Synthesis and bio-activity evaluation of tetraphenyl(phenylamino) methylene bisphosphonates as antioxidant agents and as potent inhibitors of osteoclasts in vitro

Article abstract of DOI:10.1016/j.ejmech.2011.02.038

A new series of tetraphenyl bisphosphonates have been elegantly synthesized by one-pot method and were characterized by elemental analysis, FTIR, ¹H, ¹³C, ³¹P NMR, mass spectra and evaluated for their in vitro antibone res

Full text of DOI:10.1016/j.ejmech.2011.02.038

European Journal of Medicinal Chemistry 46 (2011) 1798e1802
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and bio-activity evaluation of tetraphenyl(phenylamino) methylene
bisphosphonates as antioxidant agents and as potent inhibitors of osteoclasts
in vitro
A. Balakrishna a, M. Veera Narayana Reddy a, P. Visweswara Rao a, M. Anil Kumar a, B. Siva Kumar a,
,
a,
*
S.K. Nayak b , C. Suresh Reddy
*
a Department of Organic Chemistry and Bio-technology, Sri Venkateswara University, Tirupati-517 502, India
b Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai-400 085, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of tetraphenyl bisphosphonates have been elegantly synthesized by one-pot method and
were characterized by elemental analysis, FTIR, 1H, 13C, 31P NMR, mass spectra and evaluated for their in
vitro antibone resorptive activity by inhibiting growth of osteoclasts. Two bisphosphonates 3g and 3f
showed marked inhibition ratio (8 mM and 10 mM) and emerged as lead compounds. All compounds were
tested for their antioxidant (DPPH scavenging, reducing power and inhibition of lipid peroxidation). They
exhibited potent in vitro antioxidant activity dose-dependently.
Received 24 June 2010
Received in revised form
11 February 2011
Accepted 16 February 2011
Available online 23 February 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
TPM-BPs
Anti-bone resorptive activity
Anti-oxidant activity
Spectral analysis
1. Introduction
osteoclasts-like cells in long-term human bone marrow cultures [2].
In organ cultures also some bisphosphonates can inhibit the gener-
Osteoporosis is characterized by extensive bone resorption
leading to decreased bone mass and increased fragility and suscep-
tibility to fractures. At the cellular level, osteoporosis is disturbance
in the bone micro architecture, which typically develops in women
after menopause, due to decrease in serum estrogen levels [1]. The
clinical manifestations of osteoporosis occur most often as fractures
in the spine, femoral neck and at the wrist. Its prevention and
treatment is therefore of a paramount importance. The pronounced
selective activity of bisphosphonates for bone tissues rather than
others is the basis for their value in clinical practice. Their prefer-
ential uptake and adsorption by mineral surfaces in bone bring them
into close contact with osteoclasts. Many studies have shown that
bisphosphonates affect osteoclasts mediated bone resorption in
a variety of ways, including effects on osteoclasts recruitment,
differentiation, resorptive activity, and may also induce apoptosis.
Bisphosphonates could also inhibit bone resorption by preventing
osteoclasts formation, in addition to affecting mature osteoclasts in
vitro. Bisphosphonates inhibit dose-dependently the formation of
ation of mature osteoclasts, possibly by preventing the fusion of
osteoclasts precursors [3,4].
Nitrogen-containing bisphosphonates are the mainstay of anti-
resorptive therapy for osteoporosis [5,6]. They inhibit bone
resorption by reducing osteoclastic activity and induce osteoclasts
apoptosis [7]. Over the past two decades, several bisphosphonate
derivatives have been developed and used in the treatment of bone
skeletal disorders such as Pagets disease, myeloma and bone
metastases and reduce bone fracture risks associated with post-
menopausal osteoporosis in women [8,9]. Nitrogen-containing
bisphosphonates are also reported to inhibit farnesyl diphosphate
synthase, a key enzyme in the mevalonate pathway, and decrease
prenylation of essential GTP-binding proteins.
The latest third generation bisphoshonates having nitrogen
moiety as one of the side chains are usually preferred over non-
nitrogen side chain preparations because of their high potency [10].
In human rheumatoid arthritis (RA), osteoclasts are found at
sites of tissues invasion and emerge from synovial inflammatory
tissue, which provides signals allowing their differentiation from
monocytic precursors [11]. Bone loss in experimental models of
arthritis resembling human RA depends on osteoclasts formation.
Inhibition of osteoclastogenesis is a powerful approach to prevent
* Corresponding authors.
E-mail address: csrsvu@gmail.com (C.S. Reddy).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.038

A. Balakrishna et al. / European Journal of Medicinal Chemistry 46 (2011) 1798e1802
1799
arthritic bone destruction. In addition reactive oxygen species
(ROS) have been proved to play a major role in enhancing inflam-
matory process in rheumatoid arthritis (RA) and also contribute to
the destruction of cartilage and bone [12]. In the inflamed bone
joints, superoxide, hydrogen peroxide, hydroxyl and hypochlorous
acid radicals may be produced by macrophages, neutrophils and
chondrocytes and cause hyper-fusion cycle in inflamed joints
leading to increased generation of ROS [13,14]. Consequently,
antioxidants may have a therapeutic role in RA by suppressing the
inflammation. In this study, we accomplished synthesis of tetra-
phenyl(phenylamino)methylene bisphosphonates (TPM-BPs) and
investigated their influence on inhibition of osteoclasts and their
antioxidant profile in vitro.
pot synthesis of TPM-BPs is the nucleophillic addition of an amine
to triethyl orthoformate followed by addition of a phosphite to the
resulting imine [15].
IR absorptions for 3aej appeared for P]O in the region
1232e1224 cmꢀ1 confirms the presence of P]O group. The stretching
frequencies of NeH and PeCaliphatic groups are observed respectively at
3436e3312 cmꢀ1 and 764e752 cmꢀ1 [16]. The aromatic protons
showed multiplets in the region of
multiplet at 5.52e6.90 due to its coupling with phosphorus [16]. The
eNH signal was observed at 5.10e6.31.The carbon chemical shifts for
d 6.10e8.36. The PeCeH exhibited
d
d
PeCeH, PeOC6H5 in the title compounds were observed in their
expected regions. Their 31P NMR chemical shifts were observed at
d
24.26e28.04 for 3aej [17]. Chemical ionization mass spectra of 3a,
3b, and 3e gave molecular ions and diagnostic daughter ion peaks at
their respective expected m/z values.
2. Chemistry
3. Pharmacology
Tetraphenyl(phenylamino) methylene bisphosphonates (TPM-
BPs) 3aej are synthesized in moderate yields (Scheme 1) by one-
pot reaction by reacting an amine with triethyl orthoformate and
diphenyl phosphite. In this particular reaction, triethyl ortho-
formate performs dual role as a reagent and solvent. The reaction
conditions in the present protocol are mild and do not require any
catalyst to facilitate the reaction. Another important feature of this
reaction is the survival of a variety of functional groups such as,
chloro, fluoro, methoxy and nitro under these reaction conditions.
The reactions were clean and completed within 3 h. All the reac-
tions were monitored by thin layer chromatography using ethyl-
acetateehexane (4:6) as eluent. Evaporation of the solvent from the
reaction mixture under reduced pressure afforded pure products in
55e72% yields which are obviously dependent on the position and
nature of the substituents on the amine 1. The key step in the one-
The antioxidant activity was evaluated by Diphenyl picryl
hydrazyl method (DPPH), Reducing power and Lipid peroxidation
(LPO) methods and their inhibitory concentration (IC50) varied
according to substitution on the phenyl ring and Vitamin C was
measured as standard for antioxidant activity. To the best of our
knowledge a systematic antioxidant profile of TPM-BPs has not
been studied. Anti-bone resorption activity, in terms of inhibitory
ratios of osteoclasts were measured by MTT assay.
4. Results and discussion
According to our predicted results, the TMP-BPs 3aej have the
ability to scavenge the DPPH radical by donating one electron. In
the lipid peroxidation process iron plays an important key role in
complex formation via Fenton’s reagent. Since BPs are known for
their affinity for divalent cations, the high reactive hydroxyl radicals
formed in the inflamed joints are responsible to cause lipid per-
oxidation and tissue damage. Since phosphorus atom has affinity
towards oxygen, it can easily bind and scavenge ROS and has ability
to scavenge effectively. Our synthesized compounds 3aej, con-
taining phosphorus, oxygen, nitrogen are expected to be more
active due to the presence of hetero atoms containing non-bonded
electron pairs that serve as binding sites in the bio-matrix. 3e, 3g
and 3h displayed appreciable antioxidant activity. 3g showed the
highest activity because both fluorine and eNO2 substituents
which affect the electron and hydrogen donating capacities appears
to be useful in inducing antioxidant activity. Since fluorine shows
highly negative inductive effect and eNO2 is highly electron
withdrawing moiety, thereby electron density around P]O moiety
decreases and increases affinity towards oxygen derived free radi-
cals and mobilizes ROS to be scavenged out of living system.
Besides, the presence of chlorine on aryl ring in 3c and 3f can also
strongly polarize that parent molecule and seems to have profound
effect on biological properties of title compounds. This points to the
fact electron withdrawing substituent in 3aej appears to prevent to
some extent oxidative metabolic pathways in the living cells.
O
P
O
P
O
P
CH(OEt)3
130°C, 3h
OC6H5
OC6H5
C6H5O
C6H5O
OC6H5
OC6H5
R
NH2 + 2 H
N
(1a-j)
(2)
R
H
(3a-j)
R1 = OC6H5
Entry
R
Entry
R
F
3a
3f
Cl
F
MeO
3b
3g
O2N
Cl
F
3c
S
H3C
3h
N
4.1. DPPH radical scavenging activity
S
3d
O2N
The scavenging activity of TMP-BPs against DPPH radical was
performed in accordance with Choi et al. [18]. 85 mM of DPPH was
3i
N
added to a medium containing different TMP-BPs concentrations.
The medium was incubated for 30 min at room temperature. The
decrease in absorbance was measured at 518 nm. Ascorbic acid was
used as standard reference to record maximal decrease in DPPH
radical absorbance. The values are expressed in percentage of
inhibition of DPPH radical absorbance with those of the standard
3e
S
N
3j
O2N
Scheme 1. Synthesis of tetraphenyl(phenylamino) methylene bisphosphonates 3aej.

1800
A. Balakrishna et al. / European Journal of Medicinal Chemistry 46 (2011) 1798e1802
control values without the title compounds (Fig. 1) (ascorbic acid
maximal inhibition was considered 100% of inhibition).
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ðAcont ꢀ Atest
Þ
DPPH Scavenged ð%Þ ¼
ꢁ 100
Acont
In the case of TMP-BPs 3aej, fluoro-nitro substituted compound 3g
showed the highest DPPH radical scavenging activity with IC50 at
30.1
compounds exhibited DPPH radical scavenging activity in the
following order: 3f (IC50 32.9 g/mL), 3e (IC50 39.6 g/mL), 3b (IC50
40.7 g/mL), 3c (IC50 41.5 g/mL), 3j (IC50 47.9 g/mL), 3d (IC50 48.2 g/
mL), 3i (IC50 48.6 g/mL), 3h (IC50 58.4 g/mL), 3a (IC50 80.6 g/mL)
mg/mL when compared with other compounds. The remaining
m
m
m
m
m
m
m
m
m
and when compared with ascorbic acid (IC50 32.2 mg/mL).
3a 3b 3c 3d 3e
3f
3g 3h
3i 3j Vit
C
4.2. Reducing power assay
Compounds
The reducing power of synthesized compounds 3aej was
Fig. 2. Reducing power activity of compounds 3aej.
determined according to the method of Oyaizu [19]. The
compounds having 50e100 mM were mixed with 2.5 mL of phos-
According to the results obtained, 3h (IC50 75.2
mg/mL) (Fig. 3)
phate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricya-
nide and incubated at 50 ꢂC for 20 min. To this mixture 2.5 mL of
10% trichloroacetic acid (TCA) was added and the mixture was
centrifuged at 3000 rpm for 20 min. The upper layer (2.5 mL) was
mixed with 2.5 mL of deionised water and 0.5 mL of 0.1% ferric
chloride and the UV absorbance was measured at 700 nm using
a spectrophotometer. Increases of absorbance of the reaction
mixture indicate higher reducing power. Mean values from three
independent samples were calculated for each compound and
standard deviations were less than 5%.
significantly inhibited the ferric ion plus ascorbic acid in rat
liver cells. The remaining compounds exhibited hydroxyl radical
scavenging activity in the following order: 3g (IC50 78.5
(IC50 80.6 g/mL), 3j (IC50 84.9 g/mL), 3e (IC50 85.7 g/mL), 3d (IC50
g/mL), 3c (IC50 87.5 g/mL), 3b (IC50 89.8 g/mL), 3a (IC50
g/mL), 3i (IC50 97.9 g/mL) and when compared with ascorbic
g/mL).
mg/mL), 3f
m
m
m
86.2
96.0
m
m
m
m
m
acid (IC50 81.4
m
4.4. Bone anti-resorptive activity e inhibition of osteoclasts by MTT
assay
In the case of TMP-BPs (Fig. 2) 3aej, derivatives 3e showed the
highest reducing power with IC50 of 2.09
with other compounds. The remaining compounds exhibited
reducing power activity in the following order: 3f (IC50 2.14 g/mL),
3g (IC50 2.27 g/mL), 3j (IC50 2.44 g/mL), 3c (IC50 2.71 g/mL), 3b
g/mL), 3d (IC50 2.86 g/mL), 3h (IC50 3.01 g/mL), 3a
g/mL), 3i (IC50 3.54 g/mL) and when compared with
g/mL).
mg/mL when compared
Reduced osteoblasts survival along with increased osteoclasts
functions are the two key cellular mechanisms observed in osteo-
porosis. Osteoclasts are responsible for dissolving both the mineral
and organic bone matrix [21] and represent terminally differenti-
m
m
m
m
m
(IC50 2.73
(IC50 3.12
m
m
m
m
ated cells expressing
a unique polarized morphology with
specialized membrane areas and several membrane and cyto-
plasmic markers, such as tartrate resistant acid phosphatase (TRAP)
[22]. The effects of TMP-BPs 3aej and alendronate (one of the most
potent bone resorption inhibitor) as reference compound on bone
resorption were examined against osteoclasts on the basis of
inhibitory ratio of osteoclasts. The results are presented in Table 1.
Their inhibitory activity was related to the nature of the substitu-
ents such as fluoro, chloro, nitro and methoxy groups present in the
ascorbic acid (IC50 2.51
m
4.3. Lipid peroxidation assay
Lipid peroxidation was induced by Fe2þ ascorbate complex
system in rat liver cells and was estimated as thiobarbituric acid
reacting substances (TBARS) by the method of Ohkawa et al. [20].
Experiments in vitro lipid peroxidation were carried out to clarify
the mode of the protective effect of the tetraphenyl bisphospho-
nates against oxidative stress-induced cell damage. The inhibition
of lipid peroxidation has been used as a model to elucidate anti-
oxidant activity.
aryl ring. Osteoclasts treated with 10, 50, 100 and 250 mM of title
compounds 3aej were harvested after 24, 48 and 72 h and were
100
80
60
40
20
0
80
70
60
50
40
30
20
10
0
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j Vit C
3a 3b 3c 3d 3e
3f 3g 3h
3i
3j V it C
Compounds
Compounds
Fig. 1. DPPH radical scavenging activity of compounds 3aej.
Fig. 3. Lipid peroxidation of compounds 3aej.

A. Balakrishna et al. / European Journal of Medicinal Chemistry 46 (2011) 1798e1802
1801
Table 1
structures were characterized by physical and analytical data. In the
MTT assay, tetraphenyl(4-fluoro-3-nitro phenylamino) methylene
diphosphonate is highly effective in inhibiting osteoclasts cells. Its
activity is much higher than that of standard bone anti-resorption
drug. These results encourage further in vivo studies and explore
their possible therapeutic applications. This is the first report on the
anti-bone resorptive/anti-oxidant activity of TMP-BPs.
Osteoclast inhibitory activity of 3aej by MTT assay.
Compounds
Osteoclast inhibitory ratio mean ꢃ SDa
24 h
48
72 h
3a
3b
3c
3d
3e
3f
3g
3h
3i
201.6 ꢃ 1.97
138.7 ꢃ 1.85
191.4 ꢃ 0.73
126.1 ꢃ 1.44
49.6 ꢃ 1.42
32.7 ꢃ 0.76
31.2 ꢃ 1.22
88.4 ꢃ 0.49
106.6 ꢃ 1.20
125.9 ꢃ 1.62
52.7 ꢃ 1.55
168.2 ꢃ 1.35
102.6 ꢃ 1.06
140.3 ꢃ 0.98
89.1 ꢃ 0.76
28.8 ꢃ 1.08
21.6 ꢃ 0.66
19.1 ꢃ 0.95
64.2 ꢃ 0.76
87.7 ꢃ 0.51
95.9 ꢃ 1.06
29.6 ꢃ 1.32
104.4 ꢃ 1.05
67.7 ꢃ 0.68
71.8 ꢃ 0.59
56.4 ꢃ 0.50
14.6 ꢃ 0.54
10.4 ꢃ 0.97
8.1 ꢃ 0.58
6. Experimental protocols
48.8 ꢃ 0.74
51.6 ꢃ 0.80
54.5 ꢃ 0.63
20.4 ꢃ 1.29
6.1. Chemistry
3j
Alenb
Melting points were recorded on Buchi R-535 apparatus and are
uncorrected. IR spectra were recorded on a PerkineElmer FTIR 240-
c spectrophotometer using KBr optics. 1H, 13C and 31P NMR spectra
were recorded on AMX 400 MHz NMR spectrometer operating at
400 MHz for 1H NMR, 100 MHz for 13C and 161.89 MHz for 31P NMR.
NMR data recorded in DMSO were referenced to TMS (1H and 13C)
a
The inhibitory ratio (%) of TPM-BPs
24, 48 and 72 h.
m of 3a-j were analyzed by MTT assay after
b
Alendronate (standard reference).
subjected to MTT assay. Inhibitory ratio for 3e was 49
28 M at 48 h, and 14 M at 72 h of treatment. Inhibitory ratio for 3f
was 32 M at 24 h, 21 M at 48 h and 10 M at 72 h of treatment.
Inhibitory ratio for 3g was 31 M at 24 h, 19 M at 48 h and 8 M at
72 h of treatment. Results showed that cell viability was affected
upon treatment with compounds 3aej at 100 and 250 M, espe-
mM at 24 h,
and 85% H3PO4 (
31P). Mass spectra were recorded on a Finnigan
m
m
MAT 1020/Micro-Mass Q-T of micro AMPS MAX 10/6A, Hz 60/50
system fitted with a built-in inlet system. Elemental analyses were
performed using Perkin Elmer 2400 instrument at the Central Drug
Research Institute (CDRI), Lucknow, India.
m
m
m
m
m
m
m
Synthesis of tetraphenyl(phenylamino) methylene diphosphonate
(3a): A mixture of aniline 1a (0.45 g, 0.005 mol), triethyl ortho-
formate (30 mL) and 2 mol of diphenyl phosphite (1.91 g, 0.01 mol)
was stirred at 130 ꢂC for 2.5 h with continuous removal of ethanol
formed. The reaction was monitored by TLC on silica gel using
petroleum ether and ethyl acetate (1:2 v/v). After cooling, the
volatiles were removed in vacuo. The residue was chromatographed
on silica gel using CHCl3-MeOH (9:1). The other compounds 3bej
were prepared employing the above procedure.
cially after 72 h. Although, it is difficult to explain the observed
variations in the activity with respect to substituents in growth
inhibition. Compounds 3e, 3f and 3g showed remarkable decrease
osteoclastic bone resorption than other compounds and even than
the standard alendronate. These results suggest that inhibition of
osteoclasts is greatly influenced by nitro and halo substituted,
secondary amine moiety at the a-carbon of TPM-BPs. This may be
attributed to their interference in the osteoclasts unique polarized
morphology and decreases their metabolic rate.
During the process of bone resorption, the sub-cellular space
beneath the osteoclast is acidified by the action of vacuolar-type
proton pumps in the ruffled border of the osteoclast membrane [23].
The acidic PH normally favors the dissolution of the bone mineral
(hydroxyapatite) and provides optimal conditions for the action of
the proteinases secreted by osteoclasts. When the osteoclasts stops
resorption and moves away from the resorption lacuna, phagocytes
clean up the remains and make room for osteoblasts to begin bone
formation in the newly formed resorption cavity [24]. Therefore,
there is a need to interrupt the function of osteoclasts. The results
demonstrated that the secondary nitrogen atom bearing a suffi-
ciently bulky substituent-bearing halogens and nitro group are
effective in inhibiting osteoclasts. Among the potent compounds 3e,
3g and 3f are selected for further studies and are being investigated
on survival of osteoblasts and promotion of bone formation.
6.1.1. Tetraphenyl(phenylamino) methylene diphosphonate (3a)
Yield 66%, mp 145e147 ꢂC; IR (KBr): 3436,1225, 758 cmꢀ1 1H
;
NMR (DMSO-d6; 400 MHz): d 6.81e8.20 (m, 25 H, Ar-H), 6.40e6.75
(m,1H, CHP), 6.15 (s,1H, NH). 13C NMR (DMSO-d6): 52.4,114.8,116.7,
127.6, 139.8 (C-1eC-7); 126.6, 127.03, 129.7, 135.2 (C-10eC-60 and C-
100eC-600); 126.7, 127.7, 129.8, 135.37 (C-1a0eC-6a0 and C-1a00eC-
6a00). 31P NMR (DMSO-d6):
d
27.52. CIMS: (m/z) 572[MH]þ.
Elemental analysis: Calcd. C31H27NO6P2: C, 65.15; H, 4.76; N, 2.45.
Found C, 65.04; H, 4.71; N, 2.40
6.1.2. Tetraphenyl(4-methoxyphenyl amino) methylene
diphosphonate (3b)
Yield 62%, mp 160e162 ꢂC; IR (KBr): 3318, 1228, 757 cmꢀ1
;
1H
NMR (DMSO-d6; 400 MHz):
d 7.10e8.22 (m, 24 H, Ar-H), 6.45e6.85
(m, 1H, CHP), 6.14 (s, 1H, NH). 13C NMR(DMSO-d6): 53.2, 115.2, 117.8,
129.4, 132.9, 140.2, 142.3 (C-1eC-7); 126.6, 127.7, 129.7, 135.2 (C-
10eC-60 and C-100eC-600); 126.7, 127.3, 129.8, 135.3 (C-1a0eC-6a0 and
4.5. Structure-activity relationship and mechanism of action
C-1a00eC-6a00). 31P NMR (DMSO-d6):
d
27.04. CI (m/z): 602[MH]þ.
The PeCeP moiety of bisphosphonate is responsible for the
strong affinity and for binding to hydroxyapatite and the methylene
carbon allows for a number of structural variations with the help of
substituents R (2ꢂ-amine) and R1(eOC6H5). The ability of the
bisphosphonates to bind to hydroxyapatite crystals and to prevent
both crystal growth and dissolution was enhanced when the R side
chain is a secondary amine with aromatic ring having substituent
with electronwithdrawing groups such as halogens and nitro.
Elemental analysis: Calcd. C32H29NO7P2: C, 63.89; H, 4.86; N, 2.33.
Found C, 63.77; H, 4.81; N, 2.27.
6.1.3. Tetraphenyl(4-chlorophenylamino) methylene diphosphonate
(3c)
Yield 61%, mp 154e156 ꢂC; IR (KBr): 3316, 1224, 752 cmꢀ1
;
1H
NMR (DMSO-d6; 400 MHz):
d 6.46e8.21 (m, 24 H, Ar-H), 6.10e6.32
(m, 1H, CHP), 5.85 (s, 1H, NH). 13C NMR (DMSO-d6): 53.4, 116.2,
118.1, 128.4, 131.1, 139.2, 141.3 (C-1eC-7); 126.6, 127.7, 129.3, 136.3
(C-10eC-60 and C-100eC-600); 126.8, 127.8, 129.7, 136.4 (C-1a0eC-
5. Conclusion
6a0and C-1a00eC-6a00). 31P NMR (DMSO-d6):
d 27.24. Elemental
An effective and simple method for synthesis of novel tetra-
phenyl(phenylamino) bisphosphonates 3ae3j is reported. Their
analysis: Calcd. C31H26ClNO6P2: C, 61.45; H, 4.32; N, 2.31. Found C,
61.30; H, 4.26; N, 2.26.

1802
A. Balakrishna et al. / European Journal of Medicinal Chemistry 46 (2011) 1798e1802
6.1.4. Tetraphenyl(4-fluoro phenylamino) methylene diphosphonate
(3d)
6.2. Isolation and culture of osteoclasts
Yield 64%, mp 162e164 ꢂC; IR (KBr): 3342, 1226, 764 cmꢀ1
;
1H
Osteoclasts from 1- or 2-day-old SpragueeDawley rat pup long
bones were scraped mechanically into DMEM (Dulbecco’s modified
Eagle’s medium) buffered with 20 mM Hepes and containing 0.84 g
NMR (DMSO-d6; 400 MHz): d 6.24e8.24 (m, 24 H, Ar-H), 5.82e5.94
(m, 1H, CHP), 5.72 (s, 1H, NH). 13C NMR (DMSO-d6): 53.3, 117.2,
119.2, 129.2, 130.2, 138.2, 141.1 (C-1eC-7); 125.7, 126.4, 128.3, 136.2
(C-10eC-60 and C-100eC-600); 125.9, 126.8, 128.7, 136.4 (C-1a0eC-6a0
of sodium bicarbonate/liter, 2 mM
L-glutamine, 100 IU of penicillin/
ml, 100 g of streptomycin/ml and 7e10% heat-inactivated fetal calf
m
and C-1a00eC-6a00). 31P NMR (DMSO-d6):
d
25.24. Elemental analysis
serum (FCS). The cells were allowed to attach to sonicated bovine
cortical bone slices for 30 min. After the attachment period, non-
attached cells were rinsed away, and the bone slices with the
remaining cells were transferred into 24-well plates containing
fresh medium with appropriate substances to be tested. Cells were
cultured at 37 ꢂC (5% CO2, 95% air) for up to 48 h. Medium was
changed every 3 days. Cell viability was measured using an MTT
Cell Proliferation Assay [25]. Experiments were carried out in
triplicates and absorbance readings were taken at 492 nm.
Calcd. C31H26FNO6P2: C, 63.16; H, 4.45; N, 2.38. Found C, 63.04; H,
4.40; N, 2.32.
6.1.5. Tetraphenyl(3-nitro phenylamino) methylene diphosphonate
(3e)
Yield 72%, mp 152e154 ꢂC; IR (KBr): 3320, 1230, 755 cmꢀ1
;
1H
NMR (DMSO-d6; 400 MHz):
d 7.21e8.22 (m, 24 H, Ar-H), 6.88e6.90
(m,1H, CHP), 6.31 (s,1H, NH). 13C NMR (DMSO-d6): 52.1,112.7,113.8,
118.0, 132.9, 139.4, 142.3 (C-1eC-7); 125.7, 126.9, 128.7, 134.2 (C-
10eC-60 and C-100eC-600); 126.4, 127.9, 129.4, 134.3 (C-1a0eC-6a0 and
C-1a00eC-6a00). 31P NMR (DMSO-d6): 28.04. CIMS (m/z): 617 [MH]þ.
d
Acknowledgements
Elemental analysis: Calcd. C31H26N2O8P2: C, 60.39; H, 4.25; N, 4.54.
Found C, 60.28; H, 4.21; N, 4.49.
The authors express their grateful thanks to Prof. C.D. Reddy,
Department of Chemistry, Sri Venkateswara University, Tirupati, for
his helpful discussions and also thank BRNS, BARC, Mumbai for
providing financial assistance (2010/37C/26/BRNS/1424).
6.1.6. Tetraphenyl(3-chloro-4-fluoro phenylamino) methylene
diphosphonate (3f)
Yield 65%, mp 156e158 ꢂC; IR (KBr): 3312, 1231, 762 cmꢀ1 1H
;
NMR (DMSO-d6; 400 MHz):
d
6.21e8.34 (m, 23 H, Ar-H), 5.80e5.92
26.72.
(m, 1H, CHP), 5.64 (s, 1H, NH). 31P NMR (DMSO-d6):
d
References
Elemental analysis: Calcd. C31H25ClFNO6P2: C, 59.68; H, 4.04; N,
2.24. Found C, 59.56; H, 3.98; N, 2.19.
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Thesing, O.C.M. Bijvoet, Bone Mineral 1 (1986) 27e39.
[5] C.W.G.M. Lowik, G. Van der plujim, L.J.A. Van der wee-pals, H. Bloys van
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Res. 11 (1993) 196e214.
6.1.7. Tetraphenyl(4-fluoro-3-nitro phenylamino) methylene
diphosphonate (3g)
Yield 66%, mp 152e154 ꢂC; IR (KBr): 3316, 1228, 764 cmꢀ1 1H
;
NMR (DMSO-d6; 400 MHz):
d
6.20e8.36 (m, 23 H, Ar-H), 5.62e5.78
26.24.
(m, 1H, CHP), 5.34 (s, 1H, NH). 31P NMR (DMSO-d6):
d
Elemental analysis: Calcd. C31H25FN2O8P2: C, 58.68; H, 3.97; N, 4.42.
Found C, 58.55; H, 3.91; N, 4.35.
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B.A. Kashemirov, F.H. Ebetino, J.T. Triffitt, R.G.G. McKenna, J. Med. Chem. 50
(2007) 5967e5975.
[10] O. Bijvoet, H. Fleisch, R.E. Canfield, R.G.G. Russell (Eds.), Bisphosphonates on
Bones, Elsevier, Amsterdam, Holland, 1995.
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(1993) 26e31.
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2083e2087.
[16] Y.B. Kiran, C.D. Reddy, D. Gunasekhar, C. Suresh Reddy, C. Barbosaa,
L.C.A. Annette Leon, Eur. J. Med. Chem. 43 (2008) 885e892.
[17] L.D. Quin, J.G. Verkade, Phosphorus-31 NMR Spectral Properties Compound
Charcaterization and Structural Analysis. VCH Publisher, New York, 1994.
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Plant Sci. 153 (2002) 1161e1168.
6.1.8. Tetraphenyl(2-methyl thiazol-5-yl amino) methylene
diphosphonate (3h)
Yield 57%, mp 168e170 ꢂC; IR (KBr): 3342, 1232, 756 cmꢀ1
;
1H
6.15e8.10 (m, 23 H, Ar-H), 5.54e5.63
(m, 1H, CHP), 5.12 (s, 1H, NH). 31P NMR (DMSO-d6):
26.12.
NMR (DMSO-d6; 400 MHz):
d
d
Elemental analysis: Calcd. C29H26N2O6P2S: C, 58.78; H, 4.42; N, 4.73.
Found C, 57.55; H, 4.39; N, 4.68.
6.1.9. Tetraphenyl(2-nitro thiazol-5-yl amino) methylene
diphosphonate (3i)
Yield 59%, mp 142e144 ꢂC; IR (KBr): 3338, 1228, 759 cmꢀ1
;
1H
6.10e8.10 (m, 23 H, Ar-H), 5.64e5.75
(m, 1H, CHP), 5.10 (s, 1H, NH). 31P NMR (DMSO-d6):
24.28.
NMR (DMSO-d6; 400 MHz):
d
d
Elemental analysis: Calcd. C28H23N3O8P2S: C, 53.94; H, 3.72; N, 6.74.
Found C, 53.84; H, 3.67; N, 6.68.
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J.J. Jeffrey, Biochem. J. 290 (Pt 3) (1993) 873e884.
[25] A. Bala Krishna, Kishore K. Chiruvella, M.V.N. Reddy, Rajesh Reddy, S.K. Nayak,
Sathees C. Raghavan, C. Suresh Reddy, Lett. Drug Design Discov. 10 (4) (2010)
250e259.
6.1.10. Tetraphenyl(benzo [d] thiazol-7-yl amino) methylene
diphosphonate (3j)
Yield 55%, mp 158e160 ꢂC; IR (KBr): 3324, 1226, 754 cmꢀ1
;
1H
6.10e8.24 (m, 26 H, Ar-H), 5.52e5.62
(m, 1H, CHP), 5.15 (s, 1H, NH). 31P NMR (DMSO-d6):
24.26.
NMR (DMSO-d6; 400 MHz):
d
d
Elemental analysis: Calcd. C32H26N2O6P2S: C, 61.15; H, 4.17; N, 4.46.
Found C, 61.02; H, 4.12; N, 4.40.