Cytotoxicity of some azines of acetophenone derived mono-mannich bases against jurkat cells

Article abstract of DOI:10.1248/bpb.26.631

Acetophenone derived mono-Mannich bases (Ig1-Ig4), 1-aryl-3-amino-1- propanone hydrochlorides, which are known to have cytotoxicity in Jurkat cells, were synthesized. Then, they were converted to corresponding azine derivatives (D1-D4), N,N′-bis(3-amino-1

Full text of DOI:10.1248/bpb.26.631

May 2003
Biol. Pharm. Bull. 26(5) 631—637 (2003)
631
Cytotoxicity of Some Azines of Acetophenone Derived Mono-Mannich
Bases against Jurkat Cells
d
Halise Inci GUL, ,a,b Mustafa GUL,c,d Jouko VEPSÄLAINEN,b Ercin ERCIYAS,e and Osmo HÄNNINEN
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ataturk University; TR-25240 Erzurum, Turkey:
b Department of Chemistry, University of Kuopio; 70211 Kuopio, Finland: c Department of Physiology, Faculty of
Medicine, Ataturk University; TR-25240 Erzurum, Turkey: d Department of Physiology, Faculty of Medicine, University of
e
Kuopio; 70211 Kuopio, Finland: and Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ege University;
TR-35100 Izmir, Turkey. Received October 22, 2002; accepted January 8, 2003
Acetophenone derived mono-Mannich bases (Ig1—Ig4), 1-aryl-3-amino-1-propanone hydrochlorides, which
are known to have cytotoxicity in Jurkat cells, were synthesized. Then, they were converted to corresponding
azine derivatives (D1—D4), N,N؅-bis(3-amino-1-aryl-propylidene)hydrazine dihydrochlorides, which are bifunc-
tional agents. The aryl part was replaced by phenyl in Ig1, Ig2, Ig3, D1, D2, and D3, and by p-hydroxyphenyl in
Ig4 and D4. The amine part was replaced by dimethylamine in Ig1, D1, Ig4 and D4, by piperidine in Ig2 and D2,
and by morpholine in Ig3 and D3. The aim of this study was to investigate whether the modification in chemical
structure, converting the mono-Mannich base to a corresponding azine derivative, improves the cytotoxicity. In
addition, the effect of the representative compound, D3, N,N؅-bis(3-morpholine-4-yl-1-phenylpropylidene)hy-
drazine dihydrochloride, on cellular glutathione level after 1 h exposure in phosphate buffer at 37 °C was also de-
termined to provide information on a possible mechanism of cytotoxic action. Compounds D2—D4 are reported
for the first time in this study. Except for Ig2 and D2, the cytotoxicity of mono-Mannich bases, Ig1, Ig3 and Ig4
and corresponding azine derivatives, D1, D3 and D4 were higher than the reference compound 5-FU. Azine de-
rivatives D1 and D4 had almost equal cytotoxic potency with corresponding mono-Mannich bases Ig1 and Ig4,
respectively. On the other hand, azine derivatives D2 and D3, had 1.28 and 1.90-times less cytotoxicity in Jurkat
cells compared with the mono-Mannich bases, Ig2 and Ig3, respectively, from which they are derived. Azine de-
rivative D3 dose-dependently decreased the total cellular glutathione level, suggesting that azine derivatives may
exert cytotoxicity by thiol alkylation. Azine derivatives with equal or less cytotoxic potency compared to the
mono-Mannich bases they are derived from seemed to be less suitable derivatives for the development of new cy-
totoxic compounds.
Key words Mannich base; azine; cytotoxicity; Jurkat cell; glutathione; thiol alkylation
Mannich bases have several biological activities such as transportation of the compound to the site of action.
antimicrobial,1—5) cytotoxic,6—9) anticancer,10) analgesic,11)
Production of thiol adducts in the stability studies,1,3,6) car-
anti-inflammatory,12,13) diuretic14,15) and anticonvulsant16—19) ried out under in vitro physiological conditions (in phosphate
activities. An amino ketone, Mannich base, containing at buffer with pH 7.4, at 37 °C), with 2-mercaptoethanol, vari-
least one activated hydrogen atom at the b position to an ous unsaturated ketones or Mannich bases, suggests that a,b-
amino function, can undergo deamination in vivo20) or under unsaturated ketones and Mannich bases producing unsatu-
simulated in vitro conditions1,3,6) to liberate the corresspond- rated ketones by deamination to exert their antimicrobial ac-
ing a,b-unsaturated ketones. The a,b-unsaturated ketone is tivities by thiol alkylation of unsaturated ketones.
an active center for nucleophilic attack. The biological activi-
Clark and co-workers31) found that the thiol adduct had the
ties of Mannich bases, such as antimicrobial,1—3,21,22) cyto- greatest fungitoxicity, which was probably due to the same
toxic and anticancer6,9,23) activities, have been attributed to reaction. Dimmock et al. (1994) have shown that Mannich
these liberated a,b-unsaturated ketones which can alkylate bases of conjugated styryl ketones inhibit one or more of the
nucleophiles, especially thiol groups1,6,24,25) rather than amino following enzymes in the glutathione metabolic pathway:
and hydroxyl groups,26) by Michael type addition reactions of namely, glutathione S-transferases, glutathione reductase,
nucleophiles to a,b-unsaturated ketones.27) In addition, en- gamma-glutamyl transpeptidase and glutathione peroxidase
zyme-catalyzed alkylation of the thiol group of cystein with in Candida albicans.32) They concluded that the antifungal
a,b-unsaturated carbonyl compounds has been observed.28) activity of the Mannich bases of conjugated styryl ketones
The antimicrobial activity of a,b-unsaturated ketones is due may be due, at least in part, to interference with the GSH
to their reactions with essential thiol groups in bacteria and metabolic pathway. In one of our previous studies, we ob-
fungi, resulting in b-ketothioethers.29) Electron densities on served that mono-Mannich bases and bis Mannich bases de-
b-carbons are lower30) than on a-carbons in various unsatu- crease the total glutathione level in a dose-dependent manner
rated ketones, and thus the attack of the nucleophils should when Jurkat cells are exposed to the compounds in phosphate
occur at the b-carbon atoms. It was also shown that increased buffered saline for 1 h at 37 °C with gentle shaking.25) How-
antimicrobial activity was associated with an increased ever, in our subsequent study, where the Jurkat cells were ex-
breakdown of the Mannich bases.29) Similarly, a number of posed to the Manncih bases in culture conditions, the glu-
a,b-unsaturated ketones have alkylating ability against bio- tathione level was increased, possibly because of the stimula-
logically important nucleophiles to produce cytotoxicity.21,23) tion of feed-back mechanisms regulating the cellular glu-
The amino group in Mannich bases would increase the water tathione level.24) These findings also suggested that thiol
solubilizing property of the molecule, which may assist in alkylation may be an important mechanism responsible for
* To whom correspondence should be addressed. e-mail: incigul@mailcity.com
© 2003 Pharmaceutical Society of Japan

632
Vol. 26, No. 5
the cytotoxic activity of Mannich bases. A number of bifunc-
Synthesis of Mono-Mannich Bases (Ig1—4, Table 1)
tional agents display antitumor properties and are often for- Synthesis of the mono-Mannich bases, Ig1—3, was reported
mulated as prodrugs, e.g. the administration of clinically use- previously.9) Ig4 was synthesized as described.35)
ful nitrogen mustards produces aziridines, which are bioac-
tive species alkylating various cellular constituents.33)
Synthesis of the Corresponding Azine Derivatives
(D1—D4, Table 1) A solution of hydrazine hydrate (1.75 g,
The objectives of the present investigation were as follows: 0.035 mol) in ethanol (15 ml) was added to a solution of 3-
First, to synthesize a small number of prototype molecules, amino-1-aryl-1-propanone hydrochloride type of mono-Man-
mono-Mannich bases which are known cytotoxic activities nich base (0.07 mol) in ethanolic acetic acid (3% w/v, 70 ml).
against Jurkat cells,9) and then to convert them to the corre- 3-Dimethylamino-1-phenyl-1-propanone hydrochloride for
sponding azine derivatives, which have the potential for lib- D1, 3-piperidino-1-phenyl-1-propanone hydrochloride for
erating bifunctional alkylating species in living tissues. Sec- D2, 3-morpholino-1-phenyl-1-propanone hydrochloride for
ond, to investigate how the cytotoxic activity is affected by D3, and 3-dimethylamino-1-(p-hydroxyphenyl)-1-propanone
the conversion of mono-Mannich bases to the corresponding hydrochloride for D4 were used as mono-Mannich bases in
azine derivatives. The effect of the representative compound, the reactions to synthesize suitable azine derivatives. The
D3, on the most abundant cellular thiol, glutathione,34) in Ju- mixtures were stirred at room temperature for 24 h. After re-
rkat cells was also determined to provide information about moval of the solvent, the residue was washed with chloro-
their mechanism of action.
form three times (3ϫ20 ml), dried and crystallized from
ethanol to give the corresponding azine drivatives. The com-
pounds were dried by heating under vacuum for 24 h prior to
submission for elemental analysis. Melting points of the
EXPERIMENTAL PROCEDURES
Materials All chemicals used in syntheses were pur- compounds and yields of reactions were as follows, respec-
chased from Aldrich Chemical Co. (Munich, Germany). tively: D2 (172.5—175 °C (dec.), 81.96%), D3 (173—175
Melting points were determined on a Thomas Hoover appa- °C, 57.27%), D4 (185—186 °C, 57.56%). Elemental analy-
ratus (Philadelphia, PA, U.S.A.) and were uncorrected. UV ses (C, H, N) results of D2 (C28H40Cl2N4), D3
spectra were recorded in H2O by a Shimadzu double-beam (C26H36Cl2N4O2), and D4 (C22H32Cl2N4O2) were within 0.4%
spectrometer UV-150-02. (Tokyo, Japan). 1H-NMR (400 of the calculated values.
MHz) and 13C-NMR (100.6 MHz) spectra were obtained on a
Spectral Data of New Compounds: D2: lmax (H2O) nm
1
Bruker AM 400 WB Instrument (Karlsruhe, Germany) using (log emax): 290 (4.03). H-NMR (D2O/CD3OD; 60/40) d:
tetramethylsilane (TMS) as an internal standard (chemical 1.70 (2H, m), 1.74—1.86 (10H, m), 3.18 (4H, m), 3.29 (4H,
shift in d, ppm). Electrospray ionization (ESI) mass spectra m), 3.36 (4H, bs), 3.50 (4H, m), 7.57—7.65 (6H, m), 7.98
were recorded using a LCQ quadruple ion trap mass spec- (4H, m). Signal 3.36 ppm was very broad and hardly detected
trometer (Finnigan, San Jose, CA, U.S.A.). The spray needle from the baseline. This is due to chair-boat tautomers of the
was set at 5.5 kV in the positive ion mode. The spray was sta- piperidine ring. 13C-NMR (D2O/CD3OD; 60/40) d: 23.9 (t),
bilized by a nitrogen sheath flow and the value was set at 25.6 (t), 26.7 (t), 47.4 (t), 56.1 (t), 130.1 (d), 132.0 (d), 134.4
100. The inlet capillary temperature was 200 °C. The sam- (s), 138.2 (s), 166.4 (s). ESI-MS m/z: 431.2 (MϩϩH).
ples were dissolved in 50% methanol in water (10 mg/ml),
and 5 ml samples were injected. The eluent consisted of 50%
methanol in water, and the flow was set to 20 ml/min. Ele-
mental analyses were performed with a CHN-rapid elemental
analyzer (Perkin Elmer Instruments of Norwalk, CT, U.S.A.).
The purity of the compounds was assessed by TLC on silica
gel HF254-366 (E. Merck, Darmstadt, Germany). Develop-
ing solvents for TLC were chloroform–methanol (8 : 2) for
the compounds.
D3: lmax (H2O) nm (log emax): 285 (4.19). 1H-NMR
Table 1. Mono-Mannich Bases and Corresponding Azine Derivatives Syn-
thesized
Mono-Mannich bases
Jurkat cells (American Type Culture Collection, TIB-152,
Jurkat, clone E6-1, T cell leukemia, Human, Rockville,
Maryland) were kindly provided by Reitu Agrawal, Ph.D.,
A.I. Virtanen Institute, Kuopio, Finland. RPMI 1640 medium
W/L-glutamine (purchased in powder form and prepared and
sterile filtered according to the manufacturer’s instruction),
fetal bovine serum, and penicillin–streptomycin–glutamine
(100ϫ, containing 10000 U/ml penicillin G sodium, 10000
mg/ml streptomycin sulfate, 29.2 mg/ml L-glutamine, and
10 mM sodium citrate in 0.14% NaCl) were obtained from
Gibco BRL, Life Technologies (Paisley, Scotland). Multidish
6 and other culture dishes used were obtained from Nunclon,
Nunc (Roskilde, Denmark). Trypan blue, GSH, GSSG reduc-
tase, NADPH, and 5,5Ј-dithiobis (2-nitrobenzoic acid) were
obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
All other chemicals and reagents used were of the available
analytical grade.
X was H for the compounds Ig1, Ig2, and Ig3 and X was OH
for the compound Ig4
Z was N(CH3)2 for the compounds Ig1 and Ig4, N(CH2)5 for
Ig2, and N(CH2)4O for Ig3
Corresponding azine derivatives of mono-Mannich bases
X was H for the compounds D1, D2, and D3 and X was OH for
the compound D4
Z was N(CH3)2 for the compounds D1 and D4, N(CH2)5 for D2,
and N(CH2)4O for D3

May 2003
633
Table 2. Cytotoxic Activities of the Compounds Synthesized against Ju-
rkat Cells
(CD3OD) d: 3.22—3.40 (12H, m), 3.56 (4H, m), 3.86 (8H,
bs), 7.53 (6H, m), 8.03 (4H, m). 13C-NMR (CD3OD) d: 24.7
(t), 53.2 (t), 55.0 (t), 65.1 (t), 128.6 (d), 130.2 (d), 132.4 (d),
137.2 (s), 164.9 (s). ESI-MS m/z: 435.2 (MϩϩH).
Compounds
Molecular weight
LC50 (mmol/ml)
D4: lmax (H2O) nm (log emax): 280 (4.43). 1H-NMR
(D2O/CD3OD; 80/20) d: 2.96 (12H, s), 3.58 (8H, m), 6.99
(4H, d), 7.98 (4H, d, 3JHHϭ9.0 Hz). 13C-NMR (D2O/CD3OD;
80/20) d: 33.9 (t), 44.4 (q), 54.54 (t), 129.3 (s), 132.6 (d),
163.1, 167.0 (d), 199.7 (s). Large 3JHHϭ9.0 Hz and 13C-NMR
chemical shift 199.67 s indicated that the molecule is rather
possibly in quinone-form. ESI-MS m/z: 194.1 (The base
peak, C14H10O, MϩϪ2ϫ(CH2CH2NMe3)ϪOH), 295 (MϩϪ
CH2CH2Me3ϪMe).
Ig1
Ig2
Ig3
Ig4
D1
D2
D3
D4
5-FU
213.5
253.5
255.5
229.5
423
503
507
455
130
0.027
0.036
0.010
0.014
0.025
0.046
0.019
0.014
0.033
Cytotoxicity of Compounds against Jurkat Cells Ju-
rkat cells were maintained as a suspension culture in RPMI-
1640, supplemented with 10% fetal bovine serum (FBS) with
penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37 °C
under a humidified atmosphere of 95% air and 5% CO2. To
test the cytotoxicity of the compounds with Jurkat cells,
Phillips et al.’s method36) was used with slight modifica-
tions.9) A stock solution of Jurkat cells was prepared in cul-
ture medium, and the cells were counted with a Model DN
Coulter Counter. The test compound was dissolved in double
distilled water to give 10 mg/ml concentration. This stock so-
lution of the test compound was sterilized by filtration
(0.2 mM, Schleicher and Schuell, FP 030/3). Then, serial
three-fold dilution series was prepared (6—7 steps), and
20 ml from each concentration was added to test wells con-
taining suspended Jurkat cells (105 cells/ml culture medium).
Only the vehicle, double-distilled filter sterilized water, was
added to the control wells. The cells were incubated at 37 °C
under a humidified atmosphere of 95% air and 5% CO2 for
48 h, then counted. 5-Fluorouracil was tested as a reference
compound. All tests and controls were conducted in dupli-
cate with 24-well plates in two independent experiments.
The percentage of cell survival was calculated using the
formula: % survivalϭ(T48ϪT0)/(C48ϪC0)ϫ100, where T48
was the mean number of living cells/ml for each drug con-
centration at 48 h, T0 was the mean number for test wells at
time zero (normally 105), C48 was the mean number for the
control at 48 h, and C0 was the mean number for the control
at time zero (normally, T0ϭC0ϭ105 cells/ml). Lethal concen-
tration 50 (LC50) value was calculated by linear regression
analysis in the linear part of the concentration and % of sur-
vival plot.
scribed.37) Briefly, cell supernatant was mixed with 5.5 N per-
chloric acid (100 ml/10 ml), and the mixture was centrifuged
at 10000 g for 2 min at 4 °C to remove the precipitated pro-
tein. It was neutralized by 0.5 N KOH and centrifuged again
at 3000 g for 3 min at 4 °C to remove the salt precipitated.
TGSH was measured from the supernatant. The rate of for-
mation of thiolate anion arising from continuous reduction of
5,5Ј-dithiobis (2-nitrobenzoic acid) in the reaction medium
also containing NADPH, glutathione reductase, EDTA, and
cell supernatant in 0.1 M phosphate buffer (pH 7.5), was mea-
sured at 412 nm at room temperature. The sample was di-
luted by distilled water to adjust the rate to within the range
of reference. For each set of determinations, the reference
rate was established by using freshly prepared GSH standard
(30 mM).
The protein amount of the cell supernatant was determined
by Bio-Rad protein assay (Bio-Rad Lab., Richmond, CA,
U.S.A.) using bovine serum albumin as a standard according
to the method described by Bradford.38) A Shimadzu UV-240
double-beam spectrophotometer (Kyoto, Japan) was used for
protein and total glutathione measurements.
RESULTS
Compounds D2—D4 have been reported for the first time
by this study. Cytotoxic activities of the compounds are
shown in Table 2. Of them, the cytotoxic activities of Ig1—3
were also reported previously.9)
Except for Ig2 and D2, the cytotoxicity of mono-Mannich
bases, Ig1, Ig3 and Ig4 and correponding azine derivatives,
D1, D3 and D4 were higher than the reference compound 5-
FU. Conversion of mono-Mannich bases Ig1 and Ig4 to their
corresponding azine derivatives D1 and D4 did not affect the
cytotoxicity. However, conversion of mono-Mannich bases
Ig2 and Ig3 to their corresponding azine derivatives D2 and
D3 decreased the cytotoxicity 1.28 and 1.90 times, respec-
tively.
Exposure Protocol Jurkat cells (106 cells/ml) were ex-
posed to the compounds in phosphate buffered saline (PBS)
for 1 h in a 37 °C room with slight shaking, at concentrations
covering their LC50 values. Viability of the cells was over
90% during these experiments, as determined by the trypan
blue dye exclusion test.
Determination of Total Glutathione (TGSH) Content of
the Cells After exposure, the cells were washed with ice-
cold PBS twice by centrifuging at 200 g for 5 min at 4 °C.
Then, the cells were lysed by resuspending them in PBS with
0.05% Triton X-100 (v/v) and ultrasonicated in short bursts
on ice for 90 s. After that, the suspension was centrifuged at
10000 g at 4 °C, and the supernatant was collected in aliquots
and stored at Ϫ80 °C for TGSH and protein measurements.
TGSH and protein amounts were measured within a week.
The TGSH level of the cell supernatant was measured as de-
The effect of the representative compound D3, on the most
abundant cellular thiol, glutathione, has been shown in Fig.
1.
DISCUSSION
Mono-Mannich bases (Ig1—4) undergo deamination to
give a,b-unsaturated ketone in vivo, or in simulated physio-
logical conditions in vitro. Thus, it is possible to generate an
active center for nucleophilic attack for thiol alkylation (Fig.

634
Vol. 26, No. 5
2). Cytotoxicity increases by an increase in the deamination (Fig. 3). 2. Azine derivatives generate mono-Mannich bases
ratio.22) The higher the deamination ratio, the higher the cyto- by undergoing hydrolysis (1 mole azine gives two moles of
toxicity of the compounds.22)
corresponding mono-Mannich bases). These mono-Mannich
Two pathways may be suggested for the cytotoxicity of the bases undergo an elimination-addition reaction, as mentioned
azines: 1. Azine compounds (D1—D4), which are bifunc- above, to give thiol adducts, 2 (Fig. 4). Both pathways men-
tional agents, undergo deamination to give compound 3, tioned suggest that azines will probably show higher cytotox-
which contains biologically active unsaturated groups. Then, icity than mono-Mannich bases.
intermediate 3 produces the thiol adduct, 4. This mechanism
This controversy observed in this study for the azine com-
could be responsible for the cytotoxic activity of series D pounds, especially for compounds D2 and D3, may be ex-
plained by another pathway, as shown in Fig. 5. It is possible
that a great amount of compound 8, which does not have a
suitable center for alkylation, was formed, while small
amounts of compounds 3 and 1, which have suitable centers
for alkylation, were also formed in this pathway. It is re-
ported that a small amount of compound 4 type adduct along
with a great amount of compound 8, were formed in a stabil-
ity study using compound D1 in phosphate buffer at pH 7.4,
at 37 °C with 2-mercaptoethanol.35)
While the theoretically calculated enthalpy difference
(DH, calculated using ChemOffice 2002 software, U.S.A.)
for the reaction to form a mono-Mannich base by splitting a
Fig. 1. The Effect of Compound D3 on the Cellular Glutathione Level of
hydrazine group from compound 5 is ϩ3.62 kcal/mol (Fig.
Jurkat Cells after 1 h Exposure in Phosphate Buffered Saline in a 37 °C
6), it is ϩ10.12 kcal/mol for the reaction to form compound
Room with Slight Shaking
6 by splitting an amine group from compound 5 (Fig. 7a).
Cellular glutathione level of control cells was 36.98Ϯ1.99 nmol/mg protein. The re-
However, the calculated enthalpy difference for the formation
sults are meanϮS.D. of the two experiments in duplicate.
Fig. 2. Deamination of Mono-Mannich Bases, Ig1—4, to Produce a,b-Unsaturated Ketones, Which Have Suitable Centers for Thiol Alkylation
Fig. 3. Deamination of Azines to Produce a,b-Unsaturated Ketones, Which Have Suitable Centers for Thiol Alkylation

May 2003
635
Fig. 5. Suggested Possible Pathway for the Cytotoxic Activity of Azine Compounds,
D1—D4
Fig. 4. Hydrolysis of Azine Compounds to Produce Mono-Mannich
Bases, and Deamination of Them to Form a,b-Unsaturated Ketones, Which
Have Suitable Centers for Thiol Alkylation
of compound 7 by cyclization of compound 6 is lower,
ϩ2.4 kcal/mole (Fig. 7b). This path has lower energy than the
path shown in Fig. 6, and shows that the reaction is less en-
dothermic. On the other hand, compound 8 is more stable
compared with its precursors, compounds 7 and 1, from the
point of thermodynamics. The enthalpy for the formation of
compound 8 is Ϫ14.19 kcal/mol, and it is more stable (Fig.
7c). Theoretically calculated thermodynamic enthalpy results
seem to support the formation of compound 8 rather than
compounds 1 and 3, where the cytotoxicity was either not
affected or decreased in azine derivatives compared to the
mono-Mannich bases they are derived from, contrary to the
expectation. Equal cytotoxic potency in mono-Mannich
bases Ig1 and Ig4 and corresponding azine derivatives sug-
gests that the same amount of a,b-unsaturated ketones, 1 or
3, are being formed, which are responsible for the cytotoxic-
ity. However, unchanged or decreased cytotoxicity in azine
derivatives, contrary to our expectation (increase in cytotoxi-
city), may be attributed to the production of compound 8
(Fig. 5) by more or less all azine derivatives.
Fig. 6. Theoretical Enthalpy Change by Splitting of Hydrazine from Com-
pound 5
In this study, the representative azine compound D3 de-
creased the total cellular glutathione level dose-dependently,
when Jurkat cells were exposed to the compound in phos-
phate buffer for 1 h at 37 °C (Fig. 1). We have previously
shown that the mono-Mannich base of D3 also decreases the
cellular glutathione level.25) These findings support the thiol
alkylation mechanism for the cytotoxicity of the compounds
studied.
The cytotoxic activity of the new azine compounds D2—

636
Vol. 26, No. 5
Fig. 7. Enthalpy Changes for Some Compounds: a (for the Formation of Compound 6 from Compound 5), b (for the Formation of Compound 7 from
Compound 5), c (for the Formation of Compound 8 from Compounds 1 and 7)
Arzneimittelforschung, 51, 72—75 (2001).
5) Manavathu E. K., Vashishtha S. C., Alangaden G. J., Dimmock J. R.,
Can. J. Microbiol., 44, 74—79 (1998).
6) Gul H. I., Gul M., Erciyas E., Arzneimittelforschung, 52, 628—635
D4 is reported for the first time in this study. The cytotoxicity
of azines may result from the formation of great amount of
compound 8, along with the formation of some intermediate
compounds 1 or 3, with a center suitable for alkylation. In
conclusion, azine derivatives with equal or less cytotoxic po-
tency compared to the mono-Mannich bases they are derived
from seemed to be less suitable derivatives for the develop-
ment of new cytotoxic compounds.
(2002).
7) Gul H. I., Gul M., Erciyas E., J. Appl. Toxicol., 23, 53—57 (2003).
8) Gul H. I., Gul M., Hänninen O., Arzneimittelforschung, 52, 840—843
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Acknowledgement The authors thank S. Peräniemi,
Ph.D., University of Joensuu, Joensuu, Finland, for elemental
analyses of the compounds synthesised, and Seppo Auriola,
Ph.D., University of Kuopio, Kuopio, Finland, for mass spec-
tra of the azines. The authors are also thankful to Prof. Hasan
Secen from Ataturk University, Erzurum, Turkey, for his
comments on the theoretical thermodynamic evaluation of
the reactions.
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