Nonpeptide αvβ3 antagonists. Part 10: In vitro and in vivo evaluation of a potent 7-methyl substituted tetrahydro-[1,8]naphthyridine derivative

Article abstract of DOI:10.1016/j.bmcl.2004.06.040

Subtle modifications were incorporated into the structure of clinical candidate 1. These changes were designed to maintain potency and selectivity while inducing changes in physical properties leading to improved pharmacokinetics in three species. This ap

Full text of DOI:10.1016/j.bmcl.2004.06.040

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4515–4518
Nonpeptide avb3 antagonists. Part 10: In vitro and in vivo
evaluation of a potent 7-methyl substituted
tetrahydro-[1,8]naphthyridine derivative
Michael J. Breslin,a,* Mark E. Duggan,a Wasyl Halczenko,a George D. Hartman,a
Le T. Duong,b Carmen Fernandez-Metzler,c Michael A. Gentile,b Donald B. Kimmel,b
Chih-Tai Leu,b Kara Merkle,c Thomayant Prueksaritanont,c Gideon A. Rodan,b
Sevgi B. Rodanb and John H. Hutchinsona,
aDepartment of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bDepartment of Bone Biology and Osteoporosis Research, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 11 February 2004; accepted 14 June 2004
Available online 17 July 2004
Abstract—Subtle modifications were incorporated into the structure of clinical candidate 1. These changes were designed to
maintain potency and selectivity while inducing changes in physical properties leading to improved pharmacokinetics in three
species. This approach led to the identification of 4 as a potent, selective avb3 receptor antagonist that was selected for clinical
development based on an improved PK profile and efficacy demonstrated in an in vivo model of bone turnover.
Ó 2004 Elsevier Ltd. All rights reserved.
Osteoporosis is a disease most commonly found in post-
menopausal women, where the combination of estrogen
deficiency and aging results in the loss of a significant
portion of the peak bone mass attained as a young
adult. The characteristic skeletal fragility and high
incidence of fracture are due to a decrease in mechanical
bone strength resulting from an imbalance between the
activities of bone-resorbing osteoclast cells and bone-
depositing osteoblast cells.1–4 One potential approach to
combatting this disease is to reduce the rate of osteo-
clast-mediated bone resorption by antagonism of the
integrin avb3.5 avb3 is highly expressed in osteoclasts and
is thought to be involved in the adhesion, activation,
and migration of osteoclasts on the bone surface as well
as osteoclast polarization.6;7 Several proteins that pos-
sess the three-amino acid sequence arginine–glycine–
aspartic acid (RGD), including osteopontin, bone
sialoprotein, vitronectin, and fibrinogen, bind with high
affinity to avb3.8 Antibodies to avb3, RGD-peptides, and
small molecule mimetics of RGD have been shown to be
efficacious in in vitro and in vivo models of bone
resorption.9 These results suggest that antagonism of the
RGD binding domain of avb3 by high affinity nonpep-
tidic RGD mimetics may result in a treatment for
osteoporosis.
Previous reports from this laboratory have described the
chain-shortened imidazolidinone 1 as an orally bio-
available zwitterionic antagonist that was selected for
evaluation in human clinical trials.10 Compound 1 was
projected to display a pharmacokinetic profile in hu-
mans to support twice daily oral dosing. Satisfied with
the in vitro potency and selectivity of 1, we sought to
identify a related compound with improved pharmaco-
kinetics that could be effective in humans following
once-a-day administration. Herein, we describe modifi-
cations to the N-terminus of 1 that were designed to
increase lipophilicity and block a potential site of human
metabolism. These efforts resulted in compound 4,
which showed an enhanced pharmacokinetic profile and
was chosen for clinical development.
* Corresponding author. Tel.: +1-215-652-7928; fax: +1-215-652-7310;
e-mail: michael_breslin@merck.com
Compound 1 was selected as a clinical candidate for the
prevention and treatment of osteoporosis based on
Present address: Merck & Co., Inc. MRLSDB2, 3535 General
Atomics Court, San Diego, CA 92121, USA.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.040

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M. J. Breslin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4515–4518
excellent in vitro potency and selectivity, a good phar-
macokinetic profile and efficacy shown in three different
in vivo models of bone resorption.10 After extensive
SAR studies around aryl substituents on the b-alanine
C-terminus of the imidazolidinone core series, 2-meth-
oxypyridine was found to possess a desirable balance
between basicity, polarity, and the degree of binding to
human plasma proteins.11;12 Typically, polar compounds
in the imidazolidinone series, as defined by a log P value
approaching zero, suffered from poor pharmacokinetic
profiles due to low bioavailability. Lipophilic com-
pounds with a log P value approaching 1.0 usually dis-
played good oral bioavailability and half-life, but high
protein binding reduced in vivo efficacy due to low free
drug concentration.
Although in vitro metabolism studies on 1 using liver
microsomes from several species showed minimal
metabolism, the C-7 methylene position of the THN
ring was identified as a site of oxidative metabolism. The
C-7 hydroxylated metabolite could undergo further
oxidation to yield a lactam or eliminate and aromatize
to give the fully unsaturated naphthyridine.10 We
focused on placing a methyl substituent at the C-7 position
of the THN ring knowing that this substitution was
tolerated in a related series and that blocking a site of
metabolism could improve pharmacokinetics. The first
synthesis of a 7-methyl substituted THN ring in the
imidazolidinone series yielded a racemic compound,
which was separated by chiral chromatography to give 3
and 4.14 Both 3 and 4 were equipotent in the avb3
binding assay (SPAV3) and their physical properties
were similar.15 Later asymmetric synthesis established
the absolute stereochemistry at the C-7 methyl position
to be S for compound 3 and R for compound 4. We
concentrated further studies on diastereomer 4.
We sought to introduce conservative modifications to
the N-terminus of 1 that would not adversely alter the in
vitro potency and selectivity, but would induce favor-
able changes in physical properties. The methyl group
was chosen as a small substituent that could enhance
lipophilicity without having a dramatic effect on free
drug concentration. In a related series, 7-methyl sub-
stitution on the tetrahydro-[1,8]naphthyridine (THN)
ring was found to be 2-fold more potent than 6-methyl
substitution. In this series, substitution at the C-3 posi-
tion of the THN ring was extensively studied showing
that electron withdrawing groups decreased binding
affinity while electron donating groups increased
potency.13 Substitution on the unsaturated ring of the
THN may largely affect the binding affinity and physical
properties of the N-terminus by modulation of the pKa.
When 3-methyl substitution was employed in the
imidazolidinone series, the substitution had the desired
effect on in vitro potency and physical properties.
Compound 2 possessed comparable potency to 1 while
having less protein binding in spite of being more lipo-
philic (Table 1). This substitution had detrimental effects
on the pharmacokinetic profile of 2; clearance increased
dramatically while half-life decreased. Substitution with
slightly larger aliphatic groups such as ethyl caused a
10–20-fold loss of selectivity versus the related integrin
avb5 when compared with 1 and 2.
Like compound 1, 4 displayed good selectivity against
several related integrins. Compound 4 displayed a 50-
fold selectivity for avb3 over avb5 as demonstrated by an
IC50 of 5 nM ðn ¼ 7Þ in an avb5 binding assay.14 Affinity
for the fibrinogen receptor aIIBb3, as measured by a
platelet aggregation assay, was found to be weak with
an IC50>10 lM.16 While the addition of the C-7 methyl
group on the THN ring did not adversely alter the vitro
potency and selectivity, the physical properties of 4 were
changed. As compared to compound 1, LogP increased
favorably from 0.21 to 0.69 while protein binding in-
creased slightly from 88% to 95%. This compound dis-
played improved pharmacokinetics in three species
(Table 2). Clearance remained high in rats, but slightly
improved over compound 1. Half-life decreased slightly,
but oral bioavailability doubled (50% vs 26%). The
pharmacokinetic profile in dog and monkey was char-
acterized by low clearance (1.7 and 3.5, respectively) and
high oral bioavailability (83% and 75%) while t1=2 was
unchanged from that observed for 1. The pharmaco-
kinetic profile of compound 4 was highlighted by reduced
clearance, comparable half-life, and improved bioavail-
ability.
On the basis of its excellent in vitro potency, selectivity,
and improved pharmacokinetic profile, 4 was chosen for
study in a rodent model of bone resorption. Compound
4 was administered in two different experiments to
Table 1. In vitro binding affinity and physical properties of THN
substituted avb3 antagonists
OMe
N
O
H
R
N
N
CO2H
7
N
N
3
Table 2. Pharmacokinetics in several speciesa
R'
Species
Rat
Compd
CL (mg/min/kg) T1=2 (h)
F (%)
Compd
R
R0
SPAV3a
IC50 (nM)
PB (%)b LogP
1
4
1
4
1
2
4
47
41
3
26
50
74
75
64
55
83
1–2
2
1
2
3
4
H
H
H
C
0.08
0.1H3
0.11
0.11
88
78
92
95
0.21
0.62
0.56
0.69
Rhesus monkey
Dog
9.0
3
3.5
6.4
2
(S)-CH3
(R)-CH3
H
H
3.5
2.2
4
18
1.7
a Binding to the avb3 receptor using a scintillation-proximity assay
(SPAV3), n ¼ 235 for 1, n ¼ 2 for 2 and 3, n ¼ 7 for 4.
b Human plasma protein binding.
a Compounds dosed at 0.2 mpk iv and 1 mpk po in water.

M. J. Breslin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4515–4518
Table 3. Effect of 1 and 4 on growing young male rats
4517
O
a
Compd
Free Css
(nM)
DFMBMDb
(mg/cm2)
% Increase
over vehicle
D-Ala
methyl ester
Br
KN
O
Vehicle
––
112.5 5.8
––
e
1
4
4
4
170
190
560
1440
133.0 7.5c
137.0 10.0c
147.9 7.3c;d
161.0 13.7c;d
+18
+22
+32
+43
a,b
O
CH3
N
OH
TrHN
Vehicle
––
118.6 9.2
––
O
1-1
1-3
1
1
4
4
390
1550
170
1100
145.0 10.0c
157.2 12.3c;e
141.1 9.7c
156.6 11.8c;f
+22
+33
+19
+32
c,d
f
O
a Free Css (nM)––free steady state serum concentration.
b DFMBMD––distal femoral metaphysis bone mineral density.
c >Vehicle ðP < 0:0001Þ.
N
CH3
N
TrHN
Br
O
d >190 nM 4 ðP < 0:0001Þ.
1-2
1-4
e >390 nM 1 ðP < 0:002Þ.
f >170 nM 4 ðP < 0:0001Þ.
g,h
O
N
N
TrHN
O
young, rapidly growing male rats by minipump infusion
over ten days (Table 3). Compound 1 was used as a
positive control and at the end of the experiment bone
mineral density at the distal femoral metaphysis was
measured and compared to that of vehicle-treated ani-
mals. Both compounds produced a significant increase
in distal femoral metaphysis bone mineral density
(DFMBMD), which was significantly different than the
vehicle control group. In the first experiment, compound
4 increased DFMBMD in an exposure-dependent fash-
ion, where free steady state serum concentration (Css)
varied from 190 to 1440 nM with a maximum effect at
+43% above the vehicle. The DFMBMD increase
achieved with 4 at 190 nM (+22%) was approximately
the same as seen with 1 at 170 nM (+18%). In the second
experiment, both 1 and 4 produced statistically signifi-
cant, exposure-dependent increases in DFMBMD. The
Css of 1 varied from 390 to 1550 nM with a maximum
effect at +33% while a lower Css of 4 (170–1100 nM) was
able to produce similar effects (maximum at +32%).
These data suggest that 4 is at least as efficacious as 1
for inhibiting bone resorption in rats.
1-5
CH3
i,j
N
NH2
H2N
1-6
CH3
k
H
H3C
N
N
NH2
1-7
Scheme 1. Reagents and conditions: (a) trityl chloride, Et3N, CH2Cl2;
(b) LiAlH4, THF; (c) pyridine–SO3, Et3N, DMSO, CH2Cl2; (d) methyl
triphenylphosphonium bromide, n-BuLi, THF, 0 °C; (e) DMF, 70 °C;
(f) 9-BBN, 2,5-dibromopyridine, K2CO3, DPPF, Pd(OAc)2, DMF; (g)
9-BBN, THF; (h) 1–4, K2CO3, DPPF, Pd(OAc)2, DMF; (i) HCl,
EtOAc; (j) hydrazine, EtOH, MTBE; (k) NaH, xylenes.
The C-terminal intermediate 2–1 was converted to the
chlorocarbamate with triphosgene and then reacted di-
rectly with 1–7 to give an unsymmetric urea that was
cyclized to the imidazoline-2-one 2–2 by the addition of
2 M sulfuric acid to the reaction mixture.17 Catalytic
hydrogenation smoothly reduced the olefin and this was
followed by hydrolysis of the ester with 6 M sulfuric acid
to give 4 (Scheme 2). The original chiral resolution of 3
and 4 was performed on the tert-butyl ester following
hydrogenation. Analysis of the tert-butyl ester of 4
showed 98.8% de was obtained, which is comparable to
The asymmetric synthesis of 4 was accomplished using a
three part convergent synthesis, which utilized the
C-terminal intermediates developed for the process
scale preparation of compound 1.17 The synthesis of the
N-terminus of 4 (Scheme 1) is accomplished in 10 steps
from commercially available materials.
D-Alanine me-
thyl ester was trityl protected and reduced to the alcohol
1–1 with LAH. Oxidation by pyridine–SO3 yielded the
aldehyde, which was immediately used in a Wittig
reaction with methyl triphenylphosphonium bromide to
give 1–2 in 93% yield for four steps.18 Allyl bromide was
coupled to potassium phthalimide by heating in DMF
followed by cross-coupling to 2,5-dibromopyridine to
give 1–4 in 50% yield. Heck coupling of 1–2 and 1–4
gave 1–5, which was fully deprotected and subsequently
cyclized via an intramolecular Chichibabin reaction to
yield the key intermediate 1–7 in 60% yield for the last
four steps.
the ee of the
D-alanine methyl ester starting material.
In summary, we have incorporated subtle changes to the
structure of clinical development candidate 1 to effect
changes in physical properties and improve pharmaco-
kinetics. A strategically placed methyl group on the
N-terminus of the molecule was found to slightly
increase lipophilicity without dramatically increasing

4518
M. J. Breslin et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4515–4518
OMe
son, J. H. Exp. Opin. Ther. Pat. 2000, 10, 1367; (d)
Coleman, P. J.; Duong, L. T. Exp. Opin. Ther. Pat. 2002,
12, 1009.
OMe
N
N
O
H
N
a,b,c
d,e
10. Hutchinson, J. H.; Halczenko, W.; Brashear, K. M.;
Coleman, P. J.; Duong, L. T.; Fernandez-Metzler, C.;
Gentile, M. A.; Fisher, J. E.; Hartman, G. D.; Huff, J. R.;
Kimmel, D. B.; Leu, C.; Meissner, R. S.; Merkle, K.;
Nagy, R.; Pennypacker, B.; Perkins, J. J.; Prueksaritanont,
T.; Rodan, G. A.; Varga, S. L.; Wesolowski, G. A.;
Zartman, A. E.; Rodan, S. B.; Duggan, M. E. J. Med.
Chem. 2003, 46, 4790.
CO2tBu
OMe
OMe
H3C
N
CO2tBu
4
HN
N
N
2-2
2-1
Scheme 2. Reagents and conditions: (a) triphosgene, Et3N, THF; (b)
1–7, Et3N, THF, 40 °C; (c) 2 M H2SO4; (d) Pd(OH)2, EtOH, AcOH,
H2 (70 psi); (e) 6 M H2SO4.
11. LogP: Measured from octanol, pH 7.4 buffer.
12. Protein binding: Human plasma binding measured in a
high-throughput manner using ultrafiltration of diluted
plasma in a pH 7.4 buffer. The analysis is conducted at
room temperature, and solutions are analyzed by HPLC.
13. Wang, J.; Breslin, M. J.; Coleman, P. J.; Duggan, M. E.;
Hunt, C. A.; Hutchinson, J. H.; Leu, C.; Rodan, S. B.;
Rodan, G. A.; Duong, L. T.; Hartman, G. H. Bioorg.
Med. Chem. Lett. 2004, 14, 1049.
protein binding. On the basis of an improved pharma-
cokinetic profile in three species and efficacy demon-
strated in an in vivo model of bone turnover, 4 was
selected for clinical development.
14. Merck and Co. US Patent 6,472,403 B2, 2002.
15. SPAV3 is a scintillation-proximity assay that measures the
displacement of 4-[2-(2-aminopyridin-6-yl)ethyl]benzoyl-
2(S)4-125Iodophenylsulfonylamino-b-alanine from purified
human recombinant avb3. A similar SPA assay (SPAV5)
utilizes the same ligand to measure its displacement from
avb5. For protocol, see: Duggan, M. E.; Hartman, G. D.;
Hoffman, W. F.; Meissner, R. S.; Perkins, J. J.; Askew, B.
C.; Coleman, P. J.; Hutchinson, J. H.; Naylor-Olsen, A.
M. U.S. Patent 5,981,546, 1999; For preparation of the
radioligand, see: Hamill, T. G.; Duggan, M. E.; Perkins,
J. J. J. Label. Comp. Radiopharm. 2001, 44, 55.
16. Marguerie, G. A.; Plow, E. F.; Edgington, T. S. J. Biol.
Chem. 1979, 254, 5357.
17. Yasuda, N.; Hsiao, Y.; Jensen, M. S.; Rivera, N. R.;
Yang, C.; Wells, K. M.; Yau, J.; Palucki, M.; Tan, L.;
Dormer, P. G.; Volante, R. P.; Hughes, D. L.; Reider, P. J.
J. Org. Chem. 2004, 69, 1959.
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