The discovery of anthranilic acid-based MMP inhibitors. Part 3: Incorporation of basic amines

Article abstract of DOI:10.1016/S0960-894X(01)00601-1

Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9 and MMP-13.

Full text of DOI:10.1016/S0960-894X(01)00601-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2975–2978
The Discovery of Anthranilic Acid-based MMP Inhibitors.
Part 3: Incorporation of Basic Amines
J. I. Levin,a,* J. M. Chen,a M. T. Du,a F. C. Nelson,a T. Wehr,a J. F. DiJoseph,b
L. M. Killar,b S. Skala,b A. Sung,b M. A. Sharr,b C. E. Roth,b G. Jin,a R. Cowling,a
L. Di,b M. Sherman,b Z. B. Xu,b C. J. March,c K. M. Mohler,c R. A. Blackc
and J. S. Skotnickia
aWyeth-Ayerst Research, 401N. Middletown Rd., Pearl River, NY 10965, USA
bWyeth-Ayerst Research, PO Box CN-8000, Princeton, NJ 08543, USA
cImmunex Corporation, Seattle, WA 98101, USA
Received 28 June 2001; accepted 5 September 2001
Abstract—Anthranilic acid derivatives bearing basic amines were prepared and evaluated in vitro and in vivo as inhibitors of
MMP-1, MMP-9, MMP-13, and TACE. Piperazine 4u has been identified as a potent, selective, orally active inhibitor of MMP-9
and MMP-13. # 2001 Elsevier Science Ltd. All rights reserved.
In an effort to find novel therapies for the treatment of
diseases such as atherosclerosis,1 rheumatoid arthritis,
osteoarthritis,2 and cancer3 we have recently disclosed a
series of sulfonamide hydroxamic acid inhibitors of
MMP-1, MMP-9, MMP-13, and TACE, based on an
anthranilic acid scaffold.4 It has been postulated that
the aberrant control of specific MMPs and TACEis a
causative factor in the etiology of these pathologies. The
SAR of the anthranilic acid 3- and 5-positions (4, R3
and R5) as well as the P10 moiety (4, R1), leading to
compounds exemplified by 1 (Fig. 1), with nanomolar
level in vitro activity, and oral bioavailability, has been
discussed.5
4, containing basic amine groups at R2 or R3. Analysis
of NMR and molecular modeling studies of anthrani-
late hydroxamates bound to the active site of MMP-13
suggested that polar functionality at these two positions
would be solvent exposed.7
Chemistry
In general, the desired sulfonamide hydroxamic acids
were prepared as previously described (Scheme 1).4,8
Sulfonamide formation was followed by attachment of
the desired basic moieties and the fully functionalized
anthranilate sulfonamides were then converted into the
hydroxamic acids. Analogues 4a–4e were obtained after
alkylation of 3a (R1=OMe; R2=H; R3, R4=Me;
R5=H) or 3b (R1=OMe; R2=H; R3, R4=Me;
R5=Br) with the appropriate picolyl or benzylic chlo-
ride using potassium carbonate in DMF. The synthesis
of benzylic amine 4f began with the alkylation of 3a
with 4-carbomethoxybenzyl bromide, followed by
selective ester hydrolysis (NaOH) and reduction of the
In the course of our work on the anthranilate hydrox-
amates, it became clear that oral activity was greatly
enhanced by the incorporation of a basic amine moiety
into the inhibitor molecule. In fact, few compounds in
this series lacking a basic amine displayed any activity in
an in vivo bioactivity model. A similar circumstance has
been reported for the sulfonamide-hydroxamates,
exemplified by the oncology clinical candidate CGS-
27023A.6
We now report on the synthesis and evaluation of a
series of anthranilate hydroxamates, of general structure
*Corresponding author. Tel.: +1-845-602-3053; fax: +1-845-602-
5561; e-mail: levinji@war.wyeth.com
Figure 1. Sulfonylated anthranilate hydroxamic acids.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00601-1

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2975–2978
Biology
All of the anthranilate hydroxamic acids were tested in
vitro9 for their ability to inhibit MMP-1, MMP-9,
MMP-13, and TACE10 (Table 1). Inhibitors of MMP-9
are potentially valuable as inhibitors of tumor metas-
tasis,3 while MMP-13 inhibitors may offer protection
from the cartilage degradation associated with osteoar-
thritis.2 Inhibitors of TACEare potentially valuable for
the treatment of rheumatoid arthritis, Crohn’s disease
and other inflammatory diseases.11 Selectivity over
MMP-1 was sought in order to examine whether the
inhibition of MMP-1 is a possible source of the muscu-
loskeletal side effects seen in clinical trials of broad
spectrum MMP inhibitors.12
Scheme 1. (i) 4-R1PhSO2Cl, TEA; (ii) R2X, NaH or K2CO3; (iii)
NaOH; (iv) (COCl)2, DMF, NH2OH.
resulting acid to the alcohol with borane–THF. The
alcohol was next converted into the corresponding bro-
mide (PPh3/CBr4) and then displaced with dimethyl-
amine. Aryl piperazine derivative 4g resulted from
alkylation of 3a with 4-bromobenzyl bromide followed
by Buchwald amination of the aryl bromide with 1-
methylpiperazine. Propargylic amines 4h–4j were
obtained by alkylating 3a or 3b with propargyl bromide
and subsequent Mannich alkylation. Variants 4k–4r
bearing amines at the anthranilate 3-position were pre-
pared by amine displacement of the benzylic bromides
resulting from NBS bromination of N-methyl sulfona-
mides 3c (R1=OMe; R2=Me; R3, R4=Me; R5=H) or
3d (R1=OMe; R2=Me; R3, R4=Me; R5=Br). The 5-
aryl derivatives 4s–4t were obtained by Suzuki cou-
plings of the aryl boronic acids with 3e (R1=OMe;
R2=Me; R3=CH2N[(CH2)2]2NMe; R4=Me; R5=Br).
The biaryl ether analogues 4u and 4v arose from 3f
(R1=OPh-4-Cl; R2=Me; R3, R4=Me; R5=H or Br).
The in vitro potencies against the MMPs and TACEfor
the series of anthranilate hydroxamic acid analogues in
which a variety of basic amines have been linked to the
sulfonamide nitrogen (4a–4j) are shown in Table 1. All
of these compounds are potent inhibitors of MMP-9
and, with the exception of 4d, MMP-13. They are com-
parable in vitro to CGS-27023A. The picolyl analogues,
4a–4c, and the benzyl derivatives 4f–4g, are essentially
equipotent (3–20 nM) with similar selectivity profiles.
Incorporation of a 5-bromo substituent into the gelati-
nase selective compound 4d provides 4e, a 3 nM MMP-
13 inhibitor with enhanced potency against all four
enzymes screened and no selectivity for MMP-9. The
propargylic amines 4h–4j are also excellent (6–19 nM)
inhibitors of MMP-9 and MMP-13 with weak selectivity
Table 1. In vitro potency of substituted anthranilate hydroxamic acids
IC50, (nM)
Compd
R1
R2
R3
R5
MMP-1
MMP-9
MMP-13
TACE
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Ph-4-Cl
Ph-4-Cl
—
CH2-3-Py
CH2-3-Py
CH2-3-Py
CH2Ph-4-O(CH2)2NC5H10
CH2Ph-4-O(CH2)2NC5H10
CH2Ph-4-CH2NMe2
CH2Ph-4-N[(CH2)2]2NMe
CH2CCCH2NEt2
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
Br
H
H
H
Br
Br
H
H
H
H
H
Br
143
329
391
176
35
5
7
8
7
2
3
7
11
14
10
8
5
5
10
1
2
3
7
2
2
8
18
18
56
3
11
20
19
7
231
356
645
277
108
243
146
218
36
213
142
470
150
129
744
608
517
781
306
194
444
674
745
343
155
82
Me
Me
CH2CCCH2NEt2
CH2CCCH2N[(CH2)2]2NMe
Me
CH2NEt2
CH2N[(CH2)2]2O
CH2ProMe
CH2Im
CH2N[(CH2)2]2NMe
CH2N[(CH2)2]2NMe
CH2N[(CH2)2]2NPh
CH2N[(CH2)2]2NBoc
CH2N[(CH2)2]2NMe
CH2N[(CH2)2]2NMe
CH2N[(CH2)2]2NMe
CH2N[(CH2)2]2NMe
—
6
26
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
—
84
14
6
43
12
5
3
6
4
3
210
174
200
157
154
26
87
63
210
49
4p
4q
4r
4s
4t
4u
Br
Br
Ph-4-OCF3
2-Naphthyl
H
Br
—
1
0.5
9
0.8
0.7
8
122
80
231
4v
CGS-27023A
15

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2975–2978
2977
over MMP-1. Thus, compound 4h, unsubstituted at the
anthranilate 5-position, is a 19 nM MMP-13 inhibitor.
Its 5-bromo analogue, 4i, is a slightly more potent (7
nM) MMP-13 inhibitor and a substantially more potent
TACEinhibitor (36 nM). Excellent TACEactivity is
also displayed by propargylic piperazine 4j (26 nM),
which retains activity against MMP-9 and MMP-13.
However, as we had seen before,5 despite their potency
against isolated enzyme, compounds 4i and 4j did not
display significant TNF-a inhibitory activity in a THP-1
cellular assay at 3 mM.13
The in vivo bioactivity after oral dosing of some of the
anthranilate-hydroxamates (4a, 4d, 4g–4j, 4l, 4n–4q, and
4s–4v) against MMP-13 was initially assessed through
the use of a dialysis tubing implant assay.14 All of the
compounds tested were compared to Novartis’ sulfona-
mide-hydroxamate clinical lead, CGS-27023A, in the
same experiment. Of these, 4g (50 mpk), 4p (50 mpk), 4u
(25 mpk), and 4v (25 mpk) are 10–30% more potent
than an equal dose of CGS-27023A, with 4u and 4v
being the best.
Anthranilate-hydroxamates 4g, 4p, 4u, and 4v were also
tested side-by-side with CGS-27023A in a bovine
articular cartilage explant assay.15 At a concentration of
1 mM, compounds 4p and 4v provided a level of inhibi-
tion of cartilage degradation 60 and 20% higher than
CGS-27023A, respectively. Compounds 4g and 4u were
roughly equivalent to CGS-27023A at 1 mM in this
assay.
The feasibility of incorporating amines at the 3-position
of the anthranilic acid ring was explored with benzylic
amines 4k–4v (Table 1). The diethylamine 4k and imi-
dazole 4n were the least active members of this series
against MMP-1 and MMP-13, but remained potent
inhibitors of MMP-9 (8–10 nM). The proline methyl
ester 4m displayed selectivity approaching 100-fold for
MMP-13 over MMP-1.
Hydroxamates 4p, 4u, and 4v were next evaluated on
oral dosing in an in vivo rat sponge-wrapped cartilage
model.16 All three compounds provided significant
inhibition of collagen degradation in this model. How-
ever, while 4p and 4v at 50 mg/kg/bid were equipotent
with a 50 mg/kg/bid dose of CGS-27023A, 4u surpassed
CGS-27023A. Oral dosing of 4u at 50 mg/kg/bid pro-
vided a 73% inhibition of collagen degradation com-
pared to a 55% inhibition by CGS-27023A at the same
dose. Even a 25 mg/kg/bid dose of 4u provided better
protection from cartilage degradation than a 50 mg/kg/
bid dose of CGS-27023A (38 vs 30%).
The piperazines 4o–4v all proved to be potent inhibitors
of MMP-13. The initial analogue, 4o, is an excellent
MMP-9 (1 nM) and MMP-13 (12 nM) inhibitor. Addi-
tion of a 5-bromo substituent gives 4p with good activ-
ity versus MMP-13 (5 nM) and TACE(26 nM), but
reduced MMP-1 selectivity. Larger substituents on the
piperazine, as in the N-phenyl and N-Boc piperazines,
4q and 4r, retained MMP-13 activity and provided the
desired selectivity over MMP-1. Similarly, substitution
of 5-aryl groups (4s–4t) for the 5-bromo moiety of 4p
also provided potent MMP-13 inhibitors (3–4 nM) with
more than 100-fold selectivity over MMP-1. Extension
of the P10 group with the biaryl ethers 4u and 4v boosted
potency against all four enzymes relative to their shorter
P10 analogues 4o and 4p, but the greater increase for
MMP-13 made these compounds more than 100-fold
selective over MMP-1.
In summary, we have synthesized a series of anthrani-
late-hydroxamic acid MMP and TACEinhibitors bear-
ing basic amine moieties. Many of these compounds are
potent inhibitors of MMP-9 and MMP-13 in vitro.
Four of these compounds were evaluated in vivo to
assess their potential for treating osteoarthritis. Com-
pounds 4g, 4p, 4u, and 4v are active in an in vitro carti-
lage degradation assay, display oral activity in an in
It should be noted that at room temperature biaryl ether
4u exists as a 1:1 mixture of atropisomers due to
restricted rotation about the sulfonamide N–C bond.
The racemate can be separated by chiral HPLC on a
Chiralpak AD column eluting with ethanol. A 9:1 mix-
ture of atropisomers is converted into a 3:2 mixture on
heating for 0.5 h in refluxing ethanol. We have been
unable to resolve these atropisomers in large quantity
by either resolution or chemical synthesis. Compound
4u was the only anthranilate hydroxamic acid that we
investigated by chiral HPLC.
A single crystal X-ray structure of 4v bound to the
active site of MMP-13 was determined and is shown in
Figure 2. As expected from our previous NMR struc-
ture determinations,6 the anthranilate phenyl ring lies
near the S20 subsite, the biaryl ether is buried in the S10
pocket and the piperazine ring is solvent exposed. The
potency of 4v versus MMP-1, despite its lengthy P10
group, is consistent with previous data from this series5
and indicates that the arginine residue that forms the
bottom of the MMP-1 S10 pocket is pushed aside to
some degree by the P10 substituent, extending the depth
of the pocket.
Figure 2. X-ray structure of compound 4v in the active site of MMP-
13.

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2975–2978
vivo mouse bioactivity model and also demonstrated
oral activity in a rat sponge-wrapped cartilage model. In
particular, we have identified piperazine 4u as a 0.8 nM
MMP-13 inhibitor, with almost 200-fold selectivity over
MMP-1, that is twice as potent as CGS-27023A in this
efficacy model.
Carroll, B. J.; Goldstein, R.; Justice, M. R.; Zhu, L.; Hu, S.;
Melton, R. A.; Fryer, L.; Goldberg, R. L.; Doughty, J. R.;
Spirito, S.; Blancuzzi, V.; Wilson, D.; O’Byrne, E. M.; Ganu,
V.; Parker, D. T. J. Med. Chem. 1997, 40, 2525.
7. (a) Moy, F. J.; Chanda, P. K.; Chen, J. M.; Cosmi, S.;
Edris, W.; Levin, J. I.; Powers, R. J. Mol. Biol. 2000, 302, 671.
(b) Moy, F. J.; Chanda, P. K.; Cosmi, S.; Edris, W.; Levin,
J. I.; Powers, R. J. Biomol. NMR 2000, 17, 269.
8. All new compounds gave satisfactory 1H NMR, IR, and
MS data in accord with the assigned structure.
References and Notes
9. (a) Weingarten, H.; Feder, J. Anal. Biochem. 1985, 147, 437.
(b) Inhibitor concentrations were run in triplicate. MMP IC50
determinations were calculated from a 4-parameter logistic fit
of the data within a single experiment.
10. Jin, G.; Black, R.; Wolfson, M.; Rauch, C.; Ellestad, G.
A.; Cowling, R., Biochemistry. Submitted for publication.
11. Newton, R. C.; Decicco, C. P. J. Med. Chem. 1999, 42,
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