Synthesis and activity of tryptophan sulfonamide derivatives as novel non-hydroxamate TNF-α converting enzyme (TACE) inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.04.033

A novel series of non-hydroxamate tryptophan sulfonamide derivatives containing a butynyloxy P1′ moiety was identified as inhibitors of TNF-α converting enzyme (TACE). The structure-activity relationship of the series was examined via substitution on the

Full text of DOI:10.1016/j.bmc.2009.04.033

Bioorganic & Medicinal Chemistry 17 (2009) 3857–3865
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and activity of tryptophan sulfonamide derivatives as novel
non-hydroxamate TNF-a converting enzyme (TACE) inhibitors
a
a
a
b
c
Kaapjoo Park a, , Ariamala Gopalsamy , Alexis Aplasca , John W. Ellingboe , Weixin Xu , Yuhua Zhang ,
*
Jeremy I. Levin a
a Chemical Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
b Chemical Sciences, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
c Inflammation, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA
a r t i c l e i n f o
a b s t r a c t
A novel series of non-hydroxamate tryptophan sulfonamide derivatives containing a butynyloxy P10 moi-
Article history:
Received 25 February 2009
Revised 15 April 2009
Accepted 16 April 2009
Available online 22 April 2009
ety was identified as inhibitors of TNF-a converting enzyme (TACE). The structure–activity relationship of
the series was examined via substitution on the tryptophan indole ring. Of the compounds investigated,
2-(4-(but-2-ynyloxy)phenylsulfonamido)-3-(1-(4-methoxybenzyl)-1H-indol-3-yl)propanoic acid (12p)
has the best in vitro potency against isolated TACE enzyme with an IC50 of 80 nM. Compound 12p also
shows good selectivity over MMP-1, -13, -14.
Keywords:
Non-hydroxamate
Ó 2009 Elsevier Ltd. All rights reserved.
TNF-a converting enzyme (TACE) inhibitor
Tryptophan sulfonamide derivatives
Martix metalloprotease (MMP)
1. Introduction
structural similarities between the active sites of MMPs and TACE,
and many of these inhibitors show limited selectivity for TACE over
TNF-
and pro-inflammatory cytokine and is responsible for the initiation
of protective inflammatory reactions.1 Also, TNF-
has been impli-
a
(tumor necrosis factor-
a
) is a major immunomodulatory
MMPs.8 While TACE inhibitors with significant activity against
some MMPs may provide an advantage in treating RA, since vari-
ous MMPs are over-expressed in RA synovial tissue and contribute
to joint destruction,9 most broad spectrum MMP inhibitors have
suffered from dose-limiting toxicity in clinical trials.10 More re-
cently, a number of inhibitors of TACE with excellent selectivity
over MMPs have been reported.11 While most of these inhibitors
have incorporated a hydroxamic acid moiety as a bidentate chela-
tor of the TACE active site zinc, certain liabilities of hydroxamates,
such as poor pharmacokinetics (PK) due to hydrolysis, reduction
and O-glucuronidation of this functional group, have led to the
search for alternative zinc chelators.12 In the past several years,
non-hydroxamate TACE inhibitors have been reported13 (Fig. 1)
wherein the ubiquitous hydroxamate zinc binding group (ZBG)
has been replaced by other ZBGs such as a pyrimidinetrione (1,
2), hydantoin (3), triazolone (4), imidazolone (5) and triazolethione
(6). Herein, we report the synthesis and SAR of a series of carbox-
ylic acid TACE inhibitors derived from tryptophan.
a
cated in several autoimmune disorders including rheumatoid
arthritis (RA),2 Crohn’s disease, and psoriasis.3 Biological agents
that modulate TNF levels, such as etanercept (EnbrelÒ),4 a soluble
TNF-
a
receptor, and infliximab (RemicadeÒ),5 and adalimumab
(HumiraÒ),6 monoclonal TNF-
a
antibodies, have been proven to
be clinically efficacious in treating these diseases. The efficacy of
these biological agents has led to the search for novel, orally active,
and selective small molecule inhibitors of TACE for the treatment
of these diseases. TACE (TNF-a converting enzyme) is a zinc endo-
peptidase and a member of the ADAM (a disintegrin and metallo-
protease-containing enzyme) family of proteases (ADAM17). TACE
is the primary enzyme responsible for cleaving 26 kDa membrane-
bound TNF-a, pro-TNF-a . The
, to generate 17 kDa soluble TNF-a 7
potential for inhibitors of TACE to treat RA by affecting levels of
soluble TNF has made the development of orally active small mol-
ecule TACE inhibitors the subject of intense interest for the last
decade.
Many TACE inhibitors have been derived from the modification
of matrix metalloprotease (MMP) inhibitor scaffolds because of the
2. Results and discussion
2.1. Chemistry
Structure-based design at Wyeth previously led to the discov-
ery of sulfonamide14 and sulfone hydroxamate11a–d,15 TACE inhib-
* Corresponding author. Tel.: +1 845 602 4331; fax: +1 845 602 3045.
E-mail addresses: ParkK3@wyeth.com, Kaapjoo@hotmail.com (K. Park).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.033

3858
K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
N
O
N
N
R NH
O
N
HN
O
O
O
N
H
O
1
2-6
R =
O
Ms
N
N
O
N
O
O
O
N
NH
HN
HN
NH
HN
HN
N
HN
NH
O
O
N
H
N
H
O
S
O
2
4
6
3
5
Figure 1. Reported non-hydroxamate TACE inhibitors.
itors bearing a novel butynyloxy P10 group. The butynyloxy P10
group provides varying levels of selectivity for TACE, and cellular
activity, depending on the scaffold employed. In order to search
for sulfonamide carboxylate TACE inhibitors, 4-butynyloxyben-
zenesulfonyl chloride 714b was reacted with 73 commercially
available amino acids (Fig. 2). The resulting sulfonamide carboxyl-
tionalized as shown in Scheme 1. Thus, mono-substituted
tryptophan 10 reacts with 4-butynyloxybenzenesulfonyl chloride
714b in the presence of triethylamine to give tryptophan sulfon-
amides 11a–n in high yield (88–93%). N-Alkylation of 9 with
alkyl halides in the presence of sodium hydride gives the desired
tryptophan sulfonamides 12a–q in good yield (70–85%
yield).
An alternative route, used for the synthesis of di- and tri-substi-
tuted tryptophan sulfonamide derivatives 19a–w, is shown in
Scheme 2. Indoles 13 react with oxime 14,16 prepared from ethyl
3-bromo-2-oxopropanoate, in the presence of sodium carbonate
at room temperature to give indole-oxime 15 as a 3:1 mixture of
regioisomers. Reduction of 15 with zinc powder in acetic acid then
provides tryptophanyl esters 16. Sulfonylation of 16 in the pres-
ence of triethylamine at room temperature gives benzenesulfon-
amide ester 17 (55–70% yield for three steps), which is then
ates were tested in a TACE FRET assay and the
sulfonamide derivatives, 8 and 9, were identified as the only single
digit micromolar inhibitors. Interestingly, -isomer 9 is three-fold
-isomer 8, with IC50s of 2.5 M and 8.2 M,
D and L tryptophan
D
more potent than
respectively.
L
l
l
In an effort to further explore the SAR of the tryptophan sul-
fonamides, analogs of 8 were prepared via two synthetic routes
depending on the availability of starting materials and targeted
molecules. In the first route, commercially available tryptophans
substituted on the phenyl ring of the indole group were func-
P1' Group
73 commercially
O
O
available
O
S
amino acids
O
S
Cl
DIPEA/THF/DMF
HN
O
O
7
O
R
OH
P1 Group
O
O
O
HO
HN
HN S
O
8 (L): TACE FRET IC50 = 8.2 µM
9 (D): TACE FRET IC50 = 2.5 µM
Figure 2. Screening the sulfonamides prepared by the reaction of 73 commercially available amino acids with 4-butynyloxybenzenesulfonyl chloride 7.

K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
3859
O
H
O
O
N S
O
O
O
S
O
HO
NH2
+
HO
Cl
O
a
NH
O
NH
7
R
R
O
N S
O
11a-n
10
O
H
HO
b
9
N
R
12a-q
Scheme 1. Preparation of 11a–n and 12a–q. Reagents and conditions: (a) triethylamine, rt, 20 h, H2O–dioxane, 88–93%; (b) NaH, 0 °C, 10 h, DMF, R2X (X = I, Br, Cl) 70–85%.
reacted with alkyl or benzyl halides in the presence of NaH at 0 °C
to give N-substituted tryptophanyl esters 18 in good to moderate
yields (65–85%). Finally, esters 18 are hydrolyzed with 2 N sodium
hydroxide solution in MeOH–THF at room temperature to give the
desired tryptophan sulfonamide derivatives 19a–w in high yield
(90–96% yield).
2.2. Biological assays
The prepared compounds 11a–n, 12a–q and 19a–w were tested
in a FRET assay using the catalytic domain of TACE17 and selected
analogs were then profiled for selectivity against MMP-1, MMP-2,
MMP-13 and MMP-14. Also, selected compounds were evaluated
Br
O
R1
O
a
R2
+
OEt
Br
OEt
O
N
H
N
HO
O
O
13
14
EtO
EtO
OH
N
NH2-HCl
R2
c
R1
R1
b
R2
N
H
N
H
16
15
O
d
O
N S
H
EtO
O
S
O
O
e
Cl
O
O
O
R2
R1
NH
7
17
O
O
O
N S
H
HO
H
EtO
O
N S
O
f
O
O
R2
R2
R1
R1
N
N
R3
R3
19a-w
18, R3 = alkyl, benzyl
Scheme 2. Preparation of 19a–w. Reagents and conditions: (a) (NH2OH)2–H2SO4, rt, 24 h, 90%; (b) Na2CO3, CH2Cl2, rt, 24 h; (c) (i) Zn, AcOH, rt, 12 h; (ii) HCl (d) TEA, rt, 20 h,
dioxane–H2O, 55–70% in three steps; (e) 2 equiv NaH, R3X (X = Br or Cl), 0 °C, 10 h, DMF, 65–85%; (f) NaOH, rt, 12 h, THF–MeOH, 90–96%.

3860
K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
Table 2
for their ability to inhibit LPS-stimulated TNF production in
Raw cells.17
Inhibition of N-substituted indole sulfonamide carboxylates in TACE assay
O
2.3. Structure–activity relationships
O
N S
HO
H
O
*
As shown in Table 1, racemic tryptophan analogs mono-substi-
tuted at the 5- or 6-position of the indole (11a, 11d, 11g, 11j–n) ex-
hibit better TACE enzyme activity than initial lead compound 9,
with the exception of the 6-fluoro derivative 11m. The 5-methyl
and 5-methoxy substituents provide the most activity. As for enan-
tiomers 8 and 9, chiral separation of 11a, 11d, and 11g, provided
one enantiomer (11c, 11f, 11i) that was greater than 10-fold more
potent than the other enantiomer (11b, 11e, 11h).
The activity of a series of N-substituted indolyl carboxylic acids
is shown in Table 2. Thus, most N-alkyl and N-benzyl substituents
are tolerated, showing activity similar to or better than 9, with the
notable exception of the ortho-trifluoromethylbenzyl analog 12j. In
particular, compound 12p bearing a p-methoxybenzyl group is by
O
N
R
Compound
R
Stereochem.
TACE IC50 (lM)
12a
12b
12c
12d
12e
12f
12g
12h
12i
BOC
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
L
3.67
1.43
2.31
1.71
1.25
2.15
0.97
0.54
0.98
2.4%a
2.30
4.40
3.59
1.90
3.45
0.08
1.18
Me
n-Pentyl
Cyclobutylmethyl
4-PhO-Bu
Bnb
o-Ph-Bn
p-Allyloxy-Bn
3,5-Dimethoxy-Bn
o-CF3-Bn
far the most active compound in this series with an IC50 of 0.08
31-fold more potent than initial lead 9. Also, as expected, isomer
12q is 15-fold less active than isomer 12p.
lM,
12j
12k
12l
L
m-CF3-Bn
p-CF3-Bn
D
A variety of racemic tryptophan derivatives di- and tri-substi-
tuted on the indole moiety were also prepared and evaluated
in vitro, as shown in Table 3. Of the substituents surveyed at the
5-position (R1) only the carboxylic acid group was not tolerated
(19a–c). Comparison of the di-substituted analog 19f with the cor-
responding mono-substituted compounds 11a and 12p indicates
that 2,5-disubstitution diminishes activity relative to the parent
mono-substituted analogs. Furthermore, no analog di-substituted
12m
12n
12o
12p
12q
p-Me-Bn
p-F-Bn
p-Cl-Bn
p-MeO-Bn
p-MeO-Bn
a
% Inhibition at 1 lM.
b
Bn = benzyl.
at R1 and R3 has an IC50 less than 0.89
analogs with activity less than 0.40
l
M. However, tri-substituted
Table 3
lM can be obtained. Thus, a
Inhibition of di- or tri-substituted indole sulfonamide carboxylates in TACE assay
significant effect on activity is seen with substitution at the 2-posi-
tion (R2), that increases potency more than four-fold (19j vs 19k,
19l vs 19m, and 19p vs 19q). When R1 and R2 are held constant
as methoxy and methyl, respectively, the best activity is seen from
the R3 benzyl derivative 19i, 3.5-fold more potent than the corre-
O
O
H
HO
N S
O
R2
O
R1
N
Table 1
R3
Inhibition of mono-substituted indole sulfonamide carboxylates in TACE assay
Compound
R1
R2
R3
TACE IC50 (lM)
O
O
19a
19b
19c
19d
19e
19f
19g
19h
19i
CO2H
CO2H
CO2H
MeO
Cl
H
H
H
H
Me
H
Me
>100b
>100b
>100b
1.23
0.30
0.89
0.19
0.90
0.25
1.50
0.33
2.24
0.28
0.50
0.49
2.24
0.30
0.34
0.32
0.29
0.33
0.41
0.47
H
HO
Et
N S
O
Bna
2-Methylpropyl
2-Methylpropyl
p-MeO-Bn
p-MeO-Bn
Me
O
Me
Cl
NH
Me
Me
Me
H
Me
H
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
Cl
R
Bn
19j
19k
19l
3,4-Methylenedioxy-Bn
3,4-Methylenedioxy-Bn
m-MeO-Bn
m-MeO-Bn
o-CF3-Bn
p-CF3-Bn
o-F-Bn
o-F-Bn
m-F-Bn
p-F-Bn
p-Cl-Bn
p-Cl-Bn
m-CN-Bn
Compound
R
TACEIC50 (lM)
19m
19n
19o
19p
19q
19r
19s
19t
8
9
H, (
H, (
L
)
8.20
2.53
0.28
27%a
0.14
0.36
10.1
0.14
0.75
6.98
0.37
1.56
1.61
0.63
2.96
1.57
D)
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
5-Me (racemic)
5-Me (enantiomer-1)
5-Me (enantiomer-2)b
5-MeO (racemic)
5-MeO (enantiomer-1)
5-MeO (enantiomer-2)
5-BnO (racemic)
5-BnO (enantiomer-1)
5-BnO (enantiomer-2)
5-F (racemic)
5-OH (racemic)
5-Br (racemic)
6-F (racemic)
6-Me (racemic)
19u
19v
19w
MeO
MeO
p-CN-Bn
a
Bn = benzyl.
Not active @ 100 lM concentration.
b
sponding R3 methyl derivative 19h. Substituents on the R3 benzyl
group are tolerated (19n, 19o, 19q–w) but do not improve binding
activity relative to 19i.
a
% Inhibition at 1
l
M.
b
D isomer based on X-ray complex structure with TACE.

K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
3861
Table 4
IC50s (
cells assays
micromolar levels of cell activity, perhaps due to permeability is-
sues stemming from the carboxylate moiety.
l
M) of selected tryptophan derivatives 11c, 11f, 11i, and 12p in MMP and Raw
O
O
2.5. X-ray co-complex structure
O
S
O
O
S
O
H
N
HO
H
N
H
HO
O
O
As shown in Figure 3, an X-ray structure of compound 11c
bound to TACE was successfully obtained at a resolution of
2.1 Å.18 As expected, the carboxylate of 11c is ligated to the active
site zinc ion that is also coordinated to three histidine residues,
His405, His409 and His415. The 5-methyl indole moiety is in-
volved in hydrophobic interactions with Ala351 and Val353 and
H
R
NH
N
O
11c (R = Me)
11f (R = MeO)
11i (R = BnO)
12p
may participate in a
p interaction with His 409. As with the other
Compound
MMP-1
MMP-2
MMP-13
MMP-14
TACE
Raw cells
butynyloxy phenyl sulfonamide inhibitors previously disclosed,
the butynyloxy P10 group rests in the narrow hydrophobic channel
connecting the S10 and S30 pockets of TACE. Based on this crystal
structure of 11c it is assumed that the eutamers of the separated
enantiomeric pairs shown in Table 1 are also derived from the D
amino acids, as the enzyme would not be able to efficiently accom-
11c
11f
11i
12p
>50
>50
>50
>50
0.27
1.27
3.27
1.44
1.81
13.6
4.44
4.56
6.82
20.7
19.7
13.0
0.14
0.14
0.37
0.08
7.8
57
>100
modate the indole group of the corresponding
L isomers.
2.4. Selectivity and cellular activity
3. Summary and conclusion
Four tryptophan carboxylate compounds (11c, 11f, 11i, and
12p) that exhibited good TACE activity were selected for selectivity
profiling against MMP-1, MMP-2, MMP-13 and MMP-14. As shown
in Table 4, all four compounds show greater than 100-fold selectiv-
ity against MMP-1, more than 50-fold selectivity against MMP-14,
and greater than 10-fold selectivity against MMP-13. Selectivity
over MMP-2 is the most difficult to achieve, with only compound
12p exceeding 10-fold. Compounds, 11c, 11f, and 12p, were also
assayed for their ability to inhibit LPS-induced TNF production in
Raw cells. Unfortunately, none of these derivatives display sub-
In summary, we have identified a novel series of tryptophan sul-
fonamides as non-hydroxamate inhibitors of TACE and explored the
effect of substitution around indole core on TACE activity. It was
found that 5-methyl or 5-methoxy substituents at the 5-position
of the indole and N-p-methoxybenzyl indole derivatives signifi-
cantly increase inhibitory activity by providing additional hydro-
phobic interactions and/or
p interactions with neighboring amino
acid residues in the TACE active site, resulting in IC50s of less than
Leu350
Met345
Ala351
Val353
Leu348
His409
Zn
His405
Glu414
Ala413
Ala439
Pro437
His415
Tyr436
Figure 3. An X-ray co-complex structure of 11c bound to TACE enzyme (resolution of 2.1 Å). Three histidine residues and a carboxylate coordinate to the active site zinc ion
(yellow sphere). The butynyloxy P10 group is located between the S10 and S30 pockets of TACE.

3862
K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
0.15
l
M. A stereochemical preference was found for derivatives of
stereoisomers. Of the com-
(s, 3H), 3.00 (dd, J = 14.4, 6.4 Hz, 1H), 2.82 (dd, J = 14.4, 7.6 Hz,
D tryptophan over the analogous
L
1H), 1.83 (t, J = 2.0 Hz, 3H); HRMS: calcd for C22H24N2O6S (ESI+,
[M+H]1+), 443.1271; found 443.1265; LC–MS m/z (ESI) [M+H]
443, retention time = 2.50 min.
pounds in this series assayed for selectivity over various MMPs,
50- to 100-fold selectivity was achieved over MMP-1 and MMP-
14, while lesser selectivity was seen for MMP-13 and MMP-2. Based
on these data, the tryptophan sulfonamide scaffold is deemed a use-
ful lead for novel non-hydroxamate TACE inhibitiors.
4.1.4. Chiral separation of 11d to give 11e and 11f
Preparative HPLC of 11d using Chiralpak-OJ (20 ꢀ 250 mm) and
15% ethanol in heptane (0.1% TFA) as eluant (flow rate: 7 mL/min)
gave
D and L enantiomers as white solids. Analytical chiral HPLC
4. Experimental
4.1. Chemistry
(Chiralcel-OJ, 4.6 ꢀ 250 mm, ethanol/heptane (containing 0.02%
TFA) = 85:15, 1.0 mL/min, 215 nm) retention time (TR): 4.86 min
(1st peak: 11e) and 6.32 min (2nd peak: 11f). The absolute stereo-
chemistry of the two enantiomers was not determined.
All reactions were performed under N2 atmosphere using anhy-
drous solvents. Purification of products was carried out by flash
chromatography using EM Silica Gel 60 (230–400 mesh). Semi-pre-
parative HPLC was used under these conditions: A = 0.02% TFA in
water, B = 0.02% TFA in acetonitrile, 10–95% B in 8 min, 34 mL/
4.1.5. 2-(4-But-2-ynyloxy-benzenesulfonyl-amino)-3-(5-
benzyloxy-1H-indol-3-yl)-propionic acid (11g)
The title compound was prepared by using same procedure for
the synthesis of 11a except using 5-benzyloxy-DL-tryptophan in-
stead of 5-methyl-DL-tryptophan. 1H NMR (DMSO-d6): 400 MHz d
12.55 (br s, 1H), 10.64 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.50–7.33
(m, 7H), 7.19 (d, J = 12.0 Hz, 1H), 7.03 (d, J = 4.0 Hz, 1H), 6.97 (d,
J = 4.0 Hz, 1H), 6.87 (d, J = 8.0 Hz, 2H), 6.76 (dd, J = 8.0, 4.0 Hz,
1H), 5.04 (s, 2H), 4.74 (q, J = 2.4 Hz, 2H), 3.86 (q, J = 8.0 Hz, 1H),
3.00 (dd, J = 14.4, 6.4 Hz, 1H), 2.83 (dd, J = 14.4, 8.0 Hz, 1H), 1.82
(t, J = 2.4 Hz, 3H); HRMS: calcd for C28H27N2O6S (ESI+, [M+H]1+),
519.1584; found 519.1588; LC–MS m/z (ESI) [M+H] 519, retention
time = 2.99 min.
TM
min, 50 °C, 215 nm detection, Waters Xterra 20 ꢀ 50 mm column.
1H NMR spectra were recorded at 300 MHz. Tetramethylsilane
(d = 0 ppm) was used as an internal standard, and CDCl3, ace-
tone-d6 or DMSO-d6 were used as solvents. Chiral separation was
performed by preparative chiral HPLC using CHIRALPACK-AD
(250 ꢀ 20 mm) and the chiral purity of the different enantiomers
was determined by chiral HPLC using CHIRALPACK-AD (250 ꢀ
4.6 mm).
4.1.1. 2-(4-But-2-ynyloxy-benzenesulfonyl-amino)-3-(5-
methyl-1H-indol-3-yl)-propionic acid (11a)
4.1.6. Chiral separation of 11g to give 11h and 11i
Triethylamine (0.38 mL, 2.75 mmol) was added to a solution of
5-methyl-DL-tryptophan (0.20 g, 0.92 mmol) and 4-but-2-ynyloxy-
benzenesulfonyl chloride (0.25 g, 1.01 mmol) in H2O–dioxane
(1.6 mL:2.4 mL) at room temperature. The reaction mixture was
stirred for 20 h at room temperature. The mixture was then con-
centrated and acidified with HCl. The mixture was extracted with
ethyl acetate (3 ꢀ 10 mL). The organic solution was washed with
H2O and brine, dried over Na2SO4, and concentrated. The residue
was purified by RP-HPLC to give 0.32 g (81%) of the title compound
Preparative HPLC of 11g using Chiralpak-OJ (20 ꢀ 250 mm) and
15% ethanol in heptane (0.1% TFA) as eluant (flow rate: 13 mL/min)
gave
D and L enantiomers as white solids. Analytical chiral HPLC
(Chiralcel-OJ, 4.6 ꢀ 250 mm, ethanol/heptane (containing 0.02%
TFA) = 85:15, 1.0 mL/min, 215 nm) retention time (TR): 6.76 min
(1st peak: 11h) and 13.1 min (2nd peak: 11i). The stereochemistry
of the two enantiomers was not determined.
4.1.7. 3-(5-Bromo-1H-indol-3-yl)-2-(4-but-2-ynyloxy-
benzenesulfonylamino)-propionic acid (11l)
as
a
pale yellow solid. Mp = 45–46 °C; 1H NMR (DMSO-d6):
400 MHz d 12.54 (br s, 1H), 10.64 (s, 1H), 8.05 (d, J = 8.0 Hz, 1H),
7.53 (m, 2H), 7.17 (d, J = 8.0 Hz, 1H), 7.00 (m, 2H), 6.95 (m, 2H),
6.86 (dd, J = 8.0, 4.0 Hz, 1H), 4.78 (q, J = 2.4 Hz, 2H), 3.81 (q,
J = 7.6 Hz, 1H), 3.00 (dd, J = 14.4, 6.8 Hz, 1H), 2.79 (dd, J = 14.4,
7.8 Hz, 1H), 2.33 (s, 3H), 1.82 (t, J = 2.4 Hz, 3H); HRMS: calcd for
C22H23N2O5S (ESI+, [M+H]1+), 427.1322; found 427.1327; LC–MS
m/z (ESI) [M+H]1+ 427, retention time = 2.69 min.
4.1.7.1. Synthesis of 3-bromo-2-hydroxyimino-propionic acid
ethyl ester16b
. A solution of hydroxylamine sulfate (5.00 g,
31 mmol) in water (30 mL) was added to a solution of ethylbrom-
pyruvate (90%) (6.50 g, 30 mmol) in CHCl3 (10 mL). The two-phase
system was rapidly stirred for 24 h at room temperature. The mix-
ture was extracted with CHCl3 (3 ꢀ 30 mL). The combined extracts
were dried over Na2SO4 and concentrated to give 7.00 g of the title
compound as a white solid. 1H NMR (CDCl3): 300 MHz d 10.08 (br s,
1H), 4.38 (q, J = 7.2 Hz, 2H), 4.27 (s, 2H), 1.39 (t, J = 7.2 Hz, 3H).
4.1.2. Chiral separation of 11a to give 11b and 11c
Preparative HPLC of 11a using Chiralpak-OD (20 ꢀ 250 mm)
and 15% isopropyl alcohol in heptane (0.02% TFA) as eluant (instru-
4.1.7.2. Synthesis of 2-hydroxyimino-3-(5-bromo-1H-indol-3-
yl)-propionic acid ethyl ester. A solution of 3-bromo-2-hydrox-
yimino-propionic acid ethyl ester (3.21 g, 15.3 mmol) in CH2Cl2
(20 mL) was slowly added dropwise to a stirring mixture of 5-bro-
moindole (3.0 g, 15.3 mmol) and Na2CO3 (8.92 g, 84.2 mmol) in
CH2Cl2 (30 mL) at room temperature. The mixture was stirred for
20 h, filtered through Celite and concentrated to give 4.94 g of
the title compound. LC–MS m/z (ESI) [M+H]+ calcd for
C13H14BrN2O3 (Br79) 325.2, found 325.2.
ment: JAI-908, flow rate: 19 mL/min) gave
D and L enantiomers as
white solids. Analytical chiral HPLC (Chiralcel-OD, 4.6 ꢀ 250 mm,
ethanol/heptane (containing 0.02% TFA) = 13:87, 1.0 mL/min,
225 nm) retention time (TR): 23.0 min (1st peak: 11b) and
26.2 min (2nd peak: 11c). The absolute stereochemistry of 11c
was determined by X-ray crystallography.
4.1.3. 2-(4-But-2-ynyloxy-benzenesulfonyl-amino)-3-(5-
methoxy-1H-indol-3-yl)-propionic acid (11d)
The title compound was prepared using the same procedure for
the synthesis of 11a except using 5-methoxy-DL-tryptophan in-
stead of 5-methyl-DL-tryptophan. 1H NMR (DMSO-d6): 400 MHz d
12.55 (br s, 1H), 10.63 (s, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.53 (dd,
J = 8.0, 4.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H),
6.92 (d, J = 8.0 Hz, 2H), 6.82 (d, J = 2.0 Hz, 1H), 6.68 (dd, J = 8.0,
2.0 Hz, 1H), 4.78 (q, J = 2.0 Hz, 2H), 3.87 (q, J = 7.6 Hz, 1H), 3.72
4.1.7.3. Synthesis of 2-amino-3-(5-bromo-1H-indol-3-yl)-propi-
onic acid ethyl ester. Zn dust (3.30 g, 50.4 mmol) was added por-
tionwise to a stirred solution of 2-hydroxyimino-3-(5-bromo-1H-
indol-3-yl)-propionic acid ethyl ester (4.10 g, 12.61 mmol) in ace-
tic acid (100 mL) over 30 min. After stirring overnight, the mixture
was filtered through Celite and concentrated. The residue was dis-
solved in 1 N HCl and re-evaporated to give 4.30 g of the title com-

K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
3863
pound. LC–MS m/z (ESI) [MꢁHCl+H]+ calcd for C13H15BrN2O2 (Br79
)
crude 1-cyclobutylmethyl-
D
-tryptophan (0.657 mmol) and 4-but-
312.2, found 312.2.
2-ynyloxy-benzenesulfonyl chloride (0.177 g, 0.723 mmol) in
H2O–dioxane (2 mL:3 mL) at room temperature. The reaction mix-
ture was stirred for 20 h at room temperature. The mixture was
then concentrated and acidified with HCl. The mixture was ex-
tracted with ethyl acetate (3 ꢀ 10 mL). The organic solution was
washed with H2O and brine, dried over Na2SO4, and concentrated.
The residue was purified by RP-HPLC to give 0.152 g (48% in three
steps) of the title compound as a yellow solid. 1H NMR (DMSO-d6):
400 MHz d 12.57 (br s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.49 (d,
J = 7.2 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H),
7.08 (t, J = 7.4 Hz, 1H), 7.02 (s, 1H), 6.96–6.88 (m, 3H), 4.77 (q,
J = 2.4 Hz, 2H), 4.04 (d, J = 6.8 Hz, 2H), 3.84 (q, J = 6.8 Hz, 1H),
3.02 (dd J = 14.4, 6.4 Hz, 1H), 2.82 (dd, J = 14.4, 8.4 Hz, 1H), 2.67
(p, J = 7.6 Hz, 1H), 1.91 (m, 2H), 1.83 (m, 5H), 1.75 (m, 2H); HRMS:
calcd for C26H29N2O5S (ESI+, [M+H]1+), 481.1792; found 481.1786;
LC–MS m/z (ESI) [M+H] 481, retention time = 3.22 min.
4.1.7.4. Synthesis of 2-(4-but-2-ynyloxy-benzenesulfonylami-
no)-3-(5-bromo-1H-indol-3-yl)-propionic acid ethyl ester. Tri-
ethylamine (4.81 mL, 34.5 mmol) was added to a mixture of
2-amino-3-(5-bromo-1H-indol-3-yl)-propionic acid ethyl ester
(4.00 g, 11.51 mmol) and 4-but-2-ynyloxy-benzenesulfonyl chlo-
ride (3.10 g, 12.66 mmol) in H2O–dioxane (40 mL:60 mL) at room
temperature. The reaction mixture was stirred for 20 h at room
temperature. The mixture was concentrated and diluted with ethyl
acetate. The organic solution was washed with H2O and brine,
dried over Na2SO4 concentrated. The residue was purified by flash
column chromatography (silica, 40% ethyl acetate in hexanes) to
give 3.67 g of the title compound as a pale yellow solid. 1H NMR
(DMSO-d6): 400 MHz d 11.05 (s, 1H), 8.31 (d, J = 8.8 Hz, 1H), 7.56
(d, J = 8.8 Hz, 2H), 7.45 (d, J = 1.6 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H),
7.14 (m, 2H), 6.99 (d, J = 8.8 Hz, 2H), 4.80 (q, J = 2.4 Hz, 2H), 3.88
(q, J = 7.6 Hz, 1H), 3.75 (q, J = 7.2 Hz, 2H), 3.00 (dd, J = 14.4,
7.2 Hz, 1H), 2.88 (dd, J = 14.4, 7.6 Hz, 1H), 1.82 (t, J = 2.4 Hz, 3H),
0.92 (t, J = 7.2 Hz, 3H); HRMS: calcd for C23H24BrN2O5S (ESI+,
Br79, [M+H]1+), 519.0584; found 519.0589.
4.1.9. 2-(4-But-2-ynyloxy-benzenesulfonyl-amino)-3-[1-(2-
trifluoromethyl-benzyl)-1H-indol-3-yl]-propionic acid (12j)
The title compound was prepared using the same procedure for
the synthesis of 12d except using 2-(trifluoromethyl)benzyl bro-
mide instead of (bromomethyl)-cyclobutane. 1H NMR (DMSO-d6):
400 MHz d 12.57 (br s, 1H), 8.13 (d, J = 8.4 Hz, 1H), 7.78 (d,
J = 6.8 Hz, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.43 (m, 2H), 7.37 (d,
J = 7.6 Hz, 1H), 7.19 (s, 1H), 7.12–6.99 (m, 3H), 6.93 (d, J = 8.8 Hz,
2H), 6.44 (d, J = 6.4 Hz, 1H), 5.51 (s, 2H), 4.77 (q, J = 2.0 Hz, 2H),
3.91 (q, J = 7.6 Hz, 1H), 3.08 (dd, J = 14.4, 7.2 Hz, 1H), 2.88 (dd,
J = 14.4, 7.6 Hz, 1H), 1.82 (t, J = 2.4 Hz, 3H); HRMS: calcd for
C29H26F3N2O5S (ESI+, [M+H]1+), 571.1509; found 571.1505; LC–
MS m/z (ESI) [M+H] 571, retention time = 3.37 min.
4.1.7.5. Synthesis of 3-(5-bromo-1H-indol-3-yl)-2-(4-but-2-ynyl-
oxy-benzenesulfonylamino)-propionic acid. 2-(4-But-2-ynyl-
oxy-benzenesulfonylamino)-3-(5-bromo-1H-indol-3-yl)-propionic
acid ethyl ester (0.100 g, 0.193 mmol) was dissolved in 0.5 mL tet-
rahydrofuran. To the solution were added MeOH (0.5 mL) and 1 N
NaOH (0.4 mL, 0.4 mmol). The reaction mixture was placed in a
shaker for 10 h at room temperature. The solution was acidified
with 1 N HCl and extracted with ethyl acetate (3 ꢀ 3 mL). The or-
ganic solution was concentrated and the residue was purified by
RP-HPLC to give 0.089 g (94%) of the title compound as a pale yel-
low solid. 1H NMR (DMSO-d6): 400 MHz d 12.65 (br s, 1H), 11.01 (s,
1H), 8.08 (d, J = 8.8 Hz, 1H), 7.48 (m, 3H), 7.26 (d, J = 8.8 Hz, 1H),
7.13 (m, 2H), 6.89 (d, J = 9.2 Hz, 2H), 4.77 (q, J = 2.8 Hz, 2H), 3.82
(q, J = 8.4 Hz, 1H), 3.01 (dd J = 14.6, 6.4 Hz, 1H), 2.82 (dd, J = 14.6,
8.0 Hz, 1H), 1.83 (t, J = 2.4 Hz, 3H); HRMS: calcd for C21H20BrN2O5S
(ESI+, Br79, [M+H]1+), 491.0271; found 491.0287; LC–MS m/z (ESI)
[MꢁH] 489 (Br79), retention time = 2.75 min.
4.1.10. 2-(4-But-2-ynyloxy-benzenesulfonyl amino)-3-[1-(4-
methoxy-benzyl)-5-methyl-1H-indol-3-yl]-propionic acid (19f)
NaH (60% in mineral oil) (0.033 g, 0.831 mmol) was added to a
solution
of
2-(4-but-2-ynyloxy-benzenesulfonylamino)-3-(5-
methyl-1H-indol-3-yl)-propionic acid (11a) (0.100 g, 0.234 mmol)
in anhydrous N,N-dimethylformamide (2 mL) at 0 °C. The reaction
mixture was placed in a shaker for 20 min at 0 °C. 4-Methoxy-ben-
zyl chloride (0.032 mL, 0.234 mmol) was added to the solution and
allowed to react for 10 h at 0 °C. The reaction mixture was
quenched with H2O and acidified with HCl. The solution was ex-
tracted with EtOAc, washed with H2O and brine, dried over Na2SO4,
and concentrated. The residue was purified by RP-HPLC to give
0.086 g (67%) of the title compound as a pale yellow solid. 1H
NMR (DMSO-d6): 400 MHz d 12.58 (br s, 1H), 8.09 (d, J = 8.4 Hz,
1H), 7.53 (d, J = 11.6 Hz, 2H), 7.20 (d, J = 8.0 Hz, 1H), 7.09 (s, 1H),
7.07 (d, J = 8.4 Hz, 2H), 7.00 (s, 1H), 6.92–6.80 (m, 5H), 5.17 (s,
2H), 4.76 (q, J = 2.4 Hz, 2H), 3.82 (q, J = 8.0 Hz, 1H), 3.69 (s, 3H),
3.00 (dd J = 14.4, 6.8 Hz, 1H), 2.79 (dd, J = 14.4, 7.6 Hz, 1H), 2.32
(s, 3H), 1.82 (t, J = 2.4 Hz, 3H); HRMS: calcd for C30H31N2O6S
(ESI+, [M+H]1+), 547.1897; found 547.1896; LC–MS m/z (ESI)
[MꢁH] 545, retention time = 2.76 min.
4.1.8. 2-(4-But-2-ynyloxy-benzenesulfonyl-amino)-3-(1-
cyclobutylmethyl-1H-indol-3-yl)-propionic acid (12d)
4.1.8.1. Synthesis of N-(tert-butoxycarbonyl)-1-cyclobutylm-
ethyl-
D
-tryptophan. NaH (60% in mineral oil) (0.092 g, 2.30
-tryp-
mmol) was added to a solution of N-(tert-butoxycarbonyl)-
D
tophan (0.200 g, 0.657 mmol) in anhydrous N,N-dimethylformam-
ide (4 mL) at 0 °C. The reaction mixture was placed in a shaker for
20 min at 0 °C. (Bromomethyl)-cyclobutane (0.074 mL, 0.657
mmol) was added to the solution and allowed to react for 10 h at
0 °C. The reaction mixture was quenched with H2O and acidified
with 1 M NaHSO4 until pH 3. The solution was extracted with
CH2Cl2, washed with brine, dried over Na2SO4, and concentrated
to give the title compound.
4.2. TACE FRET assay
4.1.8.2. Synthesis of 1-cyclobutylmethyl-D-tryptophan. The
dried crude product from Section 4.1.8.1 was dissolved in
CH2Cl2-TFA (1 mL/1 mL) and the solution was stirred for 10 h at
room temperature. The solution was concentrated and the result-
ing product was dissolved in CH2Cl2. The solution was washed with
H2O and brine, dried over Na2SO4, and concentrated to give the ti-
tle compound.
4.2.1. Materials and methods
The proprietary pro-TNF-a substrate peptide Abz-LAQAVRSSSR-
Dpa (AnaSpec, WARC-1) was prepared as a 2 mM stock solution in
the TACE assay buffer (50 mM Tris–HCl, pH 7.4, 25 mM NaCl, 4%
Glycerol, 0.005% Brij 35). The catalytic domain of the recombinant
TACE protein was expressed and purified in-house. The TACE pro-
tein (1 lg/ml) was pre-treated with the inhibitors at various con-
4.1.8.3. Synthesis of 2-(4-but-2-ynyloxy-benzenesulfonylami-
no)-3-(1-cyclobutyllmethyl-1H-indol-3-yl)-propionic acid. Tri-
ethylamine (0.275 mL, 1.97 mmol) was added to a solution of
centrations for 10 min at room temperature. The reaction was
initiated by the addition of the substrate peptide. The increase in
fluorescence was monitored at an excitation of 320 nM and an

3864
K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
emission of 420 nM over a period of 10 min. The initial rate (slope)
of the reaction was determined using a fluorescence plate reader
(Molecular Devices, Spectra Max Gemini XS).
4.4.2. Analysis of results
Plots of the inhibitor concentration versus the percent inhibi-
tion were fit to the following equation: y = (a ꢁ d)/[1 + (x/c)b] + d,
a general sigmoidal curve with Hill slope, a to d. x is the inhibitor
concentration under test. y is the percent inhibition. a is the limit-
ing response as x approaches zero. As x increases without bound, y
tends toward its limit d. c is the inflection point (IC50) for the curve.
That is, y is halfway between the lower and upper asymptotes
when x = c. b is the slope factor or Hill coefficient.20
4.2.2. Analysis of results
The basic measurement of the assay is the % of inhibition of the
cleavage of the fluorogenic pro-TNF peptide by TACE or the IC50
determination by a fitting with the model-39 of LSW data analysis
tool.19
4.3. Cell-based assay for inhibition of TNF-
cells
a secretion in Raw
Acknowledgments
We would like to thank Discovery Analytical Chemistry group
for spectral data and Dr. Eric Feyfant for modeling effort. We thank
Drs. Jerry Skotnicki and Tarek Mansour for their support of this
work.
4.3.1. Materials and methods
Raw 264.7 cells (ATCC Cat No. TIB-71) are maintained in DMEM
medium containing 10% of serum, P/S, and glutamine. Cells are
split twice a week by scraping and 1–10 or 20 dilutions. For testing
compounds, cells are cultured to confluence and are seeded the day
before the experiment in 24 well culture dishes at 0.5–1 million/
ml/well. On the next morning, the medium from the overnight cul-
ture is replaced with the fresh growth medium. The compounds
are added at various concentrations at the 0.2% of final DMSO con-
centrations (10 ll of a 20% DMSO solution containing testing com-
pounds is added to each well). The cells are pre-incubated with
compounds for 1 h. LPS (Sigma, L2262) is added to cells at a final
concentration of 100 ng/ml (10 ll of a 10 lM LPS solution freshly
References and notes
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diluted from a 1 mg/ml LPS stock solution in PBS is added to each
well) and cells are further incubated for 4 h at 37 °C. 1 ml of the
supernatant was collected, cells are spun down and the superna-
tants are frozen at ꢁ80 °C until use.
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A continuous assay was used in which the substrate is a
synthetic peptide containing a fluorescent group (7-methoxy-
coumarin; Mca) which is quenched by energy transfer to a 2,4-
dinitrophenyl group. When the peptide was cleaved by MMP, a
large increase in fluorescence was observed. The source of enzyme
in the assay was the recombinant human catalytic domain of
MMP-1 prepared at Wyeth-Research in Cambridge. The substrate
used was Mca-PQGL-(3-[2,4-dinitrophenyl]-L-2,3-diaminopropio-
nyl)-AR-OH (denoted as Wammp-5, custom synthesized by Ana-
Spec, Inc.). The assay buffer consisted of 50 mM Hepes (pH 7.4),
100 mM NaCl, 5 mM CaCl2, and 0.005% Brij-35. Each well of black
polystyrene 96-well plates contained a 200 lL reaction mixture
consisting of assay buffer, purified MMP (final concentration of
25 ng/ml, prepared by diluting with the assay buffer), and varied
concentrations of inhibitor (prepared by serially diluting a given
inhibitor in DMSO in 96-well polypropylene plate). The plates were
then incubated at 30 °C for 15 min. The enzymatic reactions were
initiated by adding the substrate to a final concentration of
20 lM, and mixing 10 times with a pipette. The final DMSO con-
centration in the assay was 6.0%. The initial rate of the cleavage
reaction was determined at 30 °C temperature with a fluorescence
plate reader (excitation filter of 330 nm and emission filter of
395 nm) immediately after substrate addition.

K. Park et al. / Bioorg. Med. Chem. 17 (2009) 3857–3865
3865
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