Stable analogues of geranylgeranyl diphosphate possessing improved geranylgeranyl versus farnesyl protein transferase inhibitory selectivity

Article abstract of DOI:10.1016/j.bmcl.2003.09.035

Phosphonoacetamido(oxy) groups have proven to be good mimics of the diphosphate portion in geranylgeranyl protein transferase I (GGTase I) inhibitors. The introduction of small alkyl groups (Me, Et) into the diphosphate mimic moiety caused a further decrease in collateral farnesyl protein transferase (FTase) inhibitory activity, thereby improving GGTase I over FTase selectivity.

Full text of DOI:10.1016/j.bmcl.2003.09.035

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4405–4408
Stable Analogues of Geranylgeranyl Diphosphate Possessing
Improved Geranylgeranyl versus Farnesyl Protein Transferase
Inhibitory Selectivity
Filippo Minutolo,a Simone Bertini,a Laura Betti,b Romano Danesi,c
Gianbattista Gervasi,d Gino Giannaccini,b Chiara Papi,a Giorgio Placanica,a
Silvia Barontini,a Simona Rapposellia and Marco Macchiaa,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
b
`
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, via Bonanno 6, 56126 Pisa, Italy
cDipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Divisione di Farmacologia e Chemioterapia,
`
Universita di Pisa, Via Roma 55, 56126 Pisa, Italy
dLaboratori Baldacci SpA, Via S. Michele degli Scalzi 73, 56124 Pisa, Italy
Received 28May 2003; revised 30 July 2003; accepted 12 September 2003
Abstract—Phosphonoacetamido(oxy) groups have proven to be good mimics of the diphosphate portion in geranylgeranyl protein
transferase I (GGTase I) inhibitors. The introduction of small alkyl groups (Me, Et) into the diphosphate mimic moiety caused a
further decrease in collateral farnesyl protein transferase (FTase) inhibitory activity, thereby improving GGTase I over FTase
selectivity.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
seemed to be exclusively activated by farnesylation.
Several strategies have been followed: (a) designing
compounds that mimic the carboxy-terminal CAAX
motif of Ras proteins; (b) using molecules that compete
with the farnesyl diphosphate (FPP analogues); (c)
making drugs that act both as FPP and CAAX mimics.3
Several compounds have proven to efficiently inhibit
farnesylation of Ras proteins in vitro, showing some
reversal of tumor mass when tested in animal models,
accompanied by an impressively low toxicity. Unfortu-
nately, the same success was not achieved in early
attempts to treat human patients affected by cancer. In
fact, it was later found that in malignant cells, some
mutant forms of Ras (K- and N-Ras) can be ger-
anylgeranylated by GGTase I when FTase is blocked,
circumventing the antiproliferative effect of FTase inhi-
bitors.4 Therefore, new GGTase I inhibitors have been
synthesized5 and some of them have shown a potent
anticancer activity.6
Signal transduction of extracellular stimuli activating
cell proliferation is brought about by small intracellular
G-proteins belonging to the Ras superfamily, which
become activated by anchorage to intracellular and
plasma membranes via a posttranslationally added
lipophilic isoprenyl group, such as farnesyl (Ras pro-
teins) and geranylgeranyl (Rho, Rac, and Cdc42 pro-
teins).1 The enzymes involved in this activation step are
farnesyl transferase (FTase) and geranylgeranyl trans-
ferase (GGTase), respectively. Aberrant signalling
through Ras pathways occurs in several types of cancer,
where mutated Ras accumulates in its GTP-bound
active form and causes uncontrolled cell proliferation.2
For these reasons, molecules able to target the Ras
pathway in any of its stages are potentially useful in
anti-cancer therapies.1 Over the past few years, a great
deal of attention has been devoted to FTase inhibitors,
since Ras proteins involved in cell hyper-proliferation
Recently, GGTase I inhibitors have displayed other
interesting therapeutic properties.7 They have proven to
be effective in reducing geranylgeranylation of small G-
proteins in osteoclast, thus reducing bone resorption
*Corresponding author. Tel.: +39-050-500209; fax: +39-050-40517;
e-mail: mmacchia@farm.unipi.it
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.035

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F. Minutolo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4405–4408
and showing a potential for treatment of osteopor-
osis.7b,c Besides, they have also shown an inhibitory
effect on unwanted smooth muscle cell proliferation
accompanying reocclusion of arteries occurring in
atherosclerosis.7d All these aspects make the search for
new and selective GGTase inhibitors an on-going chal-
lenge for medicinal chemists.
hydrolysis: treatment with bromotrimethylsilane and
2,4,6-collidine, and then aqueous potassium hydroxide,
gave compounds 1a–c as the dipotassium salts.9
Compounds 2a–c were synthesized as shown in Scheme
2. Geranylgeranyl bromide 8 was used to alkylate
potassium phthalimide, to give N-(geranylger-
anyl)phthalimide 9. Removal of the phthalimido group
by hydrazinolysis yielded geranylgeranylamine 10,
which was then condensed with the appropriate phos-
phonoacetic acid derivative (6a–c) under the same con-
ditions described above. Final hydrolysis of the diethyl
phosphonate derivatives 11a–c10 afforded the target
compounds 2a–c as the dipotassium salts.11
Our research has been focused on the design of stable
analogues of the diphosphate moiety in GPP, attached
to a geranylgeranyl isoprenoid portion. We had pre-
viously found that the (phosphonoacetamido)oxy
group, as in 1a, was one of the most effective for this
purpose.5h We then performed a more detailed structure/
activity relationship investigation within this class of
molecules, and we herein report the development of new
selective GGTase I inhibitors (1b,c and 2a–c), structu-
rally related to 1a.
Biological Results and Discussion
The in vitro inhibition assays of GGTase I and FTase
were performed as previously described12 with some
modifications.13 The inhibitory activity of compounds
1a–c and 2a–c is reported in Table 1 as their IC50, the
concentration at which GGTase and FTase activity was
inhibited by 50%.
An analysis of the results shows that, in the ‘(phosphono-
acetamido)oxy’ series (1a–c), the introduction of small
Compounds 1b and 1c derive from 1a by replacement of
one of the methylene hydrogen atoms of the
(phosphonoacetamido)oxy group by a methyl or an
ethyl group, respectively. These substitutions should
make it possible to verify whether the increase in steric
hindrance within this portion of the molecule might
increase enzyme selectivity without affecting the inhibi-
tion potency. Moreover, the related series 2a–c, where
the (phosphonoacetamido)oxy groups of 1a–c have been
replaced by phosphonoacetamido ones, then lacking an
oxygen atom in the phosphate mimic portion, should
also prove to be an important means for the optimization
of the polar moiety in this class of molecules.
Scheme 1. Reagents and conditions: (a) N-hydroxyphthalimide, Ph3P,
.
DEAD, THF, rt; (b) NH2NH2 H2O, EtOH, rt; (c) 1-Hydro-
xybenzotriazole, EDC, THF, rt; (d) (1) TMS-Br, 2,4,6-collidine,
CH2Cl2, rt; (2) 1 N aqueous KOH.
Table 1. Inhibitory activity of compounds 1a–c and 2a–c on ger-
anylgeranyl protein transferase I (GGTase I) and farnesyl protein
transferase (FTase)
Chemistry
Compd
IC50 (nM)a
Selectivityb
The synthetic route for the preparation of com-
pounds 1a–c (Scheme 1) started from geranylgeraniol
3, which was subjected to a Mitsunobu reaction with
N-hydroxyphthalimide to give N-(geranylgeranyloxy)-
phthalimide 4. Hydrazinolysis of the phthalimido por-
tion afforded free oxyamine 5, which was then subjected
to a condensation reaction, promoted by 1-[3-(dimethyla-
mino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
and 1-hydroxybenzotriazole, with the appropriate
phosphonoacetic acid derivative (6a–c). The diethyl
phosphonates thus obtained (7a–c)8 were submitted to
GGTase I
FTase
1a
1b
1c
2a
2b
2c
66Æ83500
151Æ30
146Æ20
275Æ40
111Æ15
566Æ75
Æ700
53
>66
>68
18
>90
>18
>10,000
>10,000
5000Æ1000
>10,000
>10,000
aValues are reported as the meanÆrange or SD of 2–3 independent
experiments.
bFold (GGTase over FTase).

F. Minutolo et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4405–4408
4407
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Biol. Chem. 1997, 272, 14459.
5. (a) Some examples are given in: Tucker, T. J.; Abrams,
M. T.; Buser, C. A.; Davide, J. P.; Ellis-Hutchings, M.; Fer-
nandes, C.; Gibbs, J. B.; Graham, S. L.; Hartman, G. D.;
Huber, H. E.; Liu, D.; Lobell, R. B.; Lumma, W. C.; Robin-
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Marson, C. M.; Rioja, A. S.; Brooke, G.; Coombes, R. C.;
Vigushin, D. M. Bioorg. Med. Chem. Lett. 2002, 12, 255. (d)
Zahn, T. J.; Whitney, J.; Weinbaum, C.; Gibbs, R. A. Bioorg.
Med. Chem. Lett. 2001, 11, 1605. (e) Bergman, J. M.; Abrams,
M. T.; Davide, J. P.; Greenberg, I. B.; Robinson, R. G.; Buser,
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Graham, S. L.; Hartman, G. D.; Williams, T. M.; Dinsmore,
C. J. Bioorg. Med. Chem. Lett. 2001, 11, 1411. (f) Gibbs, B. S.;
Zahn, T. J.; Mu, Y. Q.; Sebolt-Leopold, J. S.; Gibbs, R. A. J.
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M.; Jannitti, N.; Gervasi, G.; Danesi, R. J. Med. Chem. 1996,
39, 1352.
Scheme 2. Reagents and conditions: (a) potassium phthalimide,
DMF, rt; (b–d) see Scheme 1.
alkyl groups, such as a methyl (1b) or an ethyl (1c), pre-
serves a good inhibitory potency on GGTase when com-
pared with the reference compound 1a, whereas the
activity on FTase is completely lost, thus affording better
selectivity ratios for both 1b and 1c, with respect to 1a.
6. (a) Sun, J.; Blaskovich, M. A.; Knowles, D.; Qian, Y.;
Ohkanada, J.; Bailey, R. D.; Hamilton, A. D.; Sebti, S. M.
Cancer Res. 1999, 59, 4919. (b) Lobell, R. B.; Omer, C. A.;
Abrams, M. T.; Bhimnathwala, H. G.; Brucker, M. J.; Buser,
C. A.; Davide, J. P.; deSolms, S. J.; Dinsmore, C. J.; Ellis-
Hutchings, M. S.; Kral, A. M.; Liu, D.; Lumma, W. C.;
Machotka, S. V.; Rands, E.; Williams, T. M.; Graham, S. L.;
Hartman, G. D.; Oliff, A. I.; Heimbrook, D. C.; Kohl, N. E.
Cancer Res. 2001, 61, 8758. (c) Di Paolo, A.; Danesi, R.;
Caputo, S.; Macchia, M.; Lastella, M.; Boggi, U.; Mosca, F.;
Marchetti, A.; Del Tacca, M. Br. J. Cancer 2001, 84, 1535.
7. (a) Gibbs, R. A. Curr. Opin. Drug Discov. Dev. 2000, 3, 585.
(b) Coxon, F. P.; Helfrich, M. H.; Larijani, B.; Muzylak, M.;
Dunford, J. E.; Marshall, D.; McKinnon, A. D.; Nesbitt, S.
A.; Horton, M. A.; Seabra, M. C.; Ebetino, F. H.; Rogers, M.
J. J. Biol. Chem. 2001, 276, 48213. (c) Coxon, F. P.; Helfrich,
M. H.; Van’t Hof, R.; Sebti, S.; Ralston, S. H.; Hamilton, A.;
Rogers, M. J. J. Bone Miner. Res. 2000, 15, 1467. (d) Cohen,
L. H.; Pieterman, E.; van Leeuwen, R. E. W.; Overhand, M.;
Burm, B. E. A.; van der Marel, G. A.; van Boom, J. H. Bio-
chem. Pharmacol. 2000, 60, 1061.
Removal of one oxygen atom from the polar moiety, as
in the ‘phosphonoacetamido’ series (2a–c), did not cause
any dramatic changes in the biological properties of
these compounds. As a matter of fact, the smaller
homologue 2a shows decreased inhibitory properties on
both enzymes with respect to its oxygenated counter-
part. This decrease is more pronounced on GGTase,
resulting in relatively low selectivity. As seen before,
also in this series the introduction of a methyl group
such as in 2b causes a dramatic decline in the inhibition
of FTase (IC50>10,000 nM) accompanied by a good
inhibition potency on GGTase (IC50=111 nM), pro-
viding 2b with a selectivity ratio higher than 90. In this
series, the introduction of a more hindered ethyl group,
as in 2c, causes significant reduction in the inhibition
ability on GGTase (IC50=566 nM), and preserves
complete inactivity on FTase.
These results show that a slight increase in the steric
hindrance in the diphosphate mimic portion of the
reference compound 1a improves GGTase selectivity. In
fact, the introduction of a methyl (1b) or an ethyl (1c)
substituent led to selectivity ratios of about 70. Among
the ‘phosphonoacetamido’ series, the most active and
selective compound proved to be the methyl-substituted
2b, with a remarkable selectivity ratio of more than 90. To
the best of our knowledge, compounds 1b, 1c and 2b are
among the most active and selective GGTase I inhibitors
possessing a GGPP-like chemical structure, and might
therefore represent a good starting-point in the design of
new selective GGTase inhibitors which may potentially be
developed as drugs in the therapy of cancer and of other
pathologies related to uncontrolled cell proliferation.
1
8. For example, compound 7b: H NMR (CDCl3) d 1.38(m,
9H), 1.59 (s, 9H), 1.67 (s, 3H), 1.70 (s, 3H), 2.02 (m, 12H), 2.56
(m, 1H), 4.04 (q, 2H, J=7.2 Hz), 4.06 (q, 2H, J=7.2 Hz), 4.41
(d, 2H, J=7.2 Hz), 5.09 (br, 3H), 5.41 (t, 1H, J=7.2 Hz); MS
(FAB+) m/e 498(M+H) +
.
9. For example, compound 1b: 1H NMR (CD3OD) d 1.35
(dd, 3H, J=15.2, 7.2 Hz), 1.60 (s, 9H), 1.67 (s, 3H), 1.72 (s,
3H), 2.04 (m, 12H), 2.51 (dq, 1H, J=20.0, 7.2 Hz), 4.40 (d,
2H, J=7.2 Hz), 5.10 (br, 3H), 5.41 (t, 1H, J=7.2 Hz); MS
(FAB+) m/e 518(M+H) +
.
10. For example, compound 11b: 1H NMR (CDCl3) d 1.37
(m, 9H), 1.59 (s, 9H), 1.68(s, 6H), 2.01 (m, 12H), 2.78(m,
1H), 3.85 (d, 2H, J=7.2 Hz), 4.03 (q, 2H, J=7.2 Hz), 4.05 (q,
2H, J=7.2 Hz), 5.12 (br, 3H), 5.23 (t, 1H, J=7.2 Hz); MS
(FAB+) m/e 482 (M+H)+.
11. For example, compound 2b: 1H NMR (CD3OD) d 1.35
(dd, 3H, J=15.2, 7.2 Hz), 1.59 (s, 9H), 1.66 (s, 6H), 2.01 (m,
12H), 2.54 (dq, 1H, J=20.0, 7.2 Hz), 3.79 (d, 2H, J=7.2 Hz),
5.13 (br, 3H), 5.23 (t, 1H, J=7.2 Hz); MS (FAB+) m/e 502
(M+H)+.
References and Notes
1. (a) Downward, J. Nat. Rev. Cancer 2002, 3, 11, and refer-
ences therein. (b) Symons, M. TIBS 1996, 21, 178.
12. (a) Zhang, F. L.; Moomaw, J. F.; Casey, P. J. J. Biol.
Chem. 1994, 269, 23465. (b) Quian, Y.; Blaskovich, M. A.;

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13. Enzymes were incubated with [3H]GGPP and recombi-
nant H-Ras-CVLL (GGTase I), or with [3H]FPP and H-Ras-
CVLS (FTase), in the presence of different concentrations of
inhibitors. After incubation the reaction was stopped and fil-
tered through glass fibre filters to separate free from incorpo-
rated label.