Thiazole- and imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase

Article abstract of DOI:10.1016/j.bmcl.2011.07.003

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing internal dipeptide AA with 4-amino-2-phenylbenzoic acid and cysteine (C) with 2-amino-4-thiazolyl-, 2-mercapto-4-thiazolyl-, 2-mercapto-4-imidazolyl- and 2-methylmercapto-4-thiazolyl-acetic or propionic acid. The compound in which C is replaced by 2-amino-4-thiazolylacetic acid inhibited FTase activity in the low nanomolar range and showed antiproliferative effect on rat aortic smooth muscle cells interfering with Ras farnesylation. On the basis of these results, 2-aminothiazole can be considered as an alternative to heterocycles, such as pyridine and imidazole, normally used in FTase inhibitors designed as non-thiol CAAX mimetics.

Full text of DOI:10.1016/j.bmcl.2011.07.003

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5408–5412
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Thiazole- and imidazole-containing peptidomimetic inhibitors of protein
farnesyltransferase
Cristiano Bolchi a, Marco Pallavicini a, Sergio K. Bernini b, Giuseppe Chiodini a, Alberto Corsini b, Nicola Ferri b,
Laura Fumagalli a, Valentina Straniero a, Ermanno Valoti a,
⇑
a Dipartimento di Scienze Farmaceutiche ‘Pietro Pratesi’, Università di Milano, via Mangiagalli 25, I-20133 Milano, Italy
b Dipartimento di Scienze Farmacologiche, Università 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 internal dipeptide
AA with 4-amino-2-phenylbenzoic acid and cysteine (C) with 2-amino-4-thiazolyl-, 2-mercapto-4-thiaz-
olyl-, 2-mercapto-4-imidazolyl- and 2-methylmercapto-4-thiazolyl-acetic or propionic acid. The com-
pound in which C is replaced by 2-amino-4-thiazolylacetic acid inhibited FTase activity in the low
nanomolar range and showed antiproliferative effect on rat aortic smooth muscle cells interfering with
Ras farnesylation. On the basis of these results, 2-aminothiazole can be considered as an alternative to
heterocycles, such as pyridine and imidazole, normally used in FTase inhibitors designed as non-thiol
CAAX mimetics.
Received 27 May 2011
Revised 30 June 2011
Accepted 3 July 2011
Available online 13 July 2011
Keywords:
Farnesyltransferase
Peptidomimetic inhibitors
Antiproliferative agents
Antitumors
Ó 2011 Elsevier Ltd. All rights reserved.
Prenylation inihibitors
The farnesylation of the cysteine residue of the C-terminal tet-
rad CAAX of Ras protein by the zinc metalloenzyme farnesyltrans-
ferase (FTase) allows Ras to be localized, after cleavage of the AAX
triad, to the inner surface of cell membrane and to transmit cellular
proliferation signals.1 Therefore, FTase inhibitors (FTis) are re-
garded as antiproliferative agents and actively pursued as antineo-
plastics, though the exact biological mechanism by which they
exert antitumor activity is still controversial.2 Their combination
with some cytotoxic antineoplastic drugs offers promising per-
spectives for cancer treatment.3–5 Moreover, the inhibition of the
proliferation of smooth muscle cells (SMCs) has suggested the po-
tential of FTis in the therapy of vascular disorders such as athero-
sclerosis and restenosis after angioplasty.6–8 The use of CAAX as a
template is one of the classical approaches to the design of FTis.9,10
Based on this strategy, we have recently developed the SS stereo-
isomers of 2-o-tolyl substituted 4-hydroxybenzamide of methio-
nine methyl ester, etherified at the phenolic function with
pyridodioxan-2-ylmethyl (I) or benzodioxan-2-ylmethyl (II),
which exert a significant antiproliferative effect on SMCs (30 and
increases the antiproliferative activity and the RAS prenylation
inhibition.
Compound I had been designed rigidifying the 3-pyridyloxym-
ethyl substructure used to replace the metabolically labile cysteine
in CAAX mimicking inhibitors of FTase such as III,9 while com-
pound II had been conceived as an isoster of I to eliminate the var-
iable of nitrogen position in the aromatic ring of the bicyclic
system (Chart 1).
In a continuation of our strategy to identify new FTis, we con-
sidered the substitution of 3-pyridyl with other aromatic heterocy-
H
N
H
N
O
COOMe
S
O
Y
COOH
S
Me
Me
O
O
6.6
l
M IC50, respectively) interfering with Ras farnesylation.11,12
SAR analysis of two series of I and II analogues indicates that
*
O
N
the nature of the linker to the biphenyl core and the o-substitution
on the latter, but not the dioxane stereochemistry, are critical fea-
tures and that replacement of pyridodioxane with benzodioxane
X
III Y = O
IV Y = NBn
I X = N
II X = CH
⇑
Corresponding author.
E-mail address: ermanno.valoti@unimi.it (E. Valoti).
Chart 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.003

C. Bolchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5408–5412
5409
cles. The 1,3-thiazole ring is a classical isostere of pyridine and
thiazolyl analogues of IV have been described in literature.13 They
have been prepared in an attempt to improve the pharmacokinetic
profile of IV by lowering the basicity of the heterocycle. In partic-
ular, the 3-pyridyl residue of IV has been replaced with 2-, 4- and
5-thiazolylmethyl registering enzyme inhibitions and cellular
potencies sensibly lower than those of IV and, in the only case of
the 5-thiazolylmethyl analogue, comparable to those of III.
Based on these preceding findings, 2-aminothiazole, which is as
basic as pyridine and has pronounced complexation properties to-
wards metal ions, was selected as an interesting candidate to pyri-
dine replacement in alternative to unsubstituted thiazole.
Therefore, we designed compounds 1 and 2, where 2-amino-4-
thiazolylacetic acid and 3-(2-amino-4-thiazolyl)propionic acid
are, respectively, linked to the biphenyl spacer through an amidic
bond. The importance of the interaction cysteine thiol-zinc for Ftase
activity suggested to further modify the heterocycle replacing ami-
no with thiol substituent. In compounds 3 and 4, 2-mercapto-4-
thiazolyl residue is the substructure which intends to mimick the
cysteine chain. Contrarily to its 2-amino analogue, 2-mercapto-
thiazole exhibits a weakly acidic behavior and is implicated in
thiol-thione tautomerism. That’s why we designed also compounds
7 and 8, analogues of 3 and 4 in which thiol is methylated and tau-
tomerism abolished. Finally, we were drawn to consider the 2-mer-
capto-4-imidazolyl analogues 5 and 6, because imidazole is a
thiazole bioisostere and the use of imidazole ring as a metal ligand
in metalloenzyme inhibition is documented (Chart 2).14–16
mercially available (2-amino-4-thiazolyl)acetic acid. This acid only
needed to be esterified, tritylated at the amino function and
desesterified to (2-tritylamino-4-thiazolyl)acetic acid 13 before
reacting with 12 to give 21, namely the tritylamino analogue
of 1a.20–23 Subsequent detritylation afforded compound 1a.24 The
homologous 3-(2-amino-4-thiazolyl)propionic acid was synthe-
sized from methyl 5-bromolevulinate and thiourea.25 Tritylation
and hydrolysis provided the N-protected aminothiazolyl propionic
acid 14, which was used to amidify 12.26–28 The resultant interme-
diate 22, namely the tritylamino precursor of 2a, was detritylated
to give 2a (Scheme 2).29
Cyclization of ethyl 4-chloroacetoacetate and methyl 5-bromo-
levulinate with ammonium dithiocarbamate afforded ethyl (2-
mercapto-4-thiazolyl)acetate and methyl 3-(2-mercapto-4-thiaz-
olyl)propionate, respectively, which were successively converted
into acids 15 and 16 and then condensed with 12 to yield the cor-
responding amides 3a and 4a (Scheme 2).30–35 The methylthio ana-
logues of 3a and 4a, namely the compounds 7a and 8a, were
obtained by the same strategy, but replacing (2-mercapto-4-thiaz-
olyl)acetic acid and 3-(2-mercapto-4-thiazolyl)propionic acid with
the respective S-methyl derivatives 17 and 18 (Scheme 2).36–41
The mercaptoimidazolyl acetic acid 19 and its propionic homo-
logue 20 were, respectively, obtained by (a) conversion of ethyl 4-
chloroacetoacetate and of methyl 5-bromolevulinate into the cor-
responding azides,42,43 (b) reduction to 4-aminoacetoacetate and
5-aminolevulinate, isolated as tosylic acid salts,44,45 (c) cyclization
with potassium thiocyanate giving the mercaptoimidazole
ring,46,47 and (d) ester hydrolysis.48,49 Condensation of 19 and 20
with 12 led to the amides 5a and 6a, respectively (Scheme 2).50,51
The isopropyl esters 1a–8a were hydrolyzed obtaining the cor-
responding acids 1–8 (Scheme 2).52–59
The isopropyl esters 1a–8a were prepared as prodrugs of the
acids 1–8 in order to facilitate cell membrane penetration and to
evaluate the cell activity of the acids exhibiting FTase inhibition.
To synthesize compounds 1–8, we firstly condensed the isopro-
pyl ester of
L
-methionine with 2-o-tolyl-4-nitrobenzoic acid (10),
The effect on protein farnesylation was first tested using a FTase
fluorescent assay and FTI-276 as a reference compound.60 FTI-276
has been reported to inhibit FTase with an IC50 of 0.5 nM;61 in our
study, its IC50 was 9 nM. We found that the compounds 1 and 5 sig-
nificantly inhibit FTase catalytic activity in vitro with an IC50 value
equal to 49 and 122 nM, respectively, while the compounds 2–4
and 8 exhibited lower inhibitory activities and 6 and 7 were not ac-
tive (Table 1). The concentration dependent inhibition of FTase
activity of 1 is shown in Figure 1.
obtained by hydrolysis of the previously reported methyl ester
9.12,17 The resultant N-nitrobenzoylated methionine ester 11 was
hydrogenated to the corresponding aminobenzoylamide 12
(Scheme 1).18,19
All the heteroaryl acetic and heteroaryl propionic acids required
for amidifying 12 had to be prepared from 4-chloroacetoacetate
and 5-bromolevulinate, respectively, with the exception of com-
None of these compounds, having a carboxylic acid group, ex-
erted significant effects on cell growth, as indicated by a cellular
assay measuring inhibition of rat aortic SMC proliferation.62 The
lack of cellular activity could be explained by poor cell penetration.
Therefore, the corresponding isopropyl esters 1a–8a, prepared to
ameliorate membrane permeability, were tested in the same assay
and, as shown in Table 1, most of them proved to inhibit rat SMC
proliferation with IC50 values in the micromolar range. In particu-
lar, compound 1, the most potent FTase inhibitor of the series, re-
duced rat SMC growth in a concentration dependent manner with
O
COOR
O
N
H
(CH2)n
N
H
S
Het
NH2
N
N
N
1
R = H
n = 1
n = 2
Het =
Het =
Het =
Het =
S
S
1a R = CHMe2
an IC50 value of 107
l
M (Fig. 2).
2
R = H
2a R = CHMe2
SH
SH
3
R = H
3a R = CHMe2
R = H
4a R = CHMe2
n = 1
n = 2
COO
O
COO
O
4
b
c
HN
HN
COOY
5
R = H
5a R = CHMe2
R = H
6a R = CHMe2
n = 1
n = 2
S
S
NH
6
O2N
H2N
O2N
a
S
9
Y = Me
11
12
N
Me
7
R = H
7a R = CHMe2
R = H
8a R = CHMe2
n = 1
n = 2
10 Y = H
S
8
Scheme 1. Synthesis of the isopropyl ester of N-(2-o-tolyl-4-aminobenzoyl)-
methionine. Reagents and conditions: (a) 2.5 N NaOH, methanol, room temperature,
12 h, 97%; (b) -methionine isopropyl ester, HOBt, DCC, DMF, room temperature,
15 h, 94%; (c) H2-Pd/C, 10% methanol, 4 h, 68%.
L-
L
Chart 2.

5410
C. Bolchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5408–5412
COOH
HOOC
N
14
N
COOH
15
S
Ph3CHN
a,b,c
S
HS
l,e
N
N
16
S
HS
COOMe
HOOC
EtOOC
d,e
l,f,e
COOH
17
O
N
S
O
d,f,g
MeS
Cl
18
Br
h,m,j,n
h,i,j,k
COOH
MeS
S
HOOC
N
Figure 1. Concentration dependent inhibition of FTase activity by 1.
19
N
H
HS
N
20
N
H
HS
p
q
q
q
q
o
o
o
o
12 + 13
12 + 14
21
1a
1
2
12 + 19
12 + 20
5a
6a
5
6
p
22
2a
3
q
q
q
q
o
o
o
o
12 + 15
12 + 16
3a
4a
7
8
12 + 17
12 + 18
7a
8a
4
Scheme 2. Synthesis of the intermediate heteroarylacetic and heteroarylpropionic
acids and their condensation with 12 to give the compounds 1–8. Reagents and
conditions: (a) thiourea, methanol, room temperature, 15 h, 78% [methyl 3-(2-
amino-4-thiazolyl)propionate]; (b) trityl chloride, TEA, DCM, room temperature,
15 h, 72% ([methyl 3-(2-tritylamino-4-thiazolyl)propionate]); (c) 3 N NaOH, meth-
anol, room temperature, 1 h, 85% (14); (d) ammonium dithiocarbamate, H2O, room
temperature 15 h and then reflux 2 h, crystallization from diisopropyl ether, 50%
[methyl 3-(2-mercapto-4-thiazolyl)propionate]; (e) 2.5 N NaOH, 55 °C, 1 h, 91%
(16), 81% (15) and 97% (17); (f) CH3I, NaH, DME, THF, room temperature, 15 h, 95%
[methyl 3-(2-methylthio-4-thiazolyl)propionate] and 93% [ethyl (2-methylthio-4-
thiazolyl)acetate]; (g) 2.5 N NaOH, methanol, room temperature, 15 h, 95% (18); (h)
NaN3, H2O, THF, 2 °C and then room temperature, 3 h (17 h for the chloroacetoac-
etate), 92% (methyl 5-azidolevulinate) and 95% (ethyl 4-azidoacetoacetate); (i) H2
(4 bar), 10% Pd/C, TsOH, MeOH, room temperature, 4 h, crystallization from
isopropanol, 83% (methyl 5-aminolevulinate tosylic acid salt); (j) KNCS, H2O, reflux,
2 h, 54% [methyl 3-(2mercapto-4-imidazolyl)propionate] and 32% [ethyl (2-mer-
capto-4-imidazolyl)acetate]; (k) 4 N NaOH, 55 °C, 2 h, 72% (20); (l) ammonium
dithiocarbamate, H2O, room temperature 15 h and then reflux 2 h, crystallization
from toluene, 46% [ethyl (2-mercapto-4-thiazolyl)acetate]; (m) H2 (4 bar), 10%
Pd(C), TsOH, EtOH, room temperature, 4 h, crystallization from isopropanol 28%
(ethyl 4-aminoacetoacetate tosylic acid salt); (n) 3 N NaOH, MeOH, reflux, 1 h,
crystallization from isopropanol, 47% (19); (o) DCC, HOBt, TEA, DMF, room
temperature, 15 h, 35–50%;(p) HCOOH, DCM, room temperature, 2 h, 90%; (q) 1 N
KOH, MeOH, room temperature, 15 h, quantitative yield.
Figure 2. Concentration dependent effect of 1 on rat SMC proliferation.
To study whether the ability of compound 1 to interfere with
FTase activity in vitro was maintained also in cultured cells, we
investigated the Ras prenylation by SDS–PAGE from a total cell
lysates of rat SMCs.63 Cells were incubated for 72 h in the presence
and in the absence of compound 1 at a concentration of 50 and
100
lM and in the presence of the 3-methyl-3-glutaryl-coenzyme
A (HMG-CoA) reductase inhibitor simvastatin (2
lM) as positive
control. The compound 1 interfered with Ras farnesylation in a
concentration dependent manner as demonstrated by the appear-
ance of a slower migrating band corresponding to the unprenylat-
ed form of the protein, also detected after incubation with
simvastatin (Fig. 3).
Of the four designed replacements of 3-pyridyl with new het-
erocycles, that with 2-amino-4-thiazolyl gave the best result. In
particular, compound 1, which has a methylenecarbonylamino lin-
ker between aminothiazole and biphenyl core, exhibited a signifi-
cant FTase inhibition both in vitro and in cells exerting
antiproliferative effect. Comparison of FTase activity in vitro on
the basis of the same fluorescent assay indicates that 1 is only little
Table 1
Inhibition of FTase activity and rat SMC proliferation
Compound FTase activity IC50
,
Compound Cell proliferation IC50
,
(nM)
(lM)
1
2
3
4
5
6
7
8
49
1a
2a
3a
4a
5a
6a
7a
8a
107
n.a.
21
25
226
34
390
538
526
122
n.a.
n.a.
415
9
n.a.
n.a.
Figure 3. After 72 h incubation of the cells with indicated concentrations of
compound 1 and 2 lM simvastatin (Simva), total cell lysates were prepared and Ras
FTI-276
prenylation evaluated by Western blotting analysis with a specific antibody anti Ras
(clone RAS10, Millipore). The slower migrating band represents the unprenylated
form of Ras, while the faster migrating band is prenylated Ras.
n.a.: not active. The IC50 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. 21 (2011) 5408–5412
5411
12. Bolchi, C.; Pallavicini, M.; Fumagalli, L.; Ferri, N.; Corsini, A.; Rusconi, C.; Valoti,
E. Bioorg. Med. Chem. Lett. 2009, 19, 5500.
less potent than FTI-276 (49 vs 9 nM IC50) and, consequently, than
III, which is reported equipotent to FTI-276 in FTase inhibition.13
Similar basicity of 2-amino-4-thiazole to pyridine64 and improved
complexation properties65 could account for such results and, at
the same time, explain why, on the contrary, replacement with
2-mercapto-4-thiazolyl is detrimental. Compounds 3 and 4 show
a modest FTase inhibition suggesting that their moderate antipro-
liferative activity is not due to interference with Ras prenylation. It
is evident that the introduction of SH substituent instead of NH2
into the 2 position of 4-thiazolyl is pejorative and that the macro-
scopic difference between 2-aminothiazole and 2-mercaptothiaz-
ole resides in the weak basicity of the former opposed to the
weak acidity of the latter. Such a difference cannot be considered
uninfluential, especially in molecules with a carboxylic head, and
it is not accidental that the fragment mimicking cysteine in the
known CAAX mimetics preferably has weakly basic or neutral char-
acter rather than acidic. Exemplary is the modest FTase inhibition
of the CAAX mimetics with phenolic or thiophenolic groups in place
of cysteine.66,67 Moreover, the tautomeric thione-thiol equilibrium
might further justify the remarkable decrease of activity.68 Obvi-
ously, this does not mean that SH methylation is sufficient to re-
store the FTase activity, as demonstrated by compounds 7 and 8,
which are also modestly or totally inactive. Though 2-methylmer-
capto-4-thiazole doesn’t substantially differ from unsubstituted 4-
thiazole in basicity and complexation properties, compounds 7 and
8 are much less potent than thiazolic CAAX mimetics reported in
literature. Therefore, the negative effect of the methylthio substitu-
ent on thiazole interaction should be of different nature from that
of sulfhydryl.
13. O’Connor, S. J.; Barr, K. J.; Wang, L.; Sorensen, B. K.; Tasker, A. S.; Sham, H.;
Ng, S.-C.; Cohen, J.; Devine, E.; Cherian, S.; Saeed, B.; Zhang, H.; Lee, J. Y.;
Warner, R.; Tahir, S.; Kovar, P.; Ewing, P.; Alder, J.; Mitten, M.; Leal, J.; Marsh,
K.; Bauch, J.; Hoffman, D. J.; Sebti, S. M.; Rosenberg, S. H. J. Med. Chem. 1999,
42, 3701.
14. Cecchi, R.; Ciabatti, R.; Favara, D.; Barone, D.; Bandoli, E. Farmaco Ed. Sci. 1985,
40, 541.
15. Hunt, J. T.; Lee, V. G.; Leftheris, K.; Seizinger, B.; Carboni, J.; Mabus, J.; Ricca, C.;
Yan, N.; Manne, V. J. Med. Chem. 1996, 39, 353.
16. Vasudevan, A.; Qian, Y.; Vogt, A.; Blaskovich, M. A.; Ohkanda, J.; Sebti, S. M.;
Hamilton, A. D. J. Med. Chem. 1999, 42, 1333.
17. Compound 10: DCM/water (pH 2) extraction; mp 166 °C; 1H NMR (CDCl3) d
8.26 (dd, J = 8.5, 2.2 Hz, 1H), 8.14 (m, 2H), 7.27 (m, 3H), 7.07 (d, J = 7.4, 1H), 2.09
(s, 3H).
18. Compound 11: LC on silica gel (eluent: cyclohexane/EtOAc 70:30);
½
a 2D5
ꢀ
¼ þ29:6 (c 1, CHCl3); 1H NMR (CDCl3) d 8.27 (dd, J = 8.5, 2.5 Hz, 1H),
8.05 (m, 2H), 7.30 (m, 3H), 7.17 (d, J = 6.6 Hz, 1H), 6.03 (d, J = 7.4 Hz, 1H), 4.94
(septet, J = 6.05 Hz, 1H), 4.54 (m, 1H), 2.20 (s, 1H), 2.09 (s, 1H), 2.01 (s, 3H), 1.85
(m, 1H), 1.60 (m, 1H), 1.41 (s, 3H), 1.18 (m, 6H).
19. Compound 12: LC on silica gel (eluent: cyclohexane/EtOAc 60:40);
½
a 2D5
ꢀ
¼ þ18:7 (c 1, CHCl3); 1H NMR (CDCl3) d 7.90 (m, 1H), 7.28 (m, 3H), 7.15
(d, J = 7.9 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.78 (s, 1H), 5.85 (m, 1H), 4.95
(septet, J = 6.6 Hz, 1H), 4.55 (m, 1H), 2.20 (s, 2H), 2.04 (m, 6H), 1.80 (m, 1H),
1.57 (m, 1H), 1.20 (m, 6H).
20. Methyl 2-amino-4-thiazolylacetate: obtained in 91% yield as
a white
crystalline solid by treatment of 2-amino-4-thiazolylacetic acid with SOCl2 in
methanol for 12 h; mp 126 °C; 1H NMR (CDCl3) d 6.31 (s, 1H), 5.56 (br s, 2H),
3.77 (s, 3H), 3.58 (s, 2H).
21. Methyl 2-tritylamino-4-thiazolylacetate: obtained in 96% yield as a white
crystalline solid by treatment of methyl 2-amino-4-thiazolylacetate with trityl
chloride and TEA in DCM for 15 h and successive crystallization of the crude
product from diisopropyl ether; mp 177 °C; 1H NMR (CDCl3) d 7.25–7.34 (m,
15H), 6.22 (s, 1H), 3.76 (s, 3H), 3.66 (s, 2H).
22. Compound 13: obtained in 95% yield as a white solid by treatment of methyl 2-
tritylamino-4-thiazolylacetate with 3 N NaOH in boiling methanol for 1 h; mp
152 °C; 1H NMR (CDCl3) d 7.25–7.34 (m, 15H), 6.05 (s, 1H), 3.58 (s, 2H).
23. Compound 21: 1H-NMR (COCl3) d 8.63 (br s, 1H), 7.90–7.82 (m, 1H), 7.41–7.06
(m, 21H), 6.41 (s, 1H), 6.02 (s, 1H), 5.88 (t, J = 7.15 Hz, 1H), 4.99–4.91 (m, 1H),
4.57–4.50 (m, 1H), 2.88–2.84 (m, 2H), 2.65–2.61 (m, 2H), 2.05–1.97 (m, 6H),
1.84–1.70 (m, 2H), 1.56–1.49 (m, 2H), 1.21–1.17 (m, 6H).
The presence of SH substituent seems deleterious also in the
imidazole derivatives 5 and 6, though less than in the thiazole
derivatives 3 and 4, if we consider compound 5. The use of imidaz-
ole to mimick cysteine in CAAX mimetics is widely exemplified and
is generally associated with high FTase activity.5,69,70 Here, 5 shows
only a moderate FTase inhibition and a modest antiproliferative
activity. The reasons, we think, could be analogous to those in-
24. Compound 1a: 1H NMR (DMSO-d6) d 10.16 (s, 1H), 7.95 (d, J = 7.43 Hz, 1H),
7.65 (dd, J = 1.93 Hz, J = 2.21 Hz, 1H), 7.51–7.44 (m, 2H), 7.19–7.10 (m, 4H),
6.29 (s, 1H), 4.90–4.97 (m, 1H), 4.28–4.24 (m, 1H), 3.47 (s, 2H), 2.39–2.15 (m,
2H), 2.12 (s, 3H), 2.05 (s, 3H), 1.89–1.69 (m, 2H), 1.21–1.16 (m, 6H).
25. Methyl 3-(2-amino-4-thaizolyl)propionate: mp 191.33 °C; 1H NMR (DMSO-d6)
d 9.09 (br, 2H), 6.54 (s, 1H), 3.59 (s, 3H), 2.77–2.63 (m, 4H).
voked comparing
3 with 1 and with literature thiazolyl
26. Methyl 3-(2-tritylamino-4-thaizolyl)propionate: mp 149.50 °C; 1H NMR
(CDCl3) d 10.64 (br s, 1H), 7.39–7.29 (m, 15H), 5.93 (s, 1H), 3.68 (s, 3H), 2.96
(t, J = 7.15 Hz, 2H), 2.76 (t, J = 7.15 Hz, 2H).
derivatives.13
In conclusion, we have demonstrated that 2-aminothiazole can
successfully replace cysteine in Ras CAAX mimetics and deserves to
be considered, when designing non-thiol-FTis, an alternative to
unsubstituted heterocycles, such as pyridine, imidazole and thia-
zole. On the contrary, the introduction of sulfhydryl into the 2 po-
sition of these two latter heterocycles seems to prejudice their
ability of mimicking terminal cysteine.
27. Compound 14: 1H NMR (CDCl3) d 10.38 (br s, 1H), 7.37–7.29 (m, 15H), 5.95 (s,
1H), 2.95–2.90 (m, 2H), 2.76–2.71 (m, 2H).
28. Compound 22: 1H NMR (CDCl3) d 8.60 (br s, 1H), 7.95–7.87 (m, 1H), 7.44–7.11
(m, 21H), 6.47 (s, 1H), 6.02 (s, 1H), 5.84 (t, J = 7.15 Hz, 1H), 4.97–4.92 (m, 1H),
4.57–4.50 (m, 1H), 2.88–2.84 (m, 2H), 2.65–2.61 (m, 2H), 2.05–1.97 (m, 6H),
1.86–1.78 (m, 2H), 1.58–1.51 (m, 2H), 1.21–1.17 (m, 6H).
29. Compound 2a: ½a D25
ꢀ
¼ þ18:3 (c 1, CHCl3); 1H NMR (CDCl3) d 8.79 (br s, 1H),
7.96–7.87 (m, 1H), 7.51–7.13 (m, 6H), 6.18 (s, 1H), 5.87 (t, J = 7.15 Hz, 1H),
5.20–5.05 (br s, 2H), 4.99–4.90 (m, 1H), 4.59–4.48 (m, 1H), 2.93–2.89 (m, 2H),
2.73–2.69 (m, 2H), 2.06–1.94 (m, 6 H), 1.86–1.74 (m, 2H), 1.60–1.52 (m, 2H),
1.21–1.16 (m, 6H).
References and notes
30. Ethyl 2-mercapto-4-thiazolylacetate: mp 143 °C; 1H NMR (CDCl3) d 6.46 (s,
1H), 4.23 (q, J = 3.3 Hz, 2H), 3.58 (s, 2H), 1.29 (t, J = 3.3 Hz, 3H).
31. Compound 15: mp 166 °C; 1H NMR (DMSO-d6) d 6.70 (s, 1H), 3.52 (s, 2H), 3.29
(s, 1H).
1. Macaluso, M.; Russo, G.; Cinti, C.; Bazan, V.; Gebbia, N.; Russo, A. J. Cell. Physiol.
2002, 192, 125.
2. Sousa, S. F.; Fernandes, P. A.; Ramos, M. J. Curr. Med. Chem. 2008, 15, 1478.
3. Adjei, A. A.; Davis, J. N.; Bruzek, L. M.; Erlichman, C.; Kaufmann, S. H. Clin.
Cancer Res. 2001, 7, 1438.
4. Taylor, S.; Marrinan, C. H.; Liu, G.; Nale, L.; Bishop, W. R.; Kirshmeier, P.; Liu, M.;
Long, B. J. Gynecol. Oncol. 2008, 109, 97.
5. Shi, B.; Yaremko, B.; Hajian, G.; Terracina, G.; Bishop, W. R.; Liu, M.; Nielsen, L.
L. Cancer Chemoth. Pharm. 2000, 46, 387.
6. Ross, R. N. Eng. J. Med. 1999, 340, 115.
7. Ueno, H.; Yamamoto, H.; Ito, S.; Li, J. J.; Takeshita, A. Arterioscler. Thromb. Vasc.
Biol. 1997, 17, 898.
8. Indolfi, C.; Avvedimento, E. V.; Rapacciuolo, A.; Di Lorenzo, E.; Esposito, G.;
Stabile, E.; Feliciello, A.; Mele, E.; Giuliano, P.; Condorelli, G.; Chiariello, M. Nat.
Med. 1995, 1, 541.
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.
32. Methyl 3-(2-mercapto-4-thiazolyl)propionate: mp 127 °C; 1H NMR (CDCl3) d
11.80 (s, 1H), 3.71 (s, 3H), 2.84 (t, J = 6.6 Hz, 2H), 2.66 (t, J = 6.6 Hz, 2H).
33. Compound 16: mp 192 °C; 1H NMR (DMSO-d6) d 12.26 (s, 1H), 6.58 (s, 1H), 3.35
(s, 1H), 2.70 (m, 2H), 2.51 (m, 2H); 1H NMR (D2O) d 6.57 (s, 1H), 2.78 (t,
J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz, 2H).
34. Compound 3a: LC on silica gel (eluent: cyclohexane/EtOAc 50:50); ½a D25
¼ þ9:9
ꢀ
(c 1, CHCl3); 1H NMR (DMSO-d6) d 10.32 (s, 1H), 8.08 (d, J = 8.0 Hz, 1H), 7.62 (d,
J = 1.9 Hz, 1H), 7.52–7.42 (m, 2H), 7.16–7.05 (m, 4H), 6.73 (s, 1H), 4.84 (septet,
J = 6.1 Hz, 1H), 4.19–4.15 (m, 1H), 3.65 (s, 2H), 2.22–1.98 (m, 5H), 1.94 (s, 3H),
1.82–1.68 (m, 2H), 1.18–1.10 (m, 6H).
35. Compound 4a: LC on silica gel (eluent: cyclohexane/EtOAc 50:50);
½
a 2D5
ꢀ
¼ þ12:1 (c 1, CHCl3); 1H NMR (DMSO-d6)
d 10.16 (s, 1H), 8.04 (d,
J = 6.9 Hz, 1H), 7.60 (dd, J = 8.5, 1.9 Hz, 1H), 7.54–7.40 (m, 2H), 7.18–7.02 (m,
4H), 6.54 (s, 1H), 4.84 (septet, J = 6.3 Hz, 1H), 4.21–4.17 (m, 1H), 2.78–2.68 (m,
2H), 2.67–2.60 (m, 2H), 2.16–1.85 (m, 8H), 1.80–1.62 (m, 2H), 1.18–1.05 (m,
6H).
10. Sakowski, J.; Böhm, M.; Sattler, I.; Dahse, H.-M.; Schlitzer, M. J. Med. Chem.
2001, 44, 2886.
11. 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.
36. Ethyl 2-methylmercapto-4-thiazolyl acetate: 1H NMR (CDCl3) d 7.08 (s, 1H),
4.18 (q, J = 3.9 Hz, 2H), 3.78 (s, 2H), 2.68 (s, 3H), 1.24 (t, J = 3.9 Hz, 3H).
37. Compound 17: mp 122 °C; 1H NMR (DMSO-d6) d 7.34 (s, 1H), 3.65 (s, 2H), 2.64
(s, 3H).

5412
C. Bolchi et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5408–5412
38. Methyl 3-(2-methylmercapto-4-thiazolyl)propionate: 1H NMR (CDCl3) d 6.83
(s, 1H), 3.67 (s, 3H), 3.03 (t, J = 7.7 Hz, 2H), 2.73 (t, J = 7.7 Hz, 2H), 2.66 (s, 3H).
39. Compound 18: mp 97 °C; 1H NMR (DMSO-d6) d 12.14 (br s, 1H), 7.18 (s, 1H),
2.85 (t, J = 7.4 Hz, 2H), 2.63 (s, 3H), 2.56 (t, J = 7.4 Hz, 2H).
58. Compound 7: ½a D25
ꢀ
¼ ꢁ9:3 (c 0.5, MeOH); 1H NMR (DMSO-d6) d 10.38 (s, 1H),
7.78–7.68 (br s, 1H), 7.59–7.51 (m, 1H), 7.48–7.41 (m, 1H), 7.36 (s, 1H), 7.18–
7.03 (m, 4H), 4.09 (m, 1H), 3.76 (s, 2H), 2.63 (s, 3H), 2.20–1.98 (m, 6H), 1.85–
1.58 (m, 4H).
40. Compound 7a: LC on silica gel (eluent: cyclohexane/EtOAc 50:50);
59. Compound 8: ½a D25
ꢀ
¼ ꢁ2:6 (c 0.25, MeOH); 1H NMR (DMSO-d6) d 10.17 (s, 1H),
½
a 2D5
ꢀ
¼ þ12:4 (c 1.2, CHCl3); 1H NMR (DMSO-d6) d 10.35 (s, 1H), 8.08 (d,
8.27 (s, 1H), 7.60–7.57 (m, 1H), 7.50–7.44 (m, 2H), 7.33 (br s, 1H), 7.18–7.12
(m, 4H), 3.94–3.85 (m, 1H), 2.96–2.91 (m, 2H), 2.71–2.63 (m, 2H), 2.62 (s, 3H),
2.10–1.91 (m, 6H), 1.70–1.58 (m, 4H).
J = 6.7 Hz, 1H), 7.62 (m, 1H), 7.49 (m, 2H), 7.36 (m, 1H), 7.15 (m, 4H), 4.84
(septet, J = 6.3 Hz, 1H), 4.18 (m, 1H), 3.76 (s, 2H), 2.63 (s, 3H), 2.04 (m, 8H), 1.75
(m, 2H), 1.14 (m, 6H).
60. In vitro FTase assay: In vitro IC50 data were determined against FTase using an
in vitro Fluorescence Assay as previously described with minor modifications
(Krzysiak, A. J.; Scott, S. A.; Hicks, K. A.; Fierke, C. A.; Gibbs, R. A. Bioorg. Med.
Chem. Lett. 2007, 17, 5548). In brief, the substrates used were Dansyl-Gly-Cys-
Val-Leu-Ser-OH peptide (Calbiochem) and farnesyl-pyrophosphate (FPP)
41. Compound 8a: LC on silica gel (eluent: cyclohexane/EtOAc 60:40);
½
a 2D5
ꢀ
¼ þ14:6 (c 1.2, CHCl3); 1H NMR (DMSO-d6) d 10.13 (s, 1H), 8.03 (d,
J = 6.9 Hz, 1H), 7.61 (d, J = 6.9 Hz, 1H), 7.46 (m, 2H), 7.15 (m, 5H), 4.83 (septet,
J = 6.3 Hz, 1H), 4.18 (m, 1H), 2.95 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.62
(s, 3H), 2.07 (m, 8H), 1.74 (m, 2H), 1.15 (m, 6H).
(SIGMA). Each reaction mixture contained 0.9
concentration 3 M), 1.3 l of FPP (final concentration 10
Assay buffer (MTT 5.8 mM, MgCl2 12 mM, ZnCl2 12 mM, Tris pH 7.5 52 mM)
aliquoted in a 96 wells plate (200 l each well). The reaction was started by
adding of rat recombinant FTase (7.5 per reaction, Calbiochem) and
l
l
of Dansyl peptide (final
42. Ethyl 4-azidoacetoacetate: 1H NMR (DMSO-d6) d 4.26 (s, 2H), 4.07 (q, J = 3.6 Hz,
2H), 3.60 (s, 2H), 1.17 (t, J = 3.6 Hz, 3H).
l
l
l
M) in 300 l of
l
43. Methyl 5-azidolevulinate: 1H NMR (CDCl3) d 4.00 (s, 2H), 3.64 (s, 3H), 2.69(m,
2H), 2.64 (m, 2H).
l
lg
44. Ethyl 4-aminoacetoacetate tosylate: mp 111 °C; 1H NMR (DMSO-d6) d 8.07 (br
s, 3H), 7.46 (d, J = 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 4.10 (q, J = 7.15 Hz, 2H),
3.98 (s, 2H), 3.70 (s, 2H), 2.15 (s, 3H), 1.10 (t, J = 7.15 Hz, 3H).
45. Methyl 5-aminolevulinate tosylate: mp 130 °C; 1H NMR (DMSO-d6) d 7.99 (s,
3H), 7.46 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 3.96 (d, J = 4.4 Hz, 2H), 3.58
(s, 3H), 2.78 (t, J = 6.6 Hz, 2H), 2.50 (t, J = 6.6 Hz, 2H).
conducted in the presence or absence of tested compounds and the FTase
inhibitor FTI-286 as a reference. The fluorescent signal was recorder by using
the Fluoroskan Ascent Microplate Fluorometer (Labsystem) every 15 s for
5 min. The IC50 value was calculated from the fluorescent signal at the steady
state.
61. Lerner, E. C.; Qian, Y.; Blaskovich, M. A.; Fossum, R. D.; Vogt, A.; Sun, J.; Cox, A.
D.; Der, C. J.; Hamilton, A. D.; Sebti, S. M. J. Biol. Chem. 1995, 270, 26802.
62. Cell proliferation assay: Rat aortic SMCs were seeded at a density of 2 ꢂ 105
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
46. Ethyl (2-mercapto-4-imidazolyl)acetate: mp 152 °C; 1H NMR (DMSO-d6) d
11.86 (s, 1H), 11.75 (s, 1H), 6.63 (s, 1H), 4.07 (q, J = 7.2 Hz, 2H), 3.47 (s, 2H),
1.17 (t, J = 7.2 Hz, 3H).
47. Methyl 3-(2-mercapto-4-imidazolyl)propionate: mp 161 °C; 1H NMR (DMSO-
d6) d 11.85 (s, 1H), 11.64 (s, 1H), 6.51 (s, 1H), 3.57 (s, 3H), 2.58 (s, 4 H); 1H NMR
(D2O) d 6.57 (s, 1H), 3.56 (s, 3H), 2.70 (m, 2H), 2.57 (m, 2H).
48. Compound 19: mp 177 °C; 1H NMR (DMSO-d6) d 11.82 (s, 1H), 11.70 (s, 1H),
6.60 (s, 1H), 3.38 (s, 2H).
49. Compound 20: mp 203 °C; 1H NMR (D2O) d 6.60 (s, 1H), 2.70 (t, J = 7.4 Hz, 2H),
2.58 (t, J = 7.4 Hz, 2H).
50. Compound 5a: LC on silica gel (eluent: DCM/MeOH 95:5); ½a D25
¼ þ4:7 (c 1.3,
ꢀ
CHCl3); 1H NMR (DMSO-d6) d 11.87 (s, 1H), 11.73 (s, 1H), 10.23 (s, 1H), 8.07 (d,
J = 7.15 Hz, 1H), 7.61 (dd, J = 5.5, 3.3 Hz, 1H), 7.45 (m, 2H), 7.14 (m, 4H), 6.64 (s,
1H), 4.85 (septet, J = 3.6 Hz, 1H), 4.19 (m, 1H), 3.51 (s, 2H), 2.12 (m, 4H), 1.94 (s,
3H), 1.75 (m, 2H), 1.21 (s, 1H), 1.14 (m, 6H).
concentration of DMSO at
a maximum of 0.5%. The 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.
63. Ras prenylation assay: Rat SMCs 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
51. Compounud 6a: LC on silica gel (eluent: DCM/MeOH 95:5); ½a D25
¼ þ0:55 (c 1,
ꢀ
acetone); 1H NMR (DMSO-d6) d 10.13 (s, 1H), 8.03 (d, J = 6.88 Hz, 1H), 7.61 (d,
J = 6.90 Hz, 1H), 7.46 (m, 2H), 7.15 (m, 5H), 4.83 (septet, J = 6.33 Hz, 1H), 4.18
(m, 1H), 2.95 (t, J = 7.15 Hz, 2H), 2.66 (t, J = 7.15 Hz, 2H), 2.62 (s, 3H), 2.07 (m,
8H), 1.74 (m, 2H), 1.15 (m, 6H).
with compound
phosphate buffer saline (PBS) and lysed in 200
7.5, 150 mM NaCl, 0.5% NP-40, 1 mM PMSF, 1 mM NaVO4, 1
g/ml Leupeptin and 1 g/ml Pepstatin) for 30 min on ice. Cell lysates were
1
and simvastatin, cells were washed twice with cold
l buffer (50 mM Tris HCl, pH
g/ml Aprotinin,
l
52. Compound 1: ½a D25
ꢀ
¼ ꢁ8:9 (c 0.5, MeOH-d6); 1H NMR (DMSO-d6) d 10.18 (s,
l
1H), 7.95–7.82 (m, 1H), 7.65–7.59 (m, 1H), 7.51–7.48 (m, 2H), 7.21–7.10 (m,
4H), 6.90 (s, 2H), 6.29 (s, 1H), 4.18–4.14 (m, 1H), 3.45 (s, 2H), 2.22–1.59 (m,
10H).
1
l
l
cleared by centrifugation at 15,000g 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 (Clone
RAS10, Millipore), 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).
53. Compound 2: ½a D25
ꢀ
¼ ꢁ4:4 (c 0.5, MeOH-d6); 1H NMR (DMSO-d6) d 10.13 (br s,
1H), 7.96–7.85 (m, 1H), 7.63–7.60 (m, 1H), 7.49–7.44 (m, 2H), 7.18–7.05 (m,
4H), 6.81 (s, 2H), 6.13 (s, 1H), 4.21–4.15 (m, 1H), 2.69–2.60 (m, 4H), 2.26–1.94
(m, 6H), 1.86–1.60 (m, 4H).
54. Compound 3: ½a D25
ꢀ
¼ ꢁ17:9 (c 0.5, 1 M NaOH); 1H NMR (DMSO-d6 100 °C) d
10.35 (s, 1H), 7.62–7.51 (m, 2H), 7.42 (s, 1H), 7.19–7.03 (m, 4H), 6.63–6.58 (m,
2H), 4.14 (m, 1H), 3.60 (s, 2H), 2.21–1.98 (m, 6H), 1.82–1.58 (m, 4H).
64. Ög˘retir, C.; Demirayak, S.; Tay, N. F.; Duran, M. J. Chem. Eng. Data 2008, 53, 422.
65. Basak, A. K.; Banerjea, D. J. Indian Chem. Soc. 1978, 55, 853.
66. Patel, D. V.; Patel, M. M.; Robinson, S. S.; Gordon, E. M. Bioorg. Med. Chem. Lett.
1994, 4, 1883.
67. Kowalczyk, J. J.; Ackermann, K.; Garcia, A. M.; Lewis, M. D. Bioorg. Med. Chem.
Lett. 1995, 5, 3073.
68. Makarova, L. G.; Usatenko, Y. I.; Barkalov, V. S.; Krasovskii, V. A. Zhurnal
Prikladnoi Spektroskopii 1979, 31, 174.
69. Breslin, M. J.; deSolms, S. J.; Giuliani, E. A.; Stoker, G. E.; Graham, S. L.;
Pompliano, D. L.; Mosser, S. D.; Hamilton, K. A.; Hutchinson, J. H. Bioorg. Med.
Chem. Lett. 1998, 8, 3311.
55. Compound 4: ½a D25
ꢀ
¼ ꢁ10:7 (c 0.5, MeOH); 1H NMR (DMSO-d6) d 10.21 (s, 1H),
7.97–7.85 (m, 1H), 7.60–7.52 (m, 1H), 7.50–7.39 (m, 1H), 7.22–7.01 (m, 4H),
6.54 (s, 1H), 6.22 (br s, 1H), 4.21–4.12 (m, 1H), 2.81–2.62 (m, 4H), 2.16–1.63
(m, 10H).
56. Compound 5: ½a D25
ꢀ
¼ ꢁ7:0 (c 0.5, MeOH); 1H NMR (DMSO-d6) d 11.89 (br s, 1H),
11.74 (br s 1H), 10.28 (s, 1H), 7.81–7.77 (m, 1H), 7.60–7.58 (m, 1H), 7.51–7.49
(m, 2H), 7.22–7.01 (m, 4 H), 6.60 (s, 1H), 4.18–4.10 (m, 1H), 3.76 (s, 2H), 2.20–
1.88 (m, 6H), 1.76–1.60 (m, 4H).
57. Compound 6: ½a D25
ꢀ
¼ þ0:5 (c 0.5, MeOH); 1H NMR (DMSO-d6) d 11.88 (br s,
0.5H), 11.64 (0.5H), 10.15 (s, 1H), 7.59–7.63 (m, 4H), 7.22–7.05 (m, 4H), 6.49 (s,
70. Ohkanda, J.; Strickland, C. L.; Blaskovich, M. A.; Carrico, D.; Lockman, J. W.;
Vogt, A.; Bucher, C. J.; Sun, J.; Qian, Y.; Knowles, D.; Pusateri, E. E.; Sebti, S. M.;
Hamilton, A. D. Org. Biomol. Chem. 2006, 4, 482.
1H), 4.10–3.98 (m, 1H), 2.66–2.58 (m, 4H), 2.20–1.88(m, 6H), 1.76–1.58 (m,
4H)