Discovery and SAR of hydantoin TACE inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.148

We disclose inhibitors of TNF-α converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are d

Full text of DOI:10.1016/j.bmcl.2010.01.148

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1877–1880
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and SAR of hydantoin TACE inhibitors
a,
a
a
a
b
a
Wensheng Yu a, , Zhuyan Guo , Peter Orth , Vincent Madison , Lei Chen , Chaoyang Dai , Robert J. Feltz ,
Vinay M. Girijavallabhan a, Seong Heon Kim a, Joseph A. Kozlowski a, Brian J. Lavey a, Dansu Li b,
Daniel Lundell c, Xiaoda Niu c, John J. Piwinski b, Janeta Popovici-Muller b, Razia Rizvi a, Kristin E. Rosner b,
Bandarpalle B. Shankar a, Neng-Yang Shih b, M. Arshad Siddiqui b, Jing Sun c, Ling Tong a, Shelby Umland c,
Michael K. C. Wong a, De-yi Yang a, Guowei Zhou a
*
*
a Department of Medicinal Chemistry, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, NJ 07033, United States
b Department of Medicinal Chemistry, Merck Research Laboratories, 320 Bent Street, Cambridge, MA 02141, United States
c Department of Inflammation, Merck Research Laboratories, 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:
We disclose inhibitors of TNF-
a converting enzyme (TACE) designed around a hydantoin zinc binding
Received 17 November 2009
Revised 28 January 2010
Accepted 29 January 2010
Available online 4 February 2010
moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydan-
toin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first
reported X-ray structures of TACE with a hydantoin zinc ligand.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
TACE
TNF-
TNF-
a
a
convertase
TACE inhibitor
Hydantoin
Hydantoin zinc ligand
Anti-TNF
Rheumatoid arthritis
Tumor necrosis factor-
mune response. Dysregulation and, in particular, overproduction of
a
(TNF-
a
) is a key cytokine for innate im-
non-hydroxamic acid TACE inhibitors. Previously we reported tar-
taric acid based TACE inhibitors and carboxylic acid based TACE
inhibitors.8,9 Herein, we report a new series of hydantoin based
TACE inhibitors.
Several hydantoin containing compounds were identified as
weak inhibitors, such as 1 (Ki = 6 lM), from our screening collec-
tion. Soaking experiments of these hydantoin compounds into
crystals of the catalytic domain of TACE (V353G)10 resulted in
the X-ray crystal structure of 2 shown in Figure 2. The structure re-
vealed two molecules of 2 coexisting in the active site, but occupy-
ing different subsites (Fig. 2A). The first molecule of 2 occupies the
S10 pocket whereas the second molecule of 2 binds to the S1 region.
Unlike hydroxamates and tartrates which exhibit bidentate and
tridentate zinc binding, respectively,8,11 monodentate zinc coordi-
nation was observed with the S1 hydantoin nitrogen.7b,12 The S1
hydantoin also forms multiple hydrogen bonds with the protein
as indicated in Figure 2B. The amide nitrogen of the hydantoin
interacts with the carbonyl oxygen of Gly349 and the oxygen of
the adjacent carbonyl forms a bidentate hydrogen bond to the
Glu406 side chain. In addition, the phenyl group occupies the
hydrophobic non-prime site defined by the side chains of Val314,
TNF-
a have been implicated in a variety of autoimmune diseases
such as rheumatoid arthritis, Crohn’s disease, and psoriasis.1,2 Cur-
rent treatments include injectable anti-TNF biologics such as En-
brelÒ and RemicadeÒ. However, the advantages of an orally
active drug, such as patient convenience and cost reduction, make
a small molecule drug highly desirable.
TNF-
proteolytic cleavage by the TNF-
There has been significant interest in the development of TACE
inhibitors to modulate the level of TNF- which has long been
a
is released from membrane-bound pro-TNF-
a through
a
converting enzyme (TACE).3
a
viewed as having potential to treat related inflammatory
diseases.4,5
The majority of known TACE inhibitors incorporate a hydroxa-
mic acid as the zinc chelating group.6 Non-hydroxamic acid TACE
inhibitors, including tartrates and hydantoins, have also been re-
ported.7,8 In the course of our program, we aimed to identify
* Corresponding authors.
E-mail addresses: wensheng.yu@spcorp.com (W. Yu), Zhuyan.guo@spcorp.com
(Z. Guo).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.148

1878
W. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1877–1880
Thr347, Leu350, and Lys315 (Fig. 2A). The molecule of 2 in the S10
site revealed hydrogen bond interactions between the hydantoin
carbonyl and the backbone NH of Leu348 and Gly349. The phenyl-
acetamide group is embedded in the S10 pocket.
This crystal structure served as a starting point in our structure-
based discovery of potent hydantoin TACE inhibitors. Our strategy
was to design a scaffold based on the binding modes shown in
Figure 2B, which would maintain the zinc binding of the hydan-
toin, and at the same time capture other key interactions, namely,
hydrogen bonding to the backbone NH of Leu348, hydrogen bond-
ing to Gly349, and filling the S10 pocket.
Scaffolds containing these key features were evaluated using
molecular modeling techniques. The results indicated that 1H-
indazol-3(2H)-one scaffold 3 (Fig. 1) would best satisfy the above
criteria. Specifically, the hydantoin could maintain all the interac-
tions near the catalytic zinc as observed in 2. The carbonyl oxygen
of the indazolinone moiety would make hydrogen bonds with the
backbone NH of Leu348 and Gly349; the methylene linker would
ensure proper orientation of the scaffold for interactions with the
S10 pocket. Furthermore, substituents on the benzene ring would
reach further into the S10/S30 pocket to establish additional interac-
tions with the protein. The indazole NH could make a hydrogen
bond with the carbonyl of Pro437. The methyl group on the hydan-
toin points into the non-prime side, into an area open for further
substitutions.
Figure 3. (A) Docked model of 3 (magenta) in the TACE active site (surface). The
X-ray structure is shown in yellow (PDB code: 3L0V). (B) Stick representations
showing the detailed interactions of 3 with the TACE active site observed in the
X-ray.
Table 1
TACE Ki of 1H-indazol-3(2H)-ones analogs ( )
O
R2
6
R1
N
HN
N
5
R3
H
O
O
N
H
Compound
R1
R2
R3
H
MeO
H
TACE Kia (nM)
3
8
9
10
11
12
13
Me
Me
Me
Me
Me
Ph
MeO
H
EtO
BnO
H
23
28
209
391
100
53
The synthetic route to 3 is illustrated in Scheme 1 and was used
for the synthesis of analogs. Compound 4 was treated with diben-
H
BnO
MeO
H
MeO
MeO
Ph
6
O
OCH3
O
a
The compounds were tested in a FRET assay using the catalytic domain of TACE.
O
H3C
HN
H
NH
N
N
N
H
O
N
N
O
O
NH
Table 2
H
H
O
N
O
H
TACE Ki of analogs with different ring system
1
2
3
R1
R2
Figure 1. Hydantoin based TACE inhibitors
HN
O
O
N
H
Compound
R1
R2
TACE Kia (nM)
O
( )-17
4-F-Ph
4
N
O
O
S
( )-18
( )-19
4-F-Ph
4-F-Ph
1048
2729
N
O
N
N
Figure 2. Modes of binding of compound 2 to the active site of TACE. (A) Two
binding modes (cyan and yellow carbons, respectively) of 2 to the active site of
TACE (shown as surface with zinc colored orange, PDB code: 3L0T). The S1 and S10
subsites are also indicated. (B) Stick representation showing the zinc binding of 2
and hydrogen bonding interactions (dashed lines).
H
O
N
( )-20
4-F-Ph
>50,000
N
H
O
O
O
O
N
N
b, c
a
NH
( )-21
( )-22
( )-23
Me
4818
24,900
5
Cl
Bn2N
HN
H2N
N
O
CH3
CH3
CH3
4
5
6
O
O
N
H3C
OCH3
Me
O
e
d
H
N
NH
O
3
HN
O2N
7
O
OMe
4-F-Ph
Scheme 1. Reagents and conditions: (a) Bn2NH, Et3N, THF, rt, 3 days, 89%; (b) KCN,
(NH4)2CO3, EtOH/H2O (1:1), 70 °C 8 h, 86%; (c) H2, 50 psi, Pd(OH)2/C, EtOH/MeOH
(5:1), rt, 48 h, 95%; (d) 5-methoxy-2-nitrobenzoic acid, EDCI, HOBT, NMM, DMF, rt,
16 h, 85%; (e) Zn, NaOH, MeOH/H2O (1:1), 75 °C, 20 h, 14–69%.
N
N
a
The compounds were tested in a FRET assay using the catalytic domain of TACE.

W. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1877–1880
1879
zylamine to afford 5 which was converted to a hydantoin via the
Bucherer–Bergs reaction. Reductive debenzylation yielded the de-
sired amine 6. Compound 6 was coupled with 5-methoxy-2-nitro-
benzoic acid to afford amide 7 which was converted to 3 in the
presence of zinc and NaOH in low to moderate yield.13
Compound 3 has a TACE Ki of 23 nM and the X-ray structure re-
vealed that 3 bound in the manner expected from modeling
(Fig. 3A). The stereochemistry of the hydantoin was determined
to be R. The corresponding C5 methoxy analog 8 was equipotent
to 3. Increasing steric bulk of the C5 or C6 substituent resulted in
a 5–10-fold loss in potency (compounds 9–11). 5,6-Dimethoxy
analog 12 was also less active. Phenyl substitution on the hydan-
toin (13) showed a fourfold improvement of activity compared
with 3 (6 nM vs 23 nM).
The length of the hydrogen bond between the indazole NH and
the C@O of Pro437 was determined to be 3.2 Å in the X-ray struc-
ture (Fig. 3A), suggesting that this may not be a key interaction.
This lead us to explore other ring systems (shown in Table 2) as
the core structure of the hydantoin TACE inhibitors. Compounds
in Table 2 were prepared as described in Scheme 2.14 Compound
14 was prepared as described in Scheme 1. Benzyl bromide 15
was prepared by bromination of the corresponding toluene deriv-
atives.15 The target compounds 16 were obtained in moderate to
good yields.
As shown in Table 2, isoindolin-1-one 17 showed excellent activ-
ity. It confirmed that the H-bond between the indazole NH and the
C@O of Pro437 in 3 is not a key interaction and suggested that a
methylene group might fit better in this position. The sulfonamide
18 is a much weaker TACE inhibitor. The hexahydroimidazo[1,5-
a]pyridin-3(5H)-one analog 19 and tetrahydro-1H-pyrrolo[1,2-
c]imidazol-3(2H)-one analog 20 are also much weaker than 17.
Pyridyl analogs 21 and 22 are less active whereas 23 showed good
activity. In 21, the pyridyl nitrogen (N7) reaches into a hydrophobic
pocket, and in 22, there is a repulsion between the pyridyl nitrogen
(N4) and the carbonyl oxygen of Tyr 436. However, in 23, the pyridyl
nitrogen (N5) forms a hydrogen bound to a conserved water mole-
cule at the bottom of the S10 pocket.
Since isoindolin-1-one analog 17 showed the best TACE activity,
additional analogs (shown in Table 3) were prepared. Compounds
in Table 3 were prepared based on Schemes 2 or 3. In Scheme 3,
compound 24 was demethylated and protected with SEMCl to give
25. Compound 25 was resolved on a ChiralpakÒ AD column16, and
the resulting, desired (R)-enantiomer was alkylated with the
appropriate alkyl bromides or alkyl iodides. The SEM group was re-
moved to give the final products (R)-26.
O
R1
Y
NH2 ·HCl
R1
HN
Y
N
+
HN
CO2Me
O
O
N
As shown in Table 3, methyl hydantoin analog 27 showed
slightly improved activity compared with the 1H-indazol-3(2H)-
one counterparts (Tables 1 and 3). Larger alkyl substituents on
the hydantoin moiety led to a loss in potency (e.g., 28) and this ef-
fect was most pronounced with compound 29. Consistent with
O
H
O
N
Br
H
(
)-14
15
( )-16
Scheme 2. Reagents and conditions: DIPEA, DMF, rt, 16 h, 54–72%.
Table 3
TACE Ki of isoindolin-1-one analogs
O
6
5
R2
R3
R1
N
HN
O
O
N
H
Compound
R1
R2
R3
TACE Ki (nM)
( )-27
(R)-28
(R)-29
( )-30
( )-31
( )-32
(R)-33
(R)-34
( )-35
(R)-36
(R)-37
(R)-38
(R)-39
( )-40
(R)-41
( )-42
Me
Et
Benzyl
Ph
20-OH-Ph
30-OH-Ph
40-OH-Ph
40-F-Ph
20,40-diF-Ph
40-F-Ph
20-Pyrazinyl
20-Furanyl
10-Methyl-1H-pyrazol-40-yl
Ph
40-F-Ph
4-F-Ph
MeO
MeO
MeO
MeO
F
Cl
MeO
Cl
H
H
H
H
H
H
H
H
H
H
H
H
10
56
>500
3.3
9
10
1.6
0.4
5.5
0.8
0.7
0.7
1.1
3.0
0.5
7.0
Cl
MeO
MeO
MeO
MeO
H
H
MeO
OH
Cl
O
H
H
(R)-43
( )-44
4-F-Ph
4-F-Ph
H
H
2.4
9
O
N
O
O
(R)-45
( )-46
4-F-Ph
4-F-Ph
H
H
332
1.7
N

1880
W. Yu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1877–1880
F
F
F
O
O
O
OR
OMe
OH c - e
a, b
N
N
N
HN
HN
HN
O
O
O
O
N
O
N
O
N
H
H
SEM
( )-24
( )-25
(R)-26
Scheme 3. Reagents and conditions: (a) pyridineꢀHCl, 200 °C, 1 h, 85%; (b) SEMCI, DIPEA, DMF, rt, 5 h, 80%; (c) chiral HPLC separation (see Ref. 16); (d) RI or RBr, Cs2CO3, DMF,
rt, 16 h, 50–95%; (e) HCl, MeOH, 90 °C, 16 h, then TEA, rt, 75–90%.
3. Black, R. A.; Rauch, C. T.; Kozlosky, C. J.; Peschon, J. J.; Slack, J. L.; Wolfson, M. F.;
Table 4
Selectivity data of 3 and 34
Castner, B. J.; Stocking, K. L.; Reddy, P.; Srinivasan, S.; Nelson, N.; Boiani, N.;
Schooley, K. A.; Gerhart, M.; Davis, R.; Fitzner, J. N.; Johnson, R. S.; Paxton, R. J.;
March, C. J.; Cerretti, D. P. Nature 1997, 385, 729.
4. Bemelmans, M. H. A.; van Tits, L. J. H.; Buurman, W. A. Crit. Rev. Immunol. 1996,
16, 1.
5. Aggarwal, B. B.; Natarajan, K. Eur. Cytokine Netw. 1996, 7, 93.
6. For a recent review of TACE inhibitors, see: DasGupta, S.; Murumkar, P. R.;
Giridhar, R.; Yadav, M. R. Bioorg. Med. Chem. 2009, 17, 444.
Compound
3
34
TACE Ki (nM)
MMP1 (nM)
MMP2 (nM)
MMP3 (nM)
MMP7 (nM)
MMP9 (nM)
MMP13 (nM)
MMP14 (nM)
ADAM 9 (nM)
ADAM 10 (nM)
28.0
0.43
176
1208
>40,000
>40,000
>40,000
>40,000
1196
—
3300
4599
10,000
—
100,000
12,860
31,910
10,000
—
7. (a) Sheppeck, J. E., II; Gilmore, J. L.; Yang, A.; Chen, X.-T.; Xue, C.-B.; Roderick, J.;
Liu, R.-Q.; Covington, M. B.; Decicco, C. P.; Duan, J. J.-W. Bioorg. Med. Chem. Lett.
2007, 17, 1413; (b) Sheppeck, J. E., II; Gilmore, J. L.; Tebben, A.; Xue, C.-B.; Liu,
R.-Q.; Decicco, C. P.; Duan, J. J.-W. Bioorg. Med. Chem. Lett. 2007, 17, 2769; (c)
Sheppeck, I. I. J. E.; Tebben, A.; Gilmore, J. L.; Yang, A.; Wasserman, Z. R.;
Decicco, C. P.; Duan, J. J.-W. Bioorg. Med. Chem. Lett. 2007, 17, 1408.
8. Rosner, K. E.; Guo, Z.; Orth, P.; Shipps, G. W., Jr.; Belanger, D. B.; Chan, T. Y.;
Curran, P. J.; Dai, C.; Deng, Y.; Girijavallabhan, V. M.; Hong, L.; Lavey, B. J.; Lee, J.
F.; Li, D.; Liu, Z.; Popovici-Muller, J.; Ting, P. C.; Vaccaro, H.; Wang, L.; Wang, T.;
Yu, W.; Zhou, G.; Niu, X.; Sun, J.; Kozlowski, J. A.; Lundell, D. J.; Madison, V.;
McKittrick, B.; Piwinski, J. J.; Shih, N.-Y.; Siddiqui, M. A.; Strickland, C. O. Bioorg.
Med. Chem. Lett. 2010, 20, 1189.
9. Guo, Z.; Orth, P.; Wong, S.-C.; Lavey, B. J.; Shih, N.-Y.; Niu, X.; Lundell, D. J.;
Madison, V.; Kozlowski, J. A. Bioorg. Med. Chem. Lett. 2009, 19, 54.
10. Ingram, R. N.; Orth, P.; Strickland, C. L.; Le, H. V.; Madison, V.; Beyer, B. M.
Protein Eng., Des. Selection 2006, 19, 155.
11. Maskos, K.; Fernandez-Catalan, C.; Huber, R.; Bourenkov, G. P.; Bartunik, H.;
Ellestad, G. A.; Reddy, P.; Wolfson, M. F.; Rauch, C. T.; Castner, B. J.; Davis, R.;
Clarke, H. R. G.; Petersen, M.; Fitzner, J. N.; Cerretti, D. P.; March, C. J.; Paxton, R.
J.; Black, R. A.; Bode, W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3408.
12. Cross, J. B.; Duca, J. S.; Kaminski, J. J.; Madison, V. S. J. Am. Chem. Soc. 2002, 124,
11004.
245
what we observed in the indazole series, phenyl substitution on
the hydantoin (30) showed a threefold improvement of activity
compared to 27. Substitution on the phenyl ring is tolerated (31–
36) and C4’ substitution seems to be preferred (33, 34). Replacing
the phenyl group with heterocycles afforded compounds with sim-
ilar activities (37–39).17 Both enantiomers of ( )-4016 were pre-
pared. The active isomer (Ki = 1.7 nM) was determined to be the
(R)-isomer by X-ray structure.
As was observed in the 1H-indazol-3(2H)-one series, small sub-
stituents on either C5 or C6 offered potent TACE inhibitors (e.g., 34,
36, 40–43). As the steric bulk of the substituents increases (44, 45),
a 4–100-fold loss in affinity was observed. However, the bulky
quinolinyl analog 46 was tolerated as its binding causes a confor-
mational change of the Ile438-Ser441 loop, to allow the quinolinyl
group to be accommodated within the S1’/S3’ pockets.18
Both the 1H-indazol-3(2H)-one series and isoindolin-1-one ser-
ies were selective against a group of MMPs as shown by represen-
tative compounds 3 and 34 in Table 4. Selected compounds from
each series (compounds 13, 34, and 36) were also evaluated for
their ability to inhibit LPS-induced TNF-
whole blood,19 an indicator of in vivo potency.20 Relatively weak
activities (IC50s of 3–16 M) were observed, suggesting that fur-
13. Bruneau, P.; Delvare, C.; Edwards, M. P.; McMillan, R. M. J. Med. Chem. 1991, 34,
1028.
14. Compounds 19 and 20 were made by treating amine 6 with tert-butyl 2-
formylpyrrolidine-1-carboxylate or 2-formylpiperidine-1-carboxylic acid tert-
butyl ester with titanium isopropoxide and NaBH3CN followed by deprotection
of the Boc protecting group. The resulting material was then subjected to
carbonyl diimidazole in the present of Hünig’s base to give 19 and 20,
respectively.
15. For the synthesis method of methyl 2-(bromomethyl)-5-methoxybenzoate,
please see patent: Yu, W. et al. WO 2007084415, 2007.
16. For chiral HPLC separation conditions, see Ref. 15.
17. For the synthesis of the corresponding 2-amino-1-heteroaryl-ethanone, (a)
from heteroaryl halide, see Chen, Z. et al. WO 2005056562, 2005.; from 2-
bromo-1-heteroaryl-ethanone, see: (b) Loughlin, W. A.; Henderson, L. C.; Elson,
K. E.; Murphy, M. E. Synthesis 2006, 1975.
a shedding in human
l
18. This is based on a X-ray crystal structure of the corresponding R1 = phenyl
compound within the active site of the TACE enzyme and additional X-ray data
on compounds within the hydantoin series that we have not discussed in detail
due to space limitations. The shift of this loop is very similar to that previously
described in Niu, X.; Umland, S.; Ingram, R.; Beyer, B. M.; Liu, Y.-H.; Sun, J.;
Lundell, D.; Orth, P. Arch. Biochem. Biophys. 2006, 451, 43.
ther improvement is required.
In summary, we have discovered a new series of selective and
potent TACE inhibitors using the hydantoin moiety as a zinc-bind-
ing ligand. We report the first X-ray structures of inhibitors with a
hydantoin zinc ligand bound to TACE. Using structure-based design
our initial optimization improved weak micromolar screening hits
into sub-nanomolar TACE inhibitors. Further optimization of our
lead compounds will be disclosed in future publications.
19. Human whole blood is diluted 1:1 with serum free medium (RPMI,
glutamine, Pen-Strep, HEPES) and incubated with compound in 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-
a
a
l
a
20. Please see the following reference for a discussion of the correlation of whole
blood activity with in vivo efficacy: Qian, M.; Bai, S. A.; Brogdon, B.; Wu, J.-T.;
Liu, R.-Q.; Covington, M. B.; Vaddi, K.; Newton, R. C.; Fossler, M. J.; Garner, C. E.;
Deng, Y.; Maduskuie, T.; Trzaskos, J.; Duan, J. J.-W.; Decicco, C. P.; Christ, D.
Drug Metab. Dispos. 2007, 35, 1916.
References and notes
1. Cerretti, D. P. Biochem. Soc. Trans. 1999, 27, 219.
2. Vassalli, P. Annu. Rev. Immunol. 1992, 10, 411.