Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents

Journal Pre-proofs Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents Olga I. Yarovaya, Anastasiya S. Sokolova, Iliya Ya. Mainagashev, Alexandrina S. Volobueva, Khristina Lantseva, Sophia S. Borisevich, Anna A. Shtro, Vladimir V. Zarubaev, Nariman F. Salakhutdinov PII: DOI: Reference:

S0960-894X(19)30708-5 https://doi.org/10.1016/j.bmcl.2019.126745 BMCL 126745

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Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

1 March 2019 30 September 2019 8 October 2019

Please cite this article as: Yarovaya, O.I., Sokolova, A.S., Mainagashev, I.Y., Volobueva, A.S., Lantseva, K., Borisevich, S.S., Shtro, A.A., Zarubaev, V.V., Salakhutdinov, N.F., Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126745

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Synthesis and structure-activity relationships of novel camphecene analogues as anti-influenza agents Оlga I. Yarovayaa,b*, Anastasiya S. Sokolovaa, Iliya Ya. Mainagasheva, Alexandrina S. Volobuevac, Khristina Lantsevac, Sophia S. Borisevichd, Anna A. Shtroe, Vladimir V. Zarubaevc, Nariman F. Salakhutdinova,b aN.N.

Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch, Russian Academy of

Sciences, Lavrentjev Avenue 9, 630090 Novosibirsk, Russia bNovosibirsk cPasteur

State University, Pirogova St. 2, 630090, Novosibirsk, Russia

Institute of Epidemiology and Microbiology, 14 Mira str., 197101 St. Petersburg, Russia

dLaboratory

of Chemical Physics, Ufa Institute of Chemistry, Ufa Federal Research Center, Russian

Academy of Sciences, 71 Octyabrya pr., 450054, Ufa, Russia eDepartment

of Chemotherapy, Influenza Research Institute, 15/17 Prof. Popova St., 197376 St.

Petersburg, Russia Corresponding author:* Dr. Olga I. Yarovaya, PhD, Tel.: +7-383-330-88-70, e-mail: [email protected]

Abstract A chemical library was constructed based on the scaffold of camphecene (2-(E)-((1R,4R)-1,7,7trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol). The modifications included introduction of mono-and bicyclic heterocyclic moieties in place of the terminal hydroxyl group of camphecene. All compounds were tested for cytotoxicity and anti-viral activity against influenza virus A/Puerto 1

Rico/8/34 (H1N1) in MDCK cells. Among 15 tested compounds 11 demonstrated a selectivity index (SI) higher than 10 and IC50 values in the micromolar range. The antiviral activity and toxicity were shown to strongly depend on the nature of the heterocyclic substituent. Compounds 2 and 14 demonstrated the highest virus-inhibiting activity with SIs of 106 and 183, and bearing pyrrolidine and piperidine moieties, correspondingly. Compound 14 was shown to interfere with viral reproduction at early stages of the viral life cycle (0–2 h post-infection). Taken together, our data suggest potential of camphecene derivatives in particular and camphor-based imine derivatives in general as effective anti-influenza compounds. KEYWORDS: Antivirals, Camphor, Imine derivatives, Influenza, Camphecene.

Influenza viruses belong to the Orthomyxoviridae family and include A, B, C, and D types, which differ in host range and pathogenicity. In particular, influenza A viruses infect a wide range of avian and mammalian hosts, while influenza B viruses infect almost exclusively humans. A number of novel virus-based anti-influenza strategies are being developed, which include improving the potency of currently available drugs, discovering new classes of compounds that target different viral proteins, and the application of combination therapy.[1] Among the viral proteins, the following potential targets have been identified: M2 ion channel; neuraminidase (NA); hemagglutinin (HA); RNA-dependent RNA polymerase; nucleoprotein; and nonstructural protein 1 (NS1). Moreover, antiviral strategies can involve host factors that are involved in virus attachment, entry, and release of virus particles. Only two classes of antiviral drugs are currently FDA-approved for clinical use against circulating influenza viruses: two NA inhibitors (oseltamivir, zanamivir) and older drugs such as amantadine and rimantadine that target the M2 ion channel. Resistance is already widespread for M2 blockers and is increasingly recognised for the NA inhibitors.[2] Thus, there is an urgent need for new anti-influenza drugs with novel targets and alternative mechanisms of activity. Camphecene (Fig. 1) is a compound based on (+)-camphor and aminoethanol – 2-(E)((1R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-ylidene-aminoethanol that were developed as effective inhibitors of influenza virus H1N1 with a selectivity index (SI) value of 500.[3] The inhibitory activity of camphecene is based on the inhibition of viral HA and is therefore the most prominent in early stages of virus replication.[4] By fusion inhibition assay, camphecene was shown to decrease the HA activity of influenza A and B viruses. Camphecene activity was further 2

confirmed in experiments with influenza virus-infected mice. We have selected and characterised an influenza virus variant resistant to camphecene. A 160-fold decrease in virus susceptibility was observed after six passages in cells. This was associated with the emergence of a V458L mutation in the HA2 subunit of HA and with a decrease in viral pathogenicity. Molecular modelling predicts that this substitution results in a more stable HA molecule compared to wild-type HA and an altered camphecene-binding site. Therefore, despite the relatively rapid development of resistance, camphecene remains promising as a potential antiviral due to the low pathogenicity of resistant viruses that may arise.[5] Later, we developed and validated a method for the quantification of camphecene in whole rat blood, using dried blood spots and LC-MS/MS[6], and determined the main metabolites of camphecene formed following its oral administration in rats and excretion in the urine.[7] Based on the structure of camphecene, we attempted to identify other clinical candidates with comparable potency.[8] According to our previous analyses of structure-activity relationships (SARs), hydrophobic regions, such as 1,7,7-trimethylbicyclo[2.2.1]heptan, are critical for antiviral activity.[9] The structure-activity analysis also showed that modification of the imino group leads to reduced efficacy in suppression of virus replication and increased toxicity.[10] Thus, for more detailed study of SARs the possible direction of transformation is introduction of a variety of structural fragments by substitution of the hydroxyl group. In addition, we previously showed that introduction of different heterocycles has a beneficial effect on antiviral activity.[11] It has been shown that the addition of saturated N-heterocyclic fragments to the naturally occurring molecule can significantly increase antiviral activity.[12] Many studies have been published on the antiviral activity of compounds containing aromatic N-heterocycles.[13] Therefore, in the present study, as a continuation of our previous work, we introduced heterocyclic moieties into the camphecene skeleton and evaluated the in vitro antiviral activity and cytotoxicity of the target compounds.

3

Direction of modification

Hydrophobic region and imino group are important for antiviral activity

N

NHet

N

S-Het

OH

N

Camphecene CTD50 (mM) 2500 IC50 (mM) 5 SI 500

Figure 1. Design strategy for heterocyclic-containing compounds. Target compounds bearing a heterocyclic moiety were synthesized as shown in Scheme 1. The starting material camphecene was prepared as previously described.[3] Subsequently, camphecene was treated with PBr3 to produce key intermediate 1 that was used immediately in further reactions. Key compounds 2–16 were prepared by nucleophilic substitution of secondary amines or thiols with bromide 1 in the presence of potassium carbonate and 1,8diazabicyclo[5.4.0]undec-7-ene (DBU). The structures of target compounds were confirmed using various spectroscopic methods, including 1H and

13C

NMR (see ESI). As a result of synthetic

modifications of camphecene, we obtained compounds containing the key pharmacophore structural block imine camphor’s and saturated N-heterocycles: pyrrolidine 2, piperidine 3, 4methylpiperidine 3, piperidin-4-ol 5, piperazine 6, N-methyl- 7, and N-ethylpiperazine 8 fragments. We synthesised agents containing five-membered heterocyclic fragments (4,5-dihydrothiazole 9, N-methyl-imidazole 10, and 1,2,4-triazole fragments 11), separated from the imino group by the sulphur atom via an aliphatic linker. We have described compounds containing six-membered aromatic fragments – 4-chlorobenzene 12, pyridine 13, and pyrimidine 14 fragments and compounds with benzothiazole 15 and benzoimidazole 16 aromatic heterocyclic scaffolds. Synthesis of new substances, spectral characteristics and biological methods are described in SI.

4

NHet

N i

ii

OH

N

2-8

Br

N 1

Camphecene

S Het, Ar

N 9-16

N

N

N

N

OH 2

N

S

9

N HN

N

10

5

4 S

S

S

S

3

11

N Cl 12

13

N

N

N H

N

N

6

7

8

N

N

14

S

S

S

S

N N

N

N

S

15

N

HN

16

Scheme 1. (i) PBr3, Et2O, room temperature (r.t.); (ii) appropriate amine or thiol, K2CO3, DBU, CH3CN, r.t. The prepared compounds 2–16 were studied as potential antiviral agents. Adamantane and bornane-based derivatives were used as reference compounds due to the close similarity of their rigid cage fragments to those in the tested compounds. The results of in vitro analysis of their cytotoxic and anti-viral properties are summarised in Table 1. Table 1. Antiviral activity of camphecene-based compounds 2–16 against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells. Compound CTD50a, µM IC50b, μM SIc 2

> 1260

12 ± 2

106

3

> 1141

27 ± 4

43

4

294 ± 14

25 ± 2

12

5

> 1031

NA

1

6

1196 ± 90

37 ± 5

33

7

1079 ± 82

45 ± 6

24

5

8

222 ± 19

101 ± 12

2

9

753 ± 62

33 ± 4

23

10

1126 ± 63

341 ± 46

3

11

197 ± 15

24 ± 3

8

12

170 ± 12

47 ± 6

4

13

503 ± 38

6±1

91

14

> 1025

6±1

183

15

383 ± 22

19 ± 2

20

16

406 ± 29

21 ± 3

20

Rimantadine

335 ± 27

67.0 ± 4.9

5

Amantadine

284 ± 21

64 ± 5

4

Deitiforin

1266 ± 82

209 ± 15

6

Ribavirin

> 2000

25

> 81.0

aCTD , cytotoxic concentration; the concentration resulting 50 bIC , effective concentration; the concentration resulting in 50 cSI, selectivity index, ratio CTC /IC . 50 50

in 50% death of cells. 50% inhibition of virus replication.

It should be noted that direct comparison of the values of SIs for compounds of different structural types, as a rule, does not make sense, since they can have fundamentally different pharmacokinetic properties in vivo. When determining promising compounds for further research, the most active compounds of each structural type should be selected, the SIs of which exceed 10.[14] Among the nitrogen-containing heterocyclic derivatives, compound 2 with the pyrrolidine fragment was the most efficacious against influenza virus (IC50 = 12 μM). Derivative 2 exhibited potent viral inhibitory activity together with low toxicity, which indicates a high therapeutic index (SI = 106). The antiviral activities of compounds with piperidine 3 and 4-methyl-piperidine 4 fragments were almost the same; however, compound 3 was much less toxic. The presence of a hydroxyl group in the piperine ring of compound 5 resulted in a loss of antiviral activity, although the compound remained non-toxic. Substances with piperazine 6 and N-methylperazine 7 fragments showed comparable antiviral activity and exhibited low toxicity. Among compounds containing a five-membered nitrogen-containing moiety linked via a sulphur atom to the natural camphor backbone 9–11, agents 9 and 11 exhibited antiviral activity, although compound 9 was significantly less toxic than 11. The highest activity was exhibited by agents containing pyridine 13 (IC50 = 6 μM) and pyrimidine fragment 14 (IC50 = 6 μM). It should be 6

noted that replacement of the pyridine-2-thiol fragment for the pyrimidine-2-thiol radical in compound 14 significantly reduced toxicity, while antiviral activity remained high. In the series of sulphides bearing benzothiazole 15 and benzoimidazole 16 rings, the observed toxicity and antiviral activity were the same. To determine the suggestive target for virus-inhibiting activity of the most active compound (14) in the virus life cycle, time-of-addition experiments were performed. The results are summarised in Figure 2. As suggested from the data, compound 14 appeared the most effective when added 0–2 h post-infection. Over time, the efficacy of the drug decreased, and starting from 4 h after infection, the infectious activity of the virus did not differ statistically from the control values. Based on these results, it can be assumed that derivative 14 acts in the initial stage of the life cycle of the influenza virus, which involves adsorption of virions on the surface of the target cell and penetration of the virion inside the host cell. At this stage, two viral proteins are essential. First, viral HA allows for attachment of virions to the cell surface and fusion of the viral envelope with the endosomal membrane. Second, the virus-specific proton channel M2 conducts protons into the virion interior, thus providing acidification of the core and allowing dissociation of viral ribonucleoproteins from envelope structures. Further studies will be necessary to clarify the exact mechanism of anti-influenza activity of camphecene-related molecules.

7

Figure 2. Time-of-addition activity of 14 against influenza virus A/Puerto Rico/8/34 (H1N1)pdm09. Influenza virus was absorbed onto MDCK cells for 1 h at +4°C, unbound virions were removed by washing, and the plate was transferred to CO2-incubator (36°C) (t = 0). Compound 14 was added at the indicated time points. The infectious activity of viral progeny was tested by further titration in MDCK cells. hpi, hours post-infection. Based on the results of the biological studies, HA and proton M2 channel may be a biological targets for compounds 2–16. We considered a binding site of HA located the field of proteolysis near

valine

at

position

615

(PDB

code

1RU7

((2004)

Science

303:

1838–1842,

10.1126/science.1093155)). According to previous work,[5] camphecene binds in this active site and is the cause of mutation V615L. The assumption that camphecene analogues are located at the same site seems logical. In the case of M2, we examined the active site located inside the channel (PDB code 2LY0 ((2013) Proc. Natl. Acad. Sci. USA 110: 1315-1320). We performed docking analysis of the most active compounds 2–16 (5, 8 and 10 were excluded as inactive) into the above-described active site and compared the results of the molecular docking and biological tests using a regression model. The dependence of the docking score on the IC50 value was considered. All stages of theoretical calculations (ligand and protein preparation, docking procedure) were carried out using Small-Molecule Drug Discovery Suite 2018-1 (Schrödinger, LLC, New York, NY, 2018). All technical details of calculation were shown in SM. Molecular docking in the binding site of HA demonstrated good correlation between the experimental data and theoretical calculations (figure 3C). The correlation index did not exceed 74%. This value can be considered as evidence that compounds 2–16 may be located in the HA2 subunit near the fusion peptide. The most active compounds 13 and 14 form π-cation stacking interactions with tyrosine at position 619. Additionally, compound 14 forms π-π stacking with phenylalanine at position 638 (figure 3B).

8

Figures 3. Molecular docking of camphecene analogues to influenza virus proteins HA and M2. A,B,D and E locations of compounds 13 (violet molecule) and 14 (green molecule) in the binding sites of HA2 and M2, respectively: π-cation interaction is shown as a green dotted line, π-π stacking in blue and H-bridge is presented as yellow. C and F are correlation between IC50 values and the results of docking of the active compound. The affinity of the compounds 2-16 to the M2 channel is lower than to HA. The values of docking score dispersions are from -4.2 to -7.2 for M2 and from-6.1 to -8.6 for HA. In this case the correlation index is the lowest (40%). Of course, we cannot state for sure that hemagglutinin is the only potential biological target. However, based on the analysis of theoretical and experimental data, we suggest that the antiviral activity of leader compounds is most likely associated with inhibition of HA. The possibility of multi-targeting is, however, not excluded. In summary, we have developed an effective method to synthesise derivatives of the novel antiviral agent camphecene against influenza virus. This method was based on the synthesis of a bromo-derivative of camphecene by reaction of imino-alcohol with phosphorus tribromide and further interaction with sulphur- and nitrogen-containing heterocycles. Minimal toxicity with highest anti-viral activity was demonstrated by compounds bearing a pyrrolidine fragment and compounds with pyridine and pyrimidine heterocycles adjusted via sulphur bridge. The most active compound 14 demonstrated virus inhibition in early stages of the viral cycle (0–2 h postinfection). According to the docking results, the lead compound 14 is located in the active site of the fusion peptide and can affect HA function. Acknowledgments 9

Authors would like to acknowledge the Multi-Access Chemical Service Centre SB RAS for spectral and analytical measurements. This work has been supported by Russian Scientific Foundation grant 15-13-00017. 1

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