Design of new arylamino-2-ethane-1,1-diyl- and benzoxazole-2-methylene- bisphosphonates vs cytotoxicity and chronic inflammation diseases. from hydrophobicity prediction to synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.09.032

A general synthetic approach to two new series of methylenebisphosphonates: arylamino-2-ethane-1,1-diyl- and benzoxazole-2-methylenebisphosphonates is presented. Acid hydrolysis of selected BPs was undertaken to give the corresponding bisphosphonic acid (

Full text of DOI:10.1016/j.ejmech.2012.09.032

European Journal of Medicinal Chemistry 57 (2012) 362e372
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design of new arylamino-2-ethane-1,1-diyl- and benzoxazole-2-methylene-
bisphosphonates vs cytotoxicity and chronic inflammation diseases. From
hydrophobicity prediction to synthesis and biological evaluation
,
a
b
Wafaa M. Abdou a , Reham F. Barghash , Ashraf A. Sediek
*
a Chemical Industries Division, National Research Centre, Elbohouth St., D-12311 Dokki, Cairo, Egypt
b Chemistry Department, Faculty of Sciences and Arts, Al-Kamil Branch, King Abdulaziz University, Jeddah, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
A general synthetic approach to two new series of methylenebisphosphonates: arylamino-2-ethane-1,1-
diyl- and benzoxazole-2-methylenebisphosphonates is presented. Acid hydrolysis of selected BPs was
undertaken to give the corresponding bisphosphonic acid (BP-acid). Next, the prediction of the
permeability (hydrophobicity) of the target compounds was measured, by a combination of RP-HPLC and
computational techniques, to study the capacity of transporting the molecule through cellular
membranes. Cytotoxicity/growth inhibition of 50% (GI50, mg/L) and antichronic inflammation properties
of the products were evaluated. Later on, a comparison of the pharmacological results with water
eoctanol partition coefficients (log KOW) of the compounds was also reported.
Received 26 June 2012
Received in revised form
19 September 2012
Accepted 24 September 2012
Available online 2 October 2012
Keywords:
Bisphosphonates
Bisphosphonic acids
Benzoxazoles
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antitumor activity
Antichronic inflammation
Lipophilicity measurement
1. Introduction
vehicle has a high affinity for only bone tissue, its therapeutic effect
on bone related disease is maximized and its distribution to other
Bisphosphonates (BPs) are very powerful inhibitors of bone
resorption and their main therapeutic use being in diseases with
high bone turnover such as Paget’s disease and osteoporosis [1e5].
This strong affinity of BPs for bone minerals allows the rapid and
selective targeting of bone in vivo [5e8]. In addition to inhibiting
bone resorption, BPs have also been shown to exhibit antiin-
flammatory and antitumor effects. In vivo, BPs inhibit proliferation
and induce apoptosis in cultured human breast cancer cells, and in
turn the chance of development of metastases is much less [9e13].
Bone is a common site of breast and prostate cancer metastasis.
Metastatic bone disease is, however, often associated with bone
pain, pathologic fractures, and nerve compression syndromes. Such
complications result in decreasing the quality of life [9]. Unfortu-
nately, until now there is no cure for patients with bone metastasis.
Therefore, effective therapies to inhibit the progression of bone
metastasis would have important clinical benefits. The ideal drug
delivery system is thought to be one that restricts its pharmaco-
logical activity solely to target sites. Thus, if the drug or delivery
sites is minimized. Hydroxyapatite [HA, Ca10(PO4)6(OH)2] is the
major inorganic mineral phase found in bone and teeth, and it is not
present in other tissue under normal circumstances, therefore,
targeting drug delivery to HA is expected to result in a sole delivery
to bone tissue. In sequel, BPs, which are considered to be analogs of
endogenous pyrophosphate, have a significant affinity for HA could
be excellent effective therapies for inhibiting the progression of
bone metastasis. Notwithstanding, the cellular and biochemical
mechanisms of action of BPs are yet to be completely unraveled,
nevertheless, over the last two decades, there was a significant
progress in elucidating the mechanism of action of BPs, which in
general can shorten the life span of osteoclasts by inducing pro-
grammed cell death (apoptosis). Moreover, some important struc-
ture activity relationship (SAR) trends of this class of agents have
emerged [14,15], e.g., by incorporating a hydroxyl group and/or
amino group close from eCP2, increases the affinity to hydroxy-
apatite, by at least 10 fold [1,3].
In a previous work, we reported the synthesis of a series of
tetrazoloquinoline-based ethylene bisphosphonic acids of remark-
able antitumor activity against breast (especially MDA-MB-23/ATCC),
and prostate cancer (PC-3 and DU-145) [16]. Later on, we extended
this work and a series of substituted isoquinoline-bisphosphonates
* Corresponding author. Tel.: þ20 233371615; fax: þ20 233370931.
E-mail addresses: wabdou@link.net, wabdou@intouch.com (W.M. Abdou).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.032

W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
363
of potent and promising antineoplastic activity was prepared. Four
BPs cause a dose dependent increase in sister chromatid exchange
(SCE) frequency followed by a decrease of proliferation rate [17]. A
group of heterocyclic-N, S-BPs of more proliferation rate index (PRI)
compared tothe control drugwererecentlyreportedtocomparetheir
chemistry and their activity with that of their oxygen counterparts as
antitumor agents [18]. In view of the above mentioned facts and in
continuationof our interestin both chemistryand biological activities
of BPs, it was of interest to prepare two series of methylenebi-
sphosphonates: arylamino-2-ethane-1,1-diyl- and benzoxazole-2-
methylene-bisphosphonates and their relevant BP-acids to evaluate
their antitumor- and chronic inflammation properties. The approach
is based on the application of methylenebisphosphonate reagent, 5 to
a group of Schiff-bases, 4aei. Lipophilicity (permeability) of the
studied compounds was measured before the pharmacological eval-
uation. Drugs cross biological barriers most frequently through
passive transfer, which strongly depends on their lipophilicity. The
prediction of the permeability, which expresses the transport of the
molecule through cellular membranes is therefore, considered one
of the most important physical properties of biologically active
compounds [19,20].
products 6aed. Nevertheless, slight homo-oxidation of 6 resulted in
the formation of oxazoles 7 via an intramolecular cyclization in
tandem of extrusion of a hydrogen molecule. The latter process was
previously discussed for the transformation of 3,5-di-tert-butyl-2-
hydroxyphenylamino derivatives to the corresponding benzoxazoles
[21]. Furthermore, when the above reaction [4 þ 5] was allowed to
proceed in methanol solution containing MeONa and catalytic amount
of 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ) [23] oxazoles 7ae
d were obtained as enantiomer products in z75% yields.
Identification of structures 6 and 7 was confirmed by combus-
tion analysis, MS, IR, and NMR-spectroscopy. Compounds 6ae
d [dP z 30, 25 (2d, JPeP ¼ 26.4 Hz)] have sharp melting points
and did not show any isomerism in the spectroscopic data. The 1H
NMR (500.6 MHz) of 6a revealed two types of methine protons
2
with two different chemical shifts at 3.73 (dt, JHeH ¼ 9.3, JPe
3
3
¼ 14.4 Hz, HaCeP2) and 4.88 (dt, JHeH ¼ 9.3, JPeH ¼ 6.2 Hz,
H
HbeC*eY). This large coupling constant (JHeH) of Hb with Ha indi-
cates that Ha is in anti-configuration to Hb-C*. The enantiospecific
isomer 6 was also verified by careful inspection of a model in terms
of the Newman projection, which confirmed the staggered anti-
conformation of Hb and Ha.
On the other hand, oxazole-2-methylenediphosphonate 7a was
analyzed correctly for C31H49NO8P2: m/z (%): 624 (17) [Mþ ꢁ 1]. In
the NMR spectra of 7a, the presence of the exocyclic methine
moiety (CHeP2) was found at dH 3.06 (t, 2JPeH ¼ 16.8 Hz) and at dc
38.7 (t, 1JPeC ¼ 168.3 Hz). These data excluded any possible cycli-
zation reaction involving the methylenediphosphonate moiety
(structure 8), and confirmed that the intramolecular cyclization
proceeded via the other HC-Y-moiety.
In contrast to the above result, applying the reagent 5 to the
Schiff bases 4eei in MeONa solution at r.t., using the same amounts
did not show any appreciated reaction, and the starting material
4eei was recovered practically unchanged. However, the reactions
between 4eei and 5 were completed smoothly, only on heating the
reaction mixture under reflux temperature and in the presence of
DDQ in the medium, to yield the respective oxazole-2-
methylenediphosphonates 7eei (z76%) (Scheme 3).
2. Chemistry
The required Schiff bases, 4aei were generated in high yields
from the condensation of 2-aminophenol 2 with the appropriate
aromatic aldehydes 3aei. However, 2-amino-4,6-di-tert-butyl-
phenol,
2 is not commercially available, and was therefore
synthesized by aminolysis of the parent 3,5-di-tert-butyl-1,2-
benzoquinone (1, Scheme 1) [21,22]. The reaction procedures for
preparing the target diphosphonate derivatives as well as the
course of the reactions were depicted in Schemes 2e4.
The substrates 4aed was treated with phosphorus reagent, 5 in
ethanol solution containing EtONa. The reaction mixture was stirred at
r.t. for the proper time (TLC) to give, after the usual workup, tetraethyl
2-(3,5-di-tert-butyl-2-hydroxyphenylamino)-2-(aryl)ethane-1,1-diyl-
diphosphonates, 6aed as major products (w71%) together with tet-
Recent advances in pharma laboratories have impressively iden-
tified remarkable therapeutics from 1,1-bisphosphonic acid to 1,1-
bisphosphonate ester counterparts [1]. Consequently, based on
evaluation results of the permeability of the synthesized BPs, hydro-
lysis of representative BPs 6b,c, 7b,c, and 7feh was undertaken to give
the corresponding BP-acids 9a,b and 10aee (Scheme 4).
raethyl
(5,7-di-tert-butyl-2-(aryl)-2,3-dihydrobenzo[doxazol-2-yl)
methylenediphosphonates, 7aed (ꢀ7%). Obviously, nucleophilic
addition of the methylene-C in 5 to the imino-C of 4 gave rise to the
The structures suggested for all new compounds are in good
agreement with their analytical and spectral data (see
Experimental section).
3. Pharmacological evaluation
3.1. Prediction and lipophilicity
Hydrophobicity is one of the most important physical properties
of biologically active compounds. This thermodynamic parameter
describes the partitioning of a compound between an aqueous and
an organic phase and is characterized by the partition coefficient
(log P). Reversed phase high performance liquid chromatography
(RP-HPLC) methods have become popular and are widely used for
lipophilicity measurement [19,20]. The general procedure consists
of the measurement of the directly accessible retention time under
isocratic conditions with varying amounts of an organic modifier in
the mobile phase using end-capped non-polar C18 stationary RP
columns and calculating the capacity factor K. Log K, calculated
from the capacity factor K, is used as the lipophilicity index and
then converted to log P scale [24].
Scheme 1. Synthesis of Schiff-bases 4aei.

364
W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
Scheme 2. Synthesis of BPs 6aed and 7aed.
Hydrophobicities of the new compounds 6aed, 7aei, 9a,b, and
3.2. Biological assessment
10aee, Log P, i.e. the logarithm of the partition coefficient for n-
octanol/water, was calculated using the programs CS ChemOffice
Ultra ver. 9.0 (CambridgeSoft, Cambridge, MA, U.S.A., Clog P) and
ACD/LogP ver. 1.0 (Advanced Chemistry Development Inc. Toronto,
Canada), softwares. The results are shown in Table 1. The displayed
data showed that the experimentally determined log K values
correlate relatively poorly with all computed lipophilicity data
using ACD/Log P program. Nevertheless, log P values calculated by
the ChemOffice software (Clog P) agree better with both targets BPs
and BP-acids. On the other hand, obviously, the dimethoxy
substituent 7i possessed, as expected, the lowest lipophilicity (log
K) while the phenolic N-BP-acid, 9a showed the highest lip-
ophilicity. Generally, BP-acid series has higher measured perme-
ability than their BP-counterparts. Also, amino-substituted BPs 6ae
d display, in contrast with our expectations, better hydrophobicities
(log P/Clog P values) than the corresponding oxazole analogs.
Nevertheless, looking insight one series, e.g., oxazole-BPs 7aei, the
substituent effect on the activity is in the order of: dimethylamine
7b > flouro 7c > hydroxyl 7e > chloro 7feh > methoxy 7a > nitro
7d > dimethoxy 7i. Finally, only slight differences were observed in
the permeability’s (log K) between the isomers 7f, 7g or 7h of the
same substituent (Cl) in one molecule.
3.2.1. Antitumor activity screening
Antitumor activity of selected BPs 6b,c, 7b,c and BP-acids 9a,b
and 10aec was carried out at a dose of 10 mM utilizing 16 different
human tumor cell lines; representing breast, ovarian, prostate, as
well as liver, according to the previously reported standard method
[24,25]. Substrates 4a and 4g were also biologically tested in a trial
to reflect the effect of introducing BP-moiety.
The results were displayed in Table 2 and showed an interesting
activity for several compounds. For the purpose of the study, cell
line growth inhibition with >50% at a concentration of 10 mM was
considered to be a noticeable activity. Other than the substrates 4a
and 4g, all synthesized compounds reflect remarkable antitumor
activity against breast (especially MDA-MB-231/ATCC and BT-549),
and prostate carcinoma cell lines (PC-3 and DU-145), whereas
a moderate to good effect was observed on ovarian and liver cancer
cells. The four most active compounds are 9a > 9b > 10a > 6b. SARs
correlation for these reported observations revealed that the
presence of dialkylamino group as a substituent to the aryl-moiety
is usually associated with the enhancement in antitumor properties
as indicated in compounds 6b, 7b, 9a, and 10a. Furthermore, the
data showed that the BP-acid series has higher activity than their
Scheme 3. Synthesis of BPs 7eei.

W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
365
Scheme 4. Synthesis of BP-acids 9a,b and 10aee.
BP-counterparts. Finally, it could be assumed, in general, that the
four compounds of the most antitumor activity are the same of the
highest lipophilicity. However, a straight correlation between the
activity and lipophilicity of BPs or BP-acids was not found, and the
antitumor activity seems to be independent of lipophilicity and
dependent on chemical structure. Further studies on experimental
tumors in vivo for evaluating the possible antineoplastic potential
of the most promising compounds are in progress.
surgical implantation of sc in the dorsum of the mice in order to
generate granuloma. All compounds were prepared as solutions or
emulsions and the pH was adjusted to 7.4. Each mouse received the
tested compound in a volume of 0.l/10 g of body weight sc in the
scruff of the neck. Dosing (25e100 mg/kg) commenced on the day
of implantation of the soaked discs and was continued thereafter
on a daily basis until day nine, when the mice were euthanized. The
granulomatous lesions were then excised and measurements were
taken for both wet and dry tissue weights. The data is displayed in
Table 3 and Fig. 1. The results were analyzed by Student’s paired t
test. Structure 11
The data in Table 3 shows that 11 reproducibly inhibited gran-
uloma wet- and dry weights and served as a positive control in
other experiments. Other than the substrates 4a and 4g, ten of
twenty tested new compounds have moderate to good antiin-
flammatory properties when compared with the standard drug 11,
without toxic side-effects toxicity (evaluation result is provided as
Supplementary document). Compounds 6b,c, 7b,c, and 7feh
3.2.2. Antiinflammatory screening
The in vivo antiinflammatory activity of all BP-products, 6ae
d and 7aei, as well as BP-acids 9a,b and 10aee was determined
by using a delayed-type hypersensitivity granuloma model [26],
and were compared with that of the BP-drug, risedronate, 11 at the
same dose. The substrates 4a and 4g were also tested to reflect the
effect of introducing the phosphorus moiety. BP-11 was selected for
its optimal potency and safety in early screening of similar assays
[1]. Furthermore, this model is unaffected by traditional nonste-
roidal antiinflammatory drugs, such as indomethacin, aspirin or
ibuprofen [27].
significantly inhibited the granuloma in
a dose-dependent
manner, while 6d, 9a, and 9b displayed an inhibitory effect,
which is almost equivalent to that of risedronate at 100 mg/kg. BPs
6a, 7i, and BP-acids 10aec showed marginal activity against the dry
weight granuloma. In contrast, the substrates 4a and 4g were
inactive at all doses.
A delayed-type hypersensitivity granuloma assay is a model of
chronic inflammation, in which mice were previously sensitized to
methylated bovine serum albumin (mBSA) and subjected to
Table 1
Structure-activity correlation based on the obtained results
indicates that the presence of the bisphosphonate moiety is an
essential factor in developing the total pharmacological properties
for these compounds. Compounds 6b,c 7b,c, and 7feh are the most
active structures among all products, and the presence of the free or
alkylatedamino group highly enhanced the efficacy of the
compounds, 6b, 7b, 9b, and 10b. In contrast, introducing the two
methoxy groups at the phenyl moiety (e.g., see compound 7i) results
incomplete loss of the activity. Conversion of 6b and 6c to the cor-
responding bisphosphonic acids 9a and 9b results in decreasing the
activity. This observation is not surprising as it is previously reported
insimilaroccasions[26]. Theresults of thebioassayalsoindicatethat
the activity of the tested BPs on chronic inflammation is dose
dependent. Finally, the results also show that the experimentally
determined log K values (Table 1) are in moderate agreement with
the antiinflammatory properties for the same compounds.
Comparison of the determined log K values with the calculated lipophilicity (log P/
Clog P) and ACD/LogP ver. 1.0, of 6aed, 7aei, 9a,b, and 10aee.
Cpd
Y
Log P (Clog P/
ChemOffice)
Log P ACD/LogP
ver. 1.0
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
7g
7h
7i
4-MeOeC6H4
4-(Me)2NeC6H4
4-FeC6H4
4- O2NeC6H4
4-MeOeC6H4
4-(Me)2NeC6H4
4-FeC6H4
4-O2NeC6H4
4-HOeC6H4
4-CleC6H4
7.49/3.06544
8.01/3.31144
7.88/3.28944
NT/2.88944
8.19/4.92904
8.71/5.17504
8.58/5.15304
NT/4.75304
8.16/3.43404
8.96/5.72304
8.96/5.72304
8.96/5.72304
7.94/5.01804
6.51/0.7106
6.38/0.68864
7.21/2.57424
7.08/2.55224
7.46/3.12224
7.46/3.12224
7.46/3.12224
7.05 ꢂ 0.51
7.24 ꢂ 0.52
7.18 ꢂ 0.57
6.86 ꢂ 0.51
6.70 ꢂ 0.73
6.90 ꢂ 0.73
6.84 ꢂ 0.77
6.52 ꢂ 0.73
6.05 ꢂ 0.73
7.38 ꢂ 0.73
7.38 ꢂ 0.73
7.38 ꢂ 0.73
6.67 ꢂ 0.75
2.63 ꢂ 0.72
2.57 ꢂ 0.76
2.29 ꢂ 0.89
2.23 ꢂ 0.92
2.78 ꢂ 0.88
2.78 ꢂ 0.88
2.78 ꢂ 0.88
3-CleC6H4
2-CleC6H4
2,5-MeOeC6H4
4-(Me)2NeC6H4
4-FeC6H4
4-(Me)2NeC6H4
4-FeC6H4
4-CleC6H4
3-CleC6H4
2-CleC6H4
9a
9b
10a
10b
10c
10d
10e
4. Conclusion
In summary, the present investigation offered two new series of
BPs: 2-hydroxyphenylamino-2-ethane-gem-diphosphonates and

366
W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
Table 2
Concentrations resulting in growth inhibition of 50% (GI50, mg/L) of in vitro human tumor cell lines of 6b,c, 7b,c, 9a,b, and 10aec.
Panel/cell line
Compounds
4a/4g
6b
6c
7b
7c
9a
9b
10a
9.80
6.80
8.9
20.2
8.84
NT
10b
10c
10.64
18.05
11.5
24.12
14.5
NT
Breast cancer
McF7
NCl/ADRRES
MDA-MB-231/ATCC
HS578T
MDA-MB-435
BT-549
T-47D
> 46
> 46
> 46
> 46
> 46
> 46
> 46
17.3
18.32
10.6
14.80
14.60
13.3
26.80
9.37
14.88
17.8
12.74
26.10
16.02
5.05
25.21
20.1
14.72
24.2
16.4
11.97
22.18
8.40
6.20
4.83
8.92
5.32
15.7
14.58
12.41
10.12
8.36
13.4
32.1
6.45
5.68
10.6
9.85
14.6
11.93
26.92
18.08
18.13
14.12
5,05
11.08
14.06
17.05
11.65
13.57
13.42
13.55
Ovarian cancer
IGROVI
> 46
> 46
> 46
> 46
> 46
> 46
15.3
7.41
9.8
16.5
14.06
16.5
14.8
11.4
10.6
25.6
28.4
16.76
20.2
26.1
18.9
10.52
NT
NT
17.13
7.9
4.4
3.6
30.6
11.4
11.53
11.6
7.4
4.32
16.3
13.5
13.6
16.8
14.5
4.83
10.3
12.66
12.6
20.6
10.7
8.65
40.04
13.7
13.68
24.8
NT
10.18
36.6
28.6
16.5
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
16.55
10.18
17.8
21.41
14.46
Prostate cancer
PC-3
DU-145
> 46
> 46
6.98
6.54
8.6
9.42
14.2
11.23
14.2
11.6
3.82
2.38
5.6
4.52
10.82
8.75
19.6
15.3
>52
10.5
Liver cancer
HEPG2
> 50
7.96
7.97
15.5
16.7
3.16
3.42
8.52
24.23
32.42
NT: not tested.
benzoxazole-2-methaylenediphosphonates as antitumor and anti-
inflammatory drugs. We have also attempted in this research work
to utilize the high bone specificity of BP-acids with other chemical
moieties of potential anticatabolic pharmacology to test the new
compounds for treating tumor proliferation diseases. The obtained
results indicated percentage growth inhibition of 50% (GI50, mg/L)
for several new BP-acids.
Biological evaluation versus chronic inflammation was also
tested whereby the screening result indicated the presence of free
phenolic (OH), amine and/or alkylatedamino group in the BPs
would enhance their potency. These groups may facilitate the
binding to the metal atom in the active site of the matrix metal-
loproteinases (MMPs) [19,20]. In parallel, the hydrophobicity
evaluation of the target compounds was tested as a prediction and
prerequisite for pharmacological screening and drug development.
The prediction of the measured permeability (lipophilicity) was
measured by means of RP-HPLC and a computational technique
was also reported in this communication. Unfortunately, the results
showed that the experimentally determined log K values (Table 1)
correlate relatively poorly with the evaluated pharmacological
potency.
regulations concerning the care and use of laboratory animals were
considered during the pharmacological screening. The substrates
4aei were prepared according to the reported method [21,22].
5.1. General procedure of bisphosphonates (BPs) 6aed and 7aed
A solution of 4.2 mmol of tetraethyl methylenebiphosphonate, 5
in 10 mL of absolute ethanol containing 9.12 mmol sodium (Na) was
stirred at 0 ꢃC for about 0.5 h. A solution of 3.8 mmol of the Schiff-
base 4aed in 10 mL of EtOH was then added in one portion, and the
reaction was stirred at r.t., for z12 h (TLC). The product mixture
was cooled, poured into ice-water, and acidified with conc HCl to
pH z6, followed by extraction with AcOEt (3 ꢄ 50 mL), and the
combined organic phase was dried over anh. Na2SO4. After removal
of the volatile materials under vacuum, the resulting residue was
chromatographed on silica gel with n-hexane/CHCl3 to give the
respective BPs 6aed and 7aed.
5.2. Reaction of 4a with 5
Reagents: (E)-2,4-Di-tert-butyl-6-(4-methoxybenzylideneamino)
phenol, 4a (1.3 g, 3.8 mmol), BP-reagent 5 (1.2 mL, 4.2 mmol), EtONa
(0.2 g of Na, 9.1 mmol), and EtOH (20 mL). The product residue was
chromatographed with n-hexane/CHCl3 (6:4 v/v) to give 7a, followed
with (1:1 v/v) to give 6a.
5. Experimental section
Melting points (uncorrected) were determined with open
capillary tube on an Electrothermal (variable heater) melting point
apparatus. IR spectra were recorded on a JASCO FT-IR 6100 using
KBr disc. NMR spectra were measured with a JEOL E.C.A-500 MHz
5.2.1. Tetraethyl (5,7-di-tert-butyl-2-(4-methoxyphenyl)-2,3-
dihydrobenzo[d]oxazol-2-yl)methylenediphosphonate, 7a
(
13C: 125.8 MHz, 1H: 500.6 MHz, 31P: 200.7 MHz) spectrometer. 31
P
Colorless crystals, yield: z7%, mp 152e154 ꢃC (from CH2Cl2). IR
(cmꢁ1, KBr): nmax 3334w (NH), 1235, 1221 (2P]O, bonded), 1179,
1064 (2PeOeC). 1H NMR [CDCl3] ppm: 1.26, 1.34 (2dt, JHeH ¼ 6.6,
4JPeH ¼ 4.6 Hz, 2ꢄ 6H, 4H3CCOP), 1.35, 1.40 (2s, 2ꢄ 9H, 2(H3C)3C),
NMR spectra were recorded with H3PO4 (85%) as external refer-
ence. 1H and 13C NMR spectra were recorded with trimethylsilane
as internal standard in CDCl3 or DMSO-d6. Chemical shifts (d) are
given in ppm. The mass spectra were performed at 70 eV on an MS-
50 Kratos (A.E.I.) spectrometer provided with a data system. The
2
3.06 (t, JPeH ¼ 16.8 Hz, 1H, HCeP2), 3.46 (s, 3H, H3CO), 4.14, 4.24
(2dq, JHeH ¼ 6.6, 3JPeH ¼ 7.2 Hz, 2ꢄ 4H, 4H2COP), 7.15e7.68, 8.27 (m,
6H, HeAr), 8.88 (s (br), 1H, HN). 13C NMR [CDCl3] ppm: 158.4, 146.2,
137.4, 134.8, 133.3, 127.1, 122.3, 118.6, 116.5, 110.5 (CeAr), 61.2 (d,
appropriate
precautions
in
handling
moisture-sensitive
compounds were observed. The purity of all new samples was
verified by microchemical analysis (H/C/N/P) and spectroscopy.
Solvents were dried by standard techniques. TLC: Merck 0.2 mm
silica gel 60 F254 analytic aluminum plates. Column chromatog-
raphy (CC): silica gel (Kieselgel 60 mesh, particle size 0.2e0.5 mm;
E. Merck, Darmstadt). All international principles and local
1
2JPeC ¼ 10.5 Hz, CH2OP), 55.2 (CH3O), 38.7 (t, JPeC ¼ 168.3 Hz,
3
CeP2), 37, 31.7 (2C(CH3)3), 31.2, 30.4 (2(CH3)3C), 15.4 (d, JPe
2
¼ 7.5 Hz, CH3COP). 31P NMR [CDCl3] ppm: 24.3, 28.6 (2d, JPe
C
¼ 24.4 Hz, P2eC). EI-MS: in m/z (%): 624 (17) [Mþ ꢁ 1], 593 (36)
P
[Mþ ꢁ 32(H þ OMe)], 319 (48) [Mþ ꢁ 306 (H þ OMe þ 2

W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
367
Table 3
Delayed-type hypersensitivity granuloma results of BPs 6aed, 7aei, and BP-acids 9a,b and 10aee.
No
Dose (mg/kg) scb
[% Inhibtn/granuloma
Dry wt
Potency(%) dose
(100 mg/kg (dry)
Lipophicity
prediction log K
Coincidence P/Ec
Wet wt
11
100
60
25
48***a
44**
33
44***
40
30
100
e
e
d
6a
6b
6c
6d
100
60
25
100
60
25
100
60
25
100
60
46***
42**
30
55***
53
42
50**
45
40***
34*
22
42*
43
30
41***
33
95.8
114.6
104.2
93.8
0.5726
0.9981
0.9254
0.5331
ꢁ/þ
þ/þ
þ/þ
ꢁ/þ
38
45*
50
23
42**
28
25
36
25
7a
7b
100
50
100
60
32***
16
49*
38
25**
15
44***
32
66.7
0.5044
0.8445
ꢁ/ꢁ
þ/þ
102.1
25
22
13
7c
100
60
44**
37
40**
28
91.7
0.7515
þ/þ
25
24
20
e
e
e
7d
7e
7f
100
50
100
50
100
50
100
50
100
50
100
50
100
50
100
60
32**
22
42***
30
41**
32
40**
30
40*
28
18*
14
42***
29
44***
40
24*
19*
36*
23
36*
21
24*
20
34*
21
16*
10
33**
< 10
34***
31
66.7
87.5
85.4
83.3
83.3
37.5
87.5
91.7
0.4065
0.6972
0.6723
0.8507
0.8445
0.3281
1.0021
1.0005
ꢁ/ꢁ
ꢁ/þ
ꢁ/þ
þ/þ
þ/þ
ꢁ/ꢁ
þ/þ
þ/þ
7g
7h
7i
9a
9b
25
32
22
10a
10b
10c
10d
10e
4a
100
50
100
50
100
50
100
50
100
50
100
50
100
50
38**
26
44***
33
30*
24
31*
26
30*
27
15**
12
<10**
<10
33**
18
39**
24
28**
18
30**
20
30**
21
30**
16
<10**
<10
79.2
91.7
62.5
64.6
62.5
31.3
e
0.9245
0.9118
0.8543
0.8555
0.8478
e
þ/þ
þ/þ
þ/ꢁ
þ/ꢁ
þ/ꢁ
e
4g
e
e
a
(***) p < 0.001, (**) p < 0.01, (*) p < 0.05; p is the percentage of inhibition.
sc: subcutaneously.
b
c
(P/E): P e prediction (lipophilicity); E ꢁ experiment (antiinflammatory); P/E e accuracy of prediction.
d
þ/þ means that both lipophilicity prediction and antiinflammatory give positive results; ꢁ/ꢁ means that both lipophilicity prediction and antiinflammatory give negative
results; þ/ꢁ means that the lipophilicity prediction gives positive result but antiinflammatory gives negative results; ꢁ/þ means that the lipophilicity prediction gives
negative results but antiinflammatory gives positive results.
[P(O)(OEt)2)]), 306 (100) [Mþ ꢁ 319 (H þ OMe þ CH [P(O)(OEt)2]2)],
287 (58) (CH[P(O)(OEt)2]2), 137 (46) (P(O)(OEt)2), 77 (5). Anal. Calcd
for C31H49NO8P2 (625.7): C, 59.51; H, 7.89; N, 2.24; P, 9.90. Found: C,
59.54; H, 7.84; N, 2.20; P, 9.95.
bonded),1161,1144 (2PeOeC). 1H NMR [CDCl3] ppm: 1.22,1.31 (2dt,
4
JHeH ¼ 7.2, JPeH ¼ 5.6 Hz, 2ꢄ 6H, 4H3CCOP), 1.35, 1.38 (2s, 2ꢄ 9H,
(H3C)3C), 3.36 (s, 3H, H3CO), 3.73 (dt, JHeH ¼ 9.3, 2JPeH ¼ 14.4 Hz, 1H,
HaCeP2), 4.00, 4.34 (2dq, JHeH ¼ 7.2, 3JPeH ¼ 5.6 Hz, 2ꢄ 4H, 4H2COP),
4.88 (dt, JHeH ¼ 9.3, 3JPeH ¼ 6.2 Hz, 1H, HbeC*), 7.05e7.62, 8.23 (m,
6H, HeAr), 9.32 (s, br, 1H, HN), 9.98 (s (br), 1H, HO).13C NMR [CDCl3]
ppm: 158.8, 151.5, 146.1, 138.8, 136.7, 134, 130.9, 122.9, 118.2, 113.1
5.2.2. Tetraethyl 2-(3,5-di-tert-butyl-2-hydroxyphenylamino)-2-
(4-methoxyphenyl)ethane-1,1-diyl-diphosphonate, 6a
2
2
Colorless crystals yield: 71%, mp 193e195 ꢃC (from MeOH). IR
(cmꢁ1, KBr): nmax 3464, 3348w (OH & NH), 1234, 1228 (2P]O,
(CeAr), 64.5 (d, JPeC ¼ 16.5 Hz, C*HCP), 60.6 (d, JPeC ¼ 10.5 Hz,
1
CH2OP), 55.1 (OCH3), 44.2 (t, JPeC ¼ 168.3 Hz, CeP2), 37.4, 35.3

368
W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
Fig. 1. Antiinflammatory activity of BPs 6aed and 7aei, and BP-acids 9a,b and 10aee.
3
(2C(CH3)3), 31.2, 29.5 (2(CH3)3C),15.3 (d, JPeC ¼ 7.5 Hz, CH3COP).
(H þ N(Me)2 þ CH[P(O)(OEt)2]2)], 287 (40) (CH[P(O)(OEt)2]2),
137 (46) (P(O)(OEt)2), 77 (77). Anal. Calcd for C32H52N2O7P2 (638.7):
C, 60.17; H, 8.21; N, 4.39; P, 9.70. Found: C, 60.13; H, 8.15; N, 4.33;
P, 9.77.
2
31P NMR [CDCl3] ppm: 26.6, 31.3 (2d, JPeP ¼ 26.4 Hz, P2eC).
EI-MS: in m/z (%): 625 (13) [Mþ ꢁ 2], 607 (23) [Mþ ꢁ 20
(2H þ H2O)], 576 (18) [Mþ
ꢁ 51(2H þ H2O þ OMe)], 439 (34)
[Mþ ꢁ 188 (2H þ H2O þ OMe þ P(O) (OEt)2)], 302 (100)
[Mþ ꢁ 325(2H þ H2O þ OMeþ2{P(O)(OEt)2})], 287 (46) (CH
[P(O)(OEt)2]2), 137 (66) (P(O)(OEt)2), 77 (56). Anal. Calcd for
C31H51NO8P2 (627.7): C, 59.32; H, 8.19; N, 2.23; P, 9.87. Found: C,
59.39; H, 8.14; N, 2.18; P, 9.92.
5.3.2. Tetraethyl 2-(3,5-di-tert-butyl-2-hydroxyphenylamino)-2-
(4-dimethylaminophenyl)ethane-1,1-diyldiphosphonate, 6b
Colorless crystals, yield: 70%, mp 171e173 ꢃC (from EtOH). IR
(cmꢁ1, KBr): nmax 3444, 3367 (OH & NH),1242,1219 (2P ¼ O, bonded),
1050,1034(2PeOeC).1HNMR[CDCl3]ppm:1.22,1.29(2dt, JHeH ¼ 6.6,
4JPeH ¼ 4.8 Hz, 2ꢄ 6H, 4H3CC$OP),1.37,1.52(2s, 2ꢄ 9H, 2(H3C)3C), 3.53
(s, 6H, Me2N), 3.77 (dt, JHeH ¼ 9.5, 2JPeH ¼ 15.6 Hz, 1H, HaCeP2), 4.05,
5.3. Reaction of 4b with 5
3
4.23 (2dq, JHeH ¼ 6.6, JPeH ¼ 7.2 Hz, 2ꢄ 4H, 2H2COP), 5.04 (dt, JHe
Reagents: (E)-2,4-Di-tert-butyl-6-(4-(dimethylamino)benzyli-
deneamino)phenol, 4b (1.3 g, 3.8 mmol), BP-reagent 5 (1.2 mL,
4.2 mmol), EtONa (0.2 g of Na, 9.1 mmol), and EtOH (20 mL). The
product residue was chromatographed with n-hexane/CHCl3 (6:4
v/v) to give 7b, followed with n-hexane/CHCl3 (1:1 v/v) to give 6b.
¼ 9.5,3JPeH ¼ 6.2 Hz, 1H, Hbe*C), 7.02e7.67, 8.23 (m, 6H, HeAr),
H
9.03 (s (br), 1H, HN), 10.19 (s (br), 1H, OH). 13C NMR [CDCl3] ppm:
151.2, 148.6, 146.6, 138.4, 136.8, 131.6, 129.5, 122.7, 114.4, 113.6 (CeAr),
64.7(d, 2JPeC ¼ 13.6 Hz, C*HCP), 62.4 (d,2JPeC ¼ 11.3Hz, CH2OP), 47.6(t,
1JPeC ¼ 174.2 Hz, CeP2), 39.5[(CH3)2N], 37.2, 35.3 (2C(CH3)3), 31.6, 30.3
3
(2(CH3)3C),15.6 (d, JPeC ¼ 7.5 Hz, CH3COP). 31P NMR [CDCl3]
5.3.1. Tetraethyl (5,7-di-tert-butyl-2-(4-dimethylaminophenyl)-2,3-
dihydrobenzo[d]oxazol-2-yl)methylenediphosphonate, 7b
2
ppm: 23.8, 27.7 (2d, JPeP ¼ 32.4 Hz, P2eC). EI-MS: in m/z (%): 638
(40) [Mþ ꢁ 2], 620 (28) [Mþ ꢁ 20(2H þ H2O)], 576 (23) [Mþ ꢁ 64
(2H þ H2O þ NMe2)], 439 (54) (Mþ ꢁ 201 (2H þ H2O þ NMe2þP(O)
(OEt)2)], 302 (100) (Mþ ꢁ 338 (2H þ H2O þ NMe2þ2{P(O)(OEt)2})],
287 (16) (CH[P(O) (OEt)2]2),137 (36) (P(O)(OEt)2), 77 (66). Anal. Calcd
for C32H54N2O7P2 (640.7): C, 59.99; H, 8.49; N, 4.37; P, 9.67. Found: C,
60.05; H, 8.43; N, 4.31; P, 9.74.
Colorless crystals yield: z7%, mp 133e135 ꢃC (from cyclo-
hexane). IR (cmꢁ1, KBr): nmax 3350w (NH), 1236, 1226 (2P]O,
bonded), 1164, 1079, (2PeOeC). 1H NMR [CDCl3] ppm: 1.12, 1.28
4
(2dt, JHeH ¼ 6.7, JPeH ¼ 4.5 Hz, 2ꢄ 6H, 4H3CCOP), 1.33, 1.47
2
(2s, 2ꢄ 9H, 2(H3C)3C), 3.36 (t, JPeH ¼ 17 Hz, 1H, HCeP2), 3.83,
3
(s, 6H, (H3C)2N), 4.11, 4.22 (2dq, JHeH ¼ 6.7, JPeH ¼ 5.8 Hz, 2ꢄ 4H,
4H2COP), 6.97e7.76, 8.33 (m, 6H, HeAr), 8.96 (s(br), 1H, HN). 13C
NMR [CDCl3] ppm: 149.1,146.2,137.4,134.7,130.6,125.3,118.8,117.8,
115.4, 110.6 (CeAr), 61.2 (d, 2JPeC ¼ 10.5 Hz, CH2OP), 40.6 (N(CH3)2),
5.4. Reaction of 4c with 5
1
38 (d, JPeC ¼ 173.3 Hz, CeP2), 37, 31.7 (2 C(CH3)3), 31.2, 30.4
Reagents: (E)-2,4-Di-tert-butyl-6-(4-fluorobenzylideneamino)
phenol, 4c (1.2 g, 3.8 mmol), BP-reagent 5 (1.2 mL, 4.2 mmol), EtONa
(0.2 g of Na, 9.1 mmol), and EtOH (30 mL). The product residue was
chromatographed with n-hexane/CHCl3 (6:4 v/v) to give 7c, fol-
lowed with n-hexane/CHCl3 (1:1 v/v) to give 6c.
(2(CH3)3C), 15.4 (d, JPeC ¼ 7.5 Hz, CH3COP). 31P NMR [CDCl3]
3
2
ppm: 24.8, 28.7 (2d, JPeP ¼ 22.4 Hz, P2eC). EI-MS: in m/z (%):
637 (14) [Mþ ꢁ 1], 593 (56) [Mþ ꢁ 45(H þ N(Me)2], 319 (28)
[Mþ ꢁ 319(H þ N(Me)2 þ 2{P(O)(OEt)2)}), 306 (100) [Mþ ꢁ 332
5.4.1. Tetraethyl (5,7-di-tert-butyl-2-(4-fluorophenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl)methylenediphosphonate, 7c
Colorless crystals, yield: z5%, mp 94e96 ꢃC (from pentane). IR
(cmꢁ1, KBr): nmax 3340w (NH), 1236, 1226 (2P]O, bonded), 1154,
1097 (2PeOeC). 1H NMR [CDCl3] ppm: 1.13, 1.27(2dt, JHeH ¼ 6.5,
4JPeH ¼ 4.3 Hz, 2ꢄ 6H, 4H3CC$OP), 1.36, 1.57 (2s, 2ꢄ 9H, (H3C)3C),
2.96 (t, 2JPeH ¼ 18.8 Hz, 1H, HCeP2), 4.06, 4.17 (2dq, JHeH ¼ 6.5, 3JPe
¼ 5.6 Hz, 2ꢄ 4H, 4H2COP), 7.17e7.86, 8.15, 8.17 (m, 6H, HeAr), 9.04
H
Structure 11. . Risedronate.
(s (br), 1H, HN). 13C NMR [CDCl3] ppm: 164.6, 158.5, 146.2, 137.4,

W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
369
132.3, 127.6, 120.2, 119.9, 118.6, 110.3 (CeAr), 61.4 (d, 2JPeC ¼ 10.5 Hz,
CH2OP), 38.2 (t, 1JPeC ¼ 158.3 Hz, CeP2), 37.3, 31.7 (2C(CH3)3), 31.2,
4JPeH ¼ 4.5 Hz, 2ꢄ 6H, 4H3CC$OP), 1.37, 1.40 (2s, 2ꢄ 9H, (H3C)3C),
2
3.50 (dt, JHeH ¼ 9.0, JPeH ¼ 15.6 Hz, 1H, HaCeP2), 4.07, 4.12 (2dq,
3
3
30.4 (2(CH3)3C), 15.4 (d, JPeC ¼ 7.5 Hz, CH3COP). 31P NMR [CDCl3]
JHeH ¼ 6.6, JPeH ¼ 6.0 Hz, 2ꢄ 4H, 2H2COP), 5.44 (dt, JHeH ¼ 9.0,
ppm: 26.6, 29.3 (2d, 2JPeP ¼ 30.2 Hz, P2eC). EI-MS: in m/z (%): 612
(21) [Mþ ꢁ 1], 593 (66) [Mþ ꢁ 20 (H þ F)], 319 (33) (Mþ ꢁ 294
(H þ Fþ2{P(O)(OEt)2})], 306 (100) [Mþ ꢁ 307 (H þ F þ CH
{P(O)(OEt)2}2)], 287 (16) (CH[P(O)(OEt)2]2), 137 (36) (P(O)(OEt)2),
77 (86). Anal. Calcd for C30H46FNO7P2 (613.6): C, 58.72; H, 7.56; F,
3.10; N, 2.28; P, 10.10. Found: C, 58.76; H, 7.49; F, 3.15; N, 2.24; P,
10.17.
3JPeH ¼ 6.4 Hz, 1H, Hbe*C), 7.37e7.69, 8.34 (m, 6H, HeAr), 8.88
(s (br), 1H, HN), 10.49 (s (br), 1H, OH). 13C NMR [CDCl3] ppm: 151.8,
145.6, 137.6, 134.6, 133.4, 129.4, 127.4, 123.5, 122.8, 114.4 (CeAr),
66.3 (d, 2JPeC ¼ 14.7 Hz, C*HCP), 61.6 (d, 2JPeC ¼ 9.7 Hz, CH2OP), 45.7
(t, 1JPeC ¼ 188.4 Hz, CeP2), 37.5, 36.6 (2C(CH3)3), 31.8, 30.4 (2(CH3)3C),
15.6 (d, 3JPeC ¼ 8.2 Hz, CH3COP). 31P NMR [CDCl3] ppm: 26.3, 28.6 (2d,
2JPeP ¼ 34.6 Hz, P2eC). EI-MS: in m/z (%): 640 (30) [Mþ ꢁ 2], 622 (28)
[Mþ ꢁ 20(2H þ H2O)], 576 (20) [Mþ ꢁ 66(2H þ H2O þ NO2)], 439 (46)
(Mþ ꢁ 203 (2H þ H2O þ NO2þP(O)(OEt)2)], 302 (100) (Mþ ꢁ 340
(2H þ H2O þ NO2þ2{P(O)(OEt)2)}], 287 (25) (CH[P(O)(OEt)2]2), 137
(32) (P(O)(OEt)2), 77 (72). Anal. Calcd for C30H48N2O9P2 (642.6): C,
56.07; H, 7.53; N, 4.36; P, 9.64. Found: C, 56.11; H, 7.47; N, 4.32; P, 9.69.
When the above reactions (4aed þ 5) were carried out in
methanol solution containing sodium (Na) using the same
amounts, and in the presence of 4.1 mmol of 2,3 dichloro-5,6-
dicyanobenzoquinne (DDQ) at r.t. for z6e10 h (TLC), BPs 7ae
d were obtained after the usual working up, as a sole reaction
products. Yields: 7a (76%), 7b (73%), 7c (76%), and 7d (80%).
5.4.2. Tetraethyl 2-(3,5-di-tert-butyl-2-hydroxyphenylamino)-2-
(4-fluorophenyl)ethane-1,1-diyldiphosphonate, 6c
Colorless crystals, yield: 72%, mp 138e140 ꢃC (from EtOH). IR
(cmꢁ1, KBr): nmax 3427, 3335w (OH & NH),1238,1222 (2P]O, bonded),
1085,1066 (2PeOeC).1H NMR [CDCl3] ppm: 1.16,1.32 (2dt, JHeH ¼ 7.2,
4JPeH ¼ 5.6 Hz, 2ꢄ 6 H, 4H3CCOP),1.36,1.52 (2s, 2ꢄ 9H, 2(H3C)3C), 3.23
(dt, JHeH ¼ 9.6, 2JPeH ¼ 16.1 Hz, 1H, HaCeP2), 4.11, 4.14 (2dq, JHeH ¼ 7.2,
3JPeH ¼ 5.9 Hz, 2ꢄ 4H, 4H2COP), 5.24 (dt, JHeH ¼ 9.6, 3JPeH ¼ 7.6 Hz,
1H, HbeC*), 7.06e7.77, 8.24 (m, 6H, HeAr), 8.92 (s, br, 1H, HN), 9.58
(s (br),1H, HO). 13C NMR [CDCl3] ppm: 163.8,151.8,146.1,138.8,136.4,
130.2,123.4,120.3,119.8,113.6 (CeAr), 65.7 (d, 2JPeC ¼ 11.8 Hz, C*HCP),
2
1
60.4 (d, JPeC ¼ 11.3 Hz, CH2OP), 46.8 (t, JPeC ¼ 155.2 Hz,
CeP2), 37.2, 35.3 (2C(CH3)3), 31.6, 30.3 (2(CH3)3C),16.2 (d,
3JPeC ¼ 7.2 Hz, CH3COP). 31P NMR [CDCl3] ppm: 23.7, 25.8 (2d,
2JPeP ¼ 31.1 Hz, P2eC). EI-MS: in m/z (%): 613 (18) [Mþ ꢁ 2], 595 (21)
[Mþ ꢁ 20 (2H þ H2O)], 576 (63) [Mþ ꢁ 39(2H þ H2O þ F)], 439 (24)
[Mþ ꢁ 176 (2H þ H2O þ F þ P(O)(OEt)2)], 303 (100) (Mþ ꢁ 313
(2H þ H2O þ Fþ2P(O)(OEt)2)], 287 (16) (CH[P(O)(OEt)2]2), 137 (36)
(P(O)(OEt)2), 77 (86). Anal. Calcd for C30H48FNO7P2 (615.6): C, 58.53;
H, 7.86; F, 3.09; N, 2.28; P, 10.06. Found: C, 58.49; H, 7.82; F, 3.11; N,
2.24; P, 10.12.
5.6. General procedure of 7eei
Following the general procedure, a mixture of 9.12 mmol of Na,
4.2 mmol of 5, 4.1 mmol of DDQ, and 3.8 mmol of the Shiff-bases 4ee
i in 20 mL of MeOH was heated under reflux for 8e10 h. The product
mixture was chromatographed with n-hexane/CHCl3 to give 7e (6:4
v/v), 7f (3:7 v/v), 7g (8:2 v/v), 7h (3:7 v/v), and 7i (8:2 v/v).
5.6.1. Tetraethyl (5,7-di-tert-butyl-2-(4-hydroxyphenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl)methylene-diphosphonate, 7e
Colorless needles, yield: 75%, mp 148e150 ꢃC (from EtOH). IR
(cmꢁ1, KBr): nmax 3350 (br) (OH, NH), 1233, 1225 (2P]O, bonded),
5.5. Reaction of 4d with 5
1075, 1024 (2PeOeC). 1H NMR [CDCl3] ppm: 1.26, 1.32 (2dt, JHe
4
Reagents:
(E)-2,4-Di-tert-butyl-6-(4-nitrobenzylideneamino)
¼ 7.4, JPeH ¼ 4.3 Hz, 2ꢄ 6H, 4H3CC$OP), 1.33, 1.44 (s, 2ꢄ 9H,
H
2
phenol, 4d (1.3 g, 3.8 mmol), BP-reagent 5 (1.2 mL, 4.2 mmol),
EtONa (0.2 g of Na, 9.1 mmol), and EtOH (30 mL). The product
residue was chromatographed with n-hexane/CHCl3 (6:4 v/v) to
give 7d, followed with n-hexane/CHCl3 (1:1 v/v) to give 6d.
(H3C)3C), 3.14 (t, JPeH ¼ 17.5 Hz, 1H, HCeP2), 4.07, 4.11 (2dq, JHe
3
¼ 7.4, JPeH ¼ 6.5 Hz, 2ꢄ 4H, 4H2COP), 7.39e7.45, 7.81 (m, 6H,
H
HeAr), 8.95, 9.24 (2s (br), 2ꢄ 1H, HN&OH). 13C NMR [CDCl3] ppm:
156.3, 148.2, 146.2, 137.4, 131.5, 126.7, 120.6, 118.6, 115.3, 110.6
(CeAr), 62.4 (d, 2JPeC ¼ 10.5 Hz, CH2OP), 44.8 (t, 1JPeC ¼ 178.3 Hz, Ce
P2), 37, 31.7 (2C(CH3)3), 31.2, 30.4 (2(CH3)3C), 15.9 (d, 3JPeC ¼ 7.5 Hz,
CH3COP). 31P NMR [CDCl3] ppm: 30.3, 31.7 (2d, 2JPeP ¼ 20.6 Hz, P2e
5.5.1. Tetraethyl (5,7-di-tert-butyl-2-(4-nitrophenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl) methylenediphosphonate, 7d
Straw yellow crystals, yield: w5%, mp 164e165 ꢃC (from EtOH). IR
(cmꢁ1, KBr): nmax 3334w (NH),1242,1226 (2P]O, bonded),1056,1044
C). EI-MS: in m/z (%): 610 (33) [Mþ
ꢁ
1], 593 (26)
[Mþ ꢁ 18(H þ OH)], 319 (28) [Mþ ꢁ 292(H þ OHþ2{P(O) (OEt)2)}),
306 (100) [Mþ ꢁ 305(H þ OH þ CH[P(O)(OEt)2]2)], 287 (29) (CH
[P(O) (OEt)2]2), 137 (55) (P(O)(OEt)2), 77 (52). Anal. Calcd for
C30H47NO8P2 (611.6): C, 58.91; H, 7.75; N, 2.29; P, 10.13. Found: C,
59.09; H, 7.79; N, 2.25; P, 10.16.
4
(2PeOeC). 1H NMR [CDCl3] ppm: 1.14, 1.17 (2dt, JHeH ¼ 7.2, JPe
¼ 4.3 Hz, 2ꢄ 6H, 4H3CC$OP), 1.35, 1.41 (2s, 2ꢄ 9H, 2(H3C)3C), 3.26
H
2
3
(t, JPeH ¼ 18.5 Hz, 1H, HCeP2), 3.97, 4.04 (2dq, JHeH ¼ 7.2, JPe
¼ 5.5 Hz, 2ꢄ 4H, 4H2COP), 7.37, 7.69, 8.37, 8.42 (m, 6H, HeAr),
H
9.06 (s(br), 1H, HN). 13C NMR [CDCl3] ppm: 148.3, 146.2, 137.4, 134.7,
132.6, 127.5, 126.4, 118.6, 115.4, 110.3 (CeAr), 61.8 (d, 2JPeC ¼ 10.5 Hz,
5.6.2. Tetraethyl (5,7-di-tert-butyl-2-(4-chlorophenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl)methylene-diphosphonate, 7f
1
CH2OP), 42.6 (t, JPeC ¼ 182.6 Hz, CeP2), 37, 31.7 (2C(CH3)3), 31.2,
3
30.4 (2(CH3)3C), 15.4 (d, JPeC ¼ 7.8 Hz, CH3COP). 31P NMR [CDCl3]
Colorless needles, yield: 82%, mp 102e104 ꢃC (from cyclo-
hexane). IR (cmꢁ1, KBr): nmax 3350w (NH), 1236, 1223 (2P]O,
bonded), 1055, 1029 (2PeOeC). 1H NMR [CDCl3] ppm: 0.88, 1.25
(2dt, JHeH ¼ 6.2, 4JPeH ¼ 3.8 Hz, 2ꢄ 6H, 4H3CC$OP), 1.46, 1.53 (s, 2ꢄ
ppm: 25.6, 26.5 (2d, 2JPeP ¼ 24.5 Hz, P2eC). EI-MS: in m/z (%): 639 (27)
[Mþ ꢁ1], 593(68)[Mþ ꢁ47(H þ NO2],319(28)[Mþ ꢁ321(HþNO2þ2
{P(O)(OEt)2)}), 306 (100) [Mþ ꢁ 334(H þ NO2þCH[P(O)(OEt)2]2)], 287
(44) (CH[P(O)(OEt)2]2), 137 (36) (P(O)(OEt)2), 77 (82). Anal. Calcd for
C30H46N2O9P2 (640.6): C, 56.24; H, 7.24; N, 4.37; P, 9.67. Found: C,
56.20; H, 7.28; N, 4.32; P, 9.73.
2
9H, (H3C)3C), 3.32 (t, JPeH ¼ 20.4 Hz, 1H, HCeP2), 4.29, 4.42 (2dq,
JHeH ¼ 6.2, 3JPeH ¼ 6.1 Hz, 2ꢄ 4H, 4H2COP), 7.31e7.70, 8.17 (m, 6H,
HeAr), 8.96 (s (br), 1H, HN). 13C NMR [CDCl3] ppm: 148.1, 146.2,
139.6, 139.2, 137.4, 134.8, 128, 118.6, 115.4, 110.6 (CeAr), 61.6 (d, 2JPe
1
5.5.2. Tetraethyl 2-(3,5-di-tert-butyl-2-hydroxyphenylamino)-2-
(4-nitrophenyl)ethane-1,1-diyldiphosphonate, 6d
¼ 10.5 Hz, CH2OP), 40.7 (t, JPeC ¼ 144.3 Hz, CeP2), 37, 31.7
C
3
(2 C(CH3)3), 31.2, 30.4 (2(CH3)3C), 16.4 (d, JPeC ¼ 8.2 Hz, CH3COP).
Straw yellow crystals, yield: 70%, mp 160e166 ꢃC (from MeCN). IR
(cmꢁ1, KBr):nmax 3445,3331w (OH&NH),1238,1222(2P]O, bonded),
1101,1064 (2PeOeC).1H NMR [CDCl3] ppm: 1.16,1.24 (2dt, JHeH ¼ 6.6,
31P NMR [CDCl3] ppm: 27.4, 30.6 (2d, 2JPeP ¼ 22.8 Hz, P2eC). EI-MS:
in m/z (%): 631(21) [Mþþ2], 629 (7) [Mþ], 628 (32) [Mþ ꢁ 1], 593
(26) [Mþ ꢁ 36 (H þ Cl)], 319 (28) [Mþ ꢁ 310(H þ Cl þ 2{P(O)

370
W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
(OEt)2)}), 306 (100) [Mþ ꢁ 323(H þ Cl þ CH [P(O)(OEt)2]2)], 287
(31) (CH[P(O) (OEt)2]2), 137 (68) (P(O)(OEt)2), 77 (67). Anal. Calcd
for C30H46ClNO7P2 (630.1): C, 57.19; H, 7.36; Cl, 5.63; N, 2.22; P, 9.83.
Found: C, 57.24; H, 7.30; Cl, 5.58; N, 2.27; P, 9.87.
z8 h (TLC). After concentrating the product mixture to its half,
under reduced pressure, the crude material was diluted with AcOEt
and water and then stirred for 30 min. The layers were separated,
and the aqueous layer was evaporated to dryness. The precipitate
was collected and dried to give the corresponding BP-acids 9a,b
and 10aee.
5.6.3. Tetraethyl (5,7-di-tert-butyl-2-(3-chlorophenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl)methylene-diphosphonate, 7g
Colorless crystals, yield: 78%, mp 88e90 ꢃC (from pentane). IR
(cmꢁ1, KBr): nmax 3350w (NH), 1240, 1224 (2P]O, bonded), 1164,
1079 (2PeOeC). 1H NMR [CDCl3] ppm: 0.98, 1.17 (2dt, JHeH ¼ 7.2,
4JPeH ¼ 4.3 Hz, 2ꢄ 6H, 4H3CCOP), 1.38, 1.54 (2s, 2ꢄ 9H, (H3C)3C),
3.25 (t, 2JPeH ¼ 18.8 Hz, 1H, HCeP2), 4.05, 4.12 (2dq, JHeH ¼ 7.2, 3JPe
5.7.1. 2-(3,5-Di-tert-butyl-2-hydroxyphenylamino)-2-(4-dimethyl-
aminophenyl)ethane-1,1-diyldiphosphonic acid, 9a
White substance, yield: 67%, mp > 300 ꢃC (from EtOH). IR (cmꢁ1
,
KBr): nmax 3420e3332 (PeOH, NH), 1233e1219 (P]O, bonded). 1H
NMR [D2O] ppm: 1.37, 1.40 (2s, 2ꢄ 9H, 2(H3C)3C), 2.44 (dt, JHe
2
¼ 6.4 Hz, 2ꢄ 4H, 4H2COP), 7.34e7.67, 8.19e8.23 (m, 6H, HeAr),
¼ 11.4, JPeH ¼ 15.3 Hz, 1H, HaCeP2), 2.82 (s, 6H, 2(H3C)2N),
H
H
9.06 (s (br), 1H, HN). 13C NMR [CDCl3] ppm: 147.9, 146.2, 142.3,
5.26 (dt, JHeH ¼ 11.4, 3JPeH ¼ 7.2 Hz, 1H, HbeC), 7.16e7.78, 8.23 (m,
6H, HeAr). 31P NMR [D2O] ppm: 21.2, 22.7. EI-MS: in m/z (%): 522
(24) [Mþ ꢁ 6]. Anal. Calcd for C24H38N2O7P2 (528.5): C, 54.54; H,
7.25. Found: C, 54.50; H, 7.18.
2
142.2, 137.8, 134.9, 127.2, 125.6, 115.6, 109.7 (CeAr), 61.7 (d, JPe
1
¼ 10.5 Hz, CH2OP), 44.0 (t, JPeC ¼ 142.3 Hz, CeP2), 37, 31.7
C
3
(2C(CH3)3), 31.2, 30.4 (2(CH3)3C), 15.4 (d, JPeC ¼ 7.5 Hz, CH3COP).
31P NMR [CDCl3] ppm: 26.8, 30.8 (2d, 2JPeP ¼ 28.4 Hz, P2eC). EI-MS:
in m/z (%): 631(18) [Mþ þ 2], 629 (6) [Mþ], 628 (35) [Mþ ꢁ 1], 306
(100) [Mþ ꢁ 323 (H þ Cl þ CH[P(O)(OEt)2]2)]. Anal. Calcd for
C30H46ClNO7P2 (630.1): C, 57.19; H, 7.36; Cl, 5.63; N, 2.22; P, 9.83.
Found: C, 57.25; H, 7.32; Cl, 5.67; N, 2.17; P, 9.88.
5.7.2. 2-(3,5-Di-tert-butyl-2-hydroxyphenylamino)-2-(4-fluoroph-
enyl)ethane-1,1-diyldiphosphonic acid, 9b
White substance, yield: 70%, mp >300 ꢃC (from EtOH), IR: nmax,
cmꢁ1: 3420e3332 (PeOH, NH), 1235e1222 (P]O, bonded). 1H
NMR [D2O] ppm: 1.35, 1.38 (2s, 2ꢄ 9H, 2(H3C)3C), 2.48 (dt, JHe
2
3
5.6.4. Tetraethyl (5,7-di-tert-butyl-2-(2-chlorophenyl)-2,3-dihydro-
benzo[d]oxazol-2-yl)methylenediphosphonate, 7h
¼ 11.4, JPeH ¼ 18.8 Hz, 1H, HaCeP2), 5.20 (dt, JHeH ¼ 11.4, JPe
H
¼ 5.9 Hz, 1H, HbeC), 7.14e7.77, 8.20 (m, 6H, HeAr). 31P NMR
H
Colorless crystals, yield: 76%, mp 113e115 ꢃC (from pentane). IR
(cmꢁ1, KBr): nmax 3355w (NH), 1235, 1225 (2P]O, bonded), 1164,
1079 (2PeOeC). 1H NMR [CDCl3] ppm: 0.89, 1.05 (2dt, JHeH ¼ 6.8,
4JPeH ¼ 3.8 Hz, 2ꢄ 6H, 4H3CCOP), 1.38, 1.53 (2s, 2ꢄ 9H, 2(H3C)3C),
3.47 (t, 2JPeH ¼ 18.8 Hz, 1H, HCeP2), 4.12, 4.20 (2dq, JHeH ¼ 6.8, 3JPe
[D2O] ppm: 22.2, 24.7. EI-MS: in m/z (%): 497 (30) [Mþ ꢁ 6]. Anal.
Calcd for C22H32FNO7P2 (503.4): C, 52.49; H, 6.41. Found: C, 52.44;
H, 6.45.
5.7.3. (5,7-di-tert-butyl-2-(4-(dimethylamino)phenyl)-2,3-dihyd-
¼ 6.7 Hz, 2ꢄ 4H, 4H2COP), 6.99e7.67, 8.15e8.24 (m, 6H, HeAr),
robenzo[d]oxazol-2-yl)methylene-diphosphonic acid, 10a
H
9.07 (s (br), 1H, HN). 13C NMR [CDCl3] ppm: 148.1, 146.2, 137.1,
White substance, yield: 65%, mp >300 ꢃC (from EtOH). IR (cmꢁ1
,
2
134.2, 130.3, 128.4, 127.7, 118.4, 115.2, 110.3 (CeAr), 62.1 (d, JPe
KBr): nmax 3415e3329 (PeOH, NH), 1233e1220 (P]O, bonded). 1H
1
2
¼ 14.5 Hz, CH2OP), 39.2 (t, JPeC ¼ 158.3 Hz, CeP2), 37, 31.7
NMR [D2O] ppm: 1.38, 1.52 (2s, 2ꢄ 9H, 2(H3C)3C), 2.40 (t, JPe
C
(2 C(CH3)3), 31.2, 30.4 (2(CH3)3C), 16.4 (d, 3JPeC ¼ 8.7 Hz, CH3COP).
31P NMR [CDCl3] ppm: 29.5, 31.4 (2d, 2JPeP ¼ 26.3 Hz, P2eC). EI-MS:
in m/z (%): 631 (24) [Mþþ2], 629 (8) [Mþ], 628 (36) [Mþ ꢁ 1], 306
(100) [Mþ ꢁ 323(H þ Cl þ CH[P(O)(OEt)2]2)]. Anal. Calcd for
C30H46ClNO7P2 (630.1): C, 57.19; H, 7.36; Cl, 5.63; N, 2.22; P, 9.83.
Found: C, 57.13; H, 7.29; Cl, 5.56; N, 2.26; P, 9.90.
¼ 16.4 Hz, 1H, HCeP2), 2.80 (s, 6H, 2(H3C)2N), 7.14e7.72, 8.10
H
(m, 6H, HeAr). 31P NMR [D2O] ppm: 21.2, 23.5. EI-MS: in m/z (%):
520 (40) [Mþ ꢁ 6]. Anal. Calcd C24H36N2O7P2 (526.5): C, 54.75; H,
6.89. Found: C, 54.71; H, 6.84.
5.7.4. (5,7-Di-tert-butyl-2-(4-fluorophenyl)-2,3-dihydrobenzo[d]
oxazol-2-yl)methylenediphosphonic acid, 10b
5.6.5. Tetraethyl (5,7-di-tert-butyl-2-(2,5-dimethoxyphenyl)-2,3-
dihydrobenzo[d]oxazol-2-yl)methylenediphosphonate, 7i
White substance, yield: 73%, mp >300 ꢃC (from EtOH). IR (cmꢁ1
,
KBr): nmax 3424e3335 (PeOH, NH), 1230e1225 (P]O). 1H NMR
[D2O] ppm: 1.36, 1.57 (2s, 2ꢄ 9H, 2(H3C)3C), 2.50 (t, 2JPeH ¼ 18.8 Hz,
1H, HCeP2), 7.18e7.68, 8.15 (m, 6H, HeAr). 31P NMR [D2O] ppm:
23.2, 25.6. EI-MS: in m/z (%): 495 (32) [Mþ ꢁ 6]. Anal. Calcd for
C22H30FNO7P2 (501.4): C, 52.70; H, 6.03. Found: C, 52.75; H, 6.81.
Colorless crystals, yield: 79%, mp 128e130 ꢃC (from CH2Cl2). IR
(cmꢁ1, KBr): nmax 3342w (NH),1235,1224 (2P]O, bonded),1078,1045
4
(2PeOeC). 1H NMR [CDCl3] ppm: 1.13, 1.18 (2dt, JHeH ¼ 6.8, JPe
¼ 4.6 Hz, 2ꢄ 6H, 4H3CC$OP), 1.35, 1.40 (2s, 2ꢄ 9H, 2(H3C)3C), 3.22
H
(t, 2JPeH ¼ 16.5 Hz, 1H, HCeP2), 3.75, 3.81(2s, 2ꢄ 3H, 2H3CO), 4.14, 4.20
(2dq, JHeH ¼ 6.8, 3JPeH ¼ 5.8 Hz, 2ꢄ 4H, 4H2COP), 7.23e7.88, 8.34 (m,
5H, HeAr), 9.81 (s (br), 1H, HN). 13C NMR [CDCl3] ppm: 159.4, 149.9,
146.2, 137.3, 136.4, 123.1, 118.5, 115.7, 112.3, 108.9 (CeAr), 61.2 (d, 2JPe
5.7.5. (5,7-Di-tert-butyl-2-(4-chlorophenyl)-2,3-dihydrobenzo[d]
oxazol-2-yl)methylenediphosphonic acid, 10c
White substance, yield: 66%, mp >300 ꢃC (from EtOH). IR (cmꢁ1
,
¼ 10.5 Hz, CH2OP), 55.8, 55.3 (2CH3O), 39.8 (t, 1JPeC ¼ 152.3 Hz, Ce
KBr): nmax 3425e3336 (PeOH, NH), 1233e1220 (P]O). 1H NMR
[D2O] ppm: 1.35, 1.40 (2s, 2ꢄ 9H, 2(H3C)3C), 2.44 (t, 2JPeH ¼ 17.2 Hz,
1H, HCeP2), 7.20e7.60, 8.12 (m, 6H, HeAr). 31P NMR [D2O] ppm:
24.2, 26.4. EI-MS: in m/z (%): 511 (45) [Mþ ꢁ 6]. Anal. Calcd for
C22H30ClNO7P2 (517.9): C, 51.02; H, 5.84. Found: C, 51.07; H, 5.59.
C
3
P2), 37.2, 31.7 (2C(CH3)3), 31.2, 30.4 (2(CH3)3C), 15.6 (d, JPe
2
¼ 8.2 Hz, CH3COP). 31P NMR [CDCl3] ppm: 31.3, 26.4 (2d, JPe
C
¼ 30.6 Hz, P2eC). EI-MS: in m/z (%): 654 (48) [Mþ ꢁ 1], 592 (26)
P
[Mþ ꢁ 63(H þ 2{OMe})], 318 (28) [Mþ ꢁ 337(H þ 2{OMe} þ 2
{P(O)(OEt)2)}), 305 (100) [Mþ
[P(O)(OEt)2]2)], 287 (55) (CH[P(O)(OEt)2]2), 137 (34) (P(O)(OEt)2), 77
(84). Anal. Calcd for C32H51NO9P2 (655.7): C, 58.62; H, 7.84; N, 2.14; P,
9.45. Found: C, 58.67; H, 7.83; N, 2.08; P, 9.51.
ꢁ
350(H
þ
2{OMe})
þ
CHe
5.7.6. (5,7-Di-tert-butyl-2-(3-chlorophenyl)-2,3-dihydrobenzo[d]
oxazol-2-yl)methylenediphosphonic acid, 10d
White substance, yield: 72%, mp >300 ꢃC (from EtOH). IR (cmꢁ1
,
KBr): nmax 3426e3328 (PeOH, NH), 1236e1219 (P]O). 1H NMR
[D2O] ppm: 1.32, 1.40 (2s, 2ꢄ 9H, 2(H3C)3C), 2.38 (t, 2JPeH ¼ 15.4 Hz,
1H, HCeP2), 7.18e7.50, 8.10 (m, 6H, HeAr). 31P NMR [D2O] ppm:
22.2, 24.6. EI-MS: in m/z (%): 511 (35) [Mþ ꢁ 6]. Anal. Calcd for
C22H30ClNO7P2 (517.9): C, 51.02; H, 5.84. Found: C, 51.06; H, 5.82.
5.7. General procedure of BP-acids 9a,b, and 10aee
Bisphosphonate 6b,c, 7b,c, and 7feh (0.5 g) was dissolved in
15 mL of conc HCl, and the mixture was heated under reflux for

W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
371
5.7.7. (5,7-Di-tert-butyl-2-(3-chlorophenyl)-2,3-dihydrobenzo[d]
oxazol-2-yl)methylenediphosphonic acid, 10e
at the time of the tested compound addition (time zero, Tz).
Experimental tested compounds are solubilized in dimethyl
sulfoxide at 400-fold the desired final maximum test concentra-
tion and stored frozen prior to use. At the time of the tested
compound addition, an aliquot of frozen concentrate is thawed
and diluted to twice the desired final maximum test concentra-
tion with complete medium containing 50 mg/mL gentamicine.
Additional four, 10-fold or ½ log serial dilutions are made to
provide a total of five tested compound concentrations (10ꢁ4 to
10ꢁ8 M concentrations) plus control. Aliquots of 100 mL of these
different tested compound dilutions are added to the appropriate
White substance, yield: 72%, mp >300 ꢃC (from EtOH). IR (cmꢁ1
KBr): nmax 3426e3327 (PeOH, NH), 1230e1220 (P]O). 1H NMR
[D2O] ppm: 1.37, 1.53 (2s, 2ꢄ 9H, 2(H3C)3C), 2.42 (t, 2JPeH ¼ 15.6 Hz,
1H, HCeP2), 6.80e7.40, 8.10 (m, 6H, HeAr). 31P NMR [D2O] ppm:
24.5, 26.6. EI-MS: in m/z (%): 511 (42) [Mþ ꢁ 6]. Anal. Calcd for
C22H30ClNO7P2 (517.9): C, 51.02; H, 5.84. Found: C, 51.04; H, 5.80.
,
5.8. Bioassays
5.8.1. Lipophilicity HPLC determination (capacity factor K/
calculated log K)
microtiter wells already containing 100 mL of medium, resulting
in the required final concentrations.
The HPLC separation module Waters Alliance 2695 XE and
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, U.S.A.) were used. The Chromatographic Column SymmetryÒ
Following the tested compound addition, the plates are incu-
bated for an additional 48 h at 37 ꢃC, 5% CO2, 95% air and 100%
relative humidity. For adherent cells, the assay is terminated by
the addition of cold TCA. Cells are fixed in situ by the gentle
C18
5
m
m, 4.6 ꢄ 250 mm, Part No. WAT054275, (Waters Corp.,
Milford, MA, U.S.A.) was used. The HPLC separation process was
monitored by Millennium32Ò Chromatography Manager Software,
Waters 2004 (Waters Corp., Milford, MA, U.S.A.). The mixture of
MeOH p.a. (90.0%) and H2O-HPLC e Mili-Q Grade (10.0%) was used
as a mobile phase. The total flow of the column was 1.0 ml/min,
addition of 50 mL of cold 50% (w/v) TCA (final concentration, 10%
TCA) and incubated for 60 min at 4 ꢃC. The supernatant is dis-
carded, and the plates are washed four times with tap water and
air dried. Sulforhodamine B (SRB) solution (100 mL) at 0.4% (w/v)
in 1% acetic acid is added to each well, and plates are incubated for
10 min at room temperature. After staining, unbound dye is
removed by washing four times with 1% acetic acid and the plates
injection 30 m
l, column temperature 45 ꢃC and sample temperature
10 ꢃC. The detection wavelength 210 nm was chosen. The KI
methanolic solution was used for the dead time (TD) determination.
Retention times (TR) were measured in minutes. The capacity
factors K were calculated using the Millennium32Ò Chromatog-
raphy Manager Software according to the formula K ¼ (TR ꢁ TD)/TD,
where TR is the retention time of the solute, whereas TD denotes the
dead time obtained via an unretained analyte. The log K values of
the individual compounds, calculated from the capacity factor K,
are shown in Table 1. All values displayed as mean ꢂ s.d. Superscript
letters denote equivalent means determined by pairwise compar-
ison (Dunnett T3, p < 0.05).
are air dried. Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated plate
read at a wavelength of 515 nm. For cells suspension, the meth-
odology is the same except that the assay is terminated by fixing
settled cells at the bottom of the wells by gently adding 50 mL of
80% TCA (final concentration, 16% TCA). Using the seven absor-
bance measurements (Tz), control growth (C) and test growth in
the presence of the tested compound at the five concentration
levels (Ti), the percentage growth is calculated at each of the
tested compound concentration levels.
Percentage growth inhibition is calculated as follows:
5.8.2. Lipophilicity calculations
Converting the logarithm of the partition coefficient for n-
octanol/water (log K) to the lipophilcity coefficient (Log P) was
calculated using the programs CS ChemOffice Ultra ver. 10.0
(CambridgeSoft, Cambridge, MA, USA) and ACD/LogP ver. 12.0
(Advanced Chemistry Development Inc., Toronto, Canada). Clog P
values (the logarithm of n-octanol/water partition coefficient based
on established chemical interactions) were generated by means of
CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA,
U.S.A.) software. The results are shown in Table 1.
½ðTi ꢁ TzÞ=ðC ꢁ TzÞꢅ ꢄ 100
for concentrations for which Ti ꢆ Tz.
[(Ti ꢁ Tz)/Tz)] ꢄ 100 for concentration for which Ti < Tz.
Growth inhibition of 50% (GI50) is calculated from:
[(Ti ꢁ Tz)/[(C ꢁ Tz) ꢄ 100 ¼ 50% (GI50), which is the tested
compound concentration resulting in a 50% reduction in the net
protein increase (as measured in SRB staining) in control cells
during the compound incubation. Table 2 represents the observed
percentage growth of each cell line treated with a certain tested
compound relative to control cell line experiments.
5.9. Pharmacology
5.9.1. Antitumor activity screening
Anti-tumor potency of the new BPs 6aed, 7aei, and BP-acids
9a,b, and 10aee, in addition to the substrates 4a and 4g was
tested at a dose of 10 mM utilizing 16 different human tumor cell
5.9.2. Delayed type hypersensitivity granuloma
Groups of 10 female albino mice (25 g) were sensitized with an
emulsion of methylated bovine serum albumin (mBSA) in saline
with Freund’s incomplete adjuvant and dextran by sc injection over
the inguinal lymph node. Three weeks later, hydroxyapatite (HA)
discs 6-mm diameter) soaked in mBSA solution (30 mg/mL saline)
were implanted sc in the dorsum of the mice (two discs, bilaterally).
All drugs were prepared as solutions, suspensions, or emulsions,
and the pH was adjusted to 7.4 with 0.1 M NaOH. Each mouse
received compound in a volume of 0.1 mL/10 g body weight sc in
the scruff of the neck. Dosing commenced on the day of implan-
tation of the mBSA soaked discs and was continued thereafter on
a daily basis until day nine, when the mice were euthanized. The
granulomatous lesions were then excised and both wet and dry
tissue weights measured. Results were analyzed by Student’s
paired t test. The results are displayed in Table 3.
lines, representing breast, ovarian, prostate and liver cancer using
adriamycin as a reference standard according to the reported
methods [24,25]. The human tumor cell lines of the cancer
screening panel are grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mL-glutamine. For a typical screening
experiment, cells are inoculated in 96-well-microtiter plates in
100 mL at plating densities ranging from 5000 to 40,000 cells/
well depending on the doubling time individual cell lines. After
cell inoculation, the microtiter plates are incubated at 37 ꢃC, 5%
CO2, 95% air and 100% relative humidity for 24 h prior to addition
of experimental tested compounds. After 24 h, two plates of each
cell lines are fixed in situ with trichloroacetic acid (TCA), to
represent a measurement of the cell population for each cell line

372
W.M. Abdou et al. / European Journal of Medicinal Chemistry 57 (2012) 362e372
[10] A. Lipton, R.L. Theriault, G.N. Hortobagyi, J. Simeone, R.D. Knight, K. Mellars,
The antiinflammatory activity was expressed as percentage
D.J. Reitsma, M. Heffernan, J. Seaman, Pamidronate prevents skeletal
complications and is effective palliative treatment in women with breast
carcinoma and osteolytic bone metastases, J. Cancer 88 (2000) 1082e1090.
[11] T. Yoneda, A. Sasaki, C. Dustan, P.J. William, F. Bauss, Y.A. De Clerck,
G.R. Mundy, Inhibition of osteolytic bone metastasis of breast cancer by
combined treatment with the bisphosphonate ibandronate and tissue inhib-
itor of the matrix metalloproteinase-2, J. Clin. Invest. 99 (1997) 2509e2517.
[12] M. Busch, M. Rave-Frank, A. Hille, E. Duhmke, Influence of clodronate on
breast cancer cells in vitro, Eur. J. Med. Res. 3 (1998) 427e431.
[13] J.E. Brown, E.C. Robert, The role of bisphosphonates in breast cancer: the
present and future role of bisphosphonates in the management of patients
with breast cancer, Breast Cancer Res. 4 (2002) 24e29.
inhibition of edema volume in the treated animals in comparison
with the control group.
ðVc ꢁ VtÞ
% Inhibition of edema ¼
ꢄ 100
Vc
where Vc and Vt are the volumes of edema for the control and tested
substance-treated animal groups, respectively, while potency of the
tested compounds was calculated regarding risedronate, reference
standard, treated group according to the following equation:
[14] R.A. Baron, R. Tavaré, A.C. Figueiredo, K.M. Baewska, B.A. Kashemirov,
C.E. McKenna, F.H. Ebetino, A. Taylor, M.J. Rogers, F.P. Coxon, M.C. Seabra,
Phosphono-carboxylates inhibit the second geranylgeranyl addition by rab
geranylgeranyl transferase, Biol. Chem. 284 (2009) 6861e6868.
[15] L.I. Plotkin, J.I. Aguirre, S. Kousteni, S.C. Manolagas, T. Bellido, Bisphosphonates
and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms
downstream of extracellular signal-regulated kinase activation, J. Biol. Chem.
280 (2005) 7317e7325.
%Potency
%Edemainhibitionof testedcompoundtreatedgroup
¼
ꢄ100
%Edemainhibitionof risedronatetreatedgroup
[16] W.M. Abdou, R.E. Khidre, A.A. Kamel, Elaborating on efficient anti-
proliferation agents of cancer cells and anti-inflammatory-based on N-
bisphosphonic acids, Arch. Pharm. Chem. Life Sci. 345 (2012) 123e136.
[17] W.M. Abdou, A.A. Kamel, R.E. Khidre, A. Geronikaki, M.T. Ekonomopoulou,
Synthesis of 5- and 6-N- heterocyclic methylenebisphosphonate derivatives of
cytogenetic activity in normal human lymphocyte cultures, Chem. Biol. Drug
Des. 79 (2012) 719e730.
[18] A.A. Kamel, W.M. Abdou, A. Geronikaki, Inhibitory effect of novel S, N-
bisphosphonates on some carcinoma cell lines, osteoarthritis, and chronic
inflammation, Eur. J. Med. Chem. 51 (2012) 239e249.
[19] T. Hartmann, J. Schmitt, Liphilicity-beyond octanol/water: a comparison of
modern technologies, Drug Disc. Today Technol. 1 (2004) 431e439.
[20] P. Wils, A. Warnery, V. Phung-Ba, S. Legrain, D. Scherman, High lipophilicity
decreases drug transport across intestinal epithelial cells, J. Pharmacol. Exp.
Ther. 269 (1994) 654e658.
Acknowledgments
Authors like to thank the Central Lab, School of Pharmacy,
Alexandria University, Egypt, and NY Cancer Institute, NY, USA for
carrying out the pharmacological screening.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.032.
[21] J. Vinsová, K. Cermáková, A. Tomecková, M. Cecková, J. Jampílek, P. Cermák,
J. Kunes, M. Dolezal, F. Staud, Synthesis and antimicrobial evaluation of new 2-
substituted 5,7-di-tert-butylbenzoxazoles, Bioorg. Med. Chem. 14 (2006)
5850e5865.
[22] J. Jampilek, J. Vinsova, J. Kaderavkova, in: J.A. Seijas, V.T.M. Pilar (Eds.), From
Proceedings of the International Electronic Conference on Synthetic Organic
Chemistry, 10th, November 1e30 (2006), a003/10.
[23] D. Walker, J.D. Herbert, 2,3-Dichloro-5,6-dicyanobenzoqinone and its reac-
tions, Chem. Rev. 67 (1966) 153e195.
[24] T. Kitamura, M. Itoh, T. Noda, M. Matsuura, K. Wakabayashi, Combined effects
of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal
tumorigenesis in adenomatous polyposis coli gene knockout mice, Int. J.
Cancer 109 (2004) 576e580.
References
[1] H. Fleisch, Bisphosphonates: a new class of drugs in diseases of bone and
calcium metabolism, in: P.F. Baker (Ed.), In Handbook of Experimental Phar-
macology, vol. 83, Springer, Berlin/Heidelberg, 1988, pp. 441e465.
[2] H. Fleisch, Bisphosphonates in Bone Disease: From the Laboratory to the
Patient, The Parathenone Publishing Group Inc, New York, 1995.
[3] A.J. Yates, G.A. Rodan, Alendronate and osteoporosis, Drug Discov. Today 3
(1998) 69e78.
[4] T. Anada, Y. Takeda, Y. Honda, K. Sakurai, O. Suzuki, Synthesis of calcium
phosphate-binding liposome for drug delivery, Bioorg. Med. Chem. Lett. 19
(2009) 4148e4150.
[25] M.R. Boyd, K.D. Paull, Some practical considerations and applications of the
National Cancer Institute in vitro anticancer drug discovery screen, Drug Dev.
Res. 34 (1995) 91e109.
[26] R.A. Nugent, M. Murphy, S.T. Schlachter, C.J. Dunn, R.J. Smith, N.D. Staite,
L.A. Galinet, S.K. Shields, D.G. Aspar, K.A. Richard, N.A. Rohloff, Pyrazoline
bisphosphonate esters as novel antiinflammatory and antiarthritic agents,
J. Med. Chem. 36 (1993) 134e139.
[5] W.K. Siestema, F.H. Ebetino, Bisphosphonates in development for metabolic
bone disease, Expert Opin. Investig. Drugs 3 (1994) 1255e1276.
[6] S.A. Gittens, G. Bansal, R.F. Zernicke, H. Uludag, Designing proteins for bone
targeting, Adv. Drug Deliv. Rev. 57 (2005) 1011e1036.
[7] H. Hirabayashi, J. Fujisaki, Phosphonated rifamycins and uses thereof for the
prevention and treatment of bone and joint infections, Clin. Pharmacokinet.
42 (2003) 1319e1330.
[27] C.J. Dunn, L.A. Galinet, A.J. Gibbons, S.K. Shields, Murine delayed type hyper-
sensitivity granuloma: an improved model for the identification and evalua-
tion of different classes of antiarthritic drugs, Int. J. Immunopharmacol. 12
(1990) 899e904.
[8] D. Wang, S.C. Miller, P. Kopeckova, J. Kopecek, Bone-targeted macromolecular
therapeutics, Adv. Drug Deliv. Rev. 57 (2005) 1049e1076.
[9] T.V. den Wyngaert, M.T. Huizing, E. Fossion, J.B. Vermorken, Bisphosphonates
in oncology: rising stars or fallen heroes, The Oncologist 14 (2009) 181e191.