Abietic, maleopimaric and quinopimaric dipeptide Ugi-4CR derivatives and their potency against influenza A and SARS-CoV-2

Abstract A set of 12 abietane diterpene derivatives have been synthesised by the Ugi-four component reaction (Ugi-4CR) and tested for cytotoxicity and activity against influenza virus A/Puerto Rico/8/34 (H1N1) and SARS-CoV-2 pseudovirus. Five dipeptide derivatives demonstrated a selectivity index (SI) higher than 10 and IC50 values from 2 to 32 μM against influenza virus. Compound 11 was found to be a lead with SI of 200, and time-of-addition experiments showed the viral entry into the cell and the binding of the virus to the receptor as a possible target. Compound 7 was the only one showed weak anti-SARS-CoV-2 activity with EC50 value of 80.96 µM. Taken together, our data suggest the potency of diterpene acids-Ugi products as new effective anti-influenza compounds. Graphical Abstract


Introduction
Multicomponent reactions (MCRs) continue to attract the attention of researchers engaged in the targeted synthesis of substances with potential biological activity and high bioavailability, due to the simplicity of their practical implementation and a wide range of variation in the structure of the reaction products (D€ omling et al. 2012). The Ugi reaction, as the most striking example of such MCRs, is the efficient condensation of a carbonyl compound, a primary or secondary amine, an isocyanide, and a carboxylic acid in the presence of an acid catalyst to form a dipeptoid structure (D€ omling 2006). The similarity of the Ugi reaction products with a variety of endogenous peptide ligands of cell receptors (Fouad et al. 2020) is largely the reason that a wide spectrum of biological activity has been described for many products obtained by this reaction, mainly as modulators of receptors associated with G-protein (G-protein coupled receptors, GPCRs) (Chen et al. 2000).
Thus, this approach is used in the one-step synthesis of a-aminoamides and imides Wang et al. 2017), cyclic imines (Nazeri et al. 2020), heteroaromatic (Abdelraheem et al. 2017) and heterocyclic (Farhid et al. 2021) systems with various pharmacological properties, including antitumor, psychotropic, anticonvulsant, anti-tuberculosis, antimicrobial, and also included in clinically used drugs in the treatment of various diseases (Paton 2002). Among the natural compounds involved in enantioselective synthesis using the multicomponent Ugi reaction, the alkaloid quinine should be noted, on the basis of which chiral spiropyrazolones and spirooxindoles were obtained (Nunes et al. 2020). Peptides obtained by the Ugi reaction based on the ecdysteroid 6-amino-20-hydroxyecdysone exhibit cytotoxic activity against leukemia T cells (Silvani et al. 2016).
Despite the availability of multicomponent Ugi and Passerini reactions, which allow one stage to synthesise complex organic molecules, as well as a wide range of biological activity of MCR products, systematic studies of terpene derivatives as potential components in these reactions have not been carried out. There are few literature data on diand triterpene molecules obtained using the multicomponent Ugi and Passerini reactions. Thus, the library of betulinic, fusidic and cholic acids -TEMPO radical conjugates with potent cytotoxic effects have been prepared using Ugi-MCR protocol (Sultani et al. 2021). The set of Ugi and Passerini products was synthesised starting from triterpenic oleanolic and maslinic acids and biological evaluation of the novel a-acylaminocarboxamides and the a-acyloxycarboxamides to determine their strong cytotoxic potential ). The glycyrrhetinic acid was modified by Ugi-4CR reactions (Czollner et al. 2009) and several lupane triterpenoids were subject of Passerini reactions (Gurrapu et al. 2012) but no biological evaluations of the products have been reported.
The number of references covering Ugi-MCRs and diterpenes is restricted to two examples Veena et al. 2022). The non-toxic Ugi MCR products were synthesised on the basis of labdan diterpenoid coronarin D and were able to reduce the LPS stimulated NO production in RAW cells similar to that of the standard anti-inflammatory drug dexamethasone (Veena et al. 2022). The dehydroabietylamine-Ugi-4CR derivatives exhibited cytotoxic activity against some human tumor cells and nonmalignant mouse fibroblasts .
Our own research was focused to modify abietane diterpene acids by MCR to increase the antiviral activity of the parent compounds. Here we present the Ugi reaction using abietic acid and levopimaric acid diene adducts as the acidic component and assessment of the antiviral activity of the target compounds against influenza virus A (H1N1) and SARS-CoV-2 pseudovirus.
The complete identification of the structure of compounds 4-15 was made by the means of one-and two-dimensional NMR spectroscopy and mass spectra. In the 13 C NMR spectra, the CON-and CONH-carbon atom signals appeared at the d 178.07 À 183.87 ppm and d 166.31-172.67 ppm respectively, while the signals of the dipeptide chain terminal amino acid keto groups at the d 160.11 À 171.30 ppm were observed. The 1 H NMR spectra of compounds 4-15 NH atom signals as broadened signals at d 8.22-9.72 ppm were contained. The aromatic proton signals of the 2,6-Scheme 1. Reagents and conditions: Ugi 4CR: n-butylamine for 4, 8, 12; glycine methyl ester hydrochloride for 5, 9, 13; L-phenylalanine methyl ester hydrochloride for 6, 10, 14; L-tyrosine methyl ester hydrochloride for 7, 11, 15; 2,6-dimethoxyphenylisocyanide, paraform, MeOH, rt, 5-7 days. dimethylphenyl substituent and L-phenylalanine (compounds 6, 10, 14) and L-tyrosine (compounds 7, 11, 15) proton atom signals resonated at the d 6.64-7.32 ppm. The characteristic signals of the amino acid methyl group protons and n-butyl methyl atom protons were appeared as singlets at the d 3.78-3.85 ppm and d 1.01-1.39 ppm, respectively. The mass spectra of 4-15 exhibited molecular ions peaks corresponding to molecular masses of the compounds (see Supplemental Materials).

Antiviral activity against influenza virus A
The prepared compounds 4-15 were studied as potential antiviral agents against influenza virus A/Puerto Rico/8/34 (H1N1). Oseltamivir carboxylate was used as reference compound. The results of in vitro analysis of their cytotoxic and antiviral properties are summarised in Table 1. For evaluation of therapeutic potential of novel compounds, the relation between activity and toxicity is important issue in addition to their absolute values. Based on the values of CC 50 's and IC 50 's, selectivity index (SI) was calculated for each compound. The most active compounds should be selected, the SIs of which exceed 10 (Smee et al. 2017).
In the present study 12 compounds were tested, among them 5 (41.7%) were considered as prospective with selectivity index (SI) higher than 10 and IC 50 values from 2 to 32 lM. Thus, abietic acid derivatives 4, 7 bearing butylamine and L-tyrosine fragments do not have any identified antiviral activity. The dihydroquinopimaric acid analogue with L-phenylalanine 10, as well as the entire set of maleopimaric acid Ugi products 12-15, showed a weak activity with IC 50 from 65.3 to 18.7 lM and relatively high toxicity (CC 50 ¼ 140-30 lM). At the same time, abietic acid L-phenylalanine derivative 6 and dihydroquinopimaric acid butylamine derivative 8 had low toxicity, but exhibited moderate antiviral activity with a SI of 10.1 and 34.32, respectively. On the contrary, abietic and dihydroquinopimaric acids derivatives with glycine residue 8, 9 showed pronounced antiviral activity, but were more toxic, especially compound 9. Among the diterpenic acids-Ugi derivatives, compound 11 with the L-tyrosine fragment was the most efficacious against influenza virus (IC 50 3.5 lM), which is 1.8 times higher than the activity of the reference drug. Dihydroquinopimaric acid derivative 11 exhibited potent viral inhibitory activity together with low toxicity, which indicates a high therapeutic index (SI ¼ 200).

Time-of-addition experiments
To determine the possible target for virus-inhibiting activity of the most active compound 11 in the virus life cycle, time-of-addition experiments were performed ( Figure  S13, Supplementary Materials). The greatest activity was established when compound was added at time point 0-10 and lessat time point (-1)-10. These results indicate that a possible target for compound 11 may be the viral entry into the cell and the binding of the virus to the receptor. Thus, a possible target of the compounds may be HA-protein. Also, it is impossible to exclude some cellular proteins, especially sialic acid.

Primary screening against SARS-CoV-2 pseudovirus
The primary anti-SARS-CoV-2 pseudovirus activity was evaluated in BHK-21-hACE2 cells. As shown in Table 2, compounds 4, 6 and 15 showed certain cytotoxicity to BHK-21-hACE2 cells with cell viability about 50% at the concentration of 20 mM and were not evaluated for inhibition of virus replication. It is worth mentioning that compound 11 only also showed weak anti-SARS-CoV-2 pseudovirus at the same concentration (46.2%), which unlike its strong anti-influenza activity as described above (IC 50 3.5 lM).
Interesting, that compounds 4 and 15 showed similar cytotoxicity to MDCK cells as described in Table 1. Amodiaquine, an effective inhibitor of viral entry of SARS-CoV-2 (Si et al. 2021), was utilised as positive control. Compared with amodiaquine (EC 50 3.17 mM), only compound 7 displayed weak antiviral activity against SARS-CoV-2 pseudovirus with EC 50 value of 80.96 mM ( Figure S14, Supplementary Materials) and SI over 2.5. These results indicated that these Ugi reaction products with abietic, maleopimaric, and dihydroquinopimaric acids may be not good lead compounds for anti-SARS-CoV-2 inhibitors. Our previous study suggests that the high conservation of HR2 might be the potential target of hydrophobic terpenoid fusion inhibitors in variety of viruses (Si et al. 2018). More research is needed in this area to search for high potent anti-SARS-CoV-2 inhibitors.

Experimental
All experimental procedures are described in the supplementary materials.

Conclusion
In general, we have demonstrated that antiviral activity of abietic, maleopimaric and dihydroquinopimaric acids against influenza A can be improved by modification of carboxylic group by the multicomponent Ugi reaction. Diterpenic dipeptides 5, 6, 8, 9, and 11 possessed the pronounced anti-influenza activity. Modification dihydroquinopimaric acid with the L-tyrosine fragment appeared to be the most effective for inhibiting virus reproduction. At the same time, compounds demonstrated a weak antiviral activity against the SARS-CoV-2 pseudovirus, which is apparently associated with a different mechanism of virus-inhibiting action. Thus, further optimisation of the structure of diterpene acids by varying the components according to the Ugi reaction can afford new effective anti-influenza compounds with better antiviral activity profiles and lower IC 50 value. The study here presented should provide valuable information for further understanding of the diterpene structure relationship and its antiviral activity, as well as pave the way for longer and in depth-investigations on the activity of abietane diterpenoids in preventing influenza infection in vivo.

Disclosure statement
No potential conflict of interest was reported by the authors.