Isoxazolines as potent antagonists of the integrin α(v)β3

Article abstract of DOI:10.1021/jm9900321

Starting with lead compound 2, we sought to increase the selectivity for α(v)β₃-mediated cell adhesion by examining the effects of structural changes in both the guanidine mimetic and the substituent α to the carboxylate. To prepare some of the desired aminoimidazoles, a novel reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found that guanidine mimetics with a wide range of pK(a)'s were potent antagonists of α(v)β₃. In general, it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity for α(v)β₃-mediated adhesion versus α(IIb)β₃-mediated platelet aggregation, with selectivity of approximately 3 orders of magnitude observed for compounds 3g and 3h. It was also found in this series that the α- substituent was required for potent activity and that 2,6-disubstituted arylsulfonamides were optimal. In addition, the selective α(v)β₃ antagonist 3h was found to be a potent inhibitor of α(v)β₃-mediated cell migration.

Full text of DOI:10.1021/jm9900321

J . Med. Chem. 2000, 43, 27-40
27
Articles
Isoxa zolin es a s P oten t An ta gon ists of th e In tegr in rvâ3
William J . Pitts,† J ohn Wityak,*,† J oanne M. Smallheer, A. Ewa Tobin, J ames W. J etter, J ennifer S. Buynitsky,
Patricia P. Harlow, Kimberly A. Solomon, Martha H. Corjay, Shaker A. Mousa, Ruth R. Wexler, and
Prabhakar K. J adhav
DuPont Pharmaceuticals Company, P.O. Box 80500, Wilmington, Delaware 19880-0500
Received J anuary 21, 1999
Starting with lead compound 2, we sought to increase the selectivity for Rvâ3-mediated cell
adhesion by examining the effects of structural changes in both the guanidine mimetic and
the substituent R to the carboxylate. To prepare some of the desired aminoimidazoles, a novel
reductive amination utilizing a trityl-protected aminoimidazole was developed. It was found
that guanidine mimetics with a wide range of pKa’s were potent antagonists of Rvâ3. In general,
it appeared that an acylated 2-aminoimidazole guanidine mimetic imparted excellent selectivity
for Rvâ3-mediated adhesion versus RIIbâ3-mediated platelet aggregation, with selectivity of
approximately 3 orders of magnitude observed for compounds 3g and 3h . It was also found in
this series that the R-substituent was required for potent activity and that 2,6-disubstituted
arylsulfonamides were optimal. In addition, the selective Rvâ3 antagonist 3h was found to be
a potent inhibitor of Rvâ3-mediated cell migration.
In tr od u ction
hyperplasia; extracellular matrix deposition; or unfavor-
able vascular remodeling.5 The relative importance of
these factors is believed to be dependent on the indi-
vidual patient, lesion, and revascularization procedure.
The Rvâ3 integrin is expressed in migrating SMCs
during restenosis in the process of neointimal hyper-
plasia.6 Several studies in animal models have shown
that selective inhibition of Rvâ3 leads to the inhibition
of neointimal hyperplasia and stenosis.7
Earlier, we disclosed a series of isoxazolines repre-
sented by XR299 (1a ) and DMP 754 (1b) that are potent
and selective GPIIb/IIIa antagonists (Figure 1).8 As
shown in Table 1, XR299 shows a greater than 800-fold
selectivity for the inhibition of platelet aggregation in
the PRP assay, an RIIbâ3-driven process, versus the
adhesion of 293 cells engineered to express the Rvâ3
receptor to immobilized fibrinogen,9 a process mediated
by this integrin. Using a strategy that relied upon solid-
phase synthesis of a small library of 3,5-disubstituted
isoxazolines and in vitro screening via ELISA format,
optimization of tether lengths and isoxazoline orienta-
tion led to the identification of 2 (Figure 1) as a potent
inhibitor of the binding of vitronectin to purified Rvâ3
receptor.10 However, selectivity for the Rvâ3 integrin
relative to RIIbâ3 was still an issue. In this work, we
disclose our efforts to reverse the integrin selectivity
observed with agents such as XR299 and DMP 754, with
the goal of discovering a new series of potent, selective
Rvâ3 antagonists.
Integrins are heterodimeric transmembrane receptors
that mediate cell adhesion, migration, and cellular
signaling. Pharmacological modulation of integrin-medi-
ated processes is of current interest. The integrin Rvâ3
is distributed on numerous cell types, such as platelets,
endothelial cells, melanoma, smooth muscle cells (SMCs),
and osteoclasts. On most cell types, it is expressed at
relatively low levels; however, up-regulation of the
receptor occurs under pathophysiological conditions.1
Like GPIIb/IIIa (RIIbâ3), Rvâ3 binds a variety of RGD-
containing adhesive proteins such as vitronectin, fi-
bronectin, von Willebrand factor (vWF), fibrinogen,
osteopontin, bone sialo protein II, and thrombospondin.
Recently, small molecule antagonists to the Rvâ3 recep-
tor have been reported to be effective when tested in
animal models of osteoclast-mediated bone resorption.2
The discovery of selective antagonists to the Rvâ3 recep-
tor could, in principle, offer new therapeutic strategies
for the treatment of osteoporosis, restenosis, angiogenic
ocular disorders, and cancer.
Since its introduction in 1978,3 percutaneous trans-
luminal coronary angioplasty (PTCA) has been trans-
formed into a routine procedure that benefits thousands
of coronary artery disease patients annually. Despite
the clinical benefit and advances in techniques, rest-
enosis occurs typically 3-6 months after the procedure
at an incidence of 30-50%.4 The pathogenesis of rest-
enosis is a multicomponent process dependent upon one
or more of the following factors: inadequate initial
luminal cross section due to elastic recoil; neointimal
Ch em istr y
The majority of the isoxazolines 3 were synthesized
from intermediate 4 using a variety of synthetic strate-
gies. Isoxazoline 4 was prepared in 70-90% yield from
the 1,3-dipolar cycloaddition of 1-hexen-6-ol (5) with
* To whom correspondence should be addressed. Tel: 609-252-4832.
Fax: 609-252-6804. E-mail: john.wityak@bms.com.
† Present address: Bristol-Myers Squibb Pharmaceutical Research
Institute, P.O. Box 4000, Princeton, NJ 08543-4000.
10.1021/jm9900321 CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/10/1999

28 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
21 to diaminopropionate 8a followed by protecting group
cleavage afforded the desired aminopyridine.
Syn th esis of (Im id a zol-2-yla m in o)a lk yl Der iva -
tives. Two potential strategies for the introduction of
the imidazol-2-ylamino moiety were known in the
literature. The first methodology relies upon variations
on the de novo synthesis of the imidazole ring.14
A
second method demonstrates the utility of the Mit-
sunobu reaction of 2-(allyloxycarbonylamino)imidazole
with a carbapenam-derived alcohol.15 This latter method
was viewed as a potentially efficient entry into the
series; however, its application met with incomplete
reaction and an arduous chromatographic separation
unsuitable for scale-up. As an alternative, reductive
amination of aldehyde 16 with 1-trityl-2-aminoimidazole
(22) was investigated (Scheme 6). Imidazole 22 was
prepared from 2-aminoimidazole (14) by protection of
the 2-amino functionality as the phthalimide, followed
by tritylation and hydrazinolysis. Under Maryanoff
conditions,16 reaction of 22 with 16 resulted not in the
desired amine but instead reduction of the aldehyde to
afford alcohol 4a . It was apparent that imine formation
was the rate-determining step; under the best conditions
(toluene at reflux), the half-life for this reaction was 1.5
h.17 After 7 half-lives, the mixture was cooled to room
temperature and NaBH(OAc)3 added, resulting in the
desired aminoimidazole adduct. Saponification then
gave the desired acid 23 in 66% overall yield from 16.
Coupling to 8, followed by protecting group cleavage,
afforded the desired aminoimidazoles 3d , 3m -q, 3t, 3v,
3w , 3z, and 3bb.
F igu r e 1. Early leads toward a selective Rvâ3 antagonist.
ethyl chlorooximidoacetate (6). The simple guanidine
and guanidine mimetics 3a , 3b, 3i, and 3j were pre-
pared from 4 using the route depicted in Scheme 1.
Tosylation of 4, followed by displacement using azide
and saponification gave the acid 7. Coupling of 7 to
diaminopropionate 8a followed by Staudinger reduction
afforded amine 9a . Reaction of 9a with reagents such
as 10,11 trimethylsilyl isocyanate, or chlorosulfonyl
isocyanate followed by protecting group cleavage af-
forded the desired analogues.
The 2-aminoimidazol-4-yl 3c was prepared as shown
in Scheme 2. The alcohol 4a was subjected to J ones
oxidation to provide acid 11. This acid was converted
to the acyl chloride, reacted with diazomethane, and
treated with HBr gas to provide bromoketone 12.
Reaction of 12 with acetylguanidine, in a manner
analogous to that published by Little and Webber,12
followed by careful saponification yielded carboxylic acid
13. Coupling of 13 to diaminopropionate 8a followed by
protecting group cleavage afforded the desired ami-
noimidazole.
The imidazol-2-ylaminoacyl bearing isoxazoline 3g
was prepared by coupling acid 11 with 2-aminoimida-
zole sulfate 14 at elevated temperature, followed by
hydrolysis with lithium hydroxide to form acid 15
(Scheme 3). Coupling of 15 to diaminopropionate 8a
followed by protecting group cleavage afforded the
desired acylaminoimidazole.
The pyridin-2-ylamino derivatives 3e, 3r , 3s, 3u , 3x,
3y, and 3a a were prepared according to the protocol
outlined in Scheme 4. Oxidation of alcohol 4 to aldehyde
16 followed by reductive amination using 2-aminopyri-
dine under Maryanoff conditions, Boc protection, and
ester saponification gave the carboxylate 17. Coupling
of 17 to diaminopropionate 8b provided the bis(carbam-
ate) ester 18 which was converted to 3a a . Alternatively,
intermediate 18 underwent chemoselective hydrogenol-
ysis of the Cbz group to afford amine 19 using conditions
adapted from Nikam.13 Refunctionalization of this amine
using a series of sulfonyl chlorides, followed by protect-
ing group cleavage, afforded the desired aminopyridines.
The isoquinolines 3k and 3l were prepared from alde-
hyde 16 in a similar manner.
The urea 3h was prepared from 9b via reaction with
phosgene followed by the addition of 22 to the interme-
diate isocyanate. Protecting group cleavage then af-
forded the desired target.
Resu lts a n d Discu ssion
The screening strategy used to discover potent and
selective Rvâ3 antagonists involved measuring the rela-
tive activity and selectivity of compounds using whole-
cell assays. Human 293 cells were engineered to over-
express the Rvâ3 receptor and were used to assess the
potency of antagonists as measured by the inhibition
of their binding to immobilized fibrinogen. Compounds
were also assayed for relative selectivity of antagonism
of the Rvâ3 versus RIIbâ3 receptor by comparing this
result with the IC50 values obtained for the inhibition
of platelet aggregation in human platelet-rich plasma
(PRP)18 and/or in human gel-purified platelets contain-
ing 1 mg/mL fibrinogen (GPP + Fg).19 Since the protein
concentration differs greatly between these two platelet
aggregation assays, the potencies obtained could provide
a rough estimate of the protein binding of the test
compounds. These data are summarized in Tables 1 and
2. In addition, a selective Rvâ3 antagonist was further
assayed using a microporous filter assay for the ability
to inhibit the migration of transfected 293 cells in
response to vitronectin.
Synthesis of the 6-aminopyridin-2-yl 3f is depicted in
Scheme 5. The synthesis began with the protection of
2-amino-6-methylpyridine (20) as the Boc derivative.
Lithiation and alkylation of the dianion using 4-bromo-
1-butene followed by dipolar cycloaddition with 6 and
saponification gave the isoxazoline acid 21. Coupling of
Gu a n id in e Mim etic SAR. A working hypothesis
regarding selectivity for Rvâ3- versus RIIbâ3-mediated
effects was that selectivity could be optimized through
careful choice of the pKa, geometry, and hydrogen-
bonding characteristics of the guanidine mimetic (Table
1). As a starting point, guanidine 3a , which has an

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 29
Sch em e 1. Synthesis of the Guanidine Mimetics 3a , 3b, 3i, and 3ja
a
Reagents: (a) NaHCO3(aq), THF, 0 °C-rt; (b) p-TsCl, pyridine; (c) NaN3, DMF; (d) NaOH, H+; (e) BOP, 8a , Hunig’s base; (f) Ph3P,
dioxane, NH4OH(aq); (g) 10, 80 °C, dioxane; (h) TFA; (i) TMSNCO; (j) ClSO2NCO, t-BuOH.
Sch em e 2. Synthesis of Imidazole 3ca
Sch em e 3. Synthesis of the Acylaminoimidazole 3ga
a
Reagents: (a) 14, BOP, Hunig’s base, 70 °C; (b) LiOH,
dioxane(aq), H+; (c) BOP, 8a , N-methylmorpholine; (d) TFA.
tural motif had a modest positive effect on both potency
and selectivity when compared to 3a . The aminoimida-
zoles 3c and 3d further probed geometry and pKa.
Aminoimidazole 3d has an estimated pKa of 8.6 and was
envisioned as a less basic alternative to 3b. It has
excellent potency in the 293 cell adhesion assay and
moderate selectivity versus RIIbâ3 receptor-driven plate-
let aggregation. In contrast, the isomeric aminoimida-
zole 3c was approximately 2-3-fold less potent and
appeared to be less selective. The decreased selectivity
a
Reagents: (a) J ones reagent; (b) oxalyl chloride, cat. DMF; (c)
diazomethane; (d) HBr(g); (e) acetylguanidine; (f) NaOH, HCl; (g)
BOP, 8a , N-methylmorpholine; (h) H2SO4, 60 °C.
estimated pKa of 12,20 had modest potency against the
Rvâ3-mediated adhesion of 293 cells to fibrinogen and
was an equipotent inhibitor of platelet aggregation in
the PRP assay. Embedding the guanidine in an imida-
zoline ring as in 3b to hinder possible binding modes
involving participation of its imino(aminomethyl) struc-

30 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
Sch em e 4. Synthesis of 2-Aminopyridines 3a a , 3e, 3r ,
3s, 3u , 3x, and 3ya
might be reasonably expected that such interaction
could be more readily accommodated by aminoimidazole
3c than aminoimidazole 3d . The aminopyridines were
selected as examples of guanidine mimetics having a
pKa of approximately 7. Both aminopyridine 3e and its
isomer 3f were approximately equipotent to 3d in the
293 cell adhesion assay and appeared to have enhanced
selectivity versus RIIbâ3 receptor-driven platelet ag-
gregation. The acylated aminoimidazoles 3g and 3h
(pKa < 4) are analogues having lower basicity than the
aminopyridines. These were also potent Rvâ3 antagonists
and had the best selectivity yet observed in the series.
Two other nonbasic guanidine mimetics, urea 3i and
sulfamide 3j, were inactive when tested at a concentra-
tion of 1 µmol. The isomeric isoquinolines 3k and 3l
were also inactive, suggesting that additional lipophi-
licity or steric bulk adjacent to the guanidine mimetic
is not tolerated.
The empirical results suggest that a positively charged
guanidine mimetic is not a prerequisite for potency
against Rvâ3 mediated adhesion. The separation of Rvâ3
mediated adhesion and antiplatelet effects was readily
achieved by the incorporation of a less basic guanidine
mimetic such as an acylated aminoimidazole. As ob-
served with aminoimidazoles 3c and 3d , selectivity
could also be gained through an orientation of the
guanidine mimetic that discouraged certain bidentate
binding arrangements involving both a δ-like nitrogen
(arginine numbering) and an amino(iminomethyl) ω′-
nitrogen.
a
Reagents: (a) oxalyl chloride, DMSO, Et3N, -70 °C; (b) sodium
triacetoxyborohydride, 2-aminopyridine; (c) Boc2O, pyridine, cat.
DMAP; (d) LiOH, dil H+; (e) BOP, 8b, N-methylmorpholine; (f)
TFA; (g) H2, 40 psi, Pd/BaSO4; (h) arylsulfonyl chloride, pyridine.
Sch em e 5. Synthesis of the 2-Aminopyridin-6-yl 3fa
Effect of th e R-Su bstitu en t. Much of the SAR with
respect to the substituent R to the carboxylate was
elucidated via analogues having the aminoimidazole and
aminopyridine guanidine mimetics (Table 2). Aminoimi-
dazoles which possessed a 2,6-disubstituted arylsul-
fonamide (3d , 3m -p ) had approximately 5-fold in-
creased potency relative to phenylsulfonamide 3q .
However, this finding was not observed with the ami-
nopyridines (3e, 3r versus 3s). The 1-naphthylsulfona-
mides 3t and 3u and the 3,5-dimethylisoxazol-4-yl
analogue 3v were approximately equipotent with their
respective phenylsulfonamide analogues. Likewise, the
presence of a phenyl or isopropyl substituent at the
4-position of the arylsulfonamide in 3w -y appeared to
confer neither increased potency nor selectivity for Rvâ3-
mediated adhesion when compared to their respective
phenylsulfonamide analogues. However, as shown with
3q, the potency of 3w could be increased through the
addition of substituents at the 2- and 6-positions (3o,
3p ). A prerequisite for potent activity in this series of
isoxazolines appears to be the derivatization of the
R-amino moiety as a sulfonamide. As shown with
analogues 3z and 3a a , compounds lacking this struc-
tural feature were inactive against Rvâ3-mediated adhe-
sion.
a
Reagents: (a) Boc2O, 40 °C; (b) LDA, -78 °C, then 4-bro-
mobutene; (c) 6, NaHCO3, THF(aq), 0 °C-rt; (d) NaOH, dil H+;
(e) BOP, 8a , N-methylmorpholine; (f) TFA.
Sch em e 6. Synthesis of the 2-Aminoimidazoles 3d ,
3m -q, 3t, 3v, 3w , 3z, and 3bba
bb
a
Reagents: (a) NaOCH3, -78 °C-rt; (b) phthalic anhydride,
melt; (c) triphenylmethyl chloride, pyridine; (d) N2H4, EtOH,
reflux; (e) 16, toluene, reflux; (f) sodium triacetoxyborohydride;
(g) LiOH, H+; (h) TBTU, 8, N-methylmorpholine; (i) TFA, reflux.
Effect of Over a ll Len gth a n d Ster eoch em istr y.
In an earlier study, we had observed that activity in an
Rvâ3 ELISA was optimal when the guanidine and
isoxazoline moieties were separated by a tether length
of either 2 or 4 methylene units.10 This curious finding
was repeated by the observation of submicromolar
activity for both 3d and 3bb in our cell-based assay. It
was initially hypothesized that compounds having a
shorter overall distance between the guanidinium mi-
observed with aminoimidazole 3c hints at the possibility
that the nitrogen at position 2 of the imidazole ring may
mimic an interaction normally made by the ω′ nitrogen
of the arginine guanidine moiety (Figure 2). Assuming
that this interaction is a more important component
toward the binding of ligands to RIIbâ3 than to Rvâ3, it

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 31
Ta ble 1. In Vitro Activity of Isoxazolines 3 with Respect to Guanidine Mimetic
IC50, nM
PRPb
240
compd
R1
n
293â3z-Fg adhesiona
GPP+Fgb
GPP+Fg/293â3z-Fg
<1
1a
3a
3b
3c
3d
3e
3f
3g
3h
3i
>100000
169 ( 87
62 ( 43
46 ( 1.0
15 ( 4.3
54 ( 38
26 ( 24
31 ( 16
34 ( 12
>1000
140
H2NCdNH(NH)
4
4
3
4
4
3
3
2
4
4
4
4
160
690
280
850
16000
78000
imidazolin-2-ylNH
2-aminoimidazol-4-yl
imidazol-2-ylNH
pyridin-2-ylNH
2-aminopyridin-6-yl
imidazol-2-ylNHCO
imidazol-2-ylNHCONH
H2NCdO(NH)
600
180
10
4
6500
120
61000
120000
>100000
32000
6300
>3200
940
3j
3k
3l
H2NSO2(NH)
isoquinolin-1-ylNH
isoquinolin-3-ylNH
>1000
>100000
>1000
78000
11000
6500
>1000
7700
a
b
Number of measurements ) 3. Number of measurements ) 1. The error in the GPP and PRP measurements is (10%.
Ta ble 2. In Vitro Activity of Isoxazolines 3 with Respect to R-Substituent, Stereochemistry, and Overall Length
IC50, nM
compd
R1
R2
n
293â3z-Fg adhesiona
PRPb
GPP+Fgb
GPP+Fg/293â3z-Fg
3d
3e
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
imidazol-2-ylNH
pyridin-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
pyridin-2-ylNH
pyridin-2-ylNH
imidazol-2-ylNH
pyridin-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
pyridin-2-ylNH
pyridin-2-ylNH
imidazol-2-ylNH
pyridin-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
imidazol-2-ylNH
2,4,6-(CH3)3C6H2SO2
2,4,6-(CH3)3C6H2SO2
2,6-(Cl)2C6H3SO2
2-Cl-6-(CH3)C6H3SO2
2,6-(Cl)2-4-(Ph)C6H2SO2
2,6-(CH3)2-4-(Ph)C6H2SO2
C6H5SO2
2,6-(CH3)2-4-(Ph)C6H2SO2
C6H5SO2
1-C10H7SO2
1-C10H7SO2
3,5-(CH3)2isoxazol-4-ylSO2
4-(Ph)C6H4SO2
4-(Ph)C6H4SO2
4-(i-Pr)C6H4SO2
H
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
2
4
4
4
15 ( 4.3
54 ( 38
850
16000
350
390
1600
6500
190
120
10
18 ( 14
7.1 ( 3.9
4.6 ( 4.0
8.8 ( 3.2
66 ( 7.3
44 ( 19
81 ( 6.4
51 ( 10
3100
1000
>100000
30000
7800
690
930
80
10
18000
570
220
10
160 ( 59
73 ( 42
>100000
1500
240 ( 140
130 ( 68
170 ( 70
>1000
43000
70000
>100000
84000
>100000
4700
5000
20
3y
3z
47000
85000
3000
890
3a a
3bb
3cc
3d d
3ee
(C6H5CH2O)CO
2,4,6-(CH3)3C6H2SO2
3s isomer 1
3s isomer 2
(R)-2,4,6-(CH3)3C6H2SO2
NDc
106 ( 39
10.6 ( 1.1
4.5 ( 3.6
2800 ( 580
30
80
980
1100
68000
690
28000
150
10
a
b
Number of measurements ) 3. Number of measurements ) 1. The error in the GPP and PRP measurements is (10%. c Not
determined. The IC50 against an Rvâ3 ELISA is 23 nM. For comparison, the IC50 for 3d in this ELISA is <1 nM.
metic and the carboxylate moiety might have enhanced
inherent selectivity for inhibiting Rvâ3-mediated adhe-
sion relative to RIIbâ3-mediated antiplatelet activity;
however, the data for compounds 3d and 3bb did not
support this hypothesis, and shorter tether length
compounds such as 3bb were not studied further. The
inherent conformational flexibility of the compounds
precluded a more detailed analysis.
The majority of compounds prepared in this study
were diastereomeric mixtures having the (S)-absolute
configuration about the diaminopropionate stereocenter.
Holding the diaminopropionate R-substituent of 3d in
the (S)-configuration, two isoxazoline epimers 3cc and
3d d were separated using chiral supercritical fluid
chromatography. As with a related series of RIIbâ3
antagonists,8 the absolute configuration of this stereo-
center did not have a significant effect on either potency
or selectivity. Conversely, inversion of the diaminopro-
pionate R-substituent as in 3ee resulted in a large
decrease in potency for both Rvâ3- and RIIbâ3-mediated
effects. Apart from the Rvâ3 activity, it is interesting to
note that in the related series targeted to RIIbâ3, the
absolute configuration of this stereocenter had very little
effect on antiplatelet potency,8a while in the present
series, it appears to be critical to both Rvâ3- and RIIbâ3-
mediated activities.

32 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
mediated chemotaxis in response to vitronectin in a
migration assay.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on an Elec-
trothermal melting point apparatus and are uncorrected.
Unless otherwise stated, preparative HPLC separations were
accomplished on a Vydac C18 column operated at room
temperature and eluted at a 10 mL/min flow rate, using a
linear gradient of 100% water containing 0.05% TFA-20%
water/acetonitrile containing 0.05% TFA over 50 min, with UV
detection at 254 nm. 1H and 13C NMR data were obtained
using Varian Unity 300, Unity 400, or VXR400 spectrometers
and were referenced to TMS, CDCl3, or residual HOD. Mass
spectral data were obtained on either VG 70-VSE (FAB, high-
res FAB, high-res DCI) or Finnigan MAT 8230 (DCI) mass
spectrometers. Combustion analyses were performed by Quan-
titative Technologies, Inc., Bound Brook, NJ . Solvents and
reagents were used as purchased from Aldrich Chemical Co.
unless otherwise stated. The yields quoted in this paper were
isolated yields.
ter t-Bu tyl 3-Am in o-N-(2,4,6-tr im eth ylp h en yl)su lfon yl-
L-a la n in e (8a ). L-Asparagine (20.0 g, 0.15 mol) was suspended
in a mixture of THF (130 mL) and water (250 mL). Triethy-
lamine (49 g, 0.48 mol) was added followed by mesitylene-
sulfonyl chloride (49.7 g, 0.227 mol). The reaction mixture
became slightly exothermic and the solids dissolved over a
period of 0.5 h to yield a yellow solution. The mixture was
stirred for 3 h at room temperature and washed with Et2O
and CH2Cl2. The aqueous layer was separated and acidified
to a pH of 1.5 using concentrated HCl, during which time a
heavy precipitate formed. After 0.5 h, the product was filtered,
washed with water, and dried (MgSO4) to yield a white solid
F igu r e 2. Orientation of the guanidine mimetic may relate
to integrin selectivity.
rvâ3-Med ia ted Migr a tion . During the process of
neointimal hyperplasia, Rvâ3 integrin expression is up-
regulated in migrating SMCs. In a chemotaxis assay,
the selective Rvâ3 inhibitor 3h inhibited the migration
of Rvâ3-transfected 293 cell migration in response to
vitronectin with an IC50 value of 140 ( 60 nM (n ) 4).
It has been suggested that different activation states
of Rvâ3, each possessing its own unique conformation of
the integrin, are important in relation to functional
activity.21 We have demonstrated that a selective ex-
ample within the current series of isoxazolines is a
potent antagonist of both Rvâ3-mediated adhesion and
migration. Migration is thought to require Rvâ3-medi-
ated outside-in signaling. It is believed that both adhe-
sion and migration play important roles in the processes
of vascular remodeling and restenosis.
1
(34 g, 72%): mp 193.5-195 °C; H NMR (300 MHz, DMSO-
d6) δ 12.58 (bs, 1H), 7.82 (d, J ) 9.2 Hz, 1H), 7.32 (bs, 1H),
6.99 (s, 2H), 6.88 (bs, 1H), 3.98 (m, 1H), 2.55 (s, 6H), 2.45
(apparent dd, J ) 15.4, 7.0 Hz, 1H; partially coincident with
solvent), 2.28 (dd, J ) 15.8, 6.2 Hz, 1H), 2.24 (s, 3H); MS (ESI)
m/z 315.2 [(M + H)+ 100]. Anal. Calcd for C13H18N2O5S: C,
49.67; H, 5.77; N, 8.91. Found: C, 49.79; H, 5.90; N, 8.84.
Sodium hydroxide (32 g, 0.80 mol) was dissolved in water
(200 mL) and cooled in an ice bath. Bromine (19.2 g, 0.12 mol)
was added dropwise over 5 min and the mixture allowed to
stir for 15 min. N-(2,4,6-Trimethylphenyl)sulfonyl-L-aspar-
agine (31.44 g, 0.10 mol) was added in several portions over a
period of 10 min, during which time the yellow color faded.
The reaction mixture was gently heated on a steam bath
during which time the internal temperature rose to ca. 85 °C.
After 1 h, the reaction mixture was allowed to cool to room
temperature; then it was cooled in an ice bath. After cautiously
acidifying to pH 6 with concentrated HCl, a solid formed and
gas was evolved. The solid was filtered, washed with cold
water, and dried overnight to give the product as a white solid
(23.9 g, 83%): 1H NMR (300 MHz, DMSO-d6) δ 7.06 (s, 2H),
3.07 (dd, J ) 11.4, 4.4 Hz, 1H), 2.94 (dd, J ) 9.5, 4.4 Hz, 1H),
2.80 (m, 1H), 2.59 (s, 6H), 2.26 (s, 3H); MS (ESI) m/z 287.2
[(M + H)+ 100].
Con clu sion s
In the present work, the effect of structural changes
in both the guanidine mimetic and the substituent R to
the carboxylate of guanidine 2 led to the observation of
increased selectivity and potency for the adhesion of 293
cells expressing Rvâ3 to fibrinogen. To prepare some of
the desired aminoimidazoles, a novel reductive amina-
tion utilizing a trityl-protected aminoimidazole was
developed. It was found that guanidine mimetics with
a wide range of pKa’s were potent antagonists of Rvâ3-
mediated adhesion of transfected 293 cells to fibrinogen.
In general, it appeared that the less basic guanidine
mimetics imparted greater selectivity for Rvâ3-mediated
adhesion versus RIIbâ3-mediated platelet aggregation. In
particular, compounds 3g and 3h had excellent potency
and selectivity of approximately 3 orders of magnitude.
It was also found that the R-substituent was required
for potent activity in this series and that 2,6-disubsti-
tuted arylsulfonamides appeared optimal in this regard.
The absolute stereochemistry of the diaminopropionate-
derived stereocenter was also determined to be a critical
factor affecting potency. In addition, aminoimidazoly-
lurea 3h was found to be a potent inhibitor of Rvâ3-
Into a Parr bottle was placed a suspension of 3-amino-N-
(2,4,6-trimethylphenyl)sulfonylamino-L-alanine (11.45 g, 0.04
mol) in dioxane (170 mL). Into the cooled reaction mixture (dry
ice-acetone bath) was condensed isobutylene (ca. 185 mL) and
concentrated sulfuric acid (11 mL) was added. The bottle was
sealed and agitated for 114 h. The bottle was depressurized
and purged with nitrogen for a brief time. The reaction mixture
was poured into a rapidly stirred mixture of water (225 mL)
containing NaOH (17 g) and Et2O (600 mL) which had been
precooled in an ice bath. The layers were separated and the
aqueous layer was extracted with additional Et2O. The pH of
the aqueous layer was carefully adjusted to 11 with concen-
trated HCl and the mixture extracted four times with Et2O.
The organic layers from the pH 11 adjusted extraction were
combined, dried (Na2SO4), filtered, and evaporated to yield the
product as a viscous oil which solidified upon standing (8.64
g, 63%): 1H NMR (300 MHz, CDCl3) δ 6.95 (s, 2H), 3.69 (m,

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 33
1H; partially coincident with residual dioxane), 2.98 (dd, J )
13.6, 4.4 Hz, 2H), 2.88 (dd, J ) 13.6, 5.5 Hz, 1H), 2.67 (s, 6H),
2.28 (s, 3H), 1.28 (s, 9H); MS (ESI) m/z 343 [(M + H)+ 100].
Anal. Calcd for C16H26N2O4S: C, 56.12; H, 7.65; N, 8.18.
Found: C, 56.13; H, 7.45; N, 7.98.
mixture washed with 1 M citric acid. The organic layer was
dried over MgSO4 and evaporated and the residue purified
using flash chromatography to afford the desired amide (2.42
g, 94%): 1H NMR (300 MHz, CD3OD) δ 6.96 (s, 2H), 4.80-
4.68 (m, 1H), 4.00-3.92 (m, 1H), 3.58-3.36 (m, 2H), 3.34-
3.20, (m, 3H), 2.82-2.70 (m, 1H), 2.60 (s, 6H), 2.24 (s, 3H),
1.78-1.38 (m, 6H). 1.20 (d, J ) 3 Hz, 9H); MS (ESI) m/z 537.4
[(M - H)-, 81].
The above amide (2.42 g, 4.5 mmol) was dissolved in dioxane
(anhyd, 3 mL), triphenylphosphine (1.99 g, 7.5 mmol) added,
and the mixture stirred at room temperature for 18 h. 1 N
Ammonium hydroxide (5 mL) was added and the reaction
mixture stirred for an additional 6 h. The solvents were
evaporated and the residue purified using flash chromatog-
raphy (89:10:1 ) CH2Cl2:MeOH:1 M NH4OH) to give the
desired amine 9a (1.92 g, 83%) as a white solid: 1H NMR (300
MHz, CD3OD) δ 6.96 (s, 2H), 4.80-4.68 (m, 1H), 4.00-3.92
(m, 1H), 3.58-3.30 (m, 2H), 3.20-3.08, (m, 3H), 2.80-2.68 (m,
1H), 2.58 (s, 6H), 2.24 (s, 3H), 1.78-1.38 (m, 6H), 1.20 (d, J )
3 Hz, 9H); MS (ESI) m/z 511.3 [(M + H)+, 100]. Anal. Calcd
for C24H38N4O6S‚1.20CF3CO2H: C, 48.97; H, 6.10; N, 8.65; F,
10.56. Found: C, 49.22; H, 6.19; N, 9.01; F, 10.84.
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(2-am i-
n oet h yl)isoxa zolin -3-ylca r b on yl]a m in op r op ion ic Acid
ter t-Bu tyl Ester (9b). This material was prepared in an
analogous manner to 9a and was isolated as a white solid:
1H NMR (300 MHz, CDCl3) δ 6.94 (s, 2H), 6.91-6.83 (b, 1H),
4.93-4.83 (m, 1H), 3.89-3.86 (m, 1H), 3.72-3.54 (m, 2H), 3.28
(dd, J ) 10.8, 2.4 Hz, 1H), 2.92-2.81 (m, 3H), 2.64 (s, 6H),
2.28 (s, 3H), 1.95-1.84 (m, 1H), 1.79-1.70 (m, 1H), 1.32 (s,
9H); MS (ESI) m/z 483.3 [(M + H)+, 100].
3-Eth oxyca r bon yl-5-(4-h yd r oxybu tyl)isoxa zolin e (4a ).
To a solution of 5-hexen-1-ol (5; 5.0 g, 0.05 mol) in THF (30
mL) was added a solution of NaHCO3 (29.4 g, 0.35 mol) in
water (20 mL) and the reaction mixture was cooled in an ice
bath. Ethyl chlorooximidoacetate (6; 11.4 g, 0.075 mol) was
added over 15 min in several portions. The reaction mixture
was stirred while still immersed in an ice bath for 6 h. An
additional amount of 6 (7.75 g, 0.05 mol) was added and the
reaction mixture was allowed to warm to room temperature
overnight. The mixture was partitioned between EtOAc and
water; the organic layer was separated, dried (MgSO4), filtered,
and evaporated. The crude product was purified using flash
chromatography (3:1 ) hexane:EtOAc) to yield the desired
isoxazoline as a colorless oil (9.22 g, 86%): 1H NMR (300 MHz,
CDCl3) δ 4.90-4.78 (m, 1H), 4.34 (q, J ) 7 Hz, 2H), 3.66 (bs,
2H), 3.54-3.20 (m, 1H), 2.94-2.80 (m, 1H), 1.90-1.40 (m, 6H),
1.3 (t, J ) 7 Hz, 3H); MS (NH3-CI) m/z 233 [(M + NH4)+, 100],
216 (M + H)+. Anal. Calcd for C10H17NO4: C, 55.80; H, 7.96;
N, 6.51. Found: C, 55.50; H, 7.72; N, 6.37.
3-Eth oxyca r bon yl-5-(2-h yd r oxyeth yl)isoxa zolin e (4b).
The title compound was synthesized from 3-buten-1-ol follow-
ing the general procedure for 4a : 1H NMR (300 MHz, CDCl3)
δ 4.90-4.78 (m, 1H), 4.34 (q, J ) 7 Hz, 2H), 3.66 (bs, 2H),
3.54-3.20 (m, 1H), 2.94-2.80 (m, 1H), 1.90-1.85 (m, 1H),
1.80-1.70 (m, 1H), 1.3 (t, J ) 7 Hz, 3H); MS (NH3-CI) m/z
205 [(M + NH4)+, 100], 188 (M + H)+.
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(4-am i-
n obu tyl)isoxa zolin -3-ylca r bon yl]a m in op r op ion ic Acid
ter t-Bu tyl Ester (9a ). To an ice-cold solution of 4a (6.45 g,
30 mmol) in CH2Cl2 (anhyd, 200 mL) was added pyridine (2.57
g, 33 mmol) followed by p-toluenesulfonyl chloride (6.27 g, 33
mmol). After 0.5 h, the ice bath was removed and the reaction
mixture stirred at room temperature for 18 h. The reaction
mixture was washed with water (2 × 200 mL), dried over
MgSO4, filtered, and evaporated to yield the crude product as
a colorless oil. Purification using flash chromatography (2%
MeOH-CH2Cl2) yielded 10.0 g (90%) of the desired tosylate:
1H NMR (300 MHz, CD3OD) δ 7.80 (d, J ) 12 Hz, 2H), 7.36
(d, J ) 12 Hz, 2H), 4.80-4.68 (m, 1H), 4.36 (q, J ) 7 Hz, 2H),
4.02 (t, J ) 6 Hz, 2H), 3.30-3.20, (m, 1H), 2.84-2.72 (m, 1H),
2.46 (s, 3H), 1.88-1.40 (m, 6H), 1.3 (t, J ) 7 Hz, 3H).
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(4-(am i-
n oim in om eth yl)am in obu tyl)isoxazolin -3-ylcar bon yl]am i-
n op r op ion ic Acid Tr iflu or oa ceta te (3a ). To a solution of
9a (160 mg, 0.31 mmol) in dioxane (anhyd, 5 mL) was added
2-(N,N′-di-tert-butyloxycarbonylguanyl)-3,5-dimethylpyra-
zole (10a ; 127 mg, 0.37 mmol), and the reaction mixture heated
at 80 °C for 16 h. The solvent was evaporated and the residue
dissolved TFA (4 mL). The reaction mixture was stirred for 1
h, the solvent evaporated, and the residue purified using
reverse-phase HPLC. Concentration of the appropriate frac-
tions in vacuo, trituration with Et2O, and drying under vacuum
gave the desired guanidine 3a (50 mg, 26% overall from 9a ):
1H NMR (300 MHz, CD3OD) δ 6.96 (s, 2H), 4.80-4.68 (m, 1H),
4.08-4.00 (m, 1H), 3.64-3.56 (m, 1H), 3.42-3.30, (m, 1H),
3.22-3.02 (m, 3H), 2.78-2.68 (m, 1H), 2.58 (s, 6H), 2.24 (s,
3H), 1.78-1.38 (m, 6H); MS (ESI) m/z 497.3 [(M + H)+, 100].
Anal. (C21H32N6O6S‚1.4CF3CO2H) C, H, N, F.
The tosylate (10.0 g, 27 mmol) was dissolved in DMF (15
mL) and cooled in an ice bath. Sodium azide (4.5 g, 69 mmol)
was added, and the reaction mixture stirred for 18 h, during
which time the reaction mixture warmed to room temperature.
After dilution with water (150 mL), the mixture was extracted
with EtOAc. The organic layer was dried over Na2SO4, filtered,
and evaporated to yield the azido ester as a colorless oil (5.96
g, 92%): 1H NMR (300 MHz, CD3OD) δ 4.86-4.76 (m, 1H),
4.36 (q, J ) 7 Hz, 2H), 3.34-3.22, (m, 3H), 2.90-2.80 (m, 1H),
1.82-1.40 (m, 6H), 1.3 (t, J ) 7 Hz, 3H). Anal. Calcd for
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(4-(im -
id a zolin -2-yl)a m in ob u t yl)isoxa zolin -3-ylca r b on yl]a m i-
n op r op ion ic Acid Tr iflu or oa ceta te (3b). To a solution of
9a (180 mg, 0.35 mmol) and 2-(3,5-dimethylpyrazolyl)-4,5-
dihydroimidazole hydrobromide (10b; 95 mg, 0.39 mmol) was
added 4-(dimethylamino)pyridine (5 mg, 0.04 mmol), and the
resulting mixture heated at 80 °C for 16 h. The solvent was
evaporated and the residue dissolved in TFA (4 mL). The
reaction mixture was stirred for 1 h, the solvent evaporated,
and the residue purified using reverse-phase HPLC. Concen-
tration of the appropriate fractions in vacuo, trituration with
Et2O, and drying under vacuum afforded 50 mg (20% overall
from 9a ) of the desired guanidine: 1H NMR (300 MHz, CD3-
OD) δ 6.94 (s, 2H), 4.80-4.68 (m, 1H), 4.08-4.00 (m, 1H), 3.64
(s, 4H), 3.62-3.56 (m, 1H), 3.42-3.30, (m, 1H), 3.22-3.04 (m,
3H), 2.80-2.70 (m, 1H), 2.58 (s, 6H), 2.24 (s, 3H), 1.78-1.38
C
10H16N4O3: C, 49.99; H, 6.71; N, 25.32. Found: C, 49.78; H,
6.61; N, 25.02.
The azido ester (5.96 g, 25 mmol) was dissolved in 70%
EtOH (20 mL) and 1 N NaOH (4 mL) was added. After stirring
for 2 h at room temperature, the solvent was evaporated under
reduced pressure (bath temperature < 25 °C), the residue
acidified with a 20% solution of citric acid, and the product
extracted with EtOAc. The organic layer was dried over
MgSO4, filtered, and evaporated to yield the azido acid 7a as
a white solid (4.52 g, 86%): 1H NMR (300 MHz, CD3OD) δ
4.82-4.72 (m, 1H), 3.34-3.20, (m, 3H), 2.90-2.74 (m, 1H),
1.82-1.38 (m, 6H); MS (ESI) m/z 211.3 [(M - H)-, 25].
The azido acid 7a (1.03 g, 4.9 mmol), 8a (1.82 g, 5.3 mmol),
N-methylmorpholine (1.08 g, 10.6 mmol), and (benzotriazol-
1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate
(BOP; 3.22 g, 7.2 mmol) were dissolved in dioxane (4 mL) and
stirred at room temperature for 18 h. The solvent was
evaporated, the residue dissolved in EtOAc, and the resulting
(m, 6H); MS (ESI) m/z 523.3 [(M + H)+, 100]. Anal. (C23H34
N6O6S‚1.4CF3CO2H) C, H, N, F.
-
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(4-(am i-
n oca r bon yla m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in o-
p r op ion ic Acid (3i). To a solution of 9a (100 mg, 0.20 mmol)
in CH2Cl2 (0.5 mL) was added trimethylsilyl isocyanate (50
mg, 0.44 mmol), and the reaction mixture was allowed to stir
at room temperature for 18 h. The mixture was diluted with
CH2Cl2 (5 mL) and washed with 0.1 N HCl (5 mL). The organic
layer was dried over MgSO4, filtered, evaporated, and purified

34 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
using flash chromatography (2% MeOH-CH2Cl2) to yield the
tert-butyl ester (67 mg, 62%): 1H NMR (300 MHz, CD3OD) δ
6.96 (s, 2H), 4.80-4.64 (m, 1H), 4.00-3.92 (m, 1H), 3.60-3.30
(m, 2H), 3.20-3.04, (m, 3H), 2.80-2.68 (m, 1H), 2.58 (s, 6H),
2.24 (s, 3H), 1.78-1.38 (m, 6H). 1.20 (d, J ) 1.5 Hz, 9H).
The above tert-butyl ester (67 mg, 0.12 mmol) was dissolved
in TFA (2 mL) and stirred for 1 h. The solvent was evaporated
and the residue purified using reverse-phase HPLC to yield
3i as a white solid (51 mg, 81%): 1H NMR (300 MHz, CD3OD)
δ 6.96 (s, 2H), 4.80-4.68 (m, 1H), 4.08-3.98 (m, 1H), 3.64-
3.54 (m, 1H), 3.42-3.30, (m, 1H), 3.22-3.02 (m, 3H), 2.78-
2.68 (m, 1H), 2.58 (s, 6H), 2.24 (s, 3H), 1.78-1.38 (m, 6H); MS
(ESI) m/z 496.2 [(M - H)-, 100]. Anal. (C23H34N6O6S‚1.5CF3-
CO2H) C, H, N, F.
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-(4-(am i-
n osu lfon yla m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in o-
p r op ion ic Acid (3j). Chlorosulfonyl isocyanate (55 mg, 0.39
mmol) was dissolved in CH2Cl2 (anhyd, 2 mL) and cooled in
an ice bath, and tert-butyl alcohol (29 mg, 0.39 mmol) was
added. After 1 h, 9a (200 mg, 0.39 mmol) was added, the
cooling bath was removed, and the reaction stirred for 4 h.
The mixture was washed with water and the organic layer
was dried (MgSO4), filtered, and evaporated. The residue was
purified using reverse-phase HPLC to yield the tert-butyl ester
(50 mg, 20%): 1H NMR (300 MHz, CDCl3) δ 7.0 (m, 1H), 6.92,
(s, 2H), 5.74 (m, 1H), 5.30 (bs, 1H), 4.78-4.70 (m, 1H), 3.92-
3.84 (m, 1H), 3.68-3.54 (m, 2H), 3.30-3.04, (m, 2H), 2.90-
2.78 (m, 1H), 2.60 (s, 6H), 2.24 (s, 3H), 1.78-1.42 (m, 6H), 1.45
(s, 9H), 1.24, (s, 9H).
was bubbled gaseous HBr until the starting material was no
longer present (approximately 5 min). The mixture was
cautiously added to an ice-cold solution of saturated NaHCO3.
After the gas evolution had subsided, the mixture was ex-
tracted with Et2O (2 × 100 mL). The combined organic layers
were dried over Na2SO4, filtered, and evaporated to yield the
title product as a white solid (4.0 g, 37% overall from 11): 1H
NMR (300 MHz CDCl3) δ 4.80-4.70 (m, 1H), 4.28 (q, J ) 8
Hz, 2H), 3.82 (s, 2H), 3.28-3.18, (m, 1H), 2.84-2.74 (m, 1H),
2.70 (m, 2H), 1.78-1.58 (m, 4H), 1.30 (t, J ) 8 Hz, 3H). Anal.
Calcd for C11H16BrNO4: C, 43.15; H, 5.27; N, 4.58. Found: C,
43.44; H, 5.07; N, 4.52.
5-[3-(2-Acetyla m in oim id a zol-4-yl)p r op yl]isoxa zolin e-
3-ca r boxylic Acid Hyd r och lor id e (13). To a solution of 12
(2.00 g, 6.5 mmol) in DMF (20 mL) was added acetylguanidine
(1.97 g, 19.5 mmol). The reaction mixture was stirred at room
temperature for 4 days and poured into water, and the
resulting precipitate was collected and dried under vacuum
to yield the desired acetylaminoimidazole (700 mg, 35%): 1H
NMR (300 MHz, CD3OD) δ 6.50 (s, 1H), 4.84-4.74 (m, 1H),
4.28 (q, J ) 8 Hz, 2H), 3.30-3.20, (m, 1H), 2.90-2.78 (m, 1H),
2.54 (m, 2H), 1.78-1.58 (m, 4H), 1.28 (t, J ) 8 Hz, 3H); MS
(ESI) m/z 309.3 [(M + H)+, 100].
To the acetylaminoimidazole (700 mg, 2.2 mmol) in 95%
EtOH (20 mL) was added 20% NaOH (1 mL). After stirring
for 5-10 min, a heavy precipitate formed. Water (1 mL) was
added and the reaction mixture filtered to remove undissolved
material. The solvent was evaporated and the residue redis-
solved in water (20 mL). The pH was adjusted to 6 using 1 N
HCl, causing a copious precipitate to form. This was filtered
and washed with a small amount of water. The solid was
resuspended in MeOH (20 mL) and 1 N HCl (7 mL) was added.
After stirring for 1 h, the mixture was filtered and evaporated.
The residue was dissolved in a small amount of water and
placed on a column of C18 reverse-phase silica, washed with
water, and eluted with acetonitrile. Evaporation of the ap-
propriate fractions provided the carboxylic acid (652 mg,
91%): 1H NMR (300 MHz, CD3OD) δ 6.90 (s, 1H), 4.88-4.78
(m, 1H), 3.36-3.22, (m, 1H), 2.90-2.80 (m, 1H), 2.64 (m, 2H),
1.78-1.62 (m, 4H).
The above tert-butyl ester Boc derivative was dissolved in
TFA (0.5 mL) and stirred for 1 h. The solvent was evaporated
and the residue purified using reverse-phase HPLC. Concen-
tration of the appropriate fractions in vacuo, trituration with
Et2O, and drying under vacuum gave the desired sulfamide
3j (32 mg, 76%): 1H NMR (300 MHz, CD3OD) δ 6.96 (s, 2H),
4.80-4.68 (m, 1H), 4.08-3.98 (m, 1H), 3.64-3.54 (m, 1H),
3.42-3.30, (m, 1H), 3.22-3.02 (m, 3H), 2.78-2.68 (m, 1H), 2.58
(s, 6H), 2.24 (s, 3H), 1.78-1.38 (m, 6H); MS (ESI) m/z 534.4
[(M + H)+, 100]; HRMS (FAB) m/z 534.1682 [(M + H)+ calcd
for C20H32N5O8S2 534.1692].
4-(3-Eth oxyca r bon ylisoxa zolin -5-yl)bu tyr ic Acid (11).
To a solution of 4a (5.0 g, 22 mmol) in acetone (100 mL) was
added J ones reagent until an orange color persisted for 10 min.
2-Propanol (2 mL) was added and the reaction mixture was
poured into water (200 mL) and extracted with Et2O (3 × 100
mL). The organic layer was dried (Na2SO4) and evaporated,
and the residue was purified using flash chromatography (10%
MeOH-CH2Cl2) to yield the title compound (3.1 g, 62%): 1H
NMR (300 MHz CDCl3) δ 4.90-4.78 (m, 1H), 4.34 (q, J ) 7
Hz, 2H), 3.34-3.20, (m, 1H), 2.94-2.82 (m, 1H), 2.20 (t, br,
2H), 1.90-1.40 (m, 4H), 1.30 (t, J ) 7 Hz, 3H).
3-E t h oxyca r b on yl-5-(5-b r om o-4-oxop e n t yl)isoxa zo-
lin e (12). To a solution of 11 (5.0 g, 21.8 mmol) in CH2Cl2 (15
mL) was added DMF (2 drops) followed by the dropwise
addition of oxalyl chloride (2.03 g, 15.9 mmol). The reaction
mixture was stirred at room temperature for 16 h, and the
solvent was removed in vacuo to yield the crude acid chloride:
1H NMR (300 MHz, CDCl3) δ 4.88-4.68 (m, 1H), 4.34 (q, J )
8 Hz, 2H), 3.38-3.24, (m, 1H), 3.02-2.80 (m, 3H), 1.90-1.60
(m, 4H), 1.38 (t, J ) 8 Hz, 3H).
The crude acid chloride was dissolved in Et2O (100 mL) and
cooled in an ice bath. An ether solution of diazomethane (300
mL) was added, and the ice bath was removed after 1 h. The
reaction mixture was allowed to stand at room temperature
for 2 h. The solvent was degassed by passing a stream of
nitrogen through the solution. The resulting solvent evapora-
tion and cooling caused the diazoketone to precipitate. When
the volume had been reduced to approximately 100 mL, the
product was filtered to afford the crude diazoketone (4.0 g) as
a solid: 1H NMR (300 MHz, CDCl3) δ 5.26 (bs, 1H), 4.84-
4.68 (m, 1H), 4.34 (q, J ) 8 Hz, 2H), 3.38-3.24, (m, 1H), 2.90-
2.80 (m, 1H), 2.38 (m, 2H), 1.84-1.64 (m, 4H), 1.38 (t, J ) 8
Hz, 3H).
2-[(S)-2,4,6-Tr im eth ylp h en ylsu lfon yla m in o]-3-[5-[3-(2-
a m in oim id a zol-4-yl)p r op yl]isoxa zolin -3-ylca r bon yl]a m i-
n op r op ion ic Acid Tr iflu or oa ceta te (3c). To a solution of
13 (474 mg, 1.5 mmol), 8a (512 mg, 1.5 mmol), and BOP (729
mg, 1.6 mmol) in DMF (1 mL) was added N-methylmorpholine
(605 mg, 6.0 mmol), and the resulting mixture stirred at room
temperature for 16 h. The solvent was evaporated in vacuo
and the residue purified using flash chromatography (3%
MeOH-CH2Cl2) to give the sulfonamide (700 mg, 79%): 1H
NMR (300 MHz, CD3OD) δ 6.94 (s, 2H), 6.62 (s, 1H), 4.80-
4.70 (m, 1H), 4.00-3.92 (m, 1H), 3.60-3.50 (m, 1H), 3.42-
3.30 (m, 1H), 3.24-3.10, (m, 1H), 2.84-2.74 (m, 1H), 2.65-
2.52 (m, 14H), 2.24 (s, 3H), 1.84-1.60 (m, 4H), 1.22 (d, J )
0.5 Hz, 9H).
To a suspension of the above sulfonamide (350 mg, 0.60
mmol) in water (7 mL) was added concentrated sulfuric acid
(15 drops), and the reaction mixture was heated at 60 °C for
16 h. The solvent was evaporated in vacuo, and the residue
was dissolved in TFA (2 mL), evaporated in vacuo, and purified
using reverse-phase column chromatography. Concentration
of the appropriate fractions in vacuo and drying under vacuum
afforded 3c as a white powder (314 mg, 86%): 1H NMR (300
MHz, CD3OD) δ 6.94 (s, 2H), 6.52 (s, 1H), 4.84-4.76 (m, 1H),
4.06-3.98 (m, 1H), 3.66-3.58 (m, 1H), 3.40-3.28 (m, 1H),
3.24-3.08, (m, 1H), 2.82-2.70 (m, 1H), 2.62-2.50 (m, 8H), 2.24
(s, 3H), 1.74-1.60 (m, 4H); MS (ESI) m/z 507.4 [(M + H)+,
100]; HRMS (FAB) m/z 507.2026 [(M + H)+ calcd for
C
22H30N6O6S 507.2038].
5-[4-(Im idazol-2-ylam in o)-4-oxobu tyl]isoxazolin e-3-car -
boxylic Acid (15). To a solution of acid 11 (2.29 g, 10 mmol),
2-aminoimidazole sulfate (14,; 3.96 g, 30 mmol), and N,N-
diisopropylethylamine (6.35 g, 50 mmol) in DMF (10 mL) was
added BOP (6.63 g, 15 mmol). The resulting mixture was
stirred at 70 °C for 16 h. The reaction mixture was cooled to
To a suspension of the crude diazoketone in Et2O (100 mL)

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 35
room temperature, poured into water (200 mL), and allowed
to stand for 1 h, resulting in the formation of a precipitate.
The product was filtered and dried under vacuum to yield the
acylaminoimidazole (1.20 g, 41%): 1H NMR (300 MHz, DMSO-
d6) δ 6.64 (s, 2H), 4.80-4.70 (m, 1H), 4.20 (q, J ) 8 Hz, 2H),
3.30-3.20 (m, 1H), 2.84-2.74 (m, 1H), 2.32 (m, 2H), 1.90-
1.40 (m, 4H), 1.22 (t, J ) 7 Hz, 3H); MS (ESI) m/z 295.4 [(M
+ H) +, 100].
To a solution of the above ester (1.00 g, 34 mmol) in 50%
aqueous dioxane (10 mL) was added LiOH (214 mg, 50 mmol),
and the reaction mixture stirred at room temperature for 0.5
h. The mixture was passed through a short column of C18
reverse-phase silica, and the product eluted using acetonitrile.
The solvent was evaporated in vacuo and the residue dissolved
in water (3 mL) and acidified with N HCl. The solid was
filtered and dried under vacuum to afford 15 (0.84 g, 92%):
1H NMR (300 MHz, DMSO-d6) δ 6.64 (s, 2H), 4.80-4.70 (m,
1H), 3.30-3.20 (m, 1H), 2.84-2.74 (m, 1H), 2.32 (m, 2H), 1.90-
1.40 (m, 4H); MS (ESI) m/z 267.3 [(M + H)+, 100].
2-[(S)-2,4,6-Tr im eth ylph en ylsu lfon ylam in o]-3-[5-[4-(im -
id a zol-2-yla m in o)-4-oxob u t yl]isoxa zolin -3-ylca r b on yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3g). To a solution
of 15 (266 mg, 1.0 mmol), 8a (342 mg, 1.0 mmol), and BOP
(486 mg, 1.1 mmol) in DMF (1 mL) was added N-methylmor-
pholine (404 mg, 4.0 mmol). After 16 h at room temperature,
the mixture was diluted with water and the crude product
filtered. The product was purified using prep TLC to yield the
desired amide (170 mg, 29%): 1H NMR (300 MHz, CD3OD) δ
6.94 (s, 2H), 6.74 (bs, 2H), 4.80-4.70 (m, 1H), 4.00-3.92 (m,
1H), 3.58-3.50 (m, 1H), 3.42-3.30 (m, 1H), 3.24-3.10 (m, 1H),
2.84-2.74 (m, 1H), 2.58 (s, 6H), 2.42 (bs, 2H), 2.24 (s, 3H),
1.84-1.60 (m, 4H), 1.20 (d, J ) 0.5 Hz, 9H); MS (ESI) m/z
591.4 [(M + H)+, 12].
The above amide (150 mg, 0.26 mmol) was dissolved in TFA
(2 mL) and stirred at room temperature for 1 h. The solvent
was evaporated in vacuo and the residue purified using
reverse-phase HPLC. Concentration of the appropriate frac-
tions in vacuo, trituration with Et2O, and drying under vacuum
gave 3g (140 mg, 84%): 1H NMR (300 MHz, CD3OD) δ 7.10
(d, J ) 3 Hz, 2H), 6.94 (s, 2H), 4.82-4.72 (m, 1H), 4.06-3.98
(m, 1H), 3.64-3.58 (m, 1H), 3.42-3.30 (m, 1H), 3.28-3.10 (m,
1H), 2.84-2.72 (m, 1H), 2.58 (s, 6H), 2.48 (bs, 2H), 2.24 (s,
3H), 1.84-1.60 (m, 4H); MS (ESI) m/z 535.3 [(M + H)+, 100].
Anal. (C23H30N6O7S‚0.3CF3CO2H) C, H, N, F.
Eth yl 5-(4-Oxobu tyl)isoxazolin e-3-car boxylate (16). Ox-
alyl chloride (7.30 g, 0.0575 mol) was dissolved in CH2Cl2
(anhyd, 100 mL) and cooled to -60 °C in a dry ice/CHCl3 bath.
To this solution was added dropwise over 30 min a solution of
DMSO (9.38 g, 0.12 mol) in CH2Cl2 (anhyd, 50 mL). After an
additional 30 min, a solution of alcohol 4a (10.75 g, 0.05 mol)
in CH2Cl2 (anhyd, 30 mL) was added dropwise to the reaction
mixture over 45 min and allowed to stir for an additional 30
min. To this mixture was added Et3N (25.25 g, 0.25 mol)
dropwise over 15 min. The cooling bath was removed and the
reaction mixture allowed to warm to room temperature. The
reaction mixture was diluted with CH2Cl2 (100 mL) and
washed with water, 1 N HCl, water, and brine. The organic
layer was separated, dried (MgSO4), and evaporated to yield
aldehyde 16 as a colorless oil (9.54 g, 90%): 1H NMR (300
MHz, CDCl3) δ 9.58 (s, 1H), 4.88-4.74 (m, 1H), 4.36 (q, J ) 7
Hz, 2H), 3.36-3.22 (m, 1H), 2.92-2.80 (m, 1H), 2.52 (t, J ) 6
Hz, 2H), 1.92 (m, 6H), 1.37 (t, J ) 7 Hz, 3H).
5-[4-(N-(P yr idin -2-yl)-N-(ter t-bu tyloxycar bon yl)am in o)-
bu tyl]isoxa zolin e-3-ca r boxylic Acid (17). A solution of 16
(9.40 g, 0.044 mol) in CH2Cl2 (100 mL) was cooled in an ice
bath and 2-aminopyridine (4.57 g, 0.048 mol) added followed
by the addition of sodium triacetoxyborohydride (14.0 g, 0.066
mol). The ice bath was removed and the reaction mixture
allowed to stir at room temperature for 4 h. The reaction
mixture was cautiously poured into a saturated solution of
NaHCO3 (200 mL) and extracted with EtOAc. The organic
layer was dried over anhydrous Na2CO3, filtered, and evapo-
rated to yield a semisolid. The crude product was triturated
with a mixture of Et2O and hexane and the resulting powder
collected by filtration (8.93 g, 70%): 1H NMR (300 MHz, CDCl3)
δ 8.04 (d, J ) 4 Hz, 1H) 7.42 (t, J ) 6 Hz, 1H), 6.48 (t, J ) 6
Hz, 1H), 6.30 (d, J ) 9 Hz, 1H), 4.90-4.70 (m, 2H), 4.34 (q, J
) 7 Hz, 2H), 3.34-3.20 (m, 2H), 2.80-2.92 (m, 1H), 1.90-
1.40 (m, 6H), 1.30 (t, J ) 7 Hz, 3H).
The above aminopyridine (8.93 g, 0.031 mol) was dissolved
in CH2Cl2 (100 mL) and DMAP (374 mg, 0.003 mol) and di-
tert-butyl dicarbonate (14.73 g, 0.067 mol) were added. The
reaction mixture was allowed to stir at room temperature
overnight. The mixture was diluted with water and the organic
layer separated, dried (MgSO4), filtered, and evaporated. The
crude product was purified using flash chromatography (3:1
) hexane:EtOAc) to afford the ester (9.7 g, 81%): 1H NMR
(300 MHz, CDCl3) δ 8.36 (d, J ) 4 Hz, 1H), 7.86-7.54 (m, 2H),
7.01 (t, J ) 6 Hz, 1H), 4.82-4.70 (m, 1H), 4.32 (q, J ) 7 Hz,
2H), 3.93 (t, J ) 7 Hz, 2H), 3.30-3.18 (m, 1H), 2.86-2.76 (m,
1H), 1.90-1.40 (m, 15H), 1.38 (t, J ) 7 Hz, 3H).
A solution of the above ester (10.7 g, 0.027 mol) in a mixture
of THF (20 mL) and water (20 mL) was cooled in an ice bath.
To this was added a solution of LiOH (1.73 g, 0.41 mol) in
water (5 mL). The reaction mixture was stirred for 45 min. A
1 M solution of citric acid (40 mL) was added and the mixture
extracted several times using EtOAc (until TLC of the organic
layer showed no product). The combined organic layers were
dried (MgSO4), filtered, evaporated, and dried under high
vacuum to give 17 as a light yellow semisolid (9.9 g, 99%): 1H
NMR (300 MHz, CDCl3) δ 8.42 (t, J ) 4 Hz, 1H), 7.70 (t, J )
6 Hz, 1H), 7.52 (d, J ) 8 Hz, 1H), 7.10 (t, J ) 6 Hz, 1H), 4.90-
4.78 (m, 1H), 3.90 (t, J ) 7 Hz, 2H), 3.32-3.20 (m, 1H), 2.78-
2.69 (m, 1H), 1.82-1.24 (m, 12H); MS (ESI) m/z 364.3 [(M +
H)+, 100].
ter t-Bu t yl 2(S)-Ben zyloxyca r b on yla m in o-3-[5-(4-(N-
(p yr id in -2-yl)-N-(ter t-b u t yloxyca r b on yl)a m in o)b u t yl)-
isoxa zolin -3-ylca r bon yl]a m in op r op ion a te (18). tert-Butyl
N2-benzyloxycarbonyl-2(S)-2,3-diaminopropionate22 (8b; 2.59
g, 8.81 mmol) and 17 (3.20 g, 8.81 mmol) were dissolved in
DMF (20 mL). N-Methylmorpholine (2.72 g, 26.9 mmol) and
BOP (4.09 g, 9.25 mmol) were added and the reaction mixture
stirred for 48 h at room temperature. Concentration in vacuo
gave an orange oil which was purified using flash chromatog-
raphy (2:1 ) hexane:EtOAc) to yield the title compound as a
pale yellow syrup (4.56 g, 83%): 1H NMR (300 MHz, CDCl3) δ
8.39-8.35 (dd, J ) 4.7, 1.8 Hz, 1H), 7.67-7.58 (td, J ) 7.0,
1.8 Hz, 1H), 7.59-7.51 (td, J ) 7.0, 1.8 Hz, 1H), 7.37-7.28
(m, 5H), 7.06-6.98 (dd, J ) 4.8, 1.8 Hz, 1H), 6.97-6.94 (t, J
) 5.4 Hz, 1H), 5.69-5.66 (d, J ) 7.0 Hz, 1H), 5.11 (s, 2H),
4.79-4.67 (m, 1H), 4.41-4.35 (m, 1H), 3.95-3.90 (t, J ) 7.0
Hz, 2H), 3.79-3.68 (m, 2H), 3.24-3.14 (ddd, J ) 17.6, 8.0, 1.1
Hz, 1H), 2.83-2.74 (dd, J ) 17.6, 8.0 Hz, 1H), 1.50 (s, 9H),
1.48 (s, 9H), 1.47-1.35 (m, 6H); MS (ESI) m/z 640.4 [(M +
H)+, 60].
2(S)-Ben zyloxycar bon ylam in o-3-[5-(4-(pyr idin -2-ylam i-
n o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in op r op ion ic Acid
Tr iflu or oa cet a t e (3a a ). The ester carbamate 18 (240 mg,
0.37 mmol) was dissolved in TFA (2 mL) and stirred at room
temperature for 1 h. The solvent was evaporated in vacuo and
the residue purified using reverse-phase HPLC. Concentration
of the appropriate fractions in vacuo, trituration with Et2O,
and drying under vacuum afforded the desired compound as
an amorphous solid (158 mg, 87%): 1H NMR (300 MHz, CDCl3)
δ 7.85 (t, J ) 5 Hz, 1H), 7.78 (d, J ) 4 Hz, 1H), 7.28 (s, 5H),
7.02 (d, 6 Hz, 1H), 6.83 (m, 1H), 5.02 (t, J ) 8 Hz, 2H), 4.55
(bs, 1H), 4.40 (bs, 1H), 3.80-3.55 (m, 3H), 3.40-3.20 (m, 3H),
2.83-2.74 (m, 2H), 1.77-1.35 (m, 6H); MS (ESI) m/z 484.3 [(M
+ H)+, 100]. Anal. (C24H29N5O6‚1.25CF3CO2H) C, H, N.
ter t-Bu tyl 2(S)-Am in o-3-[5-(4-(N-(ter t-bu tyloxyca r bo-
n yl)pyr idin -2-ylam in o)bu tyl)isoxazolin -3-ylcar bon yl]am i-
n op r op ion a te (19). To 18 (1.5 g, 2.408 mmol) in MeOH (50
mL) was added Pd/BaSO4 unreduced (0.300 g, 0.482 mmol).
The reaction mixture was placed under an atmosphere of H2
(41 psi) at room temperature for 15 h. The mixture was filtered
through a Celite pad and concentrated in vacuo to afford the
amine as a light yellow syrup (1.22 g, quantitative): 1H NMR
(300 MHz, CDCl3) δ 8.38-8.36 (dd, J ) 4.8, 1.8 Hz, 1H), 7.64-

36 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
7.58 (td, J ) 7.0, 1.8 Hz, 1H), 7.56-7.51 (td, J ) 7.0, 1.8 Hz,
1H), 7.14-7.12 (t, J ) 5.4 Hz, 1H), 7.02-6.98 (dd, J ) 4.8, 1.8
Hz, 1H), 4.78-4.69 (m, 1H), 3.91-3.86 (t, J ) 7.0 Hz, 2H),
3.66-3.59 (m, 2H), 3.78-3.81 (m, 1H), 3.28-3.19 (ddd, J )
17.6, 8.0, 1.1 Hz, 1H), 2.87-2.78 (dd, J ) 17.6, 8.0 Hz, 1H),
2.48-2.29 (bs, 2H), 1.76-1.34 (m, 6H), 1.48 (s, 9H), 1.46 (s,
9H); MS (ESI) m/z 506.4 [(M + H)+, 40].
MHz, CD3OD) δ 7.90-7.75 (m, 2H), 7.70-7.62 (m, 2H), 7.50-
7.30 (m, 5H), 7.00 (d, J ) 4.4, 1H), 6.85-6.78 (m, 1H), 4.82-
4.74 (m, 1H), 4.06-4.00 (m, 1H), 3.70-3.58 (m, 1H), 3.42-
3.18 (m, 4H), 3.20-2.90 (m, 2H), 2.70 (s, 6H), 1.70-1.30 (m,
6H); MS (ESI) m/z 594.3 [(M + H)+, 100]; HRMS (FAB) m/z
594.2386 [(M + H)+ calcd for C30H36N5O6S 594.2386].
2-[(S)-1-Na p h th ylen esu lfon yla m in o]-3-[5-(4-((p yr id in -
2-yl)a m in o)b u t yl)isoxa zolin -3-ylca r b on yl]a m in op r op i-
on ic Acid Tr iflu or oa ceta te (3u ): 1H NMR (300 MHz,
CD3OD) δ 8.62 (d, J ) 10.0 Hz, 1H), 8.18 (d, J ) 8.0 Hz, 1H),
8.10 (d, J ) 10.0 Hz, 1H), 7.96 (d, J ) 8.0 Hz, 1H), 7.85-7.50
(m, 5H), 7.03-6.98 (m, 1H), 6.84-6.76 (m, 1H), 4.78-4.65 (m,
1H), 4.14-4.06 (m, 1H), 3.70-3.58 (m, 1H), 3.42-3.24 (m, 4H),
3.10-2.96 (m, 2H), 2.76-2.60 (m, 1H), 1.80-1.40 (m, 7H); MS
(ESI) m/z 540.1 [(M + H)+, 100]; HRMS (FAB) m/z 540.1922
[(M + H)+ calcd for C26H30N5O6S 540.1917].
2-[(S)-4-P h en ylben zen esu lfon ylam in o]-3-[5-(4-((pyr idin -
2-yl)a m in o)b u t yl)isoxa zolin -3-ylca r b on yl]a m in op r op i-
on ic Acid Tr iflu or oa ceta te (3x): 1H NMR (300 MHz,
CD3OD) δ 7.90-7.64 (m, 7H), 7.50-7.34 (m, 4H), 7.00 (d, J )
4.8 Hz, 1H), 6.85-6.80 (m, 1H), 4.82-4.74 (m, 1H), 4.20-4.10
(m, 1H), 3.70-3.58 (m, 1H), 3.42-3.18 (m, 4H), 3.00-2.78 (m,
2H), 1.75-1.40 (m, 6H); MS (ESI) m/z 566.1 [(M + H)+, 100];
HRMS (FAB) m/z 566.2074 [(M + H)+ calcd for C28H32N5O6S
566.2073].
2-[(S)-4-Isop r op ylben zen esu lfon yla m in o]-3-[5-(4-((p y-
r id in -2-yl)a m in o)b u t yl)isoxa zolin -3-ylca r b on yl]a m in o-
p r op ion ic Acid Tr iflu or oa ceta te (3y): 1H NMR (300 MHz,
CD3OD) δ 7.90-7.70 (m, 4H), 7.36 (d, J ) 6.0 Hz, 2H), 7.03-
6.98 (dd, J ) 4.4, 0.05 Hz, 1H), 6.85-6.78 (dd, J ) 5.8, 0.7
Hz, 1H), 4.82-4.74 (m, 1H), 4.06-4.00 (m, 1H), 3.70-3.58 (m,
1H), 3.42-3.18 (m, 4H), 3.00-2.78 (m, 2H), 1.79-1.49 (m, 7H),
1.14 (d, J ) 10.0 Hz, 6H); MS (ESI) m/z 532.4 [(M + H)+, 100];
HRMS (FAB) m/z 532.2235 [(M + H)+ calcd for C25H34N5O6S
532.2230].
2(S)-Ben zen esu lfon yla m in o-3-[5-(4-((p yr id in -2-yl)a m i-
n o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in op r op ion ic Acid
Tr iflu or oa ceta te (3s). To a solution of 19 (250 mg, 0.50
mmol) and pyridine (202 mg, 2.58 mmol) in CH2Cl2 (5 mL)
was added benzenesulfonyl chloride (87 mg, 0.50 mmol) and
the mixture stirred at room temperature under nitrogen for 8
h. Saturated NaHCO3 (15 mL) was added and the organic layer
was separated, dried over anhydrous Na2CO3, and concen-
trated in vacuo to a syrup. The product was dissolved in TFA
(5 mL), stirred at room temperature for 1 h, and then
concentrated in vacuo to an oil. Toluene (10 mL) was added
and the mixture again concentrated in vacuo. The resulting
viscous oil was purified using reverse-phase HPLC. The
fractions containing product were concentrated in vacuo and
placed on a lyophilization apparatus overnight, to afford 3s
as a white powder (108 mg, 45% over two steps): 1H NMR
(300 MHz, CD3OD) δ 7.87-7.76 (d, J ) 6.96, 4H), 7.59-7.57
(t, J ) 7.33 Hz, 1H), 7.51-7.46 (t, J ) 7.7 Hz, 2H), 7.03-7.00
(dd, J ) 9.2, 2.6 Hz, 1H), 6.85-6.79 (d, J ) 4.8 Hz, 1H), 5.86-
5.80 (dd, J ) 7.7, 3.4 Hz, 1H), 4.75-4.70 (m, 1H), 4.12-4.06
(m, 1H), 3.69-3.62 (m, 2H), 3.36-3.30 (t, J ) 7.3 Hz, 2H),
3.25-2.88 (ddd, J ) 17.6, 10.6, 3.7 Hz, 1H), 2.96-2.88 (d, J )
22.3 Hz, 1H), 2.83-2.71 (ddd, J ) 17.6, 8.4, 2.2 Hz, 1H), 2.68-
2.59 (dd, J ) 22.3, 9.5 Hz, 1H), 1.77-1.34 (m, 6H); MS (ESI)
m/z 490.2 [(M + H)+, 100]. Anal. (C22H27N5O6S‚1.1CF3CO2H‚
0.5H2O) C, H, N, F.
Also isolated as white powders using the general procedure
reported for 3s were 3e, 3k , 3l, 3r , 3u , 3x, and 3y.
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
((p yr id in -2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m i-
n op r op ion ic Acid Tr iflu or oa ceta te (3e): 1H NMR (300
MHz, CD3OD) δ 7.86-7.81 (t, J ) 4.4 Hz, 1H), 7.78-7.75 (t, J
) 5.9 Hz, 1H), 7.03-6.98 (dd, J ) 4.4, 0.05 Hz, 1H), 6.94 (s,
2H), 6.85-6.78 (dd, J ) 5.9, 0.7 Hz, 1H), 4.82-4.74 (m, 1H),
4.06-4.00 (m, 1H), 3.65-3.31 (dd, J ) 13.6, 5.1 Hz, 2H), 3.36-
3.31 (t, J ) 7.0 Hz, 2H), 3.16-3.07 (ddd, J ) 17.6, 8.0, 1.1 Hz,
1H), 2.82-2.74 (dd, J ) 17.6, 8.0 Hz, 1H), 2.58 (s, 9H), 2.25
(s, 3H), 1.79-1.49 (m, 6H); MS (ESI) m/z 532.2 [(M + H)+,
100]; HRMS (FAB) m/z 532.2230 [(M + H)+ calcd for
5-[3-(N-(2-Am in opyr idin -6-yl)-N-(ter t-bu tyloxycar bon yl)-
a m in o)p r op yl]isoxa zolin e-3-ca r boxylic Acid (21). To a
solution of 2-amino-6-picoline (20; 15 g, 0.14 mol) in CH2Cl2
(450 mL) was added di-tert-butyl dicarbonate (33.3 g, 0.152
mol), and the reaction mixture was heated at reflux overnight
(18 h) with stirring under N2. The solvent was removed in
vacuo and the residue purified using filtration chromatography
through silica gel (CH2Cl2 as eluent) to provide the carbamate
as a white waxy solid (23.7 g, 82%): 1H NMR (300 MHz, CD3-
Cl) δ 7.70 (d, J ) 8.4 Hz, 1H), 7.54 (app t, J ) 8.1, 7.7 Hz,
1H), 7.16 (bs, 1H), 6.81 (d, J ) 7.7 Hz, 1H), 2.42 (s, 3H), 1.51
(s, 9H). Anal. Calcd for C11H16N2O2: C, 63.44; H, 7.74; N, 13.45.
Found: C, 63.51; H, 7.62; N, 13.40.
C
25H34N5O6S 532.2243].
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
((isoqu in olin -1-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3k ). The title
compound was synthesized from 16 following the general
procedure for reductive amination and ester cleavage reported
for 3a a : 1H NMR (300 MHz, CD3OD) δ 8.42 (d, J ) 6.0 Hz,
1H), 8.00-7.70 (m, 3H), 7.50 (t, J ) 5.0 Hz, 1H), 7.18 (t, J )
6.0 Hz, 1H), 6.90, (s, 2H), 4.82-4.74 (m, 1H), 4.06-4.00 (m,
1H), 3.65-3.31 (m, 2H), 3.30-3.10 (m, 3H), 2.84-2.74 (m, 1H),
2.58 (s, 9H), 2.25 (s, 3H), 1.79-1.49 (m, 6H); MS (ESI) m/z
582.2 [(M + H)+, 100]. Anal. (C29H35N5O6S‚1.2CF3CO2H) C,
H, N, F.
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
((isoqu in olin -3-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3l). The title
compound was synthesized from 16 following the general
procedure for reductive amination and ester cleavage reported
for 3a a : 1H NMR (300 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.30-
8.18 (m, 1H), 8.00-7.82 (m, 2H), 7.66-7.52 (m, 2H), 7.20 (t, J
) 5.0 Hz, 1H), 6.90, (s, 2H), 6.84 (s, 1H), 4.78-4.70 (m, 1H),
3.92-3.82 (m, 1H), 3.42-3.00 (m, 5H), 2.78-2.60 (m, 1H), 2.48
(s, 9H), 2.20 (s, 3H), 1.79-1.49 (m, 6H); MS (ESI) m/z 582.2
[(M + H)+, 100]. Anal. (C29H35N5O6S‚1.2CF3CO2H) C, H, N,
F.
A solution of the above carbamate (4 g, 19.2 mmol) in THF
(40 mL) was cooled to -23 °C under N2 and treated with a 2
M solution of LDA in THF (24 mL, 48 mmol). After stirring
for 30 min, the resulting red solution was cooled to -78 °C
and 4-bromobutene (2.9 mL, 28.8 mmol) was added dropwise.
The resulting mixture was stirred for 15 min, quenched by
the addition of saturated NH4Cl solution, and allowed to
assume room temperature. Extraction with EtOAc and sepa-
ration of product from unreacted starting material using flash
chromatography (Biotage 40S cartridge, 9:1 ) hexane:Et2O)
provided the alkene as a light yellow oil (0.29 g, 5.8%): 1H
NMR (300 MHz, CD3Cl) δ 7.71 (d, J ) 8.4 Hz, 1H), 7.55 (app
t, J ) 8.1, 7.7 Hz, 1H), 7.14 (bs, 1H), 6.80 (d, J ) 7.7 Hz, 1H),
5.77-5.90, (m, 1H), 4.94-5.07 (m, 2H), 2.65 (t, J ) 7.7 Hz,
2H), 2.05-2.13 (m, 2H), 1.72-1.84 (m, 2H), 1.51 (s, 9H).
To a solution of the above alkene (0.29 g, 1.1 mmol) in THF
(3 mL) were added water (2 mL) and NaHCO3 (0.65 g, 7.7
mmol) followed by 6 (0.25 g, 1.66 mmol). The reaction mixture
was stirred at 0 °C under N2 for 5 h, then warmed to room
temperature overnight (18 h). The mixture was again cooled
to 0 °C and a second aliquot of 6 added followed by stirring
for 4 h at 0-25 °C. The reaction mixture was diluted with
water and extracted with EtOAc (3×). The combined extracts
were washed with brine, dried (Na2SO4), filtered, and evapo-
rated. Purification using flash chromatography (Biotage Flash
40S cartridge, 4:1 then 3:1 ) hexane:EtOAc) afforded the
2-[(S)-2,6-Dim eth yl-4-p h en ylben zen esu lfon yla m in o]-3-
[5-(4-((p yr id in -2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3r ): 1H NMR (300

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 37
isoxazoline as a yellow oil (0.3 g, 72%): 1H NMR (300 MHz,
CDCl3) δ 7.73 (d, J ) 8.4 Hz, 1H), 7.56 (app t, J ) 8., 7.7 Hz,
1H), 7.14 (bs, 1H), 6.79 (d, J ) 6.5 Hz, 1H), 4.88-4.75 (m,
1H), 4.34 (q, J ) 7.3 Hz, 2H), 3.25 (dd, J ) 17.6, 11 Hz, 1H),
2.85 (dd, J ) 17.3, 8.5 Hz, 1H) 2.69 (t, J ) 7 Hz, 2H), 1.60-
1.90 (m, 4H), 1.52 (s, 9H), 1.36 (t, J ) 7.3 Hz, 3H); MS (ESI)
m/ z 400.2 (M + Na)+, 378.2 (M + H)+, 322.2 [(M + H - t-Bu)+,
100].
A solution of the above isoxazoline (0.3 g, 0.8 mmol) in THF
(3.6 mL) was treated with 1 M LiOH solution (1.2 mL, 1.2
mmol). MeOH (0.5 mL) was added to achieve a homogeneous
solution, which was stirred for 2 h at room temperature. The
reaction mixture was diluted with water, adjusted to pH 4.0
with 5% aqueous citric acid solution, and then extracted with
EtOAc (3×). The combined extracts were washed with brine,
dried (Na2SO4), filtered, and evaporated to yield acid 21 as a
white foam (0.25 g, 89%): 1H NMR (300 MHz, CDCl3) δ 8.06
(d, J ) 8.4 Hz, 1H), 7.75 (dd, J ) 8.6, 7 Hz, 1H), 6.88 (d, J )
7 Hz, 1H), 4.93-4.80 (m, 1H), 3.33 (dd, J ) 17.4, 11 Hz, 1H),
2.85 (dd, J ) 17.7, 8.1 Hz, 1H) 2.83-2.70 (m, 2H), 1.65-1.94
(m, 4H), 1.54 (s, 9H).
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(3-
((2-a m in op yr id in -6-yl)a m in o)p r op yl)isoxa zolin -3-ylca r -
bon yl]a m in op r op ion ic Acid Tr iflu or oa ceta te (3f). A mix-
ture of acid 21 (0.23 g, 0.66 mmol), 8a (0.25 g, 0.73 mmol),
N-methylmorpholine (0.16 mL, 1.4 mmol), and BOP (0.44 g,
0.99 mmol) in DMF (5 mL) was stirred under N2 at room
temperature for 3 days. The reaction mixture was poured into
water (50 mL) and extracted with EtOAc (3×). The combined
extracts were washed with saturated NaHCO3 and brine, dried
(Na2SO4), filtered, and evaporated. Purification of the residue
using flash chromatography (Biotage Flash 40S cartridge, 1:1
) EtOAc:hexane) gave the amide (380 mg, 85%): 1H NMR (300
MHz, CD3Cl) δ 7.73 (d, J ) 8 Hz, 1H), 7.56 (t, J ) 8, 7.7 Hz,
1H), 7.15 (bs, 1H) 6.94 (s, 2H) 6.85 (t, J ) 7.5 Hz, 1H) 6.80 (d,
J ) 7 Hz, 1H), 5.58 (d, J ) 7.3 Hz, 1H), 4.83-4.71 (m, 1H),
3.92-3.83 (m, 1H), 3.77-3.50 (m, 2H), 3.81 (dd, J ) 18, 11
Hz, 1H), 2.81 (dd, J ) 18, 8.4 Hz, 1H), 2.69 (t, J ) 7.5 Hz,
2H), 2.64 (s, 6H), 2.28 (s, 3H), 1.63-1.88 (m, 4H), 1.52 (s, 9H),
1.30 (s, 9H); MS (ESI) m/z 672.3 [(M + H)+, 100].
A solution of the above amide (380 mg, 0.56 mmol) in CH2-
Cl2 (6 mL) was treated with TFA (1.5 mL), and the resulting
solution stirred at room temperature under N2 overnight (18
h). Evaporation in vacuo and trituration with Et2O gave 3f as
a white solid (344 mg, 100%): 1H NMR (300 MHz, CD3OD) δ
7.84-6.97 (m, 1H), 6.95 (s, 2H), 6.79 (dd, J ) 8, 5.6 Hz, 1H),
6.75 (d, J ) 7.5 Hz, 1H), 4.05-4.00 (m, 1H), 3.63 (dt, J ) 15,
3.5 Hz, 1H), 3.10-3.37 (m, 3H), 2.74-2.85 (m, 3H), 2.58 (s,
6H), 2.26 (s, 3H), 1.91-1.65 (m, 4H); MS (ESI) m/z 540.1 (M
+ Na)+, 518.2 [(M + H)+, 100]. Anal. (C24H31N5O6S‚1.25CF3-
CO2H) C, H, N, S, F.
desired product (2.74 g, 27.5% yield): mp 212.6-215.0 °C; 1H
NMR (300 MHz, CDCl3) δ 7.28 (d, 6H, 7.64, s 4H), 7.17 (t, J )
7.7 Hz, 7H), 7.06 (t, J ) 7.3 Hz, 3H), 6.80 (d, J ) 1.1 Hz, 1H);
MS (NH3-DCI) m/z 456 (M + H)+.
To a solution of the above phthalimide (2.60 g, 5.7 mmol)
in EtOH (anhyd, 250 mL) was added freshly distilled hydra-
zine (1.83 g, 57 mmol) and the resulting mixture was heated
at reflux for 1 h. The reaction mixture was cooled and the
solvent removed in vacuo. The solid residue was purified using
flash chromatography (10:1 ) CHCl3:MeOH) to yield 1.8 g
1
(97%) of 22 as a yellow solid: mp 207-208 °C; H NMR (300
MHz, DMSO-d6) δ 7.44-7.33 (m, 9H), 7.13 (d, J ) 7.0 Hz, 6H),
6.51 (d, J ) 1.8 Hz, 1H), 6.26 (d, J ) 1.8 Hz, 1H); 13C NMR
(75 MHz, CDCl3) δ 149.13, 141.74, 130.02, 129.29, 128.08,
127.94, 127.30, 123.03, 117.27, 74.08; MS (NH3-DCI) m/z 326
(M + H)+. The analytical sample was crystallized from EtOH/
EtOAc. Anal. Calcd for C22H19N3‚0.33H2O: C, 79.75; H, 5.98;
N, 12.68. Found: C, 79.80; H, 5.97; N, 12.56.
5-[4-(1-Tr iph en ylm eth ylim idazol-2-ylam in o)bu tyl]isox-
a zolin e-3-ca r boxylic Acid (23). A solution of 22 (3.00 g, 9.23
mmol) and aldehyde 16 (2.16 g, 10.2 mmol) was stirred in
toluene (250 mL) at reflux for 27 h. After cooling to room
temperature, sodium triacetoxyborohydride (3.74 g, 17.65
mmol) was added and the mixture stirred for an additional
20 h. Water (100 mL) was added, the organic layer was
separated, and the aqueous layer was extracted with EtOAc.
The combined organic layers were concentrated and purified
using flash chromatography (CHCl3-5% MeOH/CHCl3) to
yield the product as a brown oil (5.56 g, >100%): 1H NMR
(300 MHz, DMSO-d6) δ 7.50 (d, J ) 1.5 Hz, 1H), 7.44-7.33
(m, 6H), 7.09 (m, 6H), 6.26 (d, J ) 1.5 Hz, 1H), 4.6 (m, 1H),
4.25 (q, J ) 7.3 Hz, 2H), 3.07 (bs, 1H), 3.20 (dd, J ) 11, 17.6
Hz, 1H), 2.90 (q, J ) 2.3 Hz, 2H), 2.70 (dd, J ) 8.4, 17.6 Hz,
1H), 1.48-1.31 (m, 2H), 1.26 (t, J ) 7.3 Hz, 3H), 1.0 (m, 2H),
0.92-0.84 (m, 2H); MS (NH3-DCI) m/z 523 (M + H)+.
To a solution of the crude ester (5.56 g, 9.23 mmol) in THF
(50 mL) was added a 0.5 M solution of LiOH (37 mL, 18.5
mmol). The reaction mixture was stirred for 1 h and adjusted
to pH 2 using 1 N HCl, and the solvent was evaporated. The
crude product was purified using flash chromatography (10:1
) CHCl3:MeOH) to afford 23 as a white solid (3.00 g, 66%):
1H NMR (300 MHz, DMSO-d6) δ 7.44-7.34 (m, 9H), 7.10 (appt
d, 6H), 6.52 (d, J ) 1.6 Hz, 1H), 6.26 (d, J ) 1.8 Hz, 1H), 4.36
(m, 1H), 4.15-4.0 (bs, 1H), 3.07 (dd, J ) 17.6, 10.6 Hz, 1 H),
2.89 (q, J ) 6.6 Hz, 1H), 2.57 (dd, J ) 17.6, 8.4 Hz, 1H), 1.31-
0.84 (m, 6H); MS (NH3-DCI) m/z 451 (M + H - CO2H)+.
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
((im idazol-2-yl)am in o)bu tyl)isoxazolin -3-ylcar bon yl]am i-
n op r op ion ic Acid Tr iflu or oa ceta te (3d ). To a solution of
acid 23 (98 mg, 0.198 mmol) and amine 8a (68 mg, 0.198 mmol)
in DMF (2 mL) were added 2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU; 76 mg, 0.24
mmol) and Et3N (69 mL, 0.495 mmol). After stirring at room
temperature for 1.5 h, the solvent was evaporated in vacuo
and the residue purified using flash chromatography (10:1 )
CHCl3:MeOH) to yield the amide as a white solid (135 mg,
83%): MS (ESI) m/z 819.4 [(M + H)+ 100].
To the above amide (130 mg, 0.16 mmol) was added a 1:1
mixture of TFA and CH2Cl2 (2 mL) and the resulting solution
stirred at room temperature for 5 h. The solvent was evapo-
rated in vacuo and the crude product purified using reverse-
phase HPLC to provide the desired trityl-protected imidazole
(75 mg, 73%): 1H NMR (300 MHz, acetone-d6) δ 8.03 (s, 1H),
7.70-7.66 (m, 1H), 7.48 (m, 9H), 7.33 (m, 6H), 7.14 (d, J ) 2.6
Hz, 1H), 6.98 (s, 2H), 6.85 (d, 1H), 6.70 (d, J ) 2.6 Hz, 1H),
5.25 (bs, 1H), 4.60 (m, 1H), 4.14 (m, 1H), 3.74-3.52 (m, 1H),
3.31 (q, J ) 6.6 Hz), 3.21-3.04 (m, 1H), 2.67 (m, 1H), 2.62 (s,
6H), 2.27 (s, 3H), 1.48-0.99 (m, 6H); MS (NH3-DCI) m/z 763.3;
HRMS (NH3-CI) m/z 763.3278 [(M + H)+ calcd for C42H46N6O6S
763.3291].
2-Am in o-1-(tr iph en ylm eth yl)im idazole (22). 2-Aminoim-
idazole sulfate (14; 2.64 g, 20 mmol) was dissolved in MeOH
(anhyd, 200 mL) and cooled to -78 °C. A 25% solution of
sodium methoxide in methanol (4.57 mL, 20 mmol) was added
dropwise. The reaction mixture was allowed to warm to room
temperature and stirred for an additional 3 h. The solution
was filtered and concentrated in vacuo to afford a brown oil
(1.6 g, 96.4%): 1H NMR (300 MHz, DMSO-d6) δ 6.32, (s, 2H),
5.0 (bs, 2H).
Phthalic anhydride (freshly recrystallized, 4.14 g, 29.2
mmol) and 2-aminoimidazole (2.32 g, 29.2 mmol) were quickly
blended and placed in a heating bath at a temperature of 170
°C for 15 min. After cooling to room temperature, the crude
reaction mixture was purified using flash chromatography
(95:5 ) CHCl3:MeOH, 80:20 ) CHCl3:MeOH) to yield 4.66 g
(75%) of a brown solid: 1H NMR (300 MHz, DMSO-d6) δ 12.35
(bs, 1H), 8.06-7.94 (m, 4H), 7.16 (bs, 2H); MS (ESI) m/z 214.2
(M + H)+.
To a solution of the above (4.66 g, 21.9 mmol) in pyridine
(anhyd, 100 mL) was added triphenylmethyl chloride (9.15 g,
32.82 mmol). The reaction mixture was heated at reflux for
24 h. Pyridine was removed and the residue purified using
flash chromatography (5-10% CHCl3/MeOH) to yield the
The above trityl-protected imidazole (20 mg, 27 µmol) was
dissolved in TFA (2 mL). Water (0.01 mL) was added and the
reaction mixture was heated at reflux for 1 h. The solvent was
evaporated in vacuo and the crude product purified using

38 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
Pitts et al.
reverse-phase HPLC. Concentration of the appropriate frac-
tions in vacuo and lyophilization of the remaining aqueous
solution afforded 3d as a white powder (10 mg, 58%): 1H NMR
(300 MHz, DMSO-d6) δ 12.8-12.5 (bs, 1H), 11.9 (s, 2H), 8.23
(m, 1H), 7.94 (m, 2H), 6.97 (s, 2H), 6.95 (s, 2H), 6.50 (bs, 1H),
4.70 (m, 1H), 3.91 (q, J ) Hz, 1H), 3.48-3.30 (m, 2H), 3.20
(m, 2H), 3.14-3.06 (m, 1H), 2.76-2.67 (m, 1H), 2.53 (s, 6H),
2.24 (s, 3H), 1.66-1.34 (m, 6H); IR (KBr) 3294, 2944, 1680,
1204, 1156, 1136, cm-1; MS (ESI) m/z 521.4 [(M + H)+ 100];
HRMS (NH3-CI) m/z 521.2182 [(M + H)+ calcd for C23H32N6O6S
521.2190]. Anal. (C23H32N6O6S‚1.3CF3CO2H) C, H, N.
Also prepared using the general precedures described for
3d were 3m -q, 3t, 3v, 3w , 3z, 3bb, and 3cc.
2-[(S)-2,6-Dich lor oben zen esu lfon yla m in o]-3-[5-(4-((im -
id a zol-2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in o-
p r op ion ic Acid Tr iflu or oa ceta te (3m ): 1H NMR (300 MHz,
DMSO-d6) δ 13.2-12.5 (bs, 1H), 11.98 (s, 2H), 8.85 (d, J ) 8.8
Hz, 1H), 8.30-8.22 (m, 1H), 7.96 (m, 1H), 7.60-7.51 (m, 3H),
6.96 (s, 2H), 4.75-4.63 (m, 1H), 4.18-4.10 (m, 1H), 3.57-3.32
(m, 2H), 3.23-3.08 (m, 3H), 2.78-2.69 (m, 1H), 1.69-1.50 (m,
4H), 1.47-1.28 (m, 2H); MS (NH3-DCI) m/z 326 [(M + H)+ 100].
Anal. (C20H24Cl2N6O6S‚1.8CF3CO2H) C, H, N, S.
2-[(S)-3,5-Dim eth ylisoxazolesu lfon ylam in o]-3-[5-(4-((im -
id a zol-2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in o-
p r op ion ic Acid Tr iflu or oa ceta te (3v): 1H NMR (400 MHz,
DMSO-d6) δ 13.2-12.8 (bs, 1H), 12.04-11.93 (bs, 2H), 8.53
(dd, J ) 9.3, 1.7 Hz, 1H), 8.31 (t, J ) 5.6 Hz, 1H), 7.94 (t, J )
5.9 Hz, 1H), 6.95 (s, 2H), 4.76-4.66 (m, 1H), 4.03-3.98 (m,
1H), 3.49-3.42 (m, 2H), 3.33-3.28 (m, 1H), 3.23-3.14 (m, 3H),
2.79-2.71 (m, 1H), 2.53 (s, 3H), 2.30 (s, 3H), 1.69-1.53 (m,
4H), 1.44-1.35 (m, 2H); MS (ESI) m/z 498.3 [(M + H)+ 100].
Anal. (C19H27N7O7S‚2.2CF3CO2H) C, H, N, S.
2-[(S)-4-P h en ylben zen esu lfon yla m in o]-3-[5-(4-((im id a -
zol-2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]a m in op r o-
p ion ic Acid Tr iflu or oa ceta te (3w ): 1H NMR (400 MHz,
DMSO-d6) δ 12.0-11.95 (bs, 2H), 8.27 (dt, J ) 18.6, 5.9 Hz,
1H), 8.19 (dd, J ) 12.9, 8.6 Hz, 1H), 7.92 (t, J ) 5.6 Hz, 1H),
7.87-7.82 (m, 4H), 7.76-7.73 (m, 2H), 7.53-7.49 (m, 2H),
7.45-7.41 (m, 1H), 6.96 (s, 2H), 4.65-4.46 (m, 1H), 4.06-3.98
(m, 1H), 3.34-3.00 (m, 4H), 2.74-2.62 (m, 1H), 1.58-1.43 (m,
4H), 1.41-1.28 (m, 2H); MS (ESI) m/z 555.3 [(M + H)+ 100];
HRMS (FAB) m/z 555.2026 [(M + H)+ calcd for C26H30N6O6S
555.2026]. Anal. (C26H30N6O6S‚1.2CF3CO2H‚H2O) C, H, N, F.
2(S)-Am in o-3-[5-(4-((im id a zol-2-yl)a m in o)b u t yl)isox-
a zolin -3-ylca r bon yl]a m in op r op ion ic Acid Tr iflu or oa c-
eta te (3z): H NMR (400 MHz, CD3OD) δ 6.82 (s, 2H), 4.2-
4.1 (bs, 1H), 3.87-3.81 (m, 2H), 2.87 (dd, J ) 17.1, 8.3 Hz,
1H), 1.72-1.64 (m, 4H), 1.57-1.48 (m, 2H); MS (ESI) m/z 339.3
[(M + H)+ 75], 170.2 (100). Anal. (C14H22N6O4‚2.6CF3CO2H‚
2H2O) C, H, N.
2-[(S)-2-Ch lor o-6-m et h ylben zen esu lfon yla m in o]-3-[5-
(4-((im id a zol-2-yl)a m in o)bu tyl)isoxa zolin -3-ylca r bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3n ): 1H NMR (400
MHz, DMSO-d6) δ 12.05-11.95 (bs, 2H), 8.29-8.22 (m, 1H),
8.15 (d, J ) 8.8 Hz, 1H), 7.96 (t, J ) 5.6 Hz, 1H), 7.44-7.39
(m, 2H), 7.34-7.30 (m, 1H), 6.95 (s, 2H), 4.71-4.67 (m, 1H),
4.11-4.05 (m, 1H), 3.52-3.31 (m, 2H), 3.23-3.09 (m, 3H),
2.77-2.69 (m, 1H), 2.60 (s, 3H), 1.69-1.53 (m, 4H), 1.41-1.34
(m, 2H); MS (ESI) m/z 527.3 [(M + H)+ 100], 529 (40). Anal.
(C21H27ClN6O6S‚2CF3CO2H‚H2O) C, H, N; S: calcd, 4.15;
found, 5.16.
2-[(S)-2,6-Dich lor o-4-p h en ylben zen esu lfon yla m in o]-3-
[5-(4-((im idazol-2-yl)am in o)bu tyl)isoxazolin -3-ylcar bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3o): 1H NMR (300
MHz, DMSO-d6) δ 12.1-12.0 (bs, 2H), 8.56 (dd, J ) 8.8, 3.3
Hz, 1H), 8.33-8.27 (m, 1H), 8.00 (t, J ) 5.1 Hz, 1H), 7.98 (s,
2H), 7.89-7.81 (m, 2H), 7.52-7.47 (m, 3H), 6.96 (s, 2H), 4.59-
4.48 (m, 1H), 4.38-4.18 (m, 1H), 3.21-3.04 (m, 4H), 2.76-
2.67 (m, 1H), 1.6-1.2 (m, 6H); MS (ESI) m/z 623.2 [(M + H)+
100]. Anal. (C26H28Cl2N6O6S‚1.3CF3CO2H) C, H, N.
2-[(S)-2,6-Dim eth yl-4-p h en ylben zen esu lfon yla m in o]-3-
[5-(4-((im idazol-2-yl)am in o)bu tyl)isoxazolin -3-ylcar bon yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3p ): 1H NMR (400
MHz, DMSO-d6) δ 12.9-12.6 (bs, 1H), 12.0-11.9 (bs, 2H),
8.27-8.21 (m, 1H), 8.08 (d, J ) 9.5 Hz, 1H), 7.96-7.90 (b, 1H),
7.75-7.72 (m, 2H), 7.43-7.39 (m, 1H), 6.96 (s, 2H), 4.63-4.45
(m, 1H), 4.02-3.96 (m, 1H), 3.50-3.43 (m, 1H), 3.20-2.98 (m,
3H), 2.68-2.60 (m, 1H), 2.66 (s, 6H), 1.57-1.46 (m, 4H), 1.35-
1.27 (m, 2H); MS (ESI) m/z 583.5 [(M + H)+ 100]; HRMS (FAB)
m/z 583.2339 [(M + H)+ calcd for C28H34N6O6S 583.2326]. Anal.
(C28H34N6O6S‚1.7CF3CO2H) C, H, N.
2-[(S)-Ben zen esu lfon yla m in o]-3-[5-(2-((im id a zol-2-yl)-
a m in o)b u t yl)isoxa zolin -3-ylca r b on yl]a m in op r op ion ic
Acid Tr iflu or oa ceta te (3q): 1H NMR (300 MHz, DMSO-d6)
δ 12.2-12.05 (bs, 2H), 8.34-8.26 (m, 1H), 8.20 (t, J ) 8.4 Hz,
1H), 8.05 (t, J ) 5.5 Hz, 1H), 7.77-7.74 (m, 2H), 7.64-7.50
(m, 3H), 6.96 (s, 2H), 4.74-4.64 (m, 1H), 4.02-3.92 (m, 1H),
3.46-3.31 (m, partially coincident with H2O), 3.29-3.06 (m,
4H), 2.77-2.66 (m, 1H), 1.72-1.45 (m, 4H), 1.45-1.25 (m, 2H);
MS (ESI) m/z 479.3 [(M + H)+ 100]. Anal. (C20H26N6O6S‚1.4CF3-
CO2H) C, H, N.
1
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(2-
((im idazol-2-yl)am in o)eth yl)isoxazolin -3-ylcar bon yl]am i-
n op r op ion ic Acid Tr iflu or oa ceta te (3bb): 1H NMR (300
MHz, DMSO-d6) δ 12.2-12.1 (bs, 2H), 8.34-8.27 (m, 1H),
8.08-7.99 (m, 2H), 6.98 (d, J ) 3.3 Hz, 2H), 4.77-4.74 (m,
1H), 3.95-3.88 (m, 1H), 3.34-3.08 (m, partially coincident with
H2O), 2.53 (s, 6H), 2.23 (s, 3H), 1.92-1.79 (m, 2H); MS (ESI)
m/z 493.4 [(M + H)+ 100]. Anal. (C21H28N6O6S‚1.3CF3CO2H)
C, H, N.
2-[(R)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
((im idazol-2-yl)am in o)bu tyl)isoxazolin -3-ylcar bon yl]am i-
n op r op ion ic Acid Tr iflu or oa ceta te (3cc): 1H NMR (400
MHz, DMSO-d6) δ 13.0-12.1 (bs, 1H), 11.98 (s, 2H), 8.22 (t, J
) 5.9 Hz, 1H), 7.96 (m, 1H), 7.96 (t, J ) 5.7 Hz, 1H), 7.92 (d,
J ) 9.0 Hz, 1H), 6.97 (s, 2H), 6.94 (s, 2H), 4.71-4.66 (m, 1H),
3.94-3.88 (m, 1H), 3.7-3.4 (bs, 2H), 3.45-3.30 (m, 2H), 3.23-
3.14 (m, 3H), 2.70 (dd, J ) 17.6, 8.5 Hz, 1H), 2.54 (s, 6H), 2.24
(s, 3H), 1.65-1.53 (m, 4H), 1.42-1.34 (m, 2H); MS (ESI) m/z
543[(M+Na)+80],521[(M+H)+100].Anal.(C23H32N6O6S‚1.4CF3-
CO2H) C, H, N.
2-[(S)-2,4,6-Tr im et h ylb en zen esu lfon yla m in o]-3-[5-(4-
(im id a zol-2-yla m in o)-4-oxo-3-a za b u t yl)isoxa zolin -3-yl]-
a m in op r op ion ic Acid Tr iflu or oa ceta te (3h ). To a solution
of 9b (82 mg, 0.17 mmol) in CH2Cl2 (anhyd, 10 mL) was added
triethylamine (0.05 mL, 0.36 mmol) followed by a 20% solution
of phosgene in toluene (0.097 mL, 0.19 mmol). After stirring
for 3 h at room temperature, 22 (60 mg, 0.17 mmol) was added,
and the mixture stirred an additional 18 h. Following by
partitioning between water and EtOAc, the combined organic
fraction was washed with water and saturated NaCl, dried
(MgSO4), filtered, and evaporated in vacuo. Using a combina-
tion of reverse-phase HPLC and mass spectral analysis, two
components were identified: the first component (tR ) 14.2
min) was determined to be the detritylated product: MS (ESI)
m/z 592 (M + H)+. The second component (tR ) 18.8 min) was
the desired product: MS (ESI) m/z 834 (M + H)+. The desired
material was placed in 20% HOAc/MeOH overnight, combined
with the detritylated material, and purified using reverse-
phase HPLC. Combination of the appropriate fractions, con-
centration in vacuo, and lyophilization resulted in the detri-
tylated tert-butyl ester (60 mg, 43%). Cleavage of the ester in
TFA as described for 3s gave 3h : 1H NMR (400 MHz, DMSO-
d6) δ 13.1-11.8 (bs, 2H), 11.5-10.0 (bs, 1H), 8.27-8.21 (m,
1H), 7.93 (bd, J ) 9.0 Hz, 1H), 7.42-7.33 (bs, 1H), 7.05 (s,
2H), 6.97 (s, 2H), 6.60-6.40 (bs, 2H), 4.80-4.68 (m, 1H), 3.95-
3.86 (m, 1H), 2.83-2.73 (m, 1H), 2.54 (s, 3H), 2.53 (s, 3H), 2.24
2-[(S)-1-Na p h th a len esu lfon yla m in o]-3-[5-(4-((im id a zol-
2-yl)a m in o)b u t yl)isoxa zolin -3-ylca r b on yl]a m in op r op i-
on ic Acid Tr iflu or oa ceta te (3t): 1H NMR (400 MHz,
DMSO-d6) δ 12.85-12.48 (bs, 1H), 12.0-11.9 (bs, 2H), 8.63
(d, J ) 8.6 Hz, 1H), 8.49 (t, J ) 10.0 Hz, 1H), 8.22-8.18 (m,
1H), 8.16-8.05 (m, 2H), 7.93 (t, J ) 5.4 Hz, 1H), 7.71-7.59
(m, 3H), 6.95 (s, 2H), 6.6-6.4 (bs, 2H), 4.71-4.60 (m, 1H),
4.02-3.97 (m, 1H), 3.42-3.17 (m, coincident with water),
3.09-2.96 (m, 1H), 2.64 (dd, J ) 17.6, 8.6 Hz, 1H), 1.68-1.5s
(m, 4H), 1.46-1.27 (m, 2H); MS (ESI) m/z 529.3 [(M + H)+
100].

Isoxazolines as Antagonists of Integrin Rvâ3
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1 39
(s, 3H), 1.88-1.72 (m, 2H); MS (ESI) m/z 558.1 [(M + Na)+
100], 536.1 [(M + H)+ 60]. Anal. (C20H29N7O7S‚1.9CF3CO2H)
C, H, N.
mM HEPES buffer such that the DMSO concentration was
constant (0.2%). Dilutions of a control compound and negative
DMSO control were run on each plate. Log phase 293â3z cells
were harvested and adjusted to 106 viable cells/mL of MCDB131
media/20 mM HEPES. The 293â3z cells were added to the
compound dilutions in the polypropylene plates and preincu-
bated for 15 min at 37 °C in a 5% CO2 incubator. The
compound/cell mixture was then added to the fibrinogen-coated
plate (105 cells/well) and incubated for 1 h at 37 °C in a 5%
CO2 incubator. After incubation, the nonadhered cells were
removed and the wells washed once with PBS using an
automated multichannel pipet. The adherent cells (10% of
input in absence of inhibitors) were lysed and an aliquot was
taken for determination of â-galactosidase activity using a
Galactolight Plus assay (Tropix) and a Dynatech Microlite 1
luminometer. Nonspecific binding was negligible and deter-
mined using 1 µM of a compound having an IC50 below 20 nM.
The specificity of the assay was indicated by >95% inhibition
of 293â3z adhesion by the Rvâ3 neutralizing antibody LM609
(Chemicon) and the negligible adhesion of untransfected 293
cells to purified fibrinogen under the conditions used in the
assay. The mass of â-galactosidase per well was determined
by comparison to a standard concentration curve of purified
â-galactosidase (Sigma) assayed simultaneously with the
samples.
5. 293â3z-Vn Migr a tion Assa y. Vitronectin was purified
from human plasma by heparin affinity chromatography.25
Migration media (Dulbecco minimal essential media (Gibco)
containing 0.1% bovine serum albumin was added beneath the
microporous membrane inserts of Costar Transwell plates (6.5
mm, 8-µm pore). Compounds were diluted in migration media
such that the concentration of DMSO was constant (0.5%).
DMSO controls in the absence and presence of vitronectin were
run in each assay along with dilutions of a control compound.
Log phase 293â3z cells were harvested and adjusted to 106
viable cells/mL of migration media. The 293â3z cells (105 cells/
well) were added to the compound dilutions in the polypropy-
lene plates and preincubated for 15 min at room temperature.
The compound/cell mixture was then added into the inserts
of the prepared plates. Vitronectin was added to the media
below the insert and the plates were incubated for 17-19 h
at 37 °C in a 5% CO2 incubator. The amount of vitronectin
used to induce migration in the assay was that concentration
that induced half of the maximal migration observed in
titration with that batch of vitronectin (typically 1 µg/mL
vitronectin final concentration for the best preparations). After
incubation, the nonmigrated cells still within the insert were
removed with a cotton swab. The cells that had migrated to
the opposite side of the filter were fixed and stained using a
Diff-Quick kit (Baxter). Excess stain was removed by several
rinses with water. The inserts were inverted and allowed to
dry overnight. The membrane was removed from the insert
and mounted on a microscope slide. The number of migrated
cells per membrane was quantified using an Optomax Domino
image analyzer (Hollis, NH). Generally, 7.5% of the input
293â3z cells migrated in the absence of inhibitors to the
opposite side of the filter in the presence of vitronectin, while
less than 0.3% migrated in the absence of vitronectin. Specific-
ity of the assay was indicated by >95% inhibition of migration
by LM609 and minimal inhibition (<30%) by antibodies to Rvâ5
(P1F6, Chemicon) and R5â5 (J SB5, Chemicon) in comparison
to isotype controls. Additionally, 293 cells that had not been
transfected with the â3 contruct showed negligible (0.3%)
migration toward vitronectin.
In Vitr o P h a r m a cology. 1. Hu m a n P RP -Ligh t Tr a n s-
m itta n ce Aggr egom etr y Assa y. Venous blood was obtained
from healthy human donors who were aspirin-free for at least
2 weeks prior to blood collection or from other species as
previously described.18b Blood was collected into citrate Va-
cutainer tubes. The blood was centrifuged for 10 min at 150g
in a Sorvall RT6000 tabletop centrifuge with H-1000 B rotor
at room temperature, and PRP was removed. The remaining
blood was centrifuged for 10 min at 2500 rpm at room
temperature, and platelet-poor plasma (PPP) was removed.
Samples were assayed on a PAP-4 platelet profiler, using PPP
as the blank (100% transmittance). PRP (200 µL, 2 × 108
platelets/mL) was added to each micro test tube, and transmit-
tance was set to 0%. The platelet agonist ADP (20 µL, 100 µM
final concentration) was added to each tube, and the aggrega-
tion profiles were plotted (% transmittance versus time).
Maximal aggregation was obtained with ADP at 10 µM final
concentration. The test agent (20 µL) was added at different
concentrations 8 min prior to the addition of ADP (10 µM).
The IC50 values (µM) for the different test agents were then
calculated.
2. GP P + F ibr in ogen Aggr ega tion . Human PRP (h-PRP)
or PRP obtained from healthy volunteers was applied to a
sepharose column to prepare GPP as previously described
(Mousa et al., 1994). Aliquots of GPP (2 × 108 platelets/mL)
along with 1 mM calcium chloride and 1 mg/mL fibrinogen
with or without the test agent at different concentrations were
then added to generate an inhibition curve from which IC50
values were calculated.
3. Gen er a tion of 293â3z Cells. The 293 human embryonic
kidney cells were obtained from ATCC and cultured in
Dulbecco minimal essential media (Gibco) containing 10% fetal
bovine serum and pen/strep at 37 °C, 5% CO2. The cDNA for
the human â3 integrin subunit was obtained by PCR ampli-
fication of RNA from HEL cells and cloned into the expression
vector pcDNAneo (Invitrogen). After characterization of the
cloned DNA, 293 cells were transfected with the â3 expression
construct using calcium phosphate. Transfected cells were first
selected in media containing 600 µg/mL active G418 (Gibco)
and then sorted via FACS (Becton-Dickinson) using the Rvâ3
complex-specific antibody, LM609 (Chemicon). A population
of cells expressing the highest levels of Rvâ3 were plated at
low density and a single clone was selected. These 293â3 cells
were transfected by electroporation with the SVzeo/lacZ
construct from Invitrogen and selected in media containing
50 µg/mL zeocin (Invitrogen). Individual clones were picked
and assayed for â-galactosidase specific activity. Clones ex-
pressing the highest â-galactosidase activity were character-
ized by FACS analysis for integrin receptor number, Rvâ3, Rv,
â3, Rvâ5, R4â1, and R5â1 using a Quantum Simply Cellular
microbead kit (Sigma). The parent 293â3 and the clone (293â3z)
selected for use in the adhesion and migration assays ex-
pressed 1.1 × 105 Rvâ3, <2.5 × 103 Rvâ5, 1.2 × 105 Rv, 1.0 × 105
â3, 2.6 × 104 R5â1, and 2.0 × 105 â1 receptors per cell. In
contrast, the untransfected 293 cells expressed <2.5 × 103 Rvâ3,
1.2 × 104 Rvâ5, 1.6 × 104 Rv, <2.5 × 103 â3, 2.6 × 104 R5â1, and
1.9 × 105 â1 receptors per cell. Although the 293â3 cells were
always cultured in the presence of 300 µg/mL G418 and 50
µg/mL zeocin, continuous monitoring of cells indicated de-
creases in both Rvâ3 receptor and â-galactosidase expression
with time in culture. New batches of cells were then started
when expression decreased by 50%.
Ack n ow led gm en t. We express our thanks to F.
Wayne Rankin for technical assistance; Alfred J . Mical
and Andrew M. Blum for performing preparative chiral
HPLC separations; Gregory A. Nemeth and Ernest M.
Schubert for assistance in acquiring NMR data; and
Carl Schwarz and Michael J . Haas for providing low-
and high-resolution mass spectral data. We also thank
Frank A. Barbera, J effrey M. Bozarth, Sandra K. Flint,
Mark S. Forsythe, Lori L. Foster, Maureen A. Kearney,
4. 293â3z-F g Ad h esion Assa y. Human fibrinogen (Enzyme
Research Laboratories Inc.) was further purified by precipita-
tion with glycine.23 Polystyrene EIA plates (Costar 3590) were
coated with 25 µg of fibrinogen overnight at 4 °C. The plates
were washed two times with phosphate-buffered saline without
calcium and magnesium (PBS), blocked with 5% bovine serum
albumin in PBS for 2 h, and washed two times prior to the
assay. Using polypropylene plates (Costar 9482), compounds
were diluted in MCDB131 media24 adjusted to pH 7.0 with 20

40 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 1
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