Novel, potent P2-P3 pyrrolidine derivatives of ketoamide-based cathepsin K inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.11.101

Starting from a potent pantolactone ketoamide cathepsin K inhibitor discovered from structural screening, conversion of the lactone scaffold to a pyrrolidine scaffold allowed exploration of the S³ subsite of cathepsin K. Manipulation of P³

Full text of DOI:10.1016/j.bmcl.2005.11.101

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1735–1739
Novel, potent P2–P3 pyrrolidine derivatives of
ketoamide-based cathepsin K inhibitors
David G. Barrett,a, John G. Catalano,a,* David N. Deaton,a Anne M. Hassell,b
Stacey T. Long,c Aaron B. Miller,b Larry R. Miller,d John A. Ray,a Vicente Samano,a
Lisa M. Shewchuk,b Kevin J. Wells-Knecht,e,à Derril H. Willard, Jr.f,§ and Lois L. Wrightg
aDepartment of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
bDiscovery Research Computational, Analytical, and Structural Sciences, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
cDepartment of World Wide Physical Properties, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
dDepartment of Molecular Pharmacology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
eDepartment of Research Bioanalysis and Drug Metabolism, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
fDepartment of Gene Expression and Protein Purification, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
gDiscovery Research Biology, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
Received 26 October 2005; revised 29 November 2005; accepted 30 November 2005
Available online 11 January 2006
Abstract—Starting from a potent pantolactone ketoamide cathepsin K inhibitor discovered from structural screening, conversion of
the lactone scaffold to a pyrrolidine scaffold allowed exploration of the S3 subsite of cathepsin K. Manipulation of P3 and P1 groups
afforded potent inhibitors with drug-like properties.
0
Ó 2005 Elsevier Ltd. All rights reserved.
Continual structural remodeling of skeletal tissue is nec-
essary to maintain strong healthy bone. Osteoclasts and
osteoblasts are specialized cells that affect remodeling
through an integrated process of bone resorption and
bone formation.1 Bone density and structural integrity
deteriorate when this remodeling begins to favor bone
resorption over bone formation, resulting in susceptibil-
ity to fracture. Osteoclasts utilize secreted acid and pro-
teolytic enzymes to degrade the mineral and matrix
components of bone, respectively. The main proteolytic
enzyme secreted is cathepsin K, a cysteine protease
which hydrolyzes type I collagen, the primary constitu-
ent of bone matrix.2 Small molecule inhibitors of
cathepsin K have proven efficacious in attenuating bone
resorption in humans,3 as well as in animal models of
osteoporosis.4
This group has previously reported potent a-ketoamide
inhibitors of cathepsin K.5–9 The P2 pantolactone moie-
ty of ketoamide 1 (IC50 = 3.0 nM) was discovered
through structural screening of the available chemical
directory (ACD) as part of an ongoing structure/activity
relationship analysis.10 Although quite potent, inhibitor
1 exhibited poor pharmacokinetic (PK) properties in
rats with a low terminal half-life and no oral bioavail-
ability. Poor PK parameters were presumably due, at
least in part, to the observed instability of the lactone
in plasma. A second liability of the pantolactone moiety
was the lack of suitable functionality for rapid diversifi-
cation. In order to overcome the PK liabilities of panto-
lactone 1, and also to facilitate SAR progression, a
plasma-stable P2 substituent readily amenable to deriva-
tization was sought. Reasoning that the geminal dimeth-
yl substituted pyrrolidine P2 moiety of 2 would be
equally effective in filling the hydrophobic S2 binding
pocket of the enzyme, without the instability observed
with the pantolactone, a synthetic route to the pyrroli-
dine functionality was developed. The nucleophilic
Keywords: Cathepsin K; Inhibitor; Osteoporosis; Ketoamide;
Synthesis.
*
Corresponding author. Tel.: +1 919 483 6264; e-mail: john.g.
catalano@gsk.com
Present address: Lilly Forschung GmbH, Essener Str. 93, 22419
Hamburg, Germany.
à
Present address: Schering-Plough Research Institute, 2015 Galloping
Hill, Kenilworth, NJ 07033, USA.
Deceased.
§
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.101

1736
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1735–1739
nitrogen of the pyrrolidine also offered a handle for
ready substitution.
carbamates, ureas, and sulfonamides, respectively. The
a-hydroxy amides were then oxidized with Dess–Martin
periodinane to afford a-ketoamides 2a–2y.
O
O
O
H
N
H
H
N
H
Inhibitor 2z was synthesized via mixed carbonate 12 as
shown in Scheme 2. Pyrrolidinol 6 was deprotected via
acidic hydrogenolysis to give pyrrolidine 9, which was
coupled with 2-chlorobenzothiazole 10 to give the
substituted pyrrolidinol 11. Treatment of 11 with 4-
nitrophenylchloroformate gave the mixed carbonate
12, which was then coupled with the amino alcohol 7b.
Dess–Martin oxidation of the a-hydroxyamide then
yielded the ketoamide 2z.
O
N
O
N
R2
R1
O
N
O
nBu
O
Ph
O
nBu
O
1
2
The synthesis of inhibitors 2a–2y is represented in
Scheme 1. (S)-(+)-Pantolactone 3 was reduced with
sodium borohydride and the resulting triol 4 treated
with methanesulfonyl chloride to afford the disulfonate
5. Treatment of 5 with benzylamine afforded the cyclized
pyrrolidinol 6. The chloroformate formed by treating 6
with phosgene was coupled with known amino alcohols
7a7 or 7b9 to give a carbamate, which after the removal
of the benzyl-protecting group afforded the amines
8a–8b. The resulting pyrrolidines 8a–8b were combined
with a variety of acid chlorides, chloroformates,
isocyanates, and sulfonyl chlorides to obtain amides,
Inhibitor 2aa was synthesized similarly to 2z via the
mixed carbonate 16 as shown in Scheme 3. First,
commercially available 5-(trifluoromethyl)-1,3,4-thia-
diazol-2-amine 13 was converted to the bromide 14 via
diazotization. Then, the bromide 14 was coupled with
the pyrrolidinol 9 to give the substituted pyrrolidinol
15. Finally, after the carbonate 16 was formed by treat-
ing pyrrolidinol 15 with 4-nitrophenylchloroformate, it
O
O
a
b
OH
OMs
3
OH
OH
OH
OH
OMs
OH
OH R2
NH
H2N
nBu
N
c
d, e
+
Ph
O
7a R2 = (R)-α-methylbenzyl
7b R2 = 3-pyrazole
6
OH R2
NH
O
R2
R1
H
H
X
O
N
O
N
NH
Y
f, g
HN
N
O
nBu
O
O
nBu
O
8a-b
2a-y
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, 0 °C to rt, 99%; (b) MsCl, pyridine, 0 °C to rt, 52%; (c) benzylamine, EtOH, 120 °C, sealed
tube, 90%; (d) 6, 1.93 M phosgene in toluene, pyridine, CH2Cl2, 0 °C to rt; then 7a or 7b, Et3N, THF, 41%; (e) H2, 10% Pd/C, EtOH, 75%;
(f) R1COCl or R1OCOCl or R1N@C@O or R1SO2Cl; (g) Dess–Martin periodinane, CH2Cl2, 27–96% (2 steps).
Ph
OH
OH
N
S
N
HN
+
Cl
0
a
b
6
O
OH
O
N
S
N
N
N
O
O
NO2
c
S
11
12
H
N
H
H
N
O
N
N
d, e
N
N
S
O
nBu
O
2z
Scheme 2. Reagents: (a) H2, 10% Pd/C, 1 M HCl (aq), EtOH, 99%; (b) 10, NaHCO3, i-PrOH, H2O, 96%; (c) 4-nitrophenylchloroformate, pyridine,
CH2Cl2, 73%; (d) 7b, iPr2NEt, DMF, 77%; (e) Dess–Martin periodinane, CH2Cl2, 67%.

D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1735–1739
1737
a few were less than 100-fold selective versus these
cathepsins. However, these inhibitors were much less
selective against the closely related endopeptidase
cathepsins S and V.
NH2
S
Br
N
N
N
N
OH
N
S
S
a
b
N
N
F3C
CF3
CF3
13
14
15
Inhibitors 2b–2y were significantly less potent against
rat cathepsin K than against the human isoform.
Although rat cathepsin K functions similarly to human
cathepsin K in respect to bone resorption activity, there
are significant structural differences in the active sites of
rat and human cathepsin K.2 Indeed, the lack of potency
of such inhibitors versus rat cathepsin K has hampered
efforts to progress inhibitors via rodent-based assays
such as the ex vivo rat calvarial resorption assay11,12
and the thyroid-parathyroidectomized hypocalcemic
(TPTX) rat assay.13
O
O
O
N
S
N
c
d, e
N
O
NO2
F3C
N
16
H
H
N
H
N
N
N
O
N
N
F3C
S
O
nBu
O
2aa
Scheme 3. (a) HBr, Br2, NaNO2, NaOH (aq), 54%; (b) 9, iPr2NEt,
isopropanol, reflux, 88%; (c) 4-nitrophenylchloroformate, pyridine,
CH2Cl2; (d) 7b, iPr2NEt, DMF, 21% (2 steps); (e) Dess–Martin
periodinane, CH2Cl2, 84%.
An X-ray co-crystal structure of cathepsin K with inhib-
itor 2x (Table 2) is shown in Figure 1. As with previous-
ly solved X-ray co-crystal structures of cathepsin K and
ketoamide inhibitors,5,8,9 a covalent hemithioketal inter-
mediate is formed between the 25Cys of the enzyme and
the a-keto moiety of the inhibitor. The oxyanion hole is
occupied by the carbonyl of the amide rather than the
hemithioketal hydroxyl group. As predicted, the gemi-
nal dimethyl substitution of the pyrrolidine inhibitors
effectively fills the S2 pocket of the enzyme. Unlike our
previously reported ketoamide inhibitors, however, this
inhibitor does not extend toward the S3 subsite of the
enzyme. Rather, the P3 substitution attached to the pyr-
rolidine nitrogen extends into solvent, possibly explain-
ing the similar potency of linker extended P3 analogs in
was coupled to amine 7b and oxidized to provide the
ketoamide 2aa.
An initial set of analogs 2a–2l was synthesized to inves-
tigate both the linker and spatial requirements for the
P2–P3 substituent. A phenyl group was extended 2, 3,
4, or 5 bond lengths from the pyrrolidine core and at-
tached via an amide, carbamate, urea, or sulfonamide
linker. As shown in Table 1, the urea analogs 2f, 2i,
and 2l (IC50s = 3.8–6.6 nM) were amongst the more po-
tent inhibitors of the initial set and their potencies did
not show a significant dependence on the chain length
between the pyrrolidine nitrogen and the phenyl substi-
tuent. The potencies of the carbamate derivatives 2e, 2h,
and 2k (IC50s = 24–32 nM) were also not appreciably
affected by chain length, but these analogs were ꢀ5-fold
less potent than the corresponding urea derivatives. In
contrast, the activity of analogous amide derivatives 2d
(IC50 = 3.0 nM) and 2j (IC50 = 27 nM) was much more
dependent on the chain length. Sulfonamide 2g
(IC50 = 17 nM) was roughly 5-fold less potent than its
analogous amide 2d. Although phenylamide 2b
(IC50 = 65 nM) was the least potent of the series, larger
aryl and heteroaryl amides 2o–2r (IC50s = 1.6–6.9 nM)
were quite potent. These larger aryl amides may act as
conformationally restricted analogs of 2d and were in-
deed equipotent. Similarly, carbamate 2s (IC50 = 58 nM)
is a conformationally restricted analog that shows simi-
lar potency to the unrestricted analog 2h. Carbamate
analogs 2t (IC50 = 5.0 nM) and 2u (IC50 = 2.1 nM) in-
clude a 4-biphenyl substituent that further extends the
phenyl group toward P3 in a conformationally restricted
manner. These carbamate inhibitors showed significant-
ly improved potencies compared to those of other carba-
mate derivatives. Biphenyl amide 2r (IC50 = 6.9 nM)
also exhibited good potency, but was larger and no more
potent than 2d.
the urea series. A hydrogen bond between 161Asn and
0
the P1 amide nitrogen is accomplished via a bridging
water molecule (red sphere).
Compounds 2c, 2d, and 2i were potent examples of the
sulfonamide, amide, and urea classes, respectively, and
were therefore chosen for progression into PK studies
(Table 2). Amide derivative 2d exhibited very good per-
meability (PAPP = 410 nm/s) as measured in a Madin–
Darby canine kidney (MDCK) permeability model as
well as moderate solubility (0.068 mg/mL) as measured
in fasted state-simulated intestinal fluid (FS-SIF). The
oral bioavailability of 2d (F = 29%) was similar that of
urea analog 2i (F = 21%), which had lower permeability
(PAPP = 210 nm/s) and slightly lower solubility (FS-
SIF = 0.053 mg/mL). Sulfonamide 2c had a longer half-
life and comparable oral bioavailability (F = 25%) to
2d and 2i, although permeability and solubility were
not measured. All three inhibitors exhibited good vol-
umes of distribution, moderate clearances, and relatively
short half-lives in male Han Wistar rats. Inhibitors 2v,
2w, and 2x were slightly less potent than 2d, but incorpo-
rated heterocyclic ring substitution in an attempt to
improve solubility and oral bioavailability. Despite sim-
ilar permeability and solubility properties, the isoxazole
analog 2v was less orally bioavailable (F = 8.6%) than
2d and had a shorter half-life with a lower volume of dis-
tribution. The basic pyridyl derivative 2w exhibited an
apparently longer half-life than 2d and was slightly more
soluble, which may contribute to the greater oral
bioavailability (F = 45%) observed for this inhibitor.
Inhibitors of this series generally exhibited excellent
selectivity against the exopeptidases cathepsin B and
cathepsin H, as well as against the endopeptidase
cathepsin L, as shown in Table 1. In fact, most of the
inhibitors were greater than 1000-fold selective and only

1738
D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1735–1739
Table 1. Inhibition of human cathepsin K and selectivity ratios versus other cathepsins
O
H
H
O
N
N
R2
Y
N
R1
O
nBu
O
X
Compound R1
X
Y
R2
Cat K IC50 (nM) rK/hK B/Kb
H/Kc
>500
L/Kd S/Ke V/Kf
410 13 79
a
1
3.0
21
65
8.3
3.0
29
4.3
17
24
3.8
—
—
>500
2a
2b
2c
2d
2e
2f
Ph
Ph
—
—
—
CH2 (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
SO2 (R)-CH(Me)Ph
—
—
—
—
—
—
370 43
74 16
76 17
—
470
3800
110
79
Ph
Ph
110
160
7100 >13,000
4300 >13,000
—
CH2 CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
140
—
Ph
Ph
O
—
—
—
NH CO (R)-CH(Me)Ph
CH2 SO2 (R)-CH(Me)Ph
890
490
2900 >13,000 1400 45
5200 >13,000 910 12
120
79
2g
2h
2i
Ph
PhCH2
PhCH2
Ph(CH2)2
Ph(CH2)2
Ph(CH2)2
1-Morpholine
O
CO (R)-CH(Me)Ph
—
>10,000 >10,000 1300 83
4600 >13,000 1200 46
170
130
—
NH CO (R)-CH(Me)Ph
CH2 CO (R)-CH(Me)Ph
870
2j
27
32
—
—
—
—
—
—
—
—
—
—
2k
2l
O
CO (R)-CH(Me)Ph
NH CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
—
6.6
16
2.1
930
5200 >13,000
760 26
81 2.8
>120 >120 11
>7900 890 62
>4500 1000 16
85
15
2m
2n
2o
2p
2q
2r
—
76
>120
510
430
290
54
210
>120
760
>800
4-Trifluoromethylphenyl NH CO (R)-CH(Me)Ph
43
56
2-Naphthalene
2-Benzothiophene
5-Indole
—
—
—
—
O
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
CO (R)-CH(Me)Ph
1.6
2.8
1.7
6.9
540
450
56
81
>7200
>1800
490 21
320 5.8
4-Biphenyl
2-Naphthalene
4-Biphenyl
250
8.7
2s
58
—
—
—
— —
200 13
—
2t
O
5.0
2.1
8.9
240
430
79
650
100
>2500
>6000
>1400
>890
42
2u
2v
2w
2x
2y
2z
2aa
4-Biphenyl-CH2
5-Isoxazolyl
3-Pyridyl
O
960
130
52
9.1
81
11
11
—
—
360
360
3.2
3.7
2.6
6.7
6.6
14
15
0.42
55
42
3,5-Dimethyl-isoxazolyl NH CO (R)-CH(Me)Ph
4-Trifluoromethylphenyl NH CO 3-Pyrazole
270
>810
39
9.1
240
—
—
>600
>310
>250
>600 >600
>310 >310
36
32
36
2-Benzthiazolyl
5-CF3-1,3,4-thiadiazolyl
—
—
CO 3-Pyrazole
CO 3-Pyrazole
0.81
1.0
>250 >250 17
a Inhibition of recombinant human cathepsin K activity in a fluorescence assay using 400 pM cathepsin K with 10 lM Cbz-Phe-Arg-AMC as
substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5. The IC50 values are means of two or three inhibition assays, individual data
points in each experiment were within a 3-fold range of each other.
b B/K = IC50 against cathepsin B/IC50 against cathepsin K. Inhibition of recombinant human cathepsin B activity in a fluorescence assay using
250 pM cathepsin B with 10 lM Cbz-Phe-Arg-AMC as substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
c H/K = IC50 against cathepsin H/IC50 against cathepsin K. Inhibition of recombinant human cathepsin H activity in a fluorescence assay using
107 nM cathepsin H with 50 lM L-Arg-b-naphthalamide as substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
d L/K = IC50 against cathepsin L/IC50 against cathepsin K. Inhibition of recombinant human cathepsin L activity in a fluorescence assay using
300 pM cathepsin L with 5 lM Cbz-Phe-Arg-AMC as substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
e S/K = IC50 against cathepsin S/IC50 against cathepsin K. Inhibition of recombinant human cathepsin S activity in a fluorescence assay using 400 pM
cathepsin S with 10 lM Cbz-Val-Val-Arg-AMC as substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
f V/K = IC50 against cathepsin V/IC50 against cathepsin K. Inhibition of recombinant human cathepsin V activity in a fluorescence assay using
150 pM cathepsin V with 2 lM Cbz-Phe-Arg-AMC as substrate in 100 mM NaOAc, 10 mM DTT, and 120 mM NaCl, pH 5.5.
Table 2. Pharmacokinetics of cathepsin K inhibitors
Sol. FS-SIFa (mg/mL)
Clc (mL/min/kg)
Fe (%)
b
t1/2 (min)
d
VSS (mL/kg)
Compound
MDCK PAPP (nm/s)
1
—
—
0.240
—
11
150
63
460
27
26
32
18
34
83
77
65
79
3400
2100
1500
1400
500
0.0
25
2c
2d
2i
410
210
480
210
12
0.068
0.053
0.068
0.085
0.320
0.14
29
21
61
36
2v
2w
2x
2y
2z
2aa
8.6
45
94
35
550
1400
2600
2200
3300
8.8
26
18
75
250
120
0.160
—
180
81
14
13
a FS-SIF is the equilibrium solubility in fasted state-simulated intestinal fluid at pH = 6.8. The values are means of two measurements.
b t1/2 is the iv terminal half-life dosed as a solution in male Han Wistar rats. All in vivo pharmacokinetic values are means of two experiments.
c Cl is the total clearance.
d VSS is the steady-state volume of distribution.
e F is the oral bioavailability.

D. G. Barrett et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1735–1739
1739
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Figure 1. Active site of the X-ray co-crystal structure of compound 2x
complexed with cathepsin K. The cathepsin K carbons are colored
magenta with inhibitor 2x carbons colored green. The semi-transpar-
ent white surface represents the molecular surface, while hydrogen
bonds are depicted as yellow dashed lines. The red sphere is a water
molecule. The coordinates have been deposited in the Brookhaven
Protein Data Bank, Accession No. 2BDL. This figure was generated
using PYMOL version 0.97 (Delano Scientific, www.pymol.org).
Isoxazole analog 2x was very soluble (FS-SIF =
0.320 mg/mL), but suffered from low permeability
(PAPP = 12 nm/s), which may contribute to the low oral
bioavailability (F = 8.8%) observed for this compound.
Compounds 2y, 2z, and 2aa incorporated a pyrazole at
0
P1 , which has been shown to impart improved potency,
solubility, and oral bioavailability in previously studied
series8 These compounds were indeed the most potent
of the current series and were quite soluble. Unfortunate-
ly, an improvement in oral bioavailability was not real-
ized. In general, this entire series exhibited moderate to
high clearances, relatively short half-lives, and moderate
to good oral bioavailabilities.
In conclusion, this report summarizes the progression of
P2–P3 pyrrolidineketoamide-basedcathepsinKinhibitors
that were derived from a P2 pantolactone lead structure.
Potent inhibitors of cathepsin K with moderate PK were
obtained. A co-crystal structure of an inhibitor bound to
cathepsin K suggests that substitution of the pyrrolidine
nitrogen is not optimal to interact with the S3 subsite.