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W. Goldeman, A. Nasulewicz-Goldeman / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
P(O)(OH)2
OH
P(O)(OH)2
P(O)(OH)2
OH
P(O)(OH)2
H2N
P(O)(OH)2
OH
P(O)(OH)2
N
H2N
pamidronate
alendronate
ibandronate
H
N
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
OH
P(O)(OH)2
P(O)(OH)2
OH
P(O)(OH)2
N
N
N
incadronate
Figure 1. Selected examples of the commercial bisphosphonates.
risedronate
zoledronate
O
O
POCl3(2.4eq), Et3N(7.2eq)
CH2Cl2, 0oC, 1 h, rt, 2 h
HCOOH (4eq), toluene
C
C
N+
N+
X
X
X
H
N
H
N
H
H
H2N
NH2
reflux, 8h
1
2
3
X: aryl or alkyl linker
Scheme 1. Synthesis of isonitriles 3.
1H, 13C NMR, IR and, in the case of new compounds (3i and 3l), by
HRMS spectroscopy. All isonitriles showed a characteristic strong
sharp band on the FTIR spectra (in the 2125–2135 cmꢀ1 region
for aromatic and 2140–2153 cmꢀ1 for aliphatic), due to the vibra-
tion of the isonitrile group triple bond. On the 13C NMR spectra
the characteristic signal was present at about 155–170 ppm from
the isonitrile carbon. It was a sharp 1:1:1 triplet in the case of
the aliphatic isonitrile and a very broad unresolved triplet in the
case of the aromatic isonitrile. The yields of the resultant isonitriles
are depicted in Supplementary data (Table 1 at page 3).
Having the diisonitriles 3 in hand, a series of aromatic and ali-
phatic octaethyl bis[aminomethylidene(bisphosphonates)] 4 were
synthesized by the reaction of isonitrile 3 with four equivalents
of triethylphosphite in the presence of six equivalents of dry
hydrogen chloride in 1,4-dioxane (Scheme 2). The aliphatic octa-
ethyl tetraphosphonates 4k–n were purified by washing the crude
reaction mixture with an aq. solution of NaHCO3 and the products
were obtained as a thick oil. Aromatic tetraphosphonates 4a–j
were separated as solids by crystallization from an appropriate sol-
vent (see Table 1 in Supplementary data). All octaethyl tetra-
phosphonates 4 were characterized by 31P, 1H, 13C NMR, FTIR and
by HRMS spectroscopy. In most cases, the reaction was almost
quantitative, that is, the yields of tetraphosphonates 4 assayed by
31P NMR were >90% and the only by-products were traces of the
diethyl phosphonate resulting from dealkylation of the tri-
ethylphosphite and the volatile ethyl chloride (which was easily
removed by evaporation).
Next, the octaethyl bis[aminomethylidene(bisphosphonates)] 4
were transformed into free acids 5 by the acid hydrolysis (in the
case of the aliphatic tetraphosphonates 4k–n) with refluxing 6 M
hydrochloric acid or by dealkylation (in the case of the aromatic
tetraphosphonates 4a–j) with bromotrimethylsilane (Scheme 2).
A mild dealkylation method was chosen instead of hydrolysis
because a cleavage of the Aryl-N bond and formation of aminome-
thylidenebisphosphonic acid (H2NCH(PO3H2)2) was observed in
many cases of aromatic aminomethylidenebisphosphonates. For
example, after a refluxing of the octaethyl naphthyl-1,5-diamin-
obis[aminomethylidene(bisphosphonate)] (4j) with 6 M hydro-
chloric acid, aminomethylidenebisphosphonic acid was obtained
with a 96% yield. All bis[aminomethylidene(bisphosphonic)] 5
acids were obtained as crystalline, stable solids with high yields
(see Table 1 in Supplementary data).
Among all bis[aminomethylidene(bisphosphonic)] acids 5
synthesized and described in this paper, only benzene-1,4-bis
[aminomethylidene(bisphosphonic)] acid (5a) is known in the lit-
erature.16 Xie et al. described in a Chinese patent its synthesis in
the reaction of triethyl orthoformate, p-phenylenediamine and
dimethyl phosphonate.17 It is worth mentioning that this is the
only example of the adoption of the diamine in the most widely
used ‘orthoformate method’. Surprisingly, we found only a few
examples of other tetraphosphonic acids (with general formula
(H2O3P)2CHNH-X-NHCH(PO3H2)2) in the literature. From aliphatic
tetraphosphonic acids, only a derivative of ethylenediamine ((H2-
O3P)2CHNHCH2CH2NHCH(PO3H2)2) had been described as an envi-
ronmentally friendly bleach fixing stabilizer chelating agent.18
From aromatic derivatives, Lecercle et al. published a synthesis of
four aromatic tetraphosphonates via double N–H insertion of dia-
mines into tetraethyl diphosphonodiazomethane in the presence
of Rh2(NHCOCF3)4. The resultant octaethyl esters were dealkylated
to free acids which showed interesting uranyl-binding proper-
ties.19 Petrov et al. described a preparation of the benzene-1,3-
bis[aminomethylidene(bisphosphonic)] acid in the reaction of
m-phenylenebisformamide with a mixture of PCl3/H3PO3 as phos-
phorylation agent and used it as monomer for synthesis of ion
exchange resins.20
All bis[aminomethylidene(bisphosphonic)] acids 5a–n were
evaluated for their antiproliferative activity against MCF-7 human
breast adenocarcinoma cells, HL-60 human promyelocytic leuke-
mia cells and J774E mouse macrophages.21 Since bisphosphonates
accumulate in bones, a strong antiproliferative effect on bone met-
astatic and hematopoietic tumors can be expected, suggesting
their possible clinical application as anticancer drugs. The MCF-7
cell line is a well-established model of breast cancer, which prefer-
entially metastasizes to bone forming predominantly osteolytic
lesions.22 HL-60 cells are derived from hematopoietic lineage
and, moreover, under specific culture conditions can differentiate
into cells of osteoclast phenotype. Because of a difficulty in isolat-
ing and culturing large numbers of osteoclasts, many studies char-
acterizing the pharmacologic properties of bisphosphonates
in vitro are performed in osteoclast surrogates, in particular macro-
phages. J774E macrophages and osteoclasts are derived from the
same hematopoietic lineage as well as are highly endocytic and
capable of demineralizing bone particles.23 J774E macrophages
are also a model in studies on the influence of bisphosphonates
Please cite this article in press as: Goldeman, W.; Nasulewicz-Goldeman, A. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/
Goldeman, Waldemar
