Synthesis of aniline-type analogues of farnesyl diphosphate and their biological assays for prenyl protein transferase inhibitory activity

Article abstract of DOI:10.1016/j.farmac.2003.08.002

Stable analogues of farnesyl diphosphate, possessing an aniline-type portion in the prenyl-mimic moiety and phosphonoacetamido(oxy) groups in the place of the metabolically unstable diphosphate unit, were synthesised and submitted to biological assays. Th

Full text of DOI:10.1016/j.farmac.2003.08.002

Il Farmaco 58 (2003) 1277Á1281
/
www.elsevier.com/locate/farmac
Synthesis of aniline-type analogues of farnesyl diphosphate and their
biological assays for prenyl protein transferase inhibitory activity
Filippo Minutolo a, Simone Bertini a, Laura Betti b, Valeria Di Bussolo c,
Gino Giannaccini b, Giorgio Placanica a, Simona Rapposelli a, H. Peter Spielmann d,
Marco Macchia a,*
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
c Dipartimento di Chimica Bioorganica e Biofarmacia, Universita` di Pisa, Via Bonanno 33, 56126 Pisa, Italy
d Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536-0084, USA
Received 22 June 2003; accepted 4 August 2003
Abstract
Stable analogues of farnesyl diphosphate, possessing an aniline-type portion in the prenyl-mimic moiety and phosphonoaceta-
mido(oxy) groups in the place of the metabolically unstable diphosphate unit, were synthesised and submitted to biological assays.
The enzyme inhibition tests performed on FTase and GGTase I show that the newly synthesised compounds based on a
combination of the aniline-containing portions with (phosphonoacetamido)oxy groups do not afford potent inhibitors.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Farnesyldiphosphate; FTase; Aniline; Hexafluoroaniline; Phosphonoacetamido(oxy)
1. Introduction
therapy of molecules able to block the prenylation of
Ras proteins, by means of the inhibition of the key
enzymes responsible for this process (FTase, GGTaseI)
[3,4]. So far the most widely studied target enzyme has
undoubtedly been FTase, for which inhibitors have been
designed as: (i) carboxy-terminal (CAAX-motif) mimics
of Ras proteins; (ii) FdP analogues; and (iii) ‘‘dual
mimics’’ possessing molecular portions similar to both
the CAAX-motif and the FdP structure [5].
Small G-proteins belonging to the Ras superfamily
are actively involved in signal transduction processes.
These proteins are initially activated by an
isoprenylation which makes them more lipophilic and,
therefore, capable of anchoring to the inner part of
cellular membrane, where the remaining activation
of signal transduction takes place [1]. The isoprenylation
Recently, new farnesyl side chain mimics were identi-
fied in an aniline-type moiety [6]. In fact compound 1
(Fig. 1) proved to be an inhibitor of FTase with an IC50
value (0.50 mM) comparable to the natural substrate
FdP. Moreover, 1 proved to be inactive on GGTase I
process is carried out mostly by
protein transferase (FTase)
a
which
farnesyl-
uses
farnesyldiphosphate (FdP) as the isoprenylating por-
tion, or by a geranylgeranyl-protein transferase (GGTa-
seI) which uses geranylgeranyldiphosphate (GGdP). The
observation that inhibitors of the enzyme hydroxy-
methylglutaryl-CoA reductase (designed as a choles-
terol-lowering agent that reduces the production of
isoprenylating intermediates), also reduced cell prolif-
eration [2], suggested a possible application in cancer
(IC50ꢀ20 mM), thus showing a good FTase over
/
GGTase selectivity [6]. Another efficient farnesyl mimic
group was found in a pentafluoroaniline-type portion,
as shown in compound 2 (Fig. 1), which proved to be
effectively recognized by FTase [7].
The presence of a diphosphate unit in compounds 1
and 2 makes them metabolically unstable and, therefore,
hardly suitable for future development. As a matter of
fact, di-phosphate derivatives are quickly hydrolysed by
* Correspondence and reprints.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.08.002

1278
F. Minutolo et al. / Il Farmaco 58 (2003) 1277Á1281
/
phosphoesterase enzymes, before they can exert their
pharmacological action. We therefore focused our
attention on modifying the polar portion of compounds
of type 1 with diphosphate mimics, stable to phosphoes-
terases, which could afford ‘‘drug-like’’ molecules. We
considered the (phosphonoacetamido)oxy portion, al-
ready successful in a previously developed FTase
inhibitor (FTP II, Fig. 1) [8], and its ethyl-substituted
analog, as appropriate candidates.
In an attempt to generate more potent and selective
FTase inhibitors, we designed and synthesised com-
pounds 3a,b and 4a,b (Fig. 1) deriving from a combina-
tion of the isoprenyl-mimic portion of 1 and 2 and the
polar portion, or its ethyl-substituted analog, of com-
pound FTP II.
to a Mitsunobu reaction with N-hydroxyphthalimide to
give phthalimide derivatives 7 and 8. Hydrazinolysis of
the phthalimido portion afforded free oxyamines 9 and
10, respectively. A subsequent condensation reaction,
promoted by 1-[3-(dimethylamino)propyl]-3-ethylcarbo-
diimide hydrochloride (EDC) and 1-hydroxybenzotria-
zole, with diethylphosphonoacetic acid (11a) afforded
diethylphosphonates 12a and 13a, which were
submitted to an ester hydrolysis reaction involving
an initial treatment with bromotrimethylsilane and
2,4,6-collidine, followed by aqueous potassium hydrox-
ide, to give compounds 3a and 4a as the dipotassium
salts.
Condensation of oxyamines
2-(diethylphosphono)butyric acid
9
and 10 with
(11b) [9],
under the same conditions described above,
afforded the ethyl-substituted diethylphoshonates
12b and 13b. Final ester hydrolysis of 12b
and 13b, using the same procedure described
for 12a and 13a, afforded 3b and 4b as the dipotassium
salts.
2. Chemistry
Compounds 3a,b and 4a,b were synthesized as shown
in Scheme 1. Allylic alcohols 5 [6] or 6 [7] were submitted
Fig. 1. Structural derivation of FPP analogues 3a,b and 4a,b.

F. Minutolo et al. / Il Farmaco 58 (2003) 1277Á
/
1281
1279
Scheme 1. aReagents and conditions: (a) N-Hydroxyphthalimide, Ph3P, DEAD, THF, r.t. (b) NH2NH2×
triazole, EDC, THF, r.t. (d) 1) TMS-Br, 2,4,6-collidine, CH2Cl2, r.t; 2) 1N aqueous KOH.
/H2O, EtOH, r.t. (c) 1-Hydroxybenzo-
An analysis of the results shows that none of the new
compounds is endowed with any remarkable inhibition
potencies on either of the two enzymes tested. The only
compound showing a certain inhibition of FTase
Table 1
Inhibitory activity of compounds 3a,b and 4a,b on farnesyl protein
transferase (FTase) and geranylgeranyl protein transferase I (GGTase
I), together with a reference FTase inhibitor FTP II and aniline
derivative 1
(IC50ꢀ3.5 mM) was aniline-type derivative 3b, which
/
Comp.
IC50 (mM) a
FTase
possesses an ethyl substituent on its phosphonoaceta-
mido(oxy) portion. This level of inhibition, albeit
modest, is accompanied by a total absence of GGTaseI
GGTase I
1 b
0.5
ꢀ
/
20
inhibition (IC50ꢀ
FTase selectivity.
/
10 mM) which confers on 3b a certain
FTP II c
0.0439
10.0
3.590.4
/0.0045
ꢀ
/
10.0
10.0
10.0
3a
3b
4a
4b
ꢀ
/
ꢀ
/
The introduction of five fluorine atoms in the
aromatic portions, as in compounds 4a and 4b, caused
a complete loss of the FTase inhibitory properties.
Compound 4a showed some activity on GGTase I,
whereas 4b is almost inactive also on this enzyme.
In conclusion, we have designed and synthesised new
stable aniline-type FdP-analogues. The biological results
show that the combination of the aniline-containing
portions as farnesyl side chain mimics [6,7], together
with (phosphonoacetamido)oxy groups [8] as dipho-
sphate stable mimics, do not afford potent inhibitors of
the FTase enzyme.
/
ꢀ
/
ꢀ
ꢀ/
/
10.0
10.0
4.59
8.59
/
0.6
/
1.0
a
Values are reported as the mean9
/
range or SD of 2Á3 independent
/
experiments.
b
Reference [6].
Reference [8].
c
3. Results and discussion
The in vitro inhibition assays of FTase and GGTase I
were carried out by measuring the [3H]GGdP and
[3H]FdP incorporated into H-Ras-CVLL and H-Ras-
CVLS, respectively, as described in Section 4.2.2. The
activity of the inhibitors is reported in Table 1 as their
IC50, the concentration at which FTase and GGTase I
activity was inhibited by 50%. As reference compounds
of this class of structures, we have also reported the
inhibitory properties of FTP II [8] and aniline derivative
1 [6].
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined on a Kofler hot-stage
1
apparatus and are uncorrected. H NMR spectra of all

1280
F. Minutolo et al. / Il Farmaco 58 (2003) 1277Á1281
/
compounds were obtained with a Varian Gemini-200
instrument operating at 200 MHz; the data are reported
as follows: chemical shift (in ppm) from the Me4Si line
4.19 (m, 2H), 5.25Á
/5.40 (m, 2H). MS (EI, 70eV): m/z
350 (Mꢁ).
as the external standard, multiplicity (brꢀ
/
broad, sꢀ
/
4.1.4. Synthesis of derivatives 12a and 13a
singlet, dꢀdoublet, tꢀtriplet, mꢀmultiplet). Mass
/
/
/
A solution containing 0.83 mmol of the appropriate
precursor (9 or 10), diethylphosphonoacetic acid 11a
(0.180 g, 0.92 mmol), and 1-hydroxybenzotriazole (0.170
g, 1.25 mmol) in anhydrous THF (15 ml) was treated
spectra were recorded on a VG 70-250S mass spectro-
meter or a HP-5988 A spectrometer. Analytical TLCs
were carried out on 0.25-mm layer silica gel plates
containing a fluorescent indicator; spots were detected
under UV light (254 nm). Column chromatography was
with
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (0.190 g, 0.996 mmol). The mixture was
stirred at r.t. for 24 h, and the solvent was evaporated
under reduced pressure. The residue was purified by
performed using 230Á400 mesh silica gel. Sodium sulfate
/
was always used as the drying agent. Evaporations were
performed in vacuo (rotating evaporator). Commer-
column chromatography on 230Á400 mesh silica gel
/
cially available chemicals were purchased from SigmaÁ
Aldrich.
/
eluting with CH2Cl2/acetone (9:1). The appropriate
fractions were combined, evaporated, and pump-dried
to give the corresponding product.
1
12a (0.16 g, 44%) as an oil; H NMR (CDCl3) d 1.36
4.1.2. Synthesis of derivatives 7 and 8
(t, 6H, Jꢀ
(m, 4H), 2.82 (d, 2H, Jꢀ
1H), 4.05Á4.20 (m, 4H), 4.41Á
/
7.1 Hz), 1.66 (s, 3H), 1.70 (s, 3H), 2.00Á
20 Hz), 3.64 (s, 2H), 3.95 (br,
4.46 (m, 2H), 5.29Á5.39
7.20 (m, 2H), 9.50 (br,
/
2.15
A solution containing 4.1 mmol of the appropriate
precursor (5 [6] or 6 [7]) in 50 ml of anhydrous THF was
treated with N-hydroxyphthalimide (1.4 g, 8.8 mmol),
triphenylphosphine (2.6 g, 9.9 mmol) and diethylazodi-
carboxylate (1.6 ml, 9.9 mmol) and the resulting mixture
was stirred for 24 h at r.t. under an inert atmosphere.
The solvent was then evaporated under a vacuum and
the residue was purified by column chromatography on
/
/
/
/
(m, 2H), 6.61Á
/
6.74 (m, 3H), 7.12Á
/
1H). MS (EI, 70eV): m/z 438 (Mꢁ).
1
13a (0.16 g, 37%) as an oil; H NMR (CDCl3) d 1.35
(t, 6H, Jꢀ
/
7.1 Hz), 1.65 (s, 3H), 1.72 (s, 3H), 1.97Á
/2.17
(m, 4H), 2.84 (d, 2H, Jꢀ
/
20 Hz), 3.80 (s, 2H), 4.09Á
/4.24
(m, 4H), 4.42Á
/
4.45 (m, 2H), 5.30Á5.42 (m, 2H), 9.57 (br,
/
230Á400 mesh silica gel eluting with Hex/CH2Cl2. The
/
1H). MS (EI, 70eV): m/z 528 (Mꢁ).
appropriate fractions were combined and evaporated to
give the corresponding products.
7 (1.45 g, 97%) as an oil; 1H NMR (CDCl3) d 1.64 (s,
4.1.5. Synthesis of derivatives 12b and 13b
A solution containing 0.83 mmol of the appropriate
precursor (9 or 10), was submitted to the same treatment
described above (Section 4.1.4) using 2-(diethylpho-
sphono)butyric acid 11b [9] (0.205 g, 0.92 mmol). The
crude product was purified by column chromatography
3H), 1.71 (s, 3H), 2.03Á
4.75 (m, 2H), 5.35Á5.56 (m, 2H), 6.57Á
7.11Á7.19 (m, 2H), 7.70Á7.93 (m, 4H). MS (EI, 70eV):
m/z 366 (Mꢁ).
8 (1.86 g, 94%) as an oil; 1H NMR (CDCl3) d 1.62 (s,
3H), 1.71 (s, 3H), 2.06Á2.15 (m, 4H), 3.76 (s, 2H), 4.71Á
4.75 (m, 2H), 5.27Á5.56 (m, 2H), 7.71Á7.85 (m, 4H). MS
(EI, 70eV): m/z 480 (Mꢁ).
/
2.28 (m, 4H), 3.61 (s, 2H), 4.71Á
/
/
/6.71 (m, 3H),
/
/
on 230Á400 mesh silica gel eluting with hexane/EtOAc
/
/
/
(2:8). The appropriate fractions were combined, evapo-
rated, and pump-dried to give the corresponding
product.
/
/
1
12b (0.12 g, 30%) as an oil; H NMR (CDCl3) d 1.02
4.1.3. Synthesis of derivatives 9 and 10
(t, 3H, Jꢀ
1.67 (s, 3H), 1.73Á
1H), 4.03Á4.16 (m, 4H), 4.40Á
(m, 2H), 6.60Á6.75 (m, 3H), 7.08Á
70eV): m/z 466 (Mꢁ).
13b (0.21 g, 45%) as an oil; 1.00 (t, 3H, Jꢀ
1.32 (t, 6H, Jꢀ
2.16 (m, 7H), 3.77 (s, 2H), 4.10Á
(m, 2H), 5.29Á
(Mꢁ).
/
7.1 Hz), 1.40 (t, 6H, Jꢀ
2.15 (m, 7H), 3.58 (s, 2H), 3.92 (br,
4.44 (m, 2H), 5.28Á5.40
7.15 (m, 2H). MS (EI,
/
7.0 Hz), 1.62 (s, 3H),
Hydrazine monohydrate (1.5 ml, 31 mmol) was added
to a solution containing 3.8 mmol of the appropriate
precursor (7 or 8) in EtOH (60 ml) and the resulting
mixture was stirred at r.t. for 40 h. After removal upon
filtration of the white solid formed, the resulting
solution was evaporated and the oily crude residue
/
/
/
/
/
/
/7.1 Hz),
/
7.0 Hz), 1.63 (s, 3H), 1.68 (s, 3H), 1.86Á
/
was purified by column chromatography on 230Á
/
400
/
4.21 (m, 4H), 4.36Á
/4.40
mesh silica gel eluting with CH2Cl2/Et2O. The appro-
priate fractions were combined and evaporated to give
the corresponding products.
9 (0.90 g, 91%) as an oil; 1H NMR (CDCl3) d 1.67 (s,
/5.36 (m, 2H). MS (EI, 70eV): m/z 556
4.1.6. Synthesis of the dipotassium salts of derivatives 3a
and 4a
A solution containing 0.25 mmol of the appropriate
precursor (12a or 13a), was treated with bromotri-
methylsilane (0.17 ml, 1.2 mmol) and 2,4,6-collidine
(0.07 ml, 0.5 mmol) in anhydrous CH2Cl2 (3 ml); the
6H), 2.05Á
5.34Á5.42 (m, 2H), 6.57Á
2H). MS (EI, 70eV): m/z 260 (Mꢁ).
10 (1.2 g, 89%) as an oil; 1H NMR (CDCl3) d 1.65 (s,
3H), 1.66 (s, 3H), 2.01Á2.18 (m, 4H), 3.79 (s, 2H), 4.14Á
/
2.23 (m, 4H), 3.63 (s, 2H), 4.16Á
/
4.21 (m, 2H),
/
/
6.72 (m, 3H), 7.12Á
/
7.20 (m,
/
/

F. Minutolo et al. / Il Farmaco 58 (2003) 1277Á
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1281
1281
resulting mixture was stirred at r.t. for 18 h. After
concentration of the solution, the residue was treated
with an aqueous solution of KOH 1 N (3 ml) and then
stirred at r.t. for 4 h. The solution was evaporated and
the resulting crude residue was purified by column
chromatography on reverse phase silica gel (Merck
Lichroprep† RP-18) eluting with MeOH/H2O (6:4).
The appropriate fractions were combined, evaporated,
lyophilised, and pump-dried to give the corresponding
4.2.2. GGTase I and FTase activity assay
In vitro inhibition studies were performed as pre-
viously described [10,11], with some modifications.
Briefly, GGTase (250 ng) and FTase (20 ng) were
incubated in 50 mM TrisÁHCl, pH 7.7, 25 mM ZnCl2,
/
20 mM KCl, 5 mM MgCl2, 1 mM DTT and 0.5 mM
Zwittergent 3-12, in the presence of different concentra-
tions of inhibitors in a final volume of 50 ml. The
reactions were incubated at 30 8C for 30 min with
recombinant H-Ras-CVLL (2.5 mM) and [3H]GGdP
(0.1 mM) for GGTase I, and recombinant H-Ras-CVLS
(2.5 mM) and [3H]FdP (0.6 mM) for FTase. After
incubation the reaction was stopped and filtered on
glass fibre filters to separate free from incorporated
label. The activity of the inhibitors is reported in Table 1
as their IC50, the concentration at which GGTase and
FTase activity was inhibited by 50%.
compound as the dipotassium salt.
1
3a×
1.54 (s, 3H), 1.60 (s, 3H), 1.94Á
2H, Jꢀ 19 Hz), 3.56 (s, 2H), 4.22Á
5.34 (m, 2H), 6.69Á6.78 (m, 3H), 7.12Á
(FAB): m/z 459 (Mꢁ
Hꢁ).
4a×
2Kꢁ (0.13 g, 92%) as a solid; H NMR (D2O) d
1.37 (s, 3H), 1.43 (s, 3H), 1.78Á1.88 (m, 4H), 2.33 (d,
2H, Jꢀ 19 Hz), 3.48 (s, 2H), 4.16Á4.20 (m, 2H), 5.08Á
5.16 (m, 2H). MS (FAB): m/z 549 (Mꢁ
Hꢁ).
/
2Kꢁ (0.090 g, 78%) as a solid; H NMR (D2O) d
/
2.10 (m, 4H), 2.28 (d,
4.28 (m, 2H), 5.28Á
7.22 (m, 2H). MS
/
/
/
/
/
/
1
/
/
/
/
/
/
References
4.1.7. Synthesis of the dipotassium salts of derivatives 3b
and 4b
[1] W.A. Maltese, Posttranslational modification of proteins by
isoprenoids in mammalian cells, FASEB J. 4 (1990) 3319Á3328.
/
A solution containing 0.25 mmol of the appropriate
precursor (12b or 13b), was submitted to the same
treatment described above (Section 4.1.6). The crude
product was purified by column chromatography on
reverse phase silica gel (Merck Lichroprep† RP-18)
eluting with MeOH/H2O (1:1). The appropriate frac-
tions were combined, evaporated, lyophilised, and
pump-dried to give the corresponding compound as
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6593.
/
the dipotassium salt.
1
3b×
0.66 (t, 3H, Jꢀ
(q, 2H, Jꢀ7.3 Hz), 1.70Á
4.11Á4.15 (m, 2H), 5.07Á5.13 (m, 2H), 6.40Á
3H), 6.87Á6.95 (m, 2H). MS (FAB): m/z 489 (Mꢁ
4b×
2Kꢁ (0.11 g, 80%) as a solid; H NMR (D2O) d
0.62 (t, 3H, Jꢀ7.1 Hz), 1.32 (s, 3H), 1.37 (s, 3H), 1.55
(q, 2H, Jꢀ7.0 Hz), 1.68Á2.06 (m, 5H), 3.43 (s, 2H),
4.10Á4.13 (m, 2H), 4.99Á5.13 (m, 2H). MS (FAB): m/z
577 (Mꢁ
Hꢁ).
/
2Kꢁ (0.067 g, 55%) as a solid; H NMR (D2O) d
/
/
7.1 Hz), 1.32 (s, 3H), 1.40 (s, 3H), 1.58
2.08 (m, 5H), 3.30 (s, 2H),
6.52 (m,
Hꢁ).
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(2000) 3027Á3033.
/
/
/
1
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/
/
/
farnesyl transferase, J. Am. Chem. Soc. 124 (2002) 8206Á8219.
/
/
/
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/
4.2. Biological assays
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/
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[3H]GGPP (specific activity 22 Ci/mmol) and [3H]FdP
(specific activity 16 Ci/mmol) were purchased from
Chem. 276 (2001) 11624Á11630.
/
PerkinÁ
/
Elmer Life Science; GGTase I and FTase were
[10] F.L. Zhang, J.F. Moomaw, P.J. Casey, Properties and kinetic
mechanism of recombinant mammalian protein geranylgeranyl-
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CVLL, H-Ras-CVLS (Wild type), FPT inhibitor II
(FTP II) [8] and Zwittergent 3-12 were purchased from
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normal commercial sources.
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/
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/