Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K

Article abstract of DOI:10.1016/j.bmcl.2005.07.071

The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds are 10- to 20-f

Full text of DOI:10.1016/j.bmcl.2005.07.071

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4741–4744
Trifluoroethylamines as amide isosteres in inhibitors of cathepsin K
W. Cameron Black,a,* Christopher I. Bayly,a Dana E. Davis,c Sylvie Desmarais,a
a
Jean-Pierre Falgueyret, Serge Leger, Chun Sing Li, Frederic Masse,
a
a
a
´
´ ´
´
Daniel J. McKay,a James T. Palmer,c M. David Percival,a Joe¨l Robichaud,a
Nancy Tsoub and Robert Zambonia
aMerck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Que., Canada H9R 4P8
bDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
cCelera Genomics, Inc, 180 Kimball Way, South San Francisco, CA 94080, USA
Received 13 June 2005; revised 15 July 2005; accepted 25 July 2005
Available online 9 September 2005
Abstract—The P2-P3 amide of dipeptide cathepsin K inhibitors can be replaced by the metabolically stable trifluoroethylamine
group. The non-basic nature of the nitrogen allows the important hydrogen bond to Gly66 to be made. The resulting compounds
are 10- to 20-fold more potent than the corresponding amide derivatives. Compound 8 is a 5 pM inhibitor of human cathepsin K
with >10,000-fold selectivity over other cathepsins.
Ó 2005 Elsevier Ltd. All rights reserved.
Cathepsin K (Cat K) is a lysosomal cysteine protease
thought to be the principal enzyme responsible for deg-
radation of type I collagen in osteoclastic bone resorp-
tion. Thus, an inhibitor of Cat K may be effective as
an anti-resorptive for treatment of osteoporosis. A
number of Cat K inhibitors have been previously
reported, many of which contain multiple amide bonds.
replace the carbonyl of an amide and generate a meta-
bolically stable, non-basic amine that maintains the
excellent hydrogen bond of an amide.
Replacement of this functional group has been previous-
ly explored by Zanda and coworkers2 who have used it
to replace both a glycine amide bond and a malonamide
of a partially modified retropeptide.3 These authors suc-
cinctly note that the trifluoroethylamino group features
ꢀlow NH basicity, a CH(CF3)ANHACH backbone
angle close to 120°, [and] a CACF3 bond substantially
isopolar with the C@Oꢁ. A report on the use of a
CH(CN)ANH group to replace an amide bond in
CCK-B has also been presented.4
Replacement of amide bonds with appropriate isosteres
is a classic approach in medicinal chemistry. Many
amide replacements are known which retain the geome-
try of the amide bond or maintain the hydrogen bond-
accepting properties of the amide.1 However, there are
a few functional groups, which are capable of preserving
the hydrogen bond-donating properties of the amide.
Sulfonamides, anilines, secondary alcohols, hydrazines,
and certain heterocycles constitute this list. The problem
of identifying an NH amide replacement can be recast as
how to minimize the basicity of an NH donor so that a
Much of the published literature on Cat K inhibitors de-
scribe peptide-based molecules. Dipeptide nitrile inhibi-
tors have been reported with good pharmacokinetics in
preclinical species and anti-resorptive activity in animal
models of osteoporosis.5 Nonetheless, we felt that the
overall properties of these inhibitors could be improved
by decreasing their peptidic nature.
+
NH2 moiety is not formed in the biological milieu.
Such charged groups are poorly tolerated deep in the ac-
tive site of a protein where binding interactions cannot
compensate for the energetic cost of desolvation. In this
paper, we describe that a trifluoromethyl group can
X-ray crystallography of the irreversible dipeptide inhib-
itor 1 bound to Cat K shows that the P1-P2 amide forms
hydrogen bonds to the protein with both its NH and
carbonyl components (Fig. 1). In contrast, the P2-P3
amide bond forms only one hydrogen bond to Gly66
*
Corresponding author. Tel.: +1 514 428 3073; fax: +1 514 428
4939; e-mail: blackc@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.071

4742
W. C. Black et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4741–4744
Br
O
a
S
H
HN
H
N
SO2Ph
OH
N
N
H2N
MgBr
OH
N
CN
O
N
H
F3C
b
HN
O
Ph
H
4
O
1
CF3
HN
CF3
3
H
N
CN
N
c, d
N
N
N
H
H
H
O
N
CN
Br
Br
6
5
N
H
O
2
e, f, g
CF3
H
Figure 1. Structures of peptidomimetic cathepsin K inhibitors.
N
CN
N
H
O
with the NH, while the carbonyl projects into solution.6
This implies that the P2-P3 amide bond might be re-
placed with an isostere without dramatic loss of potency.
Our first attempt at such a replacement was to introduce
a phenyl ring in place of the amide.7 The resulting com-
pound 2 had modest potency but excellent pharmacoki-
netics, justifying further pursuit of amide replacements.
The potency of these compounds could be improved by
using an aminothiophene group as in compound 3 to
take advantage of the hydrogen bond acceptor at
Gly66.8 Other methods of introducing the H-bond (alco-
hols, sulfonamides) were attempted without success.
+ R,S diastereomer
8
9
N
HN
Scheme 1. Reagents and conditions: (a) trifluoroacetaldehyde ethyl
hemiacetal, benzene, Dean-Stark, 18 h; (b) THF, 0 °C, 1 h; (c) H5IO6
(3 equiv), CrO3, CH3CN, 2 h; (d) PyBOP, aminoacetonitrile Æ HCl,
Et3N, DMF, 14 h; (e) BocPipPhB(OH)2, PdCl2dppf, Na2CO3, DMF,
90 °C, 6 h; (f) MsOH, THF, 14 h; (g) HPLC separation.
Table 1. Potency of inhibitors in purified enzyme assays
Compound
IC50 (nM)a
Cat Bc Cat Lc
3950
Cat Kb
Cat Sc
2010
In the context of developing the piperazine SAR of the
aminothiophene derivatives, we found that an N-triflu-
oroethyl group resulted in a dramatic decrease in the
basicity of the piperazine. It became quickly apparent
that this could be a method for replacing an amide car-
bonyl, while maintaining a neutral nitrogen H-bond.
2
3
11
3725
352
1.0
1.3
123
3353
102
121
6
34
84
7
7.3
60.005d
4.6
0.015d
0.6
5.0
2477
1111
145
451
8
47
68
9
>10,000
344
1050
902
120
10
12
13
14
26
The prototype molecule 6 was prepared by condensation
of trifluoroacetaldehyde with leucinol to form oxazoline
4 as a 3:1 mixture of diastereomers.9 This oxazoline was
added to three equiv of 4-bromophenyl magnesium bro-
mide to give trifluoroethylamino alcohol 5 as a 1:1 mix-
ture of diastereomers. More recently, an efficient
diastereoselective method for the synthesis of this type
of molecule has appeared.10 Alcohol 5 was oxidized to
the carboxylic acid using periodic acid catalyzed by
chromic acid.11 Coupling of the aminoacetonitrile was
carried out using conventional methods to give 6
(Scheme 1). Trifluoroethylamine 6 behaved as a neutral
molecule on TLC (Rf = 0.40, 40% EtOAc/1% HOAc/
hexanes) and could not be extracted from ethyl acetate
with dilute acid. Titration against Cat K showed that
6 was 5-fold more potent than the corresponding dipep-
tide derivative 7, even as a 1:1 mixture of diastereomers
(Table 1).
3903
>10,000
2274
>10,000
>10,000
>10,000
>10,000
614
0.4
a IC50s are an average of at least two independent titrations. See Ref 15
for assay conditions.
b Humanized rabbit enzyme.
c Human enzyme.
d Low enzyme conditions—see Ref 12.
A direct comparison of 8 to the corresponding dipeptide
10 (not shown) has demonstrated that both potency and
selectivity were increased by replacing the amide bond
by a trifluoroethylamine moiety (Table 1).
To determine the stereochemistry of the active diastereo-
mer, we turned to X-ray crystallography. The diastereo-
mer of intermediate 5, which led to the active
compounds, was derivatized as an imidazole carboxylic
ester 11. A single crystal X-ray structure revealed that
the CF3 center was in the S configuration (Fig. 2).13
Previous work in other series had shown that a phenyl-
piperazine provides optimal binding in S3.7 This P3
fragment was added to 6 using a Suzuki coupling, as
previously described.8 The resulting compound was sep-
arated by HPLC into its pure diastereomers 8 and 9.
Compound 8 has a Cat K IC50 of 65 pM, the most po-
tent compound ever obtained in this program. Only an
upper bound for this IC50 could be determined due to
limitations of the assay.12 Diastereomer 9 was 1000-fold
less potent in this assay with poor selectivity over Cat L.
In the dipeptide nitrile series of inhibitors, the optimal
P2 substituent had been found to be a 1,1-cyclohexyl
group, as exemplified by 12 (L-006235/CRA-013783).5
When this P2 group was used to replace leucine in the
trifluoroethylamine compounds, the resulting com-
pound 13 (Fig. 3) was found to be 1000-fold less active

W. C. Black et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4741–4744
Table 2. Comparison of CF3 replacements
4743
F3
C13
F2
Br1
C14
R
H
C12
C8
F1
N
CN
N
H
O
C11
C9
C7
Br
C10
Compound
IC50 (nM)
Cat Ka
N3
c
Rfb
pKa
O1
R
C6
C1
6
15
16
17
18
19
CF3
H
0.9
802
988
2.4
0.40
0.05
0.03
0.42
0.38
0.67
1.3
6.7
6.7
1.8
4.6
0.7
N2
C5
C2
C4
C18
O2
CH3
CF2CF3
C3
C15
C16
N1
4-MeSO2Ph
CN
2.5
30
a Humanized rabbit enzyme.
C17
b TLC on SiO2 plates with 40% EtOAc/1% HOAc/hexanes.
c Calculated using ACD/pKa.
CF3
N
N
O
N
H
captured by the pKa of the amine, as calculated by
ACD/pKa.14 Most interestingly, a nitrile substituent
(19), which also gave a non-basic compound, showed a
30-fold loss in activity, implying that the substituent effect
was more complex than simply neutralizing the amine.
O
Br
11
Figure 2. ORTEP representation of 11. Non-hydrogen atoms are
represented by ellipsoids corresponding to 30% probability. Hydrogen
atoms have been drawn at an arbitrary size.
Compound 6 has been modeled into a Cat K crystal
structure (Fig. 4). This model shows that the CF3 group
does not form any lipophilic interactions with the en-
zyme, but rather is directed away from the active site
into water. The hydrogen bond closely mimics the
hydrogen bond of the amide, but due to the sp3 hybrid-
ization of the nitrogen, it is oriented better toward the
Gly66 carbonyl, potentially forming a stronger hydro-
gen bond. The aromatic substituent is situated over
the glycine shelf in essentially the identical orientation
as the aromatic group of the dipeptide inhibitors.
P3
P2
P1
O
H
N
N
N
H
O
N
S
N
N
12 (L-006235/CRA-013783)
1 R2
R
H
N
N
N
H
O
13 R1 = CF3, R2 = H
14 R1 = R2 = O
The sum of the above data allows us to propose a ration-
ale for the increased potency of the trifluoroethylamine,
N
HN
Figure 3. Optimized dipeptide inhibitor L-006235/CRA-013783 and
P2-cyclohexyl derivatives 13 and 14.
than the corresponding amide derivative 14. Molecular
modeling analysis indicated that a steric clash between
the CF3 group and the cyclohexyl ring prevents the
molecule from adopting the conformation required for
binding. This suggests that only a single substituent
will be tolerated at P2 when a CF3 group replaces
the amide carbonyl.
The role of the CF3 group was explored in a brief SAR
study (Table 2). No substitution (15) or a simple methyl
substituent at this position (16) resulted in a dramatic
loss of activity. Pentafluoroethyl (17) gave similar poten-
cy to CF3. Aromatic substituents were generally quite
potent, as illustrated by the phenyl sulfone substituent
(18). With the exception of the hydrogen and methyl
cases, these amine derivatives tended to have neutral
properties, as determined by their non-polar behavior
on TLC analysis. This behavior was qualitatively
Figure 4. Compound 6 modeled into the active site of human Cat K
(pdb entry 1MEM) showing the interaction of the P2-P3 trifluoroeth-
ylamine moiety.

4744
W. C. Black et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4741–4744
relative to the amide. The sp2 atoms of the amide impose
a suboptimal conformational restraint on the aromatic
ring and the hydrogen bond. In contrast, the CF3 group
attached to an sp3 carbon orients itself perpendicular to
the aromatic ring and stabilizes the aromatic ring in its
bioactive conformation. The sp3 hybridized nitrogen al-
lows the simultaneous formation of an optimal hydro-
gen bond. In the case of compound 19, the nitrile
substituent is considerably smaller than the CF3 group
resulting in a low barrier to rotation for the aromatic
ring. As long as a substituent in this position is large en-
ough to enforce the conformational preference of the
aromatic ring, and can prevent protonation of the amine
group at physiological pH, a potent compound will be
obtained.
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This replacement of an amide with a trifluoroethylamine
leads to advantages other than potency and selectivity.
In the dipeptide nitriles, while the P2-P3 amide bond
was found to be metabolically stable, the P1-P2 amide
bond can be hydrolyzed to give the carboxylic acid. This
was especially apparent in dipeptide inhibitors contain-
ing leucine in P2. Replacing the leucine with cyclohexyl
effectively blocked this route of metabolism. When the
P2-P3 amide is replaced by the phenyl ring of compound
2, this P1-P2 amide hydrolysis is not observed. Similarly,
in the case of trifluoroethylamine derivatives described,
we found no evidence of a P1-P2 amide cleavage, even
though these compounds contain leucine in P2.
11. Zhao, M.; Li, J.; Song, Z.; Desmond, D.; Tschaen, D. M.;
Grabowski, E. J. J.; Reider, P. J. Tetrahedron Lett. 1998,
39, 5323.
In conclusion, we have found that trifluoroethylamine is
an excellent surrogate for the P2 amide bond in the
inhibitors of Cat K. Not only does this functional group
enhance potency and selectivity over other cathepsins,
but the resulting compounds are stable to P1-P2 amide
bond cleavage that is observed in analogous dipeptide
inhibitors. The fully elaborated inhibitor 8 is a ꢀ5 pM
inhibitor of Cat K and is >10,000-fold selective over
other cathepsins.
12. Inhibitory potencies were determined in a version of the
published Cat K assay in which the enzyme concentration
was reduced to 10 pM—the lowest enzyme concentration
at which activity could be reliably measured. At this
concentration, IC50s below 5 pM cannot be determined.
13. Crystallographic data (excluding structure factors) for the
structures in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplemen-
tary publication number CCDC 274083 for 11. Copies of
the data can be obtained, free of charge, on application to
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
[e-mail: deposit@ccdc.cam.ac.uk].
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