Discovery of an Orally Bioavailable Pan αv Integrin Inhibitor for Idiopathic Pulmonary Fibrosis

Article abstract of DOI:10.1021/acs.jmedchem.9b00962

The heterodimeric transmembrane αv integrin receptors have recently emerged as potential targets for the treatment of idiopathic pulmonary fibrosis. Herein, we describe how subtle modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the vitronectin receptors αvβ3 and αvβ5 significantly change the biological activities against αvβ6 and αvβ8. This resulted in the discovery of a pan αv antagonist (compound 39, 4-40 nM for the integrin receptors named above) possessing excellent oral pharmacokinetic properties in rats (with a clearance of 7.6 mL/(min kg) and a bioavailability of 97%).

Full text of DOI:10.1021/acs.jmedchem.9b00962

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Article
The discovery of an orally bioavailable pan-#v
integrin inhibitor for idiopathic pulmonary fibrosis.
Niall A Anderson, Sebastien Campos, Sharon Butler, Royston C. B. Copley, Ian Duncan,
Stephen Harrison, Joelle Le, Rosemary Maghames, Aleix Pastor-Garcia, John M. Pritchard,
James E. Rowedder, Claire E. Smith, Jack Thomas, Giovanni Vitulli, and Simon J. F. Macdonald
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00962 • Publication Date (Web): 09 Sep 2019
Downloaded from pubs.acs.org on September 9, 2019
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The discovery of an orally bioavailable pan αv integrin inhibitor for idiopathic pulmonary fibrosis.
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Niall A. Anderson,* Sebastien Campos,≠ Sharon Butler, Royston C. B. Copley, Ian Duncan, Stephen
Harrison, Joelle Le, Rosemary Maghames, Aleix Pastor-Garcia, John M. Pritchard, James E. Rowedder,
Claire E. Smith, Jack Thomas, Giovanni Vitulli, Simon J.F. Macdonald
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*GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
KEYWORDS Integrin, pan v, oral bioavailability, IPF
ABSTRACT: The heterodimeric transmembrane αv integrin receptors have recently emerged as
potential targets for the treatment of idiopathic pulmonary fibrosis. Herein we describe how subtle
modifications of the central aromatic ring of a series of phenylbutyrate-based antagonists of the
vitronectin receptors αvβ3 and αvβ5 significantly change the biological activities against αvβ6 and
αvβ8. This resulted in the discovery of a pan αv antagonist (compound 39 – 4 to 40 nM for the integrin
receptors named above) possessing excellent oral pharmacokinetic properties in rats (with of
clearance 7.6 mL/min/kg and bioavailability 97%).
INTRODUCTION
It is estimated around 45% of deaths in the industrialised world are attributable to some sort of fibrotic
disease.1 One of these, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of the
interstitial lung thought to be caused by repeated injury or insult.2,3 After diagnosis, the average life
expectancy varies from three to five years depending on the severity4,5 and the mortality rate exceeds
that of many cancer indications.6,7 Current estimates suggest it is the seventh biggest killer in the UK,
killing around 5,000 people every year and there has been a six–fold increase in deaths since 1979.8
Pirfenidone and nintedanib are the only two drugs specifically approved for the treatment of IPF. Both
drugs have demonstrated to significantly slow the progression of IPF,9,10 but neither is a cure. There
is therefore, a pressing need for novel efficacious treatments for IPF.
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Integrins are a family of heterodimeric transmembrane receptors, each consisting of an α and β
subunit.11 The genome encodes eighteen α subunits and eight β subunits that heterodimerise to form
twenty four αβ integrin combinations. Of these twenty four integrins, eight (including all five αv
containing integrins: αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8) bind ligands through an arginine-glycine-
aspartic acid (RGD) sequence.12 The αvβ6 receptor is overexpressed in the lung tissue of fibrotic
patients and inhibition of the receptor with a monoclonal antibody has been shown to prevent
pulmonary fibrosis in animal models.13 Moreover, whilst mice that do not express the αvβ6 receptor
develop mild inflammation in the lungs and skin, they are protected from bleomycin induced
pulmonary fibrosis.14 The integrin αvβ1 may also play a role based on it's inhibitory effect in the
bleomycin model although the reported doses administered were large and the compound used has
been shown to inhibit other integrins.15 In contrast, there is evidence that the integrins αvβ3 and
αvβ5 are not involved in lung fibrosis16 although they may play a role in asthma and COPD.17 Obtaining
exquisite selectivity for any particular integrin can be difficult due to similarities in some of the binding
sites so importantly, pan αv inhibitors (compounds that inhibit the αv integrins namely αvβ3, αvβ5,
αvβ6 and αvβ8 within 1 log unit) such as CWHM12 (Figure 1) have also been shown to be protective
in the bleomycin fibrosis model.18 Consequently, the αv integrins and αvβ6 in particular have been
identified as a key player in the development of IPF and inhibitors could represent efficacious new
treatments for this disease. This has led to renewed interest in integrin inhibitors12,19, 20, 21 and a
selective inhaled αvβ6 small molecule GSK3008348 clinical candidate has recently been described.22
However only a few small molecule pan αv compounds have been disclosed (such as CWHM12) which
typically feature high numbers of H-bond donors/acceptors or have molecular weight in excess of 500
Daltons.12 Described here is the discovery of a pan αv compound with good oral bioavailability and
more commensurate drug-like properties.
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RESULTS AND DISCUSSION
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With the aim of developing drugs for the treatment of restenosis following percutaneous transluminal
coronary angioplasty, a research group at GlaxoSmithKline previously reported the discovery of
phenyl-butyrate-based antagonists of the vitronectin receptors αvβ3 and αvβ5 with excellent rat
pharmacokinetics following oral administration (1, Figure 1).23
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O
H
H
N
N
N
OH
N
H
NH
O
O
HO
OH
Br
CWHM12
H
N
N
O
CO2H
1
Figure 1. Structure of CWHM12 and 1
Further profiling of the racemate of 1 (analog 2) in fluorescence polarisation (FP) αvβ3, αvβ5, αvβ6
and αvβ8 binding assays (see Supporting Information) confirmed the good activity of this template for
the vitronectin receptors αvβ3 and αvβ5 (Table 1). Moreover, it demonstrated that this compound
possessed significant selectivity (>600 fold) over the integrin receptors αvβ6 and αvβ8. Cross-
screening of a selection of compounds prepared as part this phenyl-butyrate-based antagonist
programme revealed that introduction of an aniline nitrogen in the central aromatic ring and a fluorine
in the meta position of the right hand side aryl ring (3, Table 1) increased the αvβ6 binding affinity
approximately 10-fold whilst retaining the αvβ3 and αvβ5 activities. Improvement of the αvβ6 binding
affinity by substituting a similar aryl ring of a pan αv antagonist template with a meta fluorine has
already been reported.24 However, as the increase of activity in the literature example is only 2.5-fold
and it was accompanied by an increase of activity for αvβ3 and αvβ5, we postulated that the aniline
nitrogen of the central aryl ring may also contribute to the interesting structure activity relationship
(SAR) differences between 2 and 3. Finally, the increased inhibition of 3 against αvβ6 was confirmed
in a cell adhesion assay22 with potency in the region of 1 µM in contrast to the activity of 2 which is
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below the assay threshold. In this communication, we report detailed SAR around the central
aromatic ring of 3, describing how subtle changes significantly affect the αvβ3, αvβ5, αvβ6 and αvβ8
activity and selectivity profiles. Combination of a suitable central ring and the correct aryl substitution
on the right-hand side aryl ring resulted in the discovery of a pan αv antagonist with low clearance
and excellent bioavailability in rat pharmacokinetic studies which is now described.
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Table 1: Initial SAR comparing the phenylbutyrate and the phenylaminopropanoate templates (all
compounds racemic)
F
H
H
N
N
O
N
N
O
CO2H
CO2H
N
H
2
3
a
pIC50
Cmpd
αvβ6 FP
αvβ3 FP
αvβ5 FP
αvβ8 FP
αvβ6 cell adhesion
2
3
5.9 ± 0.09 (6)
7.0 ± 0.09 (6)
8.7 ± 0.08 (6)
8.7 ± 0.15 (6)
8.7 ± 0.11 (6)
8.8 ± 0.12 (6)
5.8 ± 0.08 (6)
6.4 ± 0.10 (6)
<5.0 (2)
5.9 ± 0.07 (20)b
aThe pIC50 is the negative log of the IC50. Data are reported as the mean values ± SEM (standard error
of the mean). The number in brackets refers to the number of test occasions. See the Supporting
Information for assay experimental protocols. Cell adhesion data in this assay of standard compounds
b
are described in references 12 and 22. Tested <5.0 on one occasion.
Docking of 3 into an αvβ6 crystal structure: To generate binding poses, compounds were docked into
vx X-ray crystal structures using Glide25,26,27 (Schrödinger).
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Figure 2. Docking of 3 into a crystal structure of v6 (PDB code:4UM9) – see text for commentary.
The left-hand part of the figure is the αv subunit and the right-hand part the  subunit.
The docked pose of 3 in an v6 X-ray crystal structure (PDB code:4UM928) (Figure 2) shows a
bidentate H-bond between the 1,8-tetrahydronaphthyridine and αv-Asp218. In the -subunit, the
carboxylic acid motif is coordinated with the magnesium ion (cyan sphere) in the metal ion dependent
adhesion site (MIDAS) and forms H-bonds with Ala126 and Asn218. The aniline NH forms a H-bond
with Ile219 backbone and the meta-substituted aryl group is positioned in proximity to the specificity
determining loop (the top right quadrant of the figure).
Synthesis: The antagonists from the phenylaminopropanoate templates were prepared using the
following synthetic routes. The first route involved an SNAr reaction between a heteroaromatic core
(5) with the tetrahydronaphthyridine ethyl alcohol (4) (general procedure A in SI). This was followed
by a Buchwald-Hartwig coupling reaction between the aryl halide product (6) and phenyl azetidin-2-
one (7) under palladium catalysed conditions. The resulting lactam ring (8) was then opened with
aqueous lithium hydroxide to afford the desired product (9) in three steps (Scheme 1).
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O
5
6
7
8
HN
H
Br
N
H
N
(i)
(ii)
N
N
O
N
N
N
OH
O
Br
Br
9
Cl
6
4
8
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H
N
N
Cl
O
H
(iii)
N
N
O
N
N
CO2H
N
H
9
Cl
Scheme 1. General Procedure A: Reagents and conditions: (i) NaH, DMF, 0 oC (ii) PPh3, DIAD, THF (iii)
Cs2CO3, Xantphos, Pd2(dba)3, 1,4-dioxane, 130 oC (iv) 1M LiOH, THF.
The second route involved a Mitsunobu reaction between the tetrahydronaphthyridine ethyl alcohol
(4) and heteroaromatic alcohol (10) (procedure B). The resulting heterocycle (11) was then also
coupled with lactam (7) and ring opened as before to afford the desired product (13) (Scheme 2).
Cl
H
N
HO
H
(i)
(ii)
N
N
N
O
N
N
OH
N
N
HN
Cl
N
Cl
N
N
Cl
O
4
10
11
7
H
H
N
N
N
O
O
N
(iii)
O
CO2H
N
N
N
H
12
13
Cl
Scheme 2. General Procedure B: Reagents and conditions: (i) PPh3, DIAD, THF (ii) Cs2CO3, Xantphos,
Pd2(dba)3, 1,4-dioxane, 130 oC (iii) 1M LiOH, THF.
Variations to the right-hand side aromatic ring were achieved via an alternative synthetic route.
Following a Mitsunobu reaction between the Boc protected tetrahydronaphthyridine ethyl alcohol
(14) and 6-nitropyridin-3-ol (15), and subsequent reduction, the 2-amino pyridine intermediate (16)
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was obtained. It was then refluxed to dryness in the presence of a substituted benzaldehyde (17) to
generate the imine which was then cooled and treated with (2-(tert-butoxy)-2-oxoethyl)zinc(II)
chloride to give the tert-butyl ester product (18) (procedure C). This fully protected compound could
be deprotected in the presence of aqueous acid to afford the desired product (19), or further reacted
in a cross-coupling reaction with a trifluoroborate salt (procedure D) or boronic acid29 (procedure E)
to give (20), and then deprotected to give (21) (Scheme 3).
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Br
Boc
N
Boc
N
HO
(i, ii)
(iii)
N
N
O
N
OH
N
O
NO2
NH2
O
N
H
17
14
15
16
Br
Br
O
Boc
N
H
N
N
O
N
(iv)
N
O
N
H
O
N
H
OH
18
19
Procedure D or E
Boc
(v)
R
O
R
O
H
N
(vi)
N
N
O
N
O
N
N
N
H
O
N
H
OH
20
21
Scheme 3. General Procedure C: Reagents and conditions: (i) PPh3, DIAD, THF (ii) Pd/C, H2, EtOH:EtOAc
(5:1) (iii) 120 oC, THF, then (2-(tert-butoxy)-2-oxoethyl)zinc(II) chloride (0.5M in THF), THF (iv) 4M HCl
o
in 1,4-dioxane, DCM, H2O (v) Procedure D for R-BF3K, P(tBu)3, Xphos, Cs2CO3, Pd(OAc)2, THF, 130 C.
Procedure
E
for R-B(OH)2, K3PO4, 2′-(dimethylamino)-2-biphenylyl-palladium(II) chloride
dinorbornylphosphine complex, 1,4-dioxane, H2O, 130 oC. (vi) 4M HCl in 1,4-dioxane, DCM, H2O
Structure activity relationships – central ring modifications: The modifications of the central
aromatic ring were initially investigated with a 3-Cl substituted phenyl right-hand side (R1 = Cl, Table
2) as legacy and initial SAR suggested this is a preferred group for improving v6 activity. This
observation was subsequently also reported in the literature on a β-phenylalanine template.24
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Significant differences in activity and selectivity were observed for the six 6-member heteroaromatic
analogues 9, 13, 22-25. The positions of the nitrogen(s) appeared to be crucial for the v6 potency,
with for instance, the 5-aminopyridine 9 being nearly 10-fold less potent than the 2-aminopyridine 22
(Table 2). The 5- and 2-aminopyrimidines 23 and 13 are less effective at binding v6 than the 2-
aminopyridine 22, while the aminopyrazine 25 is surprisingly nearly inactive (pIC50 4.9 but 3 values out
of 6 are below the 4.3 threshold of the assay). The aminopyridazine 24 is the most active antagonist
of this initial set of 6 compounds. The overall selectivity profiles of these molecules are all similar,
with the activities for all four αv receptors increasing or decreasing in synchrony. More specifically,
these analogues are almost equipotent between v6 and v8 and whilst a similar effect is observed
for v3 and v5, the inhibitory activities for the latter two integrins are typically at least 10-fold
higher.12 The superior activity of the 2-aminopyridine and aminopyridazine rings at binding to v3,
v5, v6 and v8 was confirmed with the data for the 4-Cl-phenyl right hand-side analogues 26-
31 (R2 = Cl, Table 2). Similar SAR data were observed for these antagonists although there is a trend
towards slightly higher selectivity for v3 and v5 over v6 suggesting, as one could have
expected, that small modifications of the right hand-side aryl do not significantly affect the binding
conformation of the central aromatic heterocycles. None of the modifications of the central aromatic
ring produced more pan-like antagonists with the potencies of the molecules always remaining higher
for v3 and v5. The increased activity of 22 and 27 for v6 compared to 9 and 26 may be related
to changes in the basicity of the pyridine ring – the calculated pKa’s (Chemaxon) are quite different
for 2-methoxy-5-methylaminopyridine (3.36) and 2-methylamino-5-methoxypyridine (5.99) and there
is literature that v6 inhibitors with a basic group in this region of the binding site are highly potent.22
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Table 2. Activity and selectivity of the different aromatic central ring analogs (all compounds racemic)
R2
R1
H
N
N
O
O
N
OH
H
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a
pIC50
5
Cmpd
Core
R1
R2
6
αvβ6 FP
αvβ3 FP
αvβ5 FP
αvβ8 FP
7
8
9
O
O
O
O
O
O
O
O
O
O
O
O
N
9
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
6.4 ± 0.26 (6)
7.3 ± 0.04 (4)
6.2 ± 0.47 (6)
6.8 ± 0.07 (6)
7.7 ± 0.04 (5)b
4.9 ± 0.07 (6)c
6.0 ± 0.28 (6)
7.4 ± 0.11 (6)
6.6 ± 0.27 (3)
6.9 ± 0.23 (3)
7.6 ± 0.05 (6)
4.7 (1)d
7.9 ± 0.31 (6)
8.2 ± 0.17 (4)
7.3 ± 0.50 (6)
8.3 ± 0.07 (6)
8.5 ± 0.04 (5)b
5.3 ± 0.27 (5)
7.4 ± 0.39 (6)
8.5 ± 0.09 (6)
8.1 ± 0.13 (3)
8.9 ± 0.18 (3)
8.7 ± 0.11 (6)
4.9 ± 0.22 (7)b
7.8 ± 0.36 (6)
8.2 ± 0.08 (4)
7.3 ± 0.45 (6)
8.3 ± 0.07 (6)
8.5 ± 0.11 (5)b
5.3 ± 0.22 (6)
7.3 ± 0.41 (6)
8.4 ± 0.10 (6)
8.2 ± 0.03 (3)
9.0 ± 0.08 (3)
8.5 ± 0.09 (6)
6.0 ± 0.33 (6)
7.1 ± 0.12 (4)
5.6 ± 0.51 (6)
6.6 ± 0.13 (6)
7.6 ± 0.08 (5)b
4.9 ± 0.05 (3)c
5.6 ± 0.22 (6)
6.9 ± 0.10 (6)
6.1 ± 0.07 (3)
7.2 ± 0.11 (3)
7.5 ± 0.08 (6)
4.5 (1)d
N
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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N
22
23
13
24
25
26
27
28
29
30
31
N
H
N
H
N
N
H
N
N
H
N
N
N
H
H
N
H
N
H
N
N
N
H
Cl
Cl
Cl
Cl
Cl
Cl
N
H
N
H
N
H
N
H
N
N
H
N
N
H
N
N
N
H
H
N
H
N
N
H
4.9 ± 0.15 (7)b
N
H
aSee the general footnotes in Table 1. Tested <4.08 on one occasion. Tested <4.3 on 3 occasions.
dTested <4.3 on 7 occasions.
b
c
Structure activity relationships – the preferred stereochemistry: The enantiomers of the most potent
analogue, the aminopyridazine 24 (referred to as 32 and 33 and depicted in Table 3) were obtained
by chiral HPLC separation of the racemic mixture.§ A small molecule crystal structure was obtained
for a DMSO solvate of single enantiomer 32.§§ The study showed the molecule to exist as a zwitterion,
with the absolute configuration unambiguously determined [(R)- at the C3 benzylic position (Figure
3)].
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22
23
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26
27
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31
32
33
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35
36
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39
40
41
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3. A molecule of 32 from its DMSO solvate crystal structure. Anisotropic atomic displacement
ellipsoids for the non-hydrogen atoms are shown at the 50% probability level. Hydrogen atoms are
displayed with an arbitrarily small radius.
§ The chiral separation method is 40% EtOH/heptane with a flow-rate = 1 mL/min on a column 4.6mm
id x 25 cm Chiralcel OJ monitoring at a wavelength of 230 nm. Isomer 1 (32) eluted ca 10.5 min and
isomer 2 (33) ca 18 min.
§§
Crystal data and refinement summary for 32 (CCDC 1919504): C23H24ClN5O3 · C2H6OS; M = 532.05;
colourless plate from the slow evaporation of a solution of 32 in DMSO; 0.74 x 0.50 x 0.03 mm;
monoclinic; space group P21 (#4); a = 9.3008(3), b = 8.5375(2), c = 16.9166(5) Å, β = 104.657(3) °, V =
1299.56(6) Å3; T = 150(2) K; Z = 2; Dcalc = 1.360 Mgm-3; λ = 0.71073 Å; θmax = 28.57 °; reflections
collected = 14493; independent reflections = 5493; Rint = 0.0267; S = 1.041; R1 [I>2σ(I)] = 0.0306; wR2
(all data) = 0.0752; Flack = 0.01(5).
The (S) enantiomer 33 is the more active configuration to bind to αvβ6 (pIC50 8.2, Table 3) and
considering the high sequence homology between αvβ6, αvβ3, αvβ5 and αvβ8, it is not surprising to
observe that it is the more active enantiomer against all four integrin receptors. These data are in
agreement with previous reports in the literature on β-phenylalanine or phenylbutyrate templates,
which also disclosed the (S) configuration as the more active enantiomer,23,24,30 suggesting that all
these antagonists bind the β subunits with similar conformations.
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Table 3: Data for the enantiomers of 24 confirming that the (S) configuration is preferred for binding
to the αv integrin receptors.
7
8
9
Cl
Cl
H
H
N
N
N
O
N
N
O
N
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12
13
14
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17
18
19
20
21
22
23
24
25
26
27
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30
31
32
33
34
35
36
37
38
39
40
41
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N
N
CO2H
CO2H
N
N
(S)
(R)
H
H
32
33
a
pIC50
Cmpd
32
αvβ6 FP
αvβ3 FP
αvβ5 FP
αvβ8 FP
6.1 ± 0.03 (5)
8.2 ± 0.09 (5)
6.8 ± 0.02 (5)
7.9 ± 0.03 (5)
8.9 ± 0.07 (5)
5.7 ± 0.04 (5)
8.1 ± 0.07 (5)
33
9.1 ± 0.06 (5)
Structure activity relationships – right hand ring substitution: To improve the overall pan αv profile,
substitutions of the right-hand aryl ring were next investigated. This ring binds in the specificity
determining loop of the β subunit where there are differences in the amino-acid residues between
the αv integrins. From Table 2 the meta-chloro 2-aminopyridine 22 and aminopyridazine 24 are the
most potent analogues across the integrins (alongside their para-chloro comparators 27 and 30). The
pyridine 22 has higher permeability through an artificial membrane (see the Supporting Information
for experimental details) than the pyridazine 24 (33 nm/s and 8.4 nm/s, respectively), which may
reflect its slightly higher lipophilicity (chrom logD pH7.4 2.74 and 2.50 for 22 and 24 respectively).22
This, together with the increased tractability of pyridines and their greater occurrence in drugs led us
to focus on exploring the right hand-side aryl ring with the 2-aminopyridine core to identify more pan
like profiles and establish the suitability of the template for oral bioavailability.
The data for 22 and 27 indicate that meta- and para-chloro substitution does not significantly affect
the activity and selectivity profiles. Reducing the size of the halogen (m-F analogue 34, Table 4) in
comparison to the chloro analogue 22 marginally reduces the αvβ6 and αvβ8 binding potencies, while
the αvβ3 activity increases slightly and αvβ5 remains unchanged. It is interesting to note that although
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the activity changes are minimal, this exact SAR profile has already been reported by Macdonald et al.
in their investigation of the substitution of the aryl ring of the β-phenylalanine motif.24 Increasing the
size of the halogen (m-Br analogue 19, Table 4) did not significantly alter the integrin activities
compared to the 22 and neither does the para substitution with the electron withdrawing trifluoro-
methyl group (analogue 35). The trifluoromethyl 35 data contrast with those reported for the same
analogue on the β-phenylalanine motif24 where it slightly decreases the αvβ3 and αvβ5 activities to
produce an unambiguous pan αv antagonist. The para phenoxy substituent 36 increases the αvβ6
potency over the para chloro substituent 27 (albeit at the expense of increased molecular weight and
lipophilicity) but the meta or para methyl morpholines 37 and 38 respectively do not systematically
improve the αvβ6 potency nor significantly change the selectivity profile. In contrast, the smaller m-
cyclopropyl analogue 39 has the best pan-like profile between the αv isoforms driven by increased
activity at the αvβ6 receptor (pIC50 = 7.8). The 3,5-bis-cyclopropyl substitution of the aryl ring is
however detrimental for the binding affinities at the receptors (40). This ring binds into the specificity
determining loop where subtle structural changes through variation in the substituents are known to
alter binding to the different αv integrins.22,24
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22
23
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Table 4. Activity and selectivity of aryl substituted analogues of 22 and 27 (all compounds racemic)
H
N
N
O
N
O
N
OH
H
a
pIC50
Com-
pound
Aryl Group
αvβ6 FP
αvβ3 FP
αvβ5 FP
αvβ8 FP
F
34
19
7.1 ± 0.06 (6)
7.5 ± 0.10 (3)
8.6 ± 0.08 (6)
8.3 ± 0.23 (3)
8.4 ± 0.08 (6)
8.4 ± 0.08 (3)
6.8 ± 0.07 (6)
Br
7.2 ± 0.16 (3)
6.7 ± 0.22 (3)
CF3
35
7.1 ± 0.13 (3)
8.1 ± 0.27 (3)
8.1 ± 0.20 (3)
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OPh
4
5
6
7
36
37
7.9 ± 0.07 (6)
7.2 ± 0.23 (3)
8.7 ± 0.09 (6)
8.4 ± 0.13 (3)
8.5 ± 0.11 (6)
8.5 ± 0.05 (3)
7.1 ± 0.14 (6)
7.2 ± 0.05 (3)
N
O
8
9
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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30
31
32
33
34
35
36
37
38
39
40
41
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N
38
7.5 ± 0.04 (6)
8.5 ± 0.08 (6)
8.2 ± 0.07 (6)
6.9 ± 0.08 (6)
39
40
7.8 ± 0.09 (4)
6.5 ± 0.51 (3)
8.4 ± 0.11 (4)
6.9 ± 0.40 (3)
8.4 ± 0.10 (4)
7.0 ± 0.49 (3)
7.4 ± 0.11 (4)
6.9 ± 0.72 (3)
aSee the general footnotes in Table 1.
Overall, 39 has the best profile for a pan αv antagonist from this series of compounds being more pan-
like and with better physicochemical properties than 36. The increase in activity for the αvβ6 receptor
compared to the original lead 2 (Table 1) was confirmed in a αvβ6 cell adhesion assay, with 39 having
a pIC50 of 6.9 ± 0.2 (6) (tested <5.0 on one occasion).
Docking of 39 into αvβ6 and αvβ3 crystal structures: Similarly to as described above, 39 was docked
into the crystal structures of αvβ6 and αvβ3 (alongside 3 for the latter for comparison). The ligand
interactions of 39 in the binding site of αvβ6 (Figure 4) are essentially as described for 3 except for the
meta-substituted cyclopropyl group on the aryl ring which is close to lipophilic residues (Ile183,
Ala217) of the specificity determining loop (the top right quadrant of the figure).
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Figure 4. Docking of 39 into a crystal structure of v6 (PDB code:4UM9) – see text for commentary.
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Figure 5. The binding modes of 3 (orange) and 39 (cyan) docked into an v3 X-ray crystal structure
(PDB code:1L5G) See text for commentary.
The predicted binding modes of 3 (orange) and 39 (cyan) in an v3 X-ray crystal structure (PDB
code:1L5G31) are shown in Figure 5.
For both ligands, H-bonds between the 1,8-
tetrahydronaphthyridine and αv-Asp218, and acid coordination with a manganese ion of the MIDAS
and H-bond with Asn215 backbone are consistent. Due to the salt-bridge between Asp179 and
Arg214, the meta-substituted fluoro and cyclopropyl groups on the aryl ring are directed away from
these larger side-chain residues due to the reduction in the -subunit binding site size (compared with
v6).
Oral pharmacokinetics The artificial membrane permeability of 39 is 80 nm/s, suggesting that it had
the potential to be absorbed following oral administration. In fact, in vivo pharmacokinetic studies32
in CD rats revealed that racemic 39 has a low plasma clearance of 8 mL/min/kg, a low to moderate
volume of distribution of 0.5 L/kg, a moderate half-life of 1.8 h and very high bioavailability of 98%
(Table 5). These pharmacokinetic data are similar to that reported for the lead compound 1 from the
phenylbutyrate template,23 indicating that the improvement in αvβ6 and αvβ8 activities obtained by
modifying the central aromatic ring and the substitution of the right hand-side aryl have not been
obtained at the expense of good oral profile.
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Table 5. Rat pharmacokinetic data for 39a
5
6
7
8
9
Cl
Vss
T1/2
Cmpd
Oral F (%)
(h)
(mL/min/kg)
(L/kg)
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
39
8
0.5
1.8
98
aSee reference 22 for typical experimental details.
Conclusion
In conclusion, we have described how small structural changes on a phenylbutyrate template - mainly
based on the aromatic central core - affect the activity and selectivity for the αvβ3, αvβ5, αvβ6 and
αvβ8 integrin receptors. With the aim of targeting a pan αv profile for the treatment of IPF, we have
improved the αvβ6 and αvβ8 activities at these receptors by ~100 fold compared with 2, without
compromising on the good oral pharmacokinetic profile. Indeed, the new lead compound 39 from
this series possess binding activities at all four integrin receptors between 4 and 40 nM as well as low
clearance and an excellent bioavailability (98 %) in rats. Although our target here is IPF, a pan αv
antagonist has already been reported18 as being efficacious in a number of models of fibrotic diseases.
Nevertheless, current studies are aimed at improving the overall profile and the results of our
investigations in this new area will be reported in due course.
EXPERIMENTAL SECTION
Assays:
Artificial membrane permeability assay
This technique measures the permeability of a compound in a phospholipid bilayer system. The lipid
is egg phosphatidyl choline (1.8%) and cholesterol (1%) dissolved in n-decane. This is applied to the
bottom of the microfiltration filter inserts in a Transwell plate. Phosphate buffer (50 mM Na2HPO4
with 0.5% 2-hydroxypropyl-b-cyclodextrin), pH 7.4 is added to the top and bottom of the plate. The
lipids are allowed to form bilayers across the small holes in the filter. Permeation experiment is
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initiated by adding the compound to the bottom well and stopped at a pre-determined elapsed time.
The compound permeates through the membrane to enter the acceptor well. The compound
concentration in both the donor and acceptor compartments is determined by liquid chromatography
after 3 hours incubation at room temperature. The permeability (logPapp) measuring how fast
molecules pass through the black lipid membrane is expressed in nm/s. See Veber D.F.; Johnson S.R.;
Chen H-Y.; Smith B. R.; Ward K. W.; Kopple D. Molecular properties that influence the oral
bioavailability of drug candidates, J. Med. Chem. 2002, 45, 2615-2623; and Kansy M, Senner F,
Gubernator K. Physicochemical high throughput screening: parallel artificial membrane permeation
assay in the description of passive absorption processes. J. Med. Chem. 1998, 41, 1007–1010.
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60
Fluoresence polarization assays
All purified soluble protein preparations (recombinantly derived from Chinese Hamster Ovary cells)
for the human αvβ1, αvβ3, αvβ5, αvβ6 and αvβ8 integrin proteins used in fluorescence polarization
(FP) assays were purchased from R&D Systems Inc. (Minneapolis, MN, USA). Briefly, FP assays were
performed in low volume 384-well plates (Greiner Bio-One, Firckenhausen, Germany) at room
temperature (20-22°C) in assay buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid,
150 mM NaCl and 1 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at pH 7.4
(NaOH). Assay buffer was supplemented with 0.1 mM MnCl2 for αvβ1/αvβ5/αvβ8 and 0.4 mM MgCl2
for αvβ3/αvβ6. Experiments were completed in a total volume of 6.1 µL consisting of 0.1 µL/well of
either compound at varying concentrations or vehicle (dimethylsulfoxide), 3 µL/well of purified
integrin (final assay concentration (FAC) of 2 nM αvβ1, 1 nM αvβ3, 4 nM αvβ5 or 8 nM αvβ6/αvβ8)
and 3 µL/well of Cy3B-RGD probe (1 nM FAC) (Fig. 6). Compounds were pre-incubated with integrin
protein for 15 mins prior to addition of Cy3B-RGD probe. FP was then measured using an EnVision
multilabel plate reader (PerkinElmer LAS UK Ltd., Beaconsfield, UK) following a further 2 h incubation.
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N
O
O
S
OH
O
H3C
OH
CH3
N
N
or1
CH3
CH3
N
O
N
H
N
O
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H
N
N
O
O
Figure 6: Cy3B-RGD probe used in fluorescence polarization experiments.
In-vivo rat pharmacokinetics
The intravenous pharmacokinetics of 39 was investigated in two male CD rats (BW: 288-312g) at 1
mg/kg. The compound was dissolved in DMSO:PEG200:water (5:45:50 v/v/v) at a concentration of 1
mg/mL and dosed as a bolus via the femoral vein. Blood samples were collected in heparinised tubes
up to 7h via a tail vein canula and with additional sampling up to 24h via direct tail venipuncture.
The oral pharmacokinetics of 39 was investigated in two male CD rats (BW: 303-308g) at 1 mg/kg. The
compound was dissolved in 0.5% aq. Methyl cellulose containing 0.2% Tween 80 at a concentration of
0.1 mg/mL and dosed via oral gavage. Blood samples were collected up to 7h via a tail vein canula and
with additional sampling up to 24h via direct tail venipuncture.
Blood samples from both arms were centrifuged to obtain plasma, which was stored frozen at -20 oC
prior to analysis. Plasma samples were mixed with acetonitrile containing internal standard and
centrifuged. Supernatants were evaporated to dryness under nitrogen and reconstituted in
acetonitrile:water 10:90 (v/v). The concentration of 39 was determined by LC-MS/MS.
Synthetic details
General Methods: All solvents were of analytical grade, purchased from Sigma-Aldrich in anhydrous
form. Unless otherwise stated, reagents were purchased from the following suppliers: Sigma-Aldrich,
Lancaster, Fluorochem, TCI and used without further purification. NMR Spectroscopy: 1H NMR and 13C
NMR spectra were recorded on Bruker DPX-400 spectrometers at either 500, 400 or 100 MHz.
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Chemical shifts (δ) are reported in parts per million (ppm) and referenced to the residual solvent peak.
Coupling constants (J) are measured in hertz (Hz). Advanced experiments were recorded on an AVC-
600 spectrometer and were obtained by Sean Lynn or Stephen Richards of the Analytical Chemistry
Department, GlaxoSmithKline, Stevenage. Mass Spectrometry: High resolution mass spectra (HRMS)
were recorded on a Micromass Autospec 500 OAT spectrometer. HRMS was recorded by Bill Leavens,
Analytical Chemistry Department, GlaxoSmithKline, Stevenage. Mass spectra (LC-MS) were recorded
using one of five methods:
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1) Acq~2min_TFA:
TFA Generic Analytical UPLC Open Access LC/MS 2 Minute Method. The UPLC analysis was conducted
on an Acquity UPLC BEH C18 column (50 mm x 2.1 mm i.d. 1.7 μm packing diameter) at 40 °C. The
solvents employed were: A = 0.1% trifluoroacetic acid in H2O, B 0.1% trifluoroacetic acid in MeCN.
2) Acq~2min_For
Formic Acid Generic Analytical UPLC Open Access LC/MS 2 Minute Method. The UPLC analysis was
conducted on an Acquity UPLC BEH C18 column (50 mm x 2.1 mm i.d. 1.7 μm packing diameter) at 40
°C. The solvents employed were: A = 0.1% v/v solution of formic acid in H2O, B = 0.1% v/v solution of
formic acid in MeCN.
3) XBR~5min_HpH
High pH Generic Analytical HPLC Open Access LC/MS 5 Minute Method. The HPLC analysis was
conducted on an XBridge C18 column (50 mm x 4.6 mm i.d. 3.5 μm packing diameter) at 30 °C. The
solvents employed were: A = 10 mM ammonium bicarbonate in water adjusted to pH 10 with
ammonia solution, B = Acetonitrile.
4) Sun~5min_For
Formic Acid Generic Analytical HPLC Open Access LC/MS 5 Minute Method. The HPLC analysis was
conducted on an Sunfire C18 column (50 mm x 4.6 mm i.d. 3.5 μm packing diameter) at 30 °C. The
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solvents employed were: A = 0.1% v/v solution of formic acid in H2O, B = 0.1% v/v solution of formic
acid in MeCN.
7
8
9
5) Sun~5min_TFA
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TFA Generic Analytical HPLC Open Access LC/MS 5 Minute Method. The HPLC analysis was conducted
on an Sunfire C18 column (50 mm x 4.6 mm i.d. 3.5 μm packing diameter) at 30 °C. The solvents
employed were: A = 0.1% trifluoroacetic acid in H2O, B 0.1% trifluoroacetic acid in MeCN.
Synthetic procedures
All compounds ≥ 95% purity by LC-MS and 1H NMR unless otherwise stated.
General procedure A: SNAr, Buchwald-Hartwig cross coupling, hydrolysis
7-(2-((5-Bromopyridin-2-yl)oxy)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine (6) To a stirred solution
o
of 2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethanol (300 mg, 1.683 mmol) in DMF (5 mL) at 0 C
and under N2 was added NaH (60% dispersion in mineral oil, 81 mg, 2.020 mmol) and the reaction
mixture was stirred at 0 oC for 30 mins. The reaction mixture was warmed to rt and 2,5-
dibromopyridine (478 mg, 2.020 mmol) was added. The reaction mixture was then stirred at rt and
under N2 for a further 19 h. Further NaH (60% dispersion in mineral oil, 81 mg, 2.020 mmol) was added
and the reaction mixture stirred for a further 4 h. The reaction mixture was partitioned between DCM
(15 ml) and H2O (15 mL) the layers were separated and the aq. layer washed with further DCM (15
mL). The combined organic phases were dried by passing through a hydrophobic frit and concentrated
in vacuo to afford the crude product as a brown solid. The sample was purified by silica
chromatography, eluting with EtOAc:cyclohexane (0 - 100% EtOAc). The appropriate fractions were
1
combined and concentrated in vacuo to give the title compound (314 mg, 56%) as a white solid. H
NMR (400 MHz, DMSO) δ ppm = 8.27 (d, J=2.5 Hz, 1H), 7.87 (dd, J=2.8, 8.8 Hz, 1H), 7.05 (d, J=7.3 Hz,
1H), 6.78 (d, J=8.8 Hz, 1H), 6.36 - 6.31 (m, 2H), 4.50 (t, J=6.9 Hz, 2H), 3.27 - 3.20 (m, 2H), 2.88 (t, J=6.9
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Hz, 2H), 2.61 (t, J=6.3 Hz, 2H), 1.75 (quin, J=5.9 Hz, 2H). LC-MS (Acq~2min_For) tR = 0.80 min, [M+H+]
= 334/336.
7
8
9
3-(3-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-3-
yl)amino)propanoic acid (9) A solution of 7-{2-[(5-bromo-2-pyridinyl)oxy]ethyl}-1,2,3,4-tetrahydro-
1,8-naphthyridine (100 mg, 0.299 mmol), 4-(3-chlorophenyl)-2-azetidinone (65.2 mg, 0.359 mmol),
Cs2CO3 (292 mg, 0.898 mmol), Xantphos (17.31 mg, 0.030 mmol) and Pd2(dba)3 (13.70 mg, 0.015
mmol) in 1,4-Dioxane (5 mL) was heated in a Biotage Initiator microwave at 130 °C for 30 min. The
reaction mixture was partitioned between DCM (15 mL) and H2O (15 mL), the layers were separated
and the aq. layer washed with further DCM (15 ml). The combined organic phases were then dried
through a hydrophobic frit and concentrated in vacuo to afford a yellow oil. The sample was dissolved
in THF (3 mL) and 1M aq. LiOH (3 mL, 3.00 mmol) was added. The reaction mixture was stirred at rt
for 16 h. The reaction mixture was then concentrated in vacuo before being dissolved in MeOH:DMSO
(1:1, 1 mL) and purified by mass directed autoprep on Sunfire C18 column using MeCN:H2O with a
formic acid modifier. The solvent was dried under a stream of N2 in the Radleys blowdown apparatus
to give the title compound (43 mg, 32%) as an orange solid. 1H NMR (400 MHz, DMSO) δ ppm = 7.50
(s, 1H), 7.42 - 7.37 (m, 2H), 7.32 (t, J=7.8 Hz, 1H), 7.27 - 7.23 (m, 1H), 7.04 - 6.99 (m, 2H), 6.49 (d, J=8.8
Hz, 1H), 6.34 - 6.31 (m, 1H), 6.29 (d, J=7.3 Hz, 1H), 6.05 (br. s., 1H), 4.74 (t, J=6.7 Hz, 1H), 4.31 (t, J=6.9
Hz, 2H), 3.25 - 3.19 (m, 3H), 2.79 (t, J=6.9 Hz, 2H), 2.75 - 2.56 (m, 4H), 1.78 - 1.70 (m, 2H). LC-MS
(Acq~2min_For) tR = 0.79 min, [M+H+] = 453.
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General procedure B: Mitsunobo, Buchwald-Hartwig cross coupling, hydrolysis
7-(2-((2-chloropyrimidin-5-yl)oxy)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine (11) To a solution of
2-chloro-5-pyrimidinol (1.266 g, 9.70 mmol) and 2-(5,6,7,8-Tetrahydro-1,8-naphthyridin-2-yl)ethan-1-
ol (1.73 g, 9.70 mmol) in THF (5 mL) at 0 oC and under N2 was added triphenylphosphine (2.54 g, 9.70
mmol) and DIAD (1.886 mL, 9.70 mmol) and the reaction mixture stirred for 18 h during which time it
warmed to rt. The reaction was then partitioned between DCM (100 mL) and H2O (100 mL), the aq.
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phase was washed with further DCM (100 mL) and the combined organics concentrated in vacuo to
give the crude product. This was purified by silica chromatography, eluting with EtOAc:cyclohexane (0
- 100% EtOAc), then MeOH in DCM (0 - 15% MeOH). The appropriate fractions were concentrated in
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vacuo to give the target compound (680 mg, 24%). H NMR (400 MHz, CDCl3) δ ppm = 8.32 (s, 2H),
7.30 (d, J=7.3 Hz, 1H), 7.25 (br. s., 1H), 6.49 (d, J=7.3 Hz, 1H), 4.50 (t, J=6.1 Hz, 2H), 3.52 - 3.46 (m, 2H),
3.19 (t, J=6.1 Hz, 2H), 2.76 (t, J=6.1 Hz, 2H), 2.00 - 1.90 (m, 2H). LC-MS (Acq~2min_For) tR = 0.59 min,
[M+H+] = 291
3-(3-chlorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyrimidin-2-
yl)amino)propanoic acid (13) In a microwave vial was 7-{2-[(2-chloro-5-pyrimidinyl)oxy]ethyl}-1,2,3,4-
tetrahydro-1,8-naphthyridine (100 mg, 0.344 mmol), 4-(3-chlorophenyl)-2-azetidinone (75.0 mg,
0.413 mmol), Pd2(dba)3 (15.75 mg, 0.017 mmol), cesium carbonate (336 mg, 1.032 mmol) and
Xantphos (19.90 mg, 0.034 mmol) in 1,4-Dioxane (5 mL) and the reaction mixture was heated to 130
°C for 30 min. The reaction mixture was then partitioned between DCM (20 mL) and H2O (20 mL), the
aq. phase was separated and washed with further DCM (20 mL). The combined organic phases were
then concentrated in vacuo to give an orange gum. This was dissolved in THF:H2O (6 mL, 1:1) and aq.
lithium hydroxide (3 mL, 3.00 mmol) was added and the mixture stirred at 20 °C for 72 h. The reaction
mixture was then concentrated in vacuo and purified by mass directed autoprep on Sunfire C18
column using MeCN:H2O with a TFA modifier. The solvent was dried under a stream of N2 in the
Radleys blowdown apparatus to give the title compound (5.7 mg, 4%) as a yellow gum. 1H NMR (400
MHz, MeOD) δ ppm = 8.07 (s, 2H), 7.61 (d, J=7.3 Hz, 1H), 7.44 - 7.42 (m, 1H), 7.37 - 7.33 (m, 1H), 7.29
(t, J=7.7 Hz, 1H), 7.25 - 7.20 (m, 1H), 6.74 (d, J=7.5 Hz, 1H), 5.40 (dd, J=6.0, 8.0 Hz, 1H), 4.27 (t, J=6.0
Hz, 2H), 3.54 - 3.49 (m, 2H), 3.13 (t, J=6.0 Hz, 2H), 2.95 - 2.77 (m, 4H), 2.00 - 1.92 (m, 2H). two
exchangeable protons not observed. LC-MS (XBR~5min_HpH) tR = 2.14 min, [M+H+] = 454
General procedure C: Mitsunobu, reduction, condensation, organo zinc addition, hydrolysis
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Tert-butyl 7-(2-((6-aminopyridin-3-yl)oxy)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate
(16) To a solution of 6-nitro-3-pyridinol (2g, 14.28 mmol) and 1,1-dimethylethyl 7-(2-hydroxyethyl)-
3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (3.97 g, 14.28 mmol) in THF (50 mL) under N2 at 0 oC
was added DIAD (4.16 mL, 21.41 mmol) and triphenylphosphine (5.62 g, 21.41 mmol) and the reaction
mixture was stirred for 18 h. The reaction mixture was then concentrated in vacuo and partitioned
between DCM (100 mL) and H2O (100 mL). The aq. phase was washed with further DCM (100 mL) and
the combined organics were concentrated in vacuo and purified by silica chromatography, eluting with
EtOAc:cyclohexane (0 – 60 % EtOAc). The appropriate fractions were concentrated in vacuo to give a
yellow soild. The solid was dissolved in EtOH (100 mL) and EtOAc (20 mL), 10% Pd/C (1.519g, 1.428
mmol) was added and the reaction mixture stirred under an atmosphere of H2 for 18 h. The reaction
was then filtered through celite and concentrated in vacuo to give an orange oil. The oil was then
purified by silica chromatography, eluting with EtOAc:cyclohexane (0 – 100 % EtOAc) then with
DCM:MeOH (0 - 25% MeOH). The appropriate fractions were concentrated in vacuo to give the target
compound (2.1 g, 40%) as a yellow oil. 1H NMR (400 MHz, DMSO) δ ppm = 7.62 (d, J=3.0 Hz, 1H), 7.44
(d, J=7.5 Hz, 1H), 7.09 (dd, J=3.0, 8.8 Hz, 1H), 6.99 (d, J=7.5 Hz, 1H), 6.40 (d, J=8.8 Hz, 1H), 5.44 (s, 2H),
4.23 (t, J=6.8 Hz, 2H), 3.66 - 3.61 (m, 2H), 3.02 (t, J=6.8 Hz, 2H), 2.70 (t, J=6.7 Hz, 2H), 1.82 (quin, J=6.3
Hz, 2H), 1.43 (s, 9H) LC-MS (XBR~5mins_HpH) tR = 2.52 mins, [M+H+] = 371
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Tert-butyl 7-(2-((6-((1-(3-bromophenyl)-3-(tert-butoxy)-3-oxopropyl)amino)pyridin-3-yl)oxy)ethyl)-
3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate
(18)
1,1-dimethylethyl
7-{2-[(6-amino-3-
pyridinyl)oxy]ethyl}-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (500 mg, 1.350 mmol) and 3-
bromobenzaldehyde (0.157 mL, 1.350 mmol) were dissolved in THF (5 mL) and the reaction mixture
was then evaporated to dryness at 120 oC for 2 h. The reaction mixture was then cooled to rt and re-
dissolved in THF (15 mL), to this was added (2-(tert-butoxy)-2-oxoethyl)zinc(II) chloride (0.5 M in THF,
8.10 mL, 4.05 mmol) and the mixture stirred at rt for 18 h. The reaction mixture was then concentrated
in vacuo and re-dissolved in DCM (50 mL) and washed with sat. aq. NH4Cl (50 mL), the aq. phase was
separated and washed with further DCM (50 mL), the combined organic fractions were then
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concentrated in vacuo and purified by silica chromatography, eluting with EtOAc:cyclohexane (0 - 50
% EtOAc), the appropriate fractions were concentrated in vacuo to give the target compound (650 mg,
66%) as a yellow oil. 1H NMR (400 MHz, DMSO) δ ppm = 7.67 (d, J=2.8 Hz, 1H), 7.57 (s, 1H), 7.46 - 7.36
(m, 3H), 7.26 (d, J=7.5 Hz, 1H), 7.10 (dd, J=3.0, 9.0 Hz, 1H), 6.97 (d, J=7.5 Hz, 1H), 6.73 (d, J=8.8 Hz, 1H),
6.47 (d, J=8.8 Hz, 1H), 4.22 (t, J=6.8 Hz, 2H), 3.65 - 3.60 (m, 2H), 2.99 (t, J=6.7 Hz, 2H), 2.79 - 2.56 (m,
5H), 1.81 (quin, J=6.3 Hz, 2H), 1.39 (s, 9H), 1.28 (s, 9H) LCMS (XBR~5mins_HpH) tR = 3.86 mins, [M+H+]
= 653 / 655
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3-(3-Bromophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (19) To a solution of tert-butyl 7-(2-((6-((1-(3-bromophenyl)-3-(tert-butoxy)-
3-oxopropyl)amino)pyridin-3-yl)oxy)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (44 mg,
0.067 mmol) in DCM (2 mL) was added 4M HCl in 1,4-dioxane (0.5 ml, 2.000 mmol) and two drops of
H2O and the reaction mixture stirred at rt for 18 h. The reaction mixture was then concentrated in
vacuo and dissolved in MeOH:DMSO (1:1, 1 mL) and purified by mass directed autoprep on Xbridge
column using MeCN:H2O with an ammonium carbonate modifier. The solvent was dried under a
stream of N2 in the Radleys blowdown apparatus to give the title compound (17.7 mg, 53%). 1H NMR
(400 MHz, MeOD) δ ppm = 7.62 - 7.56 (m, 2H), 7.42 - 7.37 (m, 1H), 7.36 - 7.30 (m, 1H), 7.21 (dd, J=7.7,
10.9 Hz, 2H), 7.10 (dd, J=3.0, 9.0 Hz, 1H), 6.48 (d, J=7.3 Hz, 1H), 6.42 (d, J=9.0 Hz, 1H), 5.08 (t, J=6.9 Hz,
1H), 4.14 (t, J=6.5 Hz, 2H), 3.42 - 3.35 (m, 3H), 2.94 (t, J=6.5 Hz, 2H), 2.72 (t, J=6.1 Hz, 2H), 2.67 (d, J=7.0
Hz, 2H), 1.92 - 1.85 (m, 2H). Two exchangeable protons not observed, LC-MS (XBR~5mins_HpH) tR =
2.22 mins, [M+H+] = 497 / 499
3-(3-Chlorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (22), Prepared by general procedure C using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, CDCl3) δ ppm = 8.47 (br. s., 1H), 7.62 (br. s., 1H), 7.42 (s, 1H), 7.35 - 7.17 (m,
4H), 7.11 - 7.04 (m, 1H), 6.36 (d, J=7.3 Hz, 1H), 6.22 (d, J=9.0 Hz, 1H), 4.88 (t, J=6.4 Hz, 1H), 4.19 - 4.09
(m, 2H), 3.45 (t, J=5.0 Hz, 2H), 3.06 (t, J=5.4 Hz, 2H), 2.84 - 2.75 (m, 2H), 2.72 (t, J=6.0 Hz, 2H), 1.96 -
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1.86 (m, 2H), two exchangeable protons not observed, LC-MS (XBR~5min_HpH) tR = 2.2 min, [M+H+]
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yl)amino)propanoic acid (23), prepared by general procedure C using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 7.93 (s, 2H), 7.53 - 7.51 (m, 1H), 7.42 - 7.38 (m, 1H), 7.34 (t,
J=7.8 Hz, 1H), 7.29 - 7.25 (m, 1H), 7.03 (d, J=7.3 Hz, 1H), 6.34 - 6.27 (m, 2H), 4.84 - 4.76 (m, 1H), 4.37
(t, J=6.9 Hz, 2H), 3.27 - 3.17 (m, 4H), 2.81 (t, J=6.9 Hz, 2H), 2.76 - 2.64 (m, 2H), 2.60 (t, J=6.4 Hz, 2H),
1.74 (quin, J=5.9 Hz, 2H), LC-MS (Acq~2min_For) tR = 0.76 min, [M+H+] = 454
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3-(3-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridazin-3-
yl)amino)propanoic acid (24), prepared by general procedure C using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, CDCl3) δ ppm = 10.58 (br. s., 1H), 8.41 (s, 1H), 7.45 - 7.41 (m, 1H), 7.35 - 7.31
(m, 1H), 7.26 - 7.21 (m, 2H), 7.19 (d, J=9.3 Hz, 1H), 6.71 - 6.65 (m, 2H), 6.34 (d, J=7.3 Hz, 1H), 5.24 (dd,
J=4.4, 8.4 Hz, 1H), 4.59 - 4.46 (m, 2H), 3.44 (t, J=5.5 Hz, 2H), 3.17 - 3.03 (m, 2H), 2.86 - 2.74 (m, 2H),
2.71 (t, J=6.0 Hz, 2H), 1.94 - 1.86 (m, 2H), one exchangeable proton not observed, LC-MS
(SUN~5min_For) tR = 2.14 min, [M+H+] = 454
3-(3-Chlorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyrazin-2-
yl)amino)propanoic acid (25), prepared by general procedure C using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 7.47 (d, J=3.0 Hz, 1H), 7.46 (s, 1H), 7.37 - 7.25 (m, 3H), 7.24
(d, J=3.3 Hz, 1H), 7.08 - 7.02 (m, 2H), 6.42 - 6.38 (m, 2H), 5.49 - 5.42 (m, 1H), 4.55 (t, J=7.2 Hz, 2H), 3.24
(t, J=5.4 Hz, 2H), 3.01 - 2.93 (m, 3H), 2.80 (dd, J=6.0, 15.8 Hz, 1H), 2.62 (t, J=6.1 Hz, 2H), 1.75 (quin,
J=5.8 Hz, 2H), one exchangeable proton not observed, under H2O peak. LC-MS (Acq~2min_For) tR =
2.14 min, [M+H+] = 454
3-(4-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-3-
yl)amino)propanoic acid (26), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 7.45 - 7.42 (m, 2H), 7.39 - 7.33 (m, 3H), 7.04 - 6.97 (m, 2H),
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6.48 (d, J=8.8 Hz, 1H), 6.32 (br. s., 1H), 6.29 (d, J=7.3 Hz, 1H), 6.04 (br. s., 1H), 4.72 (t, J=6.9 Hz, 1H),
4.31 (t, J=6.9 Hz, 2H), 3.60 - 3.30 (m, 2H), 2.78 (t, J=7.0 Hz, 2H), 2.75 - 2.66 (m, 1H), 2.64 - 2.55 (m, 3H),
1.74 (quin, J=5.8 Hz, 2H), one exchangeable proton not observed, under H2O peak, LC-MS
(SUN~5min_For) tR = 1.70 min, [M+H+] = 453
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3-(4-Chlorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (27), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 13.58 (br. s., 1H), 12.32 (br. s., 1H), 8.04 (br. s., 1H), 7.63 (d,
J=3.0 Hz, 1H), 7.61 (d, J=7.5 Hz, 1H), 7.41 (d, J=8.0 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 7.29 (br. s., 1H), 6.70
(d, J=7.3 Hz, 1H), 6.63 - 6.67 (m, 1H), 5.19 - 5.27 (m, 1H), 4.18 (t, J=6.1 Hz, 2H), 3.40 (t, J=6.0 Hz, 2H),
3.06 (t, J=6.0 Hz, 2H), 2.78 (dd, J=16.1, 8.5 Hz, 1H), 2.73 (t, J=6.0 Hz, 2H), 2.70 (dd, J=16.0, 5.5 Hz, 1H),
1.81 (quin, J=6.0 Hz, 2H), LC-MS (Sun~5min_TFA) tR = 1.84 min, [M+H+] = 453
3-(4-Chlorophenyl)-3-((2-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyrimidin-5-
yl)amino)propanoic acid (28), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, CDCl3) δ ppm = 10.89 (br. s., 1H), 7.95 (s, 2H), 7.37 - 7.31 (m, 2H), 7.30 - 7.21
(m, 4H), 6.47 (d, J=7.0 Hz, 1H), 4.65 - 4.51 (m, 3H), 3.42 (t, J=5.1 Hz, 2H), 3.18 - 3.12 (m, 2H), 2.77 - 2.66
(m, 3H), 2.65 - 2.54 (m, 1H), 1.93 - 1.84 (m, 2H), LC-MS (Acq~2min_For) tR = 0.71 min, [M+H+] = 454
3-(4-Chlorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyrimidin-2-
yl)amino)propanoic acid (29), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, MeOD) δ ppm = 8.32 (s, 2H), 8.01 (s, 2H), 7.45 (d, J=7.3 Hz, 1H), 7.38 (d, J=8.3
Hz, 2H), 7.28 - 7.23 (m, 2H), 6.61 (d, J=7.3 Hz, 1H), 5.38 (t, J=7.0 Hz, 1H), 4.22 (t, J=6.0 Hz, 2H), 3.47 -
3.41 (m, 2H), 3.07 (t, J=6.1 Hz, 2H), 2.89 - 2.73 (m, 4H), 1.95 - 1.88 (m, 2H), one exchangeable proton
not observed, LC-MS (SUN~5min_For) tR = 1.55 min, [M+H+] = 454
3-(4-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridazin-3-
yl)amino)propanoic acid (30), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
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one. H NMR (400 MHz, MeOD) δ ppm = 12.53 (br. s, 1H), 8.15 (s, 1H), 7.44 - 7.33 (m, 4H), 7.08 (d,
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J=7.8 Hz, 1H), 7.04 (d, J=7.3 Hz, 1H), 6.93 - 6.88 (m, 1H), 6.84 - 6.80 (m, 1H), 6.37 (br. s., 1H), 6.32 (d,
J=7.3 Hz, 1H), 5.35 - 5.27 (m, 1H), 4.47 (t, J=6.9 Hz, 2H), 3.63 - 3.30 (m, 1H), 2.89 - 2.76 (m, 3H), 2.71 -
2.64 (m, 1H), 2.61 (t, J=6.1 Hz, 2H), 1.74 (quin, J=5.9 Hz, 2H), LC-MS (Acq~2min_For) tR = 0.62 min,
[M+H+] = 454
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yl)amino)propanoic acid (31), prepared by general procedure C using 4-(4-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 7.46 (d, J=3.3 Hz, 1H), 7.43 - 7.31 (m, 4H), 7.23 (d, J=3.0 Hz,
1H), 7.06 (d, J=7.3 Hz, 1H), 6.98 (d, J=8.5 Hz, 1H), 6.39 (d, J=7.0 Hz, 2H), 5.48 - 5.41 (m, 1H), 4.54 (t,
J=7.2 Hz, 2H), 3.27 - 3.22 (m, 2H), 3.01 - 2.90 (m, 4H), 2.82 - 2.74 (m, 1H), 2.62 (t, J=6.3 Hz, 2H), 1.75
(quin, J=5.8 Hz, 2H), LC-MS (Acq~2min_For) tR = 0.78 min, [M+H+] = 454
(R)-3-(3-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridazin-3-
yl)amino)propanoic acid (32), prepared by general procedure A using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 12.32 (br. s., 1H), 7.44 (s, 1H), 7.39 - 7.31 (m, 2H), 7.30 - 7.25
(m, 1H), 7.11 (d, J=8.0 Hz, 1H), 7.04 (d, J=7.3 Hz, 1H), 6.95 - 6.89 (m, 1H), 6.86 - 6.80 (m, 1H), 6.37 (br.
s., 1H), 6.32 (d, J=7.3 Hz, 1H), 5.37 - 5.28 (m, 1H), 4.48 (t, J=6.9 Hz, 2H), 3.26 - 3.20 (m, 2H), 2.86 (t,
J=6.9 Hz, 2H), 2.84 - 2.67 (m, 2H), 2.61 (t, J=6.1 Hz, 2H), 1.74 (quin, J=5.8 Hz, 2H), LC-MS
(XBR~5mins_HpH) tR = 2.12 mins, [M+H+] = 454
(S)-3-(3-Chlorophenyl)-3-((6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridazin-3-
yl)amino)propanoic acid (33), prepared by general procedure A using 4-(3-chlorophenyl)azetidin-2-
one. 1H NMR (400 MHz, DMSO) δ ppm = 12.29 (br. s., 1H), 7.44 (s, 1H), 7.38 - 7.31 (m, 2H), 7.30 - 7.25
(m, 1H), 7.11 (d, J=8.3 Hz, 1H), 7.04 (d, J=7.3 Hz, 1H), 6.94 - 6.90 (m, 1H), 6.85 - 6.81 (m, 1H), 6.36 (br.
s., 1H), 6.32 (d, J=7.3 Hz, 1H), 5.36 - 5.28 (m, 1H), 4.48 (t, J=6.9 Hz, 2H), 3.26 - 3.20 (m, 2H), 2.86 (t,
J=6.8 Hz, 2H), 2.83 - 2.66 (m, 2H), 2.61 (t, J=6.1 Hz, 2H), 1.74 (quin, J=5.8 Hz, 2H), LC-MS
(XBR~5mins_HpH) tR = 2.12 mins, [M+H+] = 454.
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(S)-3-(3-Fluorophenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (34), prepared by general procedure C using 4-(3-fluorophenyl)azetidin-2-
one. 1H NMR (400 MHz, CDCl3) δ ppm = 10.13 (br. s, 1H), 7.61 - 7.57 (m, 1H), 7.36 - 7.27 (m, 3H), 7.21
(d, J=7.8 Hz, 1H), 7.17 - 7.11 (m, 1H), 7.00 - 6.92 (m, 1H), 6.41 (d, J=7.3 Hz, 1H), 6.38 (d, J=9.5 Hz, 1H),
4.90 (dd, J=4.6, 8.9 Hz, 1H), 4.22 - 4.13 (m, 2H), 3.52 - 3.46 (m, 2H), 3.11 (t, J=5.9 Hz, 2H), 2.94 - 2.79
(m, 2H), 2.75 (t, J=6.0 Hz, 2H), 1.93 (quin, J=5.9 Hz, 2H). Two exchangeable protons not observed, LC-
MS (XBR~5mins_HpH) tR = 2.11 mins, [M+H+] = 437
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3-((5-(2-(5,6,7,8-Tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-yl)amino)-3-(4-
(trifluoromethyl)phenyl)propanoic acid (35), prepared by general procedure C using 4-
(trifluoromethyl)benzaldehyde. 1H NMR (400 MHz, DMSO) δ ppm = 7.72 - 7.54 (m, 5H), 7.09 (dd, J=2.9,
8.9 Hz, 1H), 7.04 (d, J=7.3 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 6.49 (d, J=9.0 Hz, 1H), 6.43 - 6.27 (m, 2H),
5.36 - 5.27 (m, 1H), 4.11 (t, J=6.9 Hz, 2H), 3.51 - 3.30 (m, 2H), 2.93 - 2.64 (m, 5H), 2.60 (t, J=6.0 Hz, 2H),
1.79 - 1.69 (m, 2H), LC-MS (Acq~2min_TFA) tR = 0.71 mins, [M+H+] = 487
3-(4-Phenoxyphenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
1
yl)amino)propanoic acid (36), prepared by general procedure C using 4-phenoxybenzaldehyde. H
NMR (400 MHz, MeOD) δ ppm = 7.62 - 7.58 (m, 1H), 7.40 (d, J=8.8 Hz, 2H), 7.33 (s, 2H), 7.26 (d, J=7.3
Hz, 1H), 7.16 - 7.06 (m, 2H), 6.95 (d, J=7.8 Hz, 2H), 6.91 (d, J=8.5 Hz, 2H), 6.49 (dd, J=8.3, 16.6 Hz, 2H),
5.12 (t, J=6.8 Hz, 1H), 4.17 (t, J=6.5 Hz, 2H), 3.43 - 3.38 (m, 2H), 2.96 (t, J=6.4 Hz, 2H), 2.78 - 2.70 (m,
4H), 1.94 - 1.86 (m, 2H). Three exchangeable protons not observed, LC-MS (XBR~5min_HpH) tR = 2.42
mins, [M+H+] = 511
General procedure D: Trifluoroborate salt coupling, hydrolysis
3-(3-(Morpholinomethyl)phenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-
2-yl)amino)propanoic acid (37) In a microwave vial was 1,1-dimethylethyl 7-(2-{[6-({1-(3-
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bromophenyl)-3-[(1,1-dimethylethyl)oxy]-3-oxopropyl}amino)-3-pyridinyl]oxy}ethyl)-3,4-dihydro-
1,8-naphthyridine-1(2H)-carboxylate (120 mg, 0.184 mmol), tri-tert-butylphosphane (0.092 ml, 0.092
mmol), XPhos (26.3 mg, 0.055 mmol), Cs2CO3 (179 mg, 0.551 mmol), Pd(OAc)2 (4.12 mg, 0.018 mmol)
and (morpholinium-4-ylmethyl)trifluoroborate internal salt (114 mg, 0.551 mmol) in THF (4 mL) and
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o
the reaction mixture was heated to 130 C for 30 mins. The reaction mixture was then partitioned
between DCM (50 mL) and H2O (50 mL), the aq. phase was washed with further DCM (50 mL) and the
combined organic phases were concentrated in vacuo and purififed by silica chromatography, eluting
with EtOAc:cyclohexane (0 – 100 % EtOAc). The appropriate fractions were then concentrated in vacuo
to give an oil. This was dissolved in DCM (3 mL) and 4M HCl in 1,4-dioxane (1 mL, 4.00 mmol) and two
drops of H2O were added and the reaction mixture stirred at rt for 18 h. The reaction mixture was then
concentrated in vacuo and dissolved in DMSO:MeOH (1 mL) and purified by mass directed autoprep
on Xbridge column using MeCN:H2O with an ammonium carbonate modifier. The solvent was dried
under a stream of N2 in the Radleys blowdown apparatus to give the title compound (3.5 mg, 4%). 1H
NMR (400 MHz, MeOD) δ ppm = 7.60 - 7.56 (m, 1H), 7.40 - 7.26 (m, 4H), 7.19 (d, J=7.3 Hz, 1H), 7.09
(dd, J=3.0, 9.0 Hz, 1H), 6.53 (d, J=7.3 Hz, 1H), 6.42 (d, J=9.0 Hz, 1H), 5.13 (t, J=6.8 Hz, 1H), 4.14 (t, J=6.4
Hz, 2H), 3.64 (t, J=4.6 Hz, 4H), 3.60 (s, 2H), 3.44 - 3.38 (m, 2H), 2.99 (t, J=6.4 Hz, 2H), 2.79 - 2.71 (m,
4H), 2.53 - 2.45 (m, 4H), 1.90 (quin, J=5.9 Hz, 2H). Three exchangeable protons not observed, LC-MS
(XBR~5min_HpH) tR = 2.00 mins, [M+H+] = 518
3-(4-(Morpholinomethyl)phenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-
2-yl)amino)propanoic acid (38), prepared by general procedure D using (morpholinium-4-
ylmethyl)trifluoroborate internal salt. 1H NMR (400 MHz, MeOD) δ ppm = 7.61 - 7.54 (m, 1H), 7.42 -
7.36 (m, 2H), 7.33 - 7.26 (m, 3H), 7.07 (dd, J=3.0, 9.0 Hz, 1H), 6.52 (d, J=7.3 Hz, 1H), 6.41 (d, J=9.0 Hz,
1H), 5.13 (t, J=6.9 Hz, 1H), 4.13 (t, J=6.4 Hz, 2H), 3.73 - 3.66 (m, 4H), 3.61 (s, 2H), 3.44 - 3.37 (m, 2H),
2.98 (t, J=6.4 Hz, 2H), 2.76 - 2.69 (m, 4H), 2.57 (d, J=4.0 Hz, 4H), 1.89 (quin, J=5.9 Hz, 2H). Three
exchangeable protons not observed, LC-MS (XBR~5min_HpH) tR = 1.99 mins, [M+H+] = 518
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3-(3-Cyclopropylphenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (39), prepared by general procedure C using 3-cyclopropylbenzaldehyde. 1H
NMR (400 MHz, DMSO-d6) δ = 12.19 (br. s, 1H), 7.65 (d, J = 3.4 Hz, 1H), 7.13 - 7.09 (m, 3H), 7.07 (dd, J
= 3.2, 9.0 Hz, 1H), 7.03 (d, J = 7.3 Hz, 1H), 6.87 - 6.83 (m, 1H), 6.60 (br s, 1H), 6.44 (d, J = 8.8 Hz, 1H),
6.33 (d, J = 7.3 Hz, 1H), 6.29 (br s, 1H), 5.18 (br s, 1H), 4.11 (t, J = 6.8 Hz, 2H), 3.26 - 3.19 (m, 2H), 2.80
(t, J = 6.8 Hz, 2H), 2.73 (dd, J = 8.3, 15.7 Hz, 1H), 2.63 - 2.54 (m, 3H), 1.85 (tt, J = 5.1, 8.4 Hz, 1H), 1.74
(quin, J = 5.9 Hz, 2H), 0.95 - 0.87 (m, 2H), 0.65 - 0.57 (m, 2H), 13C NMR (101 MHz, DMSO-d6) δ = 172.18,
155.83, 153.73, 153.06, 146.91, 143.75, 143.36, 135.93, 133.66, 127.94, 125.86, 123.81, 123.55,
123.34, 112.82, 110.63, 108.75, 68.32, 51.88, 42.17, 40.62, 37.02, 25.98, 20.93, 15.05, 9.32, 9.30, LC-
MS (XBR~5min_HpH) tR = 2.24 mins, [M+H+] = 459, HR-MS: calc for C27H31N4O3 (M+H)+ 459.2396,
found 459.2400.
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General procedure E: Suzuki cross coupling
3-(3,5-Dicyclopropylphenyl)-3-((5-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethoxy)pyridin-2-
yl)amino)propanoic acid (40), A mixture of cyclopropylboronic acid (70.4 mg, 0.819 mmol), tert-butyl
7-(2-((6-((3-(tert-butoxy)-1-(3,5-dibromophenyl)-3-oxopropyl)amino)pyridin-3-yl)oxy)ethyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxylate (100 mg, 0.137 mmol), tripotassium phosphate (174 mg,
0.819 mmol), 2′-(dimethylamino)-2-biphenylyl-palladium(II) chloride dinorbornylphosphine complex
(15.30 mg, 0.027 mmol) in 1,4-Dioxane (5 mL) and H2O (0.5 mL) was heated in a sealed tube in a
Biotage Initiator microwave using initial normal absorption level setting to 130 °C for 30 min. 4M HCl
in 1,4-dioxane (0.068 mL, 0.273 mmol) was added and the reaction mixture was stirred for 4 h. H2O (5
mL) and DCM (5 mL) were then added and the organic layer was extracted. 4M HCl in 1,4-dioxane (0.5
mL) was added to the organic layer. The reaction mixture was then stirred for 2 h at rt before being
concentrated in vacuo to give a brown gum. This was dissolved in DMSO (1 mL) and purified by Mass
Directed AutoPrep on Xbridge column using MeCN: H2O with an ammonium carbonate modifier. The
solvent was dried under a stream of N2 in the Radleys blowdown apparatus to give the target
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