3-Amido-4-anilinoquinolines as CSF-1R kinase inhibitors 2: Optimization of the PK profile

Article abstract of DOI:10.1016/j.bmcl.2008.12.044

The optimization of compounds from the 3-amido-4-anilinoquinolines series of CSF-1R kinase inhibitors is described. The series has excellent activity and kinase selectivity. Excellent physical properties and rodent PK profiles were achieved through the introduction of cyclic amines at the quinoline 6-position. Compounds with good activity in a mouse PD model measuring inhibition of pCSF-1R were identified.

Full text of DOI:10.1016/j.bmcl.2008.12.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 701–705
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Amido-4-anilinoquinolines as CSF-1R kinase inhibitors 2: Optimization
of the PK profile
*
David A. Scott , Kirsten J. Bell, Cheryl T. Campbell, Donald J. Cook, Les A. Dakin, David J. Del Valle, Lisa Drew,
Thomas W. Gero, Maureen M. Hattersley, Charles A. Omer, Boris Tyurin, Xiaolan Zheng
Cancer Research, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The optimization of compounds from the 3-amido-4-anilinoquinolines series of CSF-1R kinase inhibitors
is described. The series has excellent activity and kinase selectivity. Excellent physical properties and
rodent PK profiles were achieved through the introduction of cyclic amines at the quinoline 6-position.
Compounds with good activity in a mouse PD model measuring inhibition of pCSF-1R were identified.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 15 October 2008
Revised 5 December 2008
Accepted 9 December 2008
Available online 14 December 2008
Keywords:
CSF-1R
Kinase inhibitor
Quinoline
In the preceding paper, we reported 3-amido-4-anilinoquino-
lines as highly selective inhibitors of CSF-1R kinase, along with
our initial efforts to optimize the series.1 Examples from the 6,7-
dimethoxyquinoline series were very potent in our enzyme and
cell assays, but typically had poor physical properties and rat PK,
with short half-lives. Activity in our mouse PD model was achieved
with compound 1 (Fig. 1), but only at the high dose of 100mpk.
With the goal of optimizing the physical properties and PK profile
of the series, basic side chains were installed at the 6- and 7-posi-
tions of the quinoline scaffold, linked through oxygen and nitrogen
atoms. Compounds of this type (e.g., 2, Fig. 1) retained the good
enzyme and cellular potency of the dimethoxyquinoline leads,
and also possessed high aqueous solubility and low levels of plas-
ma protein binding. For these compounds, however, the in vivo
clearance in rats was extremely high, and oral exposure was min-
imal. The amidoquinolines displayed a remarkable kinase selectiv-
ity profile, however, with examples found to have activity against
almost no kinases other than CSF-1R. This observation encouraged
our continued efforts to optimize the series.
In the 7-MeO series, the dichloroanilino compounds, including
the very potent 2,3-Cl analogue 5, were highly cleared, but still
showed an improvement over compounds in this series with basic
side chains. Encouragingly, the 2,4-F example 6 had excellent rat
PK, including good oral bioavailability, although it had an IC50 of
only 1 l
M in our cell proliferation assay.4 From a small number
of matched pairs, and influenced by earlier data on related com-
pounds,1 we felt that potency was better with the piperazine sub-
stituent at the 6-position rather than the 7-position, and focused
subsequent efforts on compounds of this type.
We speculated that oxidation of the electron-rich quinoline ring
might contribute to the high rate of clearance, and compounds were
prepared without the 7-methoxy group, and with a 7-F substituent
in its place. Compounds without the 7-MeO substituent (7-10)
showed reduced activity in both enzyme and cell. The two 7-F com-
pounds (11, 12) did in fact show moderate in vivo clearance, but
were also less potent. The 2,4-F example 12 was only weakly active
The introduction of a basic side chain has been used to improve
the physical properties and PK profiles of other series of kinase
inhibitors, to deliver marketed drugs such as Gleevec2 and Iressa.3
However, this structural feature proved to be a metabolic liability
for the amidoquinoline scaffold. We next installed cyclic amines
such as methylpiperazine directly at the 6- and 7-positions. Biolog-
ical and pharmacokinetic data for these compounds are given in
Table 1.
Cl
F
F
F
NH
O
N
NH
N
O
MeO
MeO
N
O
NH2
NH2
N
MeO
2
1
* Corresponding author. Tel.: +1 781 839 4343.
Figure 1. CSF-1R inhibitors from the amidoquinoline series.
E-mail address: david.scott@astrazeneca.com (D.A. Scott).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.044

702
D. A. Scott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 701–705
Table 1
Potency, physical property data and rat PK upon iv (3 mpk) and po (10 mpk) dosing for 1 and 3–17
R1
NH
N
O
R2
NH2
R3
Compound
R1
R2
R3
IC50
(l
M)
Cell (l
M)
Sol (
lM)
PPB (% free)
F (%)
Cl (ml/min/kg)
Vss (L/kg)
T1/2 (h)
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2,4-F
MeO
MeO
MeO
MeO
MeO
MeO
H
H
H
0.025
0.003
0.002
<0.003
0.006
0.129
0.059
0.008
0.110
0.003
0.054
0.024
0.023
0.011
<0.003
0.006
0.40
0.64
0.38
0.06
1.0
8.9
2.7
0.71
3.9
0.44
4.2
0.55
0.17
0.72
0.06
0.23
54
5.6
3.2
3.6
4.5
25
1.8
5.8
4.6
26
100
ND
ND
110
90
23
72
50
76
15
0.9
8.7
3.8
6.8
2.3
0.6
1.7
1.2
2.5
1.8
2,4-Cl
3,4-Cl
2,3-Cl
2,4-F
2,4-Cl
3,4-Cl
2,3-Cl
2,4-F
2,3-Cl
2,4-F
3,4-Cl
2,3-Cl
2,4-F
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
MeO
160
ND
810
>1000
180
720
44
970
8
71
ND
21
ND
13
190
45
23
5.9
7.6
1.1
1.4
0.7
1.9
0.8
1.1
H
F
F
4.4
24
20
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
EtO
ND
ND
ND
ND
2.0
11
MeO
MeO
220
>1000
50
2,3-Cl
2,4-F
N-Methylpiperazine
N-Methylpiperazine
100
100
57
12
3.5
2.5
0.9
2.1
EtO
290
in our cell assay (4.2
level of potency we felt was required (0.44
significantly less active than the corresponding 7-MeO compound 5.
We had observed previously improved potency for 6-amino-
quinolines when 7-MeO was replaced by 7-EtO. The 2,4-F analogue
17 achieved both good in vitro activity, 0.23 lM in the cell, and
excellent in vivo PK. The 2,3-Cl compound 16 had higher clearance
in rats than the 2,4-F example, but was very potent in the cell assay
(0.06 lM).
l
M). The 2,3-Cl example 11 was closer to the
Compounds were screened for their activity against the hERG
lM in the cell), but still
channel,5 with the 2,3-Cl compounds more active here than the 2,4-
F compounds. The homopiperazine examples (20, 21, 28, 32) showed
increased hERG activity relative to the corresponding piperazines.
The cell data for the 6,7-dimethoxyquinoline compounds1
showed that other anilines were also tolerated. In addition to
determining potency, the choice of aniline also influenced the
overall physical properties and PK. For example, compounds with
a 2,4-substituted aniline typically had the best PK profiles. To
explore further the relative importance of in vitro potency,
physical properties (especially protein binding) and PK on in vivo
activity, the aniline substituent was re-evaluated with the 6-meth-
ylpiperazine, 7-ethoxy scaffold (33–38, Table 2).
With a combination of good cell potency and encouraging PK,
compounds 16 and 17 represented excellent advanced leads for
further optimization of the series. We next explored a range of cyc-
lic amines at the 6-position, with the goal of identifying com-
pounds with the best overall balance of potency and PK, as
determined by our mouse PD model. Sets of compounds were pre-
pared with both 2,3-Cl aniline 18–25 (best in vitro potency) and
2,4-F aniline 26–32 (best physical properties and good PK). Data
for these compounds is shown in Table 2.
To assess the in vivo CSF-1R activity of the amidoquinolines,
compounds were dosed orally in a mouse pharmacodynamic
(PD) model. 3T3 cells were engineered to express human mutant
full length CSF-1R (301–969) (3T3/CSF-1RMT) in which the kinase
activity was constitutively on. Female nude mice were implanted
with 5 Â 106 3T3/CSF-1RMT cells subcutaneously and grown
in vivo until tumors were >250 mm3 in size. Tumors were analyzed
for pCSF-1R levels by ELISA 2 and 6 h after dosing, and blood plas-
ma samples were assessed for drug concentrations. Earlier com-
pounds had been dosed at 50 or 100 mpk, but the potency and
other properties of these amidoquinolines allowed us to drop the
screening dose to 25 mpk. Compounds with 2,3-Cl aniline were
typically ꢀ10-fold more potent in the cell assay than the 2,4-F ani-
line examples, but had higher levels of plasma protein binding, and
higher rat in vivo clearance. The choice of amine at the 6-position
had relatively little effect on cell potency, but a significant impact
on physical properties and PK. Basic groups here typically resulted
in compounds with a superior profile to those with neutral substit-
uents such as morpholine (29), or the piperazine amides (24, 25).
Examples with both 2,3-Cl and 2,4-F anilines showed good PD
activity out to 6 h.
Enhanced in vitro potency had been observed upon moving
from 7-MeO to 7-EtO, and a set of 7-iPrO examples (39–42) was
also prepared, to see if this trend would continue. While tolerated
in terms of potency, there was no further improvement to justify
the increase in lipophilicity. Introducing a 7-methoxyethoxy group
(43–47), however, gave compounds that retained the potency of
the 7-EtO analogues, but with higher aqueous solubility and
reduced plasma protein binding. Several examples from Table 2,
including the 7-methoxyethoxy compounds, showed excellent
activity in the PD model at both 2 and 6 h.
Compounds in Tables 1 and 2 were prepared as shown in Scheme
1. For the 7-MeO (3–6) and 7-EtO compounds (16–17 and 33–38)
condensation of the appropriate 4-bromoaniline with diethyleth-
oxymethylene malonate followed by a one-pot cyclization/chlorina-
tion gave the chloroquinoline esters. For the 7-F quinolines (11–12),
it was necessary to perform the cyclization step at 250 °C in diphenyl
ether, followed by a separate chlorination step. Aniline additions
were typically straightforward, but required higher temperatures
for the less nucleophilic anilines such as 2,3-Cl. The methylpipera-
zine was introduced under standard Buchwald–Hartwig condi-
tions,6 and final compounds were prepared by conversion of the
quinoline ester to the amide by treatment with formamide and then
methoxide.7 6-Methoxy, 7-piperazine compounds (13–15) were
prepared in a similar fashion from 3-bromo-4-methoxyaniline.8 An
alternative route was used to prepare compounds with either no
substituent (7–10), isopropoxy (39–42) or methoxyethoxy (43–47)

Table 2
Cell potency, physical property data, rat PK upon iv (3 mpk) and po (10 mpk) dosing, mouse PD and hERG activity for 16–47
R1
NH
O
R2
R3
NH2
N
Compound
R1
R2
R3
Cell (
lM)
Sol (lM)
PPB (% free)
F (%)
Cl (ml/min/kg)
Vss (L/kg)
T1/2(h)
% Inhibition of pCSF-1R at 2 and 6 h
hERG (lM)
16
18
19
20
21
22
23
24
25
17
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,3-Cl
2,4-F
2,4-F
2,4-F
2,4-F
2,4-F
2,4-F
2,4-F
2,4-F
3-Cl, 4-F
3,4-Cl
2-F, 3-Cl
2-F, 4-Me
2-F, 5-Me
2,5-F
2,3-Cl
2,4-F
2-F, 3-Cl
3,4-Cl
2,3-Cl
2,4-F
N-Methylpiperazine
N-Ethylpiperazine
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
EtO
0.06
0.02
0.02
0.05
0.02
0.03
0.03
0.07
0.09
0.23
0.17
0.17
0.26
0.48
0.31
0.66
0.27
0.13
0.06
0.11
0.05
0.09
0.11
0.05
0.17
0.21
0.12
0.01
0.24
0.10
0.14
0.17
50
11
250
130
420
67
13
2
29
290
320
2.0
1.9
1.5
1.9
4.9
4.8
1.7
9.6
16
100
19
66
57
86
120
63
3.5
6.8
11
0.9
1.2
1.2
8.6
75, 45
95, 75
85, 65
90, 95
90, 75
8.4
3.2
7.6
4.0
3.1
8.1
—
8.2
—
23
19
—
5.9
>30
17
20
6.9
6.1
4.3
8.4
15
13
26
4.9
28
N-Isopropylpiperazine
N-Methylhomopiperazine
N-Isopropylhomopiperazine
N-Hydroxyethylpiperazine
N-Methoxyethylpiperazine
N-Acetylpiperazine
N-Hydroxyacetylpiperazine
N-Methylpiperazine
N-Ethylpiperazine
100
40
11
6
100
46
62
72
12
19
2.3
2.2
2.5
3.6
0.65
0.66
2.1
65, 30
90, 65
11
4.6
14
2.3
N-Isopropylpiperazine
N-Methylhomopiperazine
Morpholine
>1000
530
64
70, 50
90, 55
31
16
25
32
36
16
1.1
6.6
0.6
8.4
8.9
1.4
2.2
3.4
0.8
7.1
6.0
6.0
7.3
1.4
1.7
4.9
1.0
10
4-Hydroxypiperidine
N-Cyclopropylpiperazine
N-Cyclopropylhomopiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
N-Methylpiperazine
2
150
100
ND
3
10
54
66
71
200
52
790
250
380
720
910
63
34
21
0.6
0.9
70, 10
EtO
EtO
EtO
EtO
EtO
EtO
55
42
54
46
88
33
53
47
24
10
28
28
9.3
3.7
1.9
1.8
5.3
2.9
2.2
1.2
1.1
2.3
2.3
1.3
70, 55
80, 40
100, 90
85, 60
90, 80
75, 45
70, 35
100, 50
EtO
iPrO
iPrO
40
7
1.4
2.3
iPrO
iPrO
6.8
7.6
>30
22
>30
21
MeO(CH2)2O
MeO(CH2)2O
MeO(CH2)2O
MeO(CH2)2O
MeO(CH2)2O
32
100
290
68
21
18
0.83
3.8
100, 80
100, 70
80, 75
100, 80
85, 70
46
39
16
16
2-F, 3-Cl
2-F, 4-Me
2-F, 5-Me
100
45
33
60
8.6
4.7
3.4
4.2
ND

704
D. A. Scott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 701–705
R1
Br
Cl
EtO2C
Br
Br
R3
NH
CO2Et
(b) R3 = MeO, EtO
or (c) R3 = F
CO2Et
Br
R3
(d)
(a)
CO2Et
R3
NH2
N
H
N
N
R3
(e)
R1
N
N
N
N
Cl
N
N
NH
EtO2C
N
CO2Et
N
N
CO2Et
(b) R3 = iPrO or
CO2Et
(a)
(d)
(c) R3 = H, MeO(CH2)2O
R3
R3
R3
N
H
NH2
N
R3
(f)
(i)
R1
N
N
Cl
Cl
(g)
(h)
N
NH
N
N
CONH2
R3
NO2
NO2
HO
NO2
R3
R3 = iPrO, MeO(CH2)2O
R3
Scheme 1. Preparation of examples 3–17 and 33–47. Reagents and conditions: (a) diethylethoxymethylene malonate, CH3CN, rt; (b) POCl3, Tol, 100 °C; (c) Ph2O, 250 °C; then
POCl3, 105 °C; (d) aniline, cat. AcOH, EtOH, 80 °C or DMF, 100 °C; (e) N-methylpiperazine, Pd2(dba)3, BINAP, Cs2CO3, Tol, 100 °C; (f) HCONH2, DMF, NaOMe, 100 °C; (g)
i
MeO(CH2)2Br, or PrI, DMF, K2CO3, 40 °C; (h) N-methylpiperazine, DMF, 130 °C; (i) 10% Pd/C, H2 (50 psi), MeOH.
R1
Br
R1
R2
R1
NH
N
NH
N
NH
N
R2
CO2Et
CO2Et
CONH2
(a)
(b)
EtO
EtO
EtO
R2 = Boc
homopiperazine
R2 = Boc
piperazine
(b), (c)
(b), (c)
(d)
(h)
22
21
32
R1
R1
N
HN
NH
N
NH
HN
(e)
CONH2
N
CONH2
24
25
(g)
(g)
(f)
EtO
EtO
N
31
Scheme 2. Preparation of examples 16–32. Reagents and conditions: (a) amine, Pd2(dba)3, BINAP, Cs2CO3, Tol, 100 °C; (b) HCONH2, DMF, NaOMe, 100 °C; (c) TFA/DCM; (d)
HCOCH2OH, Tol/MeOH, , then NaBH(OAc)3, AcOH, THF,
; (e) Ac2O, Et3N, DCM; (f) HOCH2CO2H, HATU, iPr2NEt, DMF; (g) (CH2)2C(OEt)OSiMe3, AcOH, 4 Å mol. Sieves,
NaBH3CN, MeOH, ; (h) (CH3)2CO, NaBH(OAc)3, AcOH, THF,
D
D
D
D.
at the 7-position, in which the amine was installed prior to the cycli-
zation step. Quinolines unsubstituted at the 7-position were pre-
pared from commercially available 4-(4-methylpiperazino)aniline.
The other alkoxy substituents were derived from 2-chloro-5-nitro-
phenol, via alkylation, nucleophilic substitution of the chloronitro-
benzene with N-methylpiperazine, and reduction of the nitro
group to give the required aniline.
As with example 1,1 compound 17 was found to have an
extremely selective profile when evaluated in a panel of ꢀ85
kinases (Table 3).10 Apart from moderate activity versus ARK5,
the compound was essentially inactive against all the other
kinases screened, including the other class III RTKs: c-Kit, Flt3
and PDGFR
a.
Where the amine substituent was commercially available,
compoundsinTable 2 wereprepared fromtheappropriate6-bromo-
quinolineester by standard Buchwald–Hartwig couplingconditions,
followed by amidation of the ester. Where the amine substituents
were either unavailable, or unsuitable for the Pd-coupling step, com-
pounds were prepared by derivatization of the unsubstituted piper-
azine or homopiperazine amides, accessed in turn from coupling of
the Boc protected amines, as shown in Scheme 2. A reductive amina-
tion reaction with [(1-ethoxycyclopropyl)oxy]trimethylsilane9 gave
the cyclopropyl derivatives 31 and 32.
Table 3
Kinase selectivity of 17 at 1 lM
Kinase
% Inhibition
CSF-1R
ARK5
ALK
Rsk1
80 kinases
96
51
23
21
<15

D. A. Scott et al. / Bioorg. Med. Chem. Lett. 19 (2009) 701–705
705
In conclusion, 3-amido-4-anilinoquinolines are potent inhibi-
References and notes
tors of CSF-1R, with excellent kinase selectivity. The introduc-
tion of cyclic amines such as N-methylpiperazine at the
6-position gives compounds with attractive physical properties
and PK profiles. Several of these compounds show good activity
in our mouse PD model. The amidoquinoline series may there-
fore be able to test the hypothesis that a CSF-1R inhibitor will
impact tumor progression through an effect on tumor-associ-
ated macrophages. Examples have been selected for further
biological profiling, the results of which will be published in
due course.
1. Scott, D. A.; Balliet, C. L.; Cook, D. J.; Davies, A. M.; Gero, T. W.; Omer, C. A.;
Poondru, S.; Theoclitou, M.-E.; Tyurin, B.; Zinda, M. J. Bioorg. Med. Chem. Lett.
2008, preceding paper.
2. Capdeville, R.; Buchdunger, E.; Zimmermann, J.; Matter, A. Nat. Rev. Drug
Discov. 2002, 1, 493.
3. Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M. P.;
Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards, L.
Bioorg. Med. Chem. Lett. 2001, 11, 1911.
4. Scott, D. A.; Aquila, B. M.; Bebernitz, G. A.; Cook, D. J.; Dakin, L. A.; Deegan, T. L.;
Hattersley, M. M.; Ioannidis, S.; Lyne, P. D.; Omer, C. A.; Ye, M.; Zheng, X. Bioorg.
Med. Chem. Lett. 2008, 18, 4794.
5. hERG activity was assessed using the Ionworks assay.
6. (a) Wolfe, J. P.; Wagaw, S.; Marcoux, J.-F.; Buchwald, S. L. Acc. Chem. Res. 1998,
31, 805; (b) Hartwig, J. F. Angew. Chem., Int. Ed. 1998, 37, 2046.
7. Jagdmann, G. E.; Munson, H. R.; Gero, T. W. Synth. Commun. 1990, 20, 1203.
8. Liu, Y.-Y.; Minich, M. J. Labelled Compd. Radiopharm. 1981, 18, 791.
9. Gillaspy, M. L.; Lefker, B. A.; Hada, W. A.; Hoover, D. J. Tetrahedron Lett. 1995, 41,
7399.
Acknowledgments
The authors thank our colleagues Minwei Ye, Galina Repik and
Tony Cheung for biological data, Dominique Custeau for PK studies,
and Michael Block and Paul Lyne for assistance with the manuscript.
10. KinaseProfiler Department, Millipore UK Limited, Gemini Crescent, Dundee
Technology Park, Dundee DD2 1SW, UK.