Palladium catalyzed transformation and antimicrobial screening of novel angular azaphenothiazines

Article abstract of DOI:10.14233/ajchem.2017.20220

Base mediated condensation reaction between 2-amino-5-bromopyrazine-3[4H]-thione and 7-chloro-5,8-quinolinequinone under anhydrous condition gave 9-bromo-1,8,11-triaza-5H-benzo[a]phenothiazin-5-one. Palladium catalyzed cross-coupling reaction between 9-bromo-1,8,11-triaza-5H-benzo[a]phenothiazin-5-one and four arylated halogeno compounds utilizing Heck-Mizoroki protocol furnished 6-substituted derivatives of the angular tetracyclic heterocycle. Structures were assigned based on spectroscopic and elemental analytical data. Antimicrobial screening of these compounds showed they were biologically active.

Full text of DOI:10.14233/ajchem.2017.20220

Asian Journal of Chemistry; Vol. 29, No. 4 (2017), 742-748
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2017.20220
Palladium Catalyzed Transformation and Antimicrobial
Screening of Novel Angular Azaphenothiazines
1,*
2
2
3
2
E.U. GODWIN-NWAKWASI , U.C. OKORO , A.O. IJEOMAH , I. AGBO and M.A. EZEOKONKWO
1
2
3
Department of Chemistry, College of Natural & Applied Sciences, Gregory University, Uturu, Abia State, Nigeria
Department of Pure & Industrial Chemistry, Faculty of Physical Sciences, University of Nigeria, Nsukka, Nigeria
Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Nigeria
Corresponding author: Tel: +234 8038737824; E-mail: evelynnwakwasi@yahoo.com
Received: 29 July 2016; Accepted: 21 January 2017; Published online: 31 January 2017;
*
AJC-18236
Base mediated condensation reaction between 2-amino-5-bromopyrazine-3[4H]-thione and 7-chloro-5,8-quinolinequinone under anhydrous
condition gave 9-bromo-1,8,11-triaza-5H-benzo[a]phenothiazin-5-one. Palladium catalyzed cross-coupling reaction between 9-bromo-
1
,8,11-triaza-5H-benzo[a]phenothiazin-5-one and four arylated halogeno compounds utilizing Heck-Mizoroki protocol furnished 6-
substituted derivatives of the angular tetracyclic heterocycle. Structures were assigned based on spectroscopic and elemental analytical
data. Antimicrobial screening of these compounds showed they were biologically active.
Keywords: Phenothiazine, Palladium catalysis, Arylated halogeno compounds, Angular azaphenothiazines.
type 3 are known [6,9-11]. Also grossly under studied are
angular phenothiazines possessing annular nitrogen atoms
simultaneously in rings A and D, 4; only a few cases have
been reported [12-14] albeit their reported uses in medicine
INTRODUCTION
The chemistry of phenothiazine (1), dates more than a
century ago [1], yet interest in the synthesis of its derivatives
especially the aza analogues remains unabated because they
have been found to have better biological and chemical
properties than their carboxylic counterparts [2-4] which have
been attributed to the basic nitrogen of the ring donating
electrons to the biological receptors by a charge-transfer
mechanism and the stabilities of the charge transfer complexes
have been determined [3].
[
15,16], agriculture [17] and industry [5,18-20]. At present,
only two isomeric forms of angular triazaphenothiazine of type
out of forty-five possible isomeric forms of this ring system
4
have been reported [14].
H
N
H
N
H
N
N
N
N
H
N
S
S
S
2
3
4
In furtherance of studies of triaza analogues of type 4, we
now report the synthesis of new angular triazaphenothiazine
ring systems where the annular nitrogen atoms are located in
ringsA and D and their biological activities. Besides, the route
to the synthesis of the 6-substituted derivatives utilized Heck-
Mizoroki catalysis which is an entirely new and a relatively
unexplored process [21,22].
S
1
Although benzo[a]phenothiazine (2), the prototype of
angular phenothiazines has been known for over ten decades.
Its chemistry remains poorly developed [5] especially the
aza analogues of this ring system. In spite of early reports
on phenothiazines [1,5b], little is relatively known about the
chemistry of the angular systems. Only recently did the synthesis
of a few aza analogues possessing annular nitrogen atom(s)
appear in the literature [3,6-8]. Only few of aza-analogues of
EXPERIMENTAL
Solvents and chemicals used were of analytical grade from
Sigma-Aldrich. The reactions were monitored by TLC using
Merck pre-coated TLC silica gel plates. Pure products were

Vol. 29, No. 4 (2017)
Palladium Catalyzed Transformation and Antimicrobial Screening of Novel Angular Azaphenothiazines 743
obtained by recrystallization. Melting points were taken using
Fisher-Johns melting point apparatus and they remain un-
Preparation of 5-amino-7-chloro-8-hydroxyquinoline
(13): 7-Chloro-8-hydroxy-5-nitroquinoline (22.4 g, 0.1 mol)
was ground in a mortar with 1 M KOH (110 mL) to ensure
complete reduction of the insoluble potassium salt. The suspen-
sion was transferred to a 1 L three-necked flask equipped with
a long magnetic stirrer with the aid of water (280 mL). The
mixture was heated in a water-bath with vigorous stirring. 8
M KOH (70 mL) was added at 50 °C, the reaction mixture
was treated with sodium dithionite (70 g). The mixture was
then reheated and maintained at 80 °C for 10 min with rapid
stirring while a stream of nitrogen was passed into the flask.
After 10 min, more sodium dithionite (10 g) was added and
the reaction allowed to continue 10 min longer. At the end of
this period, the resulting suspension was cooled in ice under
nitrogen and the precipitate was filtered off, washed with cold
water containing a trace of sodium dithionite and dried rapidly
in a vacuum oven to furnish 5-amino-7-chloro-8-hydroxy-
quinoline (17.4 g, 77 %) as a gold-yellow solid [(m.p.: 171-
173 °C, dec) (lit. 172-173 °C [24]; 173-174 °C [23] dec.)].
Preparation of 7-chloro-5,8-quinolinequinone (6): 5-
Amino-7-chloro-8-hydroxyquinoline (17.9 g, 0.09 mol) was
suspended in water (600 mL) in a 1 L flask equipped with a
long magnetic stirring bar. One equivalent of 6 M sulphuric
acid (18 mL) was added to dissolve the amine and vigorous
stirring continued while the solution was cooled to 2 °C and the
salt precipitated in a finely divided form. An ice-cold solution
made up of 10 % potassium dichromate solution (103 mL)
and 6 M sulphuric acid (71 mL) was then added. The mixture
was stirred and cooled in an ice-bath for 15 min. The
precipitated crude product was filtered on a Büchner funnel
containing ice and washed with cold water and dried. The light
tan coloured product was recrystallized from DMF treated with
activated charcoal and it furnished fine yellow crystals of 7-
chloro-5,8-quinolinequinone (12.6 g, 74.5 %); m.p.: 173-174
°C, dec. (lit. 173.5-174.5 °C [25]). UV-visible (MeOH) λmax
1
13
corrected. Compounds were characterized by H NMR,
C
NMR, IR, UV/visible spectroscopy and elemental analysis.
UV-visible spectra were recorded on a JENWAY 6405 UV/
VIS spectrophotometer using matched 1cm quartz cells, IR
spectra on a Shimadzu FTIR-8400S Fourier transform infrared
spectrophotometer (KBr Pellets), nuclear magnetic resonance
1
13
(
H NMR and C NMR) spectra were determined usingVarian
NMR Mercury-200BB. Chemical shifts were recorded on the
δ scale (neat). The elemental analyses were done on a CE440
elemental analyzer. Antimicrobial screening was done using
agar-well diffusion method.
Preparation of 8-hydroxy-5-nitrosoquinoline hydro-
chloride (10): This compound was prepared following the
procedure of Pratt and Drake [23].
To a solution of 8-hydroxyquinoline (58 g, 0.4 mol) in
2
00 mL of water, 75 mL of concentrated HCl and 200 g of ice
was added a solution of 30 g of sodium nitrite in 100 mL of
water in portions with vigorous shaking over 1 h at 0 to 4 °C.
The mixture was allowed to stand overnight at 0 °C before the
product was filtered off and washed with cold water. The product
was air-dried giving a yield of 76 g (87 %) of the hydrochloride
of 8-hydroxy-5-nitrosoquinoline (m.p. 181-183 °C, dec.) as
fine yellow compound. The compound loses its brightness in
time and darkens due to auto-oxidation.
Preparation of 8-hydroxy-5-nitroquinoline (11): Com-
pound 11 was prepared as described by Petrow and Sturgeon
[
24].
Finely powdered 8-hydroxy-5-nitrosoquinoline (3 g,
.017 mol) was added with vigorous stirring to a mixture of
concentrated HNO (9 mL) and water (6 mL) at about 17 °C.
0
3
Oxides of nitrogen were evolved and the nitrosoquinoline was
rapidly converted into insoluble 8-hydroxy-5-nitroquinoline
nitrate. After 75 min with occasional stirring, the mixture was
diluted with water (20 mL) and cooled to 0 °C. The cooled
mixture was made alkaline by the addition of cold KOH
solution. The red potassium salt was neutralized with acetic
acid (pH = 7). Filtration was done and the product washed
with water and recrystallized from aqueous ethanol to give
fine yellow needles of 8-hydroxy-5-nitroquinoline; m.p. 180-
(nm): 240 (3.79), 290 (3.95) and 395 (4.17). IR (KBr, νmax
,
-1
cm ): 3104, 1654, 1584, 1501, 1296, 752.
Preparation of 5-amino-8-hydroxyquinoline sulphate
(14): This compound was prepared from 8-hydroxy-5-nitroso-
quinoline hydrochloride, whose synthesis has been described
previously. Freshly prepared hydrochloride of 8-hydroxy-5-
nitrosoquinoline (20 g, 0.11 mol) was dissolved in a mixture
of water (80 mL) and 5 M NaOH (130 mL) placed in a three-
necked flask (500 mL) equipped with a long magnetic bar.
The entire mixture was warmed to 40 °C with stirring and sodium
dithionite (48 g) was added portion wise. The temperature of
the solution rose spontaneously to 75-80 °C and a rapid stream
of nitrogen was introduced into the solution. The solution was
allowed to cool slowly to about 50 °C and 6 M sulphuric acid
(125 mL) was then added which led to evolution of sulphur
dioxide. When the evolution of sulphur dioxide had subsided
the solution was maintained under diminished pressure
(pressure of the nitrogen gas reduced) with magnetic stirring
until most of the dissolved gases had been removed. The mixture
was cooled in an ice-bath and the resulting precipitate filtered
without washing. Reddish-brown crystals of 5-amino-8-hydroxy-
quinoline sulphate (16.5 g, 82.5 %) was obtained, m.p.: 218-
219 °C (lit. [25] 220 °C).
182 °C (lit. 180 °C [24]; 179.5-181.5 °C [23]).
Preparation of 7-chloro-8-hydroxy-5-nitroquinoline
(12): 8-Hydroxy-5-nitroquinoline (20 g, 115 mmol) suspended
in water (2 L) and one equivalent of 1 M potassium hydroxide
solution (107 mL) were vigorously stirred while 5 % excess
sodium hypochlorite solution (143 mL) was added over 90
min. During the course of the addition, all the starting material
dissolved and soon the salt of 7-chloro-8-hydroxy-5-nitro-
quinoline began to precipitate [18]. Stirring continued for
another 2 h, before the mixture was neutralized with acetic
acid (pH = 7) and stirred to permit complete conversion of the
precipitate to the free quinolinol. The product was filtered,
washed with water and air-dried. The crude product was
recrystallized from ethyl acetate (500 mL) by continuous
extraction to yield 16.9 g (86 %) of bright orange 7-chloro-8-
hydroxy-5-nitroquinoline; m.p. 239-241 °C, dec. (lit. 239-
2
40.5 °C).

744 Godwin-Nwakwasi et al.
Asian J. Chem.
Equally, satisfactory results were obtained using 12 M
mmol) were placed in a 100 mL three-necked flask equipped
with a short magnetic stirring bar, a reflux condenser and
a thermometer. A mixture of benzene (40 mL) and DMF
(5 mL) was added into the flask. The mixture was refluxed on
a water-bath at 70-75 °C for 45 min with stirring. 7-Chloro-
5,8-quinolonequinone (0.97 g, 5 mmol) was added and the
stirring continued for 7 h still maintaining the temperature of
the reaction mixture at 70-75 °C.At the end of the reflux period,
the resultant mixture was filtered and the solvent evaporated.
Analytical sample was obtained by column chromatography
using methanol and acetone (1:3) as the eluting solvent. The
reddish-yellow solid compound, 9-bromo-1,8,11-triaza-5H-
benzo[a]phenothiazin-5-one weighed 1.28 g (74 %) and melted
at 218-220 °C (dec.). UV-visible (MeOH) λmax (nm):285 (3.92),
HCl in place of 6 M H
2
SO which gave the dihydrochloride.
4
Preparation of 6,7-dibromo-5,8-quinolinequinone (7):
Compound 7 was prepared by reported methods [27-29]. 5-
Amino-8-hydroxyquinoline sulphate (54 g, 0.34 mol) was
dissolved in 24 % hydrobromic acid (400 mL) in a 1 L three-
necked flask equipped with reflux condenser, quick fit thermo-
meter, magnetic bar and a dropping funnel. The reaction mixture
was heated to 50 °C. A solution of sodium bromate (38.5 g,
0
.225 mol) in water (150 mL) was added at such a rate that
the temperature did not exceed 50-60 °C. After the addition,
the reaction mixture was heated at that temperature with gentle
stirring for 0.5 h and then cooled. The cooled mixture was
poured onto ice (500 g) and further chilled in an ice-salt mix-
ture. The resulting precipitate was filtered and recrystallized
from ethanol to give yellow precipitate of 6,7-dibromo-5,8-
quinolinequinone (71.3 g, 66 %); m.p.: 242-243 °C (lit. [27-
-1
345 (4.34), 435 (5.06). IR (KBr, νmax, cm ): 3167, 1618, 1504,
1
1290, 691. H NMR (DMSO) δ: 9.2 (3H, m), 8.2 (1H, s), 6.8
13
(1H, s). C NMR (DMSO) δ: 168 (C=O), 153 (C-2), 143 (C-
2
2
3
9] 243-245 °C). UV-visible (MeOH) λmax (nm): 265 (3.69),
10), 142 (C-9) 123 (C-3), 120 (C-6), 38.7-41.2 (DMSO).Anal.
-1
90 (3.92), 345 (3.45) and 505 (2.20). IR (KBr, νmax, cm ):
093, 1686, 1541, 1289, 664, 662.
calcld. (%) for C13
H
5
4
N BrSO: C, 45.22; H, 1.45; N, 16.23; Br,
23.19; S, 9.28. Found (%): C13
N, 16.30; Br, 23.20; S, 9.40.
H
5
N SOBr: C, 45.30; H, 1.48;
4
Preparation of 2-amino-3,5-dibromopyrazine: This
compound was prepared following Okafor and Okoro’s proce-
dure [12,26].
Preparation of 1,4-bis(2-hydroxy-3,5-di-tert-butyl-
benzyl)piperazine (C34 ) (21): The bulky ligand, 21
H
54
2
N O
2
was prepared following Balakrishna et al. method [30]. A
mixture of piperazine (2.2 g, 25.54 mmol) and 40 % aqueous
formaldehyde solution (5.30 mL, 75.36 mmol) was dissolved
in methanol (40 mL) and heated to reflux for 2 h to get a clear
solution. To the cooled solution was added 2,4-di-tert-butyl-
phenol (10.3 g, 50.41 mmol) in methanol (60 mL). The resulting
solution was refluxed for further 12 h at 60 °C. The reaction
mixture was cooled to room temperature and filtered off to
get compound 21 as colourless crystalline compound. Yield:
N
Br
Br
N
NH2
2
-Aminopyrazine (9.5 g, 100 mmol) was placed in a
reaction flask containing glacial acetic acid (70 mL) and
warmed on a steam bath until it dissolved. Sodium acetate
trihydrate (33 g, 243 mmol) was added with constant swirling.
The slurry was stirred in an ice-salt bath maintained at -5 °C
and bromine (16 mL) was added dropwise over a 4 h period
6
4 % (8.35 g, 15.98 mmol); m.p.: > 250 °C (dec.) (lit. [30]
250 °C (dec.).
>
(
if the bromine addition was speeded up the reaction became
OH
turbulent and potentially hazardous).
N
The mixture was stirred in the ice bath for 2 h and then at
room temperature for 24 h. It was then poured into ice (50 g)
and neutralized with concentrated ammonia (pH 8). The crude
product was collected and recrystallized from methanol (Norit)
to give colourless needles of 2-amino-3,5-dibromopyrazine
N
OH
(
16.8 g, 66 %), m.p.: 113-114 °C (lit. [13,26] 114-115 °C).
Preparation of 2-amino-5-bromopyrazine-3[4H]–thione
5): A mixture of 2-amino-3,5-dibromopyrazine (7.69 g, 30
2
1
(
General procedure for Heck-Mizoroki reaction [30]:
Under nitrogen atmosphere, piperazine ligand (0.005 g, 0.01
mmol), dppb/PdCl (0.006 g, 0.01 mmol) and methanol (10
mmol) and sodium hydrosulphide (13.33 g, 238 mmol) was
added to methanol (60 mL) and the mixture was refluxed for
2
4
.5 h. Methanol was then removed by distillation and the
mL) were placed in a two-necked 100 mL round bottom flask
equipped with a magnetic stirrer. After stirring for 5 min, an
intimate mixture of 9-bromo-1,8,11-triaza-5H-benzo[a]-
phenothiazin-5-one (1.04 g, 3 mmol), aryl halide (1 mmol)
residual dark moist solid was dissolved in water (150 mL),
treated with activated charcoal, boiled and filtered. The filtrate
was cooled and the product filtered. Recrystallization from
DMF (Norit) yielded pure 2-amino-5-bromopyrazine-3[4H]-
thione (3.75 g, 61 %) as a yellow solid, m.p.: 209-211 °C
and K
2
CO (0.193 g, 1.4 mmol) was added to the reaction flask.
3
The reaction mixture was heated to 60 °C for 4 h. After the
reflux period, the solvent was removed by vacuum distillation.
Crushed ice (5 g) was added to the resultant residual mixture
which precipitated out the solid product. It was further recrysta-
llized from aqueous ethanol.
(
2
dec); (lit. [13] 208-210 °C). UV-visible (MeOH) λmax (nm):
22 (2.46), 249 (4.16), 340 (3.89), 425 (1.61). IR (KBr, νmax
,
-1
cm ): 3446, 3173, 1563, 1524, 1318, 674.
Synthesis of 9-bromo-1,8,11-triaza-5H-benzo[a]pheno-
thiazin-5-one (19): 2-Amino-5-bromopyrazine-3[4H]-thione
9-Bromo-6-(4-nitrophenyl)-1,8,11-triaza-5H-benzo[a]-
phenothiazin-5-one (20a): Following the general procedure, a
(
1.03 g, 5 mmol) and anhydrous sodium carbonate (1.06 g, 10

Vol. 29, No. 4 (2017)
Palladium Catalyzed Transformation and Antimicrobial Screening of Novel Angular Azaphenothiazines 745
mixture of 1,4-bis(2-hydroxy-3,5-di-tert-butylbenzyl)piperazine
0.005 g, 0.01 mmol) dppb/PdCl (0.006 g, 0.01 mmol), 9-
bromo-1,8,11-triaza-5H-benza[a]phenothiazin-5-one (1.04 g,
mmol), 4-iodonitrobenzene (0.249 g, 1 mmol), K CO (0.193
Found (%): C19
7,34; Br, 18.40.
9-Bromo-6-(2-carboxyphenyl)-1,8,11-triaza-5H-
benzo[a]phenothiazin-5-one (20d): Following the general
procedure, a mixture of 1,4-bis(2-hydroxy-3,5-di-tert-butyl-
benzyl)piperazine (0.005 g, 0.01 mmol) dppb/PdCl (0.006 g,
0.01 mmol), 9-bromo-1,8,11-triaza-5H-benza[a]phenothiazin-
5-one (1.04 g, 3 mmol), 2-iodobenzoic acid (0.248 g, 1 mmol),
H
10
N SOBr: C, 52.30; H, 2.10; N, 16.13; S,
5
(
2
3
2
3
g, 1.4 mmol) and methanol (10 mL) was refluxed for 4 h.
After removal of the solvent under reduced pressure, crushed
ice (5 g) was added which precipitated out the solid product.
It was further recrystallized from aqueous ethanol to yield a
yellow solid (1.20 g, 85.7 %), m.p.: (114-116 °C, dec.). UV-
visible (MeOH) λmax (nm): 245.2 (4.52), 334.8 (4.25), 419
2
K
2
CO (0.193 g, 1.4 mmol) and methanol (10 mL) was refluxed
3
for 4 h. After removal of the solvent under reduced pressure,
crushed ice (5 g) was added which precipitated out the solid
product. It was further recrystallized from aqueous ethanol to
give a reddish brown solid (1.17 g, 84 %), m.p.: (118-120 °C,
dec.). UV-visible (MeOH) λmax (nm):228.4 (4.19), 246.6 (4.46),
339.4 (4.12), 443.8 (3.97). IR (KBr, νmax, cm ): 3455, 3168,
1621, 1512, 1282, 687. H NMR (DMSO) δ: 9.6 (1H, O-H),
8.6 (1H, s), 8.2 (1H, d), 7.1 (2H, d), 6.6 (2H, s). C NMR
(DMSO) δ: 168 (C=O), 153 (C-2), 146 (C-10), 134 [(C-4 &
C-6-(5)], 131 [(C-6-(3)], 123 [(C-3 & C-6-(2)], 38.5-54.5
-
1
(
6
4.13). IR (KBr, νmax, cm ) 3148, 1618, 1504, 1447, 1290,
1
92. H NMR (DMSO) δ: 9.4 (1H, d), 8.58 (1H, d), 8.32 (4H,
13
m), 7.72 (1H, d), 7.0 (1H, s), 6.75 (2H, s). C NMR (DMSO)
δ: 188 (C=O), 153 (C-2), 146 (C-10), 143 [(C-6-(1)], 136.6
-1
(
(
C-9), 134 [(C-4), 127 [(C-6-(2)], 123 [(C-9-(2)], 120 [(C-6-
1
3)], 38.7-71.4 (DMSO). Anal. calcld. (%) for C19
8 5
H N SO
3
Br:
13
C, 48.93; H, 1.72; N, 15.02; S, 6.87; Br, 17.17. Found (%): C,
9.00; H, 1.80; N, 15.00; S, 6.68; Br; 17.20.
-Bromo-6-(4-hydroxyphenyl)-1,8,11-triaza-5H-
4
9
benzo[a]phenothiazin-5-one (20b): Following the general
procedure, a mixture of 1,4-bis(2-hydroxy-3,5-di-tert-butyl-
(DMSO). Anal. calcld. (%) for C20
1.94; N, 12.04; S, 6.88; Br, 17.20. Found (%): C20
C, 51.58; H, 1.95; N, 12.10; S, 6.79; Br, 17.25.
H
9
N
4
SO
3
Br: C, 51.61; H,
SO Br:
H
9
N
4
3
benzyl)piperazine (0.005 g, 0.01 mmol) dppb/PdCl
0
5
2
(0.006 g,
.01 mmol), 9-bromo-1,8,11-triaza-5H-benza[a]phenothiazin-
-one (1.04 g, 3 mmol), 4-iodophenol (0.220 g, 1 mmol), K CO
Evaluation of synthesized angular phenothiazinones
for antibacterial activity: The assay was conducted using
agar-well diffusion method [31]. A 20 mg/mL concentration
of each compound was constituted by dissolving 0.04 g of
each in 2 mL dimethyl sulfoxide. A single colony of each test
isolate was suspended in 2 mL of sterile nutrient broth. The
suspension of each isolate was standardized by adjusting to
correspond to 0.5 McFarland turbidity standards corresponding
2
3
(
0.193 g, 1.4 mmol) and methanol (10 mL) was refluxed for 4
h.After removal of the solvent under reduced pressure, crushed
ice (5 g) was added which precipitated out the solid product.
It was further recrystallized from aqueous ethanol to yield a
dark brown solid (1.11 g, 84.7 %), m.p.: (> 300 °C, dec). UV-
visible (MeOH) λmax (nm): 221.4 (4.21), 249.4 (4.31), 341
-
1
8
(
3
4.01), 360 (3.78), 444.8 (3.87). IR (KBr, νmax, cm ): 3452,
to approximately 10 cfu/mL and used to inocu-late the surface
1
153, 1623, 1505, 1298, 695. H NMR (DMSO) δ: 9.3 (1H,
d), 8.6 (1H, s), 8.1 (1H, d), 7.5 (2H, d), 6.8 (2H, d), 5.5 (O-H).
of the iso-sensitest nutrient agar and the excess fluid drained
into discard pot containing disinfectant. The inoculated agar
surface was allowed to dry and the plates appropriately labelled.
Using a cork borer of 6 mm in diameter, wells were bored in
the inoculated iso-sensitest nutrient agar. With a micropipette,
50 µL of each test compound solution was deli-vered into each
well. The plates were left on the bench for 30 min to allow the
compound to diffuse into the agar. Thereafter, the plates were
incubated at 37 °C for 24 h. After incubation, the plates were
observed for inhibition zones around the wells. The diameters
of the zones were measured with metre rule to the nearest
whole millimetre.
13
C NMR (DMSO) δ: 190 (C=O), 153 (C-2), 143 (C-10), 131
Cβ to C-4), 124 (C-3), 116 [(C-6-(3 &5)], 38.3-41.2 (DMSO).
Anal. calcld. (%) for C19 SO Br: C, 52.17; H, 2.06; N,
2.81; S, 7.32; Br, 18.31. Found (%): C, 52.00; H, 2.04; N,
2.90; S, 7.36; Br, 18.20.
-Bromo-6-(4-anilino)-1,8,11-triaza-5H-benzo[a]-
(
H
9
N
4
2
1
1
9
phenothiazin-5-one (20c): Following the general procedure,
a mixture of 1,4-bis(2-hydroxy-3,5-di-tert-butylbenzyl)-
piperazine (0.005 g, 0.01 mmol) dppb/PdCl
mmol), 9-bromo-1,8,11-triaza-5H-benza[a]phenothiazin-5-
one (1.04 g, 3 mmol), 4-bromoaniline (0.172 g, 1 mmol), K CO
0.193 g, 1.4 mmol) and methanol (10 mL) was refluxed for
2
(0.006 g, 0.01
2
3
Determination of minimum inhibitory concentration (MIC)
(
4
h.After removal of the solvent under reduced pressure, crushed
Minimum inhibitory concentration of synthesized
angular triazaphenothiazines: This was carried out using
agar dilution following the procedure outlined by Chemical
Laboratory Standards Institute (CLSI) [32]. Sterile test tubes
were arranged on a test tube rack and 1 mL of DMSO was dis-
pensed into each of them. From the stock compound solutions,
1 mL was transferred into the first test tube and two-fold serial
dilution of each compound solution was carried out and the
resultant concentration in the test tubes were 10, 5, 2.5, 1.25,
0.625, 0.3125 and 0.15625 (mg/mL) (i.e., graded concen-
trations of the compounds).A single colony of each test isolate
was suspended in 2 mL of sterile nutrient broth. The suspension
of each isolate was standardized and used to inoculate the
ice (5 g) was added which precipitated out the solid product.
It was further recrystallized from aqueous ethanol to yield a
yellow solid (0.89 g, 68 %), m.p.: (138-140 °C, dec.). UV-
visible (MeOH) λmax (nm): 221.4 (4.26), 247.6 (4.17), 327.8
(
3.90), 360 (3.64), 441.4 (3.27), 498.4 (2.54), 656.2 (2.42).
-1
1
IR (KBr, νmax, cm ): 3475, 3145, 1628, 1500, 1283, 692. H
NMR (DMSO) δ: 9.3 (1H, d), 8.6 (1H, s), 8.2 (1H, s), 7.52
13
(
(
1
[
C
1H, s), 7.0 (2H, s), 6.5 (2H, d), 3.96 (2H, s,-NH
DMSO) δ: 172 (C=O), 153 (C-2), 147 (C-10), 145 (C-NH
34 (C-4 & C-9), 132 (C β to C-4), 131 (C-6), 124 (C-3), 118
(C-6-(3 &5)], 24.1-63.6 (DMSO). Anal. calcld. (%) for
SOBr: C, 52.29; H, 2.29; N, 16.05; S, 7.3; Br, 18.35.
2
). C NMR
2
),
19
H
10
N
5

746 Godwin-Nwakwasi et al.
Asian J. Chem.
surface of the nutrient agar and the excess fluid drained into
discard pot containing disinfectant. The inoculated agar surface
was allowed to dry and the plates appropriately labelled. Using
a cork borer of 6 mm in diameter, wells were bored in the
inoculated nutrient agar. With a micropipette, 50 µL of each
test compound solution was delivered into each well. The plates
were left on the bench for 30 min to allow the compound to
diffuse into the agar. Thereafter, the plates were incubated at
of compound 11 with sodium hypochlorite produced, 7-chloro-
8-hydroxy-5-nitroquinoline (12) which on reduction with
sodium dithionite under inert atmosphere gave 5-amino-7-
chloro-8-hydroxyquinoline (13). Oxidation of compound 13
using acidified potassium dichromate and recystallization from
DMF (Norit) furnished fine yellow crystals of 7-chloro-5, 8-
quinolinequinone, 6, in good yield.
Under similar conditions, the coupling of compounds 5
and 7-chloro-5,8-quinolinequinone (6), in the presence of
anhydrous sodium carbonate gave 9-bromo, 1,8,11-triaza-5H-
benzo[a]phenothiazin-5-one (19) (Scheme-III).
3
7 °C for 24 h. After incubation, the plates were observed for
inhibition zones around the wells. The diameters of the zones
were measured with metre rule to the nearest whole millimetre.
The molecular formula, C13
H
5
N OSBr and assigned
4
RESULTS AND DISCUSSION
structure 19 were established based on microanalysis, UV,
IR and NMR spectroscopy of compound 19. The UV-visible
(MeOH) showed signals at λmax 285 (ε = 3.92), 345 (4.34) and
435 (5.06) nm. The IR showed signals at 3167 (C–H, aromatic),
1618 (C=O), 1504 (C=C, aromatic rings), 1290 (C–N stretch)
Intermediate 5 was obtained in good yield according to
literature [13,26] as colourless needles by treating 2-amino-
pyrazine (8), with bromine in glacial acetic acid at -5 to 0 °C
followed by thiation using sodium hydrosulphide in methanol
and recrystallization using DMF (Norit) (Scheme-I).
Compound 6 was prepared by multi-step conversion [23-25]
of 8-hydroxyquinoline (9) (Scheme-II). In reaction sequence,
nitrosation of compound 9 yielded 8-hydroxy-5-nitroso-
quinoline hydrochloride (10) which was oxidized with nitric
acid to give 8-hydroxy-5-nitroquinoline (11). Haloge-nation
-1
1
and 691 (C–Br stretch) cm . The H NMR spectra showed
13
signals at δ 9.2 (3H, arom), 8.2(s, 10-H), 6.8 (s, 6-H). The C
NMR spectra showed signals at δ 168 (C=O), 153 (C-2), 143
(C-10), 142 (C-9), 123 (C-3) and 120 (C-6).
In a similar development, base mediated reaction of the
non-linear triazaphenothiazinone with aryl iodo-/bromo-
H
N
N
Br
N
Br
Br
N
N
SH
S
Br
Br2
NaSH
MeOH
N
NH2
N
NH2
AcOH
N
NH2
NH2
8
5
5
Scheme-I: Synthesis of intermediate 5
NO
NO2
HNO3
17 °C
NaOCl
r.t
NaNO2
. HCl
N
N
HCl
N
OH
OH
9
0-5 °C
10
OH
O
11
NO2
NH2
Na S O
4
2
2
K Cr O
7
2
2
Cl
N
KOH, N2
0 °C
H SO
4
Cl
N
2
8
Cl
N
OH
OH
2 °C
O
1
2
13
6
Scheme-II: Synthesis of compound 6
O
N
Na CO
3
N
2
NH2
SH
N
N
N
C H /DMF
+
Cl
6
6
Br
N
N
o
S
7
0-75 C
Br
O
O
6
5
19
Scheme-III: Synthesis of compound 19

Vol. 29, No. 4 (2017)
Palladium Catalyzed Transformation and Antimicrobial Screening of Novel Angular Azaphenothiazines 747
derivatives under the catalytic influence of palladium(II)
chloride (Heck-Mizoroki protocol) furnished the 6-substituted
derivatives (20a-d), leading to C–C bond elongation (Scheme-
IV). These reactions were successful because compound 19
has olefinic hydrogen at position 6 which is a basic condition
for Heck-Mizoroki coupling reactions to take place.
tested against some Gram-negative and Gram-positive bacteria
are shown in Table-1.
Table-1 shows the inhibition zone diameter (IZD) produced
by each compound against each bacterial organism at 20 mg/
mL concentration. The IZD ranged between 10 and 37 mm in
diameter. Higher the IZD, then sensitivity would be higher.
The compounds showed significant activity against the test
organisms except Pseudomonas aeruginosa which was only
sensitive to compounds 10, 20c and 20d with IZD of 15 mm.
G101 (Staphylococcus) was highly sensitive to all the
compounds with IZD of 35 and 37 mm. Eco 4 and Eco 12
were sensitive to all the compounds with IZD ranging between
15 and 27 mm.
2
1
N
3
X
N
H
11
12
dppb/PdCl2
N
N
4
N
N
N
S
10
9
+
5
Ligand
MeOH
N
7S
6
1
O
Br
Br
O
M
8
K CO3
2
2
1
9
6
4
0oC
h
3
20a-d
M
Minimum inhibitory concentration (MIC) of the compounds
was also determined by the agar-well diffusion method (already
described above) with various concentrations ranging between
2 2
X = I or Br; M = p-NO2, p-OH, = p-NH and = o-CO H
Scheme-IV: Synthesis of compounds 20a-d
2
0 mg/mL and 0.15625 mg/mL. Minimum inhibitory concen-
From microanalysis and spectroscopic data, the compounds
were identified as 9-bromo-6-(4-nitrophenyl)-1,8,11-triaza-
H-benzo[a]phenothiazin-5-one (20a), 9-bromo-6-(4-hydroxy-
phenyl)-1,8,11-triaza-5H-benzo[a]phenothiazin-5-one (20b),
tration (MIC) is the minimal concentration of drug that inhibits
visible growth after overnight incubation [33]. The lowest
concentration of each compound that produced no zone was
regarded as MIC. Hence, the essence of MIC is to determine
the least concentration of drug that can inhibit the growth of
the micro-organism. The graded concentrations (mg/mL) of
the compounds numbered 1-7 are 10, 5, 2.5, 1.25, 0.625,
0.3125 and 0.15625 respectively. Table-2 represents the inhi-
bition zone diameter in millimetre.
Table-3 shows the result of minimum inhibitory concen-
tration (MIC) of the synthesized compounds compared with
those of ampicillin and gentamycin (standard drugs) against
Eco 3, G84 and Baccilus spp.
The MIC for the novel compounds against Eco 3 ranged
between 0.625 and 5 mg/mL. The MIC for the test compounds
against G84 were 1.25 mg/mL and 2.5 mg/mL; for Bacillus
spp, the MIC ranges between 1.25 mg/mL and 0.15625 mg/
mL. Ampicillin and gentamycin are standard antibiotics were
used as positive control because they are standard drugs used
for treating such strains of microorganisms but DMSO which
was the solvent used served as the negative control because
it had no effect against the isolates as it showed no zone of
inhibition.
5
9-bromo-6-(4-anilino)-1,8,11-triaza-5H-benzo[a]phenothiazin-
5-one (20c) and 9-bromo-6-(2-carboxyphenyl)-1,8,11-traiza-
5H-benzo[a]phenothiazin-5-one (20d).
An effective approach of antimicrobial therapy of an infec-
tion is based on the isolation and identification of the infected
organism and determining its sensitivity to antimicrobial drugs.
The microorganisms tested were E. coli (strains used were coded
Eco 3, Eco 4 and Eco 12), Staphylococcus pseudointermedius
and Staphylococcus scuiri (G101 and G84 respectively), Bacillus
spp. and Pseudomonas aeruginosa. The assay was conducted
using agar-well diffusion method [31] in which a 20 mg/mL
concentration of each compound was constituted by dissolving
0.04 g of each compound in 2 mL of dimethyl sulfoxide. A single
colony of each test isolate was suspended in 2 mL of sterile
nutrients broth. Inoculation was done followed by incu-bation
at 37 °C for 24 h after which the plates were observed for
inhibition zones around the wells. The diameters of the zones
were measured with metre rule to the nearest whole millimetre.
The result of the preliminary screening of the novel compounds
TABLE-1
ANTIBACTERIAL SUSCEPTIBILITY TEST (TESTED WITH 20 mg/mL OF EACH COMPOUND)
Pseudomonas
aeruginosa
Compound
G101
G84
Eco 3
Eco 4
Eco 12
Bacillus spp.
1
0
37
37
35
37
35
27
24
21
28
28
29
26
30
27
30
24
18
20
23
22
27
26
15
27
26
34
33
35
36
35
15
0
0
15
15
2
0a
0b
0c
0d
2
2
2
TABLE-2
INHIBITION ZONE DIAMETER (IZD) OF THE COMPOUNDS AT DIFFERENT CONCENTRATIONS
Eco 3
G84
Bacillus spp
Compd.
1
2
3
4
0
15
–
5
–
11
–
–
0
6
–
0
–
–
–
1
2
3
4
0
0
0
12
0
5
–
–
–
0
–
6
–
–
–
–
–
7
–
–
–
–
–
1
2
3
4
5
0
6
–
7
–
1
0
22
26
22
25
22
19
24
10
15
20
15
20
0
15
20
14
20
17
12
15
12
15
14
11
13
12
14
11
32
33
30
32
35
30
30
30
32
32
26
27
28
30
30
15
25
20
30
24
2
2
0a
0b
21
14
25
18
17
0
13
–
20c
0
–
20
13
16
0
20d
18
10

748 Godwin-Nwakwasi et al.
Asian J. Chem.
TABLE-3
5. (a) C.O. Okafor, I.O. Okerulu and S.I. Okeke, Dyes Pigments, 8, 11 (1987);
https://doi.org/10.1016/0143-7208(87)85002-7.
MINIMUM INHIBITORY CONCENTRATION (mg/mL) OF THE
SYNTHESIZED COMPOUNDS AND SOME STANDARD DRUGS
(b) C. Bodea and I. Silberg, Adv. Heterocycl. Chem., 9, 321 (1968);
https://doi.org/10.1016/S0065-2725(08)60375-X.
Compound/Drug
Eco3
2.500
0.625
5.000
5.000
1.250
100.000
6.250
G84
2.50
2.50
2.50
1.25
2.50
2.50
2.50
Bacillus spp.
1.25000
0.15625
0.62500
0.15625
0.31250
1.25000
0.15625
6
.
C.O. Okafor and U.C. Okoro, Dyes Pigments, 16, 149 (1991);
10
https://doi.org/10.1016/0143-7208(91)85006-T.
7. C.O. Okafor, Tetrahedron, 42, 2771 (1986);
https://doi.org/10.1016/S0040-4020(01)90564-2.
8. U.C. Okoro, U.I. Onwuachu and F. Musa, Int. J. Chem., 19, 241 (2009).
9. U.C. Okoro and B.E. Ezema, Int. J. Chem., 16, 115 (2006).
2
2
2
2
0a
0b
0c
0d
1
1
0. U.C. Okoro and A.O. Ijeoma, Int. J. Chem., 16, 245 (2006).
1. U.C. Okoro, M.A. Nnabugwu and C.A. Nwadinigwe, Int. J. Chem., 18, 171
Ampicillin
Gentamycin
(
2008).
1
1
2. U.C. Okoro and A.U. Ofoefule, Int. J. Chem., 18, 109 (2008).
3. C.O. Okafor and U.C. Okoro, Dyes Pigments, 9, 427 (1988);
https://doi.org/10.1016/0143-7208(88)82002-3.
The MIC of the standard drugs ranges between 100 and
.15625 mg/mL. Table-3 suggested that synthesized pheno-
thiazine derivatives were active against Eco 3 even at very low
concentrations; the MIC for compound 20a, being as low as
.625 mg/mL whereas ampicillin has its MIC against Eco 3
0
1
4. (a) U.C. Okoro, E. Onabedje and E.M. Odin, Int. J. Chem., 19, 197 (2009);
(b) A.O. Ijeomah, U.C. Okoro, E.U. Godwin-Nwakwasi and K.F. Chah,
Int. J. Sci. Res., 3, 3 (2014).
0
1
5. B. Gozsy and L. Kato, J. Pharmacol., 129, 231 (1960).
as 100 mg/mL indicating that Eco 3 is highly resistant to
ampicillin. The MIC of gentamycin against Eco 3 is 6.25
mg/mL which is still higher than the MIC values for most of
the synthesized phenothiazine compounds especially
compounds 10, 20a and 20d whose MIC values ranged from
16. T.N. Tozer, L.D. Tuck and C.J. Cymerman, J. Med. Chem., 12, 294 (1969);
https://doi.org/10.1021/jm00302a022.
1
1
1
2
7. G. Szocs and N. Seiler, Acta Phytopathol. Entomol. Hung., 37, 365 (2002);
https://doi.org/10.1556/APhyt.37.2002.4.7.
8. N.L. Agrawal and R.L. Mital, J. Chem. Eng. Data, 20, 199 (1975);
https://doi.org/10.1021/je60065a027.
9. J.A. vanAllan, G.A. Reynolds and D.P. Maier, J. Org. Chem., 34, 1691 (1969);
https://doi.org/10.1021/jo01258a035.
0. B. Zinger and P. Shier, Sens. Actuators B, 56, 206 (1999);
https://doi.org/10.1016/S0925-4005(99)00020-9.
0
.625 to 2.5 mg/mL.
The MIC value of gentamycin against Bacillus spp. is
.1562 mg/mL and this is the same for compounds 20a and
0c. These results show that the synthesized phenothiazine
0
2
21. S. Messaoudi, D. Audisio, J.-D. Brion and M. Alami, Tetrahedron, 63,
0202 (2007);
1
compounds are highly biologically active.
https://doi.org/10.1016/j.tet.2007.07.085.
22. B.J. Margolis, K.A. Long, D.L.T. Laird, J.C. Ruble and S.R. Pulley, J.
Org. Chem., 72, 2232 (2007);
Conclusion
It is shown that base mediated coupling reactions under
anhydrous conditions and using benzene/DMF as solvent offer
excellent routes to the synthesis of angular (non-linear) pheno-
thiazines. Also, utility of Heck-Mizoroki palladium catalyzed
protocol in the synthesis of derivatives of these heterocycles
proved a reliable and facile route to the formation of C–C bond
as it requires shorter time than the mineral acid catalyzed
reactions. The study has opened up a new route for possible
synthesis of other isomeric non-linear polycyclic triazapheno-
thiazine compounds. These novel compounds have also shown
high level of biological activity which should not be ignored.
https://doi.org/10.1021/jo062168u.
2
2
2
6. C.O. Okafor, Dyes Pigments, 6, 405 (1985);
https://doi.org/10.1016/0143-7208(85)80022-X.
4. V. Petrow and B.J. Sturgeon, J. Chem. Soc., 570 (1954);
https://doi.org/10.1039/jr9540000570.
3. Y.T. Pratt and N.L. Drake, J. Am. Chem. Soc., 82, 1155 (1960);
https://doi.org/10.1021/ja01490a035.
25. M.A. Nnabugwu, Ph.D. Thesis, University of Nigeria, Nsukka, Nigeria,
pp. 110-128 (2009).
7. C.K. Ryu, 6,7-Disubstituted-5,8-quinolinedione Derivatives as an
Antifungal Agent, Patent WO2001012605 A1 (2001).
2
2
8. C.W. Schellhammer and S. Petersen, Ann, 624, 108 (1959); Chem. Abstr.,
53, 15211e (1960).
9. B.H. Babu and N.V.S. Rao, Chem. Abstr., 69, 10339f (1968).
0. S. Mohanty, D. Suresh, M.S. Balakrishna and J.T. Mague, Tetrahedron,
2
3
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