EP3876927A1 - Strigolactones for use in preventing and/or treating infections caused by viruses of the herpesviridae family - Google Patents

Abstract

The present invention concerns compounds of formula (I) wherein R₁ is a substituted or unsubstituted linear or branched C1-C4 alkyl, and R₂is selected from the group consisting of H, substituted or unsubstituted phenyl, bromine, heteroaryl, thiophene and functionalised thiophene, for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.

Description

"STRIGOLACTONES FOR USE IN PREVENTING AND/OR TREATING INFECTIONS CAUSED BY VIRUSES OF THE HERPESVIRIDAE FAMILY"

Cross-Reference to related applications

This patent application claims priority from Italian patent application no. 102018000010142 filed on 08/11/2018, the entire disclosure of which is incorporated herein by reference .

Technical Field

The present invention concerns strigolactones for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.

State of the Art

Strigolactones (SL) are a class of signalling molecules produced by the roots of the majority of higher plants. They play a dual role, endogenous and exogenous. At endogenous level they represent a new class of vegetable hormones which intervene in regulating the development of the root system and foliage and in general of the plant in relation to the nutritional conditions (deficiency of nutrients, in particular phosphates) . At exogenous level they represent chemical signals that are released in the rhizosphere and are perceived by microorganisms that live in association with the plant, with beneficial effects on the establishment of symbiosis.

More recently strigolactones have been applied in the medical field. Interesting results have been obtained in oncology. In fact, it has been shown that some synthetic derivatives of SL can have an anti-tumour effect in vitro on prostate, colon, lung and breast cancer. See for example EP2758392.

Herpesviruses are viruses with double-strand DNA responsible for infections, including serious infections in humans. Currently eight human Herpesviruses are known: Herpes Simplex virus type 1 (HSV-1), Herpes Simplex virus type 2 (HSV-2), Varicella-Zoster virus (VZV) , Cytomegalovirus (CMV) , human herpesvirus 6, human herpesvirus 7, Epstein-Barr Virus (EBV) , human herpesvirus 8 (or Kaposi's sarcoma-associated herpesvirus, KSHV) .

The antiviral drugs of choice for herpes infections entail the use of nucleoside analogues (for example, ganciclovir) .

However, nucleoside analogues can have significant toxicity for the host. In particular, the use of ganciclovir or similar nucleoside derivatives in children (congenital infections or bone marrow transplant) , due to its high myelotoxicity, can have serious side effects which significantly limit its use.

Furthermore, by interfering with the synthesis phase of the viral DNA, the nucleoside analogues frequently induce the appearance of resistance mutations at gene level which encode for the viral enzymes assigned to synthesis of the DNA.

The object of the present invention is therefore to provide new antiviral compounds for use in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family, in particular HCMV, which overcome the above- mentioned problems.

According to the present invention, said object is achieved by use of the compounds as defined in claim 1 in preventing and/or treating infections caused by viruses of the Herpesviridae family.

Brief description of the figures

For a better understanding of the present invention, it will be described also with reference to the attached figures, which illustrate the following:

- Figure 1A shows the results of an MTT colorimetric assay (3- (4, 5-dimethylthiazol-2-yl ) -2, 5-diphenyltetrazolium bromide; Sigma Aldrich) on human primary fibroblasts isolated from foreskin (HFF) (left-hand panel) and on VERO (right-hand panel) to evaluate the cytotoxicity of the molecules TH-EGO, EDOT, EGO-10, GR24 ;

Figure IB shows graphs of the viral titration of the supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) at the concentration of 12.5 mM and infected with HSV-1/2 (MOI 0.1) (central and right-hand panel respectively) ;

- Figure 2A shows the results of an MTT colorimetric assay on HFF cells (left-hand panel) and on VERO (right-hand panel) to evaluate the cytotoxicity of the molecules TH-ABC, EDOT-ABC;

- Figure 2B shows graphs of the dose-response titration of the viral supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) and infected with HCMV (MOI 0.1) (upper panel) and the dose- response titration of the viral supernatants obtained from cells treated with the different strigolactones or equal volume of solvent (DMSO) and infected with HSV-1 and HSV-2 (MOI 0.1) (lower panels);

- Figure 3A shows the results of attachment assays (left-hand panel) and entry assays (right-hand panel) to evaluate the inhibitory activity of the molecules during the phases of attachment or penetration of the virus (HCMV) vis-a-vis the host cell;

- Figure 3B illustrates a Western blot of infection kinetics (MOI 0.1) 24, 48 and 72 hpi on HFF cells treated with TH-EGO or EDOT at the dose of 12.5 mM;

Figure 4A illustrates the evaluation of the necrosis and cell apoptosis by labelling with propidium iodide (PI) and with annexin V in cells (HFF) treated for 24 and 48 hours with TH-EGO, EDOT, DMSO at the concentration of 12.5 mM in the absence of HCMV (left-hand upper and lower panel) or presence of HCMV (right-hand upper and lower panel);

- Figure 4B illustrates the results of an enzymatic assay to evaluate the activity of the caspase-3 in cells (HFF) treated for 24 hours with TH-EGO, EDOT, DMSO at the concentration of 12.5 mM in the absence or presence of HCMV.

Detailed disclosure of the invention

According to the present invention compounds of formula

wherein Ri is a substituted or unsubstituted linear or branched C1-C4 alkyl, and R₂ is selected from the group consisting of H, substituted or unsubstituted phenyl, bromine, heteroaryl, thiophene and functionalised thiophene, are used in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family.

Ri is preferably CH3.

R2 is preferably in position 7.

R2 is preferably thiophene or ethylenedioxythiophene .

Preferably, the viruses of the Herpesviridae family are Cytomegalovirus, Herpes Simplex virus 1 or Herpes Simplex virus 2, even more preferably Cytomegalovirus.

According to the present invention, pharmaceutical compositions comprising the above-mentioned compounds are furthermore used in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family. Preferably the above-mentioned compositions comprise an isomer of the compounds, a salt or a pharmaceutically permitted mixture, additives and/or excipients. Preferably the above- mentioned compositions comprise at least a cyclodextrin derivative, for example nanosponges or reticulated cyclodextrins .

The above-mentioned compounds or compositions are used preferably in the prevention and/or treatment of infections caused by viruses of the Herpesviridae family in paediatric patients with congenital infections or who have undergone a bone marrow transplant, or in adult patients who have undergone a transplant.

In the following examples it is shown that:

- the treatment with strigolactones , in particular with the synthesis molecules TH-EGO and EDOT illustrated below, immediately before and during the infection with HCMV, HSV-1 and HSV-2, significantly inhibits viral replication;

- the strigolactones do not influence the initial phases of interaction of the virus in the target cells, but act in the later phases of viral replication;

- the molecular mechanism underlying the inhibitory process of the strigolactones appears to be represented by the induction of an apoptotic process in the late stages of the infection .

Examples

Example 1: synthesis and characterization of strigolactone analogues .

The structural analogues of the strigolactones subject of this study are shown below in Table 1. The Table shows: the structure of the strigolactones (SL) (first left-hand column), the abbreviation (central column) and the IUPAC name (left-hand column) . Unless indicated otherwise, the molecules 1-4 were used as racemic mixtures. The molecules indicated as TH-EGO, EDOT, EGO-10 were used as racemic mixtures and were synthesized according to the procedure reported in Prandi et al . (2011), European Journal of Organic Chemistry, 3781-3793.

GR24, also used as a racemic mixture, was purchased from Strigolab srl. TH-ABC and EDOT-ABC were synthesized according to the procedure reported in Prandi et al . (2011), with the exception of the last synthetic passage.

Example 2: the strigolactones inhibit the replication of the human Cytomegalovirus and Herpes Simplex virus type 1 and 2.

To evaluate the antiviral activity of the strigolactones (SL) TH-EGO, EDOT, EGO-10, GR24, the human Cytomegalovirus (HCMV) and the Herpes Simplex virus type 1 (HSV-1) and 2 (HSV-2) were used as study models. The propagation, titration of the virus and the experiments relative to HCMV (Merlin strain) were carried out on human foreskin fibroblasts (HFF) , while the analyses relative to HSV-1 and HSV-2 were carried out on African Green Monkey cells (VERO) . HFF and VERO were grown in DMEM (Dulbecco's Modified Eagle Medium, SIGMA-ALDRICH) , in the presence of 10% fetal bovine serum (FBS), glutamine 2 mM, sodium pyruvate 1 mM, penicillin 100 U/mL and streptomycin sulphate 100 pg/rnL (SIGMA-ALDRICH) .

Firstly, the toxicity of the SL vis-a-vis the HFF and VERO was determined, using the MTT colorimetric test ( 3— ( 4 , 5— dimethylthiazol-2-yl ) -2 , 5-diphenyltetrazolium bromide; SIGMA- ALDRICH) . For this purpose, the cells were exposed to increasing concentrations of molecule or dimethyl sulfoxide (DMSO) carrier for an incubation time of 144 hours (h) . Subsequently, the supernatant was removed, the well was washed with PBS1X (SIGMA-ALDRICH) and the supernatant was incubated for 2 hours at 37 °C and 5% of C02 with DMEM medium with the addition of 500 pg/mL of MTT solution. At the end of the incubation, the supernatant was removed again and the cells solubilised in 300 pi of DMSO. One hundred pi of the above- mentioned solubilised cells were used to measure the absorbance at the wavelength of 570 nm, using the Victor3 multi-plate reader spectrophotometer (Perkin Elmer) . The absorbance value obtained from the sample treated only with the carrier at maximum dose was considered equal to 100% vitality and the other values were related to it (Fig. 1A) .

Subsequently, to determine the antiviral activity of the SL, the HFF cells were infected with HCMV, at a multiplicity of infection (MOI) of 0.1, and the VERO cells with HSV-1 or HSV-2 at MOI of 0.1. The cells were treated one hour prior to the infection and for the entire duration of the infection with the SL (12.5 mM, a non-toxic dose for the cells as can be seen from the cell cytotoxicity test) or with equal volume of DMSO solvent .

Titration of the virus present in the supernatants of the samples treated as described previously was carried out by means of plaque assay. More specifically, the supernatant of the samples under examination and the cells were collected and lysed via three nitrogen/37 °C cycles and subsequently centrifuged at 1500 rpm for 10 min to eliminate the cell debris. The supernatant thus obtained, containing the viral particles, was analysed via plaque assay for determination of the viral titre. For said purpose, HFF or VERO cells, grown in plates with 96 wells in 100 mΐ of DMEM at 10% of FBS, were infected with serial dilutions of the virus and centrifuged at 2000 rpm for 30 minutes (min), to promote adsorption. After 2 h of incubation at 37°C, 5% of CO2, the viral inoculum was removed and replaced with 100 mΐ of methylcellulose 0.8% ( SIGMA-ALDRICH) , 1% FBS. After 144 h in the case of infection with HCMV, characterized by a slower replicative cycle, or 48 h for HSV-1 and HSV-2, which instead have a faster replicative cycle, the methylcellulose was removed and substituted with a solution of crystal violet 0.1% diluted in ethanol 10% (SIGMA- ALDRICH) for 30 min in the dark. The plate was then decolorized under running water and the infection plaques counted under the inverted optical microscope (LEIKA DMIL) . The number of plaques obtained in the sample treated with the carrier at maximum dose was set to 100% (Fig. IB) . From among the SL analysed, TH-EGO and EDOT were the most promising molecules, as they were characterised by an inhibitory activity against HCMV, HSV-1 and HSV-2 greater than EGO-10 and GR24; they were therefore used for the subsequent experiments .

Example 3: the derivatives of TH-EGO and EDOT effectively inhibit the replication of HCMV and HSV-1 and HSV-2.

From among the SL analogues analysed, TH-EGO and EDOT proved to be the compounds that most effectively inhibit the replication of HCMV, HSV-1 and HSV-2.

Therefore, to clarify which functional group from among TH-EGO and EDOT was able to inhibit the viral replication more effectively, the derivatives TH-ABC and EDOT-ABC were synthesized which, compared to TH-EGO and EDOT, respectively, do not have the enol ether bridge bound to the butenolide ring .

Firstly, to determine the cell toxicity of TH-ABC and EDOT-ABC on HFF and VERO, the MTT colorimetric test was used, performed as described in Figure 1A (Fig. 2A) .

Subsequently, to evaluate the antiviral activity of the most promising molecules, the HFF cells were infected with HCMV at MOI of 0.1, and the VERO cells with HSV-1 or HSV-2 at MOI of 0.1. The cells were treated with scalar concentrations of molecule, or with equal volume of DMSO solvent, one hour prior to the infection and for the entire duration of the infection.

The virus was collected and titred from the samples treated 144 h after infection with HCMV and 48 hours after infection with HSV-1 and HSV-2 as described in the previous paragraph (Fig. 2B) . The different SL analysed highlighted a dose- dependent inhibitory activity against HCMV, HSV-1 and HSV-2. The IC50 values (inhibiting concentration necessary to inhibit 50% of the viral replication) of TH-EGO and EDOT are given below .

Example 4: determination of the antiviral activity of the SL on HCMV.

To understand the nature of the antiviral activity of the strigolactones during the viral replication, the effects of the molecules during the various HCMV replication phases in the target cell (HFF) were analysed. To clarify whether the strigolactones act on the HCMV adhesion phase, the HFF cells were cooled beforehand to 4°C, treated with scalar dilutions of SL or DMSO and subsequently infected with HCMV (MOI 0.1) . After an incubation period of 3 h at 4°C, to ensure adhesion but not entry of the virus to the cell, the viral inoculum was removed, substituted with methylcellulose 0.8% and the cells were then placed at 37°C for 72 h. At the end of the incubation, the cells were fixed, coloured with a solution of crystal violet and the viral plaques were counted. The number of plaques obtained in the sample treated with the carrier at maximum dose was set to 100%. From the data obtained, it can be seen that the SL do not significantly inhibit the process of attachment of HCMV to the target cell (Fig. 3A) .

Subsequently, to understand whether the SL acted on the phase of entry of the virus into the cell, the HFF cells were cooled beforehand to 4°C, infected with HCMV (MOI 0.1), kept at 4°C for 3 h and lastly treated with scalar concentrations of SL or DMSO and incubated for 3 h at 37 °C to allow entry of the virus. At the end of the incubation period the cells were treated with acid glycine (100 mM glycine, 150 mM NaCl pH 3) to remove the virus that had not penetrated into the cells. After thorough washing with DMEM to restore the neutral pH, the cells were incubated with methylcellulose 0.8% for 72 h. At the end of the incubation, the cells were fixed, coloured with a solution of crystal violet and the viral plaques were counted. The number of plaques obtained in the sample treated with the carrier at maximum dose was set to 100%. From the data obtained it can be seen that the SL analysed do not significantly alter the process of entry of HCMV into the target cell (Fig. 3A) .

Lastly, to clarify in greater detail which viral replication phase was inhibited by the SL, the effects of the molecules on the levels of expression of viral proteins representative of each stage of the replicative cycle, namely immediate early (IEA), early (UL44) and late (pp28), were analysed. The cell protein a-Tubulin was included as an internal loading control. The HFF cells were infected with HCMV at MOI 0.3. These cultures were treated one hour prior to infection and for the entire duration of the infection with 12.5 mM of TH-EGO or EDOT, and the control cultures with the same volume of DMSO solvent. The cells were then collected at 24, 48 and 72 h from the infection (hpi, "hours post infection") . The cells were lysed with RIPA buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Nonidet P-40; 0.1% SDS; 0.5% deoxycholate ; protease inhibitors) and then quantified with the Bradford method (BIO-RAD) . 30 pg of protein extract were collected, to which an appropriate quantity of Laemmli sample buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 2% b-mercaptoethanol ) was added. The solution thus obtained was heated to 95°C for 5 minutes and separated by means of electrophoresis on polyacrylamide gel (8%) -SDS page. At the end of the process, the gel was analysed using the ChemiDoc (BIORAD) instrument. From the results obtained, it can be seen that the inhibition of the viral replication occurs during the late stage of the infection, since a reduction is found in the expression levels of the proteins UL44 and pp28 (Fig. 3B) .

Example 5: virolysis as an antiviral activity of the SL.

To study the molecular mechanisms underlying the inhibitory process exercised by the SL on the replication of HCMV in further detail, the ability of the SL to induce apoptosis in HFF cells following cytomegalic infection was analysed. For this purpose, the HFF cells were treated with SL (12.5 mM) , one hour prior to infection and for the entire duration of the infection, or with equal volume of DMSO solvent and infected with HCMV (MOI 1) . After 24 and 48 hours the cells were processed by means of Annexin V-FITC Apoptosis Detection Kit (Calbiochem) , a technique which highlights the presence of apoptotic and necrotic cells through cytofluorimetric analysis .

This assay exploits the ability of the annexin protein to bind with the phosphatidylserine (PS) thus producing fluorescence in the green spectral region due to the conjugated fluorophore (FITC, fluorescein isothiocyanate) . PS is a phospholipid which is normally found on the inner side of the cell membrane. During the apoptosis processes, the PS is exposed towards the outside and allows the annexin V to bind with it. Since said bond can occur also during necrosis, following rupture of the plasmatic membrane, the fluorophore propidium iodide is added, a DNA intercalating agent, which intercalates stoichiometrically and emits in the red spectral region if the cell membrane is not intact, thus discriminating between live cells (negative annexin and propidiumn iodide) , apoptotic cells (positive annexin, negative propidium) and necrotic cells (negative annexin, positive propidium) . The cytofluorimetric analysis was performed with the FACSCalibur flow cytometer (BD Biosciences) (excitation at 488 nm, emission 518 nm and 400 nm for FITC and propidium respectively) and the results were analysed with the program ModFit LT software (BD Biosciences) .

A significant increase was found in the percentage of cells in apoptotic and necrotic phase in the samples treated with TH- EGO or EDOT and infected with HCMV for 48 h, compared to the cells treated with the solvent DMSO and the non-infected samples (Fig. 4A) . These data indicate that the SL induce cell death processes in infected cells, which could favour a more rapid elimination of the virus.

To confirm the activation of apoptosis processes by the SL in cells infected by HCMV, the activity of the enzyme caspase-3, the executioner caspase in the apoptotic process, was analysed. For said purpose HFF cells were treated for 24 h with the SL TH-EGO, EDOT, or with the solvent DMSO at a concentration of 12.5 mM, in the absence or presence of HCMV (MOI 1) and subsequently analysed by means of the fluorimetric assay SensoLyte AFC Caspase Sampler Kit Fluorimetric (Anaspec, CA, USA) , which measures the cleavage of a fluorescent substrate of the enzyme caspase-3. The fluorescence intensity was measured with the Victor3 multi-plate reader spectrophotometer (Perkin Elmer) (excitation 405 nm, emission 500 nm) . The RFU values (relative units of fluorescence) obtained from the treated cells were expressed as fold induction with respect to the non-infected sample treated only with DMSO solvent, the value of which was set equal to 1. The results obtained indicate that the exposure of the HFF cells to the SL and to the infection caused by HCMV significantly induces the activity of the caspase-3 enzyme with respect to the controls (Fig. 4B) .

Claims

1. A compound of formula

wherein Ri is a substituted or unsubstituted linear or branched C1-C4 alkyl, and

R is selected from the group consisting of H, substituted or unsubstituted phenyl, bromine, heteroaryl, thiophene and functionalised thiophene,

for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.

2. The compound for use according to claim 1, wherein Ri is CH₃.

3. The compound for use according to claim 1 or 2, wherein R₂ is in position 7.

4. The compound for use according to any of the preceding claims, wherein R₂ is thiophene or ethylenedioxythiophene .

5. The compound for use according to any of the preceding claims, wherein the viruses of the Herpesviridae family are Cytomegalovirus, Herpes Simplex virus 1 or Herpes Simplex virus 2.

6. The compound for use according to claim 5, wherein the virus of the Herpesviridae family is Cytomegalovirus.

7. A pharmaceutical composition comprising at least one compound of formula

wherein Ri is a substituted or unsubstituted linear or branched C1-C4 alkyl, and

R is selected from the group consisting of H, substituted or unsubstituted phenyl, bromine, heteroaryl, thiophene and functionalised thiophene,

for use in preventing and/or treating infections caused by viruses of the Herpesviridae family.

8. The pharmaceutical composition for use according to claim 7, comprising at least one cyclodextrin derivative.

9. The compound for use according to any of claims 1 to 6 or the composition for use according to claim 7 or 8, wherein preventing and/or treating infections caused by viruses of the Herpesviridae family occurs/occur in paediatric patients with congenital infections or who have undergone a bone marrow transplant .

10. The compound for use according to any of claims 1 to 6 or the composition for use according to claim 7 or 8, wherein preventing and/or treating infections caused by viruses of the Herpesviridae family occurs/occur in an adult patient who has undergone a transplant.