Synthesis, spectroscopic characterization, stability assessment and DNA-binding of new 2,6-piperidinedione derivatives

Article abstract of DOI:10.1016/S0014-827X(01)01131-4

This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione (NPD). The

Full text of DOI:10.1016/S0014-827X(01)01131-4

www.elsevier.com/locate/farmac
Il Farmaco 56 (2001) 763–770
Synthesis, spectroscopic characterization, stability assessment and
DNA-binding of new 2,6-piperidinedione derivatives
Laila Abou-Zeid a, Abdalla M. El-Mowafy b, Mohammed M. El-Kerdawy a,
Huda Hamza c, Mohammed E. Abdel-Hamid c,*
a Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura Uni6ersity, Mansoura, Egypt
b Department of Applied Therapeutics, Faculty of Pharmacy, Kuwait Uni6ersity, P.O. Box 24923, Safat 13110, Kuwait
c Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait Uni6ersity, P.O. Box 24923, Safat 13110, Kuwait
Received 3 January 2001; accepted 30 May 2001
Abstract
This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-
piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione
(NPD). These compounds were prepared by reacting N-(4-substituted benzoyl)-glutamines with N-hydroxysuccinimide to afford
the corresponding esters, which were heated to produce the corresponding 2,6-piperidinedione (PD) compounds. Non-destructive
1
analytical procedures such as H NMR and NIR analyses confirmed the postulated chemical structures of these PD compounds.
HPLC chromatograms at an ambient temperature or from solutions preheated at 30, 40 or 60 °C displayed only a single peak
for each compound. Combination of heat with pH modification had virtually no effect on the obtained peaks, thus attesting to
the stability and purity of these compounds. MS analysis displayed molecular mass ions indicative of BPD, MPD and NPD at
m/z 233.4, 263.2 and 278.3, respectively. The fragmentation patterns using MS/MS analyses conformed to the structural and
molecular formulae of the prepared compounds. Furthermore, preliminary biological assessments showed the capacity of these
compounds to bind to the DNA. NPD, but not BMP or MPD, had a superior affinity to the DNA than the prototype ANP-A10.
© 2001 Elsevier Science S.A. All rights reserved.
Keywords: 2,6-Piperidinediones; NIR; HPLC; MS/MS; DNA-binding
ducted on human cancer cells and cancer patients
showed antineoplastons as remarkable cytostatic, im-
munemodulatory and cytodifferentiating agents [10–
1. Introduction
Antineoplaston,
3-(phenylacetylamino)-2,6-pipe-
14]. In all such studies, antineoplastons acted mostly
via nuclear mechanisms that included interaction with
the DNA or modulation of apoptic rates in tumor- or
host defensive-cells [11]. Despite the numerous biologi-
cal investigations on antineoplastons, little information,
if any, is available on their structure characterization or
stability in biological fluids. Accordingly, the present
study was undertaken to characterize the structural
profiles of antineoplastons and gain insights into their
stability at more drastic pH or thermal conditions.
Recent analytical procedures [15,16] have been cur-
rently utilized to investigate such possibilities. Further-
more, we report on the capacity of these newly
ridinedione (A10) is a human endogenous cytodifferen-
tiating agent with a broad range of biological activities
[1–4]. A10 was proved to be an efficacious cancer
chemopreventive agent when tested on cancer cell lines
[5–7] or given to animals with various tumor types [8].
Moreover, A10 and its metabolites showed promising
cytostatic responses when challenged on tumor tissue
cultures [6,7,9] or administered to human patients with
hepatocellular carcinoma [6,7]. Likewise, studies con-
* Corresponding author.
E-mail address: abdel-hamid@hsc.kuniv.edu.kw (M.E. Abdel-
Hamid).
0014-827X/01/$ - see front matter © 2001 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01131-4

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L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
prepared derivatives to bind to the DNA. This assay is
reportedly predictive of the antineoplastic activity of
agents that target the genomic structure [14,17].
2.2. Instruments
Melting point determinations were recorded using
Thomas Hoover Capillary Apparatus. Elemental analy-
ses (90.4%) were performed by Atlantic Microlab Inc.,
Nocross, GA. 1H NMR spectra were measured in
DMSO-d6 using 300 MHz Bruker NMR spectrometer.
FT-NIR spectra were collected using Nexus FT-NIR
equipment (Nicolet, USA) with NIR source (10 000–
4000 cm−1). NIR spectra were run in the transmission
mode using Nicolet NIR updraft unit. All data process-
ing was controlled by the instrument build-in OMNIC
E.S.P 5.1 software (Nicolet). HPLC analyses were per-
formed using an isocratic high-performance liquid chro-
2. Experimental
2.1. Materials
All chemicals, reagents and solvents used for the
chemical synthesis of 2,6-piperidinedione compounds
were of analytical grade. HPLC grade solvents were
used for HPLC and LC/MS analyses.
Scheme 1. Chemical reactions for the synthesis of 3-(4-substituted-benzoylamino) 2,6-piperidinedione analogs.
Table 1
Melting points, elemental analysis, and 1H NMR analysis of BPD, MPD and NPD
Comp.
BPD
m.p. (°C)
222–224
Formula
Analysis (Calc./Found)
C 62.06/62.42
H 5.17/5.21
1H NMR
C12H12N2O3
2.01 (m, 2H, HCꢀCH2ꢀCH2)
2.55 (m, 2R, CH2ꢀCO)
4.70 (q, 1H, NHꢀCHꢀCH2)
7.85 (m, 5H, ArꢀH)
N 12.06/12.23
C 59.54/59.35
H 5.34/5.39
8.61 (d, 1H, CONHꢀCH)
10.89 (s, 1H, COꢀNHꢀCO)
1.98 (m, 2H, HCꢀCH2ꢀCH2)
2.45 (m, 2H, CH2ꢀCO)
3.91 (s, 3H, CH3O)
4.65 (q, 1H, NHꢀCHꢀCH2)
7.75 (m, 4H, ArꢀH)
8.75 (d, 1H, CONHꢀCH)
10.50 (s, 1H, COꢀNHꢀCO)
2.02 (m, 2H, HCꢀCH2ꢀCH2)
2.45 (m, 2H, CH2ꢀCO)
4.78 (q, 1H, NHꢀCHꢀCH2)
7.55 (d, 2H, ArꢀH)
MPD
NPD
213–215
240–241
C
13H14N2O4
N 10.68/10.65
C
12H11N3O5
C 51.98/52.07
H 3.97/4.01
N 15.16/15.09
8.25 (d, 2H, ArꢀH)
8.68 (d, 1H, CONHꢀCH)
10.60 (s, 1H, COꢀNHꢀCO)

L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
765
Fig. 1. NIR transmission spectra of BPD, MPD and NPD.
by the instrument build-in Millennium software. MS
and MS/MS analyses were performed on the LCQ mass
spectrometer (Finnigan) linked to HPLC pump P 2000
Spectra system (TSP). The HPLC column was Shim
Pack CLC-CN, 5 mm (15×6 mm id) and the mobile
phase was a mixture of methanol/acetic acid solution
matograph (Waters 2690 Separation Module) equipped
with photodiode array detector (Waters 996) and Shim
Pack CLC-SIL column (150×6 mm id, 5 mm). The
mobile phase (methanol/1% acetic acid solution 1:1)
was pumped at flow rate 1 ml/min and the eluents were
monitored at 262 nm. Analytical data were processed

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L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
1% (4:1) flowing at 1 ml/min. APCI was selected as an
ionization process. APCI conditions were: corona dis-
charge 4.5 kV, vaporization temperature 430 °C and
capillary temperature 147 °C. The scanning modes
were full MS and full MS/MS. All data processing was
controlled by the instrument LCQ software.
stirred at room temperature for 24 h. The solvent was
removed in vacuo and the residue was dissolved in
water (50 ml) and neutralized with 1N HCl solution.
The crude product was purified by flash column chro-
matography [solvent: ethyl acetate/cyclohexane (1:1)].
The solvent was evaporated in vacuo and the white
crystalline product was collected. The yield percentages
of compounds (2A–C) were 75.1, 69.7 and 64.9, respec-
tively, of the purified products (Scheme 1).
2.3. Procedures
2.3.1. Preparation of 2,6-piperidinedione deri6ati6es
2.3.1.2. Preparation of 3-(4-substituted-benzoylamino)-
2,6 piperidinediones (3A–C). A solution of 1,3-dicyclo-
hexylcarbodiimide (20 mmol) in dry DMF (50 ml) was
gradually added to a mixture of N-hydroxysuccinimide
(20 mmol) and 2A–C (20 mmol) in dry DMF (50 ml).
The reaction mixture was stirred overnight at room
temperature. A white precipitate of 1,3-dicyclohexy-
2.3.1.1. Preparation of N-(4-substituted benzoyl)-L-glu-
tamines (2A–C). The acid chloride solutions of 1A–C
(34.25 mmol) in N,N-dimethylformamide (DMF) (100
ml) were gradually added to a cooled aqueous solution
of
L-glutamine (34.25 mmol) and NaHCO3 (68.50
mmol) in water (100 ml). The reaction mixture was then
Fig. 2. Full MS spectra of BPD (a); MPD (b) and NPD (c).

L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
767
Fig. 3. MS/MS spectra of BPD (a); MPD (b) and NPD (c).
lurea was filtered off and the filtrate was heated under
reflux for 4–6 h at 100 °C. The reaction mixture was
evaporated in vacuo and the corresponding target com-
pound was purified by flash column chromatography
[solvent: chloroform/methanol (10:1)]. The yield% of
the purified products was 49.7 of BPD, 55.2 of MPD
and 50.0 of NPD.
40 or 60 °C for 6 h. The vials were cooled and solu-
tions were neutralized (when necessary) and 10 ml
aliquots were injected into HPLC.
2.3.3. LC–MS and LC/MS/MS
Aliquots (10 ml) of BPD, MPD or NPD in methanol
were injected into the mass spectrometer. The samples
were measured in MS and MS/MS scanning modes.
2.3.2. Purity and stability studies
Aliquots (50 ml) of freshly prepared solutions of
BPD, MPD or NPD in methanol (1 mg/ml) were trans-
ferred into 1 ml glass vials and diluted to volumes with
either methanol/water (1:1), 0.1 M HCl solution, or
phosphate buffer solution (pH 7.5). The solutions were
heated in thermostatically controlled water bath at 30,
2.3.4. Assessment of DNA binding affinity
DNA binding was determined according to the re-
ported procedures [17]. The principle of the assay is
that DNA/methyl green forms a stable green-colored
complex in neutral solutions (u max, 642–645 nm).
When displaced by agents that bind DNA, the liberated

768
L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
methyl green adds a molecule of water to its structure,
thereby forming a colorless ‘carbinol’. The reduction in
the absorbance of DNA/methyl green complex reflects
the DNA binding capacity of the added drug.
cm−1 (MPD) and 6632.06 and 5106.42 cm−1 (NPD).
BPD was also characterized by the appearance of a
‘split band’ at 6498.47/6373.12 cm−1. MPD also
showed a ‘split band’ at 4933.01/4892.27 cm−1
.
DNA/methyl green (20 mg/ml) was suspended in 0.05
M Tris–HCl buffer (pH 7.5) containing 7.5 mM
MgSO4, stirred overnight at 37 °C. The newly synthe-
sized piperidinediones were dissolved in dimethyl sulfox-
ide (DMSO). Solvent was removed in vacuo, and 200 ml
of the DNA/methyl green solution was added to each
well. Initial absorbance for each well was determined in
a microplate reader and samples were then incubated in
the dark at ambient temperature. After 24 h, the final
absorbance of the samples was determined. Readings
were corrected for initial absorbance and normalized as
a percentage of the untreated DNA/methyl green ab-
sorbance value. IC50 values were determined by linear
regression of data plotted on a semi-log scale (Table 2).
3.3. Purity and stability assessment
The purity of BPD, MPD and NPD was assessed by
HPLC analysis. The chromatograms showed only a
single peak for each compound, thus indicating the lack
of degradation products or reaction intermediates. Fur-
ther evidence for purity of these compounds comes from
LC/MS analyses wherein the TIC of compounds exhib-
ited single peaks that were indicative of the mass ions of
BPD, MPD and NPD.
Stability of the prepared compounds was also verified
by running HPLC analyses following application of
more drastic levels of heat or pH. The peak areas of
BPD, MPD and NPD were insignificantly changed after
acidic or alkaline incubations for 6 h at 30, 40 and
60 °C. Furthermore, no extra peaks due to degradation
products were observed in the chromatograms after
these treatments. These observations indicate the resis-
tance of the investigated compounds to hydrolysis under
conditions similar to those encountered in gastric or
intestinal fluids.
3. Results and discussion
3.1. Chemistry of
3-(4-substituted-benzoylamino)-2,6-piperidinedione
The presented 2,6-piperidinedione derivatives were
synthesized according to Scheme 1. The intermediate
compounds, N-(4-substituted benzoyl)-
(2A–C), were firstly prepared by reacting the appropri-
ate 4-substituted benzoyl chlorides (1A–C) with -glu-
L-glutamines
L
tamine in aqueous sodium bicarbonate [2]. These
intermediates (2A–C) were then reacted with N-hy-
droxysuccinimide in the presence of 1,3-dicyclohexylcar-
bodiimide to afford the corresponding N-hydroxy-
succinimide esters. The obtained esters were heated at
100 °C for 4–6 h to afford the 3-(4-substituted-ben-
zoylamino)-2,6 piperidinedione compounds 3A–C. The
yield percentages of the purified BPD, MPD and NPD
were 49.7, 55.2 and 50.0, respectively. The melting
1
points, elemental analysis and H NMR analysis sup-
ported the assigned chemical structures (Table 1).
3.2. NIR analysis
FT-NIR has now become an increasingly valuable
tool in identifying compounds with cognate chemical
structures [15]. Currently, the NIR spectra of BPD,
MPD and NPD were recorded in the transmission
scanning mode using NIR updraft unit in the range of
10 000–4000 cm−1. As shown in Fig. 1, despite the
structural similarity of investigated compounds, their
spectra can be distinguished by fine measurement of the
wave number of NIR-bands of interest. Therefore, the
4-substituted analogs, MPD and NPD, can be distin-
guished from the non-substituted compound, BPD, by
having characteristic NIR-bands at 6647.24 and 5115.89
Scheme 2. A proposed MS/MS fragmentation pattern of BPD.

L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
769
Scheme 3. A proposed MS/MS fragmentation pattern of MPD.
3.5. DNA binding of A10 analogs
3.4. LC–MS and LC–MS/MS analyses
Fig. 2a–c shows the MS spectra of BPD, MPD and
NPD, respectively. Molecular mass ions [M−H]+ at
m/z were 233.4, 263.2 and 278.3, respectively. MS/MS
analysis was further used to define the fragmentation
pattern and confirm the molecular formulae of
these compounds. Fig. 3a shows the MS/MS spectra of
BPD at m/z 233.4 and 10% of collision energy.
The major fragments of BPD, at m/z 204.9, 122.1
and 105.1, correspond to structures in Scheme 2. Fig.
3b displays the MS/MS of MPD at m/z 263.2 and
7% of collision. The fragment ions, at m/z 235.0, 152.0
and 135.0, refer to structures in Scheme 3. Fig. 3c shows
the MS/MS fragmentation of NPD at m/z 278.3
and 20% of collision. The major fragments, observed
at m/z 249.9, 235.3, 149.3 and 135.5, are congruent
to the structures in Scheme 4. In general, the predicted
and reported m/z values were in good agreement with
all proposed structures (Schemes 2–4). A mass range
difference of ꢀ0.1 amu between the expected and
experimental values of the base fragment peaks was
observed.
This assay was carried out to gain insights into the
potential of the newly prepared A10 analogs to interact
with DNA. It has been shown that the prototype
ANP-A10 may interact reversibly with DNA, thereby
competing with carcinogens that form covalent linkage
with DNA [6]. Present data, supported by statistical
analyses, revealed that compound NPD bound to the
DNA and displaced methyl green with a significantly
higher potency than the prototype A10 (PB0.05, Table
2). Compounds BPD and MPD showed lower affinity to
the DNA. Taken together, these data suggest that
interaction with DNA is a possible nuclear mechanism
for the action of A10-analogs. This dogma is in agree-
ment with A10 studies on DNA fragmentation, cell
cycle and apoptosis in various malignant cell lines [7,11].
4. Conclusions
The present study is the first to fully characterize the
spectroscopic profiles of 2,6-piperidinedione, a now
well-recognized class of antineoplastic agents. Accord-

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L. Abou-Zeid et al. / Il Farmaco 56 (2001) 763–770
ingly, three new such derivatives were synthesized for
that purpose. Their structures were confirmed by con-
ventional spectroscopic methods as well as by advanced
analyses such as LC/MS, LC/MS/MS and NIR. These
procedures permitted identification, differentiation and
confirmation of the molecular/structural formulae of
2,6-piperidinediones. Also, they provided unequivocal
evidence for the stability of these compounds under
challenging pH or thermal conditions, similar to those
encountered in biological fluids or during pharmaceuti-
cal preparations. Because the antineoplastic activity
known for the lead is largely dictated by its DNA
interaction, we verified the potential of the prepared
compounds to bind to the DNA in vitro. A reasonable
to appreciable affinity to bind the DNA was obtained.
Acknowledgements
The authors wish to thank the Faculty of Pharmacy,
Kuwait University for the research and instrument
facilities of FT-NIR and LC/MS used throughout this
work.
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Scheme 4. A proposed MS/MS fragmentation pattern of NPD.
Table 2
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A10
9997.3
10894.2
13698.7
4392.1 a
BPD
MPD
NPD
Data are presented as means9standard error means, n=4–5.
a Significantly lower than A10 value; PB0.05.