The discovery of novel tartrate-based TNF-α converting enzyme (TACE) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.12.004

A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP's. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.

Full text of DOI:10.1016/j.bmcl.2009.12.004

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1189–1193
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of novel tartrate-based TNF-a converting enzyme (TACE)
inhibitors
b
b
a
a
b
Kristin E. Rosner a, , Zhuyan Guo , Peter Orth , Gerald W. Shipps Jr. , David B. Belanger , Tin Yau Chan ,
Patrick J. Curran a, Chaoyang Dai a, Yongqi Deng a, Vinay M. Girijavallabhan b, Liwu Hong b, Brian J. Lavey b,
Joe F. Lee b, Dansu Li a, Zhidan Liu b, Janeta Popovici-Muller a, Pauline C. Ting b, Henry Vaccaro b, Li Wang b,
Tong Wang a, Wensheng Yu b, Guowei Zhou b, Xiaoda Niu c, Jing Sun c, Joseph A. Kozlowski b,
Daniel J. Lundell c, Vincent Madison b, Brian McKittrick b, John J. Piwinski a, Neng -Yang Shih a,
M. Arshad Siddiqui a, Corey O. Strickland b
*
a Department of Medicinal Chemistry, Schering-Plough Research Institute Cambridge, 320 Bent Street, Cambridge, MA 02141, United States
b Department of Medicinal Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
c Department of Inflammation, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of TNF-
These compounds are bis-amides of
dentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure
for a tartrate-based TACE inhibitor.
a
convertase (TACE) inhibitors which are non-hydroxamate have been discovered.
-tartaric acid (tartrate) and coordinate to the active site zinc in a tri-
Received 5 October 2009
Revised 30 November 2009
Accepted 2 December 2009
Available online 5 December 2009
L
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
TACE
TACE inhibitors
Tartrates
Tumor necrosis factor-
modulatory and proinflammatory cytokines. The overproduction
a
(TNF-
a
) is one of the major immuno-
that bind in the narrow S10 tunnel and large S30 pocket. As muscu-
loskeletal side effects have been associated with broad spectrum
MMP inhibitors, there is interest in selective TACE inhibitors.7
While many TACE inhibitors are hydroxamate based,8 our inter-
ests focused on the discovery of a selective non-hydroxamate TACE
inhibitor. To achieve this goal we screened our proprietary
mixture-based combinatorial library with the Automated Ligand
Identification System (ALIS).9 The screen identified four com-
pounds (1–4) with moderate TACE affinities (Table 1). Compounds
1 and 2 were equipotent and showed sub-micromolar TACE inhibi-
tion. Interestingly the structures were found to be bis-amides of
of TNF-a has been implicated in many autoimmune disorders such
as rheumatoid arthritis, Crohn’s disease and psoriasis.1 The suc-
cessful treatment of inflammatory diseases, by the reduction of
TNF-
a levels, has been validated by the biologics such as Remi-
cadeÒ, EnbrelÒ and HumiraÒ.2 The discovery of a cost-effective, or-
ally active small molecule drug which could modulate TNF-
is of high interest.
a
levels
is
One potential approach to reduce the levels of soluble TNF-
to block the release of TNF- from the cell surface by the inhibition
of TNF-
converting enzyme (TACE/ADAM17).3 TACE is a mem-
brane-bound zinc-metalloprotease which converts the 26-kD
transmembrane pro-form of TNF- to the mature 17-kD soluble
a
a
a
L
-tartaric acid (tartrates).10 To the best of our knowledge, this is
the first report of a tartrate-based inhibitor of TACE. The activity
is specific to the -tartrates as the corresponding -tartrates were
found to be inactive.11
a
L
D
form.4 Crystal structures of inhibitors bound to TACE show that
the active site of TACE shares many common features with the ma-
trix metalloproteinases (MMP’s).5 However, one distinguishing
feature of TACE is a tunnel interconnecting the S10 and S30 pockets
into a single large cavity. It has been demonstrated6 that TACE
selectivity may be achieved by appropriately incorporating groups
Herein we report the first crystal structure of a tartrate-based
inhibitor 2 bound to the TACE enzyme (Fig. 1a).12 A unique triden-
tate zinc binding mode was revealed with the tartrate scaffold and
is defined by the two hydroxyl groups and the non-prime amide
carbonyl interacting with the catalytic zinc atom (O-Zn dis-
tances = 2.0 Å, 2.4 Å and 2.5 Å, respectively) (Fig. 1b). The zinc
atom maintains its coordination with the three imidazole nitro-
gens of His405, His409 and His415. Therefore, the Zn ion takes a
* Corresponding author.
E-mail address: kristin.rosner@spcorp.com (K.E. Rosner).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.004

1190
K. E. Rosner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1189–1193
Table 1
Non-Prime Sites
R2
Prime Sites
ALIS screening hits and early SAR
O
O
OH R1
N
R1
R3
N
S
N
H
R
O
OH O
O
H
OH
Compound
R
R1
TACE Kia (nM)
Zn
His 409
N
N
His 405
His 415
1
H
H
400
O
N
N
N
N
Figure 1b. Tridentate chelation of the zinc atom with the tartrate core.
2
3
400
Cl
important interactions with the TACE protein. The ethylthiophene
group binds in the S10 pocket which is formed by Leu401, Val434
and Val440 at the bottom and Leu348, Val402, Ala439 and
His405 on the side. Besides hydrophobic interactions with the S10
residues, the thiophene group also makes a pi-stacking interaction
with His405. The 2-chlorophenylpiperazine group binds to the S1
subsite defined by Val314, Lys315, Thr347 and Leu350 and is a
rather flat hydrophobic patch that is solvent exposed. Note that
the chlorophenyl group was excluded from the model as it appears
disordered and lacks 2fofc electron density.
Due to its binding mode, the tartrate series provides a scaffold
to explore both the non-prime and prime binding interactions. In
order to explore the effect of modifications to the P1 group on
the activity of the TACE inhibitors, a general procedure was used
to prepare analogs (Scheme 1). Compound 513 was converted to
6 in 2 steps via amide bond formation and saponification of the es-
ter. Coupling of the racemic 2-phenylpyrrolidine to 6 followed by
reverse phase HPLC separation of the diastereomers and acetonide
deprotection afforded 7 and 8.
H
1100
4
9
H
H
1400
nab
H
N
N
N
N
10
Me
nab
Cl
N
11
12
13
H
H
H
2500
1300
764
N
N
N
N
14
H
1280
a
The compounds were tested in a FRET assay using the catalytic domain of TACE.
na = not active.
b
As shown in Table 1, the preference for a tertiary amide at the
non-prime site was demonstrated by the comparison of com-
pounds 9 and 13.14 The crystal structure does not provide an obvi-
ous explanation for this preference. The N-aryl piperazine was
tolerant of substitution as shown with compounds 1 and 2.
Replacement of the N-aryl ring with 2-pyridyl of 11 resulted in a
moderate loss in activity. Acyclic tertiary amides 12–14 were also
well tolerated and equipotent to the screening hit 4. The loss of
activity observed when a tertiary amide was at the prime site dem-
onstrated the preference for secondary amides (compound 2 vs
10).
In order to further explore the role of the non-prime amide, we
focused our efforts on the structure activity relationships (SAR)
around compound 13. Heteroaryl analogs 15 and 16 were equipo-
tent to the corresponding phenyl analog 13 (Table 2). A benzylic
methyl groups (17 and 18) moderately improved the potency rel-
ative to 13. We speculated that we could gain additional potency
by designing a compound that would project functionality back to-
wards the surface of the protein. Our first attempt was to constrain
the benzyl amide 13 into the ring system of ( )-2-phenylpiperidine
amide 19 (Ki = 325 nM) which had comparable potency to com-
pounds 17 and 18. The ( )-2-phenylpyrrolidine amide 20 proved
to be more potent (Ki = 96 nM) analog. The crystal structure of 20
(Fig. 2)15 confirmed our hypothesis; the compound maintained
similar interactions with the zinc and prime site observed in the
crystal structure of 2, and added a new face-edge interaction of
the 2-phenylpyrrolidine with His415. In the crystal structure only
the 2R-phenylpyrrolidine diastereomer of 20 was observed, sug-
gesting that the resolved compound would be more potent. Com-
pound 20 was resolved into diastereomers 7 and 8 as described
in Scheme 1. The stereochemical assignment was confirmed by
synthesis of 8 from 2S-phenylpyrrolidine.16 The 2R-phenylpyrroli-
dine 7 (Ki = 20 nM), as predicted by the X-ray structure of 20, was
the more active isomer. Moving the aryl substitution to the
Figure 1a. X-ray structure of 2 (green carbon, stick) bound to the TACE catalytic
domain (surface). The 2-chlorophenyl group was missing from the crystal structure.
The S10 and S30 subsites are indicated. The zinc catalytic site residues and residues
that form the hydrophobic region of the S1 subsite are shown (cyan carbon, stick).
His405 which makes pi-stacking interactions with the thiophene is also labeled
pseudo-octahedral coordination configuration in this binding
mode. The prime amide carbonyl oxygen makes hydrogen bonds
with the backbone –NH of both Leu348 and Gly349 (O–N dis-
tance = 3.1 Å and 3.4 Å). The OH near the non-prime side also
forms hydrogen bonds with the carboxylate oxygens of Glu406
(O–O distance = 2.8 Å and 3.2 Å). It is interesting to note that all
the four oxygen atoms on the tartrate core collectively make

K. E. Rosner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1189–1193
1191
S
O
O
O
O
O
O
O
OH
O
OH
O
OH
H
H
a, b
c, d
NH
N
HO
N
S
S
O
+
N
N
O
OH O
OH O
5
6
7
8
Scheme 1. Reagents and conditions: (a) 2-thiopheneethylamine, DIEA, HATU, DMF (64%); (b) 1.0 M LiOH, THF (53%); (c) racemic 2-phenyl-pyrrolidine, DIEA, HATU (70%);
90:10 TFA/H2O (87%).
Table 2
Tertiary P1 amide optimization
O
OH
H
N
S
R
OH O
Compound
R
TACE Ki (nM)
N
N
15
1020
O
16
17
943
487
N
N
N
18
19
238
325
N
Figure 2. X-ray structure of 20 (green carbon, stick) bound to the TACE catalytic
domain (surface). Residues near the zinc catalytic site are shown (cyan carbon,
stick). The face-edge interactions between the phenyl ring and His415 are indicated.
20
7
96
20
N
N
N
Table 3
2-Aryl pyrrolidine optimization
O
OH
H
N
S
R
OH O
Compound
R
TACE Ki (nM)
8
725
456
N
24
188
21
N
N
S
N
N
22
23
91
66
25
26
27
164
323
11
N
N
N
N
Cl
Cl
3-position of pyrrolidine was not as preferred (compound 20 vs
21). Modeling suggested that compound 21 would lose the face-
edge interaction which was important in the potency of compound
20. Exploration of fused bicyclic systems led to compounds 22
(Ki = 91 nM) and 23 (Ki = 66 nM). Similar to compound 2, com-
pounds 22 and 23 would bind to the S1 region and make favorable
hydrophobic contacts with Val314, Lys315, Thr347 and Leu350
sidechains.
An exploration of 2-aryl and 2-heteroarylpyrrolidines to further
probe the face-edge interaction with His415 was carried out.
(Table 3) Replacement of the phenyl ring with a heterocycle affor-
ded compounds 24 and 25 which were less active than the phenyl
analog 20. Compounds 26–28 demonstrate that meta-substitution
on the phenyl ring was preferred. Modeling indicated that this may
be due to the additional hydrophobic contacts of the meta-substi-
tuent with the protein surface residues such as Pro437. The elec-
tronic effect of the substituent had little effect on the potency
(compounds 27, 29, 30 and 31).
Cl
28
755
N
N
N
N
29
30
31
13
33
41
O
N

1192
K. E. Rosner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1189–1193
Our early efforts to explore the SAR of the secondary prime
trate series. As shown in Scheme 2, mono acid/mono ester 42
was used to prepare the mono-hydroxy analogs in 3 steps. Deletion
of either hydroxyl group from the tartrate core resulted in the com-
plete loss of TACE inhibition for compounds 43 and 44. (Table 5)
Comparison of the diamide 45 with the monothioamides 46 and
47 demonstrates the necessity of both amide carbonyls. For thio-
amide 46, the lesser activity highlights the importance of zinc
coordination of the non-prime amide carbonyl. We speculated that
the weaker affinity of compound 46 may be attributed to the
intrinsic weaker zinc binding ability of C@S versus C@O and also
potential steric conflict of the sulfur atom with the imidazole ring
of His415. The decrease in activity of thioamide 47 may be ex-
plained by sulfur being a poorer hydrogen-bond acceptor than oxy-
gen. Therefore, the hydrogen bonds between the prime amide
carbonyl and Leu348 and Gly349 are also key binding interactions.
Replacement of the non-prime amide with amide isosteres, such as
a thiazole or imidazole, afforded compounds with weaker TACE
inhibition (compounds 48 and 49) and was not advantageous.
The loss of activity was attributed mainly to the lack of interactions
with the S1 subsite or the face-edge interactions with His415.
The binding mode of the tartrate series provided the opportu-
nity to explore the effect of the prime and non-prime substituents
on selectivity. Screening hit 2 had good selectivity relative to the
MMP panel and was equipotent against ADAM10. (Table 6) Com-
pound 27 showed that optimization of the non-prime substituent
maintained the good MMP selectivity. In contrast to compound
27, compound 34 is a TACE and pan-MMP inhibitor; thus suggest-
ing the prime interactions may influence the selectivity profile
more greatly than the non-prime interactions. Extension into the
amides are shown in Table 4. Shortening of the ethylene linker of
compound 27 to the methylene linker of compound 32 resulted
in a loss of activity. The benzyl amide 33 was significantly less ac-
tive than compound 27. However, compound 34 (Ki = 160 nM) is of
comparable activity to compound 20 (Ki = 96 nM). Extension into
the S30 pocket via the addition of a benzyl group modestly im-
proved the potency for the ethylene linked thiophene (compound
35 vs 20). A greater increase in potency was observed for com-
pounds 36 (Ki = 93 nM) and 37 (Ki = 70 nM) by the addition of a
P30 substituent to the corresponding parent compounds 32 and
33. Replacement of the thiophene with either thiazole or oxazole
resulted in active compounds (compounds 35 and 36 vs 38–41)
and a preference for the methylene linked systems was observed.
Having identified TACE inhibitors with nanomolar affinities, we
focused our investigation on the key binding elements of the tar-
Table 4
Secondary P10 amide optimization
R1
O
OH
R2
N
OH O
Compound
R1
R2a
TACE Ki (nM)
44% @ 500 nMb
1440
S
N
H
32
33
3-Cl
3-Cl
N
H
O
S
S
N
H
34
35
H
H
160
66
Table 5
Tartrate core modifications
H
N
Compound
Structurea
TACE Ki (nM)
Cl
N
H
43
nab
O
N
H
N
36
37
4-F
93
70
S
S
OH O
N
H
3-Cl
Cl
H
N
44
45
nab
O
OH
O
H
N
S
N
38
39
40
41
H
H
H
H
48
8
N
Cl
S
Cl
N
H
N
13
O
OH
H
N
S
S
S
N
H
N
OH O
O
461
22
Cl
Cl
N
O
Cl
46
47
1520
1110
S
OH
H
N
N
H
N
N
OH O
a
Cl
Synthetic procedures for these amines may be found in Ref. 10.
The data is reported as % inhibition.
b
O
OH
H
N
N
OH S
Cl
N
N
OH
H
N
O
S
S
O
48
49
5530
S
N
a, b, c
CO2H
H
O
N
S
OH O
OH
N
O
OH O
43
H
N
42
10,600
OH O
Scheme 2. Reagents and conditions: (a) 2-thiopheneethylamine, DIEA, HATU, DMF
(100%); (b) 1.0 M HCl, THF (73%); (c) ( )-2-(3-chlorophenyl)-pyrrolidine, DIEA,
HATU, DMF (49%).
a
Synthetic procedures for these compounds may be found in Ref. 10.
na = not active.
b

K. E. Rosner et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1189–1193
1193
Table 6
Selectivity data
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Paxton, R. J.; Black, R. A.; Bode, W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3408; For
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Gearing, A. J. H. Chem. Rev. 1999, 99, 2735.
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Trzaskos, J. M.; Duan, J. J.-W. Bioorg. Med. Chem. Lett. 2008, 18, 1577;
(d) Zhu, Z.; Mazzola, R.; Sinning, L.; McKittrick, B.; Niu, X.; Lundell, D.;
Sun, J.; Orth, P.; Guo, Z.; Madison, V. J. Med. Chem. 2008, 51, 725; (e)
Mazzola, R. D.; Zhu, Z.; Sinning, L.; McKittrick, B.; Lavey, B.; Spitler, J.;
Kozlowski, J. A.; Shih, N.-Y.; Zhou, G.; Guo, Z.; Orth, P.; Madison, V.;
Sun, J.; Lundell, D.; Niu, X. Bioorg. Med. Chem. Lett. 2008, 18, 5809.
7. Levin, J. I. Curr. Top. Med. Chem. 2004, 4, 1289.
8. (a) Levin, J. I.; Chen, J. M.; Laakso, L. M.; Du, M.; Schmid, J.; Xu, W.; Cummons,
T.; Xu, J.; Jin, G.; Barone, D.; Skotnicki, J. S. Bioorg. Med. Chem. Lett. 2006, 16,
1605; (b) Flipo, M.; Charton, J.; Hocine, A.; Dassonneville, S.; Deprez, B.;
Deprez-Poulain, R. J. Med. Chem. 2009, 52, 6790.
9. For information about ALIS and affinity selection-mass spectrometry based
screening see: (a) Annis, D. A.; Nazef, N.; Chuang, C.-C.; Scott, M. P.; Nash, H. M.
J. Am. Chem. Soc. 2004, 126, 15495; (b) Annis, D. A.; Shipps, G. W., Jr.; Deng, Y.;
Popovici-Mueller, J.; Siddiqui, M. A.; Curran, P. J.; Gowen, M.; Windsor, W. T.
Anal. Chem. 2007, 79, 4538; (c) Annis, D. A.; Nickbarg, E.; Yang, X.; Ziebell, M. R.;
Whitehurst, C. E. Curr. Opin. Chem. Biol. 2007, 11, 518.
10. Guo, Z.; Orth, P.; Zhu, Z.; Mazzola, R. D.; Chan, T. Y.; Vaccaro, H. A.; McKittrick,
B.; Kozlowski, J. A.; Lavey, B. J.; Zhou, G.; Paliwal, S.; Wong, S.-C.; Shih, N.-Y.;
Ting, P. C.; Rosner, K. E.; Shipps, G. W. Jr.; Siddiqui, M. A.; Belanger, D. B.; Dai, C.;
Li, D.; Girijavallabhan, V. M.; Popovici-Muller, J.; Yu, W.; Zhao, L. WO 2005/
121130, 2005.
Ki (
l
M)
2
27
34
35
TACE
0.40
0.40
>90
>30
>50
>60
9
0.011
0.029
>50
33
0.160
0.734
18.7
0.3
7.7
>50
—
0.066
>25
>50
>50
—
ADAM10
MMP1
MMP2
MMP3
MMP7
MMP12
MMP13
MMP14
17
>50
>50
20
6
0.7
3.6
>30
7
>40
0.51
0.14
11
S30 pocket improved the ADAM10 selectivity while maintaining the
favorable MMP selectivity, as shown by compound 35. Encouraged
by the selectivity profile of the tartrate series we tested com-
pounds 2 and 20 in the human whole blood assay (HWBA);17
unfortunately they were inactive. Our efforts to optimize for HWBA
activity will be discussed in future publications.
In summary, we have reported the discovery of the first tartrate
scaffold that utilizes tridentate coordination to the active site zinc
of a TACE from a diverse mixture-based library. The tartrate series
is of particular interest because it has been found to be a suitable
replacement for the hydroxamate based TACE inhibitors and their
unique binding mode allows for access to both the prime and non-
prime binding pockets. Using ligand and structure-based design
our initial optimization efforts were successful in improving po-
tency and highlighting new interactions for further refinement.
This work enables the possibility of designing potent and selective
small molecule tartrate TACE inhibitors and our additional efforts
towards improving potency and selectivity will be discussed in fu-
ture publications.
Acknowledgements
The authors would like to acknowledge Doctors E. Grant and
J. Duca for insightful discussions and assistance in preparing this
manuscript.
11. Unpublished results from the ALIS screen.
12. (a) Ingram, R. N.; Orth, P.; Strickland, C. L.; Le, H. V.; Madison, V.; Beyer, B. M.
Protein Eng., Des. Sel. 2006, 19, 155; (b) RCSB protein data bank (PDB)
deposition number 3KMC.
13. Musich, J. A.; Rapoport, H. J. Am. Chem. Soc. 1978, 100, 4865.
14. No non-prime secondary amides were found in the ALIS screen. Compound 9 is
from an array of discretes made to confirm the preference for tertiary non-
prime amides (data not shown) and is the unpublished work of Dr. Zhaoning
Zhu and Ms. Lisa Sinning.
15. RCSB protein data bank (PDB) deposition number 3KME.
16. Burgess, L. E.; Meyers, A. I. J. Org. Chem. 1991, 56, 2294.
17. Human whole blood is diluted 1:1 with serum free medium (RPMI,
References and notes
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L
-glutamine, Pen-Strep, HEPES) and incubated with a compound in a final
volume of 360 L for 1 h at 37 °C. Forty microliters of LPS (10 g/mL) is then
added. Supernatant is collected after 3.5 h incubation and the concentration of
TNF- is determined by ELISA (R&D Systems).
l
l
a