Triazole-based inhibitors of geranylgeranyltransferase II

Article abstract of DOI:10.1016/j.bmcl.2012.11.089

A small set of triazole bisphosphonates has been prepared and tested for the ability to inhibit geranylgeranyltransferase II (GGTase II). The compounds were prepared through use of click chemistry to assemble a central triazole that links a polar head group to a hydrophobic tail. The resulting compounds were tested for their ability to inhibit GGTase II in an in vitro enzyme assay and also were tested for cytotoxic activity in an MTT assay with the human myeloma RPMI-8226 cell line. The most potent enzyme inhibitor was the triazole with a geranylgeranyl tail, which suggests that inhibitors that can access the enzyme region that holds the isoprenoid tail will display greater activity.

Full text of DOI:10.1016/j.bmcl.2012.11.089

Bioorganic & Medicinal Chemistry Letters 23 (2013) 764–766
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Triazole-based inhibitors of geranylgeranyltransferase II
Xiang Zhou a, Sara V. Hartman b, Ella J. Born b, Jacqueline P. Smits a, Sarah A. Holstein b,
David F. Wiemer a,c,
⇑
a Department of Chemistry, University of Iowa, Iowa City, IA 52242-1294, USA
b Department of Internal Medicine, University of Iowa, Iowa City, IA 52242-1294, USA
c Department of Pharmacology, University of Iowa, Iowa City, IA 52242-1294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small set of triazole bisphosphonates has been prepared and tested for the ability to inhibit geranylger-
anyltransferase II (GGTase II). The compounds were prepared through use of click chemistry to assemble
a central triazole that links a polar head group to a hydrophobic tail. The resulting compounds were
tested for their ability to inhibit GGTase II in an in vitro enzyme assay and also were tested for cytotoxic
activity in an MTT assay with the human myeloma RPMI-8226 cell line. The most potent enzyme inhib-
itor was the triazole with a geranylgeranyl tail, which suggests that inhibitors that can access the enzyme
region that holds the isoprenoid tail will display greater activity.
Received 10 October 2012
Revised 15 November 2012
Accepted 20 November 2012
Available online 1 December 2012
Keywords:
GGTase II
Rab GGTase
Apoptosis
Ó 2012 Elsevier Ltd. All rights reserved.
Anti-proliferative
Bioassay
Protein prenylation is a post-translational modification which
involves attachment of hydrophobic isoprenoid chains through a
reaction that is mediated by the enzymes farnesyltransferase
(FTase), geranylgeranyltransferase I (GGTase I), or geranylgeranyl-
transferase II (GGTase II).1 The latter enzyme is also known as Rab
GGTase because it mediates modification of the Rab family of small
GTPases.2 Rab proteins play critical roles in all aspects of intracellu-
lar membrane trafficking.3 Studies involving mutated versions of
Rabs or knock-out mice have demonstrated defects in protein secre-
tion,4 which may make the Rab proteins particularly attractive ther-
apeutic targets in diseases characterized by an overabundance of
secreted proteins such as multiple myeloma. Proper function of
Rab proteins depends upon correct membrane localization which
is achieved through geranylgeranylation. Mutant forms of Rabs that
are unable to be geranylgeranylated are improperly localized and
essentially nonfunctional.5 We already have demonstrated that
agents which disrupt Rab geranylgeranylation result in the inhibi-
tion of monoclonal protein trafficking in myeloma cells.6 This repre-
sents an intriguing therapeutic strategy by which to induce cellular
stress and apoptosis in cells heavily engaged in protein secretion.
At least two lines of attack may achieve diminished cellular lev-
els of Rab geranylgeranylation. The less direct approach would be to
deplete cells of the isoprenoid substrate geranylgeranyl diphos-
phate. This might be achieved by inhibition of any of the individual
steps in the isoprenoid biosynthetic pathway that lead to geranyl-
geranyl diphosphate (GGDP), from inhibition of HMGCoA reductase
by a statin7 (the first committed step in isoprenoid biosynthesis8) to
inhibition of GGPP synthase9 (the final enzyme in the assembly of
this C20 isoprenoid10). This strategy is less specific as it affects other
cellular processes requiring the isoprenoid intermediates, including
other prenyltransferases. The more direct strategy involves inhibi-
tion of GGTase II itself. While inhibitors of GGTase II are rare,11
a
few have been reported. The carboxy phosphonate 1, an analogue
of the bisphosphonate risedronate (2) known as 3-PEHPC (Fig. 1),
is one of the more readily accessible inhibitors.12 This compound
displays good selectivity for GGTase II although millimolar concen-
trations are required to observe cellular effects and the IC50 first re-
ported for the recombinant enzyme was ꢀ600
l
M.12 More recently
(+)-(S)-3-IPEHPC (3), a carboxy phosphonate analogue of minodro-
nate (4), has been shown to be more potent than 3-PEHPC, to be a
mixed-type inhibitor with respect to GGPP, and to be an uncompet-
itive inhibitor with respect to Rab.13 Efforts that have involved ac-
tual screening of a compound library that contained a significant
number of natural products14 and a virtual high-throughput
screening of a tetrahydrobenzodiazepine core15 also have led to
compounds with activity as inhibitors of GGTase II. Our own ap-
proach is based on a longstanding interest in the design of terpe-
noid analogues as inhibitors of the enzymes of isoprenoid
biosynthesis.16 In this Letter, we describe efforts to assemble a
small set of potential inhibitors of GGTase II based on a triazole
template with varied hydrophobic tails that might engage an iso-
prenoid binding region of the protein, as well as the results of bio-
assays on these new compounds.
⇑
Corresponding author.
E-mail address: david-wiemer@uiowa.edu (D.F. Wiemer).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.089

X. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 764–766
765
OH
C(O)(OH)
OH
P(O)(OH)2
HO
HO
N
C(O)(OH)
N
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
N
N
N
N
3 ( (+)-(S)-3-IPEHPC)
Figure 1. Two carboxy phosphonates that inhibit GGTase II and their parent bisphosphonates.
1 (3-PEHPC)
2 (risedronate)
4 (minodronate)
that this mixture was the result of olefin isomerization at the stage
of the intermediate azide, and not reflective of the regiochemistry
of the cycloaddition, the isomeric neryl bromide was converted to
the azide and carried through the same reaction sequence. The
resulting product was identical to that obtained from the geranyl
series, which confirms formation of E- and Z-olefin isomers
through this reaction sequence. Whether geranyl bromide or neryl
bromide was employed as the starting material, the isomer ratio
was approximately 2:1 in favor of the E-isomer, as judged by inte-
gration of the 1H NMR resonances for the methyl or vinyl hydro-
gens of the first isoprenoid unit.
H
H
N
P(O)(OR')2
P(O)(OR')2
P(O)(OR')2
P(O)(OR')2
N
N
N
+
H
N
N
H
5
R
R
6
7
Figure 2. Retrosynthesis of a family of triazole bisphosphonates.
In compounds such as 3-PEHPC and (+)-(S)-3-IPEHPC, the polar
head group appears to represent the diphosphate moiety of gera-
nylgeranyl diphosphate while the nitrogen atom may complex to
the zinc ion known to be near the active site of GGTase II. Our syn-
thetic plan was directed at compounds of the general structure 5
(Fig. 2), which might maintain features needed to engage these
sites within the enzyme but also carry a hydrophobic tail that
would mimic the isoprenoid portion of GGPP. Assembly of such
structures is readily approached through cycloaddition of an azide
and an acetylene, a process commonly referred to as click chemis-
try.17 The most convenient preparation of the desired compounds
would require an acetylene bearing the polar head group and a
set of azides with alkyl groups of different size and shape
(Fig. 2). To simplify determination of the impact of various alkyl
chains, and avoid issues of absolute stereochemistry at this time,
a bisphosphonate head group was employed as the diphosphate
mimic (Fig. 3).
In a synthetic sense, this effort began with preparation of the
known acetylenic bisphosphonate 9 from commercial tetraethyl
methylene bisphosphonate (8), formaldehyde, and acetylene
according to the two-step sequence described by Roschenthaler.18
The first cycloadduct then was obtained from reaction of acetylene
9 with benzyl azide, generated from benzyl bromide and sodium
azide, and gave the known triazole 10a18 in good yield. Standard
hydrolysis under McKenna conditions19 gave the new bisphos-
phonic acid 11a in modest yield. A parallel series of reactions with
naphthyl bromide was employed to prepare the naphthyl analogue
10b, and cleavage of the ester groups proceeded smoothly under
standard conditions to afford compound 11b.
Given the encouraging results obtained from the reactions of
benzyl and naphthyl azide, one might assume that isoprenoid
halides can be converted to triazoles just as readily, but allylic sys-
tems are more complicated than the benzylic ones. Allylic azides
are known to undergo a facile [3,3]-sigmatropic rearrangement.20
More recent studies on prenyl azide show that the primary azide
is both more reactive in the [3 + 2] cycloaddition and favored in
the equilibrium between the primary and tertiary azides.21 Unfor-
tunately this equilibrium can result in partial isomerization of a
trans olefin to a cis olefin21 which can complicate preparation of
triazoles from higher isoprenoid azides.22
To pursue preparation of the first isoprenoid triazole in this ser-
ies, geranyl bromide was obtained from geraniol through reaction
with PBr3, converted to the azide, and the resulting azide was al-
lowed to react with acetylene 9 under standard conditions to
obtain the triazole 10c. The spectral data supported formation of
the desired triazole system, but the 13C NMR spectrum of the prod-
uct suggested that a mixture of isomers was obtained. To confirm
The corresponding farnesyl triazole 10d was prepared from
commercial farnesol through a parallel series of reactions involving
sequential formation of the bromide, the azide, and a final cycload-
dition. Once again, the resulting triazole was found to be a mixture
of olefin isomers. Preparation of the geranylgeranyl analog 10e
required synthesis of geranylgeraniol from reaction of farnesyl ace-
tone with triethyl phosphonoacetate, and reduction of the result-
ing ester with LiAlH4.23 This strategy is known to favor formation
of the 2E isomer by approximately 10:1, which is not problematic
for this application given the expected isomerization after prepara-
tion of the azide. Therefore, geranylgeraniol prepared this way was
converted to the bromide, the azide, and finally to the triazole 10e
under standard conditions. In all three of these isoprenoid cases
(compounds 10c–10e), the high polarity of the bisphosphonate
head group made separation of the olefin isomers very difficult.
Therefore, the mixtures were carried on to the corresponding phos-
phonic acid salts 11c–11e through straightforward cleavage of the
ester groups, and then used directly for screening purposes.
The five new triazole bisphosphonic acids were tested for their
ability to inhibit GGTase II using radiolabeled GGPP, recombinant
enzyme, Rab substrate, and REP.24 As shown in Table 1, the length
of the alkyl chain significantly affected inhibitory activity against
GGTase II. The most active compound was the triazole bisphosph-
onate 11e, which bears a geranylgeranyl chain. The geranyl length
compound 11c displayed no activity as a GGTase II inhibitor, while
the farnesyl length 11d displayed modest activity. All five com-
pounds also were tested for their ability to induce cytotoxicity in
the human myeloma RPMI-8226 cell line following a 48 h incuba-
tion.25 Interestingly, there was not a uniform correlation between
the observed cytotoxicity and the GGTase II inhibitory activity.
The napthyl derivative 11b, was the most potent in the MTT assay.
The mechanism of action for this compound’s cytotoxic effect is
unknown at this time. Cellular assays demonstrated that this com-
pound does not affect protein farnesylation or geranylgeranylation
(data not shown).
In conclusion, click chemistry has been used to prepare a set of
five new triazole bisphosphonic acids, and these compounds have
been tested for their ability to inhibit GGTase II and to induce cyto-
toxicity in human myeloma cells. Of these five compounds, com-
pound 11e clearly is the most potent inhibitor of this enzyme.
This suggests that a long, lipophilic tail can enhance the potency
of potential GGTase II inhibitors, presumably through interaction
with the enzyme site that holds the tail of the natural substrate
geranylgeranyl diphosphate. The potency of this new compound
is not yet at the level required for clinical utility. However, now

766
X. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 764–766
1) RBr, NaN3
2) t-BuOH/H2O,
H
CuSO4 (cat)
sodium ascorbate
H
1) H2CO, base
2) NaCCH
P(O)(OC2H5)2
P(O)(OC2H5)2
P(O)(OC2H5)2
N
P(O)(OR')2
N
H
P(O)(OC2H5)2
P(O)(OR')2
N
50%
R
8
9
10a-e R = C2H5
11a-e R = Na
TMSBr then
NaOH
10c: 79%; 11c: 75%
10a: 60%; 11a: 54%
10b: 58%; 11b: 48%
10d: 67%; 11d: 76%
10e: 58%; 11e: 59%
Figure 3. Synthesis of the target triazole bisphosphonates.
4. Fukuda, M. Cell. Mol. Life Sci. 2008, 65, 2801.
Table 1
5. Gomes, A. Q.; Ali, B. R.; Ramalho, J. S.; Godfrey, R. F.; Barral, D. C.; Hume, A. N.;
Seabra, M. C. Mol. Biol. Cell 1882, 2003, 14.
6. Holstein, S. A.; Hohl, R. J. Leuk. Res. 2011, 35, 551.
Biological activity of triazoles 11a–e
Compound
GGTase II IC50
MTT IC50
7. Holstein, S. A. The isoprenoid biosynthetic pathway and statins. In The Enzymes;
Hrycyna, C. A., Bergo, M. O., Tamanoi, F., Eds.; Elsevier, 2011; pp 279–299.
8. Wiemer, A. J.; Hohl, R. J.; Wiemer, D. F. Anticancer Agents Med. Chem. 2009, 9,
526.
9. (a) Shull, L. W.; Wiemer, A. J.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 2006,
14, 4130; (b) Wiemer, A. J.; Yu, J. S.; Lamb, K. M.; Hohl, R. J.; Wiemer, D. F.
Bioorg. Med. Chem. 2008, 16, 390; (c) Chen, C. K.; Hudock, M. P.; Zhang, Y.; Guo,
R. T.; Cao, R.; No, J. H.; Liang, P. H.; Ko, T. P.; Chang, T. H.; Chang, S. C.; Song, Y.;
Axelson, J.; Kumar, A.; Wang, A. H.; Oldfield, E. J. Med. Chem. 2008, 51, 5594.
10. Wiemer, A. J.; Wiemer, D. F.; Hohl, R. J. Clin. Pharm. Ther. 2011, 90, 805.
11. El Oualid, F.; Cohen, L. H.; Van der Marel1, G. A.; Overhand, M. Curr. Med. Chem.
2006, 13, 2385.
12. (a) Coxon, F. P.; Helfrich, M. H.; Larijani, B.; Muzylak, M.; Dunford, J. E.;
Marshall, D.; McKinnon, A. D.; Nesbitt, J. E.; Horton, M. A.; Seabra, M. C.;
Ebetino, F. H.; Rogers, M. J. J. Biol. Chem. 2001, 276, 48213; (b) Coxon, F. P.;
Ebetino, F. H.; Mules, E. H.; Seabra, M. C.; McKenna, C. E.; Rogers, M. J. Bone
2005, 37, 349.
13. Baron, R. A.; Tavare, R.; Figueiredo, A. C.; Blazewska, K. M.; Kashemirov, B. A.;
McKenna, C. A.; Ebetino, F. H.; Taylor, A.; Rogers, M. J.; Coxon, F. P.; Seabra, M.
C. J. Biol. Chem. 2009, 284, 6861.
11a
11b
11c
11d
11e
2 mM
>1 mM
0.2 mM
>1 mM
0.9 mM
1 mM
>2 mM
>2 mM
0.65 mM
0.10 mM
that the importance of this hydrophobic group is apparent, it is
quite likely that modification of the polar head group and/or heter-
ocyclic system will afford even more potent inhibitors of the en-
zyme. Furthermore, given the promising activity observed with
these triazoles, synthesis of additional examples where olefin
isomerization is precluded through use of a homoallylic azide26
or through replacement of the first prenyl unit with an aromatic
analogue27 becomes a priority. Studies along these lines are under-
way and will be reported in due course.
14. Deraeve, C.; Guo, Z.; Bon, R. S.; Blankenfeldt, W.; DiLucrezia, R.; Wolf, A.;
Menninger, S.; Stigter, E. A.; Wetzel, S.; Choidas, A.; Alexandrov, K.; Waldmann,
H.; Goody, R. S.; Wu, Y. W. J. Am. Chem. Soc. 2012, 134, 7384.
15. Bon, R. S.; Guo, Z.; Stigter, E. A.; Wetzel, S.; Menninger, S.; Wolf, A.; Choidas, A.;
Alexandrov, K.; Blankenfeldt, W.; Goody, R. S.; Waldmann, H. Angew. Chem., Int.
Ed. 2011, 50, 4957.
16. (a) Holstein, S. A.; Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorg.
Med. Chem. 1998, 6, 687; (b) Kim, M. K.; Kleckley, T. S.; Wiemer, A. J.; Holstein,
S. A.; Hohl, R. J.; Wiemer, D. F. J. Org. Chem. 2004, 69, 8186; (c) Maalouf, M. A.;
Wiemer, A. J.; Kuder, C. H.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 1959,
2007, 15; (d) Wiemer, A. J.; Yu, J. S.; Shull, L. W.; Barney, R. J.; Wasko, B. M.;
Lamb, K. M.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med. Chem. 2008, 16, 3652.
17. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2004, 2001, 40;
(b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 1128.
18. Skarpos, H.; Osipov, S. N.; Vorob’eva, D. V.; Odinets, I. L.; Lork, E.;
Roschenthaler, G.-V. Org. Biomol. Chem. 2007, 5, 2361.
Acknowledgments
Financial support from the Roy J. Carver Charitable Trust (DFW),
the Institute for Clinical and Translational Science —Looking into
Clinical Connections Faculty Development Program at the Univer-
sity of Iowa (DFW), the PhRMA Foundation (SAH), and the Univer-
sity of Iowa Holden Comprehensive Cancer CSET seed Grant
program (SAH) is gratefully acknowledged.
Supplementary data
19. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C. Tetrahedron Lett.
1977, 18, 155.
20. Padwa, A.; Sa, M. M. Tetrahedron Lett. 1997, 38, 5087.
21. Feldman, A. K.; Colasson, B.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005,
127, 13444.
22. Praud-Tabaries, A.; Dombrowsky, L.; Bottzek, O.; Briand, J. F.; Blache, Y.
Tetrahedron Lett. 2009, 50, 1645.
23. Yu, J. S.; Kleckley, T. S.; Wiemer, D. F. Org. Lett. 2005, 7, 4803.
24. Seabra, M. C.; James, G. L. Methods Mol. Biol. 1998, 84, 251.
25. Holstein, S. A.; Hohl, R. J. Leuk. Res. 2001, 25, 651.
Supplementary data (representative experimental procedures,
NMR spectra, and bioassay protocols) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.11.089.
References and notes
1. Schafer, W. R.; Rine, J. L. Annu. Rev. Genet. 1992, 26, 209.
26. Bergman, J. A.; Hahne, K.; Song, J.; Hrycyna, C. A.; Gibbs, R. A. Med. Chem. Lett.
2012, 3, 15.
2. Seabra, M. C.; Goldstein, J. L.; Sudhof, T. C.; Brown, M. S. J. Biol. Chem. 1992, 267,
14497.
27. Barney, R. J.; Wasko, B. M.; Dudakovic, A.; Hohl, R. J.; Wiemer, D. F. Bioorg. Med.
Chem. 2010, 18, 7212.
3. Schultz, J.; Doerks, T.; Ponting, C. P.; Copley, R. R.; Bork, P. Nat. Genet. 2000, 25,
201.