A Highly Selective Hydantoin Inhibitor of Aggrecanase-1 and Aggrecanase-2 with a Low Projected Human Dose

Article abstract of DOI:10.1021/acs.jmedchem.7b00650

Aggrecanase-1 and -2 (ADAMTS-4 and ADAMTS-5) are zinc metalloproteases involved in the degradation of aggrecan in cartilage. Inhibitors could provide a means of altering the progression of osteoarthritis. We report the identification of 7 which had good oral pharmacokinetics in rats and showed efficacy in a rat chemical model of osteoarthritis. The projected human dose required to achieve sustained plasma levels ≥10 times the hADAMTS-5 IC₅₀ is 5 mg q.d.

Full text of DOI:10.1021/acs.jmedchem.7b00650

Subscriber access provided by HKU Libraries
Brief Article
A Highly Selective Hydantoin Inhibitor of Aggrecanase-1
and Aggrecanase-2 with a Low Projected Human Dose.
Timothy B. Durham, Jothirajah Marimuthu, James L Toth, Chin Liu, Lisa A Adams, Daniel Robert Mudra,
Craig A Swearingen, Chaohua Lin, Mark G Chambers, Kannan Thirunavukkarasu, and Michael R. Wiley
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 14 Jun 2017
Downloaded from http://pubs.acs.org on June 14, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
A Highly Selective Hydantoin Inhibitor of Aggrecanase-1 and Aggre-
canase-2 with a Low Projected Human Dose.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Timothy B. Durham*; Jothirajah Marimuthu; James L. Toth; Chin Liu; Lisa Adams; Daniel R.
Mudra; Craig Swearingen; Chaohua Lin; Mark G. Chambers; Kannan Thirunavukkarasu; and Mi-
chael R. Wiley
Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN, 46285
KEYWORDS Aggrecanase, Osteoarthritis, Hydantoin, ADAMTS-4, ADAMTS-5, Matrix Metalloprotease.
ABSTRACT: Aggrecanase 1 and 2 (ADAMTS-4 and ADAMTS-5) are zinc metalloproteases involved in the degradation of
aggrecan in cartilage. Inhibitors could provide a means of altering the progression of osteoarthritis. We report the identi-
fication of compound 7 which had good oral pharmacokinetics in rats and showed efficacy in a rat chemical model of os-
teoarthritis. The projected human dose required to achieve sustained plasma levels ≥ 10 times the hADAMTS-5 IC50 is 5
mg QD.
Introduction
In previous reports, we disclosed a class of hydantoins (ie.
1, Figure 1) found to be dual inhibitors of ADAMTS-4 and
ADAMTS-5.5 These compounds displayed high levels of
selectivity against a number of MMPs. We also showed
that compounds which were active in an acute mono-iodo
acetate (MIA) pharmacodynamic model of OA were also
active in the much longer and resource intensive menis-
cal-tear (MT) surgical model.5a These inhibitors were effi-
cacious in the MT model at the MIA ED50 dose. Using
osmotic pumps, we were able to demonstrate that a ≥50%
reduction in synovial biomarkers of aggrecan cleavage
was achieved at doses capable of sustaining plasma levels
of the inhibitors ≥10 fold their IC50 in rat plasma.5b This
suggested that selective inhibitors able to achieve a Cmin
≥10 fold the plasma IC50 with a minimal Cmax at reasonable
doses would be attractive candidates for clinical OA in-
vestigation.5b
ADAMTS-4 and ADAMTS-5 (aggrecanase 1 and aggre-
canase 2) are zinc metalloproteases known to have a spe-
cific and primary role in aggrecan degradation.1 Aggrecan
is the component of cartilage that provides compressibil-
ity. This compressibility is derived from the highly glyco-
sylated nature of the protein which results in high levels
of hydration. Aggrecan loss would be expected to increase
the mechanical trauma the joint undergoes during
movement leading to structural damage and inflamma-
tion. Thus, inhibition of ADAMTS-4 and ADAMTS-5 ac-
tion has been suggested as a potential opportunity for
therapeutic intervention in osteoarthritis (OA).2 This hy-
pothesis is supported by data generated in genetically
modified mice as well as human chondrocytes.3
A key challenge to clinical testing of aggrecanase inhibi-
tors is the need to achieve high levels of selectivity against
the related matrix metalloprotease (MMP) enzymes.4 It
has been clinically established that broad spectrum inhi-
bition of MMPs leads to musculoskeletal syndrome in
patients. The active sites of the MMPs have high degrees
of structural homology to the aggrecanase enzymes.
In this report, we share the further optimization of this
hydantoin scaffold culminating in the identification of a
molecule with an improved projected human dose and
lower risk of reactive metabolite formation compared to
our previous leading compound (1, Figure 1)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 8
man PK profile (Figure 1).5b We conducted a bile duct
1
2
3
4
5
6
7
8
cannulated rat study to understand metabolism of com-
pound 1. This experiment revealed glutathione conjuga-
tion was the sole metabolic clearance pathway (See Sup-
porting Information). The glutathione conjugates were
arising via reactive oxidation products derived from the
benzofuran moiety. Significant glutathione depletion
could result in contraindication to acetaminophen.
Chemistry
The hydantoin core was prepared according to our previ-
ously described approach.5b The chiral amides were syn-
thesized by acylation of auxiliary 9 with commercially
available acid chlorides 10-13 (scheme 1). The resulting
imide was then alkylated at -78 °C using 4-trifluoromethyl
benzyl bromide. Removal of the chiral auxiliary gave acids
22-25. Amide formation with hydantoin 265b completed
the synthesis of inhibitors 2 and 5-7. Diastereomer 3 (Fig-
ure 1) was prepared by the same route from ent-9.
Scheme 1.a
9
O
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
a
R
R
+
N
NH
Cl
O
O
O
O
10, R = Me
11, R = Et
12, R = iPr
13, R = cPr
N
H
Ph
HN
NH
Ph
F
F
F
O
14, R = Me 16, R = iPr
15, R = Et 17, R = cPr
9
O
1
O
O
b
c
O
N
O
O
R
F
N
X
H
F
F
NH
F
F
HN
Ph
18, R = Me 20, R = iPr
F
O
19, R = Et
21, R = cPr
X =
O
O
+
NH3
Cl
HO
2
5
3
6
4
7
NH
HN
R
F
F
O
F
22, R = Me 24, R = iPr
23, R = Et 25, R = cPr
26
d
HO
Me
2, 5-7
aConditions: (a) DMAP, Et3N, CH2Cl2, 0 °C to rt, 50-85%;
(b) LDA, 4-trifluoromethylbenzyl bromide, THF, -78 °C,
40-50%; (c) H2O2, LiOH, THF/water, 0 °C, quant.; (d)
HATU, i-Pr2NEt, DCM/DMF, rt, 50-80%.
O
N
S
O
F
NH
O
HO
O
Cl
As we further developed this series we wanted to mini-
mize glutathione conjugation and lower the projected
human dose to <10 mg, QD. We set out to replace the
benzofuran with a group less likely to produce reactive
metabolites but also provide improved potency in plasma
while maintaining a good PK profile with minimal peak to
trough exposure to effectively deliver efficacy.5b Com-
pound 1 had arisen from optimization of screening hit 30
which was modestly potent but not highly selective
against MMP enzymes (Scheme 3, Table 1).5a X-ray crystal
structures revealed that the binding mode involved liga-
tion of the zinc by the hydantoin imide nitrogen. This
places the phenoxy moiety in the S1’ binding site. As de-
scribed previously, cyclization of the amide sidechain into
a benzofuran provided selectivity and PK performance
8
Figure 1. Inhibitors of Aggrecanase.
Dimethyl amide 4 was prepared starting from commercial
phosphonate 27 (Scheme 2). Olefination6 followed by hy-
drogenation and subsequent alkylation with methyl io-
dide7 provided the geminally substituted ester. This was
hydrolyzed to give carboxylic acid 29.8 Carbodiimide me-
diated amide bond formation provided hydantoin 4.
Results and Discussion
In our previous report, we identified compound 1 as a lead
compound for development of a clinically useful aggre-
canase inhibitor based on its potency and projected hu-
ACS Paragon Plus Environment

Page 3 of 8
Journal of Medicinal Chemistry
ADAMTS-4 (Figure 2 & 3) using X-ray co-crystal structure
benefits. We chose to explore replacement of the oxygen
4WKE as a starting point.5a As shown in Figure 2A, the
hydantoin serves as a ligand for the catalytic zinc while
the benzofuran of compound 1 occupies the S1’ binding
site.5a The methyl substituent of compound 2 is well ac-
commodated and directed towards the solvent. As the
substituent’s size increases from methyl to cyclopropyl
(Figure 2B), it makes increased contact with the sidechain
of Met395 which forms the upper wall of the S1’ pocket.
Both MMP2 and MMP12 have high degree of sequence
and shape homology in the S1’ binding site (Figure 3).
However, in MMP29 and MMP1210, Met395 is replaced by a
more bulky and potentially less mobile tyrosine. We hy-
pothesize that the enhanced selectivity of compounds 6
and 7 arise from increased negative steric interactions
between the substituent on the amide carbon and these
tyrosine residues.
1
2
3
4
5
6
7
8
linkage with carbon. To our delight the carbon series
proved to be generally more potent than the oxygen se-
ries, likely due to a subtle, but better, placement of the
aryl ring within the S1’ binding site. Compound 31 was a
potent ADAMTS-4 and ADAMTS-5 inhibitor but only
showed modest MMP selectivity (Table 1). However, we
hypothesized that analogs bearing appropriate amide
substitution would show enhance selectivity against MMP
enzymes arising from negative steric interactions in the
S1’ binding site.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We prepared analogs 2-7 bearing methyl, ethyl, isopropyl,
and cyclopropyl substituents and profiled their activity
against hADAMTS-4 , hADAMTS-5, and a panel of repre-
sentative hMMP enzymes (Table 1). The R-stereoisomer
proved to be the more potent and selective of the dia-
stereomers 2 and 3. Gem dimethyl analogue 4 had lower
potency and selectivity compared to compound 2.
Compound 7 was evaluated in our rat MIA pharmacody-
namics model (Figure 4).11 This model simulates OA in an
acute time frame through chemical injury to the joint,
resulting in release of large amounts of proteases, includ-
ing aggrecanases. We have developed ELISA assays to
detect both fragments (NITEGE or ARGNV) of aggrecan
cleavage by ADAMTS-5.12 With oral BID dosing, com-
pound 7 lowered aggrecan degradation products in syno-
vial fluid at all doses tested, performing as robustly as the
broad spectrum metalloprotease inhibitor 8.13 Further,
consistent with the PK/PD relationship defined using
compound 1, ED50 effects were delivered by doses capable
of sustaining ≥ 10 times the plasma ADAMTS-5 IC50.
Scheme 2.a
aConditions: (a) NaH, THF, 0 °C to rt, 74%; (b) H2, Pd-C,
MeOH, 90%; (c) LDA, MeI, THF, -78 °C, 45%; (d) NaOH,
MeOH, 80 °C, 75%; (e) iPr2NEt, EDCI, HOAT, 26,
CH3CN, rt, 70%.
Increasing the size of the substituent produced com-
pounds with higher levels of selectivity. Hydantoin 6 was
the most selective compound but suffered from loss of
potency in the rat plasma assay. Analog 7 had robust se-
lectivity, good potency in the plasma assay and good PK
in rats (Table 2). Importantly, no evidence for glutathione
conjugation in vivo was observed with any of the com-
pounds lacking the benzofuran.
Scheme 3.
Figure 2. Models of Hydantoin Inhibitors Bound to
hADAMTS-4. A: Compound 1 (magenta) and Compound
2 (orange) B: Compound 7. Ligand molecular surface
shown as wire mesh.
We estimated the human PK of compound 7 (See Sup-
porting Information for details). Based on our previous
work,5b we modeled the dose at which the sustained hu-
man plasma concentration would be ~10 times the
hADAMTS-5 IC50 (a concentration equivalent to the ED50
To understand the basis for selectivity against the MMPs,
we built models of compounds 1, 2, 6 and 7 bound to
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 8
Table 1. Biochemical IC50 Values (nM).a
1
hADAMTS-5
(Rat Plasma)
2
3
Comp.
hADAMTS-4
hADAMTS-5
hMMP2
hMMP3
hMMP12
hMMP13
hMMP14
4
5
6
7
8
9
1
30
31
2
6
74
4
5
350
5
63
620
52
5800
24000
310
17400
50000
660
9
70
5
16000
7000
320
7600
>50000
3400
1
1
15
1800
280
2800
2000
13000
15000
9
1100
3900
3
12
7
13
4
140
64
20
150
18
7
340
1600
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4
4700
3000
71
4800
4200
> 100000
47000
1
15000
8000
5
2
2
26
3400
270
< 1
6
7
14
2
8
> 100000 > 100000
> 100000
50000
150
2
35000
5
> 100000
3
8
1
1
18
a = values are an average of n = 3 or more data points.
ADAMTS-5 inhibitors. Compound 7 has been identified as
a molecule with an attractive preclinical efficacy and pro-
jected human PK profile.
Compound 7 Rat MIA Assay
30000
41% 52% 53%
50%
*
20000
10000
0
*
*
*
Figure 3. Model of Compound 6 overlaid with ADAMTS-
4, MMP-12, and MMP-14. Compound
6 (orange);
ADAMTS-4 (gray with solid surface), Met395 (stick);
MMP2 (1QIB)9 = magenta with wire surface, TYR223
(line); MMP12 (1JK3)10 = cyan with wire surface, TYR240
(line). Overlays were generated using the sequence over-
lay tool in MOE.
Figure 4. Rat MIA Assay. * = p ≤ 0.05
Table 2. Rat Pharmacokinetic Data for Compound 7.
Route of Administration IV
PO
Dose (mg/kg)
Cmax (nM)
1
10
1600 6600
0.5
Tmax (h)
Plasma (nM) at 12 h
AUC(0 to 24 h) (nM*h)
T1/2 (h)
< 5
29
1300 22000
1.5
33
2.6
Cl (mL/min/kg)
Vd (L/kg)
Figure 5. Projected Human PK for Cmpound 7. Solid red
line = simulated human PK at steady state following 5 mg
PO QD; black lines = projection ±30% CV; dashed line =
hADAMTS-5 IC90.
4.3
F (%)
100
in the MIA model). As shown in Figure 5, a 5 mg QD dose
is projected to achieve this plasma level in humans, a
meaningful improvement from the projected dose of
compound 1 (45 mg, QD).
Experimental
General. Protocols for the in vitro assays are included in
the Supporting Information. All animals were maintained
in accordance with the Institutional Animal Care and Use
Committee of Eli Lilly and the National Institutes of
Health Guide for the Care and Use of Laboratory Animals.
Conclusions
The hydantoin scaffold5 has proven to be a good platform
for development of potent and selective ADAMTS-4 and
ACS Paragon Plus Environment

Page 5 of 8
Journal of Medicinal Chemistry
MIA assay. This assay has been described in detail in the
mg,
3
mmol),
2,2-dimethyl-3-[4-
literature.11 Briefly, 7-8-week-old male Lewis rats are anes-
thetized and injected intra-articularly with either 3 mg of
freshly prepared MIA (sodium salt) in 50µl of 0.9% saline
into the right knee or 0.9% saline alone in the left knee on
day 0. This induced aggrecanase activity and release of
aggrecanase specific fragments into the synovial fluid.
Aggrecanase inhibitor or vehicle [1%hydroxyethyl cellu-
lose; 0.25% Tween 80; 0.05% antifoam] are dosed PO, BID
starting from day 3. A single dose of compound is given
on day 7, the animals are sacrificed 4 h later, and the knee
joints are lavaged with 200 ꢀL of saline. The synovial lav-
age is assayed for aggrecanase-cleaved fragments of ag-
grecan using the NITEGE/ARGNV sandwich ELISA (see
Supporting Information). The amounts of aggrecan frag-
ments present in the synovial lavage are determined
based on a standard curve generated with aggrecanase-
digested rat aggrecan. Statistical analysis is performed
using Dunnett's test.
(trifluoromethyl)phenyl]propanoic acid14 (246 mg,
1
1
2
3
4
5
6
7
8
mmol), EDCI (229 mg, 1.2 mmol), and HOAT (163 mg, 1.2
mmol). The resulting mixture was stirred for 12 h at ambi-
ent temperature. Solvents were removed on a rotary
evaporator and the residue was purified by reverse phase
flash chromatography to give the title compound (282
1
mg, 70%). H NMR (DMSO): 10.63 (s, 1H), 7.62 (d, J= 8.1
Hz, 2H), 7.47 (t, J= 6.1 Hz, 1H), 7.37 (s, 1H), 7.33 (d, J= 8.0
Hz, 2H), 3.60 (dd, J= 6.9, 13.6 Hz, 1H), 3.34 (m, 1H), 2.92-
2.89 (m, 1H), 2.86-2.83 (m, 1H), 1.05 (s, 3H), 1.04 (s, 3H),
1.02 (m, 1H), 0.42-0.33 (m, 3H), 0.08 (td, J= 9.4, 5.3 Hz,
1H); 13C (DMSO): 177.04, 157.53, 143.81, 131.25, 127.45, 127.14,
126.29, 125.10 (m), 123.59, 65.90, 45.17, 44.39, 43.23, 25.38,
25.24, 14.32, 0.96, -0.87.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(R)-N-(((R)-4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-2-(4-(trifluoromethyl)benzyl) butanamide
23
1
(5). [
α]
D
= -22.10° (c = 1.0, MeOH); H NMR (DMSO):
10.56-10.54 (m, 1H), 7.89-7.87 (m, 2H), 7.59 (d, J= 8.2 Hz,
3H), 7.43-7.41 (m, 1H), 7.35-7.33 (d, J= 8.0 Hz, 2H), 3.56-
3.54 (m, 1H), 3.29-3.28 (m, 1H), 2.90-2.87 (m, 2H), 2.65-
2.64 (m, 1H), 1.53-1.48 (m, 1H), 1.33-1.30 (m, 1H), 1.03-1.01
(m, 1H), 0.72 (t, J= 7.4 Hz, 3H), 0.42-0.40 (m, 3H), 0.14-
0.10 (m, 1H); 13C (DMSO): 176.70, 174.89, 157.39, 145.75,
129.96, 127.26, 126.94, 125.47, 123.59, 65.87, 48.61, 43.52,
38.16, 25.39, 14.76, 11.99, 0.74, -0.68.
1
Chemistry General methods: H and 13C NMR spectra
were measured in deuterated solvents at 400 MHz and
100 MHz respectively on either a Varian or Bruker spec-
trometer. Data are given as δ. All solvents were dry. Rea-
gents were obtained from commercial sources and used as
received. Purity of final compounds used in biological
assays was determined by electrospray LCMS to be ≥ 95%.
(See Supporting information for method and data).
(S)-N-(((R)-4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-3-methyl-2-(4-(trifluoromethyl)benzyl)
Compounds 2, 3, 5, and 6 were prepared according to the
same general procedure as compound 7.
23
1
butanamide (6). [
α]
D
= -38.90° (c = 1.0, MeOH); H
NMR (DMSO): 10.50 (s, 1H), 7.68 (t, J= 6.0 Hz, 1H), 7.55
(d, J= 8.1 Hz, 2H), 7.37 (d, J= 0.8 Hz, 1H), 7.31 (d, J= 7.9 Hz,
2H), 3.59-3.54 (m, 1H), 3.10-3.05 (m, 1H), 2.90-2.87 (m, 1H),
2.71-2.67 (m, 1H), 2.37-2.32 (m, 1H), 1.73-1.71 (m, 1H), 0.97-
0.92 (m, 1H), 0.88 (d, J= 6.8 Hz, 2H), 0.83 (d, J= 6.7 Hz,
2H), 0.37-0.32 (m, 3H), 0.09-0.05 (m, 1H); 13C (DMSO):
176.64, 174.18, 157.32, 146.31, 129.82, 127.09, 126.77, 126.31,
125.39, 123.61, 65.69, 53.57, 43.43, 35.07, 31.02, 20.55, 20.50,
14.89, 0.69, -0.71.
(R)-N-(((R)-4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-2-methyl-3-(4-(trifluoromethyl)phenyl)
23
1
propanamide (2). [
α]
D
= -14.10° (c = 1.0, MeOH); H
NMR (DMSO): 10.58-10.56 (m, 1H), 7.91-7.86 (m, 1H), 7.59
(d, J= 8.1 Hz, 2H), 7.44 (s, 1H), 7.35 (d, J= 7.9 Hz, 2H), 3.43-
3.35 (m, 2H), 2.95-2.91 (m, 1H), 2.65-2.61 (m, 1H), 2.48-2.46
(m, 1H), 1.03-0.99 (m, 1H), 0.90 (d, J= 6.8 Hz, 3H), 0.42-
0.39 (m, 4H); 13C (DMSO) 176.76, 175.81 157.41, 145.62,
129.99, 127.31, 127.00, 125.54, 125.50 (m), 125.47, 65.93,
43.67, 78, 14.61, 0.78, -0.70
(S)-2-cyclopropyl-N-(((R)-4-cyclopropyl-2,5-
dioxoimidazolidin-4-yl)methyl)-3-(4-
(S)-N-(((R)-4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-2-methyl-3-(4-(trifluoromethyl)phenyl)
propanamide (3). Prepared according to the same se-
quence as compound 2 beginning with (R)-4-Benzyl-2-
(trifluoromethyl)phenyl) propanamide (7). Salt 265b
(2.33 g, 11.3 mmol), acid 25 (2.66 g, 10.3 mmol), and HATU
(4 g, 10.3 mmol) were dissolved in dry 5:1 CH2Cl2/DMF
under nitrogen. To the resulting mixture was added
iPr2NEt amine (5.6 mL, 51.5 mmol). After 48 h, solvents
were removed and the residue purified by chromatog-
raphy ( C18, gradient 10% to 70% CH3CN/water + 0.1%
TFA) to give compound 7 as a white solid (3.1 g, 73%).
1
oxazolidinone. H NMR (DMSO): 10.56-10.54 (m, 1H),
7.93-7.89 (m, 2H), 7.60-7.57 (m, 2H), 7.38-7.35 (m, 3H),
3.57-3.52 (m, 1H), 3.27-3.26 (m, 1H), 2.97-2.93 (m, 1H),
2.66-2.65 (m, 1H), 2.56-2.54 (m, 1H), 1.00-0.98 (m, 1H),
0.93-0.91 (d, J= 6.8 Hz, 3H), 0.52-0.46 (m, 3H), 0.13-0.08
23
1
13
[α
]
D
= 3.30° (c = 1.0, MeOH); H NMR (DMSO): 10.56-
(m, 1H); C (DMSO): 176.91, 175.87, 157.38, 145.64, 130.06,
10.54 (m, 1H), 7.71-7.66 (m, 1H), 7.57-7.55 (d, J= 8.4 Hz,
2H), 7.41-7.37 (m, 1H), 7.34-7.32 (d, J= 8.0 Hz, 2H), 3.51-
3.46 (dd, J = 6.0, 13.6 Hz, 1H), 3.28-3.24 (dd, J = 1.2, 13.6 Hz,
1H), 3.05-3.03 (dd, J = 8.0, 13.6 Hz, 1H), 2.79-2.76 (dd, J =
5.6, 13.6 Hz, 1H), 1.87-1.84 (m, 1H), 1.02-0.98 (m, 1H), 0.85-
0.83 (m, 1H), 0.39-0.37 (m, 7H), 0.11-0.09 (m, 1H), -0.08--
0.10 (m, 1H); 13C (DMSO) : 176.63, 174.41, 157.38, 145.70,
127.31, 127.00, 126.28, 125.40 (m), 123.57, 65.95, 43.55, 41.18,
39.26, 18.17, 14.39, 0.78, -0.72.
(R)-N-((4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-2,2-dimethyl-3-(4-(trifluoromethyl)-
phenyl)propanamide (4). Hydantoin 265b (205 mg, 1
mmol) was dissolved in 15 mL of dry CH3CN under nitro-
gen. To the resulting solution was added i-Pr2NH (387
5
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 8
129.96, 127.18, 126.87, 126.31, 125.34 (m), 65.77, 51.76, 43.54, warmed to ambient temperature. The THF was removed
1
2
3
4
5
6
7
8
38.18, 14.93, 14.71, 4.99, 3.64, 0.75, -0.70.
on a rotary evaporator. The aqueous solution was washed
with dichloromethane (x2). The solution was acidified
with 2M HCl and extracted with EtOAc. The extracts were
combined, washed with saturated NaCl, dried (MgSO4),
filtered, and concentrated to give the title compound as
an oil (3 g, quantitative). 1H NMR (DMSO): 12.14-12.07 (bs,
1H), 7.59 (d, J= 8.0 Hz, 2H), 7.39 (d, J= 7.9 Hz, 2H), 2.99-
2.88 (m, 2H), 1.89-1.83 (m, 1H), 0.92-0.89 (m, 1H), 0.42-
0.40 (m, 2H), 0.27-0.25 (m, 1H), 0.13-0.08 (m, 1H).
Carboxylic acids 22-24 were made via the same general
procedure as compound 25.
(R)-2-methyl-3-(4-(trifluoromethyl)phenyl)propanoic
1
acid (22). H NMR (DMSO): 12.19 (s, 1H), 7.60 (d, J= 8.1
Hz, 2H), 7.39 (d, J= 8.1 Hz, 2H), 2.97-2.91 (m, 1H), 2.72-
2.63 (m, 2H), 1.03 (d, J= 6.8 Hz, 3H).
(R)-2-(4-(trifluoromethyl)benzyl)butanoic acid (23).
1H NMR (DMSO): 12.18-12.12 (bs, 1H), 7.60 (d, J= 8.2 Hz,
2H), 7.38 (s, 2H), 2.88-2.74 (m, 2H), 2.53-2.48 (m, 1H),
1.52-1.46 (m, 2H), 0.86-0.83 (m, 3H).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(R)-N-((4-cyclopropyl-2,5-dioxoimidazolidin-4-
yl)methyl)-3-(4-(trifluoromethyl)phenyl)
propana-
mide (31). Prepared according to the same general proce-
dure as compound 7 from the commercially available car-
boxylic acid and amine 26.5b 1H NMR (DMSO): 10.59 (s,
1H), 7.93 (t, J= 6.1 Hz, 1H), 7.62 (d, J= 8.1 Hz, 2H), 7.50 (s,
1H), 7.41 (d, J= 8.0 Hz, 2H), 3.49-3.37 (m, 2H), 2.88 (at, J=
7.6 Hz, 2H), 2.45-2.41 (m, 2H), 1.08-1.01 (m, 1H), 0.46-0.37
(S)-3-methyl-2-(4-(trifluoromethyl)benzyl)butanoic
1
acid (24). H NMR (DMSO): 12.06 (s, 1H), 7.59 (d, J= 8.1
Hz, 2H), 7.38 (d, J= 8.0 Hz, 2H), 2.87-2.74 (m, 2H), 2.41-
2.36 (m, 1H), 1.87-1.78 (m, 1H), 0.96 (d, J= 6.8 Hz, 3H), 0.92
(d, J= 6.7 Hz, 3H).
13
(m, 3H), 0.10 (td, J= 9.6, 5.3 Hz, 1H); C (DMSO): 176.81,
(S)-2-cyclopropyl-3-(4-(trifluoromethyl)phenyl) pro-
panoic acid (25). To a flask under nitrogen was added 9
(49 mmoles, 8.68 g;), CH2Cl2 (160 mL) and DMAP (0.1
equiv, 4.90 mmoles, 598.63 mg). The mixture was cooled
to 0°C. Et3N (3 equiv, 147.00 mmoles, 20.49 mL) was add-
ed followed by drop-wise addition of 2-cyclopropylacetyl
chloride (49 mmoles, 5.47 mL]). The reaction was allowed
to warm to ambient temperature overnight. The reaction
was diluted with CH2Cl2, washed with water, saturated
NaCl solution, dried over MgSO4, filtered, and concen-
trated. The residue was purified by flash chromatography
(gradient 0 to 30% THF/hexanes) to give the compound
as a yellow oil (6.48 g, 51%)which was used directly in the
next reaction. To a dry flask under nitrogen was added
iPr2NH (4.6 mL, 33 mmol). To the amine was slowly add-
ed 2.5 M n-BuLi in hexanes (12.5 mL, 31 mmol). The LDA
reagent was diluted with dry THF (20 mL). In a separate
flask under N2, imide 17 (6.5 g, 25mmol) was dissolved in
THF (160 mL) and cooled to -78 °C. The LDA solution was
slowly added via syringe such that the internal tempera-
ture did not rise above -70 °C. After the addition, the mix-
ture was stirred for 1 h. To the resulting anion was added
drop-wise 4-(trifluoromethyl)bromomethyl-benzene (6.6
g, 27.5 mmol) in THF (20 mL) such that the internal tem-
perature did not rise above -70 °C. The cooling bath was
allowed to expire overnight. The reaction was quenched
with saturated NH4Cl solution and extracted with EtOAc.
The extracts were combined, washed with saturated NaCl
solution, dried over MgSO4, filtered, and concentrated.
The residue was purified by chromatography (gradient 0
to 70% EtOAc/hexanes) to give compound 21 as a yellow
oil (4.8 g, 46%) which was used directly in the next reac-
tion. Compound 21 (4.82 g, 11.6 mmol) was dissolved in 1:2
THF/water (300 mL) and cooled to 0 °C. To the resulting
solution was added 30% aqueous H2O2 (15 mL, 150 mmol)
followed by 1M LiOH solution (20 mL, 20 mmol). The
resulting mixture was stirred at 0 °C for 2 h. The reaction
was quenched via drop-wise addition of Na2S2O3 (5.82 g,
46 mmol) in water (40 mL). The reaction was slowly
172.19, 157.37, 146.81, 129.43, 127.59, 127.28, 126.96, 126.65,
126.27, 125.60 (m), 123.57, 65.76, 43.76, 36.58, 31.19, 14.63,
0.78, -0.73.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on
the ACS Publications website: Bile Duct Cannulated Rat
Study, Assay Protocols, Human PK Modeling; PDB files
for Figures 2 and 3, Molecular formula strings.
AUTHOR INFORMATION
Corresponding Author
* Email: durhamti@lilly.com; Phone: 317-433-8447
Notes
The authors declare the following competing financial inter-
est(s): All the authors are employees and shareholders of Eli
Lilly and Company.
Author Contributions
TD wrote the manuscript. All authors reviewed and approved
the manuscript. Design of Inhibitors: TD, JM, MW. Synthe-
sis: JM, TJ, CL; Design of in vivo experiments: KT; MC, MW,
TD, LA; Biochemical and biomarker assays: CS, KT; MIA ex-
periments: CL; Human PK Projection: D\M; Modeling: TD
ABBREVIATIONS
ADAMTS = a disintegrin and metalloproteinase with throm-
bospondin motifs; MMP = matrix metalloprotease; OA =
osteoarthritis; MIA = mono-iodo acetate; MT = meniscal-
tear.
REFERENCES
(1)(a) Lohmander, L. S.; Neame, P. J.; Sandy, J. D. The structure
of aggrecan fragments in human synovial fluid. Evidence that
aggrecanase mediates cartilage degradation in inflammatory joint
disease, joint injury, and osteoarthritis. Arthritis Rheum. 1993, 36,
1214ꢀ1222; (b) Sandy, J. D.; Flannery, C. R.; Neame, P. J.;
6
ACS Paragon Plus Environment

Page 7 of 8
Journal of Medicinal Chemistry
Lohmander, L. S. The structure of aggrecan fragments in human
Swearingen, C.; Lin, C.; Chambers, M. G.; Thirunavukkarasu, K.;
1
2
3
4
5
6
7
8
synovial fluid. Evidence for the involvement in osteoarthritis of a
novel proteinase which cleaves the glu 373ꢀala 374 bond of the
interglobular domain. J. Clin. Invest. 1992, 89, 1512ꢀ1516.
(2)(a) Bakali, J. E.; GrasꢀMasse, H.; Maingot, L.; Deprez, B.;
Dumont, J.; Leroux, F.; DeprezꢀPoulain, R. Inhibition of
aggrecanases as a therapeutic strategy in osteoarthritis. Future
Med. Chem. 2014, 6, 1399ꢀ1412; (b) Dancevic, C. M.;
McCulloch, D. R. Current and emerging therapeutic strategies for
preventing inflammation and aggrecanase mediated cartilage
destruction in arthritis. Arthritis Res. Ther. 2014, 16, 429; (c)
Gilbert, A. M.; Bikker, J. A.; O'Neil, S. V. Advances in the
development of novel aggrecanase inhibitors. Expert Opin. Ther.
Pat. 2011, 21, 1ꢀ12.
(3)(a) Glasson, S. S.; Askew, R.; Sheppard, B.; Carito, B.;
Blanchet, T.; Ma, H.ꢀL.; Flannery, C. R.; Peluso, D.; Kanki, K.;
Yang, Z.; Majumdar, M. K.; Morris, E. A. Deletion of active
ADAMTS5 prevents cartilage degradation in a murine model of
osteoarthritis. Nature 2005, 434, 644ꢀ648; (b) Li, J.; Anemaet,
W.; Diaz, M. A.; Buchanan, S.; Tortorella, M.; Malfait, A. M.;
Mikecz, K.; Sandy, I. D.; Plaas, A. Knockout of ADAMTS5 does
not eliminate cartilage aggrecanase activity but abrogates joint
fibrosis and promotes cartilage aggrecan deposition in murine
osteoarthritis models. J. Orthop. Res. 2011, 29, 516ꢀ522; (c)
Little, C. B.; Meeker, C. T.; Golub, S. B.; Lawlor, K. E.; Farmer,
P. J.; Smith, S. M.; Fosang, A. J. Blocking aggrecanase cleavage
in the aggrecan interglobular domain abrogates cartilage erosion
and promotes cartilage repair. J. Clin. Invest. 2007, 117, 1627ꢀ
1636; (d) Glasson, S. S.; Askew, R.; Sheppard, B.; Carito, B. A.;
Blanchet, T.; Ma, H.ꢀL.; Flannery, C. R.; Kanki, K.; Wang, E.;
Peluso, D.; Yang, Z.; Majumdar, M. K.; Morris, E. A.
Characterization of and osteoarthritis susceptibility in ADAMTSꢀ
4ꢀknockout mice. Arthritis Rheum. 2004, 50, 2547ꢀ2558; (e)
Stanton, H.; Rogerson, F. M.; East, C. J.; Golub, S. B.; Lawlor, K.
E.; Meeker, C. T.; Little, C. B.; Last, K.; Farmer, P. J.; Campbell,
I. K.; Fourie, A. M.; Fosang, A. J. ADAMTS5 is the major
aggrecanase in mouse cartilage in vivo and in vitro. Nature 2005,
434, 648ꢀ652; (f) Song, R.ꢀH.; Tortorella, M. D.; Malfait, A.ꢀM.;
Alston, J. T.; Yang, Z.; Arner, E. C.; Griggs, D. W. Aggrecan
degradation in human articular cartilage explants is mediated by
both ADAMTSꢀ4 and ADAMTSꢀ5. Arthritis Rheum. 2007, 56,
575ꢀ585.
(4)(a) Malemud, C. J. Matrix metalloproteinases (mmps) in health
and disease: An overview. Front. Biosci. 2006, 11, 1696ꢀ1701; (b)
Peterson, J. T. The importance of estimating the therapeutic index
in the development of matrix metalloproteinase inhibitors.
Cardiovasc. Res. 2006, 69, 677ꢀ687; (c) Deng, H.; O’Keefe, H.;
Davie, C. P.; Lind, K. E.; Acharya, R. A.; Franklin, G. J.; Larkin,
J.; Matico, R.; Neeb, M.; Thompson, M. M.; Lohr, T.; Gross, J.
W.; Centrella, P. A.; O’Donovan, G. K.; Bedard, K. L.; van
Vloten, K.; Mataruse, S.; Skinner, S. R.; Belyanskaya, S. L.;
Carpenter, T. Y.; Shearer, T. W.; Clark, M. A.; Cuozzo, J. W.;
AricoꢀMuendel, C. C.; Morgan, B. A. Discovery of highly potent
and selective small molecule ADAMTSꢀ5 inhibitors that inhibit
human cartilage degradation via encoded library technology
(ELT). J. Med. Chem. 2012, 55, 7061ꢀ7079; (d) Ding, Y.;
O'Keefe, H.; DeLorey, J. L.; Israel, D. I.; Messer, J. A.; Chiu, C.
H.; Skinner, S. R.; Matico, R. E.; MurrayꢀThompson, M. F.; Li,
F.; Clark, M. A.; Cuozzo, J. W.; AricoꢀMuendel, C.; Morgan, B.
A. Discovery of potent and selective inhibitors for ADAMTSꢀ4
through DNAꢀencoded library technology (ELT). ACS Med.
Chem. Lett. 2015, 6, 888ꢀ893.
Wiley, M. R. Identification of potent and selective hydantoin
inhibitors of aggrecanaseꢀ1 and aggrecanaseꢀ2 that are efficacious
in both chemical and surgical models of osteoarthritis. J. Med.
Chem. 2014, 57, 10476ꢀ10485; (b) Wiley, M. R.; Durham, T. B.;
Adams, L. A.; Chambers, M. G.; Lin, C.; Liu, C.; Marimuthu, J.;
Mitchell, P. G.; Mudra, D. R.; Swearingen, C. A.; Toth, J. L.;
Weller, J. M.; Thirunavukkarasu, K. Use of osmotic pumps to
establish the pharmacokineticꢀpharmacodynamic relationship and
define desirable human performance characteristics for
aggrecanase inhibitors J. Med. Chem. 2016, 59, 5810ꢀ5822.
(6)(a) Wang, Q.; Liu, X.; Liu, X.; Li, B.; Nie, H.; Zhang, S.;
Chen, W. Highly enantioselective hydrogenation of 2ꢀsubstitutedꢀ
2ꢀalkenols catalysed by a chenphosꢀrh complex. Chem. Commun.
(Cambridge, U. K.) 2014, 50, 978ꢀ980; (b) Schomaker, J. M.;
Bhattacharjee, S.; Yan, J.; Borhan, B. Diastereomerically and
enantiomerically pure 2,3ꢀdisubstituted pyrrolidines from 2,3ꢀ
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
aziridinꢀ1ꢀols using
a
sulfoxonium ylide:
A
oneꢀcarbon
homologative relay ring expansion. J. Am. Chem. Soc. 2007, 129,
1996ꢀ2003; (c) Malkov, A. V.; Czemerys, L.; Malyshev, D. A.
Vanadiumꢀcatalyzed asymmetric epoxidation of allylic alcohols in
water. J. Org. Chem. 2009, 74, 3350ꢀ3355.
(7)Wang, Z.; Kuninobu, Y.; Kanai, M. Copperꢀmediated direct
C(SP3)ꢀH and C(SP2)ꢀH acetoxylation. Org. Lett. 2014, 16, 4790ꢀ
4793.
(8)Durham, T. B.; Marimuthu, J.; Wiley, M. R. Preparation of Nꢀ
(imidazolidinylmethyl)propanamides as aggrecanase inhibitors.
WO2014066151A1, 2014.
(9)Dhanaraj, V.; Williams, M. G.; Ye, Q.ꢀZ.; Molina, F.; Johnson,
L. L.; Ortwine, D. F.; Pavlovsky, A.; Rubin, J. R.; Skeean, R. W.;
White, A. D.; Humblet, C.; Hupe, D. J.; Blundell, T. L. Xꢀray
structure of gelatinase a catalytic domain complexed with a
hydroxamate inhibitor. Croat. Chem. Acta 1999, 72, 575ꢀ591.
(10)Lang, R.; Kocourek, A.; Braun, M.; Tschesche, H.; Huber, R.;
Bode, W.; Maskos, K. Substrate specificity determinants of
human macrophage elastase (MMPꢀ12) based on the 1.1 Å crystal
structure1. J. Mol. Biol. 2001, 312, 731ꢀ742.
(11)Swearingen, C. A.; Chambers, M. G.; Lin, C.; Marimuthu, J.;
Rito, C. J.; Carter, Q. L.; Dotzlaf, J.; Liu, C.; Chandrasekhar, S.;
Duffin, K. L.; Mitchell, P. G.; Durham, T. B.; Wiley, M. R.;
Thirunavukkarasu, K. A shortꢀterm pharmacodynamic model for
monitoring aggrecanase activity: Injection of monosodium
iodoacetate (MIA) in rats and assessment of aggrecan neoepitope
release in synovial fluid using novel elisas. Osteoarthr. Cartil.
2010, 18, 1159ꢀ1166.
(12)Swearingen, C. A.; Carpenter, J. W.; Siegel, R.; Brittain, I. J.;
Dotzlaf, J.; Durham, T. B.; Toth, J. L.; Laska, D. A.; Marimuthu,
J.; Liu, C.; Brown, D. P.; Carter, Q. L.; Wiley, M. R.; Duffin, K.
L.; Mitchell, P. G.; Thirunavukkarasu, K. Development of a novel
clinical biomarker assay to detect and quantify aggrecanaseꢀ
generated aggrecan fragments in human synovial fluid, serum and
urine. Osteoarthr. Cartil. 2010, 18, 1150ꢀ1158.
(13)Noe, M. C.; Natarajan, V.; Snow, S. L.; WolfꢀGouveia, L. A.;
Mitchell, P. G.; LoprestiꢀMorrow, L.; Reeves, L. M.; Yocum, S.
A.; Otterness, I.; Bliven, M. A.; Carty, T. J.; Barberia, J. T.;
Sweeney, F. J.; Liras, J. L.; Vaughn, M. Discovery of 3ꢀohꢀ3ꢀ
methylpipecolic hydroxamates: Potent orally active inhibitors of
aggrecanase and MMPꢀ13. Bioorg. Med. Chem. Lett. 2005, 15,
3385ꢀ3388.
(14)Rit, R. K.; Yadav, M. R.; Ghosh, K.; Shankar, M.; Sahoo, A.
K. Sulfoximine assisted Pd(II)ꢀcatalyzed bromination and
chlorination of primary βꢀC(sp3)ꢀH bond. Org. Lett. 2014, 16,
5258ꢀ5261.
(5)(a) Durham, T. B.; Klimkowski, V. J.; Rito, C. J.; Marimuthu,
J.; Toth, J. L.; Liu, C.; Durbin, J. D.; Stout, S. L.; Adams, L.;
7
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8
ACS Paragon Plus Environment