3-Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-lipoxygenase-1

Article abstract of DOI:10.1016/j.bmcl.2015.05.004

Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.

Full text of DOI:10.1016/j.bmcl.2015.05.004

Accepted Manuscript
3
-Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-
lipoxygenase-1
Benjamin Pelcman, Andrei Sanin, Peter Nilsson, Kiyo No, Wesley Schaal, Sara
Öhrman, Christian Krog-Jensen, Pontus Forsell, Anders Hallberg, Mats Larhed,
Thomas Boesen, Hasse Kromann, Stine Byskov Vogensen, Thomas Groth,
Hans-Erik Claesson
PII:
S0960-894X(15)00454-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.05.004
BMCL 22694
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
3 March 2015
5 May 2015
6 May 2015
Please cite this article as: Pelcman, B., Sanin, A., Nilsson, P., No, K., Schaal, W., Öhrman, S., Krog-Jensen, C.,
Forsell, P., Hallberg, A., Larhed, M., Boesen, T., Kromann, H., Vogensen, S.B., Groth, T., Claesson, H-E., 3-
Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-lipoxygenase-1, Bioorganic & Medicinal
Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.05.004
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3
-Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-
lipoxygenase-1
a
b
a,b
b
a,b
Benjamin Pelcman, * Andrei Sanin, Peter Nilsson, Kiyo No, Wesley Schaal,
b
b
b
a
Sara Öhrman, Christian Krog-Jensen, Pontus Forsell, Anders Hallberg, Mats
a
d
d
d
Larhed, Thomas Boesen, Hasse Kromann, Stine Byskov Vogensen, Thomas
d
b,c
Groth, Hans-Erik Claesson
a
Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Box 574,
SE-751 23, Uppsala, Sweden
Biolipox AB, Berzelius väg 3, SE-171 65 Solna
Department of Medicine, Building A3:02, Karolinska University Hospital Solna and Karolinska
Institutet, SE-171 76 Stockholm, Sweden
MedChem ApS, Fruebjergvej 3, DK-2100 Copenhagen
b
c
d
*
Corresponding author. Tel.: +46 70 4411 261; e-mail: benjamin.pelcman@orgfarm.uu.se
Abstract: Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-
lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not
essential for activity or selectivity. Additional halogen substituents on the pyrazole
ring, however, increased activity. Further development led to triazole-4-
carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-
LOX-1 inhibitors.
Keywords: 15-lipoxygenase, inhibitors, arachidonic acid, pyrazole, triazole,
benzoxazole.
Graphical abstract:
Cl
F
F
5
O
IC50 = 99 nM (enzyme)
56 nM (cell)
N
H
Cl
F
O
N
O
N
N
N
H
N
H
N
N
N
Cl
N
H
O
O
Cl
N
N
H
3d
Cl
O
6
O
N
H
IC50 = 2 nM (enzyme)
34 nM (cell)
N
IC50 = 109 nM (enzyme)
7 nM (cell)
IC50 = 550 nM (enzyme)
118 nM (cell)
Cl
N
H
4
Lipoxygenases are a well-studied class of non-heme, iron containing oxidative
enzymes that insert molecular oxygen into long-chain unsaturated fatty acids and
derivatives thereof having a cis-cis-1,4-pentadiene motif. The human 15-
lipoxygenase enzyme has its name due to the insertion of oxygen at position 15 of
arachidonic acid ((5Z,8Z,11Z,13E)- 5,8,11,14-eicosatetraenoic acid) to give 15(S)-
hydroperoxy-5Z,8Z,11Z,13E)- 5,8,11,14-eicosatetraenoic acid (15-HPETE) and
1

1
exists in two variants, 15-LOX-1 and 15-LOX-2. Several lines of evidence indicate
that inhibition of 15-LOX-1 can be effective in the treatment of respiratory
2,3
inflammatory diseases, in particular asthma.
In the preceding paper in this series we described the discovery and synthesis of 1N-
substituted pyrazole-3-carboxanilides as potent and selective inhibitors of 15-LOX-1
3
(
Figure 1). Unfortunately, but not unexpectedly, the propensity of the pyrazole acting
as a leaving group made compounds with an alkyl or an aryl group linked to the 1-
nitrogen of the pyrazole, via a carbonyl or a sulfonyl group, were chemically unstable.
Initial studies demonstrated that the intermediates in the syntheses, i.e. pyrazoles
lacking 1-substituents (3, Scheme 1) where at least as active as the desired
products. Due to a complex patentability landscape, they were at that point not
further investigated, but when we realized that the chemical stability issue could not
be solved, all efforts in the development program were redirected to 1-H-pyrazoles.
This paper will describe how the synthetic program evolved into the development of
clinical candidates based on pyrazol-3-carboxanilides and triazole-4-carboxanilides.
F
F
O
N
O
N
O
N
H
N
H
N
H
Cl
Cl
Cl
N
O
O
N
N
N
Cl
O
O
N
H
O
N
H
Figure 1. Examples of 1N-substituted pyrazole-3-carboxamide 15-LOX-1 inhibitors.
The synthesis of the carboxanilides 3 from pyrazole-3-carboxylic acids 1 is described
3
in the preceding paper and in the patent literature, which also contains experimental
4-9
details. When pyrazole-3-carboxylic acids (1) are readily available, the preferred
route for the synthesis of 3 is the one depicted in Scheme 1, steps a and b. Simply
heating 1 in SOCl
2
with a catalytic amount of DMAP (4-dimethylaminopyridine) gives
, crude 2 is obtained which is
the diketopiperazines 2. After evaporation of the SOCl
2
used as such in the following step, where it is treated with the appropriate aniline.
This reaction is preferably performed by heating 2 with the aniline in the presence of
DMAP in CH
2
Cl , or neat with an excess of the aniline. Compounds 3 can also be
2
obtained directly from 1 and an aniline using a coupling reagent, but although not
optimized, in a lower total yield. In some cases pyrazole carboxylic acids esters (4)
are more readily obtainable than the acids 1 themselves. Although the esters were
readily hydrolyzed, we later developed a more direct route to (3) involving a
6
trimethylaluminium-mediated amidation (Scheme 1, step d).
2

X
N
O
O
Ar
O
N
N
O
OH
1
N
H
a
b
X
X
N
X
N
N
N
H
N
H
2
3
c
d
O
N
OR
4
X
R = Me, Et
N
H
2 2
Scheme 1. General route to pyrazole-3-carboxamides. (a) SOCl (or the like); (b) ArNH ; (c) NaOH; (d)
ArNH /Me Al/CH Cl , rt, 20 °C.
2
3
2
2
We wanted to investigate the influence of substituents on the pyrazole ring on the
activity, but only a few substituted pyrazole-3-carboxylic acids/esters were
commercially available in useful amounts at a reasonable price, e.g. 4- and 5-nitro-
pyrazole-3-carboxylic acid. The preparation of 4- and 5-alkyl substituted pyrazole-3-
3
carboxanilides are described in the preceding paper in this issue.
Many substituted 3-methylpyrazoles are readily available, either commercially or by
synthesis, and are excellent starting materials for the syntheses of pyrazole-3-
carboxylic acids as KMnO expediently oxidizes the methyl group to a carboxylic acid
4
10
in acceptable yields. This approach is used e.g. for the chloro substituted pyrazoles
6
3
a, 3b and 3c (Scheme 2). Chlorination of 3-methylpyrazole using Cl
2
in CCl
4
introduces a chlorine into the 4-position of the pyrazole. A chloro substituent can also
be introduced in the same starting material in the 5-position using a lithiation
approach. This however requires protection/deprotection steps, and a more
convenient method to synthesize 5-chloro-3-methylpyrazole is to N-demethylate 5-
chloro-1,3-dimethylpyrazole by heating it in pyridinium chloride. Chlorination of 5-
chloropyrazole-3-carboxylic acid in the 4-position proceeds in high yield with Cl in
2
water. 4-Bromo and 4-iodo substituted pyrazoles are readily prepared using similar
tactics (not shown). As usual, the acids are converted to the corresponding
carboxanilides using a SOCl
2
mediated dimerization to a diketopiperazine followed
by treatment of an appropriately substituted aniline.
3

F
O
N
O
N
Cl
Cl
OH
Cl
N
H
Cl
a
b
c, d
3a
N
N
N
H
N
H
N
H
N
H
e
O
OH
f
g
h
c, d
3b
N
N
N
N
Cl
Cl
Cl
N
N
S
N
H
N
H
S
O
O
Ph
Ph
O
O
j
i
O
N
Cl
OH
c, d
3c
N
Cl
N
Cl
N
H
Me
Scheme 2. Reagents and conditions: (a) Cl
2
, CCl
, rx, 3 d; (d) 2-chloro-4-fluoroaniline, 120 °C, 1 h (77 % for 3a, 39% for
b, over steps (c) and (d); (e) PhSO Cl/Et N/MeCN, rx, 30 min (86 %); (f) BuLi/THF then Cl CCCl , -78
, H O, t-BuOH, 70 °C
O, rt, 3h, then rt 18 h (86 %)
4 4 2
, -78 °C then rt overnight (98 %); (b) KMnO , H O, rt,
3
3
d, 70 °C, 5 h (22 %); (c) SOCl
2
2
3
3
3
°
C, 10 min; (g) NaOMe/MeOH, rt, 45 min (21 % over steps (f) and (g); (h) KMnO
4
2
(
64 %); (i) pyridine-HCl, 200 °C, 2 h (67 %); (j) Cl , H
2
2
The synthesis of fluoro substituted pyrazoles pose a real challenge. Fluorine has
been introduced in the 4-postion of pyrazole-3-carboxylic acid derivatives using F
2
in
11
12
anhydrous HF or AcOH but as we did not have the equipment to handle
hazardous and corrosive F we had 4-fluoropyrazole-3-carboxylic acid ethyl ester
2
ordered. We did not receive any details of the experimental procedure, but the small
amounts of the compound we obtained consisted of a 2:1 mixture of 4-
fluoropyrazole-3-carboxylic acid ethyl ester and the unsubstituted starting material,
pyrazole-3-carboxylic acid ethyl ester. Hydrolysis, dimerization with SOCl and
2
treatment with 2-chloro-4-fluoroaniline as described above, gives the desired
carboxanilide (Entry 11, Table 2) in a 10:1 mixture with corresponding 4-
unsubstituted compound (Entry 6, Table 1). We were also able to fluorinate pyrazole-
3
-carboxylic acid ethyl ester in the 4-position by using XeF
2
/ CF
3
SO Ag as the
3
fluorinating agent, but the yield was only 7 %. Attempts to synthesize 5-
fluoropyrazoles all failed. Interestingly, there are several suppliers that nowadays
catalog 5-fluoropyrazole-3-carboxylic acid ethyl ester, but its synthesis has to our
knowledge neither been described in patents nor in the scientific literature.
Routes to trifluoromethyl substituted pyrazole-3-carboxylic acid derivatives are
6
delineated in Scheme 3. 4-Trifluoromethylpyrazole-3-carboxylic acid ethyl ester (4a)
was prepared by the [2,3]-dipolar cycloaddition of trimethylsilyldiazomethane to ethyl
4
,4,4-trifluoro-2-butynate. The reaction is crafted after the analogous reaction
4

between diazomethane and 4,4,4-trifluoro-2-butynoic acid, which however gives the
desired compound (as the methyl ester) together with the corresponding 1N-methyl-
13
and 2N-methylpyrazoles. Our synthetic route toward the 5-trifluoromethyl analogue
begins with the reaction of 2-methoxypropene and trifluoroacetic anhydride. The
masked 1,3-diketone formed is heated with hydrazine hydrate in ethanol to give 3-
methyl-5-trifluoromethylpyrazole, which as described before, is oxidized to 1b using
KMnO . The synthesis of 4,5-bis(trifluoromethyl)pyrazoles employs the reaction
4
between 1-aminopyridinium iodide and a cis/trans mixture of 2,3-dichloro-1,1,1,4,4,4-
hexafluorobut-2-ene. The pyridine ring of the formed pyrazolo[1,5-a]pyridine is
14
oxidatively ruptured with KMnO to give 1c. 4-Chloro-5-trifluoromethylpyrazole-3-
4
carboxylic acid (1d) and the two 5-difluoromethyl substituted pyrazoles 1e and 1f are
obtained using the oxidation and chlorination approach described in Scheme 2. The
ester 4a is converted to the corresponding 4-fluoro-2-chlorocarboxanilide (Entry 16,
Table 2) using the trimethylaluminium-mediated amidation mentioned above. For the
acids 1b to 1f, the standard SOCl /aniline protocol was used to obtain the
2
corresponding carboxanilides (Entries 17-21, Table 2).
O
F
3
C
OEt
O
a
F
3
C
SIMe3
N
2
N
OEt
N
H
4
a
O
N
OH
MeO
O
O
MeO
O
b
c
d
+
N
F
3
C
3
F C
F
3
C
O
CF
3
CF
3
N
H
N
H
1b
O
F
3
C
F
3
C
OH
1c
Cl
CF
Cl
3
e
f
-
I
N +
N
N
F
3
C
F
C
F C
3
3
N
N
H
NH
2
O
N
Cl
Cl
OH
d
d
N
F
3
C
3
F C
N
H
N
H
1d
O
O
Cl
OH
1f
OH
g
N
N
N
F
2
HC
F
2
HC
F
2
HC
N
H
N
H
N
H
1e
Scheme 3. Reagents and conditions: (a) Et
rt, 18 h; (c) N xH O/EtOH, rx, 2 h, 79 % over steps (b) and (c); (d) KMnO
CO /THF, rt, 24 h (86 %); (f) KMnO /t-BuOH/H O, rt, 24 h (67 %); (g) Cl /H
2
2
O, 0 °C, then rt 2 h (75 %); (b) 1. pyridine, -30 °C; 2. Et
/t-BuOH/H O, 70 °C; (e)
O, rt, 3 h (87 %).
2
O,
2
H
4
2
4
2
K
2
3
4
2
2
Starting with commercially available 4- or 5-nitropyrazole-3-carboxylic acid, anilides
containing nitro, amino or methylsulfonamido were synthesized. Here, manipulation
of the substituents on the pyrazole ring was conducted after the anilide formation
(
Scheme 4). The reduction of the nitro group was performed with PtO
2
with NaBH as
4
5

15
the hydrogen donor. If H
2
was used, the chlorine on the phenyl group was replaced
by hydrogen.
F
F
F
O
N
O
N
O
O
OH
N
H
N
H
N
H
Cl
Cl
Cl
a
b
c
O
2
N
O
2
N
H
2
N
2
MeSO NH
N
N
N
H
N
H
N
H
N
H
2 2 2 2
Scheme 4. Reagents and conditions: (a) 1. SOCl , 80 °C, 18 h; 2. ArNH /CH Cl , 60 °C, 8 h; (b)
PtO /NaBH /MeOH, rt, 18 h; (c) MeSO Cl/pyridine, 50 °C, 18 h.
2
4
2
Other heterocyclic cores were explored in parallel to the investigation of the
pyrazoles (Figure 2). Only 1,2,3-triazoles show any appreciable inhibitory activity and
these compounds were investigated further (Table 3). They were prepared using the
same methodology as for the pyrazoles, i.e. dimerization followed by addition of an
16
aniline.
F
F
F
F
F
F
O
O
N
O
N
O
N
O
O
N
H
N
H
N
H
N
H
N
H
N
H
Cl
Cl
Cl
Cl
Cl
Cl
N
N
S
NH
N
H
N
H
N
O
O
Figure 2. Investigated pyrazole bioisosters
Having an anilide moiety in a drug may be problematic as anilines, present as minute
impurities, or formed by hydrolysis in the stomach or in the intestines, are potentially
mutagenic. We therefore prepared a series of benzoxazolyl-substituted pyrazoles, as
a benzoxazole can be considered to be an anilide bioisoster. The compounds are
readily prepared by condensing pyrazole-3-carboxylic acids and 2-aminophenols
17
using polyphosphoric acids (Scheme 5). The yields were at best modest, but we did
not spend time optimizing the reaction.
R
2
O
O
N
OH
N
HO
R
1
R
1
R
2
N
N
H
H N
2
N
H
Scheme 5. Reagents and conditions: polyphosphoric acid, 160 °C, 18 h.
6

In Table 1 a selection of amides of pyrazole-3-carboxylic acid, unsubstituted on the
pyrazole core, are shown. The comparison of the anilide (Entry 1) with the
benzylamide (Entry 2) clearly shows that moving the phenyl away from the pyrazole
skeleton essentially eliminates inhibitory activity. On the anilide portion fluoro and/or
chloro substituents, particularly in the 2 and 4-position, are favorable (Entries 3-6).
Replacing fluoro or chloro with trifluoromethyl decreases activity, but not by much
(Entries 3 and 6 vs. 7 and 8). The unsubstituted 2-pyridyl amide (Entry 9) is
significantly more potent than the 3- and 4-pyridyls (Entries 10 and 11), and also
here addition of halogen substituents increases activity (Entries 12-13). Some 5-
membered heterocylic as well as some bicyclic heterocyclic amides also show good
activity (Entries 14-18). The lack of activity for N-alkylated carboxanilides (Entry 19)
suggests that an amide NH is needed for activity.
Table 2 shows N-(2-chloro-4-fluorophenyl)pyrazole-3-carboxamides with various
substituents in the 4- and/or 5-positions. Compared to the unsubstituted compound
(Entry 1), alkyl substituents decrease activity (Entries 2-6). A 4-phenyl substituent
abolishes activity. Introduction of one halogen generally does not have much
influence on the inhibitory activity whereas two halogens gave stronger inhibition
(Entries 8-15). One or two trifluoromethyl groups decrease the activity, which is
restored by the addition of a chlorine (Entries 16-19). Compounds with difluoromethyl
substituents behaved similarly (Entries 20-21). 4-Nitro, and particularly, 4-amino
substitution gave decent activity, whereas the 5-substituted ones only showed low
activity (Entries 22-25). The sulfonamido-substituted compounds (Entries 26-27)
were practically inactive.
In analogy with the pyrazole SAR, the activity of triazole-3-carboxanilides is
increased by a fluoro or chloro substituents in the 2-, and particular in the 4-position,
which was further increased by 2,3-dihalo substitution (Entries 1-6, Table 3). In
contrast to the pyrazole series, the pyridyl and quinoline amides were not particularly
active (cf. Entries 7-9, Table 3 with Entries 11,12 and 16, Table 1).
Judging by the lack of activity for N-alkylated carboxanilides, Entry 18, Table 1, one
would tend to believe that the amide NH is essential for activity. However, this notion
is not correct as evident from the activity of the 2-(pyrazol-3-yl)benzoxazoles in Table
4
. A chloro substituent, particularly in the 6-positon (Entry 2) of the benzoxazole
increases activity. As before, chloro substituents on the pyrazole core are beneficial
for activity and compound 6 (Entry 7) was the most active of all tested compounds.
7

Unfortunately, all benzoxazoles of Table 4 suffered from very low metabolic stability
1/2 a few minutes) and were deemed unsuitable for further development.
(t
Table 5 exemplifies three of the compounds investigated in the selection for a
candidate drug (CD). Our first criteria was an IC50 < 100 nM in both the cell and
enzyme assays, and the chloropyrazole BLX-2481 (3d) did not fulfil that. Although
relatively potent, the bicyclic arylamide BLX-1873 (3e), showed relatively low
solubility and metabolic stability and poor pharmacokinetic properties. The triazole
BLX-2477 (5) which had a decent IC50 in the cell assay (56 nM) was more
extensively investigated. Although it is a strong inhibitor of several human
cytochrome P450's, particularly 2C9 and 2C19, this was not considered a
showstopper. Although the high metabolic stability may indicate that it inhibits its own
metabolism by being a substrate for the CYP's, this was not investigated at this point.
The compound as such did not show any genotoxicity in the SOS/umu assay, but
this does not say anything about any possible genotoxicity of its metabolites or
degradation products. As BLX-2477, despite its high plasma protein binding, had
favourable pharmacokinetic properties: a long i.v. mean residence time (MRT) of
about 1.8 h, and high exposure in the 10-day toxicology study (150 mg/kg p.o.) i.e. a
Cmax up to 589 µM, it was selected as a CD.
3
As already discussed, compounds may inhibit 15-LOX-1 and its orthologues from
other species to a different degree. It is of course important that a CD exhibits good
activity in the species that will be used in advanced in vivo studies and the activity of
some of our compounds on 15-LOX-1 orthologues were thus investigated. From
Table 5 it is evident that compound 5 does not inhibit the rat or the dog enzymes to
any large extent, but shows good activity towards the pig orthologue. Consequently,
preclinical in vivo studies needs to be conducted using a mini-pig model. In their
search for 15-LOX-1 inhibitors for use in anti-stroke therapy, Maloney and Holman
used a mouse model, but they identified their HTS hits by simultaneously screening
18
both 15-LOX-1 and the mouse ortholog. Obviously, the compounds described in
our paper would not have been considered interesting in their study. Nevertheless,
the importance of using 15-LOX-1 in (one of) the primary screen(s) must be
emphasized.
In conclusion we have developed pyrazole-3-carboxanilides, triazole-4-
carboxanilides and 2-(3-pyrazolyl)benzoxazoles that are good inhibitors of 15-LOX-1.
BLX-2477, N-(2-Chloro-4-fluorophenyl) triazole-4-carboxamide (5) was selected as a
clinical candidate, but regrettably, adverse non-target associated effects were found
8

during the toxicological studies using mini-pigs, which forced us to terminate the
program.
9

1
9,20
Table 1. 15-LOX-1 inhibitory activity of pyrazoly-3-carboxamides
O
R
N
H
IC50/(Imax
)
IC50/(Imax
cell
)
enzyme
N
N
H
Entry
1
R
nM (%)
45*
nM (%)
47*
2
3
NI
NT
F
NT
270 (80)
618
4
5
645
180
Cl
Cl
F
1320
Cl
6
7
8
81
448
24
Cl
CF3
F
519 (69)
94 (70)
168 (79)
F
3
C
1
(
207
71)
9
3410 (53)
N
1
1
1
0
1
2
NT
NT
NI
NI
N
N
F
550 (86)
118 (84)
N
N
(3d)
Cl
1
1
3
4
26 (75)
384
43 (71)
Cl
S
130 (74)
N
1
1
5
6
334 (80)
180 (89)
3740 (68)
92 (47)
N
(
3e)
N
N
1
1
7
8
970 (82)
843 (88)
837 (74)
406 (64)
N
O
N
N
Me
Cl
1
9
NI
NI
N
H
NI = no inhibition; NT = not tested; When no Imax is given it is >90%;
% inhibition @10 µM compound concentration instead of IC50
Arachidonic acid is used as a substrate.
*
;
1
0

19,20
Table 2. 15-LOX-1 inhibitory activity of 4- and/or 5-substituted pyrazoly-3-carboxanilides
F
O
R4
N
H
IC50/(Imax
enzyme
)
IC50/(Imax)
Cl
cell
R5
N
N
H
4
5
Entry
R
R
nM (%)
81
nM (%)
24
1
2
3
4
5
6
7
H
H
Me
Me
H
SiMe
H
3
672 (65)
453
1330 (60)
183
Me
H
376 (71)
NT
757 (76)
855 (69)
42 (37)
NI
n-Bu
H
t-Bu
H
NT
Ph
Cl
NT
8
9
(3a)
(3b)
H
85
33
H
Cl
Cl
H
71 (70)
32
22 (88)
14
1
0 (3c) Cl
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
F
NT
42 (80)
39 (87)
10
Br
Br
I
H
290 (75)
1
Cl
H
184 (78)
150 (89)
NT
551 (76)
27 (86)
117 (87)
112 (82)
279
H
I
CF
3
H
H
CF
CF
CF
3
3
3
1000
NT
CF
Cl
H
3
NT
37 (80)
216 (72)
39
CHF
2
NT
Cl
NO
H
CHF
2
NT
2
H
5900
NI
616
NO
H
2
3290 (81)
69
NH
H
2
641
NH
H
2
5980
NT
334 (58)
NI
NHSO
H
2
Me
NHSO
2
Me
NT
NI
NI = no inhibition; NT = not tested; When no Imax is given it is >90%;
Arachidonic acid is used as a substrate.
1
1

19,20
Table 3. 15-LOX-1 inhibitory activity of triazolyl-4-carboxanilides
O
Ar
N
H
IC50/(Imax
)
IC50/(Imax
cell
)
N
N
enzyme
N
H
Entry
1
Ar
nM (%)
NI
nM (%)
97 (42)
2
3
F
947
2426 (77)
392 (69)
6976
Cl
F
4
5
571
99
68 (81)
56
Cl
Cl
Cl
(
5)
Cl
6
22530 (82)
79 (54)
Cl
F
7
8
3965
1400
3110 (80)
1020 (47)
N
N
9
NT
596 (50)
N
F
NI = no inhibition; NT = not tested; When no Imax is given it is >90%;
Arachidonic acid is used as a substrate.
1
2

19,20
Table 4. 15-LOX-1 inhibitory activity of 2-(pyrazol-3-yl) benzoxazoles
IC50/(Imax
)
IC50/(Imax)
Entry
1
enzyme
nM (%)
cell
nM (%)
N
N
O
3273
20
638 (76)
13 (75)
N
H
Cl
N
N
O
2
3
4
5
N
H
Cl
N
N
443
NT
NT
124 (60)
O
N
H
F
N
N
O
716
N
H
F
N
N
22240
O
N
H
Cl
N
O
6
7
4
2
131
34
N
Cl
N
H
Cl
N
N
Cl
Cl
O
N
H
(
6)
NT = not tested; When no Imax is given it is >90%;
Arachidonic acid is used as a substrate.
1
3

Table 5.
19,20
Experimental data used in the evaluation of possible clinical candidates.
F
Cl
N
O
N
O
O
N
H
N
H
N
H
Cl
N
N
N
N
Cl
N
H
N
H
N
H
BLX-2481 (3d)
BLX-1873 (3e)
BLX-2477 (5)
1
1
1
5-LOX-1 enzyme (IC50, nM)
550 (Imax 86 %)
180 (Imax 89 %)
99
5-LOX-1 cell (IC50, nM)
118 (Imax 84 %)
56 (Imax 68 %)
56
5-LOX-2 enzyme (IC50, nM)
NT
<50 %
<50 %
NI
NT
NT
NI
<50 %
42 %
5-LOX cell
12-LOX enzyme
NT
COX-1 enzyme
COX-2 enzyme
9
4
NI
NI
10
1
5-LOX-1
rat
NT
NT
24 µM (Imax 79 %)
2.4 µM
78 nM
24.3
orthologs
dog
NT
NT
enzyme (IC50
)
pig
NT
NT
pH 2
pH 7.4
pH 10
1/2 (min)
NT
573.1
10.1
10.1
>100
7
Aqeous solubility
µg/mL)
3.4
NT
83.6
(
>286.6
>100
Metab. stab.
human
t
36
CLint
17
<1
microsomes
(µl/min/mg)
Permeability (CACO-2)
high, no efflux
45 %
NT
high, no efflux
high, no efflux
21.6 µM
93 %
P450
1A2
2B6
2C8
2C9
75 %
NT
inhibition
(
IC50 or
at 10 µM)
NT
NT
NI
%
26 %
6 %
NI
0.5 µM
1.4 µM
>100 µM
10 %
2
2
2
3
C19
NI
D6
E1
A4
9 %
NT
NT
NT
NI
NT
58.1 µM
negative
>99 %
NI
Genotoxicity (SOS/umu)
Plasma protein binding
hERG safety assay
negative
92.9
negative
92.8
NI
NI
NI = no inhibition; NT = not tested; When no Imax is given it is >90%;
The values are given as either IC50 or as %-inhibition at 10 µM of the indicated inhibitor.
Arachidonic acid is used as a substrate. Unless stated otherwise, all enzymes/cells are human.
Acknowledgement
The compounds described in the article were kindly provided by Hans-Erik Claesson, CEO, Biolipid AB,
hans-erik.claesson@ki.se
1
4

References and notes
1
2
3
. Kuhn, H.; O'Donnell, V. B. Prog Lipid Res 2006, 45, 334.
. Claesson, H.-E. Prostaglandins Other Lipid Mediat 2009, 89, 120.
. Pelcman, B.; Sanin, A.; Nilsson, P.; Schaal, W.; Olofsson, K.; Krog-Jensen, C.;
Forsell, P.; Claesson, H.-E.; Hallberg, A.; Larhed, M.; Boesen, T.; Kromann, H.
Bioorg Med Chem Lett 2015, preceding paper in this series.
4
5
. Pelcman, B.; Sanin, A.; Nilsson, P. WO 2007/052000 2007,
. Pelcman, B.; Sanin, A.; Nilsson, P.; Groth, T.; Kromann, H. WO 2007/051981
2007,
6
. Pelcman, B.; Sanin, A.; Nilsson, P.; Boesen, T.; Byskov Vogensen, S.; Kromann,
H. WO 2007/045868 2007,
7
8
9
. Boesen, T.; Sanin, A.; Nilsson, P.; Pelcman, B. WO 2006/032852 2006,
. Nilsson, P.; Sanin, A.; Pelcman, B. WO 2006/032851 2006,
. Hallberg, A.; Schaal, W.; Larhed, M.; Olofsson, K.; Pelcman, B.; Sanin, A. WO
04/080999 2004,
1
1
0. Fatin-Rouge, N.; Tóth, É.; Perret, D.; Backer, R. H.; Merbach, A. E.; Bünzli, J.-C.
G. J Am Chem Soc 2000, 122, 10810.
1. Storer, R.; Ashton, C. J.; Baxter, A. D.; Hann, M. M.; Marr, C. L. P.; Mason, A. M.;
Mo, C.-L.; Myers, P. L.; Noble, S. A.; Penn, C. R.; Weir, N. G.; Woods, J. M.; Coe,
P. L. Nucleosides & Nucleotides 1999, 18, 203.
1
2. Kvasyuk, E. I.; Mikhailopulo, I. A.; Pupeiko, N. E.; Tsekh, D. J Org Chem USSR
1983, 19, 403.
1
1
1
1
1
1
3. Tajammal, S.; Tipping, A. E. J Fluorine Chem 1990, 47, 45.
4. Banks, R. E.; Hitchen, S. M. J Chem Soc Perkin 1 1982, 1593.
5. Baraldi, P. G.; Vicentini, C. B.; Simoni, D.; Guarneri, M. Il Farmaco 1983, 38, 369.
6. Pelcman, B.; Sanin, A.; Nilsson, P.; Kromann, H. WO 2007/051982 2007,
7. Sanin, A.; No, K.; Kromann, H. WO 2008/129280 2008,
8. Rai, G.; Joshi, N.; Jung, J. E.; Liu, Y.; Schultz, L.; Yasgar, A.; Perry, S.; Diaz, G.;
Zhang, Q.; Kenyon, V.; Jadhav, A.; Simeonov, A.; Lo, E. H.; van Leyen, K.;
Maloney, D. J.; Holman, T. R. J Med Chem 2014, 57, 4035.
1
9. Enzyme assays. For the enzyme activity assay of human 15-LOX-1, the enzyme
was expressed in Sf9 cells. The enzyme was purified with ion exchange
chromatography. The purity of the enzyme was determined by SD-PAGE
analysis. The human 15-LOX-2 and rat and dog ortologues of 15-LOX-1 was
20
cloned into a Sf9-cell expression vector and subsequently purified. The porcine
orthologue was purchased from Cayman company (USA). Human 12-LOX was
partially purified from platelets. In all experiments, the enzyme and compound
was preincubated for 5 min prior at room temperature followed by incubation
with arachidonic acid for another period of 10 min. The LOX activity was
determined by the conversion of arachidonic acid to 15-HETE for the human 15-
LOX enzymes, or to 12-HETE for human platelet 12-LOX and the orthologues of
2
0
1
5-LOX-1. COX-1 and COX-2 assays were purchased from Cayman company
USA). The inhibition of 12-LOX and COX-1 and 2 was determined as percent
inhibition at 10 µM concentration of the indicated compound. The formation of
5-HETE/12-HETE was followed by HPLC-UV-absorbance at 235 nm. Data
(
1
analysis was perforned using GraphPad Prism software.
Cell assay. The human Hodgkin cell line L1236 has high 15-LOX-1 activity and
2
1
was used in the cell assay. The cells were suspended in phosphate buffer
PBS) and preincubated 5 min in room temperature with the compound prior to
(
addition of arachidonic acid for 5 min. The formation of 15-HETE was analyzed
as described above in the enzyme assay. The inhibition of the 5-LOX pathway
1
5

was investigated in purified human granulocytes. The cells were incubated with
calcium ionophore plus arachidonic acid and the amounts of formed LTB4 was
detected by HPLC-UV-absorbance at 270 nm.
2
2
0. Johannesson, M; Backman, L.; Claesson, H.-E.; Forsell, P. Prostaglandins
Leukot Essent Fatty Acids 2010, 82, 121.
1. Claesson, H.-E.; Griffiths, W.; J.; Brunnström, Å.; Schain F.; Andersson E.;
Feltenmark S.; Johnson, H. A.; Porwit, A.; Sjöberg, J.; Björkholm, M. FEBS J
2
008, 275, 4222.
1
6