β-N-Biaryl ether sulfonamide hydroxamates as potent gelatinase inhibitors: Part 1. Design, synthesis, and lead identification

Article abstract of DOI:10.1016/j.bmcl.2007.11.119

A new series of β-N-biaryl ether sulfonamide hydroxamates as novel gelatinase inhibitors is described. These compounds exhibit good potency for MMP-2 and MMP-9 without inhibiting MMP-1. The structure-activity relationships (SAR) reveal the biaryl ether ty

Full text of DOI:10.1016/j.bmcl.2007.11.119

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1135–1139
b-N-Biaryl ether sulfonamide hydroxamates as potent gelatinase
inhibitors: Part 1. Design, synthesis, and lead identification
Shyh-Ming Yang,* Robert H. Scannevin, Bingbing Wang, Sharon L. Burke,
Lawrence J. Wilson, Prabha Karnachi, Kenneth J. Rhodes,
Bharat Lagu and William V. Murray
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 8 Clarke Drive, Cranbury, NJ 08512, USA
Received 8 October 2007; revised 28 November 2007; accepted 30 November 2007
Available online 5 December 2007
Abstract—A new series of b-N-biaryl ether sulfonamide hydroxamates as novel gelatinase inhibitors is described. These compounds
exhibit good potency for MMP-2 and MMP-9 without inhibiting MMP-1. The structure–activity relationships (SAR) reveal the
biaryl ether type P10 moiety together with methanesulfonamide is the optimal combination that provides inhibitory activity of
MMP-9 in the single-digit nanomolar range.
Ó 2007 Elsevier Ltd. All rights reserved.
Matrix metalloproteinases (MMPs) are a family of
structurally related zinc-dependent proteolytic enzymes
that digest extracellular matrix proteins such as colla-
gen, elastin, laminin, and fibronectin. The uncontrolled
activity of the gelatinase sub-family of MMPs, gelatin-
ase A (MMP-2) and gelatinase B (MMP-9), has been
implicated in a number of pathological events leading
to cancer, inflammatory diseases, cardiovascular dis-
eases, and neurological disorders.1 Therefore, inhibiting
MMP-2 and/or MMP-9 activity as a potential treatment
in these therapeutic areas is highly desirable.2
hydroxamate moiety.4 A representative example of a
drug candidate based on this design, CGS-27023A
(Fig. 1), has advanced in clinical trials for the potential
treatment of cancer.5
In our program directed toward novel gelatinase inhib-
itors, we envisioned the b-N-biaryl ether sulfonamide
hydroxamates (Fig. 1, B) by simply switching the P10
S1 pocket
S1 pocket
N
H
P1
P1
N
H
O
O
R1
During the past two decades, the sulfonamide-based
hydroxamates, particularly the a-sulfonamide hydroxa-
mates (Fig. 1, A), have attracted a significant level of
attention in the design of MMP inhibitors.3 These syn-
thetic inhibitors contain two key features which include:
(1) a hydroxamate moiety as the Zn-binding group
(ZBG) and (2) a sulfonyl group that provides a vital
H-bonding interaction with the enzyme backbone. This
critical interaction directs the P10 group into the S10
pocket. In addition, having the P1 group at the a-posi-
tion in a favorable configuration may contribute to the
potency and/or slow down the metabolism of the
L
S
O
O
H
N
H
N
H
N
H
S
O
O
N
H
O
O
O
O
P1'
Zn
P1'
Zn
S1' pocket
R2
R2
S1' pocket
α-sulfonamide hydroxamate (A)
L= linkage groups, ex. O, N,
β-N-biaryl ether sulfonamide
O
O
H
N
hydroxamate (B)
S
HO
N
O
OMe
N
Keywords: Gelatinase; Matrix metalloproteinase; MMP inhibitor;
Hydroxamate.
CGS-27023A
*
Figure 1. a-Sulfonamide hydroxamates (A) versus b-N-biaryl ether
sulfonamide hydroxamates (B), and structure of CGS-27023A.
Corresponding author. Tel.: +1 609 409 3491; fax: +1 609 655
6930; e-mail: syang9@prdus.jnj.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.119

1136
S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1135–1139
moiety from the sulfonyl group to the nitrogen atom
based on the traditional approach illustrated by struc-
ture A.6 The sulfonamide unit was transferred from
the a- to b-position to further maintain a favorable dis-
tance between the hydroxamate moiety and P10 group.
Various substituents could be further introduced at the
a-position by using a heteroatom (e.g., O, N) linker
(L) to provide a possible interaction with the S1 subsite.
Additionally, the substituent on the sulfonamide (R1)
may also offer potential interactions with the S20 and/
or the S30 subsites. Herein, we disclose our preliminary
results in the discovery of b-N-biaryl ether, sulfon-
amide-based hydroxamates as novel MMP-2/MMP-9
inhibitors. Evaluating the enzyme structure–activity
relationships (SAR) by modifying the R1 substituent,
the P10 group, the chirality of the a-carbon center, and
the position of N-biaryl ether sulfonamide moiety facil-
itated in identifying new chemical leads.
diverse sulfonyl chlorides. Under basic conditions, sul-
fonamides 2 could promote facile ring-opening reactions
with epoxides, such as methyl (S)-glycidate, to afford
(S)-4 with the desired skeleton constructed.7 This ring-
opening process could be performed either thermally
in the presence of a catalytic amount of the phase trans-
fer reagent BnEt3NCl, or by using microwave irradia-
tion, with similar efficiency. The esters (S)-4 were
further converted to the corresponding hydroxamates
(S)-5 using standard hydroxamate formation conditions
(hydroxylamine hydrochloride salt with sodium methox-
ide in methanol).8 The carboxylic acid (S)-6 and
hydroxamate (S)-7 were also prepared from (S)-4 by
hydrolysis or by alkylation followed by hydroxamate
formation, respectively. These sequences were applicable
in the synthesis of both enantiomers, depending on the
chirality of the epoxide employed. Removal of the hy-
droxyl group of (S)-4 gave sulfonamides 8. This
extended analog could be directly compared to the a-
N-biaryl ether sulfonamides 3. Alternatively, direct
epoxide opening of methyl (S)-glycidate with 1 followed
by acylation and hydroxamate formation gave b-N-biar-
yl ether amide hydroxamates (S)-9. Compound 10 was
prepared in a similar fashion (Scheme 2).
To test the feasibility of this design and rapidly explore
the SAR of the R1 substituent and P10 group, a two-step
synthetic sequence was investigated to efficiently assem-
ble the key intermediates (S)-4 (Scheme 1). The synthesis
of (S)-4 began with the formation of sulfonamides 2
from commercially available biaryl ether anilines 1 and
Additionally, other potential P10 moieties containing an
extended linker, such as an acetylene ((S)-13) or a more
flexible OCH2 ((S)-14), were also synthesized (Scheme
3). For preparation of (S)-13, the Sonogashira coupling
was applied to introduce the linear biaryl acetylene from
(S)-12a (R = I). On the other hand, when R is a triiso-
propylsiloxy group (OTIPS, (S)-12b), the deprotection
followed by an alkylation with substituted aryl halides
and then hydroxamate formation gave the desired com-
pound (S)-14.
O
O
O
NH2
O
O
HO
S
HO
N
H
N
Me
N
H
N
R1
i,j,d
OH
X
Y
O
O
R2
1
8
(S)-9
Me
Me
Me
Cl
a
g,h,d
O
S
O
O
O
O
S
Several compounds were prepared and assayed in vitro
against MMP-9 and MMP-2 (Table 1)9 to rapidly iden-
tify suitable leads and prove the proposed concept.
Broad spectrum MMP inhibitors have been implicated
in musculoskeletal syndrome (MSS), a phenomenon ob-
served in clinical trials.10 It has been speculated that the
MSS results from long-term inhibition of MMP-1.11
Therefore, the inhibitory activities of MMP-1 were also
tested to determine the selectivity for MMP-2/-9 versus
MMP-1. In general, these compounds showed moderate
activities against MMP-2 and slightly better potency to-
O
O
O
HN
R1
S
MeO
N
R1
HO
N
Me
OH
c
OH
b
X
Y
X
Y
O
R2
R2
(S)-4
(S)-6
2
f,d
e,d
d
O
O
S
O
O
O
O
O
O
O
HO
Me
S
HO
S
R1
N
N
H
N
N
H
N
Me
ward
MMP-9,
without
inhibiting
MMP-1
NH
OH
OMe
HO
(IC50 > 10 lM). This is consistent with literature exam-
O
X
Y
O
O O
O
O O
S
R2
S
R1
R2
HO
(S)-5
(S)-7
HN
3
N
H
N
Me
HO Me
a,b
Scheme 1. Reagents and conditions: (a) R1SO2Cl, pyridine, 0 °C to rt,
1 h, 48–96%; (b) method A: methyl (S)-glycidate, K2CO3, BnEt3NCl,
dioxane, 60–90 °C, 55–91%. Method B: methyl (S)-glycidate, K2CO3,
DMF, 80–100 °C, 71–86%; (c) LiOH(aq), THF, 90%; (d) HONH2ÆHCl,
NaOMe, MeOH, 35–93%; (e) ethyl bromoacetate, K2CO3, DMF,
microwave, 120 °C, 30 min, 86–93%; (f) MeI, NaH, DMF, rt, 30 min,
55%; (g) Tf2O, 2,6-lutidine, ꢀ20 °C to 0 °C, 91%; (h) i—LiI, THF,
30 min; ii—Zn, cat. NiCl2Æ6H2O, cat. H2O, THF, 20 h, 55%; (i) methyl
(S)-glycidate, DMSO, 60 °C, 24 h, 70%; (j) Ac2O or iso-butyryl
chloride, pyridine, CH2Cl2, rt, 92–93%.
X= O, Y= CH
R1= R2= Me
X
Y
O
R2
2
10
Me
Scheme 2. Reagents and conditions: (a) methyl 2-methylglycidate,
K2CO3, DMF, 120 °C, 20 min, 88%; (b) HONH2ÆHCl, NaOMe,
MeOH, 67%.

S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1135–1139
1137
O
With this novel skeleton identified, the attention was
then focused on investigating the SAR around the sul-
fonyl group (R1) and the P10 moiety based on the lead
compound (S)-5a. Various substituents on the sulfon-
amide were initially evaluated with a fixed P10 group,
4-methyl-biphenyl ether. The significant shift of the
activity to micromolar range, observed from the re-
sults compiled in Table 2, revealed the increased steric
influence close to the SO2. For example, changing the
size from methyl ((S)-5a) to ethyl ((S)-5b), n-propyl
((S)-5c), iso-propyl ((S)-5d), NMe2 ((S)-5i), and phenyl
((S)-5e) resulted in ca. 3- to 44-fold loss of potency
against both MMP-2 and MMP-9. However, moving
the phenyl group slightly away by inserting a methy-
lene, for example by replacing the phenyl with a Bn
((S)-5f), brought the potency back to double-digit
nanomolar range against MMP-9. A similar trend
for examples (S)-5g–h was also observed. These results
suggested that altering the interaction between S20/S30
subsites is critical and that the methyl substituent
seems to be a superior functionality based on the
in vitro results.
O O
S
HO
N
H
N
Me
OH
c,d
O
(
S
)-12a
R= I
O O
S
NH2
MeO
a,b
N
Me
(S)-13
R1
OH
O
O O
S
HO
R
R
N
H
N
Me
OH
e,f,d
11
(S)-12
(
S
)-12b
R= OTIPS
O
R1
(S)-14
Scheme 3. Reagents and conditions: (a) MeSO2Cl, pyridine, 0 °C, 93–
98%; (b) methyl (S)-glycidate, K2CO3, BnEt3NCl, dioxane, 60–90 °C,
71–73%; (c) R1CꢁCH, PdCl2(PPh3)2 (10 mol%), CuI (10 mol%), Et3N,
THF, rt, 2 h, 81–93%; (d) HONH2ÆHCl, NaOMe, MeOH, 89–96%; (e)
TBAF, THF, 0 °C to rt, 1 h, 72%; (f) R1CH2Br, K2CO3, acetone, 50–
55 °C, 72–74%.
The optimization of the P10 group was next performed.
Several directions were explored, including the linker
group between the biaryl, the shape of the P10 unit,
and the substitution effect on the aryl ring. Among the
compounds tested, the oxygen linker (biaryl ether) was
found to be optimal. As shown in Table 2, replacing
the oxygen linker with a sulfur atom decreased the
IC50 around 5-fold for both MMP-2 and MMP-9 ((S)-
5n versus (S)-5a). Moreover, complete loss of activity
was found by using SO2 ((S)-5p) as the linker. The activ-
ities also dropped significantly when the more flexible
and longer OCH2 ((S)-14a–c) was employed as the lin-
ker group. The rigid, linear P10 moiety, elongated by
an acetylene unit ((S)-13a–c), provided double-digit
nanomolar activities. These results were better than
(S)-5q which contained a shorter biphenyl P10 group.
Some of the analogs represented by (S)-13 and (S)-14
showed a slight preference for the MMP-2 over the
MMP-9 enzyme. This preference may be attributed to
the tunnel-like S10 subsite of MMP-2, which may toler-
ate the longer P10 moiety better than the MMP-9 S10
ples showing evidence that the shallow S10 pocket of
MMP-1 typically favors a shorter P10 group. The b-N-
biaryl ether sulfonamide 8 has an IC50 value of 6.6 nM
against MMP-9 that is about 5-fold more potent than
3b (31 nM, an a-N-biaryl ether sulfonamide). Notably,
this b-N-biaryl ether skeleton exhibited some selectivity
(ca. 4- to 12-fold) for MMP-9 over MMP-2 as observed
in compounds 5a, 7–8, and 10. The introduction of small
a-substituents has a minor effect on IC50 (e.g., (S)-5a,
(S)-7, and 10 vs 8). Surprisingly, the chirality of the
a-position ((R)-5a vs (S)-5a and (R)-7 versus (S)-7) mar-
ginally influenced potency. These results strongly sug-
gested that small substituents, such as OH, Me, and
OMe, were well tolerated at the a-position in the present
skeleton. On the other hand, using the carboxylic acid as
the ZBG (e.g., (S)-6) eliminated activity for both MMP-
2 and MMP-9. Meanwhile, changing the sulfonamide to
an amide also caused a 120-fold loss of potency ((S)-5a
versus (S)-9a). These preliminary results clearly demon-
strated the advantage and potential of a b-N-biaryl ether
sulfonamide moiety in the design of MMP inhibitors.
Table 1. Lead structure/skeleton identification: SAR of 3, 5a, and 6–10
R1
R2
MMP-1 IC50 (lM)
a
a
MMP-2 IC50 (nM)
a
MMP-9 IC50 (nM)
Compound
3a
3b
—
—
H
>10
—
147b
73
383b
31
Me
—
8
—
>10
>10
>10
>10
—
61b
6.6b
12b
(R)-5a
(S)-5a
10c
Me
Me
—
Me
Me
—
67b
61b
7.8b
5.5b
<10%d
13
3.8b
933
242
20b
(S)-6
(R)-7
(S)-7
(S)-9a
(S)-9b
—
—
—
—
<10%d
102
36b
>10
>10
—
—
—
—
Me
i-Pr
1262
286
—
—
a Single experiment.
b Values are means of at least two experiments.
c Racemic mixture.
d % inhibition at 10 lM.

1138
S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1135–1139
Table 2. SAR of (S)-5, (S)-13–14 on substituents of sulfonamide and P10 moiety
Compound
X
Y
R1
R2
MMP-1
a
IC50 (lM)
MMP-2
a
IC50 (nM)
MMP-9
a
IC50 (nM)
Fold selectivity
MMP-2/MMP-9
(S)-5a
(S)-5b
(S)-5c
(S)-5d
(S)-5e
(S)-5f
O
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
>10
>10
>10b
>10b
>10b
>10b
>10b
>10b
>10b
>10
>10
—
61
7.8
7.8
5.1
4.1
4.3
6.9
6.3
5.8
6.4
4.3
6.3
7.4
9.9
5.4
5.5
2.7
—
O
142
28
O
n-Pr
i-Pr
509b
1233b
2382b
582b
401b
344b
1057b
247
124b
286b
344b
93b
O
O
Ph
Bn
O
(S)-5g
(S)-5h
(S)-5i
O
(pyridin-3-yl)CH2
69b
54b
243b
39
O
(pyridin-4-yl)CH2
NMe2
Me
O
(S)-5j
(S)-5k
(S)-5l
O
O
Me
Me
Cl
35
139
4.7
14
O
CF3
CF3
Me
F
(S)-5m
(S)-5n
(S)-5o
(S)-5p
(S)-5q
(S)-13a
(S)-13b
(S)-13c
(S)-14a
(S)-14b
(S)-14c
O
Me
Me
—
—
2535
297
472
54
S
CH
CH
CH
CH
—
S
Me
Me
—
—
283
104
>10 lMb
79%b,c
44
SO2
—
—
—
—
—
—
—
H
>10 lMb
87%b,c
47
Me
H
—
—
—
4-MePh
4-ClPh
4-MeOPh
4-FPh
4-ClPh
4-CF3Ph
>10
>10b
>10b
>10b
>10b
>10b
1.1
1.2
0.4
0.5
0.9
1.9
—
—
—
—
56
47
32
91
—
—
—
—
2317b
893b
2917b
4819b
984b
1489b
—
—
a Values are means of at least two experiments.
b Single experiment.
c % inhibition at 10 lM.
Table 3. PKa, protein binding, and in vitro microsomal stability of (S)-5a and 10
AUCb (lg h/mL)
Vssb (L/kg)
CLb (mL/min/kg)
HLM/RLMc (t1/2, min)
PBc (%)
b
b
t1/2 (h)
Compound
Cmax (lM)
(S)-5a
10
1.3
1.5
0.43
0.72
0.18
0.22
1.4
1.3
47.5
41.5
>2 h/14
109/10
95
83
a Mean value of 4 animals (rats) at 0.5 mg/kg iv dose.
b Cmax, plasma concentration at t = 5 min; t1/2, apparent elimination half-life; AUC, area under curve; Vss, volume of distribution at steady state; CL,
clearance level.
c HLM, human liver microsome; RLM, rat liver microsome; PB, protein binding.
pocket. The nature of the substituent (R2) on the aryl
ring is also important. For instance, without substitu-
tion at the para-position ((S)-5j, R2 = H) the compounds
were ca. 4-fold less potent. The chlorine substituent ((S)-
5k) exhibited similar activities as the methyl ((S)-5a).
Electron-withdrawing groups, such as CF3 or F, only
slightly influenced the activity, (S)-5a versus (S)-5l and
(S)-5n versus (S)-5o. Finally, replacement of the phenyl
ring with a heteroaryl ((S)-5m) caused the potency to
drop significantly. This is consistent with the fact that
the hydrophobic residues that surround the S10 pocket
typically favor a more lipophilic P10 moiety.
erate water solubility (28–70 lg/mL) at pH 7.0, but were
relatively insoluble (<5 lg/mL) at pH 2.0.
In summary, a promising series of gelatinase (MMP-2
and MMP-9) inhibitors bearing a novel b-N-biaryl ether
sulfonamide moiety has been identified. The preliminary
SAR suggested that the methanesulfonyl group together
with biaryl ether type P10 moiety is the optimal combi-
nation to afford single-digit nanomolar activities against
MMP-9. This skeleton also exhibited great selectivity for
MMP-9/MMP-2 over MMP-1. Notably, some analogs
showed low PB. The introduction and optimization of
an a-amino substituent to further increase potency and
improve the ADME properties has been investigated.
These results will be disclosed in the following
communication.
Compounds (S)-5a and 10 were initially selected and
their pharmacokinetics (PK, rats) and other ADME
properties evaluated (Table 3). In general, a relatively
poor PK profile was obtained for both analogs. The
low plasma exposure (low Cmax and AUC values) may
be attributed to the high clearance rate (CL) and poor
rat liver microsomal (RLM) stability. Notably, several
analogs exhibited low protein binding (PB), particularly
10 (PB = 83%). In addition, these compounds had mod-
Acknowledgments
Many helpful discussions and suggestions from Dr. Paul
Jackson are gratefully acknowledged. The authors also

S.-M. Yang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1135–1139
1139
celled due to lack of clear efficacy in Phase II trials, and to
side effects).
thank the Chem/Bio Support (CBS) Group for ADME
tests and the reviewers for the great comments.
6. In the design of amide- or sulfonamide-based MMP
inhibitors, typically, the biaryl ether portion (P10) was
attached to the sulfonyl group instead of on the nitrogen
atom. Few exceptions, see: (a) Cherney, R. J.; Mo, R.;
Meyer, D. T.; Hardman, K. D.; Liu, R.-Q.; Covington, M.
B.; Qian, M.; Wasserman, Z. R.; Christ, D. D.; Trzaskos,
J. M.; Newton, R. C.; Decicco, C. P. J. Med. Chem. 2004,
47, 2981; (b) Kim, S.-H.; Pudzianowski, A. T.; Leavitt, K.
J.; Barbosa, J.; McDonnell, P. A.; Metzler, W. J.; Rankin,
B. M.; Liu, R.; Vaccaro, W.; Pitts, W. Bioorg. Med. Chem.
Lett. 2005, 15, 1101.
References and notes
1. Recent reviews, see: (a) Fingleton, B. Curr. Pharm. Des.
2007, 13, 333; (b) Fingleton, B. Exp. Opin. Ther. Targets
2003, 7, 385; (c) Vihinen, P.; Ala-aho, R.; Ka¨ha¨ri, V.-M.
Curr. Cancer Drug Targets 2005, 5, 203; (d) George, S. J.
Exp. Opin. Invest. Drugs 2000, 9, 993; (e) Yong, V. W.
Exp. Opin. Invest. Drugs 1999, 8, 255; (f) Yong, V. W.;
Power, C.; Forsyth, P.; Edwards, D. R. Nat. Rev.
Neurosci. 2001, 2, 502.
2. Recent reviews, see: (a) Whittaker, M.; Floyd, C. D.;
Brown, P.; Gearing, A. J. H. Chem. Rev. 1999, 99, 2735;
(b) Matter, H.; Schudok, M. Curr. Opin. Drug Discov.
Dev. 2004, 7, 513; (c) Fisher, J. F.; Mobashery, S. Cancer
Metastasis Rev. 2006, 25, 115; (d) Sang, Q.-X. A.; Jin, Y.;
Newcomer, R. G.; Monroe, S. C.; Fang, X.; Hurst, D. R.;
Lee, S.; Cao, Q.; Schwartz, M. A. Curr. Top. Med. Chem.
2006, 6, 289.
7. (a) Yang, S.-M.; Murray, W. V. Tetrahedron Lett. 2008,
doi:10.1016/j.tetlet.2007.11.184; (b) Albanese, D.; Landini,
D.; Lupi, V.; Penso, M. Ind. Eng. Chem. Res. 2003, 42, 680
(and references cited therein).
8. The condition was adopted from Ref. 5.
9. MMP IC50s were determined using a standard peptide-
cleavage assay. The catalytic domains from MMP-1,
ꢀ9, and full-length MMP-2 were purchased from Life
Diagnostics (West Chester, PA) and PanVera/Invitrogen
(Madison, WI). Enzymes were diluted into assay buffer
(50 mM Hepes, pH 7.4, 10 mM CaCl2, and 0.05% Brij-
3. Supuran, C. T.; Casini, A.; Scozzafava, A. Med. Res. Rev.
2003, 23, 535.
4. It has been addressed that the metabolism of hydroxamic
acid might be reduced by sterically hindering this func-
tionality, see: (a) Reiter, L. A.; Robinson, R. P.; McClure,
K. F.; Jones, C. S.; Reese, M. R.; Mitchell, P. G.;
Otterness, I. G.; Bliven, M. L.; Liras, J.; Cortina, S. R.;
Donahue, K. M.; Eskra, J. D.; Griffiths, R. J.; Lame, M.
E.; Lopez-Anaya, A.; Martinelli, G. J.; McGahee, S. M.;
Yocum, S. A.; Lopresti-Morrow, L. L.; Tobiassen, L. M.;
Vaughn-Bowser, M. L. Bioorg. Med. Chem. Lett. 2004, 14,
3389; (b) Noe, M. C.; Natarajan, V.; Snow, S. L.; Mitchell,
P. G.; Lopresti-Morrow, L.; Reeves, L. M.; Yocum, S. A.;
Carty, T. J.; Barberia, J. A.; Sweeney, F. J.; Liras, J. L.;
Vaughn, M.; Hardink, J. R.; Hawkins, J. M.; Tokar, C.
Bioorg. Med. Chem. Lett. 2005, 15, 2808.
5. MacPherson, L. J.; Bayburt, E. K.; Capparelli, M. P.;
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
(further clinical trials with CGS-27023A have been can-
35) and added to
a 384-well plate that contained
appropriate dilutions of test compounds prepared in
assay buffer containing 0.1% DMSO and incubated for
1 h at room temperature. Substrate (acetyl-Cys(Eu)-Pro-
Leu-Gly-Leu-Lys-(QSY7)-Ala-Arg-amide)
was
then
added to each well and incubated for 15 min at room
temperature. Peptide-cleavage was detected on an
EnVision reader (Perkin-Elmer, Wellesley, MA), mea-
suring Europium fluorescence that had been liberated
by removal of the QSY7 quenching. Each enzyme was
individually titrated to determine the optimal concen-
tration that resulted in linear reaction velocity during
the course of the experiment. Final concentration of
DMSO in each well was 0.04%. IC50s were determined
in triplicate for each compound using 16-point concen-
tration–response curves and fit using nonlinear regres-
sion analysis in Graphpad Prism 4.0 (Graphpad
Software, San Diego, CA).
10. Peterson, J. T. Heart Failure Rev. 2004, 9, 63.
11. Brown, P. D. APMIS 1999, 107, 174.