Polysubstituted Pyrimidines as Potent Inhibitors of Prostaglandin E2 Production: Increasing Aqueous Solubility

Article abstract of DOI:10.1002/cmdc.202100263

Water solubility is one of the key features of potential therapeutic agents. In order to enhance the low water solubility of the parent 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine, a potent inhibitor of prostaglandin E₂ (PGE₂) production, we synthesized and evaluated a new series of derivatives in which the butyl group at the C5 position of the pyrimidine ring was replaced with a less lipophilic substituent, preferably with a hydrophilic aliphatic moiety. Except for the 5-cyanopyrimidine derivative, all target compounds exhibited increased (2.7 – 87-fold) water solubility relative to the parent compound. Although nontoxic in mouse peritoneal cells, the prepared compounds were either equipotent or weaker inhibitors of PGE₂ production than the parent compound. The most promising compound from the series was found to be the 5-(2,5,8,11-tetraoxadodecyl)pyrimidine derivative (with three polyethylene glycol units at the C5 position), which exhibited 32-fold higher water solubility and only slightly weaker inhibitory activity (22 % of remaining PGE₂ production) compared with the parent compound (15 % of remaining PGE₂ production).

Full text of DOI:10.1002/cmdc.202100263

Accepted Article
Title: Polysubstituted Pyrimidines as Potent Inhibitors of Prostaglandin
E2 Production: Increasing Their Aqueous Solubility
Authors: Filip Kalčic, Viktor Kolman, Zdeněk Zídek, and Zlatko Janeba
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To be cited as: ChemMedChem 10.1002/cmdc.202100263
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01/2020

10.1002/cmdc.202100263
ChemMedChem
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Polysubstituted Pyrimidines as Potent Inhibitors of Prostaglandin E2 Production:
Increasing Their Aqueous Solubility
Filip Kalčic,[a,b] Viktor Kolman,[a] Zdeněk Zídek,*[c] and Zlatko Janeba*[a]
[a]
[b]
[c]
RNDr. F. Kalčic, Dr. V. Kolman, 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
RNDr. F. Kalčic
Department of Organic Chemistry, Faculty of Science
Charles University
Hlavova 8, 128 43, Prague 2, Czech Republic
Dr. Z. Zídek
Institute of Experimental Medicine of the Czech Academy of Sciences
Vídeňská 1083, 142 20, Prague 4, Czech Republic
Supporting information for this article is given via a link at the end of the document.
Abstract: Water-solubility is one of the key features of potential
quantified by log P which can be predicted in silico, giving
medicinal chemists some initial hints for the design of novel
molecules.[2]
therapeutic agents. In order to enhance poor water-solubility of parent
5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-2-amine,
a
potent
inhibitor of prostaglandin E2 (PGE2) production, we synthesized and
evaluated a new series of derivatives where the butyl group in the C5
There are various strategies, exploited by pharmaceutical
industry, how to increase compound’s solubility. These include
position of pyrimidine ring was replaced with less lipophilic substituent, salt formation, determination of the proper polymorph,
preferably with a hydrophilic aliphatic moiety. Except for the 5-
cyanopyrimidine derivative, all target compounds exhibited increased
(2.7 – 87-fold) water-solubility compared to the parent compound.
Although non-toxic in mouse peritoneal cells, the prepared
compounds were either equipotent or weaker inhibitors of PGE2
production than the parent compound. The most promising compound
from the series was 5-(2,5,8,11-tetraoxadodecyl)pyrimidine derivative
(with three polyethylene glycol units in the C5 position), which
exhibited 32-fold higher water-solubility and only slightly weaker
inhibitory activity (22% of remaining PGE2 production) compared to
the parent compound (15% of remaining PGE2 production).
introduction of a prodrug moiety, utilization of co-solvents, co-
crystals, or surfactants.[1,3–6] A useful way to enhance water-
solubility is to form suitable bioisosters via an introduction of a
heteroatom into a molecule. For instance, the decrease of log P
from 3.36 to 0.77 can be achieved by replacing the CH2 (X) group
for oxygen atom in the RCH2-X-CH2R segment.[7]
We have recently discovered substituted pyrimidines with potent
anti-inflammatory properties. The compounds were shown to be
inhibitors of immune-activated nitric oxide (NO) production[8,9] or
dual inhibitors of NO and prostaglandin E2 (PGE2) production.[10,11]
Later, series of 2-amino-4,6-diarylpyrimidines were synthesized
and studied as potent inhibitors of PGE2 production.[12,13]
Moreover, the compounds exhibited strong anti-inflammatory in
vivo effects in the rat model of acute inflammation.[13]
Among the polysubstituted pyrimidines studied, 5-butyl-4-(4-
methoxyphenyl)-6-phenylpyrimidin-2-amine (1, Figure 1) was
identified as a potential lead structure with anti-inflammatory
properties,[12,14] but a significant drawback of this compound was
its low water-solubility. Thus, boosting the solubility was the next
logical step in further development of polysubstituted pyrimidines
as anti-inflammatory agents.
Introduction
High in vitro biological potency of new drug candidates is not the
only key prerequisite for a successful transformation into a drug.
Other important physicochemical and pharmacokinetic properties
need to be seriously taken into account as well. Nowadays, the
poor water-solubility of small molecules represents a common
problem as approximately 40% of marketed drugs and up to 75%
of molecules in development stages are poorly water-soluble.[1]
Drug solubility is considered to be a fundamental property that has
to be evaluated already in the early stages of drug discovery.
Fine-tuning of the solubility is crucial since both adequate
lipophilicity and hydrophilicity are important factors for the
pharmacokinetics of the active compound. Hydrophilicity is pivotal
for the solubility in an aqueous environment, which is essential for
appropriate absorption in the case of highly desirable oral
bioavailability of the drug candidate. Lipophilicity, on the other
hand, is crucial for the penetration of the molecule through
biomembranes of the target cells. Lipophilicity is usually
Figure 1. The goal of the current study: a replacement of the 5-butyl group with
more hydrophilic aliphatic chain R.
1
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Herein we present structural modifications of compound 1 (Figure
1) with the aim to increase its water-solubility. Previous data[15]
suggested that modification of the substituent in the C5 position
of pyrimidine moiety should have minimal impact on anti-
inflammatory properties. Thus, we decided to replace the 5-butyl
group with various aliphatic chains containing heteroatom(s) in
order to increase water-solubility of such derivatives. An efficient
synthetic approach towards analogues of compound 1 modified
at the C5 position with polar substituents was developed and their
biological activity as well as solubility were determined.
Ozonolysis of 5-vinylpyrimidine 6 afforded 5-formylpyrimidine 7 in
a 29% yield and its subsequent reduction with NaBH4 in DMF
gave 5-(hydroxymethyl)pyrimidine derivative 8 in an 80% yield
(Scheme 1).
In view of the fact, that the next synthetic steps were based on
alkylation of the 5-(hydroxymethyl)pyrimidine group, a protection
of the 2-aminopyrimidine group was necessary. At first, benzyl
was selected as a suitable protecting group with regard to its
stability under the reaction conditions, however, its standard
removal using catalytic hydrogenation with Pd/C failed.
Afterwards, p-methoxybenzyl (PMB) group was employed in
order to achieve easier deprotection.[18] Nevertheless, even the
PMB group proved to be relatively stable as it was resistant to
catalytic (Pd/C) hydrogenation in glacial acetic acid as well as to
treatment with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
or cerium(IV) ammonium nitrate (CAN).[18–20] The efficient PMB
group removal was ultimately achieved by treatment with
trifluoroacetic acid (TFA).
Results and Discussion
Synthesis
Analogues of compound 1 (Figure 1) bearing a hydrophilic
aliphatic chain (instead of lipophilic 5-butyl group) were designed
and 10 target molecules were selected for the synthesis, based
on calculated log P values (see SI) using Virtual Computational
Chemistry Laboratory (VCCLAB).[16] The synthetic route started
from compound 2 (Scheme 1), which was prepared in a 69% yield
from acetophenone and p-anisaldehyde using the one-pot
assembly reported by Nimkar and co-workers.[17] Compound 2
was brominated using bromine in chloroform to give
5-bromopyrimidine derivative 3 (67%), which was subsequently
converted into 5-cyanopyrimidine derivative 4 (by reaction with
zinc cyanide in a 28% yield) and into 5-(1-ethoxyvinyl)pyrimidine
derivative 5 (via the Heck reaction with ethyl vinyl ether in a 61%
yield). 5-Vinylpyrimidine 6, the key intermediate for further
modifications, was obtained in a 58% yield by the Suzuki-Miyaura
The protection of the 2-aminopyrimidine group with PMB was
performed at the stage of 5-vinylpyrimidine 6 (Scheme 2). Thus,
treatment of compound 6 with p-methoxybenzyl chloride, followed
by ozonolysis/reduction afforded the desired protected
intermediate 9 in a 69% yield (over 2 steps).
coupling reaction of 5-bromopyrimidine
vinylboronate.
3
with pinacol
Scheme 2. Synthesis of target compounds 10–15. Reagents and conditions: (a)
NaH, 0 °C, DMF, then p-methoxybenzyl chloride, 16 h, 65 °C; (b) O3, DCM, -
20 °C, then NaBH4, 168 h, 25 °C; (c) NaH, 0 °C, DMF, MeI, 19 h, 80 °C; (d) TFA,
16 h, 50 °C; (e) NaH, 0 °C, DMF, EtBr, 19 h, 80 °C; (f) NaH, 0 °C, DMF, 2-
chloroethyl methyl ether, 19 h, 80 °C; (g) NaH, 0 °C, DMF, 1-bromo-2-(2-
methoxyethoxy)ethane, 19 h, 80 °C; (h) NaH, 0 °C, DMF, 1-(2-bromoethoxy)-2-
(2-methoxyethoxy)ethane, 19 h, 80 °C; (i) TFA, n-PrOH, 5 h, 50 °C.
Key 5-(hydroxymethyl)pyrimidine intermediate 9 (Scheme 2) was
treated with NaH in DMF and then with the corresponding
alkylating agent, followed by the PMB groups removal with TFA,
to afford target derivatives 10–14 in 15-52% yields (over two
steps). Interestingly, partial decomposition of the 5-alkoxymethyl
moiety was observed during the removal of PMB groups under
acidic conditions (TFA), leading to a partial formation of 5-
(hydroxymethyl) pyrimidine 8 in the reaction mixture (observed in
UPLC-MS). In order to corroborate this phenomenon, compound
13 was treated with TFA and in the UPLC-MS, a mixture of
compounds 13 and 8 (as the product of decomposition) was
observed in MeCN while a mixture of 13 and 10 (as the product
of re-etherification) was observed in MeOH (Figure S39 in SI).
This observation was subsequently exploited for the synthesis of
5-(propoxymethyl)pyrimidine derivative 15 (Scheme 2).
Treatment of compound 9 with TFA, in n-PrOH afforded target
compound 15 in a 35% yield.
Scheme 1. Synthesis of compounds 4–8. Reagents and conditions: (a) NaOH,
EtOH, 30 min, 25 °C; (b) guanidine hydrochloride, 24 h, 85 °C; (c) Br2, CaCO3,
CHCl3, 5 h, 25 °C; (d) Zn(CN)2, Pd(t-Bu3P)2, DMF, 16 h, 110 °C; (e) ethyl vinyl
ether, Et3N, Pd(t-Bu3P)2, DMF, 16 h, 110 °C; (f) pinacol vinylboronate, Cs2CO3,
Pd(t-Bu3P)2, 1,4-dioxane/H2O, 15 h, 85 °C; (g) O3, DCM, -20 °C, Me2S, 3 h,
25 °C; (h) NaBH4, DMF, 1 h, 0 °C.
2
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Since the prepared derivatives were more soluble compared to
compound 1 (see water-solubility determination below), we
decided to expand this approach and we designed novel
compound 17 (Figure 2) as a combination of soluble derivative 14
and previously reported13 potent inhibitor of PGE2 production,
compound 16 (IC50 = 31 nM, 99.7% inhibition of PGE2 production),
which was approx. 150-fold more potent compared to compound
1 (IC50 = 4.83 μM, 87.7% inhibition of PGE2 production).
Scheme 3. Design and synthesis of compound 17, as a more soluble PGE2
production inhibitor of 16. Reagents and conditions: (a) NaOH, EtOH, 30 min,
25 °C; (b) guanidine hydrochloride, 24 h, 85 °C; (c) Br2, CaCO3, CHCl3, 5 h,
25 °C; (d) pinacol vinylboronate, Cs2CO3, Pd(t-Bu3P)2, 1,4-dioxane/H2O, 15 h,
85 °C; (e) NaH, 0 °C, DMF, 4-methoxybenzyl chloride, 16 h, 65 °C; (f) O3, DCM,
-70 °C, then NaBH4, 168 h, 25 °C; (g) NaH, 0 °C, DMF, 1-(2-bromoethoxy)-2-
(2-methoxyethoxy)ethane, 19 h, 80 °C; (h) TFA, 16 h, 50 °C.
Figure 2. Design of compound 17 based on previously published derivative 16.
Compound 17 was prepared in analogous way to compound 14.
Pyrimidine 18 (Scheme 3) was prepared in a 73% yield from
acetophenone and 4-(benzyloxy)benzaldehyde by the one-pot
assembly.[17] Bromination of compound 18 with bromine in
chloroform gave 5-bromopyrimidine derivative 19 quantitatively.
5-Vinylpyrimidine 20 was obtained in a 35% yield by the Suzuki-
Miyaura coupling of compound 19 with pinacol vinylboronate.
Introduction of PMB groups followed by ozonolysis/reduction
afforded derivative 21 in a 43% yield (2 steps). Finally, alkylation
350
300
250
200
150
100
50
of
compound
21
with
1-(2-bromoethoxy)-2-(2-
methoxyethoxy)ethane and with subsequent PMB groups
removal gave the target compound 17 in a 28% yield (2 steps).
0
Water-solubility
1
4
5
7
8
10 11 12 13 14 15 16 17
The water-solubility of studied compounds was determined by a
method exploiting the compounds absorption at 254 nm using
HPLC (for details, see SI, p. S17). The area under the curve
(AUC) was correlated to the corresponding calibration curve in
order to calculate the concentration of dissolved compound.
All target compounds proved to be more soluble compared to the
parent compound 1 (Figure 3), with the exception of 5-
cyanopyrimidine 4 which had similar solubility as compound 1.
The insertion of one extra oxygen atom into the aliphatic moiety
of 5-(propoxymethyl)pyrimidine 15 (Scheme 2) to form 5-
(methoxyethoxymethyl)pyrimidine 12 led 5-fold increase in the
solubility. Interestingly, despite the calculated log P values (see
SI, p. S16), the most soluble derivative from the series was
compound 13 (with two PEG units in the C5 position), and not 14
(with three PEG units in the C5 position). This result suggests that
C5 substituents consisting of 9 or more atoms might start to coil
and form micelle-like structures, thus, reducing the polar surface
of the molecule available for interactions with water molecules.
The water-solubility of benzyloxyphenyl analogue 17 (Scheme 3)
was one order of magnitude higher compared to its parent
compound 16, but compound 17 exhibited significantly lower
potency to inhibit PGE2 production (Figure 4).
Figure 3. Experimentally determined water-solubility of target compounds. Bars
are means ± SEM obtained by averaging results of three experiments for each
compound.
Biological data
The target molecules were evaluated in vitro for inhibition of PGE2
production and viability using C57BL6 mouse peritoneal cells
(Figure 4). The effects of target compounds on the PGE2
production were expressed as a percentage change (remaining
production of PGE2) relative to the response of LPS stimulated
cells (positive control, 100 %) or unstimulated cells (negative
control, 2.50 %). The newly prepared derivatives exhibited
comparable (compounds 4, 5, 10, and 14) or worse activity
(compounds 7, 8, 11, 12, 13, and 15) than compound 1. Hence,
replacement of the 5-butyl group for more hydrophilic and longer
aliphatic moiety did not lead to an increased potency compared to
parent compounds 1 or 16. The cell viability was expressed as %
of control untreated cells and the studied compounds did not
exhibit cytotoxic effects within the exposure time of 6 h.
3
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Acknowledgements
PGE2 level
Viability
This work was supported by a grant for the development of
research organization (Institute of Organic Chemistry and
Biochemistry, Czech Academy of Sciences, RVO: 61388963) and
by the Technology Agency of the Czech Republic (TE01020028).
100%
80%
60%
40%
20%
0%
Author Information
Corresponding Author
*Z.J.: email, janeba@uochb.cas.cz
*Z.Z.: email, zdenek.zidek@iem.cas.cz
ORCID
Zlatko Janeba: 0000-0003-4654-679X
Filip Kalčic: 0000-0002-4391-8319
Figure 4. Effect of prepared compounds on in vitro production of PGE2 and on
viability of mouse peritoneal cells (interval of 6 h). Effects are expressed as a
percentage change relative to the response of LPS stimulated (100%, positive
control) or unstimulated (2.5%, negative control) cells. Bars are means ± SEM
obtained by averaging results of two to four experiments for each compound.
PC – positive control, NC – negative control.
Notes
The authors declare no competing financial interest.
Keywords: solubility • anti-inflammatory • pyrimidines •
prostaglandin E2 • synthesis
Conclusion
[1]
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The structure of 5-butyl-4-(4-methoxyphenyl)-6-phenylpyrimidin-
2-amine (1), a potent inhibitor of PGE2 production with anti-
inflammatory properties, was modified in order to increase its poor
water-solubility. A series of compounds was designed and
synthesized, where the lipophilic n-butyl substituent in the C5
position of pyrimidine moiety of compound 1 was replaced with
more hydrophilic substituent, preferably an aliphatic chain. Except
for 5-cyanopyrimidine 4 (which had a similar solubility as 1), all
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See Supporting Information for general experimental information, standard
procedures, spectral data, characterization, calculated log P values,
solubility measurements details, calibration curves, PGE2 assay, viability
assay, and copies of spectra of the prepared compounds.
4
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Entry for the Table of Contents
In this study, the goal was to increase water solubility of previously reported inhibitors of prostaglandin E2 production. The most soluble
derivative exhibited 2 orders of magnitude higher solubility although much lower potency compared to the parent compound. The most
promising compound (14) reached 32-fold higher solubility with only slight decrease in potency.
5
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