Polysubstituted Pyrimidines as mPGES-1 Inhibitors: Discovery of Potent Inhibitors of PGE2 Production with Strong Anti-inflammatory Effects in Carrageenan-Induced Rat Paw Edema

Article abstract of DOI:10.1002/cmdc.202000258

We report an extensive structure-activity relationship optimization of polysubstituted pyrimidines that led to the discovery of 5-butyl-4-(4-benzyloxyphenyl)-6-phenylpyrimidin-2-amine, and its difluorinated analogue. These compounds are sub-micromolar inh

Full text of DOI:10.1002/cmdc.202000258

Accepted Article
Title: Polysubstituted Pyrimidines as mPGES-1 Inhibitors: Discovery
of Potent Inhibitors of PGE2 Production with a Strong Anti-
inflammatory Effects in Carrageenan-Induced Rat Paw Edema
Authors: Filip Kalčic, Viktor Kolman, Haresh Ajani, Zdeněk Zídek, and
Zlatko Janeba
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To be cited as: ChemMedChem 10.1002/cmdc.202000258
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01/2020

10.1002/cmdc.202000258
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Polysubstituted Pyrimidines as mPGES-1 Inhibitors: Discovery of
Potent Inhibitors of PGE2 Production with a Strong Anti-
inflammatory Effects in Carrageenan-Induced Rat Paw Edema
Filip Kalčic,[a,b] Dr. Viktor Kolman,[a] Dr. Haresh Ajani,[a] Dr. Zdeněk Zídek,*[c] and Dr. Zlatko Janeba*[a]
[a]
[b]
[c]
F. Kalčic, Dr. V. Kolman, Dr. H. Ajani, and Dr. Z. Janeba
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
E-mail: janeba@uochb.cas.cz
F. Kalčic
Department of Organic Chemistry, Faculty of Science
Charles University
Hlavova 8, 128 43 Prague 2, Czech Republic
Dr. Zdeněk Zídek
Institute of Experimental Medicine of the Czech Academy of Sciences
Vídeňská 1083, 142 20 Prague 4, Czech Republic
E-mail: zdenek.zidek@iem.cas.cz
Supporting information for this article is given via a link at the end of the document.
Abstract: We report an extensive structure-activity relationship
optimization of polysubstituted pyrimidines that led to a discovery of
5-butyl-4-(4-benzyloxyphenyl)-6-phenylpyrimidin-2-amine, and its
difluorinated analogue. These compounds are submicromolar
inhibitors of PGE2 production (IC50 as low as 12 nM). In order to
identify the molecular target of anti-inflammatory pyrimidines, we
performed extensive studies including enzymatic assays, homology
modeling and docking. The difluorinated analogue simultaneously
inhibits two key enzymes of the arachidonic acid cascade, namely
mPGES-1 and COX-2, where the mPGES-1 inhibition represents the
principal mechanism of action. Other pyrimidines studied are potent
drawback of traditional NSAIDs (t-NSAIDs) is the occurrence of
serious gastrointestinal (GI) complications,[5] namely gastric
bleeding, ulceration, perforation and dyspepsia. Thus, selective
COX-2 inhibitors (coxibs) were developed and commercialized
as promising anti-inflammatory agents in order to avoid the GI
adverse events.[6,7] However, coxibs were later related to the
increased risk of cardiovascular events like myocardial infarction,
stroke, and systemic and pulmonary hypertension.[8]
Due to a wide spectrum of adverse events of t-NSAIDs as
well as cardiovascular toxicity of coxibs, researchers intensively
searched for new approaches to treat inflammation, pain, and
mPGES-1 inhibitors with no observed inhibition of COX-1/2 enzymes. fever. As most t-NSAIDs contained a free carboxylic acid group
Moreover, two most potent compounds proved to be significantly
effective in vivo in a model of acute inflammation, suppressing the
carrageenan-induced rat paw edema by 36% and 46%. Promising
results of this study warrant further preclinical evaluation of selected
anti-inflammatory candidates.
in their molecule, the GI toxicity seemed to be closely related to
their acidic nature. Thus, structural modification of some NSAIDs
like indomethacin and meclofenamic acid (mostly by conversion
of their free carboxylic acid moiety into ester or amide
derivatives) led to a generation of highly selective COX-2
inhibitors with eliminated GI side effects compared to the parent
compounds.[9-12] Also synthesis of analogues with lower
membrane permeabilization activity produced fewer gastric
lesions after their oral administration.[13,14] Other attempts how to
enhance gastric safety profile of anti-inflammatory drugs were
based on their association with cytoprotective mediators (for
Introduction
Prostaglandin E2 (PGE2) is a naturally occurring lipid mediator,
which plays an important role in various inflammatory processes,
fever, pain, and cancer. PGE2 belongs to the most predominant
pro-inflammatory prostanoids. PGE2 biosynthesis consists of the
release of arachidonic acid (AA) from membrane phospholipids
by phospholipases (PLAs), oxygenation of AA to prostaglandin
G2 (PGG2) and subsequent reduction of PGG2 to prostaglandin
H2 (PGH2) by cyclooxygenase (COX) enzymes, and conversion
of PGH2 to PGE2 by inducible microsomal PGE2 synthase-1
(mPGES-1).
Non-steroidal anti-inflammatory drugs (NSAIDs),[1] which
today belong to the most widely used therapeutic agents to treat
inflammation, fever, and pain, exert their biological activity via
non-selective COX enzymes inhibition, i.e. inhibition of both
constitutive COX-1 and inducible COX-2.[2-4] The major
comprehensive
reviews
see[1,15,16]),
such
as
phosphatidylcholine,[17,18] dialkylphosphate,[19] nitric oxide[20-34] or
nitroxyl,[35] and hydrogen sulfide.[36-42] Nevertheless, it has been
speculated,[43] that the NO release is not required to exert the
cytoprotective effects of modified NSAIDs, while the simple
formation of NSAIDs prodrugs is a sufficient condition to develop
a safer alternative to unprotected NSAIDs.
Relatively recent strategy in development of new anti-
inflammatory agents is
a design of compounds targeting
downstream and/or multiple enzymes of the AA cascade. In that
matter, selective inhibitors of mPGES-1 have been recently
identified,[44-61] as well as selective inhibitors of 5-lipoxygenase
(5-LOX),[62] dual COX/5-LOX inhibitors,[34,63-67] dual mPGES-1/5-
LOX inhibitors,[53,68-73] and dual thromboxane antagonists–COX-
1
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10.1002/cmdc.202000258
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2 inhibitors.[74] Similarly, dual inhibition of fatty acid amide
hydrolase (FAAH) and the COX enzymes led to enhanced
analgesic effects of NSAIDs with decreased GI side effects.[75,76]
All these findings have raised high expectations for development
of novel and safer anti-inflammatory drugs.
We previously reported polysubstituted pyrimidines as
potent inhibitors of prostaglandin E2 (PGE2) production with
potential anti-inflammatory properties.[77–81] Out of them, lead
quantitatively.[82] Subsequent lithiation of intermediate 3,
followed by transmetallation using pinacol diboronate,[83]
afforded pinacol ester 4 (Scheme 1) in a 12% yield. However,
the desired product 4 was obtained in a 66% yield when we
employed conditions of catalytic borylation reported by Harada
et al.[84]
compound
5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-
amine (1, Figure 1) was selected for further evaluation as a
preclinical candidate for treatment of inflammation. Moreover,
compound 1 served as a starting point for further structure-
activity relationship (SAR) studies with the aim to identify even
more potent inhibitors of PGE2 production and, eventually,
superior anti-inflammatory agents.
Scheme 1. Synthesis of boronic acid pinacol ester 4. Reagents and
conditions: (i) NBS, MeCN, 98%; (ii) BuLi, B2pin2, THF, -78 °C, 12%; (iii) B2pin2,
K3PO4, Pd(PPh3)4, DMF, 120 °C, 66%.
Secondly, commercially available o-vanillin (2-hydroxy-3-
methoxybenzaldehyde, 5) was used as a starting material in
order to prepare novel (7-methoxybenzofuran-4-yl)boronic acid
pinacol ester (10, Scheme 2). The condensation of o-vanillin
with ethyl 2-chloroacetate afforded ethyl carboxylate 6 in an 84%
yield,[85,86] although Yamaguchi et al.[87] have reported the
carboxylic acid derivative as the only product of the
aforementioned condensation. Intermediate 6 was selectively
brominated with NBS to yield quantitatively bromo derivative 7,
which was subsequently hydrolyzed to give carboxylic acid 8 in
a 94% yield. Microwave-assisted decarboxylation of acid 8 was
performed according to Musser et al.[88] to afford intermediate 9
(Scheme 2). Finally, catalytic borylation[84] of bromo derivative 9
gave the novel pinacol ester 10 in a 75% yield.
Figure 1. Structure of preclinical anti-inflammatory candidate 1 and a general
structure of target compounds prepared within this study.
Herein, we describe a systematic structure-activity relationship
optimization of compound 1 via preferential introduction of
electron-donating groups (e.g. alkyls, amines or ethers) at the
phenyl moiety in C4 position of the pyrimidine ring. Other
substituents (at positions C2, C5, and C6 of pyrimidine) were
kept intact in order not to introduce too many variables. Based
on our previous research,[80-81] such compounds were expected
to exhibit an increased potential to inhibit PGE2 production. The
potency of prepared compounds to inhibit PGE2 production was
evaluated in vitro on mouse peritoneal cells with induced
immune response provoked by lipopolysaccharide (LPS) from
Escherichia coli. The extensive SAR study led to the discovery
of very potent inhibitors of PGE2 production and their efficacy
was verified in vivo using carrageenan-induced rat paw edema
experiments.
Scheme 2. Synthesis of boronic acid pinacol ester 10. Reagents and
conditions: (i) ethyl 2-chloroacetate, Cs2CO3, DMF, 80 °C, 84%; (ii) NBS,
MeCN, 99%; (iii) NaOH, acetone-H2O (4:1), AcOH, 94%; (iv) Cu0, quinoline,
MW 180 °C, 89%; (v) B2pin2, K3PO4, Pd(PPh3)4, DMF, 120 °C, 75%.
Results and Discussion
Chemistry. In our laboratory, Suzuki-Miyaura cross-coupling
represents
a major tool to obtain target polysubstituted
pyrimidines bearing two aromatic moieties in the C4 and C6
positions of the pyrimidine ring.[80-81] Various arylboronic acids
(or their pinacol esters) can be nowadays purchased from
Having all the desired arylboronic acids and pinacol boronates in
hand, the previously reported synthetic methodology[80,81] was
employed for the synthesis of target 2-amino-5-butyl-6-
phenylpyrimidines bearing a substituted/modified phenyl group
commercial suppliers as
a
starting material for the
Suzuki-Miyaura cross-coupling reactions. Nevertheless, two
arylboronic acid pinacol esters had to be prepared in order to be
able to synthesize some of the desired final compounds.
Firstly, synthesis of (4-methoxynaphthalen-1-yl)boronic
acid pinacol ester (4, Scheme 1) started with a selective
in the C4 position of the pyrimidine moiety. Firstly, 2-
[81]
amino-5-butyl-4-chloro-6-phenylpyrimidine
(11, Scheme 3)
was treated with the selected arylboronic acids (and prepared
pinacol boronates 4 and 10), Pd(PPh3)4 and Cs2CO3 in a 1,4-
dioxane-H2O (4:1) mixture at 110 °C to obtain compounds 12–28
in 26–85% yields (Table 1).
bromination
of
1-methoxynaphthalene
using
N-
bromosuccinimide (NBS) to give bromo derivative
3
2
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Being encouraged by the promising data generated by biological
evaluation of the first series of compounds (12–28, Table 1, see
the discussion below), the second series of compounds was
designed based on the most potent 4-(benzyloxy)phenyl
derivative 18. Compounds 29–39 (Scheme 3, Table 2) were
then prepared in 36–99% yields starting from 4-chloropyrimidine
derivative 11 and from various commercially available
arylboronic acids under the above described Suzuki-Miyaura
cross-coupling conditions. It should be noted that the determined
yields were usually based on a single experiment and the
reactions were not optimized.
Scheme 3. Synthesis of target compounds 12–39. Reagents and conditions:
(i) arylboronic acid or pinacol boronate, Pd(PPh3)4, Cs2CO3, 1,4-dioxane-H2O
(4:1), 110 °C, yields are summarized in Table 1 and Table 2.
Table 1. Structures and yields of prepared compounds 12–28 (the 1st series) and their effect on in vitro production of PGE2 and viability of mouse peritoneal cells.
inhibition of PGE2
viability [%][b]
Ar
yield
[%]
entry
compd
Scheme 1
remaining production
IC50 (µM)
(%)[a]
control
untreat
1
2
NA
NA
NA
NA
NA
68
76
63
83
81
74
71
2.58±1.96[c]
NA
100.00±1.99[c]
control LPS
100.00±4.41
12.25±4.48*
18.60±1.37*
18.09±2.63*
26.35±0.12*
12.28±0.50*
16.36±3.77*
10.07±2.62*
NA
NA
4.83[c]
/3.70-6.32/
3
1
102.00±0.89
85.00±14.4
104.10±1.90
104.65±1.30
71.17±1.91*
105.15±0.49
102.58±0.67
105.40±0.23
4
12
13
14
15
16
17
18
ND
ND
ND
5
6
9.05
/5.53-14.80/
7
8
ND
6.58
/3.90-11.11/
9
0.031
/0.018-0.056/
10
0.33±0.17*
3
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11
12
13
14
15
16
17
18
19
20
19
20
21
22
23
24
25
26
27
28
85
77
57
30
68
77
28
50
26
71
30.55±9.77*
25.60±0.11*
15.35±0.34*
34.62±1.24*
32.51 ±2.48*
2.69±0.24*
ND
ND
ND
ND
ND
99.92±1.02
103.99±0.92
45.16±1.25*
35.59±0.84*
36.68±2.42*
102.91±0.32
99.25±1.61
97.63±1.21
101.00±0.46
88.87±0.83
1.09
/0.26-4.67/
56.11±12.69*
64.32±6.14*
53.70±7.34
7.92±1.05
ND
ND
ND
2.70
/1.32-5.55/
[a] Effects recorded for 50 µM concentration of compounds and expressed in % of control LPS response; [b] Cell viability expressed in % of control untreated
cells; [c] ± S.E.M. Results represent means of three to four experiments. Statistical significance: * P < 0.001, not significant in other values. NA: not applicable.
ND: not determined.
Structure–activity relationship study and structural optimization.
All prepared polysubstituted pyrimidines were evaluated for their
ability to inhibit in vitro PGE2 production using C57BL6 mouse
peritoneal cells. The effects of the pyrimidines on the PGE2
production are expressed as a percentage change (remaining
production of PGE2) relative to the response of LPS stimulated
cells (positive control, 100%, Table 1 and 2, entry 2) or
unstimulated cells (negative control, 2.78%, Table 1 and 2, entry
1).
The first series of tested compounds (12–28, Table 1,
entries 4–20) are direct analogues of preclinical candidate 1
(Figure 1, Table 1, entry 3). It was found that majority of new
derivatives exhibited similar potency to inhibit PGE2 production
However, compound 18 (Table 1, entry 10), the derivative
bearing 4-(benzyloxy)phenyl moiety in the C4 position of
pyrimidine, excelled in the first series of compounds as the most
potent inhibitor of PGE2 production. Compound 18 almost
completely (less than 1% of remaining production of PGE2)
inhibited PGE2 production with no apparent toxicity. This
promising result encouraged us to design and synthesize
another series of polysubstituted pyrimidines derived from
compound 18 and bearing various benzyloxyphenyl-like moieties
in the C4 position of the pyrimidine ring.
In vitro evaluation of the second series of compounds (29–
39, Table 2, entries 3–13) revealed compound 32 (Table 2, entry
6), the difluorinated derivative of compound 18, as the most
potent inhibitor of PGE2 production from the entire SAR study.
Compound 32 exhibited a remarkable inhibitory activity with only
about 0.04% of remaining production of PGE2 in vitro.
compared to parent compound
1
(with 12% remaining
production of PGE2). Moreover, the compounds did not affect
the viability of the mouse peritoneal cells, with the exception of
compounds 21–23 (Table 1, entries 13–15) which decreased the
viability of the cells significantly.
4
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Table 2. Structures of prepared compounds 29–39 (the 2nd series) and their effect on in vitro production of PGE2 and viability of mouse peritoneal cells.
inhibition of PGE2
Ar
yield
[%]
entry
compd
viability [%][b]
Scheme 1
remaining production
(%)[a]
IC50 (µM)
control
untreat
1
2
NA
NA
NA
NA
2.78±1.02[c]
100.00±3.15
NA
100.00±2.83[c]
control LPS
NA
NA
1.64[c]
/1.38-1.94/
3
4
29
78
71
4.01±1.53*
0.73±0.53*
104.24±0.35
101.25±0.84
0.131
/0.073-0.235/
30
5
6
31
32
85
70
51.13±0.83*
0.04±0.20*
ND
103.20±0.52
103.66±0.44
0.012
/0.010-0.017/
7.03
/4.27-11.56/
7
33
98
11.74±2.27*
101.58±0.74
8
34
99
20.44±1.30*
ND
103.57±0.70
1.65
/1.17-2.33/
9
35
36
97
94
7.12±1.95*
5.54±0.41*
103.57±0.32
102.82±0.46
1.31
/0.97-1.77/
10
11
37
36
26.29±5.33*
ND
104.82±0.44
5
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0.45
/0.24-0.85/
12
13
38
39
89
40
2.95±1.86*
101.83±0.21
97.00±0.60
9.84
/7.18-13.48/
12.86±1.04*
[a] Effects recorded for 50 µM concentration of compounds and expressed in % of control LPS response; [b] Cell viability expressed in % of control untreated
cells; [c] ± S.E.M. Results represent means of three to four experiments. Statistical significance: * P < 0.001, not significant in other values. NA: not applicable.
ND: not determined.
Changes in production of PGE2 may be associated with changes
in production of another important mediator of inflammation,
nitric oxide (NO), although the crosstalk between them is
controversial showing both positive and negative influence.[89,90]
Our previously reported data suggested that some pyrimidine
derivatives might act as dual inhibitors of PGE2 and NO
production.[80,91] Thus, the ability of studied pyrimidines to inhibit
in vitro production of NO was also evaluated (data not shown).
Mostly insignificant NO suppressive potential was observed in
the present set of pyrimidine analogues (including strong PGE2
inhibitors 18 and 32) compared to the previous series of
compounds,[80] with the exception of compounds 19, 22, and 31,
which mildly inhibited NO production. Thus, we have
concentrated on the mode of a predominant effect of pyrimidines,
i.e. inhibition of PGE2 production, although some synergistic
anti-inflammatory effects in vivo can be expected and these will
be addressed in our ongoing research.
Several important conclusions can be deducted from the
overall SAR study: a) an introduction of 4-(benzyloxy)phenyl
moiety at the C4 position of pyrimidine ring was beneficial for the
increased inhibition of PGE2 production (compound 18, Table 1,
entry 10); b) further substitution of the benzyloxy moiety in the
para position of 18 was well-tolerated (compounds 29 and 30,
Table 2, entries 3 and 4); c) an introduction of the methyl group
into ortho position of the C4 phenyl ring of 18 resulted in a
substantial decrease of biological potency (compound 31, Table
2, entry 5); d) an introduction of fluorine atoms into the phenyl
ring of the 4-(benzyloxy)phenyl moiety in compound 18 resulted
in almost one-order of magnitude more potent inhibitor 32 (Table
2, entry 6); e) moving the benzyloxy moiety from para to meta
position of the C4 phenyl ring led to a decreased inhibition of
PGE2 production (compounds 33 and 34, Table 2, entries 7 and
8); f) a replacement of an oxygen atom in compound 18 for
sulfur resulted in a decrease of biological potency (compound 39,
Table 2, entry 13).
Finally, for the set of selected compounds, namely
compounds 1, 15, 17, 18, 24 and 28 (Table 1) and 29, 30, 32, 33,
35, 36, 38 and 39 (Table 2), the IC50 values were determined.
The data showed that most potent compounds 18 (IC50 = 0.031
µM) and 32 (IC50 = 0.012 µM) were approx. 150-fold and 400-
fold more potent inhibitors of PGE2 production, respectively,
compared to the parent compound 1 (IC50 = 4.83 µM).
Evaluation of the mechanism of action. The mechanism of
the PGE2-inhibitory effects of pyrimidines was studied using the
most potent compound 32 at first. Applied at the concentration of
20 µM, 32 did not influence the activity of COX-1, whereas it
significantly suppressed (by 47%, P < 0.001) the activity of COX-
2 (Figure 2A). Interestingly, other compounds from this study
(including 1, 18, and 28) did not exhibit any noticeable activity
against COX-1/2 enzymes (data not shown). On the other hand,
compound 32 (at 20 µM) virtually completely inhibited (by 98%)
the activity of human mPGES-1 (Figure 2A). It was inhibited in a
dose-dependent manner (Figure 2B), the IC50 estimate being
0.066 µM, with 0.047– 0.082 µM 95% limits of confidence. The
data indicate that 32 is a dual inhibitor of COX-2 and mPGES-1,
although its interaction with mPGES-1 can be considered as the
major mechanism underlying the inhibition of PGE2 production.
A)
B)
Control
Compound 32
Control
Compound 32
ns
Reference standard
Reference standard
100
75
50
25
0
100
75
50
25
0
**
***
***
***
***
Controls
COX-1
COX-2
PGES1
0.001
0.01
0.1
1
10
Concentration (M)
Figure 2. A) Interaction of compound 32 (20 µM) with COX-1/2 and human mPGES-1. The effects are compared to those of reference standards, i.e.
indomethacin (5 µM) for COX enzymes and CAY10678 (2 µM) for mPGES-1. The bars are means ± S.E.M. obtained from two COX and six mPGES-1
experiments, each run in duplicate. Statistical significance against the control groups: ** P < 0.01, *** P < 0.001. B) Inhibition of mPGES-1 activity is dependent on
the concentration of 32. The curve represents amalgamated effects of two experiments, each done in duplicate. The points are means ± S.E.M.
6
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a promising candidate for further development as a systemic
anti-inflammatory agent.
Table 3. Direct inhibition of mouse and human mPGES-1 with selected
compounds.
mPGES-1 inhibition[a]
IC50 (µM) (95% limits of confidence)
125
100
75
Entry
Comp
1
mouse
human
1
2
3
4
5
12.780 (9.600 - 54.120)
0.060 (0.045 - 0.307)
6.280 (1.760 - 15.010)
0.700 (0.360 - 2.130)
2.730 (1.760 - 7.400)
5.065 (4.071 - 6.303)
0.117 (0.111 - 0.259)
2.423 (1.341 - 4.378)
0.248 (0.160 - 0.385)
0.066 (0.047 - 0.082)
18
28
30
50
32
reduced
by 36.3%
reduced
by 20.7%
reduced
by 45.5%
25
0
[a] The enzyme activity reflects changes in transformation of PGH2 to of
PGE2. Effects of pyrimidine derivatives were recorded for recombinant
enzymes as described in Experimental Procedures. The results represent two
independent experiments.
P<0.0001
P=0.0014
P<0.0001
18
30
32
Carrageenan
alone
Carrageenan
+ solvent
Carrageenan + compounds (25 mg/kg p.o.)
In order to verify that mPGES-1 inhibition is the key mechanism
of action common to the whole series of pyrimidine analogues
studied (since only 32 partially inhibits also COX-2), we decided
to evaluate activity in the mPGES-1 assay with other
compounds, namely 1, 18, 28, and 30. Interestingly, previously
described inhibitors of human mPGES-1 were not, in general,
potent inhibitors of the mouse or rat enzyme, and only recently,
dual inhibitors, active against both human and mouse enzymes,
have been reported.[92] In order to correlate our data obtained on
mouse peritoneal cells (Table 1 and 2) with those obtained with
human mPGES-1 (Fig. 2), selected compounds 1, 18, 28, 30,
and 32 were evaluated on both mouse and human enzymes
side by side (Table 3).
All tested compounds are potent inhibitors of both mouse
and human mPGES-1 (Table 3), though the human enzymes
seems to be more susceptible to the inhibition with
polysubstituted pyrimidines. Coumpound 18 (IC50 = 60 nM) is the
most potent inhibitor of the mouse mPGES-1, while compound
32 (IC50 = 66 nM) is the most potent inhibitor of the human
enzyme. The obtained data support the notion that mPGES-1
inhibition represents the main mechanism of action common to
anti-inflammatory pyrimidines studied in this work.
In vivo evaluation of selected compounds in the model of
acute inflammation. For the most potent compounds, namely 18,
30, and 32, we carried out the carrageenan-induced rat paw
edema experiments to verify whether the compounds are active
also in vivo when administered orally (gastric intubation). The rat
model is preferred to mouse one for its higher accuracy and
reproducibility. Since the aqueous solubility of most of the
studied polysubstituted pyrimidines is low, the compounds were
solubilized using a mixture of DMSO (1.5%) in TWEEN® 80
before dilution in the physiological saline solution. The results
are depicted in Figure 3. It was shown that solvent alone did not
have any significant effect on the development of edema
induced by carrageenan while solubilized compounds 18, 30,
and 32 reduced the carrageenan-enhanced paw volumes by
36.3%, 20.7%, and 45.5%, respectively. It can be concluded,
that the compounds are bioavailable after oral administration.
The most potent compound 32 substantially suppresses the
development of rat paw edema validating its potential to become
Figure 3. Effect of compounds 18, 30, and 32 on the development of the acute
carrageenan-induced rat paw edema. P, statistical significance against the
positive control group (carrageenan alone). Solvent: DMSO (1.5
wt%)/TWEEN® 80 mixture (100 mg) in sterile 1% saline solution (1 mL).
Compound samples: 5 mg of compound in 1 mL of solvent.
Computational Studies. The detailed structure-activity
relationship (SAR) of anti-inflammatory pyrimidines was
discussed above. In order to elucidate and better understand the
binding mode of mPGES-1 inhibitors, we decided to perform
molecular docking studies with some of our compounds. High
resolution X-ray crystal structure (PDB: 4BPM) of human
mPGES-1 with potent inhibitor is available,[93,94] while mouse
mPGES-1 structure is so far unavailable. Human mPGES-1 is,
however, highly homologous to the mouse enyzme, with
sequence identity being 79% and the sequence similarity being
84%. Thus, docking experiments of compounds 1 and 32 were
carried out using Glide based on the X-ray crystal structure of
human mPGES-1 (PDB: 4BPM) and on homology model of
mouse mPGES-1 developed by using the human mPGES-1
structure as template (Figure 4). The molecular docking
revealed that both compounds 1 and 32 can favorably bind in
conserved region of the active site of human and mouse
mPGES-1 enzymes. The conserved region around S127 has
mainly hydrophobic pocket surrounded by Y28, I32 (V32 in
mouse), G35, L39, Y130, T131 (V131 in mouse), Q134, L135
and A138 (F138 in mouse) for human (mouse) mPGES-1.[95] As
shown in Figure 4, the phenyl ring in C6 position of pyrimidine
ring stays at bottom of the substrate-binding pocket of mPGES-1
enzymes. 4-Methoxyphenyl (compound 1) and 4-benzyloxy(3,2-
difluoro)phenyl (compound 32) moiety in C4 position of
pyrimidine are involved in a π–π stacking interaction with the
side chain of Tyr130 of human subunit A and the side chain of
Tyr130 of mouse subunit C, respectively. The computational
studies support strong binding of studied derivatives to both
mouse and human mPGES-1 enzymes.
7
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10.1002/cmdc.202000258
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Figure 4. Docked binding pose of A) compound 1 and B) compound 32 in human mPGES-1. Docked binding pose of C) compound 1 and D) compound 32 in
modeled mouse mPGES-1. The trimer protein structure of human and modeled mouse mPGES-1 is depicted in ribbon diagram. Human and modeled mouse
mPGES-1 interacting residues are represented green stick form and ligand structure in yellow stick form (colored by atom type: C: green or yellow, N: blue, O: red,
F: cyan, H: white). The cyan dashed lines indicate π–π interaction. Figure was prepared with Maestro, Schrodinger, LLC.
development of carrageenan-induced rat paw edema, confirming in
vivo potency in the treatment of acute inflammation. An extensive
Conclusion
study of mechanism of action revealed that the studied compounds
(1, 18, 28, 30 and 32) are potent inhibitors of mPGES-1, both
mouse and human. Therefore, these inhibitors can be used as tool
compounds to study the effects of mPGES-1 inhibition in mice and
rats, with potential applications in humans. Moreover, it has been
suggested, that the specific inhibition of mPGES-1 is extremely
interesting for drug development with respect to the side-effects
observed with COX-1/2 inhibitors. The pyrimidines studied did not
inhibit COX-1/2 enzymes, only derivative 32 weakly inhibited
COX-2. Although the overall mode of anti-inflammatory action of
the studied compounds seems to be relatively complex, mPGES-1
evidently is their principal anti-inflammatory target. These exciting
data strongly encourage future evaluation of the most potent
candidates in various chronic inflammation models (e.g. ulcerative
colitis and rheumatoid arthritis) as well as subsequent lead
optimization in order to develop novel potent anti-inflammatory
and anti-cancer therapeutic agents.
Compound 1 was identified previously as a potent inhibitor of
prostaglandin E2 (PGE2) production with confirmed anti-
inflammatory properties and is being evaluated as a preclinical
candidate for the treatment of ulcerative colitis and rheumatoid
arthritis. The goal of the current extensive SAR study was further
structural
optimization
of
polysubstituted
pyrimidines,
identification of more potent anti-inflammatory agents, as well as
elucidation of their mechanism of action. Synthesis of 28 new
analogues derived from compound 1 bearing substituted and/or
modified phenyl moiety in the C4 position of the pyrimidine ring,
led to the discovery of several superior inhibitors of PGE2
production. Firstly, compound 18 (IC50 = 31 nM) with the 4-(4-
benzyloxy)phenyl arm in C4 position of pyrimidine was found to
be a potent inhibitor of PGE2 production. Subsequently, compound
32, the difluorinated derivative of compound 18, was identified as
the most potent inhibitor from the whole SAR series. Compound
32 (IC50 = 12 nM) was 400-fold more potent inhibitor of PGE2
production compared to parent compound 1 (IC50 = 4.83 µM).
Moreover, after oral administration, compounds 18 and 32
significantly (by 36% and 46%, respectively) suppressed the
8
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10.1002/cmdc.202000258
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This work was supported by the subvention for development of
research organization (Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, RVO: 61388963),
by the Technology Agency of the Czech Republic (TE01020028
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Abbreviations
cyclooxygenase;
dehydrogenase;
used.
GI,
LPS,
AA,
arachidonic
acid;
LDH,
NBS,
COX,
lactate
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gastrointestinal;
lipopolysaccharide;
bromosuccinimide; NO, nitric oxid, NSAIDs, non-steroidal anti-
inflammatory drugs; PGE2, prostaglandin E2; mPGES-1,
microsomal prostaglandin E2 synthase; t-NSAIDs, traditional
non-steroidal anti-inflammatory drugs.
Keywords: pyrimidines • Suzuki Miyaura reaction • mPGES-1 •
prostaglandin E2 • anti-inflammatory
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10.1002/cmdc.202000258
ChemMedChem
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Anti-inflammatory drugs belong to the mostly used therapeutic agents, but both traditional NSAIDs and coxibs suffer from serious
side effects. Thus, development of anti-inflammatory drugs with novel mechanisms of actions is desirable and inhibitors of mPGES-1
may be good approach. We designed and synthesized potent mPGES-1 inhibitors based on polysubstituted pyrimidines, which
showed strong anti-inflammatory effects in vivo.
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