New non-hydroxamic ADAMTS-5 inhibitors based on the 1,2,4-triazole-3-thiol scaffold

Article abstract of DOI:10.1016/j.bmcl.2010.08.108

In this Letter we describe the design, synthesis, screening, and optimization of a new family of ADAMTS-5 inhibitors. These inhibitors display an original 1,2,4-triazole-3-thiol scaffold as a putative zinc binding-group. In vitro results are rationalized

Full text of DOI:10.1016/j.bmcl.2010.08.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6213–6216
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New non-hydroxamic ADAMTS-5 inhibitors based on the
1,2,4-triazole-3-thiol scaffold
Lucie Maingot a,b,c,d, Florence Leroux a,b,c,d,e, Valérie Landry a,b,c,d, Julie Dumont a,b,c,d, Hideaki Nagase g,
a,b,c,d,e,
Bruno Villoutreix e,f, Olivier Sperandio e,f, Rebecca Deprez-Poulain a,b,c, , Benoit Deprez
⇑
⇑
a INSERM U761 Biostructures and Drug Discovery, Univ. Lille Nord de France,à 3 rue du Pr Laguesse, Lille F-59006, France
b Faculté de Pharmacie, Univ. Lille Nord de France, 3 rue du Pr Laguesse, Lille F-59006, France
c Institut Pasteur de Lille, IFR 142, Lille F-59021, France
d PRIM,§ Lille F-59006, France
e CDithem Platform/IGM, Paris, France
f INSERM UMR-S 973/MTi, University Paris Diderot, Paris, France
g Kennedy Institute of Rheumatology, Imperial College, London SW7 2AZ, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2010
Revised 19 August 2010
Accepted 21 August 2010
Available online 27 August 2010
In this Letter we describe the design, synthesis, screening, and optimization of a new family of ADAMTS-5
inhibitors. These inhibitors display an original 1,2,4-triazole-3-thiol scaffold as a putative zinc binding-
group. In vitro results are rationalized by in silico docking of the compounds in ADAMTS-5’s crystal
structure.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
ADAMTS-5
1,2,4-Triazol-3-thiol
Triazol-5-thiol
Metalloprotease
Osteoarthritis (OA) is a progressive disease of the joints charac-
terized by the degradation of the articular cartilage. The glycopro-
tein aggrecan is the major component of the cartilage extracellular
matrix and its extensive degradation leads to further breakdown of
other extracellular matrix macromolecules.1 Aggrecanase-2, also
called ADAMTS-5 (A Disintegrin and Metalloproteinase with
Thrombospondin motifs 5) is a metalloprotease that degrades
components of the extracellular matrix, and in particular aggre-
can.2,3 In that context, inhibitors of this enzyme could result in
treatments for osteoarthritis.
When targeting zinc metalloproteases, a zinc binding-group
(ZBG) is helpful with respect to facilitating inhibitor orientation,
and hence binding, in the enzyme’s active site. First described
inhibitors were hydroxamates that resulted from research on the
related MMPs (Matrix MetalloProteinases).4
oxothiazolidinones.8 Our team has recently published
examples of squaric acid N-hydroxylamide amides inhibitors.9
a few
To continue our work on original ZBGs, we described earlier the
synthesis of 1,2,4-triazole-3-thiols (Fig. 1).10,11 We hypothesized
that this heterocycle can bind zinc thanks to its exocyclic sulfur
atom as already shown for a series of TACE (ADAM17) inhibitors.12
We have designed and synthesized a focused library of 500
1,2,4-triazole-3-thiols. The compounds were prepared from the
amine and di-2-pyridylthionocarbonate followed by reaction with
the required hydrazide and subsequent cyclization into 1,2,4-tria-
zole-3-thiols under basic conditions (Scheme 1).
Amines and most hydrazides were commercially available.13
Some derivatives of 1,2,4-oxadiazol-5-yl-acetic acid hydrazide
were synthesized to complete the hydrazide set (Scheme 2). Ami-
doximes 1a–c were prepared as previously described by reaction of
hydroxylamine with the corresponding nitrile.14 Then the 1,2,4-
oxadiazole ring was obtained by reaction with acetyl chloride.
The resulting ethyl esters were converted to hydrazides by direct
reaction with hydrazine.
Recent research on ADAMTS-5 describes other ZBGs such as car-
boxylic acids,5 hydroxyquinolines,6 spirothiazolones,7 or thi-
⇑
Corresponding authors. Tel.: +33 (0)320 964 947; fax: +33 (0)320 964 709.
E-mail addresses: rebecca.deprez@univ-lille2.fr (R. Deprez-Poulain), benoit.de-
prez@univ-lille2.fr (B. Deprez).
The screening of the library was performed using previously
published conditions at 30 l
M.15 The hit rate was 2.5% for a 80%
Home pages: U761, http://www.u761.lille.inserm.fr.
PRES Université Lille Nord de France, http://www.univ-lille-nord-de-france.fr.
PRIM, http://www.drugdiscoverylille.org.
inhibition threshold. The hits and close inactive analogs
(compounds 4–19) were resynthesized at a larger scale, fully
à
§
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.108

6214
L. Maingot et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6213–6216
R'
N
K+
S
R
N
N
Figure 1. 1,2,4-Triazole-3-thiol scaffold.
R'
N
O
a.
Table 2
NH2
K+
NH2
S
R
R'
+
Inhibition on ADAMTS-5 for compounds 13–29
R
N
H
N
N
R'
N
Scheme 1. Reagents and conditions: (a) (i) dipyridylthionocarbonate 0.25 M in
DMF (1.05 equiv), amine (free base) 0.1 M in DMF (1 equiv), 55 °C, 1.5 h; (ii)
hydrazide (free base), 0.1 M in DMF (1 equiv) 55 °C, 1.5 h, solvent evaporation, then
(iii) 1 equiv KOH 0.1 M in H2O/EtOH (40/60) 85 °C, 5 h.
S-K+
( )n
Ar
N
N
Compd
n
Ar–
R0
ADAMTS-5
a
IC50 (lM)
H
N
X
a.
b.
X
N
OH
13
1
–(3-Phenyl)propyl
38
CN
O
N
N
X
X
NH
N
O
O
n
1a-c
14
15
16
17
18
1
1
1
1
1
–(3-Phenyl)propyl
–(3-Phenyl)propyl
–CH2-o-biphenyl
47
22
O
Cl
O
N
N
N
N
O
n
X
O
O
X
O
c.
N
N
NH2
N
N
N
22
X
OEt
X
n
H
O
3a : n=1; X=CH
3b: n=2; X=CH
3c: n=2; X=N
2a-c
–CH2-o-biphenyl
>100
6
N
Scheme 2. Reagents and conditions: (a) 1 equiv NH2OH/HCl, DIEA, EtOH, 3 h,
reflux. (b) DIEA, dioxane, reflux, 4 h then TBAF, 75%. (c) N2H4, ethanol, 18 h, 95%.
–3-(N-Imidazolyl)propyl
O
O
N
N
19
2
–3-(N-Imidazolyl)propyl
>100
characterized and their IC50 on the target measured. All the results
are shown in Tables 1 and 2.
N
a
Values are means of two experiments minimum, standard deviations are 10%.
In chlorophenoxylmethyl series (Table 1), para- and ortho-
biphenyl ring are slightly better than meta-derivative (4–6). The
biphenyl system is preferred to phenylalkyl group (4 vs 7–9). Inter-
plays an activity of 22 lM similar to 13 but isosteric replacement
estingly, 3-(N-imidazolyl)propyl displays an IC50 of 11 lM whereas
of 1,2,4-oxadiazole by a phenyl in 17 leads to complete loss of
activity. This could be attributed to the high lipophilicity of the
compound precluding its solubility in the assay. Interestingly, like
in the chlorophenoxymethyl series, 3-(N-imidazolyl)propyl 18 is
the most active compound (6 lM). Introduction of a methylene
moiety completely abolishes activity (19) evidencing an optimal
its (3-phenyl)propyl analog is inactive (9 vs 10). Interestingly, isos-
teric replacement of the chlorine atom by a fluorine (compound
11) decreases activity, consistently with the less hydrophobic
properties of fluorine. Cyclization in dihydrobenzoxazine (com-
pound 12) resulted in a loss of activity. This could be attributed
to both the steric constraint and the introduction of a NH group.
In the biaryl series (Table 2), the 3-phenylpropyl derivatives are
equipotent (13–15). Introduction of an o-biphenyl system, like in 6,
gives similar results (compound 15). o-Biphenyl derivative 16 dis-
spacer between the 1,2,4-oxadiazole ring and the ZBG.
Also, to evaluate our primary hypothesis on binding to the tar-
get by the exocyclic sulfur atom, we synthesized a few S-methyl-
ated analogs (Scheme 3). Table 3 gathers inhibition results for
the S-methylated compounds 20–22. As expected, these com-
pounds do not inhibit ADAMTS-5 due to the absence of ZBG.
The catalytic domain of ADAMTS-5 is known to be flexible in
several regions of the binding site.16 X-ray structures co-crystal-
lized with different ligands (e.g., pdb codes: 3B8Z, 2RJQ, and
3HYG) show that the His373-loop is capable to adapt to the ligand
by closing onto the binding site as for the highly hydrophobic S10
pocket through the Ser441-Ile442-Leu443-loop and the His403-loop.
Hydrophobic moieties within the ADAMTS-5 ligands are known
to plunge into the S10 pocket while nucleophilic groups such as
Table 1
Inhibition on ADAMTS-5 for compounds 4–12
R'
N
H
R'
N
N
X
S-K+
X
S-K+
O
O
N
N
N
N
4-11
12
a
R0
ADAMTS-5 IC50 (lM)
the often observed hydroxamate function complement the Zn2+
-
Compd
X
4
5
6
7
8
9
10
11
12
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
Cl–
F–
–CH2-p-biphenyl
–CH2-m-biphenyl
–CH2-o-biphenyl
–Benzyl
13
27
13
>100
>100
>100
11
>100
>100
chelating residues His410, His414, and His420
.
R'
R'
N
–Phenethyl
K+
a.
N
S
S
R
R
–(3-Phenyl)propyl
–3-(N-Imidazolyl)propyl
–CH2-p-biphenyl
–CH2-p-biphenyl
N
N
N
N
4,6,19
20-22
Cl–
a
Values are means of two experiments minimum, standard deviations are 10%.
Scheme 3. Reactions and conditions: (a) MeI (5 equiv), MeOH 18 h (quant.).

L. Maingot et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6213–6216
6215
Table 3
where it binds ADAMTS-5 through hydrophobic contact to
Leu379, Ile442, and Leu443. This may explain why compound 11
bearing a fluorine is less active than the more hydrophobic Chlo-
rine analogs.
Table 4 shows selectivity results on ADAMTS-4, measured log D
between 1-octanol and PBS buffer (pH 7.4) and solubility in PBS for
some selected compounds.
Both structurally close ADAMTS-4 and five cleave aggrecan.
ADAMTS-5 has been shown to be the major aggrecanase in a model
of arthritis.2 Interestingly, differences in four residues in the cata-
lytic site result in a roomier S10 pocket of ADAMTS-4. It may thus
be difficult to reach selectivity on ADAMTS-5 explaining why only
a few examples of selective compounds have been described in the
literature.7 In our series, some selectivity was obtained. Com-
pounds 10 and 18 have the best selectivity ratio for ADAMTS-5
and 14 displays on the contrary a lower IC50 on ADAMTS-4.
Compounds 10, 14, and 18 display reasonable Log D in compar-
ison with 6, that is, highly hydrophobic and poorly soluble. Com-
pound 18 is the most promising compound both in terms of
activity and physico-chemical properties.
Inhibition on ADAMTS-5 for compounds 20–22
R'
N
R
X
N
N
Compd
R
R0
X
ADAMTS-5
a
IC50
(l
M)
O
O
20
4
–SMe
–SꢀK+
>100
13
Cl
Cl
21
6
–SMe
–SꢀK+
>100
13
N
N
N
22
18
–SMe
–SꢀK+
>100
6
O
N
a
Values are means of two experiments minimum, standard deviations are 10%.
In conclusion, we have developed a series of ADAMTS-5 inhibi-
tors containing a 1,2,4-triazole-3-thiol metal binding-group. These
molecules display IC50s of 6–47 lM in a similar range to other non-
hydroxamic acid inhibitors reported in the literature. Based on the
potency, selectivity, and physico-chemical properties, we consider
the 3-(N-imidazolyl)propyl derivative 18 as a suitable starting
point for further optimization of this chemical series.
Acknowledgments
The authors thank G. Deglane, E. Carasso, and S. Delaroche for
technical support. We are grateful to the institutions that support
our laboratory (INSERM, Université Lille Nord de France, and Insti-
tut Pasteur de Lille). Data management was performed using Pipe-
line Pilot™ from Accelrys Inc. This project was supported by the
Fondation pour la Recherche Medicale; Nord-Pas-de-Calais (RA-
D07001EEA) and PRIM (Pôle de Recherche Interdisciplinaire pour
le Médicament). We thank the LARMNS NMR laboratory, Faculty
of Pharmacy Lille.
Figure 2. Binding mode of 1,2,4-triazole-3-thiol. ADAMTS-5 structure as in pdb
code 3HYG with docked compound 18.
Based on these observations, compounds 6, 10, and 18 were
docked into ADAMTS-5 (pdb code: 3HYG) in an attempt to ratio-
nalize their binding mode. The combination of several docking pro-
grams,17 has allowed the identification of a consensual binding
mode for the three compounds (Fig. 2 and Supplementary data).
Thus, the binding modes show that the thiol function complements
the chelating of the zinc ion by His410, His414, and His420. This con-
firms the poor activity of the S-methylated analogs. The 1,2,4-tria-
zole ring forms a Hydrogen bond with the backbone nitrogen of
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.108.
References and notes
Leu379 18 The S10 pocket is occupied by either the –3-(N-imidazo-
.
1. Mankin, H. J.; Lippiello, L. J. J. Bone Joint Surg. 1970, 52-A, 424.
lyl)propyl moiety (compounds 10 and 18) or the –CH2-o-biphenyl
moiety (compound 6). For the former, a Hydrogen bond is formed
with the backbone Nitrogen of Thr444. This may explain the better
inhibition of 10 versus 9, that is, devoided of any H-bond acceptor
in that position. Finally, the R moiety either a 1,2,4-oxadiazole-
based biaryl (compound 18) or a chlorophenoxymethyl group
(compounds 6 and 10) always occupied the tip of the S10 pocket
2. Stanton, H.; Rogerson, F. M.; East, C. J.; Golub, S. B.; Lawlor, K. E.; Meeker, C. T.;
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Table 4
Compd
IC50
(lM) TS5
IC50
(
lM) TS4
Log Da (pH 7.4)
Solubilitya
g/mL)
(
l
6
13
11
>30
6
12
>30
8
3.6
1.6
1.7
1.1
0.4
10
14
18
14
49
64
>30
a
Solubility and Log D are measured from a DMSO stock solution.

6216
L. Maingot et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6213–6216
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13. See Supplementary data. To ensure complete cyclization, primary amines were
selected among R-CH2-NH2 (with R = Alk or Ar). The hydrazides were selected
to maximize their diversity using PipelinePilot™ from Accelrys™ Inc.
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15. See Supplementary data. Briefly, an immobilized 41-residue peptide on
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streptavidin-coated microplates was used as
a substrate for ADAMTS-5.
Aggrecanase-mediated hydrolysis at the Glu373-Ala374 bond resulted in an
immobilized product that reveals a N-terminal epitope (NH3+-Ala-Arg-Gly-Ser-
Val), recognized by the specific antibody BC-3.
16. (a) Mosyak, L.; Georgiadis, K.; Shane, T.; Svenson, K.; Hebert, T.; McDonagh, T.;
Mackie, S.; Olland, S.; Lin, L.; Zhong, X.; Kriz, R.; Reifenberg, E. L.; Collins-Racie,
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11. Protocol for the synthesis at 15-
63 L of a solution of 0.25 M di-pyridylthionocarbonate in DMF (1.05 equiv)
was added 150 L of 0.1 M amine (free base) in DMF (1 equiv). The reaction
mixture was heated at 55 °C for 1.5 h. Then 150 L of a solution of 0.1 M
lmol scale using DPT in 1.5 mL PP-tubes: to
l
l
l
hydrazide (free base) in DMF (1 equiv) was added. The reaction mixture was
heated at 55 °C for 1.5 h to allow the synthesis of the thiosemicarbazide.
17. See Supplementary data.
Solvent was removed under reduced pressure; 300
lL of 0.1 M KOH in H2O/
18. Similarly the Oxygen of the amide function of the side chain of the
hydroxamate-based inhibitor co-crystallized in structure pdb code 3HYG,
makes a Hydrogen bond with the backbone of Leu379. See Supplementary data.
EtOH (40/60) was added. The reaction mixture was heated at 85 °C for 5 h until
complete cyclization. The solvents were then removed under reduced pressure.
12. Gilmore, J. L.; King, B. W.; Asakawa, N.; Harrisson, K.; Tebben, A.; Sheppeck, J.
E.; Liu, R.-Q.; Covington, M.; Duan, J. J.-W. Bioorg. Med. Chem. Lett. 2007, 17,
4678.