Synthesis of a 200-member library of squaric acid N-hydroxylamide amides

Article abstract of DOI:10.1016/j.bmcl.2008.08.025

We report here the parallel synthesis of 200 compounds based on squaric acid template. These compounds are obtained via a one-step solution-phase procedure starting from three squaric acid N-hydroxylamide esters precursors. The set of diverse reagents qualified (amines, anilines, amino-alcohols and amino-esters) makes this strategy suitable for the search of biologically active compounds. The library was screened on the zinc metalloenzyme ADAMTS-5 and hits with IC₅₀ in the range of 1-50 μM were identified.

Full text of DOI:10.1016/j.bmcl.2008.08.025

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4968–4971
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of a 200-member library of squaric acid N-hydroxylamide amides
*
Julie Charton , Benoît P. Déprez, Rébecca F. Déprez-Poulain
INSERM U761, Biostructures and Drug Discovery, 3 rue du Professeur Laguesse, Nord, Lille F-59006, France
Faculty of Pharmacy, Lille2 University, Lille F-59006, France
Institut Pasteur de Lille, IFR142, Lille F-59000, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here the parallel synthesis of 200 compounds based on squaric acid template. These com-
pounds are obtained via a one-step solution-phase procedure starting from three squaric acid N-hydrox-
ylamide esters precursors. The set of diverse reagents qualified (amines, anilines, amino-alcohols and
amino-esters) makes this strategy suitable for the search of biologically active compounds. The library
Received 19 June 2008
Revised 7 August 2008
Accepted 10 August 2008
Available online 14 August 2008
was screened on the zinc metalloenzyme ADAMTS-5 and hits with IC50 in the range of 1–50
identified.
lM were
Keywords:
Squaric acid
Ó 2008 Elsevier Ltd. All rights reserved.
Squaric acid N-hydroxylamide amide
Zinc-binding group
Metalloprotease
ADAMTS-5
The squaric template has been introduced in chemistry by the
pioneering work of Cohen in 1959.1 Since then, several examples
of the use of squaric template have been reported in the fields of bio-
organicandmedicinalchemistry.2 Theconjugate base ofsquaricacid
can serve as a mimic of negatively charged groups that are common
in biology, including carboxylates and phosphate mono- and di-es-
ters and of growing importance in medicinal chemistry such as
hydroxamic acids. As a result, derivatives of squaric acid have been
used as a replacement for these groups in medicinal applications.
Squaric acid itself is an inhibitor of glyoxylaseI, and semisquaric acid
(3-hydroxy-3-cyclobutenedione) is an inhibitor of pyruvate dehy-
drogenase and transketolase.3,4 Recently, Sekine and co-workers
have used a diamide of squaric acid as a replacement for one of the
phosphate diester linkages in an oligodeoxynucleotide.5
Our group focuses on the synthesis of acidic functions and zinc
chelating groups like hydroxamates.6,7 In that context, we investi-
gated the synthesis of squaric acid N-hydroxylamide amides as
acidic groups. We describe here the preparation of a library of such
derivatives, based on a convergent solution-phase synthesis.
Many metalloenzyme inhibitors contain a hydroxamic acid to
chelate the zinc ion present in the catalytic site of hydrolases.
However with the exception of vorinostat (SAHA, suberoylanilide
hydroxamic acid), most of them did not live up to expectations
in the development phases. There has been considerable interest
in discovering alternative groups to the hydroxamic acid.8 Bruck-
ner and co-workers have observed that vinylogous hydroxamic
acids that are derived from squaric acids (the squaric acid N-
hydroxylamide amide) bind iron (III) in aqueous system, without
demonstrating the binding mode (Fig. 1).9
Inhibitors of matrix metalloproteases and analogues of SAHA
bearing squaric acid- based moiety have been synthesized but they
revealed to be poorly active.10,11 In all, only few squaric acid N-
hydroxylamines amides, mainly peptidic, have been prepared
and described until now.12
Target compounds can be obtained from squaric acid N-substi-
tuted hydroxylamides esters. These non symmetrical building
blocks are synthesized using a squaric acid diester precursor. The
diester used is usually symmetrical. Indeed, the use of disymmet-
rical ester is not required because squarate bis-N-hydroxylamides
could not be prepared by nucleophilic displacement of diester as
reported by Bruckner9 and Grünefeld.12 For this study, dibutyl
squarate was used as the starting point because of its good solubil-
ity in most classical organic solvents and its lower cost compared
with other diesters.
Reaction of the dibutyl squarate with a series of N-substituted
hydroxylamines was conducted as described in Scheme 1. Vinylo-
O
N
O
N
M
M
O
O
R
O
R"
N
R'
R
R'
* Corresponding author. Tel.: +33 (0)3 20 96 49 28; fax: +33 (0)3 20 96 47 09.
Figure 1. Known binding mode of hydroxamic acid and putative binding mode of
E-mail address: julie.charton@univ-lille2.fr (J. Charton).
squaric acid (six membered zinc chelation model) to metal ion.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.025

J. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4968–4971
4969
Table 1
O
O
O
O
O
O
O
N
O
O
Puritya of target compound in reaction mixture
a.
b.
OH
R'
N
R"
N
OH
Class
Representative amine
Squaric precursor
R
R
1(x)
1(1)
1(2)
1(3)
3(x,y)
1(1) : R = Me 70%
1(2) : R = Bz 67%
1(3) : R = iPr 90%
O
A
B
C
D
E
98
96
94
93
98
93
97
97
93
94
NH
2
O
Scheme 1. Reagents and conditions: (a) R-NH-OHꢁHCl, 1.5 equiv, KOH, 1.5 equiv,
MeOH, rt, 5 h; (b) amine R0R00NH 2(y) (1.1 equiv).
87
90
98
100
98
95
85
92
95
99
82
93
72
72
NH2
gous hydroxamic acids 1(1–3) were obtained with good to excel-
lent yields. On the contrary, squaric acid N-hydroxylamide ester
(R = H) was not obtained although the substitution of the butyl es-
ter by the hydroxylamine was complete.13 Onaran et al. did de-
scribe the synthesis of methyl analogue from dimethylester in
reasonable yield (47%) but failed to obtain final products in yields
compatible with library synthesis.14
Compounds 1(1–3) could then be engaged in reaction with var-
ious amines using a general solution-phase method as described in
Scheme 1.
We looked for the best conditions for the synthesis of target
compounds 3(x,y) from 1(x) and amines 2(y) in microplates at a
15 lmol scale. Using precursor 1(1) and N-methyl-phenethyl-
amine we rationalized reported reaction conditions by testing sev-
eral parameters such as solvent, final concentration, temperature
and reaction time (Scheme 2).
Concentration did not prove critical for synthesis and we
decided to work at the lowest tested concentration to recruit the
largest number of amines. Heating the reaction mixture did not im-
prove results or even reduced purity in some cases. For ease of set-
ting we chose to work at 20 °C. Shorter time of reaction (5 h vs
overnight) slightly increased the yield. At last, effect of the solvent
was not critical and we decided to select MeOH which is a better
solvent than EtOH for most drug relevant amines.
Using these optimized conditions, we evaluated the scope of the
reaction by reacting compounds 1(1–3) with 8 various amines rep-
resentative from different classes. Classification was based on both
expected reactivity and chemical origin (Table 1). Among primary
amines (classes A–F) were selected non conjugated amines (classes
A–E) or anilines (class F) were selected. N-Alkylamines were either
benzylamine derivatives (class A) that display high nucleophilicity
but have a high sensitivity to oxidation and other N-alkylamines
like phenethylamines that display medium to good nucleophilicity
and a good stability (class B). Derivatives of amino-acids are repre-
O
O
NH2
Bu
NH2
HO
O
N
NH2
Bu
F
O
NH2
O
G
N
NH
BzO
H
N
H
a
HPLC monitoring at 215 nM.
The library members were synthesized in polypropylene deep-
well plates at a 15 mol scale as described in Scheme 3. The squaric
acid N-hydroxylamides esters 1(1–2) and 1(3) were reacted, respec-
tively, with amines 2(1–74) and 2(1–54) to yield 200 squaric acid N-
hydroxylamide amides (3(1, 1–74), 3(2, 1–74), 3(3, 1–54), Scheme
3).
l
All compounds were characterized by LC/MS analysis (detection
at 215 nm). Positive and negative electrospray (ESI+ and ESIꢀ) MS
showed the presence of a single parent ion, which confirmed the
identity of the library members. Out of the 200 library members,
86% displayed purity (215 nM) above 80%. Figure 4 represents
mean and standard deviation of purity in each series of compounds
sorted by both the squaric precursor and the class of amines incor-
porated in the library. As can be seen, our conditions are very ro-
bust. Purity for all classes is excellent and only low variability is
observed. When using isopropyl 1(3) precursor, nevertheless, a
greater variability in purity must be expected depending on the
substituents of on the amine. To further characterize the library,
11 structurally diverse compounds were analyzed by 1H and 13C
NMR and displayed very good spectra.
Many groups work on the systematic assessment of bioavailabil-
ity for molecules early in the drug discovery cycle.15 We calculated
the physical properties that are traditionally been considered to be
related to membrane permeability or bioavailability: molecular
weight, logP, polar surface area, number of rotatable bonds and
number of hydrogen bond donors and acceptors, using PipelinePi-
lotTM from Scitegic for all members of the library.16 All compounds
pass the Rule of Five17 and 97.5% of the library complies with Veber’s
criteria.18,19
sented by classes C and D (a-amino-esters and b-amino-alcohols,
respectively). Other N-alkylamines that bear a second function
(like a tertiary amine) are gathered in class E, called ‘bifunctional
amines’. Secondary amines are divided into two classes: cyclic
amines that are very good nucleophiles (class G) and N-meth-
ylalkylamines (class H) that display lower nucleophilicity. As can
be seen in Table 1, with squaric precursors 1(1) and 1(2) the reac-
tion tolerates a broad diversity of amines, the desired product
being formed quantitatively in all cases. In the case of precursor
1(3), only primary amines gave the desired products with good
purity. Consequently for the library synthesis, only primary amines
have been selected to react with this precursor. Figures 2 and 3
show the primary amines 2(1–54) and the secondary amines
2(55–74) selected for library synthesis.
O
N
O
O
O
O
N
H
Itisofteninterestingtodetermineexperimentallythekeyphysico-
chemical parameters for a prototypal compound of the library.20 The
partition coefficient (log D) between 1-octanol and PBS buffer (pH
7.4), pKa and solubility in PBS buffer were measured for compound
3(1,22) (Table 2). Results are compatible with drug-like properties
and pKa is in the range expected for analogues of hydroxamates.
N
+
OH
N
OH
purity (215 nM) : 71-85%
Scheme 2. Reagents and conditions: solvent: MeOH or EtOH, concentration: 0.1 M
or 0.25 M, temperature: 20 or 40 °C, reaction time: 5 or 18 h.

4970
J. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4968–4971
NH2
NH2
O
NH2
NH2
NH2
N
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N
O
N
N
O
O
Cl
F
F
tBu
12
OCF3
13
OMe
14
NBoc
NH2
1
2
3
4
5
6
7
8
9
10
11
15
16
17
18
y=
NH2
NH2
NH2 NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
N
N
N
S
F
NH
F
NH
MeO
NH
Cl
NH
BuO
N
N
H
MeO
BzO
F
Cl
25
OMe
26
O
21
19
20
22
23
NH2
24
NH2
27
28
29
30
31
32
33
34
35
NH2
36
y=
y=
H2N
NH2 NH2
H2N
N
NH2
OH
OH
NH2
OH
NH2 NH2
NH2
NH2
NH2
Bu
NH2
MeO
NH2
H N
2
O
O
O
O
OH
O
H
NH2
O
O
BzN
NH2
OMe
OMe
OtBu
OtBu
N
N
NBz
N
NBz
43
Trt
OtBu
iPr
37
38
39
40
41
42
44
45
46
47
48
49
50
51
52
53
54
Figure 2. Set of primary amines 2(1–54) for the library. Class A 2(5–18); Class B (4,22–39,44); Class C (49–54); Class D (45–48); Class E (1–3,40–43); Class F (19–21).
H
H
N
H
H
N
H
N
H
N
H
N
H
N
H
N
H
N
N
N
N
N
N
N
N
OH
OMe CO2Et
N
O
N
H
F
F
y=
55
56
57
58
59
60
61
62
63
64
CF3
H
H
H
H
N
H
N
HN
HN
HN
HN
HN
N
N
N
N
N
N
N
N
N
Cl
NO2
CF3
O
O
CF3
70
71
72
73
74
CF3
NO2
CF3
y=
65
66
67
68
69
Figure 3. Set of secondary amines 2(55–74) for the library. Class G 2(55–69); Class H 2(70–74).
120
100
80
60
40
20
0
3{1,1-74} (R=Me)
3{2,1-74} (R=Bz)
3{3,1-74} (R=iPr)
R'
NH 2(1-74)
O
O
O
O
N
O
3(1,1-74) : R = Me
3(2,1-74) : R = Bz
3(3,1-54) : R = iPr
R"
1(1) : R = Me
1(2) : R = Bz
1(3) : R = iPr
a.
OH
R'
N
R
N
OH
R"
R
Scheme 3. Reagents and conditions: (a) amines (1.1 equiv, 16.5
5 h, evaporation (Genevac TM, T = 30 °C).
lmol), MeOH, rt,
This library was screened on ADAMTS-5,21 a Zinc metallopro-
tease that degrades, like MMPs, the extracellular matrix.22,23 Inhib-
itors of this enzyme could result in efficient treatments for
osteoarthritis. Only a few inhibitors, mainly micromolar, with the
noteworthy exception of hydroxamates and some thioxothiazolid-
inone, have been published so far.24,25 The screening of our library
led to the identification of two original, structurally related,
ADAMTS-5 inhibitors with IC50 in low micromolar range (respec-
Amine
class
A
B
C
D
E
F
G
H
tively, 2.6 and 37 lM). IC50 were confirmed on resynthesized and
purified compounds (Table 3). Interestingly, these compounds are
derived from p-butoxyaniline. Preferably, the substituent of the
hydroxylamine should not be sterically hindered like iPr and
should bear an aromatic ring. In particular, compound 3(2,19)
seems to be a good starting point for hit-to-lead optimization.
We developed a practical and efficient strategy to synthesize a li-
brary of squaric acid N-hydroxylamide amides. The scope of the
amine set that can be used has been considerably extended relative
Figure 4. Purity of library members according to the amine input and the squaric
precursor (% at 215 nm).
a totally new micromolar inhibitor (IC50 = 2.6 lM). Our goal is yet
the optimization of this primary hit to obtain a lead with a submi-
cromolar activity and good pharmacokinetic properties.
to earlier data. Each compound was obtained in a 15 lmol scale suit-
able for biological screeningand with verygood purityand yield. Our
library was screened on ADAMTS-5 and allowed the identification of

J. Charton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4968–4971
4971
Table 2
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Woods, M. J. Med. Chem. 2000, 43(6), 1187.
pKa, logD and solubility measured for compound 3(1,22)
O
O
N
H
N
OH
3. Douglas, K. T.; Nadvi, I. N. FEBS Lett 1979, 106, 393.
pKa
LogD (pH 7.4)
Solubility (
lg/mL)
4. Burka, L. T.; Doran, J.; Wilson, B. J. Biochem. Pharmacol. 1982, 31, 79.
5. Sato, K.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2002, 124, 12715.
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8.5
0.5
24
7. Flipo, M.; Beghyn, T.; Charton, J.; Leroux, V. A.; Deprez, B. P.; Deprez-Poulain, R.
F. Bioorg. Med. Chem. 2007, 15, 63.
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B.; Natchus, M. G.; Hsieh, L. C.; Janusz, M. J.; Peng, S. X.; Branch, T. M.; King, S.
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13. The product was completely degraded during evaporation of the solvent under
reduced pressure after the purification step on silica gel. A one pot, sequential
reaction was attempted. The dibutyl squarate was reacted with hydroxylamine
during 1 h 30 min, the amine was then added and the reaction mixture was
stirred at room temperature overnight. The very low conversion observed
(<20%) was consistent with the yields (<15%) obtained by Onaran.10,14
14. The two compounds described by Onaran et al. (Ref. 10) were obtained in 13%
and 12% using, respectively, 2.2 and 1.2 equiv of amine.
Table 3
Inhibition on ADAMTS-5
O
O
R'
N
N
R
OH
R"
a
Compound
R0R00N-
R=
ADAMTS-5 IC50
(
lM)
3(1,19)
3(2,19)
3(3,19)
p(Bu-O)-C6H4-NH–
p(Bu-O)-C6H4-NH–
p(Bu-O)-C6H4-NH–
CH3–
Ph-CH2–
i-Pr–
37.0
2.6
>100.0
a
IC50 were measured on resynthesized and purified compounds.21
Acknowledgements
We are grateful to the institutions that support our laboratory
(Inserm, Université de Lille2 and Institut Pasteur de Lille). Data
management was performed using Pipeline PilotTM from Scitegic.
We thank also the following institutions or companies: CAMPLP
and VARIAN.inc. This project was supported by the Fondation pour
la Recherche Medicale Nord-Pas-de-Calais. NMR spectra were re-
corded in the «Laboratoire d’Application RMN » (LARMN) at Univer-
sité de Lille2.
15. Martin, C. M. J. Med. Chem. 2005, 48, 3164.
16. Results are presented in Supporting information.
17. Lipinski, C. A.; Lombardo, F.; Dominiy, B. W.; Feeney, P. J. Adv. Drug Deliv. Rev.
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18. Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
19. Verber’s rule suggests that compounds which meet only the two criteria of (1)
10 or fewer rotatable bonds and (2) polar surface area equal to or less than
140 Å2 (or 12 or fewer H-bond donors and acceptors) will have
probability of good oral bioavailability in the rat.
a high
20. Jacoby, E.; Schuffenhauer, A.; Popov, M.; Azzaoui, K.; Havill, B.; Schopfer, U.;
Engeloch, C.; Stanek, J.; Acklin, P.; Rigollier, P.; Stoll, F.; Koch, G.; Meier, P.;
Orain, D.; Giger, R.; Hinrichs, J.; Malagu, K.; Zimmermann, J.; Roth, H. J. Curr.
Top. Med. Chem. 2005, 5, 397.
Supplementary data
21. See Supporting information for experimental conditions.
22. Apte, S. S. Int. J. Biochem. Cell. Biol. 2004, 36, 981.
23. Gendron, C.; Kashiwagi, M.; Lim, N. H.; Enghild, J. J.; Thogersen, I. B.; Hughes,
C.; Caterson, B.; Nagase, H. J. Biol. Chem. 2007, 282(25), 18294.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.08.025.
24. (a) Xiang, J. S.; Hu, Y.; Rush, T. S.; Thomason, J. R.; Ipek, M.; Sum, P.-E.; Abrous,
L.; Sabatini, J. J.; Georgiadis, K.; Reifenberg, E. Bioorg. Med. Chem. Lett. 2006, 16,
311; Cherney, R. J.; Mo, R.; Meyer, D. T.; Wang, L.; Yao, W.; Wasserman, Z. R.;
Liu, R.-Q.; Covington, M. B.; Tortorella, M. D.; Arner, E. C. Bioorg. Med. Chem.
Lett. 2003, 13, 1297.
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