Synthesis and biological evaluation of potential bisubstrate inhibitors of protein farnesyltransferase. Design and synthesis of functionalized imidazoles

Article abstract of DOI:10.1039/b709854e

A novel series of compounds, derived from 2,5-functionalized imidazoles, have been synthesized as potential bisubstrate inhibitors of protein farnesyltransferase (FTase) using structure-based design. These compounds have a 1,4-diacid chain and a tripeptid

Full text of DOI:10.1039/b709854e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of potential bisubstrate inhibitors of
protein farnesyltransferase. Design and synthesis of functionalized
imidazoles†
Renata Marcia de Figueiredo, Lae¨titia Coudray and Joe¨lle Dubois*
Received 28th June 2007, Accepted 20th July 2007
First published as an Advance Article on the web 28th August 2007
DOI: 10.1039/b709854e
A novel series of compounds, derived from 2,5-functionalized imidazoles, have been synthesized as
potential bisubstrate inhibitors of protein farnesyltransferase (FTase) using structure-based design.
These compounds have a 1,4-diacid chain and a tripeptide connected by an imidazole ring. The
synthetic strategy relies on the functionalization at the C-2 position of the heterocycle with the diacid
side chain and peptide coupling at the C-5 position. Several new compounds were synthesized in good
yields. Kinetic experiments on the most active compounds revealed different binding modes depending
on the diacid chain length.
Introduction
Since its identification, the protein farnesyltransferase (FTase)
has emerged as a promising target in cancer therapy.1 Recently,
it has also appeared as a potential target for the treatment of
parasitic diseases.2 FTase is a zinc metalloenzyme3 that catalyses
the addition of the lipid 15-carbon isoprenyl farnesyl moiety from
farnesylpyrophosphate (FPP) to a sulfhydryl of a cysteine residue
embedded in the C-terminal CaaX sequence motif of cellular
signal transduction proteins.4 The CaaX box is defined by an
invariant cysteine residue (C), two aliphatic residues (aa) and the
C-terminal residue (X), which contributes to substrate specificity.
The farnesylation reaction is part of a series of modifications
necessary for plasma membrane association, extracellular signal
transmission and cell proliferation.4,5
Fig. 1 Transition state of FTase (1) and our design of transition state
analogues (2).
A broad range of FTase inhibitors (FTIs) that mimic the C-
terminal CaaX tetrapeptide and the farnesylpyrophospate (FPP)
have been already described,6 however only a few compounds
designed to mimic both substrates have been reported so far.7 This
kind of compound is classified as a bisubstrate derivative, which
is expected to exhibit better specificity and affinity for the enzyme
than either substrate alone due to its similarity with the transition
state of FTase.
In this paper, we wish to report our initial results on the synthesis
and the biological evaluation of higher-substituted imidazoles
with the aim of obtaining a novel class of FTIs. Based on the
studies reported by the Fierke,8 Poulter9 and Beese10 groups on
the transition state 1 of this enzyme, we designed a new class of
compounds with the general structure 2 outlined in Fig. 1 in order
to connect the CaaX- and pyrophosphate-binding sites.
The principal scaffold of our analogues is the imidazole ring. In
spite of the difficulty of working with this heterocycle due to its
particular reactivity, imidazole is a very important moiety of FTIs
because of the strong interaction of its basic nitrogen with the
zinc atom in the FTase catalytic binding site. Numerous FTIs bear
an imidazole ring such as tipifarnib11 and BMS-214662,12 which
are under clinical trials against cancer. It has been shown by X-
ray crystallography of these anticancer candidates complexed with
FTase that the imidazole is effectively bound to the zinc atom.13
A 1,4-diacid was chosen to go into the pyrophosphate binding
site because FPP competitive inhibitors bearing a 1,4-diacid, such
as chaetomellic acid A, have shown very good affinities for the
enzyme.14 Finally, the peptidic side chain is believed to function
as a mimic of the CaaX box. In order to obtain better interaction
with the enzyme, the chain length between the heterocycle and the
diacid has been varied.
The originality of these compounds relies on the 1,4-diacid
scaffold as well as the highly functionalized imidazole ring.
In fact, all the described imidazole-containing FTIs are either
monosubstituted in the C-4/5 position (like BMS-214662) or
disubstituted in the N-1 and C-5 positions (like tipifarnib). To
the best of our knowledge, no compound with a structure related
to our general model 2 has been described to date. Finally, this
work could also provide additional information about the exact
transition state of this enzyme that remains currently unknown.
Institut de Chimie des Substances Naturelles, CNRS, Avenue de la Terrasse,
91198, Gif-sur-Yvette cedex, France. E-mail: joelle.dubois@icsn.cnrs-gif.fr
† Electronic supplementary information (ESI) available: Experimental
data for selected compounds and Lineweaver–Burk plots of the kinetic
data. See DOI: 10.1039/b709854e
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This article describes the synthesis and the biological activity
of the first compounds of this new class of FTIs with the peptide
chain linked at the C-5 position of the imidazole ring.
Results and discussion
1. Chemistry
Scheme 1 Retrosynthetic analysis.
Our first approach relied on the introduction of the diacid side
chain at the C-2 position of the imidazole moiety followed by the
coupling of the peptide at the C-5-substituted position. Among
the reactions of 2-lithioimidazoles with electrophiles,15 only a few
methods have so far been described for the direct introduction of a
functionalized alkyl chain at the C-2 position of this heterocycle.16
In fact, our initial efforts to execute the C-2 functionalization by
reaction between 2-lithioimidazole and bromoesters were unsuc-
cessful. Therefore, we looked into palladium-catalyzed coupling
reactions with 2-iodoimidazole derivatives under Heck conditions.
No C-2-substituted compound could be isolated under these
conditions, but these attempts allowed us to find a new way to
synthesize imidazoisoindole derivatives, and this new reaction has
been the subject of another study reported elsewhere.17
Therefore, we planned another retrosynthetic route, depicted in
Scheme 1. The peptide chain could be introduced at the end of the
syntheses by reductive amination of the aldehydes 3 obtained after
deprotection and oxidation of the C-5 hydroxymethyl moiety of
compounds 4. The key step of the syntheses is the introduction of
the 1,4-diester moiety by a Horner–Wadsworth–Emmons reaction
on the aldehydes 5 obtained by functionalization at the C-2
position of the 1-methyl-5-hydroxymethylimidazole derivative 6
synthesized from 1,3-dihydroxyacetone dimer 7.
Scheme 2 Reagents and conditions: a) KSCN, CH3NH2·HCl, AcOH,
1-butanol, RT, 3 days, 62%; b) 2.4 N HNO3, NaNO2 cat., RT, 6 h, 75%;
c) TBDMSCl, imidazole, DMF, RT, 2 h, 91%; d) nBuLi, THF, −78 ◦C,
45 min, then DMF, −78 ◦C → RT, 2 h 30 min, 83%.
core and the 1,4-diester chain. Our approach was accomplished
by using a Horner–Wadsworth–Emmons reaction22 involving a
phosphonosuccinic ester followed by the hydrogenation of the
double bond (Scheme 3). The 2-(diethoxyphosphoryl)succinic
acid diethyl ester 9 has previously been synthesized in one step
using a slightly modified Linke protocol.23 Under typical Horner–
Wadsworth–Emmons conditions, compound 10 was obtained in
Starting from the commercially available 1,3-dihydroxyacetone
dimer 7, the N-methyl-5-(hydroxymethyl)imidazole 8 was readily
obtained in two steps (47% overall yield) using the Marckwald18
procedure slightly modified by Rapoport19 and Collman20
(Scheme 2). Compound 8 was easily converted into aldehyde 5a
(nꢀ = 0) via TBS-protection followed by formylation21 of the C-
2 position (75% yield after two steps). Next, this compound 5a
was used as a common intermediate for the syntheses of all our
analogues.
With aldehyde 5a in hand, we started the synthesis of the
intermediate 4a bearing only one carbon between the imidazole
Scheme 3 Reagents and conditions: a) 9, NaH, THF, RT, 2 h, (100% E
isomer), 69%; b) H2 (1 atm), Pd/C (10%), EtOAc, RT, 4 h, 100%.
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69% yield, and following catalytic hydrogenation gave the expected
compound 4a in quantitative yield.
With 4a synthesized, we turned our attention to the intermediate
4c with a three-carbon chain between the imidazole ring and
the 1,4-diester part (Scheme 4). The aldehyde 5a was submitted
to Horner–Wadsworth–Emmons olefination with triethyl phos-
phonoacetate. The double bond of the resulting ester was then
hydrogenated and the ester was reduced to the corresponding
aldehyde 5c (nꢀ = 2). An additional Horner–Wadsworth–Emmons
olefination with 9 afforded the required compound 11 in 70% yield,
which was further hydrogenated to give the analogue 4c in 92%
yield.
Scheme 5 Attempts at the synthesis of aldehyde 5b.
Scheme 4 Reagents and conditions: a) (EtO)2P(O)CH2CO2Et, NaH, THF,
0
◦C → RT, 3 h, (E/Z 9 : 1), 77%; b) H2 (1 atm), Pd/C (10%), EtOAc,
RT, 3 h, 100%; c) DIBAL-H, CH2Cl2, −78 ◦C, 2 h, 82%; d) 9, NaH, THF,
0
◦C → RT, 1 h 40 min, (E/Z 4 : 1), 70%; e) H2 (1 atm), Pd/C (10%),
EtOAc, RT, 3 h, 92%.
Whereas the syntheses of the intermediates 4a and 4c were
quite straightforward by application of the Horner–Wadsworth–
Emmons procedure with 9, it was more troublesome to obtain
compound 4b. In fact, all our attempts to synthesize the aldehyde
5b (nꢀ = 1) were unsuccessful (Scheme 5). The alkylation–
formylation sequence24 failed because methylation of 6 afforded
only compound 13. Though the formation of the enol ether 14
was straightforward, no reaction or decomposition of the starting
material was observed when compound 14 was submitted to
different deprotection conditions.25 Finally, the hydroboration of
the vinyl derivative 15, easily obtained by Wittig condensation on
5a, was unsuccessful whatever the conditions employed. Though
the formation of the organoborane was observed, the following
addition of NaOH/H2O2 gave back the starting material, likely
by simple deprotonation and elimination of the borane, since 15 is
strongly stabilized by the conjugation of the double bond with the
imidazole ring. Following these results, we changed our strategy
and investigated a novel way to synthesize compound 4b.
Scheme 6 Reagents and conditions: a) (EtO)2P(O)CH2CO2Et, NaH, THF,
0
◦C → RT, 2 h 40 min, 94%; b) DIBAL-H, CH2Cl2, −78 ◦C, 4 h,
79%; c) H3CC(OEt)3, H+, 140 ◦C, 1 h, 91%; d) KMnO4, NaIO4, K2CO3,
tBuOH–H2O, RT, 26 h, 53% at 68% conversion; e) TMSCHN2, MeOH,
0
◦C, 2 h, 72%.
responding allylic alcohol 18, a Johnson–Claisen rearrangement
afforded compound 19 in 91% yield. Application of the Sharpless
procedure27 (RuCl3·nH2O and a catalytic amount of NaIO4) on
compound 19 provided the expected acid 20 in very low yield. Then
compound 19 was submitted to Lemieux–von Rudloff conditions28
following the Hesse and Detterbeck protocol29 with NaIO4 and a
catalytic amount of KMnO4. By application of these conditions,
Finally, compound 4b was successfully obtained using a
Johnson–Claisen rearrangement26 (Scheme 6). Starting from the
aldehyde 5c, a Horner–Wadsworth–Emmons reaction led to the
a,b-unsaturated ester 17 in 94% yield. After reduction to the cor-
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the acid 20 was isolated in 53% yield at 68% conversion of the
starting material. Esterification of 20 with TMSCHN230 in MeOH
afforded the intermediate 4b required for the synthesis of the n =
2 analogue.
our target molecules 2a–f (Scheme 8). The reductive amination
was carried out with NaBH3CN,32 and the saponification was
performed with LiOH·H2O in THF–MeOH–H2O mixture, leading
to the expected 2,5-difunctionalized imidazoles in very good yields.
Having synthesized compounds 4a–c, the corresponding alde-
hydes 3a–c were prepared using a TBS-deprotection–oxidation
sequence (Scheme 7). Two tripeptides were chosen as the aaX
part of our new derivatives 2. Valine-phenylalanine-methionine
(VFM) is the aaX part of CVFM, a highly active FTI, and valine-
isoleucine-alanine (VIA) is the C-terminal part of yeast H-Ras
protein.1a These two tripeptides were synthesized in very good
overall yields according to the classical peptide chemistry using
EDCI and HOBt peptide coupling reactions.31
2. Biological evaluation
Compounds 24a–f and 2a–f were evaluated for their inhibitory
activity against recombinant yeast FTase33 using a fluorescent-
based assay34 and adapted to 96-well plate format. Results are
reported in Table 1.
Generally, VFM-bearing compounds are more active than VIA-
containing imidazoles. In the VIA series, the acids 2d–f are all
more active than the corresponding esters 24d–f, and the most
active compound in this series is 2e bearing 2 carbons between
the imidazole ring and the succinic moiety. In the VFM series,
Table 1 Activity on yeast FTase
Compound
n
Peptide
IC50/lM
24a
24b
24c
24d
24e
24f
2a
1
2
3
1
2
3
1
2
3
1
2
3
VFM
VFM
VFM
VIA
VIA
VIA
VFM
VFM
VFM
VIA
VIA
VIA
>1000
190 20
35
3
Inactive
>1000
340 10
Scheme 7 Reagents and conditions: a) TBAF, THF, 0 ◦C → RT, 30 min
(21a: 85%, 21b: 80%, 21c: 83%); b) MnO2, CHCl3, reflux, 32–48 h (3a:
86%, 3b: 92%, 3c: 97%).
150
400 10
80
5
2b
2c
5
2d
270 10
80 15
180 20
Having successfully prepared the required aldehydes 3a–c and
the protected tripeptides 22 and 23, reductive amination followed
by saponification of the ester functions was performed, giving
2e
2f
◦
˚
Scheme 8 Reagents and conditions: a) i) TFA–CH2Cl2, 0 C, 20 min, ii) 4 A MS, CH2Cl2–MeOH, RT, 10 min, then aldehyde 3a–c, iii) NaBH3CN,
MeOH–AcOH (10 : 1), RT (24a: 94%, 24b: 77%, 24c: 79%, 24d: 56%, 24e: 80%, 24f: 72%); b) LiOH·H2O, THF–MeOH–H2O, 0 ◦C → RT, 2–18 h (2a:
86%, 2b: 82%, 2c: 82%, 2d: 89%, 2e: 75%, 2f: 85%).
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the results are quite different. Except for n = 1, the esters are
more active than the corresponding acids, and the best activity is
obtained for compound 24c with n = 3. In order to understand
these differences and to check if these new compounds were
our first 2,5-disubstituted imidazoles to fit this requirement could
be due to the relative position of the succinic acid and of the
peptide. Furthermore, a hydrophobic alkyl chain might be needed
to mimic the farnesyl group and to position the acid moiety in
the pyrophosphate binding site. Therefore, we are pursuing our
investigation into highly substituted imidazoles by the synthesis
of 1,2- and 2,4-disubstituted imidazoles as well as compounds
bearing a hydrophobic chain near the 1,4-diacid moiety.
bisubstrate inhibitors, the most active derivatives, 24c (IC50
=
35 lM), 2c (IC50 = 80 lM) and 2e (IC50 = 80 lM), were subjected to
kinetic studies. The ester derivative 24c is noncompetitive to both
substrates whereas compound 2c is competitive to the peptide
but not to FPP. The inhibition constant of compound 2c (Ki =
60 lM) was deduced from these kinetic experiments. Surprisingly,
compound 2e exhibits a different binding mode. It appears to
be noncompetitive to the peptide substrate and uncompetitive to
FPP, suggesting that it binds at a location that does not prevent
FPP binding.
Experimental
General method
Unless otherwise indicated, all reactions were carried out with
magnetic stirring and, in the case of air- or moisture-sensitive com-
pounds, reactions were carried out in oven-dried glassware under
argon. Syringes were used to transfer the reagents and the solvents
were purged with argon prior to use. Tetrahydrofuran (THF) was
distilled over sodium/benzophenone. Dichloromethane (CH2Cl2),
triethylamine (Et3N), diisopropylamine and toluene were dis-
tilled over calcium hydride. N,Nꢀ-Dimethylformamide (DMF)
was dried over MgSO4 followed by distillation under reduced
pressure. Analytical thin-layer chromatography was carried out
on precoated silica gel glass plates (Merck TLC plates, silica
gel 60F254). The silica gel (silica gel 60 (35–70 lm)) used for
Some known inhibitors bearing a 1,4-diacid moiety are compet-
itive to FPP.14 However, in contrast to our designed compounds,
they all bear an aliphatic chain that mimics the farnesyl moiety.
The lack of interaction of our imidazole derivatives with the FPP
binding site may be due to the absence of such a hydrophobic
chain in that part of the molecule.
The difference of activity between the VFM- or VIA-derivatives
2b–c and 2e–f is surprising. However it is known that CVFM
adopts a binding conformation in the FTase active site different
from that of the other peptides (like CVIM) due to the presence
of the phenyl ring.35 Because of this aromatic ring, it can similarly
be assumed that the VFM-containing compounds 2b–c adopt a
different conformation than the VIA-derivatives 2e–f, and that the
length of the diacid chain is more or less detrimental to binding
according to the conformational behaviour of these derivatives.
In contrast to most of the reports on FTIs, our biological results
are based on the determination of the inhibitor binding mode.
In our case, these experiments have allowed us to demonstrate
different binding modes (dependent on the nature of the tripeptide
and of the carbon chain carried by the imidazole ring) that were
hardly foreseeable on the basis of the IC50 values.
1
column chromatography was purchased from SDS. H and 13C
NMR spectra were recorded on Bruker AC-250/300 and DPX-
1
300 spectrometers at 250 and 300 MHz. H chemical shifts are
reported in delta (d) units in parts per million (ppm) relative
to the singlet at 7.26 ppm for d-chloroform (residual CHCl3)
and the singlet (0.00 ppm) for TMS. 13C Chemical shifts (d)
are reported in ppm relative to the central line of the triplet at
77.0 ppm for d-chloroform. Splitting patterns are designated as: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broad
and combinations thereof. Coupling constants J are reported
in hertz (Hz). Melting points were measured on a Bu¨chi b-450
apparatus and are uncorrected. IR spectra were recorded with an
FTIR Perkin-Elmer Spectrum BX spectrometer. Low- and high-
resolution mass spectra were recorded by a navigator LC/MS
(source AQA) instrument for electron spray ionisation (ESI – base
resolution) and electron ionisation (EI – high resolution). Optical
rotations were measured with a JASCO 1010 polarimeter in a 1 dm
cell using the sodium D line (589 nm) at the temperature, solvent,
and concentration indicated. Elemental analyses were performed
by the Microanalytical Laboratory of the ICSN-Gif-sur-Yvette.
Conclusions
In summary, we have synthesized different higher-substituted
imidazoles with a succinic acid unit at C-2 and a tripetide at
C-5 of this heterocycle. The key transformation of our C-2
functionalisation sequence was a Horner–Wadsworth–Emmons
reaction that permitted us to achieve our syntheses in good overall
yields. This protocol was discovered following the failure of a
more traditional alkylation and of palladium-catalyzed reactions
due to the particular reactivity of the imidazole ring. The target
compounds were obtained from the commercially available 1,3-
dihydroxyacetone dimer 7 in a linear sequence with 14.5% and
9.0% (2a and 2d, n = 1, 10 steps), 2.4% and 2.4% (2b and 2e,
n = 2, 16 steps), 7.6% and 7.2% (2c and 2f, n = 3, 13 steps)
yields with VFM and VIA tripeptides respectively. Investigations
of the biological profile of our target compounds have shown that
VFM-containing compounds are generally more active than VIA
derivatives, and that the carbon chain length has an influence on
the activity. However, the kinetic experiments have demonstrated
that 2,5-disubstituted imidazoles are not bisubstrate analogues,
and only compound 2c is competitive to the CaaX motif.
5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazole
(6)
To a stirred solution of 8 (1.70 g, 15.2 mmol) in DMF
(17 mL) were added imidazole (3.10 g, 45.5 mmol) and tert-
butyldimethylchlorosilane (2.50 g, 16.7 mmol). The reaction
mixture was stirred for 2 h at room temperature. Then, the
reaction was quenched with H2O (30 mL) and extracted with
Et2O (3 × 40 mL). The combined organic layers were dried over
MgSO4. Concentration followed by column chromatography on
silica gel (EtOAc–MeOH 9.5 : 0.5) afforded 6 (3.13 g, 91%) as
1
The aim of this work was to create new inhibitors that can bind
to both CaaX and pyrophosphate binding sites. The failure of
an amorphous colorless solid. H NMR data identical to those
previously reported36; 13C NMR (62.5 MHz, CDCl3) d 138.3,
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130.5, 127.3, 54.6, 31.1, 25.3 (3C), 17.7, −5.8 (2C); IR (KBr)
1061, 1256, 1471 cm−1; HRMS calcd for C11H23N2OSi [M + H]+:
227.1580; found: 227.1547.
1.5, 7.5 Hz, 6H), 0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (62.5 MHz,
CDCl3) d173.7, 171.7, 146.4, 131.0, 126.3, 60.9, 60.6, 55.5, 39.9,
35.3, 30.3, 28.3, 25.8 (3C), 18.2, 14.1 (2C), −5.3 (2C); IR (KBr)
1057, 1256, 1472, 1736 cm−1; HRMS calcd for C20H37N2O5Si [M +
H]+: 413.2472; found: 413.2460.
5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazole-2-
carbaldehyde (5a)
(E,Z)-3-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]acrylic acid ethyl ester
n-Butyllithium (17.6 mL, 28.2 mmol, 1.6 N in hexane) was added
dropwise to a solution of 6 (5.30 g, 23.5 mmol) in THF (60 mL)
at −78 ◦C. After 40 min at this temperature, DMF (5.60 mL,
70.5 mmol) was added. The reaction mixture was allowed to
warm to room temperature and after 2 h 30 min, CH2Cl2 (90 mL)
was added. The organic phase was successively washed with brine
(80 mL) and H2O (80 mL), and dried over MgSO4. Concentration
afforded the crude product as a yellow oil that was purified
by column chromatography on silica gel (heptane–EtOAc 3 :
2) followed by crystallization (heptane) to afford the required
aldehyde 5a (5.00 g, 83%) as white crystals. Mp 60.7–62.5 ◦C;
1H NMR (250 MHz, CDCl3) d 9.77 (s, 1H), 7.16 (s, 1H), 4.72 (s,
2H), 4.01 (s, 3H), 0.89 (s, 9H), 0.08 (s, 6H); 13C NMR (62.5 MHz,
CDCl3) d 182.3, 144.3, 137.4, 130.4, 55.0, 32.1, 25.7 (3C), 18.1,
−5.5 (2C); IR (KBr) 1072, 1256, 1484, 1689 cm−1; MS (ESI+): m/z
255 [M + H]+; Elemental analysis calcd (%) for C12H22N2O2Si: C
56.65, H 8.72, N 11.01; found: C 56.52, H 8.69, N 11.04.
A suspension of sodium hydride (1.29 g, 32.1 mmol) in THF
(15 mL) was treated with triethyl phosphonoacetate (6.00 mL,
30.1 mmol) at 0 ◦C. The solution was stirred for 30 min at room
temperature before addition of the aldehyde 5a (2.55 g, 10.0 mmol)
in THF (10 mL). After 3 h at room temperature, the reaction
mixture was diluted with CH2Cl2 (30 mL) and extracted with
brine (20 mL) and H2O (20 mL). The organic layer was dried
over MgSO4 and concentrated. The crude product was purified
by column chromatography on silica gel (heptane–EtOAc 7 :
3) to afford (E,Z)-3-[5-(tert-Butyl-dimethylsilanyloxymethyl)-1-
methyl-1H-imidazol-2-yl]acrylic acid ethyl ester (2.40 g, 77%, E/Z
9 : 1) as a white amorphous solid. E isomer: 1H NMR (300 MHz,
CDCl3) d 7.53 (d, J = 15.5 Hz, 1H), 7.03 (s, 1H), 6.82 (d, J =
15.5 Hz, 1H), 4.67 (s, 2H), 4.27 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H),
1.32 (t, J = 7.0 Hz, 3H), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR
(75 MHz, CDCl3) d 167.1, 144.2, 133.9, 129.4, 128.7, 120.7, 60.7,
55.4, 30.6, 25.7 (3C), 18.3, 14.4, −5.2 (2C); IR (KBr) 1036, 1279,
1469, 1631, 1706, 3058 cm−1; HRMS calcd for C16H29N2O3Si [M +
2-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-ylmethylene]succinic acid diethyl ester (10)
1
H]+: 325.1947; found: 325.1927. Z isomer: H NMR (300 MHz,
A suspension of sodium hydride (0.54 g, 13.4 mmol) in THF
(5 mL) was treated with triethyl phosphonosuccinate 927 (3.66 g,
11.8 mmol) at 0 ◦C. The solution was stirred for 30 min at room
temperature before addition of the aldehyde 5a (2.55 g, 10.0 mmol)
in THF (10 mL). After 3 h at room temperature, the reaction
mixture was diluted with CH2Cl2 (40 mL) and extracted with
brine (30 mL) and H2O (30 mL). The organic layer was dried
over MgSO4 and concentrated. The crude product was purified
by column chromatography on silica gel (heptane–EtOAc 4 : 1)
to afford 10 (2.00 g, 69%, only the E isomer as d◦etermined by
NOE experiments) as white crystals. Mp 52.2–53.9 C; 1H NMR
(250 MHz, CDCl3) d 7.58 (s, 1H), 7.05 (s, 1H), 4.66 (s, 2H), 4.28
(s, 2H), 4.26 (q, J = 7.0 Hz, 2H), 4.13 (q, J = 7.0 Hz, 2H), 3.72
(s, 3H), 1.31 (t, J = 7.0 Hz, 3H), 1.23 (t, J = 7.0 Hz, 3H), 0.88
(s, 9H), 0.05 (s, 6H); 13C NMR (62.5 MHz, CDCl3) d 171.4, 167.5,
144.0, 132.9, 129.2, 127.4, 124.0, 61.2, 60.6, 55.3, 33.6, 30.6, 25.8
(3C), 18.2, 14.2 (2C), −5.3 (2C); IR (KBr) 1062, 1268, 1463, 1637,
1709, 1736 cm−1; MS (ESI+): m/z 411 [M + H]+, 433 [M + Na]+.
Elemental analysis calcd (%) for C20H34N2O5Si: C 58.51, H 8.35,
N 6.82; found: C 58.21, H 8.26, N 6.75.
CDCl3) d 7.61 (d, J = 12.5 Hz, 1H), 6.97 (s, 1H), 6.28 (d, J =
12.5 Hz, 1H), 4.73 (s, 2H), 4.32 (q, J = 7.0 Hz, 2H), 3.86 (s, 3H),
1.34 (t, J = 7.0 Hz, 3H), 0.91 (s, 9H), 0.07 (s, 6H); 13C NMR
(75 MHz, CDCl3) d 168.7, 144.7, 135.4, 129.6, 128.9, 120.1, 60.8,
55.7, 31.0, 25.7 (3C), 18.4, 14.1, −5.3 (2C); IR (KBr) 1040, 1245,
1466, 1617, 1695, 3377 cm−1; HRMS calcd for C16H28N2O3SiNa
[M + Na]+: 347.1767; found: 347.1755.
3-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-yl]propionic acid ethyl ester
Pd/C (0.21 g) was added to a solution of (E,Z)-3-[5-(tert-
butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-2-yl]acrylic
acid ethyl ester (2.10 g, 6.73 mmol) in EtOAc (15 mL). The
mixture was allowed to react at room temperature under hydrogen
atmosphere (1 atm) for 3 h. The reaction was filtered through a
pad of Celite with EtOAc and the filtrate was concentrated to give
3-[5-(tert-butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-yl]propionic acid ethyl ester (2.10 g, 100%) as a colorless oil.
1H NMR (300 MHz, CDCl3) d 6.82 (s, 1H), 4.61 (s, 2H), 4.15 (q,
J = 7.0 Hz, 2H), 3.59 (s, 3H), 2.90 (m, 4H), 1.25 (t, J = 7.0 Hz,
3H), 0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (75 MHz, CDCl3) d
172.9, 147.9, 131.0, 126.1, 60.6, 55.5, 31.7, 30.2, 25.9 (3C), 22.1,
18.2, 14.2, −5.3 (2C); IR (KBr) 1057, 1256, 1472, 1736 cm−1; MS
(ESI+): m/z 327 [M + H]+.
2-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-ylmethyl]succinic acid diethyl ester (4a)
Pd/C (0.10 g) was added to a solution of 10 (1.00 g, 2.44 mmol)
in EtOAc (15 mL). The mixture was allowed to react at room
temperature under hydrogen atmosphere (1 atm) for 4 h. The
reaction was filtered through a pad of Celite with EtOAc and the
filtrate was concentrated to give 4a (0.97 g, 100%) as a yellowish
oil. 1H NMR (250 MHz, CDCl3) d 6.80 (s, 1H), 4.62 (s, 2H), 4.13
(m, 4H), 3.59 (s, 3H), 3.36 (m, 1H), 3.13 (dd, J = 6.0, 15.0 Hz,
1H), 2.88 (dd, J = 6.0, 15.0 Hz, 1H), 2.74 (m, 2H), 1.23 (td, J =
3-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-yl]propionaldehyde (5c)
Diisobutylaluminium hydride (5.99 mL, 5.99 mmol, 1 N in
heptane) was added to a solution of 3-[5-(tert-butyldimethyl-
silanyloxymethyl)-1-methyl-1H-imidazol-2-yl]propionic
acid
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ethyl ester (1.30 g, 3.99 mmol) in CH2Cl2 (12 mL) at −78 ◦C. The
reaction was stirred at this temperature for 2 h, and 3 drops of
MeOH were added to neutralize the excess of diisobutylaluminium
hydride. The mixture was warmed to room temperature and
quenched with a saturated aqueous solution of potassium sodium
tartrate (20 mL). After stirring for 30 min, the mixture was
filtered through a pad of Celite. The organic layer was separated,
washed with brine (10 mL) and H2O (10 mL), dried over
MgSO4 and concentrated. The residue was purified by column
chromatography on silica gel (EtOAc–MeOH 9.7 : 0.3) to give 5c
1H), 2.63 (m, 3H), 2.41 (dd, J = 5.0, 16.0 Hz, 1H), 1.73 (m, 3H),
1.59 (m, 1H), 1.21 (m, 6H), 0.85 (s, 9H), 0.04 (s, 6H); 13C NMR
(75 MHz, CDCl3) d 174.8, 172.0, 148.9, 130.8, 126.0, 60.7 (2C),
55.6, 41.1, 36.2, 31.6, 30.3, 26.9, 25.9 (3C), 25.1, 18.3, 14.3 (2C),
−5.2 (2C); IR (KBr) 1055, 1258, 1472, 1735 cm−1; HRMS calcd
for C22H41N2O5Si [M + H]+: 441.2785; found: 441.2822.
4-(tert-Butyldimethylsilanoxymethyl)-1,2,3-trimethyl-3H-
imidazol-1-ium iodide (13)
n-Butyllithium (4.31 mL, 6.90 mmol, 1.6 N in hexane) was added
dropwise to a solution of 6 (1.30 g, 5.75 mmol) in THF (15 mL) at
−78 ◦C. After stirring for 45 min at this temperature, iodomethane
(0.55 mL, 8.63 mmol) was added. After stirring for 2 h at this
temperature and for 1 h at room temperature, CH2Cl2 (20 mL)
was added. The organic phase was successively washed with a
saturated aqueous solution of Na2S2O3 (15 mL), brine (15 mL),
H2O (15 mL), and dried over MgSO4. Removal of the solvent
afforded the crude product as a yellow solid. The residue was
purified by crystallization (pentane–EtOAc 4 : 1)◦to afford 13
(1.50 g, 68%) as a yellowish solid. Mp 122.1–124.8 C; 1H NMR
(250 MHz, CDCl3) d 7.44 (s, 1H), 4.70 (s, 2H), 3.97 (s, 3H), 3.86 (s,
3H), 2.87 (s, 3H), 0.90 (s, 9H), 0.13 (s, 6H); 13C NMR (62.5 MHz,
CDCl3) d 145.3, 132.6, 120.4, 54.8, 36.6, 33.7, 25.8 (3C), 18.2, 12.4,
−5.3 (2C); MS (ESI+): m/z 255 [M]+.
1
(0.93 g, 82%) as a colorless oil. H NMR (300 MHz, CDCl3) d
9.87 (s, 1H), 6.76 (s, 1H), 4.59 (s, 2H), 3.56 (s, 3H), 2.97 (m, 4H),
0.85 (s, 9H), 0.04 (s, 6H); 13C NMR (75 MHz, CDCl3) d 201.1,
147.6, 131.2, 125.9, 55.5, 40.8, 30.2, 25.8 (3C), 19.4, 18.2, −5.3
(2C); IR (KBr) 1077, 1248, 1479, 1679 cm−1; MS (ESI+): m/z 283
[M + H]+, 315 [M + H + MeOH]+.
2-{3-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]propylidene}succinic acid diethyl ester (11)
A suspension of sodium hydride (0.16 g, 3.94 mmol) in THF
(10 mL) was treated with triethyl phosphonosuccinate 927 (1.02 g,
3.28 mmol) at 0 ◦C. After stirring for 30 min at room temperature,
the aldehyde 5c (0.93 g, 3.28 mmol) in THF (3 mL) was
added. After stirring for 1 h 40 min at room temperature, the
reaction mixture was diluted with CH2Cl2 (15 mL) and extracted
with H2O (10 mL). The organic layer was dried over MgSO4
and concentrated. The crude product was purified by column
chromatography on silica gel (EtOAc–MeOH 9.8 : 0.2) to give
11 (1.00 g, 70%, E/Z 8 : 2) as a colorless oil. E isomer: 1H NMR
(300 MHz, CDCl3) d 6.97 (t, J = 7.0 Hz, 1H), 6.77 (s, 1H), 4.57
(s, 2H), 4.15 (t, J = 7.0 Hz, 2H), 4.08 (q, J = 7.0 Hz, 2H), 3.54
(s, 3H), 3.32 (s, 2H), 2.75 (m, 2H), 2.66 (m, 2H), 1.23 (t, J =
7.0 Hz, 3H), 1.20 (t, J = 7.0 Hz, 3H), 0.84 (s, 9H), 0.03 (s, 6H);
13C NMR (75 MHz, CDCl3) d 170.8, 166.8, 147.9, 143.5, 131.1,
126.9, 126.1, 60.8 (2C), 55.5, 32.4, 30.2, 26.9, 25.9, 25.8 (3C), 18.2,
14.3 (2C), −5.3 (2C); IR (KBr) 1076, 1256, 1487, 1643, 1706,
1735 cm−1; HRMS calcd for C22H39N2O5Si [M + H]+: 439.2628;
found: 439.2627. Z isomer: 1H NMR (300 MHz, CDCl3) d 6.80 (s,
1H), 6.17 (t, J = 7.0 Hz, 1H), 4.61 (s, 2H), 4.20 (q, J = 7.0 Hz, 2H),
4.13 (q, J = 7.0 Hz, 2H), 3.55 (s, 3H), 3.25 (s, 2H), 2.97 (m, 2H),
2.82 (m, 2H), 1.35 (t, J = 7.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H),
0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (75 MHz, CDCl3) d 171.4,
166.3, 148.4, 145.2, 130.9, 126.7, 126.4, 60.5 (2C), 55.5, 40.2, 30.3,
27.6, 26.5, 25.8 (3C), 18.2, 14.2 (2C), −5.3 (2C); IR (KBr) 1062,
1259, 1471, 1648, 1706, 1735 cm−1; HRMS calcd for C22H39N2O5Si
[M + H]+: 439.2628; found: 439.2632.
5-(tert-Butyldimethylsilanoxymethyl)-2-(2-methoxyvinyl)-1-
methyl-1H-imidazole (14)
To a suspension of (methoxymethyl)triphenylphosphonium chlo-
◦
ride (1.14 g, 3.33 mmol) in THF (6 mL) at 0 C potassium tert-
butoxide (5.54 mL, 5.54 mmol, 1 N in THF) was added dropwise.
After stirring for 30 min at this temperature, a solution of the
aldehyde 5a (705 mg, 2.77 mmol) in THF (8 mL) was added
slowly to the suspension. After 5 h at room temperature, H2O
(3 mL) was added and the reaction mixture was concentrated. The
residue was diluted with CH2Cl2 (20 mL) and washed with H2O
(15 mL). The organic phase was separated, dried over MgSO4
and concentrated. The phosphine oxide formed was crystallized
(heptane–EtOAc 4 : 1) and filtered. Then, the residue was purified
by column chromatography on silica gel (CH2Cl2–MeOH 9.5 : 0.5)
1
to give 14 (0.61 g, 78%) as a yellowish oil. H NMR (300 MHz,
CDCl3) d 7.46 (d, J = 12.0 Hz, 1H), 6.80 (s, 1H), 5.61 (d, J =
12.0 Hz, 1H), 4.61 (s, 2H), 3.69 (s, 3H), 3.54 (s, 3H), 0.88 (s, 9H),
0.04 (s, 6H); 13C NMR (75 MHz, CDCl3) d 153.3, 145.9, 131.5,
126.6, 92.4, 57.3, 55.4, 30.1, 25.8 (3C), 18.2, −5.3 (2C); IR (KBr)
1054, 1231, 1469, 1642, 3072 cm−1; HRMS calcd for C14H27N2O2Si
[M + H]+: 283.1842; found: 283.1855.
2-{3-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]propyl}succinic acid diethyl ester (4c)
5-(tert-Butyldimethylsilanoxymethyl)-1-methyl-2-vinyl-1H-
imidazole (15)
Pd/C (95.0 mg) was added to a solution of 11 (950 mg, 2.17 mmol)
in EtOAc (15 mL). The mixture was allowed to react at room
temperature under hydrogen atmosphere (1 atm) for 3 h. The
reaction was filtered through a pad of Celite with EtOAc and
the filtrate was concentrated. The residue was purified by column
chromatography on silica gel (EtOAc–MeOH 9.8 : 0.2) to give 4c
To a suspension of sodium hydride (173 mg, 4.33 mmol) in
THF (20 mL), methyltriphenylphosphonium bromide (1.41 g,
3.94 mmol) was added at room temperature. After stirring for
2 h at this temperature, a solution of the aldehyde 5a (1.00 g,
3.94 mmol) in THF (10 mL) was added. The mixture was refluxed
for 5 h followed by 14 h of stirring at room temperature. Then,
the reaction mixture was filtered, washed with ether (3 × 15 mL)
and concentrated. The phosphine oxide formed was crystallized
1
(875 mg, 92%) as a colorless oil. H NMR (300 MHz, CDCl3)
d 6.75 (s, 1H), 4.57 (s, 2H), 4.10 (m, 4H), 3.51 (s, 3H), 2.82 (m,
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(heptane–EtOAc 4 : 1) and filtered. Then, the residue was purified
by column chromatography on silica gel (ether) to afford 15
(715 mg, 72%) as a white amorphous solid. 1H NMR (300 MHz,
CDCl3) d 6.91 (s, 1H), 6.60 (dd, J = 11.0, 17.0 Hz, 1H), 6.17 (dd,
J = 1.5, 17.0 Hz, 1H), 5.41 (dd, J = 1.5, 11.0 Hz, 1H), 4.64 (s, 2H),
3.64 (s, 3H), 0.88 (s, 9H), 0.05 (s, 6H); 13C NMR (75 MHz, CDCl3)
d 146.4, 131.6, 127.5, 122.8, 118.1, 55.4, 30.3, 25.8 (3C), 18.2, −5.3
(2C); IR (KBr) 1038, 1257, 1468, 1625, 3096 cm−1; HRMS calcd
for C13H25N2OSi [M + H]+: 253.1736; found: 253.1765.
ethanol. The solution was refluxed for 3 h, and then the triethyl
orthoacetate was evaporated. The mixture was diluted with EtOAc
(15 mL) and filtered through a pad of Celite (EtOAc–MeOH 9.5 :
0.5). The organic layer was evaporated and the residue was purified
by column chromatography on silica gel (heptane–EtOAc 1 : 9)
to afford 19 (1.66 g, 91%) as a colorless oil. 1H NMR (300 MHz,
CDCl3) d 6.77 (s, 1H), 5.66 (m, 1H), 5.08 (m, 2H), 4.69 (s, 2H), 4.10
(q, J = 7.5 Hz, 2H), 3.51 (s, 3H), 2.62 (m, 3H), 2.38 (m, 2H), 1.93
(m, 1H), 1.76 (m, 1H), 1.23 (t, J = 7.5 Hz, 3H), 0.87 (s, 9H), 0.03
(s, 6H); 13C NMR (75 MHz, CDCl3) d 172.2, 149.1, 140.2, 130.7,
126.0, 116.0, 60.3, 55.5, 40.4, 40.1, 32.0, 30.1, 25.8 (3C), 24.7,
18.2, 14.3, −5.3 (2C); IR (KBr) 1052, 1252, 1471, 1640, 1731,
3079 cm−1; HRMS calcd for C20H37N2O3Si [M + H]+: 381.2573;
found: 381.2546.
5-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-yl]pent-2-enoic acid ethyl ester (17)
To a suspension of sodium hydride (341 mg, 8.53 mmol) in THF
(10 mL), triethyl phosphonoacetate (1.50 mL, 7.26 mmol) was
added at 0 ◦C. After stirring for 30 min at room temperature, the
aldehyde 5c (1.85 g, 6.60 mmol) in THF (5 mL) was added. After
stirring 2 h 40 min at room temperature, the reaction mixture was
diluted with CH2Cl2 (20 mL) and washed with brine (15 mL)
and H2O (15 mL). The organic layer was dried over MgSO4
and concentrated. The crude product was purified by column
chromatography on silica gel (EtOAc–MeOH 9.8 : 0.2) to give
17 (2.15 g, 94%) as a white amorphous solid. 1H NMR (300 MHz,
CDCl3) d 7.04 (m, 1H), 6.81 (s, 1H), 5.89 (d, J = 16.0 Hz, 1H),
4.62 (s, 2H), 4.18 (q, J = 7.0 Hz, 2H), 3.55 (s, 3H), 2.66–2.84
(m, 4H), 1.28 (t, J = 7.0 Hz, 3H), 0.88 (s, 9H), 0.04 (s, 6H); 13C
NMR (75 MHz, CDCl3) d 166.5, 147.8, 147.4, 131.0, 126.2, 122.2,
60.3, 55.5, 30.2, 29.9, 25.8 (3C), 25.6, 18.2, 14.3, −5.3 (2C); IR
(KBr) 1049, 1253, 1471, 1655, 1715, 3385 cm−1; HRMS calcd for
C18H33N2O3Si [M + H]+: 353.2260; found: 353.2267.
2-{2-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]ethyl}succinic acid 4-ethyl ester (20)
To a solution of 19 (1.00 g, 2.63 mmol) in tBuOH (4.3 mL) were
added an aqueous solution of K2CO3 (1.09 g in 8.7 mL of H2O,
7.89 mmol), NaIO4 (1.69 g, 7.89 mmol) and KMnO4 (333 mg,
2.10 mmol). After stirring for 48 h at room temperature, the
mixture was extracted with EtOAc (6 × 5 mL). The combined
organic layers were filtered through a pad of Celite (EtOAc–
MeOH 9 : 1) and concentrated. The residue was purified by column
chromatography on silica gel (EtOAc then EtOAc–MeOH–AcOH
8.5 : 1.5 : 0.1) to afford 20 (0.37 g, 53% at 68% conversion) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d 9.53 (bs, 1H), 6.87 (s,
1H), 4.61 (s, 2H), 4.12 (q, J = 7.0 Hz, 2H), 3.59 (s, 3H), 2.90 (m,
4H), 2.42 (m, 1H), 2.16 (m, 1H), 1.88 (m, 1H), 1.24 (t, J = 7.0 Hz,
3H), 0.88 (s, 9H), 0.06 (s, 6H); 13C NMR (75 MHz, CDCl3) d 176.9,
172.6, 148.5, 131.2, 122.8, 60.5, 55.2, 40.9, 36.8, 30.4, 29.4, 25.8
(3C), 24.6, 18.2, 14.2, −5.3 (2C); IR (KBr) 1024, 1253, 1453, 1723,
3373 cm−1; HRMS calcd for C19H35N2O5Si [M + H]+: 399.2315;
found: 399.2329.
5-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-imidazol-
2-yl]pent-2-en-1-ol (18)
Diisobutylaluminium hydride (18.3 mL, 18.3 mmol, 1 N in
heptane) was added to a solution of 17 (2.15 g, 6.10 mmol) in
CH2Cl2 (20 mL) at −78 ◦C. The reaction was stirred at this
temperature for 4 h, and 5 drops of MeOH were added to
neutralize the excess of diisobutylaluminium hydride. The mixture
was warmed to room temperature and quenched with a saturated
aqueous solution of potassium sodium tartrate (50 mL). After
stirring for 1 h, the mixture was filtered through a pad of Celite. The
organic layer was separated, washed with brine (20 mL) and H2O
(20 mL), dried over MgSO4 and concentrated. The crude product
was purified by column chromatography on silica gel (heptane–
EtOAc 9 : 1) to afford 18 (1.50 g, 79%) as white crystals. Mp
2-{2-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]ethyl}succinic acid 4-ethyl ester 1-methyl ester (4b)
To a solution of 20 (0.20 g, 0.50 mmol) in MeOH (4 mL) was
added trimethylsilyldiazomethane (0.75 mL, 1.50 mmol, 2 N in
ether) at 0 ◦C. After stirring for 2 h at this temperature, the reaction
mixture was concentrated and the residue was purified by column
chromatography on silica gel (heptane–EtOAc 1 : 9 → EtOAc) to
1
afford 4b (0.15 g, 72%) as a colorless oil. H NMR (300 MHz,
◦
CDCl3) d 6.78 (s, 1H), 4.60 (s, 2H), 4.12 (q, J = 7.0 Hz, 2H), 3.70
(s, 3H), 3.54 (s, 3H), 2.95 (m, 1H), 2.65–2.82 (m, 3H), 2.52 (dd,
J = 16.5, 5.0 Hz, 1H), 2.07 (m, 2H), 1.24 (t, J = 7.0 Hz, 3H),
0.87 (s, 9H), 0.03 (s, 6H); 13C NMR (75 MHz, CDCl3) d 174.8,
171.6, 148.1, 131.0, 125.8, 60.7, 55.4, 51.9, 40.7, 36.2, 30.2, 29.4,
25.8 (3C), 24.6, 18.2, 14.2, −5.3 (2C); IR (KBr) 1051, 1253, 1471,
1731 cm−1; HRMS calcd for C20H37N2O5Si [M + H]+: 413.2472;
found: 413.2477.
1
74.3–77.5 C; H NMR (300 MHz, CDCl3) d 6.79 (s, 1H), 5.73
(m, 2H), 4.61 (s, 2H), 4.08 (d, J = 5.5 Hz, 2H), 3.55 (s, 3H), 2.96
(sl, 1H), 2.76 (t, J = 7.0 Hz, 2H), 2.47 (q, J = 7.0 Hz, 2H), 0.87
(s, 9H), 0.04 (s, 6H); 13C NMR (75 MHz, CDCl3) d 148.7, 130.8,
130.6, 130.5, 125.8, 63.2, 55.6, 30.2, 30.1, 26.8, 25.8 (3C), 18.2,
−5.3 (2C); IR (KBr) 1054, 1249, 1471, 1645, 3175 cm−1; HRMS
calcd for C16H31N2O2Si [M + H]+: 311.2155; found: 311.2140.
3-{2-[5-(tert-Butyldimethylsilanyloxymethyl)-1-methyl-1H-
imidazol-2-yl]ethyl}pent-4-enoic acid ethyl ester (19)
General procedure A: Deprotection of the TBS group
◦
The allylic alcohol 18 (1.50 g, 4.84 mmol) was heated at 140 C
in the presence of triethyl orthoacetate (6.20 mL, 33.9 mmol) and
propionic acid (22.0 lL, 0.29 mmol) with concomitant removal of
A solution of tetrabutylammonium fluoride (1 N in THF) was
added to a THF solution of compounds 4a–c at 0 ◦C. After
stirring for 30 min at room temperature, the reaction mixture was
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evaporated. The residue was purified by column chromatography
on silica gel to afford the required primary alcohols 21a–c.
methylmorpholine (0.40 mL, 3.27 mmol). After stirring for 2 h
30 min at room temperature, the reaction was quenched with water
(20 mL) and extracted with CH2Cl2 (3 × 20 mL). The combined
organic layers were dried over MgSO4 and concentrated. The
residue was purified by crystallization (MeOH) to give 22 (1.33 g,
80%) as white crystals. Mp 161.8–163.7 ◦C; 1H NMR (300 MHz,
CDCl3) d 7.15–7.34 (m, 5H), 6.70 (bs, 1H), 6.61 (bd, J = 7.5 Hz,
1H), 4.95 (bd, J = 7.0 Hz, 1H), 4.72 (m, 1H), 4.60 (m, 1H), 3.91
(m, 1H), 3.70 (s, 3H), 3.09 (m, 2H), 2.40 (t, J = 7.5, 2H), 2.13 (m,
2H), 2.05 (s, 3H), 1.92 (m, 1H), 1.42 (s, 9H), 0.92 (d, J = 7.0 Hz,
3H), 0.82 (d, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 171.7,
171.6, 170.6, 156.0, 136.4, 129.4, 128.9 (2C), 127.3 (2C), 80.4, 60.4,
54.3, 52.6, 51.8, 38.1, 31.5, 30.6, 30.0, 28.4 (3C), 19.4, 17.5, 15.5;
MS (ESI+): m/z 532 [M + Na]+.
2-(5-Hydroxymethyl-1-methyl-1H-imidazol-2-ylmethyl)succinic
acid diethyl ester (21a). Prepared according to general procedure
A on compound 4a (1.00 g, 2.42 mmol) in THF (12 mL) with
TBAF (4.85 mL, 4.85 mmol). After work-up and column
chromatography (EtOAc–MeOH 9.5 : 0.5), 21a (617 mg, 85%)
1
was isolated as a white amorphous solid. H NMR (300 MHz,
CDCl3) d 6.68 (s, 1H), 4.54 (s, 2H), 4.11 (m, 4H), 3.62 (s, 3H),
3.30 (m, 1H), 3.11 (dd, J = 7.0, 15.0 Hz, 1H), 2.85 (dd, J = 7.5,
15.0 Hz, 1H), 2.69 (m, 2H), 1.23 (m, 6H); 13C NMR (75 MHz,
CDCl3) d 173.6, 171.7, 146.3, 131.9, 126.0, 61.0, 60.7, 54.1, 39.9,
35.3, 30.3, 28.2, 14.2, 14.1; IR (KBr) 1487, 1726, 3139 cm−1; MS
(ESI+): m/z 299 [M + H]+, 321 [M + Na]+; Elemental analysis
calcd (%) for C14H22N2O5: C 56.36, H 7.43, O 26.81; found: C
56.12, H 7.37, O 26.76.
Bocvalinyl-isoleucinyl-alanine methyl ester (23)
General procedure B: Oxidation of the primary alcohol
Prepared according to the protoco1 used for 22 with Boc-
L-isoleucine (2.15 g, 9.31 mmol), CH2Cl2 (35 mL), L-alanine
methyl ester (1.30 g, 9.31 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (2.12 g, 11.2 mmol), 1-
hydroxybenzotriazole hydrate (1.48 g, 11.2 mmol) and N-
methylmorpholine (1.25 mL, 9.31 mmol). After work-up, column
chromatography on silica gel (heptane–EtOAc 7 : 3) followed by
crystallization (heptane–EtOAc 9 : 1) BocIle-Ala-OMe (2.21 g,
75%) was isolated as white crystals. Deprotection of BocIle-Ala-
OMe (1.12 g, 3.54 mmol) was carried out with 75% TFA–CH2Cl2
(10 mL) at 0 ◦C for 20 min. Reaction with Boc-L-valine (769 mg,
3.54 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hy-
drochloride (807 mg, 4.25 mmol), 1-hydroxybenzotriazole hy-
drate (563 mg, 4.25 mmol) and N-methylmorpholine (0.48 mL,
3.54 mmol) afforded after work-up and crystallization (pentane–
EtOAc 9 : 1) compound 23 (1.20 g, 82%) as white crystals. Mp
176.9–180.2 ◦C; [a]D23 −42.6 (c 1.22 CHCl3); 1H NMR (300 MHz,
CDCl3) d 6.90 (bd, J = 7.5 Hz, 1H), 6.81 (bd, J = 7.5 Hz, 1H),
5.26 (bd, J = 7.5 Hz, 1H), 4.56 (m, 1H), 4.35 (m, 1H), 3.96 (m,
1H), 3.74 (s, 3H), 2.11 (m, 1H), 1.90 (m, 1H), 1.44 (s, 9H), 1.39
(d, J = 7.0 Hz, 3H), 0.91 (m, 14H); 13C NMR (75 MHz, CDCl3)
d 173.1, 172.0, 170.7, 156.1, 80.1, 60.4, 57.8, 52.5, 48.2, 37.1, 30.8,
28.6 (3C), 24.9, 19.4, 18.2, 18.1; 15.5, 11.4; MS (ESI+): m/z 438
[M + Na]+; Elemental analysis calcd (%) for C20H37N3O6: C 57.81,
H 8.98, N 10.11, O 23.10; found: C 57.69, H 8.79, N 10.05, O
23.13.
Manganese dioxide (6.5 equiv.) was added to a CHCl3 solution
of 21a–c. The mixture was refluxed for 32–48 h. After cooling to
room temperature, the reaction was filtered through a pad of Celite
(EtOAc) and concentrated. The residue was purified by column
chromatography to afford the required aldehydes 3a–c.
2-(5-Formyl-1-methyl-1H-imidazol-2-ylmethyl)succinic acid di-
ethyl ester (3a). Prepared according to general procedure B on
compound 21a (350 mg, 1.18 mmol) in CHCl3 (15 mL) with
MnO2 (671 mg, 7.67 mmol). After refluxing for 48 h, filtration
followed by column chromatography (CH2Cl2–MeOH 9.9 : 0.1)
1
afforded 3a (300 mg, 86%) as a yellowish amorphous solid. H
NMR (300 MHz, CDCl3) d 9.66 (s, 1H), 7.68 (s, 1H), 4.13 (m,
4H), 3.91 (s, 3H), 3.44 (m, 1H), 3.18 (dd, J = 7.0, 15.0 Hz, 1H),
2.96 (dd, J = 7.0, 15.0 Hz, 1H), 2.77 (m, 2H), 1.23 (m, 6H); 13C
NMR (75 MHz, CDCl3) d 179.0, 173.1, 171.4, 153.3, 143.0, 132.0,
61.2, 60.8, 39.4, 34.5, 32.3, 27.7, 14.2, 14.1; IR (KBr) 1469, 1657,
1725 cm−1; HRMS calcd for C14H20N2O5Na [M + Na]+: 319.1270;
found: 319.1251.
Boc-valinyl-phenylalaninyl-methionine methyl ester (22)
To a solution of Boc-L-phenylalanine (1.99 g, 7.51 mmol) in
CH2Cl2 (25 mL) were added at room temperature L-methionine
methyl ester (1.50 g, 7.51 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (1.72 g, 9.00 mmol), 1-
hydroxybenzotriazole hydrate (1.20 g, 9.00 mmol) and N-
methylmorpholine (1.00 mL, 7.51 mmol). After stirring for 2 h
30 min, the reaction was quenched with water (25 mL) and
extracted with CH2Cl2 (3 × 20 mL). The combined organic layers
were dried over MgSO4 and concentrated. The residue was purified
by column chromatography on silica gel (heptane–EtOAc 3 : 2)
followed by crystallization (heptane–EtOAc 9 : 1) to afford the
required dipeptide BocPhe-Val-OMe (2.92 g, 95%) as white crys-
tals. BocPhe-Val-OMe (1.34 g, 3.27 mmol) was deprotected with
a solution of 75% TFA–CH2Cl2 (8 mL) at 0 ◦C for 20 min. Evap-
oration with ether gave the required deprotected dipeptide as a
white solid, which was diluted in CH2Cl2 (25 mL) and treated with
Boc-L-valine (0.71 g, 3.27 mmol), 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (748 mg, 3.92 mmol), 1-
hydroxybenzotriazole hydrate (523 mg, 3.92 mmol) and N-
General procedure C: Reductive amination
Tripeptides 22 and 23 (1.00 mmol) were deprotected with a
solution of 75% TFA–CH2Cl2 at 0 ◦C for 20 min. Evaporation
with ether gave the required deprotected tripeptide as a white solid
which was diluted in MeOH–CH2Cl2 and treated with powdered
˚
4 A molecular sieves and triethylamine (1 equiv.). After stirring
for 10 min at room temperature, a solution of 3a–c in MeOH–
CH2Cl2 was added to the mixture. After 4 h, a solution of sodium
cyanoborohydride (1.5–2 equiv.) in MeOH–AcOH (1 : 0.1) was
added. The solution was stirred for 18 h at room temperature.
Then, the mixture was filtered through a pad of Celite (CH2Cl2)
and concentrated. The mixture was taken into H2O and extracted
with CH2Cl2 (3×). The combined organic layers were dried over
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MgSO4 and concentrated. The residue was purified by column
chromatography on silica gel to afford compounds 24a–f.
per well, 20 repeats) at 30 ◦C with an excitation filter at 340 nm and
an emission filter at 486 nm. Each measurement was performed
twice as duplicate or triplicate.
Compound 24a. Prepared according to general procedure
C. Deprotection: 22 (200 mg, 0.39 mmol) in a solution 75%
TFA–/CH2Cl2 (3 mL). Reductive amination: To a solution of
the deprotected tripeptide in MeOH–CH2Cl2 1 : 1 (4 mL) were
Acknowledgements
The authors thank J.-B. Cre´chet for a generous gift of the strain of
E. coli expressing the yeast FTase, Dr J. Ouazzani and S. Cortial
for the production and purification of recombinant yeast FTase,
Drs D. Gue´nard and F. Gue´ritte for their constant interest in this
work and CNRS for financial support.
˚
added: powdered 4 A molecular sieves (450 mg) and triethylamine
(55.0 lL, 0.39 mmol), 3a (116 mg, 0.39 mmol) and a solution
of sodium cyanoborohydride (50.0 mg, 0.79 mmol). After work-
up the residue was purified by column chromatography on silica
gel (CH2Cl2–MeOH 9.5 : 0.5) to afford 24a (225 mg, 94%) as a
1
colorless oil. H NMR (300 MHz, CDCl3) d 7.18–7.42 (m, 6H),
6.82 (bs, 1H), 6.74 (s, 1H), 4.78 (m, 1H), 4.62 (m, 1H), 4.13 (m,
4H), 3.73 (s, 3H), 3.63 (s, 3H), 3.60 (d, J = 14.0 Hz, 1H), 2.93–
3.49 (m, 6H), 2.80 (m, 3H), 2.45 (t, J = 7.0 Hz, 2H), 2.11 (m,
1H), 2.06 (s, 3H), 1.96 (m, 2H), 1.24 (m, 6H), 0.85 (d, J = 7.0 Hz,
3H), 0.80 (d, J = 7.0 Hz, 3H); 13C NMR (75 MHz, CDCl3) d
173.9, 173.0, 171.9, 171.6, 171.0, 146.2, 130.6, 136.5, 129.3 (2C),
128.8 (2C), 127.1, 122.6, 67.6, 61.4, 61.0, 54.0, 52.6, 51.6, 42.2,
40.0, 38.0, 35.4, 31.6, 31.3, 31.0, 29.8, 27.4, 19.4, 18.2, 15.4, 14.2,
14.1; HRMS calcd for C34H52N5O8S [M + H]+: 690.3537; found:
690.3521.
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To a solution of 24a–f in THF–MeOH–H2O 1 : 1 : 1 was added
lithium hydroxide monohydrate (3.6 equiv.) at 0 C. After 1–2 h
◦
at this temperature and 1–16 h at room temperature, Amberlite
IRC 50 resin (H+) was added and the mixture was stirred until
pH = 7.0. Then, the reaction was filtered and the resin was washed
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compounds 2a–f.
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24a (90.0 mg, 0.13 mmol) in THF–MeOH–H2O 1 : 1 : 1 (1.5 mL)
and lithium hydroxide monohydrate (20.0 mg, 0.47 mmol) at
0
◦C for 1 h and at room temperature for 1 h. After work-up,
1
2a (69.5 mg, 86%) was isolated as a colorless foam. H NMR
(250 MHz, CD3OD) d 7.13–7.38 (m, 5H), 6.68 (s, 1H), 4.86 (m,
1H), 4.33 (m, 1H), 3.67 (s, 3H), 3.61 (d, J = 14.0 Hz, 1H), 2.89–
3.34 (m, 6H), 2.74 (m, 3H), 2.49 (t, J = 8.0 Hz, 2H), 2.16 (m, 1H),
2.07 (s, 3H), 1.98 (m, 2H), 0.85 (m, 6H); 13C NMR (62.5 MHz,
CD3OD) d 181.6, 180.0, 177.9, 176.6, 172.8, 149.2, 138.0, 132.2,
130.5 (2C), 129.7 (3C), 122.3, 68.7, 55.7, 45.3, 42.2, 41.3, 38.7,
34.1, 32.8, 31.7, 31.2, 29.5, 28.0, 20.2, 19.4, 15.4; HRMS calcd for
C29H42N5O8S [M + H]+: 620.2754; found: 620.2762.
Yeast FTase assay
Assays were carried out on 96-well plates, prepared with Biomek
NKMC and Biomek 3000 from Beckman Coulter and read on
Wallac Victor fluorimeter from Perkin-Elmer. In each well, 20 lL
of farnesyl pyrophosphate (10 lM) was added to 180 lL of
a solution containing 2 lL of varied concentrations of 24a–
f and 2a–f (dissolved in DMSO) and 178 lL of a solution
composed of 0.1 mL of partially purified recombinant yeast FTase
(2.2 mg mL−1) and 7.0 mL of Dansyl-GCVLS peptide (in the
following buffer: 5.8 mM DTT, 12 mM MgCl2, 12 lM ZnCl2
and 0.09% (w/v) CHAPS, 53 mM Tris·HCl, pH 7.5). Then the
fluorescence development was recorded for 15 min (0.7 seconds
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