Synthesis of novel anilinoquinolines as c-fms inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.09.009

A novel series of potent substituted anilinoquinolines were discovered as c-fms inhibitors. The potency could be manipulated upon modification of the C4 aniline and C7 aryl functionality. Pharmacokinetic analysis identified a metabolically stable analog suitable for further investigative work.

Full text of DOI:10.1016/j.bmcl.2007.09.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6257–6260
Synthesis of novel anilinoquinolines as c-fms inhibitors
*
Terrence L. Smalley, Jr., Stanley D. Chamberlain, Wendy Y. Mills, David L. Musso,
Sab A. Randhawa, John A. Ray, Vicente Samano and Lloyd Frick
GlaxoSmithKline, Inc., Five Moore Drive, Research Triangle Park, NC 27709, USA
Received 15 August 2007; revised 27 August 2007; accepted 4 September 2007
Available online 7 September 2007
Abstract—A novel series of potent substituted anilinoquinolines were discovered as c-fms inhibitors. The potency could be manip-
ulated upon modification of the C4 aniline and C7 aryl functionality. Pharmacokinetic analysis identified a metabolically stable ana-
log suitable for further investigative work.
Ó 2007 Elsevier Ltd. All rights reserved.
The role of inflammation in the progression of human
1
H
diseases is well documented. Osteoarthritis in particular
has been shown to be caused in part by the over-produc-
tion of macrophages in the synovial fluid of joints, lead-
N
N
O
IC50= 16 nM
IC50 (MCSF)= 800 nM
IC50 (NSO)= 12 μM
NH2
2
ing to inflammation, cartilage loss, and pain. The
discovery of progenitors of inflammation has sparked
a great deal of interest from the scientific community
in regards to potential treatments for diseases. Macro-
phage colony stimulating factor (MCSF) has been impli-
cated as playing a role in several diseases, including
N
1
Figure 1. Structure and assay data for 1.
3
inflammation. Most notable is the role of MCSF in
model (carrageenan challenge) showed that 1 produced a
5% inhibition of inflammation, as measured by paw
cancer, particularly angiogenesis. Due to its ability to
promote tumor growth and survival, research into
down-regulating MCSF has been an area of intense
interest. The receptor for MCSF activation, c-fms, is
5
a potential target for drug discovery efforts.
9
size, relative to controls when dosed at 40 mg/kg IV.
While these results were promising, we wanted to
increase the potency in the cell assay while maintaining
or improving the binding potency. We were also inter-
ested in improving the in vivo exposure of 1 by substitut-
ing the C4 aryl group to prevent metabolic degradation
arising from oxidative processes and by replacing the
C7 pyridine, which can potentially generate an undesir-
able N-oxide.
4
Our interest in c-fms inhibitors began with the identifica-
tion of compound 1 through a high throughput screening
effort of the GSK compound collection (Fig. 1). The
measured in vitro potency in the binding assay is excel-
6
lent, with an IC50 = 16 nM. In our cell assays the po-
tency was not quite as good, showing an IC50
=
00 nM against a cell line expressing MCSF and 14-fold
The synthesis of the anilinoquinolines is depicted in
7
8
Scheme 1. 3-Iodoaniline is treated with diethyl eth-
oxymethylenemalonate to afford enamine 2. Cyclization
selectivity when compared to a non-MCSF expressing
cell line (NSO), which was used as a measure of toxicity.
In addition, pharmacokinetic (PK) experiments indi-
cated that 1 was cleared rapidly from plasma (Cl =
occurred upon heating in phenyl ether and the product
3) was isolated by filtration upon cooling. Saponifica-
(
tion of the ester was followed by conversion to the 4-
chloro-7-iodoquinoline-3-carboxylic acid chloride with
SOCl . Treatment of the acid chloride with NH gave
3
9 mL/min/kg). In vivo tests in the rat paw inflammation
2
3
4. Nucleophilic displacement of the 4-chloro moiety
with substituted anilines was accomplished in refluxing
dioxane to provide compounds of type 5. Subsequent
Keywords: Kinase; Inhibitors; Inflammation.
*
Corresponding author. Tel.: +1 919 483 1054; fax: +1 919 315
430; e-mail: terry.l.smalley@gsk.com
0
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.009

6258
T. L. Smalley et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6257–6260
OH
Cl
N
O
EtO C
CO Et
2
2
CO Et
a
2
b
c,d
NH2
I
NH2
I
N
I
N
I
H
2
3
4
R
I
R
H
O
H
N
N
N
N
O
e
f
NH2
NH2
R'
5
6
Scheme 1. Reagents and conditions: (a) diethyl ethyoxymethylenemalonate, EtOH, 100%; (b) Ph
SOCl , reflux, then NH3(g), 67%; (e) substituted aniline, dioxane, reflux; (f) arylboronic acid, 2 M aq K
2
O, 250 °C, 90%; (c) NaOH, MeOH, reflux, 95%; (d)
CO , PdCl (PPh , PhCH , EtOH, reflux.
2
2
3
2
3
)
2
3
Suzuki coupling with substituted arylboronic acids gave
the desired compounds (6).
We identified two areas of diversity that would allow for
rapid analog synthesis, the C4 aniline and the C7 aryl
positions. Computational analysis of 1 bound to the ac-
tive site of c-fms suggested that the C4 aryl group resides
in a lipophilic pocket while the C7 group points toward
8
the protein/solvent interface (Fig. 2). As a result com-
pounds containing lipophilic residues were targeted as
potential C7 derivatives, while substituents containing
more hydrophilic residues were proposed as potential
C4 targets.
Several analogs differing at C4 were made and the re-
sults are shown in Table 1. In general, introduction of
lipophilic substituents at the C4 aryl group gave com-
pounds with excellent binding potency but reduced cell
potency (7c–e, 7n–p). We were excited to discover that
the 3,4-dimethyl analog (7f) exhibited excellent cell
potency and selectivity. The potent binding activity of
Figure 2. Stick model of 1 (in green) bound in the active site of c-fms.
a
Table 1. Structure–activity relationships of 1 with modification of the C4 aniline
Ar
H
N
O
NH2
N
N
7
Ar
IC50 (lM) IC50 MCSF (lM) IC50 NSO (lM) Ar
IC50 (lM) IC50 MCSF (lM) IC50 NSO (lM)
H (1)
0.016
0.063
0.10
0.80
0.80
0.80
10
12
16
3-Cl, 4-Me (7j)
3-Me, 4-Cl (7k)
3-F, 4-OMe (7l)
3-Cl, 4-OMe (7m)
0.040
0.16
0.040
0.032
0.032
0.10
1.0
0.16
2.0
> 32
32
32
4
4
4
4
4
3
3
3
3
-OMe (7a)
-F (7b)
>40
>50
10
0.32
0.20
>20
4.0
-SMe (7c)
-NMe2 (7d)
-i-PrO (7e)
0.13
0.063
0.63
>50
>32
>16
13
6.3
40
3
3-Cl, 4-OCF (7n)
>50
>50
>32
>20
>50
3-F, 4-OEt (7o)
3-F, 4-O-i-Pr (7p)
3-Isoxazolyl (7q)
,4-di-Me (7f) 0.080
,4-di-F (7g) 0.032
0.1
2.5
5.0
1.0
8.0
3.2
>50
0.80
1.30
40
-F, 4-Me (7h) 0.080
-Me, 4-F (7i) 0.050
3-Benzopyrazolyl (7r) 0.32
4-Benzopyrazolyl (7s) 0.16
>50
>32
a
All results are reported as an average of at least two separate assays.

T. L. Smalley et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6257–6260
6259
a
Table 2. Structure–activity relationships of 1 with modification of the C7 aryl group
H
N
N
O
NH2
R
8
R
IC50 (lM) IC50 MCSF (lM) IC50 NSO (lM)
R
IC50 (lM) IC50 MCSF (lM) IC50 NSO (lM)
H (8a)
0.25
10
32
6.3
25
16
50
32
25
8.0
25
20
50
4-NHSO Me (8l)
2
0.063
(8m) 0.10
2.0
4.0
10
10
4
4
4
4
4
-Me (8b)
-OMe (8c)
-F (8d)
0.050
0.025
0.032
0.13
4-C(O)-c-C
4-C(O)-c-C
3
H
H
5
5
9
(8n)
0.032
0.025
0.050
0.20
>20
5.0
>20
>20
8.0
8.0
3-CH
4-CH
2
SO
SO
2
Me (8o)
Me (8p)
-CF
3
(8e)
2
2
1.6
-Br (8f)
0.016
4-SO
2
Et (8q)
0.80
O
N
4
-CN (8g)
0.05
0.32
4.0
8.0
32
10
0.016
0.080
0.40
0.20
8.0
8.0
(
8r)
O
N
3-CN (8h)
O
(8s)
4
4
4
-NH
2
(8i)
(8j)
0.16
0.040
1.6
6.3
1.3
10
32
4.0
4-SO
4-SO
2
NH (8t)
Me (8u)
2
0.080
0.016
3.2
0.10
13
20
-NMe
2
2
-NHAc (8k) 0.080
a
All results are reported as an average of at least two separate assays.
the 3,4-difluoro analog 7g led to the synthesis of a series
of analogs having a combination of a halogen with a
methyl group (7h–j) showing good potency. Unfortu-
nately those compounds were significantly less potent
in the cell assay. A methoxy group at the 4-position
was also well tolerated in the series (7l, 7m), however
increasing the bulk to 4-ethoxy (7o) or 4-isopropoxy
in both the binding and cell assays and had acceptable
cell selectivity. Substitution at the meta-position of the
aryl ring provided compounds that were equipotent in
the binding assay but less potent in the cell assay. Based
Table 3. Optimization of C4 Anilines in C7 methylsulfonyl-containing
a
compounds
(
7p) led to a loss in potency. Heterocyclic replacements
of the benzene ring (7q–s) provided compounds with lit-
tle or no advantage over 1.
R
H
Upon optimizing the C4 position, we turned our atten-
tion to the C7 analogs. The results are summarized in
Table 2. Initially several substitutions were made with
small lipophilic groups (8a–f), which gave compounds
with reasonable binding potency, but limited cell
potency. Based on this data we proposed that lack of
compound solubility could be a key factor in cell
potency, and the synthesis of a series of compounds with
more hydrophilic residues at C7 was initiated. As such,
substitution of an amino group in the para position (8i)
provided some cell activity that was reduced upon meth-
ylation (8j). In addition, we saw a gradual increase in
cell potencies in compounds with increasing hydrophilic-
ity, culminating with the synthesis of the piperidine (8r)
and morpholine (8s) amides. Interestingly, the para-
substituted methyl sulfonyl analog (8u) was very active
N
N
O
NH2
S
O O
9-14
R
IC50
(lM)
IC50 MCSF
IC50 NSO
(lM)
(lM)
9
10
11
12
3,4-di-Me
3-F-4-Me
3,4-di-F
0.008
0.063
0.20
0.16
ND
0.10
3.2
10
>20
>20
10
2.0
4-F
3-Cl-4-OMe
3-F-4-OMe
0.40
0.10
1.0
1
1
3
4
13
20
0.10
a
All assays are reported as an average of at least two separate assays.

6260
T. L. Smalley et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6257–6260
on the data observed and accounting for any potential
metabolic liability of the amides, we selected the 4-meth-
ylsulfonyl substituent as the optimal C7 substituent.
Ann. N.Y. Acad. Sci. 2006, 1089, 472; (b) Suganami, T.;
Mieda, T.; Itoh, M.; Shimoda, Y.; Kamei, Y.; Ogawa, Y.
Biochem. Biophys. Res. Commun. 2007, 354, 45.
. Seitz, M.; Loetscher, P.; Fey, M. F.; Tobler, A. Br. J.
Rheumatol. 1994, 33, 613.
. (a) Kirma, N.; Hammes, L. S.; Liu, Y.-G.; Nair, H. B.;
Valente, P. T.; Kumar, S.; Flowers, L. C.; Tekmal, R. R.
Cancer Res. 2007, 67, 1918; (b) Dewar, A. L.; Zannetinno,
A. C. W.; Hughes, T. P.; Lyons, A. B. Cell Cycle 2005, 4,
851, and references cited therein.
2
3
With both positions, C4 and C7, independently opti-
mized we devised a strategy to maximize the cell potency
of our inhibitors by combining the most active substitu-
ents at both positions. The striking feature that the most
potent substituent at C7 was the 4-methylsulfonyl moi-
ety led us to retain that group and modify the C4 posi-
tion with substituents from Table 1. The results of this
exercise are summarized in Table 3. Of the C4 substitu-
ents tested, 9 was the most interesting as the binding po-
tency and cell potency were the greatest, with 100-fold
selectivity. The cell potencies of 10, 11, and 14 were
not enough to elicit any further interest in these com-
pounds. The binding potencies of 12 and 13 were lower
than that of 7, but the cell potencies and selectivities
were comparable. From these results, we chose to assess
4. (a) Illig, C. R.; Ballentine, S. K.; Chen, J.; Meegalla, S. K.;
Rudolph, M. J.; Wall, M. J.; Wilson, K. J.; Desjarlais, R.
L.; Manthey, C. L.; Flores, C.; Molloy, C. J. U.S. patent
#
2006189623, Chem. Abstr., 2006, 145, 271780; (b) Iman,
E. H. D.; Gallet, M.; Mentaverri, R.; Sevenet, N.; Brazier,
M.; Kamel, S. Eur. J. Pharmacol. 2006, 551, 27.
5
. The results described herein were previously presented at
the 230th ACS National Meeting. Smalley, T. L.; Mills, W.
Y.; Chamberlain, S. D.; Kuyper, L.; Randhawa, S. A.; Tay,
J. A.; Samano, V.; Frick, L. Abstract of Papers, 230th
National American Chemical Society meeting, MEDI-376.
9
the in vivo PK profile of compounds 9 and 12. When
6. Compounds were assayed in the primary binding assay
3
3
dosed in rats at 3 mg/kg IV, compound 9 showed a plas-
ma clearance of 51 mL/min/kg while 12 showed a clear-
ance of 4.5 mL/min/kg.
using activated c-fms protein and P-labeled ATP as a
substrate in 96-well plates. Inhibition was determined by
scintillation counting. The cell assays were conducted in
mouse myeloid M-NSF-60 cells containing 20 ng/mL of
mouse MCSF and a MCSF-independent mouse myeloid
NSO cell line. Activity was determined by the difference in
cell growth upon treatment with inhibitors between the cell
lines. For detailed descriptions of the assays, please see:
Conway, J. G.; McDonald, B.; Parham, J.; Keith, B.;
Rusnak, D. W.; Shaw, E.; Jansen, M.; Lin, P.; Payne, A.;
Crosby, R. M.; Johnson, J. H.; Frick, L.; Lin, M.-H. J.;
Depee, S.; Tadepalli, S.; Votta, B.; James, I.; Fuller, K.;
Chambers, T. J.; Kull, F. C.; Chamberlain, S. D.; Hutchins,
J. T. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16078.
. (a) de laCruz, A.; Elguero, J.; Goya, P.; Martinez, A.;
Pfleiderer, W. Tetrahedron 1992, 48, 6135; (b) Koga, H.;
Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T. J. Med.
Chem. 1980, 23, 1358.
In conclusion a series of potent anilinoquinoline inhibi-
tors of c-fms have been discovered. Modification of the
C4 aryl group showed that lipophilic substituents pro-
vided an increase in potency. Modification of the C7
aryl group with potential H-bond acceptors gave com-
pounds with increased cell potency (ꢀ5–10 times) than
those without H-bond acceptors. By optimizing the C4
substituent while retaining the C7 methylsulfonyl moi-
ety, we were able to generate compounds that possessed
nanomolar binding and cell potency, along with up to
7
8
1
00-fold selectivity for the c-fms-expressing cell line.
Identification of a lead compound for in vivo studies
was made by in vivo PK experiments, and compound
. An X-ray crystal structure of the c-fms kinase domain was
used as the basis for this analysis Schubert, C.; Springer, B.
A.; Deckman, I.; Patch, R. J.; Struble, G. T.; Ma, H.;
Schalk-Hihi, C.; Brandt, B. M.; Petrounia, I. Chem. Abstr.
12 is currently being evaluated as a treatment for
osteoarthritis.
2
006, 144, 447109, patent # WO2006047505.
References and notes
9. Although compound 13 showed excellent cell potency and
acceptable cell selectivity, this compound was not included
in the PK study due to the anticipated metabolic liability of
the 4-methoxy group at C4.
1
. (a) Candore, G.; Balistreri, C. R.; Grimaldi, M. P.; Vasto,
S.; Listi, F.; Chiapelli, M.; Licastro, F.; Lio, D.; Caruso, C.