Structurally Simple, Readily Available Peptidomimetic 1-Benzyl-5-methyl-4-(n-octylamino)pyrimidin-2(1 H)-one Exhibited Efficient Cardioprotection in a Myocardial Ischemia (MI) Mouse Model

Article abstract of DOI:10.1021/acs.jmedchem.8b01471

TLR4, a member of the Toll-like receptor (TLR) family, serves as a pattern recognition receptor in the innate immune response to microbial pathogens. TLR4 also regulates the inflammatory reaction to ischemic injury in the heart. The TRIF-related adaptor molecule (TRAM) is an adapter that recruits the Toll/interleukin 1 receptor (TIR) domain, which contains adapter-inducing IFN-β (TRIF), to activate TLR4, following TRIF-dependent cytokine gene transcription. On the basis of a known TRAM-derived decoy peptide, 10 of its peptidomimetics were synthesized. One of them, 1-benzyl-5-methyl-4-(n-octylamino)pyrimidin-2(1H)-one (21), exhibited high potency and efficacy in vitro. In vitro results and in silico analysis provided evidence for the possible direct interaction of 21 with the TLR4 complex. Administered in mice, 21 was able to block the pathophysiological manifestation of MI, restoring the concomitant tissue damage, with a 100% survival rate. Thus, inhibition of TLR4-mediated inflammation in postischemic myocardium could be used as an approach for developing cardioprotective drugs.

Full text of DOI:10.1021/acs.jmedchem.8b01471

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Article
Structurally Simple, Readily Available Peptidomimetic 1-Benzyl-5-
methyl-4-(n-octylamino)pyrimidin-2(1H)-one Exhibited Efficient
Cardioprotection in a Myocardial Ischemia (MI) Mouse Model
Lena Trifonov, Vadim Nudelman, Michael Zhenin, Erez Matsree, Michal Afri, Bruria
Schmerling, Guy Cohen, Krzysztof Jozwiak, Michal Weitman, Edward Korshin,
Hanoch Senderowitz, Asher Shainberg, Edith Hochhauser, and Arie Gruzman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01471 • Publication Date (Web): 03 Dec 2018
Downloaded from http://pubs.acs.org on December 9, 2018
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Structurally Simple, Readily Available Peptidomimetic 1-Benzyl-5-methyl-4-(n-
octylamino)pyrimidin-2(1H)-one Exhibited Efficient Cardioprotection in a Myocardial
Ischemia (MI) Mouse Model
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⊥
#
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Lena Trifonov, Vadim Nudelman, Michael Zhenin, Erez Matsree, Michal Afri, Bruria
⊥
&
†
⊥
⊥
Schmerling, Guy Cohen, Krzysztof Jozwiak, Michal Weitman, Edward Korshin, Hanoch
⊥
$
*,#
,⊥
Senderowitz, Asher Shainberg, Edith Hochhauser, and Arie Gruzman*
⊥
$
Department of Chemistry, Faculty of Exact Sciences and Faculty of Life Sciences, Bar-Ilan
University, Max and Anna Webb St., Ramat-Gan 5290002, Israel.
#Cardiac Research Laboratory, Felsenstein Research Center, Rabin Medical Center, Sackler
Faculty of Medicine, Tel Aviv University, Jabotinsky St., Petach Tikva 49100, Israel.
&
The Skin Research Institute, The Dead-Sea & Arava Science Center, Masada 86910, Israel.
†Department of Biopharmacy, Medical University of Lublin, W. Chodzki 4a, Lublin 20-093,
Poland.
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ABSTRACT
TLR4, a member of the toll-like receptor (TLR) family, serves as a pattern recognition receptor
in the innate immune response to microbial pathogens. TLR4 also regulates the inflammatory
reaction to ischemic injury in the heart. The TRIF-related adaptor molecule (TRAM) is an
adapter that recruits the Toll/interleukin 1 receptor (TIR) domain, which contains adapter-
inducing IFN-β (TRIF), to activate TLR4, following TRIF-dependent cytokine gene
transcription. Based on a known TRAM-derived decoy peptide, ten of its peptidomimetics were
synthesized. One of them, 1-benzyl-5-methyl-4-(n-octylamino)pyrimidin-2(1H)-one (21),
exhibited high potency and efficacy in vitro. In vitro results and in silico analysis provided
evidence for the possible direct interaction of 21 with the TLR4 complex. Administered in mice,
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1 was able to block the pathophysiological manifestation of MI, restoring the concomitant
tissue damage, with a 100% survival rate. Thus, inhibition of TLR4-mediated inflammation in
post-ischemic myocardium could be used as an approach for developing cardioprotective drugs.
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INTRODUCTION
At the end of the 1980s, C. Janeway proposed a revolutionary option for molecular
recognition of the immune system, based on its specific receptors: pathogen-associated
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molecular patterns (PAMPs). A decade later, the molecules that are presented on the surface of
microbes, namely, lipopolysaccharides (LPS), were identified as ligands for activation of
TLR4.3-6 This receptor has several conserved structural elements, including an extracellular
leucine-rich repeat (LRR) domain, a trans-membrane domain, and an intracellular TIR domain.7
The extracellular LRR domain functions as a receptor that recognizes LPS. The transmembrane
domain anchors TLR4 in the plasma membrane with functional topography and the TIR domain
is responsible for transduction generated by changes in TLR4 conformation signals from the
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outside of the cell to intracellular space. Activation of TLR4 by its agonists leads to massive
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recruitment of TIR domain-containing adapter proteins. To date, four such adapter proteins have
been discovered: Myeloid differentiation primary response protein 88 (MyD88), TIR domain-
containing adapter protein (TIRAP), TRIF, and TRAM.10 Activated TLR4 forces the
dimerization of TIR domains located in the sequence of two receptors, which consequently
allows two distinct pairs of adapter proteins (TIRAP and MyD88 or TRAM and TRIF) to bind to
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TLR4. Two adaptor proteins (TIRAP and TRAM) lack signaling properties per se, and act to
facilitate the recruitment of MyD88 or TRIF. The latter proteins are responsible for transferring
the signal pathway, and rapidly activate immune responses by inducing the functionality of
nuclear factor kB (NF-kB), mutagen-activated protein kinases (MAPKs) and cytokine
biosynthesis. The common structural domain, which is present in all TLRs and in TRAM, is the
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TIR motif. It has been proposed that the TLR4 TIR domain induces interactions resulting in
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dimerization via interaction between the BB loop and E helix. Noteworthy, blocking even one
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of the signaling adaptors of TLR4 (MyD88 or TRIF) reduces the inflammatory response and
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releases cytokine. Thus, this key physiological phenomenon might be capitalized as a basis for
developing inflammatory response inhibitors via suppressing only one pathway. We have
previously shown that knockout (KO) mice for TLR4 were resistant to both ischemic and LPS
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damage. Using SiRNA against TLR4 was also beneficial against ischemic damage in isolated
cardiomyocytes.15
In 2013, Piao W. et al. described the design of a decoy peptide derived from the TRAM
TIR BB loop.16 The peptide [IVFAEMPCG] was conjugated with a 16-amino acid cell-
permeating
sequence
(TM4-ΔC)
from
Antennapedia
homeodomain
[RQIKIWFQNRRMKWKK].17 TM4-ΔC exhibited a significant inhibition of MyD88- and
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TRIF-dependent activation of cytokine release. TM4-ΔC blocked the co-immunoprecipitation
(IP) of TRAM and TLR4 . According to authors, this data provided an evidence that the peptide
was able to suppress the recruitment of TLR4 adapters in specific manner. TM4-ΔC also blocked
the LPS-dependent binding between MyD88 and TLR4 in primary murine macrophages. The
mechanism by which this decoy peptide inhibits TLR4 signaling is presumed to be related to the
fact that peptide blocks the docking site in TLR4’s prototype protein (TRAM), thereby
preventing functional protein–protein interactions. Importantly, TM4-ΔC was active in vivo and
displayed an ability to suppress LPS-induced systemic inflammatory responses in mice. Based
on the [IVFAEMPCG] sequence, the authors identified a minimal length of the peptide
(conjugated to the cell penetration domain) that retains it’s biologically activity. The shortest
active [IVFAE] peptide was found to contain only 5 amino acids sequence.16
Although peptides are an integral part of any living creation and are essential for any
biological systems, their drug delivery ability is very limited because of significant problematic
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properties including immunogenic effects, oral instability, poor pharmacokinetic profile, and
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nonspecific protein binding. Peptidomimetics, which might imitate the 3D structure of starting
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peptides, can be biologically active without the weaknesses of peptides. The classical way to
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mimic peptide structures is to use moieties of chemically modified amino acids and to conjugate
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them using different amide bond linkers usually by creating restricted conformations. Not all
amino acids or amide bonds must be changed in certain peptides. Frequently, even small changes
in an entire peptide might lead to the development of biologically stable and active
peptidomimetcs.21-22
This work is directed toward elaboration of novel efficient cardioprotective
peptidomimetics acting through a suppression of TLR4-mediated inflammation. TLR4 was
shown to be a key player that regulates the severity and survival after myocardial damage during
MI.15,23 Overactivation of TLR4 in leucocytes and myocardium led to a significant elevation of
proinflammatory cytokines such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β).
In addition, TLR4 activation in myocardium causes an upregulation of NADPH oxidase 4
(
NOX4), which leads to generating an enormous burst of ROS.24-26 We and others also reported
that blocking (even partially) the TLR4 transduction mechanism during MI dramatically reduces
the activation of the inflammatory response.14,15,26,27 In addition, the relationship between
inflammatory processes and the development of late left ventricular pathophysiological
symptoms have been well studied. It was shown that a direct positive correlation exists between
the level of the TLR4 activation in peripheral monocytes and TLR4 activation in left ventricular
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tissue samples obtained from patients. This correlation even mirrored the degree of myocardial
dysfunction. Overall, this evidence led us to propose that blocking the TLR4-associated
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myocardial inflammatory response during MI might lead to minimization of morphological and
functional pathological changes in the heart.
Herein, we report on our findings that the peptide P6 [IVFAEMPCG], itself, without the
cell penetration moiety, can significantly impact cardiomyocytes and displayed an impressive
biological effect. We also validated the minimal effective sequence of P6 as a linear tetrapeptide,
and the 4 amino acids scaffold of the most active short peptide was used for designing a set of
cyclic peptidomimetics. One of the developed compounds, namely 1-benzyl-5-methyl-4-(n-
octylamino) pyrimidin-2(1H)-one (21), displayed considerable cardioprotective effect in vitro
and in vivo experiments. It was clearly demonstrated that the peptidomimetic 21 in
pharmacological doses could attenuate experimental MI in vitro and in vivo, resulting in a 100%
high survival rate and blockage of ischemia-induced pathological events in myocardium.
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RESULTS and DISCUSSION
Design and biological evaluation of short peptides. Piao W. et al. described a
peptide [IVFAEMPCG], which was derived from the TRAM TIR BB loop and conjugated with
the cell penetration moiety (TM4-ΔC); blocked cytokines were released from primary murine
macrophages.16 In addition, TM4-ΔC significantly suppressed the immune response in LPS-
treated mice. We aimed to further evaluate the beneficial effects of IVFAEMPCG in a different
in vitro system using rat cardiomyocytes. However, P6 is a relatively long peptide (9 amino
acids), and after the its N-terminus was acetylated, it was insoluble in water. P6 was only soluble
in DMSO or in a mixture of water and acetonitrile. We thought that such an elevated level of
lipophilicity might allow P6 to penetrate the cardiomyocyte’s membrane without conjugation
with Antennapedia homeodomain peptide. Thus, we planned to test first the effect of the non-
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conjugated peptide and if P6 would not be active, to conjugate it with the membrane penetration
domain. The compound was tested in cardiomyocyte cultures subjected to hypoxia for 3 h. The
ability of P6 at concentrations of 10 µM and 20 µM to decrease the release of two well-known
cell damage markers, creatine kinase (CK) and lactate dehydrogenase (LDH), attributed to
cardiomyocyte protection, was investigated. As expected, the level of both enzymes significantly
increased under the hypoxic conditions (Figure 1A). Although we did not detect any effect at a
concentration of 10 µM, at 20 µM of P6 a significant reduction in the levels of both enzymes by
approximately 5.5-fold and 1.8-fold, respectively, was observed (Figure 1A). Since the project’s
goal consisted of elaboration an active peptidomimetic compound, the minimal effective
sequence of the peptide was first investigated. This step was of major importance, since
attempting to mimic a longer than necessary sequence (25 amino acids, including Antennapedia
homeodomain for cellular penetration) is not desirable and any modification should be restricted
and synthesized only after ascertaining the biologically active sequence. Thus, the minimal
effective sequence of P6 was also validated by synthesizing short peptides containing sequences
of only 4 amino acids, representing different areas of [IVFAEMPCG]. Six such peptides, namely
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[
Ac-IVFA-NH ] (1), [Ac-VFAE-NH ] (2), [Ac-FAEM-NH ] (3), [Ac-AEMP-NH ] (4), [Ac-
2 2 2 2
EMPC-NH ] (5), and [Ac-MPCG-NH ] (6) were synthesized (Scheme 1). In addition, three
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peptides containing only 3 amino acids: [Ac-FAE-NH ] (7), [Ac-VFA-NH ] (8), and [Ac-IFA-
2
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NH ] (9) were also prepared. All the peptides 1-9 were synthesized using Rink amide resin, in
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which the resulting peptide is always released with amidated C-terminus. In addition, an acetyl
group was attached at the N-terminus to mimic the amide bond of the original peptide (such a
simple manipulation does not allow the C- and N-terminus to become negatively and positively
charged respectively under physiological conditions). We assumed that tetrapeptides would be
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small enough to successfully penetrate into the cardiomyocytes without the need for an
Antennapedia homeodomain, which contains 16 amino acids. These nine peptides were screened
in primary cardiomyocyte cultures. After preliminary validation of the activity of non-conjugated
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[IVFAEMPCG] in hypoxia, a second experimental condition (LPS) was also applied in the
experiment with these nine peptides. Those two models imitate myocardial damage after direct
activation of the immune system by applying TLR4 agonist (LPS), or even by indirect activation
of the immune system by ischemia. Indeed, MI is accompanied by a widespread inflammatory
response, central to the pathology of this disease.29 Both central pathophysiological events,
namely, acute MI, followed by reperfusion and as a result, chronic ischemic damage, activate the
immune system.30 This inflammatory response promotes cardiomyocyte damage/apoptosis in
addition to hypoxia and reperfusion. Four tetrapeptides (1-4) out of nine were found to be cell
protective. They decreased the release of both enzymes in a dose-dependent manner (CK and
LDH) under hypoxia, as shown in Figure 1B and 1C. Noteworthy, the tetrapeptide 1 revealed an
impressive cell protective effect (77% and 70%, respectively) at concentration 1 µM.
Compounds 2 and 3 displayed activity at much higher concentrations (10-20 µM) compared to 1.
The peptide 4 was found to be poorly active: it significantly reduced CK level at the maximal
tested concentration (20 µM) only. Figure 1D summarizes the results of the biological validation
of all four active tetrapeptides 1-4 at concentration 10 µM, which was selected for elucidation of
any appreciable differences in the efficacy of these peptides. The other prepared tetrapeptides 5
and 6 and all the tripeptides 7-9 were not active in the physiological range of concentrations
(data not shown). Two the most potent in this assay tetrapeptides 1 and 2 were taken for further
evaluation. Here, cardiomyocytes were challenged by LPS administration (5 µg/ml) for 5 h. The
inflammatory stimuli enhanced CK and LDH levels (Figure 1E). Interestingly, both peptides 1
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and 2 blocked LPS-induced hypersecretion of these apoptotic markers at both 10 and 20 µM.
Once more, 1 displayed a higher potency in comparison with 2, being restored by both enzymes
to normal levels at a concentration of 10 µM. In fact, the most active peptide (1), compared with
another four active peptides, has no charge and is soluble only in DMSO. Thus, it might have
allowed it to penetrate into cells without a cell penetration domain, which was originally used by
Piao et al.16
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Next, the effect of the two most active primary cardiomyocites (peptides 1 and 2) in an
immune system cell was tested. Usually, following a LPS challenge, TLR4 is highly expressed in
the leukocyte–immune bone marrow-derived macrophages. Thus, these cells were chosen as a
model for determining the possible effect of compounds 1 and 2 in immune system cells. Human
macrophages were stimulated by two doses of LPS (100 ng/ml and 20 µg/ml), followed by flow
cytometric analysis (FACS) of the TLR4 expression in treated and non-treated immune system
cells by 1 and 2 cells. P6 was used as a positive control. All three peptides were administered 30
min before introducing LPS to the medium for another 4 h. Suppl. Figure 1 A, B, and C show
that administering both test compounds in the presence and absence of LPS shifts the TLR4
expression level to the left and lowers the intensity of the signal under the baseline level.
However, no difference was observed between low and high concentrations of LPS. The main
result of this experiment validates that 1 and 2 (and P6 as a positive control) abolish
physiological TLR4 elevation following LPS introduction and also decrease the TLR4
expression in non-stimulated macrophages (Suppl. Figure 1).
An almost identical effect of both compounds in LPS stimulated and non-stimulated
macrophages might be explained by the suggestion that 1 and 2 bind to TLR4, which is activated
at a basic level by other factors, for example, oxidative and environmental stress. However,
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keeping human macrophages out of the blood stream using tissue cultures does not represent
physiological conditions. Thus, the observed TLR4 stimulation occurred even without the
presence of LPS. Therefore, we concluded that the effect of both peptides might be not mediated
by LPS activation. Moreover, the lack of any appreciable difference between the inhibitory
effects of 1 and 2 in macrophages treated by two LPS doses indicates that the effect is not
correlated and is not associated with the level of LPS activation.
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Design and synthesis of peptidomimetics. Both the most active tetrapeptides
[
Ac-IVFA-NH ] (1) and [Ac-VFAE-NH ] (2) have a very similar amino acid sequence. Thus, we
2 2
used [VFA] and [I-X-FA] sequences that are present in 1 and [VFA], which is presented in 2, as
the basis for designing peptidomimetics of general type A (Scheme 2). Based on visual similarity
with side chains of isoleucine, valine, phenylalanine, and alanine, these moieties were initially
“cyclized” to the nine-membered cyclic α-tripeptides B, which in the second iteration could be
transformed to the cytosines A. Taking into account that 9-membered monocyclic α-tripeptides
of type B bearing 3 different alkyl substituents in the ring are scarcely known, hardly available31
and, probably, are metabolically unstable, this stage of the study was omitted in favor of the
faster access to more promising cytosine peptidomimetics of type A.
A straightforward synthesis of peptidomimetics A is shown in Scheme 3. In the first step,
a commercially available, inexpensive 2,4-dichloro-5-methyluracil (10) was refluxed with
sodium ethoxide (2 equiv.) in dry ethanol for 15 min to give 2,4-diethoxy-5-methyluracil (11) as
3
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32
described by D. Pluskota et al. However, in our hands the original procedure provided only a
poor yield of 11 (18%). Accordingly, the original procedure was slightly modified by increasing
the amount of EtONa to 3 equiv and elongation the reflux time to 60 min. Under these
conditions, the common for all the peptidomimetics A precursor 11 was obtained in reasonable
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7% yield. In the next step, 2,4-diethoxy-5-methyluracil (11) was subjected to N(1)-benzylation
with benzyl, p-trifluoromethylbenzyl, and p-nitrobenzyl bromides as well as with 2-
bromomethylnaphthalene according to a classical Hilbert-Johnson reaction33-34 to give the
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corresponding 1-benzyl-4-ethoxy-5-methylpyrimidin-2(1H)-ones 12-15 in variable yields (27-
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5%). The benzylations discussed here were carried out in dry THF at 50 C or in acetonitrile at
o
5 C (for the least reactive p-nitrobenzyl bromide only) in the presence of excess lithium iodide
(3 equiv) which considerably facilitated the reactions. Perhaps, being a source of iodide-anion
lithium iodide played a dual role in these processes: (i) it activated benzylating reagents through
in situ bromine to iodine exchange; and (ii) it promoted more facile dealkylation in the 1-benzyl-
2,4-diethoxypyrimidinium intermediates by a more nucleophilic iodide-anion compared to
bromide-anion. Either applying of elevated temperature or lacking of iodide-anion source was
found to be counterproductive favoring the competitive rearrangement of 11 to 1-ethyl-4-ethoxy-
5-methylpyrimidin-2(1H)-one.
Surprisingly, neither classical Hilbert-Johnson-type33-34 N(1)-benzylation of 2,4-
35
dialkoxypyrimidines with simple benzyl bromides nor N(1)-allylation with allyl bromides have
been explored sufficiently.36,37 Even the simplest N-benzylation of 2-methoxypyridine with
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benzyl bromides to give the 1-benzylpyrid-2(1H)-one has been reported once only. In practice,
the classical Hilbert-Johnson-type33,34 N(1)-alkylations of 2-alkoxypyridines proceed
successfully with primary alkyl iodides, but are rather invalid with less reactive alkyl bromides.39
Accordingly, our efforts on N(1)-alkylation of 11 with benzyl and propargyl chlorides, even in
the presence of LiI, failed.
40
Ammonolysis and, less frequently, aminolysis of N(1)-substituted 4-alkoxy- and 4-
aryloxypyrimidin-2(1H)-ones serve as efficient method for synthesis of cytosines in nucleosides
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41
and acyclic nucleosides. Indeed, in the last step of our synthetic route, aminolysis of N(1)-
benzylated-4-ethoxypyrimidin-2(1H)-ones 12-15 with primary amines smoothly afforded the
designed 4-alkylamino-derivatives 16-25 through aromatic nucleophilic substitution of ethoxy-
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group (Scheme 3). For achieving efficient aminolysis, the reactions were conducted for 3 days at
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30 C in pressure tubes in methanol with a big excess of the amine as a co-solvent. The NMR-
and HPLC-pure target peptidomimetics 16-18, 21, 23 and 24 were isolated by means of flash
chromatography on silica gel in high yield (84-90%). The aminolysis of 12 with n-decylamine
proceeded efficiently as well, however some troubles associated with its purification
significantly decreased the yield of the pure n-decyl-cytosine 22 (23%). As nucleophilicity of
benzylamine is lower than that of alkylamines, the aminolysis of 12 with BnNH was not
2
o
completed even after 3 days at 130 C, and the N-benzylamino-derivative 20 was isolated in 60%
yield only.
In vitro evaluation of peptidomimetics. All synthesized peptidomimetics were
tested in vitro as described above. They were unable to protect cardiomyocytes robustly and to
reduce the levels of CK and LDH in the medium under LPS stimulation (data not shown). We
believe that such a phenomenon might be explained by the fact that robust and significant
stimulation of the cells by LPS was not obtained even in several of identical experiments.
Namely, LPS did not work properly and repeatedly even with prolonged cell exposures of up to
24 and 48 h. In contrast, in the cardiomyocytes when a minimal inflammatory response to LPS
was detected, the protective effect of compounds 17 and 21, by reducing the CK level, was also
detected (Suppl. Figure 2). Thus, we focused on the research direction that robustly provided
modest, but significant and reproducible results: a cell protective effect under hypoxia. Two
compounds, 17 and 21, displayed moderate efficacy in cardiomyocyte protection and reduced the
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level of both enzymes under hypoxic conditions at a concentration of 10 µM. Compound 21 led
to a further significant decrease of CK and LDH also at 20 µM (Figure 2A). At a 5 µM
concentration, compound 21 was inactive; however, at 30 µM, the effect was identical to 20 µM,
and at a 50 µM concentration, compound 21 negatively affected the vitality of the tissue cultures.
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The mRNA levels of TLR4 and IL-1β were measured in cardiomyocytes pretreated with
1
7 or 21 under hypoxic conditions. Hypoxia caused a significant elevation in the level of mRNA
for TLR4 and IL-1β. In addition, a significant reduction in the mRNA levels of TLR4 and IL-1β
was observed only in those cells treated by compound 21 (Figure 2B and C, respectively). The
latter inhibited the hypoxia induced elevation of mRNA levels of TLR4 and IL-1β,
approximately by 3.1 and by 15-fold, respectively. Compound 17 was less effective: 1.5- and
1
.4-fold inhibition, respectively. Thus, the possible effect of only 21 on the protein expression of
TLR4, adaptor protein TRAM (interaction with this protein was targeted by the design of
compound 21) and its downstream targets: interferon response factor 3 (IRF3) and nuclear factor
of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB-α) - the inhibitor of NF-κB
under identical experimental conditions was investigated. Figure 2D shows that indeed, 21
significantly decreased the expression of TLR4, TRAM, and IRF3 proteins ~2.7-fold, ~2.8-fold,
and ~2.7-fold, respectively, while increasing the expression of IκB-α by ~3.3-fold. Such a
prominent elevation of IκB-α expression will result in increased inhibition of NF-κB
translocation to the nucleus; therefore, it has both an anti-inflammatory and anti-apoptotic effect.
In Figure 2E the mechanism underlying cellular transduction of the TLR4 receptor is shown
schematically. Based on these in vitro results, only 21 was chosen as a leading compound for
further evaluation. We also stopped investigating the possible effect of peptidomimetics in the
LPS-stimulated immune cells, encouraged by their promising results in cardiomyocytes under
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hypoxia conditions. We also took into consideration that the effects of peptides 1 and 2 were not
directly mediated by LPS activation.
The direct interaction between 21 and TLR4 in vitro was investigated by using a co-IP
approach (Figure 3). An antibody against TLR4 was used to pull down the receptor from
cardiomyocytes that were first treated by 21. After washing and elution, the level of 21 was
evaluated using liquid chromatography / tandem mass spectrometry (LC-MS/MS) for analysis.
The analysis of the parent molecule (327 g/mole), followed by collision-induced dissociation
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(CID), serves as a valuable tool for identifying desired target molecules in a complex matrix.
Combining: a) retention time; b) parent ion; and c) fragments provides a definitive method for
compound identification.
+
Optimization of the LC-MSMS parameters for the pure 21 (328 [M+H] ) dissolved in
acetonitrile-water displayed a retention time of 5.8 min, and the CID led to fragmentation at the
benzylic position and the release of a benzylic ion of 91 m/z and 236 m/z. Injection of the treated
cell sample under the same conditions resulted in the same parameters of retention time and
masses, as shown by EIC of MS/MS of 328 to 236 to 91 (Figure 3A&C).
The exact amount/fraction of 21 that binds to TLR4 was not determined due to the small
concentration of IP samples. Based on the IP/MS results, we concluded that 21 might directly
interact with the TLR4 complex. However, we cannot rule out the possibility that some
adaptor/regulatory proteins can precipitate through weak interactions with TLR4. Thus, binding
of 21 to adapters or to other indirectly precipitated proteins may account for the positive MS
signal.
In vivo evaluation of 21. Since 21 has not shown robust effect in LPS-treated
cardiomyocytes, in vivo validation of the effect of 21 was conducted in an MI animal model. MI
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in mice was mimicked by left anterior descending coronary artery ligation (LAD), a well-
established model. Compound 21 was dissolved in 40% polyethylene glycol (PEG) in water and
was administered IV to C57BL mice, 1 h before LAD. It was given to mice in three doses: 0.05,
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.3, and 1 mg/kg. The blood levels of four serum markers were investigated. The levels of three
markers (CK, LDH, and troponin T) were correlated with MI severity. The blood level of the
fourth; protein aspartate aminotransferase (AST) was used as a marker of normal liver functions.
All three specific MI markers were significantly reduced by 21 by 0.3 mg/kg dose (Figure 4A).
In addition, it is important to mention that 21 did not affect the AST level in all three doses used;
therefore, no damage to the liver function was observed by the treatment. Moreover, the heart
infarct size was significantly reduced, as shown in Figure 4B. Next, a time course experiment
with one very effective dose of 21: 0.3 mg/kg was conducted. Three time intervals before LAD
were chosen for 21 administration: 30 min, 1 h, and 2 h. The design of the experiment was
identical to the previously one described above. The results shown in Figure 4C indicated that
the optimal time for 21 administration was indeed 1 h. Only at this time point were all three MI
blood markers significantly reduced. In addition, at this time point the compound reduced the
infarct size (Figure 4D).
Typically, MI is a pathological situation, which occurs suddenly and without a real possibility to
provide preventive cardioprotective treatment to patients. Thus, any cardioprotective drug must
be administered only after MI is diagnosed. Therefore, after finding the optimal dose (0.3 mg/kg)
and time interval (1 h before the ligation), the next step was to determine whether 21 will also be
cardioprotective following LAD, by mimicking the real MI in humans. Thus, 21 (0.3 mg/kg) was
injected IV 1 h after ligation and blood CK and LDH levels were tested both following 24 h and
7
days. Figure 5A shows very impressive down regulation of blood levels of cardiac damage
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markers (CK and LDH) at both time points. Interestingly, the infarct size in treated mice was not
positively affected after 24 h. However, following 7 days, there was a significant reduction of the
infarct size in the treated group, as shown in Figure 5B. In addition, a histological evaluation of
the myocardial tissue using Hematoxylin/Eosin (HE) and Masson’s Trichrome (MT) staining
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(left ventricle) revealed massive damage, as seen by leukocyte infiltration (Figure 5D - I, II) and
fibrosis (Figure 5D - III, IV) observed in control mice, which did not develop in the treated
group.
Finally, the most impressive result obtained using this system was the marked increase in
the survival rate of compound 21-treated animals (100% survival rate). All mice treated by the
compounds were alive after 7 days following LAD. In contrast, 3 out of 6 mice died in the first
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4 h in the non-treated group. In addition, 2 mice died during days 5-7. In total, 5 out of 12 mice
died, which gives a 58% survival rate of the non-treated group (Figure 5E).
In silico evaluation of possible binding mode of 21. Finally, to support the
experimental findings we used computational tools to search for putative binding sites for
compounds 21 on the TIR domain of TLR4. Since no information is available on the binding site
of the adapter protein on TLR4 (recall that we wish to disrupt the adapter protein – TLR4
interaction), we used the SiteMap program to search the entire TIR domain for potential sites.
Since protein-protein interactions typically involve surface complementarity rather than deep
buried cavities, we focused our search on the surface of TLR4. Five potential sites were
identified (see Figure 6A) which together cover almost the entire protein.
Docking all the compounds studied in this work into all sites, we found that only one site
(marked in red in Figure 6A) led to a good correlation between docking scores and biological
activities (Table 1 and Suppl. Figure 3). Interestingly, this site is located in close vicinity to the
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BB loop, which is known to play a critical role in TLR4’s association with adapter proteins and
in the activation of TLRs in general, and of TLR4 in particular.16 Therefore, binding of
compounds to this particular site will likely block adapter proteins from binding to TLR4, and
consequently, may inhibit its signaling pathway.
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Figure 6B presents the binding modes of the active compounds, peptidomimetic 21 and
peptide 1 to their suggested site (Figure 6D). It is evident that the two compounds possess
different binding modes; however, they share the same region in the vicinity of a small cavity.
Compound 21 (Figure 6C) completely occupies this cavity with its long linear aliphatic chain
(left panel), whereas peptide 1 (Figure 6C) cannot occupy the cavity due to its larger function
groups (right panel).
For this binding site, both GlideScore and Emodel score are reasonably well correlated
with the biological activities (R2 values of 0.56 and 0.57 (following the removal of two data
points in the latter case), respectively). The overall more negative (i.e., favorable) scores
obtained for the peptides in comparison with the peptidomimetics likely reflects their larger
number of functional groups and consequently the larger number of interactions they make with
the binding site. Nonetheless, it is clear that Glide docking can separate active from inactive
molecules at this site. We therefore hypothesize that this site represents the best option for the
binding of the compounds studied in this work to TLR4. We are aware that some recent in silico
models present a different mode of the assembly of TLR signaling complexes42,43 that suggest
that adapter recruitment to activated TLRs is not through a symmetrical, mutual interaction of
BB loops of two interacting TIR domains. However, we started this project in 2013 and used for
the modeling models that were the most updated for that time, with relatively high TLR4 TIR
and TLR10 TIR domains that share ~32% sequence identity.
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Table 1. Biological activities and docking scores to the most probable site for the compounds
studied in this work.
Compound Glide gscore
Glide Emodel Inhibition %
R²
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21
17
-3.42
-3.09
-2.91
-2.68
-2.51
-1.19
-45.11
-40.64
-48.58
-35.55
-46.87
-38.11
35.00
28.00
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[
[
[
[
[
[
IVFA] (1)
VFAE] (2)
VFA] (8)
IFA] (9)
-6.30
-5.72
-5.15
-5.11
-4.78
-4.75
-4.69
-4.59
-3.54
-71.86
-68.72
-59.18
-55.13
-65.60
-50.52
-64.92
-58.82
-74.41
79.00
57.00
19.00
38.00
54.00
34.00
21.00
0.00
0.69
FAEM] (3)
FAE] (7)
[AEMP] (4)
[EMPC] (5)
[MPCG] (6)
0.00
CONCLUSIONS
Based on the structure of the TLR4 inhibitory peptide, a novel potent, structurally simple
cardioprotector 21 was discovered. The readily available 1-benzyl-5-methyl-4-(n-
octylamino)pyrimidin-2(1H)-one (21) exhibited an impressive cardioprotective effect in mice
using the MI model. The peptidomimetic 21 considerably decreased MI blood markers and
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caused 100% surveillance of treated animals. Compound 21 might be used as a starting point for
developing a novel cardioprotective chemotherapy which relies on inhibition of TLR4 in
cardiomyocytes.
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EXPERIMENTAL SECTION
General Chemistry Methods. The H, 13C, 19F NMR, and 2D spectra were
1
recorded at 25 °C on a Bruker Avance NMR spectrometer (Vernon Hills, IL) operating at 400
and 600 MHz and were in accordance with the assigned structures. The spectra are reported in
ppm units (δ) and are referenced to TMS at 0 ppm. The samples were prepared by dissolving the
synthesized compounds in CDCl (δ = 7.26 ppm), D O (δ = 4.79 ppm), DMSO-d (δ = 2.50
3
H
2
H
H
4
4
ppm), acetone-d (δ = 2.05 ppm), and CD CN (δ = 1.94 ppm). The splitting pattern
6
H
3
H
abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; qui, quintet; m,
unresolved multiplet, and br, broad. High-resolution mass spectra (HRMS) were obtained on a
QTOF instrument (Agilent), using electrospray ionization (ESI). Melting points were measured
with a Fisher–Johns melting point apparatus (Waltham, MA, USA). Column flash
chromatography (FC) was performed on silica gel 60 (230–400 mesh ASTM) from Merck.
Analytical TLC was carried out on pre-coated Silica gel 60F254 (Merck) sheets using UV
absorption, iodine, and vanillin physical adsorption for visualization.
Analytical and preparative HPLC (Young Lin Instruments, Anyang, Korea) were
performed on LUNA C18(2) (10 μm, 250 mm × 21.2 mm) column, from Phenomenex, Inc.
(Torrance, CA). The final biological tested compounds displayed ≥95% purity (confirmed using
analytical HPLC).
Acetonitrile and doubly distilled water were used as an eluent in different ratios. Dry
THF was prepared by reflexing over sodium/benzophenone following distillation. Regular
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commercially available reagents and solvents were purchased from several companies: Sigma-
Aldrich, Holand-Moran, Acros Organic, Alfa Aesar, Bio-Lab Ltd, Merck, and IU-CHEM LTD,
and were used without additional purification.
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Rink resin-based solid-state synthesis. The peptides were synthesized by using a
solid phase Fmoc strategy. Synthesis was performed using 0.40 mmol Rink amide resin (0.52
mmol/ g loading), which was shaken at rt for 30 min. The resin was loaded with the first amino
acid (5 equiv) using O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate
(HBTU) (480 mg/ 1.25 mmol) and N,N-Diisopropylethylamine (DIEA) (0.22 ml/ 1.25 mmol)
and was shaken at rt for 2 h. Coupling yield was checked by a ninhydrine test that was performed
after each coupling set. Fmoc protecting groups were removed by treatment with a 20%
piperidine/DMF solution for 20 min. The remaining amino acid (5 equiv) was coupled using
HBTU (480 mg/ 1.25 mmol) and DIEA (0.22 ml/ 1.25 mmol) and was shaken at rt for 1 h.
Finally, deprotection of Boc and OtBu and the release of the crude peptides were carried out by
using a solution containing 95% TFA, 2.5% t-isopropanol-silane, and 2.5% H O. To ensure that
2
during the synthesis the desired compounds were obtained, we performed MS. All peptides were
purified using preparative HPLC and were characterized by additional HRMS. The most in vitro
active peptides (1 and 2) were also characterized by NMR.
(2S,3S)-2-acetamido-N-((S)-1-(((S)-1-(((S)-1-amino-1-oxopropan-2-yl)amino)-1-oxo-
1
3
-phenylpropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3-methylpentanamide (1): HNMR:
(
600 MHz, DMSO-d), δ (ppm): 0.72-0.74 (m, 9H, CH(CH ) , CH(CH )Et), 0.78 (t, J= 7.2 Hz,
H 3 2 3
3
H, CH(CH )CH CH ), 1.01-1.07 (m, 1H, CH(CH )CH CH ), 1.18 (d, J= 6.6 Hz, 3H,
3 a,b 3 3 a,b 3
NHCHCH ), 1.35-1.45 (m, 1H, CH(CH )CH CH ), 1.63-1.69 (m, 1H, CH(CH )Et), 1.84 (s, 3H,
3
3
a,b
3
3
COCH ), 1.84-1.90 (m, 1H, CH(CH ) ), 2.79 (dd, J= 14.1, 9.0 Hz, 1H, CH Ar), 3.01 (dd, J=
3
3 2
a,b
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4.1, 4.8 Hz, 1H, CH Ar), 4.09 (dd, J= 7.8, 7.8 Hz, 1H, NHCHPr-i), 4.13-4.18 (m, 2H,
a,b
NHCHBu-sec, NHCHMe), 4.52-4.56 (m, 1H, CHCH Ar), 6.99 (br s, 1H, NH ), 7.13 (br s, 1H,
a,b
2
NH ), 7.14-7.17 (m, 1H, p-H ), 7.22-7.23 (m, 4H, o,m-H ), 7.70 (br d, J= 8.4 Hz, 1H,
2
Ar
Ar
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
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3
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NHCHPr-i), 7.92-7.95 (m, 2H, NHCHBn, NHAc), 7.98-7.99 (br d, J= 7.8 Hz, 1H, NHCHMe).
1
3
CNMR: (150 MHz, DMSO-d) δ, ppm, 10.82 (CH(CH )CH CH ), 15.24 (CH(CH )Et), 18.01
3
a,b
3
3
(
CH(CH3)2),
18.21
(NHCHCH3),
18.99
(CH(CH3)2),
22.35
(COCH3),
24.31
(CH(CH )CH CH ), 30.43 (CH(CH ) ), 36.05 (CH(CH )Et), 37.05 (CH Ar), 47.92
3 a,b 3 3 2 3 a,b
(NHCHMe), 53.42 (CHCH Ar), 56.85 (NHCHBu-sec), 57.54 (NHCHPr-i), 126.08 (p-C ),
a
,
b
A
r
1
27.89 (o-C ), 128.94 (m-C ), 137.56 (C ), 169.08 (COCH ), 170.23 (COCHBn), 170.61
Ar Ar Ar 3
(COCHPr-i), 170.98 (COCHBu-sec), 173.78 (CONH ). HRMS (ESI): calcd. for C H N O [M
2
25 40
5
5
+
+
H ]: 490.3024; found: 490.3028.
(4S,7S,10S,13S)-7-benzyl-13-carbamoyl-4-isopropyl-10-methyl-2,5,8,11-tetraoxo-
1
3,6,9,12-tetraazahexadecan-16-oic acid (2): HNMR: (400 MHz, DMSO-d), δ (ppm): 0.70 (d,
H
J= 6.8 Hz, 3H, CH(CH ) ), 0.76 (d, J= 6.8 Hz, 3H, CH(CH ) ), 1.16 (d, J= 7.2 Hz, 3H,
3
2
3 2
NHCHCH ), 1.82-1.85 (m, 5H, COCH , COCH CH ), 1.90-1.94 (m, 1H, CH(CH ) ), 1.95-2.18
3
3
2
2
3 2
(m, 2H, COCH CH ), 2.88-2.94 (m, 1H, CH Ar), 3.14-3.19 (m, 1H, CH Ar), 3.92-3.98 (m,
2 2 a,b a,b
1
1
H, NH COCH), 3.99-4.03 (m, 1H, NHCHPr-i), 4.10-4.18 (m, 1H, NHCHMe), 4.46-4.52 (m,
2
H, NHCHAr), 6.91 (br s, 1H, NH ), 7.13 (br s, 1H, NH ), 7.14-7.16 (m, 1H, p-H ), 7.20-7.27
2
2
Ar
(m, 4H, o,m-H ), 8.20 (d, J= 7.2 Hz, 1H, NHCHCH ), 8.84 (br s, 1H, NHCOCH ), 9.20 (br s,
Ar 3 3
1
3
1H, NHCHAr), 9.57 (br s, 1H, NHCHCONH ). CNMR: (100 MHz, DMSO-d) δ, ppm, 17.13
2
(NHCHCH ), 18.47 (CH(CH ) ), 19.21 (CH(CH ) ), 22.44 (COCH ), 27.51 (COCH CH ), 30.16
3 3 2 3 2 3 2 2
(CH(CH ) ), 34.63 (COCH CH ), 36.41 (CH Ar), 48.94 (NHCHMe), 54.13 (NH COCH), 54.99
3
2
2
2
a,b
2
(NHCHAr), 59.09 (NHCHPr-i), 126.02 (p-C ), 127.93 (o-C ), 129.17 (m-C ), 138.17 (C ),
Ar
Ar
Ar
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69.41 (COCH ), 170.42 (COCHBn), 171.43 (COCHPr-i, COCHCH ), 173.85 (CONH ), 177.46
3
3
2
+
(COOH). HRMS (ESI): calcd. for C H N O [M + H ]: 506.2609; found: 506.2618.
24 36 5 7
(S)-4-((S)-2-((S)-2-acetamido-3-phenylpropanamido)propanamido)-5-(((S)-1-amino-
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2
3
4
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9
0
4
-(methylthio)-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (3): HRMS (ESI): calcd. for
+
C H N O S [M + H ]: 538.2330; found: 538.2330.
2
4
36
5
7
(4S)-4-((S)-2-acetamidopropanamido)-5-(((2S)-1-(2-carbamoylpyrrolidin-1-yl)-4-
(methylthio)-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (4): HRMS (ESI): calcd. for
+
C H N O S [M + H ]: 488.2174; found: 488.2173.
2
0
34
5
7
(4S)-4-acetamido-5-(((2S)-1-(2-(((S)-1-amino-3-mercapto-1-oxopropan-2-
yl)carbamoyl)pyrrolidin-1-yl)-4-(methylthio)-1-oxobutan-2-yl)amino)-5-oxopentanoic acid
+
(5): HRMS (ESI): calcd. for C H N O S [M + H ]: 520.1894; found: 520.1892.
2
0
34
5
7 2
1
-(acetyl-L-methionyl)-N-((S)-1-((2-amino-2-oxoethyl)amino)-3-mercapto-1-
oxopropan-2-yl)pyrrolidine-2-carboxamide (6): HRMS (ESI): calcd. for C H N O S [M +
1
7
30
5
5 2
+
H ]: 448.1683; found: 448.1682.
(S)-4-((S)-2-((S)-2-acetamido-3-phenylpropanamido)propanamido)-5-amino-5-
+
oxopentanoic acid (7): HRMS (ESI): calcd. for C H N O [M + H ]: 407.1925; found:
19 27
4
6
4
07.1926.
N-terminus acetylation. A mixture of 1.90 ml of acetic anhydride, 0.90 ml DIEA,
and 0.60 ml of 1M of 1-Hydroxybenzotriazole (HOBt) in NMP solution, and an additional 6.60
ml of NMP were mixed in a flask. The Fmoc-protecting group was removed from the N-terminus
by a 20% piperidine/DMF solution. The degree of deprotection was evaluated by a ninhydrine
test. Next, 5 ml of the acetylating solution was added to the solid-state synthesis syringe and it
was shaken for 30 min at rt. This procedure was carried out twice (with another 5 ml of the
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acetylation mix). Then, other protecting groups were deprotected as described in the peptide
synthesis procedure and crude peptides were released from the resin.
Synthesis of peptidomimetics:
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
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3
3
4
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5
5
5
5
5
5
5
5
5
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1
2
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5
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2
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2
,4-Diethoxy-5-methyluracil (11). Compound 10 (12.35 mmol, 2.00 g) was dissolved in
10 ml of dry ethanol and a solution of sodium ethoxide (37.04 mmol) in 27 ml ethanol was
slowly added. The reaction mixture was warmed up to about 60 °C for 1 h. Next, the sodium
chloride was filtered off; ethanol was removed by reduced pressure to afford a crude material.
The crude material was dissolved again in ethyl ether, washed with water and with brine, dried,
and the solvent was removed under reduced pressure to yield 11 (1.73 g, 77%). A beige solid,
1
mp 123-125 °C; R = 0.15 (DCM/hexane, 6:4). HNMR: (400 MHz, CDCl ), δ (ppm): 1.30 (t,
f
3
H
J= 7.2 Hz, 3H, COCH CH ), 1.32 (t, J= 7.2 Hz, 3H, MeCCOCH CH ), 1.95 (d, J= 0.8 Hz, 3H,
2
3
2
3
CH -Ar), 4.27 (q, J= 7.2 Hz, 2H, MeCCOCH CH ), 4.34 (q, J= 7.2 Hz, 2H, COCH CH ), 7.86
3
2
3
2
3
(
(
d, J= 0.8 Hz, 1H, CH-Ar). 13CNMR: (100 MHz, CDCl ) δ, ppm, 11.81 (CH -Ar), 14.35
3 3
MeCCOCH CH ), 14.49 (OCH CH ), 62.31 (OCH CH ), 62.97 (MeCCOCH CH ), 110.80
2
3
2
3
2
3
2
3
(MeC), 156.83 (NCH), 163.58 (NCN), 169.21 (OCCMe). HRMS (ESI): calcd. for C H N O
2
9
15
2
+
[
M + H ]: 183.1128; found: 183.1133.
1
-Benzyl-4-ethoxy-5-methylpyrimidin-2(1H)-one (12). To the 11 (5.49 mmol, 1 g),
lithium iodide (16.47 mmol) and benzyl bromide (17.02 mmol) in 40 ml of dry THF were
quickly added, under an inert atmosphere. The reaction mixture was warmed up to about 50 °C
overnight. Then the reaction mixture was poured into a 300 ml solution of saturated sodium
bicarbonate and stirred for 1h. Thereafter, the solution was extracted with DCM and to the
organic layer, sodium thiosulfate (8.20 g) and sodium sulfate were added and the suspension was
stirred overnight. Next, the mixture was filtered and the solvent was removed under reduced
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2
2
2
2
2
2
2
2
3
3
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3
3
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3
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pressure to yield an oily residue. Purification of the residue by SiO chromatography yielded 12
2
1
(
1.27 g, 95%). A white solid, mp 123-125 °C; R = 0.41 (DCM/ethanol, 97:3). HNMR: (400
f
MHz, CDCl ), δ (ppm): 1.37 (t, J= 6.8 Hz, 3H, CH CH ), 1.895 (d, J= 0.8 Hz, 3H, CH Ar), 4.45
3
H
2
3
3
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
(
q, J= 7.2 Hz, 2H, CH Me), 5.02 (s, 2H, CH Ar), 7.11 (d, J= 0.8 Hz, 1H, NCH), 7.29-7.35 (m,
2 2
1
3
5
H, CH Ar). CNMR: (100 MHz, CDCl ) δ, ppm, 12.26 (CH Ar), 14.38 (CH CH ), 52.35
2 3 3 2 3
(CH Ar), 63.37 (CH Me), 105.20 (MeC), 128.31 (p-C ), 128.40 (o-C ), 129.10 (m-C ), 136.34
2 2 Ar Ar Ar
(
C ), 143.37 (NCH), 157.13 (C=O), 170.38 (COEt). HRMS (ESI): calcd. for C H N O [M +
Ar
14 17
2
2
+
H ]: 245.1285; found: 245.1283.
4
-Ethoxy-5-methyl-1-(4-nitrobenzyl)pyrimidin-2(1H)-one (13). To the 11 (4.62 mmol,
0.84 g), lithium iodide (13.87 mmol) and 4-nitrobenzyl bromide (14.33 mmol) in 40 ml of dry
MeCN were quickly added, under an inert atmosphere. The reaction mixture was warmed up to
about 75 °C overnight. Then the reaction mixture was poured into a 300 ml solution of saturated
sodium bicarbonate and stirred for 1h. Thereafter, the solution was extracted with DCM and to
the organic layer, sodium thiosulfate (8.20 g) and sodium sulfate were added and the suspension
was stirred overnight. Next, the mixture was filtered and the solvent was removed under reduced
pressure to yield a yellow solid. Purification of the residue by SiO chromatography yielded 13
2
1
(
0.37 g, 27%). A white solid, mp 78-80 °C; R = 0.55 (DCM/ethanol, 97:3). HNMR: (400 MHz,
f
CDCl ), δ (ppm): 1.39 (t, J= 7.2 Hz, 3H, CH CH ), 1.93 (s, 3H, CH Ar), 4.48 (q, J= 7.2 Hz, 2H,
3
H
2
3
3
CH Me), 5.11 (s, 2H, CH Ar), 7.17 (s, 1H, NCH), 7.48 (d, J= 8.4 Hz, 2H, CH CCH ), 8.20 (d,
2
2
2
Ar
1
3
J= 8.4 Hz, 2H, CH CNO ). CNMR: (100 MHz, CDCl ) δ, ppm, 12.30 (CH Ar), 14.34
Ar
2
3
3
(CH CH ), 52.09 (CH Ar), 63.69 (CH Me), 106.03 (MeC), 124.26 (CH CNO ), 128.80
2 3 2 2 Ar 2
(
CH CCH ), 143.12 (NCH), 143.60 (CH C ), 147.88 (CNO ), 156.82 (C=O), 170.76 (COEt).
2
Ar
2
Ar
2
+
HRMS (ESI): calcd. for C H N O [M + H ]: 290.1135; found: 290.1139.
1
4
16
3
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-Ethoxy-5-methyl-1-(4-(trifluoromethyl)benzyl)pyrimidin-2(1H)-one (14). To the
oily residue of 11 (1.65 mmol, 0.30 g), lithium iodide (4.94 mmol) and 4-(trifluoromethyl)benzyl
bromide (5.11 mmol) in 10 ml of dry MeCN were quickly added, under an inert atmosphere. The
reaction mixture was warmed up to about 75 °C overnight. Then the reaction mixture was poured
into a 300 ml solution of saturated sodium bicarbonate and stirred for 1h. Thereafter, the solution
was extracted with DCM and to the organic layer, sodium thiosulfate (8.20 g) and sodium sulfate
were added and the suspension was stirred overnight. Next, the mixture was filtered and the
solvent was removed under reduced pressure to yield an oily residue. Purification of the residue
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
by SiO chromatography yielded 14 (0.38 g, 74%). A beige solid, mp 99-101 °C; R = 0.27
2
f
1
(hexane/ethyl acetate, 1:1). HNMR: (400 MHz, CDCl ), δ (ppm): 1.40 (t, J= 7.2 Hz, 3H,
3
H
CH CH ), 1.91 (d, J= 1.2 Hz, 3H, CH Ar), 4.47 (q, J= 7.2 Hz, 2H, CH Me), 5.08 (s, 2H, CH Ar),
2
3
3
2
2
7
.15 (d, J= 1.2 Hz, 1H, NCH), 7.43 (d, J= 8.0 Hz, 2H, CH CCH ), 7.60 (d, J= 8.0 Hz, 2H,
2 Ar
1
3
CH CCF ). CNMR: (100 MHz, CDCl ) δ, ppm, 12.25 (CH Ar), 14.34 (CH CH ), 52.10
Ar
3
3
3
2
3
(
CH Ar), 63.53 (CH Me), 105.68 (MeC), 124.07 (q, J= 272.1 Hz, CF ), 126.01 (q, J= 3.6 Hz,
2
2
3
CH CCF ), 128.43 (CH CCH ), 130.55 (q, J= 32.6 Hz, CCF ), 140.40 (CH C ), 143.23
Ar
3
2
Ar
3
2
Ar
1
9
(
NCH), 156.92 (C=O), 170.60 (COEt). FNMR: (376 MHz, CDCl ), δ (ppm): -63.12. HRMS
3 H
+
(
ESI): calcd. for C H F N O [M + H ]: 313.1158; found: 313.1171.
1
5
16
3
2
2
4
-Ethoxy-5-methyl-1-(naphthalen-2-ylmethyl)pyrimidin-2(1H)-one (15). To the oily
residue of 11 (1.10 mmol, 0.20 g), lithium iodide (3.30 mmol) and 2-(bromomethyl)naphthalene
(3.40 mmol) in 10 ml of dry MeCN were quickly added, under an inert atmosphere. The reaction
mixture was warmed up to about 50 °C overnight. Then the reaction mixture was poured into a
3
00 ml solution of saturated sodium bicarbonate and stirred for 1h. Thereafter, the solution was
extracted with DCM and to the organic layer, sodium thiosulfate (8.20 g) and sodium sulfate
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were added and the suspension was stirred overnight. Next, the mixture was filtered and the
solvent was removed under reduced pressure to yield an oily residue. Purification of the residue
by SiO chromatography yielded 15 (0.13 g, 41%). An orange solid, mp 92-96 °C; R = 0.26
2
f
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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8
9
0
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9
0
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8
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1
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8
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0
1
(
toluene/ethyl acetate, 1:1). HNMR: (400 MHz, CDCl ), δ (ppm): 1.25 (t, J= 7.2 Hz, 3H,
3 H
CH CH ), 1.73 (s, 3H, CH Ar), 4.34 (q, J= 7.2 Hz, 2H, CH Me), 5.04 (s, 2H, CH Ar), 7.08 (d, J=
2
3
3
2
2
1
.2 Hz, 1H, NCH), 7.30 (dd, J= 8.4, 1.6 Hz, 1H, 3-CNaPhH), 7.33-7.38 (m, 2H, 6,7-CNaPhH), 7.62
1
3
(
s, 1H, 1-CNaPhH), 7.67-7.69 (m, 3H, 4,5,8-CNaPhH). CNMR: (100 MHz, CDCl ) δ, ppm, 11.99
3
(
CH Ar), 14.17 (CH CH ), 52.19 (CH Ar), 63.10 (CH Me), 105.04 (MeC), 125.81 (3-CNaPhH),
3
2
3
2
2
1
26.23 (6-CNaPhH), 126.37 (7-CNaPhH), 127.17 (1-CNaPhH), 127.62 (5-CNaPhH), 127.77 (8-
CNaPhH), 128.82 (4-CNaPhH), 132.89 (10-CNaPh), 133.18 (9-CNaPh), 133.68 (2-CNaPh), 143.38
+
(
NCH), 156.99 (C=O), 170.18 (COEt). HRMS (ESI): calcd. for C H N O [M + H ]:
1
8
19
2
2
2
95.1441; found: 295.1437.
General procedure for 16-25:
1
2/13/14/15 (0.50 mmol) was placed in a pressure tube and dissolved in 10 ml of
methanol. Next, 15 ml of appropriate amine (see Scheme 3) was added and the reaction mixture
was warmed up to about 130 °C for 3 days. In the next step, the reaction was allowed to reach rt,
and the methanol was removed under reduced pressure, yielding an oily residue. Distillation and
purification of the residue by SiO chromatography yielded 16-25.
2
1
-Benzyl-4-(sec-butylamino)-5-methylpyrimidin-2(1H)-one (16): (0.12 g, 90%). A
1
beige solid, mp 96-100 °C; R = 0.15 (hexane/ethyl acetate, 1:4). HNMR: (400 MHz, CDCl ),
f
3
δ (ppm): 0.93 (t, J= 7.6 Hz, 3H, CH CH ), 1.19 (d, J= 6.4 Hz, 3H, CHCH ), 1.50-1.60 (m, 2H,
H
2
3
3
CH CH ), 1.82 (d, J= 0.8 Hz, 3H, CH Ar), 4.36-4.46 (m, 1H, CHMe), 4.50 (br d, 1H, NH), 4.99
2
3
3
13
(
s, 2H, CH Ar), 6.92 (d, J= 0.8 Hz, 1H, NCH), 7.26-7.36 (m, 5H, CH Ar). CNMR: (100 MHz,
2
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CDCl ) δ, ppm, 10.24 (CH CH ), 12.96 (CH Ar), 20.39 (CHCH ), 29.53 (CH CH ), 47.35
3
2
3
3
3
2
3
(
CHMe), 51.80 (CH Ar), 101.56 (MeC), 127.87 (p-C ), 128.26, 128.83 (o,m-C ), 137.00
2 Ar Ar
(CH C ), 141.16 (NCH), 157.32 (C=O), 162.59 (CNH). HRMS (ESI): calcd. for C H N O [M
2
Ar
16 22
3
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
+
+
H ]: 272.1757; found: 272.1758.
1
-Benzyl-4-(isopentylamino)-5-methylpyrimidin-2(1H)-one (17): (0.12 g, 84%). A
1
white solid, mp 101-103 °C; R = 0.13 (hexane/ethyl acetate, 1:4). HNMR: (400 MHz, Acetone-
f
D), δ (ppm): 0.91 (d, J= 6.8 Hz, 6H, CH(CH ) ), 1.50 (dt, J= 7.6 Hz, 7.2 Hz, 2H, CH CH CH),
H
3 2
2
2
1
4
.60-1.70 (m, 1H, CH(Me) ), 1.88 (d, J= 1.2 Hz, 3H, CH Ar), 3.45-3.50 (m, 2H, CH CH CH),
2 3 2 2
1
3
.92 (s, 2H, CH Ar), 7.23-7.37 (m, 1H, NCH), 7.23-7.37 (m, 5H, CH Ar). CNMR: (100 MHz,
2
2
Acetone-D) δ, ppm, 13.10 (CH Ar), 23.00 (CH(CH ) ), 26.75 (CH(Me) ), 39.04 (CH CH CH),
3
3 2
2
2
2
3
9.83 (CH CH CH), 52.28 (CH Ar), 102.37 (MeC), 128.31 (p-C ), 128.89 (o-C ), 129.43 (m-
2 2 2 Ar Ar
C ), 139.55 (CH C ), 142.86 (NCH), 157.17 (C=O), 164.46 (CNH). HRMS (ESI): calcd. for
Ar
2
Ar
+
C H N O [M + H ]: 286.1914; found: 286.1916.
1
7
24
3
1
-Benzyl-4-(cyclopentylamino)-5-methylpyrimidin-2(1H)-one (18): (0.13 g, 90%). A
1
beige solid, mp 152-154 °C; R = 0.10 (hexane/ethyl acetate, 1:4). HNMR: (600 MHz, DMSO),
f
δ (ppm): 1.46-1.54 (m, 4H, CHCH CH ), 1.64-1.72 (m, 2H, CHCH CH ), 1.83 (s, 3H,
H
a,b
a,b
a,b
a,b
CH Ar), 1.86-1.93 (m, 2H, CHCH CH ), 4.30-4.40 (m, 1H, CHNH), 4.82 (s, 2H, CH Ar),
3
a,b
a,b
2
1
3
6
.68 (d, J= 7.2 Hz, 1H, NH), 7.25-7.33 (m, 5H, CH Ar), 7.47 (s, 1H, NCH). CNMR: (150
2
MHz, DMSO) δ, ppm, 12.85 (CH Ar), 23.52 (CHCH CH ), 31.76 (CHCH CH ), 50.88
3
a,b
a,b
a,b
a,b
(CH Ar), 51.54 (CHNH), 101.31 (MeC), 127.20 (p-C ), 127.45 (o-C ), 128.39 (m-C ), 138.15
2
Ar
Ar
Ar
(
CH C ), 142.22 (NCH), 155.70 (C=O), 162.69 (CNH). HRMS (ESI): calcd. for C H N O [M
2 Ar 17 22 3
+
+
H ]: 284.1757; found: 284.1761.
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-Benzyl-4-((3-(dimethylamino)propyl)amino)-5-methylpyrimidin-2(1H)-one (19):
1
(
0.14 g, 90%). A yellow oil; R = 0.14 (TEA/methanol/ethyl acetate, 5:20:75). HNMR: (400
f
MHz, CD CN), δ (ppm): 1.77-1.83 (m, 2H, CH CH CH ), 1.83 (d, 3H, J= 1.2 Hz, CH Ar), 2.37
3
H
2
2
2
3
0
1
2
3
4
5
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7
8
9
0
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2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
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4
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7
8
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0
1
2
3
4
5
6
7
8
9
0
(
s, 6H, N(CH ) ), 2.59 (t, J= 6.4 Hz, 2H, CH N(Me) ), 3.47 (m, 2H, NHCH ), 4.86 (s, 2H,
3 2 2 2 2
CH Ar), 7.22 (d, J= 0.8 Hz, 1H, NCH), 7.25-7.36 (m, 5H, CH Ar). 13CNMR: (100 MHz,
2
2
CD CN) δ, ppm, 12.97 (CH Ar), 25.93 (CH CH CH ), 40.72 (NHCH ), 44.82 (N(CH ) ), 52.61
3
3
2
2
2
2
3 2
(
CH Ar), 58.16 (CH N(CH ) ), 103.27 (MeC), 128.46 (p-C ), 128.54 (o-C ), 129.59 (m-C ),
2 2 3 2 Ar Ar Ar
1
39.14 (CH C ), 142.95 (NCH), 157.56 (C=O), 164.69 (CNH). HRMS (ESI): calcd. for
2 Ar
+
C H N O [M + H ]: 301.2023; found: 301.2024.
17 25
4
1-Benzyl-4-(benzylamino)-5-methylpyrimidin-2(1H)-one (20): (0.09 g, 60%). A beige
1
solid, mp 120-122 °C; R = 0.21 (hexane/ethyl acetate, 1:4). HNMR: (400 MHz, DMSO), δ
f
H
(
ppm): 1.88 (s, 3H, CH Ar), 4.56 (d, J= 6.0 Hz, 2H, NHCH ), 4.83 (s, 2H, NCH ), 7.20-7.34 (m,
3 2 2
1
3
10H, CH Ar), 7.53 (s, 1H, NCH), 7.58 (br t, J= 8.0 Hz, 1H, NH). CNMR: (100 MHz, DMSO)
2
δ, ppm, 12.72 (CH Ar), 43.08 (NHCH ), 50.95 (NCH ), 101.26 (MeC), 126.52 (NH-p-C ),
3
2
2
Ar
1
1
27.02 (NH-o-C ), 127.21 (N-p-C ), 127.48 (N-o-C ), 128.10 (NH-m-C ), 128.38 (N-m-C ),
Ar Ar Ar Ar Ar
38.02 (NCH C ), 139.54 (NHCH C ), 142.57 (NCH), 155.64 (C=O), 163.05 (CNH). HRMS
2
Ar
2
Ar
+
(
ESI): calcd. for C H N O [M + H ]: 306.1601; found: 306.1610.
19 20 3
1
-Benzyl-5-methyl-4-(n-octylamino)pyrimidin-2(1H)-one (21): (0.15 g, 88%). A
1
yellow oil; R = 0.15 (hexane/ethyl acetate, 1:4). HNMR: (400 MHz, DMSO), δ (ppm): 0.85 (t,
f
H
J= 7.2 Hz, 3H, CH CH ), 1.25 (m, 10H, (CH ) Me), 1.50 (m, 2H, NHCH CH ), 1.81 (s, 3H,
2
3
2 5
2
2
CH Ar), 3.29 (m, 2H, NHCH ), 4.81 (s, 2H, CH Ar), 7.00 (t, J= 5.6 Hz, 1H, NH), 7.24-7.34 (m,
3
2
2
1
3
5
H, CH Ar), 7.46 (s, 1H, NCH). CNMR: (100 MHz, DMSO) δ, ppm, 12.77 (CH Ar), 13.93
2 3
(CH CH ), 22.06 (CH Me), 26.49, 28.59, 28.65, 28.79 (NHCH (CH ) ), 31.22 (CH CH Me),
2
3
2
2
2 4
2
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0.16 (NHCH ), 50.90 (CH Ar), 101.32 (MeC), 127.22 (p-C ), 127.46 (o-C ), 128.41 (m-C ),
2
2
Ar
Ar
Ar
38.19 (CH C ), 142.14 (NCH), 155.78 (C=O), 162.97 (CNH). HRMS (ESI): calcd. for
2
Ar
+
C H N O [M + H ]: 328.2383; found: 328.2386.
2
0
30
3
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
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3
3
4
4
4
4
4
4
4
4
4
4
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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
-Benzyl-4-(n-decylamino)-5-methylpyrimidin-2(1H)-one (22): (0.04 g, 23%). A
1
yellow oil; R = 0.43 (hexane/ethyl acetate, 1:4). HNMR: (400 MHz, CDCl ), δ (ppm): 0.80 (t,
f
3
H
J= 6.4 Hz, 3H, CH CH ), 1.15-1.25 (m, 14H, (CH ) Me), 1.51 (m, 2H, NHCH CH ), 1.76 (s, 3H,
2
3
2 7
2
2
CH Ar), 3.43 (m, 2H, NHCH ), 4.89 (s, 2H, CH Ar), 5.05 (br t, J= 4.8 Hz, 1H, NH), 6.84 (s, 1H,
3
2
2
1
3
NCH), 7.17-7.25 (m, 5H, NCH Ar). CNMR: (100 MHz, CDCl ) δ, ppm, 12.96 (CH Ar), 14.12
2
3
3
(CH CH ), 22.68 (CH Me), 27.01 (NHCH CH CH ), 29.31 (NHCH CH ), 29.34, 29.39, 29.58
2 3 2 2 2 2 2 2
((CH ) CH CH Me), 31.89 (CH CH CH ), 41.20 (NHCH ), 51.76 (CH Ar), 101.98 (MeC),
2
4
2
2
2
2
3
2
2
1
1
27.80 (p-C ), 128.09, 128.77 (o,m-C ), 137.08 (CH C ), 141.03 (NCH), 157.31 (NC=O),
Ar
Ar
2
Ar
+
63.16 (CNH). HRMS (ESI): calcd. for C H N O [M + H ]: 356.2696; found: 356.2708.
2
2
34
3
5-Methyl-1-(4-nitrobenzyl)-4-(n-octylamino)pyrimidin-2(1H)-one (23): (0.06 g,
1
3
0%). A yellow oil; R = 0.43 (DCM/ethanol, 97:3). HNMR: (400 MHz, CDCl ), δ (ppm): 0.86
f
3
H
(t, J= 7.2 Hz, 3H, CH CH ), 1.20-1.30 (m, 10H, (CH ) Me), 1.59 (m, 2H, NHCH CH ), 1.87 (s,
2 3 2 5 2 2
3
H, CH Ar), 3.52 (m, 2H, NHCH ), 4.98 (br s, 1H, NH), 5.04 (s, 2H, CH Ar), 6.96 (s, 1H,
3 2 2
1
3
NCH), 7.44 (d, J= 8.4 Hz, 2H, CH CCH ), 8.14 (d, J= 8.4 Hz, 2H, CH CNO ). CNMR: (100
2
Ar
Ar
2
MHz, CDCl ) δ, ppm, 12.99 (CH Ar), 14.16 (CH CH ), 22.72 (CH Me), 27.06
3
3
2
3
2
(NHCH CH (CH ) ), 29.29 (NHCH CH ), 29.39, 29.79 (NHCH CH (CH ) ), 31.87
2 2 2 3 2 2 2 2 2 3
(CH CH Me), 41.39 (NHCH ), 51.69 (CH Ar), 102.57 (MeC), 124.04 (CH CNO ), 128.63
2
2
2
2
Ar
2
(CH CCH ), 141.04 (NCH), 144.59 (CH C ), 147.61 (CNO ), 157.02 (C=O), 163.42 (CNH).
2 Ar 2 Ar 2
+
HRMS (ESI): calcd. for C H N O [M + H ]: 373.2234; found: 373.2231.
2
0
29
4
3
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