New Ras CAAX mimetics: Design, synthesis, antiproliferative activity, and RAS prenylation inhibition

Article abstract of DOI:10.1016/j.bmcl.2009.07.065

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing cysteine (C) by 2-hydroxymethylbenzodioxane or 2-aminomethylbenzodioxane, respectively etherified and amidified with 2′-methyl or 2′-methoxy substituted 2-carboxy-4-hydrox

Full text of DOI:10.1016/j.bmcl.2009.07.065

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5500–5504
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New Ras CAAX mimetics: Design, synthesis, antiproliferative activity,
and RAS prenylation inhibition
Cristiano Bolchi a, Marco Pallavicini a, Laura Fumagalli a, Nicola Ferri b, Alberto Corsini b, Chiara Rusconi a,
Ermanno Valoti a,
*
a Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Università degli Studi di Milano, via Mangiagalli 25, I-20133 Milano, Italy
b Dipartimento di Scienze Farmacologiche, Università degli Studi di Milano, via Balzaretti 9, I-20133 Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing cysteine (C) by 2-
hydroxymethylbenzodioxane or 2-aminomethylbenzodioxane, respectively etherified and amidified with
20-methyl or 20-methoxy substituted 2-carboxy-4-hydroxybiphenyl and 2,4-dicarboxybiphenyl. These
pluri-substituted biphenyl systems, used as internal spacer and AA dipeptide bioisoster, were linked to
Received 25 May 2009
Revised 9 July 2009
Accepted 10 July 2009
Available online 17 July 2009
the methyl ester of L-methionine, glycine or L-leucine by an amide bond. The resultant twelve pairs of
stereoisomers at the dioxane C-2 were tested for antiproliferative effect finding the maximum activity
for derivatives with methyleneoxy linker between benzodioxane and 20-methylbiphenyl. Of these com-
pounds, the one with terminal methionine and S configuration proved a good Ras prenylation inhibitor
in a cell-based assay.
Keywords:
Peptidomimetic inhibitors
Antiproliferative agents
Antitumors
Ó 2009 Elsevier Ltd. All rights reserved.
Prenylation inhibitors
A vast number of proteins in animals are subject to prenylation,
that is the addition of a hydrophobic isoprenoid catalyzed by en-
zymes such as farnesyltransferase (FTase) and geranylgeranyl-
transferase (GGTase). This process has proven to be critical for
the biological function of several proteins involved in signal trans-
duction, since it strongly influences their association to cellular
membrane and protein–protein interactions.1 Great interest in
inhibition of protein prenylation, in particular of Ras proteins farn-
esylation, arises from the finding that farnesylation is essential for
the role of these proteins in mitogenesis and of their mutated
forms in oncogenesis. Moreover, proliferation of smooth muscle
cells (SMCs) in the arterial wall in response to vascular injury is
an important pathogenetic factor of vascular disorders such as ath-
erosclerosis and restenosis after angioplasty.2 The importance of
H-Ras in SMC proliferation in response to vascular injury has been
shown by the adenoviral delivery of a dominant negative mutant of
H-Ras, which effectively inhibits SMC accumulation after balloon
injury of rat carotid artery.3
tion, as indicated by the appearance of unprenylated Ras in the
total protein extract of aortic SMCs incubated with I stereoisomers
under the same conditions utilized for evaluating cell proliferation.4
O
COOH
Me
O
N
H
*
S
O
O
*
N
I
Compound I and its analogues with methoxyl in lieu of methyl
or methyleneamido in place of methyleneoxy linker or with both
these variations had been designed using the Ras C-terminal
tetrapeptide CAAX as a template, since such a tetrapeptide is the
minimum length co-substrate accepted by FTase for in vitro reac-
tion.5–9 Applying this commonly practiced strategy, we had re-
placed cysteine with pyridodioxane and the internal dipeptide AA
with 4-hydroxy- or 4-aminocarbonyl-2-biphenylcarboxylic acid.
Pyridodioxane had been chosen as rigidified analogue of 3-pyridyl-
oxymethyl, a substructure successfully used to replace cysteine in
FTase inhibitors,9 with improved hydrogen bond acceptor capabil-
ity and additional presence of a stereocenter with respect to 3-pyr-
idyloxymethyl. Based on the encouraging biological data of I and of
its analogues,4 we designed compounds 1–24, whose salient fea-
ture is the bioisosteric replacement of pyridodioxane with benzo-
Recently, we have reported that the stereoisomers of 2-o-tolyl
substituted 4-hydroxybenzamides of methionine, pyridodioxan-2-
ylmethyl etherified at the phenolic function, (I) have a significant
antiproliferative effect on human aortic smooth muscle cells (SMCs)
demonstrating that the two stereoisomers of I with higher antipro-
liferative activity (17 and 59 lM IC50) interfere with Ras farnesyla-
dioxane,
a modification we have previously experienced as
* Corresponding author.
E-mail address: ermanno.valoti@unimi.it (E. Valoti).
ameliorating in other classes of biologically active compounds.10
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.065

C. Bolchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5500–5504
5501
Br
Br
COOH
COOMe
a
R1
X
O
COOMe
R2
O2N
O2N
N
H
27
26
O
O
b or e
*
1, 2 R1 = Me
R2
= CH2CH2SMe X = O
= CH2CH2SMe X = O
Y
Y
Y
3, 4
5, 6
= OMe
= Me
COOMe
COOMe
COOMe
= H
= H
= CH2CHMe2
= CH2CHMe2
X = O
X = O
X = O
X = O
7, 8
= OMe
= Me
= OMe
= Me
= OMe
= Me
= OMe
= Me
O2N
H2N
HO
9, 10
11, 12
13, 14
15, 16
17, 18
19, 20
21, 22
23, 24
c
d
d
= CH2CH2SMe X = NHCO
= CH2CH2SMe X = NHCO
30 Y = Me
31 Y = OMe
28 Y = Me
29 Y = OMe
32 Y = Me
33 Y = OMe
c
= H
= H
= CH2CHMe2
= CH2CHMe2
X = NHCO
X = NHCO
X = NHCO
X = NHCO
Scheme 1. Synthesis of the methyl esters of 2-o-tolyl- and 2-o-anisyl-4-hydroxy-
benzoic acid: (a) methanol, methyl orthoformate, H2SO4 conc., reflux, 12 h, 87%; (b)
tolyl boronic acid, Pd(PPh3)4, 2 M Na2CO3, ethanol, toluene, reflux, 12 h, 86%; (c) H2-
Pd/C, methanol, 3 h, 83% (30) and 64% (31); (d) NaNO2, 2 N H2SO4, acetone, ꢀ10 °C
and then room temperature, 12 h, 84% (32) and 85% (33); (e) o-methoxyphenyl
boronic acid, Pd(PPh3)4, 2 M Na2CO3, ethanol, toluene, reflux, 12 h, 82%.
= OMe
Indeed, 2-substituted 2,3-dihydro-1,4-dioxino[2,3-b]pyridine
represents only one of the two pyridodioxane systems containing
conformationally blocked 3-pyridyloxymethyl substructure, the
other being 3-substituted 2,3-dihydro-1,4-dioxino[2,3-c]pyridine,
which we have not yet evaluated (Fig. 1). However, before under-
taking the synthesis of 2,3-dihydro-1,4-dioxino[2,3-c]pyridine
derivatives, it seemed worthwhile to first pass through the less
troublesome bioisosteric replacement of 2,3-dihydro-1,4-dioxi-
no[2,3-b]pyridine with benzodioxane thus eliminating the variable
of nitrogen position in the aromatic ring of the bicyclic system.
Such a simplifying modification could have beneficial effects, we
reasoned, in the case of unfavorable position of nitrogen in the
ester to give the target compounds 1, 3, 5, 7, 9 and 11 with S config-
uration at the dioxane stereocenter (Scheme 2).23–28 The corre-
sponding stereoisomers with
stereocenter, namelythe target compounds2, 4, 6, 8, 10 and 12, were
synthesized from (S)-25 and -methionine or -leucine or glycine
R configuration at the dioxane
L
L
methyl ester by the same sequence of reactions illustrated in
Scheme 2.29–38
The synthesis of compounds 13–24, characterized by methyle-
neamido linker between benzodioxane and biphenyl, required
unichiral aminomethylbenzodioxane 38 and methyl 2-o-tolyl-4-
carboxybenzoate or its o-anisyl analogue. The two methyl
benzoates and the enantiomers of 38 were prepared according to
previously reported procedures.4,39,40 To prepare 13, 17 and 21,
2,3-dihydro-1,4-dioxino[2,3-b]pyridine system of
I
and its
analogues.
Compounds 1–24 were designed on this hypothesis, but also
considering alternative aminoacids to terminal methionine such
as glycine and leucine, which are characterized by quite different
steric bulkiness and lipophilicity. Finally, only in vitro cellular tests
being available to us for biological evaluation, we decided to en-
hance cell permeability esterifying the aminoacid carboxyl.
To synthesize compounds 1–12, characterized by methyleneoxy
linker between terminal benzodioxane and biphenyl core, we
firstly prepared the two non-aminoacidic building blocks, namely
2-mesyloxymethylbenzodioxane 25, in both the enantiomeric
forms, and methyl 4-hydroxybenzoate, o-tolyl or o-anisyl substi-
tuted at position 2 (32 and 33, respectively). The synthesis of
(S)- and (R)-25 was accomplished according to procedures we
had previously reported,11 while 32 and 33 were prepared from
2-bromo-4-nitrobenzoic acid by a sequence of reactions identical
to that employed for the corresponding ethyl esters (Scheme
1).12–18
O
OMs
(R)-25
O
a or f
R1
R1
O
O
COOMe
R2
COOX
N
H
O
O
O
O
O
1 = Me
1
5
9
R
R
R
R
R
R
(S)-34 R1 = Me
2 = (CH2)2SMe
1 = Me
c
X
= Me
b
d
2 = H
(S)-36 R1
e
Nucleophilic displacement of mesylate group of (R)-25 by 32 and
33 yielded (S)-34 and (S)-35, respectively.19,20 Both these methyl es-
ters were hydrolyzed and the resultant carboxylic acids (S)-3621 and
= Me
= H
1 = Me
X
2 = CH2CHMe2
(S)-3722 condensed with
L
-methionine or
L
-leucine or glycine methyl
3
7
R
R
1 = OMe
(S)-35 R1 = OMe
2 = (CH2)2SMe
c
d
e
X
= Me
R1 = OMe
b
2 = H
R
O
1 = OMe
= H
(S)-37 R
11 R1 = OMe
X
2 = CH2CHMe2
R
N
A
O
O
N
Scheme 2. Synthesis of compounds 1, 5, 9, 3, 7 and 11: (a) 32, NaH, DME, ꢀ20 °C
and then reflux, 72 h, 69%; (b) 3 N KOH, acetone, 50 °C, 12 h, 80% ((S)-36) and 76%
((S)-37); (c) L-methionine methyl ester hydrochloride, HOBt, EDAC, DMF, 15 min,
room temperature and then TEA, 24 h, room temperature, 90% (1) and 94% (3);
(d) glycine methyl ester hydrochloride, HOBt, EDAC, DMF, 15 min, room temper-
O
N
O
O
C
B
O
D
ature and then TEA, 24 h, room temperature, 88% (5) and 69% (7); (e) L-leucine
Figure 1. 2-Substituted 2,3-dihydro-1,4-dioxino[2,3-b]pyridine (B), 3-substituted
2,3-dihydro-1,4-dioxino[2,3-c]pyridine (C) as rigid systems containing 3-pyridyl-
oxymethyl substructure (A) and 1,4-benzodioxane (D) as bioisoster of both.
methyl ester hydrochloride, HOBt, EDAC, DMF, 15 min, room temperature and then
TEA, 24 h, room temperature, 90% (9) and 85% (11); (f) 33, NaH, DME, ꢀ20 °C and
then reflux, 72 h, 74%.

5502
C. Bolchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5500–5504
O
O
lar, most of compounds showing 1–100 lM IC50. The twelve com-
NH2
pounds with oxymethylene linker, the ‘ethers’, are all more active
than those with methyleneamido linker, the ‘amides’. Moreover,
out of the ‘ethers’, but not out of the less potent ‘amides’, the methyl
substituted compounds are invariably more potent than the corre-
sponding methoxy substituted analogues. Such a result is consistent
with what we have reported for the pyridodioxane analogues,4
whose most active derivatives also present the biphenyl core methyl
substituted and linked to dioxane through oxymethylene bridge. On
the contrary, coherent activity trends cannot be recognized with ref-
erence both to the configuration of the benzodioxane stereocenter,
as previously found for the pyridodioxane stereocenter, and to ter-
minal aminoacid residue. Rather surprisingly, methionine, one of
the typical terminal amino acid of CAAX motif, is not associated with
the highest activities excepting 1 and more than one leucine or gly-
cine derivative shows lower IC50 values. However, it is significant
what resulted when the effect on protein prenylation was evaluated
for the compounds with the highest antiproliferative activity,
namely 1, 5, 6, 7, 8, 9, 10, 11 and 12, by Western blot analysis of
Ras from a total protein extract of cells incubated under the same
experimental conditions utilized for evaluating cell proliferation.62
Only the methionine derivative 1 proved to directly interfere with
(S)-38
a or f
R1
R1
O
O
O
O
O
O
O
COOX
COOMe
R2
NH
NH
HN
13 1 = Me
(S)-39 R1 = Me
R
2 = (CH2)2SMe
R
X
= Me
c
b
17 1 = Me
R
d
e
(S)-41 R1
= Me
= H
R2 = H
X
21 1 = Me
R
R
2 = CH2CHMe2
15 R1 = OMe
(S)-40 R1 = OMe
2 = (CH2)2SMe
= Me
R
X
c
b
19 R1 = OMe
d
= OMe
= H
2 = H
R
(S)-42 R1
e
23 R1 = OMe
X
2 = CH2CHMe2
R
Scheme 3. Synthesis of compounds 13, 17, 21, 15, 19 and 23: (a) methyl
2-o-tolyl-4-carboxybenzoate, HOBt, EDAC, DMF, 15 min, room temperature and
then TEA, 12 h, room temperature, 80%; (b) 3 N KOH, methanol, reflux, 12 h, 95%
Ras prenylation (IC50 2.8 lM), as indicated by the accumulation of
unprenylated Ras into the cell (Fig. 2);63 the other compounds, with
terminal glycine (5–8) or leucine (9–12), had no effect on Ras prenyl-
ation, including the most antiproliferative compound 6, or showed
much lower prenylation inhibition than 1.64
((S)-41) and 96% ((S)-42); (c) L-methionine methyl ester hydrochloride, HOBt,
EDAC, DMF, 15 min, room temperature and then TEA, 24 h, room temperature, 68%
(13) and 78% (15); (d) glycine methyl ester hydrochloride, HOBt, EDAC, DMF,
15 min, room temperature and then TEA, 24 h, room temperature, 63% (17) and
62% (19); (e) L-leucine methyl ester hydrochloride, HOBt, EDAC, DMF, 15 min, room
The final question is whether the replacement of 2,3-dihydro-
1,4-dioxino[2,3-b]pyridine by benzodioxane is productive.
Appropriate comparison can be made only on the basis of antiprolif-
erative activity, determined for both the classes of compounds, ben-
zodioxanes and pyridodioxanes, and considering the methyl esters,
rather than the free carboxylic acids, since the common biological
test was an in vitro cellular assay. Therefore, we prepared the methyl
ester of the SS isomer of I, that is, the pyridodioxane analogue of 1.
The SS isomer of I (free acid) had previously proved to exert antipro-
temperature and then TEA, 24 h, room temperature, 84% (21) and 70% (23); (f)
methyl 2-o-anisyl-4-carboxybenzoate, HOBt, EDAC, DMF, 15 min, room tempera-
ture and then TEA, 12 h, room temperature, 81%.
methyl 2-o-tolyl-4-carboxybenzoate was reacted with (S)-38
obtaining (S)-39.41 Successive hydrolysis of methyl ester yielded
the carboxylic acid (S)-41,42 which was condensed with
L
-methio-
nine or glycine or
L-leucine methyl ester to give the target com-
pounds 13, 17 and 21 with S configuration at the dioxane
stereocenter.43–45 Their methoxy analogues 15, 19 and 23 were
obtained by the same synthetic route, using methyl-2-o-anisyl-4-
carboxybenzoate as a biphenyl synthon (Scheme 3).46–50 The
corresponding stereoisomers with R configuration at the dioxane
stereocenter, namely the target compounds 14, 16, 18, 20, 22 and
liferative effect with 158
methyl ester increases antiproliferative activity lowering IC50 from
158 to 30 M and produces direct interference with Ras prenylation
at 50 M. Considering that 6.6 M and 2.8 M concentrations of 1
lM IC50. We found that conversion into
l
l
l
l
are required for 50% inihibition of cellular proliferation and of Ras
prenylation respectively (Fig. 2), affirmative answer to the above
questionseemsreasonable. Furthermore, inthecase of previouspyr-
idodioxanes, FTase inhibition had been determined and preliminary
results, obtained by using a specific antibody for unprenylated Rap1,
a GGTase I substrate, indicated that such inhibition was FTase spe-
cific. Though further analyses will be necessary on both the series
24, were synthesized from (S)-38 and L-methionine or L-leucine or
glycine methyl ester by the same sequence of reactions illustrated
in Scheme 3.51–60
Compounds 1–24 were tested in a cellular assay that measured
inhibition of human aortic SMC proliferation.61 As listed in the
Table 1, the IC50 values ranged from submicromolar to submillimo-
Table 1
Antiproliferative effect of compounds 1–24 on human aortic SMCs
R1
X
O
aa
O
*
O
X
O
Terminal aminoacid methyl R1 = Me (S tolyl
IC50
(
l
M)
R1 = Me (R tolyl
derivs)
IC50
(
l
M)
R1 = OMe (S anisyl
derivs)
IC50
(
l
M)
R1 = OMe (R anisyl IC50
(lM)
derivs)
ester (aa)
derivs)
L-Met
L-Gly
L-Leu
L-Met
L-Gly
L-Leu
1
5
9
13
17
21
6.6 3.8
27 8.2
3.0 2.3
87 42.3 14
113 59.7 18
103 23.1 22
2
6
10
193 61.4
0.7 0.4
5.2 4.3
533 89.3 15
89 26.6 19
88 21.5 23
3
7
11
230 24.5
71 3.4
32 12.5 12
252 65.3 16
122 29.2 20
185 35.6 24
4
8
204 76.7
35 12.6
17 9.9
NHCO
647 89.5
76 43.7
469 89.0
The IC50
(l
M) values were determined by linear regression analysis of the logarithm of the concentration versus the percentage of the inhibitory effect.

C. Bolchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5500–5504
2. Ross, R. N. Eng. J. Med. 1999, 340, 115.
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Lorenzo, E.; Esposito, G.; Stabile, E.; Feliciello, A.; Mele, E.; Giuliano, P.;
Condorelli, G.; Chiariello, M. Nat. Med. 1995, 1, 541.
4. Bolchi, C.; Pallavicini, M.; Rusconi, C.;Diomede, L.; Ferri, N.; Corsini, A.; Fumagalli,
L.; Pedretti, A.; Vistoli, G.; Valoti, E. Bioorg. Med. Chem. Lett. 2007, 17, 6192.
5. Goldstein, J. L.; Brown, M. S.; Stradley, S. T.; Reiss, Y.; Gierasch, L. M. J. Biol.
Chem. 1991, 266, 15575.
6. Reiss, Y.; Stradley, S.; Gierasch, L.; Brown, M. S.; Goldstein, J. L. Proc. Natl. Acad.
Sci. U.S.A. 1991, 88, 732.
7. Sun, J.; Qian, Y.; Hamilton, A. D.; Sebti, S. M. Cancer Res. 1995, 55, 4243.
8. Qian, Y.; Vogt, A.; Sebti, S. M.; Hamilton, A. D. J. Med. Chem. 1996, 39, 217.
9. Augeri, D. J.; O’Connor, S. J.; Janowick, D.; Szczepankiewicz, B.; Sullivan, G.;
Larsen, J.; Kalvin, D.; Cohen, J.; Devine, E.; Zhang, H.; Cherian, S.; Saeed, B.; Ng,
S. C.; Rosenberg, S. J. Med. Chem. 1998, 41, 4288.
10. 2-(2-Pyrrolidinyl)benzodioxanes (Pallavicini, M.; Bolchi, C.; Binda, M.; Cilia, A.;
Clementi, F.; Ferrara, R.; Fumagalli, L.; Gotti, C.; Moretti, M.; Pedretti, A.; Vistoli,
G.; Valoti, E. Bioorg. Med. Chem. Lett. 2009, 19, 854) are much more potent
a
4b2 nicotinic ligands than 2-(2-pyrrolidinyl)-2,3-dihydro-1,4-dioxino[2,3-
b]pyridines (unpublished data). WB4101, namely 2-[2-(2,6-dimethoxyphen-
oxy)ethyl]aminomethyl-1,4-benzodioxane is more potent a1 -adrenergic
a
ligand than 2-[2-(2,6-dimethoxyphenoxy)ethyl]aminomethyl-2,3-dihydro-
1,4-dioxino[2,3-b]pyridine (Valoti, E. Abstracts of papers, XVI Convegno
Nazionale Divisione di Chimica Farmaceutica Società Chimica Italiana,
Sorrento, Italy, September 2002; Abstract L18).
11. Bolchi, C.; Pallavicini, M.; Fumagalli, L.; Rusconi, C.; Binda, M.; Valoti, E.
Tetrahedron:Asymmetry 2007, 18, 1038.
12. Compound 27: crystallization from cold methanol; mp 83–84 °C; 1H NMR
(CDCl3) d 8.49 (d, J = 2.2 Hz, 1H), 8.19 (dd, J = 8.8, 2.2 Hz, 1H), 7.91 (d, J = 8.8 Hz,
1H), 3.98 (s, 3H).
13. Compound 28: LC on silica gel (eluent:cyclohexane/EtOAc 90/10); 1H NMR
(CDCl3) d 8.26 (dd, J = 8.4, 2.2 Hz, 1H), 8.12 (d, J = 2.2 Hz, 1H), 8.07 (d, J = 8.4 Hz,
1H), 7.32–7.23 (m, 3H), 7.07 (dd, J = 7.6, 6.6 Hz 1H), 3.65 (s, 3H), 2.09 (s, 3H).
14. Compound 29: crystallization from diisopropyl ether; 1H NMR (CDCl3) d 8.23 (d,
J = 2.2 Hz, 1H), 8.20 (dd, J = 2.3, 2.2 Hz, 1H), 7.97 (d, J = 9.15 Hz, 1H), 7.4 (m, 1H),
7.28 (m, 1H), 7.07 (m, 1H), 6.93 (d, J = 8.05 Hz, 1H), 3.73 (s, 3H), 3.70 (s, 3H).
15. Compound 30: crystallization from diisopropyl ether; 1H NMR (CDCl3) d 7.88 (d,
J = 9.2 Hz, 1H), 7.16–7.25 (m, 3H), 7.05 (d, J = 6.6 Hz, 1H), 6.62–6.66 (m, 1H),
6.43 (d, J = 2.2 Hz, 1H), 4.02 (br s, 2H), 3.57 (s, 3H), 2.07 (s, 3H).
Figure 2. Concentration dependent effect of
proliferation in human aortic SMCs. Human SMCs were incubated with increasing
concentrations of compound for 72 h, total cell lysates were prepared and
Ras prenylation evaluated by Western blotting analysis with a specific antibody anti
Ras (upstate). The slower migrating band represents the unprenylated form of Ras
(unpre. Ras), while the faster migrating band is prenylated Ras (pre. Ras). The
proliferation assay was performed under the same experimental conditions and cell
number determined after 72 h incubation with indicated concentrations of 1. CNT:
control.
1 on Ras prenylation and cell
16. Compound 31: crystallization from diisopropyl ether; 1H NMR (CDCl3) d 7.79 (d,
J = 8.4 Hz, 1H), 7.28–7.34 (m, 1H), 7.18 (dd, J = 7.3, 1.8 Hz, 1H), 6.97–7.03 (m,
1H), 6.88 (d, J = 8.1 Hz, 1H), 6.62 (dd, J = 8.4, 2.2 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H),
3.8–4.2 (br s, 2H), 3.71 (s, 3H), 3.59 (s, 3H).
1
17. Compound 32: LC on silica gel (eluent:cyclohexane/EtOAc 70/30); 1H NMR
(CDCl3) d 7.95 (d, J = 8.6 Hz, 1H), 7.08–7.23 (m, 3H), 6.95–7.03 (m, 1H), 6.78 (d,
J = 8.0 Hz, 1H), 6.6 (s, 1H), 6.3 (s, 1H), 3.59 (s, 3H), 2.05 (s, 3H).
18. Compound 33: LC on silica gel (eluent:cyclohexane/EtOAc 70/30); 1H NMR
(CDCl3) d 7.71 (d, J = 8.4 Hz, 1H), 7.27–7.33 (m, 1H), 7.16 (dd, J = 7.3, 1.8 Hz,
1H), 6.96–7.02 (m, 1H), 6.87 (d, J = 8.05 Hz, 1H), 6.77 (dd, J = 8.4, 2.6 Hz, 1H),
6.71 (d, J = 2.2 Hz, 1H), 5.84 (s, 1H), 3.69 (s, 3H), 3.62 (s, 3H).
of pyridodioxane and benzodioxane derivatives, we can hypothesize
that the present compounds affect only FTase.
19. (S)-34: LC on silica gel (eluent:cyclohexane/EtOAc 60/40); ½a D25
= ꢀ5.80 (c 1,
ꢁ
CHCl3); 1H NMR (CDCl3) d 7.99 (d, J = 8.8 Hz, 1H), 7.17–7.23 (m, 3H), 7.05 (d,
J = 7.0 Hz, 1H), 6.84–6.97 (m, 5H), 6.76 (d, J = 2.9 Hz, 1H), 4.56–4.59 (m, 1H),
4.39 (dd, J = 11.4, 3.1 Hz, 1H), 4.19–4.30 (m, 3H), 3.60 (s, 3H), 2.06 (s, 3H).
In conclusion, we have demonstrated that CAAX mimetics, in
which C-terminal methionine is esterified or replaced by esterified
glycine or leucine and cysteine is replaced by a benzodioxanem-
ethyl residue etherifying the known AA bioisostere 2-o-tolyl-4-
hydroxybenzoic acid exhibit, in whole cell tests, antiproliferative
activity at submicromolar or low micromolar level. However, such
an activity is associated to a remarkable inhibition of Ras prenyla-
tion only in the case of the most potent methionine derivative 1.
Analogously to previous results obtained for pyridodioxane deriv-
atives, critical features are the nature of the linker between diox-
ane and biphenyl core and the o-substitution on the latter, and
not the dioxane stereochemistry. Though comparison of the pres-
ent compounds with the previous ones is limited to 1 and its pyr-
idodioxane analogue, it is significant that, in this case, replacement
of pyridodioxane by benzodioxane not only potentiates but also
maintains the activity at the highest level in the respective series.
20. (S)-35: LC on silica gel (eluent:cyclohexane/EtOAc 60/40); ½a D25
= ꢀ4.0 (c 1,
ꢁ
CHCl3); 1H NMR (CDCl3) d 7.90 (d, J = 8.8 Hz, 1H), 7.31–7.37 (m, 1H), 7.21 (dd,
J = 7.3, 1.8 Hz, 1H), 7.00–7.05 (m, 1H), 6.84–6.94 (m 7H), 4.56–4.59 (m, 1H),
4.39 (dd, J = 11.35, 2.2 Hz, 1H), 4.17–4.25 (m, 3H), 3.71 (s, 3H), 3.63 (s, 3H).
21. (S)-36: crystallization from isopropyl alcohol; ½a D25
ꢁ
= ꢀ32.0 (c 1, methanol); 1
H
NMR (DMSO-d6) d 12.19 (br s, 1H), 7.87 (d, J = 9.0 Hz, 1H), 7.02–7.22 (m, 5H),
6.78–6.99 (m, 4H), 6.71 (d, J = 2.6 Hz, 1H), 4.52–4.59 (m, 1H), 4.25–4.42 (m,
3H), 4.12 (dd, J = 11.7, 7.3 Hz, 1H), 2.00 (s, 3H).
22. (S)-37: crystallization from diisopropyl ether/isopropyl alcohol 1/1;
½
a 2D5
ꢁ
= ꢀ28.5 (c 1, methanol); 1H NMR (DMSO-d6) d 12.07 (s, 1H), 7.76 (d,
J = 8.8 Hz, 1H), 7.25–7.31 (m, 1H), 7.16 (dd, J = 7.7, 1.8 Hz, 1H), 6.78–7.02 (m,
8H), 4.54–4.57 (m, 1H), 4.41 (dd, J = 11.35, 2.2 Hz, 1H), 4.27–4.36 (m, 2H), 4.12
(dd, J = 11.7, 7.3 Hz, 1H), 3.62 (s, 3H).
23. 2(S)-[2-(o-tolyl)-4-(2(S)-1,4-benzodiossan-2-ylmethoxy)benzamido]-4-methyl-
thiobutyric acid methyl ester (1): LC on silica gel (eluent:cyclohexane/ethyl
acetate 60/40); ½a D25
ꢁ
= +8.9 (c 1, CHCl3); 1H NMR consistent with that of 2.
24. [2-(o-tolyl)-4-(2(S)-1,4-benzodioxan-2-ylmethoxy)benzamido]acetic acid methyl
ester (5): LC on silica gel (eluent:cyclohexane/ethyl acetate 60/40); ½a D25
= ꢀ6.6
ꢁ
(c 1, CHCl3); 1H NMR consistent with that of 6.
25. 2(S)-[2-(o-tolyl)-4-(2(S)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-methylva-
leric acid methyl ester (9): LC on silica gel (eluent:cyclohexane/ethyl acetate 70/
Acknowledgment
30); ½a 2D5
ꢁ
= +8.5 (c 1, CHCl3); 1H NMR consistent with that of 10.
26. 2(S)-[2-(o-methoxyphenyl)-4-(2(S)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
methylthio-butyric acid methyl ester (3): LC on silica gel (eluent:cyclohexane/
Financial support provided by Ministero dell’Istruzione,
dell’Università e della Ricerca is gratefully acknowledged.
ethyl acetate 1/1); ½a D25
ꢁ
= +18.4 (c 1, CHCl3); 1H NMR consistent with that of 4.
27. [2-(o-methoxyphenyl)-4-(2(S)-1,4-benzodioxan-2-ylmethoxy)benzamido]acetic
acid methyl ester (7): LC on silica gel (eluent:cyclohexane/ethyl acetate 40/60);
References and notes
½
a 2D5
ꢁ
= ꢀ4.30 (c 1, CHCl3); 1H NMR consistent with that of 8.
28. 2(S)-[2-(o-methoxyphenyl)-4-(2(S)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
methylvaleric acid methyl ester (11): LC on silica gel (eluent:cyclohexane/ethyl
1. (a) Casey, P. J.; Seabra, M. C. J. Biol. Chem. 1996, 271, 5289; (b) Basso, A. D.;
Kirschmeier, P.; Bishop, W. R. J. Lipid Res. 2006, 47, 15; (c) Sousa, S. F.;
Fernandes, P. A.; Ramos, M. J. Curr. Med. Chem. 2008, 15, 1478.
acetate 60/40); ½a D25
ꢁ
= +10.7 (c 1, CHCl3); 1H NMR consistent with that of 12.

5504
C. Bolchi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5500–5504
29. (R)-34: ½a 2D5
ꢁ
= +5.0 (c 1, CHCl3); 1H NMR identical to that of (S)-34.
55. 2(S)-[2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethylaminocarbonyl)benzamido]-
30. (R)-35: ½a 2D5
ꢁ
= +4.4 (c 1, CHCl3); 1H NMR identical to that of (S)-35.
= +30.7 (c 1, methanol); 1H NMR identical to that of (S)-36.
= +29.1 (c 1, methanol); 1H NMR identical to that of (S)-37.
4-methylthio-butyric acid methyl ester (14): ½a D25
ꢁ
= +46.8(c 1, CHCl3); 1H NMR
31. (R)-36: ½a 2D5
ꢁ
(CDCl3) d 7.95–8.03 (m, 1H), 7.82 (d, J = 8.05 Hz, 1H), 7.63 (s, 1H), 7.16–7.36 (m,
3H), 6.82–6.90 (m, 4H), 6.66–6.70 (m, 1H), 5.96–6.00 (m, 1H), 4.58–4.65 (m,
1H), 4.31–4.42 (m, 2H), 4.01 (dd, J = 11.35, 7.3 Hz, 1H), 3.84–3.93 (m, 1H),
3.69–3.75 (m, 1H), 3.66 (s, 3H), 1.99–2.17 (m, 8H), 1.82–1.89 (m, 1H), 1.59–
1.65 (m, 1H).
32. (R)-37: ½a 2D5
ꢁ
33. 2(S)-[2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
methylthiobutyric acid methyl ester (2): ½a D25
ꢁ
= +19.0 (c 1, CHCl3); 1H NMR
(CDCl3) d 7.93 (dd, J = 18.75, 8.8 Hz, 1H), 7.09–7.27 (m, 5H), 6.92 (dd, J = 8.8,
2.6 Hz, 1H), 6.75–6.84 (m, 4H), 6.63 (d, J = 2.6 Hz, 1H), 5.74 (d, J = 7.3 Hz, 1H),
4.47–4.55 (m, 2H), 4.32 (dd, J = 11.4, 2.35 Hz, 1H), 4.12–4.24 (m, 3H), 3.57 (s,
3H), 1.93–2.11 (m, 7H), 1.71–1.82 (m, 1H), 1.42–1.54 (m, 2H).
56. [2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethylaminocarbonyl)benzamido]acetic
acid methyl ester (18): ½a D25
ꢁ
= +31.2 (c 1, CHCl3); 1H NMR (CDCl3) d 8.02 (d,
J = 8.2 Hz, 1H), 7.82 (dd, J = 8.2, 1.8 Hz, 1H), 7.64 (d, J = 1.8 Hz, 1H), 7.19–7.33
(m, 4H), 6.82–6.90 (m, 4H), 6.67 (br s, 1H), 5.95 (br s, 1H), 4.38–4.41 (m, 1H),
4.33 (dd, J = 11.4, 2.3 Hz, 1H), 4.01 (dd, J = 11.4, 7.3 Hz, 1H), 3.91 (d, J = 5.3 Hz,
2H), 3.85–3.88 (m, 1H), 3.68–3.75 (m, 1H), 3.66 (s, 3H), 2.11 (s, 3H).
34. [2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]acetic acid methyl
ester (6): ½a 2D5
ꢁ
= +4.3 (c 1, CHCl3); 1H NMR (CDCl3) d 8.01 (d, J = 8.8 Hz, 1H),
7.20–7.32 (m, 4H), 6.99 (dd, J = 8.8, 2.6 Hz, 1H), 6.83–6.92 (m, 4H), 6.71 (d,
J = 2.6 Hz, 1H), 5.81 (br s, 1H), 4.56–4.59 (m, 1H), 4.39 (dd, J = 11. 35, 2.2 Hz,
1H), 4.19–4.28 (m, 3H), 3.99 (d, J = 5.1 Hz, 2H), 3.65 (s, 3H), 2.12 (s, 3H).
35. 2(S)-[2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
57. 2(S)-[2-(o-tolyl)-4-(2(R)-1,4-benzodioxan-2-ylmethyl aminocarbonyl)benzamido]-
4-methylvaleric acid methyl ester (22): ½a D25
ꢁ
= +40.9 (c 1, CHCl3); 1H NMR (CDCl3)
d 7.99–8.05 (d, J = 8.6 Hz, 1H), 7.80 (dd, J = 8.1, 1.8 Hz, 1H), 7.63 (s, 1 H), 7.15–7.33
(m, 4H), 6.81–6.89 (m, 4H), 6.73 (t, J = 5.9 Hz, 1H), 5.76 (d, J = 7.7 Hz, 1H), 4.31–
4.42 (m, 3H), 3.97–4.03 (m, 1H), 3.83–3.92 (m, 1H), 3.66–3.75 (m, 1H), 3.64(s, 3H),
2.05 (s, 3H), 1.25–1.33 (m, 1H), 0.97–1.06 (m, 2H), 0.76 (pseudo t, 6H).
methylvaleric acid methyl ester (10): ½a D25
ꢁ
= +17.9 (c 1, CHCl3); 1H NMR (CDCl3) d
8.05 (dd, J = 22.3, 8.8 Hz, 1H), 7.16–7.33 (m, 3H), 6.99 (dd, J = 88.8, 2.6 Hz, 1H),
6.83–6.92 (m, 5H), 6.70 (t, J = 2.2 Hz, 1H), 5.59 (br s, 1H), 4.37–4.60 (m, 3H), 4.08–
4.31 (m, 3H), 3.63 (s, 3H), 2.05 (s, 3H), 0.92–1.26 (m, 3H), 0.74–0.80 (m, 6H).
36. 2(S)-[2-(o-methoxyphenyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
58. 2(S)-[2-(o-methoxyphenyll)-4-(2(R)-1,4-benzodioxan-2-ylmethylaminocarbonyl)-
benzamido]-4-methylthio-butyric acid methyl ester (16): ½ ꢁ = +54.7 (c 1,
a 2D5
methylthio-butyric acid methyl ester (4): ½a D25
ꢁ
= +25.3 (c 1, CHCl3); 1H NMR
CHCl3); 1H NMR (CDCl3) d 7.79 (d, J = 0.7 Hz, 1H), 7.65 (d, J = 1.1 Hz, 1H),
7.37–7.43 (m, 1H), 7.22 (d, J = 6.6 Hz, 1H), 7.01–7.07 (m, 1H), 6.97 (d, J = 8.4 Hz,
1H), 6.81–6.89 (m, 3H), 6.70 (m, 1H), 6.50 (br s, 1H), 4.63–4.68 (m, 1H), 4.30–
4.39 (m, 2H), 3.96–4.10 (m, 1H), 3.82–3.90 (m, 1H), 3.77 (s, 3H), 3.69–3.75 (m,
1H), 3.66 (s, 3H), 1.87–2.16 (m, 5H), 1.65–1.67 (m, 2H).
(CDCl3) d 7.78 (d, J = 8.4 Hz, 1H), 7.36–7.41 (m, 1H), 7.21 (d, J = 6.2 Hz, 1H),
6.83–7.03 (m, 7H), 6.76 (d, J = 2.6 Hz, 1H), 6.25 (br s, 1H), 4.55–4.68 (m, 2H),
4.39 (dd, J = 11.7, 2.6 Hz, 1H), 4.18–4.31 (m, 3H), 3.78 (s, 3H), 3.66 (s, 3H).
37. [2-(o-methoxyphenyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]acetic
acid methyl ester (8): ½a D25
ꢁ
= +4.0 (c 1, CHCl3); 1H NMR (CDCl3) d 7.80 (d.
59. [2-(o-methoxyphenyll)-4-(2(R)-1,4-benzodioxan-2-ylmethylaminocarbonyl)benz-
J = 8.8 Hz, 1H), 7.26–7.39 (m, 1H), 7.23 (dd, J = 7.3, 1.8 Hz, 1H), 6.80–7.06 (m,
8H), 6.09 (br s, 1H), 4.53–4.58 (m, 1H), 4.26–4.31 (m, 1H), 4.11–4.24 (m, 3H),
3.94 (d, J = 5.1 Hz, 2H), 3.76 (s, 3H), 3.66 (s, 3H).
amido]acetic acid methyl ester (20): ½a D25
ꢁ
= ꢀ33.0 (c 1, CHCl3); 1H NMR (CDCl3) d
7.69–7.76 (m, 2H), 7.61 (d, J = 1.8 Hz, 1H), 7.28–7.34 (m, 1H), 7.17 (dd, J = 6.95,
1.8 Hz, 1H), 6.94–6.99 (m, 1H), 6.82–6.88 (m, 1H), 6.74–6.81 (m, 4H), 6.63 (t,
J = 5.9 Hz, 1H), 6.21 (t, J = 4.8 Hz, 1H), 4.23–4.34 (m, 2H), 3.75–4.05 (m, 4H),
3.68 (s, 3H), 3.60 (s, 3H).
38. 2(S)-[2-(o-methoxyphenyl)-4-(2(R)-1,4-benzodioxan-2-ylmethoxy)benzamido]-4-
methylvaleric acid methyl ester (12): ½a D25
ꢁ
= +18.5 (c 1, CHCl3); 1H NMR (CDCl3) d
7.74 (d, J = 8.8 Hz, 1H), 7.27–7.33 (m, 1H), 7.13 (d, J = 2.05 Hz, 1H), 6.75–6.97
(m, 7H), 6.68 (d, J = 1.2 Hz, 1H), 5.94 (br s, 1H), 4.42–4.51 (m, 2H), 4.31 (dd,
J = 11.4, 2.35 Hz, 1H), 4.08–4.23 (m, 3H), 3.69 (s, 3H), 3.56 (s, 3H), 0.96–1.37 (m,
3H), 0.67–0.73 (dd, J = 12.0, 6.4 Hz, 6H).
60. 2(S)-[2-(o-methoxyphenyll)-4-(2(S)-1,4-benzodioxan-2-ylmethylaminocarbonyl)-
benzamido]-4-methyl valeric acid methyl ester (24): ½a D25
ꢁ
= +51.7 (c 1, CHCl3); 1
H
NMR (CDCl3) d 7.73 (d, J = 8.05 Hz, 1H), 7.78 (dd, J = 8.05, 1.5 Hz, 1H), 7.65 (d,
J = 1.5 Hz, 1H), 7.36–7.42 (m, 1H), 7.20–7.23 (m, 1H), 7.03 (t, J = 7.3 Hz, 1H),
6.94 (d, J = 8.4 Hz, 1H), 6.91–6.96 (m, 4H), 6.65 (br s, 1H), 6.20 (br s, 1H), 4.51–
4.57 (m, 1H), 4.30–4.39 (m, 2H), 3.97–4.03 (m, 1H), 3.83–3.91 (m, 1H), 3.75 (s,
3H), 3.65–3.73 (m, 1H), 3.64 (s, 3H), 1.33–1.37 (m, 1H), 1.09–1.16 (m, 2H),
0.74–0.80 (2 d, 6H).
39. Sebti, S. M.; Hamilton, A. D.; Augeri, D. J.; Barr, K. J.; Donner, J. B.; Fakhoury, S.
A.; O’Connor, S. J.; Rosenberg, S. H.; Shen, W.; Szczepankiewicz, B. G. U.S. patent
2002193596, 2002 ,Chem. Abstr. 2002, 138, 39539.
40. Bolchi, C.; Catalano, P.; Fumagalli, L.; Gobbi, M.; Pallavicini, M.; Pedretti, A.;
Villa, L.; Vistoli, G.; Valoti, E. Bioorg. Med. Chem. 2004, 12, 4937.
61. Cell proliferation assay. Human aortic SMCs were seeded at
a density of
41. (S)-39: LC on silica gel (eluent:cyclohexane/EtOAc 70/30); ½a D25
ꢁ
= ꢀ35.6 (c 1,
8 ꢂ 104 cells/Petri dish (35 mm), and incubated with DMEM supplemented
with 10% FCS. Twenty-four hours later, the medium was changed to one
containing 0.4% FCS to stop cell growth, and the cultures were incubated for
72 h. At this time (time 0), the medium was replaced with one containing 10%
FCS in the presence or absence of known concentrations of the tested
compounds, and the incubation was continued for a further 72 h at 37 °C.
Cell proliferation was evaluated by cell counting after trypsinization of the
monolayers with use of a Coulter Counter model ZM (Corsini, A.; Mazzotti, M.;
Raiteri, M.; Soma, M. R.; Gabbiani, G.; Fumagalli, R.; Paoletti, R. Atherosclerosis
1993, 101, 117). All the compounds were dissolved in DMSO prior to dilution,
being the final concentration of DMSO at a maximum of 0.5%. The final
concentration of DMSO was normalized across all the different conditions. The
CHCl3); 1H NMR (CDCl3) d 8.01 (d, J = 8.05 Hz, 1H), 7.83 (dd, J = 8.05, 1.8 Hz,
1H), 7.62 (d, J = 1.5 Hz, 1H), 7.18–7.31 (m, 3H), 7.06 (d, J = 7.3 Hz, 1H), 6.81–
6.89 (m, 5H), 6.68 (t, J = 5.9 Hz, 1H), 4.37–4.42 (m, 1H), 4.32 (dd, J = 11.35,
2.2 Hz, 1H), 4.03 (dd, J = 11.35, 7.3 Hz, 1H), 3.82–3.88 (m, 1H), 3.65–3.75 (m,
1H), 3.63 (s, 3H), 2.06 (s, 3H).
42. (S)-41: crystallization from diisopropyl ether; ½a D25
ꢁ
= ꢀ34.5 (c 1, CHCl3); 1H
NMR (DMSO-d6) d 12.83 (br s, 1H), 8.93 (t, J = 5.9 Hz, 1H), 7.88–7.96 (m, 2H),
7.71 (d, J = 1.1 Hz, 1H), 7.17–7.24 (m, 3H), 7.06 (d, J = 6.95 Hz, 1H), 6.77–6.86
(m, 4H), 4.28–4.34 (m, 2H), 3.96–4.00 (m, 1H), 3.51–3.57 (m, 2H), 2.01 (s, 3H).
43. 2(S)-[2-(o-tolyl)-4-(2(S)-1,4-benzodioxan-2-ylmethylaminocarbonyl)benzamido]-
4-methylthio-butyric acid methyl ester (13): LC on silica gel
(eluent:cyclohexane/ethyl acetate 1/1);
consistent with that of 14.
44. [2-(o-tolyl)-4-(2(S)-1,4-benzodioxan-2-ylmethylamino carbonyl)benzamido]acetic
acid methyl ester (17): LC on silica gel (eluent:cyclohexane/ethyl acetate 40/60);
½
a 2D5
ꢁ
= ꢀ4.8 (c 1, CHCl3); 1H NMR
concentration of compounds required to inhibit 50% of cell proliferation (IC50)
was calculated by linear regression analysis of the logarithm of the
concentration versus logit by using the SigmaPlot 8.0 software.
62. Ras prenylation assay. HSMCs were seeded at a density of 4 ꢂ 105 cells/Petri
dish (60 mm) and incubated under the same experimental conditions
described for the cell proliferation assay. At the end of the 72 h of incubation
with the tested compounds, cells were washed twice with cold phosphate
½
a 2D5
ꢁ
= ꢀ40.1 (c 1, CHCl3); 1H NMR consistent with that of 18.
45. 2(S)-[2-(o-tolyl)-4-(2(S)-1,4-benzodioxan-2-ylmethyl aminocarbonyl)benzamido]-
4-methylvaleric acid methyl ester (21): LC on silica gel (eluent:cyclohexane/ethyl
acetate 60/40); ½a D25
ꢁ
= ꢀ11.0 (c 1, CHCl3); 1H NMR consistent with that of 22.
buffer saline (PBS) and lysed in 200
NaCl, 0.5% NP-40, 1 mM PMSF, 1 mM NaVO4,
Leupeptin and 1 g/ml Pepstatin) for 30 min on ice. Cell lysates were cleared
l
l buffer (50 mM Tris HCl, pH 7.5, 150 mM
46. (S)-40: LC on silica gel (eluent: cyclohexane/EtOAc 70/30); ½a D25
ꢁ
= ꢀ32.3 (c 1,
1
l
g/ml Aprotinin, g/ml
1 l
CHCl3); 1H NMR (CDCl3) d 7.90 (d, J = 7.7 Hz, 1H), 7.78 (dd, J = 8.1, 1.8 Hz, 1H),
7.71 (d, J = 1.8 Hz, 1H), 7.32–7.38 (m, 1H), 7.24–7.27 (m, 1H), 7.02–7.07 (m,
1H), 6.82–6.91 (m, 5H), 6.69 (br s, 1H), 4.30–4.40 (m, 2H), 4.00 (dd, J = 11.35,
6.95 Hz, 1H), 3.82–3.91 (m, 1H), 3.70 (s, 3H), 3.67 (s, 3H).
l
by centrifugation at 15,000 g for 10 min, and protein concentrations were
determined using the BCA protein assay (Pierce). Lysates were separated by
SDS-PAGE under reducing conditions, transferred to Immobilon PVDF
(Millipore) and subsequently immunoblotted with anti Ras antibody
(Upstate), prior to visualization by enhanced chemiluminescence (ECL,
Amersham Corp.) (Ferri, N.; Colombo, G.; Ferrandi, C.; Raines, E.W.; Levkau,
B.; Corsini, A. Arterioscler. Thromb. Vasc. Biol. 2007, 27, 1043).
47. (S)-42: crystallization from diisopropyl ether; m.p. 72 °C; ½a D25
= ꢀ31.5 (c 1,
ꢁ
CHCl3); 1H NMR (DMSO-d6) d 12.64 (br s, 1H), 8.93 (pseudo t, 1H), 7.78–7.90
(m, 3H), 7.25–7.35 (m, 2H), 6.98–7.04 (pseudo t, 1H), 6.78–6.87 (m, 4H), 4.29–
4.35 (m, 2H), 3.95–4.01 (m, 1H), 3.64 (s, 3H), 3.52–3.60 (m, 2H).
48. 2(S)-[2-(o-methoxyphenyll)-4-(2(S)-1,4-benzodioxan-2-ylmethylaminocarbonyl)-
benzamido]-4-methylthio-butyric acid methyl ester (15): LC on silica gel (eluent:
63. Human aortic SMCs express all three isoforms of Ras (H-Ras, K-Ras, and N-Ras).
The antibody utilized for the detection of Ras by western blotting analysis
recognizes all three isoforms and therefore the isoform specific effect of 1 on
Ras prenylation could not be studied. Nonetheless, the almost complete
inhibition of Ras prenylation by 1 indicates that all three isoforms were
affected by this compound.
cyclohexane/ethyl acetate 1/1); ½a D25
ꢁ
= +6.6 (c 1, CHCl3); 1H NMR consistent with
that of 16.
49. [2-(o-methoxyphenyll)-4-(2(S)-1,4-benzodioxan-2-ylmethylaminocarbonyl)benz-
amido]acetic acid methyl ester (19): LC on silica gel (eluent:cyclohexane/ethyl
acetate 40/60); ½a D25
ꢁ
= ꢀ33.0 (c 1, CHCl3); 1H NMR consistent with that of 20.
64. Compounds 5–12 were tested for their ability to inhibit Ras prenylation at
50. 2(S)-[2-(o-methoxyphenyll)-4-(2(S)-1,4-benzodioxan-2-ylmethylaminocarbonyl)-
benzamido]-4-methyl valeric acid methyl ester (23): LC on silica gel
a
fixed concentration that elicited the highest non-toxic antiproliferative
activity (10 for and 12; 25 for 6, 10 and 11; 50 for 5;
75 for and 8). At this concentration, none of the compounds was
l
7
M
9
l
M
lM
(eluent:cyclohexane/ethyl acetate 60/40); ½a D25
ꢁ
= ꢀ4.5 (c 1, CHCl3); 1H NMR
l
M
consistent with that of 24.
found to significantly affect Ras prenylation excepting 5 (35% inhibition).
Since the incubation with higher concentrations would elicit a toxic effect,
we could not determine Ras prenylation inhibition and respective IC50
value for these compounds.
51. (R)-39: ½a 2D5
ꢁ
= +36.7 (c 1, CHCl3); 1H NMR identical to that of (S)-39.
52. (R)-40: ½a 2D5
ꢁ
= +36.9 (c 1, CHCl3); 1H NMR identical to that of (S)-40.
53. (R)-41: ½a 2D5
ꢁ
= +33.4 (c 1, CHCl3); 1H NMR identical to that of (S)-41.
= +32.3 (c 1, CHCl3); 1H NMR identical to that of (S)-42.
54. (R)-42: ½a 2D5
ꢁ