An efficient synthesis and biological activity of substituted p-benzoquinones

Article abstract of DOI:10.1016/j.bmc.2006.08.036

An efficient synthesis of 2,5-diarylamino-3,6-dichloro-1,4-benzoquinone derivatives has been achieved by condensing mono substituted anilines with tetrachloro-p-benzoquinone in presence of fused sodium acetate as condensing agent under microwave irradiati

Full text of DOI:10.1016/j.bmc.2006.08.036

Bioorganic & Medicinal Chemistry 14 (2006) 8519–8526
An efficient synthesis and biological activity
of substituted p-benzoquinonesq
Manoj Batra,* Prashant Kriplani, Chhavi Batra and K. G. Ojha
Department of Pure and Applied Chemistry, Maharshi Dayanand Saraswati University, Ajmer 305 009, India
Received 26 June 2006; revised 22 August 2006; accepted 24 August 2006
Available online 12 September 2006
Abstract—An efficient synthesis of 2,5-diarylamino-3,6-dichloro-1,4-benzoquinone derivatives has been achieved by condensing
mono substituted anilines with tetrachloro-p-benzoquinone in presence of fused sodium acetate as condensing agent under micro-
wave irradiation without any solvent. All the synthesized compounds were tested for their antibacterial and antitumour activity
using standard drugs.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
reactions are accelerated because of selective absorption
of microwave by polar molecules.14 As a part of our
programme towards the non traditional approach to
the experimental setup of organic reactions, the concept
of ‘microwave induced organic reaction enhancement’
(MORE) chemistry has been utilized for rapid, sustain-
able and efficient synthesis. Microwave-assisted organic
synthesis has attracted attention in recent years due to
enhanced reaction rates,high yields, improved purity,
ease of work up after the reaction and ecofriendly reac-
tion conditions compared to the conventional methods.
Quinones make up a large class of compounds with di-
verse biological activity. They can be found in many ani-
mal and plant cells and are widely used as anticancer,
antibacterial or antimalarial drugs as well as fungicides.1,2
Quinones are involved in various bioenergetic processes
as important transport agents. These compounds have
also attracted considerable attention because of their bio-
logical activity and chemotherapeutic value. Various qui-
nones containing oxygenated aromatic rings have been
reported to present biological activities.3–5
The present work reveals the comparative aspects of syn-
thesis of substituted-p-benzoquinones (conventional and
microwave) and their characterization. The compounds
were tested for AST (Antimicrobial Susceptibility Test)
against Staphylococcus aureus (ATCC 25923), Escherich-
ia coli (ATCC 10536) and Pseudomonas aeruginosa
(ATCC 25619) using Kirby-Bauers’ disc diffusion meth-
od.15 The synthesized compoundswerestudiedfor invitro
antitumour screening by multidrug resistance on mouse
lymphoma cells transfected with human mdrl gene. The
cell line was L-5178 mouse T-cell lymphoma cell line,
which was infected with the pHa MDR-1/A retrovirus.16
A variety of 1,4-benzoquinones and their nitrogen ana-
logues have been reported for their antitumour activi-
ties.6–8 Quinones that act against animal tumours
are thought to function as bio-reductive alkylating
agents.9–11
They play important role in biological functions includ-
ing a role in oxidative phosphorylation and electron
transfer.12,13
Microwave irradiation is well known to promote the
synthesis of a variety of compounds, where chemical
Keywords: Benzoquinone; Microwave irradiation; Antibacterial activ-
ity; Antitumour activity.
2. Results and discussion
q
Presented at the IUPAC Second International Symposium on
Green/Sustainable Chemistry (P-18), 10–13 January 2006, Delhi,
India.
2.1. Comparative profile of synthesis
2,5-Diarylamino-3,6-dichloro-1,4-benzoquinone deriva-
tives (3) were prepared by condensing mono substituted
*
Corresponding author. Tel.: +91 09414383561; fax: +91 0145
2670545; e-mail: manojbatra05@rediffmail.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.036

8520
M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
R1
R1
O
Cl
H
N
NH2
Cl
Cl
Cl
Cl
O
R2
EtOH
+
Fused Sod. Acetate
R2
R2
O
N
H
Cl
O
R1
(1a-i)
(2)
(3a-i)
a
b
c
d
F
H
e
f
g
h
i
Substituents
R1 =
H
H
CH3
H
OCH3
H
H
Cl
H
H
H
H
F
R2 =
Br
CH3
OCH3
Scheme 1. Synthesis of 2,5-diarylamino-3,6-dichloro-1,4-benzoquinones (3a–i).
anilines (1) (2 mol) with tetrachloro-p-benzoquinone (2)
(1 mole) in ethanolic medium in presence of fused sodi-
um acetate using methods reported earlier17 (Scheme 1).
the reactants remain unchanged, even on extended time
(4 h),thus demonstrating that the effect of microwave is
not simply thermal.19 The rate acceleration under micro-
wave irradiation was due to specific microwave effect.20
Under microwave irradiation enhanced dipole–dipole
interactions between the activated reaction intermedi-
ates caused the instantaneous condensation of reactants
to afford substituted-1,4-benzoquinones (3) without the
use of any solvent in a very short time.
In view of long reaction time, moderate yields (Table 1),
tedious work up after the reaction and requirement of
large quantity of solvent associated with conventional
method, a relatively more versatile yet simplified proce-
dure was perceived, in which substituted anilines (1) and
tetrachloro-p-benzoquinone (2) could be made to react
in the presence of fused sodium acetate under micro-
wave irradiation without using any solvent. Microwave
synthesis has received attention as a new strategy for
organic synthesis due to the fact that many reactions
seem to proceed with much alacrity under such condi-
tions as opposed to the corresponding thermal-assisted
reactions.18 The strategy worked well affording the de-
sired product in improved yields in significantly lower
reaction time (Table 1). The result obtained under
microwave irradiation was extrapolated to conventional
heating. Reactions mentioned in Table 1 were examined
by simply heating in a preheated oil-bath under the same
conditions (time, temperature, pressure and medium) as
mentioned in the typical procedure with microwave irra-
diation. It was found that no reaction occurred and that
After analysis of the results summarized in Table 1, we
found the reaction of substituted anilines (1) and tetra-
chloro-p-benzoquinone (2) in the presence of fused sodi-
um acetate afforded 3 as needles of various colours. The
elements (C, H, and N) found for all the molecular for-
mulas (3a–i) were in close agreement with the calculated
one, the UV spectrum of 3 showed a broad unstructured
band at about 400 nm characteristic of p-benzoquinon-
es.The IR spectrum of 3 exhibited a strong absorption
band due to NH stretching at 3240 cmꢀ1 and CO
stretching band at 1650 cmꢀ1in addition to other bands.
1
The H NMR spectrum of 3 revealed characteristic sig-
nals for methyl protons at d 2.28–2.42 (s, 6H, 2CH3),
methoxy protons at d 3.87 (s, 6H, 2OCH3), aromatic
protons in d 6.8–7.5 (m, 8–10H, ArH) and NH proton
Table 1. Comparative study for the synthesis of 2,5-diarylamino-3,6-dichloro-1,4-benzoquinones (3a–i)
Compound
Conventional method
Microwave irradiationa
Conventional heatingd
Time (h)
Temp (ꢁC)
Yield (%)
Time (min)
Tempb (ꢁC)
Yieldc (%)
Timee (h)
Yield (%)
3a
3b
3c
3d
3e
3f
4
4
4
4
4
4
4
4
4
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
reflux
70
65
62
60
86
75
75
80
70
1.5
1.5
1.5
2.0
1.5
2.0
2.0
1.5
2.5
68
72
70
69
73
67
66
68
65
90
86
84
83
90
85
85
89
83
4
4
4
4
4
4
4
4
4
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
Nil
3g
3h
3i
a Reaction mixture in microwave oven was irradiated at power output of 160 W.
b The final internal temperature of the reaction mixture was measured by non-contact IR thermometer.
c Isolated yield of purified compounds that exhibited physical and spectral properties in accordance with assigned structure.
d Conventional heating was conducted in thermally preheated oil-bath under identical reaction conditions and temperature used under microwave.
e Extended reaction time for reaction in thermally preheated oil-bath under identical reaction conditions and temperature used under microwave.

M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
8521
at d 8.30 (s, 1H, NH, exchanged with deuterium). For-
mation of the 3 was further confirmed on the basis of
13C NMR spectroscopy and mass spectroscopy. In the
13C NMR spectrum of 3, sharp signals were observed
at 17.6 (CH3), 55.9 (OCH3), 110.6–152.2 (aromatic ring
carbons), 172.5 (C@O).The FAB MS of 3a (Scheme 2)
showed the molecular ion peak at m/z 359 (30%, M+)
and also isotopic abundance peak at m/z 361 (16%,
M++2). Molecular ion (M+) after losing one chlorine
atom produces a fragment at m/z 324 (10%, M+ꢀCl)
along with isotopic abundance at m/z 326 (6.5%,
M++2ꢀCl). All the fragments containing two chlorine
atoms exhibit isotopic abundance at m/z M+, M++2,
M++4 in the intensity ratio of 100:65:10.6% supporting
the presence of two chlorine atoms in 3a. Fragment at
m/z 358 (28%, M+ꢀH), formed by the loss of one proton
from M+, produces a fragment at m/z 266 (9%, M+ꢀH–
C6H5N) by the loss of C6H5N. Fragment at m/z 91
(30%, C6H5N+) is further stabilized by the loss of N
frag.CO). Similarly by the unsymmetrical rDA fragmen-
tation of 3a around central ring a fragment at m/z 208
(72%, unsym. rDA frag.) is formed. Thus both the frag-
mentation pathways establish the para positioning of
two CO groups in the central ring. Existence of fragment
ion peak at m/z 329 along with 307 (10% M+ꢀ2C2H2),
which is formed due to the loss of one acetylene frag-
ment each from both the aromatic rings, provides suffi-
cient evidence in favour of two anilino (C6H5NH)
groups attached to the central ring of 3a. Thus MS frag-
mentation of 3a along with other spectral studies clearly
suggests that two chlorine and two keto groups are at-
tached to central ring at 3,6 and 2,5 positions, respec-
tively, while the two anilino (C6H5NH) groups are
attached to the central ring at the remaining 1,4 posi-
tions. Similarly mass spectrum of 3b (Scheme 3) also
support the proposed structure of the compound.
Thus on the basis of spectral data all products 3a–i have
been identified.
atom to generate
a
fragment at m/z 77 (40%,
C6Hþ5 ).The appearance of the fragment ion peak at m/
z 329 (25%, M+ꢀ2NH) due to elimination of two NH
fragments along with fragments at m/z 358, 266, 91and
77 supports the presence of two NH groups positioned
in-between two aromatic side rings and central ring of
3a. Fragment at m/z 329 undergoes symmetrical retro-
Diels-Alder (rDA) cleavage around central ring to pro-
duce rDA fragment at m/z 165 (15%, sym. rDA frag.)
which stabilizes further by losing one CO molecule to
produce another fragment at m/z 136 (70%, sym. rDA
2.2. Antibacterial screening
As we see from Table 2 compound 3c was found active
against all the strains of bacteria under study (above
50% as compared to the standard drug Gentamycine).
Compound 3e show maximum activity against S. aureus
while compound 3b exhibits minimum against it.
Against E. coli maximum activity is exhibited by com-
pound 3f and minimum by compound 3a. While against
+
H
O
N
Cl
Unsym. r.D.A. fragmentation
–Cl
H
O
N
O
N
H
Cl
O
324 (10%)
208 (72%)
Cl
Cl
H
+
N
O
Cl
+
N
O
–H
O
–2NH
O
N
H
O
N
H
O
Cl
Cl
358 (28%)
Cl
359 (30%)
329 (25%)
–H
–N
O
–C H N
6
5
C H
6 5
77(40%)
sym.
–2CH=CH
91(30%)
r.D.A.
fragmentation
Cl
+
Cl
+
H
N
Cl
N
O
O
O
N
H
O
Cl
Cl
165 (15%)
266 (9%)
307 (10%)
–CO
–C H
4
3
H
C
Cl
H
N
O
O
C
Cl
136 (70%)
N
H
Cl
256 (8%)
Scheme 2. Mass fragmentation pattern of 2,5-dianilino-3,6-dichloro-1,4-benzoquinone (3a).

8522
M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
Cl
H C
3
N
O
O
N
CH
3
Cl
233 (17%)
–2C H
6
5
CH
+
3
CH
3
+
CH
3
Cl
Cl
Cl
CH
Cl
+
3
N
O
O
N
H
+
N
O
O
O
H
O
H
O
N
O
–HCl
–HCl
–2HCN
–2C H
6
6
N
H
O
O
N
H
CH
3
Cl
CH
Cl
3
Cl
CH
3
315 (15%)
351 (35%)
387 (100%)
CH
333 (17%)
177 (25%)
3
–2H
–H
Cl
–2CO
Cl
CH
CH
3
3
+
CH
3
N
O
N
O
N
O
N
H
O
Cl
Cl
Cl
349 (20%)
CH
CH
2
3
386 (15%)
CH
3
CH
2
277 (11%)
H
107 (20%)
O
Cl
Cl
CH
3
Cl
O
O
H
N
N
O
N
O
O
Cl
–Cl
r.D.A
fragmentation
–HCN
N
H
N
H
O
N
CH
3
Cl
CH
CH
3
3
177 (25%)
279 (10%)
242 (8%)
215 (10%)
Scheme 3. Mass fragmentation pattern of 2,5-bis(20-methylanilino)-3,6-dichloro-1,4-benzoquinone (3b).
Table 2. Zone of inhibition for antibacterial activity of 2,5-dianilino-3,6-dichloro-1,4-benzoquinone
Compound
Diameter of zone of inhibition
S. aureus ATCC 25923
E. coli ATCC 10536
P. aeruginosa ATCC 25619
a
%
a
%
a
%
Mean
Mean
Mean
3a
3b
08.53 0.57
08.50 0.32
17.75 0.87
12.33 0.66
18.13 0.88
13.52 0.43
12.62 0.84
16.18 0.45
12.45 0.68
27.65 1.47
06.00 0.00
11.68
08.55 0.33
09.11 0.74
15.34 0.77
13.45 0.00
13.86 0.43
16.75 0.55
11.98 0.49
12.11 0.49
14.18 0.45
24.12 0.54
06.00 0.00
14.07
09.11 0.87
09.89 0.32
16.42 0.65
13.21 0.62
15.85 0.76
18.88 0.33
14.08 0.44
14.70 0.31
16.23 0.22
26.12 0.97
06.00 0.00
15.46
11.55
54.27
29.24
56.03
34.73
30.58
47.02
29.79
17.16
51.55
41.11
43.38
59.32
33.00
33.71
45.14
19.33
51.78
35.83
48.96
64.02
40.16
43.24
50.84
3c
3d
3e
3f
3g
3h
3i
Gent
DMF
100
00
100
00
100
00
Gent, gentamycine.
Mean, mean value of diameter of inhibition zone with standard error in millimeters.
a Percentage was calculated after subtracting disc diameter (6 mm) from all observations.
P. aeruginosa antibacterial action of compound 3f is
maximum. Other compounds 3c and 3i also show good
inhibition (above 50%) against P. aeruginosa. Minimum
activity against this strain of bacteria is shown by com-
pound 3a. Based on the above finding it is very difficult
to draw correlation between structures and activity (Ta-
ble 2).
gene. As we see from Table 3 compound 3a is very effec-
tive because its fluorescence activity ratio is compara-
tively higher than other compounds and verapamil,
although compound 3c has some marginal effects. But
compounds 3b and 3e do not affect cell size, but the
cytoplasmic granulation was enhanced.
2.3. Antitumour screening
3. Conclusion
In the present investigation, p-benzoquinone com-
pounds were studied for reversal of multi drug resistance
on mouse lymphoma cells transfected with human mdrl
We have developed an economical, solvent free, very
efficient microwave-assisted protocol for the synthesis
of 2,5-dianilino-3,6-dichloro-1,4-benzoquinone which

M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
Table 3. Flourescence activity ratio of 2,5-dianilino-3,6-dichloro,1,4-benzoquinone
8523
Compound
Concentration (lg/ml)
FSC
SSC
FL-1
Flourescence activity ratio
PAR control
MDR control
VERAPAMIL
409.25
425.01
420.89
467.44
346.50
457.14
363.44
405.29
100.33
120.64
117.21
149.20
142.65
226.44
161.20
138.08
1135.85
35.73
5
20
20
20
20
20
183.97
19.12
5.15
0.54
6.10
0.53
1.63
0.48
DMSO
3a
3b
218.02
19.03
58.26
3c
3e
17.17
can be a viable alternative to the conventional synthesis.
In all cases, a comparison of the reactions using either
thermal or microwave heating under the same condi-
tions shows clearly a specific (non-thermal) microwave
effect.
(100 ml) was added an alcoholic solution of (0.02 mol)
mono substituted aryl amines (1a–i). The resultant mix-
ture was refluxed for 4 h. The reaction was hot filtered
and residue was first washed with hot water and
finally with 30% ethanol to give the desired products.
Products were recryztallised from acetone/benzene
to give 2,5-diarylamino-3,6-dichloro-1,4-benzoquinones
(3a–i)17 (Scheme 1).
The antibacterial activity in terms of zone of inhibition
is exhibited by all the compounds against all the strains
of bacteria under study (S. aureus, E. coli and P. aeru-
ginosa). Maximum antibacterial activity is shown
against P. aeruginosa by the compound 3f. In the in vitro
antitumour screening compound 3a was found to be
very effective while compound 3c was marginally effec-
tive. There is no such permanent structure–activity rela-
tionship between the design of molecule under study and
its biological activity.
(b) The synthesis of title compounds (3a–i) was attempt-
ed by condensing (2 mmol) mono substituted anilines
(1a–i) with (1 mmol) tetrachloro-p-benzoquinones (2)
in presence of (1 mmol) anhydrous sodium acetate using
a preheated oil-bath under near identical conditions of
time, temperature, pressure and medium as under
microwave. TLC indicated the unchanged reactants for
heating up to 2.5 min. Even extended heating for 4 h
did not lead to formation of any product.
4. Experimental
4.1. Equipment
4.2.2. Under microwave irradiation. The title compounds
(3a–i) were synthesized by condensing (2 mmol) mono
substituted anilines (1a–i) with (1 mmol) tetrachloro-p-
benzoquinones (2) in presence of (1 mmol) anhydrous
sodium acetate. The reaction was carried out in Erlen-
meyer flask capped with a funnel under microwave irra-
diation at 160 W in microwave oven, at the marked hot
spots, for the specific period of time. The completion of
the reaction was monitored by silica gel TLC. (Benzene/
acetone 60:40). To ensure the reproducibility every reac-
tion was carried out five times in microwave. The final
temperature of the reaction mixture was measured by
non-contact IR thermometer specifically fitted for mea-
suring the internal temperature. After cooling the resi-
due was washed with hot water and 30% ethanol to
give 3a–i.
Reagent-grade chemicals were used without further
purification. The substrates and solvents were used as
received. All the melting points are uncorrected. The
purity of synthesized compounds was checked by TLC
studies. UV/vis spectra were recorded in Perkin-Elmer
Lamda 15 UV/vis spectrophotometer. IR spectra were
measured on FT IR Perkin-Elmer (Spectrum RX1) spec-
trometer (mmax in cmꢀ1) using KBr disc. H NMR and
1
13C NMR were recorded in DMSO with TMS as the
internal standard at 300 MHz on a Bruker DRX-300
spectrometer. Fast atom bombardment mass spectra
(FAB MS) were recorded at room temperature on a Jeol
SX-102/DA-6000 Mass spectrometer/Data System using
Aragon/Xenon (6 kV,10 mA) as the FAB gas. The accel-
erating voltage was 10 kV. C, H and N elements were
analyzed by Elementar Vario EL III Carlo Erba 1108
elemental analyzer. Reactions under microwave irradia-
tions were carried out in microwave oven model LG
MS-194W operating at 160 W generating 2450 MHz fre-
quency. Internal temperature of reaction mixture was
measured on Mini Gun Type Non-Contact IR ther-
mometer, Model No. 8868.
4.3. Spectral data of synthesized compounds (3a–i)
4.3.1. 2,5-Dianilino-3,6-dichloro-1,4-benzoquinone (3a).
Yield: 323 mg, 90%; as yellowish green needles, mp
248 ꢁC (from dimethylformamide/benzene); Anal. Calcd
for C18H12Cl2N2O2: C, 60.16; H, 3.34; N, 7.79. Found:
C, 60.13; H, 3.31; N, 7.72%. UV/vis kmax (cmꢀ1) 388;
mmax (cmꢀ1) (KBr) 3239 (N–H), 1651 (C@O), 1571,
1353, 1248, 1194, 1111, 1026; dH (300 MHz; DMSO)
8.302 (1H, s, NH, D2O exchangeable) 7.125–7.401
(10H, m, ArH (20, 30, 40, 50, 60, 200, 300, 400, 500 and 600));
dc (300 MHz; DMSO) 124.8, 125.3, 127.9, 172.9
(C@O); FAB MS (298 ꢁC) m/z (%, fragment) 359
(30%, M+), 361 (16%, M++2), 358 (28%, M+ꢀH) 329
(25%, M+ꢀ2NH), 324 (10%, M+ꢀCl), 326 (6.5%,
4.2. Investigation of the synthesis of 2,5-diarylamino-3,6-
dichloro-1,4-benzoquinones (3a–i)
4.2.1. Under conventional condition. (a) To a stirred sus-
pension of (0.01 mol) tetrachloro-p-benzoquinone(2)
and (0.01 mol) anhydrous sodium acetate in ethanol

8524
M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
M++2ꢀCl), 307 (10%, M+ꢀ2C2H2), 266 (9%, M+ꢀH–
C6H5N), 208 (72%, unsym. rDA frag.), 165 (15%, sym.
rDA frag.), 136 (70%, rDA frag.-CO), 91 (30%,
C6H5N+), 77 (40%, C6H5+).
4.3.5. 2,5-Bis(40-chloroanilino)-3,6-dichloro-1,4-benzoqui-
none (3e). Yield: 385 mg, 90%; as dark green needles mp
350 ꢁC (from dimethylformamide/benzene); Anal. Calcd
for C18H10Cl4N2O2: C, 50.46; H, 2.33; N, 6.54. Found:
C, 50.42; H, 2.31; N, 6.49%. UV/vis kmax (cmꢀ1) 385;
mmax in cmꢀ1 (KBr) 3230 (N–H), 1650 (C@O), 1590,
1322, 1250, 1190, 1045; dH (300 MHz; DMSO) 8.26
(1H, s, NH, D2O exchangeable), 7.505–7.534 (2H, d,
J = 8.7 Hz, ArH (30 and 300)), 7.375–7.404 (2H, d,
J = 8.7 Hz, ArH (50 and 500)), 7.082–7.111 (2H, d,
J = 8.7 Hz, ArH (20 and 200)), 7.033–7.062 (2H, d,
J = 8.7 Hz, ArH (60 and 600)); dc (300 MHz; DMSO)
124.6, 125.2, 127.2, 128.9, 134.8, 136.7, 140.6, 145.4,
160.1, 170.2 (C@O); FAB MS (298 ꢁC) m/z (%, frag-
ment), 428 (77, M+), 430 (100, M++2), 432 (44,
M++4), 434 (10, M++4), 427 (25, M+ꢀ1), 357 (40,
M+ꢀ2Cl), 392 (46, M+ꢀHCl), 336 (11), 321 (17), 289
(88), 265 (37), 235 (26), 205 (15), 189 (76), 157 (50)
136 (35) 105 (65), 77 (40).
4.3.2. 2,5-Bis (20-methylanilino)-3,6-dichloro-1,4-benzo-
quinone (3b). Yield: 333 mg, 86%; as brown needles mp
198 ꢁC (from dimethylformamide/benzene); Anal. Calcd
for C20H16Cl2N2O2: C, 61.97; H, 4.16; N, 7.23; C, 60.13;
H, 3.31; N, 7.72. Found: C, 61.92; H, 4.12; N, 7.19%.
UV/vis kmax (cmꢀ1) 367.2; mmax in cmꢀ1 (KBr) 3242
(N–H), 1652 (C@O), 1564, 1328, 1236, 1195, 1112,
1022; dH (300 MHz; DMSO) 8.192 (1H, s, NH, D2O
exchangeable), 7.235–7.260 (2H, d, J = 7.5 Hz, ArH (60
and 600)), 7.181–7.212 (4H, dd, J = 4.5 Hz, J = 4.8 Hz,
ArH (40, 50, 400 and 500)) 7.086–7.107 (2H, d,
J = 6.3 Hz, ArH (30 and 300)), 2.280 (6H, s, 2CH3); dc
(300 MHz; DMSO) 17.6 (CH3), 125.2, 126.9, 127.8,
129.5, 134.6, 137.6, 139.5, 144.7, 172.5 (C@O) FAB
MS (298 ꢁC) m/z (%, fragment), 387 (100, M+), 389
(33, M++2), 386 (15, M+ꢀ1), 351 (35, M+ꢀHCl), 315
(15, M+ꢀ2HCl), 307 (48), 289 (25), 279 (10), 277 (11),
242 (8), 233 (17, M+ꢀHCN), 215 (10), 203 (15), 191
(8), 177 (25), 107 (20).
4.3.6. 2,5-Bis(40-bromoanilino)-3,6-dichloro-1,4-benzoqui-
none (3f). Yield: 439 mg, 85%; as yellowish-green needles
mp 325 ꢁC (D) (from dimethylformamide/benzene);
Anal. Calcd for C18H10Cl2Br2N2O2: C, 41.77; H, 1.93;
N, 5.41. Found: C, 41.70;H, 1.90;N, 5.36%. UV/vis kmax
(cmꢀ1) 390; mmax in cmꢀ1 (KBr) 3230 (N–H), 1650
(C@O), 1595, 1318, 1245, 1175, 1100, 1035; dH
(300 MHz; DMSO) 8.24 (s, 1H, NH, D2O exchange-
able), 7.583–7.612 (2H, d, J = 8.7 Hz, ArH (30 and
300)), 7.439–7.468 (2H, d, J = 8.7 Hz, ArH (50 and 500)),
7.380–7.409 (2H, d, J = 8.7 Hz, ArH (20 and 200)),
7.089–7.118 (d, 2H, J = 8.7 Hz, ArH (60 and 600)); dc
(300 MHz; DMSO) 124.3, 125.2, 127.2, 128.2, 130.2,
134.6, 137.2, 142.6, 159.8, 171.2 (C@O); FAB MS
(298 ꢁC) m/z (%, fragment), 517 (38, M+), 519 (100,
M++2), 521 (89, M++4), 523 (32, M++4), 482 (45,
M+ꢀCl), 446 (85, M+ꢀ2Cl), 437 (25, M+ꢀBr), 357
(40, M+ꢀ2Br), 337 (15), 313 (26), 291 (28), 256 (84),
218 (23), 200 (17), 188 (65), 165 (45), 136 (70), 108
(43), 77 (35).
4.3.3. 2,5-Bis(20-methoxyanilino)-3,6-dichloro-1,4-benzo-
quinone (3c). Yield: 352 mg, 84%; as green needles mp
283 ꢁC (from dimethylformamide/benzene); Anal. Calcd
for C20H16Cl2N2O4: C, 57.29; H, 3.84; N, 6.68. Found:
C, 57.26; H, 3.81; N, 6.65%. UV/vis kmax (cmꢀ1) 390; mmax
in cmꢀ1 (KBr) 3240 (N–H), 1660 (C@O), 1580, 1340,
1200, 1140, 1040; dH (300 MHz; DMSO) 8.240 (1H, s,
NH, D2O exchangeable), 7.221–7.246 (2H, dd,
J = 1.5 Hz, J = 4.2 Hz, ArH (30 and 300)), 7.050–7.076
(2H, dd, J = 6.6 Hz, J = 1.2 Hz, ArH (60 and 600)),
6.902–6.984 (4H, m, ArH (40, 50, 400 and 500)), 3.87
(6H, s, 2OCH3); dc (300 MHz; DMSO) 55.9 (OCH3),
110.6, 119.6, 125.8, 126.9, 127.8, 139.7, 143.5, 152.2,
172.8 (C@O); FAB MS (298 ꢁC) m/z (%, fragment)
419 (100, M+), 421 (33, M++2), 418 (50, M+ꢀ1),
388 (25, M+ꢀOCH3), 387 (20, M+ꢀCH3OH), 357
(38, M+ꢀ2OCH3), 336 (25), 315 (5), 273 (12), 260 (18),
245 (8) 228 (11), 209 (12), 178 (10), 152 (20), 120 (35),
107 (60), 76 (20).
4.3.7. 2,5-Bis(40-methylanilino)-3,6-dichloro-1,4-benzo-
quinone (3g). Yield: 329 mg, 85%; as reddish brown nee-
dles mp 275 ꢁC (from dimethylformamide/benzene);
Anal. Calcd for C20H16Cl2N2O2: C, 62.03; H, 4.16; N,
7.23. Found: C, 62.01; H, 4.12; N, 7.19%. UV/vis kmax
(cmꢀ1) 420; mmax in cmꢀ1 (KBr) 3240 (N–H), 1650
(C@O), 1562, 1325, 1233, 1193, 1111, 1020; dH
(300 MHz; DMSO) 8.323 (1H, s, NH, D2O exchange-
able) 7.158–7.185 (4H, d, J = 8.1 Hz, ArH (20, 60, 200
and 600)), 7.013–7.040 (4H, d, J = 8.1 Hz, ArH (30, 50,
300 and 500)), 2.42 (6H, s, 2CH3); dc (300 MHz; DMSO)
17.9 (CH3), 125.6, 127.2, 127.5, 129.9, 134.8, 137.1,
144.6, 170.2 (C@O); FAB MS (298 ꢁC) m/z (%, frag-
ment), 387 (100, M+), 389 (63, M++2), 391 (10,
M++4), 386 (72, M+ꢀH), 351 (25, M+ꢀHCl), 349 (10),
333 (11) 315 (10, M+ꢀ2HCl), 307 (45), 279 (10) 277
(14), 242 (18), 233 (15), 215 (8), 203 (15), 191 (10), 177
(25), 154 (80), 152 (24), 136 (62), 135 (15), 107 (20).
4.3.4. 2,5-Bis(20-fluoroanilino)-3,6-dichloro-1,4-benzoqui-
none (3d). Yield: 328 mg, 83%; as brown needle mp
318 ꢁC (from dimethylformamide/benzene); Anal. Calcd
for C18H10F2Cl2N2O2: C, 54.71; H, 2.55; N, 7.08.
Found: C, 54.70; H, 2.52; N, 7.05%. UV/vis kmax
(cmꢀ1) 360; mmax in cmꢀ1 (KBr) 3240 (N–H), 1656
(C@O), 1576, 1331, 1221, 1105, 1035; dH (300 MHz;
DMSO) 8.280 (1H, s, NH, D2O exchangeable), 7.548–
7.646 (2H, dd, J = 1.5 Hz, J = 7.5 Hz, ArH (30 and
300)), 7.374–7.480 (2H, dd, J=1.2 Hz, J = 6.8 Hz, ArH
(60 and 600)), 7.210–7.327 (4H, m, ArH (40, 50, 400 and
50)); dc (300 MHz; DMSO) 126.6, 127.2, 128.2, 128.8,
133.8, 137.7, 139.6, 144.4, 162.1, 171.2 (C@O); FAB
MS (298 ꢁC) m/z (%, fragment), 395 (48, M+), 397 (16,
M++2), 394 (15, M+ꢀ1), 376 (10), 370 (9), 365 (38,
M+ꢀ2NH), 361 (17, M+ꢀ2F), 329 (50), 317 (10), 307
(20), 289 (20), 273 (7), 254 (70), 232 (40), 228 (9), 214
(10), 176 (90), 154 (89), 136 (85), 101 (80), 89 (35).
4.3.8. 2,5-Bis(40-methoxyanilino)-3,6-dichloro-1,4-benzo-
quinone (3h). Yield: 373 mg, 89%; as dark brown needles
mp 240 ꢁC (from dimethylformamide/benzene); Anal.

M. Batra et al. / Bioorg. Med. Chem. 14 (2006) 8519–8526
8525
Calcd for C20H16Cl2N2O4: C, 57.29; H, 6.65; N, 6.68.
Found: C, 57.25; H, 6.60; N, 6.16%. UV/vis kmax
(cmꢀ1) 400; mmax in cmꢀ1 (KBr) 3239 (N–H), 1654
(C@O), 1575, 1337, 1251, 1195, 1138, 1040; dH
(300 MHz; DMSO) 8.320 (1H, s, NH, D2O exchange-
able) 7.060–7.090 (4H, d, J = 9.0 Hz, ArH (30, 50, 300
and 500)), 6.870–6.900 (4H, d, J = 9.0 Hz, ArH (20, 60,
200 and 600)), 3.88 (6H, s, 2OCH3); dc (300 MHz; DMSO)
55.7, 111.1, 119.7, 126.6, 127, 127.9, 136.5, 143.6, 153.8,
172.8 (C@O); FAB MS (298 ꢁC) m/z (%, fragment), 419
(83, M+), 421 (49, M++2), 418 (50, M+ꢀ1), 413 (10), 388
(18, M+ꢀOCH3), 387 (12, M+ꢀCH3OH), 356 (9,
M+ꢀ2OCH3), 353 (10), 336 (5), 315 (8), 273 (13), 260
(10), 245 (9), 228 (8), 215 (7), 209 (12), 183 (9), 178
(12), 120 (48), 107 (56), 88 (45), 76 (30), 72 (20).
Peripheral human blood (PBL) samples were obtained
from volunteer cancer patients and PBL was prepared
by Ficoll-Hypaque density gradient centrifugation. The
sensitive leukaemia cell lines L5178 and its MDR1 gene
transfected resistant pair were obtained. MDR express-
ing cells were cultured in the presence of colchine up to
48 h before being used in the drug uptake assay.
4.5.1. Cell and fluorescence uptake, mdrl reversal effect.
The L5178 mouse T lymphoma cell line was infected
with the pHa MDR1/A retrovirus. MDR1 expressing
cell lines were selected by culturing the infected cells
with 60 mg/ml colchine to maintain expression of the
MDR phenotype. The L5178 MDR cell line and the
L5178Y parent cell line were grown in McCoy’s 5A
medium with 10% heat-inactivated horse serum, L-gluta-
mine and antibiotics. The cells were adjusted to a con-
centration of 2 · 106/ml and resuspended in serum free
McCoy’s 5A medium and the cells were distributed into
0.5 ml aliquot to Eppendorf centrifuge tubes. Then the
tested compounds were added in 20 lg/ml of the
1.0 mg/ml stock solutions and the samples were incubat-
ed for 10 min at room temperature. Then 10 ll (5.2 lM
final concentration) indicator Rhodamine 123 was add-
ed to the samples and the cells were incubated for fur-
ther 20 min at 37 ꢁC, washed twice and resuspended in
0.5 ml phosphate-buffered saline (PBS) for analysis.
The fluorescence of cell population was measured by
flow cytometry using Becton–Dickinson FACScan
instrument. Verapamil has been used as a positive con-
trol in the Rhodamine 123 exclusion experiments, Ep-
stein and Shafron calculated for parental and mdr cell
lines as compared to untreated cells. An activity ratio
was calculated by the following equation on the basis
of measured fluorescence values.
4.3.9. 2,5-Bis(40-fluoroanilino)-3,6-dichloro-1,4-benzoqui-
none (3i). Yield: 327 mg, 83%; as green needles
mp260 ꢁC (from dimethylformamide/benzene); Anal.
Calcd for C18H10F2Cl2N2O2: C, 54.68; H, 6.65; N,
4.08. Found: C, 54.63;H, 6.60;N, 4.05%. UV/vis kmax
(cmꢀ1) 390; mmax in cmꢀ1 (KBr) 3239 (N–H), 1651
(C@O), 1573, 1326, 1216, 1151, 1099, 1031; dH
(300 MHz; DMSO) 8.33 (1H, s, NH, D2O exchangeable)
7.175–7.200 (4H, d, J = 7.5 Hz, ArH (30, 50, 300 and 500)),
7.137–7.169 (4H, d, J = 6.9 Hz, ArH (20, 60, 200 and 600));
dc (300 MHz; DMSO) 123.5, 126.7, 128.2, 128.9, 135.2,
138.7, 140.2, 145.2, 170.8 (C@O); FAB MS (298 ꢁC)
m/z (%, fragment), 395 (63, M+), 397 (40, M++2), 394
(45, M+ꢀ1), 370 (10), 365 (15, M+ꢀ2NH), 361 (56,
M+ꢀ2F), 317 (8), 289 (15), 273 (6), 254 (62), 232 (38),
198 (8), 176 (85), 165 (11), 154 (82), 136 (80), 120 (18),
101 (90), 89 (32).
4.4. Antibacterial screening
The Antimicrobial Susceptibility Testing (AST) was
accomplished by the Kirby-Bauers’ disc diffusion meth-
od. All the synthesized compounds were screened for
their antibacterial activity against S. aureus (ATCC
25923), E. coli (ATCC 10536) and P. aeruginosa (ATCC
25619) using Muller Hinton Agar media (Hi Media).
The sample solution was prepared by dissolving 10 lg
of each of the compound in 1.0 ml of DMF. Gentamy-
cine (10 mcg) was used as standard drug for compari-
son. The sterilized Whatman filter paper discs of
approximately 6 (No. 1) mm were dipped in sample
solution and dried in oven. These discs were placed on
the medium previously seeded with the organisms in pet-
ri dishes at suitable distances. The petri dishes were
stored in an incubator at 30 2 ꢁC for 24 h. The zone
of inhibition thus formed around each disc containing
the test compound was measured accurately in
millimeters.
The results were analyzed by using cell quest software
(Becton–Dickinson) and presented as arbitrary units of
size (FSC) and granularity (SSC) and as the average
fluorescence intensity (FL-1).
Mdr treated=Mdr control
R ¼
parental treated=parentel control
Acknowledgments
We are thankful to SAIF, CDRI, Lucknow, for the spec-
tral (UV, IR,1H NMR,13C NMR and Mass) and elemen-
tal analysis. We are also thankful to Microbiology
department, J.L.N. Medical College, Ajmer, for provid-
ing antibacterial screening facility. Authors are grateful
to Prof. Joseph Molnar, University of Szeged, Hungary,
for his help in antitumour screening in his laboratories.
4.5. Antitumour screening
References and notes
All the compounds were studied for in vitro antitumour
screening. The compounds were screened for reversal of
multidrug resistance on mouse lymphoma cells transfec-
ted with human mdrl gene. The cell line used was L-5178
mouse T-cell lymphoma cell line which was infected with
the pH a MDR-1/A retrovirus.
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