Study and synthesis of biologically active phenothiazines, their sulfones, and ribofuranosides

Article abstract of DOI:10.1080/15257770.2012.719657

The present article describes the synthesis of new 10H-phenothiazines using the Smiles rearrangement. These synthesized phenothiazines on oxidation with 30% hydrogen peroxide in glacial acetic acid yield sulfones, and when treated with sugar give ribofura

Full text of DOI:10.1080/15257770.2012.719657

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Study and Synthesis of Biologically Active
Phenothiazines, their Sulfones, and
Ribofuranosides
Nishidha Khandelwal a , Abhilasha Yadav a , Naveen Gautam a & D.
C. Gautam a
a Department of Chemistry , University of Rajasthan , JLN Marg,
Jaipur , India
Published online: 24 Sep 2012.
To cite this article: Nishidha Khandelwal , Abhilasha Yadav , Naveen Gautam & D. C. Gautam (2012)
Study and Synthesis of Biologically Active Phenothiazines, their Sulfones, and Ribofuranosides,
Nucleosides, Nucleotides and Nucleic Acids, 31:9, 680-691, DOI: 10.1080/15257770.2012.719657
To link to this article: http://dx.doi.org/10.1080/15257770.2012.719657
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Nucleosides, Nucleotides and Nucleic Acids, 31:680–691, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.719657
STUDY AND SYNTHESIS OF BIOLOGICALLY ACTIVE
PHENOTHIAZINES, THEIR SULFONES, AND RIBOFURANOSIDES
Nishidha Khandelwal, Abhilasha Yadav, Naveen Gautam,
and D. C. Gautam
Department of Chemistry, University of Rajasthan, JLN Marg, Jaipur, India
The present article describes the synthesis of new 10H-phenothiazines using the Smiles rearrange-
2
ment. These synthesized phenothiazines on oxidation with 30% hydrogen peroxide in glacial acetic
acid yield sulfones, and when treated with sugar give ribofuranosides. These compounds are evalu-
ated for their anthelmintic and antimicrobial activities. The structural assignment of the synthesized
compounds is made on the basis of elemental analysis and spectroscopic data.
Keywords Smiles rearrangement; sulfones; ribofuranosides; anthelmintic activities;
antimicrobial activities
INTRODUCTION
The study of synthesized, biologically active molecules, such as phenoth-
iazines and their sulfones, has attracted interest in recent years due to their
theoretical and structural implications. The diversity of synthetic methods
as well as their biological, pharmacological, and industrial significance also
makes them important for research. These compounds are of immense im-
portance and possess a wide spectrum of pharmacological activities such
as analgesic, antihypertensive, antipsychotic, antibacterial, anti-AIDS., etc.
A slight change in substitution pattern may cause significant differences in
their biological activity.[1–9] Therefore, these observations encourage us to
synthesize new phenothiazines.
Received 29 January 2012; accepted 3 August 2012.
The authors are thankful to the Department of Chemistry, University of Rajasthan, Jaipur, for
providing laboratory facilities and spectral data analysis. The authors are also thankful to Rajiv Academy
for Pharmacy, Mathura, for assistance in carrying out the biological activities. UGC (Major Research
Project) and UGC Research Award Scheme, UGC, New Delhi are duly acknowledged for financial
support.
Address correspondence to Nishidha Khandelwal, Department of Chemistry, University of
Rajasthan, JLN Marg, Jaipur 302004, Rajasthan, India. E-mail: nishidha.khandelwal@gmail.com
680

Study and Synthesis of Biologically Active Phenothiazines
681
The phenothiazines serve as heterocyclic base for the formation of ribo-
furanosides for treatment with sugar. On refluxing with hydrogen peroxide
in glacial acetic acid, 10H-phenothiazines yielded 10H-phenothiazine-
5,5-dioxides. Prepared ribofuranosides and sulfones too possess similar
chemotherapeutic activities.[10–15] The synthesized compounds were
screened for their antimicrobial and antihelmintic activities. The structures
of the synthesized compounds were determined on the basis of their
spectral data and elemental analysis.
MATERIALS AND METHODS
All the melting points were determined in open capillary tubes and
1
are uncorrected. H NMR and 13C NMR spectra (by broadband proton
decoupling technique) were recorded on JEOL AL-300 spectrometer at fre-
quencies of 300.40 MHz and 75.45 MHz, respectively, in Me2SO-d6/CDCl3
using Tetramethylsilane (TMS) as an internal standard. Infrared (IR) spec-
tra were recorded in KBr on SHIMADZU 8400 S FTIR spectrophotometer.
Mass spectra were recorded on WATERS (micromass MS technologies) Q-T
by electron spray ionization. 19F NMR spectra were recorded in CDCl3 using
CF3COOH as standard compound. The purity of compounds was checked
by thin layer chromatography (TLC) using silica gel “G” as an adsorbent,
visualizing these by UV light or in an iodine chamber.
General Procedure for the Synthesis of 2-Amino-2ꢀ-
nitrodiphenylsulfides (3a–c)
To a solution of 2-amino-6-chloro-3-methoxybenzenethiol (1a) or 2-
amino-3-chloro-benzenethiol (1b) (0.01 mol) in ethanol (20 mL) contain-
ing anhydrous sodium acetate (0.01 mol) in a 50-mL round bottom flask,
a solution of halonitrobenzene, 2-chloro-3,5-dinitrobenzotrifloride (2a) or
2,4-dichloro-5-nitrobenzotrifluoride (2b) (0.01 mol) in ethanol (10 mL) was
added. The reaction mixture was refluxed for 4–5 hours. The resultant so-
lution was cooled and kept overnight in an ice chamber. The solid obtained
was filtered, washed with 30% ethanol, and recrystallized with methanol.
General Procedure for the Synthesis of 2-Formamido-2ꢀ-
nitrodiphenylsulfides (4a–c)
The 2-amino-2ꢁ-nitrodiphenylsulfide (3a–c) (0.01 mol) obtained above
was refluxed for 4 hours in 90% formic acid (20 mL). The contents were
then poured into crushed ice and the solid was filtered, washed with water,
and recrystallized from benzene.

682
N. Khandelwal et al.
General Procedure for the Synthesis of 10H-Phenothiazines (5a–c)
To the formyl derivative (4a–c) (0.01 mol), acetone (15 mL) and an
alcoholic solution of potassium hydroxide (0.2 g in 5-mL ethanol) were
added and the resulting mixture was heated at 20◦C for about 30 minutes.
Then a second lot of potassium hydroxide (0.2 g in 5-mL ethanol) was added
and refluxing was continued for about 4 hours. The reaction mixture was
poured into crushed ice, filtered, washed with minimum amount of cold
water and then with ethanol (20 mL), and crystallized from benzene.
6-Chloro-1-trifluoromethyl-9-methoxy-3-nitro-10H-phenothiazine (5a)
Brown solid; m.p.: 165◦C; yield: 65%, IR (KBr): v 3320 (N–H), 1560 and
1370 (–NO2), 1315 and 1120 (–CF3), 2960 and 2872 (–OCH3), 1220 and
1
1050 (C–O–C), and 780 cm−1 (C-Cl); H NMR spectral data (300.40 MHz,
Me2SO-d6, δ ppm from TMS): δ 8.74 (s, 1H, N-H), 8.15–6.36 (m, 4H, Ar-H),
3.73 (s, 3H); 13C NMR (75.45 MHz, CDCl3, δ ppm from TMS): 121.8 (C-1),
109.2 (CF3 at C-1), 119.5 (C-2), 138.8 (C-3), 130.3 (C-4), 129.5 (C-6), 120.2
(C-7), 115.4 (C-8), 150.5 (C-9), 55.8 (OCH3 at C-9); 19F NMR (282.65 MHz,
CDCl3): δ –62.865 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.): 376 [M]+,
378 [M+2]+, 375 (28), 330 (66), 211 (100), 261 (76), 165 (54); Anal. Calcd.
for C14H8N2SO3ClF3: C, 44.68; H, 2.13; N, 7.45; Found: C, 44.73; H, 2.16; N,
7.44.
1,8-Dichloro-7-trifluoromethyl-10H-phenothiazine (5b)
Red solid; m.p.: 162◦C; yield: 64%, IR (KBr): v 3310 (N–H), 1310 and
1
1130 (–CF3) and 775 cm−1 (C-Cl); H NMR spectral data (300.40 MHz,
Me2SO-d6, δ ppm from TMS): δ 8.78 (s, 1H, N-H), 7.78–6.47 (m, 5H, Ar-H);
13C NMR (75.45 MHz, CDCl3, δ ppm from TMS): 123.5 (C-1), 128.5 (C-2),
119.8 (C-3), 130.2 (C-4), 129.8 (C-6), 121.4 (C-7), 109.7 (CF3 at C-7), 130.1
(C-8), 119.5 (C-9); 19F NMR (282.65 MHz, CDCl3): δ –61.55 (s, 3F, CF3); MS
(FAB) 10 kV, m/z (rel. int.): 336 [M]+, 338 [M+2]+, 335 (28), 265 (65), 181
(100), 232 (56); Anal. Calcd. for C13H6NSCl2F3: C, 46.43; H, 1.78; N, 4.17;
Found: C, 46.48; H, 1.80; N, 4.16.
9-Chloro-1-trifluoromethyl-3-nitro-10H-phenothiazine (5c)
Brown solid; m.p.: 74◦C; yield: 68%, IR (KBr): v 3340 (N–H), 1345
1
and 1120 (–CF3), 1550 and 1315 (–NO2) and 800 cm−1 (C–Cl); H NMR
spectral data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.56 (s, 1H,
N–H), 8.11–6.69 (m, 5H, Ar-H); 13C NMR (75.45 MHz, CDCl3, δ ppm from
TMS): 121.8 (C-1), 109.3 (CF3 at C-1), 119.6 (C-2), 138.6 (C-3), 130.1 (C-4),
129.8 (C-6), 120.2 (C-7), 128.0 (C-8), 123.5 (C-9); 19F NMR (282.65 MHz,
CDCl3): δ –61.745 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.): 346 [M]+,
348 [M+2]+, 345 (25), 181 (100), 300 (46), 277 (59), 253 (63); Anal. Calcd.

Study and Synthesis of Biologically Active Phenothiazines
683
for C13H6N2O2SCl F3: C, 45.08; H, 1.73; N, 8.09; Found: C, 45.14; H, 1.72;
N, 8.11.
General Procedure for the Synthesis of 10H-phenothiazine-
5,5-dioxides (6a–c)
A mixture of substituted 10H-phenothiazines (5a–c) (0.01 mol), 20-mL
glacial acetic acid, and 30% hydrogen peroxide (5 mL) in round bottom
flask was refluxed for 15 minutes at 50–60◦C, then additional hydrogen per-
oxide (5 mL) was added. The reaction mixture was refluxed for 4 hours. The
contents were then poured into a beaker containing crushed ice. Residue
obtained was filtered, washed with water, and crystallized from ethanol.
6-Chloro-1-trifluoromethyl-9-methoxy-3-nitro-10H-phenothiazine-5,5-
dioxide (6a)
Brown solid; m.p.: 220◦C; yield: 48%, IR (KBr): v 3323 (N–H), 1561
and 1377 (–NO2), 1318 and 1122 (–CF3), 2960 and 2875 (–OCH3), and
780 cm−1 (C-Cl), 1170 and 1155 (SO2 sym), and 1070 cm−1 (C–S); 1H NMR
spectral data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.96 (s, 1H,
N-H), 8.58–6.51 (m, 4H, Ar-H), 3.76 (s, 3H, OCH3); 13C NMR (75.45 MHz,
CDCl3, δ ppm from TMS): 122.3 (C-1), 109.7 (CF3 at C-1), 126.1 (C-2), 139.0
(C-3), 126.0 (C-4), 125.1 (C-6), 120.3 (C-7), 121.2 (C-8), 150.3 (C-9), 55.9
(OCH3 at C-9); 19F NMR (282.65 MHz, CDCl3): δ –62.23 (s, 3F, CF3); MS
(FAB) 10 kV, m/z (rel. int.): 408 [M]+, 410 [M+2]+, 407 (24), 362 (65),
243 (100), 293 (70), 197 (59); Anal. Calcd. for C14H8N2SO5ClF3: C, 41.17;
H, 1.96; N, 6.86; Found: C, 41.23; H, 1.98; N, 6.85.
1,8-Dichloro-7-trifluoromethyl-10H-phenothiazine-5,5-dioxide (6b)
Black solid; m.p.: 211◦C; yield: 51%, IR (KBr): v 3320 (N–H), 1318 and
1121 (–CF3), 778 (C-Cl), 1180 and 1149 (SO2 sym), and 1080 cm−1 (C–S); 1H
NMR spectral data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 9.13 (s, 1H,
N-H), 8.12–6.73 (m, 5H, Ar-H); 13C NMR (75.45 MHz, CDCl3, δ ppm from
TMS): 124.1 (C-1), 134.9 (C-2), 120.5 (C-3), 125.6 (C-4), 125.7 (C-6), 121.9
(C-7), 110.3 (CF3 at C-7), 136.6 (C-8), 119.7 (C-9); 19F NMR (282.65 MHz,
CDCl3): δ –61.79 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.): 368 [M]+,
370 [M+2]+, 367 (27), 333 (46), 298 (50), 213 (100), 264 (74); Anal. Calcd.
for C13H6NSCl2O2F3: C, 42.39; H, 1.63; N, 3.80: Found: C, 42.43; H, 1.60; N,
3.85.
9-Chloro-1-trifluoromethyl-3-nitro-10H-phenothiazine-5,5-dioxide (6c)
Brown solid; m.p.: 196◦C; yield: 56%, IR (KBr): v 3345 (N–H), 1344
and 1140 (–CF3), 1558 and 1321 (–NO2), 1344 and 1146 (–CF3), 800 cm−1
1
(C–Cl), 1175 and 1164 (SO2 sym) and 1095 cm−1 (C–S); H NMR spec-
tral data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.56 (s, 1H, N–H),

684
N. Khandelwal et al.
8.11–6.69 (m, 5H, Ar-H); 13C NMR (75.45 MHz, CDCl3, δ ppm from TMS):
121.8 (C-1), 109.9 (CF3 at C-1), 126.8(C-2), 139.6 (C-3), 126.5 (C-4), 124.9
(C-6), 121.1 (C-7), 135.5 (C-8), 124.9 (C-9); 19F NMR (282.65 MHz, CDCl3):
δ –62.39 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.): 378 [M]+, 380
[M+2]+, 377 (29), 213 (100), 332 (46), 309 (64), 285 (66); Anal. Calcd.
for C13H6N2O4SCl F3: C, 41.26; H, 1.58; N, 7.41; Found: C, 41.34; H, 1.60;
N, 7.40.
General Procedure for the Synthesis of Substituted
N-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl) Phenothiazines (7a–c)
To the solution of (5a-c) (0.002 mol) in 20-mL toluene, 1-O-acetyl-2,3,5-
tri-O-benzoyl-β-D-ribofuranose (0.002 mol) was added and the contents were
refluxed in vacuum with stirring on an oil bath at 155–160◦C for 15 minutes.
The vacuum was removed and the reaction was protected from moisture
through a guard tube. Stirring was further continued for 10 hours and a
vacuum was applied for 10 minutes after every 1 hour. The viscous mass
thus obtained was dissolved in methanol, boiled for 10 minutes, and cooled
to room temperature. The reaction mixture was filtered and the methanol
was removed by distillation under reduced pressure. The viscous residue
thus obtained was dissolved in ether, filtered, concentrated, and kept in
refrigerator overnight to get crystalline ribofuranosides.
N-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-6-chloro-1-trifluoromethyl-9-
methoxy-3-nitro-10H-phenothiazine (7a)
Red solid; m.p.: 220◦C, yield: 81%, IR (KBr): v 1565 and 1380 (–NO2),
1320 and 1125 (–CF3), 2961 and 2872 (–OCH3) and 1145 cm−1 (C–O–C); 1H
NMR spectral data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.67–6.70
(m, 19H, Ar-H), 3.74 (s, 3H, OCH3), 4.34 (s, 2H, CH2 near C-4ꢁ of sugar),
5.52 (s, 1H, H at C-1ꢁ of sugar), 5.22 (s, 1H, H at C-2ꢁ of sugar), 4.85 (s, 1H,
H at C-3ꢁ of sugar), 5.01 (s, 1H, H at C-4ꢁ of sugar); 13C NMR (75.45 MHz,
CDCl3, δ ppm from TMS): 122.6 (C-1), 109.9 (CF3 at C-1), 120.5 (C-2), 139.4
(C-3), 130.8 (C-4), 130.0 (C-6), 120.7 (C-7), 114.0 (C-8), 150.5 (C-9), 56.4
(OCH3 at C-9), 88.4 (C-1ꢁ), 74.8 (C-2ꢁ), 71.1 (C-3ꢁ), 72.3 (C-4ꢁ), 65.8 (CH2
near C-4ꢁ of sugar); 19F NMR (282.65 MHz, CDCl3): δ –62.865 (s, 3F, CF3);
MS (FAB) 10 kV, m/z (rel. int.): 820 [M]+, 822 [M+2]+, 751 (41), 774 (69),
375 (58), 210 (100); Anal. Calcd. for C40H28N2SO10ClF3: C, 58.54; H, 3.4; N,
3.41; Found: C, 58.65; H, 3.40; N, 3.44.
N-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1,8-dichloro-7-
trifluoromethyl-10H-phenothiazine (7b)
Brown solid; m.p.: 186◦C; yield: 76%, IR (KBr): v 1350 and 1156 (–CF3)
1
and 1165 cm−1 (C–O–C); H NMR spectral data (300.40 MHz, Me2SO-d6,
δ ppm from TMS): 8.28–7.01 (m, 20H, Ar-H), 4.36 (s, 2H, CH2 near C-4ꢁ

Study and Synthesis of Biologically Active Phenothiazines
685
of sugar), 5.55 (s, 1H, H at C-1ꢁ of sugar), 5.18 (s, 1H, H at C-2ꢁ of sugar),
4.88 (s, 1H, H at C-3ꢁ of sugar), 5.06 (s, 1H, H at C-4ꢁ of sugar); 13C NMR
(75.45 MHz, CDCl3, δ ppm from TMS): 124.6 (C-1), 129.2 (C-2), 120.7 (C-3),
129.5 (C-4), 131.3 (C-6), 121.9 (C-7), 110.4 (CF3 at C-7), 130.6 (C-8), 120.4
(C-9), 87.3 (C-1ꢁ), 74.7 (C-2ꢁ), 71.5 (C-3ꢁ), 70.4 (C-4ꢁ), 65.6 (CH2 near C-4ꢁ
of sugar); 19F NMR (282.65 MHz, CDCl3): δ –62.18 (s, 3F, CF3); MS (FAB)
10 kV, m/z (rel. int.): 780 [M]+, 782 [M+2]+, 745 (31), 711 (53), 335 (64),
180 (100); Anal. Calcd. for C39H26NSCl2O7F3: C, 60; H, 3.33; N, 1.79; Found:
C, 60.2; H, 3.3; N, 1.80.
N-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-9-chloro-1-trifluoromethyl-3-
nitro-10H-phenothiazine (7c)
Red solid; m.p.: 179◦C; yield: 85%, IR (KBr): v 1350 and 1170 (–CF3),
1
1580 and 1346 (–NO2) and 1160 cm−1 (C–O–C); H NMR spectral data
(300.40 MHz, Me2SO-d6, δ ppm from TMS): δ s8.28–7.01 (m, 20H, Ar-H),
4.41 (s, 2H, CH2 near C-4ꢁ of sugar), 5.50 (s, 1H, H at C-1ꢁ of sugar), 5.21
(s, 1H, H at C-2ꢁ of sugar), 4.91 (s, 1H, H at C-3ꢁ of sugar), 4.97 (s, 1H, H
at C-4ꢁ of sugar); 13C NMR (75.45 MHz, CDCl3, δ ppm from TMS): 122.6
(C-1), 110.2 (CF3 at C-1), 119.3 (C-2), 139.4 (C-3), 131.1 (C-4), 130.4 (C-6),
119.4 (C-7), 128.8 (C-8), 124.3 (C-9), 87.6 (C-1ꢁ), 74.6 (C-2ꢁ), 71.3 (C-3ꢁ),
70.8 (C-4ꢁ), 65.7 (CH2 near C-4ꢁ of sugar); 19F NMR (282.65 MHz, CDCl3): δ
–62.74 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.) : 790 [M]+, 792 [M+2]+,
744 (68), 721 (56), 345 (71), 180 (100); Anal. Calcd. for C39H26N2O9SCl F3:
C, 59.24; H, 3.29; N, 3.54; Found: C, 59.36; H, 3.28; N, 3.56.
Biological Activity
In Vitro Antibacterial and Antifungal Activity
Antibacterial Activity. Antibacterial activity was tested against S. aureus,
B. subtilis (gram +ve), and E. coli, Pseudomonas aeruginosa (gram –ve) mi-
croorganisms using paper disc diffusion method in nutrient agar medium.
The paper disc diffusion method of assay of drug potency is based on the
measurement of the zone of microbial growth inhibition surrounding discs
containing various concentrations of test compounds, which are placed on
the surface of a solid nutrient previously inoculated with the culture of suit-
able microorganism. Inhibition produced by the test drug is compared with
that produced by known concentration of reference standard.
In this method, paper disc impregnated with compounds dissolved in
solvent DMF at concentrations of 25, 50, and 100 µg mL−1. Then the disc
impregnated with the solution was placed on the surface of the media inocu-
lated with the bacterial strain. The plates were incubated at 35◦C for 24 hours
for bacterial cultures. After incubation, the zones of inhibition around the
disc were observed. Each testing is done in triplicate. Ciprofloxacin at a
concentration of 50 µg mL−1 was used as standard drug for antibacterial

686
N. Khandelwal et al.
activity. Results were interpreted in terms of diameter (mm) of zone of inhi-
bition. The % Activity Index for the complex was calculated by the following
formula:
Zone of inhibition by test compound (diameter)
% Activity Index=
×100.
Zone of inhibition by standard (diameter)
Antifungal Activity. Antifungal activity of synthesized compounds was
tested on fungal strains: A. niger, C. albican using the disc diffusion method.
In the disc-diffusion method, disc impregnated with compounds dissolved
in solvent DMF at concentrations of 25, 50, and 100 µg mL−1 was spread
over microorganism culture in nutrient agar medium. The plates were incu-
bated at 25◦C for 48 hours for fungal strains. After incubation, the growth
inhibiting zones around the disc were observed. Growth inhibiting zone
indicates that the compounds inhibit growth of microorganism. Each exper-
iment is done in triplicate. Griseofulvin at a concentration of 50 µg mL−1
was used as standard drug for antifungal activity. Results were interpreted in
terms of diameter (mm) of zone of inhibition. The percentage inhibition
was calculated by the following equation:
% Inhibition = (C − T) 100/C,
where C and T are the diameters of the fungal colony in the control and the
test plates respectively.
Minimal Inhibitory Concentrations. Minimum inhibitory concentrations
(MIC) are defined as the lowest concentration of antimicrobials that in-
hibit the visible growth of a microorganism after overnight incubation at
37◦C. Determination of the MIC is a semi-quantitative test, which gives an
approximate idea of the least concentration of an antimicrobial (test) solu-
tion needed to parent microbial growth. The MIC was determined by the
liquid dilution method. Two gram +ve bacteria (Staphylococcus aureus and
Bacillus subtilis) and two gram –ve bacteria (Escherichia coli and Pseudomonas
aeruginosa) were used as quality control strains. For the antifungal activities
of the compounds Candida albicans and Aspergilius niger were tested.
Ciprofloxacin and Griseofulvin were used as standard antibacterial and
antifungal agents respectively. The stock solutions of test compounds with 1
to 20-µg mL−1 concentrations were prepared with aqueous methanol. Inocu-
lums of the overnight culture were prepared. In a series of tubes, 1 mL each
of stock solutions of test compound with different concentrations was taken
and 0.4 mL of the inoculums was added to each tube. Further, 4.0 mL of ster-
ile water was added to each of the test tubes. These test tubes were incubated
for 22–24 hours and observed for the presence of turbidity. The absorbance
of the suspension of the inoculums was observed with spectrophotometer at

Study and Synthesis of Biologically Active Phenothiazines
687
555 nm. The end result of the test was the minimum concentration of an-
timicrobial (test) solutions, which gave clear solution, i.e., no visual growth.
Anthelmintic Activity
Anthelmintic studies were carried out against the Eudrilus species of
earthworm by the Garg and Atal method[16] at 4-µg mL−1 concentration.
Suspensions of samples were prepared by triturating synthesized compounds
(200 mg) with Tween 80 (0.5%) and distilled water, and the resulting mix-
tures were stirred for 30 min using a mechanical stirrer. The suspensions
were diluted to contain 0.4% w/v of test samples. Suspension of reference
drug mebendazole was prepared with same concentration in a similar way.
Three sets of five earthworms of almost similar sizes (3 inch in length) were
placed in petri plates of 4-inch diameter containing 50 mL of suspension
of test sample and reference drug at room temperature. Another set of five
earthworms was kept as control in 50-mL suspension of distilled water and
Tween 80 (0.5%). The paralyzing and death times (in minutes) were noted
and their mean was calculated for triplicate sets. The death time was ascer-
tained by placing the earthworms in warm water (50◦C), which stimulated
movement if the worms were alive.
RESULTS AND DISCUSSION
The synthesis of various substituted 10H-phenothiazines (5a–c)
has been carried out by the Smiles rearrangement of 2-formamido-
2ꢁ-nitrodiphenylsulfides (4a–c). The formyl derivatives were synthe-
sized by the formylation of 2-amino-2ꢁ-nitrodiphenylsulfides (3a–c),
which in turn were prepared by condensation of 2-amino-6-chloro-3-
methoxybenzenethiol (1a) with 2-chloro-3,5-dinitrobenzotrifloride (2b) and
2-amino-3-chlorobenzenethiol (1b) with o-halonitrobenzene [2,4-dichloro-
5-nitrobenzotrifluoride (2a) and 2-chloro-3,5-dinitrobenzotrifluoride (2b)]
in ethanolic sodium acetate solution. Compounds (5a–c) on refluxing with
30% hydrogen peroxide in glacial acetic acid were converted into their cor-
responding sulfones (6a–c). Treatment of the mixture of (5a–c) in toluene
with 1-O-Acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose in vacuum gave the cor-
responding ribofuranosides (7a–c) (Scheme 1).
The structures proposed for the synthesized compounds were well sup-
ported by elemental analysis and spectral data. The synthesized compounds
were also screened for antimicrobial and anthelmintic activities.
Antimicrobial Activity
A series of novel heterocyclic compounds (3a–d) and (4a–d) were syn-
thesized. All the compounds were tested against for their in vitro antibac-
terial activity against the four strains of bacteria (two gram –ve, E. coli and

688
N. Khandelwal et al.
R1
R1
R2
NH2
SH
NO2
+
R5
C2H5OH
NaOH
NH2 NO2
R5
R4
CH3COONa
C2H5OH
R4
X
S
R2
R3
2a-b
R3
3a-c
1a-b
Formylation
90% HCOOH
CHO
H
N
R1
R1
9
R3
1
10
NH NO2
R5
2
8
7
R4
Acetone
3
R5
KOH/EtOH
S
R4
S
5
6
R2
4
R2
R3
5a-c
4a-c
BzO
OC
OCH3
O
B
zO
30% H2O2
Bz = Benzoyl group
gl. Acetic Acid
Toluene under vaccum
155-160oC
R2
6
H
N
5
R1
9
R3
1
4
S
3
R5
R4
7
10
R4
2
8
2
8
7
3
R5
N
S
5
10
6
R2
9
R1
1
R3
4
O
O
BzO
4´
O
6a-c
1´
2´
3´
BzO OBz
7a-c
Scheme--1
Compound
5a, 6a, 7a
5b, 6b, 7b
5c, 6c, 7c
R1
OCH3
Cl
R2
Cl
H
R3
R4
H
R5
CF3
H
NO2
CF3
Cl
SCHEME 1

Study and Synthesis of Biologically Active Phenothiazines
689
TABLE 1 Minimum inhibitory concentrations (µg mL–1) of synthesized compounds
Minimal inhibitory concentrations (µgm mL–1) of bacterial strains
Cpd. No.
E. coli
B. subtilis
P. aeruginosa
S. aureus
5a
5b
5c
6a
6b
6c
7a
7b
11.42 0.25
12.30 0.17
11.10 0.11
09.60 0.18
11.00 0.37
09.20 0.09
10.80 0.22
11.36 0.08
10.26 0.32
04.10 0.10
13.30 0.14
14.20 0.18
13.10 0.20
11.25 0.33
11.90 0.19
09.00 0.12
11.96 0.23
12.30 0.31
10.11 0.22
04.90 0.13
13.34 0.32
15.20 0.27
11.40 0.17
11.24 0.18
12.05 0.26
10.10 0.35
11.60 0.10
12.64 0.33
10.55 0.19
03.85 0.15
11.20 0.29
11.82 0.33
10.94 0.29
10.39 0.23
11.16 0.32
09.80 0.81
10.85 0.23
11.54 0.31
10.41 0.28
04.90 0.11
7c
Ciprofloxacin
Pseudomonas aeruginosa, and two gram +ve, Bacillus subtilis and Staphylococcus
aureus) and antifungal activity against the two strains of fungi (C. albicans and
A. niger). The antibacterial and antifungal activities increase with an increase
in the concentration of test compounds. The MIC of the compounds varies
from 9.00–15.20 µg mL−1 (Tables 1 and 2). Compounds (6a), (6c), (7a),
and (7c) show good activity and the remaining compounds show moderate
activity against bacteria E. coli. Compounds (6c) and (7c) show good activity
and the remaining compounds show moderate activity against bacteria B.
subtilis. For bacteria P. aeruginosa, compound (6c) shows good activity and
the remaining compounds show moderate activity. Compounds (6a) and
(7c) show good activity and the remaining compounds show moderate ac-
tivity against bacteria S. aureus. Compounds (6c) and (7c) show good activity
against fungi C. albicans and A. niger and the remaining compounds show
moderate activity.
TABLE 2 Minimum inhibitory concentration (MIC; µg mL–1) of synthesized compounds
MIC (µgm mL–1) of fungal strains
Cpd. No.
C. albicans
A. niger
5a
5b
5c
6a
6b
6c
7a
7b
12.25 0.17
12.57 0.20
11.00 0.21
11.47 0.25
11.79 0.31
10.50 0.19
11.96 0.33
12.05 0.29
10.90 0.28
03.10 0.80
11.99 0.17
13.00 0.25
11.25 0.21
11.34 0.24
11.71 0.19
10.54 0.35
11.68 0.32
12.08 0.34
10.90 0.22
04.80 0.10
7c
Griseofulvin

690
N. Khandelwal et al.
TABLE 3 Anthelmintic activity of synthesized compounds
Paralyzing time (in minutes)
Death time (in minutes)
Cpd. No.
200 mg mL–1 100 mg mL–1 50 mg mL–1 200 mg mL 100 mg mL–1 50 mg mL–1
5a
5b
5c
6a
6b
6c
7a
7b
7c
14.83 0.83 17.33 1.52 23.36 1.00 19.31 1.00 25.16 0.76
15.00 1.09 18.69 0.79 25.00 0.70 20.16 1.22 27.16 0.76
13.26 1.08 16.50 1.20 21.55 1.00 17.33 1.52 22.20 1.00
13.50 1.00 16.66 0.94 22.50 0.50 18.16 0.76 24.65 0.79
15.00 0.77 18.00 0.78 23.45 0.93 20.16 0.90 25.88 1.00
12.00 1.00 15.00 1.00 20.03 0.68 15.40 0.77 21.11 0.77
14.00 1.10 17.00 1.00 22.20 1.00 19.12 0.60 24.13 1.09
15.16 1.24 18.55 1.05 24.56 0.70 19.50 0.50 26.06 0.76
13.16 0.75 16.00 1.10 21.00 1.00 15.53 0.69 24.13 1.09
35.23 0.91
40.21 0.25
34.50 0.50
35.23 0.91
37.53 0.55
32.22 0.63
35.50 0.50
38.43 1.00
34.12 0.60
30.55 1.00
Mebendazole 12.50 1.04 16.00 0.98 21.80 1.34 15.26 0.84 22.65 0.90
Anthelmintic Activity
The above-screened compounds were tested for anthelmintic activity.
The results have been summarized in Table 3. The results show that com-
pounds (5c), (6a), (7a), and (7c) have longer paralyzing periods than the
standard drug mebendazole at given concentrations. The synthesized com-
pounds have somewhat better activity against the Eudrilus species of earth-
worm than does mebendazole.
CONCLUSION
The structures proposed for the synthesized compounds were well
supported by elemental analysis and spectroscopic data. The MIC values of
antibacterial and antifungal screening show that the compounds bearing
electron withdrawing groups, such as nitro and trifluoromethyl, exhibit
excellent antibacterial and antifungal activities against all the four strains
of bacteria and fungi respectively. In general, the presence of electron
withdrawing group on the aromatic ring increases the antimicrobial activity
of tested compounds compared with compounds having electron-donating
groups. The literature shows that the compounds having methoxy, ni-
tro groups represent good anthelmintic activity. Thus, the synthesized
compounds show significant anthelmintic activity.
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