Design, Synthesis, and Biological Evaluation of Itaconic Acid Derivatives as Potential Anti-Influenza Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b01683

Influenza A viruses (IAVs) have caused worldwide epidemics and pandemics by reassortment and generation of drug-resistant mutants, which render antivirals and current vaccinations no longer usable. In this study, an itaconic acid derivative 1 was identified from a chemical library of 20 000 compounds, by performing a cell-based screening assay, as a lead agent exhibiting anti-influenza A activity. Accordingly, a series of itaconic acid derivatives were designed and synthesized by adopting a rational design strategy to obtain more potent anti-influenza agents. The results of an in vitro pharmacological study showed that compounds 4 and 8 exhibited the most potent anti-IAV effect with half-maximal effective concentration values of 0.14 and 0.11 μM, respectively, in Madin-Darby canine kidney cells. The mechanism of action studies showed that lead agents 1 and 4 reduced virus replication by directly targeting IAV nucleoproteins and disrupting virus ribonucleoprotein export from the nucleus to the cytosol. On the basis of its high potential as an anti-IAV agent and its selectivity index >785, compound 4 was found to be a promising candidate for further development against IAVs.

Full text of DOI:10.1021/acs.jmedchem.8b01683

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Article
Design, synthesis, and biological evaluation of itaconic
acid derivatives as potential anti-influenza agents
Bidyadhar Sethy, Chung-Fan Hsieh, Ta-Jen Lin, Po-Yuan Hu, Yu-Li Chen,
Chia-Yi Lin, Sung-Nain Tseng, Jim-Tong Horng, and Pei-Wen Hsieh
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01683 • Publication Date (Web): 12 Feb 2019
Downloaded from http://pubs.acs.org on February 13, 2019
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Design, synthesis, and biological evaluation of itaconic acid derivatives as
potential anti-influenza agents
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‡
‡
‡
‡
‡
Bidyadhar Sethy , Chung-Fan Hsieh , Ta-Jen Lin , Po-Yuan Hu , Yu-Li Chen , Chia-Yi Lin ,
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Sung-Nain Tseng , Jim-Tong Horng‡,§,║,#,*, Pei-Wen Hsieh†,┴,#,∇,
§
*
†Graduate Institute of Biomedical Sciences, Division of Natural Products, College of Medicine,
Chang Gung University, Taoyuan, 33302, Taiwan.
‡Department of Biochemistry and Molecular Biology, Chang Gung University, Taoyuan, 33302,
Taiwan.
§Research Center for Emerging Viral Infections, Chang Gung University, Taoyuan, 33302,
Taiwan.
║Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital, Chang Gung
University, College of Medicine, Linkou, 333, Taiwan.
┴Graduate Institute of Natural Products, School of Traditional Chinese Medicine, College of
Medicine, Chang Gung University, Taoyuan, 33302, Taiwan.
#Research Center for Chinese Herbal Medicine, Chang Gung University of Science and
Technology, Taoyuan, 33303, Taiwan.
∇Department of Anesthesiology, Chang Gung Memorial Hospital, Linkou, 333, Taiwan.
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Abstract
Influenza A viruses (IAVs) have caused worldwide epidemics and pandemics by reassortment
and generation of drug-resistant mutants, which renders antivirals and current vaccinations no
longer usable. In this study, an itaconic acid derivative 1 was identified from a chemical library
of 20,000 compounds by performing a cell-based screening assay as a lead agent exhibiting anti-
influenza A activity. Accordingly, a series of itaconic acid derivatives were designed and
synthesized by adopting a rational design strategy to obtain more potent anti-influenza agents.
The results of an in vitro pharmacological study showed that compounds 4 and 8 exhibited the
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most potent anti-IAV effect with EC values of 0.14 and 0.11 µM, respectively, in Madin–
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Darby canine kidney cells. Mechanisms of action studies showed that lead agents 1 and 4
reduced virus replication by directly targeting IAV nucleoproteins and disrupting virus
ribonucleoprotein export from nucleus to cytosol. On the basis of its high potential as an anti-
IAV agent and its selectivity index > 785, compound 4 was found to be a promising candidate
for further development against IAVs.
Keywords: Anti-influenza A; drug discovery; itaconic acid derivatives; Michael-acceptor
moiety; nuclear export inhibitor.
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INTRODUCTION
Influenza A virus (IAV) is a negative-sense single-stranded RNA virus that belongs to the family
of Orthomyxoviridae. IAV is the most virulent human pathogen and causes severe diseases, with
infants, elderly people, pregnant women, and people with weakened immune systems at high
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risk. The symptoms of influenza virus infections include fever, muscle aches, headaches,
vomiting, and diarrhea and influenza may cause pneumonia, myocarditis, encephalitis, or
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myositis, and sometimes even death. In the past century, five notorious influenza pandemics
occurred, which not only caused >45 million human deaths but also resulted in stupendous
economic losses due to their high rates of morbidity and mortality.3-6 Currently, H1N1, H3N2,
H5N1, and H7N9 subtypes of influenza A are highly aggressive seasonal flu strains that circulate
in human populations and are a significant threat to human health.7,8 To overcome these
pathogens and other emerging mutants, development of unique therapeutic strategies or drugs is
urgently needed.7-11
To prevent or reduce the influenza A virus infections, vaccination is generally recommended.12-14
For influenza therapy, two classes of antiviral drugs have been clinically used: virion ion-channel
(
M2 protein) inhibitors (amantadine and rimantadine) and neuraminidase (NA) inhibitors
oseltamivir and zanamivir). However, IAVs have developed resistance to these anti-influenza
(
agents.6,15-18 Consequently, there is an urgent need to develop agents with different mechanisms
of action on viral life cycles.
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Since influenza viruses replicate, transcript, and generate viral proteins, such as virus
ribonucleoproteins (vRNPs) in the nuclei of infected cells, and then transport them to the
cytoplasm, transfer of nuclear viral materials is a critical step in the influenza virus life cycle.19,20
In this step, the well-known host chromosomal region maintenance 1 (CRM1; also referred to as
Exportin 1 or XPO1) can interact with nuclear export signal (NES)-containing viral nuclear
export proteins, which are then exported to the cytoplasm.21-24 This CRM1 is an important
protein involved in the nuclear export process that regulates cell proliferation, cell-cycle
progression, and apoptosis and is related to many diseases, such as cancer and viral
infection.20,21,25,26 Accordingly, development of nuclear export inhibitors (NEIs) to block this
pathway could be useful to provide a new therapeutic strategy against influenza.19,20
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In an effort to explore new lead compounds for anti-influenza A agents, large-scale screening of
a chemical library containing > 20,000 compounds against IAV was performed by using a cell-
based anti-cytopathic effect (CPE) assay. Our first hit was N,N'-bis(2,4-difluorophenyl)-2-
methylenesuccinamide (1, Figure 1), an itaconic acid derivative that exhibited anti-influenza
activity with a half-maximal effective concentration (EC ) of 2.84 µM. This compound is the
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first itaconic acid derivative to show anti-influenza activity. We then synthesized 25 itaconic acid
derivatives, and their effects against IAV were evaluated. Subsequently, structure–activity
relationships (SARs) were established. Furthermore, since the chemical structure of 1 bears a
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Michael-acceptor moiety, which is a privileged structure for NEIs, the mechanism of action of
against IAV was examined.
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RESULTS AND DISCUSSION
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The chemical structure of 1 consists of two 2,4-di-fluoro-phenyl moieties (Units A and B,
Figure 1) and an itaconic acid moiety (Unit C, Figure 1), which are linked together by two
amide bonds. Previous literature reports concluded that Unit C of 1 has a conjugated system
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that is able to react with the cysteine residue of the active site through Michael addition.21
Therefore, to discuss the correlation between the reactivity of the conjugated system in 1
toward the active site and its bioactivities, Units A and B were modified by varying their
aromatic substituents. Using an addition/elimination reaction mechanism, a one-step synthetic
route was developed (Scheme 1), which led to 25 itaconic acid derivatives. The purities of all
compounds (>96%) were evaluated by high-performance liquid chromatography (HPLC) (Suppl.
Table S1 and S2). Furthermore, the anti-IAV effects and cytotoxicities of these compounds
were determined in an influenza A (A/WSN/33) virus-infected Madin–Darby canine kidney
epithelial (MDCK) cells model.
SARs based on anti-IAV (H1N1) activity
To evaluate the antiviral activity and cytotoxicity of the itaconic acid derivatives, a CPE assay
and an MTT assay were performed in parallel (Table 1 & 2). Two nuclear export inhibitors,
leptomycin B (LMB) and verdinexor, were used as positive controls. The cytotoxic results (50%
cytotoxic concentration, CC ) indicated that, except for compounds 1, and 6-8, most of the
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derivatives were non-cytotoxic at 110 µM. Compounds 4, 7, 8, and 9 exhibited the most potent
effects on anti-IAV, with EC50 values of 0.14, 0.18, 0.11, and 0.48 µM, respectively. The
selectivity indices (SIs) of 4 and 9 were better than that of verdinexor, indicating that 4 and 9
could be good candidates against IAV. The SARs of the synthetics are discussed below on the
basis of their antiviral activities.
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Initially, we modified the para (p) position of the two aromatic rings (Units A and B) of the lead
agent 1 by introducing different electron-withdrawing groups (EWG) such as Cl, Br, or
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trifluoromethyl (–CF ), or an electron-donating group (EDG). The results showed that when p-F
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1) was replaced with p-Cl (2), p-Br (3), or p-CF (4) the antiviral activities were significantly
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improved by the presence of lipophilic (halo) groups, with an electron-withdrawing property at
the para position. Subsequently, to gain a better understanding of the electronic property at the
p-position, we introduced a methoxy group (-OCH ), which resulted in reduced activity. From
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these results, we concluded that the presence of a highly EWG (-CF ) at the p-position (Units A
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and B) improved the activity while decreasing the cytotoxicity. These effects were most likely
caused by the presence of the strong EWG at the p-position facilitating a Michael-type addition
and favoring the lipophilic interaction with the active core, which would increase the inhibitory
property with high SIs. Accordingly, we concluded that EWG substitution, compared with EDG
substitution, at the p-position improved antiviral activity.
We observed that moving the fluoro-group of Units A and B from the para to the meta position
(6) slightly enhanced antiviral activity but led to increased cytotoxicity. Similarly, replacing m-F
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6) with m-Cl (7) or m-CF (8) not only improved potency but also triggered cytotoxicity (Table
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). We also found that the compounds containing a p- or m-trifluoromethyl group (4 and 8)
exhibited 20–25-fold higher anti-IAV activity (Table 1). Since the Cys-539 residue is a prime
target located within the hydrophobic nuclear export sequences-binding region of CRM1, the
hydrophobic trifluoromethyl group may occupy the hydrophobic groove, being buried deeply
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into the active site, thereby promoting antiviral activity. To test our hypothesis, we performed
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molecular docking of compounds 4 and 8 in the active sites of LMB in CRM1 (PDB ID: 4HAT).
The results showed that the Michael-acceptor moiety (Unit C) of 4 and 8 (Figures 2A & 2B),
and the LMB lactone (Figure 2C) were closely fit at the active residue (Cys-539). Moreover, a
comparison of the docking poses of 4 (purple) and 8 (green) with the binding site of LMB (blue)
showed that an aromatic ring conjugated with the hydrophobic trifluoromethyl group of
compounds 4 and 8 was located in the hydrophobic groove of the LMB binding sites of CRM1
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(Figure 2D). Notably, 4 bound to the active site by hydrogen bonding with the Lys-579 residue
and 8 interacted with Lys-548 through hydrogen bonding, and the presence of Cys-539 helped
preserve their activities. Conversely, introducing an EDG, such as the methoxy (-OCH ) group
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(9), promoted antiviral activity with lower cytotoxicity (Table 1). Comparison of the data of 9
and 5 showed that the strong effect of a donating group at the meta position on the electron
density of the aromatic ring may concomitantly improve efficiency against IAV.
Next, to examine the effect of the substitution at the ortho position, the F group in 1 was replaced
with Br (10), which led to relatively similar activity and slightly reduced cytotoxicity (Table 1).
However, the antiviral activities completely vanished after introducing a CF group in the o-
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position (11 and 12) (Table 1). These results indicated that the presence of a strong EWG at the
o-position had a negative effect on activity. Moreover, two analogs (1 and 6) containing both m-
and p-F substitutions along with o-F on each aromatic ring exerted an inhibitory effect against
IAV, most likely because of the hydrophobic property of the F group promoting formation of pi-
alkyl hydrophobic interactions with the active site (Suppl. Figure S51A and S51B), which
would lead to activity retention.
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Extending the carbon chain between the amides and aromatic rings inhibited the effect against
IAV (Table 1), which suggested that 14 and 15 are unable to fit the active site. Furthermore, the
presence of a lone electron pair on the amide nitrogen atom may disturb the conjugate system
present on 14 (Figure 3A) or 15 by direct conjugation with the carbonyl carbon. This contrasts
with the case of 1 (Figure 3B) in which direct conjugation with the Michael-acceptor moiety
was hindered by the withdrawal property of the EWG, which resulted in a resonance effect on
the nitrogen lone pair that is drawn toward the benzene ring.
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It was previously reported that the high electronegativity of fluorine substitution can enhance
potency and affect target selectivity by affecting pK , lipophilicity, hydrophobic interactions, and
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drug metabolism. In addition, a fluoro atom at the o-position interacts with the amide proton (N–
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H), thereby leading to improved permeability. In synthetics possessing an F group at the o-
position, the interaction with N–H by intramolecular hydrogen bonding could have an effect on
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antiviral activity. Accordingly, H-NMR data showed that the NH signals were downfield shifted
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when an o-F group was present on the aromatic ring (Units A and B). The presence of different
substituents (EWG or EDG) on the aromatic ring counterbalanced the electron-withdrawing
effect of the o-F group by a resonance effect, which preserved the intramolecular hydrogen
bonding and anti-IAV activity. However, in systems containing CF where π-resonance is not
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possible, the inductive electron-withdrawing effect of fluorine could exert a powerful influence
on o-substituents and increase the chemical shift of the NH signals. On the other hand, extending
the carbon chain between the amide proton and aromatic ring could disrupt the conjugating
system and the N-H–F interaction, which might contribute to the reduced activity in 14 and 15
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IAV activities of these analogs.
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To further investigate the activity of Unit A, we synthesized 10 derivatives (1A–10A) of itaconic
acid by conjugating the aromatic ring only at the Unit A side and evaluated their anti-influenza A
activity. Additionally, the chemical structures of this series were confirmed by 2D NOESY
spectra. According to the results summarized in Table 2, all of these products were non-active,
which indicated that the presence of two aromatic rings (Units A and B) is necessary for anti-
influenza activity.
Since the two aromatic rings containing derivatives have similar structures, we initially selected
lead agent 1 to examine its effectiveness against different strains of influenza virus and other
viruses as well as to study the stage of action on influenza A virus.
Antiviral activity of compounds 1 & 4 on various strains of viruses
To evaluate the effectiveness of the itaconic acid analogues (1 and 4) against various strains of
viruses, we chose some strains especially those are clinically resistant to the drug Oseltamivir
(A/TW/7855/09 and A/TW/6663/09) and Swine Origin Influenza Virus (SOIV) strain caused
pandemic (A/TW/90206/09 and A/TW/2235/09) in last decade and also others those are endemic
in Taiwan in recent years. Cytotoxicity tests using 1 & 4 were conducted in different cell lines,
and the results were expressed as the median cytotoxic concentration (CC ). The half-maximal
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effective concentration (EC ) was used to express the inhibitory effects of 1 & 4 on different
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virus strains. Both of these compounds can effectively inhibit the replication of influenza virus in
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MDCK cells with high CC values. The inhibitory effect of 1 was satisfactory on many IAV
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strains, such as A/WSN/33 (H1N1), A/TW/90206/09 (H1N1pdm), A/TW/2235/09 (H1N1pdm),
A/TW/7855/09 (H1N1), A/TW/6663/09 (H1N1), and A/TW/3446/02 (H3N2), with EC values
5
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of approximately 2–5 μM (Table 3). It is worth noting that the A/TW/7855/09 (H1N1) and
A/TW/6663/09 (H1N1) strains are resistant to the drug oseltamivir, which suggests that 1 could
exert a therapeutic effect on virus strains that show resistance to oseltamivir. In addition, 1
exhibited a good inhibitory effect on the influenza B virus strains popular in recent years,
including B/TW/00482/13, B/TW/03384/13, B/TW/00642/14, and B/TW/01061/14, with EC50
values between 3 and 4 μM (Table 3) and greater SIs (>18). Further, the anti-influenza effect of
4 gave a five- to eight-fold higher activity for A/WSN/33 (H1N1) and A/TW/3446/02 (H3N2)
strains and slightly improved activity for A/TW/2235/09 (H1N1pdm) than those of compound 1
with higher SI values. Compounds 1 & 4 also had inhibitory effects on enterovirus A71, with an
EC value of approximately 4–7 μM, whereas it had no inhibitory effect on either enterovirus
5
0
D68 or rhinovirus, which are evaluated by using rhabdomyosarcoma (RD) cells. Although the
influenza virus has high variability, this does not affect the inhibitory effect of 1 on different
strains of influenza A and B type along with 4 on influenza A viruses. Thus, 1 & 4 show an
extensive spectrum of inhibition against influenza viruses, drug-resistant strains, and enterovirus
A71.
Microscopic observation of IAV-induced cytopathic effect inhibition by 1.
Virus-induced cytopathic changes were examined in MDCK cells, which were infected with
influenza virus A/WSN/33 with a multiplicity of infection (MOI) of 0.5 for 1 h, washed with
phosphate-buffered saline (PBS), then treated with 1 (10 M) and observed under a microscope
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post infection (pi) = 12 or 36 h (Figure 4). We found that the influenza virus caused some
cytopathic changes after 12 h and serious cytopathic effects with more round-up cells after 36 h
in the virus-only group. When co-treating drugs and viruses, we found that 1 could avoid
cytopathic phenomena caused by viral infection after 36 h, and the infection did not spread
through surrounding cells (Figure 4). With regard to toxicity, no cytotoxic effect caused by 1
was observed after 36 h. It seems reasonable to conclude that 1 has very low or no toxicity to
MDCK cells.
1
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Mechanism of action study: estimation of the inhibition period of 1 in A/WSN/33 by using
time-course test.
The influenza virus life replication/cycle occurs in multiple steps, which requires approximately
3
0
8
–10 h to complete. For the time-of-addition (TOA) assay, the following main steps are to be
considered: virus attachment, entry, uncoating, translation, replication, and release. In this study,
we performed a TOA experiment by using MDCK cells infected with A/WSN/33 (multiplicity of
infection, MOI = 0.1) and treated with 10 μM of 1. We designed this experiment according to the
indicated time points for drug treatments, and the virus was allowed to absorb at −1 to 0 h pi.
The supernatant was collected after 9 h for a plaque assay (Figure 5A). To examine whether
compound 1 targets host cell factor, the drug was treated 2 h prior to virus infection (−3 to 9 h pi;
complete cycle). The periods indicate virus attachment, and then virus entry or uncoating (−1 to
9 h and 0 to 9 h pi, respectively), the middle stage of virus cycle endocytosis, translation,
replication (3 to 9 h pi), and finally the assembly and release of progeny virus (6 to 9 h pi). From
the TOA experiment, we found that 1 mainly inhibited the virus during the early and middle
stage of replication, whereas it was less effective at the late stage relative to that in the virus only
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lane (Figure 5B). To verify the actual stage of inhibition, the following experiments, i.e.,
hemagglutinin inhibition (HAI) assay, viral RNA synthesis, and viral protein distribution test by
immunofluorescence staining, were performed.
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Effect of hemagglutinin (HA) activity in A/WSN/33 virus upon treatment with 1
In the early stage of viral replication, the virus attaches to the host cells by their surface
glycoprotein HA. From the TOA experiment, we found that 1 inhibited early steps of virus
replication. Therefore, we used the HAI assay to verify whether 1 blocked the early virus
attachment by inhibiting the hemagglutination of red blood cells (RBCs) caused by viral HA
protein. When the viral protein HA was used to contact the receptor on the surface of RBCs,
hemagglutination was generated to detect whether the drug inhibited HA activity. RBCs were
treated with various concentrations of 1 (two-fold serial dilution from 50 µM) and incubated
with influenza A/WSN/33 at 4 × HA on ice for 1 h. The results showed that 1 alone (upper row,
Figure 6) was not able to form agglutination similar to that of RBCs only (Mock, lane 12), and
virus treated with 1 could not inhibit the hemagglutination caused by viral HA (lower row,
Figure 6). Therefore, this result indicated that 1 did not inhibit the activity of HA, and there was
no significant correlation between 1 and influenza virus attachment and entry into cells.
Compound 1 did not interrupt viral RNA and protein synthesis
According to time-course experiments, 1 also inhibited the middle stages of viral replication;
therefore, it was necessary to investigate whether 1 inhibits viral RNA and protein synthesis.
After infecting MDCK cells with influenza virus (MOI = 0.1) for 1 hour, 1 was added, and cell
lysates were collected at pi = 3, 6, and 9 h for RNA and protein determination by reverse
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transcription-quantitative polymerase chain reaction (RT-qPCR) and immunoblotting assay
(
Figure 7A). From the RT-qPCR results depicted in Figure 7B, it can be seen that no significant
increase in viral RNA synthesis occurred at 0–3 h pi, whereas they showed an exponential rise at
–9 h pi. The M1 RNA levels indicated that 1 did not inhibit viral RNA synthesis. Additionally,
1
1
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the effect of 1 on viral protein synthesis was examined by western blotting as indicated by the
level of various viral proteins (Figure 7C). The results implied that 1 did not inhibit the
biosynthesis of viral proteins (NP, M1, HA, PA, NS2, and NS1). Thus, we confirmed that 1 did
not inhibit viral RNA and protein expression.
Effect of 1 on the subcellular distribution of viral proteins
We used immunofluorescence staining to detect the location of viral proteins in the cells to
examine whether 1 affected their distribution. Thus, the effect of 1 was evaluated by using
MDCK cells, which were infected with influenza virus A/WSN/33 (MOI = 0.1), and the infected
cells were treated with DMSO (mock; 0.05%) or 1 (10 μM), or leptomycin B (LMB; 10 nM) as
control group. Then, the cells were fixed after 9 h pi and stained using the specific antibodies.
From the results of IF staining, it can be seen that NP (Figures 8A & 8B) was transported into
the cytoplasm in case of influenza virus infection, but after 1 or LMB treatment, the NP of the
influenza virus was confined to the nucleus. In contrast, the viral proteins NS2 (Suppl. Figure
S52A), PA (Suppl. Figure S52B), and M1 (Suppl. Figure S52C) did not exhibit significant
differences in their nucleus/cytosol distribution after treatment with 1 or LMB compared with the
virus-only control (Suppl. Figure S52D). Notably, LMB is an NEI that acts as an antiviral and
anticancer agent, and its main function is to block the cargo/exportin transport from the nucleus
to cytoplasm by targeting the host cellular CRM1 protein. During the viral infection, the CRM1
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protein helps to localize viral proteins in appropriate positions and interrupts the cellular cargoes.
LMB efficiently binds the receptor sites of CRM1, resulting in inhibition of nuclear export of
viral NP by disturbing the nuclear export signal 3-dependent CRM1 binding. In addition, as can
be extracted from the literature, LMB has been shown to inhibit vRNP export while not affecting
the subcellular localization of other viral proteins, such as matrix protein 1 (M1) and non-
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1
structural protein 2 (NS2). As we hypothesized, 1 gave rise to a similar phenomenon (such as
NEI). The IF staining result indicated that 1 specifically inhibited the nuclear export of viral NP,
and PA also exhibited a different pattern in the presence of 1 because it surrounds the nucleus,
thereby preventing its release into the cytoplasm, but the subcellular distribution of M1 and NS2
proteins is not affected.
In addition, the mechanism of action for compound 4, which has the highest SI among all
itaconic acid derivatives presented here, was also investigated by performing the TOA assay,
HAI assay, and indirect immunofluorescence assay. TOA data indicated that 4 (1 μM) efficiently
inhibited the virus in the early and middle stage of replication, similar to 1 (10 μM) (Suppl.
Figure S53). Furthermore, 4 also did not inhibit the hemagglutination caused by viral HA, which
suggested that 4 does not inhibit the virus attachment (Suppl. Figure S54). Finally, the
immunofluorescence data revealed that the 4-treated group showed inhibition of the middle stage
of virus replication by specifically blocking subcellular distribution of viral NP to cytosol
(Figure 9), without affecting other viral proteins, such as M1 (Suppl. Figure S55). Since these
phenomena are similar to those of treatment with LMB and 1, it appears that the mechanism
underlying the activity of 4 is similar to that underlying 1.
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CONCLUSION
With the aim of developing a new class of anti-influenza agents, we designed and synthesized 25
itaconic acid derivatives based on the chemical structure of lead agent 1, and their activities
against IAV were evaluated in MDCK cells. The corresponding SARs of the derivatives were
also proposed according to the bioactivity results. Activity and cytotoxicity improved after
substitution with a highly EWG with larger size at the p-position of the aromatic rings. In
contrast, the presence of an EDG at the m-position lowered cytotoxicity. Bulky groups at the o-
position also caused a decrease in activity. Additionally, the presence of two aromatic rings
bonded to itaconic acid was crucial for activity retention. Among all compounds tested, 4
showed excellent activity (EC = 0.14 μM), with a high SI (>785). Further, the initial hit agent 1
1
1
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and its derivative 4 showed a broad spectrum of inhibition on different influenza strains along
with enterovirus A71 viruses. A preliminary mechanism study using the lead agents 1 and 4 was
conducted and showed that reduction of virus replication was caused by disruption of viral NP
export from the nucleus to the cytoplasm. This finding suggests that viral NP may be a possible
molecular target for these agents as NEIs, which presents a new opportunity for antiviral drug
development. Therefore, 4 and its active derivatives are promising anti-influenza agents that
should be further investigated in more detail.
EXPERIMENTAL SECTION
Chemistry: materials and methods
All of the chemical reagents and solvents were purchased from the commercial suppliers and
used without further purification unless stated otherwise. Thin-layer chromatography (TLC;
silica gel 60 F254 HX244089, Merck KGaA, Darmstadt, Germany) was used to monitor the
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®
chemical reactions, and the TLC plates were read under ultraviolet (UV) light (Entela UVGL-
2
5 254/365 nm; CA, USA). Compounds were purified by flash column chromatography on silica
®
gel (SiliaFlash G60, 70–230 mesh and Siliaflash P60, 230–400 mesh, Silicycle, QC, Canada). A
0
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1
Bruker AVANCE-400 MHz FT-NMR (Karlsruhe, Germany) instrument was used to measure H
1
3
and C NMR spectra at 400 MHz and 100 MHz, respectively. Deuterated solvents (CDCl and
3
acetone-d6) were used to calibrate the peaks, and trimethylsilane was used as internal standard
for CDCl . Mass spectra were measured on a TSQ quantum triple quadruple mass spectrometer,
3
Thermo Finnigan, CA, USA. A Hitachi U-2010 spectrophotometer (Tokyo, Japan) was used to
record UV spectra, and a JASCO FT/IR-4100 spectrophotometer (Tokyo, Japan) was used to
measure infrared spectra of all title compounds. Melting points were measured by using a MEL-
®
TEMP (Laboratory Devices, Inc., MA, USA) apparatus. A rotary vacuum evaporator (N-1100;
EYELA, Tokyo, Japan) was used for removal of solvents. HPLC purity analyses were conducted
by using a JASCO system: UV–VIS detector (UV-1575), autosampler (AS-1555–10), and pump
(
PU-1580), with UV detection at 230 nm, two different solvent systems (MeOH/H O and
2
MeCN/H O), and a reversed-phase column (Necleodur C18 HTec EC250/4.65 μm, Macherey-
2
Nagel, Duren, Germany). All target compounds had purities > 96% tested by HPLC.
Pan assay interference compounds (PAINS) analysis:
We examined all of the itaconic acid derivatives (1–15 and 1A–10A) for known classes of assay-
interference compounds. First, according to the “Free ADME/Tox Filtering Tool 4 (FAF-
Drugs4)” program (http://fafdrugs4.mti.univ-paris-diderot.fr/), compounds 1–15 and 1A–10A
were not perceived as PAINS. The database “Aggregator Advisor” (http://advisor.bkslab.org/)
showed that all of the synthetics were not aggregators. Further, all of the synthetics, neither as
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aggregators and/or PAINS, were verified by using the Bulk Pattern Checker tool
(http://zinc15.docking.org/patterns/home/). Thus, the activities provided herein are highly likely
not to have been caused by pan assay interference.
1
1
1
1
1
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General synthetic protocol for all title compounds (1–15) and (1A–10A)
To a solution of itaconyl chloride (1 equivalent) in anhydrous CH Cl with molecular sieve at −4
2
2
to 0C, halo-substituted aromatic aniline (2 equivalents) was added slowly by using a
micropipette. The reaction mixture was stirred at −4 to 0C for 1 h and then allowed to warm to
room temperature for 4 h to 1 d. After confirming the completion of the reaction by TLC, the
whole reaction mixture was filtered, and the filtrate was washed with CH Cl . The organic layer
2
2
was quenched with a small volume of H O, the two layers were separated by using a separation
2
funnel, and the organic layer was dried over Mg SO . Evaporation of the solvent afforded a
2
4
mixture of expected products, along with some impurities, according to TLC. The mixture was
purified by using silica gel column chromatography (hexane/ethyl acetate = 10/4) to obtain the
desired products.
N,N'-bis(2,4-difluorophenyl)-2-methylenesuccinamide (1)
ᴑ
-1
Yield of 17.1% as white solid; mp ( C): 132-133; IR ν (KBr) cm : 3385, 3247, 3232, 3090,
max
3028, 1665, 1616, 1518, 1430, 1263, 1223, 1143, 1098, 961, 853, 813, 628, 553, 490. UV λmax
MeOH
1
nm (Log Ɛ): 202.5 (4.5), 235.5 (4.1), 274.5 (3.8, sh); H-NMR (CDCl , 400MHz) δ
3
H
8
.73(1H, s, NH), 8.26-8.14 (3H, m, NH, H6ʹ, and H6ʹʹ), 6.93-6.82 (4H, m, H3ʹ, H3ʹʹ, H5ʹ and
1
3
H5ʹʹ), 5.99 (1H, s, H ), 5.83 (1H, s, H ), 3.49 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.4
b
a
3
C
(s, C4), 166.7 (s, C1), 159.3 (dd, J= 245.6, 11.5 Hz, C4ʹ), 158.9 (dd, J= 244.7, 11.4 Hz, C4ʹʹ),
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1
53.3 (dd, J= 245.7, 11.7 Hz, C2ʹ), 153.0 (dd, J= 246.1, 11.7 Hz, C2ʹʹ), 138.6 (s, C2), 123.5 (t,
C5), 123.4 (d, J= 9.1, Hz, C1ʹ), 123.2 (d, J= 9.1 Hz, C1ʹʹ), 122.8 (dd, J= 10.8, 10.8 Hz, C6ʹ),
122.3 (dd, J= 10.2, 10.2 Hz, C6ʹʹ), 111.5 (dd, J= 21.6, 3.7 Hz, C5ʹ), 111.2 (dd, J= 21.5, 3.7 Hz,
C5ʹʹ), 103.9 (dd, J= 23.0, 23.1 Hz, C3ʹ), 103.7 (dd, J= 23.1, 23.2 Hz, C3ʹʹ), 41.5 (t, C3). ESI-MS
0
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(
m/z): 375.2 [M+Na]+.
4
-((2,4-difluorophenyl)amino)-2-methylene-4-oxobutanoic acid (1A)
ᴑ
-1
Yield of 13.3% as white solid; mp ( C): 170-171; IR ν (KBr) cm : 3212, 3151, 3018, 2968,
max
2
900, 1708, 1649, 1550, 1512, 1432, 1361, 1305, 1263, 1226, 1169, 1144, 1099, 968, 910, 859,
580, 485, 437; UV λmax MeOH nm (Log Ɛ): 203.0 (4.4), 232.0 (4.1), 274.0 (3.3); H-NMR
1
(Acetone-d6, 400MHz) δ 8.99 (1H, s, NH), 8.19-8.12 (1H, m, H6ʹ), 7.06 (1H, ddd, J= 8.8, 8.4,
H
2
.8 Hz, H5ʹ), 6.96 (1H, dd, J= 8.8, 8.4 Hz, H3ʹ), 6.32 (1H, s, H ), 5.86 (1H, s, H ), 3.51 (2H, s,
b a
1
3
H3); C-NMR (Acetone-d6, 100MHz)δ 169.4 (s, C1), 168.2 (s, C4), 159.5 (dd, J= 242.2, 11.3
C
Hz, C2ʹ), 154.4 (dd, J= 245.4, 11.9 Hz, C4ʹ), 136.3 (s, C2), 128.8 (t, C5), 124.4 (dd, J= 10.9, 3.4
Hz, C1ʹ), 124.9 (d, J= 8.6 Hz, C6ʹ), 111.7 (dd, J= 21.6, 3.6 Hz, C5ʹ), 104.4 (dd, J= 23.7, 23.7 Hz,
–
–
C3ʹ), 40.8 (t, C3); ESI-MS (m/z): 239.9 [M-H] ; HRMS calculated for C H F NO , [M-H] ,
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8
2
3
2
40.0467; found 240.0476.
N,N'-bis(4-chloro-2-fluorophenyl)-2-methylenesuccinamide (2)
ᴑ
-1
Yield of 11.8% as white solid; mp ( C): 164-165; IR ν (KBr) cm : 3273, 3193, 3111, 3033,
max
3
009, 1657, 1611, 1537, 1492, 1413, 1343, 1199, 1077, 948, 901, 851, 813, 758, 575, 547, 473;
UV λmax MeOH nm (Log Ɛ): 203.5 (4.7), 246.0 (4.4), 280.0 (4.0, sh); H-NMR (CDCl , 400MHz)
1
3
δH 8.75 (1H, s, NH), 8.29 (1H, dd, J= 8.8, 8.4 Hz, H6ʹ), 8.24 (1H, dd, J= 9.2, 8.4 Hz, H6ʹʹ), 8.18
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(
1H, s, NH), 7.18-7.09 (4H, m, H3ʹ, H3ʹʹ, H5ʹ, and H5ʹʹ), 5.99 (1H, s, H ), 5.86 (1H, s, H ), 3.50
b
a
1
3
(2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.2 (s, C4), 166.6 (s, C1), 152.6 (d, J= 246.0 Hz,
3
C
C2ʹ and C2ʹʹ), 138.6 (s, C2), 129.9 (s, J= 9.9 Hz, C4ʹ), 129.2 (s, J= 9.9 Hz, C4ʹʹ), 125.4 (d, J=
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0.3 Hz, C6ʹʹ), 124.9 (d, J= 9.9 Hz, C6ʹ), 123.6 (t, C5), 122.7 (d, J= 15.5 Hz, C1ʹ, and C1ʹʹ),
16.0 (d, J= 22.6 Hz, C3ʹ), 115.8 (d, J= 22.5 Hz, C3ʹʹ), 42.3 (t, C3); ESI-MS (m/z): 407.3
1
+
+
+
[M+Na] , 409.2 [(M+2)+Na] ; HRMS calculated for C H Cl F N O Na, [M+Na] , 407.0136;
17 12 2 2 2 2
found 407.0150.
4
-((4-chloro-2-fluorophenyl)amino)-2-methylene-4-oxobutanoic acid (2A)
ᴑ
-1
Yield of 4.4% as white solid; mp ( C): 184-185; IR ν (KBr) cm : 3255, 3194, 3113, 3015,
max
2
889, 1668, 1523, 1410, 1359, 1297, 1224, 1165, 1077, 973, 907, 834, 583, 496, 466; UV λmax
MeOH
1
nm (Log Ɛ): 204.0 (4.5), 244.0 (4.2), 280.0 (3.5); H-NMR (Acetone-d6, 400MHz) δ 9.10
H
(1H, s, NH), 8.24 (1H, dd, J= 8.8, 8.4 Hz, H6ʹ), 7.27 (1H, dd, J= 11.2, 2.4 Hz, H3ʹ), 7.19 (1H,
13
ddd, J= 8.8, 2.8, 1.6 Hz, H5ʹ), 6.32 (1H, d, J=0.8 Hz, H ), 5.86 (1H, s, H ), 3.52 (2H, s, H3); C-
b
a
NMR (Acetone-d6, 100MHz)δ 169.5 (s, C1), 168.3 (s, C4), 153.7 (d, J= 253.8 Hz, C2ʹ), 136.3
C
(s, C2), 128.8 (t, C5), 127.1 (d, J= 10.6 Hz, C4ʹ), 125.4 (d, J= 3.2 Hz, C5ʹ), 124.3 (d, J= 11.9 Hz,
–
C6ʹ), 116.6 (d, J= 23.1 Hz, C1ʹ, and C3ʹ), 41.0 (t, C3); ESI-MS (m/z): 256.1 [M-H] , 258.0
–
–
[(M+2)-H] ; HRMS calculated for C H ClFNO , [M-H] , 256.0171; found 256.0181.
11 8 3
N,N'-bis(4-bromo-2-fluorophenyl)-2-methylenesuccinamide (3)
ᴑ
-1
Yield of 8.6% as white solid; mp ( C): 169-170; IR ν (KBr) cm : 3276, 3190, 3093, 3030,
max
2
997, 1677, 1657, 1608, 1534, 1487, 1408, 1340, 1271, 1194, 879, 809, 519, 476; UV λmax MeOH
1
nm (Log Ɛ): 203.0 (4.7), 248.0 (4.5), 279.5 (4.1, sh); H-NMR (CDCl , 400MHz) δ 8.77 (1H, s,
3
H
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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
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5
5
5
5
5
5
5
5
5
6
NH), 8.25 (1H, dd, J= 8.4, 8.4 Hz, H6ʹʹ), 8.20 (1H, dd, J= 8.8, 8.4 Hz, H6ʹ), 8.18 (1H, s, NH),
7
.32-7.23 (4H, m, H3ʹ, H3ʹʹ, H5ʹ and H5ʹʹ), 5.99 (1H, s, H ), 5.86 (1H, s, H ), 3.49 (2H, s, H3);
b a
1
3
C-NMR (CDCl , 100MHz) δ 168.2 (s, C4), 166.6 (s, C1), 152.6 (d, J= 246.9 Hz, C2ʹʹ), 152.4
3
C
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
(
d, J= 247.3 Hz, C2ʹ), 138.5 (s, C2), 128.1 (d, J= 3.7 Hz, C6ʹ), 127.8 (d, J= 3.6 Hz, C6ʹʹ), 125.9
d, J= 10.1 Hz, C1ʹ), 125.4 (d, J= 10.0 Hz, C1ʹʹ), 123.7 (t, C5), 123.0 (d, C5ʹ), 122.9 (d, C5ʹʹ),
(
1
18.8 (d, J= 22.2 Hz, C3ʹ), 118.6 (d, J= 22.1 Hz, C3ʹʹ), 116.8 (d, J= 9.5 Hz, C4ʹ), 116.0 (d, J= 9.2
+
+
Hz, C4ʹʹ), 42.3 (t, C3); ESI-MS (m/z): 497.1 [M+Na] , 499.1 [(M+2)+Na] ; HRMS calculated
+
for C H Br F N O Na, [M+Na] , 494.9126; found 494.9141.
1
7
12
2
2
2
2
4-((2-fluoro-4-(trifluoromethyl)phenyl)amino)-2-methylene-4-oxobutanoic acid (3A)
ᴑ
-1
Yield of 8.2% as white solid; mp ( C): 170-171; IR ν (KBr) cm : 3276, 3196, 3110, 3066,
max
3
025, 2916, 2887, 1677, 1631, 1526, 1429, 1334, 1224, 1174, 1131, 979, 914, 570, 456, 432; UV
λmax MeOH nm (Log Ɛ): 203.0 (4.5), 246.0 (4.3), 280.0 (3.5); H-NMR (Acetone-d6, 400MHz) δH
1
9
.33 (1H, s, NH), 8.53 (1H, dd, J= 8.4, 8.0 Hz, H6ʹ), 7.54-7.51 (4H, m, H3ʹ, and H5ʹ), 6.34 (1H,
1
3
d, J= 0.8 Hz, H ), 5.88 (1H, s, H ), 3.58 (2H, s, H3); C-NMR (Acetone-d6, 100MHz)δ 170.0
b
a
C
(s, C1), 168.2 (s, C4), 152.9 (d, J= 244.4 Hz, C2ʹ), 136.1 (s, C2), 131.8 (d, J= 10.3 Hz, C1ʹ),
1
1
29.1 (t, C5), 124.8 (qd, J= 269.2, 2.7 Hz, C7ʹ), 126.5-125.5 (m, C4ʹ), 122.7-122.6 (m, C5ʹ),
23.0 (d, C6ʹ), 113.3 (dq, J= 22.8, 3.7 Hz, C3ʹ), 41.1 (t, C3); ESI-MS (m/z): 289.7 [M-H]–;
–
HRMS calculated for C H F NO , [M-H] , 290.0435; found 290.0445.
12
8
4
3
N,N'-bis(2-fluoro-4-(trifluoromethyl)phenyl)-2-methylenesuccinamide (4)
ᴑ
-1
Yield of 8.8% as white solid; mp ( C): 160-161; IR ν (KBr) cm : 3305, 3261, 3116, 3025,
max
2943, 1669, 1627, 1529, 1504, 1428, 1336, 1175, 1127, 1066, 910, 884, 831, 662, 492, 447; UV
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λmax MeOH nm (Log Ɛ): 203.5 (4.8), 248.5 (4.5); H-NMR (CDCl , 400MHz) δ 8.92 (1H, s, NH),
1
3
H
8
.54 (1H, dd, J= 8.4, 8.0 Hz, H6ʹ), 8.49 (1H, dd, J= 8.4, 8.0 Hz, H6ʹʹ), 8.32 (1H, s, NH), 7.48-
1
3
7.34 (4H, m, H3ʹ, H3ʹʹ, H5ʹ, and H5ʹʹ), 6.04 (1H, s, H ), 5.92 (1H, s, H ), 3.55 (2H, s, H3); C-
b
a
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
NMR (CDCl , 100MHz) δ 168.4 (s, C4), 166.8 (s, C1), 152.0 (d, J= 244.3 Hz, C2ʹ), 151.8 (d,
3
C
J= 244.6 Hz, C2ʹʹ), 138.3 (s, C2), 129.8 (d, J= 10.3 Hz, C1ʹ), 129.3 (d, J= 9.8 Hz, C1ʹʹ), 127.3-
26.2 (m, C4ʹ, and C4ʹʹ), 123.4 (qd, J= 269.2, 2.7 Hz, C7ʹ, and C7ʹʹ), 124.2 (t, C5), 122.3-122.0
1
(
m, C5ʹ, and C5ʹʹ), 121.9 (d, C6ʹ), 121.6 (d, C6ʹʹ), 112.6 (dq, J= 22.4, 3.7 Hz, C3ʹ), 112.4 (dq, J=
–
2
2.3, 3.7 Hz, C3ʹʹ), 42.2 (t, C3); ESI-MS (m/z): 451.0 [M-H] ; HRMS calculated for
-
C H F N O , [M-H] , 451.0687; found 451.0693.
19 11
8
2
2
4
-((2,5-difluorophenyl)amino)-2-methylene-4-oxobutanoic acid (4A)
ᴑ
-1
Yield of 12.4% as white solid; mp ( C): 153-154; IR ν (KBr) cm : 3294, 3078, 3015, 2952,
max
2903, 2871, 1695, 1626, 1551, 1488, 1439, 1282, 1154, 977, 888, 808, 721, 524; UV λmax MeOH
1
nm (Log Ɛ): 203.0 (4.5), 237.0 (4.2), 275.0 (3.8); H-NMR (Acetone-d6, 400MHz) δ 9.17(1H, s,
H
NH), 8.14 (1H, ddd, J= 9.6, 6.4, 3.2 Hz, H6ʹ), 7.18 (1H, ddd, J= 9.2, 8.8, 5.2 Hz, H3ʹ), 6.86-6.80
1
3
(
1H, m, H4ʹ), 6.33 (1H, d, J=1.2 Hz, H ), 5.87 (1H, d, J=1.2 Hz, H ), 3.55 (2H, s, H3); C-NMR
b a
(
Acetone-d6, 100MHz)δ 169.8 (s, C1), 168.2 (s, C4), 159.5 (d, J= 237.1 Hz, C2ʹ), 149.7 (d, J=
C
2
9
39.7 Hz, C5ʹ), 136.1 (s, C2), 129.2 (dd, J= 12.4, 3.6 Hz, C1ʹ), 129.0 (t, C5), 116.6 (dd, J= 22.0,
.9 Hz, C3ʹ), 110.6 (dd, J= 24.6, 7.7 Hz, C4ʹ), 109.6 (dd, J= 29.3, 7.8 Hz, C6ʹ), 41.1 (t, C3); ESI-
–
–
MS (m/z): 240.2 [M-H] ; HRMS calculated for C H F NO , [M-H] , 240.0467; found 240.0475.
1
1
8
2
3
N,N'-bis(2-fluoro-4-methoxyphenyl)-2-methylenesuccinamide (5)
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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
ᴑ
-1
Yield of 8.1% as white solid; mp ( C): 144-145; IR ν (KBr) cm : 3275, 3238, 3028, 2946,
max
2
844, 1664, 1627, 1521, 1429, 1273, 1233, 1155, 1113, 1026, 941, 837, 808, 718, 595, 508, 476;
UV λmax MeOH nm (Log Ɛ): 202.0 (4.7), 242.0 (4.3, sh), 278.0 (4.1, sh); H-NMR (CDCl ,
1
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
4
00MHz) δ 8.60 (1H, s, NH), 8.12 (1H, s, NH), 8.09 (1H, dd, J= 9.6, 8.8 Hz, H6ʹʹ), 8.02 (1H,
H
dd, J= 8.8, 8.8 Hz, H6ʹ), 6.72-6.63 (4H, m, H3ʹ, H3ʹʹ, H5ʹ, and H5ʹʹ), 5.96 (1H, s, H ), 5.80 (1H, s,
b
1
3
H ), 3.79 (3H, s, H7ʹʹ), 3.76 (3H, s, H7ʹ), 3.47 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.3
a
3
C
(
s, C4), 166.6 (s, C1), 157.4 (s, J= 10.5 Hz, C5ʹ), 157.0 (s, J= 10.1 Hz, C5ʹʹ), 154.1 (d, J= 243.0
Hz, C2ʹ), 154.0 (d, J= 243.5 Hz, C2ʹʹ), 139.0 (s, C2), 123.6 (d, J= 10.5 Hz, C6ʹ, and C6ʹʹ), 122.9
t, C5), 119.5 (d, J= 11.1 Hz, C1ʹ), 118.9 (d, J= 10.9 Hz, C1ʹʹ), 109.7 (d, J= 3.0 Hz, C5ʹ), 109.4
(d, J= 3.0 Hz, C5ʹʹ), 102.0 (d, J= 22.8 Hz, C3ʹ), 101.9 (d, J= 22.7 Hz, C3ʹʹ), 55.9 (q, C7ʹ, and
(
–
–
C7ʹʹ), 42.1 (t, C3); ESI-MS (m/z): 375.3 [M-H] ; HRMS calculated for C H F N O , [M-H] ,
1
9
17
2
2
4
3
75.1151; found 375.1159.
4
-((2-fluoro-5-(trifluoromethyl)phenyl)amino)-2-methylene-4-oxobutanoic (5A)
ᴑ
-1
Yield of 9.0% as brown solid; mp ( C): 144-145; IR ν (KBr) cm : 3264, 3151, 3106, 3064,
max
3
8
4
018, 2908, 2853, 1687, 1627, 1542, 1489, 1437, 1335, 1263, 1228, 1169, 1124, 1064, 977, 918,
25, 689, 512, 487; UV λmax MeOH nm (Log Ɛ): 203.0 (4.4), 241.0 (4.0); H-NMR (Acetone-d6,
1
00MHz) δ 9.34 (1H, s, NH), 8.71 (1H, dd, J= 7.2, 2.0 Hz, H6ʹ), 7.48-7.44 (1H, m, H4ʹ), 7.40
H
(
1H, dd, J= 10.4, 8.8 Hz, H3ʹ), 6.34 (1H, d, J= 1.2 Hz, H ), 5.88 (1H, d, J= 1.2 Hz, H ), 3.57 (2H,
b
a
1
3
s, H3); C-NMR (Acetone-d6, 100MHz)δ 170.1 (s, C1), 168.2 (s, C4), 155.4 (d, J= 248.6 Hz,
C
C2ʹ), 136.1 (s, C2), 129.0 (t, C5), 128.9 (d, J= 15.5 Hz, C1ʹ), 127.6-126.8 (m, C5ʹ), 125.1 (q, J=
2
69.6 Hz, C7ʹ), 122.2 (d, C4ʹ), 119.9 (d, C6ʹ), 116.9 (d, J= 21.0 Hz, C3ʹ), 41.0 (t, C3); ESI-MS
–
–
(m/z): 289.9 [M-H] ; HRMS calculated for C H F NO , [M-H] , 290.0435; found 290.0442.
1
2
8
4
3
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N,N'-bis(2,5-difluorophenyl)-2-methylenesuccinamide (6)
ᴑ
-1
Yield of 21.2% as white solid; mp ( C): 140-141; IR ν (KBr) cm : 3393, 3301, 3285, 3255,
max
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
3
222, 3141, 3089, 3024, 2962, 1682, 1628, 1548, 1487, 1438, 1197, 967, 866, 805, 526, 462. UV
λmax MeOH nm (Log Ɛ): 202.5 (4.5), 239.0 (4.2), 277.5 (4.0); H-NMR (CDCl , 400MHz) δ 8.82
1
3
H
(1H, s, NH), 8.24 (1H, s, NH), 8.19 (1H, ddd, J= 10.4, 6.4, 3.2 Hz, H6ʹ), 8.13 (1H, ddd, J= 10.4,
6
.4, 3.2 Hz, H6ʹʹ), 7.10-6.99 (2H, m, H3ʹ and H3ʹʹ), 6.81-6.69 (2H, m, H4ʹ and H4ʹʹ), 6.00 (1H, s,
1
3
H ), 5.88 (1H, s, H ), 3.51 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.2 (s, C4), 166.6 (s,
b
a
3
C
C1), 158.7 (d, J= 243.0 Hz, C5ʹ, and C5ʹʹ) 148.8 (d, J= 235.3 Hz, C2ʹ) 148.6 (d, J= 235.3 Hz,
C2ʹʹ), 138.5 (s, C2), 127.4 (dd, J= 12.2, 12.0 Hz, C1ʹ), 126.9 (dd, J= 12.1, 11.7 Hz, C1ʹʹ), 123.7 (t,
C5), 115.7-115.2 (m, C6ʹ, and C6ʹʹ), 111.0 (dd, J= 24.4, 7.7 Hz, C3ʹ), 110.3 (dd, J= 24.4, 7.7 Hz,
C3ʹʹ), 109.1 (dd, J= 30.1, 16.5 Hz, C4ʹ, and C4ʹʹ), 42.2 (t, C3); ESI-MS (m/z): 353.1 [M+H]+,
+
+
375.2 [M+Na] ; HRMS calculated for C H F N O , [M+H] , 353.0908; found 353.0912.
1
7
13
4
2
2
4
-((5-chloro-2-fluorophenyl)amino)-2-methylene-4-oxobutanoic acid (6A)
ᴑ
-1
Yield of 9.9% as white solid; mp ( C): 171-172; IR ν (KBr) cm : 3266, 3123, 3092, 3048,
max
3
018, 2919, 2886, 1700, 1667, 1609, 1536, 1488, 1414, 1238, 1196, 963, 907, 803, 704, 523, 503;
UV λmax MeOH nm (Log Ɛ): 206.0 (4.6), 241.0 (4.2), 280.0 (3.6); H-NMR (Acetone-d6, 400MHz)
δH 9.17(1H, s, NH), 8.38 (1H, ddd, J= 6.8, 6.4, 2.4 Hz, H6ʹ), 7.20 (1H, dd, J= 10.8, 8.8 Hz, H3ʹ),
7.12-7.08 (1H, m, H4ʹ), 6.33 (1H, d, J=1.2 Hz, H ), 5.87 (1H, d, J=1.2 Hz, H ), 3.55 (2H, s, H3);
1
b
a
13
C-NMR (Acetone-d6, 100MHz)δ 169.8 (s, C1), 168.1 (s, C4), 152.2 (d, J= 242.2 Hz, C2ʹ),
C
1
36.2 (s, C2), 129.8 (d, J= 3.3 Hz, C5ʹ), 129.2 (d, J= 12.2 Hz, C1ʹ), 129.0 (t, C5), 124.5 (d, J=
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.7 Hz, C4ʹ), 122.5 (d, C6ʹ), 117.3 (d, J= 21.1 Hz, C3ʹ), 41.0 (t, C3); ESI-MS (m/z): 256.3 [M-
–
–
–
H] , 258.2 [(M+2)-H] ; HRMS calculated for C H ClFNO , [M-H] , 256.0171; found 256.0181.
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1
8
3
0
1
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4
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0
1
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0
N,N'-bis(5-chloro-2-fluorophenyl)-2-methylenesuccinamide (7)
ᴑ
-1
Yield of 11.7% as white solid; mp ( C): 140-141; IR ν (KBr) cm : 3333, 3265, 3247, 3114,
max
3
1
091, 3019, 1678, 1631, 1611, 1538, 1519, 1489, 1416, 1393, 1324, 1257, 1222, 1186, 1148,
111, 937, 875, 812, 708, 647, 462; UV λmax MeOH nm (Log Ɛ): 205.0 (4.7), 242.0 (4.3), 279.0
1
(3.9); H-NMR (CDCl , 400MHz) δ 8.73 (1H, s, NH), 8.40 (1H, dd, J= 7.6, 5.6 Hz, H6ʹ), 8.35
3
H
(1H, dd, J= 7.6, 5.6 Hz, H6ʹʹ), 8.21 (1H, s, NH), 7.07-6.99 (4H, m, H3ʹ, H3ʹʹ, H4ʹ, and H4ʹʹ), 5.99
1
3
(1H, s, H ), 5.87 (1H, s, H ), 3.50 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.2 (s, C4),
b
a
3
C
1
66.6 (s, C1), 151.3 (d, J= 242.3 Hz, C2ʹ), 151.2 (d, J= 242.5 Hz, C2ʹʹ), 138.5 (s, C2), 130.1 (d,
J= 3.3 Hz, C5ʹ), 129.8 (s, J= 3.2 Hz, C5ʹʹ), 127.4 (d, J= 11.6 Hz, C4ʹʹ), 127.0 (d, J= 11.4 Hz, C4ʹ),
124.8 (d, J= 7.7 Hz, C6ʹ), 124.2 (d, J= 7.4 Hz, C6ʹʹ), 123.7 (t, C5), 121.8 (d, J= 12.3 Hz, C1ʹ, and
C1ʹʹ), 115.9 (d, J= 20.8 Hz, C3ʹ), 115.8 (d, J= 20.8 Hz, C3ʹʹ), 42.1 (t, C3); ESI-MS (m/z): 407.3
+
+
+
[M+Na] , 409.2 [(M+2)+Na] ; HRMS calculated for C H Cl F N O Na, [M+Na] , 407.0136;
17 12 2 2 2 2
found 407.0151.
4
-((4-fluoro-2-(trifluoromethyl)phenyl)amino)-2-methylene-4-oxobutanoic acid (7A)
ᴑ
-1
Yield of 6.2% as white solid; mp ( C): 146-148; IR ν (KBr) cm : 3274, 3150, 3087, 3015,
max
2979, 2925, 2885, 1700, 1665, 1533, 1433, 1323, 1279, 1246, 1171, 1131, 1048, 964, 915, 876,
36, 668, 576, 497, 422; UV λmax MeOH nm (Log Ɛ): 203.0 (4.4), 231.0 (3.9), 272.0 (3.4); H-
1
8
NMR (Acetone-d6, 400MHz) δ 8.72 (1H, s, NH), 7.91-7.86 (1H, m, H6ʹ), 7.50-7.42 (1H, m,
H
H3ʹ, H5ʹ), 6.34 (1H, s, H ), 5.89 (1H, s, H ), 3.48 (2H, s, H3); 13C-NMR (Acetone-d6,
b
a
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00MHz)δ 169.9 (s, C1), 168.3 (s, C4), 160.3 (d, J= 243.7 Hz, C4ʹ), 136.3 (s, C2), 133.1 (brs,
C
C1ʹ), 131.2 (brd, C6ʹ), 129.0 (t, C5), 128.2-120.0 (m, C2ʹ, and C7ʹ), 120.5 (d, J= 22.0 Hz, C5ʹ),
–
114.2 (dq, J= 26.3, 5.5 Hz, C3ʹ), 40.8 (t, C3); ESI-MS (m/z): 290.2 [M-H] ; HRMS calculated
1
1
1
1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
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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
–
for C H F NO , [M-H] , 290.0435; found 290.0439.
1
2
8
4
3
N,N'-bis(2-fluoro-5-(trifluoromethyl)phenyl)-2-methylenesuccinamide (8)
ᴑ
-1
Yield of 12.3% as white solid; mp ( C): 90-92; IR ν (KBr) cm : 3268, 3163, 3094, 3027, 1670,
max
1
622, 1550, 1491, 1438, 1338, 1267, 1174, 1128, 1070, 942, 898, 823, 606, 556, 524, 457; UV
λmax MeOH nm (Log Ɛ): 203.0 (4.8), 243.5 (4.4); H-NMR (CDCl , 400MHz) δ 8.82 (1H, s, NH),
1
3
H
8.71 (1H, dd, J= 7.2, 2.0 Hz, H6ʹ), 8.65 (1H, d, J= 7.2 Hz, H6ʹʹ), 8.30 (1H, s, NH), 7.41-7.38 (1H,
m, H4ʹ), 7.34-7.31 (1H, m, H4ʹʹ), 7.24 (1H, dd, J= 9.2, 9.2 Hz, H3ʹ), 7.18 (1H, dd, J= 10.0, 9.2
1
3
Hz, H3ʹʹ), 6.03 (1H, s, H ), 5.90 (1H, s, H ), 3.55 (2H, s, H3); C-NMR (CDCl , 100MHz) δ
C
b
a
3
168.5 (s, C4), 166.8 (s, C1), 154.4 (d, J= 247.5 Hz, C2ʹ), 154.2 (d, J= 248.0 Hz, C2ʹʹ), 138.3 (s,
C2), 128.0-127.1 (m, C5ʹ, and C5ʹʹ), 127.1 (d, J= 11.2 Hz, C1ʹʹ), 126.7 (d, J= 10.8 Hz, C1ʹ),
1
1
23.7 (qd, J= 269.9, 6.8 Hz, C7ʹ, and C7ʹʹ), 124.1 (t, C5), 122.4-121.7 (m, C4ʹ, and C4ʹʹ), 119.7-
19.3 (m, C6ʹ, and C6ʹʹ), 115.6 (d, J= 20.6 Hz, C3ʹ), 115.5 (d, J= 20.6 Hz, C3ʹʹ), 42.0 (t, C3);
–
–
ESI-MS (m/z): 451.1 [M-H] ; HRMS calculated for C H F N O , [M-H] , 451.0687; found
1
9
11
8
2
2
4
51.0693.
4-((5-fluoro-2-(trifluoromethyl)phenyl)amino)-2-methylene-4-oxobutanoic acid (8A)
ᴑ
-1
Yield of 9.2% as white solid; mp ( C): 147-148; IR ν (KBr) cm : 3283, 3099, 3048, 3016,
max
2
926, 2865, 1703, 1676, 1605, 1537, 1496, 1438, 1395, 1316, 1235, 1159, 1117, 1048, 968, 944,
872, 827, 657, 585, 492, 455, 414; UV λmax MeOH nm (Log Ɛ): 204.0 (4.5), 239.0 (4.1); H-NMR
1
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(
Acetone-d6, 400MHz) δ 8.74 (1H, s, NH), 7.98-7.93 (1H, m, H6ʹ), 7.76 (1H, dd, J= 8.8, 8.8 Hz,
H
1
3
H3ʹ), 7.14-7.09 (1H, m, H4ʹ), 6.37 (1H, d, J= 0.8 Hz, H ), 5.94 (1H, s, H ), 3.52 (2H, s, H3); C-
b
a
NMR (Acetone-d6, 100MHz)δ 169.7 (s, C1), 168.2 (s, C4), 165.7 (d, J= 247.7 Hz, C5ʹ), 139.3-
C
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
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0
1
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0
1
39.1 (m, C1ʹ), 135.9 (s, C2), 129.6-129.4 (m, C5, C6ʹ), 124.8 (q, J= 270.3 Hz, C7ʹ), 118.1-117.8
(
m, C2ʹ), 113.5-113.1 (m, C3ʹ), 112.6-112.3 (m, C4ʹ), 41.4 (t, C3); ESI-MS (m/z): 290.2 [M-H]–;
–
HRMS calculated for C H F NO , [M-H] , 290.0435; found 290.0440.
12
8
4
3
N,N'-bis(2-fluoro-5-methoxyphenyl)-2-methylenesuccinamide (9)
ᴑ
-1
Yield of 7.2% as white solid; mp ( C): 121-122; IR ν (KBr) cm : 3301, 3154, 3066, 3006,
max
2946, 2835, 1672, 1618, 1541, 1482, 1428, 1353, 1316, 1252, 1211, 1038, 957, 867, 807, 716,
47, 468; UV λmax MeOH nm (Log Ɛ): 204.0 (4.7), 239.0 (4.3), 289.0 (4.1); H-NMR (CDCl ,
1
5
4
3
00MHz) δ 8.72 (1H, s, NH), 8.23 (1H, s, NH), 8.00 (1H, dd, J= 6.4, 3.2 Hz, H6ʹ), 7.95 (1H, dd,
H
J= 6.4, 3.2 Hz, H6ʹʹ), 7.01 (1H, dd, J= 9.2, 8.8 Hz, H3ʹ), 6.96 (1H, dd, J= 9.2, 8.8 Hz, H3ʹʹ), 6.60
(1H, ddd, J= 8.8, 7.2, 3.6 Hz, H4ʹ), 6.54 (1H, ddd, J= 8.8, 7.2, 3.6 Hz, H4ʹʹ), 5.98 (1H, s, Hb),
1
3
5
.84 (1H, s, H ), 3.80 (3H, s, H7ʹ), 3.77 (3H, s, H7ʹʹ), 3.51 (2H, s, H3); C-NMR (CDCl ,
a 3
1
00MHz) δ 168.3 (s, C4), 166.5 (s, C1), 156.1 (s, C5ʹ), 156.0 (s, C5ʹʹ), 147.3 (d, J= 234.8 Hz,
C
C2ʹ), 147.2 (d, J= 235.0 Hz, C2ʹʹ), 138.9 (s, C2), 127.0 (d, J= 11.6 Hz, C4ʹʹ), 126.5 (d, J= 11.5
Hz, C4ʹ), 123.2 (t, C5), 115.2 (d, J= 20.7 Hz, C3ʹ), 115.1 (d, J= 20.6 Hz, C3ʹʹ), 110.4 (d, J= 7.2
Hz, C6ʹ), 109.8 (d, J= 7.2 Hz, C6ʹʹ), 106.9 (d, J= 11.2 Hz, C1ʹ, and C1ʹʹ), 55.9 (q, C7ʹ, and C7ʹʹ),
–
–
42.3 (t, C3); ESI-MS (m/z): 375.1 [M-H] ; HRMS calculated for C H F N O , [M-H] ,
1
9
17
2
2
4
3
75.1151; found 375.1158.
2-methylene-4-oxo-4-((2-(trifluoromethyl)phenyl)amino)butanoic acid (9A)
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9
ᴑ
-1
Yield of 11.2% as white solid; mp ( C): 153-154; IR ν (KBr) cm : 3298, 3214, 3130, 3059,
max
3
013, 2910, 2864, 1703, 1676, 1637, 1593, 1537, 1451, 1393, 1320, 1290, 1235, 1163, 1122,
1051, 948, 902, 764, 653, 512, 454; UV λmax MeOH nm (Log Ɛ): 203.0 (4.4), 233.0 (3.9), 269.0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
(
3.3); H-NMR (Acetone-d6, 400MHz) δ 8.68 (1H, s, NH), 7.98-7.94 (1H, m, H3ʹ), 7.69 (1H, d,
H
J= 7.6 Hz, H6ʹ), 7.64 (1H, dd, J= 8.0, 7.6 Hz, H5ʹ), 7.36 (1H, dd, J= 7.6, 7.6 Hz, H4ʹ), 6.35 (1H,
d, J= 1.6 Hz, H ), 5.91 (1H, d, J= 1.2 Hz, H ), 3.50 (2H, s, H3); 13C-NMR (Acetone-d6,
b
a
1
00MHz)δ 169.6 (s, C1), 168.3 (s, C4), 136.8 (brs, C1ʹ), 136.3 (s, C2), 133.7 (d, C5ʹ), 129.0 (t,
C
C5), 127.8 (brd, C3ʹ), 126.9 (q, J= 5.3 Hz, C6ʹ), 126.2 (d, C4ʹ), 125.0 (q, J= 270.9 Hz, C7ʹ),
23.1-122.5 (m, C2ʹ), 41.0 (t, C3); ESI-MS (m/z): 271.8 [M-H]–.
1
N,N'-bis(2-bromo-4-fluorophenyl)-2-methylenesuccinamide (10)
ᴑ
-1
Yield of 6.6% as white solid; mp ( C): 165-166; IR ν (KBr) cm : 3263, 3193, 3110, 3033,
max
3006, 1655, 1629, 1599, 1527, 1484, 1390, 1315, 1261, 1189, 1141, 817, 657, 513, 482, 443; UV
λmax MeOH nm (Log Ɛ): 205.0 (4.7), 243.0 (4.1, sh), 277.0 (3.7, sh); H-NMR (CDCl , 400MHz) δ
1
3
H
8
.51 (1H, s, NH), 8.34 (1H, dd, J= 9.2, 5.6 Hz, H6ʹ), 8.27 (1H, s, NH), 8.22 (1H, dd, J= 9.2, 5.6
Hz, H6ʹʹ), 7.33 (1H, dd, J= 7.6, 2.8 Hz, H3ʹ), 7.28 (1H, dd, J= 8.0, 2.8 Hz, H3ʹʹ), 7.08(1H, ddd,
J= 8.0, 8.0, 2.8 Hz, H5ʹ), 7.03 (1H, ddd, J= 8.4, 8.0, 2.8 Hz, H5ʹʹ), 6.05 (1H, s, H ), 5.90 (1H, s,
b
1
3
H ), 3.55 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.1 (s, C4), 166.1 (s, C1), 159.0 (d, J=
a
3
C
2
47.6 Hz, C4ʹ and C4ʹʹ), 139.8 (s, C2), 132.5 (s, J= 3.2 Hz, C1ʹ), 131.9 (s, J= 3.2 Hz, C1ʹʹ), 123.8
(d, J= 7.7 Hz, C6ʹʹ), 123.3 (d, J= 8.2 Hz, C6ʹ), 123.1 (t, C5), 119.7 (d, J= 25.7 Hz, C3ʹ), 119.6 (d,
J= 25.3 Hz, C3ʹʹ), 115.5 (d, J= 21.8 Hz, C5ʹ), 115.2 (d, J= 21.4 Hz, C5ʹʹ), 114.4 (s, C2ʹ), 114.3 (s,
–
–
C2ʹʹ), 42.0 (t, C3); ESI-MS (m/z): 472.7 [M-H] , 474.7 [(M+2)-H] ; HRMS calculated for
–
C H Br F N O , [M-H] , 470.9150; found 470.9167.
1
7
11
2
2
2
2
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6
2
-methylene-4-oxo-4-((3-(trifluoromethyl)phenyl)amino)butanoic acid (10A)
ᴑ
-1
Yield of 12.2% as white solid; mp ( C): 132-133; IR ν (KBr) cm : 3286, 3195, 3143, 3112,
max
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
3
5
9
7
078, 3006, 2907, 1690, 1656, 1599, 1532, 1445, 1334, 1246, 1170, 1136, 1066, 978, 902, 701,
35, 493 UV λmax MeOH nm (Log Ɛ): 203.5 (4.6), 244.0 (4.3); H-NMR (Acetone-d6, 400MHz) δ
1
H
.54 (1H, s, NH), 8.16 (1H, s, H2ʹ), 7.81 (1H, d, J= 8.0 Hz, H6ʹ), 7.52 (1H, t, J= 8.0 Hz, H5ʹ),
.37 (1H, d, J= 7.6 Hz, H4ʹ), 6.32 (1H, d, J= 1.2 Hz, H ), 5.84 (1H, d, J= 1.2 Hz, H ), 3.46 (2H, s,
b
a
1
3
H3); C-NMR (Acetone-d6, 100MHz)δ 169.6 (s, C1), 167.9 (s, C4), 141.1 (s, C1ʹ), 136.1 (s,
C
C2), 131.3 (q, J= 31.7 Hz, C3ʹ), 130.6 (d, C5ʹ), 129.3 (t, C5), 125.2 (q, J= 269.9 Hz, C7ʹ), 123.3
(d, C6ʹ), 120.5-120.4 (m, C4ʹ), 116.4-116.3 (m, C2ʹ), 40.9 (t, C3); ESI-MS (m/z): 272.1 [M-H]–;
–
HRMS calculated for C H F NO , [M-H] , 272.0529; found 272.0535.
1
2
9
3
3
N,N'-bis(4-fluoro-2-(trifluoromethyl)phenyl)-2-methylenesuccinamide (11)
ᴑ
-1
Yield of 5.6% as white solid; mp ( C): 130-131; IR ν (KBr) cm : 3278, 3084, 3017, 2927,
max
2
7
846, 1671, 1628, 1531, 1505, 1433, 1319, 1277, 1251, 1204, 1167, 1129, 1049, 912, 882, 831,
41, 672, 637, 525; UV λmax MeOH nm (Log Ɛ): 204.0 (4.2), 232.0 (4.1), 272.0 (3.7); H-NMR
1
(CDCl , 400MHz) δ 8.50 (1H, s, NH), 8.15 (1H, dd, J= 9.2, 8.8 Hz, H6ʹ), 8.10 (1H, s, NH),
3 H
8
.00 (1H, dd, J= 8.8, 8.8 Hz, H6ʹʹ), 7.38-7.22 (4H, m, H3ʹ, H3ʹʹ, H5ʹ, and H5ʹʹ), 5.98 (1H, s, Hb),
1
3
5
.89 (1H, s, H ), 3.51 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.6 (s, C4), 166.6 (s, C1),
a
3
C
159.4 (d, J= 246.3 Hz, C4ʹ), 159.2 (d, J= 246.0 Hz, C4ʹʹ), 138.5 (s, C2), 131.3 (s, C1ʹ), 130.9 (s,
C1ʹʹ), 128.2 (d, J= 7.6 Hz, C6ʹʹ), 127.5 (d, J= 7.8 Hz, C6ʹ), 123.4 (t, C5), 124.6-121.7 (m, C2ʹ,
C2ʹʹ, C7ʹ, and C7ʹʹ), 119.9 (d, J= 21.8 Hz, C5ʹ), 119.6 (d, J= 21.7 Hz, C5ʹʹ), 114.0-113.5 (m, C3ʹ
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–
and C3ʹʹ), 41.8 (t, C3); ESI-MS (m/z): 451.1 [M-H] ; HRMS calculated for C H F N O , [M-
1
9
11
8
2
2
–
H] , 451.0687; found 451.0692.
1
1
1
1
1
1
1
1
1
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0
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0
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0
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0
2
-methylene-N,N'-bis(2-(trifluoromethyl)phenyl)succinamide (12)
ᴑ
-1
Yield of 6.6% as white solid; mp ( C): 131-132; IR ν (KBr) cm : 3278, 3120, 3047, 3008,
max
2
4
4
8
926, 2846, 1670, 1631, 1588, 1525, 1456, 1322, 1280, 1161, 1113, 1060, 1038, 943, 761, 654,
97, 430; UV λmax MeOH nm (Log Ɛ): 204.0 (4.6), 233.0 (4.2), 272.0 (3.7, sh); H-NMR (CDCl ,
1
3
00MHz) δ 8.55 (1H, s, NH), 8.26 (1H, d, J= 8.4 Hz, H6ʹ), 8.20 (1H, s, NH), 8.09 (1H, d, J=
H
.0 Hz, H6ʹʹ), 7.65-7.52 (4H, m, H3ʹ, H3ʹʹ, H5ʹ, and H5ʹʹ), 7.29-7.21 (2H, m, H4ʹ, and H4ʹʹ), 5.98
1
3
(1H, s, H ), 5.89 (1H, s, H ), 3.53 (2H, s, H3); C-NMR (CDCl , 100MHz) δ 168.5 (s, C4),
b
a
3
C
1
66.4 (s, C1), 138.9 (s, C2), 135.3 (s, C1ʹ), 135.0 (s, C1ʹʹ), 133.2 (d, C5ʹ), 132.8 (d, C5ʹʹ), 126.4-
26.1 (m, C3ʹ and C3ʹʹ), 125.5 (d, C4ʹʹ), 125.1 (d, C6ʹʹ), 124.9 (d, C4ʹ), 124.6 (d, C6ʹ), 124.3 (q,
1
J= 271.4 Hz, C7ʹ), 124.0 (q, J= 271.4 Hz, C7ʹʹ), 123.0 (t, C5), 121.2-120.3 (q, C2ʹ, and C2ʹʹ),
19.9 (d, J= 21.8 Hz, C5ʹ), 119.6 (d, J= 21.7 Hz, C5ʹʹ), 41.8 (t, C3); ESI-MS (m/z): 415.2 [M-H]–;
1
–
HRMS calculated for C H F N O , [M-H] , 415.0876; found 415.0891.
1
9
13
6
2
2
N,N'-bis(2,4-dibromophenyl)-2-methylenesuccinamide (13)
ᴑ
-1
Yield of 7.8% as white solid; mp ( C): 195-196; IR ν (KBr) cm : 3407, 3299, 3256, 3076,
max
3
025, 2947, 1660, 1576, 1524, 1464, 1375, 1312, 1282, 1194, 861, 819, 543, 483, 445; UV λmax
MeOH
1
nm (Log Ɛ): 206.5 (4.8), 251.0 (4.3); H-NMR (CDCl , 400MHz) δ 8.56 (1H, s, NH), 8.34
3
H
(
1H, s, NH), 8.31 (1H, d, J= 8.8 Hz, H6ʹ), 8.21 (1H, d, J= 8.8 Hz, H6ʹʹ), 7.71 (1H, d, J= 2.0 Hz,
H3ʹ), 7.67 (1H, d, J= 2.0 Hz, H3ʹʹ), 7.46 (1H, dd, J= 8.8, 2.0 Hz, H5ʹ), 7.40 (1H, dd, J= 8.8, 2.0
1
3
Hz, H5ʹʹ), 6.05 (1H, s, H ), 5.91 (1H, s, H ), 3.54 (2H, s, H3); C-NMR (CDCl , 100MHz) δ
C
b
a
3
29
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