Parallel liquid synthesis of N,N′-disubstituted 3-amino azepin-2-ones as potent and specific farnesyl transferase inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00218-9

A rapid structure-activity study was performed by parallel liquid synthesis on N,N′-disubstitution of 3-amino azepin-2-one to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities. The activities of the selected compounds were validated in vivo, and compounds 41a and 44a presented significant antitumour activity.

Full text of DOI:10.1016/S0968-0896(03)00218-9

Bioorganic & Medicinal Chemistry 11 (2003) 3193–3204
Parallel Liquid Synthesis of N,N0-Disubstituted
3-Amino Azepin-2-ones as Potent and Specific Farnesyl
Transferase Inhibitors§
Thierry Le Diguarher,a Jean-Claude Ortuno,a Gilbert Dorey,a David Shanks,a
Nicolas Guilbaud,b,y Alain Pierre,b Jean-Luc Fauchere,a John A. Hickman,b
Gordon C. Tuckerb and Patrick J. Casaraa,*
aDepartment of Medicinal Chemistry, Institut de Recherches Servier,
125 chemin de Ronde, 78290Croissy sur Seine, France
bDepartment of Experimental Oncology, Institut de Recherches Servier,
125 chemin de Ronde, 78290Croissy sur Seine, France
Received 31 January 2003; accepted 25 March 2003
Abstract—A rapid structure–activity study was performed by parallel liquid synthesis on N,N0-disubstitution of 3-amino azepin-2-one
to afford potent and specific farnesyl transferase inhibitors with low nM enzymatic and cellular activities. The activities of the
selected compounds were validated in vivo, and compounds 41a and 44a presented significant antitumour activity.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
debatable. However, access to specific inhibitors would
help to understand the relationship between the various
enzymes and different physiological pathologies. The
Protein farnesyl transferase (FTase) is a zinc-dependent
enzyme that catalyses the attachment of a farnesyl lipid
group to the sulfur atom of a cysteine residue of
numerous proteins involved in cell signalling, including
the oncogenic H-Ras protein.1 This lipid side chain is
critical for the cell membrane anchoring of these pro-
teins,2 and therefore FTase inhibition has been recog-
nized as a valuable antitumour therapeutic approach,3
although the target proteins whose farnesylation is
inhibited by FTase inhibitors are not all identified.
Another more ubiquitous prenylation of proteins can
occur via geranylgeranyl transferase (GGTase-1) pro-
cessing.1 Indeed the RhoB protein, another putative
FTase target, may be geranylgeranylated to an inactive
form after FTase inhibition.4 The issue of selectivity of
the inhibition of FTase versus GGTase has been
addressed and the optimal degree of selectivity is still
substrates of these enzymes present
a consensus
sequence of four amino acid residues (CAAX) at the
carboxylic terminal of the protein, with the cysteine in
fourth, two aliphatic residues and the terminal residue
X, being preferably methionine for FTase and leucine
for GGTase.5 In the design of FTase inhibitors, histi-
dine was found to be a good bioisosteric replacement of
the cysteine residue, and numerous non-thiol CVIM (1)
peptidomimetics were developed around an imidazol
function.6 Therefore, among the compounds in clinical
development we can distinguish those containing an imi-
dazole associated to a benzodiazepine (BMS-214662);7 a
piperazinone (L-778,123),8 or a quinolinone (R115777)9
scaffold and the tricyclic SCH66336,10 containing a
piperidine obtained from HTS. Crystal structures of the
complex between FTase and 1 are in favour of an
extended conformation of 1 in the active site cavity.11
The caprolactam derivatives 2 are proposed as the core
of constrained CVIM bioisosteres in an elongated con-
formation including an imidazole zinc-complexing
function. This scaffold can be substituted by R3 (and/or
eventually R2) on the amine function N-1 (and/or even-
tually N-4) to mimic part of the Ras protein R (and/or
§Supplementary data associated with this article can be found at
doi:10.1016/S0968-0896(03)00218-9 [Tables 7–9].
*Corresponding author. Tel.: +33-1-5572-2361; fax: +33-1-5572-
2470; e-mail: patrick.casara@fr.netgrs.com
yPresent address: Oncodesign, Parc Technologique Toison d’Or, 28 rue
Louis de Broglie, 21000 Dijon, France.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00218-9

3194
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
Figure 1. Interactions of compound 1 with FTase and structures of compounds 1 and 2.
Scheme 1. Synthesis of compounds 28–63. (i): (Boc)2O, NaOH, H2O/tBuOH, rt, 12 h, (4, 88%); (ii1) (a) BiPh3, pyridine, Cu(OAc)2, CH2Cl2, rt, 24 h
(5, 77%); (ii2) (a) NaH THF/DMF, 0 ꢀC, 30 min; (b) PhCH2Br, N(Bu)4, I, rt, 24 h (6, 97%). (ii3) (a) NaH, DMF, 0 ꢀC, 30 min; (b) Ph(CH2)2OTs,
0 ꢀC, 12 h (7, 75%); (ii4) (a) NaH DMF, 0 ꢀC, 30 min, rt, 2 h; (b) Ph(CH2)3OTs, rt, 12 h (8, 86%); (ii5) (a) LiHMDS, 0 ꢀC, 15 min; (b) PhSO2Cl, 0 ꢀC,
rt, 2 h (9, 77%); (ii6) (a) PhSCH2Cl, KOH, N(Bu)4I, THF, rt, 12 h; (b) dimethyldioxirane, acetone, 0 ꢀC, rt, 2 h, (10, 40%); (iii) TFA, CH2Cl2, 0 ꢀC,
rt, 2 h or 4 N HCl/dioxane, rt (11–16); (iv) (a) dihydroxyacetone dimer, KSCN, MeCN/EtCO2H, 60 ꢀC, 2 h; (b) H2O2, H2O, 40 ꢀC, 2 h; (c) DMSO,
SO3/pyridine, NEt3, rt, 2 h (17–27: Tables 4 and 6); (v) (a) 17–27, NaBH(OAc)3, CH2Cl2, 0 ꢀC, rt, 12 h; (b) HCl, ether, or fumaric acid, EtOH (28–33:
Table 1; 34–53: Tables 2 and 7; 54 to 63: Tables 4 and 8).
the farnesyl co-substrate) and R1 separated by a spacer
Y on the lactam nitrogen atom N-7 to replace the lipo-
philic non polar side chains of the isoleucine/methionine
residue. Moreover, a synthesis of 2 by a methodology
applicable to liquid parallel synthesis was performed
to allow an extensive and rapid investigation of the
Y/R1/R2/ R3 parts of the molecule (Fig. 1).
the corresponding sulfonyl chloride to give 9, or by
sequential alkylation of the chloromethyl sulfide and
oxidation to give 10. Then, they were deprotected
quantitatively under acidic conditions to give the corre-
sponding primary amines 11–16, and the (N-1-PCB)
imidazol-5-yl-methyl substituent was introduced by
reductive amination with the corresponding aldehyde 17
(R3=PCB), to afford the compounds 28 to 33 (Scheme
1, Table 1).
Results and Discussion
The inhibitory activities of these compounds were
examined against FTase and against GGTase-1 and the
results were compared to the various clinical references
(Table 1). Their cellular activities for FTase inhibition
were determined on an H-Ras based assay.
Investigation on the N-lactam substitution R1
A straightforward retro-synthetic analysis links 2 to
2-aminocaprolactam (3) by two selective N-alkylations.
The primary amino function of 3 was protected as a Boc
derivative to give the intermediate 5 ready for N-lactam
substitution and further substitution after deprotection.
In order to validate the caprolactam core hypothesis
and investigate the N-lactam substitution, the prox-
imal N-p-cyanobenzyl function (PCB) was used as the
imidazole substituent R3, as a well described cysteine
bioisostere.12 Then the replacement of the isoleucine/
methionine by a lipophilic side chain was first con-
sidered by using various spacer Y and a phenyl group as
R1. When Y was a bond, the N-arylation was achieved
by treatment of 4 with triphenylbismuth to give 5.13 The
N-alkylation was obtained by lactam deprotonation and
addition of the benzyl chloride to give 6. Based on the
same strategy, various alkyl spacers were also con-
sidered to complete the study to give the corresponding
compounds 7 and 8. A sulfonyl spacer was also intro-
duced either by lactam deprotonation and addition of
In the series of compounds 28–33, the introduction on
the caprolactam moiety of an aryl substituent to replace
the two terminal amino acids of CVIM afforded very
potent inhibitors of FTase when the junction was made
either by a bond or a methylene group in compounds 28
and 29, respectively. Further chain elongation decreased
the potency in the case of an ethyl group (30) and even
abolished the activity in the case of the propyl spacer
(31). The sulfone spacer, previously used in the case of
diaryl as VIM surrogate,14 was detrimental to inhibition
in our case, either alone (32) or with a methyl group
(33). These results suggest a different binding mode for
the caprolactam scaffold, possibly in a conformation
closer to that of the peptide ligand. The stereospecificity
of the enzyme inhibition was validated by using the
enantiomers of 29, respectively 29a and 29b, separated by
chiral HPLC, 29a being significantly more active than

T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
Table 1. Structures and biological data of compounds 28–33
3195
Compd
(conf.)
Y
FTase
IC50 nM
GGTase
IC50 nM
Ras Cell
IC50 nM
Compd
(conf.)
Y
FTase
IC50 nM
GGTase
IC50 nM
Ras Cell
IC50 nM
BMS-2146627
found
1.3
16
2300
7600
25
29
29a (S)
29a (S)a
CH2
CH2
9
5
16,200
12,000
10
L-778,1238
found
1.6
8
70
31
29b (R)a
2800
ND
13,200
>50,000
>50,000
SCH6633610
1.9
10
30 (RS)
30a (S)
(CH2)2
(CH2)2
69
39
5200
5900
288
160
R1157779
found
Ki 0.5
4
1.7
2
28 (RS)
28a (S)
29 (RS)
Bond
Bond
CH2
28
17
8
11,200
3300
32
17
31 (RS)
32 (RS)
33 (RS)
(CH2)3
SO2
1000
293
6200
10,600
9700
4400
260
10,600
ND
CH2SO2
168
3800
aEnantiomer obtained from the racemate by separation on chiral HPLC.
29b. To assess the C-5 configuration, a stereospecific
synthesis of the S enantiomer for the most active com-
pounds was carried out from the S caprolactam 3a. The
retention of the configuration during the synthesis was
confirmed by the synthesis of the S enantiomer of 28, 29
and 30, respectively 28a, 29a and 30a, with an enantio-
meric excess up to 85%. The conservation of activity
was observed for each of these S enantiomers versus
their racemates, which corroborated the stereospecificity
of the enzymatic and the cellular inhibition, with activ-
ities already in the same range as those of the reference
compounds (Table 1). The active configuration S of
these inhibitors is also in agreement with the hypothesis
of a close mimicry of the peptide ligand.
or even improved the enzymatic activity with an IC50 in
the low nM range (Table 2). Some exceptions included
its replacement by a 4-pyridyle or substitution by a para
trifluoromethyl group (compounds 35a and 38a,
respectively) with a 10-fold decrease in potency and the
para substitution by an ester leading to an inactive
derivative (compound 37a). However, the cellular assay
was even more discriminating, with a 3- to 5-fold
decrease in potency between enzymatic and cellular
activities for a naphtyl, cyclohexyl and dimethyl sub-
stitution (compounds 34a, 36a, 40a, respectively) and
more than a 10-fold decrease in potency for para and
poly substitution by various halogen atoms (compounds
43a and 46a–53a). No simple explanation to this phe-
nomenon could be provided, but differences in cellular
potency versus the isolated enzyme may originate at the
level of membrane crossing, protein binding or chemical
stability. However, the ortho or meta mono and di-
substitution by a methyle or an halogen maintained the
cellular activity at the low nM range IC50 (compounds
39a, 41a, 42a, 44a, 45a and 51a).
Further investigation on the primary amine substitution R1
Since the best enzymatic and especially cellular activities
were observed for compounds 28a and 29a, they could
be considered for further lead optimization, with a
slight advantage for the latter. Moreover, the benzyl
derivative 28a was also the most attractive in terms of
molecular diversity for R1 optimization, with a large
commercial access to benzylic reagents compared to
triarylbismuth derivatives. Therefore, solution phase
parallel synthesis, was performed by sequential alkyl-
ation of the S enantiomer 4a with a series of benzyl
derivatives to give, after acidic deprotection, the amines.
These intermediates were condensed, without purifica-
tion, by reductive amination with 17, to afford com-
pounds 34a to 53a. Purification of these compounds by
simple automated SPE, led average yield, but complied
with the high criteria purity necessary for the accuracy
of the biological tests (Scheme 1, Table 2).
Investigation on the amine substitution R2xv
In order to investigate the importance of the N-1 sub-
stituent on the imidazole, the unsubstituted derivative
65a (R2=R3=H) was synthesized by reductive amina-
tion of the benzyl caprolactam 12a with the 1-trityl-4-
carboxaldehyde imidazole. Since this compound was
devoid of any enzyme inhibitory activity (Table 3), the
bis PCB (R2=R3=PCB) 66a, and the mono PCB deri-
vatives (R2=H, R3=PCB) 67a were synthesized by
mono and dialkylation of 64a. Surprisingly, these com-
pounds were equipotent, but with an IC50 one order of
magnitude higher that than of 29a. To assess the
potential of this substitution R2, a series of N-4 sub-
stituted derivatives of 65a was rapidly generated by the
In this second selection, the majority of the random
substitution of the aromatic moiety in 29a maintained

3196
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
Table 2. Structures and biological data of compounds 34a–53a
Compd
R1
FTase
IC50 (nM)
GGTase
IC50 (nM)
Ras cell
IC50 (nM)
Compd
R1
FTase
IC50 (nM)
GGTase
IC50 (nM)
Ras cell
IC50 (nM)
9
16,200
10
29a
34a
35a
43
87
2500
156
103
44a
45a
3
5
5600
2
4
20,500
19,800
36a
37a
13
18,400
3600
70
46a
47a
2
9
5100
ND
64
9000
>104
160
38a
39a
113
5
4200
5500
350
4
48a
49a
20
2
ND
ND
370
120
40a
41a
7
3
24,500
7400
46
7
50a
51a
2
1
9300
44
14
25,800
42a
43a
6
10,500
ND
2
52a
53a
5
ND
ND
210
465
19
184
41
parallel liquid phase procedure by either aminoreduc-
tion with various aldehydes to give compounds 67a to
82a, or by N-acylation with various acylchlorides to give
compounds 83a to 96a (Scheme 2, Table 3).
Table 3. Biological data of compounds 65a–96a
Compd
FTase
IC50 (nM)
Compd
FTase
IC50 (nM)
In this third selection, the random amino substitution of
65a was produced, but did not allow recovery of any
significant FTase inhibitory activity (compounds 68a–
96a, Table 3). These data also diverged from the results
4
>10
255
4
>10
65a
66a
67a
298
68a–96a

T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
3197
Scheme 2. Synthesis of compounds 65a–96a. (i) NaBH(OAc)3, CH2Cl2, 0 ꢀC to rt 12 h, (64a, 88%); (ii) (a) TFA, Et3SiH, CH2Cl2, rt, 12 h; (b)
fumaric acid, EtOH (65a); (iii) (a) pCNPhCH2Br, AcOEt, 60 ꢀC, 12 h; (b) MeOH, 60 ꢀC, 12 h; (c) fumaric acid, EtOH (66a); (iv1) (a) NaBH(OAc)3,
R2CHO, CH2Cl2, rt 12 h; (b) TFA, Et3SiH, CH2Cl2, rt, 12 h (67a–82a: Fig. 2, Table 9); (iv2) (a) R2COCl polyvinylpyridine, CH2Cl2, rt 12 h; (b)
TFA, Et3SiH, CH2Cl2, rt, 18 h; (c) HCl, ether, or fumaric acid, EtOH (83a–96a: Fig. 2, Table 9).
Figure 2. Substitutents of componud 67a–96a R3¼H R2¼
reported with the aryl scaffold where the N substitution
of the imidazol-5-yl-methylamine function either on the
imidazole ring or the amine function afforded equipo-
tent compounds.12 In this case, the authors proposed a
nearby second binding pocket at this cysteine region. A
plausible explanation, in absence of X-ray data, is a
different recognition of the two scaffolds, the capro-
lactam following the peptide side chain and the aryl
being perpendicular allowing the fit of substituents in
the various amino acid pocket sites of the enzyme.
55a, respectively) led to a 10-fold loss of enzymatic
inhibition and a 100-fold loss in cellular potency com-
pared to 39a. In addition, the spacer elongation by one
or two carbons, led to inactive compounds despite fur-
ther substitution (compounds 56a, 57a and 61a–63a).
The replacement of the cyano substituent by various
halogen (compounds 58a–60a) kept the enzymatic
activity almost at the same level as those of 39a, but
induced a one order of magnitude loss in cellular
potency in spite of an increase in their CLogP. There-
fore, no better substitution for the PCB group was
found so far.
Investigation on the imidazol ring substitution R3
In the fourth selection, the effect of the modification of
the N-1 substitution of the imidazole side chain on 39a
was investigated. For this purpose, a series of N-1 sub-
stituted imidazol-5-ylcarboxaldehydes 18 to 27 were
synthesized from various primary amines by a modifi-
cation of the method described by Aulaskari.15 They
were then condensed by reductive amination with the
amine intermediate precursor of 39a, to afford com-
pounds 54a to 63a (Scheme 1, Table 4).
Pharmacological Evaluation
As a preliminary evaluation of antitumour activity,
selected compounds, with low nM IC50 in the enzy-
matic and cellular assays, were tested in an in vivo
model. H-Ras transfected fibroblasts were grafted sc
into nude mice, and compounds were administered at
100 and 200 mg/kg po for 5 days, starting from day 6
after tumour implantation. Among these compounds,
41a, 42a and 44a showed significant activity with a
marked reduction of the tumour volume (39, 85 and
In this selection, the removal of the cyano substituent or
the reduction of the aromatic ring (compounds 54a and

3198
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
Table 4. Structures of compounds 17–27 and 54a–63a and biological data of compounds 54a–63a
Compd
Compd
R3
FTase
IC50 (nM)
Ras cell
IC50 (nM)
Compd
Cpmpd
R3
FTase
IC50 (nM)
Ras cell
IC50 (nM)
17
5
4
39a
18
19
20
21
22
54a
55a
56a
57a
58a
48
81
430
730
ND
ND
210
23
24
25
26
27
59a
60a
61a
62a
63a
16
27
180
190
ND
ND
ND
3000
1700
19
3800
5300
2700
40%, respectively, at 200 mg/kg). However, these
activities remained lower than those of the reference
compound BMS-214662 which inhibited tumour
growth by 50 and 80% at 100 and 200 mg/kg,
respectively, in this model. Moreover, since signs of
toxicity occurred with 42a at the higher dose, only
41a and 44a were selected for further pharmacological
evaluation (Table 5).
Conclusion
An efficient synthetic access was developed from N,N0-
disubstituted 2-amino caprolactam to generate a new
class of inhibitors of FTase selective versus GGTase-1.
A rapid lead optimization by liquid parallel synthesis
allowed independent investigation of the substituents
and afforded very potent compounds with low nM IC50
in the enzymatic and cellular assays. It is interesting to
note that a differently substituted caprolactam scaffold
was previously used to design FTase inhibitors as
reverse-turn mimics, but led to almost inactive compounds
(IC50 in the mM range).16 The activities of the selected
compounds were validated in vivo, and compounds 41a
and 44a presented significant antitumour activity.
Table 5. In vivo antitumour activity
No.
% Control
100 mg/kg po
% Control
200 mg/kg po
BMS-214662
48
83
66
51
99
103
126
22
72
61
29a
41a
42a
44a
45a
51a
15 (Tox.)
60
71
76
Table 6. Yields and analytical data of compounds 18–27
Compd
R3
18
19
20
21
22
23
24
25
26
27
Yield%
33
27
60
37
51
46
52
18
32
26
Analysis
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR
RMN/IR

T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
3199
Experimental
Chemistry general techniques
Preparation of (R,S) 1-benzyl-2-oxo-3-(tert-butoxycar-
bonylamino)-azepine (6). To a suspension of NaH (60%
in oil, 1.9 g, 47 mmol) in THF (20 mL) was added a
solution of compound 4 (9.8 g, 43 mmol) in THF
(90 mL) at room temperature. Then this mixture was
stirred for 1 h and a solution of benzyl bromide (7.6 mL,
64 mmol) in THF (20 mL) was added. After an addi-
tional stirring of 2 h the reaction mixture was cooled
down to 0 ꢀC, treated with a satured solution of NH4Cl
and extracted with AcOEt. The organic phase was dried
over Na2SO4, filtrated and concentrated in vacuo. The
residue was purified by flash chromatography (silica gel,
heptane/AcOEt; 70:30) to give compound 6 as a solid
All reactions were carried out under nitrogen or argon
atmosphere under anhydrous conditions unless other-
wise noted.
Parallel synthesis was performed on STEM Blocks,
Workup was performed on Zymate. Yields refer to
chromatographically and spectroscopically (1H NMR)
homogenous material unless otherwise stated. All
reagents were purchased at highest commercial quality
and used without further purification unless otherwise
stated. All reactions were monitored by thin-layer
chromatography carried out on 0.2 mm Merck silicagel
plates using UV light and ethanol phosphomolybdic
acid or p-anisaldehyde and heat as developing agent.
1
(12.7 g, 93%). H NMR (200 MHz, CDCl3): d (ppm)
7.25 (m, 5H, Ph), 4.85 (d, 1H, CHPh), 4.4 (m, 2H,
CHPh, CHNH), 3.6–3.15 (2dd, 2H, CH2N), 2.2–1.8 (m,
6H, (CH2)3) 1.5 (s, 9H, tBu). IR: Vmax 3413, 1711,
1648 cmÀ1. Anal. (C18H26 N2O3): C, H, N calcd: 67.90;
8.23; 8.80. Found: 67.82; 8.21; 8.81.
Preparative flash chromatography separations were
carried out on Kiesegel 60 (0.04–0.063 mm) Merck sili-
cagel. Normal-phase solid-phase extraction (SPE) were
carried out on cartridges loaded with 8 g of Kiesegel 60
(0.04–0.063Âmm) Merck silicagel. Reversed-phase
chromatography were carried out on columns loaded
with of Kiesegel C18 (LiChroprep RP-18; 25–40 mm)
Merck. Mobile phase H2O/CH3CN+0.5% TFA.
Reverse-phase HPLC analysis was performed on an
Agilent 1100 instrument using a Waters Xterra MS C18
column with detection at 210 nm using a H2O/
CH3CN+0.1% CH3SO3H gradient over 10 min. NMR
spectra were recorded on a Brucker PPX 200 instrument
as indicated, calibrated using TMS as an internal refer-
ence. The following abbreviations were used to explain
multiplicities: s, singlet; d, doublet; t, triplet; q, quad-
ruplet; m, multiplet; b, broad. IR spectra were recorded
on a Brucker Vector 22 spectrophometer.
Preparation of (S) 1-benzyl-2-oxo-3-(tert-butoxycarbon-
ylamino)-azepine (6a). Compound 6a (0.615 g, 94%)
was prepared by the same procedure used for 6 with
compound 4a (0.5 g, 2.2 mmol), NaH (60% in oil,
0.096 g, 2.4 mmol) and benzyl bromide (0.46 mL,
3.6 mmol) in THF (5 mL). RMN; IR similar to 6.
Preparation of (R,S) 1-(2-phenylethyl)-2-oxo-3-(tert-
butoxycarbonylamino)-azepine (7). Compound 7 (0.45 g,
41%) was prepared by the same procedure used for 6
with compound 4 (1 g, 4.3 mmol), NaH (60% in oil,
0.2 g, 4.8 mmol) and 2-phenylethyltosylate (1.3 g,
4.8 mmol) in DMF (5 mL). 1H NMR (200 MHz,
DMSO-d6): d (ppm) 7.3 (m, 5H, Ph), 4.25 (m, 1H,
CHNH), 3.5–3.25 (m, 4H, CH2NCH2), 2.7 (d, 2H,
CH2Ph), 2.1–1.7 (m, 6H, (CH2)3) 1.4 (s, 9H, tBu). IR:
Vmax 3422, 1703, 1640 cmÀ1
Electrospray mass spectra were recorded in a positive
mode on a Finnigan TSQ 7000 spectrophometer, by
infusion at 15 mL/min of a 0.1 mg/mL sample solution in
a mixture of CH3CN/H2O (75:25).
Preparation of (S) 1-(2-phenylethyl)-2-oxo-3-(tert-butoxy-
carbonylamino)-azepine (7a). Compound 7a (5.1 g,
47%) was prepared by the same procedure used for 6
with compound 4a (7.4 g, 32.3 mmol), NaH (60% in oil,
1.4 g, 35 mmol) and 2-phenylethyltosylate (10.7 g,
38.6 mmol) in DMF (130 mL). RMN; IR similar to 7.
Preparation of (R,S) 1-phenyl-2-oxo-3-(tert-butoxycar-
bonylamino)-azepine (5). To a solution of 4 (1.6 g,
7 mmol), Cu(OAc)2 (1.36 g, 7 mmol) and pyridine
(0.6 mL, 7 mmol) in CH2Cl2 (20 mL) was added drop-
wise triphenylbismuth (3.4 g, 7.6 mmol), at room tem-
perature. The reaction mixture was stirred 24 h and then
filtrated over Celite and washed several times with
AcOEt. The filtrate was concentrated in vacuo, and the
residue was purified by flash chromatography (silica gel,
petroleum ether/AcOEt; 60:40) to give 5 (0.84 g, 38%)
as an oil. 1H NMR (200 MHz, DMSO-d6): d (ppm) 7.73
(m, 5H, Ph), 3.85–3.65 (m, 3H, CH2N, NCHCO), 2.2–
1.7 (m, 6H, (CH2)3), 1.5 (s, 9H, tBu). IR: Vmax 3375–
3317, 1647 cmÀ1
Preparation of (R,S) 1-(3-phenylpropyl)-2-oxo-3-(tert-
butoxycarbonylamino)-azepine (8). Compound 8 (2.6 g,
86%) was prepared by the same procedure used for 6
with compound 4 (2 g, 8.7 mmol), NaH (60% in oil,
0.384 g, 9.5 mmol) and 3-phenylpropyltosylate (3 g,
10.3 mmol) in DMF (43 mL). 1H NMR (200 MHz,
CDCl3): d (ppm) 7.4 (m, 5H, Ph), 4.4 (dd, 1H, CHNH),
3.8–3.3 (m, 4H, CH2NCH2), 2.7 (d, 2H, CH2Ph), 2.1–
1.7 (m, 6H, 2(CH2)2) 155 (s, 9H, tBu). IR: Vmax 3411,
1711, 1642 cmÀ1
.
Preparation of (R,S) 1-(benzenesulfonyl)-2-oxo-3-(tert-
butoxycarbonylamino)-azepine (9). Compound 9 (6 g,
77%) was prepared by a similar procedure used for 6
with compound 4 (5 g, 21.8 mmol), LiHMDS (1.3 M in
THF, 20 mL, 26 mmol) and benzenesulfonyl chloride
(3.3 mL, 26 mmol) in THF (50 mL) at 0 C. H NMR
(200 MHz, DMSO-d6): d (ppm) 7.95–7.55 (m, 5H, Ph),
Preparation of (S) 1-phenyl-2-oxo-3-(tert-butoxycarbon-
ylamino)-azepine (5a). Compound 5a (1.2 g, 43%) was
prepared by the same procedure used for 5 with compound
4a (2 g, 8.7 mmol), Cu(OAc)2 (1.7 g, 8.7 mmol) and pyr-
idine (0.7 mL, 8.7 mmol) and triphenylbismuth (4.2 g,
9.6 mmol) in CH2Cl2 (22 mL). RMN; IR similar to 5.
ꢀ
1

3200
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
1
4.5–4.3 (m, 2H, CHaN, NCHCO), 3.75–4.3 (dd, 1H,
CHa0N), 2.0–1.4 (m, 6H, (CH2)3), 1.35 (s, 9H, tBu). IR:
purification. H NMR (200 MHz, DMSO-d6): d (ppm)
7.3 (m, 5H, Ph), 4.7–4.5 (2d, 2H, CH2Ph), 4.35 (m, 1H,
CHNH), 3.6–3.3 (m, 2H, CH2N), 2.2–1.2 (m, 6H,
(CH2)3). IR: Vmax 3414, 2937, 1657 cmÀ1. Anal. (C13H18
N2O) HCl, 2H2O: C, H, N calcd: 53.70; 9.63; 11.9.
Found: 53.14; 7.94; 9.2.
Vmax 3426, 1702 cmÀ1
.
Preparation of (R,S) 1-(benzenesulfonylmethyl)-2-oxo-3-
(tert-butoxycarbonylamino)-azepine (10). Preparation of
(R,S)
1-(benzenesulfinylmethyl)-2-oxo-3-(tert-butoxy-
carbonylamino)-azepine. To a mixture a solution of
compound 4 (1 g, 4.36 mmol), phenylsulfinylmethyl
chloride (1.7 mL, 13 mmol) and NBu4I (0.24 g,
0.64 mmol) in THF (10 mL) was added KOH in powder
(0.73 g, 13 mmol) at room temperature. Then this mix-
ture was stirred overnight at room temperature and
treated with a saturated solution of NH4Cl and extrac-
ted with AcOEt. The organic phase was dried over
Na2SO4, filtrated and concentrated in vacuo. The resi-
due was purified by flash chromatography (silica gel,
petroleum ether/AcOEt; 85:15) to give the title com-
Preparation of (S) 1-benzyl-2-oxo-3-amino-azepine
(12a). Compound 12a (5.05 g, 95%) was prepared by
the same procedure used for 11 with compound 6
(7.74 g, 24.3 mmol) and trifluoroacetic acid (19 mL).
RMN; IR similar to 12.
Preparation of (R,S) 1-(2-phenylethyl)-2-oxo-3-amino
azepine (13). Compound 13 (1.4 g, 95%) was prepared
by the same procedure used for 11 with compound 7
(2 g, 6.05 mmol) and trifluoroacetic acid (4.7 mL). 1H
NMR (200 MHz, CDCl3): d (ppm) 7.3 (m, 5H, Ph), 4.15
(m, 1H, CHNH), 3.4–3.25 (m, 4H, CH2NCH2), 2.6 (d,
2H, CH2Ph), 2–1.3 (m, 6H, (CH2)3). IR: Vmax 3362,
1
pound as an oil (0.745 g, 49%). H NMR (200 MHz,
CDCl3): d (ppm) 7.8–7.4 (m, 5H, Ph), 5.1–4.8 (AB, 2H,
SCH2), 4.25 (m, 1H, CHNH), 3.55 (m, 2H, CH2N), 2.7
(d, 2H, CH2Ph), 1.8 (m, 6H, (CH2)3), 1.3 (s, 9H, tBu).
3297, 1645 cmÀ1
.
IR: Vmax 3415, 1711, 1654 cmÀ1
.
Preparation of (S) 1-(2-phenylethyl)-2-oxo-3-amino-aze-
pine (13a). Compound 13a (3.2 g, 91%) was prepared
by the same procedure used for 11 with compound 7a
(5.1 g, 15.2 mmol) and trifluoroacetic acid (12 mL).
RMN; IR similar to 13. Anal. (C14H20 N2O) 0.25H2O:
C, H, N calcd: 70.93; 8.65; 11.82. Found: 70.71; 8.53;
11.49.
Preparation of (10). To a solution of compound descri-
bed in step a (0.46 g, 1.3 mmol), in acetone (3 mL) was
added dropwise
a solution of dimethyldioxirane
(0.05 M, in acetone, 34 mL, 1.7 mmol) at 0 ꢀC. The
reaction mixture was stirred 2 h at room temperature
and then concentrated in vacuo to give 10 (0.32 g, 81%)
as a white solid. 1H NMR (200 MHz, DMSO-d6): d
(ppm) 7.8–7.4 (m, 5H, Ph), 5.1–4.8 (AB, 2H, SCH2),
4.25 (m, 1H, CHNH), 3.55 (m, 2H, CH2N), 2.7 (d, 2H,
CH2Ph), 1.8 (m, 6H, (CH2)3), 1.3 (s, 9H, tBu). IR: Vmax
3408, 1707, 1653, 1383, 1148 cmÀ1. Anal. (C18H26
N2O5S): C, H, N, S calcd: 56.53; 6.85; 7.32, 8.38.
Found: 56.53; 6.86; 7.04, 8.21.
Preparation of (R,S) 3-amino-2-oxo-1-(3-phenylpropyl)-
azepine (14). Compound 14 (1.2 g, 100%) was prepared
by the same procedure used for 11 with compound 8
1
(1.3 g, 3.74 mmol) and trifluoroacetic acid (2.9 mL). H
NMR (200 MHz, CDCl3): d (ppm) 7.25 (m, 5H, Ph), 4.1
(dd, 1H, CHNH), 3.7–3.3 (m, 4H, CH2NCH2), 2.55 (d,
2H, CH2Ph), 1.9–1.25 (m, 6H, 2(CH2)2). IR: Vmax 3500–
3000, 1668 cmÀ1
.
Preparation of (R,S) 1-phenyl-2-oxo-3-amino-azepine
(11). To a solution of 5 (0.69 g, 2.3 mmol) in CH2Cl2
(14 mL) was added dropwise trifluoroacetic acid
(1.7 mL) at room temperature. The reaction mixture
was stirred 2 h and then concentrated in vacuo. The
residue was purified by flash chromatography (silica gel,
CH2Cl2/MeOH; 95:5) to give 11 (0.32 g, 70%) as an oil.
1H NMR (200 MHz, CDCl3): d (ppm) 7.3 (m, 5H, Ph),
3.85–3.65 (m, 3H, CH2N, NCHCO), 2.2–1.7 (m, 6H,
Preparation of (R,S) 3-amino-2-oxo-1-(benzenesulfonyl)-
azepine (15). Compound 15 (3.45 g, 79%) was prepared
by the same procedure used for 11 with compound 9
1
(6 g, 16.2 mmol) and trifluoroacetic acid (12.5 mL). H
NMR (200 MHz, DMSO-d6): d (ppm) 7.95–7.6 (m, 5H,
Ph), 4.4 (m, 1H, CHaN, NCHCO), 3.95 (d, 1H,
NCHCO), 3.7 (dd, 1H, CHa0N), 2.1–1.8 (m, 6H,
(CH2)3). IR: Vmax 3600–2300, 1700 cmÀ1
.
(CH2)3). IR: Vmax 3375–3317, 1647 cmÀ1
.
Preparation of (R,S) 3-amino-2-oxo-1-(benzenesulfonyl-
methyl)-azepine (16). Compound 16 (0.31 g, 100%) was
obtained as a yellow gum by the same procedure used
for 11 with compound 10 (0.4 g, 1.04 mmol) and tri-
fluoroacetic acid (0.8 mL). 1H NMR (200 MHz, CDCl3):
d (ppm) 7.85 (d, 2H, o-Ph), 7.75–7.5 (m, 3H, m, p-Ph),
5.0 (AB, 2H, SCH2), 3.7 (m, 1H, CHNH), 3.5–3.3 (m,
2H, CH2N), 2.7 (d, 2H, CH2Ph), 1.8–1.2 (m, 6H,
Preparation of (S) 1-phenyl-2-oxo-3-amino-azepine
(11a). Compound 11a (0.53 g, 69%) was prepared by
the same procedure used for 11 with compound 5a
(1.2 g, 3.9 mmol) and trifluoroacetic acid (3 mL). RMN;
IR similar to 11.
Preparation of (R,S) 3-amino-2-oxo-1-benzyl-azepine
(12). To a solution of 6 (12.7 g, 40 mmol) in dioxane
(20 mL) was added dropwise a solution of 4 N HCl in
dioxane (100 mL) at room temperature. The reaction
mixture was stirred 3 h and then concentrated in vacuo.
The residue was triturated in ether to give 12 (10.6 g,
100%) which was used for the next step without further
(CH2)3). IR: Vmax 3367–3100, 1670 cmÀ1
.
Preparation of [3-(4-cyanobenzyl)-3-H-imidazol-4-yl]-
carboxaldehyde (17). Step 1: Preparation of [3-(4-cyano-
benzyl)-3-H-2-thioimidazol-4-yl]-methyl alcohol. To a
mixture of 4-cyanobenzylamine (18 g, 100 mmol), and
propionic acid (30 mL) in H2O/CH3CN (97:3, 300 mL)

T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
3201
was added sequentially KSCN (11 g, 115 mmol) and
dihydroxyacetone (dimer, 11.2 g, 115 mmol). This mix-
ture was stirred for 2 h at 60 ꢀC and overnight at room
temperature. Then the precipitate was filtrated off and
was washed with CH3CN and then H2O. The solid was
dried in vacuo to give the title compound (24 g, 98%)
which was used for the next step without further pur-
ification. 1H NMR (200 MHz, DMSO-d6): d (ppm) 12.2
(s, 1H, SH), 7.8–7.35 (2d, 4H, Ph), 6.9 (s, 1H, NCHN),
5.4 (s, 2H, PhCH2N), 4.15 (s, 1H, CH2O), 5.25 (s, 2H,
compound 28 (0.28 g, 50%) as an oil which was crys-
tallized in diethylether as the fumarate. 1H NMR
(200 MHz, CDCl3): d (ppm) 9.3 (s, 1H, NCHN), 7.85–
7.35 (m 9H, Ph), 3.85–3.65 (m, 3H, CH2N, NCHCO),
2.2–1.7 (m, 6H, (CH2)3). IR: Vmax 2800, 2229, 1702,
1655, 1561 cmÀ1. Anal. (C24H25 N5O) 1.4 C4H4 O4,
1H2O. C, H, N calcd: 61.72; 5.62; 12.06. Found: 61.40;
5.64; 11.74.
Preparation of (S) 4-{5-[(1-phenyl-2-oxo-azepan-3-yl-
amino)-methyl]-imidazol-1-ylmethyl}-benzonitrile (28a).
Compound 28a (0.22 g, 65%) was prepared by the
same procedure used for 28 with compound 11a (0.2 g,
0.98 mmol), 17 (0.21 g, 0.98 mmol) and NaBH(OAc)3
(0.31 g, 1.5 mmol). Mp: 75 ꢀC (decomposed); RMN; IR
similar to 28. Anal. (C24H25 N5O) 1.2 C4H4 O4, 1H2O:
C, H, N calcd: 61.82; 5.69; 12.5. Found: 64.15; 6.05;
11.80. The enantiomeric excess was determined by
HPLC using a Chiralcel OD column (eluant/heptane/
isopropanol:DEA; 500:500:0.5) and a UV detector at
230 nm (ee 89%).
OH). IR: Vmax 3600–3200, 2300, 2222, 1025 cmÀ1
.
MH+(246).
Step 2: Preparation of [3-(4-cyanobenzyl)-3-H-imida-
zol-4-yl]-methyl alcohol. To a solution of the thioalcohol
obtained in step 1 (23 g, 100 mmol) in diluted acetic acid
(H2O/AcOH; 80:20, 250 mL) was added dropwise an
aqueous solution of H2O2 (50%, 20 mL, 700 mmol)
during 10 min and the temperature was maintained
below 40 ꢀC. Then this mixture was stirred for 3 h at
room temperature and treated with an aqueous solution
of Na2SO3 (20%, 450 mL) and filtrated. The filtrate was
neutralized with aqueous ammonia (5%) and extracted
with AcOEt. The organic phase was dried over Na2SO4,
filtrated, concentrated in vacuo and dried to give the
title compound (14 g, 74%) which was used for the next
Preparation of (R,S) 4-{5-[(1-benzyl-2-oxo-azepan-3-yl-
amino)-methyl]-imidazol-1-ylmethyl}-benzonitrile (29).
Compound 29 was prepared by the same procedure
used for 28 with compound 12 (10.6 g, 40 mmol), 17
(11.05 g, 51.6 mmol), and NaBH(OAc)3 (8.9 g,
43 mmol). Flash chromatography (silica gel, CH2Cl2/
MeOH; 95:5) gave compound 29 as an oil which was
crystallized in diethylether as the dihydrochloride
(10.2 g, 52%). 1H NMR (200 MHz, DMSO-d6): d (ppm)
9.25 (s, 1H, NCHN), 7.85–7.3 (m 9H, PhPh), 5.85 (s,
2H, CH2Ph), 4.8–4.4 (m, 3H, CH2Ph, CHNH), 3.7–3.25
(m, 2H, CH2N), 2.35–1.2 (m, 6H, (CH2)3). IR: Vmax
3317, 2226, 1651, 1538 cmÀ1. Anal. (C25H27 N5O) 2HCl,
0.5H2O: C, H, N calcd: 60.60; 6.26; 14.14. Found: 61.06;
5.93; 14.18.
1
step without further purification. H NMR (200 MHz,
DMSO-d6): d (ppm) 7.75–7.3 (2d, 4H, Ph), 7.7 (s, 1H,
NCH¼N), 6.85 (s, 1H, NCH¼C), 5.35 (s, 2H,
PhCH2N), 5.15 (s, 2H, OH), 4.3 (s, 1H, CH2O). IR:
Vmax 3200, 2232, 1025 cmÀ1
.
Step 3: Preparation of 17. To a solution of the alcohol
obtained in step 2 (10 g, 47 mmol) and TEA (26 mL,
200 mmol) in DMSO (230 mL) was added portion wise
pyridine sulfur trioxide (50%, 18.6 g, 117 mmol) during
10 min at room temperature. Then this mixture was
stirred for 3 h at room temperature, treated with a
saturated solution of NH4Cl and extracted with AcOEt.
The organic phase was dried over Na2SO4, filtrated and
concentrated in vacuo. The residue was triturated in
diethyl ether to give 17 (9.6 g, 95%) as a white solid. 1H
NMR (200 MHz, DMSO-d6): d (ppm) 9.75 (s, 1H,
CHO) 7.9–7.8 (2s, 2H, NCH¼C, NCH¼N), 7.65–7.25
Preparation of (S) 4-{5-[(1-benzyl-2-oxo-azepan-3-yl-
amino)-methyl]-imidazol-1-ylmethyl}-benzonitrile (29a).
Compound 29a was prepared as the dihydrochloride
(0.135 g, 26%) by the same procedure used for 28 with
compound 12a (0.25 g, 1.14 mmol), 17 (0.24 g,
1.14 mmol) and NaBH(OAc)3 (0.34 g, 1.6 mmol). RMN;
IR similar to 29. Anal. (C25H27 N5O) 2HCl, 1H2O: C,
H, N calcd: 59.46; 6.14; 13.87. Found: 59.58; 6.17;
13.45. The enantiomeric excess was determined by
HPLC using a Chiralcel OD column (eluant: methanol/
DEA; 1000:1) and a UV detector at 230 nm. (ee 90%).
(2d, 4H, Ph), 5.6 (s, 2H, PhCH2N). IR: Vmax 2229 cmÀ1
.
Preparation of compounds [3-(R3)-3-H-imidazol-4-yl]-
carboxaldehyde 18–27. The aldehydes 18 to 27 listed in
Table 6 were obtained by the same procedure used for
17 by using the corresponding amine in step 1.
Preparation of the enantiomers of 2[2-(40-chlorobiphenyl-
thio)]-5-[2-(4-oxo-4H-benzo[d] [1,2,3]triazin-3-ylmethyl)]-
cyclopentanecarboxylic acid (29a/29b). The enantio-
meric mixture 29 (1.6 g) was separated by preparative
HPLC using a Chiralpack AD 250*4.6 mm column
(eluant: methanol/DEA; 1000:1) and a UV detector at
230 nm to give: Pic 1: 29a (0.6 g, elution time 7.75 min),
Preparation of (R,S) 4-{5-[(1-phenyl-2-oxo-azepan-3-yl-
amino)-methyl]-imidazol-1-ylmethyl}-benzonitrile
(28).
To a mixture of compounds 11 (0.28 g, 1.4 mmol) and
17 (0.29 g, 1.4 mmol) in DCE (5 mL), was added NaB-
H(OAc)3 (0.435 g, 2.1 mmol) at 0 ꢀC. Then this mixture
was stirred overnight at room temperature and con-
centrated in vacuo. The residue was diluted with
CH2Cl2 washed with saturated NaHCO3, brine, dried
over Na2SO4 and concentrated in vacuo. Flash chro-
matography (silica gel, CH2Cl2/MeOH; 95:5) gave
20
ee >98%, ½aꢁD =+6.69 (c 0.99; MeOH); 1H NMR
RMN; IR similar to 29. Anal. (C25H27 N5O) 2HCl,
1H2O: C, H, N calcd: 59.46; 6.14; 13.87. Found: 59.68;
6.43; 13.90. Pic 2: 29b (0.6 g, elution time 11.5 min), ee
20
1
>99%, ½aꢁD =À6.52 (c 0.89; MeOH); H NMR RMN;
IR similar to 29. Anal. (C25H27 N5O) 2HCl, 1H2O: C,

3202
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
H, N calcd: 59.46; 6.14; 13.87. Found: 59.67; 6.49;
13.88.
used for 28 with compound 16 (0.27 g, 1.04 mmol), 17
(0.2 g, 1.04 mmol) and NaBH(OAc)3 (0.44 g, 2.1 mmol).
Mp: decomposed. H NMR (200 MHz, DMSO-d6): d
1
Preparation of (R,S) 4-{5-[(1-(2-phenylethyl)-2-oxo-aze-
pan-3-ylamino)-methyl]-imidazol-1-ylmethyl}-benzonitrile
(30). Compound 30 was prepared as the dihydro-
chloride (0.155 g, 31%) by the same procedure used for
28 with compound 13 (0.25 g, 1.1 mmol), 17 (0.23 g,
1.1 mmol) and NaBH(OAc)3 (0.34 g, 1.6 mmol). Mp:
227 ꢀC; 1H NMR (200 MHz, DMSO-d6): d (ppm) 9.2 (s,
1H, NCHN), 7.95–7.25 (m, 10H, NCH, PhPh), 5.8 (AB,
2H, CH2Ph), 4.55 (m, 1H, CHNH), 4.25–3.85 (AB, 2H,
CH2N), 3.8–3.3 (m, 4H, CH2N, CH2N), 2.75 (t, 2H,
CH2Ph), 2.3–1.2 (m, 6H, (CH2)3). IR: Vmax 3600–2400,
2236, 1659, 1606 cmÀ1. Anal. (C25H27 N5O) 2HCl, 1.5H2O:
C, H, N calcd: 59.14; 5.87; 13.27. Found: 58.86; 6.28; 13.11.
(ppm) 9.2 (s, 1H, NCHN), 7.95–7.4 (m, 9H, PhPh), 7.75
(s, 1H, NCH¼), 5.75 (AB, 2H, PhCH2N), 5.05 (AB, 2H,
SO2CH2N), 4.35 (AB, 2H, CH2NH), 3.8–3.6 (2dd, 2H,
CH2NH), 3.4 (m, 2H, CH2NSO2), 2.35–1.3 (m, 6H,
(CH2)3). IR: Vmax 3407, 2800–2300, 2231, 1643, 1321,
1141 cmÀ1. Anal. (C25H27N5O3S) 2HCl, H2O: C, H, N,
S calcd: 52.77; 5.45; 12.31; 5.62. Found: 52.72; 5.55;
11.75; 5.45. MH+(478).
Preparation of compounds 34a–53a: general procedure
To a solution of compound 4a (0.23 g; 1 mmol) in THF
(5 mL) was added NaH 80% in oil (32 mg) at 0 ꢀC. After
5 min agitation, nBu4NI (37 mg; 1% mol) and the cor-
responding benzyl bromide (1.05 mmol) were added.
After 1 h agitation at room temperature the reaction
was quenched by adding of saturated NH4Cl solution
(1 mL) and H2O (4 mL) then extracted twice with
AcOEt (5 mL). The organic layer was dried over MgSO4
filtrated and evaporated to dryness. The crude com-
pound was treated with HCl (4 M) solution in dioxane
(10 mL) and agitated at room temperature until com-
plete Boc deprotection. The solvent was removed and
the residue was dissolved in CH2Cl2 (10 mL) and
washed with saturated NaHCO3 (5 mL) dried over
MgSO4 filtrated and evaporated to dryness. The residue
was dissolved in CH2Cl2 (5 mL) and aldehyde 17
(1 equiv) and NaBH(OAc)3 (1.5 equiv) were added suc-
cessively. After one night at room temperature was
added saturated NaHCO3 (5 mL) and the compound
was extracted twice with CH2Cl2 (5 mL). The organic
layer was dried over MgSO4 filtrated and evaporated to
dryness. The final compound was purified by reversed
phase SPE affording the pure corresponding compound
as a TFA salt. This salt was dissolved in CH2Cl2
(10 mL) and washed with saturated NaHCO3 solution
(5 mL). The organic layer was dried over MgSO4 fil-
trated and evaporated to dryness to afford the free base
of compounds 34a to 53a which was transformed into
hydrochloride or fumarate salt to form an amorphous
solid (%, Table 7).
Preparation of (S) 4-{5-[(1-(2-phenylethyl)-2-oxo-aze-
pan-3-ylamino)-methyl]-imidazol-1-ylmethyl}-benzonitrile
(30a). Compound 30a was prepared as the dihy-
drochloride (0.215 g, 22%) by the same procedure used
for 28 with compound 13a (0.5 g, 2.15 mmol), 17 (0.45 g,
2.15 mmol) and NaBH(OAc)3 (0.64 g, 3 mmol). Mp:
167 ꢀC; RMN; IR similar to 30. Anal. (C25H27 N5O)
2HCl, 2H2O: C, H, N calcd: 58.14; 5.96; 13.04. Found:
57.76; 6.33; 12.76. The enantiomeric excess was deter-
mined by HPLC using a Chiralcel OD column (eluant:
heptane/isopropanol/DEA; 500:500:0.5) and a UV
detector at 210 nm. (ee 85%).
Preparation of (R,S) 4-{5-[1-(3-phenylpropyl)-2-oxo-aze-
pan-3-ylamino)-methyl]-imidazol-1-ylmethyl}-benzonitrile
(31). Compound 31 was prepared as the fumarate
(0.12 g, 19%) by the same procedure used for 28 with
compound 14 (0.3 g, 1.2 mmol), 17 (0.26 g, 1.2ꢀmmol)
1
and NaBH(OAc)3 (0.52 g, 2.45 mmol). Mp: 180 C; H
NMR (200 MHz, DMSO-d6): d (ppm) 7.75–7.1 (m,
10H, NCHN PhPh), 6.8 (s, 1H, NCH), 6.6 (s, 2H,
CH¼CH), 5.45 (s, 2H, PhCH2NH), 3.5 (AB, 2H,
ImCH2N), 3.1–2.55 (m, 7H, CHNH, 2 CH2N, CH2Ph),
1.8–1.1 (m, 8H, (CH2), (CH2)3). IR: Vmax 2800–1800,
2229, 1655, 1577 cmÀ1. Anal. (C27H31 N5O) C4H4O4: C,
H, N calcd: 66.77; 6.33; 12.56. Found: 66.67; 6.38;
12.54. M-H+(440)
Preparation of (R,S) 4-{5-[(1-(benzenesulfonyl)-2-oxo-
azepan-3-ylamino)-methyl]-imidazol-1-ylmethyl}-benzoni-
trile (32). Compound 32 was prepared as the free base
(0.193 g, 37%) by the same procedure used for 28 with
compound 15 (0.3 g, 1.11 mmol), 17 (0.236 g, 1.11 mmol)
and NaBH(OAc)3 (0.33 g, 1.56 mmol). 1H NMR
(200 MHz, DMSO-d6): d (ppm) 7.95–7.25 (m, 10H,
NCHN PhPh), 6.75 (s, 1H, NCH¼), 6.5 (s, 2H,
CH¼CH), 5.35 (s, 2H, PhCH2N), 4.35 (m, 1H, CHNH),
3.8–3.6 (2dd, 2H, CH2NH), 3.4 (m, 2H, CH2NSO2), 2.0–
1.1 (m, 6H, (CH2)3). IR: Vmax 3308, 2227, 1693, 1348,
1164cmÀ1. Anal. (C24H25 N5O3S) C4H4O4: C, H, N calcd:
62.19; 5.44; 15.11. Found: 62.15; 5.44; 14.80. MH+(464).
Preparation of compounds 54a–63a
Compounds 54a–63a were prepared as dihydrochloride
or fumarate by the same procedure used for 29a by
reductive amination of the various aldehydes 18–27 with
the intermediate amine of 39a and NaBH(OAc)3 (%,
Table 8).
Preparation of (S) 1-benzyl-3-[1-trityl-imidazol-4-yl-
methyl)amino]-azepan-2-one (64a). To a mixture of
compounds 12a (7 g, 24.2 mmol) and 1-trityl-4-carbox-
aldehyde imidazol (8.2 g, 24.2 mmol) in DCE (85 mL),
was added NaBH(OAc)3 (10.2 g, 48.5 mmol) at 0 ꢀC.
Then this mixture was stirred overnight at room tem-
perature and concentrated in vacuo. The residue was
diluted with CH2Cl2 washed with saturated NaHCO3,
brine, dried over Na2SO4 and concentrated in vacuo.
Flash chromatography (silica gel, CH2Cl2/MeOH; 95:5)
Preparation of (R,S) 4-{5-[(1-(benzenesulfonylmethyl)-2-
oxo-azepan-3-ylamino)-methyl]-imidazol-1-ylmethyl}-
benzonitrile (33). Compound 33 was prepared as the
dihydrochloride (0.156 g, 27%) by the same procedure

T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
3203
1
gave compound 64a (10.5 g, 81%) as a foam. H NMR
(200 MHz, DMSO-d6): d (ppm) 7.45–7.1 (m, 20H, 4 Ph),
7.35 (s, 1H, NCHN), 6.75 (s, 1H, NCH¼), 4.55 (AB,
2H, CH2Ph), 3.8–3.5 (m, 3H, CHNH, CH2N), 1.9–1.1
Preparation of compounds 67a–96a. Preparation of com-
pounds 67a–82a. General procedure. To a solution of
compound 64a (200 mg, 0.37 mmol) in CH2Cl2 (5 mL) is
added the corresponding aldehyde (0.37 mmol) at room
temperature followed by NaBH(OAc)3 (118 mg,
0.55 mmol). After one night at room temperature is
added saturated NaHCO3 (5 mL). After 10 min the
organic layer was recovered and the aqueous solution
was extracted twice with CH2Cl2 (5 mL). The organic
layers were pooled and dried over magnesium sulphate,
filtrated and evaporated to dryness. The recovered
compound was dissolved in CH2Cl2 (2 mL), treated by
Et3SiH (0.1 mL) and TFA (2 mL). After overnight agi-
tation at room temperature, the solvent was removed
under vacuum and the final compound was purified by
reversed-phase chromatography affording the pure cor-
responding compound as a TFA salt (%, Table 9).
(m, 6H, (CH2)3). IR: Vmax 3300, 1644 cmÀ1
.
Preparation of (S) 1-benzyl-3-[3H-imidazol-4-ylmethyl)-
amino]-azepan-2-one(65a). A solution of 64a (0.5 g,
0.925 mol) in HCO2H (5 mL) was stirred 3 h at room
temperature and then concentrated in vacuo. The resi-
due was diluted in ether and extracted with 1 N aqueous
HCl. The aqueous phase was treated with 10 N aqueous
NaOH (to pH>10) and extracted with CH2Cl2. The
organic phase was dried over Na2SO4 and concentrated
in vacuo to give 65a which was crystallised as the
fumarate (0.26 g, 67%). Mp: 60 ꢀC decomposed; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 7.65 (s, 1H,
NCHN), 7.3 (m, 5H, Ph), 7.0 (s, 1H, NCH¼), 6.5 (s,
2H, CH¼CH), 4.55 (AB, 2H, CH2NH), 3.85 (AB, 2H,
CH2NH), 3.55–3.2 (m, 2H, CH2N), 1.95–1.3 (m, 6H,
(CH2)3). IR: Vmax 3600, 2200, 1705–1659, 1620,
1567 cmÀ1. Anal. (C17H22N4O) 1.25 C4H4O4, 1H2O: C,
H, N calcd: 57.28; 6.72; 12.14; 5.62. Found: 57.56; 6.70;
11.90.
Preparation of compounds 83a–96a. General procedure.
To a solution of compound 10 (170 mg, 0.31 mmol) in
THF (5 mL) is added polyvinyl pyridine (500 mg) and
the corresponding acylchloride or chloroformiate
(0.4 mmol) at room temperature. After overnight agita-
tion, the reaction mixture is filtrated and the resin is
washed twice with ethyl acetate (5 mL). The organic
layer was concentrated and the compound purified by
quick filtration on normal phase SPE (AcOEt/CH2Cl2).
The recovered compound was dissolved in CH2Cl2
(2 mL), treated by Et3SiH (0.1 mL) and TFA (2 mL).
After overnight agitation at room temperature, the sol-
vent was removed under vacuum and the final com-
pound was purified by reversed phase chromatography
affording the pure corresponding compound as a TFA
salt (%, Table 9).
Preparation of (S) 4-{[3-(4-cyanobenzyl-3H-imidazol-4-
ylmethyl)-(1-benzyl-2-oxo-azepan-3-yl)-amino]-methyl}-
benzonitrile (66a). Preparation of (S) 4-{[1-trityl-1H-imi-
dazol-4-ylmethyl)-(1-benzyl-2-oxo-azepan-3-yl)-amino]-
methyl}-benzonitrile. To a suspension of NaH (60% in
oil, 0.11 g, 2.75 mmol) in DMF (6 mL) was added a
solution of compound 64a (1 g, 1.85 mmol) in DMF
(5 mL) at 0 ꢀC. Then this mixture was stirred 1 h and a
solution of p-cyanobenzyl bromide (0.725 g, 2.7 mmol)
and NBu4I (0.07 g, 0.2 mmol) in DMF (2 mL) was
added. After an additional stirring of 2 h, the reaction
mixture was cooled down to 0 ꢀC, treated with a satu-
rated solution of NH4Cl and extracted with AcOEt. The
organic phase was dried over Na2SO4, filtrated and
concentrated in vacuo. The residue was purified by flash
chromatography (silica gel, heptane/AcOEt; 50:50) to
give the title compound as a yellow foam (0.585 g, 48%)
which was used directly for the next step.
Enzyme assays
FTase and GGTase-1 were purified from rat brains and
enzymatic activities were evaluated using the Amersham
scintillation proximity assay with the C-terminal
sequence of lamin B as a substrate for FTase (biotin-
YRASNRSCAIM) and the biotin-TKCVIL sequence
for GGTase; [3H]-farnesyl- and [3H]-geranylgeranyl-
pyrophosphate were used as donors. IC50 of each
product on each enzyme was calculated by EXCEL
software using three points in the central linear range of
fluorescence inhibition.
Preparation of (66a). To a solution of the compound
obtained in step a (0.25 g, 0.38 mmol) in AcOEt (1 mL)
was added a solution of p-cyanobenzyl bromide
(0.075 g, 0.38 mmol) in AcOEt (1 mL) at room tempera-
ture. Then the reaction mixture was heated at 60 ꢀC
overnight and concentrated in vacuo. The residue was
treated with MeOH (2 mL), heated at 60 ꢀC for 2 h and
concentrated in vacuo. The residue was purified by flash
chromatography (silica gel, AcOEt/EtOH, 90:10) to give
66a which was crystallized as a fumarate (0.036 g, 18%).
Mp: >100 ꢀC decomposed; 1H NMR (200MHz, DMSO-
d6): d (ppm) 7.8–7.0 (m, 15H, NCH¼, NCHN, 3Ph),
7.75 (s, 1H, NCH¼), 5.21 (AB, 2H, PhCH2N), 4.55
(AB, 2H, PhCH2N), 4.2–3.5 (2AB, 4H, CH2NCH2), 3.6
(m, 1H, CHN), 3.15 (m, 2H, CH2N), 2.35–1.3 (m, 6H,
Cellular assays
The rat cell line Ras#1 obtained after transfection of
fibroblastic RAT2 cells with v-H-Ras17 reverts to the
fibroblastic phenotype after treatment with FTase inhi-
bitors. This effect is accompanied by a reduction in
growth kinetics and cell viability. When quantifying cell
number as a function of dose, this translates in the for-
mation of a plateau at doses effective to inhibit FTase
eventually followed by a cytotoxic decrease at higher
doses. A serum batch was chosen to ensure that this plateau
occurred at less than 50% of the cell number so that IC50s
indicated in the tables refer to cell inhibition of FTase
when appropriate and not to unrelated cytotoxicity of
(CH2)3). IR: Vmax 3300–2300, 2228, 1700, 1645 cmÀ1
Anal. (C33H32N6O) 1C4H4O4, 0.5H2O: C, H, N calcd:
67.89; 5.66; 12.84. Found: 67.87; 5.91; 12.32.
.

3204
T. Le Diguarher et al. / Bioorg. Med. Chem. 11 (2003) 3193–3204
the compounds. Cells were treated for four days in 96-
well tissue culture plates and cell number estimated
indirectly through a tetrazolium viability assay.
Fairchild, C. R.; Lynch, M.; Monticello, T.; Kramer, R. A.;
Manne, V. Cancer Res. 2001, 61, 7507.
8. Britten, C. D.; Rowinsky, E. K.; Soignet, S.; Patnaik, A.;
Yao, S.-L.; Deutsch, P.; Lee, Y.; Lobell, R. B.; Mazina, K. E.;
McCreery, H.; Pezzuli, S.; Spriggs, D. Clin. Cancer Res. 2001,
7, 3894.
Acknowledgements
9. (a) Kelland, L. R.; Smith, V.; Valenti, M.; Patterson, L.;
Clarke, P. A.; Detre, S.; End, L.; Howes, A. J.; Dowsett, M.;
Workman, P.; Johnston, S. R. D. Clin. Cancer Res. 2001, 7,
3544. (b) End, D. W.; Smets, G.; Todd, A. V.; Applegate,
T. L.; Fuery, C. J.; Angibaud, P.; Venet, M.; Sanz, G.;
Poignet, H.; Skrzat, S.; Devine, A.; Wouters, W.; Bowden, C.
Cancer Res. 2001, 61, 131.
10. (a) Liu, M.; Bryant, M. S.; Chen, J.; Lee, S.; Yaremko, B.;
Lipari, P.; Malkowski, M.; Ferrari, E.; Nielsen, L.; Prioli, N.;
Dell, J.; Sinha, D.; Syed, J.; Korfmacher, W. A.; Nomeir,
A. A.; Lin, C.-C.; Wang, L.; Taveras, A. G.; Doll, R. J.;
Njoroge, F. G.; Mallams, A. K.; Remiszewski, S.; Catino, J. J.;
Girijavallabhan, V. M.; Kirschmeier, P.; Bishop, W. R. Cancer
Res. 1998, 58, 4947. (b) Reichert, A.; Heisterkamp, N.; Daley,
G. Q.; Groffen, J. Blood 2001, 97, 1399. (c) Peters, D. G.;
Hoover, R. R.; Gerlach, M. J.; Koh, E. Y.; Zhang, H.; Choe,
K.; Kirschmeier, P.; Bishop, W. R.; Daley, G. Q. Blood 2001,
97, 1404.
The authors wish to thank Sabine Plantier, Sophie Sci-
berras, Christel Daumas, Stephanie Dupas and Annie
Genton for their skilful technical assistance and Solange
Huet for the preparation of the manuscript. We would
also like to acknowledge the analytical department at
the IdRS for performing all the spectral analyses.
References and Notes
1. Huang, C. C.; Casey, P. J.; Fierke, C. A. J. Biol. Chem.
1997, 272, 20.
2. Lerner, E. C.; Hamilton, A. D.; Sebti, S. M. Anti-Cancer
Drug Design 1997, 12, 229.
3. (a) Prendergast, G. C.; Rane, N. Exp. Opin. Investig. Drugs
2001, 10 (12), 2105. (b) Cox, A. D.; Der, C. J. Current. Opin.
Pharm. 2002, 2, 388.
4. (a) Haluska, P.; Dye, G. K.; Adjei, A. A. Eur. J. Cancer
2002, 38, 13 1685. (b) Du, W.; Lebowitz, P. F.; Prendergast,
G. C. Mol.Cell. Biol. 1999, 19 (3), 1831 and references cited
herein.
5. Moores, S. L.; Schaber, M. D.; Mosser, S. D.; Rands, E.;
O’Hara, M. B.; Garsky, V. M.; Marshall, M. S.; Pompliano,
D. L.; Gibbs, J. B. J. Biol. Chem. 1991, 266 (22), 14603.
6. Hunt, J. T.; Lee, V. G.; Leftheris, K.; Seizinger, B.; Car-
boni, J.; Mabus, J.; Ricca, C.; Ning, Y.; Manne, V. J. Med.
Chem. 1996, 39 (2), 353 and references cited therein.
7. (a) Hunt, J. T.; Ding, C. Z.; Batorsky, R.; Bednarz, M.;
Bhide, R.; Cho, Y.; Chong, S.; Chao, S.; Gullo-Brown, J.;
Guo, P.; Kim, S. H.; Lee, F. Y. F.; Leftheris, K.; Miller, A.;
Mitt, T.; Patel, M.; Penhallow, B. A.; Ricca, C.; Rose, W. C.;
Schmidt, R.; Slusarchyk, W. A.; Vite, G.; Manne, V. J. Med.
Chem. 2000, 43 (20), 3587. (b) Rose, W. C.; Lee, F. Y. F.;
11. (a) Long, S. B.; Hancock, P. J.; Kral, A. M.; Hellinga,
H. W.; Beese, L. S. P.NA.S. 2001, 98, 12948. (b) Strickland,
C. L.; Windsor, W. T.; Syto, R.; Wang, L.; Bond, R.; Wu, Z.;
Schwartz, J.; Le, H. V.; Beese, L. S.; Weber, P. C. Biochem-
istry 1998, 37, 16601.
12. Ciccarrone, T. M.; MacTough, S. C.; Williams, T. M.;
Dinsmore, C. J.; O’Neil, T. J.; Shah, D.; Culberson, J. C.;
Koblan, K. S.; Kohl, N. E.; Gibbs, J. B.; Oliff, A. I.; Graham,
S. L.; Hartman, G. D.. Bioorg. Med. Chem. Lett. 1999, 9, 1991.
13. Chan, D. M. Tetrahedron Lett. 1996, 37, 9013.
14. Dinsmore, C. J.; Williams, T. M.; O’Neil, T. J.; Liu, D.;
Rands, E.; Culberson, J. C.; Lobell, R. B.; Koblan, K. S.; Kohl,
N. E.; Gibbs, J. B.. Bioorg. Med. Chem. Lett. 1999, 9, 3301.
15. Aulaskari, P.; Ahlgren, M.; Rouvinen, J.; Vainiotalo, P. J.
Heterocyl. 1996, 33, 1345.
16. Liu, R.; Dong, D.; Sherlock, R.; Nestler, P.. Bioorg. Med.
Chem. Lett. 1999, 9, 847.
˜
17. Martin, A.; Gomez-Munoz, A.; Waggoner, D. W.; Stone,
J. C.; Brindley, D. J. Biol. Chem. 1993, 268, 23924.