Pyrido[2,3-d]pyrimidin-5-ones: A novel class of antiinflammatory macrophage colony-stimulating factor-1 receptor inhibitors

Article abstract of DOI:10.1021/jm801406h

A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating rheumatoid arthritis and other chronic infla

Full text of DOI:10.1021/jm801406h

J. Med. Chem. 2009, 52, 1081–1099
1081
Pyrido[2,3-d]pyrimidin-5-ones: A Novel Class of Antiinflammatory Macrophage
Colony-Stimulating Factor-1 Receptor Inhibitors†
Hui Huang, Daniel A. Hutta, James M. Rinker, Huaping Hu, William H. Parsons, Carsten Schubert, Renee L. DesJarlais,
Carl S. Crysler, Margery A. Chaikin, Robert R. Donatelli, Yanmin Chen, Deping Cheng, Zhao Zhou, Edward Yurkow,
Carl L. Manthey, and Mark R. Player*
Johnson & Johnson Pharmaceutical Research and DeVelopment, Welsh and McKean Roads, Spring House, PennsylVania 19477-0776
ReceiVed NoVember 7, 2008
A series of pyrido[2,3-d]pyrimidin-5-ones has been synthesized and evaluated as inhibitors of the kinase
domain of macrophage colony-stimulating factor-1 receptor (FMS). FMS inhibitors may be useful in treating
rheumatoid arthritis and other chronic inflammatory diseases. Structure-based optimization of the lead amide
analogue 10 led to hydroxamate analogue 37, which possessed excellent potency and an improved
pharmacokinetic profile. During the chronic phase of streptococcal cell wall-induced arthritis in rats, compound
37 (10, 3, and 1 mg/kg) was highly effective at reversing established joint swelling. In an adjuvant-induced
arthritis model in rats, 37 prevented joint swelling partially at 10 mg/kg. In this model, osteoclastogenesis
and bone erosion were prevented by low doses (1 or 0.33 mg/kg) that had minimal impact on inflammation.
These data underscore the potential of FMS inhibitors to prevent erosions and reduce symptoms in rheumatoid
arthritis.
Introduction
mediators, RA also has systemic manifestations. RA patients
have a greater incidence of vascular disease6 and osteoporosis.7
Although patients seek relief from pain and stiffness, new agents
are needed that prevent progressive loss of joint function and
reduce the manifestations of systemic disease.
Macrophage colony-stimulating factor-1 receptor (FMSa)
(cellular homologue of the V-FMS oncogene product of the
Susan McDonough strain of feline sarcoma virus) is a class III
receptor tyrosine kinase and is the exclusive receptor for colony
stimulating factor-1 (CSF-1).1 CSF-1 is an important growth
factor for bone progenitor cells, monocytes, macrophages,2 and
cells of macrophage lineage such as osteoclasts3 and dendritic
cells.4 Binding of CSF-1 to the FMS extracellular domain
induces FMS dimerization and trans-autophosphorylation of the
intracellular FMS kinase domain on several tyrosine residues.
Once phosphorylated, FMS serves as a docking site for several
cytoplasmic signaling molecules the activation of which leads
to de novo gene expression and proliferation.5 Robust expression
of FMS is restricted to monocytes, tissue macrophages, and
osteoclasts. For this reason it is anticipated that FMS inhibitors
will be useful in treating diseases where osteoclasts, dendritic
cells, and macrophages are pathogenic, e.g., rheumatoid arthritis
(RA) and other autoimmune/inflammatory diseases.
Rheumatoid arthritis afflicts approximately 1% of the U.S.
population and exacts a large personal and economic burden.
It is an autoimmune disease, and several cartilage autoantigens
have been proposed but none proven to drive pathogenesis.
Acquired cellular and humoral immunity activates synovial
tissue macrophages to express cytokines that further orchestrate
leukocytic infiltration, edema, and pain. The synovial inflam-
mation is chronic and eventually organizes to form granulation
tissue known as a pannus. Pannus is a highly invasive tissue
that progressively erodes cartilage, bone, tendons, and ligaments
with irreversible loss of joint function, particularly of the hands
and feet. Because of a generalized increase in inflammatory
Low-dose methotrexate is the first-line therapy for moderate
to severe RA. Although methotrexate is economical and has
reduced a great deal of suffering, some 35-55% of patients
fail to achieve (American College of Rheumatology-20) ACR20
response rates with methotrexate alone and methotrexate does
not adequately control focal bone erosions and generalized
osteoporosis in RA.8 Patients using methotrexate must be
monitored closely for elevated liver enzymes to prevent liver
fibrosis, and use of methotrexate is associated with transient
nausea and fatigue in a majority of patients.9 Patients that do
not respond adequately to methotrexate may be prescribed one
of several biological agents that neutralize TNF (infliximab,
etanercept, adalimumab, and abatacept) or CD20 (rituxamab).
These agents effectively reduce local and systemic symptoms
and prevent joint space narrowing and bone erosions in the
majority of patients. However, they are expensive and require
parenteral administration. Thus, there remains an untapped
market for an efficacious oral drug that is better tolerated than
methotrexate, particularly one that protects the patient from joint
destruction.
There is evidence that CSF-1 may drive the macrophage
population in RA. CSF-1 levels are elevated in rheumatoid
plasma, synovium, and synovial fluid.10 Monocytes from RA
patients express elevated levels of FcγR I, IIa, and IIIa, increased
CD14 and oxygen radicals, and reduced HLA-DR.11 This
monocyte phenotype can be produced in vitro and in vivo with
recombinant CSF-1.12 Thusly, CSF-1 may drive the recruitment,
differentiation and survival of RA synovial macrophages, and
the local proliferation of myeloid progenitors. Further, CSF-1
primes macrophages for greater expression of TNF and other
cytokines.13 It has been proposed that CSF-1 is a component
of a positive feedback loop in chronic inflammation.14 In this
loop, macrophages secrete TNF and IL-1 that induce stromal
* To whom correspondence should be addressed. Phone: 610-458-6980.
Fax: 610-458-6980. E-mail: mplayer@its.jnj.com.
†
PDB ID: 3DPK.
a Abbreviations: SCF, stem cell factor; FMS, macrophage colony-
stimulating factor-1 receptor; ITD, internal tandem duplication; FLT-3, FMS-
like tyrosine kinase-3; FCS, fetal calf serum; MEM, minimal essential media;
RNase, ribonuclease; DNase, deoxyribonuclease; SEM, standard error of
the mean.
10.1021/jm801406h CCC: $40.75
2009 American Chemical Society
Published on Web 02/04/2009

1082 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Chart 1. FMS Inhibitors
Huang et al.
Chart 2. Three Novel Classes of FMS Inhibitors
cell expression of CSF-1, causing further expansion of mac-
rophage numbers and increased TNF and IL-1 expression.
Therapeutic targeting of TNF and IL-1 with biologic agents has
been successful.15 Interruption of CSF-1 signaling with oral
FMS inhibitors may provide a novel alternative approach to
break the positive feedback loop in inflammatory disease.
Further, because FMS was shown to mediate TNF-induced
osteolysis,15 we anticipate that oral FMS inhibitors will be useful
for preventing focal bone loss, deformity, and loss of joint
function in rheumatoid arthritis.
There are a number of small molecule FMS inhibitors
reported in the literature, such as 1 (Ki20227),16 2 (GW2580),17
3 (P-0728),18 4 (XLS-002),19 5 (sunitinib),20 6 (AG13736),21
and 7 (ABT869).22 While many of them have demonstrated in
vivo antiinflammatory efficacy and are in various stages of
development, 5-7 are multitargeted kinase inhibitors in clinical
trials for cancer therapies (Chart 1).
pharmacokinetic properties and potent anti-inflammatory ef-
ficacy in in vivo arthritis models.
Chemistry
The synthesis of pyrido[2,3-d]pyrimidin-5-one amide 10 is
shown in Scheme 1. 5-Aminoindan was reacted with ethyl
3-chloropropionate at elevated temperature in the presence of
an inorganic base and a catalytic amount of tetrabutylammonium
bromide, affording the aminopropionate ester 11. Compound
11 was then treated with ethyl 4-chloro-2-methylthio-5-pyrim-
idinecarboxylate to produce 4-aminopyrimidine 12. Cyclization
of this diester under Dieckmann conditions afforded bicyclic
compound 13. Subsequent halogenation with bromine followed
by dehydrohalogenation gave 14.26 The thiomethyl moiety was
then oxidized to sulfone 15, which was subsequently displaced
with an alkyl amine or aniline by nucleophilic substitution. The
resulting carboxylate ester 16 was converted to amides 10,
17a-e, and 18a-c by reaction with corresponding amines in
methanol at elevated temperatures.
We have identified several classes of novel FMS inhibitors:
arylamides (8),23 3,4,6-substituted 2-quinolones (9),24 and
pyrido[2,3-d]pyrimidin-5-ones (10),25 which have good in vitro
and in vivo activities (Chart 2). Herein is reported the structure-
based optimization of the pyrido[2,3-d]pyrimidin-5-one core,
which has led to FMS inhibitors with significantly improved
After the methyl hydroxamate group was shown to be optimal
at the C-6 position (18a, Table 1, Scheme 1), an alternate route
was developed in order to incorporate the methyl hydroxamate

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1083
Scheme 1a
a Reagents and conditions: (a) ethyl 3-chloropropionate, K2CO3, Bu4NBr (cat.), 100 °C; (b) Et3N, n-BuOH, rt; (c) t-BuONa, toluene, 90 °C; (d) Br2,
CH2Cl2, rt; (e) Et3N, CH2Cl2, rt; (f) m-CPBA, CH2Cl2, rt; (g) arylamine, i-PrOH, 90 °C; (h) R1NH2, MeOH, rt or 80-100 °C.
led to bicyclic ester 21. Acid 22, obtained through acidic
hydrolysis of 21, was coupled with methoxyamine to form 23.
Table 1. Effect of C-6 Amide Substitution on Biological Activity and
Rat Liver Microsomal Stability
The 2-methylthio group of 23 was oxidized to give methylsul-
fone 24, which was subsequently displaced with anilines R2NH2
to give final products. This route allowed rapid exploration of
the C-2 anilino substituents and facilitated evaluation of N-8
substitutions using common intermediate 20.
Results and Discussion
As previously reported, pyrido[2,3-d]pyrimidin-5-one 10 was
a potent FMS inhibitor with an IC50 of 13 nM.25 Further
FMS enzyme
IC50 (nM)
BMDM
RLM
a
b
compd
R1
X
IC50 (nM) stabilityc
optimization of the C-2 anilino substituents led to 17a with
improved potency in the enzyme assay28 and in the cellular assay
that measures proliferation of bone marrow-derived macroph-
ages (BMDM)29 in response to CSF-1 stimulation (Table 1).
Small alkyl substitution on the C-6 amide was well tolerated,
though activity diminished with increasing size (17b,d). Despite
good in vitro activity, the amide series (10, 17a, and 17c)
exhibited poor oral exposure when administered at 10 mg/kg
to male Sprague-Dawley rats (Table 2). Moreover, these
compounds showed high volumes of distribution and high
clearance rates. Because the amide series was stable in rat liver
microsomal (RLM) incubations, the lack of oral exposure was
suspected to be a result of poor absorption.
10
13
16
9
26
55
45
56
1.3
3.3
5.4
87
96
73
100
NDd
84
78
84
100
17a
17b
17c
17d
17e
18a
18b
18c
H
NCH3
NCH3
O
0.8
1.2
1.4
3.2
0.7
0.7
0.5
0.5
Me
Me
Et
NCH3
CH2CH2OH NCH3
OMe
OEt
NCH3
NCH3
OCH(CH3)2 NCH3
a Reported IC50 values are means of three experiments. Interassay
variance was <10%. b Dose-response data are the average of at least two
replicates per dose. c Percentage of compound remaining after 10 min
incubation in rat liver microsomes. d Not determined.
group earlier in the synthetic sequence (Scheme 2). Ethyl
4-chloro-2-methylthio-5-pyrimidinecarboxylate was saponified
to the corresponding acid 19. The 4-chloride was displaced by
a methoxyl group under the conditions noted. However, the
methoxyl group was found to serve the same role as the chlo-
ride in subsequent steps. Ketoester 20, prepared from an acid
chloride intermediate, was converted to an enaminoester inter-
mediate by reaction with ethyl orthoformate and acetic anhy-
The cocrystal structure of an amide compound bound in the
active site of FMS revealed that the amide NH is part of a water-
mediated hydrogen bonding network.25 It was hypothesized that
displacing the water molecule and preserving the hydrogen
bonding network might improve potency. Molecular modeling
suggested that 2-hydroxyethyl amide (17e) had the potential to
achieve this. Compound 17e was potent in the enzyme assay
but substantially less active than the corresponding amide 17a
in the BMDM assay. Alkyl hydroxamates (18a-c) were also
designed to explore this hypothesis. The hydroxamate group
dride, followed by reaction with a primary amine R3NH2.27
base-assisted cyclization reaction of the unisolated enaminoester
A

1084 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
Scheme 2a
a Reagents and conditions: (a) NaOH, methanol, rt; (b) oxalyl chloride, DMF (cat.), CH2Cl2, 0 °C to rt; (c) ethyl hydrogen malonate, MeMgBr, 0 °C to
rt; (d) triethylorthoformate, Ac2O, 130 °C; (e) aminoindan, THF, rt; (f) K2CO3, THF, rt; (g) 1 N HCl, 1,4-dioxane, 105 °C; (h) oxalyl chloride, DMF (cat.),
CH2Cl2, 0 °C to rt; (i) methoxyamine hydrochloride, Et3N, CH2Cl2, 0 °C to rt; (j) m-CPBA, CH2Cl2, rt; (k) R2NH2, AcOH, 110 °C.
mediated through the C-2 anilino NH and the pyrimidine N-3
(Figure 1A). A water-mediated hydrogen bonding network is
formed between the C-5 carbonyl oxygen, the hydroxyl group
Table 2. Pharmacokinetic Profile of Selected Analogues in
Sprague-Dawley Rats
d
Cmax
t1/2
t1/2
Cl
Vdss
F
of Thr663 and a water molecule present in the active site (Figure
1B). The hydroxamate group preserves the conformational
constraint of the intramolecular hydrogen bond between the NH
and C-5 carbonyl oxygen. This in turn enables the acetamide
carbonyl to interact with Lys616. In addition, the hydroxamate
oxygen forms hydrogen bonds with Lys616. It is believed that
this complex hydrogen bonding network contributes to the
improved potency.
The importance of the aromatic nature of the C-2 substituents
is evident, given that compounds with alkyl amino substitutions
at C-2 position (26, 27) had poor activity (Table 3). The
cocrystal structure of 25 bound in FMS shows that the pocket
for C-2 substitution is quite narrow and cannot accommodate
alkyl groups that would have a significant out-of-plane geometry.
Crystal structures also predict that the substitution at the
4-position of the C-2 anilino phenyl ring would be solvent
exposed. This region of the molecule has been used to modulate
various properties of the compound while maintaining potent
FMS inhibition.
To assess the specificity profile of FMS inhibitors as the
program progressed, compounds were routinely assessed in a
50-assay counterscreening profile (GPCRs, ion channels, and
transporters) at Cerep.31 In this screen, compound 18a inhibited
16 targets by 50% or more at a concentration of 10 µM. In
contrast, compounds with less basic substituents on the C-2
anilino phenyl inhibited fewer targets (data not shown).
Therefore, efforts refocused on replacement of the basic
piperazine tail of 18a. At first, piperazine was replaced by less
basic rings such as morpholine (29), 4,4-difluoropiperazine (30),
and piperazin-2-one (31) (Table 3). Morpholino analogue 29
was more potent than its amide counterpart 17c but was unstable
compda pob (µM) po (h) iv (h) (mL/min/kg) (L/kg) (%)
10
133
25
47
4
10
34
38
11
18.7
15.5
2.6
1.5
0.8
8.8
5.1
4.3
5.6
5.9
0
0
4
57
3
8
17a
17c
18a
31
35
36
37
39
45
7
0.13
2.7
0.08
0.13
1.8
6.5
9.7
0.7
4.5
6
3
1.7
4.6
5.2
8
0
1.6
0.4
3.4
6.9
8.1
100
28
103
20.4
8
a Formulatedin20%hydroxypropyl-ꢀ-cyclodextrin(HPꢀCD).b Administrated
at 10 mg/kg po. c Administrated at 2 mg/kg iv. d Volume of distribution at
steady state. Both Cl and Vd determined from iv dosing.
presents a distinct electrostatic surface for interaction and was
expected to displace one of the bound water molecules. Methyl
hydroxamate 18a exhibited significantly increased potency in
the BMDM assay while retaining the enzyme activity of the
amide 17a. Furthermore, 18a had an improved pharmacokinetic
profile in rat with an oral bioavailability of 57%, a Cmax of 2.7
µM at a 10 mg/kg oral dose, and a low systemic clearance rate
(4 mL/min/kg) (Table 2). Small alkyl hydroxamates were all
potent FMS inhibitors, although BMDM activity decreased with
increasing size of the alkyl group (18b,c). Some of these
analogues have kinase assay IC50s around the enzyme concen-
tration of 0.9 nM. As enzyme activity approached this limit,
the BMDM cell-based assay was more sensitive to differences
in potency.
Subsequently, one of the methyl hydroxamates (25) was
cocrystallized with FMS (Figure 1). Like previously analyzed
FMS inhibitors,25,30 the hydroxamate series interacts with the
hinge region of the kinase via hydrogen bonding interactions

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1085
Figure 1. Cocrystal structure of 25 in FMS (PDB ID: 3DPK). (A) Ball-and-stick representation of 25 (yellow) bound in the active site of cFMS
(gray). Additionally shown is the 2fo - fc electron density map (blue) contoured at 1.4σ and truncated to a radius of 2 Å beyond each atom for
clarity. Hydrogen bonds to the backbone of the protein are depicted as green dashed lines. (B) Close-up view of the water-mediated hydrogen-
bonding network in the active site. The internal hydrogen bond between the amide nitrogen and the pyridopyrimidinone carbonyl oxygen is drawn
in magenta.
in human and rat liver microsomes. While stable in HLM and
RLM and clean in the counterscreening profile, the difluorinated
piperidine analogue (30) was less active in the BMDM assay.
Piperazin-2-one analogue (31) also exhibited a clean counter-
screening profile, confirming the refocused strategy. However,
its oral bioavalability in rat was only 3% (Table 2). Methylated
analogue 32 was prepared in an attempt to improve on the
permeability of 31, but it decomposed rapidly in HLM incuba-
tions. Neutral and acidic compounds were also prepared and
had clean counterscreening profiles. However, it was difficult
to formulate the neutral compounds for in vivo assessment due
to poor solubility. Incorporation of acidic “tails” resulted in loss
of activity in the BMDM assay, as seen in acetyl sulfonamide
33.
Scheme 3a
The ethyl linker was removed to give piperidine analogues
34-37. Compound 34 was as potent as 18a and had a similar
counterscreening profile. Introduction of a hydroxyl ethyl group
(35) had minimal impact on the potency, while acetic acid
analogue (36) reduced BMDM activity more than 20-fold
compared to 34 (Table 3). Both 35 and 36 had very low oral
exposure in rat (Table 2). The N-methylated piperidine analogue
(37) stood out not only for its excellent potency, but also for
its good rat pharmacokinetic profile with an oral Cmax of 1.6
µM, a clearance rate of 11 mL/min/kg, and bioavailability of
100% (Table 2). Although 37 was not optimal in the counter-
screening profile as measured by inhibition of paradigm
compound binding in the Cerep panel, follow-up functional
assays showed that hM1 was the only target where modest
functional activity was observed.
a Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, toluene, ethanol,
110 °C; (b) MeI, acetone, 50 °C; (c) NaBH4, 0 °C to rt; (d) 6 N HCl, THF,
60 °C; (e) Deoxo-Fluor, CH2Cl2, 0 °C to rt; (f) H2, Pd/C, methanol, rt.

1086 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
Table 3. Effect of C-2 Substitution on Biological Activity and Rat/Human Liver Microsomal Stability
a Reported IC50 values are means of three experiments. Interassay variance was <10%. b Dose-response data are the average of at least two replicates
per dose. c Percentage of compound remaining after 10 min incubation in human liver microsomes. d Percentage of compound remaining after 10 min
incubation in rat liver microsomes.

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1087
Table 4. Effect of N-8 Substitution on Biological Activity and Rat/Human Liver Microsomal Stability
a Reported IC50 values are means of three experiments. Interassay variance was <10%. b Dose-response data are the average of at least two replicates
per dose. c Percentage of compound remaining after 10 min incubation in human liver microsomes. d Percentage of compound remaining after 10 min
incubation in rat liver microsomes.
Table 5. Kinase Selectivity of Selected Compounds
kinasea
17a (µM)b
18a (µM)
36 (µM)
37 (µM)
FMS
FLT3
KIT
PDGFR-ꢀ
Axl
TrkA
IRK-ꢀ
Src
0.0008
0.001
0.020
>42
>0.5
0.17
0.0007
0.036
0.006
5.3
>0.5
>0.5
19.8
0.0022
0.041
0.013
>2
>0.5
>0.5
9.3
0.0004
0.006
0.006
0.15
>1
0.5
>42
5.9
3.7
1.4
1.5
0.93
a IC50 values were determined at the respective ATP Km of each kinase.
b IC50 values are means of three experiments. Interassay variance was <10%.
Table 6. Pharmacokinetic Profiles of 37 in Multiple Species
Figure 2. In vivo inhibition of FMS 6 h after dosing mice with 37.
B6C3R1 mice were dosed orally with 37 6 h prior to a tail-vein injection
of 1 µg recombinant CSF-1. Fifteen minutes following the CSF-1
challenge, mice were sacrificed and the functional status of FMS
signaling was assessed by assay of c-fos mRNA in spleens as described
in the Experimental Section.>
Cmax
t1/2
t1/2
Cl
Vdss
F
(%)
speciesa po (µM) po (h) iv (h) (mL/min/kg) (L/kg)
rat
mouse
dog
1.6
0.9
0.12
0.06
6.9
4
4.6
1.8
9.3
6.6
11
34
30
48.7
4.3
3.6
20.3
22.6
100
51
33
16.7
monkey
a Administrated at 10 mg/kg po, 2 mg/kg iv for all species in 20%
hydroxypropyl-ꢀ-cyclodextrin (HPꢀCD).
40. It was then converted to the corresponding N-methyl
pyridinium salt, followed by borohydride reduction to give
tetrahydropyridine 41.32 Acidic treatment of 41 gave 1-meth-
ylpiperid-3-one 42, which was subsequently treated with bis-
(2-methoxyethyl)aminosulfur trifluoride to give 3,3-difluoro-
substituted piperidine 43. Hydrogenolytic deprotection of the
Cbz group provided the aniline 44. The electron withdrawing
effect of the difluoro group of 38 did not affect FMS activity
In an effort to attenuate basicity of the piperidine ring,
difluoro-substituted piperidine analogue 38 and 3-hydroxyl
piperidine analogue 39 were prepared. The aniline precursor of
38, 4-(3,3-difluoro-1-methyl-piperidin-4-yl)-phenylamine (44)
was synthesized as shown in Scheme 3. Suzuki coupling of the
boronic acid with 3-methoxy-4-bromopyridine provided biaryl

1088 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
Figure 3. Compound 37 reversed ankle swelling during the chronic phase of SCW-induced arthritis. Arthritis was induced in rats by a single ip
injection of SCW on day 0. Twice daily oral dosing with vehicle or 37 was commenced on day 19 when chronic inflammation was well established.
Ankle thickness was measured using calipers as described in the Experimental Section.
Figure 4. Compound 37 reduced paw swelling in adjuvant-induced arthritis. Adjuvant arthritis was induced in rats as described in the Experimental
Section. Twice daily oral dosing with vehicle or 37 was commenced on day 8. Calipers were used to measure ankle thickness daily (mean ( SEM).
**p < 0.001, * p < 0.01 vs arthritis + vehicle.
but resulted in poor solubility and low liver microsomal stability
(Table 3). Compounds containing 1,3-substitution patterns on
the C-2 aniline were also explored (39) and were potent FMS
inhibitors. However, 39 demonstrated a Cmax of only 0.4 µM
after oral administration in rat and had a moderate clearance
rate (20 mL/min/kg) (Table 2).
Having identified the N-methylpiperidine tail as an optimal
C-2 substitution, N-8 substitution was next investigated (Table
4). When bound to FMS, N-8 substituents occupy a hydrophobic
pocket, which can accommodate a variety of aromatic (37,
45-47) or aliphatic rings (25, 48-49). The 4-ethylphenyl
analogue 45 was equipotent to indan analogue 37 and had a
slightly improved rat pharmacokinetic profile compared to 37
in terms of oral exposure and clearance rate (Table 2). The
3-ethylphenyl analogue 46 was comparable to 37 and 45 in
activity. The loss in activity by incorporation of nitrogen into
the indan phenyl ring (47) confirmed the importance of
hydrophobic substitution at the N-8 position. Aliphatic rings
were well tolerated, and the bulkier dimethyl cyclohexyl
analogue 48 was more potent than the cyclohexyl analogue 49.

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1089
Table 7. Effect of 37 on Swelling, Bone Erosion, and Osteoclast
Counts in Adjuvant Arthritis
percent inhibition
of ankle swelling,
AUC
bone
erosion
scorea
osteoclast
countsb
treatment
Experiment no. 1c
vehicle (20% HPꢀCD)
37, 1 mg/kg
37, 3 mg/kg
4.88 (0.41)
0.6 (0.6)**
0 (0)**
29.3 (5.2)
0.2 (0.1)**
0.1 (0.1)**
0 (0)**
-0.9
-0.9
17*
37, 10 mg/kg
0 (0)**
Experiment no. 2c
vehicle (20% HPꢀCD)
37, 0.33 mg/kg
37, 1 mg/kg
4.25 (0.41
1.88 (0.23)** 9.9 (2.4)*
0 (0)**
0 (0)**
29.9 (3.2)
7
2
8
0 (0)**
0 (0)**
37, 3 mg/kg
a Based on microscopic examination of H and E stained sections and a
5-point scale described in Experimental Section. b Osteoclast counts (Five,
400× fields) were performed on ankles in the areas of greatest bone
resorption. c Groups (n ) 8) were dosed with vehicle or 37 orally twice
daily beginning on day 8 until sacrifice on day 21. Values represent group
means and (SEM). *p < 0.05 vs vehicle; **p < 0.001 vs vehicle (Student’s
t test).
Figure 6. Compound 37 reduced ankle swelling in adjuvant-induced
arthritis in combination with methotrexate. Adjuvant arthritis was induced
in rats as described in the Experimental Section. Oral dosing was
commenced on day 8. Blue squares, dosed twice daily with 20% HPꢀCD
and once daily with 1% carboxymethylcelluose (CMC); black squares,
dosed twice daily with 0.33 mg/kg 37 in 20% HPꢀCD and once daily
with 1% CMC; white squares, dosed twice daily with 20% HPꢀCD and
once daily with 0.1 mg/kg methotrexate in 1% CMC; red squares, dosed
twice daily with 0.33 mg/kg 37 in 20% HPꢀCD and once daily with 0.1
mg/kg methotrexate in 1% CMC. Calipers were used to measure ankle
thickness daily (mean ( SEM). *p < 0.05 vs methotrexate alone.
A rat tolerability study was carried out to differentiate com-
pounds 37 and 45. While 37 was well tolerated at doses of 10
and 40 mg/kg, rats treated with 45 showed nonspecific signs of
toxicity such as mucoid feces, cold body, and weight loss (data
not shown).
Selectivity profiles of some of these FMS inhibitors against
other type III receptor tyrosine kinases were obtained (Table
5). In the enzyme assays all were potent inhibitors of FLT3
and KIT but highly selective vs PDGFR-ꢀ, Axl, IRK, TrkA,
Figure 5. Compound 37 prevented the development of bone lesions in the tibial tarsal joint. Following necropsy on day 21, microradiographic
images were prepared of tibial tarsal joints. Representative images are shown obtained from a disease-free rat (A), a rat with adjuvant-arthritis
treated with vehicle (B), and a rat with adjuvant-arthritis treated with 1 mg/kg 37 (C). Note the presence of radiotranslucent pits (arrows) in the
tibia, calcanus, talus, and tarsus of the rat with adjuvant-arthritis treated with vehicle, while the tibial tarsal joint of the rat with adjuvant-arthritis
treated with 37 was difficult to discern from the disease-free rat.

1090 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
and Src. In the cell-based assays of these kinases, 37 inhibited
ITD-FLT3-dependent MV-4-11 cell growth23 and SCF/KIT-
dependent M-o7e cell growth33 with mean IC50 values of 80
nM and 20 nM, respectively (n ) 4). Relative to CSF-1/FMS-
dependent BMDM cell growth (IC50 ) 0.6 nM), this represented
modest selectivity versus KIT (33-fold) and good selectivity
over FLT3 (133-fold). Inhibition of KIT and FLT3 may have
anti-inflammatory potential due to suppression of mast cell or
dendritic cell functions, but chronic inhibition of KIT may result
in anemia and myelosuppression.34
dosed with 1 mg/kg of 37. Microradiographs of representative
paws further confirmed the impressive protection of bone
structures by 37 (Figure 5). In vehicle-treated rats, foci of
increased radiotranslucency were apparent in the distal tibia,
while calcanus, talus, and tarsus bones were frequently difficult
to delineate. In contrast, bones from animals treated with 37 (1
mg/kg) were similar to those of disease-free rats. Mean plasma
levels taken 7 h after the last dose of 0.33, 1, and 3 mg/kg
were 0.014, 0.055, and 0.189 µM, respectively.
The disease modifying activity of 37 in adjuvant arthritis was
further examined in combination with methotrexate, the standard
first line therapy for RA. A low dose of 37 (0.33 mg/kg) was
administered alone or in combination with an efficacious dose
of methotrexate and compared to methotrexate alone. As before,
0.33 mg/kg of 37 alone did not reduce ankle swelling (Figure
6). However, 37 significantly reduced to nearly undetectable
levels the mild swelling and inflammation otherwise present in
rats administered methotrexate alone. By day 20, swelling was
evident in only 2 of 16 ankles in the combination group
compared to 12 of 16 joints in the methotrexate alone group.
The pharmacokinetic profiles of 37 in mouse, dog, and
monkey were assessed (Table 6). In the mouse, 37 had good
bioavailability (51%), an oral Cmax of 0.9 µM at a 10 mg/kg
dose and a moderate clearance rate. In the dog and monkey, 37
had lower Cmax levels of 0.12 and 0.06 µM, respectively. The
clearance rates in both species were also high, in line with its
low liver microsomal stability (51% and 14% remaining after
10 min incubation in dog and monkey liver microsomes,
respectively).
Compound 37 was further evaluated in in vivo studies. A
pharmacodynamic model was developed based on the ability
of CSF-1 to elevate macrophage c-fos mRNA.35 Because
significant numbers of macrophages are present in the spleen,
spleens of mice were assessed for c-fos mRNA following
intravenous administration of 1 µg/mouse recombinant CSF-1.
Splenic c-fos mRNA was increased by approximately 10-fold
within 15 min after CSF-1 induction (Figure 2) and returned to
baseline by 30 min (data not shown). An oral dose of 3 mg/kg
of 37, administered 6 h prior to the CSF-1 challenge, reduced
c-fos mRNA induction by 90% (Figure 2). The corresponding
plasma concentration of 37 at the 6 h time point was 0.29 µM.
Compound 37 was further evaluated in rats using strepto-
coccal cell wall (SCW)-induced and adjuvant-induced models
of arthritis. Both models are responsive to prednisone and anti-
TNF therapy, and adjuvant arthritis is responsive to methotr-
exate.36 SCW-induced polyarthritis in female Lewis rats is
characterized by an acute, transient phase (days 3-7) that is
complement- and neutrophil-dependent, which evolves into a
chronic erosive phase (after day 10) that is dependent on the
development of specific T cell immunity to SCW.37 An
important characteristic of the SCW model is the predominance
of macrophages in the inflamed synovial pannus38 that ac-
curately models macrophage involvement in rheumatoid arthri-
tis.39 For this reason, the chronic phase of the SCW-induced
arthritis model was selected to investigate the ability of 37 to
reduce chronic joint inflammation and bone erosion. Twice-
daily oral dosing was initiated on day 19 when chronic
inflammation was well established, and 37 was highly effective
at reversing established ankle swelling at doses of 10, 3, or 1
mg/kg (Figure 3). The progressive increase in ankle thickness
in the vehicle-treated group was delayed in rats dosed with 0.3
mg/kg of 37.
The anti-inflammatory effects of 37 in the SCW-induced and
adjuvant-induced arthritis models are consistent with the
requirement for FMS for optimal macrophage survival and
proliferation. Indeed, mice that are genetically deficient in FMS
or CSF-1 lack synovial macrophages40 and CSF-1-deficient mice
are highly resistant to collagen-induced arthritis.41,42 Similarly,
the bone preserving effect of 37 is consistent with the require-
ment for FMS/CSF-1 for osteoclast differentiation revealed by
severe osteopetrosis of the FMS-deficient mice40 and the ability
of FMS neutralizing antibody to block TNF-induced osteolysis.15
We therefore attribute the therapeutic effects of 37 primarily to
FMS inhibition, although we cannot exclude the possibility that
inhibition of secondary targets may contribute to the pharmacol-
ogy of 37. Indeed, inhibition of KIT or FLT3 may augment the
beneficial pharmacology of 37, although at the cellular level
the selectivity of 37 for FMS was 33- and 133-fold vs KIT and
FLT3, respectively. Other FMS inhibitors (i.e., 1, 2, and 8) have
demonstrated efficacy in rodent models of arthritis despite
nonoverlapping selectivity profiles, further supporting FMS as
a promising target for the treatment of rheumatoid arthritis.16,17,23b
At present, the primary advantage of 37 may be its exemplary
potency.
Conclusion
A novel class of FMS kinase inhibitors based on the
pyrido[2,3-d]pyrimidin-5-one scaffold was developed. Introduc-
tion of a C-6 hydroxamate group resulted in significant potency
increases in the cell-based BMDM assay and an improved
pharmacokinetic profile. The cocrystal structure of 25 in FMS
revealed a complex hydrogen-bonding network between the
hydroxamate group and the kinase active site, which is believed
to contribute to the improved potency. Further optimization
afforded 37. Among known FMS inhibitors, 37 demonstated
exemplary in vitro potency together with outstanding rodent PK.
Compound 37 reversed established ankle swelling in the chronic
phase of SCW arthritis and enhanced the anti-inflammatory
activity of methotrexate in adjuvant arthritis as well as providing
profound suppression of bone erosion as a single agent.
Collectively, the data provide strong preclinical validation for
testing the therapeutic utility of FMS inhibitors in RA, both for
Compound 37 was less effective in preventing the progressive
inflammatory response in adjuvant-induced arthritis. When dosed
twice daily from the onset of arthritis (day 8), 37 reduced paw
swelling partially at 10 mg/kg, with lower doses showing no
improvement (Figure 4 and Table 7). Despite the limited effect
on inflammation, microscopic examination revealed preservation
of the bones of the tibiotarsal joint. Preservation was nearly
complete at 1 mg/kg, and good partial protection was observed
even at 0.33 mg/kg (Table 7). By day 21, osteoclasts were
abundant in the pannus and trabecular bone of adjuvant rats
treated with vehicle. In contrast, there was minimal evidence
of reactive osteoclastogenesis in the tibiotarsal joints of rats

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1091
for 30 min. The solution was cooled and poured into crushed ice.
The solution was adjusted to pH 7 using concentrated aq HCl. The
precipitates were extracted into ethyl acetate (2 × 30 mL). The
solvent was evaporated under vacuum and the product (bright-
the treatment of signs and symptoms and to prevent the loss of
function resulting from joint erosions.
Experimental Section
1
yellow solid, 4 g, 62%) was recrystallized from isopropanol. H
NMR (300 MHz, CDCl3) indicated that the presence of both enol
and keto forms in a 4:1 ratio.
Chemistry. Reagents and solvents were obtained from com-
mercial suppliers and used without further purification. Flash
chromatography was performed using Fisher Chemical Silica Gel
Sorbent (230-400 mesh, grade 60). Preparative thin-layer chro-
matography was performed on Analtech Silica Gel GF plates (1000
or 2000 µm, 20 cm × 20 cm). Preparative HPLC was performed
on a Gilson 215 HPLC system using a C-18 YMC ODS-A 5m 30
× 100 mm, 120 A column with a flow rate of 32 mL/min. The
solvents, both acetonitrile (MeCN) and water, contain 0.1% of
trifluoroacetic acid (TFA, v/v). Hydrogenation was performed in a
8-Indan-5-yl-2-methylsulfanyl-5-oxo-5,8-dihydropyrido[2,3-d]py-
rimidine-6-carboxylic Acid Ethyl Ester (14). To a solution of 13
(0.32 g, 0.84 mmol) in 5 mL of methylene chloride (CH2Cl2) was
added bromine (43 µL, 0.84 mmol) slowly under N2. The solution
was stirred at rt for 2 h. The solvent was removed under vacuum
without heating. The residue was dissolved in CH2Cl2 (2 mL), to
which was added triethylamine (234 µL, 1.68 mmol) in 1 mL of
CH2Cl2. The solution was stirred at rt for 4 h. The solvent was
evaporated and the residue was purified by flash chromatography
(eluted with ethyl acetate/hexanes, 1:5 to 2:5, v/v). The title
1
H-Cube (ThalesNano, Inc., Budapest, Hungary). H NMR spectra
were recorded on a Bruker B-ACS-120 (400 MHz) spectropho-
tometer at rt. Chemical shifts are given in ppm (δ), coupling
constants (J) are in hertz (Hz), and signals are designed as follows:
(s) singlet, (d) doublet, (t) triplet, (q) quartet, (m) multiplet, (br s)
broad singlet, (dd) doublet of doublet, (dt) doublet of triplet.
Elemental analysis was performed by Quantitative Technologies,
Inc., Whitehouse, NJ. LC-MS was performed on a system consisting
of an atmospheric pressure ionization (API) source on a Finnigan
LCQ ion trap mass spectrometer, an Agilent 1100 DAD UV detector
(214 nm wavelength), an Agilent 1100-LC binary gradient pumping
system, a Gilson 215 configured as an autosampler, and a Zorbax
SB-C18 HPLC column (5 µm, 50 mm × 2.1 mm). The purities of
the key target compounds were determined on the Finnigan LCQ
instrument, which gave both the MS and LC trace of the compound
in an 11 min run. The HPLC method used was as follows: column,
Zorbax SB-C18 HPLC column (5 µm, 50 mm × 2.1 mm); column
temperature, ambient; flow rate, 0.5 mL/min; gradient, 5% aceto-
nitrile in water to 100% acetonitrile in water in 10 min, both MeCN
and water contain 0.1% of TFA, v/v.
3-(Indan-5-ylamino)-propionic Acid Ethyl Ester (11). Tet-
rabutylammonium bromide (200 mg) was added to a mixture of
5-aminoindan (5 g, 37.6 mmol), ethyl 3-chloropropionate (4.7 mL,
37.6 mmol), and potassium carbonate (5.2 g, 37.6 mmol). The
mixture was stirred at 100 °C for 16 h. After cooling to rt, the
mixture was extracted into ethyl acetate, washed with water and
brine, and dried with sodium sulfate (Na2SO4). After evaporation
of the solvent and chromatography on silica (eluted with ethyl
acetate/hexanes, 1:20 to 1:10, v/v), the title compound was obtained
as brown oil (6.2 g, 71%). 1H NMR (300 MHz, CDCl3) δ 7.04 (d,
J ) 7.91 Hz, 1H), 6.56 (s, 1H), 6.45 (dd, J ) 1.98, 7.91 Hz, 1H),
4.15 (q, J ) 7.09 Hz, 2H), 3.44 (t, J ) 6.43 Hz, 2H), 2.82 (q, J )
7.69 Hz, 4H), 2.61 (t, J ) 6.43 Hz, 2H), 1.94 - 2.13 (m, 2H),
1.27 (t, J ) 7.09 Hz, 3H).
1
compound was obtained as a white solid (0.30 g, 94%). H NMR
(300 MHz, CDCl3) δ 9.42 (s, 1H), 8.59 (s, 1H), 7.37 (d, J ) 7.91
Hz, 1H), 7.23-7.25 (m, 1H), 7.16 (d, J ) 7.91 Hz, 1H), 4.39 (q,
J ) 7.14 Hz, 2H), 2.96-3.05 (m, 4H), 2.33 (s, 3H), 2.16-2.25
(m, 2H), 1.40 (t, J ) 7.09 Hz, 3H).
8-Indan-5-yl-2-methanesulfonyl-5-oxo-5,8-dihydropyrido[2,3-
d]pyrimidine-6-carboxylic Acid Ethyl Ester (15). To a solution of
14 (0.3 g, 0.79 mmol) in 5 mL of CH2Cl2, was added
3-chloroperoxybenzoic acid (m-CPBA, 69.5%, 431 mg, 1.73
mmol) portionwise. The solution was stirred at rt for 3 h. An
aqueous solution of 10% sodium thiosulfate (2 mL) was added
to quench the reaction. After 30 min, saturated sodium bicarbon-
ate solution (10 mL) was added, and the aqueous solution was
extracted by CH2Cl2 (2 × 10 mL). The combined organic layer
was washed with brine and dried over Na2SO4. The solvent was
evaporated and the residue was purified by flash chromatography
(eluted with ethyl acetate/hexanes, 1:3 to 2:3, v/v). The title
compound was obtained as an off-white solid (0.22 g, 67%). 1H
NMR (300 MHz, CDCl3) δ 9.74 (s, 1H), 8.70 (s, 1H), 7.39 (d,
J ) 7.91 Hz, 1H), 7.25 (s, 1H), 7.12-7.21 (m, 1H), 4.38 (q, J
) 7.25 Hz, 2H), 3.19 (s, 3H), 2.93-3.06 (m, 4H), 2.11-2.27
(m, 2H), 1.38 (t, J ) 7.25 Hz, 3H).
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)-ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Eth-
yl Ester (16a). The mixture of 4-[2-(4-methylpiperazin-1-yl)-
ethyl]-phenylamine (53 mg, 0.24 mmol) and 15 (100 mg, 0.24
mmol) in 1 mL of isopropanol was heated to 90 °C for 1 h. The
solvent was evaporated and the residue was redissolved in a
mixture of methanol and CH2Cl2 (1:1, v/v) and applied to a
preparative TLC plate (2000 micro). The plate was developed
in NH4OH/MeOH/CH2Cl2 (1:9:90, v/v/v). The title compound
1
was obtained as a yellow solid (70 mg, 53%). H NMR (400
MHz, CDCl3) δ 9.32 (br s., 1H), 8.52 (br s, 1H), 8.18 (br s,
1H), 7.40 (d, J ) 7.43 Hz, 1H), 7.23 (br s, 3H), 7.16 (d, J )
7.43 Hz, 1H), 6.90 (br s, 2H), 4.35 (q, J ) 7.04 Hz, 2H), 3.05
(t, J ) 6.85 Hz, 2H), 2.98 (t, J ) 7.24 Hz, 2H), 2.66-2.77 (m,
2H), 2.41-2.64 (m, 8H), 2.37 (br s, 2H), 2.29 (s, 3H), 2.14-2.26
(m, 2H), 1.36 (t, J ) 7.04 Hz, 3H).
4-[(2-Ethoxycarbonylethyl)-indan-5-yl-amino]-2-methylsulfa-
nylpyrimidine-5-carboxylic Acid Ethyl Ester (12). To a solution
of 11 (5 g, 21.4 mmol) and ethyl 4-chloro-2-methylthio-5-
pyrimidinecarboxylate (5 g, 21.4 mmol) in 40 mL of n-butanol was
added triethylamine (3 mL, 21.4 mmol). The solution was stirred
at rt for 2 days. The solvent was removed under vacuum. The
residue was extracted into EtOAc, washed with water and brine,
and dried with Na2SO4. The solvent was removed and the residue
was purified by flash chromatography (eluted with ethyl acetate/
hexanes, 1:10 to 1:6, v/v) to yield the title compound as a white
8-Indan-5-yl-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Ethyl Ester
(16b). Using a similar procedure described in the preparation of
16a, the title compound was prepared from 4-(2-morpholin-4-yl-
ethyl)-phenylamine (49 mg, 0.24 mmol) and 15 (100 mg, 0.24
mmol). The title compound was obtained as a yellow solid (60
1
solid (8.2 g, 90%). H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H),
7.17 (d, J ) 7.91 Hz, 1H), 6.96 (s, 1H), 6.85-6.92 (m, 1H),
4.28-4.39 (m, 2H), 4.05 (q, J ) 7.03 Hz, 2H), 3.57 (q, J ) 6.92
Hz, 2H), 2.86 (t, J ) 7.42 Hz, 4H), 2.63-2.72 (m, 2H), 2.55 (s,
3H), 2.00-2.13 (m, 2H), 1.19 (t, J ) 7.25 Hz, 3H), 1.01 (t, J )
7.25 Hz, 3H).
1
mg, 46%). H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.47 (s,
1H), 8.21 (br s, 1H), 7.35 (d, J ) 7.43 Hz, 1H), 7.20 (d, J ) 5.09
Hz, 3H), 7.11 (d, J ) 7.43 Hz, 1H), 6.85 (br s, 2H), 4.30 (q, J )
7.04 Hz, 2H), 3.68 (br s, 4H), 3.00 (t, J ) 6.85 Hz, 2H), 2.93 (t,
J ) 7.04 Hz, 2H), 2.58-2.72 (m, 2H), 2.36-2.54 (m, 6H),
2.07-2.23 (m, 2H), 1.31 (t, J ) 7.04 Hz, 3H).
8-Indan-5-yl-2-methylsulfanyl-5-oxo-5,6,7,8-tetrahydropyrido[2,3-
d]pyrimidine-6-carboxylic Acid Ethyl Ester (13). To sodium (25
wt % dispersion in paraffin wax, 1.6 g, 16.9 mmol) was added
t-butanol (30 mL) under N2 with stirring. After 10 min, a solution
of 12 (6.6 g, 15.4 mmol) in 40 mL of toluene was added to the
sodium t-butoxide solution. The mixture was then heated at 90 °C
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)-ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Amide
(17a). To the solution of 16a (20 mg, 0.036 mmol) in methanol (2
mL) was bubbled ammonia at -78 °C for 5 min in a pressure bottle.

1092 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
The bottle was then capped and warmed up to rt with stirring for
16 h. The solvent was evaporated to leave a yellow solid, which
was purified by preparative HPLC (MeCN/H2O, 1:19 to 4:1, v/v,
linear gradient over 12 min). The fractions were pooled and made
basic with sat. NaHCO3 solution and were then extracted with
CH2Cl2. The organic layer was dried over Na2SO4 and filtered. The
solvent was evaporated to leave a yellow solid, which was treated
with 1 N aq HCl solution to give a dihydrochloride salt (5.8 mg,
30%). 1H NMR (300 MHz, DMSO-d6) δ 10.48 (br s, 1H), 9.31 (s,
1H), 9.10 (s, 1H), 8.62 (s, 1H), 7.75 (m, 1H), 7.54 (m, 2H), 7.41
(m, 2H), 6.97 (br, 2H), 3.55 (m, 4H), 3.09 (t, J ) 7.31 Hz, 2H),
3.01 (t, J ) 7.42 Hz, 2H), 2.71 (m, 2H), 2.57 (m, 13H), 2.22 (m,
2H). Anal. (C30H35Cl2N7O2 ·0.35H2O) C, H, N, Cl, H2O.
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)-ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
yl Amide (17b). To the solution of 16a (20 mg, 0.036 mmol) in 1
mL methanol was added a solution of methylamine in THF (2 N,
2 mL). The mixture was stirred at 80 °C for 4 h. The solvent was
removed by vacuum to leave a yellow solid. After a preparative
HPLC purification (MeCN/H2O 1:20 to 100:0, v/v, linear gradient
over 10 min), a yellow solid was obtained (5.1 mg, 26%). 1H NMR
(400 MHz, CDCl3/CD3OD 20:1, v/v) δ 9.61 (m, 1H), 9.29 (s, 1H),
8.75 (s, 1H), 7.35 (d, J ) 7.83 Hz, 1H), 7.19 (m, 3H), 7.09 (d, J
) 7.83 Hz, 1H), 6.84 (br s, 2H), 3.00-2.90 (m, 7H), 2.64 (m, 2H),
2.47 (m, 10H), 2.24 (s, 3H), 2.15 (m, 2H). Mass spectrum (LCMS,
ESI pos.) calcd for C31H35N7O2, 538.29 (M + H); found, 538.2.
HPLC: tR ) 4.24 min (100% pure).
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
oxyamide (18a). To a mixture of 16a (10 mg, 0.018 mmol) and
methoxyamine hydrochloride (141 mg) in methanol (0.5 mL) was
added triethylamine (1 mL). The sealed vial was stirred at 100 °C
for 12 h. After cooling, water (20 mL) was added and the mixture
was extracted with CH2Cl2 (30 mL). The organic layer was washed
with brine and dried over Na2SO4. The solvent was evaporated
under vacuum and the residue was purified by preparative HPLC
(MeCN/ H2O 1:19 to 4:1, v/v linear gradient over 12 min). The
fractions were pooled, made basic with sat. NaHCO3 solution, and
were then extracted with CH2Cl2. The organic layer was dried over
Na2SO4 and filtered. The solvent was evaporated to leave a yellow
solid, which was treated with 1 N HCl solution to give a
1
dihydrochloride salt (yellow solid, 8.6 mg, 86%). H NMR (400
MHz, CDCl3) δ 11.98 (s, 1H), 9.32 (d, J ) 4.30 Hz, 1H), 8.83 (br
s, 1H), 8.01 (br s, 1H), 7.40 (d, J ) 7.43 Hz, 1H), 7.24 (br. s, 3H),
7.14 (d, J ) 6.65 Hz, 1H), 6.90 (br s, 2H), 3.88 (s, 3H), 3.05 (t, J
) 6.85 Hz, 2H), 2.97 (t, J ) 7.43 Hz, 3H), 2.66-2.78 (m, 2H),
2.41-2.65 (m, 10H), 2.31 (s, 3H), 2.21 (quin, J ) 7.34 Hz, 2H).
Mass spectrum (LCMS, ESI pos.) calcd for C31H35N7O3, 554.28
(M + H); found, 554.2. Anal. (C31H37Cl2N7O3 ·3H2O) C, H, N, Cl.
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Eth-
oxyamide (18b). Using a similar procedure described in the
preparation of 18a, the title compound was prepared from
ethoxyamine hydrochloride (150 mg) and 16a (10 mg, 0.018 mmol).
The title compound was obtained as a yellow solid (6 mg, 60%).
1H NMR (400 MHz, CDCl3) δ 11.83 (s, 1H), 9.30 (s, 1H), 8.78 (s,
1H), 7.58 (br s, 1H), 7.36 (d, J ) 7.43 Hz, 1H), 7.20 (br s, 3H),
7.10 (d, J ) 7.43 Hz, 1H), 6.85 (br s, 2H), 4.03 (q, J ) 7.04 Hz,
2H), 3.05 (t, J ) 6.85 Hz, 2H), 2.95 (t, J ) 7.42 Hz, 2H), 2.50-2.80
(m, 12H), 2.38 (s, 3H), 2.20 (m, 2H), 1.30 (t, J ) 7.04 Hz, 3H).
Mass spectrum (LCMS, ESI pos.) calcd for C32H37N7O3, 568.30
(M + H); found 568.3. HPLC: tR ) 4.41 min (100% pure).
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Iso-
propoxyamide (18c). Using a similar procedure described in the
preparation of 18a, the title compound was prepared from O-
isopropylhydroxylamine hydrochloride (170 mg) and 16a (10 mg,
0.018 mmol). The title compound was obtained as a yellow solid
(4.8 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 11.83 (s, 1H), 9.35
(br. s, 1H), 8.82 (br s, 1H), 7.70 (br s, 1H), 7.41 (d, J ) 8.22 Hz,
1H), 7.26 (br s, 3H), 7.16 (d, J ) 7.83 Hz, 1H), 6.91 (br s, 2H),
4.28 (dt, J ) 6.21, 12.23 Hz, 1H), 3.06 (t, J ) 7.24 Hz, 2H), 2.99
(t, J ) 7.24 Hz, 2H), 2.69-2.80 (m, 2H), 2.40-2.68 (m, 10H),
2.33 (s, 3H), 2.22 (quin, J ) 7.43 Hz, 2H), 1.35 (s, 3H), 1.33 (s,
3H). Mass spectrum (LCMS, ESI pos.) calcd for C33H39N7O3,
582.31 (M + H); found, 582.2. HPLC: tR ) 4.66 min (100% pure).
4-Methoxy-2-methylsulfanylpyrimidine-5-carboxylic Acid (19).
To a suspension of ethyl 4-chloro-2-methylthio-5-pyrimidinecar-
boxylate (20 g, 86 mmol) in 100 mL of MeOH was added 200 mL
of 1 N NaOH solution. After stirring at rt for 3 h, the mixture
became a homogeneous orange solution. Ethyl acetate (100 mL)
was used to extract the solution and the aqueous solution was
acidified with conc aq HCl. The precipitates were twice extracted
by ethyl acetate (200 mL). The organic layer was dried over Na2SO4
and the solvent was evaporated under vacuum to leave a white solid
8-Indan-5-yl-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methyl Amide
(17c). The ester 16b (20 mg, 0.037 mmol) was converted to the
methyl amide 17c (yellow solid, 17 mg, 87%) using a similar
1
procedure as described in the preparation of 17b. H NMR (400
MHz, CDCl3) δ 10.60 (br s, 1H), 9.29 (s, 1H), 8.78 (s, 1H), 7.75
(s, 1H), 7.60 (br s, 1H), 7.34 (d, J ) 7.83 Hz, 1H), 7.20 (m, 2H),
7.12 (d, J ) 7.43 Hz, 1H), 6.87 (br s, 2H), 3.69 (t, J ) 4.30 Hz,
4H), 3.01 (t, J ) 6.85 Hz, 2H), 2.88-3.06 (m, 5H), 2.61-2.73
(m, 2H), 2.38-2.55 (m, 6H), 2.16 (quin, J ) 7.24 Hz, 2H). Mass
spectrum (LCMS, ESI pos.) calcd for C30H32N6O3, 525.25 (M +
H); found 525.2. HPLC: tR ) 4.66 min (100% pure).
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Eth-
yl Amide (17d). To the solution of 16a (20 mg, 0.036 mmol) in 1
mL methanol was added a solution of ethylamine in THF (2 N, 2
mL). The mixture was stirred at 80 °C for 16 h. The solvent was
removed by vacuum to leave a yellow solid. After a preparative
HPLC purification (MeCN/H2O 1:20 to 100:0, v/v, linear gradient
over 10 min), a yellow solid was obtained (7.1 mg, 36%). 1H NMR
(400 MHz, CDCl3/CD3OD 20:1 v/v) δ 9.69 (br s, 1H), 9.37 (s,
1H), 8.84 (br s, 1H), 7.41 (d, J ) 7.83 Hz, 1H), 7.26 (s, 3H), 7.17
(d, J ) 7.83 Hz, 1H), 6.85 (br s, 2H), 3.50 (q, J ) 7.24 Hz, 2H),
3.05 (t, J ) 7.04 Hz, 2H), 2.98 (t, J ) 7.04 Hz, 2H), 2.66-2.82
(m, 4H), 2.38-2.64 (m, 8H), 2.31 (s, 3H), 2.12-2.28 (m, 2H),
1.27 (t, J ) 7.24 Hz, 3H). Mass spectrum (LCMS, ESI pos.) calcd
for C32H37N7O2, 553.30 (M + H); found 553.3. HPLC: tR ) 4.56
min (100% pure).
8-Indan-5-yl-2-{4-[2-(4-methylpiperazin-1-yl)ethyl]phenylamino}-
5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid (2-
Hydroxyethyl)amide (17e). To a solution of 16a (25 mg, 0.045
mmol) in methanol (1 mL) was added 2-aminoethanol (0.5 mL).
The sealed vial was heated at 110 °C overnight. The solvent was
evaporated under vacuum, and the residue was purified by prepara-
tive HPLC (MeCN/ H2O 1:19 to 4:1, v/v, linear gradient over 12
min). The title compound was obtained as an off-white solid (22.8
1
(16 g, 93%), which was dried under vacuum overnight. H NMR
(400 MHz, CDCl3/CD3OD 10:1, v/v) δ 8.95 (s, 1H), 4.30 (s, 3H),
2.77 (s, 3H).
3-(4-Methoxy-2-methylsulfanylpyrimidin-5-yl)-3-oxo-propion-
ic Acid Ethyl Ester (20). Acid 19 (16 g, 80 mmol) was suspended
in CH2Cl2 (300 mL) and cooled in an ice bath. To the stirring
solution was added a drop of DMF and then oxalyl chloride (23
mL, 258 mmol) dropwise. Upon the completion of the addition,
the mixture was allowed to stir at rt for 4 h. The solvent was
evaporated without heating to leave a brown solid, which was used
for next step without further purification.
1
mg, TFA salt, 64%). H NMR (400 MHz, CDCl3/CD3OD 10:1,
v/v) δ 9.30(s, 1H), 8.75 (s, 1H), 7.38 (d, J ) 7.83 Hz, 1H), 7.22
(m, 3H), 7.15 (d, J ) 7.83 Hz, 1H), 6.90 (br. s, 2H), 3.72 (t, J )
5.28 Hz, 2H), 3.55 (t, J ) 5.28 Hz, 2H), 2.90 (m, 4H), 2.30-2.80
(m, 15H), 2.18 (m, 2H). Mass spectrum (LCMS, ESI pos.) calcd
for C32H37N7O3, 568.30 (M + H); found, 568.3. HPLC: tR ) 4.03
min (100% pure).
To a solution of ethyl hydrogen malonate (23 mL, 214 mmol)
in THF (150 mL) was added dropwise MeMgBr in Et2O (3 M,

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1093
143 mL, 429 mmol) under nitrogen in an ice bath. After the mixture
was stirred for 20 min, a suspension of the above acid chloride in
THF (300 mL) was added slowly. After being stirred at rt for 2 h,
the reaction mixture was poured into ice water, acidified to pH
5-6 with conc aq HCl, and twice extracted with ethyl acetate (200
mL). The organic layer was dried over Na2SO4 and concentrated
under vacuum to afford a crude product, which was purified by
flash chromatography (eluted with ethyl acetate/hexanes 1:19 to
prepared similarly to 15) and 4-(2-pyrrolidin-1-yl-ethyl)phenylamine
(100 mg, 0.53 mmol) in AcOH (1 mL) was heated at 110 °C for 10
min. The reaction mixture was concentrated, and the remaining residue
was partitioned between sat. NaHCO3 and CH2Cl2. The organic layer
was dried over Na2SO4, filtered, and the solvent was evaporated. The
residue was purified by preparative HPLC (MeCN/ H2O 1:10 to 10:1,
v/v, linear gradient over 12 min). The ester obtained was converted to
the title compound using a similar procedure described in the
1
1
1:9, v/v) to give a white solid (7.0 g, 28%). H NMR (400 MHz,
preparation of 18a. A yellow solid was obtained (50 mg, 39%). H
CDCl3) indicated that the presence of both enol and keto forms in
a 1:2 ratio: δ 12.64 (s, 1H), 8.85 (s, 2H), 8.84 (s, 1H), 6.03 (s,
1H), 4.25 (q, J ) 7.17 Hz, 2H), 4.19 (q, J ) 7.17 Hz, 4H), 4.09 (s,
3H), 4.07(s, 6H), 3.91 (s, 4H), 2.60 (s, 6H), 2.59 (s, 3H), 1.33 (t,
J ) 7.04 Hz, 3H), 1.25 (t, J ) 7.04 Hz, 6H).
NMR (400 MHz, CDCl3) δ 11.95 (s, 1H), 9.30 (s, 1H), 8.75 (s, 1H),
7.75 (br s, 1H), 7.55 (d, J ) 8.61 Hz, 2H), 7.20 (d, J ) 8.61 Hz, 2H),
5.08 (t, J ) 11.93 Hz, 1H), 3.81 (s, 3H), 2.79-2.92 (m, 2H), 2.68-2.78
(m, 2H), 2.60 (br, 4H), 1.86-2.10 (m, 6H), 1.56-1.86 (m, 4H),
1.38-1.56 (m, 2H), 1.15-1.38 (m, 2H). Mass spectrum (LCMS, ESI
pos.) calcd for C27H34N6O3, 491.27 (M + H); found, 491.2. HPLC: tR
) 4.39 min (100% pure).
8-Indan-2-methylsulfanyl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimi-
dine-6-carboxylic Acid Ethyl Ester (21). A mixture of 20 (10 g,
37 mmol), acetic anhydride (10 mL, 100 mmol), and triethylortho-
formate (10 mL, 60 mmol) was heated to reflux at 130 °C for 4 h,
during which period the resulting ethyl acetate was distilled off
under atmospheric pressure. After concentration under vacuum, the
dark-brown oil that was obtained was diluted with THF (100 mL)
and 5-aminoindan (5.5 g 39 mmol) was added. The mixture was
stirred at rt for 16 h. Then K2CO3 (5.6 g, 40 mmol) was added.
The mixture was stirred at rt for 16 h. After an aqueous work up
using CH2Cl2 as extracting solvent, the organic solvent was
evaporated to leave a yellow solid, which was used in the next
step without further purification.
8-Indan-2-methylsulfanyl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimi-
dine-6-carboxylic Acid (22). To a solution of 21 in 1,4-dioxane (100
mL) was added aq HCl (1 N, 120 mL). The mixture was heated to
reflux for 4 h. The solution was cooled in an ice water bath, filtered,
and the resulting solid was washed with water. The title compound
(11.1 g, 85%) was obtained as a white solid after drying under
vacuum. 1H NMR (400 MHz, CDCl3) δ 14.02 (br s, 1H), 9.50 (br
s, 1H), 8.89 (s, 1H), 7.38 (d, J ) 7.69 Hz, 1H), 7.24 (s, 1H), 7.16
(d, J ) 7.69 Hz, 1H), 3.01 (q, J ) 8.12 Hz, 4H), 2.34 (s, 3H),
2.15-2.25 (m, 2H).
2-Benzylamino-8-indan-5-yl-5-oxo-5,8-dihydropyrido[2,3-d]py-
rimidine-6-carboxylic Acid Methoxyamide (26). A solution of 24
(30 mg, 0.075 mmol) and benzylamine (16 mg, 0.15 mmol) in acetic
acid (1 mL) was stirred at 110 °C for 10 min. A precipitate formed
upon cooling, which was filtered and washed with acetonitrile. The
white solid (8 mg, 24%) was dried and no further purification was
1
required. H NMR (400 MHz, CDCl3) δ 12.00 (s, 1H), 9.25 (s,
1H), 8.74 (s, 1H), 7.29-7.35 (m, 2H), 7.22-7.25 (m, 2H), 7.18
(s, 1H), 7.01-7.10 (m, 2H), 6.25 (br s, 1H), 4.34 (d, J ) 6.26 Hz,
2H), 3.89 (s, 3H), 2.93-3.03 (m, 4H), 2.13-2.22 (m, 2H). Mass
spectrum (LCMS, ESI pos.) calcd for C25H23N5O3, 442.18 (M +
H); found, 442.1. HPLC: tR ) 6.74 min (100% pure).
2-Butylamino-8-indan-5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyri-
midine-6-carboxylic Acid Methoxyamide (27). A solution of 24 (50
mg, 0.12 mmol) and n-butylamine (18 mg, 0.24 mmol) in acetic
acid (1 mL) was stirred at 110 °C for 10 min. A precipitate formed
upon cooling, which was filtered and washed with acetonitrile. The
white solid (15 mg, 30%) was dried and no further purification
1
was required. H NMR (400 MHz, CDCl3) δ 11.87 (s, 1H), 9.18
(s, 1H), 8.76 (s, 1H), 7.34 (d, J ) 7.83 Hz, 1H), 7.21 (s, 1H), 7.12
(d, J ) 7.83 Hz, 1H), 3.89 (s, 3H), 3.12-3.22 (m, 2H), 2.99 (q, J
) 7.83 Hz, 4H), 2.18 (t, J ) 7.43 Hz, 2H), 1.44 (t, J ) 7.24 Hz,
2H), 1.22 (dt, J ) 7.48, 15.16 Hz, 2H), 0.80 (t, J ) 7.43 Hz, 3H).
Mass spectrum (LCMS, ESI pos.) calcd for C22H25N5O3, 408.20
(M + H); found, 408.1. HPLC: tR ) 6.91 min (100% pure).
8-Indan-5-yl-2-methylsulfanyl-5-oxo-5,8-dihydropyrido[2,3-d]py-
rimidine-6-carboxylic Acid Methoxyamide (23). To a suspension
of 22 (1.30 g, 3.68 mmol) in CH2Cl2 (30 mL) at 0 °C was added a
drop of DMF followed by oxalyl chloride (0.5 mL, 5.52 mmol). The
solution was stirred at rt for 1 h. The solvent was evaporated under
vacuum without heating, and the residue was dissolved in CH2Cl2 (30
mL). To this solution was added methoxyamine hydrochloride (0.52
g, 6 mmol), followed by slow addition of triethylamine (1.4 mL, 10
mmol) at 0 °C. The solution was stirred at 0 °C for 10 min and rt for
1 h. Water was added and the aqueous solution was twice extracted
with CH2Cl2 (200 mL). The combined organic layers were dried over
Na2SO4 and concentrated under vacuum to afford a crude product,
which was purified by recrystallization from ethyl acetate/hexanes (2:
1, v/v). The title compound was obtained as an off-white solid (1.35
8-Indan-5-yl-2-(6-methoxypyridin-3-ylamino)-5-oxo-5,8-dihydro-
pyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxyamide (28). A
solution of 24 (50 mg, 0.12 mmol) and 6-methoxy-pyridin-3-
ylamine (50 mg, 0.4 mmol) in acetic acid (1 mL) was stirred at
110 °C for 10 min. The solvent was evaporated, and the residue
was purified by preparative HPLC (MeCN/ H2O 1:4 to 100:0, v/v,
linear gradient over 12 min). The title compound was obtained as
a yellow solid (37.2 mg, 68%). 1H NMR (400 MHz, CDCl3/CD3OD
19:1, v/v) δ 9.34 (s, 1H), 8.81 (br s, 1H), 8.03 (br s, 1H), 7.70 (d,
J ) 7.83 Hz, 1H), 7.41 (d, J ) 7.83 Hz, 1H), 7.25 (s, 1H), 7.13 (d,
J ) 7.43 Hz, 1H), 6.38 (d, J ) 8.61 Hz, 1H), 3.90 (s, 3H),
2.95-3.12 (m, 4H), 2.57 (br s, 3H), 2.23 (quin, J ) 7.43 Hz, 2H).
Mass spectrum (LCMS, APCI pos.) calcd for C24H22N6O4, 459.17
(M + H); found, 459.1. HPLC: tR ) 6.17 min (100% pure).
8-Indan-5-yl-2-[4-(2-morpholin-4-yl-ethyl)phenylamino]-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxy-
amide (29). A solution of 24 (20 mg, 0.048 mmol) and 4-(2-
morpholin-4-yl-ethyl)-phenylamine (18 mg, 0.087 mmol) in acetic
acid (1 mL) was stirred at 110 °C for 10 min. The solvent was
evaporated, and the residue was purified by preparative HPLC
(MeCN/ H2O 1:19 to 4:1, v/v, linear gradient over 12 min). The
title compound was obtained as a yellow solid (8 mg, HCl salt,
31%). 1H NMR (400 MHz, CDCl3/CD3OD 10:1 v/v) δ 9.30 (br s,
1H), 8.75 (br s, 1H), 7.40 (d, J ) 7.43 Hz, 1H), 7.25-7.36 (m,
2H), 7.21 (s, 1H), 7.13 (d, J ) 7.83 Hz, 1H), 6.93 (br s, 2H),
4.08-4.25 (m, 2H), 3.96 (d, J ) 10.96 Hz, 2H), 3.84 (s, 3H), 3.47
(d, J ) 12.13 Hz, 2H), 2.80-3.09 (m, 10H), 2.13-2.32 (m, 2H).
Mass spectrum (LCMS, APCI pos.) calcd for C30H32N6O4, 541.24
(M + H); found, 541.2. HPLC: tR ) 4.61 min (100% pure).
1
g, 96%). H NMR (400 MHz, CDCl3) δ 11.84 (s, 1H), 9.45 (br s,
1H), 8.88 (s, 1H), 7.36 (d, J ) 7.83 Hz, 1H), 7.23 (s, 1H), 7.12-7.17
(m, 1H), 3.90 (s, 3H), 3.00 (q, J ) 7.69 Hz, 4H), 2.33 (s, 3H), 2.19
(quin, J ) 7.43 Hz, 2H).
8-Indan-5-yl-2-methanesulfonyl-5-oxo-5,8-dihydropyrido[2,3-
d]pyrimidine-6-carboxylic Acid Methoxyamide (24). To a stirring
solution of 23 (2.4 g, 6.3 mmol)) in CH2Cl2 (100 mL) was slowly
added mCPBA (3.8 g, 15.8 mmol) at 0 °C. The mixture was then
stirred at rt for 5 h. A Na2S2O3 solution (10%, 20 mL) was added
to quench the reaction. The mixture was extracted with CH2Cl2
and the organic layer was twice washed with sat. NaHCO3 solution
and dried over Na2SO4. The solvent was evaporated to leave a pale-
yellow solid (2.3 g, 88%). 1H NMR (400 MHz, CDCl3) δ 11.61 (s,
1H), 9.85 (s, 1H), 9.05 (s, 1H), 7.39 (d, J ) 7.83 Hz, 1H), 7.23 (s,
1H), 7.15 (d, J ) 8.22 Hz, 1H), 3.91 (s, 3H), 3.22 (s, 3H), 3.01 (q,
J ) 7.43 Hz, 4H), 2.20 (t, J ) 7.63 Hz, 2H).
8-Cyclohexyl-5-oxo-2-[4-(2-pyrrolidin-1-yl-ethyl)phenylamino]-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxya-
mide (25). 8-Cyclohexyl-2-methanesulfonyl-5-oxo-5,8-dihydropyrido[2,3-
d]pyrimidine-6-carboxylic acid ethyl ester (100 mg, 0.26 mmol,

1094 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
2-{4-[2-(4,4-Difluoropiperidin-1-yl)ethyl]phenylamino}-8-indan-
5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (30). A. 4,4-Difluoro-1-[2-(4-nitro-phenyl)-ethyl]-
piperidine. To the solution of the 1-(2-bromo-ethyl)-4-nitro-benzene
(450 mg) and 4,4-difluoro-piperidine or 3,3-difluoro-piperidine (250
mg) in DMSO (5 mL) was added K2CO3 (400 mg). The mixture
was heated at 90 °C for 2 h. Water was added, and the mixture
was extracted with CH2Cl2. The combined organic extracts
were washed with brine, dried (Na2SO4), filtered, and the filtrate
concentrated and purified by flash chromatography (EtOAc/hexanes
(80 mg, 0.19 mmol) and 4-[2-(4-aminophenyl)ethyl]-1-methylpip-
erazin-2-one (60 mg, 0.26 mmol). The title compound was obtained
as a yellow solid (41.1 mg, 38%). H NMR (400 MHz, CDCl3) δ
1
11.97 (s, 1H), 9.36 (br s, 1H), 8.83 (s, 1H), 7.77 (br s, 1H), 7.42
(d, J ) 7.83 Hz, 1H), 7.27 (s, 3H), 7.10-7.20 (m, 1H), 6.92 (br s,
2H), 3.90 (s, 3H), 3.34 (t, J ) 5.48 Hz, 2H), 3.21 (s, 2H), 3.07 (t,
J ) 7.24 Hz, 2H), 2.88-3.04 (m, 5H), 2.74 (t, J ) 5.48 Hz, 4H),
2.54-2.65 (m, 2H), 2.23 (quin, J ) 7.43 Hz, 2H). Mass spectrum
(LCMS, APCI pos.) calcd for C31H33N7O4, 568.24 (M + H); found,
568.2. HPLC: tR ) 4.47 min (99.2% pure).
2-[4-(2-Acetylsulfamoyl-ethyl)phenylamino]-8-indan-5-yl-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxy-
amide (33). A. 2-(4-Nitro-phenyl)ethanesulfonyl Chloride. 1-(2-
Bromo-ethyl)-4-nitrobenzene (3 g, 13 mmol) and potassium
thioacetate (3 g, 26 mmol) in DMSO (10 mL) were stirred at rt for
3 h. EtOAc was used to dilute the reaction mixture. The organic
layer was washed with water twice (2× 100 mL) and then with
brine and dried over Na2SO4. The solvent was evaporated in vacuo
to give a brown solid (∼3 g), which was taken up in 50 mL of
acetic acid. To the stirring solution was added 20 mL of hydrogen
peroxide (30% in water). The resulting yellow solution was stirred
at rt overnight. Water (50 mL) was added, and the solvent was
evaporated in vacuo with minimal heating. The yellow residue was
dried in vacuo for two days, suspended in thionyl chloride (18 mL),
and heated at reflux (80 °C) for 6 h. The volatiles were evaporated
to give the title compound as a yellow solid, which was used in
the next step without further purification
B. 2-(4-Aminophenyl)ethanesulfonic Acid Acetylamide. A mix-
ture of 2-(4-nitro-phenyl)ethanesulfonyl chloride (200 mg, 0.8
mmol) and ammonium hydroxide (5 mL) in CH2Cl2 (10 mL) was
stirred at rt for 2 h. To the reaction mixture was added CH2Cl2
(100 mL) and water (100 mL). The organic fraction was dried over
Na2SO4, filtered, and the filtrate was concentrated in vacuo. To a
solution of the residue in CH2Cl2 (10 mL) was added pyridine (0.3
mL) and acetic anhydride (75 µL), and the reaction mixture was
stirred at rt for 3 days. After an aqueous workup, the reaction
mixture was extracted with CH2Cl2. The combined organic fractions
were dried over Na2SO4, filtered, and the filtrate was concentrated.
The residue was purified by flash chromatography (CH2Cl2/CH3OH
19:1, v/v). The resulting nitro compound was hydrogenated over
Pd/C (10%) in methanol under atmosphere pressure. The reaction
mixture was filtered, and the filtrate was concentrated to give the
title compound as a pink solid (66 mg)
C. 2-[4-(2-Acetylsulfamoyl-ethyl)phenylamino]-8-indan-5-yl-5-
oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
oxyamide (33). The title compound was prepared by reacting 2-(4-
aminophenyl)ethanesulfonic acid acetylamide (15 mg, 0.062
mmol) with 24 (20 mg, 0.048 mmol) using a similar procedure
as described in the preparation of 31. 1H NMR (400 MHz,
CDCl3/CD3OD 10:1 v/v) δ 9.32 (s, 1H), 8.77 (s, 1H), 7.40 (d,
J ) 7.81 Hz 1H), 7.30 (br, 2H), 7.25 (s, 1H), 7.15 (d, J ) 8.22
Hz, 1H), 6.90 (br, 2H), 3.85 (s, 3H), 3.58 (m, 2H), 3.00 (m,
6H), 2.20 (m, 2H), 1.95 (s, 3H). Mass spectrum (LCMS, APCI
pos.) calcd for C28H28N6O6S, 577.18 (M + H); found 577.1.
HPLC: tR ) 5.63 min (100% pure).
1
1:5 v/v) to afford a yellow solid (200 mg). H NMR (400 MHz,
CDCl3) δ 8.14 (m, 2H), 7.37 (t, 2H, J ) 7.0), 2.91 (m, 2H), 2.68
(m, 2H), 2.62 (m, 4H), 2.00 (m, 4H).
B. 4-[2-(4,4-Difluoro-piperidin-1-yl)-ethyl]-phenylamine. 4,4-
Difluoro-1-[2-(4-nitro-phenyl)-ethyl]-piperidine (200 mg) was dis-
solved in EtOH/AcOH (6 mL, 1:1 v/v). Iron powder (200 mg) was
then added. The mixture was refluxed at 100 °C for 2 h. After
cooling down, the mixture was filtered through celite and washed
with methanol. The solvent was evaporated, and the residue was
suspended in sat. NaHCO3 solution (100 mL). The suspension was
extracted with CH2Cl2 (100 mL) twice. The organic layers were
combined and washed with brine and dried (Na2SO4). The solvent
was evaporated to leave brown oil, which was used for next step
without further purification.
C. 2-{4-[2-(4,4-Difluoropiperidin-1-yl)ethyl]phenylamino}-8-in-
dan-5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (30). A solution of 24 (100 mg, 0.24 mmol) and
4-[2-(4,4-difluoropiperidin-1-yl)ethyl]phenylamine (prepared simi-
larly as 31, 125 mg, 0.52 mmol) in acetic acid (1 mL) was stirred
at 110 °C for 10 min. The solvent was evaporated, and the residue
was purified by preparative HPLC (MeCN/ H2O 1:4 to 100:0, v/v,
linear gradient over 12 min). The title compound was obtained as
1
a yellow solid (75.4 mg, 55%). H NMR (400 MHz, CDCl3) δ
11.96 (s, 1H), 9.37 (s, 1H), 8.83 (s, 1H), 7.42 (d, J ) 7.83 Hz,
1H), 7.26 (s, 7H), 7.18 (d, J ) 7.83 Hz, 1H), 6.93 (br s, 2H), 3.90
(s, 3H), 3.07 (t, J ) 7.33 Hz, 2H), 3.01 (t, J ) 7.33 Hz, 2H),
2.69-2.79 (m, 2H), 2.55-2.67 (m, 6H), 2.19-2.29 (m, 2H),
1.95-2.10 (m, 4H). Mass spectrum (LCMS, ESI pos.) calcd for
C31H32F2N6O3, 575.25 (M + H); found, 575.2. HPLC: tR ) 5.02
min (100% pure).
8-Indan-5-yl-5-oxo-2-{4-[2-(3-oxo-piperazin-1-yl)ethyl]phenyl-
amino}-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
oxyamide (31). To a solution of 1-(2-bromo-ethyl)-4-nitro-benzene
(0.6 g, 2.6 mmol) and 2-piperidinone (0.26 g, 2.6 mmol) in DMSO
(10 mL) was added K2CO3 (0.4 g, 3.0 mmol). The mixture was
stirred at 80 °C for 4 h. Water was then added, and the mixture
was twice extracted with CH2Cl2 (100 mL). The combined organic
extracts were washed with brine, dried over Na2SO4, filtered, and
the filtrate concentrated and purified by flash chromatograghy (ethyl
acetate/hexanes 1:5, v/v) to afford an off-white solid. The nitro
compound was then reduced to the aniline using H-cube (1 mL/
min, 10% Pd/C). 4-[2-(4-Aminophenyl)ethyl]piperazin-2-one was
1
obtained as a yellow solid (0.42 g, 74%). H NMR (400 MHz,
CDCl3) δ 6.97 (d, J ) 8.22 Hz, 2H), 6.62 (d, J ) 8.22 Hz, 2H),
3.51 (br. s., 2H), 3.30-3.38 (m, 2H), 3.18 (s, 2H), 2.64-2.72 (m,
4H), 2.55-2.64 (m, 2H).
8-Indan-5-yl-5-oxo-2-(4-piperidin-4-yl-phenylamino)-5,8-dihy-
dropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxyamide (34).
The title compound was prepared from 24 (100 mg, 0.24 mmol)
and 4-(4-amino-phenyl)-piperidine-1-carboxylic acid tert-butyl
ester (60 mg, 0.22 mmol) using a similar procedure to that
described in the preparation of 29. The Boc group was removed
by TFA. The title compound was obtained as a yellow solid (66
Using a similar procedure as described in the preparation of 29,
the title compound was prepared from 24 (25 mg, 0.06 mmol) and
4-[2-(4-aminophenyl)ethyl]piperazin-2-one (20 mg, 0.09 mmol). A
yellow solid (10.9 mg, HCl salt, 31%) was obtained. 1H NMR (400
MHz, CDCl3/CD3OD 19:1, v/v) δ 9.35 (br s, 1H), 8.81 (br s, 1H),
7.44 (d, J ) 7.83 Hz, 1H), 7.28 (br s, 3H), 7.18 (d, J ) 7.83 Hz,
1H), 6.91 (br s, 2H), 3.90 (s, 3H), 3.34-3.42 (m, 2H), 3.21 (s,
2H), 3.08 (t, J ) 7.24 Hz, 2H), 3.01 (t, J ) 7.43 Hz, 2H), 2.59-2.78
(m, 6H), 2.24 (quin, J ) 7.43 Hz, 2H). Mass spectrum (LCMS,
APCI pos.) calcd for C30H31N7O4, 554.24 (M + H); found, 554.2.
HPLC: tR ) 4.35 min (98.8% pure).
1
mg, 59%). H NMR (400 MHz, CDCl3) δ11.90 (br s, 1H), 9.30
(br s, 1H), 8.76 (br s, 1H), 7.54 (br s, 1H), 7.36 (d, J ) 7.83
Hz, 1H), 7.15-7.27 (m, 3H), 7.11 (d, J ) 8.22 Hz, 1H), 6.88
(br s, 2H), 3.83(s, 3H), 3.19 (d, J ) 12.13 Hz, 2H), 3.02 (t, J )
6.65 Hz, 2H), 2.94 (t, J ) 7.43 Hz, 2H), 2.63-2.76 (m, 2H),
2.49 (br s, 1H), 2.17 (quin, J ) 7.24 Hz, 2H), 1.67-1.79 (m,
2H), 1.62 (br s, 2H). Mass sSpectrum (LCMS, APCI pos.) calcd
8-Indan-5-yl-2-{4-[2-(4-methyl-3-oxo-piperazin-1-yl)ethyl]phe-
nylamino}-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxyl-
ic Acid Methoxyamide (32). Using a similar procedure as described
in the preparation of 31, the title compound was prepared from 24
for C29H30N6O3, 511.24 (M + H); found, 511.2. HPLC: tR
4.57 min (100% pure).
)

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1095
2-{4-[1-(2-Hydroxy-ethyl)piperidin-4-yl]phenylamino}-8-indan-
5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (35). To a solution of 34 (36 mg, 0.07 mmol) in
DMSO (0.5 mL) was added 2-bromoethanol (7 µL, 0.1 mmol) and
triethylamine (14 µL, 0.1 mmol). The solution was stirred at 80 °C
for 2 h. Water was added, and the mixture was extracted with
CH2Cl2 (100 mL). The organic layer was dried over Na2SO4, and
the solvent was evaporated. The product was purified by preparative
HPLC (MeCN/ H2O 1:19 to 4:1, v/v, linear gradient over 12 min).
over Na2SO4 and concentrated. The residue was purified by flash
chromatography (eluted with ethyl acetate/hexanes 2:3, v/v). A
white solid was obtained (563 mg, 79%). H NMR (400 MHz,
CDCl3) δ 8.35 (s, 1H), 8.30 (d, J ) 4.80 Hz, 1H), 7.53-7.59 (m,
J ) 8.59 Hz, 2H), 7.45-7.51 (m, J ) 8.59 Hz, 2H), 7.33-7.44
(m, 5H), 7.24 (d, J ) 4.80 Hz, 1H), 6.79 (br s., 1H), 5.23 (s, 2H),
3.91 (s, 3H).
B. [4-(5-Methoxy-1-methyl-1,2,3,6-tetrahydropyridin-4-yl)phe-
nyl]carbamic Acid Benzyl Ester (41). To a solution of 40 (563 mg,
1.68 mmol) in acetone (10 mL) was added iodomethane (158 µL,
2.5 mmol). The mixture was stirred at 50 °C for 1 h then at rt
overnight. The solvent was evaporated, and the residue was
dissolved in ethanol (10 mL). To the solution cooled in an ice water
bath was added sodium borohydride (95 mg, 2.5 mmol) portionwise.
The solution was stirred at rt for 1 h. Water was added, and the
reaction mixture was twice extracted with ethyl acetate (50 mL).
The organic layer was dried over Na2SO4 and concentrated to give
the title compound as a brown solid (460 mg, 81%). 1H NMR (400
MHz, CDCl3) δ 7.29-7.43 (m, 9H), 6.75 (br s, 1H), 5.20 (s, 2H),
3.43 (s, 3H), 3.10 (m, 2H), 2.55-2.63 (m, 2H), 2.46-2.53 (m,
2H), 2.43 (s, 3H).
1
1
A yellow solid was obtained (11 mg, 28%). H NMR (400 MHz,
CDCl3) δ 11.98 (s, 1H), 9.36 (br s, 1H), 8.83 (br s, 1H), 7.64 (br
s, 1H), 7.43 (d, J ) 7.83 Hz, 1H), 7.26 (s, 4H), 7.17 (d, J ) 7.83
Hz, 1H), 6.94 (br s, 1H), 3.90 (s, 3H), 3.67 (t, J ) 5.28 Hz, 2H),
3.08 (m, 2H), 3.00 (t, J ) 7.43 Hz, 2H), 2.57-2.66 (m, 2H), 2.47
(t, J ) 11.54 Hz, 1H), 2.16-2.30 (m, 4H), 1.67-1.90 (m, 6H).
Mass spectrum (LCMS, ESI pos.) calcd for C31H34N6O4, 555.24
(M + H); found, 555.3. HPLC: tR ) 4.48 min (99.6% pure).
{4-[4-(8-Indan-5-yl-6-methoxycarbamoyl-5-oxo-5,8-dihydropy-
rido[2,3-d]pyrimidin-2-ylamino)phenyl]piperidin-1-yl}-acetic Acid
(36). To a solution of 34 (30 mg, 0.06 mmol) in DMSO (2 mL)
was added t-butyl bromoacetate (10 µL, 0.065 mmol) and K2CO3
(11 mg, 0.08 mmol). The solution was stirred at 60 °C for 1 h.
Water was added, and the mixture was extracted with CH2Cl2 (100
mL). The organic layer was dried over Na2SO4, and the solvent
was evaporated. The solvent was removed under vacuum. The
residue was purified by preparative TLC (developed in MeOH/
CH2Cl2 1:10, v/v) to afford a yellow solid. The solid was then
treated with TFA/CH2Cl2 (5 mL, 1:1 v/v) at rt for 1 h. The solvent
was evaporated, and the residue was dissolved in CH2Cl2 (50 mL).
The organic layer was washed with sat. NaHCO3 and brine and
dried over Na2SO4. The solvent was evaporated to leave a yellow
C. [4-(1-Methyl-3-oxo-piperidin-4-yl)phenyl]carbamic Acid Ben-
zyl Ester (42). To a solution of 41 (460 mg, 1.31 mmol) in THF
(10 mL) was added aq HCl (6 N, 5 mL). The mixture was stirred
at 50 °C for 16 h. The solvent was evaporated, and the residue
was purified by flash chromatography (CH3OH/CH2Cl2 1:9, v/v).
1
A white solid was obtained (300 mg, 68%). H NMR (400 MHz,
CDCl3) δ 7.28-7.43 (m, 7H), 7.05 (d, J ) 8.59 Hz, 2H), 6.91 (br
s, 1H), 5.18 (s, 2H), 3.65 (t, J ) 6.32 Hz, 0.5H), 3.56 (t, J ) 6.57
Hz, 0.5H), 3.44 (t, J ) 9.35 Hz, 1H), 3.27-3.35 (m, 1H), 2.95-3.04
(m, 1H), 2.85 (d, J ) 13.89 Hz, 1H), 2.53 (dt, J ) 7.07, 11.62 Hz,
1H), 2.38 (s, 3H), 2.16-2.25 (m, 2H), 1.81-1.91 (m, 0.5H),
1.68-1.73 (m, 0.5H).
1
solid (10.5 mg, 31%). H NMR (300 MHz, CD3OD) δ 9.25 (s,
1H), 8.68 (s, 1H), 7.46 (d, J ) 7.83 Hz, 1H), 7.36 (br s, 3H), 7.24
(d, J ) 7.83 Hz, 1H), 6.95 (br s., 2H), 4.14 (s, 2H), 3.83 (s, 3H),
3.77 (d, J ) 12.13 Hz, 2H), 3.19-3.26 (m, 2H), 3.11 (br s, 2H),
3.01 (t, J ) 7.24 Hz, 2H), 2.83 (br s, 1H), 2.17-2.31 (m, 2H),
1.93-2.12 (m, 4H). Mass spectrum (LCMS, APCI pos.) calcd for
C31H32N6O5, 569.24 (M + H); found, 569.1. HPLC: tR ) 4.53 min
(98.9% pure).
D. [4-(3,3-Difluoro-1-methylpiperidin-4-yl)phenyl]carbamic Acid
Benzyl Ester (43). To a solution of 42 (300 mg, 0.89 mmol) in
CH2Cl2 (5 mL) cooled in an ice-water bath was added Deoxo-
Fluor (50% in toluene, 1 mL, 2.7 mmol) slowly under argon.
The mixture was stirred at rt overnight. Water was added, and
the reaction mixture was twice extracted with CH2Cl2 (20 mL).
The organic layer was dried over Na2SO4 and concentrated to a
brown oil, which was purified by flash chromatography (eluted
with CH3OH/CH2Cl2 1:9, v/v). A yellow solid was obtained (90
8-Indan-5-yl-2-[4-(1-methylpiperidin-4-yl)phenylamino]-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxy-
amide (37). To a well-ground mixture of 24 (4 g, 9.66 mmol) and
4-(1-methylpiperidin-4-yl)phenylamine (2.5 g, 13.5 mmol) was
added 20 mL of AcOH. The mixture was stirred at 110 °C for 20
min. The AcOH was evaporated under high vacuum. The residue
was dissolved in CH2Cl2 (400 mL), and the solution was washed
with sat. NaHCO3 and brine. The organic layer was dried over
Na2SO4, and the solvent was evaporated. The product was purified
by flash chromatography, followed by recrystallization from
methanol twice. The off-white solid was dissolved in CH2Cl2 and
methanol and treated with aq HCl (1 N, 1 equiv). The solvent was
evaporated in high vacuum to leave a yellow solid (3.8 g, 70%).
1H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 9.35 (br s, 1H), 8.82
(br s, 1H), 7.70 (br s, 1H), 7.41 (d, J ) 8.22 Hz, 1H), 7.26 (br s,
3H), 7.17 (d, J ) 7.83 Hz, 1H), 6.95 (br s, 2H), 3.90 (s, 3H), 3.07
(t, J ) 7.04 Hz, 2H), 2.90-3.04 (m, 4H), 2.36-2.48 (m, 1H), 2.32
(s, 3H), 2.23 (quin, J ) 7.34 Hz, 2H), 1.95-2.12 (m, 2H),
1.63-1.86 (m, 4H). Mass spectrum (LCMS, ESI pos.) calcd for
1
mg, 28%). H NMR (400 MHz, CDCl3) δ 7.36 (m, 7H), 7.25
(d, J ) 8.42, 2H), 6.82 (s, 1H), 5.18 (s, 2H), 3.16 (m, 1H), 2.99
(d, J ) 10.7, 1H), 2.82 (m, 1H), 2.38 (s, 3H), 2.33 (m, 2H),
1.84 (m, 1H).
E. 4-(3,3-Difluoro-1-methylpiperidin-4-yl)phenylamine (44). Com-
pound 43 (90 mg, 0.25 mmol) was dissolved in methanol (100 mL)
and the solution was passed through an H-Cube (1 mL/min, 10%
Pd/C). The solvent was evaporated to leave the title compound as
1
pale-yellow oil (45 mg, 80%). H NMR (400 MHz, CD3OD) δ
7.05 (d, J ) 8.22 Hz, 2H), 6.69 (d, J ) 8.61 Hz, 2H), 3.18-3.26
(m, 1H), 3.08 (d, J ) 10.17 Hz, 1H), 2.83-3.02 (m, 1H), 2.49-2.65
(m, 1H), 2.47 (s, 3H), 2.39 (t, J ) 12.13 Hz, 1H), 2.10-2.26 (m,
1H), 1.86 (ddd, J ) 2.35, 4.70, 11.35 Hz, 1H).
F. 2-[4-(3,3-Difluoro-1-methylpiperidin-4-yl)phenylamino]-8-in-
dan-5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (38). The title compound was prepared from 24 (60
mg, 0.14 mmol) and 44 (45 mg, 0.2 mmol) using the similar
procedure as described in the preparation of 29. The title compound
C30H32N6O3, 525.25 (M
(C30H33ClN6O3 ·0.3H2O) C, H, N.
+
H); found, 525.2. Anal.
1
2-[4-(3,3-Difluoro-1-methylpiperidin-4-yl)phenylamino]-8-indan-
5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (38). A. [4-(3-Methoxy-pyridin-4-yl)phenyl]car-
bamic Acid Benzyl Ester (40). 4-Bromo-3-methoxy-pyridine (400
mg, 2.13 mmol) and 4-Cbz-amino-phenyl boronic acid (576 mg,
2.13 mmol) was dissolved in a mixture of ethanol (4 mL) and
toluene (2 mL). To the solution was added 2 M Na2CO3 solution
(5 mL). The system was purged with N2. Pd(PPh3)4 (20 mg) was
added under N2. The mixture was refluxed at 110 °C for 2 h. After
cooling, water was added, and the reaction mixture was twice
extracted with ethyl acetate (30 mL). The organic layer was dried
was obtained as a yellow solid (45.3 mg, 58%). H NMR (400
MHz, CDCl3/CD3OD, 19:1, v/v) δ 9.35 (br. s, 1H), 8.83 (br s, 1H),
7.42 (d, J ) 7.83 Hz, 1H), 7.29 (br s, 2H), 7.26 (s, 1H), 7.17 (d,
J ) 7.83 Hz, 1H), 7.04 (br s, 2H), 3.89 (s, 3H), 3.12-3.25 (m,
1H), 2.92-3.11 (m, 5H), 2.71-2.92 (m, 1H), 2.41 (s, 3H),
2.26-2.39 (m, 1H), 2.10-2.26 (m, 4H), 1.79-1.91 (m, 1H). Mass
spectrum (LCMS, ESI pos.) calcd for C30H30F2N6O3, 561.23 (M +
H); found, 561.2. HPLC: tR ) 4.65 min (100% pure).
2-[3-(1-Ethyl-3-hydroxy-piperidin-3-yl)phenylamino]-8-indan-
5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (39). A. 3-(3-Aminophenyl)-1-ethylpiperidin-3-ol.

1096 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Huang et al.
To a solution of 1-ethylpiperidin-3-one (4 g, 31.4mmol) in THF
(40 mL) at -10 °C is added dropwise 3-[bis(trimethylsilyl)ami-
no]phenylmagnesium chloride (32 mL, 1 M in THF). The reaction
was stirred for 1 h with gradual warming to rt. The mixture was
then diluted with ethyl acetate, and a 0.5 M solution of aq HCl
was added. The aqueous layer was washed once more with ethyl
acetate and then was neutralized with sodium bicarbonate and
concentrated in vacuo. The resulting solid was triturated with ethyl
acetate and filtered. The filtrate was then concentrated in vacuo,
and the crude was purified by flash chromatography (CH3OH/
CH2Cl2 1:9, v/v). The title compound was obtained as yellow oil
(3.7 g, 53%). 1H NMR (400 MHz, CDCl3) δ 6.97 (t, J ) 7.83 Hz,
1H), 6.78 (s., 1H), 6.65 (d, J ) 8.02 Hz, 1H), 6.45 (dd, J ) 1.56,
8.02 Hz, 1H), 2.93 (d, 1H), 2.53 (m, 6H), 2.05 (m, 2H), 1.75 (m,
2H), 1.05 (t, J ) 7.48, 3H).
5H-[1]pyrindin-3-ylamine40) and 4-(1-methyl-piperidin-4-yl)-phe-
nylamine (20 mg, 0.1 mmol) in AcOH (1 mL) was heated at 110
°C for 10 min. The reaction mixture was concentrated, and the
remaining residue was partitioned between sat. NaHCO3 and
CH2Cl2. The organic layer was dried over Na2SO4, filtered, and
the solvent was evaporated. The residue was purified by preparative
HPLC (MeCN/H2O 1:10 to 10:1, v/v, linear gradient over 12 min).
1
A yellow solid was obtained (7.3 mg, 20%). H NMR (400 MHz,
CDCl3) δ 11.89 (s, 1H), 9.38 (s, 1H), 8.76 (s, 1H), 8.38-8.52 (m,
1H), 7.57 (s, 1H), 7.23 (br s, 2H), 7.00 (br s, 2H), 3.90 (s, 3H),
3.19 (t, J ) 7.63 Hz, 2H), 3.05 (t, J ) 7.43 Hz, 2H), 2.98 (d, J )
11.35 Hz, 2H), 2.42 (m, 1H), 2.24-2.36 (m, 5H), 2.04 (td, J )
2.93, 11.44 Hz, 2H), 1.64-1.86 (m, 4H). Mass spectrum (LCMS,
APCI pos.) calcd for C29H31N7O3, 526.25 (M + H); found, 526.3.
HPLC: tR ) 3.94 min (100% pure).
8-(4,4-Dimethylcyclohexyl)-2-[4-(1-methylpiperidin-4-yl)phenyl-
amino]-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (48). Using a similar procedure described in the
preparation of 47, the title compound was obtained as a yellow
B. 2-[3-(1-Ethyl-3-hydroxy-piperidin-3-yl)phenylamino]-8-indan-
5-yl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid
Methoxyamide (39). To a solution of 24 (50 mg, 0.12 mmol) in
DMF/1,4 dioxane (2 mL, 1:1 v/v) was added 3-(3-aminophenyl)-
1-ethylpiperidin-3-ol (42 mg, 0.144 mmol) and silver triflate (40
mg, 0.157 mmol). The mixture was stirred at 107 °C for 2 h. The
reaction mixture was diluted with ethyl acetate and ammonia (7 M
in MeOH, 2 mL) was added. The organics were washed twice with
water. The organic solvent was evaporated, and the crude was
purified on prep. TLC plate (7 N ammonia in MeOH/CH2Cl2 19:1,
v/v) affording the title compound as a yellow solid (22 mg, 33%).
1H NMR (400 MHz, CDCl3) δ 9.30 (s, 1H), 8.73 (s, 1H), 7.34 (d,
J ) 7.83 Hz, 1H), 7.19 (s, 1H), 7.06 (m, 2H), 6.86 (t, J ) 2.02 Hz,
1H), 6.75 (d, J ) 7.83 Hz, 1H), 6.535(dd, J ) 2.02, 7.83 Hz, 1H),
3.82 (s, 3H), 2.99 (t, J ) 7.33 Hz, 2H), 2.92 (t, J ) 7.33 Hz, 2H),
2.83 (d, J ) 11.11 Hz, 1H), 2.77 (m, 2H), 2.53 (m, 4H), 2.28 (q,
J ) 6.58 Hz, 2H), 2.15 (m, 2H), 1.66 (m, 2H), 1.05 (t, J ) 7.48,
3H) Mass spectrum (LCMS, ESI pos.) calcd for C31H34N6O4, 555.26
(M + H); found, 555.1. HPLC: tR ) 4.91 min (100% pure).
8-(4-Ethylphenyl)-2-[4-(1-methylpiperidin-4-yl)phenylamino]-5-
oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
oxyamide (45). 8-(4-Ethylphenyl)-2-methanesulfonyl-5-oxo-5,8-
dihydropyrido[2,3-d]pyrimidine-6-carboxylic acid methoxyamide
(1.5 g, 3.73 mmol, prepared similarly as 24 from the common
intermediate 20) and 4-(1-methyl-piperidin-4-yl)-phenylamine (1.42
g, 7.46 mmol) in AcOH (10 mL) was heated at 110 °C for 15 min.
The reaction mixture was concentrated, and the remaining residue
was partitioned between sat. NaHCO3 and CH2Cl2. The organic
layer was dried over Na2SO4, filtered, and the solvent was
evaporated. MeOH (20 mL) was added, and a fine precipitate
formed that was collected by filtration to provide the title compound
as an off-white solid (800 mg, 42%). 1H NMR (400 MHz, CDCl3)
δ 11.97 (s, 1H), 9.36 (s, 1H), 8.81 (s, 1H), 7.63 (br s, 1H),
7.38-7.46 (m, 2H), 7.30-7.36 (m, 2H), 7.23 (br s, 2H), 6.95 (br
s, 2H), 3.90 (s, 3H), 2.97 (d, J ) 11.35 Hz, 2H), 2.82 (q, J ) 7.56
Hz, 2H), 2.35-2.45 (m, 1H), 2.32 (s, 3H), 1.98-2.09 (m, 2H),
1.72-1.82 (m, 4H), 1.37 (t, J ) 7.63 Hz, 3H). Mass spectrum
(LCMS, APCI pos.) calcd for C29H32N6O3, 513.25 (M + H); found,
513.2. HPLC: tR ) 4.57 min (100% pure).
8-(3-Ethylphenyl)-2-[4-(1-methylpiperidin-4-yl)phenylamino]-5-
oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Meth-
oxyamide (46). Using a similar procedure described in the prepa-
ration of 45, the title compound was obtained as a yellow solid
(34.8 mg, 67%). 1H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 9.36
(s, 1H), 8.82 (s, 1H), 7.66 (br s, 1H), 7.42-7.55 (m, 2H), 7.17-7.26
(m, 4H), 6.94 (br s, 2H), 3.90 (s, 3H), 3.08 (d, J ) 10.17 Hz, 2H),
2.75 (q, J ) 7.43 Hz, 2H), 2.41 (m, 4H), 2.15 (br s, 2H), 1.80 (br
s, 4H), 1.27 (t, J ) 7.63 Hz, 3H). Mass spectrum (LCMS, APCI
pos.) calcd for C29H32N6O3, 513.25 (M + H); found, 513.2. HPLC:
tR ) 4.53 min (100% pure).
8-(6,7-Dihydro-5H-[1]pyrindin-3-yl)-2-[4-(1-methylpiperidin-4-
yl)phenylamino]-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-6-car-
boxylic Acid Methoxyamide (47). 8-(6,7-Dihydro-5H-[1]pyrindin-
3-yl)-2-methanesulfonyl-5-oxo-5,8-dihydropyrido[2,3-d]pyrimidine-
6-carboxylic acid methoxyamide (30 mg, 0.07 mmol), prepared
similarly to 24 from the common intermediate 20 and 6,7-dihydro-
1
solid (25.9 mg, 43%). H NMR (400 MHz, CDCl3) δ 12.02 (s,
1H), 9.36 (s, 1H), 8.82 (s, 1H), 7.68 (br s., 1H), 7.55 (d, J ) 8.61
Hz, 2H), 7.25 (d, J ) 7.04 Hz, 2H), 4.98-5.14 (m, 1H), 3.88 (s,
3H), 3.05 (d, J ) 10.96 Hz, 2H), 2.53 (quin, J ) 7.83 Hz, 1H),
2.37 (s, 3H), 2.06-2.22 (m, 2H), 1.83-2.03 (m, 8H), 1.66 (d, J )
13.30 Hz, 2H), 1.39-1.56 (m, 2H), 1.06 (s, 3H), 1.04 (s, 3H). Mass
spectrum (LCMS, APCI pos.) calcd for C29H38N6O3, 519.30 (M +
H); found, 519.2. HPLC: tR ) 4.83 min (100% pure).
8-Cyclohexyl-2-[4-(1-methylpiperidin-4-yl)phenylamino]-5-oxo-
5,8-dihydropyrido[2,3-d]pyrimidine-6-carboxylic Acid Methoxy-
amide (49). Using a similar procedure described in the preparation
of 47, the title compound was obtained as a yellow solid (34 mg,
1
38%). H NMR (400 MHz, CDCl3) δ 12.00 (s, 1H), 9.37 (s, 1H),
8.81 (s, 1H), 7.53-7.65 (m, J ) 8.22 Hz, 2H), 7.20-7.25 (m, J )
8.61 Hz, 2H), 5.16 (t, J ) 11.93 Hz, 1H), 3.89 (s, 3H), 3.10-3.28
(m, 1H), 2.72-2.84 (m, 1H), 2.58-2.70 (m, 1H), 2.33 (s, 3H),
1.99-2.12 (m, 5H), 1.72-1.97 (m, 5H), 1.47-1.71 (m, 4H),
1.21-1.39 (m, 2H). Mass spectrum (LCMS, APCI pos.) calcd for
C27H34N6O3, 491.27 (M + H); found, 491.2. HPLC: tR ) 4.37 min
(100% pure).
Biology. Kinase Assays. Assays for FMS and the other kinases
listed in Table 5 were carried out using a fluorescence polarization
competition immunoassay format and have been described else-
where in detail.23b The full cytoplasmic regions of FMS (538-972)
encompassing the tyrosine kinase domain was expressed and
purified from a baculovirus system.29 The FMS kinase assay
measured FMS-mediated phosphorylation of tyrosine residues
present on a synthetic FMS555-568 peptide (SYEGNSYTFIDPTQ).
To each well of a black 96-well microplate (cat. no. 42-000-0117,
Molecular Devices, Sunnyvale, CA), Then 5 µL of compound (in
4% DMSO) were mixed with 2 µL of 3.5 nM FMS, 25 mM MgCl2
in assay buffer (100 mM HEPES, pH 7.5, 1 mM DTT, 0.01%
Tween-20), and 2 µL of 1540 µM peptide in assay buffer. The
kinase reaction was initiated by adding 1 µL of 10 mM ATP in
assay buffer. Final concentrations in the 10 µL reaction mixture
were 100 mM HEPES, pH 7.5, 1 mM DTT, 0.01% Tween-20, 2%
DMSO, 308 µM SYEGNSYTFIDPTQ, 1 mM ATP, 5 mM MgCl2,
and 0.7 nM FMS. The plates were incubated at room temperature
for 80 min. Reactions were stopped by addition of 1.2 µL of 50
mM EDTA. Each well then received 10 µL of a 1:1:3 mixture of
10× antiphosphotyrosine antibody, 10× PTK green tracer, and
fluorescence polarization dilution buffer (cat. no. P2837, Invitrogen,
Carlsbad, CA). The plates were incubated for 30 min at room
temperature, and the fluorescence polarization was read (485 nm
excitation, 530 nm emission) on an Analyst plate reader (Molecular
Devices). Fluorescence polarization values for positive (EDTA) and
negative (DMSO) controls were approximately 290 and 160,
respectively, and were used to define 100% and 0% inhibition of
the FMS reaction.
Bone Marrow-Derived Macrophage (BMDM) Proliferation
Assay. Functional impact on cellular FMS activity was determined
by measuring compound inhibition of CSF-1-driven proliferation

Pyrido[2,3-d]pyrimidin-5-ones: FMS Inhibitors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 1097
of mouse bone marrow-derived macrophages as previously de-
scribed.29 Macrophages were derived by culturing mouse bone
marrow in R-MEM supplemented with 10% FCS and 50 ng/mL
recombinant mouse CSF-1 in bacteriologic dishes. On the sixth
day, macrophages were resuspended to 50000 cells/mL in R-MEM
containing 10% FCS, and 100 µL of cell suspension were distributed
per well into 96-well culture plates for overnight culture. Next,
wells were further supplemented with the addition of 50 µL media
containing 15 ng/mL CSF-1, 3 µM indomethacin, and a dilution
series of test compound. The cells were cultured for 30 h at 37 °C
and 5% CO2. During the final 6 h, cultures were supplemented with
an additional 30 µL of media containing a 1:500 dilution of
bromodeoxyuridine (BrDU) reagent, and incorporation of BrDU
into cellular DNA was quantified using a specific ELISA (cat. no.
X1327K) from Exalpha Corporation (Watertown, MA).
MV-4-11 and M-o7e Leukemia Cell Line Proliferation
Assays. Functional impact on cellular FLT3 activity was determined
by measuring compound inhibition of MV-4-11 (ATCC no.: CRL-
9591) cell proliferation, and M-07e (DSMZ no.: ACC 104) cells
were used to assess compound effects on cellular KIT activity. MV-
4-11 cells grew independent of growth factor due to expression of
a constitutive active FLT3 mutation, and M-07e cells were driven
to proliferate in a KIT-dependent fashion by 25 ng/mL stem cell
factor. Following a culture period of 72 h, relative cell numbers
were determined using CellTiterGlo reagent (Promega).
Biochemical Pharmacodynamic Mouse Model. Groups of six
B6C3F1 mice (Taconic Farms), 8 weeks of age, were given oral
doses of vehicle (aq 20% hydroxypropyl-ꢀ-cyclodextrin (HPꢀCD))
or 37 at 1 or 3 mg/kg. Six hours later, mice were administered
saline alone or saline containing 1 µg of recombinant mouse CSF-1
(Cell Biosciences Inc., Norwood, MA) via the tail vein. Fifteen
minutes after CSF-1 injection, mice were sacrificed and spleens
were isolated and snap frozen on dry ice. The frozen tissue was
homogenized in 1 mL of Trizol (Invitrogen) per 50 mg of tissue,
and RNA was purified according to the Trizol instructions and
treated with 6.8 Kunitz units of RNase-free DNase (Qiagen,
Valencia, CA) to degrade contaminating genomic DNA. The RNA
was purified further using RNeasy columns (Qiagen). RT-PCR was
performed in 25 µL reaction volumes using Reverse Transcriptase
qPCR Master Mix (Eurogentec) and approximately 50 ng of RNA.
Applied Biosystems, Inc., (Foster City, CA) was the source for the
primers and probes for mouse c-fos mRNA (cat. no. Mm00487425)
and 18S rRNA (cat. no. 4333760F). Amplification and detection
were performed using an ABI Prism 7900. Standard curves were
created for c-fos mRNA and for 18s rRNA using RNA isolated
from a vehicle-treated, CSF-1-induced mouse and used to calculate
relative expression levels in all other samples. c-fos mRNA values
were normalized to 18S rRNA content. Averaged, normalized c-fos
content in the saline (no CSF-1) group was assigned a value of
one and all other groups were expressed as “fold-induced”.
SCW-Induced Arthritis Model. Female Lewis rats (80-100 g
each) were purchased from Charles River. Streptococcal cell wall
peptidoglycan-polysaccharide polymers (PG-PS 10S) were pur-
chased from BD (cat. no. 210866). On day 0, rats were anesthetized
using isoflurane and injected ip with PG-PS 10S equivalent to 15
µg of rhamnose/gram (body weight) in the lower left quadrant of
the abdomen. Control rats were treated in a similar manner with
sterile saline. On day 5, rats were injected with PG-PS 10S, and
those that showed a distinct acute phase arthritic response based
on joint swelling were randomized into the treatment groups. A
chronic, T cell dependent, erosive arthritis begins in this model on
about day 10. Animals were dosed orally twice a day beginning
on day 19. Compound 37 was formulated in 20% HPꢀCD. The
dose volume was 6 mL/kg. Left and right hind ankles of each rat
were measured with calipers every day for the first six days
(postinjection) and then at least every two or three days for the
remainder of the study.
complete adjuvant (FCA, Sigma)/lipoidal amine (LA, Sigma) at
the base of the tail on day 0. Oral dosing was initiated on day 8
(the first day of clinical disease). Compound 37 or vehicle (20%
HPꢀCD) were dosed orally twice daily. Caliper measurements of
ankles were taken on days 7-21. To calculate the area under the
curve (AUC), the daily caliper measurements for each rat were
integrated vs time (treatment day). Mean AUC values for each group
were determined, and the percent inhibition from arthritis vehicle-
treated controls was calculated after subtracting the AUC of age-
matched normal control animals. Following necropsy on day 21,
digital radiographs of left paws were prepared using a VetTek
Universal AP500 (Del Medical Imaging Corporation, Franklin Park,
IL). Both hind paws were then fixed in formalin and processed for
H and E microscopy. H and E sections were scored for bone
resorption as follows: 0 ) normal; 0.5 ) normal on low magnifica-
tion but have the earliest hint of small areas of resorption in the
metaphysis with no resorption in the tarsal bones; 1 ) (minimal)
small definite areas of resorption in distal tibial trabecular or cortical
bone, or in the tarsal bones, not readily apparent on low magnifica-
tion, rare osteoclasts; 2 ) (mild) more numerous areas (e25% loss
of bone in growth plate area) of resorption in distal tibial trabecular
or cortical bone and tarsals apparent on low magnification,
osteoclasts more numerous; 3 ) (moderate) obvious resorption of
medullary trabecular and cortical bone without full thickness defects
in both distal tibial cortices, loss of some medullary trabeculae with
26-50% loss across growth plate and cortices, some loss in tarsal
bones, lesion apparent on low magnification, osteoclasts more
numerous; 4 ) (marked) full or near full thickness defects in both
distal tibial cortices, often with distortion of profile of remaining
cortical surface, marked loss of medullary bone of distal tibia
(50-100% loss across growth plate area and cortices and up to
50% loss in small tarsals if minor in tibia), numerous osteoclasts,
minor to mild resorption in smaller tarsal bones; 5 ) (severe) full
thickness defects in both distal tibial cortices with >75% loss across
growth plate and both cortices and >50% loss in tarsals, often with
distortion of profile of remaining cortical surface, marked loss of
medullary bone of distal tibia, numerous osteoclasts. Osteoclast
counts (5, 400× fields) were performed on ankles in the areas of
greatest bone resorption.
Statistical Analysis. Ankle thickness, bone erosion scores,
osteoclast counts, and c-fos expression values (mean ( SE) were
analyzed for group differences using the Student’s t test. Signifi-
cance was set at p e 0.05.
Supporting Information Available: LC-MS data for target
compounds. This material is available free of charge via the Internet
at http://pubs.acs.org.
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