Synthesis and study of complexes of the novel Russian antiviral drug Camphecene with pentacyclic triterpenes of licorice 192 D O I: 1 0. 15 82 6/ ch im te ch .2 02 0. 7. 4. 10 Khizrieva S. S., Vetrova E. V., Borisenko S. N., Maksimenko E. V., Borisenko N. I. Chimica Techno Acta. 2020. Vol. 7, no. 4. P. 192–198. ISSN 2409–5613 Synthesis and study of complexes of the novel Russian antiviral drug Camphecene with pentacyclic triterpenes of licorice S. S. Khizrieva*, E. V. Vetrova, S. N. Borisenko, E. V. Maksimenko, N. I. Borisenko Research Institute of Physical and Organic Chemistry, Southern Federal University, 194/2 Stachki Ave., Rostov-on-Don, 344090, Russia *email: hizrieva@sfedu.ru Abstract. For the first time, the complexation of pentacyclic triterpenes of licorice (glycyrrhizic acid (GA) and its aglycone, glycyrrhetinic acid (GLA)) with the novel Russian antiviral drug Camphecene (Camph) was investigated. The complexes obtained at dif- ferent molar ratios were studied using both UV/Vis spectroscopy and mass spectrometry (ESI MS). Formation of the host:guest complexes were registered: GA and GLA molecular complexes (Camph+2GA; Camph+2GLA) with stability constants К = 6.94 · 106 М–2 and К = 2.89 · 106 М–2, respectively. The research results demonstrate a considerable potential of ESI MS as a technique for simple and fast detection of formation of the complexes of GA /GLA and the novel drugs. Keywords: Camphecene; glycyrrhizic acid; glycyrrhetinic acid; antiviral activity; supramo- lecular complexes Received: 30.10.2020. Accepted: 07.12.2020. Published:30.12.2020. © Khizrieva S. S., Vetrova E. V., Borisenko S. N., Maksimenko E. V., Borisenko N. I., 2020 Introduction Influenza is known to be the most common and dangerous respiratory viral infection. It causes annual epidemics and pandemics, leading to significant increases in morbidity and mortality in all regions of the world. In connection with the grow- ing number of cases of viral infections and especially resistant viral strains, it is nec- essary to improve the available therapeu- tic methods, complementing them with the discovery of new antiviral agents. On the other fist, it is widely recognized that the  medical heritage of  plants is  a  valu- able resource for the  treatment of  infec- tious disorders. This indicates a growing interest in antiviral products based on sec- ondary plant metabolites [1–3]. The one of the unique plants used in both folk and traditional medicine is  licorice. Licorice (Glycyrrhiza glabra L.) is one of the oldest and most widely used medicinal plants [4, 5]. The major plant’s metabolite in licorice root is glycyrrhizic acid (GA, 1), pentacy- clic triterpene, consisting of  one residue of  glycyrrhetinic acid (GLA, 2) and two residues of glucuronic acid (Fig. 1). Licorice triterpenes 1 and 2 are attract- ing more and more attention of chemists and pharmacologists due to the wide spec- trum of their biological activity [6–9]. GA and GLA are active against a wide range of viruses, including herpes, corona, alpha, 193 flaviviruses and others [10]. At the same time, GA, GLA and their derivatives ex- hibit a wide spectrum of antiviral activity both in the form of individual substances [1, 10] and in combination with antiviral drugs [11]. GA 1, due to its amphiphilic- ity, can form supramolecular complexes with various hydrophobic molecules, sig- nificantly increasing their solubility. This property was used in the approach of acad. G. A. Tolstikov: to reduce therapeutic doses of drugs and prolong the action [12–13]. In this regard, the presented work considers the possibilities of synthesizing new supra- molecular complexes of GA and GLA with new antiviral drug Camphecene for the de- velopment of low-dose pharmaceutical sub- stances on their basis. The authors consider that these pharmaceutical substances can be used to suppress the multiplication of virus- es in the early stages. Camphecene 3 (Fig. 1), has a broad spectrum of antiviral activity. It is proved to be active against influenza A strains and type B virus [14]. Experimental For the  research we used GA and 18β-GLA from Aldrich. Camphecene was synthesized at  the  Novosibirsk In- stitute of  Organic Chemistry (NIOCH SB RAS) and kindly provided by  Prof. N. F. Salakhutdinov. Solvents from Merck, HPLC/MS qualifications. The complexes were obtained by traditional mixing of so- lutions with different molar ratios of GA, GLA, and Camph in  a  70% aqueous-al- coholic solution. The  founding of  com- plexes of GA and GLA with Camph was recorded by  UV/Vis spectroscopy using a  SPEKS SSP 705 spectrophotometer (190–1100  nm) (manufactured by  CJSC Spectroscopic Systems, Russia). Measure- ments were performed in  a  quartz cell. The  formation of  the  complex was con- sidered by  the  change in  optical density at a constant concentration of Camph (C = 0.5 mM). In order to exclude the contribu- tion of the absorption of triterpenes by GA and GLA, respectively, their absorption spectrum was subtracted from the  total spectrum. The  composition of  supramolecular complexes of GA and GLA with Camph was investigated with direct injection on a  Bruker Daltonics micrOTOF-Q mass spectrometer with electrospray ioniza- RO O COOH H H H 1, 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OOH OH OH OCOOH OOH OH COOH2 : R = H 1 : R = a) GA (1) b) GLA (2) c) Camph (3) Fig. 1. Formulae of a) glycyrrhetinic acid (2), С30Н46О4; b) glycyrrhizic acid (1), C42H62O16; c) Camphecene (3), C12H21NO 194 tion. The micrOTOFcontrolTM2.2 software was used to analyze the results. The mass spectrometer was calibrated using a Fluka Electrospray Calibrant Solution. Detection of negative and positive ions was performed in the range of m/z from 50 to 3000. Results and discussion In  accordance with the  objectives of the work, sets of complexes of licorice triterpenes GA and GLA with an antiviral drug Camphecene at different molar ratios “guest:host” were synthesized. In the first step, complexes of GA with Camph were obtained at molar ratios: 1:1 and 2:1 and studied by UV / Vis spectros- copy and mass spectrometry with elec- trospray ionization. As  demonstrated in Fig. 2, with an increase in the concentra- tion of GA from 0 to 1 mM, a bathochro- mic shift of  the  absorption maximum of  Camph (201 → 212  nm) is  recorded in  the  UV / Vis spectra, and a  decrease in optical density is observed. An increase in the concentration of GA (from 1.5 mM and higher) leads to the dis- appearance of  the  maximum absorption of Camph, which indicates the complete binding of Camph molecules in the pres- ence of GA (Fig. 3). From the  dependence of  the  optical density on the  concentration of  glycyr- rhizic acid (Fig. 3), over the entire range of  increase in  the  concentration of  GA, the value of the maximum optical density of Camph decreases. The study of GLA as a complexing agent demonstrated similarchanges in the UV / Vis spectra of  the  obtained complexes. When the  GLA concentration changes in  the  range from 0.05 to  0.125  mM, a  bathochromic shift of  the  absorption maximum of Camph is recorded (201 → 206 nm). To  calculate the  stability constant of  the  complexes, we used the  Benesi- Hildebrand plot (1) [15]. The  stability constant of the nGA-Camph complex was estimated from the change in the optical density of Camph (λmax1 = 201 nm) at its fixed concentration in solutions in which the GA (or GLA) concentration was var- ied. Eq. (1) allows, within the framework of  one experiment, not only to  estimate the stability constant of the complex (K), Fig. 2. Absorption spectrum of Camphecene (CCamph = 0.5 mM) at different concentrations of GA 195 but also to  determine the  stoichiometry ratio “host:guest” (n) in the complex: D/∆D – 1 = (1/[GA]n) ∙ (1/K), (1) where ∆D = ∆ε∙[СCamph]  — change in the optical density of the solution, аnd К — the constant of stability of the com- plex, determined for the reaction: [ ] [ ] [ ] Camph GA Camph GA Camph GA Camph GA, . n n n n + ‑ = ‑ = ⋅  (2) The  absorption spectrum of  Camph was recorded at a wavelength of 201 nm, while the Camph concentration was con- stant and amounted to 0.5 mM. The ob- tained dependence of the absorption in- tensity of Camphecene (λ = 201 nm) on the concentration of GA is shown in Fig. 4. From the  slope of  the  straight-line D/DD, depending on 1/[GA]2 (Fig.  4), the stability constant of the complex was calculated using Eq. (1). The stability con- stant for the Camph + GA complex is 1/K = 1.44 · 10–7 M2 or K = 6.94 · 106 M–2. Recognizing the  value of  the  binding constant, the change in the Gibbs energy was calculated. Obtained from the binding constant, the change in Gibbs energy ∆G = –38.4 kJ. Based on the obtained negative value, it can be concluded that the reaction proceeds spontaneously during the forma- tion of GA and Camph complexes. Similarly, according to the Eq. (1), the stability con- stant of the complex Camph+GLA was cal- culated, 1/K = 3.46 · 10–7 M2 or K = 2.89 · 106 M–2. Using the value of the binding constant, the change in Gibbs energy was calculated. The change in the Gibbs energy ∆G = –36.2 kJ, which allows us to conclude that the reac- tion proceeds spontaneously during the for- mation of a complex of GLA with Camph. The formation of complexes of Cam- phecene with natural triterpenoids has also been studied using electrospray ionization mass spectrometry (ESI MS). In the mass spectra of complexes of GA and GLA with Camph in the mode of positive ions, both the peaks of GA, GLA, and Camph ions and the peaks of heterocomplexes containing GA and Camph or GLA and Camph are recorded. Thus, in the GLA and Camph complex- es synthesized both at a ratio of the com- ponents of  GLA and Camph in  1:1 and at  a  ratio of  2:1, a  peak with m/z 688.73 was recorded, corresponding to  a  single charged ion of  the  complex GLA and Camph [MGLA + MCamph + Na] +. A typical mass spectrum of the GLA-Camph com- plex is shown in Fig. 5. The presence of peaks of heterocom- plexes containing GA and Camph, GLA and Camph confirm the formation of com- plexes of GA and GLA with Camph, re- spectively. The  results obtained demon- Fig. 3. Dependence of optical density on the concentration of glycyrrhizic acid Fig. 4. Dependence of the absorption intensity of Camphecene (λ = 201 nm) on the concentration of GA 196 strate the  high potential of  triterpenes GA and GLA in the development of new pharmaceutical forms using the example of Camphecene in the form of molecular complexes. Conclusions In  the  presented work, complexes of  pentacyclic triterpenes of  the  licorice (glycyrrhizic acid (GA) and glycyrrhetinic acid (GLA), its aglycone) with the antiviral drug Camphecene (Camph) were synthe- sized and studied. The  complexes obtained at  different molar ratios “host — GA (GLA): guest — Camph”: 1:1 and 2:1 were studied using both UV / Vis spectrophotometry and mass spectrometry with electrospray ionization. Formation of  the  host:guest complexes were registered: GA and GLA molecules form molecular complexes (Camph+2GA; Camph+2GLA) with stability constants K = 6.94 · 106 М–2 and K = 2.89 · 106 М–2, respectively. In the mode of positive ion, the mass spectra of mixture of GA and GLA with Camph showed peaks of  GA, GLA, and Camph ions and the peaks of heterocom- plexes containing GA and Camph, and GLA and Camph, respectively. The  re- search results demonstrated the  consid- erable potential of ESI MS as a technique for simple and fast detection of formation of the complexes of GA /GLA and the nov- el drugs. Acknowledgements This work was supported by the Russian Foundation for Basic Research (RFBR, grant no. 19-33-90211-Aspiranty). References 1. Zarubaev VV, Anikin VB, Smirnov VS. Anti-viral activity of glycyrrhetinic and glycyrrhizic acids. Rus J of Inf and Imm = Infektsiyaiimmunitet. 2016;6(3):199–206. doi:10.15789/2220-7619-20163-199-206 2. Akram M, Tahir IM, Shah SMA, Mahmood Z, Altaf A, Ahmad K, Munir N, Dani- yal M, Nasir S, Mehboob H. Antiviral potential of medicinal plants against HIV, Fig. 5. 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