Elaborating on efficient Anti-proliferation agents of cancer cells and Anti-inflammatory-based N-bisphosphonic acids

Article abstract of DOI:10.1002/ardp.201100080

Methylenebisphosponic acid tetraethyl ester (1) was added to 2-azido-7a-e and 2-chloroquinoline-3-chalcones 10a-e in boiling sodium ethanolate solution to give, via Michael addition, tetrazolo[1,5-a]quinoline-8a-d, 13a and 2-chloroquinoline-based bisphosphonates 11a-d, 14a in E-configuration. Further acid hydrolysis afforded the respective BP-acid analogues E-9a-d, 12a-d, 13b, and 14b in excellent yields. Anti-tumor activity screening for the new BP-acids at a dose of 10μM utilizing 44 different human tumor cell lines representing breast, ovary, prostate, lung, and CNS cancer as well as leukemia and melanoma was carried out. Eight of ten tested compounds exhibited remarkable anti-tumor activity against breast and prostate cancer, and a promising anti-tumor sensitivity toward ovarian cancer and melanoma. Conversely, there was only scattered activity against leukemia and no noticeable action of these BP-acids on CNS or lung cancer. Based on the prediction results (PASS program), anti-inflammatory activity of the new acids was also determined in vivo, by the acute carrageenin induced paw edema in rats. Many of these compounds showed anti-inflammatory properties at a dose of 50mg/kg body weight. New bisphosphonates and bisphosphonic acids were synthesized and screened for anti-tumor activity. The results indicate that this moiety is an essential factor in developing the total pharmacological properties for the synthesized compounds whereas the type of the substituents attached to the quinoline nucleus determines and controls the potency.

Full text of DOI:10.1002/ardp.201100080

Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
123
Full Paper
Elaborating on Efficient Anti-Proliferation Agents of Cancer
Cells and Anti-Inflammatory-Based N-Bisphosphonic Acids
Wafaa M. Abdou, Rizk E. Khidre, and Azza A. Kamel
Chemical Industries Division, National Research Centre, Cairo, Egypt
Methylenebisphosponic acid tetraethyl ester (1) was added to 2-azido-7a–e and 2-chloroquinoline-3-
chalcones 10a–e in boiling sodium ethanolate solution to give, via Michael addition, tetrazolo[1,5-
a]quinoline-8a–d, 13a and 2-chloroquinoline-based bisphosphonates 11a–d, 14a in E-configuration.
Further acid hydrolysis afforded the respective BP-acid analogues E-9a–d, 12a–d, 13b, and 14b in
excellent yields. Anti-tumor activity screening for the new BP-acids at a dose of 10 mM utilizing 44
different human tumor cell lines representing breast, ovary, prostate, lung, and CNS cancer as well as
leukemia and melanoma was carried out. Eight of ten tested compounds exhibited remarkable anti-
tumor activity against breast and prostate cancer, and a promising anti-tumor sensitivity toward
ovarian cancer and melanoma. Conversely, there was only scattered activity against leukemia and no
noticeable action of these BP-acids on CNS or lung cancer. Based on the prediction results (PASS
program), anti-inflammatory activity of the new acids was also determined in vivo, by the acute
carrageenin induced paw edema in rats. Many of these compounds showed anti-inflammatory
properties at a dose of 50 mg/kg body weight.
Keywords: Anti-inflammatory activity / Anti-tumor screening / Bisphosphonates / Chalcones / Tetrazoloquinolines
Received: March 1, 2011; Revised: April 19, 2011; Accepted: April 29, 2011
DOI 10.1002/ardp.201100080
Introduction
doses (ꢀ10 mg clinically). Over the last decade [6], there was
significant progress in elucidating the mechanism of action
of N-BPs. For all N-BPs, the molecular target is the isoprenoid
biosynthetic enzyme, farnesyl diphosphate synthase, in the
cholesterol biosynthesis pathway. Although inhibition of this
enzyme by N-BPs results in the suppression of sterol biosyn-
thesis, it is actually disruption of a branch pathway, isopre-
nylation, that is responsible for N-BP pharmacological
activity. Isoprenylation involves covalent linkage of the 15
or 20 carbon isoprene moiety farnesyl diphosphate or ger-
anyl-geranyl diphosphate, respectively, to the carboxy-termi-
nus of regulatory proteins, including the small GTPases Ras,
Rac, Rho and Cdc42. The latter three, as well as numerous
others, are geranyl-geranylated and play a rate-limiting role
in the activity of the bone-resorbing osteoclast. This targeted
osteoclast inhibition accounts for the potency of the N-BPs
and for their ability to elicit the desired therapeutic response
of suppressing bone turnover.
In addition to inhibiting bone resorption, bisphosphonates
(BPs) have also been shown to exhibit anti-tumor effects.
In vitro, BPs inhibit proliferation and induce apoptosis in
cultured human breast cancer cells [1–4]. There is now a
much greater appreciation of the benefits of introduction
of BPs in oncology, which has dramatically changed the
management of patients with metastatic bone disease.
Recently, BPs have been, however, proven to be the drugs
of choice to: i) decrease in bone resorption in tumor bone
disease; ii) decrease in hypercalcemia as a result of dimin-
ution of bone resorption that leads to a big decrease of new
osteolytic lesions; and iii) decrease of fractures, which results
in amelioration of pain and an improvement of the quality of
life [5]. The nitrogen-containing BPs (N-BPs) are the latest and
most potent addition to this family of compounds and have
the widest use. They have high potency, are specifically tar-
geted to the osteoclasts on bone and are used at very low
Our contribution to this field of BPs that started in 2002
included synthesis of a series of BPs for the treatment of bone
resorption disorder [7–14]. Indeed, all BPs have significant
potency on bone resorption. We have also utilized the high
bone/joint specificity of this class of compounds together
Correspondence: Wafaa M. Abdou, Chemical Industries Division,
National Research Centre, Elbohouth Street, D-12622, Dokki, Cairo, Egypt
E-mail: wabdou@link.net
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W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
with other chemical moieties of potential anti-catabolic
pharmacology in the design of N/S-bisphosphonic acids as
anti-inflammatory and anti-arthritic agents [7–9, 12]. Several
of the newly synthesized BP-acids have shown in vivo to be
potent agents in rat adjuvant model of inflammation.
As a sequel, the present work aimed at investigating the
synthesis of novel quinoline-based bisphosphonate deriva-
tives utilizing accessible starting materials and facile syn-
thetic approaches. Furthermore, the prospective potency of
the products for treating cancer and inflammatory diseases
was investigated. The computer-assisted molecular modeling
(CAMM), PASS program, was adopted for designing, in silico,
the structures of potentially active molecules for future syn-
thesis. Later on, in vitro the anti-tumor activity screening; and
the in-vivo anti-inflammatory properties of the synthesized
bisphosphonic acids in the rat adjuvant model were also
studied in terms of structure-activity relationships (SARs).
The interest in the synthesis of these analogues is mainly
driven by the growing use of quinolines in anti-tumor drugs
[15, 16]. Several quinoline derivatives exhibit potency as
inhibitors of the enzyme farnesyl protein transferase and
are therefore believed to be useful as anti-cancer agents [16].
Scheme 2. Preparation of BPs 8a–d and BP-acids 9a–d.
Results and discussion
In a previous investigation [8] Perkin-type condensation
between tetraethyl methylenebisphosphonate (1) and 2-azi-
doquinolines-3-carbaldehyde (3) led to tetrazoloquinoline-
based ethylene-bisphosphonates 4 and 5 with anti-inflamma-
tory properties (Scheme 1). The investigation reported not-
withstanding the azide 3 reacts with phosphorus reagents
mainly in the tautomeric-tetrazole form, the involvement
of N3 group in some reactions was also reported [8, 17].
The results reflected the versatility of the azido group and
the phosphorus reagents as well.
In parallel, the bisphosphonate reagent 1 was applied to 3-
substituted chalcones of 2-chloro- and 2-azidoquinolines, to
produce the corresponding bisphosphonate derivatives
(Scheme 2). Chalcones 7a–e were smoothly obtained in more
than 85% yield through the condensation of 3-formyl tetra-
zolo[1,5-a]-quinoline (3) with the ketone derivatives 6a–e
1
in ethyl alcohol containing 10% NaOH. Accoding to the H-
NMR data, we assigned the configuration (E) for the C–C
double bond based on the vicinal proton–proton coupling
J ꢀ 15 Hz. Similarly, (E)-isomers were also assigned for other
Scheme 1. Preparation of BPs 4 and 5.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
125
chalcones [18]. However, the selectivity in configuration may
derive from the repulsion between the two moieties bonding
on C–C double bond, which makes the geometric isomer (Z)
highly unfavorable. Compounds 7a–e were then treated with
slight excess of tetraethyl methylenebisphosphonate (1) in
ethanolate solution, and the reaction mixture was refluxed
for 4–6 h (TLC) to give the corresponding tetraethyl 4-oxo-4-
aryl-2-(tetrazolo[1,5-a]quinolin-4-yl)butane-1,1-diyl-diphosph-
onates 8a–d in about 70% yield. Obviously, compounds 8a–d
were formed via Michael addition at the exocyclic a,b-unsa-
turated system. The formation of the tetrazole ring in 8 is
however consistent with the data reported for the azide 3 [19].
Only one isomer of the respective bisphosphonates 8a–d
was identified and was assigned as (E)-isomer. The configur-
ation of the exo heterocyclic substituent was examined by
NMR analyses. The 1H-NMR spectrum of 8a, taken as an
example, revealed four types of methine protons with differ-
ent chemical shifts. The diastereotopic protons Ha and Hb
resonated at d 3.47 (dd, Ja,b ¼ 2.8, Ja,c ¼ 10.5 Hz), 3.51 (dd,
Jb,a ¼ 2.8, Jb,c ¼ 15.3 Hz) ppm, respectively. The multiplet at
d 4.98 ppm assigned to the Hc-proton, while the P-CHd-proton
resonated at d 4.17 (dd, Jd,c ¼ 16.5, 2JP-H ¼ 22.4 Hz) ppm. This
large coupling constant (JH-H) of Hd with Hc as well as its
coupling with phosphorus clearly indicates that Hd is anti-
configuration to Hc. The structure of 8a–d was also verified by
careful inspection of a model in terms of the Newman pro-
jection that confirmed the staggered anti-conformation of Hc
and Hd.
Scheme 3. Preparation of BPs 11a–d and BP-acids 12a–d.
vinylbisphosphonates 13a and 14a in E-configuration,
respectively (Scheme 4). The 1H-NMR spectrum of 13a showed
2
among others, two doublet of doublets (d 4.66, JP-H ¼ 23.8,
4
4
4JHa-Hb ¼ 2.8 Hz, and d 7.08, JP-H ¼ 4.2, JHa-Hb ¼ 2.8 Hz)
readily recognized as arising from the diastereotopic
methine protons of the allylic moiety Ha and Hb. In the
1H-NMR spectrum of 14a, the vinyl proton of C-3 appeared
as an allylic defined doublet of doublet (4JH-H ¼ 2.6,
4JP-H ¼ 3.8 Hz, Hb) at 7.12 ppm while the CHa-P2 proton
(dd, 4JH-H ¼ 2.2, 2JP-H ¼ 23.7 Hz) was deshielded at
4.62 ppm. The observation of the allylic coupling constants
H-C-C ¼ C-H (4JH-H ¼ 2.8 & 2.6 Hz) and the large P-H coupling
constants of Ha (2JP-H ¼ 23.8 & 23.7 Hz) in 13a and 14a
indicates the dominance of anti-configuration. The inspec-
tion of a model in terms of the Newman projection also
As structure-activity studies in several pharmaceutical
laboratories have identified distinct therapeutic character-
istics of bisphosphonic acid compared to bisphosphonate
analogs [20, 21], acid hydrolysis of compounds 8a–d was
therefore undertaken to give the corresponding BP-acids (E)
9a–d.
The reactions of the bisphosponate reagent 1 with the
chalcones 10a–e were then investigated, in an effort to study
the possible influence on the potency of the chlorine atom
compared to the tetrazole ring. The required chalcones 10a–e
were obtained in high yields (ꢁ85%) via the condensation of
the parent 2-chloro-3-formylquinoline (2) with the ketones
6a–e in ethanol containing NaOH (10%) solution. Treatment
of 10a–d with an equivalent amount of 1 under the same
previous experimental conditions afforded the bisphospo-
nate analogues 11a–d in reasonable yields (>70%). Acid
hydrolysis (conc. HCl) of 11a–d afforded the respective acids
12a–d (Scheme 3). Elemental analyses and spectral data sub-
stantiated the chemical structure 11. On the same bases and
by spectral analogy with structure 8, the configuration of the
exocyclic substituents in 11 was confirmed as staggered anti-
conformation.
Conversely, coupling reaction of 1 with 7e and 10e with
concomitant loss of HCN molecule was observed to give the
Scheme 4. Preparation of BPs 13a, 14, and BP-acids 13b and
14b.
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W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
confirmed this result. In the case of 13a the configuration of
the exocyclic double bond was examined by selective NOE
experiments, which were also useful for the assignment
of the 13C-NMR signals. The irradiation of CHb-proton
(7.08 ppm) resulted only in a little enhancement of 1-C-triplet
(56.8 ppm) and the C-2-doublet (133.3 ppm). Irradiation of
the CHa proton (4.66 ppm) produced a small NOE effect at the
3-C-doublet (122.8 ppm) and at 2-C-doublet (133.2 ppm), indi-
cating the anti-configuration of the Ha proton to Hb proton in
compounds 13 and 14.
extracted from sea weed has been extensively used to induce
inflammation in a number of animal species. Furthermore,
the carrageenin induced rat hind paw edema is now rou-
tinely used for the assay of anti-inflammatory agents. The
reproducibility and the fact that the edema depends entirely
on a local inflammatory reaction devoid of antigenic proper-
ties, has made carrageenin a most widely used phlogistic
agent in experimental pharmacology and a good correlation
has been shown to exist between the antiphlogistic and anti-
inflammatory effects of several drugs. In all experiments,
carrageenin was administered into the left hind paw. Anti-
inflammatory activity of the tested compounds (at a dose of
50 mg/kg body weight) was measured at successive time
intervals (1, 2, and 4 h after carrageenin injection) and com-
pared with that of the bisphosphonate drug, clodronic acid
(A) at the same dose. The reference A was used as a reference
standard on similar occasions [26, 27]. However, when the
rats were reused, carrageenin injection was given into the
right hind paw (Table 2, Figs. 1 and 2).
Biological assays
The prospective potency of our products for treating cancer
and inflammatory diseases was based on the results of the
prediction that had been carried out in the earlier stage. The
computer-assisted molecular modeling (CAMM), PASS pro-
gram, was adopted for designing, in silico, the structures of
potentially active molecules for synthesis.
Anti-tumor activity screening
The recorded data in Table 2 show that 8 of 10 new syn-
thesized BP-acids have moderate to good anti-inflammatory
properties when compared with available indomethacin-
drug, without toxic side-effects. The bisphosphonates 9c
and 9b possess maximum inhibitory effect at all detected
time intervals when compared to the standard group.
Nevertheless, the substrates 7a and 10a showed poor con-
siderable effect as anti-inflammatory agents. Other com-
pounds, i.e., 9a, 9d, 12a–d, 13b, and 14b have displayed
good to a moderate effect on inhibitory properties, at least
after 1 h.
Antitumor activity for the synthesized bisphosphonic-acids
9a–d, 12a–d, 13b, and 14b at a dose of 10 mM utilizing 44
different human tumor cell lines, representing breast, ovar-
ian, prostate, lung, and brain cancer as well as leukemia and
melanoma was carried out according to the previously
reported standard method [22, 23]. Substrates 7a and 10a
were also biologically tested in a trial to reflect the effect of
introducing BP moiety. The obtained results represent per-
centage growth of the tumor cell lines treated with com-
pounds under investigation relative to control cell.
The pharmacological results in Table 1 showed that other
than the substrates 7a and 10a, all synthesized compounds
reflect remarkable anti-tumor activity against breast (especi-
ally MDA-MB-23/ATCC), and prostate cancer whereas a mod-
erate effect was observed on ovarian cancer and melanoma.
On the other hand, only BP-acids bearing the tetrazole moiety
9a–d and 13b showed sensitivity against leukemia, brain and
lung cancer cells. However, we considered that cell line
growth inhibition with >50% at a concentration of 10 mM
usually seems to be a noticeable activity. SARs correlation for
these reported observations reveal that the presence of
the fused tetrazole moiety to the quinoline-ring usually
associates with the enhancement in anti-tumor properties
as indicated in compounds 9a–d and 13b.
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. The type of the substituent
attached on the 2-position or the 3-substituents on quinoline
nucleus is a determining element for the potency; e.g., the
presence of fused tetrazole ring highly enhanced the efficacy.
Also it could be concluded that the activity of the hetero-ring
of the chalcone on the biological potency for 9 or 12 increases
in the succession: pyrrolyl > thienyl > furanyl > phenyl. In
sequel, the BP-acids 9c and 9b, which are the most active
products as anti-inflammatory compounds are along the line
of our observations.
Toxicity of the most promising products
Anti-inflammatory screening
Toxicological studies of the most promising synthesized anti-
inflammatory active compounds 9a and 12a were performed
using LD50 standard method in mice in 500, 750 and
1000 mg/kg (body weight), i.e. 10–20-fold of the used anti-
inflammatory effective dose. However, no toxic symptoms or
mortality rates were observed after 24 h post-administrations
explaining the safe behavior of the used doses.
The anti-inflammatory activity of the synthesized bisphos-
phonic acids (BP-acids) 9a–d, 12a–d, 13b, and 14b was deter-
mined in vivo by the acute carrageenin-induced paw edema
standard method in rats; the substrates 7a and 10a were also
tested to reflect the effect of introducing the BPs moiety
[24, 25]. Carrageenin which is a sulfated polysaccharide,
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Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
127
Table 1. Concentrations resulting in growth inhibition of 50% (GI50, mg/L) of in vitro human tumor cell lines.
Panel/cell line
Compounds
13b
7a/10a
9a
9b
9c
9d
12a
12b
12c
12d
14b
Breast cancer
McF7
NCl/ADR-RES
MDA-MB-231/ATCC
HS578T
MDA-MB-435
BT-549
T-47D
>46
>46
>46
>46
>46
>46
>46
17.58
6.50
8.13
14.80
16.60
13.3
9.80
9.37
11.12
32.92
9.85
5.21
6.46
8.35
26.10
5.32
5.05
8.40
20.1
9.3
24.2
13.4
11.97
11.08
30.64
33.52
30.9
38.2
38.2
32.1
30.4
27.6
25.5
25.8
19.6
30.4
17.6
17.8
26.3
20.8
18.9
20.2
17.6
NT
24.23
22.41
17.05
8.36
36.20
NT
13.42
32.4
22.5
20.7
18.6
14.5
NT
38.55
38.05
33.36
34.2
21.0
NT
18.13
14.12
11.65
14.06
13.57
14.7
13.55
Ovarian cancer
IGROVI
>46
>46
>46
>46
>46
>46
28.8
27.41
10.18
40.04
40.6
11.6
4.4
10.6
25.6
28.4
32.3
7.9
7.4
3.6
17.8
14.6
11.5
26.1
27.2
10.5
40.6
36.5
13.6
32.3
30.3
14.6
30.6
21.4
16.7
23.6
24.2
24.8
16.3
13.5
13.6
20.6
14.5
24.6
10.3
11.4
12.6
16.8
10.7
4.3
16.5
13.7
8.4
30.8
18.9
9.8
36.6
28.6
16.5
>50
NT
>52
>52
>52
NT
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
16.5
Prostate cancer
PC-3
DU-145
>46
>46
16.3
11.4
5.6
16.42
3.82
2.38
14.2
11.6
>52
>52
19.4
11.52
10.82
8.75
19.6
15.3
18.3
14.5
>52
>52
Melanoma
LOXIMVI
MALME-3M
MI4
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Non small cell lung cancer
A549/ATCC
EKVX
>50
>50
>50
>50
>50
>50
>50
>50
36.52
18.42
33.42
29.5
11.22
16.40
9.62
34.51
14.73
15.72
27.33
14.35
11.44
8.64
8.83
8.36
5.64
11.43
14.69
17.36
10.46
11.62
13.16
11.5
9.50
14.23
26.21
12.06
10.54
9.52
33.42
28.61
40.23
30.35
18.36
40.22
19.23
22.6
29.37
37.3
24.52
31.31
27.52
26.47
40.35
37.4
24.23
22.82
19.36
23.71
24.64
28.56
28.76
18.63
32.42
21.53
27.42
12.62
34.06
27.6
>50
>50
>50
15.34
>50
36.15
NT
NT
47.82
30.73
38.92
46.42
14.33
12.53
23.2
26.67
>42
16.23
24.3
7.21
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
44.8
>50
>52
>48
39.7
>50
>50
>50
36.6
36.4
>50
>52
>52
25.3
>52
>52
28.6
>52
38.2
>52
>52
>52
15.6
>52
>52
30.7
>50
35.9
>46
>52
>52
40.6
36.9
>52
41.4
>50
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
>52
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
>50
>50
>50
>50
>50
>50
>46
36.55
34.23
NT
>46
NT
33.25
30.35
31.24
32.14
28.25
24.29
>42
30.52
17.54
38.82
NT
24.50
NT
38.9
40.0
36.9
26.52
14.25
NT
40.0
>46
35.36
28.21
NT
42.5
37.25
23.37
27.36
NT
>46
30.36
>50
NT
31.62
22.36
>46
34.27
17.39
NT
23.59
NT
>50.5
>50.3
>50.4
NT
>50
>50
33.53
30.55
40.23
41.00
28.32
20.35
18.20
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>52
>52
>50
>50
21.36
28.25
26.72
26.72
26.72
26.72
26.72
26.72
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>47
>47
>47
>47
20.32
>47
>50
>50
>50
23.11
>50
20.36
>50
>50
>50
>50
>50
>50
SNB-75
U251
Nt: not tested
Conclusion
and the related BP-acids from chalcones and methylene-
bisphosphonate. The convenience and novelty of this
work is reflected in its several advantages such as mild
reaction conditions, short reaction time, and easy workup
The present investigation offered an easy approach for the
transformation of available starting materials to the title BPs
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Table 2. Anti-inflammatory activity of 9a-d, 12a-d, 13b, 14b, 7a, and 10a in acute carrageenin induced paw edema in rats.
Cmpd
Mean swellinga volume (mL) (percentage inhibition of edema)
Potencyd (%)/4 h
1 h
2 h
4 h
Control
Ab
9a
9b
9c
0.585 ꢂ 0.192c (00.0)
0.287 ꢂ 0.031c (50.9)
0.256 ꢂ 0.026c (54.9)
0.218 ꢂ 0.023c (59.8)
0.211 ꢂ 0.025c (61.0)
0.247 ꢂ 0.032c (56.5)
0.364 ꢂ 0.039c (35.9)
0.338 ꢂ 0.055c (40.4)
0.310 ꢂ 0.028c (46.9)
0.348 ꢂ 0.034c (40.4)
0.495 ꢂ 0.045c (12.9)
0.510 ꢂ 0.034c (12.3)
0.558 ꢂ 0.055c (8.8)
0.580 ꢂ 0.055c (6.8)
0.649 ꢂ 0.067c (00.0)
0.290 ꢂ 0.011c (55.3)
0.326 ꢂ 0.039c (49.7)
0.308 ꢂ 0.024c (52.5)
0.297 ꢂ 0.032c (54.2)
0.317 ꢂ 0.037c (51.17)
0.378 ꢂ 0.102c (41.7)
0.551 ꢂ 0.092c (15.1)
0.378 ꢂ 0.102c (41.7)
0.498 ꢂ 0.062c (23.2)
0.442 ꢂ 0.062c (31.8)
0.542 ꢂ 0.077c (16.5)
0.594 ꢂ 0.077c (8.5)
0.600 ꢂ 0.077c (7.5)
0.869 ꢂ 0.058c (00.0)
0.415 ꢂ 0.022c (52.2)
0.436 ꢂ 0.048c (49.8)
0.403 ꢂ 0.030c (53.6)
0.401 ꢂ 0.033c (53.8)
0.425 ꢂ 0.045c (51.0)
0.632 ꢂ 0.094c (27.2)
0.538 ꢂ 0.048c (38.0)
0.502 ꢂ 0.012c (42.2)
0.625 ꢂ 0.052c (28.0)
0.725 ꢂ 0.052c (16.5)
0.721 ꢂ 0.102c(17.0)
0.822 ꢂ 0.066c (5.9)
0.827 ꢂ 0.052c (4.8)
–
100.0
101.2
108.9
109.3
103.6
55.3
77.2
85.7
56.9
33.5
34.5
12.0
9.7
9d
12a
12b
12c
12d
13b
14b
7a
10a
(a) Data are means of two independent determinations at least, and the deviation in absorbance values was less than 10%.
(b) A ¼ Clodronic acid (used as a reference standard), each value represents the mean ꢂ of two independent experiments with
6 animals in each group. (c) (SEM: standard error of the mean) Statistical significance of results was established using the Student’s test
from the standard at P < 0.05. (d) Potency was expressed as percentage edema inhibition of the tested compounds relative to
percentage edema inhibition of A at 4 h effect.
procedure without the need for chromatographic puri-
fication.
In parallel, screening results showed that the BP moiety is
an essential factor in developing the total pharmacological
properties for the synthesized compounds whereas the type
of the substituents attached to the quinoline nucleus deter-
mines and controls the potency. Finally, the presence of the
fused tetrazole ring markedly enhanced the anti-tumor and
the anti-inflammatory efficacy of the synthesized
bisphosphonates.
Experimental section
General
Melting points were determined with open capillary tube on
an Electrothermal (variable heater) melting point apparatus
and were uncorrected. IR spectra were recorded on a Perkin-
Elmer spectrophotometer model 297 using KBr disc. NMR
spectra were measured with a JEOL E.C.A-500 MHz (13C:
125.8 MHz, 1H: 500.6 MHz, 31P: 200.7 MHz) spectrometer.
31P-NMR spectra were recorded with H3PO4 (85%) as external
reference. 1H and 13C NMR spectra were recorded with tri-
methylsilane 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 purity of all new samples
was verified by microchemical analysis (H/C/N) and in some
cases via 13C-, 31P-, and 1H-NMR spectroscopy. Materials
and reagents were purchased from Aldrich Company. All
international principles and local regulations concerning
Figure 1. Percentage inhibition of edema for the tested compounds
at successive intervals.
Figure 2. Anti-inflammation activity of the tested compounds
relative to indomethacin.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
129
the care and use of laboratory animals were considered
during the pharmacological screening.
(13) [Mþ], 264 (100). Anal. calcd. for C16H10N4O2 (290.3):
C, 66.20; H, 3.47; N, 19.30. Found: C, 66.27; H, 3.51;
N, 19.37. For NMR-spectral data see Tables 3 and 4.
Synthesis
Preparation of 2-azidoquinoline-3-carbaldehyde (3)
To a solution of (26 mmol) of 2-chloroquinoline-3-carbalde-
hyde (4.98 g) in 15 mL of DMSO, 2 g of sodium azide
(30 mmol) was added and the resulting mixture was heated
to 908C for 4 h. The precipitated product was collected,
washed with acetone, dried, and crystallized to give the azide
3 as straw yellow crystals 4.12 g (80%); mp 260–2628C (EtOH)
([28]: mp 2608C).
(E)-2-Cyano-3-(tetrazolo[1,5-a]quinolin-4-ylacrylamide
(7e)
Yellow powder, mp 2778C (MeCN), yield 92%. IR: nmax, cmꢃ1
:
–
–
1663 (C O), 1595 (C C, olefin), 1182 (tetrazole). EI-MS:
–
–
m/z (%) ¼ 264 (100) [Mþ]. Anal. calcd. for C13H8N6O (264.2):
C, 59.09; H, 3.05; N, 31.80. Found: C, 59.14; H, 3.12; N, 31.74.
For NMR-spectral data see Tables 3 and 4.
Reaction of chalcones 7a–d and 10a–d with tetraethyl
methylenebisphosphonate (1)
Preparation of quinoline-3-chalcones 7a–e and 10a–e
A mixture of 5 mmol of the ketone derivatives 6a–e and 3-
formyl tetrazolo[1,5-a]-quinoline (3) or 2-chloroquinoline-3-
carbaldehde (2) in 20 mL ethanol containing 5 mL of
NaOH (10% aq.) was stirred at r.t. for about 6 h. The resulting
precipitate was filtered, washed with 10 mL dil. EtOH, and
dried. Crystallization of the collected residue from the proper
solvent afforded the corresponding known chalcones 10a–e
[29–31] in >85% yield, and the new products 7a–e.
Preparation of the bisphosphonate derivatives 8a–d,
11a–d
General Method
A solution 1.25 mmol of 1 in 20 mL of absolute ethanol
containing 2.4 mmol sodium ethoxide was stirred at 08C
for about 0.5 h. A solution of 1.0 mmol 7a–d (or 10a–d)
in 10 mL of absolute ethanol was then added in one portion,
and the reaction was completed by heating under reflux
for ꢄ6 h (TLC). The resulting mixture was allowed to
warm to r.t. The product mixture was cooled, poured onto
iced-water, and acidified with conc HCl to pH ꢀ6, followed by
extraction with AcOEt (3 ꢅ 50 mL), and the combined
organic phase was dried over anhydr. Na2SO4. After
removal of the solvent under vacuum, the resulting residue
was washed several times with light petroleum (40–608C),
and crystallized from the proper solvent to give the
corresponding quinolinyl-biphosphonates 8a–d, 11a–d,
respectively.
(E) 1-Phenyl-3-(tetrazolo[1,5-a]quinolin-4-yl)-
prop-2-en-1-one (7a)
Yellow substance, mp 238–2408C (MeOH), yield 93%. IR: nmax
,
cmꢃ1: 1657 (C O), 1594 (C C), 1180 (tetrazole). EI-MS: m/z (%)
–
–
–
–
300 (9) [Mþ], 274 (100). Anal. calcd. for C18H12N4O (300.3):
C, 71.99; H, 4.03; N, 18.66. Found: C, 72.05; H, 4.08; N, 18.61.
For NMR-spectral data see Tables 3 and 4.
(E) 3-(Tetrazolo[1,5-a]quinolin-4-yl)-1-(thiophen-2-yl)-
prop-2-en-1-one (7b)
Yellow crystals, mp 272–2748C (EtOH), yield 92%. IR: nmax
,
cmꢃ1: 1660 (C O), 1592 (olefin), 1184 (tetrazole). EI-MS:
(E) Tetraethyl 4-oxo-4-phenyl-2-(tetrazolo[1,5-a]-
quinolin-4-yl)butane-1,1-diyl-diphosphonat (8a)
Dark orange crystals, mp ¼ 210–2128C (MeCN), yield 75%. IR:
–
–
306 (7) [Mþ], 280 (100). Anal. calcd. for C16H10N4OS (306.3):
C, 62.73; H, 3.29; N, 18.29; S, 10.47. Found: C, 62.81; H, 3.33;
N, 18.35; S, 10.58. For NMR-spectral data see Tables 3
and 4.
nmax, cmꢃ1: 1677 (C O), 1254, 1236 (2P O), 1184 (tetrazole),
–
–
–
–
1164, 1030 (P–O–C). EI-MS: m/z (%) 588 (11) [Mþ], 276 (100).
Anal. calcd. for C27H34N4O7P2 (588.5): C, 55.10; H, 5.82; N,
9.52; P 10.53. Found: C, 55.13, H, 5.93; N, 9.56; P, 10.64. For
NMR-spectral data see Tables 3 and 4.
(E) 1-(1H-Pyrrol-2-yl)-3-(tetrazolo[1,5-a]quinolin-4-yl)-
prop-2-en-1-one (7c)
Orange crystals, mp 2788C (EtOH), yield 93%. IR: nmax, cmꢃ1
:
–
–
3434 (NH), 1659 (C O), 1590 (C C), 1182 (tetrazole). EI-MS:
(E)Tetraethyl 4-oxo-2-(tetrazolo[1,5-a]quinolin-4-yl)-4-
(thiophen-2-yl)butane-1,1-diyl-diphosphonate (8b)
Dark orange crystals, mp 234–2368C (MeCN), yield 72%. IR:
–
–
289 (10) [Mþ], 263 (100). Anal. calcd. for C16H11N5O (289.3):
C, 66.43; H, 3.83; N, 24.21. Found: C, 66.51; H, 3.89; N, 24.18.
For NMR-spectral data see Tables 3 and 4.
nmax, cmꢃ1: 1685 (C O), 1487 (tetrazole), 1268, 1234 (2 P O),
–
–
–
–
1161, 1074 (2 P–O–C). EI-MS: m/z (%) ¼ 594 (13) [Mþ], 282 (100).
Anal. calcd. for C25H32N4O7P2S (594.2): C, 50.50; H, 5.42;
N, 5.42; P, 10.42; S, 5.39. Found: C, 50.56; H, 5.47; N,
5.49; P, 10.46; S, 5.51. For NMR-spectral data see Tables 3
and 4.
(E) 1-(Furan-2-yl)-3-(tetrazolo[1,5-a]quinolin-4-yl)-
prop-2-en-1-one (7d)
Yellow powder, mp 2438C (EtOH), yield 95%. IR: nmax, cmꢃ1
:
–
–
–
1663 (C O), 1595 (C C), 1182 (tetrazole). EI-MS: m/z (%) 290
–
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

130
W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Table 3. Spectral data 31P- and 1H-NMR of BPs 7a–7e, 8a–8d, 11a–11d, 13a, 14a, and BP-acids 9a-9d, 12a-12d, 13b, and 14b.
Cmpd. #
31P-NMR d (ppm)
1H-NMR d (ppm)
7aa
7ba
7ca
–
–
–
6.31, 7.20 (2d, JH-H ¼ 15.7 Hz, 2 ꢅ 1H, HC CH, exocycl.), 7.81–8.69 (m, 10 H, H-Ar & Ph)
–
–
–
–
6.31, 7.45 (2d, JH-H ¼ 16.12 Hz, 2 ꢅ 1H, HC CH, exocycl.), 7.76 - 8.53 (m, 8 H, H-het, & Ar)
–
6.25, 7.34 (2d, JH-H ¼ 15.6 Hz, 2 ꢅ 1H, HC CH, exocycl.), 7.63 - 8.29 (m, 8 H, H-het & -Ar),
–
11.84 (s br, 1H, NH)
6.29, 7.17 (2d, JH-H ¼ 15.8 Hz, 2 ꢅ 1H, HC CH, exocycl.), 7.57–8.36 (m, 8H, H-Ar)
7da
7ea
8aa
–
–
–
–
–
21.8, 29.2
(2d, 2JP-P ¼ 34.6 Hz, P-C-P)
7.78 (s, 1H, CH, exoxcycl.), 7.82 (d, J
¼ 2.4 Hz, 2H, NH2), 7. 84, 8.21, 8.36 (3m, 5H, H-Ar)
–
H-H
1.28, 1.33 (2dt, JH-H ¼ 7.6, 4JP-H ¼ 4.3 Hz, 2 ꢅ 6H, 4 H3CCO), 3.47 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.5 Hz,
1H, HaC), 3.51 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.3 Hz, 1H, HbC), 3.94, 4.35 (2 quint, 3JP-H ¼ 12.8 Hz,
2 ꢅ 4H, 4 H2CO), 4.17 (dd, JHd-Hc ¼ 16.5, 2JP-H ¼ 22.4 Hz, 1H, Hd-C-P), 4.98 (m, 1H, HcC), 7.24–7.83, 8.13,
8.32 (m, 10 H, H-Ph & Ar)
8ba
24.6, 29.8
(2d, 2JP-P ¼ 33.4 Hz, P-C-P)
1.23, 1.69 (2dt, JH-H ¼ 7.7, 4JP-H ¼ 5.2 Hz, 2 ꢅ 6H, 4 H3CC.O), 3.47 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.2 Hz,
1H, HaC), 3.72 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC), 3.94, 4.13 (2 quint, 3JP-H ¼ 12.8 Hz,
2 ꢅ 4H, 4 H2CO), 4.20 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.18 (m, 1H, HcC), 7.24–7.88, 8.15,
8.64 (m, 8H, H-Het & Ar)
1.22, 1.52 (2dt, JH-H ¼ 7.4, 4JP-H ¼ 4.8 Hz, 2 ꢅ 6H, 4 H3CCO), 3.38 (dd, JHa-Hb ¼ 2.6, JHa-Hc ¼ 10.4 Hz,
1H, HaC), 3.65 (dd, JHb-Ha ¼ 2.6, JHb-Hc ¼ 14.8 Hz, 1H, HbC), 3.96, 4.08 (2 quint, 3JP-H ¼ 11.7 Hz,
2 ꢅ 4H, 4 H2CO), 4.42 (dd, JHd-Hc ¼ 16.2, 2JP-H ¼ 22.3 Hz, 1H, Hd-C-P), 5.48 (m, 1H, HcC), 7.24–7.77, 8.23,
8.54 (m, 8H, H-Het & -Ar), 11.74 (s br, 1H, HN)
1.23, 1.63 (2dt, JH-H ¼ 6.8, 4JP-H ¼ 5.6 Hz, 2 ꢅ 6H, 4 H3CCO), 3.38 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.4 Hz,
1H, HaC), 3.46 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC), 3.99, 4.18 (2quint, 3JP-H ¼ 12.6 Hz, 2 ꢅ 4H,
4 H2COP), 4.46 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 22.4 Hz, 1H, Hd-C-P), 5.42 (m, 1H, HcC), 7.24–7.78, 8.18, 8.35
(m, 8H, H-Het & Ar)
8ca
22.6, 28.8
(2d, 2JP-P ¼ 40.2 Hz, P-C-P)
8da
25.3, 28.2
(2d, 2JP-P ¼ 36.7 Hz, P-C-P)
11aa
11ba
11ca
11da
24.6, 27.8
(2d, 2JP-P ¼ 34.6 Hz, P-C-P)
1.28, 1.33 (2dt, JH-H ¼ 7.6, 4JP-H ¼ 4.3 Hz, 2 ꢅ 6H, 4 H3CCO), 3.43 (dd, JHa-Hb ¼ 2.8, JHa-H ¼ 10.4 Hz,
1H, HaC), 3.62 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC), 3.94, 4.35 (2quint, 3JP-H ¼ 12.8 Hz, 2 ꢅ 4H,
4 H2CO), 4.38 (dd, JHd-Hc ¼ 16.0, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.22 (m, 1H, HcC), 7.24–7.78, 8.18, 8.35
(m, 8H, H-Het & Ar)
1.22, 1.47 (2dt, JH-H ¼ 6.6, 4JP-H ¼ 6.4 Hz, 2 ꢅ 6H, 4 H3CCO), 3.38 (dd, JHa-Hb ¼ 2.6, JHa-Hc ¼ 10.4 Hz,
1H, HaC), 3.46 (dd, JHb-Ha ¼ 2.8, JHb-Hc¼ 15.6 Hz, 1H, HbC), 3.94, 4.35 (2 quint, 3JP-H ¼ 12.8 Hz, 2 ꢅ 4H,
4 H2CO), 4.38 (dd, JHd-Hc ¼ 16.0, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.22 (m, 1H, HcC), 7.24–7.78, 8.18, 8.35
(m, 8H, H-Het & Ar)
1.21, 1.58 (2dt, JH-H ¼ 7.2, 4JP-H ¼ 5.6 Hz, 2 ꢅ 6H, 4 H3CCO), 3.38 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.2 Hz,
1H, HaC), 3.72 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC), 3.94, 4.13 (2 quint, 3JP-H ¼ 12.8 Hz,
2 ꢅ 4H, 4 H2CO), 4.26 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.38 (m, 1H, HcC), 7.24–7.88, 8.15,
8.23 (m, 8H, H-Het & Ar), 11.76 (s, 1H, HN, D2O exch.)
23.5, 29.4
(2d, 2JP-P ¼ 36.8 Hz, P-C-P)
20.6, 27.9
(2d, 2JP-P ¼ 34 Hz, P-C-P)
23.6, 27.5
(2d, 2JP-P ¼ 34 Hz, P-C-P)
1.19, 1.43 (2dt, JH-H ¼ 6.7, 4JP-H ¼ 6.6 Hz, 2 ꢅ 6H, 4 H3CCO), 3.42 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 9.8 Hz,
1H, HaC), 3.62 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 16.6 Hz, 1H, HbC), 3.88, 4.06 (2 quint, 3JP-H ¼ 12.8 Hz,
2 ꢅ 4H, 4 H2CO), 4.36 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 20.6 Hz, 1H, Hd-C-P), 5.28 (m, 1H, HcC), 7.24–7.88, 8.13,
8.2 (m, 8H, H-Het & Ar)
13aa
14aa
9ab
22.7, 28.3
(2d, 2JP-P ¼ 29 Hz, P-C-P)
1.14, 1.47 (2dt, JH-H ¼ 7.3, 4JP-H ¼ 4.4 Hz, 2 ꢅ 6H, 4 H3CCO), 3.89–4.12 (m, 8H, 4 H2CO), 4.66
(dd, JHa-Hb ¼ 1.6, 2JP-Ha ¼ 23.8 Hz, 1H, HaC), 5.08 (d, JH-H ¼ 2.4 Hz, 2H, H2N), 7.08 (dd, 4JHb-Ha ¼ 1.8,
4JH-P ¼ 4.2 Hz, 1H, HbC), 7.37, 7.77, 8.23, 8.46 (3m, 5H, H-Ar)
24.7, 28.7
(2d, 2JP-P ¼ 32 Hz, P-C-P)
1.16, 1.43 (2dt, JH-H ¼ 6.5, 4JP-H ¼ 4.8 Hz, 2 ꢅ 6H, 4 H3CCO), 3.88–4.02 (m, 8H, 4 H2CO), 4.62
(dd, JHa-Hb ¼ 1.6, 2JP-Ha ¼ 23.7 Hz, 1H, HaC), 4.86 (d, JH-H ¼ 2.8 Hz, 2H, H2N), 7.12 (dd, 4JHb-Ha ¼ 1.6,
4JH-P ¼ 3.8 Hz, 1H, HbC), 7.43, 7.76, 8.25, 8.5 (3m, 5H, H-Ar)
21.2, 22.7
20.3, 22.5
20.5, 22.7
20.7, 21.8
20.2, 22.6
20.45, 22.35
3.35 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.5 Hz, 1H, HaC), 3.65 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.3 Hz, 1H, HbC),
3.98 (dd, JHd-Hc ¼ 16.5, 2JP-H ¼ 22.4 Hz, 1H, Hd-C-P), 5.12 (m, 1H, HcC), 7.62–7.86, 8.23, 8.52
(m, 10 H, H-Ar & Ph)
9bb
3.67 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.2 Hz, 1H, HaC), 3.81 (dd, JHb-Ha ¼ 2.8, JHb-Hc¼ 15.6 Hz, 1H, HbC),
4.35 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.26 (m, 1H, HcC), 7.24–7.88, 8.15, 8.64
(m, 8H, H-Het & Ar)
9cb
3.58 (dd, JHa-Hb ¼ 2.6, JHa-Hc ¼ 10.4 Hz, 1H, HaC), 3.73 (dd, JHb-Ha ¼ 2.6, JHb-Hc ¼ 14.8 Hz, 1H, HbC),
4.62 (dd, JHd-Hc ¼ 16.2, 2JP-H ¼ 22.3 Hz, 1H, Hd-C-P), 5.48 (m, 1H, HcC), 7.62–7.79, 8.23, 8.56
(3m, 8H, H-Het & Ar)
9db
3.62 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.4 Hz, 1H, HaC), 3.76 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC),
4.86 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 22.4 Hz, 1H, Hd-C-P), 5.42 (m, 1H, HcC), 7.24–7.78, 8.18, 8.35
(m, 8H, H-Het & Ar)
12ab
12bb
3.53 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.4 Hz, 1H, HaC), 3.75 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC),
4.48 (dd, JHd-Hc ¼ 16.0, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.32 (m, 1H, HcC), 7.26–7.82, 8.20, 8.35
(m, 8H, H-Ar & Ph)
3.38 (dd, JHa-Hb ¼ 2.6, JHa-Hc ¼ 10.4 Hz, 1H, HaC), 3.46 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 15.6 Hz, 1H, HbC),
4.38 (dd, JHd-Hc ¼ 16.0, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.22 (m, 1H, HcC), 7.74–7.88, 8.18, 8.35
(3m, 8H, H-Het & Ar)
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Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Table 3. (continued )
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
131
Cmpd. #
12cb
31P-NMR d (ppm)
1H-NMR d (ppm)
20.5, 22.6
3.42 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 10.2 Hz, 1H, HaC), 3.84 (dd, JHb-Ha ¼ 2.8, JHb-Hc¼ 15.6 Hz, 1H, HbC),
4.46 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 21.6 Hz, 1H, Hd-C-P), 5.45 (m, 1H, HcC), 7.64–7.88, 8.15, 8.23
(m, 8H, H-Het & Ar)
12db
21.3, 23.1
3.52 (dd, JHa-Hb ¼ 2.8, JHa-Hc ¼ 9.8 Hz, 1H, HaC), 3.73 (dd, JHb-Ha ¼ 2.8, JHb-Hc ¼ 16.6 Hz, 1H, HbC),
4.42 (dd, JHd-Hc ¼ 16.6, 2JP-H ¼ 20.6 Hz, 1H, Hd-C-P), 5.36 (m, 1H, HcC), 7.24–7.89, 8.14, 8.21
(m, 8H, H-Het & Ar)
13bb
14bb
20.9, 22.7
21.5, 22.8
4.69 (dd, JHa-Hb ¼ 1.6, 2JP-Ha ¼ 23.8 Hz, 1H, HaC), 7.18 (dd, 4JHb-Ha ¼ 1.8, 4JH-P ¼ 4.2 Hz, 1H, HbC),
7.42–8.28, 8.48 (ms, 5H, H-Ar)
4.72 (dd, JHa-Hb ¼ 1.6, 2JP-Ha ¼ 23.7 Hz, 1H, HaC), 7.12 (dd, 4JHb-Ha ¼ 1.6, 4JH-P ¼ 3.8 Hz, 1H, HbC),
7.43, 7.76, 8.25, 8.5 (3m, 5H, H-Ar)
a)
b)
ꢆSolvents of NMR: DMSO-d6; D2O.
(E) Tetraethyl 4-oxo-4-(1H-pyrrol-2-yl)-2-(tetrazolo-
[1,5-a]quinolin-4-yl)butane-1,1-diyl-diphosphonate (8c)
Dark orange crystals, mp 260–2628C (MeCN), yield 72%. IR:
1154, 1043 (2 P–O–C). EI-MS: m/z (%) ¼ 578 (13) [Mþ], 266 (100).
Anal. calcd. for C25H32N4O8P2 (578.5): C, 51.91; H, 5.58;
N, 9.68; P, 10.71. Found: C, 51.95; H, 5.64; N, 9.73; P, 10.79.
For NMR-spectral data see Tables 3 and 4.
nmax, cmꢃ1: 3423 (NH), 1676 (C O), 1561 (C N), 1263, 1237
–
–
–
–
þ
–
(2 P O), 1164, 1065 (2 P–O–C). EI-MS: m/z (%) ¼ 577 (13) [M ],
–
265 (100). Anal. calcd. for C25H33N5O7P2 (577.5): C, 51.99;
H, 5.76; N, 12.13; P, 10.73. Found: C, 52.07; H, 5.82;
N, 12.19; P, 10.81. For NMR-spectral data see Tables 3 and 4.
(E) Tetraethyl 2-(2-chloroquinolin-3-yl)-4-oxo-4-
phenylbutane-1,1-diyl-diphosphonate (11a)
Yellow crystals, mp 150–1528C (EtOH), yield 74%. IR: nmax
,
cmꢃ1: 1681 (C O), 1567 (C N), 1254, 1231 (2 P O), 1164,
–
–
–
–
–
–
(E) Tetraethyl 4-(furan-2-yl)-4-oxo-2-(tetrazolo-
[1,5-a]quinolin-4-yl)butane-1,1-diyl-diphosphonate (8d)
1030 (P–O–C). EI-MS: m/z (%) ¼ 581 (13) [Mþ], 582 (2.5)
[Mþþ1], 583 (4.4) [Mþþ2], 584 (0.8) [Mþþ3], 295 (100),
297 (32). Anal. calcd. for C27H34ClNO7P2 (581.9): C, 55.72;
H, 5.89; Cl, 6.09; N, 2.41; P, 10.64. Found: C, 55.78; H, 5.96;
Dark orange crystals, mp 250–2528C (MeCN), yield 70%. IR:
nmax, cmꢃ1: 1679 (C O), 1478 (tetrazole), 1257, 1231 (2 P O),
–
–
–
–
Table 4. 13C-NMR Spectral Data for BPs 7a-7e, 8a-8d, 11a-11d, 13a, and 14a.
Cmpd. #
13C-NMR [DMSO- d6] d (ppm)
–
–
–
7a
7b
7c
7d
7e
8a
192.2 (C O), 140.1, 138.2, 137.2, 134.6, 131, 130.3, 129.0, 128.2, 119.3, 114.6 (C-Ar & Ph), 128.4, 120.6 (C C, exocycl.)
–
–
–
–
168.7 (C O), 140.4, 138.6, 137.2, 134.8, 130.7, 129.6, 128.2, 119.3, 114.2 (C-Het, Ar), 127.4, 120.3 (C C, exocycl.)
–
–
–
–
168.9 (C O), 140.2, 138.4, 137.2, 134.6, 131.7, 129.06, 128.7, 119.3, 114.3 (C-Het, Ar), 128.1, 120.8 (C C, exocycl.)
–
–
–
168.9 (C O), 141.4, 138.5, 137.4, 134.8, 130.9, 129.1, 128.3, 119.6, 114.3 (C-Het & Ar), 127.8, 121.2 (C C, exocycl.)
–
–
–
163.2 (C O), 131.8 (HC C, exocycl.), 140.2, 139.7, 134.8, 130.9, 129.1, 124.8 (C-Het, Ar), 117.6 (CN), 101.6 (C-CN)
–
–
–
2
–
196.6 (C O), 141.2, 138.5, 135.1, 134.8, 134.6, 133.3, 129.6, 127.8, 123.8, 113.8 (C-Ar, Ph), 60.7 (t, J ¼ 9.7 Hz, 4 CH2O),
–
C-P
46.8 (t, 2JP-C ¼ 14.2 Hz, CH), 43.2 (t, 1JP-C ¼ 144.3 Hz, C-P2), 42.8 (t, 3JP-C ¼ 8.2 Hz, CH2), 15.4 (t, 3JP-C ¼ 8.8 Hz, 4 CH3)
2
–
8b
191.6 (C O), 145.3, 143.5, 136.4, 134.6, 132.3, 129.6, 126.7, 123.5, 114.3 (C-Het & Ar), 60.8 (t, J ¼ 10.3 Hz, 4 CH2O), 48.4
–
C-P
(t, 2JP-C ¼ 15.4 Hz, CHc), 42.2 (t, 1JP-C ¼ 148.4 Hz, C-P2), 41.6 (t, 3JP-C ¼ 8.2 Hz, CH2), 15.6 (t, 3JP-C ¼ 7.2 Hz, 4 CH3)
2
–
8c
188.2 (C O), 145.4, 137.5, 134.6, 132.6, 131.3, 127.8, 125.4, 121.5, 114.2 (C-Ar, Ph), 61.4 (t, J ¼ 9.7 Hz, 4 CH2O), 49.6
–
C-P
(t, 2JP-C ¼ 18.4 Hz, CHc), 43.4 (t, 1JP-C ¼ 142.4 Hz, C-P2), 41.8 (t, 3JP-C ¼ 8.2 Hz, CH2), 15.3 (t, 3JP-C ¼ 7.2 Hz, 4 CH3)
2
–
8d
190.6 (C O), 152.6, 148.2, 136.1, 134.8, 132.6, 126.8, 125.3, 123.8, 114.6 (C-Ar, Ph), 61.8 (t, J ¼ 8.8 Hz, 4 CH2O), 45.6
–
C-P
(t, 2JP-C ¼ 24.2 Hz, CHc), 42.6 (t, 1JP-C ¼ 140.4 Hz, C-P2), 41.2 (t, 3JP-C ¼ 6.2 Hz, CH2), 15.4 (t, 3JP-C ¼ 7.2 Hz, 4 CH3)
2
–
11a
11b
11c
11d
13a
14a
199.1 (C O), 145.5, 142.4, 135.4, 133.1, 130.3, 128.3, 127.7, 124.4, 114.4 (C-Ar, Ph), 62.1 (t, J ¼ 10.7 Hz, 4 CH2O), 45.2
–
C-P
(t, 2JP-C ¼ 16.5 Hz, CH), 43.7 (t, 1JP-C ¼ 168.3 Hz, C-P2), 41.8 (t, 3JP-C ¼ 8.8 Hz, CH2), 15.4 (t, 3JP-C ¼ 7.5 Hz, 4 CH3).
2
–
193.6 (C O), 145.5, 143.1, 133.4, 132.3, 129.2, 128.3, 127.4, 127.2, 123.8, 119.8 (C-Ar, Ph), 61.2 (t, J ¼ 8.7 Hz, 4 CH2O),
–
C-P
47.2 (t, 2JP-C ¼ 14.6 Hz, CH), 44.2 (t, 1JP-C ¼158.4 Hz, C-P2), 42.8 (t, 3JP-C ¼ 8.2 Hz, CH2c), 16.2 (d, 3JP-C ¼ 7.2 Hz, 4 CH3)
2
–
190.6 (C O), 146.8, 146.2, 135.4, 133.4, 127.6, 126.4, 125.7, 124,7, 123.8, 119.8 (C-Het & Ar), 61.8 (t, J ¼ 9.2 Hz, 4 CH2O),
–
C-P
48.2 (t, 2JP-C ¼ 15.6 Hz, CH), 45.6 (t, 1JP-C ¼154.5 Hz, C-P2), 44.6 (t, 3JP-C ¼ 10.7 Hz, CH2), 15.8 (t, 3JP-C ¼ 7.8 Hz, 4 CH3)
2
–
192.8 (C O), 152.5, 146.1, 135.0, 130.4, 127.4, 126.7, 125.9, 124,7, 123.8, 114.8 (C-Ar, Ph), 60.7 (t, J ¼ 9.7 Hz, 4 CH2O),
–
C-P
46.8 (t, 2JP-C ¼ 18.4 Hz, CH), 43.2 (t, 1JP-C ¼156.6 Hz, C-P2), 42.8 (t, 3JP-C ¼ 10.3 Hz, CH2c), 15.8 (t, 3JP-C ¼ 7.8 Hz, 4 CH3)
2
b
3
b
–
–
–
–
–
169.6 (C O), 133.3 (t, J ¼ 33.7 Hz, C CH ), 122.8 (t, J ¼ 16.4 Hz, CH ), 146.4, 144.7, 136.7, 130.2, 129.4, 127.4,
–
P-C
P-C
119.6 (C-Ar), 60.8 (t, 2JC-P ¼ 9.6 Hz, 4 CH2O), 56.8 (t, 1JP-C ¼ 196.6 Hz, C-P2), 16.2 (t, 3JP-C ¼ 7.8 Hz, 4 CH3)
2
b
3
b
–
–
–
–
–
169.4 (C O), 133.7 (t, J ¼ 34.7 Hz, C CH ), 122.4 (t, J ¼ 14.8 Hz, CH ), 148.4, 146.1, 134.6, 130.4, 129.4, 127.4,
–
P-C
P-C
(C-Ar), 61.6 (t, 2JC-P ¼ 10.4 Hz, 4 CH2O), 56.5 (t, 1JP-C ¼ 196.6 Hz, C-P2), 16.8 (t, 3JP-C ¼ 7.8 Hz, 4 CH3)
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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132
W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Cl, 6.19; N, 2.37; P, 10.77. For NMR-spectral data see Tables 3
and 4.
(E)-Tetraethyl 4-amino-4-oxo-2-(tetrazolo[1,5-a]quinolin-4-yl)-
but-2-ene-1,1-diyl-diphosphonate (13a)
Orange crystals, mp 234–2608C (MeCN), yield 75%. IR: nmax
,
(E) Tetraethyl 2-(2-chloroquinolin-3-yl)-4-oxo-4-
(thiophen-2-yl)butane-1,1-diyl-diphosphonate (11b)
cmꢃ1: 1686 (C O), 1616 (C C), 1480 (tetrazole), 1258, 1244
–
–
–
–
þ
–
–
(2 P O), 1182, 1045 (2 P–O–C). EI-MS: m/z (%): 525 (18) [M ], 509
Yellow crystals, mp 163–1658C (EtOH), yield 72%. IR: nmax
,
(34) [Mþꢃ16, NH2], 479 (22), [Mþꢃ44 (C(O)þNH2], 205 (100)
[Mþꢃ(44þ274) [P(O)(OEt)2]2]. Anal. calcd. for C21H29C5O7P2
(525.4): C, 48.00; H, 5.56; N, 13.33; P, 11.79. Found: C,
48.12; H, 5.63; N, 13.41; P, 11.85. For NMR-spectral data see
Tables 3 and 4.
cmꢃ1: 1675 (C O), 1552 (C N), 1259, 1236 (2 P O), 1150, 1039
–
–
–
–
–
–
(2 P–O–C). EI-MS: m/z (%) ¼ 587 (11) [Mþ], 588 (2.1) [Mþþ1], 589
(3.7) [Mþþ2], 590 (1.0) [Mþþ3], 301(100), 303 (36). Anal. calcd.
for C25H32ClNO7P2S (587.9): C, 51.07; H, 5.49; Cl, 6.03; N, 2.38;
P, 10.54; S, 5.45. Found: C, 51.15; H, 5.54; Cl, 6.11; N, 2.43;
P, 10.59; S, 5.52. For NMR-spectral data see Tables 3
and 4.
(E)-Tetraethyl 4-amino-2-(2-chloroquinolin-3-yl)-4-
oxobut-2-ene-1,1-diyl-diphosphonate (14a)
Yellow crystals, mp 258–2608C (MeCN), yield 71%. IR: nmax
,
(E) Tetraethyl 2-(2-chloroquinolin-3-yl)-4-oxo-4-
(1H-pyrrol-2-yl)butane-1,1-diyl-diphosphonate (11c)
cmꢃ1: 1679 (C O), 1618 (C C), 1262, 1238 (2 P O), 1159, 1033
– –
– –
–
–
(2 P–O–C). EI-MS: m/z (%): 518 (18) [Mþ], 519 (3.5) [Mþþ1], 520
(6.3) [Mþþ2], 521 (1.4) [Mþþ3], 502 (34) [Mþꢃ16, NH2], 474
(22), [Mþꢃ44 (C(O)þNH2], 200 (100) [Mþꢃ44þ274
[P(O)(OEt)2]2]. Anal. calcd. for C21H29ClN2O7P2 (518.8): C,
48.61; H, 5.63; Cl, 6.83; N, 5.40; P, 11.94. Found: C, 48.72;
H, 5.74; Cl, 6.76; N, 5.49; P, 12.04. For NMR-spectral data see
Tables 3 and 4.
Yellow crystals, mp 145–1478C (MeOH), yield 72%. IR: nmax
,
cmꢃ1: 3430 (NH), 1678 (C O), 1562 (C N), 1258, 1244 (2 P O),
–
–
–
–
–
–
1154, 1035 (2 P–O–C). EI-MS: m/z (%) ¼ 570 (19) [Mþ], 571(3.6)
[Mþþ1], 572 (6.5) [Mþþ2], 573 (6.5) [Mþþ3], 284 (100), 286 (32).
Anal. calcd. for C25H33ClN2O7P2 (570.9): C, 52.59; H, 5.83; Cl,
6.21; N, 4.91; P, 10.85. Found: C, 52.63; H 5.85; Cl, 6.25;
N, 4.97; P, 10.92. For NMR-spectral data see Tables 3
and 4.
Acid hydrolysis of synthesized BPs
Preparation of the bisphosphonic acids 9a–d, 12a–d, 13b,
14b
(E) Tetraethyl 2-(2-chloroquinolin-3-yl)-4-(furan-2-yl)-
4-oxobutane-1,1-diyl-diphosphonate(11d)
Yellow crystals, mp 168–1708C (MeOH), yield 73%. IR: nmax
,
General procedure
cmꢃ1: 1679 (C O), 1565 (C N), 1256, 1232 (2 P O), 1159,
0.3 g of bisphosphonate 8a–d, 11a–d, 13a or 14a was dis-
solved in 20 mL conc. HCl, and the mixture was heated under
reflux for ꢀ12 h (TLC). After concentrating in vacuo, the crude
material was diluted with AcOEt and water and then stirred
for 30 min. The layers were separated, and the aqueous layer
was evaporation to dryness. The precipitate was collected and
dried to give the respective bisphosphonic acids 9a–d, 12a–d,
13b or 14b, respectively.
–
–
–
–
–
–
1033 (2 P–O–C). EI-MS: m/z (%) 571 (15) [Mþ], 572 (2.9)
[Mþþ1], 573 (5.2) [Mþþ2], 574 (1.0) [Mþþ3], 285 (100), 287
(32). Anal. calcd. for C25H32ClNO8P2 (571.9): C, 52.50; H, 5.64;
Cl, 6.20; N, 2.45; P, 10.83. Found: C, 52.54; H, 5.71; Cl, 6.24;
N, 2.51; P, 10.87. For NMR-spectral data see Tables 3
and 4.
Reaction of chalcones 7e and 10e with tetraethyl
methylenebisphosphonate (1)
Preparation of the bisphosphonate derivatives 13a, 14a
(E) 4-Oxo-4-phenyl-2-(tetrazolo[1,5-a]quinolin-4-
yl)butane-1,1-diyl-diphosphonic acid (9a)
Dark brown substance, mp 3008C, yield 86%. IR: nmax, cmꢃ1
:
–
3450–3330 (P–OH), 1679 (C O), 1256, 1235 (P O), 1186 (tet-
–
–
General Method
–
A solution 1.25 mmol of 1 in 20 mL of absolute ethanol
containing 2.4 mmol sodium ethoxide was stirred at
0–(ꢃ5) 8C for about 0.5 h. A solution of 1.0 mmol of
(E-3-(tetrazolo[1,5-a]quinolin-3-yl)-2-cyanoacrylamide (7e) (or
its 2-chloroquinolin analog 10e) in 10 mL of absolute ethanol
was then added in one portion, and the reaction was com-
pleted by heating under reflux for ꢄ6 h (TLC). After the usual
work up and crystallization of the collected residue from the
proper solvent, biphosphonates 13a and 14a were obtained,
respectively.
razole). EI-MS: m/z (%) ¼ 472 (27) [Mþꢃ4]. Anal. calcd.
for C19H18N4O7P2 (476.32): C, 47.91; H, 3.81; N, 11.76; P,
13.01. Found: C, 47.94; H, 3.87; N, 11.83; P, 13.08. For
NMR-spectral data see Tables 3 and 4.
(E) 4-Oxo-2-(tetrazolo[1,5-a]quinolin-4-yl)-4-(thiophen-2-
yl)butane-1,1-diyl-diphosphonic acid (9b)
Brown substance, mp > 3008C, yield 91%. IR: nmax, cmꢃ1
:
–
–
3325–3248 (P–OH), 1681 (C O), 1254, 1238 (2 P O), 1188
–
–
(tetrazole). EI-MS: m/z (%) ¼ 478 (13) [Mþꢃ4]. Anal. calcd.
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Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
133
for C17H16N4O7P2S (482.34): C, 42.33; H, 3.34, N, 11.62;
P, 12.84; S, 6.57. Found: C, 42.37; H, 3.42; N, 11.59;
P, 12.92; S 6.65. For NMR-spectral data see Tables 3 and 4.
(E) 2-(2-Chloroquinolin-3-yl)-4-(furan-2-yl)-4-oxobutane-
1,1-diyl-diphosphonic acid (12d)
Dark brown substance; mp > 3008C, yield 83%. IR: nmax
,
cmꢃ1: 3425–3360 (P–OH), 1682 (C O), 1608 (C N), 1255,
–
–
–
–
þ
–
–
(E) 4-Oxo-4-(1H-pyrrol-2-yl)-2-(tetrazolo[1,5-a]quinolin-4-yl)-
butane-1,1-diyl-diphosphonic acid (9c)
1235 (2 P O). EI-MS: m/z (%) ¼ 455 (20) [M ꢃ4], 456 (4.0),
457 (6.5), 458 (<5). Anal. calcd. for C17H16ClNO8P2 (459.71):
C, 44.42; H, 3.51; Cl, 7.71; N, 3.05; P, 13.48. Found: C, 44.52; H,
3.61; Cl, 7.67; N, 3.13; P, 13.53. For NMR-spectral data see
Tables 3 and 4.
Dark brown substance; mp > 3008C (H2O/acetone); yield 72%.
IR: nmax, cmꢃ1: 3455–3328 (P–OH), 1679 (C O), 1253, 1235
–
–
þ
–
(P O), 1186 (tetrazole). EI-MS: m/z (%) ¼ 461 (9) [M ꢃ4]. Anal.
–
calcd. for C17H17N5O7P2 (465.29): C, 43.88; H, 3.68; N, 15.05;
P 13.31; Found: C, 43.94; H, 3.75; N, 15.09; P, 13.9. For
NMR-spectral data see Tables 3 and 4.
(E)-4-Amino-4-oxo-2-(tetrazolo[1,5-a]quinolin-4-yl)but-2-
ene-1,1-diyl-diphosphonic acid (13b)
Yellow powder; mp > 3008C; yield 83%. IR: nmax, cmꢃ1: 3445–
– –
3333 (OH, NH ), 1679 (C O), 1610 (C N), 1254, 1235 (P O),
– –
2
–
(E) 4-(Furan-2-yl)-4-oxo-2-(tetrazolo[1,5-a]quinolin-4-yl)-
butane-1,1-diyl-diphosphonic acid (9d)
–
1186 (tetrazole). EI-MS: m/z (%) ¼ 409 (22) [Mþꢃ4]. Anal. calcd.
for C13H13N5O7P2 (413.22): C, 37.79; H, 3.17; N, 16.95; P, 14.99.
Found: C, 37.84; H, 3.23; N, 17.04; P, 15.07. For NMR-spectral
data see Tables 3 and 4.
Dark brown crystals, mp > 3008C (H2O/acetone), yield 86%.
IR: nmax, cmꢃ1: 3435–3353 (P–OH), 1683 (C O), 1256, 1233
–
–
þ
–
–
(P O), 1186 (tetrazole). EI-MS: m/z (%) ¼ 462 (13) [M ꢃ4].
Anal. calcd. for C17H16N4O8P2 (466.28): C, 43.79; H, 3.46;
N, 12.02; P, 13.29. Found: C, 43.86; H, 3.49; N, 12.06;
P, 13.34. For NMR-spectral data see Tables 3 and 4.
(E)-4-Amino-2-(2-chloroquinolin-3-yl)-4-oxobut-2-ene-1,
1-diyl-diphosphonic acid (14b)
Brown powder; mp > 3008C; yield 83%. IR: nmax, cmꢃ1
:
–
3450–3328 (OH, NH ), 1680 (C O), 1606 (C N), 1251, 1238
–
2
–
(E) 2-(2-Chloroquinolin-3-yl)-4-oxo-4-phenylbutane-1,1-
diyl-diphosphonic acid (12a)
–
þ
–
(2 P O). EI-MS: m/z (%) ¼ 402 (16) [M ꢃ4], 403 (3), 404 (5.5),
–
Brown crystals; mp 2208C (MeOH), yield 82%. IR: nmax, cmꢃ1
:
405 (<5). Anal. calcd. for C13H13ClN2O7P2 (406.65): C, 38.40; H,
3.22; Cl, 8.72; N, 6.89; P, 15.23. Found: C, 38.53; H, 3.31; Cl,
8.79; N, 6.95; P, 15.29. For NMR-spectral data see Tables 3
and 4.
–
–
3410–3331 (P–OH), 1679 (C O), 1607 (C N), 1254, 1235
þ
–
–
–
–
(2 P O). EI-MS: m/z (%) ¼ 465 (30) [M ꢃ4], 466 (5.8), 467
(10.2), 468 (<5). Anal. calcd. for C19H18ClNO7P2 (469.75):
C, 48.58; H, 3.86; Cl, 7.55; N, 2.98; P, 13.19. Found:
C, 48.62; H, 3.89; Cl, 7.65; N, 2.95; P, 13.27. For NMR-spectral
data see Tables 3 and 4.
Anti-tumor activity screening
Anti-tumor potency of the new BP-acids 9a–d, 12a–d, 13b
and 14b in addition to the substrates 7a and 10a was
tested at a dose of 10 mM utilizing 44 different human tumor
cell lines, representing breast, ovarian, prostate lung and
brain cancer as well as leukemia and melanoma using adria-
mycin as a reference standard according to the reported
methods [22, 23]. 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 378C, 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 at the time of the tested compound
addition (time zero, Tz). Experimental tested compounds
are solubilized in dimethyl sulfoxide at 400-fold the desired
(E) 2-(2-Chloroquinolin-3-yl)-4-oxo-4-(thiophen-2-yl)-
butane-1,1-diyl-diphosphonic acid (12b)
Brown powder; mp 2318C (MeCN); yield 78%. IR: nmax, cmꢃ1
:
–
–
–
–
3433–3325 (OH), 1688 (C O), 1598 (C N), 1258, 1233 (2 P O).
–
–
EI-MS: m/z (%) ¼ 471 (27) [Mþꢃ4], 472 (15), 473 (9.0), 574 (<5).
Anal. calcd. for C17H16ClNO7P2S (475.78): C, 42.92; H, 3.39; Cl,
7.45; N, 2.94; P, 13.02; S, 6.81. Found: C, 42.95; H, 3.43; Cl,
7.51; N, 3.11; P, 13.11; S, 6.74. For NMR-spectral data see
Tables 3 and 4.
(E) 2-(2-Chloroquinolin-3-yl)-4-oxo-4-(1H-pyrrol-2-yl)-
butane-1,1-diyl-diphosphonic acid (12c)
Brown powder; mp 2258C (EtOH); yield 84%. IR: nmax, cmꢃ1
:
–
–
–
–
3428–3648 (P–OH), 1679 (C O), 1610 (C N), 1254, 1236 (P O).
–
–
EI-MS: m/z (%) ¼ 454 (17) [Mþꢃ4], 455 (3.3), 456 (5.7), 457
(<5), Anal. calcd. for C17H17ClN2O7P2 (458.73): C, 44.51; H,
3.74; Cl 7.73; N, 6.11; P, 13.50. Found: C, 44.56; H, 3.72; Cl,
7.77; N, 6.17; P, 13.57. For NMR-spectral data see Tables 3
and 4.
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W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
final maximum test concentration 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 concentration with
complete medium containing 50 mg/mL gentamicine.
Additional four, 10-fold or 1/2 log serial dilutions are made
to provide a total of five tested compound concentrations
(10ꢃ4 to 10ꢃ8 M concentrations) plus control. Aliquots of
100 mL of these different tested compound dilutions are
added to the appropriate microtiter wells already containing
100 mL of medium, resulting in the required final
concentrations.
Anti-inflammatory activity experiments in vivo;
carrageenin induced edema
Animals
All experiments have been conducted on adult Wistar
strain albino rats of either sex, weighing between 150–
200 g. Animals were kept in colony cages under
identical housing conditions at an ambient temperature
of 25 ꢂ 28C and 45–55% relative humidity with 12 h
light-dark cycle in the departmental animal room and fed
on standard diet. Animals were acclimatized for a week
before use.
Experimental model of inflammation
Following the tested compound addition, the plates
are incubated for an additional 48 h at 378C, 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 addition of 50 mL of cold
50% (w/v) TCA (final concentration, 10% TCA) and incubated
for 60 min at 48C. The supernatant is discarded, 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 are air dried.
Bound stain is subsequently solubilized with 10 mM
trizma base, and the absorbance is read on an automated
plate at a wavelength of 515 nm. For cells suspension, the
methodology 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 absorbance 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 concen-
tration levels.
Carrageenin induced paw edema was used throughout the
investigation: Standard drug clodronic acid (A), substrates
(7a, 10b), synthesized BP-acids (9a–d, 12a–d, 13b, 14b) and
solutions:
(a)
(b)
(c)
Powder of the pure carrageenin was used and fresh sus-
pension was prepared in distilled water to make 1%
carrageenin solution.
Fresh tested compound solutions were made by adding
10 mg of the compound in 500 mg carboxymethyl cellu-
lose (CMC) and 50 mL distilled water.
Experimental inflammation: It was produced by the fol-
lowing method: carrageenin induced paw edema in rats:
1% carrageenin suspension was prepared as a homo-
geneous solution in distilled water. A volume of 0.1 mL
of carrageenin solution was injected through a 26 gauge
needle into the plantar surface of the left hind paw below
the plantar aponeurosis. The volume of the paw was
measured before and at different intervals for 4 h after
injection of carrageenin. The difference in the paw vol-
ume before and after administration of the phlogistic
agent was taken as the measure of pedal edema. The
compounds whose effects have been studied on this
particular model were administered as per schedule.
Measurement of paw volume: The volume of hind paw of
the rats up to the ankle joint was measured by plathysmo-
graphically by the mercury displacement method. The
ankle joint of the rats was marked with a skin marking
pencil and the paw was dipped in the mercury, so that the
mark on the paw coincides with a prefixed line kept
constant on the syringe. The level of the mercury was
every time brought to the level of this line by adjusting
the height of the displaced mercury. The difference in the
paw volume before and after injection of the phlogistic
agents was taken as a measure of pedal edema. The
change in paw volume was expressed in ‘‘mL’’ of mercury
displaced.
Percentage growth inhibition is calculated as:
(d)
½ðTiꢃTzÞ=½ðCꢃTzÞ ꢅ 100 with Ti ꢁ Tz
½ðTiꢃTzÞ=TzÞꢇ ꢅ 100 with Ti < Tz
Growth inhibition of 50% (GI50) is calculated from:
½ðTiꢃTzÞ=½ðCꢃTzÞ ꢅ 100 ¼ 50
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 1 represents the observed percentage growth of each
cell line treated with a certain tested compound relative to
control cell line experiments.
The anti-inflammatory activity was expressed as percent-
age inhibition of edema volume in the treated animals in
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Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
Activity of Bisphosphonic Acids vs. Cancer and Inflammatory Diseases
135
[3] A. Lipton, R. L. Theriault, G. N. Hortobagyi, J. Simeone, R. D.
Knight, K. Mellars, D. J. Reitsma, M. Heffernan, J. Seaman,
J. Cancer 2000, 88, 1082–1090.
comparison with the control group.
ðVcꢃVtÞ
% Inhibition of edema ¼
ꢅ 100
[4] M. Busch, M. Rave-Frank, A. Hille, E. Duhmke, Eur. J. Med. Res.
1998, 3, 427–431.
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
clodronic acid, reference standard, treated group according
to the following equation:
[5] (a) J. E. Brown, R. E. Coleman, Breast Cancer Res. 2002, 4, 24–
29; (b) I. J. Diel, N. Marschner, M. Kindler, O. Lange, M.
Untch, H. J. Hurtz, G. P. Breitbach, B. Richter, Proc. ASCO
1999, 18, 128.
[6] (a) L. I. Plotkin, J. I. Aguirre, S. Kousteni, S. C. Manolagas, T.
Bellido, J. Biol. Chem. 2005, 280, 7317 –7325; (b) R. G. Russell,
Bisphosphonates: From bench to bedside, Ann. NY Acad. Sci.
2006, 1068, 367–401.
% Potency
%Edema inhibition of tested compound treated groups
¼
ꢅ 100
[7] W. M. Abdou, A. A. Kamel, A. A. Shaddy, Eur. J. Med. Chem.
2010, 45, 5217–5224.
%Edema inhibition of clodronic acid treated groups
[8] W. M. Abdou, R. E. Khidre, A. A. Shaddy, J. Heterocycl. Chem.
2011, accepted for publication. ((DOI!))
Toxicity evaluation
[9] W. M. Abdou, A. A. Shaddy, J. Med. Chem. Res. 2010, 19, 39–40.
[10] W. M. Abdou, A. A. Shaddy, ARKIVOC 2009, 9, 143–182.
The LD50 determination of the most promising synthesized
anti-inflammatory active agents (9c and 12c) was determined
by the standard known LD50 method in mice. Albino mice
weighing 20–25 g were divided into 6 groups of 8 mice each.
Administrations of the tested compounds (9c and 12c) dis-
solved in the same vehicle solution in 500, 750, and 1000 mg/
kg (body weight) were given intraperitoneally. The control
groups were given in buffer solution only. The toxic symp-
toms, mortality rates and postmortem findings in each group
were recorded 24 h post-administration.
[11] W. M. Abdou, N. A. Ganoub, A. Geronikaki, E. Sabry, Eur. J.
Med. Chem. 2008, 43, 1015–1024.
[12] W. M. Abdou, M. D. Khidre, A. A. Sediek, Lett. Org. Chem. 2006,
3, 634–640.
[13] W. M. Abdou, N. A. Ganoub, A. F. Fahmy, A. A. Shaddy,
Monatsh. Chem. 2006, 136, 105–116.
[14] W. M. Abdou, Y. O. Elkhoshnieh, N. A. Ganoub, SYNLETT
2002, 785–790.
[15] M. S. Shahabuddin, M. Nambiar, B. Choudhary, G. M.
Advirao, S. C. Raghavan, Invest. New Drugs 2010, 28, 35–48.
LD50 of the tested compounds were calculated according to
the following formula:
[16] (a) M. Yamato, Y. Takeuchi, K. Hashigaki, Y. Ikeda, M. R.
Chang, K. Takeuchi, M. Matsushima, T. Tsuruo, T. Tashiro,
J. Med. Chem. 1989, 32, 1295 –1300; (b) Z. Ma, Y. Hano, T.
Nomura, Y. Chen, Med. Chem. Lett. 2004, 14, 1193–1196.
X
LD50 ¼ Dmꢃ ðz ꢅ dÞ=n
where Dm ¼ the largest dose which kills all animals, z ¼ mean
of dead animals between two successive groups, d ¼ the
constant factor between two successive doses, n ¼ number
of animals in each group, S ¼ the sum of (z ꢅ d).
[17] N. M. Ibrahim, H. A. A. Yosef, J. Chem. Res. 2009, 220–224.
[18] R. Ferrer, G. Lobo, N. Gamboq, J. Rodrigues, C. Abramjuk, K.
Jung, M. Lein, J. E. Charris, Sci. Pharm. 2009, 77, 725–741.
[19] (a) A. H. Kategaonkar, S. B. Sapkal, B. R. Madje, B. B. Shingate,
M. S. Shingare, Chem. Heterocycl. Compd. 2010, 46, 933–937;
(b) P. Cmoch, L. Stefaniak, G. A. Webb, Magn. Reson. Chem.
1997, 35, 237–242.
Financial support from the Egyptian Academy of Scientific Research and
Technology is gratefully acknowledged. We thank NYU Cancer Institute,
NY, USA, and the Biology Lab, Banha University, Banha, Egypt, for
carrying out the pharmacological screening.
[20] H. Fleisch, Bisphosphonates: A New Class of Drugs in
Diseases of Bone and Calcium Metabolism, in Handbook of
Experimental Pharmacology (Ed.: P. F. Baker), Vol. 83, Springer,
Berlin/Heidelberg 1988, pp. 441–465.
The authors have declared no conflict of interest.
[21] F. H. M. Ebetino, D. Francis, M. J. Rogers, R. G. G. Russell, Rev.
Contemp. Pharmacother. 1998, 9, 233–243.
References
[22] T. Kitamura, M. Itoh, T. Noda, M. Matsuura, K. Wakabayashi,
Int. J. Cancer 2004, 109, 576–580.
[1] R. Tedesco, A. N. Shaw, R. Bambal, D. Chai, N. O. Concha,
M. G. Darcy, D. Dhanak, D. M. Fitch, A. Gates, W. G.
Gerhardt, D. L. Halegoua, C. Han, G. A. Hofmann, V. K.
Johnston, A. C. Kaura, N. Liu, R. M. Keenan, J. Lin-Goerke,
R. T. Sarisky, K. J. Wiggall, M. N. Zimmerman, K. J. Duffy,
J. Med. Chem. 2006, 49, 971–983.
[23] M. R. Boyd, K. D. Paull, Drug Dev. Res. 1995, 34, 91–109.
[24] C. A. Wintar, E. A. Risley, G. W. Nuss, Proc. Soc. Exp. Biol. Med.
1962, 111, 544–547.
[25] M. Di Rosa, J. P. Giroud, D. A. Willoughby, J. Pathol. 1971, 104,
15–29.
[26] R. A. Nugent, S. T. Schlachter, M. Murphy, C. J. Dunn, N. D.
Staite, L. A. Galinet, S. K. Shields, H. Wu, D. G. Aspar, K. A.
Richard, J. Med. Chem. 1994, 37, 4449–4454.
[2] T. Yoneda, A. Sasaki, C. Dustan, P. J. William, F. Bauss,
Y. A. De Clerck, G. R. Mundy, J. Clin. Invest. 1997, 99, 2509–
2517.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

136
W. M. Abdou et al.
Arch. Pharm. Chem. Life Sci. 2012, 345, 123–136
[27] 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, J. Med. Chem. 1993, 36,
134–139.
[29] N. J. Datta, R. C. Khunt, A. R. Parikh, Orient. J. Chem. 2002, 18,
131–134.
[30] N. M. Fathy, G. H. Elgemeie, J. Chem. Eng. Data 1988, 33, 218–
219.
[28] P. Cmoch, L. Stefaniak, G. A. Webb, Magn. Reson. Chem. 1997,
35, 237–242.
[31] V. Tiwari, P. Ali, J. Meshram, Int. J. Chem. Tech. Res. 2010, 2,
1031–1035.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com