Rapid and efficient synthesis of a novel series of substituted aminobenzosuberone derivatives as potent, selective, non-peptidic neutral aminopeptidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.06.041

Racemic 5-substituted 7-aminobenzocyclohepten-6-one were synthesized and evaluated for their ability to inhibit metalloaminopeptidase activities. Unexpectedly, 5-thio substituted compounds showed enhanced inhibition potency with K_i values in the nanomolar range against the 'one zinc' aminopeptidases from the M1 family, while most of them were rather poor inhibitors of the 'two zincs' enzymes from the M17 family. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.

Full text of DOI:10.1016/j.bmc.2012.06.041

Bioorganic & Medicinal Chemistry 20 (2012) 4942–4953
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Rapid and efficient synthesis of a novel series of substituted
aminobenzosuberone derivatives as potent, selective, non-peptidic
neutral aminopeptidase inhibitors
⇑
⇑
Sébastien Albrecht , Emmanuel Salomon, Albert Defoin, Céline Tarnus
Université de Haute Alsace, Laboratoire de Chimie Organique et Bioorganique, EA 4566, ENSCMu, 3, rue Alfred Werner, F-68093 Mulhouse Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Racemic 5-substituted 7-aminobenzocyclohepten-6-one were synthesized and evaluated for their ability
to inhibit metalloaminopeptidase activities. Unexpectedly, 5-thio substituted compounds showed
enhanced inhibition potency with Ki values in the nanomolar range against the ‘one zinc’ aminopepti-
dases from the M1 family, while most of them were rather poor inhibitors of the ‘two zincs’ enzymes
from the M17 family. This interesting selectivity profile may guide the design of new, specific inhibitors
of target mammalian aminopeptidases with one active site zinc.
Received 16 March 2012
Revised 30 May 2012
Accepted 22 June 2012
Available online 28 June 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Racemic 5-substituted
aminobenzocycloheptenone
Aminopeptidase N inhibitor
APN/CD13
1. Introduction
Recent reviews provide an excellent overview of the currently
available collection of APN/CD13 inhibitors.4 Among the most ac-
APN/CD13 is a membrane-bound, zinc-dependent homodimeric
enzyme and a member of the M1 family of aminopeptidases.1 Like
other members of this family, APN/CD13 possesses the consensus
zinc binding motif (HEXXH-(X18)-E) in its extracellular domain,
as well as the exopeptidase motif (GXMEN) for binding the free pri-
mary amino group of the N-terminal residue of its peptidic sub-
strates. APN/CD13 removes the N-terminal amino acid from
unsubstituted oligopeptides, amides or arylamides, with a broad
substrate specificity, although a significant preference for hydro-
phobic residues is observed.1 This ectoenzyme appears to be a mul-
tifunctional protein involved in the regulation of signalling
peptides as well as in various cell activation and migration pro-
cesses.2 APN/CD13 is emerging as a target of significant biological
and medical importance. Moreover, recent studies have indicated
that APN/CD13 is an active player in angiogenesis and tumor
metastasis.3 Still, the exact role played by APN/CD13 in those
pathologies, as well as the cellular pathways involved, remain to
be elucidated. To this end, a small molecular weight, drug-like,
selective inhibitor of APN/CD13 would be an invaluable biological
tool to assess the pathophysiological roles of this multifunctional
enzyme that depend solely on its catalytic activity.
tive compounds, pseudo-peptides that mimic the transition state
analogs such as -boronic acids, sulfonamide and phosphonic sur-
a
rogates of the scissile peptide bond are the most prominent. Natu-
ral products such curcumin5 and psammaplin A6 or synthetic
flavonoid derivatives,7 as well as compounds incorporating the
hydroxamic acid moiety8 have also been reported as potent APN/
CD13 inhibitors. However, while many of these compounds display
high in vitro potency, their selectivity is not always well docu-
mented. In view of the potential therapeutic relevance of some of
the APN/CD13 functions, the rational design of potent and selective
APN/CD13 inhibitors is of considerable interest, but remains a chal-
lenging endeavor and a daunting task.
3-Amino-2-tetralone 1 has been described as potent and selec-
tive inhibitor of the ‘one zinc’ APN/CD13.9,10 Nevertheless, tetra-
lone 1 was not stable under physiological conditions, and some
more stable derivatives have been later synthesized without
improvement in affinity.11 Lately, its seven-membered homolog
2a has been reported as more selective without loss of stability
and affinity against APN (Scheme 1).12 After having performed a
SAR study around this scaffold, we were delighted to find out that
the simple 4-phenyl substituted aminobenzosuberone 20 was a
nanomolar APN inhibitor (Ki 7 nM).13,14 It is noteworthy that it re-
tained a very high selectivity towards the M1 subfamily of one-zinc
aminopeptidases. However, its synthesis was problematic and gave
rise to a regioisomeric mixture (in an overall yield of 2.4–6.7% from
the commercially available starting materials) that required a
⇑
Corresponding authors. Tel.: +33 3 89 33 68 59; fax: +33 3 89 33 68 60
E-mail addresses: sebastien.albrecht@uha.fr (S. Albrecht), celine.Tarnus@uha.fr
(C. Tarnus).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.041

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
4943
chemical stability
improvement
R
R
O
O
7
O
4
1
6
O
5
SAR & scope
NH2,HCl
5
NH2,HCl
NH2,HCl
NH2,HCl
1
2a
2a R =H
b R = F
2d' R = SPh
APN Ki = 0.5 µM, LE = 0.73
selective over a panel of proteases
unstable at pH =7.4 & 37°C
APN Ki = 1 µM, LE = 0.65
e' R = (CH2)2Ph
f' R = CH2Ph
g' R = (CH2)3Ph
selective over a panel of proteases
stable at pH =7.4 & 37°C
c R = SBn
d R = SPh
e R = (CH2)2Ph
O
O
NHZ
O
NH2,HCl
2'
Y
O
4
6
5
7
NH2,HCl
3
4a Z = Cbz
5a Z = Boc
1
APN Ki = 7 nM, LE = 0.60
selective over a panel of proteases
stable at pH =7.4 & 37°C
more readily accessible derivatives ?
Scheme 2. Chemical structures of the novel APN/CD13 inhibitors.
potency improvement ?
reaction of Selectfluor™18,19 with a silyl enol ether according to
the literature20,21 (Scheme 3). The silyl enol ethers 6a and 6b were
obtained by enolization of 4a or 5a with LiHMDS, in the presence of
HMPA as chelating agent in the case of 6b, and quenching with
Me3SiCl or better with Me2tBuSiCl. The electrophilic addition of
Selectfluor™ in acetonitrile at ꢀ20 °C gave different results accord-
ing to the N-protection (scheme 3): the benzyloxycarbonyl deriva-
tive 6a led to the expected fluoro compound 4b, but the Boc-
derivative 6b gave a 1:1 mixture of 5b and its acidic transposition
product 7 (as hydrate). In the presence of aqueous NaHCO3 as base
at 0 °C, 6b led to the expected fluoroketone 5b in a 1:1 mixture
with the stable silylated hemiacetal 8, and, without separation, a
subsequent treatment with fluoride anion provided 5b as the sole
product in 52% yield. Attempts to deprotect 4b either by hydrogen-
olysis or with BBr3 led to partial defluorination in amine 2a, but
acidic N-deprotection of 5b gave easily the fluoroamine hydrochlo-
ride 2b in 37% overall yield from 5a. In D2O solution, 2b appeared
in mixture with 85% of the corresponding hydrate form 2b0. The
configuration of these compounds 2b, 4b and 5b was trans with
an axial fluorine atom.
synthesis & purification issues
Scheme 1. Strategy envisioned for the development of novel APN/CD13 inhibitors.
LE, ligand efficiency, LE = ꢀ1.4log (Ki)/N, N: number of nonH-atoms.
preparative HPLC purification to separate both regioisomers. This
method was not suitable and not satisfying for a large scale synthe-
sis needed to carry out further in vivo studies which will be re-
ported in due course.
We thus wanted to design a straightforward synthesis allowing
easy access to a large variety of derivatives retaining the phenyl
moiety, a key phamacophore feature. To this end, we decided to ex-
plore the functionalization at the 5 position of the benzosuberone
2a.
In the present work, we report the synthesis of racemic 5-
substituted analogs 2b–h of the 7-aminobenzocyclohepten-6-one
2a, and their inhibitory activities against a panel of ‘one zinc’
APs. The parent compound 3-amino-2-benzosuberone 2a and best-
atin were used as reference inhibitors.
2. Chemistry
2.2. Thio-derivatives 2c and 2d,d0
Since hydrated ketone is a structural mimic of the tetrahedral
intermediate that forms during the hydrolysis of peptide sub-
strates, the synthesis of 5-fluoro-5-susbtituted aminobenzosuber-
ones should enhance considerably the inhibitory activities
towards metalloproteases. Therefore, the simple fluoroketone 2b
was investigated at first. Secondly, an efficient and practical meth-
od for preparing 5-substituted analogs was sought out. Hence, we
turned our attention to the ring opening of epoxides with thiols, a
convenient reaction, characterized by operational simplicity, high
yield and regioselectivity (addition of thiols to the benzylic car-
bon). Then, a series of C-substituted compounds was explored
and required more complex procedures.
The preparation of the aminobenzocycloheptenone 2a has been
previously described from the known benzocycloheptanone 315–17
through the N-protected derivatives 4a or 5a12 (Scheme 2). Two
synthetic ways have been used in order to substitute the 5-posi-
tion: the trans-derivatives 2c–e were prepared from the ketone 3
via epoxide opening, the cis-derivatives 2e0-g0 and the fluoro com-
pound 2b were directly obtained from the N-protected aminoke-
tones 4a or 5a.
The synthesis of the thio-derivatives 2c and 2d,d0 was achieved
via the epoxide 11b (Scheme 4).
The silyl enol ether of ketone 3, prepared by O-silylation with
Me3SiOTf in the presence of NEt3 at 85 °C, was oxidized to the en-
one 9 in 85% yield, with O2 in DMSO and Pd(OAc)2 as catalyst
according to the Saegusa method.22 The reductive amination of
the ketone function according to already described procedures,12,23
followed by N-protection with Boc2O, provided the amide 10b. This
latter was epoxidized with m-CPBA to the cis epoxyamide 11b in
65% overall yield from ketone 3. A subsequent SN2 epoxide opening
reaction with benzylmercaptan or thiophenol as soft nucleophiles
led stereospecifically to the thioethers 12c and 12d with trans-con-
figuration for the 4-thioether and 5-hydroxy substituents. Then,
the oxidation of the alcohol function in 6-position with DMP gave
ketones 5c and 5d without modifications of the thioether function.
Their N-deprotection with HCl led to the corresponding final amine
hydrochlorides 2c and 2d,d0 in 38% and 34% yield, respectively
(overall yields calculated from ketone 3). Unlike benzylthioether
2c, which was isolated as a single isomer, the phenylthioether
amine hydrochloride appeared in D2O solution as a 40/60 mixture
of 2d-trans and 2d0-cis-isomers in equilibrium.
2.1. Fluoration of the N-protected aminoketones 4a or 5a
In order to mimic the tetrahedral intermediate produced during
peptide hydrolysis, by all metalloenzymes, we aimed at favoring
the hydrate form of ketone 2a through incorporation of a fluorine
atom. The latter was introduced at the 5-position by electrophilic
2.3. Aliphatic substitutions
Three methods were used to substitute the 5-position with
an aliphatic chain: epoxide opening of 11a by organocuprates,

4944
S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
F
O
O[Si]
a
b
NHZ
NHZ
4a, 5a
6a Z = Cbz, [Si] = SiMe3
4b Z = Cbz (68%)
5b Z = Boc (52%)
2b Z = H,HCl (74%)
6b Z = Boc, [Si] = SiMe2tBu
c
F
F
F
OH
OH
HO
HO
OSiMe2tBu
NHBoc
OH
NH2,HCl
NH
OSiMe2tBu
O
7
8
2b'
Scheme 3. Synthetic scheme for the fluoro analogs. Reagents and conditions: (a) (1) LiHMDS, HMPA, in THF, 30 min, ꢀ78 °C; 2. ClSiMe3 for 4a or ClSiMe2tBu for 5a,15 min; (b)
Selectfluor in MeCN, 2 h, ꢀ20 °C for 6a, 68% yield from 4a; Selectfluor™ in MeCN and aqueous 1 M NaHCO3, 30 min, 0 °C for 6b, then NBu4F, THF, 10 min, 52% yield from 5a. (c)
0.6 N HCl in EtOH, 12 h.
O
e
a,b
X
NHR
O
9
X = O (85%)
11a R = Z (quant.)
11b R = Boc (90%)
3
c,d
10a X = H,NHZ (80%)
10b X = H,NHBoc (76%)
RS
RS
O
OH
NHBoc
f
g
NHZ
11b
12c R = Bn (86%)
12d R = Ph (80%)
5c R = Bn Z = Boc (83%)
h
2c R = Bn Z = H,HCl (83%)
5d R = Ph Z = Boc (quant.)
PhS
PhS
PhS
O
O
OH
NH2,HCl
h 66%
5d
+
NH2,HCl
40/60
NH2,HCl
2d
2d'
2d''
Scheme 4. Synthetic scheme for the S-substituted analogs. Reagents and conditions: (a) Me3SiOTf, NEt3, toluene, 85 °C, 2 h. (b) Pd(OAc)2, O2, DMSO. (c) Ti(OiPr)4, satd NH3 in
MeOH, 6 h then NaBH4, 1 h; (d) for 10a: CbzCl, Na2CO3, THF; for 10b: Boc2O, Na2CO3, MeOH; (e) m-CPBA, CH2Cl2, 0 °C, 10 h; (f) PhSH (86%) or BnSH (80%), NEt3, EtOH, 1 h; (g)
DMP, CH2Cl2, 2 h; (h) dry HCl 2 M in Et2O, dioxane.
aldolisation–crotonisation of the amidoketone 5a or 1,4-addition
of organocuprates with the methylene ketone 4i (Scheme 5).
The cis N-benzyloxycarbonylepoxide 11a was obtained by a
similar method as for the N-Boc derivative 11b in 61% overall yield
from ketone 3. Its opening with phenethyl cuprate led to the alco-
hol 12e which was directly oxidized with DMP to give the corre-
sponding ketone 4e. Its N-deprotection by hydrogenolysis in the
presence of HCl provided the 5-trans-phenethyl amine hydrochlo-
ride 2e in 53% yield from the epoxide 11a.
The aldolisation–crotonisation of 5a with benzaldehyde was
carried out with LiHMDS in the presence of HMPA in THF, or with
DBU in toluene, to give the benzylidene derivative 5h in 50–60%
yield. Deprotection of 5h with dry HCl in dioxane–Et2O or hydrog-
enolysis over Pd-C and subsequent acidic deprotection gave the
amine hydrochlorides 2h and 2f0 in 42–45% yield from 5a.
hydrochlorides 2e0 and 2g0, having cis-configuration, from the
ketoamide 4a in 28% and 32% yield, respectively.
2.4. Stereostructure and conformation of the synthesized
compounds
The conformation of the cycloheptene ring has already been
theoretically studied26 and the more stable conformation was cal-
culated to be the chair one as depicted in Figure 1 for 2a and its
derivatives. For all derivatives of 7-aminobenzocyclohepten-6-
one, as amines or amides, except for the methylene derivatives
2h, 5h and 4i, the amino group was in equatorial position by che-
lation with the ketone function. The axial Ha-8 proton was thus
strongly coupled with the axial H-7 proton (J = 12–14 Hz) and with
the axial Ha-9 proton (J = 9–13 Hz). A characteristic feature of
these seven-membered rings was important axial-axial and equa-
torial-equatorial couplings for the H-C(8) and H-C(9) protons
(J(8a,9a) = 9–13 Hz and J(8e,9e) = 6.7–9 Hz) related to a large (ca.
195°) and to a weak dihedral angle (ca. 45°) between the corre-
sponding protons, respectively.
The Mannich
a-methylenation of the ketoamide 4a was carried
out with formaldehyde and pyrrolidine in acetic acid to give the
reactive and unstable
a-methylene ketone 4i which was used
without further purification.24,25 Reaction of crude 4i with the cup-
rates [Ph(CH2)n]2CuMgBr (n = 1 and 2) gave the addition products
4e0 and 4g0 with moderate yield (ca. ꢁ40%). N-deprotection by
hydrogenolysis in the presence of HCl led to the final amine
The fluoro, thioamines and amides 2b–d, 4b and 5b–d were
characterized by an important deshielding, ca. 0.5–0.7 ppm, for

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
Ph
4945
Ph
O
O
OH
NHCbz
a (n = 2)
b
NHZ
NHCbz
NHBoc
4e Z = Cbz (79%)
11a
12e (82%)
c
2e Z = H,HCl (82%)
Ph
O
Ph
O
O
e
d
NH2,HCl
NHBoc
5a
5h (53%)
2h (79%)
f
Ph
O
Ph
O
e
NH2,HCl
NHBoc
2f' (86%)
5f' (quant.)
Ph
n
O
O
O
g
a
NHCbz
NHCbz
NHZ
4e' (n = 1) 42%, 4g' (n = 2) 43%, Z = Cbz
2e' (66%), 2g' (75%), Z = H,HCl
4a
4i
c
Scheme 5. Synthetic scheme for the aliphatic-substituted analogs. Reagents and conditions: (a) Ph(CH2)nMgBr, ½CuBrꢂMe2S, THF, ꢀ40 °C, 2 h; (b) DMP, CH2Cl2; (c) H2, 5% Pd-
C, dioxane, 1 N HCl, 40–50 °C, 1 d; (d) (1) LiHMDS, HMPA, ꢀ78 °C in THF; (2) benzaldehyde; (e) 1 N dry HCl/Et2O–dioxane, rt, 3 d; (f) H2, Pd-C, EtOH, rt, 1 h; (g) CH2O, AcOH,
pyrrolidine (cat), reflux, 5 h.
H
7NH
Ha
O
Ha
O
O
8
these compounds were similar. This cis-configuration was in agree-
ment with the weak J(6,7) value between the equatorial H-6 proton
and the axial H-7 and with a long-range W-coupling (J(6,8e) = 1.2–
1.5 Hz) with the equatorial He-8 (Fig. 2).
Ha
He
Ha
7NHZ
7NHZ
Ha
5R
5
8
8
Ha
Ha
Ha
R
9
9
9
Ha
Ha
In the case of enones 2h, 5h and 4i, which possess an exocyclic
double bond, a change in conformation is worth noting. The axial/
equatorial nomenclature was only a convention; indeed, Ha-8
being the proton strongly coupled with H-7, and Ha-9 the proton
strongly coupled with Ha-8. Then, a large coupling J(8e,9a) = ca.
13 Hz and a middle one J(8e,9e) = ca. 7 Hz were observed between
the He-8 proton and both H-9 protons. These values indicate a boat
conformation which would correspond to a better conjugation of
the exocyclic enone moiety and to an hydrogen bond between
the equatorial NH group and the CO(6) function. In these cases,
the calculated dihedral angles26 were large (148°) for (8e,9a) and
near to 90° (82°) for (8e,9e), while it was near to 90° (82°) for
(8a,9e).
trans-compounds
2b-e Z = H
cis-compounds
2d',e'-g' Z = H
2a
4b,e Z = CO2Bn
5b-d Z = Boc
4e',g' Z = CO2Bn
5f'
Z = Boc
Figure 1. Conformation of the ketone derivatives.
the axial protons H-7 and Ha-9 by comparison with the values of
the corresponding protons of the unsubstituted 2a, 4a and 5a. This
strong effect indicated that the electronegative substituent R was
in axial position (Fig. 1), which is spatially near to Ha-7 and Ha-
9. The effect of an alkyl group was weaker (ca. 0.1 ppm) for the
trans-phenethyl compounds 2e and 4e.
In the cis-alkylated compounds 2f0, 2g0, 4e0 and 4g0 no important
spatial effect were observed in 1H NMR. Therefore NOE experi-
ments were performed in the case of the phenethyl compound
4e0 and demonstrated the cis-configuration of these compounds:
by irradiation of H-5 at 3.94 ppm, NOE values of 6% and 11% were
observed for the axial Ha-9 at 3.05 ppm and H-7 at 4.56 ppm,
respectively. These effects agreed to a spatial proximity of these
protons, and consequently, to an axial Ha-5 and thus to an equato-
rial alkyl group in cis-position toward the amino group.
The observed trans-configuration of the thio substituents in
compounds 5c and 5d corresponded for the compounds 11a,b to
an epoxide ring in cis-position of the amino group. According to
the similar J values between H-7, H-8 and H-9 protons with those
of compounds 4a and 5a, we can consider that the conformation of
Z
7
H
N
Ha
5
O
H
Ha
NHZ
R
O
8
7
Ha
He
8
He
Ha
9
9
He
Ha
Ha
enones-compounds
11a Z = CO2Bn
11b Z = Boc
2h R = CHPh, Z = H
5h R = CHPh, Z = CO2tBu
4i R = CH2, Z = CO2Bn
Figure 2. Conformation of the epoxide derivatives 11a and 11b and exocyclic
enones compounds 2h, 5h and 4i.

4946
S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
Table 1
2.5. Properties of the ketoamines 2a–h
Inhibition of selected aminopeptidases by racemic 5-substituted 7-amino-5,7,8,9-
tetrahydrobenzocyclohepten-6-one hydrochlorides
It has already been reported12 that the ketoamine 2a exhibited
two structural properties in D2O solution: firstly, a spontaneous
partial hydration of the ketone function leading to a 80/20 mixture
of the ketone form and its hydrate; secondly, a very slow enoliza-
tion led to a deuteration of the 5-position at higher temperature
(60 °C).12 In CD3OD, the hydration occurred with concomitant for-
mation of two isomeric hemiacetals.
R
O
NH2,HCl
2a-h
Compounds
R
Ki (lM)
APN (EC
3.4.11.2)
LAPc (EC
3.4.11.1)
LTA4H (EC
3.3.2.6)
In this work, all ketoamines of type 2 appeared only in prepon-
derant ketone form (in CD3OD), but two exceptions were observed:
Bestatin
1
2a
trans-2b
trans-2c
trans-2d/ cis- –SPh
2d0
trans-2e
3.5a
0.5a
–H
–F
–SBn
0.0005a
120a
0.5a
>1000
>1000
>1000
40a
– Firstly, the fluoro derivative 2b appeared (in D2O) as a ketone/
hydrate mixture, the hydrate form being strongly preponderant
(85%) and characterized by a strongly shielded H-7 at 3.9 ppm, as
for the hydrate of 2a12, instead of 5 ppm for the ketone form.
– Secondly, in the case of the thiophenyl derivative 2d, the enoli-
zation into 2d00 occurred spontaneously at room temperature lead-
ing, in D2O, to a 60/40 mixture of 2d0-cis/ 2d-trans isomers in
equilibrium and to the instantaneous deuteration of the H-5 posi-
tion which did not appear in the 1H NMR spectrum. This equilibra-
tion was observed at room temperature neither for the trans-
thiobenzyl compound 2c, nor for the other trans compounds 2e,
4e, 5c and 5d.
1a
>1000
>1000
>100
0.3a
0.083a,b
0.06a,b
>100
>100
–
88a
22a
nd
470a
>100
(CH2)2Ph
–
(CH2)2Ph
–CH2Ph
–
cis-2e0
>100
cis-2f0
cis-2g0
13a
12a
>100
>100
>100
>100
(CH2)3Ph
@CHPh
2h
80a
nd
380a
Ki values (l
M) were determined (a) from Dixon plots27 for rapid equilibrium
kinetics, (b) from Morrison plot28 for slow binding inhibition. Inactive compounds
were tested up to their solubility limit under the assay conditions. See experimental
section. LAPc: cytosolic leucine aminopeptidase from bovine kidney (EC 3.4.11.1),
APN: porcine kidney aminopeptidase-N (EC 3.4.11.2) and LTA4H: human leukotri-
ene A4 hydrolase (EC 3.3.2.6). nd: not determined.
In the case of the trans-alkylated amine 2e, an isomerization
into the more stable cis-isomer was observed, but only in basic
medium at room temperature (with Na2CO3 in CD3OD or in D2O
at pH >7.4) for 24 h. and led quantitatively to the cis-isomer 2e0.
3. Aminopeptidases inhibition and discussion
for the inhibitory potency. 2e behaved as a reversible competitive
inhibitor in rapid equilibrium and was 1000 times less potent
All compounds were evaluated as racemic mixtures. All active
compounds behaved as competitive inhibitors of the panel amino-
peptidases. The inhibition constants (Ki) are reported in Table 1.
The aminobenzosuberone 2a was previously described as a selec-
tive competitive inhibitor of the ‘one zinc’ APN that belongs to
the M1 subfamily of metalloaminopeptidases, whereas bestatin is
much more potent on the ‘two zincs’ M17 family of enzymes.9–11
These two compounds were used here as reference inhibitors.
These newly synthesized inhibitors maintained the selectivity
profile towards mammalian APN. LTA4H was not inhibited to any
significant extent and most of them were totally inactive towards
(Ki = 80 lM) than 2c. The corresponding Dixon plot is reported in
Figure 4. Similar results were obtained for compounds 2e0-g0,
which rapidly reached the steady state under our experimental
conditions, with Ki values reported in Table 1.
3.1. Structure–activity relationships: main findings
3.1.1. Fluorine substitution
Compound 2b (Ki 0.3
slightly better than the parent amino-benzosuberone 2a (Ki
M) and exhibited an excellent selectivity profile. The fluorine
lM), substituted by a fluorine atom, was
1
l
LAPc, up to a concentration of 100
lM, except for compound 2c
atom might improve the affinity by favoring the hydrate form over
(Ki = 40 M). However, this latter compound exhibited an interest-
ing selectivity profile, being 500 times more active against APN
(Ki = 83 nM).
The most potent APN inhibitors, compounds 2c and 2d, behaved
as reversible slow binding inhibitors; the time dependent inhibition
is reported in Figure 3A. For this single-substrate reaction, the rate
equation was shown to be v = vs+(vo ꢀ vs)eꢀkt where v is the
observed velocity at any time, vo is the initial velocity of the reaction
and vs is the steady state velocity.29 The exponential decay constant
l
the ketone one, and/or by undergoing multipolar C–Fꢂ ꢂ ꢂH–N,
C–Fꢂ ꢂ ꢂC@O, and C–Fꢂ ꢂ ꢂH–C interactions with the enzyme back-
a
bone.31 The latter contributions were probably more significant,
as non-fluorinated ketoamines were already hydrated in aqueous
buffer.
Thus, the synthesis of 5-fluoro disubstituted analogs was not
pursued due to the weak improvement in binding affinity.
3.1.2. Aliphatic substitutions
k
could be determined for each individual concentration of
In general, aliphatic substitutions at the 5-position of the ali-
phatic ring did not result in affinity enhancement and led to disap-
pointing results. In particular, compounds 2e and 2e0-g0 having the
key phenyl moiety pharmacophore, connected to the aminobenzo-
suberone core through an aliphatic methylene chain spacer X-
(CH2)n-Y, exhibited a 12- to 88-fold loss in activity. However,
worth noting is an interesting trend in the activity profile related
inhibitor and the values of the association (kon) and dissociation
(koff) constants were derived from the plot of k as a function of [I]
( Fig. 3B).28 For compound 2c, kon = 1.2 ꢃ 103 Mꢀ1 sꢀ1 and
koff = 10ꢀ4 sꢀ1, which correspond to a Ki value of 83 nM. A very close
time dependent inhibition was observed with compound 2d and
similar values were calculated, with kon = 2 ꢃ 103 Mꢀ1 sꢀ1
,
koff = 1.2 ꢃ 10ꢀ4 sꢀ1 and a Ki value of 60 nM. For both compounds,
the observed kinetic constants are in the range of values generally
encountered for time dependant slow binding inhibition.30
to the cis/trans conformation: cis-2e0 (Ki 22
lM) displayed a better
inhibitory activity than its trans-equivalent trans-2e (Ki 88
l
M).
Moreover, cis-aliphatic compounds 2e0-g0, regardless of spacer
length, were better than the trans-derivative 2e, suggesting that
the cis-conformation, with the substituent at the 5-position in
The comparison of the kinetic profiles of compound 2c and its
analog 2e emphasized the importance of the sulfur atom in 2c

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
4947
Figure 3. Kinetic analysis for APN inhibition by compound 2c. (A) Progress curve in absence (vo) or presence of 2c at the reported
l
M concentrations. The reaction was started
by the addition of enzyme at a final concentration of 3 nM with Leucine-p-nitroanilide as the substrate (S = Km = 0.2 mM), in Tris buffer pH 7.5 at 30 °C.13 The steady state was
reached after 1.5 h (vs). (B) Plot of k as a function of I (n = 3): k = koff + (kon/(1 + S/Km) I.28 The value of koff is given by the vertical intercept, while the value of kon can be
calculated from the slope.
equatorial position, was the more favorable conformation, allow-
ing the best fit with the enzyme active site.
expected between the bulkier S-axial substituent and the protein
binding site. The equilibrium (observed for the trans-thiophenyl
2d and assumed for the trans-thiobenzyl 2c) between the axial
and equatorial S-substituent via enolization should favor the
cis-conformation which provides a better fit to the active site and
allows metal chelation by the sulfur atom. This equilibrium may
account for the observed time dependent, slow binding inhibition
by this APN inhibitor series. However, from a chemical standpoint,
the enol intermediates 2c00 or 2d00 cannot be ruled out as the
potential active form of these molecules.
3.1.3. Thio substitutions
Compounds 2c and 2d, substituted with a sulfide moiety,
proved 12 and 20 times more active against APN/CD13 than the
parent aminobenzosuberone 2a, with Ki values of 83 nM and
60 nM, respectively. Furthermore, these 5-axial-S-substituted
derivatives were 1000 times more potent than their carbon ana-
logs, which suggested a different mode of binding or new binding
interactions. The comparison of the activities of 2c/2e and 2d/2f0
demonstrated that the sulfide moiety was crucial for strong affinity
to APN. The strongest interaction that could possibly account for
the very large increase in potency of the sulfur containing deriva-
tives as compared to their carbon analogs would be zinc chelation.
Thiols and sulfides are potent zinc-chelating group and are known
to bind tightly to zinc-dependent metalloenzymes.32
3.2. Putative binding mode
To date, the three dimensional structure of a mammalian APN is
still lacking. Nevertheless, it is possible to propose a model (Fig. 5)
for the binding of our compounds, based on highly conserved res-
idues in the active site of the M1 family of enzymes and the SAR
discovered in the course of our studies. With the thio derivatives
2c and 2d, metal binding is likely to be mediated through the sulfur
atom and the ketone in its hydrated form. According to the previ-
ously postulated binding mode of the aminobensosuberone 2a
( Fig. 5A),12 thio derivatives would have to undergo a small
The high inhibitory activities (in the nM range) of these two
5-axial-S-substituted derivatives, together with the observed slow
binding kinetics, came as an intriguing result. As mentioned before,
in the carbon series, the trans-derivatives was the less active com-
pound. Hence, a poor fit, or even a steric clash might have been
Figure 4. Dixon plot27 of APN inhibition by compound 2e. See Section 5 (d).

4948
S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
Figure 5. Proposed mode of binding for the hydrate form of aminobenzosuberone 2a (A)12 and its analog 2d substituted with a thiophenyl moiety (B) based on zinc chelators
affinity and inhibition studies. (E. coli numbering). The binding pose of the thiophenyl inhibitor might be slightly shifted with the sulfide moiety getting close to the zinc ion,
leading to an overall rotation of the suberone, with the primary amine interacting solely with one carboxylate of the GAMEN site. The key phenyl pharmacophore would fit in
the S‘1 subsite. In both cases, the hydrate form of the ketone would be stabilized by Glu 298 and Tyr 381.
rotational shift of the scaffold in order to optimize zinc-chelation,
interactions with the conserved essential catalytic residues and
optimize contacts to the protein. This shift would govern the prop-
er positioning of the phenyl ring in the S01 pocket (Fig. 5B). For the
carbon analogs 2e–h, this binding would not be possible as it
would induce close unfavorable contacts of the carbon linker to
the zinc, thus hampering optimal binding of the phenyl ring into
S01. The primary amine, usually engaged in strong electrostatic
interactions with the side-chain carboxylate of two Glu residues
(‘GAMEN’ site), conserved in the one-zinc family of aminopepti-
dases and responsible for the recognition and binding of the free
amino terminus of peptidic substrates,1 may retain an interaction
with at least one carboxylic function. Taken together these effects
(proper binding of the phenyl ring, zinc chelation and partial inter-
action of the primary amine with the GAMEN site) may well ac-
count for the 1000-fold improvement in affinity observed with
the thio substituted compounds. The time dependent inhibition
observed with the thio derivatives may be a consequence of the
trans/cis equilibrium as well as of the required rotational shift fol-
lowing the primary binding event.
corrected. IR spectra (m a]D: Schmidt-
in cmꢀ1): Nicolet 405 FT-IR. [
Haensch Polartronic Universal or Perkin–Elmer 341 LC polarimeter.
1H and 13C NMR (400 MHz and 100.6 MHz resp.) spectra at 295 K:
Bruker Avance 400, tetramethylsilane (TMS), or sodium (D4)-trim-
ethylsilylpropionate (D4-TSP) in D2O (1H NMR), CDCl3, or (in D2O)
dioxane [d(CDCl3) = 77.0, in D2O d(dioxane) = 67.4 with respect to
TMS] (13C NMR) as internal references; d in ppm and J in Hz. High
resolution MS were measured in Spectroscopy Laboratory of Stras-
bourg University or on a Waters Micromass Q-Tof Ultima API spec-
trometer in Basilea Pharmaceuticals, Basel. Microanalyses were
carried out by the Service Central de Microanalyses du CNRS, F-
69390 Vernaison.
5.2. Reagents and solvents
Dess–Martin periodinane (DMP) was obtained as a 1 M solution
in CH2Cl2 from Aldrich or synthesized according to the literature.33
Commercial products were purchased from usual suppliers. Usual
solvents were freshly distilled, dry EtOH and MeOH distilled over
Mg/MgI2, dry THF over Na and benzophenone, dry Et2O was dis-
tilled and stored over Na, CH2Cl2 was distilled over P2O5 and kept
over Na2CO3. Dioxane, NEt3 were distilled before use. Hexamethyl-
phosphoramide (HMPA) and DMSO distilled and conserved over
molecular sieves 4 Å.
4. Conclusion
In summary, we have successfully devised a straightforward
synthesis allowing access to a new series of 7-aminobenzocyclo-
hepten-6-one derivatives 2b–h. To this end, we have explored func-
tionalization at the 5-position of the aliphatic ring of 2a with either
a fluorine atom, or a phenyl moiety attached to the core scaffold
through an aliphatic spacer or via a sulfur atom. The thio derivatives
2c and 2d, which show strong (60–83 nM) inhibitory activity, are
the first examples of a new chemotype of APN inhibitors, derived
from the aminobenzosuberone scaffold. In contrast to previously
reported derivatives, which are believed to be monodentate ligands
of the active site zinc ion, these new compounds are most probably
bidentate zinc binders. As our understanding of APN biology and
pathophysiological roles is still very limited, these new compounds
have potential as chemical biology tools for shedding light into the
biological functions of this enzyme, and as lead compounds for the
development of therapeutic APN inhibitors.
The preparation of compounds 3, 4a, 5a and 9 have been previ-
ously described and fully characterized in the literature.12,13
5.3. Novel chemical entities
5.3.1. trans-7-(Benzyloxycarbonylamino)-5-fluoro-6,7,8,9-
tetrahydrobenzocyclohepten-6-one (4b)
To a stirred solution of HMDS (0.3 mL, 1.42 mmol, 2.2 equiv) in
dry THF (1.6 mL) at ꢀ78 °C, was successively added BuLi (1.6 M in
hexane, 0.89 mL, 1.42 mmol, 2.2 equiv), and after 15 min 4a
(200 mg, 65 mmol) in dry THF (3 mL). After 45 min at ꢀ78 °C,
TMSCl (0.25 mL, 1.94 mmoles, 3 equiv) was added, and the solution
stirred further for 30 min. The solution was diluted with Et2O, the
organic phase washed with brine, dried (MgSO4) and evaporated to
give crude silyl enol ether 6a.
To a solution of crude 6a in dry CH3CN (10 mL) at ꢀ20 °C under
Ar, was added Selectfluor™ (275 mg, 0.78 mmol, 1.2 equiv) and the
solution stirred for 10 h at rt. Et2O was added and the organic solu-
tion washed with 2 N aqueous NH4Cl, and with brine, dried
(MgSO4) and evaporated. Crystallization in iPrOH and washing
with Et2O gave pure 4b (145 mg, 68%).
5. Experimental part
5.1. General
Flash chromatography (FC): silica gel (Merck 60, 230–400
mesh). TLC: Al-roll silica gel (Merck 60, F254). Mp: Kofler hot bench,

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
4949
Compound 4b: colorless crystals, mp 119–121 °C (iPrOH). IR
(KBr): 547,708, 720, 752, 757, 770, 982, 1012, 1019, 1229, 1328,
J(7,8b) = 12.0,
J(8a,8b) = 13.0,
J(8a,9b) = 6.7,
J(8b,9a) = 12.6,
J(9a,9b) = 15.0, J(9b,F) = 4.0 Hz. 13C NMR (CDCl3, 100 MHz): 28.5
(CMe3); 30.6 (d, J = 2.8 Hz, C-9); 37.3 (C-8); 58.0 (C-7); 80.1
(CMe3); 98.5 (d, J = 188.6 Hz, C-5); 127.4 (C-3); 130.8 (d,
J = 6.3 Hz, C-1); 130.9 (d, J = 3.5 Hz, C-2); 131.1 (d, J = 2.8 Hz, C-
4); 131.1 (C(4a)); 142.1 (C-9a); 154.1 CON-7; 204.3 (d,
J = 30.9 Hz, CO(6)). HR-MS (ESI-Q-Tof) calcd for C16H20FNO3 [M]+:
293.1427; found: 293.1405.
1380, 1452, 1456, 1494, 1525, 1709, 2949, 2975, 3029, 3068,
3394 cmꢀ1 1H NMR (CDCl3, 400 MHz): 7.23–7.07 (m, 9H, Har);
.
5.66 (d, 1H, NH); 5.48 (d, 1H, H-5); 5.22 (m, 1H, H-7); 5.02 (s,
2H, CH2Ph); 3.42 (t, 1H, Ha-9); 2.68 (m, 1H, Hb-9); 2.61 (m, 1H,
Ha-8); 1.48 (q, 1H, Hb-8). J(5,F) = 49.8, J(7,NH) = 7.2, J(7,F) = 5.4,
J(7,8a) = 5.8,
J(7,8b) = 11.5,
J(8a,8b) = 12.8,
J(8a,9b) = 7.0,
J(8b,9a) = 13.2, J(9a,9b) = 14.2, J(9b,F) = 4.0 Hz. 13C NMR (CDCl3,
100 MHz): 30.5 (d, J = 2.5 Hz, C-9); 37.2 (C-8); 58.3 (C-7);
67.1(CH2Ph); 98.4(d, J = 185.5 Hz; C(5)); 127.5 (C(3)); 128.3;
128.4; 128.7(Cp,m,o-Ph); 130.6 (C(4a)); 130.8 (d, J = 5.1 Hz, C(2));
131.0 (d, J = 2.6 Hz, C(1)); 131.1(d, J = 2.6 Hz, C(4)); 136.3 (Cs-Ph);
142.0 (C(9a)); 155.4; 203.9 (d, J = 31.9, C(6)). Anal. calcd for
Compound 7: colorless crystals, mp 114–116 °C (iPrOH). 1H
NMR (CDCl3, 400 MHz): 0.10–0.14 (2 s, 6H, SiMe2); 0.45 (s, 9H, Sit-
Bu); 1.99 (m, 1H, H-8 eq); 2.16 (q, 1H, H-8ax); 2.88 (dd, 1H, H-
9 eq); 2.98 (t, 1H, H-9ax); 4.50 (dt, 1H, H-7); 5.27 (s, 1H, NH);
5.43 (d, 1H, H-5); 7.22 (m, 4H, Har). J(5,F) = 49.4, J(7,F) = 3,2,
J(7,8ax) = 12,0, J(7,8 eq) = 3.2, J(8ax,8 eq) = 12.6, J(8ax,9ax) = 12.3,
J(8ax,9 eq) = 2.0, J(8 eq,9ax) = 3.2, J(8 eq,9 eq) = 5.2, J(9ax,9 -
eq) = 15.0 Hz. 13C NMR (CDCl3, 100 MHz): -3.1, -3.5 (SiMe2); 17.7
(SiCMe3); 25.2 (SiCMe3); 26.2 (C(9)); 33.9 (C(8)); 59.2 (d,
J = 4.2 Hz,C(7)); 94.1 (d, J = 182.1 Hz, C(5)); 104.1 (d, J = 26.0 Hz,
C(6)); 126.9 (d, J = 1.4 Hz, C(3)); 130.1 (d, J = 2.8 Hz, C(2)); 132.3
(d, J = 2.8 Hz, C(1)); 133.4 (d, J = 17.5 Hz, C(4a)); 133.6 (d,
J = 5.6 Hz, C(4)); 140.1 (C(9a)); 157.7 CO2N. HR-MS (ESI-Q-Tof)
calcd for C18H26FNO3Si [M]+: 351.1666, found: 351.1641.
C19H18FNO3 (327.36): C, 69.71; H, 5.54; N, 4.28; F, 5.80. Found: C,
70.0; H, 5.6; N, 4.2; F, 5.5.
5.3.2. 5-Methylene-7-(benzyloxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (4i)
A solution of 4a (300 mg, 0.97 mmol), formaldehyde 36% aq.
(225 lL, 2.7 mmol, 2.8 equiv) and pyrrolidine (50 lL, 0.6 mmole,
0.6 equiv) in AcOH (10 mL) was heated at 110 °C for 5 h. The reac-
tion was neutralized with aqueous 1 N NaOH, extracted with
AcOEt, the organic phase washed with aqueous 1 N NaHCO3, dried
(MgSO4) and evaporated to give crude 4i.
5.3.4. 5-Benzylidene-7-(tertiobutoxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (5h)
Compound 4i was used without further purification and only
To a solution of LiHMDS (preparated from BuLi 1.6 M (0.5 mL,
0.8 mmol, 2.2 equiv) and HMDS (0.17 mL, 0.8 mmol, 2.2 equiv for
15 min) in THF (1 mL) at ꢀ78 °C, was added a solution of 5a
(100 mg, 0.36 mmol) and HMPA (0.19 mL, 1.1 mmol, 3 equiv) in
THF (2 mL), then stirred 20 min at ꢀ78 °C. A solution of benzalde-
characterized with 1H NMR.
1H NMR (CDCl3, 400 MHz): 7.33–7.19 (m, 9H, Har); 6.52 (d, 1H,
Ha-10); 5.70 (d, 1H, Hb-10); 5.67 (d, 1H, NH); 5.04 (s, 2H, OCH2Ph);
4.58 (q, 1H, H-7); 2.83 (dt, 1H, Ha-9); 2.72 (dd, 1H, Hb-9); 2.61
(ddd, 1H, Ha-8); 1.76 (q, 1H, Hb-8). J(10a,10b) = 1.5; J(7,NH) = 7.6;
hyde (74 lL, 0.73 mmol, 2 equiv) in THF (2 mL) was then added
J(7,8a) = 8.4;
J(7,8b) = 10.0;
J(8a,8b) = 12.2;
J(8a,9a) = 13.4;
and the mixture stirred 1 h at ꢀ78 °C, then 2.5 h at rt. Water
(2 mL) was added and the solution extracted with AcOEt, the or-
ganic phase was washed with brine, dried (MgSO4) and evapo-
rated. Purification by FC (cyclohexane /AcOEt 8/2 to 7/3) gave
pure 5h (70 mg, 53%).
J(8a,9b) = 6.8; J(8b,9a) = 6.8; J(8b,9b) = 1.4; J(9a,9b) = 13.8 Hz.
5.3.3. trans-7-(tertioButyloxycarbonylamino)-5-fluoro-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (5b), and trans-5-fluoro-7-
(dimethyl-tertiobutyl)silyloxycarbonylaminobenzocyclo
hepten-6-one, hydrate (7)
Compound 5h: colorless crystals, mp 156–158 °C (iPrOH). IR
(KBr): 3356, 2976, 2933, 1709, 1691, 1602, 1530, 1364, 1251,
1179, 1169, 1046, 764, 754, 691 cmꢀ1. 1H NMR (CDCl3,
400 MHz): 7.95 (s, 1H, Har); 7.29–7.09 (m, 9H, Har); 5.49 (dl, 1H,
NH); 4.45 (ddd, 1H, H-7); 2.99 (ddd, 1H, Ha-9); 2.76 (ddd, 1H,
Hb-9); 2.56 (dddd, 1H, Ha-8); 1.74 (m, 1H, Hb-8); 1.41 (s, 9H,
CMe3). J(7,NH) = ca. 7.6, J(7,8a) = 8.0, J(7,8b) = 10.5, J(8a,8b) = 12.5,
J(8a,9a) = 13.1, J(8a,9b) = 7.5, J(8b,9a) = 7.6, J(8b, 9b) = 1.4,
J(9a,9b) = 13.7 Hz. 13C NMR (CDCl3, 100 MHz): 198.2 (CO(6));
155.1 (NCO2); 138.8 (Car); 138.6 (C(10), 136.2, 134.9, 134.7 (3
Car); 131.2 (Co(Ph)); 130.5, 129.6, 129.4, 129.0 (4 CHar); 128.4
(Cm(Ph)); 127.2 (Car); 79.7 (CMe3); 55.8 (C(7)); 34.1 (C(8)); 29.9
(C(9)); 28.5 (CMe3). Anal. calcd for C23H25NO3 (363.45): C, 76.01;
H, 6.93; N, 3.85. Found: C, 75.4; H, 7.1; N, 3.9. HR-MS (ESI-Q-Tof)
calcd for C23H25NO3 [M]+: 363.1834; found: 363.1846.
To a stirred solution of HMDS (0.34 mL, 1.6 mmol, 2.2 equiv) in
dry THF (1.6 mL) at ꢀ78 °C, was successively added BuLi (1.6 M in
hexane, 1.0 mL, 1.6 mmol, 2.2 equiv), and after 15 min, 5a (200 mg,
73 mmol) in mixture of dry THF (3 mL) and HMPA (0.38 mL,
2.18 mmol, 3 equiv). After 45 min at ꢀ78 °C, tBuMe2SiCl (263 mg,
1.74 mmol, 2.4 equiv) was added, and the solution stirred further
for 15 min at ꢀ78 °C. Aqueous 2 M NH4Cl (2 mL) and AcOEt were
then added, the organic phase separated and washed with brine,
dried (MgSO4) and evaporated to give crude silyl enol ether 6b.
To a solution of crude 6b in dry CH3CN (8 mL) and aqueous N
NaHCO3 (2 mL) at 0 °C under Ar, was added Selectfluor™
(309 mg, 0.87 mmol, 1.2 equiv) and the solution stirred for
35 min at rt. Et2O was added and the organic solution washed with
2 N aqueous NH4Cl, then with brine, dried (MgSO4) and evaporated
to give a mixture of 5b and 8. This crude mixture was stirred in THF
(10 mL) with TBAF (76 mg, 0.29 mmol, 0.4 equiv) at 0 °C for
10 min, then extracted with AcOEt, the organic phase was washed
with brine, dried (MgSO4) and evaporated. Purification by FC
(cyclohexane/ethyl acetate 8/2) gave pure 5b (110 mg, 52%).
Without NaHCO3, a mixture of 5b (25%) and 7 (16%) was ob-
tained. Compound 8 was neither isolated nor characterized.
Compound 5b: colorless crystals, mp 127–129 °C (iPrOH). IR
(KBr): 618, 760, 876, 975, 1011, 1061, 1171, 1248, 1293, 1327,
5.3.5. N-Benzyloxycarbonyl-6,7-dihydro-5H-benzocyclohepten-
7-amine (10a)
To a solution of 9 (1 g, 6.33 mmol) in a 2–3 M solution of NH3 in
EtOH (20 mL) was added Ti(iPrO)4 (3.8 mL, 12.7 mmol, 2 equiv)
and the solution stirred for 6 h at rt. NaBH4 (280 mg, 6.6 mmol,
1.1 equiv) was then added and the solution stirred for another
1 h. After evaporation of the solvent, AcOEt (20 mL) and 1 N aque-
ous NH4OH (20 mL) were added and the mixture stirred for 1 h.
The precipitate was filtered off, washed with AcOEt/1 N aqueous
NH4OH and the solutions extracted with AcOEt. The organic phases
were dried (MgSO4) and evaporated. The obtained amine was pro-
tected by stirring in THF (20 mL) with CbzCl (1.7 mL, 11.9 mmol,
1.8 equiv) and Na2CO3 (2.5 g, 23.9 mmol, 3.5 equiv) for 1 h. 10a
1365, 1492, 1526, 1709, 1725, 2857, 2942, 2981, 3375 cmꢀ1 1H
.
NMR (CDCl3, 400 MHz): 7.33–7.16 (m, 4H, Har); 5.58 (d, 1H, H-
5); 5.50 (d, 1H, NH); 5.27 (m, 1H, H-7); 3.50 (t, 1H, Ha-9); 2.78
(m, 1H, Hb-9); 2.67 (m, 1H, Ha-8); 1.54 (q, 1H, Hb-8); 1.46 (m,
9H, CMe3). J(5,F) = 49.6, J(7,NH) = 7.0, J(7,F) = 5.7, J(7,8a) = 5.7,

4950
S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
(1.2 g, 80%) was precipitated with H2O, isolated by filtration,
washed with iPr2O and dried.
J(6,7) = 2.7, J(6,8a) = 1.3, J(NH,7) = 9.0, J(7,8a) = 4.4, J(7,8b) = 10.6,
J(8a,8b) = 13.6,
J(8a,9a) = 8.8,
J(8a,9b) = 1.5,
J(8b,9a) = 1.5,
Compound 10a: colorless crystals, mp 135–137 °C (iPrOH). IR
(KBr): 3320, 1685, 1534, 1304, 1248, 1048, 1019, 785, 743,
J(8b,9b) = 10.2, J(9a,9b) = 15.5 Hz. 13C NMR (CDCl3, 100 MHz):
155.3 (NCO2); 141.8, 132.4, (C(4a),C(9a)); 134.0, 130.0, 129.0,
126.6 (4 CHar); 79.9 (CMe3); 61.6, 60.4 (C(5),C(6)); 51.8 (C(7));
31.7 (C(8)); 30.8 (C(9)); 28.5 (CMe3). Anal. calcd for C16H21NO3
(275.34): C, 69.79; H, 7.69; N, 5.09. Found: C, 69.9; H, 7.9; N, 5.1.
697 cmꢀ1 1H NMR (CDCl3, 400 MHz): 7.38–7.34 (m, 5H, Har);
.
7.23–7.14 (m, 4H, Har); 6.46 (d, 1H, H-9); 5.76 (dd, 1H, H-8);
5.12 (s, 2H, CH2Ph); 4.90 (broad d, 1H, NH); 4.58 (broad s, 1H, H-
7); 2.86 (m, 1H, Ha-5); 2.74 (m, 1H, Hb-5); 2.06 (m, 2H, CH2(6)).
J(NH,7) = ca. 8, J(7,8) = 4.0, J(8,9) = 12.2 Hz.
5.4. Addition reactions—procedure A
5.3.6. N-tertiobutoxycarbonyl-6,7-dihydro-5H-benzocyclo
hepten-7-amine (10b)
A solution of BnMgBr (2.85 mL, 1.5 M dans Et2O, 4.27 mmol)
was added at ꢀ50 °C to a suspension of CuBr.SMe2 (440 mg,
2.13 mmol) in anhydrous THF (20 mL) under Ar, then stirred at
ꢀ50 °C for 45 min. To this suspension at ꢀ40 °C, was added drop-
wise a solution of crude epoxide 11a,b or enone 4i (1 mmol) in
anhydrous THF (10 mL) and the solution further stirred for 2 h at
ꢀ40 °C. After addition of 2 N aqueous NH4Cl solution, the mixture
was extracted with AcOEt, the organic phases washed with brine,
dried (MgSO4) and evaporated.
Same procedure as for 10a with 9 (0.50 g, 3.15 mmol). The
crude amine was protected in MeOH (6 mL) with Na2CO3
(370 mg, 3.48 mmol) and Boc2O (1.4 g, 6.33 mmol) with stirring
for 2 h at rt. 10b was isolated as above (0.70 mg, 76%).
Compound 10b: colorless crystals, mp 146–148 °C (iPrOH). IR
(KBr): 3361, 1682, 1517, 1245, 1168, 1156, 1005, 787, 747 cmꢀ1
.
1H NMR (CDCl3, 400 MHz): 7.18–7.11 (m, 4 Har); 6.44 (dd, 1H,
H-9); 5.76 (dd, 1H, H-8); 4.69 (d, 1H, NH); 4.49 (broad s, 1H, H-
7); 2.85 (m, 1H, Ha-5); 2.73 (m, 1H, Hb-5); 2.04 (m, 2H, H-6);
1.46 (s, 9H, CMe3). J(7,8) = 4.0 Hz, J(7,9) = 1.9 Hz, J(8,9) = 12.3 Hz.
13C NMR (CDCl3, 100 MHz): 155.0 (NCO2); 141.8, 134.8, 131.6,
131.4, 131.0, 129.1, 127.5, 126.3 (8 Car); 79.6 (CMe3); 51.5
(C(7)); 33.1 (C(6)); 31.5 (C(5)); 28.5 (CMe3). Anal. calcd for
5.4.1. trans-5-Phenylethyl-7-(benzyloxycarbonylamino)-
5,7,8,9-tetrahydrobenzocyclohepten-6-one (4e)
Procedure
A with BnCH2MgBr (1.7 mL, 1.3 M dans Et2O,
2.13 mmol), CuBr.SMe2 (220 mg, 11.1 mmol) in anhydrous THF
(10 mL) and epoxyde 11a (105 mg, 0.34 mmol) in anhydrous THF
(10 mL). Purification by FC (Chx/AcOEt, 9/1 then 8/2) gave crude
12e (115 mg, 82%). 12e was directly oxidized according to the Pro-
cedure B with DMP (0.2 g, 0.48 mmol, 1.4 equiv) to give 4e (90 mg,
79%).
C16H21NO2 (259.34): C, 74.10; H, 8.16; N, 5.40. Found: C, 74.2; H,
8.3; N, 5.5.
5.3.7. cis-N-Benzyloxycarbonyl-5,6-epoxy-6,7,8,9-tetrahydro-
5H-benzocyclohepten-7-amine (11a)
Compound 4e: colorless resin. IR (KBr): 3417, 3062, 3028, 2929,
To a solution of 10a (640 mg, 2.18 mmol) in CH2Cl2 (40 mL) was
added m-CPBA (860 mg, 5.0 mmol, 2.2 equiv) and the solution stir-
red at 0 °C for 16 h. Aqueous solution of Na2S2O3ꢂ5H2O (2.5 g,
10 mmol) in 1 N NaHCO3 (10 mL) was added and the mixture vig-
orously stirred for 45 min at rt, then extracted with Et2O, the or-
ganic phase washed with 1 N NaHCO3 then with brine, dried
(MgSO4) and evaporated to give 11a (610 mg, 90%).
2859, 1703, 1497, 1454, 1246, 749, 698 cmꢀ1 1H NMR (CDCl3,
.
400 MHz): 7.33–7.11 (m, 14H, Har); 5.60 (sl, 1H, NH); 5.09 (s,
2H, OCH2Ph); 4.68 (ddd, 1H, H-7); 3.75 (t, 1H, H-5); 3.17 (ddd,
1H, Ha-9); 2.88 (ddd, 1H, Hb-9); 2.64 (t, 2H, H-20); 2.56 (m, 1H,
Ha-8); 2.52 (dt, 1H, Ha-10); 2.27 (dt, 1H, Hb-10); 1.58 (dddd, Hb-
8). J(5,10a) = J(5,10b) = 7.5, J(10a,20) = J(10b,20) = 7.7, J(10a,10b) = 14.0,
J(7,NH) = 6.5,
J(8a,9a) = 3.6,
J(7,8a) = 6.5,
J(8a,9b) = 8.2,
J(7,8b) = 11.0,
J(8b,9a) = 9.5,
J(8a,8b) = 13.4,
J(8b,9b) = 3.7,
Compound 11a: colorless crystals, mp 140–141 °C (EtOH). IR
(KBr): 3336, 2941, 1688, 1537, 1315, 1250, 1041, 1016, 902, 754,
J(9a,9b) = 15.2 Hz. 13C NMR (CDCl3, 100 MHz): 207.3 (C(6)); 155.6
(NCO2); 140.9, 139.8, 136.5, 135.6 (4 Car); 130.9, 129.9, 128.7,
128.6, 128.3, 128.0, 127.6, 126.4 (Car); 67.0 (OCH2Ph); 59.2
(C(5)); 58.9 (C(7)); 34.5 (C(8)); 33.6 (C(20)); 32.2 (C(10)); 31.3
729, 699 cmꢀ1 1H NMR (CDCl3, 400 MHz): 7.51 (m, 1H, Har);
.
7,35 (m, 6H, Har); 7.24 (m, 1H, Har); 7.09 (m, 1H, Har); 5.22 (d,
1H, NH); 5.14 (s, 2H, CH2Ph); 4.44 (dddd, 1H, H-7); 4.01 (d, 1H,
H-5); 3.69 (m, 1H, H-6); 2.84 (ddd, 1H, Ha-9); 2.65 (dd, 1H, Hb-
9); 1.98 (m, Ha-8); 1.72 (ddt, Hb-8). J(5,6) = 4.2, J(6,7) = 2.8,
(C(9)). HR-MS (ESI-Q-Tof) calcd for
420.2151; found: 420.2160.
C
27H27LiNO3 [M+Li]+:
J(6,8) = 1.2,
J(8a,8b) = 13.6,
J(7,8a) = 4.5,
J(8a,9a) = 8.8,
J(7,8b) = 10.4,
J(8a,9b) = 1.3,
J(NH,7) = 9.2,
J(8b,9a) = 1.7,
J(8b,9b) = 10.6, J(9a,9b) = 15.6 Hz. 13C NMR (CDCl3, 100 MHz):
155.6 (NCO2); 141.5 (Cs(Bn)); 136.3 (C(9a)); 133.8 (CHar); 132.1
(C(4a)); 129.8, 128.9, 128.5, 128.1, 128.0, 126.4 (6 CHar); 66.8
(CH2Ph); 61.2, 60.2 (C(5), C(6)); 52.1 (C(7)); 31.3 (C(8)); 30.6
(C(9)). Anal. calcd for C19H19NO3 (309.36): C, 73.77; H, 6.19; N,
4.53. Found: C, 73.9; H, 6.2; N, 4.5.
5.4.2. cis-5-Phenylethyl-7-(benzyloxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (4e0)
Procedure A with BnMgBr (2.85 mL, 1.5–1.6 M dans Et2O,
4.27 mmol), CuBr.SMe2 (440 mg, 2.13 mmol) in anhydrous THF
(20 mL), crude 4i (ca. 0.32 g, 0.97 mmol) in anhydrous THF
(10 mL). Purification by FC (Chx/AcOEt, 9/1 then 8/2) gave 4e0
(170 mg, 42%).
Compound 4e0: colorless crystals, mp 136–138 °C (iPrOH). IR
(KBr): 3418, 3331, 3031, 2945, 2856, 1715, 1680, 1528, 1249,
5.3.8. cis-N-tert Butoxycarbonyl-5,6-epoxy-6,7,8,9-tetrahydro-5H
-benzocyclohepten-7-amine (11b)
To a solution of 10b (400 mg, 1.54 mmol) in CH2Cl2 (20 mL) was
added m-CPBA (610 mg, 2.46 mmol, 1.6 equiv) and the solution
stirred at 0 °C for 16 h. Same work-up as above gave 11b
(425 mg, quant.).
749, 696 cmꢀ1 1H NMR (CDCl3, 400 MHz): 7.36–7.11 (m, 14H,
.
Har); 5.65 (d, 1H, NH); 5.10 (s, 2H, OCH2Ph); 4.56 (m, 1H, H-7);
3.94 (t, 1H, H-5); 3.95 (dd, 1H, Ha-9); 2.84 (dd, 1H, Hb-9); 2.70–
2.60 (m, 4H, Ha-10,Ha-8, CH2(20)); 2.10 (m, 1H, Hb-10); 1.42 (m,
Hb-8). J(NH,7) = 7.0, J(7,8a) = 7.2, J(7,8b) = 11.0, J(8a,8b) = 13.0,
Compound 11b: colorless crystals, mp 170–172 °C (iPrOH). IR
(KBr): 3359, 2980, 2968, 2934, 1687, 1519, 1390, 1370, 1314,
J(8a,9a) = 2.8,
J(8a,9b) = 8.6,
J(8b,9a) = 9.6,
J(8b,9b) = 3.2,
J(9a,9b) = 14.6 Hz. 13C NMR (CDCl3, 100 MHz): 206.9 (C(6)); 155.5
(NCO2); 141.3, 140.5, 136.5, 135.3 (4 Car); 129.5, 128.7, 128.6,
128.3, 128.2, 127.7, 126.7, 126.3 (CHar); 67.0 (OCH2Ph); 62.3
(C(7)); 52.9 (C(5)); 36.3 (C(8)); 33.7 (C(20)); 31.0 (C(9)); 30.1
(C(10)). Anal. calcd for C27H27NO3 (413.51): C, 78.42; H, 6.58; N,
.
1250, 1169, 1158, 1002, 753, 614 cmꢀ1 1H NMR (CDCl3,
400 MHz): 7.50 (m, 1H, Har); 7.23 (m, 2H, Har); 7.08 (m, 1H,
Har); 4.98 (d, 1H, NH); 4.36 (m, 1H, H-7); 3.99 (d, 1H, H-5); 3.68
(dl, 1H, H-6); 2.84 (dd, 1H, Ha-9); 2.63 (dd, 1H, Hb-9); 1.95 (m,
1H, Ha-8); 1.69 (m, 1H, Hb-8); 1.47 (s, 9H, CMe3). J(5,6) = 4.2,

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
4951
3.39. Found: C, 78.2; H, 6.7; N, 3.2. HR-MS (ESI-Q-Tof) calcd for
27H27NNaO3 [M+Na]+: 436.1883; found: 436.1895.
(Cs(Ph)); 134.8, 134.6 (C(4a),C(9a)); 133.7, 132.3, 130.6, 129.1,
128.7, 128.1, 126.6 (7 CHar); 79.5 (CMe3); 72.9 (C(6)); 59.6
(C(5)); 52.6 (C(7)); 32.1 (C(9)); 28.6 (C(9),CMe3). HR-MS (ESI-
microTof) calcd for
408.1590.
C
5.4.3. cis-5-Phenylpropyl-7-(benzyloxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (4g0)
C
22H27NO3S [M+Na]+: 408.1604; found:
Procedure A with BnCH2CH2MgBr (3.1 mL, 1.3 M dans Et2O,
4.27 mmol), CuBrꢂSMe2 (440 mg, 2.13 mmol) in anhydrous THF
(20 mL), crude 4i (ca. 0.32 g, 0.97 mmol) in anhydrous THF
(10 mL). Purification by FC (Cyclohexane/AcOEt, 9/1 then 8/2) gave
4g0 (182 mg, 43%).
5.5. Oxidation of alcohol—procedure B
To a solution of amido-alcohol 12 (1 mmol) in 40 mL wet CH2Cl2
(10 mL) under Ar was added Dess-Martin periodinane (DMP)
((0.6 g, 1.4 mmol, 1.4 equiv) and the mixture stirred at rt for 2 h.
AcOEt (10 mL) and a solution of Na2S2O3ꢂ5H2O (4 mmol) in aque-
ous 1 N NaHCO3 (10 mL) were added and the mixture was vigor-
ously stirred for 1 h, then extracted with AcOEt, the organic
solutions washed with aqueous 1 N NaHCO3 then with brine, dried
(MgSO4) and evaporated.
Compound 4g0: colorless crystals, mp 106–108 °C (iPrOH). IR
(KBr): 3354, 3330, 3026, 2941, 2854, 1717, 1682, 1525, 1453,
1365, 1244, 751, 729, 695 cmꢀ1 1H NMR (CDCl3, 400 MHz):
.
7.34–7.11 (m, 14H, Har); 5.68 (d, 1H, NH); 5.08 (s, 2H, OCH2Ph);
4.58 (m, 1H, H-7); 3.91 (t, 1H, H-5); 3.06 (dd, 1H, Ha-9); 2.84
(dd, 1H, Hb-9); 2.74–2.60 (m, 3H, Ha-8, CH2(30)); 2.36 (m, 1H,
Ha-10); 1.83 (m, 1H, Hb-10), 1.63 (m, 2H, CH2(20)), 1.43 (m, 1H,
Hb-8).
J(10a,10b) = 13.0,
J(10,20) = J(20,30) = 7.7,
J(10a,5) = 6.2,
J(7,8b) = 11.2,
J(8b,9a) = 10.0,
5.5.1. trans-5-Benzylthio-7-(tertiobutoxycarbonylamino)-
5,7,8,9-tetrahydrobenzocyclohepten-6-one (5c)
J(10b,5) = 7.7,
J(NH,7) = 7.0,
J(8a,9a) = 2.5,
J(7,8a) = 7.0,
J(8a,9b) = 8.4,
J(8a,8b) = 12.6,
Procedure B with 12c (40 mg, 0.1 mmol) and DMP (47 mg,
0.11 mmol) in wet CH2Cl2 (5 mL) to give 5c (40 mg, quant.).
Compound 5c: colorless resin. 1H NMR (CDCl3, 400 MHz): 7.30–
7.03 (m, 9H, Har); 5.32 (m, 2H, NH, H-C(7)); 4.40 (s, 1H, H-5); 3.68–
3.70 (2 d, J = 13.7 Hz, 2H, SCH2Ph); 3.50 (dd, 1H, Ha-9); 2.71 (ddd,
1H, Hb-9); 2.52 (m, 1H, Ha-8); 1.38 (m, 1H, Hb-8); 1.40 (s, 9H,
J(8b,9b) = 2.8, J(9a,9b) = 14.8 Hz. 13C NMR (CDCl3, 100 MHz):
207.2 (C(6)); 155.4 (NCO2); 141.9, 140.2, 136.4, 135.3 (4 Car);
129.4, 128.6, 128.5, 128.4, 128.2, 128.1, 127.6, 127.5, 126.9, 125.9
(10 CHar); 66.9 (OCH2Ph); 61.9 (C(7)); 54.0 (C(5)); 36.2, 36.1
(C(8),C(30)); 30.8 (C(9)); 29.4 (C(20)); 28.1 (C(10)). Anal. calcd for
C28H29NO3 (427.53): C, 78.66; H, 6.84; N, 3.28. Found: C, 78.8; H,
CMe3).
J(8a,9b) = 7.8,
J(8b,9a) = 11.2,
J(8b,9b) = 1.8,
6.9; N, 3.1. HR-MS (ESI-microTof) calcd for
C28H29NNaO3
J(9a,9b) = 15.0 Hz. 13C NMR (CDCl3, 100 MHz): 204.4 (C(6)); 155.1
(NCO2); 141.1 (C(9a)); 136.8 (Cs(Ph)), 132.6 (C(4a)); 131.0, 130.7,
129.3, 128.8, 128.7, 127.5, 127.4 (7 Car); 79.8 (CMe3); 60.2
(C(5)); 57.4 (C(7)); 36.9 (CH2Ph)); 35.3 (C(8)); 31.5 (C(9)); 28.5
(CMe3). HR-MS (ESI-microTof) calcd for C23H27NO3S [M]+:
397.1712; found: 397.1703.
[M+Na]+: 450.2040; found: 450.2048.
5.4.4. r-5-Benzylthio-t-7-(tertiobutoxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-t-6-ol (12c)
A solution of 11c (52 mg, 0.19 mmol), Et3N (63
lL, 0.45 mmol,
2.4 equiv) and BnSH (30 L, 0.23 mmol, 1.2 equiv) in EtOH (1 mL)
l
was stirred at rt for 1 h. 12c was precipitated and washed with
H2O, then dried (60 mg, 80%).
5.5.2. trans-5-Phenylthio-7-(tertiobutoxycarbonylamino)-
5,7,8,9-tetrahydrobenzocyclohepten-6-one (5d)
Procedure B with 12d (230 mg, 0.60 mmol) and DMP (280 mg,
0.66 mmol) in wet CH2Cl2 (10 mL) to give 5d (190 mg, 83%), after
recristallisation in iPrOH.
Compound 12c: colorless resin. RMN 1H (CDCl3, 400 MHz): 7.32–
7.15 (m, 6H, Har); 7.15 (t, J = 7.3 Hz, 2H, Har); 6.98 (d, J = 7.3 Hz,
1H, Har); 4.99 (d large, 1H, NH); 4.22 (large s, 1H, H-7); 4.09 (t
large, 1H, H-6); 3.94 (d, 1H, H-5); 3.69 (d, J = 13.8 Hz, 1H, Ha(SBn));
3.57 (d, J = 13.8 Hz, 1H, Hb(SBn); 3.38 (t l, 1H, Hax-9); 2.67 (dd, 1H,
Heq-9); 2.00 (m, 1H, Heq-8); 1.46 (m, 1H, Hax-8), 1.45 (s, 9H,
CMe3). J(5,6) = 6.0, J(6,7) = 8.4, J(NH,7) = ca. 7.2, J(8 eq,9 eq) = 6.6,
J(8ax,9 eq) = 1,6, J(8ax,9ax) = 12.2, J(9ax,9 eq) = 14.6 Hz. RMN 13C
(CDCl3, 100 MHz): 155.0 (NCO2); 142.5 (Cs(Ph)); 137.7, 134.8
(C(4a), C(9a)); 132.2, 130.9, 129.2, 128.7, 128.5, 127.3, 126.4 (7
CHar); 79.4 (CMe3); 72.7 (C(6)); 53.0, (C(7), C(5)); 36.3 (SCH2Ph);
31.9 (C(9)); 28.6 (C(8), CMe3). HR-MS (ESI-microTof) calcd for
Compound 5d: colorless crystals, mp 121–123 °C (iPrOH). IR
(KBr): 3428, 3369, 2975, 2927, 1693, 1493, 1484, 1366, 1169,
754, 737 cmꢀ1 1H NMR (CDCl3, 400 MHz, 328 K): 7.38 (m, 2H,
.
Har); 7.30–7.09 (m, 7H, Har); 5.47 (sl, 1H, H-7); 5.41 (m, 1H, H-
7); 4.97 (s, 1H, H-5); 3.67 (tl, 1H, Ha-9); 2.86 (ddd, 1H, Hb-9);
2.66 (m, 1H, Ha-8); 1.53 (m, 1H, Hb-8); 1.45 (s, 9H, CMe3).
J(7,NH) = ca. 6.0, J(7,8a) = 6.2, J(7,8b) = 11.4, J(8a,8b) = 13.2,
J(8a,9a) = 1.6,
J(8a,9b) = 7.6,
J(8b,9a) = 11.6,
J(8b,9b) = 1.8,
J(9a,9b) = 15.2 Hz. 13C NMR (CDCl3, 100 MHz): 204.0 (C(6)); 155.0
(NCO2); 141.4, 133.4, 132.2 (3 Car); 132.4, 131.18, 131.16, 129.5,
129.2, 128.3, 127.6 (7 CHar); 79.8 (CMe3); 65.3 (C(5)); 58.0
(C(7)); 35.6 (C(8)); 31.7 (C(9)); 28.5 (CMe3). HR-MS (ESI-microTof)
calcd for C22H25NaNO3S [M+Na]+: 406.1453; found: 406.1420. For
C22H25LiNO3S [M+Li]+: 390.1715; found: 390.1684.
C
23H29NO3S [M]+: 399.1868; found: 399.1871.
5.4.5. r-5-Phenylthio-t-7-(tertiobutoxycarbonylamino)-5,7,8,9-
tetrahydrobenzocyclohepten-t-6-ol (12d)
A
solution of 11b (225 mg, 0.82 mmol), Et3N (0.275 mL,
1.96 mmol) and PhSH (100 L, 0.98 mmol) in EtOH (15 mL) was
l
stirred at rt for 1 h. 12d was precipitated with H2O, filtrated and
5.5.3. N-deprotection
washed with iPr2O to give pure product (270 mg, 86%).
Procedure C: A solution of 5a–d,h (1 mmol) in 2.2 N dry HCl in
Et2O (2 mL) and dry dioxane (2 mL) was stirred under Ar at rt for
3 d. The precipitated hydrochloride was isolated by filtration or
centrifugation, washed with dry Et2O and recrystallized in iPrOH/
Et2O.
Compound 12d: colorless crystals, mp 174–176 °C (iPr2O). IR
(KBr): 3395, 3356, 2975, 2935, 1674, 1506, 1566, 1240, 1171,
746 cmꢀ1 1H NMR (CDCl3, 400 MHz): 7.37 (m, 2H, Har); 7.25–
.
7.14 (m, 5H, Har); 7.08 (t, J = 7,5 Hz, 1H, Har); 6.97 (d, J = 7.0 Hz,
1H, Har); 5.06 (d, 1H, NH); 4.41 (s large, 1H, H-7), 4.39 (d, 1H, H-
5); 4.23 (t, 1H, H-6); 3.38 (t, 1H, Hax-9); 2.74 (dd, 1H, Heq-9);
2.07 (m, 1H, Heq-8); 1.55 (q, 1H, Hax-8); 1.46 (s, 9H, CMe3).
J(5,6) = 5.8, J(6,7) = 9.0, J(NH,7) = ca. 8.0, J(7,8ax) = J(8ax,9ax) = ca.
12.6, J(8ax,9ax) = 12.0, J(8ax,9 eq) = 1.6, J(8 eq,9 eq) = 6.8, J(9ax,9 -
eq) = 14.8 Hz. 13C NMR (CDCl3, 100 MHz): 155.1 (NCO2); 142.2
Procedure D:
A
solution of N–CO2Bn aminocetone 4e,e0,g0
(1 mmol) was hydrogenolysed over Pd-C 5% (20 mg) in dioxane
(5–20 mL) and aqueous 1 N HCl (1.1 mL, 1.1 mmol) at 40 °C for
24 h. The catalyst was centrifuged off, the solvent evaporated.
Crude 2e,e0,g0 were recrystallized in iPrOH/Et2O.

4952
S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
5.5.4. trans-7-Amino-5-fluoro-5,7,8,9-
(C(7) 2d); 32.3 (C(8) 2d0); 32.2 (C(8) 2d); 30.4 (C(9) 2d); 28.8
(C(9) 2d0).
tetrahydrobenzocyclohepten-6-one, hydrochloride (2b)
5b (19 mg, 0.65 mmol) in solution in EtOH (1 mL) and aqueous
6 N HCl (0.11 mL) was stirred at rt for 12 h. After evaporation of the
solvent, recrystallization in iPrOH/Et2O gave 2b (11 mg, 74%).
Compounds 2b/2b0: colorless crystals, mp >150 °C (dec) (iPrOH/
Et2O). IR (KBr): 408, 427, 448, 451, 620, 769, 797, 874, 955, 994,
1052, 1194, 1217, 1456, 1503, 1574, 1737, 2886, 2914, 3010,
5.5.8. trans-5-Phenylethyl-7-amino-5,7,8,9-
tetrahydrobenzocyclohepten-6-one, hydrochloride (2e)
Procedure D from 4e (85 mg, 0.21 mmol) in dioxane (1 mL) to
give the hydrochloride 2e (53 mg, 82%).
Compound 2e: colorless crystals, mp 190–192 °C (iPrOH/Et2O).
IR (KBr): 3440, 2935, 2862, 1708, 1497, 1491, 1459, 1452, 761,
3058, 3336, 3386, 3403 cmꢀ1
.
697 cmꢀ1 1H NMR (CD3OD, 400 MHz): 7.30–7.16 (m, 9H, Har);
.
5.5.5. 1H NMR (D2O, 85/15 mixture of hydrate 2b0 and ketone 2b)
Hydrate 2b0: 7.44–7.28 (m, 4H, Har); 5.48 (d, 1H, H-5); 3.90 (dt,
1H, H-7); 3.13 (m, 1H, Ha-9); 2.86 (m, 1H, Hb-9); 2.17 (m, 1H, Ha-
8); 1.77 (q, 1H, Hb-8). J(5,F) = 45.3, J(7,F) = 2.4, J(7,8a) = 3.6,
4.41 (dd, 1H, H-7); 3.82 (dd, 1H, H-5); 3.35 (ddd, 1H, Ha-9); 3.06
(ddd, 1H, Hb-9); 2.65 (t, 2H, CH2(20)); 2.54 (m, 1H, Ha-10); 2.46
(m, 1H, Ha-8); 2.32 (m, Hb-10); 1.80 (m, Hb-8). J(10a,10b) = 13.2,
J(10a,20) = J(10b,20) = 7.8, J(10a,5) = 6.7, J(10b,5) = 8.7, J(7,8a) = 6.1,
J(7,8b) = 12.3,
J(8a,8b) = 13.6,
J(8a,9a) = 1.6,
J(8a,9b) = 7.4,
J(7,8b) = 12.4,
J(8a,8b) = 12.8,
J(8a,9a) = 4.2,
J(8a,9b) = 7.8,
J(8b,9a) = 12.4, J(8b,9b) = 2.0, J(9a,9b) = 15.2, J(9b,F) = 2.0 Hz.
Ketone 2b: 7.44–7.28 (m, 4H, Har); 5.90 (d, 1H, H-5); 5.09 (m,
1H, H-7); 3.53 (m, 1H, Ha-9); 2.99 (m, 1H, Hb-9); 2.66 (m, 1H,
Ha-8); 1.88 (q, 1H, Hb-8). J(5,F) = 48.6, J(7,F) = 4.2, J(7,8a) = 6.0,
J(8b,9a) = 9.4, J(8b,9b) = 4.2, J(9a,9b) = 15.4 Hz. 13C NMR (CD3OD,
100 MHz): 205.5 (C(6)); 142.1 (Cs(Ph)); 140.4, 136.2 (C(4a),
C(9a)); 131.9, 130.8, 129.6, 129.5, 129.3, 128.8, 127.3 (9 Car);
60.0 (C(5)); 58.7 (C(7)); 34.3 (C(20)); 32.5 (C(10)); 32.3 (C(8));
31.1 (C(9)). HR-MS (ESI-microTof) calcd for C19H22NO [M+H]+:
280.1696; found: 280.1664.
J(7,8b) = 11.8,
J(8a,8b) = 13.2,
J(8a,9a) = 1.8,
J(8a,9b) = 6.4,
J(8b,9a) = 12.2, J(8b,9b) = ca. 3, J(9a,9b) = 15.0, J(9b,F) = 3.0 Hz.
HR-MS (ESI-microTof) calcd for C11H12FNO [M]+: 193.0903,
found: 193.0901; for the hydrate C11H14FNO2 [M]+: 211.1009,
found: 211.1032.
5.5.9. cis-5-Phenylethyl-7-amino-5,7,8,9-
tetrahydrobenzocyclohepten-6-one, hydrochloride (2e0)
Procedure D with 4e0 (140 mg, 0.34 mmol) in dioxane (5 mL) to
give the hydrochloride 2e0 (87 mg, 81%).
5.5.6. trans-7-Amino-5-benzylthio-5,7,8,9-
Compound 2e0: colorless crystals, mp 215–218 °C (iPrOH/Et2O).
IR (KBr): 3428, 3023, 2939, 2910, 1723, 1522, 1491, 1454, 749,
tetrahydrobenzocyclohepten-6-one, hydrochloride (2c)
Procedure C from 5c (40 mg, 0.1 mmol) was obtained the
hydrochloride 2c (20 mg, 66%).
698 cmꢀ1 1H NMR (CD3OD, 400 MHz): 7.30–7.20 (m, 9H, Har);
.
Compound 2c: yellowish crystals, mp 176–180 °C (iPrOH/Et2O).
4.35 (dd, 1H, H-7); 4.20 (m, 1H, H-5); 3.26 (m, 1H, Ha-9); 2.98
(ddd, 1H, Hb-9); 2.70–2.60 (m, 3H, CH2(20), Ha-10); 2.58 (m, 1H,
Ha-8); 2.16 (m, 1H, Hb-10), 1.66 (m, 1H, Hb-8).
J(10a,5) = J(10b,5) = 6.7, J(7,8a) = 7.2, J(7,8b) = 11.7, J(8a,8b) = 12.7,
IR (KBr): 3434, 2922, 2864, 1708, 1492, 1450, 767, 747, 668 cmꢀ1
.
1H NMR (CD3OD, 400 MHz): 7.36–7.09 (m, 9H, Har); 5.18 (dd, 1H,
H-7); 4.60 (s, 1H, H-5); 3.86 (d, 1H, Ha-C(SBn)); 3.79 (d, 1H, Hb-
C(SBn)); 3.64 (dd, 1H, Ha-9); 2.97 (ddd, 1H, Hb-9); 2.53 (m, 1H,
Ha-8); 1.75 (q, 1H, Hb-8). J(SBn) = 13.2, J(7,8a) = 6.0,
J(8a,9a) = 2.4,
J(8a,9b) = 7.9,
J(8b,9a) = 10.7,
J(8b,9b) = 2.6,
J(9a,9b) = 14.8 Hz. 13C NMR (CD3OD, 100 MHz): 205.3 (C(6));
142.9, 141.5 (C(9a), Cs(Ph)); 135.8 (C(4a)); 130.6 (CHar); 129.6,
129.4 (CHo,m(Ph)); 129.1, 129.0, 127.5, 127.2 (4 CHar); 61.6
(C(7)); 53.2 (C(5)); 34.7 (C(8)); 34.4 (C(20)); 31.1 (C(10)); 30.9
J(7,8b) = 12.1,
J(8a,8b) = 12.8,
J(8a,9a) = 2.0,
J(8a,9b) = 7.6,
J(8b,9a) = 11.5, J(8b,9b) = 2.0, J(9a,9b) = 15.0 Hz. 13C NMR (CD3OD,
100 MHz): 202.0 (C(6)); 141.5 (Cs(Ph)); 137.9, 133.1
(C(4a),C(9a)); 132.1, 131.8 (C(1),C(4)); 130.3, 129.8 (CPh); 129.0,
128.7 (C(2),C(3)); 60.7 (C(5)); 57.9 (C(7)); 37.3 (C(10)); 33.4
(C(8)); 31.6 (C(9)). HR-MS (ESI-microTof) calcd for C18H20NOS
[M+H]+: 298.1260; found: 298.1241.
(C(9)). HR-MS (ESI-microTof) calcd for
280.1696; found: 280.1693.
C
19H22NO [M+H]+:
5.5.10. cis-7-Amino-5-benzyl-5,7,8,9-
tetrahydrobenzocyclohepten-6-one, hydrochloride (2f0)
5h (30 mg, 0083 mmol) was hydrogenolysed over Pd-C 5%
(2 mg) in EtOH (5 mL) for 2 h. The catalyst was centrifugated off,
the solvent evaporated. Crude 5f0 (30 mg, 0083 mmol) was depro-
tected according to the procedure C to give 2f0 (18 mg, 72%).
Compound 2f0: colorless crystals, mp 220–222 °C (dec.). IR
5.5.7. trans and cis 5-Phenylthio-7-amino-5,7,8,9-
tetrahydrobenzocyclohepten-6-one, hydrochloride (2d and 2d0)
Procedure C with 5d (80 mg, 0.21 mmol) to give 2d/2d0 (55 mg,
83%).
Compounds 2d/2d0: yellowish crystals, mp 148–154 °C (iPrOH/
Et2O). IR (KBr): 3420, 2925, 2867, 1719, 1490, 1481, 744, 691 cmꢀ1
.
(KBr): 3425, 2923, 1722, 1496, 1490, 1453, 755, 749, 693 cmꢀ1
.
HR-MS (ESI-microTof) calcd for C17H18NOS [M+H]+: 284.1109;
found: 284.1085.
1H NMR (CD3OD, 400 MHz): 7.25–7.16 (m, 9H, Har); 4.56 (dd,
1H, H-5); 4.24 (dd, 1H, H-7); 3.62 (dd, 1H, Ha-10); 3.26 (ddd, 1H,
Ha-9); 3.22 (dd, 1H, Hb-10); 2.86 (ddd, 1H, Hb-9); 2.55 (dddd, 1H,
Ha-8); 1.69 (dddd, 1H, Hb-8). J(10a,10b) = 13.7, J(10a,5) = 8.5,
J(10b,5) = 5.6, J(7,8a) = 7.4, J(7,8b) = 11.4, J(8a,8b) = 12.6, J(8a,9a) =
2.8, J(8a,9b) = 8.6, J(8b,9a) = 9.8, J(8b, 9b) = 3.0, J(9a,9b) = 14.8 Hz.
13C NMR (CD3OD, 100 MHz): 204.9 (C(6)); 141.2, 140.2 (C(9a),
Cs(Ph)); 135.7 (C(4a)); 130.5 (CHar), 130.3, 129.4 (CHo,m(Ph)),
129.2, 128.8, 128.4, 127.5 (4 CHar); 61.0 (C(7)); 55.8 (C(5)); 35.1
(C(10)); 34.8 (C(8)); 30.7 (C(9)). HR-MS (ESI-Q-Tof) calcd for
5.5.7.1. trans-Isomer 2d, deuteriated at C(5).
RMN 1H (D2O,
400 MHz): 7.52–7.22 (m, 9H, Har); 5.29 (dd, 1H, H-7); 3.77 (tl, 1H,
Ha-9); 3.08 (m, 1H, Hb-9); 2.63 (m, 1H, Ha-8); 1.87 (m, 1H, Hb-8).
J(7,8a) = 5.8,
J(7,8b) = 12.2,
J(8a,8b) = 12.8,
J(8a,9a) = 2.2,
J(8a,9b) = 7.2, J(8b,9a) = 11.6, J(8b,9b) = 2.4, J(9a,9b) = 15.6 Hz.
5.5.7.2. cis-Isomer 2d0, deuteriated at C(5).
RMN 1H (D2O,
400 MHz): 7.52–7.22 (m, 9H, Har); 4.46 (dd, 1H, H-7); 3.01 (m,
2H, CH2(9)); 2.57 (m, 1H, Ha-8); 1.96 (m, 1H, Hb-8). J(7,8a) = 8.2,
J(7,8b) = 10.5, J(8a,8b) = 13,1 Hz.
C
18H19NO [M]+: 265.1467; found: 265.1481.
13
RMN C (D2O, 100 MHz, deuteriated at C(5)): 204.8 (C(6) 2d0);
5.5.11. cis-5-Phenylpropyl-7-amino-5,7,8,9-
tetrahydrobenzocyclohepten-6-one, hydrochloride (2g0)
Procedure D with 4g0 (139 mg, 0.325 mmol) in dioxane (20 mL)
to give the hydrochloride 2g0 (80 mg, 75%).
202.5 (C(6) 2d); 141.0, 138.8, 134.1, 133.9, 132.5, 131.7, 131.3,
130.22, 130.20, 130.16, 129.9, 129.8, 129.7, 129.6, 129.5, 129.2,
129.1, 128.5, 128.0, 127.3 (20 Car, 2d, 2d0); 58.1 (C(7) 2d0); 57.7

S. Albrecht et al. / Bioorg. Med. Chem. 20 (2012) 4942–4953
4953
Compound 2g0: colorless crystals, mp 156–158 °C (iPrOH/Et2O).
(Basel) for HR-mass spectrometry measurements. We thank the
students Bertrand Honnert and Arnaud Mignatelli for their partic-
ipation to this work.
IR (KBr): 3429, 3019, 2945, 2924, 2895, 2870, 1718, 1505, 1488,
1453, 743 cmꢀ1 1H NMR (CD3OD, 400 MHz): 7.27–7.15 (m, 9H,
.
Har); 4.32 (dd, 1H, H-7); 4.19 (dd, 1H, H-5); 3.28 (m, 1H, Ha-9);
2.95 (ddd, 1H, Hb-9); 2.70 (m, 2H, CH2(30)); 2.55 (m, 1H, Ha-8);
2.35 (m, 1H, Ha-10); 1.89 (m, 1H, Hb-10); 1.67 (tt, 2H, CH2(20));
1.63 (m, 1H, Hb-8). J(1a0,1b0) = 13.2, J(10,20) = 8.0, J(1a0,5) = 8.2,
J(1b0,5) = 6.0, J(20,30) = 7.4, J(7,8a) = 7.2, J(7,8b) = 11.8, J(8a,8b) =
12.8, J(8a,9a) = 2.4, J(8a,9b) = 7.8, J(8b,9a) = 11.0, J(8b,9b) = 2.6,
J(9a,9b) = 14.6 Hz. 13C NMR (CD3OD, 100 MHz): 205.4 (C(6));
143.4, 141.5 ((C(9a),Cs(Ph)); 136.0 (C(4a)); 130.5 (CHar); 129.5,
129.4 (CHo,m(Ph)), 128.9, 128.8, 127.5, 126.9 (4 CHar); 61.5
(C(7)); 53.7 (C(5)); 36.9 (C(30)); 34.7 (C(8)); 30.9 (C(9)); 30.3
(C(20)); 28.7 (C(1’)). HR-MS (ESI-microTof) calcd for C20H24NO
[M+H]+: 294.1852; found: 294.1828.
References and notes
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Enzymes; Elsevier Academic Press: Oxford, 2004; Vol. 2, p 233; (b) Drag, M.;
Bogyo, M.; Ellman, J. A.; Salvesen, J. S. J. Biol. Chem. 2010, 285, 3310.
2. Mina-Osorio, P. Trends Mol. Med. 2008, 14, 361.
3. (a) Bhagwat, S. V.; Lahdenranta, J.; Giordano, R.; Arap, W.; Pasqualini, R. Blood
2001, 97, 652; (b) Bhagwat, S. V.; Petrovic, N.; Okamoto, Y.; Shapiro, L. H. Blood
1818, 2003, 101; (c) Petrovic, N.; Schacke, W.; Gahagan, J. R.; O’Conor, C. A.;
Winnicka, B.; Conway, R. E.; Mina-Osorio, P.; Shapiro, L. H. Blood 2007, 110,
142; (d) Terauchi, M.; Kajiyama, H.; Shibata, K.; Ino, K.; Nawa, A.; Mizutani, S.;
Kikkawa, F. BMC Cancer 2007, 7, 140; (e) Langner, J.; Mueller, C.; Riemann, D.;
Löhn, M. Immunol. Lett. 1997, 56, 62; (f) Yamashita, M.; Kajiyama, H.; Terauchi,
M.; Shibata, K.; Ino, K.; Nawa, A.; Mizutani, S.; Kikkawa, F. Int. J. Cancer 2007,
120, 2243; (g) Fukasawa, K.; Fujii, H.; Saitoh, Y.; Koizumi, K.; Aozuka, Y.; Sekine,
K.; Yamada, M.; Saiki, I.; Nishikawa, K. Cancer Lett. 2006, 243, 135; (h) Rangel,
R.; Sun, Y.; Guzman-Rojas, L.; Ozawa, M. G.; Sun, J.; Giordano, R. J.; Van Pelt, C.
S.; Tinkey, P. T.; Behringer, R. R.; Sidman, R. L.; Arap, W.; Pasqualini, R. Proc.
Natl. Acad. Sci. 2007, 104, 4588.
5.5.12. 7-Amino 5-benzylidene-5,7,8,9-
tetrahydrobenzocyclohepten-6-one (2h)
Procedure C with 5h (20 mg, 55 lmol) in HCl 2.2 N/Et2O
(0.5 mL) and dioxane (0.5 mL) to give the hydrochloride 2h
4. a Mucha, A.; Drag, M.; Dalton, J. P.; Kafarski, P. Biochimie 2010, 92, 1509; (b)
Bauvois, B.; Dauzonne, D. Med. Chem. Rev. 2006, 26, 88; c Jacobsen, F. E.; Lewis,
J. A.; Cohen, S. M. Chem. Med. Chem. 2007, 2, 152.
(13 mg, 79%).
5. Shim, J. S.; Kim, J. H.; Cho, H. Y.; Yum, Y. N.; Kim, S. H.; Park, H. J.; Shim, B. S.;
Choi, S. H.; Kwon, H. J. Chem. Biol. 2003, 10, 695.
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8. Grzywa, R.; Oleksyszyn, J.; Salvesen, G. S.; Drag, M. Bioorg. Med. Chem. 2010, 20,
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9. Schalk, C.; d’Orchymont, H.; Jauch, M.-F.; Tarnus, C. Arch. Biochem. Biophys.
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10. d’Orchymont, H.; Tarnus, C. Eur. Patent 1990, 0378456, A1.
11. Albrecht, S.; Defoin, A.; Salomon, E.; Tarnus, C.; Wetterholm, A.; Haeggström, J.
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12. Albrecht, S.; Al-Lakkis-Wehbe, M.; Orsini, A.; Defoin, A.; Pale, P.; Salomon, E.;
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13. Maiereanu, C.; Schmitt, C.; Schifano-Faux, N.; Le Nouën, D.; Defoin, A.; Tarnus,
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14. Tarnus-Rondeau, C.; Defoin, A.; Albrecht, S.; Maiereanu, C.; Faux, N.; Pale, P.
Compound 2h: colorless resin, IR (KBr): 3407, 3019, 2984, 2943,
1702, 1591, 1510, 1482, 1188, 1096, 762, 757, 695 cmꢀ1 1H NMR
.
(CD3OD, 400 MHz): 8.05 (s, 1H)); 7.46–7.39 (m, 2H, Har); 7.32–
7.13 (m, 7H, Har); 4.00 (dd, 1H, H-C7); 3.09 (ddd, 1H, Ha-9); 2.96
(ddd, 1H, Hb-9); 2.45 (dddd, 1H, Ha-8); 2.08 (dddd, 1H, Hb-8).
J(7,8a) = 8.2,
J(7,8b) = 11.0,
J(8a,8b) = 12.4,
J(8a,9a) = 13.0,
J(8a,9b) = 7.4, J(8b,9a) = 7.4, J(8b, 9b) = 1.4, J(9a,9b) = 13.7. 13C
NMR (DMSO-d6, 100 MHz): 194.2 (C(6)); 138.6, 138.2, 135.1,
134.1, 133.8, 131.0, 130.1, 130.0, 129.6, 129.2, 128.5, 128.5, 127.3
(12 Car); 54.9 (C(7)); 30.2 (C(8)); 28.3 C(9)). HR-MS (ESI-Q-Tof)
calcd for C18H17NO [M]+: 263.1310; found: 263.1312.
5.6. Enzyme assays
French
Patent:
FR2908131,
W2008059141,
Dérivés
d’aminobenzocycloheptènes, leurs procédés de préparation et leur utilisation
en thérapeutique.
15. Allinger, N. L.; Szkrybalo, W. J. Org. Chem. 1962, 27, 722.
16. Russell, G. A.; Blankespoor, R. L.; Trahanovsky, K. D.; Chung, C. S. C.; Whittle, P.
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Soc. 1906, 1975, 97.
17. Mataka, S.; Takahashi, K.; Mimura, T.; Hirota, T.; Takuma, K.; Kobayashi, H.;
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19. Nyffeler, P. T.; Durón, S. G.; Burkart, M. D.; Vincent, S. P.; Wong, C.-H. Angew.
Chem. Int. Ed. 2005, 44, 192.
5.6.1. Enzyme source
Porcine kidney APN was purchased from Sigma Chemical Co.
Porcine kidney LAPc was purified according to a published proce-
dure.34 Human recombinant LTA4H was provided by our collabora-
tor J. Z. Haeggström.11
5.6.2. Assay conditions11
(a) All enzymes: Kinetic data were collected with an HP/Agilent
UV–Visible, diode array, spectrophotometer 8453 using the
software ‘HP chemstation’ provided with the machine. Typically,
20. Gilicinski, A. G. J. Fluorine Chem. 1992, 59, 157.
21. Banks, R. E. J. Fluorine Chem. 1998, 87, 1.
22. Larock, C. K.; Hightower, T. R.; Kraus, G. A.; Hahn, P.; Zheng, D. Tetrahedron Lett.
1995, 36, 2423.
spectrophotometric assays were performed with L-leucine-p-nitro-
anilide as the substrate for APN (Km = 0.2 mM), LAPc (Km = 2 mM)
and Alanine-p-nitroanilide for LTA4H (Km = 2 mM). All kinetic
studies were performed at 30 °C and the reactions were started
by addition of the enzyme in 1 ml assay medium. (b) APN: 1 mUn-
its per assay, in 0.02 M Tris–HCl pH 7.5. (c) LAPc: 20 Units per assay
in 0.1 M Tris–HCl, 0.1 mM ZnCl2, 5 mM MnCl2, 1 M KCl, pH 8.0 and
23. Miriyala, B.; Bhattacharyya, S.; Williamson, J. Tetrahedron 2004, 60, 1463.
24. Siqueira Filho, E. P.; Rodrigues, J. A.; Moran, P. J. Tetrahedron: Asymmetry 2001,
12, 847.
25. Kim, M. Y.; Lim, G. J.; Kim, D. S.; Kim, I. Y.; Yang, J. S. Heterocycles 1997, 45,
2041.
26. 26. Leong, M.; Mastryukov, V.; Boggs, J. J. Mol. Struct. 1998, 445, 149. From the
given dihedral angles it is possible to calculate the dihedral angles between the
different protons. For the chair conformation: (7,8a) 185°; (7,8e) 65°; (8a,9a)
195°; (8a,9e) = (8e,9a) 75°; (8e,9e) 45°. For the boat conformation: (7,8a) 176°;
(7,8e) 56°; (8e,9a) 148°; (8a,9e) 82°; (8e,9e) = (8a,9a) 28°.
27. Segel, I. H. Enzyme Kinetics; John Wiley & Son: New-York, 1993.
28. Williams, J. W.; Morrison, J. F. Methods Enzymol. 1979, 63437.
29. Cha, S. Biochem. Pharmacol. 1975, 24, 2177.
(d) LTA4H: 5
The release of p-nitroanilide (
l
g per assay in10 mM Tris–HCl, 0.1 mM KCl, pH 7.5.
e
= 10,800 Mꢀ1 cmꢀ1) at 405 nm
was measured continuously to determine initial velocities. Assays
were performed in semi microcuvettes (1 cm path).
30. Schloss, J. V. Acc. Chem. Res. 1988, 21348.
31. Paulini, R.; Müller, K.; Diederich, F. Angew. Chem. Int. Ed. 2005, 44, 1788.
32. A Ocain, T.; Rich, D. J. J. Med. Chem. 1988, 31, 2193; (b) Gordon, E. M.; Godfrey, J.
D.; Delaney, N. G.; Asaad, M. M.; Von Langen, D.; Cushman, D. W. J. Med. Chem.
1988, 31, 2199.
33. (a) Ireland, R. E.; Liu, L. J. Org. Chem. 1993, 58, 2899; (b) Dess, D.; Martin, J. J. Am.
Chem. Soc. 1991, 113, 7277.
Acknowledgements
The Swedish Research Council is gratefully acknowledged for
providing LTA4H. We also wish to thank the Université de Haute
Alsace and the ENSCMu, the INCa and the Ligue Contre le Cancer
for their financial support. We thank Pr Vandorsselear of Stras-
bourg University and Dr Mathias Wind of Basilea Pharmaceuticals
34. Himmelhoch, R.; Peterson, S. A. Biochemistry 1968, 7, 2085.