4-(3-Trifluoromethylphenyl)-pyrimidine-2-carbonitrile as cathepsin S inhibitors: N3, not N1 is critically important

Article abstract of DOI:10.1016/j.bmcl.2010.06.043

Using computer aided modelling studies, a new extended P2/S2 interaction was identified. This extended region can accommodate a variety of functional groups, such as aryls and basic amines. It was discovered that the N3 nitrogen of the pyrimidine-2-carbonitrile is critical for its cathepsin cysteine protease inhibition. N1 nitrogen also contributes to the inhibitory activity, but to a very limited degree. An 'in situ double activation' mechanism was proposed to explain these results.

Full text of DOI:10.1016/j.bmcl.2010.06.043

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4507–4510
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-(3-Trifluoromethylphenyl)-pyrimidine-2-carbonitrile as cathepsin S
inhibitors: N3, not N1 is critically important
a
b
a
a
Jiaqiang Cai a, , Xavier Fradera , Mario van Zeeland , Maureen Dempster , Kenneth S. Cameron ,
D. Jonathan Bennett a, John Robinson a, Lucy Popplestone a, Mark Baugh a, Paul Westwood a, John Bruin a,
William Hamilton a, Emma Kinghorn a, Clive Long a, Joost C. M. Uitdehaag b
*
a Merck Research Laboratories, MSD, Newhouse, Lanarkshire ML1 5SH, United Kingdom
b Merck Research Laboratories, MSD, 5340BH Oss, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Using computer aided modelling studies, a new extended P2/S2 interaction was identified. This extended
region can accommodate a variety of functional groups, such as aryls and basic amines. It was discovered
that the N3 nitrogen of the pyrimidine-2-carbonitrile is critical for its cathepsin cysteine protease inhi-
bition. N1 nitrogen also contributes to the inhibitory activity, but to a very limited degree. An ‘in situ dou-
ble activation’ mechanism was proposed to explain these results.
Received 12 May 2010
Revised 4 June 2010
Accepted 5 June 2010
Available online 10 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cathepsin S
Cysteine protease
Heteroarylnitrile
In situ activation
Lysosomotropism
Eleven members of the cysteine cathepsin family proteases
have been identified in the human genome (cathepsins B, C, H, F,
K, L, O, S, V, W and X).1 Two of these, cathepsins K and S have been
the subject of extensive effort in the pharmaceutical industry.1–3
Cathepsin S is highly expressed in antigen presenting cells and
plays a major role in the degradation of the invariant peptide chain
associated with the major histocompatibility complex and affects
antigen presentation. Selective cathepsin S inhibitors should then
be useful therapeutics for autoimmune disorders, for example,
rheumatoid arthritis (RA) and multiple sclerosis (MS). More re-
cently cathepsin S has also been indicated for neuropathic pain.4
Several cathepsin K inhibitors have progressed into human clinical
trials for osteoporosis,5 but as yet there are no reports of any
cathepsin S inhibitors in advanced stages of human clinical trials.
Several different cathepsin S inhibitor chemotypes have been re-
ported in the literature, most of which contain some kind of pep-
tide feature within the molecule and pose some challenges in
terms of pharmacokinetic property optimisation.2
becomes too reactive and could potentially cause covalent binding
to proteins in the microsomal protein binding assay.18
In our previous publication,19 we reported that 4-EtO group on
the P2 phenyl ring significantly improves cathepsin S inhibitory
activity probably through hydrophobic interactions with S2 subsite
residues Phe70, Phe211 and Val162 (Fig. 1). In view that the side
chain of Phe211 at the bottom of the S2 pocket is quite mobile
and preferentially found in an ‘open’ conformation which increases
the space available at the end of the S2 pocket by opening a narrow
passage between Phe70 and Phe211, it was suggested that the P2
region of the molecule can be further expanded to take advantage
of the Phe70 and Phe211 which could potentially have both hydro-
phobic or
p–p interactions with either lipophilic groups, p-elec-
tron-containing groups or basic nitrogen containing groups. Thus
basic nitrogen containing compounds are especially interesting
as they could potentially improve compound properties, for
We6,7 and others8–17 reported non-peptide heteroaryl nitriles as
cathepsins K and S inhibitors. One common feature of all these
arylnitrile based cysteine cathepsin inhibitors is that the nitrile
war-head is flanked between two electronically negative aromatic
nitrogen atoms. This often over-activates the nitrile group which
CN
CN
N
N
N
N
F3C
O
F3C
1
2
hCat S IC50: 6nM
hCat K IC50: 40nM
hCat S IC50 : 41nM
hCat K IC50 : 33nM
* Corresponding author. Tel.: +44 1698736133; fax: +44 1698736187.
E-mail address: jiaqiang.cai@merck.com (J. Cai).
Figure 1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.043

4508
J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4507–4510
Table 1
Cathepsin
compounds
example, solubility, cellular activity and absorption. Optimisation
of the activity will also allow the opportunity to replace the ‘reac-
tive’ pyrimidine-2-carbonitrile core with less reactive alternatives.
In this Letter, we will describe some brief SAR in the expanded S2
binding region and also our early efforts to obtain less reactive pyr-
idine-carbonitrile based cysteine protease inhibitors.
S
inhibitory activity of the pyrimidine- and pyridine-carbonitrile
CN
Y
X
F3C
R
The synthesis of the P2 expanded pyrimidine compounds is
shown in Scheme 1. Starting with commercially available 4-bro-
mo-2-trifluoromethylphenol, O-hydroxypropylation gives com-
a
Compds
R
X
Y
IC50 (nM)
Cat K
pound
4 in very high yield. Standard palladium catalyzed
Cat S
conversion of aryl bromide provided boronic ester 5 in good yield.
Suzuki coupling of 5 with 4-chloro-2-methylthiopyrimidine affor-
ded compound 6. Oxidation of sulphide using Oxone proceeded
smoothly to the desired sulphone 7 which was then converted to
the first desired pyrimidine-2-carbonitrile 8. Conversion of the
alcohol to amine mediated through methanesulphonate 9 went
smoothly to give final product 10. Results of these P2-expanded
pyrimidine-2-carbonitrile compounds 8 and 10 are shown in
Table 1.
2
8
6
40
50
HO
N
O
O
N
N
N
N
4.5
10
1.3
13
N
N
O
O
14
15
H
EtO
N
N
CH
CH
2884
1660
>10,000
>10,000
HO
N
16
N
CH
155
8900
18
N
CH
58
1660
Although the P1 propyl group was deleted from the molecule
for the purpose of easy synthesis, both compounds 8 and 10
appeared to be more potent against human cathepsin S than the
corresponding compound 2.
N
19
20
N
N
CH
CH
31
29
3090
N
O
O
O
>10,000
N
With a view to reducing the undesired war-head reactivity of
the above pyrimidine-2-carbonitrile core, a pyrimidine ring nitro-
gen deletion study was carried out in the hope that extra-binding
from this expanded P2/S2 interactions could compensate for the
loss of activity from reduced war-head reactivity as reported by
Altmann et al.13
The pyridine analogues are synthesized according to Scheme 2.
Suzuki coupling of readily available boronic ester 12 or 13 with
6- or 4-chloropyridine-2-carbonitrile gives the desired final
products.
HO
N
O
21
22
CH
CH
N
N
Inactive
Inactive
Inactive
Inactive
N
O
N
23
CH
CH
N
N
Inactive
Inactive
Inactive
Inactive
N
O
O
O
24
N
a
For assay conditions see Ref. 6; all pyridine compounds showed no activity
against human cathepsins B, L, and V.
Other pyridine compounds were synthesized according to
Scheme 3. Suzuki coupling of boronic ester 5 with 6- or 4-chloro-
pyridine-2-carbonitriles gives the corresponding pyridine-carboni-
trile compounds 16 and 21, respectively, in high yields. The alcohol
intermediates were then converted to the amine final products 18,
19, 22 and 23 mediated through the methanesulphonate interme-
diate 17.
O
F3C
HO
Br
a
F3C
R'
Br
c
F3C
B
b
O
R'
O
O
12
11
3
CN
O
B
X
Y
c
F3C
R
F3C
O
R
O
B
F3C
O
Br
F3C
O
F3C
HO
Br
a
b
13
O
14, 15, 20, 24
HO
HO
Scheme 2. Reagents and conditions: (a) R0OH, DIAD, Ph3P, 12 h; (b) bis(pinacola-
to)diboron, PdCl2(DPPF), KOAc, dioxane, 100 °C, 3 h; (c) 6- or 4-chloropyridine-2-
carbonitrile, Pd2(DBA)3, K3PO4, (c-Hex)3P, Dioxane–H2O, 100 °C, 3 h.
5
4
3
O
O
N
S
S
c
e
N
N
d
N
F3C
O
F3C
CN
HO
HO
O
O
B
X
Y
a
6
7
F3C
O
b
F3C
O
O
CN
N
CN
N
HO
HO
c
N
N
g
5
16, 21
f
F3C
O
F3C
O
O O
S
CN
Y
CN
Y
O
HO
X
X
9
8
F3C
O
F3C
R1
CN
N
O O
S
N
O
O
R2
N
F3C
17
18, 19, 22, 23
N
O
N
Scheme 3. Reagents and conditions: (a) 6- or 4-chloropyridine-2-carbonitrile,
Pd2(DBA)3, K3PO4, (c-Hex)3P, Dioxane–H2O, 100 °C, 3 h; (b) MsCl, DIPEA, DCM, rt,
2 h; (c) amine, NMP, 100–120 °C, 10–40 min.
10
Scheme 1. Reagents and conditions: (a) 3-iodopropanol, K2CO3, MeCN, 85 °C, 3h;
(b) bis(pinacolato)diboron, PdCl2(DPPF), KOAc, dioxane, 100 °C, 3 h; (c) 4-chloro-2-
methylthiopyrimidine, Pd2(DBA)3, K3PO4, (c-Hex)3P, Dioxane–H2O, 100 °C, 3 h; (d)
Oxone, MeCN–H2O, rt, 12 h; (e) KCN, DMSO, rt, 1 h; (f) MsCl, DIPEA, DCM, rt, 2 h; (g)
1-methylpiperazine, NMP, 90 °C, 20 min.
The results of all pyridine compounds together with the results
from pyrimidine derived compounds are shown in Table 1.

J. Cai et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4507–4510
4509
Removal of N1 nitrogen (Y) of the pyrimidine-2-carbonitrile
Enz
S
N
compounds results in the loss of just over 30-fold activity (16/8
and 18/10 pairs) against human cathepsin S. Extension of the ethyl
of compound 15 to a hydroxypropyl 16 resulted in a 10-fold
improvement of cathepsin S inhibitory potency. It is probable that
the extra CH2 has contributed to further hydrophobic interactions
in that region.20 The piperazine containing analogue 18 is threefold
better than the hydroxypropyloxy analogue 16. The most active
H
N
F3C
R
compounds identified are the imidazo- and
c-lactam containing
Figure 3. An ‘in situ double activation’ mechanism: electrostatic interaction
between the thiol SH proton and the ‘N3’ pyridine nitrogen activates both thiol
and nitrile simultaneously with a favourable five-membered ring transition state.
analogues 19 and 20. These results show that this extended S2
pocket is very accommodative in terms of binding interactions.
Compound 18 has an EC50 of 138 nM in the human JY cells based
Lip10 assay; however all other lower pKa or neutral analogues in
the same series are all >10 lM in the same assay likely due to
attacks the nitrile war-head from the left hand side (P2 side) of the
inhibitors. These structural data suggest that a possible ‘in situ
double activation’ mechanism with an electrostatic interaction be-
tween the active site cysteine thiol proton and the pyridine ring
nitrogen as shown in Figure 3. This is supported by our docking
studies. This interaction should make the thiol more nucleophilic
and the nitrile carbon more electrophilic. It is obvious that this
double activation is not possible for the ‘N1’ pyridine compounds.
In summary, by using X-ray structural information and com-
puter aided molecule design, a new expanded S2 pocket for
cathepsin S enzyme containing Phe70, Phe211 and Val162 was
identified. This pocket contributes nearly 100-folds towards
cathepsin S inhibitory activity in the current aryl nitrile series. It
is discovered that N3 nitrogen of the well-reported pyrimidine-2-
carbonitrile is critical for its cathepsin family cysteine protease
inhibitory activity. An ‘in situ double activation’ mechanism was
proposed to explain the results. The N1 nitrogen contributes only
30- to 50-fold towards activity. It is our intention that this in situ
activation could be used for other families of biological targets
where covalent binding is often necessary, such as lipases or other
proteases to reduce the potential risk of idiosyncratic toxicity re-
lated to non-specific irreversible covalent binding to off-target tis-
sues and proteins.
the lack of lysosomotropic effect. Removal of N3 nitrogen (X) of
the pyrimidine-2-carbonitrile compounds resulted in a total loss
of cathepsins S and K inhibitory activity as shown by analogues
21–24. The difference in the nitrile war-head chemical reactivity
between the two pyridine types should be very small. Using the
method described by Oballa et al.18 to estimate the reactivity of a
cysteine thiol towards the nitrile from the left hand side (P2 side),
we obtained values of approximately ꢀ8 kcal/mol for pyrimidine,
ꢀ5 kcal/mol for ‘N3-pyridine’ (X = N, Y = CH), and ꢀ3.5 kcal/mol
for ‘N1-pyridine’ (X = CH, Y = N). The ‘N1-pyridine’ is predicted to
be less reactive; however, the complete lack of activity is surpris-
ing. NMR measurements of nitrile reactivity towards glutathione
showed that compound 18 (N3-pyridine) is in fact slightly more
stable (t1/2 150 h) than compound 22 (N1-pyridine) (t1/2 120 h).21
However it should be pointed out that this NMR based reactivity
study does not consider the spatial direction limitation of the
thio-nitrile reaction in the enzymatic case.
Removal of N3 nitrogen should also have little impact on the
torsion angle between the pyridine ring and the P2 phenyl ring
as this biaryl normally adopts torsion angle 15–40°, consistent
with our X-ray structures of nitrile-pyrimidine and nitrile-pyridine
based inhibitors. The X-ray structure of compound 18 complexed
with human cathepsin S protein was obtained (Fig. 2). The X-ray
structure showed that this N3 nitrogen is not involved in any final
binding interactions with the cathepsin S protein. However this
structure and others of similar compounds bound to both cathep-
sins K and S6,7,22 revealed that the active site cysteine thiol always
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