Solid-Phase Synthesis of Substrate-Based Dipeptides and Heterocyclic Pseudo-dipeptides as Potential NO Synthase Inhibitors

Article abstract of DOI:10.1002/cmdc.201900659

More than 160 arginine analogues modified on the C-terminus via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO synthases (NOS). A methodology involving formation of a thiocitrulline intermediate linked through its side-chain on a solid support followed by modification of its carboxylate group was developed. Finally, the side-chain thiourea group was either let unchanged, S-alkylated (Me, Et) or guanidinylated (Me, Et) to yield respectively after TFA treatment the corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-arginine substrate analogues. They all were tested against three recombinant NOS isoforms. Several compounds containing a S-Et- or a S-Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-oxadiazole series were shown to inhibit nNOS and iNOS with IC₅₀ in the 1–50 μM range. Spectral studies confirmed that these new compounds interacted at the heme active site. The more active compounds were found to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with similar efficiency than the reference compounds L-NIL and SEIT.

Full text of DOI:10.1002/cmdc.201900659

Accepted Article
Title: Solid Phase Synthesis and Evaluation of Substrate-based
Dipeptides and Heterocyclic Pseudo-dipeptides as Potential NO
Synthases Inhibitors
Authors: Youness Touati-Jallabe, Thibault Tintillier, Elodie
Mauchauffée, Jean-Luc Boucher, Jérémy Leroy, Booma
Ramassamy, Abdallah Hamzé, Karima Mezghenna, Amine
Bouzekrini, Claudia Verna, Jean Martinez, Anne-Dominique
Lajoix, and Jean-François Hernandez
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201900659
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900659
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ChemMedChem
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Solid Phase Synthesis of Substrate-based Dipeptides and
Heterocyclic Pseudo-dipeptides as Potential NO Synthase
Inhibitors
Youness Touati-Jallabe,[a,b] Thibault Tintillier,[a,c] Elodie Mauchauffée,[a] Jean-Luc Boucher,[d] Jérémy
Leroy,[e] Booma Ramassamy,[d] Abdallah Hamzé,[a,f] Karima Mezghenna,[e] Amine Bouzekrini,[e] Claudia
Verna,[a] Jean Martinez,[a] Anne-Dominique Lajoix,*[e] and Jean-François Hernandez*[a]
[a]
Dr. Y. Touati-Jallabe, Dr. T. Tintillier, Dr. E. Mauchauffée, Dr. A. Hamzé, C. Verna, Pr. J. Martinez, Dr. J.-F. Hernandez
Institut des Biomolécules Max Mousseron
CNRS, Université Montpellier, ENSCM
Faculté de Pharmacie, 34000 Montpellier, France
E-mail: jean-francois.hernandez@umontpellier.fr
[b]
[c]
[d]
Dr. Y. Touati-Jallabe
Current address: Avara Pharmaceutical Services, Boucherville, QC J4B 7K8, Canada
Dr. T. Tintillier
Current address: Asymptote Project Management, 1 rue Edisson, 69500 Bron, France
Dr. J.-L. Boucher, B. Ramassamy
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques
UMR8601, CNRS, Université Paris-Descartes
45 rue des Saints Pères, 75270 Paris Cedex 06, France
J. Leroy, Dr. K. Mezghenna, A. Bouzekrini, Dr. A.-D. Lajoix
Centre Biocommunication en Cardio-métabolique
Université Montpellier
Faculté de Pharmacie, 34000 Montpellier, France
[e]
[f]
E-mail: anne-dominique.lajoix@umontpellier.fr
A. Hamzé
Current address: BioCIS, UMR 8076, CNRS, Université Paris Sud, Université Paris-Saclay, Faculté de Pharmacie, 92296 Châtenay-Malabry, France
Supporting information for this article is given via a link at the end of the document.
Abstract: More than 160 arginine analogues modified on the C-terminus
via either an amide bond or a heterocyclic moiety (1,2,4-oxadiazole, 1,3,4-
oxadiazole and 1,2,4-triazole) were prepared as potential inhibitors of NO
synthases (NOS). A methodology involving formation of a thiocitrulline
intermediate linked through its side-chain on a solid support followed by
modification of its carboxylate group was developed. Finally, the side-chain
thiourea group was either let unchanged, S-alkylated (Me, Et) or
guanidinylated (Me, Et) to yield respectively after TFA treatment the
corresponding thiocitrulline, S-Me/Et-isothiocitrulline and N-Me/Et-
arginine substrate analogues. They all were tested against three
recombinant NOS isoforms. Several compounds containing a S-Et- or a S-
Me-Itc moiety and mainly belonging to both the dipeptide-like and 1,2,4-
oxadiazole series were shown to inhibit nNOS and iNOS with IC50 in the 1-
50 µM range. Spectral studies confirmed that these new compounds
interacted at the heme active site. The more active compounds were found
to inhibit intra-cellular iNOS expressed in RAW264.7 and INS-1 cells with
similar efficiency than the reference compounds L-NIL and SEIT.
also expressed in pancreatic b-cells where it controls insulin
secretion.[3] By contrast, inducible NOS (iNOS) is expressed by
macrophages in response to pro-inflammatory cytokines, and
contributes to the innate immune response.[4] The three NOS
isoforms are composed of two domains: the N-terminal
oxygenase domain containing the heme prosthetic group and
the binding site for the substrate L-Arg, and the C-terminal
reductase domain. The two domains are connected by a
calmodulin binding sequence.[2,5-7]
Overproduction of NO is observed in several pathophysiological
states and mainly concerns nNOS and iNOS.[8-12] Neuronal NOS
is involved in stroke and neurodegenerative diseases,[13,14] while
iNOS has been identified as the cause of or an aggravating
factor in several human diseases involving an inflammatory state,
including septic shock, chronic inflammatory diseases, insulin
resistance, cancers, migraine.[15-20] Thus, nNOS and iNOS are
potential therapeutical targets, but the multiple roles of NO
complicate the discovery of inhibitors of pharmaceutical
interest.[21] In particular, such inhibitors must be highly selective
to preserve the essential eNOS functions. The crystal structure
of the three NOS oxygenase domains (NOSoxy) has shown a
very strong conservation of the heme active site and substrate
binding site residues.[22-25] Because of this striking similarity,
selective inhibition of a NOS isoform is highly challenging.
Several successes have been reached in the case of nNOS.[26,27]
In particular, the original strategy proposed by R. B. Silverman
was to attach, via an amide bond, an extension to the C-
terminus of a substrate-based and poorly selective inhibitor (i.e.
L-Nw-nitro-Arginine).[28] The extension can establish selective
Introduction
Nitric oxide (NO) is an important signaling molecule in the living
kingdom.[1] In mammals, it is synthesized from L-Arginine (L-Arg)
by nitric oxide synthases (NOS) comprising the neuronal,
inducible and endothelial NOS distinct isoforms.[2] Neuronal
NOS (nNOS) and endothelial NOS (eNOS) are constitutively
expressed
and
mainly
involved
respectively
in
neurotransmission, and in smooth muscle relaxation,
maintenance of blood pressure, platelet aggregation. nNOS is
1
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10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
interactions with residues of the less conserved substrate-
access channel. This strategy led to compounds exhibiting
>1000-fold selectivity toward nNOS over eNOS). X-Ray analysis
of co-crystal complexes of these inhibitors with nNOS and eNOS
allowed identification of the main sites inducing selectivity.[29]
Based on these results, a computer modelling approach led to
the design of new promising compounds.[30] In the case of iNOS,
a few highly selective inhibitors have been reported. Amidine-
type substrate analogues were shown to irreversibly inactivate
iNOS in a time-dependent manner, whereas they behave as fast
reversible inhibitors of the two other isoforms.[31-35] Combinatorial
approaches identified pyrimidine-imidazole-based inhibitors of
iNOS dimerization[36-37] and the spirocyclic quinazoline AR-
C102222. The causes of selectivity were also established by X-
ray crystallography.[38] To date, despite a huge amount of
studies, no NOS inhibitor was shown to be clinically useful
because some parameters (toxicity, bioavailability, etc…) have
prevented their therapeutic use.[39-41] For instance, efforts are still
in progress to improve the permeability of nNOS inhibitors.[42-44]
Therefore, developing a clinically useful NOS inhibitor remains a
strong challenge.
steps from the ornithine derivative Boc-L-Orn(Nd-Z)-OH
according to our previously described strategy (Scheme 1).[51]
After formation of the methyl ester 1 using MeI, the Z protecting
group was removed by catalytic hydrogenolysis in the presence
of 1.5 equiv. TFA to trap the free d-amino group and prevent its
intramolecular reaction with the methyl ester. The resulting TFA
salt 2 was converted into isothiocyanate 3 using CS2 followed by
desulfurization with H2O2.[52] Compound 3 was then loaded onto
a Rink amide solid support to yield the protected thiocitrulline
derivative 4 attached via its side-chain. The loading completion
was assessed using the TNBS (trinitrobenzenesulfonyl) test.
Finally, the methyl ester 4 was either hydrolysed with LiOH in a
mixture of THF/H2O (7/3)[53] yielding the intermediate
5
(precursor for dipeptide-like and 1,2,4-oxadiazole compounds),
or treated with hydrazine hydrate in THF leading to hydrazide 6
(precursor for 1,3,4-oxadiazole and 1,2,4-triazole compounds).
O
O
O
Boc-HN
Z-HN
Boc-HN
Z-HN
Boc-HN
+H3N
OH
OMe
OMe
a
b
1
2
-
CF3CO2
In this context, we have developed dipeptide mimetic
compounds following Silverman’s strategy from different
substrate-based inhibitors, i.e. thiocitrulline (Tci), S-alkyl-
isothiocitrullines (Itc) and N-alkyl-arginines (Figure 1).[45-48]
Whereas few S-alkyl-L-isothiocitrulline-containing dipeptides
were previously reported as human iNOS moderate selective
inhibitors,[49-50] no extensive development of these series was
undertaken yet. We first synthesized a library of thiocitrullines,
S-alkyl-L-isothiocitrullines and N-alkyl-arginines modified on their
C-terminus via an amide bond (dipeptide-like). We also replaced
the amide bond by 1,2,4- and 1,3,4-oxadiazoles and 1,2,4-
triazoles heterocyclic structures considered as amide bond
isosteric moieties. For their synthesis, we exploited a solid
O
Boc-HN
OH
f
O
O
HN
Boc-HN
Boc-HN
HN
OMe
OMe
c,d
S
e
N
H
5
N
C
S
g
O
Boc-HN
NH2
S
N
N
H
3
4
H
HN
S
N
6
H
phase synthetic strategy following
a side-chain anchoring
approach.[51] This method allowed the easy and rapid synthesis
of numerous compounds containing both substrate analogues
Scheme 1. Synthesis of the supported thiocitrulline intermediates 5 and 6: (a)
MeI, Na2CO3, DMF, rt, 5h; (b) H2, 10% Pd/C, EtOH, 1.5 equiv. TFA, 4h; (c)
CS2, TEA, THF, 0 °C, 45 min; (d) H202, 30 min addition, then 5 min, 0 °C; (e) 3
equiv. 3, Rink amide solid support, TEA, THF, 60 °C, 3h; (f) 5 equiv. LiOH,
THF/water (7/3), rt, overnight; (g) 5 equiv. hydrazine hydrate, THF, rt, 24 h..
from
a
single supported thiocitrulline intermediate, and
possessed a high combinatorial approach potential.
O
O
O
Y
H2N
H2N
H2N
H2N
OH
OH
OH
R
-
Solid phase synthesis of the inhibitors. The efficiency of all
synthetic strategies was checked on standard PS resin and
allowed the fast synthesis of several series of compounds
possessing a variable linker. In addition, in the case of dipeptide-
like and 1,2,4-oxadiazole-containing analogues, two libraries
were prepared on Synphase lanterns (Mimotopes, Australia).
Thanks to color tagging, the advantage of these solid support
units was to perform common steps (i.e. S-alkylation,
guanidinylation, see below) in only one batch for all compounds.
Two 35 µmol-loaded PS lanterns possessing a Rink amide
spacer were used for the synthesis of each compound.
0d,e
0b,c
7a-39e
0a
HN
HN
HN
HN
Alk
Alk
S
NH2
S
NH
N
H
NH
X
NH(2)
Figure 1. Structure of thiocitrulline (0a), S-alkyl-isothiocitrullines (0b, Alk = Me;
0c, Alk = Et), N-alkyl arginines (0d, Alk = Me; 0e, Alk = Et), and general
structure of the target molecules (7a-39e; X = S=, Alk-S-, or Alk-NH-; Y = CO-
NH, or heterocycle).
- Dipeptide-like inhibitors. The preparation of the dipeptide-like
compounds on either PS resin or lanterns is presented in
Scheme 2. The thiocitrulline intermediate 5 was coupled to
various amines using BOP and HOAt in the presence of DIEA to
yield the corresponding supported dipeptide-like intermediates.
Each intermediate was divided into five samples. One sample
was cleaved with TFA to give Tci-containing analogues 7a-22a.
Another sample was reacted with MeI leading to S-Me-Itc-
containing analogues 7b-21b after cleavage from the solid
Results and Discussion
Chemistry.
- Synthesis of the supported precursors. All compounds were
prepared from
a single intermediate, a Na-Boc-protected
thiocitrulline methyl ester linked to the solid support (a standard
cross-linked polystyrene (PS) resin or a PS-loaded Synphase
lantern from Mimotopes) through its thiourea group (compound
4). The solid-supported intermediate 4 was synthesized in four
2
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10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
support. A similar treatment was performed on another sample
with EtI yielding S-Et-Itc-containing analogues 7c-22c after
cleavage. Finally, the last two samples were first S-methylated
and then guanidinylated using either methylamine or ethylamine,
respectively giving the series of N-Me-Arg- and N-Et-Arg-
containing analogues 7d-21d and 7e-21e after cleavage.
Seventy-six compounds (16 different extensions x 5 substrate
analogues with the exception of 12e, 22b, 22d and 22e, which
could not be obtained in sufficient amounts) have been prepared
(see Table 1). In addition, the five substrate-based amino acids
(0a-e) were similarly prepared from 4, the methyl ester being
removed just before acidic cleavage (not shown).
oxadiazole
compounds
from
Fmoc-protected
aspartyl
derivatives.[56] Indeed, we found that sodium acetate strongly
accelerated the cyclo dehydration step. The same was true on
the solid phase and a THF/water mixture was used to keep a
good resin swelling. Two cycles of coupling/cyclo dehydration
were generally sufficient for complete conversion. Finally, the
supported thiocitrulline intermediates were treated as described
above to yield the five substrate analogues. HPLC analyses of
the crude products obtained after cleavage generally showed a
major peak with at least 80% purity (Figures S1 and S2).
N
O
O
O
H2N
HO
R
Boc-HN
Boc-HN
N
R
Boc-HN
N
R
OH
+
O
N
a
NH2
b
O
O
HN
HN
HN
Boc-HN
Boc-HN
R
N
H
OH
S
N
H
S
N
H
S
N
H
a
5
HN
HN
Scheme 3. Key steps in the synthesis of the Tci- (23a-31a), S-alkyl-Itc- (23b-
31b, 23c-31c) and N-alkyl-Arg- (23d-31d, 23e-31e) based 1,2,4-oxadiazole-
containing inhibitors: (a) 1.3 equiv. amidoxime, HOBt, DIC, NMP, rt, 4h; (b) 1.2
equiv. AcONa, THF/water (7/3), reflux, overnight. Steps a and b were repeated
one more time.
S
N
H
S
N
H
5
b
d,e,b
c,b
O
O
O
H2N
R
H2N
R
H2N
R
N
H
N
H
N
H
We also synthesized a few examples of 1,3,4-oxadiazole (13
compounds) and di-substituted 1,2,4-triazole (25 compounds)
derivatives. They were prepared on a standard cross-linked PS
resin from the hydrazide precursor 6 (Scheme 4).
HN
HN
HN
R'
R'
S
NH2
7a-22a
S
NH
N
H
NH
7b-21b (R' = Me)
7c-22c (R' = Et)
7d-22d (R' = Me)
7e-22e (R' = Et)
N
N
Scheme 2. Synthesis of the Tci- (7a-22a), S-alkyl-Itc- (7b-21b, 7c-22c) and N-
alkyl-Arg- (7d-21d, 7e-22e) containing dipeptide-like inhibitors: (a) 1.3 equiv.
H2N-R, BOP, HOAt, 1.5 equiv. DIEA, DMF, rt, 5h, repeated twice; (b)
TFA/TIS/H20 (95:2.5:2.5), rt, 1.5 h, excepted S-alkyl-Itc analogues: 40 °C, 2h;
(c) 7b-21b: 0.2 M MeI in DMF, rt, 3x1h; 7c-22c: 0.2 M EtI in DMF, 50 °C, 3x1h
(d) 0.2 M MeI in DMF, rt, 3x1h; (e) 7d-21d: 2 M MeNH2,HCl and 2 M NMM in
anhydrous DMSO, 80 °C, 15h; 7e-21e: 2.5 M EtNH2,HCl and 2.5 M NMM in
anhydrous DMSO, 80 °C, 15h.
R
Boc-HN
O
O
a,b
HN
Boc-HN
HN
NH2
N
H
S
N
H
S
N
N
HN
c
H
- Dipeptide mimetics with a heterocyclic link. Several series of
compounds containing a 5-atoms heterocyclic link (i.e. 1,2,4-
and 1,3,4-oxadiazoles, 1,2,4-triazole) were considered. The
presence of such heterocyclic structure could increase the
metabolic stability as well as the bioavailability, compared to the
amide-containing analogues. In addition, their higher rigidity
might help to obtain NOS isoform selective inhibitors. These
heterocycles contain an extra heteroatom compared to the
amide bond, which could establish an additional hydrogen bond
in the enzyme binding site. Although the three heterocyclic
moieties are similar in size and shape and present the two
substituents in very similar positions, they show variations in
aromatic, electrostatic, and hydrogen bonding properties. This
could lead to different binding modes with the target, and
therefore to variation in inhibitory potencies between compounds
only differing by the heterocyclic moiety.[54,55]
R
Boc-HN
HN
6
N
S
N
H
Scheme 4. Key steps in the synthesis of the Tci- (32a-34a and 35a-39a), S-
alkyl-Itc- (32b-34b and 35b-39b, 32c-34c and 35c-39c) and N-alkyl-Arg- (32d-
34d and 35d-39d, 32e-34e and 35e-39e) containing 1,3,4-oxadiazole- and
1,2,4-triazole-based inhibitors. aReagents and conditions: (a) 3 equiv. R-CO2H,
3 equiv. HBTU, 4 equiv. DIEA, DMF, 4 h; (b) 2.6 equiv. C2Cl6, 10 equiv. DIEA,
anhydrous MeCN, 5 min then 4 equiv. PPh3, rt, 30 min. Steps i and ii were
repeated once more. (c) 5 equiv. R-C(=NH)NH2 or R-C(=NOH)NH2, reflux 24 h.
For the 1,3,4-oxadiazole compounds, the hydrazide was coupled
to three different carboxylic acids, followed by cyclo dehydration
in the presence of C2Cl6 and PPh3.[57] The five substrate
analogues were then prepared as described above. These
compounds were obtained in moderate yields after purification.
The cyclisation was not complete and we observed formation of
a side product probably resulting from the conversion of the
thiourea group to urea as suggested by LC-MS analysis. This
Fourty-four 1,2,4-oxadiazole derivatives were synthesized and
the specific synthetic steps of their preparation on standard resin
and Synphase lanterns are shown in Scheme 3.
Intermediate
5 was coupled to various amidoximes using
DIC/HOBt. The intermediates were then cyclo dehydrated by
adapting mild conditions (80 °C and no strong base) that we
previously described for the solution synthesis of 1,2,4-
3
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10.1002/cmdc.201900659
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FULL PAPER
compound remained unchanged through the following steps (S-
alkylation and guanidinylation). However, about 70% of the
expected derivative was present in the crude after cyclisation
(Figure S3).
substituents on the amide bond and an S-Et-Itc moiety were
preferred.
In the 1,2,4-oxadiazole series (Table 2), a small substituent
was also favorable to the inhibition of the three NOS (23b and
23c). The methyl group was probably too small to establish any
significant interaction with the enzymes, suggesting that the
1,2,4-oxadiazole heterocycle could indeed interact favorably.
The micromolar activity of other compounds (25c, 26c, 27c, 29b,
29c, 31c) indicates that in this series, the enzymes could
accommodate bulky aromatic substituents.
The 1,3,4-oxadiazole series (Table 3) contains only three
different substituents. As in the 1,2,4-oxadiazole series, a methyl
substituent led to significant inhibition but surprisingly with a N-
Et-Arg (32e). All other compounds were found inactive with the
exception of the S-Et-Itc analogue 34c.
For the synthesis of 1,2,4-triazole compounds substituted at
positions
3 and 5, we first reacted hydrazide 6 with
benzamidine.[58] After 24h reflux in 2-methoxyethanol, the
condensation to the acylamidrazone intermediate followed by
cyclo dehydration proceeded cleanly and totally (Figure S4A).
However, this approach was limited by the access to various
amidines. So, we checked if the close and more easily
accessible amidoxime derivatives, which were used in the
synthesis of 1,2,4-oxadiazole analogues could replace amidines.
Indeed, the reaction of resin
6 with the corresponding
benzamidoxime in the same experimental conditions led to
similar results (Figure S4B). To our knowledge, the use of
amidoximes for the synthesis of di-substituted 1,2,4-triazoles
has never been described. Therefore, we followed this synthetic
pathway using four more amidoximes and 25 compounds were
obtained.
Finally, in the 1,2,4-triazole series (Table 4), the methyl group
was moderately favorable for iNOS and nNOS inhibition (35c).
Among other analogues, only 37b, 37c and 38c showed
moderate inhibition of nNOS and/or iNOS.
As a general observation, modification of the a-carboxylic group
of the substrate analogues resulted in the loss of important
stabilizing interactions and most of the compounds showed no
significant (< 50% inhibition at 100 µM) or modest inhibitory
activity in the 10-50 µM range, except for compounds 7b and 7c
that bore a NH2-group at the end of a short flexible chain, and for
the 1,2,4-oxadiazole derivatives 23b and 23c having a short
extension. This low activity could be explained by poor
interaction(s) of the moiety replacing the a-COOH group in the
substrate access channel. The steric hindrance was also clearly
an unfavorable factor for binding to the NOSs, but the
introduction of polar substituents (e.g. OH, COOH in 12b, 29b)
on the extension could restore some interaction(s), probably with
residues in the substrate access channel. Although the five a-
amino acids 0a-e were all potent inhibitors of the three enzymes,
the most active compounds belonged to the S-Et-Itc series,
indicating that this substrate analogue made sufficient stabilizing
interactions at the active site to compensate the loss of the
carboxylic function, and/or favors the extension interactions
within the substrate access channel. The X-ray structure of the
complex of human iNOS and S-ethylisothiourea (SEIT) has
shown that the ethyl group packed near the heme and Phe369
side-chain and appeared optimal for interacting with the NOS
distal cavity.[23] iNOS almost exclusively preferred S-Et-Itc (for
instance, 7c, 8c, 21c, 23c, 25c, 26c, 27c, 34c, 35c, 38c, with
IC50 from 7.2 to 45 µM) with very few exceptions in the S-Me-Itc
series (7b and 23b, with IC50 of 7.0 and 23.9 µM, respectively)
and only one example in the Tci and N-Et-Arg series (7a and
32e, respectively).
In the case of nNOS, S-Et-Itc and S-Me-Itc were also the most
favorable substrate analogues (for instance, 7b, 7c, 8c, 11c,
12b, 12c, 14b, 21c, 22c, 23b, 23c, 27c, 29b, 29c, 31c, 34c, 35c,
37c, 38c, with IC50 from 0.4 to 42.0 µM) with the exception of the
Tci analogue 7a and the NEt-Arg analogue 32e. Finally, only a
few compounds significantly inhibited eNOS (7a, 7b, 21c, 23b,
23c, 32e, 35c, 37c, with IC50 from 1.4 to 44.9 µM), but also
inhibited nNOS and iNOS, indicating that eNOS was less easily
inhibited by this type of compounds. This result is in agreement
with the studies published by Park et al.[50] and Huang et al.[28]
about few S-Me-Itc- and S-Et-Itc-containing dipeptides, and
Arg(NwNitro)-containing dipeptides, respectively.
The completion of each reaction was checked by LC-MS
analysis after cleavage of a small portion of the solid support
(resin or lantern). Whereas the linkage between the thiourea
group of Tci and the Rink amide support was found to be TFA
labile, the one involving the S-alk-Itc residues was much more
resistant. A prolonged TFA treatment and light warming were
necessary to cleave S-alk-Itc from the solid support. All
compounds were obtained in low to moderate yields (5-50%)
after purification (reverse-phase HPLC).
Biological evaluation.
- Inhibition of NO Synthases. The inhibitory effects of all
compounds were evaluated against the formation of NO by three
recombinant NOSs (mouse iNOS, rat nNOS, and bovine eNOS)
on 96-wells plates using the oxyhemoglobin test.[59] A first
screening at 100 and 10 µM was performed and the IC50 were
measured for compounds showing more than 50% inhibition at
100 µM (Tables 1 to 4).
The five a-amino acids 0a-e are all potent inhibitors of the three
NOSs, with IC50 values in the low micromolar range (Table 1),
close to those reported in the literature.[48,60,61]
In the dipeptide-like series (Table 1), compounds possessing
two main types of extension displayed significant activities
against at least one NOS (IC50 in the 2-50 µM range). The first
one contained an alkylamine moiety preferentially with a propyl
(7a-c) but also a butyl (8c) chain. Increasing the chain length by
one more CH2 (pentyl) led to inactive compounds (9 series)
indicating that the propyl chain was optimal. This result was
expected as Huang et al.[62] reported that the corresponding
analogue Arg(NwNO2)-NH-(CH2)3-NH2 inhibited nNOS with a Ki
of 0.46 µM. These authors proposed that the amine group
establishes a significant ionic interaction within the substrate
access channel. However, this compound was much more
selective toward iNOS and eNOS (> 200-fold selectivity) than 7c
that was only 10- and 18-fold more selective against iNOS and
eNOS respectively. The second most favorable extension was
the furfuryl group with 21c being moderately active on both
NOSs. Apart some exceptions for nNOS (11c, 12b/c, 14b, and
22c), all other more or less bulky aromatic substituents did not
lead to any significant inhibitor. In the dipeptide-like series, small
4
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Table 1. Structures of amino acids 0a-e and dipeptide-like compounds 7-22 and in vitro NOS inhibition (IC50 in µM)[a]
O
H2N
R
HN
X
NH(2)
Cpds
R
Series
X
IC50 (µM) (or % inhibition at 100 µM)
iNOS
(Mouse)
nNOS
(Rat)
eNOS
(Bovine)
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
5.1
2.2
2.9
21.9
4.1
1.45
0.23
0.64
2.9
5.0
0.8
3.1
3.5
4.5
0
7
-OH
4.5
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
41.8
7.0
47.9
NI
10.0
0.4
6.6
NI
33.3
12.5
1.4
52.4
NI
N
H
NH2
61.6
19.8
8
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
17.0
NI
NI
NI
21.7
NI
NI
NI
56.6
NI
NH2
N
H
NI
NI
NI
9
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
53% at 100 µM
N
H
NH2
All NI
All NI
All NI
All NI
NI
NI
NI
10
11
12
13
14
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
90.0
NI
All NI
N
H
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
75.3
NI
NI
OH
45% at 100 µM
32.5
NI
NI
N
H
NI
a
b
c
d
S=
ND[b]
45% at 100 µM
70% at 100 µM
NI
NI
9.6
23.0
NI
NI
45.2
NI
CO2H
Me-S-
Et-S-
Me-NH-
N
H
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
N
H
All NI
All NI
All NI
All NI
All NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
18.6
45% at 100 µM
HN
N
H
NI
NI
5
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15
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
26.6
45% at 100 µM
HN
All NI
All NI
All NI
All NI
All NI
All NI
All NI
All NI
All NI
All NI
All NI
All NI
N
H
NI
NI
OMe
16
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
Cl
Cl
N
All NI
All NI
All NI
H
17
18
19
20
21
22
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
N
H
NO2
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
HN
O
Cl
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
45% at 100 µM
55% at 100 µM
N
H
NI
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
O
O
N
H
All NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
19.1
NI
NI
NI
16.9
NI
NI
NI
27.6
ND
ND
O
N
H
NI
NI
a
c
S=
NI
All NI
All NI
N
Et-S-
40.0
N
H
[a]NI = no significant inhibition (<50% at 100 µM). [b]ND = not determined.
Some inhibitors showed moderate (10-20-fold) selectivity for
nNOS against iNOS. This was the case for 7b (IC50 of 0.4 and
7.0 µM, respectively), 7c (IC50 of 6.6 and 47.9 µM,
respectively), 23b (IC50 of 2.0 and 23.9 µM, respectively), and
29b (IC50 of 3.2 µM and NI, respectively). Compound 7c was
also 8-fold selective against eNOS. The 1,2,4-oxadiazoles
25c, 26c and 31c, and the 1,3,4-oxadiazole 34c showed
moderate selectivity (≥ 3-fold) in favor of iNOS vs nNOS and
ring being the less efficient. In contrast, when comparing the
1,2,4-oxadiazole 30c and the 1,2,4-triazole 38c (p-
nitrobenzyl), only 38c showed inhibitory activity, the
corresponding dipeptide-like analogue 17c being also inactive.
The dipeptide-like 11c, the 1,2,4-oxadiazole 29c and the
1,2,4-triazole 37c (p-hydroxybenzyl) exhibited similar
inhibitory potencies toward nNOS. Finally, 1,2,4-oxadiazoles
28a-e and 1,2,4-triazoles 39a-e (benzodioxol extension)
exhibited either low or no activity, respectively. Although the
number of examples was too low to establish a reliable
ranking between the various links, the present results
suggested that 1,2,4-oxadiazole and 1,2,4-triazole rings could
be as favorable as the peptide bond. As stated above, the
observed variation in inhibitory potencies between
compounds only differing by the heterocyclic link probably
originated from the different aromatic, electrostatic, and
hydrogen bonding properties of the three heterocycles.[54,55]
eNOS. In particular, 25c, which contains
a branched
extension, did not inhibit nNOS at 100 µM, indicating that this
bulky substituent was not favorable for nNOS inhibition.
One interesting point was to assess the importance of the link
between the substrate analogue and the extension. For
instance, it was possible to compare 23b and 23c (1,2,4-
oxadiazoles) with, respectively, 32b and 32c (1,3,4-
oxadiazoles), and 35b and 35c (1,2,4-triazoles), all bearing a
methyl substituent on the heterocycle. We observed that the
1,2,4-oxadiazole ring (compounds 23b and 23c) was the
most favorable for NOS inhibition with the 1,3,4-oxadiazole
6
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Table 2. Structures of 1,2,4-oxadiazoles 23-31 and in vitro NOS inhibition (IC50 in µM)[a]
N
O
R
H2N
N
HN
X
NH(2)
Cpds
R
Series
X
IC50 (µM) (or % inhibition at 100 µM)
iNOS
(Mouse)
nNOS
(Rat)
eNOS
(Bovine)
a
b
c
d
S=
Me-S-
Et-S-
NI
23.9
19.1
NI
NI
2.0
9.4
NI
NI
6.8
44.9
NI
23
24
-CH3
Me-NH-
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
All NI
All NI
All NI
All NI
All NI
All NI
25
26
27
28
29
30
31
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
31.0
NI
NI
NI
NI
NI
ND[b]
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
22.9
NI
All NI
O
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
39.5
NI
NI
NI
19.6
NI
O
O
NI
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
O
All NI
All NI
All NI
All NI
45% at 100 µM
All NI
All NI
NI
NI
O
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
3.2
35.9
NI
OH
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
All NI
All NI
All NI
NO2
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
34.2
ND
NI
NO2
[a]NI = no significant inhibition (<50% at 100 µM). [b]ND = not determined.
7
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Table 3. Structures of 1,3,4-oxadiazoles 32-34 and in vitro NOS inhibition (IC50 in µM)[a]
N
N
R
H2N
O
HN
X
NH(2)
Cpds
R
Series
X
IC50 (µM) (or % inhibition at 100 µM)
iNOS
(Mouse)
nNOS
(Rat)
eNOS
(Bovine)
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
NI
NI
54.3
NI
57.4
NI
NI
17.9
NDb
NI
NI
NI
12.8
32
33
34
-CH3
a
c
d
e
S=
Et-S-
Me-NH-
Et-NH-
All NI
All NI
All NI
All NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
7.2
NI
NI
NI
26.9
NI
NO2
NI
NI
[a]NI = no significant inhibition (<50% at 100 µM). [b]ND = not determined.
Table 4. Structures of 1,3,4-oxadiazoles 35-39 and in vitro NOS inhibition (IC50 in µM)[a]
N
HN
R
H2N
N
HN
X
NH(2)
Cpds
R
Series
X
IC50 (µM) (or % inhibition at 100 µM)
iNOS
(Mouse)
nNOS
(Rat)
eNOS
(Bovine)
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
NI
37.9
NI
NI
NI
42.0
NI
NI
NI
9.9
NI
35
36
-CH3
NI
NI
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
All NI
All NI
All NI
37
38
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
13.8
14.6
NI
NI
NI
20.6
NI
All NI
NI
OH
NI
NI
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
NI
45% at 100 µM
NI
16.1
NI
All NI
14.5
NI
NI
NO2
NI
8
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39
a
b
c
d
e
S=
Me-S-
Et-S-
Me-NH-
Et-NH-
O
O
All NI
All NI
All NI
[a]NI = no significant inhibition (<50% at 100 µM).
- Stability of selected compounds in inhibition test conditions.
The chemical stability of few S-alkyl-Itc compounds
inhibitory potencies measured versus iNOS and nNOS (Table
2).
a
(dipeptide-like 11b and 11c, and 1,2,4-oxadiazole 31c, all at 1
mM in 100 µM Hepes buffer, pH 7.5, at 30 °C) was checked
alone or in the presence of 10 eq. benzylamine. Indeed, the
S-alkyl-isothiourea could be converted into urea or react with
nucleophiles (here yielding N-benzyl-guanidine in the
presence of benzylamine). LC-MS analysis performed after
45 min and 3 h incubation showed no significant formation of
urea or N-benzylguanidine (data not shown).
- Spectral studies. UV-Visible difference spectroscopy allows
observation of interactions of substrates and inhibitors with
the heme active site of NOS.[63] To better understand how
some of the new compounds (dipeptide-like 7b, 7c, 19c, 21c
and 22c, 1,2,4-oxadiazoles 23b, 23c, 25c, 26c, and 31c,
1,3,4-oxadiazole 34c, 1,2,4-triazoles 35c and 37c) acted as
inhibitors, we have studied their effects on the UV-visible
spectra of the nNOS (nNOSoxy
) and iNOS (iNOSoxy)
Figure 2. Difference spectrum obtained upon stepwise addition of
increasing concentrations (from 7.5 µM to 2.0 mM) of 23c to iNOSoxy in
Hepes buffer, as described in Experimental section. Inset: plot of 1/(DA395-
425nm) vs 1/[23c].
oxygenase domains.[63] The UV–visible spectrum of nNOSoxy
and iNOSoxy in Hepes buffer displayed a broad Soret peak
with a maximum at 415 nm indicating that they predominantly
existed as hexacoordinated heme–FeIII–H2O complexes, with
water as the 6th axial ligand. When added to these proteins,
L-arginine elicited difference spectra with a peak at ~395 nm
and a trough at ~420 nm (type I interaction, data not shown).
The stepwise additions of the selected compounds to n- and
iNOSoxy gave in all cases rise to difference spectra
characterized by a trough at ~425 nm and a peak at ~395 nm
characteristic of type I interactions (Figure 2 for compound
23c). These results showed that all the studied compounds
bound in close proximity to the heme of n- and iNOSoxy and
shifted the spin state equilibrium to the pentacoordinated
heme–FeIII state in a similar manner than the L-arginine
substrate. Double reverse plotting of the differences in
absorbance between peak at ~395 nm and valley at ~425 nm
(DA395-425nm) as a function of the concentration of the added
compounds allowed to calculate apparent binding constants
(Ks) (Table 5). As a reference inhibitor, SEIT tightly bound
both proteins with high affinity for iNOSoxy (Figure S5 in
Supporting Information for SEIT). Surprisingly, compounds
7b-c poorly bound both proteins. By contrast, dipeptide-like
compounds 19c, 21c and 22c preferentially bound nNOSoxy
vs iNOSoxy with apparent affinities close to that of SEIT for
this isoform. Similarly, 1,2,4-oxadiazoles 23b, 23c, 26c, 31c,
1,3,4-oxadiazole 34c and 1,2,4-triazoles 35c and 37c
preferentially bound nNOSoxy vs iNOSoxy but with lower affinity
than the dipeptide-like compounds. Interestingly, in these
series of compounds, similar selectivity of nNOS over iNOS
was observed when comparing their inhibitory potencies
(Tables 1-4). Only the 1,2,4-oxadiazole 25c showed
preference for iNOSoxy over nNOSoxy in accordance with its
Table 5. Apparent equilibrium constants (Ks, µM) for the binding of some
dipeptides, 1,2,4-oxadiazoles, 1,3,4-oxadiazole and 1,2,4-triazoles to
[a]
nNOSoxy and iNOSoxy
.
Cpds
nNOSoxy
6.4 ± 1.5
iNOSoxy
SEIT
2.4 ± 0.4
Dipeptide-like
7b
7c
19c
21c
22c
75 ± 9
122 ± 31
11.4 ± 3.8
9.6 ± 1.2
7.1 ± 2.6
56 ± 16
144 ± 56
50 ± 5
71 ± 10
406 ± 97
1,2,4-Oxadiazoles
23b
23c
25c
26c
31c
37.4 ± 8.5
164 ± 25
253 ± 129
311 ± 59
40 ± 13
193 ± 36
178 ± 19
69 ± 13
351 ± 56
162 ± 21
1,3,4-Oxadiazole
34c
68 ± 8
> 500
1,2,4-Triazoles
35c
37c
166 ± 19
163 ± 42
487 ± 51
311 ± 141
[a] Titrations were performed by UV/Vis difference spectroscopy as
described in Experimental Section. Values
experiments.
+ SD from three different
- Inhibition of NOS in cells. To study the potential of these
compounds to inhibit NOS expressed in cells, we performed
9
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several experiments with the two more potent compounds,
the dipeptide-like 7b and the 1,2,4-oxadiazole 23c. L-N6-(1-
iminoethyl)-lysine (L-NIL)[31] and/or S-ethyl isothiourea
(SEIT),[64] two well-known potent and quite selective iNOS
inhibitors were used as positive controls.
First, cell viability was evaluated in the murine macrophage
RAW 264.7 cells using a MTT assay. This experiment was
performed with 1 µg/mL or without LPS, which induces iNOS
expression. In the absence of LPS, none of the compounds
tested modified cell viability. In the presence of LPS (1 µg/ml),
cell viability was reduced by 42% due to the inhibition of key
enzymes involved in cell metabolism (e.g. cytochrome
oxidase) by NO.[65] L-NIL and SEIT partially reversed this
effect by respectively 26% and 32% at 100 µM (P<0.05, n =
6) (Figure 3A and 3B). Cell viability was also improved by
18% and 22% for compounds 7b (P<0.05, n = 6) (Figure 3C)
and 23c (P<0.05, n = 6) (Figure 3D), respectively, at the
same concentration. These results indicated that 7b and 23c
could at least partially reverse the iNOS-derived toxicity of NO
and had significant ability to enter the cells.
Figure 4. Inhibitory effects of L-NIL, SEIT and compounds 7b and 23c on
NO production by RAW 264.7 cells stimulated by LPS (1µg/mL).
Figure 5. Inhibitory effects of L-NIL, SEIT and compounds 7b and 23c on
NO production by INS-1 cells stimulated by IL-1b (1 ng/mL).
Figure 3. Effects of L-NIL, SEIT, 7b and 23c on cell viability: MTT assay
on RAW 264.7 in the presence or not of LPS (1µg/mL). *, P<0.05; ***,
P<0,001.
In INS-1 cells, L-NIL and SEIT reduced NO production by
respectively 51% (P<0.001, n=4) and 77% (P<0.01, n = 4) at
100 µM (Figure 5). Compound 7b showed an effect
comparable to that of L-NIL (-51% at 100 µM; P<0.001, n=8)
(Figure 5). All three compounds started to be active at 5 µM
concentration (L-NIL, P<0.01; SEIT, P<0.05; 7b, P<0.001).
Compound 23c was less potent than 7b with a noticeable
inhibitory activity at 25 µM (P<0.05), but of equal efficiency in
the two cell lines (-30% at 100 µM; P<0.05, n = 4) (Figure 5).
The 1,3,4-oxadiazole 34c and the 1,2,4-triazole 35c were
also evaluated in the same experimental conditions. They
were inactive with no effect on NO production in RAW 264.7
cells (data not shown) probably due to lack of membrane
permeability.
Finally, we assessed the ability of the compounds to restore
insulin secretion after induction of iNOS. Indeed, when iNOS
is induced in INS-1 cells, insulin secretion induced by basal
(2.8 mM) and stimulating (5.6 mM) glucose was reduced by
respectively 42 and 55% (Figure 6A). L-NIL partially improved
insulin secretion in the presence of 5.6 mM glucose with a
maximum effect at 25 µM concentration. Compound 7b also
restored insulin secretion in the presence of 5.6 mM glucose
at 10 and 25 µM (P<0.05, n=5) (Figure 6B). These results are
Then, we evaluated their ability to inhibit iNOS in RAW 264.7
cells (Figure 4) and in the rat pancreatic β-cell line INS-1
(Figure 5), where iNOS was induced by LPS (1 µg/mL) or IL-
1β (1 ng/mL), respectively.
Nitrite accumulation in the supernatant of RAW 264.7 cells in
the presence of LPS was used as a measurement of iNOS
activity. We observed a dose-dependent decrease in NO
production with increasing concentrations of L-NIL and SEIT,
reaching respectively -66% and -75 % at 100 µM (P<0.001,
n=4) (Figure 4). The two compounds were already active at a
5
µM concentration (L-NIL, P<0.001; SEIT, P<0.01).
Compound 7b also reduced NO production starting at 10 µM
(P<0.01) and with a maximum of -61% at 100 µM (P<0.001,
n=8) (Figure 4). Compound 23c decreased NO production to
a lesser extent (-32% at 100 µM; P<0.001, n = 4), with a
detectable effect at 50 µM (P<0.01) (Figure 4). No compound
induced noticeable cell toxicity.
10
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of interest as iNOS inhibition was found to protect beta cells
against the deleterious effects of pro-inflammatory cytokines,
which in type 1 diabetes are a major cause of beta-cell
destruction.[66,67]
whereas eNOS was scarcely inhibited. These results are in
accordance with Silverman’s team studies on nitro-arginine-
containing dipeptides, which were shown to be highly
selective against eNOS. The observed main preference for
the S-Et-Itc analogues could be related to the study about a
series of S-alkylisothiourea developed as non-selective NOS
inhibitors, where SEIT was found to be the best inhibitor.[68]
These results, supported by the X-ray structure of hiNOS-
SEIT complex,[23] suggested that the S-ethyl moiety was
optimal for binding. While some dipeptide-like compounds
were found significantly active as expected from previous
studies,[26,50] it is noteworthy that active compounds were also
identified in the not yet described dipeptide mimetic series
containing various five-membered-heterocycles. This is an
important finding as these heterocyclic compounds are
expected to be more stable and less flexible than the
dipeptide-like analogues. Our results open the way to the
development of new heterocyclic compounds only containing
a S-Et-Itc derivative as a substrate analogue that could take
advantage of differences along the substrate access channel
for NOS isoform selectivity.
Experimental Section
- Chemistry
-
General. Protected amino acids, BOP, DIEA, TFA, piperidine,
solvents and other reagents were purchased from Iris-Biotech,
Novabiochem, Riedel-de Haën, Carlo Erba or Acros organics and
used without further purification. Fmoc Rink amide polystyrene resin
(100-200 mesh, 0.94 mmol/g) and Synphase lanterns (35 µmol
loading) were purchased from Iris-Biotech and Mimotopes,
respectively. Solvents used for RP-HPLC and LC-MS were of HPLC
grade. Amidoximes were either commercially available or have been
synthesized as previously described.[56]
Figure 6. Effect of L-NIL (A) and compound 7b (B) on insulin secretion by
INS-1 cells in the presence of 2.8 (basal) or 5.6 mM glucose and after
iNOS induction with IL-1b (1 ng/mL). *, P<0.05.
- NMR and IR spectroscopy.1H NMR spectra were recorded at 300,
400 or 500 MHz using DMSO-d6 or CDCl3 solutions. Splitting patterns
are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; br, broad. 13C NMR spectra were recorded at 75, 101 or 125
MHz using DMSO-d6 solutions.
Overall, these results support that compound 7b was able to
inhibit intracellular iNOS and to affect cellular functions.
IR spectra were collected on a Perkin Elmer Spectrum One apparatus.
- Analysis and purification. Thin-layer chromatography was performed
on aluminium backed sheets of silica gel F254 (0.2 mm), which were
visualized under 254 nm light and by spraying with a 2% EtOH
solution of ninhydrin followed by heating, or by charring with an
aqueous solution of ammonium sulfate and sulfuric acid (200 g
(NH4)2SO4 and 40 mL concentrated sulfuric acid in 1 L of water).
Conclusion
In this study, we have synthesized 164 compounds (5 amino
acids, 76 dipeptide-like compounds, 44 1,2,4-oxadiazoles, 14
1,3,4-oxadiazoles, and 25 1,2,4-triazoles) using an original
solid-phase synthetic strategy. This straightforward synthetic
strategy was implemented with generally efficient and simple
reactions, allowing the rapid preparation of numerous
compounds with satisfying crude purity. Beside the synthesis
on PS resin, we have prepared two libraries of dipeptide-like
and 1,2,4-oxadiazole analogues on Lantern supports. This
approach has great potential for the combinatorial synthesis
of many more analogues from the lead compounds
discovered in this study.
RP-HPLC analysis was performed using the following conditions:
Chromolith SpeedRod C18 column (0.46 x 5 cm), linear gradient (0-
100%) of eluent B in A over 5 min (flow rate: 3 mL/min). Eluent A:
0.1% aqueous TFA; eluent B: acetonitrile/0.1% TFA. Detection was
made at 214 nm.
Purification by preparative RP-HPLC was performed on a Waters
Delta Pak C18 column (40 x 100 mm, 15 µm, 100 Å), linear gradient
of eluent B in A at a 1%/min rate (flow rate: 28 mL/min). Eluent A:
0.1% aqueous TFA; eluent B: acetonitrile/0.1% TFA. In the case of
the libraries of dipeptide-like and 1,2,4-oxadiazole compounds, their
purification was performed on a reverse-phase preparative HPLC
All compounds were tested against three recombinant NOS
isoforms. Overall, several new compounds showed significant
inhibitory activity (IC50 about 2 to 80 µM) toward at least one
NOS, with a global preference for nNOS, followed by iNOS,
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
coupled to mass spectrometer (Autopurif system from Waters driven
by the software MassLynx 4.0 Fractonlynx) using a Waters X Bridge
Prep C18 column (19 x 100 mm, 5 µm) and a linear gradient of eluent
B in A (flow rate: 20 mL/min).
IR nmax cm-1 3373, 2977, 2926, 2182, 2096, 1742, 1704; 1H NMR (300
MHz, DMSO-d6) d 1.38 (s, 9H), 1.65-1.75 (m, 4H), 3.63 (s, 3H), 3.64-
3.68 (m, 2H), 4.00 (m, 1H), 7.34 (d, J = 7.56 Hz, 1H); 13C NMR (75
MHz, DMSO-d6) d 25.9, 27.8, 28.1, 44.4, 51.8, 52.8, 78.3, 127.3,
155.5, 172.8.
Column chromatography was performed using Merck silica gel 60 of
particle size 40-63 µm.
- Boc-Tci(Nw-Rink amide)-OMe, 4. A portion of Fmoc-protected Rink
amide resin (0.94 mmol/g substitution) was loaded in a peptide
synthesis reaction vessel and swelled in DMF for 15 min. The resin
was then treated twice (5 min + 20 min) with a solution of 20%
piperidine in DMF to remove the Fmoc-protecting group, washed with
DMF, DCM, MeOH, and dried under vacuum. The deprotected
peptide-resin was placed in a balloon and swelled in dry THF for 15
min and a solution of compound 3 (3 equiv.) and TEA (3.1 equiv.) in
THF (10 mL per g of resin) was added. The suspension was stirred at
65 °C for 3 h and at room temperature overnight. Completion of the
reaction was assessed using the TNBS test (see below). The resin
was finally filtered, washed with THF and DCM and dried.
Mass spectrometry: samples were prepared in acetonitrile/water
(50/50 v/v) mixture. The LC-MS system consisted of a Waters Alliance
2690 HPLC, coupled to
a Waters-Micromass ZQ spectrometer
(electrospray ionization mode, ESI+). All analyses were carried out
using a RP C18 monolithic Onyx Phenomenex column (25 x 4.6 mm),
linear gradient (0–100%) of eluent B in A over 3 min (flow rate: 3
mL/min). Eluent A: 0.1% aqueous formic acid; eluent B:
acetonitrile/0.1% formic acid. Positive ion electrospray mass spectra
were acquired at a solvent flow rate of 100–500 µL/min. Nitrogen was
used as both the nebulizing and drying gas. The data were obtained
in a scan mode in 0.1 s intervals; 10 scans were summed up to get
the final spectrum.
In the case of Fmoc-Rink amide-loaded Synphase lanterns (35 µmol
per unit), they were first labelled with color-coded tagging using stems
and cogs of different colors. Then, they were introduced in drilled-
capped glass bottles and conditioned in DMF. After removal of solvent,
a solution of 20% piperidine in DMF to remove the Fmoc-protecting
group was added to cover all lanterns. After 20 min without stirring,
the treatment was repeated for the same duration and the lanterns
were washed with DMF (3 x 3 min), MeOH (3 x 3 min) and DCM (3 x
3 min) and dried. Loading of compound 3 was performed in the same
conditions as described above, taking care that lanterns are covered
with solvent. Lanterns were finally washed with DMF, MeOH and
DCM (3 x 3 min each solvent) and dried. A small piece of one lantern
was cut to check reaction completion with the TNBS test.
HR-MS were registered on a JEOL JMS-SX-102A mass spectrometer.
The HPLC retention times, calculated monoisotopic mass and
measured high resolution mass of the compounds, NMR data and
yields of purified products are reported in Supporting Information
Synthesis of resin supported intermediates 4, 5 and 6.
- Boc-Orn(Z)-OMe, 1. To a solution of Boc-Orn(Z)-OH (25 g, 68.2
mmol) in DMF were added Na2CO3 (14.46 g, 136 mmol) and MeI
(8.07 mL, 129.6 mmol). After 5 h stirring at room temperature, the
reaction mixture was filtered and concentrated. The residue was taken
up in AcOEt (100 mL) and the organic phase was washed with water,
aqueous 1 M KHSO4, water, saturated aqueous NaHCO3, water and
saturated aqueous NaCl, dried over MgSO4 and concentrated in
vacuo to give a yellow solid (25.8 g, 99%), which was used without
further purification. Rf 0.45 (AcOEt/hexane, 3:7); tR 2.69 min; m/z
(ES+) 381.3 [M + H]+, 325.3 [M – tBu + H]+, 281.3 [M –Boc +H]+. 1H
NMR (300 MHz, DMSO-d6) d 1.38 (s, 9H), 1.45-1.64 (m, 4H), 2.97 (m,
2H), 3.61 (s, 3H), 3.94 (m, 1H), 5.00 (s, 2H), 7.24 (m, 2H), 7.34 (m,
5H); 13C NMR (75 MHz, DMSO-d6) d 26.0, 28.0, 28.2, 40.2, 51.7, 53.3,
65.1, 78.2, 127.7, 128.3, 137.3, 155.5, 156.1, 173.1.
- Boc-Tci(Nw-Rink amide)-OH, 5. Saponification of 4 was performed
according to Cantel S. et al..[53] A portion of the resin 4 was loaded in
a peptide synthesis reaction vessel and swelled in THF for 15 min. A
2 M aqueous solution of LiOH (5 equiv.) was then added. Volumes
were calculated on the basis of a 7/3 THF/H2O ratio. After 15 h stirring
at room temperature, the resin was washed with THF, DCM and
MeOH, and dried. Completion of the reaction was assessed by LC-
MS analysis after a cleavage test (see below).
For Synphase lanterns, saponification was similarly carried out but
without stirring and respecting 3 x 3 min duration for each solvent
washing.
- Boc-Orn-OMe, TFA salt, 2. To a solution of 1 (25.8 g, 67.9 mmol) in
ethanol, was added slowly and under strong stirring TFA (7.6 mL, 102
mmol), followed by 4 g of 10% Pd/C. After 4 h stirring at room
temperature under H2 bubbling, the suspension was filtered on celite
and the filtrate was evaporated to give a white solid (24.4 g, 100%). tR
1.50 min; m/z (ES+) 247.2 [M + H]+, 191.2 [M – tBu + H]+, 147.2 [M –
Boc +H]+. 1H NMR (300 MHz, DMSO-d6) d 1.38 (s, 9H), 1.55-1.75 (m,
4H), 2.76 (m, 2H), 3.62 (s, 3H), 3.95 (m, 1H), 7.29 (d, J = 7.56 Hz,
1H), 7.82 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 24.5, 28.2, 28.8,
39.0, 52.4, 53.6, 78.9, 156.2, 173.5.
- Boc-Tci(Nw-Rink amide)-NHNH2, 6. A portion of the resin 4 was
loaded in a peptide synthesis reaction vessel and swelled in THF for
15 min. Hydrazine hydrate (5 equiv.) was then added and the mixture
was stirred at room temperature for 24 h. The resin was washed with
DMF, DCM and MeOH, and dried. Completion of the reaction was
assessed by LC-MS analysis after a cleavage test (see below). A
TNBS test gave dark red resin beads, due to the hydrazide amine
group.
General procedures for synthesis.
-
Methyl Na-(tertbutoxycarbonyl)-Nd-(thioxomethylene)-L-ornithinate
(Boc-L-Orn(Nd=C=S)-OMe), 3. To a solution of 2 (24.4 g, 67.8 mmol)
in THF (250 mL) were added at 0°C under strong stirring CS2 (36.2
mL, 0.6 mol) immediately followed by TEA (32.6 mL, 232 mmol). The
reaction was stirred for 45 min at this temperature and 81 mL (0.9
mol) of H2O2 were added dropwise. The mixture was then diluted with
diethylether (725 mL). The organic layer was washed with aqueous 1
M KHSO4, water, saturated aqueous NaHCO3, water and saturated
aqueous NaCl, dried over MgSO4 and concentrated in vacuo to afford
after chromatographic purification over silica gel (hexane/AcOEt (5:1))
a yellowish oil (14 g, 72%). Rf 0.26 (AcOEt/hexane, 1:5); tR 2.77 min;
m/z (ES+) 289.1 [M + H]+, 233.1 [M – tBu + H]+, 189.1 [M –Boc +H]+;
A) Synthesis of supported dipeptide-like 7-22.
A portion of the
supported thiocitrulline intermediate 5 was swelled in DMF or NMP for
15 min and filtered. Were then successively added DMF or NMP
solutions of amine compound (1.3 equiv.), HOAt (1.3 equiv.), DIEA
(1.5 equiv.) and BOP (1.3 equiv.). After 12 h stirring, the resin was
filtered and washed with DMF and the coupling was repeated. The
resin was finally washed with DMF and DCM and dried.
In the case of Synphase lanterns, the same conditions were followed
and final volume of DMF or NMP was 10 mL/10 lanterns.
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
B) Synthesis of supported 1,2,4-oxadiazole derivatives 23-31. This
procedure was adapted from a solution protocol described by Hamzé
et al.[56] and was composed of a coupling step followed by a cyclo
dehydration step. A portion of the supported thiocitrulline intermediate
5 was placed in a balloon, swelled in DMF/DCM (9:1) for 15 min and
cooled to –10°C. Were then added the amidoxime derivative (1.2
equiv.), HOBt (1.2 equiv.) and DIC (1.2 equiv.). After 20 min at –10°C
and 4 h at room temperature, the resin was filtered and washed with
DMF and the coupling was repeated. The resin was finally washed
with DMF, DCM and MeOH. For the cyclo dehydration step, it was
conditioned in THF/H2O (7:3). Sodium acetate (1.1 equiv.) was then
added and the mixture was refluxed during 5 h. The resin was finally
washed with DMF, DCM and MeOH, and dried.
2 h with the exception of S-methyl and S-ethyl isothiourea analogues,
which were submitted to the cleavage mixture for 3h at 30 °C. After
resin filtration, the filtrate was concentrated under vacuum, and
compounds were precipitated by diethyl ether addition and recovered
after centrifugation. The pellet was washed twice with diethyl ether.
When no precipitation occurred, the residues were solubilized in
water/MeCN (50:50) and freeze-dried. All compounds were obtained
with an average yield of 5%-80% after reverse-phase HPLC
purification. All compounds were above 90-95% purity.
- Cleavage test: some reaction steps (loading of compound 3 onto the
solid support and its saponification, S-ethylation and guanidinylation
for selected compounds) were checked by cleaving 2-3 mg of resin
with a solution of TFA/TIS/H2O (95:2.5:2.5, v/v/v, 0.5 mL) for 30 min at
room temperature. After filtration, the mixture was evaporated under a
nitrogen stream and the residue was precipitated with diethylether.
Precipitate was collected by filtration, solubilized in 50% MeCN/H2O
and submitted to LCMS analysis.
In the case of Synphase lanterns, the same conditions were followed
and final volume of DMF/DCM or THF/H2O was 10 mL/10 lanterns.
C) Synthesis of supported 1,3,4-oxadiazole derivatives 32-34. This
procedure was adapted from James et al.[57] and was composed of a
coupling step followed by a cyclo dehydration step. A portion of the
supported thiocitrulline intermediate 6 was swelled in DMF for 15 min
and DIEA (4 equiv.), a carboxylic compound (3 equiv.) and HBTU (3
equiv.) were added. After 4 h stirring at room temperature, the resin
was filtered and washed with DMF, MeOH and DCM. Completion of
the coupling step was assessed by the TNBS test. In case of positive
test, the coupling step was repeated. For the cyclodehydration step,
the dried resin was swelled in dry acetonitrile and DIEA 10 equiv.) and
C2Cl6 (2.6 equiv.) were added, followed, after 5 min, by PPh3 (4
equiv.). After 30 min stirring at room temperature, the resin was
washed with DMF, DCM and MeOH and dried.
J) TNBS test. An aliquot portion of resin beads or a small piece of one
lantern is collected and treated with one drop of a 1 M TNBS solution
in DMF and one drop of a 10% DIEA solution in DMF (v/v). Colourless
support after 1 min at room temperature indicates the absence of free
amine groups. The presence of free amine groups gives a red colour.
In the case of the hydrazide resin 6, the free NH2 gives a dark brown
colour.
- Biological evaluation.
L-Arginine, L-citrulline, Nw-nitro-L-arginine, dithiothreitol (DTT),
hemoglobin, superoxide dismutase, catalase, bovine serum albumin,
L-NIL, SEIT, (6R)-5,6,7,8-tetrahydrobiopterin, NADPH, porcine brain
calmodulin and all common salts and buffers were purchased from
Sigma-Aldrich.
D) Synthesis of supported di-substituted 1,2,4-triazole derivatives 35-
39. A portion of the supported thiocitrulline intermediate 6 was placed
in a balloon and swelled in 2-methoxyethanol for 15 min. An amidine
or amidoxime derivative (5 equiv.) was then added and the reaction
mixture was stirred under reflux for 24 h. The resin was finally washed
with DMF, DCM and MeOH, and dried.
A) Production of recombinant NOSs. Full length recombinant rat
nNOS, mouse iNOS, bovine eNOS, and the heme domains of rat
brain nNOS and mouse iNOS were expressed in Escherichia coli and
purified as described previously.[69-73] Protein concentrations were
determined by the method of Bradford using bovine serum albumin as
a standard and the Bradford reagent from Biorad.[74] The heme
concentrations of the purified NOS were determined optically from the
[FeII-CO] – [FeII] difference spectrum using a De445-480nm of 74 mM-
1.cm-1.[75] They were estimated to be more than 95% pure by SDS-
PAGE electrophoresis.
E) Synthesis of supported S-Methyl-isothiourea derivatives.
A
supported thiourea derivative was swelled in DMF for 15 min, filtered
and a 0.2 M solution of MeI (15 equiv.) in DMF was added. The
reaction was stirred at room temperature for 1 h and was repeated
twice. The resin was then washed with DMF and DCM and dried.
F) Synthesis of supported S-Ethyl-isothiourea derivatives. S-
Ethylation of thiourea compounds was performed as S-methylation
but in a balloon at 45°C and in the presence of a 0.4 M solution of EtI
(30 equiv.) in DMF.
B) Measurement of NO production by recombinant NOSs. NOS
catalytic activity was measured using the hemoglobin capture
assay.[59] The test was performed at 30 °C in 96-well microplates
using a final volume of 200 µl. The assay mixture contained 100 µM
NADPH, 10 µM BH4, 6 µM HbO2, 100 µM DTT, 5 µM FAD, 5 µM FMN,
10 µM (nNOS and eNOS) or 20 µM (iNOS) arginine, all in 100 mM
Hepes buffer, pH 7.5, and, for constitutive n- and e-NOS, 10 µg/mL
calmodulin and 1 mM CaCl2. The studied compounds were introduced
G) Synthesis of supported N-Methyl-guanidine derivatives.
A
supported S-methyl-isothiourea derivative was treated with a 2 M
solution of MeNH2, HCl (60 equiv.) and NMM (60 equiv.) in dry DMSO
in a sealed vessel resistant to pressure. The suspension was stirred
at 80°C for 14 h, cooled and the resin was washed with water, MeOH
and DCM, and dried.
as
2 µL of x100 concentrated solutions in DMSO and control
experiments were performed with DMSO or buffer only. The kinetics
of NO production was measured at 401 nm using the Infinite F500
microplate reader (Tecan).
H) Synthesis of supported N-Ethyl-guanidine derivatives. A supported
S-methyl-isothiourea derivative was treated with a 2.5 M solution of
EtNH2, HCl (75 equiv.) and NMM (75 equiv.) in dry DMSO in a sealed
vessel resistant to pressure. The suspension was stirred at 80°C for
14 h, cooled and the resin was washed with water, MeOH and DCM,
and dried.
C) Effects of selected compounds on UV-visible spectra of n- and
iNOSoxy. Optical spectra were recorded using an Uvikon 941
spectrophotometer in 150 µL quartz cuvettes. The apparent binding
affinities of the studied compounds with the n- and iNOSoxy active
sites were determined by perturbation difference spectroscopy at
room temperature.[63] The native enzymes (~1 μM in 50 mM Hepes
buffer, pH 7.4) were equally divided into reference and sample
cuvettes and a baseline was recorded. Increasing concentrations
(usually in the 1 µM – 1 mM range) of the studied compounds in
I) Cleavage from the solid support.
- Final cleavage of compounds from the solid support was performed
with TFA/TIS/H2O (95:2.5:2.5, 10 mL/g resin) at room temperature for
13
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
buffer (or DMSO) were added to the sample cuvette and equivalent
amounts of buffer (or DMSO) were added to the reference cuvette.
Apparent dissociation constants (Ks app) were estimated by plotting the
difference in absorbance between peak (~395 nm) and valley (~425
high performance liquid chromatography; rt, room temperature; Rt,
retention time; SEIT, S-ethyl-isothiourea; tBu, tert-butyl; Tci,
thiocitrulline ; TEA, triethylamine; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; TIS, triisopropylsilane; TNBS, 2,4,6-trinitrobenzene-1-
sulfonic acid; Z, benzyloxycarbonyl.
nm) versus concentration of the added compound as
a double
reciprocal plot. Linear fittings of the data were performed by
KaleidaGraph (Version 4.1, Synergy Software).
Acknowledgements
D) Evaluation on cellular models. The macrophage cell line RAW
264.7 (a gift from Dr A. Blangy, Montpellier, France) was cultured in
Dulbecco's Modified Eagle's Medium (DMEM), supplemented with
10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin and 2 mM
L-glutamine.
T.T. was supported by the Fondation pour la Recherche
Médicale (DCM20121225755). The authors thank Dr D. J.
Stuehr (Cleveland Clinic Fundation, Cleveland, USA) for
providing NOS isozymes plasmids, and Pierre Sanchez for
performing mass spectrometry analyses.
The insulin-secreting cell line INS-1 (a gift from Prof. C. Wolheim,
Geneva, Switzerland) was cultured in RPMI-1640 supplemented with
10% FCS, 100 units/ml penicillin, 100 µg/mL streptomycin, 2 mM L-
glutamine, 10 mM HEPES, 1 mM sodium pyruvate and 50 mM 2-
mercaptoethanol, according to the method of M. Asfari et al.[76]
Conflict of Interest
The authors declare no conflict of interest.
In RAW 264.7 cells, iNOS was induced by LPS (1µg/mL, Sigma
Aldrich) during 24h, and, in INS-1 cells, by IL-1β (1 ng/mL) during 48h.
All studied compounds were incubated with cells during the last 24h.
S-ethylisothiourea (SEIT) and L-NIL were used as positive controls
(Sigma Aldrich).
Keywords: arginine • heterocycles • NO synthases inhibitors
• solid phase synthesis • thiocitrulline
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Abbreviations
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Boc,
tert-butyloxycarbonyl;
BOP,
(benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium, hexafluorophosphate; DCM,
dichloromethane;
diisopropylethylamine;
DIC,
DMF,
diisopropylcarbodiimide;
dimethylformamide;
DIEA,
DMSO,
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(dimethylamino)methylene]-N-methylmethanaminium
hexafluorophosphate; Hepes, 4-(2-hydroxyethyl)-1-piperazine-
ethanesulfonic acid; HPLC, high performance liquid chromatography;
HOBt, hydroxybenzotriazole; HRMS, high resolution mass
spectrometry; IL-1b, interleukin-1b; Itc, isothiocitrulline ; LC-MS, liquid
chromatography coupled to mass spectrometry; L-NIL, L-N -(1-
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iminoethyl)-lysine;
LPS,
lipopolysaccharide;
MTT,
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMM, N-
methyl-morpholine; NMP, N-methyl pyrrolidone; NOHA, Nw-hydroxy-L-
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14
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10.1002/cmdc.201900659
ChemMedChem
FULL PAPER
Entry for the Table of Contents
Substrate access channel
Y
O
O
H
N
H
N
H
N
Y
Boc
Boc
Boc
H2
N
R
R
OMe
OMe
H2
N
N
HN
S
HN
S
HN
C
S
164 compounds
N
H
X
NH(2)
N
H
Heme
Substrate binding pocket
N
N
N
HN
S=,
O
N
X = Me-S-, Et-S-,
Me-NH-, Et-NH-
Y = CONH,
N
O
N
164 potential NO synthases inhibitors were prepared on solid support from a single thiocitrulline intermediate anchored by its side-
chain. Most compounds inhibiting nNOS and/or iNOS with IC50 in the 1-50 µM range and with selectivity toward eNOS contained a S-
Et-Itc moiety and mainly belonged to the dipeptide-like and 1,2,4-oxadiazole series. Selected inhibitors were found to significantly
inhibit iNOS expressed in RAW264.7 and INS-1 cells.
16
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