Antioxidant activity of newly synthesized 2,7-diazaphenothiazines

Article abstract of DOI:10.1002/ardp.200900253

A series of 19 derivatives of 2,7-diazaphenothiazine was synthesized and evaluated for their antioxidant activity bearing in mind the structural similarity with "classical" phenothiazines several of which are considered powerful antioxidants. Among the ne

Full text of DOI:10.1002/ardp.200900253

268
Arch. Pharm. Chem. Life Sci. 2010, 343, 268–273
Full Paper
Antioxidant Activity of Newly Synthesized 2,7-
Diazaphenothiazines
Beata Morak-Młodawska1, Krystian Pluta1, Alexios N. Matralis2, and Angeliki P. Kourounakis2
1 Department of Organic Chemistry, The Medical University of Silesia, Sosnowiec, Poland
2 Department of Pharmaceutical Chemistry, School of Pharmacy, University of Athens, Athens, Greece
A series of 19 derivatives of 2,7-diazaphenothiazine was synthesized and evaluated for their anti-
oxidant activity bearing in mind the structural similarity with “classical” phenothiazines several
of which are considered powerful antioxidants. Among the new derivatives that inhibited in
vitro Fe2+/ascorbate-induced lipid peroxidation of rat liver microsomal membranes, several exhib-
ited significant antioxidant activity with IC50 values in the range of 64–125 lM. Although N-sub-
stitution led to a variable degree of antioxidant activity, the latter appears to correlate with the
lipophilicity (expressed as clogP values) of the substituted derivatives. Reduced lipophilicity may
also explain the relatively lower protection offered by these derivatives against lipid peroxida-
tion when compared to their “classical” phenothiazine counterparts. Thus, modification of the
phenothiazine structure by a substitution of two benzene rings with pyridine rings to form this
new type of azaphenothiazines does not enhance antioxidant activity, although it retains it.
Keywords: Lipid peroxidation / Lipophilicity / Microsomal membranes / Phenothiazine derivatives /
Received: October 22, 2009; Accepted: December 11, 2009
DOI 10.1002/ardp.200900253
type of azaphenothiazines, 2,7-diazaphenothiazines [11,
Introduction
12]. The parent compound, 10H-2,7-diazaphenothiazine
1, exhibited promising anticancer activity against
human tumor cell lines deriving from the colon, breast,
kidneys, ovaries, prostate, and CNS, and against mela-
noma and leukemia cell lines [13]. This compound was
found to strongly suppress the humoral immune
response even at low concentrations and inhibited the
Phenothiazines are an important class of heterocyclic
compounds with a variety of biological activities such as
antipsychotic, antihistaminic, antitussive, anti-emetic,
as well as interesting chemical properties [1]. Recent
reports have focused on various activities of classical and
newly synthesized phenothiazines such as anticancer,
antiplasmid, and antibacterial activities, reversal of mul-
tidrug resistance (MDR), as well as potential treatment of
Alzheimer’s, Creutzfeldt–Jakob, and AIDS [2–10]. The
most significant modifications of the phenothiazine
structure that have been performed are the introduction
of new pharmacophoric substituents at the thiazine
nitrogen atom and the substitution of the benzene ring
with an azine ring to form azaphenothiazines. Recently,
we modified the phenothiazine structure by substitution
of two benzene rings with pyridine rings to form a new
delayed-type
hypersensitivity,
lipopolysaccharide-
induced production of tumor necrosis factor, and inter-
leukin-6 in cultures of human blood cells [14].
Although the phenothiazine structure, mainly repre-
sented by chlorpromazine, is considered to possess anti-
oxidant properties [15–19], only few reports have studied
the antioxidant activity of other phenothiazine deriv-
atives [20, 21]. Due to structural similarities as well as tak-
ing into consideration the involvement of reactive oxy-
gen species (ROS) in pathological conditions such as
inflammation, degenerative diseases, and aging, we con-
sidered it of interest to further investigate our newly syn-
thesized 2,7-diazaphenothiazine derivatives for antioxi-
dant activity. Thus, in this study, we evaluated the pro-
tection offered by 10H-2,7-diazaphenothiazine 1 and
Correspondence: As. Prof. A. Kourounakis, Department of Pharmaceut-
ical Chemistry, School of Pharmacy, University of Athens, 15771 Athens,
Greece.
E-mail: angeliki@pharm.uoa.gr
Fax: +30 210 727-4747
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Arch. Pharm. Chem. Life Sci. 2010, 343, 268–273
Antioxidant Activity of 2,7-Diazaphenothiazines
269
tograms induced phosphorescence, spraying with a phe-
nothiazine detection reagent (sulfuric acid/water/etha-
nol, 1:1:8) [25] induced an oxidation process to give differ-
ent colored spots (lilac 5, orange 7, and intense yellow
12–14).
Antioxidant activity
The effect of the investigated derivatives on the non enzy-
matic peroxidation of hepatic microsomal membrane
lipids after 45 min of incubation, expressed as IC50 values
(or, for some derivatives, percent inhibition at a com-
pound concentration of 0.5 mM), is shown in Table 1.
Under the same experimental conditions, trolox ((S)-(–)-6-
hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid)
and probucol (4,49-[(1-methylethylidene) bis(thio)]bis[2,6-
bis(1,1-dimethylethyl)phenol]), known potent antioxi-
dants, exhibited IC50 values of 25 lM and >1 mM, respec-
tively [26]. Most of the studied derivatives demonstrated
antioxidant activity, although not higher than that of
Scheme 1. Synthesis of compounds 5, 7, and 12–14.
eighteen derivatives with varying 10-substitutions (with
alkyl, aryl, heteroaryl, cycloaminoalkyl, acylaminoalkyl,
and sulfonylaminoalkyl groups), against in-vitro lipid per-
oxidation of rat liver microsomal membranes.
Results and discussion
Chemistry
The structures of the synthesized compounds, as well as the unsubstituted compound 1. The time course of lipid
their lipophilicity, calculated as cLogP values by the peroxidation, as affected by several concentrations of the
method of Leo–Hansch [22], are shown in Table 1. For most active compounds 1, 5, and 12 is depicted in Fig. 1.
some derivatives, experimental logP values (estimated by
reverse phase thin layer chromatography) were available the phenothiazine structure by a substitution of two ben-
[23, 24] that correlated well with the calculated values. zene rings with pyridine rings to form this new type of
It should first be mentioned that the modification of
The parent compound 1 was prepared from 4-chloro-3- azaphenothiazines (e.g. compound 1) does not seem to
nitropyridine and 3-amino-4(1H)-pyridinethione in DMF favor an increase in antioxidant capacity when compared
according to the described procedure [11] and was trans- to the “classical” unsubstituted phenothiazine ring sys-
formed into derivatives 2–19. Reaction of compound 1 tem (tested and found to have an IC50 value of 0.35 lM
with 1-fluoro-2,4-dinitrobenzene and 2-chloro-pyrimi- under the same experimental conditions). This may be
dine in DMF in the presence of sodium hydride at room due to the different electronic properties of the aromatic
temperature led to 10-substituted derivatives 5 and 7. rings involved, e.g. the electron-withdrawing effects of
Reaction of compound 1 with cycloaminoethyl chlorides the two N atoms of the diazaphenothiazine rings that
(N-(2-chloroethyl)pyrrolidine hydrochloride, N-(2-chloro- may influence (decrease) the stabilization (via resonance)
ethyl)piperidine hydrochloride, and N-(2-chloroethyl)- of potential free radical intermediates of the aromatic
morpholine hydrochloride) in boiling dioxane with thiazine. Another difference is the relatively increased
sodium hydroxide gave 10-cycloaminoethyl derivatives lipophilicity of the unsubstituted phenothiazine struc-
12–14 (Scheme 1). Derivatives 2–4, 6, 8–11, and 15–19 ture (clogP = 3.23) compared to that of compound 1 (clogP
were obtained according to the procedures described pre- = 2.12). It is considered that the lipophilic character of
viously [11, 12].
antioxidants contributes to the offered inhibition
against peroxidation of microsomal lipids, since lipo-
philic compounds may acquire an easy access to biologi-
cal membranes and a more efficient interaction.
Chromatographic properties of 2,7-
diazaphenothiazines
The described reactions were monitored by TLC analysis.
A similar comparison may be made between com-
All chromatograms of 2,7-diazaphenothiazines showed pound 9 and the more active chlorpromazine (reported
color changing during irradiation with UV light. We IC50 values of ca. 10–75 lM [16, 20, 27]) which differ only
reported previously a color change from white to beige in the (diaza/Cl) phenothiazine system. Apart from the
and from yellowish to celadon or to intense yellow for higher lipophilic character of chlorpromazine (clogP =
compounds 1–4, 6, 8–11, and 15–19 [11, 12]. The synthe- 5.30) compared to that of compound 9 (clogP = 2.39, IC50 >
sized new compounds showed color changes from violet 500 lM), this difference in activity may also be due to the
to grey 5, orange to canary 7, and white to beige 12–14. above mentioned altered electronic/resonance effects of
Whereas UV irradiation of the azaphenothiazine chroma- the modified (i.e. diaza) phenothiazine ring structure.
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B. Morak-Mlodawska et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 268–273
Table 1. Structures, activity, and theoretical lipophilicity values (clogP) of the 10H-2,7-diazaphenothiazine derivatives.
Compound
R
Antioxidant activity
clogP/logPTLC [23, 24]
IC50 (lM)
-H
64
10%§
2.12
2.12
4.39
1.54
1
2
3
-CH3
1.69
2.57
-CH2C6H5
400
394
107
3.57
3.33
2.45
4
5
13%§
32%§
1.43
1.42
1.34
6
7
-CH2COC6H5
-(CH2)3N(CH3)2
25%§
21%§
12%§
27%§
3.08
2.39
2.79
3.14
8
1.91
1.97
1.83
9
-CH2CH(CH3)CH2N(CH3)2
-(CH2)2N(CH2CH3)2
10
11
125
325
2.49
3.05
12
13
n.a.
1.89
14
-(CH2)3NHCOCH3
-(CH2)3NHCOC6H5
6%§
293
470
8%§
n.a.
1.34
3.15
3.35
3.70
2.53
1.68
15
16
17
18
19
-(CH2)4NHCOC6H5
-(CH2)3NHSO2-C6H4CH3
-(CH2)4NHCONHCH2CH2Cl
§%-inhibition at 0.5 mM; n.a. = not active at 0.5 mM.
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Arch. Pharm. Chem. Life Sci. 2010, 343, 268–273
Antioxidant Activity of 2,7-Diazaphenothiazines
271
and extrapolated IC50 values was found, indicating a
trend, i.e. that in general the more lipophilic compounds
interacted better with the membrane substrate and
offered better protection against lipid peroxidation.
In conclusion, it is known that chlorpromazine and
some other phenothiazines act as powerful antioxidants,
inhibiting lipid peroxidation of liposomes or microso-
mal membranes [22, 25]. Several of the newly synthesized
diazaphenothiazine derivatives investigated hereby,
exhibited significant antioxidant activity, although not
higher than their “classical” phenothiazine counter-
parts, a fact that may be attributed to altered electronic/
resonance effects of the modified (i.e. diaza) phenothia-
zine ring structure as well as decreased lipophilicity.
Experimental
Chemistry
Melting points were determined in open capillary tubes on a
Boetius melting point apparatus (Franz Kꢀstner. Nachf. KG, Ger-
1
many) and are uncorrected. The H-NMR spectra were recorded
on a Varian Unity-Inova-300 (Varian, Palo Alto CA, USA) and a
Bruker DRX spectrometers (Bruker Bioscience, USA) at 300 and
500 MHz in deuteriochloroform with tetramethylsilane as the
internal standard. Electron Impact mass spectra (EI MS) and Fast
Atom Bombardment mass spectra (FAB MS, in glycerol) were run
on a Finnigan MAT 95 spectrometer at 70 eV (Thermo Electron
Corporation, Bremen, Germany). The thin layer chromatogra-
phy were performed on silica gel 60 F254 (Merck 1.05735) with
CHCl3/EtOH (5:1 and 10:1, v/v) and on aluminum oxide 60 F254
neutral (type E) (Merck 1.05581) with CHCl3/EtOH (10:1, v/v) as
eluents (Merck, Germany).
Synthesis of 10H-2,7-diazaphenothiazine 1 and the
10-substituted derivatives 2 to 4, 6, 8 to 11, and 15 to 19
10H-2,7-Diazaphenothiazine 1 was obtained (in 85% yield) in the
reaction of 4-chloro-3-nitropyridine with 3-amino-4(1H)-pyri-
dinethione in dry DMF in the presence of NaOH and was trans-
formed into derivatives 2–4, 6, 8–11 and 15–19 according to the
described procedures [11, 12].
Synthesis of 10-substituted 2,7-diazaphenothiazines
5 and 7
To a solution of 10H-2,7-diazaphenothiazine 1 (0.100 g, 0.5
mmol) in dry DMF (5 mL) NaH (0.024 g, 1 mmol, 60% NaH in min-
eral oil was washed out with hexane) was added. The reaction
mixture was stirred at room temperature for 1 h and then aryl
and heteroaryl halides (1-fluoro-2,4-dinitrobenzene, 2-chloro-
pyrimidine, 1.5 mmol) were added and the stirring was contin-
ued for 24 h. The mixture was poured into water (15 mL). After
cooling, the resulting solid was filtered off and dried. The
obtained product was purified by column chromatography (alu-
minum oxide, CHCl3/EtOH, 10:1) to give:
Figure 1. Representative graph of the time course of lipid perox-
idation as affected by various concentrations of compound 1, 5,
and 12.
The performed structural modifications, i.e. N10-substi-
tution of the diazaphenothiazines (compounds 2–19) led
to a variable degree of antioxidant profile. No specific
structure-activity relationships could be derived related
to steric or electronic effects of substitution on the N10
atom. However, it seems that antioxidant activity was
somewhat related with increased lipophilicity (expressed
as clogP values) of the substituted derivatives. A modest
correlation (r = 0.7, P = 0.016, n = 17) between cLogP values
10-(29,49-Dinitrophenyl)-2,7-diazaphenothiazine 5
Yield: 0.155 g (85%), m.p.: 169–1708C; 1H-NMR (CDCl3) d: 5.78 (d,
1H, H-9), 6.99 (d, 1H, H-4), 7.89 (d, J = 8.6 Hz, 1Hphenyl), 8.08 (m, 4H,
H-1, H-3, H-6, H-8), 8.79 (d, J = 8.6 Hz, 1Hphenyl), 9.13 (s, 1Hphenyl); EI
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Arch. Pharm. Chem. Life Sci. 2010, 343, 268–273
MS m/z: 367 [M] (100), 321 [M – NO2] (10), 275 [M – 2 NO2] (38).
Anal. calcd. for C16H9N5O4S: C, 52.31; H, 2.47; N, 19.07. Found: C,
52.09; H, 2.45; N, 19.01.
residues), ascorbic acid (0.2 mM) in Tris-HCl/KCl buffer (50 mM/
150 mM, pH = 7.4), and the studied compounds (10 lM to 1 mM)
dissolved in dimethyl sulfoxide. The reaction was initiated by
addition of a freshly prepared FeSO4 solution (10 lM), and the
mixture was incubated at 378C for 45 min. Aliquots were taken
at various time intervals and lipid peroxidation was assessed by
spectrophotometric determination (535 against 600 nm) of the
2-thiobarbituric acid reactive material [26]. All compounds and
solvents were tested and found not to interfere with the assay.
Each assay was performed in duplicate and IC50 values represent
the mean concentration of compounds that inhibit the peroxi-
dation of control microsomes by 50% after 45 min of incubation.
All standard errors are within 10% of the respective reported
values.
10-(29-Pyrimidinyl)-2,7-diazaphenothiazine 7
Yield: 0.114 g (82%), m.p.: 214–2158C; 1H-NMR (CDCl3) d: 5.98 (d, J
= 6.0 Hz, 1H, H-9), 6.56 (d, J = 6.0 Hz, 1H, H-4 ), 7.14 (t, J = 4.5 Hz,
1H, H-59), 7.85 (s, 1H, H-1), 7.91 (d, J = 6.0 Hz, 1H, H-3), 7.98 (s, 1H,
H-6), 8.20 (d, J = 6.0 Hz, 1H, H-8), 8.63 (d, J = 4.5 Hz, 2H, H-49, H-69);
EI MS m/z: 279 [M] (100), 200 [M – pyrimidinyl] (22). Anal. calcd.
for C14H9N5S: C, 60.20; H, 3.25; N, 25.07. Found: C, 60.02; H, 3.21;
N, 24.87.
General procedure for the synthesis of 10-substituted
2,7-diazaphenothiazines 12–14
Statistical analysis and calculated lipophilicity
Statistical and correlation analysis was performed with Excel
while lipophilicity was calculated as cLogP values by the method
of Leo–Hansch [22].
To a solution of 10H-2,7-diazaphenothiazine (0.100 g, 0.5 mmol)
in dry dioxane (5 mL) NaOH (0.20 g, 5 mmol) was added. The mix-
ture was refluxed 1 h and hydrochlorides of cycloaminoethyl
chloride (N-(2-chloroethyl)pyrrolidine hydrochloride, N-(2-
chloroethyl)piperidine hydrochloride, and N-(2-chloroethyl)-
morpholine hydrochloride, 1.5 mmol) were added. The reaction
mixture was refluxed for 12 h. After cooling, dioxane was evapo-
rated in vacuo and the residue was dissolved in CHCl3 (10 mL).
The extracts were washed with water, dried with anhydrous
sodium sulfate, and evaporated in vacuo. The obtained product
was purified by column chromatography (aluminum oxide,
CHCl3/EtOH, 10:1) to give:
The authors have declared no conflict of interest.
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