Structure-activity relationships of imidazole-derived 2-[ N-carbamoylmethyl-alkylamino]acetic acids, dual binders of human insulin-degrading enzyme

Article abstract of DOI:10.1016/j.ejmech.2014.12.005

Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as amyloid-a and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the understanding of its role in the physiopathology of Alzheimer's disease, amyloid-a clearance, and its validation as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we describe the structure-activity study, that required the synthesis of 48 analogues. We found that while the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical probes for the exploration of IDE's role.

Full text of DOI:10.1016/j.ejmech.2014.12.005

European Journal of Medicinal Chemistry 90 (2015) 547e567
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationships of imidazole-derived 2-[N-
carbamoylmethyl-alkylamino]acetic acids, dual binders of human
insulin-degrading enzyme
,
b
,
c
,
d
,
,
b
,
c
,
d
,
,
b
,
c
,
d
,
e
Julie Charton a
e, Marion Gauriot a
e, Jane Totobenazara a
,
Nathalie Hennuyer b
g, Julie Dumont a
e, Damien Bosc a
,
,
c
,
f
,
,
,
b
,
c
,
d,
,
b,
c
,
d,
e
Xavier Marechal a
e, Jamal Elbakali a
e, Adrien Herledan a
,
,
b,
c
,
d
,
b
,
c
,
d,
, b, c, d, e
,
b
,
c
,
d
,
,
b,
c
,
d,
, b, c, d, e
Xiaoan Wen a
e, Cyril Ronco a
e, Helene Gras-Masse a
,
Antoine Heninot a
e, Virginie Pottiez a
e, Valerie Landry a
,
,
b
,
c
,
d,
,
b
,
c
,
d,
, b, c, d, e
,
c
,
f
,
, b, c, d,
Bart Staels b
g, Wenguang G. Liang h, Florence Leroux a
e, Wei-Jen Tang h,
Benoit Deprez a
a INSERM U761 Biostructures and Drug Discovery, Lille, France
1, Rebecca Deprez-Poulain a
,
b
,
c
,
d
,
e,
*,
, b, c, d, e, *, 1
b Lille F-59000, France
c Institut Pasteur de Lille, IFR 142, Lille F-59000, France
d PRIM, Lille F-59000, France
e CDithem Platform/IGM, Paris, France
f INSERM U1011 Nuclear Receptors, Cardiovascular Diseases and Diabetes, Lille F-59000, France
g European Genomic Institute for Diabetes (EGID), FR 3508, F-59000, Lille, France
h Ben-May Institute for Cancer Research, The University of Chicago, W421 Chicago, IL, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Insulin degrading enzyme (IDE) is a zinc metalloprotease that degrades small amyloid peptides such as
Received 6 June 2014
Received in revised form
19 August 2014
Accepted 3 December 2014
Available online 4 December 2014
^
amyloid-a and insulin. So far the dearth of IDE-specific pharmacological inhibitors impacts the under-
^
standing of its role in the physiopathology of Alzheimer's disease, amyloid-a clearance, and its validation
as a potential therapeutic target. Hit 1 was previously discovered by high-throughput screening. Here we
describe the structure-activity study, that required the synthesis of 48 analogues. We found that while
the carboxylic acid, the imidazole and the tertiary amine were critical for activity, the methyl ester was
successfully optimized to an amide or a 1,2,4-oxadiazole. Along with improving their activity, compounds
were optimized for solubility, lipophilicity and stability in plasma and microsomes. The docking or co-
crystallization of some compounds at the exosite or the catalytic site of IDE provided the structural
Keywords:
Enzymes
Structureeactivity relationships
Abbreviations used: AcOH, acetic acid; ANP, Atrial natriuretic peptide; Boc, tert-
butoxycarbonyl; CH3CN, acetonitrile; DCE, dichloroethane; DCM, dichloromethane;
DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulf-
oxide; EDCI, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide; Et3N, triethylamine;
EtOAc, ethyl acetate; EtOH, ethanol; hIDE, human insulin-degrading enzyme; HOBt,
N-hydroxybenzotriazole; MeOH, methanol; PBS, phosphate buffered saline; PTSA,
para-toluenesulfonic acid; rt, room temperature; SAR, structureeactivity relation-
ship; TFAA, Trifluoroacetic anhydride; THF, tetrahydrofuran.
* Corresponding authors. INSERM U761 Biostructures and Drug Discovery, 3 rue
du Pr Laguesse, F-59000 Lille, France.
E-mail addresses: benoit.deprez@univ-lille2.fr (B. Deprez), rebecca.deprez@
univ-lille2.fr (R. Deprez-Poulain).
1
Home pages: http://www.insulysin.org, http://www.deprezlab.fr.
http://dx.doi.org/10.1016/j.ejmech.2014.12.005
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

548
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
Amyloid-beta peptides
Inhibitors
X-ray diffraction
Docking
basis for IDE inhibition. The pharmacokinetic properties of best compounds 44 and 46 were measured
in vivo. As a result, 44 (BDM43079) and its methyl ester precursor 48 (BDM43124) are useful chemical
probes for the exploration of IDE's role.
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
member library on amyloid-beta hydrolysis [22]. We showed that
these compounds are dual binding inhibitors of IDE. Indeed, they
Insulin-degrading enzyme (IDE) is a zinc protease of the M16
family that is linked to type-2 diabetes and Alzheimer's disease
[1,2]. In addition to hydrolyse insulin or IGF-II [3], IDE was shown to
degrade numerous small size peptide substrates [4] including
bind a permanently formed exosite and the catalytic site formed
upon conformational switch of the N- and C-terminal halves from
the open to closed state and stabilisation of the swinging door [22].
A few analogues leading to cell-active compounds were disclosed.
Herein, we describe the full structureeactivity relationships in the
series. We performed additional studies for the interaction of IDE
with inhibitors both by X-ray analysis and docking. Finally best
compounds were evaluated for their in vivo pharmacokinetic
properties.
amyloid-
models display elevated brain A
expression of IDE in neurons results in reduced brain A
b
(A
b
) [5] and ubiquitin [6]. Interestingly, IDE-ko rodent
[7], while transgenic over-
levels [8].
b
b
Moreover, Ide gene was linked Alzheimer's disease (AD) in humans
[9]. In addition to being involved in the clearance of peptides, IDE
may have additional functions such as the regulation of the pro-
teasome complex [10], the refolding of amyloid-forming peptides
by serving as a chaperone [11] or the elimination of Ab1-40 across
the bloodebrain barrier by capillary endothelial cells [12].
Structures of human IDE have revealed the molecular basis for
the preference of IDE to degrade amyloidogenic peptides below
8 kDa [13,14]. IDE has a sizable and enclosed catalytic chamber that
is delimited by the N-terminal and C-terminal halves joined by a
loop [15]. Upon opening, the enzyme encapsulates the substrates
that primarily bind an exosite, 30 Å away from the catalytic zinc ion.
This binding promotes a conformational change of the substrate to
2. Chemistry
A few analogues were synthesized to explore the replacement of
the imidazole ring of histidine (part A) (Fig.1). Also we explored the
benzyle replacement by either alkyl groups, homologues of benzyle
or substituted benzyle. The impact of the nature of the linker be-
tween the nitrogen and the phenyl ring was investigated, as well as
the removal of the tertiary amine function (part B) (Fig. 1). Several
analogues were designed to evaluate the importance of the car-
boxylic acid function (part C) (Fig. 1) or the methyl ester group (part
D) (Fig. 1). Finally, a few analogues that combine several modifi-
cations were synthesized.
allow the regions that can adapt the b-strand structure to enter the
catalytic cleft for zinc-ion-mediated cleavage [16,17]. While larger
substrates need to enter into the catalytic chamber via a large open-
closed conformational switch of IDE, shorter peptides could also
enter the catalytic chamber by the displacement (swinging-door) of
a subdomain of IDE that creates an 18 Å opening [18].
The first substrate-based zinc-binding hydroxamate inhibitors
of IDE [19] display both a hydroxamate group [20] and an arginine
residue that limit their use as pharmacological probes. Other
compounds that behave as activators were also published [21].
We previously reported reversible, partial, competitive in-
hibitors of IDE discovered by high-throughput screening of a 2000-
2.1. Synthesis of analogues modified at part A
The synthesis of analogues 2e4 of hit 1 derived from different L-
amino-acid methyl esters was performed using a two-step proce-
dure: cyclization of commercially available iminodiacetic precursor
with TFAA in acetic anhydride, then anhydride opening in DMF
(Scheme 1).
2.2. Synthesis of analogues modified at part B
N
The synthesis of analogues 5e23 proceeded as depicted in
Scheme 2. Non commercial iminodiacetic precursors 5ae20a were
prepared by alkylation of iminodiacetic acid with bromides.
20ae22a were prepared by acylation of the dimethyl ester of imi-
nodiacetic, using acid chlorides or activated carboxylic acids. Re-
action of iminodiacetic with Boc2O or benzylchloroformate in
2 N NaOH solution allowed diacid 17a and 23a respectively. Syn-
thesized iminodiacetic acid precursors (5ae16a, 20ae23a) and
commercial analogues (1a and 18ae19a) were converted in situ to
the corresponding cyclic anhydride with trifluoroacetic anhydride
in acetic anhydride. 17a was converted to the corresponding cyclic
anhydride with DCC (Scheme 2). The anhydride then reacted with
histidine derivatives to give final amide compounds 1, 5e23 after a
deprotection step if needed (Scheme 2). Branched analogue 15 was
NH
O
HN
O
O
1 (BDM41367)
N
Dual-site
binding
Hit-to-Lead
Optimization
O
OH
Part A
Ar
exosite
HN R2
O
Part D
catalytic
site
obtained via
a different synthetic route from 1-methyl-3-
N
phenylpropylamine and L-His(Trt)-OMe. First L-His(Trt)-OMe was
converted to chloroacetamide 15a. 1-methyl-3-phenylpropylamine
reacted with tert-butylbromoacetate to give secondary amine 15b.
Both amine 15b and halide 15a reacted in refluxed DMF in the
presence of NaHCO3 to give 15 after a deprotection step of the tert-
butyl group (Scheme 3). Cyclic analogue 24 (Scheme 3) was
Part C
R1
R3
Part B
Fig. 1. Structures of hit 1 discovered by screening, binding to hIDE (PDB code 4DTT)
and hit-to-lead optimization strategy.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
549
Scheme 1. Synthesis of analogues 1e4. Reagents and conditions: (a) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h 2) L-aminoacid methyl esters, anhydrous
DIEA, anhydrous DMF, Argon, room temp., overnight.
obtained
in
three
steps
from
1,2,3,4-Tetrahydro-3-
reduced to alcohols by LAH, then converted in tosyle group to give
25a. Reaction with KCN allowed to get the bis-nitrile 25b. Hydro-
lysis of the nitrile functions in H2SO4 provided the desired diacid
25c (Scheme 4). Analogues 26e31 required the synthesis of pre-
cursors 26ae27a, 29ae31a (Scheme 5). N-benzyl-iminodiacetic
acid was first converted to cyclic anhydride then treated either with
ammonia or with O-trityl-hydroxylamine to give respectively pre-
cursors 27a or 30a. Homologue 29a was obtained in 3 steps from N-
benzylglycine. First N-benzylglycine was converted to its methyl
ester using SOCl2 in methanol. Then, the nitrogen was alkylated
using tert-butyl-3-bromo-propionate. Finally, methyl ester was
saponified to give 29a. Compound 26a was directly obtained from
N-benzylglycine using Boc2O. Squaric derivative 31a was synthe-
sized using previously published conditions [24]. In order to avoid
the formation of squaramide, commercially available diethyl
squarate was dissymmetrized using potassium tert-butoxide in dry
THF [25]. This dissymmetric diester then reacted with N-benzyl-
glycine to give compound 31a. Intermediates 26ae27a, 29ae31a
reacted with the desired amine and gave compounds 26e31 after
deprotection if necessary (Scheme 5). Methyl ester 28 was obtained
directly from 1 by treatment of the acid by thionyl chloride in
methanol. Tetrazole analogue 32 was obtained in three steps. First
carboxylic acid 27a was reacted with amine 35a. Then the primary
amide function was converted to cyano group using trifluoroacetic
acid in pyridine. Then the nitrile function was converted to the
tetrazole using NaN3 to give 32 (Scheme 6) [26].
isoquinolinecarboxylic acid, tert-butylbromoacetate and L-
His(Trt)-OMe.
2.3. Synthesis of analogues modified at part C
Compound 25 deprived of a tertiary amine function derived
from diacid 25c obtained by chain homologation from dieth-
ylbenzylmalonate [23] (Scheme 4). Briefly, ester functions were
1/ Diacid precursors
R- = Alk-; Ar-
CO2H
CO2H
a
N
HN
R
CO2H
CO2H
5a-14a
16a
18a : R = CH3- (commercial)
19a : R = Ph- (commercial)
R- = R'CO-
b,c,d
CO2H
CO2H
20a-22a
23a
17a
HN
e
f
N
R
CO2H
CO2H
17a : R = Boc-
20a : R = PhCO
21a : R = Ph-CH2-CO-
2.4. Synthesis of analogues modified at part D
22a : R = Ph-(CH2)2-CO-
23a : R = Cbz-
Dicarboxylic acid 33 was obtained from 1 by saponification of
the methyl ester (Scheme 7). For other analogues, amine precursors
(34ae38a, 40ae42a, 46a) were synthesized as depicted in Scheme
7. First, amine 40a was obtained from L-H-His(Trt)-OMe by direct
reaction with methylamine. Then trityle protection was removed
2/ Analogues
N
NH
HN
COOMe
using TFA.
isobutanol to give the corresponding isobutyl ester 36a. Similarly,
Boc- -histidine was reacted with isopropanol in the presence of
L-histidine was activated with SOCl2 then coupled with
CO2H
O
L
N
R
N
R
EDCI to give the isopropyl ester 35a after deprotection of the amine
function. Dimethylamide (42a) and benzylamine derivatives (41a)
were obtained using classical conditions. Bioisosteric replacement
of the methyl ester with a 1,2,4-oxadiazole (compound 46a) was
performed using methylamidoxime [27]. Analogues 34e46 were
synthesized by reacting either 3-benzyl-glutaric acid-anhydride or
3-(3-phenylpropyl)-glutaric acid-anhydride generated in situ from
the corresponding iminodiacetic acids with synthesized amine
CO2H
CO2H
g
5a
10a
5
10
6a-9a
13a;16a
18a-23a
6-9
13;16
18-23
h,i
j,k
17
17a
precursors or commercial histamine 34a, -histidine(Trt)-OtBu 37a,
histidinol 38a. 39 was obtained by reaction of 1 with gaseous
ammonia in a dioxane/methanol mixture (Scheme 7).
L
Scheme 2. Synthesis of analogues 5e14, 16e23. Reagents and conditions: (a) ReBr,
MeOH, DIEA, room temp., 2e12 h, 27e76%; (b) SOCl2, MeOH, 0ꢀc then room temp.,
18 h; (c) RCOCl, DIEA, DCM or RCOOH, EDCI, HOBt, DIEA, DCM, room temp., 18 h; (d)
NaOH, MeOH, H2O, 1e6 h, room temp.; (e) benzylchloroformate, NaOH 2 N, 0 ꢀC then
room temp., 2 h, 64%; (f) Boc2O, dioxane, NaOH 2 N, 0 ꢀC then room temp., 18 h, 67%.
(g) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h 2) L-Histidine
methyl ester dihydrochloride, anhydrous DIEA, anhydrous DMF, Argon, room temp.,
overnight. (h) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h 2) H-
His(1-Trt)-OMe HCl, anhydrous DIEA, anhydrous DMF, Argon, room temp., overnight.
(i) TFA, TIS, DCM, room temp., 5min-4 h. (j) 1) DCC, THF, overnight, room temp. 2) L-
Histidine methyl ester dihydrochloride, DIEA, THF, 5 h, room temp. (k) HCl(g), DCM,
2 h, room temp.
3. Results and discussion
3.1. Structureeactivity relationships
All the compounds reported here are partial inhibitors of the
enzyme for the degradation of amyloid-beta peptide [22].

550
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
N
O
N
N
N
Trt
Trt
N
a
b
O
NH
O
O
O
H2N
N
O
H
c,d
Cl
O
N
H
15a
N
O
15
H
N
HO
O
NH2
CO2tBu
N
15b
N
O
NH
NH
O
O
O
O
O
b,e
N
d
OH
N
H
H
O
N
NH
N
24
24a
tBuO
O
HO
O
Scheme 3. Synthesis of analogues 15 and 24. Reactants and conditions a) chloroacetylchloride, NaHCO3, DCM, room temp., 10 min; b) tert-butylbromoacetate, DIEA, THF, 0 ꢀC then
room temp., overnight; c) NaHCO3, DMF, reflux, 24 h; d) TFA, TIS, DCM, room temp. 2 h; e) L-Histidine methyl ester dihydrochloride, EDCI, HOBt, DIEA, DCM, room temp., 18 h.
Analogues were assayed for their ability to inhibit amyloid-b16-23
hydrolysis by hIDE (Tables 1e5).
Also removal of the methylene of the benzyl moiety (19) results in
complete loss of activity. This could be due to both steric constrains
or the loss of the charge on the adjacent nitrogen. Also, all amide or
carbamate analogues with various chain lengths (20e23) are
inactive. These results show the critical role of the tertiary amine
function. Finally, cyclization of 1 into a tetrahydroquinoleine result
in slightly equipotent compound 24.
3.1.1. Impact of imidazole ring on activity
We first evaluated the replacement of the imidazole ring of 1 by
a phenyl (2) or an indole (3). Such modification in Part A of the hit is
deleterious for activity while introducing a guanidine like in argi-
nine derivative (4) allows retaining some activity (Table 1).
3.1.3. Isosteric replacement of methyl ester and impact on plasma
stability
3.1.2. Impact of tertiary amine and chain elongation on activity
Table 2 shows the impact of modifications at Part B of hit 1 such
as substitution, chain elongation, charge deletion of the benzyla-
mino group. First, substitution in para with either electroattractor
or electrodonor substituents did not have a large impact on the
activity (5e8). Isosteric replacement of the phenyl ring by a pyri-
dine leads to a loss of activity (9) whereas hydrophobic naphthyl or
indole analogues 10e11 are slightly more active. Best activities are
Methyl ester function of 1 (Part D) was hydrolyzed rapidly in
mouse plasma giving the corresponding carboxylic acid 33 which is
inactive on the target. We evaluated the impact of both isosteric
replacement of the methyl ester and increasing ester size on ac-
tivity and stability (Table 3). Complete removal of the methyl ester
results in inactive compound 34. Interestingly, bulkier esters 35e37
are 1.7e4.7 more active than 1, iso-propyl ester (35) being the most
obtained
with
longer-chain
homologues
(12e14)
active in the series with an IC50 of 0.3 mM. Their plasma half-life was
(phenylbutyl > phenylpropyl > phenethyl > benzyl). A branched
chain is tolerated, as exemplified with 15. Interestingly, replace-
ment of the phenylalkyl group (like in 14) by an n-hexyl group
provides equipotent compound 16. Consistent with the positive
effect of chain elongation, truncated analogues are less active.
Indeed, secondary amine 17 and methyl derivative 18 are inactive.
ranked as tert-butyle > iso-butyle > methyl > iso-propyle, consis-
tently with reported metabolism of esters by carboxyesterases [28].
Thus tert-butyle ester 37 is the best compromise between a good
activity and a good stability compared to 1.
Alcohol 38 analogue is equipotent to 1 and stable in plasma.
Interestingly, primary amide 39 or dimethyl amide 42 are less
1) Diacid precursor
OTs
CN
CO2H
CO2Et
a,b
d
c
CO2Et
OTs
25a
CN
CO2H
25c
25b
N
2) Analogue
NH
HN
CONHMe
CO2H
e
O
CO2H
CO2H
25c
25
Scheme 4. Synthesis of analogue 25. Reagents and conditions: (a) LAH, THF, 0 ꢀC then reflux, 18 h, 70%; (b) TsCl, TEA, DCM, room temp., 18 h, 44%; (c) KCN, DMSO, 80 ꢀC, 3.5 h; (d)
H2SO4, H2O, reflux, 3 days, 40% (e) 1) trifluoroacetic anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h or DCC, THF, 5 h, room temp. 2) L-H-Histidine-methylamide, anhydrous DIEA,
anhydrous DMF, Argon, room temp., overnight.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
551
3.1.5. Identification of lead 44 (BDM43079)
Chain elongation at Part B was beneficial for activity. As well,
isosteric replacement of methylester at Part D proved to enhance
both plasma stability of the compounds and activity. Several ana-
logues with these two modifications (43e45; Table 5) are more
active than their respective benzyl analogues (37e41), with IC50
values ranging from 100 nM to 1.6
mM. Best compound in the series
is 44 (BDM43079), with an IC50 of 0.1
mM on the target. We also
evaluated in the phenylpropyl series two more isosteric re-
placements of the methyl ester by the corresponding 1,2,4-
oxadiazole (46) or methanol analogue (47). Interestingly, while
alcohol analogue is less active than amide counterpart 44, oxadia-
zole analogue is almost equipotent.
3.2. Binding of analogues
We previously disclosed the binding of 1 obtained by X-Ray
diffraction of crystallized IDE-1 complex [22]. Surprisingly, 1
(BDM41367) has two possible binding sites in the crypt: either the
exosite or the catalytic site. This binding mode is consistent with
observed SAR.
At the exosite (Fig. 2), the imidazole ring forms a hydrogen bond
with Glu341 side chain a key residue that binds the N-terminus of
IDE substrates. The backbone of Leu359 also interacts with the
imidazole ring. The amide function of inhibitors interacts with
Gly361 while the hydrophobic benzyl or phenylpropyl is in the
hydrophobic region of Ile374 and Lys364. Interestingly, inactive
analogues 22 and 33 bind to the exosite.
Scheme 5. Synthesis of analogues 26e31. Reagents and conditions: (a) trifluoroacetic
anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h (b) ammoniac, DMF 100%; (c) TritylO-
NH2, DIEA, DMF, room temp., 18 h, 93%; (d) 20% SOCl2/MeOH, room temp., 18 h, 100%;
(e) Br(CH2)2COOtBu, K2CO3, KI, acetone, reflux, 36 h; (f) NaOH, MeOH, 5 days, 40 ꢀC; (g)
Boc2O, NaOH 2 N, dioxane, room temp.overnight; (h) potassium tert-butylate 1 M in
THF, diethyl squarate, THF, 4 ꢀC, 15 min, 55%; (i) N-Benzylglycine hydrochloride, TEA,
MeOH, room temp., 18 h, 70%. (j) EDCI, HOBt, DIEA, DCM, L-His-OMe.2HCl, room temp.,
18 h, or i. chlorure d'oxalyle, DCM, DMF cat., 0 ꢀC, 30 min; ii. L-(1-Trt)-His-OMe.HCl,
DIEA, DCM, room temp., 18 h, 38%; (k) TFA, TIS, DCM, room temp., 5 min-4 h. (l) SOCl2,
MeOH, room temp., 2 h.
At the catalytic site, the carboxylic acid function of all three
inhibitors (1, 44, 46) completes the zinc coordination sphere
formed by His108, His112 and Glu198 (Fig. 3). Interestingly they
interact with residues from both N- and C-terminal domains,
keeping the enzyme in a closed conformation. The amide function
is implicated in two hydrogen bonds with the side chains of Tyr831
(with NHCO) and Asn139 (with NHCO). Furthermore, the imidazole
ring interacts with the backbone of Val833. Finally, the phenyl ring
makes stacking with Phe115. The negatively charged E111 and E182
are in the vicinity of the imino function of the inhibitors. This ex-
plains why analogue 24 and 22 deprived of this positively nitrogen
are inactive. In particular 22 binds the exosite (Fig. 2) but should not
be able to adequately bind the catalytic site resulting in inactivity.
The critical involvement of E111-amine interaction is also sup-
ported by the fact that when the IDE E111Q mutant is used for co-
crystallisation, the compounds only bind the exosite [22]. The
better activities of analogues 44 and 46 (Fig. 3, panels B and C
respectively) over hit 1 can be explained by the better fitting of the
phenylpropyl ring into the hydrophobic pocket defined by Phe115
and Phe820. Also, the carbonyl of amides is a better hydrogen bond
acceptor than the carbonyl of esters [29] resulting in stronger
hydrogen bond between Asn139 and the carbonyl group in 44. The
oxygen of 1,2,4-oxadiazole 46 is a good acceptor too [30]. 46 is 5
times less active than 44 but this could be attributed to the extra-
methyl group. Finally, diacid 33, though binding the exosite
(Fig. 2) may be inactive because the anionic charge in the vicinity of
the imidazole ring may prevent the imidazole to correctly interact
with Val833 in the C-term domain.
active than methylamide 40. This shows the importance of an
acceptor of hydrogen bond at this position. Interestingly, intro-
ducing a larger benzyle group on the amide decreases activity (41
vs 40).
3.1.4. Impact of carboxylic acid isosteric replacement on activity
Compounds 25e32 (Table 4) were synthesized to evaluate the
importance of the amino-methyl-carboxylic acid function (Part C)
for the activity of 1 and methylamide analogue 40. Isosteric
replacement of the nitrogen by a CH (25 vs 40) abolishes activity,
illustrating, as shown in Table 2, the critical role of the tertiary
amine function. Deletion of the acetic acid resulted in inactive
compound 26. Replacement of the carboxylic function by amide
(27) or methyl ester (28) is deleterious for activity. Elongation of
the chain is not efficient (29). Introduction of hydroxamate or
squaric isosters allows retaining some activity (30 and 31 vs 1).
Replacement of the carboxylic function of 40 by a tetrazole moiety
resulted in inactive compound 32. All these data suggest an
important interaction between the carboxylic acid function and
the target.
3.3. Vitro and vivo pharmacokinetic properties
As shown in Table 5, compounds in the series are highly hy-
drophilic with LogD ranging from ꢁ2.29 to ꢁ0.58 and aqueous
solubilities above 150 mM. As expected chain elongation enhances
Log D (13 vs 1, and 44 vs 40). Replacement of the methyl ester group
by a methyl amide surprisingly lowers LogD, this may be due to the
Scheme 6. Synthesis of 32. Reagents and conditions: (a) EDCI, HOBt, DIEA, DMF, L-H-
Histidine-methylamide, room temp., 18 h, 80%; (b) TFAA/pyridine, THF, 0 ꢀC; (c) NaN3,
ammonium chloride, DMF, 90 ꢀC, 60 h.
introduction of
a new hydrogen-bond donor while having

552
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
Scheme 7. Synthesis of compounds 33e47. Reactants and conditions: (a) i. MeNH2, EtOH, MeOH, reflux; (b) SOCl2, iBuOH, 60 ꢀC, 48 h; (c) EDCI, DMAP, isopropanol, DCM, reflux,
24 h, 56% (d) HClg, DCM, 1 h, 100%; (e) amine, EDCI, HOBt, TEA, DMF, room temp., 16 h; (f) i. TBTU, DIEA, DMF, 10 min, room temp.; ii. amidoxime, DMF, room temp. 4.5 h then reflux
1.5 h (g) i) N-benzyl-iminodiacetic acid, TFAA 2% in acetic anhydride, 50e70 ꢀC, 5 h, ii) amine, anhydrous DIEA, anhydrous DMF, argon, room temp., overnight; (h) i) diacid 13a, TFAA
2% in acetic anhydride, 50e70 ꢀC, 5 h, ii) amine, anhydrous DIEA, anhydrous DMF, argon, room temp., overnight, (i) TFA/DCM 50/50, TIS; (j) NH3g, dioxane/MeOH (5/2), room temp.
10 h, (k) NaOH, H2O, MeOH, room temp., 4 h.
moderate impact on solubility (40 vs 1; 44 vs 13). Further
replacement of the methyl ester function of 1 by either the corre-
sponding alcohol (47) or oxadiazole (46) has no dramatic impact on
solubility and LogD. As expected all compounds except methyl es-
ters are stable in plasma. Some compounds were evaluated for
microsome stability and showed a good half-life (>40 min).
Methyl ester precursors 48 and 49 were obtained in two steps
from 3-(3-phenylpropyl)-glutaric acid 13a (Scheme 8). At last,
suppression of the acid function (28, 48, 49) leads to inactive
compounds and allows to reach moderate LogD that may be better
for cell permeation. As expected, these compounds give readily the
active carboxylic acid in plasma and/or microsomes.
Given the preliminary in vitro ADME data and activity, both
carboxylic acids 44 and 46 were selected, along with their methyl
esters precursors 48 (BDM43124) and 49, for pharmacokinetic
experiment in mice following intraperitoneal dosing at 10 mg/kg
(n ¼ 3/group). Results are presented in Table 6. All 4 compounds
Table 1
Impact of imidazole replacement on inhibition of Ab16-23 hydrolysis by hIDE (1e4).
R
O
N
display good AUC ranging from 0.2 to 3.6 h mg/mL. Surprisingly,
mice are better exposed to the carboxylic derivatives that the
methyl esters. This may suggest the implication of transporters for
the highly hydrophilic compounds 44 and 46. Nevertheless, in the
series, half-lives of the series are short for both methyl esters and
carboxylic acids. This may be due to rapid elimination due to high
polarity. Also, mice that were given methyl esters 48 or 49 were
dosed also for carboxylic acid metabolites. These were detected
instantaneously. This is coherent with in vitro data on plasmatic
stability.
O
OH
Cpd
-R
IC50 (m
M)a
1
2
3
4
-L-H-His-OMe
-L-H-Phe-OMe
-L-H-Trp-OMe
-L-H-Arg-OMe
2.9
_b
_b
36.3
a
Values are means of 2 experiments minimum, standard deviations are 10%.
b
%inhibition below 10 at 100 mM.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
553
Table 2
Table 4
Impact of tertiary amine replacement and chain elongation on inhibition of Ab16-23
hydrolysis by hIDE (1, 5e24).
Impact of carboxylic acid replacement on inhibition of Ab16-23 hydrolysis by hIDE for
compounds (1, 25e32, 40).
N
N
N
NH
NH
NH
O
O
X
HN
HN
HN
O
O
O
25: X=N; Y=CH
26-31: X=O; Y=N
40: X=N; Y=N
O
O
O
N
N
Y
R
R
1, 5-23
OH
24
O
O
OH
Y-R
IC50 (m
M)a
Cpd
R-
IC50
(m
M)a
Cpd
R-
IC50
0.6
(m
M)a
1
-N-CH2COOH
-N-CH2COOH
-CH-CH2COOH
-NH
-NCH2CONH2
-NCH2COOMe
-N(CH2)2COOH
2.9
0.6
40
25
26
27
28
29
30
31
1
2.9
15
b
_
b
_
b
5
6
F
2.5
2.0
16
17
0.4
_
_
b
b
_
b
H-
_
-NCH2CONHOH
3.2
12.5
O
b
7
8
1.7
2.9
18
19
CH3-
_
N
O
b
_
HO
b
32
_
N
b
9
6.3
20
_
N
N
N
N N
O
H
b
10
11
1.2
1.2
21
22
_
a
O
Values are means of 2 experiments minimum, standard deviations are 10%.
%inhibition below 10 at 100 mM.
b
b
_
b
4. Conclusion
12
1.4
23
24
_
The effect of lead 44 (BDM43079) on the hydrolytic profile of
IDE was further assessed using full unlabelled native substrates
[22]. Expectedly 44 inhibits the hydrolysis of amyloid-b1-40 by IDE.
However, it moderately promotes the hydrolysis of insulin, in
13
14
1.0
0.6
e
3.6
a
Values are means of 2 experiments minimum, standard deviations are 10%.
%inhibition below 10 at 100 mM.
Table 5
b
Inhibition of Ab16-23 hydrolysis by hIDE and LogD, solubility, plasmatic and micro-
some stability for some analogues of 44.
N
NH
Table 3
Impact of isosteric replacement of methyl ester on inhibition of Ab16-23 hydrolysis by
hIDE and plasmatic stability (1, 33e42).
HN
R1
O
N
N
NH
n
O
OR2
HN
R
b
O
Cpd
n
-R1
-R2 IC50
Mouse plasma LogD7.4 Sol.
Mouse liver
M)a stability t1/2 (h)
(m
M)b microsome
N
(
m
1, 33-42
t1/2 (min)
O
OH
c
1
1
1
3
3
3
3
3
-CO2Me
-CONHMe -H 0.6
-CO2Me
-CO2tBu
-CONHMe -H 0.1
-CONHBz -H 0.4
-H 2.9
6.4
>24
1.3
>24
>24
>24
>24
ꢁ2.05
ꢁ2.29
ꢁ0.78
154
151
199
_
Cpd
-R
IC50
(
m
M)a
Mouse plasma
stability t1/2 (h)
40
13
43
44
45
46
>40
c
-H 1.0
-H 0.4
_
_
c
c
c
_
_
1
-CO2Me
-CO2H
-H
2.9
6.4
b
c
ꢁ1.36
ꢁ0.58
ꢁ0.84
170
179
198
>40
33
34
35
36
37
38
39
40
41
42
_
_
_
c
b
c
_
_
N
-H 0.5
>40
-CO2iPr
-CO2iBu
-CO2tBu
-CH2OH
-CONH2
-CONHMe
-CONH-Bz
-CON(Me)2
0.3
0.6
0.8
4.1
1.4
0.6
1.4
6.9
0.2
8.7
O
N
>24
>24
>24
>24
47
28
48
49
3
1
3
3
-CH2OH
-CO2Me
-CONHMe -Me
-Me
-H 1.6
>24
_
ꢁ1.30
0.82
1.07
1.52
180
191
179
172
>40
c
c
-Me >100
_
c
_
_
0.1
26
19
c
c
N
_
c
_
_
O
N
c
a
a
Values are means of 2 experiments minimum, standard deviations are 10%.
Values are means of 2 experiments minimum, standard deviations are 10%.
Solubility and LogD are measured from a DMSO stock solution.
Not determined.
b
c
b
c
% inhibition below 10 at 100
Not determined.
mM.

554
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
Fig. 2. Binding of 1 and analogues 22, 33 and 44 at the exosite of hIDE-CF-E111Q. O (red); N (blue); C (blue for IDE N-terminal domain, orange for IDE C-terminal domain, grey for
inhibitors); hydrogen contacts in dotted lines. A) 1: X-Ray structure PDB code: 2YPU; B) 33 X-Ray structure PDB code: 4QIA; C) 22 X-Ray structure PDB code: 4GSF; D) 44 X-Ray
structure PDB code: 4GS8. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
contrast with pan-inhibitors like EDTA [22]. 44 (BDM43079) was
shown to be selective of hIDE versus a panel of metalloproteases
including hNEP [31] and hECE two other metalloproteases impli-
5. Experimental section
5.1. Biology
cated in the hydrolysis of amyloid-b. Cellular effects of 48
(BDM43124) methyl ester precursor of the lead 44 were assessed in
SH-SY5Y [22]. Cells treated with 48, display a dose-dependent in-
5.1.1. In vitro IDE activity assay
In vitro IDE activity was measured with a quenched substrate
ATTO 655- Cys-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Trp. Human re-
combinant IDE was cloned and purified as previously described.
Briefly, human IDE (1.87 ng/mL) was incubated 10 min with com-
pound in Hepes 50 mM, NaCl 100 mM, pH 7.4 and the enzymatic
crease in levels of both amyloid-b1-40 and b1e42
.
Our compounds differentiate from the pan-substrate inhibitor
[19a] and are deprived of guanidinium function known to slow
down cell membrane permeation. Fig. 4 summarizes the structur-
eeactivity relationships in the series in the light of the binding of
compound 1 to IDE. We evidenced the key role of chain length and
methyl ester replacement both on activity and plasma stability. In
particular, compound 44 (IC50 ¼ 100 nM) is a promising analogue
that showed cellular activity. This series is highly soluble in
aqueous media, and both methyl amide and 1,2,4-oxadiazole de-
rivatives displayed interesting bioavailability. Further optimization
of pharmacokinetic parameters in the series will focus on opti-
mizing both cell permeation and elimination half-life. In summary,
we have successfully developed and optimized a series of inhibitors
of amyloid-beta hydrolysis by Insulin-degrading enzyme.
reaction is started by adding the substrate (final concentration
5 m
M). After 30 min at 37 ꢀC, samples (1% DMSO final) are excited at
635 nm and fluorescence emission at 750 nm is measured on a
Victor3 V1420 Perkin Elmer spectrophotometer. All measurements
were carried out as 8-point dose response curves and are reported
as the average of at least two independent measurements. EDTA
was used as a reference inhibitor (100% inhibition at 2 mM). Results
were expressed as percentage of inhibition and IC50 values were
calculated from concentration-response curves by a non-linear
regression analysis at four parameters (Hill equation) using
XLfit™ software.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
555
Fig. 3. Binding of 1 and analogues 44 and 46 at the catalytic site of hIDE-CF. Zn (magenta sphere); O (red); N (blue); C (blue for IDE N-terminal domain, orange for IDE C-terminal
domain, grey for inhibitors); hydrogen contacts in dotted lines. A) 1: X-Ray structure PDB code: 4DTT; B) Docking of 44 in PDB code: 4DTT; C) Docking of 46 in PDB code: 4DTT; D)
Structures of 1, 44 and 46. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5.1.2. Solubility/LogD measurements
diluted in 180
mL of MeOH. The solubility is determined by the ratio
The analysis was performed using an LC-MS/MS system (Varian
1200L) under SIM detection using the parameters optimized for
each compounds. HPLC analysis was performed using a Luna C18
of mass signal area PBS/organic solvent. 40
in DMSO of the compound were diluted in 1.960 mL of a 1/1
octanol/PBS at pH 7.4 mixture. The mixture was gently shaken
m
L of a 10 mM solution
(50*2.1 mm, 5
m
m); the gradient and the mobile phase (flow rate
2 h at room temperature. 20 mL of each phase was diluted in 480 mL
600
m
L minꢁ1) used are determined in order to detect the com-
of MeOH and analyzed by LC-MS. Each compound is tested in
triplicate. Log D was determined as the logarithm of the ratio of
concentration of product in octanol and PBS respectively, deter-
mined by mass signals.
pound of interest with satisfying retention time and peak shape.
Acquisition and analysis of data were performed with MS Work-
stationTM software (version 6.3.0 or higher). 10
solution in DMSO of the compound are diluted either in 490
PBS pH 7.4 or in organic solvent MeOH in a 700 L-microtube (in
mL of a 10 mM
mL of
m
5.1.3. Stability in plasma
triplicate). The tubes are gently shaken 24 h at room temperature,
then centrifuged for 5 min at 4000 rpm. The mixtures are filtered
Incubations were performed in duplicate in Eppendorf tubes.
The mouse plasma (Mouse Plasma Lithium Heparine from Sera
Laboratories International Ltd) or the human plasma (mixed
over 0.45
mm filters (Millex-LH Millipore). 20 mL of sample are
Scheme 8. Synthesis of 48e49. Reagents and conditions: (a) trifluoroacetic anhydride 2% in acetic anhydride, 50e70 ꢀC, 5 h; (c) MeOH, DIEA, reflux, overnight; (c) amine, DMF,
HOBt, EDCI, NMM, room temp. 22 h; (d) TFA/DCM TIS, room temp. 4 h.

556
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
Table 6
Control incubations were performed with denaturated microsomes
with acetonitrile containing 1 M internal standard and sampling
Intraperitoneal pharmacokinetic parameters for 44, 46, 48, 49 in mouse (30 mpk).a
m
points were taken at 0 min and 40 min (to evaluate the compound
chemical stability in the experimental conditions). Remaining
compound was quantified by converting the corresponding ana-
lyte/internal standard peak area ratios to percentage drug
remaining, using the initial ratio values in control incubations as
100%. The degradation half-life (t1/2) values were calculated using
the following equation: t1/2 ¼ 0.693/k where k is the first-order
degradation rate constant. The degradation rate constant (k) was
estimated by one-phase exponential decay non-linear regression
analysis of the degradation time course data using Xlfit™ software
(version 2.1.2 or higher) from IDBS.Ltd.
N
N
NH
NH
N
HN
HN
CONHMe
O
N
O
O
N
N
O
OR
O
OR
44: R = H
48: R = Me
46: R = H
49: R = Me
Cpd
Cmax (
m
g/mL)
Tmax (min)
T1/2 (min)
AUC (h.mg/mL)
48
49
44
46
3.8
1.0
11.0
5.8
10
10
10
10
7
6
9
1.0
0.2
3.6
1.9
5.1.5. Pharmacokinetics in mice
14
Compounds were dissolved in either in 100% PBS (48 and 49) or
in 10% cyclodextrin HP in PBS (Keptose, Roquette) (44 and 46), and
were administered at 30 mg per kg body weight by intraperitoneal
route to male C57Bl6/N 8-week old mice (env 25e30 g) (Charles
River). Three mice per time point were anesthetized with isoflurane
and aliquots taken from retroorbital sinus sampling at 10 min,
20 min, 30 min,1 h, 2 h, and 4 h after administration of a single dose
of ligands were put in heparinated tubes (4 ꢀC). The blood samples
were centrifuged (5000 g, 15 min) for plasma separation.
a
Analogues were dosed in male Black mice (n ¼ 3) as a PBS solution.
gender) was pre-incubated 5 min at 37 ꢀC before the addition of test
compounds to a final concentration of 10 M (1% DMSO maximum).
At the defined time points, 50 L from each tube were removed to
another tube containing 450
L of cold CH3CN þ internal standard
(1 M). After centrifugation (10 min at 10,000 rpm), supernatants
are analyzed. Analysis and quantification used an LC-MS/MS triple-
quadrupole system (Varian 1200L) under MRM detection using the
parameters optimized for each compounds. HPLC analysis was
m
m
m
m
Plasma samples were thawed on ice. Aliquots of 50
mL were
precipitated with 450 L of ice cold acetonitrile containing 1 (1
m
mM)
used as internal standard. The samples were vigorously mixed with
a Vortex and centrifuged at 10,000 rpm at 4 ꢀC for 10 min, and the
supernatants were transferred into Matrix tubes for LCꢁMSeMS
analysis (Table S2). Spiked standard solutions (10, 50, 100, 500,
1000, 5000, 10 000, and 50 000 nM) were prepared the same way.
The half-life (t1/2) values were calculated using the following
equation: t1/2 ¼ 0.693/k where k is the first-order degradation rate
constant. The rate constant (k) was estimated by one-phase expo-
nential decay non-linear regression analysis of the degradation
time course data using Xlfit™ software (version 2.1.2 or higher)
from IDBS.Ltd. AUC is the area under the concentration versus time
curve extrapolated to infinity.
performed using a Luna C18 (50*2.1 mm, 5
mm); the gradient and
the mobile phase (flow rate 600 L/min-1) used are determined in
m
order to detect the compound of interest with satisfying retention
time and peak shape. Acquisition and analysis of data were per-
formed with MS Workstation™ software (version 6.3.0 or higher).
The degradation half-life (t1/2) values were calculated using the
following equation: t1/2 ¼ 0.693/k where k is the first-order
degradation rate constant. The degradation rate constant (k) was
estimated by one-phase exponential decay non-linear regression
analysis of the degradation time course data using Xlfit™ software
(version 2.1.2 or higher) from IDBS.Ltd.
5.2. Molecular modelling
5.1.4. Microsomal stability
Male murine liver microsomes were purchased from BD Gentest
(Le Pont de Claix, France). All incubations were performed in trip-
licates in a shaking water bath at 37 ꢀC. The incubation mixture
The analogues 44 and 46 were docked into the binding pocket of
hIDE using MOE32 using the Protein DataBank (PDB) under pdb
code 4DTT. Protein protonation was made with the Protonate3D
process within MOE 10.2012. The docking used was based on the
Induced Fit protocol of MOE, using a Triangle Matcher placement, a
first rescoring process using the London dG routine and a force
field-based refinement process and a second rescoring process
using GBVI/WSA dG keeping in the end the best docking pose for
each ligand. No restraints were used during docking but the bind-
ing modes were chosen based on both the most favoured predicted
interaction energy with IDE and a proper binding to the Zinc ion by
the carboxylic acid.
were prepared in polypropylene tubes and contained 1
mM test
compound (1% acetonitrile), human liver microsomes (0.6 mg of
microsomal protein/ml), 5 mM MgCl2, 1 mM NADP, 5 mM glucose-
6-phosphate, 0.4 U/mL glucose-6-phosphate dehydrogenase and
50 mM potassium phosphate buffer pH 7.4 in a final volume of
0.5 mL. Sampling points were taken at 10, 20, 30 and 40 min and
reactions were terminated by adding ice-cold acetonitrile con-
taining 1 mM internal standard (4 vol). The samples were centri-
fuged for 10 min at 10,000 g, 4 ꢀC to pellet precipitated microsomal
protein, and the supernatant was subjected to LC-MS/MS analysis.
5.3. Crystallization and data process
The complexes of IDE-CF-E111Q with compounds 1, 22, 33 were
crystallized by hanging drop vapour diffusion at 18 ꢀC, using 1
m
l of
10 mg/ml IDE and 1
mL of mother liquor (10e13% PEG MME 5000,
100 mM HEPES pH 7.0, 4e14% Tacsimate, 10% dioxane). IDE-CF in
complex with 1 (BDM41367) was crystallized under the same
condition except the addition of 200 mM of given compound in the
crystallization drop. IDE crystals were equilibrated in cryo-
protective buffer containing mother liquor with 30% glycerol and
flash frozen in liquid nitrogen. Diffraction data were collected at
Fig. 4. Summary of structureeactivity relationships.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
557
100 K at the Advance Photon Source 19-ID beamline at Argonne
National Laboratory. The data sets were processed using HKL2000
[33] and the structures were solved by molecular replacement
using software Phaser [34] and the IDE-CF-E111Q portion of
(5 mmol) was dissolved in trifluoracetic anhydride 2% in acetic
anhydride (5 mL). The reaction mixture was stirred for 4 h at room
temperature or 70 ꢀC if product was not soluble at room temper-
ature and then evaporated under reduced pressure. The crude
product was directly used in the next step. In the case of iminodi-
acetic acids 9a and 18a, a stirred solution of diacid (5 mmol) in THF
(10 mL) was added DCC (6 mmol). The resulting mixture was stirred
at room temperature for 5 h and evaporated. 2) anhydride opening
by amines: The crude anhydride (1 mmol) was solubilised in
anhydrous DMF (5 mL). Then corresponding amine (1 mmol) (free
base) and DIEA (4 mmol) was added. The mixture is stirred at room
temperature under argon overnight. The solvent is removed under
reduced pressure and the crude product is purified by preparative
HPLC. Trityle protections were removed using TFA/DCM/TIS (5/90/5
v/v) mixtures and final compounds were purified by preparative
HPLC if needed.
insulin-bound IDE-CF-E111Q structure as
a
search model
(PDB:2WBY). Structure refinement and rebuilding were performed
using software REFMAC and Coot [32,35]. The extra electron den-
sity at the catalytic chamber of IDE-CF in the structures of IDE in
complex with compound were clearly visible based on sA
-
weighted Fo-Fc map calculated by software CNS [36] and manually
built. The refinement statistics are summarized in supplemental
information Table S3. Figures were generated using software
PyMol [37].
5.4. Chemistry
5.4.1. General information
NMR spectra were recorded on a Bruker Avance 300 spec-
trometer. Chemical shifts are in parts per million (ppm) and were
referenced to the residual proton peaks in deuterated solvents. The
assignments were made using one dimensional (1D) 1H and 13C
spectra (classical or Jmod) and two-dimensional (2D) HSQC, HMBC,
ROESY and COSY spectra. Mass spectra were recorded with an
LCꢁMSeMS tripleꢁquadrupole system (Varian 1200ws) or an
LCMS (Waters Alliance Micromass ZQ 2000). LCMS analysis was
5.4.3. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid methyl ester (1)
BDM41367 as its formiate salt was synthesized following gen-
eral procedure A from commercial N-benzyliminodiacetic acid and
L
-Histidine methyl ester dihydrochloride. White solid. Yield 20%. 1H
NMR (DMSO-d6)
d
ppm:, 8.34 (d, 1H, NH), 8.26 (d, J ¼ 1.1 Hz, 1H),
7.30 (m, 5H), 7.09 (d, J ¼ 1.1 Hz, 1H), 4.65 (m, 1H), 3.77 (d,
J ¼ 13.4 Hz, 1H), 3.71 (d, J ¼ 13.4 Hz, 1H), 3.59 (s, 3H), 3.27 (s, 2H),
3.23 (s, 2H), 2.99 (d, J ¼ 6.3 Hz, 2H), LC tR ¼ 2.30 min, MS (ESIþ): m/
performed using a C18 TSK-GEL Super ODS (2
mm particle size
column, dimensions 50 mm ꢂ 4.6 mm). A gradient starting from
100% H2O/0.1% formic acid and reaching 20% H2O/80% CH3CN/0.08%
formic acid within 10 min at a flow rate of 1 mL/min was used.
Preparative HPLC were performed using a Varian PRoStar system
using an OmniSphere 10 column C18 250 mm ꢂ 41.4 mm Dynamax
from Varian, Inc. A gradient starting from 20% CH3CN/80% H2O/0.1%
formic acid and reaching 100% CH3CN/0.1% formic acid at a flow
rate of 80 mL/min or 20% MeOH/80% H2O/0.1% formic acid reaching
100% MeOH/0.1% formic acid was used. Purity (%) was determined
by reversed phase HPLC, using UV detection (215 nm), and all
compounds showed purity greater than 95%. Melting points were
determined on a Bu€chi B-540 apparatus and are uncorrected. All
commercial reagents and solvents were used without further pu-
rification. Organic layers obtained after extraction of aqueous so-
lutions were dried over MgSO4 and filtered before evaporation
under reduced pressure. Purification yields were not optimized.
The LC-MS/MS system consisted of a Varian 1200L (Varian, Les Ulis,
France) a Prostar 430 autosampler, a (Varian, Les Ulis, France) triple
quadrupole mass spectrometry equipped with an electrospray
ionisation source, with a Prostar 325 detector. The HPLC separation
z ¼ 375 (M þ H)þ. 13C NMR (DMSO-d6)
d ppm: 172.3, 171.8, 170.4,
138.2, 137.9, 129.0, 128.4, 127.3, 115.9, 57.5, 56.7, 53.6, 52.1, 51.7, 28.4.
tR,LCMS ¼ 2.30 min; Purity 100%; MS (ESIþ): m/z ¼ 375 (MþH)þ;
HRMS (m/z): (Mþ) calcd. for C18H23O5N4, 375.1668; found,
375.1665.
5.4.4. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
phenyl-propionic acid methyl ester (2)
Compound (2) was synthesized from L-phenylalanine methyl
ester hydrochloride and N-benzyliminodiacetic acid following
general procedure A. Colourless oil. Yield 40%. 1H NMR (DMSO-d6)
d
ppm: 8.13 (d, J ¼ 8.10 Hz, 1H), 7.31e7.16 (m, 10H), 4.58 (m, 1H),
3.69 (d, J ¼ 13.2 Hz,1H), 3.64 (d, J ¼ 13.2 Hz,1H), 3.61 (s, 3H), 3.22 (s,
2H), 3.16 (s, 2H), 3.08 (dd, J ¼ 5.4 and 13.8 Hz, 1H), 2.96 (dd, J ¼ 8.7
and 13.8 Hz, 1H); tR,LCMS ¼ 4.39 min, Purity 100%; MS (ESIþ): m/
z ¼ 385 (MþH)þ.
5.4.5. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(1H-indol-3-yl)-propionic acid methyl ester (3)
Compound (3) was synthesized following general procedure A
uses a gel TSK C18 Super-ODS, 5
m
m, 50 ꢂ 4.6 mm column (Inter-
from
L-tryptophan methyl ester dihydrochloride and N-benzyli-
chim, Montluçon France) with mobile phase solvents: (A) 0.01%
formic acid in water; (B) 0.01% formic acid in acetonitrile for with
the following mobile phase gradient: 0% B during 30 s, 0e98% (B) in
30; hold at 98% (B) for 1 min; 98%e0% B in 0.30 min; 0% B hold for
minodiacetic acid. White solid. Yield: 25%. 1H NMR (DMSO-d6)
d
ppm: 10.9 (s, 1H), 8.10 (d, J ¼ 7.8 Hz, 1H), 7.48 (d, J ¼ 7.8 Hz, 1H),
7.36 (d, J ¼ 8.1 Hz, 1H), 7.20e7.22 (m, 3H), 7.16 (d, J ¼ 2.1 Hz, 1H),
7.08e7.10 (m, 2H), 7.08 (td, J ¼ 1.2 and 7.8 Hz, 1H), 6.98 (td, J ¼ 1.2
and 8.1 Hz, 1H), 4.60 (dd, J ¼ 7.2 and 15.0 Hz, 1H), 3.68 (d, J ¼ 13.2,
1H), 3.62 (d, J ¼ 13.2, 1H), 3.59 (s, 3H), 3.14e3.27 (m, 6H), 13C NMR
1 min, at a flow rate of 1 mL/min, or Luna C18, 5
mm, 50 ꢂ 2.1 mm
column (Phenomenex) with mobile phase solvents: (A) 5 mM
ammonium formate pH 9,2 in water; (B) 5 mM ammonium formate
pH 9,2 in acetonitrile with the following mobile phase gradient: 0%
B during 30 s, 0e98% (B) in 2030; hold at 98% (B) for 1 min; 98%e0%
(DMSO-d6)
d ppm: 172.8, 172.6, 170.7, 138.4, 136.7, 128.8, 128.2,
127.1,124.2,121.7,119.2,118.2,111.9,109.3, 58.0, 57.2, 54.2, 52.4, 52.1,
27.4. tR,LCMS ¼ 4.42 min, Purity 99%; MS (ESIþ): m/z ¼ 424 (MþH)þ.
B in 0010; 0% B hold for 1 min, at a flow rate of 600
injection volume was 40
m
L/min. The
mL. The pressures of drying and nebulising
5.4.6. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-5-
guanidino-pentanoic acid methyl ester (4)
gas are respectively 19 and 50 psi; the curtain gas pressure is
2 mTorr. Source temperature, declustering potential, collision en-
ergy and observed transitions were respectively individually opti-
mized for each compound.
Compound (4) was synthesized following general procedure A
from commercial N-benzyliminodiacetic acid and
methyl ester dihydrochloride. White solid. Yield 98%. 1H NMR
(DMSO-d6)
L-Arginine
d
ppm: 8.39 (d, J ¼ 7.8 Hz, 1H), 7.97 (m, 1H), 7.26e7.35
5.4.2. General procedure A for anhydride opening by amines
(m, 5H), 4.30 (ddd, J ¼ 4.8 Hz, 8.4 Hz and 8.1 Hz, 1H), 3.78 (s, 2H),
1) Obtention of anhydrides: iminodiacetic acid analogue
3.63 (s, 3H), 3.34 (s, 2H), 3.26 (s, 2H), 3.07e3.14 (m, 2H), 1.70e1.79

558
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
(m, 2H), 1.44e1.51 (m, 2H). 13C NMR (DMSO-d6)
d
ppm: 172.7, 172.2,
5.4.17. (Carboxymethyl-(4-phenyl-butyl)-amino)-acetic acid (14a)
White solid. Yield 76%. NMR (DMSO-d6) ppm: 7.26e7.12 (m,
5H), 3.39 (s, 4H), 2.65 (t, J ¼ 7.1 Hz, 2H), 2.55 (t, J ¼ 7.1 Hz, 2H), 1.54
(m, 2H), 1.39 (m, 2H). tR,LCMS ¼ 3.59 min; Purity 99%; MS (ESIþ): m/
z ¼ 266 (MþH)þ.
170.6,157.0,138.2,128.8,128.2,127.2, 57.6, 56.4, 54.5, 51.9, 51.2, 41.5,
28.1, 24.9. tR,LCMS ¼ 2.51 min, Purity 99%; MS (ESIþ): m/z ¼ 394
(MþH)þ.
d
5.4.7. General procedure for the synthesis of N-substituted
iminodiacetic acids from corresponding halides (5ae14a, 16a)
To a solution of iminodiacetic acid hydrochloride (2 mmol) in
MeOH (10 mL) were added NEt3 (6 mmol) and the corresponding
bromide derivative (2 mmol). The mixture was stirred overnight at
room temperature. The crude product was purified by preparative
HPLC.
5.4.18. (Carboxymethyl-hexyl-amino)-acetic acid (16a)
Oil directly used in the next step tR,LCMS ¼ 2.90 min; MS (ESIþ):
m/z ¼ 218 (MþH)þ.
5.4.19. (tert-Butoxycarbonyl-carboxymethyl-amino)-acetic acid
(17a)
To a stirred solution of iminodiacetic acid (2 g, 15 mmol) in a
dioxane/H2O (3/1, 40 mL) mixture, were added at 0 ꢀC, Boc2O
(3.93 g, 18 mmol) and 10 mL of 2 N NaOH solution. The mixture was
stirred at room temperature and dioxane was evaporated under
reduced pressure. The aqueous layer was acidified with a 20% citric
acid solution and extracted with AcOEt. The organic layer was dried
over MgSO4 and evaporated to give 17a (67%), as a white solid
directly used in the next step.
5.4.8. (Carboxymethyl-(4-fluoro-benzyl)-amino)-acetic acid (5a)
White solid. Yield 55%. 1H NMR (DMSO-d6)
d ppm: 7.30 (m, 2H),
7.12 (m, 2H), 3.77 (s, 2H), 3.31 (s, 4H). tR,LCMS ¼ 1.93 min, Purity
100%; MS (ESIþ): m/z ¼ 242 (M þ H)þ.
5.4.9. (Carboxymethyl-(4-trifluoromethyl-benzyl)-amino)-acetic
acid (6a)
White solid. Yield 44%. 1H NMR (DMSO-d6)
d ppm: 7.68 (d,
J ¼ 7.8 Hz, 2H), 7.58 (d, J ¼ 7.8 Hz, 2H), 3.91 (s, 2H), 3.42 (s, 4H).
5.4.20. (Benzoyl-carboxymethyl-amino)-acetic acid (20a)
tR,LCMS ¼ 3.17 min; Purity 98%; MS (ESIþ): m/z ¼ 292 (MþH)þ.
Iminodiacetic acid was stirred overnight in an 80/20 MeOH/
SOCl2 mixture. The diester obtained after evaporation under
reduced pressure (300 mg, 1.52 mmol) was dissolved in CH2Cl2
5.4.10. (Carboxymethyl-(4-methyl-benzyl)-amino)-acetic acid (7a)
White solid. Yield 31%. 1H NMR (DMSO-d6)
d
ppm: 12.27 (s, 3H),
(10 mL) and DIEA (792 mL, 4.56 mmol) and benzoylchloride (176 mL,
3.34 (s, 2H), 7.10 (d, J ¼ 7.8 Hz, 2H), 7.20 (d, J ¼ 7.8 Hz, 2H).
1.52 mmol) were added. The mixture was stirred overnight at room
temperature and washed with HCl 0.5 N and NaHCO3 10% solutions.
The organic layer was dried over MgSO4 and evaporated to give the
desired diester as a colourless oil (99%). The former compound
(400 mg, 1.5 mmol) was saponified with NaOH (6 mmol) in MeOH
(5 mL) and H2O (1 mL). After evaporation of MeOH under reduced
pressure, HCl 1 N was added to the residue and the aqueous layer
was extracted 3 times with AcOEt to give (benzoyl-carboxymethyl-
amino)-acetic acid (20a) directly used in the next step. White
powder. Yield 98 Purity 98%; MS (ESIþ): m/z ¼ 238 (MþH)þ.
tR,LCMS ¼ 2.38 min; Purity 90%; MS (ESIþ): m/z ¼ 238 (MþH)þ.
5.4.11. ((4-Tert-Butyl-benzyl)-carboxymethyl-amino)-acetic acid
(8a)
White solid. Yield 43%. 1H NMR (DMSO-d6)
d ppm: 7.34 (d,
J ¼ 8.4 Hz, 2H),7.23 (d, J ¼ 8.4 Hz, 2H), 3.76 (s, 2H), 3.37 (s, 4H),1.28 (s,
9H). tR,LCMS ¼ 3.88 min; Purity 90%; MS (ESIþ): m/z ¼ 280 (MþH)þ.
5.4.12. Carboxymethyl-pyridin-4-ylmethyl-amino)-acetic acid (9a)
White solid. Yield 95%. 1H NMR (DMSO-d6)
d ppm: 8.48 (d,
J ¼ 5.7 Hz, 2H), 7.29 (d, J ¼ 5.7 Hz, 2H), 3.83 (s, 2H), 3.35 (s, 4H);
5.4.21. (Carboxymethyl-phenylacetyl-amino)-acetic acid (21a)
Iminodiacetic acid was stirred overnight in an 80/20 MeOH/
SOCl2 mixture and evaporated. To the diester obtained (591 mg,
3.0 mmol) in solution in CH2Cl2 (15 mL) were added DIEA (2.08 mL,
12 mmol), phenylacetic acid (490 mg, 3.6 mmol), EDCI.HCl (747 mg,
3.9 mmol), HOBt (597 mg, 3.9 mmol). The mixture was stirred
overnight at room temperature and washed with HCl 1 N solution,
NaHCO3 10% solution and H2O. The organic layer was dried over
MgSO4 and evaporated to give the diester as a colourless oil. The
diester was saponified with NaOH (15 mmol) in MeOH (10 mL) and
H2O (1 mL). MeOH was evaporated, H2O was added and the
aqueous layer was extracted with CH2Cl2 (3 times). The aqueous
layer was acidified to pH 2 and extracted 4 times with AcOEt to give
the 21a. White solid. Yield 70% over two steps. 1H NMR (DMSO-d6)
tR,LCMS ¼ 0.87 min; Purity 98%; MS (ESI-): m/z ¼ 223 (MꢁH)ꢁ.
5.4.13. (Carboxymethyl-naphthalen-2-ylmethyl-amino)-acetic acid
(10a)
White solid. Yield 57%. 1H NMR (DMSO-d6)
d ppm: 7.89e7.85 (m,
3H), 7.77 (s, 1H), 7.57 (dd, J ¼ 1.5 and 8.4 Hz, 1H), 7.51e7.45 (m, 2H).
tR,LCMS ¼ 3.13 min; Purity 95%; MS (ESIþ): m/z ¼ 274 (MþH)þ.
5.4.14. (Carboxymethyl-(2-(1H-indol-3-yl)ethyl)-amino)-acetic
acid) (11a)
White solid. Yield 58%. 1H NMR (DMSO-d6)
d ppm: 10.77 (s, NH),
7.49 (d, J ¼ 7.7 Hz, 1H), 7.31 (d, J ¼ 8.04 Hz, 1H), 7.16 (d, J ¼ 2.16 Hz,
1H), 7.04 (dt, J ¼ 1.1 Hz, 7.1 Hz and 8.04 Hz, 1H), 6.94 (dt, J ¼ 1.1 Hz,
7.1 Hz and 7.7 Hz, 1H), 3.50 (s, 4H), 2.88 (m, 4H); tR,LCMS ¼ 3.28 min;
Purity 98%; MS (ESIþ): m/z ¼ 277 (MþH)þ.
d
ppm: 7.27 (m, 5H), 4.22 (s, 2H), 3.99 (s, 2H), 3.65 (s, 2H).
tR,LCMS ¼ 2.83 min, Purity 98%; MS (ESIþ): m/z ¼ 252 (MþH)þ.
5.4.15. (Carboxymethyl-phenethyl-amino)-acetic acid (12a)
5.4.22. (Carboxymethyl-(3-phenyl-propionyl)-amino)-acetic acid
(22a)
White solid. Yield 36%. 1H NMR (DMSO-d6)
d ppm: 7.22 (m, 5H),
3.46 (s, 4H), 2.86 (m, 2H), 2.70 (m, 2H). tR,LCMS ¼ 1.12 min, Purity
Was synthesized following procedure described for 20a from
iminodiacetic acid and hydrocinnamoyl chloride. White solid. Yield
100%; MS (ESIþ): m/z ¼ 238 (MþH)þ.
99%. 1H NMR (DMSO-d6)
d ppm: 7.23 (m, 5H), 4.18 (s, 2H), 3.98 (s,
5.4.16. (Carboxymethyl-(3-phenyl-propyl)-amino)-acetic acid
2H), 2.79 (t,
J
¼
8.1 Hz, 2H), 2.56 (t,
J
¼
8.1 Hz, 2H).
(13a)
tR,LCMS ¼ 3.41 min, Purity 97%; MS (ESIþ): m/z ¼ 266 (MþH)þ.
White solid. Yield 60%. 1H NMR (DMSO-d66)
d ppm: 7.13e7.26
(m, 5H), 3.41 (s, 4H), 2.65 (t, J ¼ 7.5 Hz, 2H), 2.56 (t, J ¼ 7.5 Hz, 2H),
1.67 (dt, J ¼ 7.5 Hz, 2H). tR,LCMS ¼ 2.76 min; Purity 99%; MS (ESIþ):
m/z ¼ 252 (MþH)þ.
5.4.23. (Benzyloxycarbonyl-carboxymethyl-amino)-acetic acid
(23a)
To a solution of iminodiacetic acid (266 mg, 2.0 mmol) in 2 N

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
559
NaOH solution in an ice bath, was added benzylchloroformate
(337 L, 2.4 mmol). The mixture was stirred at room temperature
55.5, 53.6, 52.4, 50.9, 26.2, 20.7. tR,LCMS ¼ 2.86 min, Purity 99%; MS
(ESIþ): m/z ¼ 389 (MþH)þ.
m
for 2 h and extracted twice with Et2O. The aqueous layer was
acidified to pH 2 and extracted with Et2O (3 times). The organic
layers were dried over MgSO4 and evaporated under reduced
pressure to give 24a as a colourless oil directly used in the next step.
Yield 64%. tR,LCMS ¼ 3.49 min, Purity 98%; MS (ESIꢁ): m/z ¼ 266
(MꢁH)ꢁ.
5.4.27. (S)-2-(2-((4-tert-Butyl-benzyl)-carboxymethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (8)
First N-4-(tert-butyl)benzyliminodiacetic acid 8a and H-His(1-
trt)-OMe HCl reacted as described in general procedure A to give
the protected intermediate (S)-2-(2-((4-tert-butyl-benzyl)-carbox-
ymethyl-amino)-acetylamino)-3-(1-trityl-1H-imidazol-4-yl)-pro-
pionic acid methyl ester. White solid. Yield 79%. 1H NMR (DMSO-d6)
5.4.24. (S)-2-(2-(Carboxymethyl-(4-fluoro-benzyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (5)
Compound (5) was synthesized according to general procedure
d
ppm: 8.47 (d, J ¼ 8.1 Hz, 1H), 7.37 (m, 10H), 7.26 (d, J ¼ 8.4 Hz, 2H),
7.20 (d, J ¼ 8.4 Hz, 2H), 7.03 (m, 6H), 6.65 (s, 1H), 4.62 (m, 1H), 3.74
A from 5a and
L
-histidine methyl ester dihydrochloride. White
ppm: 8.38 (d, J ¼ 8.1 Hz, 1H),
(s, 2H), 3.52 (s, 3H), 3.22 (s, 2H), 3.16 (s, 2H), 2.92 (m, 2H), 1.19 (s,
solid. Yield 37%. 1H NMR (DMSO-d6)
d
9H). 13C NMR (DMSO-d6)
d ppm: 172.1, 171.6, 170.1, 149.5, 142.1,
7.58 (d, J ¼ 0.9 Hz,1H), 7.35e7.30 (m, 2H), 7.15e7.09 (m, 2H), 6.84 (d,
J ¼ 0.9 Hz, 1H), 4.57 (m, 1H), 3.75 (d, J ¼ 13.2 Hz, 1H), 3.69 (d,
J ¼ 13.2 Hz, 1H), 3.58 (s, 3H), 3.23 (s, 2H), 3.22 (s, 2H), 2.98 (dd,
J ¼ 6.9 and 15 Hz, 1H), 2.92 (dd, J ¼ 5.4 and 14.7 Hz, 1H)).
tR,LCMS ¼ 2.54 min, Purity 100%; MS (ESIþ): m/z ¼ 393 (MþH)þ.
137.9, 136.3, 135.0, 129.2, 128.6, 128.2, 128.0, 124.9, 119.1, 74.5, 56.6,
56.5, 52.6, 34.1, 31.1, 29.9. tR,LCMS ¼ 6.32 min Purity 94%; MS (ESIþ):
m/z ¼ 673 (M þ H)þ. Deprotection using TFA/DCM in the presence
of triisopropylsilane allowed 8 as colourless oil (TFA salt). Yield 84%.
1H NMR (DMSO-d6)
d
ppm: 8.96 (s, 1H), 8.40 (d, J ¼ 8.1 Hz, 1H), 7.36
(s, 1H), 7.33 (d, J ¼ 8.4 Hz, 2H), 7.20 (d, J ¼ 8.4 Hz, 2H), 4.70 (m, 1H),
3.71 (s, 2H), 3.65 (s, 3H), 3.30 (s, 2H), 3.26 (s, 2H), 3.19 (dd, J ¼ 5.6
and 15.4 Hz, 1H), 3.09 (dd, J ¼ 8.8 and 15.4 Hz, 1H),1.27 (s, 9H) 13C
5.4.25. (S)-2-(2-(Carboxymethyl-(4-trifluoromethyl-benzyl)-
amino)-acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl
ester (6)
NMR (DMSO-d6) d ppm: 172.5,171.3, 170.7,150.2,134.9,134.2,129.6,
First N-4-(trifluoromethyl)benzyliminodiacetic acid 6a and H-
His(1-trt)-OMe HCl reacted as described in general procedure A to
give the trityle protected intermediate (S)-2-(2-(carboxymethyl-(4-
trifluoromethyl-benzyl)-amino)-acetylamino)-3-(1-trityl-1H-imi-
dazol-4-yl)-propionic acid methyl ester. White solid. Yield 75%. 1H
129.3, 125.5, 117.6, 57.5, 56.7, 54.1, 52.8, 51.2, 34.7, 31.6, 26.7.
tR,LCMS ¼ 4.13 min, Purity 95%; MS (ESIþ): m/z ¼ 431 (MþH)þ.
5.4.28. (S)-2-(2-(Carboxymethyl-pyridin-4-ylmethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (9)
First trityle protected intermediate was synthesized from 9a and
H-His(1-trt)-OMe HCl following general procedure A. Yield 41%. 1H
NMR (DMSO-d6)
d
ppm: 8.50 (d, J ¼ 8.3 Hz, 1H), 7.61 (d, J ¼ 8.1 Hz,
2H), 7.5 (d, J ¼ 8.1 Hz, 2H), 7.36 (m, 9H), 7.25 (d, J ¼ 1.2 Hz, 1H),
7.00e7.04 (m, 6H), 6.65 (d, J ¼ 1.2 Hz, 1H), 4.59 (m, 1H), 3.88 (s, 2H),
3.51 (s, 3H), 3.29 (s, 4H), 2.95 (dd, J ¼ 6.5 and 14.6 Hz, 1H), 2.87 (dd,
NMR (DMSO-d6)
d
ppm: 8.65 (d, J ¼ 4.8 Hz, 0.3 CONH), 8.62 (d,
J ¼ 4.8 Hz, 0.6 CONH), 8.48 (d, J ¼ 5.7 Hz,1H), 8.36 (d, J ¼ 5.7 Hz,1H),
8.2 (s, HCOOH), 7.36e7.18 (m, 15H(Trt)þ1H), 7.04e7.01 (m, 2H),
6.85 (s, 0.3H), 6.65 (s, 0.6H), 4.56 (m, 1H), 3.81 (s, 2H), 3.55 (s,
CO2Me), 3.27 (s, 2H), 3.24 (s, 2H), 2.95 (m, 2H); tR,LCMS ¼ 4.59 min,
Purity 95%; MS (ESIþ): m/z ¼ 618 (M þ H)þ. Deprotection using
TFA/DCM/TIS mixture allowed 9 as ist formiate salt after prepara-
J ¼ 5.4 and 14.6 Hz, 1H). 13C NMR (DMSO-d6)
d ppm: 172.0, 171.6,
169.9, 143.3, 142.1, 137.9, 136.4, 129.9, 129.6, 128.6, 128.5, 126.41,
125.4, 119.2, 74.5, 56.9, 56.8, 53.3, 51.8, 29.5 tR,LCMS ¼ 5.87 min,
Purity 99%; MS (ESIþ): m/z ¼ 685 (MþH)þ. Deprotection using TFA/
DCM in the presence of triisopropylsilane allowed 6 as an oil (TFA
salt). Yield 100%. 1H NMR (DMSO-d6)
d
ppm: 8.97 (s, 1H), 8.42 (d,
tive HPLC purification. Yield 75%. 1H NMR (DMSO-d6)
d ppm:
J ¼ 8.3 Hz, 1H), 7.68 (d, J ¼ 8.2 Hz, 2H), 7.54 (d, J ¼ 8.2 Hz, 2H), 7.37
(s, 1H), 4.80 (m, 1H), 3.85 (s, 2H), 3.63 (s, 3H), 3.35 (s, 2H), 3.28 (s,
2H), 3.18 (dd, J ¼ 5.3 and 15.3 Hz, 1H), 3.08 (dd, J ¼ 9.1 and 15.3 Hz,
8.5e8.47 (m, CONHþ2H), 8.2 (s, HCOOH), 7.59 (d, J ¼ 1.2 Hz, 1H),
7.33 (d, J ¼ 5.7 Hz, 2H), 6.85 (d, J ¼ 1.2 Hz, 1H), 4.55 (m, 1H), 3.80 (s,
2H), 3.58 (s, CO2Me), 3.27 (s, 2H), 3.24 (s, 2H), 2.96 (m, 2H); 13C
1H) 13C NMR (DMSO-d6)
d
ppm: 172.5, 171.3, 170.6, 143.4, 132.4,
NMR (DMSO-d6) d ppm: 172.5, 171.9, 170.3, 163.8, 149.6, 147.8, 135.6,
130.0, 129.6, 126.4, 125.5, 117.5, 57.4, 56.6, 54.4, 52.8, 51.2, 26.6
123.8, 116.8, 57.0, 56.6, 54.3, 52.1, 52.0, 28.8; tR,LCMS ¼ 0.79 min
tR,LCMS ¼ 4.68 min, Purity 99%; MS (ESIþ): m/z ¼ 443 (MþH)þ.
(5 min gradient), Purity 99%; MS (ESIþ): m/z ¼ 376 (MþH)þ.
5.4.26. (S)-2-(2-(Carboxymethyl-(4-methyl-benzyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (7)
Compound (7) was synthesized following general procedure A
from 7a and H-His(1-trt)-OMe HCl to give the trityle protected
5.4.29. (S)-2-(2-(Carboxymethyl-naphthalen-2-ylmethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (10)
Compound (10) was synthesized according to general procedure
A from 10a and
L
-histidine methyl ester dihydrochloride. White
intermediate as a white solid. Yield 40%. 1H NMR (DMSO-d6)
d
ppm:
solid. 45%. 1H NMR (DMSO-d6)
d
ppm: 8.44 (d, J ¼ 8.1 Hz, 1H), 8.14
8.57 (d, J ¼ 8.4 Hz, 1H), 7.35e7.39 (m, 9H), 7.26 (d, J ¼ 1.2 Hz, 1H),
7.21 (d, J ¼ 7.9 Hz, 2H), 7.01e7.04 (m, 6H), 6.95 (d, J ¼ 7.9 Hz, 2H),
6.64 (d, J ¼ 1.2 Hz, 1H), 4.58e4.64 (m, 1H), 3.71 (s, 2H), 3.51 (s, 3H),
3.25 (s, 2H), 3.18 (s, 2H), 2.95 (dd, J ¼ 6.2 and 14.5 Hz, 1H), 2.87 (dd,
(s, 1H, HCOOH), 7.82e7.90 (m, 3H), 7.78 (s, 1H), 7.71 (d, J ¼ 0.9 Hz,
1H), 7.49e7.56 (m, 3H), 6.91 (d, J ¼ 0.9 Hz, 1H), 4.59 (m, 1H), 3.95 (d,
J ¼ 13.5 Hz, 1H), 3.90 (d, J ¼ 13.5 Hz, 1H), 3.59 (s, 3H), 3.32 (s, 2H),
3.29 (s, 2H), 2.99 (m, 2H), tR,LCMS ¼ 3.46 min, Purity 100%; MS
(ESIþ): m/z ¼ 425 (MþH)þ.
J ¼ 5.0 and 14.5 Hz, 1H), 2.21 (s, 3H). 13C NMR (DMSO-d6)
d ppm:
172.3, 171.6, 170.2, 142.1, 137.7, 136.2, 135.1, 129.2, 128.9, 128.7, 128.3,
128.2, 119.1, 74.5, 57.1, 57.0, 53.2, 51.8, 29.6, 20.7. tR,LCMS ¼ 5.28 min
(10 min gradient), Purity 95%; MS (ESIþ): m/z ¼ 631 (M þ H)þ. Then
deprotection using TFA/DCM/TIS allowed 7 as ist TFA salt. White
5.4.30. (S)-2-(2-(Carboxymethyl-(2-(1H-indol-3-yl)-ethyl)-
amino)-acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl
ester (11)
The compound was synthesized according to general procedure
A starting from 11a and H-His(1-trt)-OMe HCl to give protected
intermediate (S)-2-(2-(Carboxymethyl-(2-(1H-indol-3-yl)-ethyl)-
amino)-acetyl amino)-3-(1-trityl-1H-imidazol-4-yl)-propionic acid
methyl ester. White solid. Yield 45%. tR,LCMS ¼ 6.93 min, Purity 98%;
MS (ESIþ): m/z ¼ 670 (M þ H)þ. Deprotection using TFA/DCM in the
solid. Yield 100%. 1H NMR (DMSO-d6)
d
ppm: 8.98 (d, J ¼ 1.2 Hz, 1H),
8.54 (d, J ¼ 8.1 Hz,1H), 7.38 (d, J ¼ 1.2 Hz,1H). 7.20 (d, J ¼ 8.1 Hz, 2H),
7.15 (d, J ¼ 8.1 Hz, 2H), 4.70 (m, 1H), 3.83 (s, 2H), 3.65 (s, 3H), 3.45 (s,
2H), 3.41 (s, 2H), 3.19 (dd, J ¼ 5.4 and 15.3 Hz, 1H), 3.08 (dd, J ¼ 9.0
and 15.3 Hz, 1H), 2.29 (s, 3H), 13C NMR (DMSO-d6)
d ppm: 171.0,
170.7, 169.7, 158.0, 137.1, 133.7, 132.7, 129.5, 129.1, 129.0, 117.1, 57.3,

560
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
presence of triisopropylsilane allowed 11 as a white solid (TFA salt).
Yield 86%. 1H NMR (DMSO-d6)
ppm: 10.92 (sl, NH), 8.97 (s, 1H),
(DMSO-d6)
d
ppm: 8.63 (d, J ¼ 7.8 Hz,1H), 7.66 (s, 1H), 7.47e7.36 (m,
d
9H), 7.11e7.06 (m, 6H), 6.82 (s,1H), 4.55 (m,1H), 4.09 (s, 2H), 3.58 (s,
1H), 2.96 (dd, J ¼ 5.7 Hz, J ¼ 14.7 Hz, 1H), 2.88 (dd, J ¼ 8.3 Hz,
J ¼ 14.7 Hz, 1H); tR,LCMS ¼ 2.81 min, Purity 97%; MS (ESIþ): m/
z ¼ 488 (M þ H)þ.
8.93 (m, CONH), 7.53 (d, J ¼ 7.8 Hz, 1H), 7.40 (s, 1H), 7.35 (d,
J ¼ 8.1 Hz, 1H), 7.17 (d, J ¼ 1.8 Hz, 1H), 7.08 (t, J ¼ 7.2 Hz, 1H), 6.98 (t,
J ¼ 7.8 Hz, 1H), 4.71 (m, 1H), 3.96 (s, 2H), 3.87 (s, 2H), 3.62 (s,
CO2Me), 3.22e2.98 (m, 6H); 13C NMR (DMSO-d6)
d ppm: 170.5,
169.4, 166.6, 136.3, 134.6, 128.8, 126.8, 123.3, 121.3, 118.5, 118.2,
117.3, 111.6, 109.5, 55.7, 54.4, 54.9, 52.5, 51.4, 26.1, 21.0;
tR,LCMS ¼ 4.17 min (at pH 3.8), Purity 97%; MS (ESIþ): m/z ¼ 428
(MþH)þ.
5.4.35. (1-Methyl-3-phenyl-propylamino)-acetic acid tert-butyl
ester (15b)
To a stirred solution of 1-methyl-3-phenylpropylamine (321
2 mmol) and DIEA (870 L, 5 mmol) in THF (5 mL) was added
dropwise at 0 ꢀC tert-butylbromoacetate (322
L, 2 mmol). The
mL,
m
m
5.4.31. (S)-2-(2-(Carboxymethyl-phenethyl-amino)-acetylamino)-
3-(1H-imidazol-4-yl)-propionic acid methyl ester (12)
resulting mixture was stirred at room temperature overnight. The
insoluble was filtrated and filtrate was washed with brine, dried
over MgSO4 and evaporated under reduced pressure. The crude
product was purified by flash chromatography to give 15b. Yield
Compound (12) was synthesized from 12a and L-histidine
methyl ester dihydrochloride following general procedure A, as its
formiate salt. White solid. Yield 52%. 1H NMR (DMSO-d6)
63%. 1H NMR (DMSO-d6)
d ppm: 7.25e7.15 (m, 5H), 3.30 (m, 1H),
d
ppm:8.22 (d, J ¼ 7.8 Hz, 1H), 8.14 (s, 1H, HCOOH), 7.56 (d,
3.20 (s, 2H), 2.58 (m, 2H), 1.71e1.51 (m, 2H), 1.41 (s, 9H), 0.99 (d,
J ¼ 6.2 Hz, 2H); tR,LCMS ¼ 4.73 min, Purity 97%; MS (ESIþ): m/z ¼ 264
(M þ H)þ.
J ¼ 1.2 Hz, 1H), 7.28e7.22 (m, 2H), 7.18e7.13 (m, 3H), 6.82 (d,
J ¼ 1.2 Hz, 1H), 4.52 (m, 1H), 3.58 (s, 3H), 3.38 (s, 2H), 3.25 (s, 2H),
2.92 (m, 2H), 2.76 (m, 2H), 2.66 (m, 2H); tR,LCMS ¼ 2.63 min, Purity
96%; MS (ESIþ): m/z ¼ 389 (M þ H)þ. HRMS (m/z): (Mþ) calcd. for
5.4.36. (S)-2-(2-(Carboxymethyl-(1-methyl-3-phenyl-propyl)-
amino)-acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl
ester (15)
C
19H25O5N4, 389.1825; found, 389.1820.
5.4.32. (S)-2-(2-(Carboxymethyl-(3-phenyl-propyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (13)
The compound was synthesized according to general procedure
A starting from 13a and H-His(1-trt)-OMe HCl to give the protected
To a stirred solution of 31 (102 mg, 0.2 mmol) and NaHCO3
(66 mg, 0.6 mmol) in anhydrous DMF (1 mL) was added 33 (61 mg,
0.23 mmol). The mixture was stirred at room temperature for 1 h
then refluxed 20 h. KI (15 mg, 0.09 mmol) was then added and the
resulting mixture was refluxed 1 h. The solvent was evaporated
under reduced pressure and the residue was precipitated in water.
The resulting solid was purified by preparative HPLC to give pro-
intermediate. Yield 57%. 1H NMR (DMSO-d6)
d ppm: 8.49 (d,
J ¼ 8.2 Hz, 1H), 6.99e7.38 (m, 21H), 6.62 (s, 1H), 4.58 (ddd, J ¼ 6 Hz,
8.1 Hz, 1H), 3.51 (s, 3H), 3.28 (s, 2H), 3.19 (s, 2H), 2.88 (m, 2H), 2.59
(t, J ¼ 7.4 Hz, 2H), 2.5 (m, 2H), 1.62 (dt, J ¼ 7.4 Hz, 2H). 13C NMR
tective intermediate as an oil. Yield 45%. 1H NMR (DMSO-d6)
d ppm:
(DMSO-d6)
d
ppm: 172.7,171.7,170.6,142.1, 137.6,136.3,129.2,128.2,
8.43 (d, J ¼ 8.4 Hz, 1H), 7.37e6.99 (m, 21H), 6.60 (s, 1H), 4.60 (m,
1H), 3.69e3.61 (m, 7H), 3.20e3.09 (m, 3H), 2.61 (m, 2H), 1.80e1.46
(m, 2H), 0.97 (m, 3H); tR,LCMS ¼ 3.94 min, Purity 98%; MS (ESIþ): m/
z ¼ 715 (M þ H)þ. Then protections were removed using TFA/DCM
(50/50) in the presence of triisopropylsilane. Solvents were evap-
orated under reduced pressure and the crude product was
precipitated in Et2O/pentane to give quantitatively 15 as its TFA salt.
128.0, 127.8, 127.5, 125.6, 119.0, 57.8, 55.1, 53.8, 51.8, 32.7, 29.8, 29.3,
tR,LCMS ¼ 5.21 min, Purity 92%; MS (ESIþ): m/z ¼ 645 (M þ H)þ.
Deprotection of the trityle group using TFA/DCM/TIS mixture
allowed 13 as its formiate salt after purification by preparative
HPLC. White solid. Yield 55%. 1H NMR (DMSO-d6)
d ppm: 8.98 (s,
1H), 8.86 (d, J ¼ 7.2 Hz, H), 7.41 (s, 1H), 7.17e7.30 (m, 5H), 4.70 (m,
1H), 3.78 (s, 2H), 3.70 (s, 2H), 3.62 (s, 3H), 3.11 (m, 2H), 2.90 (m, 2H),
1H NMR (DMSO-d6)
d
ppm: 8.98 (s, 1H), 8.92 (d, J ¼ 8.1 Hz, 1H), 7.4
2.57 (m, 2H), 1.79 (m, 2H). 13C NMR (DMSO-d6)
d
ppm: 170.6, 169.7,
(s, 1H), 7.37e7.17 (m, 5H), 4.72 (m, 1H), 3.69e3.61 (m, 7H),
3.20e3.01 (m, 3H), 2.57 (t, J ¼ 7.9 Hz, 2H), 1.85 (m, 1H), 1.59 (m, 1H),
167.5. 141.1, 133.7, 128.9, 128.4, 128.3, 126.0, 117.2, 55.7, 54.8, 54.7,
52.4, 51.2, 32.2, 26.8, 26.1. tR,LCMS ¼ 3.06 min; Purity 99%; MS (ESIþ):
m/z ¼ 403 (M þ H)þ; HRMS (m/z): (Mþ) calcd. for C20H27O5N4,
403.1981; found, 403.1978.
1.13 (m, 3H); 13C NMR (DMSO-d6)
d ppm: 170.5, 158.6, 158.1, 141.4,
135.14, 128.7, 128.3, 128.2, 125.9, 117.2, 52.4, 51.2, 33.7, 31.8, 26.3,
26.4, 14.3; tR,LCMS ¼ 2.22 min, Purity 96%; MS (ESIþ): m/z ¼ 417
(M þ H)þ.
5.4.33. (S)-2-(2-(Carboxymethyl-(4-phenyl-butyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (14)
5.4.37. (S)-2-(2-(Carboxymethyl-(n-hexyl)-amino)-acetylamino)-
3-(1H-imidazol-4-yl)-propionic acid methyl ester (16)
Was prepared from 14a and
chloride following general procedure A. White solid. Yield 73%. 1H
NMR (DMSO-d6)
L-histidine methyl ester dihydro-
The compound was prepared using procedure A starting from
16a and H-His(1-trt)-OMe HCl to give trityle-protected intermedi-
ate as a white solid. Yield 52%. tR,LCMS ¼ 4.93 min; Purity 83%; MS
(ESIþ): m/z ¼ 611 (M þ H)þ. Then deprotection using a TFA/DCM/
TIS mixture allowed 16 as its TFA salt. Yield 100%.
d
ppm: 8.28 (d, J ¼ 8.6 Hz, 1H), 8.04 (d, J ¼ 1.2 Hz,
1H), 7.28e7.12 (m, 5H), 7.01 (d, J ¼ 1.2 Hz, 1H), 4.59 (m, 1H), 3.59 (s,
1H), 3.30 (s, 2H), 3.19 (s, 2H), 3.02 (dd, J ¼ 6.1 Hz, J ¼ 15.2 Hz, 1H),
2.93 (dd, J ¼ 7.3 Hz, J ¼ 15.2 Hz, 1H), 2.56 (m, 4H), 1.52 (m, 2H), 1.40
(m, 1H); 13C NMR (DMSO-d6)
d
ppm: 172.5, 171.4, 170.6, 142.2, 134.7,
1H NMR (DMSO-d6)
d ppm: 8.99 (s, 1H), 8.80 (m, CONH), 7.41 (s,
131.9, 128.3,128.2, 125.6, 116.3, 57.6, 55.0, 54.0, 52.0, 51.5, 35.0, 28.5,
28.0, 26.7. tR,LCMS ¼ 3.41 min, Purity 99%; MS (ESIþ): m/z ¼ 417
(M þ H)þ.
1H), 4.70 (m, 1H), 3.76 (s, 2H), 3.65 (s, 5H(Ph)), 3.19 (dd, J ¼ 5.7 Hz
and 15.3 Hz, 1H), 3.07(dd, J ¼ 9.3 Hz and 15.3 Hz, 1H), 2.85 (m, 2H),
1.45 (m, 2H), 1.22 (m, 6H), 0.85 (t, J ¼ 6.3 Hz, 3H); 13C NMR (DMSO-
d6) d ppm: 171.1, 170.6, 168.5, 134.5,129.0,117.1, 56.1, 54.9, 54.7, 52.4,
5.4.34. (S)-2-(2-Chloro-acetylamino)-3-(1-trityl-1H-imidazol-4-
yl)-propionic acid methyl ester (15a)
To a stirred solution of L-His(Trt)-OMe (895 mg, 2 mmol) and
NaHCO3 (420 mg, 5 mmol) in DCM (10 mL) was added dropwise
51.1, 30.9, 26.5, 26.1, 25.9, 21.9, 13.9; tR,LCMS ¼ 3.03 min, Purity 98%;
MS (ESIþ): m/z ¼ 369 (M þ H)þ.
5.4.38. (S)-2-(2-(Carboxymethyl-amino)-acetylamino)-3-(1H-
imidazol-4-yl)-propionic acid methyl ester (17)
chloroacetylchloride (159 mL, 2 mmol). The mixture was stirred at
room temperature 10 min. The insoluble was filtered and the
To 17a (1 g, 4.29 mmol) in solution in dry THF (20 mL) was added
DCC (0.88 g, 4.29 mmol). The mixture was stirred overnight at room
filtrate evaporated to give the halide 15a quantitatively. 1H NMR

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
561
temperature and
L
-histidine methyl ester dihydrochloride (1.14 g,
0.8H), 4.02 (s, 1.2H), 3.88 (s, 2H), 3.64 (s, 1.8H), 3.59 (s, 1.2H),
2.85e3.02 (m, 2H). tR,LCMS ¼ 2.48 min; Purity 98%; MS (ESIþ): m/
z ¼ 389 (M þ H)þ.
4.67 mmol) and DIEA (2.1 mL) were added. After stirring at room
temperature during 5 h, the precipitate was filtrated and the filtrate
was evaporated. The crude product was purified by preparative
HPLC to give (S)-2-(2-(tert-butoxycarbonyl-carboxymethyl-
amino)-acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl
ester as a white solid. Yield 40% over 2 steps. 1H NMR (DMSO-d6)
5.4.42. (S)-2-(2-(Carboxymethyl-phenylacetyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (21)
Compound (21) was synthesized following general procedure A
from 21a and L-H-Histidine methyl ester dihydrochloride as a
yellow solid. Yield 77%. 2 conformers (30/70). 1H NMR (DMSO-d6)
d
ppm: 8.95 (d, J ¼ 7.4 Hz, 0.5H), 8.90 (d, J ¼ 7.4 Hz, 0.5H), 7.74 (d,
J ¼ 0.9 Hz, 0.5H), 7.72 (d, J ¼ 0.9 Hz, 0.5H), 6.88 (s, 1H), 4.50 (m, 1H),
3.85 (m, 2H), 3.83 (s, 2H), 3.58 (s, 3H), 2.95 (dd, J ¼ 5.7 and 14.9 Hz,
1H), 2.87 (dd, J ¼ 8.1 and 14.9 Hz, 1H), 1.33 (s, 4.5H), 1.28 (s, 4.5H);
tR,LCMS ¼ 2.65 min, Purity 99%; MS (ESIþ): m/z ¼ 385 (M þ H)þ. The
latter compound (300 mg, 0.78 mmol) was deprotected in presence
of HClg in DCM for 2 h at room temperature. DCM was evaporated
and the product was precipitated in Et2O to give 17 as its white
d
ppm: 8.87 (d, J ¼ 7.5 Hz, 0.7H, CONH), 8.84 (d, J ¼ 7.5 Hz, 0.3
CONH), 7.80 (s, 0.4H), 7.68 (s, 0.6H), 7.30e7.13 (m, 5H(Ph)), 6.92 (s,
0.3H), 6.89 (s, 0.7H), 4.56 (m, 0.7H), 4.46 (m, 0.3H), 4.13 (s, 0.6H),
4.10 (s, 1.4H), 3.95 (s, 2H), 3.63 (s, 0.6H), 3.61 (s, 2.1 CO2Me), 3.57 (s,
0.9 CO2Me), 3.56 (1.4H), 2.96 (m, 2H); 13C NMR (DMSO-d6)
d ppm:
171.6, 171.3, 171.1, 168.7, 135.2, 134.9, 134.7, 132.9, 129.3, 128.1, 126.3,
116.5, 52.2, 52.0, 51.8, 51.1, 49.3, 38.8, 38.7, 28.5. tR,LCMS ¼ 2.81 min,
Purity 99%; MS (ESIþ): m/z ¼ 403 (MþH)þ.
dihydrochloride salt. Yield 77%. 1H NMR (DMSO-d6)
d ppm: 9.36 (d,
J ¼ 7.5 Hz, 1H), 9.09 (d, J ¼ 1.2 Hz, 1H), 7.48 (s, 1H), 4.66 (m, 1H), 3.85
(s, 2H), 3.82 (s, 2H), 3.65 (s, 3H), 3.20 (dd, J ¼ 5.2 and 15.2 Hz, 1H),
3.10 (dd, J ¼ 9.0 and 15.4 Hz, 1H); tR,LCMS ¼ 0.68 min, Purity 100%;
MS (ESIþ): m/z ¼ 285 (M þ H)þ.
5.4.43. (S)-2-(2-(Carboxymethyl-(3-phenyl-propionyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester
(22)
5.4.39. (S)-2-(2-(Carboxymethyl-methyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid methyl ester (18)
H-His(trityl)OMe.HCl and commercial N-methyliminodiacetic
acid (18a) reacted following general procedure A to give interme-
diate (S)-2-(2-(Carboxymethyl-methyl-amino)-acetylamino)-3-(1-
trityl-1H-imidazol-4-yl)-propionic acid methyl ester as a white
Compound (22) was obtained from 22a as depicted for 20, as a
white solid. Yield 36%.1H NMR (DMSO-d6)
d
ppm: 8.89 (d, J ¼ 7.2 Hz,
1H), 7.74 (s, 0.4H), 7.61 (s, 0.6H), 7.28e7.13 (m, 5H), 6.89 (s, 0.4H),
6.84 (s, 0.6H), 4.48 (m, 1H), 4.06 (s, 2H), 3.93 (s, 2H), 3.58 (s, 1.2H),
3.54 (s, 1.8H), 2.95 (m, 2H), 2.82 (m, 2H), 2.50 (m, 2H). 13C NMR
(DMSO-d6) d ppm: 172.7, 172.5,171.6, 171.5, 168.9, 141.2, 134.9, 134.7,
solid. Yield 33%. 1H NMR (DMSO-d6)
d
ppm: 7.43 (d, J ¼ 1.5 Hz, 1H),
128.3, 128.2, 125.8, 116.8, 116.5, 52.5, 52.4, 51.9, 51.8, 49.5, 33.5, 30.3,
28.5, 28.4. tR,LCMS ¼ 3.25 min, Purity 98%; MS (ESIþ): m/z ¼ 417
(MþH)þ.
7.42e7.37 (m, 9H), 7.16e7.11 (m, 6H), 6.76 (d, J ¼ 1.5 Hz, 1H), 4.73
(m, 1H), 3.65 (s, 3H), 3.61 (s, 2H), 3.48 (s, 2H), 3.09 (dd, J ¼ 5.4 and
15.0 Hz, 1H), 2.98 (dd, J ¼ 7.8 and 14.7 Hz, 1H), 2.64 (s, 3H).
tR,LCMS ¼ 4.21 min, Purity 98%; MS (ESIþ): m/z ¼ 541 (M þ H)þ.
Deprotection allowed 22 as the TFA salt. Colourless oil. Yield 100%.
5.4.44. (S)-2-(2-(Benzyloxycarbonyl-carboxymethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester
(23)
The diacid 23a (1.2 mmol) was dissolved in trifluoracetic an-
hydride 2% in acetic anhydride (5 mL). The reaction mixture was
stirred for 4 h at room temperature then evaporated. To a stirred
1H NMR (DMSO-d6)
d
ppm: 9.16 (d, J ¼ 7.8 Hz, 1H), 8.99 (d,
J ¼ 1.2 Hz, 1H), 7.43 (d, J ¼ 1.2 Hz, 1H), 4.70 (m, 1H), 4.03 (s, 2H), 3.97
(s, 2H), 3.66 (s, 3H), 3.19 (dd, J ¼ 5.4 and 15.3 Hz, 1H), 3.06 (dd,
J ¼ 8.7 and 15.3 Hz, 1H), 2.80 (s, 3H), tR,LCMS ¼ 0.70 min, Purity 98%;
MS (ESIþ): m/z ¼ 299 (M þ H)þ.
solution of
L-histidine methyl ester dihydrochloride (290 mg,
1.2 mmol) in DMF (10 mL) were added DIEA (834
mL) and the an-
5.4.40. (S)-2-(2-(Carboxymethyl-phenyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid methyl ester (19)
hydride (1.2 mmol) in DMF (2 mL). The mixture was stirred over-
night at room temperature and the solvent was evaporated. The
crude product was purified by preparative HPLC to yield 23 as a
Compound (19) was synthesized from commercial N-phenyl-
iminodiacetic acid (19a) and L-H-histidine methyl ester dihydro-
white solid. Yield 32%. 1H NMR (DMSO-d6)
d
ppm: 9.00 (J ¼ 7.2 Hz,
chloride as a white solid. Yield 33%. 1H NMR (DMSO-d6)
d
ppm: 9.05
0.5H), 8.93 (d, J ¼ 7.5 Hz, 0.5H), 7.79 (s, 0.5H), 7.75 (s, 0.5H),
7.25e7.34 (m, 5H), 6.91 (s, 0.5H), 6.88 (s, 0.5H), 5.02 (s, 1H), 5.01 (s,
1H), 4.50 (m, 1H), 3.92 (m, 4H), 3.58 (s, 1.5H), 3.54 (s, 1.5H),
2.88e2.97 (m, 2H). tR,LCMS ¼ 3.15 min, Purity 98%; MS (ESIþ): m/
z ¼ 419 (M þ H)þ.
(d, J ¼ 7.6 Hz, CONH), 7.63 (d, J ¼ 1.1 Hz, 1H), 7.15 (dd, J ¼ 7.2 Hz and
8.6 Hz, 2H), 6.77 (s, 1H), 6.68 (t, J ¼ 7.2 Hz, 1H), 6.42 (d, J ¼ 8.1 Hz,
2H), 4.50 (m, 1H), 4.13 (s, 2H), 4.02 (s, 2H), 3.57 (s, CO2Me), 2.94 (dd,
J ¼ 5.3 Hz and 14.6 Hz, 1H), 2.83 (dd, J ¼ 8.7 Hz and 14.6 Hz, 1H); 13
C
NMR (DMSO-d6) d ppm: 173.5, 171.9, 171.6, 147.5, 135.1, 133.2, 129.4,
117.4, 116.7, 111.9, 56.0, 54.6, 52.3, 49.0, 29.0; tR,LCMS ¼ 2.94 min,
5.4.45. 3-(((S)-1-Methoxy-1-oxo-3-imidazol-2-yl)carbamoyl)-
1,2,3,4-tetrahydroisoquinoline-2-ethanoic acid (24)
Purity 98%; MS (ESIþ): m/z ¼ 361 (MþH)þ.
To
isoquinolinecarboxylic acid hydrochloride (426 mg, 2 mmol) and
DIEA (695 L, 2 eq) in H2O (10 mL) and dioxane (5 mL) was added
dropwise at 0 ꢀC tert-butylbromoacetate (322
L, 2 mmol) in
a
stirred
solution
of
1,2,3,4-Tetrahydro-3-
5.4.41. (S)-2-(2-(Benzoyl-carboxymethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid methyl ester (20)
The diacid 20a (0.74 mmol) was dissolved in trifluoracetic an-
hydride 2% in acetic anhydride (3 mL). The reaction mixture was
stirred for 4 h at room temperature then evaporated. To a stirred
m
m
dioxane (5 mL). The resulting mixture was stirred at room tem-
perature overnight. The insoluble was filtrated and filtrate was
washed with brine, dried over MgSO4 and evaporated under
reduced pressure. The crude product was directly used in the next
step. To a solution of carboxylic acid (2 mmol) in DCM (10 mL) were
added EDCI (422 mg, 2.2 mmol), HOBt (334 mg, 2.2 mmol), DIEA
solution of
L-histidine methyl ester dihydrochloride (188 mg,
0.77 mmol) in DMF (8.0 mL) were added DIEA (515
mL) and the
anhydride (0.74 mmol) in DMF (1.0 mL). The mixture was stirred
overnight at room temperature and the solvent was evaporated.
The crude product was purified by preparative HPLC to give 20 as a
(1390
mL, 8 mmol) and L-histidine methyl ester dihydrochloride
white solid. Yield: 26%. 1H NMR (DMSO-d6)
d
ppm: 8.89 (J ¼ 7.2 Hz,
(482 mg, 2 mmol). The mixture was stirred at room temperature
overnight. Then, the mixture was washed with a 5% NaHCO3
aqueous solution. The organic layer was dried over MgSO4 and
0.4H), 8.78 (d, J ¼ 7.5 Hz, 0.6H), 7.74 (s, 0.4H), 7.69 (s, 0.6H),
7.31e7.44 (m, 5H), 6.91 (s, 0.4H), 6.86 (s, 0.6H), 4.55 (m, 1H), 4.08 (s,

562
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
evaporated under reduced pressure. The crude product was puri-
fied by preparative HPLC to give the tert-butyl protected interme-
diate 24a. Yield 33% (two-steps). tR,LCMS ¼ 2.79 and 2.87 min, Purity
96%; MS (ESIþ): m/z ¼ 443.1 (MþH)þ. Then tert-butyl protection
was removed using TFA/DCM (50/50) in the presence of triisopro-
pylsilane. Solvents were evaporated under reduced pressure and
the crude product was precipitated in Et2O/pentane to give quan-
titatively 24 as a mixture of diastereoisomers (TFA salts). Yield: 95%.
0.55 mmol), DIEA (412 mL, 2.5 mmol) and L-histidine methyl ester
dihydrochloride(145mg, 0.6 mmol). The mixturewasstirredat room
temperature overnight. Then, the mixture was washed with a 5%
NaHCO3 aqueous solution. The organic layer was dried over MgSO4
and evaporated under reduced pressure. The crude product was
purified by preparative HPLC. Tert-butyl, Boc- and Trityle protections
were removed using TFA/DCM/TIS (5e50% TFA v/v) mixtures and
final compounds were purified by preparative HPLC if needed.
1H NMR (DMSO-d6) ppm: 1H NMR (DMSO-d6)
d d ppm: 9.1 (br s, 1H,
CONH), 9.0 (d, J ¼ 3.0 Hz, 1H); 7.33 (d, J ¼ 6.0 Hz, 1H); 7.12e7.25 (m,
5H), 4.64e4.68 (m, 1H), 4.15e4.36 (m, 3H), 3.84 (dd, J ¼ 7.50 Hz,
J ¼ 10.5 Hz, 2H), 3.64 (s, 3H), 2.99e3.22 (m, 4H). 13C NMR (DMSO-
5.4.48. (S)-2-(2-Benzylamino-acetylamino)-3-(1H-imidazol-4-yl)-
propionic acid methyl ester (26)
To a stirred solution of N-benzylglycine hydrochloride (402 mg,
2 mmol) in a H2O/dioxane mixture (1/4, 5 mL) were added, at 0 ꢀC,
Boc2O (524 mg, 2.4 mmol) and 2 mL of 2 N NaOH solution. The
mixture was stirred overnight at room temperature and dioxane
was evaporated. The aqueous layer was acidified with a 20% citric
acid solution and extracted by AcOEt. The organic layer was dried
over MgSO4 and evaporated to give (Benzyl-tert-butoxycarbonyl-
amino)-acetic acid (26a) as a colourless oil. Yield 100%. 1H NMR
d6)
d ppm: 170.5, 158.4, 158.3, 128.9, 128.1, 127.5, 126.7, 126.6, 114.1,
60.7, 52.5; 51.3, 17.9. tR tR,LCMS ¼ 1.74 and 1.84 min, Purity 98%; MS
(ESIþ): m/z ¼ 387 (MþH)þ.
5.4.46. 3-Benzyl-4-((S)-2-(1H-imidazol-4-yl)-1-methylcarbamoyl-
ethylcarbamoyl)-butyric acid (25)
To a stirred solution of LAH (600 mg, 15 mmol) in THF (4 mL) at
0
ꢀC were added dropwise a solution of diethyl benzylmalonate
(DMSO-d6) d ppm: 7.23e7.35 (m, 5H), 4.4 (s, 1.1H), 4.38 (s, 0.9H),
(940
m
L, 4 mmol) in THF (4 mL). The mixture was then refluxed
3.83 (s, 0.9H), 3.74 (s, 1.1H), 1.38 (s, 1.8H), 1.34 (s, 9.9H);
overnight. The excess of LAH was quenched with wet Na2SO4. The
solid was eliminated and the filtrate evaporated to give 2-benzyl-
propane-1,3-diol. Yield 70%. tR,LCMS ¼ 1.93 min, Purity 98%; MS
(ESIþ): m/z ¼ 167 (MþH)þ. To a stirred solution of the diol (623 mg,
3.75 mmol) in DCM (15 mL) were added p-toluenesulfonyl chloride
tR,LCMS ¼ 5.4 min, Purity 99%; MS (ESI-): m/z ¼ 264 (MꢁH)-.26a
reacted with L-Histidine methyl ester dihydrochloride following
general procedure B to give Boc-protected intermediate (S)-2-(2-
(Benzyl-tert-butoxycarbonyl-amino)-acetylamino)-3-(1H-imida-
zol-4-yl)-propionic acid methyl ester as a white solid. Yield 51%. 1H
(2.86 g, 15 mmol) and triethylamine (1.56
mL, 11.25 mmol). The
NMR (DMSO-d6) d ppm: 11.84 (s, 1H), 8.30 (s, 1H), 7.52 (s, 1H),
mixture was stirred at roomtemperature overnight andwashed with
water. The organiclayerswere thenwashed with a saturated solution
of Na2CO3. The organic layers were dried over MgSO4 and the solvent
was removed under reduced pressure. The residue was purified by
silica gel chromatography to yield the 2-benzyl-propane-1,3-
7.33e7.18 (m, 5H), 6.82 (s, 1H), 4.52 (m, 1H), 4.35 (m, 2H), 3.74 (m,
2H), 3.59 (s, 3H), 2.88 (m, 2H), 1.34 (s, 9H); tR,LCMS ¼ 4.06 min, Purity
100%; MS (ESIþ): m/z ¼ 417 (M þ H)þ. The latter compound was
deprotected in presence of HClg in dioxane during 30 min at room
temperature. Dioxane was evaporated and the product was
precipitated in Et2O to give the 26 as its white hydrochloride salt.
ditosylate 25a. Yield 44%. 1H NMR (DMSO-d6)
d ppm:7.70 (d,
J ¼ 8.4 Hz, 4H), 7.46 (d, J ¼ 8.4 Hz, 4H), 7.19e7.155 (m, 3H), 6.95e6.92
(m, 2H), 3.91 (dd, J ¼ 10.2 Hz and 4.8 Hz, 2H), 3.80 (dd, J ¼ 10.2 Hz and
6 Hz, 2H), 2.52 (m, 2H), 2.43 (s, 6H), 2.25 (m, 1H), tR,LCMS ¼ 3.45 min
(5 min gradient), Purity 98%; MS (ESIþ): m/z ¼ 475 (M þ H)þ. To a
stirred solution of 25a (792 mg, 1.6 mmol) in DMSO (6 mL) were
added KCN (458 mg, 7 mmol). The mixture was heated at 80 ꢀC for
3.5 h. The mixture was diluted with a solution of NaCl and extracted
with DCM. The organic layers were dried over MgSO4 and the solvent
evaporated to give 3-benzyl-pentanedinitrile 25b directly in the next
reaction. tR,LCMS ¼ 2.58 min (5 min gradient), Purity 97%; MS (ESIþ):
m/z ¼ 186 (M þ H)þ. To a stirred solution ofdinitrile 25b(1.6mmol)in
water (4 mL) were added H2SO4 96% (4 mL). The mixture was
refluxed 3 days. The mixture was then extracted with DCM. The
organic layers were extracted with a diluted solution of NH4OH. The
aqueous layers were frozen dry to give 3-benzyl-pentanedioic acid
25c as an orange powder, directly used in the next step. Yield 40%.
tR,LCMS ¼ 2.18 min (5 min gradient), Purity 98%; MS (ESIþ): m/z ¼ 223
(MþH)þ. The compound was prepared using general procedure A
starting from 25c (134 mg, 0.6 mmol) and L-H-Histidine-methyl-
amide 40a (145 mg, 0.6 mmol). The crude product was purified by
preparative HPLC to give 25 as a white powder. Yield 45%. 1H NMR
Yield 74%. 1H NMR (DMSO-d6)
d
ppm: 9.26 (d, J ¼ 7.5 Hz, 1H), 9.04
(d, J ¼ 1.2 Hz, 1H), 7.52e7.38 (m, 6H), 4.67 (m, 1H), 4.11 (s, 2H), 3.71
(d, J ¼ 15.9 Hz, 1H), 3.70 (d, J ¼ 15.9 Hz, 1H), 3.65 (s, 3H), 3.20 (dd,
J ¼ 5.4 and 15.3 Hz, 1H), 3.08 (dd, J ¼ 8.4 and 15.0 Hz, 1H).
tR,LCMS ¼ 1.91 min, Purity 100%; MS (ESIþ): m/z ¼ 317 (M þ H)þ.
5.4.49. (S)-2-(2-(Benzyl-carbamoylmethyl-amino)-acetylamino)-
3-(1H-imidazol-4-yl)-propionic acid methyl ester (27)
N-benzyliminodiacetic acid (342 mg, 1.53 mmol) was dissolved
in trifluoracetic anhydride 2% in acetic anhydride (5 mL). The re-
action mixture was heated for 15 min and then stirred for 5 h at
room temperature and evaporated. The crude product was dis-
solved in DMF (3 mL). The mixture was saturated with gaseous
ammoniac and stirred overnight at room temperature. Then the
solvent was evaporated to yield (Benzyl-carbamoylmethyl-amino)-
acetic acid (27a) as yellow oil. Yield 98%. 1H NMR (DMSO-d6)
d ppm:
7.36e7.25 (m, 5H þ 1 CONH2), 7.15 (s, 1 CONH2), 3.76 (s, 2H), 3.32 (s,
2H), 3.16 (s, 2H). tR,LCMS ¼ 2.14 min (5 min gradient), Purity 96%; MS
(ESIþ): m/z ¼ 223 (M þ H)þ. 27a reacted with
L-histidine methyl
ester dihydrochloride following general procedure B as a white
solid (formiate salt). Yield 10%. 1H NMR (DMSO-d6)
d ppm: 8.73 (d,
(CD3OD)
d
ppm: 8.54 (s, CONH), 7.61 (s, 1H) 7.26e7.14 (m, 5H (Ph))
J ¼ 7.8 Hz, 1H), 7.63 (s, 1H), 7.59 (s, 1H), 7.30 (m, 5H), 7.23 (s, 1H),
6.88 (s, 1H), 4.59 (m, 1H), 3.11e2.86 (m, 2H), 2.70 (s, 3H), 2.60e2.49
6.89 (s, 1H), 4.57 (m, 1H), 3.65 (d, J ¼ 13.4 Hz, 1H), 3.57 (d,
(m, 2Hþ1H), 2.31e2.06 (m, 4H); 13C NMR (CD3OD)
d
ppm:175.2,
J ¼ 13.4 Hz, 1H), 3.57 (s, 3H), 3.08 (s, 2H), 3.02e2.96 (m, 4H). 13
C
173.9,141.4, 136.2,130.4,129.2,127.0, 118.0, 54.741.3, 41.0, 36.8, 36.6,
30.1, 26.4; tR,LCMS ¼ 1.92 min (5 min gradient), Purity 98%; MS (ESIþ):
m/z ¼ 373 (M þ H)þ.
NMR (DMSO-d6) d ppm: 172.2, 171.7, 169.9, 137.3, 135.2,133.8, 129.2,
128.2,127.3,115.8, 57.9, 57.5, 56.9, 51.9, 51.8, 28.6. tR,LCMS ¼ 2.77 min,
Purity 99%; MS (ESIþ): m/z ¼ 374 (MþH)þ.
5.4.47. General procedure B for the synthesis of compounds 26e7,
29e31
To a solution of carboxylic acid 26ae27a, 29ae31a (0.5 mmol) in
DCM (10 mL) were added EDCI (105 mg, 0.55 mmol), HOBt (85 mg,
5.4.50. (S)-2-(2-(Benzyl-methoxycarbonylmethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester (28)
1 (60 mg, 0.16 mmol) was stirred overnight at room temperature
in a 20% SOCl2 solution in MeOH (2 mL) and the solvent was

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
563
evaporated. The crude product was purified by preparative HPLC to
give 28 as colourless oil. Yield 73%. 1H NMR (CD3OD)
ppm: 7.87 (s,
under argon and the solvent was evaporated. The crude product
was dissolved in DCM and washed with water. Intermediate 30a
was obtained as orange oil and used directly in the next step. Yield
d
1H), 7.33e7.25 (m, 5H), 6.97 (s, 1H), 7.75 (m, 1H), 3.77 (d,
J ¼ 13.2 Hz, 1H), 3.71 (d, J ¼ 13.2 Hz, 1H), 3.73 (s, 3H), 3.67 (s, 3H),
3.36 (s, 2H), 3.31 (s, 2H), 3.21 (dd, J ¼ 4.5 and 14.1 Hz, 1H), 3.13 (dd,
J ¼ 7.5 and 15.0 Hz, 1H). tR,LCMS ¼ 3.42 min, Purity 100%; MS (ESIþ):
m/z ¼ 389 (MþH)þ.
93%. Then 30a was reacted with L-histidine methyl ester dihydro-
chloride following general procedure B to give the protected in-
termediate. White solid. Yield 51%. tR,LCMS ¼ 5.9 min, Purity 100%;
MS (ESIþ): m/z ¼ 633 (MþH)þ. Deprotection using a TFA 2%/TIS 5%/
DCM mixture allowed 30 as its TFA salt. Yield 98%. 1H NMR (DMSO-
5.4.51. (S)-2-(2-(Benzyl-(2-carboxy-ethyl)-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid methyl ester (29)
d6)
d
ppm: 10.66 (s, 1H), 8.97 (d, J ¼ 1.2 Hz, 1H), 8.68 (d, J ¼ 8.1 Hz,
1H), 7.38e7.30 (m, 5H), 4.71 (m, 1H), 3.72 (s, 2H), 3.64 (s, 3H),
To a stirred solution of N-benzylglycine (820 mg, 4 mmol) and
methanol (8 mL) was added thionyle chloride (2 mL) dropwise at
0
3.22e3.15 (m, 5H), 3.08 (dd,
J
¼
9.2 and 15.3 Hz, 1H).
tR,LCMS ¼ 2.6 min, Purity 100%; MS (ESIþ): m/z ¼ 390 (MþH)þ.
ꢀC. The mixture was stirred for 18 h. Then the solvent was
evaporated under reduced pressure to give the compound
benzylamino-acetic acid methyl ester as a white solid. Yield 100%.
5.4.53. (S)-2-(2-(Benzyl-(2-hydroxy-3,4-dioxo-cyclobut-1-enyl)-
amino)-acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl
ester (31)
1H NMR (DMSO-d6)
d ppm: 7.55e7.58 (m, 2H), 7.41e7.43 (m, 3H),
4.16 (s, 2H), 3.99 (s, 2H), 1.72 (s, 3H). tR,LCMS ¼ 2.01 min; Purity 99%;
MS (ESIþ): m/z ¼ 180 (M þ H)þ. To a stirred solution of
benzylamino-acetic acid methyl ester (222 mg, 1.03 mmol) in
acetone (10 mL) were added tert-butyl 3-bromopropionate
A solution of potassium tert-butylate (17.6 mL, 17.6 mmol, 1 eq.),
1 M in tetrahydrofuran was added at once to a solution of diethyl
squarate (3 g, 17.6 mmol, 1 eq.) in 50 mL of tetrahydrofuran at 4 ꢀC.
After 15 min of stirring, the reaction was quenched and acidified
with 1 N HCl (1 eq., pH after hydrolysis: 2e3). The mixture was
extracted three times with ether. The combined organic layers were
washed with saturated aqueous NaCl, dried over magnesium sul-
phate, filtered and solvent was evaporated under reduced pressure.
The crude product was purified by silica gel chromatography
(eluent: cyclohexane/AcOEt (97/3)) to give ethyl tert-butyl squarate
(3*834
mL, 3*5 mmol), K2CO3 (3*290 mg, 3*2.09 mmol) and KI
(174 mg, 1.04 mmol) and the mixture was refluxed overnight. The
solvent was evaporated and the residue was solubilized in ethyle
acetate. The organic layer was washed with water and brine then
dried over MgSO4 and concentrated under reduced pressure to give
a white powder, directly used in the next step. tR,LCMS ¼ 4.76 min;
Purity 99%; MS (ESIþ): m/z ¼ 308 (M þ H)þ. To a stirred solution of
the methyl ester (349 mg, 1.13 mg) in methanol (5 mL) was added
NaOH (74 mg, 1.85 mmol). The mixture was stirred at room tem-
perature for 5 days. The solvent was evaporated and the residue
was solubilised in water, then the aqueous layer was acidified with
a solution of HCl 1 N, and extracted with DCM. The organic layers
were washed with brine, dried over MgSO4 and concentrated to
give the corresponding 3-(benzyl-methoxycarbonylmethyl-
amino)-propionic acid tert-butyl ester (29a), directly used in the
next step. Yield 45%. tR,LCMS ¼ 3.76 min; Purity 95%; MS (ESIþ): m/
z ¼ 294 (MþH)þ. To a solution of the latter carboxylic acid 29a
(1.95 g, 55%). 1H NMR (CDCl3)
d
ppm: 4.762 (q, J ¼ 7.2 Hz, 2H), 1.60
(s, 9H), 1.48 (t, J ¼ 7.2 Hz, 3H). tR,LCMS ¼ 5.06 min, Purity 99%; MS
(ESIþ): m/z ¼ 143 (M-tBuþ2H)þ and 115 (M-tBu-Etþ2H)þ. To a
solution of N-Benzylglycine hydrochloride (305 mg,1.51 mmol) and
NEt3 (424 mL, 302 mmol) in MeOH (10 mL), was added ethyl tert-
butyl squarate (300 mg, 1.51 mmol). The mixture was stirred at
room temperature overnight and MeOH was evaporated. The crude
product was purified by preparative HPLC to give 31a as a colour-
less oil. Yield 70%. tR,LCMS ¼ 5.28 min, Purity 100%; MS (ESI-): m/
z ¼ 316 (MꢁH)ꢁ. Then 31a was reacted with
L-histidine methyl
ester dihydrochloride following general procedure B. The crude
product was purified by preparative HPLC to give protected inter-
mediate as a white solid. Yield 37%. tR,LCMS ¼ 4.3 min, Purity 100%;
MS (ESIþ): m/z ¼ 469 (MþH)þ. The latter intermediate was dis-
solved in dichloromethane (2 mL) and cooled at 4 ꢀC in an ice/water
bath. After 15 min of stirring, trifluoroacetic acid (2 mL) was added
(132 mg, 0.4 mmol) and oxalyl chloride (50 mL, 0.58 mmol) in DCM
(3 mL) was added at 0 ꢀC one drop of DMF. The mixture was stirred
for 30 min. Then the solvent was evaporated and the crude com-
pound was solubilized in DCM (3 mL). Then L-His-(Trt)-OMe
(181 mg, 0.4 mmol) and DIEA (313 mL, 1.8 mmol) were added, the
mixture was stirred overnight. The solvent was evaporated and the
and the mixture was stirred at 4 ꢀC for 30 min then 10
mL of distilled
crude product was purified by preparative HPLC to give the trityle-
water were added. The reaction mixture was concentrated under
reduced pressure and the crude product was precipitated in Et2O
and filtrated to give 31 as a purple solid. Yield 99%. 1H NMR (DMSO-
protected intermediate. Yield 40%. 1H NMR (DMSO-d6)
d ppm: 8.43
(d, J ¼ 8.10 Hz, 1H), 7.39e7.32 (m, 11H), 7.27 (d, J ¼ 1.2 Hz, 1H), 7.17
(m, 3H), 7.02 (m, 6H), 6.63 (d, J ¼ 1.2 Hz, 1H), 4.60 (m, 1H), 3.62 (d,
J ¼ 13.5 Hz, 1H), 3.54 (d, J ¼ 13.5 Hz, 1H), 3.51 (s, 3H), 3.32 (s, 2H),
3.03e2.89 (m, 2H þ2H), 2.64 (m, 2H), 1.31 (s, 9H);
tR,LCMS ¼ 6.96 min; Purity 99%; MS (ESIþ): m/z ¼ 687 (M þ H)þ.
Deprotection with a TFA/DCM/TIS mixture allowed 29 as its white
d6)
d
ppm: 8.97 (d, J ¼ 1.5 Hz, 1H), 8.57 (d, J ¼ 8.1 Hz, 1H), 7.40e7.22
(m, 5H), 4.70e4.66 (m, 1H), 4.65 (d, J ¼ 14.7 Hz, 1H), 4.58 (d,
J ¼ 14.7 Hz, 1H), 4.00 (d, J ¼ 16.5 Hz, 1H), 3.95 (d, J ¼ 16.5 Hz, 1H),
3.67 (s, 3H), 3.16 (dd, J ¼ 5.1 and 15.0 Hz, 1H), 3.03 (dd, J ¼ 9.0 and
15.0 Hz, 1H). tR,LCMS ¼ 3.02 min, Purity 100%; MS (ESIþ): m/z ¼ 413
(MþH)þ.
TFA salt. Yield 82%. 1H NMR (DMSO-d6)
d ppm: 8.98 (s, 1H), 8.90 (s,
1H), 7.40 (m, 6H), 4.68 (m, 1H), 4.1, (s, 2H), 3.65 (s, 3H), 3.56 (s, 2H),
3.16e3.03 (m, 2Hþ2H), 2.67 (t, J ¼ 7.5 Hz, 2H). 13C NMR (DMSO-d6)
5.4.54. (S)-2-(2-(Benzyl-(1H-tetrazol-5-ylmethyl)-amino)-
d
ppm: 172.3, 170.5, 133.9, 130.5, 128.9, 128.7, 117.3, 57.2, 53.8, 52.4,
acetylamino)-3-(1H-imidazol-4-yl)-N-methyl-propionamide (32)
First, (S)-2-(2-(Benzyl-carbamoylmethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-N-methyl-propionamide (32a) was synthesized
from 27a and L-H-Histidine-methylamide following general pro-
51.3, 49.3, 29.7, 26.1. tR,LCMS ¼ 2.39 min; Purity 99%; MS (ESIþ): m/
z ¼ 389 (M þ H)þ.
5.4.52. (S)-2-(2-(Benzyl-hydroxycarbamoylmethyl-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid methyl ester
(30)
To a stirred solution of O-trityl hydroxylamine (550 mg,
2.0 mmol) in DMF (10 mL) were added DIEA (660 mL) and N-ben-
cedure B as a yellow oil. Yield 80%. 1H NMR (DMSO-d6)
d (ppm):
8.46 (d, J ¼ 8.3 Hz, CONH), 7.71 (q, J ¼ 4.6 Hz, CONHMe), 7.52 (d,
J ¼ 1.0 Hz, 1H); 7.36e7.23 (m, 5H(Ph)), 6,76 (s, 1H), 4.42 (m, 1H),
3.64 (d, J ¼ 13.4 Hz, 1H), 3.57 (d, J ¼ 13.4 Hz,1H), 3.14e2.85 (m,
CONHMe), 2.54 (d, J ¼ 4.6 Hz, 3H); tR,LCMS ¼ 1.47 min (5 min
gradient), Purity 95%; MS (ESIþ): m/z ¼ 373 (M þ H)þ.To a stirred
solution of 32a (588 mg, 1.57 mmol) in THF (10 mL) at 0 ꢀC were
zyliminodiacetic anhydride acid (410 mg, 2.0 mmol) in DMF
(2.0 mL). The mixture was stirred overnight at room temperature

564
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
added pyridine (319
m
L, 3.94 mmol) and TFAA (241
m
L, 1.73 mmol)
(35a) as its white dihydrochloride salt. Yield 77%. 1H NMR (DMSO-
d6)
in three times over 3 h. Water was added and the mixture was
extracted with ethyl acetate. The combined organic layers were
washed successively with a saturated solution of NaHCO3 and brine
and dried over MgSO4. The solvent was evaporated and the crude
material was purified by preparative HPLC to give (S)-2-(2-(Benzyl-
cyanomethyl-amino)-acetylamino)-3-(1H-imidazol-4-yl)-N-methyl-
propionamide (32b) as an orange oil. Yield 58%. 1H NMR (CD3OD)
d
ppm: 9.08 (d, J ¼ 1.2 Hz, 1H), 7.53 (d, J ¼ 1.2 Hz, 1H), 4.93 (sept,
J ¼ 6.2 Hz, 1H), 4.13 (m, 1H), 2.83 (m, 2H), 1.35 (s, 9H(Boc)), 1.13 (d,
J ¼ 6.3 Hz, 3H),1.09 (d, J ¼ 6 Hz, 3H); tR,LCMS ¼ 0.65 min; Purity: 98%;
MS: (ESIþ) m/z ¼ 198 (M þ H)þ.N-Benzyliminodiacetic acid and 35a
reacted as described in general procedure A to give 35 as a col-
ourless oil. Yield 40%. 1H NMR (DMSO-d6)
d ppm: 8.41 (d,
J ¼ 8.10 Hz, CONH), 7.65 (s, 1H), 7.33e7.27 (m, 5H(Ph)), 6.87 (s, 1H),
4.83 (sept, J ¼ 6.30 Hz, 1H), 4.51 (m, 1H), 3.80 (d, J ¼ 13.2 Hz, 1H),
3.74 (d, J ¼ 13.2 Hz, 1H), 3.27 (s, 2H), 3.24 (s, 2H), 2.98 (dd, J ¼ 6.9 Hz
and 14.7 Hz, 1H), 2.92 (dd, J ¼ 5.4 Hz and 14.7 Hz, 1H), 1.08 (m, 6H);
d
(ppm): 7.91 (d, J ¼ 1.1 Hz, 1H), 7.36e7.30 (m, 5H(Ph)), 6.97 (s, 1H),
4.67 (ddd, J ¼ 8.2 Hz and 5.4 Hz, 1H), 3.72 (d, J ¼ 12.9 Hz, 1H), 3.66
(d, J ¼ 12.9 Hz, 1H), 3.62 (d, J ¼ 17.5 Hz, 1H), 3.54 (d, J ¼ 17.5 Hz, 1H),
3.16 (dd, J ¼ 14.9 Hz and 5.3 Hz,1H), 3,03 (dd, J ¼ 14.9 Hz and 8.2 Hz,
1H), 3.28 (s, 2H), 2.73 (s, CONHMe); tR,LCMS ¼ 1.77 min (5 min
gradient), Purity 100%; MS (ESIþ): m/z ¼ 355 (M þ H)þ. To a stirred
solution of 32b (108 mg, 0.3 mmol) in DMF were added sodium
azide (198 mg, 3.04 mmol) and ammonium chloride (163 mg,
3.04 mmol). The mixture was heated at 90 ꢀC for 60 h. The solvent
was evaporated and the crude material was diluted in EtOH and
filtered. The filtrate was concentrated under vacuum to give yellow
13C NMR (DMSO-d6)
d ppm: 172.2, 170.6, 170.1, 138.1, 135.1, 133.5,
128.8, 128.3, 127.2, 68.0, 57.4, 56.6, 53.3, 51.8, 28.7, 21.4;
tR,LCMS ¼ 3.23 min; Purity: 98%; MS: (ESIþ) m/z ¼ 403 (M þ H)þ.
5.4.58. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(3H-imidazol-4-yl)-propionic acid isobutyl ester (36)
First, to a stirred solution of L-histidine (463 mg, 2.98 mmol) in
iBuOH (5 mL) was added at 0 ꢀC thionyl chloride (2 mL). The
resulting solution was heated at 60 ꢀC for 2 days and the solvent
was evaporated to give 36a directly used in the next step. N-ben-
zyliminodiacetic acid and 36a reacted following general procedure
oil. Yield 97%. 1H NMR (CD3OD)
7.32e7.25 (m, 5H(Ph) þ 1H), 6.95 (s, 1H), 4.63 (dd, J ¼ 8.2 Hz and
5.2 Hz, 1H), 3.95 (d, J ¼ 14.1 Hz, 1H), 3.88 (d, J ¼ 14.1 Hz, 1H), 3.65 (s,
2H), 3.21e3.04 (m, 4H), 2.74 (s, CONHMe); 13C NMR (CD3OD)
d
(ppm): 8.07 (d, J ¼ 1.1 Hz, 1H),
A to give 36.White solid. Yield 8%. 1H NMR (DMSO-d6)
d ppm: 8.44
d
(ppm): 172.2, 171.8, 167.6, 158.4, 137.2, 134.4, 131.5, 129.1, 128.1,
(d, J ¼ 7.7 Hz, 1H), 7.60 (s, 1H), 7.29e7.22 (m, 5H), 6.85 (s, 1H), 4.57
(m, 1H), 3.78 (m, 2Hþ2H), 3.26 (s, 2H), 3.24 (s, 2H), 3.00 (d, J ¼ 6.7
and 14.7 Hz,1H), 2.93 (dd, J ¼ 5.4 and 14.7 Hz,1H),1.78 (m,1H), 0.79
127.2, 117.2, 58.6, 56.4, 52.5, 28.2, 25.1; tR,LCMS ¼ 1.95 min (5 min
gradient), Purity 95%; MS (ESIþ): m/z ¼ 398 (M þ H)þ.
(d, J ¼ 7.16 Hz, 6H). 13C NMR (DMSO-d6)
d ppm: 172.2, 171.3, 170.1,
5.4.55. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid (33)
138.1, 135.4, 134.0, 129.9, 128.3, 127.2, 115.6, 70.3, 57.5, 56.6, 53.4,
51.9, 28.9, 27.2, 18.7. tR,LCMS ¼ 3.52 min, Purity 99%; MS (ESIþ): m/
z ¼ 417 (M þ H)þ.
To a stirred solution of 1 (125 mg, 0.30 mmol) in MeOH (3 mL)
were added NaOH (36 mg, 0.90 mmol) and 50 mL of distilled water.
The mixture was stirred overnight at room temperature. The sol-
vent was removed under reduced pressure to give 33 as its white
5.4.59. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid tert-butyl ester (37)
sodium salt. Yield 99%. 1H NMR (CD3OD)
d
ppm: 7.45 (d, J ¼ 0.9 Hz,
N-Benzyliminodiacetic acid and commercial L-H-Histidine-(1-
trityl) tert-butylester 37a reacted as described in general proce-
dure A to give the trityle eprotected intermediate as a white
powder. Deprotection using TFA/DCM/TIS mixture allowed 37 as an
oil (formiate salt oil) after preparative HPLC. Yield 41%. 1H NMR
1H), 7.33e7.21 (m, 5H), 6.81 (s, 1H), 4.52 (dd, J ¼ 4.5 and 7.2 Hz, 1H),
3.78 (d, J ¼ 13.2 Hz, 1H), 3.66 (d, J ¼ 13.2 Hz, 1H). 3.25 (m, 2H), 3.19
(dd, J ¼ 11.1 and 15.6 Hz, 1H), 3.13 (d, J ¼ 16.5 Hz, 1H), 3.09 (dd,
J ¼ 7.8 and 15.3 Hz, 1H) 3.05 (d, J ¼ 16.5 Hz, 1H). tR,LCMS ¼ 1.86 min,
Purity 94%; MS (ESIþ): m/z ¼ 361 (M þ H)þ.
(DMSO-d6)
5H(Ph)), 7.14 (s, 1H), 4.50 (m, 1H), 3.76 (s, 2H), 3.29 (s, 2H), 3.22 (s,
2H), 3.02 (m, 2H), 1.33 (s, 9H(tBu)); 13C NMR (DMSO-d6)
ppm:
d
ppm: 8.38 (s, 1H), 8.32 (d, J ¼ 8.1 Hz, CONH), 7.28 (m,
5.4.56. (Benzyl-((2-(1H-imidazol-4-yl)-ethylcarbamoyl)-methyl)-
amino)-acetic acid (34)
d
172.3, 170.2, 169.9, 138.1, 134.7, 131.2, 128.9, 128.3, 127.3, 116.5, 81.1,
57.5, 56.5, 53.7, 51.8, 27.5, 27.4; tR,LCMS ¼ 3.41 min, Purity 98% MS
(ESIþ): m/z ¼ 417 (M þ H)þ.
Compound (34) was obtained following general procedure A
from histamine 34a and N-benzyliminodiacetic anhydride acid.
Colourless oil. Yield 47%. 1H NMR (DMSO-d6)
d ppm: 7.94 (t,
J ¼ 5.7 Hz, 1H), 7.67 (d, J ¼ 1.2 Hz, 1H), 7.33e7.22 (m, 5H), 6.85 (d,
J ¼ 0.6 Hz, 1H), 3.72 (s, 2H), 3.32 (m, 2H), 3.26 (s, 1H), 3.18 (s, 1H),
2.63 (t, J ¼ 6.9 Hz, 2H). tR,LCMS ¼ 1.99 min, Purity 99%; MS (ESIþ): m/
z ¼ 317 (M þ H)þ.
5.4.60. (Benzyl-(((S)-2-hydroxy-1-(1H-imidazol-4-ylmethyl)-
ethylcarbamoyl)-methyl)-amino)-acetic acid (38)
Compound (38) was synthesized from N-Benzyliminodiacetic
acid and histidinol 38a following general procedure A, as its for-
miate salt. Yield 47%. 1H NMR (DMSO-d6)
d
ppm: 7.81 (d, J ¼ 8.7 Hz,
5.4.57. (S)-2-(2-(Benzyl-carboxymethyl-amino)-acetylamino)-3-
(1H-imidazol-4-yl)-propionic acid isopropyl ester (35)
First to a stirred solution of Boc-His-OH (1.27 g, 5 mmol) in DCM
(30 mL) were added iPrOH (5 mL, 65 mmol), DMAP (671 mg,
1H), 7.71 (s, 1H), 7.27 (m, 5H), 6.83 (s, 1H), 3.98 (m, 1H), 3.71 (s, 2H),
3.37 (dd, J ¼ 4.5 and 10.5 Hz, 1H), 3.29 (dd, J ¼ 6.0 and 10.5 Hz, 1H),
3.24 (s, 2H), 3.16 (s, 2H), 2.77 (dd, J ¼ 6.0 and 15.0 Hz, 1H), 2.67 (dd,
J ¼ 7.5 and 15.0 Hz, 1H); 13C NMR (DMSO-d6)
d ppm: 172.5, 169.7,
5.5 mmol), EDCI (1.05 g, 5.5 mmol) and DIEA (956
mL, 5.5 mmol).
138.2, 134.6, 133.4, 128.9, 128.4, 127.3, 117.0, 62.4, 57.7, 57.1, 54.3,
50.2, 28.0.; tR,LCMS ¼ 2.24 min, Purity 98% MS (ESIþ): m/z ¼ 347
(M þ H)þ.
The mixture was heated at 50 ꢀC for 28 h. Then the mixture was
washed with a saturated solution of NaHCO3. The organic layer was
dried with MgSO4 and evaporated. The crude product was purified
by preparative HPLC to yield L-Boc-histidine isopropyl ester as
5.4.61. (Benzyl-(((S)-1-carbamoyl-2-(1H-imidazol-4-yl)-
ethylcarbamoyl)-methyl)-amino)-acetic acid (39)
colourless oil. Yield 56%. 1H NMR (DMSO-d6)
d
ppm: 7.58 (s, 1H),
7.14 (d, J ¼ 7.5 Hz, CONH), 6.80 (s,1H), 4.83 (sept, J ¼ 6.2 Hz,1H), 4.13
(m, 1H), 2.83 (m, 2H), 1.35 (s, 9H(Boc)), 1.13 (d, J ¼ 6.3 Hz, 3H), 1.09
(d, J ¼ 6 Hz, 3H); tR,LCMS ¼ 3.60 min; Purity: 98%; MS: (ESIþ) m/
z ¼ 298 (M þ H)þ. Then deprotection using HClg in DCM during
30 min at room temperature allowed L-H-Histidine isopropyl ester
To a stirred solution of 1 (150 mg, 0.5 mg) in dioxane/MeOH
(5 mL/2 mL) were added by portion gaseous NH3. When the reac-
tion is total, the solvent was evaporated. The crude product was
purified by preparative HPLC to give 39 as its formiate salt. Yield
79%. 1H NMR (DMSO-d6)
d
ppm: 8.95 (d, J ¼ 1.4 Hz, 1H), 8.34 (d,

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
565
J ¼ 8.7 Hz, 1H), 7.54 (s, 2H), 7.32 (m, 5H), 7.29 (s, 1H), 4.57 (m, 1H),
5.4.64. (Benzyl-(((S)-1-dimethylcarbamoyl-2-(1H-imidazol-4-yl)-
ethylcarbamoyl)-methyl)-amino)-acetic acid (42)
3.88 (s, 2H), 3.47 (s, 2H), 3.45 (s, 2H), 3.15 (dd, J ¼ 4.3 and 15.4 Hz,
1H), 2.96 (dd, J ¼ 8.5 and 15.4 Hz, 1H). 13C NMR (DMSO-d6)
d
ppm:
To a stirred solution of L-Boc-Histidine (765 mg, 3 mmol) in DMF
(20 mL) were added dimethylamine. HCl (293 mg, 3.6 mmol), HOBt
(551 mg, 3.6 mmol), EDCI (690 mg, 3.6 mmol) and TEA (1 mL,
6.8 mmol). The mixture was stirred overnight. The solvent was
evaporated. The crude product was solubilised in DCM and washed
with a saturated solution of NaHCO3. The organic layers were dried
over MgSO4 and evaporated to give L-Boc-histidine dimethyl
amide. The latter compound (198 mg, 0.7 mmol) was deprotected
in presence of gaseous HCl in dichloromethane during 30 min at
171.5, 158.6, 158.1, 135.4, 134.4, 129.8, 129.4, 128.5, 128.0, 116.7, 57.8,
55.8, 53.7, 51.1, 27.2. tR,LCMS ¼ 1.98 min (5 min gradient), Purity 98%
MS (ESIþ): m/z ¼ 360 (M þ H)þ.
5.4.62. (Benzyl-(((S)-2-(1H-imidazol-4-yl)-1-methylcarbamoyl-
ethylcarbamoyl)-methyl)-amino)-acetic acid (40)
To a stirred solution of L-H-histidine-(1-trityl) methyl ester
hydrochloride (2.5 g, 5.5 mmol) in methanol (10 mL) were added
4 mL of a solution of methylamine in ethanol (33%). The mixture
was refluxed overnight. The solvent was evaporated and the crude
product was precipitated in water and filtrated. The crude product
was purified by preparative HPLC to give L-H-Histidine-(1-trityl)-
methyl amide (40a) as a white powder. Yield 63%. 1H NMR (DMSO-
room temperature. The solvent was evaporated to give L-histidine
dimethyl amide 42a used directly in next step. 42a and N-Benzy-
liminodiacetic acid reacted following general procedure A to give
42 as a colourless oil (formiate salt) after preparative HPLC purifi-
cation. Yield 25%. 1H NMR (DMSO-d6)
d ppm: 8.17 (s, 2HCOOH), 8.12
d6)
d
ppm: 8.30 (s, 1H), 7.95 (q, J ¼ 4.8 Hz, CONHMe), 7.06e7.43 (m,
(d, J ¼ 8.4 Hz, CONH), 7.62 (s, 1H), 7.32e7.23 (m, 5H (Ph)), 6.78 (s,
1H), 4.96 (m, 1H), 3.72 (s, 2H), 3.26 (s, 2H), 3.18 (s, 2H), 2.93 (s, 3H
(CONMe2)), 2.90e2.70 (m, 3H (CONMe2) þ 2H);13C NMR (DMSO-
15H (Trt)), 6.63 (s, 1H), 3.53 (dd, J ¼ 5.7 Hz and 7.2 Hz, 1H), 2.77
(dd, J ¼ 5.7 Hz and 14.4 Hz, 1H), 2.63 (dd, J ¼ 5.7 Hz and 14.4 Hz,
1H), 2.54 (d, J ¼ 4.8 Hz, CONHMe); tR,LCMS ¼ 4.79 min; Purity: 74%;
MS: (ESIþ) m/z ¼ 411 (M þ H)þ. 40 was synthesized following
general procedure A from N-Benzyliminodiacetic acid and 40a to
give first the trityle protected intermediate after precipitation in
water and filtration. Yield 100%. tR,LCMS ¼ 4.5 min, Purity 97% MS
(ESIþ): m/z ¼ 616 (M þ H)þ. Deprotection using a TFA/DCM/TIS
mixture allowed 40 as an oil (formiate salt) after purification by
d6) d ppm: 172.3, 170.5, 169.5, 138.1, 134.7, 132.6, 128.9, 128.3, 127.2,
117.0, 57.6, 56.7, 54.0, 48.4, 36.4, 35.2, 29.5; tR,LCMS ¼ 2.60 min,
Purity 98% MS (ESIþ): m/z ¼ 388 (M þ H)þ.
5.4.65. (S)-2-(2-(Carboxymethyl-(3-phenyl-propyl)-amino)-
acetylamino)-3-(1H-imidazol-4-yl)-propionic acid tert-butyl ester
(43)
preparative HPLC. Yield 70%. 1H NMR (DMSO-d6)
d
ppm: 8.95 (s,
Compound (43) was synthesized from commercially available L-
H-histidine-(1-trityl)tert-butylester 37a and 13a following general
procedure A. First trityle protected intermediate was obtained.
1H), 8.39 (d, J ¼ 8.1 Hz, CONH), 8.00 (q, J ¼ 4.5 Hz, CONHMe), 7.33
(m, 5H(Ph)), 7.28 (s, 1H), 4.57 (m, 1H), 3.91 (s, 2H), 3.51 (s, 2H),
3.49 (s, 2H), 3.14 (dd, J ¼ 5.4 Hz and 15.3 Hz, 1H), 2.93 (dd,
J ¼ 8.1 Hz and 15.3 Hz, 1H), 2.60 (d, J ¼ 4.5 Hz, CO2Me); 13C NMR
Yield 48%. 1H NMR (DMSO-d6)
d
ppm: 8.72 (d, J ¼ 8.1 Hz, CONH),
8.40 (s, 4HCOOH), 7.36e6.99 (m, 15H(Trt)þ5H(Ph)þ1H), 6.65 (s,
(DMSO-d6)
d ppm: 170.8, 169.8, 168.5, 133.9, 135.3, 129.3, 129.8,
1H), 4.42 (m, 1H), 3.16 (s, 2H), 3.12 (s, 2H), 2.85 (m, 2H), 2.60e2.54
128.5, 128.1, 116.8, 57.9, 55.9, 53.7, 51.3, 27.3, 25.7;
(m, 4H), 1.60 (qt,
J
¼
7.5 Hz, 2H7), 1.29 (s, 9H(tBu));
tR,LCMS ¼ 2.55 min, Purity 98% MS (ESIþ): m/z ¼ 374 (M þ H)þ;
tR,LCMS ¼ 6.02 min, Purity 97% MS (ESIþ): m/z ¼ 686 (M þ H)þ.
Deprotection using a TFA/DCM/TIS mixture allowed 43 as ist for-
miate salt after preparative HPLC purification. Yield 68%. 1H NMR
HRMS (m/z): (Mþ) calcd. for
C18H24O4N5, 374.1828; found,
374.1823.
(DMSO-d6)
d
ppm: 8.54 (d, J ¼ 8.6 Hz, CONH), 8.32 (s, 2HCOOH),
5.4.63. (Benzyl-(((S)-1-benzylcarbamoyl-2-(1H-imidazol-4-yl)-
ethylcarbamoyl)-methyl)-amino)-acetic acid (41)
7.48 (s, 1H), 7.27e7.14 (m, 5H(Ph)), 6.81 (s, 1H), 4.41 (m, 1H), 3.24 (s,
2H), 3.16 (s, 2H), 2.88 (m, 2H), 2.60e2.54 (m, 4H),1.60 (qt, J ¼ 7.5 Hz,
First, to
a
stirred solution of L-Boc-Histidine (500 mg,
L,
2H), 1.29 (s, 9H(tBu)); 13C NMR (DMSO-d6)
d ppm: 173.2, 170.7,
1.95 mmol) in DMF (10 mL) were added benzylamine (213
m
170.3, 164.6, 142.1, 134.9, 133.0, 128.3, 128.2, 125.6, 116.5, 80.5, 58.0,
56.1, 54.2, 52.5, 32.7, 29.3, 29.0, 27.6; tR,LCMS ¼ 4.19 min, Purity 99%
MS (ESI-): m/z ¼ 443 (MꢁH)-.
1.95 mmol), HOBt (264 mg, 1.95 mmol), EDCI (455 mg, 2.38 mmol)
and DIEA (2 mL, 11.7 mmol). The mixture was stirred overnight. The
solvent was evaporated. The residue was solubilised in DCM and
washed with a saturated solution of NaHCO3 then with brine. The
organic layers were dried over MgSO4 and evaporated. The crude
product was purified by preparative HPLC to give L-Boc-Histidine
5.4.66. ((((S)-2-(1H-imidazol-4-yl)-1-methylcarbamoyl-
ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic acid
(44)
benzyl amide. Yield 64%. 1H NMR (DMSO-d6)
d
ppm: 8.28 (t,
(BDM43079) was synthesized as depicted for 40, from 13a and
J ¼ 6 Hz, CONHBz), 7.57 (s, 1H), 7.29e7.11 (m, 5H(Ph)), 6.98 (d,
J ¼ 8.1 Hz, CONHBoc), 6.79 (s,1H), 4.25 (d, J ¼ 6 Hz, 2H), 4.18 (m,1H),
2.89e2.75 (m, 2H), 1.36 (s, 9H(Boc)); tR,LCMS ¼ 2.54 min, Purity 98%
MS (ESIþ): m/z ¼ 345 (M þ H)þ. Deprotection was performed using
HClg in dichloromethane during 30 min at room temperature.
Solvent was evaporated to give L-H-Histidine benzyl amide 41a as
its dihydrochloride salt directly in the next step. tR,LCMS ¼ 0.75 min
(5 min gradient), Purity 98% MS (ESIþ): m/z ¼ 245 (M þ H)þ. Then
41a and N-Benzyliminodiacetic acid reacted following general
procedure A to give 41 as an oil (formiate salt). Yield 10%. 1H NMR
40a, as its TFA salt. Yield 58%. 1H NMR (DMSO-d6)
d ppm: 9.05 (d,
J ¼ 8.7 Hz, 1H), 9.01 (s, 1H), 8.22 (q, J ¼ 4.8 Hz, 1H), 7.38 (s, 1H),
7.32e7.19 (m, 5H), 4.58 (m, 1H), 4.13 (m, 2Hþ1H), 4.08 (d, J ¼ 6.6 Hz,
1H), 3.17e3.13 (m, 2Hþ1H), 2.96 (dd, J ¼ 8.7 Hz and J ¼ 14.8 Hz,
1H),2.60e2.57 (m, 3Hþ2H), 1.92 (qt, J ¼ 7.8 Hz, 2H); 13C NMR
(DMSO-d6)
d ppm: 169.5, 167.7, 165.2, 140.5, 134.1, 129.2, 128.4,
128.3, 126.1, 116.8, 55.3, 54.9, 54.1, 52.1, 31.8, 27.0, 25.7, 25.3;
tR,LCMS ¼ 3.35 min, Purity 98% MS (ESIþ): m/z ¼ 402 (M þ H)þ;
HRMS (m/z): (Mþ) calcd. for C20H28O4N5, 402.2141; found,
402.2137.
(DMSO-d6)
d
ppm: 8.37 (t, J ¼ 5.96 Hz, CONHBz), 8.32 (d, J ¼ 7.95 Hz,
CONH), 7.56 (s, 1H), 7.39e7.11 (m, 10H(Ph)), 6.78 (s, 1H), 4.54 (m,
1H), 4.26 (d, J ¼ 5.7 Hz, 2H), 3.75 (s, 2H), 3.33e3.20 (m, 4H), 2.92 (m,
5.4.67. ((((S)-1-Benzylcarbamoyl-2-(1H-imidazol-4-yl)-
ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic acid
(45)
2H); 13C NMR (DMSO-d6)
d ppm: 172.3, 170.8, 170.0, 139.3, 138.1,
134.7, 128.9, 128.2, 128.1, 127.2, 126.8, 126.6, 116.3, 57.6, 57.0, 53.8,
52.5, 41.9, 29.8; tR,LCMS ¼ 1.82 min (5 min gradient), Purity 97% MS
(ESIþ): m/z ¼ 450 (M þ H)þ.
Compund (45) was obtained following the procedure of
obtention of 41, as its formiate salt. Yield 84%. 1H NMR (DMSO-d6)
d
ppm: 8.38 (t, J ¼ 5.5 Hz, CONHBz), 8.30 (d, J ¼ 8.5 Hz, CONH), 8.28

566
J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
(s, HCOOH), 7.5 (s, 1H), 7.11e7.29 (m, 10H(Ph)), 6.76 (s, 1H), 4.55 (m,
1H), 4.24 (d, J ¼ 5.5 Hz, 2H), 3.27 (s, 2H), 3.21 (d, J ¼ 16.6 Hz, 1H),
3.14 (d, J ¼ 16.6 Hz, 1H), 2.94 (dd, J ¼ 5.5 Hz and 15 Hz, 1H), 2.87 (dd,
5.4.70. ((((S)-2-(1H-imidazol-4-yl)-1-methylcarbamoyl-
ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic acid
methyl ester (48)
J ¼ 7.4 Hz and 15 Hz, 1H), 2.52 (m, 4H), 1.64 (dt, J ¼ 7.5 Hz, 2H); 13
C
13a (751 mg, 2.99 mmol) was dissolved in trifluoracetic anhy-
dride 2% in acetic anhydride (7.5 mL). The reaction mixture was
stirred for 4 h at 70 ꢀC and then evaporated. The residue was dis-
solved in methanol (6 mL) and DIEA (1.56 mL, 8.97 mmol) was
added. The mixture was stirred overnight at reflux under argon.
The solvent was evaporated under reduced pressure. The crude
product was dissolved in DCM and washed with water. The organic
layer was dried with MgSO4 and evaporated to give (Methox-
ycarbonylmethyl-(3-phenyl-propyl)-amino)-acetic acid 48a as an
orange oil. Yield 78%. tR,LCMS ¼ 3.96 min, Purity 97%; MS (ESIþ): m/
z ¼ 266 (M þ H)þ. To a stirred solution of L-H-Histidine-(1-trityl)
methyl amide 40a (738 mg,1.47 mmol) in DMF (7.5 mL) were added
acid 48a (390 mg, 1.47 mmol), HOBt (397 mg, 2.94 mmol), EDCI
NMR (DMSO-d6) d ppm: 173.0, 170.9, 170.6, 164.2, 142.1, 139.3, 134.7,
133.4, 128.3, 128.25, 128.2, 126.8, 126.6, 125.7, 125.6, 58.1, 55.9, 54.3,
52.9, 41.9, 32.7, 29.8, 29.7; tR,LCMS ¼ 5.02 min (10 min gradient),
Purity 98% MS (ESIþ): m/z ¼ 478 (MþH)þ.
5.4.68. ((((S)-2-(1H-imidazol-4-yl)-1-(3-methyl-(1,2,4)oxadiazol-
5-yl)-ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic
acid (46)
To a stirred solution of hydroxylamine hydrochloride (3.82 mg,
55 mmol) in a solution of ethanol/water (80/20) were added 2.6 mL
(50 mmol) of acetonitrile and 2.2 mg (55 mmol) of NaOH. The
mixture was refluxed overnight. The solvent was evaporated and
the residue was solubilized in 80 mL of ethanol, the suspension was
filtrated and the filtrate was evaporated. The crude product was
recrystallized from iPrOH to give N-Hydroxy-acetamidine as a
(338 mg,1.76 mmol) and N-methylmorpholine (484 mL, 4.41 mmol).
The mixture was stirred 22 h. The solvent was evaporated. The
crude product was purified by preparative HPLC to give the trityle
protected compound. Yield 30%. tR,LCMS ¼ 2.00 min, Purity 81%; MS
(ESIþ): m/z ¼ 658 (MþH)þ. To a stirred solution of TIS and DCM
(1.0 mL/8.2 mL) was added the protected compound (339 mg,
0.52 mmol). Then 1.0 mL of TFA was added. The mixture was stirred
at room temperature during 16 h. Then the solvent was evaporated
and the crude product was purified by preparative HPLC to give 48
(BDM43124) as a yellow oil (formiate salt). Yield 94%. 1H NMR
white powder. Yield 47%. Purity: 99%; 1H NMR (DMSO-d6)
d ppm:
8.66 (sl, 1H), 5.34 (sl, 2H), 1.61 (s, 3H). To a stirred solution of L-Boc-
Histidine (510 mg, 2 mmol) in DMF (10 mL) were added TBTU
(702 mg, 2.2 mmol) and DIEA (1 mL, 6 mmol). The resulting yellow
mixture was stirred for 5 min. N-Hydroxy-acetamidine (150 mg,
2 mmol) was added and the mixture was stirred for 4.5 h since the
intermediate product was formed. The solution was then refluxed
1.5 h. The solvent was evaporated and the residue was solubilized in
AcOEt washed with a saturated solution of NaHCO3. The organic
layers were dried with MgSO4 and evaporated. The crude product
was purified by preparative HPLC to give ((S)-2-(1H-Imidazol-4-yl)-
1-(3-methyl-(1,2,4)oxadiazol-5-yl)-ethyl)-carbamic acid tert-butyl
ester as a brown oil. Yield 73%. tR,LCMS ¼ 3.56 min; Purity: 98%;
(CD3OD)
d ppm: 8.63 (1, H, HCOOH), 7.27e7.15 (m, 6H), 4.74 (dd,
J ¼ 5.0 Hz and J ¼ 9.0 Hz, 1H), 3.70 (s, 3H), 3.49 (s, 2H), 3.30 (s, 2H),
3.27 (dd, J ¼ 5.0 Hz and J ¼ 15.0 Hz, 1H), 3.10 (dd, J ¼ 9.0 Hz and
J ¼ 15.0 Hz, 1H), 2.73 (s, 3H), 2.64 (t, J ¼ 7.5 Hz, 2H), 2.57 (t,
J ¼ 7.5 Hz, 2H), 1.70 (m, 2H); 13C NMR (CD3OD)
d ppm: 172.4, 172.1,
171.0, 141.7, 129.8, 128.0, 125.5, 118.8, 117.0, 114.9, 58.0, 55.5, 55.3,
51.7, 51.0, 32.8, 29.0, 27.2, 25.1; tR,LCMS ¼ 2.00 min, Purity 100%; MS
(ESIþ): m/z ¼ 416 (MþH)þ. HRMS (m/z): (Mþ) calcd. for C21H30O4N5,
416.2298; found, 416.2284.
MS: (ESIþ) m/z ¼ 294 (M þ H)þ; 1H NMR (DMSO-d6)
d ppm: 8.19 (s,
1H), 7.67 (d, J ¼ 7.8 Hz, 1H), 7.54 (s, 1H), 6.76 (s, 1H), 5.03 (m, 1H),
3.02 (m, 2H), 2.29 (s, 3H), 1.35 (s, 9H). Tert-butoxycarbonyl groups
were removed in presence of gaseous HCl in dichloromethane
during 30 min at room temperature. Solvent was evaporated to give
the expected product (S)-2-(1H-Imidazol-4-yl)-1-(3-methyl-(1,2,4)
oxadiazol-5-yl)-ethylamine.2HCl (46a) as its dihydrochloride salt
used directly in next reaction. tR,LCMS ¼ 0.66 min (5 min gradient);
Purity: 98%; MS: (ESIþ) m/z ¼ 194 (M þ H)þ. 46 was synthesized as
its formiate salt from 13a and 46a following general procedure A.
5.4.71. ((((S)-2-(1H-imidazol-4-yl)-1-(3-methyl-(1,2,4)oxadiazol-
5-yl)-ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic
acid methyl ester (49)
Compound (49) was synthesized as described for compound 48,
from 46a and acid 48a. Yield 40%. 1H NMR (CD3OD)
d ppm: 7.73 (s,
1H), 7.28e7.14 (m, 5H), 6.95 (s, 1H), 5.51 (dd, J ¼ 6.0 Hz and
Yield 50%.1H NMR (DMSO-d6)
d
ppm: 8.97 (t, J ¼ 7.8 Hz, 1H), 8.27 (s,
J ¼ 8.0 Hz, 1H), 3.70 (s, 3H), 3.45 (s, 2H), 3.30 (m, 4H), 2.65e2.55 (m,
4H), 2.32 (s, 3H), 1.71 (m, 2H); 13C NMR (DMSO-d6)
d ppm: 178.3,
1H), 7.5 (s, 1H), 7.28e7.12 (m, 5H), 6.79 (s, 1H), 4.23 (m, 1H), 3.28 (s,
2H), 3.22 (s, 2H), 3.12 (m, 2H), 2.69e2.50 (m, 4H), 2.26 (s, 3H), 1.64
172.8, 172.4, 167.2, 141.9, 135.1, 132.2, 128.1, 125.5, 116.8, 58.0, 55.3,
54.7, 50.8, 46.4, 32.9, 29.7, 29.4, 10.0; tR,LCMS ¼ 2.28 min, Purity 98%;
MS (ESIþ): m/z ¼ 441 (M þ H)þ.
(m, 2H); 13C NMR (DMSO-d6)
d ppm: 178.8, 173.1, 170.9, 166.1, 164.1,
142.0, 135.0, 132.9, 128.2, 125.6, 119.1, 57.9, 55.9, 46.3, 32.7, 32.6,
30.3, 29.1, 11.0; tR,LCMS ¼ 3.68 min, Purity 98% MS (ESIþ): m/z ¼ 427
(M þ H)þ.
Acknowledgements
The authors acknowledge financial support from INSERM, Uni-
versity of Lille 2, Pasteur Institute of Lille, Region Nord Pas de Calais,
EDFR, Etat (0823007, 0823008, 07-CPER 009-01, 2007-0172-02-
CPER/3), ANR (11-JS07-015-01), FRM (DCM20111223046), and NIH
GM81539 (to WJT). The authors thank Qing Guo for assistance for
X-ray experiments. M.G. is a recipient of a fellowship from Minis-
5.4.69. ((((S)-2-Hydroxy-1-(1H-imidazol-4-ylmethyl)-
ethylcarbamoyl)-methyl)-(3-phenyl-propyl)-amino)-acetic acid
(47)
Compound (47) was obtained as depicted for compound 38 from
13a and 38a, as a colourless oil (formiate salt) after preparative
HPLC purification. Yield 30%. 1H NMR (DMSO-d6)
d ppm: 8.31 (s,
ꢀ
ꢁ
tere de la Recherche et de l’Enseignement Superieur.
2HCOOH), 8.07 (d, J ¼ 8.5 Hz, CONH), 7.50 (d, J ¼ 1.2 Hz, 1H),
7.28e7.15 (m, 5H (Ph)), 6.76 (d, J ¼ 1.2 Hz, 1H), 3.92 (m, 1H), 3.35
(dd, J ¼ 4.8 Hz and 10.6 Hz,1H), 3.28 (dd, J ¼ 6.1 Hz and 10.6 Hz,1H),
3.19 (s, 2H), 3.10 (s, 2H), 2.75 (dd, J ¼ 5.7 Hz and 14.6 Hz, 1H), 2.63
(dd, J ¼ 7.3 Hz and 14.6 Hz, 1H), 2.5 (m, 4H), 1.62 (qt, J ¼ 8.1 Hz, 2H);
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.12.
005. These data include MOL files and InChiKeys of the most
important compounds described in this article.
13C NMR (DMSO-d6)
d ppm: 173.2, 170.3, 164.5, 142.1, 134.6, 133.4,
128.3, 128.2, 125.6, 117.8, 62.4, 58.5, 56.6, 54.4, 50.4, 32.7, 29.1, 28.2;
tR,LCMS ¼ 3.08 min, Purity 98% MS (ESIþ): m/z ¼ 375 (M þ H)þ.

J. Charton et al. / European Journal of Medicinal Chemistry 90 (2015) 547e567
567
[18] L.A. McCord, W.G. Liang, E. Dowdell, V. Kalas, R.J. Hoey, A. Koide, S. Koide, W.-
J. Tang, Conformational states and recognition of amyloidogenic peptides of
human insulin-degrading enzyme, PNAS 110 (2013) 13827e13832.
[19] (a) M.A. Leissring, E. Malito, S. Hedouin, L. Reinstatler, T. Sahara, S.O. Abdul-
Hay, S. Choudhry, G.M. Maharvi, A.H. Fauq, M. Huzarska, P.S. May, S. Choi,
T.P. Logan, B.E. Turk, L.C. Cantley, M. Manolopoulou, W.-J. Tang, R.L. Stein,
G.D. Cuny, D.J. Selkoe, Designed inhibitors of insulin-degrading enzyme
regulate the catabolism and activity of insulin, PLoS One 5 (2010) e10504;
(b) S.O. Abdul-Hay, A.L. Lane, T.R. Caulfield, C. Claussin, J. Bertrand, A. Masson,
S. Choudhry, A.H. Fauq, G.M. Maharvi, M.A. Leissring, Optimization of peptide
hydroxamate inhibitors of insulin-degrading enzyme reveals marked sub-
strate-selectivity, J. Med. Chem. 56 (2013) 2246e2255.
[20] M. Flipo, J. Charton, A. Hocine, S. Dassonneville, B. Deprez, R. Deprez-Poulain,
Hydroxamates: relationships between structure and plasma-stability, J. Med.
Chem. 52 (2009) 6790e6802.
[21] (a) F. Hoffman, La Roche AG. WO 2006066847. (b) C. Cabrol, M.A. Huzarska,
C. Dinolfo, M.C. Rodriguez, L. Reinstatler, J. Ni, L.A. Yeh, G.D. Cuny, R.L. Stein,
D.J. Selkoe, M.A. Leissring, Small-molecule activators of insulin-degrading
enzyme discovered through high-throughput compound screening, PLoS
One 4 (2009) e5274;
References
[1] A. Fernandez-Gamba, M.C. Leal, L. Morelli, E.M. Castano, Insulin-degrading
enzyme: structure-function relationship and its possible roles in health and
disease, Curr. Pharm. Des. 15 (2009) 3644e3655.
[2] L.B. Hersh, The insulysin (insulin degrading enzyme) enigma, Cell. Mol. Life
Sci. 63 (2006) 2432e2434.
[3] (a) R.A. Roth, M.L. Mesirow, K. Yokono, S. Baba, Degradation of insulin-like
growth factors I and II by a human insulin degrading enzyme, Endocr. Res.
10 (1984) 101e112;
(b) Q. Guo, M. Manolopoulou, Y. Bian, A.B. Schilling, W.-J. Tang, Molecular
basis for the recognition and cleavages of IGF-ii, TGF-[alpha], and Amylin by
human insulin-degrading enzyme, J. Mol. Biol. 395 (2010) 430e443.
[4] (a) C. Ciaccio, G.R. Tundo, G. Grasso, G. Spoto, D. Marasco, M. Ruvo, M. Gioia,
E. Rizzarelli, M. Coletta, Somatostatin: a novel substrate and a modulator of
insulin-degrading enzyme activity, J. Mol. Biol. 385 (2009) 1556e1567;
(b) L.A. Ralat, Q. Guo, M. Ren, T. Funke, D.M. Dickey, L.R. Potter, W.-J. Tang,
Insulin-degrading enzyme modulates the natriuretic peptide- mediated
signaling response, J. Biol. Chem. 286 (2011) 4670e4679;
(c) M. Ren, Q. Guo, L. Guo, M. Lenz, F. Qian, R.R. Koenen, H. Xu, A.B. Schilling,
C. Weber, R.D. Ye, A.R. Dinner, W.-J. Tang, Polymerization of MIP-1 chemokine
(CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme, EMBO
J. 29 (2010) 3952e3966.
(c) B. Cakir, O. Dagliyan, E. Dagyildiz, I. Baris, I.H. Kavakli, S. Kizilel, M. Türkay,
Structure based discovery of small molecules to regulate the activity of human
insulin degrading enzyme, PLoS One 7 (2012) e31787;
(d) S.S. Kukday, S.P. Manandhar, M.C. Ludley, M.E. Burriss, B.J. Alper,
W.K. Schmidt, Cell-permeable, small-molecule activators of the insulin-
degrading enzyme, J. Biomol. Screen. 17 (2012) 1348e1361.
[5] I.V. Kurochkin, S. Goto, Alzheimer's beta-amyloid peptide specifically interacts
with and is degraded by insulin degrading enzyme, FEBS Lett. 345 (1994)
33e37.
[6] L.A. Ralat, V. Kalas, Z. Zheng, R.D. Goldman, T.R. Sosnick, W.-J. Tang, Ubiquitin
is a novel substrate for human insulin-degrading enzyme, J. Mol. Biol. 406
(2011) 454e466.
[7] (a) W. Farris, S. Mansourian, M.A. Leissring, E.A. Eckman, L. Bertram,
C.B. Eckman, R.E. Tanzi, D.J. Selkoe, Partial loss-of-function mutations in
insulin-degrading enzyme that induce diabetes also impair degradation of
amyloid beta-protein, Am. J. Pathol. 164 (2004) 1425e1434;
[22] J. Charton, M. Gauriot, Q. Guo, N. Hennuyer, X. Marechal, J. Dumont,
M. Hamdane, V. Pottiez, V. Landry, O. Sperandio, M. Flipo, L. Buee, B. Staels,
F. Leroux, W.-J. Tang, B. Deprez, R. Deprez-Poulain, Imidazole-derived 2-[N-
carbamoylmethyl-alkylamino]acetic acids, substrate-dependent modulators
of insulin-degrading enzyme in amyloid-b hydrolysis, Eur. J. Med. Chem. 79
(2014) 184e193.
[23] R.G. Wilkinson, T.L. Fields, J.H. Boothe, Total synthesis of tetracyclines. 1 III.2
synthesis of a tricyclic model system, J. Org. Chem. 26 (1961) 637e643.
€
(b) H. Fakhrai-Rad, A. Nikoshkov, A. Kamel, M. Fernstrom, J.R. Zierath,
ꢁ
ꢁ
[24] (a) J. Charton, F. Leroux, S. Delaroche, V. Landry, B.P. Deprez, R.F. Deprez-
Poulain, Synthesis of a 200-member library of squaric acid N-hydroxylamide
amides, Bioorg. Med. Chem. Lett. 18 (2008) 4968e4971;
S. Norgren, H. Luthman, J. Galli, Insulin-degrading enzyme identified as a
candidate diabetes susceptibility gene in GK rats, Hum. Mol. Genet. 9 (2000)
2149e2158.
(b) J. Charton, L. Charruault, R. Deprez-Poulain, B. Deprez, Alkylsquarates as
key intermediates for the rapid preparation of original drug-inspired com-
pounds, Comb. Chem. High. Throughput Screen. 11 (2008) 294e303.
[25] M.C. Pirrung, H. Han, J. Chen, O-Alkyl hydroxamates as metaphors of enzyme-
bound enolate intermediates in hydroxy acid dehydrogenases. Inhibitors of
isopropylmalate dehydrogenase, isocitrate dehydrogenase, and tartrate de-
hydrogenase, J. Org. Chem. 61 (1996) 4527e4531.
[26] R. Deprez-Poulain, N. Cousaert, P. Toto, N. Willand, B. Deprez, Application of
Ullmann and UllmanneFinkelstein reactions for the synthesis of N-aryl-N-
(1H-pyrazol-3-yl) acetamide or N-(1-aryl-1H-pyrazol-3-yl) acetamide de-
rivatives and pharmacological evaluation, Eur. J. Med. Chem. 46 (2011)
3867e3876.
[27] R.F. Poulain, A.L. Tartar, B.P. Deprez, Parallel synthesis of 1,2,4-oxadiazoles
from carboxylic acids using an improved, uronium-based, activation, Tetra-
hedron Lett. 42 (2001) 1495e1498.
[28] H. Sun, Capture hydrolysis signals in the microsomal stability assay: molecular
mechanisms of the alkyl ester drug and prodrug metabolism, Bioorg. Med.
Chem. Lett. 22 (2012) 989e995.
[8] M.A. Leissring, W. Farris, A.Y. Chang, D.M. Walsh, X. Wu, X. Sun, M.P. Frosch,
D.J. Selkoe, Enhanced proteolysis of beta-amyloid in APP transgenic mice
prevents plaque formation, secondary pathology, and premature death,
Neuron 40 (2003) 1087e1093.
[9] (a) L. Bertram, D. Blacker, K. Mullin, D. Keeney, J. Jones, S. Basu, S. Yhu,
M.G. McInnis, R.C. Go, K. Vekrellis, D.J. Selkoe, A.J. Saunders, R.E. Tanzi, Evi-
dence for genetic linkage of Alzheimer's disease to chromosome 10q, Science
290 (2000) 2302e2303;
(b) J.A. Prince, L. Feuk, H.F. Gu, B. Johansson, M. Gatz, K. Blennow, A.J. Brookes,
Genetic variation in a haplotype block spanning IDE influences Alzheimer
disease, Hum. Mutat. 22 (2003) 363e371;
(c) W.Q. Qiu, M.F. Folstein, Insulin, insulin-degrading enzyme and amyloid-
beta peptide in Alzheimer's disease: review and hypothesis, Neurobiol. Ag-
ing 27 (2006) 190e198.
[10] R.G. Bennett, J. Fawcett, M.C. Kruer, W.C. Duckworth, F.G. Hamel, Insulin in-
hibition of the proteasome is dependent on degradation of insulin by insulin-
degrading enzyme, J. Endocrinol. 177 (2003) 399e405.
[11] M.B. de Tullio, V. Castelletto, I.W. Hamley, P.V. Martino Adami, L. Morelli,
E.M. Castano, Proteolytically inactive insulin-degrading enzyme inhibits am-
yloid formation yielding non-neurotoxic Ab peptide aggregates, PLoS One 8
(2013) e59113.
[29] S. Deechongkit, P.E. Dawson, J.W. Kelly, Toward assessing the position-
dependent contributions of backbone hydrogen bonding to b-sheet folding
thermodynamics employing amide-to-ester perturbations, J. Am. Chem. Soc.
126 (2004) 16762.
[12] S. Ito, S. Ohtsuki, S. Murata, Y. Katsukura, H. Suzuki, M. Funaki, M. Tachikawa,
T. Terasaki, Involvement of insulin-degrading enzyme in insulin- and atrial
[30] R.D. Brown, B.A.W. Coller, J.E. Kent, 1,2,5-, 1,3,4- and 1,2,4-oxadiazoles: a
theoretical study of electric dipole moments, Theor. Chim. Acta 10 (1968)
435e446.
[31] J. El Bakali, L. Maingot, J. Dumont, H. Host, A. Hocine, N. Cousaert,
S. Dassonneville, F. Leroux, B. Deprez, R. Deprez-Poulain, Novel selective in-
hibitors of neutral endopeptidase: discovery by screening and hit-to-lead
optimisation, MedChemComm 3 (2012) 469e474.
natriuretic peptide-sensitive internalization of amyloid-b peptide in mouse
brain capillary endothelial cells, J. Alzheimers Dis. 38 (2014) 185e200.
[13] Y. Shen, A. Joachimiak, M.R. Rosner, W.J. Tang, Structures of human insulin-
degrading enzyme reveal a new substrate recognition mechanism, Nature
443 (2006) 870e874.
[14] E. Malito, R.E. Hulse, W.J. Tang, Amyloid beta-degrading cryptidases: insulin
degrading enzyme, presequence peptidase, and neprilysin, Cell. Mol. Life Sci.
65 (2008) 2574e2585.
[15] H. Im, M. Manolopoulou, E. Malito, Y. Shen, J. Zhao, M. Neant-Fery, C.Y. Sun,
S.C. Meredith, S.S. Sisodia, M.A. Leissring, W.J. Tang, Structure of substrate-free
human insulin-degrading enzyme (IDE) and biophysical analysis of ATP-
induced conformational switch of IDE, J. Biol. Chem. 282 (2007)
25453e25463.
[16] N. Noinaj, S.K. Bhasin, E.S. Song, K.E. Scoggin, M.A. Juliano, L. Juliano,
L.B. Hersh, D.W. Rodgers, Identification of the allosteric regulatory site of
insulysin, PLoS One 6 (2011) e20864.
[32] MOE-Dock: http://www.chemcomp.com.
[33] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Refinement of macromolecular
structures by the Maximum-Likelihood method, Acta Crystallogr. Sect. D. 53
(1997) 240e255.
[34] E. Potterton, S. McNicholas, E. Krissinel, K. Cowtan, M. Noble, The CCP4
molecular-graphics project, Acta Crystallogr. Sect. D. 58 (2002) 1955e1957.
[35] P. Emsley, K. Cowtan, Coot: model-building tools for molecular graphics, Acta
Crystallogr. Sect. D. 60 (2004) 2126e2132.
[36] A.T. Brunger, P.D. Adams, G.M. Clore, W.L. DeLano, P. Gros, R.W. Grosse-
Kunstleve, J.-S. Jiang, J. Kuszewski, M. Nilges, N.S. Pannu, R.J. Read, L.M. Rice,
T. Simonson, G.L. Warren, Crystallography & NMR system: a new software
suite for macromolecular structure determination, Acta Crystallogr. Sect. D. 54
(1998) 905e921.
[17] M. Manolopoulou, Q. Guo, E. Malito, A.B. Schilling, W.J. Tang, Molecular
basis of catalytic chamber-assisted unfolding and cleavage of human in-
sulin by human insulin-degrading enzyme, J. Biol. Chem. 284 (2009)
14177e14188.
€
[37] The PyMOL Molecular Graphics System, Version 1.3, Schrodinger, LLC.