Synthesis of pyrazoles and isoxazoles as potent αvβ3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.03.045

We describe a series of pyrazole and isoxazole analogs as antagonists of the α_vβ₃ receptor. Compounds showed low to sub-nanomolar potency against α_vβ₃, as well as good selectivity against α_IIbβ_3<

Full text of DOI:10.1016/j.bmcl.2006.03.045

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3156–3161
Synthesis of pyrazoles and isoxazoles as potent
avb3 receptor antagonists
Thomas D. Penning,a,* Albert Khilevich,a Barbara B. Chen,a Mark A. Russell,a
Mark L. Boys,b Yaping Wang,a Tiffany Duffin,c,d V. Wayne Engleman,c,d
Mary Beth Finn,c,d Sandra K. Freeman,c,d Melanie L. Hanneke,c,d Jeffery L. Keene,c,d
Jon A. Klover,c,d G. Allen Nickols,c,d Maureen A. Nickols,c,d Randall K. Rader,c,d
Steven L. Settle,c,d Kristen E. Shannon,c,d Christina N. Steininger,c,d
Marisa M. Westlinc,d and William F. Westlinc,d
aDepartment of Medicinal Chemistry, Pfizer Global Research & Development, 4901 Searle Parkway, Skokie, IL 60077, USA
bDepartment of Chemistry, Pfizer Global Research & Development, 2800 Plymouth Rd., Ann Arbor, MI 48105, USA
cDepartment of Discovery Pharmacology, Pfizer Global Research & Development,
700 Chesterfield Village Parkway, Chesterfield, MO 63198, USA
dDepartment of Oncology, Pfizer Global Research & Development, 700 Chesterfield Village Parkway, Chesterfield, MO 63198, USA
Received 9 February 2006; accepted 17 March 2006
Available online 18 April 2006
Abstract—We describe a series of pyrazole and isoxazole analogs as antagonists of the avb3 receptor. Compounds showed low to
sub-nanomolar potency against avb3, as well as good selectivity against aIIbb3. In HT29 cells, most analogs also demonstrated sig-
nificant selectivity against avb6. Several compounds showed good pharmacokinetic properties in rats, in addition to anti-angiogenic
activity in a mouse corneal micropocket model. Compounds were synthesized in a straightforward manner from readily available
glutarate precursors.
Ó 2006 Elsevier Ltd. All rights reserved.
The integrin avb3 is a heterodimeric transmembrane
receptor found on the surface of activated endothelial
cells, smooth muscle cells, and many tumor cells. avb3
recognizes the RGD tripeptide sequence on numerous
extracellular matrix proteins1 allowing endothelial cells
and tumor cells to interact with these proteins. A num-
ber of antagonists of avb3 have been reported to demon-
strate anti-angiogenic activity, and thus have potential
utility in inhibiting tumor growth.2 avb3 is also the
predominant integrin found on the surface of osteo-
clasts, which are responsible for cellular attachment,
and subsequent bone resorption.3 Antagonists of avb3
have been shown to block the degradation of bone in
animal models of osteoporosis4 and thus may also have
utility in the treatment of this disease.
We previously described an RGD-based peptidomimetic
avb3 antagonist, SC-68448, which demonstrated anti-
tumor efficacy in a mouse Leydig cell tumor model.5
We later reported a series of conformationally restricted
cinnamic acid analogs such as 1 in which the glycine-3-
aminopropionic acid functionality was replaced by the
bioisosteric 4-aminocinnamic acid moiety.6 In two re-
cent papers, this work was extended to a series of oxa-
diazole- and thiazole-containing analogs (2 and 3).7,8
These compounds were shown to be potent and selective
antagonists of avb3 with a reasonable pharmacokinetic
profile in rats. To further expand upon this work, two
additional heterocyclic series, pyrazoles and isoxazoles,
were investigated using intermediates available from
the previously described heterocyclic series.7,8
The glutarate portion of the molecule was synthesized
from readily available aryl or heteroaryl aldehydes
(Scheme 1). Condensation with ethylacetoacetate pro-
vided the cyclic compound 26, which could be converted
to either glutaric diacid 27 or diester 28. Treatment of 27
Keywords: Integrin antagonist; avb3; RGD peptidomimetic.
*
Corresponding author at present address: Abbott Laboratories, 100
Abbott Park Road, Abbott Park, IL 60064-6101, USA. Tel.: +1 847
938 6707; e-mail: thomas.penning@abbott.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.045

T. D. Penning et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3156–3161
3157
O
tion, hydroxypyrazole 36. Mitsunobu reaction with 35
or alkylation with bromide 37 provided ester 38.
Saponification gave the desired pyrazole acids (5, 11–
19, and 22–24). Hydroxypyrazole 36 could also be con-
verted to thiol 39 using Lawesson’s reagent. Alkylation
with 37 gave ester 40, which was saponified to acid 20
or oxidized with OxoneÒ and saponified to give acid
21. The (S)-isomer of pyrazole 11 was prepared under
somewhat modified conditions (Scheme 4). Chiral acid
30 was condensed with Meldrum’s acid to give, after
refluxing in ethanol, diester 41. Condensation with
methylhydrazine, Mitsunobu reaction with 32, and
saponification gave (S)-11.
H
H
H2N
N
N
N
H
CO2H
Cl
SC-68448
NH
O
Cl
OMe
CO2H
1
O
H
N
H
N
N
H
N
Ar
CO2H
H
N
N
X
Z
2 X=N, Y=N, Z=O
3 X=N,Y=S, Z=CH
Y
To expand upon the previously described oxadiazole
and thiazole series of inhibitors,7,8 related pyrazole
and isoxazole series were investigated. The initial
synthesis focused on analogs that maintained a 3,4-
methylenedioxyphenyl b-substituent and the 1,8-tetra-
hydronaphthyridine guanidine isostere (4 and 5).
Isoxazole 4 and unsubstituted pyrazole 5 (Tables 1
and 2) both showed very similar low-nanomolar potency
in the avb3 solid-phase receptor assay (SPRA). Com-
pounds 4 and 5 also demonstrated reasonable selectivity
against the other b3 integrin, aIIbb3. In addition, these
compounds showed excellent cellular activity against
avb3 in 293 cells11 and were selective versus avb6 in
HT29 cells. Based on the promising potency and selec-
tivity profiles of 4 and 5, as well as ease of synthesis,
more detailed SAR studies were carried out on these
two heterocyclic classes. Table 1 describes the SAR of
a series of isoxazole analogs. b-substituents that had
previously demonstrated good potency against avb3 in
other heterocyclic series were chosen for further study.
In addition to the relatively optimized tetra-
hydronaphthyridine group, several other guanidine isos-
teres were also investigated. These included additional
groups with reduced basicity relative to a simple guani-
with acetic anhydride gave glutaric anhydride 29, while
enzymatic resolution followed by chiral chromatogra-
phy provide chiral mono-ester 30. The 1,8-tetra-
hydronaphthyridine guanidine mimetic 32 was
synthesized using conditions somewhat modified from
what was previously described10 (Scheme 2). 2-Amino-
3-pyridine carboxaldehyde was treated with acetone un-
der Friedlander conditions to provide 31, which was
deprotonated and reacted with diethyl carbonate. Cata-
lytic hydrogenation followed by LAH reduction provid-
ed alcohol 32. The synthesis of other guanidine isosteres
detailed in the tables has been described previously.7–9
Scheme 3 describes the general synthesis of racemic pyr-
azoles and isoxazoles. The lithium enolate of ethyl ace-
tate was added to anhydride 29 to give b-ketoester 33.
Condensation with hydroxylamine, followed by esterifi-
cation, gave hydroxyisoxazole 34. Mitsunobu reaction
with a guanidine-containing alcohol 35, followed by
saponification, gave the desired isoxazole acids (4 and
6–10). Alternately, condensation of 33 with hydrazine
or a mono-substituted hydrazine gave, after esterifica-
R
R
d
HO2C
CO2H
b
O
O
O
R
27
a
29
EtO2C
O
CO2Et
RCHO
b, c
OH
R
R
e
26
EtO2C
CO2Et
HO2C
CO2Et
28
30
Scheme 1. Reagents: (a) ethyl acetoacetate, piperidine; (b) i—10% NaOH, EtOH, D; ii—HCl; (c) EtOH, HCl(g); (d) Ac2O, D; (e) i—Chirazyme L-2,
ii—chiral chromatography.
H
H
N
N
OH
N
NH2
N
N
d
a
b, c
N
N
OEt
O
CHO
31
32
Scheme 2. Reagents and conditions: (a) acetone, L-proline, EtOH, D; (b) LiHMDS, THF, diethyl carbonate, À40 to 0 °C; (c) 10% Pd/C, H2, EtOH;
(d) LAH, THF.

3158
T. D. Penning et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3156–3161
O
R1
OH
HO
R1
O
O
e, f
G
N
b, c
d
N
OEt
O
O
R
R1
O
a
34
O
O
4, 6-10
EtO
OH
O
O
O
HO
HS
29
33
R1
O
i
R1
O
N
N
OEt
OEt
R2
N
R2
N
39
36
e or g
g
O
O
O
R1
R1
R1
OH
OEt
OEt
O
O
S
G
f or h
G
G
N
N
N
N
N
R2
N
R2
R2
38
40
5, 11-19, 22-24
h
j, h
R1
O
R1
Br
OH
OH
O
G
G
S
OH
O2
G
37
S
35
N
G
N
R2
N
N
21
R2
20
Scheme 3. Reagents and conditions: (a) LDA, EtOAc, À78 °C; (b) NH2OH–HCl, NaOH, H2O; (c) 4 N HCl, dioxane, EtOH; (d) R2NHNH2,
EtOH, D; (e) diethyl azodicarboxylate, Ph3P, 35, THF; (f) 1 N NaOH, THF; (g) Na2CO3, 37, DMF; (h) HCl, H2O, acetone, D; (i) Lawesson’s
reagent; (j) OxoneÒ.
O
O
O
O
O
O
O
O
OH
H
HO
c, d
N
N
O
b
a
O
O
O
30
N
N
Me
OEt
N
N
EtO
OEt
Me
42
41
(S)-11
Scheme 4. Reagents: (a) i—diethyl cyanophosphonate, Meldrum’s acid, Et3N, DMF, ii—EtOH, D; (b) MeNHNH2, EtOH, D; (c) 32, diisopropyl
azodicarboxylate, Ph3P, THF; (d) HCl, H2O, acetone, D.
dine group, as well as a more basic cyclic guanidine
(i.e., 10). These analogs demonstrated excellent poten-
cy, both in SPRA and cellular assays. In addition,
good selectivity versus avb6 in cells was observed, par-
ticularly with aminopyridine 9 and cyclic guanidine 10.
In general, significant selectivity versus avb5 was not
obtained.
substituted analogs, selectivity versus aIIbb3 was some-
what diminished, while selectivity versus avb6 was gen-
erally maintained. Since 11 was identified as one of the
more potent analogs, particularly in 293 cells, the (S)-
enantiomer (in previous series, consistently the more
potent isomer) was synthesized, and as expected,
showed a modest increase in potency. Based on the
excellent potency of 11, further modifications of this
N-methyl analog were investigated, focusing on alter-
nate b-substituents and guanidine isosteres (Table 3).
In addition, a thioether (20) and sulfonyl (21) linker
were explored in place of the ether linkage. All were
very potent inhibitors of avb3, in both SPRA and 293
whole cell assays. As before, aminopyridine and cyclic
N-substituted analogs of pyrazole 5 were also investigated
(Table 2). Several diverse substituents on the pyrazole
ring nitrogen were well tolerated, providing analogs
with low to sub-nanomolar potency in both avb3 SPRA
and cellular assays. Both sterics and electronics seemed
to have minimal effect on potency. However, for the

T. D. Penning et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3156–3161
Table 1. In vitro data for isoxazole analogs
3159
R
CO2H
O
G
N
O
a
IC50 (nM)
Compound
G
R
avb3 SPRA
avb3 293
avb5 293
avb6 HT29
O
H
O
N
N
4
1.0
0.34
1.2
350
OMe
N
H
N
N
N
6
7
0.9
1.1
—
—
—
—
66.4
Cl
H
N
131
N
S
O
H
N
N
N
O
8
9
1.2
2.4
0.9
0.69
2.49
0.58
1.47
17.5
9.4
323
N
O
O
H
N
O
O
8225 (2)
6830
H
N
N
10
NH
a Average of at least three determinations except where noted.
Table 2. In vitro data for N-substituted pyrazole analogs
Rat pharmacokinetic data, along with anti-angiogenic
efficacy data in a mouse corneal micropocket model12
are shown for select analogs in Table 4. In general, most
compounds in these series demonstrated good pharma-
cokinetic properties in rats, with excellent oral bioavail-
ability and a reasonable half-life. The exception was for
those analogs that contained a more basic guanidine
moiety, such as 10, which was poorly absorbed orally
and had a relatively short half-life.
O
O
CO2H
H
N
N
O
R
N
N
a
IC50 (nM)
Compound
R
avb3
SPRA SPRA 293 293 HT29
aIIbb3 avb3 avb5 avb6
Compounds 4, 10, and 11 all showed good efficacy in the
micropocket model when dosed by osmotic mini-pump
(OMP) for 5 days at doses from 50 to 100 mg/kg/day.
Although 4 and 11 showed good oral bioavailability in
the rat, oral dosing was not investigated in this model.
However, 23 was dosed orally at 30 mg/kg/day and dem-
onstrated reasonable efficacy.
5
11
–H
1.2
1.1
0.44
2.7
1.5
81.9
23.8
—
0.71 2.1
0.21 0.77 754
347
1633 (2)
1.09 1.03 465
674
–Me
–Me
–n-Bu
(S)-11
12
13
—
—
39.4
32.7
—
1.25 3.0
–CH2CF3
14
15
–CH2CH2OH 1.0
—
—
0.9
—
—
25.9
491
–CH2CO2H
–CH2Ph
–Ph
0.6
2.6
1.4
—
16
17
13.5
24.4
1.84 2108
In conclusion, we have described a new series of avb3
receptor antagonists with excellent potency in a solid-
phase receptor assay as well as in a cellular assay. These
analogs also showed reasonable selectivity versus aIIbb3
and avb6, and a very good rat pharmacokinetic profile.
In addition, several analogs also demonstrated efficacy
in an in vivo model of angiogenesis, further supporting
the concept of using avb3 receptor antagonists for select
disease states.
1.18 3.27 352
a Average of at least three determinations except where noted.
guanidine analogs (24 and 25) also demonstrated excel-
lent selectivity against avb6 in HT29 cells, along with
thioether analogs 20 and 21.

3160
T. D. Penning et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3156–3161
Table 3. In vitro data for pyrazole analogs
R
CO2H
X
G
N
N
Me
a
IC50 (nM)
Compound
G
X
R
avb3 SPRA
aIIbb3 SPRA
avb3 293
avb5 293
avb6 HT29
OMe
N
H
N
N
N
18
–O–
0.8
—
—
—
164
Cl
H
N
19
–O–
1.4
57.6
0.9
18
189
N
S
O
O
O
O
O
O
H
N
O
O
O
O
O
O
N
20
21
22
23
24
25
–S–
1.5
2.6
0.83
1.2
2.6
21
37.6 (1)
0.8
5.43
1.86
—
4623 (2)
1394 (2)
216
H
N
N
N
–SO2–
–O–
–O–
–O–
–O–
—
1.69
—
H
N
—
N
H
H
N
N
N
36.2
0.75
1.77
—
2.58
1433
N
H
N
80.3 (1)
16.2 (1)
11
4762 (2)
>10,000 (2)
H
N
N
—
a Average of at least three determinations except where noted.
Table 4. Pharmacokinetic and in vivo efficacy data
Yin, Z.; Tanghetti, E.; Yaremko, B.; Nechuta, T.; Varner,
J.; Liu, M.; Smith, E. M.; Neustadt, B.; Presta, M.;
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Slee, A. M.; Mousa, S. A. Expert. Opin. Investig. Drugs
2000, 9, 1271; (d) Mitjans, F.; Meyer, T.; Fittschen, C.;
Goodman, S.; Jonczyk, A.; Marshall, J. F.; Reyes, G.;
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Cheresh, D. A. Mol. Med. 1998, 4, 741; (f) Brooks, P. C.;
Stro¨mblad, S.; Klemke, R.; Visscher, D.; Sarkar, F. H.;
Cheresh, D. A. J. Clin. Invest. 1995, 96, 1815; (g) Brooks,
P. C.; Clark, R. A. F.; Cheresh, D. A. Science 1994, 264,
569.
Compound
Rat PK
Mouse CMP
F (%)
86
8.5
t1/2 (h)
(% inhib of angiogenesis)
4
10
11
23
1.3
0.5
4.9
5.0
25% at 100 mpk/d (OMP)
50% at 50 mpk/d (OMP)
41% at 100 mpk/d (OMP)
33% at 30 mpk/d (po,bid)
95
100
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