The discovery of anthranilic acid-based MMP inhibitors. Part 1: SAR of the 3-position.

Article abstract of DOI:10.1016/S0960-894X(00)00642-9

A novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, and MMP-13 was prepared and evaluated both in vitro and in vivo. The most potent compound, 6e, has in vivo activity in a rat sponge-wrapped cartilage model.

Full text of DOI:10.1016/S0960-894X(00)00642-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 235±238
The Discovery of Anthranilic Acid-Based MMP Inhibitors.
Part 1: SAR of the 3-Position
Jeremy I. Levin,a,* Mila T. Du,a John F. DiJoseph,b Loran M. Killar,b Amy Sung,b
Thomas Walter,b Michele A. Sharr,b Catherine E. Roth,b Franklin J. Moy,a
Robert Powers,a Guixian Jin,a Rebecca Cowlinga and Jerauld S. Skotnickia
aWyeth-Ayerst Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth-Ayerst Research, PO Box CN-8000, Princeton, NJ 08543, USA
Received 7 September 2000; accepted 8 November 2000
AbstractÐA novel series of anthranilic acid-based inhibitors of MMP-1, MMP-9, and MMP-13 was prepared and evaluated both
in vitro and in vivo. The most potent compound, 6e, has in vivo activity in a rat sponge-wrapped cartilage model. # 2001 Elsevier
Science Ltd. All rights reserved.
The matrix metalloproteinases (MMPs), comprised of
collagenases, stromelysins, gelatinases and membrane-
type MMPs, are a family of over 20 zinc-containing
enzymes that play a role in the normal remodeling and
degradation of extracellular matrix proteins. The aberrant
control of MMP levels has been implicated in the etiology
of a variety of disease states including atherosclerosis,1
rheumatoid arthritis and osteoarthritis,2 and cancer.3 The
potential exists for potent, orally bioavailable small
molecule inhibitors of MMPs to treat a broad spectrum
of pathologies, and has been investigated in the clinical
trials of agents such as marimastat,4 Ro 32-3555,5 CGS-
27023A,6 and AG33407 (Fig. 1).
In that regard the discovery of sulfonamide-based
hydroxamic acid inhibitors of stromelysin (MMP-3),
exempli®ed by CGS-27023A, is the seminal work in the
rapidly expanding area of nonpeptide MMP inhibitors.8
The disclosure of numerous sulfonamide analogues rela-
ted to CGS-27023A, including the biaryl sulfonamides,9
piperazine,10 thiazine,11 thiazepine,11 and diazepine12
ring systems, now prompts us to report on the synthesis
and biological evaluation of a novel series of anthranilic
acid-based MMP inhibitors.
Although a substantial number of nonpeptide sulfona-
mide hydroxamate MMP inhibitors have been studied,
almost all of these compounds have been derived from
a-amino acids, with a single carbon linking the sulfona-
mide nitrogen and the zinc chelating hydroxamic acid
moiety.13 We were interested in ascertaining whether
novel, potent MMP inhibitors could be made by using
an aromatic ring as the linker between the sulfonamide
nitrogen and the hydroxamate (Fig. 2).
Figure 1. MMP inhibitors in clinical trials.
*Corresponding author. Tel.:+1-845-732-3053; fax: +1-845-732-5561;
e-mail: levinji@war.wyeth.com
Figure 2. Sulfonylated anthranilate hydroxamic acids.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00642-9

236
J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 235±238
In the event, the anthranilic acid derivative 1 had IC50s
below 1 mM versus MMP-1, MMP-9, and MMP-13 (Table
1). In contrast, the 3- and 4-aminobenzoic acid derivatives,
2 and 3, had no appreciable activity versus MMP-1 at
1 mM. With this result in hand we set out to synthesize a
series of sulfonylated anthranilic acid analogues of 1.
than 6a. A series of analogues of 6a in which the 3-sub-
stituent was varied was therefore targeted next.
Compounds 6f and 6g (Table 2) were synthesized from
the commercially available 3-substituted anthranilic
acids in the same general manner as shown in Scheme 1.
The 3-nitro derivative, 6h, was synthesized via nitration
of 5 (R1=H, R2=Me, R3=Bn). The N,N-dimethyl ani-
line 6i was prepared via tin chloride reduction of the nitro
aryl intermediate 5 (R1=3-NO2, R2=Me, R3=Bn), fol-
lowed by methylation of the resulting aniline with iodo-
methane/potassium carbonate in DMF and subsequent
ester hydrolysis and hydroxamate formation. The synth-
esis of the 3-tri¯uoromethyl analogue 6j commenced with
the sulfonylation of benzylamine to give 7. Reaction of 7
with 2-¯uoro-3-tri¯uoromethylbenzonitrile in the pre-
sence of sodium hydride then provided 8. The nitrile
was then converted into the corresponding hydroxamic
acid via carboxamide 8 as shown in Scheme 2.
Table 1. In vitro potencies of compounds 1, 6a±6d
Compound R1 R2 R3 R4 MMP-1a MMP-9a MMP-13a
1
H
Me
H
H
H
H
H
Me
H
H
H
H
Me
H
H
H
H
H
Me
639
115
884
553
650
23
346
353
440
555
50
982
728
717
6a
6b
6c
6d
Additional 3-substituted anthranilic acid analogues
were readily prepared according to Scheme 3. Thus,
sulfonylation of 3-hydroxy methyl anthranilate gave 9,
which was elaborated as shown to give compounds 6k
and 6l.
H
1573
aIC50, nM.
Chemistry
The 3-carbomethoxy derivative was synthesized from
aldehyde 11 as shown in Scheme 4. Bis-bromination of
the 3-methyl derivative 5c, followed by hydrolysis gave
aldehyde 11. Oxidation to the carboxylic acid was
accomplished with sulfamic acid and sodium chlorite.
The desired sulfonamide hydroxamic acids were prepared
as shown in Scheme 1.14 Sulfonylation of the appropriate
anthranilic acid with 4-methoxybenzenesulfonyl chloride
provided the sulfonamides 5a (R2, R3=H). The sulfona-
mides were then concomitantly N- and O-alkylated to
provide compounds 5b (R2, R3=Bn). Hydrolysis of the
ester followed by acid chloride formation and conversion
into the hydroxamic acid gave compounds 6.
Biology
All of the anthranilate hydroxamic acids were tested in
vitro15 for their ability to inhibit MMP-1, MMP-9, and
MMP-13 (Table 1). Inhibitors of MMP-9 are potentially
valuable as inhibitors of tumor metastasis,3 while MMP-
13 inhibitors may o€er protection from the cartilage
degradation associated with osteoarthritis.2 Selectivity
for MMP-9 and MMP-13 over MMP-1 was sought in
order to examine whether the inhibition of MMP-1 is
the source of musculoskeletal side e€ects seen in clinical
trials of broad spectrum MMP inhibitors.16
Scheme 1. (i) 4-MeOPhSO2Cl, TEA; (ii) R3X, NaH; (iii) NaOH; (iv)
(a) (COCl)2, DMF; (b) NH2OH.
The 3-substituted anthranilate hydroxamic acids shown
in Table 2 are potent in vitro inhibitors of both MMP-9
and MMP-13. In particular, the 3-methyl derivative 6e,
methyl ester 6m, and the bis-hydroxamic acids, 6k and
6l, are less than 10 nM against both MMP-9 and MMP-
13. Compounds 6k and 6m are also potent inhibitors of
TNF-a converting enzyme (TACE) with IC50 values of
81 and 38 nM, respectively.17 Compounds 6l and 6m are
the only inhibitors of the series that are at least 50-fold
selective for MMP-13 over MMP-1, with the geminal
dimethyl group of 6l providing a 25-fold reduction in
potency versus MMP-1, relative to 6k. The 3-methoxy
analogue 6f and the 3-tri¯uoromethyl derivative, 6j, are
potent gelatinase inhibitors with greater than 20-fold and
5-fold selectivity over MMP-1 and MMP-13, respec-
tively. Dimethylaniline 6i is a less potent MMP-9 inhi-
bitor that displays a similar gelatinase-selective pro®le.
In order to examine the e€ect of substitution on the
anthranilic acid ring on inhibitory potency, analogues of
6 derived from 3-, 4-, 5-, and 6-methyl 2-amino-benzoic
acid were synthesized. The IC50 values of methyl analo-
gues 6a±6d (Table 1) clearly demonstrate that while the 4-,
5-, and 6-methyl compounds (6b±6d) are no more active
than the parent compound, the 3-methyl derivative 6a is
an order of magnitude more potent than the unsub-
stituted anthranilate. Furthermore, compound 6e, the
N-3-picolyl analogue of 6a, is as potent as the CGS-
27023A standard (Table 2).
The carboxylic acid analogue of 6a, and the NH-sulfon-
amide analogue are approximately 100-fold less active

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 235±238
Table 2. In vitro potency of 3-substituted anthranilate hydroxamic acids
237
Compound
R1
R2
MMP-1a
MMP-9a
MMP-13a
1
6a
6e
6f
6g
6h
6i
6j
6k
6l
6m
H
CH3
CH3
OCH3
Cl
NO2
N(CH3)2
CF3
CH2Ph
CH2Ph
CH2-3-Py
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2-3-Py
Ð
639
115
143
520
398
202
19%b
41%b
24
650
23
5
23
31
13
64
27
2
555
50
8
138
NT
42
531
136
1
OCH2CONHOH
OC(CH3)2CONHOH
CO2CH3
597
207
15
4
6
9
6
4
8
CGS-27023A
Ð
aIC50, nM. Inhibitor concentrations were run in triplicate. IC50 determinations were calculated from a 4-parameter logistic ®t of the data within a
single experiment.
b% Inhibition at 1 mM.
nilate aromatic ring occupies the S21 pocket of the active
site. This binding scheme is quite similar to that of
CGS-27023A, with the anthranilate aryl ring taking the
place of the isopropyl group of CGS 27023A.19
The in vivo bioactivity of several of the anthranilate-
hydroxamates (6a, e, g, k, and m) was assessed through
the use of a dialysis tubing implant assay.20 All of the
compounds tested were compared to CGS-27023A in
the same experiment. None of the N-benzyl substituted
sulfonamides had signi®cant in vivo activity. The most
active of the compounds tested in this assay is the N-
picolyl derivative 6e, which is essentially equipotent to
CGS-27023A at 50 mg/kg, po against MMP-9. It has an
ED50 of 33 mg/kg, po in this assay. However, against
MMP-13 in the dialysis implant model, compound 6e has
only 60% of the activity of CGS-27023A at the same oral
dose. Ester 6m, with 30% of the activity of CGS 27023A
versus MMP-13 at 50 mg/kg ip, was signi®cantly less
potent than 6e.
Scheme 2. (i) NaH, 2-F-3-CF3PhCN; (ii) NaOH; (iii) NOBF4; (iv) (a)
(COCl)2, DMF; (b) NH2OH.
Scheme 3. (i) TBDMSCl, imidazole; (ii) BnBr, NaH; (iii) Bu4NF; (iv)
BrC(R)2CO2Et, NaH; (v) NaOH; (vi) (a) (COCl)2, DMF; (b) NH2OH.
The 3-methyl anthranilate-hydroxamate 6e was also tested
in a bovine articular cartilage explant assay.5b At a dose of
1 mM, 6e provides a level of inhibition of collagen degra-
dation comparable to CGS 27023A (49%). Evaluation of
6e in an in vivo rat sponge-wrapped cartilage model21
demonstrated that the administration of 50 mg/kg/day ip
of 6e via osmotic pump gave a 51% inhibition of collagen
degradation. Oral dosing of 6e at 50mg/kg/bid provided a
27% inhibition of collagen degradation, compared to a
52% inhibition of CGS-27023A at the same dose.
Scheme 4. (i) NBS; (ii) NaOH; (iii) 3-Picolyl chloride-HCl, K2CO3;
(iv) NaClO2, H2NSO3H; (v) (a) (COCl)2, DMF; (b) NH2OH.
In conclusion, we have synthesized a series of anthrani-
late-hydroxamic acid MMP inhibitors. These com-
pounds are potent inhibitors of MMP-9 and MMP-13 in
vitro. Compound 6e is also active in an in vitro cartilage
degradation assay and displays oral activity in an in
vivo mouse bioactivity model as well as ip activity in a
rat sponge-wrapped cartilage model. The further
exploration of the SAR of these novel MMP inhibitors
will be reported in due course.
Structural analysis by NMR of the solution structure of
compound 6e bound in the active site of MMP-13 indi-
cates that the para-methoxyphenyl group lies in the S11
pocket of the enzyme and that the pyridine ring is solvent
exposed.18 The sulfonyl oxygens are within hydrogen
bonding distance to the peptide backbone. The anthra-

238
J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 235±238
A. G.; Picard, J. A.; Sliskovic, D. R.; Roth, B. D.; Dyer, R. D.;
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