Identification of 4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine Derivatives as Potent, Selective, and Orally Efficacious Inhibitors of the Copper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2)

Article abstract of DOI:10.1021/acs.jmedchem.7b00345

LOXL2 catalyzes the oxidative deamination of ε-amines of lysine and hydroxylysine residues within collagen and elastin, generating reactive aldehydes (allysine). Condensation with other allysines or lysines drives the formation of inter- and intramolecular cross-linkages, a process critical for the remodeling of the ECM. Dysregulation of this process can lead to fibrosis, and LOXL2 is known to be upregulated in fibrotic tissue. Small-molecules that directly inhibit LOXL2 catalytic activity represent a useful option for the treatment of fibrosis. Herein, we describe optimization of an initial hit 2, resulting in identification of racemic-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone 28, a potent irreversible inhibitor of LOXL2 that is highly selective over LOX and other amine oxidases. Oral administration of 28 significantly reduced fibrosis in a 14-day mouse lung bleomycin model. The (R,R)-enantiomer 43 (PAT-1251) was selected as the clinical compound which has progressed into healthy volunteer Phase 1 trials, making it the “first-in-class” small-molecule LOXL2 inhibitor to enter clinical development.

Full text of DOI:10.1021/acs.jmedchem.7b00345

Subscriber access provided by HKU Libraries
Article
The Identification of 4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine
Derivatives as Potent, Selective, and Orally Efficacious Inhibitors of
the Copper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2)
Martin W. Rowbottom, Gretchen Bain, Imelda Calderon, Taylor Lasof, David Lonergan, Andiliy Lai, Fei Huang,
Janice Darlington, Patricia Prodanovich, Angelina M. Santini, Christopher D. King, Lance Goulet, Kristen
E. Shannon, Gina L. Ma, Katherine Nguyen, Deidre A. MacKenna, Jilly F. Evans, and John H. Hutchinson
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 04 May 2017
Downloaded from http://pubs.acs.org on May 4, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The Identification of 4ꢀ(Aminomethyl)ꢀ6ꢀ
10
11
12
13
14
15
16
17
18
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
(
trifluoromethyl)ꢀ2ꢀ(phenoxy)pyridine Derivatives
as Potent, Selective, and Orally Efficacious
Inhibitors of the CopperꢀDependent Amine Oxidase,
Lysyl OxidaseꢀLike 2 (LOXL2)
∗
Martin W. Rowbottom, Gretchen Bain, Imelda Calderon, Taylor Lasof, David Lonergan,
Andiliy Lai, Fei Huang, Janice Darlington, Patricia Prodanovich, Angelina M. Santini,
Christopher D. King, Lance Goulet, Kristen E. Shannon, Gina L. Ma, Katherine Nguyen, Deidre
A. MacKenna, Jilly F. Evans, and John H. Hutchinson
PharmAkea Therapeutics, San Diego Science Center, 3030 Bunker Hill Street, Suite 300, San
Diego, California 92109, United States
KEYWORDS Copperꢀdependent amine oxidase, lysyl oxidase, lysyl oxidaseꢀlike 2, LOX,
LOXL2, LOXL2 inhibitors, collagen crossꢀlinking, fibrosis, lung fibrosis, idiopathic pulmonary
fibrosis, IPF, bleomycinꢀinduced pulmonary fibrosis, 4ꢀ(aminomethyl)pyridines.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
LOXL2 catalyzes the oxidative deamination of εꢀamines of lysine and hydroxylysine residues
within collagen and elastin, generating reactive aldehydes (allysine). Condensation with other
allysines or lysines drives formation of interꢀ and intramolecular crossꢀlinkages, a process critical
for remodeling of the ECM. Dysregulation of this process can lead to fibrosis and LOXL2 is
known to be upregulated in fibrotic tissue. Smallꢀmolecules that directly inhibit LOXL2 catalytic
activity represent a useful option for the treatment of fibrosis. Herein, we describe optimization
of an initial hit 2, resulting in identification of racemicꢀtransꢀ(3ꢀ((4ꢀ(aminomethyl)ꢀ6ꢀ
(trifluoromethyl)pyridinꢀ2ꢀyl)oxy)phenyl)(3ꢀfluoroꢀ4ꢀhydroxypyrrolidinꢀ1ꢀyl)methanone 28, a
potent irreversible inhibitor of LOXL2 that is highly selective over LOX and other amine
oxidases. Oral administration of 28 significantly reduced fibrosis in a 14ꢀday mouse lung
bleomycin model. The (R,R)ꢀenantiomer 43 (PATꢀ1251) was selected as the clinical compound
which has progressed into healthy volunteer Phase 1 trials, making it the ‘firstꢀinꢀclass’ smallꢀ
molecule LOXL2 inhibitor to enter clinical development.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
INTRODUCTION
The lysyl oxidase (LOX) family of enzymes are copperꢀdependent amine oxidases consisting of
1,2
five distinct isoforms, namely lysyl oxidase (LOX) and lysyl oxidaseꢀlike (LOXL) 1 through 4.
The LOX family is divided into two subgroups based on structural differences in the Nꢀterminal
domain. Both LOX and LOXL1 contain a highly basic propeptide that requires cleavage in order
to generate active enzyme, whereas LOXL2, 3, and 4 contain four scavenger receptor cysteineꢀ
rich (SRCR) domains which are thought to mediate proteinꢀprotein interactions and cell
2
adhesion. All LOX family members possess a conserved carboxyꢀterminal catalytic domain
ACS Paragon Plus Environment

Page 3 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
containing both a HisꢀXꢀHisꢀXꢀHis copper binding motif and a postꢀtranslationally modified
lysine tyrosylquinone (LTQ) coꢀfactor. These enzymes localize to the extracellular matrix
(ECM) where they catalyze the oxidativeꢀdeamination of the terminal εꢀamine of lysine and
10
11
12
13
14
15
16
17
18
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
hydroxylysine residues in both collagen and elastin. This results in the generation of highly
reactive aldehydes (allysine) that can further condense with other allysines or intact lysines to
form interꢀ and intramolecular crossꢀlinkages, a process critical for normal remodeling of the
2
,3
ECM. Dysregulation of this process can lead to increased crossꢀlinking, resulting in abnormal
and uncontrolled ECM deposition in tissue. If left unchecked, this can lead to pathological
fibrosis characterized by excessive scaring, impairment of organ function, and ultimately organ
4
failure. Examples include fibrosis of the lung (such as idiopathic pulmonary fibrosis or IPF),
5
6
7
liver, kidneys, and skin (systemic sclerosis). Unfortunately, current treatment options for
patients diagnosed with fibrosis are extremely limited. Only for IPF have new antiꢀfibrotic
8
medications been recently approved, these being nintedanib and pirfenidone. However, these
9
agents offer only modest benefit to patients, so there is a need for additional therapies for both
IPF and other types of fibrotic diseases. Given that aberrant ECM deposition is a driver in the
progression of fibrotic disease, and that LOX family enzymes are key mediators of this process,
inhibition of one or more LOX proteins may delay or even halt disease progression. One family
member in particular, LOXL2, has been extensively studied and shown to be upregulated in both
1
0
fibrotic tissue and certain cancers. In addition to its role in the ECM, an intracellular role has
been proposed and LOXL2 has been reported to be involved in the induction of epithelial to
mesenchymal transition (EMT), a process central to tumor cell invasion and metastasis. LOXL2
can mediate EMT via either stabilization of the transcription factor Snail1, or catalytic oxidativeꢀ
1
1
deamination of the trimethylated lysine 4 of histone H3 (H3K4me3). Although deamination of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H3K4me3 to give the corresponding allysine requires catalytically competent LOXL2, the
mechanism is not well understood, but has been suggested to involve release of trimethylamine,
1
1b
followed by oxidation to give the aldehyde (allysine). As such, an inhibitor of LOXL2 capable
of cell and nuclear penetration could be effective in modulating EMT, and both extracellular and
intracellular inhibition of LOXL2 may be more efficacious in the treatment of both fibrosis and
metastatic cancers. Simtuzumab is a LOXL2ꢀspecific monoclonal antibody that only partially
inhibits catalytic activity via binding to a distal allosteric site located in the fourth SRCR
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0b
domain.
Although the corresponding mouse antibody (AB0023) demonstrated efficacy in
10b,d
several preclinical models of fibrosis and cancer,
subsequent Phase 2 trials with simtuzumab
in patients with pancreatic cancer, IPF, and liver fibrosis failed to demonstrate efficacy and
1
2
development was recently discontinued. We envision that a smallꢀmolecule approach toward
selective inhibition of LOXL2 will have significant advantages over an antibody, for instance, (i)
complete inhibition of catalytic activity via direct binding to the catalytic active site, (ii) greater
penetration into the fibrotic matrix and (iii) access to both extraꢀ and intracellular compartments,
allowing for inhibition of LOXL2 activity in the ECM and within cells. There are currently no
published structures of selective inhibitors of LOXL2, and the only published smallꢀmolecule
13
inhibitor is the panꢀLOX(L) inhibitor βꢀaminopropionitrile (BAPN) 1 (Figure 1a). However,
LOX is a ubiquitous enzyme and mice deficient in LOX show severe cardiovascular and
1
4
pulmonary defects. Therefore, our goal was to identify inhibitors of LOXL2 that are selective
over LOX. Given the paucity of selective smallꢀmolecule leads and the lack of NMR or Xꢀray
structures for any of the LOX(L) enzymes, our approach started with the examination of the
proposed mechanism by which LOX(L) proteins enzymatically convert lysines to allysines. A
1
0a
simplified proposed mechanism is shown in Figure 1b.
Briefly, the εꢀamine (of lysine
ACS Paragon Plus Environment

Page 5 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
residues) reversibly binds one of the carbonyl groups of the LTQ coꢀfactor (of I) to yield the
substrate Schiff base intermediate (II). Subsequent irreversible conversion of the substrate Schiff
base to the product Schiff base intermediate (III) occurs via proton abstraction (postulated to be
mediated by a nearby active site basic residue) and concomitant reduction/aromatization of the
quinonoid ring (of II). Hydrolysis of III yields the aldehyde (allysine) and the intermediate
10
11
12
13
14
15
16
17
18
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
aminophenol (IV), which is then recycled, via a number of steps involving O and Cu, to
2
regenerate the catalytically active species (I) with release of both hydrogen peroxide and
1
5
ammonia. Based upon the proposed mechanism, we hypothesized that a substrateꢀlike
aminomethylꢀcontaining small molecule should be able to compete with lysine in binding
directly to the LTQ carbonyl, thus forming a small moleculeꢀcontaining substrate Schiff base (II)
which is presumably converted to the product Schiff base (III). Furthermore, whereas the lysineꢀ
containing Schiff base (III) readily undergoes hydrolysis releasing allysine, the small moleculeꢀ
containing Schiff base may be able to form a tightly bound Schiff base complex which could be
1
6
more resistant to hydrolytic cleavage. In addition LOXL2 is glycosylated at the active site
while LOX is not, and this structural difference may provide an opportunity to design inhibitors
2
,10a
selective for LOXL2 versus LOX.
With this in mind we executed a focused screening
campaign and rapidly identified a series of 2ꢀsubstituted pyridineꢀ4ꢀylmethanamines as
moderately selective LOXL2 inhibitors. The best compound, 4ꢀ(aminomethyl)ꢀ2ꢀchloropyridine
2 (Figure 1a) was shown to have ~30ꢀfold selectivity over LOX and was also selective against a
16
panel of amine oxidases. Herein we describe the SAR and lead optimization campaign around 2
which led to the identification of racemicꢀtransꢀ(3ꢀ((4ꢀ(aminomethyl)ꢀ6ꢀ(trifluoromethyl)pyridinꢀ
2ꢀyl)oxy)phenyl)(3ꢀfluoroꢀ4ꢀhydroxypyrrolidinꢀ1ꢀyl)methanone 28, a highly potent LOXL2
inhibitor with significantly improved selectivity over LOX. Compound 28 is an orally
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
bioavailable, irreversible inhibitor of LOXL2 that exhibits efficacy in a mouse bleomycin lung
fibrosis model with onceꢀdaily oral dosing. Subsequent profiling of the individual enantiomers of
1
7
2
8 ultimately led to the selection of the (R,R)ꢀenantiomer 43 (PATꢀ1251) as the lead
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
compound.
CHEMISTRY
All compounds described herein were prepared as illustrated in Schemes 1−4. Compounds 6, 9
and 10 were obtained in the following manner (Scheme 1). Reaction of 2ꢀchloroꢀ4ꢀ
pyridinecarbonitrile 4 with phenol in the presence of Cs CO gave 2ꢀphenoxypyridine derivative
2
3
5
. Subsequent nitrile reduction followed by treatment with HCl afforded compound 6. Treatment
of 4 with methyl 4ꢀhydroxybenzoate in the presence of Cs CO , gave paraꢀsubstituted 2ꢀ
2
3
phenoxypyridine derivative 7. Reduction of the nitrile followed by NꢀBoc protection of the
resulting amine, then baseꢀmediated hydrolysis of the ester, afforded carboxylic acid derivative
8
. Subsequent HATUꢀmediated coupling of 8 with the appropriate amine followed by NꢀBoc
deprotection, gave final compounds 9 and 10 as HCl salts. Final compounds 13 – 20 (Scheme 1)
were obtained in analogous fashion via reaction of 2ꢀchloroꢀ4ꢀpyridinecarbonitrile 4 with methyl
3ꢀhydroxybenzoate to afford the metaꢀsubstituted 2ꢀphenoxypyridine derivative 11. Subsequent
nitrile reduction, NꢀBoc protection, and then ester hydrolysis gave acidꢀderivative 12, which was
readily converted to compounds 13 – 20. Coupling of acidꢀderivative 12 with methyl 4ꢀ
aminobenzoate, followed by ester hydrolysis and NꢀBoc deprotection, gave final compound 21
(Scheme 1). 4ꢀ(Methylamino)ꢀ6ꢀ(trifluoromethyl)pyridine derivatives 25 – 33 were prepared as
described in Scheme 2. Treatment of 2ꢀchloroꢀ6ꢀ(trifluoromethyl)isonicotinonitrile 22 with
methyl 3ꢀhydroxybenzoate in the presence of K CO3 gave 2ꢀ(phenoxy)ꢀ6ꢀ
2
(trifluoromethyl)pyridine derivative 23. Reduction of the nitrile to the corresponding
ACS Paragon Plus Environment

Page 7 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
methylamine via treatment with NaBH and CoCl , followed by NꢀBoc protection then ester
4
2
hydrolysis, afforded acidꢀcontaining derivative 24. Desired compounds 25 – 33 were obtained
via previously described amideꢀcoupling procedures followed by NꢀBoc deprotection. Piperidine
phenylureaꢀcontaining derivatives 36 and 37 were prepared as shown in Scheme 2. Treatment of
2ꢀchloroꢀ6ꢀ(trifluoromethyl)isonicotinonitrile 22 with either (R)ꢀ or (S)ꢀ1ꢀNꢀBocꢀ3ꢀ
hydroxypiperidine and NaH, followed by NꢀBoc deprotection, afforded NHꢀpiperidine
derivatives 34 and 35, respectively. Subsequent reaction with phenyl isocyanate followed by
reduction of the nitrile, afforded final compounds 36 and 37. In addition, the 3ꢀphenoxyphenylꢀ
containing derivative 39 was prepared from 2ꢀchloroꢀ6ꢀ(trifluoromethyl)isonicotinonitrile 22 and
10
11
12
13
14
15
16
17
18
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
3
ꢀphenoxyphenol via a simple twoꢀstep S Ar/reduction procedure (Scheme 2). Optically pure
N
transꢀ4ꢀfluoroꢀ3ꢀpyrrolidinolꢀcontaining derivatives 43 and 44 were prepared as shown in
Scheme 3. The aforementioned acid derivative 24 was coupled with racemic transꢀ4ꢀfluoroꢀ3ꢀ
pyrrolidinol to give the racemate 40. At the time this work was performed, only racemic transꢀ4ꢀ
fluoroꢀ3ꢀpyrrolidinol was commercially available. Thus, the individual enantiomers (of 40) were
obtained via preparative reverseꢀphase chiral HPLC separation, wherein the (R,R)ꢀenantiomer 41
and (S,S)ꢀenantiomer 42 were the first and second to elute, respectively. Subsequent NꢀBoc
deprotection of 41 and 42 afforded optically pure final compounds 43 and 44, respectively. The
absolute stereochemistry of 41 was confirmed via its preparation from 24 and optically pure
(R,R)ꢀtransꢀ4ꢀfluoroꢀ3ꢀpyrrolidinol 2,3ꢀdibenzoylꢀDꢀtartrate 47 (under identical HATU mediated
conditions used previously). Optically pure 47 was readily obtained in 94.4% ee from racemate
46 via diastereomeric salt crystallization with 2,3ꢀdibenzoylꢀDꢀtartaric acid (Scheme 4), and the
(R,R)ꢀconfiguration of the 4ꢀfluoroꢀ3ꢀpyrrolidinol 47 was confirmed via Xꢀray crystallography
(see Figure 2).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
RESULTS AND DISCUSSION
All compounds (Tables 1 through 3) were profiled for inhibitory activity against human LOXL2
and counterꢀscreened against human LOX. The LOXL2 assay was carried out using serumꢀfree
concentrated conditioned media (CCM) from CHO cells stably expressing human LOXL2, and
amine oxidase activity was determined via measurement of hydrogen peroxide released during
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
8
the oxidative deamination of the artificial substrate 1,5ꢀdiaminopentane (DAP). The panꢀ
LOX(L) inhibitor 1 exhibits timeꢀdependent inhibition of LOXL2 with IC values shifting from
50
0.24 to 0.066 ꢀM when the preꢀincubation period is increased from 15 min to 2 hour. We
observed no further significant change in potency upon extending the preꢀincubation out to 4
1
6
hours. In order to allow for any timeꢀdependent inhibition with our compounds, amine oxidase
activity for both LOXL2 and LOX was measured after a 2 hour preꢀincubation with test
compounds. To assess selectivity of compounds versus human LOX, a slightly modified assay
was used which required the addition of BSA to the serumꢀfree media from HEK cells stably
expressing human LOX in order to obtain measurable activity. The corresponding LOXL2 assay
(+ BSA in serumꢀfree conditioned media) was also run in order to generate an accurate headꢀtoꢀ
head comparison of selectivity. In addition, LOXL2 activity was assessed in a more clinically
relevant human whole blood assay wherein recombinant human LOXL2 and test compounds
were incubated for 2 hours in whole blood prior to addition of DAP and subsequent
measurement of hydrogen peroxide release.
Structure-Activity Relationships of LOXL2 Inhibitors. Compound 2 (Figure 1a and Table
1
6
1), is an attractive lead for several reasons including good in vitro potency for LOXL2 (IC =
5
0
0.13 ꢀM), selectivity for LOXL2 over LOX (~30ꢀfold vs 6ꢀfold for 1), and a modest 10ꢀfold shift
ACS Paragon Plus Environment

Page 9 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
in potency in a human whole blood assay (IC = 1.5 ꢀM). In addition, the molecular properties
5
0
1
9
of 2 fall well within the ‘rule of five’ (RO5) criteria, thus an attractive starting point for
allowing efficient access to compounds with excellent physicoꢀchemical and drugꢀlike
properties. The goals for our SAR campaign included improving LOXL2 potency and selectivity,
generating a compound with acceptable bioavailability to demonstrate in vivo efficacy in a
mouse model of fibrosis and, ultimately, selecting a clinical development candidate. Initial
studies focused on the 2ꢀposition of the pyridine, as summarized in Table 1. We first investigated
replacement of the 2ꢀCl moiety (of 2) with other small groups of varying electronic and
10
11
12
13
14
15
16
17
18
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
lipophilic character such as CF (48), OMe (49), or Oꢀcyclohexyl (50). This led to a 6 to 9ꢀfold
3
drop in potency for LOXL2, although selectivity over LOX was improved for the more
electronegative CF ꢀcontaining derivative 48 (40ꢀfold selective). Potency in human whole blood
3
was also significantly reduced, particularly for both OMe (49, hWB IC50 = 35 ꢀM) and Oꢀ
cyclohexyl (50, hWB IC = 29 ꢀM) derivatives. However, replacement of Oꢀcyclohexyl with an
5
0
aromatic Oꢀphenyl (6) led to improved potency at LOXL2 (versus 49 and 50), particularly in
blood where 6 (hWB IC = 6.7 ꢀM) was ~4ꢀfold more potent than 50. One hypothesis is that the
5
0
phenyl group is picking up a favorable interaction with the enzyme, so we explored substitution
of the phenyl ring of 6. Numerous derivatives were initially prepared incorporating a variety of
substituents on the phenyl ring (not shown). From these, it was found that substitution with a
functionalized amide moiety proved particularly productive and that the position of the
substituent amide is important for potency. Derivatives incorporating the amide substituent meta
to the pyridine Oꢀlinker exhibited improved potency for LOXL2 compared to the corresponding
paraꢀsubstituted analogs (compare 9 and 10 with 13 and 14, respectively). This was particularly
apparent in the blood assay. For example, the metaꢀbenzamide derivative 14 (LOXL2 CCM IC50
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
=
0.056 ꢀM; hWB IC = 1.3 ꢀM) was 30ꢀfold more potent in human whole blood than the
5
0
corresponding paraꢀbenzamide derivative 10 (LOXL2 CCM IC = 0.27 ꢀM; hWB IC = 39
5
0
50
ꢀ
M). Additional metaꢀamide containing analogs demonstrated that substituents of varying sizes
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and lipophilicities may be incorporated while maintaining good potency for LOXL2. For
example, benzyl 15 (LOXL2 CCM IC50 = 0.062 ꢀM) and 2ꢀ(methylene)benzothiophene 17
(LOXL2 CCM IC50 = 0.079 ꢀM) derivatives were equipotent to benzamide derivative 14.
Furthermore, substitution of the benzamide ring (of 14) with groups of varying electronic
character proved well tolerated as, for example, incorporation of either an electronꢀdonating
OMe (18) or an electronꢀwithdrawing fluoro (19) had little effect on overall LOXL2 potency or
selectivity. Even the incorporation of ionizable groups such as amidine (20) or carboxylic acid
(21) proved well tolerated by the enzyme. Overall, these data suggest that the amide is important
for potency; however, substituents on the amide nitrogen are not directly involved in critical
binding interactions with the enzyme. Rather, this area of the molecule probably sits in a
relatively open (solvent exposed) region next to the LTQꢀcontaining active site, which is
presumably the same region normally occupied by the protein backbone immediately adjacent to
the active lysine or hydroxylysine residues of both collagen and elastin substrates (illustrated in
2
0
Figure 1c). This is the case for the related topaquinone (TPQ) containing copper amine oxidase,
Pichia pastoris LOX, which has a broad funnel leading to the active site, thus allowing access to
2
1
(and subsequent oxidation of) lysine residues in polypeptides. The identification of a region
within our small molecule scaffold amenable to significant structural variation proved an
important finding, and ultimately allowed for the efficient modulation and optimization of key
physicochemical properties of this series without affecting potency at LOXL2 (vide infra).
Despite having identified a series of potent pyridineꢀderived LOXL2 inhibitors (in both serumꢀ
ACS Paragon Plus Environment

Page 11 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
free and human whole blood assays), we felt that the selectivity over LOX (~30ꢀfold) could be
1
4
further improved, particularly given the physiological role of LOX in normal healthy tissue.
Key structural differences between the catalytic domains of LOX and LOXL2 are known. In
10
11
12
13
14
15
16
17
18
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
particular, the catalytic domain of LOXL2 contains a predicted Nꢀglycosylated asparagine
2
,10a
residue (N644) close to the LTQꢀCu active site, which is absent in LOX.
This may result in
structural differences between the active site binding pockets of LOX and LOXL2, leading to
differential binding of smallꢀmolecule inhibitors. Using our hypothesis that the pyridineꢀ4ꢀ
ylmethylamino group (or warhead) of our inhibitors covalently binds to the quinone carbonyl of
the active site LTQ (Figure 1b), we reasoned that targeted modification around the warhead may
further reduce LOX activity while maintaining potency for LOXL2. Substitution at either the
warhead amine nitrogen (ꢀCH NHR) or the adjacent carbon (ꢀCHRNH ) resulted in significant
2
2
loss in potency for LOXL2 (data not shown), further supporting our hypothesis that inhibition is
driven via Schiff base formation between the primary amine and the LTQ carbonyl. We had
previously identified the 2ꢀCF (48) derivative as being the most selective versus LOX (~40ꢀ
3
fold) and reasoned that the combination of this with the OPh of 6 may be beneficial, i.e.
incorporation of both CF (at Cꢀ6 of the pyridine) and a phenoxy moiety (at Cꢀ2) may allow
3
access to potent LOXL2 compounds with an improved selectivity profile. Gratifyingly, this idea
proved successful, as the 6ꢀCF ꢀpyridineꢀcontaining derivatives 25 and 26 (Table 2) maintained
3
potency at LOXL2 and were 177ꢀ and 110ꢀfold selective over LOX, respectively. The
corresponding 6ꢀHꢀpyridineꢀcontaining derivatives 14 and 17 were only ~25ꢀfold selective. This
observed improvement in selectivity over LOX may be due to the increase in steric bulk at Cꢀ6
of the pyridine (H → CF ), or a combination effect with the reduced basicity/HBA capability of
3
the pyridine ring nitrogen. This represented a major finding and resulted in the 6ꢀCF ꢀpyridine
3
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
moiety being incorporated into subsequent molecules. Unfortunately, both 25 and 26 exhibited a
significant reduction in potency in the human blood assay (69 and 291ꢀfold, respectively),
2
2
presumably as a result of high protein binding. The calculated LogP (cLogP) values for 25 and
6 are 4.3 and 5.1, respectively, and it is known that highly lipophilic molecules often exhibit
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
high blood protein binding. In order to identify suitable candidates for in vivo efficacy
evaluation, whole blood potency needed to be improved, presumably by reducing overall
2
3
lipophilicity and therefore protein binding. This was achieved by fine tuning of the overall
lipophilicity through modification of the pendant amide substituents and the results are shown in
Table 2 where compounds are listed in order of decreasing lipophilicity (cLogP of 5.1 to 0.8).
Most compounds proved potent for LOXL2 under serumꢀfree conditions with IC50 values
between 0.034 and 0.11 ꢀM. An outlier was compound 36 (CCM IC50 = 0.57 ꢀM) which
contains the (3R)ꢀpiperidine ring in place of phenyl. However, potency was restored with the
corresponding (3S)ꢀpiperidine enantiomer 37 (CCM IC = 0.13 ꢀM), suggesting that relative
5
0
spatial orientation of both pyridine and piperidine rings in the enzyme binding pocket is
important. As predicted, the decrease in lipophilicity was accompanied by a significant
improvement in whole blood potency for LOXL2. For instance, for cLogP values ~5
(
compounds 26 and 39) the blood shift is ~300ꢀfold. When cLogP values are reduced by just 1
log unit to 4.3 (25) the blood shift is reduced to ~70ꢀfold and for compounds with cLogP values
3 the human blood potency is shifted only ~10 fold (compounds 27 through 33). The observed
<
improvement in whole blood potency correlates with the reduction in protein binding as, for
example, compound 28 (cLogP = 2.0; hWB IC50 = 0.87 ꢀM) had measured human proteinꢀ
binding of 68%, compared with 96% for the more lipophilic 25 (cLogP = 4.3; hWB IC = 2.9
5
0
2
4
ꢀM). Importantly, variation of overall lipophilicity did not erode selectivity versus LOX,
ACS Paragon Plus Environment

Page 13 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
further supporting the role of the 6ꢀCF ꢀpyridine as a key selectivity driving motif. Having
3
identified a series of potent compounds we wanted to understand if these were irreversible
inhibitors of LOXL2, especially given that they contain a primary amine similar to that for the
mechanismꢀbased inhibitor BAPN 1. It has been hypothesized that formation of the irreversible
complex between LOX and 1 proceeds via a product Schiff base intermediate (see III, Figure
10
11
12
13
14
15
16
17
18
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
1b), which can rearrange to a more stable enamine product (V, Figure 1b) potentially allowing
for an additional covalent interaction between the nitrile and a nearby nucleophilic residue in the
1
3b
LOX active site.
Our inhibitors are likely to form a Schiff base complex with LOXL2;
however, these inhibitors are unlikely to form additional covalent bonds with the enzyme as
proposed in V (Figure 1b). Instead, we believe multiple nonꢀcovalent binding interactions help
drive formation of a tightly bound Schiff base complex and irreversibly lock down the enzyme in
an inactive form. A major advantage of irreversible inhibition is the potential for an extended
pharmacodynamic (PD) response that outlasts systemic drug levels (PK). This assumes that the
target protein is not rapidly regenerated in vivo at the site of action. Such minimization of overall
exposure in vivo helps limit any offꢀtarget activities and can lead to an improved safety profile
25
and higher therapeutic index. Select compounds, including 1, were assessed in a human
LOXL2 reversibility assay and the results are shown in Figure 3a. Briefly, purified recombinant
human LOXL2 bound to the bottom of 96ꢀwell plates was incubated with inhibitor compound
(1000 × IC concentration) for 2 hours at 37 °C. After repeated washing with PBS to remove
5
0
any unbound inhibitor, incubation was continued for a further 2 hours at 37 °C to allow for any
bound compound to dissociate. Total LOXL2 activity was then measured and % recovery of
LOXL2 activity was obtained via direct comparison with the corresponding nonꢀwashed
controls. As shown in Figure 3, a 2 hour preꢀincubation with test compounds results in >90%
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
inhibition of LOXL2 activity (solid bars, Figure 3). Washing, followed by a 2 hour equilibration,
in most cases led to minimal (10 to 20%) recovery of LOXL2 activity (open bars, Figure 3) when
compared with the corresponding nonꢀwashed controls, thus indicating that these are irreversible
inhibitors of LOXL2. The exception was the initial lead 4ꢀ(methylamino)pyridine 2 which was
found to be largely reversible at LOXL2, exhibiting ~65% recovery of enzyme activity after a 2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
h postꢀwash equilibration. We speculate that the observed difference in reversibility between 2
and the 2ꢀ(phenoxy)ꢀ6ꢀ(trifluoromethyl)pyridine containing inhibitors is due to the presence of
additional bulky substituents, that upon formation of the initial substrate Schiff base complex,
can participate in additional nonꢀcovalent binding interactions further facilitating the formation
16
and stabilization of a more tightly bound product Schiff base complex. Given that increased
levels of LOXL2 have been observed in lung tissue and serum samples from patients with IPF
and that LOXL2 has been implicated as a proꢀfibrotic mediator of fibrosis in murine lung fibrosis
1
0b,26
models,
the mouse oropharyngeal bleomycin lung fibrosis model was chosen as our primary
in vivo efficacy screen. A key consideration for advancing candidates into in vivo studies is
ensuring that appropriate drug concentrations are achieved in tissues of interest. Normal
C57BL/6 mice were administered compound as a single oral dose (30 mg/kg) and both lung and
plasma concentrations were assessed over time (Cmax data is shown in Table 2). Compounds 28
and 37 showed the greatest concentrations in lung tissue with Cmax values of 21 and 30 ꢀM,
respectively, with compounds 25, 27 and 29 also exhibiting reasonable lung concentrations
(Cmax ~ 10 ꢀM). However, compounds 30 through 33 exhibited significantly lower lung
concentrations; in particular, 33 resulted in an average lung concentration of only 0.54 ꢀM. In
general, the lung to plasma ratios proved greater for compounds of a more lipophilic nature.
Compounds with cLogP values ≥ 2 (25, 27, 28, and 37) displayed between 7 to 16ꢀfold higher
ACS Paragon Plus Environment

Page 15 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
maximal concentrations in lung tissue versus plasma, whereas compounds with cLogP < 2 (29
through 33) had lung concentrations only 0.5 to 1.8 times that seen in plasma. It is known that
the lungs are a site for both uptake and accumulation of certain drugs. In particular, lipophilic
10
11
12
13
14
15
16
17
18
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
amines with pK values ≥ 8 often exhibit significant pulmonary uptake (for example fentanyl and
a
2
7
28
propranolol). The pK of the aminomethyl group of these 6ꢀCF ꢀpyridine derivatives is ~ 8
a
3
which is in line with known lungꢀpenetrable compounds. The above data indicate that for this
series of basic amineꢀcontaining inhibitors, a cLogP of at least 1.8 is required to ensure good
distribution to mouse lung. Furthermore, the combined dataset presented in Table 2 shows that
by ‘tuning’ overall lipophilicity, one can readily access compounds with both optimal lung
exposure and high potency in human whole blood. This suggests that, for this series, the
lipophilic sweet spot sits in a narrow range between cLogP of ~1.8 to 3 and compound 28
(cLogP = 2.0) emerged as the lead compound suitable for advancement in to the mouse lung
bleomycin model. Additionally, we measured the potency of 28 in mouse blood to ensure that
the concentrations observed in mouse lung tissue would be sufficient to fully inhibit lung
LOXL2. The mouse blood LOXL2 IC and IC values were 0.86 ꢀM and 5.6 ꢀM, respectively,
50
90
which is very similar to the human blood IC50 and IC90 values of 0.87 ꢀM and 5.4 ꢀM,
respectively. Therefore, we reasoned that a single 30 mg/kg dose should be sufficient to cover
the LOXL2 IC in mouse lung for at least 2 hours, thus allowing adequate time for irreversible
9
0
binding to the enzyme.
Evaluation of Compound 28 in the Mouse Bleomycin Lung Fibrosis Model. Lung fibrosis
was induced in 6 to 7ꢀweek old male C57BL/6 mice via the oropharyngeal administration of a
single dose of bleomycin (1.5 U/kg). Compound 28 was administered orally (PO) once daily
(QD) at 3, 10, 30, or 60 mg/kg for a total of 15 days, beginning 1 day prior to instillation of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
bleomycin (prophylactic dosing regimen). At day 14 postꢀbleomycin administration, lungs from
each animal were removed for histological examination of lung slices stained with H&E and
2
9
Masson’s trichrome and an Ashcroft score of fibrosis and inflammatory damage determined.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All doses were well tolerated with no mortality or significant body weight loss (<5% relative to
vehicle matched controls) observed during treatment. Fibrosis was consistently present in the
mice administered bleomycin and treated with vehicle and the mean fibrotic index of this group
was 3.6. Prophylactic treatment with 28 doseꢀdependently reduced lung fibrosis in mice as
evidenced by a reduction in mean Ashcroft score (Figure 4). Dosing at 3 mg/kg QD had little
effect on fibrosis compared with the bleomycin only group (mean Ashcroft score = 3.2);
however, dosing at 10 or 30 mg/kg QD did result in significantly reduced lung fibrosis with
mean Ashcroft scores for those groups of 2.1 and 1.5, respectively. No further antiꢀfibrotic
efficacy was seen at 60 mg/kg, therefore, the maximally efficacious dose was determined to be
30 mg/kg QD.
Profiling of the Individual Enantiomers of 28. Given the robust antiꢀfibrotic efficacy
observed with compound 28 (racemate), we prepared and profiled the individual (R,R; 43) and
(S,S; 44) enantiomers with the aim of selecting a single isomer for further preꢀclinical evaluation
(
see Table 3 and Figure 3). The racemate and its enantiomers are equipotent (CCM IC values ~
50
80 nM) irreversible inhibitors of LOXL2, showing significant inhibition of enzyme activity out
to 24 h (as illustrated by compound 43, Figure 3b). That the enzyme does not differentiate
between either enantiomer further supports our hypothesis that the pendent amide substituent
does not participate in key binding interactions with the active site. In the human blood assay, the
(R,R)ꢀenantiomer (43) is slightly more potent than the (S,S)ꢀenantiomer (44), with average IC50
values of 0.71 and 1.1 ꢀM, respectively. Both enantiomers were also assessed for selectivity
ACS Paragon Plus Environment

Page 17 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
across a broader panel of related human amine oxidases (Table 3). As expected, both 43 and 44
proved highly selective for LOXL2 over LOX (> 400ꢀfold). For the more closely related LOXL
isoforms, activity was only evaluated against LOXL3, where both compounds exhibit IC values
5
0
10
11
12
13
14
15
16
17
18
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
of ~ 1.2 ꢀM. Attempts to generate stable cell lines for LOXL1 and LOXL4 were unsuccessful, so
potencies against these two enzymes were not determined. Compounds 43 and 44 also proved
highly selective for LOXL2 over other key members of the amine oxidase family, including the
copperꢀdependent amine oxidases semicarbazideꢀsensitive amine oxidase (SSAO) and diamine
oxidase (DAO), as well as the flavinꢀdependent monoamine oxidases A (MAOꢀA) and B (MAOꢀ
B). They all showed <10% inhibition at 10 ꢀM. In addition, after oral administration (30 mg/kg)
to C57BL/6 mice, both enantiomers exhibited very similar lung and plasma concentrations to
that observed with the corresponding racemate 28 (Table 2). Although the enantiomers (43 and
44) have a very similar profile, the (R,R)ꢀenantiomer 43 was chosen for further profiling based
upon its slightly improved whole blood potency. In line with the observed high potency for
human LOXL2, 43 was also potent against mouse, rat, and dog LOXL2 (average IC values of
5
0
0.10, 0.12, and 0.16 ꢀM, respectively) which are key efficacy and toxicological species.
Compound 43 also exhibits a reasonable CYP450 inhibition profile, with measured IC values
5
0
of 1.8, 1.7, 2.4, 12, and 30 ꢀM versus 1A2, 2C9, 2C19, 2D6, and 3A4, respectively. In addition,
3 was inactive when screened against a panel of 55 diverse receptors, ion channels, and
4
3
0
transporters (all <50% at 10 ꢀM). Also, given that 43 contains a basic amine flanked by an
3
1
aromatic moiety, a known pharmacophore for inhibitors of the hERG potassium channel, it was
screened for activity in a patchꢀclamp assay. Compound 43 proved inactive against hERG (<10%
inhibition at 10 ꢀM), thus limiting the potential for cardiotoxicity. Similarly, compound 43
proved negative in both in vitro genetox (Ames mutagenicity; ± Sꢀ9 metabolic activation) and
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hepatotoxicity (glutathione trapping in HLM) assays, suggesting minimal risk for the formation
of reactive metabolites. Given its very attractive profile, 43 was selected for advanced efficacy,
DMPK, and toxicological profiling, the results of which will be the subject of subsequent
publications.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CONCLUSION
We have described the SAR studies leading to the identification of potent, selective, and orally
efficacious smallꢀmolecule inhibitors of LOXL2. Commencing with 4ꢀ(aminomethyl)ꢀ2ꢀ
chloropyridine 2, the introduction of the 3ꢀ(amido)phenoxy moiety improved potency at LOXL2,
and further substitution with CF at Cꢀ6 of the pyridine ring allowed access to compounds with
3
very high selectivity for LOXL2 over LOX. Lastly, ‘tuning’ overall cLogP to between ~1.8 to 3
through variation of the amide substituent yielded compounds with both good blood potency and
distribution to lung. The compound with the best overall profile, racemicꢀtransꢀ(3ꢀ((4ꢀ
(aminomethyl)ꢀ6ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)oxy)phenyl)(3ꢀfluoroꢀ4ꢀhydroxypyrrolidinꢀ1ꢀ
yl)methanone (28) significantly reduced lung fibrosis in a 14ꢀday mouse lung bleomycin model
when dosed orally (QD). The individual (R,R)ꢀenantiomer 43 (LOXL2 IC = 0.074 ꢀM; >400ꢀ
5
0
fold selective over LOX) was ultimately selected as the clinical compound and has since
progressed into healthy volunteer Phase 1 trials, thus making it the ‘firstꢀinꢀclass’ smallꢀmolecule
LOXL2 inhibitor to enter clinical development with the potential to treat a number of fibrotic
diseases.
ACS Paragon Plus Environment

Page 19 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
EXPERIMENTAL SECTION
Chemistry. General Methods. All commercially available solvents and reagents were used as
received. Where appropriate, reactions were carried out under an argon atmosphere. Reactions
were monitored by either analytical thinꢀlayer chromatography (TLC) or by analytical liquid
chromatography mass spectrometry (LCꢀMS) recorded on a Shimadzu LCꢀMS 2020 instrument
using reverseꢀphase conditions (acetonitrile/ water, containing 0.1% formic acid). Flash column
chromatography was conducted under medium pressure either on silica (Merck silica gel 40ꢀ63
10
11
12
13
14
15
16
17
18
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
ꢀm) or on prepacked silica gel cartridges using a flash chromatography system (Biotage,
Uppsala, Sweden), and eluents were monitored by UV light (λ = 254 nm). Unless otherwise
stated, preparative reverseꢀphase HPLC was performed using Shimadzu HPLC systems
employing a Waters XTerra 5 ꢀm preparative C OBD 50 × 100 mm column, eluting with
1
8
acetonitrile/water (containing 0.1% TFA) at a flow rate of 30 mL/min, and eluents monitored by
1
UV light (λ = 254 nm). H NMR spectra were recorded using either a Varian Mercury Plus 300
MHz or Bruker 400 MHz NMR spectrometer. Chemical shifts (δ) are reported as parts per
million (ppm) relative to tetramethylsilane (TMS; internal standard), and coupling constants (J)
are reported in Hz. The following abbreviations are used for multiplicities: s = singlet; br s =
broad singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br m = broad multiplet. Low
resolution mass spectra (MS) were obtained as electrospray ionization (ESI) mass spectra, which
were recorded on a Shimadzu LCꢀMS 2020 instrument using reverseꢀphase conditions
(
acetonitrile/ water, containing 0.1% formic acid). The purity of all tested compounds
synthesized and purchased) was determined to be ≥95% by analytical reverseꢀphase LCꢀMS,
(
which was performed on a Shimadzu LCꢀMS 2020 EV (ESI probe) instrument, using an Agilent
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Zorbax SBꢀC8 5 ꢀm 2.1 x 50 mm column, eluting with a gradient of 5ꢀ100% acetonitrile in
water (containing 0.1% formic acid) over 7 min (at 27 °C), with a flow rate of 0.5 mL/min and
visualized using a wavelength of 254 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
-Aminopropionitrile ½fumarate salt (1). Obtained from a commercial source and used without
1
further purification. H NMR (300 MHz, D O): δ 6.50 (s, 2 × ½H), 3.40 (m, 2H), 2.95 (m, 2H).
2
1
-(2-Chloro-4-pyridinyl)methanamine hydrochloride (2). Obtained from a commercial source
1
and used without further purification. H NMR (300 MHz, DMSOꢀd ): δ 8.71 (br s, 3H), 8.45
6
+
(
m, 1H), 7.70 (m, 1H), 7.55 (m, 1H), 4.11 (s, 2H). LCꢀMS m/z 143.0 (M +1).
2
-Phenoxyisonicotinonitrile (5). A mixture of phenol (679 mg, 7.22 mmol), Cs CO (3.53 g,
2 3
10.83 mmol), and DMA (10 mL) was stirred at rt for 30 min. 2ꢀChloroꢀ4ꢀpyridinecarbonitrile 4
(1.0 g, 7.22 mmol) was added and the mixture heated at 80 °C for 20 h. After cooling the mixture
to rt, water (100 mL) was added followed by brine (15 mL) and aq 2M HCl (15 mL). The
mixture was extracted with EtOAc (2 × 50 mL) and the combined organic layers were dried
(
MgSO ), filtered, and then concentrated under reduced pressure to afford the title compound as
4
1
a white solid (1.34 g, 95%) which did not require further purification. H NMR (300 MHz,
DMSOꢀd ): δ 8.35 (m, 1H), 7.62 (m, 1H), 7.55 (m, 1H), 7.40 – 7.46 (m, 2H), 7.24 (m, 1H), 7.14
6
+
–
7.17 (m, 2H). LCꢀMS m/z 197.0 (M +1).
(2-Phenoxypyridin-4-yl)methanamine hydrochloride (6). To
a
solution of 2ꢀ
phenoxyisonicotinonitrile 5 (250 mg, 1.27 mmol) in a 1:1 mixture of MeOH: EtOAc (4 mL), was
added 10% palladium on activated carbon (5 mol%). The mixture was stirred under hydrogen (1
atmosphere) at rt for 2 h. After removal of hydrogen, the mixture was filtered through a celite
pad and the filtrate was concentrated under reduced pressure to afford (2ꢀphenoxypyridinꢀ4ꢀ
ACS Paragon Plus Environment

Page 21 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
yl)methanamine as a yellow oil (231 mg, 91%) which did not require further purification. H
NMR (300 MHz, DMSOꢀd ): δ 8.02 (m, 1H), 7.34 – 7.44 (m, 2H), 6.99 – 7.21 (m, 5H), 3.71 –
6
3
.73 (m, 2H). A mixture of (2ꢀphenoxypyridinꢀ4ꢀyl)methanamine (231 mg, 1.15 mmol), DCM (2
10
11
12
13
14
15
16
17
18
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
mL) and 2M HCl in Et O (2 mL, 4.0 mmol) was stirred at rt for 15 min. The mixture was
2
concentrated under reduced pressure to afford the title compound as a white solid (272 mg,
1
1
00%). H NMR (300 MHz, DMSOꢀd ): δ 8.61 (m, 1H), 8.17 (m, 1H), 7.00 – 7.50 (m, 6H), 4.00
6
+
–
4.30 (m, 2H). LCꢀMS m/z 201.0 (M +1).
Methyl 4-((4-cyanopyridin-2-yl)oxy)benzoate (7). To a stirred solution of 2ꢀchloroꢀ4ꢀ
pyridinecarbonitrile 4 (1.0 g, 7.22 mmol) and methyl 4ꢀhydroxybenzoate (1.10 g, 7.22 mmol) in
DMA (10 mL), was added Cs CO (3.53 g, 10.83 mmol). The reaction mixture was heated at 80
2
3
°C for 1h. After cooling to rt, the mixture was partitioned between water (200 mL) and EtOAc
100 mL). The organic layer was separated and the aq layer was reꢀextracted with EtOAc (100
(
mL). The combined organic layers were dried (MgSO ), filtered, and then concentrated under
4
reduced pressure. The residue was purified (silica gel; eluting with 0ꢀ60% EtOAc in hexanes) to
1
afford the title compound as a solid (1.07 g, 58%). H NMR (300 MHz, DMSOꢀd ): δ 8.39 (m,
6
1H), 7.99 – 8.03 (m, 2H), 7.74 (m, 1H), 7.63 (m, 1H), 7.28 – 7.32 (m, 2H), 3.84 (s, 3H). LCꢀMS
+
m/z 255.0 (M +1).
4-((4-(((tert-Butoxycarbonyl)amino)methyl)pyridin-2-yl)oxy)benzoic acid (8). To a solution of
methyl 4ꢀ((4ꢀcyanopyridinꢀ2ꢀyl)oxy)benzoate 7 (1.07 g, 4.21 mmol) in a 1:1 mixture of MeOH:
EtOAc (60 mL), was added 10% palladium on activated carbon (5 mol%). The mixture was
stirred under hydrogen (1 atmosphere) at rt for 18 h. After removal of hydrogen, the mixture was
filtered through a celite pad and the filtrate was concentrated under reduced pressure to afford
methyl 4ꢀ((4ꢀ(aminomethyl)pyridinꢀ2ꢀyl)oxy)benzoate as a solid (1.08 g, 100%) which did not
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
require further purification. H NMR (300 MHz, DMSOꢀd ): δ 8.08 (m, 1H), 7.96 – 8.00 (m,
6
+
2
H), 7.15 – 7.22 (m, 3H), 7.10 (m, 1H), 3.83 (s, 3H), 3.78 (s, 2H). LCꢀMS m/z 259.0 (M +1).
A mixture of methyl 4ꢀ((4ꢀ(aminomethyl)pyridinꢀ2ꢀyl)oxy)benzoate (1.08 g, 4.18 mmol), DIEA
1.36 g, 10.54 mmol), diꢀtertꢀbutyl dicarbonate (1.38 g, 6.32 mmol) and THF (30 mL), was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
stirred at rt for 2.5 h. The mixture was partitioned between DCM and aq 0.5M HCl. The organic
layer was separated, dried (MgSO ), filtered, and then concentrated under reduced pressure. The
4
residue was purified (silica gel; eluting with 0ꢀ60% EtOAc in hexanes) to afford methyl 4ꢀ((4ꢀ
1
(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoate as an oil (1.01 g, 67%). H
NMR (300 MHz, DMSOꢀd ): δ 8.10 (m, 1H), 7.96 – 8.00 (m, 2H), 7.52 (m, 1H), 7.19 – 7.22 (m,
6
2
H), 7.04 (m, 1H), 6.89 (m, 1H), 4.15 – 4.18 (m, 2H), 3.84 (s, 3H), 1.37 (s, 9H). LCꢀMS m/z
+
359.0 (M +1). A mixture of methyl 4ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀ
yl)oxy)benzoate (1.0 g, 2.79 mmol), THF (18 mL) and aq 2M LiOH (17 mL), was stirred at rt for
1
6 h. The mixture was diluted with water (25 mL) and adjusted to pH 3 – 4 via careful addition
of aq 1M HCl followed by saturated aq citric acid solution. The mixture was extracted with
EtOAc (2 × 30 mL) and the combined organic layers were dried (Na SO ), filtered, and then
2
4
concentrated under reduced pressure to afford the title compound as a white solid (800 mg, 83%)
1
which did not require further purification. H NMR (300 MHz, DMSOꢀd ): δ 12.83 (s, 1H), 8.08
6
(
m, 1H), 7.96 – 7.99 (m, 2H), 7.52 (m, 1H), 7.15 – 7.17 (m, 2H), 7.02 (m, 1H), 6.88 (s, 1H), 4.13
+
–
4.18 (m, 2H), 1.36 (s, 9H). LCꢀMS m/z 345.0 (M +1).
4
-(Aminomethyl)pyridin-2-yl)oxy)phenyl-benzamides.
General
Procedure
A.
4-((4-
(Aminomethyl)pyridin-2-yl)oxy)-N-(2-methoxyethyl)benzamide hydrochloride (9). To a stirred
solution of 4ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 8 (80 mg,
0
.232 mmol) in DCM (2 mL), was added HATU (132 mg, 0.348 mmol) and the mixture was
ACS Paragon Plus Environment

Page 23 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
stirred at rt for 10 min. 2ꢀMethoxyethylamine (23 mg, 0.302 mmol) and DIEA (90 mg, 0.697
mmol) were added and the mixture stirred at rt for 20 h. The mixture was concentrated under
reduced pressure and the residue partitioned between water (20 mL) and EtOAc (20 mL). The
organic layer was separated, dried (Na SO ), filtered, and then concentrated under reduced
10
11
12
13
14
15
16
17
18
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
2
4
pressure. The residue was purified (silica gel; eluting with 0ꢀ100% EtOAc in hexanes) to afford
tertꢀbutyl ((2ꢀ(4ꢀ((2ꢀmethoxyethyl)carbamoyl)phenoxy)pyridineꢀ4ꢀyl)methyl)carbamate as a
1
white solid (81 mg, 87%). H NMR (300 MHz, DMSOꢀd ): δ 8.48 (m, 1H), 8.08 (m, 1H), 7.85 –
6
7.90 (m, 2H), 7.54 (m, 1H), 7.13 – 7.18 (m, 2H), 7.02 (m, 1H), 6.84 (m, 1H), 4.12 – 4.17 (m,
+
2
H), 3.37 – 3.44 (m, 4H), 3.25 (s, 3H), 1.36 (s, 9H). LCꢀMS m/z 402.0 (M +1). A mixture of
tertꢀbutyl ((2ꢀ(4ꢀ((2ꢀmethoxyethyl)carbamoyl)phenoxy)pyridineꢀ4ꢀyl)methyl)carbamate (81 mg,
0.202 mmol), DCM (2 mL), and 2M HCl in Et O (2.0 mL, 4.0 mmol) was stirred at rt for 16 h.
2
The mixture was concentrated under reduced pressure to afford the title compound as a white
1
solid (51 mg, 75%). H NMR (300 MHz, DMSOꢀd ): δ 8.52 – 8.65 (br m, 4H), 8.17 (m, 1H),
6
7.87 – 7.92 (m, 2H), 7.14 – 7.28 (m, 4H), 4.05 – 4.12 (m, 2H), 3.35 – 3.44 (m, 4H), 3.24 (s, 3H).
+
LCꢀMS m/z 302.0 (M +1).
4-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-phenylbenzamide hydrochloride (10). Prepared from
4ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 8 (80 mg, 0.232
mmol) and aniline (28 mg, 0.302 mmol) according to General Procedure A to afford the title
1
compound as a white solid (69 mg, 84%). H NMR (300 MHz, DMSOꢀd ): δ 10.31 (s, 1H), 8.68
6
(br s, 3H), 8.21 (m, 1H), 7.98 – 8.04 (m, 2H), 7.74 – 7.80 (m, 2H), 7.22 – 7.37 (m, 6H), 7.08 (m,
+
1
H), 4.05 – 4.14 (m, 2H). LCꢀMS m/z 320.0 (M +1).
Methyl 3-((4-cyanopyridin-2-yl)oxy)benzoate (11). To a stirred solution of 2ꢀchloroꢀ4ꢀ
pyridinecarbonitrile 4 (1.0 g, 7.22 mmol) and methyl 3ꢀhydroxybenzoate (1.10 g, 7.22 mmol) in
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
DMA (10 mL), was added Cs CO (3.53 g, 10.83 mmol). The reaction mixture was heated at 80
2
3
°
C for 1h. After cooling to rt, the mixture was partitioned between water (200 mL) and EtOAc
(100 mL). The organic layer was separated and the aq layer was reꢀextracted with EtOAc (100
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mL). The combined organic layers were dried (MgSO ), filtered, and then concentrated under
4
reduced pressure. The residue was purified (silica gel; eluting with 0ꢀ60% EtOAc in hexanes) to
1
afford the title compound as a solid (1.1 g, 60%). H NMR (300 MHz, DMSOꢀd ): δ 8.35 (m,
6
1H), 7.83 (m, 1H), 7.67 – 7.71 (m, 2H), 7.57 – 7.62 (m, 2H), 7.48 (m, 1H), 3.83 (s, 3H). LCꢀMS
+
m/z 255.0 (M +1).
3-((4-(((tert-Butoxycarbonyl)amino)methyl)pyridin-2-yl)oxy)benzoic acid (12). To a solution of
methyl 3ꢀ((4ꢀcyanopyridinꢀ2ꢀyl)oxy)benzoate 11 (1.1 g, 4.32 mmol) in a 1:1 mixture of MeOH:
EtOAc (30 mL), was added 10% palladium on activated carbon (5 mol%). The mixture was
stirred under hydrogen (1 atmosphere) at rt for 5 h. After removal of hydrogen, the mixture was
filtered through a celite pad and the filtrate was concentrated under reduced pressure to afford
methyl 3ꢀ((4ꢀ(aminomethyl)pyridinꢀ2ꢀyl)oxy)benzoate as a colorless oil (1.05 g, 94%) which did
+
not require further purification. LCꢀMS m/z 259.0 (M +1).
A mixture of methyl 3ꢀ((4ꢀ(aminomethyl)pyridinꢀ2ꢀyl)oxy)benzoate (1.05 g, 4.06 mmol), DIEA
(1.31 g, 10.15 mmol), diꢀtertꢀbutyl dicarbonate (1.33 g, 6.09 mmol) and THF (30 mL), was
stirred at rt for 16 h. The mixture was concentrated under reduced pressure. The residue was
partitioned between DCM and aq 0.5M HCl. The organic layer was separated, dried (MgSO4),
filtered, and then concentrated under reduced pressure. The residue was purified (silica gel;
eluting
with
0ꢀ70%
EtOAc
in
hexanes)
to
afford
methyl
3ꢀ((4ꢀ(((tertꢀ
1
butoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoate as a colorless oil (1.17 g, 80%). H
NMR (300 MHz, DMSOꢀd ): δ 8.06 (m, 1H), 7.79 (m, 1H), 7.50 – 7.61 (m, 3H), 7.42 (m, 1H),
6
ACS Paragon Plus Environment

Page 25 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
.01 (m, 1H), 6.87 (s, 1H), 4.15 – 4.20 (m, 2H), 3.83 (s, 3H), 1.37 (s, 9H). LCꢀMS m/z 359.0
+
(M +1).
A
mixture of methyl 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀ
yl)oxy)benzoate (1.17 g, 3.26 mmol), THF (20 mL) and aq 2M LiOH (20 mL), was stirred at rt
for 16 h. The mixture was diluted with water (25 mL) and adjusted to pH 3 – 4 via careful
addition of aq 1M HCl followed by saturated aq citric acid solution. The mixture was extracted
10
11
12
13
14
15
16
17
18
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
with EtOAc (2 × 30 mL) and the combined organic layers were dried (MgSO ), filtered, and then
4
concentrated under reduced pressure to afford the title compound as a white solid (1.12 g, 100%)
1
which did not require further purification. H NMR (300 MHz, DMSOꢀd ): δ 12.90 (s, 1H), 8.06
6
(m, 1H), 7.76 (m, 1H), 7.44 – 7.60 (m, 3H), 7.37 (m, 1H), 7.00 (m, 1H), 6.86 (s, 1H), 4.13 – 4.20
+
(m, 2H), 1.37 (s, 9H). LCꢀMS m/z 345.0 (M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-(2-methoxyethyl)benzamide
hydrochloride
(13).
Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12 (80
mg, 0.232 mmol) and 2ꢀmethoxyethylamine (26 mg, 0.345 mmol) according to General
1
Procedure A to afford the title compound as a white solid (49 mg, 63%). H NMR (300 MHz,
DMSOꢀd ): δ 8.56 – 8.70 (br m, 4H), 8.16 (m, 1H), 7.72 (m, 1H), 7.57 (m, 1H), 7.50 (m, 1H),
6
7.23 – 7.29 (m, 3H), 4.03 – 4.07 (m, 2H), 3.34 – 3.46 (m, 4H), 3.23 (s, 3H). LCꢀMS m/z 302.0
+
(M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-phenylbenzamide hydrochloride (14). Prepared from
3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12 (80 mg, 0.232
mmol) and aniline (32 mg, 0.345 mmol) according to General Procedure A to afford the title
1
compound as a white solid (40 mg, 48%). H NMR (300 MHz, DMSOꢀd ): δ 10.33 (s, 1H), 8.70
6
(br s, 3H), 8.17 (m, 1H), 7.84 (m, 1H), 7.72 – 7.80 (m, 2H), 7.69 (m, 1H), 7.57 (m, 1H), 7.27 –
+
7.36 (m, 5H), 7.08 (m, 1H), 4.05 – 4.13 (m, 2H). LCꢀMS m/z 320.0 (M +1).
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-benzylbenzamide hydrochloride (15). Prepared from
3
ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12 (80 mg, 0.232
mmol) and benzylamine (37 mg, 0.345 mmol) according to General Procedure A to afford the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
title compound as a white solid (42 mg, 49%). H NMR (300 MHz, DMSOꢀd ): δ 9.15 (m, 1H),
6
8.63 (br s, 3H), 8.16 (m, 1H), 7.77 (m, 1H), 7.61 (m, 1H), 7.52 (m, 1H), 7.20 – 7.30 (m, 8H),
+
4
.42 – 4.48 (m, 2H), 4.04 – 4.14 (m, 2H). LCꢀMS m/z 334.0 (M +1).
(3-((4-(Aminomethyl)pyridin-2-yl)oxy)phenyl)(3,4-dihydroisoquinolin-2(1H)-yl)methanone
trifluoroacetate (16). Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀ
yl)oxy)benzoic acid 12 (80 mg, 0.232 mmol) and 1,2,3,4ꢀtetrahydroisoquinoline (46 mg, 0.345
mmol) according to General Procedure A. Additional purification via preparative reverseꢀphase
1
HPLC afforded the title compound as a white solid (38 mg, 35%). H NMR (300 MHz, DMSOꢀ
d6): δ 8.67 (br s, 3H), 8.17 (m, 1H), 7.43 – 7.58 (m, 2H), 7.12 – 7.30 (m, 8H), 4.52 – 4.78 (br m,
2
H), 4.02 – 4.14 (m, 2H), 3.80 (br m, 1H), 3.57 (br m, 1H), 2.78 – 2.88 (m, 2H). LCꢀMS m/z
+
3
60.0 (M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-(benzo[b]thiophen-2-ylmethyl)benzamide
hydrochloride (17). Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀ
yl)oxy)benzoic acid 12 (80 mg, 0.232 mmol) and 1ꢀbenzothiophenꢀ2ꢀylmethanamine (56 mg,
0
.345 mmol) according to General Procedure A to afford the title compound as a white solid (75
1
mg, 76%). H NMR (300 MHz, DMSOꢀd ): δ 9.38 (m, 1H), 8.70 (br s, 3H), 8.16 (m, 1H), 7.82
6
(
m, 1H), 7.72 – 7.80 (m, 2H), 7.62 (m, 1H), 7.53 (m, 1H), 7.24 – 7.35 (m, 6H), 4.67 – 4.74 (m,
+
2
H), 4.06 – 4.12 (m, 2H). LCꢀMS m/z 390.0 (M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-(4-methoxyphenyl)benzamide hydrochloride (18).
Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12 (100
ACS Paragon Plus Environment

Page 27 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mg, 0.290 mmol) and 4ꢀmethoxyaniline (54 mg, 0.436 mmol) according to General Procedure A
1
to afford the title compound as a white solid (64 mg, 57%). H NMR (300 MHz, DMSOꢀd ): δ
6
10.19 (s, 1H), 8.60 (br s, 3H), 8.18 (m, 1H), 7.82 (m, 1H), 7.54 – 7.67 (m, 4H), 7.33 (m, 1H),
10
11
12
13
14
15
16
17
18
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
7
.24 – 7.26 (m, 2H), 6.88 – 6.92 (m, 2H), 4.05 – 4.12 (m, 2H), 3.72 (s, 3H). LCꢀMS m/z 350.0
+
(M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-(4-fluorophenyl)benzamide
hydrochloride
(19).
Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12 (100
mg, 0.290 mmol) and 4ꢀfluoroaniline (48 mg, 0.436 mmol) according to General Procedure A to
1
afford the title compound as a white solid (71 mg, 66%). H NMR (300 MHz, DMSOꢀd ): δ
6
10.39 (s, 1H), 8.65 (br s, 3H), 8.18 (m, 1H), 7.76 – 7.85 (m, 3H), 7.68 (m, 1H), 7.57 (m, 1H),
7.34 (m, 1H), 7.25 – 7.30 (m, 2H), 7.14 – 7.21 (m, 2H), 4.03 – 4.13 (m, 2H). LCꢀMS m/z 338.0
+
(M +1).
3-((4-(Aminomethyl)pyridin-2-yl)oxy)-N-(3-carbamimidoylbenzyl)benzamide dihydrochloride
(20). Prepared from 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid
12 (100 mg, 0.290 mmol) and 3ꢀ(aminomethyl)benzamidine dihydrochloride (97 mg, 0.436
mmol) according to General Procedure A to afford the title compound as a white solid (10 mg,
1
9%). H NMR (300 MHz, DMSOꢀd ): δ 9.35 (m, 1H), 8.78 (br s, 3H), 8.49 (m, 1H), 8.14 (m,
6
1H), 7.70 – 7.82 (m, 3H), 7.64 (m, 1H), 7.46 – 7.58 (m, 3H), 7.26 – 7.32 (m, 3H), 4.49 – 4.51
+
(m, 2H), 4.05 – 4.15 (m, 2H). LCꢀMS m/z 376.0 (M +1).
4
-(3-((4-(Aminomethyl)pyridin-2-yl)oxy)benzamido)benzoic acid hydrochloride (21). To a
stirred solution of 3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridinꢀ2ꢀyl)oxy)benzoic acid 12
200 mg, 0.58 mmol) in DMF (4 mL), was added HATU (442 mg, 1.16 mmol) and the mixture
(
was stirred at rt for 10 min. Methyl 4ꢀaminobenzoate (132 mg, 0.87 mmol) and DIEA (224 mg,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 74
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
.74 mmol) were added and the mixture stirred at rt for 16 h. The mixture was diluted with water
(15 mL) and brine (1 mL) then extracted with EtOAc (3 × 10 mL). The combined organic layers
were separated, dried (Na SO ), filtered, and then concentrated under reduced pressure. The
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
residue was purified (silica gel; eluting with 0ꢀ80% EtOAc in hexanes) to afford methyl 4ꢀ(3ꢀ((4ꢀ
(((tertꢀbutoxycarbonyl)amino)methyl)pyridineꢀ2ꢀyl)oxy)benzamido)benzoate as a yellow foam
1
(189 mg, 68%). H NMR (300 MHz, DMSOꢀd ): δ 10.56 (s, 1H), 8.07 (m, 1H), 7.88 – 7.98 (m,
6
4H), 7.82 (m, 1H), 7.71 (m, 1H), 7.50 – 7.60 (m, 2H), 7.36 (m, 1H), 7.00 (m, 1H), 6.88 (m, 1H),
+
4.15 – 4.18 (m, 2H), 3.82 (s, 3H), 1.38 (s, 9H). LCꢀMS m/z 422.0 (MH ꢀC H ). A mixture of
4
8
methyl
4ꢀ(3ꢀ((4ꢀ(((tertꢀbutoxycarbonyl)amino)methyl)pyridineꢀ2ꢀyl)oxy)benzamido)benzoate
(153 mg, 0.320 mmol), THF (1.5 mL) and aq 2M LiOH (1.5 mL) was stirred at rt for 4 h. The
mixture was diluted with water (4 mL) and adjusted to pH 3 – 4 with saturated aq citric acid. The
mixture was extracted with EtOAc (2 × 30 mL) and the combined organic layers were dried
(MgSO ), filtered, and then concentrated under reduced pressure. The residue was purified (silica
4
gel; eluting with 0ꢀ100% EtOAc in hexanes) to afford 4ꢀ(3ꢀ((4ꢀ(((tertꢀ
butoxycarbonyl)amino)methyl)pyridineꢀ2ꢀyl)oxy)benzamido)benzoic acid as an offꢀwhite solid
1
(87 mg, 59%). H NMR (300 MHz, DMSOꢀd ): δ 12.70 (br s, 1H), 10.52 (s, 1H), 8.07 (m, 1H),
6
7.80 – 7.94 (m, 5H), 7.71 (m, 1H), 7.50 – 7.60 (m, 2H), 7.36 (m, 1H), 7.00 (m, 1H), 6.88 (m,
+
1H), 4.15 – 4.17 (m, 2H), 1.38 (s, 9H). LCꢀMS m/z 408.0 (MH ꢀC H ). A mixture of 4ꢀ(3ꢀ((4ꢀ
4
8
(((tertꢀbutoxycarbonyl)amino)methyl)pyridineꢀ2ꢀyl)oxy)benzamido)benzoic acid (87 mg, 0.188
mmol), DCM (2 mL), and 2M HCl in Et O (2 mL, 4 mmol) was stirred at rt for 16 h. The
2
mixture was concentrated under reduced pressure to afford the title compound as a white solid
1
(39 mg, 52%). H NMR (300 MHz, DMSOꢀd ): δ 10.60 (s, 1H), 8.55 (br s, 3H), 8.19 (m, 1H),
6
ACS Paragon Plus Environment

Page 29 of 74
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
.82 – 7.96 (m, 5H), 7.70 (m, 1H), 7.60 (m, 1H), 7.37 (m, 1H), 7.22 – 7.27 (m, 2H), 4.08 – 4.15
+
(m, 2H). LCꢀMS m/z 364.0 (M +1).
Methyl 3-((4-cyano-6-(trifluoromethyl)pyridin-2-yl)oxy)benzoate (23). To a stirred solution of
10
11
12
13
14
15
16
17
18
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
2
ꢀchloroꢀ6ꢀ(trifluoromethyl)isonicotinonitrile 22 (4.0 g, 19.4 mmol) and methyl 3ꢀ
hydroxybenzoate (3.24 g, 21.3 mmol) in a mixture of THF/DMF (4:1, 55 mL), was added K CO
2
3
(8.0 g, 58 mmol). The reaction mixture was heated at 60 °C for 2h. The THF was evaporated
under reduced pressure and the remaining mixture was partitioned between water (200 mL) and
EtOAc (100 mL). The organic layer was separated and the aq layer was reꢀextracted with EtOAc
(100 mL). The combined organic layers were dried (Na SO ), filtered, and then concentrated
2 4
under reduced pressure. The residue was purified (silica gel; eluting with 0ꢀ50% EtOAc in
1
hexanes) to afford the title compound as a light yellow solid (5.63 g, 91%). H NMR (300 MHz,
DMSOꢀd ): δ 8.21 (m, 1H), 8.07 (m, 1H), 7.87 (m, 1H), 7.77 (m, 1H), 7.64 (m, 1H), 7.55 (m,
6
+
1
H), 3.85 (s, 3H). LCꢀMS m/z 323.0 (M +1).
3-((4-(((tert-Butoxycarbonyl)amino)methyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)benzoic acid
(24). To a stirred solution of methyl 3ꢀ((4ꢀcyanoꢀ6ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)oxy)benzoate
23 (1.5 g, 4.65 mmol) in THF/MeOH (1:1, 140 mL) at 0 °C, was added portionꢀwise CoCl (1.8
2
g, 13.98 mmol) followed by NaBH (1.77 g, 46.5 mmol). The reaction mixture was stirred at 0
4
°C for 20 min. The mixture was diluted with EtOAc (100 mL) and filtered through celite. The
filtrate was concentrated and the resulting residue was partitioned between water (200 mL) and
EtOAc (200 mL). The waterꢀorganic mixture was filtered through celite and the organic layer
was separated, dried (Na SO ), filtered, and then concentrated under reduced pressure to obtain
2
4
methyl 3ꢀ((4ꢀ(aminomethyl)ꢀ6ꢀ(trifluoromethyl)pyridinꢀ2ꢀyl)oxy)benzoate as an amber oil (1.38
1
g, 92%) which did not require further purification. H NMR (300 MHz, DMSOꢀd ): δ 7.83 (m,
6
ACS Paragon Plus Environment