Tetrahydroisoquinoline-3-carboxylate based matrix-metalloproteinase inhibitors: Design, synthesis and structure-activity relationship

Article abstract of DOI:10.1016/S0968-0896(02)00215-8

The design, synthesis and structure-activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase (MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy was directed to complement major protein-ligand interaction regions mainly in the S1′ hydrophobic specificity pocket close to the catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates and carboxylates as typical zinc complexing functionalities, with a broad variety of S1′ directed mono- and biaryl substituents consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site. The effect of different S1′ directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S1′ directed substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.

Full text of DOI:10.1016/S0968-0896(02)00215-8

Bioorganic & Medicinal Chemistry 10 (2002) 3529–3544
Tetrahydroisoquinoline-3-carboxylate Based
Matrix-Metalloproteinase Inhibitors: Design, Synthesis and
Structure–Activity Relationship
Hans Matter,* Manfred Schudok*, Wilfried Schwab, Werner Thorwart,
Denis Barbier, Gunter Billen, Burkhard Haase, Bernhard Neises,
Klaus-Ulrich Weithmann and Theo Wollmann
Aventis Pharma Deutschland GmbH, D-65926 Frankfurt am Main, Germany
Received 21 February 2002; accepted 7 June 2002
Abstract—The design, synthesis and structure–activity relationship (SAR) of a series of nonpeptidic 2-arylsulfonyl-1,2,3,4-tetra-
hydro-isoquinoline-3-carboxylates and-hydroxamates as inhibitors of the matrix metalloproteinase human neutrophil collagenase
(MMP-8) is described here. Based on available X-ray structures of MMP-8/inhibitor complexes, our structure-based design strategy
was directed to complement major protein-ligand interaction regions mainly in the S10 hydrophobic specificity pocket close to the
catalytic zinc ion. Here, the rigid 1,2,3,4-tetrahydroisoquinoline scaffold (Tic) provides ideal geometry to combine hydroxamates
and carboxylates as typical zinc complexing functionalities, with a broad variety of S10 directed mono- and biaryl substituents
consisting of aromatic rings perfectly accommodated within this more hydrophobic region of the MMP-8 inhibitor binding site.
The effect of different S10 directed substituents, zinc-complexing groups, chirality and variations of the tetrahydroisoquinoline ring-
system is investigated by systematic studies. X-ray structure analyses in combination with 3D-QSAR studies provided an additional
understanding of key determinants for MMP-8 affinity in this series. The hypothetical binding mode for a typical molecule as basis
for our inhibitor design was found in good agreement with a 1.7 A X-ray structure of this candidate in complex with the catalytic
domain of human MMP-8. After analysis of all systematic variations, 3D-QSAR and X-ray structure analysis, novel S10 directed
substituents were designed and synthesized and biologically evaluated. This finally results in inhibitors, which do not only show
high biological affinity for MMP-8, but also exhibit good oral bioavailability in several animal species.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
Stromelysin (MMP-3), as well as particularly the intest-
inal fibroblast collagenase (MMP-1) and the neutrophil
collagenase (MMP-8) are responsible for the cleavage of
type I-, II- and III-collagen, and therefore regarded as
key enzymes in the pathology of matrix degradation.
Matrix metalloproteinases (MMPs) are a family of zinc
endopeptidases which are capable of degrading the
extracellular matrix of connective tissues.1 They are
broadly implicated in a number of degenerative diseases
in which there is a slow matrix degradation rate,
including cartilage loss in osteoarthritis2 and rheuma-
toid arthritis,3 bone matrix degradation in osteoporosis
or remodeling in Alzheimer disease.4 All the MMPs
possess three discrete domains: a propeptide (ꢀ80 AS)
cleaved during activation, a catalytic domain (ꢀ180
AS) including a conserved HEXXHXXGXXH zinc
binding motif, and a hemopexin-like domain (ꢀ250
AS), presumably responsible for substrate recognition.5
Thus, selective inhibitors targeted against these col-
lagenases have become attractive for rational drug
design. At an early stage, peptidic inhibitors were
designed based on the knowledge of the natural sub-
strate located at the catalytic site of the enzyme. Com-
bined with
a thiol-, phosphonic-, carboxylic- or
hydroxamic-acid group as zinc chelating function, they
achieve in vitro inhibitory activity in the nanomolar
range.6 However, those substrate-orientated inhibitors
show low oral bioavailibility, which might be related to
their peptidic nature. Hence, our design was directed
towards conformationally restricted nonpeptidic inhibi-
tors combining high affinity, selectivity, and improved
pharmacokinetic properties.
*Corresponding authors. Tel.: +49-69-305-84329; fax: +49-69-
e-mail:
331399; e-mail: hans.matter@aventis.com (H. Matter);
manfred.schudok@aventis.com (M. Schudok).
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00215-8

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H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
In this study the design, synthesis, and structure–activ-
ity relationship (SAR) for a novel inhibitor series is
presented. Stimulated by X-ray structures of human
MMP-8 with low molecular weight ligands,7ꢁ11 the rigid
1,2,3,4-tetrahydroisoquinoline scaffold (Tic) was chosen
as central scaffold. Consequently, a series of 2-arylsul-
fonyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates 1a,
hydroxamates 1b and their corresponding isoquinolines
analogues targeting MMP-8 was designed, synthesized
and biologically evaluated (Scheme 1). The systematic
variation of different mono- and biaryl ring systems
directed towards the hydrophobic MMP S10 specificity
pocket,12 zinc-complexing groups, chiralities at the Tic-
C3 carbon and variations at the aromatic moiety of the
tetrahydroisoquinoline scaffold is described in this pub-
lication and led us to establish a detailed SAR prior to a
second design cycle in search for more potent analogues
in this series (cf. Fig. 1).
In order to unravel the underlying SAR for this system,
all molecules were docked into the binding site of
MMP-8, as defined using 3D structures of the MMP-8
Scheme 1. Synthesis of 2-orylsulfonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylates and -hydroxamates.
Figure 1. Schematic view to the MMP-8 binding site with key residues indicated. The bicyclic 1,2,3,4-tetrahydroisoquinoline scaffold provides ideal
geometry to link S10 directed hydrophobic substituents with zinc complexing groups. Hydrogen bond acceptors are favourable at the S10 entrance,
while the aromatic 1,2,3,4-tetrahydroisoquinoline ring is situated close to a hydrophobic region.

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3531
catalytic domain with different peptides.9,13 This led to a
binding hypothesis for all compounds from this series
by superimposing them onto the template and minimiz-
ing them within the MMP-8 binding site. The resulting
superpositions produced consistent 3D-QSAR models
using CoMFA and related methods,14ꢁ18 allowing to
rationalize ligand–enzyme interactions by deriving a
significant and predictive relationship between mole-
cular property fields and biological activities. Details of
the 3D-QSAR-studies have been published else-
where,19,20 while the qualitative structure–activity rela-
tionship reported in this contribution in combination
with those results plus X-ray data19 formed the basis for
further structure-based design of potent, bioavailable
candidates in a second iterative design cycle. This finally
led to the discovery of potent and bioavailable inhibitors
targeted against MMP-8 presented here.
N2 are added to complementary interactions at the S10
entrance. Furthermore this linkage allows for many
synthetic variations to explore S10 pocket binding
requirements (Fig. 3C) Hydroxamates and carboxylates
were chosen as zinc complexing groups. Although car-
boxylates do not exhibit optimal zinc binding proper-
ties, it has been demonstrated earlier that they relate to
higher bioavailability.23,24 Thus is was considered by
us19,20 and others23ꢁ26 to compensate any expected loss
in binding affinity by adding substituents with optimal
complementarity to the S10 pocket.
The aromatic sidechains of Tyr219 and His197 at
opposite sides of the S10 wall here suggest that favour-
able contribution to binding energy could result from
protein–ligand aryl–aryl interactions within the S10
pocket.27 Thus, aromatic residues (monoaryl, biaryl and
biarylether substituents) were incorporated, which, at
the same time, are replacing crystallographically con-
served water molecules.
Structure-Based Design of MMP-8 Inhibitors
All inhibitors were designed based on active site geo-
metries from public domain MMP-8 X-ray stuctures (cf.
Figs 2 and 3). The specificity of collagenases to cleave at
medium-sized hydrophobic residues is tightly connected
to size, electrostatic and hydrophobic properties of the
S10 specificity pocket close to the catalytic Zn2+ ion.
This pocket’s wall is formed by Pro217-Asn218-
Tyr219,21 while its entrance is built by Gly158 to Ala161
and the aromatic sidechain from Tyr219, opening into a
hydrophobic interior. The S10 bottom is occupied by the
polar Arg222, its side chain being flexible, adopting dif-
ferent conformations in different X-ray structures. The
terminal guanidine is hydrogen bonded to the carbonyl
groups of Pro211, Gly212 (via solvent) and Ala213. In
most X-ray structures the S10 pocket is filled with two or
three solvent molecules interacting with the Arg222
guanidine and other polar backbone atoms.
The postulated binding mode for inhibitor design was
later validated using an 1.7 A X-ray structure analysis
(
xix) of the MMP-8/62 complex with a biphenyl sub-
stituent directed towards S10. The experimental binding
mode of compound 62 is shown in Figure 2D (green
carbons) compared to 10 best docking modes (white
carbons) obtained using the program FlexiDock.28 Both
oxygens of the 3-carboxyl group are in coordination
with the catalytic zinc, one sulfonamide oxygen is
exposed to the solvent, while the other is hydrogen
bonded to Leu160-NH and Ala161-NH. The biphenyl
inside S10 is stacked on top of the His197 imidazole, and
the loop from Leu214 to Ala220 wraps around the
inhibitor. As obvious the experimental binding mode is
similar to our template for design. The 3D-QSAR
analyses are discussed elsewhere,19,20 their results in
combination with X-ray structure investigations then
led to a successful second design cycle (see below).
Several hydrophobic and hydrogen-bonding regions in the
MMP-8 binding site (Figs 2 and 3) were identified using
GRID with appropriate probe atoms22 and by visual
inspection of protein-ligand complexes. It was found that a
rigid 1,2,3,4-tetrahydroisoquinoline provides ideal geo-
metry to connect zinc binding groups with S10 directed
substituents. Its heterocyclic ring is situated at the
entrance to the active site between Ile159 and His207,
with appropriate bond vectors at C3 and N2 pointing
towards the catalytic zinc and the S10 pocket, respec-
tively. The hydrophobic aromatic ring is close to a
hydrophobic region identified using GRID (dry probe)
at the surface of the cavity (Fig. 2B and C) comprising of
the plane of the His207 imidazole ring, and, for example,
being occupied by an thiophene in the inhibitor present
in the PDB structure 1mmb.10 When chirality is inverted
at C3, the biological activity decreases, as this favour-
able hydrophobic interaction is not longer possible.19
Chemistry
The target series29 of 2-arylsulfonyl-1,2,3,4-tetrahydro-
isoquinoline-3-carboxylates 1a and -hydroxamates 1b
were synthesized according to Scheme 1. Biaryl-sub-
stituents were selected according to the design strategy
and taking synthetic availability as additional con-
straints into account. This reaction sequence was
applied for the majoritiy of candidate molecules. Most
syntheses proceeded either under standard or slightly
modified conditions, as reported below.
Sulfonyl chlorides were commercially available or pre-
pared by treatment of the corresponding sulfonic acids
or their salts with chlorination agents (PCl5, POCl3 or
SOCl2) under standard procedures. Alternatively, an
aromatic ring system was sulfochorinated in para-posi-
tion using chlorosulfonic acid. The synthesis of the
chlorobiphenylethyl-sulfonylchloride building block as
an example for an alkyl sulfonamide resulting from the
second design cycle and is outlined in Scheme 2. The
Hydrogen bonds to ligands involving the amide protons
of Leu160 and Ala161 at the S10 entrance are to be
found in these X-ray structures.7ꢁ11 In our design, the
sulfonyl-oxygen atoms of the sulfonamide attached to
the rigid tetrahydroisoquinoline scaffold via nitrogen

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H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
formation of tetrahydroisoquinoline-sulfonamides 1a
was done using standard Schotten–Baumann condi-
tions. Preferred solvents were THF or THF/DMF-mix-
tures, NaOH or organic bases like DIPEA were applied.
The conversion to the corresponding hydroxamates 1b
was achieved by activation of the acids as mixed anhy-
dride (isobutyl or ethyl chloroformate and NMM or
NEM) or acid chloride (oxalyl chloride) and reaction
Figure 2. (A) Human neutrophil collagenase (MMP-8) fold from a 1.7 A X-ray structure of the MMP-8/62 complex,19 indicated by a magenta
ribbon-tube encoding secondary structure elements, while compound 62 is displayed with green carbons. The MMP-8 inhibitor binding site is
highlighted by a GRID22 interaction energy contour map based on a methyl probe, drawn at +1 kcal/mol. Depth cueing is used to allow for a view
into S10, where the biphenyl substituent is located. Similar models were obtained for public domain inhibitor complexes, serving as basis for our
design. (B) Detailed MMP-8 binding site containing compound 62 with green carbons in comparison to public domain inhibitors from PDB files
1jan, 1jao, 1jap, 1jaq, 1mnc, 1kbc, 1mmb,13 shown with grey carbons. Magenta carbons indicate selected protein residues from the above X-ray
structure. Grey spheres indicate structurally conserved zinc ions. The MMP-8 binding site is highlighted by a GRID interaction energy contour (+1
kcal/mol) using a methyl probe, while hydrophobic regions are indicated by orange contours at ꢁ0.3 kcal/mol. (C) Detailed MMP-8 binding site
containing compound 62 with green carbons. Regions with favourable hydrophobic interactions are indicated with orange contours at ꢁ0.3 kcal/
mol from GRID interaction energies using a hydrophobic (DRY) probe. (D) 1.7 A X-ray structure19 of the MMP-8/62 complex (green carbons)
compared to 10 best docking modes using FlexiDock (white carbons) after manual placement of a template in the MMP-8 cavity. Similar docking
modes were used as basis for inhibitor design.

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3533
with O-trimethylsilyl hydroxylamine in THF, followed
by treatment with aqueous HCl for deprotection.
structure of the MMP-8/62 complex,19,32 (b) 3D-QSAR
for 90 analogues19 and (c) 2-D chemical substitutions. It
outlines the rational for designing MMP inhibitors with
improved selectivity and pharmacokinetic properties in
a second design cyclus.
Modified 3-Tic (tetrahydroisoquinoline-3-carboxylic
acid) derivatives and hetero-analogues were prepared by
standard Pictet–Spengler cyclization.30 Additionally, the
6- or 7-nitro and amino-3-Tic derivates were synthesized
by a new method,31 outlined in Scheme 3.
The tetrahydroisoquinoline ring of 62 is located at the
entrance to the active site between Ile159 and His207
with its aromatic part exposed to the solvent. This
position was modified during the chemical optimization
to improve affinity and physicochemical properties. No
significant affinity change was observed upon substitu-
tion of the solvent exposed tetrahydroisochinoline moi-
ety, listed as IC50 value for MMP-8 in nM. This is
obvious from comparison of the compound 21 (4 nM)
to 30 with an amino substituent at R3 (2 nM). Further-
more the unsubstituted 49 with an 4-Cl-biphenylsulfo-
nyl group directed towards S10 is equally active (5 nM) to
the corresponding 7-amino derivative 60 at R4 (7 nM).
Starting from commercially available 1,2,3,4-tetra-
hydroisoquinoline-3-carboxylic acid, the nitration with
KNO3/H2SO4 was found to be the best method with
respect to regioselectivity and purity. Under strict tem-
perature control, no di-nitration products could be
observed, while the excess for the 7-nitration product
30a is 9-fold. For the synthesis of regiopure compounds,
three different ways were used: (a) flash chromato-
graphy on silica gel of the carboxylic acid sulfonamides
(nitro-stage). However, this was tedious because of the
chemical similarity of both isomers. (b) The 2-Boc-6-/7-
nitro-Tic could be transformed to the DCHA-salt,
which easily could be recrystallized resulting in the pure
7-isomer 30c. (c) Reduction of the nitro group to the
amine. Here, the 6-isomer is slighly better soluble in hot
acetonitrile. As expected, the chemistry of the nitro-Tic
derivatives was similar to unsubstituted analogues. All
compounds in Table 1 were prepared in analogy, com-
pound IDs correspond to ref 19.
One of the sulfonamide oxygens is solvent exposed,
while the other is involved in hydrogen bonds to
Leu160-NH and Ala161-NH and thus essential to
maintain biological activity in this series. The biphenyl
system attached to this sulfonamide is deeply buried
inside the S10 pocket, stacked on top of the His197 imi-
dazole ring. At the other side, the loop between Leu214
and Ala220 wraps around the inhibitor. This experi-
mental binding mode is similar to that used as template
for defining the alignment rule for 3D-QSAR. It was
also shown to be consistent with the SAR of this series
according to docking studies.
Structure–Activity Relationship
The discussion of the structure–activity relationship for
2-arylsulfonyl-1,2,3,4-tetrahydro-isoquinolines (Scheme
1, Table 1) is based on a combined analysis of (a) X-ray
Introducing bulky substituents at the bottom of the
MMP-8 S10 pocket increases biological activity, as
Figure 3. (A) Postulated binding mode of compound 96 within MMP-8 binding site. Only selected amino acid residues are shown. (B) Observed
19
versus predicted MMP-8 affinities for utilized CoMFA model
derive this model are indicated by diamonds, while predictions for five new candidates are indicated by crosses.
with crossvalidated r2 value of 0.569 for five components. Compounds used to

3534
H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
exemplified by comparing the following hydroxamates.
While the parent compound 9 with phenyl substitution
at R2 directed towards the S10 pocket shows an activity
of 20 nM, a 4-methoxy substituent at this phenyl
increases activity (21, 4 nM). Increasing the size of this
substituent by replacing 4-methoxy by 4-phenoxy (8, 2
nM) or 4-(4-dimethylamino)-phenoxy (22, 2 nM) also
improved MMP-8 affinity. Replacing the biarylether
moiety by biphenyl substituents attached to the sulfo-
namide at R1 does not affect biological activity, as
exemplified in compounds 23 with 4-biphenyl (2 nM)
and 50 with 4-(4-chloro)-biphenyl (3 nM). This corre-
sponds to carboxylates with otherwise unchanged sub-
stituents at the central scaffold. However, here the
parent compound carrying a 4-methoxy substitution at
the S10 directed phenyl ring shows moderate activity
(29, 7000 nM) compared to 4-phenoxy or 4-(4-dimethy-
lamino)-phenoxy substitutions (52, 9 nM and 63, 1 nM),
respectively. Again the replacement of biarylethers
against biphenyls does not significantly affect biological
activity, as obvious from compound 62 with 4-biphenyl
(10 nM). However, for carboxylates, the effect of steric
bulk in the S10 pocket is more pronounced, as small
modifications in that hydrophobic area compensate for
the loss of binding affinity in the parent compounds
with 4-methoxyphenyl substitution at R2 (21, 4 nM as
hydroxamate versus 29, 7000 nM as carboxylate), not
optimally accomodating the hydrophobic environment
in the lower part of the S10 pocket in MMP-8. If a sec-
ond aromatic ring is introduced, differences between
zinc binding groups decrease. This observation is in
agreement to X-ray structural data and 3D-QSAR, both
highlighting a hydrophobic cleft in S10 formed by the
MMP-8 sidechains of Tyr219, Leu193 and Val194. This
Scheme 2. Synthesis of the chlorobiphenylethyl-sulfonylchloride building block.
Scheme 3. Synthesis of nitro and amino-3-Tic derivatives.

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3535
area is filled with structurally conserved water in several
MMP-8–inhibitor complexes, if not replaced by hydro-
phobic inhibitor parts.
that inhibitors with weaker zinc binding groups require
a compensation by an optimal fit of hydrophobic sub-
stituents into the S10 pocket.25,26
From X-ray structural data and 3D-QSAR, the pre-
ferred zinc binding geometry is highlighed, as the opti-
mal distance of two oxygens for zinc coordination is
better realized in hydroxamates (2.7 A) than in carbox-
ylates (2.2 A). The X-ray structure of 62 showed both
carboxylate oxygens interacting with the catalytic zinc
ion, while several other X-ray stuctures of different zinc-
binding motifs for matrix metalloproteinases support
the bidental interaction of these zinc binding motifs.
These differences in favourable protein–ligand interac-
tions are exemplified by comparison of compound 29
(carboxylate, 7000 nM) to 21 (hydroxamate, 4 nM) with
4-methoxybenzenesulfonyl substituents, 52 (carboxylate
9 nM) to 8 (hydroxamate, 2 nM) with 4-phenox-
ybenzenesulfonyl substituents and 49 (carboxylate, 5
The analysis of the 3D-QSAR model at the first biphe-
nylether ring indicates a close proximity of low-affinity
ligands to Pro217, His207, Zn2+, His197 or Val194,
which correspond to the narrow, polar S10 entrance.
Hence, any substitution at this aromatic ring directly
attached to the sulfonamide should reveal a certain
shape in order not to undergo unfavourable interactions
with those residues flanking this narrow S10 entrance.
At the edge of this S10 pocket unfavourable steric inter-
actions at the Asn218-Tyr219 peptide backbone are also
identified, which is causing rigid S10 directed substit-
uents to have less favourable MMP-8 affinity, as observed,
for example, for 56 with an IC50 value of 500 nM.
The presence of the Arg222 side chain at the bottom of
S10 impose restrictions on the tolerable size of ligands,
as it cannot freely rotate. This size-related effect is
exemplified in 2-arylsulfonyl-1,2,3,4-tetrahydro-iso-
quinoline-3-carboxylates with different phenoxyphenyl
substituents at R2 directed towards S10. While com-
pound 52 without substitution at this biarylether moiety
shows an IC50 value of 9 nM against MMP-8, any
additional substitution causes the affinity to decrease: 68
(4-chloro substitution: 20 nM), 81 (4-dimethylamino sub-
stitution: 30 nM), and 85 (4-(methylamido) substitution:
40 nM).
nM) to 50 (hydroxamate
3 nM) with 4-chlor-
obiphenylsulfonyl substituents. Again, it is obvious that
reduced binding affinity due to replacement of hydro-
xamic acids by carboxylic acids can be compensated by
S10-directed substituents with optimal protein–ligand
interactions. Such a sterically favourable substitution
optimally interacting with the S10 pocket can lead to
carboxylate-based inhibitors with comparable binding
affinity to hydroxamates. This has important implica-
tions for inhibitor design, as carboxylates are known to
exhibit higher oral bioavailability.23,24 We showed here
Table 1. Selected 2-arylsulfonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylates and hydroxamates as inhibitors of the matrix metalloproteinase
MMP-8 to derive a structure–activity relationship
No.a
Isomer
R1
R2
R3
R4
MMP-8
IC50 (nMol/l)
8
9
R
R
S
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
NH–OH
OH
NH–OH
NH–OH
OH
4-Phenoxybenzene
Benzene
4-Methoxybenzene
H
H
H
H
H
H
H
NH2
H
NH2
H
H
H
H
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2
20
1000
4
20
21
22
23
29
30
36
48
49
50
52
56
60
62
63
67
68
80
81
85
89
R
R
R
R
R
R
R
R
R
R
R
R
R
R
S
4-Methoxybenzene
4-(4-Dimethylaminophenoxy)-phenyl
4-Biphenyl
4-Methoxybenzene
2
2
7000
2
100
2000
5
3
9
500
7
10
1
3000
20
90
30
40
10
4-Methoxybenzene
4-Morpholinobenzene
4-Methoxybenzene
4-Chlorobiphenyl
4-Chlorobiphenyl
4-Phenoxybenzene
OH
NH–OH
OH
OH
OH
OH
OH
OH
OH
OH
2-Dibenzofuran
4-Chlorobiphenyl
4-Biphenyl
4-(4-Dimethylaminophenyl)-benzene
4-Chlorobiphenyl
4-(4-Chlorophenoxy)-benzene
2-(2H-[1,2,3]triazole)
4-(4-Dimethylamino)-phenoxybenzene
4-(N-Methyl-4-phenoxy-benzamide)
4-(4-Trifluoromethyl)-biphenyl
R
R
R
R
R
OH
OH
COOH
H
H
H
aThe compound ID numbering refers to ref 19.

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H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
If the distal ring in S10 directed biaryl-substituents is
polar or sterically demanding, the biological activity
also decreases. This is exemplified by compound 80 with
a distale triazole ring system (90 nM) and compound 36
with a morpholine system (100 nM). This latter finding
is even more surprising, as the parent Tic scaffold car-
ries a hydroxamate in 36. In contrast, this detrimental
effect for affinity is not so pronounced when changing
the para substitution in the distal aromatic ring. The
polar 4-dimethylamino-biphenyl-derivative 63 shows
very good activity due to additional favourable interac-
tions of the basic group, whereas a lipophilic CF3 (89,
10 nM) is disfavoured compared to the sterically less
demanding 5-chloro derivative 49 (5 nM).
conducted for selected compounds. The pharmacokinetic
profiles discussed below result from analysis of rabbit
blood plasma levels after oral administration of the cor-
responding inhibitor. The maximum drug concentration
in plasma (Cmax), the halflife (t1/2) and area under the
curve (AUC) from those studies were compared to iden-
tify the optimal candidate. Relevant pharmacokinetic
parameters for selected compounds after the first design
cycle plus newly designed analogues from the second
cycle are summarized in Table 2. The following section
provides a discussion of structural elements which are
correlated with oral bioavailability in this series, based
on a comparative analysis of data from Table 2.
In order to arrive at inhibitors with acceptable bioa-
vailability, the use of 1,2,3,4-tetrahydro-isoquinoline-3-
carboxylates was seen as prerequisite, while a compen-
sation for the loss of MMP-8 binding affinity was
achieved by appropriate substituents within the S10
pocket. Although the parent compound from the
1,2,3,4-tetrahydroisoquinoline-3-carboxylate series 29
with 4-methoxybenzene sulfonyl substitution in S10
shows low MMP-8 activity, excellent oral bioavail-
ability indicated by maximum plasma levels (Cmax) of
188 mg/mL from rabbit plasma after oral administration
of 100 mg/kg was detected. On the other hand, the cor-
responding hydroxamic acid derivative 21 is a potent
MMP-8 inhibitor without significant oral bioavail-
ability: at a dose of 100 mg/kg maximal plasma levels
(Cmax) of only 2.3 mg/mL were detected. Please note
that only for these compounds, the corresponding stud-
ies were conducted with doses of 100 mg/kg, while for all
other candidates below, this dose was reduced to 15 mg/
kg (cf. Table 2). Those unfavourable pharmacokinetic
properties, apparently associated with rapid metabolism
and excretion of 21, prevented this compound from
further evaluation. Similar observations were made for
related hydroxamic acid derivatives (no data given).
This prompted us to search for corresponding carbox-
ylates with increased enzyme activity, while maintaining
their favourable pharmacokinetic properties.
Finally, the preferred stereochemistry of the 1,2,3,4-tet-
rahydroisoquinoline ring is R, while chirality inversion
lead to a loss of binding affinity. This inversion of chir-
ality is causing the Tic aromatic ring to move away from
a favourable region for hydrophobic protein–ligand
interactions. This location at the MMP-8 protein sur-
face agrees perfectly to favourable interactions of a
hydrophobic probe atom to the binding cavity identified
using the program GRID.22 This affinity difference
might be caused by hydrophobic interactions to the plane
of the His207 imidazole. While, for example, the 4-chlor-
obiphenylsulfonyl-substituted Tic derivative 49 shows an
IC50 value of 5 nM in (R)-configuration, its enantiomer
67 is much less active (3000 nM, S-configuration).
This combined analysis leads to a detailed under-
standing of relevant protein–ligand interactions for fur-
ther ligand design. In addition, the availability of a
validated and predictive 3D-QSAR model was the basis
for rank-ordering novel synthetic candidates. These 3D-
QSAR results19 based on CoMFA and CoMSIA ana-
lyses allow to focus on those regions, where steric, elec-
tronic or hydrophobic effects play a dominant role in
ligand–receptor interactions.
Pharmacokinetic Studies
Enlargement of the S10-directed substituent resulted in
MMP-8 activity enhancement for 1,2,3,4-tetrahydro-
isoquinoline-3-carboxylates. In addition, oral bioavail-
To obtain information on oral bioavailability, relevant
in vivo pharmacokinetic (PK) studies in rabbits were
Table 2. Additional data on enzyme selectivity and PK-properties of some of the most important compounds during the optimization cycles; PK-
data are based on rabbit studies at 15 mg/kg po dosing (details are given in the Experimental)
Example
IC50
MMP-1
(nM)
IC50
MMP-3
(nM)
IC50
MMP-13
(nM)
Cmax
(mg/mL)
AUC
(mg/mLꢂh)
Remarks
21
29
49
52
68
81
94
95
96
nd
nd
7000
600
2000
500
>10,000
>10,000
>10,000
nd
nd
nd
nd
40
50
40
nd
100
10
2.3a
188a
nd
4.7
15.5
0.3
1.9
1.6
29
2.3a
nda
nd
49.9
267
nd
nd
nd
887
Unfavourable properties
Lack of MMP-8-activity
Study cancelled
100
200
500
600
30
Selectivity versus MMP-1 not sufficient
Unfavourable elimination profile
Selectivity and PK-data not sufficient
Cmax not sufficient
13
20
Cmax not sufficient
Best compound
20
nd, not determined.
aAt 100 mg/kg.

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3537
ability could be improved after introduction of ade-
quate ring systems within the S10 pocket, for example
the biphenyl ether substituent. The representative car-
boxylate 52 with this S10 directed substitution exhibited
a Cmax value of 4.7 mg/mL at a dose of 15 mg/kg in
rabbits. However, this compound lacks sufficient selec-
tivity with respect to the undesirable metalloproteinase
MMP-1. Modifications at the distal phenyl ring in para
position was found to enhance selectivity by a factor of
4 for the 4-chlorine derivative 68 with a 3-carboxylate
attached to the central scaffold, while in general activity
against all relevant MMPs (MMP-3, -8, -13) decreases.
Nevertheless, to support the design rational, pharmaco-
kinetic studies were conducted for this lipophilic com-
pound, resulting in high maximum plasma levels with a
Cmax value of 15.5 mg/mL at a dose of 15 mg/kg in
rabbits. However, a more careful analysis revealed a
non-reproducible and unfavourable elimination profile,
which prevented us from additional studies on 68.
Further variations of substituents at the distal phenyl
ring in biphenyl-ether S10 directed substituents did not
further improve the pharmacokinetic profile. Although
the more polar 4-dimethylaminophenyl substituent in 81
lead to increased water solubility, it shows inacceptable
enzyme activity and selectivity. The pharmacokinetic
Table 3. 2-Arylsulfonyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylate and-hydroxamate MMP-8 inhibitors after second design cycle
No.
91
R2
Isomer
R
1H NMR (DMSO-d6, 300 MHz)
MS
(M+H)
MMP-8 MMP-3
IC 50 (nM) IC 50 (nM)
2.9–3.6 (4 m, 6H, CH2);
4.6 (dd, 2H, CH2;
4.8 (m, 1H, CH);
6.9 (dd, 4H, ar); 7.1–7.4
(m, 11H, ar)
472.6
3000
7000
500
2.9–3.5 (3 m, 6H, CH2);
4.32 (m, 1H, CH);
4.5; 4.72 (2 d, 2H, CH2);
7.0 (dd, 4H, ar);
92
93
R
R
438.2
2000
7.1 (‘s’, 4H, ar);
7.35 (2 d, 4H, ar)
2.9–3.6 (4 m, 6H, CH2);
4.65 (dd, 2H, CH2; 4.75 (m, 1H, CH);
6.9 (d, 2H, ar); 7.0 (m, 2H, ar)
7.2 (m, 8H, ar)
456.3
438.2
2000
300
3.0–3.6 (m, 6H, CH2);
4.6 (m, 3H, CH, CH2);
6.83 (d, 2H, ar);
7.15 (‘s’, 4H, ar);
7.45 (d, 2H, ar);
7.47 (dd, 4H, ar);
9.5 (s, br, 1H, OH)
40
30
94
95
R
3.0–3.6 (m, 9H, CH2, CH3);
3.8 (m, 2H, CH2);
4.11 (m, 2H, CH2);
4.6 (m, 3H, CH, CH2);
6.01 (d, 2H, ar);
R
496.2
30
10
7.15 (‘s’, 4H, ar);
7.30 (d, 2H, ar);
7.52 (dd, 4H, ar
3.03 (m, 2H, CH2);
3.17 (‘d’, 2H, CH2);
3.52 (m, 2H, CH2);
4.66 (dd, 2H, CH2);
4.83 (m, 1H, CH);
96
R
494.1
(+ FAB/K)
10
20
7.20 (m, 4H, ar);
7.35; 7.45; 7.58; 7.66 (4 d, 8H, ar)

3538
H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
parameters of this less lipophilic compound also did not
fulfill the desired profile: A Cmax value of only 0.3 mg/
mL was observed in rabbits after oral administration of
a dose of 15 mg/kg. The evaluation of corresponding
hydroxamic acids led to similar conclusions as described
above for the analogues 21/29. As representative mole-
cules of other subseries also showed insufficient phar-
macokinetic properties, a new design cycle was initiated
to retain MMP-8 activity and selectivity, while improv-
ing pharmacokinetic properties of the candidate mole-
cules.
bed.19 The chemical structures, analytical data and cor-
responding IC50-values are summarized in Table 3.
From this design cycle, compound 96 with a 2-(40-
chloro-biphenyl-4-yl)-ethanesulfonamide side chain
attached to the 1,2,3,4-tetrahydroisoquinoline-3-car-
boxylate scaffold has highest MMP-8 affinity (IC50 10
nM, cf. Table 3). The postulated binding mode for 96
after docking into MMP-8 is displayed in Figure 3A.
When comparing the experimentally determined bind-
ing mode of compound 62 with only a biaryl substituent
to the postulated binding mode of this analogue, a slight
movement of Arg222 at the bottom of the S10 pocket
has to be assumed in order to accommodate the larger
substituent in this specificity pocket. This sidechain
flexibility is reproduced by the employed docking pro-
tocol outlined below and also observed upon compara-
tive analysis of in-house and public domain MMP-8 X-
ray structures. The binding mode of 96 is characterized
by a location of the hydrophobic portion of the 2-(40-
Novel MMP-8 Inhibitors from Second Design Cycle
These previously outlined SAR results were applied to
design novel synthesis candidates based on the 1,2,3,4-
tetrahydroisoquinoline-3-carboxylate scaffold with high
affinity and improved oral bioavailability (Table 3). To
take advantage of favourable hydrophobic interactions at
the bottom of the S10 specificity pocket for MMP-8 affi-
nity, biaryl- and biarylether substituents were oriented
deeper within this pocket by introducing an ethyl spacer
between the aromatic moiety and the sulfonamide at the
1,2,3,4-tetrahydro-isoquinoline-3-carboxylate scaffold.
Thus, a variety of substituted 2-(biphenyl-4-yl)-ethane-
sulfonamides were designed, evaluated by flexible dock-
ing into the MMP-8 binding pocket and CoMFA based
affinity predictions. The most promising candidate mole-
cules were synthesized and subjected to further profiling.
chloro-biphenyl-4-yl)-ethanesulfonamide
substituent
deeper in the S10 pocket, while no unfavourable inter-
actions close to the narrow entrance into the S10 pocket
were observed. Hence, this derivative presents another
example for the observation that a loss of favourable
zinc binding affinity in hydroxamates can be compen-
sated by adequate substituents in this S10 specificity
pocket.
Additional biological and pharmacological profiling
ultimatively reveals compound 96 as best member from
this series. This compound is not only a potent MMP-8
inhibitor, it also shows a high predicted human absorp-
tion (>95%, as estimated from Caco-2 cell permeability
assays)34 metabolic stability in S9 liver fractions in ade-
quate in vitro assays. No significant inhibition on a
variety of cytochrome P 450 isoforms was detected in
additional in vitro profiling assays.35 Moreover, 96
shows favourable in vivo pharmacokinetic properties, as
found in corresponding animal experiments. In rats
(male Wistar rats) a maximal plasma concentration
Cmax36 after oral administration of 21 mg/mL was
obtained with a half-life of 4 h after 15 mg/kg single oral
dosing. In rabbits the observed Cmax value was 29 mg/
mL, with a halflife of 29 h after administration of a
For ranking of virtual synthesis candidates, their MMP-
8 affinities were predicted after docking using the pre-
viously reported CoMFA model19 with 2 A grid spacing
based on 90 MMP-8 inhibitors, showing a cross-
validated r2 value of 0.569 for five PLS components and
a conventional r2 of 0.905. To demonstrate the ranking
of compounds by this model, a graph of observed versus
predicted biological activities33 is shown in Figure 3B.
Compounds included in the training set are indicated by
dots, while a priori affinity predictions for five newly
synthesized candidates with this ethyl spacer described
below are indicated by crosses. All compounds were
evaluated for inhibitory activities on MMP-8 and
MMP-3 using an amidolytic assay previously descri-
Figure 4. Mean concentration–time curve (mg/mL vs h) showing the plasma elimination profile of compound 96. Administration of a single oral
dose of 6 mg/kg in healthy beagle dogs (n=2; vehicle: 1% CMC, 0.05% DONSS) gave a maximal plasma concentration Cmax of 17 mg/mL, with a
half life of 7 h.

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3539
single oral dose of 15 mg/kg. In all studies, 1% CMC
and 0.05% DONSS36 were used as vehicle. Monitoring
was done by RP-HPLC analysis of plasma samples
taken at distinct time points. Absolute bioavailabilities
in both animal species after analysis of the correspond-
ing iv experiments were determined to be 53% (rat) and
64% (rabbit). These promising pharmacokinetic prop-
erties of 96 were underlined by conducting in vivo
studies in beagle dogs. Here, a Cmax value of 17 mg/mL,
with a half-life of 7 h is obtained after administration of
a single oral dose of 6 mg/kg (same vehicle as above),
resulting in an absolute oral bioavailability of 100%
after analysis of corresponding iv experiments. The
pharmacokinetic profile after oral administration in
dogs is shown in Figure 4 as time versus concentration
graph. The maximum drug concentration in plasma
(Cmax) is reached at about 1 h after single oral dosing.
The graphical inspection of Figure 4 also suggests a
favourable elimination profile with similar character-
istics in all animal species.
expected loss of binding affinity after replacement of
hydroxamates against carboxylates by adequate choice
of elongated S10 directed substituents. The novel MMP-
8 inhibitors carrying substituted 2-(biphenyl-4-yl)-etha-
nesulfonamides attached to the 1,2,3,4-tetrahydro-iso-
quinoline scaffold did not only result in highly active
compounds, but also in compounds with improved
pharmacokinetic properties. In particular, compound 96
shows an interesting in-vitro profile and also exhibits
significant oral bioavailability in three animal species.
Hence, a novel class of orally available MMP-inhibitors
emerged from this study. This iterative procedure
towards the discovery of novel MMP-8 inhibitors with
high affinity and good pharmacokinetic properties was
only possible after a successful combination of medic-
inal chemistry, structure-based design, reliable affinity
predictions using 3D-QSAR models and pharmacoki-
netic profiling. It is expected that additional new inhi-
bitor classes directed towards MMP-8 may emergy from
the combined application of these techniques for ther-
apy of several MMP-related diseases.
The comparison of 96 to 94 and 95 suggests that the
lipophilic S10 directed substituent carrying a 4-chlorine
substituent is responsible for the acceptable pharmaco-
kinetic profile. Replacing this 4-Cl by a more polar
phenolic hydroxyl group in 94 or a methoxy–ethoxy
substituent in 95 still resulted in acceptable MMP-8
affinity, while lower plasma levels (Cmax values 1.9 mg/
mL for 94 and 1.6 mg/mL for 95) and shorter half-life
were observed.
Experimental
Enantiomerically pure 1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid is commercially available (e.g., Nutras-
weet company). Sulfonic acid chlorides are commer-
cially available or were prepared by literature
1
procedures. H NMR spectra were recorded at room
temperature on a 200 MHz spectrometer from Varian or
a 400 MHz spectrometer from Bruker with tetra-
methylsilane (TMS) as internal standard in DMSO-d6.
Final products were characterized by NMR and mass
spectroscopic methods (FAB-, ESI-MS). Temperature
data in degrees Celsius, rt means room temperature (22–
25 ꢃC). Melting points were determined with a Buchi
capillary melting-point apparatus, and they are uncor-
rected. All abbreviations are explained or correspond to
conventions.
These pharmacokinetic data of 96 let us to consider this
compound as a promising candidate for further biolo-
gical profiling towards the treatment of MMP-related
diseases. Hence, the modification of an unwanted che-
mical functionality, which nevertheless is required for
high binding affinity to the binding cavity of MMP-8,
against a more tolerable but weaker substrate was
achieved by a combination of structure-based design, 3-
D-QSAR-studies and pharmacokinetic profiling.
Synthesis
Conclusion
2-(4-Chlorobiphenylethanesulfonyl)-1,2,3,4-tetrahydro-
isoquinoline-3-carboxylic acid (96) (cf. Scheme 2). 1-(2-
Bromoethenone)-4-(4-chlorophenyl)-benzene 96a. To a
stirred suspension of AlCl3 (34.7 g, 0.26 mol) and bro-
moacetyl bromide (25.2 g, 0.125 mol) in 400 mL CS2 4-
chlorobiphenyl (23.6 g, 0.125 mol) was gradually added
at 0ꢃ C and then heated at reflux temperature for 3 h.
The reaction mixture was slowly added to crushed ice,
extracted with EtOAc and washed with a saturated
aqueous NaHCO3 solution and water. The organics
were dried over anhydrous sodium sulfate and con-
centrated under reduced pressure. The residue was
recrystallized from dichloromethane. Yield: 24.2 g (62%
The systematic evaluation of nonpeptidic 2-arylsulfo-
nyl-1,2,3,4-tetrahydro-isoquinoline-3-carboxylates and-
hydroxamates as inhibitors of the matrix metalloprotei-
nase MMP-8 led us to establish their structure–activity
relationship. In combination with a high-resolution
X-ray structure for one derivative plus a predictive 3D-
QSAR model to explain affinity towards MMP-8, a
better understanding of relevant protein–ligand interac-
tions responsible for high affinity ligands was generated.
This knowledge provided essential information for a
subsequent structure-based design cycle to arrive at
inhibitors with an improved pharmacokinetic profile
and high activity against the target enzyme MMP-8. In
this process the 3D-QSAR model was successfully
applied to rank-order candidates during lead optimiza-
tion. Although carboxylates exhibit weaker zinc binding
properties than hydroxamates, they are known to show
better oral bioavailability and are less prone to meta-
bolic degradation. It was possible to compensate the
of theory). Mp: 127–128 ꢃC; H NMR: (300 MHz) 5.0
1
(s, 2H, CH2); 7.5–8.1 (4 d, 8H, ar); MS: 311.1
(M+H).
4-Chlorobiphenylethane bromide 96b. To a stirred sus-
pension of AlCl3 (20.0 g, 0.15 mol) in dichloromethane
(500 mL) was added tert-butylamine borane (27.5 g,

3540
H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
0.31 mol) at 0 ꢃC. After stirring the resulting mixture at
0ꢃ C for 15 min a solution of the bromo ketone 96a
(16.0 g, 50 mmol) in dichloromethane (150 mL) was
added and stirred at 0 ꢃC for additional 4 h. Cold dilute
HCl (1 N, 30 mL) was added dropwise to the reaction
mixture, following by extraction with EtOAc. The
combined organic extracts were washed with dilute HCl
and then with brine. The concentration of the organic
extracts gave an oil which was purified by flash chro-
matography on silica. Yield: 15 g (quant). Mp: 142 ꢃC;
1H NMR: (300 MHz) 3.2; 3.78 (2 t, 4H, CH2); 7.4–7.7 (4
d, 8H, ar); MS: 296.2 (M+H).
and cooled to 0 ꢃC, were then added dropwise during
1.5 h. The internal temperature should not exceed
ꢁ10 ꢃC in this process. After completion of the addition
of nitrate, the mixture was additionally stirred for 10
min at ꢁ10 ꢃC and for 1 h without external cooling. The
mixture was poured onto ice and neutralized with con-
centrated aqueous ammonia solution with cooling; con-
sumption approximately 1.8 L of the 25% strength
solution. Before filtering off the precipitating amino
acid, the mixture was diluted with the same volume of
water. The solid obtained was again suspended in water
and filtered off to remove residual ammonium salts. It
was washed with plenty of cold water and dried at 60 ꢃC
under reduced pressure. Yield: 110.1 g (88% of theory).
Mp: from 245 ꢃC (slow discoloration), 272–275 ꢃC
Sodium salt of 4-chloro-biphenylethane sulfonic acid 96c.
Compound 96b (14.8 g, 50 mmol) was dissolved in a
mixture of ethanol and water (1:1, 200 mL). After add-
ing sodium sulfite (9.5 g, 75 mmol) and tetra-
butylammonium jodide (1.8 g, 5 mmol) the mixture was
heated at reflux temperature for 16 h. From a small
amount of a solid was decanted, the solvent partly
removed and on cooling the product crystallizes. It was
filtered off, recrystallized from MeOH/H2O and dried in
vacuo. Yield: 13.9 g (94% of theory). 1H NMR:
(300 MHz) 2.6; 2.95 (2 m, 4H, CH2); 7.3–7.7 (4 d, 8H, ar).
1
(melts with decomposition); H NMR: (400 MHz, DCl/
D2O) 3.05 (dd, 1H, 7-isomer); 3.30 (2 dd, superimposed,
2H, 6- and 7-isomer); 3.44 (dd, 1H, 6-isomer); 4.25 (m,
3H); 7.20; 7.80 (2 m, 3H); proportion of 6-isomer: 13%.
Elemental analysis: C 53.9 (theor. 54.06), H 4.50 (theor.
4.55), N 12.6 (theor. 12.61); IR: 1640 (s), 1540 (s), 1400
(s), 1350 (s) cmꢁ1
.
tert-Butoxycarbonyl-(6/7)-nitro-1,2,3,4-tetrahydroisoqui-
noline-(R)-3-carboxylic acid. 13.3 g (59.9 mmol) of the
nitro imino acid mixture 30a/b were dissolved or sus-
pended in 300 mL of dioxane/water 1:1; 13.1 g (60
mmol) of di-tert-butyl dicarbonate and 12.72 g (120
mmol) of sodium carbonate was added and the mixture
was stirred at room temperature for 16 h. The dioxane
was then distilled off on a rotary evaporator and the
residual aqueous suspension covered with a layer of 200
mL of ethyl acetate. The mixture was cooled to 5 ꢃC,
acidified to pH 3 (1 N HCl) and the organic phase is
separated off. It was then washed twice with saturated
NaCl solution and dried over sodium sulfate. After fil-
tering off the drying agent, the filtrate was evaporated
under reduced pressure. Yield: 18.1 g (94% of theory).
Purity/isomer distribution: HPLC determination:
Nucleosil RP 18, 125ꢂ4 mm, 254 nm, acetonitrile/0.1 M
phosphoric acid 5:95–70:30; 6-isomer: retention time
14.19 min, 7-isomer: retention time 14.72 min. Ratio
approximately 1:9; purity: 99.0%; 1H NMR: (200 MHz)
1.4 (2 s, 9H); 3.3 (m, 2H); 4.4–5.0 (3 m, 3H); 7.4–8.2 (5
m, 3H); 12.7 (s, 1H).
4-Chloro-biphenylethanesulfonyl chloride 96d. To a sus-
pension of the sodium sulfonate 96c (4.8 g, 15 mmol) in
phoshorus oxychloride (50 mL) was added phoshorus
pentachloride (3.2 g, 15 mmol). After heating at 60 ꢃC
for 6 h the solution was poured into ice water after
methylene chloride was added. Saturated sodium
hydrogen carbonate solution was added to neutralize
the mixture, the organic phase was separated, dried and
the solvent removed. Yield: 5 g (quant). 1H NMR:
(300 MHz) 2.9 (m, 4H, CH2); 7.3–7.7 (4 d, 8H, ar).
2-(40-Chloro-biphenyl-4-ethylsulfonyl)-1,2,3,4-tetrahydro-
isoquinoline-3-carboxylic acid (96). To a suspension of
D-Tic (1.4 g, 8 mmol) in 30 mL dry acetonitrile bis-(tri-
methylsilyl) trifluoroacetamide (2.3 g, 9 mmol) was
added under Ar and then refluxed for 2 h. To this solu-
tion, 4-chloro-biphenylethanesulfonyl chloride 96d (2.84
g, 9 mmol) in 30 mL acetonitrile was added and refluxed
for 3 h. After cooling to rt, 9 mL 1 M HCl was added to
the reaction mixture and stirred for 1 h. The solvent was
removed, water and chloroform added, the organic
phase separated and dried. After removal of the solvent
the product was precipitated from toluene and recrys-
tallized twice from MeOH. Yield: 1.1 g (30 of theory;
purity >98%; mother lipuors could be recycled to a
totaꢃl yield of 65% with a purity >94%). Mp: 211–
Dicyclohexylammonium 2-tert-butoxycarbonyl-7-nitro-
1,2,3,4-tetrahydroisoquinoline-(R)-3-carboxylate 30c. To
separate the regioisomers, 10 g of the mixture of the
Boc-iminoacids were dissolved in 300 mL of ethyl acetate
and then treated at room temperature with 1 equiv (6.2
mL) of dicyclohexylamine in 10 mL of ethyl acetate. In
the cold, after addition of n-heptane, the dicyclohex-
ylammonium salt slowly crystallizes out, was filtered off
after 16 h and dried. After two further recrystallizations,
the proportion of the 6-isomer was less than 1.0% with
a total purity of greater than 99%. Further material can
be obtained from mother liquors. Yield: 6.1 g (1st frac-
tion). Purity/isomer distribution: HPLC determination:
Nucleosil RP 18, 125ꢂ4 mm, 254 nm, acetonitrile/0.1 M
phosphoric acid 5:95 to 70:30; 6-isomer: retention time
13.51 min, 7-isomer: retention time 14.23 min. Ratio
1
212 C; H NMR: (300 MHz) 3.0–3.6 (3 m, 6H, CH2);
4.6 (‘q’ 2H, CH2); 4.82 (t, 1H, CH); 7.2–7.7 (mm, 12H,
ar); MS: (M+H).
2-(4-Methoxybenzenesulfonyl)-7-amino-1,2,3,4-tetrahy-
droisoquinoline-(R)-3-(N-hydroxy)carboxamide
(30).
(6/7) - Nitro - 1,2,3,4 - tetrahydroisoquinoline - (R) - 3 - car-
boxylic acid 30a/b. 100 g of 1,2,3,4-tetrahydroisoquino-
line-3-carboxylic acid (564 mmol) were dissolved or
suspended in 500 mL of sulfuric acid (98%, d 1.84) at
ꢁ10 ꢃC and cooled to ꢁ30 ꢃC. 59 g (584 mmol) of
potassium nitrate, dissolved in 200 mL of sulfuric acid
1
>1:99. H NMR: (200 MHz) 0.9–1.9 (several m, about

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3541
30H); 2.7–3.05; 3.4; 4.6 (5 m, about 5H); 7.4; 8.0 (2 m,
3H). Specific rotation: ꢁ23.6ꢃ (MeOH, c=1).
removed in vacuo. Yield: 0.41 g (91% of theory). 1H
NMR: (200 MHz) 3.0–3.5 (m, 2H); 4.2–4.5 (m, 3H);
7.1–7.4 (2 m, 3H); 10.0 (s, broad, 1H). MS: 193.0
(M+H). Specific rotation: +86.3ꢃ (c=1, methanol).
2-tert-Butoxycarbonyl-7-nitro-1,2,3,4-tetrahydroisoqui-
noline-(R)-3-carboxylic acid. To liberate the protected
amino acid 30c, the DCHA salt was dissolved in ethyl
acetate and extracted by shaking with an excess of aqu-
eous, 10% strength citric acid solution. The organic
phase was extracted by shaking with saturated NaCl
solution, dried over sodium sulfate and evaporated
2-(4-Methoxybenzenesulfonyl)-7-amino-1,2,3,4-tetrahydro-
isoquinoline-(R)-3-(N-hydroxy)carboxamide (30). As
outlined in Scheme 3 7-nitro-1,2,3,4-tetrahydroiso-
quinoline-(R)-3-carboxylic acid hydrochloride was
transformed to the sulfonamide under standard Schot-
ten–Baumann conditions followed by reduction of the
nitro group to the amino group (as outlined above,
resulting in compound 48 in Table 1) and introduction
of the Boc protective group (described above, resulting
in compound 30e), all steps are giving the products in
very high yields each (cf. Scheme 3). To prepare the
hydroxamic acid, 10 g (22 mmol) of 2-(4-methoxy-
benzenesulfonyl)-7-(tert-butoxycarbonyl)-amino-1,2,3,4-
tetrahydroisoquinoline-(R)-3-carboxylic acid 30e was
dissolved in 100 mL of THF, cooled to ꢁ15 ꢃC and
treated successively with 2.1 mL (22 mmol) of ethyl
chloroformate, 4.8 mL (44 mmol) of N-methylmorpho-
line and, after 45 min at this temperature, with 13.5 mL
(110 mmol) of O-trimethylsilylhydroxylamine. The
mixture was additionally stirred for 3 h at rt, the solvent
was removed under reduced pressure, the residue taken
up in ethyl acetate and extracted by shaking successively
with 10% strength citric acid solution, 10% sodium
carbonate solution and saturated NaCl solution, dried
over sodium sulfate and evaporated in a rotary eva-
porator, and solvent residues were removed. 2.6 g of
this compound (total yield 9.1 g) was purified by silica
gel chromatographic, resulting in example 30 (Table 1);
this compound was then treated with 50 mL of HCl in
diethyl ether and the mixture was stirred at rt for 30 min. It
was then evaporated under reduced pressure and the resi-
due was coevaporated with toluene. Yield: 1.97 g (89% of
theory). 1H NMR: 2.75 (m, 2H); 3.8 (s, 3H); 4.40 (m, 3H);
6.9–7.3 (m, 3H); 7.0; 7.7 (2 d, 4H); 8.8; 9.3; 10.7 (3 s, 3H).
1
under reduced pressure. Yield: 87–95%. H NMR: The
characteristic signals of dicyclohexylamine are absent.
The compound liberated was immediately further
processed.
7-Nitro-1,2,3,4-tetrahydroisoquinoline-(R)-3-carboxylic
acid hydrochloride 30d. 0.5 g of the above mentioned
compound (1.55 mmol) were treated with 19 mL of HCl
in ether and the mixture was stirred at rt for 30 min,
evaporated to dryness, coevaporated several times with
toluene and dried under reduced pressure. Yield: 0.385 g
(96% of theory). 1H NMR: (200 MHz) 3.2–3.6 (m, 2H);
4.3–4.6 (m, 3H); 7.6 (d, 1H); 8.1 (dd, 1H); 8.3 (d, 1 h);
10.5 (s, br., 1H); MS: 223.1 (M+H). Specific rotation:
+143.5ꢃ (c=1, MeOH).
2-tert-Butoxycarbonyl-7-amino-1,2,3,4-tetrahydroisoqui-
noline-(R)-3-carboxylic acid. 38 g of the regiopure Boc-
nitro compound (117 mmol) were hydrogenated in a
Parr apparatus at rt and a slight excess pressure for 7 h
with 2 g of 10% Pd on C in methanol. After evaporating
the solvent, the residue was washed with diisopropyl
ether and recrystallized from water/ethanol and finally
dried under reduced pressure. Yield: 33 g (95% of the-
1
ory). H NMR: (200 MHz) 1.4 (2 s, 9H); 2.9 (m, 2H);
4.2–4.8 (several m, 3H); 6.4 (m, 2H); 6.8 (m, 1H). MS:
293.1 (M+H). Specific rotation: +28.33ꢃ (c=1,
methanol).
2-tert-Butoxycarbonyl-(6/7)-amino-1,2,3,4-tetrahydroiso-
quinoline-(R)-3-carboxylic acid. For reduction of the
Boc-nitro-mixture, the procedure was as above. The
crude product was evaporated under reduced pressure.
1H NMR: (200 MHz) 1.4 (2 s, 9H); 2.9 (m, 2H); 4.2–4.8
(several m, 3H); 6.4 (m, broad, 2H); 6.8 (m, 1H). MS:
293.1 (M+H).
Compound 60 was prepared accordingly.
2-(40 -Dimethylamino-biphenyl-4-sulfonyl)-1,2,3,4-tetra-
hydro-isoquinoline-3-carboxylic acid hydrochloride (63).
4-Dimethylaminobiphenyl 63a. 10.15 g (60 mmol) 4-ami-
nobiphenyl was dissolved together with 10.09 g (80
mmol) of dimethylsulfate in 120 mL tetrahydrofurane
under stirring, 11.06 g (80 mmol) of potassium carbon-
ate, dissolved in 100 mL of water were slowly added and
stirring is continued until completion of the reaction
(ca. 24 h, TLC control). After addition of ethyl acetate,
the phases were separated, the aqueous phase was reex-
tracted and the combined organic phases were washed
with water and brine, dried over sodium sulfate and
evaporated under reduced pressure. Purification from
byproducts (monoalkylated and quat. ammonium) was
performed by flash-chromatography on silica gel (ethyl
acetate/petrol ether 1:9). Yield: 6.9 g (58.3% of theory).
2-tert-Butoxycarbonyl-7-amino-1,2,3,4-tetrahydroisoqui-
noline-(R)-3-carboxylic acid (alternative process for se-
paration of regioisomers). The isomer mixture from the
above mentioned example was treated with a small
amount of acetonitrile at boiling heat. After cooling, it
was filtered off. This treatment is carried out 2–3 times.
1H NMR: (200 MHz) 1.4 (2 s, 9H); 2.9 (m, 2H); 4.2–4.8
(several m, 3H); 6.4 (m, 2H); 6.8 (m, 1H); no difference
from Example 6. MS: 293.1 (M+H). Specific rotation:
+28.13ꢃ (c=1, methanol).
7-Amino-1,2,3,4-tetrahydroisoquinoline-(R)-3-carboxylic
acid dihydrochloride. 0.5 g (1.7 mmol) of the Boc-pro-
tected amino imino acid were treated with HCl in ether for
30 min at rt. After evaporating under reduced pressure, the
residue is coevaporated with toluene and residual solvent
40-Dimethylamino-biphenyl-4-sulfonic acid 63b. 6.8 g
(43.5 mmol) of compound 63a was dissolved in 30 mL
of concd sulfuric acid, and heated to 130 ꢃC, resulting in
a deep purple solution. After 30 min of stirring at

3542
H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
130 ꢃC the mixture was cooled to rt and poured into 200
mL of ice-water. The product was isolated by filtration,
carefully washed with water finally and dried in vacuo.
Yield: 6.5 g (68% of theory).
lic acid was suspended in 30 mL tetrahydrofurane and
10 mL water, 1.21 g (5 mmol) of step 2 compound and
then slowly 5 mL 1 N of sodium hydroxide solution was
added under intensive stirring at rt. After 20 h, the
reaction was complete (HPLC control). The pH was
adjusted to 2 using aqueous HCl and the product was
twice extractet with ethyl acetate. The organic phase
was washed with aqueous sodium bicarbonate and
concd sodium chloride solution, then partly evaporated
and filtered over silica gel for decolorization. After
removal of ethyl acetate under reduced pressure, the
crude product was taken up in a small volume of
methanol, treated in a ultrasonic bath for several min,
then isolated by filtration and dried in vacuo. Yield:
1.05 g (60.7% of theory). 1H NMR (DMSO-d6): 3.0–3.2
(m, 2H, CH2), 4.45–4.75 (dd, 2H, CH2-CHN), 4.9 (m,
1H, CHN), 7.05–7.25 (m, 4H, ar), 8.05 (AA0, 2H, ar),
8.15–8.3 (m, 4H, ar and triazole).
40-Dimethylamino-biphenyl-4-sulfonyl chloride 63c. 0.28
g (1 mmol) of 63b was dissolved in 1 mL of phosphorus
oxycloride and 0.21 g (1 mmol) phosphorus pen-
tachloride. On heating to 100 ꢃC, the product dissolved
under gas evolution. After 5 h, the reaction was com-
plete (HPLC control). The reaction mixture was poured
into ice water and the pH adjusted to 8 (sodium bicar-
bonate). The product was isolated by repeated extrac-
tion with dichloromethane, washing with water and brine,
then evaporation to dryness under reduced pressure.
Yield: 0.25 g (84.5% of theory).
2-(40-Dimethylamino-biphenyl-4-sulfonyl)-1,2,3,4-tetrahy-
dro-isoquinoline-(R)-3-carboxylic acid hydrochloride
(63). 0.11 g (0.6 mmol) of 1,2,3,4-tetrahydroisoquino-
line-(R)-3-carboxylic acid was suspended in 30 mL tet-
rahydrofurane, 0.21 g (0.7 mmol) of 63c and then 0.28 g
(2 mmol) of potassium carbonate, dissolved in 15 mL
water under intensive stirring at rt. After 24 h, the
reaction was complete (HPLC control). The pH was
adjusted to 4 using aqueous citric acid, the product was
extractet with ethyl acetate. The organic phase was
washed with water and concd sodium chloride solution,
then evaporated to give an oily residue. Trituration with
methyl-tert-butylether resultes in 0.16 g (61.1% yield) of
an amorphous product (mp 194–196 ꢃC). This product
was transferred into the hydrochloride salt by dissolving
100 mg in 5 mL tetrahydrofurane, adding 0.3 mmol of
an 1 N HCl solution in diethylether and then slowly
methyl-tert-butylether until completion of crystal-
lization. The final product was isolated by filtration and
dried in an vacuum oven at 40 ꢃC. Yield: 85 mg (46% of
Enzyme assays
The catalytic domain of human recombinant MMP-8
(hMMP-8cd; Gly99-Gln271; Mr 19.9 kD) was obtained
as described previously,38 biological activities were
determined in 96 well titer plate format in a lumines-
cence spectrometer (LS 50B, Perkin-Elmer, Langen,
FRG) using the quenched fluorigenic substrate39 (7-
methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-3-(20,40-
dinitrophenyl)-l-2,3-diaminopropionyl-Ala-Arg-NH2
[l 328 nm (ex)/l 393 nm (em)]. Each well contained 70
mL of buffer (0.1 mol/l Tris–HCl, pH 7.5; 0.1 mol/L
NaCl; 0.01 mol/L CaCl2; 0.05% Brij 35), 10 mL of
enzyme, and 10 mL of drug in 19% DMSO. After pre-
incubation for 15 min at rt the reaction was started by
addition of 10 mL of substrate (1 mmol/L in 10%
DMSO). The initial velocity of the enzymatic reaction
was determined without ligand (=100%) and with
seven different ligand concentrations, ranging from 10ꢁ4
to 10ꢁ10 mol/L. Each measurement was done in dupli-
cate to ensure statistically significant results (p<0.05).
The ligand-dependent inhibition of the intial velocity
was plotted, and the IC50 values were determined using
standard software.
1
theory). H NMR (MeOH-d4): 3.05–3.25 (m, 2H, CH2
and s, 6H, NMe2), 4.5–4.75 (dd, 2H, CH2–CHN), 4.95
(m, 1H, CHN), 7.05–7.2 (m, 4H, ar), 7.45–8.0 (m, 8H, ar).
2-(4-[1,2,3]Triazol-2-yl-benzenesulfonyl)-1,2,3,4-tetrahy-
dro-isoquinoline-(R)-3-carboxylic acid (80). 2-Phenyl-
2H-[1,2,3]-triazole 80a. The synthesis was performed in
close analogy to ref 37. A mixture of 2 g (8.4 mmol)
glyoxyl-bis-phenylhydrazone and 5.5 g (22 mmol)
CuSO4ꢂ5H2O in water were heated to 75–80 ꢃC under
stirring for 6.5 h and then steam-distilled to remove
from salts and byproducts. The triazole-water phase is
saturated with NaCl and the product extracted twice
with diethylether. The combined organic phase was
washed with 10% aqueous HCl and water, dried and
evaporated under reduced pressure. Yield: 1.15 g (94%
of theory).
Computational procedures
All modelling work was performed using the program
SYBYL,40 energy calculations were based on the TRI-
POS 6.0 force field41 including Gasteiger–Marsili char-
ges.42 Conformations of ligands and protein–ligand
complexes were minimized using quasi-Newton–Raph-
son (BFGS) or conjugate gradient (CG) procedures.
For docking, X-ray structures of catalytic domains of
MMP-8 provided starting geometries. After analysis of
protein–ligand interactions using the program GRID,22
candidate molecules were manually docked into the
active site. Subsequently the protein–ligand complex
was minimized treating all ligand atoms plus all protein
residues within a sphere of 4 A as flexible, while the
remaining receptor was only used to compute non-bon-
ded interactions. All other compounds were built
4-[1,2,3]Triazol-2-yl-benzenesulfonyl chloride 80b. 1.1 g
(7.6 mmol) of step 1 compound 80a was chlor-
osulfonated using 10 equivalents of chlorosulfonic acid
according to ref 40. Yield: 1.3 g (70.4% of theory).
2-(4-[1,2,3]Triazol-2-yl-benzenesulfonyl)-1,2,3,4-tetrahy-
dro-isoquinoline-(R)-3-carboxylic acid (80). 0.8 g (4.5
mmol) of 1,2,3,4-tetrahydroisoquinoline-(R)-3-carboxy-

H.Matter et al./ Bioorg.Med.Chem.10 (2002) 3529–3544
3543
accordingly, docked into MMP-8 and minimized. Some
4. Peress, N.; Perillo, E.; Zucker, S. J.Neuropathol.Exp.
Neurol. 1995, 54, 16.
5. Bode, W.; Reinemer, P.; Huber, R.; Kleine, T.; Schnierer,
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genetic algorithm docking procedure implemented in
the program FlexiDock,28 which did not result in alter-
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dates was done by inspection of the complementarity of
protein–ligand interactions after minimization plus
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Rabbit PK studies
Rabbit PK studies were performed using a standard
drug concentration of 15 mg drug/kg animal body
weight in 0.1 M sodium phosphate buffer solution pH
8.0, except if noted otherwise. Data sampling was per-
formed by taking blood samples from the ear vein into
heparinized syringes according to the following time
schedule: predose (=0), 30, 60, 120, 180, 240, 360 and
1440 min post administration. Subsequently all plasma
samples were treated by combining 500 mL plasma with
50 mL acetonitril and 50 mL internal drug standard
solution with 100 mL 1 N HCl and 2.5 mL dichloro-
methane. The internal drug standard solution was pre-
pared by generating a 50 mL solution in acetonitril
containing 0.5 mMol drug substance. The subsequent
extraction of the organic layer was done for 15 min by
using a CENCO mixer. After centrifugation (3500 rpm),
the organic layer was dried under a gentle stream of
nitrogen at 40 ꢃC. The residue was taken up in 100 mL
acetonitril and 100 mL water. HPLC: Merck I-6200A and
L-7200 Autosampler; processing: Waters Maxima chro-
matography workstation; column: Nucleosil C18; col-
umn size: 125ꢂ4 mm; particle size: 5 mm; detection:
UV–absorbance (246 nm) GAT LCD 503; flow rate: 1.5
mL/min; inj. solut.: 200 mL acetonitril+100 mL water;
inj. vol.: 20 mL; limit of detection: <10 ng/mL plasma;
elution: start with: 10% acetonitril/90% 0.1 M H3PO4;
go to 70% acetonitril after 10 min, keep constant for 5
min. The chemical purity of the compound was usually
>96% and checked immediate prior to performing the
in vivo PK study.
12. Schlechter, I.; Berger, A. Biochem.Biophys.Res.Commun.
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Acknowledgements
We thank H. Schreuder, V. Wehner, S. Ruf and B. Pir-
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