Microwave-assisted catalytic amination of phenothiazine; Reliable access to phenothiazine analogues of Troeger's base

Article abstract of DOI:10.1002/ejoc.201300480

Efficient protocols for microwave-assisted catalyzed amination of halogeno-phenothiazines are described. Phenothiazine analogues of Troeger's base (PTB) were obtained by condensation of amino-phenothiazines with formaldehyde in HCl/EtOH/H₂O and

Full text of DOI:10.1002/ejoc.201300480

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FULL PAPER
Heterocycles
Microwave-assisted catalyzed amination of
halogeno-phenothiazines provides precur-
sors for phenothiazine analogues of
Tröger’s base. DFT and HF computational
analysis, electronic properties (fluorescence
emission in solution and solid state), and
binding to proteins and DNA with effects
on prooxidant reactivity were investigated.
L. I. G a˘ in a˘ , L. N. M a˘ t a˘ râng a˘ -Popa,
E. Gal, P. Boar, P. Lönnecke,
E. Hey-Hawkins, C. Bischin,
R. Silaghi-Dumitrescu, I. Lupan,
C. Cristea,*
L. Silaghi-Dumitrescu ..................... 1–10
Microwave-Assisted Catalytic Amination
of Phenothiazine; Reliable Access to
Phenothiazine Analogues of Tröger’s Base
Keywords: Amination / Microwave chemis-
try / Fluorescence / Cyclic voltammetry /
Molecular modeling
0000, 0–0
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1

Job/Unit: O30480
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C. Cristea et al.
FULL PAPER
DOI: 10.1002/ejoc.201300480
Microwave-Assisted Catalytic Amination of Phenothiazine; Reliable Access to
Phenothiazine Analogues of Tröger’s Base
[
a]
[a]
[a]
[a]
Luiza I. G a˘ in a˘ , Larisa N. M a˘ t a˘ râng a˘ -Popa, Emese Gal, Paul Boar,
[b]
[b]
[a]
[a]
Peter Lönnecke, Evamarie Hey-Hawkins, Cristina Bischin, Radu Silaghi-Dumitrescu,
[
c]
[a]
[a]
Iulia Lupan, Castelia Cristea,* and Lumini t¸ a Silaghi-Dumitrescu
Keywords: Amination / Microwave chemistry / Fluorescence / Cyclic voltammetry / Molecular modeling
Efficient protocols for microwave-assisted catalyzed amin- phenothiazines and PTB were assessed on the basis of cyclic
ation of halogeno-phenothiazines are described. Phenothi-
azine analogues of Tröger’s base (PTB) were obtained by
condensation of amino-phenothiazines with formaldehyde in
voltammetry and UV/Vis absorption/emission spectroscopy
data. The compounds exhibit blue fluorescence emission
characterized by extremely large Stokes shifts (8900–
10300 cm ) in both dilute solutions and aggregated states.
Interactions with different types of biomolecules show the ca-
pacity of binding to proteins and DNA, with effects on proox-
idant reactivity including lipid peroxidation.
–
1
HCl/EtOH/H
2
O and structurally characterized by NMR and
XRD analyses. The formation of PTB isomers was predicted
by computational analysis based on theoretical methods
(DFT and HF). Electronic properties of the parent amino-
Introduction
Amino-phenothiazines have been used as versatile key in-
termediates in the preparation of many synthetic targets
containing the phenothiazine core (functional group deriva-
tives, mixed azaheterocyclic structures) and, thus, a specific
interest directed towards new synthetic methodologies ap-
plicable to the selective preparation of regioisomers has
been maintained. Nitro-phenothiazine derivatives have
most often been used as starting materials, based on strate-
Since 1883, when the first synthesis of phenothiazine was
[
1]
described, derivatives containing this heterocyclic core
have constantly attracted scientific interest and, over the
[
2]
years, major applications have been developed in dyes and
[
3]
pharmaceuticals, and as additives for lubricants and poly-
[
4]
mers. More recently, old and new compounds containing
[5]
redox-active phenothiazine units were found to exhibit
unusual physical properties that were suitable for applica-
tions in materials science (electrically conducting charge-
[11]
[12]
gies using reducing agents such as Zn/ZnCl ,
Sn,
or
2
[13]
SnCl2,
drate,
and HCl, Ni and acetic acid or hydrazine hy-
as well as catalytic hydrogenations with catalysts
[14]
[6]
transfer composites, materials for photoinduced electron
[15]
[16]
such as Raney nickel, or palladium on carbon. Other
[
7]
[8]
transfer, redox-active fluorophores, or electrode materi-
strategies involved the hydrolysis of a pending N-acyl-sub-
[9]
als ). Detailed investigations of the electronic properties of
phenothiazine and oligophenothiazine derivatives per-
formed by means of cyclic voltammetry, and absorption
and fluorescence spectroscopy, illustrated the possibility of
fine-tuning the typical properties associated with this het-
erocyclic core; shifts of the oxidation potential, bathoch-
romic shifts of the absorption band, varying emission quan-
[17]
[18]
stituent (carbamate, phthalimide ) of an aromatic unit
of the phenothiazine core previously obtained by ring clo-
sure of the correspondingly protected amino-diphenylamine
[11]
[19]
with sulfur, or the rearrangement of an acylazide. Te-
dious syntheses were developed for chloro-phenothiazine
transformations into amino-phenothiazine derivatives by
[20]
using sodium in liquid ammonia
or amines in the pres-
[
10]
tum yields, and producing exceedingly large Stokes shifts.
[21]
ence of alkali. New strategies were developed to comple-
ment these traditional methods, which were suggested by
[
a] Faculty of Chemistry and Chemical Engineering, Babes-Bolyai the efficient preparation of a wide variety of arylamines by
University,
the amination of (hetero)aryl halides based on either Pd-
1
1, Arany Janos street, 400028 Cluj-Napoca, Romania
[
22]
[23]
catalyzed,
or copper-mediated
coupling reactions.
Fax: +40-264-590818
E-mail: castelia@chem.ubbcluj.ro
Homepage: www.chem.ubbcluj.ro
These catalyzed C–N bond formation reactions, which typi-
cally require long reaction times to reach completion under
traditional convective heating conditions (often in inert at-
mosphere), can be significantly accelerated by microwave
[
[
b] Institut für Anorganische Chemie, Leipzig University,
04103 Leipzig, Germany
c] Institute for Interdisciplinary Research Babes-Bolyai University
4
00271 Cluj-Napoca, Romania
[
24]
heating.
Microwave-assisted aminations of bromoaryl
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201300480.
derivatives with nitrogen-containing heterocycles in the
2
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Access to Phenothiazine Analogues of Tröger’s Base
presence of CuI, or copper,[26] as well as palladium-medi- ation of aryl halides[22] was applied, using halogeno-10H-
[
25]
[
27]
ated aminations were successfully performed.
phenothiazines 1a–f as substrates and LiN(SiMe ) as am-
3 2
A range of heterocyclic analogues of Tröger’s base (TB) monia equivalent, in the presence of a palladium complex
[
28]
[29]
[30]
have been reported. Phenanthroline, porphyrin, and catalyst [Scheme 1, path (i)]. This synthetic protocol gave
[
31]
acridine analogues of TB were designed as chiral solvat- unsatisfactory yields of amino-10H-phenothiazines 2a and
ing agents, hydrogen-bond receptors, ligands in metal com- 2b (explicable by the steric hindrance induced by the N-
plexes, and DNA intercalators. The general procedure for alkyl substituent in 1a, or the competitive reactivity of the
the preparation of TB derivatives involves acid-induced heterocyclic NH group in 1b, respectively), but afforded
condensation of an aromatic amine with formaldehyde or various yields of amines 2c–f when starting with regioiso-
[
32]
formaldehyde equivalents such as paraformaldehyde,
mers 1c–e; the experimental results are summarized in
hexamethylene-tetramine[
33]
or dimethoxymethane.
[34]
A
Table 1. The regioselective substitution reaction indicates
range of yields of heterocyclic analogues of TB were ob- that, at the reaction temperature (140 °C), the elimination–
tained, depending on the selected reaction conditions and addition mechanism does not appear to be competitive with
the reactivity of the heterocyclic amine.[
35]
the catalytic cycle.
As part of our efforts directed to the synthesis and inves-
tigation of the chemical, physical and biological properties Table 1. Experimental conditions for the microwave-assisted amin-
ation of halogeno-phenothiazines; (i) Pd-catalyzed, (ii) copper(I)-
mediated.
of new organic compounds containing the phenothiazine
core, we here describe a protocol for the convenient micro-
wave-assisted preparation of primary phenothiazinyl-
amines and their further use as substrates in the synthesis
of phenothiazine analogues of Tröger’s base (PTB). The
structural features of the new PTB combine the chiral V
2
Temp. [°C]
Time [h]
Yield [%]
i)[a]
(ii)[b]
(i)[a]
(ii)[b]
(i)[a]
(ii)[b]
(
2
2
a
b
140
140
140
140
140
140
110
110
110
110
110
110
1
1
1
1
1
1
2
–
33
–
23
93
26
62
10
10
2
2
2
87
90
28
–
shaped TB scaffold induced by the pyramidal geometry of 2c
2
2
2
d
e
f
the two nitrogen atoms in the methanobenzo-diazocine ri-
gid core, with two folded heterocyclic 1,4-benzothiazine
units (which contribute to the molecular complexity with
additional stereogenic nitrogen centers and also impart the
specific electronic properties of the phenothiazine core).
The structural assessments based on computational, analyt-
ical, and electrochemical studies presented below, qualify
the described phenothiazine derivatives as potential candi-
dates for applications in materials science and molecular
biology.
43
[
[
a] Reaction conditions: LiN(SiMe
Pd (dba) ] (0.5 mol-%), DCPB (1.2 mol-%). [b] Reaction condi-
(10 mmol), Cu O (5 mol-%), H O/NMP (1:1).
3 2
) (1 m in THF; 1.2 equiv.),
2
3
tions: NH
3
2
2
The copper-catalyzed coupling of aryl halides with aque-
[36]
ous ammonia
wave-assisted amination of halogeno-10H-phenothiazines
a–f, based on a protocol using Cu O as catalyst in the
was alternatively applied for the micro-
1
2
presence of N-methyl-2-pyrrolidinone [NMP; Scheme 1,
path (ii)]. Very good yields of 3-amino-10-methyl-10H-
phenothiazine (2d) were isolated after 2 h irradiation at
Results and Discussion
1
10 °C in a sealed vessel, as well as moderate yields of sym-
Amination of Halogeno-phenothiazine
metrical 3,7-diamino-10-methyl-10H-phenothiazine (2f;
Two alternative microwave-assisted catalytic amination Table 1). A very long reaction time (10 h) was required for
routes were applied for the regioselective preparation of pri- the substitution of 2-chloro-10-methyl-10H-phenothiazine
mary 10H-phenothiazinyl-amines starting from the corre- 1c and only small amounts of 2c were obtained.
sponding 1-, 2-, 3- and 4-halogeno-10H-phenothiazine
Palladium-catalyzed amination appears to be a conve-
(Scheme 1). A modified protocol for Pd-catalyzed amin- nient route for the preparation of 2-amino-10-alkyl-pheno-
Scheme 1. Catalytic amination of halogeno-phenothiazines. Reagents and conditions: (i) LiN(SiMe
acetone)dipalladium [Pd (dba) ] (0.5 mol-%), 2-(dicyclohexylphosphanyl)biphenyl (DCPB; 1.2 mol-%); (ii) 35% aqueous ammonia, Cu
5 mol-%), NMP (5 mol-%).
3 2
) (1 m in THF), tris(dibenzylidene-
2
3
2
O
(
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thiazine derivatives, whereas the Cu-catalyzed pathway is and N-ethyl- (3a) substituted substrates (above 90% yields),
a less expensive alternative method that is suitable for the whereas bulkier substituents such as n-hexyl or n-octyl in
preparation of 3-amino-10-alkyl-phenothiazine derivatives.
3b and 3c, respectively, induced increasing steric hindrance
The regioselectivity of the amination reaction appears to and consequently slightly lower yields. Similar reactivity
be well-correlated with the electron-density distribution in was observed in the bis-amination of symmetrical 1,6-bis(2-
the substituted benzene ring of the phenothiazine core, as chloro-phenothiazin-10-yl)hexane (3d), which afforded
suggested by the electrostatic, natural, or Mulliken charges good yields of the new 1,6-bis(2-amino-phenothiazin-10-
[
37]
calculated using SPARTAN ‘06 Semiempirical program
yl)hexane (4d). The structures of amines 2 and 4 were con-
1
13
and presented in Table 2. In substrate 1c, lower charge den- firmed by spectroscopic methods (FTIR, H and C NMR
sity at C2 indicates an electron-poor aryl chloride (typical spectroscopic and MS analyses).
substrates for Buchwald–Hartwig reactions), whereas in
substrate 1d, a higher charge density at C3 suggests an elec-
tron-rich aryl bromide (with preference for Ullmann–Gold- Phenothiazine Analogues of Tröger’s Base
berg reaction). Unfortunately, both synthetic methods pro-
The formation of the methano[1,5]diazocine unit during
vided only small amounts of 1- and 4-amino-substituted
the condensation of 2-amino-10-alkyl-phenothiazine with
regioisomers (Table 1), which may be due to steric hin-
formaldehyde under acidic conditions is based on electro-
drance and coordination effects induced by the two hetero-
philic aromatic substitution involving either C1 or C3 (atom
atoms in the phenothiazine core. No secondary N,N-bis-
labeling in Scheme 1) and thus, three possible regioisomers
(phenothiazinyl)amines were formed during either catalysis
of the phenothiazine analogues of Tröger’s base (PTB) can
be formed: one asymmetric (1,3Ј) and two symmetric (1,1Ј
and 3,3Ј) (Figure 1).
conditions.
Table 2. Charge-density distribution in the substituted benzene unit
of the lowest energy conformer of halogeno-phenothiazines 1c and
1
d and amino-phenothiazines 2c and 2d obtained after molecular
mechanics optimization of the conformers generated within the
Confanal module of SpartanЈ06, followed by semiempirical (PM3)
and 6-31G(d) B3LYP DFT calculations.
Charge[a] Position
1c
–0.29438 –0.24010 –0.43922 –0.18504
0.16595 –0.08281 0.44925 –0.33753
–0.24929 –0.05143 –0.38109 0.39218
–0.08339 –0.07407 –0.09123 –0.32270
–0.28744 –0.25868 –0.31747 –0.25019
1d
2c
2d
E
C1
C2
C3
C4
C1
C2
C3
C4
C1
C2
C3
C4
N
M
–0.03163 –0.23732
–0.26250 –0.12553 –0.29652
0.18107 –0.27337
0.16102
–0.21712 –0.24259 –0.21116 –0.27518
–0.17148 –0.16555 –0.22874 –0.17684
–0.08225 –0.16314
–0.12032
0.30870 –0.19030
0.32558
0.02192 –0.16680
Figure 1. PTB regioisomers.
–0.17747 –0.19694 –0.18249 –0.22616
Condensation of several 2-amino-10-alkyl-10H-pheno-
thiazines with formaldehyde and hydrochloric acid in eth-
[a] E: electrostatic, N: natural, M: Mulliken.
The successful microwave-assisted palladium-catalyzed anol afforded PTB 5a–d (Scheme 3) in moderate yields (45–
amination protocol was further applied to the preparation 55%). Experimental results show a preference for substitu-
of several new 2-amino-10-alkyl-10H-phenothiazines 4 tion at C3 (3,3Ј-PTB). In the case of amine 2c, a compari-
(Scheme 2), containing variable alkyl chain lengths. Com- son of the computed charge densities at C1 and C3 (shown
parable results were obtained in the case of N-methyl- (1c) in Table 2) indicates higher values at C1, supporting the as-
Scheme 2. Palladium-catalyzed amination of 2-chloro-10-alkyl-10H-phenothiazines.
4
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Access to Phenothiazine Analogues of Tröger’s Base
Scheme 3. PTB formation by condensation of 2-amino-10-alkyl-phenothiazine with formaldehyde.
sumption of steric control of the electrophilic attack at the NOEDIFF spectra of 5b show spatial interactions between
less sterically crowded C3. 2-Amino-10H-phenothiazine 2b, aromatic protons in the 4-position of the phenothiazine (δ
which does not contain a protective N-alkyl group, gave a = 6.64 ppm, s) and the endo CH protons of the diazocine
2
complex mixture containing only small amounts of PTB. unit, thus providing evidence for the regioselectivity of the
Other methods involving formaldehyde equivalents such as cyclization.
paraformaldehyde or hexamethylenetetramine in strong ac-
The stereoisomers of PTB 5a, induced by the chirality of
the methano[1,5]diazocine unit and the pyramidal geometry
ids[ were not successful for the preparation of PTB.
38]
Long alkyl chains attached to the heterocyclic nitrogen of the nitrogen atoms in the folded phenothiazine units,
atom dramatically lowered the melting points of the PTB were analyzed by using computational tools. DFT optimi-
[
cf. m.p. 312 (5a) and 68 (5d) °C].
zations (M062x/6-31G**) reveal that three pairs of dia-
The core structures of PTB 5a–d were identical, as estab- stereoisomers are feasible (Figure 2). The conformational
1
13
lished by H, C, 1D NOEDIFF, HMQC, HMBC, and analysis of the phenothiazine unit (folded along an axis
1
COSY NMR experiments. For example, in the H NMR passing through the heteroatoms N and S) indicates a pref-
spectrum of 5b, the protons in the diazocine unit appear as erence for a quasi-equatorial orientation of the methyl sub-
a singlet (δ = 4.3 ppm for the methylene bridge) and a well- stituent, but minima with one or both methyl groups in
separated AB spin system for the endo (δ = 4.1 ppm, d) and quasi-axial orientation were also identified (Figure 3),
exo (δ = 4.6 ppm, d) methylene protons in the equivalent which are higher in energy by 5–12 kcal/mol, depending on
positions 4 and 8 (atom labeling in Scheme 3). The equiva- the method employed. On the methodological side, we note
lence of the two alkyl-phenothiazine units generates a re- that minima identifiable by classical HF theory [as well as
duced number of well-separated signals that are located in by semiempirical AM1 (data not shown)] could not be iden-
the aromatic (δ = 6.6–7.2 ppm) and aliphatic (δ = 1.5 ppm, tified as proper local minima when using a DFT method
3
H, t, and 4 ppm, 2 H, q) region, respectively. The recorded particularly adapted for dealing with weak supramolecular
Figure 2. Structural models, computed relative energies, and dipole moments for diastereoisomers of 5a characterized by S,S configuration
at the nitrogen atoms in the diazocine bridge, quasi-equatorial positions of the methyl groups, and variable configurations at the
stereogenic nitrogen atoms in the folded phenothiazine units: (a) R,R; (b) R,S; (c) S,S.
Figure 3. Structural models and computed relative energies for conformers of 5a with S,S configuration at nitrogen atoms in the diazocine
bridge and R,R configurations in the phenothiazine units: (a) quasi-equatorial orientation of the N-methyl groups; (b) quasi-axial orienta-
tion of the N-methyl groups.
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FULL PAPER
Figure 4. ORTEP representations of the molecular structures of PTB (a) 5a and (b) 2c. Selected angles between the planes of aromatic
rings are presented.
interactions, which gives particularly good results in confor-
mational and supramolecular issues.[
A different packing is observed for orthorhombic 5a and
monoclinic 5b (Figure 5). In both compounds, a pair of
39]
To elucidate the preferred molecular structures in the so- enantiomers with similar configurations at the four ste-
lid state, single crystals of PTB 5a and 5b that were suitable reogenic nitrogen atoms is present; S,S,S,S and R,R,R,R
for X-ray analysis were obtained from ethanol. The molecu- enantiomer, with S,S or R,R configuration for the two
lar structures of 5a and its amino-substituted precursor 2c nitrogen atoms located in the diazocine unit and S,S or R,R
are given in Figure 4. In 5a, a central TB scaffold and a for the two nitrogen atoms in the adjacent phenothiazine
pair of adjacent 1,4-benzothiazine rings is present. The ri- units.
gid central methylene bridge in the diazocine moiety im-
poses a twisted V shape with C symmetry on the molecule.
2
The dihedral angle between the two adjoining benzene rings
is 80.0° (close to the values reported for TB analogues),
whereas the phenothiazine units show folding angles of
Electronic Properties
The electronic properties of the new PTB compounds
5
2
a–d and their amino-10H-phenothiazines precursors 2b,
c, and 4a–d, determined by UV/Vis absorption spec-
146.4° and quasi-equatorial orientations of the methyl sub-
stituents (typical for 10-methyl-phenothiazine deriva-
[
40]
troscopy, fluorescence spectroscopy, and cyclic voltamme-
try, are summarized in Table 3.
tives). A quasi-equatorial orientation of the methyl sub-
stituents was also observed in the structure of the amino-
substituted precursor 2c (Figure 4, b), which is charac-
terized by a folding angle of 140.9°, a value slightly smaller
[
40]
than that reported for 10-methyl-phenothiazine (143.7°).
Table 3. Electronic properties of PTB and amino-10H-pheno-
thiazines (UV/Vis absorption/emission data and cyclic voltammetry
parameters).
Neighboring molecules of 2c are associated by NH···N
hydrogen bonds [2.70(3) Å], NH···S hydrogen bonds
0
/+1
+1/+2
[
2.75(3) Å] and offset parallel π–π interactions (3.39 Å) be-
λ
max,abs
λ
max,em
Stokes
shift
[cm ]
Quantum
E
0
E
0
yield
tween aromatic units.
–
1
[a]
[mV][b] [mV][b]
[nm]
[nm]
Φ
f
[%]
2
b
272, 324
272, 315
275, 317
275, 315
275, 315
272, 315
271, 315
271, 317
274, 321
267, 321
276, 321
276, 321
487
457
444
449
463
457
457
465
451
455
447
457
10300
9800
8900
9400
10000
9800
9800
10200
8900
9100
8600
9100
9.50
6.20
5.43
9.60
6.18
4.55
4.85
12.30
6.90
6.23
5.12
6.00
7.75
755
897
524
–
781
692
841
–
902
938
992
978
–
1085
1211
883
–
2
c
2
d
2
f
4
a
1180
1109
1255
–
4
b
4
c
4
d
[
b]
b]
b]
b]
5
a
1449
1716[
1772[
1912[
–
5
b
5
c
5
d
6
268, 324 458, 520 (sh) 9000
a] The fluorescence quantum yield Φ
perylene as standard (Φ = 94%). [b] In CH
[
f
was determined by using
Cl , 20 °C, v = 100 mV/
, Pt working electrode, Pt counter elec-
trode, Ag/AgCl reference electrode.
f
2
2
+
–
4 6
s, electrolyte: nBu N PF
Figure 5. Crystal packing diagram of PTB (a) 5a and (b) 5b.
6
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Access to Phenothiazine Analogues of Tröger’s Base
In the UV/Vis absorption spectra of PTB compounds oxidation potentials of PTB, pointing towards a higher sta-
5a–d and amino-10H-phenothiazines 2b–d, 2f, and 4a–d, bility, but also an independent participation of the two
two absorption bands with maxima at 268–275 and 315– equivalent phenothiazine units, as indicated by the in-
324 nm are observed due to electronic transitions in the creased current intensities (see for example the CV of PTB
phenothiazine unit. Upon UV excitation by irradiation with 5c in Figure 7).
the corresponding longest wavelength, all compounds emit
blue fluorescence due to extremely large Stokes shifts
–
1
(
8600–10300 cm ), with 4–12% fluorescence quantum
yields. The fluorescence emissions observed in diluted solu-
tions are also present in the solid states of PTB 5a–d, which
is supported by the lack of strong face-to-face π-π stacking
interaction between the aromatic units of the phenothiazine
fluorophore (which are usually responsible for fluorescence
quenching in the solid state). Figure 6 shows the UV/Vis
absorption and fluorescence emission spectra of dichloro-
methane solutions of PTB 5b and amine precursor 2c as
well as the emission properties of the crystalline solids; the
latter show a small hypsochromic shift.
Figure 7. Cyclic voltammograms of 4a and 5c (recorded in CH
2
Cl
2
,
Figure 6. UV/Vis absorption and emission spectra in dilute di-
+
–
2
0 °C, v = 100 mV/s, electrolyte: nBu N PF , Pt working elec-
4 6
chloromethane solution (10–5 m) and the crystalline state for PBT
trode, Pt counter electrode, Ag/AgCl reference electrode) using fer-
5b and amino-substituted precursor 2c.
+
rocene/ferrocenium (Fc/Fc ) as internal standard.
Electrochemical data were obtained by cyclic voltamme-
try studies on PTB 5a–d and amino-substituted precursors
b–d and 4a–c in dichloromethane using ferrocene/ferrocen-
2
+
ium (Fc/Fc ) as internal standard, with scanning in the an- Interaction with Biomolecules
odic (up to 2 V) and cathodic (up to –0.2 V) region.
In comparison with the parent 10H-phenothiazine,
Several experiments were performed to test the capacity
which was characterized by the first one-electron oxidation of the synthesized amino-substituted phenothiazines and
0
/+1
potential E0
= 624 mV, alkylation of the heterocyclic PTBs to interact with proteins and DNA. Autoxidation of
nitrogen atom results in an anodical shift (10-Me-10H- the physiologically-useful oxy form of hemoglobin and
0
/+1
phenothiazine E0
= 767 mV), which increases in the case myoglobin into the toxic ferric (“met”) forms, monitored
of the synthesized amines [e.g., 2-amino-10-Me-10H-pheno- by UV/Vis absorption spectroscopy, indicated that amino-
0
/+1
thiazine (2c) E0
dation peak appears at higher anodic potential (ca. oxidation rates of hemoglobin (Hb) (Figure 8) and myoglo-
500 mV). bin (Mb), accelerating this process in a qualitatively similar
A comparison between the CV patterns recorded for manner for both proteins. On the other hand, the corre-
= 897 mV]. A second irreversible oxi- substituted phenothiazines have a strong effect on the auto-
1
PTB and the corresponding amino-substituted phenothi- sponding PTB showed much smaller or even negligible ef-
azine precursors revealed not only an anodic shift of both fects (Table 4).
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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C. Cristea et al.
FULL PAPER
Figure 8. UV/Vis spectra of hemoglobin, collected at the initial and
final time points during the autooxidation experiments in the pres-
ence of 2c and 4c.
Figure 9. Lipid peroxidation induced by cytochrome c (1.5 μm) in
liposomes (5 μg/mL) in the presence/absence of PTB (10 μm), in
phosphate buffer (20 mm), pH 7.4.
Table 4. Increases in autooxidation rates induced in hemoglobin
and myoglobin, and inhibition of lipid peroxidation by amino-sub-
stituted phenothiazines and PTBs.
Hb [%][a]
Mb [%][a]
Lipid inhibition[b]
2d
2c
4a
4b
4c
5a
5b
5c
5d
+362.1
+358.4
+358.9
+118.3
+170.4
+8.3
–1.9
+10.7
–35.2
+71.9
+76.9
+73.4
+72.1
+70.0
+15.6
+29.0
n.d.
–
+
+
+
+
+
+
–
n.d.
–
[
a] Percentage of increase relative to the native form of Hb/Mb un-
der the same conditions. [b] Compounds marked with “–” showed
no difference from the control over a 10 h reaction time; com-
pounds marked with “+” showed significant delays in the ab-
sorbance increase associated with lipid peroxidation in this experi-
ment.
Figure 10. Electrophoresis data for (a) 2c and (b) 5a. The lanes con-
tain 250 ng plDNA with 0, 0.5, 1, 2, 4, 6, 8, 10, and 0 μL of each
compound. In the case of compound 5a, the first and last lanes
show the linear-DNA control.
applications involving biological activity with respect to
more than one class of biomolecules – proteins, lipids, and
nucleic acids.
Liposome oxidation experiments, monitored by re-
cording the typical absorbance band situated at 235 nm for
10 h, indicated that some of the synthesized amino-pheno-
thiazines and PTB inhibit the lipid peroxidation induced
by cytochrome c (Figure 9). Both types of protein-related
reactivity reported in Table 4 can be related to free radical
stress, as free radicals (superoxide, peroxides) are known to
Conclusions
Microwave-assisted catalyzed C–N bond formation reac-
be involved in accelerating protein autooxidation and lipid tions can be successfully applied in the preparation of pri-
[
41]
peroxidation.
mary amino-10-alkyl-10H-phenothiazines. The Pd-cata-
DNA electrophoresis experiments using plasmid lyzed amination is suitable for the preparation of 2-amino-
pTZ57R treated with dimethyl sulfoxide (DMSO) solutions phenothiazine, whereas the Cu-catalyzed amination conve-
of amino-phenothiazines and PTBs respectively at different niently generates the 3-amino-phenothiazine regioisomer.
ratios, indicated that several of the compounds examined
New PTB compounds, combining the chiral V shaped
can not only bind to DNA, but also cause detectable TB scaffold with the folded 1,4-benzothiazine unit, are
changes in its physical properties. Thus, the amino-substi- formed by regioselective condensation of 2-amino-10-alkyl-
tuted phenothiazine 2c and the corresponding PTB 5a in- 10H-phenothiazines with formaldehyde. Computational
duce relaxation of the circular plasmid, as illustrated in Fig- analysis of the stereoisomers of PTB revealed three possible
ure 10.
pairs of diastereoisomers. A pair of enantiomers each con-
The experiments shown here, illustrating binding to pro- taining similar configurations at the four stereogenic nitro-
teins with effects on prooxidant reactivity including lipid gen atoms was evidenced by XRD.
peroxidation and binding to DNA, suggest that the family
The UV/Vis absorption/emission and redox processes in-
of phenothiazine derivatives described here is amenable to volving PTBs appear to be rather insensitive to the effects
8
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Pages: 10
Access to Phenothiazine Analogues of Tröger’s Base
of the rigid methano-diazocine bridge, which electronically Acknowledgments
decouples the two phenothiazine units in the PTB. Com-
pounds 5a–d exhibit blue fluorescence emission in dilute
Financial support from the Romanian Ministry of Education, Re-
search Youth and Sports (grant number ID PCCE 140/2008) and
tremely large Stokes shifts (8900–10300 cm ) and moderate from the Deutscher Akademischer Austausch-Dienst (DAAD)
solution and the solid state, and are characterized by ex-
–
1
quantum yields (4.5–12.3%).
(SOE programme) is gratefully acknowledged.
The experiments shown here, illustrating the supramolec-
ular assemblies, chirality, redox, and fluorescence properties
of amino-substituted phenothiazines and their related PTB,
qualify this family of phenothiazine derivatives for further
studies in materials science; their capacity to bind to pro-
teins or nucleic acids, and additional prooxidant reactivity
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