Substituted 3-cyanoquinolines for the prevention and treatment of sexually transmitted diseases

Abstract

The present invention refers to substituted 3-cyanoquinolines which are useful for preventing and treating sexually transmitted diseases.

Description

Substituted 3-cyanoquinolines for the prevention and treatment of sexually transmitted diseasesFIELD OF THE INVENTIONThe present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a sexually transmitted infection or disease, particularly a sexually transmitted infection or disease which is caused by and / or associated with a sexually transmitted pathogen capable of entering and surviving within a mammalian cell.BACKGROUND OF THE INVENTIONMore than 1 million sexually transmitted infections (STIs) are acquired every day. In 2020, WHO estimated 374 million new infections with 1 of 4 STIs: chlamydia (129 million), gonorrhoea (82 million), syphilis (7.1 million) and trichomoniasis (156 million). More than 490 million people were estimated to be living with genital herpes in 2016, and an estimated 300 million women have an HPV infection, the primary cause of cervical cancer and anal cancer among men who have sex with men. An estimated 296 million people are living with chronic hepatitis B globally (WHO FACT Sheets on STIs, July 2013). More than 30 different bacteria, viruses and parasites are known to be transmitted through sexual contact, including vaginal, anal, and oral sex. Some STIs can also be transmitted from mother-to-child during pregnancy, childbirth, and breastfeeding. STIs have a profound impact on sexual and reproductive health worldwide.A sexually transmitted infection (STI) is a virus, bacteria, fungus, or parasite people can get through sexual contact. Many STIs have no symptoms, so people can have an infection but not know it. A sexually transmitted disease (STD) develops because of an STI and the termimplies that the infection has led to some symptom of disease. People sometimes use the terms in one another's place. The primary goal of public health and healthcare is to prevent and treat infections before they develop into disease. As a result, many - including CDC - are using the term STI more often. However, STD is still used when referring to data or information from sources that use the term.Although highly effective, condoms do not offer protection for STIs that cause extra-genital ulcers (i.e., syphilis or genital herpes). Safe and highly effective vaccines are available for 2 viral STIs: hepatitis B and HPV. These vaccines have represented major advances in STI prevention. By the end of 2020, the HPV vaccine had been introduced as part of routine immunization program in 111 countries, primarily high- and middle-income countries. To eliminate cervical cancer as a public health problem globally, high coverage targets for HPV vaccination, screening and treatment of precancerous lesions, and management of cancer must be reached by 2030 and maintained at this high level for decades. Research to develop vaccines against genital herpes and HIV is advanced, with several vaccine candidates in early clinical development. More research into vaccines for chlamydia, gonorrhoea, syphilis and trichomoniasis is needed. Such approach requires the immunization of the patient for each STIs available on the market.Another approach to provide protection for STIs is the use of conventional antivirals which are instead targeting specifically one virus at the time. The best example is pre-exposure prophylaxis used for HIV, which reduces the risk of contracting HIV through sex by 99%. However, these compounds do not show broad-spectrum activity. Moreover, the antivirals targeting the virus are more at risk of development of resistance.There is thus a need for a compound which is efficacious against various sexually transmitted infections or diseases.Substituted 3-cyanoquinolines have been tested for their inhibitory effects in non-sexually transmitted Dengue virus (DENV), which is spread by mosquitos (S. J. F Kaptein et al., ChemMedChem, 2018, 13, 1371-1376; P. Vincetti et al., J. Med. Chem. 2015, 58(12): 4964-75).WO 00 / 18740 Al and EP 2253 620 Al disclose substituted 3-cyanoquinolines which are useful as antineoplastic agents (anticancer drugs) and in the treatment of polycystic kidney disease.It is an object of the present invention to provide compounds which are useful in the treatment or prevention of sexually transmitted infections or diseases.The compounds of the present invention are particularly advantageous because they exhibit efficacy against a wide range of different sexually transmitted infections and diseases.SUMMARY OF THE INVENTIONThe present invention provides substituted 3-cyanoquinolines for use in the prevention or treatment of a sexually transmitted infection or disease.Previously, substituted 3-cyanoquinolines have only been known to be useful as antineoplastic agents (anticancer drugs) and in the treatment of polycystic kidney disease. Previous studies further suggested that substituted 3-cyanoquinolines were either cytotoxic or inactive against dengue virus (DENV-2). The inventors of the present invention surprisingly found that the compounds of the invention show a broad-spectrum activity against multiple different sexually transmitted pathogens. Without wishing to be bound by theory, it is believed that the compounds of the invention show efficacy against several sexually transmitted infections and diseases, since they do not target specifically the pathogen but the host cell. Due to their broad-spectrum activity against multiple different sexually transmitted pathogens, the compounds of the invention are broadly applicable as topical microbicides. Formulated as a gel, cream, lotion, aerosol spray, film, sponge, lubricant or time-released suppository, the compounds of the invention can be useful as vaginal or rectal microbicides in order to provide primary protection to women and men and couples who cannot or are unwilling to use condoms.Various embodiments of the invention are described herein. Provided herein is a compound of formula (I):or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, for use in the prevention or treatment of a sexually transmitted infection or disease.In another aspect, the invention provides a method of preventing or treating a sexually transmitted infection or disease, comprising administering a therapeutically effective amount of the compound of formula (I) as disclosed herein to a subject in need thereof.In another aspect, the invention provides a use of the compound of formula (I) as disclosed herein for the preparation of a medicament for preventing or treating a sexually transmitted infection or disease.In a preferred aspect, the sexually transmitted infection or disease is selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum (Poxvirus), Gonorrhea (Neisseria gonorrhoeae), Mycoplasma genitalium, Ebola virus, Marburg virus and Lassa virus; wherein the sexually transmitted infection or disease is preferably selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum (Poxvirus), Ebola virus, Marburg virus and Lassa virus; wherein the sexually transmitted infection or disease is more preferably selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), and Chlamydia (Chlamydia trachomatis).DEFINITIONSFor the purpose of interpreting this specification, the following definitions will apply unless specified otherwise, and when appropriate, terms used in the singular will also include the plural and vice versa. It is also noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the compound" includes reference to one or more compounds; and so forth.The term "amino" refers to a group of the formula N(R*)(R**) wherein R* and R** are independently selected from H and C1-C4 alkyl.The term "C1-C4 alkyl" refers to a saturated straight or branched hydrocarbon chain consisting solely of carbon and hydrogen atoms containing no unsaturation, having from one to four carbon atoms, and which is attached to the rest of the molecule by a single bond. Examples of suitable alkyl groups having 1 to 4 carbon atoms include, but are not limited to, methyl, ethyl, propyl, isopropyl, 1-methylethyl, butyl, tert-butyl. The C1-C4 alkyl is preferably C1-C3 alkyl, more preferably C1-C2 alkyl, even more preferably Ci alkyl.The term "C1-C4 alkoxy" refers to a C1-C4 alkyl group which is bonded to oxygen (alkyl-O-). Examples of suitable alkoxy groups having 1 to 4 carbon atoms include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, and tert-butoxy. The C1-C4 alkoxy is preferably C1-C3 alkoxy, more preferably C1-C2 alkoxy, even more preferably Ci alkoxy.The term "Hal" or "halogen" or "Halo" refers to F, Cl, Br, and I, preferably F or Cl.The term "Hal-Ci-C4 alkyl" refers to a C1-C4 alkyl group which is subsituted by one or more halogen atoms. Examples thereof include trifluoromethyl, difluoromethyl, dichloromethyl, chloromethyl and fluoromethyl, particularly trifluoromethyl.Unless defined otherwise, the term "carbocyclic group" refers to any 5- to 10 -membered (e.g. unsaturated or saturated) carbocyclic group such as aromatic groups. Peferably, thecarbocyclic group is 5- or 6-membered. Examples of carbocyclic groups include, for instance, phenyl, naphthyl, cyclopentyl or cyclohexyl, preferably phenyl.Unless defined otherwise, the heterocyclic group refers to any 5- or 10-membered (e.g., unsaturated or saturated) heterocyclic group such as heteroaryl groups. The heteroatom can be selected from N, O and S, preferably N or O, more preferably N.Non-limiting examples of suitable heterocyclic groups include:5-membered heterocycles: pyrrole, pyrrolidine, imidazole, imidazolidine, pyrazole, pyrazolidine, oxazole, isoxazole, thiazole, isothiazole, oxazoline, oxazolidine, isoxazoline, imidazoline, pyrazoline, thiophene, furan, tetrahydrofuran, dioxolane, triazole (1,2,3- and 1,2,4-), tetrazole, oxadiazole (e.g., 1,2,4-oxadiazole), thiadiazole (e.g., 1,3,4-thiadiazole), thiazoline, 1,3-dioxole.6-membered heterocycles: pyridine, piperidine, piperazine, diazine (e.g., pyridazine, pyrimidine, pyrazine), morpholine, thiomorpholine, triazine (e.g., 1,3,5-triazine), oxazine, thiazine.Fused and benzo-fused heterocycles: indole, benzimidazole, benzoxazole, benzothiazole, benzofuran, benzothiophene, benzotriazole, quinoline, isoquinoline, phthalazine, quinazoline, cinnoline, 1,3-benzodioxole (methylenedioxybenzene), 1, 2,3,4- tetrahydroquinoline.Others (7- to 10-membered and bridged rings): azepine, oxepane, thiepane, azocane, diazepine, diazocane, dioxane, dioxepane, crown ethers with heteroatoms (e.g., 12-crown-4 with N or O), spiro-heterocycles containing at least one heterocyclic ring attached via a spiro carbon to a second ring.The heterocyclic group can contain 1 to 3 heteroatoms. Examples include pyrrolidine, pyrrol, pyrimidine, tetra hydrofuran, furan, imidazolidine, pyrazolidine, imidazole, benzimidazole, benzothiazole, pyrazole, oxazolidine, dioxolane, triazole, tetrazole, piperidine, pyridine,diazine, morpholine, triazine, thiazole, pyridazine, benzoxazole, oxazole, thiophen, 1,3- benzodioxolane, isoxazole, 1,3,4-thiadiazole, 1,2,4-oxadiazole, benzoxazole, and benzotriazole.Preferably, pyrrolidine, pyrrol, pyrimidine, tetra hydrofuran, furan, imidazolidine, pyrazolidine, imidazole, benzimidazole, benzothiazole, pyrazole, oxazolidine, dioxolane, triazole, tetrazole, piperidine, pyridine, diazine, morpholine, triazine, thiazole, pyridazine, benzoxazole, and oxazole.The carbocyclic group or heterocyclic group can be optionally substituted. Examples of possible substituents include one or more (e.g., 1 to 3, such as 1 or 2) substituents selected from halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.Alternatively, any two substituents which are adjacent to each other can be connected to form an optionally substituted carbocyclic moiety or an optionally substituted heterocyclic moiety. The optionally substituted carbocyclic moiety or the optionally substituted heterocyclic moiety can be 5- or 6-membered. It can be annealed to the carbocyclic group or be attached in a spiro manner (preferably annealed).Unless specified otherwise, the term "compound(s) of the invention" refers to a compound of formula (I), or a pharmaceutically acceptable salt thereof.Compounds of the invention may have one or more optically active carbons and can be thus provided as racemates, stereoisomers (including diastereomers, enantiomers, and conformers), etc. All isomeric forms are included in the present invention.The term "polymorphs" refers to crystalline forms of the compounds of the invention.Solvates, hydrates as well as anhydrous forms of the salt are also encompassed by the invention. The solvent included in the solvates is not particularly limited and can be any pharmaceutically acceptable solvent. Examples include C1-4 alcohols (such as methanol or ethanol).The compound of the invention can also be provided in the form of a prodrug, i.e., a compound of the present invention which is metabolized in vivo to the active metabolite."Pharmaceutically acceptable salts" are defined as derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as, but not limited to, acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like. The pharmaceutically acceptable salts of the compounds of the present invention and their precursors can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Organic solvents include, but are not limited to, nonaqueous media like ethers, ethyl acetate, ethanol, isopropanol, or acetonitrile. Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 18thed., Mack Publishing Company, Easton, PA, 1990, p. 1445, the disclosure of which is hereby incorporated by reference."Pharmaceutically acceptable" is defined as those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.The terms "disease" or "infection" are used interchangeably herein.The term "sexually transmitted infection (STI)", also referred to as "sexually transmitted disease (STD)" herein, refers to an infection that is transmitted through sexual contact, including vaginal, anal and oral sex. The sexually transmitted infection may pass from person to person in blood, semen, or vaginal bodily fluids. Some STIs can also be transmitted during pregnancy, childbirth and breastfeeding. The sexually transmitted infection or disease is in particular caused by and / or associated with a pathogen which is capable of entering and surviving within a mammalian cell. The pathogen can be a virus or a bacterium (such as a bacterium which is capable of growing, proliferating or reproducing within a mammalian cell, in particular a bacterium which must enter a mammalian cell to reproduce). The pathogen, virus and bacterium are capable of being sexually transmitted.The term "subject" refers to mammals, such as primates (e.g., humans, male orfemale), dogs, rabbits, guinea pigs, pigs, rats and mice. Preferably, the subject is a human or an animal. More preferably, the subject is a human.As defined herein, a subject is "in need of" a prevention or treatment if such subject would benefit biologically, medically or in quality of life from such prevention or treatment.The preferred definitions given in the "Definition"-section apply to all of the embodiments described below unless stated otherwise. Various embodiments of the invention are described herein, it will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.BRIEF DESCRIPTION OF THE FIGURESFigure 1. Broad-spectrum antiviral activity of the compoundsA) VeroE6 cells were infected with 100 pfu / well of HSV-2, lhpi the inoculum was removed and cells were treated with serial dilutions of compounds in medium supplemented with methylcellulose. Cells were fixed and plaques were counted 24 hpi.B) Cells were infected with HSV-l expressing GFP for lh at 37°C, after the removal of the viral inoculum cells were treated with serial dilutions of the compounds. The number of infected cells was counted 24h piC-D) THP-1 cells were treated with 10 pM of the compounds and infected with HIV expressing VSV-G protein. The infection was evaluated 48 hpi through flow cytometry (C) or by measurement of the p24 in the supernatant (D) * p<0.05 **p<0.01, ***p<0.001. The results are the mean of three independent experiments.Figure 2. Activity against Chlamydia. Vero cells were treated with compounds MR56 and MR58 (10 pM) and infected with C. trachomatis at MOI 1. Infection was evaluated either by qPCR (A) or by determining the number of released infectious units (IFU) by immunofluorescence (B). Results are presented as fold change in comparison with DMSO treated wells and are the mean of three independent experiments. ****p<0.0001Figure 3. Antiviral activity in EpiVaginal. Tissues were infected apically with lxlO5pfu of MPXV for 3 hours, the inoculum was removed, and the tissues treated with the compounds. 24hpi 200 pl of medium was added apically for 20 min and subsequently collected. The supernatants were then titrated on Vero6 cells by plaque assay. The results are mean and SEM of a single independent experiment. UT: untreatedFigure 4. Time of addition experiment for cidofovir and compound MR59. Cells were infected with VACV and treated with cidofovir and MR59 either lh before infection, or during infection, or at different time post infection (1, 2 or 4 hpi). 24hpi the number of infected cells was evaluated, and the 50% inhibitory concentration was calculated with graphpad prism. The results are mean and SEM of three independent experiments.Figure 5. Early, intermediate and late gene expression of VACV in presence of treatment. VeroE6 cells were infected and treated with MR56, MR59 or cidofovir. Cells were lysed either at 4, 8, 12 or at 24hpi and a specific RT-qPCR for an early, intermediate and a late gene was performed. The results are mean and SEM of a three independent experiments.Figure 6. MR59 and MR56 synergism with cidofovir. Serial dilutions of MR56 and cidofovir or MR59 and cidofovir were used to treat cells infected with VACV (MOI 0.5). 24hpi the number of infected cells was evaluated, and the synergy score was calculated with SynergyFinder 3.0. The results are mean and SEM of two independent experiments.Figure 7. Serial passage in the presence of antiviral compounds did not result in resistance development. VACV was grown in presence or absence of MR59 for 10 passages. After each passage the virus was titered to re-infect cells at a constant multiplicity of infection. Drug concentrations were increased until passage 3 and then held constant (A). After 10 passages, viruses were re-tested for sensitivity by performing a dose-response assay and measuring EC50values for MR59 (B).DETAILED DESCRIPTION OF THE INVENTIONThe present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a sexually transmitted infection or disease, in particular a sexually transmitted infection or disease which is caused by and / or associated with a sexually transmitted pathogen capable of entering and surviving within a mammalian cell.Various embodiments of the invention are described herein, it will be recognized that features specified in each embodiment may be combined with other specified features to provide further embodiments of the present invention.The present invention relates to a compound of formula (I):or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, for use in the prevention or treatment of a sexually transmitted infection or disease.Ri is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, amino and -O-(CH2)qC=CH, wherein q is 0 to 2, preferably Ri is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, and amino. In one embodiment, Ri is selected from C1-C4 alkoxy, hydrogen, and C1-C4 alkyl. Preferably Ri is selected from C1-C4 alkoxy and hydrogen. More preferably Ri is C1-C4 alkoxy. Even more preferably Ri is methoxy.R2 is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, and amino. Preferably R2 is selected from C1-C4 alkoxy, hydrogen, and C1-C4 alkyl. More preferably R2 is selected from Ci- C4 alkoxy and hydrogen. Even more preferably R2 is hydrogen.Alternatively, either Ri or R2 is -O-linker-protein-binding moiety. When Ri is -O-linker- protein-binding moiety, the compound of the invention functions as a proteolysis targeting chimera (PROTAC). A PROTAC is a heterobifunctional molecule composed of two active domains and a linker.The protein-binding moiety can be any protein-binding moiety which is capable of binding with an E3 ubiquitin ligase, in particular those which are capable of binding to IAP (e.g., ILM), MDM2 (e.g., MLM), cereblon (e.g., CLM) and VHL (e.g., VLM).A wide range of protein-binding moieties are known in the art. Examples thereof are disclosed, for instance, in the definitions of ULM WO 2016 / 197114, for example, in paragraphs

[0066] et seq. and particularly in paragraphs

[0082] to

[0113] as well as claim 3 of WO 2016 / 197114 which are incorporated by reference. Further examples thereof are disclosed, for instance, in the definitions of ULM in WO 2018 / 102725, for example, in paragraphs

[0086] et seq. and claims 2 to 12 of WO 2018 / 102725 which are incorporated by reference.In one embodiment, the protein-binding moiety may be selected from thalidomide, pomalidomide, Von Hippel-Lindau E3 ubiquitin ligase (VHL) binding moiety (VLM), a cereblon E3 ubiquitin ligase binding moiety (CLM), a mouse double minute 2 homolog (MDM2) E3 ubiquitin ligase binding moiety (MLM), and an IAP E3 ubiquitin ligase binding moiety (ILM), preferably pomalidomide.One example of such a protein-binding moiety isA linker binds the protein-binding moiety to the core of the compound of formula (1). Therefore, the linker is not particularly limited. The linker can be a bond or any moiety which contains from 1 to 80 atoms (e.g., 1 to 50 atoms) selected from C, N, O, S, Si and Hal (e.g., selected from C, N, O, S and Hal). The linker can be saturated or unsaturated. Examples of possible linkers include a bond and:wherein n is an interger of 1 to 5. Examples of further possible linkers can be found in paragraphs

[0114] to

[0122] and claims 4 to 6 of WO 2016 / 197114 which are incorporated herein by reference. Examples of further possible linkers can be found in the chapter entitled "Exemplary Linkers" in paragraphs

[0277] et seq. and in claims 13 to 21 of W 2018 / 102725 which are incorporated herein by reference.X is selected from N(R3) and O, wherein R3 is H or C1-C4 alkyl. Preferably X is N(R3), more preferably X is NH. In one embodiment, X is N(R3). In another embodiment, X is O. Preferably R3is Hanother embodiment F,wherein Rio is an optionally substituted heterocyclic group. q is an integer of O to 2. p is an integer of O to 2.Rs is selected from halogen, hydrogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro. Preferably R5 is selected from halogen, hydrogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, and nitro. More preferably R5 is selected from halogen, Hal-Ci-C4 alkyl, and hydrogen. Even more preferably R5 is halogen.Re is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro. Preferably Re is selected from halogen and hydrogen. Even more preferably Re is hydrogen.R7 is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro. Preferably R7 is selected from halogen, Hal-Ci-C4 alkyl, nitro, hydrogen, Ci- C4 alkyl, C1-C4 alkoxy, and hydroxy. More preferably R7 is selected from halogen, Hal-Ci-C4 alkyl, hydrogen, and nitro. Even more preferably R7 is selected from halogen, Hal-Ci-C4 alkyl and nitro.Rs is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro. Preferably Rs is selected from halogen and hydrogen. Even more preferably Rs is hydrogen.Rg is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro. Preferably R9 is selected from halogen, hydrogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, and nitro. More preferably R9 is selected from halogen, Hal-Ci-C4 alkyl, and hydrogen. Even more preferably R9 is hydrogen.Rio is a carbocyclic group or a heterocyclic group, wherein the carbocyclic group or the heterocyclic group can be optionally substituted.In one embodiment, Rio ipreferred aspect of this embodiment,Rs is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;Re is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;R7 is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;Rs is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;R9 is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.In a further preferred aspect of this embodiment, any two groups selected from Rs, Re, R7, Rs, and R9 which are adjacent to each other can be connected to form an optionally substituted carbocyclic moiety or an optionally substituted heterocyclic moiety, wherein the optional substituent is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy,hydroxy, amino, and nitro. Examples of the carbocyclic moiety include an annealed phenyl (i.e., Rio is an optionally naphthyl).In one aspect, Rio is naphthyl which is optionally substituted by a substituent selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.In a further aspect, Rio is phenyl which is substituted by halogen and / or nitro, preferably phenyl which is substituted by halogen and optionally nitro (such as 4-chlorophenyl or 2- chloro-4-nitro-phenyl).In another embodiment, Rio is a heterocyclic group, wherein the heterocyclic group can be optionally substituted by hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.If the carbocyclic or hetercyclic group / moeity is substituted, it can have one or more (e.g., 1 to 3, such as 1 or 2) substituents.In one embodiment, the compound of formula (I) is selected from 4-((2,4- dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR15), 4-((4- chlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR54), 4-((2-chloro-4- nitrophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR56), 7-methoxy-4-((4- (trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile (compound MR58), 7-methoxy-4- (naphthalen-2-ylamino)quinoline-3-carbonitrile (compound MR59), 7-methoxy-4-((2-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile (compound MR55), 4-((3-chloro-4- fluorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR57), 4-((2,4- dichlorophenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile (compound MR19), 7- methoxy-4-(methyl(3-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile (compound MR691), 4-(cyclohexylamino)-7-methoxyquinoline-3-carbonitrile (compound MR692), 7- methoxy-4-(thiophen-3-ylamino)quinoline-3-carbonitrile (compound MR694), 4-((2-chloro-4- nitrophenyl)amino)-6-methoxyquinoline-3-carbonitrile (compound MR731), 4-((2-chloro-4- nitrophenyl)(methyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR735), 4-((2- chloro-4-nitrophenyl)(methyl)amino)-6-methoxyquinoline-3-carbonitrile (compoundMR740), 4-(benzo[d][l,3]dioxol-5-ylamino)-7-methoxyquinoline-3-carbonitrile (compound MR744), 4-((l / 7-benzo[d][l,2,3]triazol-5-yl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR745), 7-methoxy-4-((4-(trifluoromethyl)benzyl)amino)quinoline-3-carbonitrile (compound MR690), 4-((4-chlorobenzyl)amino)-7-methoxyquinoline-3-carbonitrile(compound MR693), 4-((2-chloro-4-nitrophenyl)amino)-7-(prop-2-yn-l-yloxy)quinoline-3- carbonitrile (compound MR773), and 4-(naphthalen-2-ylamino)-7-(prop-2-yn-l- yloxy)quinoline-3-carbonitrile (compound MR774), or a pharmaceutically acceptable salt thereof.In one embodiment, the compound of formula (I) is selected from 4-((2,4- dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR15), 4-((4- chlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR54), 4-((2-chloro-4- nitrophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR56), 7-methoxy-4-((4- (trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile (compound MR58), 7-methoxy-4- (naphthalen-2-ylamino)quinoline-3-carbonitrile (compound MR59), 7-methoxy-4-((2-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile (compound MR55), 4-((3-chloro-4- fluorophenyl)amino)-7-methoxyquinoline-3-carbonitrile (compound MR57), 4-((2,4- dichlorophenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile (compound MR19), or a pharmaceutically acceptable salt thereof.Preferably, the compound of formula (I) is selected from 4-((2,4-dichlorophenyl)amino)-7- methoxyquinoline-3-carbonitrile (compound MR15), 4-((4-chlorophenyl)amino)-7-methoxy- quinoline-3-carbonitrile (compound MR54), 4-((2-chloro-4-nitrophenyl)amino)-7-methoxy- quinoline-3-carbonitrile (compound MR56), 7-methoxy-4-((4-(trifluoromethyl)phenyl)amino)- quinoline-3-carbonitrile (compound MR58), 7-methoxy-4-(naphthalen-2-ylamino)quinoline- 3-carbonitrile (compound MR59), 4-((2-chloro-4-nitrophenyl)amino)-7-(prop-2-yn-l- yloxy)quinoline-3-carbonitrile (compound MR773) or a pharmaceutically acceptable salt thereof.The chemical structures of the preferred compounds are shown in Table 1 below.The compounds of the invention are for use in the prevention or treatment of a sexually transmitted infection or disease. The prevention or treatment of a sexually transmitted infection or disease can be conducted in mammals, preferably in humans and animals, more preferably in humans, even more preferably in females.The compounds of the invention show a broad-spectrum activity against multiple different sexually transmitted pathogens. Without wishing to be bound by theory, it is believed that the compounds of the invention show efficacy against several sexually transmitted pathogens, since they do not specifically target the pathogen but the host cell. Due to their broadspectrum activity against multiple different sexually transmitted pathogens, the compounds of the invention are broadly applicable as topical microbicides.A sexually transmitted infection or disease includes, but is not limited to, a sexually transmitted infection or disease which is caused by and / or associated with a sexually transmitted pathogen capable of entering and surviving within a mammalian cell. In particular, the sexually transmitted infection or disease can be caused by and / or associated with a sexually transmitted pathogen capable of entering and surviving within a mammalian cell. More particularly, the sexually transmitted infection or disease can be caused by and / or associated with a sexually transmitted virus or a sexually transmitted bacterium capable of entering and surviving within a mammalian cell. Even more particularly, the sexually transmitted bacterium can be capable of growing, proliferating or reproducing within a mammalian cell. In one embodiment, the sexually transmitted bacterium can be a bacterium which must enter a mammalian cell to reproduce.The pathogen can be a virus or a bacterium.The pathogen can be an intracellular pathogen.Examples of a sexually transmitted infection or disease include Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum (Poxvirus), Gonorrhea (Neisseriagonorrhoeae), Mycoplasma genitalium, Ebola virus, Marburg virus and Lassa virus. In a preferred embodiment, a sexually transmitted infection or disease includes Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum (Poxvirus), Ebola virus, Marburg virus and Lassa Virus; more preferably, Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), and Chlamydia (Chlamydia trachomatis).The compounds of the invention can be administered by any suitable route. Preferably they can be administered topically such as in the form of a gel, cream, lotion, aerosol spray, film, sponge, lubricant or time-released suppository. The compounds can be administered, for instance, by application to the vagina, labia, anus, rectum or penis (in particular the meatus), particularly the vagina, anus, or rectum.The compounds of the present invention may be prepared by the routes described in the examples and Scheme 1 below.The use of any and all examples, or exemplary language (e.g. "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. Unless otherwise stated, starting materials are either commercially available or are prepared by known methods.The invention is illustrated by the following examples which, however, should not be construed as limiting.EXAMPLESAbbreviations Abbreviations used are those conventional in the art.CC50 50% cytotoxic concentrationDMF dimethylformamideDMSO dimethylsulfoxideEC50 50% effective concentrationHBV hepatitis B virusHCV hepatitis C virusHIV human Immunodeficiency VirusHPV human PapillomavirusHSV herpes Simplex VirusMOI multiplicity of infectionMPXV monkeypox virusNMR nuclear magnetic resonance o.n. overnightRT-qPCR real time - quantitative PCRSI selectivity indexExample 1- Chemistry GeneralAll commercially available chemicals were purchased from both Sigma-Aldrich and Alfa Aesar and, unless otherwise noted, used without any prior purification. Solvents used for work-up and purification procedures were of technical grade. TLC was carried out using Sigma-Aldrich TLC plates (silica gel on Al foils, SUPELCO Analytical). Where indicated, products were purified by silica gel flash chromatography on columns packed with Merck Geduran Si 60 (40-63 pm).TH and13C NMR spectra were recorded on BRUKER AVANCE 300 MHz and BRUKER AVANCE400 MHz spectrometers. Chemical shifts (6 scale) are reported in parts per million relative toTMS.1H-NMR spectra are reported in this order: multiplicity and number of protons; signals were characterized as: s (singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), m (multiplet), bs (broad signal). Low resolution mass spectrometry measurements were performed on an Agilent InfinityLab LC / MSD iQ, Single Quadropole analyzer (ESI) and are reported in the form of (m / z). Elemental analyses were performed on a FlashSmart CHNS analyzer (Thermo Fisher) with gas-chromatographic separation. All final compounds were >95% pure as determined by elemental analysis (within 0.4% of the theoretical values). Melting points were taken using a Gallenkamp melting point apparatus and were uncorrected.Cidofovir was purchased from Sigma.Microwave Irradiation ExperimentsMicrowave reactions were conducted using a CEM Discover Synthesis Unit (CEM Corp., Matthews, NC). The machine consists of a continuous focused microwave power delivery system with an operator-selectable power output from 0 to 300 W. The temperature inside the reaction vessel was monitored using a calibrated infrared temperature control mounted under the reaction vessel. All experiments were performed using a stirring option whereby the reaction mixtures were stirred by means of a rotating magnetic plate located below the floor of the microwave cavity and a Teflon-coated magnetic stir bar in the vessel.Scheme 1. Reagents and conditions: i. neat, pW, 120 °C, 5 min; ii. Ph2O, 230 °C, 5-7 min; iii. POCI3, reflux, 2h; iv. substituted aniline, NaH, DMF, reflux, 2h.Scheme 2. Reagents and conditions: i. neat, nW, 120 °C, 5 min; ii. Ph2O, 230 °C, 5-7 min; iii. POCI3, reflux, 2h; iv. appropriate amine, NaH, DMF, reflux, l-16h.Scheme 3. Reagents and conditions: i. AICI3, DCE, reflux, o.n.; ii. propargyl bromide, K2CO3, DMF, reflux, 5h; iii. substituted aniline, NaH, DMF, reflux, 2h.General Procedure for the Synthesis of 4a-cEthyl 2-cyano-3- ethoxyacrylate 2 (275 mg, 1.62 mmol) and the opportune substituted aniline (la-c) (1.62 mmol) were heated at 120 °C for 5 minutes in a microwave oven (max pW power input: 250 W; ramp time: 1 minute; reaction time: 5 minutes; power max: off; maximum pressure: 190 psi). The solid formed at the bottom of the tube was separated by filtration,suspended in 5 mL of diphenyl ether and irradiated at 230 °C for 5-7 minutes (max pW power input: 250 W; ramp time: 3 minutes; reaction time: 5-7 minutes; power max: off; maximum pressure: 180 psi). At the end of the irradiation, petroleum ether was added to the reaction mixture, and the solid obtained was filtered over a Buchner funnel, washed thoroughly with petroleum ether, and used in the following step without any further purification.Compound 4a: 7-methoxy-4-oxo-3,4-dihydroquinoline-3-carbonitrileYield: 75%.XH NMR (300 MHz DMSO-d6): 6 3.87 (s, 3H); 6.99 (d, 1H, J = 2.34 Hz); 7.06 (dd, 1H, J = 8.94, 2.46 Hz); 8.02 (d, 1H, J = 8.94 Hz); 8.65 (s, 1H).Compound 4b: 6,7-dimethoxy-4-oxo-l,4-dihydroquinoline-3-carbonitrileYield: 70%. 1H NMR (300 MHz DMSO-d6) 6 3.87 (s, 3H), 3.89 (s, 3H), 7.03 (s, 1H), 7.44 (s, 1H), 8.59 (s, 1H), 12.57 (s, 1H)Compound 4c: 6-methoxy-4-oxo-l,4-dihydroquinoline-3-carbonitrileYield: 87%.TH NMR (600 MHz DMSO-d6) 6 3.83 (s, 3H), 7.37 (m, 1H), 7.47 (m, 1H), 7.56 (dd, 1H, J= 4Hz, J= 8Hz), 8.62 (s, 1H).General Procedure for the Synthesis of 5a-c:Under nitrogen atmosphere intermediates 4a-c (0.99 mmol) and freshly distilled POCI3 (5 mL) were heated at reflux for 2 hours. The reaction mixture was cooled down to room temperature and volatile residues were removed under vacuum. The solid obtained was cooled down to 0 °C and then CH2CI2, H2O, and solid K2CO3 were added until pH reached 11. The aqueous phase was extracted twice with CH2CI2 and the combined organic phases were dried over Na2SO4, filtered and dried under vacuum. Intermediates 5a-c were used in the next step without any further purification.Compound 5a: 4-chloro-7-methoxyquinoline-3-carbonitrileYield: 95%. MS (ESI) [M+H]+: 219.3 m / z, [M+2+H]+: 221.2 m / z.TH NMR (400 MHz CDCI3): 64.01 (s, 3H); 7.41 (dd, 1H, J = 9.26, 2.4 Hz); 7.48 (d, 1H, J = 2.36 Hz); 8.18 (d, 1H, J = 9.24 Hz); 8.88 (s, 1H).Compound 5b: 4-chloro-6,7-dimethoxyquinoline-3-carbonitrileYield: 95%.XH NMR (400 MHz DMSO-d6) 6 4.02 (d, 6H, J = 2.8 Hz), 7.44 (s, 1H), 7.55 (s, 1H),8.99 (s, 1H)Compound 5c: 4-chloro-6-methoxyquinoline-3-carbonitrileYield: 98%.TH NMR (400 MHz DMSO-d6) 6 4.01 (s, 3H), 7.53 (s, 1H), 7.72 (dd, 1H, J= 4Hz, J=8 Hz), 8.13 (d, 1H, J = 8 Hz); 9.06 (s, 1H).General Procedure for the Synthesis of 7a-t:An opportunely substituted aniline or appropriate amine(0.40 mmol) was added to a suspension of NaH (60% dispersion in mineral oil) (16 mg, 0.4 mmol) in dry DMF (5 mL). The reaction mixture was stirred at room temperature for 1 hour, followed by the addition of intermediate 5a-b (0.2 mmol). The mixture was heated at reflux for 1 h for 7m, 2 h for 7a-h, 3 h for 7i, q, r, 4 h for 7s, 5 h for 7f, I, n and 16 h for 7o, p, t. Then H2O and ethyl acetate were added and the reaction mixture was cooled down to room temperature. The organic phase was washed with an aqueous solution of LiCI (5% w / w), brine, dried over Na2SO4 and concentrated under vacuum. The crude was purified by flash chromatography: DCM / acetone (from 100 / 0 to 98 / 2) for 7a; DCM / acetone (from 99 / 1 to 9 / 1) for 7b; DCM / acetone (from 99 / 1 to 98 / 2) for 7c; DCM / acetone (from 99 / 1 to 97 / 3) for 7d; DCM / acetone (from 99 / 1 to 96 / 4) for 7e; DCM / acetone (from 99 / 1 to 96 / 4) for 7f; DCM / acetone (from 100 / 0 to 95 / 5) for 7g; petroleum ether / ethyl acetate (7 / 3) for 7h; petroleum ether / ethyl acetate (8 / 2) for 7t and for lOa-b; DCM / MeOH (99 / 1) for 7s; DCM (100) for 7i-m; DCM / MeOH (98 / 2) for 7n-r.Compound 7a (MR15): 4-((2,4-dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrileYield: 64%. Mp 194-195 °C. MS (APCI) [M+H]+: 344.3, [M+2+H]+: 346.2 m / z.TH NMR (400 MHz CDCh): 6 3.99 (s, 3H); 6.98 (d, 1H, J = 8.64 Hz); 7.17 (dd, 1H, J = 9.3, 2.44 Hz); 7.21 (dd, 1H, J = 8.7, 2.0 Hz); 7.44 (d, 1H, J = 2.44 Hz); 7.53 (d, 1H, J = 2.2 Hz); 7.71 (d, 1H, J = 9.28 Hz); 8.76 (s, 1H).13C NMR (100 MHz CDCI3): 6 56.04, 56.42, 94.74, 101.13, 109.13, 115.06, 116.30, 122.12, 126.44, 127.60, 129.66, 129.87, 136.67, 147.18, 147.68, 147.82, 149.67, 150.14, 154.50.Compound 7b (MR54): 4-((4-chlorophenyl)amino)-7-methoxyquinoline-3-carbonitrileYield: 64%. MS (APCI) [M+H]+: 310.3 m / z.XH NMR (300 MHz CDCI3): 6 3.9 (s, 3H); 7.07 (m, 3H); 7.27 (m, 4H); 7.86 (d, 1H, J = 9.39 Hz); 8.49 (s, 1H).13C NMR (75 MHz CDCI3): 6 55.62, 88.98, 107.97, 113.69, 116.79, 119.01, 124.06, 124.87, 129.39, 131.20, 138.46, 151.30, 152.92, 162.75.Compound 7c (MR55): 7-methoxy-4-((2-(trifluoromethyl)phenyl)amino)quinoline-3- carbonitrileYield: 56%. MS (APCI) [M+H]+: 344.3 m / .TH NMR (400 MHz CDCI3): 6 3.97 (s, 3H); 7.14 (m, 3H); 7.38 (m, 1H); 7.41 (d, 1H, J = 2.56 Hz); 7.53 (t, 1H, J = 7.8 Hz); 7.73 (d, 1H, J = 9.28 Hz); 7.78 (d, 1H, J = 7.72 Hz); 8.70 (s, 1H).13C NMR (100 MHz CDCI3): 655.78, 91.36, 108.86, 113.74, 116.20, 119.82, 123.49, 125.23, 126.04, 127.14, 132.99, 138.32, 150.36, 152.02, 152.54, 162.76.Compound 7d (MR56): 4-((2-chloro-4-nitrophenyl)amino)-7-methoxyquinoline-3- carbonitrileYield: 58%. MS (APCI) [M+H]+: 355.4 m / z, [M+Na]+: 377.4.TH NMR (400 MHz CDCI3): 64.03 (s, 3H); 6.78 (d, 1H, J = 9.04 Hz); 7.27 (dd, 1H, J = 9.24, 2.56 Hz); 7.53 (d, 1H, J = 2.4 Hz); 7.75 (d, 1H, J = 9.28 Hz); 8.05 (dd, 1H, J = 9.04, 2.52 Hz); 8.44 (d, 1H, J = 2.52 Hz); 8.95 (s, 1H).13C NMR (100 MHz CDCI3): 6 56.00, 98.05, 108.86, 115.52, 115.98, 116.46, 121.64, 122.22, 123.59, 124.24, 125.96, 142.10, 144.13, 147.36, 151.67, 152.75, 163.52.Compound 7e (MR57): 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinoline-3- carbonitrileYield: 61%. MS (APCI) [M+H]+: 328.4 m / z.TH NMR (400 MHz Acetone): 64.00 (s, 3H); 7.24 (dd, 1H, J = 9.28, 2.64 Hz); 7.34 (m, 2H); 7.38 (d, 1H, J = 2.68 Hz); 7.51(m, 1H); 8.24 (d, 1H, J = 9.24 Hz); 8.58 (s, 1H); 8.98 (bs, 1H).13C NMR (100 MHz Acetone): 6 55.28, 88.95, 108.76, 114.00, 116.84, 118.69, 120.42, 123.80, 124.35, 125.93, 137.54, 150.69, 151.73, 153.22, 154.36, 156.79, 162.64.Compound 7f (MR58): 7-methoxy-4-((4-(trifluoromethyl)phenyl)amino)quinoline-3- carbonitrileYield: 54%. MS (APCI) [M+H]+: 344.2 m / z.XH NMR (400 MHz Acetone): 64.02 (s, 3H); 7.26 (dd, 1H, J = 9.28, 2.56 Hz); 7.38 (d, 2H, J = 8.4 Hz); 7.44 (d, 1H, J = 2.52 Hz); 7.69 (d, 2H, J = 8.52 Hz); 8.20 (d, 1H, J = 9.32 Hz); 8.75 (s, 1H); 9.15 (bs, 1H).13C NMR (100 MHz Acetone): 6 55.35, 93.16, 108.69, 115.58, 116.38, 119.34. 120.29, 124.49, 126.30, 145.11, 149.68, 152.25, 152.76, 162.88.Compound 7g (MR59): 7-methoxy-4-(naphthalen-2-ylamino)quinoline-3-carbonitrileYield: 66%. MS (APCI) [M+H]+:326.3, [M+Na]+: 348.3 m / z.TH-NMR (400 MHz CDCI3): 6 3.96 (s, 3H); 6.98 (dd, 1H, J = 9.36, 2.64 Hz); 7.20 (bs, 1H); 7.27 (m, 1H); 7.41 (d, 1H, J = 2.61 Hz); 7.5 (m, 2H); 7.56 (d, 1H, J = 1.8 Hz); 7.67 (d, 1H, J = 9.36 Hz); 7.75 (m, 1H); 7.86 (m, 1H); 8.75 (s, 1H).13C NMR (100 MHz Acetone): 6 55.70, 92.15, 108.76, 113.55, 116.92, 118.96, 119.56, 122.06, 124.98, 125.8, 127.03, 127.41, 127.90, 129.66, 131.32, 133.73, 138.13, 150.94, 152.22, 152.42, 162.56.Compound 7h (MR19): 4-((2,4-dichlorophenyl)amino)-6,7-dimethoxyquinoline-3- carbonitrileYield: 57%. Mp 243-244 °C. MS (ESI) [M+H]+: 374.4 m / z.TH NMR (400 MHz CDCI3): 6 3.79 (s, 3H); 4.06 (s, 3H); 6.78 (bs, 1H); 6.84 (d, 1H, J = 8.67 Hz); 6.89 (s, 1H); 7.17 (dd, 1H, J = 8.64, 2.28 Hz); 7.43 (s, 1H); 7.52 (d, 1H, J = 2.28 Hz); 8.71 (s, 1H).13C NMR (100 MHz CDCI3): 6 44.52, 47.38, 53.88, 56.08, 85.68, 109.09, 113.37, 116.18, 117.71, 118.01, 127.30, 131.29, 149.38, 151.24, 152.10, 154.38, 162.34.Compound 7i (MR691): 7-methoxy-4-(methyl(3-(trifluoromethyl)phenyl)amino)quinoline-3- carbonitrileYield: 10%. LC-MS (ESI) [M+H]+: 358.1 m / z.TH-NMR (400MHz CDCI3): 6 3.62 (s, 3H); 4.02 (s, 3H); 7.64 (dd, 1H, J=4 Hz, J=2 Hz); 7.97 (s, 1H); 7.16 (d; 1H; J=4 Hz); 7.20 (dd, 1H, J= 4 Hz, J= 2 Hz); 7.32 (t, 1H, J= 4 Hz); 7.52 (d, 1H, J=2 Hz); 9.01 (s, 1H); 9.68 (d, 1H, J= 4 Hz).13C NMR (100MHz CDCI3): 6 44.95; 55.98; 103.35; 108.72; 110.89; 116.04; 116.78; 118.11; 119.96; 121.87; 124.96; 125.67; 129.96; 147.75; 152.64; 153.54; 156.44; 163.33.Compound 71 (MR692): 4-(cyclohexylamino)-7-methoxyquinoline-3-carbonitrile:Yield: 68%. LC-MS (ESI) [M+H]+: 282 m / z; [M+H]’: 280 m / z.XH-NMR (600MHz CDCI3): 6 1.25 (m, 1H); 1.32 (m, 2H); 1.41 (m, 2H); 1.70 (m, 1H, J=4Hz, J=2Hz); 1.81 (dt, 2H, J=4Hz, J=2Hz); 2.21 (dd, 2H, J=4Hz, J=2Hz); 4.38 (m, 1H); 3.91 (s, 3H); 5.38 (d, 1H, J= 2Hz); 7.08 (dd,lH, J= 4Hz, J=2 Hz); 7.27 (d, 1H, J=4Hz); 7.98 (d, 1H, J=4Hz); 8.51 (s, 1H).13C NMR (151 MHz CDCI3): 624.31; 25.40; 34.26; 52.57; 55.74; 83.40; 109.31; 112.15; 118.43; 119.93; 121.01; 150.87; 154.31; 162.24.Compound 7m (MR694): 7-methoxy-4-(thiophen-3-ylamino)quinoline-3-carbonitrile:Yield: 34%. LC-MS (ESI) [M+H]+: 282.05 m / z. H -NMR (400MHz CDCI3): 6 3.97 (s, 3H); 6.99 (m, 2H); 7.09 (m, 2H); 7.40 (m, 2H); 7.70 (d, 1H, J= 4Hz); 8.65 (s, 1H).13C NMR (100MHz CDCI3): 6 55.72; 88.51; 108.85; 112.47; 116.70; 118.42; 118.90; 123.53; 125.42; 126.42; 137.99; 151.93; 152.81; 162.50.Compound 7n (MR731): 4-((2-chloro-4-nitrophenyl)amino)-6-methoxyquinoline-3- carbonitrile:Yield: 68%. LC-MS (ESI) [M+H]+: 355.4 m / z.TH-NMR (400MHz MeOD): 6 3.99 (s, 3H); 7.31 (m, 2H); 8.12 (m, 1H); 8.74 (s, 1H).13C NMR (100MHz MeOD) 6: 56.13; 85.65; 102.28; 103.16; 114.23; 116.03; 120.88; 125.35; 125.29; 128.95; 132.08; 136.93; 152.12; 158.86.Compound 7o (MR735): 4-((2-chloro-4-nitrophenyl)(methyl)amino)-7-methoxyquinoline-3- carbonitrile:Yield: 14%. LC-MS (ESI) [M+H]+: 380.0 m / z.TH-NMR (400 MHz CDCI3): 6 3.05 (s, 3H); 3.88 (s, 3H); 7.01 (m, 2H); 7.12 (t, 1H, J= 8Hz); 7.36 (m, 2H); 8.08 (dd, 1H, J= 4Hz, J= 8Hz); 8.73(s, 1H).13C NMR (100MHz CDCI3): 643.00; 55.50; 89.31; 100.72; 103.18; 108.80; 117.85; 118.89; 123.33; 124.94; 125.37; 127.18; 129.74; 137.41; 149.77; 150.47; 155.12; 159.50; 163.84.Compound 7p (MR740): 4-((2-chloro-4-nitrophenyl)(methyl)amino)-6-methoxyquinoline-3- carbonitrile:Yield: 33%. LC-MS (ESI) [M+H]+: 380.0 m / z.TH-NMR (400 MHz CDCI3): 6. 3.05 (s, 3H); 3.89 (s, 3H); 6.98 (m, 2H); 7.13 (m, 1H); 7.54 (m, 2H); 8.11 (dd, 1H, J= 4Hz, J= 8Hz); 8.55(s, 1H).Compound 7q (MR744): 4-(benzo[c / ][l,3]dioxol-5-ylamino)-7-methoxyquinoline-3- carbonitrile:Yield: 11%. LC-MS (ESI) [M+H]+: 320.0 m / z.XH-NMR (400 MHz MeOD): 6 3.87 (s, 3H); 5.76 (s, 2H); 6.54 (d, 1H, J= 8 Hz); 6.83 (m, 1H); 6.99 (m, 1H); 7.37 (t, 1H, J= 8Hz); 7.92 (m, 1H); 8.14 (s, 1H); 8.84 (s, 1H).13C NMR (100MHz MeOD) 6: 55.01; 85.50; 98.12; 100.38; 107.31; 107.94; 111.37; 114.20; 114.38; 114.70; 117.18; 119.04; 120.74; 138.26; 140.43; 141.98; 157.42.Compound 7r (MR745): 4-((lH-benzo[d][l,2,3]triazol-5-yl)amino)-7-methoxyquinoline-3- carbonitrile:Yield: 23%. LC-MS (ESI) [M+H]+: 317.4 m / z. H -NMR (400 MHz MeOD): 64.03 (s, 3H), 6.83 (m, 1H); 6.94 (d, 1H, J= 4Hz); 7.36 (m, 1H); 7.45 (d, 1H, J = 8Hz); 7.64 (d, 1H, J= 4Hz); 7.83 (d, 1H, J= 8 Hz); 7.95 (s, 1H); 9.20 (s, 1H).Compound 7s (MR690): 7-methoxy-4-((4-(trifluoromethyl)benzyl)amino)quinoline-3- carbonitrile:Yield: 13%. LC-MS (ESI) [M+H]+: 358.1m / z. H -NMR (400MHz CDCI3): 6 3.97 (s, 3H); 5.19 (m, 2H); 5.71 (bs, 1H); 7.17 (dd, 1H, J= 4 Hz, J= 2 Hz); 7.37 (bs, 1H); 7.56 (d, 2H, J= 4 Hz); 7.70 (m, 3H); 8.59 (s, 1H).13C NMR (100MHz CDCI3): 6 48.66; 55.75; 84.14; 109.24; 111.93; 119.02; 119.44; 121.06; 122.54; 125.25; 126.22; 128.03; 130.51; 130.83; 141.16; 150.76; 151.85; 153.88; 162.47.Compound 7t (MR693): 4-((4-chlorobenzyl)amino)-7-methoxyquinoline-3-carbonitrile:Yield: 41%. LC-MS (ESI) [M+H]+: 324.1 m / z; [M+H]’: 322 m / z.TH-NMR (CDCI3400MHz): 6 3.94 (s, 3H); 5.06 (d, 2H, J=2Hz); 5.77 (t, 1H, J=2Hz); 7.13 (dd; 1H; J=4Hz, J=2Hz); 7.38 (m, 5H); 7.69 (d, 1H, J= 4 Hz); 8.56 (s, 1H).13C NMR (100MHz CDCI3): 648.65; 55.77; 84.04; 109.29; 112.06; 118.87; 119.67; 121.29; 129.27; 129.45; 134.37; 135.67; 150.82; 151.94; 154.01; 162.45.General Procedure for the synthesis of 8a: 4-chloro-7-hydroxyquinoline-3-carbonitrile5a (0.91 mmol; 200mg) dissolved in DCE (2 mL) was heated at reflux, followed by addition of anhydrous aluminium chloride (3.2 mmol; 3.5 eq) portionwise over a period of five minutes. The reaction mixture was cooled down to room temperature after stirring overnight andvolatile residues were removed under vacuum. NH4OH and NH4CI were added to the reaction mixture. A fume was formed and the reaction mixture was stirred under heating for 1 h until a jelly-like solid was formed. Then the aqueous phase was extracted with EtOAc several times (x 7), and the combined organic phases were dried over Na2SO4, filtered and dried under vacuum. The crude product was used for the next reaction without further purification. Yield: 96%. LC-MS (ESI) [M+H]+: 205.0 m / z.XH-NMR (400MHz MeOD): 6 7.38 (d, 1H, J= 4Hz); 7.42 (dd, 1H, J= 4 Hz, J=12 Hz); 8.25 (d, 1H, J= 12Hz); 8.90 (s, 1H).General Procedure for the Synthesis of Compound 9a: 4-chloro-7-(prop-2-yn-l-yloxy)quinoline-3-carbonitrile8a was solubilized in dry DMF. K2CO3 and propargyl bromide were added and the reaction mixture was refluxed for 5 h. Then H2O and ethyl acetate were added and the reaction mixture was cooled down to room temperature. The organic phase was washed with an aqueous solution of LiCI (5% w / w) and brine, dried over Na2SO4 and concentrated under vacuum. The crude product was purified by flash chromatography petroleum ether / ethyl acetate (7 / 3). Yield: 85%. LC-MS (ESI) [M+H]+: 243.0 m / z. H -NMR (400MHz CDCI3): 6 2.59 (m, 1H); 4.88 (s, 1H); 7.42 (d, 1H, J= 4Hz); 7.57 (s, 1H); 8.20 (d, 1H, J= 4Hz); 8.89 (s, 1H).Compound 10a (MR-773): 4-((2-chloro-4-nitrophenyl)amino)-7-(prop-2-yn-l-yloxy)- quinoline-3-carbonitrileYield: 64%. LC-MS (ESI) [M+H]+: 379.0 m / z, [M+H]’: 377.0 m / z. H -NMR (400 MHz CDCI3): 6 2.64 (s, 1H); 4.93 (s, 2H); 6.80 (d, 1H, J= 8Hz); 7.17 (s, 1H); 7.33 (dd, 1H, J= 4Hz, J= 8Hz); 7.66 (d, 1H, J= 4Hz); 7.79 (d, 1H, J= 8Hz); 8.06 (dd, 1H, J= 4 Hz, J= 8 Hz); 8.44 (d, 1H, J= 4Hz); 8.98 (s, 1H).Compound 10b (MR774): 4-(naphthalen-2-ylamino)-7-(prop-2-yn-l-yloxy)quinoline-3- carbonitrileYield: 42%. LC-MS (ESI) [M+H]+: 350.0 m / z, [M+H]’: 348.0 m / z. H -NMR (400 MHz CDCI3): 6 2.57 (s, 1H); 4.82 (s, 2H); 6.99 (dd, 1H, J= 4Hz, J= 8Hz); 7.15 (s, 1H); 7.24 (d, 1H, J= 4Hz); 7.46 (m, 1H); 7.49 (m, 1H); 7.54 (d, 1H, J= 4 Hz); 7.66 (d, 1H, J= 8 Hz); 7.72 (d, 1H, J= 8Hz); 7.84 (m, 2H); 8.73 (s, 1H).Table 1. List of compoundsExample 2VirusesMonkeypox virus (MPXV) was isolated from anonymized clinical samples from the University Hospital of Lausanne tested positive by PCR for the virus. Herpes simplex virus type 2 (HSV-2) is a clinical isolate obtained from the University of Pisa, VACV WT was obtained from Labor Spiez, HSV-1 expressing GFP and VACV GFP Western Reserve EL EGFP were obtained from the University of Lausanne. The viruses were grown on VeroE6 cells, when the cells displayed extensive cytopathic effect the supernatants were harvested, clarified, aliquoted and stored at -80°C. Titration was performed with plaque assay on VeroE6 cells in medium supplemented with 0.5% methylcellulose.CellsVero C1008 (clone E6) (ATCC ref.: CRL-1586) were obtained from Prof Gary Kobinger. Cells were propagated in DMEM High Glucose + Glutamax supplemented with 10% fetal bovine serum (FBS) and and penicillin / streptomycin (pen / strep) 100 Ul / ml.Viability AssayCell viability was measured by the MTT assay or MTS assay (Promega) for tissues. Confluent cell cultures seeded in 96-well plates were incubated with different concentrations of compounds in duplicate under the same experimental conditions described for the antiviral assays. Absorbance was measured using a Microplate Reader at 570 nm. The effect on cell viability at different concentrations was expressed as a percentage, by comparing the absorbance of treated cells with the one of cells incubated with equal concentrations of solvent in medium. The 50 % cytotoxic concentrations (CC50) and 95 % confidence intervals (Cis) were determined using Prism software (Graph-Pad Software, San Diego, CA).Plaque reduction assay for Monkeypox virus and HSV-2.Vero E6 were seeded in 24 wp with a density of 100k cells / well. The following day 100 pfu / well of viruses diluted in medium 2.5% FBS were added on cells for 1 hour at 37°C. After infection serial dilutions of compounds in DMEM 2.5% FBS supplemented with 0.5% methylcellulose were added on cells. Cells were fixed with a solution containing crystal violet and ethanol 24hpi for HSV-2 and 48hpi for MPXV. Percentage of inhibition were calculated by comparing treated and untreated wells and the 50% inhibitory concentration was determined by Prism software (Graph-Pad Software, San Diego, CA).Antiviral assays for VACV and HSV-1Vero E6 were seeded in 96 wp with a density of 10k cells / well. The following day 1000 ffu / well of VACV diluted in medium were added on cells for 1 hour at 37°C. After infection, serial dilutions of compounds in DMEM 2.5 % FBS were added on cells. 24hpi cells were fixed with 4 % formaldehyde. For VACV WT an immunostaining procedure was followed while for VACV and HSV-1 GFP cells were only fixed and stained with DAPL Infected cells were counted by ImageXpress PICO microscope. EC50 values were determined using GraphPad Prism 9.Preparation of NL4.3-deltaEnv-GFP / VSV-GFor the HIV-based lentiviral vector NL4.3-deltaEnv-GFP / VSV-G production, 2.5 million of HEK293T cells per 10cm dishes were transfected with 7.5 pg of NL4.3-deltaEnv-GFP (NIH AIDS Research and Reference Reagent program, 11100) and 2.5 pg pMD2G (Addgene, 12259) coding for the VSV-G envelope, using jetPrime (Polyplus transfection), following manufacturer's instructions. Viral particles were then harvested 48h after transfection, filtered through 0.45 pm filters and concentrated by filtration on Centricon units (Centricon Plus-70 / 100K, Millipore).Viral titers were measured by HIV-1 p24 Enzyme-linked immunosorbent assay (ELISA) (Innotest HIV Antigen mAb, FUJ I REBIO).Differentiation and infection of THP-1THP-1 were maintained in RPMI + 10% FBS + 50 pg ml-1 gentamycin (R10) and incubated at 37°C 5% CO2. For the experiment, 0.5*106THP-1 were differentiated into macrophages; cells were incubated during 48h in R10 supplemented with 25nM PMA (Merk, P8139) in a well of a 24-well plate at 37°C, 5% CO2 , as previously described (Lund ME, To J, O'Brien BA, Donnelly S. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus. J Immunol Methods. 2016 doi: 10.1016 / j.jim.2016.01.012). After 48h, culture medium was replaced with fresh R10 and cells were let to recover for 24 h before being exposed to drug treatment and infection. Differentiated THP-1 were treated with each compound at the concentration of 10 pM during 1 h and subsequentially infected with 50 ng p24 equivalent of HIV_NL4.3-deltaEnv-GFP / VSV- G in a total volume of 500 pL of R10 containing 4 pg / mL of polybrene. Infected cells were spinoculated for 90 min at 1500 g, the infection media was removed and replaced with 500 pL of R10 culture medium containing drugs and incubated at 37°C during 48h. Supernatant of infected cells were collected and neutralized with 0.5% Nonidet-P-40 substitute (Sigma- Aldrich) and viral particle release was assessed by p24 ELISA according to manufacturer instructions (Innotest HIV Antigen mAb, FUJ I REBIO). Infected cells were detached with TrypLE (Thermo Fisher Scientific), washed once in Robosep (Stemcell) and fixed in PBS 1% PFA(Thermo Fisher Scientific). HIV encoded GFP expression was assessed by flow cytometry using a Cytoflex S or Cytoflex LX. Data were analyzed using FlowJo_V10_CL software.Bacterial strainsC. trachomatis serovar LGV II strain 434 (ATCC ref.:VR-902B) was routinely propagated in McCoy cells (ATCC ref.: CRL-1696) and stored at -80°C in sucrose-phosphate-glucose (SPG) medium as described in Scidmore MA. Cultivation and Laboratory Maintenance of Chlamydia trachomatis. Curr Protoc Microbiol. 2005 Jul; Chapter 11: Unit 11A.1. doi:10.1002 / 9780471729259. mcllaOlsOO.Chlamydia infection procedureVero cells were seeded the day before infection in 24-well plates at a density of 2.5xl05cells. One hour prior to infection, culture medium was replaced by medium containing 10 mM of inhibitors diluted in DMSO. Cells were then infected with frozen C. trachomatis diluted 1 / 1000 in medium containing 10 mM inhibitors, an inoculum that corresponds to a Multiplicity Of Infection (MOI) of about 1. To synchronize infection, cells were centrifuged at 900g for 10 minutes and incubated 30 minutes at 37°C, 5% CO2. Non-internalized bacteria were removed by culture medium exchange and cells were incubated further at 37°C and 5% CO2 for48 hours in presence of inhibitors.Chlamydia infection focus unit determinationForty-eight hours post infection, cells were detached, mixed with glass beads (Sigma-Aldrich, Buchs, Switzerland) and lysed in a Precellys Evolution (Bertin Technologies, Montigny-le- Bretonneux, France) at 4500rpm, during 3 x 15 sec with 30 sec pause between the cycles. Tenfold serial dilutions of lysed cells were then used to infect fresh McCoy cells, seeded on glass coverslips, as described above except that 1 pg / mL cycloheximide (Sigma-Aldrich, Buchs, Switzerland) was added to the culture medium. Cells were further grown at 37°C with 5% CO2 for 16 hours before being fixed with ice-cold methanol for 5 minutes. Fixed cells were washed3 times with PBS, blocked in PBS + 0.1% saponin + 5% FCS + 0.01 NaNs (blocking solution) for at least two hours and stained for immunofluorescence.ImmunofluorescenceCells on glass coverslips were stained for 2 hours with a polyclonal goat anti-C. trachomatis MOMP antibody (LSBio, LubioScience, Zurich, Switzerland) diluted 1:500 in blocking solution. After three washes in PBS + 0.1% saponin, coverslips were incubated one hour at room temperature in blocking solution with 1.6 pg / ml DAPI dilactate (Molecular Probes, Thermo Fisher Scientific, Waltham, USA), 100 pg / ml Texas red conjugated-Concanavalin A (Invitrogen, Thermo Fisher Scientific, Waltham, USA) and Alexa-488 conjugated chicken anti-goat antibodies diluted 1:500 (Life Technologies, Thermo Fisher Scientific, Waltham, USA) in blocking solution. Coverslips were finally washed 2 times in PBS + 0.1% saponin, once in PBS and once in water and embedded in Moewiol (Sigma-Aldrich, Buchs, Switzerland). Images were taken with a confocal microscope Zeiss LSM 900 (Zeiss, Feldbach, Switzerland) and the number of inclusions and cells were counted with Image J.Quantitative PCRGenomic DNA was extracted from 100 pL of infected cells following the manufacturer's instructions (Wizard SV Genomic DNA purification kit, Promega, Madison, Wl, USA) and analyzed with a specific C. trachomatis qPCR targeting the 16SrRNA gene.Ex vivo testingThe EpiVaginal tissues (VEC-100) were purchased from MatTek Corporation (Ashland, MA, USA). According to the manufacturer's instructions, EpiVaginal cultures were seeded with the apical surface exposed to air in six-well plates containing 5 ml of MatTek assay medium (VEC- 100-ASY) per well. Plates were incubated overnight at 37°C in 5% CO2. On the following day, the tissues were infected with MPXV at lxlO5PFU for 3 hours at 37°C. The inoculum was then removed, and the tissues were treated apically with 35 pl of compounds (10 pM of 59, 20 pM of 56 and 50 pM of cidofovir). 24 hpi 200 pl of assay medium was applied to the tissue apicallyfor 20 min, and then the collected medium was used for DNA extraction or subsequent titration on Vero cells.Time of addition experimentsTo elucidate which stage of the VACV life cycle was targeted, compounds were serially diluted in high glucose DMEM + GlutaMAX, supplemented with 2.5% FBS + 1% P / S. For the pretreatment, compounds were incubated with the cells for either 1 hour at 37°C, followed by a wash and subsequent infection with VACV at 37°C for 1 hour. In the co-treatment protocol, virus and serial dilution of compounds were added on cells for 1 hour, followed by a washout of the inoculum. In the post-treatment protocol, the virus-containing medium was replaced with 100 pL of high glucose DMEM + GlutaMAX, supplemented with 2.5% FBS + 1% P / S and the compounds were introduced to the cells after 1, 2, and 4 hours. After one day, the viruscontaining medium was aspirated, the cells were fixed with 50 pL of formaldehyde, followed by addition of 40 pL of 4',6-diamidino-2-phenylindole (DAPI) and 100 pL of PBS. Infected cells were counted by ImageXpress PICO microscope. EC50 values were determined using GraphPad Prism 9.Real-time quantitative PCR (RT-qPCR)RNA from infected cells was isolated using EZNA total RNA extraction kit and eluted in 20 pL RNase-free water. Retro transcription and the amplification was performed with KAPA SYBR FAST One-Step (Sigma Aldrich). The targeted genes were VACV E3L with forward primer 5'-CGC AGA GAT TGT GTG TGA GGC-3' and reverse primer 5'-GGA GG A ATA TCG TCG GAG CTG- 3', VACV G8 with forward primer 5'-AAT GTA GAC TCG ACG GAT GAG TTA-3' and reverse primer 5'-TCG TCA TTA TCC ATT ACG ATT CTA GTT-3' and VACV F17 using forward primer 5'-ATT CTC ATT TTG CAT CTG CTC-3' and reverse primer 5'-AGC TAC ATT ATC GCG ATT AGC-3'. The fold change was calculated with the delta CT method in comparison with GADPH gene expression.Synergy evaluationVero E6 cells were seeded on 96-well plates at a density of 105cells and incubated overnight at 37 °C and 5 % CO2. The following day cells were infected (MOI 0.5) and treated with serial dilutions of compound MR59 and cidofovir or MR56 and cidofovir. The following day the number of infected cells were counted by ImageXpress PICO microscope (Molecular Devices). The synergy scores were calculated with SynergyFinder 3.0.Resistance selectionVero E6 cells were seeded on 6-well plates at a density of 3 x 105cells and incubated overnight at 37 °C and 5 % CO2. The following day cells were infected and either left untreated or treated with compound MR59 at an initial concentration corresponding to the EC50. When the cells displayed extensive cytopathic effect (2 to 3 days post infection), cells were mechanically detached and centrifuged at 2000 rpm for 5 min. The resultant culture supernatants were stored at -80 °C and titrated using a plaque assay in a 96-well plate. For subsequent passages, cells were infected with a volume of virus adjusted to the titer obtained from the previous passage's conditions at a 0.05 MOI for 1 h at 37 °C. The concentration of the antiviral compound was doubled if the viral titer was comparable to the untreated condition or kept unchanged.ResultsSelection of compounds on Monkeypox virusDue to limited therapeutic options for MPXV and the prevalent sexual transmission of the last epidemic, the inventors tested various compounds against this virus. The cells were infected, and the compounds added in dose response 1 hour post-infection. Cidofovir was included in the analysis as a reference compound. In parallel the compounds were tested to evaluate their cytotoxic potential on the same cell line. The results are shown in Table 3.Table 3. Antiviral activity against MPXVn.a. not assessable, EC50 50% effective concentration, CC50 50% cytotoxic concentration, SI selectivity indexAmong the tested compounds the control compound (cidofovir) showed activity, and compounds of the invention proved to be active against the virus. The control compound, cidofovir, showed as expected a large selectivity index, however its mechanism of action restrains its activity against DNA viruses and in vivo is reported to exert side effects.Antiviral testing on other sexually transmitted virusesTo evaluate the potential broad-spectrum activity of the compounds for sexually transmitted viruses the inventors evaluated their inhibitory potential against HSV-2, HSV-l and HIV (Figure 1). For HSV-2 (Figure 1A) and HSV-l (Figure IB), multiple cycles of replication were measured, therefore the compounds were added post-infection. In contrast for HIV a single round of replication was evaluated either by flow cytometry (Figure 1C) or by quantification of p24 in the supernatant (Figure ID). Therefore, to evaluate if the compounds could have an effect on any of the phases of the replication, the treatment at a fix dose of 10 pM was started before infection.For HSV-2, the three compounds showing inhibitory activity are MR15, IVIR56 and MR773, with the latter being more potent. For HSV-l only compounds IVIR56 and IVIR59 were tested, with compound MR56 showing a better inhibitory activity. For HIV, compounds MR54 and MR56 are significantly more active against HIV than DMSO in the reduction of the number of infected cells, but all show inhibitory activity (Figure 1C). When the supernatant of HIV infected cells was tested for HIV antigen presence, compound MR15 is the one showing a significative reduction if compared to DMSO, while also compound MR56 shows a reduction trend.The results evidence that the compound showing the larger spectrum of activity is the compound MR56. It is active against HSV-2 with an EC50 of 6.17 pM and against HSV-l with an EC50 of 5.57 pM. Moreover, it shows a significative inhibitory activity against HIV in the percentage of infected cells with 18.7% of residual infection, while the reduction of the release of p24 is not significative. However, from the Figure ID it is possible to see that in 2 out of 3 independent experiments also the release of p24 is inhibited, in concordance with the results of Figure 1C. Importantly in parallel to the antiviral activity, viability was measured in the same experimental conditions of the experiments and the compounds did not show any cytotoxicity at the effective doses.Antibacterial testing on chlamydia trachomatisTo evaluate if the broad-spectrum activity was maintained against other sexually transmitted pathogens, the inventors included in our analysis the intracellular bacterium Chlamydia Trachomatis. Cells were treated with compounds MR56 and MR58 and infected. The inhibition was evaluated either by evaluation of intracellular qPCR (Figure 2A) or by measuring infectious chlamydia in cell supernatants (Figure 2B). For both analysis it is possible to observe a significative inhibition of Chlamydia Trachomatis.Antiviral activity on a human derived tissue modelCompounds MR56 and MR59, in parallel with Cidofovir, were then evaluated in a vaginal tissue model (EpiVaginal, Mattek, USA) to evaluate the activity against MPXV in a more relevant model, considering the possible use of the compounds as vaginal microbicides. The treatment was started 3hpi and the supernatant of the tissues was collected at 24hpi. As it is possible to see in Figure 3, compounds MR56, MR59 and cidofovir, included as a positive control, reduced the release of infectious viruses also in this model.Investigation of the mechanism of actionCompounds MR56 and MR59 were tested on cell lines infected with Vaccinia virus (VACV) due to its genetic similarity to MPXV. The compounds, tested in parallel with cidofovir, were first evaluated for their ability to inhibit VACV across different strains, showing inhibitory activity at non-toxic doses (Table 4). These results demonstrate a conserved activity against MPXV and VACV and suggest that also other viruses of the Poxviridae family can be inhibited by the compounds.Table 4. Antiviral efficacy against VACVn.t. not testedThe mechanism of action of compound MR59 was then evaluated using time-of-addition studies. MR59 and cidofovir were added either before, during, or at various times postinfection. The results shown in Figure 4 indicate that MR59 retains antiviral activity even when added 4 hours post-infection, whereas cidofovir loses inhibitory activity. This suggests that the compounds have distinct mechanisms of action.To further investigate the stage of the viral cycle affected, RT-qPCR analysis was performed to evaluate the expression of early, intermediate, and late VACV genes at different time points post-infection. The results confirm that compounds IVIR59 and IVIR56 act via a different mechanism than cidofovir, which impairs viral DNA replication resulting in the inhibition of the intermediate and late genes. In contrast IVIR56 and IVIR59 do not show the same profile of gene expression inhibition shown by cidofovir (Figure 5). The different mechanism of action supports the possible use in combination of IVIR56 and IVIR59 with cidofovir, therefore a synergy test was performed. The results show areas of synergism for both IVIR56 and cidofovir (Figure 6a) and IVIR59 and cidofovir (Figure 6b), with a more marked synergism between IVIR59 and cidofovir highlighted by a Loewe score strictly above 10 (i.e synergism) (Figure 6).Finally, a key consideration in antiviral development is the evaluation of resistance potential. VACV was therefore passaged in the presence of increasing concentrations of IVIR59 (Figure 7a). After ten passages (plO), viruses grown with or without the compound were tested in a dose-response assay, which showed no emergence of resistance (Figure 7b).

Claims

CLAIMS1. A compound of formula (I):or a stereoisomer, polymorph, racemic mixture, tautomer, pharmaceutically acceptable salt, prodrug, hydrate, or solvate thereof, for use in the prevention or treatment of a sexually transmitted infection or disease, whereinRi is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, amino and -O-(CH2)qC=CH, wherein q is 0 to 2, preferably Ri is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, and amino;R2 is selected from C1-C4 alkoxy, hydrogen, C1-C4 alkyl, hydroxy, and amino; alternatively, either Ri or R2 can be -O-linker-protein-binding moiety;X is selected from N(Rs) and O, wherein R3 is H or C1-C4 alkyl, preferably X is N(Rs), more preferably X is NH;wherein p is 0 to 2;Rio is a carbocyclic group or a heterocyclic group, wherein the carbocyclic group or the heterocyclic group can be optionally substituted by hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.

2. A compound for use according to claim 1, whereinwherein q is 0 to 2;Rs is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;Re is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;R7 is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;Rs is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro;R9 is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro; and optionally, any two groups selected from Rs, Re, R7, Rs, and R9 which are adjacent to each other can be connected to form an optionally substituted carbocyclic moiety or an optionally substituted heterocyclic moiety, wherein the optional substituent is selected from hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro; or whereinwhereinRio is a heterocyclic group, wherein the heterocyclic group can be optionally substituted by hydrogen, halogen, C1-C4 alkyl, Hal-Ci-C4 alkyl, C1-C4 alkoxy, hydroxy, amino, and nitro.

3. The compound for use according to claim 1 or 2, whereinRi is C1-C4 alkoxy.

4. The compound for use according to claim 1 or 2, wherein the linker of the -O-linker-protein-binding moiety is selected from:wherein n is 1 to 5; and / or wherein the protein-binding moiety of the -O-linker-protein-binding moiety is selected from thalidomide, pomalidomide, Von Hippel-Lindau E3 ubiquitin ligase (VHL) binding moiety (VLM), a cereblon E3 ubiquitin ligase binding moiety (CLM), a mouse double miniute 2 homolog (MDM2) E3 ubiquitin ligase binding moiety (MLM), and an IAP E3 ubiquitin ligase binding moiety (I LM), preferably wherein the protein-binding moiety is pomalidomide.

5. The compound for use according to any one of claims 1 to 4, wherein R2 is selected from C1-C4 alkoxy and hydrogen.

6. The compound for use according to any one of claims 2 to 5, wherein Rs is selected from hydrogen, Hal-Ci-C4 alkyl and halogen.

7. The compound for use according to any one of claims 2 to 6, wherein Re is selected from hydrogen and halogen.

8. The compound for use according to any one of claims 2 to 7, wherein R? is selected from halogen, Hal-Ci-C4 alkyl, hydrogen, and nitro.

9. The compound for use according to any one of claims 2 to 8, wherein Rs is selected from halogen and hydrogen.

10. The compound for use according to any one of claims 2 to 9, wherein R9 is hydrogen.

11. The compound for use according to claim 1, wherein the compound is selected from 4-((2,4-dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((4-chlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 7-methoxy-4-((4-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile, 7-methoxy-4-(naphthalen-2-ylamino)quinoline-3-carbonitrile, 7-methoxy-4-((2-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile, 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((2,4-dichlorophenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile, 7-methoxy-4-(methyl(3-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile, 4-(cyclohexylamino)-7-methoxyquinoline-3-carbonitrile, 7-methoxy-4-(thiophen-3-ylamino)quinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)amino)-6-methoxyquinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)(methyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)(methyl)amino)-6-methoxyquinoline-3-carbonitrile, 4-(benzo[d][l,3]dioxol-5-ylamino)-7-methoxyquinoline-3-carbonitrile, 4-((l / 7-benzo[d][l,2,3]triazol-5-yl)amino)-7-methoxyquinoline-3-carbonitrile, 7-methoxy-4-((4-(trifluoromethyl)benzyl)amino)quinoline-3-carbonitrile, 4-((4-chlorobenzyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)amino)-7-(prop-2-yn-l-yloxy)quinoline-3-carbonitrile, and4-(naphthalen-2-ylamino)-7-(prop-2-yn-l-yloxy)quinoline-3-carbonitrile; preferably 4-((2,4-dichlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((4-chlorophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 4-((2-chloro-4-nitrophenyl)amino)-7-methoxyquinoline-3-carbonitrile, 7-methoxy-4-((4-(trifluoromethyl)phenyl)amino)quinoline-3-carbonitrile, and 7-methoxy-4-(naphthalen-2-ylamino)quinoline-3-carbonitrile.

12. The compound for use according to any one of claims 1 to 11, wherein the sexually transmitted infection or disease is selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum (Poxvirus), Gonorrhea (Neisseria gonorrhoeae), Mycoplasma genitalium, Ebola virus, Marburg virus and Lassa virus; wherein the sexually transmitted infection or disease is preferably selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), Chlamydia (Chlamydia trachomatis), Human Papillomavirus (HPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Molluscum Contagiosum, Ebola virus, Marburg virus and Lassa virus (Poxvirus); wherein the sexually transmitted infection or disease is more preferably selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV) - type 1 and type 2, Monkeypox virus (MPXV), and Chlamydia (Chlamydia trachomatis).

13. The compound for use according to any one of claims 1 to 11, wherein the compound is to be administered topically.

14. The compound for use according to any one of claims 1 to 11, wherein the compound is to be administered to the vagina, anus or rectum.

15. A method of preventing or treating a sexually transmitted infection or disease, comprising administering a therapeutically acceptable amount of the compound of formula (I) as defined in any one of claims 1 to 11 to a subject in need thereof.

16. Use of the compound of formula (I) as defined in any one of claims 1 to 11 for the preparation of a medicament for preventing or treating a sexually transmitted infection or disease.

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