Benzodiazepine inhibitors of the MMPs and TACE. Part 2

Article abstract of DOI:10.1016/j.bmcl.2004.06.031

A series of benzodiazepine MMP/TACE inhibitors bearing polar moieties has been synthesized in an effort to optimize inhibitory activity against LPS-stimulated TNF production in human monocytes and oral activity in a murine LPS model.

Full text of DOI:10.1016/j.bmcl.2004.06.031

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4147–4151
Benzodiazepine inhibitors of the MMPs and TACE. Part 2
Jeremy I. Levin,a,* Frances C. Nelson,a Efren Delos Santos,a Mila T. Du,a
Gloria MacEwan,a James M. Chen,a Semiramis Ayral-Kaloustian,a Jun Xu,a
Guixian Jin,a Terri Cummonsb and Dauphine Baronec
aWyeth Research, 401N. Middletown Road, Pearl River, NY 10965, USA
bWyeth Research, PO Box CN-8000, Princeton, NJ 08543, USA
cAmgen, Seattle, WA 98101, USA
Received 21 May 2004; accepted 9 June 2004
Available online
Abstract—A series of benzodiazepine MMP/TACE inhibitors bearing polar moieties has been synthesized in an effort to optimize
inhibitory activity against LPS-stimulated TNF production in human monocytes and oral activity in a murine LPS model.
Ó 2004 Elsevier Ltd. All rights reserved.
TNF-a converting enzyme (TACE) is the enzyme
responsible for cleaving 26 kDa membrane-bound TNF
to provide 17 kDa soluble TNF, a pro-inflammatory
cytokine.1 The modulation of levels of both membrane-
bound and soluble TNF through the use of biologics
such as TNF antibodies or soluble TNF receptors has
been shown to be an effective treatment for rheumatoid
arthritis (RA),2 and TACE expression has been found to
be up-regulated in the synovium of RA patients.3 Small
molecule inhibitors of TACE therefore have the poten-
tial to provide an orally active treatment for TNF-
mediated diseases including RA, and this has led to a
number of reports on these agents.4 Of the reported
inhibitors of TACE,4 most have also been potent
inhibitors of one or more matrix metalloproteinases
(MMPs), a related family of enzymes that have been
implicated in arthritis, tumor metastasis and other
pathologies.5 Recently more selective TACE inhibitors
have been disclosed that take advantage of the difference
in shape between the TACE S10 pocket and that of the
MMPs.6 Since levels of several MMPs involved in car-
tilage degradation are over-expressed in the synovial
tissue of RA patients,7 dual MMP/TACE inhibitors may
have clinical advantages over inhibitors of TACE alone,
if they can avoid the dose limiting toxicities that have
plagued this class of compounds.
We have previously disclosed our work on benzodiaze-
pine-sulfonamide hydroxamic acids with potent activity
against MMPs (1, Fig. 1).8 In addition, the use of a
butynyl ether P10 moiety on the benzodiazepine scaffold
enhanced the MMP-1 selectivity of these compounds as
well as their ability to inhibit both cell-free TACE and
TNF production in human monocytes (2, Fig. 1). We
now present our efforts to optimize the cellular potency
and oral activity of our initial set of benzodiazepine
MMP/TACE inhibitors. Since the incorporation of
basic amines into sulfonamide hydroxamate MMP/
TACE inhibitors had previously been shown to be
beneficial for cell and in vivo activity,9 we have inves-
tigated the effect of appending basic amines and other
polar functionality onto solvent exposed portions of the
benzodiazepine scaffold.
The synthesis of the benzodiazepine analogs 3 varied
at R1 (Scheme 1) commenced with aniline-alcohol 4,
O
O
O
S
N
O
S
N
O
O
O
O
HO
HO
N
N
H
H
N
N
O
O
1
2
* Corresponding author. Tel.: +1-845-602-3053; fax: +1-845-602-5561;
e-mail: levinji@wyeth.com
Figure 1. Benzodiazepine MMP/TACE inhibitors.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.031

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4147–4151
O
S
O
S
O
S
O
10
O
O
Br
O
O
O
O
i, ii
O
O
O
NO2
i, ii
N
N
NH
CH3O
HO
CH3O
HO
CH3O
HO
4
NH2
NH2
OTBS
4
5
9
OTBS
iii, iv
vi, vii, v
or
iii, iv
O
S
O
S
viii, ix, vi, vii
O
S
O
S
O
O
or
x-xiii
O
O
v, vi, vii
O
O
O
O
O
O
O
N
N
N
HOHN
N
H3CO
HOHN
H3CO
N
N
H
N
N
O
R1
3a-3d
R3
O
R3
O
6
11a R3 = CH2OTBS
11b R3 = CH2OH
3k-3p
v
Scheme 1. Reagents and conditions: (i) 2-NO2PhCH2Br, Bu4NI, NaH,
DMF; (ii) SnCl2, EtOH; (iii) BnOCOCl, TEA; (iv) NaHCO3, MeOH,
45°; (v) R1COCl, NaH, THF; (vi) LiOH, THF, MeOH; (vii) a.
(COCl)2, DMF, b. NH2OH–HCl, TEA, DMF.
Scheme 3. Reagents and conditions: (i) NaH, DMF; (ii) Fe, NH4Cl,
EtOH, H2O; (iii) AcCl/TEA; (iv) NaHCO3, MeOH, rt; (v) HF, ACN;
(vi) LiI, EtOAc, reflux; (vii) a. EDC, HOBt, DMF, b. NH2OH, H2O;
(viii) Ms2O, TEA; (ix) amine, Bu4NI, THF, rt; (x) a. PCC, DMF; b.
NaH2PO4, NaClO2, t-BuOH, (CH3)2CCHCH3; (xi) a. (COCl)2, DMF,
b. amine; (xii) LiOH, THF, MeOH; (xiii) a. (COCl)2, DMF, b.
NH2OH–HCl, TEA, DMF.
prepared in three steps from
D,L
-serine methyl ester.8;10
Thus, serine methyl ester was sulfonylated with butynyl-
oxyphenylsulfonyl chloride to give sulfonamide 4. This
sulfonamide was then alkylated with 2-nitrobenzyl
bromide followed by reduction of the nitro aryl to the
requisite aniline, 5. Reaction of 5 with benzyl chloro-
formate in the presence of triethylamine followed by
sodium bicarbonate-mediated ring closure provided
NH-benzodiazepine 6 directly in 35% yield, rather than
the expected benzyl carbamate. Acylation of 6, followed
by ester hydrolysis and subsequent hydroxamate for-
mation via the acid chloride afforded analogs 3a–d.
aniline 7b. Acylation of the aniline with concomitant
elimination of the serine hydroxyl moiety followed by
formation of the benzodiazepine ring via intramolecular
Michael addition into the a,b-unsaturated ester gave
ester 8. Compound 8 was then converted into the desired
hydroxamates, 3e–j.
Derivatives functionalized at R3 were synthesized as
shown in Scheme 3. Thus, sulfonamide 4 was first
alkylated with benzylic bromide 9, which had been
synthesized from commercial 2-nitro-4-carboxybenzyl
bromide by reduction with borane and silylation of the
resulting alcohol, and the aromatic nitro group was then
reduced to give aniline 10. As before, acetylation of 10
and intramolecular Michael reaction yielded benzodi-
azepine 11a. Conversion of ester 11a into the corre-
sponding carboxylic acid was followed by hydroxamate
formation and desilylation to give 3k. Desilylation of
11a gave alcohol 11b, which was mesylated followed by
displacement with amines to give 3l–n. Secondary
amines resulting from this displacement were protected
as the corresponding t-butyl carbamates prior to
installation of the hydroxamic acid moiety. Displace-
ment of the mesylate of 11b with sodium azide and
subsequent reduction gave the primary amine that was
next acylated to ultimately afford amide 3o. Alcohol 11b
could also be oxidized to the corresponding carboxylic
acid and then converted into the N-methylamide, fol-
lowed ultimately by hydroxamate formation to provide
3p.
Analogs varied at R2 were prepared according to
Scheme 2. Alkylation of 4 with 2-nitro-4-fluorobenzyl
bromide followed by SNAr displacement of the fluorine
with the desired amine or alkoxide provided 7a.
Reduction of the nitro group with tin(II) chloride gave
O
S
O
O
Br
O
i, ii
N
NO2
CH3O
4
X
F
HO
R2
7a X = NO2
7b X = NH2
iii
iv, v
O
S
O
O
O
O
vi, vii
S
O
O
O
N
N
HOHN
H3CO
R2
R2
N
N
O
O
3e-3j
8
The inhibitors synthesized as shown in Schemes 1–3
were evaluated in vitro against MMP-1, MMP-13, and
TNF-a converting enzyme (TACE) and the data are
shown in Table 1.11 Additionally, all compounds were
tested for their ability to inhibit LPS-stimulated TNF
Scheme 2. Reagents and conditions: (i) Bu4NI, NaH, DMF; (ii) R2H,
(iPr)2NEt, DMF or R2H, NaH, DMF; (iii) SnCl2, EtOH; (iv) AcCl/
TEA; (v) NaHCO3, MeOH, 45°; (vi) LiOH, THF, MeOH; (vii) a.
(COCl)2, DMF, b. NH2OH–HCl, TEA, DMF.

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4147–4151
Table 1. In vitro activity of substituted benzodiazepine hydroxamates
4149
O
O
S
N
O
O
HO
N
H
R2
N
R3
O
R1
2, 3a-p
Compd
R1
R2
R3
TACEa
THPb
MMP-1a
MMP-13a
2
CH3
H
H
H
H
H
H
H
H
H
H
H
H
16
81
60
73
74
33
41
33
73
75
82
44
83
63
81
70
72
46
64
835
853
77
97
7
3a
3b
3c
3d
3e
3f
CH2OCH3
2-Furyl
3-Pyridyl
Pyrazine
CH3
H
H
33
89
125
238
H
19
20
17
33
21
53
47
28
20
32
67
17
12
42
H
N(CH3)2
69
356
682
197
130
92
CH3
CH3
N[(CH2)2]2O
N[(CH2)2]2NCH3
1255
852
3g
3h
3i
CH3
CH3
N(CH3)(CH2)2-2-pyridyl
NH(CH2)2-2-pyridyl
O(CH2)2Ph
121
49
19% (1)
30% (1)
45% (1)
500
3j
CH3
CH3
36
12
3k
3l
H
H
H
H
H
H
CH2OH
CH2NHCH3
CH2N(CH3)2
CH3
CH3
14
18
456
904
3m
3n
3o
3p
CH3
CH3
CH2N[(CH2)2]2NH
CH2NHCOCH3
CONHCH3
14
31
483
578
CH3
41
813
a IC50, nM or % inhibition (lM).
b % Inhibition at 3 lM.
production in THP-1 cells.12 Activity in human whole
blood was not assessed.
not parallel the activity of these derivatives against cell-
free TACE. Thus, 3f–h, which range from approxi-
mately 6-fold to 12-fold less potent than 2 against iso-
lated TACE enzyme, are all superior to 2, as well as
secondary amine 3i, in terms of cellular activity. Ether 3j
is also relatively potent in cells, equivalent to the tertiary
amines. Since TACE is located intracellularly as well as
on the cell surface,13 correlations have been suggested
between the cell permeability of a compound, as mea-
sured in Caco-2 cells, and its potency in inhibiting TNF
production in cells.14 However, measurement of Caco-2
permeability for aniline 3e and morpholine derivative 3f,
which have comparable activity against cell-free TACE,
showed that 3e was highly permeable and had relatively
poor activity in THP-1 cells, while 3f had poor perme-
ability but was among the most potent of the benzo-
diazepine series in THP-1 cells. For comparison,
compound 2 is also highly permeable in the Caco-2 as-
say, and 10-fold more potent than 3e and 3f in the
TACE FRET assay, but less potent than 3f at 3 lM in
the THP assay. Also, the small amount of protein
present in the THP-1 cellular assay is not sufficient for
protein binding to be a significant factor in these dif-
ferences.
The replacement of the acetyl R1 moiety of 2 with more
polar functionality does not result in increased potency
against cell-free TACE. Thus, the methoxyacetyl deriv-
ative 3a is fivefold less potent than the acetyl analog
against TACE and fivefold less selective over MMP-1.
Interestingly, despite its reduced enzyme activity, 3a
gives slightly better inhibition at 3 and 1 lM (73% and
47%, respectively) than 2 in the THP-1 cellular assay.
Furan derivative 3b is similar in activity to the acetyl
derivative against cell-free TACE and in cells, but it has
no significant selectivity over MMP-1 and is a more
potent inhibitor of MMP-13 than TACE. Pyridyl and
diazine analogs, 3c and 3d, show the same selectivity
profile as furyl derivative 3b, but are much less potent
inhibitors of TACE, particularly in LPS-stimulated
THP-1 cells.
Compounds 3e–h with a tertiary amine substituent at R2
are significantly less potent in the TACE FRET assay
compared to the parent analog 2. However, these
inhibitors have enhanced potency against MMP-13 rel-
ative to 2. Removal of the N-methyl group of 3h results
in a more than twofold increase in TACE potency for
secondary amine 3i, although it is still somewhat less
potent than 2. Phenethyl ether 3j is slightly more potent
than the isosteric secondary amine 3i versus both TACE
and MMP-13, but still less potent than 2, lacking sub-
stitution at R2. Again, the cellular activity of 3e–i did
Benzodiazepines 3k–n, substituted at R3, are essentially
equipotent to the parent compound 2 against TACE
enzyme and significantly more potent than analogs 3e–j
substituted at R2. These analogs are also highly active
against MMP-13 and 30–40-fold selective for TACE
over MMP-1. In contrast to the R2 substituted analogs,

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J. I. Levin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4147–4151
little difference is seen in TACE potency between alcohol
3k, secondary and tertiary amines 3l and 3m, and the
dibasic piperazine 3n. Amide derivatives 3o and 3p are
less potent TACE inhibitors than 2 and have a reduced
level of selectivity over MMP-1 relative to 2 and the R2
substituted analogs. In LPS-stimulated THP-1 cells
compounds 3k–n were all equal to or better than 2, with
N-methyl amine 3l the most active, providing 81%
inhibition at 3 lM. The amides 3o and 3p are also both
less potent in cells than the compounds bearing a basic
amine moiety at R3.
Acknowledgements
We thank R. Cowling, R. Mulvey, R. Ley, A. Sung, and
Wyeth DSM for technical support, and J. Skotnicki, R.
A. Black, and K. M. Mohler for their insight and
guidance during the course of this work.
References and notes
1. (a) Moss, M.; Becherer, J. D.; Milla, M.; Pahel, G.;
Lambert, M.; Andrews, R.; Frye, S.; Haffner, C.; Cowan,
D.; Maloney, P.; Dixon, E. P.; Jansen, M.; Vitek, M. P.;
Mitchell, J.; Leesnitzer, T.; Warner, J.; Conway, J.;
Bickett, D. M.; Bird, M.; Priest, R.; Reinhard, J.; Lin, P.
In Metalloproteinases as Targets for Anti-inflammatory
Drugs; Bottomley, K. M. K., Bradshaw, D., Nixon, J. S.,
Eds.; Birkhauser: Basel, 1999; pp 187–203; (b) Killar, L.;
White, J.; Black, R.; Peschon, J. Ann. N.Y. Acad. Sci.
1999, 878, 442–452.
The in vivo activity of the benzodiazepine analogs was
assessed by measuring their ability to inhibit LPS-stim-
ulated TNF production in mice.15 In this assay com-
pound 2 provided 71% inhibition at an oral dose of
50 mg/kg, but was not dosed down further. None of
compounds 3a–d afforded greater than 35% inhibition at
25 mg/kg po. Of the analogs bearing a substituent at R2
(3e–j) the most potent on oral dosing were dimethylani-
line 3e and piperazine 3g with 88% (n ¼ 1) and 67%
(n ¼ 2) inhibition, respectively, at 50 mg/kg, and ether
3j, which gave 42% inhibition at 25 mg/kg.
2. Feldmann, M.; Maini, R. N.; Bondeson, J.; Taylor, P.;
Foxwell, B. M.; Brennan, F. M. Adv. Exp. Med. Biol.
2001, 490, 119–127.
3. Takahi, K.; Tomita, T.; Nakase, T.; Kaneko, M.; Takano,
H.; Myoui, A.; Hashimoto, J.; Ochi, T.; Yoshikawa, H.
J. Musculoskelet. Res. 2002, 6, 63–71.
The pharmacokinetics of 2 and 3g were examined after a
5 mg/kg iv dose in Balb/c mice. Piperazine 3g had a
longer half-life than 2, 1.3 and 0.2 h, respectively, but
both were rapidly cleared (6.0 vs 8.3 L/h kg). The met-
abolic stability of 3g after incubation with CD-1 mouse
microsomes was also evaluated and compared to 2.
Piperazine 3g was far more stable than 2 and had only
one significant metabolite, resulting from conversion of
the hydroxamate into the corresponding carboxylic acid.
Products resulting from cleavage of the butynyl ether
were not detected for 3g. In contrast, 2 had three major
metabolites, including oxidation of the phenyl sulfon-
amide ring, cleavage of the butynyl ether P10 group and,
primarily, reduction of the hydroxamate to the corre-
sponding amide.
4. For reviews on small molecule TACE inhibitors, see: (a)
Skotnicki, J. S.; Levin, J. I. TNF-a Converting Enzyme
(TACE) as a Therapeutic Target. In Annual Reports in
Medicinal Chemistry; Bristol, J. A., Ed.; Academic: San
Diego, 2003; pp 153–162; (b) Moss, M. L.; White, J. M.;
Lambert, M. H.; Andrews, R. C. Drug Discovery Today
2001, 6, 417–426; (c) Nelson, F. C.; Zask, A. Exp. Opin.
Invest. Drugs 1999, 8, 383–392.
5. Skiles, J. W.; Gonnella, N. C.; Jeng, A. Y. Curr. Med.
Chem. 2001, 8, 425–474.
6. Wasserman, Z. R.; Duan, J. J.-W.; Voss, M. E.; Xue, C.-
B.; Cherney, R. J.; Nelson, D. J.; Hardman, K. D.;
Decicco, C. P. Chem. Biol. 2003, 10, 215–223.
7. (a) Yoshihara, Y.; Nakamura, H.; Obata, K.; Yamada,
H.; Hayakawa, T.; Fujikawa, K.; Okada, Y. Ann. Rheum.
Dis. 2000, 59, 455–461; (b) Konttinen, Y. T.; Ainola, M.;
Valleala, H.; Ma, J.; Ida, H.; Mandelin, J.; Kinne, R. W.;
Santavirta, S.; Sorsa, T.; Lopez-Otin, C.; Takagi, M. Ann.
Rheum. Dis. 1999, 58, 691–697.
Compounds 3l, 3n, and 3p, all substituted at R3, were
each tested at 25 mg/kg po and unfortunately none gave
better than 25% inhibition of LPS-stimulated TNF
production.
8. Nelson, F. C.; Delos Santos, E.; Levin, J. I.; Chen, J. M.;
Skotnicki, J. S.; DiJoseph, J. F.; Sharr, M. A.; Sung, A.;
Killar, L. M.; Cowling, R.; Jin, G.; Roth, C. E.; Albright,
J. D. Bioorg. Med. Chem. Lett. 2002, 12, 2867–2870.
9. (a) Levin, J. I.; Chen, J. M.; Du, M. T.; Nelson, F. C.;
Killar, L. M.; Skala, S.; Sung, A.; Jin, G.; Cowling, R.;
Barone, D.; March, C. J.; Mohler, K. M.; Black, R. A.;
Skotnicki, J. S. Bioorg. Med. Chem. Lett. 2002, 12, 1199–
1202; (b) Levin, J. I.; Chen, J. M.; Cheung, K.; Cole, D.;
Crago, C.; Delos Santos, E.; Du, X.; Khafizova, G.;
MacEwan, G.; Niu, C.; Salaski, E. J.; Zask, A.; Cummons,
T.; Stratman, T.; Killar, L. M.; Mulvey, R.; Skala, S.;
Sung, A.; Xu, J.; Zhang, Y.; Xu, W.; Ayral-Kaloustian, S.;
Jin, G.; Cowling, R.; Barone, D.; Mohler, K. M.; Black,
R. A.; Skotnicki, J. S. Bioorg. Med. Chem. Lett. 2003, 13,
2799–2803.
In conclusion, we have developed efficient synthetic
routes to a variety of functionalized racemic benzodi-
azepine-sulfonamide hydroxamic acids. Many of these
analogs have been shown to be potent inhibitors of
TACE and MMP-13 and some demonstrate selectivity
over MMP-1. The incorporation of polar functionality
into the benzodiazepine scaffold at any of three different
positions was also found to provide greatly increased
aqueous solubility (>60 lg/mL) for all of the compounds
that were assessed (3c–e, 3i, 3n, and 3p) relative to
compound 2 (5 lg/mL). Furthermore, three members of
this series, 2, 3e, and 3g, were shown to be effective at
inhibiting LPS-stimulated TNF production on oral
dosing in mice at 50 mg/kg. The isolation of the active
enantiomer of each of these racemates should further
enhance activity and the potential utility of these com-
pounds for the treatment of TNF-mediated inflamma-
tory diseases.
1
10. All new compounds gave satisfactory H NMR, IR, and
MS in accord with their assigned structure.
11. (a) Weingarten, H.; Feder, J. Anal. Biochem. 1985, 147,
437; (b) Inhibitor concentrations were run in triplicate.
MMP IC50 determinations were calculated from a four-
parameter logistic fit of the data within a single experi-

J. I. Levin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4147–4151
4151
ment; (c) Jin, G.; Huang, X.; Black, R.; Wolfson, M.;
Rauch, C.; McGregor, H.; Ellestad, G.; Cowling, R. Anal.
Biochem. 2002, 302, 269–275.
Christ, D. D.; Hardman, K. D.; Ribadeneira, M. D.;
Newton, R. C.; Trzaskos, J. M.; Decicco, C. P. Abstracts
of Papers, 226th National Meeting of the American
Chemical Society, New York, NY, Sept 7–11, 2003;
American Chemical Society: Washington, DC, 2003; Medi
201.
12. Levin, J. I.; Chen, J. M.; Cole, D. C. WO 0044709, 2000;
Chem. Abstr. 2000, 133, 150908.
13. Schlondorff, J.; Becherer, J. D.; Blobel, C. P. Biochem. J.
2000, 347, 131–138.
14. Duan, J. J.-W.; Ott, G. R.; King, B. W.; Maduskuie, T. P.;
Xue, C.-B.; Chen, L.; Lu, Z.; Gilmore, J. L.; Asakawa, N.;
Mercer, S. E.; Xu, M.; Harris, C. M.; Wasserman, Z. R.;
Liu, R.-Q.; Covington, M. B.; Qian, M.; Vaddi, K. G.;
15. Mohler, K. M.; Sleath, P. R.; Fitzner, J. N.; Cerretti, D.
P.; Alderson, M.; Kerwar, S. S.; Torrance, D. S.; Otten-
Evans, C.; Greenstreet, T.; Weerawarna, K.; Kronheim, S.
R.; Petersen, M.; Gerhart, M.; Kozlosky, C. J.; March, C.
J.; Black, R. A. Nature 1994, 370, 218–220.