Design, synthesis, and biological evaluation of novel potent and selective αvβ3/αvβ5 integrin dual inhibitors with improved bioavailability. Selection of the molecular core

Article abstract of DOI:10.1021/jm049725u

A novel series of potent and selective α_vβ ₃/α_vβ₅ dual inhibitors was designed, synthesized, and evaluated against several integrins. These compounds were synthesized through a Mitsunobu reaction between the gua

Full text of DOI:10.1021/jm049725u

926
J. Med. Chem. 2005, 48, 926-934
Design, Synthesis, and Biological Evaluation of Novel Potent and Selective
rvâ3/rvâ5 Integrin Dual Inhibitors with Improved Bioavailability. Selection of
the Molecular Core
Juan Jose´ Maruga´n,* Carl Manthey, Beth Anaclerio, Lou Lafrance, Tianbao Lu, Tom Markotan,
Kristi A. Leonard, Carl Crysler, Stephen Eisennagel, Malini Dasgupta, and Bruce Tomczuk
Johnson and Johnson Pharmaceutical Research & Development, L.L.C., 665 Stockton Drive, Suite 104,
Exton, Pennsylvannia 19341
Received April 9, 2004
A novel series of potent and selective Rvâ3/Rvâ5 dual inhibitors was designed, synthesized, and
evaluated against several integrins. These compounds were synthesized through a Mitsunobu
reaction between the guanidinium mimetics and the corresponding central templates. Guani-
dinium mimetics with enhaced rigidity (i.e., (2-pyridylamino)propoxy versus the 2-(6-methyl-
amino-2-pyridyl)ethoxy) led to improved activity toward Rvâ3. Exemplary oral bioavailability
in mice was achieved using the indole central scaffold. Although, oral bioavailability was
maintained when the indole molecular core was replace with the bioisosteric benzofuran or
benzothiophene ring systems, it was found to not significantly impact the integrin activity or
selectivity. However, the indole series displayed the best in vivo pharmacokinetic properties.
Thus, the indole series was selected for further structure-activity relationships to obtain more
potent Rvâ3/Rvâ5 dual antagonist with improved oral bioavailability.
Chart 1
Introduction
Integrins are a family of heterodimeric cell surface
receptors responsible for the regulation of cell attach-
ment to the extracellular matrix.1 The noncovalent
union of two different subunits, called unit R and unit
â, forms these heterodimeric receptors. Both subunits
are type I membrane proteins with large extracelullar
segments.1 In mammals, there are 19 R and 8 â
subunits, which assemble into 26 different receptors.2
Several integrins have been the focus of attention in the
past decade due to their capacity to regulate cell
mobility and cell-cell interaction. In this sense, IIbIIIa,
the platelet fibrinogen receptor, has been involved in
thrombosis,3 or R4â1, the very late antigen-4 (VLA-4),
has been involved in inflammatory response through the
adhesion, migration, and activation of leukocytes.4 Two
additional integrins that have received considerable
attention have been Rvâ3, the vitronectin receptor5 that
is up-regulated on endothelial cells during tumor angio-
genesis and on smooth muscle cells mobility during
proliferation, and Rvâ5, involved also in angiogenesis.6
These receptors represent an interesting therapeutic
target because of their important role in pathologies
as diverse as osteoporosis,7 restenosis,8 acute renal
failure,9 ocular diseases,10 tumor-induced angiogen-
esis,11 metastasis formation,12 and sickle cell anemia.13
Many of the receptors from the integrin family
recognize the same core amino acid sequence Arg-Gly-
Asp (RGD)14 contained in a number of matrix proteins
(fibronectin, fibrinogen, vitronectin, osteopontin, etc.),
and as consequence, some of the integrin receptors, such
as Rvâ3, are promiscuous to a number of these matrix
proteins.15
A number of groups have reported small molecule
inhibitors designed as RGD mimetics.16 The difficulty
in obtaining Rvâ3 inhibitors with good oral properties
has been recognized for many years as one of the major
hurdles to be overcome.17 One of the reasons is the
zwiterionic character of these RGD mimetics. We have
recently reported a first series of potent Rvâ3/Rvâ5
integrin inhibitors with an O-guanidine moiety as a
basic ending (Chart 1).18 However, the poor bioavail-
ability of these compounds hampered their development
as drug candidates and prompted us to develop other
templates to identify a novel series of compounds with
improved oral bioavailability. A number of teams have
already reported Rvâ3/Rvâ5 inhibitors with good oral
bioavailability.17 The majority of these compounds
incorporate basic ending groups with relatively low pKa
* To whom correspondence should be addressed. Phone: (610) 458-
5264 ext. 6539. Fax: (610) 458-8249. E-mail: JMARUGA1@
prdus.jnj.com.
10.1021/jm049725u CCC: $30.25 © 2005 American Chemical Society
Published on Web 01/28/2005

Potent and Selective Integrin Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 927
Scheme 1. Synthesis of Indole 6a
a Reagents and conditions: (i) NaHCO3, tert-amyl alcohol, reflux; (ii) H2, Pd/C, cyclohexanes-ethanol, room temperature; (iii) NaH/
DMF, ethyl 3-bromopropionate, room temperature; (iv) H2, Pd/C, EtOH, room temperature; (v) n-Bu3P/THF, 1,1-(azodicarbonyl)dipiperidine,
room temperature; (vi) LiOH, MeOH-H2O, room temperature.
Scheme 2. Synthesis of Indole 13a
a Reagents and conditions: (i) di-tert-butyl dicarbonate, 60 °C; (ii) NaH, MeI, DMF, 0 °C to room temperature; (iii) LDA, diethyl carbonate,
THF, -78 °C to 0 °C; (iv) TFA, CH2Cl2, 0 °C to room temperature; (v) LiAlH4, THF, 0 °C to room temperature; (vi) 4, PPh3, DIAD, THF,
0 °C to room temperature; (vii) NaOH, MeOH-H2O, room temperature.
values (e.g., aminopyridine and analogues). The reduc-
tion of the total number of heteroatoms in the molecule,
for example by avoiding sulfonamide substitution near
of the acidic ending, is another approach that has
successfully led to compounds with improved pharma-
cokinetic (PK) properties. In view of these promising
results, we decided to incorporate those premises in our
lead series, while studying the possibility of improving
the druglike properties of the resulting compounds by
modifying the central molecular core. In this paper, we
report the synthesis and biological evaluation of a novel
series of Rvâ3/Rvâ5 dual inhibitors, which incorporate
different bioisosteric ring bicycles (indole, benzoxazole,
and benzothiophene) and which have ultimately led to
the identification of compounds with a better PK profile
and an improved selectivity profile toward R5â1 and
bonate produces 2-[(3-hydroxypropyl)amino]pyridine N-
oxide (1) (Scheme 1), which was reduced by catalytic
hydrogenation to afford 2-[(3-hydroxypropyl)amino]-
pyridine (2) (88% yield). The indole scaffold was pro-
duced by N-alkylation of 5-benzyloxyindole with ethyl
3-bromopropionate in the presence of sodium hydride
in dimethylformamide (DMF). The benzyloxy-protected
group was cleaved under standard hydrogenolysis con-
ditions leading to 4 (53% yield), which was subsequently
coupled with 2 through a Mitsunobu reaction to give
the propionate ester 5 in 60% yield. Hydrolysis of the
ester 5 with lithium hydroxide provided the target 3-{5-
[3-(2-pyridylamino)propoxy]indol-1-yl}propionic acid (6).
Indole 13 was synthesized as outlined in Scheme 2.
2-Amino picoline was first Boc protected and subse-
quently N-alkylated to give 8 in 57% yield. The car-
bonylation of 8 with diethyl carbonate and lithium
diisopropyl amide (LDA) provided the ethyl 2-{6-
[N-(tert-butoxycarbonyl)-N-methylamino]-2-pyridyl}-
acetate 9 in 60% isolated yield. After removal of the Boc
protection, compound 10 was reduced with lithium
RΙΙbâΙΙΙa intergins.
Chemical Synthesis
Reaction of 2-chloropyridine-N-oxide hydrochloride
and 3-aminopropanol in the presence of sodium bicar-

928 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Scheme 3. Synthesis of Benzothiophene 20a
Maruga´n et al.
a Reagents and conditions: (i) acetyl chloride, K2CO3, DMF, room temperature; (ii) triisopropylsilanethiol, NaH, Cl2Pd(Ph3)2,toluene/
THF, reflux; (iii) TBAF, THF, room temperature; (iv) H2SO4 concentrated, 0 °C; (v) 11, N-methylmopholine, Ph3P, DIAD, THF, room
temperature; (vi) NaOH, H2O/THF, room temperature.
Scheme 4. Synthesis of Benzofurane 26a
a Reagents and conditions: (i) TIPSiCl, LiN(SiMe3)2, THF, -78 °C to room temperature; (ii) NaOH, H2O/THF, room temperature; (iii)
11, Ph3P, DIAD, THF, 0 °C to room temperature; (iv) 16, TBAF, THF, room temperature; (v) H2SO4 concentrated, 0 °C; (vi) NaOH, H2O,
THF, room temperature.
aluminum hydride to produce the basic ending chain
11 in 70 % yield. The condensation of the alcohol 11
with the phenol 4 was realized by a Mitsunobu reaction
to afford the protected ester 12, which was eventually
deprotected using sodium hydroxide at the last step of
the reaction sequence, to produce the target indole 13.
The benzothiophene derivative was prepared accord-
ing to the synthesis depicted in Scheme 3. Protection of
3-iodophenol with acetyl chloride followed by a pal-
ladium-mediated cross-coupling reaction with triiso-
propylsilanethiol and iodo 14 afforded the correspond-
ing silanethiol acetate 15 in 52% yield. After the in situ
deprotection of the triisopropylsylil moiety with tetra-
butylammonium fluoride, the thio intermediate was
subsequently S-alkylated with the R-bromoketone 16 to
furnish the diester 17 in 73% yield. Cyclization and
deprotection of 17 were performed by treatment with
concentrate sulfuric acid to give 18 (19% yield). Inter-
mediate 18 was then coupled with alcohol 11 through
a Mitsunobu reaction to produce 19, which was ulti-
mately deprotected to afford the final thiophene 20.
In the case of the benzofurane 26, the resorcinol
monoacetate was reacted with triisopropylsilyl chloride
in the presence of lithium bis(trimethylsilyl)amide to
give 21 (Scheme 4). Hydrolysis of the acetyl protecting
group followed by the reaction with alcohol 11 under
Mitsunobu general conditions yielded ether 23 (28%
yield). Intermediate 23 was then deprotected in situ
with tetrabutylammonium fluoride and was subse-
quently reacted with ethyl 5-bromo-4-oxovalerate to give
the ester 24. Cyclization of 24 using concentrated
sulfuric acid furnished the benzofurane 25, which was
eventually hydrolyzed by treatment with sodium hy-
droxide to afford the target compound 26 in 74% yield.
Biological Activity
All of the compounds synthesized have been evaluated
on a panel of integrins (Rvâ3, Rvâ5, R5â1, and RΙΙbâΙΙΙa
)
for their ability to antagonize integrin-ligand interac-
tions. To predict their propensity for GI absorption, a
CACO-2 permeability evaluation was also determined.
All of these data are depicted in Table 1.

Potent and Selective Integrin Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 929
Table 1. In Vitro Inhibition of Protein Binding and CACO-2
Cell Permeability of Compounds 6, 13, 20, and 26
CACO-2
permeability
(AB cm/s)
ELISA IC50 (µM)a
compound
number
Rvâ3
Rvâ5
R5â1 ΙΙbΙΙΙa
6
0.47
0.0025
((0.0007)
0.21
>20
>20
>20
>20
>20
6 × 10-6
((0.03)
((1 × 106)
6 × 10-6
13
20
26
0.049
((0.012) ((0.04)
0.030
((0.003) ((0.05)
0.033 0.42
((0.002) ((0.24)
15
((3)
7.8
((2.5)
>20
((1 × 106)
16 × 10-6
((1 × 106)
17 × 10-6
((1 × 106)
0.14
Figure 1.
a In vitro inhibition (n g 3, mean (SD)), as measured by ELISA,
of Rvâ3-vitronectin, Rvâ5-vitronectin, R5â1-fibronectin, and ΙΙbΙΙΙa-
fibrinogen protein interactions.
As seen by comparison of compounds 6 and 13,
switching the position of the pyridine ring has a
beneficial impact on the activity against Rvâ3 (IC50-Rvâ3
) 0.47 and 0.049 µM, respectively) but considerably
decreases the activity against Rvâ5 (IC50-Rvâ5 ) 0.0025
and 0.21 µM, respectively). Interestingly, the selectivity
profile toward the other integrins (R5â1, RΙΙbâΙΙΙa) is not
significantly altered by this modification. Further bio-
isosteric replacement of the indole template by the
bicyclic benzothiophene or benzofurane ring systems
does not dramatically affect the activity against Rvâ3 or
the selectivity profile of the resulting compounds (20 and
26, respectively). As seen in Table 1, all of these
compounds (6, 13, 20, and 26) exhibited a very similar
CACO-2 permeability profile. However, the benzo-
thiophene 20 and benzofurane 26 derivatives appeared
to be slightly more permeable that the indoles 6 and
13. No evidence of efflux activity has been seen with
these four compounds (data not shown).
Figure 2.
furan was the worst due to a small volume of distribu-
tion and high clearance. Overall, the pharmacokinetic
behavior highlighted the indole-containing derivatives
as the most promising series for the development of
novel potent, selective, and oral bioavailable Rvâ3/Rvâ5
antagonists. Further studies identified the indole scaf-
fold as superior in a second species, dog (data not
shown).
To assess whether the bioisosteric replacement of the
central molecular core of these Rvâ3 antagonists would
affect their pharmacokinetic profile, a full mouse PK
study was undertaken. These data are reported in Table
2 and illustrated in Figures 1 and 2.
In summary, a novel series of Rvâ3/Rvâ5 inhibitors was
synthesized and evaluated in vivo and in vitro for their
potency and selectivity profiles against Rvâ5 and RIIbâIIIa
.
Consistent with the very promising in vitro CACO-2
permeability data, all three compounds (13, 20, and 26)
exhibited rapid and nearly complete absorption with
Tmax values between 15 and 30 min and good Cmax
values (38.3, 27.5, and 16.2 µg/mL, respectively) after
oral administration. The indole analogue 13 showed the
best overall pharmacokinetic profile after iv and oral
administration with low systemic clearance in relation
to liver blood flow (liver blood flow ) 90 mL/(min/kg)),19
moderate volume of distribution (total body water )
725 mL/kg),19 and the highest AUCinf value. The benzo-
Interestingly, it was observed that the switch of the
position of the pyridine ring of the basic ending has a
positive effect in terms of activity against Rvâ3. Although
the bioisosteric replacement of the indole ring with
benzofuran or benzothiophene is well-tolerated in vitro
in terms of Rvâ3 potency or in the CACO-2 permeability,
the indole 13 appears to have a much better pharma-
cokinetics profile than the benzothiophene (20) or the
benzofuran (26). Thus, we have selected the indole
series to perform further SAR studies (manuscript in
preparation).
Table 2. PK Parameters Determination for Compounds 13, 20, and 26a
compound number
13
20
26
5 mpk iv
50 mpk po
5 mpk iv
50 mpk po
5 mpk iv
50 mpk po
Cmax (µg/mL) (n ) 3)
Tmax (h) (n ) 3)
t1/2 (h)
CL (mL/(min/kg))
Vz (L/kg)
AUCinf (µg/(mL‚min))
% ext AUC
38.3 ((3.7)
0.25-0.50
2.39
27.5 ((8.4)
0.25-0.50
2.34
16.2 ((8.8)
0.25-0.50
1.92
1.56
11.74
1.58
426.0
3
0.74
16.5
1.05
302.2
1
0.09
65.1
0.50
76.9
2
3837.6
7
2201.8
5
877.3
1
MRT (h)
F
1.72
0.56
0.11
90%
73%
114%
a PK parameters were calculated from the mean curve prepared with data from three animals at each time point.

930 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Maruga´n et al.
mineral oil (0.60 g, 15 mmol). After being stirred 1 h at
ambient temperature, the reaction mixture was treated with
ethyl 3-bromopropionate (1.00 mL, 6.96 mmol) and stirred an
additional 18 h. The reaction was then treated with additional
sodium hydride (0.3 g, 7.5 mmol) and stirred two more hours,
and the solvent removed in vacuo. The crude product was
dissolved in methylene chloride, washed with 10% aqueous
HCl, water, and brine, dried over anhydrous sodium sulfate,
and filtered. The filtrate was evaporated, and the residue was
purified by flash column chromatography (1:1 methylene
chloride/ethyl acetate eluant) giving compound 3 as a yellow
Experimental Section
Chemical Synthesis. Reagents used for synthesis were
purchased from Sigma-Aldrich (Milwaukee, WI) and Lancaster
(Windham, NH). All of the solvents were obtained from
commercial suppliers and used without further purification.
Flash chromatography was performed on Geduran Silical gel60
(40-63 µm, Merck). The spots were visualized either under
UV light (λ ) 254 nm) or by spraying with molybdate reagent
(H2O/concentrated H2SO4/(NH4)6Mo7O24‚4H2O/ (NH4)Ce(SO4)4‚
2H2O, 90/10/25/1 v/v/w/w) and charring at 140 °C for a few
minutes. All of the chemical yields are unoptimized and
generally represent the result of a single experiment.
1H NMR spectra were recorded on a Brucker B-ACS-120
(400 MHz) spectrophotometer at room temperature. Chemical
shifts are given in ppm (δ), coupling constants (J) are in hertz
(Hz), and signals are designed as follows: s, singled; d, doublet;
t, triplet; q, quadruplet; quint, quintuplet; m, multiplet; br s,
broad singlet.
LC-MS/ELS was performed on a system consisting of an
electrospray source on a Finnigan LCQ ion trap mass spec-
trometer, a SEDEX 75C evaporative light scattering detector,
a Shimadzu LC-10ADvp binary gradient pumping system, a
Gilson 215 configured as an autosampler, and a Princeton
Chromatography HTS HPLC columm (5 µm, 50 mm × 3.0
mm).
1
oil (0.96 g, 51%). H NMR (400 MHz, CDCl3) δ: 7.47 (br d, J
) 7.2 Hz, 2H), 7.37 (m, 2H), 7.32 (m, 1H), 7.24 (br d, J ) 8.8
Hz, 1H), 7.15 (d, J ) 2.4 Hz, 1H), 7.10 (m, 1H), 6.96 (dd, J )
8.8 Hz, 2.4 Hz, 1H), 6.38 (m, 1H), 5.09 (s, 2H), 4.44 (t, J ) 6.9
Hz, 2H), 4.21 (q, J ) 7.1 Hz, 2H), 2.92 (t, J ) 6.9 Hz. 2H),
1.26 (m, 3H).
Ethyl 3-(5-Hydroxyindolyl)propanoate (4). A solution
of 3 (0.94 g, 2.90 mmol) and 10% palladium(0) on carbon (97
mg) in reagent ethanol (40 mL) was stirred under hydrogen
at ambient pressure and temperature for 18 h. The reaction
was filtered over Celite, and the evaporated filtrate was
purified by flash column chromatography (10% ethyl acetate
in methylene chloride eluant) to yield 4 as colorless oil. (0.36
1
g, 53%). H NMR (400 MHz, CDCl3) δ: 7.18 (d, J ) 8.7 Hz,
1H), 7.10 (d, J ) 3.0 Hz, 1H), 7.01 (d, J ) 1.9 Hz, 1H), 6.78
(dd, J ) 8.7 Hz, 2.2 Hz, 1H), 6.34 (d, J ) 3.0 Hz, 1H), 4.86 (s,
1H), 4.43 (t, J ) 6.9 Hz, 2H), 4.22 (q, J ) 7.1 Hz, 2H), 2.92 (t,
J ) 6.9 Hz, 2H), 1.27 (t, J ) 7.1 Hz, 3H).
Accurate mass analysis was performed using a Micromass
Autospec OATOF high-resolution magnetic sector mass spec-
trometer. Compounds were ionized using fast atom bombard-
ment (FAB) ionization with poly(ethylene glycol) (PEG) matrix.
Samples were prepared by dissolving approximately 0.2 mg
of compound into 200 µL of PEG deposited on the FAB target.
Mass measurement was performed using voltage scanning and
bracketing the molecular ion of interest with two PEG refer-
ence peaks.
Ethyl 3-{5-[3-(2-Pyridylamino)propoxy]indolyl}pro-
panoate (5). A solution 4 (0.35 g, 1.51 mmol) and 2 (0.24 g,
1.58 mmol) in anhydrous tetrahydrofuran (25 mL) was treated
with tri-n-butylphosphine (0.43 mL, 1.72 mmol) and 1,1-
(azodicarbonyl)dipiperidine (0.43 g, 1.70 mmol) at ambient
temperature. After 18 h, the reaction was concentrated in
vacuo, and the crude product purified by flash column chro-
matography (1:1 methylene chloride/ethyl acetate eluant)) to
The purity of the final compounds was analyzed with a 218
PrepStar Varian HPLC system connected to a ProStar Varian
UV detector and following the peaks at λ ) 254 nm. The flow
was 1 mL/min, and the gradient was from 5% acetonitrile/
water (with 0.1% of trifluoroacetic acid), until 95% over a
period of 23 min. The columns for the analysis were Betabasic
C18 (5 µm, 150 Å, 150 mm × 4.6 mm) and Betabasic C8 (5
µm, 150 Å, 150 mm × 4.6 mm). All of the the final compounds
had 95% or greater purity (Supporting Information).
2-(3-Hydroxypropyl)aminopyridine N-Oxide (1). A mix-
ture of 2-chloropyridine-N-oxide hydrochloride (3.32 g, 20
mmol), 3-amino-1-propanol (3.06 mL, 40 mmol), and NaHCO3
(8.4 g, 100 mmol) in tert-amyl alcohol (20 mL) was heated to
reflux. After being stirred overnight, the reaction mixture was
cooled, diluted with methylene chloride (100 mL), and suction
filtered to remove the insoluble materials. The filtrate was
concentrated and reconcentrated from methylene chloride
twice. The residue was recrystallized from ethyl acetate and
hexane, collected by filtration, washed with ethyl acetate, and
dried under high vacuum to give the title compound as a pale
1
yield compound 5 as a yellow oil (0.33 g, 60%). H NMR (400
MHz, CDCl3) δ: 8.08 (dd, J ) 5 Hz, 1 Hz, 1H), 7.40 (m, 1H),
7.24 (d, J ) 8.8 Hz, 1H), 7.11 (d, J ) 3.1 Hz, 1H), 7.09 (d, J )
2.4 Hz, 1H), 6.89 (dd, J ) 8.8 Hz, 2.4 Hz, 1H), 6.55 (m, 1H),
6.41 (d, J ) 8.4 Hz, 1H), 6.39 (d, J ) 3.0 Hz, 1H), 4.76 (br m,
1H), 4.45 (t, J ) 6.9 Hz, 2H), 4.22 (q, J ) 7.1 Hz, 2H), 4.12
(m, 2H), 3.53 (dd, J ) 12.6 Hz, 6.5 Hz, 2H), 2.93 (t, J ) 6.9
Hz, 2H), 2.12 (quint., J ) 6 Hz, 2H), 1.27 (m, 3H).
3-{5-[3-(2-Pyridylamino)propoxy]indolyl}propanoic
Acid Ammonium Salt (6). Compound 5 (0.33 g, 0.90 mmol)
was dissolved in methanol (10 mL) and treated with 1 N
aqueous LiOH (2 mL) at ambient temperature. After 18 h, the
reaction was acidified with 10% aqueous HCl and concentrated
in vacuo, and the crude product was purified by flash column
chromatography (15% methanol in methylene chloride eluant),
giving a very hygroscopic solid. This was dissolved in a mixture
of methylene chloride and methanol (saturated with ammonia
gas) and filtered, and the filtrate was concentrated in vacuo
to yield 6 as a stable, pale yellow solid (0.14 g, 42%). 1H NMR
(400 MHz, DMSO-d6) δ: 7.92 (m, 1H), 7.59 (m, 1H), 7.37 (d, J
) 8.9 Hz, 1H), 7.28 (d, J ) 3.1 Hz, 1H), 7.04 (d, J ) 2.3 Hz,
1H), 6.78 (dd, J ) 8.9 Hz, 2.3 Hz, 1H), 6.75 (d, J ) 9.7 Hz,
1H), 6.63 (br t, J ) 6.3 Hz, 1H), 4.34 (t, J ) 6.8 Hz, 2H), 4.05
(t, J ) 6.2 Hz, 2H), 3.45 (dd, J ) 12.5 Hz, 6.6 Hz, 2H), 2.71 (t,
J ) 6.8 Hz, 2H), 2.02 (quint., J ) 6.5 Hz, 2H). Mass spectrum
(LCMS, ESI+) Calcd for C19H21N3O3: 339.4 (M + H). Found:
340.1. HSMS (FAB+) Calcd for C19H22N3O3: 340.166117
(MH+). Found: 340.166819.
(tert-Butoxy)-N-[6-methyl-(2-pyridyl)]carboxamide (7).
A mixture of 2-amino-picoline (6.0 g, 5.5 mmol) and di-tert-
butyl dicarbonate (13.3 g, 6.0 mmol) was heated to 60 °C
overnight (16 h). The reaction was cooled, poured into satu-
rated NH4Cl (250 mL), and extracted with ethyl acetate
(2 × 250 mL). The combined organic layers were washed with
brine, dried (Na2SO4), filtered, and concentrated to give a
yellow oil (crude 12.3 g), which was used directly in the next
reaction.
1
yellow solid (3.2 g, 95%). H NMR (400 MHz, CDCl3) δ: 8.07
(d, J ) 6.5 Hz, 1H), 7.32 (br s, 1H), 7.21 (t, J ) 8.6 Hz, 1H),
6.64 (d, J ) 8.5 Hz, 1H), 6.53 (t, J ) 6.7 Hz, 1H), 3.75 (t, J )
5.8 Hz, 2H), 3.47 (q, J ) 6.2 Hz, 2H), 1.86 (t, J ) 6.0 Hz, 2H).
2-(3-Hydroxypropyl)aminopyridine (2). A mixture of 1
(3.0 g, 17.9 mmol), as prepared in the preceding step, cyclo-
hexene (10 mL, 100 mmol), and 10% palladium(0) on carbon
(300 mg) in ethanol (50 mL) was heated to reflux. After 2 days,
the reaction mixture was cooled. The catalyst was removed
by filtration through Celite, and the filtrate was concentrated.
The residue was purified by flash column chromatography
(silica gel, 5% methanol in methylene chloride) to give 2 as a
1
colorless oil (2.4 g, 88%). H NMR (400 MHz, CDCl3) δ: 8.02
(d, J ) 5.0 Hz, 1H), 7.37 (t, J ) 7.8 Hz, 1H), 6.54 (d, J ) 6.0
Hz, 1H), 6.39 (t, J ) 8.0 Hz, 1H), 4.69 (br s, 2H), 3.65 (t, J )
5.5 Hz, 2H), 3.53 (q, J ) 5.9 Hz, 2H), 1.77 (t, J ) 5.6 Hz, 2H).
Ethyl 3-(5-Benzyloxy)indolylpropanoate (3). A solution
of 5-benzyloxyindole (1.30 g, 5.82 mmol) was dissolved in
anhydrous N,N-dimethylformamide (25 mL) under nitrogen
and treated with a 60% suspension of sodium hydride in

Potent and Selective Integrin Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 931
(tert-Butoxy)-N-methyl-N-[6-methyl-(2-pyridyl)]car-
boxamide (8). To a suspension of NaH (2.63 g 6.6 mmol) in
200 mL of N,N-dimethylformamide at 0 °C was added a
solution of (tert-butoxy)-N-[6-methyl-(2-pyridyl)]carboxamide
7 (12.3 g, crude), as prepared in the preceding step, in 50 mL
of N,N-dimethylformamide. The reaction stirred at 0 °C for
15 min then at ambient temperature for 1 h. Then, iodo-
methane (10.22 g, 7.2 mmol) was added, and the mixture was
stirred at ambient temperature overnight (16 h). The reaction
mixture was concentrated in vacuo, diluted with saturated
NH4Cl (400 mL), and extracted with ethyl acetate (2 × 250
mL). The combined organic layers were washed with brine,
dried (Na2SO4), filtered, and concentrated. The residue was
purified by flash chromatography on silica gel (10% ethyl
ambient temperature overnight (16 h), the reaction was
concentrated and the residue was purified by flash chroma-
tography on silica gel (20%-30% ethyl acetate in hexane) to
yield 12 as yellow oil (0.023 g, 15%). 1H NMR (400 MHz,
CDCl3) δ: 7.39 (t, J ) 7.3 Hz, 1H), 7.20 (d, J ) 8.9 Hz, 1H),
7.11 (d, J ) 2.3 Hz, 1H), 7.07 (d, J ) 3.1 Hz, 1H), 6.87 (dd, J
) 2.4, 8.9 Hz, 1H), 6.56 (d, J ) 7.2 Hz, 1H), 6.37 (d, J ) 3.1
Hz, 1H), 6.24 (d, J ) 8.2 Hz, 1H), 4.56 (br s, 1H), 3.40 (t, J )
6.9 Hz, 2H), 4.34 (t, J ) 7.0 Hz, 2H), 3.65 (s, 3H), 3.10 (t, J )
7.0 Hz, 2H), 2.89 (d, J ) 4.8 Hz, 2H), 2.80 (t, J ) 6.9 Hz, 2H).
3-(5-{2-[6-(Methylamino)-2-pyridyl]ethoxy}indolyl)-
propanoic Acid (13). To a solution of methyl 3-(5-{2-[6-
(methylamino)-2-pyridyl]ethoxy}indolyl)propanoate 12 (0.023
g, 0.65 mmol) in methanol (3 mL) was added sodium hydroxide
(0.15 g, 3.8 mmol) in H2O (0.5 mL), and the reaction was
stirred for 6 h at ambient temperature. After the solvent was
evaporated in vacuo, the residue is taken up in H2O (5 mL)
and acidified to pH 4-5 with 10% HCl and extracted with a
mixture of ethyl acetate and butanol (2 × 50 mL) and the
combined organic layers were washed with brine, dried
(Na2SO4), filtered, and concentrated to yield 13 as a solid (0.018
g, 82%). 1H NMR (400 MHz, CDCl3 + CD3OD) δ: 7.52 (t, J )
7.3 Hz, 1H), 7.25 (d, J ) 8.9 Hz, 1H), 7.14 (d, J ) 3.1 Hz, 1H),
7.06 (d, J ) 2.3 Hz, 1H), 6.81 (dd, J ) 8.9, 2.4 Hz, 1H), 6.60
(d, J ) 7.3 Hz, 1H), 6.38 (d, J ) 8.6 Hz, 1H), 6.33 (d, J ) 3.2
Hz, 1H), 4.38 (t, J ) 7.0 Hz, 2H), 4.24 (t, J ) 6.6 Hz, 2H), 3.06
(t, J ) 6.6 Hz, 2H), 2.89 (s, 3H), 2.77 (t, J ) 6.9 Hz, 2H). Mass
spectrum (LCMS, ESI+) Calcd for C19H21N3O3: 340.3 (M +
H). Found: 340.9. HSMS (FAB+) Calcd for C19H22N3O3:
340.166117 (MH+). Found: 340.166546.
3-Iodophenyl Acetate (14). A solution of 3-iodophenol (3
g, 13.6 mmol), acetyl chloride (2.9 mL, 40.9 mmol), and
potassium carbonate (9.42 g, 68.2 mmol) in N,N-dimethyl-
formamide (75 mL) was stirred for 16 h at room temperature.
The mixture was partitioned between water and ethyl acetate.
The organic layer was washed with 1N NaOH, dried over
magnesium sulfate, and evaporated under vacuum. The crude
product was chromatographed over silica gel, eluting with 20%
ethyl acetate/hexanes to yield 2.3 g (65%) of 14. 1H NMR (400
MHz, CDCl3) δ: 7.57 (m, 1H), 7.46 (m, 1H), 7.08 (m, 2H) 2.29
(s, 3H).
3-[1,1-Bis(methylethyl)-2-methyl-1-silapropylthio]phen-
yl Acetate (15). Triisopropylsilanethiol (2.91 mL, 13.5 mmol)
was added dropwise to a suspension of sodium hydride (325
mg, 13.5 mmol) in THF (10 mL). After the evolution of
hydrogen ceased, a solution of 14 (2.37 g, 9.0 mmol) and
tetrakis(triphenylphosphine)palladium(0) (1.04 g, 0.9 mmol)
in toluene (90 mL) was added. After being refluxed for 16 h
under argon, the reaction was cooled to room temperature, and
the solvent was evaporated under vacuum. The resulting
residue was dissolved in ethyl acetate, washed with 1 N NaOH
and brine, dried with sodium sulfate, filtered, and evaporated
under vacuum. The crude product was chomatrographed over
silica gel to yield 1.53 g (52%) of 15. 1H NMR (400 MHz, CDCl3)
δ: 7.34 (m, 2H), 7.23 (t, J ) 2.4 Hz, 1H), 6.94 (dd, J ) 1.2, 8.4
Hz, 1H), 2.28 (s, 3H), 1.25 (m, 3H), 1.08 (d, J ) 7.2 Hz, 18 H).
1
acetate in hexane) to give 8 as a yellow oil (7.56 g, 57%). H
NMR (400 MHz, DMSO-d6) δ: 7.63 (t, J ) 7.2 Hz, 1H), 7.37
(d, J ) 8.0 Hz, 1H), 6.97 (d, J ) 6.9 Hz, 1H), 3.27 (s, 2H), 2.42
(s, 3H), 1.45 (s, 9H).
Ethyl 2-{6-[(tert-butoxy)-N-methylcarbonylamino]-2-
pyridyl}acetate (9). Lithium diisopropylamide (6.6 mmol)
was prepared in tetrahydrofuran (60 mL) and cooled to -78
°C, and (tert-butoxy)-N-methyl-N-[6-methyl-(2-pyridyl)]car-
boxamide (8) (7.56 g, 3.3 mmol) was dissolved in tetrahydro-
furan (100 mL) and added dropwise over 30 min. The mixture
was stirred for 15 min then diethyl carbonate (6.24 g, 5.3
mmol) was added. The solution was stirred for an additional
15 min, and then allowed to warm to 0 °C over 2 h. The
reaction was quenched with saturated NH4Cl solution (200
mL). The mixture was allowed to warm to ambient tempera-
ture and extracted with ethyl acetate (2 × 100 mL). The
combined organic layers were washed with brine, dried
(Na2SO4), filtered, and concentrated. The residue was purified
by flash chromatography (silica gel, 10% ethyl acetate in
1
hexane) to yield 9 as yellow oil (5.51 g, 60%). H NMR (400
MHz, DMSO-d6) δ: 7.71 (t, J ) 7.9 Hz, 1H), 7.49 (d, J ) 8.2
Hz, 1H), 7.07 (d, J ) 7.4 Hz, 1H), 4.09 (q, J ) 7.1 Hz, 2H),
3.78 (s, 2H), 2.54 (s, 3H), 1.46 (s, 9H), 1.18 (t, J ) 7.1 Hz, 3H).
Ethyl 2-[6-(methylamino)-2-pyridyl]acetate (10). A so-
lution of 9 (5.51 g, 1.9 mmol) in methylene chloride (25 mL)
was stirred in an ice bath at 0 °C. Trifluoroacetic acid (10 mL)
was then added, and the solution was allowed to warm to
ambient temperature and stirred overnight (16 h). The reac-
tion mixture was concentrated, 10% aqueous K2CO3 (300 mL)
was added, and the mixture was extracted with ethyl acetate
(2 × 100 mL). The combined organic layers were washed with
brine, dried (Na2SO4), filtered, and concentrated to yield 10
as bright yellow oil (3.4 g, 100%). 1H NMR (400 MHz, DMSO-
d6) δ: 7.32 (t, J ) 7.2 Hz, 1H), 6.40 (d, J ) 7.0 Hz, 1H), 6.29
(d, J ) 8.3 Hz, 1H), 4.07 (q, J ) 7.1 Hz, 2H), 3.56 (s, 2H), 2.71
(d, J ) 4.9 Hz, 3H), 1.17 (t, J ) 7.1 Hz, 3H).
2-[6-(Methylamino)-2-pyridyl]ethan-1-ol (11). To a sus-
pension of lithium aluminum hydride (1.8 g, 4.9 mmol) in
tetrahydrofuran (50 mL) was added dropwise a solution of 10
(3.5 g, 1.9 mmol) in tetrahydrofuran (50 mL) at 0 °C. After
the addition was completed, the reaction mixture was stirred
at 0 °C for 30 min and then stirred at ambient temperature
for 2 h. The reaction mixture was then cooled back to 0 °C,
quenched with H2O (1.8 mL), 10% NaOH (1.8 mL), and H2O
(3.0 mL), and allowed to warm back to ambient temperature.
The solids were removed by filtration through Celite and
washed with tetrahydrofuran (100 mL). The filtrate was dried
(Na2SO4), filtered, and concentrated. The residue was purified
by flash chromatography on silica gel (3% methanol in meth-
ylene chloride) to yield 11 as yellow oil (2.1 g, 70%). 1H NMR
(400 MHz, CDCl3) δ: 7.36 (t, J ) 7.8 Hz, 1H), 6.41 (d, J ) 7.2
Hz, 1H), 6.26 (d, J ) 8.3 Hz, 1H), 4.51 (br s, 1H), 3.96 (t, J )
5.2 Hz, 2H), 2.89 (d, J ) 5.1 Hz, 3H), 2.84 (t, J ) 5.4 Hz, 2H).
Ethyl 5-Bromo-4-oxopentanoate (16). (Trimethylsilyl)-
diazomethane (34 mL, 67 mmol, 2.0 M solution in hexanes)
was added dropwise to a solution of ethyl succinyl chloride (5
g, 30.3 mmol) in acetonitrile (60 mL) over a period of 30 min.
After the mixture was stirred for 2 h, hydrogen bromide (14
mL, 30% solution in acetic acid) was slowly added over 15 min.
After the reaction was stirred for an additional 1 h, the solvent
was evaporated under vacuum. The residue was dissolved in
ethyl acetate and washed with 1 N NaOH and brine. The
organic layer was dried with sodium sulfate, filtered, and
evaporated under vacuum to yield 4.3 g (64%) of 16. 1H NMR
(400 MHz, CDCl3) δ: 4.13 (c, J ) 7.2 Hz, 2H), 3.96 (s, 2H),
2.95 (t, J ) 6.4 Hz, 2H), 2.65 (t, J ) 6.4 Hz, 2H), 1.24 (t, J )
7.2 Hz, 1H).
Methyl 3-(5-{2-[6-(Methylamino)-2-pyridyl]ethoxy}-
indolyl)propanoate (12). Diisopropyl azodicarboxylate (0.19
g, 0.94 mmol) was added to a solution of 2-[6-(methylamino)-
2-pyridyl]ethan-1-ol 11 (0.10 g, 0.66 mmol), methyl 3-(5-
hydroxyindolyl)propanoate (4) (0.10 g, 0.46 mmol), and tri-
phenylphosphine (0.24 g, 0.92 mmol) in tetrahydrofuran (5.0
mL) at 0 °C in an ice bath. After the mixture was stirred at
Ethyl 5-(3-Acetyloxyphenylthio)-4-oxopentanoate (17).
Tetrabutylammonium fluoride (7 mL, 7.0 mmol, 1 M in THF)
was added to a solution of 15 (1.53 g, 4.7 mmol) in THF (10
mL) under argon at room temperature. The reaction was

932 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4
Maruga´n et al.
stirred for 15 min, followed by addition of a solution of 16 (1.15
g, 5.17 mmol) in THF (5 mL). After the reaction was stirred
for 3 h, the solvent was removed under vacuum, and the crude
product was chromatographed over silica gel to yield 920 mg
(73%) of 17. 1H NMR (400 MHz, CDCl3) δ: 7.29 (t, J ) 8.0 Hz,
1H), 7.18 (dd, J ) 0.8, 7.6 Hz, 1H), 7.07 (t, J ) 1.6 Hz, 1H),
6.94 (dd, J ) 1.2, 8.0 Hz, 1H), 4.12 (c, J ) 7.2 Hz, 2H), 3.75 (s,
2H), 2.89 (t, J ) 6.8 Hz, 2H), 2.60 (t, J ) 6.8 Hz, 2H), 2.29 (s,
3H), 1.24 (t, J ) 7.2 Hz, 1H).
Ethyl 3-(6-Hydroxybenzo[b]thiophen-3-yl)propanoate
(18). Concentrated sulfuric acid (20 mL) was cooled in an ice-
water bath to 0 °C and added to a flask containing 17 (920
mg, 3.4 mmol) at 0 °C. The reaction was stirred at 0 °C for 15
min and then poured over ice. The mixture was extracted with
ethyl acetate, dried, filtered, and evaporated under vacuum
to yield 700 mg (82%) of 18. 1H NMR (400 MHz, DMSO-d6) δ:
9.59 (s, 1H), 7.59 (d, J ) 8.4 Hz, 1H), 7.25 (d, J ) 2.4 Hz, 1H),
7.08 (s, 1H), 6.89 (dd, J ) 2.4, 8.4 Hz, 1H), 4.06 (c, J ) 7.2 Hz,
2H), 2.99 (t, J ) 6.8 Hz, 2H), 2.70 (t, J ) 6.8 Hz, 2H), 1.16 (t,
J ) 7.2 Hz, 1H).
the reaction mixture was partitioned between ethyl acetate
and water. The organic layer was washed with brine, dried,
filtered, and evaporated under vacuum. The crude product was
chromatographed over silica gel to yield 3.89 g (90%) of 22.
1H NMR (400 MHz, CDCl3) δ: 7.06 (t, J ) 8.0 Hz, 1H), 6.42
(m, 3H), 1.28 (m, 3H), 1.10 (d, J ) 7.0 Hz, 18H).
[6-(2-{3-[1,1-Bis(methylethyl)-2-methyl-1-silapropoxy]-
phenoxy}ethyl)(2-pyridyl)]methylamine (23). To a stirred
solution of 22 (200 mg, 0.75 mmol), 11 (104 mg, 0.68 mmol),
triphenylphosphine (199 mg, 0.75 mmol), and THF (25 mL)
was added diethyl azodicarboxylate (0.12 mL, 0.75 mmol) at
0 °C. The reaction was stirred overnight under argon. The
solvent was removed under vacuum, and the crude product
was chromatographed over silica gel to yield 76 mg (28%) of
1
23. H NMR (400 MHz, CDCl3) δ: 7.39 (m, 1H), 7.07 (t, J )
8.0 Hz, 1H), 6.5 (m, 3H), 6.25 (d, J ) 8 Hz, 1H), 4.27 (t, J )
6.8 Hz, 2H), 3.06(t, J ) 6.8 Hz, 2H), 2.90 (d, J ) 8.0 Hz, 3H),
1.28 (m, 3H), 1.10 (d, J ) 7.0 Hz, 18H).
Ethyl
5-(3-{2-[6-(Methylamino)(2-pyridyl)]ethoxy}-
phenoxy)-4-oxopentanoate (24). To a solution of 23 (1.60
g, 4.0 mmol) in THF (30 mL) under argon at room temperature
was added tetrabutylammonium fluoride (4.4 mL, 4.4 mmol,
1 M in THF). After the mixture was stirred for 15 min, a
solution of 16 (0.98 g, 4.4 mmol) in THF (5 mL) was added.
The mixture was stirred for an additional 3 h. The solvent was
removed under vacuum, and the remaining residue was
chromatographed over silica gel to yield 860 mg (56%) of 24.
1H NMR (400 MHz, CDCl3) δ: 7.38 (m, 1H), 7.16 (t, J ) 8.2
Hz, 1H), 7.08 (t, J ) 7.9 Hz, 1H), 6.64 (m, 1H), 6.45 (m, 3H),
6.26 (dd, J ) 8.2, 2.3 Hz, 1H), 4.57 (s, 2H), 4.30 (t, J ) 6.8 Hz,
2H), 4.13 (c, J ) 7.2 Hz, 2H), 3.07 (m, 2H), 2.91 (m, 5H), 2.63
(t, J ) 6.6 Hz, 2H), 1.24 (t, J ) 7.2 Hz, 3H).
Ethyl 3-(6-{2-[6-(Methylamino)-2-pyridyl]ethoxy}benzo-
[b]thiophen-3-yl)propanoate (19). Ethyl 3-(6-hydroxybenzo-
[b]thiophen-3-yl)propanoate (18) (100 mg, 0.4 mmol) and
4-methylmorpholine (0.05 mL, 0.44 mmol) were dissolved in
THF (5 mL) and stirred for 5 min. 2-[6-(Methylamino)-2-
pyridyl]ethan-1-ol (11) (91 mg, 0.6 mmol), triphenylphosphine
(210 mg, 0.8 mmol), and diisopropyl azodicarboxylate (0.16 mL,
0.8 mmol) were added to the mixture sequentially. After being
stirred overnight under argon, the reaction mixture was
partitioned between ethyl acetate and water. The organic layer
was dried with sodium sulfate, filtered, and evaporated under
vacuum. The crude product was chromatographed over silica
1
gel to yield 30 mg (19%) of 19. H NMR (400 MHz, CDCl3) δ:
Ethyl 3-(6-{2-[6-(Methylamino)-2-pyridyl]ethoxy}benzo-
[b]furan-3-yl)propanoate (25). Concentrated sulfuric acid
(3 mL) was cooled in an ice-water bath to 0 °C and added to
a flask containing 24 (190 mg, 0.5 mmol) at 0 °C. The reaction
was stirred 15 min and then poured over ice. The solution was
neutralized with solid sodium hydrogencarbonate (pH ) 7),
and the product was extracted with ethyl acetate. The organic
layer was dried, filtered, and evaporated under vacuum to yield
7.60 (d, J ) 8.8 Hz, 1H), 7.38 (dd, J ) 7.2, 8.0 Hz, 1H), 7.35
(d, J ) 2.0 Hz, 1H), 7.07 (dd, J ) 2.4, 8.8 Hz, 1H), 6.93 (m,
1H), 6.56 (d, J ) 7.2 Hz, 1H), 6.25 (d, J ) 8.0 Hz, 1H), 4.40 (t,
J ) 6.8 Hz, 2H), 4.15 (c, J ) 7.2 Hz, 2H), 3.10 (t, J ) 6.4 Hz,
2H), 2.90 (m, 5H), 2.74 (t, J ) 6.4 Hz, 2H), 1.25 (t, J ) 7.2 Hz,
1H).
3-(6-{2-[6-(Methylamino)-2 -pyridyl]ethoxy}benzo[b]-
thiophen-3-yl)propanoic Acid (20). NaOH (10 mL, 1 N) was
added to a solution of 19 and THF (10 mL). The reaction was
stirred at room temperature for 16 h. The mixture was diluted
with water and ethyl acetate. The separated aqueous layer
was neutralized with 1 N HCl to pH ) 6.5. The resulting
precipitate was filtered, washed with distilled water, and dried
to yield 74 mg (55%) of 20 as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ: 7.67 (d, J ) 8.0 Hz, 1H), 7.58 (d, J ) 2.4 Hz,
1H), 7.31 (dd, J ) 7.2, 8.0 Hz, 1H), 7.18 (s, 1H), 7.00 (dd, J )
2.4, 8.0 Hz, 1H), 6.45 (d, J ) 7.2 Hz, 1H), 6.37 (m, 1H), 6.27
(d, J ) 8.0 Hz, 1H), 4.36 (t, J ) 6.4 Hz, 2H), 2.99 (t, J ) 6.4
Hz, 2H), 2.90 (d, J ) 8.0 Hz, 3H), 2.64 (t, J ) 6.4 Hz, 2H).
Mass spectrum (LCMS, ESI) Calcd for C19H21N2O3S: 357.1 (M
+ H). Found: 357.3. HSMS (FAB+) Calcd for C19H21N2O3S:
357.127290 (MH+). Found: 357.128119.
3-[1,1-Bis(methylethyl)-2-methyl-1-silapropoxy]phen-
yl Acetate (21). Lithium bis(trimethylsilyl)amide (73 mL, 1
M solution in THF) was added dropwise to a solution of
resorcinol monoacetate (10 g, 65.7 mmol) in THF (100 mL) at
78 °C under argon. The solution was stirred for 10 min, and
then triisopropylsilyl chloride (15.5 mL, 73 mmol) was added
via syringe. After being stirred at room temperature overnight,
the mixture was partitioned between water and ethyl acetate.
The organic layer was dried, filtered, and evaporated under
vacuum to yield 13 g of crude 3-[1,1-bis(methylethyl)-2-methyl-
1-silapropoxy]phenyl acetate (21), which was used in the next
step without further purification. 1H NMR (400 MHz, CDCl3)
δ: 7.19 (t, J ) 8.0 Hz, 1H), 6.75 (m, 1H), 6.68 (m, 1H), 6.63 (t,
J ) 4.0 Hz, 1H), 2.29 (s, 3H), 1.25 (m, 3H), 1.11 (d, J ) 7.0
Hz, 18H).
1
104 mg (57%) of 25. H NMR (400 MHz, CDCl3) δ: 7.36 (m,
3H), 7.01 (d, J ) 2.1 Hz, 1H), 6.87 (dd, J ) 8.5, 2.1 Hz, 1H),
6.54 (d, J ) 7.2 Hz, 1H), 6.23 (d, J ) 8.2 Hz, 1H), 4.68 (br s,
1H), 4.15 (c, J ) 7.4 Hz, 2H), 3.09 (t, J ) 6.9 Hz, 2H), 2.97 (t,
J ) 6.9 Hz, 2H), 2.87 (d, J ) 5.1 Hz, 3H), 2.68 (t, J ) 6.9 Hz,
2H), 1.26 (t, J ) 7.4 Hz, 1H).
3-(6-{2-[6-(Methylamino)-2-pyridyl]ethoxy}benzo[b]-
furan-3-yl)propanoic Acid (26). NaOH (4 mL, 1 N) was
added to a solution of 25 in THF (4 mL) and stirred for 16 h.
The reaction mixture was partitioned between ethyl acetate
and water. The aqueous layer was neutralized with 1 N HCl
(pH ) 6.5). The resulting precipitate was filtered, rinsed with
distilled water, and dried to yield 70 mg (74%) of 26 as a white
solid. 1H NMR (400 MHz, DMSO-d6) δ: 7.54 (dd, J ) 7.3, 8.6
Hz, 1H), 7.34 (m, 2H), 6.99 (d, J ) 2.0 Hz, 1H), 6.77 (dd, J )
2.0, 8.6 Hz, 1H), 6.53 (d, J ) 7.1 Hz, 1H), 6.37 (d, J ) 7.1 Hz,
1H), 6.27 (d, J ) 8.5 Hz, 1H), 4.19 (t, J ) 6.5 Hz, 2H), 3.09 (t,
J ) 6.5 Hz, 2H), 2.94 (m, 2H), 2.87 (s, 3H), 2.69 (t, J ) 6.5 Hz,
2H). Mass spectrum (LCMS, ESI) Calcd for C19H21N2O4: 341.1
(M + H). Found: 341.4. HSMS (FAB+) Calcd for C19H21N2O4:
341.150132 (MH+). Found: 341.150855.
In Vitro Inhibition of Protein-Protein Binding. RIIbâIIIa
-
Fibrinogen Assay. The assay is based on the method of
Dennis.20 Costar 9018 flat-bottom 96-well ELISA plates were
coated overnight at 4 °C with 100 µL/well of 10 µg/mL human
fibrinogen (Calbiochem) in 20 mM Tris-HCl pH 7.5, 150 mM
NaCl, 2 mM CaC12, and 0.02% NaN3 (TAC buffer). Plates were
subsequently emptied and blocked for 1 h at 37 °C with 150
µL/well of TAC buffer containing 0.05% Tween 20 and 1%
bovine serum albumin (TACTB buffer). After being washed
three times with 300 µL/well of 10 mM Na2HPO4 pH 7.5, 150
mM NaCl, and 0.01% Tween 20 (PBST buffer), controls or test
compounds (0.027-20.0 µM) were mixed with 40 µg/mL human
GPIIbIIIa (Enzyme Research Laboratories) in TACTB buffer,
3-[1,1-Bis(methylethyl)-2-methyl-1-silapropoxy]phe-
nol (22). An aqueous (50 mL) solution of NaOH (3.25 g, 81
mmol) was added to a solution of 21 (5 g, 16.2 mmol) in THF
(50 mL). After being stirred overnight at room temperature,

Potent and Selective Integrin Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 4 933
and 100 µL/well of these solutions was incubated for 1 h at 37
°C. The plate was then washed five times with PBST buffer,
and 100 µL/well of a monoclonal anti-GPIIbIIIa antibody in
TACTB buffer (1 µg/mL, Enzyme Research Laboratories) was
incubated at 37 °C for 1 h. After the plate was washed 5 times
with PBST buffer, 100 µL/well of goat anti-mouse IgG conju-
gated to horseradish peroxidase (Kirkegaard & Perry) was
incubated at 37 °C for 1 h (25 ng/mL in PBST buffer), followed
by a 6-fold PBST buffer wash. The plate was developed by
adding 100 µL/well of 0.67 mg of o-phenylenediamine dihy-
drochloride per milliliter of 0.012% H2O2, 22 mM sodium
citrate, and 50 mM sodium phosphate, pH 5.0 at room
temperature. The reaction was stopped with 50 µL/well of 2
M H2SO4, and the absorbance at 492 nm was recorded. Percent
(%) inhibition was calculated from an average of three separate
determinations relative to buffer controls (no test compound
added), and a four parameter fit was used to estimate the half-
maximal inhibition concentration (IC50).
rvâ3-Vitronectin Assay. The assay was based on the
method of Niiya,21 and all steps were performed at room
temperature. Costar 9018 flat-bottom 96-well ELISA plates
were coated overnight at room temperature with 100 µL/well
of 0.4 µg/mL human Rvâ3 (Chemicon) in TS buffer (20 mM Tris-
HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1
mM MnCl2). Plates were subsequently emptied and blocked
for 2 h with 150 µL/well of TS buffer containing 1% BSA (TSB
buffer) and washed three times with 300 µL/well of PBST
buffer. Controls or test compounds (0.0001-20.0 µM) were
mixed with 1 µg/mL of human vitronectin (Chemicon) that had
been biotinylated in-house with sulfo-NHS-LC-LC-biotin (Pierce,
20:1 molar ratio), and 100 µL/well of these solutions (in TSB
buffer) were incubated for 2 h. The plate was then washed
five times with PBST buffer, and 100 µL/well of 0.25 µg/mL
NeutrAvidin-horseradish peroxidase conjugate (Pierce) in
TSB buffer was added to the plate and incubated for 1 h. After
a 5-fold PBST buffer wash, the plate was developed, and
results were calculated as described for the IIbIIIa-fibrinogen
assay.
rvâ5-Vitronectin Assay. The assay is similar to the Rvâ3-
vitronectin assay, and all steps were performed at room
temperature. Costar 9018 flat-botom 96-well ELISA plates
were coated overnight at room temperature with 100 µL/well
of 1 µg/mL human Rvâ5 (Chemicon) in TS buffer. Plates were
blocked for 2 h with 150 µL/well of TSB buffer and washed
three times with 300 µL/well of PBST buffer. Controls or test
compound (0.0001-20 µM) were mixed with 1 µg/mL of human
vitronectin (Chemicon) that had been biotinylated in-house
with sulfo-NHS-LC-LC-biotin (Pierce, 20:1 molar ratio), and
100 µL/well of these solutions (in TSB buffer) was incubated
for 2 h. The plate was then washed five times with PBST
buffer, and 100 µL/well of 0.25 µg/mL NeutrAvidin-horse-
radish peroxidase conjugate (Pierce) in TSB buffer was added
to the plate and incubated at for 1 h. After a 5-fold PBST buffer
wash, the plate was developed, and results were calculated
as described for the IIbIIIa-fibrinogen assay.
r5â1-Fibronectin Assay. Costar 9018 flat-botom 96-well
ELISA plates were coated overnight at room temperature with
100 µL/well of 3 µg/mL human R5â1 (Chemicon) in TS buffer.
Plates were subsequently emptied, blocked for 2 h at 30 °C
with 150 µL/well of TSB buffer, and washed 3 times with 300
µL/well of PBST buffer. Controls or test compounds (0.0001-
20 µM) were mixed with 1 µg/mL of human fibronectin
(Chemicon) that had been biotinylated in-house with sulfo-
NHS-LC-LC-biotin (Pierce, 20:1 molar ratio), and 100 µL/well
of these solutions (in TSB buffer) was incubated for 2 h at 30
°C. The plate was then washed three times with PBST buffer,
and 100 µL/well of 0.25 µg/mL NeutrAvidin-horseradish
peroxidase conjugate (Pierce) in TSB buffer was added to the
plate and incubated at for 1 h at 30 °C. After a 6-fold PBST
buffer wash, the plate was developed, and results were
calculated as described for the IIbIIIa-fibrinogen assay.
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