2
V. Straniero et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
In a continuation of our effort to identify new FTIs, we consid-
MeO
Br
O
MeO
X
O
HO
O
MeO
Br
O
ered modifying compounds 2 and 3. The portion mimicking
cysteine, containing the benzodioxane or the 2-aminothiazole
substructure, was retained unaltered as well as the X residue
methionine, whereas the central spacer 2-o-tolylbenzoyl, which
is responsible for correctly positioning the two side fragments,
was replaced with other rigid systems. Docking studies on the
CAAX mimetic FTI-2148, a potent non-thiol FTI,19 have shown that
the 2-o-tolylbenzoyl is not only a mere spacer replacing AA, but
also a substructure interacting with a hydrophobic pocket of FTase
through its tolyl residue and that such a pocket can accommodate
larger groups than tolyl.20 Therefore, we planned the replacement
of tolyl with a heteroaromatic ring, such as thiophene and furan, or
with a larger aromatic system, such as naphthalene and isoquino-
line, in order to verify if the extra interaction potential due to the
additional heteroatom or ring would improve the enzyme inhibi-
tion. In particular, we designed the six analogues 4–9 of 2 and
the six analogues 10–15 of 3, where 2-o-tolyl is replaced by 1-
naphthyl, 4-isoquinolinyl, 2- or 3-thienyl, and 2- or 3-furanyl,
and the corresponding isopropyl esters 4a–15a (Chart 2).
The isopropyl esters 4a–15a were prepared as prodrugs of the
acids 4–15 in order to facilitate cell membrane penetration and
to evaluate the cell activity of the acids exhibiting FTase inhibition.
To synthesize compounds 4a–9a, the mesylate group of racemic
2-mesyloxymethyl-1,4-benzodioxane21,22 was displaced by methyl
2-bromo-4-hydroxybenzoate.23–25 Subsequent Suzuki coupling of
the resultant bromophenyl ether with the suitable aryl or hetero-
aryl boronic acid gave the intermediate methyl esters 16–21,
which were converted into the corresponding carboxylic acids
X
b
c
a
O
O
O
O
O
O
OH
MsO
O
+
O
O
O
O
16-21
22-27
H
O
N
COOiPr
S
X
e
d
4-9
O
16, 22, 4a X = 1-naphtyl
O
17, 23, 5a
18, 24, 6a
19, 25, 7a
20, 26, 8a
21, 27, 9a
4-isoquinolinyl
2-thienyl
3-thienyl
2-furanyl
3-furanyl
O
4a-9a
Scheme 1. Synthesis of compounds 4–9 and of the corresponding isopropyl esters
4a–9a. Reagents and conditions: (a) K2CO3, DMF, 30 min, room temperature and,
after addiction of the mesylate, 60 °C, 18 h, 76%; (b) Pd(PPh3)4, XB(OH)2, DMF or
THF or toluene/methanol, Na3PO4Á12H2O or Na2CO3/H2O or K2CO3/H2O, room
temperature or 100 °C, 18 h, 58–95%; (c) NaOH 1 M at 60 °C or NaOH 4 M at room
temperature, methanol, 18 h, 60–100%; (d) L-methionine isopropyl ester, HOBt,
22–27 and then condensed with the isopropyl ester of L-methio-
nine to yield the desired esters 4a–9a. Hydrolysis of these esters
afforded the carboxylic acids 4–9 (Scheme 1).
EDACÁHCl, DIPEA, DMF, 18 h, room temperature, 67–91%; (e) NaOH 1 M, MeOH,
18 h, room temperature, 100%.
A different synthetic strategy was adopted to prepare com-
pounds 10–15. Firstly, we synthesized the methyl 2-aryl- or 2-het-
eroaryl-4-nitrobenzoates 28–33 by Suzuki coupling of methyl 2-
bromo-4-nitrobenzoate with the suitable aryl or heteroaryl boro-
nic acid. The methyl benzoates 28–33 were then hydrolyzed and
inhibition with an IC50 equal to 5.0 and 2.8 lM, while the others
exhibited lower inhibitory activities or were not active.
The isopropyl esters 4a–15a, prepared to ameliorate membrane
permeability and to study the effects on cell growth, were tested
by a cellular assay measuring inhibition of rat aortic SMC prolifer-
ation. As shown in Table 2, most of them proved to inhibit rat SMC
proliferation with IC50 values in the micromolar range. In particu-
lar, compound 15a, the ester of the most potent FTase inhibitor of
the series, reduced rat SMC growth in a concentration dependent
manner with an IC50 value of 250 lM, whereas compound 7a,
whose free acid 7 is only little less potent than 15 as a FTI, showed
no antiproliferative activity.
condensed with the isopropyl ester of L-methionine. The NO2 group
was reduced to NH2 and the resulting anilines 46–51 converted
into the anilides 52–57 by reaction with 2-tritylamino-4-thiazolyl
acetic acid.18 Subsequent N-detritylation provided the esters 10a–
15a, which were hydrolyzed to the acids 10–15 (Scheme 2) (See
Supplementary data for reactions conditions and work-up, yields
and spectral data).
The inhibition of FTase activity was first determined using a
FTase fluorescent assay25 and FTI-276 (IC50 9 nM)18 and 3 (IC50
49 nM)18 as reference compounds. In the initial screening, the free
acids 4–15 were tested at a 20 lM and the percentual inhibition at
such concentration was determined. Results are summarized in
To study whether the ability of compound 15 to interfere with
FTase activity in vitro was maintained also in cultured cells by its
ester 15a, we investigated the Ras prenylation by western blot
analysis from total cell lysates of rat SMCs. Cells were incubated
for 72 h in the presence or absence of 15a at concentrations rang-
Table 1. Two compounds, namely 7 and 15, showed a near 100%
ing from 25 to 400
aryl-coenzyme
(2 M) as positive control. The compound 15a interfered with
Ras farnesylation in a concentration-dependent manner with
approximately 100 M IC50, as demonstrated by the appearance
l
M and in the presence of the 3-methyl-3-glut-
A
(HMG-CoA) reductase inhibitor simvastatin
l
OH
OH
H
N
H
N
O
O
l
O
O
of a slower migrating band corresponding to the unprenylated
form of the protein, also detected after incubation with simvastatin
(Fig. 1) (See Supplementary data for FTase inhibition tests and for
cell proliferation and Ras prenylation assays).
In terms of FTase inhibition, two of the six designed replace-
ments of o-tolyl with new aryl or heteroaryl systems are invariably
detrimental, namely those with 4-isoquinolinyl and 2-furanyl. On
the contrary, the other four tolyl replacements result in a moderate
FTase inhibition but depending on the compounds series: 1-naph-
thyl and 3-furanyl in the 2-aminothiazoles (compounds 10 and
15), 2- and 3-thienyl in the benzodioxanes (compounds 6 and 7).
X
X
S
S
O
O
HN
O
4, 10
5, 11
6, 12
7, 13
8, 14
9, 15
X = 1-naphtyl
4-isoquinolinyl
2-thienyl
3-thienyl
2-furanyl
3-furanyl
S
O
N
H2N
4-9
10-15
Chart 2.
However, of the two thienyl substituted esters, one has
a