Synthesis and Screening of Anti-HSV-1 Activity of Thioglucoside Derivatives of Natural Polyhydroxy-1,4-Naphthoquinones

Abstract

Four 1,4-naphthoquinone dithioglucoside derivatives based on natural polyhydroxy-1,4-naphthoquinones were synthesized. These thioglucosides were screened for their antiradical and antiviral activity in vitro. Antiradical activity of tested compounds was determined by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. The anti-herpes simplex virus type 1 (anti-HSV-1) activity of thioglucosides was analyzed by the cytopathic effect inhibition assay and mode of antiviral action was determined by the addition of the tested compounds to uninfected cells, to the virus prior to infection, or to herpes-infected cells. Most effective inhibition of HSV-1 replication was observed at pretreatment of virus by the compounds (direct virucidal effect). The dithioglucoside conjugate with the single β-OH group and lipophilic ethyl substituent in naphthoquinone core showed the greatest antiviral activity.

Keywords

antiradical activity herpes simplex virus (HSV-1)antiviral activity

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen, with a worldwide prevalence rate of approximately 90%.¹ Usually HSV-1 infection is acquired within the first 2 decades of life and is characterized by latent infection in human peripheral nervous system. Approximately in 25% of infected individuals the periodic viral reactivations are followed by such common clinical manifestation as facial herpes, also known as herpes labialis or cold sores. However, HSV-1 can also cause such severe diseases as recurrent keratitis, encephalitis, and systemic disease in neonates and immunocompromised patients.² The most effective drugs for the selective treatment of HSV-1 are acyclovir and its nucleoside analogs targeting the virus DNA polymerase, but their prolonged use for the treatment of the disease can lead to the formation of drug-resistant strains.^3,4

The problem of drug resistance poses a current and future threat that should be addressed through the development of new effective antivirals.^5,6 It is well known that oxidative stress plays an important role in the pathogenesis of viral infections^7,8 promoting virus replication in infected cells, decreasing cell proliferation, and inducing cell apoptosis.⁹

It was established that the sea urchin pigment echinochrome (1) and its mixtures with other antioxidants directly affect the HSV-1 virus particles and improve the antioxidant defense mechanisms in the infected cells.¹⁰ These results demonstrate that echinochrome 1 and related natural hydroxy 1,4-naphthoquinones 2 to 5 (Figure 1) are the promising starting compounds for the development of new antiviral drugs.

Figure 1

Natural hydroxylated 1,4-naphthoquinones.

Echinochrome 1 is the active substance of ophthalmological and cardioprotective drug Gistochrome, known to prevent injury by trapping active oxygen radicals and enhancing mitochondrial biogenesis.^11

-14 In general, biological effects of naphthoquinone core are associated with the generation of reactive oxygen species and modulation of redox signaling radical reactions¹⁵ as prooxidants, antioxidants, or electrophiles due to the formation of covalent bonds with tissue nucleophiles.¹⁶ Unfortunately, poor solubility of naphthoquinones limited their practical application. The quinone solubility could be enhanced by the conjugation of naphthoquinones with nontoxic carbohydrates,^17
-19 providing an access to the novel structures with various types of biological activity.^20

-24

The aim of our study was the synthesis of new thioglucoside derivatives of polyhydroxynaphthoquinones structurally similar to echinochrome and evaluation of their antiradical and anti-HSV-1 effects in vitro.

Conjugation of Natural Naphthoquinones with Thioglucose

Conjugation of natural naphthoquinones 2 to 5 with 1-thioglucose was performed via the reaction of dichloronaphthoquinones 6 to 9 with available 1-thioglucose tetra-O-acetyl derivative 10 at basic condition (Scheme 1). Dichloroquinones 6 to 9 were readily condensed with thioglucose derivative 10 in CH₃CN solution with K₂CO₃ and gave acetylthioglucoside derivatives 11 to 13 in excellent yields of 81% 87%. Subsequent saponification of acetyl derivatives 11 to 13 under MeONa/MeOH treatment (method A) led to the targeted thioglucosides 15 to 17 in good yields of 75% to 82%.²⁵ Dichloroquinone 9 with 2 acidic β-hydroxyl groups in the quinone part did not react with thioglucose 10 under these conditions. Apparently, the ionization of 2 quinonoid hydroxyl groups significantly reduced the activity of chlorine atoms to substitution. The desired dithioglucoside 14 was obtained in 80% yield, when the condensation was carried out in DMSO solution.

Scheme 1

Preparation of thioglucosides of 1,4-naphthoquinones 11 to 18.

Dithioglucoside 14 was deacetylated in acidic HCl/MeOH solution (method B) in order to avoid side reactions associated with the base cleavage of 2,3-dihydroxy-1,4-naphthoquinoid structure. Dithioglucoside 18 was obtained in 80% yield. The structures of new compounds were assigned by NMR, IR spectroscopy, and HR mass spectrometry. The 6,7-dithioglucoside structure of conjugate 14 was evidenced by the disappearance of 2 chlorine atoms in MS and the presence of the signal of β-OH protons in ¹H spectra at 6.81 ppm, together with the signals of the other phenolic α-hydroxyl groups in a weak field at 12.67 ppm. The 1′,2′-trans(β)-configuration of glucosidic bond was confirmed by the value of anomeric proton doublets (J _1′ _,2′ =10 Hz). Spectral data and properties of the other deacetylated glucosides 15 to 17 were previously described in our work.²⁵

Antiradical Activity of Compounds

For the best understanding of the features of structure-activity relationship of synthesized quinone compounds, we evaluated the antiradical activity of diglucosides 15 to 18 in 2,2-diphenyl-1-picrylhydrazyl (DPPH) test against echinochrome and ascorbic acid. The results are presented in Table 1.

Table 1

Antioxidant Activities in DPPH Assay of Thioglucosides 15 to 18 Compared with Echinochrome (Ech) and Ascorbic Acid (VC)^a.

DPPH radical scavenging activity (%)
Compds	Concentration (μM)					IC₅₀ (μM)
Compds	5	10	20	30	40	IC₅₀ (μM)
15	11.8 ± 0.6	15.9 ± 1.5	24.3 ± 1.2	32.5 ± 2.1	40.4 ± 0.9	60.1 ± 3.1*
16	14.3 ± 0.5	23.4 ± 0.9	32.4 ± 1.1	40.1 ± 0.4	45.0 ± 0.8	53.4 ± 2.6*
17	16.5 ± 2.2	28.5 ± 1.9	40.8 ± 1.1	55.7 ± 1.8	66.1 ± 0.6	28.2 ± 1.8*
18	27.4 ± 0.8	37.6 ± 2.1	55.6 ± 0.7	77.6 ± 1.4	–	16.1 ± 2.3*
Ech	51.7 ± 0.1	71.5 ± 0.3	98.0 ± 0.2	–	–	4.7 ± 0.1
VC	10.8 ± 0.2	17.8 ± 0.3	35.9 ± 0.2	56.5 ± 0.6	75.3 ± 0.4	26.1 ± 1.7

DPPH, 2,2-diphenyl-1-picrylhydrazyl.

^aEach value is presented as the mean ± standard deviation (n = 3).

^*Statistically significant differences between echinochrome and other compound (P ≤ 0.05).

Our studies revealed that thioglucosides 15 to 18 were less active than echinochrome 1. Thus, replacing of 2 β-OH groups of the echinochrome core with thioglucoside moieties increases the solubility of naphthoquinone, but leads to a decreasing of antiradical activity (Table 1). This fact can be explained by the negative inductive effect of sulfur atoms and decreasing of the number of β-OH groups responsible for radical scavenging.^13,14 Thioglucoside 18 with 2 β-OH groups showed highest antiradical properties among tested compounds. Thioglucoside 17 bearing the single β-OH group in the quinone nucleus and ethyl substituent resembling echinochrome showed similar antiradical activity to glycoside 18 and vitamin C.

Cytotoxicity and Antiviral Activity of Compounds

Cytotoxicity assay was carried out to determine the concentration range of compounds for the subsequent study of its anti-HSV-1 activity in the nontoxic range for Vero cells. Echinochrome was used as reference compound. Acyclovir was used as standard antiviral for HSV-1. Based on the results of methylthiazolyltetrazolium bromide (MTT) assay the 50% cytotoxic concentrations (CC₅₀) were calculated for all tested compounds (Table 2). The results showed that the conjugation of 1,4-naphthoquinones with thioglucose led to the formation of low toxic substances 15 to 18 that were 1.5 to 2 times less toxic than the reference compound echinochrome 1 (P ≤ 0.05). Further antiviral activity assay was performed on Vero cells at the concentrations of compounds 15 to 18 below 1000 µM.

Table 2

Cytotoxic Effect (CC₅₀), Anti-HSV-1 Activity (IC₅₀), and Selectivity Index (SI) of Thioglucosides 15 to 18, Echinochrome, and Acyclovir.

Compounds	CC₅₀ (μM)	Treatment
		Simultaneous		Virucidal
		IC₅₀ (μM)	SI	IC₅₀ (μM)	SI
15	318.0 ± 63.8*	399.8 ± 88.0*	0.8 ± 0.1	232.5 ± 51.2	1.4 ± 0.2
16	385.0 ± 88.5*	353.5 ± 68.9*	1.1 ± 0.2	367.6 ± 0.09*	1.1 ± 0.2
17	416.2 ± 83.1*	219.0 ± 46.0	1.9 ± 0.3*	86.2 ± 12.9*	4.8 ± 0.7*
18	477.7 ± 100.3*	285.6 ± 59.8	1.7 ± 0.3*	177.2 ± 32.0	2.7 ± 0.5*
Echinochrome	199.6 ± 39.6	246.8 ± 44.5	0.8 ± 0.1	214.7 ± 36.6	0.9 ± 0.2
Acyclovir	2750 ± 550	20.5 ± 4.5	134 ± 25	>200	<15

The results are given as mean ± standard deviation (n = 3).

^*Statistically significant differences between echinochrome and other compounds (P ≤ 0.05).

The HSV-antiviral activity of glucosides 15 to 18 was examined by the inhibition of virus-induced cytopathic effect (CPE) on Vero cells using MTT test. Tested compounds were added at different stages (before, during, and after) of virus infection and the virucidal effect was assayed by pretreatment of virus with glucosides 15 to 18. It was demonstrated that treatment of infected cells and pretreatment of uninfected cells showed no anti-HSV-1 activity. By contrast glucosides 15 to 18 used in the two other modes of treatment (pretreatment of virus and simultaneous treatment of cells with the virus and tested compounds) showed obvious antiviral effect (Table 2). In the case of simultaneous treatment of cells with the HSV-1 virus and thioglucosides15 to 18 (early stage of virus infection), compounds 17 (selectivity index [SI] 1.9) and 18 (SI 1.7) demonstrated the antiviral activity significantly higher than echinochrome (SI 0.8) (P ≤ 0.05). Pretreatment of virus by tested compounds (direct virucidal effect) resulted in more pronounced anti-HSV-1 activity of all compounds, especially for compound 17 (SI 4.8) and 18 (SI 2.7). At that time inhibitory concentration (IC₅₀) of compound 17 was 2 times lower than IC₅₀ of echinochrome, and SI value was 5 times higher in comparison with echinochrome (SI 0.9). By contrast, Acyclovir demonstrated only minor virucidal effect (IC₅₀ >200) against HSV-1, as noted by other researchers.^10,26,27 We can assume that the mechanisms of action of thioglucoside 17 are the direct inactivation of virus particles and partially the inhibition of virus attachment and virus binding. It is well known that natural polyphenols are targeted to the HSV-1 glycoproteins, which interact with cell surface glycosaminoglycans (GAGs).²⁸ This interaction of polyphenols with the virus glycoproteins interferes in the connection of virus with GAG as initial attachment receptors during infection of host cells. We believe that quinone glycosides 15 to 18 can bind some proteins of virus envelope (including glycoproteins), hereby blocking adsorption of virus to cells. It was reported that some naphthoquinones have the similar mechanism of action against, for example, dengue virus (DEN-4 and DEN-2),²⁹ HIV-1,³⁰ and measles virus.³¹ It can be assumed that increasing of quinone core lipophilicity will lead to enhancement of antiviral activity and give new promising antiviral naphthoquinone thioglucosides.

In conclusion, 4 hydroxy- and alkyl dithioglucoside derivatives 15 to 18 based on natural naphthoquinones were synthesized and were screened for antiradical and antiviral activity in vitro. Among tested thioglucosides, compound 17 with a lipophilic ethyl substituent exhibited the most effective virucidal action and high antiradical activity similar to vitamin C. Accordingly, this compound can directly affect virus particles and protect host cells against radical damage induced by HSV-1 virus.

Experimental

General

Melting points (uncorrected) were measured with a Boetius apparatus. IR spectra were recorded on Bruker Vector-22 FT-IR spectrophotometer. ¹H NMR spectra were recorded on Bruker AVANCE-500 and Bruker AVANCE-700 at frequencies 500 and 700 MHz for ¹H spectra and 125 and 176 MHz for ¹³C spectra, respectively. 2D NMR experiments {¹H−¹H} COSY, {¹H−¹³C} HMBC-qs, and {¹H−¹³C} HSQC were used where necessary in assigning NMR spectra. Spin-spin coupling constants (J) were reported in hertz. Chemical shifts were referenced to TMS (δ = 0.00 ppm). ESI high-resolution mass spectra were recorded on an Agilent 651 Q-TOF LC/MS instrument. Silufol UV-VIS TLC plates treated by hydrochloric acid vapor or ammonia (basic plates) were used for analytical TLC. Preparative TLC was performed on silica gel 60 (Merck, 40–63 µm). TLC was developed in system A: hexane-benzene-acetone, 2:1:2 and system B: benzene-ethyl acetate-methanol, 2:1:1.

Reagents

DPPH and ascorbic acid were purchased from Alfa Aesar, Germany. Tetra-O-acetyl-β-D-thioglucose was purchased from Lachema, Czech Republic. MTT was purchased from Sigma, Saint-Louis, MO, USA. Acyclovir, pharmaceutical grade, was purchased from Belmedpreparation, Republic of Belarus. All solvents were purchased from A.O. Ecos-1, Russia, and distilled before use. Starting dichloronaphthoquinones 6 to 9 were synthesized as published in papers.^25,32

Preparation of 14

Dichloronaphthoquinone 9 (100 mg, 0.343 mM) and acetylthioglucose 10 (300 mg, 0.82 mM) were dissolved in DMSO (4 mL), added finely powdered K₂CO₃ 95 mg (0.69 mM) and mixed for 1 hour until complete conversion of quinone 9 with R _f 0.72(A) into a new red compound with R_f 0.33(A). Reaction mixture was acidified by 2N hydrochloric acid, diluted with CHCl₃ (15 mL), organic layer washed with water (2 × 10 mL), dried with Na₂SO₄, evaporated, and the residue subjected to preparative TLC on SiO₂, eluting with system A, yielding 14 as a dark red solid (276 mg, 85%).

6,7-Di(tetra-O-Acetyl-β-D-Glucopyranosyl-1-Thio)-5,8-Dihydroxy-Naphthalene-1,4-Dione (14)

MP: 235°C to 238°C.

IR ν _max/cm^-−1 (CHCl₃): 3438 (OH), 2954, 1756 (CH₃CO), 1691, 1624, 1597 (C = O), 1379, 1310, 1292, 1244, 1194, 1123, 1092, 1043.

¹H NMR (500 MHz, CDCl₃): 2.00 (6H, s, 2 × OAc), 2.01 (6H, s, 2 × OAc), 2.02 (6H, s, 2 × OAc), 2.11 (6H, s, 2 × OAc), 3.64 (2H, ddd, 2 × H-5′, J = 2.5, 4.8, 9.8 Hz), 4.01 (2H, d.d, 2 × H-6′(a), J = 2.5, 12.5 Hz), 4.19 (2H, d.d, 2 × H-6′(b), J = 4.8, 12.5 Hz), 5.11 (2H, m, 2 × H-4′), 5.12 (2H, m, 2 × H-2′), 5.25 (2H, m, 2 × H-3′), 5.40 (2H, d, 2 × H-1′, J = 10.0 Hz), 6.81 (2H, br.s, 2 × β-OH), 12.67 (2H, s, 2 × α-OH).

¹³C NMR (125 MHz, CDCl₃): 20.52 (2 × OCOCH₃), 20.57 (2 × OCOCH₃), 20.59 (2 × OCOCH₃), 20.67 (2 × OCOCH₃), 61.9 (2 × C-6′), 68.2 (2 × C-4′), 71.0 (2 × C-2′), 73,9 (2 × C-3′), 75.9 (2 × C-5′), 83.9 (2 × C-1′), 108.0 (C-9, C-10), 138.1 (2 × C_β-OH), 138.9 (2 × CS), 157.0 (2 × C_α-OH), 169.3 (2 × OCOCH₃), 169.4 (2 × OCOCH₃), 170.2 (2 × OCOCH₃), 170.5 (2 × OCOCH₃), 182.8 (2 × C = O).

HRMS (ESI): m/z [M-H]^- calcd 945.1435 for C₃₈H₄₂O₂₄S₂, found 945.1426.

Preparation of 18

To partially dissolved diacetylthioglucoside 14 (47 mg, 0.05 mM) in dry MeOH (8 mL) under vigorous stirring acetyl chloride (1 mL) was added dropwise. The reaction mixture was closed and stirred for 6 hours at room temperature until the total conversion of acetylthioglucoside 14 with R _f 0.75 (B) into a new violet compound with R _f 0.35 (B). The reaction mixture was evaporated in vacuum with toluene to remove HCl, the residue was dissolved in dry methanol (5 mL), toluene (2 mL) was added, and the solution was slowly evaporated in vacuum until crystal formation. The violet precipitate was filtered, washed with toluene, and dried giving 18 as a dark red solid (24.2 mg, 80%).

6,7-Di(β-D-Glucopyranosyl-1-Thio)-2,3,5,8-Tetrahydroxynaphtha-Lene-1,4-Dione (18)

MP: >360°C.

IR ν _max/cm^-1 (KBr): 3414 (OH), 2920, 1619, 1599 (CO), 1535, 1450, 1382, 1261, 1181, 1098, 1075, 1044, 884, 801.

¹H NMR (500 MHz, DMSO-d ₆): 3.06 (m, 2 H, 2 × H-5′), 3.05 (m, 2 H, 2 × H-4′), 3.11 (m, 2 H, 2 × H-2′), 3.18 (m, 2 H, 2 × H-3′), 3.36 (d.d, 2 H, J = 5.5, 12.0 Hz, 2 × H-6′(a)), 3.49 (d, 2 H, J = 12.0 Hz, 2 × H-6′(b)), 4.21 (br.s, 2 H, 2 × CHOH), 4.93 (br.s, 2 H, 2 × CHOH), 5.09 (br.s, 2 H, 2 × CHOH), 5.31 (d, 2 H, J = 9.2 Hz, 2 × H-1′), 5.34 (br.s, 2 H, 2 × CHOH), 10.52 (br.s, 2 H, 2 × β-OH), 11.31 (s, 2 H, 2 × α-OH).

¹³C NMR (DMSO-d ₆, 125 MHz): 60.8 (2 × C-6′), 69.9 (2 × C-4′), 74.9 (2 × C-2′), 78.2 (2 × C-3′), 81.3 (2 × C-5′), 84.2 (2 × C-1′), 108.0 (C-9, C-10), 138.1 (2 × CS), 141.3 (2 × C_β-OH), 157.9 (2 × C_α-OH), 181.0 (2 × C = O).

HRMS (ESI): m/z [M-H]⁻ calcd 607.0590 for C₂₂H₂₆O₁₆S₂, found 607.0582.

DPPH Radical Scavenging Assay

The determination of DPPH antiradical activity of thioglucosides was established according to the published method³³ with adaptation to colored quinones. Solutions of pure compounds in EtOH at different concentrations (0, 200 µM final concentrations) were prepared and adjusted to 2 mL total volume with 0.7 mL of DPPH-EtOH solution (6 mg/50 mL; 0.1 mM final concentration). The absorbance at 517 nm was determined after 20 minutes, and the percent free radical inhibition was calculated as follows: Inhibition (%) =100 – [(A _reaction – A _compound)/A _control × 100], where A _reaction is the absorbance of the reaction mixture, A _compound is the absorbance at 517 nm of the test compound at test concentrations, and A _control is the absorbance of 0.1 mM DPPH solution. EtOH was used as a blank. A correction for reaction absorbance at 517 nm is necessary due to the absorbance of the compounds in this region. The percentage of inhibition was plotted to obtain the IC₅₀ value. Echinochrome and ascorbic acid were used as positive control standards. The IC₅₀ value denotes the concentration of the compound required to scavenge 50% DPPH free radical.

Virus, Cell Culture, and Dissolution of Tested Compounds

The DNA-containing herpes virus (HSV-1, strain L2) was obtained from the State Collection of Viruses (Moscow, Russia). HSV-1 was grown in African green monkey kidney (Vero) cells using Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL gentamicin, and glutamine. Viral titers were determined by CPE assay and expressed as the 50% tissue culture infectious dose (TCID₅₀/mL). The titer of HSV-1 was 10^6.2 TCID₅₀/mL. Tested compounds 14 to 18 were dissolved in maintenance cell culture medium (DMEM with 1% FBS). Echinochrome was dissolved in DMSO.

Determination of the Cytotoxic Activity by MTT Assay

The cytotoxicity of the tested compounds was estimated by MTT assay in Vero cell lines.³⁴ A monolayer of cells (1 × 10⁴ cells/well) grown in 96-well plates was treated with different concentrations of tested compounds (from 0 to 1000 µM) and cultured at 37°C in a CO₂-incubator for 3 days; untreated cells were used as controls. Cell viability was assayed using МТТ reagent. Optical density (OD) was measured at 540 nm using an ELISA microplate reader (Labsystems Multiskan RC, Vantaa, Finland) with a reference absorbance at 620 nm. The viability of the cells was calculated as (OD_t)/(OD_c) × 100%, where OD_t and OD_c correspond to the absorbance of treated and control cells, respectively. Cytotoxicity was expressed as CC₅₀ of the tested compound that reduced the viability of treated cells by 50% compared with control cells. Experiments were performed in triplicate and repeated 3 times.

Determination of the Antiviral Activity

The anti-HSV-1 activity of tested compounds was evaluated by inhibition of virus-induced CPE using MTT test. Vero cells monolayers on 96-well microplates (1 × 10⁴ cells/well) were infected with 100 TCID₅₀/mL of HSV-1 and tested compounds in the concentrations from 30 to 1000 µM were used for experiments. Untreated cells were used as a cells control, and cells treated only with a virus were used as a virus control. The plates were kept at 37°C in CO₂incubator for 3 days until 80% to 90% CPE was observed in virus control compared with cells control. To investigate the mode of anti-HVS-1 action of tested compounds the following procedures were performed.

Co-treatment of Cells with Virus and Compound

Cell monolayer was simultaneously treated with virus and tested compound at a ratio 1:1 (v/v) in triplicate. After 1 hour adsorption at 37°C, cells were washed and maintenance medium was added followed by incubation for 3 days.

Pretreatment of Virus with Compounds

The virus was mixed with tested compound at a ratio 1:1 (v/v), incubated for 1 hour at 37°C, then applied to monolayer of cells in triplicate. After 1 hour adsorption at 37°C, cells were washed and maintenance medium was added followed by incubation for 3 days.

Pretreatment of Cells with Compounds

Monolayer of cells was pretreated with tested compounds in triplicate and incubated at 37°C for 1 hour. Thereafter, the cells were washed and infected with virus at 37°C for 1 hour. Unabsorbed virus was removed by washing and cells were incubated under maintenance medium for 3 days.

Treatment of Infected Cells

Monolayer of cells was infected with the virus at 37°C for 1 hour, then washed, and treated with tested compounds in triplicate for 3 days.

MTT test was used for assessing the antiviral activity of compounds, and the viral inhibition rate (IR, %) was calculated according to the formula,³⁵ IR = (OD_tv − OD_cv) / (OD_cd − OD_cv) × 100, where OD_tv represents the OD of cells infected with virus and treated with the test compound; OD_cv corresponds to the OD of the untreated virus-infected cells, and OD_cd is OD of control (untreated and noninfected) cells. The concentration of compound that reduced the virus-induced CPE by 50% was determined as IC₅₀. SI was calculated as the ratio of CC₅₀ to IC₅₀. Experiments were repeated 3 times.

Statistical Analysis

CC₅₀ and IC₅₀ were calculated by regression analysis of the dose-response curve. Statistical processing of the data was performed using Statistica 10.0 software. The results are given as mean ± standard deviation. The differences between parameters of control and experimental groups were estimated using the Wilcoxon test and were considered significant at P ≤ 0.05.

Footnotes

Acknowledgments

The authors acknowledge Dr R.S. Popov, Dr V.P. Glazunov, and Dr V.A. Denisenko for MS, IR, and NMR measurements.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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