Structure activity relationships of αv integrin antagonists for pulmonary fibrosis by variation in aryl substituents

Article abstract of DOI:10.1021/ml5002079

Antagonism of α_vβ₆ is emerging as a potential treatment of idiopathic pulmonary fibrosis based on strong target validation. Starting from an α_vβ₃ antagonist lead and through simple variation in the nature and position of the aryl substituent, the discovery of compounds with improved α_vβ₆ activity is described. The compounds also have physicochemical properties commensurate with oral bioavailability and are high quality starting points for a drug discovery program. Compounds 33S and 43E1 are pan α_v antagonists having ca. 100 nM potency against α_vβ_3, α_vβ_5, α_vβ₆, and α_vβ₈ in cell adhesion assays. Detailed structure activity relationships with these integrins are described which also reveal substituents providing partial selectivity (defined as at least a 0.7 log difference in pIC₅₀ values between the integrins in question) for α_vβ₃ and α_vβ₅.

Full text of DOI:10.1021/ml5002079

Letter
pubs.acs.org/acsmedchemlett
Structure Activity Relationships of αv Integrin Antagonists for
Pulmonary Fibrosis by Variation in Aryl Substituents
James Adams,† Edward C. Anderson,† Emma E. Blackham,† Yin Wa Ryan Chiu,† Thomas Clarke,†
Natasha Eccles,† Luke A. Gill,† Joshua J. Haye,† Harvey T. Haywood,† Christian R. Hoenig,†
Marius Kausas,‡ Joelle Le,‡ Hannah L. Russell,† Christopher Smedley,† William J. Tipping,†
Tom Tongue,† Charlotte C. Wood,† Jason Yeung,† James E. Rowedder,‡ M. Jonathan Fray,†
Thomas McInally,† and Simon J. F. Macdonald*,‡
†University of Nottingham, School of Chemistry, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
‡GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage SG1 2NY, U.K.
S
* Supporting Information
ABSTRACT: Antagonism of αvβ6 is emerging as a potential
treatment of idiopathic pulmonary fibrosis based on strong
target validation. Starting from an αvβ3 antagonist lead and
through simple variation in the nature and position of the aryl
substituent, the discovery of compounds with improved αvβ6
activity is described. The compounds also have physicochem-
ical properties commensurate with oral bioavailability and are
high quality starting points for a drug discovery program.
Compounds 33S and 43E1 are pan αv antagonists having ca.
100 nM potency against αvβ3, αvβ5, αvβ6, and αvβ8 in cell adhesion assays. Detailed structure activity relationships with these
integrins are described which also reveal substituents providing partial selectivity (defined as at least a 0.7 log difference in pIC50
values between the integrins in question) for αvβ3 and αvβ5.
KEYWORDS: Integrins, antagonist, pulmonary, fibrosis, αvβ6, αvβ3, β-amino acids
nce diagnosed, life expectancy for patients with idiopathic
antagonists of the αvβ3 receptor for osteoporosis and other
Opulmonary fibrosis (IPF) is usually only a few years and indications,10−13 there is remarkably little literature on potent
the mortality rate exceeds that of many cancer indications.1,2
A
and selective small molecule antagonists of αvβ6. The selectivity
profiles of the αvβ3 antagonists in the literature rarely include
cross-screening data against αvβ6 or other αv integrins, with the
exception of a Pfizer series of compounds for which a broad
range of integrin data is reported (although the potencies are
quite weak as αvβ6 antagonists).14 Other exceptions are the
Merck KGa,15 Merck AG,16 Merck,17 and Monsanto18
compounds (1−4, Figure 1) and JNJ-2607671319 (where the
Arg guanidine and Asp acid mimetics are apparent at either end
of a chain) and very recently αv integrin data in patents from
Ruminski and Griggs at the University of St. Louis.20
We describe here detailed structure activity relationship
(SAR) studies of novel analogues of 4 describing for the first
time their profiles against αvβ3, αvβ5, αvβ6, and αvβ8 in cell
adhesion assays. By varying the substituent pattern on the aryl
ring, we have discovered pan αv antagonists such as 33S and
43E1 (“pan” defined here as having antagonism against all the
αv integrins tested, namely αvβ3, αvβ5, αvβ6, and αvβ8),
antagonists with partial selectivity (defined as at least a 0.7
log difference in pIC50 values between the integrins in
recent study in the U.K. also suggests that the incidence of IPF
is rising, with >5,000 new cases diagnosed each year.3 Although
pirfenidone is now marketed for the treatment of IPF in some
countries,4 there remains an urgent need for effective new
medicines. Recent research into IPF together with estimated
peak sales for an effective treatment at around $2 billion per
annum have driven significant commercial activity around
potential biological and small molecule fibrosis assets in the
pharmaceutical sector.5
There is now reasonable evidence that the RGD (arginine−
glycine−aspartic acid) integrin receptor αvβ6 may play an
important role in the initiation and progression of IPF inter
alia.6,7 This receptor is predominantly expressed in injured lung
tissue, and inhibition of the receptor with αvβ6 antibodies or its
absence in knockout mice leads to substantial protection from
the development of fibrosis in both bleomycin8 and radiation9
induced animal models. Selective αvβ6 antibodies are in
development for fibrotic diseases.5
The αvβ6 receptor is a cell surface heterodimeric receptor
composed of a noncovalently bound complex between the αv
and β6 proteins. Three closely related integrins where only the
β subunit is varied are αvβ3, αvβ5, and αvβ8. Despite substantial
research and drug discovery over the past decade on
Received: May 21, 2014
Accepted: September 19, 2014
© XXXX American Chemical Society
A
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from 3-formylpyridine. Indane-5-carboxaldehyde was prepared
in 38% yield by treatment of indane with hexamethylenetetr-
amine/TFA, as described by Arora et al.23
Hydrogenation of amino ester hydrochloride 7 (R = H) gave
the cyclohexyl derivative 8. The 4-cyano analogue 7 (R = 4-
CN) was obtained in low yield after the esterification, owing to
a competing Pinner reaction. To circumvent this problem in
the case of the 3-cyano analogue, the amino group was
protected as the benzyl carbamate (CBZ), followed by
esterification under mildly basic conditions and CBZ-group
deprotection to give 9.
Amide coupling of (1,8-naphthyridin-2-yl)pentanoic acid
1117 to amino esters 7 (R = 2-OMe, 3-Me, 3-OCF3, and 3,4-
(OMe)2, Scheme 2) gave the corresponding esters 12 in 31−
67% yields. Selective hydrogenation of the naphthyridine ring
gave the corresponding compounds 13 (R = 2-OMe, 3-Me, 3-
OCF3, and 3,4-(OMe)2) (44−96%). For all the other
compounds, the tetrahydronaphthyridine pentanoic acid 1417
could be coupled directly to the amino esters 7−9 to give
compounds 13. Hydrolysis of the esters 13 under basic
conditions then afforded the target acids (see Table 1 for
numbering).
For the preparation of the single enantiomers of 33,
commercially available assigned (R) and (S) enantiomers of
the 3-CF3 amino ester 7 were used. For the enantiomers of 37
and 43, the precursor ethyl esters 13 (R = 3,5-Cl2 and 3-CF3,4-
Cl, respectively) were separated by preparative chiral HPLC
and then hydrolyzed to afford the target compounds.
The compounds were tested in αvβ6, αvβ3, αvβ5, and αvβ8 cell
adhesion assays as previously described.24 Lipophilicity was
determined using an HPLC chromatographic logD protocol.21
Preliminary unpublished work led to the selection of
template 4 as suitable for exploring αvβ6 activity and in
particular investigating what the impact of different aryl
substituents might be. Although structure activity relationships
(SAR) have been described by Merck for this template,17 these
predominantly relate to αvβ3 with little, if any, αvβ6 data
published. Data from the present study are presented in Table
1. In order to establish preliminary αv SAR rapidly, fluoro,
chloro, methyl, and methoxy analogues were prepared in the
ortho, meta, and para positions.
Figure 1. Exemplar αv integrin antagonists from the literature together
with selected reported activities and activities in the assays described
herein.
question) for αvβ3 (such as 27), and partial selectivity for αvβ5
(such as 35). The profiles of antagonists with partial dual
selectivity for αvβ3 and αvβ5 (such as the known compounds 417
and 4217) are also described. Seemingly fairly subtle changes on
just the aryl ring of the structures exert a profound effect on the
overall selectivity profile, and in this series, it appears
considerably more marked than in the Merck KGa series.15
Measured lipophilicity values for compounds (chrom. logD)21
are also provided. (When the sum of chrom. logD plus aromatic
ring count is <6, there is an increased likelihood of a compound
being more developable as an oral drug.21)
The combination of the αvβ6 activity of these compounds
with their physicochemical properties, which are commensurate
with potential oral bioavailability, identifies them as high quality
starting points for a drug discovery program and among the
best currently known in the literature.
Derivatives of ethyl or methyl 3-amino-3-phenylpropanoate 7
were prepared in two steps from the appropriate aldehydes 5 by
the Rodionov reaction22 followed by esterification to 7, or were
purchased from commercial sources (Scheme 1). Ethyl 3-
amino-3-(3-pyridyl)propanoate 10 was made in the same way
The parent phenyl compound 15 is a micromolar αvβ6
antagonist and substantially more potent against αvβ3 and
αvβ5.25,26 In every case, the fluoro, chloro, methyl, and methoxy
compounds are similarly more potent against αvβ3 and αvβ5
than αvβ6, with activity against αvβ8 being similar to or less than
the αvβ6 values. The αvβ3 and αvβ5 values are generally similar
to each other. The superior antagonism against αvβ3 is perhaps
unsurprising given the series emanates from one designed as
αvβ3 antagonists.
Scheme 1. Synthesis of 3-Aryl-3-aminopropanoic Ester
a
Intermediates
The SARs are idiosyncratic, although there are compounds
with approximately 10-fold selectivity for αvβ3 and αvβ5 over
αvβ6 and αvβ8, such as R = H (15), m-F (4), m-OMe (22), and
p-OMe (26). More intriguingly, perhaps, given the relative lack
of such compounds in the literature, some show suggestions of
selectivity for αvβ5 with the o-F (16), m-Cl (20), and m-Me
(21) having 0.5 log selectivity over αvβ3 and about a log
selectivity over αvβ6 and αvβ8. Recent studies suggest there may
be a role for αvβ5 antagonists in the treatment of sepsis.27 Based
on these data, the next iteration of analogues focused on further
monosubstituents in the meta and para positions, as these
showed more consistent αvβ6 activity and are more synthetically
accessible compared to the ortho analogues.
a
Reagents and conditions. (a) malonic acid, ammonium acetate,
i
MeOH or EtOH or PrOH, reflux; (b) SOCl2, EtOH, −15 °C then
reflux; (c) H2 (4 bar), 5% Rh/Al2O3, EtOH, 80 °C; (d) N-
(benzyloxycarbonyloxy)succinimide, EtNPri2, CH2Cl2, room temp., 2
h; (e) N,N′-carbonyldiimidazole, THF, room temp., then EtOH; (f)
H2 (1 bar), 10% Pd/C, EtOH, room temp., 24 h. All compounds are
racemic unless indicated in the text.
B
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a
Scheme 2. Synthesis of Integrin Antagonists
a
Reagents and conditions. (a) N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide, N-methylmorpholine, 1-hydroxybenzotriazole hydrate, 20 °C;
(b) H2 (1 bar), 10% Pd/C, EtOH, 20 °C; (c) 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate,
Et3N, CH2Cl2, room temp.; (d) NaOH(aq), EtOH, 20 °C; (e) LiOH, THF/H2O, 20 °C. All compounds are racemic unless indicated in the text.
a
Table 1. Activity of Aryl Substituted Analogues in αv Integrin Cell Adhesion Assays
b
c
Compd
R
αvβ6 cell pIC50
αvβ3 cell pIC50
αvβ5 cell pIC50
αvβ8 cell pIC50
Chrom. log D
Partial selectivity for ...
15
H
5.7
6.0
<5.0
6.4
<5.0
6.1
6.6
6.4
6.5
6.3
6.4
6.1
6.3
6.4
6.2
6.2
6.2
6.4
6.6
7.0
7.1
5.2
6.7
5.1
6.7
6.6
7.0
6.5
5.7
7.3
6.3
7.5
7.1
6.8
7.4
7.0
6.9
7.4
7.6
7.1
6.6
6.8
6.9
7
7.1
7.0
5.8
7.7
6.3
7.8
7.6
7.3
7.2
7.2
6.9
7.0
7.3
6.4
6.5
6.3
6.4
6.2
7.4
7.4
7.2
6.1
5.4
<5.0
5.9
<5.0
5.8
6.3
6.2
6.3
6.3
6.2
6.0
6.3
6.1
6.1
6.1
6.1
6.5
6.0
6.8
6.9
1.56
1.88
1.92
1.85
1.83
1.92
2.30
1.95
1.66
1.75
2.14
1.97
1.64
1.47
2.72
2.85
1.08
3.10
1.67
2.73
2.72
2.61
3.11
2.73
2.62
2.93
2.97
2.80
2.29
2.52
2.43
1.37
1.47
3.1
αvβ3 and αvβ5
16
2-F
17
2-Cl
18
2-Me
2-OMe
3-F
αvβ3 and αvβ5
αvβ3 and αvβ5
19
d
4
αvβ3 and αvβ5
20
3-Cl
21
3-CH3
3-OMe
4-F
22
αvβ3 and αvβ5
23
24
4-Cl
25
4-Me
4-OMe
4-CN
4-CF3
4-OCF3
4-SO2Me
4-Ph
αvβ3 and αvβ5
αvβ3 and αvβ5
αvβ3
26
27
28
pan
29
30
31
32
3-CN
3-CF3
6.7
6.7
7.0
αvβ5
33
33S
33R
34
(S)-3-CF3
pan, αvβ6 lead
d
d
d
(R)-3-CF3
<5
5.7
<5
3-OCF3
6.4
5.4
7.3
6.4
7.1
6.3
7.0
6.7
6.6
5
35
2,3-Cl2
αvβ5
36
3,4-Cl2
6.7
6.6
<5
pan
37
3,5-Cl2
d
d
d
37E1
37E2
38
3,5-Cl2 (Ent. 1)
3,5-Cl2 (Ent. 2)
3,4-Me2
5.2
<5
6.0
6.8
6.7
6.8
6.3
6.5
6.6
7.0
7.2
<5
6.9
7.2
6.9
6.4
7
6.6
7.2
7.1
6.4
7.2
7.9
6.9
7.2
7.0
6.3
6.4
6.2
6.4
6.1
6.6
6.9
pan
39
3,4-CH2CH2CH2
3,5-Me2
40
41
3,4-(OMe)2
3,4-OCH2O-
3-CF3-4-Cl
3-CF3-4-Cl (Ent. 1)
3-CF3-4-Cl (Ent. 2)
d
42
7.8
6.5
6.8
αvβ3 and αvβ5
43
pan
43E1
43E2
3.28
3.25
pan, αvβ6 lead
e
e
e
5.8
6.2
<5
a
All compounds racemic unless shown. All biological data are means from at least n = 2 and within 0.42 of the mean. pIC50 values are the negative
b
c
log of the IC50. The lower limit of the assays is around pIC50 5. Chromatographic log D; see ref21. Defined as at least a 0.7 log difference in pIC50
values between the integrins in question; See ref 17; Values are n = 1.
d
e
Data from further mono-para substituents were explored
(Table 1, 27−31). Activities against αvβ6 are similar (pIC50
6.1−6.4) despite varying size and electronic properties. In
contrast, there is a 10-fold range in activity against αvβ3 (pIC50
6.6−7.6) with a p-OMe 26 showing the most potent activity
and a p−CF3 28 the least, suggesting the electronic properties
of the substituent may play a role. A similar pattern is seen with
αvβ5 whereas antagonism of αvβ8 is flat and essentially similar to
αvβ6. The p-CN analogue 27 suggests some selectivity for αvβ3.
However, these data do not indicate monosubstitution in the
para position is useful for increasing αvβ6 activity, and so no
further analogues were prepared.
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In contrast, monosubstitution in the meta-position (Table 1,
32−34) proved to be more influential on αvβ6 antagonism, with
around a 10-fold range of activity (pIC50 6.1−7.1). The size of
the substituent is more influential (compare m-F 4 (pIC50 6.1)
with m-CF3 33 (pIC50 7.0)) than electronic properties
(compare m-MeO 22 and m-Me 21 with m-CN 32 and m-Cl
20 (pIC50 values of 6.5 and 6.4 vs 6.6 and 6.6, respectively)).
The enantiomers of the most potent m-CF3 analogue 33
(racemate pIC50 7.0) were prepared from commercially
available pure (S) and (R) enantiomers of 7, thus allowing
33S and 33R to be assigned as the (S) and (R) trifluoromethyl
enantiomers, respectively, and with corresponding αvβ6 pIC50
values of 7.1 and 5.2. Given the high sequence homology
between αvβ3 and αvβ6, this is consistent with the more potent
m-fluoro analogue 4 against αvβ3 also having (S) stereo-
chemistry, as described elswhere.28
An approximately 10-fold range in activity for m-substituents
is also seen for αvβ3 (pIC50 6.4−7.5) and αvβ5 (pIC50 7.1−7.8),
with antagonist activities generally being greater than those
observed for αvβ6. The SAR is idiosyncratic, suggesting that
multiple low energy binding conformations may be possible. As
seen with the para-substituents, the αvβ8 SAR essentially tracks
the αvβ6 SAR.
The m-CN analogue 32 shows an approximate 0.7−1.4 log
selectivity for αvβ5 (pIC50 7.4) over the other αv integrins. The
m-CF3 analogue 33S is the most potent antagonist against αvβ6
(pIC50 7.1) and equipotent against αvβ3, αvβ5, and αvβ8 (pIC50
7.0, 7.2, and 6.9). Meta-substitution is clearly highly influential
on the selectivity profile, and further work is ongoing.
Given the impact of monoaryl substitution on potency and
selectivity, we decided to explore also the impact of
disubstitution because if there are multiple binding pockets in
this region of the active site, then synergistic effects might be
observed. As the meta position had proved most sensitive, we
preserved substitution at this position while varying the
position of the second substituent, selecting analogues (35−
43) which could be prepared from readily available starting
materials.
A greater range in potency was seen with disubstituted
analogues compared with monosubstituted analogues. Prepara-
tion of substitution patterns of dichloro analogues gives an
interesting range of selectivity profiles. The 2,3-dichloro 35
shows micromolar potency for αvβ5 and 10-fold selectivity over
the other αv integrins. The 3,4-dichloro 36 and the 3,5-dichloro
37 restore αvβ6 activity (pIC50 6.7 and 6.6, respectively), with
the 3,5 analogue 37 being a pan αv antagonist. Separation of the
3,5-Cl2 enantiomers29 gave αvβ6 pIC50 activities of 6.8 (37E2)
and 5.2 (37E1), with 37E2 remaining predominantly a pan
antagonist.
42 have previously been described as αvβ3 antagonists,17 the
studies described here show a more detailed picture of their
profile with both compounds potent against αvβ3 but also being
equipotent against αvβ5. The SARs presented here clearly show
that, by simple variation of the position and nature of the aryl
substituent, the cell adhesion potency against αvβ6 can be
increased and, comparatively, potency against αvβ3 and αvβ5
reduced. Comparison of the lead compounds described here
(e.g., 33S and 43E1) with the standards 1 and 3 from the
literature (cf. Figure 1) shows they have similar αvβ6 activity but
with structural features perhaps more commensurate with oral
bioavailability properties. Their lipophilicities are reasonable
(chrom. logD values of 2.72 for 33S and 3.28 for 43E1), and
they possess good permeability and solubility (data not shown).
Indeed, analogues of these compounds prepared by Merck have
been shown to have good oral bioavailability in dog.17,28
Although a crystal structure of αvβ6 is currently not available,
a homology model is available,30 and the αvβ3 crystal structure
has been described.31 From the latter, it is known that the RGD
motif (or ligand mimetic) binds at the interface between the αv
and the β3 (or β6) subunits, with the arginine (or mimetic)
binding to the αv unit and the aspartic acid (or mimetic)
binding to the β3 (or β6) subunit. There is considerable
sequence similarity between the β3 and β6 subunits, so the gross
topology is also likely to be similar. Based on this, an extended
conformation for these ligands in αvβ6 is likely to be the
conformation for antagonism and is also consistent with why
small differences in aryl group substitution might have a
profound impact on the selectivity between the different αv
integrins observed.
Further studies to improve αvβ6 potency are underway.
Clearly sufficient potency is required to drive an antifibrotic
response at a realistic clinical dose, but a compromise may be
required between the ideal potency and ideal selectivity.
Although our focus here is IPF, a pan αv antagonist has
recently been reported32 as being efficacious in a number of
models of fibrotic diseases (see Supporting Information).
Described here are SAR relationships against αvβ3, αvβ5, αvβ6,
and αvβ8 with the aim of ultimately identifying an orally
bioavailable selective and potent αvβ6 for treating IPF. The lead
compounds 33S and 43E1 have been derived from αvβ3
antagonists to pan αv antagonists with antagonisms around
pIC50 7 (100 nM). This has been achieved while maintaining
physicochemical properties commensurate with oral bioavail-
ability. Compounds which are partially selective for αvβ5 have
also been identified. Further studies will be reported in due
course.
ASSOCIATED CONTENT
■
As expected, the known 3,4-methylenedioxy analogue 4217
has approximately nanomolar potency and greater than 10-fold
selectivity for αvβ3 and αvβ5 over αvβ6 and αvβ8; it also has
increased potency by 0.6−0.7 log units over the 3,4-dimethoxy
derivative 41, which is less selective for αvβ3 and αvβ5. The
presence of the oxygens is important, as both the
corresponding indane 39 and 3,4-dimethyl derivative 38 is
almost a log unit less potent against αvβ3 and αvβ5 and are also
less selective. The same applies to the 3,4-dichloro analogue 36.
The 3-trifluoromethyl-4-chloro analogue 43 is also a pan αv
antagonist, with the more active enantiomer 43E129 having a
similar profile.
S
* Supporting Information
Experimental details and spectroscopic data for the compounds
described in this paper. This material is available free of charge
via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: simon.jf.macdonald@gsk.com. Telephone: +44 (0)
1438 768249.
Author Contributions
Presented here are SAR studies of a series of integrin
All authors have given approval to the final version of the
antagonists against αvβ3, αvβ5, αvβ6, and αvβ8. Although 4 and
manuscript.
D
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Discovery of (+)-(2-{4-[2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-
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Nanomolar small molecule inhibitors for αvβ6, αvβ5 and αvβ3 integrins.
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Funding
M.J.F. holds a University of Nottingham Teaching Fellowship
funded by GSK, who also provided funds for consumables
associated with this work.
Notes
The authors declare no competing financial interest.
The results in this paper are from fourth year undergraduate
research projects at the University of Nottingham in
collaboration with GlaxoSmithKline with the aim of giving
students experience of industrial medicinal chemistry. The
selection of the targets and the syntheses were carried out
predominantly by the students under the guidance of the last
three authors.
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ACKNOWLEDGMENTS
■
The authors wish to thank Dr. Simon Peace for helpful
discussions and the following project students whose
compounds are not described here: Christine Butcher, Adam
Eason, Nick Herbert, Dominic Howgego, William Restorick,
and Jack Sorrell.
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ACS Medicinal Chemistry Letters
Letter
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