Bioactive phenylenediamine derivatives of dehydroacetic acid: Synthesis, structural characterization and deuterium isotope effects

Article abstract of DOI:10.5562/cca1825

Several phenylenediamine derivatives of dehydroacetic acid, 3-acetyl-4-hydroxy-6-methyl-2Hpyran-2-one have been synthesized and their structure elucidated by using NMR and IR spectroscopies and mass spectrometry. Spectral analyses have pointed toward the

Full text of DOI:10.5562/cca1825

CROATICA CHEMICA ACTA
CCACAA, ISSN 0011-1643, e-ISSN 1334-417X
Croat. Chem. Acta 84 (2) (2011) 203–209.
CCA-3467
Original Scientific Article
Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid:
Synthesis, Structural Characterization and Deuterium Isotope Effects†
a
b
Tomislav Jednačak,a Predrag Novak,a, Krunoslav Užarević, Igor Bratoš,
*
Jelena Marković,a and Marina Cindrića
aDepartment of Chemistry, Faculty of Natural Science, University of Zagreb,
Horvatovac 102a, HR-10000 Zagreb, Croatia
bPLIVA Croatia Ltd., TAPI Research & Development, Prilaz baruna Filipovića 25, HR-10000 Zagreb, Croatia
RECEIVED JANUARY 18, 2011; REVISED MARCH 24, 2011; ACCEPTED MARCH 29, 2011
Abstract. Several phenylenediamine derivatives of dehydroacetic acid, 3-acetyl-4-hydroxy-6-methyl-2H-
pyran-2-one have been synthesized and their structure elucidated by using NMR and IR spectroscopies
and mass spectrometry. Spectral analyses have pointed toward the localized keto-amine form as the pre-
dominant tautomeric form in solution and solid-state which was in agreement with X-ray data. The rela-
tively broad N–H stretching bands observed in IR spectra and significant N–H proton down-field and keto
C=O up-field shifts in NMR spectra indicated the presence of intra-molecular hydrogen bonds in all stu-
died compounds. In order to gain further insights into nature of these interactions in solution deuterated
isotopomers have been prepared and deuterium isotope effects in 13C NMR spectra have been determined
and analyzed. The magnitude and sign of isotope effects have confirmed the existence of intra-molecular
hydrogen bonds. Observed differences in isotope effects reflected different hydrogen bond structure and
dynamics in studied compounds. The results presented in this paper might help in better understanding of
biological properties of the related compounds.(doi: 10.5562/cca1825)
Keywords: dehydroacetic acid, phenylenediamine derivatives, IR, NMR, MS, hydrogen Bonds, tautomer-
ism, deuterium isotope effects
INTRODUCTION
study possible hydrogen bonding interactions and tau-
tomeric keto-enol equilibria these compounds might
undergo which in turn could affect their biological
properties.
Generally, enaminones might exist in two tauto-
meric forms: keto-amine and enol-imine (Figure 2).12
They have conjugated N–C=C–C=O moiety with two
electron-deficient centers at atoms C-1 and C-3, electron
rich atom C-2 and an amino/imino group. Hence, they
may act as both electrophiles and nucleophiles. A strong
intramolecular resonance assisted hydrogen bond
(RAHB) was found to be present in both tautomeric
forms (Figure 2).13,14
Hydrogen bonding is one of the major factors that
govern the overall structure and functionality of biologi-
cally important molecules.15 Hydrogen bonds in solution
can successfully be studied by the combination of various
spectroscopic and theoretical methods.16−18 Furthermore,
X-ray structural analysis proved the existence of keto-
amine tautomeric form and intramolecular hydrogen
bonds in 1 in solid state.5 The same tautomer was ob-
Dehydroacetic acid, 3-acetyl-4-hydroxy-6-methyl-2H-
pyran-2-one possesses interesting biological properties
such as fungicide and antibacterial activities and it can
also act as a complexing ligand.1,2 Furthermore, this
compound has several reactive functional groups and
can serve as a reagent in organic synthesis.3−6 Schiff
bases derived from dehydroacetic acid and phenylene-
diamines have a great potential as bioactive com-
pounds, too. Chelate complexes with Schiff base li-
gands have found applications in antibacterial therapy
based on selective DNA cleveage ability,7 brain imag-
ing8 and catalysis.9 They were also reported to be used
in polymerization reactions, as biosensor and as chelat-
ing ligands for coordination of transition metal ca-
tions.10,11
In this paper we report on preparation of ortho (1)-,
meta (2)- and para (3)- N,N´-phenylene-diylbis[3-(1-a-
minoethyl)-6-methyl-2H-pyran-2,4(3H)-diones] (Figure
1) and their deuterated isotopomers (4–6) in order to
†
This article belongs to the Special Issue Chemistry of Living Systems devoted to the intersection of chemistry with life.
* Author to whom correspondence should be addressed. (E-mail: pnovak@chem.pmf.hr)

204
T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
Figure 1. The structures and atom numbering of 1–3.
served in the asymmetrical Schiff bases derived from
dehydroacetic acid and p-phenylenediamine.19 Since the
position of tautomeric equilibria can be crucial for reac-
tivity of these compounds, one of the most important
aims of this work was to study solvent influence on
tautomeric equilibria and nature of hydrogen bonds in
1–3.
Isotope effects have proven useful to study tauto-
merism and hydrogen bonds in solution.20−27 Many
isotopes have been investigated, but the most important
is deuterium owing to the largest change in mass upon
proton/deuterium substitution. Secondary deuterium
isotope effects in 13C NMR spectra (DIE) can be deter-
mined as the difference in chemical shifts between car-
bon substituted with hydrogen δ13C(H) and carbon subs-
tituted with deuterium δ13C(D), where n represents a
Figure 3. Deuterated isotopomers of dehydroacetic acid de-
rivatives.
number of bonds between the investigated nucleus and
deuterium:
n 13C D  δ13C H – δ13C D




 
DIE can be intrinsic and equilibrium in origin.
They differ in magnitude, sign and temperature depen-
dence. Intrinsic isotope effects can be observed in sys-
tems which are not in chemical equilibrium and they are
described with a potential energy curve with one mini-
mum. Equilibrium isotope effects, which are described
with a two-minimum potential energy curve, exist when
isotope exchange alters chemical equilibrium.13,27 The
measured deuterium isotope effects can help in elucidat-
ing the reactivity of certain functional groups in studied
compounds, the nature and strength of possible hydro-
gen bonds and influence of the solvent. Hence, we pre-
pared deuterated isotopomers 4–6 (Figure 3) and rec-
orded their 13C NMR spectra. An attention was also paid
to conditions of deuteration which might occur at differ-
ent sites in the molecule yielding various isotopomers.
Figure 2. Tautomeric structures of enaminones: a) keto-
amine, b) enol-imine.
Croat. Chem. Acta 84 (2011) 203.

T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
205
EXPERIMENTAL
Materials
nol/chloroform mixture (v:v = 2:1, 2.0 ml) and by add-
ing a few drops of deuterium oxide. The solution was
refluxed for 5 h, evaporated under vacuo to 1/3 of initial
volume and the reaction product was rinsed and dried.
Yield: 11 mg, 73.3%.
Partially deuterated isotopomer of (3), owing to its
low solubility in methanol, was prepared by dissolving
3 (10 mg) in solution of deuterated methanol and chlo-
roform (v:v = 2:1, 2.0 ml) by adding a few drops of
deuterium oxide. The solution was refluxed for 5 h and
then evaporated under vacuo to 1/3 of the initial vo-
lume. Crystals were filtered and dried. Yield: 11 mg,
66.7 %.
Dehydroacetic acid, ortho-, meta- and para- phenyle-
nediamine, methanol-d4, chloroform-d1 and deuterium
oxide were purchased from commercial sources and
used without further purification. Chloroform and
dichloromethane were dried using P2O5. Methanol was
dried using iodine and magnesium turnings and then
distilled.
Synthesis of Non-deuterated Compounds
Ortho-N,N´-phenylene-diylbis[3-(1-aminoethyl)-6-meth-
yl-2H-pyran-2,4(3H)-dione] (1) was prepared as pub-
lished in the literature.5 2.5 mmol of ortho-
phenylenediamine was added in a hot methanolic solu-
tion (15 ml) of dehydroacetic acid (5.0 mmol). The
reaction mixture was refluxed and temporally stirred for
10 h. After staying for 6 days at approximately 4 °C
crystals were filtered, rinsed with cold ethanol and dried
under vacuo. Yield: 0.53 g, 52%.
Meta-N,N´-phenylene-diylbis[3-(1-aminoethyl)-6-
methyl-2H-pyran-2,4(3H)-dione] (2) was prepared by a
similar procedure. Solution of meta-phenylenediamine
(0.27 mmol in 5 ml) was added in a hot methanolic
solution (6 ml) of dehydroacetic acid (0.13 mmol). The
reaction mixture was refluxed and temporally stirred for
4 h. After staying for one day at room temperature,
crystals were filtered, rinsed with cold methanol and
dried on the air. Yield: 35.3 mg, 65%.
Para-N,N´-phenylene-diylbis[3-(1-aminoethyl)-6-
methyl-2H-pyran-2,4(3H)-dione] (3) was prepared in a
hot methanolic solution (6 ml) of deydroacetic acid
(2.85 mmol) with addition of para-phenylenediamine
(0.71 mmol in 4 ml of methanol). The reaction mixture
was refluxed and temporally stirred for 6 h. After stay-
ing for approximately 2 days, crystals were filtered,
rinsed with cold methanol and dried on the air. Yield:
20.7 mg, 71.4%.
To the best of our knowledge, the synthesis of
compounds 2–6 has not yet been reported in the litera-
ture.
IR Spectroscopy
IR spectra of compounds 1–3 and deuterated isotopo-
mers 4–6 were recorded on Perkin-Elmer Spectropho-
tometer 502, with a resolution of 2 cm−1 and 4 scans per
spectrum. Samples were prepared by KBr pellets.
Mass spectrometry
Mass spectra were recorded on a Waters Micromass Q-
TOF micro instrument equipped with an electrospray
ionization (ESI) source and operating in positive ion
mode. The instrument parameters were as follows: ca-
pillary voltage 3.0 kV, sample cone voltage 18 V, ex-
tractor voltage 1 V, source temperature 100 °C, desolva-
tion temperature 150 °C and desolvation gas flow 350
L/h.
Samples were dissolved either in a 1:1 (v:v) mix-
ture of acetonitrile and water or in methanol, to a con-
centration of about 10 µg/mL and introduced into the
ESI source from a gastight 250 µL syringe driven by a
syringe pump at a flowrate of 10 µL/min.
NMR spectroscopy
Synthesis of Deuterated Isotopomers
One-dimensional (1H and APT) and two-dimensional
(COSY, HSQC and HMBC) NMR spectra were record-
ed on a Bruker Avance 600 spectrometer equipped with
an inverse probe and z – gradient accessories with con-
stant magnetic field operating at 14.2 T. Samples were
measured at 298 K in 5 mm tubes. CDCl3 was used as
solvent and TMS as internal standard.
Partially deuterated isotopomer of (1) was prepared by
dissolving 1 (30 mg) in deuterated methanol (1.0 ml)
and by adding a few drops of deuterium oxide. The
solution was refluxed for 3 h, evaporated under vacuo to
1/3 of initial volume and the reaction product was rinsed
and dried. Yield: 21.5 mg, 71 %.
Partially deuterated isotopomer of (2) was pre-
pared by dissolving 2 (15 mg) in deuterated methanol
(1.0 ml) and by adding a few drops of deuterium oxide.
Solvent was almost completely evaporated under vacuo
and the reaction product at the bottom of the reaction
flask was rinsed and dried. Yield: 10.5 mg, 70 %.
Partially deuterated isotopomer of (2) was also
prepared by dissolving 2 (15 mg) in the metha-
Proton spectra with spectral width of 12019.23
Hz, acquisition time of 1.36 s and digital resolution of
0.55 Hz per point were measured with 32 scans. A
number of points in time domain was 32 K.
APT spectra with spectral width of 35971.22 Hz,
acquisition time of 0.91 s and digital resolution of 0.55
Hz per point were measured with 51791 scans per spec-
trum. Number of points in time domain was 64 K.
Croat. Chem. Acta 84 (2011) 203.

206
T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
Table 1. Characteristic IR vibrational modes (cm−1) of 1–3
−1

ν / cm
Vibrational mode
1
2
3
ν(N–H)
3450
3050
3400
3050
3459
–
ν(C–H)aromatic
ν(C–H)aliphatic
ν(C=O)lactone
2950
2950
2950
1700
1559
800
1706
1708
ν(C=O)ketone
1570
1563
ν(C–H)o.o.p.* bending, aromatic
* out of plane
998, 950, 840
999, 846
In the gCOSY experiment, 2048 points in F2
dimension and 512 increments in F1 dimension were
used. Spectra were measured with spectral width of
12019.23 Hz, 4 pulses per increment and relaxation
delay of 1 s. Digital resolution was 5.87 Hz per point in
F2 and 23.44 Hz per point in F1 dimension.
For gHSQC and gHMBC spectra 2048 points in F2
dimension and 512 increments in F1 dimension were
used. Spectral width was 12019 Hz in F2 dimension and
36216 Hz in F1 dimension. Relaxation delay was 1 s and
number of scans was 128. Digital resolution was 5.87 and
141.48 Hz per point in F2 and F1 dimension, respective-
ly. A delay for long-range coupling was set to 65 ms.
trometry. IR spectra revealed characteristic vibrational
modes of phenylenediamines and diones. These are
displayed in Table 1.
Owing to structural similarity, the IR spectra of
1–3 are very similar in the range 3500–1100 cm−1. The
C–H aromatic stretching vibration in 3 is partially over-
lapped by a broad N–H stretching vibration band and
hence could not be unambiguously assigned in the spec-
trum. Differences in out of plane bending vibrations are
due to different substitution at the benzene ring. The
broadening of secondary amine N–H stretching vibra-
tion band at ≈ 3450 cm−1 pointed towards the presence
of intramolecular hydrogen bonds. This is in agreement
with the results obtained by X-ray diffraction analysis of
1.5 The existence of C=O stretching band of conjugated
ketone and lack of O–H stretching mode in 1–3 indi-
cated that the predominant form in solid state is the
keto-amine.
RESULTS AND DISCUSSION
Structural Characterization
The structures of prepared compounds 1–3 were charac-
terized by IR and NMR spectroscopies and MS spec-
The ESI mass spectra of 1–3 showed the existence
of precursor ion [M+H]+ at m/z = 409 and the expected
Table 2. 1H and 13C NMR chemical shifts (ppm) for 1–3 in CDCl3
1
2
3
Atom
1H δ / ppm
13C δ / ppm
97.42
1H δ / ppm
13C δ / ppm
97.19
1H δ / ppm
13C δ / ppm
97.28
1(1`)
2(2`)
184.51
106.39
163.22
162.46
19.83
184.53
106.46
163.42
162.59
19.47
184.57
106.54
163.40
162.72
19.72
3(3`)
5.73
5.77
5.79
4(4`)
5(5`)
6(6`)
2.16
2.55
2.18
2.63
2.20
2.66
7(7`)
19.94
19.85
19.89
8(8`)
175.65
174.89
174.82
9(9`)
16.12
16.07
16.04
7.29
10(10`)
11(11`)
12(12`)
13(13`)
132.19
128.60
127.09
137.32
124.65
130.31
122.36
135.56
126.27
7.49
7.31
7.24
7.57
7.07
Croat. Chem. Acta 84 (2011) 203.

T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
207
Figure 5. The methyl region of 13C NMR APT spectrum of 6.
Figure 4. Precursor ion peaks in the mass spectrum of 6.
elucidate structure and dynamics of possible hydrogen
bonds and influence of the non-polar solvent we pre-
pared deuterated isotopomers of 1–3 in solution of deu-
terated methanol. According to NMR and MS spectra,
deuteration of N–H group was successful for 1 and 2
giving isotopomers 4 and 5a (Figure 3).
fragmentation pattern. The precursor ion signal at m/z =
411 in the mass spectra of deuterated compounds 1
(isotopomer 4) and 2 (isotopomer 5a) obtained in me-
thanol indicated the exchange of both N–H groups with
deuterium. Surprisingly, precursor ion peaks of deute-
rated compounds 5b and 6 prepared in a mixture of
deuterated methanol and chloroform pointed towards
multiple deuteration. This finding will be discussed later
on.
An attempt to prepare deuterated compound 3 (iso-
topomer 6) in deuterated methanol was unsuccessful due
to its very low solubility. Hence, isotopomer 6 was pre-
pared in a mixture of deuterated methanol and chloroform
(Figure 3). Deuteration of 2 was also performed under
these conditions yielding isotopomer 5b (Figure 3).
1H NMR and mass spectra of isotopomers 4 and
5a revealed that the deuteration occurred at both amino
groups. Furthermore, in mass spectra of isotopomers 5b
and 6 precursor ion peaks at m/z = 409.2, 410.2, 411.1
and 412.2 were observed (Figure 4). These findings
pointed towards the formation of multiple deuterated
species. The percentage of particular species can be
estimated from the peak intensities. Peaks at m/z 411.1
and 412.2 corresponded to three and four deuterium
atoms in the molecule which could not be obtained only
by deuteration of amino groups. This issue was resolved
by a close inspection of 13C NMR spectra.
1
The analysis of H and 13C NMR spectra con-
firmed the structures of 1–3 in solution and corroborated
the presence of the keto-amine form (Table 2). The
chemical shifts of C-2(2`) atoms assigned at ≈184.5
ppm corresponded to the keto group of dehydroacetic
acid involved in the RAHB. The amino group has been
found in proton spectrum at 16.1 ppm. Proton chemical
shifts at ≈ 2.2 ppm and ≈ 2.6 ppm were observed for
methyl protons H-6(6`) and H-7(7`), respectively. The
COSY spectra of 1–3 exhibited correlation peaks cor-
responding to different substitutions at the benzene ring.
Two correlated aromatic proton signals between H-
11(11`) and H-12(12`) with equal integrals revealed
ortho- substitution in 1. The H-13(13`) proton singlet at
7.07ppm confirmed the meta- substitution in 2. The
absence of correlation peaks between aromatic protons
in COSY spectrum of 3 indicated the symmetric para-
substitution at the benzene ring.
Namely, the 13C NMR spectra of deuterated com-
pounds 5b and 6 prepared in a mixture of deuterated
methanol and chloroform exhibited C–D triplets around
19.7 ppm indicating the presence of monodeuterated
methyl groups in α-position to amino group (Figure 5).
The 13C NMR spectra recorded with more scans re-
vealed quintets and septets corresponding to two and
three deuteriums in the methyl groups, respectively.
According to these findings, the following mechanism
of deuteration shown in Figure 6 can be proposed.
The mechanism involves hyperconjugation of po-
lar resonant form (c) and the deuteration of methyl
groups. The resonant form (c) is stabilized by the partial
double bond character of C–N bond, present both in
amides and their higher homologues enaminones. The
catalytic D+ ions might come from the dissociation of
CD3OD or as a consequence of the decomposition of
The HMBC spectra of 1–3 exhibited correlation
peaks between C-4(4`) and H-6(6`) as well as between
C-8(8`) and H-7(7`). Significant line-broadening and
deshielding effects observed for amine N–H protons and
shielding of keto C-2(2`) carbons reflected interactions
of these groups via intramolecular hydrogen bonds.
Deuterium Isotope Effects
Deuterium isotope effects have found practical applica-
tions as a structural tool for analysis of hydrogen bonds
and tautomeric equilibria in solution.15,20 In order to
Croat. Chem. Acta 84 (2011) 203.

208
T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
Figure 7. Determination of a deuterium isotope effect at C-
2(2`) in 5a. The narrow region APT spectrum of the partially
deuterated 5a (a) before and (b) after addition of the non-
deuterated isotopomer.
Hence, the site and selectivity of deuteration depend on
Figure 6. The mechanism of the methyl group deuteration at
solvent properties and reaction conditions.
position 7.
Subsequently, DIEs were determined from mix-
tures of partially deuterated 4−6 and non-deuterated 1−
3 samples using the equation (1). The sign of DIE was
determined by quantitative addition of the non-
deuterated sample in the NMR tube of the deuterated
sample, as demonstrated in Figure 7. DIEs are given in
Table 3.
In 4 and 5a generally high values of DIE were ob-
served. The high positive values at C-8(8`) in 4 (+320.9
ppb) and 5a (+250.0 ppb) provided further evidence for
the localized keto-amine form in solution stabilized by
intra-molecular N–H···O hydrogen bonds. Relatively
CDCl3 yielding D+ and CCl3− which can be accelerated
by heating.28
Owing to the higher pKa value of the mixture of
deuterated methanol and chloroform in comparison with
the pure deuterated methanol, the deuteration of N–H
groups is less possible and the main reaction products
are 5b and 6. This is in accordance with much smaller
deuterium isotope effects measured for these com-
pounds in comparison with 4 and 5a as seen in Table 3.
Table 3. Deuterium isotope effects (ppb) on 13C NMR chemical shifts for 4 − 6 in CDCl3
nΔ13C (D) / ppb
Atom
5b(a)
6(a)
4
5a
(b)
1(1`)
2(2`)
600.0
700.0
−187.5
375.0
−150.0
100.0
−350.0
250.0
−7.7
−8.0
−7.8
(b)
3(3`)
−193.1
4(4`)
100.1
5(5`)
108.0
(b)
−9.4
6(6`)
(b)
7(7`)
231.2
22.9
230.7
24.1
8(8`)
320.9
9(9`)
(b)
10(10`)
11(11`)
12(12`)
13(13`)
500.3
(b)
−132.5
−107.9
7.7
7.4
9.2
500.5
(b)
(a) prepared in a mixture of deuterated methanol and chloroform
(b) broad signal
Croat. Chem. Acta 84 (2011) 203.

T. Jednačak et al., Bioactive Phenylenediamine Derivatives of Dehydroacetic Acid
209
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at C-7(7`). The triplets arising from a scalar coupling
between methyl carbon and directly attached deuterium
were observed in 13C NMR spectra (Figure 5). High
values of DIE (22.9 and 24.1 ppb) at atom C-8(8`) in 5b
and 6 could be attributed to the proximity of deuterium
and are intrinsic in nature. Negative DIEs were ob-
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whereas no such effects were detected in 5b. It is not
quite clear what the reason for this observation is but
inter-molecular interactions with other molecules and
influence of the solvent which can easily approach these
C=O groups cannot be excluded.30
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equilibrium and intrinsic contributions. Generally,
equilibrium DIEs have higher magnitude than intrinsic
ones.15,20,27 Much larger values of DIEs in isotopomers
4 and 5a with respect to those observed in 5b and 6
can be rationalized by an efficient transmission path-
way through intramolecular N−H(D)···O hydrogen
bonds.
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Acknowledgements. This study was supported by the Ministry
of Science, Education and Sports of the Republic of Croatia
(Project Nos. 119-1191342-1083 and 119-1191342-1082). We
thank Prof. Hrvoj Vančik and Katarina Čuljak for helpful
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