Potent farnesyltransferase inhibitors with 1,4-diazepane scaffolds as novel destabilizing microtubule agents in hormone-resistant prostate cancer

Article abstract of DOI:10.1021/jm101067y

A new class of potent farnesyltransferase inhibitors based on a 1,4-diazepane scaffold was synthesized with protein farnesyltransferase inhibition potencies in the low nanomolar range. The compounds block the growth on two hormone-resistant tumor prostati

Full text of DOI:10.1021/jm101067y

ARTICLE
pubs.acs.org/jmc
Potent Farnesyltransferase Inhibitors with 1,4-Diazepane Scaffolds
as Novel Destabilizing Microtubule Agents in Hormone-Resistant
Prostate Cancer
Nicolas Wlodarczyk,† Delphine Le Broc-Ryckewaert,‡ Pauline Gilleron,† Amꢀelie Lemoine,† Amaury Farce,†
Philippe Chavatte,† Jo€elle Dubois,§ Nicole Pommery,‡ Jean-Pierre Hꢀenichart,† Christophe Furman,‡ and
Rꢀegis Millet*,†
†Institut de Chimie Pharmaceutique Albert Lespagnol, Universitꢀe Lille-Nord de France, EA4481, IFR114, 3 Rue du Pr Laguesse,
B.P. 83, F-59006 Lille, France
‡Facultꢀe des Sciences Pharmaceutiques et Biologiques de Lille, Universitꢀe Lille-Nord de France, EA4483, IFR114, 3 Rue du Pr Laguesse,
B.P. 83, F-59006 Lille, France
§Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Centre de Recherche de Gif, Avenue de la Terrasse,
F-91198 Gif-sur-Yvette Cedex, France
S Supporting Information
b
ABSTRACT: A new class of potent farnesyltransferase inhibitors
based on a 1,4-diazepane scaffold was synthesized with protein
farnesyltransferase inhibition potencies in the low nanomolar range.
The compounds block the growth on two hormone-resistant tumor
prostatic cell lines (DU145 and PC3). The advanced cellular evalua-
tion of the morepotentfarnesyltransferase inhibitors was explored and
revealed a disorganization of tubulin in PC3 cells.
’ INTRODUCTION
the BMS-214662 (Figure 1). Although efficient with low sys-
temic toxicity in preclinical and animal models, no significant
benefit has been observed for FDA approbation. However,
synergistic or additive effects of combination of various FTis
with some cytotoxic antineoplastic drugs (e.g., taxane, cisplatine)
offer new perspectives for cancer treatment.5-9 The exact
biological mechanism by which FTis exert their antitumor
activity remains controversial. Nevertheless, the discoveries of
several farnesylated proteins with potent antitumor activity
suggest that the mechanism results in the combination of effects
of numerous proteins. For example, the centromere-associated
proteins CENP-E constitute one pertinent target of FTis, since
the elucidation of a functional association of CENP-E with
microtubules requires farnesylation.10
Prostate cancer is the most frequently diagnosed malignancy
in men in Europe and the United States.11 Antiandrogenic
treatments are often effective in the treatment of hormone-
dependent prostate cancer. However, resistance against treat-
ment often occurs as tumors acquire a hormone-refractory
phenotype that does not respond to chemotherapy. This recur-
rent problem highlights the necessity to develop novel drugs for
The maturation of many proteins requires one or more post-
translational modifications. Around 60 proteins are activated by a
series of post-translational modifications: prenylation, proteoly-
sis, and carboxymethylation.1 The prenylation reaction is cata-
lyzed by a zinc metalloenzyme: the farnesyltransferase (FTase)
or geranylgeranyltransferase-I (GGTase-I).2 This reaction
results in the addition of a farnesyl (C15) or a geranylgeranyl
(C20) moiety to the cysteine residue located at the C-terminal
tetrapeptidic sequence called “CA1A2X box” (A1A2, aliphatic
amino acids; X, methionine or serine for farnesyltransferase, leucine,
or isoleucine for geranylgeranyltransferase I). The farnesylation
provides sufficient hydrophobicity for the adequate cellular localiza-
tion of proteins overactivated in some pathologies like cancer (Ras,
Rheb, RhoB, CENP-E, -F). This overactivation can be induced
either directly by mutation of the farnesylated protein or by
mutation of an activator or an inactivator of farnesylated proteins.
Therefore, the development of FTase inhibitors (FTis) represents a
promising strategy in cancer therapy.3
FTis have demonstrated efficiency in the treatment of 70% of
cancers cell lines in vitro.4 To date, many compounds have been
developed for clinical trials for the treatment of solid tumors and
hematological malignancies. The three most studied are the
R115777 (or tipifarnib), the SCH66336 (or lonafarnib), and
Received: August 17, 2010
Published: February 07, 2011
r
2011 American Chemical Society
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the treatment of hormone-refractory prostate cancer. We there-
fore decided to focus the study of our FTis on whole cell assays
using hormone-resistant prostate cancer cell lines derived from
brain metastasis (DU145) and bone metastasis (PC3).
was used to design the novel, potent FTis structures shown in
Figure 2. The p-cyanobenzylimidiazole zinc chelator was retained
in this study, since ALIFT126 has exhibited low in vivo toxicity in
our previous work.19 The 1,4-diazepane scaffold confers the
steric constraint with minimal peptidic features and higher
metabolic stability to obtain the correct fit with the zinc and
the “A2 binding site”.
The pharmacomodulations take place around the hydropho-
bic fragment. For the exploration of the structure-activity
relationships (SARs), 19 different hydrophobic substituents
(series A) were introduced on the 1,4-diazepane scaffold and
SAR studies were completed by the synthesis of two further series
of compounds (series B and C) bearing an additional phenyl
group on the 1,4-diazepane scaffold. In light of these considera-
tions, our efforts to identify new FTis have revealed three novel
series of 1,4-diazepane and 5-phenyl-1,4-diazepane derivatives.
Cellular activity against prostatic cell lines (DU145 and PC3)
was determined as well as their effects on tubulin organization.
One of the possible approaches in the development of FTis is
the design of peptidomimetics of the “CA1A2X box”. Cocrystal-
lization of FTase with numerous FTis or substrate has enabled
the complete elucidation of FTase mechanism of action.12-15
Thereafter, the design of FTis evolved from thiol-containing
peptidomimetics16 to non-thiol, non-peptidic molecules.17-19
On the basis of these data and molecular modeling studies, we
designed a pharmacophoric model based on a spacer that
positioned correctly in the enzyme’s active site a zinc chelator
and a hydrophobic fragment. Molecular docking provided inter-
esting features17 in terms of key interactions between the
p-cyanobenzylimidazole part with zinc and interaction between a
hydrophobic partwith a subsite called the“A2 bindingsite”. As part
of our continuing effort to identify novel FTis, this information
’ CHEMISTRY
The syntheses of diazepane derivatives are outlined in
Schemes 1 and 2; structures of target amides (3-21, 28-33,
and 46-51) are summarized in Tables 1, 2, and 3. The
p-cyanobenzylimidazole moiety was introduced into target mole-
cules according to a standard procedure of N-alkylation with the
5-chloromethyl-1-(4-cyanobenzyl)imidazole initially prepared.20
Compounds 3-21 were prepared in three steps from com-
mercially available Boc-homopiperazine (series A, Scheme 1).
Briefly, N-alkylation of Boc-homopiperazine with chloromethyl
Figure 1. FTis developed to clinical trials.
Figure 2. (A) Docking of compound 20 (IC50 = 5 nM) in the active site of the FTase. (B) Superimposition of compounds 32 (blue) and 50 (yellow)
docked in the FTase binding site.
Scheme 1. Syntheses of Series Aa
a Reagents and conditions: (a) 5-chloromethyl-1-(4-cyanobenzyl)imidazole, DIEA, ACN, 80 °C, 4 h; (b) HCl/iPrOH, rt, 4 h; (c) R-COOH, PyBroP,
PS-HOBt (HL), DIEA, DMF, rt, 20 h.
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Scheme 2. Syntheses of Series B and Ca
a Reagents and conditions: (a) xylene, reflux, 8 h; (b) H2, 50 bar, 10% Pd/C, MeOH, 60 °C, 24 h; (c) (Boc)2O, DIEA, dioxane/water (4:1), rt, 48 h;
(d) LiAlH4, THF, rt, 24 h; (e) 5-chloromethyl-1-(4-cyanobenzyl)imidazole, DIEA, ACN, 80 °C, 4 h; (f) iPrOH/HCl (5-6 N), rt, 2 h; (g) R-COOH,
EDCI, DMAP, pyridine, DMF, rt, 18 h.
p-cyanobenzylimidazole gave compound 1. After deprotection,
the amine 2 was next coupled with a set of different carboxylic
acids. Chemical diversity around the 1,4-diazepane scaffold was
easily obtained by a solid-phase procedure using previously
reported polystyrene-supported HOBt as coupling agent.21
The reaction was performed on a Quest 205 synthesizer accord-
ing to the general reaction pathway generating compounds
3-21. This was a two-step procedure consisting of (i) formation
of the polymer bound activated ester of the carboxylic acid using
PyBrop and (ii) release of the target amide in solution by
addition of the amine. Optimization of both steps was performed
to obtain a solution of amide without byproducts. Optimum
conditions for the activation step were found to be two periods of
2 h in DMF. The coupling step was achieved in 20 h with 0.6
equiv of the amine 2 in DMF.22 The target compounds 3-21
were purified by preparative TLC.
Synthesis of 5-phenyl-1,4-diazepane derivatives (Scheme 2)
was performed by the use of two different synthetic routes (series
B and C) that both included the formation of the 4-Boc-5-
phenyl-1,4-diazepane scaffold 25 previously described by our
team.23 Condensation of ethyl benzoylacetate with ethanedia-
mine was carried out in xylene to yield enaminone 22 by
azeotropic distillation. Reduction of the olefin by catalytic
hydrogenation (H2, 50 bar) gave 2-phenyl-1,4-diazepan-5-one
23. After carbamate protection of the secondary amine, the 1,4-
diazepane scaffold 25 was obtained by lactam reduction with
LiAlH4.
The target compounds 28-33 (series B) and 46-51 (series C)
were finally obtained by classical methodology including alkyla-
tion of amines with chloromethylimidazole, cleavage of the Boc
group, and acylation under peptide coupling conditions (EDCI/
DMAP).
’ PHARMACOLOGICAL EVALUATION
All of the synthesized compounds were tested in a preliminary
enzymatic assay and in a cytotoxicity assay on hormone-resistant
prostate cancer DU145 and PC3 cell lines. The FTase assays
were determined by measurement of the inhibition of human
recombinant FTase which catalyzed the transfer of the FPP
moiety to a fluorescent dansyl-GCVLS.24 This fluorescent assay
is easier and more rapid than conventional methods using
radioactive substrates such as [3H]FPP. Commercially available
L-744,832 ((2S)-2-[[(2S)-2-[(2S,3S)-2-[(2R)-2-amino-3-mercap-
topropyl]amino]-3-methylpentyl]oxy]-1-oxo-3-phenylpropyl]-
amino]-4-(methylsulfonyl)-butanoic acid 1-methylethyl ester)
was used as a reference compound.25 L-744,832 has been
reported to exhibit good pharmacological activity in hormone-
resistant prostate cancer cell lines. Moreover, an in vivo study
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Table 1. Structures and Biological Data of Compounds in Series A
a Values are the mean of three determinations (standard deviation lower than 10%). b Cell proliferation was measured from at least three independent
determinations (standard deviation lower than 10%).
with L-744,832 on xenograft mice at 30 mg/kg showed a
decrease of 85% of tumor on DU145 and 87% on PC3.27 In
our study, the IC50 values for the growth inhibitions were 75 μM
(PC3) and 78 μM (DU145). Indeed, there is ∼300-fold dif-
ference of cytotoxicity between the IC50 (PC3) evaluated in our
cell culture conditions and that referenced in the literature (225
nM versus 75 μM, respectively).26 No data were referenced for
IC50 on DU145 cell lines.
We started our investigations by evaluating the influence on
enzymatic and cellular activity of the hydrophobic moiety at the
4-N-position of the 1,4-diazepane spacer (series A). The results
are summarized in Table 1. All compounds inhibited FTase
activity in the nanomolar range. These results allowed character-
ization of a first FTase inhibitory fragment based on the 1,4-
diazepane linked to 4-cyanobenzylimidazole. These observations
suggested that the diazepane core constitutes a convenient linker.
Compound 3, with a benzoyl substituent, is one of the most
potent compounds of the series A with an IC50 of 8 nM. The
influence of the electronic character was then studied. No
correlation can be established between the FTase inhibition
and the electronic character of the aroyl moiety. The modifica-
tion of the electronic density of the aroyl either by electron-
donating level like methyl (9, IC50 = 15 nM) and methoxy (10,
IC50 = 43 nM) or by withdrawing groups like fluorine (4, IC50
140 nM), chlorine (5, IC50 = 25 nM), and bromine (6, IC50
=
=
64 nM) led to a decrease of FTase inhibition. Moreover, the
displacement of fluorine from ortho (4) to para (7, IC50
69 nM) enhances the activity and the introduction of a second
=
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Table 2. Structures and Biological Data of Compounds in
Series B
Table 3. Structures and Biological Data of Compounds in
Series C
a Values are the mean of three determinations (standard deviation lower
than 10%). b Cell proliferation was measured from at least three
independent determinations (standard deviation lower than 10%).
a Values are the mean of three determinations (standard deviation lower
than 10%). b Cell proliferation was measured from at least three
independent determinations (standard deviation lower than 10%).
chlorine in compound 5 reduces affinity (8, IC50 = 41 nM). From
this observation, it seems that only the steric character of these
groups modifies the activity rather than their electronic effect on
the benzoyl.
Next, we investigated the modification of the phenyl group in
3 by bioisosters such as pyridinyl, thienyl, naphthyl, or biphenyl
groups. Results confirmed that this modification affects affinity
toward FTase. For example, the pyridinyl derivative 11 (IC50=
142 nM) and thienyl derivative 12 (IC50= 42 nM) displayed
lower inhibitory potencies than 3. A similar result was obtained
group, have no antiproliferative activity on DU145 or PC3
cell lines. These eight compounds all exhibited a better cellular
eficiency than L-744,832. The best cellular activities were
observed with the 2,4-dichlorophenyl (8) (IC50(DU145) = 45 μM,
IC50(PC3) = 26 μM) and the adamantyl (20) (IC50(DU145) =
44 μM, IC50(PC3) = 21 μM). No correlation in the series A was
observed between the FTase inhibition and the cell growth
inhibition on the two cell lines. Among many factors influencing
the cell-based assay activity, the cell membrane penetration of
FTis is certainly important. The low cellular activity of many of
the compounds studied here could be explained by a lack of
lipophilicity. The addition of a chlorine atom (8) to compound 5
decreased the FTase activity but increased the whole cell assay.
This observation could only be explained by the improvement of
cell penetration facilitated by increased lipophilicity (5, CLogP =
2.73; 8, CLogP = 3.47).28 It is worth pointing out that all
compounds exhibited antiproliferative activity in excess of their
intrinsic activity against FTase. Though rare, this phenomenon
has been reported in other publications.29,30
Next, we decided to retain the diazepane scaffold and to
develop two series (B and C) of potent FTase inhibitors bearing a
phenyl group on the scaffold in order to improve their lipophilic
character and to procure an additional interaction with the
enzyme. Two alkyl groups (cyclohexyl and adamantyl) and
four aryl groups (phenyl, 2,4-dichlorophenyl, naphthyl, and
o-biphenyl) were introduced. The enzymatic studies revealed
different results between series B and C derivatives and showed
that the position of the phenyl group around the spacer was
important for optimal interaction with the active site. The FTase
activity of series B derivatives (IC50 = 17-503 nM) was less than
those of series C (IC50 = 6-27 nM). In addition, when looking at
compounds 28-33, we found that series B compounds had a
for naphthyl derivatives 13 (R, IC50 = 39 nM) and 14 (β, IC50
=
35 nM). However, when the naphthyl moiety was positioned
further away from the diazepane core (by a methylene) leading to
compound 15 (IC50 = 13 nM), inhibition of FTase was of the
same magnitude as 3. We also synthesized three biphenyl
derivatives: FTase inhibitory activity of compounds 16 (IC50
=
153 nM), 17 (IC50 = 43 nM), and 18 (IC50 = 26 nM) suggests
that the hydrophobic cavity could be wide and shallow.
Different results were observed when the diazepane core was
substituted by an aliphatic substituent. The isobutyl group (21,
IC50 = 205 nM) decreased markedly the interaction with the
active site, whereas the adamantyl group (20, IC50 = 5 nM)
induced a slight increase in affinity and afforded the best FTase
inhibitory potency of the series A. As a net result, the aromatic
character of the terminal group does not appear to be critical for
good affinity, while the steric parameter of this group is clearly of
great importance. Any substitution on a benzoyl group tends to
decrease the affinity in direct relation to the bulk introduced. On
the other hand, a shorter moiety, such as the isobutyl of
compound 21, does not lead to a good affinity, presumably
because it lacks a sufficient contact with the FTase pocket.
Only eight compounds (5, 8, 13, 14, 16, 17, 18, and 20)
among the 19 prepared exhibited cellular activity. Thus, some
powerful inhibitors of FTase, like compound 3 with a phenyl
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slightly lower affinity compared with their nonsubstituted analo-
gues in series A (3, 8, 13, 18, 19, and 20).
As expected, the cellular activity of compounds in both series B
and C generally followed the FTase assay results. The majority of
these FTis exhibited better cell growth inhibition than the
reference inhibitor L-744,832. The cyclohexyl derivative 50
displayed the best cellular activities (IC50(DU145) = 29 μM
and IC50(PC3) = 18 μM). The addition of a phenyl group at the
C-5-position of diazepane improved only slightly the FTase
inhibition (50, IC50 = 7 nM) but significantly enhanced the cell
growth inhibition on both cell lines. This observation supports
the hypothesis that the low lipophilicity of many compounds in
series A limited their cellular activity. The phenyl bearing at the
C-5-position of the diazepane scaffold increases the lipophilicity
which is necessary for membrane penetration and subsequent
inhibition of cell proliferation.
Figure 3. Immunofluorescent visualization of the tubulin network in
control (A) and in treated (B-F) PC3 cells. Cells were treated for 24 h
both in the absence and in the presence of 21, 18, and 32 μM compounds
20, 50, and 51, respectively (D, E, F), and microtubules were visualized
by immunofluorescence labeling using an antibody against R-tubulin.
Treatment with CA-4 (3 nM (B)) and L-744,832 (75 μM (C)) were
included as positive controls.
’ PHARMACOKINETIC STUDIES IN VITRO
Pharmacokinetic data for compounds 20, 50, and 51 were
collected on Caco2 cells to evaluate their absorption potency in
an in vitro model and in human liver microsomes to assess their
metabolic stability.31 Compounds 20 and 50 revealed an appar-
ent permeability coefficient (Papp) value of 28.8 ꢀ 10-6 and
25.1 ꢀ 10-6 cm/s, respectively, and are therefore considered
highly permeable. Evaluation of compound 51 revealed a Papp
value of 3.6 ꢀ 10-6 cm/s, which is indicative of a medium
permeable molecule. In this assay, propranolol was used as a
highly permeable reference (Papp = 41.1 ꢀ 10-6 cm/s), labetalol
as a medium permeable reference (Papp = 7.3 ꢀ 10-6 cm/s), and
ranitidine as a low permeable reference (Papp = 0.7 ꢀ 10-6 cm/s),
according to the FDA guidance.32 Furthermore, metabolic
stability of these compounds were evaluate in human liver
microsomes and showed a poor stability as the percentage of
remaining parent drug at the end of the assay were 1%, 2%, and
3% for compounds 20, 50, and 51, respectively. For metabolic
stability assays, propanolol, terfenadine, and verapamil were used
as references (percentage of remaining parent drug at the end of
the assay: 60, 6 and 9% respectively).
of the pocket. From an atomic point of view, the best conforma-
tions of these three compounds were very similar to each other
(19, 32, and 50), with the positioning of the cyclohexyl group
nearly superimposable. It is also of interest to note that the
adamantyl of compounds 20 and 33 also occupies the same
position in the binding site, implying that a bulky aliphatic group
is more favorably bound in the portion of the pocket, delineated
by Trp102β, Trp106β, and Tyr361β (the “A2 binding site”). At
the other end of the compounds, the imidazole nitrogen co-
ordinates with the zinc of the enzyme while the cyano moiety is
engaged in a hydrogen bond with Arg202. The most numerous
conformations of compounds 32 and 50 offer an explanation for
the better potency of series C. Although the best-ranked con-
formation of 32 binds in a conformation very close to that of
compound 50, it is also very uncommon in the pool of solutions.
The most numerous binding mode of 32 is shown in Figure 2. It
is characterized by the placement of the phenyl and the terminal
cyclohexyl at the very gate of the pocket, where they do not
contribute to the affinity of the compound. As there is no high
energy interaction present to anchor the inhibitor to the enzyme,
such a binding mode can only be described as potentially
unstable and weak.
’ MOLECULAR MODELING
Docking simulations,33 using GOLD software, were carried
out in order to clarify the binding mode of FTis at the active site
of FTase (Figure 2). Thirty-one solutions were generated for
each compound, then ranked according to an in-house scoring
function. The consistency of the best ranked solution was
assessed by manually inspecting the conformations. The phar-
macological results were concordant with molecular modeling
which provides a reasonable explanation for their inhibitory
activity.
Compound 19 is too small to have a well-defined binding
mode. Its small size enables it to adopt a large number of
conformations, none of them being predominant among the
best ranked solutions. Only one solution displayed the same U
shape, as the ligand used crystallized previously with the
enzyme.34 For compound 50, the benzyl group on the diazepane
is oriented toward the opening of the binding site in a conforma-
tion that is clearly poorer because it is unable to form any
interaction. Its terminal cyclohexyl occupies the depth of the
pocket, surrounded by Trp 102β and 106β. Compound 32
displayed evenly distributed conformations in the expected U
shape and the other conformations, directing it toward the entry
’ IMPACT OF FTIS ON TUBULIN ORGANIZATION
Some recent studies have revealed that FTis such as lonafarnib
(or SCH66336) were involved in the destabilization of the
tubulin network.35 We wanted to know, in this part of the study,
if the more potent FTis defined earlier (compounds 20, 50, and
51) were able to affect microtubule organization in PC3 cells.
Thus, PC3 cells were treated with the three compounds of
interest for 24 h. After fixing and permeabilization, microtubules
were visualized by indirect immunofluorescence microscopy
using an antibody against R-tubulin. Cells treated with DMSO
(control cells) exhibited a well-organized microtubule network
throughout the cells (Figure 3A). In contrast, cells treated with
CA-4 (positive control, Figure 3B), known to bind tubulin at the
colchicine site and to destabilize microtubules by the inhibition
of assembly of tubulin molecules,36 showed a disruption of the
tubulin network and the appearance of a bundle at a pole of the
cell compared with DMSO treated cells. Treatment with 75 μM
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network by FTis. These results are in agreement with previous
observed data. Except for the classical targets of FTis like Ras and
RhoB, involved in the antiproliferative and cytotoxic activities of
these compounds, the function of CENP-E, which mediates cell
cycle progression by chromosome capture and alignment and is a
known farnesylated mitotic protein, was also investigated in this
study. Depletion of CENP-E in our cells, by transfecting the
appropriate siRNA, led to a disruption of the tubulin network
similar to that observed with treatment of PC3 by the com-
pound 20. We therefore hypothesize that in PC3 cells, inhibi-
tion of CENP-E farnesylation by compound 20 led to a loss
of CENP-E activity and tubulin network disruption. Since
another farnesylated mitotic protein, CENP-F, is known to be
required in cell cycle progression, it would be interesting to test
the ability of our compounds to block the cell cycle in G2-M.
Furthermore, studies concerning the combined use of our potent
FTis with microtubule interfering agents are in progress, as this
association presents an effective antiproliferative and antimitotic
synergy.
Figure 4. Immunofluorescent visualization of the tubulin network in
CENP-E knockdown PC3 cells (C) and control siRNA-transfected cells
(B) compared to DMSO treated cells (A).
of L-744,832 (FTis positive control, Figure 3C) led to the
formation of similar microtubule bundles observed in the CA-4
treated cells. Cells treated with compounds 20, 50, and 51 at
concentrations equal to their IC50 values displayed a completely
disorganized tubulin network restricted to the periphery of PC3
cells (Figure 3C, Figure 3E, and Figure 3F, respectively). More-
over, with these compounds, the microtubule mass appeared to
be decreased compared with control cells, especially for com-
pounds 50 and 51.
’ EXPERIMENTAL SECTION
Aberrant spindle formation following treatment with FTis was
related, in previous work,37,38 to the disruption of farnesylated
mitotic proteins’ functionality, CENP-E and CENP-F, known to
be strongly involved in kinetochore-microtubule interactions. To
assess the role of CENP-E in PC3 cells as one of the targets
associated with the tubulin network destabilization following
FTis treatment, we suppressed CENP-E expression by using
CENP-E siRNA. This approach led to a 90% decrease in CENP-E
expression in our cells (data not shown). Compared to controls
or to GAPDH siRNA transfected cells, CENP-E siRNA trans-
fected cells exhibited a completely disorganized tubulin network
(Figure 4) similar to PC3 cells treated with the compound 20.
Chemistry. General Remarks. All commercial reagents and
solvents were used without further purification. Analytical thin-layer
chromatography was performed on precoated Kieselgel 60F254 plates
(Macherey Nagel); the spots were visualized by UV (254 and 366 nm)
and/or with iodine. Silica gel grade 60 (230-400 mesh) purchased from
Macherey Nagel was used to carry out chromatographic separation.
Preparative thin layer chromatography (TLC) was performed using
silica gel from Macherey Nagel, the compounds being extracted from the
silica using CH2Cl2/MeOH (8:2 v/v). All melting points were deter-
mined with a B€uchi 535 capillary apparatus and remained uncorrected.
1H NMR spectra were obtained using a Bruker 300 MHz spectrometer.
Chemical shifts (δ) were expressed in ppm relative to tetramethylsilane
used as an internal standard. J values are in hertz, and the splitting
patterns were designated as follows: s singlet, d doublet, t triplet, m
multiplet. HPLC-MS analyses were accomplished using a HPLC
instrument combined with a Surveyor MSQ (Thermo Electron)
equipped with an APCI source (ODS-30 column, mobile phase of
water/acetonitrile/formic acid, gradient mode). Elemental analyses for
all compounds were performed by the “Service Central d’Analyses” at
the CNRS, Vernaison, France. All tested target compounds possessed a
purity ofg95% as verified by HPLC-MSorelementalanalysis(C, H, N).
N-1-tert-Butoxycarbonyl-N-4-[1-(4-cyanobenzyl)-1H-5-
imidazolyl]methyl-1,4-diazepane (1). Boc-homopiperazine (3.09 mL,
15.7 mmol) was added to a solution of 2-chloromethyl-1(4-cyanobenzyl)-
imidazole (3.5 g, 13.1 mmol) and DIEA (6.82 mL, 39.2 mmol) in 40 mL of
ACN. The reaction mixture was stirred for 4 h at 80 °C and then concentrated
under vacuum. The residue was dissolved in EtOAc and washed with water
and brine. The organic layer was dried over MgSO4, filtered, and evaporated.
The residue was purified by column chromatography on silica gel (CH2Cl2/
MeOH 96:4) to give a white powder (3.73 g, 72% yield). Rf = 0.65 (CH2Cl2/
MeOH 9:1). Mp: 131-133 °C. IR (neat): ν 2229 (CN), 1683 (CO). 1H
NMR (DMSO-d6): δ 1.45 (s, 9 H), 2.45-2.60 (m, 4 H), 3.31 (s, 2 H),
3.31-3.46 (m, 6 H), 5.38 (s, 2 H), 6.97 (s, 1 H), 7.16 (d, J=7.9Hz,2H),7.56
(s, 1 H), 7.63 (d, J = 7.9 Hz, 2 H). MS (APCIþ) m/z = 396 (M þ Hþ).
4-[1-(4-Cyanobenzyl)-1H-5-imidazolyl]methyl-1,4-diaze-
pane Hydrochloride (2). A solution of 5-6 N HCl in isopropanol
was added to a solution of compound 1 (1 g, 2.53 mmol) in MeOH
(2 mL). After the mixture was stirred for 4 h at room temperature, the
solvent was removed under vacuum. The solid was washed with diethyl
ether and recrystallized in absolute EtOH to give a white powder (0.97 g,
95% yield). Rf = 0.35 (CH2Cl2/MeOH 9:1). Mp: 152-154 °C. IR
(neat): ν 2228 (CN). 1H NMR (DMSO-d6): δ 3.10-3.90 (m, 10 H),
’ CONCLUSION
This study has shown that the 1,4-diazepane scaffold consti-
tutes an effective platform for the development of novel inhibi-
tors. Thirty-one compounds were synthesized and most of them
exhibited nanomolar affinity for an isolated FTase enzyme as well
as cellular activity on two hormone refractory prostate cancer cell
lines (PC3 and DU145).
Examination of the SAR of these compounds revealed inter-
esting information about their interactions with the active site of
the FTase. In series A (with no phenyl on the spacer), modula-
tion of 19 substituents enabled us to demonstrate that this group
FTis fits principally by hydrophobic interactions with a pocket
delineated by Trp102β, Trp106β, and Tyr361β. Moreover, the
correct addition of a phenyl group on the diazepane scaffold
(series C vs series B) proved to be necessary to increase the
cellular activity without loss of FTase inhibition and to increase
the lipophilicity of the compounds.
The permeation ability of compounds 20, 50, and 51 through
biological membranes was assessed on Caco2 cells to evaluate
their potential for gastrointestinal absorption. 20 and 50 were
predicted to be “highly permeable” and therefore conveniently
enter blood circulation. 51 was predicted to have “medium
permeability”. However, these compounds exhibited a poor
metabolic stability, suggesting that a peroral administration is
to be avoided because of the first-pass effect. These preliminary in
vitro results are the first step for a fully in vivo evaluation.
In this study, we have shown for the first time in the prostatic
hormone-independent PC3 cell line a disruption of the tubulin
1184
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Journal of Medicinal Chemistry
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4.43 (s, 2 H), 5.82 (s, 2 H), 7.57 (d, J = 7.9 Hz, 2 H), 7.92 (d, J = 7.9 Hz, 2
H), 8.11 (s, 1H), 9.31 (s, 1 H), 9.64 (bs, 1 H). MS (APCIþ) m/z = 296
(M þ Hþ).
9.26 (s, 1 H), 12.22 (bs, 1 H). MS (APCIþ) m/z = 468.0 (M þ Hþ).
Anal. (C24H23Cl2N5O 2HCl 1.5H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(3-toluyl)-1,4-
diazepane Hydrochloride (9). 61% yield. Rf = 0.65 (CH2Cl2/MeOH
9:1). Mp: 156-157 °C. IR (neat): ν 2228 (CN), 1623 (CO). 1H NMR
(DMSO-d6): δ 2.34 (s, 3 H) ; 3.15-3.85 (m, 10 H), 4.51 (s, 2 H), 5.84
(s, 2 H), 7.17-7.39 (m, 4 H,), 7.41-7.57 (m, 2 H), 7.83-7.95 (m, 2 H),
8.18 (bs, 1 H), 9.12 (s, 1 H), 12.13 (bs, 1 H); MS (APCIþ) m/z = 414
(M þ Hþ); Anal. (C25H27N5O 2HCl 1.5H2O) C, H, N, Cl.
General Procedure for Synthesis of 1-[1-(4-Cyanobenzyl)-
1H-imidazol-5-yl]methyl-1,4-diazepanes (Compounds 3-21).
An amount of 1.18 g of PS-HOBt (HL) resin was added to a 100 mL
polypropylene Quest 205 reaction vessel and swelled in anhydrous DMF
(10 mL) at room temperature. After 24 h, the solvent was filtered and a
solution of carboxylic acid (2.32 mmol), DIEA (0.81 mL, 4.64 mmol),
and PyBroP (1.08 g, 2.32 mmol) in anhydrous DMF (20 mL) was
added. The mixture was stirred at room temperature for 2 h and filtered.
The resin was washed successively with DMF (2 ꢀ 10 mL), DCM (2 ꢀ
10 mL), and DMF (2 ꢀ 10 mL). The same activation procedure was
repeated a second time. Then a solution of amine 2 (500 mg, 1.24 mmol)
and diisopropylethylamine (0.81 mL, 4.64 mmol) in DMF (20 mL) was
added, and the reaction mixture was stirred for 20 h at room tempera-
ture. The supernatant was then separated from the resin, and the
polymer beads were washed with DMF (2 ꢀ 10 mL), DCM (2 ꢀ 10
mL), and DMF (2 ꢀ 10 mL). The combined solutions were concen-
trated, and the residue was purified by preparative TLC on silica gel
(DCM/MeOH 95:5). The product was then added to a solution of 5-6
N HCl in iPrOH and stirred for 1 h at room temperature. After addition
of diethyl ether, the solid was filtered off, washed with diethyl ether, and
recrystallized from EtOH/Et2O to give a white powder.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2,3-dimethoxy-
benzoyl)-1,4-diazepane Hydrochloride (10). 60% yield. Rf = 0.44
(CH2Cl2/MeOH 9:1). Mp: 143-145 °C. IR (neat): ν 2230 (CN),
1623 (CO). 1H NMR (DMSO-d6): δ 2.98-3.69 (m, 10 H), 3.71 (s, 3
H), 3.83 (s, 3 H), 4.42 (s, 2 H), 5.80 (s, 2 H), 6.82-6.92 (m, 1 H),
7.06-7.15 (d, J = 8.0 Hz, 2 H), 7.49-7.61 (m, 2 H), 7.92 (d, J = 8.2 Hz,
2 H), 8.15 (s, 1 H), 9.18 (bs, 1 H), 12.12 (bs, 1 H). MS (APCIþ) m/z =
460.1 (M þ Hþ). Anal. (C26H29N5O3 2HCl 1.5H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(4-pyridinoyl)-
1,4-diazepane Hydrochloride (11). 52% yield. Rf = 0.17 (CH2Cl2/
MeOH 9:1). Mp: 210-212 °C. IR (neat): ν 2229 (CN), 1627 (CO). 1H
NMR (DMSO-d6): δ 3.10-3.85 (m, 10 H), 4.48 (s, 2 H), 5.82 (s, 2 H),
7.28 (d, J = 8.0 Hz, 2 H), 7.55-7.78 (m, 4 H), 8.13 (s, 1 H), 8.92 (m,
2 H), 9.23 (s, 1 H), 12.32 (bs, 1 H). MS (APCIþ) m/z = 401.2 (M þ Hþ).
Anal. (C23H24N6O 3HCl 3H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(3-thenoyl)-1,4-
diazepane Hydrochloride (12). 54% yield. Rf = 0.33 (CH2Cl2/MeOH
9:1). Mp: 214-216 °C. IR (neat): ν 2230 (CN), 1631 (CO). 1H NMR
(DMSO-d6): δ 3.18-3.92 (m, 10 H), 4.48 (s, 2 H), 5.80 (s, 2 H),
7.02-7.10 (m, 1 H), 7.46 (s, 1 H), 7.52 (d, J = 8.0 Hz, 2 H), 7.76 (m, 1 H),
7.92 (d, J = 7.9 Hz, 2 H), 8.13 (s, 1 H), 9.22 (s, 1 H), 12.15 (bs, 1 H).
MS (APCIþ) m/z = 406.2 (M þ Hþ). Anal. (C22H23N5OS 2HCl
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-benzoyl-1,4-dia-
zepane Hydrochloride (3). 60% yield. Rf = 0.36 (CH2Cl2/MeOH 9:1).
1
Mp: 168-171 °C. IR (neat): ν 2228 (CN), 1623 (CO). H NMR
(DMSO-d6): δ 3.15-3.85 (m, 10 H), 4.51 (s, 2 H), 5.84 (s, 2 H),
7.32-7.50 (m, 5 H), 7.57 (d, J = 7.3 Hz, 2 H), 7.91 (d, J = 7.0 Hz, 2 H),
8.18 (s, 1 H), 9.29 (s, 1 H), 12.13 (bs, 1 H). MS (APCIþ) m/z = 400.2
(M þ Hþ). Anal. (C24H25N5O 2HCl 2.5H2O) C, H, N, Cl.
3
3
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2-fluorobenzoyl)-
1,4-diazepane Hydrochloride (4). 56% yield. Rf = 0.63 (CH2Cl2/
MeOH 9:1). Mp: 162-163 °C. IR (neat): ν 2228 (CN), 1623 (CO).
1H NMR (DMSO-d6): δ 3.15-3.85 (m, 10 H), 4.51 (s, 2 H), 5.84 (s, 2
H), 7.17-7.58 (m, 5 H), 7.83-7.95 (m, 3 H), 8.18 (s, 1 H), 9.12 (s, 1
H), 12.13 (bs, 1 H). MS (APCIþ) m/z = 418 (M þ Hþ). Anal.
(C24H24FN5O 2HCl 1.5H2O) C, H, N, Cl.
2H2O) C, H, N, Cl.
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(1-naphthoyl)-1,4-
diazepane Hydrochloride (13). 68% yield. Rf = 0.42 (CH2Cl2/MeOH
9:1). Mp: 228-230 °C. IR (neat): ν 2228 (CN), 1641 (CO). 1H NMR
(DMSO-d6): δ 3.15-3.95 (m, 10 H), 4.48 (s, 2 H), 5.82 (s, 2 H), 7.40-
7.60 (m, 5 H), 7.85-8.05 (m, 6 H), 8.21 (s, 1 H), 9.27 (s, 1 H), 12.22
(bs, 1 H). MS (APCIþ) m/z = 450.1 (M þ Hþ). Anal. (C28H27N5O
3
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2-chlorobenzoyl)-
1,4-diazepane Hydrochloride (5). 52% yield. Rf = 0.47 (CH2Cl2/
MeOH 9:1). Mp: 139-141 °C. IR (neat): ν 2230 (CN), 1623 (CO).
1H NMR (DMSO-d6): δ 3.19-3.88 (m, 10 H), 4.50 (s, 2 H), 5.85 (s, 2
H), 7.48-7.71 (m, 6 H), 7.85 (s, 1 H), 7.89 (d, J = 7.2 Hz, 2 H), 9.26 (s, 1
H), 12.18 (bs, 1 H). MS (APCIþ) m/z = 433.9 (M þ Hþ). Anal.
(C24H24ClN5O 2HCl 2H2O) C, H, N, Cl.
2HCl 3H2O) C, H, N, Cl.
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2-naphthoyl)-
1,4-diazepane Hydrochloride (14). 62% yield. Rf = 0.39 (CH2Cl2/
MeOH 9:1). Mp: 224-226 °C. IR (neat): ν 2229 (CN), 1627 (CO). 1H
NMR (DMSO-d6): δ 3.15-3.85 (m, 10 H), 4.48 (s, 2 H), 5.82 (s, 2 H),
7.50-7.65 (m, 5 H), 7.85-8.08 (m, 6 H), 8.13 (s, 1 H), 9.18 (s, 1 H),
12.13 (bs, 1 H). MS (APCIþ) m/z = 450.2 (M þ Hþ). Anal.
(C28H27N5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2-bromobenzoyl)-
1,4-diazepane Hydrochloride (6). 46% yield. Rf = 0.55 (CH2Cl2/
MeOH 9:1). Mp: 178-180 °C. IR (neat): ν 2228 (CN), 1625 (CO).
1H NMR (DMSO-d6): δ 3.28-3.92 (m, 10 H), 4.50 (s, 2 H), 5.85 (s, 2
H), 7.44-7.62 (m, 6 H), 7.82 (s, 1 H), 7.90 (d, J = 7.2 Hz, 2 H), 9.25 (s, 1
H), 12.20 (bs, 1 H). MS (APCIþ) m/z = 478.1 (M þ Hþ). Anal.
(C24H24BrN5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-[2-(1-naphtyl)acetyl]-
1,4-diazepane Hydrochloride (15). 53% yield. Rf = 0.45 (CH2Cl2/
MeOH 9:1). Mp: 216-218 °C. IR (neat): ν 2229 (CN), 1614 (CO). 1H
NMR (DMSO-d6): δ 3.22-3.82 (m, 10 H), 3.88 (s, 2 H), 4.48 (s, 2 H),
5.82 (s, 2 H), 6.88 (m, 1 H), 7.50-7.65 (m, 4 H), 7.85-8.08 (m, 6 H),
8.13 (s, 1 H), 9.16 (s, 1 H), 12.18 (bs, 1 H). MS (APCIþ) m/z = 464.3
(M þ Hþ). Anal. (C29H29N5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(4-fluorobenzoyl)-
1,4-diazepane Hydrochloride (7). 43% yield. Rf = 0.45 (CH2Cl2/
MeOH 9:1). Mp: 95-97 °C. IR (neat): ν 2230 (CN), 1638 (CO).
1H NMR (DMSO-d6): δ 3.30-3.90 (m, 10 H), 4.52 (s, 2 H), 5.82 (s, 2
H), 7.48-7.68 (m, 6 H), 7.75 (s, 1 H), 7.92 (d, J = 7.5 Hz, 2 H), 9.22 (s, 1
H), 12.22 (bs, 1 H). MS (APCIþ) m/z = 418.0 (M þ Hþ). Anal.
(C24H24FN5O 2HCl 1H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(4-phenylbenzoyl)-
1,4-diazepane Hydrochloride (16). 56% yield. Rf = 0.42 (CH2Cl2/
MeOH 9:1). Mp: 231-233 °C. IR (neat): ν 2230 (CN), 1635 (CO). 1H
NMR (DMSO-d6): δ 3.12-3.85 (m, 10 H), 4.48 (s, 2 H), 5.83 (s, 2 H),
7.38-7.62 (m, 7 H), 7.67-7.79 (m, 4 H), 7.92 (d, J = 7.9 Hz, 2 H),
8.15 (s, 1 H), 9.22 (s, 1 H), 12.17 (bs, 1 H). MS (APCIþ) m/z = 476.2
(M þ Hþ). Anal. (C30H29N5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2,4-dichlorobenzoyl)-
1,4-diazepane Hydrochloride (8). 40% yield. Rf = 0.45 (CH2Cl2/
MeOH 9:1). Mp: 192-193 °C. IR (neat): ν 2230 (CN), 1635 (CO).
1H NMR (DMSO-d6): δ 3.28-3.96 (m, 10 H), 4.38-4.51 (m, 2 H),
5.85 (s, 2 H), 7.48-7.68 (m, 5 H), 7.75 (s, 1 H), 7.90 (d, J = 7.2 Hz, 2 H),
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(3-phenylbenzoyl)-
1,4-diazepane Hydrochloride (17). 55% yield. Rf = 0.42 (CH2Cl2/
MeOH 9:1). Mp: 247-248 °C. IR (neat): ν 2230 (CN), 1630 (CO). 1H
NMR (DMSO-d6): δ 3.15-3.85 (m, 10H), 4.44 (s, 2 H), 5.83 (s, 2 H),
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Journal of Medicinal Chemistry
ARTICLE
7.34-7.60 (m, 7 H), 7.67-7.79 (m, 4 H), 7.92 (d, J = 7.9 Hz, 2 H), 8.15
(s, 1 H), 9.16 (s, 1 H), 12.20 (bs, 1 H). MS (APCIþ) m/z = 476.3
(M þ Hþ). Anal. (C30H29N5O 2HCl 2H2O) C, H, N, Cl.
134-135 °C. IR (neat): ν 1681 (CO), 1672 (CO). 1H NMR (CDCl3):
δ 1.38 (s, 9 H), 2.59 (d, J = 14.0 Hz, 1 H), 2.95-3.00 (m, 2 H),
3.17-3.22 (m, 2 H), 3.47-3.55 (m, 1 H), 3.91 (d, J = 10.0 Hz, 1 H),
7.25-7.30 (m, 1 H), 7.28-7.37 (m, 5 H). MS (ESI) m/z = 290 (Mþ).
4-tert-Butoxycarbonyl-5-phenyl-1,4-diazepane (25). To a
solution of lactam 24 (25 g, 86 mmol) in anhydrous THF (200 mL),
LiAlH4 (6.5 g, 172 mmol) was added slowly at 0 °C. After the mixture
was stirred for 24 h at room temperature, the reaction was quenched by
the dropwise addition of water (50 mL) and sodium hydroxide (7 g, 172
mmol) at 0 °C. The precipitate was filtered off, and the filtrate was
concentrated. The residue was purified by chromatography on silica gel
(CH2Cl2/MeOH 95:5) to give a yellow oil (1.9 g, 40% yield). Rf = 0.62
(CH2Cl2/MeOH 9/1). IR (neat): ν 1697 (CO). 1H NMR (CDCl3): δ
1.50 (s, 9 H), 1.85-1.89 (m, 1 H), 2.03-2.12 (m, 1H), 2.13 (m, 1H,),
2.82-2.95 (m, 1H), 3.13-3.19 (m, 1H), 3.36-3.75 (m, 5H),
7.21-7.45 (m, 5 H). MS (APCIþ) m/z = 277 (M þ Hþ).
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(2-phenylbenzoyl)-
1,4-diazepane Hydrochloride (18). 55% yield. Rf = 0.42 (CH2Cl2/
1
MeOH 9:1). Mp: >250 °C. IR (neat): ν 2230 (CN), 1627 (CO). H
NMR (DMSO-d6): δ 3.12-3.77 (m, 10 H), 4.45 (s, 2 H), 5.77 (s, 2 H),
7.38-7.60(m, 11H), 7.92 (d, J = 7.9 Hz, 2 H), 8.09 (s, 1 H), 9.18(s, 1 H),
12.09 (bs, 1 H). MS (APCIþ) m/z = 476.2 (M þ Hþ). Anal.
(C30H29N5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-cyclohexanoyl-
1,4-diazepane Hydrochloride (19). 58% yield. Rf = 0.45 (CH2Cl2/
MeOH 9:1). Mp: 212-214 °C. IR (neat): ν 2229 (CN), 1630 (CO). 1H
NMR (DMSO-d6): δ 1.15-1.40 (m, 6 H), 1.55-1.80 (m, 5 H),
2.95-3.15 (m, 2H), 3.30-3.65 (m, 6H), 3.85-4.20 (m, 2 H), 4.44
(s, 2 H), 5.80 (s, 2 H), 7.54 (d, J = 8.3 Hz, 2 H), 7.92 (d, J = 8.1 Hz, 2 H),
8.14 (s, 1H), 9.24 (s, 1 H), 12.10 (bs, 1 H). MS (APCIþ) m/z = 406.2
(M þ Hþ). Anal. (C24H31N5O 2HCl 2H2O) C, H, N, Cl.
4-tert-Butoxycarbonyl-1-{[1-(4-cyanobenzyl)-1H-imidazol-
5-yl]methyl}-5-phenyl-1,4-diazepane (26). Amine 25 (1 g, 3.6
mmol), 5-chloromethyl-1-(4-cyanobenzyl)imidazole (1.1 g, 3,6 mmol),
and diisopropylethylamine (2.4 mL, 2.4 mmol) were added to 50 mL of
acetonitrile and stirred at 80 °C. After 4 h, the solvent was concentrated
under vacuum. The residue was diluted with EtOAc and washed by
water and brine. The organic layer was dried over MgSO4, filtered, and
evaporated. The residue was purified by column chromatography on
silica gel (CH2Cl2/MeOH 97:3) to obtain a yellow oil (0.93 g, 55%
yield). Rf = 0.65 (CH2Cl2/MeOH 9:1). IR: ν 2229 (CN), 1689 (CO).
1H NMR (CDCl3): δ 1.50 (s, 9 H,), 1.85-1.89 (m, 1 H,), 2.03-2.12
(m, 1 H), 2.82-2.95 (m, 1 H), 3.13-3.19 (m, 1 H), 3.36-3.75 (m, 7
H), 5.47 (s, 2 H), 6.78-6.80 (m, 1H), 7.21-7.58 (m, 10 H).
1-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}-5-phen-
yl-1,4-diazepane Hydrochloride (27). An amount of 2 mL of a
solution of hydrochloric acid (5-6 N) in iPrOH were added to carbamate
26(1 g, 2.12 mmol) in 3 mL of MeOH. After the mixture was stirred for 2 h
at room temperature, the solvent was concentrated under vacuum. An
amount of 100 mL of EtOAc was added to the residue dissolved in
minimum EtOH. The precipitate was filtered to obtain a white powder
(0.97 g, 95% yield). Rf = 0.05 (CH2Cl2/MeOH 9:1). Mp: > 250 °C. IR
(neat): ν 2229 CN). 1H NMR (D2O): δ 1.85-1.89 (m, 1 H), 2.03-2.12
(m, 1 H), 2.82-2.95 (m, 1 H), 3.13-3.19 (m, 1 H), 3.36-3.75 (m, 7 H),
5.45 (s, 2 H), 6.93-6.96 (m, 1 H), 7.19-7.59 (m, 10 H).
General Procedure for the Preparation of 1-{[1-(4-Cy-
anobenzyl)-1H-imidazol-5-yl]methyl}-5-phenyl-1,4-diaze-
panes (Compounds 28-33). To 30 mL of a solution of amine 27
in dry N,N-dimethylformamide were added carboxylic acid, EDCI-HCl,
pyridine, and DMAP. After the mixture was stirred for 18 h at room
temperature, the solvent was concentrated under vacuum. The residue
was extracted by EtOAc and washed successively with K2CO3 (10% aq),
water, and brine. After drying over MgSO4, the organic layer was
evaporated under vacuum. The residue was purified by chromatography
on silica gel (CH2Cl2/MeOH 95:5). The product obtained was then
added to a solution of 5-6 N HCl in iPrOH and stirred for 1 h at room
temperature. After addition of diethyl ether, the solid was filtered,
washed with diethyl ether, and recrystallized by EtOH/Et2O to give a
white powder.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(1-adamantanoyl)-
1,4-diazepane Hydrochloride (20). 58% yield. Rf = 0.29 (CH2Cl2/
MeOH 9:1). Mp: 206-207 °C. IR (neat): ν 2229 (CN), 1631 (CO). 1H
NMR (DMSO-d6): δ 1.68 (s, 6 H), 1.80-2.00 (m, 9 H), 3.00-3.12 (m,
2 H), 3.30-3.60 (m, 6 H), 3.85-4.15 (m, 2 H), 4.46 (s, 2 H), 5.82 (s, 2
H), 7.56 (d, J = 8.4 Hz, 2 H), 7.92 (d, J = 8.1 Hz, 2 H), 8.19 (s, 1 H), 9.29
(s, 1 H), 12.11 (bs, 1 H). MS (APCIþ) m/z = 458.3 (M þ Hþ). Anal.
(C28H35N5O 2HCl 2H2O) C, H, N, Cl.
3
3
1-[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl-4-(3-methylbutyroyl)-
1,4-diazepane Hydrochloride (21). 63% yield. Rf = 0.65 (CH2Cl2/
MeOH 9:1). Mp: 160-161 °C. IR (neat): ν 2228 (CN), 1623 (CO). 1H
NMR (DMSO-d6): δ 0.91 (d, J = 7.1 Hz, 6 H,), 2.07-2.21 (m, 1 H),
2.40 (d, J = 6.0 Hz, 2 H), 3.15-3.85 (m, 10 H), 4.51 (s, 2 H), 5.84 (s, 2
H), 7.41-7.57 (m, 2 H), 7.83-7.95 (m, 2 H), 8.18 (bs, 1 H), 9.12 (s, 1
H), 12.13 (bs, 1 H). MS (APCIþ) m/z = 380 (M þ Hþ). Anal.
(C22H29N5O 2HCl 1.5H2O) C, H, N, Cl.
3
3
7-Phenyl-2,3,4,5-tetrahydro-1H-1,4-diazepin-5-one (22).
Ethanediamine (4.7 g, 78 mmol) was added to ethyl benzoylacetate
(15 g, 78 mmol) in dry xylene (50 mL). The mixture was heated at
135 °C with an azeotropic Dean-Stark apparatus until the end of
water-EtOH distillation. After cooling to room temperature, the reac-
tion mixture was concentrated under vacuum. The residue was washed
with diethyl ether, filtered, and dried to give a white powder (8.8 g, 60%
yield). Rf (CH2Cl2/MeOH 9:1): 0.25. Mp: 206-207 °C. IR (neat):
ν 1626 (CO), 1550 (CdC). 1H NMR (DMSO-d6): δ 3.49-3.53 (m,
2 H), 3.64-3.68 (m, 2 H), 4.91 (m, 1 H), 5.00 (s, 1 H), 6.10 (m, 1 H),
7.38-7.41 (m, 3 H), 7.51-7.53 (m, 2 H).
7-Phenyl-1,4-diazepan-5-one (23). Palladium (10%) on acti-
vated carbon (0.5 g, 0.47 mmol) was added to diazepinone 22 (4.4 g, 2.3
mmol) in MeOH (55 mL), and the mixture was stirred with H2 (50 bar)
in an autoclave. After 24 h at 60 °C, the catalyst was filtered off and the
filtrate was concentrated under vacuum. The residue was purified by
chromatography (CH2Cl2/MeOH 9:1) to give a white powder (4.10 g,
1
95% yield). Rf = 0.15. Mp: 45 °C. IR (neat): ν 1670 (CO). H NMR
(CDCl3): δ 2.00 (m, 1 H), 2.59 (d, J = 14.0 Hz, 1 H), 2.95-3.00 (m, 2
H), 3.17-3.22 (m, 2 H), 3.47-3.55 (m, 1 H), 3.91 (d, J = 10.0 Hz, 1 H),
7.25 (m, 1 H), 7.28-7.37 (m, 5 H). MS (APCIþ) m/z = 191 (M þ Hþ).
1-tert-Butoxycarbonyl-7-phenyl-1,4-diazepan-5-one (24).
Diazepanone 23 (20 g, 88.5 mmol) and DIEA (38 mL, 220 mmol) were
dissolved in 300 mL of a mixture of dioxane/water (4:1). Di-tert-butyl
dicarbonate (22 g, 97 mmol) was added slowly to the solution, stirred for
48 h at room temperature, and concentrated under vacuum. The residue
was extracted with EtOAc and then washed with water and brine. The
organic layer was dried over MgSO4 and concentrated under vacuum.
The residue was purified by chromatography on silica gel (CH2Cl2/
MeOH 95:5) to give a white powder (2.45 g, 95% yield). Rf = 0.65. Mp:
4-Benzoyl-1-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-5-phenyl-
1,4-diazepane Hydrochloride (28). 85% yield. Rf = 0.70 (CH2Cl2/
MeOH 9:1). Mp: 185-186 °C. IR (neat): ν 2231 (CN), 1652 (CO). 1H
NMR (D2O): δ 1.78-2.15 (m, 2 H,), 2.78-2.89 (m, 1 H), 3.07-3.16
(m, 1 H), 3.36-3.75 (m, 7 H), 3.95 (s, 2 H), 5.25 (s, 2 H), 7.01-7.75
(m, 16 H). MS (APCIþ) m/z = 476 (M þ Hþ). Anal. (C30H29N5O
3
2HCl 1.5H2O) C, H, N, Cl.
3
4-(2,4-Dichlorobenzoyl)-1-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]-
methyl}-5-phenyl-1,4-diazepane Hydrochloride (29). 82% yield. Rf =
0.70 (CH2Cl2/MeOH 9:1). Mp: 188-189 °C. IR (neat): ν 2229 (CN),
1186
dx.doi.org/10.1021/jm101067y |J. Med. Chem. 2011, 54, 1178–1190

Journal of Medicinal Chemistry
ARTICLE
1649 (CO). 1H NMR (DMSO-d6): δ 1.88-2.21 (m, 2 H), 2.82-2.95
(m, 1 H), 3.08-3.15 (m, 1 H), 3.33-3.81 (m, 5 H), 4.01 (s, 2 H), 5.18
(s, 2 H), 7.01-7.72 (m, 13 H), 8.81 (s, 1 H), 12.11 (bs, 1 H). MS
(APCIþ) m/z = 510 (M þ Hþ), Anal. (C30H27Cl2N5O 2HCl 1H2O)
2.82-2.95 (m, 1 H), 3.13-3.19 (m, 1 H), 3.38-3.73 (m, 5 H),
7.09-7.38 (m, 5 H).
1-(1-Adamantanoyl)-4-tert-butoxycarbonyl-5-phenyl-1,4-diazep-
ane (39). 88% yield. Rf = 0,85 (CH2Cl2/MeOH 9:1). IR (neat): ν 1690
3
3
1
C, H, N, Cl.
(CO), 1649 (CO). H NMR (DMSO-d6): δ 1.55-1.72 (m, 15 H),
4-(2-Biphenoyl)-1-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-
5-phenyl-1,4-diazepane Hydrochloride (30). 78% yield. Rf = 0.78
(CH2Cl2/MeOH 9:1). Mp: >250 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.81-1.87 (m, 1 H), 2.07-2.16 (m, 1 H),
2.84-3.73 (m, 7 H), 3.97 (s, 2 H), 5.44 (s, 2 H), 6.92-6.99 (m, 1 H),
7.06-7.70 (m, 19 H). MS (APCIþ) m/z = 552 (M þ Hþ) . Anal.
(C36H33N5O 2HCl 1.5H2O) C, H, N, Cl.
1.75-1.95 (m, 9 H), 1.98-2.05 (m, 1 H), 2.03-2.12 (m, 1 H),
2.81-2.93 (m, 1 H), 3.10-3.17 (m, 1 H), 3.36-3.75 (m, 5 H),
7.11-7.45 (m, 5 H).
General Method for the Preparation of 5-Phenyl-1,4-
diazepanes (Compounds 40-45). Corresponding Boc deriva-
tives (34-39) were used for the synthesis of target compounds 40-45.
The procedure was the same as for the synthesis of compound 2.
1-Benzoyl-5-phenyl-1,4-diazepane Hydrochloride (40). 95% yield.
Rf = 0.15 (CH2Cl2/MeOH 9:1). Mp: 175 °C. IR (neat): ν 1653 (CdO
amide). 1H NMR (DMSO-d6): δ 1.88-1.98 (m, 1 H), 2.09-2.21 (m, 1
H), 2.76-2.97 (m, 1 H), 3.13-3.19 (m, 1 H), 3.36-3.75 (m, 5 H),
7.11-7.55 (m, 10 H), 8.53 (bs, 2 H).
3
3
1-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}-4-(1-naphthoyl)-
5-phenyl-1,4-diazepane Hydrochloride (31). 75% yield. Rf = 0.75
(CH2Cl2/MeOH 9:1). Mp: 205 °C. IR (neat): ν 2227 (CN), 1649
(CO). 1H NMR (DMSO-d6): δ 1.85-1.89 (m, 1 H), 2.03-2.12 (m, 1
H), 2.87-3.13-3.21 (m, 2 H), 3.36-3.75 (m, 7 H), 5.41 (s, 2 H) ;
6.93-6.96 (m, 1 H), 7.15-8.15 (m, 16 H), 8.84 (s, 1 H), 12.01 (bs, 1
H). MS (APCIþ) m/z = 526 (M þ Hþ). Anal. (C34H31N5O 2HCl
1-(2,4-Dichlorobenzoyl)-5-phenyl-1,4-diazepane Hydrochloride
(41). 95% yield. Rf = 0.20 (CH2Cl2/MeOH 9:1). Mp: 205 °C. IR
3
3
1
(neat): ν 1649 (CO). H NMR (DMSO-d6): δ 1.7-1.94 (m, 1 H),
1.5H2O) C, H, N, Cl.
2.01-2.10 (m, 1 H), 2.80-2.91 (m, 1 H), 3.11-3.15 (m, 1 H),
3.38-3.77 (m, 5 H), 7.11-7.55 (m, 8 H), 8,48 (bs, 2 H).
1-(2-Biphenoyl)-5-phenyl-1,4-diazepane Hydrochloride (42). 95%
yield. Rf = 0.20 (CH2Cl2/MeOH 9:1). Mp: 188 °C. IR (neat): ν 1649
(CO). 1H NMR (DMSO-d6): δ 1.79-1.86 (m, 1 H), 2.05-2.16 (m, 1
H), 2.86-2.97 (m, 1 H), 3.14-3.21 (m, 1 H), 3.33-3.71 (m, 5 H),
7.11-7.65 (m, 14 H), 8,61 (bs, 2 H).
1-(1-Naphthoyl)-5-phenyl-1,4-diazepane Hydrochloride (43). 95%
yield. Rf = 0.20 (CH2Cl2/MeOH 9:1). Mp: 188 °C. IR (neat): ν 1649
(CO). 1H NMR (DMSO-d6): δ 1.87-1.91 (m, 1 H), 2.05-2.14 (m, 1
H), 2.84-2.96 (m, 1 H), 3.16-3.22 (m, 1 H), 3.41-3.81 (m, 5 H),
7.17-7.62 (m, 11 H), 8.83 (s, 1 H).
1-{[1-(4-Cyanobenzyl)-1H-imidazol-5-yl]methyl}-4-cyclohexanoyl-
5-phenyl-1,4-diazepane Hydrochloride (32). 68% yield. Rf = 0.62
(CH2Cl2/MeOH 9: 1). Mp: 125 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.15-1.40 (m, 6 H), 1.55-1.80 (m, 5 H),
1.85-1.89 (m, 1 H), 2,03-2,12 (m, 1 H), 2.82-2.95 (m, 1 H),
3.13-3.19 (m, 1 H), 3.36-3.75 (m, 7 H), 5.45 (s, 2 H), 6.93-6.96
(m, 1 H), 7.17-7.63 (m, 10 H). MS (APCIþ) m/z = 482 (M þ Hþ).
Anal. (C30H35N5O 2HCl 1,5H2O) C, H, N, Cl.
3
3
4-(1-Adamantanoyl)-1-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]me-
thyl}-5-phenyl-1,4-diazepane Hydrochloride (33). 67% yield. Rf =
0.64 (CH2Cl2/MeOH 9:1). Mp: >250 °C. IR (neat): ν 2229 (CN),
1
1649 (CO). H NMR (D2O): δ 1.68 (s, 6 H), 1.80-2.00 (m, 9 H),
1-Cyclohexanoyl-5-phenyl-1,4-diazepane Hydrochloride (44). 95%
yield. Rf = 0.10 (CH2Cl2/MeOH 9:1). Mp: 195 °C. IR (neat): ν 1649
(CO). 1H NMR (DMSO-d6): δ 1.15-1.40 (m, 6 H), 1.55-1.80 (m, 5 H),
1.85-1.89 (m, 1 H), 2.03-2.12 (m, 1 H), 2.82-2.95 (m, 1 H), 3.13-3.19
(m, 1 H), 3.36-3.75 (m, 5 H), 7.11-7.45 (m, 5 H), 8,32 (bs, 2H).
1-(1-Adamantanoyl)-5-phenyl-1,4-diazepane Hydrochloride (45). 95%
yield. Rf= 0.10 (CH2Cl2/MeOH 9:1). Mp: 188 °C. IR (neat): ν 1649
1.85-1.89 (m, 1 H), 2.03-2.12 (m, 1 H), 2.82-2.95 (m, 1 H), 3.13-
3.19 (m, 1 H), 3.36-3.75 (m, 7 H), 5.45 (s, 2 H), 6.93-6.96 (m, 1 H),
7.21-7.65 (m, 10 H). MS (APCIþ) m/z = 534 (M þ Hþ). Anal.
(C34H39N5O 2HCl 1.5H2O) C, H, N, Cl.
General Procedure for the Preparation of 4-tert-Butoxy-
carbonyl-5-phenyl-1,4-diazepanes (Compounds 34-39).
Amine 25 was used for the acylation step. The procedure was adapted
from the synthesis of compounds 28-33.
3
3
1
(CO). H NMR (DMSO-d6): δ 1.68 (s, 6 H), 1.82-2.05 (m, 9 H),
1.98-2.05 (m, 1 H), 2.08-2.17 (m, 1 H), 2.91-3.03 (m, 1 H),
3.17-3.29 (m, 1 H), 3.14-3.66 (m, 5 H), 7.17-7.49 (m, 5 H), 8,36
(bs, 2 H).
1-Benzoyl-4-tert-butoxycarbonyl-5-phenyl-1,4-diazepane (34). 77%
yield. Rf = 0.87 (CH2Cl2/MeOH 9:1). IR (neat): ν 1682 (CO), 1649
1
(CO). H NMR (DMSO-d6): δ 1.33 (s, 9 H), 1.55-1.61 (m, 1 H),
General Procedure for the Preparation of 4-{[1-(4-Cy-
anobenzyl)-1H-imidazol-5-yl]methyl}-5-phenyl-1,4-diaze-
panes (Compounds 46-51). Corresponding diazepanes 40-45
were used for the preparation of target compounds (46-51). The
procedure was adapted from the methodology used for the synthesis of
compound 1. The residues were purified by chromatography on silica gel
(CH2Cl2/MeOH 95:5).
2.13-2.21 (m, 1 H), 2.71-3.77 (m, 6 H), 3.82-3.99 (m, 1 H),
7.11-7.55 (m, 8 H), 7.72-7.91 (m, 2 H).
1-(2,4-Dichlorobenzoyl)-4-tert-butoxycarbonyl-5-phenyl-1,4-dia-
zepane (35). 95% yield. Rf = 0.95 (CH2Cl2/MeOH 9:1). IR (neat): ν
1691 (CO), 1649 (CO). 1H NMR (DMSO-d6): δ 1.38 (s, 9 H),
1.85-1.89 (m, 1 H), 2.03-2.12 (m, 1 H), 2.82-2,95 (m, 1 H),
3.13-3.19 (m, 1 H), 3.36-3.75 (m, 5 H), 7.11-7.55 (m, 8 H).
1-(2-Biphenoyl)-4-tert-butoxycarbonyl-5-phenyl-1,4-diazepane
(36). 95% yield. Rf = 0.95 (CH2Cl2/MeOH 9:1). IR (neat): ν 1680
(CO), 1649 (CO). 1H NMR (DMSO-d6): δ 1.50 (s, 9 H), 1.85-1.89
(m, 1 H), 2.03-2.12 (m, 1 H), 2.82-2.95 (m, 1 H), 3.13-3.19 (m,
1 H), 3.34-3.72 (m, 5 H), 7.11-7.80 (m, 14 H).
1-(1-Naphthoyl)-4-tert-butoxycarbonyl-5-phenyl-1,4-diazepane
(37). 95% yield. Rf = 0.95 (CH2Cl2/MeOH 9:1). IR (neat): ν 1685
(CO), 1649 (CO). 1H NMR (DMSO-d6): δ 1.50 (s, 9 H,), 1.85-1.89
(m, 1 H), 1.98-2.02 (m, 1 H), 2.84-2.97 (m, 1 H), 3.15-3.20 (m,
1 H), 3.36-3.75 (m, 5 H), 7.11-7.65 (m, 12 H).
1-Benzoyl-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-5-phe-
nyl-1,4-diazepane Hydrochloride (46). 55% yield. Rf = 0.69 (CH2Cl2/
1
MeOH 9:1). Mp: 185 °C. IR (neat): ν 2229 (CN), 1649 (CO). H
NMR (D2O): δ 1.45-1.57 (m, 1 H), 1.73-1.85 (m, 1 H2), 2.55-2.68
(m, 1 H), 3.01-3.12 (m, 1 H), 3.22-3.61 (m, 7 H), 5.11 (s, 2 H),
6.93-6.96 (m, 1 H), 7.13-7.62 (m, 15 H). MS (APCIþ) m/z = 476
(M þ Hþ). Anal. (C30H29N5O 2HCl 1.5H2O) C, H, N, Cl.
3
3
1-(2.4-Dichlorobenzoyl)-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]-
methyl}-5-phenyl-1,4-diazepane Hydrochloride (47). 95% yield. Rf =
0.70 (CH2Cl2/MeOH 9:1). Mp: 188 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.91-1.99 (m, 1 H), 2.01-2.09 (m, 1 H),
2.76-2.91 (m, 1 H), 3.13-3.19 (m, 1 H), 3.36-3.75 (m, 7 H), 5.30 (s, 2
H), 7.05-7.63 (m, 14 H). MS (APCIþ) m/z = 510 (M þ Hþ). Anal.
1-Cyclohexanoyl-4-tert-butoxycarbonyl-5-phenyl-1,4-diazepane
(38). 70% yield. Rf = 0.85 (CH2Cl2/MeOH 9:1). IR (neat): ν 1686
(CO), 1649 (CO). 1H NMR (DMSO-d6): δ 1.10-1.38 (m, 6 H), 1.39
(s, 9 H),1.45-1.78 (m, 5 H), 1.81-1.92 (m, 1 H), 2.08-2.21 (m, 1 H),
(C30H27N5OCl2 2HCl 1H2O), C, H, N, Cl.
3
3
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dx.doi.org/10.1021/jm101067y |J. Med. Chem. 2011, 54, 1178–1190

Journal of Medicinal Chemistry
ARTICLE
1-(2-Biphenoyl)-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-
5-phenyl-1,4-diazepane Hydrochloride (48). 62% yield. Rf = 0.70
(CH2Cl2/MeOH 9:1). Mp: >250 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.70-2.03 (m, 2 H), 2.55-3.55 (m, 5 H),
4.17-4.26 (m, 1 H), 4.48 (s, 2 H), 5.45 (s, 2 H), 7.05-7.70 (m, 19 H).
MS (APCIþ) m/z = 552 (M þ Hþ). Anal. (C36H35N5O 2HCl
Invitrogen Corp.), 2 mM glutamine, and 5 units/mL of streptomycin
and 5 units/mL of penicillin. All cells were cultured at 37 °C in a
humidified incubator with 5% CO2.
MTT Cytotoxicity Assay. Cytotoxicity of selected drugs was
tested using the MTT (thiazolyl blue tetrazolium bromide) assay based
on the cellular uptake of MTT (Sigma Aldrich) and its subsequent
reduction in the mitochondria of living cells to dark blue MTT formazan
crystals (Mosmann, 1983). Cells were seeded on 96-well plates
((1.6-1.8) ꢀ 104 cells/well) in the corresponding medium supplemen-
ted with 10% FBS. Seventy-two hours later, cells were deprived of FBS.
The next day, they were treated with various concentrations of each
tested compound. Following the 72 h exposure to the cytotoxic drugs,
cell survival was determined by incubation with MTT (4 mg/mL) for 4 h
(37 °C, 5% CO2). The cells were then lyzed in SDS/HCl and plates left
in the incubator to dissolve the purple formazan crystals. The color
intensity reflecting cell viability was read with a spectrophotometer Elx
800 (Biotek Instruments Inc.) at 570 nm.
3
3
1.5H2O), C, H, N, Cl
1-(1-Naphthoyl)-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-
5-phenyl-1,4-diazepane Hydrochloride (49). 51% yield, Rf = 0.81
(CH2Cl2/MeOH 9:1). Mp: 208 °C. IR (neat): ν 2227 (CN), 1654
(CO). 1H NMR (DMSO-d6): δ 1.78-1.86 (m, 1 H), 1.91-2.06 (m, 1
H), 2.75-2.82 (m, 1 H), 3.01-3.11 (m, 1 H), 3.36-3.75 (m, 5 H), 4.12
(s, 2 H), 5.05 (s, 2 H), 6.93-6.96 (m, 1 H), 7.05-8.17 (m, 17 H), 8.84
(s, 1 H), 10.32 (bs, 2 H). MS (APCIþ) m/z = 526 (M þ Hþ). Anal.
(C34H31N5O 2HCl 1.5H2O), C, H, N, Cl.
3
3
1-Cyclohexanoyl-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-
5-phenyl-1,4-diazepane Hydrochloride (50). 82% yield. Rf = 0.70
(CH2Cl2/MeOH 9:1). Mp: 125 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.17-1.41 (m, 6 H), 1.63-1.96 (m, 6 H),
2.02-2.10 (m, 1 H), 3.12-3.75 (m, 7 H), 4.17 (s, 2 H), 5.15 (s, 2 H),
6.95-6.99 (m, 1 H), 7.18-7.65 (m, 10 H). MS (APCIþ) m/z = 482
(M þ Hþ). Anal. (C30H35N5O 2HCl 1.5H2O) C, H, N, Cl.
siRNA transfection. siRNA transfection was realized with CENP-E
siRNA 10616 (Ambion) and transfection reagent DharmaFECT2
(Dharmacon RNA Technologies) according to the manufacturer’s
instructions. Cells were incubated at 37 °C for 48 h.
Immunofluorescence. Cells were plated on poly-D-lysine coated
coverslips (BD-Biocoat) and incubated overnight at 37 °C and 5% CO2.
After synchronization, cells were treated with FTIs for 24 h. The cells
were fixed in 4% paraformaldehyde and then permeabilized with 0.5%
Triton X-100/PBS. After blocking for 45 min in 5% BSA/PBS, cells were
washed with PBS and incubated with anti-R-tubulin antibody conju-
gated with Alexa 488 (dilution 1/100) and with anti-CENP-E antibody
(dilution 1/100) followed by addition of a secondary antibody con-
jugated with Alexa 568 (dilution 1/400). The samples were then washed
and mounted in Mowiol. Cells were visualized using a fluorescence
microscope (Nikon, Eclipse 80i).
3
3
1-(1-Adamantanoyl)-4-{[1-(4-cyanobenzyl)-1H-imidazol-5-yl]methyl}-
5-phenyl-1,4-diazepane Hydrochloride (51). 57% yield. Rf = 0.59
(CH2Cl2/MeOH 9:1). Mp: >250 °C. IR (neat): ν 2229 (CN), 1649
(CO). 1H NMR (D2O): δ 1.59 (s, 6 H), 1.78-2.08 (m, 10 H),
3.36-3.75 (m, 7 H), 4.10-4.18 (m, 1 H), 4.28 (s, 2 H), 5.30 (s, 2
H), 6.93-6.96 (m 1 H), 7.21-7.45 (m, 10 H). MS (APCIþ) m/z = 534
(M þ Hþ). Anal. (C34H39N5O 2HCl 1.5H2O) C, H, N, Cl.
3
3
Molecular Modeling. Molecular modeling studies were per-
formed using SYBYL39 software, version 6.92, running on a Silicon
Graphics workstation. Three-dimensional models of selected com-
pounds were built from a standard fragments library, and their geometry
was subsequently optimized using the Tripos force field including the
electrostatic term calculated from Gasteiger and H€uckel atomic charges.
Powell’s method, available in the Maximin2 procedure, was used for
energy minimization until the gradient value was smaller than 0.001
’ ASSOCIATED CONTENT
S
Supporting Information. Results from elemental analysis.
b
This material is available free of charge via the Internet at http://
pubs.acs.org.
kcal/(mol Å). The structure of the human FTase was obtained from its
3
complexed X-ray crystal structure of the RCSB Protein Data Bank
(1LD7) with FPP and the inhibitor molecule described by Bell.40
Flexible docking of FTis into the enzyme’s active site was performed
using GOLD software.39 The distance observed in the crystal structure
between the distal nitrogen imidazole and the zinc cation was applied as
a constraint. For each compound, the most stable docking model was
selected according to the best conformation predicted by the GoldScore
and X-Score scoring functions.41 The complexes were energy-minimized
using the Powell method available in the Maximin2 procedure with the
Tripos force field and a dielectric constant of 4.0 until the gradient value
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ33-3-2096-4374. Fax: þ33-3-2096-4906. E-mail:
rꢀegis.millet@univ-lille2.fr.
’ ACKNOWLEDGMENT
We gratefully acknowledge the financial support of the “ARC”
(to J.-P.H). We thank Dr. L. Heliot for his technical assistance
concerning the immunocytochemistry studies (IRI, Lille 1 Uni-
versity) and M. Howsam and J. Hargreaves for checking the
manuscript.
reached 0.01 kcal/(mol Å).
3
FTase Fluorescent Assay Procedure. Assays were realized on
96-well plates, prepared with Biomek NKMC and Biomek 3000 from
Beckman Coulter and read on a Wallac Victor fluorimeter from Perkin-
Elmer. To each well, 20 μL of farnesyl pyrophosphate (10 μM) was
added to 180 μL of a solution containing 2 μL of varied concentrations
of potent inhibitor (dissolved in DMSO) and 178 μL of a solution
composed of 100 μL of partially purified recombinant yeast FTase (2.2
mg/mL) and 7.0 mL of dansyl-GCVLS peptide (2 μM) in the following
buffer: 5.5 mM DTT, 10 mM MgCl2, 10 μM ZnCl2 and 0.08% (w/v)
CHAPS, and 50 mM Tris/HCl, pH 7.5. Then the fluorescence devel-
opment was recorded for 15 min (0.7 s per well, 20 repeats) at 30 °C
with an excitation filter at 340 nm and an emission filter at 486 nm.
Cell Culture. The human prostate cancer lines PC3 and DU145
were obtained from ATCC. Cells were grown in RPMI 1640 (Gibco,
Invitrogen Corp.) supplemented with 10% fetal bovine serum (Gibco,
’ ABBREVIATIONS USED
ACN, acetonitrile; CA-4, combretastatin A-4; CENP-E, centro-
mere protein E; CENP-F, centromere protein F; CLogP, calcu-
lated log P; DCM, dichloromethane; DIEA, N,N-diisopropyl-
ethylamine; DMAP, 4-(N,N-dimethylamino)pyridine; DMF,
N,N-dimethylformamide; DMSO, dimethylsulfoxide; EDCI, 1-eth-
yl-3-(3-dimethylaminopropyl)carbodiimide; EtOAc, ethyl acet-
ate; EtOH, ethanol; FPP, farnesylpyrophosphate; FTase, protein
farnesyltransferase; FTis, farnesyltransferase inhibitors; GADPH, gly-
ceraldehyde 3-phosphate dehydrogenase; GGPP, geranylgeranyl
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pyrophosphate; GGTase-I, protein geranylgeranyltransferase I;
HOBt, 1-hydroxybenzotriazole; HPLC, high performance
liquid chromatography; IC50, half-maximal inhibitory concentra-
tion; iPrOH, isopropanol; PBS, phosphate buffered saline;
PyBroP, bromotrispyrrolidinophosphonium hexafluorophosphate;
rt, room temperature; SAR, structure-activity relationship; TLC,
thin layer chromatography
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