Small-molecule inhibitors of protein geranylgeranyltransferase type I

Article abstract of DOI:10.1021/ja070274n

Small molecules that inhibit the geranylgeranylation of K-Ras4B and RhoA by protein geranylgeranyltransferase type I (GGTase-I) were identified from chemical genetic screens of heterocycles synthesized through phosphine catalysis of allenes. To further improve the efficacy of the GGTase-I inhibitors (GGTIs), 4288 related compounds bearing core dihydropyrrole/pyrrolidine and tetrahydropyridine/piperidine scaffolds were synthesized on SynPhase lanterns in a split-pool manner through phosphine-catalyzed [3 + 2] and [4 + 2] annulations of resin-bound allenoates. Testing of the 4288 analogues resulted in several GGTIs exhibiting submicromolar IC₅₀ values. Because proteins such as Ras and Rho GTPases are implicated in oncogenesis and metastasis, these GGTIs might ultimately lead to the development of novel antitumor therapeutics. Copyright

Full text of DOI:10.1021/ja070274n

Published on Web 04/17/2007
Small-Molecule Inhibitors of Protein Geranylgeranyltransferase Type I
Sabrina Castellano,† Hannah D. G. Fiji,† Sape S. Kinderman,† Masaru Watanabe,‡ Pablo de Leon,†
Fuyuhiko Tamanoi,‡ and Ohyun Kwon*,†
Department of Chemistry and Biochemistry, Department of Microbiology, Immunology and Molecular Genetics,
Jonsson ComprehensiVe Cancer Center and Molecular Biology Institute, UniVersity of California,
Los Angeles, California 90095-1569
Received January 13, 2007; E-mail: ohyun@chem.ucla.edu
Although library approaches to the discovery of small-molecule
enzyme inhibitors or receptor ligands are well-established,1 many
reactions continue to pose challenges when applied to solid-phase
synthesis for the generation of compound libraries. From our
Figure 1. Protein GGTase-I inhibitors 1 and 2.
development of phosphine catalysis of allenoates,2 we envisioned
that these reactions might be adaptable to solid-phase synthesis for
the generation of heterocycle libraries using resin-bound allenoates.
Before embarking on the potentially time-consuming development
of solid-phase processes, however, we decided to screen our model
compounds synthesized through solution-phase reactions. If we
could identify a biologically important molecule from the prelimi-
nary screen, it would then be worthwhile pursuing a library
generated through solid-phase split-pool synthesis. Herein, we report
the first example of phosphine catalysis of polymer-bound allenoates
and a combinatorial library approach to the development of potent
inhibitors of protein geranylgeranyltransferase type I (GGTase-I).
Protein prenylation, a post-translational modification of nascent
proteins by either the farnesyl or geranylgeranyl isoprenoid at the
C-terminus cysteine residue, is a key event in the regulation of
many protein functions.3 Of particular interest is the farnesylation
of the oncogenic forms of Ras proteins, which is required for their
membrane association and cell transforming activity.4 Constitutively
activated mutant Ras proteins are found in ca. 30% of human
tumors.5 Consequently, the development of FTase inhibitors (FTIs)
as anticancer agents has been the focus of much academic and
industrial research.6 Upon blocking FTase, however, the human
oncogenic Ras isoform K-RasB is geranylgeranylated by protein
GGTase-I.7 Geranylgeranylation functionally substitutes the farne-
sylation of Ras proteins. This phenomenon suggests that to
effectively block Ras processing, the development of selective
inhibitors of GGTase-I (GGTIs) is required just as importantly as
the development of FTIs.8
With this premise in mind, we screened a collection of 138
heterocycles9 for their ability to inhibit the activity of human
GGTase-I to geranylgeranylate K-Ras4B or RhoA. Purified
GGTase-I was incubated with its substrate protein K-Ras4B or
RhoA, [3H]GGPP, and the 138 compounds. After 30 min, the degree
of incorporation of tritiated geranylgeranyl groups was measured
using a scintillation counter. We identified a number of compounds
as GGTIs (Figure 1).
This discovery of promising lead GGTI compounds and their
moderate activity warranted the development of efficient and rapid
syntheses and evaluations of analogous structures in the search for
better inhibitors; we envisioned a short, modular synthetic route
(Scheme 1), using SynPhase lanterns as the solid support.10
Validation of the synthetic route on the polymer support commenced
with formation of resin-bound allenoates 5. The loading of allenoic
Scheme 1. Solid-Phase Syntheses of Dihydropyrroles 8 and
Tetrahydropyridines 9
acids onto solid supports has not been reported previously. The
allenoic acids 4 were coupled to the benzyl alcohol units of the
SynPhase-PS lanterns grafted with Wang resin 3 using Mukaiya-
ma’s reagent and Hu¨nig’s base for 4a/b or Et3N for 4c/d.11 The
direct use of an unmodified Wang resin minimizes the number of
synthetic operations run on solid support. In addition, our strategy
enabled simple trifluoroacetic acid (TFA)-mediated cleavage to
release the carboxylic acid group, a key functional group in our
GGTIs.
Because we were unaware of any previous examples of phos-
phine catalysis of solid-bound allenoates, we were pleased to
discover that the phosphine-catalyzed annulation between polymer-
supported allenoates 5 and N-tosylimines proceeded smoothly. The
allenoates 5a and 5b were treated with N-tosyltolualdimine and 50
mol % of PPh3 (for 5a) or PBu3 (for 5b) in benzene at 60 °C to
provide the polymer-bound dihydropyrroles 6.2b Tetrahydropyridines
7 were formed from the reactions of 5c and 5d with N-tosyl-
tolualdimine in the presence of 50 mol % of PBu3 at room
temperature for 2 and 4 days, respectively.2a Heterocycles 6 and 7
were cleaved from the resin using 2.5% TFA in DCM to provide
the carboxylic acids 8 and 9 in 91-94% yield (based on a
theoretical loading of 15 µmol/lantern) with high diastereoselec-
tivities (dr ) 99:1 for 8b; 93:7 for 9b) after chromatographic
purification.
The R,â-unsaturated enoate functionalities in 6 and 7 were
utilized to further increase the modularity and number of analogues.
For example, the Michael additions of thiols to 6 and 7 using
n-butyllithium as base12 provided 10 and 11, respectively, which
† Department of Chemistry and Biochemistry.
‡ Department of Microbiology, Immunology and Molecular Genetics.
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J. AM. CHEM. SOC. 2007, 129, 5843-5845
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C O M M U N I C A T I O N S
Scheme 2. Solid-Phase Diastereoselective Michael Additions
Chart 1. Eleven γ-Substituted Allenoic Acids A, 12 R-Substituted
Allenoic Acids B, 46 N-Sulfonimines C, and 32 Thiols D
Scheme 3. Building Block Preparation
upon TFA-mediated cleavage yielded 12 and 13, respectively, in
77-95% yield (Scheme 2). These two-step sequences occurred with
high diastereoselectivities, providing the pentasubstituted pyrrolidine
12 and the tetrasubstituted piperidine 13 as single diastereoisomeric
products.13 Apparently, thiols added opposite to the pre-existing
substituents in both the dihydropyrrole 6 and the tetrahydropyridine
7.15 Interestingly, however, the protonation of the resulting R-car-
banions occurred anti (for 10) and syn (for 11) to the added
â-mercapto groups.
Having successfully established the solid-phase reaction condi-
tions, we next prepared the R- and γ-substituted allenoic acid
building blocks (Scheme 3). The reactions between the phosphorane
14 and the acid chlorides 15 (1 equiv) in the presence of Et3N (1
equiv) provided the allenoates 16,16 which were hydrolyzed into
γ-substituted allenoic acids A.17 Phosphorane 14 was treated with
the alkyl halides 17 to give the phosphonium salts 18, which we
converted to the R-substituted allenoates 19 upon treatment with
Et3N (2 equiv) and acetyl chloride (1 equiv).18 R-Substituted allenoic
acids B were prepared through saponification of the esters 19. The
N-sulfonylimines C were formed simply through azeotropic removal
of water from a mixture of the appropriate sulfonamide 20, aldehyde
21, and BF3‚OEt2 under reflux in toluene.19 Chart 1 presents the
building blocks synthesized as illustrated in Scheme 3 and the
commercially available thiol building blocks D.
46) imine building blocks for allenoate A01, 21 for A05, 25 for
B01, and 31 for B05.
For Michael addition of the thiols, all three building blocks were
tested as follows: To select suitable allenoic acids, the 23 annulation
products synthesized above were subjected to Michael addition
using benzenethiol, toluenethiol, or benzyl thiol. Analysis of the
cleaved products indicated that eight (of 23) allenoic acids were
suitable candidates. For imine selection, the annulation products
of 46 imines with A05 and B01 were tested because the allenoic
acids A01-04 and B02-B12 were excluded from the Michael
addition sequence. The number of imines selected was 25 (of 46)
for A05 and 21 for B01. The annulation products 6 and 7 (Scheme
2) were used to select the thiols; the various thiols required different
reaction times and temperatures. Upon extensive optimization, 19
(of 32) thiols for dihydropyrrole 6 and 17 for tetrahydropyridine 7
were selected. The combination of these selected building blocks
resulted in the preparation of 4288 compounds.14
Using the chosen building blocks, we commenced the split-pool
syntheses of the 4288 GGTI analogues on the SynPhase lanterns.
Tagging was performed by inserting colored spindles and cogs into
the lanterns prior to synthesis of the library.14 Twenty-three allenoic
acids A and B were loaded onto the Wang resin 3 using
Mukaiyama’s reagent (Scheme 1). The resulting allenoate-loaded
lanterns 5 were pooled and split into a number of flasks corre-
sponding to the number of imines for each group of allenoic acids
The building blocks were tested so that only the ones that
provided high purity and stereoselectivity (as judged from 1H NMR
spectra and LCMS analyses) for their crude cleavage products
would be used in the synthesis of the GGTI analogue library.14
Eleven γ-substituted allenoic acids A and 12 R-substituted allenoic
acids B were loaded and reacted with imine C02 in the presence
of a catalytic amount of phosphine, as indicated in Scheme 1. After
cleavage with TFA and analysis (1H NMR and LCMS spectra) of
the purity and diastereoselectivity of the 23 annulation products 6
and 7, we found that each of the allenoic acids, except for A10,
1
yielded a single identifiable compound (dr g12:1; H NMR) in
high purity (72-100%; LCMS/UV210). The resin-bound allenoates
derived from allenoic acids A01, A05, B01, and B05 were selected
to assess the reactivity and stereoselectivity of 46 imines in
phosphine-catalyzed annulations (because A01, A02-A11, B01,
and B02-B12 required different annulation reaction conditions).
Using the criteria of >70% purity and >9:1 dr, we selected 30 (of
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C O M M U N I C A T I O N S
Acknowledgment. We are grateful to UCLA, Amgen, Pfizer,
UCLA-JCCC, the Center for Biological Modulators of the 21st
Century Frontier R&D Program (CBM-01-B-5) of the Korean
Ministry of Science and Technology (O.K.), NIH (CA32737), and
the Susan E. Riley Family Foundation (F.T.) for financial support.
S.C. and S.S.K. thank the Italian Government (MIUR) for a research
grant and the Nederlandse Organisatie voor Wetenschappelijk
Onderzoek for a TALENT fellowship, respectively. We thank Drs.
Matt Renner and Saeed Khan for performing the LCMS and the
X-ray crystallographic analyses, respectively. O.K. thanks Professor
Chulbom Lee for editorial assistance.
Figure 2. (a) GGTIs 22 and 23 that exhibited the highest potency. (b)
Western blot of the cell (treated with 22 or 23 at 25 µM concentration)
lysate qualitatively detecting the amount of geranylgeranylated Rheb. The
descriptors P and U designate the processed and unprocessed Rheb,
respectively.
Supporting Information Available: Representative experimental
1
procedures and spectral data for all new compounds (PDF). H NMR
(A01, A02-A11, B01, and B02-B12). Sets of 240 dihydropyrrole-
loaded lanterns 6 and 366 tetrahydropyridine-loaded lanterns 7 were
placed aside for cleavage. Sets of 3325 dihydropyrrole-bound
lanterns 6 and 357 tetrahydropyridine-bound lanterns 7 were further
divided into 19 and 17 flasks, respectively, and subjected to the
thiol Michael reactions (Scheme 2).
The 4288 lanterns were inserted into 4288 vials and treated with
2.5% TFA in CH2Cl2 for 12 h; the lanterns were then removed
and rinsed with CH2Cl2. The resulting solution was concentrated
and further co-evaporated with CHCl3 to effectively remove TFA.
The cleaved compounds were weighed and redissolved in CHCl3;
a portion (2 µmol) of each compound was transferred into 54 96-
well plates (80 compounds per well; two columns of wells in each
plate were left empty to accommodate controls in subsequent
assays), and the solvents were left to evaporate. The products were
redissolved in DMSO and analyzed in the same assay for activity
against GGTase-I.
In the in vitro assay, active compounds were sought for their
ability to inhibit the geranylgeranylation of both RhoA and
K-Ras4B. Figure 2a displays the two compounds (22 and 23) that
exhibited the highest activities obtained so far. These compounds
exhibit specific inhibition of GGTase-I; that is, they did not inhibit
FTase at concentrations at which they inhibited GGTase-I by more
than 90%.14
spectroscopic and LCMS data for 251 library members (PDF).
Crystallographic data for compounds 12 and 13a (CIF). This material
is available free of charge via the Internet at http://pubs.acs.org.
References
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CSVL construct that expresses the geranylgeranylated form of the
Rheb protein.20 Inhibition of the geranylgeranylation of this protein
can be detected from a shift in its mobility on SDS polyacrylamide
gel; the unprocessed form appears as a slowly migrating band.
Figure 2b indicates that treatment of the cells with 22 or 23 resulted
in the appearance of a slowly migrating band (cf. the DMSO lane
with lanes 22 and 23); known GGTIs (GGTI298, GGTI2166) were
used as controls. These results suggest that compounds 22 and 23
inhibit geranylgeranylation within the cell. These dihydropyrrole
and tetrahydropyridine-based GGTIs differ from the previously
reported GGTIs, which, with the exception of Casey’s GGTI-DU40,
have been peptidomimetic compounds.8
In conclusion, small-molecule inhibitors of GGTase-I were
identified through chemical genetic screens of the heterocycles
produced through allene phosphine catalysis. This discovery
instigated the development of the first solid-phase phosphine
catalysis of resin-bound allenoates. To further improve the efficacy
of the GGTIs and to explore their structure-activity relationships,
4288 GGTI analogues were synthesized on SynPhase lanterns in a
split-pool fashion. Screening the 4288 analogues resulted in the
identification of GGTIs 22 and 23 having submicromolar IC50
values. These powerful GGTIs should be useful for studies of the
protein geranylgeranylation process and might ultimately lead to
novel therapeutic leads.
(9) The structures of 138 compounds are provided in the Supporting
Information. For the synthesis of the compounds, see ref 2.
(10) The SynPhase lantern consists of a mobile surface polymer (e.g.,
polystyrene) grafted onto a rigid and unreactive base polymer of cylindrical
shape. The rigid polymeric support beneath the grafted mobile phase makes
weighing unnecessary and handling easier than that of resins. The
SynPhase lanterns are available in three different sizes, with loadings of
15, 35, and 75 µmol, providing more material for a given compound than
beads when used in a split-pool library synthesis. Nonchemical tagging
methods, such as radiolabeling (similar to the IRORI system) or color-
coding using colored spindles and cogs, are available, alleviating the need
for chemical encoding.
(11) Mukaiyama, T.; Usui, M.; Shimada, E.; Saigo, K. Chem. Lett. 1975, 1045.
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(13) The relative stereochemistry of 12 and 13a was confirmed through X-ray
crystallographic analyses.14
(14) See the Supporting Information for details.
(15) Dihydropyrroles 6 with C5-H, C5-Me, and C5-Ph substituents provided
mixtures of diastereoisomers. On the other hand, dihydropyrroles with
C5-t-Bu substituents were recalcitrant to the 1,4-addition conditions. For
the tetrahydropyridines 7, only those lacking a C2 substituent underwent
the 1,4-addition of the thiol. Remarkably, the remote C6 substituent
directed the Michael addition from the opposite side.
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1990; Collect. Vol. 7, p 232.
(17) The saponification of allenoates 16 provided mixtures of allenoic and
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allenoates.14
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