Synthesis and Inhibition of Influenza H1N1 Virus by Indolo-Glycyrrhetic Acid Cyanoesters

Abstract

Two new indolo-glycyrrhetic acid derivatives containing cyano-substitutent at C30 have been synthesized, and their anti-influenza activity has been evaluated in vitro. The resulting data demonstrated moderate inhibitory activity against the H1N1 virus with IC₅₀ 29 and 23 μM, respectively. The value of SI 18 and 21 confirm low toxicity and potential of new compounds for following research and development of new biological agents.

1. Introduction

As the pathogen that caused the first influenza virus pandemic in this century, the influenza A (H1N1) virus has caused continuous harm to human public health. At present, amantadine, rimantadine, oseltamivir and zanamivir are the main antiviral drugs used to suppress influenza viruses. However, it has been reported that the influenza A virus’s drug resistance to oseltamivir is gradually increasing [1].

Hence, the development of new antiviral compounds is desperately needed, so a lot of articles have been dedicated to the synthesis of this type of drug [2,3,4]. Among them, much attention has been paid to medicinal plants, which exhibit antiviral activities and therapeutic effects. Extensive research has demonstrated that natural oleanane-type pentacyclic triterpenes, for example, 3β-hydroxy-11-oxo-olean-12(13)-en-30-oic acid (GA), possess several beneficial pharmacological activities including anti-viral, anti-cancer, anti-inflammatory, anti-microbial, antifungal and hepatoprotective properties [5]. Moreover, different methods of modification of GA have been widely used to synthesize many derivatives for many pharmacological applications. [6,7].

Thus, the oxidized analog of GA (3-oxo-GA) has been shown to possess the most potent anti-HCV activity with an IC₅₀ value of 0.79 μM, which is nearly 200 times more than that of GA (IC₅₀ 159.4 μM) [8].

GA derivatives bearing substituted indoles or N-phenylpyrazoles fused to their A-ring showed a 50% inhibitory concentration of PTP1B (protein-tyrosine phosphatase 1B enzyme) in a range of 2.5 to 10.1 µM. For example, the GA derivative containing a trifluoromethyl group in the 5′ carbon atom of the indole cycle exhibited non-competitive inhibition of PTP1B as well as higher potency (IC₅₀ = 2.5 µM) [9]. An analog of GA with ortho-methoxybenzyl pyrozole in the A-ring showed a strong anti-inflammatory effect and high affinity for HMGB1 with a K_d value of 12.5 μM [10] (Figure 1).

A series of amide derivatives of GA were investigated for their antiviral activity [11,12]. Thus, a compound containing a propanol-2 fragment at C30 was active (EC₅₀ 4.95 µM, SI 38.38) against TK+ and TK-strains of Herpes simplex virus type 1 (HSV-1) and sensitive and resistant to acyclovir (ACV) treatment [11], whereas an amide containing an aminopyridine moiety had a strong inhibitory activity on ZIKV-induced CPE and viral protein translation in infected cells (IC₅₀ 0.13 μM, TI > 384) [12].

On the other hand, cyano-fragment also has a positive influence on the biological profile of the molecule. Thus, methyl soloxolone (analogous to CDDO-OMe) contains a cyano group at the C2 position and has high antitumor activity, including against multidrug-resistant cell lines [13].

In the present study, two new indolo-GA derivatives with cyano-substitution at C30 were synthesized and their cytotoxicity and anti-influenza properties were evaluated.

2. Results and Discussion

2.1. Chemistry

The compounds were synthesized, as outlined in Scheme 1. At first, 2,3-indolo-derivative 1 was prepared by oxidation of starting 3-hydroxo-glycyrrhetinic acid followed by treatment with PhNHNH2 (yield 89%) [9]. Then compound 1 was treated with chloroacetonitrile or 4-bromobutyronitrile to afford 2 and 3 (yield 90% and 92%, respectively). The structure of the newly synthesized compounds was confirmed by ¹H- and ¹³C-NMR spectroscopy data. The signal of the carbon atom of the amide bond was observed at δ 176 ppm (¹³C), the CN-group at δ 114 (for 2) and 118 (for 3) ppm. (See Supplementary Material).

2.2. Biological Assay

Cytotoxicity and anti-influenza properties of the derivatives 2 and 3 were studied in MDCK cell culture against influenza virus A/PuertoRico/8/34 (H1N1). Compounds at a range of concentrations (3.7–300 µg/mL) were tested followed by the calculation of their CC50′s and IC50′s, converting these values from µg/mL to micromoles and the calculation of selectivity indices. Oseltamivir carboxylate was used as a reference compound. As can be seen in

Table 1. Cytotoxicity and anti-influenza data of the derivatives 2 and 3.

Compound	CC50, μM a	IC50, μM b	SI c
2	>517	29 ± 3	>18
3	>493	23 ± 2	>21
Rimantadine	344 ± 19	61 ± 8	>6
Oseltamivir	>200	0.3 ± 0.01	>667

^a CC₅₀—50% cytotoxic concentration; ^b IC₅₀–50% inhibitory concentration; ^c SI—Selectivity index is defined as: SI CC₅₀/IC₅₀.

, the study of cytotoxic activity demonstrated a complete absence of cytotoxicity activity even at the highest concentration used (300 µg/mL). At the same time, cyano-ester derivatives 2 and 3 exhibited moderate inhibitory activity against the H1N1 virus with IC₅₀ 29 and 23 μM, respectively. The values of SI 18 and 21 were moderate, indicating low toxicity and the potential for new compounds with further modifications to obtain more active and non-toxic compounds.

Derivatives of triterpenes were previously shown to be effective in the inhibition of influenza virus reproduction. Indeed, they were demonstrated to bind to viral hemagglutinin, thus disrupting the interaction of HA with the cellular receptor and further penetration of viruses into host cells. In an in vivo model, in addition to direct virus-inhibiting activity, these compounds exert anti-inflammatory effects as well as several indirect effects, including reduced activation of NF-kB, JNK and p38, redox-sensitive signaling events that are known to be relevant for influenza A virus replication [14]. Further studies, therefore, should decipher the exact targets and mechanisms of the anti-viral activity of lead compounds identified in our work and determine the range of their virus-inhibiting effects.

The nature of triterpene scaffold modification can play two roles. First, these modifications can be considered as having an indirect effect, i.e., facilitation of membrane penetration or prolongation of the half-life of a compound. Otherwise, they may exert direct and specific inhibiting functions in binding to viral and/or cellular targets. Further studies should be devoted to these issues.

3. Materials and Methods

The spectra were recorded at the Center for the Collective Use ‘Chemistry’ of the Ufa Institute of Chemistry of the UFRC RAS and RCCU “Agidel” of the UFRC RAS. ¹H- and ¹³C-NMR spectra were recorded on a “Bruker Avance-III” (Bruker, Billerica, MA, USA, 500 and 125.5 MHz, respectively, δ, ppm, Hz) in CDCl₃, internal standard—tetramethylsilane. Mass spectra were obtained on a liquid high-resolution chromatograph–mass spectrometer Agilent LC/Q-TOF 6530 (Santa Clara, CA, USA). Melting points were detected on a microtable «Rapido PHMK05» (Nagema, Dresden, Germany). Optical rotations were measured on a polarimeter Perkin–Elmer 241 MC (PerkinElmer, Waltham, MA, USA) in a tube length of 1 dm. Elemental analysis was performed on a Euro EA-3000 CHNS analyzer (Eurovector, Milan, Italy), the main standard is acetanilide. Thin-layer chromatography analyzes were performed on Sorbfil plates (Sorbpolimer, Krasnodar, Russia), using the solvent system chloroform–ethyl acetate, 40:1. Substances were detected by a 10% solution of sulfuric acid solution with subsequent heating at 100–120 °C for 2–3 min. All chemicals were of reagent grade (Sigma-Aldrich). 2,3-Indolo- glycyrrhetic acid 1 was synthesized by Fisher reactions as described in [9].

3.1. General Method for Synthesis of Compounds 2 and 3

The solution of 1 (500 mg, 1 mmol), K₂CO₃ (135 mg, 0.98 mmol), and chloroacetonitrile (0.08 mL, 1.3 mmol) or 4-bromobutyronitrile (0.13 mL, 1.3 mmol) in DMF (20 mL) was stirred at 60 °C for 2 h. After cooling to room temperature, the mixture was poured into ice-cold water and filtered. The residue was filtered off, washed with water and dried, then purified by column chromatography on SiO₂ using a mixture of petroleum ether—EtOAc (10:1) as eluent.

3.1.1. Cyanomethyl Ester 2,3-Indolo-olean-11-oxo-12(13)-en-30-oat (2)

90%, yellow powder. mp: 227–229 °C, [α]²⁰ _D +7 (c 0.75, CH₂Cl₂). ¹H-NMR (δ, ppm, CDCl₃, 500 MHz): 7.89 (1H, br. s, NH), 7.50–7.03 (4H, m, H_arom), 5.74 (1H, s, H-12), 4.24–3.91 (2H, m, OCH₂), 2.69–1.44 (19H, m, CH, CH₂), 1.42, 1.32, 1.25, 1.22, 1.19, 0.85 (21H, s, 7CH₃). ¹³C-NMR (δ, ppm, CDCl₃, 125.5 MHz): 199.8 (C-11), 174.9 (C-30), 168.5 (C-13), 140.1 (C-3), 136.2 (C-arom), 128.9 (C-arom), 128.3 (C-12), 120.9 (C-arom), 118.8 (C-arom), 118.5 (C-arom), 114.3 (CN), 110.3 (C-arom), 107.0 (C-2), 60.5, 53.0, 48.3, 48.2, 45.3, 44.2, 43.3, 40.9, 38.0, 37.7, 37.6, 34.0, 32.0, 31.8, 31.0, 28.5, 27.9., 26.5, 26.4, 23.5, 23.3, 18.5, 18.3, 16.0. Anal. Calcd for C₃₈H₄₈N₂O₃: C, 78.58; H, 8.33; N, 4.82. Found: C, 78.66; H, 8.39; N, 4.77. HRMS (ESI-MS) m/z: [M + H]⁺ 581.3752, calcd for C₃₈H₄₈N₂O₃: 580.8130.

3.1.2. 3-Cyanopropyl Ester Ester 2,3-Indolo-olean-11-oxo-12(13)-en-30-oat (3)

92%, yellow powder. mp: 219–221 °C, [α]²⁰ _D +12 (c 0.75, CH₂Cl₂). ¹H-NMR (δ, ppm, CDCl₃, 500 MHz): 7.78 (1H, br. s, NH), 7.41–6.93 (4H, m, H_arom), 5.66 (1H, s, H-12), 4.61–3.84 (2H, m, OCH₂), 2.56 (2H, m, OCH₂CH₂), 2.19–1.32 (21H, m, CH, CH₂), 1.31, 1.21, 1.15, 1.11, 1.18, 0.75 (21H, s, 7CH₃). ¹³C-NMR (δ, ppm, CDCl₃, 125.5 MHz): 200.0 (C-11), 176.2 (C-30), 169.2 (C-13), 140.2 (C-3), 136.2 (C-arom), 128.9 (C-arom), 128.2 (C-12), 121.0 (C-arom), 118.9 (CN), 118.7 (C-arom), 118.5 (C-arom), 110.3 (C-arom), 107.1 (C-2), 62.3 (OCH₂), 60.6, 53.0, 48.5, 45.4, 44.1, 43.5, 43.4, 41.1, 38.1, 37.8, 37.6, 34.1, 32.1, 32.0, 31.1, 28.7, 28.4, 26.6, 26.4, 24.8, 23.5, 23.4, 18.6, 18.4, 16.1, 14.4. Anal. Calcd for C₄₀H₅₂N₂O₃: C, 78.91; H, 8.61; N, 4.60. Found: C, 78.85; H, 8.74; N, 4.72. HRMS (ESI-MS) m/z: [M + H]⁺ 609.4057, calcd for C₄₀H₅₂N₂O₃: 608.8670.

4. Conclusions

Two new glycyrrhetinic acid derivatives with A-fused ring nitrogen-heterocycle- and cyano-substitutes at C30 were synthesized using the consequent modifications (oxidation, Fisher reaction, reaction with chloroacetonitrile or 4-bromobutyronitrile). The in vitro anti-influenza activity showed low toxicity (SI 18 and 21) and moderate inhibitory activity against the H1N1 virus with an IC₅₀ 29 and 23 μM, respectively. The obtained compounds are promising for the synthesis of tetrazolyl derivatives with potential strong biological activity.