2-Aminomethylene-5-sulfonylthiazole Inhibitors of Lysyl Oxidase (LOX) and LOXL2 Show Significant Efficacy in Delaying Tumor Growth

Article abstract of DOI:10.1021/acs.jmedchem.9b01112

The lysyl oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing the formation of cross-links in fibrillar elastin and collagens leading to extracellular matrix (ECM) stabilization. These enzymes have also been implicated in tumor progression and metastatic disease and have thus become an attractive therapeutic target for many types of invasive cancers. Following our recently published work on the discovery of aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors, we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz) scaffold. Incorporation of a thiazole core leads to improved potency toward LOXL2 inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved pharmacokinetic properties and excellent antitumor efficacy, with significantly reduced tumor growth in a spontaneous breast cancer genetically engineered mouse model.

Full text of DOI:10.1021/acs.jmedchem.9b01112

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Article
2-Aminomethylene-5-Sulfonylthiazole Inhibitors of Lysyl Oxidase
(LOX) and LOXL2 Show Significant Efficacy in Delaying Tumor Growth
Deborah A Smithen, Leo Leung, Mairi Challinor, Rae Lawrence, HaoRan Tang, Dan
Niculescu-Duvaz, Simon P Pearce, Robert Mcleary, Filipa Lopes, Mohammed Aljarah,
Michael Brown, Louise Johnson, Graeme Thomson, Richard Marais, and Caroline Springer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01112 • Publication Date (Web): 20 Aug 2019
Downloaded from pubs.acs.org on August 21, 2019
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2-Aminomethylene-5-Sulfonylthiazole Inhibitors
of Lysyl Oxidase (LOX) and LOXL2
Show Significant Efficacy in
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Delaying Tumor Growth
a,b
a,b
a
a
Deborah A. Smithen, Leo M. H. Leung, Mairi Challinor, Rae Lawrence,
c
a,b
d
a,b
HaoRan Tang, Dan Niculescu-Duvaz, Simon P. Pearce, Robert McLeary, Filipa
a,b
a,b
a,b
b
a
Lopes, Mohammed Aljarah, Michael Brown, Louise Johnson, Graeme Thomson,
c
a,b
Richard Marais, Caroline Springer *
a. Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester,
Alderley Park, Macclesfield, SK10 4TG, United Kingdom
b. Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research,
1
5 Cotswold Road, London, SM2 5NG, United Kingdom
c. Molecular Oncology Team, Cancer Research UK Manchester Institute, University of
Manchester, Alderley Park, Macclesfield, SK10 4TG, United Kingdom
d. Clinical and Experimental Pharmacology, Cancer Research UK Manchester Institute,
University of Manchester, Alderley Park, Macclesfield, SK10 4TG, United Kingdom
ABSTRACT
The Lysyl Oxidase (LOX) family of extracellular proteins plays a vital role in catalyzing
the formation of crosslinks in fibrillar elastin and collagens leading to extracellular matrix
(ECM) stabilization. These enzymes have also been implicated in tumor progression and
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metastatic disease, and have thus become an attractive therapeutic target for many types of
invasive cancers. Following on from our recently published work on the discovery of
aminomethylenethiophenes (AMTs) as potent, orally bioavailable LOX/LOXL2 inhibitors,
we report herein the discovery of a series of dual LOX/LOXL2 inhibitors, as well as a
subseries of LOXL2-selective inhibitors, bearing an aminomethylenethiazole (AMTz)
scaffold. Incorporation of a thiazole core leads to improved potency towards LOXL2
inhibition via an irreversible binding mode of inhibition. SAR studies have enabled the
discovery of a predictive 3DQSAR model. Lead AMTz inhibitors exhibit improved
pharmacokinetic properties and excellent anti-tumor efficacy, with significantly reduced
tumor growth in a spontaneous breast cancer genetically engineered mouse model.
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INTRODUCTION
The Lysyl Oxidase (LOX) family of copper-dependent extracellular proteins comprises
1
-6
the founder member enzyme, LOX, and four LOX-like enzymes (LOXL1-4). Whilst there
is greater than 50% sequence identity between the isoforms, which includes a conserved C-
terminal catalytic domain across the family containing the copper binding site and the lysine
tyrosylquinone (LTQ) co-factor, the enzymes can be divided into two sub-groups based on
differences to the N-terminal structure. Indeed, LOX and LOXL1 contain a variable N-
terminal propeptide that undergoes proteolytic cleavage to form the active enzyme
extracellularly. LOXL2-4 differ in that they do not possess this propeptide region and instead
contain four scavenger receptor cysteine-rich (SRCR) domains at the N-terminus, which are
thought to mediate protein-protein interactions in the ECM. In the case of LOXL2, the
protein undergoes proteolytic cleavage of the first two SRCR domains upon secretion;
7
-8
however, unlike LOX, processing is not required for catalytic activation.
The most widely studied function of the LOX enzymes is their ability to form cross-links
in fibrillar elastin and collagens through oxidative deamination of specific lysyl residues, thus
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stabilizing the ECM. However, recent reports suggest that these enzymes have a multitude of
biological functions, which include cell proliferation and epithelial-mesenchymal transition
3
, 5, 9
(EMT).
Consequently, the more widely studied LOX and LOXL2 isoforms have been
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implicated in tumor progression, where they are highly expressed and actively involved in
10-16
remodeling the tumor microenvironment.
The LOX family has thus become an attractive therapeutic target for the treatment of
many types of invasive cancers, particularly those with poor patient outcomes. Targeting
LOXs with small molecule inhibitors is very challenging owing to the lack of crystal
structures useful for drug design for any of the isoforms (the only reported LOXL2 structure
1
7
is a precursor state without cofactor formed), and difficulties associated with isolating
several of the enzymes in an active form. Nevertheless, in recent years several LOXL2-
selective inhibitors have been reported, including haloallylamine-based inhibitors PXS-S1A
1
8
and the highly potent PXS-S2A (full structures not disclosed), as well as dual
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LOXL2/LOXL3 inhibitors PXS-5153A (1) and aminomethylenepyridine 2 (Figure 1) . Our
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1-22
dual LOX/LOXL2 inhibitor CCT365623 (3a)
is an orally efficacious
aminomethylenethiophene- (AMT) based inhibitor, which has been used to help elucidate
mechanisms by which LOX drives tumor progression. These novel inhibitors offer significant
2
3-24
advantages over the prototypical pan-LOX inhibitor β-aminopropionitrile (BAPN),
whose lack of sites amenable for chemical modification precludes preclinical optimization.
Figure 1. Small molecule inhibitors of LOX family enzymes
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We recently reported the discovery of AMT inhibitor CCT365623 (3a) following a
significant medicinal chemistry campaign to elucidate the structure activity relationship
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(
SAR) of this class of compound with respect to LOX inhibition. Systematic modifications
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were made to a hit compound identified following a high-throughput screen (HTS), leading to
development of sub-micromolar half maximal inhibitory concentration (IC ) inhibitors
5
0
possessing desirable selectivity and pharmacokinetic (PK) properties.
Figure 2. LOX and LOXL2 activity of analogous AMT and AMTz inhibitors.
During the course of these studies, the 2,5-substituted thiophene core was replaced with
various other 5-membered heterocyclic rings to ascertain the importance of this moiety on
activity. Of those assessed, only a 2-aminomethylene-5-sulfonyl thiazole core retains activity,
with naphthalenesulfonyl-substituted thiazole 6 showing comparable levels of LOX
2
2
inhibition to the analogous thiophene compound 4 (Figure 2). By contrast, thiazole
regioisomer 5 is relatively inactive. Interestingly, in the case of the active thiazole compound
(
6) we also observe a modest increase in potency towards LOXL2 inhibition, with 6 proving
equipotent against both isoforms. These observations prompted parallel investigations into
the development of 2-aminomethylene-5-sulfonylthiazoles (AMTz) as dual LOX/LOXL2
inhibitors and, given the commercial availability of purified LOXL2 enzyme, these studies
were carried out using LOXL2 enzyme, both as a target and as a surrogate to assess LOX
activity.
RESULTS AND DISCUSSION
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Time-Dependent Inhibition. Initial studies concerned the enzyme-inhibitor preincubation
time in our biochemical assay, whereby we assessed whether our compounds inhibit enzyme
activity in a time-dependent manner. Dual LOX/LOXL2 inhibitors 4 and 6, along with the
less active 5-aminomethylene-2-sulfonyl thiazole regioisomer 5, were assessed using longer
preincubation times of 1 hour and 3 hours, and the activity compared to previously obtained
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0 minute preincubation data (Table 1). The results of this study demonstrate that longer
preincubation times result in increased levels of enzyme inhibition with the greatest
difference in effect observed when the time is increased from 20 minutes to 1 hour, upon
which up to a fivefold increase in potency is observed. Further increasing the time to 3 hours
has a smaller positive effect on activity, whilst remaining within twofold of the 1 hour data.
Based on these findings we decided to employ a 1 hour preincubation time for the remainder
of the studies described herein, which provides an optimal determination of enzyme
inhibition at a physiologically relevant time point.
Table 1. Effect of Preincubation Time on LOXL2 Potency
LOXL2
LOXL2
1 hour
IC50
LOXL2
3 hour
IC50
2
0 min
Entry Cmpd
X
Y
IC50
a,b
a,c
a,d
(µM)
(µM)
(µM)
3
4
2
2
0.86
0.69
.40
.76
6.8
7.0
0.81
0.85
6.41
6.02
0.28
0.47
0.59
3.54
3.19
0.12
0.12
1
2
3
4
5
6
CH
CH
N
CH
N
CH
[0.22, 0.36]
a
Reported IC50 values were determined in at least 2 separate experiments (n ≥ 2). When n = 2, individual IC50
values are shown. When n > 2, the values are reported as the geometric mean with the corresponding 95%
b
c
confidence interval in square brackets; Compound was preincubated with enzyme for 20 minutes; Compound
d
was preincubated with enzyme for 1 hour; Compound was preincubated with enzyme for 3 hours.
Thiophene vs Thiazole Core. Optimization studies began with our recently published
2
2
AMT inhibitor (3a), whereby we modified the core to the analogous 1,3-thiazole compound
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(
7a) and assessed LOXL2 activity inhibition (Table 2, compare entries 1 and 4). Pleasingly,
this gives a modest increase in potency, with an IC of 0.086 µM. We then assessed whether
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this increase in activity, attributed to the presence of a nitrogen atom in the ring, could be
mimicked through the incorporation of an electronegative halogen atom on the thiophene
ring, which could also engender the formation of an intramolecular hydrogen bond to
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improve ligand binding: 3-chlorothiophene (entry 2) was found to be less potent by >
tenfold compared to the parent compound (entry 1), whilst a fluorine substituent was fourfold
less active (entry 3). A second matched pair was synthesized to confirm this trend, and again
the potency achieved with the thiazole analogue was slightly greater than that of the
thiophene compound (compare entries 6 and 7). This suggests that the presence of a nitrogen
atom in the heterocyclic core is advantageous, providing additional stabilization to the
protein-inhibitor complex either through resonance mechanisms or non-covalent interactions.
In order to confirm that the aminomethylene group was still essential for inhibition, we
assessed the effect of an N-methyl substituent group (entry 5). As expected this results in
significant loss of activity, which is consistent with previous conclusions that the
aminomethylene group is required to form a stable Schiff base between the inhibitor and the
LTQ cofactor that is a feature of both LOX and LOXL2.
Table 2. Effects of Thiophene vs Thiazole Cores on LOXL2 Potency
1
2
SO
Position
2
Me
LOXL2
Entry
Cmpd
3a
X
R
R
a
IC (µM)
5
0
0.176
0.105, 0.295]
1
CH
H
H
3
[
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0
0
.488
.164
.671
.714
2
3
4
5
6
7
3b
3c
7a
7b
8
CCl
CF
N
H
H
H
H
3
3
3
3
4
4
0.086
H
H
[
[
[
[
0.061, 0.119]
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
2
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0
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0
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0
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8
9
0
1
2
3
4
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7
8
9
0
6
8.26
43.77, 106.5]
.686
N
CH3
H
H
0
CH
N
CH3
CH3
0.456, 1.031]
0.423
0.302, 0.593]
9
H
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets.
Modification of Thiazole C-5 Group. We next assessed the C-5 substituent group of the
thiazole-based inhibitors, to determine whether the same SAR trends were observed as in the
thiophene series, and whether the lead scaffold remained optimal. An aryl sulfonyl group is
preferred to either an alkyl or cycloalkyl group (Table 3, compare entries 1-3). Focusing on
the aryl substituents, we find that mono- substitution with either a methane sulfonyl (entry 4)
or a phenyl group (entry 6) is tolerated since these are equipotent with the unsubstituted
example (entry 3), but a di-substituted aryl group (entry 8) remains preferred for anti-LOXL2
activity.
We assessed the effect of the oxidation level of the sulfonyl linker and find that use of a
sulfoxide or sulfide linker results in less active compounds (compare entries 8-10). This is
expected, based on previous studies; however, it is noted that the impact resulting from a
decrease in oxidation state is less significant in the AMTz series herein than it was in the
2
2
AMT series. Removal of the sulfonyl linker is tolerated but results in partial loss of activity,
with the most significant impact observed in the biaryl systems (compare entries 4 and 5; 6
and 7; 8 and 11). As explored later (Table 6), this increased tolerance to a range of linkers
appears to be a feature of the thiazole compared to thiophene core, whereby the presence of
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3
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5
5
5
5
5
6
2
6-27
the nitrogen atom in the ring increases the electron-withdrawing properties,
mitigating the need for an electron-withdrawing linker.
thus
Table 3. Effect of Thiazole C-5 Linker (L) and Substituent (R) Groups on LOXL2 Potency
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
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9
0
1
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9
0
LOXL2
IC (µM)
Entry
Cmpd
L
R
a
50
0
0
0
0
.554
.590
.628
.606
1
2
3
4
5
6
10
11
12
13
14
15
0.316
0
0
0
0
.310
.184
.246
.414
0.394
0.366
0
.342
1
1
.751
.596
7
16
0
.086
8
9
7a
7c
7d
17
[
0.061, 0.119]
0
0
.244
.236
0.233
1
0
[
0.19, 0.28]
0
0
.652
.716
11
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets.
Variation of the Phenylsulfonyl Ring Substituents. Incorporation of a bis-sulfonyl biaryl
C-5 group is advantageous for activity and was previously found to improve oral in vivo PK
2
2
exposure in the AMT series of LOX inhibitors. We subsequently looked to ascertain if there
was scope to optimize the substituent effects further (Table 4). Varying the methanesulfonyl
2
group (R ) to an ethyl or isopropyl group is tolerated, albeit resulting in a drop in potency;
however, a larger tert-butyl group proves detrimental to activity (compare entries 1-4). A
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methoxy substituent is similarly well tolerated (entry 5), whilst a methylamino group appears
to be less favorable (entry 6).
1
Small modifications to the aryl substituent (R ) do not result in a significant change in
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6
7
8
9
0
potency. Inhibitors bearing either an electron-donating p-methyl group, or an electron-
withdrawing p-fluoro or p-trifluoromethyl substituent demonstrate comparable levels of
activity to the parent compound (compare entries 7-9 to entry 1). Replacing the phenyl group
with an N-methyl pyrazolyl group is found to be favorable in conjunction with an ethyl group
2
(
compare entries 2 and 11), though does not appear to confer additional potency when R is a
methanesulfonyl group (compare entries and 1 and 10). Further modification of the ethyl
group of 21b to an electron-withdrawing trifluoromethyl or chloro group have a slightly
negative effect on activity, though a fluoro substituent is well tolerated (compare entries 11-
1
4). Overall, from these results we conclude that the parent inhibitor 7a is well optimized,
whilst gaining further understanding of the SAR and identifying additional scaffolds for
further study.
Table 4. Effects of 3,5-Disubstituted Phenylsulfonyl Groups on LOXL2 Potency
1
2
LOXL2
Entry
Cmpd
R
R
a
IC (µM)
5
0
0
.086
[0.061, 0.12]
.38
1
2
3
4
5
7a
7e
7f
Ph
Ph
Ph
Ph
Ph
SO Me
2
0
Et
[
0.27, 0.54]
i
0.343
Pr
0
.370
1.04
[0.62, 1.7]
t
7g
7h
Bu
0
0
.270
.255
OMe
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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
0
0
.417
.397
.103
6
7
8
9
7i
18
19
20
Ph
NHMe
p-Tol
SO Me
2
[
[
0.083, 0.13]
0.174
0.085, 0.35]
p-CF -Ph
SO Me
3
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
0
0
.197
.192
p-F-Ph
SO Me
2
0
.109
10
21a
21b
21c
21d
21e
SO Me
2
[
0.066, 0.18]
0.151
[0.12, 0.19]
1
1
Et
CF3
Cl
0
0
.259
.374
12
0.280
0.16, 0.49]
1
1
3
4
[
0
.143
F
[
0.057, 0.36]
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets.
Activity of AMTz Regioisomers. Final SAR studies concerned the substitution pattern
around the thiazole core, whereby we compared the anti-LOXL2 activity of selected 2,5-
substituted AMTz inhibitors to that of their 2,4-substituted regioisomers (Table 5). Although
there is a trend favoring 5-substitution vs 4-substitution, it is again less pronounced than that
2
2
previously observed in the thiophene series against LOX (compare entries 1 and 2; 3 and 4;
and 6; 7 and 8).
5
Table 5. Effects of 2,4- vs 2,5-Substitution of Thiazole Core on LOXL2 Potency
LOXL2
Entry
Cmpd
Position
R Structure
a
IC (µM)
5
0
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5
6
7
8
9
0
.28
1
2
3
6
5
4
5
[
0.22, 0.36]
0
0
.895
.828
22a
7a
0.086
[
[
0.061, 0.12]
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
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
0.302
4
22b
4
[
0.22, 0.42]
.109
0
5
6
21a
22c
5
4
0.066, 0.18]
0.37
[0.23, 0.596]
.151
0
7
8
21b
22d
5
4
[0.12, 0.19]
.425
0.35, 0.51]
0
[
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets.
Selectivity Studies
We assessed the selectivity profiles of our AMTz inhibitors against LOX and LOXL3
isoforms (Table 6), and against common amine oxidases and the potassium ion channel
hERG (Table 7).
Activity Profiles against LOX and LOXL3 Isoforms. Whilst use of LOXL2 as a surrogate
for LOX was a practical and informative approach, it was necessary to assess the activity of a
range of AMTz inhibitors against LOX to confirm our belief that these are in fact dual
LOX/LOXL2 inhibitors. A diverse set of compounds were selected, encompassing 2-
aminomethylene-5-sulfonyl- (7a, 7e, 21a and 21b) and 2-aminomethylene-4-sulfonyl- (22b,
2
2c and 22d) AMTz regioisomers, sulfide-linked 7d, and 17 with no linker, as well as
thiophene 3a and BAPN. From this study we were able to confirm that 2-aminomethylene-5-
sulfonyl thiazoles 7a, 21a and 21b, thiophene 3a and BAPN exhibit good anti-LOX activity
2
1-22, 28
that is in accordance with previously obtained data.
A slight decrease in potency
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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
towards LOX inhibition is observed with compound 7e, which is consistent with LOXL2
activity. Removal of the sulfonyl linker (17) is detrimental to activity; however, a sulfide
linker (7d) is well tolerated which, as discussed previously, can be attributed to the presence
of a nitrogen atom in the thiazole core which presumably increases the ability of these
compounds to form a stable covalent bond upon binding. Interestingly, 2,4-AMTz
regioisomers (22b, 22c and 22d) are found to be inactive against LOX, which follows a
similar trend to the thiophene series in which 2,4-regioisomers were demonstrated to be 15-
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
22
fold less potent. As seen previously, all AMTz inhibitors demonstrate increased potency
towards LOXL2 inhibition versus LOX.
Table 6. Activity of AMT and AMTz Inhibitors against LOX, LOXL2 and LOXL3
LOXL2
LOX
LOXL3
Cmpd
BAPN
a
a
a
IC (µM)
IC (µM)
IC (µM)
50
50
50
0
.665
0.57, 0.77]
.176
[0.105, 0.295]
.086
0.061, 0.12]
.233
3.14
0.31
[
[1.79, 5.49]
0.898
0.728
1.77
[0.22, 0.45]
13.49
18.54
0.92
0
3
7
a
a
0
[
1.49
1.31
1.13
0.86
4.70
3.85
0
7
d
[
0.19, 0.28]
0.38
[0.27, 0.54]
7.17
6.91
6.81
6.78
7
e
0
0
0
.652
.716
.109
51.96
46.49
3.42
2.65
3.3
9.31
9.52
1.22
1.51
1.25
1.43
3.86
4.26
7.56
6.69
24.24
36.86
1
7
2
1a
1b
22b
2c
2d
[
0.066, 0.18]
0.151
2
[0.12, 0.19]
.302
0.22, 0.42]
.37
1.86
0
>100
>100
>100
>100
>100
>100
[
0
2
[
0.23, 0.596]
0.425
0.35, 0.51]
2
[
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets.
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5
6
7
8
9
As discussed previously, literature compounds 1 and 2 are in fact dual LOXL2/LOXL3
19-20
inhibitors,
whilst selectivity data concerning LOXL isoforms has not been reported for
other literature inhibitors. As a member of the LOX family, LOXL3 is known to modulate the
29-31
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
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
ECM,
though there is tissue expression variance compared to other LOX proteins, and it
has been shown to play a significant role in muscular, skeletal and lung development in mice.
3
2-34
More recently, studies have demonstrated an involvement of LOXL3 in cancer and
3
5-36
metastasis, suggesting it to be a potential therapeutic target for malignant disease.
With regards to LOXL3 inhibition, it is interesting to observe that our AMT and AMTz
compounds exhibit moderate to high selectivity towards LOXL2 in all cases (Table 6), unlike
the non-selective LOX-family inhibitor BAPN and reported literature compounds 1 and 2
1
9-20
(activity within threefold for LOXL2 and LOXL3 in all cases).
2,5-AMTz inhibitors 7a,
7
e, 21a and 21b demonstrate ≥ tenfold selectivity towards LOXL2, with IC50 values of
approximately 1 µM; compound 7e is consistently less potent against all isoforms.
Modification of the sulfonyl linker to a sulfide (7d) or direct aryl-linked compound (17)
results in a decrease in potency, comparable to that observed against LOXL2. Increased
selectivity is observed with 2,4-AMTz regioisomers (22b-d), in particular compound 22d
which does not possess a bis-sulfonyl group. AMT inhibitor 3a is found to be a weak
inhibitor of LOXL3, providing selectivity in excess of 100-fold. This study demonstrates that
2
-aminomethylene-5-sulfonyl thiazoles are potent inhibitors of three LOX isoforms, whilst
selectivity towards LOXL2, particularly 2,4-AMTz regioisomers, supports their use as
valuable tool compounds to study the biology and functions of LOXL2. Owing to the lack of
20
availability and difficulties involved in obtaining other LOXL enzymes in an active form,
we have been unable to assess selectivity of our compounds over other LOX-family
members.
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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
Selectivity over Common Amine Oxidases and hERG. A selection of compounds including
our most potent AMTz inhibitors were assessed for their selectivity over the flavin-containing
monoamine oxidases (MAO) A and B, copper-containing diamine oxidase (DAO) and
semicarbazide-sensitive amine oxidase (SSAO), and the hERG channel (Table 7). In general,
our inhibitors show excellent selectivity over MAO-A and B, and DAO. Bis-sulfonyl
compounds 7a, 18 and 21a, along with biphenyl compound 7e, demonstrate moderate SSAO
inhibition; N-Methyl pyrazolyl inhibitors 21b and 21e show improved selectivity, and further
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
22
studies carried out with 21b indicate that it is not a substrate of SSAO, unlike 3a. Bis-
sulfonyl compounds 7a and 18 are also found to be moderate inhibitors of the hERG channel,
whilst others compounds assessed show good selectivity. Based on the in vitro activity and
selectivity profiles, 2-aminomethylene-5-sulfonylthiazole inhibitors 6, 7e, 21b and 21e, along
with 7a for direct comparison with thiophene 3a and 22d as an exemplar of the 2,4-AMTz
subseries, were advanced to metabolic stability assessment and in vivo mouse PK studies.
Table 7. Potency and Selectivity of AMTz Inhibitors over Common Amine Oxidases and
hERG
LOXL2
MAO-A
IC50a
MAO-B
IC50a
DAO
IC50a
(M)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
21.3
22.5
SSAO
IC50a
(M)
47.2
47.1
3.2
1.2
5.2
3.6
2.7
1.3
13.3
17.2
28.6
19.6
hERG
IC50b
(M)
Cmpd
6
IC50
(M)
a
(M)
>100
(M)
>100
0.28
0.22, 0.36]
51
10
36
3.2
nd
47
66
nd
[
>100
63.8
59.3
29.2
31.4
100
88.3
>100
>100
>100
>100
61.7
66.8
>100
>100
>100
64.3
46.2
16.4
20.0
>100
>100
>100
>100
>100
>100
>100
>100
0
.086
7
a
[
0.061, 0.12]
0.38
7
e
[
0.27, 0.54]
.103
0
18
1a
21b
1e
22b
[
[
0.083, 0.13]
0.109
0.066, 0.18]
2
0
.151
[
0.12, 0.19]
0.143
36.3
40.3
>100
>100
2
[0.057, 0.36]
.302
0.22, 0.42]
0
>100
>100
>100
>100
[
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4
5
6
7
8
9
0
.37
0.23, 0.596]
.425
[0.35, 0.51]
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
102.2
74.3
22c
2d
nd
29
[
0
2
a
Reported IC50 values were determined using 1 hour preincubation in at least 2 separate experiments (n ≥ 2).
When n = 2, individual IC50 values are shown. When n > 2, the values are reported as the geometric mean with
the corresponding 95% confidence interval in square brackets. nd – not determined.
b
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
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
Pharmacokinetic Evaluation
All compounds assessed in vivo demonstrate good metabolic stability against mouse liver
microsomes (MLM) and inhibitor exposure following oral administration in mouse PK
studies (Table 8). Naphthalenesulfonyl compound 6 shows low to moderate plasma exposure
(
AUC 1-10 µM), as does 3-ethyl-5-phenyl inhibitor 7e and 21e bearing N-methyl pyrazolyl
and fluoro substituents. Pleasingly, a number of compounds assessed demonstrate desirable
PK profiles, achieving greater plasma exposure levels than that of our previously published
22
inhibitor (3a), and excellent oral bioavailability. Indeed, compounds 7a, 21b and 22d have
AUC’s above 18 µM.hr, achieving Cmax concentrations of up to 32 µM. These compounds
also exhibit good permeability in the Caco-2 assay, used to model adsorption of orally
administered drugs in the small intestine, and present lower efflux levels than those seen
previously for thiophene 3a. Given our observations that these compounds demonstrate time-
dependent inhibition (see Tables 1 and 9), it is likely that efficacy would be driven by Cmax
.
Table 8. In Vitro Mouse Liver Microsome (MLM) Stability, in Vivo PK Properties and
Caco-2 Permeability of AMTz Inhibitors
e,f
Caco-2 P
app
MLM
stability
-6
C
AUC(PO)
t1/2(PO)
max(PO)
e
(10 cm/s)
Cmpd
6
F (%)
c
d
a
(M)
(M.hr)
(hr)
(
%)
A→B B→A
89
6.35
9.75
4.20
4.48
9.97
0.8
2.0
0.6
nd
nd
nd
nd
nd
nd
8.5
nd
nd
nd
35
2
1e
90
70
7
e
10.16
3
a
i) Mouse
i) 64
i) 16.83
i) 14.89
i) 0.45
i) 45
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5
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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
b
ii) Rat
1b
i) Mouse
ii) Rat
ii) 35
ii) 0.34
ii) 0.22
ii) 0.5
ii) 0.2
2
i) 76
i) 26.71
ii) 6.5
i) 18.5
ii) 18.5
i) 1.12
ii) 1.3
i) 98
ii) 68
31
29
b
ii) 82
2
2d
97
27.22
20.98
1.0
68
23
23
51
33
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
7
a
100
32.03
21.61
0.89
nd
a
Mouse liver microsome (MLM) stability values represents the percentage of compound remaining after 30
minutes; Rat liver microsome (RLM) stability values represents the percentage of compound remaining after 30
minutes; Mouse plasma PK parameters were determined following a single PO dose at 50 mg/kg or IV dose at
0 mg/kg; Rat plasma PK parameters were determined following a single PO dose at 20 mg/kg or IV dose at 4
mg/kg. C – maximum concentration. AUC – area under curve. nd – not determined. P – permeability
b
1
c
d
e
f
max
app
coefficient.
Further PK studies were carried out using AMT compound 3a and AMTz 21b in a rat
model. Metabolic stability against rat liver microsomes (RLM) is moderate in 3a and good in
2
1b; however, a significant difference in oral bioavailability is observed between the
compounds, with 3a demonstrating very poor levels of plasma exposure. In contrast, 21b
exhibits comparable AUC’s of 18 µM.hr between the species, and maintains a good Cmax
concentration (6.5 µM) and oral bioavailability (68%). Based on the balance of potency,
selectivity profile, PK and permeability, compound 21b is determined to have the best profile
overall and thus was chosen for further in vivo anti-tumor efficacy evaluation.
Evaluation of Anti-Tumor Efficacy
In vivo efficacy studies involving compound 21b were carried out using a genetically
engineered mouse model (GEMM) that functions as a LOX-driven spontaneous breast cancer
21
model. Mice were dosed daily via oral gavage (70 mg/kg) from around 60 days post birth,
once primary tumors become palpable. Inhibitor 21b was very well tolerated, with no
observed body weight loss.
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9
Figure 3. a) Anti-tumor efficacy of compound 21b in MMTV-PyMT GEMM model. Control
animals (n = 5; grey) or treated with compound 21b at 70 mg/kg qd (n = 3; green). Day 91
*
p = 0.0367, Welch’s t-test; b) Kaplan-Meier survival analysis.
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
1
2
3
4
5
6
7
8
9
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Pleasingly, we observe a delay in primary tumor development and a significant reduction
in tumor growth rate in the 21b-treated group, compared to that of the controls (Figure 3a),
with no deaths due to tumor volume reaching ethical size limits necessitated in the inhibitor-
treated group during the course of this study (Figure 3b).
Potency and Mode of Inhibition (MOI) of AMT and AMTz Inhibitors
We assessed the time-dependent inhibitory activity of select AMT and AMTz compounds
(Table 9) and investigated the mode of enzyme inhibition for key series examples (Figure 4).
Time-Dependent Inhibition of Lead Compounds. We previously demonstrated that series
exemplars of AMT and AMTz compounds inhibit LOXL2 in a time-dependent manner
(
Table 1); we now sought to ascertain if this trend remained true for the series in general. As
such, a range of AMTz inhibitors including potent 2-aminomethylene-5-sulfonyl compounds
a, 21a and 21b, 2,4-regioisomers 22b, 22c and 22d, along with AMT compound 3a and
7
BAPN, were assessed for anti-LOXL2 activity following different enzyme preincubation
times (Table 9). All compounds were found to exhibit time-dependent inhibition, with the
most significant effect again observed on increasing the time from 20 minutes to 1 hour,
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whereupon up to a sevenfold increase in activity is observed; further increasing the
preincubation time to 3 hours provides a small additional increase in activity, with IC ’s <
5
0
0
.1 µM attained for all 2-aminomethylene-5-sulfonylthiazoles (7a, 21a, and 21b) assessed.
0
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Table 9. Time-Dependent Activity of Lead AMTz Inhibitors
LOXL2
LOXL2
1 hour
IC50
LOXL2
3 hour
IC50
2
0 min
Cmpd
IC50
a,b
a,c
a,d
(µM)
(µM)
(µM)
4.26
3.63, 5.00]
0.665
[0.57, 0.77]
0.176
0.372
[0.31, 0.45]
0.157
BAPN
3a
[
0
0
0
0
.439
.460
.587
.648
[0.105, 0.295]
0.126
0.069
0.075
0.072
0.069
0.078
0.079
0.090
0.085
0.146
0.157
0.199
0.086
[0.061, 0.12]
0.109
7
a
0.544
2
1a
21b
2b
2c
22d
0
0
0
1
.331
.710
.687
.252
[0.066, 0.18]
0.151
[0.12, 0.19]
0.302
2
0.530
0.795
[0.22, 0.42]
0.37
[0.23, 0.596]
0.425
2
0
1
1
.787
.238
.373
[0.35, 0.51]
0.176
a
Reported IC50 values were determined in at least 2 separate experiments (n ≥ 2). When n = 2, individual IC50
values are shown. When n > 2, the values are reported as the geometric mean with the corresponding 95%
b
c
confidence interval in square brackets; Compound was preincubated with enzyme for 20 minutes; Compound
d
was preincubated with enzyme for 1 hour; Compound was preincubated with enzyme for 3 hours.
MOI Studies. Given the time-dependent inhibition observed with these compounds we
wanted to clarify the mechanism of LOXL2 inhibition; as such, we set up a jump dilution
assay, which readily distinguishes between reversible and irreversible modes of enzyme
3
7
inhibition (Figure 4). Leading AMT and AMTz inhibitors 3a and 21b, respectively, along
with BAPN were assessed, whereby the enzyme was preincubated for 1 hour with 10 x IC50
of these compounds, at which concentration we see complete inhibition of the enzyme in all
cases. The enzyme/inhibitor mixture was then diluted 100-fold into a solution containing all
enzyme reaction components and the activity of the enzyme was assessed.
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Figure 4. LOXL2 jump dilution assay to assess MOI of key LOXL2 inhibitors
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The resulting curve obtained for 3a displays about 80% recovery of activity following
dilution, as compared to the DMSO control, which suggests that this compound behaves as a
reversible inhibitor under these assay conditions, unlike BAPN which can be characterized as
an irreversible inhibitor. In the case of 21b a more modest regain in activity of around 30% is
observed following the jump dilution. This is suggestive of either a slowly reversible
compound or an irreversible inhibitor whereby a residual amount of enzyme has not
committed to forming a stable covalent bond during the 2-step inhibition process. The results
obtained from this study suggest that the electronic nature of the heterocyclic core affects the
binding mechanism of these inhibitors, with the AMTz inhibitors better able to form stable,
irreversible Schiff base intermediates following initial reversible binding to the LTQ co-
2
0, 38
factor.
One possible explanation for this is that the initial Schiff-base formed is more
susceptible to hydrolysis in the case of 3a compared with 21b, which is better able to
rearrange to form a more stable intermediate either as a result of increased resonance
stabilization or non-covalent electrostatic interactions. Alternatively, this could also be the
result of differences in enzyme-inhibitor kinetics, with initial rates of binding affecting the
ability of these inhibitors to form covalent bonds.
Pharmacophore and QSAR Modeling
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Binding Mode/Pharmacophore Hypothesis Generation. With the dearth of protein-ligand
crystal structures for LOXL2 in its active form, we decided to embark upon a ligand-based
approach to propose a legitimate binding conformation for SAR analysis and modeling.
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FieldTemplater
was used to derive a pharmacophore model, by comparing
conformational ensembles of molecules using their electrostatic and hydrophobic characters
to identify common motifs. The field point pattern for a conformer of 7a is shown in Figure
5.
Figure 5. Sample conformation of 7a showing electrostatic fields and 3D field point pattern,
which provides a concise 3D pharmacophore.
The field-based alignment can be independent of chemical structure, allowing alignment
of molecules from different series. The compounds reported in this article are thus augmented
2
0
with a set of LOXL2 inhibitors obtained from the literature, along with in-house inhibitors
not explicitly described in this article, with only those compounds with pIC >5 included (see
50
supporting information). The collection of 54 molecules (with IC50 data) were visually
inspected, with the four most active, most structurally diverse compounds selected for
2
0
pharmacophore modelling (7a, pIC = 7.07; 21e, pIC = 6.84; JMC2017-31, pIC = 6.55;
5
0
50
50
20
and JMC2017-33, pIC = 6.51).
5
0
The FieldTemplater experiment was run using Normal (large molecules) conformation
+
hunt settings, with a group constraint placed on the cationic NH3 groups to force their
alignment in the templated result, since these compounds are all believed to follow the same
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mechanism of inhibition via covalent binding of the aminomethylene group to the LTQ co-
factor. The best scoring template consisting of all four compounds was taken forward to
4
0-41
Forge,
and the 7a and 21e conformations were used as references for the field-based
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alignment of the other molecules in the dataset.
Structure-Activity Relationship Modelling. We attempted to calculate statistically-relevant
mathematical models to predict activities of new compounds. Pleasingly, we were rewarded
with two predictive, complementary models as described below.
4
0-41
Field-based 3DQSAR.
Cresset’s approach to 3DQSAR is similar to traditional
4
2-43
CoMFA,
however, there are some striking differences around how sampling points are
selected and the use of irregular grids, such that calculation speed is greatly improved. The 54
compound data set was randomly partitioned to put 15% in the test set (8 molecules), leaving
4
6 in the training set, upon which the model was built; tested with 50 y-scrambles, followed
2
by Leave-One-Out (LOO) cross-validation. The resulting 3-component model features an r
2
44
of 0.856, q of 0.665, RMSE of 0.147, and a Kendall’s tau value of 0.760 – a model we
4
5
believe to be statistically relevant and able to predict rank order of activity.
Forge can be used to visualize the field/steric contributions to predicted activities, as
demonstrated in Figure 6c; these plots were helpful in rationalizing the SAR observed for the
reported compounds. The details of this field-based 3DQSAR model are shown in Figures 6a
and 6b, with the model details included in the supplementary information.
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Figure 6. (a) Plot of predicted vs. actual activity for training, training cross-validation, and
test sets. (b) 7a with the electrostatic and steric coefficient positions/values displayed. (c)
Example of the field/steric contributions to predicted activity for 7a.
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As a follow up, and to build confidence in the model and binding mode hypothesis, an
alternate set of machine-learning algorithms: k-Nearest Neighbor (kNN), Random Forest
(RF), Support Vector Machine (SVM), and Relevance Vector Machine (RVM) regression
4
1
models were used to model the data. The statistics for RVM for the full model on the
2
training set were r =0.852, RMSE=0.150, and Kendall’s tau=0.757. For the cross-validation,
the model statistics were q2=0.649, RMSE=0.230, Kendall’s tau=0.638. This model is
similarly predictive to the field-based 3DQSAR model described earlier, and the calculation
of multiple predictive models from a set of molecules aligned to a common binding mode
hypothesis lends support to our having derived a sensible binding mode in the absence of
protein-ligand crystallographic information. While not reported here, it is noteworthy that the
other machine learning models also have statistics that suggest that they are predictive.
Activity Atlas Analysis: Activity Cliff Summary. In tandem with the predictive 3DQSAR
4
0-41
model, Activity Atlas/Activity Miner
was used to conduct a qualitative assessment of the
SAR for the data set. The summary of the calculated electrostatic and steric activity cliffs are
shown in Figure 7, and compare favorably to the 3DQSAR model visualizations. This
information is built up from doing a pair-wise analysis of all molecules in their aligned
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conformation and automatically examining activity cliffs – highly similar pairs where there is
a large change in activity. The most potent and least potent compounds are shown in the
context of the activity cliff summary.
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Figure 7. Activity Atlas/Activity Cliff Summary plots for LOXL2 activity shown with (a) 7a
around thiophene/thiazole core, (b) 7a, 7e and 7g to illustrate steric and electrostatic
contributions (using field points, as described above) that rationalize the observed SAR. All
isosurfaces are shown at ≥2.0 confidence level.
The predicted SAR around the thiazole moiety (Figure 7a) is consistent with the observed
decrease in potency when the thiazole nitrogen is replaced by the larger CX groups (3b, 3c),
suggesting that this position is sterically restricted, and requires negative electrostatics. In
addition, the requirement for negative electrostatics is consistent with the moderate decrease
in potency of the thiophene analogues (3a) and the 2,4 substituted thiazoles (22b).
Examination of the SAR around the thiazole C-5 substituent using Activity Atlas suggests
1
that the meta-position of the phenyl group (R ) is predicted to favor large groups; in 21b,
replacement of phenyl with N-methyl pyrazolyl substituent meets both the desired steric and
electrostatic conditions near the 5-position. With regard to the 3-position, the activity cliff
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summary (Figure 7b) can be used to rationalize the drop in potency on varying the
methanesulfonyl group of 7a to an ethyl (7e) or tert-butyl (7g) group, which increases the
number of cliff violations (i.e., a mismatching of field points with activity cliff summary): In
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a, the 3-sulfonyl group has favorable electrostatics and is able to thread the needle of a
sterically unfavorable region; a larger tert-butyl group has unfavorable electrostatics and
multiple steric clashes that result in diminished activity. Further, in the context of the model,
the central phenyl group is predicted to have a less electron-rich π-cloud, in line with the
observed improvement in potency when small electron-withdrawing groups are present in the
3
-position.
We have developed predictive field-based 3DQSAR and visual qualitative models of
activity based on the LOXL2 inhibition data acquired during the development of the
aminomethylenethiazole inhibitors. This will be a useful tool to aid further development of
the series as well as to design new chemical inhibitors in the future.
SYNTHETIC CHEMISTRY
Sulfone-linked AMTz analogues were synthesized from N-Boc-protected (5-
bromothiazol-2-yl)methanamine 23 and the appropriate thiol, using palladium-catalyzed
coupling methods, as shown in Scheme 1. In the case of aryl thiols,
tris(dibenzylideneacetone)dipalladium catalyst and XantPhos ligand were used in conjunction
with sodium tert-butoxide base in a 4:1 solvent mixture of toluene:tert-butanol. In the case of
alkyl thiols, DIPEA was found to be preferable as a base and toluene was used as the solvent.
Oxidation of the sulfide was achieved using m-CPBA to afford the desired sulfone, and
subsequent Boc-removal using 4 M HCl in dioxane gave rise to the target AMTz inhibitors.
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Scheme 1. General Route to Sulfur-Linked 2,5-AMTz Analogues
t
t
Reagents and conditions: (a) Pd dba , XantPhos, NaO Bu, PhMe, BuOH, 110 °C; (b) Pd dba , XantPhos,
2
3
2
3
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DIPEA, PhMe, 110 °C; (c) m-CPBA, DCM, rt; (d) 4 M HCl in dioxane, rt.
Substituted aryl thiols that were not commercially available were synthesized according to
46
the method shown in Scheme 2. Palladium-catalyzed coupling of an aryl halide with 2-
ethylhexyl 3-mercaptopropanoate afforded a thiol surrogate, that also functions as a thiol
protecting group for further chemical modification. The thiol surrogate could then be used
directly in a palladium-catalyzed coupling reaction, whereby deprotection was achieved in
situ, or the protecting group can be removed using sodium ethoxide to afford the desired aryl
thiol.
Scheme 2. General Synthesis of Non-Commercially Available Thiols
Reagents and conditions: (a) 2-Ethylhexyl 3-mercaptopropanoate, Pd dba , XantPhos, DIPEA, PhMe, 110 °C;
2
3
t
(
b) NaOEt, PhMe, EtOH, rt; then acid; (c) KO Bu, THF, rt; then acid.
AMTz analogues bearing aryl groups directly attached to the thiazole ring were
synthesized according to the method shown in Scheme 3. Suzuki reaction of N-Boc-protected
5-bromothiazol-2-yl)methanamine (23) with a boronic acid or ester, followed by acid-
(
mediated deprotection, afforded the desired phenyl-linked target compounds.
Scheme 3. General Route to Phenyl-Linked AMTz Analogues
Reagents and conditions: (a) Pd(PPh ) , K CO , 1,2-DME, H O, 95 °C; (b) 4 M HCl in dioxane, rt.
3
4
2
3
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Synthesis of the thiol surrogate used in the synthesis of 7a and close analogues was
achieved starting from 1,3-dibromo-5-(methylsulfonyl)benzene, Scheme 4. Palladium cross
coupling with 2-ethylhexyl 3-mercaptopropanoate installed the protected thiol group and
Suzuki coupling with phenyl boronic acid at the remaining bromo-position completed the
synthesis of the required thiol reactant. In situ deprotection followed by coupling with a 5-
bromo-2-AMTz intermediate (23 or 24) resulted in the corresponding sulfide-linked
compound. Direct N-Boc-deprotection provided the sulfide-linked final compound 7d, or
oxidation using either 1 or 2 equivalents of m-CPBA prior to deprotection resulted in the
sulfoxide or sulfone-linked inhibitors, 7c and 7a or 7b, respectively.
0
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Scheme 4. AMTz Sulfide, Sulfoxide and Aminomethylene Modifications
Reagents and conditions: (a) 2-Ethylhexyl 3-mercaptopropanoate, Pd dba , XantPhos, DIPEA, PhMe, 110 °C;
2
3
t
t
(b) PhB(OH) , Pd(PPh ) , K CO , 1,2-DME, H O, 95 °C; (c) Pd dba , XantPhos, NaO Bu, PhMe, BuOH, 110
2
3 4
2
3
2
2
3
°
C; (d) 4 M HCl in dioxane, rt; (e) m-CPBA (1 equiv.), DCM, 0 °C (f) m-CPBA (2.2 equiv.), DCM, rt.
Thiophene analogues 3b and 3c, bearing a halogen atom in the 3-position, were
synthesized from the corresponding 3-halo-5-bromo-AMT precursors (25 and 26), which
4
7-48
were synthesized according to literature procedures,
Scheme 5. Palladium coupling was
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then carried out with the thiol surrogate, and m-CPBA oxidation of the resulting bis-sulfide
followed by treatment with HCl afforded the desired halogenated thiophene analogues.
Scheme 5. General Route to Sulfur-Linked AMT Analogues
1
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t
Reagents and conditions: (a) NaOEt, PhMe, EtOH, rt; then acid; (b) KO Bu, THF, rt; then acid; (c) Pd dba ,
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t
t
XantPhos, NaO Bu, PhMe, BuOH, 110 °C; (d) m-CPBA, DCM, rt; (e) 4 M HCl in dioxane, rt.
Synthesis of the 2,4-thiazole regioisomers was achieved in the same manner as described
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previously, starting from N-Boc-protected (4-bromothiazol-2-yl)methanamine (27), Scheme
6. Palladium-catalyzed coupling with the respective thiol, followed by subsequent oxidation
using m-CPBA and Boc-deprotection using HCl afforded the desired regioisomers.
Scheme 6. General Route to Sulfur-Linked 2,4-AMTz Regioisomer Analogues
t
t
Reagents and conditions: (a) Pd dba , XantPhos, NaO Bu, PhMe, BuOH, 110 °C; (b) m-CPBA, DCM, rt; (c) 4
2
3
M HCl in dioxane, rt.
CONCLUSION
Following our recent discovery of a potent, selective and orally bioavailable LOX
inhibitor we observed that replacement of the thiophene core with a 2-aminomethylene-5-
sulfonyl thiazole core leads to potent, irreversible LOXL2 inhibitors. SAR investigations
revealed similar trends as seen in the analogous AMT series, resulting in potencies of < 0.1
µM achieved, and enabled development of a predictive LOXL2 3DQSAR model. Selectivity
studies concerning LOX and LOXL3 isoforms revealed a modest selectivity towards LOXL2
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in our main series inhibitors, whilst 2-aminomethylene-4-sulfonyl thiazole regioisomers
exhibit excellent selectivity for LOXL2, thus have the potential to be used as probe
compounds. Further selectivity and ADME assessment leads to the discovery of 21b, which
has an improved PK profile and demonstrates excellent anti-tumor efficacy in a LOX-driven
GEMM breast cancer model.
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EXPERIMENTAL
Synthesis of Inhibitors. All chemicals, reagents and solvents were purchased from
commercial sources and were used as received. Flash chromatography was performed on a
Biotage Isolera or Combiflash Rf+ UV-Vis flash purification system using pre-packed silica
gel cartridges (Biotage) with HPLC grade solvents. Thin layer chromatography (TLC)
analysis was performed using silica gel 60 F-254 thin layer plates and visualized using UV
light (254 nm) and/or developed with vanillin stain. LCMS and HRMS analyses of chemical
compounds were performed on an Agilent 1200 series HPLC and diode array detector
coupled to a 6210 time of flight mass spectrometer with a multimode ESI source; or a Waters
Acquity UPLC or I-Class UPLC with a diode array detector coupled to a Waters G2 QToF,
1
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SQD or QDa mass spectrometer fitted with a multimode ESI/APCI source. H, F and C
NMR spectra were recorded using a Bruker Avance 500, 400 or 300 MHz spectrometer using
an internal deuterium lock. Chemical shifts are expressed in parts per million (ppm), and
splitting patterns are indicated as follows: br, broad; s, singlet; d, doublet; t, triplet; q, quartet;
p, pentet; h, hextet; m, multiplet. All coupling constants (J) are reported in Hertz (Hz). All
final inhibitors submitted for biological evaluation were at least 95% pure by HPLC-MS.
22
Synthesis of inhibitors 3a, 4, 5, 6 and 8 has previously been described in the literature.
Below is a representative synthesis of compound 7a, and analytical data for all final
inhibitors. All tested inhibitors have purity >95% (LCMS/UV).
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(
3-Chloro-5-((5-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)sulfonyl)thiophen-2-
1
yl)methanamine hydrochloride (3b). Pale yellow solid (>99% purity). H NMR (CDCl /
3
Methanol-d , 500 MHz) δ 8.52 – 8.45 (m, 3H), 7.94 (s, 1H), 7.76 – 7.70 (m, 2H), 7.58 – 7.53
4
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(
m, 2H), 7.52 – 7.47 (m, 1H), 4.37 (s, 2H), 3.27 (s, 3H) ppm; C NMR (CDCl / Methanol-d ,
3 4
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1
25 MHz) δ 146.03, 144.53, 144.28, 143.73, 138.08, 137.97, 135.13, 131.81, 131.23, 130.39,
+
30.35, 129.33, 128.23, 125.55, 44.17, 36.07 ppm; LCMS m/z 424.9734 found (M-NH ) ,
2
424.9737 calculated for C H S O Cl.
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3
4
(3-Fluoro-5-((5-(methylsulfonyl)-[1,1'-biphenyl]-3-yl)sulfonyl)thiophen-2-
1
yl)methanamine hydrochloride (3c). White solid (>99% purity). H NMR (DMSO-d , 500
6
MHz) δ 8.65 (br s, 3H), 8.59 – 8.49 (m, 2H), 8.39 (s, 1H), 8.27 (s, 1H), 7.99 – 7.81 (m, 2H),
13
7.68 – 7.45 (m, 3H), 4.20 (s, 2H), 3.46 (s, 3H) ppm; C NMR (DMSO-d , 125 MHz) δ
6
1
1
55.78 (d, J = 265.9 Hz), 144.21, 143.82, 142.92, 139.39 (d, J = 7.6 Hz), 137.00, 131.30,
30.08, 129.88, 129.81, 128.00, 124.96 (d, J = 17.6 Hz), 124.57 (d, J = 25 Hz), 124.28,
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43.51, 32.77 ppm; F NMR (DMSO-d , 471 MHz) δ -122.29 ppm; LCMS m/z 409.0026
6
+
found (M-NH ) , 409.0033 calculated for C H S O F.
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3
4
(5-((5-(Methylsulfonyl)-[1,1'-biphenyl]-3-yl)sulfonyl)thiazol-2-yl)methanamine
hydrochloride (7a). 2-Ethylhexyl 3-((3-bromo-5-(methylsulfonyl)phenyl)thio)propanoate. A
solution of 1,3-dibromo-5-(methylsulfonyl)benzene (500 mg, 1.59 mmol) and DIPEA (0.55
mL, 3.18 mmol) in toluene (10 mL) was degassed with nitrogen for 5 minutes. Pd dba (36
2
3
mg, 2.5 mol%), XantPhos (46 mg, 5 mol%) and 2-ethylhexyl-3-mercaptopropionate (0.36
mL, 1.59 mmol) were then added with stirring, and the solution was bubbled with nitrogen
for a further 5 minutes before sealing the flask and heating to 110 °C, with stirring for 18
hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate
(
60 mL) and washed with water (50 mL) and brine (50 mL), dried over anhydrous
magnesium sulfate, filtered and concentrated to give the crude product, which was purified
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