S-farnesyl-thiopropionic acid triazoles as potent inhibitors of isoprenylcysteine carboxyl methyltransferase

Article abstract of DOI:10.1021/ml200106d

We report the design and synthesis of novel FTPA-triazole compounds as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt), through a focus on thioether and isoprenoid mimetics. These mimetics were coupled utilizing a copper-assisted cycloaddition to assemble the potential inhibitors. Using the resulting triazole from the coupling as an isoprenyl mimetic resulted in the biphenyl-substituted FTPA triazole 10n. This lipid-modified analogue is a potent inhibitor of Icmt (IC₅₀ = 0.8 ± 0.1 μM; calculated K_i = 0.4 μM).

Full text of DOI:10.1021/ml200106d

Letter
pubs.acs.org/acsmedchemlett
S-Farnesyl-Thiopropionic Acid Triazoles as Potent Inhibitors of
Isoprenylcysteine Carboxyl Methyltransferase
Joel A. Bergman,† Kalub Hahne,‡ Jiao Song,† Christine A. Hrycyna,‡ and Richard A. Gibbs*,†
‡
†Department of Medicinal Chemistry and Molecular Pharmacology and the Center for Cancer Research, and Department of
Chemistry and the Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, United States
S
* Supporting Information
ABSTRACT: We report the design and synthesis of novel FTPA-triazole compounds
as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt), through a
focus on thioether and isoprenoid mimetics. These mimetics were coupled utilizing a
copper-assisted cycloaddition to assemble the potential inhibitors. Using the resulting
triazole from the coupling as an isoprenyl mimetic resulted in the biphenyl-substituted
FTPA triazole 10n. This lipid-modified analogue is a potent inhibitor of Icmt (IC50
0.8 0.1 μM; calculated Ki = 0.4 μM).
=
KEYWORDS: Isoprenylcysteine carboxyl methyltransferase (Icmt), Ras, prenylcysteine, dipolar cycloaddition,
S-farnesyl-thiopropionic acid (FTPA), triazole
he post-translational processing of members of the Ras
protein superfamily is under active investigation because
Rando et al. identified the minimal substrate for Icmt as
farnesyl thiopropionic acid (FTPA, 1).8 Using this information
as a starting point, inhibitors of Icmt were developed from
studies of rotationally restricted prenylcysteines,9 library screen-
ing efforts,10 and amide-11,12 and isoprenyl-modified13 prenyl-
cysteines. In recent studies, we have established the prenyl-
cysteine analogue POP-3MB as a low micromolar inhibitor of
human Icmt.12
T
approximately 20% of human cancers result from mutated Ras
proteins.1 These proteins contain a −CaaX motif that is first
isoprenylated on the cysteine thiol with a farnesyl group by
farnesyltransferase (FTase) or a geranylgeranyl group by
geranylgeranyltransferase-1 (GGTase-1). Following lipidation,
two critical modifications occur sequentially at the endoplasmic
recticulum (ER) by membrane-associated enzymes. First, the
endoprotease Ras converting enzyme-1 (Rce-1) cleaves the
terminal −aaX residues, resulting in a newly exposed
prenylcysteine at the C terminus of the protein. Second,
isoprenylcysteine carboxylmethyltransferase (Icmt) methyl
esterifies the carboxylate using S-adenosyl-methionine (SAM)
as the methyl donor. The increased hydrophobicity imparted by
these modifications is thought to allow for proper localization
of the Ras proteins to the plasma membrane and thus their
function.2
We have now utilized FTPA as a starting point for nonamino
acid Icmt inhibitor scaffolds, incorporating a triazole as a
thioether or isoprene mimetic. This approach allowed us to
utilize the synthetic advantages of copper-assisted dipolar
cycloaddition (click chemistry) for rapid and facile analogue
assembly.14 Herein, we report the synthesis and evaluation of
FTPA analogues assembled utilizing click chemistry. Triazole
10n represents our most potent Icmt inhibitor to date, with an
in vitro IC50 of 0.8 0.1 μM.
Because blocking Ras localization should prevent its
functioning in tumor progression, this post-translational
modification pathway has been the target for the development
of therapeutics against mutant Ras-based tumors. To this end,
potent FTase inhibitors were developed that showed promise
against some tumors but proved ineffective against K-Ras
driven tumors,3 due in part to alternative geranylgeranylation of
K-Ras. We chose to focus on developing inhibitors of Icmt, the
only known enzyme that catalyzes the methyl esterification of
the terminal prenylcysteine.4 Genetic knockout studies have
provided support for the importance of Icmt-mediated
methylation in Ras signaling.5
In the initial design, a 1,4-disubstituted 1,2,3-triazole was
positioned as the thioether replacement and cysteine backbone
modifier that would join the lipid and carboxylate motifs of the
FTPA analogues (Scheme 1). This approach had two key
advantages: (a) It replaced the potentially labile allylic
thioether, and (b) the building blocks needed for the reaction
were readily available. Azido-esters 3a−d underwent cyclo-
addition with alkynes 4a−c,15,16 followed by saponification to
afford 5a−g in acceptable yields. This synthetic sequence
generated analogues with varying flexibility between the
carboxylate, the triazole, and the lipid motifs.
Substrate specificity studies revealed several key elements
necessary for recognition by Icmt, including a C15 or C20
isoprenyl lipid and a requirement for a thioether bridge
between the lipid and the carboxylate motifs.6,7 Early work by
Received: June 7, 2011
Accepted: November 14, 2011
Published: November 28, 2011
© 2011 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Previous efforts examined isoprenyl-modified AFC analogues
as Icmt inhibitors.13 These efforts demonstrated that the
correct (E)-geometry of the first isoprene must be maintained
as well as inclusion of the sulfur heteroatom.7 Furthermore,
saturated lipids were also not mimetics,13 suggesting that
specific “druglike” lipid structural motifs will interact with the
active site of Icmt. We also took compounds developed
previously as ligands for other isoprenyl binding pockets into
consideration when designing our analogues. Aryl mimetics of
isoprenoids have been utilized in the development of
nanomolar squalene synthase18 and FTase ligands,19−21 and
thus, we focused on this approach in our subsequent set of
potential Icmt inhibitors.
Scheme 1. Synthesis of Triazole for Sulfur Analogues
The synthesis of the FTPA-triazoles began from iodide 6,
which underwent Zr-assisted carboalumination,22 affording the
desired trisubstituted olefin 7 (Scheme 2). Corey-Kim
chlorination23 and alkylation with methyl 3-mercaptopropio-
nate resulted in ester 8. Sodium azide displaced the primary
iodide, affording the desired cycloaddition partner 9. Copper-
mediated cycloaddition with a variety of terminal alkynes (with
a focus on aromatic moieties) provided the FTPA-triazole
analogues with good yields. These compounds were evaluated
as both methyl esters (10a−m) and carboxylates (11a−m)
following saponification.
These FTPA-triazole analogues were evaluated using the
vapor diffusion assay. Neither the methyl esters 10a−m nor the
carboxylates 11a−m were Icmt substrates. The alkyl analogues
10a−d and 11a−d were modest inhibitors, and the esters
showed inhibitory activity comparable to the acids. However,
aryl rings attached to the triazole (10e−i and 11e−i) resulted
in both ester and acid compounds that were poor inhibitors of
Icmt. These data suggest that neither saturated lipids nor rigid
aryl groups directly joined to the triazole satisfied the structural
requirements necessary for inhibition.
a
Reagents and conditions: (a) Sodium ascorbate, cupric sulfate,
tBuOH/H2O, room temperature. (b) LiOH, MeOH; 20−80% over
two steps. The number in parentheses indicates Icmt specific activity
as percent of AFC control in the presence of 10 μM inhibitor.
These analogues (Scheme 1) were evaluated for activity using a
vapor diffusion assay.17 Compounds 5a−g were modest inhibitors
of Icmt but were not substrates. Structure−activity relationships
(SAR) determined an optimal two-methylene bridge (5b) between
the carboxylate and the triazole. Triazole-containing analogues based
on the FTPA structural motif thus warranted further exploration, as
this approach could generate diverse compounds quickly and easily.
a
Scheme 2. Synthesis of Triazole Substitutions in FTPA
a
Reagents and conditions: (a) Me3Al, Cp2ZrCl2, (HCO)n, 0 °C−room temperature, 60%. (b) (i) N-Chlorosuccinimide, Me2S, DCM, −40 °C−
room temperature; (ii) methyl thiopropionate, DIEA, DCM, 0 °C−room temperature, 55% over two steps. (c) NaN3, DMF, 80 °C, 78%.
(d) Sodium ascorbate, cupric sulfate, tBuOH/H2O, room temperature, 35−97%. (e) LiOH, MeOH, room temperature, 40−90%. The number in
parentheses indicates Icmt specific activity as percent of AFC control in the presence of 10 μM inhibitor.
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 3. Triazole Substitutions in FTPA: Use of Biphenyl Alkynes
a
Reagents and conditions: (a) Sodium ascorbate, cupric sulfate, tBuOH/H2O, room temperature; 36−78%. (b) LiOH, MeOH, room temperature,
50−96%. The number in parentheses indicates Icmt specific activity as percent AFC control with 10 μM inhibitor.
Natural isoprenylcysteine substrates have a high degree of
flexibility, and the aryl analogues described above possessed an
inflexible motif. Increased inhibition of Icmt was afforded with
increased spacing between the triazole and the aryl motifs.
Analogue 10k was the most potent analogue in this series, with
an IC50 of 41 5 μM. Alternatively, 11m, with both a polar
in the terminal aromatic ring (10s, 10t) were approximately
twice as potent as the electron rich analogue 10u.
Table 1. Cellular Activities (IC50) of Select FTPA-Triazole
Analogues
MEF
and a nonaromatic tail, displayed poor Icmt inhibition (IC50
>
cell-free
Icmt−/−
ac
Icmt+/+
ac
PaTu-8902
bc
,
100 μM). These data suggest that incorporation of aryl groups
within the isoprenyl region via a trizole must be accompanied
by a concomitant introduction of flexibility.
,
,
compd
(μM)
(μM)
>100
(μM)
96 0.01
(μM)
10g
10n
10o
FTS
ND
ND
0.8 0.1
>100
>100
33
55
1
2
8
3
Because Icmt can also efficiently methylate geranylgerany-
lated substrates,6 we hypothesized that further extension into
the region of the terminal isoprene could be utilized for
enhanced binding of our FTPA-triazole analogues. To examine
this hypothesis, a series of biphenyl alkynes were utilized, as we
have previously shown that the biphenyl group is an effective
isoprene mimetic.12,13,19 These analogues also included
incorporation of increased spacing between the triazole and
the biphenyl motifs to afford flexibility. Of these analogues, the
most potent inhibitor of Icmt in vitro was 10n. In our single
point assay, 10n (10 μM) decreased the specific activity of Icmt to
14% of the no inhibitor control (Scheme 3). We experimentally
29
5
ND
25.5
2
14.3 2.2
15.3 1.8
7.8 1.7
a
MEFs are plated in 96-well plates at a density of 1000 cell/well in
DMEM supplemented with 10% FBS. After incubation for 24 h, the
media are replaced with DMEM supplemented with 5% FBS and 10n
(various concentrations) or DMSO (0.1%). The cell viability is deter-
mined after 5 days using MTT. Twenty microliters of MTT (5 mg/mL)
is added to each well and incubated for 4 h at 37 °C. Afterward, the
media are removed, and 150 μL of DMSO are added to each well. The
absorbance is determined at 590 nm using a Molecular Devices
b
VERSAmax microplate reader. IC50 determinations were performed in
c
triplicate and calculated with Graphpad Prism V4. ND, not
determined.
determined an in vitro IC50 of 0.8
0.1 μM for 10n and a
calculated Ki of 0.4 μM (Cheng and Prusoff method24). Further
biochemical evaluation found 10n to be a competitive inhibitor.
Because the methyl ester 10n was more potent than the
corresponding carboxylate, 11n, modification of the carboxylate
group may represent a new approach for inhibitor design.25
One particularly attractive feature of 10n is its ease of syn-
thesis; it was prepared in five linear steps from homopropargyl
iodide 6 in an unoptimized 14% overall yield. This route
facilitated rapid additional SAR efforts through the synthesis of
modified biphenyl analogues. Analogues with alterations to the
orientation of the biphenyl moiety (10o−p) decreased Icmt
inhibition. The ester 10o was nearly 30 times less potent than
10n, with an IC50 of 29 5 μM (Table 1), and the ortho-10p
analogue possessed further reduced inhibitory activity, indicating
the importance of the para-biphenyl motif. Furthermore,
inclusion of an oxygen linker (10q) was also detrimental to
inhibition. Compounds containing electron-withdrawing groups
The lead compound (10n) was tested for efficacy in vitro in
several cell line model systems. First, MEF cells derived from
Icmt+/+ and Icmt−/− animals4 were treated with increasing
concentrations of 10n for 24 h, and cell viability was measured
using a standard MTT assay (Table 1). In MEF Icmt−/− cells,
10n demonstrated an IC50 of >100 μM, whereas an IC50 of
33 μM was observed for wild-type MEF Icmt+/+ cells. Two
analogues that are structurally related to 10n but are not Icmt
inhibitors in vitro were also examined in this assay. Analogues
10g and 10o had IC50 values in MEF Icmt+/+ cells of 96 and
55 μM, respectively, and did not affect viability in MEF Icmt−/−
cells (Table 1). Furthermore, treatment with FTS (S-farnesylth-
iosalicylic acid), an Icmt inhibitor that also possesses other cellular
effects27 and has clinical potential,28 showed equivalent activity in
both cell lines. Finally, as activating point mutations in K-Ras can
be found in approximately 90% of pancreatic cancers,29 the in vitro
efficacy of 10n was examined in the highly metastatic and “K-Ras
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ACS Medicinal Chemistry Letters
Letter
addicted”30 pancreatic cancer cell line PaTu-8902. An IC50 of 8
μM was determined for 10n, which is comparable to the known
Icmt inhibitor FTS (Table 1).
AUTHOR INFORMATION
Corresponding Author
*E-mail: rgibbs@purdue.edu.
■
Proper posttranslational processing is a prerequisite for Ras
membrane localization and function. To determine the cellular
effects of our most potent inhibitor 10n, the membrane
localization of fluorescently tagged K-Ras was visualized in the
presence and absence of 10n.26−31 Jurkat T-cells that were
transfected with GFP-K-Ras and treated with either delivery
vehicle alone, the statin drug simvastatin (which blocks K-Ras
prenylation), or 10n were fixed, and GFP-K-Ras localization
was visualized using fluorescent microscopy. After 24 h of
treatment, GFP-K-Ras localization was categorized into three
groups: normal plasma membrane localization, partial mis-
localization, and complete mislocalization. This classification
was determined via visual inspection of randomly selected fields
containing 100 cells. The results demonstrated that 10n led to a
decrease in normal membrane localization (Figure 1). These
Funding
Funding was provided by the NCI [R01CA112483 (R.A.G.)
and P30CA21328 (Purdue University Center for Cancer
Research CCSG)].
ACKNOWLEDGMENTS
■
We thank Mark Phillips (NYU) for the generous gift of GFP-
KRas and Marietta Harrison (Purdue University) for helpful
advice.
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10n exhibits significantly improved ligand efficiency32 and is
thus a superior starting point for drug development efforts.
ASSOCIATED CONTENT
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