Anti-metastatic Inhibitors of Lysyl Oxidase (LOX): Design and Structure-Activity Relationships

Article abstract of DOI:10.1021/acs.jmedchem.9b00335

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that cross-links collagens and elastin in the extracellular matrix and is a critical mediator of tumor growth and metastatic spread. LOX is a target for cancer therapy, and thus the search for therapeutic agents against LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High-throughput screening provided the initial hits. Structure-activity relationship (SAR) studies led to the discovery of AMT inhibitors with sub-micromolar half-maximal inhibitory concentrations (IC₅₀) in a LOX enzyme activity assay. Further SAR optimization yielded the orally bioavailable LOX inhibitor CCT365623 with good anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy.

Full text of DOI:10.1021/acs.jmedchem.9b00335

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Article
Anti-metastatic Inhibitors of Lysyl Oxidase (LOX):
Design and Structure-Activity Relationships
Leo Leung, Dan Niculescu-Duvaz, Deborah Smithen, Filipa Lopes, Cedric Callens, Robert
McLeary, Grazia Saturno, Lawrence Davies, Mohammed Aljarah, Michael Brown, Louise
Johnson, Alfonso Zambon, Tim Chambers, Delphine Ménard, Natasha Bayliss, Ruth Knight,
Laura Fish, Rae Lawrence, Mairi Challinor, HaoRan Tang, Richard Marais, and Caroline Springer
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00335 • Publication Date (Web): 09 May 2019
Downloaded from http://pubs.acs.org on May 9, 2019
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Springer, Caroline; Cancer Research UK Manchester Institute, Drug
Discovery Unit
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Anti-metastatic Inhibitors of Lysyl Oxidase (LOX):
Design and Structure-Activity Relationships
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a,b
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Leo Leung, Dan Niculescu-Duvaz, Deborah Smithen, Filipa Lopes, Cedric Callens, Robert
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McLeary, Grazia Saturno, Lawrence Davies, Mohammed Aljarah, Michael Brown, Louise
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Johnson, Alfonso Zambon, Tim Chambers, Delphine Ménard, Natasha Bayliss, Ruth Knight, Laura
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Fish, Rae Lawrence, Mairi Challinor, HaoRan Tang, Richard Marais, Caroline Springer *
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a. Drug Discovery Unit, Cancer Research UK Manchester Institute, University of Manchester, Alderley
Park, Macclesfield, SK10 4TG, United Kingdom
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b. Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, 15 Cotswold
Road, London SM2 5NG, United Kingdom
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c. Molecular Oncology Team, Cancer Research UK Manchester Institute, University of Manchester,
Alderley Park, Macclesfield, SK10 4TG, United Kingdom
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ABSTRACT. Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase that crosslinks
collagens and elastin in the extracellular matrix (ECM) and is a critical mediator of tumor growth and
metastatic spread. LOX is a target for cancer therapy and thus the search for therapeutic agents against
LOX has been widely sought. We report herein the medicinal chemistry discovery of a series of LOX
inhibitors bearing an aminomethylenethiophene (AMT) scaffold. High throughput screening (HTS)
provided the initial hits. Structure-activity relationship (SAR) studies led to the discovery of AMT
inhibitors with sub-micromolar half maximal inhibitory concentrations (IC ) in a LOX enzyme activity
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assay. Further SAR optimization yielded the orally bioavailable LOX inhibitor CCT365623 with good
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anti-LOX potency, selectivity, pharmacokinetic properties, as well as anti-metastatic efficacy.
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INTRODUCTION
Lysyl oxidase (LOX) and its family members LOX-like (LOX-L) 1-4 are copper-dependent amine
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oxidases that covalently crosslink collagens and elastin in the tumor extracellular matrix.1–4 LOX is
secreted as a catalytically inactive 50 kDa pro-protein, which is cleaved to an active 32 kDa enzyme by
proteases such as procollagen C-proteinase. LOX and LOXL1-4 have variable N-termini and they share
a highly conserved C-terminus, where the catalytic domain is located. The catalytic site comprises a
copper binding motif and a covalently bound lysine tyrosylquinone (LTQ) cofactor, where peptidyl
lysine residues (H NCH R) are converted to the corresponding α-aminoadipic-δ-semialdehyde
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(O=CHR) in an oxidative deamination reaction. The newly formed aldehyde residues undergo
spontaneous crosslinking with adjacent nucleophilic functionalities, leading to the insoluble
extracellular protein matrices.
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LOX and LOXL2 also have important roles in promoting tumor growth in many types of cancer.5–12
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In particular, LOX has been demonstrated to be a critical mediator of cancer metastasis. Therapeutic
agents targeting the activity of LOX are thus proposed as cancer treatments, especially against
metastasis where no effective therapeutic methods are currently available.
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Figure 1. Small Molecule Inhibitors of LOX Family Enzymes
O
O
O
S
O
O
H N
2
S
S
N
H2N
CF3
CN
O
N
OH
H N
2
F
BAPN
1
CCT365623
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Until recently, no drug-like small molecule inhibitors of LOX itself have been reported. Noticeably,
the irreversible inhibitor β-aminopropionitrile14,15 (BAPN) has found widespread applications in LOX-
family-related biological studies (Figure 1), although the lack of amenable sites for chemical
modification has prevented its development into a clinically optimal drug. More recently,
haloallylamine-based inhibitor PXS-S1A and PXS-S2A (full structure not disclosed),16 and
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trifluoromethyl (CF ) substituted aminomethylenepyridine 1 were reported to be potent selective
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inhibitors of one of the family members, LOXL2, the latter also showed weak inhibiton against
LOX.17,18 Intriguingly, analogs of pyridine 1 without the CF functionality were less selective towards
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LOXL2, with low micromolar IC s against LOX.
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breast cancer, and that treatment with the aminomethylenethiophene (AMT) inhibitor CCT365623
(9f) led to significant reduction in tumor growth and importantly, in metastastic burden too, in a LOX
dependent breast tumor transgenic mouse model. In our current study, we present the medicinal
chemistry development leading to the discovery of the orally efficacious AMT inhibitor 9f.
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RESULTS AND DISCUSSION
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LOX Inhibition – initial SAR. We ran a high throughput screen (HTS) at Evotec, of 267,000 diverse
compounds and 5000 fragments, on LOX which yielded a hit rate of 0.4%. (5-(Piperidin-1-
ylsulfonyl)thiophen-2-yl)methanamine 2a was identified as a positive hit with a mean IC of 19 µM.
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Since no crystal structure of LOX is available, the design of inhibitors could not be aided by
crystallographic or in silico methods. Therefore, the SAR of enzyme inhibition is largely elucidated by
introducing systematic modifications to different regions of the hit molecule.
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Substitutions at the 5-Sulfonyl Linker – Sulfonamides. SAR exploration commenced with the
investigation of sulfonamide substitutions on LOX inhibition (Table 1). Acyclic sulfonamides show no
improvement (2b and 2c vs 2a), while 2-amido- and 2-hydroxymethylpyrrolidine substitutions exhibit
comparable or better LOX potencies (2d and 2e vs 2a). 2-Phenylpyrrolidine 2f is also effective against
LOX, as is the bicyclic indoline 2g which is ~10 fold more potent than the piperidine hit 2a. Similarly,
tetrahydroquinoline 2h is equipotent to indoline 2g. Replacement of the piperidine ring with morpholine
does not improve LOX inhibition (2i vs 2a), whereas homopiperazine (2j) substitution leads to ~2 fold
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improvement in IC . Functionalization of the free homopiperazine nitrogen with small groups leads to
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gains in potency compared to the initial hit, as exemplified in N-methyl analog 2k, ethyl urea 2l and, in
particular, sulfonamide 2m.
Table 1. Effects of Sulfonamide Substitution on LOX Potency
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O
O
S
S
H N
2
R
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LOX
LOX
R
IC50
M)
R
IC50
(M)
a
a
(
N
N
2
a
17, 21
2h
2.5, 2.7
N
N
N
2
b
70, 72
27, 31
2i
2j
20, 25
O
N
H
NH
2c
9.4, 12.7
O
N
N
N
N
N
2d
11, 13
2k
2l
3.1, 3.1
NH2
OH
O
2
.1
N
2
e
3.1, 3.3
HN
[1.3, 3.2]
O
S
0
.93
N
O
2
f
3.8, 3.8
1.9
2m
N
N
[
0.74, 1.17]
2
g
N
[1.2, 3.1]
a
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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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
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Substitution at the 5-Sulfonyl Linker – Sulfones. The effect of alkyl and aryl substitutions at the 5-
sulfonyl linker on LOX inhibition was investigated next (Table 2). Replacements of the piperidine
moiety on HTS hit 2a with cyclohexyl (3a) and phenyl groups (3b) are beneficial, as are pyridine (3c)
and thiophene (3d). Biphenylsulfone is a weaker inhibitor than the phenyl analog (3e vs 3b), while the
2-naphthalylsulfone 3g is more potent than the 1-regiosiomer 3f. Methanesulfonamido-phenyl analog 3h
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moderately inhibits LOX, while methanesulfonylphenyl sulfone 3i is an excellent LOX inhibitor with an
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IC of 0.26 M, ~70 fold more potent than the HTS hit 2a. Replacement of the phenyl moiety of
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inhibitor 3i with an alkyl group (3j) leads to a reduction in potency. The SAR data illustrate that the
attachment of cyclic alkyl or aryl groups to the sulfonyl linker greatly improves LOX potency and the
inhibitory effect is further enhanced by the addition of a second sulfonyl group.
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Table 2. Effects of Sulfonyl-Alkyl and -Aryl Substitutions on LOX Potency
O
O
S
S
H N
2
R
8
LOX
LOX
R
IC50
R
IC50
(M)
a
a
(M)
.1
0.65, 7.1]
.7
1.4, 2.2]
.3
1.2, 4.1]
2
4.7
[3.3, 6.8]
3
a
3f
[
1
0.69
[0.093, 5.1]
3b
3g
3h
[
N
O
O
2
2.8
[1.8, 4.4]
3
c
S
[
N
H
S
0.26
0.15, 0.44]
3
d
0.89, 1.3
5.5, 7.3
3i
3j
S
[
O
O
S
3
e
12, 13
O O
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0
1
2
a
1
1
1
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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
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3
1
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Modification of the 5-Sulfonyl Linker. The impact of the sulfonyl linker was subsequently examined
(Table 3). A noticeable correlation can be observed between the electron withdrawing ability of the
linker and the LOX IC , with the most electron withdrawing sulfonyl group achieving the most potent
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inhibition (LOX IC : 3b < 4a < 4b). Carboxamide substitution does not improve LOX inhibition (4d vs
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0
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4c). Finally, exchanging the sulfonyl linker and the phenyl ring leads to reduction in potency (4e vs 3b).
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It is thus apparent that the sulfonyl moiety is the optimum linker for the AMT core, and that the electron
withdrawing effect of the linker is likely to play an important role in the mechanism of inhibition even
though the possibility of H-bond and dipole-dipole interactions cannot be ruled out.
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6
Table 3. Effects of Sulfonyl Linker (X) Modification on LOX Potency
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S
X
H N
2
R
6
LOX
X
R
IC50
a
(M)
.7
1.4, 2.2]
.9
O
1
O
3
b
S
[
[
O
6
4a
S
5.6, 8.6]
S
4
b
14, 27
O
N
O
H
4
c
15, 15
20, 25
S
O
N
H
4
d
O
1
3
O
4
e
S
[
7.4, 22]
a
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8
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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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
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15
Modification of the Thiophene Ring. Aminomethylenepyridine 5a is ~10 fold weaker than the
aminomethylenethiophene counterpart (Table 4; 5a vs 5b). 1,4-thiazole 5c is considerably less potent as
an inhibitor than the 1,3-regiomer 5d, while 1,3-thiazole 5d demonstrates a potency that is similar to the
thiophene 3g. Furan replacement does not improve the effectiveness of the inhibitors (5e vs 4d).
Additional substitutions on the thiophene ring can potentially be a useful handle for the development of
the series, but the introduction of a small methyl group is highly unfavorable (5f vs 2g). Overall,
although the replacements of the thiophene core with some unsubstituted 5-membered heterocycles are
tolerated for LOX inhibition, they are not superior to thiophene itself.
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1
1
7
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Table 4. Effects of Thiophene Modifications on LOX Potency
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1
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2
2
2
2
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5
5
5
5
5
5
6
R
IC50
M)
a
(
5
a
25, 25
N
R
S
S
S
1.5
0.47, 4.5]
O
H N
5b
2
O
[
5
c
24, 27
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0
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0
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0
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9
0
N
O
O
S
S
H N
2
R
5d
3g
1.0, 1.2
0.69
N
S
O
S
[
0.093, 5.1]
O
5
e
40, 45
H N
2
R
R
N
H
4
d
20, 25
1.9
S
S
O
2g
O
N
[
1.2, 3.1]
S
H N
2
5
f
12, 13
A
1
2
3
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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
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3
Modification of the Aminomethylene Moiety. All the inhibitors contain the aminomethylene moiety
(H NCH ), which forms part of the core AMT scaffold. We therefore investigate a series of
2
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modifications to this group where the replacement moieties are sufficiently diverse to probe non-
covalent interactions such as H-bond, electrostatic and dipolar interactions, while small enough to
minimize unfavorable steric clashes. The SAR data reveals that all substitutions or modifications at this
site result in total loss of activity (Table 5, 6a – 6f). It is thus apparent that the aminomethylene moiety
has a unique role in LOX inhibition; it is likely to be involved in the formation of a Schiff base similar
to that of the natural lysyl substrates.
1
1
1
1
1
1
1
4
5
Table 5. Effects of Aminomethylene Modifications (R ) on LOX Potency
O
O
S
R1
S
R2
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LOX
1
2
3
4
5
6
7
8
9
Compound
R1
R2
IC50
a
(
M)
>100,
100
100,
>100
100,
100
N
6
a
H
>
>
6b
H N
2
>
N
6
c
>
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
HN
H N
>100,
>100
6
d
2
O
>
100,
>100
100,
100
6
e
H N
2
>
6
f
HON
>
a
1
2
3
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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
4
5
6
7
8
9
Next, regiosiomers on the thiophene ring were investigated (Table 6). Both 2-aminomethyl-3-
sulfonylthiophene 7a and 3-aminomethyl-4-sulfonylthiophene 7b show no inhibitory activity against
LOX, while 2-aminomethyl-4-sulfonylthiophene 7c is a weak inhibitor. Therefore, from these
modifications, the thiophene ring is the optimal ring type, and the aminomethylene and sulfonyl groups
are the most effective substituents when placed on the 2- and 5- positions of the ring respectively.
1
1
0
1
Table 6. Effects of Regioisomers on the Thiophene Ring on LOX Potency
LOX
IC50
R
a
(M)
0
.69
[0.093,
.1]
100,
S
3
7
g
a
5
S
S
2
5
>
S
>100
H N
3
4
2
O
O
S
>100,
7
b
>
100
S
11
[8.0, 18]
7
c
a
12
13
14
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 error in square brackets expressed as the 95%
confidence interval of the geometric mean.
1
5
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1
2
3
4
5
6
7
8
9
Whilst the exact mode of binding of these AMT inhibitors remains unclear due to the absence of a co-
crystal structure or a homology model, the observed SAR suggests that a stable Schiff base formed from
the inhibitor and the LTQ cofactor (Figure 2) is likely to be involved. While the formation of the Schiff
base is reversible, its stabilization by the sulfonyl substituent on the thiophene ring by resonance
stabilization and/or direct binding to the enzyme by non-covalent processes such as H-bonding,
electrostatic, dipolar or van der Waals interactions can lead to a tightly bound enzyme-inhibitor
complex. This can potentially rationalize the improvement in potency observed in Table 3. The SAR
also suggests an additional non-covalent interaction between the methanesulfonylphenyl moiety and the
enzyme, which further enhances the potency of bis-sulfonyl inhibitors such as compound 9f.
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
1
2
3
4
5
6
7
8
9
0
1 10
2
3
1
1
Figure 2. Proposed Binding Mode of AMT inhibitors
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
HN
H
H
N
N
O
N
O
S
O
O
S
OH
R
1
2
3
1
Optimization Towards in vivo-Compatible Inhibitors. As our aim is to discover LOX inhibitors
that can be administered orally, metabolic stability and pharmacokinetics (PK) studies were performed
on the most promising AMT inhibitors described above. The highly potent bis-sulfonylphenyl analog 3i
has good stability against mouse microsomal (MLM) degradation (Table 9), but its poor detectability by
mass spectrometry renders it unsuitable for in vivo studies. Bis-sulfonylhomopiperazine 2m also
exhibits good anti-LOX potency, but it cannot be progressed further due to poor stability against
microsomal metabolism. Naphthalenesulfone 3g demonstrates good MLM stability but only a moderate
plasma exposure (AUC = 4.2 M.hr) when administered orally in mice at 50mg/kg. It was apparent that
further medicinal chemistry development was necessary to achieve both potent LOX inhibition and oral
plasma drug exposure compatible with in vivo studies.
1
1
1
1
1
4
5
6
7
8
8 19
9
0
1
2
3
4
2
2
0
1
5 22
6
7
8
9
0
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1
Due to its superior LOX potency and ease of synthesis, inhibitor 3i was chosen as the platform for the
1
2
2
3
4
5
6
7
next phase of discovery. Initial SAR established that modifications of the aminomethylene group, the
thiophene ring and the sulfonyl linker were unfavorable to target inhibition. Therefore, optimization to
improve PK properties focused on the aryl ring and its side chain substituents. The aim of the
subsequent studies therefore targeted improvement to oral in vivo PK exposure whilst
maintaining/improving LOX potency.
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
1
2
3
4
5
6
7
8
9
0
1
8
9
Side Chain Substitutions. Further SAR studies began with the investigation of the sulfonyl side chain
substitutions (Table 7). The attachment of the sulfonyl side chain through the 3- position (with respect
to the thiophene sulfone) affords inhibitors with similar LOX IC as the 4-regioisomers (8a vs 3i, 8d vs
2 10
5
0
3
4
1
1
1
1
2
3
8c), whilst 2- substitution is disfavored (8b vs 3i). Sulfonylpyrrolidines exhibit similar potencies as the
methyl sulfone counterparts (8c–8e vs 3i and 8a), but phenyl sulfone 8f is a weaker inhibitor. Small
alkyl substituents on the sulfonyl group are generally well tolerated (8g–8k). Inhibitors 8d, 8e and 8j
show good anti-LOX potency and MLM stability (Table 9) and were thus selected for in vivo PK
evaluation. Unfortunately, they have poor plasma exposure in mice when dosed orally.
5
6
7
8
9
0
1 14
2
3
1
1
5
6
4
5
6
7
8
9
0
1
2
Table 7. Effects of Side Chain Substitutions on LOX Potency of Phenylsulfonyl-AMT Analogs
O
O
S
S
2
R
H N
2
3
4
3 17
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LOX
LOX
IC50
(M)
Position Structure
Position Structure of
Compound
IC50
M)
Compound
8f
of R
of R
of R
R
a
a
(
O
O
O
O
0.26
0.15, 0.44]
S
3i
4
4
1.6, 2.0
S
[
O
O
O
O
0.39
0.27, 0.58]
1.4
O
O
0.29
[0.11, 0.78]
8a
3
2
8g
8h
4
4
S
S
S
[
OH
O
O
8
b
0.44, 0.46
S
OH
[1.2, 1.7]
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O
O
O
O
O
O
0
.92
0.59
[0.47, 0.73]
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
S
S
S
8
c
4
3
3
N
8i
8j
4
4
4
N
[0.78, 1.1]
O
O
O
0
.61
0.72
[0.58, 0.91]
8
d
N
S
N
H
[0.48, 0.77]
OH
O
O
0.42
[0.34, 0.53]
O O
S
8
e
8k
S
1.3, 2.1
N
O
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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 error in square brackets expressed as the 95%
confidence interval.
Phenyl Ring Substitution. 1,3- and 1,4-bis-sulfonylphenyl-AMT inhibitors containing an additional
substituent on the 5- position of the phenyl ring were evaluated against LOX inhibition (Table 8). tert-
Butyl, tert-butoxy, trimethylsilylethynyl, N-methylpyrazolyl and pyridinyl substitutions on the 1,3-
bisulfonylphenyl motif all lead to moderate reduction in LOX potency (9a–9e vs 8a), whilst a phenyl
substituent is well tolerated (9f). Replacement of the phenyl group with p-tolyl affords an equipotent
LOX inhibitor (9g vs 9f), but m-xylyl (9h) and o-ethylphenyl (9i) substitutions are disfavored. For the
1,4-bis-sulfonylphenyl-AMT motif, the N-methylpyrazolyl (9j), phenyl (9k) and p-tolyl (9l) analogs all
exhibit sub-micromolar LOX IC values.
8
9
0
1
5 11
6
7
8
9
0
1
1
1
2
3
2 14
5
0
3
4
5
6
7
8
1
1
5
6
In vivo mouse PK studies were then conducted for selected 5-substituted compounds. The tert-butoxy
(9b) and N-methylpyrazolyl (9j) analogs both exhibit low AUCs of 2.3 M.hr and 2.8 M.hr
9 17
respectively (Table 9), although this improves to 6.5 M.hr when the oxygen atom of the tert-butoxy
group is removed from the parent compound (9a vs 9b). Pleasingly, the 5-p-tolyl substituted bis-
sulfones have greatly improved and therapeutically relevant plasma exposures (11 M.hr and 12 M.hr
for 9g and 9l respectively). While the exposure for 5-phenyl-1,4-bis-sulfone 9k is disappointing (AUC =
1.4 M.hr), the 5-phenyl-1,3-bis-sulfonyl-AMT 9f achieves a desirable in vivo PK profile, with the
highest AUC, Cmax, longest half-life, lowest clearance of the series (albeit this is still moderate) and a
respectable intermediate oral bioavailability (F) of 45%.
0
1
1
1
2
2
8
9
0
1
2
3
4
5
6
7
8
9
0
1 22
2
3
2
3
4
5
6
7
8
9
0
2
2
4
5
Table 8. Effects of 5-Substitutions on LOX Potency of Bis-Sulfonylphenyl-AMT Analogs
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O
O
O
O
S
1
2
3
S
2
S
H N
2
1
3
4
5
4
1
R
5
6
7
8
9
Position
of
Position
of
LOX
IC50
LOX
Compound
R
Compound
R
IC50
O
O
O
O
a
a
(M)
(M)
S
S
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
1
.2
1.1
[0.75, 1.62]
9
a
3
9g
9h
9i
3
[0.88, 1.75]
1
.9
3.7
[1.7, 8.2]
9b
3
3
O
3
3
[
1.5, 2.3]
Et
2.5
1.6, 3.9]
6.5
[3.9, 11]
9
c
[
Si
1
.7
9
d
3
3
9j
4
4
0.71, 0.76
0.80, 0.81
N
N
[
1.56, 1.75]
N
N
9
e
1.4, 1.7
9k
N
0
.90
9
f
3
9l
4
0.76, 0.86
[
0.55, 1.48]
2
3
4
5
Table 9. In Vitro Mouse Liver Microsome (MLM) Stability and In vivo Pharmacokinetic (PK)
Properties of AMT Inhibitors
MLM
C
(
AUC(PO)
(M.hr) ((mL/min/kg))
CL(IV)
t
F
max(PO)
M)b
1/2(IV)
(hr)e
Compound stability
c
d
f
(%)
a
(
%)
2
m
37
-
-
3
i
67
91
59
63
41
67
90
ND
0.38
0.83
1.5
1.6
6.3
9.6
15
ND
8
j
0.36
8
d
0.22
8
e
0.46
9k
9b
9j
1.4
2.3
2.8
3g
100
68
4.2
9
a
16
6.5
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Page 14 of 68
1
2
3
4
5
6
7
8
9
0
1
2
3
4
9
g
60
95
65
6.7
9.4
17
11
12
15
67
106
49
1.0
0.4
1.2
39
74
45
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
9
l
9
f
1
1
1
1
1
a
Mouse liver microsome (MLM) stability values represents the percentage of compound remaining after 30 minutes; Mouse
plasma PK parameters were determined following a single dose by oral gavage (PO) or intravenous injection (IV) at 50
mg/kg or 10 mg/kg respectively: Cmax – maximum concentration. AUC – area under curve. CL – clearance; t1/2 – half-
0
1
2
3
4
b
c
d
e
f
life; F – bioavailability. ND - Could not be detected by MS.
1
5
5 16
Profile of Orally Available AMT Inhibitors. We have discovered three AMT inhibitors 9g, 9l and
9f that are highly effective in inhibiting LOX activity as well as possessing therapeutically relevant PK
profiles. These compounds are equally potent inhibitors of LOXL2 (Table 10), which is as expected due
to the highly conserved catalytic site across the LOX family members. Pleasingly, all three inhibitors
are inactive against common amine oxidases including the copper-containing diamine oxidase (DAO),
semicarbazide-sensitive amine oxidase (SSAO), and the flavin-containing monoamine oxidases (MAO)
A and B. It is noteworthy that AMT 9f is a substrate of SSAO, thus SSAO-catalyzed metabolism could
be a potential mechanism of degradation in vivo.
6
7
1
1
1
2
2
2
7
8
9
0
1
2
8
9
0
1
2
3
4
5
6
7
8
9
0
1 23
2
3
4
5
6
7
2
4
5
AMT 9g is a weak inhibitor of the human potassium ion channel hERG, whilst inhibitors 9l and 9f are
2
highly selective. All three inhibitors exhibit high permeability through colon Caco cells, which is
2
8 26
reflected in their good oral availabilities in mice (Table 9). AMT inhibitor 9f achieves the most
favorable overall profile and was therefore chosen for in vivo efficacy studies.
9
0
2
7
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
8
9
Table 10. Potency, Selectivity and Permeability of Optimized AMT inhibitors
b
MAO-A
MAO-B
Caco P
(10 cm/s)
2
app
LOX
LOXL2
DAO
IC50
(M)
SSAO
hERG
IC50
(M)
-6
Inhibitor
9g
IC50
(M)
IC50
(M)
IC50
(M)
IC50
M)
a
a
a
a
A→B B→A
a
(
A - >100,
>100
B - 87, 89
A – 33, 33
B - >100,
1
.1
2.0
[1.3, 3.0]
>100,
>100
49, 59
4.6
8.0
17
[
0.75, 1.62]
2
.2
>100,
>100
>100,
100
9
l
0.76, 0.86
0.90
68
25
18
25
35
[1.4, 3.7]
>
100
A - >100,
9
f
1.5
>100,
48, 90
8.5
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Journal of Medicinal Chemistry
[
0.55, 1.48] [0.28, 8.1]
>100
>100
B - >100,
1
2
3
>
100
a
1
2
3
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 error in square brackets expressed as the 95%
confidence interval of the geometric mean; Papp – permeability coefficient;
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
b
4
5
6
7
8
9
0
Evaluation of Anti-metastatic Efficacy. Compound 9f was assessed in a LOX-driven genetically
0
1
2
3
4
5
6
7
8
9
0
1
2
19
engineered mouse model (GEMM) of breast cancer that metastasizes to the lungs. Mice were dosed
daily by oral gavage (70 mg/kg) from day 60 when primary tumors start to be palpable (Figure 3). The
metastatic nodules in the lungs are measured when the primary tumors reach ethical size limit.
Pleasingly, compound 9f reduces lung metastasis significantly as measured by total surface area (Figure
3B).
1
3 11
4 12
5
Figure 3. Anti-metastatic Efficacy of Compound 9f in LOX-Driven GEMM Model. Animals treated
with vehicle (black) or compound 9f at 70 mg/kg qd (blue). All values are reported as the arithmetic
mean with the error expressed as the standard error of the means. (A) Number of lung metastasis -
1
1
3
4
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
2
vehicle, n = 7; 9f treated, n = 5; (B) Lung metastasis area in µm - vehicle, n = 7; 9f treated, n = 5.
1
5
6
1
1
7
0 18
SYNTHETIC CHEMISTRY
1
2
3
4
5
6
7
1
2
2
9
0
1
AMT-sulfonamides. All sulfonamide analogs were synthesized from the sulfonyl chloride
intermediates 11a and 11b by condensation with the corresponding amines in dichloromethane (Scheme
8 22
1). Subsequent trifluoroacetamide hydrolysis or methanolysis using aqueous NaOH or 7 N NH in
3
9
0
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Page 16 of 68
1
2
3
4
5
6
7
8
9
methanol furnishes the desired amines 2a–2i. 3-Methylthiophene sulfonamide 5f (Table 4) was also
synthesized by this method. Sulfonyl chlorides 11a and 11b were derived from the commercially
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
available
thiophen-2-ylmethanamines
10a/10b
in
straightforward
steps.
Access
to
sulfonylhomopiperazine analogs with additional functionalization at the free amino group could be
achieved via the amine hydrochloride intermediate 12, where N-substituted sulfonylhomopiperazines
2k, 2l and 2m was obtained in 3 steps. The unsubstituted sulfonylhomopiperazine 2j was synthesized
from sulfonyl chloride 11a by condensation with N-Boc-homopiperazine, trifluoroacetamide hydrolysis
followed by Boc removal.
0
1
2
3
4
5
6
7
8
9
0
1
2 10
Scheme 1. General Synthetic Routes to AMT-Sulfonamide Analogs 2a–n and 5f
3
4
5
6
7
8
9
0
1
2
3
4
O
O
O
O
O
S
S
S
a - c
S
d, e
S
H N
2
F3C
N
H N
2
Cl
NR2
H
(28-30%)
(14-86%)
Y
Y
Y
1
1
0a, Y = H
0b, Y = CH3
11a, Y = H
11b, Y = CH3
2a-2i, 5f
f, e, g
(68%) f, g
(63%)
O
O
O
O
O
O
O
2
HCl
S
S
S
S
S
HCl
NH
h, e
21-88%)
S
H N
N
F3C
N
N
H N
2
N
2
NH
NR'
H
(
5
2j
12
2k-2m
6 11
7
8
o
1
2
Reagents and conditions: (a) TFAA, Et N, DCM, rt; (b) ClSO H, DCM, -78 C to rt, then H O; (c)
3 3 2
9 13
0 14
1
oxalyl chloride, DMF, DCM, rt; (d) R NH, Et N, DCM, rt; (e) aq. NaOH, MeOH, rt or 7 N NH in
2
3
3
MeOH, rt; (f) N-Boc-homopiperazine, Et N, DCM, rt; (g) 2 M HCl in Et O, rt; (h) R’-Cl (EtNCO for
3
2
1
5
6
2l), Et N, DCM, rt.
3
2
3
4
5
6
7
8
9
1
1
7
8
AMT-sulfones. Synthesis of 2-pyridinesulfonyl 3c proceeded by the lithiation of 2-methylthiophene
TM
1
14 followed by condensation with aldrithiol -2 to afford the corresponding sulfide, which underwent
0 19
S-oxidation with mCPBA to afford sulfone 15 (Scheme 2). Subsequent methyl bromination was
followed by the displacement of the bromide by sodium azide to give the resulting alkyl azide, which
was catalytically hydrogenated to afford the desired 2-pyridinesulfonyl-AMT 3c. Similarly, ring
opening of 1,2-dithiane with lithiated 2-methylthiophene followed by condensation with iodomethane
1
2
3
4
5
6
2
2
0
1
7 22
8
9
0
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Page 17 of 68
Journal of Medicinal Chemistry
1
2
3
4
5
afforded the corresponding bis-sulfide, which underwent subsequent S-oxidation to afford bis-sulfone
16. This intermediate was subsequently converted to bis-sulfonylbutyl-AMT 3j by the method described
above.
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
Scheme 2. Synthetic Routes to AMT-sulfone Analogs 3c and 3j
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
O
S
O
O
S
O
S
S
S
a, b
N
c, d, e
(36%)
N
H N
2
(87%)
14
15
3c
(82%) f, b
O
O
O
O
S
S
S
c, d, e
(3%)
S
S
H N
2
S
O
O
O
O
16
3j
6
n
o
TM
o
7
8
9
Reagents and conditions: (a) BuLi, THF, -40 C, then aldrithiol -2, -40 C to rt; (b) mCPBA, DCM,
o
n
o
rt; (c) NBS, Bz O , DCE, 70 C; (d) NaN , DMF, rt; (e) H , Pd/C, THF, rt; (f) BuLi, THF, -40 C, then
2
2
3
2
o
1,2-dithiolane, -40 C to rt, then MeI, rt.
1
1
1
0
1
2
The commercially available 5-bromo-2-thiophenecarbonitrile 17a (interchangeable with 5-chloro-2-
thiophenecarbonitrile) and 2-bromothiazole-5-carbonitrile 17b served as valuable building blocks for
the AMT-sulfone inhibitors (Scheme 3). Nucleophilic aromatic substitution with a range of thiols
afforded the corresponding sulfides, which were oxidized with mCPBA to afford sulfone intermediates
18. Subsequent nitrile reduction with borane-tetrahydrofuran complex afforded AMT-sulfones 3a, 3b,
3d, 3f, 3g, 3i, 5b, 8a, 8b and 9b. For sulfonylaniline 3h, the product from the initial condensation 19
underwent an additional sulfonamide formation step. Subsequent mCPBA-mediated oxidation afforded
the usual sulfone intermediate 18, which was converted to the desired target. Analogs 3e and 5c were
synthesized from carbamates 20, which were derived from nitriles 17a and 17b respectively by
condensation with the corresponding thiols, followed by nitrile reduction and subsequent Boc
protection.
5 13
6
7
8
9
0
1
1
1
4
5
2 16
3
4
5
6
7
8
1
1
7
8
9 19
0
1
2
2
2
0
1
2
2
3
4
5
6
7
8 23
Scheme 3. Synthetic Routes to AMT-sulfone Analogs 3a, 3b, 3d–3i, 5b, 5c, 8a, 8b and 9b
9
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Journal of Medicinal Chemistry
Page 18 of 68
S
S
NC
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
NH2
1
9
Z = CH; 3a-3b, 3d-3i,
b, 8a, 8b, 9b;
Z = N; 5c
a
d, b
5
(
88%) (39%)
O
O
O
S
O
R
S
a, b
S
c
S
S
NC
Br
NC
R
H N
(3-63%) nHCl
2
Z
(7-68%)
Z
Z
18
1
7a, Z = CH
17b, Z = N
a, c, e
(7-17%)
b, f
(37-42%)
S
S
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R
BocHN
Z
20
1
2
3
Reagents and conditions: (a) RSH, K CO , DMF, heat; (b) mCPBA, DCM, rt; (c) BH THF, THF, rt;
(d) MsCl, Et N, DCM, rt; (e) Boc O, Et N, DCM, rt; (f) 4 M HCl in dioxane, rt.
2
3
3
3
2
3
4
5
Sulfonamide-substituted phenylsulfonyl-AMT analogs 8c–8e were synthesized from the
corresponding thiols 22 (Scheme 4). Hence, condensation of 3- or 4-fluorophenylsulfonyl chloride 21
6
7
8
9
with the desired amines (HNR ) afforded the corresponding sulfonamides, which were thiolated by
2
treatment with sodium thiomethoxide to afford intermediate thiols 22. The thiols were condensed with
5-chloro-2-thiophene-carbonitrile to yield sulfides 23. The nitrile group of sulfides 23 was then
converted to the corresponding trifluoroacetamide by nitrile reduction and amide formation. S-oxidation
of sulfides 24 and subsequent amine deprotection affords the desired AMT targets 8c–8e.
1 10
2
3
1
1
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
3
Scheme 4. Synthetic Route to Sulfonamide-Substituted Phenylsulfonyl-AMTs 8c–8e
O
O
O
O
O
O
a, b
41-
c
S
S
NR2
S
S
S
NC
Cl
NR2
F
HS
(
1
(25-43%)
00%)
21
22
23
(72-78%) d, e
O
O
S
O
O
O
O
O
S
S
f, g
49-51%)
S
S
S
NR2
NR2
H N
F3C
N
2
(
H
24
8
c-e
1 14
2
3 15
4
5
6
7
8
9
0
o
Reagents and conditions: (a) HNR , DCM; (b) NaSMe, DMF, 165 C; (c) 5-chloro-2-thiophene-
carbonitrile, K CO , DMF, 120-130 C; (d) BH THF, THF, rt; (e) TFAA, Et N, DCM; (f) mCPBA,
2
o
1
1
1
6
7
8
2
3
3
3
DCM, rt; (g) 7N NH in MeOH, rt.
3
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Page 19 of 68
Journal of Medicinal Chemistry
1
The sulfonyl side chains of AMT analogs 8g–8k were introduced through the nucleophilic
1
2
2
3
4
5
6
7
8
9
substitution of the chlorine atom on intermediate 26 (Scheme 5), which could be obtained in
straightforward steps using methods previously described in Scheme 3. Condensation of intermediate 26
with 2-mercaptoethanol or 3-mercaptopropanol followed by sulfide oxidation led to alkanol
intermediates 27. After Boc removal, these intermediates afforded AMT targets 8g and 8h.
Alternatively, treatment of hydroxyethylsulfone 27 (n = 1) with methanesulfonyl chloride led to
concomitant sulfonylation and elimination, furnishing vinyl sulfone 28. Addition of pyrrolidine to vinyl
sulfone 28 followed by Boc removal gives AMT target 8i. Ammonia addition to intermediate 28
provided the corresponding aminoethyl sulfone, which was treated with acetic anhydride to afford the
corresponding AMT-acetamide 8j after Boc removal. Methoxyethylsulfone 8k was also obtained by
from vinyl sulfone 28 by methoxide addition followed by Boc removal.
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
1
2
3
4
5
6
7
8
9
0
1 10
2
3
1
1
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
1
1
2
3
Scheme 5. Synthetic Routes to Phenylsulfonyl-AMTs with Sulfonyl Side Chain Modifications (8g–8k)
O
S
O
S
O
HS
O
S
S
N
S
NC
Br
Scheme 3
(38%)
H N
BocHN
2
2HCl
S
Cl
Cl
O O
17a
25
26
45-61%) a, b
8i
85%) e, c
(
(
O
S
O
S
O
O
O
n = 1, 2
O
n = 1, 2
S
S
S
OH
OH
S
c
d
H N
2
BocHN
BocHN
54%)
(
1
87-
00%)
(quant.)
HCl
S
O O
S
O O
S
O O
8
g, 8h
27
28
55%) f, g, c
h, c
(
(
O
O
S
O
O
S
O
S
S
O
NH
H N
2
H N
2
HCl
HCl
S
S
O O
O O
8k
8j
1
4
5
o
1
Reagents and conditions: (a) 2-Mercaptoethanol or 3-mercaptopropanol, K CO , DMF, 50 C; (b)
2
3
2 16
mCPBA, DCM, rt (45–61% over 2 steps); (c) 4 M HCl in dioxane, rt; (d) MsCl, Et N, DCM, rt (quant.);
3
3
4
5
6
7
8
9
0
1
1
7
8
(e) Pyrrolidine, DCM, rt (85% for steps e + c); (f) 7 N NH in MeOH, rt; (g) Ac O, Et N, DCM, rt; (h)
3
2
3
K CO , MeOH, rt.
2
3
1
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Journal of Medicinal Chemistry
Page 20 of 68
1
2
3
4
5
6
Phenyl sulfide 29, obtained from methods previously described in Scheme 3, underwent a Friedel-
Crafts sulfonylation to afford the corresponding phenyl sulfonyl (Scheme 6), which was subsequently
oxidized to bis-sulfone 30 using mCPBA. Reduction of the nitrile group with borane-tetrahydrofuran
complex afforded the bis-sulfonylphenyl-AMT analog 8f.
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
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
Scheme 6. Synthetic Route to Bis-sulfonylphenyl-AMT 8f
O
O
O O
S
S
S
S
a, b
NC
S
NC
S
c
H N
2
(25%)
(70%)
Ph
Ph
S
S
O
O
O
O
29
30
8f
7
o
8
9
Reagents and conditions: (a) PhSO Cl, FeCl , neat, 100 C; (b) mCPBA, DCM, rt (c) BH THF, THF,
2
3
3
rt.
1
1
0
1
tert-Butyl-bis-sulfonylphenyl-AMT 9a was synthesized from 1,3-dibromo-5-(tert-butyl)benzene 31
8 12
(Scheme 7). tert-Butyllithium-mediated lithium-bromine exchange of dibromide 31 and treatment of the
resultant lithium phenylate with dimethyl disulfide afforded the corresponding methyl sulfide, which
was oxidized to sulfone 32 using mCPBA. This underwent Pd-catalyzed cross coupling with thiophene-
2-thiol to yield the corresponding thiophene sulfide, which was formylated under Vilsmeier-Haack
conditions to give aldehyde 33. After reduction of the aldehyde with sodium borohydride and
subsequent sulfide oxidation, the hydroxyl group was substituted with azide using diphenylphosphoryl
azide (DPPA) before it was subsequently reduced to the desired AMT target 9a.
9
0
1
1
1
1
1
1
3
4
5
6
7
8
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5 19
6
7 20
Scheme 7. Synthetic Route to tert-Butyl-bis-Sulfonylphenyl-AMT 9a
8
2
1
9
0
1
2
3
4
5
6
7
8
9
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Page 21 of 68
Journal of Medicinal Chemistry
O
O
O
O
O
1
2
3
4
5
6
7
8
9
Br
Br
Br
S
S
S
S
a, b
c, d
H
(83%)
(27%)
31
32
33
(41%) e,b,f,g
O
O
O
O
S
S
S
H N
2
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
9a
1
t
o
o
2
3
4
Reagents and conditions: (a) BuLi, THF, -78 C then MeSSMe, -78 C to rt; (b) mCPBA, DCM, rt; (c)
thiophene-2-thiol, Pd (dba) , Xantphos, K CO , p-xylene; 140 C; (d) POCl , DMF, 50 C; (e) NaBH ,
THF, rt; (f) DPPA, PPh , DIAD, THF, rt; (g) H , Pd/C, EtOH, rt.
o
o
2
3
2
3
3
4
3
2
5
6
19
5-Substituted 1,3-bis-sulfonylphenyl-AMTs 9c–9i were synthesized from aryl bromide 34. Hence,
Pd-catalyzed cross coupling of aryl bromide 34 with trimethylsilylacetylene under Sonogashira
conditions afforded the corresponding aryl-alkyne (Scheme 8), which is subjected to HCl-mediated Boc
deprotection to afford AMT analog 9c. Suzuki-Miyaura coupling with boronic acids gave the
corresponding biaryl products, which underwent Boc removal to furnish the desired AMT inhibitors 9d–
9i.
7
8
9
0
1
3 11
4
5
6 12
7
8 13
9
0
1
2
3
4
5
6
7
Scheme 8. Synthetic Routes to Substituted Bis-sulfonylphenyl-AMTs 9c–9i
O
O
O
O
S
S
S
H N
2
nHCl
a, b
O
O
O
O
S
S
S
(88%)
BocHN
9c
TMS
c, b
Br
O
O
O
O
3
4
S
8
9
0
1
(24-71%)
S
S
H N
2
nHCl
R
9d-9i
2 14
3
4
1
5
Reagents and conditions: (a) Pd(PPh ) , CuI, Et N, trimethylsilylacetylene, dioxane, rt; (b) 4 M HCl in
3 4 3
dioxane, DCM, rt; (c) Pd(PPh ) , RB(OR’) Cs CO , dioxane, 100 C.
o
5 16
3 4 2, 2 3
6
7 17
8
9
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Journal of Medicinal Chemistry
Page 22 of 68
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2
3
4
5
6
7
5-Substituted 1,4-bis-sulfonylphenyl-AMTs 9j–9l were synthesized from bromofluorobenzene
intermediate 36 (Scheme 9), which could be obtained from the commercially available 5-
bromothiophene-2-carbonitrile 17a and 3-bromo-4-fluorobenzenethiol 35 by methods previous
described in Scheme 3. Selective substitution of the fluorine atom of intermediate 36 with
thiomethoxide afforded the corresponding sulfide, which was oxidized to sulfone 37 using mCPBA.
Suzuki-Miyaura coupling with boronic acids and subsequent Boc removal furnished the desired AMT
inhibitors 9j–9l.
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
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
8
9
Scheme 9. Synthetic Routes to Substituted Bis-sulfonylphenyl-AMTs 9j–9l
O
O
HS
Br
F
S
S
Scheme 3
46%)
S
Br
F
NC
Br
BocHN
(
17a
35
36
(
68%) a, b
O
S
O
S
O
O
S
S
R
S
Br
S
c, d
(62-95%)
H N
BocHN
2
nHCl
O
O
O
O
9i-9l
37
1
0
3 11
4 12
5
Reagents and conditions: (a) NaSMe, DMF, rt; (b) mCPBA, DCM, rt; (c) Pd(PPh ) , RB(OR’)
3 4 2,
o
Cs CO , dioxane, 100 C; (d) 4 M HCl in dioxane, DCM, rt.
2
3
6 13
7
8 14
9
AMT-sulfide, -sulfoxide and aminomethylene modifications. AMT-sulfide 4b and -sulfoxide 4a
(Scheme 10) were derived from intermediate sulfide 29 (see Scheme 3; R = Ph). Mono S-oxidation of
sulfide 29 with 1 equivalent of mCPBA and subsequent nitrile reduction leads to AMT-sulfoxide 4a.
Reduction of intermediate 29 with borane-tetrahydrofuran complex, followed by Boc protection affords
carbamate 38. Removal of Boc from carbamate 38 led to AMT-sulfide 4b, whereas N-methylation using
sodium hydride/iodomethane and subsequent oxidation with mCPBA affords intermediate 39 which was
deprotected to afford the N-methyl AMT analog 6a. Alternatively, sulfone 6c can be obtained from the
condensation of bromothiophene 17a and sodium benzenesulfinate in DMF. This intermediate was
0
1
5
1
2
3
4
1
6
5 17
6
7
8
9
0
1
1
8
9
1
2 20
3
4
2
1
5
6
7
8
9
0
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Page 23 of 68
Journal of Medicinal Chemistry
1
2
3
4
converted to the corresponding thiopheneacetimidamide 6d and thiophenecarboxamide 6e by treatment
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
with indium trichloride (InCl ) and lithium hexamethyldisilazide (LiHMDS) respectively.
3
Scheme 10. Synthetic Routes to Compounds 4a, 4b, 6a, 6c–6e
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
O
S
S
NC
Br
O
O
O
S
S
S
S
H N
2
H N
2
17a
4
a
(
51%)
i
d
e
6e
S
(
50%)
(
34%) b, c
HN
O
O
O
O
S
S
S
NC
NC
S
H N
2
(16%) HCl
29
6c
6d
(
58%) c, f
O
O
O
O
S
S
S
S
S
g, a
S
h
(quant.)
BocHN
BocN
N
H
(
54%)
HCl
38
39
6a
(60%)
S
h
HCl
H N
S
2
4b
5
6
7
8
9
0
Reagents and conditions: (a) mCPBA (2 equiv.), DCM, rt; (b) mCPBA (1 equiv.), DCM, rt (c)
BH THF, THF, rt; (d) InCl , acetaldoxime, toluene, reflux; (e) LiHMDS, Et O, rt, then 2 M HCl, rt; (f)
3
3
2
Boc O, Et N, DCM, rt; (g) NaH, MeI, THF, rt; (h) 4 M HCl in dioxane, DCM, rt; (i) Sodium
2
3
o
benzenesulfinate, DMF, 135 C.
1
1
1
2
3
Condensation of 1-(5-chlorothiophen-2-yl)ethan-1-one 40 with thiophenol afforded the corresponding
sulfide (Scheme 11), which was oxidized to sulfone 41 using mCPBA. Subsequent oxime formation
followed by reduction using zinc powder in trifluoroacetic acid furnished C-methylated AMT 6b.
Sulfide 42 was obtained in straightforward steps by methods previously illustrated in Scheme 3.
Oxidation of sulfide 42 with mCPBA afforded the corresponding sulfone 43. This was reduced to the
corresponding aldehyde with DIBAL-H before conversion to oxime 6f by treatment with hydroxylamine
hydrochloride.
1
1
6 14
7
8
9
0
1
2
1
5
6
1
3 17
4
5
6
7
8
9
0
1
1
8
9
Scheme 11. Synthetic Routes to Compounds 6b and 6f
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O
O
O
O
O
O
S
S
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
S
a, b
S
c, d
S
N
Cl
H N
2
(
26%)
(18%)
4
0
41
6b
O
O
O
O
S
S
S
NC
S
NC
S
HO
S
b
e, f
(38%)
(
92%)
4
2
43
6f
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
o
2
3
4
5
Reagents and conditions: (a) Thiophenol, K CO , DMF, 120 C; (b) mCPBA, DCM, rt; (c)
2 3
H NOH.HCl, DIPEA, EtOH, rt; (d) Zn, TFA, rt; (e) DIBAL-H, DCM, 45 C; (f) H NOH.HCl, K CO ,
o
2
2
2
3
o
EtOH, 70 C.
6
7
8
9
0
AMT- and Aminomethylenefuran-amides. Synthesis of AMT-amide 4d and aminomethylenefuran-
amide 5f were accomplished via methyl esters 44 (Scheme 12; A = S or O).20,21 Esters 44 first
underwent saponification with aqueous hydroxide and the resultant carboxylic acids were then
converted to acid chlorides 45 using oxalyl chloride. Condensation with benzylamine in
dichloromethane followed by catalytic hydrogenation using 10% Pd/C in tetrahydrofuran furnished the
desired amides 4d and 5e.
1
8 11
9
0
1
1
2
3
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
Scheme 12. Synthetic Routes to Amide Analogs 4d and 5e
O
O
O
A
A
a, b
A
c, d
H N
2
N
N3
N3
OMe
Cl
H
(75-81%)
(39-45%)
4
4
45
4d, A = S
5e, A = O
1
4
1
5
6
Reagents and conditions: (a) 1 M NaOH, MeOH, rt; (b) (COCl) , DMF, DCM, rt; (c) Benzylamine,
2
1
DCM, rt; (d) H , Pd/C, THF, rt.
2
1
1
7
8
Aminomethylene-pyridine, -1,3-thiazole and -imidazole. Aminomethylenepyridine 5a was
obtained from the commercially available tert-butyl ((6-chloropyridin-3-yl)methyl)carbamate 46.
Condensation with sodium benzylthiolate afforded the corresponding sulfide 47 (Scheme 13). Sulfide
oxidation with mCPBA and subsequent HCl-mediated Boc removal furnished the desired target 5a. 2-
(Aminomethylene)-1,3-thiazole 5d was obtained from sulfide intermediate 49 in a similar manner.
1
9
2 20
3
4
2
1
2
5
6
7
8
9
0
2
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2
3
4
Sulfide 49 was synthesized from tert-butyl ((5-bromothiazol-2-yl)methyl)carbamate22 48 and
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
naphthalene-2-thiol by a Pd-catalyzed cross coupling reaction.
Scheme 13. Synthetic Routes to Aminomethylene-Pyridine, -Thiazole and -Imidazole 5a and 5d
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
O
a
b, c
HCl
H N
O
Cl
S
S
(
66%)
(77%)
BocHN
BocHN
N
BocHN
BocHN
N
N
2
46
47
5a
O
S
O
S
S
S
S
b, c
Br
d
H N
2
N
N
N
(52%)
(52%)
HCl
48
49
5d
5
o
6
7
8
Reagents and conditions: (a) NaH, benzylmercaptan, DMF, 70 C; (b) mCPBA, DCM, rt; (c) 2 M HCl
t
in Et O or 4 M HCl in dioxane, rt; (d) Pd (dba) , Xantphos, naphthalene-2thiol, NaO Bu,
BuOH/toluene, 100 C.
2
2
3
t
o
9
0
1
2
1
1
1
AMT-sulfone regiosiomers. The AMT regioisomers 7a-7c were synthesized by 2 different methods
(Scheme 14). The synthesis of 2,3-regioisomer 7a began with nucleophilic aromatic substitution
between 3-bromothiophene-2-carbonitrile 51 and naphthalene-2-thiol. The resultant sulfide underwent
subsequent sulfide oxidation followed by nitrile reduction to afford the desired target 7a. The initial
nucleophilic aromatic substitution step was replaced with a Pd-catalyzed cross coupling for the
synthesis of the 3,4- and 2,4-regioisomers 7b and 7c, starting from bromothiophenes 53 and 55. The
resultant sulfides were oxidized to sulfones 54 and 56, which were subsequently converted to the
corresponding AMT targets 7b and 7c by nitrile reduction.
3 13
4
5
6
7
8
9
1
1
4
5
0 16
1
2
1
1
1
7
8
9
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 14. Synthetic Routes to AMT Regioisomers 7a-7c
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H N
2
S
S
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
NC
S
NC
Br
a, b
c
(81%)
S
(48%)
S
O
O
O
O
5
1
52
7a
S
S
S
d, b
c
NC
NC
H N
(64-
S
(47-51%)
2
S
O
O
Br
100%)
O
O
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
53, 3-CN
5, 2-CN
54, 3-CN
56, 2-CN
7b, 3-CH NH2
7c, 2-CH NH2
2
5
2
1
o
2
3
Reagents and conditions: (a) Naphthalene-2-thiol, K CO , DMF, 120 C; (b) mCPBA, DCM, rt; (c)
BH THF, THF, rt; (d) Pd dba , Xantphos, naphthalene-2-thiol, NaO Bu, toluene, 110 C.
2
3
t
o
3
2
3
4
5
6
7
8
9
0
Finally, the Suzuki-Miyaura coupling between 5-bromothiophene-2-carbonitrile 17a and (4-
(methylthio)phenyl)boronic acid 57 affords phenyl thiophene 58 (Scheme 15). This was converted to
carbamate 59 by nitrile reduction, Boc protection and sulfide oxidation. Subsequent Boc removal
furnished the phenyl-linked AMT analog 4e.
1
Scheme 15. Synthetic Route to Phenyl-linked AMT 4e
S
S
a
S
HO
NC
Br
S
NC
B
(80%)
HO
17a
57
58
33%) b, c, d
(
O
O
O
O
S
e
S
HCl
S
S
(
64%)
H N
BocHN
2
4
e
59
1
1
2
o
1
Reagents and conditions: (a) Pd(PPh ) , Cs CO , 1,4-dioxane, 100 C; (b) BH THF, THF, rt; (c)
3 4
2
3
3
6 13
Boc O, Et N, DCM, rt; (d) mCPBA, DCM, rt; (e) 4 M HCl in dioxane, DCM, rt.
2 3
7
8 14
9
0
1
1
1
5
6
7
CONCLUSION
1
2
3
4
5
6
We described herein a series of LOX inhibitors containing a 2-aminomethylene-5-sulfonyl thiophene
core. The attachment of a sulfonylphenyl side chain to the core scaffold via the 5-sulfonyl linker
7 18
8
9
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
furnishes inhibitors with submicromolar LOX IC values. Further SAR optimization leads to the
50
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
discovery of inhibitor 9f with potent anti-LOX activity as well as desirable selectivity and PK profile
making it a valuable asset for LOX research. More importantly, we have described its ability to reduce
19
the growth of spontaneous breast tumor lung metastasis in a GEMM, thus demonstrating the promise
of 9f as a drug candidate.
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL
Synthesis of Inhibitors. Commercial building blocks, reagents and solvents for reactions were
reagent grade and used as purchased. Flash chromatography was performed on a Biotage Isolera 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. 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 and diode array detector coupled to a Waters G2 QToF mass
1 10
2
3
4
5
6
7
1
1
1
2
8 13
9
0
14
15
16
17
18
19
1
2
3
4
5
6
7
8
9
0
1
2
3
1
13
spectrometer fitted with a multimode ESI/APCI source. H and C NMR spectra were recorded on a
Bruker Avance 500 MHz or a 300 MHz spectrometer using an internal deuterium lock. NMR data is
given as follows: chemical shift (δ) in ppm, multiplicity, coupling constants (J) given in Hz and
integration. All final inhibitors submitted for biological evaluation were at least 95% pure by HPLC,
apart from compound 2g, which has a purity of 94%.
4 20
5
6
7
8
9
0
2
2
1
2
General procedures GP1: Alkylamine was added to solution of sulfonyl chloride 11a in DCM and the
1 23
reaction mixture was stirred at rt for 1 - 16 h. 1 M HCl was added and the aqueous phase was extracted
2
3
2
2
2
4
5
6
with DCM (3 ×). The combine organic layer was dried over MgSO , filtered and the solvent was
4
4
5
6
7
8
9
0
removed under reduced pressure. MeOH and aq. NaOH were then added and the mixture was stirred at
rt for 16 h (MeOH and aq. NaOH can be replaced by 7 N NH in MeOH). H O was added and the
3
2
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1
2
3
4
5
6
7
8
9
aqueous phase was extracted with DCM (3 ×). The combined organic phase was dried over MgSO4,
filtered and the solvent was removed under reduced pressure to afford the desired AMT-sulfonamide,
which could be further purified if necessary.
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
General procedures GP2: 4 M HCl in dioxane or 2 M HCl in Et O was added to tert-butyl carbamate
2
(neat or as a solution in DCM) and the mixture was stirred at rt for 1 - 16 h. EtOAc was added to
precipitate the solids if necessary. The solids were collected by filtration or centrifugation, washed with
EtOAc and dried under vacuum to afford the desired amine hydrochloride. If necessary, this can be
further purified by chromatography in its free amine form, which can be obtained by treatment with 7 N
0
1
2
3
4
5
6
7
8
9
0
NH in MeOH.
3
1 10
General procedures GP3: BH .THF was added to a solution of heteroaryl nitrile in THF and the
3
2
3
4
5
6
7
1
1
1
2
mixture was stirred at rt for 1 - 5 h. EtOH (equal volume to reaction mixture) was then carefully added
o
to quench the reaction. The solution was subsequently heated at 70 C for 1 h to aid borane
8 13
decomplexation. The solvent was removed under reduced pressure to afford the desired amine, which
could be purified if necessary.
9
0
14
15
16
17
18
19
1
2
3
4
5
6
7
8
9
0
1
2
3
General procedures GP4: A mixture of 5-bromothiophene-2-carbonitrile 17a (interchangeable with 5-
chorothiophene-2-carbonitrile) or 2-bromothiazole-5-carbonitrile 17b, alkyl or aryl thiol, K CO and
2
3
o
o
DMF was stirred at 50 C - 140 C. After cooling to rt, the mixture was diluted with EtOAc. The organic
phase was washed with 1:1 H O/brine (3 ×), dried over MgSO and filtered. The solvent was removed
2
4
under reduced pressure to afford the desired sulphide product, which could be further purified if
necessary.
4 20
5
6
7
8
9
0
2
1
2
General procedures GP5: mCPBA (>2 equiv. for sulfones, 1.0 equiv. for sulfoxides) was added in
o
o
2
small portions to a solution of sulfide in DCM at 0 C and the mixture was stirred at rt (0 C for
sulfoxides) for 3 - 16 h. When complete conversion was achieved, EtOAc was added. The organic phase
was washed with sat. NaHCO (3×) and sat. Na S O (until no color detected on starch iodide paper),
1 23
2
3
2
2
2
4
5
6
3
2
2
3
4
5
6
7
8
9
0
dried over MgSO and filtered. The solvent was removed under reduced pressure to afford the desired
4
sulfone/sulfoxide, which could be further purified if necessary.
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General procedures GP6: The alkylamine was dissolved in DCM. Et N followed by Boc O were
3
2
1
2
2
3
4
5
6
7
8
9
added and the mixture was stirred at rt for 16 h. When complete conversion was achieved, DCM was
added. The organic phase was washed with H O and brine, dried over MgSO and filtered. The solvent
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
2
4
was removed under reduced pressure to afford the desired tert-butyl-carbamate, which could be further
purified if necessary.
0
1
2
3
4
5
6
7
8
9
0
General procedures GP7: A mixture of aryl bromide, Pd(PPh ) , ArB(OR’) (boronic acid or
3 4
2
o
pinacolatoboronate), Cs CO and 1,4-dioxane was degassed with argon and then stirred at 100 C for 16
2
3
h. After cooling to rt, the mixture was filtered through celite and washed with EtOAc. The solvent was
removed under reduced pressure to afford the desired biaryl, which could be further purified if
necessary.
1 10
2
3
4
5
6
7
t
1
1
1
2
General procedures GP8: A mixture of aryl bromide, Pd (dba) , Xantphos, alkyl or aryl thiol, NaO Bu
2
3
t
o
and BuOH/toluene (1:4) was degassed with argon and then stirred at 100 C for 16 h. After cooling to
rt, the suspension was filtered through celite, washed with DCM. The solvent was removed under
reduced pressure to afford the desired sulfide, which could be further purified if necessary.
8 13
9
0
1
1
1
1
1
1
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
0
1
2
3
(5-(Piperidin-1-ylsulfonyl)thiophen-2-yl)methanamine (2a). The titled compound was synthesized
according to general procedures GP1 - from i) sulfonyl chloride 11a (150 mg, 0.487 mmol), piperidine
(96.3 µL, 0.975 mmol) and DCM (2.4 mL); 16 h, rt. ii) 1 M NaOH (2 mL) and MeOH (2 mL); rt, 16 h.
Compound 2a was obtained as a white crystalline solid (109 mg, 86%) and didn’t require further
1
4 20
purification. H NMR (500 MHz, Chloroform-d) δ 7.37 (d, J = 3.6 Hz, 1H), 6.92 (d, J = 3.6 Hz, 1H),
5
6
7
8
9
0
13
2
2
1
2
4.20 (br, 2H), 3.11 – 2.96 (m, 4H), 1.82 – 1.12 (m, 8H). C NMR (126 MHz, Chloroform-d) δ 134.51,
+
132.45, 123.57, 47.10, 25.23, 23.58. HRMS (ESI) for C H N O S ([M+H] ): Calculated 261.0726;
1
0
17
2
2 2
1 23
Observed 261.0743; Error = 4.8 ppm.
2
3
24
25
26
5-(Aminomethyl)-N,N-dimethylthiophene-2-sulfonamide (2b). The titled compound was
synthesized according to general procedures GP1 - from i) sulfonyl chloride 11a (100 mg, 0.325 mmol),
4
5
6
7
8
9
0
dimethylamine (40% in H O; 0.206 mL, 1.62 mmol) and DCM (1.63 mL); 16 h, rt. ii) 30% NaOH (0.5
2
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