cis-enone resorcylic acid library,[7,11] and
KIT, which represents a less inhibited kinase. As shown in
Table 1, fluoroenones 1 and 2 are less active than the natural
Table 1. Inhibition of VEGF-R2 and KIT (biochemical assay).
Scheme 2. Synthesis of fluoroenones 1 and 2 from key intermediate 14.
Reagents and conditions: a) 1. LDA (2.0 equiv), THF/HMPA, ꢀ788C, 10 min;
2. 14 (1.0 equiv), THF, ꢀ788C, 20 min, 80%; 3. H2O2 (2.0 equiv), THF, 238C,
3 h, 92%; b) K2CO3 (2.0 equiv), 14 (1.0 equiv), DMF, 808C, 12 h, 99%; c) TBAF
(10 equiv), THF, 238C, 48–56 h; d) PPh3 (2.0 equiv), DIAD (2.0 equiv), PhMe,
238C, 2-4 h, 70–74% (two steps); e) 1% NaOH in MeOH, 508C, 2–2.5 h, 69–
76%; f) DMP (1.5 equiv), CH2Cl2, 238C, 14 h, 80–88%; g) 40% aq HF in CH3CN
(1:10), 238C, 6.5 h, 65%; h) PS-SO3H (10 equiv), MeOH, 238C, 9 h, 70%. Ab-
breviations: DIAD, diisopropyl azodicarboxylate; LDA, lithiumdiisopropyl-
amide; HMPA, hexamethylphophoramide; DMF, N,N-dimethylformamide;
DMP, Dess–Martin periodinane; TBAF, tetrabutylammoniumfluoride; PS,
polystyrene supported.
Compound
VEGF-R2 [nm]
KIT [nm]
LL-Z1640-2
fluoroenone 1
fluoroenone 2
2.63
6.8
60.5
57.8
221
1600
Assays were performed in duplicate
product LL-Z1640-2, however, they maintain an inhibition level
that is interesting: 6.8 nm and 60.5 nm for 1 and 2, respective-
ly, against VEGF-R2 compared with 2.63 nm for LL-Z1640-2, and
their selectivity for VEGF-R2 relative to KIT remains unaffected.
The products originating from the minor diastereoisomers of
the dihydroxylation were significantly less active.
versely, alkylation of phenol 16 with fragment 14 yielded the
ether analogue 18 in excellent yield. Global deprotection of
the silyl groups (TMSE and TBDPS) followed by Mitsunobu
macrocyclization, selective benzoate deprotection and oxida-
tion of the resulting alcohol afforded products 19 and 20 from
17 and 18, respectively, in good overall yield. Global deprotec-
tion of 19 and 20 using aqueous hydrofluoric acid and resin-
The cellular activity of fluoroenones 1 and 2 against VEGF-
R2 was tested in HUE cells, a spontaneously immortalized
HUVEC clone known to overexpress VEGF-R2. The results paral-
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leled those of the enzyme inhibition assays. As shown in
Figure 3, the natural product LL-Z1640-2 is a very potent inhib-
itor with an IC50 value of 6.5 nm. Direct comparison with fluo-
roenone 2 revealed an IC50 value of 17 nm, while the analogue
containing the synthetically more accessible ether macrocycle
remained very potent with an IC50 value of 70 nm.
shown that the addition of a methyl substituent at the g-posi-
tion relative to the enone (C3, numbering shown in Figure 2)
enhanced the metabolic stability with a tolerable loss of activi-
ty (tenfold).[12] It should be noted, however, that several cis-
enone have already been shown to be effective in vivo. As the
inhibition is irreversible, the relative rate of metabolic instabili-
ty and irreversible inhibition coupled to target turn over
should be considered.
Experimental Section
Detailed procedures for the synthesis of compounds 1 and 2 are
described in the Supporting Information.
Fluoroenone 1: Rf =0.19 (CH2Cl2/MeOH, 9:1); 1H NMR (CD3OD,
400 MHz): d=6.99 (d, J=15.3 Hz, 1H), 6.40 (d, J=2.4 Hz, 1H), 6.28
(d, J=2.4 Hz, 1H), 6.09 (ddd, J=15.1, 8.9, 6.4 Hz, 1H), 5.95 (ddd,
J=21.9, 11.6, 2.7 Hz, 1H), 5.28–5.20 (m, 1H), 4.90 (br s, 1H), 4.09–
4.04 (m, 1H), 3.50–3.39 (m, 1H), 2.62–2.53 (m, 1H), 2.31–2.17 (m,
2H), 1.50 ppm (d, J=6.2 Hz, 3H), n.b., 4OH signals not visible;
2
13C NMR (CD3OD, 100 MHz, 258C): d=196.4 (d, JC,F =36.8 Hz, C=O),
172.8 (C=O), 166.9 (C), 164.1 (C), 155.2 (2ꢁd, 1JC,F =254.6 Hz, C),
2
145.0 (C), 133.9 (CH), 132.3 (CH), 123.9 (d, JC,F =18.4 Hz, CH), 109.4
3
(CH), 103.5 (C), 102.9 (CH), 81.7 (d, JC,F =3.8 Hz, CH), 74.8 (CH), 74.2
3
(CH), 36.6 (CH2), 33.4 (d, JC,F =6.1 Hz, CH2), 20.9 ppm (CH3); HRMS
(MALDI-TOF): m/z [M+Na]+ calcd for C18H19FO7Na: 389.1013;
found: 389.1024.
Fluoroenone 2: Rf =0.18 (CH2Cl2/MeOH, 9:1); 1H NMR (CD3OD,
400 MHz): d=6.02 (d, J=1.9 Hz, 1H), 5.98 (d, J=1.9 Hz, 1H), 5.88
(ddd, J=21.8, 11.8, 2.7 Hz, 1H), 5.44–5.29 (m, 1H), 5.03 (d, J=
4.3 Hz, 1H), 4.24–4.18 (m, 2H), 3.97–3.88 (m, 1H), 3.11–3.00 (m,
1H), 2.55–2.44 (m, 1H), 1.94–1.86 (m, 1H), 1.74–1.61 (m, 1H),
1.38 ppm (d, J=5.9 Hz, 3H), n.b., 4OH signals not visible; 13C NMR
(CD3OD, 100 MHz, 258C): d=196.8 (d, 2Jc,F =35.9 Hz, C=O), 172.6
1
(C=O), 167.1 (C), 165.4 (C), 163.6 (C), 155.6 (2xd, JC,F =255.3 Hz, C),
2
119.5 (d, JC,F =17.6 Hz, CH), 96.6 (CH), 96.4 (C), 92.8 (CH), 80.8 (d,
3
3JC,F =2.9 Hz, CH), 73.1 (CH), 69.5 (CH), 65.8 (CH2), 33.1 (d, JC,F
=
5.9 Hz, CH2), 31.3 (CH2), 20.8 ppm (CH3); HRMS (MALDI-TOF): m/z
[M+Na]+ calcd for C17H19FO8Na: 393.0962, found: 393.0942.
Enzymatic inhibition of VEGF-R2 and KIT: A radiometric protein
kinase assay (33PanQinaseꢂ Activity Assay) was used for measuring
the kinase activity of VEGF-R2 and KIT kinases (Proqinase, Freiburg,
Germany). All kinase assays were performed in 96-well Flash-
Platesꢃ from Perkin–Elmer (Boston, USA) using 50 mL of assay
buffer (60 mm HEPES-NaOH, pH 7.5, 3 mm MgCl2, 3 mm MnCl2,
3 mm Na3VO4, 1.2 mm DTT, 50 mgmLꢀ1 PEG2000, 1 mm [g-33P]ATP),
20 ng of kinase and a generic substrate (polyGluTyr) with 1%
DMSO. The test compound concentration ranged from 10 mm to
0.1 nm (semilog dilution). The assays were performed by premixing
the ATP solution with the test compound and addition of this solu-
tion to the kinase/substrate solution. After 60 min at 308C, the re-
action was stopped with 50 mL of 2% H3PO4, plates were aspirated
and treated with 200 mL of 0.9% NaCl (2ꢁ) and the level of 33P in-
corporation was determined with a microplate scintillation counter
(MicroBeta, Wallac). Assays were run in duplicate.
Figure 3. Cellular inhibition of VEGFR2 by a) LL-Z1640-2 (IC50 =6.5 nm), b) flu-
oroenone 1 (IC50 =17 nm) and c) fluoroenone 2 (IC50 =70 nm). Assays were
performed in triplicate.
Thus the fluoroenone modification is well tolerated but does
not enhance the cellular efficacy of VEGF-R2 inhibition. During
the course of our investigation, a similar modification was re-
ported by researchers from Esai,[12] however, their medicinal
chemistry efforts focused on the inhibition of inflammation-re-
lated pathways (MEKs and their down-stream regulatory effect
on cytokines).[12,17] A selected analogue (E6201) indeed shows a
>60- and >200-fold selectivity[17] for MEK1 relatively to VGF-
R2 and PDGFRb, which is a significantly different pattern rela-
tively to the parent natural product (LL-Z1640-2) for which
VEGF-R2 and PDGFRb are most inhibited.[11] It was further
Cellular VEGF-R2 inhibition assay: HUE cells, a spontaneously im-
mortalized HUVEC clone known to overexpress VEGF-R2, were
plated in ECGM (PromoCell, Germany), supplemented with 10%
fetal calf serum (FCS), with 25.000 cells per well in 48-well cell cul-
ture dishes. After 6 h, the medium was exchanged against ECBM
(PromoCell, Germany) without FCS, and the cells were starved
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in half logarithmic steps) were added to the cell culture medium
(1:100) resulting in a final DMSO concentration of 1%. After incu-
bation for 90 min at 378C, cells were stimulated for 7 min at room
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Acknowledgements
This work was funded by grants from the Agence National de la
Recherche (ANR) and Conectus.
Keywords: fluorine · irreversible inhibitors · kinases · resorcylic
acid lactones · VEGF
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Published online on March 5, 2010
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