Substituted α-mercaptoketones, new types of specific neprilysin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.050

New neprilysin inhibitors containing an α-mercaptoketone HSC(R¹R²)CO group, as zinc ligand were designed. Two parameters were explored for potency optimization: the size of the inhibitor which could interact with the S₁, S

Full text of DOI:10.1016/j.bmcl.2017.06.050

Accepted Manuscript
Substituted α-mercaptoketones, new types of specific neprilysin inhibitors
Hervé Poras, Rémi Patouret, Simon Leiris, Tanja Ouimet, Marie-Claude
Fournié-Zaluski, Bernard P. Roques
PII:
S0960-894X(17)30647-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.050
BMCL 25084
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
27 April 2017
15 June 2017
17 June 2017
Please cite this article as: Poras, H., Patouret, R., Leiris, S., Ouimet, T., Fournié-Zaluski, M-C., Roques, B.P.,
Substituted α-mercaptoketones, new types of specific neprilysin inhibitors, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.050
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Mercaptoketone vf2_tracked
Substituted mercaptoketones, new types of specific neprilysin inhibitors
a
a
a
a
a
Hervé Poras Rémi Patouret , Simon Leiris , Tanja Ouimet , Marie-Claude Fournié-Zaluski ,
,
a, b, *
Bernard P. Roques
Pharmaleads, Paris BioPark, 11 rue Watt, 75013 Paris, France
Université Paris-Descartes, 4 Avenue de l’Observatoire, 75006 Paris, France
a
b
Keywords:Neprilysin inhibitor, Substituted mercaptoketone; Zinc bidentation
ABSTRACT
1
2
New neprilysin inhibitors containing an mercaptoketone HSC(R R )CO group, as zinc
ligand were designed. Two parameters were explored for potency optimization: the size of the
inhibitor which could interact with the S
1
, S
1
’ or S
2
’ domain of the enzyme and the nature of
1
2
1
the substituents R , R of the mercaptoketone group. Introduction of a cyclohexyl chain in R ,
2
3
R position and a (3-thiophen)benzyl group in position R ( compound 12n) yielded to the
most potent inhibitor of this series with a Ki value of 2 ± 0.3 nM. This result suggests that this
new inhibitor interacts within the S
catalytic Zn ion by the mercaptoketone moiety.
1
, S ’ domain of NEP allowing a pentacoordination of the
1
2
+
Abbreviations: BTFA, Boron trifluoroacetate, DIEA, Diisopropylethylamine; EtOH, Ethanol;
HPLC, High Performance Liquid Chromatography; LC/MS, Liquid Chromatography/Mass
Spectroscopy; LDA, Lithium diisopropyl amidure; NEP, Neprilysin; NMM, N-
Methylmorpholine; PMBSH, para-Methoxybenzenethiol; TFA, Trifluoroacetic acid.
*
Corresponding Author. E-mail address: bernard.roques@pharmaleads.com
1

Mercaptoketone vf2_tracked
Neprilysin also called neutral endopeptidase (NEP, EC 3.4.24.11), enkephalinase or
atriopeptidase, is a zinc dependent type II integral membrane protein belonging to the M13
family of metalloproteases. It is involved in the catabolism of biologically active peptides
1
,2
such as various vasoactive peptides and essentially in the inactivation of enkephalins (Met-
ENK: TyrGlyGly↓PheMet and Leu-ENK: TyrGlyGly↓PheLeu), the endogenous ligands of
1
the mu and delta opioid receptors . Neprilysin is an endopeptidase hydrolyzing peptide
substrates at the N terminus of aromatic or large hydrophobic amino acids which constitute
3
the P’
1
residue in the P
2
-P
1
-P’
1
-P’
2
commonly accepted Schechter and Berger nomenclature .
It also possesses dipeptidyl carboxypeptidase activity thus releasing the C terminal
hydrophobic dipeptides of small peptides such as enkephalins (Phe-Leu or Phe-Met). In all
these cases, specificity is the result of specific interactions within the S’
1
and S’ subsites of
2
the enzyme active site. The development of selective neprilysin inhibitors firstly required the
4
characterization of these enzyme subsite specificities using various peptide substrates and
secondly the characterization of the best ligand among the different chemical entities able to
chelate the catalytic zinc ion. Together these studies have led to the synthesis of very efficient
1
inhibitors with inhibitory constant (Ki) values in the nanomolar range .
5
The co-crystallization of neprilysin with phosphoramidon later allowed confirmation of
the main hypotheses proposed for the active-site characteristics and also highlighted the
stabilizing interactions (hydrogen bonds and hydrophobic interactions) between the inhibitor
6
and the enzyme active site . Two types of inhibitors have been co-crystallized with neprilysin:
7
a modified dipeptide such as thiorphan which was found to interact only with the S
1
’ and S ’
2
5
8
8
subsites of the enzyme and phosphoramidon , aminophosphinic and mercaptoacyl dipeptides
which recognized the S , S ’ and S ’ subsites (Figure 1).
In these two-last series of molecules, the enzyme-inhibitor complex was characterized by a
1
1
2
2
+
bidentation of the catalytic Zn through two oxygens of the phosphinic moiety or the SH and
8
CO groups of the mercapto acyl moiety .
These inhibitors possessed one or two amide groups which interacted respectively with the
5
43
542
carbonyl of NEP Ala and with the CO and NH
2
of the terminal amide group of NEP Asn
(
Figure 1). These stabilizing interactions increased the enzyme recognition by these molecules
but their polar groups could be unfavorable to their crossing the blood brain and/or
9
gastrointestinal barriers . Moreover, the presence of carboxamide bonds in these inhibitors
could render these molecules sensitive proteolytic degradation thus decreasing their half-life.
It could be also underlined that the amide bond planarity orients the spatial position of the
inhibitor side chains and their adjustment within the enzyme active site.
Taking into account all these parameters, we decided to test the replacement of the amide
1
0
bond by a ketone group COCH , “a classical replacement method” in the synthesis of
2
pseudopeptides. Starting from mercaptoacyl amino-acids or mercaptoacyl dipeptides NEP
8
blockers , we have developed a series of mercaptoketones as new high affinity inhibitors of
this enzyme.
The firstMercaptoketones have previously been described as matrix metalloproteinases
1
1,12
inhibitors
and some of them were very efficient with nanomolar inhibitory potency against
MMP1, MMP3 or MMP9. However, these molecules possessed an unsubstituted
2
+

mercaptoketone group HSCH CO, to interact with the Zn ion in the catalytic site.
2
2

Mercaptoketone vf2_tracked
For NEP inhibition, the modification of the mercaptoacyl- aminoacid or -dipeptide led to
1
2
the synthesis of compounds bearing substituted mercaptoketones HS-CHR R -CO where
2
+
the thiol and the carbonyl would be interacting with the catalytic Zn and R would fit within
1
2
a binding subsite of the enzyme. The sequence HS-CHR R -CO presents the disadvantage to
be chiral and consequently to induce the formation of two supplementary stereoisomers this
possible disadvantage could always be circumvented by stereoisomer separation.
The aim of this study was to characterize the impact of two parameters on inhibitory
potency: i) the importance of the amide bond replacement, ii) the optimization of the
hydrophobic interactions in each subsite.
Synthesis of pseudo-tripeptide inhibitors
Compounds 11 were obtained using a linear multi step scheme from an amino acid 1 of
defined absolute configuration (R) (Scheme 1).
The amino acid 1 was converted to brominated derivative 2 by a reaction of deamination-
1
3
halogenation, which was usually performed with retention of configuration .
The bromide derivative 2 was transformed into thioether 3 by nucleophilic substitution,
with inversion of configuration, by reaction with 4-Methoxy-benzyl thiol.
The ketene 4, obtained by addition of diazomethane to the mixed anhydride (prepared by
the reaction of 3 with isobutyl chloroformate in presence of N-methylmorpholine), was then
converted to halomethylketone 5 by reaction with bubbling gaseous HCl or HBr in 1,4-
dioxane, diethyl ether or ethyl acetate.
The halomethylketone 5, treated with NaI, underwent a halogen exchange and then reacted
5
with the dialkyl malonate sodium salt (R may be a methyl, ethyl or tert-butyl group) leading
to compound 6. The basic conditions used in this reaction resulted in a racemization of
compound 6.
3
3
The R substituent was introduced by reaction of the brominated derivative R Br with the
malonate 6, in situ deprotonated with NaH, to yield compound 7.
5
After deprotection by action of TFA (when R was a tert-butyl group) or hydrolysis of
5
diester 7 (when R was a methyl or ethyl group), a decarboxylation in refluxing toluene
occurred leading to compound 8.
A usual amino-acid coupling reaction could be performed on compound 8, as described in
the literature, with the aminoester 9 leading to compound 10 (Scheme 1).
The ester 10 was then hydrolyzed and the thioether protection was removed by the action
1
4
of BTFA (prepared from BBr in trifluoroacetic acid) , to yield the expected compound 11.
3
3

Mercaptoketone vf2_tracked
Scheme 1. Synthesis of pseudo-tripeptide inhibitor 11 starting from compound 1.
The biaryl group was obtained by Suzuki-Miyaura reaction on the 4-bromobenzyl 8 analog
15
with boronic acid in presence of Pd catalyst as described in litterature (Scheme 2). The
following steps were as described in Scheme 1.
Scheme 2. Suzuki-Miyaura reaction
Synthesis of pseudo-dipeptide inhibitors
Starting from compound 8, the thioether protection was removed by the action of BTFA
1
4
(
prepared from BBr in trifluoroacetic acid) , to yield the expected compound 12 (Scheme 3).
3
Scheme 3. Synthesis of pseudo-dipeptide inhibitor 12 starting from compound 8.
4

Mercaptoketone vf2_tracked
Synthesis of geminal inhibitors
Alternatively, in case of geminal compounds, the chloromethylketones 16 were prepared
from a reaction between bromo-acetic acid and 4-methoxy benzyl mercaptan followed by the
double alkylation leading to 15. The synthesis of the chloromethylketones 16 was obtained by
1
6
action of ICH Cl in presence of LDA as described in litterature (Scheme 4).
2
Scheme 4. Synthesis of geminal chloro mercaptoketones 16
The following steps leading to geminal inhibitors were those described in the previous
Scheme 1 and 3.
Synthesis of cyclic core inhibitors
The bromomethylketone 19 was synthesized from the cyclic methyl ester 17 by alkylation
1
7
with PMB-S-S-DNP , leading to compound 18. Saponification followed by reaction with
1
8
19
oxalyl chloride gave the acid chloride which could react with trimethylsilyldiazonium and
HBr in acetic acid to give the bromomethylketone 19 (Scheme 5).
Scheme 5. Synthesis of geminal bromo -mercaptoketones 19
The following steps leading to geminal inhibitors were those described in the previous
Scheme 1 and 3.
Inhibitory potency
Ki values of the different compounds towards neprilysin (NEP) were determined as
2
0
described by Poras et al (Tables 1-3). All these compounds were tested on NEP-2, ACE,
ECE-1 and ECE-2 and were shown to be at least 20-fold more selective on NEP.
5

Mercaptoketone vf2_tracked
Biochemical and structural results
7
Compound I (Figure 1) has been crystallized in NEP and was shown to fully occupy the
enzyme active site. The three inhibitor side chains interacted respectively with the S , S ’ and
’ binding subsites and the Zn was bidentated by the mercapto acyl group leading to a
1
1
2
+
S
2
2
+
pentacoordinated Zn in this complex. The two amide NH groups are hydrogen bonded with
the CO of Ala and Asn respectively and the C-terminal carboxylate interacted with the
side chains of Arg , Asp and Arg at the surface of the enzyme. A new series of
inhibitors based on this structure is reported in Table 1. These compounds (11a-k) contain
three asymmetric carbons and only the C terminal aminoacid is optically pure. Consequently,
they were synthesized as mixtures of four stereoisomers, which were initially not separated.
5
43
542
1
02
107
110
7
Figure 1: Positioning of compound I in Neprilysin
The replacement of the mercapto amide in Compound I by an mercaptoketone in
compound 11a (Ki 95 ± 8 nM) significantly increased the Ki value by 40-fold. This reflects
5
43
both the loss of the hydrogen bond involving Ala (Figure 1), but also the presence of four
diastereoisomers in 11a whose Ki values could be extremely different.
1
3
4
To improve NEP recognition, the side chains R , R and R were successively modified
with the aim of optimizing their interaction with their respective subsites (Table 1). When the
3
R benzyl group was replaced by a bulkier aromatic side chain, 4-bromo benzyl in 11b or 4-
phenyl benzyl in 11c, a notable increase in their potencies (78 ± 8 and 46 ± 4 nM
respectively) was observed. This result is in accordance with the known preference of the
NEP S
NEP inhibitors .
1
’ subsite for large aromatic residues as illustrated in the aminophosphinic series of
2
1
4
The introduction of a tryptophan residue in R position (compound 11d (Ki 18 ± 1 nM) and
1e (Ki 30 ± 4 nM) was also favorable (Table 1). This is in complete accordance with the
1
6

Mercaptoketone vf2_tracked
5
41
ability of the Trp indole NH to create a hydrogen bond with the carbonyl of Val in the S
2
’
5
subsite as shown in the crystallized NEP-phosphoramidon complex .
1
The R side chain interacts with the S
1
subsite of NEP. As shown in the crystallographic
5
,8
1
structures , the R chain is at the surface of the enzyme and is not in direct contact with the
protein, reflecting the minor stabilizing role of the S subsite in NEP. Consequently, as
1
1
-5
expected, a large increase in the size of hydrophobic R (compound 11f, Ki>10 M) was
unfavorable, while decreasing side chain size ie aromatic (11g, Ki 22 ± 4 nM) or aliphatic
(
11h, Ki 12 ± 2 nM and 11i, Ki 7 ± 0.5 nM) was well accepted. However, too small a side
chain, like a CH (compounds 11j and 11k), did not improve S subsite recognition.
3
1
Taken together, these different results allowed structural parameters for NEP active site
occupancy by these -mercapto-ketones to be proposed: an aliphatic moiety such as an
isobutyl chain in position S
aliphatic residue in S’
1
, a large aromatic residue as biphenyl methyl in S’ and a small
1
2
.
In a second series of inhibitors, the C-terminal amino-acid has been deleted leading to a
5
smaller molecule (compound II , Table 2) with a Ki of 23nM, 10-fold higher than that of
compound I (Figure 1). Based on compound II structure, a series of molecules in which the
CO-NH group was replaced by a CO-CH
asymmetric carbons and were studied as a mixture of 4 diastereoisomers.
Compound II has only one amide bond and its reduction into COCH
without peptide bond. This opens the question of the positioning of the analogues 12a-g in
NEP active site: i) interactions inside S ' and S ' subsites such as observed for thiorphan and
its analogues or ii) interactions inside S and S ' subsites, favored by bidentation of zinc, as
previously described with compounds 11a-k.
2
is reported in Table 2. These molecules contain two
2
leads to a derivative
1
2
2
2
1
1
Compound 12a (Ki 140 ± 10 nM) showed a 7-fold decrease in inhibitory potency as
5
compared to II (Ki 23 nM) , suggesting a relatively weak participation of the amide NH
group in the stabilization of the inhibitor-enzyme complex. Interestingly, an increase in size of
3
the aromatic R residue (12b, Ki 50 ± 8 nM) significantly favored NEP inhibition, while the
1
1
3
same modification for R (12c, Ki 1750 ± 80 nM) was detrimental. When both R and R were
bulky aromatic groups (12d and 12e), intermediate inhibitory potencies were observed (Ki
1
2
60 ± 40 and 252 ± 50 nM respectively). The replacement of the R aromatic side chain in
1
2a by an aliphatic one in 12f led to an efficient, high affinity inhibitor with a Ki of 20 ± 5
nM, but the amidification of the C terminal carboxylic group by a morpholine in 12g (Ki 1650
±
40 nM) was not well accepted. All these results are in favor of an interaction of these
2
+
inhibitors within the S
1
, S ’ domain of the enzyme, enlightening the importance of Zn
1
pentacoordination for the complex formation.
1
To simplify the study of these mercaptoketones, the R chain was modified in order to
suppress one asymmetric center by introducing a symmetrical aliphatic geminal derivative
(
compounds 12h and 12i) (Table 3) or a cyclic residue based on symmetrical cyclopentyl or
cyclohexyl structure (compounds 12j-n) (Table 3). Such poorly flexible cycles have
2
0,23
previously been introduced in NEP inhibitors in position S
this subsite. Moreover, it was interesting to test the ability of these cycles to fit within the
largely solvent exposed S subsite.
As shown in Table 3, the introduction of small aliphatic residues gem-dimethyl in 12h (Ki
1
’
and were well recognized by
1
-7
4
70 ± 40 nM) and gem-diethyl in 12i (Ki 86 ± 9 nM) yielded inhibitory potencies in the 10
7

Mercaptoketone vf2_tracked
M range. Conversely, the cyclic structures were more efficient. A comparison between 12j-m
revealed that the cyclohexyl moiety (12l and 12m) in the S
inhibition than with the cyclopentyl one (12j and 12k) and in all these cases, the biphenyl
methyl S’ residue (12k and 12m) is preferred to a bromobenzyl (12j and 12l).
Due to this preference for large aromatic residues in S’ , we tested a para-(3-
thiophen)benzyl group in this position (12n). The inhibitory potency of this compound (Ki 4.6
1
position leads to a better
1
1
±
0.5 nM) was the best obtained for a racemic in this series of NEP inhibitors.
Compound 12n was therefore resolved and the absolute configuration of the (3-
thiophen)benzyl group determined by 1H-NMR spectroscopy after coupling with a (S)-Ala
2
4
residue as described in the litterature . Interestingly, the (R) stereoisomer (Ki 2 ± 0.3 nM)
was found to be 30-fold more potent than the (S) stereoisomer (Ki 61 ± 2 nM).
In order to try and optimize the enzyme-inhibitor interactions within the S
1
subsite,
modifications were introduced in position 4 of the cyclohexyl moiety of 12l or 12m.
1
2
Inhibitory potencies obtained with these latter compounds, with an increased size of the R -R
cycle residue (12p to 12t as compared to 12l or 12o, 12u as compared to 12m), showed that
the cyclohexyl group is significantly preferred even if the inhibitory potencies of 12o to 12u
-8
remained relatively low with Ki values in the 10 molar range (Table 3).
The 3D structure of NEP, shows that the essential parameters for a strong interaction of a
2
+
small inhibitory molecule inside the active site are the Zn ion and the S’
All the other possible interactions within the S and S’ subsites, hydrogen bonds and ionic
1
subsite.
1
2
interactions are energetically less efficient but are essential for the correct positioning of
peptide substrates or inhibitors inside the enzyme.
Potent inhibitors of NEP have systematically been obtained with compounds interacting
2
+
with the Zn ion and the hydrophobic S ’ subsite, associated with either occupancy of the
1
7
,22
6,21,25
S’
2
or the S
Thus, with compounds interacting only with the S
recognized by a benzyl group as well as by a biphenyl group, due, for the latter, to the
1
subsite
.
1
’- S
2
’ domains, the S ’ subsite is well
1
5
79
693
8
displacement of Met and Trp , increasing the size of S’
recognizing also the S domain, a biphenyl moiety is preferred to a benzyl one
According to these results, we demonstrated in this paper that it is possible to obtain high
affinity NEP inhibitors with compounds recognizing only the S and S’ domains provided
1
cavity . With compounds
8
,21
1
.
1
1
2
+
that Zn bidentation was insured by suitable ligands such as -mercapto ketones groups.
The pharmacological properties of these novel inhibitory molecules will be explored
further using animal models.
8

Mercaptoketone vf2_tracked
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1
0

Mercaptoketone vf2_tracked
Table 1
1
2
3
4
Compound
R
R
R
R
NEP Ki (nM)
95 ± 8
1
1
1
1
1
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
CH
CH
CH
CH
CH
CH
Ph
2
2
2
2
2
2
Ph
H
H
H
H
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
2
2
2
2
Ph
(S)-Ala
(S)-Ala
(S)-Ala
(S)-Trp
(S)-Trp
(S)-Leu
(S)-Ala
(S)-Ala
(S)-Ala
(S)-Trp
(S)-Ala
Ph
(4-Br-Ph)
(4-Ph-Ph)
Ph
78 ± 8
Ph
46 ± 4
Ph
18 ± 1
Ph
(4-Br-Ph)
Ph
30 ± 4
-
5
(4-Br-Ph)
> 10
1g
1h
1i
(4-Br-Ph)
(4-Ph-Ph)
(4-Br-Ph)
(4-Br-Ph)
(4-Ph-Ph)
22 ± 4
12 ± 2
7 ± 0.5
162 ± 8
40 ± 5
iBu
iBu
CH
CH
1j
3
3
1k
Table 2
1
2
3
Compound
2a
2b
R
R
R
R’
NEP Ki (nM)
140 ± 10
50 ± 8
1
CH
CH
CH
CH
CH
iBu
iBu
2
2
2
2
2
Ph
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
2
Ph
OH
1
Ph
2
2
2
2
2
2
(4-Br-Ph)
Ph
OH
1
1
1
1
1
2c
2d
2e
2f
(4-Br-Ph)
(4-Br-Ph)
(4-Ph-Ph)
OH
1750 ± 80
260 ± 40
252 ± 50
20 ± 5
(4-Br-Ph)
(4-Br-Ph)
(4-Br-Ph)
(4-Br-Ph)
OH
OH
OH
2g
Morpholine
1650 ± 40
1
1

Mercaptoketone vf2_tracked
Table 3
1
2
3
Compound Stereochemistry
R
,
R
R
NEP Ki (nM)
470 ± 40
86 ± 9
1
1
1
2h
2i
Racemic
Racemic
Racemic
CH
3
CH
CH
CH
2
2
2
(4-Br-Ph)
(4-Br-Ph)
(4-Br-Ph)
2 5
C H
2j
52 ± 4
1
1
1
1
1
1
1
1
1
2k
2l
Racemic
Racemic
Racemic
Stereo 1
Stereo 2
Racemic
Stereo 1
Stereo 2
Racemic
CH
CH
CH
2
2
2
(4-Ph-Ph)
(4-Br-Ph)
(4-Ph-Ph)
29 ± 5
32 ± 7
9 ± 1
2m
2m
2m
2n
2n
2n
2o
(R)-CH
2
(4-Ph-Ph)
36 ± 1
4.2 ± 0.1
4.6 ± 0.5
61 ± 6
2 ± 0.3
61 ± 2
2
(S)-CH (4-Ph-Ph)
2
CH (3-Thiophen-Ph)
2
(R)-CH (3-Thiophen-Ph)
2
(S)-CH (3-Thiophen-Ph)
CH
CH
CH
2
2
2
(4-Ph-Ph)
1
1
2p
2q
Racemic
Racemic
(4-Br-Ph)
(4-Br-Ph)
54 ± 1
54 ± 14
1
1
2r
2s
Racemic
Racemic
CH
CH
2
2
(4-Br-Ph)
(4-Br-Ph)
49 ± 2
95 ± 13
1
1
2t
Racemic
Racemic
CH
CH
2
2
(4-Br-Ph)
(4-Ph-Ph)
36 ± 3
26 ± 1
2u
1
2

Mercaptoketone vf2_tracked
Graphical abstract
1
3