Probing the isoprenylcysteine carboxyl methyltransferase (Icmt) binding pocket: Sulfonamide modified farnesyl cysteine (SMFC) analogs as Icmt inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.01.078

Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is important for the post-translational modification of oncogenic Ras proteins. We herein report the synthesis and biochemical activity of 41 farnesyl-cystei

Full text of DOI:10.1016/j.bmcl.2011.01.078

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2616–2620
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Probing the isoprenylcysteine carboxyl methyltransferase (Icmt) binding
pocket: Sulfonamide modified farnesyl cysteine (SMFC) analogs as Icmt
inhibitors
Jaimeen D. Majmudar a, Kalub Hahne b, Christine A. Hrycyna b, , Richard A. Gibbs a,
⇑
⇑
a Department of Medicinal Chemistry and Molecular Pharmacology and the Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
b Department of Chemistry and the Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Human isoprenylcysteine carboxyl methyltransferase (hIcmt) is a promising anticancer target as it is
important for the post-translational modification of oncogenic Ras proteins. We herein report the synthe-
sis and biochemical activity of 41 farnesyl-cysteine based analogs versus hIcmt. We have demonstrated
that the amide linkage of a hIcmt substrate can be replaced by a sulfonamide bond to achieve hIcmt inhi-
bition. The most potent sulfonamide-modified farnesyl cysteine analog was 6ag with an IC50 of
Received 2 November 2010
Revised 14 January 2011
Accepted 19 January 2011
Available online 22 January 2011
8.8 0.5 lM for hIcmt.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Inhibitor
Methyltransferase
Enzymes
Isoprenoid
Signal transduction
Anti-cancer agents
Proteins that contain a C-terminal CaaX motif (where C-cys-
teine, aa-any aliphatic amino-acid, X being L, S, M, F or Q) are
post-translationally modified by a series of reactions that enable
their appropriate cellular targeting, most commonly membrane
association (Fig. 1). K-Ras, one of the >120 proteins having a CaaX
box undergoes farnesylation followed by endoproteolyic cleavage
of the –aaX residues by Rce-1 and finally methyl esterification of
the free carboxylate of the prenylated cysteine by isoprenylcys-
teine carboxyl methyltransferase (Icmt). Mutant K-Ras, stabilized
in the constitutively active GTP-bound conformation, signals con-
tinuously resulting in tumorogenesis. Mutant Ras is implicated in
15–20% of all human malignancies and greater than 90% of pancre-
atic cancers.1
hIcmt inhibitors have also identified and vary in structure ranging
from indoloacetamides,7 thiosalicylic analogs8 and prenylated
thioacetic acid9 to halogenated natural products.10
Due to its membrane localization and its lack of homology with
other methyltransferases, there is no structural information on
hIcmt. This poses a fundamental challenge to designing Icmt inhib-
itors. In an effort to understand the requirements for hIcmt inhibi-
tion, we hypothesized that isosteric replacements of the amide
bond in AMFCs will result in molecules that will retain their inter-
action with hIcmt and provide more information on inhibition
requirements. As a first step to realize this goal, we have focused
our attention on evaluating metabolically stable amide bond
replacements. Figure 2 shows the structure of the most potent
AMFCs synthesized by Donelson et al.6
Replacement of the amide bond with a metabolically stable and
more drug-like isostere is an important goal of our research. Its
biological stability and the ease of synthesis led us to explore the
sulfonamide bond in AFC based analogs. Herein, we demonstrate
that the sulfonamide bond is a viable amide surrogate and that sul-
fonamide-modified farnesyl cysteine analogs (SMFCs) are low-
micromolar inhibitors of hIcmt.
Inhibition of human Icmt (hIcmt), a 33 kDa integral ER mem-
brane methyltransferase,2 has been postulated to block the activity
of oncogenic Ras. The validity of Icmt as a viable drug target was
supported through a knock-out study performed by Bergo et al.3
and later by Michaelson et al.4 who showed that ablation of Icmt
had a selective effect on Ras signaling. N-Acetyl-S-farnesyl-L-cys-
teine (AFC) is a substrate for human Icmt (hIcmt) and recent work
in our laboratory has shown that amide modified farnesyl cysteine
analogs (AMFCs) are low-micromolar inhibitors of hIcmt.5,6 Other
We synthesized 41 SMFCs through a facile two-step synthetic
route from easily available starting materials as depicted in
Scheme 1. L-Cysteine was S-farnesylated with farnesyl chloride in
⇑
7 N ammonia/methanol.11 The resulting lipidated amino acid was
coupled with various commercially available sulfonyl chlorides
Corresponding authors.
E-mail address: rag@pharmacy.purdue.edu (R.A. Gibbs).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.078

J. D. Majmudar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2616–2620
2617
plasma
membrane
SH
Ras
Cys-aaX-OH
farnesyl transferase
CYTOSOL
S
-Cys-OMe
Ras
Ras
Xaa-Cys-
S
translocation to
plasma membrane
S
Ras
HO-Cys-
Icmt
S
Ras
MeO-Cys-
Fully post-translationally
modified KRas
ER membrane
Figure 1. Post-translational modifications of Ras and transport from the ER to the plasma membrane.
using aqueous sodium carbonate as the base and dioxane as
solvent.
Our preliminary goal in using this SMFC library was to determine
SH
OH
+
Cl
H2N
4
3
O
the structural requirements for hIcmt inhibition by prenylated cys-
teine derivatives containing the sulfonamide unit replacement.
Chemically diverse sulfonyl chlorides were chosen for the synthesis
of the SMFCs. Sulfonyl chlorides with different electronic character,
steric bulk and alkyl/aromatic scaffolds were incorporated. All syn-
thesized SMFCs were evaluated as substrates and inhibitors of
human Icmt using the vapor diffusion assay (VDA).12–14 The struc-
tures of the synthesized SMFCs are shown below in Figure 3.
The biochemical evaluation using the VDA revealed that no
SMFCs are effective substrates, and are instead moderate inhibitors
of hIcmt. The percent inhibition profiles of the SMFCs range from
a
S
70%
OH
H2N
5
O
b
S
O
22% to 60% hIcmt inhibition at 10
lM. Compounds 6a–q inhibited
O
R
30-76%
S
OH
human Icmt poorly, exhibiting less than 45% inhibition at 10
l
M.
N
H
6
All other analogs showed greater that 45% inhibition at the test
concentration and their percent inhibition values are listed in Fig-
ure 3. Compounds 6r–ab inhibited hIcmt between 45% and 55% at
O
Scheme 1. Reagents and conditions: (a) 7 N NH3/MeOH, 0 °C – rt, overnight; (b)
10% Na2CO3/dioxane (1:1), then RSO2Cl, rt, overnight.
10
10
l
M and compounds 6ac–ag inhibited hIcmt more than 55% at
lM.
IC50 determinations were carried out for a select group of com-
pounds 6r, 6s, 6ae, 6af and 6ag. Given below in Table 1 are the IC50
values for the selected compounds.
Our data establish that all SMFCs are relatively poor to moder-
ate inhibitors of hIcmt. Surprisingly, none of the SMFCs are sub-
strates for hIcmt (data not shown). We hypothesize that the
presence of the amide bond, or a carbonyl carbon may be necessary
for molecules to exhibit substrate activity. All SMFCs inhibit hIcmt,
but vary in the strength of inhibition. Highly bulky or rigid analogs
(6a, b and h) are poor hIcmt inhibitors, although 6ab and 6y are
S
S
O
O
O
O
OH
OH
N
N
H
H
O
O
2. POP-3MB-FC
1. POP-FC
Figure 2. Phenoxyphenyl-farnesyl cysteine (POP-FC) and phenoxy-phenyl-3-methylbutenyl-biphenyl farnesyl cysteine (POP-3MB-FC).6 The metabolically labile amide bond
is highlighted.

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J. D. Majmudar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2616–2620
S
O
O
S
OH
R
N
6
H
O
O
S
6
N
H
Br
a (22%)
b (26%)
c (28%)
NO2
d (34%)
e (35%)
f (37%)
g (37%)
h (37%)
F
i (38%)
F
F
F
F
H3CO
H3CO
CF3
Br
Cl
N
F
j (38%)
k (40%)
F
l (40%)
m (43%)
n (43%)
w (50%)
o (44%)
NO2
p (45%)
q (45%)
r (46%)
OCH3
N
Cl
F
H3CO
O
s (47%)
t (47%)
u (49%)
v (49%)
x (50%)
y (53%)
af (63%)
z (53%)
aa (54%)
H3CO
H3CO
O2N
S
F
O
O2N
ab (54%)
ac (56%)
ad (56%)
ae (59%)
ag (64%)
Figure 3. Structures of the sulfonamide-modified farnesyl cysteine (SMFC) analogs synthesized. The numbers in the parentheses indicate the percent inhibition by the SMFC
analog in the vapor diffusion assay at 10
l
M using 25
lM AFC as the substrate.
Table 1
Table 2
IC50 values of selected SMFCs
Percent inhibition values for prenyl modified SMFCs
Percent inhibitiona
a
Compound
IC50
(
lM)
Compound
6r
6y
6ag
6ae
6af
–
17.0 1.6
11.6 1.3
8.8 0.5
13.4 1.5
15.5 1.8
–
7a
7b
7c
8a
8b
–
22
18
23
17
32
–
a
a
IC50 values are mean of three experiments, were
determined using the VDA and calculated using GRAPH-
PAD PRISM 5.0. Concentration of the substrate used (AFC)
Percent hIcmt inhibition at 10 lM inhibitor con-
centration. AFC is used as a normalizing control and
specific activities are converted to percent inhibition.
was 25 lM.
O2N
R =
S
exceptions. We determined that compounds containing electron
withdrawing groups were generally better as hIcmt inhibitors as
compared to the ones with electron donating functionalities (6af,
ae, ac vs 6c, j, k, l); although there are exceptions (e.g., 6p and
6q). Planar bulk next to the sulfonamide bond also enhances inhi-
bition (6s, w and y).
O
O
R
F
S
OH
a
b
N
H
9
O
Figure 5. Structures of D-SMFCs synthesized.
We next wanted to evaluate the effect of the prenyl group in
SMFCs on hIcmt inhibition. To achieve this goal, we synthesized
three sulfonamide-modified geranyl cysteine (SMGC) analogs and
two analogs where the prenyl chain was replaced by an alkyl chain.
These analogs were synthesized in a similar manner to the SMFCs.
O2N
S
R =
O
O
R
7
F
S
OH
N
H
b
c
a
O
O
S
n
8a n = 4
8b n = 9
O
S
O2N
OH
8
N
H
O
Figure 4. Structures of prenyl-modified SMFCs.

J. D. Majmudar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2616–2620
2619
O
O
S Cl
S
SH
O
O
a
b
O
12
11
13
c
S
O
O
O
S
OH
N
H
O
14
Scheme 2. Reagents and conditions: (a) Li wire, DTTB, THF, À78 °C to room temp, 45%; (b) NCS (4 equiv), 2 M HCl/ACN (1:1), 20 °C, 92%; (c) farnesyl cysteine, 2N Na2CO3/
dioxane, 63%.
For the analogs that contain the alkyl chain, the base in the
S-alkylation step was changed to 2 M sodium hydroxide in ethanol
as described before (see Supplementary data for more detail).15,16
Although racemization at the alpha carbon is certainly a possibil-
ity, we did not investigate for this possibility. The structures of
these analogs are shown in Figure 4. These analogs were evaluated
as inhibitors and substrates of hIcmt using the VDA.
inhibitory activity although more analogs need to be evaluated to
corroborate this result.
Finally, we wanted to incorporate the phenoxyphenyl motif
(Fig. 2) that is a part of the most potent AMFCs into the SMFC scaf-
fold. As 2-phenoxy-phenyl sulfonyl chloride is not commercially
available, we synthesized it and then coupled it to farnesyl cys-
teine. Scheme 2 shows the synthesis of POP-SMFC that uses a
lithium mediated homolytic C–S bond cleavage in phenoxathin17
followed by oxidative chlorination18 to yield the sulfonylchloride
13 of interest.
The percent inhibition of compounds 7a–c and 8a–b are shown
in Table 2. None of these compounds exhibited substrate activity at
25
l
M.
Analogs 7a–c and 8a–b are relatively poor inhibitors of hIcmt as
Compound 14, the SMFC analog of POP-FC (1) inhibits hIcmt by
compared to their farnesyl analogs (compounds 6r, 6ae and 6af).
These data illustrate that the farnesyl group is highly important
for hIcmt inhibition by SMFCs. Increased chain length of the prenyl
group appears to be a key factor in hIcmt inhibition. Also, the fact
that analog 8a exhibits very similar inhibition to 7b illustrates that
the geranyl and the hexyl chains make little difference in the bio-
logical activities of these analogs. The fact that compound 8b only
showed marginal improvement in inhibition over 8a also strength-
ens our hypothesis that a longer prenyl chain is a key for hIcmt
inhibition. Overall, these data suggest that a determining factor
for hIcmt inhibition by SMFCs is the presence of the farnesyl chain,
imparting a specific binding interaction rather than simply hydro-
phobic bulk.
55% at 10 lM and has an IC50 of 18.4 1.8 lM as determined by
the VDA. Although most SMFCs are better, or equal inhibitors of
hIcmt as compared to AMFCs, POP-SMFC (14) is a poorer inhibitor
of hIcmt as compared to its amide counterpart.
Overall, we have shown that the sulfonamide bond is a viable
replacement for the amide bond in AFC derived hIcmt inhibitors.
We have also explored the structure–activity relationship of SMFCs
as hIcmt inhibitors. The most potent analog, 6ag has an IC50 of
8.8 0.5 lM. We have demonstrated that the farnesyl group of
SMFCs is a necessary structural motif for hIcmt inhibition and that
stereochemistry of the alpha carbon does not play a significant role
in inhibiting hIcmt. We have also highlighted the importance of the
carboxylate motif in SMFCs for hIcmt inhibition, but this result
needs further evaluation.
In comparison to the AMFCs, the SMFC library is superior for a
number of reasons. Notably, the lead SMFC 6ag exhibits superior
ligand efficiency19 to POP and POP-3-MB. While the AMFC library
yielded several substrates for hIcmt, the SMFC library showed no
substrate activity. Our lead, 6ag is a step in the right direction be-
cause it has a lower molecular weight, a lower C Log P, a higher
tPSA and overall a more ‘drug-like’ character. These encouraging
data are fueling our efforts in designing and evaluating novel scaf-
folds as amide isosteres for achieving potent hIcmt inhibition.
Having determined the role of the farnesyl group in hIcmt inhi-
bition by SMFCs, we next wanted to evaluate the effect of stereo-
chemistry at the alpha carbon of the SMFCs. To achieve this goal,
we synthesized the (S) enantiomers of compounds 6ae and 6af.
These were synthesized in a manner similar to the one shown in
Scheme 1, using
of these analogs are shown in Figure 5.
Interestingly, analogs 9a–b exhibited identical inhibition pro-
files at 10 M as compared to compounds 6ae and 6af. There
D-cysteine in place of L-cysteine. The structures
l
was little to no difference in the inhibition profiles of the
two enantiomers (data not shown). The enantiomers also did
not exhibit any substrate activity. The fact that stereochemistry
at the alpha carbon did not play a significant role in hIcmt
inhibition by SMFCs coupled with the knowledge that the far-
nesyl group appeared to be a key factor for inhibition leads
us to hypothesize that the farnesyl chain in either enantiomer
is able to adopt a favorable conformation and enables the SMFC
to inhibit hIcmt.
Next, we wanted to evaluate the importance of the carboxylate
motif of SMFCs for hIcmt inhibition. Toward this aim, we synthe-
sized the methyl ester analog (compound 10) of 6ag (details for
synthesis in Supplementary data). Strikingly, this analog exhibited
Acknowledgements
This work was supported by NIH/NCI Grant R01CA112483 (to
R.A.G.), and by NIH/NCI P30CA21328 (to the Purdue University
Center for Cancer Research).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.078.
only 10% hIcmt inhibition at 10 lM in the VDA. Although com-
References and notes
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a deviation from the carboxylate group greatly reduces hIcmt
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