Design, synthesis, and preliminary biological evaluation of pyrrolo[3,4-c]quinolin-1-one and oxoisoindoline derivatives as aggrecanase inhibitors

Article abstract of DOI:10.1002/cmdc.200900523

A small set of aggrecanase inhibitors based on the pyrrolo[3,4-c]-quinolin- 1-one or oxoisoindoline frameworks bearing a 4-(benzyloxy) phenyl substituent and different zinc binding groups were rationally designed and evaluated in silico by docking studies using the crystal structure of the ADAMTS-5 catalytic domain (PDB code: 3B8Z). The designed compounds were synthesized via straightforward routes and tested for their potential inhibitory activity against ADAMTS-5 and ADAMTS-4. Among the compounds containing the pyrrolo[3,4-c]quinolinone tricyclic system, hydroxamate derivative 2b inhibited both ADAMTS-5 and ADAMTS-4, with IC₅₀ values in the submicromolar range and an inhibitory profile very similar to that of reference carboxylate derivative 11. Conversely, the corresponding carboxylate derivative 2a was significantly less active and unable to discriminate between ADAMTS-5 and -4. The structure-activity relationship analysis of pyrroloquinolinone derivatives 2a-i suggests that the carboxylate or hydroxamate groups of compounds 2a,b play a key role in the interaction of these compounds with ADAMTS-5 and -4. On the other hand, the oxoisoindoline derivatives 3a,b lack significant ADAMTS-4 inhibitory activity and inhibit ADAMTS-5 showing IC₂₅ values in the micromolar range.

Full text of DOI:10.1002/cmdc.200900523

DOI: 10.1002/cmdc.200900523
Design, Synthesis, and Preliminary Biological Evaluation of
Pyrrolo[3,4-c]quinolin-1-one and Oxoisoindoline
Derivatives as Aggrecanase Inhibitors
Andrea Cappelli,*[a] Chiara Nannicini,[a] Salvatore Valenti,[a] Germano Giuliani,[a]
Maurizio Anzini,[a] Laura Mennuni,[b] Antonio Giordani,[b] Gianfranco Caselli,[b] Luigi
Piero Stasi,[b] Francesco Makovec,[b] Gianluca Giorgi,[c] and Salvatore Vomero[a]
A small set of aggrecanase inhibitors based on the pyrrolo[3,4-c]-
quinolin-1-one or oxoisoindoline frameworks bearing a 4-(ben-
zyloxy)phenyl substituent and different zinc binding groups
were rationally designed and evaluated in silico by docking
studies using the crystal structure of the ADAMTS-5 catalytic
domain (PDB code: 3B8Z). The designed compounds were syn-
thesized via straightforward routes and tested for their poten-
tial inhibitory activity against ADAMTS-5 and ADAMTS-4.
Among the compounds containing the pyrrolo[3,4-c]quinoli-
none tricyclic system, hydroxamate derivative 2b inhibited
both ADAMTS-5 and ADAMTS-4, with IC50 values in the submi-
cromolar range and an inhibitory profile very similar to that of
reference carboxylate derivative 11. Conversely, the corre-
sponding carboxylate derivative 2a was significantly less active
and unable to discriminate between ADAMTS-5 and -4. The
structure–activity relationship analysis of pyrroloquinolinone
derivatives 2a–i suggests that the carboxylate or hydroxamate
groups of compounds 2a,b play a key role in the interaction
of these compounds with ADAMTS-5 and -4. On the other
hand, the oxoisoindoline derivatives 3a,b lack significant
ADAMTS-4 inhibitory activity and inhibit ADAMTS-5 showing
IC25 values in the micromolar range.
Introduction
Osteoarthritis is a debilitating disease caused by degradation
of aggrecan and collagen in the articular cartilage matrix lead-
ing to progressive and chronic inflammation, pain, and re-
duced mobility in the affected joint.[1] While collagen is the
structural component of cartilage and provides strength to the
tissue, aggrecan is the major cartilage proteoglycan, which
forms a complex network of aggregates with hyaluronic acid
and collagen to provide flexibility, elasticity, and resistance to
compression in the articular cartilage.[2] Under physiologic con-
ditions, the cartilage matrix is constantly remodeled through
degradation followed by synthesis of type II collagen and ag-
grecan to maintain the integrity of the cartilage.[3] The loss of
glycosaminoglycan-rich aggrecan fragments via proteolysis is
the primary event leading to the destruction of the cartilage
and is attributable to the activity of aggrecanase.[4] Current os-
teoarthritis treatments (e.g., corticosteroids, glucosamine, non-
steroidal anti-inflammatory drugs, intra-articular injections of
hyaluronic acid conjugates) provide only symptomatic relief
with no pharmacological therapy available to stop and/or re-
verse the progression of this disease.[5] In the end, the articular
cartilage is eroded and joint replacement by surgery is
required.
which the catalytic domain of ADAMTS-5 was deleted, indicat-
ed that aggrecanase is the primary enzyme responsible for in
vivo aggrecan degradation in both mechanical and inflamma-
tory osteoarthritis murine models.[7]
Comparison of the features of ADAMTS-4 and ADAMTS-5
showed that, in terms of gene regulation, they are antithetical
to each other. In fact, in most cases ADAMTS-5 is constitutively
expressed in human condrocytes and synovial fibroblasts,
whereas ADAMTS-4 expression is induced by proinflammatory
cytokines.[8] Thus, selective aggrecanase inhibitors have
become attractive for rational drug design, and a number of
hydroxamate inhibitors were reported in very recent years.[9]
However, the hydroxamate group is known to be a strong
zinc-chelating moiety, which generally confers poor pharmaco-
kinetic properties and low selectivity in the interaction with
metalloproteases.[10] Structural modifications by the introduc-
[a] Prof. A. Cappelli, Dr. C. Nannicini, Dr. S. Valenti, Dr. G. Giuliani,
Prof. M. Anzini, Prof. S. Vomero
Dipartimento Farmaco Chimico Tecnologico and the European Research
Centre for Drug Discovery and Development, Universitꢀ di Siena
Via A. Moro, 53100 Siena (Italy)
Fax: (+39)057-723-4333
E-mail: cappelli@unisi.it
Aggrecanases are members of the “a disintegrin and metal-
loprotease with thrombospondin motifs” (ADAMTS) family of
zinc metalloproteases. Both ADAMTS-4 (aggrecanase-1) and
ADAMTS-5, (aggrecanase-2) have been shown to cleave aggre-
can at the Glu373ÀAla374 peptide bond in the interglobular
domain (IGD).[6] Experiments with genetically modified mice, in
[b] Dr. L. Mennuni, Dr. A. Giordani, Dr. G. Caselli, Dr. L. P. Stasi, Dr. F. Makovec
Rottapharm S.p.A.
Via Valosa di Sopra 7, 20052 Monza (Italy)
[c] Prof. G. Giorgi
Dipartimento di Chimica, Universitꢀ di Siena
Via A. Moro, 53100 Siena (Italy)
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A. Cappelli et al.
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tion of polar groups in the region of the hydroxamate moiety
were reported to show favorable effects (via an intramolecular
hydrogen bond and steric hindrance) on absorption and clear-
ance.[9a] Moreover, different zinc-binding groups (ZBGs) other
than the hydroxamate moiety have been investigated to
obtain matrix metalloproteinase inhibitors with improved ab-
sorption and clearance. For instance, a carboxylate moiety has
become a common replacement for a hydroxamate group in
the design of aggrecanase inhibitors, as well as other matrix
metalloproteinase modulators.[11]
This paper describes the results of the initial investigation
on ADAMTS inhibitors 2 and 3. In this structure–activity rela-
tionship study, the effect of hydroxamate masking and replace-
ment was evaluated in compounds based on planar pyrrolo-
[3,4-c]quinolin-1-one or oxoisoindoline frameworks.
Figure 1. Superimposition of reference hydroxamate 1 (green) in its
ADAMTS-5-bound conformation with compound 2b (grey, van der Waals
surfaces are given in order to show the funnel shape of the molecule). The
structures of pyrrolo[3,4-c]quinolin-1-one derivatives 2a,b are included for
clarity.
Results and Discussion
ZBGs such as carboxylate and hydroxamate through the forma-
tion of an intramolecular hydrogen bond.
Design of ADAMTS inhibitors
The recently published high-resolution crystal structure[12] of
the ADAMTS-5 catalytic domain in the presence of a hydroxa-
mate inhibitor 1 (PDB code: 3B8Z) shows a funnel shape for
In order to evaluate the fitting of compounds 2a,b and 3a,b
within the ADAMTS-5 catalytic domain, docking simulations
were performed by means of AutoDock 4.0 (for details see the
Experimental Section). Quinolinone derivatives 2a,b appear to
be capable of fitting the catalytic site of the ADAMTS-5 with
good effectiveness (Figure 2) and similar short-range contacts,
but with a subtle difference in the polar interactions. In partic-
ular, docking results suggest that hydroxamate derivative 2b
could establish an additional polar interaction (e.g., with
Glu411) compared with carboxylate derivative 2a, which leads
to the prediction of a higher potency for compound 2b. The
docking result obtained with quinoline derivative 2a suggest-
ed the replacement of its polar carboxylate substituent with
the different groups of compounds 2c–i in order to obtain
direct information about the polar interaction of compounds 2
with ADAMTS enzymes.
the catalytic site, which is open at the zinc site and terminates
with a L-shaped hydrophobic channel at the opposite end.[12a]
The enzyme–inhibitor complex exhibits a three-point interac-
tion in which the hydroxamate group plays a key role by che-
lating the zinc atom and interacting with Glu411. Furthermore,
an additional hydrogen-bonding interaction and a specific
strong hydrophobic interaction (within the S1’ pocket) are also
established.
Our initial target was the design of aggrecanase inhibitors
showing structurally simple, fairly rigid, and easily accessible
scaffolds bearing substituents capable of establishing suitable
interactions with the ADAMTS catalytic domain.
On the basis of its shape and dimension, a planar pyrrolo-
[3,4-c]quinolinone tricyclic system bearing the 4-(benzyloxy)-
phenyl substituent of compounds 2 (Figure 1) was considered
to represent an appropriate scaffold for the funnel-shaped
ADAMTS-5 catalytic site that could be “decorated” with suita-
ble ZBGs (e.g., COOH as in compound 2a, or CONHOH as in
compound 2b).
In our design hypothesis, the quinoline nitrogen of com-
pounds 2 was considered to be capable of masking (i.e.,
during the drug transport across membranes) polar ZBGs by
forming an intramolecular hydrogen bond. On the other hand,
compounds 3a,b represent simplified analogues of pyrroloqui-
nolinone derivatives 2a,b in which the carbonyl oxygen atom
should assist the ZBG in the chelating effect and mask polar
Similarly, the in silico predicted interaction of oxoisoindoline
derivative 3a with ADAMTS-5 catalytic site appears to be as ef-
fective as that predicted for 2a. In fact, in the docked confor-
mations (Figure 3), compound 3a seems to be capable of coor-
dinating the zinc ion by means of a bidentate interaction
through the lactam carbonyl oxygen and the hydroxy group of
the carboxylic moiety. Moreover, the docking results suggest
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Aggrecanase Inhibitors
presence of palladium on carbon to obtain the un-
substituted pyrroloquinolinone derivative 2g. Suzuki
cross-coupling of 2-chloroquinoline derivative 2h
with N-phenyldietanolamine 2-pyridilboronate[14]
gave compound 2d.
Imidoylchloride 2h (Scheme 2) was subjected to
Stille-type coupling reaction[15] with trimethylalumi-
num to give compound 2i, which provided oxime 2c
via oxidation with SeO2 to aldehyde 2e and subse-
quent reaction with hydroxylamine hydrochloride. Al-
ternatively, oxidation of aldehyde 2e with hydrogen
peroxide gave carboxylic acid 2a. The conversion of
2a into the corresponding hydroxamate 2b was ac-
complished by coupling with O-(tert-butyldimethyl-
silyl)hydroxylamine in the presence of Castro’s re-
agent (BOP)[16] and spontaneous deprotection.
Figure 2. Pyrroloquinolinone derivatives a) 2a and b) 2b docked into the ADAMTS-5 cat-
alytic site. The zinc ion is shown as a grey sphere. Coordinating and hydrogen bonds are
represented as red dotted lines.
Finally, compound 2i was treated with meta-chloro-
peroxybenzoic acid (m-CPBA) to obtain quinoline N-
oxide 6 in high yield (Scheme 3). Reaction of 6 with
p-toluenesulfonyl chloride (TsCl) gave tosylate 7,
which provided methyl ether 2 f by reaction with
magnesium turnings in methanol.[17]
Isoindoline derivatives 3a,b were prepared from
commercially available 3-methylphtalic anhydride 8
as shown in Scheme 4. Anhydride 8 was brominated
with N-bromosuccinimide (NBS) in the presence of di-
benzoyl peroxide following the literature[18] and cy-
clized with 4-benzyloxyaniline to give the expected
carboxylic acid 3a. The structure of carboxylic acid
3a was confirmed by crystallographic studies, which
showed the existence of an unexpectedly strong in-
tramolecular hydrogen-bonding interaction between
Figure 3. Oxoisoindoline derivatives a) 3a and b) 3b docked into the ADAMTS-5 catalytic
site. The zinc ion is shown as a grey sphere. Coordinating and hydrogen bonds are repre-
sented as red dotted lines.
that the hydroxy group is involved in a hydrogen
bond with Glu411. Similarly to pyrroloquinolinone
derivatives 2a,b, hydroxamate derivative 3b is pre-
dicted to be capable of establishing a stronger inter-
action than the carboxylate derivative 3a. However, it
should be noted that in the docked conformations of
2a,b, and 3a,b, no intramolecular hydrogen bond is
present. Therefore, our masking hypothesis could
represent a critical point in the design of these com-
pounds, because a too strong intramolecular hydro-
gen bond could prevent
inhibitor interaction.
a favorable enzyme–
Chemistry
Target pyrrolo[3,4-c]quinolin-1-one derivatives 2a–f
were synthesized by means of a multistep procedure
described in Scheme 1–3. Pyrrolo[3,4-c]quinolin-1-one
derivatives 2 were synthesized using chemistry previ-
ously developed for the synthesis of 5-HT3 receptor li-
gands.[13] Compound 4[13] was brominated and cy-
clized with 4-benzyloxyaniline (Scheme 1) to give the
Scheme 1. Preparation of compounds 2d, g, h. Reagents and conditions: a) NBS, benzoyl
peroxide, CCl4, reflux, 2.5 h, 79%; b) 4-benzyloxyaniline hydrochloride, K2CO3, EtOH,
reflux, 1 h, 62%; c) H2, Pd/C, CHCl3, EtOH, Et3N, RT, 30 h, 16%; d) PPh3, Pd(OAc)2, K2CO3,
intermediate 2h, which was hydrogenated in the CuI, N-phenyldietanolamine 2-pyridilboronate, THF, reflux, 10 min, 9%.
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A. Cappelli et al.
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the acid proton and lactam carbonyl oxygen atom (Figure 4).
1
The H NMR spectrum of compound 3a, performed in both
deuterated chloroform and deuterated dimethyl sulfoxide,
showed the peak attributable to acid proton to resonate at
15.5–15.7 ppm, indicating that the hydrogen-bonding interac-
tion is rather strong in the organic solvents used. This result
supports the postulated masking effect in the hydrophobic en-
vironment, but it raises questions about the hydrogen bond
stability in the aggrecanase active site that leads to the forma-
tion of a pseudocycle and could prevent a favorable interac-
tion with the zinc atom (see Figure 3). Similarly to 3a, crystallo-
graphic studies of 3b showed that its NH proton interacts with
the lactam carbonyl oxygen atom in the solid-state structure
(Figure 5).
Also, in compound 10 (Figure 6), the carboxylic proton ap-
pears to interact strongly with the imidate nitrogen atom, and
1
the H NMR spectra of 10 show a very low-field resonance at
18.8 ppm in both deuterated chloroform and deuterated di-
methyl sulfoxide.
Therefore, the X-ray crystal structures of compounds 3a,b
and 10 show, as a common feature, the occurrence of strong
intramolecular hydrogen-bonding interactions involving the
COOH and the lactam carbonyl oxygen atom (3a), the hy-
droxamic NH group and the lactam carbonyl oxygen atom
(3b), the COOH and the imidate nitrogen atom in 10 (Table 1
and Table 2). In all the three compounds, the existence of
these interactions causes the formation of a seven-membered
ring in which one bond is due to the proton···acceptor interac-
tion that, in the three compounds, averages 1.68 ꢁ (in the
range of the partially covalent hydrogen bonds[19]).
The presence of a rigid bicyclic planar system does
not facilitate the formation of a reasonable proto-
n···acceptor distance; this hydrogen bond can only
be obtained by distortion of the bond angles involv-
ing the acidic moiety. Indeed, significant distortions
of the C(4)ÀC(5)ÀC(10) and C(6)ÀC(5)ÀC(10) bond
angles from the theoretical value of 1208 are ob-
served. In particular, the former is wider (mean=
128.18) so as to allow a longer distance proton···ac-
ceptor interaction, while, as a consequence, the latter
is narrower (mean=115.48) than the theoretical value
(1208). Moreover, compound 10 also shows two
strong intermolecular hydrogen-bonding interac-
tions: O(4)ÀH(x,y,z)···O2(-x,1-y,2-z) and O(2)(x,y,z)···H-
Scheme 2. Preparation of compounds 2a–c,e,i. Reagents and conditions:
a) Al(CH3)3, Pd(PPh3)2Cl2, LiCl, DMF, 858C, 2 h, 47%; b) SeO2, dioxane, reflux,
45 min, 71%; c) NH2OH·HCl, pyridine, RT, 30 min, 78%; d) H2O2, H2O, HCOOH,
RT, overnight, 96%; e) O-(tert-butyldimethylsilyl)hydroxylamine, BOP, Et3N,
DMF, RT, overnight, 48%.
Scheme 3. Preparation of compound 2 f. Reagents and conditions: a) m-CPBA, CH2Cl2, RT,
overnight, 52%; b) TsCl, K2CO3, CHCl3, H2O, reflux, overnight, 54%; c) Mg, MeOH, reflux,
2 h, 18%.
O(4)(-x,1-y,2-z) with H···O distance equal to 1.82 ꢁ
In vitro ADAMTS inhibitory
activity
Table 1. Intramolecular hydrogen-bonding interactions and significant geometrical features in the X-ray crystal
structures of compounds 3a,b and 10.
Potential aggrecanases inhibitors
2a–i and 3a,b were screened for
protease action at the “signa-
ture” cleavage site Glu373–
Ala374 in the IGD. In fact, this
cleavage releases the entire gly-
cosaminoglican-containing por-
tion of aggrecan and may have
Compd
Donor (D)
Acceptor (A)
DÀH [ꢁ][a]
H···A [ꢁ][a]
D···A [ꢁ][a]
DÀH···A [8][a]
3a
O(3A)ÀH
O(3B)ÀH
N(3)ÀH
O(1A)
O(1B)
O(1)
0.82
0.82
1.10(5)
0.82
1.74
1.71
1.56(6)
1.72
2.541(6)
2.524(6)
2.618(5)
2.521(5)
164
177
159(5)
164
3b
10
O(4)ÀH
N(1)
[a] Numbers in parentheses are the standard deviations.
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Aggrecanase Inhibitors
the most deleterious effects on the loss of the me-
chanical properties of cartilage.[20] Moreover, in vivo
studies showed that blocking aggrecanolysis in the
aggrecan IGD alone protects against cartilage erosion
and may promote cartilage repair.[6a] Thus, com-
pounds 2a–i and 3a,b were tested for their potential
inhibitory activity against ADAMTS-4 and ADAMTS-5
enzymes by means of the InviLISA aggrecanase assay
(Invitek GmbH, Berlin) in comparison with reference
compounds 11[21] and 12.[9d] A recombinant fragment
of human aggrecan IGD (aggrecan–IGD; Thr331–
Gly458) was used as a substrate, and the assay was
carried out in two steps according to Will and co-
workers[22] (for details see the Experimental Section).
The results, summarized in Table 3, show that car-
boxylate derivative 2a appears to be capable of in-
hibiting both ADAMTS-5 and ADAMTS-4, showing
IC50 values (half maximal inhibitory concentration) in
the micromolar range, and is
Scheme 4. Preparation of compound 3a,b and 10. Reagents and conditions: a) NBS, ben-
zoyl peroxide, CCl4, reflux, 4 h, 66%; b) 4-benzyloxyaniline hydrochloride, K2CO3, EtOH,
reflux, 45 min, 29%; c) O-(tert-butyldimethylsilyl)hydroxylamine, BOP, Et3N, DMF, RT, over-
night, 51%; d) NH2C6H4OCH2CH2CH3, K2CO3, EtOH, reflux, 45 min, 14%.
about one order of magnitude
less potent than the reference
carboxylate derivative 11 at in-
hibiting ADAMTS-5. On the other
hand, the corresponding hy-
droxamic acid 2b is significantly
more potent than carboxylic
acid 2a and appears to discrimi-
nate
appreciably
between
ADAMTS-5 and -4. However, the
inhibitory potencies shown by
2b are different from those ob-
tained with reference hydroxa-
Figure 4. Two molecules form the asymmetric unit of 3a. Ellipsoids enclose 50% probability.
mate 12. The remaining quinoli-
none derivatives 2c–i (bearing less polar substitu-
ents) show inhibition values below 40% at the maxi-
mum concentration tested (10 mm). Thus, the results
obtained with pyrroloquinolinone derivatives 2a–i
confirmed the key role of the carboxylate group, or
hydroxamate group of compounds 2a,b, in the inter-
action with ADAMTS-5 and -4.
The results obtained with oxoisoindoline deriva-
tives 3a,b show that these compounds lack signifi-
cant ADAMTS-4 inhibitory activity and show a partic-
ular behavior in inhibiting ADAMTS-5. In fact, the in-
hibition curves of compounds 3a,b show an appar-
ent plateau at ~50% inhibition. The IC25 values calcu-
lated show that ADAMTS-5 inhibitory activity is in the
Figure 5. Crystal structure of compound 3b. Ellipsoids enclose 50% probability.
micromolar range and slightly higher in the hydroxamate de-
rivative 3b with respect to the corresponding carboxylate 3a,
as anticipated by the docking studies. The behavior shown by
3a,b in inhibiting ADAMTS-5 may be related to solubility prob-
lems, probably caused by the presence of planar moieties
(prone to forming stacked aggregates as suggested by
Figure 4) and the particularly strong intramolecular hydrogen
bonds originally designed to mask polar ZBGs. Specifically, the
dilution of the clear dimethyl sulfoxide stock solution to obtain
the first working solution (DMSO concentration of ~10%)
Table 2. Significant bond angles in the X-ray crystal structures of com-
pounds 3a,b, and 10.[a]
Compd
C(4)ÀC(5)ÀC(10)
C(6)ÀC(5)ÀC(10)
C(4)ÀC(5)ÀC(6)
3a
128.4(6)
128.3(6)
128.8(4)
127.2(5)
115.7(6)
115.2(6)
113.8(4)
117.1(4)
115.9(6)
116.5(6)
117.3(4)
115.7(4)
3b
10
[a] Numbers in parentheses are the standard deviations.
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A. Cappelli et al.
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(compounds 3) frameworks bearing a 4-(benzyloxy)-
phenyl substituent and different ZBGs have been de-
signed and evaluated in silico by docking studies
using the crystal structure of the ADAMTS-5 catalytic
domain (PDB code: 3B8Z). Molecular modeling pre-
dicts the good fit of compounds 2a,b and 3a,b
within the enzyme cleft, with hydroxamate deriva-
tives 2b and 3b predicted to show slightly superior
binding features with respect to the corresponding
carboxylate derivatives 2a and 3a.
Compounds 2a–i and 3a,b were synthesized via
straightforward procedures, and crystallographic
studies revealed that the polar (carboxylic or hy-
droxamic) groups of compounds 3a,b are involved in
unexpectedly strong intramolecular hydrogen bonds.
The newly synthesized compounds were evaluated
for their potential to inhibit ADAMTS-4 and ADAMTS-5
(InviLISA aggrecanase assay). Among the compounds contain-
ing the pyrrolo[3,4-c]quinolinone tricyclic system, hydroxamate
derivative 2b appears to be capable of inhibiting both
ADAMTS-5 and ADAMTS-4, with IC50 values in the submicromo-
lar range. It is worth remarking that the inhibitory profile
shown by 2b is very similar to that found for reference carbox-
ylate derivative 11. Conversely, the corresponding carboxylate
derivative 2a is significantly less active and unable to discrimi-
nate between ADAMTS-5 and 4. The structure–activity relation-
ships within the pyrroloquinolinone derivatives (2a–i) prove
the key role played by the contacts established between the
polar groups of compounds 2a,b and the ADAMTS-4 and -5
enzymes. The simplified analogues 3a,b show different
enzyme interactions compared with the corresponding pyrro-
loquinolinone derivatives 2a,b. In fact, compounds 3a,b lack
significant ADAMTS-4 inhibitory activity but inhibit ADAMTS-5
in the micromolar range.
Figure 6. Crystal structure of compound 10. Ellipsoids enclose 50% probability.
Table 3. In vitro inhibitory activity of compounds 2a–i, 3a,b and reference
compounds 11 and 12 against ADAMTS-4 and ADAMTS-5.
Compd
R
IC50 [mm]
ADAMTS-4
ADAMTS-5
2a
2b
2c
2d
2e
2 f
2g
2h
2i
COOH
15Æ2.1
12Æ1.8
C(O)NHOH
CH=NOH
2-pyridyl
CHO
CH2OCH3
H
Cl
CH3
COOH
C(O)NHOH
0.21Æ0.05
0.95Æ0.11
In summary, this study represents a successful approach to
the design of ADAMTS inhibitors possessing simple structures
and syntheses (especially in the case of oxoisoindoline deriva-
tives 3a,b) for which unanticipated solubility features have
prevented further development.
NA[b]
34%[a]
NA[b]
NA[b]
NT[c]
NA[b]
NA[b]
27%[a]
NA[b]
NA[b]
32%[a]
NA[b]
NA[b]
32%[a]
3a
3b
11
NA[b]
7.5Æ3.1[d]
2.5Æ0.44[d]
1.1Æ0.21
0.0014Æ0.00020
NA[b]
Experimental Section
0.21Æ0.04
0.0011Æ0.00030
12
Chemistry
[a] Values expressed as a percentage represent inhibition of enzyme activity
at 10 mm. [b] NA: not active at 10 mm. [c] NT: not tested. [d] IC25 values (mm)
given because compounds 3a,b showed a plateau inhibition at ~50%.
All chemicals used were reagent grade. Yields refer to purified
products and are not optimized. Melting points were determined
in open capillaries on a Gallenkamp apparatus and are uncorrect-
ed. Merck silica gel 60 (230–400 mesh) was used for column chro-
matography. Merck TLC plates, silica gel 60 F254 were used for TLC.
1H NMR spectra were recorded at 200 MHz (Bruker AC200 spec-
trometer) or at 400 MHz (Bruker DRX-400 AVANCE spectrometer) in
the indicated solvents (TMS as internal standard). Chemical shifts
(d) are expressed in parts per million (ppm), and the coupling con-
stants (J) are given in Hz. An Agilent 1100 LC/MSD operating with
a electrospray source was used in mass spectrometry experiments;
unless otherwise stated, experiments were carried out in the posi-
tive mode. Microanalyses were carried out using a Perkin–Elmer
Series II CHNS/O Analyzer 2400.
caused opalescence, which suggests that a partial precipitation
occurred. The incomplete solubility of 3a,b in the conditions
used in the enzymatic tests may lead to an underestimation of
the ADAMTS activity of these compounds.
Conclusions
A small series of aggrecanase inhibitors based on the planar
pyrrolo[3,4-c]quinolin-1-one (compounds 2) or oxoisoindoline
744
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ChemMedChem 2010, 5, 739 – 748

Aggrecanase Inhibitors
2-[4-(Benzyloxy)phenyl]-4-chloro-2,3-dihydro-1H-pyrrolo[3,4-c]-
quinolin-1-one (2h): A mixture of 5[13] (1.31 g, 4.0 mmol), 4-(benzyl-
oxy)aniline hydrochloride (1.04 g, 4.4 mmol) and K2CO3 (0.61 g,
4.4 mmol) in EtOH (20 mL) was refluxed for 1 h. The reaction mix-
ture was allowed to cool to RT and the precipitate was collected
by filtration, and washed in sequence with H2O and EtOH/EtOAc to
give compound 2h as a pale yellow solid (1.0 g, 62%); mp: 214–
dioxane (10 mL) was treated with SeO2 (0.30 g, 2.7 mmol) and re-
fluxed for 45 min. The reaction was partitioned between CH2Cl2
and H2O and separated. The organic layer was washed with H2O,
dried over Na2SO4, and evaporated in vacuo. Purification of the res-
idue by flash chromatography (CH2Cl2/EtOAc; 95:5) gave 2e as a
1
thick yellow oil (0.132 g, 71%); H NMR (200 MHz, CDCl3): d=5.09
(s, 2H), 5.25 (s, 2H), 7.06 (m, 2H), 7.31–7.42 (m, 5H), 7.79–7.94 (m,
4H), 8.36 (m, 1H), 9.22 (m, 1H), 10.34 ppm (s, 1H); MS (ESI): m/z
395 [M+H]+.
1
2158C; H NMR (200 MHz, CDCl3): 4.91 (s, 2H), 5.11 (s, 2H), 7.07 (d,
J=8.8, 2H), 7.33–7.43 (m, 5H), 7.70–7.87 (m, 4H), 8.14 (d, J=8.3,
1H), 9.11 ppm (d, J=7.7, 1H); 13C NMR (100 MHz, CDCl3): d=49.8,
70.3, 115.6, 121.9, 122.9, 123.8, 127.5, 128.1, 128.7, 131.1, 131.8,
133.3, 136.8, 138.2, 144.8, 148.6, 156.5, 165.6 ppm; MS (ESI): m/z
423 [M+Na]+; Anal. calcd for C24H17ClN2O2·43H2O: C 69.57, H 4.50, N
6.76, found: C 69.40, H 4.18, N 6.53.
(Z)-2-[4-(Benzyloxy)phenyl]-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-c]-
quinoline-4-carbaldehyde oxime (2c): A solution of 2e (0.030 g,
0.076 mmol) in pyridine (2 mL) was treated with hydroxylamine hy-
drochloride (0.014 g, 0.20 mmol) and stirred for 30 min at RT. The
solvent was removed in vacuo and the residue washed in se-
quence with H2O and with CH2Cl2 to give compound 2c as a
yellow solid (24 mg, 78%); mp: 225–2268C; 1H NMR (200 MHz,
[D6]DMSO): d=5.10 (s, 2H), 5.19 (s, 2H), 7.11 (d, J=8.8, 2H), 7.30–
7.46 (m, 5H), 7.73–7.89 (m, 4H), 8.15 (d, J=8.1, 1H), 8.39 (s, 1H),
9.01 (d, J=8.2, 1H), 12.22 ppm (s, 1H): The chemical shift values of
the signal attributed to aldoxime OH (12.22 ppm) and CH
(8.39 ppm) groups were used in the configuration assignment ac-
2-[4-(Benzyloxy)phenyl]-2,3-dihydro-1H-pyrrolo[3,4-c]quinolin-1-
one (2g): A mixture of 2h (0.10 g, 0.25 mmol), Et3N (0.1 mL) and
10% Pd/C (0.03 g) in CHCl3/EtOH (30 mL; 1:1) was hydrogenated
under atmospheric pressure at RT for 30 h. The catalyst was filtered
off, and the filtrate was evaporated in vacuo. The residue was parti-
tioned between CHCl3 and H2O. The organic layer was dried over
Na2SO4 and concentrated in vacuo. Purification of the residue by
flash chromatography (CH2Cl2/EtOAc; 8:2) gave 2g as a pale yellow
solid (0.015 g, 16%); mp: 194–1968C; 1H NMR (200 MHz, CDCl3):
d=4.92 (s, 2H), 5.07 (s, 2H), 7.03 (d, J=8.9, 2H), 7.28–7.41 (m, 5H),
7.66–7.83 (m, 4H), 8.20 (d, J=8.2, 1H), 9.04–9.18 ppm (m, 2H); MS
(ESI): m/z 389 [M+Na]+; Anal. calcd for C24H18N2O2·31H2O: C 77.40, H
5.05, N 7.52, found: C 77.35, H 4.84, N 7.28.
cording to Kleinspehn et al.[23] 13C NMR (100 MHz, [D6]DMSO): d=
;
52.4, 69.9, 115.7, 122.4, 122.7, 123.2, 128.2, 128.3, 128.9, 129.3,
129.7, 130.7, 132.6, 133.6, 135.3, 137.5, 147.5, 148.3, 150.4, 156.0,
166.0 ppm; MS (ESI): m/z 410 [M+H]+; Anal. calcd for
C25H19N3O3·2H2O: C 71.25, H 4.86, N 9.97, found: C 71.11, H 4.57, N
3
9.81.
2-[4-(Benzyloxy)phenyl]-2,3-dihydro-4-(pyridin-2-yl)-1H-pyrrolo-
[3,4-c]quinolin-1-one (2d): A mixture of compound 2h (0.20 g,
0.50 mmol), N-phenyldiethanolamine 2-pyridylboronate (0.27 g,
1.0 mmol), PPh3 (0.030 g, 0.11 mmol), Pd(OAc)2 (6.0 mg,
0.027 mmol), K2CO3 (0.14 g, 1.0 mmol) and CuI (0.040 g, 0.21 mmol)
in THF (10 mL) was heated to reflux under N2 for 10 min. The reac-
tion mixture was then cooled to RT and the solvent removed in
vacuo. The residue was partitioned between H2O and CHCl3 and
the organic layer was dried over Na2SO4 and evaporated in vacuo.
Purification of the residue by flash chromatography (CH2Cl2/EtOAc;
8:2) gave 2d as a yellow solid (20 mg, 9%); mp: 205–2068C;
1H NMR (200 MHz, CDCl3): d=5.11 (s, 2H), 5.53 (s, 2H), 7.08 (m,
2H), 7.31–7.47 (m, 6H), 7.68–7.95 (m, 5H), 8.26 (d, J=8.4, 1H),
8.73–8.79 (m, 2H), 9.25 ppm (d, J=7.6, 1H); MS (ESI): m/z 444
[M+H]+; Anal. calcd for C29H21N3O2·31H2O: C 77.49, H 4.86, N 9.35,
found: C 77.29, H 4.56, N 9.22.
2-[4-(Benzyloxy)phenyl]-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-c]quin-
oline-4-carboxylic acid (2a): A solution of 2e (0.13 g, 0.33 mmol)
in formic acid (7.0 mL) was treated with H2O2 (4.0 mL, 3% w/v in
H2O). The reaction mixture was stirred overnight at RT and extract-
ed with CH2Cl2. The organic layer was dried over Na2SO4 and
evaporated in vacuo. Purification of the residue by washing with
1
Et2O gave 2a as a red solid (0.13 g, 96%); mp: 197–1998C; H NMR
(200 MHz, CDCl3): d=5.14 (s, 2H), 5.34 (s, 2H), 7.12 (d, J=9.0, 2H),
7.32–7.47 (m, 5H), 7.84 (d, J=9.0, 2H), 7.89–7.98 (m, 2H), 8.30 (d,
J=8.2, 1H), 9.10 (d, J=8.0, 1H), 13.81 ppm (br s, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=51.5, 69.4, 115.2, 122.0, 122.7, 123.9,
127.6, 127.8, 128.4, 130.2, 130.4, 130.6, 132.0, 135.6, 135.8, 137.0,
143.6, 146.7, 155.6, 165.3, 165.6 ppm; MS (ESIÀ): m/z 409 [MÀH]À;
Anal. calcd for C25H18N2O4·1CHCl3: C 68.88, H 4.18, N 6.36, found: C
4
68.96, H 4.24, N 5.96.
2-[4-(Benzyloxy)phenyl]-2,3-dihydro-N-hydroxy-1-oxo-1H-
pyrrolo[3,4-c]quinoline-4-carboxamide (2b): A solution of 2a
(0.060 g, 0.146 mmol) in dry DMF (10 mL) was treated with Castro’s
reagent (BOP; 0.065 g, 0.147 mmol), Et3N (60 mL), and O-(tert-butyl-
dimethylsilyl)hydroxylamine (0.035 g, 0.24 mmol) and stirred over-
night at RT. The solvent was evaporated in vacuo, and the residue
was purified by washing with H2O, CH2Cl2, and Et2O to give 2b as
a yellow solid (30 mg, 48%); mp: 1638C (decomp); 1H NMR
(200 MHz, [D6]DMSO): d=5.13 (s, 2H), 5.36 (s, 2H), 7.12 (d, J=9.0,
2H), 7.34–7.43 (m, 5H), 7.80–7.94 (m, 4H), 8.22 (d, J=8.0, 1H), 9.08
(d, J=7.8, 1H), 9.30 (s, 1H), 11.73 ppm (s, 1H); MS (ESIÀ): m/z 424
[MÀH]À; Anal. calcd for C25H19N3O4·43CHCl3: C 60.06, H 3.87, N 8.16,
found: C 59.89, H 4.08, N 7.88.
2-[4-(Benzyloxy)phenyl]-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]-
quinolin-1-one (2i): A solution of 2h (1.0 g, 2.5 mmol) in dry DMF
(10 mL) under Ar was treated with Al(CH3)3 (2.0m in toluene;
1.2 mL, 2.4 mmol), LiCl (0.30 g, 7.1 mmol) and Pd(PPh3)2Cl2 (0.080 g,
0.11 mmol). The resulting mixture was heated at 858C for 2 h, then
poured into ice, neutralized with HCl (3n), and extracted with
CH2Cl2. The organic layer was dried over Na2SO4 and evaporated in
vacuo. The residue was washed with EtOH to give 2i as a light
brown solid (0.45 g, 47%); mp: 183–1858C; 1H NMR (200 MHz,
CDCl3): d=2.78 (s, 3H), 4.85 (s, 2H), 5.09 (s, 2H), 7.08 (m, 2H),
7.31–7.46 (m, 5H), 7.61–7.80 (m, 4H), 8.12 (d, J=8.3, 1H), 9.08 ppm
(d, J=7.9, 1H); 13C NMR (100 MHz, CDCl3): d=21.8, 49.8, 70.3,
115.4, 121.7, 122.4, 123.4, 127.5, 127.6, 128.0, 128.6, 128.8, 129.9,
132.3, 133.4, 134.8, 136.8, 148.2, 153.2, 156.2, 166.9 ppm; MS (ESI):
m/z 381 [M+H]+; Anal. calcd for C25H20N2O2·41H2O: C 78.00, H 5.37,
N 7.28, found: C 78.04, H 5.18, N 7.14.
2-[4-(Benzyloxy)phenyl]-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]-
quinolin-1-one 5-oxide (6): A mixture of 2i (0.60 g, 1.6 mmol) in
10 mL of CH2Cl2 with 0.66 g (3.8 mmol) of m-CPBA was stirred over-
night at RT. After this time, the reaction mixture was washed in se-
quence with a 5% aq solution of K2CO3 and H2O. The organic layer
was dried over Na2SO4 and evaporated in vacuo. Purification of the
2-[4-(Benzyloxy)phenyl]-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-c]quin-
oline-4-carbaldehyde (2e): A solution of 2i (0.18 g, 0.47 mmol) in
ChemMedChem 2010, 5, 739 – 748
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745

A. Cappelli et al.
MED
residue by flash chromatography (CH2Cl2/EtOAc; 1:1) gave 6 as a
yellow solid (0.33 g, 52%); mp: 150–1528C; 1H NMR (200 MHz,
CDCl3): d=2.62 (s, 3H), 4.78 (s, 2H), 5.06 (s, 2H), 7.01 (d, J=8.8,
2H), 7.27–7.40 (m, 5H), 7.62–7.78 (m, 4H), 8.68 (d, J=8.7, 1H),
9.04 ppm (d, J=8.2, 1H); 13C NMR (100 MHz, CDCl3): d=14.3, 49.5,
70.3, 115.5, 119.7, 121.6, 123.2, 124.2, 124.3, 127.5, 128.1, 128.6,
129.4, 130.5, 132.1, 135.3, 136.8, 140.7, 141.8, 156.2, 165.9 ppm; MS
(ESI): m/z 419 [M+Na]+.
was removed in vacuo and the residue partitioned between H2O
and EtOAc. The organic layer was dried over Na2SO4 and evaporat-
ed in vacuo. Purification of the residue by flash chromatography
(EtOAc/MeOH; 9:1) and subsequent recrystallization (acetone/
CHCl3) gave 10 as pale-yellow crystals (36 mg, 14%); mp: 206–
1
2078C; H NMR (200 MHz, CDCl3): d=1.04 (t, J=7.4, 3H), 1.72–1.90
(m, 2H), 3.93 (t, J=6.6, 2H), 5.66 (s, 2H), 6.91 (d, J=8.8, 2H), 7.53–
7.77 (m, 4H), 8.52 (d, J=7.6, 1H), 18.77 ppm (s, 1H); MS (ESI): m/z
312 [M+H]+.
[2-[4-(Benzyloxy)phenyl]-2,3-dihydro-1-oxo-1H-pyrrolo[3,4-c]qui-
nolin-4-yl]methyl 4-methylbenzenesulfonate (7): A solution of
TsCl (0.11 g, 0.58 mmol) and 6 (0.18 g, 0.45 mmol) in CHCl3 (5.0 mL)
was treated with a 10% aq solution of K2CO3 (5.0 mL). The result-
ing mixture was refluxed overnight with vigorous stirring. The reac-
tion was partitioned between CH2Cl2 and H2O, and the organic
layer was washed with H2O, dried over Na2SO4, and evaporated in
vacuo. Purification of the residue by flash chromatography (petro-
leum ether/EtOAc; 3:7) gave 7 as a glassy solid (0.134 g, 54%);
1H NMR (200 MHz, CDCl3): d=2.39 (s, 3H), 5.01 (s, 2H), 5.11 (s, 2H),
5.47 (s, 2H), 7.07 (m, 2H), 7.24–7.47 (m, 7H), 7.68–7.89 (m, 6H),
8.08 (d, J=8.8, 1H), 9.14 ppm (d, J=8.0, 1H); MS (ESI): m/z 573
[M+Na]+.
X-ray crystallography
Single crystals of 3a,b and 10 were submitted for X-ray data collec-
tion on a Siemens P4 four-circle diffractometer with graphite mon-
ochromated Mo-Ka radiation (l=0.71073 ꢁ) at 293 K. The struc-
tures were solved by direct methods implemented in SHELXS-97.[24]
The refinements were carried out by full-matrix anisotropic least-
squares on F2 for all reflections for non-hydrogen atoms using
SHELXL-97.[25]
Crystallographic data (excluding structure factors) for the structures
of 3a, 3b, and 10 have been deposited with the Cambridge Crys-
tallographic Data Centre. CCDC 722320 (3a), 722321 (3b) and
722322 (10) contain the supplementary crystallographic data for
this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk.
2-[4-(Benzyloxy)phenyl]-4-(methoxymethyl)-2,3-dihydro-1H-
pyrrolo[3,4-c]quinolin-1-one (2 f):
A mixture of 7 (0.050 g,
0.091 mmol) in MeOH (8.0 mL) and Mg turnings (0.025 g,
0.10 mmol) was refluxed for 2 h. The cooled reaction mixture was
neutralized with HCl (3n) and partitioned between CH2Cl2 and
H2O. The organic layer was dried over Na2SO4 and evaporated in
vacuo. Purification of the residue by flash chromatography (petro-
leum ether/EtOAc; 1:1) gave 2 f as a light brown solid (6.7 mg,
Biology
1
A recombinant fragment of human aggrecan IGD (aggrecan–IGD;
Thr331–Gly458) was first digested with aggrecanase. Proteolytic
cleavage of the substrate released an aggrecan peptide with the
N-terminal sequence AlaArgGlySerValIleLeu (ARGSVIL-peptide),
which was then identified by means of two monoclonal antibodies
(ELISA module).[22] ARGSVIL-peptide standard curves were run in
parallel.
18%); mp: 195–1978C; H NMR (200 MHz, CDCl3): d=3.50 (s, 3H),
4.92 (s, 2H), 5.04 (s, 2H), 5.09 (s, 2H), 7.06 (m, 2H), 7.29–7.42 (m,
5H), 7.61–7.80 (m, 4H), 8.15 (d, J=7.8, 1H), 9.15 ppm (m, 1H); MS
(ESI): m/z 411 [M+H]+; Anal. calcd for C26H22N2O3·32H2O: C 73.92, H
5.57, N 6.63, found: C 73.84, H 5.30, N 6.27.
2-[4-(Benzyloxy)phenyl]-3-oxoisoindoline-4-carboxylic acid (3a):
A solution of 9 (0.20 g, 0.83 mmol) in EtOH (15 mL) was treated
with K2CO3 (0.13 g, 0.94 mmol) and 4-(benzyloxy)aniline hydrochlo-
ride (0.22 g, 0.93 mmol) and refluxed for 45 min. The solvent was
removed in vacuo and the residue partitioned between H2O and
EtOAc. The organic layer was dried over Na2SO4 and evaporated in
vacuo. Purification of the residue by washing with EtOH and subse-
quent recrystallization (acetone/EtOH) gave 3a as colorless crystals
suitable for crystallographic studies (87 mg, 29%); mp: 180–1818C;
1H NMR (200 MHz, CDCl3): d=4.96 (s, 2H), 5.09 (s, 2H), 7.05 (d, J=
8.5, 2H), 7.35–7.41 (m, 5H), 7.60–7.79 (m, 4H), 8.43 (m, 1H),
15.60 ppm (br s, 1H); MS (ESI): m/z 382 [M+Na]+.
Proteolysis of aggrecan–IGD by aggrecanase: Aggrecan–IGD was in-
cubated with standard recombinant human aggrecanase 1
(ADAMTS-4 amino acids Phe213–Ala579 with a C-terminal tag) or
standard recombinant human aggrecanase 2 (ADAMTS-5 amino
acids Ser262–Gly625 with a C-terminal tag) in the absence (total
enzymatic activity) or presence (modulated enzymatic activity) of
the test compounds. Enzymes were pre-incubated with the inhibi-
tors or the appropriate concentration of vehicle as a control
(DMSO final concentration of 0.25% in proteolysis), at 48C for
30 min prior to assaying their activity. 5 mL of mix enzyme–inhibitor
were added to the substrate (final concentration=0.1 mm) in a
total volume of 100 mL and incubated for 15 min at 378C. The reac-
tion was terminated with EDTA-containing buffer. The inhibitors
were tested at 10 mm final concentration, and compounds achiev-
ing> 50% inhibitory effect were evaluated for IC50 calculation, in a
range generally extended from 0.1 to 10 mm final concentration.
2-[4-(Benzyloxy)phenyl]-N-hydroxy-3-oxoisoindoline-4-carbox-
amide (3b): A solution of 3a (0.030 g, 0.083 mmol) in dry DMF
(5.0 mL) was treated with BOP (0.040 g, 0.090 mmol), Et3N (28 mL),
and O-(tert-butyldimethylsilyl)hydroxylamine (0.015 g, 0.10 mmol)
and stirred overnight at RT. The solvent was evaporated in vacuo,
and the residue was purified by washing with CH2Cl2 and subse-
quent recrystallization (CHCl3/MeOH) by slow evaporation of the
solvents to give 3b as pale yellow crystals (16 mg, 51%); mp: 193–
Aggrecan peptide ELISA: ARGSVIL-peptide standard, proteolytic di-
gestion of aggrecan–IGD with standard aggrecanases and test
samples were incubated in microtiter wells pre-coated with anti-
ARGSVIL-neoepitope antibody. ARGSVIL-peptide was bound to the
coated antibody, while other components were removed by wash-
ing and aspiration. The bound ARGSVIL-peptide was detected with
a second peroxidase-labeled antibody. Any excess of the conjugate
was removed by washing and aspiration. The amount of perox-
idase bound to different wells was determined by reaction with
peroxidase substrate TMB. The reactions were stopped by addition
1
1948C; H NMR (200 MHz, [D6]DMSO): d=5.03 (s, 2H), 5.13 (s, 2H),
7.09 (m, 2H), 7.34–7.42 (m, 5H), 7.70–7.78 (m, 4H), 8.07 (m, 1H),
9.24 (s, 1H), 12.95 ppm (s, 1H); MS (ESI): m/z 397 [M+Na]+.
(Z)-3-(4-Propoxyphenylimino)-1,3-dihydroisobenzofuran-4-car-
boxylic acid (10): A solution of 9 (0.20 g, 0.83 mmol) in EtOH
(15 mL) was treated with K2CO3 (0.13 g, 0.94 mmol) and 4-propoxy-
aniline (0.14 g, 0.93 mmol) and refluxed for 45 min. The solvent
746
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ChemMedChem 2010, 5, 739 – 748

Aggrecanase Inhibitors
ngham, A. J. Fosang, J. Dudhia, Eur. J. Clin. Chem. Clin. Biochem. 1994,
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of sulfuric acid solution and absorbance was read at 450 nm in a
microtiter plate spectrophotometer.
[3] a) J. Martel-Pelletier, C. Boileau, J. P. Pelletier, P. J. Roughley, Best Pract.
Res., Clin. Rheumatol. 2008, 22, 351–384; b) T. Hardingham, Curr. Rheu-
matol. Rep. 2008, 10, 30–36; c) R. J. Lories, Best Pract. Res. Clin. Rheuma-
tol. 2008, 22, 209–220; d) M. Hashimoto, T. Nakasa, T. Hikata, H. Asa-
hara, Med. Res. Rev. 2008, 28, 464–481.
[4] a) Y. Rengel, C. Ospelt, S. Gay, Arthritis Res. Ther. 2007, 9, 221; b) G. S.
Firestein, Curr. Opin. Rheumatol. 1992, 4, 348–354; c) M. W. Lark, E. K.
Bayne, J. Flanagan, C. F. Harper, L. A. Hoerrner, N. I. Hutchinson, T. T.
Singer, S. A. Donatelli, J. R. Weidner, H. R. Williams, R. A. Mumford, L. S.
Lohmander, J. Clin. Invest. 1997, 100, 93–106; d) J. D. Sandy, C. Verscha-
ren, Biochem. J. 2001, 358, 615–626.
[5] a) K. D. Brandt, Am. J. Ther. 2000, 7, 75–90; b) H. R. Schumacher, L. X.
Chen, Am. J. Med. 2005, 118, 1208–1214; c) A. J. Matheson, C. M. Perry,
Drugs Aging 2003, 20, 1041–1060; d) M. Fajardo, P. E. Di Cesare, Drugs
Aging 2005, 22, 141–161; e) G. Singh, Surg. Technol. Int. 2003, 11, 287–
292; f) J. P. Pelletier, Osteoarthritis Cartilage 2004, 12, Suppl. A, S63–68;
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2008, 4, 420–427.
[6] a) C. B. Little, C. T. Meeker, S. B. Golub, K. E. Lawlor, P. J. Farmer, S. M.
Smith, A. J. Fosang, J. Clin. Invest. 2007, 117, 1627–1636; b) J. D. Sandy,
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1512–1516.
Molecular modeling
Inhibitor setup: The structures of the inhibitors used in the docking
simulations were generated by means of Discovery Studio software
(v 1.5), then minimized using a CHARMm force field.[26] Minimiza-
tions were carried out by means of 50 steps of steepest descent
and 10000 steps of conjugated gradient as minimization algo-
rithms, with an RMS convergence criterion of 0.01 ꢁ. Partial atomic
charges were assigned by means of the Gasteiger–Marsili formal-
ism.[27] All the relevant torsion angles were treated as rotatable
during the docking process, thus allowing a search of the confor-
mational space.
Enzymes setup: The ADAMTS-5 protein was set up for docking as
follows: polar hydrogens were added by means of Discovery
Studio software, and Kollman united-atom partial charges were as-
signed; ab initio calculated charges were introduced for the pros-
thetic group.[28] The ADDSOL utility of AutoDock was used to add
solvation parameters to the protein structures, and the grid maps
representing the proteins in the docking process were calculated
using AutoGrid. The grids, one for each atom type in the inhibitor
plus one for the electrostatic interactions, were chosen to be large
enough to include not only the hypothetical catalytic sites but also
a significant part of the protein around it. As a consequence, the
dimensions of grid maps were 68 ꢁ ꢂ 60 ꢁ ꢂ 52 ꢁ with a grid-
point spacing of 0.375 ꢁ for ADAMTS-5 for all docking calculations.
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Acknowledgements
The authors thank Prof. Stefania D’Agata D’Ottavi (Universitꢀ per
Stranieri di Siena, Siena, Italy) for the careful reading of the
manuscript, Dr. Roberto Beretta (Rottapharm, Monza, Italy) for
combustion analyses, the Consorzio Interuniversitario Nazionale
per la Scienza e la Tecnologia dei Materiali (INSTM) for the
access to Accelrys software, and Prof. Maria Cristina Menziani
(Universitꢀ di Modena e Reggio Emilia, Modena, Italy) for helpful
discussions.
Keywords: ADAMTS · aggrecanase · inhibitors · osteoarthritis ·
oxoisoindolines · pyrroloquinolinones
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