Synthesis and biological activity of substituted 3-fluoro/3-trifluoromethyl 10H-phenothiazines, its ribofuranosides and sulfones

Article abstract of DOI:10.1016/j.jfluchem.2011.04.012

The present article describes the synthesis of new fluorinated 10H-phenothiazines by Smiles rearrangement. The reaction of these synthesized phenothiazines with sugar (beta-d-ribofuranose 1-acetate-2,3,5-tribenzoate) afforded the new ribofuranosides and t

Full text of DOI:10.1016/j.jfluchem.2011.04.012

Journal of Fluorine Chemistry 132 (2011) 420–426
Contents lists available at ScienceDirect
Journal of Fluorine Chemistry
journal homepage: www.elsevier.com/locate/fluor
Synthesis and biological activity of substituted 3-fluoro/3-trifluoromethyl
10H-phenothiazines, its ribofuranosides and sulfones
a
b
b
a
Naveen Gautam a, , Kshamta Goyal , Omprakash Saini , Ashok Kumar , D.C. Gautam
*
a Department of Chemistry, University of Rajasthan, Jaipur 302004, India
b Department of Zoology, University of Rajasthan, Jaipur 302004, India
A R T I C L E I N F O
A B S T R A C T
Article history:
The present article describes the synthesis of new fluorinated 10H-phenothiazines by Smiles
rearrangement. The reaction of these synthesized phenothiazines with sugar (beta- -ribofuranose 1-
Received 15 December 2010
Received in revised form 15 April 2011
Accepted 20 April 2011
D
acetate-2,3,5-tribenzoate) afforded the new ribofuranosides and the oxidation of these synthesized
phenothiazines with 30% hydrogen peroxide in glacial acetic acid resulted in the formation of 10H-
phenothiazine sulfones. These compounds were evaluated for their antioxidant and antimicrobial
activities (using broth microdilution method). The structural assignments of the synthesized compounds
were made on the basis of elemental analysis and spectroscopic data.
Available online 27 April 2011
Keywords:
Smiles rearrangement
Ribofuranosides
Sulfones
ß 2011 Elsevier B.V. All rights reserved.
Antioxidant activities
Antimicrobial activities
1. Introduction
2. Results and discussion
Two series of fluorinated 10H-phenothiazines were prepared by
treating 2-aminobenzenethiols (1a–b) with o-halonitrobenzenes
(2a–c). In first series, substituted 10H-phenothiazine (5a) can be
accomplished via Smiles rearrangement of substituted 2-forma-
mido-20-nitrodiphenylsulfide (4a). The formyl derivative was
prepared by the formylation of 2-amino-20-nitrodiphenylsulfide
(3a), which in turn was prepared by condensation of 2-amino-5-
methoxybenzenethiol (1a) with 1,4-difluoro-2-nitrobenzene (2a)
in ethanolic sodium acetate solution. In second series, 10H-
phenothiazines (5b,c) were prepared by condensation of 2-amino-
The vogue of constructing biologically active molecules such as
phenothiazines, their ribofuranosides and sulfones by molecular
modification has been of enormous interest in recent years because
of their theoretical and structural implications, the diversity of
synthetic methods used in their preparation, and their biological,
pharmacological and industrial significance.
These moieties are extensively used as tranquilizers, sedatives,
antibacterial, antifungal, antitumor, anticancer, CNS depressants,
etc. A slight change in the substitution pattern in phenothiazine
nucleus causes distinguishable difference in their biological
activities [1–7]. Therefore, these observations prompted us to
synthesize new fluorinated phenothiazines. Fluorinated phe-
nothiazines serve as heterocyclic base for the formation of
ribofuranosides on treatment with sugar. On refluxing with
hydrogen peroxide in glacial acetic acid, 10H-phenothiazines
yielded 10H-phenothiazine-5,5-dioxides. These prepared ribofur-
anosides and sulfones too possess similar chemotherapeutic
activities. The synthesized compounds were screened for their
antioxidant [8–12] and antimicrobial activities [13–16].
5-methoxybenzenethiol
(1a)/2-amino-5-bromo-3-ethylbenze-
nethiol (1b) with reactive halonitrobenzenes [2,4-dichloro-3,5-
dinitrobenzotrifluoride (2b) and 4-chloro-3,5-dinitrobenzotri-
fluoride (2c)] respectively, (which have two nitro groups ortho
to the reactive halogen atom) in ethanolic sodium hydroxide,
where Smiles rearrangement occured in situ. Oxidation of 10H-
phenothiazines (5a–c) with 30% hydrogen peroxide in glacial
acetic acid yielded sulfones (6a–c). All synthesized phenothiazines
(5a–c) were treated with
b-D-ribofuranose-1-acetate-2,3,5-tri-
benzoate in toluene stirred in vacuum on an oil bath at 155–160 8C
for 10 h to form ribofuranosides (7a–c) (Scheme 1).
The structures proposed for the synthesized compounds were
well supported by elemental analysis and spectral data. The
synthesized compounds were also screened for their antioxidant
and antimicrobial activities (Scheme 2).
* Corresponding author. Fax: +91 141 2719008.
E-mail addresses: ngautam70@yahoo.co.in (N. Gautam), kshamtaa@gmail.com
(K. Goyal), drdcgautam@yahoo.co.in (O. Saini), opsaini08@gmail.com (A. Kumar),
mamsjpr@gmail.com (D.C. Gautam).
0022-1139/$ – see front matter ß 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2011.04.012

N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
421
R1
R1
NH2
SH
NO2
R5
R4
C2H5OH
NaOH
NH2 NO2
R5
R4
CH3COONa
C2H5OH
+
R2
X
R2
S
X
= Cl(2b,c),
X = F (2a)
R3
2a-c
R3 = NO2
R3
R3 = H
1a-b
3a
Formylation
90% HCOOH
H
R1
9
R3
CHO
R1
1
10
2
R4
8
7
N
Acetone
KOH/EtOH
NH NO2
R5
3
R5
R2
S
5
6
4
R2
S
R4
c
5a-
R3
4a
5
S
4
6
R2
3
R5
R4
7
BzO
OCOCH3
O
H
N
R1
9
R3
1
2
10
R4
8
8
BzO
Where Bz = benzoyl group
N
10
30% H2O2
9
2
1
R3
R1
Tol
uen
e und
er va
cuum
gl. Acetic Acid
R2
7
3 R5
BzO
4´
S
5
O
O
6
4
155-160°C
O
1´
2´
BzO OBz
3´
6a-c
Compound
5a, 6a, 7a
5b, 6b, 7b
5c, 6c, 7c
R
R2
R3
R4
R5
1
H
H
7a-c
OCH3
OCH3
Br
H
H
F
NO2
NO 2
Cl
H
CF 3
CF 3
C2H5
Scheme 1.
R1
R6
R1
R6
NH2
SH
NO2
R5
R4
C2H5OH
NaOH
NH2 NO2
R5
R4
CH3COONa
C2H5OH
+
R2
Cl
R2
S
R3 = NO2 (2c)
R
3 = H (2a,b)
R3
2a-c
R3
1a-b
3a,b
Formylation
90% HCOOH
H
R1
9
R3
1
10
CHO
R1
R6
2
R4
8
7
N
Acetone
NH NO2
R5
3
R5
KOH/EtOH
R2
S
5
6
4
R6
R2
S
R4
8a-c
R3
4a,b
R6
5
4
6
9
R2
S
3
R5
R4
7
BzO
OCOCH3
O
H
N
R1
9
R3
1
2
10
R4
8
8
BzO
Where Bz = benzoyl group
N
10
30% H2O2
gl. Acetic Acid
2
1
R3
R1
Tolu
ene u
nder
vacu
um
R2
7
3 R5
BzO
4´
S
5
O
O
6
4
155-160°C
R6
O
1´
2´
3´
9a-c
BzO OBz
Compound
8a, 9a, 10a
8b, 9b, 10b
8c, 9c, 10c
R1
H
R2
R3
H
R4
H
R5
NO2
H
R6
H
Cl
H
10a-c
OCH3
OCH3
Br
H
H
Cl
H
C2H5
NO2
COOH
Scheme 2.

422
N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
2.1. Antioxidant activity
activity as compared to their phenothiazines 5a–c respectively due
to electron withdrawing effect of oxygen in 6a–c. Compounds 5a–c
showed good antioxidant activity as compared to their ribofurano-
sides 7a–c, respectively due to slight electron withdrawing effect
of ribofuranosyl moiety in the framework of 7a–c. In general, the
presence of electron donating groups on the aromatic ring increase
the antioxidant activities of tested compounds as compared to
compounds having electron withdrawing groups. Regarding
antimicrobial activity, the MIC values of antibacterial and
antifungal screening suggest that compounds 6a–c exhibited
significant antibacterial and antifungal activities as compared to
their phenothiazines 5a–c, respectively due to electron withdraw-
ing effect of oxygen in 6a–c. Compounds 5b, 7b and 5a, 7a bearing
electron donating group like methoxy group exhibited moderate
antibacterial and antifungal activities as compared to compounds
5c and 7c which showed good activity against all the four strains of
bacteria and fungi, respectively. In general the presence of electron
withdrawing group on the aromatic ring increase the antimicrobial
activities of tested compounds as compared to compounds having
electron donating groups.
The present study demonstrated that the synthesized com-
pounds showed mixed radical scavenging activity in both DPPH
and ABTSꢀ+ assays. These compounds showed good chemopreven-
tive and antigenotoxic effect.
(a) Compounds 5a and 7a showed strong radical scavenging
activity in DPPH assay that have DPPH% inhibition ꢁ 50.
(b) Compounds 5b, 5c and 7b showed moderate radical scavenging
activity in DPPH assay that have DPPH% inhibition ꢁ 30.
(c) Compounds 6a, 6b, 6c and 7c showed mild radical scavenging
activity in DPPH assay that have DPPH% inhibition < 30.
(d) Compounds 5a, 5b, 5c, 7a and 7b were found to be more active
in ABTSradicalradicalꢀ+ assays which showed much decline in
graph.
2.2. Antimicrobial activity
The present paper is focused on the synthesis of novel
fluorinated phenothiazine derivatives as possible antibacterial
and antifungal agents. A series of novel compounds 5a–c, 6a–c and
7a–c were prepared and tested for their in vitro antibacterial
activity against the four strains of bacteria (two gram positive and
two gram negative) and antifungal activity against the four strains
of fungi. Three compounds of the obtained series showed high in
vitro antimicrobial activity. Compounds 6a–c showed excellent
activity against all the four strains of bacteria (i.e. Bacillus cereus,
Staphylococcus aureus, E. coli DH5 alpha and Pseudomonas
aeruginosa). This indicated that all these three compounds showed
in vitro antibacterial activity lower than that of ampicillin sodium
salt. Compounds 5c and 7c showed good activity against all the
four strains of bacteria indicating in vitro antibacterial activity
slightly lower than that of ampicillin sodium salt. Compounds 5b,
7b and 5a, 7a showed moderate activity against all the four strains
of bacteria. This indicated that these compounds showed in vitro
antibacterial activity higher than ampicillin sodium salt.
4. Experimental
All the melting points were determined in open capillary
tubes and are uncorrected. 1H NMR and 13C NMR spectra (by
broad band proton decoupling technique) were recorded on JEOL
AL-300 spectrometer at frequencies of (300.40 MHz and
75.45 MHz) respectively, in Me2SO-d6/CDCl3 using TMS (Tetra-
methyl silane) as an internal standard. IR spectra were recorded
in KBr on SHIMADZU 8400 S FTIR spectrophotometer.19F NMR
spectra were recorded in CDCl3 using CF3COOH as standard
compound. The FAB mass spectra were recorded on a JEOL SX
102/DA-600 mass spectrometer using Ar/Xe as FAB(Fast atom
bombardment) gas. Purity of compounds were checked by TLC
using silica gel ‘‘G’’ as adsorbent, visualizing these by UV light or
in an iodine chamber.
4.1. General procedure for the synthesis of 2-amino-20-
Compounds 6a–c showed excellent activity against all the four
strains of fungi (Aspergillus niger, Aspergillus flavus, Fusarium
oxysporum and Rhizopus stolonifer). This indicated that all these
three compounds showed in vitro antifungal activity lower than
that of Ketoconazole. Compounds 5c and 7c showed good activity
against all the four strains of fungi indicating in vitro antifungal
activity slightly lower than that of ketoconazole. Compounds 5b,
7b and 5a, 7a showed moderate activity against all the four strains
of fungi. This indicated that these compounds showed in vitro
antifungal activity higher than that of ketoconazole.
nitrodiphenylsulfide (3a)
2-Amino-5-methoxybenzenethiol (1a) (0.01 mol) was dis-
solved in EtOH (20 mL) containing (0.01 mol) of anhydrous NaOAc
in a 50 mL round bottom flask and 1,4-difluoro-2-nitrobenzene
(2a) (0.01 mol) in 10 mL EtOH was added. The reaction mixture
was refluxed for 4–5 h. The resultant solution was cooled and kept
overnight in an ice chamber. The solid obtained was filtered,
washed with 30% EtOH and recrystallised with MeOH.
4.2. General procedure for the synthesis of 2-formamido-20-
2.3. Structure activity relationships
nitrodiphenylsulfide (4a)
Structure activity relationships between fluorinated derivatives
(6a–c) and the corresponding non-fluorinated derivatives (9a–c)
have been expressed in terms of pMIC by drawing comparison
plots against different bacteria and fungi. Fluorinated compounds
(6a–c) have superior antimicrobial properties (i.e. biologically
more active) than their corresponding non-fluorinated compounds
(9a–c) as it is evidenced by their antimicrobial activities expressed
in terms of MIC (Table 3, and Tables 4–6 and Fig. 2).
The 2-amino-20-nitrodiphenylsulfide (3a) (0.01 mol) obtained
above was refluxed for 4 h in 90% HCOOH (20 mL). The contents
were then poured into crushed ice and the solid was filtered,
washed with H2O and recrystallized from C6H6.
4.3. General procedure for the synthesis of fluorinated 10H-
phenothiazine (5a)
Formyl derivative (4a) (0.01 mol) in acetone (15 mL) was
refluxed and an alcoholic solution of KOH (0.2 g in 5 mL EtOH) was
added. These were then heated for 30 min. After then a second lot
of KOH (0.2 g in 5 mL EtOH was added and further refluxed for 4 h.
The contents were poured into a beaker containing crushed ice and
filtered. The residue was washed with cold H2O, then with 30%
EtOH and then crystallized from C6H6.
3. Conclusion
The structures proposed for the synthesized compounds were
well supported by elemental analysis and spectroscopic data.
Regarding both DPPH and ABTS assays, compounds 5a and 7a
showed excellent activity. Compound 6a–c showed moderate

N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
423
4.3.1. 3-Fluoro-7-methoxy-10H-phenothiazine (5a)
Black solid; m.p.: 90 8C; Yield: 67%, IR (KBr): 3360 (N–H), 1260
and 1040 (C–O–C), 2950 and 2835 (–OCH3), 1072 (C–S), and
1270 cmꢂ1 (C–F); 1H NMR spectral data (300.40 MHz, Me2SO-d6,
ppm from TMS): 8.92 (s, 1H, N–H), 7.01–6.35 (m, 6H, J1,2 = 7.9 Hz,
J8,9 = 8 Hz, Ar–H), 3.72 (s, 3H, –OCH3); 13C NMR (75.45 MHz, CDCl3,
bottom flask were refluxed for 15 min at 50–60 8C, then additional
H2O2 (5 mL) was added. The reaction mixture was refluxed for 4 h.
The contents were then poured into a beaker containing crushed
ice. Residue obtained was filtered, washed with H2O and
crystallized from EtOH.
v
d
d
d
ppm from TMS):
d
120.2 (C-1), 114.6 (C-2), 151.4 (C-3, d,
4.5.1. 3-Fluoro-7-methoxy-10H-phenothiazine-5,5-dioxide (6a)
1JCF = 249.0 Hz), 118.2 (C-4), 117.6 (C-6), 152.4 (C-7), 113.2 (C-8),
Brown solid; m.p.: 250 8C; Yield: 45%, IR (KBr): v 3360 (N–H),
118.9 (C-9), 123.1 (C-4a), 122.6 (C-5a), 137.8 (C-9a), 141.0 (C-10a),
2955 and 2841 (–OCH3), 1278 (C–F), 1175 and 1140 (SO2 sym), 580
and 560 (SO2 bend), 1338, 1280 and 1260 (SO2 asym), and
1080 cmꢂ1 (C–S); 1H NMR spectral data (300.40 MHz, Me2SO-d6,
d
55.7 (OCH3 at C7); 19F NMR (282.65 MHz, CDCl3):
d–109.367 (s, 1F, C–
F); MS (FAB) 10 kV, m/z(rel.int.): 247 [M]+ (100), 246 [M–H]+ (60),216
[M–OCH3]+ (45), 177 [M–C4H3F]+ (75); ‘‘Anal. Calcd for C13H10NOFS:
C, 63.15; H; 4.04; N, 5.66. Found: C, 63.38, H, 4.01; N, 5.61’’.
ppm from TMS):
d 9.15 (s, 1H, N–H), 8.01–6.40 (m, 6H,
J1,2 = 7.98 Hz, J8,9 = 8.2 Hz, Ar–H), 3.72 (s, 3H,–OCH3); 13C NMR
(75.45 MHz, CDCl3,
d ppm from TMS): d 120.7 (C–1), 121.6 (C-2),
4.4. General procedure for the synthesis of 1-nitro-10H-
151.9 (C-3, d, 1JCF = 249.3 Hz), 113.7 (C-4), 113.4 (C-6), 152.8 (C-7),
phenothiazines (5b,c)
119.7 (C-8), 119.3 (C-9), 129.8 (C-4a), 129.3 (C-5a), 133.3 (C-9a),
136.5 (C-10a), 55.7 (OCH3 at C7); 19F NMR (282.65 MHz, CDCl3):
d –
A mixture of 2-amino-5-methoxybenzenethiol (1a)/2-amino-
5-bromo-3-ethylbenzenethiol (1b) (0.01 mol), NaOH (0.01 mol),
20 mL absolute alcohol and 0.01 mol of substituted reactive o-
halonitrobenzene (2b,c) were taken in 50 mL round bottom flask
fitted with reflux condensor. The colour of the solution darkened
immediately. The reaction mixture was refluxed for 2 h, concen-
trated, cooled and filtered. The precipitate was washed well with
hot H2O, then with EtOH and crystallized from acetone.
108.967 (s, 1F, C–F); MS (FAB) 10 kV, m/z (rel. int.): 279 [M]+ (100),
278 [M–H]+ (34), 215 [M–SO2]+ (41), 188 [M–SO2–HCN]+ (52);
‘‘Anal. Calcd for C13H10NO3FS: C, 55.91; H; 3.58; N, 5.01. Found: C,
55.84, H, 3.52; N, 5.07’’.
4.5.2. 2-Chloro-3-trifluoromethyl-7-methoxy-1-nitro-10H-
phenothiazine-5,5-dioxide (6b)
Black solid; m.p.: 220 8C, yield: 53%, IR (KBr):
v 3281 (N–H),
1565 and 1340 (–NO2), 1355 and 1150 (–CF3), 2970 and 2881 (–
OCH3), 1180 and 1150 (SO2 sym), 570 and 555 (SO2 bend), 1340,
1292 and 1265 (SO2 asym), and 1095 cmꢂ1 (C–S); 1H NMR spectral
4.4.1. 2-Chloro-3-trifluoromethyl-7-methoxy-1-nitro-10H-
phenothiazine (5b)
Red solid; m.p.: 80 8C, yield: 82%, IR (KBr):
and 1320 (–NO2), 1340 and 1140 (–CF3), 2962 and 2872 (–OCH3),
1252 and 1030 (C–O–C), 1084 (C–S), and 800 cmꢂ1 (C–Cl); 1H NMR
v
3280 (N–H), 1550
data (300.40 MHz, Me2SO-d6,
8.32–6.58 (m, 4H, J8,9 = 8.16 Hz, Ar–H), 3.76 (s, 3H,–OCH3); 13C
NMR (75.45 MHz, CDCl3, ppm from TMS): 139.8 (C-1), 131.8 (C-
d ppm from TMS): d9.88 (s, 1H, N–H),
d
d
spectral data (300.40 MHz, Me2SO-d6,
1H, N–H), 7.78–6.45 (m, 4H, J8,9 = 8 Hz, Ar–H), 3.75 (s, 3H,–OCH3);
13C NMR (75.45 MHz, CDCl3,
ppm from TMS): 138.9 (C-1), 125.3
d
ppm from TMS):
d
9.79 (s,
2), 122.9 (C-3), 131.9 (C-4), 113.1 (C-6), 152.7 (C-7), 120.2 (C-8),
119.8 (C-9), 127.5 (C-4a), 129.1 (C-5a), 133.3 (C-9a), 140.7 (C-10a),
1
d
d
109.0 (CF3 at C3, q, JCF = 270.84 Hz), 56.2 (OCH3 at C7); 19F NMR
(C-2), 122.5 (C-3), 136.4 (C-4), 117.5 (C-6), 152.3 (C-7), 113.2 (C-8),
119.4 (C-9), 120.8 (C-4a), 122.4 (C-5a), 137.8 (C-9a), 145.2 (C-10a),
(282.65 MHz, CDCl3): d –61.465 (s, 3F, CF3); MS (FAB) 10 kV, m/z
(rel. int.): 408.0 [M]+ (100), 410.0 [M+2]+ (33.33), 391 [M–OH]+
(82), 378 [M–NO]+ (61), 362 [M–NO2]+ (41), 361 [M–HNO2]+ (51),
344 [M–SO2]+ (38); ‘‘Anal. Calcd for C14H8N2O5ClF3S: C, 41.17; H,
1.96; N, 6.86, Found: C, 41.29; H, 1.93; N, 6.79’’.
1
108.5 (CF3 at C3, q, JCF = 270.8 Hz), 56.2 (OCH3 at C7); 19F NMR
(282.65 MHz, CDCl3):
d –61.865 (s, 3F, CF3); MS (FAB) 10 kV, m/z
(rel. int.): 376 [M]+ (100), 378 [M+2]+ (33.33), 359 [M–OH]+ (85),
346 [M–NO]+ (62), 330 [M–NO2]+ (43), 329 [M–HNO2]+ (52); ‘‘Anal.
Calcd for C14H8N2O3ClF3S: C, 44.68; H, 2.12; N, 7.44, Found: C,
44.73; H, 2.15; N, 7.50’’.
4.5.3. 7-Bromo-9-ethyl-3-trifluoromethyl-1-nitro-10H-
phenothiazine-5,5-dioxide (6c)
Black solid; m.p.: 190 8C, yield: 59%, IR (KBr):
v 3320 (N–H),
4.4.2. 7-Bromo-9-ethyl-3-trifluoromethyl-1-nitro-10H-
1580 and 1346 (–NO2), 1360 and 1130 (–CF3), 2955 and 2887 (–
CH3), 1177 and 1165 (SO2 sym), 590 and 570 (SO2 bend), 1345,
1300 and 1262 (SO2 asym), and 1090 cmꢂ1 (C–S); 1H NMR spectral
phenothiazine (5c)
Red solid; m.p.: 86 8C, yield: 78%, IR (KBr):
v 3320 (N–H), 1570
and 1340 (–NO2), 1345 and 1120 (–CF3), 2941 and 2880 (–CH3),
1079 (C–S), and 590 cmꢂ1 (C–Br); 1H NMR spectral data
data (300.40 MHz, Me2SO-d6, dppm from TMS): d9.80 (s, 1H, N–H),
8.33–6.95 (m, 4H, Ar–H), 2.64 (q, 2H, 3JHH = 6.51 Hz, CH2 of C2H5 at
3
(300.40 MHz, Me2SO-d6,
d
ppm from TMS):
d
9.01 (s, 1H, N–H),
C9) 1.30 (t, 3H, JHH = 6.51 Hz, CH3 of C2H5 at C9); 13C NMR
8.12–6.74 (m, 4H, Ar–H), 2.62 (q, 2H, 3JHH = 6.5 Hz, CH2 of C2H5 at
(75.45 MHz, CDCl3, d ppm from TMS): d 139.6 (C-1), 126.7 (C-2),
3
C9), 1.29 (t, 3H, JHH = 6.5 Hz, CH3 of C2H5 at C9); 13C NMR
118.1 (C-3), 130.6 (C-4), 127.4 (C-6), 113.9 (C-7), 137.4 (C-8), 133.3
(C-9), 128.6 (C-4a), 130.3 (C-5a), 140.1 (C-9a), 140.3 (C-10a), 122.8
(CF3 at C3, q, 1JCF = 271.6 Hz), 19.5 (CH2 of C2H5 at C9), 15.8 (CH3 of
(75.45 MHz, CDCl3,
d ppm from TMS): d 138.9 (C-1), 120.2 (C-2),
122.4 (C-3), 135.1 (C-4), 131.9 (C-6), 113.5 (C-7), 130.9 (C-8), 132.9
(C-9), 121.9 (C-4a), 123.6 (C-5a), 144.5 (C-9a), 144.8 (C-10a), 117.6
(CF3 at C3,q, 1JCF = 271.0 Hz) 19.1 (CH2 of C2H5 at C9), 15.8 (CH3 of
C2H5 at C9); 19F NMR (282.65 MHz, CDCl3):
d –62.052 (s, 3F, CF3);
MS (FAB) 10 kV, m/z (rel. int.): 451 [M]+ (100), 453 [M+2]+ (100),
434 [M–OH]+ (74), 421 [M–NO]+ (38), 405 [M–NO2]+ (52), 404 [M–
HNO2]+ (59), 387 [M–SO2]+ (44); ‘‘Anal. Calcd for C15H10N2O4F3BrS:
C, 39.91; H, 2.21; N, 6.20, Found: C, 39.85; H, 2.16; N, 6.12.
C2H5 at C9); 19F NMR (282.65 MHz, CDCl3):
d –62.452 (s, 3F, CF3);
MS (FAB) 10 kV, m/z (rel. int.): 419 [M]+ (100), 421 [M+2]+ (100),
402 [M–OH]+ (77), 389 [M–NO]+ (40), 373 [M–NO2]+ (54), 372 [M–
HNO2]+ (63); ‘‘Anal. Calcd for C15H10N2O2F3BrS: C, 42.95; H, 2.38;
N, 6.68, Found: C, 43.15; H, 2.33; N, 6.61’’.
4.5.4. 7-Methoxy-3-nitro-10H-phenothiazine-5,5-dioxide (9a)
IR (KBr): v3335 (N–H), 2932 and 2830 (–OCH3), 1556 and 1350
4.5. General procedure for the synthesis of fluorinated 10H-
(–NO2), 1160 and 1145 (SO2 sym), 570 and 565 (SO2 bend), 1352,
1282 and 1260 (SO2 asym), and 1034 cmꢂ1 (C–S); 1H NMR spectral
phenothiazine-5,5-dioxide (sulfones) (6a–c)
data (300.40 MHz, Me2SO-d6,
7.56–6.25 (m, 6H, J1,2 = 8.12 Hz, J8,9 = 8.12 Hz, Ar–H), 3.71 (s, 3H,–
OCH3); 13C NMR (75.45 MHz, CDCl3,
ppm from TMS): 119.3 (C-
d ppm from TMS): d8.89 (s, 1H, N–H),
A mixture of substituted fluorinated 10H-phenothiazines (5a–
c) (0.01 mol), 20 mL glacial HOAc and 30% H2O2 (5 mL) in round
d
d

424
N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
1), 128.9 (C-2), 139.7 (C-3), 123.6 (C-4), 113.3 (C-6), 152.1 (C-7),
121.2 (C-8), 119.5 (C-9), 128.4 (C-4a), 129.5 (C-5a), 135.1 (C-9a),
146.7 (C-10a), 55.8 (OCH3 at C7); MS (FAB) 10 kV, m/z (rel. int.): 306
[M]+ (100), 305 [M-H]+ (26), 242 [M-SO2]+ (43), 215 [M-SO2-HCN]+
(60).
4.6.2. N-(20,30,50-tri-O-benzoyl -
trifluoromethyl-7-methoxy-1-nitro-10H-phenothiazine (7b)
Grey solid; m.p.: 120 8C, yield: 41%, IR (KBr): 1558 and 1315 (–
NO2), 1120 (C–O–C), 1344 and 1146 (–CF3), and 810 cmꢂ1 (C–Cl);
1H NMR spectral data (300.40 MHz, Me2SO-d6,
ppm from TMS):
7.89–6.50 (m, 4H, J8,9 = 8.05 Hz, Ar–H), 3.75 (s, 3H, –OCH3); 13C
NMR (75.45 MHz, CDCl3, ppm from TMS): 139.0 (C-1), 125.5 (C-
b-D-ribofuranosyl)-2-chloro-3-
v
d
d
4.5.5. 2,4-Dichloro-7-methoxy-10H-phenothiazine-5,5-dioxide (9b)
d
d
IR (KBr):
v
3352 (N–H), 2950 and 2836 (–OCH3), 1185 and 1145
2), 122.8 (C-3), 136.3 (C-4), 117.7 (C-6), 152.3 (C-7), 113.7 (C-8),
119.6 (C-9), 121.1 (C-4a), 122.6 (C-5a), 138.0 (C-9a), 145.2 (C-10a),
69.9 (C–10), 76.3 (C–20), 72.5 (C–30), 71.6 (C–40), 108.5 (CF3 at C3, q,
1JCF = 270.81 Hz), 56.2 (OCH3 at C7); 19F NMR (282.65 MHz, CDCl3):
(SO2 sym), 585 and 560 (SO2 bend), 1348, 1285 and 1260 (SO2
asym), and 1054 cmꢂ1 (C–S); 1H NMR spectral data (300.40 MHz,
Me2SO-d6,
J8,9 = 8.3 Hz, Ar–H), 3.75 (s, 3H, –OCH3); 13C NMR (75.45 MHz,
CDCl3, ppm from TMS): 118.1 (C-1), 140.8 (C-2), 120.6 (C-3),
dppm from TMS): d9.07 (S, 1H, N–H), 7.69–6.35 (m, 5H,
d
–61.854 (s, 3F, CF3); MS (FAB) 10 kV, m/z (rel. int.): 820.5 [M]+
d
d
(100), 822.5 [M+2]+ (33.33), 803.5 [M–OH]+ (76), 790.5 [M–NO]+
(56), 774.5 [M–NO2]+ (45), 773.5 [M–HNO2]+ (50); ‘‘Anal. Calcd for
135.2 (C-4), 113.0 (C-6), 152.2 (C-7), 121.4 (C-8), 120.2 (C-9),
125.9 (C-4a), 129.7 (C-5a), 134.8 (C-9a), 144.2 (C-10a), 56.0 (OCH3
at C7); MS (FAB) 10 kV, m/z (rel. int.): 330 [M]+ (100), 332 [M+2]+
(33.33), 329 [MꢂH]+ (39), 266 [MꢂSO2]+ (61), 239 [MꢂSO2–HCN]+
(48).
C40H28N2O10ClF3S: C, 58.50; H, 3.41; N, 3.41, Found: C, 58.59; H,
3.35; N, 3.44’’.
4.6.3. N-(20,30,50-tri-O-benzoyl -
b-D-ribofuranosyl)-7-bromo-9-
ethyl-3-trifluoromethyl-1-nitro-10H-phenothiazine (7c)
Brown solid; m.p.: 110 8C, yield: 53%, IR (KBr): 1576 and 1330
(–NO2), 1160 (C–O–C), 1350 and 1124 (–CF3), 600 cmꢂ1 (C–Br); 1H
4.5.6. 7-Bromo-3-carboxy-9-ethyl-1-nitro-10H-phenothiazine-5,5-
v
dioxide (9c)
IR (KBr):
v
3330 (N–H), 1565 and 1337 (–NO2), 2960 and 2868
NMR spectral data (300.40 MHz, Me2SO-d6, d ppm from TMS): d
(–CH3) 1155 and 1142 (SO2 sym), 558 and 545 (SO2 bend), 1348,
1280 and 1266 (SO2 asym), and 1092 cmꢂ1 (C–S); 1H NMR
8.20–6.83 (m, 4H, Ar–H), 2.62 (q, 2H, 3JHH = 6.5 Hz, CH2 of C2H5 at
3
C9) 1.29 (t, 3H, JHH = 6.5 Hz, CH3 of C2H5 at C9); 13C NMR
spectral data (300.40 MHz, Me2SO-d6,
d
ppm from TMS):
d
9.69
(75.45 MHz, CDCl3, d ppm from TMS): d 139.1 (C-1), 120.6 (C-2),
(s, 1H, N–H), 8.48–6.90 (m, 4H, Ar–H), 11.20 (s, 1H, COOH), 2.68
(q, 2H, JHH = 6.76 Hz, CH2 of C2H5 at C9), 1.35 (t, 3H,
3JHH = 6.76 Hz, CH3 of C2H5 at C9); 13C NMR (75.45 MHz, CDCl3,
122.3 (C-3), 135.1 (C-4), 132.0 (C-6), 113.5 (C-7), 131.2 (C-8), 132.9
(C-9), 121.8 (C-4a), 123.6 (C-5a), 144.4 (C-9a), 144.8 (C-10a), 70.1
(C-10), 76.3 (C-20), 72.5 (C-30), 71.7 (C-40), 117.6 (CF3 at C3, q,
1JCF = 271.2 Hz), 19.4 (CH2 of C2H5 at C9), 15.8 (CH3 of C2H5 at C9);
3
d
ppm from TMS): d 139.5 (C-1), 130.4 (C-2), 123.1 (C-3), 136.2
(C-4), 127.3 (C-6), 113.9 (C-7), 135.8 (C-8), 133.7 (C-9), 129.1 (C-
4a), 130.0 (C-5a), 140.4 (C-9a), 141.6 (C-10a), 170.1 (COOH at
C3), 19.2 (CH2 of C2H5 at C9), 16.2 (CH3 of C2H5 at C9); MS (FAB)
10 kV, m/z (rel. int.): 427 [M]+ (100), 429 [M+2]+ (100), 410
[MꢂOH]+ (76), 397 [MꢂNO]+ (26), 381 [MꢂNO2]+ (55), 380
[MꢂHNO2]+ (44), 363 [MꢂSO2]+ (39).
19F NMR (282.65 MHz, CDCl3):
d –62.445 (s, 3F, CF3); MS (FAB)
10 kV, m/z (rel. int.): 863 [M]+ (100), 865 [M+2]+ (100), 846 [M–
OH]+ (75), 833 [M–NO]+ (47), 817 [M–NO2]+ (51), 816 [M–HNO2]+
(62); ‘‘Anal. Calcd for C41H30N2O9F3BrS: C, 57.01; H, 3.47; N, 3.24,
Found: C, 57.05; H, 3.41; N, 3.26’’.
4.7. Antioxidant activity
4.6. General procedure for the synthesis of substituted fluorinated N-
(20,30,50-tri-O-benzoyl-
b
-D
-ribofuranosyl) phenothiazines (7a–c)
All the synthesized compounds were screened for their
antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging assay (Table 1) and 2,2-azinobis (3-ethylben-
zothiazoline-6-sulfonic acid) ABTSꢀ+ radical cation decolorization
assay (Table 2 and Fig. 1). In antioxidant activity assays (both DPPH
ABTSꢀ+ and assays), ascorbic acid was used as a positive control.
To the solution of (5a–c) (0.002 mol) in 5 mL C6H5CH3,
b-D-
ribofuranose-1-acetate-2,3,5-tribenzoate (0.002 mol) was added
and the contents were refluxed under vacuum with stirring on an
oil bath at 155–160 8C for 15 min. The vacuum was broken and
reaction mixture was protected from moisture by fitting a guard
tube. Stirring was further continued for 10 h and a vacuum was
applied for 10 min after every 1 h. The viscous mass obtained was
dissolved in MeOH, boiled for 10 min and cooled to room
temperature. The reaction mixture was filtered and the filtrate
was evaporated to dryness. The viscous residue thus obtained was
dissolved in Et2O, filtered, concentrated and kept in refrigerator
overnight to afford crystalline ribofuranosides.
4.7.1. DPPH radical scavenging assay
Radical scavenging activity of synthesized compounds against
stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was deter-
mined spectrophotometrically by the method described by Braca
Table 1
Antioxidant activity of synthesized compounds (DPPH assay).
Compd. no.
DPPH % inhibitiona of 1 mg/mL of compound
4.6.1. N-(20,30,50-tri-O-benzoyl-
methoxy-10H-phenothiazine (7a)
Red solid; m.p.: 96 8C; Yield: 68%, IR (KBr):
1273 cmꢂ1 (C–F); 1H NMR spectral data (300.40 MHz, Me2SO-d6,
ppm from TMS): 7.42–6.48 (m, 6H, J1,2 = 7.92 Hz, J8,9 = 8.04 Hz,
Ar–H), 3.72 (s, 3H,–OCH3); 13C NMR (75.45 MHz, CDCl3,
ppm
from TMS): 120.5 (C-1), 113.9 (C-2), 151.7 (C-3, d,
1JCF = 249.2 Hz), 118.4 (C-4), 117.5 (C-6), 152.5 (C-7), 113.9 (C-
8), 118.9 (C-9), 123.5 (C-4a), 122.9 (C-5a), 137.6 (C-9a), 140.8 (C-
10a), 70.1 (C-10), 76.3 (C-20), 72.5 (C-30), 71.7 (C-40), 55.7 (OCH3 at
b-D-ribofuranosyl)-3-fluoro-7-
5a
64.27 ꢃ 0.08
45.56 ꢃ 1.50
31.24 ꢃ 0.08
27.46 ꢃ 0.26
22.73 ꢃ 1.02
19.36 ꢃ 1.20
59.86 ꢃ 0.07
39.39 ꢃ 0.18
29.87 ꢃ 0.05
98.37 ꢃ 0.02
5b
v
1145 (C–O–C), and
5c
d
6a
d
6b
d
6c
d
7a
7b
7c
Ascorbic acid
a
C7); 19F NMR (282.65 MHz, CDCl3):
d –109.359 (s, 1F, C–F); MS
Inhibition (%) of DPPH radical scavenging activity of various compounds at
particular concentration. Stock solution of crude compound was prepared as 1 mg/
(FAB) 10 kV, m/z (rel. int.): 691 [M]+ (100), 660 [M–OCH3]+ (33),
621 [M–C4H3F]+ (72); ‘‘Anal. Calcd for C39H30NO8FS: C, 67.72; H;
4.34; N, 2.02. Found: C, 67.84, H, 4.29; N, 2.07’’.
mL in CH3OH. 10 mL of samples of particular concentration were added to 3 mL of
0.1 mM CH3OH solution of DPPH.. After 30 min incubation in dark at room
temperature, the absorbance was read against a blank at 517 nm.

N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
425
Table 2
Antioxidant activity of synthesized compounds (ABTSꢀ+assay).
Compd. no.
ABTSꢀ+ activity at different intervals (min)
Initial
0 min
1 min
2 min
4 min
6 min
5a
0.702
0.710
0.709
0.710
0.711
0.715
0.704
0.705
0.707
0.717
0.636
0.698
0.676
0.682
0.702
0.712
0.645
0.658
0.665
0.34
0.337
0.415
0.382
0.469
0.595
0.629
0.396
0.280
0.435
0.049
0.049
0.225
0.204
0.350
0.496
0.575
0.159
0.235
0.315
0.039
0.039
0.175
0.198
0.286
0.328
0.466
0.056
0.205
0.226
0.03
0.037
0.105
0.191
0.225
0.290
0.325
0.049
0.182
0.210
0.028
5b
5c
6a
6b
6c
7a
7b
7c
Ascorbic acid
et al. [10]. DPPH is stable free radical by virtue of delocalization of
electron over the whole molecule. For hydrogen atom transfer,
antioxidants quench the free radicals by donating hydrogen, while
for single electron transfer, antioxidants transfer one electron to
the radical.
A stock solution containing 1 mg/mL of the compound was
prepared in MeOH. 10 L of the solution were added to 3 mL of a
0.1 mM solution of DPPH in MeOH. After 30 min incubation in the
dark at room temperature, the absorbance was read against a blank
at 517 nm.
m
ABTS activity (at different time intervals) of 10H-phenothiazines (5a-c)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
5c
5b
5a
Initial
0 min.
1 min.
2 min.
4 min.
5c
6 min.
Time (min)
Ascorbic acid
5a
5b
ABTS activity (at different time intervals) of 10H-phenothiazine sulfones (6a-c)
0.8
0.7
0.6
0.5
0.4
6c
0.3
0.2
0.1
0
6b
6a
Initial
0 min.
1 min.
2 min.
4 min.
6c
6 min.
Time (min)
Ascorbic acid
6a
6b
ABTS activity (at different time intervals) of 10H-phenothiazine ribofuranosides (7a-c)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
7c
7b
7a
Initial
0 min.
1 min.
2 min.
4 min.
7c
6 min.
Time (min)
Ascorbic acid
7a
7b
Fig. 1. The effect of time on the suppression of absorbance of ABTS by synthesized compounds. After addition of 1 mL of diluted ABTS solution (A 734 nm = 0.700 ꢃ 0.020) to
˚
10 mL of the compound the absorbance reading was taken at 30 C exactly 1 min., after initial mixing and every min. up to 6 min. All determinations were repeated three times.

426
N. Gautam et al. / Journal of Fluorine Chemistry 132 (2011) 420–426
Table 3
Antimicrobial activity of synthesized fluorinated compounds (in terms of MIC).
Compounds
Minimal inhibition concentrations of bacterial strains (MIC) in
mg/mL
Minimal inhibition concentrations of fungal strains (MIC) in
g/mL
m
Bacillus cereus
Staphylococcus
aureus MTCC
3160
E. coli DH 5
alpha MTCC
1652
Pseudomonas
Aspergillus
niger MTCC
282
Aspergillus
flavus MTCC
2456
Fusarium
Rhizopus
MTCC 4317
aeruginosa MTCC
oxysporum MTCC
6659
stolonifer MTCC
2591
4676
5a
50.1
46.1
35.1
25.2
21.4
19.7
49.4
45.2
34.6
38
78.9
73.6
58.6
46.7
42.6
39.4
76.8
72.9
57.9
64
79.7
74.1
62.1
49.2
43.1
40.3
78.1
73.4
61.3
64
74.8
69.1
57.4
42.4
36.5
33.6
73.5
67.8
56.6
60
53.1
46.9
36.9
24.5
20.2
18.9
51.2
46.3
36.2
–
112.8
102.1
96.1
66.8
61.1
58.2
111.6
101.2
95.4
–
84.3
80.2
74.2
51.3
48.1
45.7
82.4
79.4
73.8
–
63.5
51.1
46.9
28.6
24.2
22.6
61.2
49.7
46.2
–
5b
5c
6a
6b
6c
7a
7b
7c
Ampicillin sodium salt
Ketoconazole
–
–
–
–
38
95
74
46
The assay was carried out in triplicate and the percentage of
inhibition was calculated using the following formula.
ðAB ꢂ AAÞ
compound. Antibacterial activities of the bacterial strains were
carried out in Luria broth (Himedia) medium and all fungi were
cultivated in Sabouraud Dextrose Agar (Himedia), at pH 6.9, with an
inoculum of 108 cfu/mL by the spectrophotometric method and an
Inhibition ¼
ꢄ 100
AB
aliquot of 10
mL was added to each tube of the serial dilution and
where AB = Absorption of blank, AA = Absorption of test.
incubated on a rotary shaker at 37 8C for 24 h at 150 rpm. At the end
of incubation period, MIC values were recorded. The minimal
inhibitory concentrations (MICs,
5. ABTS radical cation decolorization assay
m
g mLꢂ1) of tested compounds
against certain bacteria and fungi are shown in Table 3.
The 2,2-azinobis(3-ethybenzothiazoline-6-sulphonic acid) radi-
cal cation (ABTSꢀ+) decolorization test was also used to assess the
antioxidant activity of the synthesized compounds. The ABTSꢀ+
assay was carried out using the improved assay of Re et al. [9] ABTSꢀ+
was generated by oxidation of ABTS with K2S2O8. For this purpose,
ABTS was dissolved in deionized H2O at a concentration of 7 mM,
and K2S2O8 was added to a concentration of 2.45 mM. The reaction
mixture was left at room temperature overnight (12–16 h) in the
dark before use; the ABTSꢀ+ solution then was diluted with EtOH to
an absorbance of 0.700 ꢃ 0.020 at 734 nm. After addition of 1 mL of
the diluted ABTS solution to 10 mL of compound and mixing,
absorbance readings were taken at 30 8C at intervals of exactly 1–
6 min, later. All determinations were carried out in triplicate.
Acknowledgements
The authors are grateful to the Department of Chemistry,
University of Rajasthan, Jaipur for providing necessary facilities.
UGC Research Award Scheme and UGC, New Delhi are duly
acknowledged for financial support. Authors are also thankful to
Institute of Applied Sciences and Biotechnology (Chemind
Biosolutions), Jaipur and Department of Zoology, University of
Rajasthan for assistance in carrying out antimicrobial and
antioxidant activities respectively.
Appendix A. Supplementary data
5.1. Antimicrobial activity
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jfluchem.2011.04.012.
The minimal inhibition concentrations (MICs,
m
g mLꢂ1) of the
chemicalcompoundsassayswerecarriedoutbybrothmicrodilution
method as per NCCLS-1992 manual. Two Gram-positive (Bacillus
cereus MTCC 4317 and Staphylococcus aureus MTCC 3160) and two
Gram-negative (Escherichia coli DH5 alpha MTCC 1652 and
Pseudomonas aeruginosa MTCC 4676) bacteria were used as quality
control strains. For determining antifungal activities of the
compounds, the following reference strains were tested: A. niger
MTCC 282, A. flavus MTCC 2456, F. oxysporum MTCC 6659 and R.
stolonifer MTCC 2591. Ampicillin Na salt and Ketoconazole were
used as standard antibacterial and antifungal drugs, respectively.
Solutions of test compounds and standard drugs were prepared in
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to obtain 100, 50, 25, 20, 15
mg/mL concentrations. The highest
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dilution showing at least 99% inhibition was taken as MIC. This
means that the lowest concentration of each chemical compound in
the tube with no growth (i.e. no turbidity) of inoculated bacteria/
fungi was recorded as minimal inhibitory concentration of that