First Structure-Activity Relationship Study of Potent BLT2 Agonists as Potential Wound-Healing Promoters

Article abstract of DOI:10.1021/acs.jmedchem.0c00588

The first potent leukotriene B4 (LTB4) receptor type 2 (BLT2) agonists, endogenous 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT), and synthetic CAY10583 (CAY) have been recently described to accelerate wound healing by enhanced keratinocyte migration and indirect stimulation of fibroblast activity in diabetic rats. CAY represents a very valuable starting point for the development of novel wound-healing promoters. In this work, the first structure-activity relationship study for CAY scaffold-based BLT2 agonists is presented. The newly prepared derivatives showed promising in vitro wound-healing activity.

Full text of DOI:10.1021/acs.jmedchem.0c00588

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Article
First structure-activity relationship study of potent
BLT2 agonists as potential wound healing promoters
Victor Hernandez-Olmos, Jan Heering, Viktoria Planz, Ting Liu, Alexander Kaps,
Rinusha Rajkumar, Matthias Gramzow, Astrid Kaiser, Manfred Schubert-Zsilavecz,
Michael J. Parnham, Maike Windbergs, Dieter Steinhilber, and Ewgenij Proschak
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00588 • Publication Date (Web): 18 Sep 2020
Downloaded from pubs.acs.org on September 19, 2020
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First structure-activity relationship study of potent
BLT2 agonists as potential wound healing promoters
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a †
a †
b
a,c
Victor Hernandez-Olmos , Jan Heering , Viktoria Planz , Ting Liu , Alexander
a,c
a,c
a,c
c
Kaps , Rinusha Rajkumar , Matthias Gramzow , Astrid Kaiser , Manfred Schubert-
c
a
b
a,c
Zsilavecz , Michael J. Parnham , Maike Windbergs , Dieter Steinhilber , and Ewgenij
Proschaka,,c*
a Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Branch for
Translational Medicine and Pharmacology TMP, Theodor-Stern-Kai 7, 60596 Frankfurt
am Main
b Institute of Pharmaceutical Technology and Buchmann Institute for Molecular Life
Sciences, Goethe University, Max-von-Laue-Str. 15, 60438 Frankfurt am Main,
Germany.
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c Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str.
9, 60438 Frankfurt am Main, Germany
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These authors contributed equally.
KEYWORDS
Leukotriene B4 receptor type 2, BLT2, Wound healing, Diabetes, Cay10583,
Keratinocyte, Fibroblast, SAR
ABSTRACT
The first potent Leukotriene B4 (LTB4) receptor type 2 (BLT2) agonists, endogenous
12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and synthetic CAY10583
(CAY) have been recently described to accelerate wound healing by enhanced
keratinocyte migration and indirect stimulation of fibroblast activity in diabetic rats. CAY
represents a very valuable starting point for development of novel wound healing
promoters. In this work, the first structure-activity relationship study for CAY scaffold-
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based BLT2 agonists is presented. The newly prepared derivatives showed promising in
vitro wound healing activity.
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Introduction
The leukotriene B4 type 1 (BLT1) and type 2 receptors (BLT2) are members of the
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rhodopsin-like G-protein-coupled receptor superfamily. BLT2 was first described in
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2000 by Yokomizo et al. The two transmembrane GPCR receptors differ in their affinity
and specificity for their ligands. BLT1 is the high-affinity receptor for leukotriene B4
(LTB4, Figure 1A) (BLT1, pK = 8.5) and is predominantly expressed in leukocytes in
d
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humans. BLT2 is the low affinity receptor for LTB4 (BLT2, pK = 7.64) and is expressed
d
ubiquitously in humans, with highest mRNA expression level in the spleen, liver,
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ovaries, and leukocytes. In mice, BLT2 is found predominantly in epidermal
keratinocytes and small intestine tissues, but also in human epidermal keratinocytes
BLT2 is detected in high concentrations.5–7 12-Hydroxyheptadecatrenoic acid (12-HHT,
Figure 1B) was identified in 2008 as the endogenous potent agonist of BLT2 in humans
8
and mice leaving BLT1 unaffected. While the role of BLT1 as a potent chemotactic
receptor for inflammatory cells has been well-studied, and the benefits of BLT1
inhibition in inflammatory diseases such as bronchial, chronic inflammatory bowel
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disease, arthritis, dermatitis, and osteoporosis are well known, there are only a few
studies examining the physiological and pathophysiological role of the BLT2 receptor.7; 8
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BLT2 activation has also been associated with a reduction of neuropathic pain. BLT2
activation results in a significantly reduced perception of pain as a result of a reduction
in the sensitivity of the transient receptor potential cation channel subfamily V member 1
(TRPV1). In experiments in rats, TRPV1 antagonists have been shown to significantly
reduce inflammation and neuropathic pain, yet TRPV1 inhibition leads to hyperthermia
as a side effect.10; 11 However, BLT2 activation does not alter the basal activity of
TRPV1 but reverses the sensitization of nociceptive inhibitors,10 making it an interesting
target for the development of new analgesics.
12
In addition, BLT2 has been implicated in diseases such as carcinogenesis and
13
inflammatory arthritis, and has also been considered as a potent therapeutic target for
new drugs for the treatment of asthma.8; 11
Among the potential therapeutic applications of BLT2 activation, wound healing
acceleration stand out. Chronic and slow healing wounds are a serious global problem
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nowadays. Only in the USA, chronic wounds are reported to affect 6.5 million patients
resulting in more than US$25 billion yearly expenses to the healthcare system on
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treating wound-related complications. The cause of disturbed wound healing is
manifold, but usually one consequence of chronic underlying diseases such as Diabetes
mellitus (DM).15–17 According to the World Health Organization, the number of people
with DM has risen from 108 million in 1980 to 422 million in 2014. Around 2-10% of
people affected with diabetes suffer from chronic wounds, such as diabetic foot
18
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syndrome, with half of them suffering infections leading to amputation in 1 out of 5
cases.16; 17
Natural wound healing is a complex, multi-stage, injury-initiated process that restores
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the integrity of normal tissue. Traditionally, the wound healing process is subdivided
into the exudation, granulation, and epithelialization phases. These phases are the
result of numerous complex processes involving different cell types such as
keratinocytes, epithelial cells, growth factors, cytokines, and extracellular matrix
components. Epithelialization, in which the keratinocytes at the edge of the wound
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increasingly divide and migrate into the wound, and the fibroblast-mediated wound
contraction, which ensures continuous centripetal reduction of the wound surface,
represent two distinct mechanisms leading to direct wound closure. If the individual
biological processes in wound healing are disturbed, wounds cannot heal at all or only
with delay. Patients with diabetes often develop wound healing disorders due to
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endothelial dysfunction and impaired angiogenesis. Consequently, reduced migration
and proliferation of keratinocytes and fibroblasts at the wound edges of non-healing
diabetic wounds is observed. BLT2 activation is reported to accelerate wound healing
by enhanced keratinocyte migration in diabetic mouse models stimulated using 12-HHT
as BLT2 agonist.22; 4
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A study by Luo et al. showed that synthetic BLT2 agoinst CAY10583 (1, Figure 1C)
significantly enhances granulation in addition to stimulating epithelialization. Moreover,
the first evidence for the indirect stimulation of fibroblast activity by increased
expression of growth factors TGF-β1 and bFGF in keratinocytes treated with CAY was
provided and in wounds treated with CAY, complete wound closure was observed.
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Additionally, a very recent publication presented CAY-promoted BLT2 activation as a
possible treatment of Crohn's disease internal intestinal lesions via acceleration of
epithelial cell proliferation.23
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A
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O
O
OH
OH
OH
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OH
OH
LTB4
12-HHT
CAY10583 (1)
Figure 1. Structures of LTB4 (A), 12-HHT (B) and Cay10583 (C)
All in all, BLT2 activation constitutes an attractive strategy for the development of new
therapeutics, which may be especially promising in chronic persistent diabetic wounds
and the cure of diabetic foot syndrome. However, with just a few agonists known to
date, there is a need to understand the structural features that lead to potent
compounds. This knowledge should allow the development of new agonists that can
reach the clinic. In this work, the first structure-activity-relationship study of a BLT2
receptor agonist based on the CAY scaffold is presented. Some of the newly prepared
compounds were tested in a scratch assay showing comparable activity to CAY.
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Chemistry
Most CAY10583 (1) derivatives were prepared according to the synthetic route
depicted in Scheme 1. First, a Suzuki reaction catalyzed by tetrakis
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(triphenylphosphine)palladium(0) took place coupling the corresponding boronic acids to
bromoaryl starting materials. The resulting aldehydes 2-12 were converted to secondary
amines via reductive amination, with different amines and sodium borohydride as a
reducing agent, to provide intermediates 13-37. Then, acylation with the corresponding
acid chloride in presence of triethylamine gave products 39-75. Finally, in the case that
R is a methyl ester group, hydrolysis of the ester under alkaline conditions provided the
2
final products 76-107.
R1
R1
R1
R2
R4
O
R2
HN
R2
R4
O
Br
R5
+
OH
B
b
a
OH
R3
R3
R3
2-12
13-37
R4
O
R6
R1
R6
R1
O
OH
O
N
R2
N
R = COOMe
2
R5
R5
d
c
R3
R3
39-75
76-107
R4
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Scheme 1. General synthetic route for CAY derivatives. Conditions: a) Pd(PPh
3
)
4
, Cs
2
CO
3
, DME, Toluene, H
2
O,
8
0 °C, 20-96 % b) 1. R
5
NH
2
, CHCl
3
, MgSO
4
2. NaBH
4
, THF/EtOH, 50 °C, 9-99% c) R
6
COCl, Et
3
N, CH Cl , 21-
2
2
99% d) LiOH, THF, H
2
O, MeOH, 60 °C, 19-99 %.
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In the case that R = 4-benzonitrile, the nitrile group was reduced to methyl group in
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the reductive amination step. For this reason, an alternative synthetic route was
designed to obtain intermediate 38 (Scheme 2).
NH2
HN
CO Me
2
Br
CO Me
2
+
a
CN
CN
3
8
3
Scheme 2. Alternative synthetic route for the synthesis of intermediate 38. Conditions: TBAB, CH CN, reflux, 45%
The structures of all intermediates are shown in Tables 1-3 for clarity.
Table 1. Structure of aldehyde intermediates 2-12.
R1
O
O
OMe
OEt
O
R2
R4
O
O
N
N
R3
N
2-9, 11
10
12
Entry Compd. R1
R2
R3 R4
1
2
2
3
H
H
CO Me
H
H
H
2
CO
Me
H
2
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F
CO Me
H
H
H
F
H
H
H
H
F
2
H
H
H
H
H
H
SO CH
2
3
6
SO NH
2
2
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CO Me
2
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CO Me
H
H
H
2
9
NHSO Me
H
F
2
11
CN
Table 2. Structure of secondary amine intermediates 13-37.
R1
O
HN
R2
HN
X
N
HN
OEt
R5
N
N
R3
R4
1
3-25, 27-29, 31-36
26: X = CH OH
30: X = CO Et
37
2
2
Entry Compd. R1
R2
R3 R4
R5
Ph
1
2
3
4
5
6
7
8
13
14
15
16
17
18
19
20
H
H
H
F
CO Me
H
H
F
H
H
H
H
H
H
H
H
2
CO
Me
4-ClPh
Ph
2
CO Me
2
CO Me
H
H
H
H
H
Ph
2
H
H
H
H
CO Me
nBu
Ph
2
SO Me
2
SO NH
Ph
2
2
CO Me
4-MeOPh
2
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34
35
36
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CO Me
F
H
F
Ph
Ph
2
1
0
1
2
3
4
5
6
7
8
9
0
1
2
CO Me
H
H
H
H
H
H
H
H
H
H
H
H
H
2
1
1
1
1
1
1
1
1
1
2
2
2
CO Me
H
H
H
H
H
H
H
H
H
H
H
F
3-ClPh
Ph
2
NHSO Me
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
CO Me
Bn
2
CO Me
iPr
2
CO Me
Cy
2
CO Me
4-PhOPh
2
CO Me
4-CF OPh
2
3
CO Me
4-FPh
2
CO Me
4-CF Ph
2
3
CO Me
3-FPh
3,4-diFPh
4-FPh
2
CO Me
2
CN
Table 3. Structure of amide intermediates 43-75.
R6
R1
O
O
N
R2
R4
O
N
X
N
O
N
OEt
R5
N
N
R3
43-71, 75
72
7
X = CH OCOnBu
2
74
3 X = CO Et
2
Entry Compd. R1
43
R2
CO Me
R3 R4
R5
R6
Ph
1
H
H
H
nBu
2
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
CO
2
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
nBu
nBu
Bn
Bn
CO Me
PhCH CH
2
2
2
CO Me
nBu
nPr
Et
2
CO Me
Ph
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
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
CO Me
Ph
2
CO Me
Ph
Me
2
CO Me
Ph
CH OCH CH
3 2 2
2
CO Me
Ph
Bn
2
1
0
1
2
3
4
5
6
7
8
CO Me
Ph
iPr
2
1
1
1
1
1
1
1
1
CO Me
Ph
iBu
Ph
2
CO Me
Ph
2
CO Me
iPr
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
nBu
2
CO Me
Cy
2
CO Me
4-ClPh
4-MeOPh
3-ClPh
4-PhOPh
2
CO Me
2
CO Me
2
CO Me
2
19
20
21
22
23
24
25
26
27
CO Me
4-CF OPh
2
3
CO Me
3-CH Ph
2
3
CO Me
4-FPh
2
CO Me
4-CF Ph
2
3
CO Me
3-FPh
3,4-diFPh
4-CNPh
Ph
2
CO Me
2
CO Me
2
CO Me
2
H
CO Me
Ph
2
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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
2
3
8
9
0
70
71
75
H
H
H
CO Me
F
H
H
H
F
F
Ph
Ph
nBu
nBu
iBu
2
CO Me
2
CN
4-FPh
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
Alternative synthetic routes were also used to prepare final compounds 114-117 as
depicted in Scheme 3. CAY scaffold was constructed in one step from the
corresponding amides and benzyl bromides to obtain compounds 109-112. Methylation
with methyl iodide and sodium hydride of compound 111 was used to obtain
intermediate 113. Final products 114, 115 and 117 were prepared by hydrolysis of the
corresponding methyl esters 110, 111 and 113 with lithium hydroxide. Compound 116
was obtained by deprotection of the tert-butyl ester derivative 112 with trifluoroacetic
acid.
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9
O
OH
O
OH
O
N
O
N
H
114
1
15
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
c
c
R1
O
R1
Br
R3
+
O
OH
NH
R2
O
N
R3
O
N
N
a
R2
d
109-112
R1
R2
R3
CN
116
b
1
1
1
09 nPr
Ph
Ph
10
COOMe
COOMe
O
OH
O
OMe
c
O
N
nPr
H
O
N
11
CH3
CH3
nPr
4-Py COOtBu
112
1
13
117
Scheme 3. Alternative synthetic route for the synthesis of 114-117. Conditions: a) NaH, DMF 0°C to rt, 25-98% b)
MeI, NaH, DMF, 50% c) LiOH, THF, H O, MeOH, 60 °C, 34-56% d) CF CO H, CH Cl , 0°C to rt, 80%
2
3
2
2
2
Compound 109, obtained according to Scheme 3, was further transformed to obtain
compounds 118, 120 and 121 as shown in Scheme 4. Tetrazole derivative 118 was
obtained by reaction of 109 with Bu SnN . On the other hand, reaction of 109 with
3
3
ammonium hydroxide solution in ethanol provided desired intermediate 119 and 120 as
a side product, which was also isolated and tested. Further reaction of intermediate 119
with CDI in presence of DBU gave derivative 121.
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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
OH
N
b
NH2
O
NH2
O
N
+
O
N
O
N
CN
1
09
119
120
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
a
c
O
N N
O
N
NH
N
NH
O
N
O
N
121
1
18
3 3 2
Scheme 4. Synthesis of final compounds 118, 120-121. Conditions: a) Bu SnN , xylene, 160 °C, 45% b) NH OH,
EtOH, reflux, 42% for 119 and 5% for 120 c) CDI, DBU, dioxane, reflux, 56%.
Compounds 122 and 123 were obtained by late stage functionalization reactions from
1 and 93 respectively as shown in Scheme 5. Amide coupling conditions between 1 and
methanesulfonylamide provided derivative 122 while compound 123, bearing a phenol
group, was obtained by deprotection of the methoxy group of 93 with boron tribromide.
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9
H
O
N
CH3
O
OH
O
N
S
O
N
a
b
O O
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
1
22
O
OH
O
OH
O
N
O
N
OMe
OH
93
123
Scheme 5. Synthesis of compounds 122-123. Conditions: a) CH
3
2 2 3
SO NH , EDCl, DMAP, DCM, 57 % b) BBr ,
CH Cl , 78°C to rt, 39%.
2
2
In order to investigate the importance of the amide carbonyl group on the activity,
derivative 125 was prepared according to Scheme 6. Intermediate 124 was obtained by
reductive amination from aniline 13 and valeraldehyde. Then, 125 was obtained, as in
the general synthetic route, by hydrolysis of the ester group.
O
OMe
O
OMe
O
OH
HN
a
N
b
N
13
124
125
Scheme 6. Synthesis of compound 125. Conditions: a) Valeraldehyde, H
O, MeOH, 60 °C, 43%.
2
SO
4
, NaBH
4
, THF, 80% b) LiOH, THF,
H
2
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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
Finally, urea and sulfonamide derivatives 128 and 129 were obtained according to
Scheme 7. Once more, aniline 13 was used as the starting material for both
compounds. Reaction of 13 with butyl isocyanate and trimethylamine provided urea
intermediate 126, while reaction of 13 with 1-butanesulfonyl chloride in pyridine gave
sulfonamide intermediate 127. Hydrolysis of the ester groups gave, as usual, the
desired final products.
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
NH
N
NH
O
OMe
O
O
OH
c
O
N
a
b
O
OMe
128
HN
126
13
O
S
O
OMe
c
O
N
S
O
OH
O
N
O
127
129
Scheme 7. Synthesis of compounds 128 and 129. Conditions: a) CH
3
(CH
2
)
3
NCO, Et
3
N, CH
2
Cl
2
, rt, 3 days, 60 % b)
CH (CH SO Cl, pyridine, rt, 74% c) LiOH, THF, H O, MeOH, 60 °C, 90% for 128 and 44% for 129.
3
2
)
3
2
2
Results and discussion
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For EC determination, the compounds were tested for their potency in a functional
50
cell-based assay with detection of accumulation of inositol monophosphate (IP-1) as a
measure for activation of BLT2. Efficacy was compared to the reference agonist CAY.
Compounds were also tested for off-target activity on BLT1 using the same assay
principle and LTB4 as reference agonist for BLT1.
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
Results are summarized in Table 4.
Table 4. Final product structures and EC values for BLT2 activation.
50
O
N N
NH
O
OH
N
N
NH
O
N
R
O
N
O
N
O
N
N
1, 39-42, 109, 120, 122
118
121
105
BLT2
EC ±SEM (nM)
Efficacy compared
to cpd #1 ± SEM
off-target test
on BLT1
Entry Compd.
R
5
0
1
2
3
1
COOH
28.9 ± 2.2
~ 570 *| ˅
inactive
100%
< 16% ˅
-
inactive
inactive
39
40
COOMe
SO CH
not tested
2
3
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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
4
5
6
7
8
41
42
SO NH
inactive
inactive
-
inactive
inactive
inactive
inactive
inactive
inactive
2
2
NHSO CH
-
2
3
105
109
118
CH OH
~ 4260 *
inactive
~ 12%
-
2
CN
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
tetrazole
23.2 ± 2.3
82 ± 3%
9
120
CONH2
~ 1730 *| ˅
< 30% ˅
1
0
121
122
1,2,4-oxadiazole
CONHSO CH
48.2 ± 6.1
77 ± 3%
52 ± 2%
inactive
inactive
11
2060 ± 480
2
3
* Compound was tested in triplicate but the experiment was not conducted two or more times, and hence,
SEM was not determined.
˅
Pronounced reverse effect at compound concentrations > 10 µM. Therefore, efficacy was estimated
based on the maximal IP-1 concentration reached, and data points from higher concentrations were not
taken into account for determination of the EC50
.
We first studied the effect of diversifications at the carboxylic acid position of compound 1.
Different bioisosters of the original carboxylic acid were studied. The presence of a relatively
acidic hydrogen in this position seems to be key for the activity since derivatives bearing no
acidic hydrogen were inactive (40, 109) or showed only moderate/poor activity (39, EC = 570
50
nM; 105, EC = 4260 nM; 120, EC = 1730 nM). On the other hand, bioisosters with relatively
5
0
50
acidic hydrogens presented high activity (1, EC = 28.9 nM; 118, EC = 23.2 nM; 121, EC =
5
0
50
50
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4
8.2 nM) with exception of sulfonamide derivatives (41 and 42, inactive and 122 EC = 2060
5
0
nM). Selectivity against BLT1 was not affected by diversification at this position.
Modifications on the amide substitution were studied next (Table 5). Exchange of R and R2
1
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
substituents on the amide group in comparison to 1 was tolerated but brought a considerable
decrease in activity (76, EC = 216 nM). Longer aromatic substituents on R (Bn, 77;
5
0
1
PhCH CH , 78) were even slightly less active in combination with R = nBu. In a similar way,
2
2
2
exchange of phenyl group in R for a Bn group gave result to a more dramatic decrease in
2
activity (79, EC = 2650 nM). A number of different groups were further studied on R (80-87).
5
0
1
Substituents with at least 3 carbons in length were well tolerated (80, 84, 86-87, 114) with the
exception of ether 83. On the other hand, shorter substituents (81, 82, 85) decreased the activity.
Exchange of the phenyl group in R for aliphatic groups (88, 89, 117) or no substituent (115)
2
resulted in poorly active compounds, meaning that the presence of the phenyl ring is of high
importance for the potency. Therefore, substituents on the phenyl ring were studied next (90-100,
1
16, 123). Small substituents in para position with electron withdrawing (90, 96, 100) or weak
electron donating properties (95) were preferred. On the contrary, bigger substituents (93, 94, 97)
or strong electron donating substituents (91, 123) provided less potent compounds. A derivative
with a meta chloro substitution (92) was much weaker than its para equivalent, indicating that
there might be a size restriction in this position of the molecule. Smaller substituents like fluorine
were better tolerated (98-99). A derivative with 4-pyridine substitution instead of the phenyl ring
(116) was however only weakly active.
To complete our study on this part of the scaffold, variations on the amide functional group
were introduced. A compound lacking the carbonyl group (125) was totally inactive, indicating
that this group plays an essential role in the binding to the protein. Exchange of the amide for a
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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
urea derivative (128) brought just a small decrease in activity but a sulfonamide functional group
was inactive (129).
Table 5. Final product structures and EC values for BLT2 activation
50
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
R1
O
OH
O
OH
O S
O
O
OH
O
N
N
N
R2
1, 76-100, 114-117, 123, 128
125
129
BLT2
Efficacy
compared to
off-target
test on
Entry Compd.
R1
R2
EC ±SEM
50
(nM)
cpd #1 ± SEM
BLT1
1
2
3
4
5
6
7
8
9
1
76
77
78
79
80
81
82
83
114
84
nBu
Ph
Ph
nBu
nBu
nBu
Bn
Ph
28.9 ± 2.2
216 ± 43
789 ± 55
404 ± 2%
2650 ± 30
42.2 ± 9.2
390 ± 37
2400 ± 360
3060 ± 630
103 ± 1
100%
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
not tested
inactive
inactive
54 ± 2%
53 ± 11%
41 ± 2%
59 ± 1%
85 ± 2%
65 ± 1%
59 ± 3%
48 ± 2%
89 ± 5%
84 ± 5%
Bn
PhCH CH
2
2
nBu
nPr
Et
Ph
Me
Ph
CH OCH CH
2
Ph
3
2
1
0
1
cyclopropylmethyl
Bn
Ph
1
Ph
38.3 ± 3.0
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2
3
4
5
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7
8
9
1
1
1
1
2
3
4
5
85
86
87
88
iPr
iBu
Ph
Ph
Ph
Ph
iPr
398 ± 13
36.3 ± 1.7
52.0 ± 11.5
6460 ± 700
78 ± 2%
81 ± 4%
88 ± 2%
48 ± 4%
inactive
inactive
inactive
nBu
inactive
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
7
3 ± 14%
agonism at
16
89
nBu
Cy
2960 ± 500
≥
10 µM
1
1
1
2
2
2
7
8
9
0
1
2
117
115
90
nBu
nBu
nBu
nBu
nBu
nBu
Me
H
≥ 7600 □
658 ± 135
47.5 ± 7.4
229 ± 58
720 ± 140
inactive
~ 55%
73 ± 14%
69 ± 6%
82 ± 3%
89 ± 3%
-
inactive
inactive
inactive
inactive
inactive
inactive
4-ClPh
4-MeOPh
3-ClPh
4-PhOPh
91
92
93
4
-
-
agonism at
≥ 10 µM
2
3
94
nBu
inactive
CF OPh
3
max ~ 115% ˅
inactive
2
2
4
5
95
96
nBu
nBu
4-CH Ph
26.0 ± 3.0
27.3 ± 5.5
3
4-FPh
88 ± 5%
-
inactive
agonism at
26
97
98
99
nBu
nBu
nBu
4-CF Ph
inactive
51.8 ± 9.1
48.8 ± 6.1
3
≥
10 µM
2
2
7
8
3-FPh
102 ± 5%
84 ± 6%
inactive
inactive
3
,4-
diFPh
4-NCPh
4-Py
2
3
3
9
0
1
100
116
123
125
128
129
nBu
nBu
nBu
134 ± 12
~ 1670 *
~ 750 *
120 ± 1%
~ 44%
inactive
inactive
inactive
4-HOPh
~ 53%
32
33
34
inactive
-
inactive
inactive
inactive
NHnPr
Ph
96.0 ± 12.0
inactive
95 ± 3%
-
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Journal of Medicinal Chemistry
Page 24 of 115
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2
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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
*
Compound was tested in triplicate but the experiment was not conducted two or more times, and hence,
SEM was not determined.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
˅
The compound showed pronounced reverse effect at concentrations ≥ 50 µM.
□
The potency was too low for a more accurate determination of EC50
.
Variations on the middle and right aromatic rings were also investigated (Table 6).
Introduction of a fluorine group on R (101) was tolerated with no change on the activity.
1
Regarding the right aromatic ring, fluorine substitution performed again better than the
bigger chlorine in R . Furthermore, a fluorine substituent in R was also beneficial and
3
2
brought a small increase in activity. Exchange of the phenyl group for other
heterocycles like pyridine (106) or N-methylimidazole (107) resulted in no improvement
of potency.
Table 6. Final product structures and EC values for BLT2 activation
50
R1
O
OH
O
OH
O
OH
O
N
O
N
O
N
N
N
R3
N
R2
1, 101-104
106
107
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Page 25 of 115
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9
BLT2
Efficacy
compared to
cpd #1 ±SEM
off-target
test on
BLT1
Entry
Compd.
R1
R2
R3
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
EC ±SEM
50
(nM)
1
2
3
4
5
6
7
1
H
F
H
H
H
H
F
H
H
Cl
F
28.9 ± 2.2
48.4 ± 5.6
428 ± 47
60.7 ± 11.5
22.1 ± 3.0
357 ± 22
inactive
100%
75 ± 2%
44 ± 1%
76 ± 7%
85 ± 4%
96 ± 5%
-
inactive
inactive
inactive
inactive
inactive
inactive
not tested
101
102
103
104
106
107
H
H
H
H
Finally compound 108, featuring some of the well-tolerated modifications discovered
in our SAR study (isobutyl group like in 86, 4-FPh like in 96, R =F like in 104 and the
2
presence of tetrazole group like in 118) was prepared resulting in a highly potent
compound (EC = 25.0 ± 0.8 nM; efficacy = 93.0 ± 3.4% compared to CAY); Table 7.
50
Table 7. EC values for BLT2 activation
50
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Journal of Medicinal Chemistry
Page 26 of 115
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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
N N
N
NH
O
N
F
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
F
108
BLT2
Efficacy
compared to
cpd #1 ±SEM
off-target
test on
BLT1
Entry
Compd.
EC ±SEM
5
0
(
nM)
pronounced
agonism at
≥ 20 µM
108
25.0 ± 0.8
93.0 ± 3.4%
The activation of BLT2 by compound 108 as determined in the IP-One assay was
characterized by a steeper slope, an upper plateau of maximal activation comparable to
that seen for CAY, but also a pronounced inhibitory effect at higher µM concentrations;
(
Supporting Information, Figure S9). At 20 µM accumulation of IP-1 was back to base level,
and 50 µM of compound 108 even causes a reduction down to circa 40% below the base level
detected on untreated cells. No compound dependent modulation of IP-1 accumulation was
observed when cells that lack the BLT2 expression cassette and overexpress only GNA14 (G
protein subunit alpha 14) were stimulated with 108, CAY or 12-HHT (data not shown). Therefore,
the inhibitory effect of high µM concentrations of 108 in cells that express BLT2 and GNA14
cannot be explained by the higher cellular toxicity of 108; Figure S10. This leaves the
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phenomenom of ligand dependent ‘biased signaling’ as the most likely explanation. 108 may differ
from CAY in terms of bias for different G proteins and/or promotion of ß-arrestin recruitment to
the activated receptor. A thorough assessment of biased G-protein signaling would require
additional assays (e.g. cAMP assay for G and G ) and is not possible based on data derived solely
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
s
i
from IP-One. Alternatively, the transforming growth factor-α (TGF-α) shedding assay recently
published by Inoue et al. would enable a comprehensive examination of ligand bias towards
individual GNAs in a single assay platform that is based on G chimeras with subtype specific C-
q
terminal sequences. 24
Signaling of most GPCRs is terminated by phosphorylation of the agonist-bound receptor by
specific kinases and subsequent binding of ß-arrestins. This uncouples the receptor from G protein
signaling and promotes endocytosis of the receptor.25; 26
The higher hillslope observed for compound 108 in comparison to CAY suggests that 108 differs
in terms of active conformations stabilized by the ligand and/or the dwell time of BLT2 in the
active state. For the β2-adrenergic receptor conformational exchange kinetics have recently been
described to differ between an agonist with balanced activation of G protein signaling and β-
2
7
arrestin recruitment in comparison to another agonist that dominantly activates β-arrestin. In
further development of optimized BLT2 agonists the phenomenon of ‘biased signaling’ has to be
taken into account in order to ensure a broad therapeutic window. 28; 29
The SARs of the CAY scaffold are summarized in Figure 2.
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Page 28 of 115
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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
O
N
N
Alkyl chain of at least 3 C long
needed for high activity
Aromatic tolerated
O
N
NH
OH
NH
F tolerated
N
O
=
>> non-
acidic
groups
>
carbonyl needed
O
N
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
aromatic > aliphatic
Ph > Py > pyrazole
H = F > Cl
H =F > Cl
H = F = CH > Cl > CN >>
3
MeO, OH, 4-Py, OPh
Figure 2. SAR summary of CAY scaffold.
Aqueous solubility and metabolic stability
In order to achieve sufficient exposure following administration of the compound,
aqueous solubility and metabolic stability are two important factors for good absorption
and low clearance of the drug. Solubility of the compounds in PBS buffer and in vitro
metabolic half-life using rat liver microsomes were determined. The results are shown in
Table 8. Selected compounds 96 and 108 showed a slightly improved metabolic stability
and a similar solubility as 1.
Table 8. Aqueous Solubility and Metabolic Stability of Selected Compounds
Aq. Solubilitya,
µM)
Rat liver microsomes ( remaining
after 60 min)
b
Cmpd.
(
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9
66 %c
1
250-300
9
6
250-300
250-300
82 %c
73 %c
1
08
aSolubility of compound in PBS buffer, pH 7.4, containing 1% DMSO.
bMicrosome mix from the liver of Sprague-Dawley rats.
cPercentage of remaining compound after 60 min
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
Cell toxicity studies
Selected compounds were tested on human hepatocellular carcinoma cells (HepG2) in
order to gain a first estimate of their toxicological profiles. ATP was taken as global
measure for metabolic activity and cell survival. A reduction in the ATP dependent
®
CellTiterGlo assay compared DMSO control was interpreted as a reduction in cell
survival. After 72 h of treatment the ATP concentration was determined and percent cell
survival was calculated; Figure S10. Compound 108 showed the most pronounced effect
on cell survival. 62.5 µM reduced cell survival to about 60% and higher concentrations
turned out to be even more toxic. However, in this range also CAY and the reference
agonist 12-HHT caused a reduction of cell survival. None of the compounds significantly
ACS Paragon Plus Environment
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