The discovery of potent, orally bioavailable pyrimidine-5-carbonitrile-6- alkyl CXCR2 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.06.011

A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrimidine 2-((2,6-dichlorobenzyl)thio)-5-isocyano-6- phenylpyrimidin-4-ol 4, resulting in the discovery of CXCR2 receptor antagonist 2-((2,3-difluorobenzyl)thio)-

Full text of DOI:10.1016/j.bmcl.2014.06.011

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3285–3290
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery of potent, orally bioavailable pyrimidine-5-
carbonitrile-6-alkyl CXCR2 receptor antagonists
⇑
David W. Porter , Michelle Bradley, Zarin Brown, Steven J. Charlton, Brian Cox, Peter Hunt, Diana Janus,
Sarah Lewis, Paul Oakley, Des O’Connor, John Reilly, Nichola Smith, Neil J. Press
Novartis Institutes for BioMedical Research, Horsham Research Centre, Wimblehurst Road, Horsham, West Sussex RH12 5AB, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A hit-to-lead optimisation programme was carried out on the Novartis archive screening hit, pyrimidine
2-((2,6-dichlorobenzyl)thio)-5-isocyano-6-phenylpyrimidin-4-ol 4, resulting in the discovery of CXCR2
receptor antagonist 2-((2,3-difluorobenzyl)thio)-6-(2-(hydroxymethyl)cyclopropyl)-5-isocyanopyrimi-
din-4-ol 24. The SAR was investigated by systematic variation of the aromatic group at c-6, the linker
between c-2 and the halogenated ring, and the c-5 nitrile moiety.
Received 13 March 2014
Revised 4 June 2014
Accepted 5 June 2014
Available online 14 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
CXCR2
Chemokines
Pyrimidine
Neutrophils
COPD
Chemokines are small 8–10 kDa cytokines characterised by four
conserved cysteine residues linked via disulfide bonds.1 Chemo-
kines act through G-protein-coupled receptors (GPCRs) to regulate
a variety of effects, including cell migration and inflammatory
events. To date, numerous small molecule chemokine antagonists
have been described and claimed in patents.2,3
COPD patients in 201110 and a Phase II trial in bronchiectasis
patients in 201211 (Fig. 1).
Our previous paper described hit-to-lead efforts for a series of
pyrazolo and triazolopyrimidines.12 In this Letter, we present
efforts directed towards a second, chemically distinct hit that led
to another series of CXCR2 antagonists.
Chronic obstructive pulmonary disease (COPD) is a major cause
of morbidity and mortality throughout the world and is a chronic
inflammatory disease characterised by neutrophilic inflammation,
tissue damage and mucus hypersecretion.4 There is a significant
body of evidence linking the chemokine IL-8 in the pathogenesis
of COPD.5,6 Given the unmet medical need, this has prompted
interest in the development of antagonists of the IL-8-activated
chemokine receptors CXCR1 and/or CXCR2. The first non-peptide
CXCR2 antagonist SB-225002 was described in 1998. Subsequent
optimisation led to the CXCR2 antagonist danirixin 1, which has
since progressed into clinical trials for the treatment of COPD.7
Several other pharmaceutical companies have disclosed CXCR2
antagonists and amongst these, navarixin 2,8 and AZD-5069 3
(structure not disclosed9) are noteworthy of mention. Navarixin
progressed into Phase II studies for the treatment of COPD but
Merck omitted to list it in their updated development pipeline of
January 2012. AZD-5069 recently completed a Phase II trial in
A HTS was undertaken to identify functional antagonists that
blocked the binding of GRO-
(hrCXCR2) expressed in CHO membranes using a [35S]-GTP
assay. In this assay the amount of accumulated [35S]-GTP
S is
directly proportional to the degree of receptor activation. At the
hit evaluation stage, compounds with inhibition >50% at10
were re-confirmed as inhibitors in a 10 point IC50
35S]-GTP
membrane assay and a more conventional filter wash hrCXCR2
125I]-GRO- or [125I]-IL-8 binding assay.13 A good correlation
a to human recombinant CXCR2
cS
c
lM
[
cS
[
a
between the binding and functional assays was noted and binding
affinity was not routinely measured during hit-to-lead. On-target
activity was confirmed by showing a lack of functional antagonism
in a SDF-1 stimulated hrCXCR4 [35S]-GTP
cS selectivity assay and
periodic spot checking revealed selectivity not to be an issue.
Pyrimidine 4 was discovered from the CXCR2 HTS and although
inactive in the functional assay, it displayed a modest level of
potency in the binding assay. This activity translated into poor
ligand-lipophilicity efficiency (LLE)14 and the profile of this CXCR2
hit is shown in Table 1.
⇑
At the time of investigation, more potent thiazolo15 and
imidazololylpyrimidine16 structures had been reported. As such,
Corresponding author. Tel.: +44 0 1403 324443.
E-mail address: david.porter@novartis.com (D.W. Porter).
http://dx.doi.org/10.1016/j.bmcl.2014.06.011
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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D. W. Porter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3285–3290
Me
N
Me
OH
OH
Cl
O
N
O
O
HN
Me
O
HN
Me
N
F
O
O
S
N
H
N
H
F
S
F
O
O
Me
OH
Me
N
H
N
N
H
S
O
O
OH
Danirixin 1
Navarixin 2
AZD-5069 3
Proposed structure
Figure 1. Publicly identified CXCR2 antagonists danirixin and navarixin; compound 3 is believed to be AZD-50699.
Table 1
clearance following conjugation of the phenol with glucuronic acid.
A limited investigation into variation of the phenol at the 4-position
was therefore carried out and representative examples of this are
shown in Figure 2. Several AZD-5069 analogues have been
disclosed containing a hydroxyl or reverse sulfonamide at the
4-position of the pyrimidine ring and the ionisation state of the
phenol has been shown to be critical for binding affinity. In our
hands, methanesulfonation of 5 to give 6 yielded an inactive
compound, consistent with observations from the pyrazolo/
triazolopyrimidine series that the presence of larger acidic substit-
uents might interfere with binding to the receptor. Substituting the
phenol with an alkyl substituted hydroxylamine also gave an inac-
tive compound 7. This was an important observation since a series
of potent, non-acidic imidazolylpyrimidine antagonists appear to
have a similar spatial orientation of functionality.16
Hit profile of pyrimidine-5-carbonitrile 4
NC
N
Cl
HO
N
S
Cl
Measure
Pyimidine 4
4.3
hrCXCR2 [125I]-GRO-
a
IC50
lM
[
35S]-GTP
c
S IC50
>30
388
6.1
lM
Molecular weight
cLogP
Binding LLE (pIC50—cLogPa)
À0.7
Having established that the pyrimidinol moiety was important
for activity, variation of the 6-phenyl head group was investigated
next. Routes to these compounds are shown in Scheme 1. Thiouro-
nium salt 8 was obtained in quantitative yield by treating 2,3-diflu-
orobenzyl bromide with thiourea in ethanol at 65 °C. The synthesis
of pyrimidinol 5 and analogues 9–21 was accomplished in two
steps via Knoevenagel condensation of the aldehyde with ethyl
2-cyanoacetate and reaction of the resulting acrylates [predomi-
nantly (E)-] with thiouronium salt 8. Numerous aldehydes were
tolerated including electron-withdrawing/electron-rich aromatics
and simple aliphatics.
a
Compound 4 is weakly acidic and for the purposes of this calculation one can
assume LogP = LogD.
the plan was to examine in sequence the four pyrimidine substituents
aiming to improve potency and lower cLogP. Isosteres and amine
substitutions for the pyrimidinol functionality, aromatic/non-aro-
matic groups at c-6, alternative linkers between c-2 and the halo-
genated ring and replacements for the c-5 nitrile were explored.
Preference for a thiol linked 2,3-difluorobenzyl had already
been noticed elsewhere in a pyrazolo/triazolopyrimidine and
thiazolopyrimidine series of antagonists.12,15 Replacement of the
2,6-dichlorobenzyl with 2,3-difluoro gave compound 5 (Fig. 2)
which had measurable activity in the functional assay. This com-
pound was used as a starting point for variation of the other pyrim-
idine substituents.
CXCR2 receptor potency of the new 6-phenyl containing ligands
was initially guided by the application of the Topliss tree approach
with the goal of gaining maximum information from the least
number of analogues—a selection of these is shown in Table 2.
As discussed in our previous Letter,12 there was some concern
that pyrimidinol compounds like 5 would have high in vivo
The Topliss approach takes into account the electronic (
r), lipo-
philic ( ), and steric factors (Es) for substitution on a phenyl ring
p
NC
NC
HO
Methanesulfonation
N
F
N
F
F
F
HN
S
N
S
N
S
O
Me
O
5
6
GTPγS IC50 = 3.70μM
γ
μ
GTP S IC50 = >30 M
NC
HN
NC
HO
Alkyl hydroxyamine
N
F
N
F
F
F
N
S
N
S
HO
5
7
GTPγS IC50 = 3.70μM
γ
μ
GTP S IC50 = >30 M
Figure 2. Alternative c-4 groups.

D. W. Porter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3285–3290
3287
F
+ Br-
F
NH2
S
i
F
F
HS
H2N
99%
8
R
N
R
NC
HO
ii
(26-100)%
iii
N
F
NC
CHO
R
F
(51-80)%
S
CO2Et
5, 9-21
Scheme 1. Reagents and conditions: (i) thiourea, EtOH, 65 °C, 3–4 h; (ii) NCCH2CO2Et, NH4OAc, AcOH, toluene, 4 Å molecular sieves, reflux, 3–4 h; (iii) 8, DIPEA, EtOH, rt.
Table 2
CXCR2 antagonist binding potencies
R
NC
N
F
F
HO
N
S
a
Compound
R
CXCR2 [35S]-GTP
3.7
c
S IC50
(
lM)
cLogP
Functional LLE (pIC50—cLogPb)
5
4.9
0.4
9
10
11
12
7.4
5.7
5.4
5.0
5.8
À0.6
0.5
1.1
0
Cl
1.4
Me
OMe
0.71
1.6
O
Me
13
4.6
5.5
À0.2
OMe
14
15
0.78
4.7
4.5
5.4
1.6
OH
Me
À0.1
16
17
4.3
3.5
4.4
1.9
1.6
Me
0.90
18
4.9
5.0
0.3
19
1.7
4.5
3.9
4.8
1.3
3.0
0.6
20
0.14
3.7
21
a
Mean of n P2.
b
Compounds 5, 9–21 are weakly acidic and for the purposes of this calculation one can assume LogP = LogD.
using basic Hansch principles in a non-computerised manner.17 4-
Chloro substitution of the phenyl ring gave compound 9 of compa-
rable activity to the parent compound 5. According to the Topliss
decision tree, equal potency would most probably result from a
dependency, whilst lower potency would most likely indicate that
activity is À controlled. Synthesis and comparison of the 4-Me 10
r
and 4-OMe 11 analogues confirmed that a À
r effect was dominant.
This prompted follow-on synthesis of 4-OiPr 12 and 3-Me, 4-OMe
13 in an attempt to further reinforce this effect [the 4-N(Me)2
favourable +
p
effect counter balanced by an unfavourable À
r

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D. W. Porter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3285–3290
Table 3
CXCR2 antagonist binding potencies
R
NC
N
F
F
HO
N
S
a
Compound
R
CXCR2 [35S]-GTP
c
S IC50
(lM)
cLogP
Functional LLE (pIC50—cLogPb)
rac-22
CO2Et
CO2Et
CO2Et
CO2H
CH2OH
CONH2
1.5
0.32
1.3
0.01
0.006
0.007
3.7
3.7
3.7
2.8
2.4
1.9
2.1
2.8
2.2
5.2
5.8
6.3
ent-22a
ent-22b
ent-23a
ent-24a
ent-25a
a
Mean of n P2.
b
22–25 are weakly acidic and for the purposes of this calculation one can assume LogP = LogD.
CO2Et
CO2H
CO2Et
NC
HO
NC
HO
i
ii
83%
N
F
N
F
F
F
CHO
97%
N
S
N
S
23
22
O
OH
NH2
NC
HO
iii
51%
iv
N
F
NC
HO
23
N
F
F
73%
N
S
F
N
S
24
25
Scheme 2. Reagents and conditions: (i) NCCH2CO2Et, 8, K2CO3, EtOH, °C, 3–4 h; (ii) 1 M NaOH, EtOH, rt, 18 h; (iii) 1 M BH3, THF, MeOH, À5 °C (10 min) to rt, 18 h; (iv) HATU,
NH3, Et3N, DMF, rt, 18 h.
and 4-NH2 analogues were avoided due to potential for mutagenic-
ity18]. No additional increase in activity signalled some
effect
suggesting synthesis of the 4-OH 14 and 3-Me 15 analogues. These
compounds showed no improvement in activity relative to 11
implying that optimum electronic and hydrophobicity contribu-
tions had already been obtained.
A similar Topliss operational scheme for side-chain alkyl substi-
tution has also been reported.17 By and large the cases covered are
all those other than direct substitution on an aromatic nucleus. We
wished to probe the replacement of the 6-phenyl substituent with
a 6-alkyl and recognised that the electronic nature of the pyrimi-
dine ring and steric influence of the 5-CN group may complicate
application of the approach. Nonetheless, starting with methyl 16
as the base compound, the iso-propyl substituent was chosen first
SAR studies with a related series of thiazolopyrimidines sug-
gested that a hydroxylated alkyl substituent would be beneficial
for potency.15 With this in mind and using 20 as a starting point,
we designed a series of analogues to explore additional parameters
of the cyclopropyl motif – a selection of these is shown in Table 3
and the synthesis of these compounds is shown in Scheme 2. Syn-
thesis was accomplished via a one-pot modified Biginelli reaction
with ( )-trans ethyl 2-formyl-1-cyclopropanecarboxylate and thio-
uronium salt 8 to give ethyl ester 22. Alkaline hydrolysis in EtOH
gave intermediate acid 23 whilst treatment with borane in THF
proceeded smoothly to yield alcohol 24. HATU mediated coupling
of 23 with ammonia in DMF gave carboxamide 25. In addition to
the racemic ( )-trans-mixtures generated, compounds 23–25 were
also isolated as single enantiomers following chiral HPLC purifica-
tion. Data for the racemate and single enantiomers is presented for
compound 22, whilst data for the most potent enantiomer is
shown for compounds 23–25. Confirmation of absolute stereo-
chemistry was not determined at this stage.
p
on the premise that a +
in activity was obtained with 17. Cyclopentyl 18 was then synthes-
ised on account of its larger value with minimal change in steric
factor Es. However, failure of cyclopentyl to show an increase in
potency indicated that the optimum value had already been
p effect was most probable and an increase
p
p
Comparison of the racemic ethyl ester rac-22 with the sepa-
rated enantiomers ent-22a and ent-22b revealed that there was a
chiral preference for one enantiomer over the other. This was fur-
ther qualified with analogues 23–25—removal of the ethyl group
led to a dramatic increase in potency with acid ent-23a showing
a 30-fold improvement over ethyl ester ent-22a. Additional manip-
ulation of ent-23a was well tolerated as exemplified by the
exceeded. Cyclobutyl 19 and cyclopropyl 20 were made next and
gratifyingly, cyclopropyl 20 displayed a significant improvement
in activity and LLE relative to 5. The second possibility of activity
increasing with increasing À
r values was ruled out following syn-
thesis of the tert-butyl analogue 21 which displayed comparable
activity to cyclopentyl 18.
⁄

D. W. Porter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3285–3290
3289
Table 4
Table 5
CXCR2 antagonist binding potencies
Lead profile of pyrimidine-5-carbonitrile 20
R
NC
HO
N
N
F
Y
F
HO
N
X
F
Ar
F
N
S
a
Compound
R
X
S
Y
Ar
CXCR2 [35S]-GTP
M)
cS IC50
(l
Measure
Triazolopyrimidine 20
hrCXCR2 [125I]-GRO-
[35S]-GTP
a
IC50
0.04
0.14
45
l
l
M
M
cS IC50
20
CN
CH2
0.14
Rat microsome Cl (ml/min/kg)
Human microsome Cl (ml/min/kg)
Rat iv Cl (ml/min/kg)
Rat iv Vss (l/kg)
14
1.6
0.2
6
26
27
28
29
CN
CN
CN
S
S
S
CH2
0.51
4.8
Rat iv t½ (h)
Rat po bioavailability (%)
Rat plasma protein binding (%)
Molecular weight
>100a
>99
319
0.01
3.9
CHMe
C(Me)2
S
HT-eq Solubility pH 6.8 (g/l)
cLogP
6.4
Functional LLE (pIC50—LogPb)
3.0
CN CH2
CN CH2
>30
a
Plasma concentrations in many samples were outside the range of the cali-
bration curve—PK parameters should therefore be viewed as approximations.
Compound 20 is weakly acidic and for the purposes of this calculation one can
assume LogP = LogD.
F
F
F
F
b
F
F
F
F
30
31
32
33
S
10
profile suggests that clearance via glucuronidation is not an issue.
Metabolite identification studies indicated the formation of sulfone
and sulfoxide metabolites in vitro and in vivo but further studies
are required to determine the levels of each.20 LLE is improved sig-
nificantly relative to the HTS hit 4 and compound 20 was inactive
(IC50 >10lM) against a broader panel of 49 GPCRs. Pyrimidines
exemplified by compound 20 formed the basis for on-going studies
with this series of compounds.
In conclusion, HTS identified novel pyrimidine-5-carbonitrile
chemotypes as selective CXCR2 antagonists. The SAR of the scaffold
was explored, resulting in identification of pyrimidine-5-carboni-
trile-6-cyclopropyl 20 which is a functional antagonist of the
human CXCR2 receptor and shows good oral bioavailability in
the rat.
H
Br
I
S
S
S
CH2
CH2
CH2
0.32
1.4
3.2
a
Mean of n P2.
(hydroxymethyl)cyclopropyl derivative ent-24a and cyclopropane-
carboxamide ent-25a.
References and notes
As with the pyrazole/triazolopyrimidine series of antagonists,12
the lipophilic S-benzyl was seen as a potential site of metabolic
instability and the c-5 nitrile is in an appropriate position to
enhance this. As such, a set of alternative linkers at c-2 and
replacements for the nitrile at c-5 were synthesised and assessed
for potency in comparison to compound 20 (Table 4).
1. Uguccioni, M.; D’Apuzzo, M.; Loetscher, M.; Dewald, B.; Baggioloini, M. Eur. J.
Immunol. 1995, 25, 64.
2. Busch-Peterson, J.; Wang, Y. Expert Opin. Ther. Patents 2008, 18, 629.
3. Pease, J. E.; Horuk, R. Expert Opin. Ther. Patents 2009, 19, 199.
4. Chung, K. F. Curr. Drug Targets Inflamm. Allergy 2005, 4, 619.
5. Barnes, P. J. J. Allergy Clin. Immunol. 2007, 119, 1055.
6. Stockley, R. A. Curr. Med. Res. Opin. 2009, 25, 1235.
Benzylic substitution may be a strategy for hindering displace-
ment at c-2 but (1-phenylethyl)thio 27 and gem-dimethyl 28 were
found to be less active than unsubstituted 26. Reversing the C–S
linker between c-2 and the phenyl ring gave inactive 29. Re-intro-
ducing the favourable 2,3-difluoro substitution gave analogue 30
but this was 70-fold less potent than the parent compound 20.
Turning our attention to the c-5 nitrile position, unsubstituted 31
was comparable in activity to 20 but weaker deactivating groups
such as bromo 32 and iodo 33 led to a 10-fold drop in potency
when compared to 31.
7. Busch-Petersen, J. U.S. Pat. Appl. US 20070249672, 2007.
8. Nair, P.; Gaga, M.; Zervas, E.; Alagha, K.; Hargreave, F. E.; O’Byrne, P. M.;
Stryszak, P.; Gann, L.; Sadeh, J.; Chanez, P. Clin. Exp. Allergy 2012, 42, 1097.
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10. NCT01233232 A 4 Week Study to Investigate the Safety and Tolerability of
AZD5069 in Patients With Moderate to Severe Chronic Obstructive Pulmonary
Disease (COPD) (CIRRUS). Available from: http://www.clinicaltrial.gov/ct2/.
11. NCT01255592 Evaluation of the Effect of AZD5069 in Patients With
Bronchiectasis (STRATUS). Available from: http://clinicaltrials.gov/ct2/show/.
12. Porter, D. W.; Bradley, M.; Brown, Z.; Canova, R.; Charlton, S.; Cox, B.; Hunt, P.;
Kolarik, D.; Lewis, S.; O’Connor, D.; Reilly, J.; Spanka, C.; Tedaldi, L.; Watson, S.
J.; Wermuth, R.; Press, N. J. Bioorg. Med. Chem. Lett. 2014, 24, 72.
In vitro and in vivo pharmacokinetic data was obtained for key
compounds throughout this study and the results for compound
2019 are shown in Table 5. This compound has a reasonable lead
like profile with acceptable potency in the binding and functional
assays. The in vitro rat and human microsomal clearance is low
to moderate and the compound is a weak acid (pKa 8.9) with mod-
erate to high lipophilicity. Plasma protein binding as a conse-
quence is high and the compound has a suitable rat in vivo
profile with low clearance and good oral bioavailability. The PK
13. For full details of the biological assays used see: Porter, D.; Press, N. J.; Spanka,
C. U.S. Pat. Appl. US 20100069407, 2010; (i) receptor binding assay: [125I]-IL-8
hrCXCR2 was performed in a 96-well micro plate format with each reaction
mixture containing 0.05 mg/ml CXCR2 membrane protein. Compounds of
interest were pre-dissolved in DMSO so as to reach a final concentration of
between 10
was initiated by addition of 0.02 nM [125I]-IL-8; (ii) [35S]-GTP
for hrCXCR2 receptor using SPA technology: assay was performed in duplicate
in 96 well Optiplate™ microplates in final volume of 250 per well.
l
M and 0.0005
l
M [final concentration of DMSO 2% (v/v)]. Binding
c
S binding assay
a
ll
Compounds were diluted in DMSO (0.5% final concentration). Data were
expressed as the% response to 100 nM IL-8 minus basal.

3290
D. W. Porter et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3285–3290
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19. Selected data for 20: dH (500 MHz, DMSO-d6) 13.59 (1H, br), 7.38 (1H, m), 7.27
(1H, m), 7.23 (1H, m), 4.46 (2H, s), 2.15 (1H, m), 1.17 (2H, m), 1.07 (2H, m);
C15H11F2N3OS requires C, 56.42; H, 3.47; N, 13.16; S, 10.04. Found: C, 55.52; H,
16. Ho, K-K.; Auld, D. S.; Bohnstedt, A. C.; Conti, P.; Dokter, W.; Erickson, S.; Feng,
D.; Inglese, J.; Kingsbury, C.; Kultgen, S. G.; Liu, R-Q.; Masterson, C. M.;
Ohlmeyer, M.; Rong, Y.; Rooseboom, M.; Roughton, A.; Samama, P.; Smit, M-J.;
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3.46; N, 12.88; S, 9.56.
20. Glutathione conjugates were detected in vitro following incubation with
reduced glutathione (GSH) in rat and human liver microsomes. Incubation was
carried out for 60 min with 0.1 mg/ml microsomal protein and 5 mM GSH +/À
oxidation co-factor NADPH. Samples were analysed by full scan LC–MS/MS
following precipitation with MeCN and detection was confirmed by accurate
mass measurement.