2019 Volume 2 Issue 2

^a Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow, 119991 Russia
^b Mendeleev University of Chemical Technology of Russia, Miusskaya pl. 9, Moscow, 125047 Russia
 

Corresponding author:  V. S. Romanova, e-mail: roman@ineos.ac.ru; K. A. Kochetkov, e-mail: const@ineos.ac.ru
Received 2 July 2018; accepted 27 December 2018

Abstract

For a number of years, our group research has been focused on the synthesis of biologically active compounds based on transition metal complexes that are able to catalyze the processes which involve biological substrates under conditions close to physiological ones. Transition metal complexes are known to serve as efficient catalysts for a range of chemical reactions that resemble the processes in living organisms which occur under action of metalloenzymes [1].

According to the concept suggested by M. E. Vol'pin [2], catalytic sources of reactive oxygen species (ROS), which tend to accumulate selectively in tumors, can significantly inhibit the growth of cancer cells. Of particular interest are natural macrocyclic cobalt complexes, for example, vitamin В₁₂ and its derivatives (cobalamins, in particular, vitamin B₁₂ е-carboxylic acid: e-COOH-Cbl-CN 1). Our recent investigations on the catalytic activity of related complexes in oxidation of natural substrates [3], formation of ROS [4], and oxidative cleavage of nucleic acids [5–7] as well as medical and biological assays on anticancer activity of corrin cobalt complexes and their compositions with l-ascorbic acid confirmed that the catalytic systems based on cobalt complexes with corrin ligands and l-ascorbic acid, which generate reactive oxygen species, can be used as efficient anticancer and related agents [8].

Mono(amino acid) and peptide derivatives of fullerene, synthesized for the first time at INEOS RAS [9], appeared to be nontoxic and exhibited a broad spectrum of biological activity. The most promising compounds with anticancer and antimetastatic properties were found to be the compounds that include nitro groups or carnosine residues [10, 11].

We assumed that the introduction of an active form of vitamin В₁₂ into a molecule of a pharmacophoric fullerene derivative would essentially improve the biological activity of the resulting complexes. For this purpose, it is important to show the principal possibility of synthesis of these compounds.

Results and discussion

The first step of our investigations was concerned with e-COOH-Cbl-CN 1 and N-(monohydrofullerenyl)-ε-aminocaproic acid methyl ester HC₆₀NH(CH₂)₅COOMe 2.

It is known that fullerene derivatives have an active hydrogen atom in a С₆₀ core which can be readily substituted for the halogen atoms [12–14]. Therefore, for direct attachment of e-COOH-Cbl-CN 1 to a fullerene core, we firstly converted it to the corresponding chloride upon refluxing with a 1.5-fold excess of thionyl chloride in dimethylformamide for 2 h (Scheme 1). The excess of thionyl chloride was removed under vacuum, and the resulting solution was added to a 1.5-fold excess of compound 2 in pyridine. The reaction mixture was stirred at room temperature for 8 h and dialyzed against water. Product 3, CN-Cbl-C(O)-C₆₀NH(CH₂)₅COOMe, appeared to be soluble in water, therefore, the excess of starting ester 2, which does not dissolve in water, can be readily removed by filtration, whereas unreacted compound 1 moves into a dialysis bag.

Scheme 1. Synthesis of cobalamin–fullerene complex 3.

The second derivative was obtained by the attachment of 1 to 2 after introduction of a spacer, an ethylene unit, which afforded compound 4 with the following formula: CN-Cbl-C(O)OCH₂CH₂-C₆₀NH(CH₂)₅COOMe (Scheme 2). For this purpose, the carboxyl group of compound 1 was firstly modified with 2-chloroethanol. The reaction was carried out in the presence of N,N'-dicyclohexylcarbodiimide (DCC) under stirring at room temperature for 8 h. The resulting solution was added to a solution of a 1.5-fold excess of compound 2 in pyridine. The reaction mixture was stirred at room temperature for 8 h and dialyzed to give compound 4. The latter also appeared to be soluble in water; therefore, its purification was carried out analogously to the above-described procedure.

Scheme 2. Synthesis of cobalamin–fullerene complex 4.

The structures of the compounds obtained were elucidated using IR and UV-vis spectroscopies as well as circular dichroism (CD).

The comparison of the CD spectrum of compound 1 with those of fullerene derivatives 3 and 4 revealed some changes (Figs. 1, 2). Thus, the spectrum of compound 3 display, along with the extreme points typical for starting compound e-COOH-Cbl-CN 1, new bands at 471 and 542 nm, which are characteristic for p–p* electronic transitions of fullerene derivatives [15, 16]. Furthermore, the Cotton effect (CE) changes the sign to a negative one in the short-wave region. The negative value of CE was also observed in the CD spectrum of compound 4 at the wavelength below 400 and above 470 nm. The extreme points at 471 and 542 nm, attributed to p–p* electronic transitions of fullerene derivatives, were also detected in the CD spectrum of 4, but were less expressed than in the spectrum of compound 3. This is likely to be connected with a very strong conjugation between the cobalamin moiety and the fullerene core in compound 3 unlike product 4, where the cobalt complex is attached through the ethylene bridge. At the same time, the band at 430 nm, which refers to vitamin B₁₂ е-carboxylic acid, is well expressed in the spectra of all three compounds. The above-mentioned data confirm the attachment of the vitamin В₁₂ derivative to the fullerene core.

Figure 1. CD spectra of compounds 1 (a) and 3 (b).

Figure 2. CD spectra of compounds 1 (a) and 4 (b).

Figure 3. IR spectra of compounds 1 (a), 3 (b) and 4 (c).

Figure 4. UV-vis spectra of compounds 1 (a), 2 (b), 3 (c) and 4 (d).

Table 1. Characteristic absorption bands in the IR spectra of compounds 1, 3 and 4 (cm^–1)

The IR spectra of compounds 3 and 4 show almost all the absorption bands characteristic for compound 1, except for the band at 1216 cm^–1 which refers to ν(C–O) of the free carboxyl group (Fig. 3, Table 1). Furthermore, the IR spectrum of compound 1 displays strong absorption bands at 3350 cm^–1 (with a shoulder at 3401 cm^–1) and 3195 cm^–1, whereas the IR spectra of compounds 3 and 4 contain only the broad bands at about 3400 cm^–1, which confirms the assignment of the mentioned bands to stretching vibrations of the H-bonded OH groups in compound 1. The IR spectrum of 1 demonstrates a strong absorption band at 1668 cm^–1. The latter is observed also in the IR spectra of products 3 and 4 and can be attributed to the stretching vibrations of the С=О bond in the cobalamin moiety._. The IR spectrum of compound 3 in the range of carbonyl vibrations displays, along with the absorption band at 1668 cm^–1, a weak shoulder at 1719 cm^–1, which can be attributed to the C=O stretching vibrations of the ester group in the fullerene moiety. A difference in the electronic structures of compounds 3 and 4 consists in the mode of attachment of complex 1 to the fullerene core. In the case of compound 3, where complex 1 is directly attached to the fullerene moiety, the carbonyl group is conjugated with the С₆₀ core. As a result, the absorption band of the C=O bond stretching appears to be shifted to the low-frequency region and coincides with ν(С=О) of the cobalamin moiety. This conclusion is in accordance with the appearance of a strong band at 256 nm in the electronic spectrum of compound 3 (Fig. 4). When the complex is attached to the fullerene core through the ethylene unit as in compound 4, there is no conjugation and the ester group does not differ from that in the fullerene moiety. As a result, the IR spectrum shows a strong band at 1719 cm^–1, and the electronic spectrum demonstrates only a weak band at 252 nm.