Novel inhibitors of the αvβ3 integrin-lead identification strategy

Article abstract of DOI:10.1016/j.bmcl.2009.06.041

A novel approach to inhibition of the αvβ3 integrin is described, which uses compounds designed to generate nM potency without using the arginine binding site.

Full text of DOI:10.1016/j.bmcl.2009.06.041

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4832–4835
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel inhibitors of the
avb3 integrin—lead identification strategy
*
David Elliot, Eleanor Henshaw, Philip A. MacFaul, Andrew D. Morley , Peter Newham, Keith Oldham,
*
Ken Page, Neil Rankine, Paul Sharpe, Attilla Ting , Christine M. Wood
AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel approach to inhibition of the avb3 integrin is described, which uses compounds designed to gen-
erate nM potency without using the arginine binding site.
Received 3 April 2009
Revised 8 June 2009
Accepted 10 June 2009
Available online 13 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Integrin
Inhibitor
Lead identification
Integrins are a family of cell surface heterodimeric adhesion
receptors composed of and b chains. Ligands include cell surface
(MIDAS) located within the integrin b subunit, while the arginine
side chain of physiological ligands inserts into a narrow groove
a
counter receptors (e.g., the cell adhesion molecules or CAMs),
ligands of the vasculature (e.g., fibrinogen) and extracellular matrix
macromolecules (e.g., collagens and fibronectin). Integrin–ligand
interactions are critical for cell positioning within tissues, for pro-
viding traction and guidance during cell migration, for mechano-
transduction and for the organization of signaling complexes that
modulate cell phenotype.1
on the
vb3 MIDAS-binders that achieved high binding affinity through
alternative interactions on the interface. The strategy is
depicted schematically in Figure 1. Thus our hit-finding approach
centered on a directed screen of low molecular weight carboxylic
acids and isosteres (MW <350).
a subunit. Our hit identification strategy was to identify
a
ab
a
vb3 acts as a regulator of disease pathology associated with
cancer, osteoporosis and rheumatoid arthritis.2–4 For example,
endothelial cell vb3 regulates the survival of angiogenic vessels,
osteoclast vb3 supports firm adhesion to the bone matrix, facili-
tating bone remodeling, while macrophage vb3 appears to play
a role in modulating pro-inflammatory cytokine expression.
Although numerous small molecule ligands of vb3 are
a
a
a
a
known,5,6 all contain motifs that mimic the arginine and aspartate
of the RGD tripeptide, present in endogenous protein ligands.
Whilst these zwitterionic inhibitors provide potency, they are fre-
quently large, and highly flexible, invariably leading to sub-optimal
in vivo pharmacokinetic profiles. Although a significant number of
small molecule antagonist structures have been published for this
target, very few compounds have progressed into clinical develop-
ment. At the outset of our work we decided to follow an alternative
antagonist development approach. The carboxylate present in all
known avb3 inhibitors mimics the acidic moiety of the RGD tripep-
tide and thus competes with physiological ligands. These acid
motifs coordinate the Mg2+ present in the metal ion adhesion site
Figure 1. CycloRGDFV bound to
a
vb313 (RCSB code:1L5G). Carboxylate and
* Corresponding authors.
arginine motifs are circled in blue. The strategy is to explore potential binding
E-mail address: andy.morley@astrazeneca.com (A.D. Morley).
sites (green circles 1–3) at the interface of the integrin dimer.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.041

D. Elliot et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4832–4835
4833
Table 2
SAR of the
CO2H
O
S O
CO2H
O
S O
H2N
H2N
b
H2N
CO2H
NH2
a
-amino motif
a
N
O
N
H
O
H
O
O
CO2H
R
CO2H
R
N
N
H
H
N
N
R
CO2H
O
R1
R1
N
H
c
N
S
O
R isomer
S isomer
Compd
Isomer
R
R1
avb3 IC50 (lM)
1
10
11
12
13
14
15
16
17
R
S
(2,4,6-Trimethylphenyl)sulfonyl
(2,4,6-Trimethylphenyl)sulfonyl
Phenylsulfonyl
Benzylsulfonyl
Benzoyl
2,6-Dichlorobenzoyl
Phenoxycarbonyl
tert-Butyloxycarbonyl
(2,4,6-Trimethylphenyl)sulfonyl
H
H
H
H
H
H
H
H
0.8
8
R
R
R
R
R
R
R
9.1
>50
>50
>50
>50
>50
>50
Scheme 1. Reagents: (a) mesityl chloride, NaOH, dioxane; (b) Br2, NaOH/H2O; (c)
HATU, acid, DMF, DIPEA or chlorofomate, DIPEA, THF, or PhCHO, NaCNBH3, AcOH,
MeOH/CH2Cl2.
The strength of this approach is that it has the potential to
identify novel scaffolds with superior pharmacokinetic profiles
over standard RGD mimics. However, since it is unclear if alterna-
tive binding modes are consistent with high affinity interactions,
there was a risk that compounds of this type may only possess
modest potency.
Me
In parallel to the work above, the initial exploration of the SAR
around the
in Table 2.
a-amino substituent was also undertaken and is shown
One of the hits from screening was 1, which had an IC50 of
The R enantiomer (1) of the amino acid is 10-fold more potent
than the S (10). The removal of the methyl substituents from the
aromatic ring of the sulfonamide (11) also resulted in a 10-fold loss
of potency. Further derivatization of the molecule in this region by
replacement of the sulfonamide with small sub sets of amides and
carbamates abolished activity in all cases, as did methylation of the
sulfonamide (17).
800 nM in inhibiting fibrinogen binding to
the start point for lead identification.
avb3 and was used as
O
CO2H
N
H
O
S
HN
O
1
As the benzamide analogues are truncated versions of known
integrin inhibitors,5,6,8,9 a library of aromatic amide analogues,
based on (7) was synthesized and tested to explore the SAR in
more detail. Data for key compounds is shown in Table 3.
A variety of aromatic rings can be incorporated in this region,
the majority maintaining potency in the 10–300 nM range. Further
derivatization is tolerated. SAR can be quite specific, but no signif-
icant increase in potency was observed through elaboration with
additional substituents.
The synthesis of 1 and analogues is outlined in Scheme 1. This
was achieved using commercial R-asparagine as a start point, fol-
lowing known literature procedures.7
Preliminary derivatization focused on the exploration of the
SAR around the acetamide motif of 1. Data for key compounds is
highlighted below in Table 1.
Most modifications in this region dramatically reduced potency.
The removal of the acetyl (2) or its replacement with methyloxy-
carbonyl (5) or methylsulfonyl (6) completely abolished
avb3
Compounds 18 and 25 are a matched pair, showing that larger
motifs are tolerated, but result in loss of potency (ꢀ8-fold). The
modeling of known RGD mimetics and project compounds support
activity. Further derivatization of the amide was specific, 3-meth-
lypropanoyl (3) losing >30-fold activity and most other modifica-
tions being inactive. The only positive variation was the
incorporation of a benzoyl motif (7) which increased potency by
ꢀ7-fold. Expansion of these results showed that the addition of a
linker between the aromatic and the amide (8) or the removal of
the carbonyl (9) significantly reduced potency.
the observed SAR.10 The overlay of L73975811,12 into the
avb3 crys-
tal structure13 suggests that the carboxylate binds to the MIDAS
site, along with the sulfonamide oxygen and nitrogen forming H-
bonds to backbone amides. The basic piperidine extends towards
to the alpha subunit, forming another H-bond. The central portion
of the molecule acts purely as a spacer that links the molecular
fragments that bind into the
a
and b subunits. Modeling suggested
Table 1
SAR of the acetamide
Table 3
SAR of the aromatic amide
R
CO2H
N
H
O
O
S
N
H
CO2H
Ar
N
H
O
O
N
H
S
a
O
Compd
R
avb3IC50 (lM)
1
2
3
4
5
6
7
8
9
Acetyl
Hydrogen
0.8
>50
30
>50
>50
>50
0.12
>50
>50
Compds
Ar
avb3 IC50 (nM)
3-Methlypropanoyl
tert-Butyloxycarbonyl
Methyloxycarbonyl
Methylsulfonyl
Benzoyl
18
19
20
21
22
23
24
25
3-Thienyl
3-Furyl
2-Methylpyrazol-3-yl
2-Methyloxazol-4-yl
3-Pyridyl
5-Pyrimidinyl
4-Pyridyl
Benzo[b]thiophene
44
32
11
41
81
63
301
350
2-Phenylacetyl
Benzyl
a
Values are means of three experiments.

4834
D. Elliot et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4832–4835
Table 5
CO2H
HN
O
O
Profiles of key lead generation compounds
S
N
N
H
O
O
CO2H
O
Ar1
N
H
S
S
N
H
S
O
Ar2
HN
Compd
20
21
33
Ar1
Ar2
2-Methylpyrazol-3-yl 2-Methyloxazol-4-yl 3-Thienyl
L739758
(2,4,6-Trimethyl-
(2,4,6-Trimethyl-
2,3-Dichloro-
phenyl)sulfonyl
11
394
>3000
16.6
1.85
12.5
phenyl)sulfonyl
41
395
>2000
4.9
phenyl
19
423
>3000
2.7
Figure 2. Overlay of Merck compound L73975811,12 in orange (RCSB code: 1TY7)
a
vb3IC50 (nM)
Mol Wt (g/mol)
Solubility ( M)
and potential binding mode of compound 18 (cyan) in
1L5G), showing molecular surface of the protein.
a
vb3 crystal13 (RCSB code:
l
Rat PB (% free)
Log P
Rat heps
1.5
4.8
2
9.9
(l
l/min/106 cells)
O
O
CO2Me
CO2Me
CO2H
a,b
O
c
R
N
H
Hu mics
l/min/mg)
Cyp ( M)
PAMPA
(106 cm/s)
14.6
4.7
<2
R
N
H
H2N
NHCBZ
NH2
(l
NHCBZ
l
>10 (5/5)
0.021
>10 (5/5)
0.1
>10 (5/5)
0.046
CO2H
O
d,e
R
N
H
HN
S
O
R1
Scheme 2. Reagents: (a) SOCl2, MeOH; (b) HATU, acid, CH2Cl2, DIPEA; (c) HBr,
AcOH; (d) sulfonyl chloride, DIPEA, CH2Cl2; (e) 1 M NaOH, MeOH.
analogues tolerated. Variations around the sulfonamide were
generally consistent with any changes to Ar1.
The series showed promising potency without an arginine mi-
metic and key compounds were profiled more widely (Table 5).
Physicochemical and in vitro DMPK profiles are encouraging for
Lead Identification.
that the benzo[b]thiophene of 25 can bind in a similar manner to
the thienothiophene ring in L739758, while the thiophene ana-
logue (18) is small enough to bind more deeply into the hydropho-
bic grove and make additional interactions with the protein, as
shown in Figure 2. Further analysis of the predicted binding mode
of 18 indicated that the carboxylate would bind to the MIDAS site
while the sulfonamide would form an H-bond to the backbone as
observed in L739758. The amide orientation of 18 overlays well
with the crystal structure of cyclo RGD mimetics, and positions
the thiophene into the hydrophobic grove.
Fixing the amide substituent as phenyl, 3-thienyl and 3-furyl,
initial evaluation of the SAR around the sulfonamide was under-
taken. Compounds were synthesized as highlighted in Scheme 2
and SAR is detailed in Table 4.
Lipophilic motifs were preferred at the ortho position of Ar2.
The addition of a second ortho substituent did not offer any signif-
icant improvement in potency. The incorporation of functionality
in the 3 and 4-positions was encouraging with a diverse set of
Compound 21 was evaluated in a rat PK study and found to
have an iv clearance of 4.7 ml/min/kg, a Vdss of 0.8 l/kg and a termi-
nal T1/2 of 7 h. Oral dosing showed it to have bioavailability of 15%.
Whilst this is a modest profile, it appears to be superior to the
related diaminopropionic acid templates which incorporate an
arginine mimic.14 Bioavailability appears to be permeability lim-
ited, as 21 possesses solubility in excess of 1 mM, whilst PAMPA
experiments showed a Papp value of 0.1 Â 106 cm/s. This lies just
within the low permeability classification (<30% predicted Fabs
)
as defined by internal validation data comparing human Fabs and
Papp for a set of around 70 oral drugs.15 In an attempt to increase
the inherent series permeability, we explored modification of the
amide in more detail. Results are detailed in Table 6.
The replacement on the secondary amide (19) with a tertiary
analogue (39) maintained a similar lipophilicity value (clog P), en-
hanced permeability by threefold (PAMPA), but lost >300-fold in
potency. This is supported by modeling, where the tertiary amide
would clash with the furan ring, unless a different amide orienta-
tion is adopted. The five-membered lactam (40) also lost activity,
but not as dramatically, presumably because it is conformationally
constrained. The incorporation of an ortho methoxy substituent
Table 4
SAR of the sulfonamide
O
CO2H
Ar1
N
H
(41) slightly increased
a
vb3 activity, compared to 7, whilst sub-
O
S O
N
H
Ar2
Table 6
Effect of amide modification
Compds
Ar1
Ar2
o-Tolyl
2-Chlorophenyl
2-Iodophenyl
2-Methylsulfonylphenyl
2-Methoxyphenyl
2,3-Dichlorophenyl
2,6-Dichlorophenyl
2,3-Dichlorophenyl
2,4-Dichlorophenyl
2,4,6-Trichlorophenyl
3-Cyanophenyl
avb3 IC50 (nM)
26
27
28
29
30
31
32
33
34
35
36
37
38
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
3-Thienyl
3-Thienyl
3-Thienyl
3-Furyl
3-Furyl
3-Furyl
320
260
25
4600
729
62
118
19
8
24
98
208
793
CO2H
R
O
N
H
S
O
Compd
R
a
(nM)
vb3 IC50
clog P
PAMPA
(106 cm/s)
19
39
7
40
41
Furan-3-carbonylamino
Furan-3-carbonyl-methyl-amino
Benzamido
1-Oxoisoindolin-2-yl
2-Methoxybenzamide
32
8000
120
975
78
1.9
1.98
2.5
2.1
2.5
0.14
0.45
0.15
0.8
3-Methoxyphenyl
3-Pyridyl
4.6

D. Elliot et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4832–4835
4835
tioned unfavorably close to Tyr190 in
a
ii, whilst the absence of ‘in-
sert3’ and subsequent differing conformation of Tyr 178 in
tolerated.
This approach described above demonstrates that for
a
v is
a
vb3, low
nM potency is achievable without the need to incorporate an
arginine mimetic. The observed SAR and overall profiles of the
compounds produced in Lead Generation are different from those
previously published for this target. Broader SAR exploration of
target areas 2 and 3 of Figure 1 through derivatizing the ortho
position of the sulfonamide should improve selectivity and
possibly potency and would form the key strategy for Lead
Optimization.
Figure 3. (a) Binding mode of compound 43 (cyan) in
1L5G). (b) Overlay binding mode of compound 43 (cyan) into aiib3a crystal (RCSB
avb3 crystal (RCSB code:
Acknowledgement
code: 1TY7).
The authors would like to acknowledge the input and advice of
Dr. Peter Kenny in the preparation of this Letter.
Table 7
Selectivity comparisons
References and notes
O
CO2H
N
H
1. Hynes, R. O. Cell 2002, 110, 673.
O
S O
N
O
2. Cai, W.; Chen, X. Anticancer Agents Med. Chem. 2006, 6, 407.
3. Nakamura, I.; Duong, le T.; Rodan, S. B.; Rodan, G. A. J. Bone Miner. Metab. 2007,
25, 337.
H
R
4. Wilder, R. L. Ann. Rheum. Dis. 2002, 61(Suppl 2), ii96.
5. Coleman, P. J.; Duong, Le T. Expert Opin. Therapeutic Patent 2002, 12, 1009.
6. Henry, C.; Moitessier, N.; Chapleur, Y. Mini-Rev. Med. Chem. 2002, 2, 531.
7. Jones, R. C. F.; Dickson, J. J. Pept. Sci. 2000, 6, 621.
8. Kubota, D.; Ishikawa, M.; Yamamoto, M.; Murakami, S.; Hachisu, M.; Katano, K.;
Ajito, K. Bioorg. Med. Chem. 2006, 14, 2089.
Compd
R
a
vb3 IC50 (nM)
aiib3a IC50 (nM)
42
43
Me
OMe
260
700
2380
>100,000
9. Duggan, M. E.; Duong, L. T.; Fisher, J. E.; Hamill, T. G.; Hoffman, W. F.; Huff, J. R.;
Ihle, N. C.; Leu, C.-T.; Nagy, R. M.; Perkins, J. J.; Rodan, S. B.; Wesolowski, G.;
Whitman, D. B.; Zartman, A. E.; Rodan, G. A.; Hartman, G. D. J. Med. Chem. 2000,
43, 3736.
stantially increasing the overall permeability, making this a useful
observation to develop further.
10. Models of the binding modes of compounds 18, 25 and 43 with
avb3 were
generated from the RCSB (home.rcsb.org) Protein Data Bank (www.rcsb.org/
pdb/home/home.do), with entry reference code 1L5G (Ref. 13) using the
Maestro molecular modeling program. The models were docked using Glide
and then energy-minimized using OPLS 2005 force field. Glide, Maestro and
MarcoModel were licensed from Schrödinger, LGG (www.schrodinger.com).
11. Xiao, T.; Takagi, J.; Coller, B. S.; Wang, J.-H.; Springer, T. A. Nature, 2004. 59.
12. Since completion of the work described above, a re-refinement of the L-739758
Disappointingly, most analogues showed only modest selectiv-
ity for
avb3 over aiib3a (5–10-fold). Analysis of the X-ray struc-
tures showed differences in the regions highlighted in Figure 3,
and modeling suggested that the ortho position of the sulfonamide
might be a good area to target to improve selectivity. Broader test-
ing of ortho substituted analogues highlighted the following
matched pair comparison (Table 7).
in
aiib3a complex has been deposited in the RCSB PDB with Refcode: 2vc2
replacing the previous version Refcode: 1TY7 (the coordinates are still
available). There are subtle difference observed between the two versions,
but this did not impact significantly on our findings and our strategy going into
Lead Optimization.
Compound 43 demonstrates >130-fold selectivity for
iib3a. This improvement in selectivity can be rationalized by the
presence of ‘insert 3’11,12 in
ii. This significantly modifies the
avb3 over
a
13. Xiong, J-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.;
Arnaout, M. A. Science 2002, 296, 151.
14. Ishikawa, M.; Hiraiwa, Y.; Kubota, D.; Tsushima, M.; Watanabe, T.; Murakami,
S.; Ouchi, S.; Ajito, K. Bioorg. Med. Chem. 2006, 14, 2131.
15. Unpublished AstraZeneca data.
a
interface for
aiib3a compared to avb3 in this region and also re-
stricts the conformational freedom of Tyr 190. Modeling10 suggest
the 2-methoxy substituent of compound 43 (Fig. 3b) will be posi-