Copper(II) and gallium(III) complexes of trans -Bis(2-hydroxybenzyl) cyclen derivatives: Absence of a cross-bridge proves surprisingly more favorable

Article

pubs.acs.org/IC

Copper(II) and Gallium(III) Complexes of trans-Bis(2-hydroxybenzyl)

Cyclen Derivatives: Absence of a Cross-Bridge Proves Surprisingly

More Favorable

Catarina V. Esteves, Joana Madureira, Luís M. P. Lima, Pedro Mateus, Isabel Bento, and Rita Delgado*

́

Instituto de Tecnologia Química e Biologica, Universidade Nova de Lisboa, Av. da Republica 2780−157 Oeiras, Portugal

S

* Supporting Information

ABSTRACT: Two cyclen (1,4,7,10-tetraazacyclododecane) deriva-

tives bearing trans-bis(2-hydroxybenzyl) arms, the 1,7-(2-hydrox-

ybenzyl)-1,4,7,10-tetraazacyclododecane (H₂do2ph) and its cross-

bridged counterpart (H₂cb-do2ph), have been synthesized, aiming

toward the possible use of their copper(II) and gallium(III)

complexes in nuclear medicine. The protonation of both compounds

was studied in aqueous solution as well as their complexes with Cu²⁺

and Ga³⁺cations. The complexes of both ligands with Ca²⁺and Zn²⁺

metal ions were also studied due to the abundance of these cations

in biological media. In mild conditions the complexes of Ca²⁺and

Ga³⁺with H₂cb-do2ph did not form. The behavior of the two

ligands and their complexes was compared by the values of the

equilibrium constants, the data of varied spectroscopic techniques,

the values of redox potentials of their copper(II) complexes, and the resistance of the complexes to acid dissociation. It was

expected that, as found for related pairs of cyclen and cyclam (1,4,8,11-tetraazacyclotetradecane) derivatives, the cross-bridged

macrocyclic derivative could be an excellent ligand for the complexation of copper(II). Additionally, the N-2-hydroxybenzyl

groups were chosen due to their known ability to coordinate the gallium(III) cation. Due to the small size of the latter cation and

its particular propensity to form hexacoordinate complexes, it was also expected that there would be a good ability of both ligands

for the uptake of Ga³⁺. Surprisingly, the results revealed that the cyclen derivative H₂do2ph is the best ligand for the coordination

of Cu²⁺and Ga³⁺cations, not only from their thermodynamic stability as expected but also from their kinetic inertness, when

compared with its cross-bridged counterpart.

chelates (BFC) used in noninvasive imaging techniques such

as positron emission tomography (PET) and single photon

emission computed tomography (SPECT), or in radioimmu-

notherapy (RIT) of tumors. The BFC is composed of a metallic

radioisotope complex to which a targeting biomolecule, capable

of binding to speciﬁc receptors overexpressed in certain tumors

or organs in the body, is covalently bound to a suitable point of

the ligand framework.^3,13,14This is especially important when

short-lived radionuclides are used, as is the case of the most

INTRODUCTION

■

The search for ligands capable of forming metal complexes with

high thermodynamic stability, fast complexation kinetics, and

high inertness toward dissociation for safe use in medicinal

applications continues to be a challenging research ﬁeld.¹⁻⁴

Metal complexes of noncyclic ligands usually form rapidly but

are not very inert systems toward dissociation, while complexes

of macrocyclic ligands are more kinetically inert and have larger

thermodynamic stability but display slower rates of complex-

ation.^1,3−5On the other hand, macrobicyclic ligands, namely

the cross-bridged ones derived from tetraazamacrocycles, form

complexes even slower but that are much more inert toward

dissociation,⁶⁻¹⁰and most of them are selective for copper(II)

metal ions.¹⁰⁻¹²Generally, the more preorganized the ligand

is, the slower the formation and dissociation of its metal

complexes is.

Therefore, to avoid premature in vivo demetalation of the

complexes or metal transchelation to proteins, cyclic ligands

(macrocycles or macrobicycles) have been preferred, especially

those that are preorganized, in which the size of the cavity and

the number and the type of donor atoms match the size, the

geometrical, and the stereochemical requirements of the metal

ion. These features are particularly important for bifunctional

used positron emitters ⁶⁸Ga (t_1/2= 1.13 h) and ⁶⁴Cu(t_1/2

=

12.8 h).^3,4,15,16Other isotopes are also suitable for nuclear

imaging, such as ⁶⁷Ga (t_1/2= 78.3 h; γ-emitter) used in SPECT

and therapy, and ⁶⁶Ga (t_1/2= 9.4 h; β⁺-emitter).^1,3,16,17In

addition to ⁶⁴Cu, other interesting copper isotopes are ⁶²Cu

(t_1/2= 0.16 h, β⁺-emitter) that is generator produced, and ⁶⁷Cu

(t_1/2= 62.01 h, β⁻-emitting) that is only produced at high-

energy accelerators.^2,3,18Therapeutic radiopharmaceuticals for

RIT deliver cytotoxic nonpenetrating radiation (Auger elec-

trons and β⁻or α particles) doses to diseased sites, resulting in

the death of the cancer cells. Therapeutic nuclear medicine is

Received: December 27, 2013

Published: April 22, 2014

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less advanced than imaging techniques but has enormous

potential if the radionuclide is accurately targeted.¹⁸

adequate from the chemical point of view for applications in

nuclear medicine.

The aim of the present work is to compare the behavior of

two novel cyclen derivatives with trans-bis(2-hydroxybenzyl)

pendant arms and diﬀerent degrees of preorganization, the

H₂do2ph and its cross-bridged counterpart (H₂cb-do2ph), see

Chart 1. Until very recently, cyclen derivatives with phenolic

RESULTS AND DISCUSSION

■

Synthesis of the Ligands. The compound H₂cb-do2ph

was prepared by slight adaptation of the method used to syn-

thesize its previously reported methylnitrophenolic analogue,²⁰

as illustrated in Scheme 1. Bisquaternarization of cyclen-glyoxal

Chart 1. Structures of Compounds Discussed in This Work

a

Scheme 1. Synthetic Procedures

pendant arms had not been used for complexation of

copper(II), except in the case of H₂do2nph2me.¹⁹Last year

we reported the study of two cyclen derivatives H₂do2nph and

H₂cb-do2nph containing two 2-methyl-4-nitrophenol pendant

arms, where it was found that they present a good chelation

ability for copper(II), especially the cross-bridged one that

yielded a distorted octahedral coordination mode.²⁰However,

the rather low solubility of the ligands and their complexes

in aqueous media limited the range of studies that could be

performed. We were nonetheless convinced of the promise of

such ligand structure for eﬃciently binding copper(II) and

predictably also gallium(III).

Therefore, in this work 2-hydroxybenzyl arms replaced the

2-methyl-4-nitrophenol ones used in the previous report,

resulting in compounds with excellent water solubility. It was

expected that the cross-bridged macrocyclic derivative could be

an excellent ligand for the complexation of copper(II) and also

gallium(III), due to its rigid backbone and the appropriate size

of the cavity for the inclusion of these cations. The phenolic

pendant arms were selected due to their known ability to co-

ordinate the gallium(III) and also copper(II) cations. The

properties of the copper(II) and gallium(III) complexes were

thoroughly studied, including the thermodynamic, kinetic, and

electrochemical stabilities of the complexes in aqueous solution,

and their structural properties in solution and in the solid state.

The behavior of the pair of ligands now studied will be com-

pared with the very few available data of other pairs of cyclen

derivatives bearing diﬀerent pendant arms, see Chart 1.^10,11,20

These studies also allowed us to evaluate if the compounds are

a

Synthetic procedure used to obtain H₂do2ph and H₂cb-do2ph: (i)

glyoxal trimer, MeOH, 0 °C, rt, 2.5 h; (ii) 2-(bromomethy1)phenyl

acetate, MeCN, rt, 7 d; (iii) NaBH₄, EtOH/H₂O (9:1), rt, 24 h; 1 M

KOH, 70 °C, 12 h; (iv) CbzCl, CHCl₃, 0 °C; (v) 2-(bromomethy1)-

phenyl acetate, MeCN, 40 °C, 3 d and 60 °C, 4 d; (vi) 33% HBr, rt,

1 h; 0.1 M NaOH, rt, 12 h.

(1)²¹with 2 equiv of 2-(bromomethy1)phenyl acetate pro-

duced the cyclen-glyoxal trans-bis(2-methylphenyl acetate)

diammonium bromide (2) in nearly quantitative yield. Sodium

borohydride reduction in ethanol/water²²followed by hydrol-

ysis of the acetate groups in basic medium aﬀorded the desired

compound. Care must be taken not to expose the compound to

highly acidic hydrochloric or nitric media due to the formation

of intractable insoluble compounds, probably resulting from

electrophilic aromatic substitution reactions.

Attempts to synthesize the compound H₂do2ph by depro-

tection of cyclen-glyoxal trans-bis(2-methylphenyl acetate)

diammonium bromide (2) with hydrazine monohydrate,²³

hydroxylamine,²⁴or NaOH²⁵failed. Alternatively, the benzy-

loxycarbonyl (Cbz) trans-N-diprotected cyclen derivative 3²⁶

(see Scheme 1) was dialkylated at the remaining trans positions

with 2-(bromomethy1)phenyl acetate to yield compound 4.

The removal of the Cbz groups in acidic medium followed by

basic hydrolysis of the acetate functions aﬀorded the H₂do2ph

compound in reasonable yield.

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X-ray Diﬀraction Structures of H₂do2ph and Its

Copper(II) and Gallium(III) Complexes. Structure of

H₂do2ph. The asymmetric unit of the H₂do2ph compound,

presented in Figure 1 with the labeling scheme adopted, shows

compound (H₃do2nph2me)Br²⁰(see Chart 1 for the molecular

representation of the compound); however, in this case the

protonation state of the ligand is diﬀerent.

Structure of [Cu(Hdo2ph)]ClO₄·H₂O. The asymmetric unit

of the copper(II) complex of H₂do2ph is composed of a

[Cu(Hdo2ph)]⁺cation, one ClO₄counterion, and a crystal-

−

lization water molecule, giving rise to the molecular formula

[Cu(Hdo2ph)]ClO₄·H₂O. Selected bond distances and angles

in the coordination sphere of [Cu(Hdo2ph)]⁺were collected

in Table 3. The copper center exhibits a distorted square

Table 3. Selected Bond Distances (Å) and Angles (deg) in

the Coordination Spheres of the [Cu(Hdo2ph)]⁺and

[Ga(do2ph)]⁺Cation Complexes

complex

[Cu(Hdo2ph)]⁺

[Ga(do2ph)]⁺

Bond Lengths/Å

M−N1

M−N4

M−N7

M−N10

M−O20

M−O28

2.038(4)

2.035(4)

2.031(4)

2.038(4)

2.101(4)

2.095(2)

2.091(2)

2.104(2)

2.116(2)

1.963(4)

1.892(1)

Figure 1. ORTEP view of H₂do2ph with the atomic labeling scheme

and thermal ellipsoids drawn at the 50% probability level.

a secondary nitrogen atom (N4) and one phenolic oxygen

atom (O20) protonated as revealed by the last diﬀerence

Fourier maps calculated along the structure reﬁnement. The

cyclen ring exhibits the usual [3333] conformation²⁷with all

nitrogen atoms pointing to the same side of the macrocycle,

with a distance between the trans tertiary amine nitrogen atoms

of 4.360(7) Å, while the secondary ones are 4.013(7) Å apart

(Table 1). The protonated phenolic oxygen atom lies 2.438 Å

Bong Angles/deg

N1−M−N7

N4−M−N10

N1−M−N10

N4−M−N1

N7−M−N4

N7−M−N10

O20−M−N7

O20−M−N10

O20−M−N1

O20−M−N4

N4−M−N1

N4−M−O28

N1−M−O28

N10−M−O28

N7−M−O28

O20−M−O28

153.3(1)

145.8(1)

85.8(2)

86.5(1)

86.4(1)

85.7(1)

110.9(1)

110.5(1)

95.7(1)

103.4(1)

157.33(7)

94.90(7)

80.86(7)

84.34(7)

83.57(7)

81.15(7)

105.69(6)

172.87(7)

92.01(7)

84.03(6)

84.34(7)

174.15(7)

99.66(7)

89.97(7)

94.01(6)

91.54(6)

Table 1. Intramolecular N···N and Selected N···O Distances

(Å) on the Macrocyclic Backbone of H₂do2ph

N1···N4

N1···N7

N1···N10

N4···N7

N4···N10

N7···N10

N1···O20

N4···O20

N7···O20

N10···O20

2.858(7)

4.360(7)

2.926(8)

3.047(7)

4.013(7)

3.027(6)

2.942(6)

3.111(6)

3.591(6)

3.309(8)

pyramidal coordination environment with the four nitrogen

atoms of the cyclen ring deﬁning the basal plane and an oxygen

atom of one of the 2-methylphenol arms occupying the apical

position (Figure 2). The copper atom is positioned above the

N₄coordination plane by 0.534 Å, pointing toward the apical

above the mean plane deﬁned by the four nitrogen atoms and

receives two N−H···O hydrogen bonds from the secondary

amines of the macrocycle, exhibiting N···O distances of

3.309(8) and 3.111(6) Å, see Table 2. In addition, this

phenolic OH group establishes a strong hydrogen bond with

the oxygen atom of the phenolate pendant arm, with an O···O

distance of 2.553(5) Å and O−H···O angle of 168.4(3)°. A

strong hydrogen bond between both phenolic arms was also

observed in the structure of the related methylnitrophenolic

Table 2. Hydrogen Bond Dimensions of H₂do2ph

D−H···A*

H···A/Å

D···A/Å

D−H···A/deg

N4−H4b···O20

N10−H10···O20

O20−H20a···O28

2.185(4)

2.40(7)

3.111(6)

3.309(8)

2.553(5)

159.4(3)

164(5)

1.743(3)

168.4(3)

Figure 2. ORTEP view of [Cu(Hdo2ph)]⁺with the atomic labeling

*

D = N or O; A = O.

scheme and thermal ellipsoids drawn at the 50% probability level.

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oxygen atom from the coordinated 2-methylphenol arm. The

trigonal distortion calculated using the index structural param-

eter τ (τ = 0 for a perfect square-pyramidal geometry and τ = 1

for an ideal trigonal-bipyramidal geometry, as previously

deﬁned by Addison et al.)²⁸assumes a value of 0.125 which

is consistent with a distorted square pyramidal coordination

sphere. The cyclen ring adopts the [3333] conformation

already observed in the free ligand H₂do2ph, which suggests

that only a small conformational rearrangement occurs upon

coordination.

The structure of this complex is very similar to those

of the related complexes [Cu(Hdo2nph)]⁺,²⁰and [Cu-

(Hdo2nph2me)]⁺.¹⁹Indeed, the mean Cu−N and Cu−O

distances of 2.036 and 2.101 Å, respectively, compare well with

the mean Cu−N and Cu−O distances of 2.031 and 2.079 Å

for [Cu(Hdo2nph)]⁺,²⁰and 2.046(14) and 2.095 Å for

[Cu(Hdo2nph2me)]⁺.¹⁹

The coordination mode of [Ga(do2ph)]⁺is similar to those

found in the structures of all reported gallium(III) complexes of

related dota-like ligands (cyclen derivatives with acetate

pendant arms),²⁹⁻³³although some diﬀerences are noteworthy.

In all cases two nitrogen atoms of the macrocycle occupy the

apical positions, although in the [Ga(do2ph)]⁺complex the

d(Ga−N)_axis slightly below the range found in the dota-like

complexes (2.117−2.187 Å) and the N_ax−Ga−N_axangle is

slightly above (156.3−157.1°). Interestingly, although in all

cis-octahedral gallium(III) complexes of dota-like ligands the

macrocyclic framework adopts a [2424] conformation with a

type I arrangement (Bosnich nomenclature),³⁴and having the

coordinated carboxylate pendant arms bound to the equatorial

macrocyclic nitrogen atoms, in [Ga(do2ph)]⁺the macrocycle

displays the type II arrangement with the coordinated

phenolate pendant arms attached on the axial nitrogen atoms.

In this regard the structure reported here bears higher similarity

to the [Ga(cb-do2a)]⁺one, due to the ethylenic cross-bridge

displaying a [2424] conformation with a type V arrangement

and coordinated carboxylate pendant arms attached to the axial

instead of equatorial nitrogen atoms.³⁵

Structure of [Ga(do2ph)]NO₃·2H₂O. The asymmetric unit of

the gallium(III) complex of H₂do2ph is composed of the

[Ga(do2ph)]⁺complex cation, one NO₃⁻anion, and two crys-

tallization water molecules, giving rise to the molecular formula

[Ga(do2ph)]NO₃·2H₂O. The complex cation [Ga(do2ph)]⁺

is shown in Figure 3 along with the relevant atomic notation

Acid−Base Properties of the H₂do2ph and

H₂cb-do2ph Compounds. The compounds H₂do2ph and

H₂cb-do2ph have six basic centers consisting of the four amines

and the two phenol functions. From potentiometric titrations in

aqueous solution at 298.2 0.1 K and ionic strength of 0.10

0.01 M in KNO₃, ﬁve protonation constants of H₂do2ph and

only three of H₂cb-do2ph could be accurately determined. The

overall (β_i^H) and stepwise (K_i^H) protonation constants are

compiled in Table 4, together with reported values for

H₂do2a,³⁶H₄dota,^37,38do2py, and cb-do2py¹¹for comparison.

Cross-bridged derivatives of tetraaza macrocycles are well-

known for their strongly basic properties, where the ﬁrst pro-

tonation constant usually assumes very large values and is

frequently outside the possible scope of determination by

potentiometric and sometimes even spectroscopic tech-

niques,^10,11as was also found for H₂cb-do2ph. A H NMR

1

titration of this compound in D₂O solution in the range of

pD 3−15 was performed, but even in this solvent the ﬁnal de-

protonation step of the compound is barely starting at pD > 14.

Thus, in order to allow for comparison of the stability constants

of H₂cb-do2ph with H₂do2ph and literature compounds, a

value of 15.0 (in log units) was postulated for the mentioned

ﬁrst protonation constant of H₂cb-do2ph. A ¹H NMR titration

was also performed for H₂do2ph in the range pD 3−14. The

two NMR titrations (Figures S1−S2 of Supporting Informa-

tion) allowed for determination of protonation constants for

both compounds (presented in Table 4 in square brackets), and

in spite of the higher standard deviations arising from the

experimental limitations, they are in very good agreement with

the values determined by potentiometry.

The two compounds, H₂do2ph and H₂cb-do2ph, diﬀer

enormously in protonation constant values, the ﬁrst constant

being much larger and the fourth one much smaller for the

cross-bridged macrocycle. These diﬀerences can be observed in

Figure S3 of Supporting Information where the speciation

diagrams of both compounds as a function of pH are rep-

resented. In Supporting Information Figure S3 (left) it can be

seen that the maximum percentage of H₃do2ph⁺is formed at

pH 9 and the total deprotonation of this compound is found at

pH about 12.5, while for the cross-bridged derivative the

maximum percentage of H₃cb-doph⁺species exists at pH 7, and

at pH 14 only a very small amount of the cb-doph²⁻species is

Figure 3. ORTEP view of [Ga(do2ph)]⁺complex cation with the

atomic labeling scheme and thermal ellipsoids drawn at the 50%

probability level.

adopted. The complex displays a cis-distorted octahedral

geometry comprising the four nitrogen atoms of the macrocycle

and two phenolic oxygen atoms. The N1 and N7 donors

occupy the axial positions as the Ga−N1 and Ga−N7 bond

lengths add up to the highest possible sum [4.199(2) Å] and

the respective angle of 157.33(7)° gives a measure of the extent

of distortion from a perfect octahedral geometry. The two

secondary amine nitrogen atoms and the two phenolate oxygen

atoms occupy the equatorial positions forming an almost

perfect plane with a maximum mean plane deviation of 0.100 Å,

with the Ga center positioned at 0.031 Å from this plane. The

average Ga−N and Ga−O bond lengths (see Table 3) are

within the expected range for complexes of amine phenol type

ligands [d(Ga−N) = 2.090−2.183 Å and d(Ga−O) = 1.854−

1.954 Å].¹⁵

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Table 4. Overall (β_i^H) and Stepwise (K_i^H) Protonation Constants, in log Units, of H₂do2ph and H₂cb-do2ph at 298.2 K in

0.10 M KNO₃with Values for Some Related Compounds Also Included

a

b

c

d

e

equilibrium reaction

H₂do2ph

H₂cb-do2ph

H₂do2a

H₄dota

do2py

cb-do2py

log β_i^H

f

L + H⁺⇄ HL

11.32(3)

22.48(2)

32.41(2)

39.97(2)

41.63(3)

15.0

11.45

20.99

24.99

27.35

12.09

21.85

26.41

30.50

9.90

18.30

22.05

24.55

13.8

L + 2 H⁺⇄ H₂L

L + 3 H⁺⇄ H₃L

L + 4 H⁺⇄ H₄L

L + 5 H⁺⇄ H₅L

25.34(1)

34.95(1)

39.55(1)

19.11

23.07

25.53

log K_i^H

f

L + H⁺⇄ HL

11.32 [11.36]

11.16 [11.35]

9.93 [10.07]

7.56 [7.83]

1.66

15.0

11.45

9.54

4.00

2.36

12.09

9.76

4.56

4.09

9.90

8.40

3.75

2.50

13.8

5.31

3.96

2.46

HL + H⁺⇄ H₂L

H₂L + H⁺⇄ H₃L

H₃L + H⁺⇄ H₄L

H₄L + H⁺⇄ H₅L

10.34 [10.72]

9.61 [9.72]

4.60 [4.8]

a

b

L denotes the ligand in general; charges of the species are omitted for clarity. This work; values in parentheses are standard deviations in the last

signiﬁcant ﬁgures, and in square brackets were included the values determined by ¹H NMR titrations. T = 298.2 K, I = 0.10 M in NEt₄ClO₄.³⁶T =

c

d

298.2 K, I = 0.10 M in NMe₄NO₃.^37,38T = 298.2 K, I = 0.10 M in NMe₄NO₃.¹¹Value impossible to determine accurately, see text.

e

f

Figure 4. Spectral variations with pH of the compound H₂do2ph: (a) ¹H NMR experimental chemical shifts as a function of pD upon titration in D₂O

with KOD at 298.2 K; (b) ﬁrst derivative of the calculated chemical shifts; (c) UV spectra in aqueous solution at increasing pH values. Inset: values of the

absorbance at 238 and 294 nm as a function of pH. The plots a), b), and the inset were drawn over the corresponding speciation diagram.

formed in solution. However, these results do not give infor-

mation about the basic center that is protonated at a given pH.

This information, which is crucial for the interpretation of the

values, can be retrieved from the NMR titrations and, due to

the presence of the phenolic arms, assisted by UV spectra in

aqueous solutions in the range pH 2.8−12.4 (Figures S4−S5 of

Supporting Information), as detailed below.

Actually, the acid−base behavior of H₂do2ph displays some

particularities that are worthy of a deeper discussion. The ﬁrst

two log K_i^Hvalues are rather similar (11.32 and 11.16), and

according to the determined crystal structure (see above), it is

expected that also in solution one of the protonation constants

must be assigned to the protonation of a secondary amine and

the other to the protonation of one phenolate group. Evidence

that the same happens in solution is provided by the NMR

spectra recorded in the pD range 11.6−14.0 which shows

considerable shifts of the resonances corresponding to both

cyclen and phenol protons (Figure 4a,b).

In addition, the decrease of intensity of the bands at 238 and

294 nm in the UV spectra, assigned to the π−π* transitions of

phenolate, and concomitant increase of a band at 274 nm

attributable to the phenol form clearly show the protonation

of a phenolate group occurring at the pH 12.4−10.5 region

(Figure 4c).

Although the available data does not allow unambiguous

assignment of each protonation constant to the respective basic

center, it is clear that the protonation of the ﬁrst secondary

amine occurs at the pH that is typical in cyclen derivatives while

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1

Figure 5. Spectral variations with pH of the cross-bridged compound, H₂cb-do2ph: (a) H NMR experimental chemical shifts as a function of pD

upon titration in D₂O with KOD at 298.2 K; (b) ﬁrst derivative of the calculated chemical shifts; (c) UV spectra in aqueous solution at increasing pH

values. Inset: values of the absorbance at 238 and 294 nm as a function of pH. The plots a), b) and the inset were drawn over the corresponding

speciation diagram.

the basicity of the phenol pendant is very high when compared

to the log K_i^Hof phenol (9.79) or of 2-(aminomethyl)phenol

(8.63).³⁹This unusual fact may be explained by the formation

of a strong hydrogen bond between the phenol pendant and

the phenolate one (O−H···⁻O), and additional hydrogen

bonds between both secondary ring amines and the phenol

(N−H···OH), as evidenced also in the crystal structure. The

third log K_i^His assigned to the other secondary amine as

evidenced by the pronounced downﬁeld shift of the resonances

of cyclen protons 7, 8, and 9 as the pD drops from 11.6 to 9.6

and by the small shift observed for the phenol proton reso-

nances in the same pD region (Figure 4a). The UV spectra

show almost no changes in the bands at the pH 8.8−10.5

region (Figure 4c) which conﬁrms that the remaining pheno-

late is not being protonated in this pH range. Below pH 8.8 the

two UV bands at 238 and 294 nm disappear to give rise to a

single band at 274 nm, providing unequivocal evidence for the

protonation of the second phenolate arm. The log K_i^Hvalue of

7.56 is quite lower than that of phenol or of 2-(aminomethyl)-

phenol because this protonation causes disruption of the strong

hydrogen bond between the phenol and phenolate pendant

arms (O−H···⁻O). Interestingly, the NMR spectra for pD < 9.6

showed only small shifts in the phenol proton resonances and

larger shifts in the cyclen ones (Figure 4). These odd resonance

shift variations may be explained by the disruption of the strong

O−H···⁻O hydrogen bond and concomitant movement of one

of the phenols to a position away from the macrocyclic cavity,

as observed in the crystal structure of the related H₄do2nph²⁺

compound. Simultaneous rearrangements of the macrocyclic

backbone can also account for this behavior.

delocalization of the proton by strong hydrogen bonds with all

amines, and it could not be fully observed. Both H NMR and

1

UV spectra (Figure 5) clearly show the three remaining

protonation steps, with the two highest log K_i^Hassigned to the

consecutive protonations of both phenolate pendant arms

without any noticeable hydrogen bonding eﬀects, unlike for

H₂do2ph. The lowest log K_i^Hcorresponds to the protonation of

a second ring amine, and its signiﬁcantly lower value is a con-

sequence of the necessary disruption of the hydrogen bonding

pattern involving all ring amines and the stretched cavity shape

that are usual in cross-bridged tetraaza macrocycles.^10,11,19

Stability Constants of Metal Complexes. The ability of

H₂do2ph and H₂cb-do2ph to form complexes with Ca²⁺, Cu²⁺,

Zn²⁺, and Ga³⁺cations was evaluated in aqueous solution, and

their thermodynamic stability constants were determined in

conditions similar to those used for the determination of pro-

tonation constants. The complexes of Zn²⁺and Ca²⁺cations

were also studied due to its abundance in biological media.

The obtained values were compiled in Table 5 together with

those for the complexes of related ligands for comparison

reasons.^{11,33,36−38,40}The corresponding species distribution

diagrams are shown in Figure 6 for the complexes of Cu²⁺and

Ga³⁺with H₂do2ph, and in Figure 7 for the complex of

copper(II) with H₂cb-do2ph (in Supporting Information

Figures S6−S7 for the remaining complexes).

The complexation reactions of H₂do2ph are generally slow

on the acidic pH range except with the Ca²⁺cation. For Cu²⁺it

was necessary to perform a batch titration at the acidic pH

range in order to complement the in-cell titrations. In the case

of Ga³⁺, the stability constants had to be determined by OH⁻

competition on the basis of the dissociation equilibrium of the

complex at basic pH and formation of the Ga(OH₄)⁻

species,^41,42which could nonetheless be followed by in-cell

In contrast with H₂do2ph, the acid−base behavior of

H₂cb-do2ph displays the features expected for a cross-bridged

cyclen derivative with phenol pendant arms. The ﬁrst protona-

tion occurs on the amines at very high pH, with probable

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Table 5. Overall (β_{MH L}) and Stepwise (K_{MH L}) Stability Constants, in log Units, of Metal Complexes of H₂do2ph, H₂cb-do2ph,

i

and Other Related Ligands at 298.2 K in 0.10 M KNO₃

a

b

c

d

equilibrium reaction

H₂do2ph

H₂cb-do2ph

H₂do2a

H₄dota

do2py

cb-do2py

log β_{MH L}

i

f

Ca²⁺+ L + H⁺⇄ CaHL

Ca²⁺+ L ⇄ CaL

Ca²⁺+ L ⇄ CaLOH + H⁺

Cu²⁺+ L + 2H⁺⇄ CuH₂L

Cu²⁺+ L + H⁺⇄ CuHL

Cu²⁺+ L ⇄ CuL

14.50(4)

5.07(1)

e

20.77

f

17.23

−6.57(1)

37.81(2)

32.34(3)

22.83(4)

f

34.08(5)

25.06(4)

24.1

21.1

26.03

22.25

23.11

20.3

13.0

22.86

19.03

Cu²⁺+ L ⇄ CuLOH + H⁺

Zn²⁺+ L + 2H⁺⇄ ZnH₂L

Zn²⁺+ L + H⁺⇄ ZnHL

Zn²⁺+ L ⇄ ZnL

Zn²⁺+ L ⇄ ZnLOH + H⁺

Ga³⁺+ L + H⁺⇄ GaHL

Ga³⁺+ L ⇄ GaL

33.19(9)

28.88(1)

19.33(2)

7.57(3)

f

27.48(1)

20.79(2)

11.09(4)

35.19(2)

31.82(1)

22.2

18.2

25.28

21.10

20.73

17.51

10.67

17.10

g

34.5(1)

25.33

g

h

31.16(3)

21.33; 26.05

log K_{MH L}

i

f

CaL + H⁺⇄ CaHL

Ca²⁺+ L ⇄ CaL

9.43

5.07

3.54

f

17.23

CaLOH + H⁺⇄ CaL

CuHL + H⁺⇄ CuH₂L

CuL + H⁺⇄ CuHL

Cu²⁺+ L ⇄ CuL

Cu²⁺+ L ⇄ CuLOH + H⁺

ZnHL + H⁺⇄ ZnH₂L

ZnL + H⁺⇄ ZnHL

Zn²⁺+ L ⇄ ZnL

11.64

5.47

f

9.51

9.02

3.0

3.78

2.81

20.3

7.3

3.83

f

22.83

25.06

21.1

22.25

19.03

4.31

9.55

f

6.69

20.79

9.70

e

4.0

4.18

3.22

17.51

6.84

f

19.33

11.76

3.34

18.2

21.10

17.10

ZnLOH + H⁺⇄ ZnL

GaL + H⁺⇄ GaHL

Ga³⁺+ L ⇄ GaL

g

h

4.00; 3.64

g

h

31.16

e

21.33; 26.05

a

b

L denotes the ligand; charges of the species are omitted for clarity. This work; values in parentheses are standard deviations in the last signiﬁcant

ﬁgures. T = 298.2 K, I = 0.1 M in NEt₄ClO₄.³⁶T = 298.2 K, I = 0.10 M in NMe₄NO₃.¹¹No complex formation at the experimental conditions.

c

d

e

f

g

h

T = 298.2 K, I = 0.10 M in NMe₄NO₃.³⁷T = 298.2 K, I = 0.1 M in KCl.⁴⁰T = 298.2 K, I = 0.1 M in NMe₄Cl; the authors report also constants for

GaH₂L (log K = 2.43) and GaH₃L (log K = 1.84).³³

Figure 6. Species distribution diagrams for the complexes of Cu²⁺and Ga³⁺with H₂do2ph in aqueous solution at c_L= c_M= 1.0 × 10⁻³M.

titrations using long equilibration times. For the complexes of

Zn²⁺and Ca²⁺, the usual in-cell titrations were used.

The cross-bridged ligand, H₂cb-do2ph, does not form

complexes with Ga³⁺and Ca²⁺in the mild conditions used.

Evidence of the gallium(III) complex formation was only found

upon heating; however, some degradation of the complex was

also observed along time, as revealed by mass spectrometry.

In the case of Cu²⁺, the slow equilibrium of formation and

dissociation of the complex demanded batch titrations at the

entire pH region.

The stability constants of the complexes of H₂do2ph and

H₂cb-do2ph are high with Cu²⁺and somewhat less high with

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values reported for the gallium(III) complex of H₄dota.^33,40

However, until now triazamacrocyclic derivatives have been

revealed to be the best chelators for the Ga³⁺ion, especially

when three additional arms with oxygen or sulfur donor atoms

are appended in order to fulﬁll the coordinative demand of

that metal ion. Indeed, the pGa for the complex of H₃nota is

very high (26.41, following Martell et al.,⁴³or 23.82, Hermann

et al.⁴⁴).

Spectroscopic Characterization of Metal Complexes.

The UV−vis spectra of the copper(II) complex of H₂do2ph

display a marked pH dependence, see Figure 8. The d−d tran-

sition band in the visible region, which is centered at 550 nm at

acidic pH, appears at 650 nm at neutral pH and is only slightly

intensiﬁed at basic pH. At the same time, a shoulder at 395 nm

(LMCT band) appears from around neutral pH. On the UV

range, the single band at 276 nm at acidic pH gives rise to two

separate bands at 234 and 282 nm at neutral pH and then at

238 and 294 nm at basic pH. These results point to the Cu

center being bound only by the macrocyclic ring and eventually

by a water or solvent molecule at acidic pH, and to the addi-

tional coordination of one phenolate group at around neutral

pH, suggesting also that the second pendant arm is not bound

to the copper center even at basic pH while deprotonating at a

value expected for an uncoordinated phenol group.

In contrast with the previous complex, the UV−vis spectra of

the copper(II) complex of H₂cb-do2ph display no signiﬁcant

change of λ_maxof bands in the visible range with the increase of

pH (from 5 to 12.2), indicating that at least one phenolate is

already coordinated to the metal center at low pH. However,

above pH = 9 there is a small increase of absorbance at 410 nm

and a small red shift of the band at 605 to 615 nm with a

shoulder at 650 nm (the band in the NIR region at 960 nm is

maintained along pH change), see Figure 9. In the UV range

there are always two bands, one shifting very slightly from 235

to 240 nm and another one shifting from 280 to 293 nm, clearly

indicating the deprotonation of the second phenol. These com-

bined features may be the consequence of a slight rearrange-

ment of the ligand around the metal center for the ﬁnal

geometry. This also indicates that the deprotonation of the last

phenolate group only occurs at high pH (pK_a= 9.02), sug-

gesting only a weak interaction with the metal center.

Figure 7. Species distribution diagram for the complexes of Cu²⁺with

H₂cb-do2ph in aqueous solution at c_L= c_M= 1.0 × 10⁻³M.

Zn²⁺, as expected. The most interesting result, however, is the

very large stability constant for the complex of H₂do2ph with

Ga³⁺relative to the values of other complexes of the same

ligand, and especially when compared to other known ligands

such as H₄dota.^33,40This feature can be explained by the

presence of the phenol groups on H₂do2ph, which are known

to be particularly suitable for binding hard cations such as

Ga³⁺.^16,41The high basicity of phenol groups is also responsible

for the presence of protonated complex species in solution,

and their dominance at neutral or acidic pH, see speciation

diagrams in Figures 6 and 7.

However, as is well-known, the comparison of stability

constants of metal complexes with ligands of diﬀerent basicities

can be erroneous, and the more correct way consists of

comparing them by the conditional constants or the pM = log

[Mⁿ⁺] values at a given pH.⁴²Consequently, the pM values

were determined at physiological pH on the basis of all the

constants given in Table 5, and they are collected in Table 6.

a

Table 6. Calculated pM Values for the Complexes of

H₂do2ph, H₂cb-do2ph, and Literature Values of Related

Complexes for Comparison

The X-band EPR spectra of both complexes exhibit three

well-resolved lines of the four expected ones at low ﬁeld

resulting from the interaction of the unpaired electron spin with

the copper nucleus, and no superhyperﬁne splitting due to the

coupling with the four nitrogen atoms of the macrocycle was

observed. The fourth copper line is partially overlapped by the

strong and unresolved band at the high ﬁeld part of the spectra,

see Figure 10. The hyperﬁne coupling constants (A_i) and g_i

values of frozen solutions of the complexes at diﬀerent pH

values, obtained by simulation of the spectra,⁴⁵are listed in

Table 7. For all of them the parameters are typical of copper(II)

b

c

d

cation H₂do2ph H₂cb-do2ph H₂do2a

H₄dota

do2py cb-do2py

e

Cu²⁺

Zn²⁺

Ga³⁺

14.33

10.86

20.56

13.94

8.12

14.91 15.19

12.01 14.04

17.11

14.63

12.63

10.70

e

f

g

17.83, 19.05

a

Values calculated at pH = 7.4 for 100% excess of ligand with [M²⁺]_tot

b

= 1.0 × 10⁻⁵M, based on the reported stability constants. This work.

c

d

e

f

g

From ref 36. From ref 11. From ref 37. From ref 40. From ref 33.

2

complexes in axially elongated rhombic symmetry and a d_{x −y}

These values highlight the relative strength of the complexes

and clearly indicate that the H₂do2ph is a better ligand in com-

parison to the corresponding cross-bridged one for the com-

plexation of all the studied metal ions, being especially eﬃcient

for Ga³⁺cation. A decrease of stability is expected for complexes

of Zn²⁺relative to those of Cu²⁺following the trend of Irving−

Williams, but the decrease in pM values is more accentuated for

the cross-bridged ligand (see the pM values in Table 6).

ground state, consistent with elongated rhombic-octahedral,

tetragonal, or distorted square-based pyramidal stereochemis-

tries.⁴⁶⁻⁵³In fact, for all complexes the values for the g_iparam-

eters are g_z> g_y≈ g_xand g_x≥ 2.04.

In the complex of H₂do2ph at pH 4 (species [Cu-

(H₂do2ph)]²⁺), the parameters point to a near axial symmetry,

and they are typical of N4O square-pyramidal geometry, fol-

lowing the diagrams of Peisach and Blumberg,⁵⁴as well as the

similar values of [Cu(cyclen)(NO₃)](NO₃)⁵⁵and of other

complexes of cyclen derivatives bearing one pendant arm

In conclusion, the [Ga(do2ph)]⁺complex presents a surpris-

ingly high pM, to the best of our knowledge the highest one for

a complex of a tetraaza macrocyclic derivative, higher than the

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Figure 8. UV and vis spectra of the copper(II) complex of H₂do2ph in aqueous solution at variable pH. (UV: 1−4.1, 2−5.5, 3−7.0, 4−9.5, 5−11.4.

Vis: 1−4.0, 2−5.4, 3−7.0, 4−9.2, 5−11.0.)

Figure 9. UV and vis−NIR spectra of the copper(II) complex of H₂cb-do2ph in aqueous solution at increasing pH values. (UV: 1−6.2, 2−8.8,

3−9.6, 4−10.0, 5−12.3. vis−NIR: 1−5.0, 2−9.0, 3−9.4, 4−10.3, 5−12.2.)

known as adopting a distorted square pyramidal geometry.²⁰

Increasing the pH to 7 (species [Cu(Hdo2ph)]⁺) causes an in-

crease of the g_zand a decrease of the A_zvalues, which according

to the ligand ﬁeld theory means that the axial ligand ﬁeld be-

comes stronger. This fact, together with the simultaneous red-

shift of the d−d absorption bands in the electronic spectra, indi-

cates that a coordinated water molecule at the axial position of

[Cu(H₂do2ph)(H₂O)]²⁺ion complex is replaced by the oxygen

of a phenolate pendant arm upon deprotonation. Indeed, by

potentiometric titration the pK_avalue of 5.47 was determined

for this deprotonation. This low pK_avalue indicates a strong

interaction of this phenolate oxygen with the Cu center. Addi-

tionally, no signiﬁcant change of the EPR spectra was observed

above pH 7, indicating that the complex preserves the distorted

square pyramidal geometry and the same environment. Al-

though from potentiometric and spectrophotometric results it

was observed that another phenol group deprotonates at basic

pH (pK_a= 9.51), it clearly does not coordinate to the metal

center. These structural features of the copper(II) complex of

H₂do2ph in aqueous solution are in good agreement with the

X-ray diﬀraction crystal structure determined for the complex

[Cu(Hdo2ph)]ClO₄·H₂O (see above).

For the copper(II) complex of H₂cb-do2ph, the EPR param-

eters are identical in the entire pH range studied, indicating that

the structure of the complex is very similar at any given pH.

The potentiometric studies revealed that the complex is formed

at low pH and only a deprotonation of one phenolate was ob-

served at high pH (pK_a= 9.02, see Table 5), indicating that one

of the phenol groups deprotonates at low pH and is strongly

bound to the metal center. In a comparison of the EPR param-

eters of this complex with those of [Cu(Hdo2ph)]⁺or [Cu-

(do2ph)], an increase of the g_zand a decrease of the A_zvalues

are observed, indicating a stronger axial ligand ﬁeld or/and a

weaker equatorial ligand ﬁeld. These features suggest a dis-

torted tetragonal geometry with a water molecule completing

the hexacoordination at low pH, which is replaced by the

second phenolate group weakly bound at pH > 9 (pK_a= 9.02,

which is lower than the corresponding pK_a= 10.34 of the free

ligand, suggesting a weak interaction). In spite of our eﬀorts, it

was unfortunately not possible to obtain crystals of this com-

plex with appropriate quality for X-ray determination. However,

the structure of the complex with the related ligand H₂cb-

do2nph [Cu(cb-do2nph)]²⁰exhibited a distorted axial elongated

octahedral geometry with a N3O equatorial plane and NO axial

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To get an insight into the structures of zinc(II) and gallium(III)

complexes of H₂do2ph, NMR spectra of deuterated water solu-

1

tions were recorded at relevant pH values. The H spectra of

the zinc(II) complex are quite simple and similar at pH 7 and

11, which is indicative of a single and very symmetric species in

solution, see Figure S8 of Supporting Information. They display

only three resonances for the macrocyclic ring and another

ﬁve for the two pendant arms, indicating that the arms are

magnetically equivalent. The sharp singlet at 3.83 ppm assigned

to the methylene (N−CH₂−Ar) protons of the pendant arms

means that they are in fast exchange on the NMR time scale

and thus suggests that there probably only exists a weak binding

of the phenolate groups to the cation. For the gallium(III) com-

1

plex, the H spectra are much more complicated, as there are

many multiplets for the macrocyclic ring and the pendant arms

both at pH 4 and 9, see Figure S9 of Supporting Information.

The most distinctive features of the spectra are three diﬀerent

multiplets in the range 3.9−5.0 ppm that are assigned to the

methylene protons of the pendants (three pairs of doublets

from the AB spin systems formed by inequivalent geminal

protons), which means that all the phenolate groups bind

strongly to the cation. The ¹³C spectrum of the complex is also

complicated, displaying two sets of resonances apparently

corresponding to one major (M) and one minor (m) isomers

(Figure S10 of Supporting Information), which could be

identiﬁed with the help of standard 2D COSY and HMQC

experiments (Figures S11 and S12 of Supporting Information).

The M isomer has 7 diﬀerent carbon atoms assigned to the

macrocyclic ring (41−59 ppm range) and 2 assigned to the

methylene carbon atoms of the pendant arms (∼61 ppm) while

the m isomer has only 4 macrocyclic carbon atoms and one for

the pendants, which points to an eﬀective C₁symmetry for the

former isomer and a C₂symmetry for the latter one. The com-

bined NMR data for the gallium(III) complex point to two

isomers coexisting in solution such that one of them has mag-

netically inequivalent pendant arms yielding two of the methy-

lene multiplets, and the other one has equivalent pendants

1

yielding a single multiplet. H spectra were also performed

at variable temperatures up to 90 °C in D₂O and 110 °C in

DMSO-d₆(Figures S13 and S14 of Supporting Information),

but although an increase of the linewidths of the multiplets of

the pendants multiplets was visible, that reﬂects conformational

exchange processes taking place, and no ﬁnal interconversion

between the two isomers was observed. This indicates a

dynamic process with high activation barrier.

Unfortunately, it was not possible to perform NMR measure-

ments of the diamagnetic Zn²⁺complexes of H₂cb-do2ph due

to its slow formation. However, it is interesting to note that the

complex of this ligand with Zn²⁺has a deprotonation at lower

pH than the one of the copper(II) complex (pK_a= 6.69), sug-

gesting the coordination of both phenolate groups to the Zn

center to form a 6-coordinate complex which is completely

formed around pH 8, see Supporting Information Figure S7.

Kinetic Iinertness of Copper(II) and Gallium(III)

Complexes of H₂do2ph and H₂cb-do2ph. The kinetic

inertness of metal complexes is one of the most important

properties to consider for the possible application in nuclear

medicine, since it is crucial that complexes do not dissociate

during their residence time in the patient body. Determination

of the half-life times for complexes in acidic or basic media is a

commonly accepted method to ascertain their inertness in

comparison with other known complexes.

Figure 10. X-band EPR spectra of the copper(II) complexes of

H₂do2ph (top) and H₂cb-do2ph (bottom) in frozen water/ethylene

glycol (1:1 v/v) solutions of variable pH recorded at 90 K, and the corre-

sponding simulated spectra in dashed lines. Microwave power of 2.0 mW,

modulation amplitude of 1.0 mT, and frequency (υ) of 9.5 GHz.

bonds, with the axial O-phenolate being at a distance of 2.343 Å.

The EPR parameters of [Cu(do2ph)] being intermediate be-

tween those of [Cu(cb-do2nph)]²⁰and [Cu(do2ph)] point to

a structure similar to that of [Cu(cb-do2nph)] with a longer

distance to the axial phenolate.

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Table 7. Visible and X-Band EPR Spectroscopic Data of the Copper(II) Complexes of H₂do2ph and H₂cb-do2ph

b

X-band EPR

a

c

complex

pH

UV−vis−NIR λ_max/nm (ε/M⁻¹cm⁻¹

276 (6.6 × 10³); 550 (312)

234 (9.0 × 10³); 282 (7.2 × 10³); 650 (254)

)

g_z

g_y

g_x

A_z

A_y

A_x

[Cu(H₂do2ph)(H₂O)]²⁺

[Cu(Hdo2ph)]⁺

4

7

2.191

2.207

2.233

2.053

2.071

2.061

2.040

2.042

2.037

191

180

173

25

13

16

22

<10

[Cu(do2ph)]

11 238 (1.5 × 10⁴); 294 (8.4 × 10³); 395 (266); 650 (298)

[Cu(Hcb-do2ph)]⁺

[Cu(cb-do2ph)]

6

235 (8.8 × 10³); 280 (8.9 × 10³); 410 (546); 605 (134); 960 (39)

10 239 (12.3 × 10³); 285 (8.3 × 10³); 410 (610); 615 (141); 650 (sh);

960 (39)

d

[Cu(cyclen)(NO₃)]NO₃

500 (250)

2.198

2.057

184

24

a

b

In aqueous solution, recorded at 298 K. In H₂O/ethylene glycol (1:1 v/v) solution, recorded at 90 K at microwave power 2.0 mW and frequency

c

d

(ν) 9.5 GHz; parameters obtained by spectral simulation. Values of A × 10⁴in cm⁻¹. From ref 55.

The acid-assisted dissociation of the copper(II) complexes of

H₂do2ph and H₂cb-do2ph was studied by vis spectropho-

tometry under pseudo-ﬁrst-order conditions in 1 M HCl solu-

tions at diﬀerent temperatures. The calculated half-life times

(t_1/2) are presented in Table 8 together with literature values

other cyclen derivatives (Table 9).^33,56Nonetheless, it is of a

magnitude similar to the half-life of the ⁶⁸Ga isotope (67.7 min)

that is useful for applications in PET imaging.

Table 9. Half-Life Times for the Base-Assisted Dissociation

of the Gallium(III) Complex of H₂do2ph and Literature

Values for Related Complexes

Table 8. Half-Life Times (t_1/2) for the Acid-Assisted

Dissociation of the Copper(II) Complexes of H₂do2ph and

H₂cb-do2ph and Literature Values for Complexes of Related

Ligands

ligand

pH (T/K)

t_1/2/min

ref

H₂do2ph

H₄dota

11 (298.2)

10 (298.2)

11 (298.2)

54

372

43

this work

33

56

copper(II) complex

of L

conditions

[HCl]/M (T/K)

a

H₃do3a1am

t

_1/2/min

ref

b

H₆trap-Pr

3600

H₂do2ph

1 (298.2)

1 (310.2)

1 (333.2)

1 (363.2)

1 (298.2)

1 (303.2)

1 (310.2)

1 (333.2)

1 (363.2)

1296

358

28.5

2.3

this work

20

a

1,4,7,10-Tetraazacyclododecane-1,4,7-tris-acetic acid-10-acetamide.

1,4,7-Triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic

b

acid].

H₂cb-do2ph

393

214

86.1

6.6

Electrochemical Behavior of the Copper(II) Com-

plexes of of H₂do2ph and H₂cb-do2ph. The eﬃciency

of copper complexes for radiopharmaceutical applications can

also be limited by bioreduction to copper(I) complex followed

by demetalation in case the ligand is not able to stabilize the

reduced copper species. However, most copper(II) complexes

of polyazamacrocyclic derivatives have reduction potentials that

are well below the estimated −0.40 V (NHE) threshold for

typical bioreductants.³Therefore, the copper(II) complexes of

H₂do2ph and H₂cb-do2ph were investigated by cyclic volta-

mmetry (CV) in 0.10 M aqueous NaOAc at pH 10.0 and 9.4,

respectively, see Figure S16 of Supporting Information. The

reduction of both complexes is irreversible with the cathodic

waves at −912 and −941 mV (Ag/AgCl, scan rate 100 mV/s)

for [Cu(do2ph)] and [Cu(cb-do2ph)], respectively. However,

for [Cu(do2ph)] one anodic peak at −568 mV (Ag/AgCl, scan

rate 100 mV/s) on the reverse sweep was observed. It seems

that the reduced complex may exist as a mixture of at least two

species in fast equilibrium. These species may consist of four-

and ﬁve-coordinate Cu(I) complexes, as also observed for other

macrobicyclic complexes.^57,58Such irreversibility is generally

attributed to the lack of ability of cyclen derivatives to accom-

modate and stabilize the Cu⁺ion in its cavity, in contrast with

their cyclam counterparts.⁹Consequently, upon reduction of

the Cu²⁺center to Cu⁺in the studied complexes, a topological

reorganization, demetalation, and/or disproportionation may

occur, indicating that their rigid backbones and small cavities

do not appear to be able to adapt well to the coordination

requirements of Cu⁺in order to stabilize it.

0.5

a

H₂do2nph

H₂cb-do2nph

H₂cb-do2a

cb-do2py

cyclen

1 (298.2)

10.9

46.1

240

52

a

1 (298.2)

20

1 (303.2)

5 (303.2)

9

11

<3

3

H₄dota

<3

3

a

Determined in DMSO−H₂O (9:1 v/v) solutions due to poor

solubility of the complexes in water.

for related complexes. The ﬁrst striking observation from

the results obtained is that the complex of the cross-bridged

H₂cb-do2ph derivative is surprisingly much less inert than that

of H₂do2ph. That is even more signiﬁcant taking into account

that the inertness of the complex of H₂cb-do2ph is reasonably

high when compared to other complexes of cross-bridged

cyclen derivatives, thus indicating that it is actually the complex

of H₂do2ph that is more inert than expected. The temperature

dependences of the rate constants (k_obs) determined for both

complexes are shown in the Arrhenius plots obtained for each

dissociation reaction (Figure S15 of Supporting Information).

The base-assisted dissociation of the gallium(III) complex

of H₂do2ph was also studied by ⁷¹Ga NMR spectroscopy in

buﬀered aqueous solutions at pH = 11. The dissociation was

followed by integration of the increasing resonance of the

[Ga(OH)₄]⁻species that is formed at basic pH by the metal

released from the complex. The half-life of 54 min found for

the complex is much lower than the best known gallium(III)

complex, but still moderately high when compared to those of

CONCLUSIONS

In this work two novel cyclen-based metal chelators are

reported containing two trans-methyl phenol pendant arms,

■

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on a Bruker EMX 300 spectrometer equipped with continuous-ﬂow

cryostat for liquid nitrogen, operating at the X-band.

Caution! Although no problems were encountered during this work with

the perchlorate salts, these compounds should be considered potentially

explosive.

H₂do2ph and H₂cb-do2ph, where the latter one contains also

an ethylene cross-bridge, aiming for possible use of their metal

complexes in nuclear medicine. Studies of the acid−base pro-

perties of both compounds showed that H₂do2ph displays

unusual dissimilar basicity of the phenolic pendant arms, while

the cross-bridged H₂cb-do2ph derivative has a marked “proton

sponge” behavior that is common in small polyamine cages.

These basicity features were found to play a crucial role in the

metal binding properties of the compounds.

The copper(II) complexes of H₂do2ph and H₂cb-do2ph have

similarly high thermodynamic stability, which in the case of

H₂do2ph is somewhat lower than for other cyclen derivatives

such as H₄dota. This may be assigned to the coordination role

of the phenol pendants in the structure of both complexes, as

the spectroscopic studies and the X-ray structure of the com-

plex of H₂do2ph showed that one phenolate arm is not coor-

dinated to the copper center while for the H₂cb-do2ph the

spectroscopic data suggest a hexacoordinated complex in which

one of the phenolate groups is only weakly bound to the metal

center. More important and rather surprising is the fact that

the copper complex of H₂do2ph is approximately 4 times

more inert that that of H₂cb-do2ph, being more inert than the

complexes of most other cyclen derivatives. All of the above

features indicate that H₂do2ph may be an interesting ligand

for the complexation of copper(II) radioisotopes in medicinal

applications.

The gallium(III) complex of H₂do2ph has a very high ther-

modynamic stability, which translates to a pM value (20.79)

that is better than for H₄dota. This means that H₂do2ph is the

best known tetraaza macrocyclic derivative for complexation of

Ga³⁺cation. The octahedral complex [Ga(do2ph)]⁺has addi-

tionally shown an appropriate inertness toward base-assisted

dissociation, making it a promising candidate for applications

with gallium(III) radioisotopes in medicine if the problem of a

rather slow complexation rate can be overcome.

In contrast with the behavior of all reported cross-bridged

compounds, our studies revealed that the trans-bismethylphe-

nol cyclen derivative H₂do2ph is the best ligand for the coordi-

nation of both Cu²⁺and Ga³⁺cations in aqueous solution, not

only from the larger pM values but also from the kinetic

inertness of its complexes when compared with its correspond-

ing cross-bridged one. In fact, to the best of our knowledge this

is the ﬁrst case where the copper(II) complex of the cross-

bridged derivative (H₂cb-do2ph) is much less inert than that of

its non-cross-bridged counterpart.

Cyclen-glyoxal trans-Bis(2-methylphenyl acetate) Diammonium

Bromide, 2. A solution of 2-(bromomethy1)phenyl acetate (2.133 g,

9.3 mmol) in MeCN (15 mL) was added dropwise over 30 min to a

magnetically stirred solution of cyclen glyoxal 1 (0.611 g, 3.1 mmol) in

MeCN (15 mL). The mixture was left under stirring at room

temperature (rt) for 7 days. A precipitate formed which was ﬁltered

and washed with MeCN, followed by diethyl ether. The compound

was dried in an oven at 55 °C and used without puriﬁcation in the next

1

step (1.950 g, 95%). H NMR (400 MHz, D₂O): δ 7.75 (2 H, d, J =

8.0 Hz, 3-H, Ar), 7.71 (2 H, t, J = 8.0 Hz, 5-H, Ar), 7.51 (2 H, t, J =

8.0 Hz, 2-H, Ar), 7.42 (2 H, d, J = 8.0 Hz, 4-H, Ar), 5.03, 4.73 (4 H,

ABq, J_AB= 13 Hz, CH₂ph) 4.90 (2 H, s, CH-bisaminal), 4.35−3.10 (16

H, m, CH₂−ring), 2.48 (6 H, s, CH₃) ppm. ¹³C NMR (100 MHz,

D₂O): δ 172.1 (CO), 150.2, 134.5, 133.2, 127.4, 124.0, 118.7 (Ar),

78.3 (CH-bisaminal), 61.0 (CH₂ph), 55.7, 55.1, 45.9, 43.1 (C-ring),

21.0 (CH₃) ppm.

H₂cb-do2ph. The diammonium bromide salt 2 (1.950 g, 2.83 mmol)

was suspended in EtOH/H₂O 90:10 (50 mL), and solid NaBH₄

(1.071 g, 28.3 mmol) was added in small portions. After the addition

was completed, the mixture was left under stirring at rt for 1 day. The

suspended solid was ﬁltered and discarded. The mother liquor was

evaporated to dryness, and the residue was redissolved in 1 M KOH

(40 mL) and left stirring overnight at 70 °C. The solution was trans-

ferred to a separating funnel and the pH adjusted to 9−10 with diluted

HClO₄. The white precipitate formed was extracted with CHCl₃(4 ×

50 mL), and the organic phases were dried with anhydrous Na₂SO₄,

ﬁltered, and evaporated to dryness. The solid was suspended in water

acidiﬁed to pH 3−4 with diluted HClO₄after which the mixture was

heated to 65 °C and left stirring for 3 days (at this temperature the

solid is completely dissolved). The solution was allowed to reach rt,

and the white precipitate formed was ﬁltered and dried under vacuum

(0.639 g, 37%). Mp 170 °C (decomp). ¹H NMR (400 MHz, D₂O): δ

7.43−6.95 (8 H, m, Ar), 4.47 (4 H, s, CH₂ph), 3.80−2.74 (16 H,

NCH₂CH₂N-ring), 2.86 (4 H, s, NCH₂CH₂N-bridge) ppm. ¹³C NMR

(100 MHz, D₂O): δ 155.3 (C1), 132.8 (C4, C5), 132.2 (C6), 121.1,

116.7 (C2, C3), 56.4, 53.2 (CH₂-ring), 55.6 (CH₂ph), 46.8 (CH₂-

bridge) ppm. Anal. Calcd for C₂₄H₃₆N₄O₂·2ClO₄: C, 47.1; H, 5.9; N,

9.2%. Found: C, 47.1; H, 6.2; N, 9.0%. ESI-MS (H₂O/MeOH, 1:1)

m/z: 411.1 (100) [M + H]⁺, 305.1 (4) [M − C₇H₇O⁺+ 2H]⁺

1,7-Bis(benzyloxycarbonyl)-4,10-bis(2-methylphenyl acetate)-tet-

raazacyclododecane, 4. A solution of 2-(bromomethy1)phenyl acetate

(2.5 g, 10.9 mmol) in MeCN (5 mL) was added dropwise to a mag-

netically stirred mixture of 1,7-bis(benzyloxycarbonyl)-tetraazacyclodo-

decane 3 (1.922 g, 4.4 mmol) and K₂CO₃(1.33 g, 9.6 mmol) in MeCN

(15 mL). The mixture was left under stirring at 40 °C for 3 days and at

60 °C for 4 days. The precipitate formed upon cooling to rt was ﬁltered,

dissolved in CHCl₃, and washed with H₂O. The organic phase was dried

with anhydrous Na₂SO₄, ﬁltered, and evaporated to dryness. The pure

compound was obtained after ﬂash column chromatography on silica gel

EXPERIMENTAL SECTION

■

1

Materials and Methods. Cyclen (1,4,7,10-tetraazacyclododecane)

was obtained from CheMatech. All solvents and chemicals were

obtained from commercial sources as reagent grade quality and used

as received. 1,7-Bis(benzyloxycarbonyl)-tetraazacyclododecane (2),

cyclen-glyoxal (1), and (bromomethy1) phenyl acetate were prepared

using CHCl₃/ethyl acetate (8:2) as eluent (1.750 g, 55%). H NMR

(400 MHz; CDCl₃): δ 7.40−6.95 (16 H, m, Ar), 4.90 (4 H, s, CH₂bz),

3.51 (4 H, s, CH₂phac), 3.38 (8 H, m, NCH₂CH₂N), 2.65 (8 H, m,

NCH₂CH₂N), 2.28 (6 H, s, CH₃) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ 169.3 (CO), 156.5, 149.6, 136.8, 128.5, 128.2, 128.0, 126.1,

122.4 (Ar), 67.1 (CH₂bz), 60.5 (CH₂phac), 54.7, 46.9 (C-ring), 21.1

(CH₃) ppm.

H₂do2ph. 1,7-Bis(benzyloxycarbonyl)-4,10-bis(2-methylphenyl ac-

etate)-tetraazacyclododecane 4 (1.750 g, 2.4 mmol) was suspended in

33% HBr (10 mL) and left stirring for 1 h at rt. Diethyl ether was

added (50 mL), and the precipitate formed was ﬁltered. The solid was

dissolved in NaOH 0.1 M (170 mL) and left stirring at rt overnight.

The solution was transferred to a separating funnel and extracted with

CHCl₃(5 × 60 mL). The aqueous phase was concentrated to about

40 mL, the pH was adjusted to ∼9 with diluted HClO₄, and a second

extraction was carried out with CHCl₃(4 × 20 mL). The combined

by literature procedures.^26,59,60The H and ¹³C{¹H} NMR spectra

1

were recorded on a Bruker Avance II+ 400 (¹H at 400.13 MHz and

¹³C at 100.61 MHz) or a Bruker Avance DRX 300 (¹H at 300.13 MHz)

spectrometer at probe temperature of 298.2 K. Chemical shifts (δ) are

given in ppm and coupling constants (J) in Hz. 3-(Trimethylsilyl)

propionic acid sodium salt was used as internal references for ¹H NMR

spectra in D₂O. Assignments are based on peak integration and multi-

plicity and on 2D HMQC experiments (see Supporting Information).

Microanalyses were carried out by the ITQB Analytical Services Unit.

The electronic absorption spectra were recorded on a UNICAM

UV−vis spectrophotometer model UV-4. EPR spectra were recorded

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Article

organic phases were dried with anhydrous Na₂SO₄, ﬁltered, and

evaporated to dryness. The product was recrystallized from boiling

MeCN. The desired compound was obtained as a crystalline solid

(0.940 g, 56%). Mp 169 °C (decomp). ¹H NMR (400 MHz, CDCl₃):

δ 7.10 (2 H, td, J = 8.0 Hz, 1.7 Hz, 3-H, Ar), 6.91 (2 H, dd, J = 7.4 Hz,

1.3 Hz, 5-H, Ar), 6.86 (2 H, dd, J = 8.1 Hz, 1.0 Hz, 2-H, Ar), 6.70

(2 H, td, J = 7.4 Hz, 1.0 Hz, 4-H, Ar), 4.94 (bs, OH), 3.71 (s, 4 H,

CH₂ph), 2.70−2.60 (16 H, NHCH₂CH₂N) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ 157.8 (C1), 129.0 (C3, C5), 122.9 (C6), 119.2 (C4), 116.9

(C2), 60.4 (CH₂ph), 53.6 (NHCH₂), 47.6 (CH₂N) ppm. Anal. Calcd

for C₂₂H₃₂N₄O₂: C, 68.7; H, 8.4; N, 14.6%. Found: C, 68.7; H, 8.5; N,

14.2%. ESI-MS (H₂O/MeOH, 1:1) m/z: 107.0 (5) [C₇H₇O]⁺, 279.1

(25) [M − C₇H₇O⁺+ 2H]⁺, 385.1 (100) [M + H]⁺.

[Cu(do2ph)]. An aqueous 0.1 M solution of Cu(ClO₄)₂(0.52 mL,

0.052 mmol) was added to a solution of H₂do2ph (20 mg, 0.052 mmol)

in water (15 mL), the pH was neutralized with diluted aqueous KOH,

and the blue solution was stirred overnight at rt. The solution was

evaporated to dryness, the complex was dissolved in absolute ethanol,

and the insoluble matter was ﬁltered oﬀ. The previous step was re-

peated until no more insoluble matter was observed. The resulting

solution was evaporated, and the solid obtained was dried under

vacuum to yield the complex in the monoprotonated form (21 mg,

71%). Anal. Calcd for CuC₂₂H₃₁N₄O₂·ClO₄·3H₂O: C, 44.0; H, 6.2; N,

9.3%. Found: C, 44.0; H, 5.8; N, 9.2%. ESI-MS (H₂O/MeOH, 1:1)

m/z: 446.1 (100) [Cu(Hdo2ph)]⁺, 223.4 (10) [Cu(H₂do2ph)]²⁺,

554.9 (10) ([Cu(H₂do2ph)]ClO₄)⁺.

[Cu(cb-do2ph)]. An aqueous 0.1 M solution of Cu(ClO₄)₂(1.68 mL,

0.168 mmol) was added to a solution of H₄cb-do2ph(ClO₄)₂(103 mg,

0.168 mmol) in water (50 mL), the pH was neutralized with diluted

aqueous KOH, and the dark green solution was stirred at 50 °C for

2 h. The solution was evaporated to dryness, the complex was dis-

solved in methanol, and the insoluble matter was ﬁltered oﬀ. This

procedure was repeated until no more precipitation was observed. The

dark green solid obtained was dried in vacuum until constant mass

(71 mg, 74%). Anal. Calcd for CuC₂₄H₃₃N₄O₂·ClO₄: C, 50.3; H, 5.8;

N, 9.8%. Found: C, 50.0; H, 5.9; N, 10.1%. ESI-MS (H₂O/MeOH,

1:1) m/z: 472.1 [CuH(cb-do2ph)]⁺.

[Ga(do2ph)]NO₃. An aqueous 0.022 M Ga(NO₃)₃(4.6 mL)

solution was added to H₂do2ph (38.4 mg, 0.1 mmol) dissolved in

water (30 mL), the pH was slowly raised to 9.0 by addition of 0.1 M

KOH, and the solution was left stirring at 60 °C overnight. The pH

was readjusted to 9.0, the solution was concentrated in the evaporator

and was left cooling overnight. The precipitate obtained was ﬁltered

and dried in vacuum until constant mass (27 mg, 53%). Anal. Calcd

for GaC₂₂H₃₀N₄O₂·NO₃: C, 51.4; H, 5.9; N, 13.6%. Found: C, 51.3;

H, 6.2; N, 13.5%. ESI-MS (H₂O) m/z: 451.1 [Ga(do2ph)]⁺.

X-ray Crystallography. Crystals of H₂do2ph were prepared by

slow overnight crystallization of a concentrated solution of the

compound prepared in hot MeCN. Crystals of [Cu(Hdo2ph)]ClO₄·

H₂O were obtained by addition of 5 M NaClO₄to an aqueous solution

of the complex, after 2 days of slow evaporation at rt. Crystals of

[Ga(do2ph)]NO₃·2H₂O were prepared by slow evaporation at rt over

2 days of a concentrated solution of the complex. Single crystal X-ray

data of H₂do2ph was collected on an in-house Bruker AXS Microstar

imaging plate detector (X8 PROTEUM diﬀractometer), with graphite

monochromated Cu-Kα radiation (λ = 1.54 Å). The selected crystal

was positioned at 50 mm from the CCD and exposed for 20s per frame.

Data reduction of the data set was carried out using the SAINT-NT

from Bruker AXS, with a multiscan absorption correction (SADABS).

The crystal only diﬀracted up to 1.25 Å resolution, with a reﬂection

to parameters ratio of 4.8, which resulted in a low bond precision for

C−C. Attempts were made to collect higher resolution data, either by

improving crystal quality and/or by collecting data on a Bruker AXS-

KAPPA APEX II diﬀractometer with graphite monochromated Mo-Kα

radiation, but were unsuccessful. Nevertheless, it was possible to solve

and determine the structure of the compound. The structure was

solved with SHELXS⁶¹by direct methods and reﬁned on F²using full-

matrix least-squares with SHELXL,⁶¹included in the WINGX-Version

1.70.01⁶²package of programs. All non-hydrogen atoms were reﬁned

with anisotropic thermal parameters. The majority of the hydrogen

atoms were introduced at calculated positions and reﬁned using the

riding model, except for the hydrogen atoms bound to the macrocycle

nitrogens and to oxygen atoms that were positioned according to

electron density peaks identiﬁed in the diﬀerence Fourier maps and

reﬁned with individual isotropic temperature parameters. Single crystal

X-ray data of [Cu(Hdo2ph)]ClO₄·H₂O and of [Ga(do2ph)]NO₃·

2H₂O were collected on a Bruker AXS-KAPPA APEX II diﬀracto-

meter, with graphite monochromated Mo-Kα radiation (λ = 0.710 69 Å).

The selected crystals of each compound,were positioned at 40 mm

from the CCD and exposed for 10 s per frame. Data reduction of each

data set was carried out using the SAINT-NT from Bruker AXS, with a

multiscan absorption correction (SADABS). The structures were