How Does the Replacement of Acetate Arms with Acetamide Ones in the Structure of Chelators Affect Complexation with Pb²⁺ ?

Abstract

The growing interest in the application of Pb²⁺ complexes in fields such as radiopharmaceuticals encourages a more thorough study of the coordination chemistry of this metal ion and the search for a desirable chelating agent. In this work, a comparative study of the complexing properties of various acetate and acetamide chelators was carried out to determine which pendant arms are preferable for binding the Pb²⁺ ion. The study focused on chelators containing the same pyridine bisamide fragment in their structure, while the type of chelator, the size of the macrocyclic cavity, and the number of pendant arms were varied. To better understand the impact of specific factors on Pb²⁺ binding, the discussion was supplemented with literature data on the complexation of the well-known ligands DOTA and DTPA, along with their acetamide analogues DOTAM and DTPAM. Potentiometric titration was employed to determine the basicity of the ligands and the thermodynamic stability of their Pb²⁺ complexes. Their structures were characterized by single-crystal X-ray diffraction and NMR spectroscopy. The results revealed that macrocyclic chelators with flexible structures and acetate pendant arms form the most thermodynamically stable complexes with Pb²⁺, exhibiting shorter and more uniform coordination bond lengths.

Introduction

From a medical point of view, until recently lead­(II) was considered only as one of the most toxic and widespread inorganic environmental pollutants, since it is easily absorbed into the human body and can cause many diseases. In this regard, chelating agents have been developed to remove Pb²⁺ ions from tap water and to treat lead poisoning. − With the development of nuclear medicine technologies, some lead radionuclides have attracted great interest. The radionuclide ²⁰³Pb (t _1/2 = 51.9 h) is a γ-emitter, which can be used for cancer diagnosis by single-photon emission computed tomography (SPECT). , A decay chain of ²¹²Pb (t _1/2 = 10.6 h) resulting in the release of both β^– and α-particles via the “in vivo generation” of the daughter ²¹²Bi (t _1/2 = 60.6 min) determines its therapeutic application. , At the same time, the theranostic pair ²⁰³Pb/²¹²Pb is a promising candidate for use in personalized theranostics of oncological diseases. − All this has prompted efforts aimed at finding effective chelators for Pb²⁺ ions. It should be emphasized that for medical use of chelators it is necessary that the binding of the metal ion occurs quickly and under mild conditions, and the resulting complex is inert and does not dissociate in biological environments.

Although DOTA is the standard chelator for most medical radionuclides, it is not effective enough for²¹²Pb. , Instead, the chelator of choice for ^203/212Pb-based radiopharmaceuticals is DOTAM, also known as TCMC (Figure ). − Despite the fact that acetate ligands typically exhibit higher thermodynamic stability constants for their metal complexes compared to their acetamide analogues, the replacement of “hard” carboxyl groups with “softer” amide groups, according to Pearson’s HSAB theory, radically improves the kinetic inertness of Pb²⁺ complexes. − For example, CHX-PYTAM proved to be a promising chelator for [²⁰³Pb]­Pb²⁺, whereas CHX-PYTA and PYTA formed kinetically labile complexes with [²⁰³Pb]­Pb²⁺ (Figure ). , However, there are opposite examples, such as with the chelators PADA and PADAM (Figure ): the Pb²⁺ complex with acetate PADA remained stable in the presence of serum proteins for 1 day, in contrast to the Pb²⁺ complex of acetamide PADAM, which rapidly dissociated within the first minutes of incubation.

1.

Acetate and acetamide derivatives of macrocyclic and acyclic chelators discussed herein.

Complexation of the Pb²⁺ ion with the amide analogue DTPAM of another popular chelator DTPA (Figure ), was also investigated and demonstrated the affinity of DTPAM for Pb²⁺, although inferior to the “gold standard” DOTAM. Nevertheless, the kinetics of complexation were much faster for DTPAM, as radiolabeling required only 15 min, as opposed to 60 min for DOTAM. Such differences in properties between acyclic and macrocyclic chelators are characteristic, and the complexation of the first one occurs faster, but the complexes of the second one are more stable to rechelation.

In our earlier studies, we showed that the triacetate macrocycle Py3a (Figure ) successfully chelates the Bi³⁺ ion, including within nanoconjugates. − Similar to DOTAM, we hypothesized that replacing the acetate groups in the Py3a structure with acetamide ones would be preferable for binding the Pb²⁺ ion. To fully assess the influence of cavity size, number of pendant arms and chelator type on Pb²⁺ complexation, in this study we compared macrocyclic and acyclic chelators Py2am, Py3am, and aPytam containing acetamide groups with the analogous acetate derivatives Py2a, Py3a, and aPyta (Figure ). ,− The chelator Py3am was previously prepared and its complexation with Zn²⁺ and Ni²⁺ ions was studied. It was shown that it is not ideally suited for such small ions. We thus hypothesized that binding to the larger Pb²⁺ ion would be more efficient. The series of chelators under investigation contains the pyridine bisamide moiety that imparts structural rigidity to the ligand molecule. This is known to increase the rate of complex formation due to preorganization of the coordinating center and can improve the kinetic inertness of the complexes. Furthermore, this fragment can be easily modified to introduce the chelator into various conjugates with peptides, antibodies, nanoparticles, and other carrier molecules. It is also known that the introduction of rigid fragments into the ligand structure often leads to a decrease in the thermodynamic stability constants of the resulting complexes, but at the same time can improve the kinetic inertness of the complexes. It is also known that the introduction of rigid fragments into a macrocyclic structure often leads to a decrease in the thermodynamic stability constants of the resulting complexes, but at the same time can improve the kinetic inertness of the complexes.

Acetamide chelators clearly have potential for Pb²⁺ binding, but the lack of comprehensive research on them leads to an incomplete understanding of the effect of replacing acetate groups in macrocyclic and acyclic chelators with acetamide ones. Here we report the synthesis of new acetamide chelators and comparative studies with their acetate analogues on their basicity, thermodynamic stability of their Pb²⁺ complexes, as well as structural features of these complexes in solution and in crystalline form.

Results and Discussion

Synthesis of the Ligands

The synthesis of both macrocyclic and acyclic chelators started with dimethyl 2,6-pyridinedicarboxylate 1, which reacted with long polyamines (triethylenetetramine and tetraethylenepentamine) to give 15- and 18-membered azacrown compounds 2 and 3, respectively, and reacted with short ethylenediamine to give podand 4 (Scheme ). Both types of products were prepared in methanol at room temperature according to previously described procedures. ,

1. Synthesis of Acetate (Py2a, Py3a, aPyta) and Acetamide (Py2am, Py3am, aPytam) Chelators .

^a Conditions: (a) triethylenetetramine or tetraethylenepentamine, MeOH, r.t.; (b) ethylenediamine, MeOH, r.t.; (c) (1) BrCH₂COOtBu, K₂CO₃, MeCN, reflux, (2) H₂O, reflux; (d) BrCH₂CONH₂, K₂CO₃, MeCN, reflux.

Acetate chelators Py2a, Py3a, and aPyta were prepared as described in our previous studies , via a step of formation of the corresponding tert-butyl esters followed by thermal deprotection without the addition of any acid catalysts. This method is quite simple and convenient due to the ease of isolation of the target product and is suitable for compounds of this type.

Acetamide chelators Py2am, Py3am, and aPytam were synthesized from compounds 2-4 similarly to acetate chelators Py2a, Py3a, and aPyta, but bromoacetamide was used as the alkylating agent. Macrocyclic chelator Py2am was obtained in similar yield to the larger macrocycle Py3am (58% and 65%, respectively). However, acyclic chelator aPytam could only be isolated in 38% yield due to difficulties in purification.

Constants of Ligand Protonation and Stability of Pb²⁺ Complexes

The protonation constants of Py2am, Py3am, aPytam, and aPyta were determined at 25 °C in 0.1 M KNO₃ by potentiometric titration in a wide range of pH from 2 to 12. The distribution curves of the protonated and deprotonated forms of the ligands as a function of pH are shown in Figures S18–S21 in ESI. Table presents the calculated stepwise protonation constants for these ligands and the previously described chelators for comparison. It can be noted that for all the ligands studied, the replacement of acetate chelating groups with acetamide ones leads to a decrease in the protonation constants. The same patterns are observed for DOTA and DOTAM, DTPA and DTPAM (Table ). This phenomenon can be explained if we take into account that the acceptor effect of the acetate group on the basicity of the tertiary amino group to which it is attached is less than that of the acetamide group, which is due to the anionic nature of the carboxylate.

1. Stepwise Protonation Constants (Log K) of the Ligands and Stability Constants (Log β) of Their Pb²⁺ Complexes (T = 25.0 °C; I = 0.1 M KNO₃).

Ion	Species	Py2a	Py2am	Py3a	Py3am	aPyta	aPytam	DOTA	DOTAM	DTPA	DTPAM
H+	HL	9.22	5.1 (1)	9.91	7.0 (1)	10.29 (2)	3.94 (3)	12.09	7.70	10.48	5.99
	H2L	3.59	-	6.8	2.9 (1)	8.16 (4)	3.06 (2)	9.76	6.21	8.60	2.33
	H3L	2.3	-	2.8	-	3.1 (1)	-	4.56	-	4.28	-
	H4L	-	-	2.9	-	2.2 (1)	-	4.09	-	2.6	-
	H5L	-	-	-	-	-	-	-	-	2.0	-
Pb2+	H3LPb	-		-	-	-		-		2.75	-
	H2LPb	-		-	-	-		2.72		3.40	-
	HLPb	-		3.04	-	5.42 (2)		3.98		4.05	2.22
	LPb	8.7		14.12	9.16 (4)	12.86 (3)		25.3	>19	19.10	8.79
	LPb(OH)	15.3		16.25	15.4 (1)	16.68 (4)		-		-	10.74
	LPb(OH)2	20.0		-	19.3 (1)	-		-		-	-
	pPb	8.1		12.4	10.1	10.0		20.0	>19.5	19.0	9.7

^a0.1 M NaClO₄, 25 °C.

^b0.1 MMe₄NNO₃, 25 °C.

^c0.1 M NaCl, 25 °C.

^d0.1 M NaNO₃, 25 °C.

^e0.1 M KNO₃, 25 °C.

^f0.2 M NaClO₄, 25 °C.

^g0.16 M NaCl, 25 °C.

^hpPb = −log­[Pb_free], when [L] = 10 μM, [Pb] = 1 μM at pH 7.4.

ⁱNot determined due to precipitation.

For the acetamide ligands Py2am and Py3am, only nitrogen atoms more than one ethylene bridge away can be simultaneously protonated due to the repulsion of closely spaced positive charges, as for DOTAM. Accordingly, only one protonation constant was determined for Py2am, and two constants for Py3am. For the acetate ligands Py2a and Py3a stepwise protonation of closely located tertiary amines is possible due to the compensation of the positive charge by deprotonated carboxylate groups with the formation of zwitterions. Thus, two protonation constants were determined for Py2a. It is interesting to note the difference of two orders in the values of the first protonation constants for Py2am and Py3am which turns out to be quite insignificant when comparing Py2a and Py3a.

Four protonation constants have been determined for the acyclic ligand aPyta. The first two constants relate to the protonation of backbone amines, and the third and fourth constants relate to the protonation of the acetate groups. Acyclic ligands are characterized by greater flexibility compared to macrocycles which can facilitate the addition of a proton since the repulsion factor of closely located positive charges is less pronounced. The protonation constants of aPyta are close to those of DTPA. Thus, the introduction of the pyridine bisamide moiety does not significantly affect the protonation of aPyta.

For acyclic ligands aPyta and aPytam, containing acetate or acetamide groups, respectively, the difference in the first protonation constants reaches 6.4 orders of magnitude, which is due to a decrease in the basicity of tertiary nitrogen atoms with acetamide substituents (Table ). The values of the first two protonation constants are close for aPytam (log K ₁ = 3.94 and log K ₂ = 3.06) indicating almost independent protonation of tertiary backbone amines. These nitrogen atoms are sufficiently distant from each other due to the pyridine bisamide fragment, in contrast to the same tertiary nitrogen atoms in DTPAM, the structure of which is not fixed.

To detect complex formation of the studied ligands with the Pb²⁺ ion, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of aqueous solutions of the ligands in the presence of Pb­(ClO₄)₂ was primarily used. It was shown that mononuclear Pb²⁺ complexes are formed with all acetate and acetamide ligands, and binuclear complexes were also detected for the large macrocyclic Py3a and acyclic aPyta ligands (Figures S12–S17). In the case of acetamide ligands Py2am and aPytam, low-intensity peaks of the complexes are present in the spectra. It can be noted that there are peaks corresponding to the matrix complex (α-cyano-4-hydroxycinnamic acid) with Pb²⁺ in the mass spectra of the acetate ligands Py2a, Py3a and aPyta. This can be explained by the competition for the ion between the matrix and the ligands due to the presence of similar groups in their structure.

Potentiometric titrations of the ligands in the presence of the Pb²⁺ ion over the pH range from 2 to 12 at 25 °C in 0.1 M KNO₃ were carried out to determine the stability constants of Pb²⁺ complexes (Figures S22 and S23 in ESI). Stability constants were calculated using ligand protonation constants, Pb²⁺ hydrolysis constants (Table S1), and the ionic product of water in the HyperQuad2013 program (Table ). In addition, the values of pPb = −log­[Pb_free] were calculated to quantify the metal-binding ability of the ligands when c(L) = 10 μM, c(Pb²⁺) = 1 μM at pH = 7.4. pM values present a more adequate indicator of metal complex formation, when ligands with different basicity and denticity are compared, although pM values often correlate with log β.

Potentiometric titration of acetamide ligands Py2am and aPytam in the presence of Pb²⁺ was complicated due to the formation of a precipitate. Therefore, it was not possible to calculate the stability constants of these complexes due to the lack of stable forms of the complexes for detection by potentiometric titration. For another acetamide ligand, Py3am, the pPb value turned out to be 2 orders of magnitude lower than that for its acetate analog, Py3a (pPb = 10.1 for Py3am vs log pPb = 12.4 for Py3a). A significant advantage of Py3am is the chelation of Pb²⁺ at low pH values: there is almost no free Pb²⁺ ion already at pH = 3 (Figure S22).

The search for similar patterns when considering Pb²⁺ complexes with DOTA and DOTAM is complicated by the lack of an exact value of the complexation constant for DOTAM. However, a dramatic decrease in the stability constants of the complexes is obvious when moving from DOTA to Py3a and from DOTAM to Py3am. The reasons for this may be the smaller number of chelating groups in the ligands Py3a and Py3am (three instead of four), as well as their structural feature caused by the presence of a rigid pyridine bisamide fragment in the macrocyclic framework. The formation of hydroxo complexes with Py3a and Py3am in the solution, unlike DOTA (Table ), indicates insufficient saturation of the Pb²⁺ coordination sphere. However, it is worth mentioning that rigid fragments introduced into the macrocyclic framework promote structural preorganization of the ligand cavity for rapid interaction with the metal ion, which leads to acceleration of the kinetics of complexation. This is extremely important for many applications, including radiopharmaceuticals. A well-known disadvantage of DOTA and DOTAM is their slow complexation, so in most cases prolonged heating is required to fully bind the metal ion. ,,,,

The acyclic ligand aPyta forms a less stable complex with the Pb²⁺ ion compared to DTPA (pPb = 10.0 for aPyta vs pPb = 19.0 for DTPA). Such a significant difference in stability constants can be attributed to several factors, including the presence of an additional chelating group in DTPA compared to aPyta and, accordingly, a larger number of donor centers (eight vs six), as well as the greater structural flexibility of DTPA allowing it to better adapt to the coordination geometry of the metal ion without steric strain in the structure.

If we compare the acyclic ligand aPyta with its macrocyclic analogues Py2a and Py3a, we can see that the pPb values of their complexes decrease in the following order: [Pb­(Py3a)]⁻ > [Pb­(aPyta)]^2– > [Pb­(Py2a)]. The lowest stability of the Py2a complex can be explained by the smallest number of donor centers in the ligand molecule, which is not enough to coordinate the Pb²⁺ ion. This is evidenced by the presence of hydroxo complexes of different composition in an aqueous solution. It is interesting that aPyta, which contains more chelating groups compared to Py3a (four vs. three), but the same number of donor centers (six), forms a less stable complex with Pb²⁺ and its pPb value is 2.4 orders of magnitude lower. Most likely, the higher stability of the Py3a complex compared to aPyta is associated with the macrocyclic effect. Comparison of DOTA with DTPA, and DOTAM with DTPAM shows the same pattern in the complexing properties of macrocyclic and acyclic ligands.

Structure of the Ligands and Their Pb²⁺ Complexes in Solid State

The structures of macrocycle Py2am and complexes [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ were established with single-crystal X-ray diffraction (Figure ). The compounds Py2am, [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ crystallize in centrosymmetric space groups P2₁/c, P2₁/n and P1̅ respectively. The structures of complexes [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ contain water molecules (four molecules in [Pb­(Py2am)]²⁺ and two molecules in [Pb­(Py3am)]²⁺) and two perchlorates as counterions. Bond lengths and angles in all structures are within normal ranges with few exceptions, that was confirmed by the analysis with the Mogul program, which compares geometric parameters of molecules with corresponding values for similar structural fragments in the Cambridge Structural Database (CSD). The exceptions are some coordination bonds, which are discussed below, as well as some angles that are considered to be unusual based on a small data set. Experimental details and crystallographic data are provided in ESI (Table S2).

2.

General view of crystal structures Py2am (A), [Pb­(Py2am)]²⁺ (B) and [Pb­(Py3am)]²⁺ (C) in thermal ellipsoid representation for non-hydrogen atoms (p = 50%), heteroatoms are labeled, hydrogen atoms connected to carbon atoms, uncoordinated water molecules and perchlorate anions are omitted for clarity. The atom O1a in [Pb­(Py2am)]²⁺ is generated by a 2-fold screw axis to complete the coordination environment of the Pb²⁺ ion.

In crystal, the ligand Py2am (Figure A) has a flattened macrocyclic part with the pendant arms located above and below the macrocycle cavity. Amide groups of substituents are rotated unequally so that either the carbonyl atom O4 or the amide atom N6 are located closer to the center of the macrocycle. The carbonyl atom O4 is involved in the intramolecular bifurcated hydrogen bonding with the amide H­(N) atoms of the macrocycle (N ··· O 3.0446(12) and 3.0751(12), Å; NHO 146 and 160°, respectively; N–H distances normalized hereinafter to 1.015 Å) and the amide atom N6 forms N3 ··· N6 intramolecular hydrogen bond (2.7311(12) Å, NHO 109°) thus stabilizing this conformation. In turn, intermolecular hydrogen bonds define main supramolecular units (Figure S24).

The conformation of the macrocyclic part is similar in the structures of Py2am and [Pb­(Py2am)]²⁺ (Figure S25). Thus, the coordination of the ligand Py2am with the Pb²⁺ ion has a minor effect on its conformation, because of the specific coordination geometry. The metal ion is located above the macrocyclic cavity, as its relatively small size does not allow the encapsulation of the large Pb²⁺ ion (r = 1.19–1.49, CN = 6–12). The size of the ring available for such a coordination can be estimated as the mean distance between heteroatoms of the macrocycle and their centroid and is equal to 2.365 Å for Py2am and 2.418 Å for [Pb­(Py2am)]²⁺ (Table S3). The resulting distance between Pb²⁺ and the mean plane of five heteroatoms of the macrocycle showing its shift out of the ring is equal to 1.653 Å. The minor difference between the conformation of the macrocycle in Py2am and [Pb­(Py2am)]²⁺ stems from the change in geometry required for the effective coordination with the pendant arms. To bring the arms in the cis- orientation (to the same side of the macrocyclic ring), the flexible C8–N4–C9 fragment must be “turned out”, and the bonds with pendant arms play significant role in stabilization of the complex.

Thus, the first coordination sphere of the Pb²⁺ ion in [Pb­(Py2am)]²⁺ (Figures B and S26) is formed by four heteroatoms of the ligand (two amino N atoms of the macrocycle and two carbonyl O atoms of the acetamide groups), an O atom of a water molecule, and, in addition, the “outer” macrocyclic carbonyl atom O1 of the adjacent complex cation, which makes the structure polymeric. The distances range from 2.3883(12) to 2.6840(13) Å for Pb–O and from 2.6734(14) to 2.7352(14) Å for Pb–N (Table ). The choice of the first coordination sphere in this complex is straightforward, as the six coordination bond lengths are significantly shorter than the secondary interaction with the N atom of the pyridine fragment (3.0461(14) Å) and, potentially N atoms of the amide fragment (>3.1 Å), although they, being protonated, cannot participate in strong coordination with the Pb²⁺ ion. Note that the limiting distances for the first and secondary coordination can be defined in different ways with the use of combination of ionic, covalent and van der Waals radii, , so it is customary in many cases to include or not the long distances in the primary and secondary coordination. Also, despite the nature of a “bond” can be ambiguous in case of secondary coordination sphere, the relatively short distances (less than sum of van der Waals radii) still assume a possibility of a bonding interaction and therefore must be considered. As for the coordination polyhedron of Pb²⁺, the first-order sphere of six heteroatoms is a distorted pentagonal pyramid. However, the coordination geometry and the short Pb–O4 bond length suggest the presence of the stereochemically active lone electron pair (LP) in the [Pb­(Py2am)]²⁺, so that the complex is hemidirected. Therefore, the coordination polyhedron of the Pb²⁺ ion can also be described as a ψ-pentagonal bipyramid (in terms of the valence shell electron-pair repulsion (VSEPR) model, with the LP being in the axial position.

2. Interatomic Distances (Å) in Metal Coordination Spheres in Complexes [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ and Two Similar Previously Reported Structures [Pb­(Py2py)]²⁺ and [Pb­(Py3a)]⁻ .

	[Pb(Py2am)]2+		[Pb(Py2py)]2+ (CSD code: RAGFIR)		[Pb(Py3am)]2+		[Pb(Py3a)]− (CSD code: RAGFUD)
Primary bonds	Pb1–O4	2.3883(12)	Pb1–N6	2.479(3)	Pb1–O5	2.3753(13)	Pb1–O4	2.429(5)
	Pb1-O3	2.5273(12)	Pb1-N4	2.573(3)	Pb1-O3	2.5647(14)	Pb1-N4	2.646(7)
	Pb1–O5	2.6258(14)	Pb1–N3	2.580(3)	Pb1–N5	2.7234(14)	Pb1–N3	2.661(5)
	Pb1-N4	2.6734(14)	Pb1-N7	2.613(3)	Pb1-N3	2.7316(14)	Pb1-O5	2.670(5)
	Pb1–O1#	2.6840(13)	Pb1–O6	2.795(3)	Pb1–N4	2.7345(15)	Pb1–O7	2.681(5)
	Pb1–N3	2.7352(14)	Pb1-O3	2.814(9)	Pb1-O6	2.8027(14)	Pb1-N5	2.687(6)
Secondary coordination	Pb1–N1	3.0461(14)	Pb1–N1	3.078(3)	Pb1–O4	2.9127(14)	Pb1–O9	2.858(8)
					Pb1-N1	3.0862(14)	Pb1-N1	3.160(6)

^aO1# is the atom of the neighboring complex cation of the polymeric structure.

^bData from ref. .

A similar coordination of the Pb²⁺ ion has been observed in the complex with the analogous macrocyclic ligand Py2py bearing two pyridyl pendant arms instead of the acetamide ones in Py2am (CSD code: RAGFIR). In the complex [Pb­(Py2py)]²⁺, the pendant arms are also cis-located, so that the metal ion is coordinated by N atoms of the pyridine rings and two amine atoms of the macrocycle. However, the remaining two positions in the first coordination sphere (not counting the activated LP) are occupied by two O atoms of the perchlorate anion. Note that the number of carbon atoms between the coordinating heteroatoms in the chelating fragments in [Pb­(Py2am)]²⁺ and [Pb­(Py2py)]²⁺ coincides. Therefore, both ligands are able to form complexes of very similar geometry (see Tables and S2). The comparison also shows that this type of coordination, where the ligand atoms occupy only a part of the coordination sphere, provides a certain degree of flexibility for the coordination with the neighboring complexes, counterions, and solvate molecules, which can be important for the behavior of the compound in solution.

The backbone of the ligand in the complex [Pb­(Py3am)]²⁺ (Figures C and S27) has an additional fragment −CH₂–N­(R)–CH₂– (R = acetamide) compared to [Pb­(Py2am)]²⁺. This difference seems to be crucial, as it has a complex effect on the coordination behavior of the ligand: it provides two additional coordination sites, increases the cavity size, and improves flexibility. The larger size of the macrocyclic cavity, estimated as 2.759 Å (Table S3), facilitates the encapsulation of Pb²⁺, which is located much closer to the mean plane of the macrocycle (at 0.981 Å). In the complex [Pb­(Py3am)]²⁺, two pendant arms are located on the same side of the macrocycle, but the central arm is located at the opposite side. During complex formation, the ligand Py3am fills more coordination sites of the Pb²⁺ ion, leaving only one position available and reducing the probability of coordination with large anions, solvate molecules or the formation of the polymeric structures. Therefore, Py3am has a stronger chelating ability than Py2am, suggesting a higher stability of its complex with the Pb²⁺ ion. The observed geometry of the complex [Pb­(Py3am)]²⁺ is also stabilized by intramolecular hydrogen bonds of the H atoms of the macrocyclic amide groups with the O atom of one central pendant arm (O4 ··· N2 and O4 ··· N6 3.064(2) and 2.852(2) Å), only possible with the mixed cis- and trans- orientation of the arms.

Nevertheless, the polyhedron of the first coordination sphere of [Pb­(Py3am)]²⁺ is very similar to that of [Pb­(Py2am)]²⁺, being a pentagonal pyramid with vertices occupied by three N atoms of tertiary amino groups, two O atoms of chelating acetamide groups and one of a water molecule, while the remaining O atom of the third acetamide arm must be classified as an secondary-sphere due to a long Pb–O distance (Table ). Similarly, within the VSEPR model, the coordination polyhedron geometry is a ψ-pentagonal bipyramid with the LP occupying the axial position. In addition to the above-mentioned secondary interaction with the O atom, the [Pb­(Py3am)]²⁺ exhibits a weak coordination with the pyridine N1 atom. It is important to note that despite the similar coordination environment, the bond lengths in the complexes of Py2am and Py3am are inevitably different, being generally slightly longer in the latter.

The complex with the macrocycle of the same size but with acetate pendant arms (Py3a) has also been described previously (CSD code: RAGFUD). Although in general the coordination geometry of [Pb­(Py3a)]⁻ resembles [Pb­(Py3am)]²⁺ (Figure S28), the distribution of coordination bond lengths differs significantly, apparently due to the anionic nature of the carboxylate groups in the former structure, and its polymeric structure. Namely, the distribution of Pb–O bond lengths is more even in [Pb­(Py3a)]⁻, so that all O atoms of the acetate arms fall in the first coordination sphere. As a result, the first-sphere coordination polyhedron (defined by the distance cutoff of 2.8 Å, is so deformed compared to [Pb­(Py3am)]²⁺, that it cannot be described by a proper polyhedron other than a very distorted trigonal antiprism with the metal center lying nearly on one of the triangular faces. However, a more uniform distribution of bond lengths and shorter Pb–N bonds in the Py3a complex indicate of its higher stability, which is confirmed by data on thermodynamic stability constants.

Structural Investigation of the Pb²⁺ Complexes in Aqueous Solutions

The metal-chelator interaction was studied in D₂O solution using NMR spectroscopy. ¹H NMR spectra of Pb²⁺ complexes were recorded at slightly acidic or close to neutral pH to evaluate complex formation. The proton resonances of the obtained spectra are compared to those of a free ligand at a pH, where the fully deprotonated form is observed according to potentiometric titration. The assignment of proton resonances was produced using two-dimensional NMR techniques including homonuclear ¹H–¹H COSY and ¹H–¹H NOESY or ROESY experiments, which give cross-peaks between the geminal and vicinal methylene protons with different signal intensities. These studies were performed not only to confirm metal chelation, but also to gain insight into the approximate ligand configurations in solution, the symmetry of the complexes, and their fluxionality. Furthermore, this series of macrocyclic and acyclic chelators provides an opportunity to study how the size of the macrocycle cavity and the mobility of the donor framework impact chelation chemistry.

The chelators Py2a and Py2am in the presence of Pb²⁺ display a certain degree of broadening of proton resonances in the ¹H NMR spectra (Figure S29), indicating metal ion binding, although the positions of the chemical shifts of the signals undergo minor changes. The broad proton resonances in ¹H NMR spectra are not uncommon for Pb²⁺ complexes, and a similar behavior has been observed with other chelating agents. ,,, Noteworthy, diastereotopic splitting patterns are absent, and the proton signals of the methylene groups of the pendant arms are observed as singlets. The crystal structure of [Pb­(Py2am)]²⁺ described above (Figure B) provides a static coordination of the ligand, while the ¹H NMR spectrum reveals a more dynamic behavior of the of the ligand upon complexation. It is likely that the Pb²⁺ complexes with Py2a and Py2am are labile in aqueous solutions, and their geminal proton signals remain magnetically equivalent as a result of dissociation and recoordination. The high degree of fluxionality in solutions is a result of a poor size-matching between the binding sites offered by these 15-membered macrocyclic chelators and the size of the Pb²⁺ ion.

The expansion of the macrocycle framework from 15-membered to 18-membered has a beneficial effect on the resolution of proton resonances (Figure ). The size of the Pb²⁺ ion is more suitable for the macrocyclic cavity of Py3a and Py3am, so the ¹H NMR spectra show sharply resolved peaks, which corresponds to the complexes [Pb­(Py3a)]⁻ and [Pb­(Py3am)]²⁺ with rigid conformations. In contrast to Pb²⁺ complexes of Py2a and Py2am, metal coordination with both Py3a and Py3am leads to significant shifts of the most proton signals of methylene groups to the downfield region with values varying from 0.2 to 1.0 ppm as a result of the ligand-to-metal electron density donation. The phenomenon of diastereotopic splitting of methylene protons is also a distinctive feature of metal complex formation due to the strong interaction between donor atoms of chelators and the metal center. The protons of two pendant arms appear as two distinct nonequivalent H_9x and H_9y signals with large ² J _AB coupling constants (about 16.5–17.6 Hz). At the same time, the H₁₁ protons of the central pendant arm are magnetically equivalent and appear as a singlet signal in the spectrum. This indicates that the central acetate or acetamide group lies in a plane of symmetry and, therefore, the geminal protons have the same chemical surroundings. The effect has been previously described in literature for similar three-armed macrocyclic chelators. , Moreover, the values of the integrated intensities of individual proton signals in the aliphatic region of the ¹H NMR spectra confirm the symmetric conformation of the complexes [Pb­(Py3a)]⁻ and [Pb­(Py3am)]²⁺. For both chelators, there is no strong interaction between the Pb²⁺ ion and the pyridine nitrogen, which is revealed by the unchanged chemical shifts of the H₁ and H₂ protons in the complexes compared to those for free ligands.

3.

¹H NMR spectra of: (a) Py3a at pH = 12.5; (b) Py3a in the presence of Pb²⁺ at pH = 5.9; (c) Py3am at pH = 9.2; (d) Py3am in the presence of Pb²⁺ at pH = 5.5 in D₂O.

The acyclic chelators aPyta and aPytam have the same number of coordination centers as macrocyclic ones Py3a and Py3am, but their structures are more flexible, which affects the dynamics of complexation on the NMR time scale. The¹H NMR spectra of the Pb²⁺ complexes with both acyclic chelators show a strong line-broadening of aromatic and aliphatic resonances at room temperature (Figure S30). This behavior causes significant hindrance in the assignment of proton resonances. It was hypothesized that multiple isomers are formed in solution due to the fluxional coordination of Pb²⁺ ions with the chelators. As for aPyta, the ¹H NMR spectrum was recorded in the presence of Pb­(NO₃)₂ at pH = 6.2. According to potentiometric titration data, a high percentage of the complex [Pb­(Py3a)]^2– is observed at this pH value (Figure S23). The ¹H NMR spectrum of aPytam in the presence of Pb­(NO₃)₂ was recorded at a lower pH = 4.8. The higher pH value resulted in the precipitation of Pb²⁺ in the form of insoluble hydroxides, which greatly reduced the percentage of the complex [Pb­(Py3am)]²⁺ in solution, and proton resonances of the free chelator Py3am were observed. Nevertheless, in the ¹H NMR spectra of both acetate Py3a and acetamide Py3am chelators in the presence of Pb²⁺, noticeable downfield shifts of all proton signals in the aliphatic region occurred, while the positions of signals in the aromatic region remained almost unchanged relative to those for free ligands. The resulting spectra indicate that the acyclic chelators aPyta and aPytam, as well as the macrocyclic Py3a and Py3am, coordinate Pb²⁺ only via pendant groups and tertiary amino groups without substantial participation of the pyridine nitrogen atom. This feature of interaction with metal ions is characteristic of chelators containing a pyridine bisamide fragment in their structure and has also been demonstrated previously with other metal ions. ,− ,

Conclusions

To investigate whether acetamide groups are more effective than acetate groups in chelating the Pb²⁺ ion, the complexation behavior of various known and newly synthesized chelators containing either acetate or acetamide pendant arms was compared. In addition to the nature of the pendant arms, the effects of the chelator type, the size of the macrocyclic cavity, the number of chelating groups, and the presence of a rigid fragment in the ligand structure on complex formation were analyzed. It is obvious that the introduction of acetamide groups instead of acetate ones into the composition of chelators leads to a significant decrease in the thermodynamic stability of the resulting complexes with Pb²⁺. The explanation may lie not only in the anionic nature of carboxylate, which stabilizes the structure of the protonated form of the ligand and compensates for the positive charge of the metal ion, but also, apparently, structural aspects are important. Although at first glance, from the point of view of structure, the resulting complexes of acetate and acetamide ligands are similar, which was shown in aqueous solutions using NMR spectroscopy, but in the crystalline form, structural differences are noticeable, and acetate groups provide shorter and more even bond lengths in contrast to acetamide ones.

It was shown that macrocyclic chelators are more preferable for binding Pb²⁺ and give more stable complexes compared to acyclic ones with the same number of coordinating centers. This pattern is the same for chelators with both types of pendant groups. Moreover, the incorporation of a rigid fragment into the structure of both macrocyclic and acyclic chelators has a negative effect on the thermodynamic stability of the resulting complexes, and for all chelators containing pyridine bisamide, much lower pPb values were obtained compared to those for more flexible analogues

Thus, this study sheds additional light on the coordination chemistry of the Pb²⁺ ion, which is of great importance given its growing popularity for various applications, especially radiopharmaceuticals.

Experimental Section

Materials and Equipment

All the reagents were purchased from commercial sources and used without purification. The compounds 1–4 and the ligands Py3am and aPyta were prepared as we described earlier. ,, The ¹H and ¹³C, NMR spectra were registered on Varian Inova 400 and Bruker Avance 400 spectrometers at ambient temperature using deuterated solvents (CDCl₃, D₂O). Chemical shifts were reported in parts per million (δ) relative to deuterated solvent as an internal reference. Coupling constants (J) are given in hertz. Numbering of the hydrogen and carbon nuclei used to describe the NMR spectra is given in Figures in the Electronic Supporting Information. The mass spectra (ESI) were registered on a Shimadzu LCMS-2020 High Performance Liquid Chromatograph Mass Spectrometer with a single quadrupole detector, desolvation line/heat block temperature 250/400 °C and an ionization voltage at 4.5 kV. Electrospray full scan spectra was obtained by infusion at 0.4 mL/min of aqueous or MeCN solutions of the compounds. MALDI-TOF measurements were performed using a Shimadzu AXIMA Confidence MALDI-TOF MS in reflectron mode, with an α-cyano-hydroxycinnamic acid matrix in 10-fold excess. The melting points were determined on a Mel-temp II apparatus in open capillary tubes. Reaction progress was followed by TLC using aluminum oxide (Merck, 60 F254, neutral). The elemental analyses were performed on a Carlo Erba 1108 elemental analyzer at the Laboratory of Microanalysis of A. N. Nesmeyanov Institute of Organoelement Compounds of RAS, Moscow, Russia.

Synthesis and Characterization Data

2-[9-(Carbamoylmethyl)-2,13-dioxo-3,6,9,12,18-pentaazabicyclo­[12.3.1]­octadeca-1­(17),14­(18),15-trien-6-yl]­acetamide (Py2am)

The macrocycle 2 (204 mg, 0.736 mmol) and K₂CO₃ (406 mg, 2.943 mmol) were dissolved in dry MeCN (20 mL) and 2-bromoacetamide (207 mg, 1.501 mmol) was added. The reaction mixture was refluxed for 30 h. The solvent was evaporated in vacuum. The product was extracted with cold EtOH from the dry residue and purified by column chromatography (SiO₂, ethyl acetate-MeOH). The product was obtained as a white solid (174 mg, 58%); mp 212–214 °C. ¹H NMR (D₂O, 400 MHz): 2.69 (s, 4H, H(7)), 2.84 (br.s, 4H, H(6)), 3.18 (s, 4H, H(8)), 3.42 (br.s, 4H, H(5)), 8.09 (m, 3H, H­(1,2). ¹³C NMR (D₂O, 400 MHz): 37.03 (C-5), 54.13 (C-6), 55.44 (C-8), 56.68 (C-7), 124.26 (C-2), 140.33 (C-1), 147.52 (C-3), 165.12 (C-4), 177.89 (C-9). MS (ESI), m/z: calcd for C₁₇H₂₅N₇O₄ + H⁺: 392.2 [M + H]⁺; found: 392.1; calcd for C₁₇H₂₅N₇O₄ + Na⁺: 414.2 [M + Na]⁺; found: 414.1; Anal. Calcd for C₁₇H₂₅N₇O₄ ·0.5H₂O: C, 50.99; H, 6.54; N, 24.49. Found: C, 50.88; H, 6.36; N, 23.40.

2-[(2-{[6-({2-[Bis­(carbamoylmethyl)­amino]­ethyl}­carbamoyl)­pyridin-2- yl]­formamido}­ethyl)­(carbamoylmethyl)­amino]­acetamide (aPytam)

The compound was obtained analogously to Py2am using the compound 4 (166 mg, 0.661 mmol), 2-bromoacetamide (382 mg, 2.775 mmol) and K₂CO₃ (584 mg, 4.229 mmol) in MeCN (15 mL). The product was extracted with cold EtOH from the dry residue and recrystallized from methanol and diethyl ether and purified by column chromatography (Al₂O₃, ethyl acetate-MeOH). The product was obtained as a yellow solid (141 mg, 38%); mp 88–90 °C. ¹H NMR (D₂O, 400 MHz): 2.82 (t, 4H, H(6), J = 6.1), 3.33 (s, 8H, H­(7,9)), 3.51 (t, 4H, H(5), J = 6.1), 8.17 (m, 3H, H­(1,2)). ¹³C NMR (D₂O, 400 MHz): 37.51 (C-5), 53.59 (C-6), 57.42 (C-7, C-9), 124.86 (C-2), 139.88 (C-1), 147.79 (C-3), 165.56 (C-4), 176.56 (C-8, C-10). MS (ESI), m/z: calcd for C₁₉H₂₉N₉O₆ + H⁺: 480.2; found: 480.3; calcd for C₁₉H₂₉N₉O₆ + Na⁺: 502.2 [M + Na]⁺; found: 502.3; Anal. Calcd for C₁₉H₂₉N₉O₆ · 4.5H₂O: C, 40.71; H, 6.83; N, 22.49. Found: C, 40.87; H, 6.69; N, 22.60.

Mass Spectrometric Analysis of Complexes

The samples of the Pb²⁺ complexes for MALDI-TOF-experiments were prepared by mixing the solution of corresponding ligand (1 mM, 50 μL) in H₂O and the solution of Pb­(ClO₄)₂ (1 mM, 100 μL) in H₂O. Then a small amount of each sample was mixed with a solution of 10-fold excess of α-cyanohydroxycinnamic acid matrix in a mixture of MeCN and H₂O (2.3/1 v/v). A 1.5 μL portion of the resulting solution was pipetted onto a stainless steel MALDI plate, and was dried at room temperature for a few minutes to evaporate the solvent. The sample on the dried plate was then analyzed by MALDI-TOF MS.

Potentiometric Titration

Potentiometric titration experiments were performed using 808 Titrando autotitrator (Metrohm) equipped with a 10 mL autoburet, a combination glass-body pH electrode (model HI 1330B, Hanna Instruments), and a water-jacketed titration vessel maintained at 25.0 ± 0.1 °C with a circulation thermostat (model 12108-15, Cole-Parmer). Argon flow through the titration vessel was used in all experiments. A carbonate-free NaOH titrant solution (approximately 0.1 M) was prepared in deionized water (18.2 Megohm/cm) and standardized by potentiometric titration with 0.1 M HClO₄. 0.1 M HClO₄ was prepared by diluting a 70% aqueous solution and standardized with Na₂B₂O₇ titrant solution prepared from fixanal. The combination pH electrode was calibrated as a hydrogen-ion concentration probe by titration of standardized HClO₄ with standardized NaOH solution and determining the equivalent point by the Gran’s method using the GLEE program, which gives the standard electrode potential, E ₀, the slope of electrode function, S, and protolytic impurity level. The ionic product of water pKw = 13.78 at 25.0 °C for 0.1 M KNO₃ was kept constant. Ligand stock solutions (0.01 M) were prepared in deionized water. The stock solution of Pb­(NO₃)₂ was prepared in deionized water. To determine the protonation constants, the 16 mL of about 1 mM solution of the ligand and 5–9 mM HClO₄ were titrated potentiometrically with NaOH, using 0.1 M KNO₃ as background electrolyte. The potentiometric titrations of the same solutions but in the presence of Pb­(NO₃)₂ were performed to determine the stability constants of Pb²⁺ complexes of the ligands. In all cases the titration solutions were purged with argon throughout the experiment, stirred with magnetic stirrer, and thermostated at 25.0 ± 0.1 °C. After each titrant addition, equilibrium was considered established if the potential change was <0.5 mV/min. Data were collected over the pH range of 2–12. The number of experimental points used to calculate the constants varied from 55 to 130. To calculate protonation constants of ligands and stability constants of complexes HYPERQUAD2013 program was used. The hydrolysis constants of the aqueous Pb²⁺ ion included in the calculations were taken from the HYDRA database (Table S1).

X-ray Crystallographic Data and Refinement Details

Caution! Perchlorate salts are considered potentially explosive and should be handled with care. Suitable crystals of Py2am for X-ray diffraction studies were grown from an aqueous solution by slow evaporation method at room temperature. Crystals of the complexes [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ were obtained as follows. A solution of Pb­(ClO₄)₂ · 6H₂O (33 mg, 45 μmol) in H₂O (1 mL) was added to a solution of the corresponding ligand (∼15 μmol) in H₂O (2 mL). The mixture was kept at room temperature for slow evaporation until single crystals suitable for X-ray diffraction studies were formed. Data collection for sample Py2am was performed on a Bruker SMART APEX II diffractometer, equipped with Photon-II area-detector (graphite monochromator, φ- and ω-scans), and for samples [Pb­(Py2am)]²⁺ and [Pb­(Py3am)]²⁺ on a Bruker Quest D8 diffractometer equipped with a Photon-III area-detector (graphite monochromator, shutterless φ- and ω-scan technique), using Mo Kα-radiation (λ = 0.71073 Å). The intensity data were integrated by the Bruker SAINT software package and were corrected for absorption by the multiscan method implemented in the SADABS program. The structures were solved using the SHELXT program and refined by the full-matrix least-squares technique against F ² _hkl in anisotropic approximation for non-hydrogen atoms with the SHELXL program. Hydrogen atoms connected to heteroatoms were found from difference Fourier synthesis and refined isotropically without constrains in Py2am and in the riding model for water molecules in [Pb­(Py2am)]²⁺ as well as for all atoms in [Pb­(Py3am)]²⁺. Hydrogen atoms connected to carbon atoms were placed in calculated positions and refined in the riding model in all structures. In the riding model the U_iso(H) parameters were set to be 1.2 U_eq(C, O, N) of the corresponding connected atom. The water molecule in [Pb­(Py2am)]²⁺ was found to be disordered by two positions with refined relative occupancies 0.73:0.27. SHELXTL and Mercury programs were employed for visualization.

Detailed crystallographic information is given in Table S2. CCDC 2447461–2447463 contain supplementary crystallographic data for this paper. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/structures.

NMR Study

The samples of the Pb²⁺ complexes for ¹H NMR measurements were prepared in situ by dissolving the ligand and 1.2 eq. Pb­(NO₃)₂ in D₂O (600 μL), followed by adjustment of the desired pH with small volumes of concentrated HClO₄ or NaOH. The concentration of ligands for Pb²⁺ complexes was about 20–45 mM. Accurate pH measurements in D₂O were obtained by direct reading in a D₂O solution using a combined glass/AgCl electrode after appropriate calibration procedures using standard buffers. The signals were assigned using two-dimensional spectra of ¹H–¹H COSY and ¹H–¹H NOESY or ROESY.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c10550.

• NMR, mass spectra, potentiometric curves, and X-ray crystallographic data (PDF)

• Py2am (CIF)

• [Pb­(Py2am)]²⁺ (CIF)

• [Pb­(Py3am)]²⁺ (CIF)

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.