Influence of the C-5 substitution in polysubstituted pyrimidines on inhibition of prostaglandin E2 production

Article abstract of DOI:10.1016/j.ejmech.2018.07.010

As a part of a broader structure-activity relationship study of substituted 2-aminopyrimidines, the influence of the C-5 substitution on inhibition of prostaglandin E₂ (PGE₂) production was studied. Thirty compounds were prepared sta

Full text of DOI:10.1016/j.ejmech.2018.07.010

Accepted Manuscript
Influence of the C-5 substitution in polysubstituted pyrimidines on inhibition of
prostaglandin E production
2
Viktor Kolman, Filip Kalčic, Petr Jansa, Zdeněk Zídek, Zlatko Janeba
PII:
S0223-5234(18)30566-X
10.1016/j.ejmech.2018.07.010
EJMECH 10545
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 May 2018
Revised Date: 4 July 2018
Accepted Date: 5 July 2018
Please cite this article as: V. Kolman, F. Kalčic, P. Jansa, Zdeně. Zídek, Z. Janeba, Influence of the
C-5 substitution in polysubstituted pyrimidines on inhibition of prostaglandin E production, European
2
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.010.
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ACCEPTED MANUSCRIPT
Influence of the C-5 substitution in polysubstituted pyrimidines on inhibition of
prostaglandin E2 production
Viktor Kolmana, Filip Kalčica, Petr Jansa,a,‡ Zdeněk Zídekb,*, Zlatko Janebaa,*
a
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences,
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
b Institute of Experimental Medicine of the Czech Academy of Sciences, Vídeňská 1083, 142 20,
Prague 4, Czech Republic
Abstract: As a part of a broader structure-activity relationship study of substituted 2-
aminopyrimidines the influence of the C-5 substitution on inhibition of prostaglandin E2 (PGE2)
production was studied. Thirty compounds were prepared starting from the corresponding 2-
amino-4,6-dichloropyrimidines using Suzuki cross-coupling. It was shown previously that 2-
amino-4,6-dichloropyrimidines with smaller C-5 substituent (hydrogen and methyl) were devoid
of significant activity, while 5-butyl derivatives exhibited prominent potency. In this study, on
the other hand, both monoaryl- and bisarylpyrimidines were potent inhibitors of PGE2
production regardless the length of the C-5 substituent (hydrogen, methyl, n-butyl). Moreover,
the shorter the C-5 substituent the higher potency to inhibit PGE2 production was observed. 2-
Amino-4,6-diphenylpyrimidine was the best inhibitor of PGE2 production with IC50 = 3 nM and
no cytotoxicity. The most potent inhibitors deserve further preclinical evaluation as potential
anti-inflammatory agents.
Keywords: pyrimidines; Suzuki-Miyaura reaction; prostaglandin E2; inhibitor
1. Introduction
Prostaglandins (PGs) are lipid mediators and end products of fatty acid metabolism, that are
produced from the key precursor, arachidonic acid (AA), via the cyclooxygenase (COX)
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pathway: either by constitutively expressed COX-1 or by, in response to cell-specific stimuli,
highly inducible COX-2.1-4
Prostaglandin E2 (PGE2) is a naturally occurring eicosanoid derived from AA, produced by an
action of COX enzymes and prostaglandin E synthases (PGES).5,6 PGE2, as a lipid mediator, is
known to regulate various important proceses including inflammation, pain and pyrexia.7
Nonsteroidal anti-inflammatory drugs (NSAIDs)1,8-10 are agents that, by inhibiting the activity of
COX enzymes (and thus inhibiting PGs production), efficiently reduce pain and fever, prevent
blood clots, and at higher doses, decrease inflammation. The traditional (non-selective) NSAIDs
belong to the most widely used drugs around the world, but they carry the risk of serious
gastrointestinal (GI), as well as cardiovascular (CV) and renal adverse effects.8-10 In search for
safer medication, COX-2 selective inhibitors (coxibs)11,12 were developed with reduced upper GI
toxicity, nevertheless still exhibiting CV adverse events.10,13 Thus, there is a persisting need for
development of novel PGE2 production inhibitors lacking the negative side effects of currently
used therapeutics.
A pyrimidine moiety represents one of the most important medicinal chemistry scaffolds.14,15
Numerous compounds based on the pyrimidine scaffold are known to exhibit anti-inflammatory
activity,16-19 as well as properties immunomodulating,20 anti-leishmanial,21 antibacterial,22-24
antifungal,23 analgesic,25 anticonvulsant,26 and anticancer.27-31
Within previous extensive structure-activity relationship studies, we discovered a series of
polysubstituted pyrimidines as potent inhibitors of immune-activated nitric oxide (NO)
production,32-36 as well as inhibitors of production of prostaglandin E2 (PGE2).34-36 It has been
demonstrated, that in the case of 2-amino-4,6-dichloropyrimidines, at least a 3-carbon-long
aliphatic group in the C-5 position of the pyrimidine moiety was required to inhibit PGE2
production substantially, while derivatives bearing either hydrogen or methyl in the C-5 position
were not PGE2 production inhibitors.34 Based on these results novel series of compounds was
prepared, 2-amino-4,6-di(hetero)arylpyrimidines having a n-butyl group in the C-5 position, as
an optimal substituent.36 Several compounds in this series were found to be low micromolar dual
inhibitors of NO and PGE2 production.36 Since non-toxic small-molecule inhibitors of PGE2
production may have great therapeutic potential in treatment of serious inflammatory diseases,
we decided to verify the influence of the C-5 substitution in the series of selected 2-amino-4-
aryl-6-chloropyrimidines and 2-amino-4,6-diarylpyrimidines.
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In this work, we prepared novel types of symmetrical and unsymmetrical 2-amino-4,6-
diarylpyrimidines (where aryl is either phenyl, p-tolyl or 4-methoxylphenyl) bearing in the C-5
position either hydrogen, methyl or n-butyl group. The prepared compounds were subsequently
tested for their ability to inhibit PGE2 production in mouse peritoneal cells.
2. Results and discussion
2.1. Synthesis
Starting 2-amino-4,6-dichloropyrimidines 1a-1c (Scheme 1) were prepared according to the
previously published procedure.32 Compounds 1a-1c were subsequently treated with
commercially available arylboronic acids (phenyl-, p-tolyl-, and 4-methoxyphenylboronic acid)
under Suzuki-Miyaura cross-coupling conditions.37 Depending on the reaction conditions,36 we
were able to prepare both monoarylated products 2-4 (Method A: Pd(PPh3)4, Na2CO3,
toluene/ethanol (3:1), 80 °C, 12 h) or symmetrical diarylated products 5-7 (Method B: Pd(PPh3)4,
Cs2CO3, dioxane/water (4:1), 100 oC, 12 h).
Treatment of 2-amino-4-aryl-6-chloropyrimidines 2-4 with another arylboronic acid using the
latter conditions (Method B) afforded unsymmetrical 4,6-diarylpyrimidine analogues 8-10
(Scheme 1). Structures of all prepared compounds are summarized in Table 1.
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Scheme 1. General synthesis of target polysubstituted pyrimidines. Ar1, Ar2 – phenyl, p-tolyl, 4-
methoxyphenyl; R – hydrogen, methyl, n-butyl. Reaction conditions: a) arylboronic acid,
Pd(PPh3)4, Na2CO3, toluene/ethanol (3:1), 80 °C, 12 h; b) arylboronic acid, Pd(PPh3)4, Cs2CO3,
dioxane/water (4:1), 100 °C, 12 h.
Table 1. List of the prepared compounds. Me, methyl; Bu, n-butyl; Ph, phenyl; Tol, p-tolyl;
MeOPh, 4-methoxyphenyl.
Compound R
Ar1
-
Ar2
H
-
-
-
-
-
-
-
-
-
1a
1b
1c
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
Me -
Bu
H
-
Ph
Me Ph
Bu Ph
H
Tol
Me Tol
Bu Tol
H
MeOPh -
Me MeOPh -
Bu MeOPh -
H
Ph
-
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Me Ph
Bu Ph
-
-
-
-
-
5b
5c
H
Tol
6a
6b
6c
Me Tol
Bu Tol
H
MeOPh -
7a
7b
7c
Me MeOPh -
Bu MeOPh -
H
Tol
Ph
Ph
Ph
8a
8b
8c
Me Tol
Bu Tol
H
MeOPh Ph
9a
9b
9c
Me MeOPh Ph
Bu MeOPh Ph
10a
10b
10c
H
MeOPh Tol
Me MeOPh Tol
Bu MeOPh Tol
2.2. Biological evaluation
All prepared polysubstituted pyrimidines were evaluated for their ability to inhibit production
of PGE2 in vitro, using C57BL6 mouse peritoneal cells. High output PGE2 production was
activated by lipopolysaccharide (LPS) and assayed at the interval of 5 h of culture. Compounds
(as 50 µM solutions) were applied concomitantly with LPS. The effects of pyrimidines were
expressed as a percent change related to the response of LPS-stimulated control cells. With the
exception of 2-amino-4,6-dichloropyrimidine analogues bearing hydrogen or methyl at C-5
position (1a and 1b, respectively), all tested compounds were discovered to be potent inhibitors
of PGE2 production (Fig. 1A). The extent of the inhibition of PGE2 production clearly depended
on the character of the C-5 substituent. Contrary to the previously studied 2-amino-4,6-
dichloropyrimidine analogues,32 the current series of 2-amino-4-arylpyrimidines and 2-amino-
4,6-diarylpyrimidines no longer required prolonged aliphatic chain in the C-5 position to exert
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their biological activity. The most potent pyrimidines were those bearing hydrogen at C-5
position (i.e. compounds 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, and 10a), while prolongation of the
substituent in this position led to attenuation of the capability to reduce PGE2 production (Fig.
1B). Namely, analogues bearing methyl in the C-5 position are statistically less efficient to
inhibit PGE2 production compared to those bearing hydrogen at that position and the compounds
bearing n-butyl group in the C-5 position are even less effective in comparison to the 5-methyl
derivatives (Fig. 1B).
For the selected potent compounds, the IC50 values were also determined in comparison with
two commonly used NSAIDs as control compounds, indomethacin38-40 and aspirin40,41 (Table 2).
The compounds were found to be potent at low micromolar to submicromolar levels. The most
effective inhibitors of PGE2 production were compounds 5a, 6a, 8a, and 9a with IC50s in the
range of 3-33 nM (Table 2). Again, all of them were the C-5 unsubstituted analogues, i.e.
derivatives with hydrogen at the C-5 position.
Most importantly, the high inhibitory activity of the studied compounds cannot be explained as
a result of possible cytotoxic effects, as no changes in viability of cells could be detected in the
presence of the compounds (Table 2). Cell death was analyzed in the same cells using the lactate
dehydrogenase (LDH) assay which is based on the determination of LDH activity released from
the cytosol of damaged cells into the supernatant.
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A)
100
Controls
c (C-5 = Bu)
b (C-5 = Me)
a (C-5 = H)
75
50
25
0
Statistical significance against LPS alone:
1a, 1b: not significant (P > 0.05)
all others: highly significant (P < 0.001)
1
2
3
4
5
6
7
8
9
10
Group
B)
40
30
20
10
0
P = 0.001
P < 0.01
P < 0.01
b (C-5 = methyl) c (C-5 = butyl)
a (C-5 = H)
Figure 1. Effects of individual pyrimidine derivatives (50 µM solutions) (A) and medians of
grouped compounds according to the C-5 substituents (B) on in vitro production of PGE2 in
mouse peritoneal cells. Bars are means ± S.E.M. that were obtained by averaging results of two
to four experiments for each compound.
Previously reported data36 suggested that post-translation mechanism(s) are plausible explanation
for the PGE2-inhibitory action of the pyrimidine derivatives. Their interaction with activity of
enzymes involved in biosynthesis of PGE2 is under current investigation.
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Table 2. Concentration of selected pyrimidines inhibiting PGE2 production in C57BL6 mouse
peritoneal cells by 50% (IC50), and effects on cell viability of the same cell type evaluated as a
percent change against untreated control cells.
Compound
IC50 (µM)
Viability (%)
99.82±1.33b
102.6±0.4
101.4±1.7
103.2±0.7
96.6±1.8
97.4±0.9
102.8±1.2
101.7±0.7
99.1±0.6
103.1±0.7
102.2±0.6
99.9±1.7
102.0±0.9
n.d.c
1c
3a
3c
4a
4c
5a
6a
7a
7c
8a
8c
9a
9c
5.34 (4.65-6.12)a
2.32 (1.46-3.42)
1.86 (1.27-2.74)
2.79 (1.98-3.92)
2.16 (1.87-2.49)
0.003 (0.002-0.004)
0.033 (0.024-0.046)
0.119 (0.068-0.202)
3.71 (2.81-4.90)
0.009 (0.007-0.01)
2.46 (1.91-3.16)
0.009 (0.005-0.016)
3.40 (2.55-4.55)
indomethacin 0.005 (0.003-0.007)
aspirin
DMSO
4.08 (3.09-5.39)
n.d.
n.d.
98.9±1.6
a95% limits of confidence; b ± S.E.M. Results represent means of two identical experiments. cn.d.
= not determined.
3. Conclusion
In summary, we have identified novel potent and non-toxic inhibitors of prostaglandin E2
(PGE2) production using C57BL6 mouse peritoneal cells. The selected monoaryl- and
diarylsubstituted 2-aminopyrimidines exhibited single-digit micromolar to submicromolar
potency to inhibit PGE2 production. By contrast to the previously studied 2-amino-4,6-
dichloropyrimidines, herein reported arylpyrimidines were most potent inhibitors when bearing
hydrogen atom in the C-5 position. At least twelve of the prepared compounds were more potent
than still popular aspirin. 2-Amino-4,6-diphenylpyrimidine was the most potent inhibitor in the
whole series, with IC50 = 3 nM and no observed cytotoxicity. This compound was shown to be
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equipotent to indomethacin, a nonsteroidal anti-inflammatory drug commonly used as a
prescription medication to treat inflammation symptoms. Indomethacin, as an inhibitor of both
COX-1 and COX-2, nevertheless, exhibits many serious adverse effects and superior drugs are
needed. Although the exact mode of action of the reported compounds remains to be elucidated,
the preliminary findings encourage further preclinical studies of the selected candidates as
potential anti-inflammatory agents.
4. Experimental section
4.1. Chemistry
Unless otherwise stated, solvents were evaporated at 40 °C/2 kPa and the compounds were dried
1
over P2O5 at 2 kPa. H NMR and 13C NMR were recorded using Bruker Avance 400 MHz
spectrometer operating at 9.39 T in DMSO-d6 solution. The 1H and 13C spectra were acquired at
400.00 MHz and 100 MHz, respectively. Chemical shifts (δ) are reported in ppm and interaction
constants (J) in Hz. The spectra were referenced to tetramethylsilane (TMS) or to the residual
solvent signal. High-resolution mass spectra of the products were obtained using GCT Premier
(Waters, USA) spectrometer for electron ionization (EI) and LTQ Orbitrap XL (Thermo Fisher
Scientific) spectrometer for electrospray ionization (ESI). Starting 2-amino-4,6-
dichloropyrimidines were prepared according to the published procedure.32 Purity of the
prepared compounds was ≥95%; the purity was determined by the combination of HPLC
(H2O/CH3CN,linear gradient), NMR, and HR-MS.
4.1.1. Method A. General procedure for synthesis of 2-amino-4-aryl-6-chloropyrimidines 2a-c,
3a-c, 4a-c. Starting 2-amino-4,6-dichloropyrimidine derivative (1.36 mmol), aryl boronic acid
(1.51 mmol), tetrakis(triphenylphosphine) palladium (58 mg, 0.05 mmol) and sodium carbonate
(213 mg, 2.01 mmol) were suspended in a toluene/ethanol mixture (3:1, 50 mL). The reaction
o
was heated to 80 C for 12 h. After cooling to room temperature, the solid was filtered off and
the solvents were removed in vacuo. The residue was dissolved in ethyl acetate (100 mL) and
adsorbed on silica gel. After removal of organic solvent in vacuo the product was purified on
silica gel column chromatography using a hexane/ethyl acetate mixture (4:1) as the eluent and
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subsequently on C-18 reverse chromatography column using water/methanol eluent (0-100%
gradient).
1
4.1.1.1. 4-Chloro-6-phenylpyrimidin-2-amine (2a). Yield 76%, white solid. H NMR δ (400
H
MHz, DMSO-d6) 8.13 – 8.08 (m, 2H), 7.57 – 7.48 (m, 3H), 7.26 (s, 1H), 7.19 (s, 2H). 13C NMR
δ
(100 MHz, DMSO-d6) δ 166.45, 163.99, 161.60, 136.33, 131.57, 129.22, 127.52, 105.15.
C
HRMS (EI) m/z [M]+ calcd for C10H8ClN3: 205.0407, found: 205.0406.
4.1.1.2. 4-Chloro-5-methyl-6-phenylpyrimidin-2-amine (2b). Yield 50%, white solid. 1H NMR δ
13
(400 MHz, DMSO-d6) 7.56 – 7.42 (5 H, m), 6.89 (2 H, s), 2.11 (3 H, s). C NMR δ (100
H
C
MHz, DMSO-d6) 168.27, 161.88, 161.51, 138.74, 129.49, 129.04, 128.52, 113.57, 15.70. HRMS
(EI) m/z [M]+ calcd for C11H10ClN3: 219.0563, found: 219.0566.
4.1.1.3. 5-Butyl-4-chloro-6-phenylpyrimidin-2-amine (2c). Yield 50%, white solid. 1H NMR δ H
(400 MHz, DMSO-d6) 7.53 – 7.45 (3 H, m), 7.44 – 7.40 (2 H, m), 6.93 (2 H, s), 2.49 – 2.42 (2 H,
m), 1.42 – 1.28 (2 H, m), 1.14 (2 H, q, J = 7.2 Hz), 0.72 (3 H, t, J = 7.3 Hz). 13C NMR δ C (100
MHz, DMSO-d6) 169.08, 161.56, 161.41, 139.00, 129.18, 128.54, 128.42, 118.41, 31.64, 27.96,
22.28, 13.82. HRMS (EI) m/z [M]+ calcd for C14H16ClN3: 261.1033, found: 216.1031.
1
4.1.1.4. 4-Chloro-6-(p-tolyl)pyrimidin-2-amine (3a). Yield 41%, white solid. H NMR δ (400
H
MHz, DMSO-d6) 8.01 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 7.22 (s, 1H), 7.14 (s, 2H),
2.37 (s, 3H). 13C NMR NMR δ C (100 MHz, DMSO-d6) 166.36, 163.96, 161.50, 141.54, 133.56,
129.81, 127.47, 104.77, 21.44. HRMS (EI) m/z [M]+ calcd for C11H10ClN3: 219.0563, found:
219.0565.
4.1.1.5. 4-Chloro-5-methyl-6-(p-tolyl)pyrimidin-2-amine (3b). Yield 32%, white solid. 1H NMR
δ H (400 MHz, DMSO-d6) 7.43 (2 H, d, J = 8.2 Hz), 7.38 – 7.20 (2 H, m), 6.87 (2 H, s), 2.37 (3
H, s), 2.12 (3 H, s). 13C NMR δ C (100 MHz, DMSO-d6) 168.19, 161.50, 161.22, 161.01, 139.07,
135.90, 129.10, 113.48, 21.38, 15.80. HRMS (ESI) m/z [M+H]+ calcd for C12H13ClN3:
234.0793, found: 234.0793.
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4.1.1.6. 5-Butyl-4-chloro-6-(p-tolyl)pyrimidin-2-amine (3c). Yield 7%, white solid. 1H NMR δ H
(400 MHz, DMSO-d6) 7.33 (2 H, d, J = 8.2 Hz), 7.28 (2 H, d, J = 7.9 Hz), 6.91 (2 H, s), 2.49 –
2.43 (2 H, m), 2.37 (3 H, s), 1.46 – 1.29 (2 H, m), 1.23 – 1.08 (2 H, m), 0.75 (3 H, t, J = 7.3 Hz).
13C NMR δ (100 MHz, DMSO-d6) 169.00, 161.57, 161.39, 138.65, 136.18, 129.06, 128.46,
C
118.38, 31.69, 28.06, 22.33, 21.35, 13.89. HRMS (ESI) m/z [M+H]+ calcd for C15H19ClN3:
276.1262, found: 276.1262.
4.1.1.7. 4-Chloro-6-(4-methoxyphenyl)pyrimidin-2-amine (4a). Yield 34%, white solid. 1H NMR
δ H (400 MHz, DMSO-d6) 8.11 – 8.06 (m, 2H), 7.20 (s, 1H), 7.09 (s, 2H, NH2), 7.08 – 7.02 (m,
13
2H), 3.84 (s, 3H). C NMR δ C (100 MHz, DMSO-d6) 165.98, 163.89, 162.17, 161.35, 129.21,
128.58, 114.55, 104.25, 55.84. HRMS (EI) m/z [M]+ calcd for C11H10ClN3O: 235.0512, found:
235.0514.
4.1.1.8. 4-Chloro-6-(4-methoxyphenyl)-5-methylpyrimidin-2-amine (4b). Yield 44%, white solid.
1H NMR δ H (400 MHz, DMSO-d6) 7.51 (2 H, d, J = 8.8), 7.03 (2 H, d, J = 8.8), 6.84 (2 H, s),
3.82 (3 H, s), 2.15 (3 H, s). 13C NMR δ C (100 MHz, DMSO-d6) 167.76, 161.86, 161.46, 160.35,
130.95, 130.84, 113.86, 113.30, 55.70, 15.94. HRMS (EI) m/z [M]+ calcd for
C12H12ClN3O: 249.0669, found: 249.0673.
4.1.1.9. 5-Butyl-4-chloro-6-(4-methoxyphenyl)pyrimidin-2-amine (4c). Yield 52%, white solid.
1H NMR δ H (400 MHz, DMSO-d6) 7.51 (2 H, d, J = 8.9), 7.12 (2 H, d, J = 8.9), 6.99 (2 H, s),
3.91 (3 H, s), 2.66 – 2.53 (2 H, m), 1.56 – 1.39 (2 H, m), 1.34 – 1.20 (2 H, m), 0.86 (3 H, t, J
7.3). 13C NMR δ (100 MHz, DMSO-d6) 168.58, 161.60, 161.38, 160.08, 131.29, 130.12,
C
118.30, 113.89, 55.66, 31.69, 28.11, 22.33, 13.90. HRMS (EI) m/z [M]+ calcd for
C15H18ClN3O: 291.1138, found: 291.1136.
4.1.2. Method B. General procedure for synthesis of symmetrical 4,6-diarylpyrimidines 5a-c, 6a-
c, 7a-c. Starting 2-amino-4,6-dichloropyrimidine derivative (1.36 mmol), boronic acid (2.05
mmol), tetrakis(triphenylphosphine)palladium (58 mg, 0.05 mmol) and cesium carbonate
(213 mg, 2.01 mmol) were dissolved in a dioxane/water mixture (4:1, 50 mL). The reaction was
o
heated to 100 C for 12 h. After cooling to room temperature the solvents were removed in
11

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vacuo. The residue was dissolvedin ethyl acetate (100 mL) and adsorbed on silica gel. After
removal of the organic solvent in vacuo the product was purified on silica gel column
chromatography using a hexane/ethyl acetate mixture (4:1) as the eluent and subsequently on C-
18 reverse chromatography column using water/methanol eluent (0-100% gradient).
1
4.1.2.1. 4,6-Diphenylpyrimidin-2-amine (5a). Yield 81%, white solid. H NMR δ (400 MHz,
H
DMSO-d6) 8.26 – 8.19 (m, 4H), 7.71 (s, 1H), 7.53 (p, J = 3.8, 3.3 Hz, 6H), 6.76 (s, 2H). 13C
NMR δ (100 MHz, DMSO-d6) δ 165.32, 164.48, 137.81, 130.90, 129.07, 127.44, 102.30.
C
HRMS (EI) m/z [M]+ calcd for C16H13N3: 247.1109, found: 247.1108.
1
4.1.2.2. 5-Methyl-4,6-diphenylpyrimidin-2-amine (5b). Yield 44%, white solid. H NMR δ
H
(400 MHz, DMSO-d6) 7.63 – 7.54 (4 H, m), 7.52 – 7.39 (6 H, m), 6.51 (2 H, s), 2.04 (3 H, s).
13C NMR δ (100 MHz, DMSO-d6) 167.54, 161.90, 139.73, 129.20, 129.04, 128.43, 113.28,
C
16.72. HRMS (EI) m/z [M]+ calcd for C17H15N3: 261.1266, found: 261.1269.
1
4.1.2.3. 5-Butyl-4,6-diphenylpyrimidin-2-amine (5c). Yield 69%, white solid. H NMR δ H (400
MHz, DMSO-d6) 7.55 – 7.39 (10 H, m), 6.52 (2 H, s), 2.49 – 2.42 (2 H, m), 1.02 – 0.91 (2 H, m),
0.88 – 0.76 (2 H, m), 0.45 (3 H, t, J = 7.3). 13C NMR δ C (100 MHz, DMSO-d6) 168.08, 161.49,
158.68, 140.10, 128.70, 128.62, 128.45, 118.60, 32.24, 26.92, 22.02, 13.55. HRMS (EI) m/z
[M]+ calcd for C20H21N3: 303.1735, found: 303.1740.
1
4.1.2.4. 4,6-Di-p-tolylpyrimidin-2-amine (6a). Yield 37%, white solid. H NMR δ (400 MHz,
H
DMSO-d6) 8.13 (4H, d, J = 8.2 Hz), 7.65 (1 H, s), 7.33 (4H, d, J = 7.9 Hz), 6.67 (2H, s), 2.39 (s,
6H). 13C NMR δ (100 MHz, DMSO-d6) 165.06, 164.40, 140.61, 135.07, 129.65, 127.34,
C
101.57, 21.43. HRMS (EI) m/z [M]+ calcd for C18H17N3: 275.1422, found: 275.1420.
1
4.1.2.5. 5-Methyl-4,6-di-p-tolylpyrimidin-2-amine (6b). Yield 40%, white solid. H NMR δ
H
(400 MHz, DMSO-d6) 7.49 (4 H, d, J = 7.8), 7.28 (4 H, d, J = 7.8), 6.43 (2 H, s), 2.37 (6 H, s),
2.05 (3 H, s). 13C NMR δ (100 MHz, DMSO-d6) 167.39, 161.89, 138.46, 136.93, 129.26,
C
128.95, 115.46, 113.22, 21.37, 16.99. HRMS (EI) m/z [M]+ calcd for C19H19N3: 289.1579,
found: 289.1574.
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4.1.2.6. 5-Butyl-4,6-di-p-tolylpyrimidin-2-amine (6c). Yield 49%, white solid. 1H NMR δ H (400
MHz, DMSO-d6) 7.38 (4 H, d, J 8.2), 7.27 (4 H, d, J = 7.7), 6.45 (2 H, s), 2.54 – 2.46 (2 H, m),
13
2.37 (6 H, s), 1.03 – 0.89 (2 H, m), 0.89 – 0.77 (2 H, m), 0.47 (3 H, t, J = 7.3). C NMR δ
C
(100 MHz, DMSO-d6) 167.96, 161.48, 137.97, 137.38, 128.97, 128.65, 118.61, 32.22, 27.03,
21.36, 13.66. δ C (101 MHz, DMSO-d6) 167.96, 161.48, 153.31, 137.98, 137.38, 128.96, 128.65,
118.63, 32.22, 27.03, 22.01, 21.35, 13.66. HRMS (ESI) m/z [M+H]+calcd for C22H26N3:
332.2121, found: 332.2122.
4.1.2.7. 4,6-Bis(4-methoxyphenyl)pyrimidin-2-amine (7a). Yield 25%, white solid. 1H NMR δ H
(400 MHz, DMSO-d6) 8.22 – 8.17 (m, 4H), 7.60 (s, 1H), 7.09 – 7.04 (m, 4H), 6.58 (s, 2H), 3.85
(s, 6H). 13C NMR δ (100 MHz, DMSO-d6) 164.55, 164.27, 161.59, 130.23, 128.94, 114.35,
C
100.71, 55.78. HRMS (EI) m/z [M]+ calcd for C22H17N3O2: 307.1321, found: 307.1320.
1
4.1.2.8. 4,6-Bis(4-methoxyphenyl)-5-methylpyrimidin-2-amine (7b). Yield 33%, white solid. H
NMR δ H (400 MHz, DMSO-d6) 7.57 (4 H, d, J = 9.0), 7.03 (4 H, d, J = 9.0), 6.38 (2 H, s), 3.82
(6 H, s), 2.10 (3 H, s). 13C NMR δ (100 MHz, DMSO-d6) 166.94, 161.84, 160.00, 132.06,
C
130.93, 113.73, 113.00, 55.66, 17.36. HRMS (ESI) m/z [M+H]+ calcd for C19H20N3O2:
322.1550, found: 322.1551.
1
4.1.2.9. 5-Butyl-4,6-bis(4-methoxyphenyl)pyrimidin-2-amine (7c). Yield 14%, white solid. H
NMR δ H (400 MHz, DMSO-d6) 7.45 (4 H, d, J = 8.6), 7.02 (4 H, d, J = 8.8), 6.41 (2 H, s), 3.81
(6 H, s), 2.61 – 2.44 (2 H, m), 1.02 – 0.91 (2 H, m), 0.91 – 0.80 (2 H, m), 0.49 (3 H, t, J = 7.2).
13C NMR δ (100 MHz, DMSO-d6) 167.57, 161.48, 159.66, 132.60, 130.22, 118.57, 113.78,
C
55.61, 32.14, 27.14, 22.02, 13.71. HRMS (ESI) m/z [M+H]+ calcd for C22H26N3O2: 364.2020,
found: 364.2019.
4.1.3. Method C. General procedure for synthesis of unsymmetrical 4,6-diarylpyrimidines 8a-c,
9a-c, 10a-c. Starting 2-amino-4-aryl-6-chloropyrimidine derivative (1.36 mmol), boronic acid
(1.51 mmol), tetrakis(triphenylphosphine)palladium (58 mg, 0.05 mmol) and cesium carbonate
(213 mg, 2.01 mmol) were dissolved in a dioxane/water mixture (4:1, 50 mL). The reaction was
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o
heated to 100 C for 12 h. After cooling to room temperature the solvents were removed in
vacuo. The residue was dissolved in ethyl acetate (100 mL) and adsorbed on silica gel. After
removal of the organic solvent in vacuo the product was purified on silica gel column
chromatography using a hexane/ethyl acetate mixture (4:1) as the eluent and subsequently on C-
18 reverse chromatography column using water/methanol eluent (0-100% gradient).
1
4.1.3.1. 4-Phenyl-6-(p-tolyl)pyrimidin-2-amine (8a). Yield 75%, white solid. H NMR δ (400
H
MHz, DMSO-d6) 8.24 – 8.19 (m, 2H), 8.14 (d, J = 8.2 Hz, 2H), 7.68 (s, 1H), 7.55 – 7.50 (m,
3H), 7.33 (d, J = 7.7 Hz, 2H), 6.71 (s, 2H), 2.39 (s, 3H). 13C NMR δ (100 MHz, DMSO-d6)
C
165.21, 165.17, 164.44, 140.69, 137.88, 135.01, 130.83, 129.67, 129.05, 127.41, 127.37, 101.93,
21.44. HRMS (EI) m/z [M]+ calcd for C17H15N3: 261.1266, found: 261.1269.
1
4.1.3.2. 5-Methyl-4-phenyl-6-(p-tolyl)pyrimidin-2-amine (8b). Yield 59%, white solid. H NMR
δ
(400 MHz, DMSO-d6) 7.57 (2 H, d, J = 6.1), 7.52 – 7.42 (5 H, m), 7.29 (2 H, d, J = 8.1),
H
6.47 (2 H, s), 2.37 (3 H, s), 2.04 (3 H, s). 13C NMR δ C (100 MHz, DMSO-d6) 167. 52, 167.47,
161.89, 139.80, 138.51, 136.86, 129.25, 129.20, 129.01, 128.96, 128.41, 113.25, 21.38, 16.86.
HRMS (EI) m/z [M]+ calcd for C18H17N3: 275.1422, found: 275.1425.
4.1.3.3. 5-Butyl-4-phenyl-6-(p-tolyl)pyrimidin-2-amine (8c). Yield 52%, white solid. 1H NMR δ
H (400 MHz, DMSO-d6) 7.53 – 7.42 (5 H, m), 7.39 (2 H, d, J = 8.1), 7.27 (2 H, d, J = 7.8), 6.49
(2 H, s), 2.49 – 2.45 (2 H, m), 2.37 (3H, s), 1.02 – 0.89 (2 H, m), 0.89 – 0.74 (2 H, m), 0.46 (3 H,
t, J = 7.2). 13C NMR δ C (100 MHz, DMSO-d6) 168.05, 167.99, 161.49, 140.18, 138.03, 137.29,
130.14, 128.98, 128.65, 128.43, 118.61, 115.46, 32.24, 26.98, 22.02, 21.35, 13.60. HRMS (EI)
m/z [M]+ calcd for C21H23N3: 317.1892, found: 317.1895.
4.1.3.4. 4-(4-Methoxyphenyl)-6-phenylpyrimidin-2-amine (9a). Yield 47%, white solid. 1H NMR
δ
(400 MHz, DMSO-d6) 8.23 – 8.18 (m, 4H), 7.66 (s, 1H), 7.55 – 7.47 (m, 3H), 7.09 – 7.03
H
(m, 2H), 6.68 (s, 2H), 3.84 (s, 3H). 13C NMR δ C (100 MHz, DMSO-d6) 164.83, 164.68, 161.53,
138.26, 130.57, 130.42, 128.95, 128.92, 127.33, 114.31, 100.21, 55.77. HRMS (EI) m/z [M]+
calcd for C17H15N3O: 277.1215, found: 277.1217.
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4.1.3.5. 4-(4-Methoxyphenyl)-5-methyl-6-phenylpyrimidin-2-amine (9b). Yield 49%, white
solid.1H NMR δ H (400 MHz, DMSO-d6) 7.62 – 7.54 (4 H, m), 7.51 – 7.41 (3 H, m), 7.03 (2 H,
d, J = 8.8), 6.45 (2 H, s), 3.82 (3 H, s), 2.07 (3 H, s). 13C NMR δ (100 MHz, DMSO-d6)
C
167.51, 166.98, 161.87, 160.03, 139.86, 131.92, 130.91, 129.23, 128.98, 128.40, 113.76, 113.14,
55.66, 17.04. HRMS (ESI) m/z [M+H]+ calcd for C18H18N3O: 292.1444, found: 292.1446.
4.1.3.6. 5-Butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine (9c). Yield 92%, white solid.
1H NMR δ (400 MHz, DMSO-d6) 7.54 – 7.37 (7 H, m), 7.02 (2 H, d, J = 8.8), 6.48 (2 H, s),
H
3.81 (3 H, s), 2.57 – 2.46 (2 H, m), 1.07 – 0.92 (2 H, m), 0.90 – 0.74 (2 H, m), 0.47 (3 H, t,
13
J = 7.2). C NMR δ (100 MHz, DMSO-d6) 168.07, 167.58, 161.49, 159.70, 140.26, 132.43,
C
130.20, 128.64, 128.42, 118.58, 113.80, 55.61, 32.20, 27.03, 22.02, 13.63. HRMS (ESI) m/z
[M+H]+ calcd for C21H24N3O: 334.1914, found: 334.1914.
1
4.1.3.7. 4-(4-Methoxyphenyl)-6-(p-tolyl)pyrimidin-2-amine (10a). Yield 89%, white solid. H
NMR δ H (400 MHz, DMSO-d6) 8.20 (2 H, d, J = 9.0), 8.12 (2 H, d, J = 8.3), 7.62 (1 H, s), 7.33
13
(2 H, d, J = 7.8), 7.06 (2 H, d, J = 9.0), 6.61 (2 H, s), 3.85 (3 H, s), 2.39 (3 H, s). C NMR δ C
(100 MHz, DMSO-d6) 164.90, 164.73, 164.34, 161.65, 140.53, 135.15, 130.16, 129.63, 128.98,
127.32, 114.38, 101.16, 55.79, 21.43. HRMS (ESI) m/z [M+H]+ calcd for C18H18N3O: 292.1444,
found: 292.1445.
4.1.3.8. 4-(4-Methoxyphenyl)-5-methyl-6-(p-tolyl)pyrimidin-2-amine (10b). Yield 43%, white
solid. 1H NMR δ H (400 MHz, DMSO-d6) 7.57 (2 H, d, J = 8.8), 7.49 (2 H, d, J = 8.3), 7.29 (2
H, d, J = 7.8), 7.03 (2 H, d, J = 8.8), 6.40 (2 H, s), 3.82 (3 H, s), 2.38 (3 H, s), 2.08 (3 H, s). 13C
NMR δ (100 MHz, DMSO-d6) 167.41, 166.93, 161.86, 160.01, 138.45, 137.00, 132.00,
C
130.91, 129.28, 128.94, 113.74, 113.11, 55.66, 21.38, 17.18. HRMS (ESI) m/z [M+H]+ calcd for
C19H20N3O: 306.1601, found: 306.1602.
4.1.3.9. 5-Butyl-4-(4-methoxyphenyl)-6-(p-tolyl)pyrimidin-2-amine (10c). Yield 36%, white
1
solid. H NMR δ H (400 MHz, DMSO-d6) 7.45 (2 H, d, J = 8.8), 7.38 (2 H, d, J = 8.3), 7.27 (2
H, d, J = 7.6), 7.02 (2 H, d, J = 9.0), 6.42 (2 H, s), 3.81 (3 H, s), 2.56 – 2.48 (2 H, m), 2.37 (3 H,
13
s), 1.02 – 0.91 (2 H, m), 0.90 – 0.79 (2 H, m), 0.49 (3 H, t, J = 7.2). C NMR δ (100 MHz,
C
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DMSO-d6) 166.75, 161.49, 159.69, 159.25, 137.96, 137.45, 132.54, 130.21, 128.96, 128.67,
118.60, 113.78, 55.62, 32.17, 27.08, 22.01, 21.35, 13.68. HRMS (ESI) m/z [M+H]+ calcd for
C22H26N3O: 348.2070, found: 348.2071.
4.2. Biology
Stock solutions (200 mM) of pyrimidine analogues were prepared in tissue-culture tested
dimethylsulfoxide (DMSO, Sigma-Aldrich). Required working solutions were freshly obtained
by the dilution in complete RPMI-1640 culture medium. It contained 10% heat-inactivated foetal
bovine serum, 2 mM L
-glutamine, 50 µg/ml gentamicin, and 5 x 10-5 M 2-mercaptoethanol (all
Sigma-Aldrich). Effects of studied pyrimidine analogues on production of prostaglandin E2
(PGE2) were evaluated in vitro, using mouse (C57BL6, Charles River Deutschland, Sulzfeld,
Germany) peritoneal cells. Animals, killed by cervical dislocation, were i.p. injected with 8 ml of
sterile saline. Pooled peritoneal cells collected from mice (n = 5-8 in individual experiments)
were washed, re-suspended in complete culture medium, and seeded into 96-well round-bottom
microplates (Costar) in 100-µl volumes, 2 x 105 cells/well. Cultures were maintained at 37 °C,
5% CO2 in humidified incubator (Sanyo Electric Biomedical, Osaka, Japan). Possible effects of
the vehiculum (DMSO) were evaluated using the 0.025% dilution corresponding to the highest
molarity of tested pyrimidines (i.e. 50 µM). This amount of DMSO was found to be devoid of
any interference with the PGE2 and viability assays (see Fig. 1A, and Table 2, respectively).
Experimental protocols were approved by the institutional ethics committee.
4.2.1. PGE2 and cytotoxicity assays in mouse C57BL6 peritoneal cells. The cells were cultured in
presence of test compounds applied concomitantly with lipopolysaccharide (LPS from E. coli
0111:B4, Sigma; 10 ng/ml). Amount of PGE2 was determined at the interval of 5 h of culture
using ELISA kit (R&D Systems), following manufacturer instruction. The effects of compounds
were evaluated as a percentage of LPS-induced (control) concentrations of PGE2.
4.2.3. LDH assay. Viability of cells was analyzed using the lactate dehydrogenase (LDH) assay.
It is based on the determination of LDH activity released from the cytosol of damaged cells into
the supernatant. The cell supernatants were harvested at the interval of 5 h of culture, diluted 1:1
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and mixed with an aliquot of the LDH kit. After 30-min incubation in the dark at ambient
temperature, the reaction was stopped with 2 N HCl. Differences between absorbances at 492
and 690 nm were evaluated. The percentage cytotoxicity of test samples was related to the
control samples and to the samples with 100% dead cells evoked by 1% Triton, according to the
formula: [(exp. value – control value) / (Triton value – control value)] x 100. All control and
experimental variants were run in triplicate.
4.2.4. Statistical analysis. Analysis of variance (ANOVA) with subsequent Bonferroni´s multiple
comparison test, Wilcoxon signed rank test for comparison of medians, and graphical
presentation of data were done using the Prism program (GraphPad Software, San Diego, CA).
Acknowledgment
This work was supported by the subvention for development of research organization (Institute
of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, RVO: 61388963)
and Technology Agency of the Czech Republic (TE01020028).
Abbreviations
COX, cyclooxygenase; LPS, lipopolysaccharide; NSAIDs, nonsteroidal anti-inflammatory drugs;
NO, nitric oxide; PGE2, prostaglandin E2.
AUTHOR INFORMATION
Corresponding Authors
*(Z.Z.) E-mail: zidekz@biomed.cas.cz.
*(Z.J.) E-mail: janeba@uochb.cas.cz.
Orcid
Zlatko Janeba: 0000-0003-4654-679X
Present Addresses
‡ Gilead Sciences Inc., 333 Lakeside Drive, Foster City, CA 94404, USA.
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ACCEPTED MANUSCRIPT
Graphical abstract
Highlights
• Synthesis of polysubstituted pyrimidines using Suzuki-Miyaura cross-coupling reaction
• Potent inhibitors of prostaglandin E2 production
• No cytotoxicity observed at the concentrations tested
• Potential anti-inflammatory agents
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