Aminobisphosphonates based on cyclohexane backbone as coordinating agents for metal ions. Thermodynamic, spectroscopic and biological studies

Article abstract of DOI:10.1039/c8nj01158c

Single and double amino-bisphosphonates were synthesized and tested for coordination capabilities towards Ca²⁺, Mg²⁺, Cu²⁺ and Ni²⁺ metal ions by means of potentiometry, UV-vis spectroscopy, mass spectrometry (ESI-MS) and isothermal titration calorimetry (ITC), as well as for cytotoxic activity by MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] assay. Half minimal inhibitory concentrations (IC₅₀) were determined with respect to two cell lines (human melanoma A375 and human colorectal adenocarcinoma HT29). Basing the structure of compounds on a cyclohexane ring allowed for a slight reduction of high hydrophilic character of the studied bisphosphonates (BPs). The ligands efficiently bind the examined metal ions forming complex equilibria with diversified stoichiometry of equimolar, polynuclear species and biscomplexes. Both ligands as well as their Ca²⁺ and Mg²⁺ complexes show selective antiproliferative activity toward the studied cancer cell lines. Given the thermodynamic and biological data, it can be assumed that ligands are good candidates for linking compounds that may be used in the design of new drug delivery systems. In this approach, one bisphosphonate moiety acts as a bone-targeting molecule, while another molecule can be readily attached to the second donor function (primary amine or bisphosphonate).

Full text of DOI:10.1039/c8nj01158c

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Aminobisphosphonates based on cyclohexane backbone as
coordinating agents for metal ions. Thermodynamic,
spectroscopic and biological studies.
Received 00th January
20xx,
Accepted 00th January
20xx
J. Gałęzowska,*a H. Czapor-Irzabek,b E. Chmielewska,c P. Kafarskic and T. Janeka
Single- and double-amino-bisphosphonates were synthesized and tested for coordination capabilities
towards Ca2+, Mg2+, Cu2+ and Ni2+ metal ions by means of potentiometry, UV-vis spectroscopy, mass
spectrometry (ESI-MS) and isothermal titration calorimetry (ITC), as well as for cytotoxic activity by MTT [(3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Half minimal inhibitory concentrations
(IC50) were determined with respect to two cell lines (human melanoma A375 and human colorectal
adenocarcinoma HT29). Bearing the structure of compounds on cyclohexane ring allowed for a slight
reduction of high hydrophilic character of studied bisphosphonates (BPs). The ligands efficiently bind
examined metal ions forming complex equilibria with diversified stoichiometry of equimolar, polynuclear
species and biscomplexes. Both ligands as well as their Ca2+ and Mg2+ complexes show selective
antiproliferative activity toward studied cancer cell lines. Given thermodynamic and biological data, it can be
assumed that ligands are good candidates for linking compounds that may be used in the design of new drug
delivery systems. In such approach one bisphosphonate moiety acts as a bone-targeting molecule, while
another molecule can be readily attached to the second donor function (primary amine or bisphosphonate).
DOI: 10.1039/x0xx00000x
www.rsc.org/
related diseases. They are first choice drugs for osteoporosis,14 act
as therapeutic drug carriers,15 imaging bone-targeting tools16 and
bone-pain palliation relievers.17–19 However, in every aspect of use;
Introduction
Bisphosphonates (BPs) are a group of compounds with two
phosphonate functions attached to a geminal carbon atom that has
found various medical applications.1–8 Thanks to such features like;
1) structural similarity of phosphonic group to phosphate, 2) low
biodegradability,9 3) possibility to accommodate metal ions with
various ionic radii10–12 and 4) presence of P-C-P backbone, that
provides bone delivery properties followed by antiresorptive
effects,213 BPs have become particularly important drugs in bone-
BPs reveal side effects among which osteonecrosis of the jaw (95%
of patients treated with the most potent heterocyclic N-BPs),20,21
atrial fibrillation,22 hypocalcemia, over-suppression of bone
turnover and other complications23 can be listed. These side-effects
along with low bioavailability of BP are the reason why there is still
a need to design new ligands with better characteristics. However,
nowadays research goals are rather aimed at design of locally
administrated drugs, materials and conjugated delivery tools24–29
than designing new bisphosphonates of subsequent generations,30
although such BPs are also desired especially with lower binding
affinity which produce e.g. more rapid decrease in fracture risk.31
At the same time clinical findings suggest that some of BPs
reduce the burden of bone cancer and increase the survival rate
among patients. Most probably BP have a direct effect on some
types of cancer cells and reveal antitumor effect.32–34
We have synthesized three, based on a cyclohexane skeleton,
aminobisphosphonic acids (amino-BP) which chemical structures
are depicted in Fig. 1. L1 contain one (single-amino-BP) and L2, L3
two bisphosphonic moieties (double-amino-BP). Ligands were
shown previously in our patent.35 L3 was also prepared almost in
parallel by Goldeman et al.,36 however using a different synthetic
approach. For all studied ligands we used three-component
reaction of cyclic diamines with triethyl orthoformate and diethyl
phosphite. It is perhaps the most general and common procedure
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J. Name., 2013, 00, 1-3 | 1
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P(O)(OH)2
NH
that chemical structure is the closest to the structure of studied
ligands.
(HO)2(O)P
P(O)(OH)2
(HO)2(O)P
P(O)(OH)2
P(O)(OH)2
NH2
NH
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
P(O)(OH)2
N
N
N
H
H
H
Results and Discussion
Fig. 1. Chemical structures of studied ligands
Acid-base properties of ligands
L1 as a fully protonated ligand possess six dissociable protons (H6L);
four belong to phosphonic functions, one to primary amine and one
to tertiary amine NH+R. Under applied conditions four of
protonation constants could be determined via potentiometry and
the results are given in Table 1. All estimated values can be
attributed to deprotonation of phosphonic groups. This assumption
agrees with literature data for similar ligands; pKs of 1-hydroxy-4-
to synthesize N-substituted aminomethylenebisphosphonic acids.37–
39 Since this reaction usually gives the mixtures of products, that are
difficult to separate, the crude esters are commonly hydrolyzed
with concentrated hydrochloric acid and the desired bisphosphonic
acids are isolated in moderate to good yields. Introducing
cyclohexane as a backbone for the ligands had three purposes: 1) to
reduce the hydrophilicity of phosphonic groups, 2) to provide a
steric barrier that prevents the coordination of metal ion by more
than one functional group and 3) to allow to exhibit cytotoxic
aminopropilydenediphosphonic acid are as follows: 10.95amine
,
43
9.80phosphonate, 6.01phosphonate, 2.56phosphonate
amino-1-hydroxypropylidene-1,1-bisphosphonate):
activity, since it has been proven that aromatic BPs reveal 10.30phosphonate, 5.85phosphonate, 1.80phosphonate, <1.20phosphonate
,
pamidronic acid (3-
13.06amine
,
42
.
For a
ligand with an analogous structure, however with no NH+R amine;
N-cycloheptyl-alkylaminomethane-1,1-diphosphonic acid,
significantly higher activity than aliphatic ones.36 Antiproliferative
activity of L3 has been previously studied with respect to MCF-7
human breast cancer cells, J774E mouse macrophages and HL-60
human promyelocytic leukemia cells lines, revealing differentiated,
mild activity.36 The aim of this work was to study the physico-
chemistry of interactions between synthesized ligands and various
metal ions. We focused on metal ions of biological importance: Ca2+,
Mg2+ but because of the wide binding capacity of BPs, examined
coordination properties of L1-3 towards chosen transition metals as
well. We have chosen Cu2 + and Ni2+ ions due to their relatively well-
described spectral response to amino-BPs binding.
measured pK values are: 8.72, 4.76 and 2.07,44 and all belong to
deprotonation procesess of phosphonic moieties. For L1 examined
here, it was possible to approximate the highest pK value and it was
calculated as 12.59(8). This value most probably can be assign to
the NH3+ group. However, because the measurements were carried
out only up to pH 12 this value is unreliable and was excluded from
the calculations. The last, basic proton of L1 dissociates above pH
1345 from the nitrogen atom of NHR+ function. This deprotonation
constant could not be estimated under pH titrations as well. In this
case, we use the neglect approach in the calculation of the
dissociation of NH+R group, as suggested by Kurzak et al.45 and
Szpak et al.44 for similar ligands, where it is not possible to obtain
reliable pK constants pK>13. So high pK value of NH+R (as well as
The interactions with Ca2+ ions are important in particular
because of the binding of BPs to hydroxyapatite. This binding opens
up a cascade of chemical and biological events that lead to
antiresorptive action of BPs in general. It has been found that the NH3+) was observed in similar compounds and was explained by the
existence of intramolecular N-H···Ophosphonate hydrogen bond.44 It
was proven that such H-bond stabilizes the structure of
formation of Ca2+/BP complexes is either the first event before drug
introduction into the bone structure40,41 or a competitive reaction
to bone surface absorption,41 therefore this binding and its physico-
chemistry has to be taken into account.
a
monosubstituted piperid-1-ylmethane-1,1-diphosphonic acids in
solid state and is a reason to create stabile head to head dimers by
molecules of free ligands.46 In case of L1 the possibilities to form H-
bonds are two: eighter using N(1) or N(2), both would lead to
increase the pKs of nitrogen-containg groups, what most likely
takes place. Probable zwitterionic structure of L1 is depicted on Fig.
2. The lowest deprotonation step which occurs on phosphonic
moiety (pH <<2) could not be detected, what is very common for
BPs.
An important factor of pharmacological activity of BPs is their
chemical nature/state in blood plasma (free acid, complex, charge,
polarity, etc.). Because the concentration of Ca2+ in blood plasma at
pH 7.4 is maintained at ~ 3mM and of Mg2+ at ~ 1mM,42 it is highly
likely that the majority of introduced BP is bound to Ca2+, Mg2+ and
other species e.g. Na+. Therefore, quantitative thermodynamic
studies and biological tests were performed for calcium and
magnesium cations with the studied ligands. An attempt was also
made to predict the dominant species found in the solution under
the conditions of biological studies in order to indicate active
species.
Fully protonated L2 and L3 ligands behave like H10L acids. Groups
capable of deprotonating are the following: two NH+R functions
that release two protons and two bisphosphonic moieties releasing
a total of eight protons. However, only four protonation
An additional goal, considering literature examples of
antitumor effects revealed by BPs in general, as well as
antiproliferative characteristics of L3, was to test anti-cancer activity
of both; free ligands and their Ca2+ and Mg2+ complexes in respect
to cell lines of colon adenoma and melanoma skin cancer, the most
common malignant tumors worldwide. The results were compared
+
NH3
(2)
PO3H-
HN
(1)
PO3H2
experimentally
to
incadronate
(N-(cycloheptyl)-
aminomethylenebisphosphonic acid), a commercially available BP
Fig. 2. Zwitterionic form of L1.
2 | NJC 2018, 00, 1-3
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Table 1. Protonation constants of studied ligands at 25°C and I=0.1M (KCl). All
estimated values belong to phosphonic moietes. Due to very basic constants of
amine functions, which do not deprotonate in measurable range ligands, correct
notifications for studied ligands, following literature44 should be: L1=HL, L2,L3=H2L,
Metal Complexes
Up to date numerous papers have been devoted to coordination
properties of BPs towards Ca2+/Mg2+42–44,54–57 (alkaline earth metal
ions) and Cu2+ 42,45,58–60 significantly less to Ni2+61–63 (transition metal
however here it was omitted for simplicity
.
,
ions). Only very few data can be found for complexation of ligands,
that contain more than one bisphosphonic functions, toward any
kind of metal ion.50 The available data indicate, as expected, taking
into account different chemical properties of these metal ions, that
the equilibrium in the solution varies significantly with alkaline
earth metals and transition metal ions. However, certainly a
common feature of BPs regardless of the metal ion is eagerness to
create apart from monomeric complexes also oligonuclear species
which were found both in solution12 and solid state.11 Such abilities
lead to creation of complex equilibria of co-existence of
monomeric and oligomeric species in a broad pH range,12 what is
expected for studied ligands as well. Towards this end we have used,
apart from potentiometry, mass spectrometry (ESI-MS) in order to
detect various stoichiometries of complexes, which calculations
based on potentiometric titrations cannot distinguish. All peak
assignments for ESI-MS were confirmed by comparison of
calculated and experimental isotope distribution patterns.
It is also known, that the phenomenon of precipitation often
accompany BP-complexation studies. Especially upon coordination
of Ca2+ cations, precipitations were observed in wide pH ranges for
several BPs,42 however, precipitate might also occur in solution due
to the interactions between phosphonic moieties that lead two or
more ligand molecules to aggregation.11,54,55 Herein such
precipitations were also observed, details are given in Experimental
Section, and these measurements were excluded from calculations.
Species
L1
L2
L3
logβ
logK
logβ
pK
logβ
pK
HL
9.76(7)
9.76
9.80(3)
9.80
9.54(3)
9.54
H2L
H3L
H4L
H5L
16.41(5) 6.65 18.20(3) 8.40
20.03(5) 3.62 24.89(5) 6.69
21.58(5) 1.55 29.89(5) 5.00
17.92(3) 8.38
24.13(6) 6.21
28.88(5) 4.75
-
-
31.41(7) 1.52
-
-
constants for L3 and five for L2 could be calculated and are given in
Table 1. All estimated values belong to phosphonic functions. Both;
the lowest (<pH 1) and the most basic (>pH 12) protonation
constants, as in case of L1, could not be determined due to the
limitations of the method. Studies of double BPs (tetraphosphonic
acids) in terms of their acid-base properties are rare,49,50 what
excludes any straightforward comparison to analogues studied in
literature. A comparison to a different group of multiphosphonates:
N-methylenephosphonic acids,51–53 which have gained more
scientific attention, is not possible due to the well-known fact of
significant changes in acid-base diphosphonate properties when
combined with a geminal carbon atom in comparison with ligands
possessing two phosphonic functions, but differently connected
(see Fig. S1, SI). We assume that two very basic pKs for NH+R groups
were not determined under the conditions of experiment, for the
same reason as for L1, because they lie above pH 13 due to
hydrogen bond formation involving a nitrogen atom from NH+R as a
donor and oxygen from the phosphonic function as an acceptor.
ESI-MS spectra of all studied ligands showed their high
tendency to create polimeric structures (Fig. S4, SI), that confirms
the possibility of formation of internal hydrogen bonds providing
stabilisation of aggregates in solution (Fig. S4, SI).
Complexation of Ca2+and Mg2+ . The complex formation equilibria
in L1/Ca2+ and L1/ Mg2+ systems are similar to each other (Table 2,
Fig. 3). Calculations based on potentiometric titrations revealed the
existence of equimolar species and biscomplexes that occur in
differently protonated forms in both systems. ML and ML2 species
coexist in the solution above pH 7. pK values of following
deprotonations processes; MH2L↔MHL + H, MHL↔ML + H vary
between 6.21 and 9.40 and for MHL2↔ML2 + H between 8.79 and
9.40 (Table 2). These values can be attributed to a stepwise
deprotonation and coordination of oxygen atoms from phosphonic
groups. In basic pH both ML and ML2 type of species undergo
hydrolysis to MH-1L, MH-1L2 and MH-2L2 complexes which better can
be described as; M(OH)L, M(OH)L2 and M(OH)2L2, respectively.
Since the pK values of reactions fall in a range of 10.27-11.1, they
can be attributed to a deprotonation of water molecules, that is a
common behaviour of amino-BPs complexes in high pH.44 The
presence of both; equimolar and biscomplexes, was confirmed by
ESI-MS for Mg2+ (Table S2, Fig. S5, SI). For the Ca2+/L1 system, ML3
complex was additionally detected by ESI-MS. In literature similar
amino-BPs were confirmed to create ML2 species in solution,44,56,57
although the repulsion of highly negatively charged phosphonic
groups makes such species rather unfavourable in general. Most
likely due to this reason ML3 complexes were not detected
potentiometrically. No occurrence of polymeric complexes was
detected.
Table 2. Stability constants (logβ) of M2+ complexes with L1 obtained by
potentiometry at 25°C and I=0.1M (KCl).
Species
M:H:L
L1
Ca2+
Mg2+
Cu2+
Ni2+
logβ[MH2L]
logβ[MHL]
logβ[ML]
19.89(4)
13.68(6)
-
-
-
13.19(3)
17.71(3)
11.73(4)
-
14.56(9)
10.30(2)
-
4.86(14)
5.08(4)
logβ[MH-1L]
logβ[MH2L2]
logβ[MHL2]
logβ[ML2]
-5.41(7)
-
-
-
-
18.33(13)
17.57(14)
-
23.19(10)
16.65(8)
8.04(9)
-2.52(10)
-
8.93(9)
8.78(8)
16.69(6)
6.69(6)
-3.52(5)
18.78(5)
logβ[MH-1L2]
logβ[MH-2L2]
logβ[M2L]
-
-
-
-2.32(5)
-
-
pKa[MH2L]
pKa[MHL]
pKa[ML]
6.21
8.33
-
-
-
8.11
5.98
4.26
10.27
-
-
-
pKa[MH-1L]
pKa[MH2L2]
pKa[MHL2]
pKa[ML2]
-
-
-
-
-
-
-
-
9.40
8.79
-
6.54
8.61
10.56
-
-
-
-
11.10
10.00
10.21
-
Comparison of the stability constants of fully deprotonated ML
species indicates that L1 forms complexes with stability that is
consistent with the literature data (logβCaL=4.86, logβMgL=5.08),44,55–
57 which also state that BP-Mg2+ complexes are slightly more stable
than those of Ca2+ in general. However, it must be borne in mind
that for BP type of compounds a simple
pKa[MH-1L2]
pKa[MH-2L2]
-
-
Numbers in parentheses denote statistical errors on the last significant digits.
Existence of hydroxocomplexes47,48 was considered in all calculations (M2+/L1-3).
Charges were omitted for clarity.
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Table 3. Stability constants (logβ) of M2+ complexes with L2 and L3 obtained by potentiometry at 25°C and I=0.1M (KCl).
Species
M:H:L
L2
L3
Cu2+
Ca2+
Mg2+
Cu2+
Ni2+
Ca2+
Mg2+
Ni2+
-
logβ[MH2L]
logβ[MHL]
-
-
24.69(3)
24.43(3)
21.78(5)
21.62(10)
-
16.05(4)
14.40(3)
18.89(3)
17.99(5)
13.72(6)
13.77(12)
18.09(9)
10.20(6)
-
16.54(13)
logβ[ML]
4.60(6)
4.66(4)
10.74(4)
8.77(6)
4.83(6)
4.95(10)
8.33(8)
logβ[MH-1L]
logβ[MHL2]
logβ[ML2]
-
-6.11(4)
-
-1.37(6)
-5.21(6)
-4.98(10)
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
14.87(4)
12.79(10)
-
14.52(8)
5.01(8)
-4.53(7)
65.81(14)
62.42(8)
-
-
logβ[MH-1L2]
logβ[MH-2L2]
logβ[M2H7L2]
logβ[M2H6L2]
logβ[M2H5L2]
logβ[M2H4L2]
logβ[M2H3L2]
logβ[M2HL3]
-
-
3.96(4)
-
-
0.88(15)
-
-
-
-8.67(9)
-
-9.52(9)
-
-
65.89(17)
-
-
-
-
-
62.78(3)
-
-
-
55.81(6)
50.91(5)
43.46(11)
-
53.88(8)
48.11(5)
40.19(10)
-
57.86(10)
55.91(16)
-
-
-
-
-
-
-
53.00(9)
47.27(10)
-
-
46.20(11)
-
-
45.11(7)
-
26.41(20)
pK[MH2L]
pK[MHL]
-
-
-
-
-
-
-
-
-
-
-
-
5.80
6.44
8.04
7.85
pK[ML]
11.45
9.74
8.15
9.22
8.83
8.82
7.89
8.21
pK[MH-1L]
pK[MHL2]
pK [ML2]
-
10.77
-
10.14
10.14
9.93
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
pK[MH-1L2]
pK[MH-2L2]
pK[M2H7L2]
pK[M2H6L2]
pK[M2H5L2]
pK[M2H4L2]
pK[M2H3L2]
pK[M2HL3]
-
-
10.91
9.51
-
-
-
-
9.54
10.40
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.11
3.39
4.92
-
4.90
7.45
-
5.77
7.92
-
-
-
-
-
5.73
-
L1
L3
L2
100
80
100
100
80
60
40
20
0
Ca2H4L22
CaHL2
CaH2L3
Ca2+
CaH-1L1
CaH-1L3
CaH2L1
Ca2+
CaHL1
Ca2H1L33
80
Ca2+
CaL3
CaL21
CaHL 1
2 CaL1
Ca2H3L22
60
60
CaL2
40
40
20
0
Ca2H5L22
CaHL3
20
0
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
100
100
100
Mg2+
MgH-1L3
MgL3
Mg2+
Mg2H4L22
MgH-1L2
MgL2
Mg2+
MgH2L3
80
60
40
20
0
80
60
40
20
0
80
60
40
20
0
MgHL2
MgHL1
MgHL3
MgL21
MgL1
Mg2H3L22
Mg2H5L22
MgH-1L21
MgHL21
6
2
3
4
5
6
7
8
9
10
11
2
3
4
5
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
100
80
60
40
20
0
100
80
60
40
20
0
100
80
CuHL2
Cu2H7L22
Cu2+
Cu2H7L23
Cu2H3L23
CuH-2L21
Cu2H6L23
CuH-2L23
CuL1
Cu2H4L23
Cu2H6L22
Cu2+
CuL21
CuL22
CuH2L2
Cu2L1
CuL2
CuHL1
CuL3
60
CuL23
Cu2H5L22
40
CuH-1L21
CuH-1L23
CuHL3
Cu2+
CuH-1L22
20
0
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
NiH-1L21
11
100
80
60
40
20
0
100
100
80
60
40
20
0
Ni2+
NiL1
Ni2+
Ni2+
NiL3
NiH-2L23
NiHL2
80
60
40
20
0
NiH-2L22
NiL21
NiH2L2
NiL22
Ni2H3L22
NiH-2L21
NiH-1L2
Ni(OH)2
NiH-1L23
NiHL21
Ni2H5L22
NiHL1
NiHL3
Ni2H3L23
NiL2
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
2
3
4
5
6
7
8
9
10
11
pH
Fig. 3. Species distribution profiles for M2+ complexes of L1,2,3 at 25°C and I = 0.1 M KCl, [L]=2×10-3, [M]=1×10-3.
4 | NJC 2018, 00, 1-3
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Table 4. Thermal transition parameters of studied systems (first binding site).
run
cella
syringeb
pHc
n
KITC
[M-1]
ΔGITC
[kJ mol-1]
ΔH°ITC
[kJ mol-1]
ΔS°ITC
[Jmol-1K-1]
L1-Ca2+
0.49
0.34
0.24
4.47
4.51
4.47
7.4
7.4
7.4
0.590
1.552
3.147
4.254
3.255
4.354
-26.41
-31.45
-26.46
0.95
-8.23
-2.41
91.80
77.93
80.72
L2-Ca2+
L3- Ca2+
L1-Ca2+
0.46
0.46
0.49
0.38
0.35
0.20
0.44
0.50
-
4.57
4.57
4.57
4.48
4.48
4.48
4.47
4.47
-
10.0
10.0
10.0
7.4
1.090
1.808
1.537
1.521
2.050
2.977
1.345
1.802
-
6.124
2.955
2.155
9.383
5.864
7.514
6.024
7.504
-
-15.73
-31.21
-18.82
-22.66
-9.81
-27.82
-16.52
-27.82
-
-12.87
-4.07
-12.89
2.50
48.48
91.08
58.84
84.42
26.42
74.76
51.44
64.72
-
L2- Ca2+
L3- Ca2+
L1- Mg2+
L2- Mg2+
L3- Mg2+
L1- Mg2+
L2- Mg2+
L3- Mg2+
7.4
-19.34
-5.54
-11.95
-8.53
-
7.4
10.0
10.0
10.0
aInitial concentrations of the ligands in the cell [mM]. bInitial concentrations of metal ions in the syringe
[mM]. n: the molar ratio M2+/L at which the inflection was observed on the titration curve. CSolvent
details are described in Experimental section.
comparison of stability constants values does not give
straightforward answer for the question towards which metal ion a use this simple approach. Titrations of zoledronic acid (1-hydroxy-2-
ligand binds more sufficient, due to several factors which overlap (1H-imidazol-1-yl)ethylidene-1,1-bisphosphonate) with Ca2+ 67
up to
and influence the strength and formation yields of complexes e.g. date the only example of ITC studies of interactions of BPs with
concentrations, molar ratios, pH and metal-hydrolysis.57
metal ions, were fitted using 'selective binding sites' model,
a
metal ions is more complex and in many cases it is not possible to
,
Most likely only phosphonic moiety is involved into because the isotherms have shown three inflections throughout the
coordination of alkaline earth metal ions. UV-vis titrations of L1 whole titration.67 An existence of first endothermic event was
upon addition of Ca2+ and Mg2+ did not reveal any significant followed by an exothermic one and a subsequent endothermic
changes in wide pH range (data not shown) that suggests oxygen- process. The authors attributed the results to a presence of CaL,
only coordination. According to authors knowledge there is no CaL2 and Ca2L species that exist in pH dependent equilibrium.
examples of amino-BPs that coordinate towards Ca2+ and Mg2+ with Isotherms obtained for studied herein systems; Ca2+/L1 in both
an involvement of amine moiety. Those metal ions prefer pure O studied pHs, Ca2+/L2,3, and Mg2+/L1 in pH 7.4 (Fig. 4, 5) showed one
(phosphonate) coordination.64
exothermic inflection point (ΔHoITC<0) and could be fitted with
In the solutions of L2/Ca2+, L2/Mg2+ and L3/Ca2+, L3/Mg2+ 'independent site' model. Whereas Mg2+/L1, Ca2+/L2,3 in pH 10 and
equimolar complexes were detected with no indication of Mg2+/L2,3 in both pHs, clearly showed additional endothermic
biscomplexes (in contrast to respective L1 systems), while inflection (Fig. 4, 5). The latter curves have been fitted with
polynuclear species are present for both studied ligands (Fig. 3, ‘multiple sites’ model available in NanoAnalyze program, provided
Table 3). In acidic pH range a presence of variously protonated by the producer of the calorimeter.
M2HnL2 (for L2) and M2L3 complexes for Ca2+/L3 was proposed in
In all studied cases the first (or the only existing) inflection can
potentiometric calculations and confirmed by ESI-MS for Mg2+/L2 be assigned to metal ion binding (Table 4). Those are entropy driven,
system. These species (M2L2) co-exist of monomeric (ML) forms of spontaneous processes (ΔGITC<0). ΔG of binding of simple, non-
complexes in solution (Table S2, Fig. S5, SI). Because the amino-BPs with Ca2+ was previously shown to be ~29kJ/mol.68 It
potentiometry is a method that is not able to distinguish between stays in a good agreement with presented here data (except for
these two types of species, it causes the obtained species Mg2+/L2 system for which ΔGITC is smaller) and confirms no
distributions for L2 in acidic pH (Fig. 3) to be only a presumption.
additional support from a coordination of neither amino-group nor
In order to quantify the thermodynamics of the formation of phosphonic function from the other side of the molecule (Table 4).
Ca2+ and Mg2+ complexes, a direct determination of enthalpy For the systems into which ‘multiple sites’ models needed to be
binding change (ΔHoITC) was applied by using isothermal titration applied the second inflection was in all cases fitted with n~0.1-0.4
calorimetry method (ITC). Measurements were performed in two stoichiometry. Endothermic nature of those reactions does not
pHs; 1) pH 7.4, chosen for biological reasons, and 2) pH 10.0, in allow to attribute this process to creation of new bonds (ΔHoITC>0
which all phosphonic moieties are deprotonated. Titrations mode of for the second ‘binding’ site, see Fig. 4, 5). Rather a change in the
ligand solution being an analyte and metal solution being a titrant water structure surrounding cation, i.e. the desolvation of water
was applied (M2+syringe→Lcell). The results in the form of binding might be responsible for the observed thermal behavior with an
isotherms, that depend on stoichiometry (n), binding constant (KITC
)
endothermic character.69 Nevertheless, the chemical nature of this
and change on enthalpy (ΔHoITC), are depicted in Fig. 4 and Fig. 5 inflection remains unclear since it might also be assigned to
with details of calculated fitting curves. Estimated values of KITC, n, polymerization of unbound ligand individuals, to bonds breaking
ΔHoITC and ΔSo together with calculated Gibbs free energy ΔGITC
are given in Table 4.
,
process or interactions with buffer molecules. At the same time
neither cooperative effect nor competitive replacement type of
ITC
Many classical interactions between ligand and metal ions are interactions were detected.
described by 'independent' model used to match the resulting
binding isotherms that is based on 1 for 1 approach.65 It was also
applied to describe rarely performed ITC studies for metal-
phosphonates interactions.66 However, the interaction of BP with
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Ca2+→L1
Ca2+→L2
Ca2+→L3
pH 7.4
pH 10.0
Fig. 4. ITC experiment of the titrations of Ca2+ →L1, L2, L3 titrations at pH 7.4 and pH 10.0. Raw data overlaid with calculated models.
In order to give a comprehensive thermodynamic picture of considerably higher (for approx.
5 logarithmic units) stability
studied systems and compare potentiometric data with calorimetric constants of formed complexes (Table 1, Fig. 3). ESI-MS spectra
ones, the logarithms of conditional stability constants have been exhibit peaks which could be attributed to ML, ML2 and M2L (only
calculated (logKc) at fixed pH values and summarized in Table 5. for Cu2+ coordination). Furthermore, an existence of M2L2, M3L2 and
Overall, the data stay in an agreement and are a good reflection of M3L3 was detected (Table S2, SI).
real binding strength, independent from the co-presence in the
Coordination for Cu2+ starts around pH 2, and Ni2+ ~ pH 3, what
solution of different complex species. Yet due to the complex is confirmed by d-d absorption spectra of studied systems which
equilibrium in solutions, determined stoichiometry values (n) maximum revealed blue-shifts at acidic pH (Fig. S2 and S3).
should be considered as approximate (Table 4). The fit of isotherms Formation of MHL shifts maximum of absorption from 810 nm to
was carried out using standard program options assuming a binding 685 nm for Cu2+ and from 720 nm to 728 nm, and from 652 nm to
of individuals in the solution with full saturation, which was not 635 nm for Ni2+ (Table 6). Binding via oxygen atom only in
achieved for studied ligands, in particular below pH 7.4. Here, for analogues amino-BPs was causing d-d transition ~30 nm blue-shift
almost all systems 'n' was estimated to be > 1, that would suggest only.45 Here this significant change was four times bigger, so it lead
the existence of MnL complexes. However, they were not found for us to an assumption that nitrogen atoms are involved in binding of
each case in ESI-MS studies. The affinity of the ligands expressed as the metal ion. Coordination in CuHL is most likely already
logKc value for both Ca2+ and Mg2+ ions rises with pH (see Table 4) implemented trough bidentate mode [N,O] with creation of
most likely because above pH 10 all phosphonic groups are unfavorable seven-membered ring. For Ni2+ such assumption is less
deprotonated and available for coordination. Dropping pH to the probable since the change on the spectra correspond to ~ 20 nm
value of 7.4 drops this affinity by 1-2 logarithmic units.
shift, which rather are responsible for pure oxygen coordination in
NiHL species. Since CuHL complex strongly overlaps with Cu2L this
Complexation of Cu2+and Ni2+. A coordination model for L1/Ca2+ and notable shift for Cu2+/L1 can be partially attributed to the presence
L1/Ni2+ does not differ significantly from the one of alkali earth
metal ions, except the existence of Cu2L1 species in acidic pH and
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Mg2+→L1
Mg2+→L2
Mg2+→L3
pH 7.4
pH 10.0
Fig. 5. ITC experiment of the titrations of Ca2+ →L1, L2, L3 titrations at pH 7.4 and pH 10.0. Raw data overlaid with calculated models.
Table 5. Conditional stability constants (logKc) for Ca2+ and Mg2+ complexes of studied ligands at selected pH values determined by potentiometry and ITC in 25°C.
M2+/ligand
pH
logKc
(potentiometrya)
4.00
logK ITC
(ITCb )
4.63
5.50
4.64
4.78
5.47
5.33
3.97
4.32
4.76
4.79
4.77
nd
match
Ca2+→ L1
Ca2+→ L2
Ca2+→ L3
Ca2+→ L1
Ca2+→ L2
Ca2+→ L3
Mg2+→ L1
Mg2+→ L2
Mg2+→ L3
Mg2+→ L1
Mg2+→ L2
Mg2+→ L3
7.4
7.4
+
+
+
~
+
+
+
+
~
~
+
5.54
7.4
3.88
10.0
10.0
10.0
7.4
5.92
5.85
5.11
3.50
7.4
4.02
7.4
3.75
10.0
10.0
10.0
5.76
4.87
5.17
aObtained by Hyss2009 based on traditional approach introduced by G.Schwarzenbach,70 defined as Kc=[M2+
experimental logβ values (under concentration conditions: [M2+]=2.0×10-6-1.0×10-3, [L]=2.0×10-3 and fixed pH value). bBased on direct ITC result of K ITC (Fig. 4, 5, Table
]
bound to L /[M2+ not bound to L×[M2+
] ]not bound to M2+, based on
4).
of
a
complex in which one ligand binds two metal ions. Cu2+58 or Ni2+61 was excluded in solid state. Possibly as soon as
+
Nevertheless, the involvement of NH3 throughout a whole pH phosphonic groups are deprotonated the coordination with
range is rather unfavorable due to steric reasons. Primary amine unfavourable geometry is replaced by [O,O] bidentate, purely
was reported to be involved in charge-assisted hydrogen-bonding
which stabilized the complex structure, but its binding to either
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Table 6. Spectroscopic characteristics of Cu2+ and Ni2+ complexes.
Cu2+ and Ni2+ speciation profiles for L2 and L3 are depicted on
Fig. 3 and are comparable. Coordination of Cu2+ is more favourable
and starts for both ligands below pH 2, for Ni2+ ~ pH 3.5 with a
formation of oligomeric complexes of M2L2. stoichiometry (Table 3)
Their presence was confirmed by ESI-MS studies for Cu2+/L2 and
Ni2+/L2 systems (Table S2, Fig. S5, SI). M2L2 complexes coexist in the
solution with ML species in acidic pH (Fig. 3). Since for Cu2+/L2 and
Ni2+/L2 systems upon applying 1:1 molar ratio in spectroscopic
studies revealed the presence of opalescence (below pH 5), we
assume that the oligomers aggregate in solution and create
precipitation. It should be added that polymerization of partly
deprotonated ligands may occur in parallel and probably have an
additional effect on precipitation. Precipitation in an almost whole
pH range permitted to obtain spectroscopic data for Cu2+/L3 and
Ni2+/L3 (Table 6, Fig. S3). When rising pH up to 5 in case of Cu2+/L2
and up to 7 for Ni2+/L2, only equimolar species and biscomplexes
are present in the solution. Identified by ESI-MS species are as
Ligand
Species
UV-visa parameters
λ(nm)
ε (M-1 cm-1)
pH
[Cu2L]
2.0
3.0
7.0
9.0
10.0
11.0
737
685
701
707
702
694
16
35
49
58
58
61
[CuHL]b
[CuL]
L1
[CuL2]b
[CuH-1L2]
[CuH-2L2]b
[CuH2L]b
[CuHL]b
[CuL]b
5.5
7.0
8.5
10.0
11.0
2.0-5.0
755
702
658
647
650
nd
31
40
44
46
46
-
L2
[CuL2]b
[CuH-1L2]
[Cu2HnL2]
[CuHL]
[CuL]
7.0
8.5
nd
nd
-
-
-
-
46
-
[CuL2]
9.0
9.5
11.0
2.0-8.0
nd
nd
606
nd
L3
[CuH-1L2]
[CuH-2L2]
[Cu2HnL2]
following: NiL2,3, CuL3, CuL22 and CuL2
.
3
First formed equimolar complex is MH2L which causes blue-
shifting of maximum of d-d band from 810 nm for Cu2+aqua to 755
nm. For Ni2+ a spectral change is reflected in ~ 20 nm shift (Table 6).
Coordination of M2+ in MHL can be therefore attributed to simple
bidentate coordination [O,O].60 Creation of CuHL most likely
corresponds to deprotonation only of second bisphopshonic
function (pK=5.80 for Cu2+/L2 and 6.44 for Ni2+/L2). In pH range 7-10
the d-d transition band of Cu2+/L2 system shifts significantly from
755 nm to 647 nm and suggests an involvement of nitrogen atom in
coordination [N,O,O] in CuL and later on 2× [N,O,O] in CuL2, as for
single-amino-BPs studied previously.45 Further deprotonation to
hydrolytic species (pK 10-77-10.91) can be assign to H2O→OH-
process. However, since almost no changes were detected on
Ni2+/L2 system above pH 8 (see Fig. S3, SI) we assume that the
involvement of NH+R in Ni2+ coordination is rather not possible and
the coordination mode remain purely oxygen one.63
L1
[NiHL]b
[NiL]b
4.6
6.0
6.5
8.1
9.5
10.9
728(sh), 635, 384
735(sh), 644, 383
nd
5.4, 6.1, 11
6.9, 9, 15
-
10, 22
15, 34
18, 47
[NiHL2]
[NiL2]b
651, 386
648, 384
644, 379
[NiH-1L2]b
[NiH-2L2]b
[NiH2L]
[NiHL]
[NiL]
[NiH-1L]
[NiL2]
[NiH-2L2]
[Ni2HnL2]
4.8
8.0
9.0
10.5
10.0
11.0
3.0-6.5
nd
-
755, 679, 408
4.9, 4.8, 12.5
nd
nd
-
-
L2
755(sh), 683, 405
5, 6, 15
-
-
nd
nd
[NiHL]
[NiL]
[NiH-1L2]
[NiH-2L2]
[Ni2HnL2]
8.5
9.5
10.5
11.0
6.0
nd
nd
-
-
-
L3
nd
749, 685, 406
nd
5.6, 5.5, 16
-
Absorption spectra show that all Cu2+ and Ni2+ complexes of
studied ligands are of octahedral or pseudo-octahedral geometry.
[a]d-d. For comparison; [Cu(H2O)6]2+, λ [nm](ε [M-1 cm-1])=810(17), [Ni(H2O)6]2+
,
720(3.3), 652(3), 393(6.4), under the same concentration conditions as measured
systems. The data reflect optical properties of solutions in which the given forms are
major species. Sh=shoulder. Nd=not detected. [b] Stability constants of marked
species calculated in HypSpec2014 based on spectrophotometric titrations are given
in Table S1, SI.
Liphophilicity measurements
BPs are characterized as highly hydrophilic compounds in general.
This is the reason for their low bioavailability when administered as
phosphonic with six-membered rings or [N,O] mixed coordination
mode with a participation of NH+R. Rising pH up to 7 leads to
creation of CuL which causes red-shift of maximum of d-d band
from 685 nm to 701 nm. Final deprotonation of phosphonic
function and creation of [N,O,O] tridentate favored by other amino-
BPs systems45 coordination might be responsible for this spectral
shift. Further pH rise leads to the formation of CuL2. The energy of
d-d transition for CuL2 complex equals to λ=707nm and ε=58(M-1
cm-1) and is higher in relation to the d-d absorption band of CuL. No
significant shift suggests an involvement in binding of a second
ligand oxygens only. Above pH 10 the band blue-shifts slightly from
707 nm to 694 nm. For Ni2+/L1 the changes do not exceed ~ 5 nm.
Species distribution diagrams show in this pH region a formation of
MH-1L2 [M(OH)L2] and MH-2L2 [M(OH)2L2] hydroxy-complexes (pK
range: 8.61-10.56). Calculations based on UV-vis titrations of the
Cu2+/L1 and Ni2+/L1 systems confirms proposed model and
calculated stability constants are given in Table S1, SI.
drugs and one of
a serious problems associated with the
administration of high doses of BPs during treatment. In order to
estimate lipophilicity of studied compounds, shake flask method
was applied.71 A partition coefficient (logPo/w), that provides a
measure by which to assess the ability of a drug candidate to be
absorbed across an intestinal membrane, was determined
experimentally for free ligands and their Ca2+ complexes. The
results are presented in Table 7 in comparison to alendronate (4-
amino-1-hydroxybutyl-1,1-bisphosphonate), routinely used drug for
osteoporosis treatment. All ligands exhibit logPo/w values which
indicate a hydrophilic character, as expected, notwithstanding -
smaller than alendronate. An increase of lipophilicity upon binding
via polar functions to metal ion is observed for Ca2+ complexes,
what most likely would be reflected in higher intestinal uptake of
complexes in comparison to free ligands.
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Table 7. Experimentally Determined logPo/w Values.[a]
The most abundant species under conditions of IC50 in pH 7.4 are
given in Fig. S6, SI, that depicts recalculated distribution species
diagrams in narrow pH range. The diagrams indicate complex
equilibrium out of which one active species cannot be clearly
pointed out. A majority of cases exhibits coexistence of protonated
species of complexes with protonated forms of free ligands.
Compound
log Po/w (L)
log Po/w
log Po/w (L)
(Ca2+/L, 1:2)
-0.24 ± 0.3
-0.84 ± 0.3
-1.28 ± 0.3
nd
(Ca2+/L, 1:1)
-0.53 ± 0.3
-0.88 ± 0.3
-0.77 ± 0.3
nd
L1
-1.27 ± 0.3
-2.00 ± 0.3
-2.43 ± 0.3
nd
L2
L3
incadronate
alendronate
-4.4972,73[b]
aShake-flask method; Po/w = [ ]1-octanol/[ ]HEPES; pH 7.4; 25°C, concentration
determined by UV-vis absorption. Incadronate logPo/w values cannot be
measured due to a lack of spectra in UV-vis range.bcalculated
Conclusions
In this study combined potentiometric, ESI-MS, spectroscopic and
calorimetric studies allowed us to investigate complex-formation
abilities of three amino-BPs based on cyclohexane backbone: one
single-amino-BPs (equipped in one bisphosphonic moiety) and two
double-amino-BPs (equipped in two bisphosphonic moieties) in
complexation of Ca2+, Mg2+, Cu2+ and Ni2+ ions in aqueous solution.
Both ligands and their complexes were tested in terms of cytotoxic
activity with respect to two cell lines (human melanoma A375 and
human colorectal adenocarcinoma HT29) and half minimal
In vitro anticancer activity
Colon adenocarcinoma and melanoma skin cancer are the most
common malignancy throughout the world. Both cell lines (HT29
and A375) are a well-established model of colon and skin cancer,
which preferentially metastasizes to bone.74 In vitro anticancer
activities of the ligands and Ca2+ and Mg2+ complexes were
evaluated in melanoma A375 cells and human colon
adenocarcinoma HT29 cell by the MTT assay. Incadronate was used inhibitory concentrations (IC50) were determined.
Complex equilibria in solution consist of equimolar, biscomplex
for reference purposes. Representative dose−response curves are
shown in Fig. S8, SI. Ligands; L1 and incadronate significantly
suppressed cell growth in both A375 and HT29 cell lines, whereas L2
and L3 had weaker effects. The resulting half minimal inhibitory
concentration (IC50) values were displayed in Table 8. All of the
compounds exhibited cytotoxic activity at tested concentrations
(IC50 > 600 μM). Notably, L1 and incadronate gave rise to IC50 values
of less than 350 μM and 450 μM in a case of A375 and HT29 cell
lines, respectively. Among all tested Ca2+ and Mg2+ complexes of L1
and incadronate, the species showed higher cytotoxicity on A375
and HT29 cells than the free ligands. Compounds L2 and L3
containing Ca2+ and Mg2+ showed decrease cytotoxicity in
proliferation to cell lines. As a general observation, the complexes
of single-amino-BP showed higher effectiveness against both the
cell lines than the respective complexes of double-amino-BPs.
Although the mechanism of the antitumor activity exhibited by
these ligands and their complexes remains unknown, we tentatively
suppose that they may directly induce apoptosis in cancer cell lines,
as previously reported for similar compounds.75,76
and oligomeric complexes, which is a typical feature for amino-BPs.
In a coordination of Ca2+, Mg2+ and probably Ni2+, the amino groups
of studied ligands are not involved, whereas in the case of Cu2+, a
participation of both: NH3+ (for L1, up to pH 7) and NH+R group (for
all ligands) is highly probable. L1, L2 and L3 bind biogenic metal ions
upon spontaneous, exothermic reaction that are entropy driven
processes and create stabile complexes in broad pH range. Stability
constants of ML complexes fall into well-known rank: logβCuL
>
logβNiL >> logβMgL ~ logβCaL and the magnitude of order of logβs is
consistent with literature data for single-amino-BPs, that confirms
no participation of second BP moiety in binding of one metal ion.
Estimated via potentiometry (logKc) and isothermal titration
calorimetry (logKITC) independent conditional stability constants for
pH 7.4 and pH 10.0 stay in a good agreement.
Ca2+ and Mg2+ complexes of L1 as well as incadronate showed
higher inhibitory rate than their free ligands against A375 and HT29
cell lines. However, it was impossible to indicate which complex is
active, because in the conditions of biological measurements the
equilibrium consist both: complexed and unbound ligand molecules.
In comparison, L2 and L3 complexes showed slight decrease of
cytotoxicity upon coordination against both cell lines.
Table 8. Anti-proliferative activity of studied ligands with or without Ca2+ or Mg2+
Incorporation of cyclohexane backbone increased the
liphophilicity of studied ligands, which is desirable for newly
designed ligands that are intended to potentially act in direct or
indirect way as drugs or building-blocks for drugs-related materials
Furthermore, since being equipped in a second donor function,
apart from bisphosphonic one, did not change the complexation
abilities of studied ligands in comparison to literature examples of
single-amino-BP it opens new route of thinking for such compounds
to use this second function as an anchor for a different molecule.
against melanoma A375 cells and adenocarcinoma HT29 cells.
Compounds/complexes
IC50 [μM]
A375
HT29
L1
349.24±3.42 435.19±3.26
215.28±2.04 338.91±2.47
313.31±2.11 410.78±2.02
L1-Ca2+
L1-Mg2+
L2
527.25±2.88 486.16±7.22
539.46±4.91 519.75±0.86
543.28±1.85 540.55±4.21
L2-Ca2+
L2-Mg2+
L3
577.36±5.21 541.55±3.21
603.61±1.75 587.02±2.19
647.53±0.32 695.31±4.83
L3-Ca2+
L3-Mg2+
Experimental Section
Chemicals
incadronate
328.03±2.64 423.16±1.49
243.92±1.38 282.61±5.29
273.42±5.71 382.50±2.75
All solvents and reagents were purchased from commercial
suppliers, were of analytical grade and were used without further
purification. Unless otherwise specified, solvents were removed
with a rotary evaporator. The 1H-, 31P- and 13C-NMR spectroscopic
Incadronate-Ca2+
Incadronate-Mg2+
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experiments were performed on a Bruker Avance II Ultrashield Plus = 18.88, 28.50, 37.27, 51.52, 54.82 (t, J=127.97Hz, CHP2);35,77 HRMS
(Bruker, Rheinstetten, Germany) operating at 600.58 MHz (1H), (TOF MS ESI); [M-H]- Calcd for C8H22N2O12P4: 461.0045; found:
243.12 MHz (31P{1H}) and 151.016 MHz (13C). Measurements were 461.0032.
made in solutions of D2O + NaOD at 300 K, and all solvents were
supplied by ARMAR AG (Dottingen, Switzerland). Chemical shifts are Synthesis of L3.
(trans)-Cyclohexane-1,4-di(aminomethylene-
reported in ppm relative to TMS and 85% H3PO4, used as external bisphosphonic) acid (L3) was obtained as a white solid; yield: 52%;
standards, and coupling constants are reported in Hz. Melting mp 243 0C; 31P-NMR (243.12 MHz, D2O+NaOD, ppm) δ = 18.50; H-
1
points were determined on an SRS Melting Point Apparatus NMR (600.58 MHz, D2O+NaOD, ppm) δ =1.31-1.41 (m, 4H, CH2),
OptiMelt MPA 100 (Stanford Research Systems, Sunnyvale, CA, 2.14-2.23 (m, 4H, CH2), 2.98 (t, J=16.10Hz, 2H, CHP), 3.38-3.47 (m,
USA) and are reported uncorrected. Mass spectra were recorded at 2H, CHN); 13C-NMR (151.02 MHz, D2O+NaOD, δ = 27.51 (CH2), 55.35
the Faculty of Chemistry, Wroclaw University of Science and (t, J=116.20Hz, CHP2), 56.22 (CHN);35,36,77 HRMS (TOF MS ESI); [M-
Technology using a Waters LCT Premier XE mass spectrometer H]- Calcd for C8H22N2O12P4: 461.0045; found: 461.0047.
(method of electrospray ionization, ESI) (Waters, Milford, MA, USA).
Synthesis of incadronate. N-(cycloheptyl)aminomethylene-
General procedure of the synthesis of
bisphosphonic acid (Incadronate) was obtained as a white solid;
yield: 67%. Cycloheksylamine (0.03 mol, 3.39 g), triethyl
orthoformate (0.033 mol, 5.30 mL) and diethyl phosphite (0.066
aminomethylenebisphosphonic acid (L1) and
bisaminomethylenebisphosphonic acids (L2, L3)
mol, 8.52 mL) were heated and simultaneously stirred at
a
Diamine (0.03 mol) and the appropriate amounts of diethyl
phosphite (0.126 mol, 16.32mL) and triethyl orthoformate (0.063
mol, 4.4mL) were were heated and simultaneously stirred at a
temperature of ~130 °C on a heating plate (125 °C in the reaction
medium) of a Radley’s Carousel apparatus overnight (15 h). The
mixture was cooled, and the volatile components were removed
using a rotary evaporator. The resulting mixture was dissolved in
ethyl acetate (100 mL) and purified by washing with water (100 mL),
saturated sodium chloride solution (100 mL) and again with water
(100 mL). The solution was dried over anhydrous MgSO4, and the
solvent was evaporated under vacuum. The obtained ester (0.030
mol) was refluxed for 15 h (overnight) in 40 ml 6 N aqueous
hydrochloric acid solution. After cooling, the volatile components
were removed using a rotary evaporator, and the resulting oil was
dissolved in minimal amount of water (40-50 mL), discolored with
activated charcoal and purified by crystallization from water–
ethanol mixture (80/20 v/v). The impure product was mixed with
water for few days until the dissolution of impurities was observed
and then was filtered and washed with distilled water and dried in
vacuum.
temperature of ~130 °C on a heating plate (125°C in the reaction
medium) of a Radley’s Carousel apparatus overnight (15 h, Radleys,
Essex, UK). The mixture was cooled, and the volatile components
were removed using a rotary evaporator. The resulting mixture was
dissolved in ethyl acetate (100 mL) and purified by washing with
water (100 mL), saturated sodium chloride solution (100 mL) and
again with water (100 mL). The solution was dried over anhydrous
MgSO4, and the solvent was evaporated under vacuum. Boiling in
20 mL of 6 N hydrochloric acid for 12 h hydrolysed the resulting
crude reaction product. After cooling, the volatile components were
removed using a rotary evaporator, and the resulting oil was
dissolved in minimal amount of water (30 mL), discolored with
activated charcoal and purified by crystallization from hot
water/ethanol mixture. Mp 249-250°C; 31P-NMR (243.12 MHz,
1
D2O+NaOD, ppm) δ =8.03; H-NMR (600.58 MHz, D2O+NaOD, ppm)
δ =1.49-1.63 (m, 6H, CH2), 1.67-1.80 (m, 4H, CH2), 2.11-2.21 (m, 2H,
CH2), 3.59 (t, J=18.23Hz, 1H, CHP2), 3.66-3.74 (m, 1H, CHN); 13C-
NMR (151.02 MHz, D2O+NaOD) δ= 24.49, 25.42, 31.46, 54.71 (t,
J=124.39Hz, CHP2), 56.14 (CHN);45,77,78 HRMS (TOF MS ESI); [M-H]-
Calcd for C8H19NO6P2: 286.0609; found: 286.0597.
Synthesis
of
L1.
Cyclohexane-1-amino-2-aminomethylene-
bisphosphonic acid (L1) was obtained as a white solid; yield: 64%;
The studied ligands are well soluble in aqueous solutions in the
range of concentrations used in the experiments (details below).
mp 235-2360C; 31P-NMR (243.12 MHz, D2O+NaOD, ppm) δ = 17.73
(AB system, J=17.73Hz); 1H-NMR (600.58 MHz, D2O+NaOD, ppm) δ Potentiometry
= 0.82 (qbr, J=11.50Hz, 1H), 1.01 (m, 1H), 1.08-1.12 (m, 2H), 1.49-
The chemicals were commercial products of reagent grade and
1.54 (m, 2H), 1.66 (d, J=12.55Hz, 1H), 1.99 (d, J=12.76HZ, 1H), 2.21-
2.26 (m, 1H), 2.44-2.48 (m, 1H), 2.67 (d, J=16.88Hz, CHP); 2.71 (d,
J=17.17, CHP); 13C-NMR (151.02 MHz, D2O+NaOD, δ = 24.51, 24.73,
30.57, 33.68, 55.43, 56.26 (d, J=123.06Hz, CP), 57.13 (d, J=124.50Hz,
CP);35,77 HRMS (TOF MS ESI); [M-H]- Calcd for C7H18N2O6P2:
287.0562; found: 287.0511.
were used without further purification. Solutions were prepared
with bidistilled water which were flushed with Ar to exclude CO2.
The ionic strength was fixed at I = 0.1 M with KCl (POCh). The
titrations were performed using a Metrohm 809 Titrando system
equipped in combined glass electrode (Metrohm 6.0224.100) which
was calibrated daily in hydrogen concentrations using HCl (Merck)
(~0.004M) according to the procedure of Irving et al.79 Alkali, CO2 -
free 0.1029 M KOH solution (POCh) was added from a Metrohm
800 Dosino auto burette. The purity and exact concentration of the
ligand were determined by the method of Gran.80 The stock
solutions of metal ions were prepared by dissolving the appropriate
amount of chlorides in aqueous HCl solution. All the titrations were
carried out on 2.0 ml samples in a thermostatted cell at 25±0.2°C
Synthesis of L2. Cyclohexane-1,3-di(aminomethylenebisphosphonic)
0
acid (L2) was obtained as a white solid; yield:51%; mp 269-270 C;
1
31P-NMR (243.13 MHz, D2O+NaOD, ppm) δ = 17.54&17.78; H-NMR
(600.58 MHz, D2O+NaOD, ppm) δ = 1.18-1.23 (m, 2H, CH2), 1.31-
1.39 (m, 2H, CH2), 1.46-1.54 (m, 4H, CH2), 2.67(t, 2H, J=16.77Hz,
CHP2), 3.09-3.16 (m, 2H, CHN); 13C-NMR (151.02 MHz, D2O+NaOD, δ
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under a stream of Ar. HYPERQUAD201381 computer program that concentrations, molar ratios and pH for L3 with all studied metal
use non-linear least-squares methods82 was applied to calculate the ions, it reflected with no results for L3/Ca2+, Mg2+ systems (after
stability constants. The results were obtained in the form of filtration). Except for Cu2+/L1 system (to which both data are given in
concentration overall stability constants βpqr = [MpHqLr]/[M]p[H]q[L]r, Table S1, SI) no significant differences between different molar
where M stands for metal. H is proton and L the deprotonated form ratios were noticed. Analyte solutions (70 μl) were introduced at a
of the ligand. Triplicate titrations of the free ligand and the flow rate of 3 μl/min. The instrument parameters were as follows;
complexes were carried out at metal to ligand ratios 1:1.1, 1:2, 1:3 scan range: 50–3000 m/z, drying gas: nitrogen, flow rate: 4.0 L/min,
and 1:4. Opalescence occurred in 1:1.1 solution of L2 with Cu2+ and temperature: 200 °C and potential between the spray needle and
Ni2+ in acidic pH. The precipitation in whole pH range was present in the orifice: 4.0 kV. For MS spectra analysis a Bruker Compass
1:1.1 solutions of L3 with Cu2+ and Ni2+. The titrations with DataAnalysis 4.2 software was used. A sophisticated numerical
opalescence or precipitations were excluded from calculations and annotation procedure (SNAP) algorithm was used for finding peaks.
higher molar ratios were applicated. The ligand concentration was Relative intensities (Table S1 and S2) were read directly from the
3
3
1.0×10− mol dm− in all titrations. The distribution curves of the MS spectra in the DataAnalysis 4.2 program. The Supplementary
protonated species of ligands as a function of pH were calculated Information contain raw MS spectra with intensities on the y-axis
using HySS2009 program.83
and isotopic patterns enlargements for the main species present.
Isothermal titration calorimetry
Spectrophotometric measurements and combined
potentiometric/spectrophotometric measurements.
ITC experiments were carried out using a Nano ITC calorimeter (TA
Instruments) with a standard volume of 1.0 ml cell at 25°C. In a
typical experiment the solution of ligand (0.24-0.50mM; in buffer)
was placed in the cell and the solution of; Ca2+ (4.47-4.57mM, in
buffer) or Mg2+ (4.47-4.48mM, in buffer) was taken up in a 250 μL
injection syringe. The concentration of buffers (HEPES pH 7.4, CAPS
pH 10.0) was 100mM. pH of each sample was measured before and
after the experiment to be sure of keeping it constant. Each sample
was degassed prior the titration for 5 minutes. The total number of
40 injections, 6 μL each were added after the calorimeter finalized
the primary equilibration, with 300s apart. The stirring rate was
300rpm. The calorimeter was operated using the Nano ITC Run
software and all the data obtained were analyzed with the
NanoAnalyze v. 3.1.2 program. 'Independent' or 'multiple sites'
models were used to evaluate the results obtained and the control
experiments were performed in each case; the enthalpies of
reagents dilution were subtracted from the enthalpies of binding
processes. Each ITC data was collected by two independent
measurements and reproducible data was employed. Standard
deviation fit of the data is provided in the Supplementary
Information (Fig. S7, SI).
Absorption spectra in combined potentiometric and UV-vis
titrations were collected using Varian Cary 50 spectrophotometer
equipped with immersion probe made of Quartz SUPRAZIL®300 and
removable light path tip with 1cm optical path (type 665.622) for
Cu2+ and Ni2+ complexes. The initial pH of 10 ml samples prepared
in 0.1M (KCl) ionic strength was adjusted to be acidic and the
titration of the solution was carried out by addition of known KOH
volumes dosed by Metrohm 809 Titrando system. Special care was
taken to ensure that complete equilibration was attained before
collecting the spectra. The spectral deconvolutions were refined
using the least squares fitting program HypSpec2014.84 Factor
analysis by the HypSpec software was implemented to characterize
the number of species present in the solution. The concentration of
ligands was 2.0-3.0×10−3M for Cu2+ measurements and 3.0-
5.0×10−3M for Ni2+. For L1 molar ratio M2+:L was kept 1:2 and for L2
and L3 1:1 since while having 1:2 (and 1:3) solutions disclosed slight
precipitation. For L3 for both metal ions it was possible to record
only few spectra (Fig. S2 and S3) due to a precipitation under
applied conditions, lowering concentration to 1.0×10−3M as it was
used for potentiometric studies caused the spectra to be in the
range of noise. Experimental errors on λmax=±2nm and ε=±5%.
Absorption spectra for liphophilicity studies were collected
Lipophilicity measurements
using Hitachi HALO DB-20 double beam spectrophotometr (U-2800), The shake-flask method was used to experimentally determine the
using quartz Hellma cuvettes.
log Po/w values for studied ligands and their calcium complexes. The
aqueous (50mM HEPES, pH 7.4 and IKCl=0.16M, 25°C) and 1-octanol
solvents were used in the experiment. 0.750ml of 1-octanol and
0.75ml of 1mM solution of studied ligand or complex (in molar ratio
1:1 and 1:2, [L]=2×10-3M) were mixed together. The samples were
vortexed (~2min), shaked manually (~2min) and centrifuged (~1min,
5000 rpm). The layers were separated and measured by UV-vis
spectroscopy, applying the Beer-Lambert law to determine the
ligand concentration in analyzed layer (ε i λmax are given in Table S3,
SI). Measurements were run in triplicate. Incadronates' lipophilicity
could not be measured due to a lack of spectra in measured range.
Mass spectrometry
All ESI-MS experiments were performed on a compact™ mass
spectrometer (Bruker Daltonics, Bremen, Germany) equipped with
standard ESI source. The instruments were operated in the positive-
ion mode and calibrated with the Tunemix™ mixture (Agilent
Technologies. Palo Alto. CA. USA) in a quadratic method. Spectra
were recorded for samples dissolved in MeOH/H2O 50/50 mixture
with M:L molar ratio 1:1 and 1:2 (pH~5, lowered by HCl and pH~7-8,
raised up by KOH, rising pH up to 10 in each case caused a
significant deterioration of the spectrum) and heated up to 40°C for
15 min prior to the experiment. In case when a precipitation was
formed, the solids were filtered off and the clear solution was
measured. Strong precipitation was observed regardless of
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Cell lines and culture conditions
Wrocław Centre of Biotechnology, programme The Leading
National Research Centre (KNOW) for years 2014-2018 (ECh).
The human cancer cell lines, melanoma (A375) and colon
adenocarcinoma cells (HT29) were used for in vitro screening
experiments. A375 melanoma (ATCC CRL-1619) cells and colon
adenocarcinoma cell line HT29 were obtained from Dr. Dorota
Nowak and Dr. Elżbieta Gocek (University of Wroclaw, Poland),
respectively. The cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Institute of Immunology and Experimental
Therapy (IITD), Poland) containing 10% fetal bovine serum (FBS,
Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) at 37 ºC in
5% CO2. For all the experiments, the A375 cells and HT29 cells were
used at 80% confluence following 2–6 passages.
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Authors Contributions
JG conceived and designed studies and wrote the paper. ECh
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the experiments. PK reviewed the paper. All authors read and
approved the manuscript.
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Conflicts of Interest
There are no conflicts to declare.
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ARTICLE
NJC
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Graphical Abstract
Figure
Synopsis
Complex formation equilibria of calcium, magnesium,
copper and nickel with amino-bisposphonic ligands is
described, together with a speciation study along with
calorimetric outcome and cytotocixity characteristics..