Facile synthesis, antioxidant and antimicrobial activity of amino methylene bisphosphonates

Article abstract of DOI:10.1248/cpb.60.104

A green and efficient preparation method for the amino bisphosphonates is accomplished by simple mixing and stirring of diethylphosphite, triethylorthoformate and various amines in the presence of amberlyst-15 as catalyst at room temperature under solvent free conditions. The title compounds are characterized by IR, ¹H-, ¹³C-, ³¹P-NMR and mass spectra, also studied their antimicrobial and antioxidant activity.

Full text of DOI:10.1248/cpb.60.104

—
104
Regular Article
Chem. Pharm. Bull. 60(1) 104 109 (2012)
Vol. 60, No. 1
Facile Synthesis, Antioxidant and Antimicrobial Activity of Amino
Methylene Bisphosphonates
Uma Maheswara Rao Kunda,a Satheesh Krishna Balam,a Bakthavatchala Reddy Nemallapudi,a
,a
Syama Sundar Chereddy,a Sandip Kumar Nayak,b and Suresh Reddy Cirandur*
b
a Department of Chemistry, Sri Venkateswara University; Tirupati 517 502, India: and Bio-Organic Division, BARC;
Mumbai 400-085, India. Received September 8, 2011; accepted October 17, 2011; published online October 21, 2011
A green and efficient preparation method for the amino bisphosphonates is accomplished by simple mix-
ing and stirring of diethylphosphite, triethylorthoformate and various amines in the presence of amberlyst-15
as catalyst at room temperature under solvent free conditions. The title compounds are characterized by IR,
1H-, 13C-, 31P-NMR and mass spectra, also studied their antimicrobial and antioxidant activity.
Key words amino methylene bisphosphonate; antimicrobial activity; antioxidant activity; amberlyst-15
Early research on the role of antioxidants in biology focused forming the various functional group transformations in the
on their use in preventing oxidation of unsaturated fats, which general organic synthesis.19)
is the cause of rancidity.1) However, it was the identification of
We now report a relatively simple synthesis of amino meth-
the antioxidant activity of vitamins A, C, and E that revolu- ylene bisphosphonates in good yields using amberlyst-15 as an
tionized the field and led to the realization of the importance efficient and environmentally benign catalyst under solvent-
of antioxidants in the biochemistry of living organisms.2,3) The free conditions at room temperature. There have been no
antioxidant activity was measured simply by placing the fat reports on the synthesis of amino methylene bisphosphonates
in a closed container with oxygen and measuring the rate of using amberlyst-15 as catalyst.
oxygen consumption. Excessive generation of reactive oxygen
species (ROS) induced by various stimuli leads to variety of
Experimental
pathophysiological abnormalities such as inflammation, dia-
General Melting points were recorded on Buchi R-535
betes, genotoxicity and cancer. Search for active components apparatus and are uncorrected. IR spectra were recorded on
that prevent or reduce the impact of oxidative stress on cell is a Perkin-Elmer FT-IR 240-c spectrophotometer using KBr
1
a contemporary field.
optics. H-, 13C- and 31P-NMR spectra were recorded on AMX
1
The aim of this study was to investigate the in vitro antimi- 400MHz NMR spectrometers operating at 400MHz for H-,
crobial and antioxidant profile of different nitrogen-containing 100MHz for 13C- and 161.89MHz for 31P-NMR. NMR data
bisphosphonates (N-BPS), which, the best of our knowledge were recorded in DMSO-d6 and referenced to tetramethyl-
has not yet been systematically studied. N-BPS, a class of silane (TMS) (1H and 13C) and 85% H3PO4 (31P). Mass spectra
geminal bisphosphonates (BPS) are important drugs for wide were recorded on a Finnigan MAT 1020/Micro-Mass Q-T
variety of medicinal applications.4) Besides their well-known of micro AMPS MAX 10/6A, Hz 60/50 system fitted with
anti-bone resorption properties,5) antimicrobial activity6) and a built-in inlet system. Elemental analyses were performed
anti-tumor properties,7) some of their derivatives inhibit can- using Perkin Elmer 2400 instrument at the Central Drug Re-
cer manifestations through antiangiogenic, anti-invasive and search Institute (CDRI), Lucknow, India.
—
10)
immunomodulatory actions.8
Experimental Procedure for the Preparation of Tetra-
BPS are also effective in the treatment of rheumatoid ar- ethyl(3,4-dichlorophenylamino)methylenebisphosphonate
—
13)
thritis (RA).11
Recently bisphosphonates have been proved (3a) A mixture of 3,4-dichloro aniline (1a, 0.005mol), dieth-
to prevent glucocorticoid-induced osteoporosis in RA14,15) and ylphosphite (2, 0.01mol), triethylorthoformate (0.005mol) and
to possess selective antioxidant properties in vitro.16) More- catalytic amount of amberlyst-15 (0.1g) was stirred magneti-
over, some recent clinical studies in the animals indicate anti- cally at room temperature for appropriate time. After comple-
inflammatory effects by BPS in RA.16,17)
tion of the reaction as monitored by TLC, it was purified by
—
In recent years, use of solid acid catalysts has attracted column chromatography on 60 120 mesh silica gel using
considerable attention.18) In this regard, amberlyst-15 possesses ethyl acetate–hexane (1:4) as eluent. The residue was re-
unique properties is successfully used as a solid acid catalyst crystallized from ethylacetate to afford pure 3a in 95% yield.
for the preparation of bisphosphonates from the amine, dialkyl This procedure was applied successfully for the preparation of
1
phosphite and triethylorthoformate. Amberlyst-15 is a strongly 3b j. All the compounds were characterized by IR, H-, 13C-,
—
acidic, sulfonic acid, macro reticular polymeric resin based on 31P-NMR, mass spectral and elemental analytical data.
cross linked styrene divinylbenzene copolymers. Its continu-
Tetraethyl(3,4-dichlorophenylamino)methylenebisphospho-
ous open pore structure and excellent physical, thermal and nate (3a): White solid, yield: 95%, mp 120 122 C. 1H-
—
°
3
chemical stability makes it the resin of choice in many ap- NMR (400MHz, DMSO-d6) δ: 1.06 (6H, t, JPH =8.8Hz,
plications. It also possesses greater resistance to oxidants such POCH2CH3), 1.20 (6H, t, JPH =9.2Hz, POCH2CH3), 3.70
3
—
—
as chlorine, oxygen and chromates than most other polymeric 3.89 (8H, m, P–OCH2CH3), 4.80 5.04 (1H, m, PCH), 5.90
resins. Its physical and chemical stability, non corrosive, non (1H, s, NH), 6.25 6.55 (3H, m, Ar-H). 13C-NMR (100MHz,
—
3
toxic nature, selectivity in reactions, reusability and environ- DMSO-d6) δ: 14.8 (d, JP–C =5.8Hz, P–OCH2–CH3), 57.2 (t,
2
mental compatibility make this as a versatile catalyst for per- 1JPC =156.3Hz, PCH), 59.5 (d, JP–C =7.6Hz, P–OCH2CH3),
*To whom correspondence should be addressed. e-mail: csrsvu@gmail.com
© 2012 The Pharmaceutical Society of Japan

January 2012
105
112.1 (C-6), 111.9 (C-5), 116.4 (C-2), 122.2 (C-4), 125.6 (C-3), (3f): White solid, yield: 80%, mp 60 61 C. 1H-NMR
138.5 (C-1). 31P-NMR (161.7MHz, DMSO-d6) δ: 18.26. IR (400MHz, DMSO-d6) δ: 1.08(6H, t, 3JPH =8.2Hz, POCH2CH3),
—
°
−
1
3
—
(KBr) cm : 3200 (NH), 1240 (P=O), 760 (P–Caliphatic). Elec- 1.28 (6H, t, JPH =9.2Hz, POCH2CH3), 3.60 4.10 (8H, m,
trospray ionization (ESI)-MS (m/z): 447 (M+·). Anal. Calcd for P–OCH2CH3), 4.85 5.08 (1H, m, PCH), 5.95 (1H, s, NH),
—
C15H25Cl2NO6P2: C, 40.20; H, 5.62; N, 3.12. Found C, 40.16; 7.48 8.20 (4H, m, Ar-H). 13C-NMR (100MHz, DMSO-d6) δ:
—
3
1
H, 5.58; N, 3.08.
17.5 (d, JP–C =5.6Hz, P–OCH2–CH3), 56.7 (t, JPC =162.4Hz,
2
Tetraethyl(1-phenylethylamino)methylenebis-phosphonate PCH), 63.2 (d, JP–C =7.6Hz, P–OCH2CH3), 114.5 (C-4),
(3b): White solid, yield: 70%, mp 135 137 C. 1H-NMR 120.0 (C-6), 121.5 (C-5), 157.6 (C-3), 175.6 (C-1). 31P-NMR
—
°
−
1
(400MHz, DMSO-d6) δ: 1.15 (6H, t, 3JPH =9.1Hz, POCH2CH3), (161.7MHz, DMSO-d6) δ: 24.5; IR (KBr) cm : 3390 (NH),
1.22 (3H, d, JH–H =7.4Hz), 1.25 (6H, t, JPH =9.2Hz, 1244 (P=O), 746 (P–Caliphatic). ESI-MS m/z: 397 (M+·). Anal.
2
3
—
POCH2CH3), 1.78 (1H, q, J=7.6Hz, Ph–CH–CH3), 3.82 4.05 Calcd for C15H26FNO6P2: C, 45.34; H, 6.60; N, 3.53. Found C,
—
(8H, m, P–OCH2CH3), 4.80 5.05 (1H, m, PCH), 5.65 (1H, s, 45.29; H, 6.56; N, 3.50.
NH), 6.25 7.15 (5H, m, Ar-H). 13C-NMR (100MHz, DMSO-
Tetraethyl(benzo[d]thiazol-2-ylamino)methylenebisphos-
—
3
—
°
d6) δ: 13.7, 16.2 (d, JP–C =5.8Hz, P–OCH2–CH3), 45.1, 58.3 phonate (3g): White solid, yield: 77%, mp 125 127 C.
1
2
3
(t, JPC =157.8Hz, PCH), 61.2 (d, JP–C =7.6Hz, P–OCH2CH3), 1H-NMR (400MHz, DMSO-d6) δ: 1.09 (6H, t, JPH =8.8Hz,
3
—
114.4 (C-4), 114.7 (C-2), 115.2 (C-3), 115.9 (C-5), 116.2 (C-6), POCH2CH3), 1.20 (6H, t, JPH =9.2Hz, POCH2CH3), 3.75
136.8 (C-1). 31P-NMR (161.7MHz, DMSO-d6) δ: 18.50. IR 3.99 (8H, m, P–OCH2CH3), 4.70 4.95 (1H, m, PCH), 5.60
—
−
(KBr) cm : 3200 (NH), 1225 (P=O), 775 (P–Caliphatic). ESI- (1H, s, NH), 7.85 8.15 (4H, m, Ar-H). 13C-NMR (100MHz,
1
—
3
MS (m/z): 407 (M+·). Anal. Calcd for C17H28N4O6P2: C, 50.12; DMSO-d6) δ: 16.9 (d, JP–C =5.6Hz, P–OCH2–CH3), 54.4 (d,
2
H, 7.67; N, 3.44. Found C, 50.08; H, 7.63; N, 3.39.
1JPC =162.4Hz, PCH), 65.1 (d, JP–C =7.6Hz, P–OCH2CH3),
Tetraethyl(3,4-dimethylphenylamino)methylenebisphospho- 113.8 (C-4), 117.6 (C-7), 121.6 (C-6), 122.1 (C-5), 131.6 (C-8),
1
nate (3c): White solid, yield: 75%, mp 140 142 C. H-NMR 156.7 (C-3), 174.5 (C-1). 31P-NMR (161.7MHz, DMSO-d6) δ:
—
°
−
1
(400MHz, DMSO-d6) δ: 1.13 (6H, t, 3JPH =9.0Hz, POCH2CH3), 20.5. IR (KBr) cm : 3350 (NH), 1235(P=O), 755 (P–Caliphatic).
1.22 (3H, s), 1.24 (3H, s), 1.26 (6H, t, JPH =9.2Hz, ESI-MS m/z: 436 (M+·). Anal. Calcd for C16H26 N2O6P2S: C,
3
—
—
POCH2CH3), 3.70 3.99 (8H, m, P–OCH2CH3), 4.75 4.99 44.04; H, 6.01; N, 6.42. Found C, 44.01; H, 5.96; N, 6.38.
—
(1H, m, PCH), 5.55 (1H, s, NH), 6.78 7.30 (4H, m, Ar-H).
Tetraethyl(1,5-dimethyl-3-oxo-2-phenylpyrazolidin-4-yl-
3
13C-NMR (100MHz, DMSO-d6) δ: 16.5 (d, JP–C =5.8Hz, amino)methylenebisphosphonate (3h): White solid, yield:
1
1
—
°
P–OCH22–CH3), 23.5, 26.4, 56.0 (t, JPC =163.6Hz, PCH), 70%, mp 139 141 C. H-NMR (400MHz, DMSO-d6) δ: 1.05
3
3
63.0 (d, JP–C =7.6Hz, P–OCH2CH3), 109.0 (C-2), 117.0 (C-5), (6H, t, JPH =9.2Hz, POCH2CH3), 1.20 (6H, t, JPH =9.2Hz,
123.2 (C-6), 136.5 (C-3), 144.2 (C-4), 145.2 (C-1),. 31P-NMR POCH2CH3), 1.48 (3H, d, J=7.5Hz), 1.64 1.86 (1H, m),
—
−
1
—
—
(161.7MHz, DMSO-d6) δ: 20.52. IR (KBr) cm : 3250 (NH), 1.92 2.15 (1H, m), 2.52 (3H, s, N–CH3), 3.70 3.92 (8H,
1230 (P=O), 775 (P–Caliphatic). ESI-MS (m/z): 407 (M+·). Anal. m, P–OCH2CH3), 4.85 5.10 (1H, m, PCH), 5.29 (1H, s, NH),
—
Calcd for C17H31NO6P2: C, 50.12; H, 7.67; N, 3.44. Found C, 6.86 7.91 (5H, m, Ar-H). 13C-NMR (100MHz, DMSO-d6)
—
3
50.08; H, 7.62; N, 3.41.
δ: 11.6 (CH3), 14.3 (d, JP–C =5.6Hz, P–OCH2–CH3), 38.4
1
Tetraethyl(2-methyloxazol-5-yllamino)methylenebisphos- (N–CH3), 44.6 (C-2), 56.4 (t, JPC =156.7Hz, PCH), 58.7 (C-1),
2
—
°
′
′
phonate (3d): White solid, yield: 72%, mp 160 162 C. 60.3 (d, JP–C =7.6Hz, P–OCH2CH3), 114.5 (C-3 ), 114.9 (C-5 ),
1H-NMR (400MHz, DMSO-d6) δ: 1.10 (6H, t, JPH =9.1Hz, 116.8 (C-4 ), 119.8 (C-2 ), 120.2 (C-6 ), 122.1 (C-1 ), 152.3 (C-
3
′
′
′
′
−
1
POCH2CH3), 1.20 (6H, t, JPH =9.2Hz, P–OCH2CH3), 5). 31P-NMR (161.7MHz, DMSO-d6) δ: 20.8. IR (KBr) cm :
3
—
—
1.34 (3H, s), 3.72 3.95 (8H, m, P–OCH2CH3), 4.90 5.25 3430 (NH), 1220 (P=O), 755 (P–Caliphatic). ESI-MS (m/z): 491
(1H, m, PCH), 5.50 (1H, s, NH), 8.25(1H, s Ar-H). 13C- (M+·). Anal. Calcd for C20H35N3O7P2: C, 48.88; H, 7.18; N,
3
NMR (100MHz, DMSO-d6) δ: 17.05 (d, JP–C =5.6Hz, P– 8.55. Found C, 48.84; H, 7.14; N, 8.51.
OCH2-CH3), 28.7, 55.0 (t, JPC =163.4Hz, PCH), 62.2 (d,
1
Tetraethyl(3,5-dichloro-4-hydroxyphenylamino)methyl-
2
—
=
JP–C =7.4Hz, P–OCH2CH3), 135.5 (C-5), 147.5 (C-4), 163.0 enebisphosphonate (3i): White solid, yield: 80%, mp 114
(C-2). 31P-NMR (161.7MHz, DMSO-d6) δ: 19.52. IR (KBr) 116 C. H-NMR (400MHz, DMSO-d6) δ: 1.09 (6H, t, JPH
1
3
°
−
1
3
cm : 3255 (NH), 1235 (P=O), 770 (P–Caliphatic). ESI-MS (m/z): 8.8Hz, POCH2CH3), 1.20 (6H, t, JPH =9.2Hz, POCH2CH3),
384 (M+·). Anal. Calcd for C13H26N2O7P2: C, 40.63; H, 6.82; N, 3.75 3.99 (8H, m, P–OCH2CH3), 5.60 (1H, s, NH), 5.05
7.29. Found C, 40.59; H, 6.78; N, 7.24. 5.35 (1H, m, PCH), 7.89 (1H, s, Ar-H), 8.20 (1H, s, Ar-H).
—
—
Tetraethyl(4-nitrophenylamino)methylenebisphosphonate 13C-NMR (100MHz, DMSO-d6) δ: 15.6 (d, JP–C =5.8Hz, P–
3
(3e): Yellow solid, yield: 70%, mp 205 206 C. 1H- OCH2–CH3), 58.5 (t, JPC =157.6Hz, PCH), 62.7 (d, JP–C
=
1
2
—
°
3
NMR (400MHz, DMSO-d6) δ: 1.12 (6H, t, JPH =8.7Hz, 7.6Hz, P–OCH2CH3), 113.9 (C-2), 115.7 (C-5), 115.8 (C-6),
POCH2CH3), 1.22 (6H, t, JPH =9.2Hz, POCH2CH3), 3.85
3
118.5 (C-3), 123.1 (C-4), 140.3 (C-1). 31P-NMR (161.7MHz,
—
−
1
—
4.10 (8H, m, P–OCH2CH3), 4.98 (1H, s, NH), 5.10 5.55 (1H, DMSO-d6) δ: 22.5. IR (KBr) cm : 3369 (NH), 1240 (P=O),
m, PCH), 6.52 7.13 (4H, m, Ar-H). 13C-NMR (100MHz, 750 (P–Caliphatic). ESI-MS (m/z): 463 (M+·). Anal. Calcd
—
3
DMSO-d6) δ: 16.5 (d, JP–C =5.6Hz, P–OCH2–CH3), 47.8 (t, for C15H25Cl2NO7P2: C, 38.81; H, 5.43; N, 3.02. Found C,
1JPC =161.5Hz, PCH), 62.7 (d, JP–C =7.6Hz, P–OCH2CH3), 38.77; H, 5.38; N, 2.97.
2
112.8 (C-2), 113.1 (C-6), 126.6 (C-5), 127.4 (C-3), 134.2 (C-4),
Tetraethyl(pyridin-4-ylamino)methylenebisphosphonate (3j):
1
151.2 (C-1). 31P-NMR (161.7MHz, DMSO-d6) δ: 21.5. IR (KBr) White solid, yield: 90%, mp 142 144 C. H-NMR (400MHz,
—
°
−
1
3
cm : 3255 (NH), 1230 (P=O), 765 (P–Caliphatic). ESI-MS m/z: DMSO-d6) δ: 1.10 (6H, t, JPH =8.9Hz, POCH2CH3), 1.20
424 (M+·). Anal. Calcd for C15H26N2O8P2: C, 42.46; H, 6.18; N, (6H, t, JPH =9.2Hz, POCH2CH3), 3.80 4.10 (8H, m, P–
3
—
—
6.60. Found C, 42.43; H, 6.14; N, 6.56. OCH2CH3), 4.95 5.20 (1H, m, PCH), 5.00 (1H, s, NH),
Tetraethyl(4-fluorophenylamino)methylenebisphosphonate 6.70 (2H, d, J=8.8Hz, Ar-H), 8.40 (1H, d, J=7.6Hz, Ar-H).

106
Vol. 60, No. 1
3
13C-NMR (100MHz, DMSO-d6) δ: 15.8 (d, JP–C =5.8Hz,
1
P–OCH2–CH3), 54.9 (t, JPC =163.4Hz, PCH), 63.5 (d,
2JP–C =7.6Hz, P–OCH2CH3), 159.2 (C-2), 107.2 (C-5), 110.3
(C-3), 150.5(C-6), 156.7 (C-4). 31P-NMR (161.7MHz, DMSO-
−
1
d6) δ: 18.52. IR (KBr) cm : 3252 (NH), 1235 (P=O), 770
(P–Caliphatic). ESI-MS (m/z): 380 (M+·). Anal. Calcd for
C14H26N2O6P2: C, 44.21; H, 6.89; N, 7.37. Found C, 44.17; H,
6.86; N, 7.33.
Results and Discussion
Synthesis The conventional method of this 3 component
one pot reaction requires high temperatures and long reac-
tion times to afford the corresponding bisphosphonates.20)
A new method is presently developed for the preparation of
bisphosphonates from a mixture of amine, diethyl phosphite
and triethylorthoformate in the presence of amberlyst-15 as
catalyst goes to completion at room temperature under solvent
free conditions in 90min and afforded high yields. Further
after completion of the reaction the catalyst can be separated
—
simply by filtration and reused for another 4 5 times without
losing its activity.21)
To optimize the reaction conditions, the reaction of 4-
nitroaniline, diethyl phosphite and triethylorthoformate was
selected as a model. This reaction has been performed in dif-
ferent organic solvents such as toluene, methanol, dioxane,
tetrahydrofuran, acrylonitrile, chloroform, dichloromethane,
diethyl ether in the presence of amberlyst-15 at room tem-
perature and low yields (<40%) of the bisphosphonates were
Chart 1. The Synthetic Route for Amino Methylene Bisphosphonates
obtained in all these experiments. Use of higher amount of ganisms (NCIM), National Chemical Laboratory, Pune, India.
catalyst also did not lead to significant change in the reaction Antibacterial and Antifungal Assays Preliminary anti-
—
yields. The best result was obtained when the same reaction microbial activities of compounds 3a j were tested by agar
was done under solvent free conditions with small amount of disc-diffusion method. Sterile filter paper discs (6mm diam-
catalyst.
eter) moistened with the test compound solution in DMSO of
Based on the optimized reaction conditions, a group of specific concentration 100µM and 200µM/disc were carefully
bisphosphonates were synthesized by the same reaction using placed on the agar culture plates that had been previously in-
different alkyl/aromatic/heterocyclic amines. In all the cases oculated separately with the microorganisms. The plates were
°
the reaction proceeded at room temperature in 90min and incubated at 37 C and the diameter of the growth inhibition
gave good to excellent yields. In these experiments, the cata- zones were measured after 24h in case of bacteria and after
lyst was isolated by filtration and could be reloaded with fresh 48h in case of fungi.
reagents for further runs, thus, recyclization of the catalyst is
The results of preliminary antimicrobial testing of the
possible without significant loss of activity. The products were compounds are shown in Tables 1 and 2. The results revealed
obtained as solids and purified by column chromatography us- that, both Gram-positive bacteria and Gram-negative bacteria
—
ing silica gel as adsorbent and ethyl acetate–hexane (1:4) as show similar inhibitory activity. The compounds 3a d and
—
eluent. The chemical structures of 3a j were confirmed by 3h showed excellent activity against Gram-positive (inhibitory
1
elemental analysis, IR, H-, 13C-, 31P-NMR and mass spectral zone >25mm) and Gram-negative (inhibitory zone >25mm)
data. bacteria. All the test compounds showed moderate to high
Biological Activity. Antimicrobial Activity The com- inhibitory effect towards tested fungi.
—
—
Antioxidant Activity The compounds 3a j were tested
pounds 3a j were tested for in vitro antimicrobial activ-
ity against the Gram-positive bacteria Staphylococcus aureus for antioxidant property by nitric oxide,23,24) DPPH25) and
(NCIM No. 5021), Bacillus subtilis (NCIM No. 2063), the H2O2 methods.26) The observed data on the antioxidant activ-
Gram-negative bacteria Klebsiella pneumoniae (NCIM No. ity is shown in Fig. 1.
2957), Proteus vulgaris (NCIM No. 2027) and fungi Fusarium
Determination of Nitric Oxide (NO) Scavenging Activ-
solani (NCIM No. 1330), Curvularia lunata (NCIM No. 716) ity Sodium nitropruside (5µM) in phosphate buffer pH 7.4
and Aspergillus niger (NCIM No. 596). The preliminary was incubated with 100µM concentration of test compounds
screening was carried out by agar disc-diffusion method.22) dissolved in a suitable solvent (dioxane/methanol) and tubes
°
Chloramphenicol and Ketoconazole were used as control were incubated at 25 C for 120min. Control experiment was
drugs. The observed data on the antimicrobial activity of the conducted with equal amount of solvent in an identical man-
compounds and control drugs are given in Tables 1 and 2.
ner. At intervals, 0.5mL of incubation solution was taken
Cells Bacterial strains S. aureus, B. subtilis, K. pneumo- and diluted with 0.5mL of Griess reagent (1% sulfanilamide,
niae, P. vulgaris and fungi F. solani, C. lunata and A. niger 0.1% N-naphthylethylenediamine dihydrochloride and 2% o-
were obtained from National Collection of Industrial Microor- phosphoric acid dissolved in distilled water). The absorbance

January 2012
107
—
Table 1. Antibacterial Activity of 3a
j
Zone of inhibition (mm)
Concentration
(μM/disc)
Compound
Gram-positive bacteria
Staphylococcus aureus Bacillus subtilis
Gram-negative bacteria
Klebsiella pneumoniae
Proteus vulgaris
3a
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
29
32
30
31
28
29
30
32
26
25
17
19
19
18
25
27
17
18
14
16
35
41
28
30
31
33
31
33
26
29
23
21
20
21
14
15
28
30
16
19
18
19
38
44
30
32
25
27
26
28
30
31
24
26
21
22
25
26
25
26
16
17
19
21
37
42
32
34
26
29
25
27
26
28
24
22
18
19
13
14
30
31
13
14
17
19
42
45
3b
3c
3d
3e
3f
3g
3h
3i
3j
Chloramphenicol
—
Table 2. Antifungal Activity of 3a
j
Zone of inhibition (mm)
Compound
Concentration (μM/disc)
Fusarium solani
Curvularia lunata
Aspergillus niger
3a
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
100
200
30
33
31
33
27
30
28
30
23
26
16
18
19
21
29
31
15
17
17
18
38
42
26
29
29
30
29
31
27
29
22
21
18
20
19
22
30
32
17
18
20
22
41
44
28
30
27
31
28
31
27
30
26
28
17
20
18
21
27
30
19
20
21
23
36
39
3b
3c
3d
3e
3f
3g
3h
3i
3j
Ketoconazole

108
Vol. 60, No. 1
of the chromophore formed during diazotization of nitrite
with sulfanilamide and subsequent N-naphthylethylenediamine
dihydrochloride was read at λ 546nm. The experiment was
repeated in triplicate.
(Acont − Atest
)
NO scavenged (%) =
×100
Acont
Where Acont is the absorbance of the control reaction and Atest
is the absorbance in the presence of the sample.
—
j
In the case of amino methylene bisphosphonates 3a
derivatives 3i showed the highest NO scavenging with IC50
of 56.21µM when compared with other compounds. The
remaining compounds exhibited reducing power activity in
the following order: 3g (IC50 58.79µM), 3f (IC50 63.39µM),
3j (IC50 79.72µM), 3e (IC50 79.81µM), 3d (IC50 86.51µM), 3c
(IC50 86.65µM), 3b (IC50 89.43µM), 3h (IC50 89.65µM), 3a
(IC50 90.11µM) and when compared with ascorbic acid (IC50
96.90µM).
—
Fig. 1. Antioxidant Activity of 3a
j
Determination of 1,1-Diphenyl-2-picrylhydrazyl (DPPH)
Free Radical Scavenging Activity The nitrogen centered
stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) has
—
j
In the case of amino methylene bisphosphonates 3a
often been used to characterize antioxidants. It is reversibly derivatives 3i showed the highest H2O2 scavenging activity
reduced and the odd electron in the DPPH free radical gives with IC50 of 33.03µM when compared with other compounds.
a strong absorption maximum at λ 517nm, which is purple in The remaining compounds exhibited reducing power activity
color. This property makes it suitable for spectrophotometric in the following order: 3f (IC50 36.65µM), 3j (IC50 36.80µM),
studies. A radical scavenging antioxidant reacts with DPPH 3g (IC50 39.05µM), 3e (IC50 39.65µM), 3c (IC50 46.48µM), 3b
stable free radical and converts into 1,1-diphenyl-2-picryl- (IC50 46.78µM), 3a (IC50 47.65µM), 3h (IC50 47.83µM), 3d
hydrazine. The resulting decolorization is stoichiometric with (IC50 48.98µM), and when compared with ascorbic acid (IC50
respect to the number of electrons captured. The change in the 51.13µM).
—
absorbance produced in this reaction has been used to meas-
ure antioxidant properties.
The compounds 3a j were tested for antioxidant property
by Nitric Oxide, DPPH and H2O2 methods. The compounds
The solutions of test compounds (100µM) were added to 3e, 3f, 3g, 3i and 3j exhibited high antioxidant property in all
DPPH (100µM) in dioxane/ethanol. The tubes were kept at the 3 methods at 100µM concentration. In fact, the compound
an ambient temperature for 20min. and the absorbance was having amino methylene bisphosphonates units displayed high
measured at λ 517nm. The difference between the test and the antioxidant property.
control experiments was taken and expressed as the per cent
scavenging of the DPPH radical.
Majority of the compounds showed high activities for nitric
oxide, DPPH and H2O2 inhibition as shown in Fig. 1. The
potency of these enzymes inhibition was mainly influenced
—
j
In the case of amino methylene bisphosphonates 3a
derivatives 3i showed the highest DPPH radical scavenging by the fragments attached to the amino methylene bisphos-
activity with IC50 of 62.57µM when compared with other phonates.
compounds. The remaining compounds exhibited reducing
Compounds 3e, 3f, 3g, 3i and 3j showed appreciable anti-
power activity in the following order: 3g (IC50 69.43µM), 3j oxidant activity. Fluorine, Chlorine and NO2 substituents
(IC50 72.95µM), 3f (IC50 74.57µM), 3e (IC50 81.21µM), 3h (IC50 which attract the electron density appear to enhance antioxi-
87.25µM), 3c (IC50 87.70µM), 3b (IC50 87.91µM), 3a (IC50 dant activity. Since fluorine shows highly negative inductive
89.81µM), 3d (IC50 90.01µM) and when compared with ascor- effect and NO2 is also highly electron withdrawing, the elec-
bic acid (IC50 95.37µM).
tron density around phosphonate moiety decreases and conse-
Determination of Hydrogen Peroxide (H2O2) Scaveng- quently increases affinity towards oxygen derived free radicals
ing Activity It was determined using a modified method of and mobilizes ROS to be scavenged from the living system.
Gow-Chin Yen and Hui-Yin Chen. 4mM solution of H2O2 was The presence of fluorine as a substituent also plays a determi-
prepared in phosphate-buffered saline (PBS, pH 7.4). H2O2 nant role in the inhibition of enzymes which has importance
concentration was determined spectrophotometrically from in drug discovery such as mimic and destabilization effects.
−
1
−
1
—
absorbance at 230nm using molar absorptivity 81M cm . Due to the presence of electron withdrawing groups in 3a j,
One hundred micromolar compounds solution in 4mL dis- all the title compounds exhibit significant antioxidant activity.
tilled water were added to 0.6mL H2O2-PBS solution. Absorb-
ance of H2O2 at 230nm was determined 10min later against a
blank solution containing parent compound with PBS without
Conclusion
An effective and simple method for the synthesis of new
—
—
d
H2O2. Ascorbic acid was added in place of compound in 4mL amino bisphosphonates (3a j) is reported. Compounds 3a
distilled water and the solution wad added to 0.6mL H2O2 and 3h exhibited significant antimicrobial and 3e, 3f, 3g, 3i
solution in PBS. Absorbance was determined 10min later and 3j exhibited significant antioxidant properties in nitric
against a blank solution similar to that above.
oxide, DPPH and H2O2 method inhibitory potency.

January 2012
109
Staite N. D., Aspar D. G., Elliott G. A., Essani N. A., Rohloff N. A.,
Acknowledgements The authors express their grateful
thanks to Prof. C. D. Reddy, Department of Chemistry, Sri
Venkateswara University, Tirupati, for his helpful discussions
and for BRNS, BARC, Mumbai for providing financial as-
sistance (2010/37C/26/BRNS/1424). The authors also express
their thanks to Mr. P. Ramamohan, Department of Biochem-
istry, S.V. University, Tirupati for conducting biologtical activ-
ity.
—
J. Pharmacol. Exp. Ther., 266, 1691 1698 (1993).
—
12) Flora L., Smith R. J., Arthritis Rheum., 22, 340 346 (1979).
13) Van Offel J. F., Schuerwegh A. J., Bridts C. H., Bracke P. G.,
—
Stevens W. J., De Clerck L. S., Clin. Exp. Rheumatol., 19, 13 20
(2001).
14) Singh U. S., Shankar R., Kumar A., Trivedi R., Chattopadhyay N.,
Shakya N., Palne S., Gupta S., Hajela K., Bioorg. Med. Chem., 16,
—
8482 8491 (2008).
15) Dombrecht E. J., Cos P., Vanden Berghe D., Van Offel J. F., Schuer-
wegh A. J., Bridts C. H., Stevens W. J., De Clerck L. S., Biochem.
References
—
Biophys. Res. Commun., 314, 675 680 (2004).
—
1) German J. B., Adv. Exp. Med. Biol., 459, 23 50 (1999).
16) Cantatore F. P., Acquista C. A., Pipitone V., J. Rheumatol., 26,
—
2) Jacob R. A., Subcell. Biochem., 25, 1 16 (1996).
—
2318 2323 (1999).
—
3) Knight J. A., Ann. Clin. Lab. Sci., 28, 331 346 (1998).
17) Balakrishna A., Narayana Reddy M. V., Rao P. V., Kumar M. A.,
4) Kasser J., Nazarov A. A., Hartinger C. G., Wdziekonski B., Dani C.,
Kumar B. S., Nayak S. K., Reddy C. S., Eur. J. Med. Chem., 46,
Kuznetsov M. L., Arion V. B., Keppler B. K., Bioorg. Med. Chem.,
—
1798 1802 (2011).
—
17, 3388 3393 (2009).
—
18) Ko S., Yao C. F., Tetrahedron Lett., 47, 8827 8829 (2006).
—
5) Russell G. G., Rogers M. J., Bone, 25, 97 106 (1999).
—
19) Liu Y. H., Liu Q. S., Zhang H., J. Mol. Catal., 296, 42 46 (2008).
6) Veera Narayana Reddy M., Kishore Kumar Reddy K. R., Bhupen-
dra Reddy C., Siva Kumar B., Suresh Reddy C., Devendranath
20) Takeuchi M., Sakamoto S., Yoshida M., Abe T., Isomura Y., Chem.
—
Pharm. Bull., 41, 688 693 (1993).
—
Reddy C., J. Chem. Res., 4, 258 260 (2009).
21) Tajbakhsh Md., Heydari A., Khalilzadeh Md. A., Synlett, 15,
7) Hudock M. P., Sanz-Rodríguez C. E. Y., Song Y., Chan J. M.,
Zhang Y., Odeh S., Kosztowski T., Leon-Rossell A., Concepción J.
L., Yardley V., Croft S. L., Urbina J. A., Oldfield E., J. Med. Chem.,
—
2347 2350 (2007).
22) Mangte D. V., Deshmukh S. P., Bhokare D. D., Deshponde Arti R.,
—
Indian J. Pharm., 69, 295 298 (2007).
—
49, 215 223 (2006).
23) Shirwaiker A., Rajendran K., Dinesh Kumar C., Indian J. Exp.
8) Kiran Y. B., Devendranath Reddy C., Gunasekar D., Suresh Reddy
—
Biol., 42, 803 807 (2004).
—
C., Leon A., Barbosa L. C. A., Eur. J. Med. Chem., 43, 885 892
—
24) Babu B. H., Shailesh B. S., Paddikala J., Fitotherapia, 72, 272 277
(2008).
(2001).
—
9) Green J. R., Cancer, 97 (Suppl.), 840 847 (2003).
25) Kato K., Terao S., Shimamoto N., Hirata M., J. Med. Chem., 31,
10) Clézardin P., Ebetino F. H., Fournier P. G. J., Cancer Res., 65,
—
793 798 (1988).
—
4971 4974 (2005).
—
26) Gow-Chin Y., Hui-Yin C., J. Agric. Food Chem., 43, 27 32 (1995).
11) Dunn C. J., Galinet L. A., Wu H., Nugent R. A., Schlachter S. T.,