Synthesis, structure and antimicrobial activity of 6-(propan-2-yl)-3- methyl-morpholine-2,5-dione

Article abstract of DOI:10.1016/j.molstruc.2012.02.057

Cyclodepsipeptides are known to exhibit a broad spectrum of biological activities and present a great potential for pharmacological application. A novel didepsipeptide member of the family, 6-(propan-2-yl)-3-methyl-morpholine- 2,5-dione, was synthesized and its structure was confirmed by IR, ¹H and ¹³C NMR spectral data. The structure and relative stability of the diastereoisomers, tautomers and anionic derivatives of 6-(propan-2-yl)-3- methyl-morpholine-2,5-dione were studied by DFT. Different conditions were considered by calculations in gas phase at the B3LYP/6-311++G ^** level and in polar medium utilizing PCM methods at the same level of theory. In all cases the keto forms were found to be more stable and this form should be expected to exist in real systems. Experimental evidence for this statement was found by the IR spectra measured in KBr, polar and nonpolar solvent. The IR spectroscopy was employed to monitor the formation of anion derivative of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione. The corresponding spectral and structural changes, accompanying the molecule → anion conversion were studied by IR spectra and DFT calculations. The 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione showed antimicrobial activity against four of five tested bacterial strains, being the most effective against Escherichia coli.

Full text of DOI:10.1016/j.molstruc.2012.02.057

Journal of Molecular Structure 1016 (2012) 147–154
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Synthesis, structure and antimicrobial activity of
6-(propan-2-yl)-3-methyl-morpholine-2,5-dione
a
a
a
b
Denitsa Yancheva a, , Lalka Daskalova , Emiliya Cherneva , Bozhanka Mikhova , Aleksandra Djordjevic ,
⇑
Zaklina Smelcerovic c, Andrija Smelcerovic d
a Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Build. 9, 1113 Sofia, Bulgaria
b Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia
c Center for Biomedicinal Science, Faculty of Medicine, University of Niš, Bulevar Dr. Zorana Djindjica 81, 18000 Niš, Serbia
d Department of Chemistry, Faculty of Medicine, University of Niš, Bulevar Dr. Zorana Djindjica 81, 18000 Niš, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclodepsipeptides are known to exhibit a broad spectrum of biological activities and present a great
potential for pharmacological application. A novel didepsipeptide member of the family, 6-(propan-2-
yl)-3-methyl-morpholine-2,5-dione, was synthesized and its structure was confirmed by IR, 1H and 13C
NMR spectral data. The structure and relative stability of the diastereoisomers, tautomers and anionic
derivatives of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione were studied by DFT. Different conditions
were considered by calculations in gas phase at the B3LYP/6-311++Gꢀꢀ level and in polar medium utiliz-
ing PCM methods at the same level of theory. In all cases the keto forms were found to be more stable and
this form should be expected to exist in real systems. Experimental evidence for this statement was found
by the IR spectra measured in KBr, polar and nonpolar solvent. The IR spectroscopy was employed to
monitor the formation of anion derivative of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione. The corre-
sponding spectral and structural changes, accompanying the molecule ? anion conversion were studied
by IR spectra and DFT calculations. The 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione showed antimi-
crobial activity against four of five tested bacterial strains, being the most effective against Escherichia
coli.
Received 9 December 2011
Received in revised form 21 February 2012
Accepted 22 February 2012
Available online 1 March 2012
Keywords:
Cyclodidepsipeptide
6-(Propan-2-yl)-3-methyl-morpholine-2,5-
dione
Tautomerism
Anion
Antimicrobial activity
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
erism, hydrogen bonding and their effect on the biological activi-
ties of cyclodidepsipeptide will help to characterize these
Cyclodepsipeptides are known to exhibit a broad spectrum of
biological activities [1–3]. In this way, they present a great poten-
tial for pharmacological application [3,4] yet demonstrated by the
clinical trials of Kahalalide [5,6], Romidepsin [7,8], Aplidin [9],
PF1022A and Emodepside [10,11], and the laboratory testing of
many other compounds [1–4].
Among the large family of cyclodepsipeptides, the simplest
members are the cyclodidepsipeptides which have an ester group
and an amide group in the same 6-membered ring. They contain
compounds in view of their pharmacological application.
Many studies relate the mechanism of the biological action of
cyclodepsipeptides to their ionophoric properties i.e. to the interac-
tion with metal ions [18–23]. The ability to bind to metal ions is
important also in view of the interaction of the bioactive com-
pounds with catalytic active sites of metallo-enzymes [24,25],
and DNA coordination [26]. On the other side, biological activity
of heterocycles depends on prototropic tautomerization [27–30].
All these points should be considered in the characterization of
cyclodepsipeptides in order to gain better insight into their biolog-
ical action and the molecular features governing the biochemical
processes involved.
only one residue of amino acid and one residue of lactic,
yisovaleric or other -hydroxy acid. Some reports on their immu-
nomodulating [12], anticoagulant [13], and inhibitory activity
towards acyl-CoA:cholesterol acyltransferase [14] and -glucosi-
a-hydrox-
a
a
In our previous paper, we reported that three cyclodidepsipep-
dase [15–17], were published. However, an extensive study on
their structure and biological activity was not carried out until
now. In this regard, detailed studies on the conformation and size
of the depsipeptide cycle, electronic structure properties, tautom-
tides containing residues of a-hydroxyisovaleric acid and valine,
leucine and isoleucine were found for the first time in natural
products, as potential precursors of enniatin B in the pathogenic
fungi Fusarium sporotrichioides, isolated from the stem of fresh
Hypericum barbatum Jacq. For identification and confirmation,
those compounds were synthesized and studied by IR and DFT
methods [31].
⇑
Corresponding author. Tel.: +359 2 9606106; fax: +359 2 8700225.
E-mail address: deni@orgchm.bas.bg (D. Yancheva).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.molstruc.2012.02.057

148
D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
The present work is focused on the synthesis, structure and
amino acids, may result in racemic product [32, and references
therein]. During cyclization the stereogenic center adjacent to the
halogen atom is involved leading to racemization at C6 of the het-
erocycle [32]. In our case, diastereomeric mixture is present for both
the 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione 5 and its non-
cyclic precursor 3. It is an indication that the racemization occurred
in a preceding synthetic step – the introduction of the bromine atom
in the isovaleryl chloride.
antimicrobial activity of a novel cyclodidepsipeptide, 6-(propan-
2-yl)-3-methyl-morpholine-2,5-dione (5a–6d, Scheme 1), contain-
ing an alanine moiety. It is structurally related to the three above-
mentioned cyclodidepsipeptides, isolated from Fusarium sporotri-
chioides. Its structure was confirmed by IR, 1H and 13C NMR spec-
tral data. DFT computational methods and experimental IR
spectral techniques were employed to investigate the preferred
conformations of different diastereomeric structures and the pro-
totropic tautomerism of the compound. The interaction of the
cyclodidepsipeptide with sodium ion resulting in the formation
of respective anion was monitored by IR spectra in solution. The
changes arising from the ion formation were described in terms
of structural variations, electron charge distribution over molecu-
lar fragments, and IR frequency shifting based on the experimental
and theoretical data. The in vitro antimicrobial activity of 6-(pro-
pan-2-yl)-3-methyl-morpholine-2,5-dione was tested against five
bacterial strains.
2.1.1. Synthesis of noncyclic methyl 2-(2-bromo-3-
methylbutanamido)propanoate (3)
L-Alanine methyl ester hydrochloride 1 (0.002 mol) was dis-
solved in 25 ml dry dichloromethane and 0.006 mol of triethyl-
amine was added. The solution was cooled in ice bath, and
0.003 mol of 2-bromoisovaleryl chloride 2 was added dropwise.
The mixture was stirred for 2 h, and then the temperature was al-
lowed to rise to RT. The reaction mixture is washed by 0.5 M HCl,
10% NaHCO3 and brine. The combined organic layers were dried
over sodium sulfate and the solvent was removed under reduced
pressure. The crude methyl 2-(2-bromo-3-methylbutanamido)pro-
panoate 3 was recrystallized from water–methanol mixture (1:4)
and light yellow crystals as needles were obtained.
2. Experimental
2.1. Synthesis
Methyl
2-(2-bromo-3-methylbutanamido)propanoate
(3):
C9H16BrNO3, M = 266.13; yield = 75%; m.p. 58–59 °C; IR (KBr),
cmꢁ1: 3294, 3076, 2994, 2967, 2935, 2874, 1745, 1652, 1549,
1452, 1437, 1380, 1342, 1320, 1283, 1226, 1191, 1149, 1115,
1072, 1057, 999, 979, 932, 859, 773, 767, 731, 622, 521.
The 1H spectra indicated the presence of a mixture of two dia-
stereomes in a 3:2 ratio.
The title compound was synthesized according to the general
procedure reported by us earlier [31]. The reaction conditions were
slightly modified (Scheme 1) which allowed us to isolate and char-
acterize the noncyclic precursor 3, as well as to obtain the cyclo-
didepsipeptide product 5 with higher yields.
The structures of both compounds were confirmed by IR, 1H and
13C NMR spectral data. The 1H spectra indicated the presence of a
mixture of two diastereomes in a 3:2 ratio for 3 and in a 4:3 ratio
for 5, respectively. It was previously reported that synthesis of
2.1.1.1. Major isomer. 1H NMR (250 MHz, CDCl3): dH = 7.10 (1H, br
s, NH); 4.58 (1H, m, NCH); 4.33 (1H, d, J = 5.2, CHBr); 3.78 (3H, s,
OCH3); 2.41 (1H, m, CHMe2); 1.48 (3H, d, J = 7.2, NCHCH3); 1.09
optically active morpholine-2,5-diones, via N-(a-halogenoacyl)-a-
Scheme 1. Synthesis of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione and its anion.

D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
149
(3H, d, J = 6.8, CHCH3); 1.03 (3H, d, J = 6.8 Hz, CHCH3). 13C NMR
(62.8 MHz, CDCl3): dC = 172.8 (COO); 167.9(CON); 60.9 (CHBr);
52.5 (OCH3); 48.6 (NCH); 32.5 (CMe2); 20.84, 18.3 (CMe2); 17.9
(CMe).
spectrometer at a resolution of 2 cmꢁ1 and 64 scans. The FT-IR
spectra in solid state (in KBr) of 3 and 5 were also measured.
2.3. NMR spectra measurements
2.1.1.2. Minor isomer. 1H NMR (250 MHz, CDCl3): dH = 7.00 (1H, br
s, NH); 4.59 (1H, m, NCH); 4.31 (1H, d, J = 5.2, CHBr); 3.80 (3H, s,
OCH3); 2.40 (1H, m, CHO); 1.46 (3H, d, J = 7.2, NCHCH3); 1.08
(3H, d, J = 6.8, CHCH3); 1.01 (3H, d, J = 6.8, CHCH3). 13C NMR
(62.8 MHz, CDCl3): dC = 172.9 (COO); 167.8(CON); 60.6 (CHBr),
52.6 (OCH3), 48.7 (NCH); 32.4 (CMe2); 20.79, 18.4 (CMe2); 18.2
(CMe).
The NMR spectra were recorded on a Bruker DRX250 spectrom-
eter in solvent CDCl3 using TMS as internal standard. The struc-
tures of the investigated compounds were elucidated with the
help of 1D and 2D (COSY, HMQC, HMBC) spectra. Standard Bruker
pulse sequences and software were used to record and process the
spectra.
2.4. Computations
2.1.2. Cyclization of 3 to 6-(propan-2-yl)-3-methyl-morpholine-2,5-
dione (5)
All theoretical calculations were performed using the Gaussian
09 package [33] of programs. Geometry and vibrational frequen-
cies of species studied were performed by analytical based gradi-
ent technique without any symmetry constraint. All the results
were obtained using the density functional theory (DFT), employ-
ing the B3LYP (Becke’s three-parameter non-local exchange [34]
and Lee et al. correlation [33]) potentials. In order to determine
the preferred geometry of the compound studied, a large number
of probable geometries of the neutral compound in keto form were
constructed taking into account the flexibility of the ring system
and the change-over to chair- and boat-conformations. For each
boat or chair ring conformation, all relevant combinations of axial
and equatorial positions of the 3- and 6-alkyl groups were studied.
In this way, four diastereoisomeric structures (3R,6R), (3R,6S),
(3S,6S), and (3S,6R), were optimized for each ring conformation.
Then the same procedure was applied for selecting the most prob-
able geometries for the enol form and anionic derivative. For a bet-
ter correspondence between experimental and calculated wave
values, we modified the results using the empirical scaling factors
(0.9688 for B3LYP/6-311++Gꢀꢀ), reported by Merrick and Radom
[35]. To establish the stability order for the neutral diastereoisomers
and anions in water and DMSO solution we used the Polarizable
Continuum Model (PCM) [36] on the same level of theory.
0.002 mol of methyl 2-(2-bromo-3-methylbutanamido)propan-
oate 3, dissolved in 2 ml abs. ethanol, and 3 ml 0.5 N NaOH were
mixed and cooled in ice. 0.3 ml 5 N NaOH were added and the mix-
ture was stirred for 2 h at 0 °C. The solution was acidified with
equimolar amount of 2.5 M H2SO4 (pH ꢂ 2), stirred for another
30 min, and extracted with 30 ml dichloromethane (3 portions of
10 ml). The combined organic layers were washed by brine, dried
over sodium sulfate and the solvent was removed under reduced
pressure. The evaporation yielded small amount of light yellow
oil which crystallized on being kept. The crude 6-(propan-2-yl)-
3-methyl-morpholine-2,5-dione 5 was purified by multiple recrys-
tallization from water–ethanol mixture (1:4).
6-(Propan-2-yl)-3-methyl-morpholine-2,5-dione (5): C8H13NO3,
M = 171.19; yield = 60%; m.p. 101–102 °C; IR (KBr), cmꢁ1: 3288,
3083, 2966, 2924, 2874, 2852, 1718, 1650, 1547, 1457, 1421,
1376, 1338, 1313, 1284, 1231, 1194, 1165, 1119, 1049, 927, 853,
646, 566.
The 1H spectra indicated the presence of a mixture of two dia-
stereomes in a 4:3 ratio.
2.1.2.1. Major isomer. 1H NMR (250 MHz, CDCl3): dH = 7.33 (1H, d,
J = 8.2, NH); 4.59(1H, m, NCH); 4.31 (1H, d, J = 4.3, CHO); 2.36
(1H, m, CHMe2); 1.47 (3H, d, J = 7.2, NCHCH3); 1.05 (3H, d, J = 6.5,
OCHCH3); 1.00 (3H, d, J = 6.5, OCHCH3). 13C NMR (62.8 MHz,
CDCl3): dC = 175.6 (COO); 168.7(CON); 59.7 (CHO); 48.65 (NCH);
32.3 (CMe2), 20.57, 18.51 (CMe2); 17.8 (CMe).
2.5. Assay for in vitro antibacterial activity
The in vitro antimicrobial activity of 6-(propan-2-yl)-3-methyl-
morpholine-2,5-dione sample was tested against a panel of laboratory
control strains belonging to the American Type Culture Collection
Maryland, USA (except one, belonging to National Collection of Type
Cultures, see below). Antibacterial activity was evaluated against
two Gram-positive and three Gram-negative bacteria. Gram-
positive bacteria used were: Bacillus subtilis ATCC 6633 and Staphy-
lococcus aureus ATCC 6538 while Gram-negative bacteria utilized in
the assay were: Escherichia coli ATCC 8739, Pseudomonas aeruginosa
ATCC 9027 and Salmonella abony NCTC 6017.
The minimal inhibitory concentration (MIC) of 6-(propan-2-yl)-
3-methyl-morpholine-2,5-dione, against tested bacteria was deter-
mined by using a broth microdilution method in 96 multi-well
microtitre plates [37]. After overnight cultivation, bacterial suspen-
sions were made in Mueller Hinton broth and their turbidity was
standardized to 0.5 McFarland. Dimethyl sulphoxide (10%, v/v
aqueous solution) was used to dissolve and to dilute sample. A se-
rial double dilution of the sample was prepared in 96 well micro-
2.1.2.2. Minor isomer. 1H NMR (250 MHz, CDCl3): dH = 7.22 (1H, d,
J = 8.2, NH); 4.58 (1H, m, NCH); 4.33 (1H, d, J = 4.3, CHO); 2.37
(1H, m, CHMe2); 1.50 (3H, d, J = 7.2, NCHCH3); 1.06 (3H, d, J = 6.5,
OCHCH3); 1.01 (3H, d, J = 6.5, OCHCH3). 13C NMR (62.8 MHz,
CDCl3): dC = 175.7 (COO); 168.8(CON); 60.0 (CHO); 48.60 (NCH);
32.4 (CMe2); 20.60, 18.46 (CMe2); 17.6 (CMe).
2.1.3. Conversion of 3-methyl-6-(propan-2-yl)-4-methyl-morpholine-
2,5-dione into anion (8)
The corresponding anion 8 was obtained by adding a DMSO-d6
solution of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione to ex-
cess of dry CD3ONa (CD3ONa was preliminary prepared by reacting
CD3OD (Merck, 99% at. enrichment) with Na). The reaction mixture
was filtered to remove the excess of solid CD3ONa. The filtered
solution was put immediately into a spectroscopic cell and the IR
spectra were recorded.
titre plates, using method of Sarker et al. [38].
A stock
2.2. IR spectra measurements
concentration was 33 mg/ml. The lowest concentration of the sam-
ple that inhibited visible growth was taken as the MIC value. To
determine minimal bactericidal concentration (MBC), broth was
taken from each well without visible growth and inoculated in
Mueller Hinton agar for 24 h at 37 °C. The lowest concentration
of the tested sample that killed 99.9% of bacterial cells was
evaluated as the MBC value. Two columns in each plate were used
Commercially available spectral quality tetrachlormethane,
chloroform, and DMSO-d6, were employed as solvents. The follow-
ing sample cells were used: 0.6 mm NaCl for tetraclormethane
(0.1 M) solution; and 0.125 mm CaF2 for DMSO-d6 (0.1 M)
solutions. All IR spectra were recorded on a Brucker Tensor 27 FT

150
D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
as controls. One column was used as a positive control and con-
tained a broad-spectrum antibiotic (doxycycline in a serial dilution
of 200–0.05 lg/ml) to determine the sensitivity of Gram-negative
and Gram-positive bacterial species. The other column contained
the solvent as negative control. Tests were carried out in triplicate.
values listed in Table 1, the stability of the keto diastereoisomers
(No. 1–4) in gas phase decreases in the following order:
5a > 5b > 5c > 5d. The energy difference between the most stable
and the least stable diastereoisomers is in the range 1.23–
6.53 kJ molꢁ1. The molecular structures of 5a–d were studied also
by PCM calculations in water, which is the common medium in
real bio-chemical processes, and in DMSO. It was found that the
higher polarity of the medium does not influence the stability or-
der of 5a–d, and the relative energies are in the same range.
Similarly to the keto forms, the most stable among the four cor-
responding enol diastereoisomers (7a–d, Scheme 2) is the one with
axial 3-methyl group and equatorial 6-isopropyl group. The rela-
tive energies of enol forms 7a–d calculated at the same level of
theory are given in Table 1 (No. 5–8). In all cases the keto form is
more stable with 47–56.66 kJ molꢁ1 than the enol configuration.
According to Minkin et al. [40] prototropic conversions are proba-
ble in case when the energy differences between the initial and the
final structure do not exceed 25 kJ molꢁ1, with activation barrier
not higher than 105 kJ molꢁ1. The energy difference is larger in
the present case and convinces that only keto form should be ex-
pected to exist in real systems. However, since the tautomeric
equilibria in heterocyclic compounds depend strongly on the med-
ium polarity [41,42], optimization of molecular structures of 7a–d
was also carried out in polar medium. The respective Etot. in water
and DMSO show unambiguously that enol structures are highly
energetically unfavored even in polar medium. This conclusion is
supported by the experimentally measured IR spectra, described
in Section 3.2.
3. Result and discussion
3.1. Energy analysis
Being one of the smallest cyclodepsipeptides, 6-(propan-2-yl)-
3-methyl-morpholine-2,5-dione contains in its molecule the func-
tional groups characteristic for those compounds i.e. an ester group
and a secondary amide function which make possible the forma-
tion of lactam (keto) and lactim (enol) tautomeric forms (Scheme
1). The relative stability of the different tautomeric forms is of fun-
damental importance for the prediction of the probability of muta-
tions in biomechanism [39].
On the other hand, the conformational flexibility of heterocycle
and the presence of two stereogenic centers (C3 and C6, for num-
bering see Scheme 1) give rise to a wide number of possible diaste-
reoisomers and enantiomers. In order to determine the preferred
geometry we constructed the most probable structures of keto
and enol forms and then optimized their geometries at the
B3LYP/6-311++Gꢀꢀ level of theory. The optimized species describe
all relevant combinations of boat and chair cycle conformations
as well as (R) and (S) configuration of both stereogenic centers.
In this way, 16 keto isomers and 16 enol isomers were studied
and we found that in all cases the morpholinedione ring adopts
boat conformation. Thus the possible isomers were reduced to 8
keto and 8 enol forms. The optimized geometries of the eight keto
forms are presented in Scheme 2. They correspond to four enantio-
meric pairs of molecules (5a–d and 6a–d).
Formally both tautomeric forms could be ionized and would
produce corresponding azanion or oxanion. In fact, according to
Pauling’s resonance theory the two structures are canonical forms
of the resonance hybrid 8 (Scheme 1), rather than tautomeric
forms.
As it can be seen from Table 1, the most stable among the anio-
nic diasereoisomers 8a–d (No. 9–12) is the one with equatorial
configuration of both alkyl substituents. Following the same com-
putational scheme as employed with neutral 6-(propan-2-yl)-3-
For illustration of their relative stability, we examined the mol-
ecules with (3S) configuration. Their total (Etot) and relative (
DE)
energies are summarized in Table 1. As it could be seen from the
KETO FORMS
5a (3S,6R)
6a (3R,6S)
5b (3S,6S)
6b (3R,6R)
5c (3S,6S)
6c (3R,6R)
5d (3S,6R)
6d (3R,6S)
(3S) ENOL FORMS
7a (3S,6R)
7b (3S,6S)
7c (3S,6S)
7d (3S,6R)
Scheme 2. Diastereoisomeric keto (5a–d and 6a–d) and enol (7a–d) forms of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione.

D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
151
Table 1
Calculated ZPVE-corrected total energies (B3LYP/6-311++G(d,p)) of the species studied. For their structures see Scheme 2.
No.
Species
Gas phase
Water medium
DMSO
Etot (Hartree)
D
E (kJ molꢁ1
)
Etot (Hartree)
D
E (kJ molꢁ1
)
Etot (Hartree)
D )
E (kJ molꢁ1
Keto forms
1
2
3
4
5a
5b
5c
5d
ꢁ593.095578
ꢁ593.095108
ꢁ593.093806
ꢁ593.093090
0.00
ꢁ593.130809
ꢁ593.130108
ꢁ593.128883
ꢁ593.126946
0.00
1.84a
5.06a
10.14a
ꢁ593.108487
ꢁ593.108144
ꢁ593.107246
ꢁ593.105764
0.00
1.23a
4.65a
6.53a
0.90a
3.26a
7.15a
Enol forms
5
6
7
8
7a
7b
7c
7d
ꢁ593.077677
ꢁ593.075493
ꢁ593.076704
ꢁ593.073999
47.00b
52.73b
49.55b
56.66b
ꢁ593.110431
ꢁ593.109157
ꢁ593.107764
ꢁ593.104678
53.50c
55.01c
55.45c
58.46c
ꢁ593.088855
ꢁ593.087111
ꢁ593.087575
ꢁ593.084294
51.54d
55.22d
51.65d
56.37d
Anionic forms
9
10
11
12
8a
8b
8c
8d
ꢁ592.547037
ꢁ592.547810
ꢁ592.544596
ꢁ592.544281
2.03e
0.00
ꢁ592.652463
ꢁ592.652624
ꢁ592.648620
ꢁ592.646049
0.42e
0.00
ꢁ592.629746
ꢁ592.628400
ꢁ592.629035
ꢁ592.628344
0.00
3.53e
1.87e
3.68e
8.44e
9.27e
10.51e
17.26e
a
b
c
D
D
D
D
D
E = En(diastereoisomer) ꢁ E1(diastereoismer)
.
E = Egnas
ꢁ Eg1as
.
ðenolÞ
ðketoÞ
E = Ewn ater
ꢁ Ew1 ater
.
ðenolÞ
ðketoÞ
E = EDnSMO
ꢁ ED1SMO
.
d
e
ðenolÞ
ðketoÞ
E = En(anion) ꢁ E1(anion)
.
neutral 5a
anion 8a
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers, cm-1
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers, cm-1
Fig. 1. Experimental IR spectrum (4000–500 cmꢁ1) of 6-(propan-2-yl)-3-methyl-
morpholine-2,5-dione in KBr.
Fig. 2. Theoretical (B3LYP/6-311++Gꢀꢀ) IR spectrum (4000–500 cmꢁ1) of the anion
8a and neutral 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione 5a in gas phase.
does not differ from this one in gas phase, while in DMSO the most
stable is diastereisomer 8a.
methyl-morpholine-2,5-dione, we optimized the structures of the
anions in water and DMSO. In water medium the stability order
Table 2
Selected experimental and theoretical IR data for 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione 5a and its anion 8a.
Neutral compound
Experimental
Approximate descriptione
Anion
B3LYP/6-311++Gꢀꢀ
Experimental
B3LYP/6-311++Gꢀꢀ
ma (KBr) ma (CCl4)
m
a(DMSO-d6) Arel.b(DMSO-d6) mc
Ad
ma (DMSO-d6) Arel. (DMSO-d6) mc
Ad
b
–
3288
3083
3406
–
–
–
m
s
w
3406
–
–
121.6
–
–
m
m
NH(free)
NH(assoc.)
–
–
–
–
–
–
–
–
–
–
–
–
3267
3055
amid:
Combination of dCNH and
m
m
m
CO
ester:
CO
amid:
CO
1717
1650
1546
1722
1672
1514
1722
1672
1547
s
vs
s
1729
1674
1424
499.0
850.1
35.8
1669
1610
–
vs
vs
–
1687
1543
–
651.8
1125.7
–
dCNH + mCN + dCNC
a
b
c
Frequencies in cmꢁ1
.
Relative intensities: vs very strong; s, strong; m, moderate; w, weak.
Frequencies in cmꢁ1, scaled according to [37].
d
e
Integrated intensities in km molꢁ1
.
Vibration modes: m, stretching; d, in-plane bending.

152
D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
Table 3
The spectrum of 5 in KBr shows that the molecule exists in keto
form in solid state. The evidences for this statement are:
Selected geometrical parameters (B3LYP/6-311++G(d,p)) of 6-(propan-2-yl)-3-
methyl-morpholine-2,5-dione 5a and its anion 8a.
Parametera
Keto form
Anion
Db
– The absence of the band for mOH stretching vibration in the
3600–3500 cmꢁ1 region.
Bondh lengthc
C5N4
– The presence of two intensive bands in 1700–1600 cmꢁ1 region,
1.359
1.219
1.100
1.201
1.455
1.527
1.317
ꢁ0.042
0.037
0.012
0.013
0.011
0.024
C5O8
1.256
1.112
1.214
1.466
1.551
ester
CO
amide
stretching vibrations.
CO
corresponding to
m
and
m
C3H
C2O7
C6O1
The IR spectrum of molecule 5 in DMSO-d6 solution (as in KBr)
shows very broad band near 3267 cmꢁ1 (3288 cmꢁ1 in KBr), that
corresponds to hydrogen-bonded NH-groups. Single mNH band of
free NH-groups appears under other conditions: 3406 cmꢁ1 in
C5C6
Angled
C5N4C3
O8C5N4
N4C3C2
124.54
123.81
110.62
117.20
126.94
114.25
ꢁ7.34
3.13
3.63
ester
CO
amide
are observed in
CO
CCl4 solution. The bands for
m
and
m
DMSO-d6 and CCl4 at identical positions, slightly shifted in com-
parison to the solid state spectrum. The examination of the IR spec-
tra in DMSO-d6 and CCl4 revealed no evidences for enol formation
neither in polar nor in nonpolar solvent.
We used the IR spectroscopy to monitor the formation of the
anion of 5 in DMSO-d6 solution. The spectral changes expected,
caused by the conversion of the neutral 5 into anion 8, are illus-
trated in Fig. 2 by theoretical spectra of species studied in gas
phase.
a
b
c
See Scheme 1 for numbering of atoms.
= Anion-molecule.
D
In angstroms.
In degrees.
d
3.2. Spectral analysis
The possibility of prototropic tautomerism of 6-(propan-2-yl)-
3-methyl-morpholine-2,5-dione 5 was studied by measuring IR
spectra in solid state (KBr disc), polar DMSO-d6 and nonpolar
CCl4 solvent. The IR spectrum of 5 in KBr recorded in our laboratory
is reported in Fig. 1. Selected experimentally observed and the the-
oretically calculated data are presented in Table 2. It can be seen,
there is a good agreement between experimental and scaled theo-
retical frequencies. An exception is found for dCNH vibrations in KBr
and DMSO-d6. This deviation is obviously due to the participation
of the NH-group in hydrogen bonds.
The experimental IR data for the anion as well as the corre-
sponding calculated frequencies and intensities are summarized
in Table 2. The bands of the C–H vibrations do not undergo
changes. This result is reasonable, as the CH, CH2, and CH3 groups
cannot be involved in strong interactions with anionic center.
Therefore these vibrations are not included in Table 2.
Strong changes after ionization of the molecule 1 are observed
in the range 1720–1540 cmꢁ1. The formation of anion caused de-
ester
CO
amide
stretching vibrations. The predicted
CO
crease in both
m
and
m
frequency shift for
m
is 42 cmꢁ1, experimentally measured –
ester
CO
(8a)
(5a)
Scheme 3. B3LYP/6-311++Gꢀꢀ Mulliken net electric charges of the fragments of 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione 5a and its anion 8a.
Table 4
Minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of the 6-(propan-2-yl)-3-methyl-morpholine-2,5-dione.
Microorganisms
Tested sample (5)
Doxycycline (
l
g/ml)
DMSO (10%)
MIC (mg/ml)
MBC (mg/ml)
MIC
B. subtilis
S. aureus
P. aeruginosa
S. abony
E. coli
n.a.
n.a.
1.56
0.78
12.50
6.25
n.a.
n.a.
n.a.
n.a.
n.a.
8.25
8.25
8.25
4.125
16.50
16.50
16.50
8.25
0.78
n.a. – not active.

D. Yancheva et al. / Journal of Molecular Structure 1016 (2012) 147–154
153
53 cmꢁ1, and for mCamOide: 131 cmꢁ1, measured 62 cmꢁ1. The forma-
tion of anion is also demonstrated by disappearance of the band
for dCNH in the ion spectrum.
way, cyclodidepsipeptides may give a promise to be used as anti-
bacterial agents.
After ionization the rest of the frequencies remain at unchanged
positions in the IR spectrum.
4. Conclusions
A novel cyclodidepsipeptide, 6-(propan-2-yl)-3-methyl-mor-
pholine-2,5-dione, was synthesized and its structure was con-
firmed by IR, 1H and 13C NMR spectral data. The
cyclodidepsipeptide was studied by using Infrared Spectroscopy
and Density Functional Theory, and the following conclusions were
reached:
3.3. Structural analysis
3.3.1. Steric structure
In view of the fact that single crystals of 5 or the respective an-
ionic derivative 8 could not be obtained and analyzed by X-ray
analysis, no experimental geometrical data for them are available.
In this case the quantum chemical calculations are the best tools to
study the structural features of the molecule and anion and the
changes of the geometrical parameters caused by the conversion
molecule ? anion. For this purpose we have performed full geom-
etry optimization of the studied species. The steric parameters (in
gas phase) that are the most sensitive to this conversion are given
in Table 3. We can state the following comments on the results in
this table:
(i) The most stable molecular structure of species studied in gas
phase and polar medium is 5a (3S,6R) i.e. this one with axial
3-alkyl group and equatorial 6-isopropyl group. The species
studied exist in lactam form both in polar and nonpolar
medium.
(ii) In DMSO the most stable diastereoisomeric structure of the
ion, formed as a result of the interaction with CD3ONa, is
similar to the one of the neutral compound. In gas phase
and water medium is those with equatorial configuration
of both alkyl substituents.
(iii) Ionization of the cyclodidepsipeptide studied leads to short-
ening of the C–N bond and lengthening of the C@O bond of
the amide function. 0.52eꢁ of the anionic charge are local-
ized at the amide group and 0.48eꢁ are delocalized over
the rest part of the anion.
(iv) The structural changes occurring as a result of the ionization
are characterized by strong decrease of the stretching vibra-
tions of both carbonyl groups and by disappearance of the
N–H deformation vibration.
– The strongest bond length changes take place at and next to the
azanionic center. They are: 0.042 Å shortening of the C5–N4
bond and 0.037 Å lengthening of the C5@O8 bond. These effects
are obviously due to the strong resonance between the azanion-
ic center and carbonyl group.
– The conversion into anion does not lead to significant changes
in the bond angles.
3.3.2. Electronic structure
It is known, that charge distribution play an important role to
biological activity. That is why, the next step in this study was to
determine the charge rearrangement of the species studied after
ionization. The Mulliken electronic charges qi on fragments of the
species studied can be seen in Scheme 3.
6-(Propan-2-yl)-3-methyl-morpholine-2,5-dione displays anti-
microbial activity against four bacterial strains, being the most
effective against E. coli.
The charges changes
D
qi = qi(anion) ꢁ qi(molecule) are usually
quite informative to show the distribution of the new charge over
the corresponding anion [43,44] (and references therein). So,
according to the calculations (Scheme 3), 0.52eꢁ of the anionic
charge remained localized at the amide group (0.25eꢁ of them
are localized over the carbonyl group and 0.27eꢁ at the N atom),
and 0.48eꢁ are delocalized over the rest part of the anion.
Acknowledgments
The financial support of this work by National Science Fund of
Bulgaria (Young Researchers Project DMU-03/66 and Contract
RNF01/0110) and Ministry of Education and Science of the Republic
of Serbia (Project OI 172044) is gratefully acknowledged.
3.4. Antimicrobial activity
References
The antimicrobial activity of 6-(propan-2-yl)-3-methyl-mor-
pholine-2,5-dione was evaluated against 5 bacterial strains and
the results are listed in Table 4. In general, the sample exhibited
activity against all bacterial strains (except one – B. subtilis), with
MIC values ranging from 4.125 to 8.25 mg/ml and MBC from 8.25
to 16.50 mg/ml. Both inhibitory and microbicidal concentrations
of the tested sample were the most prominent against E. coli, with
values two times lower, in comparison with the other bacteria
tested (Table 4). The assayed sample was less effective than the
antibiotic used as a referent standard (Table 4).
Recently we have shown that two cyclodidepsipeptides, 3,6-
di(propan-2-yl)-4-methyl-morpholine-2,5-dione and 3-(2-methyl-
propyl)-6-(propan-2-yl)-4-methyl-morpholine-2,5-dione, do not
induce the toxicity and mitochondrial membrane potential de-
crease in rat thymocytes and, for the first time for this group of
compounds, do not trigger the significant intracellular reactive
oxygen species production and exhibited antibacterial activity.
On the other hand, higher concentrations of two studied cyclodid-
epsipeptides were able to stimulate proliferative activity of thymo-
cytes, with mechanisms not yet known, indicating potential
stimulatory effect on the cells of the immune system [45]. In this
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