Synthesis and biological evaluation of nonpeptide integrin antagonists containing spirocyclic scaffolds

Article abstract of DOI:10.1016/j.bmcl.2003.10.057

Analogues of isoxazoline α_vβ₃ antagonist 1 designed to further restrict the four carbon alkyl tether were prepared by incorporating two spirocyclic scaffolds, 1-oxa-2-azaspiro[4,5]dec-2-ene and 1-oxa-2,7-diazaspiro[4,4]non-2-ene. Additional optimization provided potent antagonists of both α_vβ₃ and α ₅β₁ which are selective over GPIIb/IIIa.

Full text of DOI:10.1016/j.bmcl.2003.10.057

Bioorganic & Medicinal Chemistry Letters 14 (2004) 383–387
Synthesis and biological evaluation of nonpeptide integrin
antagonists containing spirocyclic scaffolds
Joanne M. Smallheer,* Carolyn A. Weigelt, Francis J. Woerner, Jennifer S. Wells,
Wayne F. Daneker, Shaker A. Mousa, Ruth R. Wexler and Prabhakar K. Jadhav
Bristol-Myers Squibb Company, Pharmaceutical Research Institute, PO Box 5400, Princeton, NJ 08543-5400, USA
Received 30 June 2003; revised 22 October 2003; accepted 31 October 2003
Abstract—Analogues of isoxazoline avb3 antagonist 1 designed to further restrict the four carbon alkyl tether were prepared by
incorporating two spirocyclic scaffolds, 1-oxa-2-azaspiro[4,5]dec-2-ene and 1-oxa-2,7-diazaspiro[4,4]non-2-ene. Additional optimi-
zation provided potent antagonists of both avb3 and a5b1 which are selective over GPIIb/IIIa.
# 2003 Elsevier Ltd. All rights reserved.
Integrins are heterodimeric transmembrane glycopro-
tein receptors which mediate various cell–cell and cell–
extracellular matrix interactions.1 As such, integrins are
attractive targets for therapeutic intervention in diseases
involving cell-adhesion processes2 including throm-
boembolic disorders,3 metastasis4 and osteoporosis.5
The glycoprotein IIb/IIIa receptor, which is ultimately
responsible for platelet aggregation during thrombosis,6
was the focus of early integrin antagonist research, and
numerous small molecule inhibitors have now been
studied in clinical trials.7 More recently, another integ-
rin, avb3, has moved into the forefront of integrin
antagonist research. The avb3 integrin or vitronectin
receptor binds to a variety of intrinsic ligands including
vitronectin, fibronectin, von Willebrand factor, throm-
bospondin and osteopontin,8 and has been implicated in
a variety of pathological conditions involving abnormal
cell adhesion including osteoporosis, restenosis and dis-
eases involving neovascularization, for example cancer
metastasis, diabetic retinopathy, and macular degenera-
tion.7 The a5b1 integrin receptor may serve as an
attachment point for cell penetration of certain viruses9
and bacteria,10 and is therefore of interest as a target for
antiinfective therapy. The a5b1 integrin has also been
shown to affect avb3-mediated endothelial cell migration
and angiogenesis via regulation of avb3 integrin func-
tion.11 A recent study with avb3 and a5b1 specific anti-
bodies further demonstrated that the simultaneous
blockade of both these integrins inhibits smooth muscle
cell spreading and invasive migration (processes impor-
tant in restenosis), whereas blocking avb3 alone was
ineffective.12 Similarly, it was reported that combined
antagonism of both avb3 and a5b1, as opposed to avb3
alone, induced apoptosis of angiogenic endothelial cells
on type I collagen.13
All of these integrins recognize a putative RGD binding
site on their respective ligands. Extremely potent inhi-
bitors of GPIIb/IIIa and avb3 have been identified
through the use of conformationally restricted cyclic
peptides.14 While generally unsuitable as drugs due to
their poor bioavailability, these cyclic peptides provided
structural insight for the design of small molecule pep-
tidomimetics that incorporate mimics of the RGD
sequence and are much more attractive as potential
therapeutics. Many heterocyclic scaffolds have been
successfully employed to provide the desired conforma-
tional constraint to maintain the acidic and basic ends
of these linear molecules at the appropriate distance and
in a conformation suitable for binding.15 An ongoing
interest in avb3 as a therapeutic target is evident in the
number of avb3 antagonists that continue to be descri-
bed in the literature.16 On the other hand, selective small
molecule antagonists of a5b1 or dual a5b1–avb3 antago-
nists have yet to be described in detail.
We have recently reported on a series of potent avb3
integrin antagonists which incorporate an isoxazoline
ring as the conformational constraint.17 An early
example in this series was compound 1.
* Corresponding author. Tel.: +1-609-818-5332; fax: 1-609-818-3450;
e-mail: joanne.smallheer@bms.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.057

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J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 383–387
tion at the carbon adjacent to the aldehyde could not be
avoided and the resulting products were obtained as
mixtures of diastereomers. Non-aromatic heterocyclic
guanidine mimics were prepared from alcohol 3 via
conversion the tosylate and displacement with sodium
azide to give 5. Hydrolysis of the ester and coupling to
an aminopropionate was carried out as described above.
Reduction of the azide21 provided an amine which was
heated with methyl-(tetrahydropyrimidin-2-ylidene)-
sulfonium iodide in DMF followed by deprotection to
provide the 2-aminotetrahydropyrimidine analogue 8.
Compounds with a carbonyl in place of the methylene
linker were also prepared from alcohol 3 by Jones oxi-
dation, followed by coupling with 2-aminoimidazole
sulfate in the presence of Castro’s reagent. Elaboration
of the acidic end as previously described provided 9.
Both diastereomers of 1 had comparable potency in an
avb3 binding assay, which suggested that the binding of
the basic group occurs within the common conforma-
tional space shared by both isomers. Based on this
observation, we felt that the introduction of an addi-
tional steric constraint in the alkylene linker to restrict
the guanidine mimic to this common space would pro-
vide more potent analogues. The steric constraint we
envisioned was a spiro-fused ring system, wherein the
open chain tether between the isoxazoline and the gua-
nidine mimic would be replaced by a spiro-fused carbo-
cyclic or heterocyclic ring.
In the initial comparison of the spirocyclic compounds
with the diastereomers of corresponding open chain
compound 1, the affinity for the avb3 integrin was
determined using a purified avb3 receptor binding assay
(avb3 ELISA).22 Isomer 7a, in which the carbon sub-
stituents on the cyclohexyl ring are trans, was compar-
able in potency (2 nM vs 1.3 and 0.9 nM, respectively,
for the isomers of 1) to both diastereomers of 1 and
about 25X more potent that the cis isomer 7b. Having
established some confidence in the design of the spiro-
cyclic linker, we proceeded to elaborate the SAR of this
series with respect to the basicity of the guanidine mimic
and the aryl sulfonamide alpha to the carboxy terminus.
These subsequent compounds were evaluated as mix-
tures of diastereomers using a functional avb3 antagon-
ism assay, involving adhesion of b3-transfected 293 cells
to fibrinogen (avb3 293b3).23 Inhibition of platelet
aggregation in human platelet-rich plasma (GPIIb/IIIa
hPRP) was used to assess the selectivity of compounds
for the avb3 over the GPIIbIIIa integrin receptor.24
Compounds were also screened for a5b1 antagonism
using an ELISA assay.25 The data are summarized in
Table 1.
Spirocyclic scaffolds which incorporate an isoxazoline
ring are readily available from 1,3-dipolar cycloaddition
of a nitrile oxide with exomethylene carbocycles or het-
erocycles. An appropriately functionalized 6,5-spiro-
isoxazoline scaffold was prepared from commercially
available 4-methylene cyclohexanol 2 as shown in
Scheme 1.18,19 The resulting alcohol 3 was obtained as a
3:2 mixture of cis/trans diastereomers around the cyclo-
hexyl ring. Swern oxidation gave the corresponding
mixture of aldehydes 4 which were separated into single
diastereomers by careful flash chromatography. Reduc-
tive amination with 2-aminopyridine in the presence of
sodium triacetoxyborohydride20 followed by hydrolysis
of the ethyl ester, coupling with t-butyl 3-amino-2-phe-
nylsulfonylaminopropionate18 and TFA deprotection
gave the initial targets 7. Synthesis of analogues with
alternate basic groups was accomplished by substitution
of other aromatic 2-aminoheterocycles for the 2-amino-
pyridine with appropriate modification of the reductive
amination conditions. The reductive amination could be
carried out with unprotected 2-aminobenzimidazole
using Ti(OiPr)4 as a catalyst for imine formation fol-
lowed by reduction in situ with NaBH4. In the case of 2-
aminoimidazole, initial formation of the intermediate
imine using 1-trityl-2-aminoimidazole in the presence of
anhydrous MgSO4 in refluxing benzene, followed by
reduction of the isolated imine was found to be superior
to the one-pot procedure.18 Unfortunately, racemiza-
Varying the pKa of the basic moiety did not significantly
alter the potency. Holding the basic group constant at
imidazole and varying the substituents on the aryl sul-
fonamide at N2 provided compounds 10–14. While
improved potency in the avb3 cell adhesion assay was
Scheme 1. Reagents and conditions: (a) ethyl oximidoacetate, NaHCO3, THF, 0 ꢀC; (b) diethyl nitromalonate, mesitylene, 175 ꢀC; (c) (COCl)2,
DMSO; (d) 2-aminopyridine, Na(OAc)3BH, DCE; (e) 1 M LiOH, THF; (f) t-butyl 3-amino-2-phenylsulfonylaminopropionate, Castro’s reagent,
NMM, DMF; (g) TFA, CH2Cl2; (h) TsCl, pyr; (i) NaN3, DMF; (j) Ph3P , HO; (k) methyl-(tetrahydropyrimidin-2-ylidene)-sulfonium iodide DMF,
2
95 ꢀC; (l) Jones reagent; (m) 2-aminoimidazole sulfate, Castro’s reagent, DMF, NMM, 70 ꢀC.

J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 383–387
385
obtained with des-methyl compound 11 and the analo-
gous 2,6-dichloro analogue 12, the selectivity over IIb/
IIIa for these compounds was only 5–15-fold. Improved
selectivity over IIb/IIIa was obtained with compounds
13 and 14 which have biphenylsulfonamide groups,
however, the addition of the second phenyl ring sig-
nificantly reduced the aqueous solubility of these com-
pounds. Also, the examples in Table 1 were 2–10Â less
potent than the corresponding open-chain compounds
previously reported.18
The (S,S,S) isomer 18a was >20Â more potent than the
(S,R,S) isomer 18b. As previously observed, varying
the basic group in compounds 19–21 did not sig-
nificantly affect the avb3 potency, however improved
selectivity over IIb/IIIa was obtained by lowering the
pKa of the basic moiety. The proline-derived com-
pounds with R1=Cbz exhibited 3–6-fold better potency
versus avb3 as compared to the analogous compounds
in Table 1 and had similar potency to the open
chain isoxazolines with increased selectivity over IIb/
IIIa. Removal of the Cbz group to provide 22 resulted
in a drop in avb3 potency as well as a 100-fold loss of
selectivity over IIb/IIIa. The avb3 potency was retained
in the methylcarbamate 23, but activity dropped off
3-fold as the size of the alkyl group was increased to
n-butyl in 24. The inverse was true for selectivity over
IIb/IIIa. Pyridylcarbamate 25 was equipotent to 19
and shows good aqueous solubility, although there
was some loss in selectivity in going from phenyl to
pyridyl.
Based on the above results, a spirocyclic scaffold based
on 3-hydroxyproline was designed to further rigidize
the molecule and set the absolute stereochemistry of the
products. The substituent on the second nitrogen in
the spirocycle was also viewed as an opportunity to fine
tune the properties of the molecules. Synthesis of a
spiro-fused 5,5-system was accomplished as shown in
Scheme 2 starting from N-Cbz-4-hydroxy-cis l-proline
15. Reduction of the acid with borane–methyl sulfide
complex26 provided the diol which was selectively pro-
tected on the primary alcohol followed by Swern oxi-
dation of the remaining secondary hydroxyl to provide
ketone 16. A Wittig reaction generated the exo-methyl-
ene compound which underwent 1,3-dipolar cycloaddi-
tion with ethyl oximidoacetate to give a 7:1 mixture of
diastereomers that were readily separated by flash
chromatography to give the single isomers. NOE
experiments on the deprotected alcohol 17b established
that the carbon substituents on the spiro system were cis
in the major diastereomer. The major diastereomer
therefore has (S,S) absolute stereochemisty, and the
minor product is the (R,S) isomer. The remaining steps
were carried out as described above on the individual
isomers to provide the compounds shown in Table 2.
The introduction of a second lipophilic substituent at
the basic end of the molecule also resulted in a sig-
nificant increase in activity against a5b1. In contrast to
the IIb/IIIa selectivity, the larger, more lipophilic car-
bamates (Cbz>CO2Bu>CO2Me>H) and less basic
guanidine mimics (benzimidazoleꢁpyridine>imidazole
>imidazoline) resulted in more potent a5b1 antagonists.
Both 20 and 27 were subnanomolar antagonists in the
a5b1 ELISA assay. When compared to the correspond-
ing open-chain analogue (example 3e in ref 18), com-
pound 20 is equipotent in the 293b3 avb3 assay but
>1000-fold more potent in the a5b1 ELISA assay.27
Consistent with its increased potency versus a5b1, com-
pound 27 was shown to inhibit the in vitro streptococcal
Table 1.
Compda
X
R
avb3 293b3
GPIIb/IIIa
hPRP IC50 (nM)
a5b1 ELISA
IC50 (nM)
IC50ÆSEM (nM)b
7c
7d
8
2-Imidazolyl NHCH2
2-Benzimidazolyl NHCH2
3,4,5,6-Tetrahydro-2-pyrimidinyl NHCH2
2-ImidazolylNHCO
SO2mesityl
SO2mesityl
SO2mesityl
SO2mesityl
SO2Ph
170Æ120 (4)
440Æ160 (3)
160Æ28 (3)
106Æ31
1000
7100
800
33,000
830
310
490
8300
8300
1500
170
NT
2700
4700
1800
3000
670
9
10
11
12
13
14
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
130Æ48 (5)
56Æ24 (4)
34Æ8.6 (4)
93Æ45 (5)
17Æ7.4 (4)
SO2(2,6-dimethylphenyl)
SO2(2,6-dichlorophenyl)
SO2(2,6-dimethyl-4-biphenyl)
SO2(2,6-dichloro-4-biphenyl)
310
a All compounds were purified by reverse-phase HPLC and isolated as TFA salts. Satisfactory spectral and analytical data were obtained for all
compounds.
b Values are means of number of experiment in parentheses.
+
Scheme 2. Reagents and conditions: (a) BH3 Me2S, THF; (b) tBuMe2SiCl, TEA, CH2Cl2; (c) (COCl)2, DMSO; (d) Ph3PCH3 BrÀ, KOtBu, THF;
.
(e) ethyl oximidoacetate, TEA, CH2Cl2; (f) HOAc/THF/H2O.

386
J. M. Smallheer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 383–387
Table 2.
Compda
X
R1
avb3 293b3
GPIIb/IIIa
hPRP IC50 (nM)
a5b1 ELISA
IC50 (nM)
IC50ÆSEM (nM)b
18a
18b
19
20
21
22
23
24
25
26
27
2-Dihydroimidazolyl NHCH2
2-Dihydroimidazolyl NHCH2
Cbz
Cbz
Cbz
Cbz
Cbz
H
57Æ35
7300
9200
34,000
13
1200
18
0.18
NT
230
21
14
39
1.4
0.39
f
>1000
e
2-Imidazolyl NHCH2
2-PyridylNHCH2
41Æ26
49Æ24
74Æ28
180Æ74
57Æ35c
130Æ70
32Æ29d
34Æ15
87Æ38
>100,000
>100,000
1400
7300
11,000
5600
>100,000
>100,000
2-Benzimidazolyl NHCH2
e
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
e
CO2Me
CO2nBu
CO2CH2(3-pyr)
CO2Me
CO2nBu
e
2-Imidazolyl NHCH2
2-Imidazolyl NHCH2
e
2-Benzimidazolyl NHCH2
2-Benzimidazolyl NHCH2
a All compounds were purified by reverse-phase HPLC and isolated as TFA salts.Satisfactory spectral and analytical data were obtained for all
compounds.
b n of 3.
c n of 4.
d n of 6.
e Mix of diastereomers at C7.
f (S,R,S) isomer.
6. Ferguson, J. J.; Waly, H. M.; Wilson, J. M. Am. Heart J.
1998, 135, S35.
7. (a) Wityak, J. Curr. Drugs 1997, 2, 185. (b) Smallheer,
J. M.; Olson, R. E.; Wexler, R. R. Ann. Rpt. Med. Chem.
2000, 35, 103.
8. Preissner, K. T.; Seiffert, D. Thromb. Res. 1998, 89, 1.
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invasion of mammalian epithelial cells, a process medi-
ated by the a5b1 integrin.28
In conclusion, we have identified two spirocycles which
can serve as novel conformational constraints in RGD
peptidomimetics. With appropriate substitutions, the
1-oxa-2,7-diazaspiro[4,4]non-2-ene scaffold provided
potent and selective antagonists of both the avb3 and
a5b1 integrin receptors.
11. Okada, N.; Watarai, M.; Ozeri, V.; Hanski, E.; Caparon,
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Acknowledgements
We would like to express appreciation to C. Joseph
Eyermann for computer modeling. We are also grateful
to Gregory Nemeth for his assistance with the NMR
experiments, Michael J. Haas for providing mass spec-
tral data, and Jeffrey M. Bozarth, Mark S. Forsythe,
Maureen A. Kearney, Frank Barbara, Peter Bouchard
and William F. Lorelli for technical assistance in per-
forming the biological assays.
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24. ELISA and selectivity results are based on single deter-