syntheses of benzothiazinophenothiazine derivatives …
TRANSCRIPT
i
SYNTHESES OF BENZOTHIAZINOPHENOTHIAZINE DERIVATIVES AND EVALUATION OF THEIR
ANTIMICROBIAL ACTIVITIES
BY
ADEKOLA, OJO EMMANUEL PG/M.SC./12/62239
DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY FACULTY OF PHYSICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA
SEPTEMBER, 2014.
i
TITLE PAGE
SYNTHESES OF BENZOTHIAZINOPHENOTHIAZINE DERIVATIVES AND EVALUATION OF THEIR ANTIMICROBIAL ACTIVITIES
ADEKOLA, OJO EMMANUEL REG. NO: PG/M.SC/12/62239
A PROJECT SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF THE MASTER OF SCIENCE DEGREE (M.SC) IN ORGANIC CHEMISTRY IN THE DEPARTMENT OF PURE AND INDUSTRIAL CHEMISTRY,
FACULTY OF PHYSICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA
SEPTEMBER, 2014.
ii
CERTIFICATION Adekola, Ojo Emmanuel, a postgraduate student in the Department of Pure and Industrial Chemistry with registration number PG/MSc/12/62239, has satisfactorily completed the requirements for research work for the degree of Master of Science in Pure and Industrial Chemistry. The work embodied in this thesis is original and has not been submitted in part or full for any other diploma or degree in this or any other university.
__________________ _________________ Dr. B.E. Ezema Dr. E.A. Ochonogor Project Supervisor (Head of Department) Date…………… Date………………..
_____________________ External Examiner
Date ……………………..
iii
DEDICATION
This project work is dedicated to Almighty God for His wisdom and inspiration which
make this work a success. May His name be praised forever.
iv
ACKNOWLEDGEMENT
I express my profound gratitude to the Almighty God, the giver of all things for his
protection, guidance and blessing throughout the research period.
I acknowledged the supervision of Dr. B.E. Ezema who enthusiastically gave me his
precious time and made invaluable encouragement, corrections, suggestions and penetrating
contributions as well as contructive criticism at every stage of this work. All these gave me a
sense of responsibility, reliability and close attention to details.
I am equally indebted to the Head of Department, Dr. E.A. Ochonogor and to all my
lecturers in the Department of Pure and Industrial Chemistry, University of Nigeria, Nsukka.
I extend my profound gratitude and love to my beloved family members, my father Chief
Ogedengbe Adekola, my beloved, God sent and caring mother Mrs. Funmilayo Adekola, my
beloved Aunty, brothers and sisters, whom in the presence of nothing, showed great love to me
financially.
I am also extremely grateful and thankful to Prof. Dauda Oladepo for his love, fatherly
advise and amiable support; morally, financially throughout my stay in Nsukka.
Finally, I extend my sincere appreciation to my colleagues in the department of Pure and
Industrial Chemistry, University of Nigeria, Nsukka, especially Ayogu Jude, Ike Christian,
Abuekwu Priscillia, Agwogie Bright, Ugwu David, Ugwuona Florence, Ezugwu James, Ugwuja
Daniel and so on. I thank them for their encouragement, words of advice and constructive
criticism during this period.
May God bless and reward you all abundantly. Amen.
Adekola O.E.
v
ABSTRACT
The syntheses of benzothiazinophenothiazine derivatives from simple heterocyclic compounds as
precursors is described. Condensation of 2-aminothiophenol with 2,3-dichloro-1,4-naphthoquinone in
an alkaline medium furnished a good yield of the intermediate, 6-chloro-5H-benzo[a]phenothiazin-5-
one. Further condensation of the intermediate with 2,4-diamino-6-hydroxypyrimidine-5-thiol
obtained by alkaline hydrolysis of 2,4-diamino-6-hydroxy-5-thiacyanatopyrimidine gave the
benzothiazinophenothiazine ring system. On the other hand, using a facile acid-catalyzed method, the
synthesis of some benzothiazinophenothiazine ring systems were achieved with improved yield and
lesser reaction time. Structures of the compounds were characterized using UV/Visible
spectrophotometry, fourier transform infra red, 1HNMR and 13CNMR spectroscopies and elemental
analysis. The antimicrobial properties of the synthesized compounds were determined against
Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli,
Klebsiella pneumoniae, Candida albican and Aspergillus niger using agar diffusion technique.
Results showed that the complex derivatives were significantly active against the microorganisms.
vi
LIST OF FIGURES
Fig 1: UV-Vis spectrum of 6-chloro-5H-benzo[a]phenothiazin-5-one…………………………..72
Fig 2: UV-Vis spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine…………………………………………………………………………73
Fig 3: UV-Vis spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-
tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine …………………………………….74
Fig 4: UV-Vis spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine. ………………………………………………………………………75
Fig 5: UV-Vis spectrum of 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-
tetrazatriphenodithiazine……………………………………………………………….76
Fig 6: UV-Vis spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-
triazatriphenodithiazine ……………………………………………………………….77
Fig 7: IR spectrum of 6-chloro-5H-benzo[a]phenothiazin-5-one…………………………… 78
Fig 8: IR spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]
phenothiazine. …………………………………………………………………………..79
Fig 9: IR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-
tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine ……………………………….80
Fig 10: IR spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]
phenothiazine. ………………………………………………………………………..81
Fig 11: IR spectrum of 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-
tetrazatriphenodithiazine… ……………………………………………………….….82
Fig 12: IR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,
8-triazatriphenodithiazine …………………………………………………………83
vii
Fig 13: 1H-NMR spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine. …………………………………………………………………… .84
Fig 14: 13C-NMR spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine………………………………………………………………………85
Fig 15: 1H-NMR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-
tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine …………………………..86
Fig 16: 13C-NMR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-
tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine …………………………….87
Fig 17: 1H-NMR spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]
phenothiazine.… ………………………………………………………………….88
Fig 18: 13C-NMR spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]
phenothiazine.……………………………………………………..……...............89
Fig 19:1H-NMR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-
triazatriphenodithiazine ……………………………………………………………...90
Fig 20:13C-NMR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-
triazatriphenodithiazine ………………………………………………………….……91
viii
LIST OF TABLES
Table 1: Results of Antimicrobial Sensitivity Test of the Synthesized Compounds………...62
Table 2: Results of Inhibition Zones Diameter of the Compounds…………………………..63
Table3: Results of Minimum Inhibitory Concentration of the Synthesized Compounds…….64
ix
LIST OF ABBREVIATIONS
DMF-Dimethylformimide
DMSO-Dimethyl sulfoxide
CPFX-Ciprofloxacin
IZD – Inhibition Zone Diameter
KTCN-Ketoconazole
MIC-Minimum Inhibitory Concentration
x
TABLE OF CONTENTS
Title page …………………………………………………………………………….i
Certification …………………………………………….………………………….ii
Dedication ……………………………………………………………………….….iii.
Acknowledgement …………………………………………………………….……iv
Abstract ……………………………………………………………………………....v
List of Figures …………………………………………………………………….…vi
List of Tables ……………………………………………………………………….vii
Abbreviations ………………………………………………………………….……vii
Table of Contents …………………………………….……………………………...viii
1.0 INTRODUCTION
1.1 Background of study …………………………………………………………....1
1.2 Statement of problem ……………………………………………………….…..7
1.3 Aims and objectives of study ……………………………………………………7
1.4 Justification of study …………………………………………………………......8
2.0 LITERATURE REVIEW
2.1 Linear Phenothiazines ………………………………………………………… 9
2.2 Aza-Analogues of Linear Phenothiazines…………………………………….. 15
2.3 Angular Phenothiazines……………………………………………………….. 18
2.4 Aza-Analogues of Angular Phenothiazines …………………………………… 22
2.5 Branched Benzothiazinophenothiazine Ring systems ………………………… 27
3.0 EXPERIMENTAL
xi
3.1 2,4-Diamino-6-hydroxypyrimidine-5-thiol…………………………………….. 37
3.2 6-chloro-5H-benzo[a]phenothiazin-5-one ……………………………………………38
3.3 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine…….. 38
3.4 4-amino-2-ethylthio-6-hydroxypyrimidine-5-thiol ……………………………….......39
3.5 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-tetraazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine …………………………………………………………………... 40
3.6 3-amino-6-methoxypyridine-2-thiol ………………………………………………. 41
3.7 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine ……. 47
3.8 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-tetrazatriphenodithiazine
…………………………………………………………………………………….. 42
3.9 4-amino-2-methyl-6-hydroxypyrimidine-5-thiol ………………………………….. 42
3.10 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-triazatriphenodithiazine
................................................................................................................................. 43
3.11 Evaluation of the Synthesized Phenothiazine Derivatives for Antimicrobial
Activities…………………………………………………………………………… 44
3.11.1 Sensitivity Test of the Compounds .……………………………………………. 44
3.11.2 Determination of Minimum Inhibitory Concentration (MIC) of the
Synthesized Derivatives………………………………………………………….... 45
4.0 RESULTS AND DISCUSSION
4.1 6-chloro-5H-benzo[a]phenothiazin-5-one …………………………………………….. 46
4.2 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
……………………………………………………………………………………… 48
xii
4.3 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-tetraazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine ……………………………………………………………………... 51
4.4 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine…………………………………………………………………… 54
4.5 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-tetrazatriphenodithiazine…
……………………………………………………………………………………. 57
4.6 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-triazatriphenodithiazine
…………………………………………………………………………………….. 59
4.7 Results of Antimicrobial Sensitivity Test of the Synthesized Compounds………. 62
4.8 Results of Inhibition Zones Diameter of the Compound……………………………. 63
4.9 Results of Minimum Inhibitory Concentration of the Synthesized Compounds…… 64
4.10 Conclusion…………………………………………………………………………... 65
REFERENCES ……………………………………………………………………………..66
1
CHAPTER ONE
1.0 INTRODUCTION
The chemistry of phenothiazine (1) and its derivatives has been of interest for over a century
due to their wide range of applications in drug, agriculture, textile, paint and other related industries.
Phenothiazine and its derivatives constitute a pharmaceutically important class of heterocycles with a
broad spectrum of pharmacological activity; they are useful in medicine as anticonvulsants,1
antitumour agents,2,3 antituberculosis,4 tranquilizers and antimalaria agents5. It also has anthelmintic
activity, 6, 7, 8 to mention a few.
N
S
H
(1)
Notable among the early phenothiazine drugs are Chlorpromazine (2) and promethazine (3) which are
broad spectrum tranquilizers with diffuse antipsychotic properties9.
N
S
NCH3CH3
Cl N
S
N
CH3
CH3CH3
(2) (3)
2
These classes of drugs were the largest and most widely investigated class of neuroleptic agents9.
Chlorpromazine, the first commercially produced phenothiazine for the management of psychosis,
was also one of the first commercially produced in the phenothiazine series shown to have anti-
tuberculosis properties both in vitro and in vivo.10,11 Promethazine and chlorpromazine, clinically
useful in the chemotherapy of mental and emotional disturbances has further stimulated an
investigation into other phenothiazine derivatives for possible central nervous system
(CNS)depressant activity.12,13
In the petroleum industry, these compounds are useful as antioxidants in gasoline, petroleum
lubricants and stabilizers.14-18 They are used as vat dyes and pigments18-22 in textile and paint
industries and in agriculture as insecticides and nematodicides.23,24
Since the discovery of the parent ring (1), a lot of structural modifications have been carried
out to enhance their pharmacological and biological activities, minimize undesirable effects and open
new areas of applications.
3
Such molecular modifications had yielded derivatives such as (4), (5),25 (6), (7), (8) and (9).26
N
S
HN
S
H
N
S
H
N
S
H
N
S
H
N
S
H
(4)(5)
(6)(7) (8)
(9)
Compounds (4), (6), (7) and (9) are described as angular phenothiazines because of the non-linear
arrangement of the ring systems27. They possess fused rings at positions a, c, h and j bonds of the
phenothiazine.
There are also systems in which two benzene rings are attached to two different positions in the
parent compound. Such structures include dibenzo[a,h]phenothiazine28 (10),
dibenzo[c,h]phenothiazine29 (11) and dibenzo[a,i]phenothiazine (12).
4
N
S
H
N
S
H
(10)(11) (12)
N
S
H
Branched phenothiazine compounds of the types (13) and (14)30 have been reported.
(13)(14)
N
S N
S
N
S
H
With regard to the aza analogues of angular phenothiazine compounds, there have been
reports on the monoaza, diaza and the triaza derivatives such as (15), (16)31, (17) and (18)
respectively
5
(15) (16)
N
S
HN
S
H
N
S
HN
S
H
N 2N
2N
N
N
N
(17)(18)
On the search for more aza analogues of angular phenothiazine ring system, the first aza
analogues of pyrrolo[3,4-a][1,4]benzothiazino[3,2-c]phenothiazine (19) was reported by Japanese
workers32.
N
S N
S
N
H
(19)
Okafor and Okoro33 also reported the synthesis of the first three-branched diazaphenothiazine
dyes of the type (20).
6
N
S N
S
N
N(20)
The diaza (21) and triaza (22) three-branched benzoxazinophenothiazine ring systems were
reported by Okafor34 and also reported was the tetraaza analogue (23) of benzothiazinophenothiazine
ring system by Ezema.35
N
O N
S
N
O N
S
N
S N
S
N
N
N
2N
2N
2N
(21) (22)
(23)
7
Other structures synthesized are the aza and non-aza analogues of dibenzotriphenodithiazine ring
systems of the types (24)36 and (25).37
N
S
N N
S
H H
(24)
N
S
S
N
(25)
1.2 Statement of the Problem
Owing to the wide range of applications of phenothiazines derivatives with highly improved
pharmacological and biological activities, several papers describing the successful synthesis of these
derivatives had been reported especially on the angular derivatives including the non-aza and the
congeneric aza analogues. However, there are still limited literatures on the complex derivatives of
this phenothiazine ring system and, hence, modification of the existing ones is necessary.
The past work done was based on their dye and pigment properties. Not much is known of
antimicrobial properties of these complex phenothiazine derivatives.
1.3 Aims and Objectives of Study
The aims and objectives of this study were to:
i. Synthesize complex aza derivatives of benzothiazinophenothiazine.
ii. Characterize the synthesized compounds by spectral analysis.
iii. Undertake antimicrobial screening on the complex derivatives.
8
1.4 Justification of the Study
The wide pharmaceutical applications of phenothiazines and the need to synthesize more
derivatives with better and more desirable pharmacological properties led to the synthesis and
antimicrobial screening of the derivative undertaken in the present work.
9
CHAPTER TWO
LITERATURE REVIEW
2.0 LINEAR PHENOTHIAZINE
Linear phenothiazines may be defined as phenothiazine derivatives in which variation in the
parent phenothiazine ring system (1) does not alter the linear structure of the ring system.
Lauth’s violet, produced in 1876, was the first reported phenothiazine dye. It was prepared by
the oxidation of benzene-1,4-diamine with iron III chloride in the presence of sulphur in acid
solution38. Methylene Blue, discovered by Caro in 1876, was prepared similarly from 4-amino–N,N-
dimethylaniline38. Better yields were obtained using Bernthsen’s thiosulphate method39 in which 4-
amino–N,N-dimethylaniline (26) was oxidized with sodium dichromate in the presence of sodium
thiosulphate in sulphuric acid to give the arenethio-sulphuric acid derivative (27). Oxidation of the
intermediate (27) in the presence of N,N-dimethylaniline gives the indaminethiosulphuric acid
derivative (28) which is subsequently oxidized with air in hot water to give Methylene Blue (29).
NH2
Me2N
NH2
Me2N SSO3H
Na2S2O3, Na2Cr2O7
H2SO4, O OC
PhNMe2
Na2Cr2O7(26)
(27)
Me2N(28)SSO3
-
N
NMe2
H
(+)O2, H2O
Heat
N
S+Me2N NMe2
Cl-
(29)
10
Craig et al40 reported the synthesis of a number of substituted phenothiazines which was
achieved by heating substituted diphenylamine (30) with powdered sulphur and iodine in 1,2-
dichlorobenzene for 1-2½ h.
S
R2R1 N
H
S, I2 Heat
R1=H, R2=Cl, F, Me, OMe(30)
(31)
R2R1 N
H
The thionation of oligomeric diphenylamines (32) and (34) with sulphur in 1,2-
dichlorobenzene in the presence of iodine to afford the corresponding phenothiazines (33) and (35) in
moderate yields was reported by Adreani and coworkers41.
11
.
S
SN
N
H
H
S
S
S
N N
H H
(35)
(34)
(33)(32)
S, I2
S, I2
1,2-Cl2C6H4
1,2-Cl2C6H4
N
N
H
H
N
N
N
H
H
H
Fries et al.42 successfully synthesized 7,14-dihydrotriphenodithiazine-6,13-dione (38) in 45%
yield. This was obtained by heating 2,5-dichloro-3,6-bis(phenylamino)-1,4-benzoquinone (36) and
sodium sulphide in ethanol for 30 min and subsequent air oxidation. The same product was obtained
by the thermolysis of suitably substituted 2,5-bis(arylamino)-3,6-dichloro-1,4-benzoquinones as
shown in the case of (38) which was obtained from 2,5-dichloro-3,6-bis[2-(methylsulphonyl)aniline]-
1,4-benzoquinone (37) as a blue powder in 77% yield43.
12
N
N
Cl
Cl
O
OH
H
N
S
S
N
O
O
H
H
1. Na2S, EtOHHeat, 0.5h2. O2
(36)(37)
N
N
Cl
Cl
O
OSMe
SMe
H
H
N
S
S
N
O
O
H
H(36)(37)
-MeCl
Heat
The fusion of aniline (39), hydroquinone (40) and sulphur gives phenothiazin-3-ol (41) in an
unspecified yield44.
NH2
+OH
OH
NH
S OH
S, 1800C
(39) (40)(41)
Terdic 45 reported the synthesis of 3H-phenothiazin-3-one by the reaction of 2-
aminothiophenol (42) with 2-methyl-1,4-benzoquinone (43) in 95% EtOH, to afford a mixture of 1-
methyl-3H-phenothiazin-3-one (44) in 13% yield and 2-methyl-3H-phenothiazine-3-one (45) in 18%
yield. As reported by the authors, the reaction of 2-aminothiophenol with 1,4-benzoquinones affords
3H-phenothiazine-3-ones in relatively low yields. Better yields are obtained using 1,4-
naphthoquinone.
13
NH2
SH+
O
CH3
O
N
S O
CH3
(42)(44)
+
N
S O
CH3
(43)
(45)
95% EtOH
Heat 200C, 2.5h
Several derivatives of 3H-phenothiazin-3-ones (47) were obtained in unspecified yields by
reacting 2-aminothiophenol with the corresponding 1,4-benzoquinones46.
NH2
SHR2
R3+
O
O
R1
95%, EtOHHeat, 1h
(42)(46)
N
S O
R1
R2
R3(47)
where R1,R2,R3 =H, CH3
The reaction of the zinc salts of 2-aminothiophenol (48) with dihalo-1,4-quinones (49) in
ethanol affords 3H-phenothiazin-3-one (50)47.
NH2
S- 2 Zn2+X
(48)
(49)
EtOH, Heat+X
O
O
N
S O
x
X= Br
(50)
14
Mital and Jain48 reported that if two moles of the zinc thiolate are reacted with one mole of
tetrahalo-1,4-benzoquinones (51) in acetic acid for 4-5 h, 6,13-dihalotriphenodithiazines (52) are
obtained.
NH2
R S- 2 Zn
2++X
X X
X
O
O
CH3COOH
Reflux, 4-5 h
N
S
S
NR
R
X
X(48)
(51)(52)
R = H, Cl X = Cl, Br
2
Nishi and coworkers49 also synthesized 6,13-bis(arylamino)triphenodithiazines (54) by
reacting one mole of 2,5-bis(arylamino)-3,6-dichloro-benzoquinones (53) with two equivalents of the
zinc salts of 2-aminothiophenol in 2-methoxyethanol in the presence of sodium ethoxide.
NH2
R1 S-/2Zn2+
(48)
(53)
+
O
O
Cl NHR2
ClR2NH
EtONa,MeO(CH2)2OH
Reflux, 6-8h
NHR2
R1NHR2
N
S
S
N
(54)
R1
According to the same authors49, if two equivalents of the zinc salts of 2-aminothiophenols are
refluxed with 2,5-dichloro-3,6-dianilino)-1,4-benzoquinone in dimethylformamide, moderate to good
yields of 7,14-dihydrotriphenodithiazine-6,13 diones (55) are obtained.
15
NH2
R1 S-/2Zn2+PhHN(48)
(53)
+2Cl NHPh
O
OCl
DMF, Reflux
12h
R2
N
S
S
NO
OH
HR1
R1
(55)
R2
R2
R1,= H, Cl, Br, CH3, OMe, R2= H, Cl, Me
2.2 AZA ANALOGUES OF LINEAR PHENOTHIAZINES
Apart from the non-aza derivatives of linear phenothiazines, there have also been reports on
the aza derivatives of linear phenothiazine ring systems.
In 1945, Petrow and Rewald50 reported the synthesis of 3-azaphenothiazine (42) with 3-nitro-
4-chloropyridine (56) with excess sodium ethoxide in the presence of sodium acetate to give (57) in
62% yield.
NH2
SH+
O2N
N
Cl
(42)(56)
NaOAc
N
SN
H
(57)
So far, there have been reported synthesis of the other isomeric mono-aza analogues of the
parent phenothiazine ring system51. In a systematic attempt to prepare the isomeric diaza analogue of
16
compound (1), Roth and Hitchings52 prepared compound (59). This was however obtained from the
reaction of 5-bromo-2,4-dihydroxypyrimidine (58) and 2-aminothiophenol(42) in dilute hydrochloric
acid to give the desired product as 2-hydroxy-1,3-diazaphenothiazine (59).
N
NBr
OHOH+
NH2
SH(58) (42)
N
SN
N OH
H
(59)
The synthesis of 1,4-diazaphenothiazine (61)53 was accomplished by condensing an alkaline
mixture of 2-aminothiophenol (42) with 2,3-dichloropyrazine (60).
NH2
SH+
N
N
Cl
Cl
R
R(60)
(42)
N
S
N
N
R
R
H
(61)
All the earlier reports on the aza analogues of phenothiazine were concerned with the
chemistry and biological properties of only the monoaza and diaza phenothiazines. There was
however no report on any of the possible twenty isomeric triazaphenothiazine system until 1973
when Okafor 54 reported the successful synthesis of the first compound in this series. The compound,
1,3,6-triazaphenothiazine (64) was obtained in varying yields from 11% to 95% by the acid-catalyzed
condensation of 3-aminopyridin-2[1H]-thiones (62) with 4,5-dihalogenopyrimidines (63). Compound
(64) has appreciable CNS-depressant activities when tested.
N S
NH2
R1
H
+ N
NCl
Br
R2
R3
N
N
SN
N
R1
R2
R3
H
(62)(63) (64)
17
Derivatives of 1,3,8-triazaphenothiazine (68), a new heterocyclic ring were reported by
Okoro55. The new system was synthesized by the acid-catalyzed condensation of 3-
aminopyridine 4[1H] thione (65) with 5,6-dihalogenopyrimidines (66).
N
S
NH2
H
+ N
N
Br
Cl
R2
R1
NHN
N
N
R2
R1
S
H
NN
SN
N R1
R2
H
(65) (66) (67)
(68)
R1 = NH2, OCH3.
R2 = Cl, OCH3, NH2
H3O+
Heat
The same author55 described the synthesis of 2,4,9-triamino-1,3,6,8-tetraza-phenothiazine (71), a
new tetraza phenothiazine ring system. Under similar reaction conditions above, 4,5-diamino
pyrimidine-6-thione (69) reacted with (70) to give compound (71) in good yield.
N
N
NH2
NH2
S
H
+ N
N
NH2
NH2
Br
Cl
H3O+
HeatN
NN
SN
N
NH2
NH2
NH2
H
(69)(70) (71)
In the search for new pharmacoactive phenothiazine compounds, the synthesis of 1,4,6,8-
tetrazabenzo[b]phenothiazine ring system was reported by Okafor and coworkers56. This was
18
accomplished by the reaction of 4,5-diaminopyrimidine-6[1H]thione (69) with 2,7,6-
trichloroquinoxaline (72) in N,N-dimethylformamide in the presence of a near stoichiometric amount
of sodium hydroxide to give a high yield of a single product identified as 9-amino,12-chloro-1,4,6,8-
tetrazabenzo[b]phenothiazine (73)
N
N
NH2
NH2
S
H
+
(69)
N
N
Cl
Cl
Cl
N
NN
S
N
N
NH2
Cl
H
(72)(73)
DMFNaOH
Okafor57 also reported the synthesis of 1,4,7,9-tetraazabenzo[b]phenothiazine (76) by the reaction of
an equimolar mixture of 4,6-diaminopyrimidine-5-thiol (74) and 2,3-dichloroquinoxaline (75) in
propylene glycol in the presence of potassium hydroxide solution to give a yellowish-green
microcrystalline solid melting above 300 0C
+
(74)
N
NR3
Cl
Cl
NH
S
N
N
R3N
N
R1
R2(75) (76)
N
N
NH2
SH
R1
R2
R1=H, R2=NH2, R3=H
KOH
2.3 ANGULAR PHENOTHIAZINES
Angular or non-linear phenothiazine compounds are the derivatives of the parent
phenothiazine ring system obtained by the fusion of benzo group or dibenzo groups onto one of the
sides of the phenothiazine ring (1).
Kym58 in 1890 reported the first synthetic angular phenothiazine ring system,
benzo[a]phenothiazine (78), which was synthesized in 40% yield by heating 1-anilinonaphthalene
19
(77) with powdered sulphur at an elevated temperature of 120 0C for 8 h. It was obtained as yellow
solid of melting point, 134 – 136 0C
Shirley and coworkers59 modified Kym’s method by introducing a catalytic amount of iodine
powder and heated to 180 – 185 0C for 25 min. and obtained a higher percentage yield of 70% of the
same compound (78)
N
HN
S
H
Heat
(77) (78)
S/I2
In a related development, thionation of di-2-naphthylamine (79) in trichlorobenzene in an
atmosphere of carbondioxide produced 7H-dibenzo[c,h]phenothiazine (80)60.
N
H
N
S
H
S/I2, Cl3C6H3
Reflux, 8h
(79)(80)
Chemical oxidation of 7H-dibenzo[c,h]phenothiazine (80) with mercury (II) oxide in xylene
affords a 50% yield of the stable radical 7H-dibenzo[c,h]phenothiazin-7-yl (81)60.
N
S
.
(80)(81)
HgO, Xylene
N
S
H
20
Akatsuka, et al61 successfully synthesized 5H-benzo[a]phenothiazin-5-one (83) by reacting
1,4-naphthoquinones (82) with 2-aminothiophenol in ethanol and 15% hydrochloric acid.
NH2
SH+
O
O
N
S O
EtOH
15% HCl
(42)
(82)(83)
The same authors61 described the reaction of 2,3-dichloro-1,4-naphthoquinone (84) with 2-
aminothiophenol in ethanol in the presence of 15% hydrochloric acid to afford 6-chloro-5H-
benzo[a]phenothiazin-5-one (85).
NH2
SH+
O
O
Cl
Cl
N
S O
Cl
15% HCl
(42)
(84)
C2H5OH
(85)
If two moles of 2-aminothiophenol are reacted with 2,3-dichloro-1,4-naphthoquinones (84),
high yields of benzo[a][1,4]benzothiazino[3,2-c]phenothiazines (86) are obtained61,62
21
NH2
SH+
O
O
Cl
Cl
R N
S
R
N
S
15% HCl
(42)
(84)
C2H5OH
(86)
2
R= NH2, H
As a result of the search for more derivatives of angular phenothiazine compounds, Agarwal
and Atal63 reported the synthesis of substituted 6-arylamino-5H-benzo[a]phenothiazin-5-ones (89) in
high yield. They achieved this by reacting substituted zinc salts of 2-aminothiophenol (87) with
substituted 2-arylamino-3-chloro-1,4-naphthoquinones (88) in pyridine for 2 h.
+O
O
R3
NH
R4
N
S O
R3
NH
R4
R2
R1
NH2
S- 2Zn2+R1
R2
C5H5N
Heat, 2h
(87)
(88)
(89)
Heating 2-aminothiophenol with 3-hydroxyphenalen-1-one (90) in dimethyl sulphoxide for 40
min produced a 47% yield of naptho[1,8-ab]phenothiazin-7[13H]-one (91)64.
22
NH2
SH
NH
S
O
+OH
O
DMSO, 1400C
40mins
(42)
(90)(91)
In a similar manner, [1]benzopyrano[3,4-b][1,4]benzothiazin-6[12H]-ones (93) (R=H, OH)
are obtained from 2-aminothiophenol and either 4-hydroxycoumarin (92, R=H) or 4,7-
dihydroxycoumarin (92, R=OH)65.
NH2
SH
N
SO
R
O
H
+ DMSO, 1400C
(42)
(92)(93)
O
OH
O
R
R=H, OH
35min
2.4 AZA-ANALOGUES OF ANGULAR PHENOTHIAZINES
The aza analogues of non-linear phenothiazine compounds which have one or more annular
nitrogen atoms have been found to possess more marked biological and pharmacological activities
due to the presence of the basic nitrogen atoms which donate electrons to the biological receptor by
charge electron transfer mechanism. As a result of these improved activities, current search for more
derivatives of angular phenothiazine compounds has been shifted towards the synthesis of these aza
analogues.
Gritsenko and coworkers66 reported the successful synthesis of the first aza angular
phenothiazine compound, 2,4-dimethylpyrido[2,3-a]phenothiazine (98). These workers obtained the
23
compound by reacting 1-aminophenothiazine (94) with pentane-2,4-dione (95) in xylene to give an
intermediate (96) whose tautomeric form (97) cyclised in the presence of poly phosphoric acid(PPA)
to give (98).
N
S
NH2H
+CH3 CH3
O O
N
S
N
CH3
CH3
O
H
N
S
N
CH3
CH3
OH
H
N
S
N
CH3
CH3
H
(94)
(95) (96)
(97)(98)
Xylene
PPA
As a continuation of search the for new pharmaco-active phenothiazine compounds and
those with improved dyeing properties, Okafor67 reported the successful synthesis of the first
monoaza and diazaphenothiazine ring systems (99) and (101). These were obtained by refluxing an
equimolar mixture of 3-aminopyridine-2[1H]thione (62) and 2,3-dichloro-1,4-naphthoquinone (84) in
chloroform in the presence of anhydrous sodium carbonate to give compound (99) identified as 6-
chlorobenzo[a]phenothiazin-5-one
NH
NH2
SR+
Cl
Cl
O
O
N
S OR
ClR = H, Cl, OMe
Na2CO3
CHCl3, Heat
(84)(62) (99)
24
Conversion of 4,5-diaminopyrimidin-6[1H]-one (100) to the corresponding thione (66) by
refluxing with P2S5 in dry pyridine followed by treatment with a stoichiometric amount of 2,3-
dichloro-1,4-naphthoquinone (84) in the presence of anhydrous sodium carbonate gave 98% yield of
11-amino-6-chloro-8,10-diazabenzo[a]phenothiazine-5-one (101).
NH
N
NH2
NH2
O NH
N
S
NH2
NH2
O
O
Cl
Cl
N
NN
S O
Cl
NH2
(100)
(84)
(101)
P2S5
pyridine
Na2CO3 Heat+
In a related development, Okafor and Okoro68 reported the synthesis of 9-bromo-6-chloro-
8,11,12-triazabenzo[a]anthracen-5-one (103), the first angular 1,4-diazaphenothiazine ring system.
Compound (103) was obtained by reacting 2-amino-5-bromopyrazine-3[4]-thione (102) with 2,3-
dichloro-1,4-naphthoquinone (84) in chloroform in the presence of anhydrous sodium carbonate.
N
N
NH2
SHBr+
O
O
Cl
Cl
N
N
N
SBr
Cl
O
(102) (103)(84)
CHCl3Na2CO3
Also reported was the successful synthesis of 1,8-diaza-5H-benzo[a]phenothiazin-5-one
(105)69 obtained by condensing 3-aminopyridine-2[1H]thione (62) with 7-chloro-5,8-dioxoquinoline
(104) in benzene/DMF in the presence of anhydrous sodium carbonate
25
+
(62) (105)(104)
Na2CO3N
S
N
ONN
NH2
SH NCl
O
O
C6H6/DMF
In a similar development, Okoro and Ijeoma70 reported a new aza non-linear polycyclic
phenothiazine, 10-methyl-1,11-diazabenzol[a]phenothiazin-5-one (107), obtained by condensing 2-
amino-6-methylpyridine-3-thiol (106) with 7-chloro-5,8-dioxoquinoline (104) in benzene/DMF as the
solvent and in the presence of anhydrous sodium carbonate.
+
(106) (107)(104)
NCH3 NH2
SH
NCl
O
O
N
S
N
O
NCH3
Na2CO3
In related development, Onoabedje71 synthesized another aza angular phenothiazine
compound, 9,11-diamino-6-chloro-8,10-diaza-5H-benzo[a]phenothiazin-5-one (109), by the
condensation of 2,4,5-triaminopyrimidine-6-thiol (108) with 2,3-dichloro-1,4-naphthoquinone (84)
in anhydrous basic medium using benzene/DMF as the solvent.
N
N
NH2
NH2
NH2 SH
+
O
O
Cl
Cl N
NN
S
NH2
NH2
Cl
O
(84)(108)
C6H6/DMFNa2CO3
(109)
26
It is important to note that these angular phenothiaziones can be reduced with sodium
hydrosulphite to give the corresponding angular phenothiazin-5-ols which cannot be isolated in a pure
form as a result of their being unstable, these derivatives quickly revert to the oxidized forms on
exposure to atmospheric oxygen71.
N
NN
S
Cl
ONH2
NH2
N
NN
S
Cl
NH2
NH2
OH
H
(109) (110)
Na2S2O4
Air
The synthesis of 6,10-dichloro-17-azadibenzo[a,n]triphenodithiazine-5-11-dione (112) has
been reported by Okoro72. The compound was obtained by treating potassium thiolate (111) with 2
mole equivalents of 2,3-dichloro-1,4-naphthoquinone (84), potassium hydroxide and sodium sulphite
in 95% dioxane.
N
S-K+
NH2
S-K+
NH2
+
O
O
Cl
Cl
N
S
N N
SO O
Cl Cl
95%
Dioxane
Na2SO3
KOH
(111)
(112)
(84)
2
27
2.5 BRANCHED BENZOTHIAZINOPHENOTHIAZINE RING SYSTEMS
The various structural modifications on the parent phenothiazine compounds have resulted in
the successful synthesis of three-branched phenothiazine derivatives which are useful vat dyes and
pigments.
Fries and Ochwat73 reported the first synthesis of three-branched phenothiazine in 1923, the
derivative, benzo[a][1,4]benzothiazino[3,2-c]phenothiazine(114) by reductive cyclisation of 2,3-bis(-
2-nitrophenylthio)-1,4-naphthoquinone(113) in acetic acid and tin(II)chloride.
S
S
O
O
NO2
NO2
N
S N
S
AcOH
SnCl2
(113)(114)
Okafor and Okoro74 reported the synthesis of substituted three-branched phenothiazine in
1991. The compounds, 15,16-dithia-1,5,10-triazabenzo[h]pentaphenes (115) were prepared by
condensing 2,3-dichloro-1,4-naphthoquinone (84) with substituted 3-aminopyridine-2[1H]-thiones
(62) in the presence of anhydrous sodium carbonate or sodium acetate to produce substituted 6-
chlorobenzo[a]phenothiazin-5-one (99). Further reaction of this product (99) with an alkaline solution
of an equimolar amount of 2-aminothiophenol under strong heat gave compound (115).
28
NH
NH2
SR+
Cl
Cl
O
O
N
N
SR
Cl
O
NH2
SH
N
N
S N
S
R
(115)
(99)
(62)(84)
Na2CO3, Heat
R=H, Cl, OMe
Na2CO3
Agarwal and Mital75, in their own contribution, reported the synthesis of another substituted
derivative (115), compound (116) by using two moles of substituted 2-aminothiophenol (42) and 2,3-
dichloro-1,4-naphthoquinone (84) under similar reaction conditions.
R
NH2
SH+
Cl
Cl
O
O
N
S N
S
R
R
(42)(84)
(116)
Na2CO32
Okafor and Okoro76 in another development, reported the synthesis of a new branched
diazaphenothiazine dye, 15,16-dithia-3,5,10,12-tetrazabenzo[h]pentaphene (119) by condensing 3,5-
29
dinitropyridine-4[1H]thione (117) with 2,3-dichloro-1,4-naphthoquionine (84) in basic medium to
give an intermediate 2,3-bis(-3-nitro-4-pyridylthio)-1,4-naphthoquionone (118) which, on reduction
with tin (II) chloride and glacial acetic acid, furnished the compound (119)
N
NO2O2N
S-Na+
+Cl
Cl
O
O
O
O
S
S
NO2N
N
NO2
O2N
NO2
NN
S N
S
N
NH2
NH2
SnCl2AcOH/Heat
Na2CO3
(117)
(84)
(118)
(119)
Okafor77, also reported the first three-branched benzoxazinophenothiazine and its aza
analogue. He obtained the first compound, benzo[a][1,4]benzoxazino[3,2-c]phenothiazine (121) by
refluxing a mixture of 2-aminothiophenol (42) and 6-chlorobenzo[a]phenoxazin-5-one (120) in
benzene/DMF and in the presence of anhydrous sodium carbonate.
30
N
O
Cl
O
+NH2
SH
N
O N
S(120)
(121)
(42)
Na2CO3
C6H6/DMF
The aza analogue, 16-oxa-15-thia-4,5,10-triazabenzo[h]pentaphene (124), was synthesized by
refluxing a mixture of 2-amino-3-pyridinol (122) and 2,3-dichloro-1-4-naphthoquinone (84) in
alkaline medium to produce 6-chloro-7-oxa-11,12-diazabenzo[a]anthracen-5-one (123) which, on
further treatment with 2-aminothiophenol in benzene/DMF and anhydrous sodium carbonate,
furnished the compound (124)77.
N NH2
OH
+Cl
Cl
O
O
N N
O
Cl
O
NH2
SH
(122)(84)
(123)
(124)
Na2CO3
Na2CO3
N N
O N
S
31
In the same development, another derivative 16-oxa-15-thia-4,5,10,14-
tetraazabenzo[h]pentaphene (125) was reported by the same author77. Compound (125) was obtained
by reacting compound (123) with 3-aminopyridine-2[1H]-thione (62) in a basic medium using
anhydrous sodium carbonate for 10 h.
N N
O
Cl
O
+NR
NH2
SH
N N
O N
S
N
R
(125)
(123)(62)
Na2CO3
Finally in the series, Okafor77 reported the synthesis of 11-amino-16-oxa-15-thia-
4,5,10,12,14-pentaazabenzo[h]pentaphene (126) by refluxing compound (123) with 4,5-
diaminopyrimidine-6[1H]thione (69) in benzene/DMF in the presence of anhydrous sodium
carbonate for 9 h.
32
N N
O
Cl
O
+NH
NNH2
S
NH2
N N
O N
S
NN
NH2
(123)
(69)
(126)
Na2CO3
Further search for more derivatives of branched phenothiazine compounds resulted in the the
successful synthesis of some new Y-shaped benzothiazinophenoxazine ring systems by Okafor et al78
in 1992. The first derivative synthesized by these workers was dibenzo[a, j][1,4]benzothiazino[3,2-
c]phenoxazine (129). This was achieved by the condensation of 2, 3-dichloro-1,4-naphthoquinone
(84) and 1-amino-2-napthol hydrochloride (127) in the presence of excess anhydrous sodium
carbonate to give a purple-red solid identified as 6-chlorodibenzo[a, j][1,4]phenoxazin-5-one (128).
Further reflux of compound (128) with 2-amino-thiophenol (42) in nitrobenzene for 11 h in the
presence of anhydrous sodium carbonate gave the compound (129) in 82% yield.
33
OH
NH2+
Cl-+
Cl
Cl
O
O
N
O
Cl
O
Na2CO3
Reflux
Na2CO3, heat
(127)(84)
(128)
(129)
NH2
SH
N
O N
S
Reported by the same authors78 was the synthesis of the aza derivative of compound (129),
11-oxa-10-thia-5,9,18-triazadibenzo[a,r]pentaphene (130). This was accomplished by refluxing a
mixture of compound (128) and 3-aminopyridine-2[1H]-thione (62) in nitrobenzene for 21 h in the
presence of anhydrous sodium carbonate.
34
NH
S
NH2
R+
N
O
Cl
O
N
O N
S
N
R
(62) (128)
(130)
Na2CO3
R=H, Cl, OMe
The same author78 reported the synthesis of The diaza derivative of (130). The compound, 6-
amino-11-oxa-10-thia-5,7,9,18-tetrazadibenzo[a,r]pentaphene (131) was obtained by heating a
mixture of 4,5-diaminopyrimidine-6-thiol (69) with 6-chlorodibenzo[a,j][1,4]phenoxazin-5-one (128)
in nitrobenzene for 25 h in the presence of anhydrous sodium carbonate.
35
N
N
SH
NH2
NH2
+N
O
Cl
O
N
O N
S
NN
NH2
(69) (128)
(131)
Na2CO3
In continuation of the chemistry of the three-branched series, Ezema et al.79 had reported the
synthesis of angular tetraaza complex phenothiazine ring system. This was achieved by condensing
2,4-diamino-6-hydroxy-pyrimidine-5-thiol (132) with 2,3-dichloro-1,4-naphthoquinone (84) in the
presence of anhydrous sodium carbonate in benzene/DMF to give diaza heterocycle named, 10-
amino-5-chloro-8-hydroxy-9,11-diazabenzo[a]phenothiazin-5-one (133). Further condensation of
(133) with another molecule of 2,4-diamino-6-hydroxypyrimidine -5-thiol (132) for 10 h gave the
tetraza heterocycle, 7,14-diamino-9,12-dihydroxy-6,8,13,15-tetraazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine (134) in good yield.
36
N
N
NH2NH2
SH
OH
+Cl
Cl
O
O
N
N
N
S
NH2
OH Cl
O
N
N
NH2NH2
SH
OH
(134)
(132) (84) (133)Na2CO3
C6H6/DMF
C6H6/DMF
Na2CO3
N
N
N
S N
SN
NOH NH2
NH2
OH
37
CHAPTER THREE
3.0 EXPERIMENTAL
All the starting materials and reagents were obtained from commercial sources (Sigma
Aldrich chemicals, Germany) and were used without further purification. The melting points were
determined with a Fischer John’s apparatus and are uncorrected. UV and Visible spectra were
recorded on UV-25500PC series spectrophotometer using matched 1cm quartz cells; absorption
maxima are given in nanometers (nm) while the figures in parentheses are the molar absorptivity
coefficient (�) values. Infrared spectra were recorded on 8400S Fourier Transform Infrared
spectrophotometer using KBr discs, unless otherwise stated, and absorptions were reported in wave
number (cm-1) in National Research Institute for Chemical Technology (NARICT), Zaria. Nuclear
magnetic resonance (1H-NMR and 13C-NMR) were determined using Jeol 400 MHz at University of
Stratchclyde, Scotland. Chemical shifts were reported on the δ scale using tetramethylsilane (TMS) as
the internal standard. Elemental analysis was carried out on a CHN rapid analyzer whereas the
antimicrobial screening was done at the Faculty of Pharmaceutical Sciences, University of Nigeria,
Nsukka.
3.1 2,4-Diamino-6-hydroxypyrimidine-5-thiol (132)
2,4-Diamino-6-hydroxy-5-thiocyanatopyrimidine (10.0 g, 55 mmol) was placed in a 500 mL
reaction flask equipped with a reflux condenser. Potassium hydroxide (30 g, 55 mmol) in water (200
mL) was added and the mixture refluxed in a sand bath for 12 h until ammonia gas ceased to evolve.
The reaction mixture was then filtered and the filtrate was allowed to cool followed by neutralization
with glacial acetic acid in an ice bath, ensuring that the temperature did not exceed 10 oC. A massive
orange precipitate was formed, which was filtered out, re-crystallized from acetone and dried in a
38
descicator to give 2,4-diamino-6-hydroxypyrimidine-5-thiol (132) as orange yellow crystals; (6.0 g,
88%); m.p > 300 oC; (lit. m.p > 300 oC).
3.2 6-chloro-5H-benzo[a]phenothiazin-5-one (85)
A mixture of 2-aminothiophenol (4.0 g; 32 mmol) and anhydrous sodium trioxocarbonate (IV)
(3.3 g; 31 mmol) was placed in 250 mL 2-necked reaction flask equipped with a magnetic stirrer,
thermometer and reflux condenser. A Solution of benzene (100 mL) and DMF (10 mL) was added
and the mixture boiled for 1 h. 2,3-Dichloro-1,4-naphthoquinone (7.26 g; 32 mmol) was later added
and the entire solution was refluxed with continuous stirring for 7 h at 78-80 oC. At the end of the
reaction period, benzene solvent was distilled off, while the slurry was poured into water and stirred
for 20 min to dissolve the inorganic materials was chilled overnight, filtered and recrystallized from
methanol-acetone mixture. 6-Chloro-5H-benzo[a]phenothiazin-5-one (85) (8.5 g, 85% yield) was
obtained as a purple microcrystalline powder, m.p 234 oC (lit. m.p 232)30. Uv-visible (MeOH) �� ��
(nm) log(�): 210 (1.9780), 221 (1.4779), 228 (1.2517), 234 (1.3543), 247 (0.2255), 255 (0.5149), 261
(1.4326), 266 (1.2138), 289 (2.3308), 314 (2.3640), 379 (2.3041), 491 (2.2271); FT-IR (KBr): Vmax
3063 cm-1 (aromatic C-H str.), 1631 cm-1 (C=O), 1502 (aromatic C=N), 1467 (aromatic C=C), 1292,
1228, 1155, 1082, 1039, 902, 765, 680, 567 and 462 cm-1.
3.3 7-Amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothizine (136)
2,4-Diamino-6-hydroxypyrimidine-5-thiol (1.0 g, 6 mmol) and anhydrous sodium
trioxocarbonate(IV) (1.5 g, 14 mmol) were place in 250 mL reaction flask equipped with a magnetic
stirrer, thermometer and reflux condenser. A solution of benzene (100 mL) and DMF (10 mL) was
added and the mixture boiled for 1 h. 6-chloro-5H-benzo[a]phenothiazin-5-one (1.8 g, 6 mmol) was
later added and the entire solution refluxed in a water bath with continuous stirring for 9 h at 78-80
39 oC. At the end of the reaction period, the benzene solvent was evaporated and the slurry added to
water (500 mL), heated to near boiling, filtered and then crystallized from equal volumes of acetone
and methanol to give 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
as purple-red crystals; m.p. > 300 oC (2.46 g 84 % yield). Uv-visible (MeOH) �� �� (nm) log(�): 780
(1.7000), 745 (1.7204), 688 (1.7704), 486 (2.5758), 380 (2.6022), 319 (2.6437), 265 (2.8636), 254
(2.7968), 247 (2.7875);IR (KBr): �� �� 3761 (OH), 3400, 3331 (N-H stretches) 3063, (aromatic C-H
str.), 1608 (arom. C=N) 1494, 1452 (arom. C=C), 1300, 1242, 1151, 1087, 1024, 877, 850, 758, 673,
642, 538 and 455 cm-1;1H-NMR (DMSO: d6) δ: 8.87 (2H, dd, J1 = 7.87, J2 = 1.36 Hz), 8.24 (2H, dd,
J1= 7.55, J2=1.61 Hz) 8.08 (4H, m, Ar-H), 7.92 -7.67 (4H, m, 1H, 2H, 3H), 5.46 (1H, s, 9-OH);13C-
NMR (DMSO) δ: Few peaks, due to the insolubility of the compound. Absence of chemical shift at
150 ppm and above indicates the absence of C=O.
Analysis: Calculated for C20H11N5OS2: C, 59.84, H, 2.76, N, 17.44 and S, 15.97; Found: C, 59.88, H,
2.81, N, 17.45 and S, 15.99.
3.4 4-Amino-2-ethylthio-6-hydroxypyrimidine-5-thiol (137)
4-Amino-2-ethylthio-6-hydroxy-5-thiocyanatopyrimidine (12 g; 77 mmol) was refluxed in
200 mL of 40% potassium hydroxide solution for 12 h. The resulting dark brown solution was filtered
hot. The filtrate was cooled and neutralized to pH 7 with glacial acetic acid while cooling in an ice
bath. The neutralized product was further chilled in a refrigerator and later filtered. On re-
crystallizing the residue from aqueous actone, green crystals of 4-amino-2-ethyltho-6-
hydroxypyrimidine-5-thiol (6.7 g, 83 % yield) were collected. m.p > 300 oC (Lit > 300 oC).80
40
3.5 7,14-Diethylthio-9,12-dihydroxy-6,8,13,15-tetraazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine. (138)
4-Amino-2-ethylthio-6-hydroxypyrimidine-5-thiol (2 g; 10 mmol) was placed in a 250 mL
two-necked flask equipped with a dropping funnel, mechanical stirrer, thermometer and a reflux
condenser. Absolute ethanol (100 mL) and 15 % HCl (10 mL) were then added and the mixture was
warmed to dissolve. 2,3-Dichloro-1,4-naphthoquionone (1.1 g; 5 mmol) was added and the mixture
refluxed on a water bath for 6 h at 78 oC. As soon as refluxing started, an orange brown precipitation
was observed. As refluxing proceeded, the orange brown colour turned blue. The later colour
observed persisted throughout the reflux period. The reaction mixture was poured in to a clean beaker
along with the thorough rinsing of the glassware with acetone and poured into the beaker containing
the reaction mixture. The solvent was then evaporated. Distilled water was added and it was then
cooled, filtered and the residue crystallized from acetone-methanol mixture. 7,14-Diethylthio-9,12-
dihydroxy-6,8,13,15-tetrazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (3.2 g; 91 % yield) was
obtained as blue powder; m.p > 310 oC. Uv-visible (MeOH) �� �� (nm) log(�): 748 (1.2533), 539
(2.4100), 328 (3.2316), 322 (3.2249), 305 (3.2634), 276 (1.5901), 266 (1.7812), 257 (1.0315), 239
(1.0895), 233 (0.9881), 288 (0.8554), 222 (1.3721) ; IR (KBr) �� �� 3755, 3400 (OH), 3184 (aromatic
C-H str.), 2937 (C-H stretch of CH3 or CH2), 1626, 1556, (aromatic C=N), 1518, 1446 (aromatic
C=C), 1325, 1276, 1224, 1130, 1095, 964, 885, 823, 779, 700, 648, 561, 447 cm-1;1H-NMR (DMSO:
d6) δ: 8.08 (4H, m, Ar-H), 7.91 (4H, td, J1 = 6.72, J2 = 5.89, J3 = 3.44 Hz, Ar-H),3.3 (2H, q, J1 = 8.16,
J2 = 7.3 Hz, -SCH2CH3) 3.06 (3H, t, -SCH2CH3), 1.33 (3H, dt, J2 = 54.74, J2 = 7.41 Hz);13C-NMR
(DMSO) δ: 142.98-127.63 (C1 – C9, Ar-C), 15.19 (aliphatic carbon).
Analysis: Calculated for C19H11N7O2S2: C, 52.65, H, 2.56, N, 22.62, S, 14.79; Found: C, 52.72, H,
2.59, N, 22.60, S, 14.90.
41
3.6 3-Amino-6-methoxypyridine-2-thiol (140)
This compound was prepared from 5-methoxy[1,3]thiazolo[5,4-b]pyridine-2-amine (139) based
on a modified method by Okafor81 as follows:
5-Methoxy[1,3]thiazolo[5,4-b]pyridine-2-amine (10.0 g, 55 mmol) was refluxed for 12 h in 40%
potassium hydroxide solution (200 mL) as described for 4-amino-2-ethylthio-6-hydroxypyrimidine-5-
thiol. Glistening yellow crystals of 3-amino-6-methoxypyridine-2-thiol (6.2 g, 90% yield) were
collected after crystallization from acetone-methanol mixture M.p > 300 oC.
3.7 8,3-Dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (141)
3-Amino-6-methoxypyridin-2-thiol (2 g; 13 mmol) in 100 mL of absolute ethanol was placed
in 2-necked reaction flask containing 10 mL of 15 % HCl. The mixture was warmed to dissolve. 2,3-
Dichloro-1,4-naphthoquinone (1.45 g; 6 mmole) was then added and the mixture refluxed on a water
bath for 6.5 h at 78 oC . The precipitate obtained was initially yellow but later turned dark-brown.
This coloration persisted throughout the reflux period. The mixture was then poured into a beaker,
heated for 15 min to evaporate the remaining solvent, diluted twice with water and cooled. The dark-
brown product was collected by filtration and re-crystallized from acetone-methanol mixture to yield
8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (3.40 g; 94 % yield) as
brown microcrystalline powder, m.p > 310 oC. Uv-visible (MeOH) �� �� (nm) log(�): 749 (1.6377),
478 (2.5529), 334 (3.0278), 296 (2.8135), 275 (1.2669), 269 (1.5525), 257 (1.0313), 246 (1.1897),
239 (1.4659), 228 (1.3409), 222 (1.5827), 212 (1.2566), 203 (1.6419) ; IR (KBr) �� �� 3088 (aromatic
C-H str.), 1587 (C=N aromatic), 1504 (aromatic C=C), 1346, 1278, 1147, 1087, 1022, 889, 831, 781,
692, 648, 555 and 462 cm-1; 1H-NMR (DMSO: d6)δ: 10.57 (2H, d), 10.31 (2H, d, J = 7.51 Hz), 7.66
(4H, s, Het. Ar-H), 7.31 (4H, s, Ar-H), 4.28 (3H, d, -OCH3); 13C-NMR (DMSO) δ: There were few
peaks due to difficulty in the solubility of the compound. 24.63-15.05 ppm (aliphatic carbon).
42
Analysis: Calculated for C22 H14N4O2S2: C, 61.38, H, 3.28, N, 13.01, S, 14.90; Found: C, 61.45, H,
3.33, N, 13.21, S, 15.10.
3.8 6,13-Dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-tetrazatriphenodithiazine (142)
A mixture of 4-amino-2-ethylthio-6-hydroxypyrimidine-5-thiol (4 g; 19 mmol), absolute
ethanol (150 mL) and 10 mL of 15 % HCl was warmed to dissolve. Tetrachloro-1,4-benzoquinone (2
g; 8 mmol) was then added and the mixture refluxed on a water bath with continuous stirring for 6 h.
The mixture went into solution initially gave a yellowish clear solution followed by a yellowish red
precipitation about an hour. At the end of the reflux period, the mixture was poured into a beaker and
heated to evaporate the solvent. 100 mL of water was poured into the beaker containing the mixture
and was allowed to cool. The product was then collected by filtration and later re-crystallized from
aqueous acetone. 6,13-Dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-tetraazatriphenodithiazine
was obtained in 73 % yield (5.3 g) as greenish yellow powder, m.p > 310 oC. Uv-Visible (MeOH)
�� �� (nm) log(�): 753 (1.1864), 671 (1.2357), 612 (1.5859), 531 (1.5920), 491 (1.6538), 322
(3.2634), 305 (3.2993), 274 (1.1819), 257 (1.4759), 251 (1.7954), 246 (1.2910) ;IR (KBr): �� ��
3751, 3375 (OH), 3196, (C-H str.), 1626 (aromatic C=N), 1548, 1437 (aromatic C=C), 1319, 1282,
1215, 1089, 962, 881, 773, 698, 545 and 451 cm-1
Analysis: Calculated for C16H8Cl2N6O2S2: C, 42.58, H, 1.79, Cl, 15.70, N, 18.62, S, 14.21;
Found: C, 42.67, H, 1.92, Cl, 15.73, N, 18.60, S, 14.26.
3.9 4-Amino-2-methyl-6-hydroxypyrimidine-5-thiol (144)
4-Amino-2-methyl-6-hydroxy-5-thiocyanatopyrimidine(143) (6.5 g, 36 mmol) was placed in a
reaction flask to which was added 20 g of potassium hydroxide in 120 mL of water. The entire
mixture was refluxed on a sand bath for 12 h. At the end of the reflux period, it was filtered while hot.
The filtrate was cooled and later neutralized with glacial acetic acid in an ice bath. Excessive frothing
43
was observed during the neutralization period. This was reduced to a minimum by ensuring that the
temperature never exceeded 10 oC during the neutralization period. It was filtered with a Buchner
funnel and the residue re-crystallized from methanol. A Glistening yellow crystals of 4-amino-2-
methyl-6-hydroxypyramidine-5-thiol (5.2 g, 82 % yield) were obtained, m.p < 320 oC, (lit. m.p > 300
oC)57.
3.10 6,13-Dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-triazatriphenodithiazine (145)
A mixtures of 3-amino-6-methoxypyridine-2-thiol (2 g; 12 mmol), 4-amino-2-methyl-6-
hydroxypyrimidine-5-thiol (2 g; 10 mmol), was placed in a reaction flask equipped with a magnetic
stirrer, thermometer and reflux condenser. Absolute ethanol (120 mL) and 10 mL of 15 % HCl were
then added. The solution was warmed to dissolve. Tetrachloro-1,4-benzoquinone (3 g, 12 mmol) was
later added and the mixture refluxed with continuous stirring for 7 h at 78 oC. At the end of the reflux
period, the mixture was poured into a clean beaker and 100 mL of water was added, it was heated for
15 min and cooled. The product was collected by filtration, dried in an oven and re-crystallized from
acetone-methanol mixture to give 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-
triazatriphenodithiazine (4.79 g, 86 % yield) as purple powder. m.p > 300 oC. Uv-Visible (MeOH)
�� ��(nm) log(�): 750 (1.5670), 583 (2.1691), 343 (2.6682), 257 (3.0207), 228 (2.3856), 223
(1.2553) ; IR (KBr) �� ��: 3751, 3346 (OH), 3119 (aromatic C-H-stretch), 2918, 2852 (C-H stretch of
CH3, CH2) 1641, 1595 (aromatic C=N), 1546, 1444 (aromatic C=C) 1354, 1273, 1155, 1085, 1012,
912, 833, 777, 723, 677, 608, 553, and 509cm-1 ;1H-NMR (DMSO-d6) δ: 11.64 (1H, s), 7.01 (2H, d, J
= 144.25 Hz, Het. Ar-H), 2.4 (3H, s, -OCH3), 1.45 (3H, s, -CH3); 13C-NMR (DMSO) δ: 165.96,
162.37, 159.79 (Ar-C), 88.53 (C=C), 21.43 (-CH3, aliphatic carbon).
Analysis: Calculated for C17H9Cl2N5O2S2: C, 45.34, H, 2.01, Cl, 15.75, N, 15.55, S, 14.24; Found: C,
45.42, H, 2.30, Cl, 15.86, N, 15.53, S, 14.33.
44
3.11 Evaluation of the Synthesized Phenothiazine Derivatives for Antimicrobial Activity
The increasing documented biological and pharmaceutical activities of phenothiazines
necessitated the evaluation of the antimicrobial activity of the synthesized derivatives. The
compounds were tested against three gram positive and three gram negative bacteria and two fungal
organisms: Bacillus subtitis, Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa,
Escherichia coli, Klebsiella pneumonia, Candida albicans and Aspergillus niger.
3.11.1 Sensitivity Test of the Compounds
The assay was conducted using the agar-well diffusion method82. 20 Mg/mL concentration of
each compound was constituted by dissolving 0.04 g of each in 2 mL of dimethyl sulfoxide (DMSO).
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 to approximately 108cfu/ml and used to inoculate the surface of the iso-sensitest
nutrients agar and the excess fluid drained into discard pot containing disinfectant. The inoculated
agar surface was allowed to dry and the plates appropriately labeled. 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 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 for antibacterial screening
were incubated at 37 oC for 24 h while the fungi were incubated at 30 oC for 48 h. After incubation,
the plates were observed for inhibition zones around the wells. The diameters of the zones were
measured with a metre rule to the nearest whole millimeter.
45
3.11.2 Determination of the Minimum Inhibitory Concentration (MIC) of the Synthesized
Derivatives
This was carried out using agar dilution following the procedure outlined by the chemical
Laboratory Standards Institute (CLSI) 83. Sterile test tubes were arranged on a test tube rack and 1 mL
of DMSO was dispensed 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 1, 0.5, 0.25, 0.125 and 0.0625 (mg/mL) (i.e.,
graded concentrations of the compound). 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
surface of the nutrient agar while the excess fluid drained into a discard pot containing disinfectant.
The inoculated agar surface was allowed to dry and the plates labeled appropriately. 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 as it was
done previously. After incubation, the plates were observed for inhibition zones around the wells. The
minimum inhibitory concentration was determined as the value of the lowest concentration that
completely suppressed growth of the organisms.
46
CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
The synthesis of angular phenothiazine derivatives was achieved by condensation reactions
between one mole of 2,3-dichloro-1,4-naphthoquinone and another mole of 2-aminothiophenol or (its
substituted derivatives) in anhydrous basic medium under refluxing. The synthesis of its aza
analogues, involved condensation reactions of 1:1 molar ratios of 2,3-dichloro-1,4-naphthoquionone
and aminopyrimidine thiol or aminopyridine thiol in basic medium.
The synthesis of three-branched phenothiazine compounds involve condensation reactions of
1:2 molar ratios of 2,3-dichloro-1,4-naphthoquinone and aminothiophenol, aminopyridinethiol or
aminopyrimidinethiol in anhydrous basic condition under refluxing.
In our search for more complex derivatives of linear and angular phenothiazine, an acid
catalyzed method was also utilized which proved facile and produced higher yields, requiring shorter
reaction time.
4.1 6-Chloro-5H-benzo[a]phenothiazin-5-one
A mixture of 2-aminothiophenol (42) and 2,3-dichloro-1,4-naphthoquinone (84) was refluxed
in benzene-DMF for 8 h in the presence of anhydrous sodium trioxocarbonate (IV). The product was
purified by recrystallization from methanol-acetone mixture to yield 6-chloro-
5H benzo[a]phenothiazin-5-one (85) as purple powder; m.p. 234 oC. Scheme 1.
NH2
SH+
Cl
Cl
O
O
C6H6/DMF
Na2CO3
N
S
Cl
O
(42)
(84) (85)
Scheme 1
47
The mechanism of the reaction proceeded by proton abstraction from the-thiol (42) by the
base converting it to a mercaptide ion (146), which initiated a nucleophitic attack on the 2,3-
dichloronaphthoquinone (84), by displacing one of the halogen atoms to form the diaryl intermediate
(147). By so doing, cyclisation took place by a second nucleophitic attack from the amino group on
the carbon atom of the carbonyl group to form a second intermediate (148), which on elimination of
water molecule yielded the expected compound(85) Scheme 2.
NH2
S-+ O
OCl
Cl
NH2
S
O
ClCl
O-
-Cl-
NH2
S
O
Cl
O
N
S
HOH
O
Cl
..
S O
Cl
N[-H2O]
(42)
(84)
(85a)(85b)
Scheme 2:
48
The assigned structures are consistent with the observed spectral characteristics which are
given as follows:
Uv-visible (MeOH) �� ��(nm) log(�): 210 (1.1978), 261 (1.4326), 379 (2.3041), 491
(2.2271). The visible maximum absorption band at 491 nm agrees with the observed purple
colouration.
The IR spectrum was interpreted as follows: the bands at 3063 (aromatic C-H stretch), 1631
cm-1 (C=O), 1502 (aromatic C=N), 1467 cm-1 (aromatic C=C), 902, 765 cm-1 (C-H bending in
substituted benzene ring), 680, 567 cm-1 (C-S-C str. of thiazine ring) are consistent with the assigned
structure.
4.2 7-Amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (136)
6-Chloro-5H-benzo[a]phenothiazin-5-one(85) was condensed with 2,4-diamino-6-
hydroxypyrimidine-5-thiol (132) in the presence of anhydrous sodium carbonate under refluxing for
10 h in a mixture of benzene/DMF as the solvent to yield the three-branched heterocyclic compound,
7-amino-9-hydroxy-6-8,diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (136) as purple-red
crystals; m.p. > 300 oC.
49
N
N
NH2 NH2
SCN
OH
1. KOH
2. CH3CO2H
N
N
NH2 NH2
SH
OH
N
S
Cl
O
Na2CO3
+
N
S N
SN
N NH2OH
(135) (85)(132)
(136)
A
BCD
E
F
12
3
4
5
6
7
8
9
10
111213
1415 16
Scheme 3:
50
N
N
NH2
SH
OH
NH2 N
N
NH2
S-
NH2
OH
+N
S
Cl
O
N
SS
O-
N
N
NH2
NH2OH
Cl
N
SNH2
SN
N
O
NH2OH
N
S N
SN
N NH2OH
HOH
N
S N
SN
N NH2OH
Na2CO3
(146)(132)
(147)
(136a) (136b)
-Cl-
..
-H2O
The mechanism of the reaction followed the same pattern of proton abstraction from 5-thiol
by the base and formation of mercaptide ion (146) wich initiated a nucleophilic attack on compound
(85) by displacing the reactive halogen group. This is followed by another nucleophilic attack of the
carbonyl carbon centre by the amino group, resulting in cyclization. Elimination of water molecule
under alkaline conditions furnished the complex compound (136b) (Scheme 4).
Scheme 4
51
The non-linear structure assigned to compound (136) is consistent with the observed
spectroscopic characteristics.
Uv-Visible (MeOH) showed absorptions at �� �� 380 nm (���� = 2.6022), 486 nm (2.5758),
688 nm (1.7704), 745 (1.72004) and 780 (1.72000). The shift in the absorption maximum band from
491 nm of compound (85) to 780 nm is consistent with the presence of an extended conjugation
system.
The infrared assignments were made as follows: �� �� 3761 (OH), 3400, 3331 (N-H stretches),
3063 (aromatic C-H str.), 1608 (aromatic C=N), 1494, 1452 (aromatic C=C), 877, 850, 758 (C-H
bend in substituted benzene ring), 673, 642, (C-S-C str. of thiazine ring), 538 and 455 cm-1. The
infrared spectrum showed no strong absorption band at 1650 cm-1 for (C=O), which is evidence for a
second condensation and cyclization leading to the formation of the complex derivative.
1H-NMR provided further evidence for the proposed structure. The chemical shift at δ 8.08
(4H, m) was assigned to the aromatic protons in ring D, the shifts at δ 7.92-7.67 (4H, m) were
assigned to the aromatic protons at positions 1, 2, 3 and 4 in ring A, while the shift at δ 5.46 (1H, s) is
for OH. In the 13C-NMR spectrum, the absence of chemical shift at 150 ppm indicated the absence of
C=O.
The elemental analysis of the complex derivative is in agreement with the molecular formula of the
compound C20H11N5OS2.
4.3 7,14-Diethylthio-9,12-dihydroxy-6,8,13,15-tetraazabenzo[a][1,4]benzothiazino[3,2-
c]phenothiazine (138)
A more complex triangular azabenzothiazinophenothiazine ring system was synthesized by
condensing 2 moles of 4-amino-2-ethylthio-6-hydroxypyrimidine-5-thiol (137) with 1 mole of 2,3-
dichloro-1,4-naphthoquinone in acid medium. This reaction led to 7,14-diethylthio-9,12-dihydroxy-
52
6,8,13,15-tetrazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (138) as a blue powder. m.p. > 300
oC.
53
N
N
EtS OH
SCN
NH2
1. KOH
2. CH3CO2H
N
N
EtS OH
SH
NH2
(137a) (137b)
2 EtOH
15% HCl
N
N
EtS OH
S-
NH2
2 +Cl
Cl
O
O
Heat
S
S
O
O
N
N
N
N
NH2
NH2
OHOH
SEt
SEt
..
..
S
NH
NN
S
NH
NN
SEt
OH
OH
SEt
OH
OH:
:
-2H2O S
N
NN
S
N
NN
SEt
OH
OH
SEt
N
N
N
S N
SN
N
OH
EtS
OH SEt(138b)
(138a)
(151)
(150)
(84)(149)
Scheme 5
54
Compound (138b) was formed by a nucleophilic attack of two moles of the mercaptide ion
(149), on 2,3-dichloro-1,4-naphthoquinone leading to the loss of two chloride ions. A second
nucleophitic attack of the amino groups on the carbonyl carbon resulted in cyclisation, which on
elimination of 2 molecules of water gave 7,14-diethylthio-9,12-dihydroxy-6,8,13,15-
tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (Scheme 5).
The proposed structure is in agreement with the following spectra data:
Uv-Visible (MeOH) �� ��(nm) log(�): 322 (3.2249), 305 (3.2634), 328 (3.2316), 539
(2.4100) and 748 (1.2533). The visible absorption maximum band at 748 nm is consistent with its
blue colour.
In the infrared spectrum, the strong bands at 3755, 3400 cm-1 (OH), 3184 cm-1 (aromatic C-H
str.), 1626, 1556 (aromatic C=N), 1518, 1446 cm-1 (aromatic C=C), 964, 885, 823, 779 cm-1 (C-H
bend in substituted benzene ring), 648, 561 cm-1 (C-S-C str. of thiazine ring) are consistent with the
proposed structure.
The 1H-NMR spectrum gave peaks at δ 8.08 (4H,m) for aromatic protons, δ 7.71 (4H, td, Ar-
H). The peaks at δ 3.3 (2H, q) was assigned to CH2 in – SCH2CH3 and δ 3.06 (3H, t) for CH3 in
-SCH2CH3.
In the 13C-NMR spectrum, peaks at δ 142.98-127.63 were for aromatic carbons while the peak
at δ 15.19 was for the aliphatic carbon.
The elemental analysis of the compound also agreed with the molecular formula, C19H11N7O2S2.
4.4 8, 13-Dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (141)
Base-catalyzed ring cleavage of 5-methoxy[1,3]thiazolo[5,4-b]pyridine-2-amine (139) gave 3-
amino-6-methoxypyridine-2-thiol (140) which was coupled in situ with 2,3-dichloro-1,4-
55
naphthoquinone in the presence of 15 % HCl and ethanol as the solvent to furnished a good yield of
another compound, 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine (141)
as a brown crystalline powder. m.p > 310 oC.
N
N
SMeO
NH21. KOH
2. CH3CO2H N SHMeO
NH2
(139) (140)
The reaction above is simply the ring cleavage of the thiacyano group to give the thiol with the
elimination of ammonia and carbondioxide. Scheme 6.
(Scheme 6)
56
N
NH2
SHMeO
(152)
2EtOH
15% HCl N
NH2
S-MeO
2 +Cl
Cl
O
O
S
S
O
O
N
N
NH2
NH2
OMe
OMe
..
..
S
NHN
S
NHN
OH
OH
OMe
OMe
:
:
-2H2O S
NN
S
NN
OMe
OMe
N
N
S N
S
N
MeO
OMe
(141b)
(141a)
(154)
(153)
(84)
Heat
(140)
A
BCD
E
F
12
3
4
5
6
78
9
101112
13
1415 16
Scheme 7
57
The mechanism of the reaction follows the usual pattern of nucleophilic attack on compound
(84) by the mercaptide ions leading to loss of two chloride ions to form the diarylsuphide. The
diarylsuphide (153) cyclizes by amino groups attack on the carbonyl carbons to form (154) which on
elimination of 2 molecules of water gave the desired compound (141) (Scheme 7).
The structure was assigned based on the following spectral information:
Uv-visible (MeOH) �� �� (nm) log(�): the bands at 296 (2.8135), 334 (3.0278), 478 (2.5529), and
749 (1.6377) agreed with the colour and extended conjugation system.
In the IR spectrum, the peaks at 3088, cm-1 (aromatic C-H str.), 1587 (C=N of aromatic ring),
1504 cm-1 (C=C of aromatic ring), 889, 831, 781 cm-1 (C-H bend in substituted benzene ring) and
692, 648 cm-1 (C-S-C of the thiazine ring) were in conformity with the proposed structure.
The chemical shifts in the 1H-NMR at δ 7.66 (4H, s) was assigned to the equivalent heterecyclic
aromatic protons in rings D & F and δ 7.31 (4H, s) for aromatc protons in ring A. The chemical shift
at δ 4.28 (3H, d) was assigned to the methoxy groups (OCH3). In the 13C-NMR (DMSO) δ: 24.63-
15.05 was for the aliphatic carbons (OCH3).
The result obtained from the elemental analysis of the complex compound is in agreement with the
molecular formular, C22H14N2O2S2.
4.5 6,13-Dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-tetraazatriphenodithiazine (142)
Further extension of the work by condensing 2 moles of 4-amino-2-ethylthio-6-
hydroxypyrimidine-5-thiol (137) with 1 mole of tetrachloro-1,4-benzoquinone (155) in an acid
medium led to the formation of 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11-
tetraazatriphenodithiazine (142) as a greenish yellow powder. m.p > 310 oC.
58
N
N
OH
NH2
EtS
SH2 +
Cl
Cl Cl
Cl
O
O
15% HCl
C2H5OH
N
N
N
S
S
N
N
N
EtS
SEt
OH
OHCl
Cl
(137) (155) (142b)
N
N
EtS
NH2
OH
SH
C2H5OH
15 % HCl
N
N
EtS
NH2
OH
S-
ClCl
Cl Cl
O
O
-2Cl-Heat
Cl
Cl
O
O
N
N
NH2
S
N
NNH2 SEt
OH
S
EtS
OH
..
..
N
N
NH
S
S
NH
N
N
OH
OH
Cl
Cl
SEt
OH
EtS
OH
..
..
N
N
N
S
S
N
N
N
Cl
Cl
SEt
OH
EtS
OH
-2H2O
2 2
(137)
(155)
(142a) (142b)
Compound (142b) was formed by a nucleophilic attack of two moles of the mercaptide ion
(149), on 2,3-dichloro-1,4-naphthoquinone leading to the loss of two chloride ions. A second
Scheme 8
Scheme 9
59
nucleophitic attack of the amino groups on the carbonyl groups resulted in cyclisation, which on
elimination of 2 molecules of water gave the desired product (Scheme 9).
Uv-Visible (MeOH) �� �� (nm) log(�): 305 (3.2993), 322 (3.2634), 491 (1.6538), 531
(1.5920), 612 (1.5859), 671 (1.2357) and 753 (1.1864). The strong visible absorption maximum band
at 753 nm is consistent with its colour.
The infrared spectrum gave signals at 3315, 3375 cm-1 (OH), 3196, cm-1 (C-H str. Of
aromatic), 1626 (aromatic C=N), 1548, 1437 cm-1 (aromatic C=C), 962, 881, 775 cm-1 (C-H bend in
substituted benzene ring), 698, 545 (C-S-C in the thiazine ring). These IR absorption are consistent
with the assigned structure.
The result of the elemental analysis of the compound is in agreement with the molecular formula,
C16H8Cl2N6O2S2.
4.6 6,13-Dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8-triazatriphenodithiazine (145)
This was accomplished by the condensation of 3-amino-6-methoxypyridine-2-thiol with
tetrachloro-1,4-benzoquinone and 4-amino-2-methyl-6-hydroxy-5-thiopyrimidine, which was
obtained by base-catalyzed hydrolysis of 4-amino-2-methyl-6-hydroxy-5-thiopyrimidine, in the
presence of 15 % HCl and ethanol as the solvent to give 6,13-dichloro-3-methyl-9-methoxy-1-
hydroxy-2,4,8-triazatriphenodithiazine(145) as purple crystalline powder. m.p > 300 oC.
60
N
N
NH2
Me OH
SH
(144)(143)
N
N
NH2
Me OH
SCN
1. KOH
2. CH3CO2H
N
NH2
SHMeO+
Cl
Cl Cl
Cl
O
O
+N
N
NH2
Me OH
SH
15% HCl C2H5OH
N
N
S
S
N
N
N
Cl
Cl
Me
OH
MeO
(145b)
(144)(155)(140)
Scheme 10
61
+N
N
Me
NH2
OH
SH
C2H5OH
15 % HCl N
NH2
MeO S-+
N
N
Me
NH2
OH
S-+
ClCl
Cl Cl
O
O
-2Cl- Heat
Cl
Cl
O
O
N
NH2
MeO S
N
NNH2 Me
OH
S
..
..
N
NH
S
S
NH
N
N
OH
OH
Cl
Cl
MeO Me
OH
..
..N
N
S
S
N
N
N
Cl
Cl
MeO Me
OH
-2H2O
N
NH2
MeO SH
(140)(155)(144)
(145a) (145b)
The formation of compound (145b) proceeds by a mechanism similar to that formulated for
compound (142b). The mercaptide ions being more nucleophilic than any of the two amino groups
preferentially mount nucleophilic attack on the tetra chlorobenzoquinone(155) leading to the
formation of the diarylsuphide. Cyclization was achieved by the internal condensation of the carbonyl
and amino groups with the loss of 2 molecules of water, leading to the isolated complex
triphenodithiazine heterocycle. (Scheme 11).
The proposed structure is in agreement with the spectral information.
The Uv-visibe; absorptions, �� �� 228 (���� = 2.3856), 257 (3.0207), 343 (���� = 2.6682),
583 (2.1691) and 750 (1.5670) were consistent with the structure.
Scheme 11
62
In the infrared spectrum, the absorptions at 3751, 3346 cm-1 (OH), 3119 (aromatic C-H
stretches), 2918, 2852 cm-1(C-H str of CH2, CH3), 1641, 1595 1 (aromatic C=N), 1546, 1444 cm-1
(aromatic C=C), 912, 833 cm-1 (CH-bend in substituted benzene ring), 777 cm-1 (C-Cl), 677, 609 cm-1
(C-S-C in the thiazine ring) were consistent with the assigned structure.
The 1H-NMR spectrum showed signals at δ 7.01 (2H, d) for Heteroaromatic protons,
δ 2.4 ( 3H, s) for –OCH3 group and δ 1.45 (3H, s,) for CH3. In the 13C-NMR, the peaks at δ 165.96,
162.37 and 159.79 (aromatic carbon), 88.53 (C=C), and 21.43 (-CH3) for the aliphatic carbon.
The elemental analysis of the product is in agreement with the molecular formula, C17H9Cl2N5O2S2.
4.7 Results of Antimicrobial Sensitivity Test of the Synthesized Compounds
Table 1
Compound
No
Gram-positive bacteria Gram-negative bacteria Fungi
Organisms
B.subtilis B. cereus S. aureus P. aeruginosa E. coli K. pneumoniae C. albican A. niger
85b + + + + - + - +
136b + ++ + + - - + +
138b +++ +++ +++ + - + +++ +++
141b + + - - + - ++ +
142b + ++ - - + - - ++
145b - + - + - + + -
CPFX ++ ++ +++ ++ + ++ - -
KTCN - - - - - - ++ +++
+ = sensitive CPFX, KTCN (reference drugs). ++ = moderately sensitive CPFX (Ciprofloxacin, antibacteria) +++ = highly sensitive KTCN (Ketoconazole, antifungi) - = resistance Table 1 above shows the range of sensitivity of the microorganisms to the compounds. The
compounds showed significant activity against the test organisms except E. coli which was only
sensitive to compounds 141b and 142b. Bacillus cereus was sensitive to all the compounds S.aureus,
63
P. aeruginosa and K. pneumonia were resistant to compounds 141b and 142b, but were sensitive to
other compounds.
Table 2: Results of the Inhibition Zones Diameter (mm)
The inhibition zone diameter was measured in millimeter to the nearest whole number
Compound No.
Gram-positive Bacteria Gram negative Bacteria Fungi Organisms
B.subtilis B.cereus S. aureus
P. aeruginosa
E.coli K pneumoniae
C.albican A. niger
85b 10 9 9 11 4 10 7 10 136b 12 15 11 9 5 - 10 9 138b 22 21 18 8 - 12 22 21 141b 8 12 - - 9 6 16 12 142b 11 17 - - 8 - 5 18 145b 5 10 - 10 - 8 11 - CPFX 17 17 20 13 8 15 - - KTCN - - - - - - 16 21 The results above are the sensitivity values. The figures greater than 8 were considered to be active against the microorganisms which then further undergo serial dilution to give the MIC. The higher the IZD values, the higher the activity. 4.8. Result of Minimum Inhibitory Concentration of the Compounds (mg/mL)
Minimum inhibitory concentration (MIC) of the synthesized compounds were also determined
by agar-well diffusion method as described above. The essence of MIC is to determine the least
concentration of the compound (drug) that can inhibit the growth of the microorganism84. The figures
showed in table 3 below were the least concentration(mg/mL) of the synthesized compounds that
inhibited the growth of the microorganisms.
64
Table 3: Minimum Inhibitory Concentration of the Compounds (mg/ml)
Compoun
d no
Gram-positive bacteria Gram-negative bacteria Fungi Organisms
B.subtilis B. cereus S. aureus P.
aeruginosa
E. coli K.
pneumoniae
C. albican A. niger
85b 0.1738 0.0871 0.1202 0.1514 - 0.1819 - 0.0912
136b 0.1254 0.1047 0.0794 0.1202 - - 0.1819 0.0912
138b 0.0633 0.0505 0.0398 0.585 - 0.1514 0.1659 0.0603
141b 0.1819 0.1738 - - 0.1445 - 0.0724 0.1738
142b 0.1445 0.1905 - - 0.1689 - - 0.1380
145b - 0.1521 - 0.1933 - 0.1831 0.1162 -
CPFX 0.0212 0.0315 0.0213 0.0323 0.1677 0.0567 - -
KTCN - - - - - - 0.0622 0.1356
Almost all the synthesized phenothiazine derivatives were active against the microorganisms
even at very low concentrations, which implies that, the lower the MIC values obtained, the higher
the activity. Compound 138b has the highest MIC values in bacteria which ranged from 0.0398
mg/mL to 0.1585 mg/mL. Compound 141b and 142b were resistant to S. aureus and P. aeruginosa
respectively. The E.coli is also resistant to compound 85b, 136b & 138b respectively. The entire
compounds were very active against B.cereus and B.substilis, having lower MIC values. In fungal
organisms, C. albicans was only resistant to compounds 85b and 142b, while other compounds were
highly active against the organisms.
All the compounds were very active against A. niger (fungal organism) except compound
145b which was resistant to it. The standard drugs used for both the bacteria and fungi were all active
against the microorganisms, having MIC lower than the synthesized compounds, except in A.niger
where the compounds have lower MIC value than the standard drug used.
65
4.4 CONCLUSION
The synthesis of complex phenothiazine derivatives were successfully carried out. The angular triaza
and tetraaza complex phenothiazines prepared in this work could likely meet the requirements to be
considered as dyes besides their antimicrobial properties. The antimicrobial activities of the
compounds revealed that virtually all the compounds synthesized showed varying activities against
the cultured bacteria and fungi. However, they were less active than the standard antibacterial drug.
Compounds 85b, 136b and 138b have stronger activity against aspergillus niger when compared to
the standard antifungal drug (Ketoconazole). Conclusively, the compounds which showed high
activity against A. niger than the ketoconazole are recommended for preclinical screening.
66
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N
S
Cl
O
Fig. 1: UV-Vis spectrum of 6-chloro-5H-benzo[a]phenothiazin-5-one
73
N
S N
SN
NOH N H 2
Fig. 2: UV-Vis spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2- c]phenothiazine.
74
N
N
N
S N
SN
N
O H
OH
EtS
SEt
Fig 3: UV-Vis spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15- tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
75
Fig 4: UV-Vis spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2- c]phenothiazine.
N
N
S N
S
N
MeO
OMe
76
N
N
N
S
S
N
N
N
OH
OH
SEt
EtS
Cl
Cl
Fig 5: UV-Vis spectrum of 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11- tetrazatriphenodithiazine
77
Fig 6: UV-Vis spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8- triazatriphenodithiazine
N
N
S
S
N
N
NMeO
Cl
Cl
OH
Me
78
N
S
C l
O
Fig 7: IR spectrum of 6-chloro-5H-benzo[a]phenothiazin-5-one
79
N
S N
SN
NOH N H 2
Fig 8: IR spectrum of 7-amino-9-hydroxy-6,8-diazabenzo[a][1,4]benzothiazino[3,2- c]phenothiazine
80
N
N
N
S N
SN
N
O H
OH
EtS
SEt
Fig 9: IR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15- tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
81
Fig 10: IR spectrum of 8,13-dimethoxy-9,12- diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
N
N
S N
S
N
MeO
OMe
82
N
N
N
S
S
N
N
N
O H
O H
SEt
EtS
Cl
Cl
Fig 11: IR spectrum of 6,13-dichloro-3,10-diethylthio-1,8-dihydroxy-2,4,9,11- tetrazatriphenodithiazine
83
Fig 12: IR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8- triazatriphenodithiazine
N
N
S
S
N
N
NMeO
Cl
Cl
OH
Me
84
N
S N
SN
NOH N H 2
Fig 13: 1H-NMR spectrum of 7-amino-9-hydroxy-6,8- diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
85
N
S N
SN
NOH N H 2
Fig 14: 13C-NMR spectrum of 7-amino-9-hydroxy-6,8- diazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
86
N
N
N
S N
SN
N
OH
OH
EtS
SEt
Fig 15: 1H-NMR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15- tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
87
N
N
N
S N
SN
N
OH
OH
EtS
SEt
Fig 16: 13C-NMR spectrum of 7,14-diethylthio-9,12-dihydroxy-6,8,13,15- tetraazabenzo[a][1,4]benzothiazino[3,2-c]phenothiazine
88
Fig 17: 1H-NMR spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2- c]phenothiazine
N
N
S N
S
N
MeO
OMe
89
Fig 18: 13C-NMR spectrum of 8,13-dimethoxy-9,12-diazabenzo[a][1,4]benzothiazino[3,2- c]phenothiazine
N
N
S N
S
N
MeO
OMe
90
Fig 19:1H-NMR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8- triazatriphenodithiazine
N
N
S
S
N
N
NMeO
Cl
Cl
OH
Me
91
Fig 20:13C-NMR spectrum of 6,13-dichloro-3-methyl-9-methoxy-1-hydroxy-2,4,8- triazatriphenodithiazine
N
N
S
S
N
N
NMeO
Cl
Cl
OH
Me