Synthesis of bis-glyoxylamide peptidomimetics derived from bis-n-acetylisatins linked at C5 by a methylene or oxygen bridge

Article abstract of DOI:10.3390/molecules24234343

The bis-glyoxylamide peptidomimetics have been synthesized from bis-N-acetylisatins linked at C5 by ring-opening with alcohols, amines, and amino acid methyl ester hydrochlorides. X-ray images of single crystals of bis-glyoxylamide peptidomimetics have been obtained.

Full text of DOI:10.3390/molecules24234343

molecules
Article
Synthesis of Bis-Glyoxylamide Peptidomimetics
Derived from Bis-N-acetylisatins Linked at C5 by a
Methylene or Oxygen Bridge
Venty Suryanti 1, Ruonan Zhang 2, Vina Aldilla 2, Mohan Bhadbhade 3, Naresh Kumar 2 and
David StC Black 2,
*
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Sebelas Maret University,
Jl. Ir. Sutami 36A Surakarta, Jawa Tengah 57126, Indonesia; venty@mipa.uns.ac.id
School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia;
zhrn_516@hotmail.com (R.Z.); vina.r.aldilla@student.unsw.edu.au (V.A.); n.kumar@unsw.edu.au (N.K.)
Solid State and Elemental Analysis Unit, Mark Wainwright Analytical Centre, Division of Research,
The University of New South Wales, Sydney, NSW 2052, Australia; m.bhadbhade@unsw.edu.au
Correspondence: d.black@unsw.edu.au; Tel.: +612-9385-4657
2
3
*
Academic Editor: Mário J.F. Calvete
Received: 29 October 2019; Accepted: 22 November 2019; Published: 27 November 2019
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The bis-glyoxylamide peptidomimetics have been synthesized from bis-N-acetylisatins
linked at C5 by ring-opening with alcohols, amines, and amino acid methyl ester hydrochlorides.
X-ray images of single crystals of bis-glyoxylamide peptidomimetics have been obtained.
Keywords: antimicrobial peptides; N-acetylisatins; glyoxylamides; peptidomimetics; quorum sensing
1. Introduction
Many peptides show strong biological activities and are considered as a potential source of
therapeutic agents. Prior work in this field has been well reviewed and describes examples of bioactive
compounds [
low bioavailability, inability to cross cell membranes, and in vivo instability [
issues, peptidomimetics are designed by mimicking existing peptide structures and/or function but
whose backbone is not only based on -amino acids. The modification of the peptide backbone by
1,2]. However, their use as therapeutics has some limitations, such as poor absorption,
3,4
]. To address these
α
incorporation of a large range of non-proteinogenic amino acids enhances the proteolytic stability of
the molecules, increasing their function as drugs against diseases, such as viral and bacterial infections,
cancer and autoimmune diseases. Thus, synthesis of such peptidomimetics is a critical and promising
approach in the development of novel pharmaceutical compounds [5–13].
Our group has reported libraries of mono-glyoxylamides derived from N-acylisatins [14–16].
Acyclic and cyclic glyoxamide derivatives resulting from ring-opening reactions of N-chloroacetylisatins
expressed quorum sensing (QS) inhibition activity against P. aeruginosa MH602 and E. coli MT102 [17].
The ring-opening reactions of N-naphthoylisatins with amines and amino acids have been examined to
give guanidine-embedded amphipathic glyoxamide-based peptidomimetics and the example of methyl
{2-[2-(2-naphthylamido)phenyl]-2-oxoacetyl}arginylargininate dihydrochloride demonstrated the greatest
disruption of established biofilms by 60–65% in S. aureus, P. aeruginosa, and S. marcescens [18]. Related
N-sulfonylphenylglyoxamide derivatives have been synthesized as small molecular antimicrobial peptide
(AMP) mimics by ring-opening reactions of N-sulfonylisatins with N,N-dimethylethane-1,2-diamine or
N,N-dimethylpropane-1,3-diamine and were converted into their hydrochloride or iodide salts [19].
In our previous research, we reported the synthesis of bis-glyoxylamide peptidomimetics from
oxalyl-bis-isatins and bis-acylisatins linked through their carbonyl groups and some analogs showed
Molecules 2019, 24, 4343; doi:10.3390/molecules24234343
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4343
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potent QS inhibitory activity against Gram-positive bacteria [20,21]. In order to expand the library of
bis-glyoxylamides, we have designed novel bis-glyoxylamide peptidomimetics from bis-N-acetylisatins
linked at C5. In this study, we synthesized bis-N-acetylisatins linked by a C5 methylene or oxygen
bridge and their nucleophilic ring-opening reactions were examined with alcohols, amines, and amino
acid methyl ester hydrochlorides.
2. Results and Discussion
2.1. Synthesis of bis-N-acetylisatins
The bis-isatyl methane
any spectroscopic information [22
here. As a starting point, the intermediate bis-isonitrosoacetanilide
the Sandmeyer isonitrosoacetanilide isatin synthesis [25]. The starting material of methylene bridged,
4,4’-methylenedianiline was initially dissolved in aqueous hydrochloric acid and heated in the
presence of chloral hydrate and hydroxylamine. The product crystallized out after being left overnight
at room temperature and was recrystallized from ethyl acetate to give bis-isonitrosoacetanilide in
5
and the bis-isatyl ether
24]. Therefore, details of our modified syntheses are included
was synthesized by applying
6 have been reported many years ago, but without
–
3
1
3
10% yield as yellow crystals. A consideration of the mechanism shows the generation of a large
amount of HCl in the reaction. Therefore, in an attempt to increase the yield, the concentrated HCl
solvent was replaced by dioxane. The 4,4’-methylenedianiline
1 was dissolved in the minimum
amount of 1,4-dioxane instead of hydrochloric acid and this led to the successful synthesis of the target
bis-isonitrosoacetanilide 3 to 55% yield (Scheme 1).
Scheme 1. Synthesis of bis-N-acetylisatins 7 and 8.
The reaction was continued by dissolving bis-isonitrosoacetanilide
3
in concentrated sulfuric
was then
in 64%
acid and stirring the solution to afford the bis-isatin in high yield (88%). The bis-isatin
5
5
suspended in acetic anhydride and heated at reflux to generate the target bis-N-acetylisatin
7
1
yield. In the H NMR spectrum of compound
7
, the two CH3 groups resonated as a singlet that
2.59, the CH2 appeared as a singlet at 4.11 and the aromatic protons resonated
as a singlet at δ 7.69, a doublet of doublet at δ 7.73, and a doublet at δ 8.85.
In the same manner, the structurally similar bis-N-acetylisatins with oxygen as the linkage was
generated from 4,4’-oxydianiline . The ring closure of intermediate bis-isonitrosoacetanilide gave
in 83% yield. The targeted bis-N-acetylisatin was obtained in 55% yield by acetylating
with acetic anhydride (Scheme 1). Following the synthesis of bis-N-acetylisatins and
integrated for 6H at
δ
δ
8
2
4
bis-isatin
bis-isatin
6
6
8
7
8,
their nucleophilic ring-opening reactions were then carried out with alcohols, amines and amino acid
methyl ester hydrochlorides, in order to generate the new class of peptide mimics.

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2.2. Ring-Opening Reactions of bis-N-acetylisatins with Alcohols
The ring-opening reactions of bis-N-acetylisatins
MeOH and EtOH. Bis-N-acetylisatin
7
and
8
were initially examined with the alcohols
7
was dissolved in anhydrous methanol and the solution was
stirred for 24 h at room temperature. The resulting yellow crystals were recrystallized with some loss
1
to give methyl bis-glyoxylacetate 9 in 34% yield (Scheme 2). H NMR spectrum of bis-glyoxylacetate
9
revealed three singlets at δ 3.89, 3.90, and 10.91 corresponding to CH3, CH2, and two NH protons,
respectively. The resonances of aromatic protons appeared as a singlet at
7.35 and 8.66.
δ 7.37 and two doublets at δ
Scheme 2. Ring-opening reactions of bis-N-acetylisatins 7 and 8 with alcohols.
The analogous reaction with ethanol did not show the formation of any new products over 24 h,
as confirmed by TLC analysis. Thus, the reaction mixture was heated at reflux for 3 h and during that
time the solution turned dark yellow. A yellow solid precipitated upon cooling to room temperature
and was recrystallized to give bis-glyoxylacetate 10 in 51% as yellow crystals. The analogous reactions
of the bis-N-acetylisatin 8 afforded the bis-glyoxylacetates 11 and 12.
2.3. Ring-Opening Reactions of bis-N-acetylisatins with Amines
Theprimaryandsecondaryamines, butylamineandpiperidine, werereactedwiththebis-N-acetylisatins
7
and
15
18 were obtained in yields of 53–98% (Scheme 3). In the 1H NMR spectrum of bis-glyoxylamide
15, the CH2 linkage was present as a singlet at 3.91, the butylamine carbon chain resonated as four
multiplets at 0.86–0.88, 1.28–1.35, 1.47–1.52, and 3.28–3.32, the aromatic protons appeared as a doublet
of doublets at 7.33 and two doublets at 8.07 and 8.50, and four NH protons resonated as two singlets
at δ 6.81 and 10.79.
8 by stirring the reaction mixtures for 5 h at room temperature. The bis-glyoxylamide products
–
δ
δ
δ
δ
Scheme 3. Ring-opening reactions of bis-N-acetylisatins 7 and 8 with amines.
The ring-opening reactions of bis-N-acetylisatins using primary amines with longer alkyl chains,
8
such as hexylamine and dodecylamine were also investigated, but these reactions did not proceed at
room temperature. However, by heating the reaction mixtures at reflux overnight the bis-glyoxylamide
products 19 and 20 were obtained as yellow solids in moderate yields.

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2.4. Ring-Opening Reactions of bis-N-acetylisatins with Amino Acid Methyl Esters
The reaction mixtures of bis-N-acetylisatins
salts and sodium hydrogen carbonate in dichloromethane or acetonitrile at room temperature overnight
gave the corresponding bis-glyoxylamide peptide mimics 21 30 in yields of 26–80% as yellow solids
(Scheme 4, Table 1). The 1H NMR spectrum of compound 25 was typical for these peptide mimics and
revealed two singlet peaks at 7.94 and 10.87, which integrated for 2H each and corresponded to the
four NH groups. The valine subunit was identified by two multiplets and one doublet at 0.86–0.92,
2.16–2.21, and 4.54 which corresponded to the four methyl groups, the -proton and the -proton,
7 and 8 with amino acid methyl ester hydrochloride
–
δ
δ
α
β
respectively. The aromatic protons appeared as a multiplet at δ 7.31–7.41 and a doublet at δ 8.51.
Scheme 4. Ring-opening reactions of bis-N-acetylisatins 7 and 8 with amino acid methyl ester
hydrochlorides.
Table 1. Bis-glyoxylamide peptidomimetics 21–32.
No
Product
X
Amino Acid Methyl Esters
Yields (%)
1
2
3
4
5
6
7
8
9
21
22
23
24
25
26
27
28
29
30
31
32
CH2
CH2
CH2
CH2
CH2
CH2
O
O
O
O
O
Gly OMe
l-Leu OMe
l-Met OMe
l-Val OMe
d-Phe OMe
l-Phe OMe
Gly OMe
l-Leu OMe
l-Met OMe
l-Val OMe
d-Phe OMe
l-Phe OMe
55
57
51
80
28
48
28
26
76
46
68
78
10
11
12
O
The bis-glyoxylamide peptidomimetics were recrystallized from a range of solvents via slow
evaporation of the solvent at room temperature. Crystals suitable for X-ray crystallography were
successfully obtained for compounds 22–24 from methanol. Single crystal X-ray structure determination
was carried out on compounds 22–24.
Figure 1 shows the Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagrams for the molecules 22
and for 23 and 24 (only one of the diastereoisomers similar to 22 is shown). In all the compounds, the
bulky substituents, probably to avoid a steric clash, adopt trans positions with respect to each other.
The side chains are orientationally disordered in 22 and 24, while this is not observed in the structure
of 23 that contains heavy sulfur atom. The core of the structure is well ordered. Strong intramolecular
N-H···O hydrogen bonds (shown as dotted lines in Figure 1) maintain the planarity of each half of the

Molecules 2019, 24, 4343
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molecule. Weaker intramolecular C-H···O (C6-H···O1) contacts restrict the planarity of these moieties.
However, the overall molecular framework is angled because of the central CH2 link.
Figure 1. ORTEP diagrams of compound 22 (a), compound 23 (b), and compound 24 (c).
There is only one molecule in the asymmetric unit of 22 in orthorhombic space group P21212,
whereas there are two molecules (diastereoisomers) in the asymmetric unit of 23 as well as in 24
in monoclinic space group C2 (Figure 2). The ring-opening reaction takes place with retention of
configuration, as shown by optical rotation measurements. One point that establishes this issue
unambiguously is the space group in which these compounds have crystallized. They are all chiral
space groups (P2(1)2(1)2 and two in C2), which by the absence of mirror (or glide) symmetry, allow
only one of the enantiomers.

Molecules 2019, 24, 4343
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Figure 2. Two molecules in the asymmetric unit of 23 (a) and 24 (b).
3. Materials and Methods
Melting points were measured using a Reichert microscope (Gallenkamp hot stage apparatus;
Mettler-Toledo Ltd, Sydney, Australia) and are uncorrected. Infrared spectra were recorded with a
Thermo Nicolet 370 FTIR spectrometer with the sample prepared as a KBr pellet. NMR data were
recorded using a Bruker DPX300 instrument (1H 300 MHz, 13C 75.6 MHz) (Bruker Pty Ltd, Preston,
Victoria, Australia) at 25 ◦C and reported as chemical shift (
δ) relative to SiMe4. High resolution mass
spectrometric analysis was carried out at the Biomedical Mass Spectrometry Facility, UNSW, and the
spectra were recorded on Q-TOF Ultima API (Micromass; Waters, Rydalmere, NSW, Australia). Gravity
column chromatography was carried out using Merck 230–400 mesh ASTM silica gel. Supplementary
Materials contains NMR spectroscopic data.
N,N’-[4,4’-bis(4,1-phenylene)] bis [2-(hydroxyimino)acetamide]methylene (3). Concentrated sodium sulfate
aqueous solution (175 mL) and a solution of 4,40-methylenedianiline 1 (10 g, 50.4 mmol) in dioxane
(15 mL) was added to a stirred solution of chloral hydrate (18.5 g, 126.1 mmol) in water (175 mL).
The reaction mixture was heated slowly to 75 ◦C until a yellow precipitate was formed. A solution of
hydroxylamine hydrochloride (8.8 g, 126.7 mmol) in water (55 mL) was then added into the resulting
suspension. The temperature was increased to 75 ◦C and the reaction mixture was stirred for a further
2 h. The reaction mixture was cooled to room temperature and left to stand overnight. The crude
product was collected by filtration and recrystallized from ethyl acetate to give the title compound as
yellow crystals (9.44 g, 55%); mp 272–274 ◦C; IR (KBr):
1443, 1412, 1252, 1100, 1023, 999, 820, 765, 622, 510 cm−1; 1H NMR (DMSO-d6, 300 MHz):
2H, 2 x OH) (disappears on D2O exchange), 10.14 (bs, 2H, 2 x NH) (disappears on D2O exchange), 7.65
(s, 2H, 2 x C NOH), 7.59 (d, J = 8.5 Hz, 4H, ArH), 7.16 (d, J = 8.7 Hz, 4H, ArH), 3.85 (s, 2H, ArC 2Ar);
13C NMR (DMSO-d6, 75.6 MHz):
160.4 ( O), 144.4 ( HNOH), 137.2 (ArC), 136.8 (ArC), 129.2 (ArC),
H2Ar); HRMS (+ESI) m/z [M + Na]+ calcd for C17H16N4NaO4: 363.1069; found:
υ
max 3194, 2919, 2613, 1675, 1606, 1547, 1510,
12.13 (bs,
δ
H
H
δ
C
C
120.3 (ArC), 40.2 (Ar
363.1055.
C

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N,N’-[4,4’-bis(4,1-phenylene)]bis [2-(hydroxyimino)acetamide]oxide (
the same method as compound
4
). This compound was prepared by
3
from 4,40-oxydianiline 2 (10 g, 50.0 mmol) as orange needles (11.8 g,
69%); mp 221–223 ◦C; IR (KBr):
υ
3185, 2931, 2622, 1669, 1596, 1521, 1510, 1433, 1410, 1232, 1096,
max
1021, 1000, 822, 764, 619 cm−1; 1H NMR (DMSO-d6, 300 MHz):
δ
12.22 (bs, 2H, 2 x OH) (disappears on
D2O exchange), 10.22 (bs, 2H, 2 x NH) (disappears on D2O exchange), 7.69 (d, J = 9.0 Hz, 4H, ArH),
7.67 (s, 2H, 2 x C 153.2 (ArC),
NOH), 6.99 (d, J = 9.0 Hz, 4H, ArH); 13C NMR (DMSO-d6, 75.6 MHz):
160.4 ( O), 144.4 (
HNOH), 137.2 (ArC), 121.9 (ArC), 119.1 (ArC); HRMS (+ESI) m/z [M + Na]+ calcd
for C16H14N4NaO5: 365.0862; found: 365.0851.
H
δ
C
C
5,5’-Methylenediindoline-2,3-dione ( ). Compound 3
(5 g, 14.7 mmol) in small portions at 60 ◦C was
5
added to concentrated sulfuric acid (20 mL). The deep red reaction mixture was stirred at 60 ◦C for a
further 30 min. Chilled water (100 mL) was added to quench the reaction. The title compound was
collected by filtration as a red solid (3.96 g, 88%); mp > 300 ◦C; IR (KBr):
υ
max 3450, 3109, 1622, 1472,
1271, 1200, 1141, 906, 833, 745, 731, 711, 660, 622 cm−1; 1H NMR (DMSO-d6, 300 MHz):
10.99 (bs, 2H,
2 x NH) (disappears on D2O exchange), 7.51 (dd, J = 1.8, 8.1 Hz, 2H, ArH), 7.42 (s, 2H, ArH), 6.86 (d,
J = 8.2 Hz, 2H, ArH), 3.89 (s, 2H, ArCH2Ar); 13C NMR (DMSO-d6, 75.6 MHz):
184.8 (CO ONH),
159.8 ( OCONH), 149.4 (ArC), 138.9 (ArC), 136.2 (ArC), 124.9 (ArC), 118.3 (ArC), 112.7 (ArC), 39.3
(ArCH2Ar); HRMS (+ESI) m/z [M + Na]+ calcd for C17H10N2NaO4: 329.0538; found: 329.0541.
δ
δ
C
C
5,5’-Oxydiindoline-2,3-dione ( ). This compound was prepared by the same method as compound 8
from compound 4 (5.0 g, 14.6 mmol) as a red solid (4.02 g, 83%); mp > 300 ◦C; IR (KBr):
3457,
3111, 1743, 1621, 1472, 1199, 1145, 907, 839, 744, 712, 660, 623 cm−1; 1H NMR (DMSO-d6, 300 MHz):
11.02 (bs, 2H, 2 x NH) (disappears on D2O exchange), 7.30 (dd, J = 2.6, 2H, 8.5 Hz, ArH), 7.09 (s,
2H, ArH), 6.93 (d, J = 8.5 Hz, 2H, ArH); 13C NMR (DMSO-d6, 75.6 MHz):
184.4 (CO ONH), 159.9
OCONH), 149.4 (ArC), 147.1 (ArC), 129.0 (ArC), 118.9 ( OCONH), 114.7 (ArC), 114.0 (ArC); HRMS
(+ESI) m/z [M + Na]+ calcd for C16H8N2NaO4: 331.0331; found: 331.0318.
Bis(1-acetylindoline-2,3-dione)methylene ( ). A suspension of compound 5 (2 g, 6.5 mmol) in acetic
6
υ
max
δ
δ
C
(C
C
7
anhydride (30 mL) was heated to reflux for 4 h. The deep red solution was cooled to room temperature.
The excess acetic anhydride was removed under vacuum. The resulting brown oily residue was
redissolved in ethyl acetate (20 mL) and washed with water (2
×
20 mL) and brine (2
×
20 mL).
The organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum. The
crude product was purified by flash column chromatography (dichloromethane) to yield the title
compound as a bright yellow solid (1.63 g, 64%); mp 289–291 ◦C; IR (KBr):
υ
1785, 1712, 1756, 1617,
max
1588, 1481, 1443, 1371, 1346, 1306, 1294, 1250, 1227, 1201, 1168, 1124, 1042, 996, 659, 598 cm−1; 1H NMR
(CDCl3, 300 MHz): 8.22 (d, J = 8.5 Hz, 2H, ArH), 7.73 (dd, J = 1.9, 8.5 Hz, 2H, ArH), 7.69 (s, 2H, ArH),
4.11 (s, 2H, ArCH2Ar), 2.59 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
180.5 (CO ONH),
170.0 ( OCH3), 158.7 ( OCONH), 146.8 (ArC), 138.3 (ArC), 124.5 (ArC), 124.5 (ArC), 120.5 (ArC),
117.8 (ArC), 39.2 (Ar H2Ar), 26.3 (CO
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C21H14N2NaO6:
413.0750; found: 413.0713.
δ
δ
C
C
C
C
C
5,5’-Bis(1-acetylindoline-2,3-dione)oxide (
8
). This compound was prepared by the same method as
(2.0 g, 6.5 mmol) as a bright yellow solid (1.40 g, 55%); mp > 300 ◦C;
3429, 1783, 1747, 1702, 1617, 1470, 1371, 1330, 1312, 1293, 1266, 1241, 1156, 850, 599,
compound
IR (KBr):
7
from compound 6
υ
max
468 cm−1; 1H NMR (CDCl3, 300 MHz):
δ
8.31 (d, J = 9.2 Hz, 2H, ArH), 7.51 (dd, J = 2.9, 8.9 Hz, 2H,
ArH), 7.31 (s, 2H, ArH), 2.57 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
179.9 (CO ONH),
170.0 ( OCH3), 158.6 ( OCONH), 154.1 (ArC), 144.4 (ArC), 128.3 (ArC), 121.8 (ArC), 119.7 (ArC),
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C20H12N2NaO7: 415.0542; found:
δ
C
C
C
113.9 (ArC), 26.2 (CO
C
415.0526.
Dimethyl 2,2’-[5,5’-bis(2-acetamido-5,1-phenylene)]bis(2-oxoacetate)methylene (9). A solution of compound 7
(0.15 g, 0.38 mmol) in anhydrous methanol (20 mL) was stirred at room temperature for 24 h. A massive
yellow precipitate formed. The crude compound was collected by filtration and recrystallized from

Molecules 2019, 24, 4343
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n-hexane/DCM to afford the title compound as a yellow solid (0.059 g, 34%); mp 162–164 ◦C; IR (KBr):
max 3303, 1747, 1733, 1702, 1652, 1595, 1528, 1420, 1340, 1295, 1250, 1226, 1160, 1013 cm−1; 1H NMR
(CDCl3, 300 MHz): 10.91 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.66 (d, J = 8.7 Hz, 2H, ArH),
7.37 (s, 2H, ArH), 7.35 (d, J = 2.1 Hz, 2H, ArH), 3.90 (s, 2H, ArCH2Ar), 3.89 (s, 6H, 2 x COOCH3), 2.18
(s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
190.4 ( OCH3), 169.8 ( OCOO), 164.1 (CO OO),
141.7 (ArC), 138.0 (ArC), 134.5 (ArC), 133.6 (ArC), 121.6 (ArC), 117.6 (ArC), 53.4 (COO H3), 40.2
(Ar H2Ar), 25.9 (CO
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C23H22N2NaO8: 477.1274; found:
477.1243.
υ
δ
δ
C
C
C
C
C
C
Diethyl 2,2’-[5,5’-bis(2-acetamido-5,1-phenylene)]bis(2-oxoacetate)methylene (10). A solution of compound
7
(0.15 g, 0.38 mmol) in absolute ethanol (20 mL) was heated to reflux for 12 h. The reaction mixture was
allowed to cool to room temperature and a massive yellow precipitate formed. The crude compound
was collected by filtration and recrystallized from n-hexane/DCM to afford the title compound as a
yellow solid (0.095 g, 51%); mp 162–164 ◦C; IR (KBr):
1399, 1367, 1295, 1243, 1211, 1089, 1007 cm−1; 1H NMR (CDCl3, 300 MHz):
υ
max 3317, 1749, 1738, 1705, 1648, 1617, 1590, 1493,
11.02 (bs, 2H, 2 x NH)
δ
(disappears on D2O exchange), 8.73 (d, J = 9.0 Hz, 2H, ArH), 7.44 (s, 2H, ArH), 7.42 (dd, J = 2.2, 9.0 Hz,
2H, ArH), 4.41 (q, J = 7.2 Hz, 4H, 2 x CH2CH3), 3.97 (s, 2H, ArCH2Ar), 2.24 (s, 6H, 2 x COCH3), 1.35 (t,
J = 7.1 Hz, 6H, 2 x CH2CH3); 13C NMR (CDCl3, 75.6 MHz):
δ
190.2 (
OO), 141.2 (ArC), 137.4 (ArC), 134.0 (ArC), 133.1 (ArC), 121.1 (ArC), 117.1 (ArC), 62.6 (
39.7 (Ar H2Ar), 25.4 (OCH3), 13.9 (CH2
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C25H26N2NaO8:
505.1587; found: 505.1572.
C
OCH3), 169.3 (
C
OCOO), 163.3
(COC
C
H2CH3),
C
C
Dimethyl 2,2’-[5,5’-bis(2-acetamido-5,1-phenylene)]bis(2-oxoacetate)oxide (11). Compound 11 was prepared
by the same method as compound
9
from compound
3456, 1750, 1737, 1704, 1655, 1587, 1520, 1488, 1410, 1286, 1257,
8 (0.15 g, 0.38 mmol) as a white solid (0.129 g,
74%); mp 159–161 ◦C; IR (KBr):
υ
max
1237, 1217, 1154, 1016, 970, 783 cm−1; 1H NMR (CDCl3, 300 MHz):
δ
10.82 (bs, 2H, 2 x NH) (disappears
on D2O exchange), 8.72 (d, J = 9.1 Hz, 2H, ArH), 7.32 (d, J = 2.7 Hz, 2H, ArH), 7.22 (dd, J = 2.9, 9.2 Hz,
2H, ArH), 3.89 (s, 6H, 2 x COOCH3), 2.19 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
189.6
OO), 151.3 (ArC), 139.2 (ArC), 127.7 (ArC), 123.2 (ArC), 122.9
δ
(C
OCH3), 169.7 (
C
OCOO), 163.7 (CO
C
(ArC), 118.6 (ArC), 53.6 (COO
C
H3), 25.9 (CO
C
H3); HRMS (+ESI) m/z [M + H]+ calcd for C22H21N2O9:
457.1247; found: 457.1230.
Diethyl 2,2’-[5,5’-bis(2-acetamido-5,1-phenylene)]bis(2-oxoacetate)oxide (12). Compound 12 was prepared
by the same method as compound 10 from compound (0.15 g, 0.38 mmol) as a yellow solid (0.143 g,
77%); mp 104–106 ◦C; IR (KBr):
3307, 2965, 1745, 1655, 1589, 1520, 1437, 1411, 1372, 1267, 1217,
8
υ
max
1282, 1160, 1011, 842 cm−1; 1H NMR (CDCl3, 300 MHz): δ 10.92 (bs, 2H, 2 x NH) (disappears on D2O
exchange), 8.79 (d, J = 9.3 Hz, 2H, ArH), 7.37 (d, J = 2.7 Hz, 2H, ArH), 7.35 (dd, J = 2.9, 9.3 Hz, 2H, ArH),
4.41 (q, J = 7.2 Hz, 4H, 2 x CH2CH3), 2.25 (s, 6H, 2 x COCH3), 1.36 (t, J = 7.1 Hz, 6H, 2 x CH2CH3); 13
C
NMR (CDCl3, 75.6 MHz):
127.3 (ArC), 122.8 (ArC), 122.4 (ArC), 118.2 (ArC), 62.9 (
(+ESI) m/z [M + Na]+ calcd for C24H24N2NaO9: 507.1380; found: 507.1360.
δ
189.6 (
C
OCH3), 169.3 (
C
OCOO), 162.9 (CO
C
OO), 151.0 (ArC), 138.8 (ArC),
C
H2CH3), 25.4 (OCH3), 14.0 (CH2
CH3); HRMS
N,N’-{4,4’-bis-[2-(2-acetamidophenyl)-2-oxoacetamide]}methylene (13). To a solution of compound
7 (0.15 g,
0.38 mmol) in dichloromethane (20 mL) was added concentrated ammonia solution (10 mL). The
reaction mixture was stirred at room temperature for 30 min. A massive white precipitate formed.
The crude product was collected by filtration and purified by recrystallization from methanol to yield
the title compound 13 as a white solid (0.127 g, 78%); mp 229–232 ◦C; IR (KBr):
υ
3310, 2933, 1692,
max
1641, 1588, 1522, 1466, 1411, 1359, 1310, 1279, 1201, 1177, 1132, 987, 900, 826, 735, 591 cm−1; 1H NMR
(DMSO-d6, 300 MHz): 10.50 (bs, 2H, 2 x NH) (disappears on D2O exchange), 7.79 (s, 4H, 2 x NH2)
(disappears on D2O exchange), 8.01 (s, 2H, ArH), 7.50 (d, J = 1.9 Hz, 2H, ArH), 7.44 (dd, J = 2.1,
8.5 Hz, 2H, ArH), 3.99 (s, 2H, ArCH2Ar), 2.04 (s, 6H, 2 x COCH3); 13C NMR (DMSO-d6, 75.6 MHz):
169.2 ( OCONH2), 192.6 ( OCH3), 166.3 (CO ONH2), 137.4 (ArC), 136.4 (ArC), 134.8 (ArC), 131.8
δ
δ
C
C
C

Molecules 2019, 24, 4343
9 of 15
(ArC), 124.3 (ArC), 122.3 (ArC), 40.0 (Ar
C
H2Ar), 24.5 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd for
C21H20N4NaO6: 447.1281; found: 447.1252.
N,N’-{4,4’-bis-[2-(2-acetamidophenyl)-2-oxoacetamide]}oxide (14). Compound 14 was prepared by the
same method as compound 13 from compound (0.15 g, 0.38 mmol) as a white solid (0.108 g, 66%);
8
mp 183–186 ◦C; IR (KBr):
υ
3309, 2940, 2859, 1701, 1639, 1588, 1521, 1463, 1416, 1368, 1326, 1285,
1237, 1215, 1181, 1165, 1142, 991, 942, 842, 753, 637 cm−1; H NMR (DMSO-d6, 300 MHz):
max
1
δ 10.36
(bs, 2H, 2 x NH) (disappears on D2O exchange), 8.00 (s, 2H, ArH), 7.74 (s, 4H, 2 x NH2) (disappears
on D2O exchange), 7.50 (d, J = 1.7 Hz, 2H, ArH), 7.12 (dd, J = 1.7, 8.0 Hz, 2H, ArH), 2.14 (s, 6H, 2 x
COCH3); 13C NMR (DMSO-d6, 75.6 MHz):
δ 193.4 (COCH3), 169.5 (COCONH2), 166.2 (COCONH2),
137.1 (ArC), 136.3 (ArC), 130.9 (ArC), 128.9 (ArC), 120.5 (ArC), 113.2 (ArC), 26.1 (OCH3); HRMS (+ESI)
m/z [M + Na]+ calcd for C20H18N4NaO7: 449.1073; found: 447.1059.
N,N’-{4,4’-bis-[2-(2-acetamidophenyl)-N-butyl-2-oxoacetamide]}methylene (15). A solution of n-butylamine
(0.20 mL, 2.0 mmol) in anhydrous dichloromethane (20 mL)was added to a stirred solution of compound
7
(0.15 g, 0.38 mmol) in anhydrous dichloromethane (30 mL). The reaction mixture was stirred at room
temperature for 12 h. The organic layer was diluted with dichloromethane (30 mL) and extracted in
aqueous hydrochloric acid (0.5 M, 2 40 mL) and water (3 x 40 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated under vacuum. The crude compound was purified by
gravity column chromatography (dichloromethane-ethyl acetate 3:1) to afford the title compound 15
×
/
as a yellow solid (0.196 g, 95%); mp 144–148 ◦C; IR (KBr):
υ
3309, 2960, 1681, 1650, 1582, 1507, 1402,
1371, 1320, 1288, 1253, 1222, 1180, 1134, 1006, 833, 700 cm−1m;a1xH NMR (CDCl3, 300 MHz):
δ 10.79 (bs,
2H, 2 x NH) (disappears on D2O exchange), 8.50 (d, J = 8.7 Hz, 2H, ArH), 8.07 (d, J = 2.0 Hz, 2H, ArH),
7.33 (dd, J = 2.0, 8.6 Hz, 2H, ArH), 6.81 (bs, 2H, 2 x NH butylamine) (disappears on D2O exchange),
3.91 (s, 2H, ArCH2Ar), 3.31 (dd, J = 6.8, 13.2 Hz, 4H, 2 x CH2CH2), 2.14 (s, 6H, 2 x COCH3), 1.47–1.52
(m, 4H, 2 x CH2CH2CH2CH3), 1.28–1.35 (m, 4H, 2 x CH2CH3), 0.87 (t, J = 7.6 Hz, 6H, 2 x CH3); 13
C
NMR (CDCl3, 75.6 MHz):
δ
192.4 (
C
OCH3), 169.5 (
C
OCONH), 163.2 (CO
CONH), 140.9 (ArC), 137.4
(ArC), 134.9 (ArC), 134.7 (ArC), 121.6 (ArC), 119.4 (ArC), 40.4 (Ar
C
H2Ar), 39.8 (CH2CH2CH2), 31.7
(CH2CH2CH2), 25.8 (OCH3), 20.4 (CH2CH2CH3), 14.1 (CH3); HRMS (+ESI) m/z [M + Na]+ calcd for
C29H36N4NaO6: 559.2533; found: 559.2513.
N,N’-{4,4’-bis-[2-(2-acetamidophenyl)-N-butyl-2-oxoacetamide]}oxide (16). Compound 16 was prepared by
the same method as compound 15 from compound
2.0 mmol) as a yellow solid (0.202 g, 98%); mp 188–191 ◦C; IR (KBr):
1513, 1406, 1370, 1324, 1286, 1264, 1222, 1181, 1162, 1008, 847, 699 cm−1; 1H NMR (CDCl3, 300 MHz):
8
(0.15 g, 0.38 mmol) and n-butylamine (0.20 mL,
υ
3311, 2960, 1686, 1656, 1589,
max
δ
10.75 (bs, 2H, 2 x NH) (disappears on D2O exchange), 7.99 (d, J = 3.0 Hz, 2H, ArH), 8.58 (d, J = 9.3 Hz,
2H, ArH), 7.25 (dd, J = 3.4, 9.1 Hz, 2H, ArH), 6.97 (bs, 2H, 2 x NH butylamine) (disappears on D2O
exchange), 3.34 (dd, J = 6.9, 13.2 Hz, 4H, 2 x CH2CH2), 2.21 (s, 6H, 2 x COCH3), 1.51–1.56 (m, 4H, 2 x
CH2CH2CH2CH3), 1.32–1.39 (m, 4H, 2 x CH2CH3), 0.91 (t, J = 7.2 Hz, 6H, 2 x CH3); 13C NMR (CDCl3,
75.6 MHz):
(ArC), 123.4 (ArC), 122.7 (ArC), 120.2 (ArC), 39.4 (
(CH2
H2CH3), 13.7 (CH3); HRMS (+ESI) m/z [M + Na]+ calcd for C28H34N4NaO7: 561.2325; found:
561.2300.
δ
191.3 (
C
OCH3), 169.0 (
COCONH), 162.4 (CO
C
ONH), 151.2 (ArC), 137.7 (ArC), 126.8
CH2CH2CH2), 31.2 (CH2
C
H2CH2), 25.3 (OCH3), 20.0
C
N,N’-[4,4’-bis-(N-{2-[2-oxo-2-(piperidin-1-yl)acetyl]phenyl}acetamide)]methylene (17). Compound 17 was
prepared by the same method as compound 15 from compound (0.15 g, 0.38 mmol) and piperidine
(0.20 mL, 2.0 mmol) as a yellow solid (0.114 g, 53%); mp 181–183 ◦C; IR (KBr):
max 2939, 2859, 1701,
1642, 1591, 1520, 1451, 1412, 1367, 1326, 1298, 1251, 1182, 1136, 988, 851, 784, 752 cm−1; 1H NMR (CDCl3,
300 MHz): 11.15 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.66 (d, J = 8.8 Hz, 2H, ArH), 7.35
7
υ
δ
(dd, J = 1.8, 8.7 Hz, 2H, ArH), 7.28 (d, J = 1.8 Hz, 2H, ArH), 3.89 (s, 2H, ArCH2Ar), 3.55 (t, J = 5.5
Hz, 4H, 2 x NCH2), 3.13 (t, J = 5.5 Hz, 4H, 2 x NCH2), 2.18 (s, 6H, 2 x COCH3), 1.51–1.57 (m, 8H,
2 x CH2CH2CH2CH2CH2), 1.29–1.38 (m, 4H, 2 x CH2CH δ 196.4
2CH2); 13C NMR (CDCl3, 75.6 MHz):

Molecules 2019, 24, 4343
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(
C
OCH3), 169.8 (
(ArC), 118.5 (ArC), 47.5 (N
(NCH2 H2), 24.6 (CH2
H2CH2); HRMS (+ESI) m/z [M + Na]+ calcd for C31H36N4NaO6: 583.2533;
found: 583.2493.
C
OCON), 164.5 (CO
C
ON), 141.3 (ArC), 137.6 (ArC), 135.0 (ArC), 133.6 (ArC), 121.4
CH2), 40.1 (ArCH2Ar), 25.7 (NCH2CH2), 25.9 (OCH3), 25.7
C
H2), 42.5 (N
C
C
N,N’-[4,4’-bis-(N-{2-[2-oxo-2-(piperidin-1-yl)acetyl]phenyl}acetamide)]oxide (18). Compound 18 was
prepared by the same method as compound 15 from compound (0.15 g, 0.38 mmol) and piperidine
(0.20 mL, 2.0 mmol) as a yellow solid (0.198 g, 92%); mp 158–160 ◦C; IR (KBr):
8
υ
max 3444, 3288, 1682,
1557, 1489, 1415, 1371, 1335, 1303, 1289, 1253, 1126, 929, 839, 669, 617, 559 cm−1; H NMR (CDCl3,
1
300 MHz):
δ 11.08 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.75 (d, J = 8.8 Hz, 2H, ArH), 7.23
(dd, J = 2.8, 9.1 Hz, 2H, ArH), 7.20 (d, J = 1.9 Hz, 2H, ArH), 3.58 (t, J = 5.3 Hz, 4H, 2 x NCH2), 3.23 (t,
J = 5.3 Hz, 4H, 2 x NCH2), 2.22 (s, 6H, 2 x COCH3), 1.63–1.65 (m, 4H, 2 x CH2CH2CH2), 1.51–1.57 (m,
8H, 2 x CH2C
(CO ON), 151.3 (ArC), 138.4 (ArC), 127.0 (ArC), 122.6 (ArC), 122.4 (ArC), 119.1 (ArC), 47.1 (N
42.2 (N H2), 26.1 (NCH2 H2), 25.4 (OCH3), 25.3 (NCH2 H2), 24.2 (CH2 H2CH2); HRMS (+ESI) m/z
[M + Na]+ calcd for C30H34N4NaO7: 585.2325; found: 585.2293.
H
2CH2CH
2CH2); 13C NMR (CDCl3, 75.6 MHz):
δ
195.2 (
COCH3), 169.3 (COCON), 163.8
C
CH2),
C
C
C
C
2,20-[Methylene-bis(2-acetamido-5,1-phenylene)]bis(N-hexyl-2-oxoacetamide) (19). A mixture of compound
7
(0.24 g, 0.61 mmol) and hexylamine (0.12 g, 1.22 mmol) in dichloromethane (30 mL) was heated at
reflux overnight. After cooling to room temperature, the solvent was evaporated in vacuo and the
crude product was purified by column chromatography using silica gel and n-hexane/DCM as eluent.
The title compound 19 was obtained as an off-white solid (0.26 g, 71%); mp 182–184 ◦C; IR (KBr):
υ
max
3305, 3053, 2928, 2855, 1694, 1650, 1585, 1518, 1465, 1411, 1369, 1293, 1223, 1176, 1006, 855, 811 cm−1; 1H
NMR (CDCl3, 300 MHz): 10.89 (s, 2H, 2 x NHCO), 8.56 (d, J = 8.6 Hz, 2H, ArH), 8.16 (d, J = 2.0 Hz,
2H, ArH), 7.42 (dd, J = 8.7, 1.9 Hz, 2H, ArH), 6.98 (t, J = 5.5 Hz, 2H, 2 x CONH), 4.00 (s, 2H, ArCH2Ar),
3.47-3.49 (q, J = 6.7, 6.3 Hz, 4H, 2 x NHC 2CH2(CH2)3CH3), 2.25 (s, 6H, 2 x COCH3), 1.55–1.66 (m, 4H,
2 x NHCH2CH2(CH2)3CH3), 1.30–1.44 (m, 12H, 2 x NHCH2CH2(CH2)3CH3), 0.93 (t, J = 6.6 Hz, 6H,
2 x NHCH2(CH2)4CH3); 13C NMR (CDCl3, 75.6 MHz):
162.8, 169.1, 192.0 (6 x C=O), 121.2, 134.5,
136.9 (6 x ArCH), 119.0, 134.3, 140.5 (6 x ArC), 40.0 (2 x NHCH2), 39.7 (Ar H2Ar), 25.4 (2 x CO H3),
31.4 (2 x CH2(
H2)4CH3), 22.5, 26.6, 29.2, 14.0 (2 x CH2(CH2)4CH3); HRMS (ESI) m/z [M + H]+ calcd
for C33H45N4O6: 593.3333; found: 593.3326.
δ
H
δ
C
C
C
2,20-[Methylene-bis(2-acetamido-5,1-phenylene)]bis(N-dodecyl-2-oxoacetamide) (20). Compound 20 was
prepared by the same method as compound 19 from compound
(0.23 g, 1.22 mmol) as an off-white solid (0.26 g, 69%); mp 162–164 C; IR (KBr):
7
(0.24 g◦, 0.61 mmol) and dodecylamine
3344, 3293,
max
υ
2919, 2850, 1694, 1650, 1586, 1519, 1467, 1412, 1370, 1294, 1224, 1177, 1014, 855, 721 cm−1; 1H NMR
(CDCl3, 300 MHz): 10.89 (s, 2H, 2 x NHCO), 8.54 (d, J = 8.5 Hz, 2H, ArH), 8.17 (d, J = 2.1 Hz, 2H,
ArH), 7.45 (dd, J = 8.6, 2.12 Hz, 2H, ArH), 6.98 (t, J = 5.9 Hz, 2H, 2 x CONH), 4.03 (s, 2H, ArCH2Ar),
3.37–3.44 (q, J = 6.7, 6.8 Hz, 4H, 2 x NHC 2CH2(CH2)9CH3), 2.28 (s, 6H, 2 x COCH3), 1.56-1.66 (m, 4H,
2 x NHCH2CH2(CH2)9CH3), 1.26–1.40 (m, 36H, 2 x CH2CH2(CH2)9CH3), 0.93 (t, J = 7.0 Hz, 6H, 2 x
CH2(CH2)9CH3); 13C NMR (CDCl3, 75.6 MHz):
162.8, 169.9, 191.7 (6 x C = O), 121.6, 134.9, 136.9 (6 x
ArCH), 120.6, 134.2, 139.9 (6 x ArC), 40.0 (2 x NHCH2), 39.6 (Ar H2Ar), 25.2 (2 x CO H3), 22.7, 26.9,
29.2, 29.2, 29.4, 29.5, 29.6, 29.6, 31.3 (2 x CH2( H2)10CH3), 14.1 (2 x CH2(CH2)10 H3); HRMS (ESI) m/z
[M + Na]+calcd for C45H68N4NaO6: 761.5211; found: 761.5207.
δ
H
δ
C
C
C
C
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]acetate}) methane (21). A mixture of the
glycine methyl ester hydrochloride (0.24 g, 1.9 mmol) and saturated sodium hydrogen carbonate
solution (3 mL) in water (7 mL) was added to a stirred solution of compound 7 (0.15 g, 0.38 mmol))
in dichloromethane (20 mL) was. The reaction mixture was stirred at room temperature for 24 h.
The organic layer was diluted with dichloromethane (20 mL) and extracted in aqueous hydrochloric
acid (0.5 M, 30 mL) and water (2
sodium sulfate and concentrated under vacuum. The crude compound was purified by gravity
×
30 mL). The combined organic extracts were dried over anhydrous

Molecules 2019, 24, 4343
11 of 15
column chromatography (dichloromethane-ethyl acetate = 4:1) to afford the title compound as a
yellow solid (0.120 g, 55%); mp 204–206 ◦C; IR (KBr):
3279, 1750, 1698, 1666, 1651, 1592, 1519,
1412, 1367, 1326, 1293, 1234, 1217, 1177, 1005, 688 cm−1; 1H NMR (CDCl3, 300 MHz):
2 x NH) (disappears on D2O exchange), 9.07 (d, J = 11.2 Hz, 2H, ArH), 7.74 (s, 2H, ArH), 7.60 (d,
J = 1.5 Hz, 2H, 2 x -NH Gly) (disappears on D2O exchange), 7.49 (dd, J = 2.0, 8.5 Hz, 2H, ArH), 3.97
(d, J = 5.7 Hz, 4H, 2 x NHCH2COOCH3), 3.91 (s, 2H, ArCH2Ar), 3.67 (s, 6H, 2 x COOCH3), 2.05 (s, 6H,
2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
189.8 ( OCH3), 169.5 ( OCONH), 168.9 (CO ONH),
163.2 ( OOCH3), 149.8 (ArC), 138.6 (ArC), 127.1 (ArC), 123.5 (ArC), 122.6 (ArC), 119.6 (ArC), 53.1
(COO H3), 40.9 (NH H2COOCH3), 38.9 (Ar
H2Ar), 24.9 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd
for C27H28N4NaO10: 591.1703; found: 591.1692.
υ
max
δ
10.49 (bs, 2H,
α
δ
C
C
C
C
C
C
C
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-4-methylpentanoate})methane
Compound 22 was prepared by the same method as compound 21 from compound (0.15 g, 0.38
mmol) and l-leucine methyl ester hydrochloride (0.30 g, 1.9 mmol) as a yellow solid (0.149 g, 57%);
mp 184–186 ◦C; IR (KBr):
3328, 2958, 1747, 1648, 1592, 1522, 1438, 1415, 1370, 1298, 1253, 1231,
1176 cm−1; 1H NMR (CDCl3, 300 MHz):
(d, J = 8.8 Hz, 2H, ArH), 8.01 (d, J = 1.9 Hz, 2H, 2 x
(dd, J = 2.1, 8.7 Hz, 2H, ArH), 7.25 (d, J = 8.4 Hz, 2H, ArH), 4.57–4.64 (m, 2H, 2 x NHC
3.91 (s, 2H, ArCH2Ar), 3.66 (s, 6H, 2 x COOCH3), 2.14 (s, 6H, 2 x COCH3), 1.56-1.69 (m, 4H, 2 x
H2CH(CH3)2), 1.18–1.20 (m, 2H, 2 x C
(CH3)2), 0.89 (dd, J = 2.6, 5.9 Hz, 12H, 2 x CH(CH3)2); 13
NMR (CDCl3, 75.6 MHz): 191.9 ( OCH3), 172.9 (CO ONH), 169.5 (CO ONH), 163.3 ( OOCH3),
141.1 (ArC), 137.7 (ArC), 134.8 (ArC), 134.6 (ArC), 121.4 (ArC), 119.0 (ArC), 52.9 (COO H3), 51.3
H(CH3)2), 23.0 ( H3),
(22).
7
υ
max
δ
10.84 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.53
-NH Leu) (disappears on D2O exchange), 7.35
COOCH3),
α
H
C
H
C
δ
C
C
C
C
C
(NH
C
HCOOCH3), 41.7 (
C
H2CH(CH3)2), 40.3 (Ar
C
H2Ar), 25.8 (OCH3), 25.3 (
C
C
22.2 (CH3); HRMS (+ESI) m/z [M + Na]+ calcd for C35H44N4NaO10: 703.2955; found: 703.2948.
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-4-(methylthio)butanoate})methane
(23).
Compound 23 was prepared by the same method as compound 21 from compound
mmol) and l-methionine methyl ester hydrochloride (0.38 g, 1.9 mmol) as a yellow solid (0.140 g, 51%);
mp 172–174 ◦C; IR (KBr):
3292, 1746, 1695, 1663, 1642, 1589, 1523, 1433, 1412, 1294, 1250, 1219,
1173 cm−1; 1H NMR (CDCl3, 300 MHz):
(d, J = 8.5 Hz, 2H, ArH), 8.01 (d, J = 1.9 Hz, 2H, 2 x
J = 8.1Hz, 2H, ArH), 7.35 (dd, J = 2.0, 8.7 Hz, 2H, ArH), 4.68–4.77 (m, 2H, 2 x NHC
(s, 2H, ArCH2Ar), 3.70 (s, 6H, 2 x COOCH3), 2.47 (t, J = 7.2 Hz, 4H, 2 x CH2CH2S), 2.15 (s, 6H, 2 x
COCH3), 2.03 (s, 6H, 2 x SCH3), 1.05 (t, J = 7.2 Hz, 4H, 2 x CH2CH2S); 13C NMR (CDCl3, 75.6 MHz):
191.6 ( OCH3), 171.9 (CO ONH), 169.6 (CO ONH), 163.3 ( OOCH3), 141.1 (ArC), 137.7 (ArC), 134.8
(ArC), 134.5 (ArC), 121.5 (ArC), 118.9 (ArC), 53.2 (COO H3), 52.0 (NH HCOOCH3), 40.3 (Ar H2Ar),
31.6 ( H2CH2S), 30.3 (CH2
H2S), 25.8 (OCH3), 15.9 (SCH3); HRMS (+ESI) m/z [M + Na]+ calcd for
7 (0.15 g, 0.38
υ
max
δ
10.80 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.52
-NH Met) (disappears on D2O exchange), 7.49 (d,
COOCH3), 3.91
α
H
δ
C
C
C
C
C
C
C
C
C
C33H40N4O10NaS2: 739.2084; found: 739.2071.
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-methylbutanoate})methane
(
24).
(0.15 g, 0.38
mmol) and l-valine methyl ester hydrochloride (0.32 g, 1.9 mmol) as a yellow solid (0.201 g, 80%); mp
204–207 ◦C; IR (KBr):
max 3251, 2962, 1740, 1696, 1666, 1644, 1588, 1521, 1469, 1438, 1413, 1369, 1295,
1245, 1225, 1211, 1176, 1141 cm−1; 1H NMR (CDCl3, 300 MHz):
10.87 (bs, 2H, 2 x NH) (disappears on
D2O exchange), 8.51 (d, J = 8.6 Hz, 2H, ArH), 7.93 (d, J = 1.9 Hz, 2H, 2 x -NH Val) (disappears on
D2O exchange), 7.39 (d, J = 8.8 Hz, 2H, ArH0), 7.32 (dd, J = 2.0, 8.8 Hz, 2H, ArH), 4.54 (dd, J = 4.9, 9.0
Hz, 2H, 2 x NHC COOCH3), 3.89 (s, 2H, ArCH2Ar), 3.67 (s, 6H, 2 x COOCH3), 2.18–2.22 (m, 2H,
2 x C
(CH3)2), 2.14 (s, 6H, 2 x COCH3), 0.89 (dd, J = 6.9, 12.7 Hz, 12H, 2 x CH(CH3)2); 13C NMR
(CDCl3, 75.6 MHz): 192.3 ( OCH3), 172.1 (CO ONH), 169.6 (CO ONH), 163.7 ( OOCH3), 141.1
(ArC), 137.7 (ArC), 134.7 (ArC), 134.5 (ArC), 121.4 (ArC), 118.9 (ArC), 57.6 (NH HCOOCH3), 52.8
(COO H3), 40.3 (Ar H2Ar), 31.8 ( H(CH3)2), 25.8 (OCH3), 19.4 (2 x H3), 18.0 ( H3); HRMS (+ESI)
m/z [M + Na]+ calcd for C33H40N4NaO10: 675.2642; found: 675.2616.
Compound 24 was prepared by the same method as compound 21 from compound
7
υ
δ
α
H
H
δ
C
C
C
C
C
C
C
C
C
C

Molecules 2019, 24, 4343
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N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-phenylpropanoate})methane
(
25).
Compound 25 was prepared by the same method as compound 21 from compound
7 (0.15 g, 0.38
mmol) and d-phenylalanine methyl ester hydrochloride (0.41 g, 1.9 mmol) as a yellow solid (0.081 g,
h
28%); mp 200–202 ◦C; α]2D2 (c 0.1 in MeOH); IR (KBr):
υ
3294, 1749, 1693, 1644, 1590, 1521, 1412,
max
1293, 1268, 1217, 1173, 701 cm−1; 1H NMR (CDCl3, 300 MHz):
δ
10.79 (bs, 2H, 2 x NH) (disappears
-NH Phe) (disappears on D2O
exchange), 7.16–7.29 (d, 12H, ArH, ArH), 7.05 (d, J = 6.4 Hz, 2H, ArH), 4.86 (dd, J = 6.6, 14.8 Hz, 2H, 2 x
NHC COOCH3), 3.84 (s, 2H, ArCH2Ar), 3.66 (s, 6H, 2 x COOCH3), 2.99-3.17 (m, 4H, 2 x CH2Ph),
2.11 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
191.7 ( OCH3), 171.5 (CO ONH), 169.6
(CO ONH), 163.0 ( OOCH3), 141.1 (ArC), 137.6 (ArC), 135.7 (ArC), 134.7 (ArC), 134.5 (ArC), 129.6
(ArC), 129.1 (ArC), 127.8 (ArC), 121.4 (ArC), 118.8 (ArC), 53.7 (NH HCOOCH3), 53.0 (COO H3),
40.3 (Ar H2Ar), 38.3 (
H2Ph), 25.8 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd for C41H40N4NaO10:
on D2O exchange), 8.50 (d, J = 9.0 Hz, 2H, ArH), 7.88 (s, 2H, 2 x
α
H
δ
C
C
C
C
C
C
C
C
771.2642; found: 771.2610.
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-phenylpropanoate})methane
(26).
Compound 26 was prepared from compound
7 (1.00 g, 2.56 mmol) and l-phenylalanine methyl ester
hydrochloride (1.21 g, 5.64 mmol) in the presence of Et3N (1.04g, 10.2 mmol) as a yellow solid (0.78
h
mg, 48%); mp 201–202 C; α]2D3 (c 0.1 in MeOH)
;
IR
υ
3291, 1742, 1690, 1642, 1594, 1523, 1418,
max
◦
1289, 1272, 1212, 1171, 705 cm−1. H NMR (CDCl3, 400 MHz):
δ
10.80 (bs, 2H, 2 x NH) (disappears
-NH Phe) (disappears on D2O
exchange), 7.14-7.30 (d, 12H, ArH, ArH), 7.06 (d, J = 6.4 Hz, 2H, ArH), 4.88 (dd, J = 6.6, 14.8 Hz, 2H, 2 x
NHC COOCH3), 3.85 (s, 2H, ArCH2Ar), 3.67 (s, 6H, 2 x COOCH3), 2.98–3.18 (m, 4H, 2 x CH2Ph),
2.12 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 101 MHz):
191.8 ( OCH3), 172.1 (CO ONH), 169.6
(CO ONH), 163.0 ( OOCH3), 141.1 (ArC), 137.6 (ArC), 135.7 (ArC), 134.7 (ArC), 135.6 (ArC), 129.9
(ArC), 129.2 (ArC), 128.1 (ArC), 121.2 (ArC), 119.2 (ArC), 54.1 (NH HCOOCH3), 53.1 (COO H3),
40.2 (Ar H2Ar), 38.4 (
H2Ph), 25.9 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd for C41H40N4NaO10:
1
on D2O exchange), 8.51 (d, J = 9.0 Hz, 2H, ArH), 7.89 (s, 2H, 2 x
α
H
δ
C
C
C
C
C
C
C
C
771.2642; found: 771.2612.
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]acetate})oxide (27). Compound 27 was
prepared by the same method as compound 21 from compound
methyl ester hydrochloride (0.24 g, 1.9 mmol) as a yellow solid (0.061 g, 28%); mp 180–182 ◦C; IR (KBr):
max 3354, 3293, 2923, 1747, 1686, 1658, 1588, 1512, 1438, 1408, 1373, 1327, 1286, 1262, 1219, 1183, 1011
cm−1; 1H NMR (CDCl3, 300 MHz):
10.69 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.60 (d,
J = 9.3 Hz, 2H, ArH), 7.93 (d, J = 3.0 Hz, 2H, 2 x -NH Gly) (disappears on D2O exchange), 7.36 (t,
J = 5.3 Hz, 2H, ArH), 7.49 (dd, J = 3.0, 9.2 Hz, 2H, ArH), 4.05 (d, J = 5.5 Hz, 4H, 2 x NHCH2COOCH3),
3.68 (s, 6H, 2 x COOCH3), 2.17 (s, 6H, 2 x COCH3); 13C NMR (CDCl3, 75.6 MHz):
190.9 ( OCH3),
169.8 (CO ONH), 169.5 (CO ONH), 163.2 ( OOCH3), 151.6 (ArC), 138.5 (ArC), 127.8 (ArC), 123.5
H2COOCH3), 25.8 (OCH3). HRMS (+ESI)
8 (0.15 g, 0.38 mmol) and glycine
υ
δ
α
δ
C
C
C
C
(ArC), 123.1 (ArC), 119.9 (ArC), 53.1 (COOCH3), 41.5 (NHC
m/z [M + Na]+ calcd for C26H26N4NaO11: 593.1496; found: 593.1472.
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-4-methylpentanoate})oxide (28). Compound
28 was prepared by the same method as compound 21 from compound
l-leucine methyl ester hydrochloride (0.30 g, 1.9 mmol) as a yellow solid (0.068 g, 26%); mp 106–108
◦C; IR (KBr):
3313, 2958, 1748, 1655, 1589, 1519, 1438, 1410, 1370, 1263, 1217, 1182, 1162, 1013, 830,
698 cm−1; 1H NMR (CDCl3, 300 MHz):
J = 9.3 Hz, 2H, ArH), 7.83 (d, J = 2.7 Hz, 2H, 2 x
J = 8.5 Hz, 2H, ArH), 7.28 (dd, J = 2.9, 9.2 Hz, 2H, ArH), 4.60–4.67 (m, 2H, 2 x NHC
6H, 2 x COOCH3), 2.21 (s, 6H, 2 x COCH3), 1.57–1.72 (m, 6H, 2 x CH2 (CH3)2), 0.93 (d, J = 5.9 Hz,
12H, CH(C 191.3 ( OCH3), 172.7 (CO ONH), 169.0 (CO ONH),
3)2); 13C NMR (CDCl3, 75.6 MHz):
162.9 ( OOCH3), 151.1 (ArC), 138.0 (ArC), 127.3 (ArC), 122.9 (ArC), 122.6 (ArC), 119.4 (ArC), 52.5
(COO H3), 50.8 (NH HCOOCH3), 41.3 ( H2CH(CH3)2), 25.3 (OCH3), 24.9 (C (CH3)2), 22.8 ( H3),
21.8 (CH3); HRMS (+ESI) m/z [M + Na]+ calcd for C34H42N4NaO11: 705.2748; found: 705.2720.
8 (0.15 g, 0.38 mmol) and
υ
max
δ
10.82 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.64 (d,
-NH Leu) (disappears on D2O exchange), 7.41 (d,
COOCH3), 3.67 (s,
α
H
C
H
H
δ
C
C
C
C
C
C
C
H
C

Molecules 2019, 24, 4343
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N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-4-(methylthio)butanoate})oxide
(
29).
Compound 29 was prepared by the same method as compound 21 from compound
8 (0.15 g, 0.38
mmol) and l-methionine methyl ester hydrochloride (0.38 g, 1.9 mmol) as a yellow solid (0.206 g,
75%); mp 102–104 ◦C; IR (KBr):
υ
3274, 1743, 1658, 1588, 1519, 1489, 1410, 1370, 1271, 1218, 1183,
max
1162 cm−1; H NMR (CDCl3, 300 MHz):
δ
10.77 (bs, 2H, 2 x NH) (disappears on D2O exchange),
-NH Met) (disappears on D2O exchange),
7.65 (d, J = 8.1 Hz, 2H, ArH), 7.29 (dd, J = 2.9, 9.2 Hz, 2H, ArH), 4.74 (dd, J = 8.8, 12.9 Hz, 2H, 2 x
NHC COOCH3), 3.72 (s, 6H, 2 x COOC 3), 2.49–2.56 (m, 4H, 2 x C 2CH2S), 2.21 (s, 6H, 2 x COC 3),
2.07 (s, 6H, 2 x SCH3), 1.03 (t, J = 6.8 Hz, 4H, 2 x CH2CH2S); 13C NMR (CDCl3, 75.6 MHz):
190.8
OCH3), 171.6 (CO ONH), 169.1 (CO ONH), 162.7 ( OOCH3), 151.1 (ArC), 138.0 (ArC), 127.3(ArC),
122.9 (ArC), 122.6 (ArC), 119.5 (ArC), 52.8 (COO H3), 51.5 (NH HCOOCH3), 31.1 ( H2CH2S), 29.9
(CH2 H2S), 25.3 (OCH3), 15.5 (S
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C32H38N4O11NaS2:
741.1876; found: 741.1854.
1
8.63 (d, J = 9.3 Hz, 2H, ArH), 7.87 (d, J = 2.9 Hz, 2H, 2 x
α
H
H
H
H
δ
(C
C
C
C
C
C
C
C
C
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-methylbutanoate})oxide (30). Compound
30 was prepared by the same method as compound 21 from compound (0.15 g, 0.38 mmol) and
l-valine methyl ester hydrochloride (0.32 g, 1.9 mmol) as a yellow solid (0.115 g, 46%); mp 99–101 ◦C;
IR (KBr): max 3215, 2992, 1743, 1701, 1660, 1591, 1518, 1485, 1417, 1370, 1289, 1256, 1215, 1210, 1016,
920, 836, 741 cm−1; 1H NMR (CDCl3, 300 MHz):
10.80 (bs, 2H, 2 x NH) (disappears on D2O exchange),
8.61 (d, J = 9.2 Hz, 2H, ArH), 7.70 (d, J = 2.8 Hz, 2H, 2 x -NH Val) (disappears on D2O exchange),
7.47 (d, J = 9.1 Hz, 2H, ArH), 7.24 (dd, J = 2.8, 9.2 Hz, 2H, ArH), 4.54 (dd, J = 4.8, 9.1 Hz, 2H, 2 x
NHC COOCH3), 3.66 (s, 6H, 2 x COOC 3), 2.16 (s, 6H, 2 x COC 3), 1.18–1.20 (m, 2H, 2 x C (CH3)2),
0.88 (dd, J = 6.9, 15.3 Hz, 12H, 2 x CH(C 192.0 ( OCH3), 172.4
3)2); 13C NMR (CDCl3, 75.6 MHz):
(CO ONH), 169.4 (CO ONH), 163.7 ( OOCH3), 151.5 (ArC), 138.5 (ArC), 127.8 (ArC), 123.1 (ArC),
123.0 (ArC), 119.7 (ArC), 57.4 (NH HCOOCH3), 52.8 (COO H3), 31.8 ( H(CH3)2), 25.7 (OCH3), 18.0
H3), 19.4 (
H3); HRMS (+ESI) m/z [M + Na]+ calcd for C33H40N4NaO10: 677.2435; found: 677.2419.
8
υ
δ
α
H
H
H
H
H
δ
C
C
C
C
C
C
C
(C
C
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-phenylpropanoate})oxide (31). Compound
31 was prepared by the same method as compound 21 from compound (0.15 g, 0.38 mmol) and
d-phenylalanine methyl ester hydrochloride (0.41 g, 1.9 mmol) as a yellow solid (0.196 g, 68%); mp
200–202 ◦C; IR (KBr):
3298, 1745, 1669, 1587, 1518, 1496, 1439, 1410, 1370, 1267, 1218, 1180, 702
cm−1; 1H NMR (CDCl3, 300 MHz):
J = 9.3 Hz, 2H, ArH), 7.79 (d, J = 2.9 Hz, 2H, 2 x
(m, 12H, ArH), 7.11 (dd, J = 1.7, 8.0 Hz, 2H, ArH), 4.86–4.93 (m, 2H, 2 x NHC
x COOC 3), 3.00–3.19 (m, 4H, 2 x C 2Ph), 2.21 (s, 6H, 2 x COC
3); 13C NMR (CDCl3, 75.6 MHz):
190.9 ( OCH3), 171.3 (CO ONH), 169.0 (CO ONH), 162.3 ( OOCH3), 151.1 (ArC), 138.1 (ArC), 135.2
(ArC), 129.2 (ArC), 128.8 (ArC), 127.4 (ArC), 127.3 (ArC), 123.0 (ArC), 122.6 (ArC), 119.3 (ArC), 53.2
(NH HCOOCH3), 52.6 (COO H3), 37.9 (2 x
H2Ph), 25.4 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd
for C40H38N4NaO11: 773.2435; found: 773.2419.
8
υ
max
δ
10.78 (bs, 2H, 2 x NH) (disappears on D2O exchange), 8.67 (d,
-NH Phe) (disappears on D2O exchange), 7.22-7.31
COOCH3), 3.70 (s, 6H, 2
α
H
H
H
H
δ
C
C
C
C
C
C
C
N,N’-(4,4’-bis-{methyl-2-[2-(2-acetamidophenyl)-2-oxoacetamido]-3-phenylpropanoate})oxide
Compound 32 was prepared by the same method as compound 21 from compound
g, 2.55 mmol), l-phenylalanine methyl ester hydrochloride (1.21 g, 5.61 mmol) and Et3N (1.03 g, 10.20
mmol) as a yellow solid (1.50 g, 78%); mp 201–202 ◦C; [
]D -12 (c 0.1 in MeOH); IR: max 3292, 1743,
1671, 1582, 1520, 1494, 1440, 1411, 1371, 1265, 1211, 1178, 701 cm−1; 1H NMR (CDCl3, 400 MHz):
10.74
(bs, 2H, 2 x NH) (disappears on D2O exchange), 8.65 (d, J = 9.2 Hz, 2H, ArH), 7.80 (d, J = 3.0 Hz, 2H, 2 x
-NH Phe) (disappears on D2O exchange), 7.21–7.34 (m, 12H, ArH), 7.10 (dd, J = 1.8, 8.0 Hz, 2H, ArH),
4.87–4.91 (m, 2H, 2 x NHC COOCH3), 3.71 (s, 6H, 2 x COOC 3), 3.00–3.20 (m, 4H, 2 x C 2Ph), 2.22 (s,
6H, 2 x COCH3); 13C NMR (CDCl3, 101 MHz):
190.8 ( OCH3), 171.2 (CO ONH), 168.5 (CO
162.0 (
(ArC), 123.0 (ArC), 122.5 (ArC), 119.0 (ArC), 53.1 (NH
(
32).
8
(1.00
α
υ
δ
α
H
H
H
δ
C
C
C
ONH),
H2Ph),
C
OOCH3), 151.2 (ArC), 137.8 (ArC), 135.1 (ArC), 129.1 (ArC), 128.7 (ArC), 127.3 (ArC), 127.1
C
HCOOCH3), 52.5 (COOCH3), 37.7 (2 x
C
25.3 (OCH3); HRMS (+ESI) m/z [M + Na]+ calcd for C40H38N4NaO11: 773.2435; found: 773.2428.

Molecules 2019, 24, 4343
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Single-Crystal X-Ray Diffraction
The X-ray diffraction measurements for compounds 22
–
24 were carried out at MX1 and MX2
beamlines at the Australian Synchrotron Facility, Melbourne. The procedure for diffraction intensity
measurements on both beamlines was similar. The crystal was mounted on the goniometer using a
cryo loop for diffraction measurements, and it was coated with paraffin oil and then quickly transferred
to the cold stream using cryo stream attachment. Data were collected using Si<111> monochromated
synchrotron X-ray radiation (λ = 0.71023 Å) at 100(2) K and were corrected for Lorentz and polarization
effects using the XDS software [23]. The structure was solved by direct methods and the full-matrix
least-squares refinements were carried out using SHELXL [26]. X-ray crystallographic information
files (CIF) for the structures 22–24 are CCDC 1505262, 1,505, 263 and 1956306. A copy of the data
can be obtained free of charge from CCDC, 12 Union Road, Cambridge CB2 1EZ, UK or email:
deposit@ccdc.cam.ac.uk.
4. Conclusions
The synthesis of a library of bis-glyoxylamide peptidomimetics was achieved by nucleophilic
ring-opening reactions of bis-N-acetylisatins linked at C5 by a methylene or oxygen bridge with alcohols,
amines, and amino acid methyl ester hydrochlorides. The bis-isatins were prepared using a modified
Sandmeyer isonitrosoacetanilide isatin synthesis using 1,4-dioxane instead of hydrochloric acid as the
reaction solvent. Amines were the most reactive reagents for ring-opening of the bis-N-acetylisatins
and the products were obtained in high yields. The addition of sodium bicarbonate was needed for
ring-opening of bis-N-acetylisatins with amino acid methyl ester hydrochlorides and the reactions
were complete in 24 h. The alcohols were slow to react and heating the reaction mixture at reflux was
required for ethanol. The biological activity of these compounds is currently under investigation.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/24/23/4343/s1,
1H and 13C NMR spectra for the synthesized compounds.
Author Contributions: D.S.B. and N.K. conceived and directed this project. The synthesized and spectroscopic
identification of the title compounds 3–32 were conducted by V.S., R.Z. and V.A. V.S. produced the single crystal of
compounds 22
22–24.
–24 and prepared the manuscript for publication. M.B. conducted the X-ray analysis of compounds
Funding: This research was funded by Directorate General of Higher Education, Ministry of Research,
Technology and Higher Education, Indonesia through the World Class Professor Program 2019, grant number:
T/85/D2.3/KK.04.05/2019.
Acknowledgments: We thank Tom Caradoc-Davies, a Principal Scientist (Australian Synchrotron), for his help in
data acquisition.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the synthesized compounds are available from the corresponding authors.
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