Synthesis, biological evaluation and structure-activity relationships of 5-arylidene tetramic acids with antibacterial activity against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1016/j.bmcl.2020.127107

The steady rise of the antimicrobial resistance is a major global threat to human health that requires the urgent need for novel antibiotics. In this work we report the synthesis of a small library of 3-subsituted-5-arylidene tetramic acids in order to in

Full text of DOI:10.1016/j.bmcl.2020.127107

Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, biological evaluation and structure-activity relationships of
5
-arylidene tetramic acids with antibacterial activity against methicillin-
resistant Staphylococcus aureus
a,⁎
a
a
a
Dimitris Matiadis , Dimitrios Tsironis , Valentina Stefanou , Spyridon Boussias ,
b
c,d
a
e,⁎
Angeliki Panagiotopoulou , Vickie McKee , Olga Igglessi-Markopoulou , John Markopoulos
a
b
c
d
National Technical University of Athens, School of Chemical Engineering, Laboratory of Organic Chemistry, Zografou Campus, Athens 15773, Greece
Institute of Biosciences and Applications, NCSR “Demokritos”, Ag. Paraskevi, 15310 Attiki, Greece
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark
School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland
e
Laboratory of Inorganic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis, 15771 Athens, Greece
A R T I C L E I N F O
A B S T R A C T
InChIKeys:
The steady rise of the antimicrobial resistance is a major global threat to human health that requires the urgent
need for novel antibiotics. In this work we report the synthesis of a small library of 3-subsituted-5-arylidene
tetramic acids in order to investigate the scope of our previously established methodology via an intermediate
oxazolone and their antimicrobial activity. From this series of 14 tetramic acids, 11 derivatives are novel and one
of them is a Schiff base, which was structurally characterized with single-crystal X-ray analysis and NMR
spectroscopy. The compounds incorporating a lipophilic acyl group at carbon-3 of the ring showed moderate to
high activity with minimum inhibitory activity of 4–32 μg/mL against methicillin-resistant Staphylococcus aureus
ITINXNLUXHSDPH-JYRVWZFOSA-N
VTIKXFQFIUTPJB-JYRVWZFOSA-N
XCWVVYBLKVYDOB-GHXNOFRVSA-N
IMNDKSNSHZZIEF-GHXNOFRVSA-N
BSKHMXHBXUVHON-GHXNOFRVSA-N
YMBVQRFRYOHPPB-GHXNOFRVSA-N
WQVBLIYLKLKKKS-RAXLEYEMSA-N
BIEUIQJXUQQCKE-VKAVYKQESA-N
PNXPLOBGFZPPOJ-MNDPQUGUSA-N
SNVPGPDGYGABMU-ATJXCDBQSA-N
HCYAWYVDZFJDEI-LVWGJNHUSA-N
IYMBBMLSXACSMH-VBKFSLOCSA-N
QIASDTRYOAULGK-UVTDQMKNSA-N
IMRAGIHYGYLUQR-VXYIRXSZSA-N
(
MRSA), accompanied by no human cell toxicity and hemolytic activity within the tested concentration range.
The substituent at para position of the aryl ring seemed to have no or little effect on the antimicrobial activity of
these compounds.
Keywords:
Pyrrolidine-2-dione
Tetramic acid
Lactam
X-ray crystallography
MRSA
Antibacterial activity
The antimicrobial resistance (AMR) or multidrug resistance (MDR)
Specifically, studies indicate the rapidly increasing rates of infec-
1
5
is one of the greatest threats for humankind. Undoubtedly, a large
tions associated with methicillin-resistant S. aureus (MRSA). Clinical
public health burden, increasing over time, is associated with infections
isolates of community-associated and healthcare-associated MRSA have
2
6
caused by antibiotic-resistant bacteria. Especially, the so-called ES-
developed resistance to many classes of antibiotics, such as β-lactams,
3
7
8
9
KAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella
macrolides, tetracyclines, quinolones and even last-resort agents,
10 11 12
pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and En-
terobacter species) represent the majority of nosocomial infections and
characteristic models of pathogenesis, transmission and resistance.4
such as vancomycin, daptomycin and linezolid. As a result, the
development of novel antibiotics having new mechanisms of action is
urgently needed to address the challenge of treating multidrug resistant
⁎
Corresponding authors.
E-mail addresses: dmatiadis@gmail.com (D. Matiadis), jmmarko@chem.uoa.gr (J. Markopoulos).
https://doi.org/10.1016/j.bmcl.2020.127107
Received 2 February 2020; Received in revised form 9 March 2020; Accepted 10 March 2020
Available online 13 March 2020
0960-894X/ © 2020 Elsevier Ltd. All rights reserved.

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
bacterial infections.
long lipophilic group attached at the nitrogen or at the C-3 of the ring.
Yet, there are only a few reports investigating the structure activity
relationships of these compounds regarding their antibacterial proper-
ties.
3
-Acyltetramic acids present a great variety of biological activities,
13 14
including antibacterial and antifungal properties. A few naturally
occurring and synthetic tetramic acid-containing antibacterials have
been found to inhibit effectively the methicillin-resistant or methicillin-
Due to their aforementioned biological activities and the challen-
1
5
15a
16
sensitive S. aureus (Fig. 1). The fungal toxin equisetin, as well as its
ging structures of natural products bearing the tetramic acid moiety
1
5b
analogue trichosetin,
are 3-acyltetramic acids bearing an aliphatic
many protocols for their synthesis have been developed, both conven-
1
7
18
19
22
bicyclic ring and show potent antibacterial activity against Gram-po-
tional and catalytic. Tetramic acid metal coordination compounds
1
5c
15d
20
21
sitive bacteria. Reutericyclin
and magnesidin,
effective inhibitors
and derivatives such as amides, peptide analogues, macrocycles
23
of S. aureus, are natural products incorporating a lipophilic alkanoyl
and hybrids with commercial antibiotics have been examined in order
to further expand the potential of this valuable skeleton. However, only
a few studies in the literature have reported the synthesis of 3-acyl or 3-
group at nitrogen and at the carbon-3 of the ring respectively. Synthetic
1
5e–g
tetramic acids with novel structures
or analogues of the above-
1
5h,i
24
mentioned natural products
have been identified as potent in-
phenyl 5-arylidenetetramic acids so far.
hibitors of S. aureus. The common point seems to be the presence of a
Our group has contributed to the synthesis and study of many
Fig. 1. Representative examples of the nature derived antibiotic tetramic acids equisetin, reutericyclin and magnesidin A, potent synthetic S. aureus inhibitors
bearing lipophilic groups, our previous work model compound and this work’s identified antimicrobial 3-alkanoyl-5-benzylidene tetramic acids. The tautomeric form
of each structure is adopted from the cited literature.
2

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
modifications on two groups were made; the para-substituent on the 5-
arylidene group (3a-f) and the 3-acyl group (4a-g). Moreover, the in-
corporation of a Schiff base component at C-3 was examined to in-
vestigate the influence of the carbonyl group, resulting in a novel
structure (5) characterized with single-crystal X-ray analysis. All the
synthesized compounds were primary-screened against 5 key ESKAPE
pathogens (E. coli, methicillin-resistant S. aureus (MRSA), K. pneumo-
niae, A. baumannii and P. aeruginosa) and 2 fungi (C. neoformans and
Candida albicans) at a concentration of 32 μg/mL and the active com-
pounds were further screened in dose response antimicrobial assays to
confirm their activity and determine their minimum inhibition con-
centration (MIC). The active compounds were tested for both human
non-cancerous cell toxicity and hemolytic activity.
The target compounds were synthesized bearing in mind the po-
tential for antimicrobial activity and the scope of our previously pub-
2
4a
lished methodology for 3-acyl-5-benzylidene tetramic acids.
The
final products were prepared using a short synthetic route (Scheme 1)
2
7
starting from N-acetylglycine . N-Acetylglycine was condensed with
the appropriate aromatic aldehyde in the presence of sodium acetate in
acetic anhydride. The resulting azlactones 1a-g proved to be ideal for
the one-pot synthesis of 5-arylidenetetramic acids 3a-f and 4 g, since
the time consuming and yield- or purity-decreasing steps of protection,
deprotection and activation are eliminated. 1a-f were subjected to
nucleophilic attack from the anion of the β-ketoester 2a-g, formed by
non-nucleophilic base (NaH) deprotonation. Final products 3a-f and 4 g
were precipitated in good purity after acidification with 10% HCl. Pure
samples for analytical and biological tests were obtained after re-
crystallization from methanol or ethanol.
Fig. 2. Structures of tetramic acids 3–5.
In a similar manner, tetramic acids 4a-f were prepared from the
oxazolone 1g and the anion of ethyl benzoylacetate or the appropriate
long hydrocarbon chain β-ketoester. The latter were prepared following
2
4a-d,25
tetramic acid derivatives,
including their coordination com-
1
4c,24a,26
pounds.
In our previous study, we published the first cadmium
28
a slightly modified literature method. 4a-f were purified by column
complex of N-acetyl-3-acetyl-5-benzylidene tetramic acid with pro-
mising antifungal activity against Cryptococcus neoformans with no
human cell toxicity or hemolytic activity within the tested concentra-
chromatography. In this case, the extraction of the aqueous phase be-
fore acidification with petroleum ether instead of diethyl ether, in-
creased the yield by 10–15% in comparison to our previously reported
1
4c
tion range.
24a
synthesis.
As a continuation of our efforts to expand the scope of our estab-
The results proved that the developed method via arylidene ox-
azolones allows wide variation of 3-acyl substituents and electron-
withdrawing or donating groups at the para position of the 5-benzyli-
dene group.
2
4a
lished methodology via an appropriate oxazolone intermediate
and
to further investigate the biological activities of this relatively under-
explored class of antibiotics, we report herein the synthesis of a small
library of N-acetyl-3-acyl-5-arylidene tetramic acids (3,4) and a novel
tetramic acid Schiff base (5) (Fig. 2). In order to discuss the struc-
ture–activity relationships of their antimicrobial activities, appropriate
In order to investigate the structure-activity relation of the carbonyl
oxygen at C-3 compared to a substituted imine analogue, we prepared
the Schiff base derived from the condensation reaction between A and
Scheme 1. Synthesis of tetramic acids 3a-f and 4a-g. Reagents and conditions: (a) acetic anhydride, CH
2a: ethyl acetoacetate).
3
COONa, 1 h, reflux; (b) NaH, dry THF, 0 °C to r.t., 10% HCl
(
3

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
Scheme 2. Synthesis of the Schiff base 5.
aniline. Compound 5 was prepared29 in satisfactory yield and high
purity by refluxing the starting materials in methanol for 2 h (Scheme
2
).
1
The structures of the synthesized compounds were verified by H,
1
3
2
Fig. 3. Perspective view of 5 showing 50% probability ellipsoids for the non-
C, and D NMR experiments and elemental analyses and the structure
hydrogen atoms. Hydrogen bond shown as a dashed red line.
of the Schiff base 5 was elucidated by X-ray diffraction studies
Table 1).
(
The protons of the phenyl group, ortho and para to electron-do-
Broadly speaking, the structure of 5 is as expected (Fig. 3), although
1
nating and electron-withdrawing R substituents of compounds 3a-f
examination of the bond lengths (Table 2) suggests some delocalisation.
The molecules have an intramolecular hydrogen bond (N1·····O1,
and 4g show distinct upfield and downfield shifts in relation to the
parent unsubstituted compound A. The chemical shift difference of the
ortho resonance signals appears up to 0.90 ppm, while the para re-
sonance signals chemical shift difference are up to 0.54 ppm, according
2
.7253(11) Å) and the same nitrogen also makes an intermolecular
hydrogen bond, linking pairs of molecules (N1·····O1, 2.9980(12) Å
under symmetry operation −x + 1/2, y, −z + 1). There is a reason-
ably convincing (aromatic)CeH····O hydrogen bond linking the mole-
cules into chains (C2·····O2, 3.5348(14) Å under symmetry operation
x−1/2, y + 1/2, z−1/2). The result is a 2D hydrogen-bonded sheet
lying parallel to the (1 0–1) plane; this appears to have large cavities
1
to the substituent effect. Thus, the H signals of the ortho and para
1
positions when R = CH
3
are found at 7.13 ppm and 7.11 ppm re-
1
spectively whereas in the case of R = NO
2
are found at 8.12 ppm and
1
3
7.42 ppm. Changes of C NMR chemical shifts follow the same trend as
1
3
(
Fig. 4) but these “dimples” are filled by the next 2D layer (Table 3).
the ortho C signal is deshielded by 2.9 ppm (signal at 133.3 ppm) in
the case of NO substitution or shielded in the case of CH substitution
(signal at 128.2 ppm).
There is no evidence of significant interaction between the sheets.
2
3
1
13
The H, C and 2D NMR spectra in DMSO‑d of the Schiff base 5
1 1
6
confirm the transfer of the hydrogen to the imine. The H– H NOESY
spectrum of the Schiff base 5 revealed the cross peaks between the ortho
hydrogens of the phenyl group which appeared at 7.45 ppm with the
The infrared (FT-IR) spectrum of the Schiff base 5 exhibits two
−1 −1
broad absorption bands at 3436 cm and 3187 cm that correspond
to the proton of the enol and the nitrogen of the imine in accordance
with the X-ray crystallographic data. The broadness of the absorptions
indicate the presence of the intramolecular hydrogen bonding and the
hydrogen of the imine at 12.84 ppm and the CH protons of the
3
CH C = N group at 2.57 ppm.
3
−
1
migration of the proton. The strong absorption band at 1574 cm
is
attributed to the partial double bond character of the iminic C]N due
to delocalization.
Table 1
Crystal data and structure refinement for compound 5.
In order to investigate the potential anti-MRSA activity of the newly
synthesized series of compounds 3a-f, 4a-g and the Schiff base analogue
Empirical formula
21 18 2 3
C H N O
5
, single point concentration (32 μg/mL; n = 2) broth antimicrobial
Formula weight
346.37
100(1)
screening was undertaken (Fig. 5). The preliminary results were eval-
Temperature/K
uated in comparison to the N-acetyl-3-acetyl-5-benzylidene tetramic
Crystal system
monoclinic
I2/a
2
4a
acid A, tested in our recently published work.
Space group
a/Å
b/Å
c/Å
α/°
β/°
γ/°
15.08930(10)
15.33770(10)
14.55900(10)
90
First, a series of tetramic acids incorporating the pharmacologically
important F, OMe, Me, Cl, CF
3
and NO , keeping at the same time the 3-
2
acetyl group of the initial compound A was evaluated. As a general
trend, these compounds showed low activity against MRSA. Compound
3e proved to be partially active at 32 μg/mL with mean %inhibition of
61.7, compared to growth and media controls. From the rest of the
series only 3c and 3f were relatively more active than A (mean %in-
hibition 45.1 and 38.6 respectively in relation to 25.5 of A).
92.2440(10)
90
3
Volume/Å
3366.88(4)
8
Z
3
ρcalc g/cm
1.367
−
1
μ/mm
0.750
Next, a series of tetramic acids bearing a long aliphatic alkanoyl
chain or a benzoyl group at C-3 of the ring was evaluated to test our
hypothesis for enhancement of S. aureus inhibition with the
F(0 0 0)
1456.0
3
Crystal size/mm
Radiation
0.165 × 0.055 × 0.04
CuKα (λ = 1.54184)
8.224–152.238
2
θ range for data collection/°
Index ranges
−18 ≤ h ≤ 18, −19 ≤ k ≤ 19, −18 ≤ l ≤ 18
Reflections collected
36,171
Table 2
Independent reflections
Data/restraints/parameters
3486 [Rint = 0.0177, Rsigma = 0.0073]
3486/0/241
Selected bond lengths (A) for 5.
2
Goodness-of-fit on F
1.027
N1–C7
N1–C4
C7–C9
1.3265 (13)
1.4301 (12)
1.4040 (14)
1.4287 (14)
1.4917 (14)
C11–C12
O1–C10
O3–C21
O2–C19
N2–C11
1.3411 (15)
1.2418 (12)
1.2139 (13)
1.2159 (13)
1.4270 (13)
Final R indexes [I > =2σ (I)]
Final R indexes [all data]
R1 = 0.0332, wR2 = 0.0846
R1 = 0.0343, wR2 = 0.0855
0.24/−0.22
−
3
Largest diff. peak/hole/e Å
CCDC
C9–C10
C10–C11
1975919
4

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
Fig. 4. H-bonded layer structure viewed perpendicular to the 1 0–1 plane.
Table 3
found totally inactive verifying our hypothesis based on the literature
15
Hydrogen-bond geometry (Å, °) for 5.
data for the significance of a 3-acetyl or 3-acyl group on tetramic acid
biological activities.
D–H···A
D–H
H···A
D···A
D–H···A
To confirm the activity of compounds 4a-e and 4 g an 8-point dose
response (0.25 μg/mL – 32 μg/mL) antimicrobial activity assay was
performed in duplicate, and activity expressed as minimum inhibitory
concentrations (MIC). These assays were performed on both the target
resulting from the primary screening results, MRSA ATCC 43300 as well
as the full panel of bacteria/fungi for a uniform directly comparable
data set for this lead candidate compound. Appropriate positive in-
hibitory antibiotic controls were included for each strain and data
provided in the Supplementary information. The minimum inhibitory
concentration (MIC) was determined following the CLSI guidelines,
identifying the lowest concentration at which full inhibition of the
bacteria or fungi was observed. Full inhibition of growth has been de-
fined at ≤20% growth (or > 80% inhibition).
N1–H1···O1
N1–H1···O1
0.906 (17)
0.906 (17)
0.95
1.966 (16)
2.368 (16)
2.61
2.7253 (11)
2.9980 (12)
3.5348 (14)
140.3 (14)
126.6 (13)
164
i
ii
C2–H2···O2
Symmetry codes: (i) −x + 1/2, y, −z + 1; (ii) x−1/2, y + 1/2, z−1/2.
incorporation of a lipophilic hydrocarbon group. All of the compounds
(
4a-g) from this series were found active with 73.1–99.2% mean in-
hibition, excluding the benzoyl derivative which was found almost as
active as A (22.9% mean inhibition).
Finally, with the aim of determining the importance of the carbonyl
presence at the C-3 acyl group, a nitrogen analogue was prepared in the
form of a Schiff base (5), derived from A and aniline. Analogue 5 was
Fig. 5. Percentage inhibition at single concentration of 32 μg/mL of all preliminary screened compounds. Mean values. (Sa: S. aureus, Ec: E. coli, Kp: K. pneumoniae,
Pa: P. aeruginosa, Ab: A. baumanii, Ca: C. albicans, Cn: C. neoformans).
5

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
Table 4
methodology regarding substituents on the benzylidene group and on
C-3 of the tetramic acid nucleus and their effects on antimicrobial ac-
tivity. Among the compounds screened, compounds 4a-e and 4g
showed interesting anti-MRSA activity. Three of the four most active
compounds (4b, 4d, 4e) with MIC: 4 μg/mL against MRSA ATCC 43300
strain were found non-hemolytic and non-toxic against human em-
bryonic kidney cells (HEK293) within the tested concentration range.
The structure-activity relationship (SAR) studies indicate that the
compounds with 8–16 carbon-chained acyl groups at C-3 of the ring
show remarkable activities over the less lipophilic groups. The varia-
tions of substituents at the para position of the benzylidene group at C-5
have no or little effect on the tested pathogens. The incorporation of an
iminic group in the form of a Schiff base instead of the carbonyl group
showed that the latter is essential for the anti-MRSA activity, or alter-
natively, that the existence of the imine hydrogen bond is canceling the
above inhibition activity.
Antimicrobial activity against S. aureus (ATCC 43300, MRSA) as Minimum
Inhibitory Concentration (MIC), cytotoxicity (CC50) and hemolytic activity
(
HC10) of compounds 4a-4e, 4g and 3e. LogP values (miLogP) were calculated
using Molinspiration platform (v2018.10).
Compound
MIC (μg/mL)
CC50
HC10
logP
MRSA
(μg/mL)
(μg/mL)
a (R1 = H, R =(CH
2
2
2
2
)
CH
CH
)
32
4
> 32
> 32
> 32
> 32
> 32
> 32
> 32
> 32
> 32
> 32
20.4
2.94
4.96
5.97
7.99
8.69
3.00
2.77
1.88
4
4
4
4
4
4
3
2
2
2
2
3
3
3
1
b (R = H, R =(CH
)
6
CH
)
1
1
c (R = H, R =(CH
d (R = H, R =(CH
e (R = H, R =(CH
)
8
)
4
2
)
12CH
3
)
4
> 32
> 32
> 32
> 32
> 32
1
1
2
2
)
14CH
3
CH
)
4
2
g (R = OMe, R =(CH
2
)
)
2
3
)
32
> 32
> 32
1
2
e (R = CF
3
, R = CH
3
1
2
24a
A (R = H, R = CH
3
)
Compounds 4a and 4g demonstrated moderate inhibitory activity
against MRSA (32 μg/mL) confirming thus the preliminary results that
showed > 3-fold increase of the %inhibition at 32 μg/mL. In principle,
these results indicate that irrespective of the substitution at the ben-
zylidene group, the introduction of a more lipophilic acyl group at C-3
has substantial effect on MRSA inhibition.
Compounds 4b, 4d and 4e are the most promising ones for further
modifications in order to develop an effective lead compound. The
developed one-pot method via azlactones combined with the potential
of tetramic acids for modifications encourage us to direct our efforts
toward an expanded structure-activity relationship study.
Confirming our expectations, compounds 4b-e exhibited excellent
equal inhibitory activity against the target bacterium (4 μg/mL)
Declaration of Competing Interest
(
Table 4). The length of the hydrocarbon acyl chain plays a key role in
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
the effectiveness of the anti-MRSA activity. The inhibition values at the
MIC suggest the following order of activity 4b > 4c = 4d < 4e
(
86.6%, 94.4%, 94.4%, 81.7% mean values respectively). Therefore, for
a further future optimization, compounds with 8 to 14 carbon chain
Acknowledgements
(
including the carbonyl carbon) should be employed.
Finally, compound 3e was tested in the same way, but the MIC was
Antimicrobial and mammalian cell toxicity screening was per-
formed by CO-ADD (The Community for Open Antimicrobial Drug
determined > 32 μg/mL, only confirming the partial activity (60–80%
inhibition value) at this concentration.
31
Discovery), funded by the Wellcome Trust (UK) and The University of
Higher logP values are related to better anti-MRSA activities
Queensland (Australia).
(
Table 4) in this series of tetramic acid compounds.
DM would like to thank the Greek State Scholarship Foundation and
SIEMENS for an IKY Fellowship of Excellence for Postdoctoral Studies
in Greece – SIEMENS Program.
The aforementioned results came as no surprise to us, given the
incorporation of a lipophilic group as we noticed in the – limited –
1
5
relevant literature. However, the determined MICs are impressive
since the tested compounds bear a new skeleton and not an optimized
known and studied one.
VMcK is grateful for funding from the Carlsberg Foundation (grant
CF15-0675) for the X-ray diffractometer in SDU.
To investigate the specificity of compounds 4a-e, 4g and 3e as a
potential antibacterial agents, they were assayed against a mammalian
cell line and human red blood cells (RBCs) to determine general cell
toxicity.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127107. These data include MOL files
and InChiKeys of the most important compounds described in this ar-
ticle.
The cytotoxicity of all the aforementioned compounds as a growth
inhibitor of human embryonic kidney cells (HEK293) was also de-
termined by a dose response (0.25 μg/mL–32 μg/mL) cell viability
assay, and found non-toxic up to the highest tested concentration of
References
3
3
2 μg/mL, i.e. CC50 deemed > 32 μg/mL.
None of the tested active compounds showed hemolytic activity at
2 μg/mL, except 4c which exhibited HC10 (10% hemolytic con-
1
.
Tacconelli E, Carrara E, Savoldi A, et al. Lancet Infect Dis. 2018;18:318.
2. (a) de Kraker MEA, Stewardson AJ, Harbarth S. PLoS Med. 2016;13:e1002184;
(
b) Tacconelli E, Sifakis F, Harbarth S, et al. Lancet Infect Dis. 2018;18:e99.
centration) of 20.4 μg/mL.
3
4
.
.
Sugden R, Kelly R, Davies S. Nat Microbiol. 2016;1:16187.
(a) Louis LB. J Infect Dis. 2008;197:1079;
Therefore, compounds 4b, 4d and 4e can be considered as the most
promising compounds for further studies against MRSA and other Gram
positive bacteria, given their high inhibitory activity and low toxicity
(b) Boucher HW, Talbot GH, Edwards JE, et al. Clin Infect Dis. 2009;48:1.
5. Rodvold KA, McConeghy KW. Clin Infect Dis. 2014;58:S20.
6
7
8
.
.
.
Henry F, Chambers MD, Engl N. J Med. 2005;352:1485.
Moran GJ, Krishnadasan A, Gorwitz RJ, Engl N. J Med. 2006;355:666.
Han LL, McDougal LK, Gorwitz RJ, Mayer KH, Patel JB. J Clin Microbiol.
2007;45:1350.
(
> 8-fold specificity of antibacterial activity over general cell toxicity).
Taking into account the compliance with Lipinski’s rule of five, among
them, 4b seems to be the best candidate, since it has molecular
weight = 355.43 Da, logP = 4.96, number of hydrogen acceptors = 5
9
.
Frazee BW, Lynn J, Charlebois ED, Lambert L, Lowery D, Perdreau-Remington F. Ann
Emerg Med. 2005;45:311.
3
0
and number of hydrogen bond donors = 1. According to this rule,
1
0. Hiramatsu K. Lancet Infect Dis. 2001;1:147.
most “drug-like” molecules have molecular weight ≤ 500 Da, logP ≤ 5,
number of hydrogen bond acceptors ≤ 10 and number of hydrogen
bond donors ≤ 5. Molecules violating one or more of these rules may
have a problem with bioavailability.
11. Boucher HW, Sakoulas G. Clin Infect Dis. 2007;45:601.
1
1
2. Tsiodras S, Gold HS, Sakoulas G, et al. Lancet. 2001;358:207.
3. (a) Schobert R, Schlenk A. Bioorg Med Chem. 2008;16:4203;
(
b) Jeong Y-C, Moloney MG. Synlett. 2009;15:2487;
(c) Mattio L, Musso L, Scaglioni L, Pinto A, Martino PA, Dallavalle S. Beilstein J Org
Chem. 2018;14:2482;
In conclusion, a series of 14 functionalized N-acetyl-5-arylidenete-
tramic acids were synthesized to investigate the scope of our
(
d) Holloway CA, Matthews CJ, Jeong Y-C, Moloney MG, Roberts CF, Yacoob M.
6

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
1
Chem Biol Drug Des. 2011;78:229;
(EtOH); R
δ: 2.44 (s, 3H, CH
J = 8.6 Hz, Ph), 6.93 (s, 1H, CH-Ph), 7.21 (d, 2H, J = 8.6 Hz, Ph); C NMR (62.5
MHz, DMSO-d ) δ: 26.1 (CH CON), 26.3 (CH COC-3), 55.2 (CH ΟPh), 101.5 (C-3),
f
: 0.26 (Petroleum ether: Ethyl acetate 9:1); H NMR (500MHz, DMSO-d
6
)
(
(
e) Klapper M, Paschold A, Zhang S, et al. Biol. 2019;14:1693;
3
CON), 2.51 (s, 3H, CH COC-3), 3.78 (s, 3H, CH OPh), 6.90 (d, 2H,
3
3
13
f) Panduwawala TD, Iqbal S, Thompson AL, et al. Org Biomol Chem. 2019;17:5615.
1
1
4. (a) Xu W-Q, Chen M, Wang K-Y, Ren Z-J, Lu A-M, Yang C-L. Molecules. 2016;21:355;
6
3
3
3
(
(
(
b) Wang S, Bao L, Song D, Wang J, Cao X, Ke S. Eur J Med Chem. 2019;179:567;
c) Matiadis D, Tsironis D, Stefanou V, et al. J Inorg Biochem. 2019;194:65;
d) Dhavan AA, Ionescu AC, Kaduskar RD, et al. Bioorg Med Chem Lett.
113.3 (PhCH=), 118.0, 127.1, 127.4, 132.4, (Ph), 132.6 (C-5), 159.5 (C-2), 167.7
(CH
3
CON), 179.5 (CH
3
COC-3), 191.9 (C-4); Anal. Calcd for C16
H
5
15NO : C, 63.78, H,
5.02, N, 4.65. Found C, 63.99, H, 4.87, N, 4.58. N-acetyl-3-acetyl-5-(4-methylben-
zylidene)-tetramic acid (3b): Yellow, Yield: 97mg (68%); Mp: 125-126°C; Rf: 0.42
(Petroleum ether: Ethyl acetate: Acetic acid 3:7:0.1); 1H NMR (500MHz, DMSO-d6)
2
016;26:1376.
5. (a) Burmeister HR, Bennett GA, Vesonder RF, Hesseltine CW. Antimicrob. Ag.
Chemother. 1974;5:634;
3 3 3
δ: 2.30 (s, 3H, CH Ph), 2.40 (s, 3H, CH CON), 2.48 (s, 3H, CH COC-3), 3.78 (s, 3H,
CH OPh), 6.85 (s, 1H, CH-Ph), 7.11 (d, 2H, J = 7.1 Hz, Ph), 7.13 (d, 2H, J = 7.1 Hz,
3
13
(
(
(
b) Marfori EC, Kajiyama S, Fukusaki E, Kobayashi A. Z Naturforsch. 2002;57c:465;
c) Gänzle MG. Appl Microbiol Biotechnol. 2004;64:326;
Ph); C NMR (62.5 MHz, DMSO-d6) δ: 20.9 (CH
3
Ph), 26.3 (CH
3
CON), 26.4
d) Gandhi NM, Nazareth J, Divekar PV, Kohl H, de Souza NJ. J Antibiot.
(CH COC-3), 101.1 (C-3), 116.7 (PhCH=), 128.2 (Ph), 128.3 (Ph), 130.2 (Ph), 130.4
3
1
973;26:797;
(Ph), 131.5 (C-5), 167.7 (C-2), 168.2 (CH
3
CON), 179.7 (CH COC-3), 191.3 (C-4);
3
(
(
(
e) Yendapally R, Hurdle JG, Carson EI, Lee RB, Lee RE. J Med Chem. 2008;51:1487;
f) Matsuo K, Kitaguchi I, Takata Y, Tanaka K. Chem Pharm Bull. 1980;28:2494;
g) Rosen T, Fernandes PB, Marovich MA, Shen L, Mao J, Pernet AG. J Med Chem.
Anal. Calcd for C16H15NO4: C, 67.36, H, 5.30, N, 4.91. Found C, 67.52, H, 5.30, N,
4.77. N-acetyl-3-acetyl-5-(4-chlorobenzylidene)-tetramic acid (3c): Yellow, Yield:
135mg (88%); Mp: 116-117°C; Rf: 0.34 (Petroleum ether: Ethyl acetate: Acetic acid
1
989;32:1062;
3
3:7:0.1); 1H NMR (500MHz, DMSO-d6) δ: 2.42 (s, 3H, CH CON), 2.48 (s, 3H,
(
h) Hurdle JG, Yendapally R, Sun D, Lee RE. Antimicrob. Ag. Chemother.
009;53:4028;
i) Jones RCF, Bullous JP, Law CCM, Elsegood MRJ. Chem Commun. 2014;50:1588.
6. For a review on naturally occurring tetramic acids see: Mo X, Li Q, Ju J. RSC Adv.
CH
Hz, Ph); 13C NMR (62.5 MHz, DMSO-d6) δ: 26.3 (CH
(C-3), 115.0 (PhCH=), 127.7, 130.5, 131.8, 132.4 (Ph), 134.2 (C-5), 167.8 (C-2),
3
COC-3), 6.87 (s, 1H, CH-Ph), 7.22 (d, 2H, J = 8.0 Hz, Ph), 7.36 (d, 2H, J = 8.0
2
3
CON), 26.4 (CH COC-3), 101.1
3
(
1
1
168.2 (CH
3
CON), 179.6 (CH
3
COC-3), 191.1 (C-4); Anal. Calcd for C15
H
12ClNO : C,
4
2
014;4:50566.
58.93, H, 3.96, N, 4.58. Found C, 58.80, H, 5.69, N, 4.78. N-acetyl-3-acetyl-5-(4-
fluorobenzylidene)-tetramic acid (3d): Off-white, Yield: 113 mg (78%); Mp: 125-
126°C; Rf: 0.33 (Petroleum ether: Ethyl acetate: Acetic acid 3:7:0.1); 1H NMR (500
7. (a) For a review on structure and synthesis of tetramic acids see: Royles BJL. Chem
Rev. 1981;1995:95;
(
(
(
(
(
b) Lacey RN. J Chem Soc. 1954:850;
3 3
MHz, DMSO-d6) δ: 2.41 (s, 3H, CH CON), 2.48 (s, 3H, CH COC-3), 6.87 (s, 1H, CH-
13
c) Jones RCF, Tankard M. J Chem Soc, Perkin Trans. 1991,;1:240;
d) Jouin P, Castro B, Nisato D. J Chem Soc, Perkin Trans. 1987,;1:1177;
e) DeShong P, Cipollina JA, Lowmaster NK. J Org Chem. 1988;53:1356;
f) Kempf K, Schmitt F, Bilitewski U, Schobert R. Tetrahedron. 2015;71:5064.
Ph), 7.14 (t, 2H, J = 7.2 Hz, Ph), 7.26 (t, 2H, J = 7.2 Hz, Ph); C NMR (125 MHz,
3 3
DMSO-d6) δ: 26.4 (CH CON), 26.6 (CH COC-3), 100.9 (C-3), 114.4 (d, Ph, JCF = 21.
6 Hz), 114.7 (d, Ph, JCF = 21.6 Hz), 115.03 (PhCH=), 129.9 (Ph), 131.8 (d, Ph, JCF
= 3.1 Hz), 131.9 (d, Ph, JCF = 3.1 Hz), 132.2 (d, Ph, JCF = 8.3 Hz), 132.3 (d, Ph, JCF
1
8. (a) For a review on metal-catalyzed synthesis of tetramic acids see: Matiadis D.
3 3
= 8.3 Hz), 159.5 (C-5), 163.4 (C-2), 167.7 (CH CON), 168.4, 179.8 (CH COC-3),
Catalysts. 2019;9:50;
191.1 (C-4).; Anal. Calcd for C15
4
H12FNO : C, 62.28, H, 4.18, N, 4.84. Found C, 62.09,
(
(
(
(
(
b) Ishida T, Kobayashi R, Yamada T. Org Lett. 2014;16:2430;
c) Xu C-P, Huang P-Q, Py S. Org Lett. 2012;14:2034;
H, 4.12, N, 5.02. N-acetyl-3-acetyl-5-(4-trifluoromethylbenzylidene)-tetramic acid
(3e): White, Yield: 112mg (66%); Mp: 110-111°C; Rf: 0.21 (Petroleum ether: Ethyl
d) Gabriele B, Plastina P, Salerno G, Costa M. Synlett. 2005;6:935;
e) Dittmer DC, Avilov DV, Kandula VS, et al. ARKIVOC. 2010;6:61;
f) Iida T, Hori K, Nomura K, Yoshii E. Heterocycles. 1839;1994:38.
3
acetate: Acetic acid 3:7:0.1); 1H NMR (500 MHz, DMSO-d6) δ: 2.33 (s, 3H, CH CON),
2.46 (s, 3H, CH
2H, J = 8.4 Hz, Ph); C NMR (62.5 MHz, DMSO-d6) δ: 27.75 (CH
100.18 (C-3), 111.99, 121.9, 131.5, 137.2 (q, CF , JCF=272.9 Hz), 124.7 (q, Ph,
CF= 3.4 Hz), 130.59 (q, C-CF , JCF = 33.2 Hz), 131.04 (C-5), 140.9 (Ph), 168.3
(C=O), 170.88 (CH -C=O-N), 180.46 (C-OH), 190.80 (CH -C=Ο); Anal. Calcd for
4
NO : C, 56.64, H, 3.57, N, 4.13. Found C, 56.88, H, 3.66, N, 3.98. N-acetyl-
3
COC-3), 6.79 (s, 1H, CH-Ph), 7.35 (d, 2H, J = 8.4 Hz, Ph), 7.62 (d,
1
3
3
3
), 28.42 (CH ),
1
2
9. (a) For a review on tetramic and tetronic acids as ligands see: Athanasellis G,
3
Igglessi-Markopoulou O, Markopoulos J. Bioinorg. Chem. Appl. 2010:11 Article ID
J
3
3
15056;
3
3
(
b) For a review on 3-acyl tetramic acids as metal binders see: Zaghouani M, Nay B.
C
16
H
12
F
3
Nat Prod Rep. 2016;33:540.
3-acetyl-5-(4-nitrobenzylidene)-tetramic acid (3f): Dark orange, Yield: 95 mg (60%);
Mp: 116-117°C; Rf: 0.36 (dichloromethane: methanol: Acetic acid 95:5:1); 1H NMR
0. (a) Peukert S, Sun Y, Zhang R, et al. Bioorg Med Chem Lett. 1840;2008:18;
(
b) Jeong Y-C, Moloney MG. Beilstein J Org Chem. 1899;2013:9.
3 3
(500 MHz, DMSO-d6) δ: 2.38 (s, 3H, CH CON), 2.48 (s, 3H, CH COC-3), 6.87 (s, 1H,
13
2
2
1. Hosseini M, Kringelum H, Murray A, Tønder JE. Org Lett. 2006;8:2103.
2. Yoshinari T, Ohmori K, Shrems MG, Pfaltz A, Suzuki K. Angew Chem Int Ed.
CH-Ph), 7.42 (t, 2H, J = 8.4 Hz, Ph), 8.12 (t, 2H, J = 8.4 Hz, Ph); C NMR (62.5
MHz, DMSO-d6) δ: 26.3 (CH CON), 26.9 (CH COC-3), 100.3 (C-3), 111.9 (PhCH=),
122.6, 130.6 (Ph), 133.4 (C-5), 143.1, 145.8 (Ph), 167.9 (C-2), 168.6 (CH CON),
3
3
2
010;49:881.
3
2
2
3. Cherian PT, Cheramia MN, Marreddy RKR, Fernando DM, Hurdle JG, Lee RE. Bioorg
Med Chem Lett. 2018;28:3105.
3 12 2 6
179.6 (CH COC-3), 190.5 (C-4); Anal. Calcd for C15H N O : C, 56.97, H, 3.82, N, 8.
86. Found C, 56.60, H, 3.72, N, 9.04. N-acetyl-5-benzylidene-3-octanoyltetramic acid
(4b): White solid, Yield: 99 mg (56%); Mp: 180 °C (dec.); Rf: 0.40 (petroleum ether/
4. (a) Matiadis D, Igglessi-Markopoulou O, McKee V, Markopoulos J. Tetrahedron.
2
014;70:2439;
ethyl acetate 1:1); 1H NMR (500 MHz, DMSO-d6) δ: 0.84 (t, 3H, CH
3
CH
CON), 2.73 (br,
COC-3), 6.68 (s, 1H, CH-Ph), 7.13-7.28 (br m, 5H, Ph); 13C NMR (62.5
MHz, DMSO-d6) δ: 13.9 [CH (CH ], 22.1, 24.7 [(CH ], 26.4 (CH CON), 27.8, 28.
7, 29.0, 31.2 [(CH ], 98.1 (C-3), 118.9 (PhCH=), 127.0, 127.1, 127.3, 127.5, 129.7
2 2
CH ), 1.24
(
(
b) Petroliagi M, Igglessi-Markopoulou O. J Chem Soc Perkin Trans. 1997;1:3543;
c) Petroliagi M, Igglessi-Markopoulou O, Markopoulos J. Heterocycl Commun.
(br, 8H, CH
3 2 4 2 2 3
(CH ) ), 1.47 (br, 2H CH CH COC-3), 2.45 (s, 3H, CH
2H, CH CH
2
2
2
000;6:157;
3
2
)
6
2
)
6
3
(
d) Athanasellis G, Gavrielatos E, Igglessi-Markopoulou O. J Heterocycl Chem.
001;38:1203;
e) Mawer IM, Kulagowski JJ, Leeson PD, Grimwood S, Marshall GR. Bioorg Med
Chem Lett. 1995;5:2643;
f) Stachel H-D, Harigel KK, Poschenrieder H, Burghard H. J Heterocycl Chem.
980;17:1195.
2 6
)
2
3 3
(Ph), 130.4 (C-5), 167.8 (C-2), 170.8 (CH CON), 179.3 (CH COC-3), 195.8 (C-4);
(
Anal. Calcd for C21
H
4
25NO : C, 70.96, H, 7.09, N, 3.94. Found C, 71.22, H, 6.88, N, 3.
82. N-acetyl-5-benzylidene-3-decanoyltetramic acid (4c): Off-white solid, Yield: 105
mg (55%); Mp: 166 °C (dec.); Rf: 0.46 (petroleum ether/ethyl acetate 1:1); 1H NMR
(
1
(500 MHz, DMSO-d6) δ: 0.84 (br s, 3H, CH
3
CH
2
CH
2
), 1.22 (br, 12H, CH
3
(CH
2 6
) ), 1.
2
2
5. (a) Matiadis D, Igglessi-Markopoulou O. Eur J Org Chem. 2010:5989;
46 (br, 2H CH
2
CH
2
COC-3), 2.46 (s, 3H, CH
3
CON), 2.72 (br, 2H CH
2
CH
2
COC-3), 6.67
1
3
(
(
b) Athanasellis G, Gavrielatos E, Igglessi-Markopoulou O. Synlett. 2001;10:1653;
c) Detsi A, Markopoulos J, Igglessi-Markopoulou O. Chem Commun. 1996:1323.
(s, 1H, CH-Ph), 7.14-7.26 (br m, 5H, Ph), C NMR (62.5 MHz, DMSO-d6) δ: 13.9
[CH
3
(CH
2
)
8
], 22.1, 24.5, 24.7 [(CH
], 98.8 (C-3), 118.6 (PhCH=), 127.1, 127.4, 128.1, 128.4 (Ph), 129.7
CON), 178.7 (CH COC-3), 194.1 (C-4); Anal. Calcd for
2 8 3
) ], 26.7 (CH CON), 28.5, 28.7, 28.9, 29.0, 31.3,
6. (a) Athanasellis G, Igglessi-Markopoulou O, Markopoulos J. Bioinorg. Chem. Appl.
33.7 [(CH )
2 8
2
010:11 Article ID 315056;
b) Gavrielatos E, Mitsos C, Athanasellis G, et al. J Chem Soc, Dalton Trans.
001:639;
(C-5), 167.8 (C-2), 170.8 (CH
3
3
(
C
23
H
4
29NO : C, 72.04, H, 7.62, N, 3.65. Found C, 71.46, H, 7.42, N, 3.91. N-acetyl-5-
2
benzylidene-3-tetradecanoyltetramic acid (4d): Off-white solid, Yield: 110 mg (50%);
(
c) Markopoulou O, Markopoulos J, Nicholls D. J Inorg Biochem. 1990;39:307.
Mp: 134°C (dec.); Rf: 0.19 (petroleum ether/ethyl acetate 8:2); 1H NMR (500 MHz,
2
7. Synthesis of tetramic acids and analytical data for novel compounds: The synthetic
procedure was adapted from our previously published protocol with proper mod-
3 2 2
DMSO-d6) δ: 0.84 (br t, 3H, CH CH CH ), 1.23 (br s, 20H, CH3(CH2)10CH2CH2), 1.
46 (br, 2H CH
(s, 1H, CH-Ph), 7.13-7.30 (br m, 5H, Ph); C NMR (62.5 MHz, DMSO-d6) 13.9
[CH (CH 12], 22.1, 24.4, 24.7[(CH 12], 26.6 (CH CON), 28.7, 29.0, 29.1, 31.3
[(CH 12], 98.3 (C-3), 115.7 (PhCH=), 127.1, 127.4, 127.6, 130.0 (Ph) 129.8 (C-5),
2 2 3 2 2
CH COC-3), 2.50 (s, 3H, CH CON), 2.73 (br, 2H CH CH COC-3), 6.65
2
4a
13
ifications.
To a suspension of NaH (60% in mineral oil, 40 mg, 1.00 mmol) in dry
THF at 0°C, the active methylene compound (β-ketoesters, 1.00 mmol) was added
dropwise or portionwise over 10 minutes. After stirring for 1h, the appropriate ox-
azolone (0.50 mmol) was added at 0°C and the mixture was left under vigorous
stirring at room temperature overnight. The resulting mixture was concentrated
at < 40°C and the thick slurry was diluted with water (1.0 mL). The suspension was
3
2
2
)
2
)
3
)
3 3
130.3 (Ph), 167.9 (C-2), 169.2 (CH CON), 182.9 (CH COC-3), 194.6 (C-4); Anal.
Calcd for C27
H
4
37NO : C, 73.77, H, 8.48, N, 3.15. Found C, 74.16, H, 8.70, N, 2.97. N-
acetyl-3-butyryl-5-(4-methoxybenzylidene)-tetramic acid (4g): Bright yellow, Yield:
109mg (66%); Mp: 89-91°C (EtOH); Rf: 0.42 (Petroleum ether: Ethyl acetate 9:1); 1H
washed carefully with small quantity of Et
2
O for 3a-f and 4g or petroleum ether for
4
3
a-f and acidified with 10% hydrochloric acid in an ice bath. The precipitated solids
a-f and 4g were filtered, washed with water (x2) and petroleum ether (x2) and
NMR (500 MHz, DMSO-d6) δ: 0.92 (t, 3H, J = 7.4 Hz, CH
3
2
CH
= 7.4 Hz, CH CH
3
CH CON), 3.79 (s, 3H, CH OPh), 6.90 (d, 2H, J = 8.4 Hz, Ph), 6.94 (s, 1H, CH-Ph), 7.
2
2
CH
2
), 1.59 (sext, 2H, J
3
CH
2
CH
2
), 2.83 (t, 2H, J = 7.4 Hz, CH
3
CH
), 2.50 (s, 3H,
recrystallized from methanol or ethanol to yield off-white to bright yellow crystals.
3
1
3
For 4a-f, the aqueous phase was extracted with DCM (x3), dried with Na
2
SO
4
and the
3 2 2
21 (d, 2H, J = 8.4 Hz, Ph); C NMR (62.5 MHz, DMSO-d6) δ: 13.8 (CH CH CH ),
organic solvent was evaporated to give the crude products, which were purified ei-
ther by flash chromatography using gradient elution petroleum ether:ethyl acetate/
dichloromethane:methanol or preparative TLC. The reaction completion was mon-
17.7 (CH CH CH ), 26.3 (CH CON), 26.4 (CH CH CH ), 55.2 (OCH ), 101.0 (C-3),
3
2
2
3
3
2
2
3
113.3 (PhCH=), 117.9, 127.2, 127.4, 132.3 (Ph), 132.6 (C-5), 159.5 (C-2), 167.7
(CH
3
CON), 179.2 (CH
3
COC-3), 195.3 (C-4); Anal. Calcd for C18
H
5
19NO : C, 65.64, H,
3
itored by TLC using UV light and FeCl stain. N-acetyl-3-acetyl-5-(4-methox-
5.82, N, 4.25. Found C, 65.45, H, 5.77, N, 4.39.
ybenzylidene)-tetramic acid (3a): Bright yellow, Yield: 137mg (91%); Mp: 123-124°C
28. Oikawa Y, Yoshioka T, Sugano K, Yonemitsu O. Org Syn. 1985;63:198.
7

D. Matiadis, et al.
Bioorganic & Medicinal Chemistry Letters 30 (2020) 127107
2
9. Synthesis and analytical data for 5: N-Acetyl-3-acetyl-5-benzylidene tetramic acid A
4a (136mg, 0.50mmol) was dissolved in 7mL of dry MeOH under reflux. Methanolic
(s), 1385 (m), 1240 (m); 1H NMR (500 MHz, DMSO-d6) δ: 2.55 (s, 3H, CH
57 (s, 3H, CH CO), 6.94 (s, 1H, CH-Ph), 7.25 (d, 2H, J = 7.6 Hz, Ph), 7.29 (d, 1H, J
= 7.4 Hz, Ph), 7.34 (t, 2H, J = 7.3 Hz, Ph), 7.45 (d, 2H, J = 7.7 Hz, Ph), 7.53 (t, 2H,
3
-C=N), 2.
2
3
solution (1mL) of aniline (93mg, 1.00mmol) was added dropwise. The brown mix-
ture was stirred under reflux for 2h when a light yellow solid precipitated. The solid
was filtered and washed with 2 portions of ethanol (2mL each). The filtrate was
evaporated under vacuum and the residual solid was washed well with ice-cold
methanol. The yellow solid was recrystallized with ethanol forming light yellow
needles. The completion of the reaction was monitored by TLC using permanganate
stain. N-acetyl-3-(1-(phenylamino)ethylidene)-5-benzylidenetetramic acid (5): Light
yellow needles. Yield: 86 mg (48%); Mp: 163-164 °C (EtOH) ; Rf: 0.65 (Petroleum
ether: Ethyl acetate 1:1); nmax/cm-1 3435 (m), 3187 (m), 1613 (s), 1574 (s), 1625
1
3
J = 7.3 Hz, Ph) 12.85 (s, 1H, NH); C NMR (62.5 MHz, DMSO-d6) δ: 16.0
(CH C=N), 26.5 (CH CO), 65.9 (C-3), 116.2 (PhCH=), 125.6, 127.7, 128.2, 129.5,
3
3
3
129.8, 130.0, 134.8 (Ph), 135.5 (C-5), 167.8 (CH CON), 169.8 (C-2), 184.0 (C-4);
Anal. Calcd for C21H18N2O3: C, 72.82, H, 5.24, N, 8.09. Found C, 73.05, H, 5.49, N,
7.87.
30. Lipinski CA, Lombardo F, Dominy BW, Feeny PJ. Adv Drug Deliv Rev. 1997;23:4.
31. Blaskovich MAT, Zuegg J, Elliott AG, Cooper MA. ACS Infect Dis. 2015;1:285.
8