Kinase inhibition by deoxy analogues of the resorcylic lactone L-783277

Article abstract of DOI:10.1021/ml1001807

The natural product L-783277 is a resorcylic lactone type covalent kinase inhibitor. We have prepared the 5′-deoxy analogue of L-783277 (1) in a stereoselective fashion. Remarkably, this analogue retains almost the full kinase inhibitory potential of natu

Full text of DOI:10.1021/ml1001807

pubs.acs.org/acsmedchemlett
Kinase Inhibition by Deoxy Analogues of the Resorcylic
Lactone L-783277
Marc Liniger,† Christian Neuhaus,† Tatjana Hofmann,† Luca Fransioli-Ignazio,† Michel Jordi,†
‡
‡
‡
,†
€
Peter Drueckes, Jorg Trappe, Doriano Fabbro, and Karl-Heinz Altmann*
†Swiss Federal Institute of Technology (ETH) Zurich, Department of Chemistry and Applied Biosciences, Institute of
Pharmaceutical Sciences, 8093 Zurich, Switzerland, and ‡Novartis Institute for Biomedical Research, Center for Proteomic
Chemistry, Expertise Platform Kinases, 4002 Basel, Switzerland
ABSTRACT The natural product L-783277 is a resorcylic lactone type covalent
kinase inhibitor. We have prepared the 50-deoxy analogue of L-783277 (1) in a
stereoselective fashion. Remarkably, this analogue retains almost the full kinase
inhibitory potential of natural L-783277, with low nanomolar IC50 values against
the most sensitive kinases, and it exhibits essentially the same selectivity profile
(within the panel of 39 kinases investigated). In contrast, removal of both the
40- and the 50-hydroxyl groups leads to a more significant reduction in kinase
inhibitory activity and so does a change in the geometry of the C70-C80 double bond
in 1 from Z to E. These findings offer new perspectives for the design of second
generation resorcylic lactone-based kinase inhibitors.
KEYWORDS Cancer, irreversible inhibitor, kinase inhibitor, natural product, SAR,
stereoselective synthesis, resorcylic lactone (RL)
he inhibition of protein kinases has emerged as a
powerful mechanistic paradigm for the treatment of
diseases that are characterized by dysregulated intra-
moiety in the macrocycle, as has been demonstrated in
biochemical8 as well as structural studies.9,10 The inhibition
of protein kinases by cis-enone-containing RL, thus, is largely
confined to kinases incorporating a properly located Cys
residue in their ATP binding pocket, corresponding to
Cys166 in ERK2,8 which provides a “built-in” selectivity
advantage over other inhibitor classes. Importantly, while
kinases containing the Cys166 equivalent of ERK2 comprise
only ca. 10% of the total kinome, they include a number of
important drug targets such as VEGFR2, MEK2, cKIT, or
FLT3.8 At the same time, the use of covalent inhibitors may
bear the potential for undesired off-target effects, due to the
unspecific modification of protein thiols. However, on the
basis of work by Kosan, the second order rate constant for
the reaction of hypothemycin with glutathione at pH 7.5 is
6.6 M-1 s-1 vs an apparent second order rate constant
(kinact/Ki) of 1.2 ꢀ 105 for the reaction with MEK1, thus
indicating a high degree of specificity.8
T
cellularsignaling or defects in cellcycle regulation.1-4 Atotalof
11 kinase inhibitors are currentlyapproved for the treatment of
various types of cancers,1,2 and kinase inhibitor drugs are
likely to be of therapeutic value also in other disease
indications.3,4 With the exception of the two rapamycin
derivatives temsirolimus and everolimus, which do not inhibit
kinase activity directly,5 all marketed kinase inhibitors are
based on different types of (poly)heterocyclic or urea
scaffolds.1-3 These structural features are also shared by the
majority of agents in clinical and preclinical development. As
an important extension of kinase inhibitor chemical space, the
resorcylic lactone (RL)-based fungal metabolites hypothemy-
cin, LL-Z1640-2, and L-783277 have recently been recognized
as a new and structurally unique group of kinase inhibitors.6,7
Structure-activity relationship (SAR) studies around
hypothemycin11 and LL-Z1640-2 (also called f152A1 or 5Z-
7-oxozeaenol)12-19 have included modifications of both the
aliphatic and the aromatic portion of the structures, and
analogues have been identified with in vivo activity in
tumor20 as well as inflammatory disease14,18 models; one
compound, Eisai's E6201, has been advanced to clinical
trials in humans13 in cancer and plaque type psoriasis
Received Date: August 2, 2010
The suppression of kinase activity by these natural pro-
ducts involves 1,4-addition of a protein thiol to the cis-enone
Accepted Date: September 23, 2010
Published on Web Date: October 20, 2010
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Scheme 1a
a Reagents and conditions: (a) Ti(OiPr)4 (2.5 mol %), (S)-BINOL (5 mol %), CH2dCHCH2SnBu3, toluene, -20 °C, 139 h, 90%, 98% ee. (b) TBDPSCl,
imidazole, DMF, room temperature, 21 h, 90%. (c) p-TsOH, THF/H2O (20:1), room temperature, 10 h, 92%. (d) (COCl)2, DMSO, NEt3, CH2Cl2, -78 °C,
1.5 h, 94%. (e) Compound 9, n-BuLi, THF, -78 °C f -10 °C, 40 min; then addition of 8, -78 °C f room temperature, 2 h, 93%, dr 1:1. (f) TBSCl,
imidazole, DMF, room temperature, 15 h, 96%. (g) Compound 11, 9-BBN (1.5 equiv), THF, room temperature, 5 h; then 12, 2 M K3PO4, Pd(OAc)2
(20 mol %), TFP (0.8 equiv), DME, reflux, 19 h, 68%. (h) 7.5 bar H2, Lindlar catalyst (5 mol %), AcOEt, 50 min, room temperature, 0.1 M, 97%. (i) 1 M
NaOH/MeOH (1:3), reflux, 21 h, 60%. (j) DEAD, PPh3, toluene, 0 °C f room temperature, 2.5 h, 49%. (k) CrO3, H2SO4, KF, H2O, acetone, -10 °C, 2 h,
64%. (l) HF Py, THF, room temperature, 9 h, 88%. BBN, 9-borabicyclo[3.3.1]nonane; DEAD, diethyl diazodicarboxylate; TBDPSCl, tert-butyldiphe-
3
nylsilyl chloride; TBSCl, tert-butyldimethylsilyl chloride; TFP, tri-2-furanylphosphine; p-TsOH, para-toluenesulfonic acid.
(according to the Thomson Reuters Integrity database). With
the exception of their clinical candidate E6201,17 however,
no kinase inhibition data have been reported by the Eisai
group on the numerous LL-Z1640-2 analogues that were
prepared as part of the work leading to the discovery of
E6201. In general, the presence of two oxygen functional-
ities at C40 and C50 and the retention of the natural config-
uration at these chiral centers have been considered as
important determinants for potent kinase inhibition by
cis-enone-containing RL.15,19 Thus, all three non-natural
epimeric diols of LL-Z1640-2 were found to be >50-fold less
active than the natural product in a cellular reporter assay for
TNFR release.19 At the same time, the structure of hypothe-
mycin bound to ERK2 does not show any significant inter-
actions between the C50-OH and the protein.9 This is in line
with the observation that methylation of the 50-OH group in
hypothemycin produces no more than a 3-5-fold loss in
antiproliferative activity;11 in contrast, the corresponding
C40-O-methyl derivative was found to be significantly less
potent, but no in vitro kinase inhibition data have been
reported for either of these methylated derivatives.11 While
the presence of a 50-OH (or -methoxy) group may be
important for the stabilization of the bioactive conformation
of RL-based kinase inhibitors, even in the absence of dis-
cernible hydrogen bonding with the protein, the structural
data leave open the possibility for the 50-OH group to be a
functionally less relevant (or even irrelevant) inborn struc-
tural feature of the natural products. In light of this and
because the presence of a chiral center R to the carbonyl had
caused synthetic problems in some of our previous work,21
we have investigated the activity of 50-deoxy L-783277 (1).
For reference purposes, we have also prepared the corre-
sponding C70-C80 E analogue 2 and dideoxy L-783277 (3)
and determined their kinase inhibitory properties.
Neither of these three analogues has been investigated pre-
viously; 40,50-dideoxy LL-Z1640-2 has been described as part of a
model study toward the total synthesis of LL-Z1640-2,22 but the
inhibition of kinase activity by this compound was not studied.
The synthesis of 1 was based on the same overall strategy
that we had followed in our recent total synthesis of
L-78327723 and involved the use of building blocks 8, 9,
and 12 for the assembly of the macrolide skeleton (Scheme 1).
Thus, the addition of the Li-acetylide derived from protected
homopropargylic alcohol 9 to aldehyde 8 followed by TBS
protection of the secondary hydroxyl group provided the
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protected C10-C100 fragment 11 (as a mixture of diastere-
oisomers at C60) in excellent overall yield. Building block 8
(which is the distinguishing element from the synthesis
of the natural product L-78327723) was prepared from
TBS-protected 3-hydroxy propanal (4) in a high-yielding
four-step sequence (69% overall yield) involving Keck
allylation24 of 4 (which proceeded with 98% ee) followed
by protecting group reshuffling and oxidation of the primary
hydroxyl group under Swern conditions. Olefin 11 was then
connected to aryl bromide 12 by Suzuki-Miyaura coupling
to produce the protected seco acid 13; subsequent partial
hydrogenation of the triple bond with Lindlar catalyst
followed by simultaneous ester saponification and TES clea-
vagewith NaOH/MeOH gave the seco acid 15 as the immediate
cyclization precursor (38% over three steps). It should be
noted here that the clean cleavage of the methyl ester group in
13 proved to be difficult and could be realized in acceptable
yield only after extensive optimization.26 Cyclization of 15
under Mitsunobu conditions gave the protected macrolide 16
in moderate but acceptable yield (49%). Treatment of 16 with
Jones reagent complemented with KF resulted in cleavage of
the allylic TBS ether and immediate oxidation to ketone 17.
desired 50-deoxy L-783277 (1) in 56% yield from 16. Upon
standing in chloroform solution for 4 days, the protected
macrolactone 17 isomerized to its C70-C80 E analogue 18 in
high yield (Scheme 2).25 Treatment of 18 with HF pyridine
3
then gave E-50-deoxy-L-783277 (2) (81%).
The synthesis of dideoxy-L-783277 (3) is summarized in
Scheme 3. Conceptually, the synthesis parallels that of 1,
except for the use of a different protecting group strategy
[i.e., MOM protection for the secondary hydroxyl group
formed in the acetylide addition step and TMS-ethyl ester
protection for the carboxyl group in the aromatic building
block 2223 (Scheme 3)]. Analogue 3 was ultimately obtained
from aldehyde 19 in eight steps and ca. 10% overall yield.
The in vitro kinase inhibitory activity of L-783277 and
compounds 1-3 were assessed against a panel of 39
kinases. Table 1 provides a listing of those kinases that were
inhibited with an IC50 value <10 μM by at least one of the
compounds; in all other cases, IC50 values were >10 μM.
As expected, L-783277 is highly selective for the inhibition
of kinases with an appropriately located Cys residue in the
ATP binding site, with nanomolar activity against VEGFR2
(IC50 = 2 nM) and PDGFRR (V651E)28 (ΙC50 = 2.4 nM). cKIT,
MK5, ERK2, and TYK2, although they do contain the requi-
site Cys residue, are inhibited with much lower potency, thus
indicating that L-783277 (as other RL-based kinase
inhibitors)8,15 does not indiscriminately inhibit all poten-
tially susceptible kinases with the same potency. IC50 values
of ca. 800 nM and 7 μM were observed against ALK and
LCK, respectively, which do not contain an ERK Cys166
equivalent; inhibition of LCK by L-783277 has been reported
previously by the Merck group with an IC50 value of 750 nM.29
The IC50 value of L-783277 for VEGFR2 is one order of
magnitude lower than has been reported by others;15 the
reasons for this discrepancy are unknown, but it may (at
least in part) reflect differences in assay format.30 L-783277
was found to inhibit MEK2 with an IC50 of 15 nM, which is in
The latter was deprotected with HF pyridine to furnish the
3
Scheme 2a
a Reagents and conditions: (a) CDCl3, room temperature, 4 days, 96%
conversion, 83%. (b) HF Py, THF, room temperature, 10 h, 81%.
3
Scheme 3a
a Reagents and conditions: (a) Compound 9, n-BuLi, THF, -10 °C, 20 min; then 19, -78 °C f -18 °C, 1.5 h, 51%, dr 1:1.1. (b) MOM-Cl, (i-Pr)2NEt,
Bu4NI, DMF, 19 h, 91%. (c) Compound 21, 9-BBN, THF, room temperature, 2 h; then 2-(trimethylsilyl)ethyl 2-bromo-6-hydroxy-4-methoxybenzoate
(22), 2 M K3PO4, Pd(OAc)2 (15 mol %), TFP (0.6 equiv), DME, reflux, 1.5 h, 83%. (d) H2, Lindlar catalyst, AcOEt, 3 h, 92%. (e) TBAF, THF, room
temperature, 2 h, 90%. (f) DEAD, Ph3P, toluene/THF, 1 h, 64%. (g) Sulfonic acid resin, MeOH, reflux, 3 h, 73%. (h) DMP, CH2Cl2, 4.5 h, 72%. DMP,
Dess-Martin periodinane (1,1,1-tris(acetyloxy)-1,1-dihydro-1,2-benziodoxol-3(1H)-one); TBAF, tetra-n-butylammonium fluoride.
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Table 1. Inhibition of Protein Kinases by RL L-783277, 1, 2, and 3a
IC50 (nM)
exhibits a very similar selectivity profile. Further modification
of 1 by a change in double bond geometry from Z to E, to
produce 2, or the removal of the OH group on C40, to give 3,
lead to a significant loss in potency, although both compounds
retain clearly submicromolar activity against VEGFR2 and
PDGFRR (V561D). The data for E analogue 2 provide broad
confirmation for the substantially reduced activity of C70-C80
E-configured RL-based kinase inhibitors, which had been re-
ported previously for the C70-C80 E isomers of L-783277 and LL-
Z1640-2 against MEK229 and TAK1,31 respectively, and also for
E-LL-Z1640-2 in a cellular reporter assay for TNFR signaling.18
There appears to be a trend for 3 to be more potent than 2, but
additional data are required to consolidate this conclusion. It
should also be noted that the significant difference in activity
between 3 and 1 does not necessarily imply the 40-(mono)-
deoxy analogue of L-783277 to be less active than 1. Unfortu-
nately, efforts to prepare 40-deoxy L-783277 by a route analo-
gous to that employed for 1 (Scheme 1) have failed so far, due to
problems with protecting group migration and isomerization
in the final oxidation and deprotection steps (Neuhaus, C.;
Altmann, K.-H. Unpublished data). An alternative synthesis of
this compound is currently under investigation.
LL-Z1640-2 has been reported to be converted to its less
active C70-C80 E isomer in mouse blood or plasma with a half-
life of roughly 20 min.18 We have independently investigated
the stability of L-783277 in human plasma and, in agreement
with the work of Du et al. on LL-Z1640-2,18 found the com-
pound to be transformed into its C70-C80 E isomer as the only
detectable metabolite (ca. 50% conversion after 20 min at
37 °C and 55 μM compound concentration; data not shown).
The compound is fully stable in pH 7.0-7.5 HEPES buffer for 24
h at 37 °C (<1% isomerization), which implicates the involve-
ment of plasma components in the isomerization process, most
likely reduced glutathione and glutathione-S-transferase.18
Unsurprisingly, the stability of 50-deoxy L-783277 (1) in human
plasma was unchanged as compared to L-783277.
Kinaseb
L-783277
1
2
3
ALK
763 ( 40
950/1300
>10000
303 ( 134
6200/7000
15d
763 ( 75
6600/6400
7400/7500
673 ( 119
4800/4700
ND
>10000
>10000
>10000
7600/8500
>10000
ND
3500/4400
>10000
>10000
>10000
>10000
6840d
ERK2
EPHB4
cKIT
LCK
MEK2c
MK5
640d
ND
150d
ND
>10000
>10000
95/100
MNK2
PDGFRRe
RET
ND
ND
2.38 ( 1.90
>10000
4100/5600
2.0 ( 1.4
7.23 ( 1.15
9900/7200
4400/5700
5.80 ( 1.01
180/220
>10000
>10000
230/190
>10000
>10000
170/190
TYK2
VEGFR2
a IC50 values from two independent experiments; for L-783277 and
1, values against ALK, cKIT, PDGFRR, and VEGFR2 are averages ( SDs
from three independent experiments. Kinase assays were essentially
carried out as described in ref 27. IC50 values for AUR A, CDK2A,
CDK4D1, COT1, ERK2, JAK1-3, PDK1, PKCR, PKN1-2, ROCK2, ABL,
and p38R were determined in a Caliper mobility shift assay; TR-FRET-
based LanthaScreen assay technology was used for all other kinases.
ND, not determined. No significant inhibition was observed at concen-
trations <10 μM forthe following kinases: AUR A (aurora kinase A), AXL,
BTK (Bruton's tyrosine kinase), CDK2A/4D1 (cyclin-dependent kinases
2A and 4D1), ZAP70 (ζ-chain-associated protein kinase), COT1 (colonial
temperature-sensitive gene-encoded kinase 1), EPHA4 (ephrin A4),
FGFR3 (fibroblast growth factor receptor 3, K651E mutant), GSK3β
(glycogen synthase kinase 3β), HER2 (human epidermal growth factor
receptor 2), HGFR (hepatocyte growth factor receptor; cMet), IGF1R
(insulin-like growth factor 1 receptor), INSR (insulin receptor), JAK1/2/3
(Janus kinases 1, 2, and 3), PDK1 (phosphoinositide-dependent protein
kinase 1), PKA/R/τ (protein kinases A, R, and τ), PKN1/2 (protein kinases
N 1 and 2), ROCK2 (rho-associated protein kinase 2), cABLT315I
(Abelson kinase, T315I mutant), and p38R (mitogen-activated protein
kinase 14). b ALK, anaplastic lymphoma receptor tyrosine kinase; ERK2,
extracellular-signal-regulated kinase 2; EPHB4, ephrin B4; LCK, lym-
phocyte-specific protein tyrosine kinase; MEK2, mitogen-activated pro-
tein kinase kinase 2; MK5, mitogen-activated kinase-activated protein
kinase 5; MNK2, mitogen-activated protein kinases-interacting kinase 2;
PDGFRR, platelet-derived growth factor receptor R; RET, “rearranged
during transfection” gene-derived kinase; TYK2, nonreceptor tyrosine
kinase 2; and VEGFR2, vascular endothelial growth factor receptor 2.
c MEK2 testing was performed at Millipore/Upstate (Charlottesville, VA).
d Single measurement. e V561D mutant.28
In summary, we have shown that 50-deoxy L-783277 (1)
retains almost the full kinase inhibitory potential of the
parent natural product L-783277. In contrast, C70-C80 E
derivative 2 and 40,50-dideoxy analogue 3 are significantly
less active. Our remarkable finding on the activity of 1
broadens the perspectives for the design of new RL-based
kinase inhibitors, which may be based on the monohydroxy-
lated scaffold of 1. Studies to determine the effects of the
removal of the 50-hydroxyl group in combination with other
modifications are ongoing in our laboratories.
good agreement with the activity originally reported by the
Merck group.29 Activity was also observed at the cellular level,
where L-783277 inhibited VEGFR2 autophosphorylation27 with
an IC50 of 57 nM.
SUPPORTING INFORMATION AVAILABLE Synthetic pro-
cedures and analytical data for all new compounds (1-3, 5-8,
10, 11, 13, and 20-27). Individual IC50 values for inhibition of ALK,
cKIT, PDGFRR, and VEGFR2 by L-783277 and 1. This material is
available free of charge via the Internet at http://pubs.acs.org.
Strikingly, the potency of L-783277 against VEGFR2 and
PDGFRR (V561D), the most sensitive kinases in our panel, is
only slightly reduced (2-3-fold) upon removal of the 50-OH
group (Table 1). Thus, 50-deoxy L-783277 (1) inhibits
VEGFR2 and PDGFRR (V561E) with IC50 values of 5.8 and
7.2 nM, respectively. Other kinases that are inhibited by 1
with submicromolar potency are ALK, MNK2 (ERK Cys166
equivalent present), and cKIT; against the latter, 1 is ca. 2-fold
less potent than L-783277, in accordance with the results
obtained for VEGFR2 and PDGFRR (V561D). Collectively, the
data indicate that 1 is almost equipotent with L-783277 and
AUTHOR INFORMATION
Corresponding Author: *Tel: þ41-44-6337390. Fax: þ41-44-
6331369. E-mail: karl-heinz.altmann@pharma.ethz.ch.
€
Funding Sources: We thank the ETH Zurich for generous
financial support. This research was also supported by a grant from
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the Swiss Federal Department of Home Affairs, State Secretariat
for Education and Research (SER) (SER Nr. SBF NR. C08.0052) in
the context of COST Action CM0602 (“Inhibitors of Angiogenesis:
Design, Synthesis, and Biological Exploitation”).
(16) Dakas, P.-Y.; Jogireddy, R.; Valot, G.; Barluenga, S.; Winssinger,
N. Divergent Syntheses of Resorcylic Acid Lactones: L-783277,
LL-Z1640-2, and Hypothemycin. Chem.;Eur. J. 2009, 15,
11490–11497.
(17) Goto, M.; Chow, J.; Muramoto, K.; Chiba, K.; Yamamoto, S.;
Fujita, M.; Obaishi, H.; Tai, K.; Mizui, Y.; Tanaka, I.; Young, D.;
Yang, H.; Wang, Y. J.; Shirota, H.; Gusovsky, F. E6201
[(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8,9,16-trihydroxy-
3,4-dimethyl-3,4,9, 19-tetrahydro-1H-2-benzoxacyclotetra-
decine-1,7(8H)-dione], a Novel Kinase Inhibitor of Mitogen-
activated Protein Kinase/Extracellular Signal-regulated Kinase
Kinase(MEK)-1and MEK Kinase-1: Invitro Characterization of
its Anti-inflammatory and Antihyperproliferative Activities.
J. Pharmacol. Exp. Ther. 2009, 331, 485–495.
(18) Du, H.; Matsushima, T.; Spyvee, M.; Goto, M.; Shirota, H.;
Gusovsky, F.; Chiba, K.; Kotake, M.; Yoneda, N.; Eguchi, Y.;
DiPietro, L.; Harmange, J.-C.; Gilbert, S.; Li, X.-Y.; Davis, H.;
Jiang, Y.; Zhang, Z.; Pelletier, R.; Wong, N.; Sakurai, H.; Yang,
H.; Ito-Igarashi, H.; Kimura, A.; Kuboi, Y.; Mizui, Y.; Tanaka, I.;
Ikemori-Kawada, M.; Kawakami, Y.; Inoue, A.; Kawai, T.;
Kishi, Y.; Wang, Y. Discovery of a Potent, Metabolically
Stabilized Resorcylic Lactone as an Anti-Inflammatory Lead.
Bioorg. Med. Chem. Lett. 2009, 19, 6196–6199.
ACKNOWLEDGMENT We are indebted to Dr. Bernhard Pfeiffer
and Fabienne Gaugaz (ETHZ) for NMR support, Kurt Hauenstein
(ETHZ) for technical advice, and Louis Bertschi (ETHZ, LOC MS-
Service) for HRMS spectra acquisition.
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only resulted in loss of the TBS group (55%). 4 M NaOH (aq)/
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