Synthesis of a Resorcylic Acid Lactone (RAL) library using fluorous-mixture synthesis and profile of its selectivity against a panel of kinases

Article abstract of DOI:10.1002/chem.200901375

A library of resorcylic acid lactones (RAL) containing a cis-enone moiety targeting kinases bearing a cysteine residue within the ATP-binding pocket was prepared using a fluorous-mixture synthesis and evaluated against a panel of 19 kinases thus providing important structure-activity trends. Two new analogues were then profiled for their selectivity against a panel of 402 kinases providing the broadest evaluation of this pharmacophores' selectivity.

Full text of DOI:10.1002/chem.200901375

DOI: 10.1002/chem.200901375
Synthesis of a Resorcylic Acid Lactone (RAL) Library Using Fluorous-
Mixture Synthesis and Profile of its Selectivity Against a Panel of Kinases
Rajamalleswaramma Jogireddy, Pierre-Yves Dakas, Gaꢀlle Valot, Sofia Barluenga, and
Nicolas Winssinger*[a]
Dedicated to Professor Jean-Marie Lehn on the occasion of his 70th birthday
Abstract: A library of resorcylic acid lactones (RAL) containing a cis-enone
moiety targeting kinases bearing a cysteine residue within the ATP-binding pocket
Keywords: combinatorial chemis-
was prepared using a fluorous-mixture synthesis and evaluated against a panel of
try · fluorous chemistry · kinases ·
19 kinases thus providing important structure–activity trends. Two new analogues
lactones · resorcylic acid
were then profiled for their selectivity against a panel of 402 kinases providing the
broadest evaluation of this pharmacophoresꢀ selectivity.
Introduction
versible kinase inhibitors[4] (L-783277[5] also named
FR265082;[6] LL-Z1640-2[7] also named 5Z-7-oxozeaenol[8]
and FR148083;[6] radicicol A[9,10] and hypothemycin[11,12]).
Two of these compounds (5Z-7-
Phosphorylation of proteins is one of the most prevalent cel-
lular mechanisms for regulating protein function in a rapid
and reversible fashion. There are approximately 518 protein
kinases encoded in the human genome[1] as well as a smaller
set of protein phosphatases[2] that can work synergistically
or concurrently at various levels in cellular pathways, thus
resulting in a tremendous combinatorial output. Virtually
every signal transduction pathway implicates kinases and as
much as 30% of all human proteins may be modified by
them. A number of pathologies ranging from oncology to in-
flammation and neurodegenerative diseases can be attribut-
ed to a dysfunctional kinase.[3] Accordingly, kinases have
become one of the most intensively pursued classes of pro-
teins for drug discovery, the vast majority being currently in-
vestigated for the treatment of cancer. To date, 11 kinase in-
hibitors have received FDA approval and there are approxi-
mately 30 distinct kinase targets being developed at the
level of phase I clinical trials.
oxozeaenol and hypothemycin)
have already been shown to be
effective in animal models.[8,11] As
such, these cis-enone RAL repre-
sent a pharmacologically validat-
ed starting point for kinase inhib-
ition. More recently, it has been
shown by Santi and co-workers
that hypothemycin irreversibly in-
activates ERK2 by forming a co-
valent Michael adduct with the
cys166 positioned in the ATP-
binding pocket of this kinase.[12]
A structure–bioinformatics analy-
sis of the kinome revealed that 46
out of the 518 putative kinases
Four natural resorcylic acid lactones (RAL) containing a
cis-enone moiety have been reported to be potent and irre-
contained a cysteine residue ade-
quately positioned to participate
in the Michael addition onto the
cis-enone of hypothemycin. Nev-
[a] R. Jogireddy, P.-Y. Dakas, G. Valot, S. Barluenga, N. Winssinger
Institut de Science et Ingꢁnierie Supramolꢁculaires (ISIS–UMR 7006)
Universitꢁ de Strasbourg—CNRS
ertheless, it was clearly shown that a significant difference in
the kinetics of inactivation existed amongst 16 out of the 46
tested kinases. Our profile of the related chloro-radicicol A
against 127 kinases showed that this compound inhibited ex-
clusively kinases from this subset of 46; however, not all
were inhibited (GSK3b, for example, was not inhibited).[10]
8 allꢁe Gaspard Monge, 67000 Strasbourg (France)
Fax. (+33)368-855-113
E-mail: winssinger@isis.u-strasbg.fr
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200901375.
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FULL PAPER
Furthermore it was shown by Nakajima and Miyake that
there was a significant difference in activity (5-fold) between
LL-Z1640-2 (FR148083) and L-783277 (FR265082) in their
inhibition of ERK2, suggesting that a good level of selectivi-
ty may be achieved within this subset of the kinome as L-
783277 was reported to be a potent MEK inhibitor (4 nm)[5]
compared to LL-Z1640-2 (411 nm).[8]
Herein we report a library of over 50 analogues based on
this resorcylic acid scaffold. The activity of a representative
subset of 31 members was evaluated against a panel of
19 kinases. Two analogues were further evaluated against a
panel of 402 kinases which includes 359 distinct kinases and
43 mutants.
deemed interesting and thus motivated the exclusive use of
the fluorous isolation technology for the purpose of the
present library. Fragment 5 was foreseen to come from the
coupling of fragment 6 and 7, with fragment 6 being either
an alkyne, which would be reduced to the cis-alkene with
Lindlar catalyst, or a cis-vinyl iodide. Of course, the general
library 1 could be further elaborated by epoxidation of the
benzylic alkene to obtain hypothemycin analogues 2 or by
methylation of the more acidic phenol and/or oxime forma-
tion on the ketone (3). Our choice of the fragments R1, R2
and aryl substitutions was based on our preliminary struc-
ture activity data coupled to reported structure activity ob-
tained by semi-synthesis of hypothemycin[17] and available
structural information.[6,18] Clearly, the nature of the func-
tionality at the benzylic position appeared to be important
in dictating the selectivity based on the afore-mentioned dif-
ference in activity between LL-Z1640-2 bearing an alkene
and L-783277 bearing an alkane for ERK2[6] and MEK1.[5,8]
While this modification at the benzylic position may seem
rather modest, preliminary modeling experiments have sug-
gested fairly different conformational landscape for both
compounds.[19] We thus felt it important to include both of
these functionalities in the library. While the epoxide at the
benzylic position imparts a similar conformation to an
alkene, difference in dipoles may be significant. Alternative-
ly, it appeared that a phenolic ether at that position should
also provide significant difference providing a conformation-
al profile more similar to the alkane, but with different
dipole moments. While structural information could rule out
certain modifications, it should be noted that the crystal
structures reported[6,18] are for the Michael adduct and the
initial recognition event between the protein and the cis-
enone may involve different
Results and Discussion
The synthetic planning of the library was based on the previ-
ously developed chemistry[10,13] which in turn was based on
the use of fluorous tags to facilitate isolation of reaction
product and to carry mixtures of products through a
common synthetic pathway.[14–16] Thus a library of the gener-
al structure 1 (Scheme 1) was envisioned to emanate from
the coupling of the key fragment 5 bearing a fluorous tag
encoding its structure and the different aromatic moieties 4
bearing either the selenide or methyl or hydroxyl at position
Y to obtain respectively an alkene, an alkane or phenolic
ether at position X in the library 1. While we have shown
that this coupling chemistry could be carried out on solid
phase using a sulfide linker at Y-position of intermediate 4
to access the alkane and alkene functionality,[13] this strategy
precluded the formation of phenolic ethers, which were
macrocyclic and/or kinase con-
formations. The modifications
which were envisioned for the
library are shown in Scheme 1
with four different fragments
for the ester moiety R1 (a–d),
three different functionalities
at position X (e–g) and three
different fragments for the
lower part of the macrocycle
R2 (h–j). The chiral methyl
substituent on the ester points
towards the surface of b-plead-
ed sheets. Our prior investiga-
tion had shown that the R chir-
ality at that center dramatically
attenuated activity; neverthe-
less, compounds lacking that
methyl group (fragment b)
may broaden specificity. An
extra hydroxyl group at the ad-
jacent position (fragment c)
was deemed interesting as it
Scheme 1. General structure and synthetic planning of a library of cis-enone RAL.
would probe whether addition-
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N. Winssinger et al.
al interactions may be achieved and whether the chiral sub-
stituent imparts an important conformational bias on the
macrocycle. The same reasoning was applied to the addition
of a hydroxyl group on the lower part of the macrocycle
(R2, fragment i). Modification of the alkene (fragment d)
was seen as crucial in modulating the rate of conjugate addi-
tion (if the conjugate addition is catalyzed by a hydrogen
bond to the carbonyl, the greater stability of a tertiary
cation vs. a secondary cation may favor the reaction). Fur-
thermore, having a methyl substituent at that position could
alleviate the issues related to cis/trans isomerization. It is
known that the trans-enones are significantly less active
than the cis-enone.[5] The homoallylic diol appeared to be
important for biological activity and it was already known
that methylation of either led to significant reduction in ac-
tivity.[17] This functionality was thus kept constant in frag-
ments h–j. Our preliminary investigations had shown that
compounds lacking the diol were indeed inactive as well as
acyclic analogues. However, a larger macrocycle appeared
interesting in providing subtle changes in conformation pro-
files and could be probed with fragment j. While each of
these modifications can be rationalized as providing a po-
tential benefit, the combination of several modifications
Scheme 2. Synthesis of RF tagged fragments 6a–d. a) PMBOC=NHCCl3
(1.0 equiv) , CSA (0.14 equiv), CH2Cl2, 238C, 12 h, 72–86%; b) DIBAL-
H (1.1 equiv), PhMe, À788C, 1 h, 45–52%; c) CBr4 (4.0 equiv), PPh3
(8.0 equiv), CH2Cl2, 08C, 45 min, 63%; d) nBuLi (2.0 equiv), THF,
À788C, 1 h, and 238C, 1.5 h, 85%; e) HC2MgBr (1.5 equiv), THF, À788C
to 238C, 12 h, 88%; f) EOM-Cl (3.0 equiv), iPr2NEt (3.0 equiv), nBu4N+I
(cat), CH2Cl2, 238C, 12 h, 89%; g) Cp2ZrCl2 (0.25 equiv), AlMe3
(3.0 equiv), CH2Cl2, 238C, 19 h and reflux 5 days; h) I2 (1.5 equiv), THF,
À308C, 15 min, 62%. CSA=camphorsulfonic acid, Cp=cyclopentadien-
yl, DIBAL-H=diisobutylaluminium hydride, EOM=ethoxymethyl,
PMB=p-methoxybenzyl, THF=tetrahydrofuran.
may provide unanticipated synergistic ACTHNURGTNEbUNG enefits.
As shown in Scheme 2, fragments 6a–d were obtained in
one to five steps through well-established chemistry. The
first step to obtain 6a–c involved a protection of the alco-
hols 8, 10, and 11 with the trichloroacetimidate of the fluo-
rous p-methoxybenzyl (PMB) 9, bearing different length of
the fluorous tag encoding the structure of the starting alco-
hol. For 10 and 11, the alcohol
MeOH fraction without further attempt to optimize individ-
ual isolation or recovered product from mixed fractions.
The synthesis of fragments 7h–j (Scheme 3) was based on
the naturally abundant chirality of deoxyribose and lyxose
by using well-established methodology. Thus, d-deoxyribose
protection was followed by a
diisobutylaluminium hydride
(DIBAL-H) reduction and a
Corey–Fuchs reaction or
a
Grignard addition of acetylene
(2:1 d.r., inseparable mixture)
followed by ethoxymethyl
(EOM) protection to afford 6a
and 6c respectively. Compound
6d was prepared from the rac-
emic 4-hydroxylpentyne using
a known procedure[20] to access
the (Z)-vinyl iodide 13, which
was protected with the fluo-
rous tagged PMB. The product
of each reaction was isolated
by flash chromatography on
fluorous silica gel. In general, a
10- to 20-fold ratio of silica to
crude product weight was used
for the isolation. The elutions
were carried out systematically
using a three step gradient (7:3
MeOH/H2O; 8:2 MeOH/H2O,
and pure MeOH) and the
product was collected from the
Scheme 3. Synthesis of fragments 7. a) 2-Methoxypropene (2.0 equiv), pTsA (0.04 equiv), CaSO4 (0.25 equiv),
DMF, 08C, 3 h, 60% or 2,2-dimethoxypropane (3.5 equiv), pTsA (0.02 equiv), acetone, 238C, 12 h, 90%; b)
LiAlH4 (1.4 equiv), THF, 08C to 238C, 2 h, 95%; c) TBDPS-Cl (0.9 equiv), imidazole (1.5 equiv), DMF, 238C,
2-12 h, 66–99%; d) SO3.Py complex (3.5 equiv), Et3N (4.9 equiv), CH2Cl2/DMSO (4:1), 08C to 238C, 0.5–1 h,
91–94%; e) BrPPh3CH3 (3.0 equiv), NaHDMS (2.8 equiv), THF, À788C to 238C, 1–12 h, 72–86%; f) PivCl
(2.0 equiv), Et3N (4.0 equiv), DMAP (0.2 equiv), CH2Cl2, 08C to 238C, 12 h, 93%; g) 9-BBN (2.2 equiv), THF,
08C to 238C, 3.5 h, then 3n NaOH/H2O2, 08C to 238C, 1.5 h, 94%; h) NaOMe (3.0 equiv), MeOH, 238C, 16 h,
83%; i) BnOH (7.2 equiv), 238C, 10 h, 96%; j) EOM-Cl (8.0 equiv), iPr2NEt (8.0 equiv), nBu4NI (cat),
CH2Cl2, 238C, 12 h, 95%; k) H2, Pd/C (5 mol%), MeOH, 238C, 5 h, 90%; l) O3, PPh3 (2.0 equiv), CH2Cl2,
À788C, 1 h, 90%. Bn=benzyl, 9-BBN=9-borabicyloACHTUNGERTN[NUNG 3.3.1]nonane, DMAP=4-dimethylaminopyridine,
DMF=N,N-dimethylformamide, DMSO=dimethylsulfoxide, EOM=ethoxymethyl, NaHDMS=sodium bis-
(trimethylsilyl)amide, Piv=pivaloyl, pTsA=p-toluene sulfonic acid, py=pyridine, TBAI=tetrabutylammoni-
um iodide, TBDPS=tert-butyldiphenylsilyl, THF=tetrahydrofuran.
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Natural Products
FULL PAPER
was selectively protected with
an acetonide[21] and reduced
with LiAlH4 to obtain diol 14,
which was selectively protect-
ed on the less hindered alcohol
with TBDPS-Cl (TBDPS=
tert-butyldiphenylsilyl)
and
oxidized to aldehyde 7h. Start-
ing with the same selective
protection of ribose but engag-
ing the resulting lactol in a
Wittig olefination[22] rather
Scheme 4. Synthesis of compounds 5 (12 permutations) by means of fluorous mixture synthesis using three
pools. a) nBuLi (1.1 equiv), THF, À788C, 10 min, then 7 (1.2 equiv), À788C, 30 to 60 min, 80%; b) BzCl
(2.5 equiv), pyridine (2.5 equiv), CH2Cl2, 08C to 238C, 4 h, 90%; c) TBAF (1.5 equiv), THF, 238C, 3 h, 86–
94%; d) H2, Pd/CaCO3 (1.72 equiv), MeOH, 238C, 45 min, 90–96%; e) PPh3 (1.5 equiv), imidazole (2.5 equiv),
I2 (1.5 equiv), THF, 08C8C, 30 min, 70–93%; f) 6d (1.0 equiv), tBuLi (2.0 equiv), Et2O, À788C, 20 min, then 7
(1.2 equiv), pentane, À788C to 08C, 7 h, >80%. Bz=benzoyl, TBAF=tetrabutylammonium fluoride, THF=
tetrahydrofuran.
than
a
reduction afforded
alkene 15 after pivaloylation
of the primary alcohol. A se-
quence involving a hydrobora-
tion, silyl protection, pivaloyl
deprotection, and oxidation af-
forded fragment 7j, which has
an additional carbon relative
to 7h. The third fragment ema-
nated from d-lyxose by protec-
tion of the anomeric position
followed by selective acetonide
protection of the cis-diol, and
EOM protection of the re-
maining hydroxyl group. De-
protection of the anomeric po-
sition and conversion of the
lactol to an alkene allowed
protection of the unique hy-
droxyl group with TBDPS-Cl
followed by ozonolysis of the
alkene to obtain fragment 7i.
While a number of alternative
strategies can be envisioned
for each of these fragments,
Scheme 5. Synthesis of fragments 4. a) TFA (20.0 equiv), TFAA (7.6 equiv), acetone, 238C, 12 h, 54%; b)
EOM-Cl (4.0 equiv), iPr2NEt (4.0 equiv), nBu4N+I (cat), CH2Cl2, 238C, 12 h, quant.; c) TMSCH2CH2OH
(4.0 equiv), NaHDMS (4.4 equiv), THF, 08C to 238C, 12 h, 69%; d) LDA (2.0 equiv), THF, À788C, 30 min,
(PhSe)2 (0.9 equiv), THF, À788C, 2 h, 75%. EOM=ethoxymethyl, LDA=lithium diisopropylamide,
NaHDMS=sodium bis(trimethylsilyl)amide, TFA=trifluoro acetic acid, TFAA=trifluoro acetic anhydride,
THF=tetrahydrofuran, TMSE=2-trimethylsilylethyl.
these procedures were found to be inexpensive, reliable and
scalable.
2,4,6-trihydroxybenzoic acid (18) by protection of the acid
and ortho-phenol with an acetonide[23] followed by protec-
tion of the two remaining phenols with EOM-Cl to obtain
19, which was treated with the alkoxide of trimethylsilyl eth-
anol to yield 4g. Compound 4 f was converted to 4e by
deprotonation with LDA and reaction with diphenyl
The synthesis of key fragments 5 is shown in Scheme 4.
Starting with a mixture of three fluorous-tagged alkynes 6,
which were deprotonated with nBuLi and added to three
vessels containing the different aldehydes 7 afforded three
pools of 17, each containing three compounds labeled with a
unique fluorous tag. Benzoyl protection of the alcohol fol-
lowed by desilylation, hydrogenation over Lindlar catalyst
and iodination afforded the three pools of three compounds
5a–c. All reactions were monitored by LC-MS and showed
greater than 90% conversion. While the isolated yield of
the product following fluorous isolation was not optimized,
acceptable yields were generally obtained. The vinyl iodide
6d was transmetalated with tBuLi and similarly added to
three different pools of aldehydes 7. A similar sequence of
benzoylation, desilylation, and iodination afforded three
pools of 5d.
AHCTUNGTRENNUNG
omatic fragment 4 was coupled to four pools of fragment 5.
Analysis of the reaction mixtures by LC-MS indicated good
to excellent conversion for all reactions, with the phenol
couplings being systematically the highest yielding followed
by the selenide and alkyl couplings. Amongst the different
pools of electrophiles, the pool containing fragment i, with
an additional EOM-protected hydroxyl adjacent to the
iodide being displaced, gave the lowest coupling efficiency.
However, the desired coupled product was obtained in all
cases. Each pool was demixed to resolve the individual com-
ponents, which were detagged (2,3-dichloro-5,6-dicyanoben-
zoquinone (DDQ)) and deprotected (tetrabutylammonium
The aromatic fragments were obtained as shown in
Scheme 5. The fragment 4g was obtained starting from
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N. Winssinger et al.
cle was reliably achieved with polymer-bound 2-iodoxyben-
zoic acid (IBX). On the other hand, for the oxidation of the
macrocycles containing an alkane or alkene at the benzylic
position, the oxidation was performed prior to deprotection
using Dess–Martin periodinane in refluxing CH2Cl2 (the oxi-
dation of one of the diastereoisomer is slower than the de-
protected macrocycle and requires more stringent condi-
tions). Removal of the acetonide and EOM by using aque-
ous HF afforded the desired macrocycles in moderate to
good yields. It should be noted that the reaction was never
allowed to reach completion as prolonged reaction time led
to product decomposition. Macrocycles 1ef bearing an
EOM group on the para-phenol were also isolated from
most reactions. All compounds were purified by preparative
TLC. The macrocycles 1e could be further elaborated by ep-
oxidation of the benzylic position using DMDO.[13] Howev-
er, the lability of the benzylic epoxide makes isolation of the
product challenging and pharmacologically undesirable.
Macrocycles could be also further derivatized by selective
methylation of the para-phenol with diazomethane. While
the reaction was quite selective for the methylation of the
para-phenol (3k and 3l; Scheme 7), to ensure completion of
the reaction, these were performed with a large excess of di-
azomethane leading to the isolation of 10–40% of the bis-
methylated product (not shown). The ketone could be con-
verted to a methyl or benzyl oxime in high yield under stan-
dard conditions. In total, 51 macrocycles were isolated from
these efforts.
The IC50 of a subset of the library (28, containing at least
one example of each modification) was assayed against a
panel of kinases (19) as representative of kinases bearing
the adequately positioned cysteine residue (VGFR-R1-3,
PDGF-Ra, FLT3, MEK1, KIT, GSKa, MAP, KAPK5), kin-
ases bearing a cysteine residue at a different position within
the ATP binding pocket (EGFR-3, JNK3, NEK2, NIK,
SRC, ZAP70), and kinases that do not bear a cysteine resi-
due (PKCa, CK2a, INS-R). It should be noted that as these
compounds are expected to be irreversible inhibitors, the
measured IC50 will depend on the procedure used and cau-
tion should be applied in comparing data from experiments
performed under different conditions. To obtain a value
which best reflects the rate of inactivation, the substrate and
inhibitor were added simultaneously to the kinase solution.
Scheme 6. Library synthesis. a) LDA (2.0 equiv), THF, À788C, 10 min,
then 5 (0.9 equiv), THF, À788C, 2.0 h, 60%; b) LDA (2.0 equiv), THF/
HMPA (10:1), À788C, 10 min, then 5 (0.9 equiv), THF, À788C, 30 min
and then H2O2 (2.0 equiv), THF, 238C, 2 h, 64–85%; c) K2CO3
(2.0 equiv),
5 (0.9 equiv), DMF, 1008C, 12 h, 94–100%; d) DDQ
(1.2 equiv), CH2Cl2/H2O (2:1), 238C, 2 h, 85–96%; e) TBAF (3.0 equiv),
THF, 238C, 2 h, quant.; f) RF-PPh3 (2.0 equiv), RF-DIAD (2.0 equiv),
PhMe, 238C, 2 h, 50–85%; g) 1% NaOH in MeOH, reflux, 12 h, 80–
90%; h) PS-SO3H (5.0 equiv), MeOH, 508C, 2 h, >90%; i) PS-IBX
(3.0 equiv), CH2Cl2/few drops of DMSO, 238C, 1–3 h (monitored by
TLC), 50%; j) DMP (1.5 equiv), CH2Cl2, reflux, 4 h, 80–90%; k) 40%
aq. HF in CH3CN (1:10), 238C, 3-6 h, 50–70%. DDQ=2,3-dichloro-5,6-
dicyanobenzoquinone, DIAD=diisopropyl azodicarboxylate, DMF=
N,N-dimethylformamide, DMP=Dess–Martin periodinane, DMSO=di-
methylsulfoxide, EOM=ethoxymethyl„ HMPA=hexamethylphophora-
mide, IBX=2-iodoxybenzoic acid, LDA=lithium diisopropylamide, PS=
polystyrene, TBAF=tetrabutylammonium fluoride, THF=tetrahydro-
ACHTUNGTRENNUNGfuran.
fluoride (TBAF)) to be engaged in a macrolactonization.
We had previously observed that these reactions could be
performed at fairly high concentration (0.1m) without dime-
rization or oligomerization. Treatment of each compound
with fluorous-tagged PPh3 and fluorous-tagged diisopropyl
azodicarboxylate (DIAD) afforded high yield in the lactoni-
zation (>85%) except for compounds containing the frag-
ment c, bearing a sterically more demanding alcohol for
which 50% conversion was
achieved. The allylic benzoate
was removed in excellent yield
for all macrocycles. We had
previously noted that a selec-
tive allylic oxidation could be
achieved in some cases, but
was unfortunately not general
and depended on the relative
geometry of the allylic alco-
hol.[13] In the cases of the mac-
rocycles bearing an ether (X=
O), the selective oxidation of
the fully deprotected macrocy-
Scheme 7. a) DMDO (4.0 equiv), CH3CN, 08C, 1–2 h, 60–70%; b) CH2N2 (5-10 equiv), Et2O, 23 8C, 3–12 h, 50–
60%; c) RNH2OH.HCl (10.0 equiv), pyridine, 408C, 12 h,>50%. DMDO=dimethyldioxirane.
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Natural Products
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The data is shown in Table 1 (kinases have been ranked
from the highest to least inhibited). As evident from
Table 1, there is very little difference in relative selectivity
of kinase inhibition throughout the library under these con-
ditions. VEGF-R2 is the most highly inhibited kinase in this
panel for all library members followed by PDGFR-a,
VEGR-R3, Flt3, VEGF-R1, MEK1 SESE (which is a consti-
tutively active form of MEK) and KIT. Overall, the trend of
structure activity relationship which emerged from this
screen is that the chiral methyl group at the ester position,
while not essential, does provide higher activity (entry 6 vs.
10, 7 vs. 11, and 8 vs. 12). Substitution at the adjacent posi-
tion with a hydroxyl group (entry 14 vs. 7) or at the b-posi-
tion of the enone with a methyl abrogated activity (15–19).
Position X of library 1 (Scheme 1), as suggested from the di-
verse natural products, is tolerant of modification and, an
oxygen is also tolerated at that position. The trend of activi-
ty for the three natural products can be ranked as follows:
alkene=epoxide>alkane. The ether in the macrocycle is
generally comparable to the alkane. The two modifications
of R2 tested were beneficial in certain combinations. For ex-
ample, the extra hydroxyl group present in fragment i does
afford a significant (tenfold) gain of activity (8 vs. 7) in com-
bination with fragment a. The same beneficial effect was not
observed in the presence of fragment b (entry 12 vs. 11).
The larger macrocycle (fragment j) was particularly benefi-
cial in conjunction with the alkane moiety at the benzylic
position (f). For example, entries 6, 10, and 25 are amongst
the most potent compounds tested, whereas a similar gain is
not observed for X=CH or oxygen (entries 4, 9, and 13).
Generally, methylation of the phenol para to the ester had
marginal changes in activity, while oxime formation abolish-
ed activity. Interestingly, none of the alcohols 23 lacking the
ketone showed significant inhibition attesting to the fairly
low affinity of the macrocycle for the ATP-binding pocket
and the importance of the Michael acceptor.
Table 2, 11 kinases out of 31 had more than 50% residual
activity given this high concentration of 1afh (MEK4 had
0% residual activity at this concentration). The kinases
which showed less than 10% residual activity relatively to
control were: Flt1&3; GAK; KIT, MEK1,2,4; MKNK 1&2,
PDGFRa&b, TAK1, and TGFR2. Interestingly, some kinas-
es in this group (CDKL5, ERK1, GSK3b, NIK, NLK,
PRP4) were not inhibited. For 1bgi, 19 out of the 31 kinases
had over 50% residual activity and MEK4 was also the
most highly inhibited kinases, with 1% residual activity.
While 1bgi was overall less potent, it does appear to show
subtle differences in reactivity compared to 1afh. For exam-
ple, while both compounds have similar activity against
GAK, 1bgi is ten times less active against Flt1 and
PDGFRb.
The two compounds were also evaluated against a series
of mutations of Flt3 and KIT. It is known that several muta-
tions in Flt3 lead to a gain of function or constitutive activi-
ty. An internal tandem duplication (ITD) in Flt3[27] is found
in 20% of patients with acute myeloid leukemia (AML),
while the most abundant mutation leading to a gain of func-
tion was found to be D835Y for which both compounds
(1afh and 1bgi, Table 3) are more active than against the
wild type.[28] Other mutants (K663Q[29] and N841 L[30]) lead-
ing to a gain of function remained equally sensitive to the
inhibitors. Likewise, the compound retained their activity
against KIT mutants which results in a gain of function
(D816 V[31] L576P[32]and V559D[33]); however, these com-
pounds are not active against the dual mutations that confer
resistance to imatinib (V559D, T670I; V559D, V654 A)[34]
From a therapeutic perspective, the kinases inhibited by
the cis-enone resorcylic acids contribute to the development,
progression, and aggressiveness of cancer. More recent in
depth characterization of the specificity of kinase inhibitors
have shown that all small molecule kinase inhibitors ap-
proved for therapeutic intervention or in clinical develop-
ment do inhibit multiple targets.[25] The pallet of kinases in-
hibited by the cis-enone resorcylides all drive tumor growth.
The fact that all compounds lacking the enone moiety failed
to show significant inhibition testifies to the importance of
the Michael acceptor and its potential as a selectivity filter.
An important question is whether a mutation in the cysteine
residue, which is so important for the activity, would be
viable. However, based on the number of kinases inhibited
along the MAP kinase cascade, a concerted evolution of
several kinases would be necessary to evade the growth in-
hibition of these resorcylic acid lactones. The irreversible
nature of the inhibition may prove to be important in ach-
ieving long-lasting inactivation of specific pathways. The
EGFR receptor has also been the target of irreversible in-
hibitors exploiting a cysteine residue. Results from phase I
clinical trials have shown that EKB-569 is safe.[35] From a
chemical biology standpoint, compounds that form covalent
bonds to their target are particularly useful as they can be
used to label the targeted kinases for activity-based[36] profil-
ing and imaging. It is interesting to note how frequently evo-
lution has resorted to irreversible inhibition in the selection
Two representative library members (1afh and 1bgi) were
then evaluated in a larger panel of kinases (402) using the
technology described by Lockhart et al.[24,25] In this panel,
31 kinases out of the 46 kinases bioinformatically identified
by Santi and co-workers are present along with numerous
other kinases bearing a cysteine residue at other positions
within the ATP-binding pocket.[3] The two compounds were
screened at 1 mm (i.e. well above their IC50 concentration for
the most potently inhibited kinases) to have a realistic per-
spective of their selectivity. Interestingly, only two kinases
outside of the set of 46 kinases predicted by Santi et al.
showed significant inhibition (STK36 and PRKD2) attesting
to the potential of exploiting cysteine residues within the
ATP-binding pocket as selectivity filters.[26] As shown in
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www.chemeurj.org
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Chem. Eur. J. 2009, 15, 11498 – 11506

Natural Products
FULL PAPER
Table 2. Residual activity as a % of control for 31 of the 46 putative
kinase bearing the suitably positioned cysteine inhibited by two resorcy-
lide containing an alkane (1afh) or ether (1bgi) at the benzylic position.
[1] G. Manning, D. B. Whyte, R. Martinez, T. Hunter, S. Sudarsanam,
Science 2002, 298, 1912–1934.
[2] L. Bialy, H. Waldmann, Angew. Chem. 2005, 117, 3880–3906;
Angew. Chem. Int. Ed. 2005, 44, 3814–3839.
Kinase
1afh
1bgi
Kinase
1afh
1bgi
AAK1
BIKE
17
26
83
69
100
94
85
5.8
7
12
2.8
70
91
76
87
52
100
95
100
57
40
MEK2
MEK3
MEK4
MEK6
MKNK1
MKNK2
NIK
0.7
49
0
28
4.4
0
100
87
4.7
0.65
100
3.4
10
20
15
11
73
1
19
23
4.4
100
100
78
37
100
27
[3] J. Zhang, P. L. Yang, N. S. Gray, Nat. Rev. Cancer 2009, 9, 28–39.
[4] N. Winssinger, S. Barluenga, Chem. Commun. 2007, 22–36; S. Bar-
luenga, P. Dakas, M. Boulifa, E. Moulin, N. Winssinger, C. R. Chim.
2008, 11, 1306–1317; T. Hofmann, K.-H. Altmann, C. R. Chim.
2008, 11, 1318–1335.
[5] A. Zhao, S. H. Lee, M. Mojena, R. G. Jenkins, D. R. Patrick, H. E.
Huber, M. A. Goetz, O. D. Hensens, D. L. Zink, D. Vilella, A. W.
Dombrowski, R. B. Lingham, L. Huang, J. Antibiot. 1999, 52, 1086–
1094.
[6] M. Ohori, T. Kinoshita, S. Yoshimura, M. Warizaya, H. Nakajima,
H. Miyake, Biochem. Biophys. Res. Commun. 2007, 353, 633–637.
[7] G. A. Ellestad, F. M. Lovell, N. A. Perkinson, R. T. Hargreaves, W. J.
McGahren, J. Org. Chem. 1978, 43, 2339–2343.
[8] J. Ninomiya-Tsuji, T. Kajino, K. Ono, T. Ohtomo, M. Matsumoto,
M. Shiina, M. Mihara, M. Tsuchiya, K. Matsumoto, J. Biol. Chem.
2003, 278, 18485–18490.
CDKL2
CDKL3
CDKL5
ERK1
ERK2
FLT1
NLK
FLT3
FLT4
GAK
PDGFRA
PDGFRB
PRP4
30
3.8
100
100
62
61
5.6
GSK3 A
GSK3B
KIT
TAK1
100
8.8
63
TGFBR2
VEGFR2
ZAK
18
77
65
MAPKAPK5
MEK1
0.35
[9] T. Kastelic, J. Schnyder, A. Leutwiler, R. Traber, B. Streit, H. Niggli,
A. Mackenzie, D. Cheneval, Cytokine 1996, 8, 751–761.
[10] P. Y. Dakas, S. Barluenga, F. Totzke, U. Zirrgiebel, N. Winssinger,
Angew. Chem. 2007, 119, 7023–7026; Angew. Chem. Int. Ed. 2007,
46, 6899–6902.
Table 3. Residual activity as a % of control for kinases bearing muta-
tions.
Kinase
1afh
1bgi
Kinase
1afh
1bgi
[11] H. Tanaka, K. Nishida, K. Sugita, T. Yoshioka, Jpn. J. Cancer Res.
1999, 90, 1139–1145.
[12] A. Schirmer, J. Kennedy, S. Murli, R. Reid, D. V. Santi, Proc. Natl.
Acad. Sci. USA 2006, 103, 4234–4239.
[13] P. Y. Dakas, R. Jogireddy, G. Valot, S. Barluenga, N. Winssinger,
Chem. Eur. J. 2009 DOI: 10.1002/chem.200901373, preceeding publi-
cation.
FLT3
7
9
0.4
3
6
40
12
2.8
26
19
4.8
KIT
8.8
0.9
5.2
7.8
68
62
13
57
38
84
FLT3
FLT3
FLT3
FLT3
FLT3
G
KIT
KIT(L576P)
KIT(V559D)
ACHTUNGTRNE(NUNG D816 V)
E
G
N
T
T
KIT(V559D,T670I)
KIT(V559D,V654 A)
A
0
66
100
[14] D. P. Curran, Handbook of Fluorous Chemistry, Wiley-VCH, Wein-
heim, 2004, pp. 101–127.
[15] Z. Luo, Q. Zhang, Y. Oderaotoshi, D. P. Curran, Science 2001, 291,
1766–1769.
[16] W. Zhang, D. P. Curran, Tetrahedron 2006, 62, 11837–11865.
[17] B. R. Hearn, K. Sundermann, J. Cannoy, D. V. Santi, ChemMed-
Chem 2007, 2, 1598–1600.
[18] G. Rastelli, R. Rosenfeld, R. Reid, D. V. Santi, J. Struct. Biol. 2008,
164, 18–23.
of bioactive secondary metabolites.[37] It should also be
noted that irreversible inhibition of kinases leveraged on a
conjugate addition is an endogenous mechanism as has been
demonstrated for the inhibition of IKB by cyclopentenone
prostaglandins.[38]
In conclusion, this library offers important structural ac-
tivity relationship in the cis-enone resorcylic acids. Two
modifications were found to independently and synergisti-
cally improve the activity of this series of compounds while
a modification which dramatically simplifies the synthetic
accessibility of these compounds was established. The fact
that several members of this family are potent inhibitors of
multiple notorious oncogenic kinases should heighten their
therapeutic interest.
[19] M. Karplus, V. Zoete, unpublished results.
[20] S. Ma, E.-i. Negishi, J. Org. Chem. 1997, 62, 784–785.
[21] J. Barbat, J. Gelas, D. Horton, Carbohydr. Res. 1983, 116, 312–316.
[22] X. Geng, S. J. Danishefsky, Org. Lett. 2004, 6, 413–416.
[23] R. G. Dushin, S. J. Danishefsky, J. Am. Chem. Soc. 1992, 114, 655–
659.
[24] M. A. Fabian, W. H. Biggs, III, D. K. Treiber, C. E. Atteridge,
M. D. Azimioara, M. G. Benedetti, T. A. Carter, P. Ciceri, P. T.
Edeen, M. Floyd, J. M. Ford, M. Galvin, J. L. Gerlach, R. M. Grotz-
feld, S. Herrgard, D. E. Insko, M. A. Insko, A. G. Lai, J. M. Lelias,
S. A. Mehta, Z. V. Milanov, A. M. Velasco, L. M. Wodicka, H. K.
Patel, P. P. Zarrinkar, D. J. Lockhart, Nat. Biotechnol. 2005, 23, 329–
336.
Experimental Section
[25] M. W. Karaman, S. Herrgard, D. K. Treiber, P. Gallant, C. E. Atter-
idge, B. T. Campbell, K. W. Chan, P. Ciceri, M. I. Davis, P. T. Edeen,
R. Faraoni, M. Floyd, J. P. Hunt, D. J. Lockhart, Z. V. Milanov, M. J.
Morrison, G. Pallares, H. K. Patel, S. Pritchard, L. M. Wodicka, P. P.
Zarrinkar, Nat. Biotechnol. 2008, 26, 127–132.
Physical characterization for all final library members, selected synthetic
intermediates, and the full profile of kinase inhibition are given in the
Supporting Information.
[26] M. S. Cohen, C. Zhang, K. M. Shokat, J. Taunton, Science 2005, 308,
1318–1321.
[27] M. Nakao, S. Yokota, T. Iwai, H. Kaneko, S. Horiike, K. Kashima,
Y. Sonoda, T. Fujimoto, S. Misawa, Leukemia 1996, 10, 1911–1918.
[28] Y. Yamamoto, H. Kiyoi, Y. Nakano, R. Suzuki, Y. Kodera, S. Miya-
waki, N. Asou, K. Kuriyama, F. Yagasaki, C. Shimazaki, H. Akiya-
ma, K. Saito, M. Nishimura, T. Motoji, K. Shinagawa, A. Takeshita,
H. Saito, R. Ueda, R. Ohno, T. Naoe, Blood 2001, 97, 2434–2439.
Acknowledgements
This work was funded by a grant from the Agence National de la Recher-
che (ANR) and Conectus. An MNRT fellowship (G.V.) and fellowship
from ARC (P.-Y.D.) are also gratefully acknowledged.
Chem. Eur. J. 2009, 15, 11498 – 11506
ꢂ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
11505

N. Winssinger et al.
[29] M. Schittenhelm, K. Yee, J. Tyner, L. McGreevey, A. Haley, A.
Town, D. Griffith, T. Bainbridge, R. Braziel, A. OꢀFarrell, J. Cher-
rington, M. Heinrich, Leukemia 2006, 20, 2008–2014.
[30] J. Jiang, J. Paez, J. Lee, R. Bo, R. Stone, D. DeAngelo, I. Galinsky,
B. Wolpin, A. Jonasova, P. Herman, E. Fox, T. Boggon, M. Eck, E.
Weisberg, J. Griffin, D. Gilliland, M. Meyerson, W. Sellers, Blood
2004, 104, 1855–1858.
[31] C. Willmore-Payne, J. Holden, B. Chadwick, L. Layfield, Mod.
Pathol. 2006, 19, 1164–1169.
[32] C. Willmore-Payne, J. Holden, B. Chadwick, L. Layfield, Mod.
Pathol. 2006, 19, 1164–1169.
[34] T. Carter, L. Wodicka, N. Shah, A. Velasco, M. Fabian, D. Treiber,
Z. Milanov, C. Atteridge, W. Biggs, P. Edeen, M. Floyd, J. Ford, R.
Grotzfeld, S. Herrgard, D. Insko, S. Mehta, H. Patel, W. Pao, C.
Sawyers, H. Varmus, P. Zarrinkar, D. Lockhart, Proc. Natl. Acad.
Sci. USA 2005, 102, 11011–11016.
[35] C. Erlichman, M. Hidalgo, J. P. Boni, P. Martins, S. E. Quinn, C. Za-
charchuk, P. Amorusi, A. A. Adjei, E. K. Rowinsky, J. Clin. Oncol.
2006, 24, 2252–2260.
[36] N. Jessani, B. F. Cravatt, Curr Opin Chem. Biol 2004, 8, 54–59.
[37] C. Drahl, B. F. Cravatt, E. J. Sorensen, Angew. Chem. 2005, 117,
5936–5958; Angew. Chem. Int. Ed. 2005, 44, 5788–5809.
[38] A. Rossi, P. Kapahi, G. Natoli, T. Takahashi, Y. Chen, M. Karin,
M. G. Santoro, Nature 2000, 403, 103–108.
[33] S. Hirota, K. Isozaki, Y. Moriyama, K. Hashimoto, T. Nishida, S. Ish-
iguro, K. Kawano, M. Hanada, A. Kurata, M. Takeda, G. Tunio, Y.
Matsuzawa, Y. Kanakura, Y. Shinomura, Y. Kitamura, Science 1998,
279, 577–580.
Received: May 22, 2009
Published online: October 9, 2009
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Chem. Eur. J. 2009, 15, 11498 – 11506