Design and synthesis of novel magnolol derivatives as potential antimicrobial and antiproliferative compounds

Article abstract of DOI:10.1016/j.ejmech.2011.12.039

A series of novel magnolol derivatives were synthesised and evaluated for in vitro antimicrobial and antiproliferative activities. We found that most of the compounds were effective inhibitors of Staphylococcus aureus, MRSA and VRE with MIC in the range of 1-64 μg/mL and MBC in the range of 1-128 μg/mL. Few derivatives also exhibited promising antifungal activity. Some magnolol analogues exhibited promising antiproliferative activity than parent magnolol when tested against three human cancer cell lines.

Full text of DOI:10.1016/j.ejmech.2011.12.039

European Journal of Medicinal Chemistry 51 (2012) 35e41
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel magnolol derivatives as potential antimicrobial
and antiproliferative compounds
Srinivas Jada a, Mahendhar Reddy Doma a, Parvinder Pal Singh b, Suresh Kumar b, Fayaz Malik b,
,
Akash Sharma c, Inshad Ali Khan c, G.N. Qazi b, H.M. Sampath Kumar a
*
a Organic Division-I, Indian Institute of Chemical Technology, Hyderabad 500007, India
b Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180001, India
c Clinical Biology Division, Indian Institute of Integrative Medicine, Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel magnolol derivatives were synthesised and evaluated for in vitro antimicrobial and
antiproliferative activities. We found that most of the compounds were effective inhibitors of Staphy-
lococcus aureus, MRSA and VRE with MIC in the range of 1e64 mg/mL and MBC in the range of 1e128 mg/
mL. Few derivatives also exhibited promising antifungal activity. Some magnolol analogues exhibited
promising antiproliferative activity than parent magnolol when tested against three human cancer cell
lines.
Received 15 March 2011
Received in revised form
9 November 2011
Accepted 23 December 2011
Available online 30 January 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Magnolol
Antibacterial
MRSA
VRE
Antifungal
Antiproliferative
1. Introduction
Magnolol possesses an unusual biphenolic structure with two
para-allyl groups [13]. Owing to the pharmacological importance
Widespread use of antibiotics led to increased bacterial resis-
tance that become a life threatening problem worldwide. Of
particular concern are the infections caused by Methicillin resistant
Staphylococcus aureus (MRSA) and Vancomycin resistant Enterococci
(VRE) [1e3] which are very dangerous especially in immune
compromised patients due to HIV, surgery or any other illness. In
view of the limited classes of antibacterial drugs currently in use
that are prone to the development of microbial drug resistance,
there is an urgent unmet need for the development of new class of
antibiotics to fight against multi-drug resistant bacteria. Magnolol
is a naturally occurring biphenolic compound (Fig. 1) isolated from
the bark of Magnoliae officinalis which has been used in traditional
eastern medicine for the relief of flu symptoms, treatment of
anxiety and stroke. Several pharmacological activities are attrib-
uted to magnolol viz., antimicrobial, antioxidative, antianxiety,
antiinflammatory, including antiproliferative activities [4e12].
of magnolol, several groups have undertaken the structural
modifications of magnolol scaffold as well as the synthesis of its
structural analogues. Our literature survey revealed that antioxi-
dant, antimicrobial and antiproliferative activities of structural
analogues of magnolol which was primarily attributed to the
hydroxyl group at the biphenolic moiety [14e17]. Thus, anti-
proliferative and proapoptotic activity of eugenol related biphenyls
that are close structural analogues of magnolol on malignant
melanoma cells has been reported. Similarly di-hydroxyl magnolol
obtained through coupling of eugenol has been reported to exhibit
mild in vitro cytotoxicity against cancer cells. Allylated magnolol
derivatives prepared through Claisen rearrangement exhibited
potent antioxidant properties [16,17]. In view of the diverse
pharmacological activities of magnolol scaffold we felt the need
for further structural manipulations towards development of
improved structures derived from magnolol. Our continued
interest in developing secondary leads based on natural product
scaffolds [18e20], we report here, the synthesis and in vitro
screening of magnolol derivatives for antimicrobial and anti-
proliferative activities.
* Corresponding author. Tel.: þ91 9912901010; fax: þ91 191 2569333.
E-mail address: hmskumar@gmail.com (H.M.S. Kumar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.039

36
S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
reflux (Suzuki coupling) in excellent yields (90%). Compound 7 on
OH
OH
reaction with AlCl3 in dimethyl sulphide, followed by the reaction
with CuCl2, LiCl in acetic acid gives compounds 9 and 11 (3:7) in
good yields (82%). Compound 7 on reaction with AlCl3 in dimethyl
sulphide, followed by the reaction with Br2 in triethylamine and
toluene afford compounds 13 and 15 (4:6) in good yields (91%).
Compound 7 on reaction with AlCl3 in dimethyl sulphide, followed
by the reaction with I2 and CAN in acetonitrile gives compounds 17
and 19 (2:8) in high yields (90%). Compound 21 was prepared
according to literature procedure [22]. As illustrated in Scheme 2,
homo peroxidative coupling of compound 20 using MTBAP gives
compound 21 in moderate yields (52%). Compound 21 on reaction
with AlCl3 in dimethyl sulphide gives compounds 22 and 23 (2:8) in
excellent yields (95%) (Scheme 2).
Fig. 1. The chemical structure of magnolol.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 1, magnolol derivatives were syn-
thesised through Pd catalysed Suzuki coupling reaction. Compound
4 was synthesised by the reaction of 2 with B(OMe)3 and nBuLi in
dry THF at ꢀ78 ꢁC. Compound 6 could be obtained by the reaction
of 4 with Pd(PPh3)4 and Na2CO3 in dry DME under reflux (Suzuki
coupling) in excellent yields (92%). Compound 6 on reaction with
AlCl3 in dimethyl sulphide, followed by the reaction with CuCl2, LiCl
in acetic acid gives compounds 8 and 10 (3:7) in good yields (80%).
Compound 6 on reaction with AlCl3 in dimethyl sulphide, followed
by the reaction with Br2 in triethylamine in toluene gives
compounds 12 and 14 (4:6) in excellent yields (90%). Compound 6
on reaction with AlCl3 in dimethyl sulphide, followed by the reac-
tion with I2 and CAN in acetonitrile gives compounds 16 and 18
(2:8) in excellent yields (93%). The synthesis of compound 18 was
reported in literature with poor yields (16%), in which homo
coupling of 3,6-diiodo-4-propyl phenol gives compound 18 [21].
Compound 5 was synthesised by the reaction of 3 with B(OMe)3
and nBuLi in dry THF at ꢀ78 ꢁC. Compound 7 could be obtained by
the reaction of 5 with Pd(PPh3)4 and Na2CO3 in dry DME under
2.2. Evaluation of biological activity
2.2.1. Evaluation of antimicrobial activity
All the compounds were screened for antimicrobial activity
(Table 1), and ciprofloxacin and erythromycin are taken as stan-
dard. Most of the compounds showed excellent antibacterial
activity against gram positive bacteria (S. aureus ATCC, MRSA 15187
and VRE) whereas none of the compounds showed activity against
gram negative bacteria. Compounds 10e15, 19, 22 and 23 showed
promising antibacterial activity against S. aureus ATCC, MRSA 15187,
VRE (MIC in the range of 1e32
1e128 g/mL). Out of all the molecules synthesised compound 13
showed best activity (MIC against S. aureus ATCC, MRSA 15187 and
VRE are 2, 1 and 1 g/mL respectively; MBC against S. aureus ATCC,
MRSA 15187 and VRE are 2, 1 and 2 g/mL respectively). From the
mg/mL and MBC in the range of
m
m
m
data (Table 1) it is clear that unsaturation at the side chain is not
essential for antibacterial activity and alkyl groups like propyl,
butyl, along with halogen substituents on the aromatic ring
increases the antibacterial activity. The results clearly reveal the
OH
OH
OH
OH
Cl
Cl
Cl
R
R
R
R
10, R = C3H7
11, R = C4H9
8, R = C3H7
9, R = C4H9
c, d
OMe
R
OMe
R
OMe
R
OMe
OH
R
OH
R
OH
R
OH
R
Br
B(OMe)3
Br
Br
Br
a
c, e
b
R
2, R = C3H7
3, R = C4H9
6, R = C3H7
7, R = C4H9
4, R = C3H7
5, R = C4H9
12, R = C3H7
13, R = C4H9
14, R = C3H7
15, R = C4H9
c, f
OH
OH
R
OH
R
OH
I
I
I
R
R
16, R = C3H7
17, R = C4H9
18, R = C3H7
19, R = C4H9
Scheme 1. Reagents and conditions: (a) nBuLi, B(OMe)3, dry THF, ꢀ78 ꢁC, 2 h; (b) Pd(PPh3)4, Na2CO3, dry DME, reflux, 6 h; (c) AlCl3, Me2S, r.t. 1 h; (d) CuCl2, LiCl, CH3COOH, reflux,
12 h; (e) Br2, triethylamine, ꢀ70 ꢁC; (f) I2, CAN, CH3CN, r.t., 2 h.

S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
37
OH
OH
OH
OH
OH
OH
OH
OMe
MeO
OMe
a
HO
OH
HO
OMe
b
20
21
23
22
Scheme 2. Reagents and conditions: (a) MTBAP, dry CH2Cl2, 0 ꢁC, 15 min; (b) AlCl3/Me2S, r.t., 1 h.
Table 2
pattern of activity with regard to degree and nature of halogen
substitution on the aromatic rings of biaryl compounds. Even
though mono-chloro and -iodo derivatives of propyl and butyl
magnolol did not show appreciable activity against MRSA, VRE and
S. aureus ATCC strains, the corresponding -monobromo derivatives
showed significant inhibitory activity against these pathogens.
However, the antibacterial activity of -dihalo derivatives of propyl
and butyl magnolol had been comparatively significant wherein
the -dibromo compounds exhibited highest activity. We have also
tested all the compounds for antifungal activity against Candida
albicans ATCC 90028 and Aspergillus fumigatus LSI-II using
Amphotericin-B as standard. Interestingly, compounds with allylic
substitution i.e., 21 and 23 exhibited significant antifungal activity
IC50 values (mM) of magnolol derivatives against a panel of cell lines.
Compound
HL-60
PC-3
MOLT-4
8
9
10
11
12
13
14
15
16
17
18
19
85
>100
>100
90
>100
25
30
>100
45
>100
>100
>100
>100
>100
74
95
>100
63
>100
>100
>100
>100
>100
35
55
>100
60
50
7
2
60
7
2
66
18
14
against C. albicans ATCC 90028 (MIC 64 and 128
compounds 21 and 23 respectively) and A. fumigatus LSI-II (MIC 32
and 64 g/mL for compounds 21 and 23 respectively) whereas
mg/mL for
21
22
23
Magnolol
5-FU
>100
>100
>100
48
>100
>100
>100
62
>100
>100
>100
58
m
other compounds did not show any significant antifungal activity.
266
2.1
3.2
2.2.2. Antiproliferative activity
In vitro antiproliferative activity of all the compounds was
evaluated against a panel of three human cancer cell lines viz., PC-3
(prostate cancer cells), HL-60 (human promyelocytic leukaemia
cells) and MOLT-4 (human acute lymphoblastic leukaemia cell line)
according to NCI guidelines and 5-FU was taken as reference
compound. Among all the compounds, only alkylated-iodo and
alkylated-bromo derivatives 13, 14, 18 and 19 exhibited better
antiproliferative activity than parent magnolol (1) (Table 2).
Compound 19, with -diiodo and butyl substitutions, is the most
2.2.3. DNA fragmentation and flow cytometric analysis
DNA fragmentation, which is a typical hallmark of the apoptotic
cell death was analysed in HL-60 cells. From the in vitro cytotoxicity
active with IC50 values 2, 2 and 10
cell lines respectively.
mM on HL-60, PC-3 and MOLT-4
Table 1
MIC and MBC of magnolol derivatives in
m
g/mL.
MRSA 15187
Compound
S. aureus ATCC 29213
VRE
MIC
MBC
MIC
MBC
MIC
MBC
8
9
10
>256
>256
>256
>256
>256
>256
4
>256
>256
4
>256
>256
8
>256
>256
8
4
4
11
12
13
8
8
2
8
8
2
8
16
1
8
16
1
8
16
1
16
32
2
14
4
4
4
4
4
8
15
8
8
8
8
8
16
16
17
18
>256
>256
64
>256
>256
64
>256
>256
64
>256
>256
64
>256
>256
64
>256
>256
64
19
4
4
4
4
4
8
21
22
23
>256
16
>256
16
>256
16
16
>256
64
16
>256
32
16
>256
128
32
8
8
Magnolol
Erythromycin
Ciprofloxacin
16
0.5
0.25
32
1
0.5
32
64
8
32
64
8
32
64
32
32
64
64
Fig. 2. Agarose gel electrophoresis of DNA extracted from HL-60 cells. The figure
represents HL-60 cells treated with compound 19 for 24 h. DNA from the cells was
extracted and electrophoresed in 1% agarose gel and visualized by ethidium bromide
staining under UV illumination. Lanes A, B, C, D and E represent compound 19 at 0, 1, 5,
MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal
concentration.
10 and 30 mM.

38
S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
Fig. 3. Flow cytometric analysis of cells treated with compound 19 (10 mM) at different time intervals.
studies it was found that compound 19 significantly inhibits the
growth of human promyelocytic leukaemia cells HL-60, and was
therefore studied further to determine the mechanism of cell death
in the same cell line. The DNA fragmentation analysis revealed that
compound 19 induced a discrete ladder pattern in HL-60 cell line at
drug resistant bacteria like MRSA and VRE. Alkylated-iodo and
alkylated-bromo substituted derivatives showed improved anti-
proliferative activity as compared to magnolol against PC-3, HL-60
and MOLT-4 human cancer cell lines. DNA laddering and flow
cytometric analysis clearly revealed the mechanism involving
apoptotic pathway to cell death induced by the novel biphenolic
derivatives. We found that some of the compounds bearing allylic
side chain exhibited significant antifungal activity against
C. albicans and A. strains.
5,10 and 30 mM after 24 h of incubation (Fig. 2). The flow cytometric
analysis of compound 19 showed a dose dependent increase in the
sub-G1 population in HL-60 cell line after 24 h. Flow cytometric
analysis showed that the ratio of apoptosis in HL-60 cells for
compound 19 at 1, 10, 30 and 100 mM were 12, 53, 67 and 78%
respectively. In order to examine DNA profiles at different time
points, flow cytometric analysis of compound 19 has been carried
4. Experimental
out at 10 mM concentration at time intervals of 0, 6, 9, 18, 24 h and
ratio of apoptosis was found to be 3.7, 21, 33, 44 and 52% respec-
tively (Fig. 3).
Melting points were recorded on Buchi Melting point apparatus
D-545 and IR spectra (KBr) on Bruker Vector 22 instrument. NMR
spectra were recorded on Bruker DPX500 instrument in CDCl3 with
TMS as an internal standard. Chemical shift values are reported in
3. Conclusions
d (ppm) and coupling constants in hertz. Mass spectra were
recorded on Q-STAR XL mass spectrometer (Applied Biosystems,
USA) instrument. The progress of all reactions was monitored by
TLC on 2 ꢂ 5 cm pre-coated silica gel 60 F254 plates of thickness
0.25 mm (Merck). The chromatograms were visualized under UV
254e366 nm and iodine.
In conclusion, a series of novel magnolol derivatives synthesised
and screened for antimicrobial and antiproliferative activity. Our
study found that most of the alkylated halogenated magnolol
derivatives exhibited excellent antibacterial activity against multi-

S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
39
4.1. Synthesis of magnolol derivatives
mixture was stirred for 1 h, the phases were separated and the
organic layer was dried over Na2SO4. After filtration, the dichloro-
methane was removed under reduced pressure and the product,
a dark purple solid, was kept under high vacuum for 1 h affording
1.277 g (90%) of the oxidant salt MTBAP.
4.1.1. General procedure for Suzuki coupling
To a solution of compound 4 in dry DME were added Pd(PPh3)4
(catalytic amount) and Na2CO3 (3 equiv). The reaction mixture was
refluxed for 6 h. After completion, the reaction mixture was diluted
with 80 mL of water and extracted with ethyl acetate (3ꢂ50 mL).
The combined extracts were washed with brine, dried over anhy-
drous Na2SO4 and evaporated in vacuum. The crude product
obtained was purified by flash chromatography to get pure
compound (6) in excellent yields (92%).
4.1.7. Typical procedure for oxidative coupling of eugenol (21)
To a solution of eugenol 20 (493 mg, 3 mmol) in dry dichloro-
methane (5 mL) under N2 atmosphere at 0 ꢁC, was added a solution
of MTBAP (479 mg, 1.5 mmol) in dry dichloromethane (10 mL).
After 15 min, water (10 mL) was added into the reaction flask fol-
lowed by the bubbling of SO2. The organic layer was separated,
washed with a 0.1 M solution of HCl (3ꢂ5 mL), dried over anhy-
drous Na2SO4, filtered and the solvent was removed under reduced
pressure. The product obtained was purified by flash chromatog-
raphy to get pure compound 21 (52%).
4.1.2. General procedure for demethylation using AlCl3/Me2S
To a solution of AlCl3 (1.2 equiv) in Me2S was added compound
(21) dissolved in Me2S drop wise at room temperature under N2
atmosphere. The reaction mixture was stirred for 1 h. Check the
TLC, two new spots will appear due to partial and complete
demethylation (compounds 22 and 23). If the reaction mixture was
stirred for 2 h, complete demethylation takes place and compound
23 form as sole product. After completion, the reaction mixture was
quenched with ice-cold water and 2 N HCL, and then extracted with
ethyl acetate. The combined extracts were washed with brine, dried
over anhydrous Na2SO4 and evaporated in vacuum. The crude
product obtained was purified by flash chromatography to get pure
compounds 22 and 23 (2:8) in good yields (95%).
4.1.8. 3-Chloro-5,50-dipropyl-2,20-biphenyldiol (8)
White colour solid; mp: 100 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.95 (t, J ¼ 7.32, 6H), 1.57e1.53 (m, 4H), 2.5 (t, J ¼ 7.3, 4H), 5.88 (s,
eOH, 1H), 6.12 (s, eOH, 1H), 6.94 (d, J ¼ 8.23, 1H), 7.02 (d, J ¼ 1.86,
1H), 7.04 (d, J ¼ 1.91,1H), 7.11 (d, J ¼ 8.2,1H), 7.20 (d, J ¼ 1.91,1H); 13C
NMR: 13.76, 13.88, 24.56, 24.79, 37.03, 37.23, 117.11, 120.65, 124.35,
125.88, 128.7, 129.81, 130.59, 130.98, 135.63, 136.60, 145.92, 151.02;
IR (KBr): 3518.38, 2954.5, 2872.24, 1239.23, 1078.7, 742.50 cmꢀ1
;
HRMS for C18H21O2Cl [M þ Na]þ Calc. 327.1127; found 327.1144;
4.1.3. General procedure for chlorination
Anal. Calc. for C18H21O2Cl: C 70.93, H 6.94; found: C 70.86, H 6.98.
To a solution of alkylated magnolol (1 equiv) in CH3COOH were
added CuCl2 (1.5 equiv) and LiCl (1.5 equiv) under O2 atmosphere.
The reaction mixture was refluxed for 12 h. After completion, the
reaction mixture was diluted with 100 mL of water and extracted
with ethyl acetate (3ꢂ50 mL). The combined extracts were washed
with brine, dried over anhydrous Na2SO4 and evaporated in vacuum.
The crude product obtained was purified by flash chromatography
to get pure compounds 8 and 10 (3:7) in good yields (80%).
4.1.9. 3-Chloro-5,50-dibutyl-2,20-biphenyldiol (9)
White colour solid; mp: 100e101 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.94 (t, J ¼ 7.2, 6H), 1.39e1.35 (m, 4H), 1.61e1.58 (m, 4H), 2.55 (t,
J ¼ 7.24, 4H), 6.96 (d, J ¼ 8.1, 1H), 7.03 (d, J ¼ 1.88, 1H), 7.05 (d,
J ¼ 1.90, 1H), 7.10 (d, J ¼ 8.12, 1H), 7.2 (d, J ¼ 1.92, 1H); 13C NMR:
13.99, 22.34, 22.44, 29.78, 33.64, 33.74, 34.68, 34.71, 115.06, 119.6,
123.6, 125.9, 126.18, 128.9, 129.33, 130.9, 136.1, 136.12, 144.5, 149.5;
IR (KBr): 3356.5, 2956.6, 2928.12, 1468.17, 1236.15, 756.6 cmꢀ1
;
4.1.4. General procedure for bromination
HRMS for C20H25O2Cl [M þ Na]þ Calc. 355.5217; found 355.5225;
To a solution of triethylamine (4 equiv) in toluene solution
at ꢀ20 ꢁC was added bromine drop wise. Then temperature was
decreased to ꢀ70 ꢁC. Then alkylated magnolol dissolved in anhy-
drous dichloromethane was added drop wise. The reaction mixture
was stirred at ꢀ70 ꢁC for 10 min then allows the reaction mixture to
room temperature. After completion, the reaction mixture was
diluted with 100 mL of water and extracted with ethyl acetate
(3ꢂ50 mL). The combined extracts were washed with brine, dried
over anhydrous Na2SO4 and evaporated in vacuum. The crude
product obtained was purified by flash chromatography to get pure
compounds 12 and 14 (4:6) in excellent yields (90%).
Anal. Calc. for C20H25O2Cl: C 72.17, H 7.57; found: C 72.12, H 7.6.
4.1.10. 3,30-Dichloro5,50-dipropyl-2,20-biphenyldiol (10)
White colour solid; mp: 103 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.95 (t, J ¼ 7.32, 6H), 1.57e1.52 (m, 4H), 2.46 (t, J ¼ 7.3, 4H), 5.86 (s,
eOH, 2H), 6.93 (d, J ¼ 2.01, 2H), 7.12 (d, J ¼ 1.98, 2H); 13C NMR: 12.7,
23.48, 35.94, 119.86, 124.49, 127.92, 129.29, 135.02, 145.39; IR (KBr):
3518.38, 2959.20, 2870.54, 1240.23, 1077.72, 742.5 cmꢀ1; HRMS for
C18H20O2Cl2 [M þ Na]þ Calc. 362.6344; found 362.6347; Anal. Calc.
for C18H20O2Cl2: C 63.73, H 5.94; found: C 63.66, H 5.98.
4.1.11. 5,50-Butyl-3,30-dichloro-2,20-biphenyldiol (11)
4.1.5. General procedure for iodination
White colour solid; mp: 105 ꢁC; 1H NMR (500 MHz, CDCl3):
To a solution of alkylated magnolol in acetonitrile were added I2
and CAN. The reaction mixture was stirred at room temperature for
2 h. After completion of the reaction, solvent was evaporated in
vacuum. Then the reaction mixture was diluted with 100 mL of
water and extracted with ethyl acetate (3ꢂ50 mL). The combined
extracts were washed with brine, dried over anhydrous Na2SO4 and
evaporated in vacuum. The crude product obtained was purified by
flash chromatography to get pure compounds 16 and 18 (4:6) in
excellent yields (90%).
d
0.93 (t, J ¼ 7.19, 6H), 1.45e1.22 (m, 4H), 1.66e1.51 (m, 4H), 2.56 (t,
J ¼ 7.19, 4H), 6.98 (d, J ¼ 2.05, 2H), 7.2 (d, J ¼ 2.05, 2H); 13C NMR:
13.89, 22.24, 33.54, 34.59, 120.87, 125.48, 128.87, 130.08, 136.25,
146.38: IR (KBr): 3518.95, 2959.20, 2927.47, 1475.14, 1243.23,
1077.72, 795.57 cmꢀ1; HRMS for C20H24O2Cl2 [M þ Na]þ Calc.
390.6466; found 390.6470; Anal. Calc. for C20H24O2Cl2: C 65.40, H
6.59; found: C 65.36, H 6.63.
4.1.12. 3-Bromo-5,50-dipropyl-2,20-biphenyldiol (12)
White colour solid; mp: 95 ꢁC; 1H NMR (500 MHz, CDCl3):
d 0.88
4.1.6. Typical procedure for preparation of MTBAP
(t, J ¼ 7.26, 6H), 1.6e1.55 (m, 4H), 2.52 (t, J ¼ 7.21, 4H), 5.8 (s, eOH,
1H), 6.01 (s, eOH,1H), 6.86 (d, J ¼ 8.12, 1H), 6.97 (d, J ¼ 1.9, 1H), 6.99
(d, J ¼ 1.92, 1H), 7.03 (d, J ¼ 6.41, 1H), 7.18 (d, J ¼ 1.92, 1H); 13C NMR:
13.28, 13.47, 23.89, 24.09, 36.22, 36.62, 110.37, 116.33, 123.71, 125.0,
129.1, 130.4, 131.2, 131.63, 134.92, 136.25, 146.12, 150.27; IR (KBr):
To a round bottom flask containing an aqueous solution (75% w/
w) of methyltributylammonium chloride (1.258 g, 4 mmol) and
dichloromethane (100 mL) was added a solution of potassium
permanganate (632 mg, 4 mmol) in water (35 mL). The resulting

40
S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
3521.3, 2962.60, 2871.54, 1472.12, 1249.23, 1080.72, 760.33,
745.60 cmꢀ1; HRMS for C18H21O2Br [M þ Na]þ Calc. 369.9727;
found 369.9732; Anal. Calc. for C18H21O2Br: C 61.9, H 6.06; found: C
61.82, H 6.09.
4.1.19. 5,50-Butyl-3,30-diiodo-2,20-biphenyldiol (19)
Light yellow colour solid; mp: 113e114 ꢁC; 1H NMR (500 MHz,
CDCl3):
d
0.95 (t, J ¼ 7. 11, 6H), 1.44e1.3 (m, 4H), 1.68e1.63 (m, 4H),
2.65 (t, J ¼ 7.56, 4H), 7.45 (d, J ¼ 2.06, 2H), 7.99 (d, J ¼ 2.03, 2H),
10.84 (s, 2H); 13C NMR: 13.94, 22.13, 32.84, 33.84, 123.5, 126.51,
131.06, 133.71, 139.19, 151.35; IR (KBr): 3518.45, 2960.0, 2870.42,
1475.14, 1077.5, 770.3 cmꢀ1; HRMS for C20H24O2I2 [M þ Na]þ Calc.
573.3456; found 573.3460; Anal. Calc. for C20H24O2I2: C 43.66, H
4.40; found: C 43.56, H 4.43.
4.1.13. 3-Bromo-5,50-dibutyl-2,20-biphenyldiol (13)
Pale brown colour solid; mp: 97e98 ꢁC; 1H NMR (500 MHz,
CDCl3):
d
0.93 (t, J ¼ 7.23, 6H), 1.38e1.22 (m, 4H), 1.67e1.45 (m, 4H),
2.58 (t, J ¼ 7.29, 4H), 5.7 (s, eOH, 1H), 5.95 (s, 1H), 6.92 (d, J ¼ 8.19,
1H), 7.01 (d, J ¼ 1.92, 1H), 7.05 (d, J ¼ 1.94, 1H), 7.14 (d, J ¼ 6.41, 1H),
7.35 (d, J ¼ 1.69, 1H); 13C NMR: 13.99, 22.34, 29.78, 33.74, 33.93,
34.71, 34.86, 114.70, 118.54, 121.3, 124.5, 125.9, 129.5, 130.6, 131.1,
135.9, 136.3, 144.0, 150.3; IR (KBr): 3526.02, 2958.30, 2869.8,
4.1.20. 3-Hydroxy-30-methoxy-5,50-dipropyl-2,20-biphenyldiol (22)
White colour solid; mp: 114e115 ꢁC; 1H NMR (500 MHz, CDCl3):
d
3.32 (d, J ¼ 6.51, 4H), 3.92 (s, 3H), 5.11e5.03 (m, 4H), 6.04e5.94 (m,
1481.27, 1468.93, 1240.81, 1093.81, 756.61 cmꢀ1
;
HRMS for
2H), 6.80e6.67 (m, 4H); 13C NMR: 39.80, 40.00, 56.11, 110.28, 114.41,
115.63, 115.99, 120.9, 121.75, 123.36, 125.98, 133.16, 133.83, 137.38,
137.63, 138.72, 139.20, 146.35, 146.50; IR (KBr): 3525.3, 2935.0,
2870.24, 1233.23, 1125.1, 1069.6, 744.54 cmꢀ1; HRMS for C19H20O4
[M þ Na]þ Calc. 335.1259; found 335.1264; Anal. Calc. for C19H20O4:
C 73.06, H 6.45; found: C 73.06, H 6.48.
C20H25O2Br [M þ Na]þ Calc. 398.0854; found 398.0859; Anal. Calc.
for C20H25O2Br: C 63.66, H 6.68; found: C 63.60, H 6.12.
4.1.14. 5,50-Dipropyl-3,30-dibromo-2,20-biphenyldiol (14)
White colour solid; mp: 96 ꢁC; 1H NMR (500 MHz, CDCl3):
d 0.95
(t, J ¼ 7.32, 6H),1.66e1.59 (m, 4H), 2.53 (t, J ¼ 7.47, 4H), 5.88 (s, eOH,
2H), 7.01 (d, J ¼ 2.03, 2H), 7.35 (d, J ¼ 2.05, 2H); 13C NMR: 13.80,
24.58, 37.06, 120.98, 125.59, 129.03, 130.25, 136.13, 146.52; IR (KBr):
4.2. Antibacterial activity
3455.5, 2957.36, 2928.27, 2870.6, 1375.6, 1245.75, 781.9 cmꢀ1
;
Antibacterial activity of the samples was performed using micro
dilution method [23] against 3 g positive strains (S. aureus ATCC
29213, Methicillin resistant S. aureus, Vancomycin resistant Entero-
coccus). Bacterial suspensions were prepared in sterile normal
saline from 24 h grown culture. The Minimum Inhibitory Concen-
tration (MIC) was performed in Muller Hinton Broth (MHB; BD
Biosciences, USA). Two-fold serial dilutions of samples were
HRMS for C18H20O2Br2 [M þ Na]þ Calc. 448.9727; found 448.9739;
C18H20O2Br2: C 50.49, H 4.71; found: C 50.41, H 4.75.
4.1.15. 5,50-Dibutyl-3,30-dibromo-2,20-biphenyldiol (15)
Brown colour solid; mp: 98 ꢁC; 1H NMR (500 MHz, CDCl3):
d 0.93
(t, J ¼ 7.22, 6H), 1.49e1.26 (m, 4H), 1.66e1.51 (m, 4H), 2.56 (t,
J ¼ 7.33, 4H), 5.83 (s, eOH, 2H), 7.01 (d, J ¼ 1.7, 2H), 7.35 (d, J ¼ 1.67,
2H); 13C NMR: 13.79, 24.60, 36.81, 36.93, 111.12, 125.52, 131.02,
132.09,136.64,147.34; IR (KBr): 3450.83, 2958.33, 2928.21, 2869.88,
1377.99, 1241.67, 787.13 cmꢀ1; HRMS for C18H20O2Br2 [M þ Na]þ
Calc. 469.0754; found 469.0769; Anal. Calc. for C20H24O2Br2: C
52.65, H 5.30; found: C 52.58, H 5.33.
prepared in MHB in 100
mL volume in a 96 well U bottom microtitre
plates (Tarson, Mumbai, India). The final concentrations of the
samples ranged from 0.25 to 256 m
g mLꢀ1. The turbidity of bacterial
suspensions was adjusted to 0.5 McFarland (w1.5 ꢂ 108 CFU mLꢀ1),
which was further diluted in MHB and, a 100 mL volume of this
diluted inoculum was added to each well of the plate, resulting in
a final inoculum of 5 ꢂ 106 CFU mLꢀ1. The plates were incubated at
37 ꢁC for 24 h and were read visually. The minimum concentration
of the sample showing no turbidity was recorded as MIC. The
Minimum Bactericidal Concentration (MBC) was also determined
4.1.16. 3-Iodo-5,50-dipropyl-2,20-biphenyldiol (16)
White colour solid; mp: 108 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.95 (t, J ¼ 7.27, 6H), 1.54e1.5 (m, 4H), 2.5 (t, J ¼ 7.36, 4H),
7.36e7.23 (m, 4H), 7.83 (d, J ¼ 1.96, 1H), 10.84 (s, eOH, 2H); 13C
NMR: 13.75, 13.82, 24.72, 24.86, 37.66, 37.73, 117.154, 121.5, 124.36,
125.93, 129.1, 130.6, 131.5, 132.9, 135.6, 136.50, 145.9, 151.02; IR
(KBr): 3524.3, 2958.5, 1472.4, 1070.3, 761.30 cmꢀ1; HRMS for
C18H21O2I [M þ Na]þ Calc. 419.1215; found 419.1244; Anal. Calc. for
C18H21O2I: C 54.56, H 5.34; found: C 54.50, H 5.37.
from the same microtitre plates but after 24 h incubation. 20 mL of
suspension from the well showing MIC value and wells containing
2ꢂ, 4ꢂ, 8ꢂ and 16ꢂ concentration of MIC value was spotted onto
the Muller Hinton Agar plate. The spotted plate was incubated for
24 h and CFU count was taken simultaneously. The minimum
concentration of the sample showing 3 log reductions in the
inoculums size as compared to the original inoculums size was
considered as the MBC.
4.1.17. 3-Iodo-5,50-dibutyl-2,20-biphenyldiol (17)
Pale brown colour solid; mp: 110 ꢁC; 1H NMR (500 MHz, CDCl3):
d
0.94 (t, J ¼ 7.21, 6H), 1.39e1.23 (m, 4H), 1.69e1.47 (m, 4H), 2.56 (t,
4.3. DNA fragmentation assay
J ¼ 7.36, 4H), 7.37e7.24 (m, 4H), 7.75 (d, J ¼ 1.73, 1H), 10.71 (s, eOH,
2H); 13C NMR: 13.86, 22.12, 30.3, 33.5, 33.66, 34.75, 34.93, 113.96,
118.42, 121.54, 124.27, 126.6, 129.75, 131.4, 131.9, 136.24, 136.57,
144.42, 150.64; IR (KBr): 3526.02, 2958.30, 2869.8, 1468.93,
1093.81, 756.61 cmꢀ1; HRMS for C20H25O2I [M þ Na]þ Calc.
447.3354; found 447.3367; Anal. Calc. for C20H25O2I: C 56.61, H
5.94; found: C 56.56, H 5.98.
DNA fragmentation was determined by electrophoresis of
extracted genomic DNA from HL-60 cells. Cells (2ꢂ106/mL medium)
in 60 mm tissue culture plate were treated with compound 19 at 1,
5, 10 and 30 mM for 24 h. Cells were harvested, washed with PBS,
pellets were dissolved in lysis buffer (10 mM EDTA, 50 mM tris pH
8.0, 0.5% w/v SDS and proteinase K (0.5 mg/mL)) and incubated at
50 ꢁC for 1 h. Finally the DNA obtained was heated rapidly to 70 ꢁC,
supplemented with loading dye and immediately resolved on to
1.5% agarose gel at 50 V for 2e3 h.
4.1.18. 5,50-Dipropyl-3,30-diiodo-2,20-biphenyldiol (18)
Light yellow colour solid; mp: 111e112 ꢁC; 1H NMR (500 MHz,
CDCl3):
d
0.98 (t, J ¼ 7.26, 6H),1.74e1.59 (m, 4H), 2.63 (t, J ¼ 7.29, 4H),
7.35 (d, J ¼ 2.16, 2H), 7.99 (d, J ¼ 2.23, 2H), 10.84 (s, 2H); 13C NMR:
4.4. Flow cytometric analysis
13.84, 22.73, 35.84, 122.54, 125.91, 130.86, 132.45, 139.1, 150.5; IR
(KBr): 3520.5, 2962.0, 2873.45, 1685.0, 1472.0, 1081.55, 763.3 cmꢀ1
;
Effect of compound 19 on DNA content by cell cycle phase
HRMS for C18H20O2I2 [M þ Na]þ Calc. 545.3352; found 545.3358;
distribution was assessed using HL-60 cells by incubating the cells
Anal. Calc. for C18H20O2I2: C 41.4, H 3.86; found: C 41.33, H 3.89.
1ꢂ106 mL/well with compound 19 (1, 10, 30 and 100
mL each) for

S. Jada et al. / European Journal of Medicinal Chemistry 51 (2012) 35e41
41
24 h. The cells were then washed twice with ice-cold PBS, har-
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