Computer-aided design, synthesis, and biological studies of anticological nitrogen-containing tetraphosphonic acids against melanoma

Article abstract of DOI:10.1007/s00706-018-2196-9

Abstract: As a part of our quest to develop new bioactive bisphosphonic acids, we synthesized a series of bis(α-aminobisphosphonic acids) in good yields (66–78%) as new entities for treating malignant melanoma. The reaction of the Schiff bases, 1,4-pheney

Full text of DOI:10.1007/s00706-018-2196-9

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ORIGINAL PAPER
Computer-aided design, synthesis, and biological studies
of anticological nitrogen-containing tetraphosphonic acids against
melanoma
Mohamed S. Bekheit1 Reham F. Barghash1 Wafaa M. Abdou1
•
•
Received: 16 January 2018 / Accepted: 3 April 2018
Ó Springer-Verlag GmbH Austria, part of Springer Nature 2018
Abstract
Asapartofourquesttodevelopnewbioactivebisphosphonicacids, wesynthesizedaseriesofbis(a-aminobisphosphonic acids) in
good yields (66–78%) as new entities for treating malignant melanoma. The reaction of the Schiff bases, 1,4-pheneylenediimines
with the Horner–Emmons–Wadsworth reagent, tetraethyl methylene-1,1-bisphosphonate in DMF/LiOH (aq) solution led
exclusively to a meso form of nitrogen-containing tetraphosphonates (NTPs) (bis(a-aminobisphosphonates)). Next, hydrolysis of
the ester moieties of the tetraphosphonate products yielded the corresponding tetraphosphonic acids, which treated with MeOH/
KOH (aq, 20%) to give the respective NTP-tetrapotassium salts. Prior to synthesis, the suggested structures (and others) were
applied to the computer-assisted molecular modeling, PASS program to investigate their prospective biological properties.
Cytotoxic properties were later evaluated against five malignant melanoma cell lines that originated from different categories of
malignant melanoma primary stage (I/II), histologically advanced stage (III/IV), and metastasized malignancy. Almost all tested
compounds showed antitumor activity though on different levels. Three NTP salts were found to have activity in the range of GI50
0.650–5.73 lM vs. control reference GI50: 1.745–6.50 lM. Structure activity relationship is also discussed.
Graphical abstract
Keywords Bis(a-aminobisphosphonic acids) Á Pudovik reaction Á Malignant melanoma Á Structure activity relationship
Introduction
of the scientific community as well as public health offi-
cials worldwide [1, 2]. Research also shows that mortality
rates among patients are increasing due to not only the
numerous side effects but also the resistance to prescribed
drugs [3, 4]. Therefore, research targeting the development
of more effective therapeutic agents has been recently
intensified. Melanoma, also known as malignant mela-
noma, is a type of cancer that originates from pigments
containing skin cells known as melanocytes [5]. Despite
melanoma is less common than other kinds of skin cancer
The alarming rates of the growing resistance to
chemotherapeutic drugs, which are used for treating dif-
ferent cancers, have become a major concern for members
& Wafaa M. Abdou
wabdou@link.net
1
Chemical Industries Research Division, National Research
Centre, Elbuhouth St., Dokki, Cairo 12622, Egypt
123

M. S. Bekheit et al.
(basal and squamous cell carcinoma), it is the most serious
type of skin malignancy. The melanoma cancerous growth
develops from unprepared mutagenized DNA of skin cells,
which is mostly caused by either intense occasional
exposure to sun-UV irradiation, over-using tanning bed
device leading to skin burn, or could be due to heredity
genetic defects. These factors trigger the skin cells to split
rapidly and multiply increase causing the DNA mutation
followed by skin cancer [6, 7]. In 2016, the WHO
announced that an estimated 76,330 melanoma cases were
recorded with about 46,810 cases reported in males and
29,510 cases in women. It is also reported that melanoma
kills an estimated 10,130 people in the USA annually.
Fortunately, melanoma malignancy is almost always cur-
able if it is diagnosed in the early stage (I/II) phase.
Otherwise the proliferation can advance rapidly generating
early metastasis. In addition, the melanoma (III/IV) phase
is characterized by its relatively high therapeutically
resistance [8] (Fig. 1 [9]) and, therefore, new treatment
strategies need to be developed.
number of investigations studied the effect of NBPs on
melanoma [25, 26]. In a detailed study, Geilen et al.
showed that the NBPs drug, pamidronate, could induce
apoptosis and inhibits the proliferation of all types of
human malignant melanoma cells [27, 28].
Building on this information, we demonstrate that
treating malignant melanoma cell lines with bis(a-amino-
bisphosphonate) tetrapotassium salts induced medium to
excellent apoptosis and inhibited the proliferation in a
concentration-dependent manner. Remarkably, the concept
of our present investigation was developed using the pre-
dicted spectrum of the biological activity of the suggested
compounds (and others). The prediction process was run in
the early stage of the work, using the computer-assisted
molecular modeling, PASS 2014 program (prediction
activity spectra of substances) [29, 30]. It is based on a
robust analysis of structure–activity relationships [30] in a
heterogeneous training set that currently includes 7000
types of biological activity. There are many examples of
successful use of the PASS approach for finding new
pharmaceutical agents [31–34].
Nitrogen-containing bisphosphonates (NBPs) are well
known as potent inhibitors of bone resorption and are
frequently used for the treatment of Paget’s disease,
osteoporosis, and cancer-induced osteolysis [10]. The main
mechanism of the inhibitory effect of NBPs on bone
metastasis relies on the inhibition of osteoclast’s growth
[11, 12]. Furthermore, clinic findings showed that NBPs
revealed a direct apoptotic effect on several types of car-
cinoma cells. This is attributed to the capability of NBPs to
inhibit farnesyl diphosphate synthase, which induces
blocking of the synthesis of higher isoprenoids [13, 14]. As
a result of this mechanism, NBPs inactivate monomeric
G-proteins of Ras and Rho families for which proliferation
is a functional requirement. Encouraged by these findings,
the beneficial use of NBPs in the treatment of different
kinds of cancer, especially breast, colon, prostate, cervix,
and multiple myeloma has been intensively reported by us
[15–20] and others [21–24]. Nevertheless, only a limited
Results and discussion
Prediction spectra of biological activity
The analysis of the biological activity spectra prediction for
NTP acids 6a–6j is a good example of in silico study of
chemical compounds before running their experimental
processes. It is available on the free website with the
internet version of PharmaExpert: http://www.pharmaex
pert.ru [29–34]. The biological activity spectrum of a
compound is a list of biological activity types for which the
probability to be revealed is represented by Pa and the
probability not to be revealed is represented by Pi. The
probabilities Pa and Pi are independent and their values
vary from 0 to 1. In this work, the biological activity
Fig. 1 a Regression in I/II stage of melanoma; b subcapsular lymph node metastasis (malignant melanoma III/IV stage)
123

Computer-aided design, synthesis, and biological studies of anticological nitrogen-…
spectra were predicated for the new acid structures 6a–6j
using the PASS.2014.1 version. By default, in PASS
Pa = Pi is chosen as a threshold and that compounds with
Pa [ Pi are suggested to be active. Table 1 summarizes
the prediction spectra of most activity types for compounds
6a–6j. Accordingly, other than bone resorption and relative
diseases, the cytotoxic activity for these specified com-
pounds was the promising probability and, therefore, was
considered.
result is expected since these compounds are achiral
compounds that have two chiral centers (C2 and C20) and
are superimposed on their mirror image. According to
previous reports [44–46], the mechanism of the formation
of the meso isomer, exclusively, is considered a two-step
reaction. The stereogenic center that created in the first
mono-aminobisphosphonates would probably control the
attack of the second bisphosphonate molecule strictly at
one face.
Next, ester cleavage of NTPs 5a–5j was carried out by
boiling the ester in chloroform solution containing
trimethylsilyl bromide (TMS-Br) to give the respective
NTP-acid counterparts 6a–6j. The resulting acids were
dramatically hygroscopic and could be kept in a vacuumed
desiccator only for few days, which were enough for
identification purposes. Subsequently, 6a–6j were stirred in
MeOH/KOH (10% aq) solution (20 cm3, 1:3 v/v) for 1 h,
followed by concentration to give the respective
tetrapotassium salts 7a–7j [40, 47]. Furthermore, in anal-
ogy to 5b–5j, the acids 6b–6j and 7b–7j were also assigned
meso form (Scheme 2).
Chemistry
The roadmap for the synthesis of new series of N-te-
traphosphonates (NTPs) and relevant acid salts is depicted
in Schemes 1 and 2. The key Schiff-base substrates, N1,N4-
[(diarylidene/dihetarylidene)phenylene]-1,4-diamines 3a–
3j were prepared by treating 1,4-phenylenediamine (1)
with the appropriate aldehydes 2a–2j according to the
reported
methods
[35–37].
Treating
N1,N4-1,4-
phenylenediimines 3a–3j with the Horner–Emmons–
Wadsworth (HEW) reagent tetraethyl methylene-1,1-bis-
phophonate (4) in dimethylformamide solution containing
aqueous lithium hydroxide (DMF/LiOH aq), our targets
Pharmacology
octaethyl
2,20-[1,4-phenylenebis(azanediyl)]bis[2-(aryl/
hetryl)ethane-2-1,1-triyl]tetraphosphonates 5a–5j were
obtained in good yields (66–78%), via Pudovik reaction
(66–78%) [38] (Scheme 1).
Reports by pharmaclinic laboratories confirmed the
remarkable therapeutic potency of bisphosphonic acids for
many pathogens as compared to the parent bisphosphonate
esters [47, 48]. Therefore, in this work only N-tetraphos-
phonic acids 6a–6j were applied to the prediction process
while their salts were applied for the biological screening
assessment.
The structures and the purity of 5a–5j were substanti-
ated by the spectrometric (MS, IR, 1H, 13C, and 31P NMR)
data and the elemental analysis. Due to the symmetry of the
molecules [39, 40], some shifts overlapped in the NMR
spectra. We determined by the NMR experiments that
[except 5a (Y = H)] the occurring products 5b–5j present
exclusively in the meso diastereoisomeric form. Com-
pounds 5b–5j revealed the presence of one sharp signal of
Antitumor evaluation
Screening of the antineoplastic properties of the synthe-
sized new salts 7a–7j was in vitro evaluated against a panel
of human malignant melanoma cell lines, Sk-MEL-2, SK-
MEL-5 (obtained from primary tumor), LOX-IMMVI
(originated from metastasized malignancy), and UACC-
257, UACC-62 (derived from patients with histologically
confirmed advanced stage by surgical intervention). The
biological process was carried out utilizing the sulforho-
damine-B (SRB) protein assay [49–51]. Each compound of
7a–7j and the reference standard pamidronate (3-amino-1-
hydroxypropylidene-1,1-bisphosphonic acid, Fig. 2) was
tested at a minimum of five concentrations at tenfold
dilution against every cell line in the panel with 48 h
continuous drug exposure protocol. The results are dis-
played in Table 2 while the flow of GI50 of the most active
NBP salts vs. the malignant melanoma carcinoma cell lines
is displayed in Fig. 3.
1
at dp * 25.3 ppm in the 31P NMR spectra. In their H
NMR spectra, they displayed two signal sets for methine
0
protons. The first was ascribed to the Ha and Ha protons,
appeared at dH * 2.5 ppm as two doublet of triplets as a
result of the coupling with the (2)31P nucleus and the CH
proton whereas the second set appeared at dH * 4.3 ppm
as a multiplet obscur0ed by the 8 CH2OP protons and
assigned to Hb and Hb protons. To exclude the formation
of the second diastereoisomeric configuration, the postre-
action crude mixture was subjected to NMR spectroscopy
in each case. The NMR analysis disclosed no presence of
the other diastereoisomeric isomer and, therefore, we can
safely state that the stereoselectivity was not formed from
the preferential crystallization of one of the isomers. Since
these molecules possess a mirror plane and in analogy to
the previous reports in similar cases [41–44], one would
obtain meso compounds. Consequently, we proposed that
bis(a-aminobisphosphonate) are meso form. Indeed, this
The findings in Table 2 show that all tested NTP salts
display significant inhibition of SK-MEL-2 and SK-MEL-5
123

M. S. Bekheit et al.
Table 1 Pa values of biological activities for the most expected potent types of activities for N-tetraphosphonic acids (NTP acids) 6a–6j
Cmpd.
Predictive activity
Pa
Cmpd.
Predictive activity
Pa
6a
Antiosteoporotic
0.850
0.750
0.613
0.482
0.571
0.693
0.750
0.580
0.513
0.582
0.511
0.139
0.750
0.724
0.421
0.512
0.703
0.571
0.641
0.533
0.550
0.537
0.550
0.537
0.693
0.537
0.484
0.368
0.278
0.750
0.124
0.651
0.512
0.326
0.312
0.504
0.519
0.442
0.780
0.415
0.614
0.410
0.342
0.480
0.751
0.194
0.451
0.251
6f
Bone formation stimulant
Acute neurologic disorders treatment
Leukopoiesis stimulant
Antineoplastic (bone cancer)
Antineoplastic (lung cancer)
Antineoplastic (ovarian cancer)
Antineoplastic (melanoma)
Antileukemic
0.543
0.358
0.390
0.527
0.432
0.504
0.761
0.418
0.273
0.414
0.442
0.331
0.596
0.529
0.294
0.683
0.555
0.270
0.694
0.535
0.875
0.270
0.375
0.470
0.842
0.780
0.451
0.294
0.510
0.503
0.684
0.773
0.426
0.571
0.212
0.842
0.780
0.551
0.394
0.510
0.503
0.484
0.473
0.424
0.610
0.503
0.424
0.118
Hypolipemic
Bone diseases treatment
Acute neurologic disorders treatment
Leukopoiesis stimulant
Antineoplastic (bone cancer)
Antineoplastic (melanoma)
Antineoplastic (lymphoma)
Antitumor (ovarian cancer)
Immunosuppressant
Antineoplastic (brain cancer)
Immunosuppressant
Antiinflammatory
Antiinflammatory
Toxicity
Toxicity
6b
Antiosteoporotic
6g
Bone formation stimulant
Antineoplastic (bone cancer)
Antineoplastic (lung cancer)
Antineoplastic (bone cancer)
Antitumor (ovarian cancer)
Antineoplastic (brain cancer)
Antineoplastic
Bone formation stimulant
Contraceptive female
Antineoplastic (bone cancer)
Antineoplastic (lung cancer)
Antitumor (ovarian cancer)
Antineoplastic (melanoma)
Antineoplastic
Antiosteoporotic
Antineoplastic (bone cancer)
Immunosuppressant
Antineoplastic (melanoma)
Immunosuppressant
Antiinflammatory
Antiinflammatory
Toxicity
Toxicity
6c
Antiosteoporotic
6h
Antiosteoporotic
Bone diseases treatment
Antiarthritic
Bone diseases treatment
Antiarthritic
Antineoplastic (lung cancer)
Antitumor (ovarian cancer)
Antitumor (ovarian cancer)
Antineoplastic (brain cancer)
Antineoplastic (melanoma)
Immunosuppressant
Antineoplastic (lung cancer)
Antineoplastic (ovarian cancer)
Antineoplastic (ovarian cancer)
Antineoplastic (brain cancer
Antineoplastic (melanoma)
Immunosuppressant
Antiinflammatory
Antiinflammatory
Toxicity
Toxicity
6d
Antiosteoporotic
6i
Bone formation stimulant
Bone diseases treatment
Hypolipemic
Bone formation stimulant
Hypolipemic
Bone diseases treatment
Acute neurologic disorders treatment
Antineoplastic (bone cancer)
Leukopoiesis stimulant
Antineoplastic (lung cancer)
Antitumor (ovarian cancer)
Antineoplastic (melanoma)
Antileukemic
Antiarthritic
Antineoplastic (bone cancer)
Antineoplastic (ovarian cancer)
Antineoplastic (lung)
Antineoplastic (gastric cancer)
Antineoplastic (ovarian cancer)
Antineoplastic (melanoma)
Antileukemic
Antiinflammatory
Antiinflammatory
Toxicity
Toxicity
123

Computer-aided design, synthesis, and biological studies of anticological nitrogen-…
Table 1 (continued)
Cmpd.
Predictive activity
Pa
Cmpd.
Predictive activity
Pa
6e
Bone diseases treatment
0.843
0.443
0.558
0.690
0.327
0.414
6j
Bone diseases treatment
Acute neurologic disorders treatment
Leukopoiesis stimulant
Contraceptive female
0.643
0.243
0.528
0.490
0.362
0.648
0.532
0.502
0.581
0.468
0.253
0.414
Acute neurologic disorders treatment
Leukopoiesis stimulant
Contraceptive female
Antineoplastic (lung cancer)
Antineoplastic (non-Hodgkin’s lymphoma)
Antineoplastic (lung cancer)
Antineoplastic (melanoma)
Antineoplastic (bone cancer)
Antineoplastic (ovarian cancer)
Immunosuppressant
Antineoplastic (melanoma)
Antitumor (ovarian cancer)
Immunosuppressant
Antiinflammatory
0.691
0.468
0.253
0.514
0.274
Antiinflammatory
Immunosuppressant
Toxicity
Toxicity
(I/II-stages) cell lines in the range of GI50 0.634–3.846 lM
vs. the control reference GI50 1.745 and 2.89 lM. The
remarkable effect of the low concentrations on I/II-phases
confirmed the importance of diagnosing the skin malignant
melanoma in an early stage. However, higher concentra-
tions of the tested salts and the control drug were needed to
decrease the cell viability of UACC-257 and UACC-62 cell
lines that originated from patients with histologically
confirmed advanced stage by surgical invention with GI50
ranging from 2.885 to 10.15 lM vs. reference drug with
GI50 4.550 and 4.454 lM. Contrary to these results, the
tumor cell line LOX-IMVI that derived from metastasized
malignancy showed highest resistance toward all tested
compounds thereof only relatively high concentrations
could affect the inhibition with GI50 (7a–7j: from 3.74 to
22.7 lM vs. the control GI50 6.5 lM). Furthermore, the
biological results of 7a–7j showed that the substituents at
C2 as well as at the aryl ring played a vital role in the
activity of these compounds, suggesting specific interac-
tions of these groups with the biological targets. Among the
ten NTP salts, 7a (Y = H) and 7e (Y = F) were the most
active, and more potent than the positive control toward all
the tested cell lines. However, the efficacy dramatically
dropped on replacing the H at C2 by phenyl (7b) or thio-
phene (7j), taking into account that the hetero ring (7j)
imposed slightly higher activity than the phenyl moiety
(7b, Y = Ph). Considering the substituents at the aryl ring,
the structure activity relationships revealed that the cyto-
toxic activity depends not only on the nature of the sub-
stituent but also on its position and number. Thus,
introduction of the fluorine atom 7e and the dimethylamino
group 7g to the parent phenyl group in 7b imposed a sig-
nificant activity on the molecule. These two substituents
were found to be the most favorable, followed by a chlo-
rine atom at 2- or 4-positions in 7c and 7d, whereby the
introduction of the chlorine in the para position caused
better inhibition than that at 2-position. Replacing the flu-
orine atom with the CF3 group (7f) resulted in an obvious
decrease in activity when compared with the salt 7e
(Y = 4-FC6H4) whereas insertion of methoxy groups 7h, 7i
in the molecule exhibited less inhibitory action (7h [ 7i)
among other substituents at the phenyl nucleus against all
tested carcinoma cells. The order of activity is:
7a
(H) [ 7e
(4-FC6H4) [ 7g
(4-Me2NC6H4 [ 7d
(4-ClC6H4) [ 7c (2-ClC6H4) [ 7f (4-F3CC6H4) [ 7h
(4-MeOC6H4) [ 7i (2,5-(MeO)2C6H3) [ 7j (thiophene) [
7b (Ph).
Conclusion
A novel class of nitrogen-tetraphosphonic acid derivatives
as new entities for malignant melanoma treatment was
developed. The cytotoxic activity was in vitro evaluated
using the sulforhodamine-B (SRB) protein assay on a panel
of five melanoma carcinoma cell lines. Almost all tested
compounds showed antitumor activity but on different
levels and this activity was even better than that of the
positive control reference Pamidronate, especially toward
SK-MEL-2 and SK-MEL-5 (I/II-stage) cell lines. Never-
theless, only five compounds (out of ten) 7a (H), 7e (4-
FC6H4), 7g (4-Me2NC6H4), 7d (4-ClC6H4), and 7c (2-
ClC6H4) displayed good activity on UACC-257, UACC-62
cell lines obtained from histologically advanced stage (III/
IV-phases). On the other hand, the cell line, LOX-IMVI,
that originated from metastasized melanoma showed high
resistance, and only compounds 7a, 7e, and 7g exhibited
significant activity on utilizing relatively higher concen-
tration. It could be concluded that these three compounds
were the most potent and even better than the positive
123

M. S. Bekheit et al.
Scheme 1
control, Pamidronate toward all tested malignant mela-
noma cell lines. The antitumor potential among the new
NMR spectra were measured with a JEOL E.C.A-500 MHz
1
(13C: 125.7 MHz, H: 500 MHz, 31P: 202.4 MHz) spec-
1
NTP
salts
7a–7j
7a [ 7e [ 7g [ 7d [ 7c [ 7f [ 7h [ 7i [ 7j [ 7b.
could
be
presented
as
trometer (JEOL, Japan). H and 13C NMR spectra were
recorded with trimethylsilane as internal standard in
DMSO solution. Chemical shifts (d) are given in ppm; 31P
NMR spectra were recorded with H3PO4 (85%) as external
reference. The mass spectra were performed at 70 eV on an
MS-50 Kratos (A.E.I.) spectrometer provided with a data
system (spectrometer (Kratos, UK). Elemental analyses
were carried out at the Microanalysis Laboratory, Cairo
University, Cairo, Egypt, using elementary Analysen-sys-
teme GmbH-vario EL III Element Analyzer, Germany;
Experimental
Melting points were determined with an open capillary
tube on an Electro-thermal (variable heater) melting point
apparatus and were corrected. IR spectra were recorded on
a JASCO FT-IR 6100 using KBr disc (JASCO, Japan).
123

Computer-aided design, synthesis, and biological studies of anticological nitrogen-…
Scheme 2
O
O
H
CHCl3 /TMS-Br
/3 h
H
P(OH)(OZ)
(ZO)(HO)P
2
5a-5j
6a-6j
2
H
H
Y
N
N
Y
H
H
6a-6j (Z = H)
(6b-6j, meso-form)
EtOH / r.t.
10-30 min
EtOH / KOH (10% aq)
7a-7j (Z = K)
(7b-7j, meso-form)
their results were in good agreement with the calculated
data. The appropriate precautions in handling moisture-
sensitive compounds were observed. The purity of all new
samples was verified by microchemical analysis (C/H/N/S)
and spectroscopy. Thin-layer chromatography (TLC):
Merck 0.2 mm silica gel 60 F254 analytic aluminum
plates. The substrates 3a–3j were prepared according to
Refs. [35–37].
NH2
O
P
O
HO
C
P
OH
OH OH OH
Fig. 2 Structure of pamidronate
Table 2 Antitumor properties of bis(a-aminobisphosphonate) salts (NTP salts) 7a–7j vs. melanoma cell lines using pamidronate drug as standard
reference
Cmpd.
Y
GI50*/lM
SK-MEL-2
SK-MEL-5
LOX-IMVI
UACC-257
UACC-62
7a
7b
7c
7d
7e
7f
H
1.393 0.11
3.650 0.47
1.618 0.92
1.729 1.67
0.634 1.51
3.846 0.24
1.590 1.83
3.536 2.66
3.970 2.52
3.294 1.82
1.745 0.18
0.77 0.26
3.74 0.35
0.650 0.26
2.57 0.78
2.36 0.68
1.76 2.56
2.53 3.06
2.41 0.88
3.30 0.07
3.23 3.27
2.89 0. 37
3.74 1.99
22.70 3.59
8.13 2.86
8.03 0.37
4.93 0.72
9.16 1.88
5.73 1.69
12.31 0.48
10.53 4.57
20.11 4.08
6.50 0.33
3.381 3.76
10.15 0.78
4.833 3.78
4.736 0.08
2.885 0.87
5.714 0.45
3.715 0.92
8.255 0.77
8.816 1.06
9.433 1.10
4.550 1.48
3.274 0.57
9.679 0.49
5.830 2.17
5.111 3.47
3.371 2.05
6.677 1.76
3.712 3.87
7.226 1.55
7.684 2.35
8.083 1.18
4.454 0.34
Ph
2-ClC6H4
4-ClC6H4
4-FC6H4
4-CF3C6H4
4-Me2N-C6H4
4-MeO-C6H4
(2,5-MeO)2-C6H3
Thiophene
7g
7h
7i
7j
Pamidronate [27]
123

M. S. Bekheit et al.
Fig. 3 Flow of GI50 of most
active NTP salts compared to
pamidronate vs. melanoma
carcinoma cell lines
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis(2-phenyl-
ethane-2,1,1-triyl)tetraphosphonate (5b, C38H60N2O12P4)
Yield: 1.3 g (69%); m.p.: 125 °C (from cyclohexane); H
General procedure to prepare nitrogen-
bisphosphonates (NBPs) 5a–5j
1
NMR (500 MHz, DMSO-d6): d = 1.18, 1.29 (2dt, 24H,
To a stirred solution of the starting materials 3a–3j
(2.2 mmol) dissolved in 20 cm3 DMF, a solution of LiOH
(6.6 mmol) dissolved into a least amount of distilled water
was added, followed by the addition of tetraethyl
methylenebiphosphonate (4, 5 mmol) in one portion. The
reaction mixture was heated under reflux temperature
for & 6–8 h (TLC). After cooling, the product mixture
was poured in ice water and acidified with HCl, followed
by extraction with AcOEt (3 9 20 cm3) and the combined
organic phase was washed and dried over anhy Na2SO4.
After removal of the volatile materials under vacuum, the
resulting residue was collected and crystallized from the
proper solvent to give the bisphosphonates 5a–5j as col-
orless materials in good to high yields.
4
3JHH = 5.9, JPH = 4.1 Hz, 8 MeCO), 2.49, 2.59 (2dt, 2H,
0
3JHH = 12.9, 2JPH = 19.2 Hz, Ha, Ha ), 3.088–4.01 (2dq (m),
16H, 8 H2CO), 4.41–4.44 (m, 2H, Hb, Hb ), 6.73 (m, 4H, H/
Ph), 7.10–7.73 (m, 10H, H/Ar), 9.06 (br, 2H, 2HN) ppm;
13C NMR (125.7 MHz, DMSO-d6): d = 145.1, 141.3,
2
130.2, 128.3, 125.3, 121.8 (C/Ar), 61.9 (t, Jpc = 12.8 Hz,
2
C*2, C*20), 58.2 (d, JPC = 12.5 Hz, 8 CH2O), 41.5 (t,
3
1JPC = 156.3 Hz, C1, C10), 14.4 (d, JPC = 8.7 Hz, 8
MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6): d = 24.7
(s, 2 P2C) ppm; IR (KBr): mꢀ = 3420 (br, 2 NH), 1259 (br,
P=O), 1097 (br, POC) cm-1; MS (EI, 70 EV): m/
z (%) = 860 (M?, 22), 286 (100).
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis[2-(2-chlor-
Octaethyl
2,20-[1,4-phenylenebis(azanediyl)]bis(ethane-
ophenyl)ethane-2,1,1-triyl]tetraphosphonate
(5c,
C38H58Cl2N2O12P4) Yield: 1.6 g (78%); m.p.: 165 °C
2,1,1-triyl)tetraphosphonate (5a, C26H52N2O12P4) Yield:
1.02 g (66%); m.p.: 147 °C (from MeCN); 1H NMR
(500 MHz, DMSO-d6): d = 1.02, 1.24 (2dt, 24H,
1
(from CH3Cl); H NMR (500 MHz, DMSO-d6): d = 0.87,
1.29 (2dt, 24H, 3JHH = 6.9, 4JPH = 4.5 Hz, 8 MeCO), 2.65,
0
2.81 (2dt, 2H, 3JHH = 13.1, 2JPH = 18.9 Hz, Ha, Ha ), 4.09–
4.18 (2dq (m), 16H, 8 H2CO), 4.39–4.02 (m, 2H, Hb, Hb’),
6.31 (m, 4H, H/Ph), 7.21–7.51 (m, 8H, H/Ar), 9.32 (br, 2H,
2 HN) ppm; 13C NMR (125.7 MHz, DMSO-d6): d = 143.2,
137.3, 135.2, 134.6, 133.8, 129.1, 126.7, 121.5 (C/Ar), 62.5
4
3JHH = 6.3, JPH = 4.5 Hz, 8 MeCO), 2.51, 2.63 (2dt, 2H,
00
3JHH = 13.4, 2JPH = 18.8 Hz, Ha, Ha ), 4.09–4.21 (2dq (m),
16H, 8 H2CO), 4.31–4.39 (2tt (m), 4H, 2 H2C), 6.69–6.75
(m, 4H, H/Ph), 8.86 (br, 2H, 2HN) ppm; 13C NMR
(125.7 MHz, DMSO-d6): d = 144.6, 119.8 (4C/Ar), 61.3
(d, 2JPC = 11.3 Hz, 8 CH2O), 43.5 (t, 2JPC = 8.4 Hz, CH2),
2
2
(t, Jpc = 12.8 Hz, C*2, C*20), 59.2 (d, JPC = 11.8 Hz, 8
1
1
3
CH2O), 42.7 (t, JPC = 157.1 Hz, C1, C10), 15.6 (d,
3JPC = 6.7 Hz, 8 MeCO) ppm; 31P NMR (202.4 MHz,
42.6 (t, JPC = 155.3 Hz, C1, C10), 14.2 (d, JPC = 8.8 Hz,
8 MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6):
ꢀ
DMSO-d6): d = 22.8 (s, P2C) ppm; IR (KBr): m = 3415 (br,
ꢀ
d = 23.6 (s, 2 P2C) ppm; IR (KBr): m = 3398 (br, 2 NH),
NH), 1256 (br, P=O), 1112 (br, POC) cm-1; MS (EI, 70
1252 (P=O), 1121 (POC) cm-1; MS (EI, 70 EV): m/
z (%) = 708 (M?, 18), 134 (100).
EV): m/z (%) = 930 ([M?2]?, 9), 928 (M?, 15), 354 (100).
123

Computer-aided design, synthesis, and biological studies of anticological nitrogen-…
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis[2-(4-chlor-
ophenyl)ethane-2,1,1-triyl]tetraphosphonate (5d,
P=O), 1119 (br, POC) cm-1
;
MS (EI, 70 EV):
m/z (%) = 996 (M?, 17), 422 (100).
C38H58Cl2N2O12P4) Yield: 1.46 g (72%); m.p.: 181 °C
Octaethyl
2,20-[1,4-phenylenebis(azanediyl)]bis[2-[4-
1
(from MeOH); H NMR (500 MHz, DMSO-d6): d = 1.08,
(dimethylamino)phenyl]ethane-2,1,1-triyl]tetraphospho-
nate (5g, C42H70N4O12P4) Yield: 1.49 g (72%); m.p.:
234 °C (from MeOH); H NMR (500 MHz, DMSO-d6):
1.21 (2dt, 24H, 3JHH = 6.8, 4JPH = 4.8 Hz, 8 MeCO), 2.61,
00
3
2
2.82 (2dt, 2H, JHH = 12.4, JPH = 19.1 Hz, Ha, Ha ),
4.12–4.21 (2dq (m), 16H, 8 H2CO), 4.31–4.38 (m, 2H, Hb,
Hb’), 6.41 (m, 4H, H/Ph), 7.34–7.92 (m, 8H, H/Ar), 8.69
(br, 2H, 2 HN) ppm; 13C NMR (125.7 MHz, DMSO-d6):
d = 141.7, 137.7, 136.1, 135.1, 133.9, 121.0 (C/Ar), 61.9
1
3
4
d = 0.88, 1.26 (2dt, 24H, JHH = 6.3, JPH = 4.4 Hz, 8
MeCO), 2.57, 2.68 (2dt, 2H, JHH = 13.8, JPH = 20.6 Hz,
3
2
0
Ha, Ha ), 3.07 (s, 12H, 2 Me2N), 3.88–4.36 (2dq (m), 16H,
0
8 H2CO), 4.37 (m, 2H, Hb, Hb ), 6.74 (m, 4H, H/Ph), 6.99–
7.70 (m, 8H, H/Ar), 8.66 (br, 2H, 2 HN) ppm; 13C NMR
(125.7 MHz, DMSO-d6): d = 145.8, 141.7, 132.4, 128.9,
2
2
(t, Jpc = 12.8 Hz, C*2, C*20), 59.5 (d, JPC = 10.5 Hz, 8
CH2O), 42.1 (t, JPC = 154.6 Hz, C1, C10), 15.3 (d,
3JPC = 8.9 Hz, 8 MeCO) ppm; 31P NMR (202.4 MHz,
1
2
121.1, 115.8 (C/Ar), 62.6 (t, Jpc = 12.4 Hz, C*2, C*20),
ꢀ
DMSO-d6): d = 23.4 (s, 2 P2C) ppm; IR (KBr): m = 3419
61.4 (d, 2JPC = 12.5 Hz,
8
CH2O), 43.3 (t,
(br, 2 NH), 1234 (br, P=O), 1098 (br, POC) cm-1; MS (EI,
70 EV): m/z (%) = 930 ([M ? 2]?, 5), 928 (M?, 18), 354
(100).
1JPC = 158.3 Hz, C1, C10), 38.8 (2 Me2N), 14.7 (d,
3JPC = 7.9 Hz, 8 MeCO) ppm; 31P NMR (202.4 MHz,
ꢀ
DMSO-d6): d = 25.3 (s, 2 P2C) ppm; IR (KBr): m = 3404
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis[2-(4-fluo-
(br, NH), 1260 (P=O), 1089 (br, POC) cm-1; MS (EI, 70
rophenyl)ethane-2,1,1-triyl]tetraphosphonate
(5e,
EV): m/z (%) = 946 (M?, 18), 372 (100).
C38H58F2N2O12P4) Yield: 1.47 g (75%); m.p.: 151 °C
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis[2-(4-meth-
1
(from EtOH); H NMR (500 MHz, DMSO-d6): d = 0.93,
oxyphenyl)ethane-2,1,1-triyl]tetraphosphonate
(5h,
1.29 (2dt, 24H, 3JHH = 6.5, 4JPH = 5.1 Hz, 8 MeCO), 2.61,
0
C40H64N2O14P4) Yield: 1.37 g (68%); m.p.: 102–104 °C
2.69 (2dt, 2H, 3JHH = 13.5, 2JPH = 18.9 Hz, Ha, Ha ), 3.92–
(from CH2Cl2); 1H NMR (500 MHz, DMSO-d6): d = 0.81,
b0
4.39 (2dq (m), 16 H, 8 H2CO), 4.37 (m, 2H, Hb, H ), 6.88
1.30 (2dt, 24H, 3JHH = 7.7, 4JPH = 4.8 Hz, 8 MeCO), 2.62,
0
(m, 4H, H/Ph), 7.32–7.75 (m, 8H, H/Ar), 8.88 (br, 2H, 2
HN) ppm; 13C NMR (125.7 MHz, DMSO-d6): d = 160.5
(d, JCF = 271.3 Hz, CF), 144.7, 136.8, 131.8 (d,
3
2
2.81 (2dt, 2H, JHH = 14.8, JPH = 21.3 Hz, Ha, Ha ), 3.65
(s, 6H, 2 MeO), 3.91–4.38 (2dq (m), 16 H, 8 H2CO), 4.42
1
b0
(m, 2H, Hb, H ), 6.45 (m, 4H, H/Ph), 7.31–7.65 (m, 8H, H/
3JCF = 10.8 Hz, CCCF), 122.8(C/Ar), 119.7 (d,
Ar), 9.01 (brs, 2H, 2 HN) ppm; 13C NMR (125.7 MHz,
2
2JCF = 28 Hz, CCF), 62.1 (t, Jpc = 12.8 Hz, C*2, C*20),
DMSO-d6): d = 154.7, 141.2, 131.8, 130.3, 121.9, 117.2
58.2 (d, 2JPC = 12.5 Hz,
1JPC = 154.6 Hz, C1, C10), 15.4 (d, JPC = 8.1 Hz, 8
MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6): d = 26.3
8
CH2O), 41.7 (t,
3
2
(C/Ar), 62.4 (t, Jpc = 11.8 Hz, C*2, C*20), 61.2 (d,
2JPC = 11.9 Hz, 8 CH2O), 52.7 (2 MeO), 43.4 (t,
3
1JPC = 148.9 Hz, C1, C10), 15.7 (d, JPC = 8.4 Hz, 8
ꢀ
(s, 2 P2C) ppm; IR (KBr): m = 3422 (br, 2 NH), 1248 (br,
MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6): d = 26.2
P=O), 1091 (br, POC) cm-1; MS (EI, 70 EV): m/
z (%) = 896 (M?, 21), 322 (100).
ꢀ
(s, 2 P2C) ppm; IR (KBr): m = 3419 (br, 2 NH), 1250 (br,
P=O), 1115 (br, POC) cm-1; MS (EI, 70 EV): m/
z (%) = 920 (M?, 21), 346 (100).
Octaethyl 2,20-[1,4-phenylenebis(azanediyl)]bis[2-[4-(triflu-
oromethyl)phenyl]ethane-2,1,1-triyl]tetraphosphonate (5f,
C40H58F6N2O12P4) Yield: 1.53 g (70%); m.p.: 175 °C
Octaethyl
2,20-[1,4-phenylenebis(azanediyl)]bis[2-(2,5-
dimethoxyphenyl]ethane-2,1,1-triyl]tetraphosphonate (5i,
1
(from MeOH); H NMR (500 MHz, DMSO-d6): d = 0.85,
C42H68N2O16P4) Yield: 1.53 g (71%); m.p.: 147 °C (from
1.19 (2dt, 3JHH = 7.2, 24H, 4JPH = 5.6 Hz, 8 MeCO), 2.54,
1
a0
MeCN); H NMR (500 MHz, DMSO-d6): d = 0.89, 1.08
2.72 (2dt, 2H, 3JHH = 11.9, 2JPH = 17.6 Hz, Ha, H ), 4.12–
(2dt, 24H, 3JHH = 6.7, 4JPH = 5.2 Hz, 8 MeCO), 2.46, 2.69
0
4.29 (2dq (m), 16H, 8 H2CO), 4.38 (m, 2H, Hb, Hb ), 6.84
(m, 4H, H/Ph), 7.31–7.61 (m, 8H, H/Ar), 8.78 (br, 2H, 2
HN) ppm; 13C NMR (125.7 MHz, DMSO-d6): d = 145.8,
0
3
2
(2dt, 2H, JHH = 12.1, JPH = 19.61 Hz, Ha, Ha ), 3.65,
3.68 (2 s, 12H, 4 MeO), 3.82–4.26 (2dq (m), 16H, 8
0
(H2CO)P), 4.42 (m, 2H, Hb, Hb ), 6.68 (m, 4H, H/Ph),
7.42–7.81 (2d, 6H, H/Ar), 8.82 (br, 2H, 2 HN) ppm; 13C
NMR (125.7 MHz, DMSO-d6): d = 156.2, 146.1, 145.7,
129.9, 121.9, 117.8, 116.4, 112.6 (C/Ar), 64.4 (t,
3
144.1, 131.8, 128.3 (q, JCF = 8.8 Hz, CCCF3), 125.2 (q,
2JCF = 28.6 Hz, CCF3), 119.3 (C/Ar), 122.5 (q,
1JCF = 269.8 Hz, CF3), 64.6 (t, 2Jpc = 11.9 Hz, C*2, C*20),
63.2 (d, 2JPC = 13.2 Hz,
1JPC = 157.2 Hz, C1, C10), 14.8 (d, JPC = 8.1 Hz, 8
MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6): d = 22.6
8
CH2O), 43.1 (t,
3
2
2Jpc = 14.2 Hz, C*2, C*20), 61.8 (d, JPC = 12.4 Hz, 8
CH2O), 55.7, 52.4 (2 MeO, 20MeO), 42.7 (t,
3
1JPC = 161.3 Hz, C1, C10), 14.2 (d, JPC = 7.3 Hz, 8
ꢀ
(s, 2 P2C) ppm; IR (KBr): m = 3410 (br, 2 NH), 1247 (br,
MeCO) ppm; 31P NMR (202.4 MHz, DMSO-d6): d = 25.7
123

M. S. Bekheit et al.
(s, 2 P2C) ppm; IR (KBr): mꢀ = 3422 (br, 2 NH), 1265 (br,
P=O), 1179 (br, POC) cm-1; MS (EI, 70 EV): m/
z (%) = 980 (M?, 19), 406 (100).
up (as described for 6a), the acids 6b–6j were obtained. They
were converted as above to their tetrapotassium salts 7b–7j
by adding in situ a solution (20 cm3) of aq KOH (10%)/
methanol (1: 3 v/v) and were stirred for 1 h, followed by
concentration. The precipitates were collected/air dried to
give the salts 7b–7j in * 68% yields.
Octaethyl
phen-2-yl)ethane-2,1,1-triyl]tetraphosphonate
2,20-[1,4-phenylenebis(azanediyl)]bis[2-(thio-
(5j,
C34H56N2O12P4S2) Yield: 1.46 g (76%); m.p.: 131–133 °C
(from CH2Cl2); 1H NMR (500 MHz, DMSO-d6): d = 0.92,
Bioassay screening
1.21 (2dt, 24H, 3JHH = 7.8, 4JPH = 4.9 Hz, 8 MeCO), 2.55,
0
2.73 (2dt, 2H, 3JHH = 12.9, 2JPH = 21.5 Hz, Ha, Ha ), 3.73–
0
The effect of NTP-tetrapotassium salts 7a–7j on melanoma
cell proliferation was studied on: SK-MEL-2, SK-MEL-5,
LOX-IMVI, UACC-257, and UACC-62 cell lines utilizing
sulforodamine-B method [43, 45]. The cells were seeded at
a density of 5000 cells per well in 96-multiwell micrometer
plate and left for 36 h at 37 °C under atmosphere of 5%
CO2 to allow the attachment of the cell to the wall of the
plate. The tested compounds 7a–7j and the positive control
pamidronate were dissolved in dimethylsulfoxide (DMSO)
99% and diluted 1000 fold in the assay. Concentrations of
the tested compounds were increased as 0, 5, 10, and 12.5
lg/cm3 and added to the cell monolayer. Triplicate wells
were used for each individual dose. The monolayer cells
were incubated with the tested compounds for 48 h at 36–
38 °C. Then, the cells were fixed, washed and stained with
sulforodamine-B (SRB). Excess stain was washed with
acetic acid while the attached stain was recovered with
Tris–EDTA buffer. Cell viability was determined by
measuring the color intensity at 750 nm using ELISA
micrometer reader (Meter-tech-R960, USA). Data were
collected as mean values for experiments that proceeded by
SRB assay. A control experiment did not exhibit significant
change compared to the DMSO vehicle. The percentage of
cell survival was calculated according to the equation:
4.23 (2dq (m), 16H, 8 H2CO), 4.57 (m, 2H, Hb, Hb ), 6.82
(m, 4H, H/Ph), 7.13–7.34 (m, 6H, H/thiophene), 8.75 (br,
2H, 2 HN) ppm; 13C NMR (125.7 MHz, DMSO-d6):
d = 146.3, 142.6, 140.1, 132.2, 126.2, 118.9 (C/Ar), 62.2
2
2
(t, Jpc = 12.4 Hz, C*2, C*20), 60.8 (d, JPC = 13.2 Hz, 8
1
CH2O), 42.3 (t, JPC = 158.6 Hz, C1, C10), 14.5 (d,
3JPC = 8.5 Hz, 8 MeCO) ppm; 31P NMR (202.4 MHz,
ꢀ
DMSO-d6): d = 24.8 (s, 2 P2C) ppm; IR (KBr): m = 3421
(br, NH), 1255 (br, P=O), 1127 (br, POC) cm-1; MS (EI,
70 EV): m/z (%) = 872 (M?, 16), 298 (100).
2,20-[1,4-Phenylenebis(azanediyl)]bis(ethane-2,1,1-triyl)te-
traphosphonic acid (6a, C10H20N2O12P4) A solution of
0.6 g bisphosphonate 5a (0.84 mmol) in 10 cm3 CHCl3
was treated with trimethylsilyl bromide (TMS-Br, 5 mmol)
and the reaction mixture was heated at 40°C for * 6 h.
After concentrating the product mixture in vacuum, the
crude residue was diluted with dist. water/AcOEt (2:1 v/v)
and stirred for 30 min. The two layers were separated and
the aqueous layer was evaporated to dryness. The precip-
itates were collected and dried to give the corresponding
hygroscopic tetraacid 6a, which could be kept under vac-
uum. Compound 6a was directed without any further
purification for the analytical purposes. White compound;
1
yield: 0.27 g (66%); m.p.: [ 300 °C; H NMR (500 MHz,
Surviving fraction ¼ Optical density ðODÞ of treated
cell/OD of control cells:
D2O):
d = 2.45,
2.57
(2dt,
2H,
3JHH = 12.4,
0
2JPH = 20.7 Hz, Ha, Ha ), 4.28–4.29 (m, 4H, 2 H2C), 6.74
(s, 4H, H/Ph) ppm; 13C NMR (125.7 MHz, D2O):
d = 143.1, 121.4 (C/Ar), 43.9 (t, 2JPC = 8.4 Hz, CH2), 42.2
(t, 1JPC = 154.0 Hz, C1, C10) ppm; 31P NMR (202.4 MHz,
The GI50 (concentration required to produce 50% inhi-
bition of cell grown compared to control experiment) was
determined using the Graph-Pad PRISM version-5 soft-
ware statistical calculations for determination of the mean
and standard error values by SPSS software [52, 53]. The
results of antimelanoma properties are displayed in Table 2
and Fig. 3.
ꢀ
D2O): d = 18.3 (s, 2 P2C) ppm; IR (KBr): m = 3416–3387
(NH, OH), 124.8 (br, P=O) cm-1; MS (EI, 70 EV):
m/z (%) = 476 ([M-8]?, 16), 136 (100).
The acid 6a was stirred for 1 h in 20 cm3 solution of
aqueous KOH (10%)/methanol (1: 3 v/v), followed by
concentration. The precipitate was collected and air dried
to give the tetrapotassium salt 7a (72% yield).
Acknowledgements The authors would like to thank the National
Research Centre, Dokki, Cairo, Egypt (project # 10010340) for the
financial support of the present work. They also are grateful to Cancer
Research Institute, NY, USA, for handling the antitumor properties.
General procedure to prepare NBP-
tetrapotassium salts 7a–7j
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