Optimization of Platelet-Derived Growth Factor Receptor (PDGFR) Inhibitors for Duration of Action, as an Inhaled Therapy for Lung Remodeling in Pulmonary Arterial Hypertension

Article abstract of DOI:10.1021/acs.jmedchem.6b00703

A series of potent PDGFR inhibitors has been identified. The series was optimized for duration of action in the lung. A novel kinase occupancy assay was used to directly measure target occupancy after i.t. dosing. Compound 25 shows 24 h occupancy of the PDGFR kinase domain, after a single i.t. dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro:in vivo correlations which link duration of action in vivo with low permeability and high basicity and demonstrate that nonspecific binding to lung tissue increases with lipophilicity.

Full text of DOI:10.1021/acs.jmedchem.6b00703

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Article
Optimization of Platelet Derived Growth Factor Receptor
(PDGFR) inhibitors for duration of action, as an inhaled
therapy for lung remodeling in pulmonary arterial hypertension
Duncan E. Shaw, Ferheen Baig, Ian Bruce, Sylvie Chamoin, Stephen P. Collingwood,
Sarah Cross, Satish Dayal, Peter Drueckes, Pascal Furet, Vikki Furminger, Deborah
Haggart, Martin Hussey, Irena Konstantinova, Jon C. Loren, Valentina Molteni, Sonia
Roberts, John Reilly, Alex M Saunders, Rowan Stringer, Lilya Sviridenko, Matthew Thomas,
Christopher G Thomson, Christine Tomlins, Ben Wen, Vince Yeh, and Andrew Pearce
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00703 • Publication Date (Web): 09 Aug 2016
Downloaded from http://pubs.acs.org on August 9, 2016
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Optimization of Platelet Derived Growth Factor
Receptor (PDGFR) inhibitors for duration of action,
as an inhaled therapy for lung remodeling in
pulmonary arterial hypertension
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Duncan E. Shaw,*^ Ferheen Baig,+ Ian Bruce,§ Sylvie Chamoin,> Stephen P.
Collingwood,§ Sarah Cross, ^ Satish Dayal,+ Peter Drückes,> Pascal Furet,> Vikki
Furminger,§ Deborah Haggart,» Martin Hussey,# Irena Konstantinova,# Jon C
Loren,» Valentina Molteni,» Sonia Roberts,# John Reilly, § Alex M. Saunders,§
Rowan Stringer,+ Lilya Sviridenko,§ Matthew Thomas, # Christopher G. Thomson,^
Christine Tomlins,# Ben Wen, » Vince Yeh,» and Andrew Pearce#
^Novartis Institutes of Biomedical Research(NIBR), 100 Technology Square, Cambridge MA 02139,
USA; +Metabolism and Pharmacokinetics, §Global Discovery Chemistry, #Respiratory Diseases, NIBR
Horsham, Wimblehurst Road, Horsham, West Sussex, UK; >NIBR Basel Fabrikstrasse 2, 4056 Basel,
Switzerland; »Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive,
San Diego, CA 92121 USA
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ABSTRACT: A series of potent PDGFR inhibitors has been identified. The series was optimized for
duration of action in the lung. A novel kinase occupancy assay was used to directly measure target
occupancy after it dosing. Compound 25 shows 24 hour occupancy of the PDGFR kinase domain, after a
single it dose and has efficacy at 0.03 mg/kg, in the rat moncrotaline model of pulmonary arterial
hypertension. Examination of PK/PD data from the optimization effort has revealed in vitro: in vivo
correlations which link duration of action in vivo with low permeability and high basicity, and
demonstrate that nonspecific binding to lung tissue increases with lipophilicity.
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INTRODUCTION
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Pulmonary arterial hypertension (PAH) is a progressive and deadly disease with a poor prognosis despite
the introduction of multiple therapeutic classes over the last decade.1 The disease is characterized by
increases in pulmonary vascular resistance (PVR) such that the resting mean pulmonary arterial pressure
rises above 25 mmHg.2 The disease is driven by aberrant smooth muscle proliferation in the pulmonary
arteries and arterioles which increasingly makes passage of blood more difficult. Characteristic plexiform
lesions, which show medial hypertrophy and fibrotic intimal lesions can envelop the entire artery wall, are
also features of the pathology. The current marketed therapies function by vasodilation and have limited
effects on the progression of the smooth muscle proliferation or on lesion formation.3
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The platelet derived growth factor (PDGF) is a powerful mitogen which signals through the receptor
tyrosine kinases (PDGFRα and PDGFRβ). Both the chemokine and its receptor have been shown to be
overexpressed in both the pulmonary artery smooth muscle cells (PASMC) and the endothelial cells of
severe PAH patients. Actively proliferating PASMC have been shown to have raised levels of
phosphorylated PDGFR.4
Oral Imatinib 1 demonstrated efficacy in the first trial to investigate the effects of a nonꢀvasodilatory
agent in PAH. The rationale for initiating the clinical trial was the activity of 1 as an inhibitor of PDGFR
receptor tyrosine kinases. The drug targeted the remodeling of the pulmonary arteries and thus attempted
to alter the course of the disease. Patients in the trial showed improvements in six minute walk distance
and reduced PVR measured by right heart catheterisation.5 The systemic administration of 1 is however
associated with side effects and these led to significant numbers of patients withdrawing from the trial.
Indeed in the IMPRES (phase III) trial, despite clear clinical benefits, a positive risk benefit ratio could
not be demonstrated.6
It was hypothesized that an inhibitor of PDGFR locally administered by inhalation might achieve efficacy
similar or better than 1 but with greatly reduced systemic side effects.
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When the desired site of action is in the lung, inhalation can be used to reduce the systemic side effects of
a medication. A key challenge to this approach is the rapid absorption of small molecules from the
airways into the systemic circulation, since it has been clearly demonstrated that the lung offers little
barrier to the absorption of soluble drugs.7 The maintenance of duration of action in the lung such that
clinical dosing can be once or twice a day is a key objective in inhaled drug design.7
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The selective inhibition of PDGFR in the lung vasculature presented a challenging objective. Whilst there
is extensive precedent for inhaled medicines demonstrating an enhanced therapeutic index, the site of
action for these drugs is typically in the airway epithelial tissue (e.g. β2 agonists, M3 antagonists).8 The
target tissue for this project was the smooth muscle surrounding the pulmonary arteries and arterioles of
the lung and, by definition this tissue is separated by only a short distance from the circulatory system.
This presented a novel drug delivery challenge, to achieve sufficient exposure in the target tissue whilst
avoiding rapid equilibration of the drug with the rest of body. Given the novel nature of the drug delivery
challenge, the characteristics shown to be desirable for traditional inhaled drugs were not necessarily
relevant to this project. The strategy taken was to profile a selection of potent PDGFR inhibitors in an in
vivo mechanistic assay to measure duration of action, and in parallel to assess an extensive range of in
vitro parameters. This data set allowed the establishment of in vitro: in vivo correlations which were then
used to prioritize subsequent optimized molecules for in vivo testing.
A search for novel chemotypes, which could display similar kinase activity, was initiated, in the Novartis
sample collection, based on the coꢀcrystal structure of 1 with cKit (PDB: IT46)9. The imidazo [1, 2ꢀa]
pyridine 2 (Rat A10 IC50 36± 16 nM) was identified as a potent inhibitor of PDGFR and cKit.
Furthermore the pyrazolo [1, 5ꢀa] pyridine 3 (Rat A10 IC50 12± 5 nM) offered an attractive alternative
chemotype (Figure 1). These molecules compared well to 1 (Rat A10 IC50 162± 39 nM) in a cell based
assay of PDGFβ driven proliferation in rat A10 PASMC, and thus offered attractive starting points.
Compounds 2 and 3 were both identified by separate Novartis project teams in search of novel and
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patentable analogues of 1. They were considered ideal starting points for this work since they maintained
a kinase selectivity profile similar to that of 1.
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Figure 1 Lead molecules identified from the Novartis sample collection
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CHEMISTRY
Compounds described in this paper were synthesized according to the general scheme (Figure 2)10
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Figure 2 General scheme for preparation of the compounds described: for specific structures of R1 and R2
refer to Tables 1 and 2
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N
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X
Q
NH2
i) Amide coupling
NH
P
W
O
W
O
ii) Ester hydrolysis
(optional)
Suzuki Reaction
O
P
O
4
5
4a W= CH, X= Me, P= H
4b W= CH, X= F, P= Me
4c W = N, X = Me, P = Me
5a W= CH, X= Me, Y= C, Z =N, P= H, Q = H
5b W= CH, X= F, Y= N, Z =C, P= Me, Q = H
5c W= CH, X= F, Y= N, Z =C, P= Me, Q = Br
5d W= CH, X= F, Y =C, Z= N, P= Me, Q= Br
5e W= N, X= Me, Y =C, Z= N, P= Me, Q= Br
N
Z
O
O
Y
X
R2
NH
P
W
O
6
6a W= CH, X= F, Y= N, Z =C, P= Me, R2 = 1-methyl-1H-pyrazol-5-yl
6b W= CH, X= F, Y= N, Z =C, P= H, R2 = 1-methyl-1H-pyrazol-5-yl
6c W= CH, X= F, Y= N, Z =C, P= H, R2 = 1-methyl-1H-pyrazol-3-yl
6d W= N, X= Me, Y =C, Z= N, P= H, R2 = 1-methyl-1H-pyrazol-5-yl
6e W= N, X= Me, Y =C, Z= N, P= H, R2 = 1-methyl-1H-pyrazol-3-yl
Amide Coupling
Amide Coupling
N
N
Z
Z
O
O
Y
X
Y
R2
X
2 W= CH, X= F, Y= N, Z =C, Q = H
3 W= CH, X= Me, Y= C, Z =N, Q = H
7c W= CH, X= F, Y= N, Z =C, Q = Br
7d W= CH, X= F, Y =C, Z= N, Q= Br
Q
Suzuki Reaction
NH
NH
W
O
W
O
NH
R1
NH
R1
7
12 - 22 W= CH, X= F, Y= N, Z =C
23 W= CH, X= F, Y= C, Z = N
24 W= N, X=Me, Y= C, Z = N
25 W= N, X=Me, Y= C, Z = N
The compounds could be prepared by standard methods in a flexible way using orthogonal chemistry
which allowed for a number of interchangeable synthetic routes. Largely commercially available 3ꢀ
aminobenzoic acids or esters 4 were used as the core and amide coupling with fused pyrazolopyridine or
imidazopyridine carboxylic acids (optionally substituted with bromine) allowed access to the central
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intermediates 5. The final compounds could then be accessed by carrying out an initial palladium
catalyzed coupling to give 6, followed by an amide coupling. Alternatively one can perform an amide
coupling reaction to give 7 and then a palladium catalyzed cross coupling reaction to give the final
products. This chemistry was employed flexibly to enable structural variation at either end of the
molecule.
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The pyridine core molecule 4c was novel and was synthesized as described in Figure 3. Chlorination of
the nitro derivative 8 allowed formation of the chloropyridine with concomitant esterification of the
carboxylic acid to furnish 9. Hydrogenation of the nitro group then allowed the core amino pyridine 4c to
be synthesized.
Figure 3 Preparation of the pyridine core
The amine groups used to form R1 were commercially available with the exception of (5, 5ꢀ
dimethyltetrahydrofuranꢀ2ꢀyl) methanamine 11 which was synthesized as shown in Figure 4. The
commercially available bromocyclisation product 10 was converted into the intermediate azide, which
was subsequently reduced using triphenylphosphine to give the desired primary amine.
Figure 4 Synthesis of (5, 5ꢀdimethyltetrahydrofuranꢀ2ꢀyl) methanamine
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RESULTS AND DISCUSSION
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PDGFRα inhibition was assessed in a biochemical assay using the isolated kinase domain and examining
the degree of phosphorylation of a probe peptide substrate in the presence of the test molecule.11 The cell
based activity of the test molecules was determined by measuring the effect of the compound on the
PDGFβ driven proliferation of Rat A10 (PASMC) over a four day period. 12 A homology model was
generated based on the the cKIT coꢀcrystal structure of 19 and this model was used to predict the binding
mode of 7c . Examination of the predicted docking modes suggested substitution of the imidazopyridine
ring of 2 at the 6ꢀposition would be tolerated since space existed at that position (Figure 5). Indeed a boost
in potency was achieved when a range of small heterocycles were incorporated at this position (Table 1).
The introduction of a 3ꢀpyridyl substituent 12 gave an increase in cell based activity. The methyl 5ꢀ
pyrazolyl 13 and methyl 3ꢀpyrazolyl 14 substituents were also particularly beneficial, with reductions in
permeability leading to a bigger disconnect between potency in the isolated enzyme system and that seen
in the cell based assay. However, by finding successively more potent inhibitors in the enzyme assay, the
desired level of cell based potency could be maintained, despite the reduced permeability. The 3ꢀ
pyrazolyl substituent 15 maintained functional activity at the enzyme but was slightly less potent in the
cell based assay suggesting that the reduction in permeability had begun to impact cell penetration (Table
1).
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Figure 5 Docking of 7c into the homology model generated from the PDB structure 1T46 (1 coꢀ
crystallized with cKit)9 A) Showing the position of 1 (cyan) compared to the proposed binding mode for
7c (orange). B) Showing the potential space at the 6ꢀposition of the imidazopyridine, by replacement of
the bromo substituent.
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A)
B)
Table 1 Effect of imidazopyridine 6ꢀsubstituent upon potency, permeability and lipophilicity
N
R
N
HN
O
F
O
NH
N
N
PAMPA
Permeability
% transcellular13
83.1
CE PDGFRα IC50
Rat A10 assay
IC50 nMb
cLog P
R
nMa
H
13±18
36±16
3.36
3.75
2
0.75±0.45
7.9±3.8
23
12
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0.18±0.02
9.2±1.6
4.8
3.57
13
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0.32±0.17
<0.1±0.01
3.6±0.7
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0
3.36
3.34
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15
18.7±6.9
a) Inhibition of probe substrate phosphorylation by PDGFRα, measured by capillary electrophoresis. Determinations based on n=
3 or more11
b) Inhibition of PDGFRβ driven proliferation of Rat A10 cells. Determinations based on n= >3 or more12
The presence of an aryl piperazine substituent in these molecules was seen as a risk in terms of potential
off target activity, since compounds containing this moiety are known to bind promiscuously to GPCRs
and ion channels.14 Indeed compound 13 showed an IC50 of binding <10 ꢀM at 9 out 26 GPCRs tested
and exhibited binding at the HERG channel (IC50 = 4 ꢀM). A search for an alternative amide substituent
with a lower lipophilicity, and MW was initiated guided by docking studies (Table 2).
The substituted difluorobenzyl compound 16 showed very high potency indicating that the size of the
amide substituent could be reduced without detrimental effects on potency. Compound 16 however,
showed poor solubility (no detectable solubility in the i.t. dosing vehicle 10% PEG 200 / 95 % D5W)
suggesting it would be beneficial to introduce polarity into the molecules. The dimethyltetrahydrofuran
17, exhibited potency equivalent to 16 with reduced lipophilicity. The introduction of a basic amine such
as 18 was also tolerated. An improved understanding of the required elements for optimal lipophilic
binding was derived from comparison of compounds 19 and 20 in which no further advantage is gained
by extension of the aliphatic side chains. This improved understanding led to the discovery of the cyclic
derivative 21. The potency could be improved further using the methyl 5ꢀpyrazolyl substituent to give 22.
Table 2 Effect of amide substituent upon potency, permeability and lipophilicity
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N
R2
N
HN
O
O
F
1 NH
R
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PAMPA
CE PDGFRα Rat A10 assay
R1
R2
Permeability
% transcellular12
cLog P
IC50 nMb
IC50 nMa
0.09±0.03
3.0±0.2
11
81
5.4
0
3.77
3.26
3.19
3.23
16
17
18
19
0.53±0.18
1.3±0.7
3.2±1.5
1.6±0.9
2.7 ± 0.7
8.7± 0.8
23±7
22±12
23
3.76
3.67
20
21
1.3±0.03
9.5±3.7
0.6
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0.19±0.06
1.7±0.7
0.7
3.46
22
7
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9
a) Inhibition of probe substrate phosphorylation by PDGFRα, measured by capillary electrophoresis. Determinations based on n=
3 or more11
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b) Inhibition of PDGFRβ driven proliferation of Rat A10 cells. Determinations based on n= >3 or more12
With the SAR established for both the amide portion and the hinge binding extension, it was considered
important to reꢀexamine the alternative core structures. Both the pyrazolopyridine 23 and the 2ꢀ
methylpyridine 24 showed good in vitro potency and allowed the direct comparison to the original
imidazo[1, 2ꢀa] pyridine core structure 13 (Table 3). The core changes were well tolerated in terms of
potency in vitro and led to modified properties allowing us to examine the effects of such changes on
duration of action in vivo (Table 4). The optimal amide and hinge extension substituents identified in the
earlier work were then transferred into the 2ꢀmethylpyridine core, using the pyrazolopyridine as the hinge
binding group; this allowed the identification of 25. This compound showed a much improved selectivity
profile with respect to binding to GPCRs (26 tested 24 >30 ꢀM, M1 IC50 5.0 ꢀM; H3 IC50 5.0 ꢀM) and
had an IC50 >30 ꢀM at the HERG channel. These data were supportive of further profiling particularly
given the low anticipated dose based on the inhaled route of delivery (Table 3)
Table 3 Effect of changes to the aniline core and the 6,5 heterocycle upon potency, permeability and
lipophilicity
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CE PDGFRα IC50
nMa
Rat A10 assay IC50
nMb
PAMPA Permeability
Compound
cLog P
% transcellular12
0.18±0.02
1.2±0.8
9.2±1.6
4.8
70
3.57
3.03
3.01
2.90
13
23
24
25
10.3±2.8
35±12
0.96±0.5
0.2±0.08
17
12.8 ± 0.9
4.7
a) Inhibition of probe substrate phosphorylation by PDGFRα, measured by capillary electrophoresis. Determinations based on n=
3 or more11
b) Inhibition of PDGFRβ driven proliferation of Rat A10 cells. Determinations based on n= >3 or more12
The identified compounds were screened in a range of in vitro assays (High Throughput Solubility,
PAMPA13, CACO2 (AꢀB/BꢀA), CACO2 Papp, RLM Clint, CHIꢀIAM membrane retention15, RSA
binding16, HSA binding16, Vss calc16 ) and in silico calculations (cLogP, PSA, cLogD) to assess their
physicochemical properties (Full data set is in the supplementary information).18 The intention of this
exercise was initially to facilitate the development of in vitro: in vivo correlations (ivivc) rather than to
screen out any particular property at this stage. Potent compounds with a range of properties were then
screened in vivo by measurement of kinase site occupancy.
Critical to the success of this strategy was the identification of a direct method for measurement of
duration of action. In this respect the identification of the PDGFRꢀβ antibody CST456419 was vital. This
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antibody has the remarkable property that it can only recognize PDGFRꢀβ in its active form. When the
kinase is in an inactive form (i.e. in the presence of a type II kinase inhibitor) the antibody does not
recognize PDGFRꢀβ in immunoprecipitation experiments. Applying this finding to exꢀvivo tissue from
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PKꢀPD experiments allowed us to determine the percentage of the PDGFRꢀβ in the given tissue sample
which was bound to compound, and therefore to directly measure kinase occupancy. Control experiments
were able to show that addition of excess test compound to the lysates during the processing and
immunoprecipitation work up did not affect the level of PDGFR occupancy measured. This finding
confirmed that our readout was directly correlated to the occupancy seen in the in vivo experiment.17
The poorly soluble analogue 16 was shown to exhibit low occupancy at 7 h after a 1 mg/kg dose, despite
very high measured lung levels. This observation indicated that the drug was very likely to be still in the
airway and not absorbing into the lung tissue itself. As a result of this early finding, in vivo testing was
confined to molecules which could be shown to have sufficient solubility in the dosing vehicle (10% PEG
200 / 95 % D5W).
The dosing of soluble compounds led to much lower apparent lung levels, but also led to superior
occupancy, compounds 13 and 18 for example exhibited duration of action to 7h. (Table 4)
Another key comparison was that between the core structures, with 23 showing relatively poor duration of
action compared to 13. The duration of action was recovered when the pyridyl core was introduced in 24.
The common factor between 13 and 24 is that they possess two basic centers at physiological pH which is
not the case for 23. This suggested that these dibasic compounds had some intrinsic advantage.
Additionally the decreased lipophilicity of 24 is a factor and an improved occupancy was demonstrated
despite somewhat lower cell based activity.
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The improved amide substituent and the optimal hinge extension, when combined with the more polar
core structure 25 further improved the in vivo profile leading to occupancy of >80% at 24 hours after a
0.1 mg/kg dose.
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Table 4 Occupancy of the PDGFR kinase domain and lung concentrations from in vivo studies
Compound
Solubilitya
mg/mL
<0.01
0.77
it dose
mg/kg
1
% Occ 7 hb
[Lung] at 7h
(nM)
% Occ
24hb
[Lung] at 24 h
(nM)
27±11
54±24
70±25
75±13
82±8
276200±143700
188±32
16
13
13
18
22
23
24
25
0.1
0.77
0.4
687±188
9±19
0
60±39
44±16
420±85
0.59
0.1
302±56
> 1
0.1
1026±255
221±144
51±9
> 1
0.4
29±22
83±8
> 1
0.4
3950±1100
1304±230
> 1
0.1
88±10
81±9
680±121
a) 10% PEG200/95% D5W. i.t. dosing vehicle , i.t. dosing of vehicles with higher organic content is not well tolerated
b) As determined by immunoprecipitation from lung tissue lysates using CST4564
For a set of 40 compounds tested in vivo18 a data set was obtained that enabled assessment of in vitro: in
vivo correlations. The compound set consisted of potent compounds (IC50 < 50 nM in the rat A10 assay)
all of which had good solubility in the dosing vehicle. These restrictions mean that the data set is not well
suited to assessing the effect of potency or solubility upon duration of action.
As an initial unbiased approach, a random forest plot was generated to show which of the in vitro
parameters measured correlated most strongly to occupancy at 8 h, and to the concentration of drug
required to reach 50% occupancy (Occ50) in the in vivo studies (Figure 6).
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When considering occupancy at 8 hours, by far the strongest correlation was that seen to permeability in
the artificial membrane PAMPA assay (x. transcellular). A correlation was also observed to clearance in
rat liver microsomes (Clint, RLM.ER), and a role for basicity was identified by correlations seen to
calculated Log D and pKa. Lipophilicity in isolation was not a good predictor of duration of action, but
had by far the strongest correlation to Occ50.
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Figure 6 Random forest comparison of in vivo occupancy to in vitro parameters
A) Relative correlation strength between Occupancy at 8 hours and various in vitro measurements
PSA
cLogP
CHI-IAM
cVss Rat
Fraction ionized
Papp CACO2
RLM ER
c highest pKa
cLogD
RLM CL int
PAMPA % transcellular
0
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B) Relative correlation strength between Occ50 and various in vitro measurements
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cLogP
Fraction ionized
c highest pKa
cVss Rat
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PSA
Papp CACO2
RLM ER
CHI-IAM
cLogD
RLM CL int
PAMPA % transcellular
0
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It was possible to show that permeability, measured in the artificial membrane PAMPA assay, correlated
with duration of action (see Figure 7). Compounds with good occupancy at 7 hours after an it dose were
shown to exhibit much lower permeability than those without occupancy at 7 hours (p = 0.00003).
Figure 7 Permeability is a key factor in determining duration of action
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Figure 8 More basic compounds exhibit longer duration of action
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Whilst low permeability was necessary for duration of action, it was not sufficient and compounds with
low permeability were also shown to lack duration of action in many cases. Absorption of low molecular
weight compounds from the airway into the systemic circulation is known to be rapid.7 One can speculate
that for soluble compounds, absorption from the airway may be too rapid to allow a significant depoting
effect to occur as part of the first pass absorption event. Low permeability may be necessary to slow the
absorption and allow the opportunity for depoting to occur in the case where a compound has sufficient
affinity for lung tissue.
A correlation with high pKa was observed, with more basic compounds showing a higher tendency to
have good duration of action (p = 0.011) (Figure 8). This empirical observation is consistent with the
known affinity of basic molecules for tissue and in this case could be a representative surrogate measure
of lung tissue affinity.
Correlations were also observed within the data, when the amount of drug required to achieve 50%
Occupancy (Occ50) was considered. A clear positive correlation between Occ50 and cLogP was observed
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(p = 0.00028) (Figure 9) suggesting a role for nonꢀspecific binding of drug to the tissue. Less lipophilic
molecules are likely to be more available to the target due to a reduction in nonꢀspecific tissue binding.
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Figure 9 cLogP correlates with Occ50
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Figure 10 Calculated in vivo halfꢀlife correlates with duration of action
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Within the set of molecules evaluated no direct correlations were seen between membrane affinity and
occupancy. Furthermore a close link was observed between PK and PD across the compound set. This
link suggests that, for these molecules at least, there is little role for variations in binding kinetics
(Kon/Koff), in determining duration of action. Variations in binding kinetics would be expected to manifest
themselves in a disconnect between PK and PD.
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Using column methods it was possible to estimate a rat volume of distribution based on Rat HSA binding
and CHIꢀIAM measurements20 using this data and measurement of CLint from rat liver microsomes it was
possible to estimate an in vivo halfꢀlife for the compound set. This estimated number correlated well with
duration of action (p = 0.003) (Figure 10) despite being essentially an estimate of systemic clearance,
which did not take into account the effects of secondary metabolism, renal or biliary clearance.
Interestingly the compounds with the best duration of action (e.g. 22 and 25) were outliers in this analysis
being the compounds with the highest predicted in vivo halfꢀlife. Purely by use of in vitro methods a
prediction of duration of action for this compound class can be made. A possible interpretation of this
empirical observation is that molecules with high predicted microsomal clearance are also metabolised in
the lung tissue itself, leading to a shorter duration of action. Measurements of lung microsomal stability
were not carried out for compounds within this series. Interestingly although systemic halfꢀlife was
predicted to be very long for 25, the molecule was primarily cleared by renal excretion of parent and had
an in vivo halfꢀlife in rat of 14±3 h. This had the effect of reducing systemic exposure without
compromising lung levels.
The comparison of the occupancy seen with 25 when dosed by the it and iv routes gives a clear indication
of the enhanced therapeutic index obtained by the it delivery route, which is a consequence of depoting in
the lung tissue as part of the absorption from the airways. The enhanced occupancy seen makes it clear
that the depoted compound is also available at the site of action. For a given systemic exposure much
more occupancy is achieved by the it route (Figure 11). Compound 25 showed high bioavailability after it
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dosing (F= 90%) but a comparison of lung levels by the it and iv routes shows that the tissue distribution
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has been altered with a much higher lung: plasma ratio enabling enhanced lung efficacy to be achieved.
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Figure 11 Comparison of Occupancy after it and iv dosing
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To confirm that the occupancy of the PDGFR kinase observed translated into inhibition of downstream
signaling, the efficacy of 25 was assessed in a the monocrotaline (MCT) model of PAH. Pulmonary artery
smooth muscle proliferation was measured in rats pretreated with monocrotaline for two weeks.21 25 was
administered twice daily by the it route for seven days in a therapeutic mode (i.e. Pathology was allowed
to develop before administration of the test compound). Oral administration of 1 was used as a positive
control in the study, since it has shown efficacy in human clinical trials.
Compound 25 showed antiꢀremodeling efficacy against MCT induced Smooth muscle cell proliferation in
the pulmonary vasculature (Figure 12). The minimum efficacious dose of 25 was 0.03 mg/kg (twice
daily), which significantly reduces the percent of fully muscularized vessels to 14.3 (SEM = 1.6%)
compared to 34.6 (SEM = 3.1%) in the MCT vehicle control group (A). Imatinib 1 significantly reduced
the muscularization of small vessels at 100 mg/kg (A). However in a separate experiment, no efficacy was
observed when administering 1 at 25 mg/kg, a dose which provides equivalent exposure to that achieved
in patients receiving a 400 mg/day dose of 1 as part of the PAH Phase III clinical trial(B)6. This
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comparative data suggest that clinical efficacy may be observed at drug exposures which were well below
those required for efficacy in the monocrotaline model.
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Figure 12 A) Efficacy of 25 (i.t.) in the rat Monocrotaline model of pulmonary hypertension in
comparison to 1 (p.o), expressed as a percentage of fully muscularised small blood vessels within the lung
upon histological evaluation B) Efficacy of a clinically relevant dose of 1 (p.o) in the rat Monocrotaline
model of pulmonary hypertension expressed as a percentage of fully muscularised small blood vessels
within the lung upon histological evaluation
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A
B
By using lower doses and it administration of compound the systemic burden in terms of blood levels is
significantly reduced compared to oral administration of 1 (Table 5).
Table 5 Systemic exposure of test compound in the MCT model
Compound
Dose (mg/kg)
Blood Conc. (ꢀM)
0.21 ± 0.081
0.75 ± 0.14
0.01
0.03
0.1
25
25
25
1
1.90 ± 0.31
100
305.5 ± 268.5
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A Stable crystalline saccharin salt of 25 was identified as suitable for micronisation and Inhaled delivery
as an aerosol. The compound was then administered to rats at 0.79 mg/kg of as a micronized dry
powder22, over 1 hour and showed occupancy consistent with that seen after it dosing given the expected
delivery efficiency of 10% (Figure 13).
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Figure 13 Occupancy seen after inhaled dosing of 25 saccharinate salt
Compound 25 showed good selectivity in a receptor panel of 85 assays at Ricerca with no significant
activity at 10 ꢀM, confirming an excellent selectivity observed in an in house screening panel of 45
GPCRs and Ion channels. A selectivity screen of 451 kinases at DiscoverX revealed 31 interactions a
selectivity score S(35) = 0.08. These interactions were as expected in the PDGFR/KIT and SRC families
of receptor tyrosine kinases and pleasingly showed a very similar pattern of selectivity to that seen with
1.23 (Figure 14).
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Figure 14 Kinome tree plot for 25
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CONCLUSION
We have established that efficacy in the whole lung can be achieved, whilst maintaining an enhanced
therapeutic index over systemic delivery. Within our series of soluble, basic molecules the desired
properties for duration of action have been highlighted. Low permeability has been shown to be important
for this series of molecules, and the beneficial effect of high pKa has also been demonstrated. The effect
of a highly basic group has been noted before in inhaled drug design7 as has the presence of a second less
basic centre7 and this study reconfirms this finding starting from an unbiased analysis of in vitro: in vivo
correlations. The size of this effect has been quantified for this series of molecules. The effect of nonꢀ
specific binding to tissues is a clear factor, and the correlation between cLogP and Occ50 is perhaps
unsurprising given that basic molecules already have an affinity for tissues. Combining basicity with high
lipophilicity is likely to lead to extensive nonꢀspecific binding to tissue and to limit the percentage of free
drug available to achieve occupancy. The wider applicability of these findings to inhaled drug design in
general would be supported by direct measurement of PKꢀPD at other targets and in more diverse
chemical space. Corroboration of these findings from the literature does exist but the importance of
directly measuring pharmacodynamics as well as pharmacokinetics when assessing therapeutic index and
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duration of action has been emphasized by problems encountered within other inhaled drug discovery
projects.7
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Indeed within this series, high drug levels in the lung have been observed for multiple molecules without
correspondingly high target occupancy (e.g. 16), by measuring the PD in the same animal at the same
time point, it can be established that drug present is binding to the target.
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By using solutions in our it dosing experiments, we have eliminated the effects of solid form and
dissolution rate from our measurements of duration of action, and have thereby assessed the intrinsic
ability of the molecules to depot in lung tissue as part of the first pass absorption. By using a soluble
molecule when dosing it, we essentially looked at the worst case scenario for duration of action. The
inhaled dosing of a solid form will generally allow for increased duration based on dissolution rate
factors. The Inhalation PK and PD of the lead molecule 25 as a saccharin salt validate our assumption that
the intrinsic depoting properties and duration of action would not be adversely affected by dosing route
and solid form factors.
This approach has allowed us to identify 25, a potent PDGFR antagonist (10 fold more potent than 1 in
cell based proliferation assays) with a selectivity profile similar to that of 1, which shows excellent
duration of action after a single low dose in our PK/PD model.
Comparison of the same dose delivered by the it route and by the iv route demonstrates clearly the
significant perturbation of the drug distribution to lung obtained by changing dosing route. This is
coupled with a clear demonstration that the extra drug levels in the lung lead to enhanced occupancy and
duration of action (80 % at 24h after it dose vs 10 % at 24h after iv dose). This finding predicts a lower
absolute dose requirement in the clinic since a far greater proportion of the dose delivered is reaching the
target tissue and is delivering occupancy.
Importantly we have shown that the occupancy obtained translates into efficacy in the MCT model of
PAH in rat and that the Minimum effective dose, 0.03 mg/kg (it /bid.) is significantly reduced compared
to the dose of 1 which is effective in the model 100 mg/kg (p.o. /q.d.).
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The IMPRESS trial with oral Imatinib successfully demonstrated efficacy in PAH but was hampered by
side effect driven patient withdrawals. As an inhaled compound 25 has the potential to deliver the
efficacy in patients with much lower systemic exposure to drug and to allow greater inhibition of the
target in the lung without similar tolerability issues. Further characterization of 25 will be reported in due
course.
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EXPERIMENTAL
General Conditions:
Compounds were confirmed to be of ≥95% purity or greater by LCMS (methods described below)
Mass spectra were run on LCMS systems using electrospray ionization, using Waters Acquity UHPLC
with SQD Mass Spectrometer. [M+H]+ refers to monoꢀisotopic molecular weights. Purity of tested
compounds was determined by LCMS 5 minute run (Method 1: ACQUITY UHPLC BEH C18, 130Å, 1.7
µm, 2.1 mm X 50 mm, Temp 50oC; flow rate 1.0 mL/min, Eluents A H2O containing 5 mM ammonium
hydroxide; B Acetonitrile containing 5 mM ammonium hydroxide; Gradient 0.06 min 2% B, 2%ꢀ98% B
in 4.40 min, 0.35 min 98% B). Intermediates were characterized using a 2 minute LCMS run (Method 2:
Column Waters BEH C18 50x2.1 mm, 1.7 µm, Temp 50oC; flow rate 0.8 mL/min, Eluents A H2O; B
MeOH containing 0.1 % TFA; Gradient 0.2 min 5% B, 5%ꢀ95% B in 1.30 min, 0.25 min 95% B).
Structural confirmation was by 1H NMR and 13C NMR in conjunction with High Resolution Mass
Spectrometry (HRMS). NMR spectra were run on Bruker AVANCE 400 NMR spectrometers using
ICONꢀNMR. Spectra were measured at 298K and were referenced using the solvent peak. HRMS was
measured on an Acquity G2 Xevo QTof machine. Melting points were determined by differential
scanning calorimetry.
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N-(2-fluoro-5-((2-(4-methylpiperazin-1-yl)benzyl)carbamoyl)phenyl)imidazo[1, 2-a]pyridine-3-
carboxamide 2; Methyl 3ꢀaminoꢀ4ꢀfluorobenzote (350 mg, 2 mmol) and imidazo[1, 2ꢀa]pyridineꢀ3ꢀ
carbonyl chloride (411 mg, 2.2 mmol) were combined in pyridine (7.5 mL) and stirred for 16 h. The
reaction mixture was diluted with chloroform and washed with water (x 2), and then the organic layer was
washed with 1M HCl, sat. aq. sodium bicarbonate, and then brine, the organic layer was then dried
(MgSO4) and evaporated. The residue was triturated with ethyl acetate and 5b (448 mg, 1.3 mmol, 65%
yield) was obtained as a white solid. 1H NMR, (D6ꢀDMSO, 400 mHz) δ 13.14 (1H, br s), 10.31 (1H, s),
9.48 (1H, d), 8.64 (1H, s), 8.28 (1H, m), 7.87 (1H, m), 7.80 (1H, d), 7.57 (1H, m), 7.46 (1H, m), 7.23 (1H,
m).
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5b (100 mg, 0.319 mmol), (2ꢀ(4ꢀmethylpiperazinꢀ1ꢀyl)phenyl)methanamine (197 mg, 0.958 mmol), and
1,5,7ꢀTriazabicyclo[4.4.0]decꢀ5ꢀene (TBD) (133 mg, 0.958 mmol) were combined in THF (3 mL), the
mixture was then heated to 70°C for 16h. The mixture was evaporated and the residue then purified by
column chromatography on silica eluting with 0ꢀ15% Methanol in DCM. The desired fractions were
collected , evaporated and precipitated with ethyl acetate to give as a white solid 2 (20mg, 0.319 mmol,
24% yield). mp = 71.2oC; LCMS Method 1; Rt. 1.85 min; [MH]+ m/z 487.2; purity 95% 1H NMR, (D6ꢀ
DMSO, 400 mHz) δ 10.30 (1H, s), 9.47 (1H, d), 9.01 (1H, t), 8.62 (1H, s), 8.21 (1H, m), 7.86 (1H, m),
7.80 (1H, d), 7.57 (1H, m), 7.50 (2H, m), 7.18 (5H, m), 4.61 (2H, d), 3.18 (3H, s), 3.01 (8H, m). 13C
NMR(CDCl3, 100 MHz) 166.0, 158.4, 155.4, 153.0, 150.4, 148.7, 137.1, 132.9, 131.4, 130.1, 129.1,
128.0, 127.9, 126.3, 125.5, 124.2, 121.4, 118.0, 117.7, 115.9, 114.6, 55.0, 55.0, 50.9, 50.9, 44.7, 41.2;
HRMS (ESI): m/z (MH+) 487.2225. Calcd. 486.2257.
N-(5-(benzylcarbamoyl)-2-methylphenyl) pyrazolo [1, 5-a] pyridine-3-carboxamide 3; 3ꢀAminoꢀ4ꢀ
methylbenzoic acid 4a (423 mg, 2.77 mmol) was dissolved in DCM, Triethylamine (385 ꢀl, 2.77 mmol)
and pyrazolo [1, 5ꢀa] pyridineꢀ3ꢀcarbonyl chloride (500 mg, 2.77 mmol) were added. The reaction
mixture was stirred for 30 min at RT then was poured into a separation funnel containing water. The
organic layer was collected and the aqueous layer was extracted with DCM (x3). The combined organic
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Page 29 of 42
Journal of Medicinal Chemistry
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layers were dried (MgSO4), filtered and evaporated to dryness. The product was recrystallized in MeOH
and after filtration 4ꢀmethylꢀ3ꢀ(pyrazolo [1, 5ꢀa] pyridineꢀ3ꢀcarboxamido) benzoic acid 5a was obtained
(716 mg, 2.4 mmol 87% yield) of pure compound. LCMS Method 1; Rt. 0.54 min; [MH]+ m/z 296.1;
purity 99% 1H NMR, (D6ꢀDMSO, 400 mHz) δ 12.82 (1H, s), 9.69 (1H, s), 8.84 (1H, d), 8.78 (1H, s), 8.22
(1H, d), 7.99 (1H, d), 7.72 (1H, dd), 7.53 (1H, dd), 7.40 (1H, d), 7.21 (1H, dd), 2.32 (3H, s).
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5a (42 mg, 0.14 mmol) was dissolved in DMF (1.0 mL) and treated with HATU (58 mg, 0.152 mmol)
and Triethylamine (59 ꢀL 0.42 mmol). To this solution was added benzyl amine (18 mg, 0.17 mmol). The
reaction mixture was stirred at room temperature overnight and then diluted with water. The aqueous was
washed with 0.5M LiCl, 0.5M citric acid and then brine. The organic layer was dried (MgSO4) and
concentrated in vacuo then purified by chromatography on silica eluting with 0ꢀ100% ethyl acetate in
heptane. 3 (22mg, 6 mmol, 38% yield) was obtained as a white solid. mp = 183oC ; LCMS Method 1; Rt.
1.83 min; [MH]+ m/z 385.2; purity 99% 1H NMR, (D6ꢀDMSO, 400 mHz) δ 9.74 (1H, s), 9.03 (1H, t),
8.82 (1H, d), 8.76 (1H, s), 8.22 (1H, d), 7.93 (1H, s), 7.87 (1H, m), 7.74 (1H, d), 7.52 (1H, m), 7.39 (1H,
d), 7.31 (3H, m), 7.23(1H, m), 7.14 (1H, t), 4.46 (2H, d), 2.31 (3H, s). 13C NMR (D6ꢀDMSO, 100 MHz)
164.6, 160.1, 140.7, 139.2, 138.7, 131.2, 129.2, 128.5, 127.1, 126.15, 126.12, 125.6, 124.6, 123.4, 117.6,
114.3, 112.9, 105.1, 41.4, 17.0; HRMS (ESI): m/z (MH+) 385.1653. Calcd. 385.1664.
methyl 5-amino-6-methylnicotinate 4c
To a suspension of 6ꢀmethylꢀ5ꢀnitroꢀ2ꢀoxoꢀ1, 2ꢀdihydropyridineꢀ3ꢀcarboxylic acid 8 (12.5 g, 63.1 mmol)
in chlorobenzene (210 mL) was added DMF (2.4 mL, 31.5 mmol) followed by POCl3 (23.5 mL, 252
mmol). The mixture was heated at 133 °C for 1 hr. After cooling to RT, the mixture was concentrated in
vacuo. The residue was cooled in an ice bath, treated with MeOH (200 mL, 4944 mmol) and stirred at RT
for 16h. The mixture was concentrated in vacuo and the residue was partitioned between water (300 mL)
and EtOAc (300 mL). The organics were dried (MgSO4) and concentrated in vacuo to afford 9 as a red
crystalline solid (11.8g, 41 mmol, 80% yield); LCMS Method 2; Rt 1.10 min; [MH]+ m/z 230.9.
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