Lipid and sulfur substituted prenylcysteine analogs as human Icmt inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.06.053

Inhibition of isoprenylcysteine carboxyl methyltransferase (Icmt) offers a promising strategy for K-Ras driven cancers. We describe the synthesis and inhibitory activity of substrate-based analogs derived from several novel scaffolds. Modifications of bot

Full text of DOI:10.1016/j.bmcl.2011.06.053

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5616–5619
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lipid and sulfur substituted prenylcysteine analogs as human Icmt inhibitors
Joel A. Bergman a, Kalub Hahne b, Christine A. Hrycyna b, Richard A. Gibbs a,
⇑
a Department of Medicinal Chemistry and Molecular Pharmacology and Purdue University Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA
b Department of Chemistry and 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:
Inhibition of isoprenylcysteine carboxyl methyltransferase (Icmt) offers a promising strategy for K-Ras
driven cancers. We describe the synthesis and inhibitory activity of substrate-based analogs derived from
several novel scaffolds. Modifications of both the prenyl group and thioether of N-acetyl-S-farnesyl-L-cys-
teine (AFC), a substrate for human Icmt (hIcmt), have resulted in low micromolar inhibitors of Icmt and
have given insights into the nature of the prenyl binding site of hIcmt.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 12 April 2011
Revised 10 June 2011
Accepted 13 June 2011
Available online 21 June 2011
Keywords:
Isoprenylcysteine
carboxylmethyltransferase
Amino acid analogs
Enzyme inhibitors
Isoprenoids
Click chemistry
Proteins possessing a -CaaX motif at their C-terminus undergo
three sequential post-translational modifications that are neces-
sary for their proper cellular localization and function.1 It is
thought that more than 120 human proteins utilize this three-step
modification process, including the small GTPases.2 The first step
in the sequential processing pathway involves attachment of a
Preliminary work on hIcmt inhibitors focused on modifications
to N-acetyl-S-farnesyl-
-cysteine (AFC),14 a minimal substrate for
L
human Icmt (hIcmt) that contains a peptide bond. Amide-modified
farnesyl cysteine analogs (AMFCs)15,16 were synthesized and stud-
ied in our laboratories to investigate tolerances and specific
requirements upstream of the prenylcysteine. The culmination of
this work resulted in low micromolar inhibitors of hIcmt (A and
B; Fig. 2) that are based on a prenylcysteine scaffold. In contrast,
our initial studies to investigate substitutions in the isoprene re-
gion led to poor hIcmt inhibitors.17
Other synthetic and screening approaches have also resulted in
inhibitors of hIcmt (Fig. 2). A small-molecule library screening ef-
fort led to cysmethynil, an indole based inhibitor.18 A small mole-
cule prenylcysteine analog, S-farnesyl-thiosalicylic acid (FTS), is
both an hIcmt inhibitor as well as an inhibitor of H-Ras driven cell
growth.19
C15-farnesyl or C20-geranylgeranyl isoprenoid to the free thiol of
the cysteine in the -CaaX motif. This prenylation event is catalyzed
by either farnesyl transferase (FTase) or geranylgeranyl transfer-
ase-I (GGTase-I), respectively.3 The next step involves the removal
of the -aaX residue by the endoprotease Ras-converting enzyme-1
(Rce1).4,5 The newly exposed carboxylate of the prenylcysteine is
then methylated by human isoprenylcysteine carboxyl methyl-
transferase (hIcmt; Fig. 1).6 The summation of these steps gener-
ates increased hydrophobicity that is necessary for association of
the protein to the target membrane and its function.7,8
This processing pathway has been investigated for potential
cancer therapeutics because approximately 20% of human cancers,
including 90% of pancreatic cancers, result from mutated Ras pro-
teins.9 Potent FTase inhibitors have been discovered, but unfortu-
nately are ineffective against K-Ras driven tumors10 due to
alternative prenylation by GGTase-I, which allows the Ras protein
to properly localize.11 However, hIcmt is the only known enzyme
capable of methylating the prenylcysteine substrate12 and is thus
an intriguing putative therapeutic target.13
Despite some initial success, there is still much room for
improvement of the characteristics of substrate-based (AFC) inhib-
itors of hIcmt. Current successful inhibitors of this nature have
thus far included the less than ideal characteristics existing in
AFC: the isoprenoid tail, the allylic thioether, and the highly lipidic
disposition. Herein, we report the investigation into lipid and thio-
ether replacements leading to structurally novel AFC-based inhib-
itors of hIcmt. This study has been approached in two parts: (1)
C15-lipid replacement with diverse aryl motifs and (2) thioether
replacement with 1,2,3-triazoles.
Previously, our group demonstrated that a biphenyl group
replacing the second and third isoprene positions was accepted
as a substrate for FTase.20 It was also shown that the third isoprene
⇑
Corresponding author.
E-mail addresses: rag@pharmacy.purdue.edu, rgibbs@purdue.edu (R.A. Gibbs).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.06.053

J. A. Bergman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5616–5619
5617
Table 1
n
a,c
Compd
Percent Inhibition of specific activitya,b
IC50 , lM
Plasma Membrane
S
AFC
1
2
3
4
100 1.2
20.7 3.3
23.1 1.5
18.8 1.7
31.1 1.4
9.8 1.7
Cys-OMe
Transport
to PM
Ras
34.6 2.4
SAM
AdoHcy
5
S
6
7
8
9
10
11
12
16a
16b
16c
16d
16e
15.5 3.3
14.5 1.8
19.4 4.8
22.4 2.1
15.7 1.4
26.6 8.1
25.3 1.5
19.5 3.1
35.9 2.4
17.0 2.5
10.1 3.6
22.7 2.9
S
n
n
Cys-OH
Cys-OMe
Ras
Ras
ER Membrane
19.4 1.0
Figure 1. Methylation of Ras by hIcmt is required for proper localization to the
plasma membrane and downstream signaling.
a
Values represent mean of three experiments.
b
Values determined at 10
lM analog and 25
l
M AFC by VDA assay previously
described38
.
c
Calculated by GraphPad Prizm 5.0.
Figure 2. Current inhibitors of hIcmt developed from amide- and prenyl-modified
AFC.
Figure 4. Depiction of ether and extended ether compounds.
closely mimics the C20 geranylgeranyl-cysteine, the product from
prenylation by GGTase-I, which is an equivalent substrate for car-
boxyl methylation.27 Analog 4 is the most potent of this series
(IC50 = 34 lM).
To further remove isoprene character from the prenyl region of
AFC, a second class of AFC analogs was prepared with an aryl ether
group. Both the central and terminal isoprene units were replaced
with aromatic moieties (5–7, Fig. 4; see Supplementary data for
synthesis). Selenium dioxide catalyzed allylic oxidation28 of prenyl
alcohol followed by bromination resulted in the first isoprene syn-
thon necessary for ether synthesis.
None of these compounds were substrates for hIcmt, nor were
they efficient inhibitors (Table 1). Despite differences in flexibility
and length, these three compounds possessed similar inhibitory
potency. The trend of 1–4 prompted us to introduce flexibility
and increased lipid resemblance by introducing a methylene
bridge between the first isoprene and ether (8–9). This modifica-
tion resulted in an approximate 2-fold increase in enzyme inhibi-
tion, (Table 1) but did not surpass that of 4. These compounds
were also poor Icmt substrates. From this limited set of compounds
we learned that specific spatial organization and positioning of aryl
substituted isoprene groups may be crucial for their ability to bind
to the hIcmt active site.
Figure 3. Depiction of aryl–alkyl analogs.
position is flexible in accepting aryl substitutions, as modified ani-
linogeranyl analogs are high-affinity substrates for FTase.21 Early
evidence from the design of squalene synthase inhibitors also dem-
onstrated the ability for isoprenoid binding pockets to tolerate aryl
ligands.22 More recently, Boger and co-workers inserted aryl motifs
in lieu of unsaturated fatty acid chains in the development of fatty
acid amide hydroxylase inhibitors.23
Using these studies as a guide, our first series of compounds in-
cluded an aryl-alkyl tail that was designed to investigate tolerances
of a prenyl binding pocket for hIcmt (Fig. 3, see Supplementarydata
for synthesis). Two key reactions were utilized in the synthesis of
this class of molecules. A Kumada coupling24 installed the required
alkyl chain to the aryl core representing a modified tail region. This
reaction was followed by a zirconium-assisted carboalumination of
the terminal alkyne25,26 to establish the required first isoprene
group in a stereocontrolled manner.
This series of compounds (Fig. 3) explored the general ability of
an aromatic residue to mimic the central isoprene unit of the far-
nesyl chain. Evaluation of 1–4 revealed an interesting trend: inhib-
itory activity increased with elongation of terminal alkyl-chain,
with the exception of 3 (Table 1). The replacement of the central
isoprene in conjunction with n-alkyl group tails led to modest inhi-
bition of hIcmt despite a deviation from an isoprene-based struc-
ture. The overall length of 1 is closest to that of AFC, and 4 more
The generation of the vinyl iodide first-isoprene synthon al-
lowed us to generate a third set of analogs (C, Fig. 5a). Substituting
the flexible farnesyl group with highly rigid and bulky replace-
ments was thus investigated (10–12, Fig. 5b, see Supplementary
Content data for synthesis). A Suzuki coupling was utilized to in-
stall the phenyl ring in the first isoprene.29 To add the tail of the
isoprene mimetic, a copper-mediated allylic –OTHP displace-

5618
J. A. Bergman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5616–5619
Figure 5. (A) New vinyl first isoprene synthon;(B) Compounds containing highly
rigid and bulky prenyl groups.
ment30 with Grignard reagents gave the desired redesigned
isoprene.
The more effective analogs contained aryl groups that could
possibly extend deeper into a prenyl binding site (11–12). Despite
the lack of the flexibility found in the other compounds, these mol-
ecules exhibited similar inhibition of hIcmt (Table 1). These results
highlight the need to further investigate structural motifs that can
mimic the necessary isoprenoid characteristics for Icmt binding.
Our previous work demonstrated inhibitory potency of hIcmt by li-
pid-modified prenylcysteines containing a 4-biphenyl group in the
isoprene moiety.16,17 Our future work will aim to elucidate other
specific motifs for hIcmt inhibition.
In previous reports by the Rando laboratory, the heteroatom
requirements for prenylcysteine recognition by AFC were investi-
gated. They found that a nucleophilic sulfur was crucial for efficient
carboxylate methylation. Sulfoxide-, seleno-, amine-, and ether-,
prenylcysteines maintained the ability to bind to the enzyme;
however, deviation from sulfur decreased enzymatic activity.31
Our groups furthered this effort through the synthesis of an ‘all-
carbon’ prenylcysteine and found that complete removal of the thi-
oether ablated hIcmt substrate and inhibitor activity.32
Despite the apparent need for a linking heteroatom, preferably
sulfur, between the peptide and farnesyl group of the substrate, it
would be beneficial to find a suitable replacement that would not
be turned over as substrate but would still bind to and thus inhibit
hIcmt. The presence of the allylic thioether is also undesirable as
they are chemically and enzymatically labile.33,34
We introduced a heteroaryl motif that could act as a surrogate
for the thioether. Utilizing solid-phase chemistry previously devel-
oped in our laboratory16 and copper-assisted cycloaddition,35 1,4-
substituted 1,2,3-triazole prenyl-‘cysteines’ were generated. Main-
taining a heteroatom proximal to the natural position of sulfur in
AFC was paramount in our design, based on the work of Rando
and co-workers.31
A solid-phase synthetic route was used for the synthesis of 16a–
e, which began by loading orthogonally protected diamine onto 2-
chlorotrityl resin generating starting material 13 (Scheme 1, de-
tailed procedure in Supplementary data). Cleavage of the alloc
group followed by diazo transfer with trifyl azide generated
in situ36 afforded azidocysteine analog 14. Cycloaddition37 with
15 followed by standard Fmoc peptide coupling afforded dually
modified derivatives 16a–e. Following cleavage from the resin,
HPLC (C8 column) was used to purify these dually modified com-
pounds. This solid phase approach allowed us to investigate the ef-
fects of prenylcysteines substituted at two important locations: the
thioether and the amide. We have previously reported on the SAR
of amide modified prenylcysteine (AMFC) compounds15,16 and in
this study we selected amide modifications that previously re-
sulted in substrates and inhibitors of hIcmt to highlight the effect
of the triazole-for-sulfur substitution.
Scheme 1. Solid phase synthesis of triazole-for-sulfur substituted analogs of
AMFCs on 2-chlorotrityl resin. Reagents and conditions: (a) PhSiH3, Pd(PPh3)4,
DCM; (b) (i) NaN3, Tf2O, Ph-H; (ii) ZnCl2, Et3N, MeOH; (c) 15, CuI, Ascorbic Acid,
DIEA, DMF; (d) (i) HOBT, HBTU, DIEA, DCM/DMF; (ii) 0.5% TFA/DCM.
results were somewhat surprising as the parental AMFC analogs
corresponding to 16d,e were found to be excellent substrates, sur-
passing that of AFC (unpublished data).15,16 Evaluation of these five
compounds revealed that all are inhibitors of the enzyme. The
most potent analog is 16b (IC50 = 19.4
lM, Table 1); note that the
parent AMFC has an IC50 = 4.6
l
M.16 These data serve as an impor-
tant comparison, as the transition from thioether to triazole leads
to a decrease in potency, but reduces undesirable instability and
decreases lipophilicity. Despite the replacement of the thioether
with a heterocycle, important structural features of known hIcmt
inhibitors can still affect biological function. This result represents
an important step forward in the rational design of non-cysteine
based inhibitors of hIcmt.
We have synthesized 12 prenylcysteine analogs bearing aro-
matic isoprenoid replacements and five analogs inserting a triazole
replacement for sulfur. The latter series resulted in low micromolar
inhibitors of hIcmt with unique and potentially beneficial physical
characteristics, including increased rigidity and stability, and de-
creased hydrophobicity. Spatial organization and positioning of
the lipid replacements must be balanced with lipidic character
for the design of hIcmt inhibitors. In summary, we have generated
interesting results from the introduction of the triazole-for-sulfur
substitution leading to a new class of AFC-based hIcmt inhibitors.
Acknowledgements
This work was support by the NIH grants R01 CA112483 (RAG)
and P30 CA21328 (Purdue University Center for Cancer Research).
Supplementary data
When evaluated as substrates of hIcmt, none of the triazole-for-
sulfur compounds exhibited any activity (data not shown). These
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.06.053.

J. A. Bergman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5616–5619
5619
17. Anderson, J. L.; Henriksen, B. S.; Gibbs, R. A.; Hrycyna, C. A. J. Biol. Chem. 2005,
280, 29454.
References and notes
18. Winter-Vann, A. M.; Baron, R.; Wong, W.; de la Cruz, J.; York, J. D.; Gooden, D.
M.; Bergo, M.; Young, S. G.; Toone, E. J.; Casey, P. J. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 4336.
19. Marciano, D.; Ben-Baruch, G.; Marom, M.; Egozi, Y.; Haklai, R.; Kloog, Y. J. Med.
Chem. 1995, 38, 1267.
20. Zhou, C.; Shao, Y.; Gibbs, R. A. Bioorg. Med. Chem. Lett. 2002, 12, 1417.
21. Subramanian, T.; Liu, S.; Troutman, J. M.; Andres, D. A.; Spielmann, H. P.
ChemBioChem 2008, 9, 2872.
22. Biller, S. A.; Abt, J. W.; Pudzianowski, A. T.; Rich, L. C.; Slusarchyk, D. A.; Ciosek,
C. P., Jr. Bioorg. Med. Chem. Lett. 1993, 3, 595.
1. Gelb, M. H.; Brunsveld, L.; Hrycyna, C. A.; Michaelis, S.; Tamanoi, F.; Van Vooris,
W. C.; Waldmann, H. Nature Chem. Biol. 2006, 2, 518.
2. Reid, T. S.; Terry, K. L.; Casey, P. J.; Beese, L. S. J. Mol. Biol. 2004, 343, 417.
3. Lane, K. T.; Beese, L. S. J. Lipid Res. 2006, 47, 681.
4. Bergo, M. O.; Ambroziak, P.; Gregory, C.; George, A.; Otto, J. C.; Kim, E.; Nagase,
H.; Casey, P. J.; Balmain, A.; Young, S. G. Mol. Cell. Biol. 2002, 22, 171.
5. Dolence, J. M.; Steward, L. E.; Dolence, E. K.; Wong, D. H.; Poulter, C. D.
Biochemistry 2000, 39, 4096.
6. Anderson, J. L.; Frase, H.; Michaelis, S.; Hrycyna, C. A. J. Biol. Chem. 2005, 280,
7336.
23. Hardouin, C.; Kelso, M. J.; Romero, F. A.; Rayl, T. J.; Leung, D.; Hwang, I.; Cravatt,
B. F.; Boger, D. L. J. Med Chem 2007, 50, 3359.
24. Hayashi, T.; Konishi, M.; Kobori, M.; Kumada, M.; Higuchi, T.; Hirotsu, K. J. Am.
Chem. Soc. 1984, 106, 158.
7. Michaelson, D.; Ali, W.; Chiu, V. K.; Bergo, M.; Silletti, J.; Wright, L.; Young, S. G.;
Philips, M. Mol. Cell. Biol. 2005, 16, 1606.
8. Michaelson, D.; Silletti, J.; Murphy, G.; D’Eustachio, P.; Rush, M.; Philips, M. J.
Cell Biol. 2001, 152, 111.
25. Rand, C. L.; Horn, D. E. V.; Moore, M. W.; Negishi, E. J. Org. Chem. 1981, 46, 4093.
26. Negishi, E.; Horn, D. E. V.; Yoshida, T. J. Am. Chem. Soc. 1985, 107, 6639.
27. Volker, C.; Lane, P.; Kwee, C.; Johnson, M.; Stock, J. FEBS Lett. 1991, 295, 189.
28. Umbreit, M. A.; Sharpless, K. B. J. Am. Chem. Soc. 1977, 99, 5526.
29. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
30. Mechelke, M. F.; Wiemer, D. F. J. Org. Chem. 1999, 64, 4821.
31. Tan, E. W.; Perez-Sala, D.; Rando, R. R. J. Am. Chem. Soc. 1991, 113, 6299.
32. Henriksen, B. S.; Anderson, J. L.; Hrycyna, C. A.; Gibbs, R. A. Bioorg. Med. Chem.
Lett. 2005, 15, 5080.
33. Gordon, E. M.; Pluscec, J. J. Am. Chem. Soc. 1992, 114, 1521.
34. Park, S. B.; Howald, W. N.; Cashman, J. R. Chem Res Toxicol 1994.
35. Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
36. Angelo, N. G.; Arora, P. S. J. Org. Chem. 2007, 72, 7963.
37. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 33, 45.
9. Downward, J. Nature Rev. Cancer 2002, 3, 11.
10. Kohl, N. E.; Omer, C. A.; Conner, M. W.; Anthony, N. J.; Davide, J. P.; DeSolms, S.
J.; Giuliani, E. A.; Gomez, R. P.; Graham, S. L.; Hamilton, K.; Handt, L. K.;
Hartman, G. D.; Koblan, K. S.; Kral, A. M.; Miller, P. J.; Mosser, S. D.; O’Neill, T. J.;
Rands, E.; Schaber, M. D.; Gibbs, J. B.; Oliff, A. Nature Med. 1995, 1, 792.
11. Whyte, D. B.; Kirschmeier, P.; Hockenberry, T. N.; Nunez-Oliva, I.; James, L.;
Catino, J. J.; Bishop, W. R.; Pai, J.-K. J. Biol. Chem. 1997, 272, 14459.
12. Bergo, M. O.; Leung, G. K.; Ambroziak, P.; Otto, J. C.; Casey, P. J.; Young, S. G. J.
Biol. Chem. 2000, 275, 17605.
13. Gelb, M. H.; Brunsveld, L.; Hrycyna, C. A.; Michaelis, S.; Tamanoi, F.; Van
Voorhis, W. C.; Waldmann, H. Nat. Chem. Biol. 2006, 2, 518.
14. Ma, Y. T.; Gilbert, B. A.; Rando, R. R. Methods Enzymol. 1995, 250, 226.
15. Donelson, J. L.; Hodges, H. B.; MacDougall, D. D.; Henriksen, B. S.; Hrycyna, C.
A.; Gibbs, R. A. Bioorg. Med. Chem. Lett. 2006, 16, 4420.
16. Donelson, J. L.; Hodges, H. B.; Henriksen, B. S.; Hrycyna, C. A.; Gibbs, R. A. J. Org.
Chem. 2009, 74, 2975.
38. Hrycyna, C. A.; Wait, S. J.; Backlund, P. S.; Michaelis, S. Methods Enzymol. 1995,
250, 251.