HETEROARYLAMIDE DERIVATIVES AS SODIUM CHANNEL INHIBITORS

Abstract

The present invention generally relates to compounds of formula (I) inhibiting sodium channel (NaV) activity; particularly, the invention relates to compounds that are heteroaryl amide derivatives, including zwitterionic forms and pharmaceutically acceptable salts thereof, methods of preparing such compounds, and therapeutic use thereof. The compounds of the invention may be useful for instance in the treatment of many disorders associated with NaV, such as respiratory diseases.

Description

[0001] HETEROARYL AMIDE DERIVATIVES AS SODIUM CHANNEL INHIBITORS

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to compounds inhibiting voltage-gated sodium channels (NaV) activity (hereinafter NaV inhibitors); the invention relates to compounds that are heteroaryl amide derivatives, including pharmaceutically acceptable salts thereof, methods of preparing such compounds, and therapeutic use thereof.

[0004] The compounds of the invention may be useful for instance in the treatment of many disorders associated with sodium channel receptors mechanisms, such as respiratory diseases.

[0005] BACKGROUND OF THE INVENTION

[0006] Sensory afferents carry impulses originated by diverse stimuli (thermal, cold, heat, mechanical and chemical) and also mediate the cough reflex.

[0007] Coughing is a defensive reflex designed to protect the airways from foreign bodies and to aid in the removal of luminal debris. Chronic cough is defined as a cough lasting longer than 8 weeks. Hyper- and allo-tussive states are often chronic in nature lasting longer than three months and can be manifested in many airway diseases states including asthma, chronic obstructive pulmonary disease (COPD), asthma-COPD overlap syndrome (ACOS), cough variant asthma, gastroesophageal reflux disease (GERD), idiopathic pulmonary fibrosis (IPF) and lung cancer. In addition, inappropriate cough reflexes can be manifested acutely and chronically following viral infection. Finally, chronic cough can be idiopathic in nature with unknown etiology (Dicpinigaitis, P.V. et al, Pharmacol Rev, 2014, 66, 468- 512; Patil, M.J et al, Pulmonary Pharmacology& Therapeutics, 2019, 56, 15- 19).

[0008] As many therapeutic strategies for cough inhibition have been unsuccessful in clinical practice, increased attention in recent years has been focused on potential inhibitors of voltagegated sodium channels (NaV) which are absolutely required for the conduction of action potentials originated in nerve terminals as a response to a variety of stimuli including many inflammatory mediators. There are currently at least nine known members of the family of voltage-gated sodium channel (NaV) alpha subunits. The NaV family has been phylogenetically divided into two subfamilies NaV 1.x (all but SCN6A) and NaV 2.x (SCN6A). The NaV 1.x subfamily can be functionally subdivided into two groups, those which are sensitive to blocking by tetrodotoxin (TTX-sensitive or TTX-s) and those which are resistant to blocking by tetrodotoxin (TTX-resistant or TTX-r). Cough is a complex process triggered and regulated by two independent nerve pathways that can interfere with each other: mechanosensitive vagal nodose AS fibres and chemosensitive vagal jugular C-fibres. From the variety of known NaV 1.x subunits, only a few are expressed in airway vagal jugular C-fibres, involving TTX-sensitive NaV 1.7 and TTX- resistantNaV 1.8 and NaV 1.9 (Kollarik, M., et al, JPhysiol, 2018, 596, 1419- 1432). The NaV 1.7 (PN 1 , SCN9A) is sensitive to blocking by tetrodotoxin and is preferentially expressed in peripheral sympathetic and sensory neurons.

[0009] Cough reflex can be negated by effective sodium channel blockers, wherein Lidocaine is an example. Lidocaine blocks all the sensory inputs (such as in local anesthesia) and mechanoreceptors and motor neurons, leading to numbness and paralysis. A spray of lidocaine is routinely used before bronchoscopies to suppress the cough reflex. Inhalation of lidocaine has been firstly reported to relieve intractable cough in 1977 (C Fletcher et al, BrMedJ, 1977 1(6077), 1645- 8), and then confirmed in a recent clinical trial, formulated as throat spray, on refractory chronic cough (RCC) patients (Abdulqawi, R. et al., The Journal of Allergy and Clinical Immunology: In Practice, 2021, 9, 4, 1640-1647). However, Lidocaine is short-acting, non- selective, weakly potent, with off-target effects (burns when injected) and its use (as antitussive) is limited due to a poor risk / benefit profile (block of cardiac NaV 1.5 channels).

[0010] Sensory neurons express several channels (TRPV1, TRPA1, ASICS, P2X3 etc.) with large pores that, when activated, enable permeation of charged molecules that normally cannot permeate the neuron membrane, differently from the neutral molecules like lidocaine, that can access to the Nav binding site in the channel pore, penetrating into the lipid membrane surrounding the channel (Frazier et al, JPET, 2015; 171, 1.; Brenneis et al, J Neuroscience., 2013, 33(1):315-26).

[0011] In 1999 Nortran Pharmaceutical filed a patent application (WO99 / 63985) relating to pharmaceutical compositions having anti-tussive activity and a method of treating warm-blooded animals affected by coughs or bronchoconstriction by administering an effective amount of the pharmaceutical compositions of the invention. The exemplified active anti-tussive agent reported therein was a charged quaternary ammonium compound, namely N,N- bis(phenylcarbamoylmethyl)dimethylammonium chloride (aka Carcainium chloride).

[0012] Carcainium chloride had been disclosed for the first time in 1962 by Astra for use in the treatment of arrhythmia (US 3255207). The anti-tussive effects of Carcainium chloride have been investigated in various animal models: in guinea pigs’ model Carcainium chloride was shown to possess higher anti-tussive potency and somewhat a different mechanism of action than Lidocaine (J. J. Adcock, British Journal of Pharmacology, 2003, 138, 407- 416). In 2013 Verona Pharma filed a patent application (WO2013 / 03490) related to Carcainium in the form of a salt having an anion of a pharmaceutically acceptable acid, for use in the treatment and / or suppression of cough, tussive attacks or tussive episodes in patients. The compound was found efficacious in a pilot study conducted in patients with chronic cough and idiopathic interstitial pneumonias (Lavorini et al, Pulmonary Pharmacology & Therapeutics, 2016, 40, 91-94) but never progressed to the market because it failed to reduce frequency and severity of cough in a larger subsequent study (Satia et al, Thorax, 2015;70 (Suppl 3): A1-A254).

[0013] Currently available antitussive drugs have often limited efficacy. The quality of life of patients with cough can be severely affected, with physical (including fatigue, breathlessness, disturbed sleep and incontinence), psychological (including anger, frustration, depression and anxiety) and social consequences. Therefore, a continuous effort in the search for new promising therapies and targets in cough treatment is essential, and chronic cough remains an unmet medical need.

[0014] The state of the art does not describe or suggest the amide derivative compounds of general formula (I) of the present invention which may represent a solution to the afore mentioned unmet medical need.

[0015] SUMMARY OF THE INVENTION

[0016] In a first aspect the invention refers to a compound of formula (I) in zwitterionic or pharmaceutically acceptable salt form, wherein at least one of Y and Z is S, and the other one is CR4;

[0017] Ri and R2 are independently -(Ci-Ce)alkyl or are fused together forming a -(C3- C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from the group consisting of halogen, -(Ci-Ce)alkyl and -(Ci-Ce)alkyl- ORs;

[0018] R3 and R4 are independently H or -(Ci-Ce)alkyl, or, wherein Y is CR4, R3 and R4 are fused together forming an aryl;

[0019] Rs and Re are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(Ci- Ce)aminoalkyl, -(Ci-Ce)alkyl-OR8, -(Ci-Ce)alkyl-NR7R8, -(Ci-C4)alkyl-NR7-C(O)OR8, -(Ci- C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, or are fused together forming a - (C3-C7)heterocycloalkyl, wherein said -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3- C7)heterocycloalkyl, -(Ci-C6)aminoalkyl or -(Ci-Ce)alkyl-NR7R8 may be optionally substituted by one or more groups selected from -(Ci-Ce)alkyl, -C(0)0R8 and -SO2R7; R? and Rs are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(C3- Ce)cycloalkyl and -(Ci-Ce)haloalkyl, or are fused together forming a -(C3-C7)heterocycloalkyl;

[0020] Li is a bond or selected from the group consisting of -(Ci-Ce)alkylene- and -(Ci- C6)cycloalkylene-;

[0021] A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl may be optionally substituted by one or more groups selected from -ORs, - C(O)ORs, -C(O)NR?R8 and -(Ci-Ce)alkyl; provided that when Li is a bond any of such aryl is not substituted by -(Ci-Ce)alkyl.

[0022] In a second aspect, the invention refers to a pharmaceutical composition comprising a compound of formula (I) in admixture with one or more pharmaceutically acceptable carrier or excipient.

[0023] In a third aspect, the invention refers to a compound of formula (I) for use as a medicament.

[0024] In a further aspect, the invention refers to a compound of formula (I) for use in treating diseases, disorders or conditions associated with dysregulation of voltage-gated sodium channels (NaV).

[0025] In a further aspect, the invention refers to a compound of formula (I) for use in the prevention and / or treatment of respiratory diseases selected from cough, sub-acute or chronic cough, treatment-resistant cough, refractory chronic cough, idiopathic chronic cough, post-viral cough, iatrogenic cough, asthma, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and cough associated with respiratory diseases such as COPD, asthma, and bronchospasm.

[0026] DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise provided, the term “compound of formula (I)” comprises in its meaning stereoisomers, tautomers, pharmaceutically acceptable salts, zwitterionic forms or solvates thereof.

[0028] The term "stereoisomer" refers to isomers of identical constitution that differ in the arrangement of their atoms in space. Enantiomers and diastereomers are examples of stereoisomers.

[0029] The term "enantiomer" refers to one of a pair of molecular species that are mirror images of each other and are not superimposable.

[0030] The term "diastereomer " refers to stereoisomers that are not mirror images.

[0031] The term "racemate" or "racemic mixture" refers to a composition composed of equimolar quantities of two enantiomeric species, wherein the composition is devoid of optical activity.

[0032] The symbols "R" and "S" represent the configuration of substituents around a chiral carbon atom(s) and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68:2193-2222 (1996)).

[0033] The term "tautomer" refers to each of two or more isomers of a compound that exist together in equilibrium and are readily interchanged by migration of an atom or group within the molecule.

[0034] The compound of formula (I) is a quaternary ammonium compound. The term “pharmaceutically acceptable salt”, as used herein, refers to a compound of formula (I), wherein such compound is provided as an addition salt with any acid conventionally intended as being pharmaceutically acceptable. Suitable examples of said salts may thus include mineral or organic acid addition salts of the quaternary ammonium moiety, such as salts of hydrochloric acid, hydrobromic acid, iodic acid, formic acid, benzoic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, fumaric acid, succinic acid, 2-naphthoic acid, tartaric acid, 1- hydroxy-2-naphthoic acid, naphthalene-2,7-disulfonic acid and citric acid.

[0035] Alternatively, the compound of formula (I) according to the invention may be provided as a “zwitterionic form”, also referred to as inner salt or dipolar ion, which is a form wherein both a positive electrical charge and a negative electrical charge are present.

[0036] The term "solvate" means a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances, the solvate might be isolated by crystallization, for example, when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The solvate may comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules.

[0037] The term “halogen” or “halogen atoms” or “halo” as used herein includes fluorine, chlorine, bromine and iodine atom.

[0038] The term "-(Cx-Cy)alkyl" wherein x and y are integers, refers to a straight or branched chain alkyl group having from x to y carbon atoms. Thus, when x is 1 and y is 6, for example, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n- hexyl.

[0039] The term "-(Cx-Cy)alkylene-" wherein x and y are integers, refers to a (Cx-Cy)alkyl radical having in total two unsatisfied valences. Example of such "-(Cx-Cy)alkylene-" groups may include for example a divalent methylene radical and -CH(CH3)-.

[0040] The term “-(Cx-Cy)haloalkyl” wherein x and y are integers, refers to the above defined “(Cx-Cy)alkyl” groups wherein one or more hydrogen atoms are replaced by one or more halogen atoms, which can be the same or different. Examples of said “-(Cx-Cy)haloalkyl” groups may thus include halogenated, poly-halogenated and fully halogenated alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, e.g. trifluoromethyl. The term "-(Cx-Cy)aminoalkyl” wherein x and y are integers, refers to the above defined “(Cx-Cy)alkyl” groups wherein one or more hydrogen atoms are replaced by one or more amino group, which can be the same or different. Examples of "-(Cx-Cy)aminoalkyl” include, for instance, aminomethyl.

[0041] The term “-(Cx-Cy)hydroxy alkyl” wherein x and y are integers, refers to the above defined “-(Cx-Cy)alkyl” groups wherein one or more hydrogen atoms are replaced by one or more one or more hydroxy (OH). Examples of said “-(Cx-Cy)hydroxyalkyl” include, for instance, hydroxymethyl.

[0042] The term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0043] The term “-(Cx-Cy)cycloalkyl” wherein x and y are integers, refers to saturated or partially unsaturated mono- or poly-cyclic hydrocarbon groups containing the indicated number of ring carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl.

[0044] The term "(Cx-Cy)cycloalkylene” wherein x and y are integers, refers to saturated or partially unsaturated mono- or poly- (Cx-Cy)cycloalkyl groups containing the indicated number of ring carbon atoms, having in total two unsatisfied valences on the same carbon atom. Examples of “- (Cx-Cy)cycloalkylene-“ include “-(Ci-C4)cycloalkylene-“ and “-(Ci-C6)cycloalkylene-“, for instance cyclopropylene.

[0045] The term “-(Cx-Cy)heterocycloalkyl” wherein x and y are integers, refers to saturated or partially unsaturated monocyclic or polycyclic or polycyclic spiro groups, containing the indicated number of ring carbon atoms, in which at least one ring carbon atom is replaced by at least one heteroatom or hetero-group (e.g. N, NH, S or O) or bears an oxo (=0) substituent group. The said heterocycloalkyl (i.e. heterocyclic radical or group) might be further optionally substituted on the available positions in the ring, namely on carbon atoms, or on heteroatoms or hetero-groups available for substitution, by one or more specific groups. When more than one substituent are present, these may involve the same or adjacent positions in the ring, e.g. the “(C3- Cio)heterocycloalkyl” may be 1,1- or 1,2- or 1,3- di substituted. Substitution on a carbon atom includes spiro di -substitution, wherein an additional spiro condensed 3- to 6-membered ring is formed. Such spiro di -substitution is included in the meaning of “(C3-Cio)heterocycloalkyl” as polycyclic spiro group. Non limiting examples of “(Cx-Cy)heterocycloalkyl” are represented by pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, dihydro- or tetrahydro- pyridinyl, oxetanyl, isoindolyl, tetrahydropyranyl, pyranyl, dihydro- or tetrahydrofuranyl. The term “-(Cx-Cy)heterocycloalkylene-“ wherein x and y are integers, refers to saturated or partially unsaturated mono- or poly- (Cx-Cy)heterocycloalkyl groups containing the indicated number of ring carbon atoms, having in total two unsatisfied valences on the same carbon atom.

[0046] A group may be optionally substituted, wherein the term “optionally substituted” refers to being substituted or unsubstituted. When the term "one or more " refers to any atoms or groups as substituents of the groups of the compound of formula (I), it is intended that from 1 to 3, preferably 1 to 2, more preferably 1 of such substituents may replace hydrogens on such variables.

[0047] The term “aryl” refers to mono- or bi-cyclic unsaturated or partially unsaturated carbon ring systems wherein at least one ring is aromatic. Examples of suitable aryl ring systems include, for instance, phenyl, dihydro- IH-indenyl or naphthyl.

[0048] The term “heteroaryl” refers to mono- or bi- or tri-cyclic ring systems with 5 to 20, preferably from 5 to 15 ring atoms, in which at least one ring is aromatic and in which at least one ring atom is a heteroatom (e.g. N, NH, S or O). Examples of “heteroaryl” include thiophenyl, benzothiophenyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl.

[0049] A bond pointing to a wavy or squiggly line, such as as used in structural formulas herein, depicts the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

[0050] The term "bond" used to define a substituent refers to the situation where the two functional groups which the substituent is connected to are directly linked to each other with no additional atoms in between.

[0051] A dash (“-”) that is not between two letters or symbols is meant to represent the point of attachment for a substituent.

[0052] The terms “sodium channel receptors” or “voltage-gated sodium channels receptors” or “NaV receptors” used to refer to the inhibitory activity of the compounds of the present invention, are meant to include the members of the family of voltage-gated sodium channel (NaV) alpha subunits which are expressed in airway vagal jugular C-fibres, i.e. NaV 1.7, NaV 1.8 and NaV 1.9.

[0053] The term “NaV inhibitor” refers to the compounds of present invention as inhibitors of NaV receptors, in particular, but not limited, NaV 1.7 receptor.

[0054] The term “ICso” refers to the half maximal inhibitory concentration as a measure of the potency of a substance in inhibiting a specific biological or biochemical function.

[0055] The term “pICso” refers to the negative logarithm of the ICso value expressed as molar concentration.

[0056] Whenever other basic amino or quaternary ammonium groups are present in the compounds of formula (I), pharmaceutically acceptable anions may be present, selected among chloride, bromide, iodide, trifluoroacetate, formate, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, p-toluenesulfonate, pamoate, emipamoate, xinafoate and naphthalene disulfonate. Likewise, in the presence of acidic groups, such as for instance COOH groups, corresponding pharmaceutically acceptable cations may be present, for instance including alkaline or alkaline earth metal ions, including sodium, potassium, calcium and the like.

[0057] As above indicated, the present invention refers to a series of compounds represented by the general formula (I), as herein below described in detail, with inhibitory activity versus voltagegated sodium channels receptors.

[0058] Inhibitory activity of the compounds of formula (I) was tested on CHO cells stably expressing human NaV 1.7 channel in an Automated Patch Clamp assay, as described in the experimental part.

[0059] For some compounds of the invention pICso values were calculated, demonstrating the intracellular inhibition of the NaV 1.7 receptor by the tested compounds (see Qian, Binbin et al, Current protocols in pharmacology, 2020, 89, 1).

[0060] Indeed, the compounds of formula (I) of the present invention are able to act as inhibitors of NaV 1.7 in a substantive and effective way. In particular, as indicated in the experimental part, the compounds of formula (I) of the invention show intracellular inhibition of NaV 1.7, as reported in Table 2 for representative compounds.

[0061] The compounds of formula (I) are heteroaryl quaternary ammonium amide derivatives in salt or zwitterionic form, having a good inhibitory activity in particular on NaV 1.7.

[0062] As indicated in the experimental part, comparative compounds section, in particular in Table 4, conversely to Carcainium chloride, which is considered as comparative Compound Ci, the presence of an ami de- substituted tiophene or benzothiophene ring in the compounds of the present invention unexpectedly and remarkably determines a relevant increase in the intracellular inhibitory activity on NaV 1.7 receptor.

[0063] Furthermore, as indicated in the same experimental part section, the reported data demonstrate that, conversely to comparative Compound C2, characterized by a different heteroaryl ring, the presence of the amide-substituted thiophene or benzothiophene ring in the compounds of the present invention unexpectedly and noteworthy determines a relevant increase in the inhibitory activity on NaV receptors.

[0064] Compounds of formula (I) in pharmaceutically acceptable salt or zwitterionic form, hereinafter alternatively referred to as compounds of the invention, are useful as medicaments. Accordingly, the invention also provides a compound of formula (I) in pharmaceutically acceptable salt or zwitterionic form for use as a medicament.

[0065] The compounds of formula (I) of the present invention are able to act as inhibitors of NaV 1.7 receptor in a substantive and effective way, particularly appreciated by the skilled person when looking for compounds for the potential treatment of respiratory diseases such as cough, asthma, IPF and COPD, and in particular chronic cough.

[0066] Accordingly, the compounds of formula (I) can be used in the treatment of respiratory diseases selected from cough, asthma, IPF and COPD, and in particular chronic cough, whenever NaV receptors are involved.

[0067] As it can be appreciated, the compounds of the present invention according to Table 2 show a potency with respect to their intracellular inhibitory activity on NaV 1.7 receptor, expressed as pICso values, equal to or higher than 5, preferably between 5 and 5.5, more preferably equal to or higher than 5.5.

[0068] More advantageously, the compounds represented by the general formula (I) are able of inhibiting one or more voltage-gated ion channels when exposed or applied into the cell but show little or no inhibition when applied outside the cell.

[0069] The compounds of formula (I) were also tested in Automated Patch Clamp assay with a similar protocol for extracellular inhibition, i.e. after exchange of the external solution with a solution containing the test compound: the obtained experimental values were used to calculate pICso values representing the extracellular inhibition of NaV 1.7 receptor.

[0070] As indicated in the experimental part, the compounds of formula (I) of the present invention have a NaV 1.7 extracellular inhibition lower than the intracellular inhibition, as shown in Table 2.

[0071] Accordingly, the compounds of the present invention according to Table 2 show a difference between intracellular and extracellular potency with respect to NaV 1.7 inhibition, defined as DELTA, which is equal to or higher than 0,5, preferably between 0,5 and 1, more preferably equal to or higher than 1.

[0072] This difference between intracellular and extracellular inhibition could result in a greater safety of the compounds on sensory neurons.

[0073] The compounds of the invention were tested also in safety pharmacological assays, in particular in human Ether-a-go-go Related Gene (hERG) channel in vitro assay. Safety pharmacological studies are non-clinical studies to test the safety of novel drugs on human from pharmacological viewpoints. Guideline on Safety Pharmacology Studies which aims at examining the safety of test substances on human and predicting adverse effects thereof has been set in the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) between Europe, Japan and the United States. According to the Guideline, examination of the arrhythmogenic effect, in particular, the presence or absence of electrocardiographic QT interval prolongation effect, of test substances is required as a part of safety pharmacological studies. The QT interval is a measurement made on an electrocardiogram used to assess some of the electrical properties of the heart and approximates to the time taken from when the cardiac ventricles start to contract to when they finish relaxing. In order to protect patients from ventricular tachycardia, torsades de pointes and lethal arrhythmia associated with QT interval prolongation induced by drug administration, it is very important in the development of drugs to detect QT interval prolongation effect which may induce such serious adverse effects.

[0074] To date, it has been known that a large number of drugs having QT interval prolongation effect inhibit delayed rectifier potassium channels in cardiomyocytes. It is believed that hERG channel is functioning as a major constituent protein in the delayed rectifier potassium channel. Therefore, in the draft guideline for non-clinical evaluation of the potential for delayed ventricular repolarization (QT Interval Prolongation) by human pharmaceuticals, ion channel assay using hERG channel-transferred cells is recommended as a non-clinical study.

[0075] In addition to being notably potent with respect to their inhibitory activity on NaV receptors, the compounds of formula (I) of the present invention were tested for in vitro inhibition of the hERG channel and showed low toxicity profile for hERG, thus reducing the possibility to observe adverse events during clinical studies.

[0076] Thus, in one aspect the present invention relates to a compound of general formula (I) in zwitterionic or pharmaceutically acceptable salt form, wherein at least one of Y and Z is S, and the other one is CR4;

[0077] Ri and R2 are independently -(Ci-Ce)alkyl or are fused together in a -(C3- C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from the group consisting of halogen, -(Ci-Ce)alkyl and -(Ci-Ce)alkyl- ORs;

[0078] RJ and R4 are independently H or -(Ci-Ce)alkyl, or, wherein Y is CR4, Rj and R4 are fused together forming an aryl; Rs and Re are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(Ci- Ce)aminoalkyl, -(Ci-Ce)alkyl-OR8, -(Ci-Ce)alkyl-NR7R8, -(Ci-C4)alkyl-NR7-C(O)OR8, -(Ci- C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, or are fused together forming a - (C3-C7)heterocycloalkyl, wherein said -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3- C7)heterocycloalkyl, -(Ci-C6)aminoalkyl or -(Ci-Ce)alkyl-NR7R8 may be optionally substituted by one or more groups selected from -(Ci-Ce)alkyl, -C(O)OR8 and -SO2R7;

[0079] R7 and Rs are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(C3- Ce)cycloalkyl and -(Ci-Ce)haloalkyl, or are fused together forming a -(C3-C7)heterocycloalkyl;

[0080] Li is a bond or selected from the group consisting of -(Ci-Ce)alkylene- and -(Ci- C6)cycloalkylene-;

[0081] A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl may be optionally substituted by one or more groups selected from -ORs, - C(O)ORs, -C(O)NR?R8 and -(Ci-Ce)alkyl; provided that when Li is a bond any of such aryl is not substituted by -(Ci-Ce)alkyl.

[0082] All the listed groups for each of the variable moieties Y, Z, Ri, R2, Rj, Rt, Rs, Re, R7, Rs, Li and A of the compounds of the invention have to be intended as alternatives and may be combined with each other in embodiments which are included in the scope of the invention.

[0083] The 5-membered ring comprising Y and Z is an aromatic ring, namely a thiophene ring, substituted by a -C(O)NRsR6 group.

[0084] Preferred halogens, as such and in -(Cx-Cy)haloalkyl substituents, are fluorine and bromine, wherein fluorine is more preferred.

[0085] In a preferred embodiment Z is S and Y is CR4. In a more preferred embodiment, Z is S and Y is CH.

[0086] In another embodiment, Y is S and Z is CR4, wherein R4 is preferably hydrogen.

[0087] In one embodiment, Ri and Rz are independently -(Ci-Ce)alkyl or are fused together in a - (C3-C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from the group consisting of halogen, -(Ci-Ce)alkyl and -(Ci-Ce)alkyl- OR8. In a preferred embodiment, Ri and Rz are independently -(Ci-Ce)alkyl. In a more preferred embodiment, Ri and Rz are independently -(Ci-C4)alkyl. In an even more preferred embodiment, Ri and Rz are methyl. In another preferred embodiment, Ri and Rz are fused together forming a - (C3-C7)heterocycloalkyl, optionally substituted by one or more groups selected from the group consisting of halogen, -(Ci-Ce)alkyl and -(Ci-Ce)alkyl-OR8. In a more preferred embodiment, Ri and Rz are fused together forming a -(C3-C7)heterocycloalkyl, optionally substituted by one or more groups selected from halogen, methyl and -(Ci-C4)alkyl-OR8. In a most preferred embodiment, Ri and Rz are fused together in an optionally substituted piperidinyl or azepanyl. In one embodiment, R3 and R4 are independently H or -(Ci-Ce)alkyl, or, wherein Y is CR4, R3 and R4 are fused together forming an aryl. In another embodiment R3 and R4 are H. In a more preferred embodiment, R3 and R4 are independently H or -(Ci-C4)alkyl; even more preferably, R3 and R4 are independently H or methyl, most preferably R3 is methyl and R4 is H. In a most preferred embodiment, Z is S and Y is CH, and R3 is H or methyl.

[0088] In another preferred embodiment, R3 and R4 are fused together forming an optionally substituted phenyl ring, which is condensed with the 5-membered ring thus forming an optionally substituted benzothiophene ring system.

[0089] In one embodiment, R5 and Re are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(Ci-C6)aminoalkyl, -(Ci-Ce)alkyl-OR8, -(Ci-Ce)alkyl-NR7R8, -(Ci-C4)alkyl-NR?- C(O)OR8, -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, or are fused together forming a -(C3-C7)heterocycloalkyl, wherein said -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, -(Ci-C6)aminoalkyl or -(Ci-Ce)alkyl-NR7R8 may be optionally substituted by one or more groups selected from -(Ci-Ce)alkyl, -C(O)OR8 and -SO2R7. In another embodiment, R5 and Re are independently H or selected from the group consisting of -(Ci- Ce)alkyl, -(Ci-Ce)aminoalkyl, -(Ci-Ce)alkyl-OR8, -(Ci-Ce)alkyl-NR7R8, -(Ci-C4)alkyl-NR?- C(O)OR8, -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, wherein said - (Ci-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, -(Ci-Ce)aminoalkyl or -(Ci- Ce)alkyl-NR7R8 may be optionally substituted by one or more groups selected from -(Ci-Ce)alkyl, -C(O)OR8 and -SO2R7. In a preferred embodiment, R5 and Re are independently H or -(Ci- Ce)alkyl. In a more preferred embodiment, Rs and Re are independently H or -(Ci-C4)alkyl. In a more preferred embodiment, Rs and Re are independently H or Me. In another preferred embodiment, Rs and Re are independently selected from the group consisting of H, -(Ci- C4)aminoalkyl, -(Ci-C4)alkyl-OR8, -(Ci-C4)alkyl-NR?R8, -(Ci-C4)alkyl-NR7-C(O)OR8, -(Ci- C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, wherein said -(Ci-C4)alkyl-(C3- C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, -(Ci-C4)aminoalkyl or -(Ci-C4)alkyl-NR?R8 may be optionally substituted by one or more groups selected from -(Ci-C4)alkyl, -C(O)OR8 and - SO2R7. In another embodiment, Rs and Re are fused together in a -(C3-C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from -(Ci-Ce)alkyl, -C(O)OR8 and -SO2R7. In a preferred embodiment, Rs and Re are fused together in a -(C3-C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from -(Ci-C4)alkyl. In a more preferred embodiment, Rs and Re are fused together in a -(C4-C7)heterocycloalkyl selected from the group consisting of pyrrolidine, piperazine and morpholine, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from -(Ci-C4)alkyl. In one embodiment, R? and Rs are independently H or selected from the group consisting of -(Ci-Ce)alkyl, -(C3-C6)cycloalkyl and -(Ci-Ce)haloalkyl, or are fused together forming a -(C3- C7)heterocycloalkyl. In a preferred embodiment, R7 and Rs are independently H or -(Ci-C4)alkyl. In another preferred embodiment, R7 and Rs are independently H or methyl. In a further preferred embodiment, R7 and Rs are H or R7 and Rs are methyl.

[0090] In one embodiment, Li is a bond or selected from the group consisting of -(Ci-Ce)alkylene- and -(Ci-C6)cycloalkylene-. In a preferred embodiment, Li is a bond or selected from the group consisting of -(Ci-C4)alkylene- and -(Ci-C4)cycloalkylene-. In another preferred embodiment, Li is a bond. In another more preferred embodiment, Li is selected from the group consisting of methylene, cyclopropylene and -CH(CH )-.

[0091] In one embodiment, A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl may be optionally substituted by one or more groups selected from -ORs, -C(O)ORs, -C(O)NR?R8 and -(Ci-Ce)alkyl; provided that when Li is a bond any of such aryl is not substituted by -(Ci-Ce)alkyl. In a preferred embodiment, A is heteroaryl, optionally substituted by one or more groups selected from -ORs, -C(O)OR8, -C(O)NR?R8 and - (Ci-Ce)alkyl. In another preferred embodiment, A is heteroaryl selected from the group consisting of optionally substituted thiophenyl, benzothiophenyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl and triazinyl. In another preferred embodiment, A is an aryl, optionally substituted by one or more groups selected from - OR8, -C(O)OR8, -C(O)NR?R8 and -(Ci-Ce)alkyl, provided that when Li is a bond any of such aryl is not substituted by -(Ci-Ce)alkyl. In a preferred embodiment, A is an aryl, wherein any of such aryl may be optionally substituted by one or more groups selected from -ORs, -C(O)OR8 and - C(O)NR?R8. In a more preferred embodiment, A is selected from the group consisting of phenyl, tiophenyl and isoxazolyl, any of which are optionally substituted by one or more groups selected from -ORs, -C(O)OR8 and -C(O)NR?R8. In other preferred embodiments, A is phenyl or selected from unsubstituted isoxazolyl or isoxazolyl substituted by one or more methyl.

[0092] All the preferred groups listed above for each of the variable moieties Y, Z, Ri, R2, R3, R>, Rs, Re, R7, Rs, Li, and A of the compounds of the invention may be combined with each other in embodiments which are included in the scope of the invention.

[0093] In another preferred embodiment, the invention refers to a compound of formula (I) as defined above wherein

[0094] Z, Y, Ri, R2, Rj, R4, Rs, Re, R7, Rs, are defined as above;

[0095] Li is selected from the group consisting of -(Ci-C4)alkylene- and -(Ci-C4)cycloalkylene-; and A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl may be optionally substituted by one or more groups selected from -OR8, - C(O)OR8, -C(O)NR7R8and -(Ci-C4)alkyl.

[0096] In a preferred embodiment, Li is selected from the group consisting of -(Ci-C4)alkylene and -(Ci-C4)cycloalkylene. In an even more preferred embodiment, Li is selected from the group consisting of methylene, cyclopropylene and -CH(CH3)-.

[0097] In another preferred embodiment, A is a ring selected from aryl and heteroaryl, wherein any of such aryl and heteroaryl may be optionally substituted by one or more groups selected from - OR8, -C(O)OR8and -C(O)NR7R8. In a more preferred embodiment, A is heteroaryl, wherein any of such heteroaryl may be optionally substituted by one or more groups selected from -OR8, - C(O)OR8, -C(O)NR7R8. In another more preferred embodiment, A is an aryl, wherein any of such aryl may be optionally substituted by one or more groups selected from -OR8, -C(O)OR8and - C(O)NR7R8.

[0098] In another embodiment, the invention refers to a compound of formula (I) wherein Li is a bond, represented by formula (la): in zwitterionic or pharmaceutically acceptable salt form, wherein

[0099] Z, Y, Ri, R2, R3, t, Rs, Re, R7and Rs are defined as above; and

[0100] A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl may be optionally substituted by one or more groups selected from -OR8, -C(O)OR8and - C(O)NR7R8, and wherein any of such heteroaryl may be optionally substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8and -(Ci-Ce)alkyl.

[0101] In a more preferred embodiment, A is heteroaryl, wherein any of such heteroaryl may be optionally substituted by one or more -OR8, -C(O)OR8, -C(O)NR7R8and -(Ci-Ce)alkyl. In an even more preferred embodiment, A is heteroaryl, wherein any of such heteroaryl is selected from the group consisting of thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl and triazinyl, and any of such heteroaryl may be optionally substituted by one or more methyl, ethyl, isopropyl or tert-butyl.

[0102] In another preferred embodiment, the invention refers to a compound of formula (I) as defined above, wherein at least one of Y and Z is S, and the other one is CR4;

[0103] Ri and R2 are independently -(Ci-C4)alkyl or are fused together in a -(C3- C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl may be optionally substituted by one or more groups selected from the group consisting of halogen, -(Ci-C4)alkyl and -(Ci-C4)alkyl- OR8;

[0104] RJ and R4 are independently H or -(Ci-Ce)alkyl, or wherein Y is CR4, Rj and R4 are fused together forming an aryl, preferably an optionally substituted aryl;

[0105] Rs and Re are independently H or selected from the group consisting of -(Ci-C4)alkyl, -(Ci- C4)aminoalkyl, -(Ci-C4)alkyl-OR8, -(Ci-C4)alkyl-NR?R8, -(Ci-C4)alkyl-NR7-C(O)OR8, -(Ci- C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, or are fused together forming a -(C3- C7)heterocycloalkyl, wherein said -(Ci-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3- C7)heterocycloalkyl, -(Ci-C4)aminoalkyl or -(Ci-C4)alkyl-NR?R8may be optionally substituted by one or more groups selected from -(Ci-C4)alkyl, -C(O)OR8and -SO2R7;

[0106] R7 and Rs are independently H or selected from the group consisting of -(Ci-C4)alkyl, -(C3- Ce)cycloalkyl and -(Ci-C4)haloalkyl, or are fused together forming a -(C6-C7)heterocycloalkyl;

[0107] Li is a bond or selected from the group consisting of -(Ci-C4)alkylene and -(Ci- C4)cycloalkylene;

[0108] A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl may be optionally substituted by one or more groups selected from -OR8, - C(O)OR8, -C(O)NR?R8and -(Ci-C4)alkyl; provided that when Li is a bond any of such aryl is not substituted by -(Ci-C4)alkyl.

[0109] In a preferred embodiment, Ri and R2 are fused together in a -(C6-C7)heterocycloalkyl, wherein said -(C6-C7)heterocycloalkyl is optionally substituted by one or more groups selected from -OR8, -(Ci-Ce)alkyl and -(C3-C7)heterocycloalkyl.

[0110] In another preferred embodiment, the invention refers to a compound of formula (I) as defined above, wherein Z is S and Y is CH or Z is CH and Y is S; Rj is H or methyl, and Ri, Rz, Rs, Re, R7, Rs, Li, A are defined as above.

[0111] In a preferred embodiment, the invention refers to at least one of the compounds listed in Table 1 below in zwitterionic form or as pharmaceutically acceptable salts thereof. Table 1 - List of preferred compounds It is to be understood that all the single enantiomers, diastereoisomers and mixtures thereof, in any proportion, of the compounds of formula (I) of the invention are encompassed within the scope of the present invention.

[0112] In a preferred embodiment, the invention refers to a compound of formula (I) as NaV inhibitor. In this respect, it has been found that the compounds of formula (I) of the present invention have an inhibitor drug potency expressed as pICso on NaV 1.7 receptor equal to or higher than 5.

[0113] Preferably, the compounds of the present invention have a pICso on NaV 1.7 between 5 and 5.5. More preferably, the compounds of the present invention have a pICso on NaV 1.7 equal to or higher than 5.5. Even more preferably the compounds of the present invention have a pICso on NaV 1.7 between 5.5 and 6.

[0114] The present invention also refers to a pharmaceutical composition comprising a compound of formula (I) in zwitterionic or a pharmaceutically acceptable salt form, in admixture with at least one or more pharmaceutically acceptable carrier and / or excipient.

[0115] As used herein, "effective amount" in reference to a compound of formula (I), in zwitterionic or pharmaceutically acceptable salt form, or other pharmaceutically active agent means an amount of the compound sufficient to treat the patient's condition but low enough to avoid serious side effects and it can nevertheless be routinely determined by the skilled artisan.

[0116] The compounds of formula (I) may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. Typical daily dosages may vary depending upon the route of administration chosen.

[0117] In one embodiment, the invention refers to a pharmaceutical composition of compounds of formula (I) in admixture with one or more pharmaceutically acceptable carrier or excipient, for example those described in Remington’s Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., N.Y., U.S.A.

[0118] In therapeutic use, the compound of formula (I) may be administered by any convenient, suitable, or effective route. Administration of the compounds of the invention and their pharmaceutical compositions may be accomplished according to patient needs, for example, orally, nasally, parenterally (subcutaneously, intravenously, intramuscularly, intrastemally and by infusion) and by inhalation.

[0119] Preferably, the compounds of the present invention are administered orally or by inhalation.

[0120] In a more preferred embodiment, the compounds of the present invention or their pharmaceutical compositions are administered by inhalation.

[0121] In another preferred embodiment, the compounds of the present invention or their pharmaceutical compositions are administered orally. In one preferred embodiment, the pharmaceutical composition comprising the compound of formula (I) in zwitterionic or pharmaceutically acceptable salt form is a solid oral dosage form such as tablets, gel caps, capsules, caplets, granules, lozenges and bulk powders.

[0122] The compounds of the invention can be administered alone or combined with various pharmaceutically acceptable carriers, diluents (such as sucrose, mannitol, lactose, starches) and known excipients, including suspending agents, solubilizers, buffering agents, binders, disintegrants, preservatives, colorants, flavorants, lubricants and the like.

[0123] In a further embodiment, the pharmaceutical composition comprising a compound of formula (I) is a liquid oral dosage form such as aqueous and non-aqueous solution, emulsion, suspension, syrup. Such liquid dosage form can also contain suitable known inert diluents such as water and suitable known excipients such as preservatives, wetting agents, sweeteners, flavorants, as well as agents for emulsifying and / or suspending the compounds of the invention.

[0124] In a further embodiment, the pharmaceutical composition comprising the compound of formula (I) is an inhalable preparation such as inhalable powders, propellant-containing metering aerosols or propellant-free inhalable formulations. Inhalable preparations may be administered through a suitable inhalation device which may be selected from dry powder inhalers, pressurized metered dosed inhalers or nebulizers.

[0125] In another embodiment the invention is also directed to a device comprising a pharmaceutical composition comprising a compound of formula (I) according to the invention, in zwitterionic or pharmaceutically acceptable salt form, obtained as described above according to the invention, in form of a single- or multi-dose dry powder inhaler or a metered dose inhaler.

[0126] For administration as a dry powder, single- or multi-dose inhalers known from the prior art may be utilized. In that case the powder may be filled in gelatine, plastic or other capsules, cartridges, or blister packs or in a reservoir.

[0127] A diluent or carrier, chemically inert to the compounds of the invention, e.g. lactose or any other additive suitable for improving the respirable fraction, may be added to the powdered compounds of the invention.

[0128] Inhalation aerosols containing propellant gas such as hydrofluoroalkanes may contain the compounds of the invention either in solution or in dispersed form. The propellant-driven formulations may also contain other ingredients such as co-solvents, stabilizers, and optionally other excipients.

[0129] The propellant-free inhalable formulations comprising the compounds of the invention may be in form of solutions or suspensions in an aqueous, alcoholic or hydroalcoholic medium and they may be delivered by j et or ultrasonic nebulizers known from the prior art or by soft-mist nebulizers. The compounds of the invention can be administered as the sole active agent or in combination with other pharmaceutical active ingredients.

[0130] The dosages of the compounds of the invention depend upon a variety of factors including among others the particular disease to be treated, the severity of the symptoms, the route of administration and the like.

[0131] In a further aspect, the invention refers to the use of the compounds of formula (I) for the preparation of a medicament. In another aspect, the present invention refers to a compound of formula (I) for use as a medicament.

[0132] Thus, the invention refers to a compound of formula (I) in the preparation of a medicament, preferably for use in the treatment of disorders associated with voltage-gated sodium channels (NaV) receptors mechanism.

[0133] In a further embodiment, the present invention refers to a compound of formula (I) for use in the prevention and / or treatment of diseases, disorders or conditions associated with dysregulation of voltage-gated sodium channels (NaV).

[0134] In one aspect, the invention also refers to a method for the prevention and / or treatment of disorders associated with NaV receptors mechanisms, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula (I).

[0135] Preferably, the compounds of the present invention are useful for the treatment and / or prevention of respiratory diseases.

[0136] In a preferred embodiment, the present invention provides a method for preventing and / or treating respiratory diseases, the method comprising administering a compound of formula (I).

[0137] In another aspect, the present invention is directed to a pharmaceutical composition comprising the compounds of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients, for use in the prevention and / or treatment of respiratory diseases.

[0138] In another embodiment, the present invention provides a method for preventing and / or treating respiratory diseases, the method comprising administering a pharmaceutical composition comprising the compounds of formula (I).

[0139] In a further aspect, the respiratory diseases mentioned above are selected from cough, subacute or chronic cough, treatment-resistant cough, idiopathic chronic cough, refractory chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

[0140] In one embodiment, the invention refers to the use of the compound of formula (I) or its pharmaceutical composition for the preparation of a medicament for the treatment and / or prevention of cough, sub-acute or chronic cough, treatment-resistant cough, idiopathic chronic cough, refractory chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

[0141] In another embodiment, the invention refers to a compound of formula (I) or a pharmaceutical composition for use in the prevention and / or treatment of a respiratory disease, wherein the respiratory disease is selected from: cough, sub-acute or chronic cough, treatmentresistant cough, idiopathic chronic cough, refractory chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

[0142] In another embodiment, the invention refers to a method for the treatment and / or prevention of respiratory diseases selected from the group consisting of cough, sub-acute or chronic cough, treatment-resistant cough, idiopathic chronic cough, refractory chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm, the method comprising administering a compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients.

[0143] In a preferred embodiment, the respiratory disease mentioned above is chronic cough.

[0144] In another preferred embodiment, the invention refers to a compound of formula (I) or its pharmaceutical composition for use in the prevention and / or treatment of chronic cough.

[0145] In another preferred embodiment, the invention refers to the use of a compound of formula (I) or its pharmaceutical composition for the preparation of a medicament for the treatment and / or prevention of chronic cough.

[0146] In another preferred embodiment, the invention refers to a method for the treatment and / or prevention of chronic cough, the method comprising administering a compound of formula (I) or a pharmaceutical composition comprising a compound of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients.

[0147] Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dosage of the compound of formula (I).

[0148] The magnitude of prophylactic or therapeutic dose of the compound of formula (I) will, of course, vary with the nature of the severity of the condition to be treated and with its route of administration, and will generally be determined by clinical trial as required in the pharmaceutical art. It will also vary according to the age, weight and response of the individual patient.

[0149] All preferred groups or embodiments described above for compounds of formula (I) may be combined with each other and apply as well mutatis mutandis.

[0150] The compounds of the present invention can be prepared in a number of ways known to the one skilled in the art of organic synthesis. It will be understood that the functionality / s present on the molecule should be consistent with the transformation proposed. This will sometimes require a modification of the order of synthetic steps in order to obtain a desired compound of the invention. While the optimal reaction conditions may vary depending on the particular reactants or solvent used, such conditions can be readily determined by those skilled in the art by routine optimization procedures. Thus, processes described below should not be viewed as limiting the scope of the synthetic methods available for the preparation of the compounds of the invention. In some cases, generally known protective groups (PG) may be employed when needed to mask or protect sensitive or reactive moieties, in accordance with general principles of chemistry (Protective group in organic syntheses, 3rd ed. T. W. Greene, P. G. M. Wuts). The compounds of formula (I), including all the compounds here above listed, can be generally prepared according to the procedures outlined in Schemes 1 to 2 shown below, wherein at least one non-limiting synthetic route is provided for the preparation of the exemplified compounds (i.e. the Examples).

[0151] Scheme 1

[0152] In one embodiment of the present invention, compound (I) may be prepared from compound (VI) and compound (VIII) according to Scheme 1. Compound (VI) may be prepared by starting from compound (II). Compound (V) may be obtained from compound (II) by a two-step sequence: step a) involves acylation reaction with a suitable haloacetyl halide (III) wherein Xi and X2 are halogens, preferably and independently chloride or bromide. Step b) involves alkylation reaction with a suitable amine NHR1R2 (IV). Compound (VI) may then be prepared from compound (V) by a two-step sequence: step c) involves ester hydrolysis reaction mediated by a base, like for instance NaOH or KOH, or an acid, like for instance HC1 or H2SO4. Subsequent step d) involves amide formation reaction with a suitable amine NHR5R6 (IX) in the presence of a suitable coupling reagent, like, for example, chloro-N,N,N',N'-tetramethylformamidinium hexafluorophosphate (TCFH). Compound (VIII) may be prepared from compound (VII) by acylation reaction with a suitable haloacetyl halide (III), wherein Xi and X2 are halogens, preferably and independently chloride or bromide. Compound (I) may then be obtained from compound (VI) and compound (VIII) by nitrogen quatemarization reaction (Menshutkin reaction).

[0153] Scheme 2

[0154] In another embodiment of the present invention, compounds of formula (I) may be prepared according to Scheme 2.

[0155] Compound (X) may be prepared from compound (V) by a two-step sequence: step a) involves ester hydrolysis reaction mediated by a base, like for instance NaOH or KOH, or an acid, like for instance HC1 or H2SO4, and step b) involves the introduction of a suitable carboxylic acid ester protective group (=PG) like, for example, a tert-butyl group. Compound (XI) may be prepared by reacting compound (X) and compound (VIII) via nitrogen quatemarization reaction (Menshutkin reaction). Compound (I) may then be obtained from compound (XI) by a two-step sequence: step c) involves carboxylic acid protective group removal reaction, and step d) involves amide formation reaction with a suitable amine NHR5R6 (IX) in the presence of a suitable coupling reagent, like, for example, TCFH. Scheme 3

[0156] In another embodiment of the present invention, compounds of formula (I) may be prepared according to Scheme 3. Compound (XIV) may be prepared from compound (XII) ) by an amide formation reaction with a suitable amine NHRsRe (IX) in the presence of a suitable coupling reagent, like, for example, TCFH. Compound (XVI) may be prepared from compound (XIV) by acylation reaction with a suitable haloacetyl halide (III) wherein Xi and X2 are halogens, preferably and independently chloride or bromide. Compound (XV) may be prepared from compound (VIII) by alkylation reaction with a suitable amine NHR1R2 (IV). Finally, compounds of formula (I) may be prepared from compounds (XV) and (XVI) by alkylation reaction.

[0157] Accordingly, the present invention provides intermediate compounds (V), (VI), (VIII), (X), (XI), (XIV), (XV) and (XVI), as defined above, and their use in the preparation of compounds of formula (I).

[0158] In another aspect, the invention provides the use of an intermediate compound selected from the group consisting of compound (V), (VIII), (X), (XI), (XV) and (XVI), as defined above, in particular in Scheme 1, 2 and 3, for the preparation of the compounds of formula (I), as defined above.

[0159] When compounds of formula (I) were isolated by precipitation from the reaction mixture and then triturated or crystallized, they were generally obtained as salt forms, with the anion being chloride or bromide, depending on the meaning of X2 in the alkylating agent, i.e. the compound (III), (VIII) and (XVI), used in the formation of the quaternary ammonium salt (see Scheme 1, 2 and 3). Compounds of formula (I) in salt form can be obtained in zwitterionic form by subsequent treatment in basic conditions, such as by purification on amino-functionalized silica gel chromatography. Zwitterionic forms of compounds of formula (I) may be treated with acids, such as hydrochloric acid or the like, to obtain the corresponding compounds of formula (I) in salt form.

[0160] Exemplified preparation processes are given in the following experimental part.

[0161] PREPARATIONS OF INTERMEDIATES AND EXAMPLES

[0162] Chemical Names of the compounds were generated with Structure-To-Name tool of PerkinElmer ChemDraw® Professional application (v. 20.0.0.41.) or are common chemical names. All reagents, for which the synthesis is not described in the experimental part, are either commercially available, or are known compounds or may be formed from known compounds by known methods by a person skilled in the art.

[0163] In the procedures that follow, some of the starting materials are identified through an “Intermediate” or “Example” number with indications on step number. This is provided merely for assistance to the skilled chemist.

[0164] When reference is made to the use of a “similar” or “analogous” procedure, as it will be appreciated by those skilled in the art, such a procedure may involve minor variations, for example reaction temperature, reagent / solvent amount, reaction time, work-up conditions or chromatographic purification conditions, that will be appreciated by those skilled in the art. All final compounds were obtained as pharmaceutically acceptable salts, unless stated otherwise.

[0165] All the Intermediates and the Examples reported were analytically characterized by LC-MS and / or 'H-NMR as described therein, the optimal proton frequency and solvent conditions may vary and can be readily determined by those skilled in the art by routine optimization procedures.

[0166] Abbreviations

[0167] ACN = acetonitrile; AMU = Atomic Mass Unit; CDCh = deuterated chloroform; CyHex = cyclohexane; DCM = dichloromethane; DMF = dimethylformamide; DMSO = dimethyl sulfoxide; DMSO-d6 = deuterated dimethyl sulfoxide; EGTA = ethylene glycol-bis(P-aminoethyl ether)-

[0168] N,N,N',N'-tetraacetic acid; Et2O = diethyl ether; EtOAc = Ethyl acetate; eq. = equivalents; FCC = flash column chromatography; h = hour / s; HATU = l-[Bis(dimethylamino)methylene]-lH-l,2,3- triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; HCOOH = formic acid; HEPES = N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC = High Performance Liquid Chromatography; MeOH = methyl alcohol; min = minute / s; NMR = nuclear magnetic resonance; RT / rt = room temperature; tR = retention time; s.s .= saturated solution; TCFH = Chloro-N,N,N',N'- tetramethylformamidinium hexafluorophosphate; TFA = trifluoroacetic acid; THF = tetrahydrofuran, LC-MS = Liquid Chromatography / Mass Spectrometry; XPhos = 2- Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl; XPhos Pd G3 = (2-dicyclohexylphosphino- 2',4',6'-triisopropyl-l,l'-biphenyl)[2-(2'-amino-l,l'-biphenyl)]palladium(II) methanesulfonate.

[0169] Analytical Methods

[0170] NMR characterization:

[0171] JH-NMR spectra were performed on 400 Agilent INOVA, 500 Agilent VNMRS, 600 Agilent INOVA spectrometers operating at 400, 500, 600 MHz (proton frequency), or on a Bruker Avance III 400 spectrometer.

[0172] Chemical shifts are reported as 8 values in ppm relative to tetramethyl silane (TMS) as an internal standard. Coupling constants (I values) are given in hertz (Hz) and multiplicities are reported using the following abbreviation (s=singlet, d=doublet, t=triplet, quin=quintet, m=multiplet, br=broad).

[0173] Information on the zwitterionic or salt form for compounds of formula (I) was obtained byJH-NMR spectroscopy: zwitterionic forms generally showed one missing amide NH signal. HBr or HC1 salts generally showed all amide NH signals. Formic acid salts (i.e. formates) were checked and quantified byJH-NMR signal of formic acid.

[0174] LC-MS:

[0175] LCMS may be recorded under the following conditions: diode array DAD chromatographic traces, mass chromatograms and mass spectra may be taken on Waters LC / PDA / MS Acquity™ system coupled with Micromass ZQ™ or Waters SQD single quadrupole mass spectrometer operated in positive and / or negative electron spray ES ionization mode.

[0176] LC / UV / MS characterization:

[0177] LC / MS retention times (t ) are estimated to be affected by an experimental error of ± 0.5 min.

[0178] LC-MS Method 1: Acquity CSH C18 2.1 x 50mm 1.7pm, the column temperature was 40 °C; mobile phase solvent A was milliQ water + 0.1% HCOOH, mobile phase solvent B ACN +

[0179] O.1% HCOOH. The flow rate was 1 mL / min. The gradient table was t=0 min 97% A 3% B, t=1.5 min 0.1% A 99.9% B, t=1.9 min 0.1% A 99.9% B and t=2 min 97% A 3% B. The UV detection range was 210-350 nm and ES+ZES- range was 100 to 1000 AMU.

[0180] LC-MS Method 2: column: Kinetex EVO C18 2.1 x 50mm 1.7 m, the column temperature was 40 °C; mobile phase solvent A was 10 mM aqueous solution of NH4HCO3 adjusted to pH=10 with ammonia, mobile phase solvent B ACN. The flow rate was 1 mL / min. The gradient table was t=0 min 97% A 3% B, t=l .5 min 0.1% A 99.9% B, t=l .9 min 0.1% A 99.9% B and t=2 min 97% A 3% B. The UV detection range was 210-350 nm and ES+ZES- range was 100 to 1200 AMU.

[0181] Purification Methods

[0182] The purification of some compounds was achieved by semipreparative HPLC by using a MDAP Waters apparatus with mass spectrometry detection (MS:ZQ2000) and DAD detection 220, equipped with CSH C18 (30 x 100 mm, 5pm) column and 1 mL injection loop. A gradient of ACN in H2O + 0.1 % HCOOH from 50% to 70% in 10 min (flow= 40.00 mL / min.) was used as mobile phase.

[0183] Other compounds were purified by FCC using Biotage® columns and conditions specified in each Example.

[0184] General Synthetic procedures

[0185] Intermediate 1: methyl 3-(2-bromoacetamido)-4-methylthiophene-2-carboxylate

[0186] To a suspension of methyl 3-amino-4-methylthiophene-2-carboxylate (10.0 g, 58.4 mmol) in water (235 mL), 2-bromoacetyl bromide (6.11 mL, 71.0 mmol) was added dropwise at 0°C observing the formation of an off-white solid. The reaction was allowed to warm to RT and stirred overnight. The solid was filtered, washed with water and dried under high vacuum to obtain the title compound (Intermediate 1 : 16.0 g, 55.0 mmol, 94 % yield) as an off-white powder.

[0187] LC-MS Method 1 : ty? = 0.81 min, MS (ESI) m / z = 292.0 / 294.0 [M+H]+

[0188] Intermediate 2: 2-bromo- / V-(isoxazol-3-yl)acetamide To a suspension of potassium carbonate (6.58 g, 47.6 mmol) and isoxazol-3-amine (1.44 mL, 19.0 mmol) in DCM (73 mL), 2-bromoacetyl bromide (1.82 mL, 20.9 mmol) was added dropwise. After 3 h the reaction mixture was washed with water (2x) and then with NaHCCh aq. sat. sol. The organic phase was separated, dried over a phase separator and concentrated under reduced pressure to afford a crude that was purified by FCC (KP Sfar silica, 10-80% EtOAc in cyclohexane) to afford title compound (Intermediate 2: 1.60 g, 7.80 mmol, 41 % yield) as an orange solid.

[0189] LC-MS Method 1 : tz? = 0.53 min, MS (ESI) m / z = 205.0 / 207.0 [M+H]+

[0190] Intermediate 3: (l?)-2-bromo-A-(l-phenylethyl)acetamide

[0191] Analogously to Intermediate 1, Intermediate 3 was prepared by starting from the suitable amine:

[0192] (7? -l-phenylethan-l-amine: 1.00 g, 8.25 mmol (1.0 eq)

[0193] Amount / yield: 1.69 g, 7.00 mmol, 84 % yield.

[0194] LC-MS Method 2: tz? = 0.83 min, MS (ESI) m / z = 242.0 / 244.0 [M+H]+

[0195] Example 1: 2-(benzylamino)- / V,7V-dimethyl-7V-(2-((4-methyl-2-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethan-l-aminium (zwitterion)

[0196] Step 1 - methyl 3-(2-chloroacetamido)-4-methylthiophene-2-carboxylate (Intermediate 4)

[0197] To a solution of methyl 3-amino-4-methylthiophene-2-carboxylate (5.00 g, 29.0 mmol) in water (117 mL), was added 2-chloroacetyl chloride (9.30 mL, 116 mmol) dropwise at 0°C. The reaction was allowed to warm to RT overnight observing the formation of a white precipitate, which was filtered, washed with water (3x) and pentane (2x), affording title compound (Intermediate 4: 7.40 g, quantitative yield) as an off-white powder.

[0198] LC-MS Method 1 : tz? = 0.82 min, MS (ESI) m / z = 248.0 [M+H]+

[0199] Step 2 - methyl 3-(2-(dimethylamino)acetamido)-4-methylthiophene-2-carboxylate (Intermediate 5)

[0200] To a stirred solution of methyl 3-(2-chloroacetamido)-4-methylthiophene-2-carboxylate Intermediate 4 (1.00 g, 4.0 mmol) and sodium hydrogen carbonate (0.68 g, 8.1 mmol) in can (10 mL) at RT, dimethylamine (2.0 M in THF) (2.42 mL, 4.84 mmol) was added. The mixture was stirred at RT overnight. Water was added and the reaction extracted with EtOAc (3x). The collected organic layers were dried over Na2SO4, filtered and concentrated to afford title compound (Intermediate 5: 0.91 g, 3.6 mmol, 88% yield) as a yellow oil.

[0201] LC-MS Method 1 : ta = 0.38 min, MS (ESI) m / z = 257.1 [M+H]+

[0202] Step 3 - 2-(dimethylamino)- / V-(4-methyl-2-(pyrrolidine-l-carbonyl)thiophen-3- yl)acetamide (Intermediate 6)

[0203] A 10 mL ball milling reactor was loaded with 4 balls, pyrrolidine (64 mL, 0.78 mmol), potassium terLbutoxide (39 mg, 0.35 mmol) and methyl 3-(2-(dimethylamino)acetamido)-4- methylthiophene-2-carboxylate (Intermediate 5, 100 mg, 0.39 mmol). The reactor was closed and shaken at 13 Hz for 4 h at rt. Then the reactor was opened and water and EtOAc were added, the reactor was closed and shaken. The organic layer was separated and the aqueous one extracted with EtOAc (2x). The collected organic layers were dried over Na2SO4, filtered and concentrated to afford title compound (Intermediate 6: 104 mg, 0.35 mmol, 90% yield).

[0204] LC-MS Method 1 : tz? = 0.43 min, MS (ESI) m / z = 296.1 [M+H]+

[0205] Step 4 - 2-(benzylamino)- / V,7V-dimethyl-7V-(2-((4-methyl-2-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethan-l-aminium (zwitterion)(Example 1)

[0206] To a stirred solution of N-benzyl-2-chloroacetamide (46 mg, 0.25 mmol) and 2- (dimethylamino)-A-(4-methyl-2-(pyrrolidine-l-carbonyl)thi ophen-3 -yl)acetamide (Intermediate 6, 70 mg, 0.23 mmol) in ACN (0.8 mL), potassium iodide (3.8 mg, 0.020 mmol) was added. The reaction was stirred at 80°C overnight. After this time, the reaction was cooled to RT and concentrated under high vacuum. The residue was purified by FCC (Sfar amino, 0-5% MeOH in DCM), affording the title compound (Example 1 : 35 mg, 0.079 mmol, 32% yield) as yellowish powder.

[0207] LC-MS Method 2: tz? = 0.98 min, MS (ESI) m / z = 443.2 [M]+ ‘HNMR (400 MHz, CDCh) 5 ppm 9.78 (br s, 1 H), 7.21 - 7.38 (m, 5 H), 6.92 (s, 1 H), 4.71 (s, 2 H), 4.45 (s, 2 H), 4.26 (s, 2 H), 3.46 (s, 6 H), 3.13 - 3.80 (m, 4 H), 2.13 (s, 3 H), 1.85 (br s, 4 H).

[0208] Example 2: 2-(benzylamino)- \-(2-((2-(dimethylcarbamoyl)-4-methyl thiophen-3- yl)amino)-2-oxoethyl)-\, \ dimethyl-2-oxoethan-l-aminium formate

[0209] Step 1 - 3-(2-(dimethylamino)acetamido)-4-methylthiophene-2-carboxylic acid sodium chloride (Intermediate 7)

[0210] To a solution of Intermediate 5 (180 mg, 0.700 mmol) in THF (2 mL), water (0.4 mL) and sodium hydroxide 2 M in water (0.42 mL, 0.84 mmol) were added and the reaction heated at 50°C for 16 h. Then the reaction cooled to 0°C and neutralized by addition of HC1 2 M in water until pH = 7. Volatiles were removed under reduced pressure to afford the title compound (Intermediate 7: 230 mg, 92% wt, quantitative yield) as an orange solid.

[0211] LC-MS Method 1: tz? = 0.32 min, MS (ESI) m / z = 243.1 [M+H]+

[0212] Step 2 - 3-(2-(dimethylamino)acetamido)-7V,7V,4-trimethylthiophene-2-carboxamide (Intermediate 8)

[0213] To a solution of Intermediate 7 (230 mg, 92% wt, 0.70 mmol) in DMF (2 mL), HATU (315 mg, 0.83 mmol) was added. After 5 min of stirring dimethylamine 2 M in THF (0.69 mL, 1.38 mmol) was added. The reaction was stirred at RT overnight. A solution of NaOH in water at pH = 9 was added and the reaction extracted with EtOAc (3x). The collected organic layers were dried over Na2SO4, filtered and concentrated to afford a crude that was purified by FCC (Sfar amino, 1% MeOH in DCM) to give the title compound (Intermediate 8: 176 mg, 0.65 mmol, 93% yield).

[0214] LC-MS Method 1 : t« = 0.36 min, MS (ESI) m / z = 270.1 [M+H]+

[0215] Step 3 - 2-(benzylamino)-7V-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)-7V,7V-dimethyl-2-oxoethan-l-aminium formate (Example 2)

[0216] Analogously to Example 1, Example 2 was prepared by starting from the suitable amine and chloride.

[0217] Intermediate 8: 176 mg, 0.65 mmol (1.0 eq) N-benzyl-2-chloroacetamide: 120 mg, 0.65 mmol (1.0 eq)

[0218] Purified by FCC (Sfar-C18, 10-30% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH).

[0219] Amount / yield: 9.9 mg, 0.021 mmol, 3% yield.

[0220] LC-MS Method 2: tz? = 0.87 min, MS (ESI) m / z = 417.5 [M]+

[0221] 'HNMR (600 MHz, CDCh) 5 ppm 11.92 (br s, 1 H), 9.13 (s, 1 H), 8.66 (br s, 1 H), 7.30 - 7.35 (m, 4 H), 7.26 - 7.29 (m, 1 H), 6.97 (s, 1 H), 4.69 (s, 2 H), 4.61 (s, 2 H), 4.45 (d, J=5.6 Hz, 2 H), 3.53 (s, 6 H), 2.69 - 3.34 (m, 6 H), 2.22 (s, 3 H).

[0222] Example 3: 2-(benzylamino)- / V-(2-((2-(tert-butylcarbamoyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)-7V,7V-dimethyl-2-oxoethan-l-aminium formate

[0223] Step 1 - N-(tert-butyl)-3-(2-(dimethylamino)acetamido)-4-methylthiophene-2- carboxamide (Intermediate 9)

[0224] Analogously to Intermediate 8, Intermediate 9 was prepared by starting from the suitable carboxylic acid and amine:

[0225] Intermediate 7: 150 mg, 0.62 mmol (1.0 eq)

[0226] 2-methyl-2-propanamine: 0.33 mL, 3.1 mmol (5 eq)

[0227] Amount / yield: 140 mg, 0.47 mmol, 76% yield.

[0228] LC-MS Method 1 : ty? = 0.45 min, MS (ESI) m / z = 298.1 [M+H]+

[0229] Step 2 - 2-(benzylamino)- / V-(2-((2-(tert-butylcarbamoyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)-7V,7V-dimethyl-2-oxoethan-l-aminium formate (Example 3)

[0230] To a stirred solution of Intermediate 9 (140 mg, 0.47 mmol) in ACN (1.7 mL), N-benzyl-2- bromoacetamide (118 mg, 0.52 mmol) was added. The reaction was stirred at 80°C overnight. The residue was purified by HPLC Semi -prep, method, to afford the title compound (Example 3: 10 mg, 0.021 mmol, 4% yield).

[0231] LC-MS Method 2: tz? = 0.66 min, MS (ESI) m / z = 445.2 [M]+

[0232] 'H NMR (400 MHz, CDCh) 5 ppm 10.37 - 12.99 (m, 1 H), 9.24 (br s, 1 H), 8.72 (s, 1 H), 7.23 - 7.37 (m, 5 H), 6.92 (s, 1 H), 5.89 (s, 1 H), 4.79 (s, 2 H), 4.61 (s, 2 H), 4.47 (br d, J=5.3 Hz, 2 H), 3.57 (s, 6 H), 2.17 (s, 3 H), 1.34 (s, 9 H).

[0233] Example 4: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(morpholine-4- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0234] Step 1 - methyl 3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2-carboxylate (Intermediate 11)

[0235] To a stirred solution of Intermediate 1 (2.0 g, 6.78 mmol) in ACN (27 mL), homopiperidine (0.99 mL, 8.81 mmol) and potassium carbonate (1.21 g, 8.81 mmol) were added, and the mixture was stirred for 16 h at RT. The reaction was filtered to remove the salts and the organic phase was concentrated. The residue was dissolved in EtOAc and water. The phases were mixed in an extractor, the organic layer was separated and the aqueous one extracted with EtOAc (2x). The collected organic layers were dried over Na2SO4, filtered and concentrated to afford the title compound (Intermediate 11 : 1.77 g, 5.69 mmol, 84% yield) as orange oil.

[0236] LC-MS Method 1 : tz? = 0.48 min, MS (ESI) m / z = 311.2 [M+H]+

[0237] Step 2 - 2-(azepan-l-yl)-7V-(4-methyl-2-(morpholine-4-carbonyl)thiophen-3- yl)acetamide (Intermediate 12)

[0238] Analogously to Intermediate 6, Intermediate 12 was prepared by starting from the suitable amine and ester:

[0239] Intermediate 11 : 200 mg, 0.64 mmol (1.0 eq)

[0240] Morpholine: 0.11 mL, 1.29 mmol (2.0 eq)

[0241] Potassium tert-butoxide: 65 mg, 0.58 mmol (0.9 eq)

[0242] Amount / yield: 182 mg, 0.5 mmol, 77% yield.

[0243] LC-MS Method 1 : tz? = 0.36 min, MS (ESI) m / z = 366.2 [M+H]+

[0244] Step 3 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(morpholine-4- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 4)

[0245] To a stirred solution of N-benzyl-2-bromo-acetamide (41 mg, 0.18 mmol) in ACN (0.9 mL), 2-(azepan-l-yl)-A-(4-methyl-2-(morpholine-4-carbonyl)thi ophen-3 -yl)acetamide (Intermediate 12, 60 mg, 0.16 mmol) was added. The reaction was stirred at 80°C overnight. The reaction was cooled down and concentrated under high vacuum. The residue was purified by FCC (Sfar amino, 1-5% MeOH in DCM), affording the title compound (Example 4: 42 mg, 0.082 mmol, 45% yield).

[0246] LC-MS Method 1 : t« = 0.63 min, MS (ESI) m / z = 513.3 [M]+ ‘H NMR (500 MHz, DMSO-d6) 5 ppm 9.13 - 10.77 (m, 1 H), 7.19 - 7.38 (m, 5 H), 7.09 (br s, 1 H), 4.48 (s, 2 H), 4.35 (d, J=5.6 Hz, 2 H), 4.08 (br s, 2 H), 3.75 - 3.83 (m, 2 H), 3.72 (br d, J=6.0 Hz, 2 H), 3.40 - 3.57 (m, 8 H), 1.77 - 2.07 (m, 7 H), 1.62 (br s, 4 H).

[0247] Example l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-

[0248] (methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0249] Step 1 - 3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2-carboxylic acid sodium chloride (Intermediate 13)

[0250] Analogously to Intermediate 7, Intermediate 13 was prepared by starting from the corresponding ester:

[0251] Intermediate 11 : 5.0 g, 16.1 mmol (1.0 eq)

[0252] Sodium hydroxide (2 M in water): 9.7 mL, 19.3 mmol (1.2 eq)

[0253] Amount / yield: 6.3 g, 90% wt, quantitative yield.

[0254] LC-MS Method 1 : tz? = 0.43 min, MS (ESI) m / z =297.2 [M+H]+

[0255] Step 2 - 3-(2-(azepan-l-yl)acetamido)-A,4-dimethylthiophene-2-carboxamide

[0256] (Intermediate 14)

[0257] Analogously to Intermediate 8, Intermediate 14 was prepared by starting from the corresponding acid and amine.

[0258] Intermediate 13: 284 mg, 0.80 mmol (1.0 eq)

[0259] Methanamine 2 M in THF: 0.80 mL, 1.60 mmol (2.0 eq)

[0260] Purified by FCC (Sfar amino, 20% EtOAc in DCM)

[0261] Amount / yield: 212 mg, 0.69 mmol, 86% yield

[0262] LC-MS Method 1 : ta = 0.38 min, MS (ESI) m / z = 310.2 [M+H]+

[0263] Step 3 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(methylcarbamoyl) thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 5)

[0264] Analogously to Example 4, Example 5 was prepared by starting from the corresponding bromide and amine.

[0265] Intermediate 14: 70 mg, 0.23 mmol (1.0 eq)

[0266] N-benzyl-2-bromo-acetamide: 52 mg, 0.23 mmol (1.0 eq) Purified by FCC (Sfar amino, 10-20% EtOAc in DCM)

[0267] Amount / yield: 34 mg, 0.074 mmol, 32% yield

[0268] LC-MS Method 1: ta = 0.59 min, MS (ESI) m / z = 457.3 [M]+

[0269] 'HNMR (400 MHz, DMSO-d6) 5 ppm 9.19 (t, J=5.7 Hz, 1 H), 8.99 (q, J=4.4 Hz, 1 H), 7.18 - 7.37 (m, 5 H), 6.97 (s, 1 H), 4.61 (s, 2 H), 4.34 (d, J=5.7 Hz, 2 H), 4.25 (s, 2 H), 3.68 - 3.88 (m, 4 H), 2.73 (d, J=4.6 Hz, 3 H), 1.80 - 2.03 (m, 7 H), 1.63 (br s, 4 H).

[0270] Example 6: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0271] Step 1 - 2-(azepan-l-yl)-7V-(4-methyl-2-(pyrrolidine-l-carbonyl)thiophen-3- yl)acetamide (intermediate 15)

[0272] Analogously to Intermediate 6, Intermediate 15 was prepared by starting from the corresponding amine and ester.

[0273] Intermediate 11 : 200 mg, 0.63 mmol (1.0 eq)

[0274] Pyrrolidine: 0.10 mL, 1.26 mmol (2.0 eq) potassium tert-butoxide: 64 mg, 0.57 mmol (0.9 eq)

[0275] Amount / yield: 134 mg, 0.38 mmol, 61% yield

[0276] LC-MS Method 1: ta = 0.51 min, MS (ESI) m / z = 350.2 [M+H]+

[0277] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 6)

[0278] Analogously to Example 4, Example 6 was prepared by starting from the corresponding bromide and amine.

[0279] Intermediate 15: 70 mg, 0.20 mmol (1.0 eq)

[0280] N-benzyl-2-bromo-acetamide: 58 mg, 0.22 mmol (1.0 eq)

[0281] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0282] Amount / yield: 60 mg, 0.10 mmol, 52% yield

[0283] LC-MS Method 1 : tz? = 0.70 min, MS (ESI) m / z = 497.2 [M]+

[0284] ‘HNMR (400 MHz, DMSO-d6) 5 ppm 10.38 (br s, 1 H), 6.96 - 7.50 (m, 6 H), 4.44 (s, 2 H), 4.35 (d, J=5.5 Hz, 2 H), 4.05 - 4.66 (m, 2 H), 3.64 - 3.91 (m, 4 H), 3.34 - 3.56 (m, 4 H), 2.00 (br s, 3 H), 1.91 (br s, 4 H), 1.75 (br s, 4 H), 1.62 (br s, 4 H).

[0285] Example 7 : l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) Analogously to Example 4, Example 7 was prepared by starting from the corresponding bromide and amine.

[0286] Intermediate 15: 70 mg, 0.20 mmol (1.0 eq)

[0287] Intermediate 2: 58 mg, 0.26 mmol (1.3 eq)

[0288] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0289] Amount / yield: 33 mg, 0.069 mmol, 35% yield.

[0290] LC-MS Method 1 : ty? = 0.58 min, MS (ESI) m / z = 474.2 [M]+

[0291] 'HNMR (400 MHz, CDC13) 5 ppm 8.20 (d, J=I .4 Hz, 1 H), 6.96 (s, 1 H), 6.89 (d, J=I .4 Hz, 1 H), 4.39 (br s, 4 H), 3.78 - 3.98 (m, 2 H), 3.58 - 3.75 (m, 4 H), 3.47 (br s, 2 H), 2.28 (s, 3 H), 1.98 (br s, 4 H), 1.86 - 1.94 (m, 4 H), 1.77 (br s, 4 H).

[0292] Example 8: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(morpholine-4- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0293] Analogously to Example 4, Example 8 was prepared by starting from the corresponding bromide and amine.

[0294] Intermediate 12: 60 mg, 0.16 mmol (1.0 eq)

[0295] Intermediate 2: 44 mg, 0.21 mmol (1.3 eq)

[0296] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0297] Amount / yield: 11 mg, 0.022 mmol, 14% yield.

[0298] LC-MS Method 1: tz? = 0.51 min, MS (ESI) m / z = 490.2 [M]+

[0299] 'HNMR (400 MHz, CDC13) 5 ppm 8.64 (br s, 1 H), 8.31 (s, 1 H), 6.99 (s, 1 H), 6.86 (s, 1 H), 4.98 (br s, 2 H), 4.70 (br s, 2 H), 3.95 - 4.26 (m, 4 H), 3.64 - 3.80 (m, 8 H), 2.23 (s, 3 H), 2.05 (br s, 4 H), 1.80 (br s, 4 H).

[0300] Example 9: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-

[0301] (methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium formate

[0302] Analogously to Example 4, Example 9 was prepared by starting from the corresponding bromide and amine.

[0303] Intermediate 14: 70 mg, 0.23 mmol (1.0 eq)

[0304] Intermediate 2: 60 mg, 0.29 mmol (1.3 eq)

[0305] Purified by FCC (Sfar -Cl 8, 10%-25% ACN + 0.1 % HCOOH in H2O+ 0.1 % HCOOH)

[0306] Amount / yield: 14 mg, 0.029 mmol, 13% yield.

[0307] LC-MS Method 1 : ta = 0.46 min, MS (ESI) m / z = 434.2 [M]+

[0308] 'HNMR (500 MHz, CDCh) 5 ppm 8.65 (s, 1 H), 8.25 (d, J=1.1 Hz, 1 H), 7.96 (br s, 1 H), 6.96 (s, 1 H), 6.83 (s, 1 H), 4.51 - 4.97 (m, 4 H), 3.91 (br s, 4 H), 2.83 (d, J=4.4 Hz, 3 H), 1.89 - 2.10 (m, 7 H), 1.65 - 1.85 (m, 4 H). Example 10: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0309] Step 1 - 3-(2-(azepan-l-yl)acetamido)-A,A,4-trimethylthiophene-2-carboxamide (Intermediate 16)

[0310] Analogously to Intermediate 8, Intermediate 16 was prepared by starting from the corresponding acid and amine.

[0311] Intermediate 13: 250 mg, 0.70 mmol (1.0 eq)

[0312] A-mehylmethanamine 2 M in THF: 0.70 mL, 1.41 mmol (2.0 eq)

[0313] Purified by FCC (Sfar amino, 20-40% EtOAc in cyclohexane)

[0314] Amount / yield: 157 mg, 0.49 mmol, 69% yield.

[0315] LC-MS Method 1 : ty? = 0.44 min, MS (ESI) m / z = 324.2 [M+H]+

[0316] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 10)

[0317] Analogously to Example 4, Example 10 was prepared by starting from the corresponding bromide and amine.

[0318] Intermediate 16: 77 mg, 0.24 mmol (1.0 eq)

[0319] N-benzyl-2-bromo-acetamide: 54 mg, 0.24 mmol (1.0 eq)

[0320] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0321] Amount / yield: 65 mg, 0.14 mmol, 58% yield

[0322] LC-MS Method 1 : tz? = 0.64 min, MS (ESI) m / z = 471.2 [M]+

[0323] 'H NMR (400 MHz, CDCh) 5 ppm 10.51 (br s, 1 H), 7.23 - 7.45 (m, 5 H), 6.90 (s, 1 H), 4.58 (br s, 2 H), 4.45 (s, 2 H), 4.23 (br s, 2 H), 3.94 - 4.09 (m, 2 H), 3.60 - 3.74 (m, 2 H), 3.02 (br s, 6 H), 2.12 (s, 3 H), 1.64 - 2.05 (m, 8 H).

[0324] Example 11 : l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0325] Analogously to Example 4, Example 11 was prepared by starting from the corresponding bromide and amine.

[0326] Intermediate 16: 77 mg, 0.24 mmol (1.0 eq)

[0327] Intermediate 2: 59 mg, 0.29 mmol (1.2 eq)

[0328] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0329] Amount / yield: 22 mg, 0.049 mmol, 20% yield. LC-MS Method 1 : ta = 0.50 min, MS (ESI) m / z = 448.2 [M]+

[0330] 'HNMR (500 MHz, CDCh) 5 ppm 12.01 (br s, 1 H), 8.29 (s, 1 H), 6.97 (s, 1 H), 6.84 (s, 1 H), 4.70 - 5.06 (m, 4 H), 3.90 - 4.14 (m, 4 H), 2.88 - 3.25 (m, 6 H), 2.19 (s, 3 H), 2.01 (br d, J=I .9 Hz, 4 H), 1.77 (br s, 4 H).

[0331] Example 13: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((2-((2-methoxyethyl) carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium formate

[0332] Step 1 - methyl 4-methyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2-carboxylate (Intermediate 17)

[0333] Analogously to Intermediate 11, Intermediate 17 was prepared by starting from the corresponding bromide and amine.

[0334] Intermediate 1 : 2.0 g, 6.85 mmol (1.0 eq)

[0335] Piperidine: 0.88 mL, 8.9 mmol (1.3 eq)

[0336] Potassium carbonate: 1.23 g, 8.9 mmol (1.3 eq)

[0337] Amount / yield: 1.3 g, 4.5 mmol, 65% yield

[0338] LC-MS Method 1 : ta = 0.45 min, MS (ESI) m / z = 297.1 [M+H]+

[0339] Step 2 - N-(2-methoxyethyl)-4-methyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2- carboxamide (Intermediate 18)

[0340] Analogously to Intermediate 6, Intermediate 18 was prepared by starting from the suitable amine and ester.

[0341] Intermediate 17: 300 mg, 0.93 mmol (1.0 eq)

[0342] 2-methoxy ethanamine: 0.28 ml, 2.1 mmol (2.20 eq) potassium tert-butoxide: 102 mg, 0.91 mmol (0.98 eq)

[0343] Amount / yield: 110 mg, 0.32 mmol, 35% yield.

[0344] LC-MS Method 1: ta = 0.39 min, MS (ESI) m / z = 340.2 [M+H]+

[0345] Step 3 - l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((2-((2-methoxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium formate (Example 13) Analogously to Example 4, Example 13 was prepared by starting from the corresponding bromide and amine.

[0346] Intermediate 18: 55 mg, 0.16 mmol (1.0 eq)

[0347] Intermediate 2: 110 mg, 0.16 mmol (1.0 eq)

[0348] Purified by FCC (Sfar-C18, 5-25% ACN + 0.1 % HCOOH in H2O+ 0.1 % HCOOH)

[0349] Amount / yield: 6.2 mg, 0.08 mmol, 7.5% yield

[0350] LC-MS Method 1 : tz? = 0.47 min, MS (ESI) m / z = 464.2 [M]+

[0351] 'H NMR (500 MHz, CDCh) 5 ppm 8.72 (s, 1H), 8.27 (d, J=I .6 Hz, 1 H), 7.35 (br s, 1 H), 6.94 (s, 1 H), 6.86 (d, J=I .6 Hz, 1 H), 4.88 (br s, 4 H), 3.90 - 4.08 (m, 4 H), 3.38 - 3.54 (m, 4 H), 3.28 (s, 3 H), 2.02 (s, 3 H), 1.91 - 2.08 (m, 4 H), 1.62 - 1.82 (m, 2 H).

[0352] Example 14: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion)

[0353] Step 1 - 4-methyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2-carboxylic acid sodium chloride (Intermediate 19)

[0354] Analogously to Intermediate 7, Intermediate 19 was prepared by starting from the corresponding ester.

[0355] Intermediate 17: 0.60 g, 2.0 mmol (1.0 eq)

[0356] Sodium hydroxide (2 M in water): 1.2 mL, 2.4 mmol

[0357] Amount / yield: 0.62 g, 1.83 mmol, 90% yield

[0358] LC-MS Method 1 : ty? = 0.39 min, MS (ESI) m / z = 283.0 [M+H]+

[0359] Step 2 - A,A,4-trimethyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2-carboxamide (Intermediate 20)

[0360] Analogously to Intermediate 8, Intermediate 20 was prepared by starting from the corresponding acid and amine.

[0361] Intermediate 19: 400 mg, 1.42 mmol (1.0 eq)

[0362] A-methylmethanamine 2 M in THF: 1.4 mL, 2.84 mmol (2.0 eq)

[0363] Purified by FCC (Sfar amino, 20-40% EtOAc in cyclohexane)

[0364] Amount / yield: 100 mg, 0.32 mmol, 23% yield LC-MS Method 1 : tz? = 0.41 min, MS (ESI) m / z = 310.1 [M+H]+

[0365] Step 3 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion) (Example 14)

[0366] Analogously to Example 4, Example 14 was prepared by starting from the corresponding bromide and amine.

[0367] Intermediate 20: 60 mg, 0.19 mmol (1.0 eq)

[0368] A-benzyl-2-bromo-acetamide: 51 mg, 0.19 mmol (1.0 eq)

[0369] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0370] Amount / yield: 34 mg, 0.074 mmol, 39% yield

[0371] LC-MS Method 1 : tz? = 0.61 min, MS (ESI) m / z = 457.2 [M]+

[0372] 'HNMR (400 MHz, CDC13) 5 ppm 9.41 (br s, 1 H), 7.25 - 7.36 (m, 5 H), 6.94 (s, 1 H), 4.72 (s, 2 H), 4.52 (br d, J=5.2 Hz, 2 H), 4.46 (s, 2 H), 4.01 - 4.16 (m, 2 H), 3.80 (br d, J=12.5 Hz, 2 H), 2.98 (br s, 6 H), 2.18 (s, 3 H), 1.38 - 2.10 (m, 6 H).

[0373] Example 15: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert-butoxycarbonyl) (methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium formate

[0374] Step 1 - tert-butyl(2-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxamido)ethyl)(methyl)carbamate (Intermediate 10)

[0375] Analogously to Intermediate 6, Intermediate 10 was prepared by starting from the suitable amine and ester.

[0376] Intermediate 11 : 600 mg, 1.72 mmol (1.0 eq)

[0377] A-(2-aminoethyl)-A-methyl carbamic acid tert-butyl ester: 600 mg, 3.44 mmol) (2.0 eq)

[0378] Potassium tert-butoxide: 193 mg, 1.72 mmol (1.0 eq)

[0379] Purified by FCC (Sfar amino, 10-30% EtOAc in cyclohexane)

[0380] Amount / yield: 110 mg, 0.32 mmol, 15% yield.

[0381] LC-MS Method 1 : ty? = 0.63 min, MS (ESI) m / z = 453.2 [M+H]+

[0382] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert- butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium formate (Example 15)

[0383] Analogously to Example 4, Example 15 was prepared by starting from the corresponding bromide and amine. Intermediate 10: 95 mg, 0.21 mmol (1.0 eq)

[0384] N-benzyl-2-bromo-acetamide: 61 mg, 0.23 mmol (1.0 eq)

[0385] Purified by FCC (Sfar-C18, 5-40% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH). Amount / yield: 29 mg, 0.045 mmol, 21% yield.

[0386] LC-MS Method 1 : tz? = 0.77 min, MS (ESI) m / z = 600.2 [M]+

[0387] ‘H NMR (500 MHz, CDCh) 5 ppm 10.66 - 13.72 (m, 1 H), 9.32 - 10.46 (m, 1 H), 8.77 (s, 1 H), 7.37 - 7.59 (m, 1 H), 7.25 - 7.35 (m, 5 H), 7.02 (s, 1 H), 4.50 - 4.98 (m, 4 H), 4.38 - 4.49 (m, 2 H), 3.76 - 4.30 (m, 4 H), 3.26 - 3.49 (m, 4 H), 2.87 (s, 3 H), 2.08 (s, 3 H), 1.89 - 2.15 (m, 4 H), 1.72 - 1.87 (m, 4 H), 1.36 (br s, 9 H).

[0388] Example 16: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-methoxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0389] Step 1 - 3-(2-(azepan-l-yl)acetamido)-7V-(2-methoxyethyl)-4-methylthiophene-2- carboxamide (Intermediate

[0390] Analogously to Intermediate 8, Intermediate 21 was prepared by starting from the corresponding acid and amine.

[0391] Intermediate 13: 350 mg, 0.99 mmol (1.0 eq)

[0392] 2-methoxy ethanamine: 133 mg, 1.78 mmol (1.8 eq)

[0393] Purified by FCC (Sfar amino, 10-40% EtOAc in cyclohexane)

[0394] Amount / yield: 200 mg, 0.57 mmol, 57% yield

[0395] LC-MS Method 1 : ty? = 0.43 min, MS (ESI) m / z = 354.2 [M+H]+

[0396] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-methoxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 16)

[0397] Analogously to Example 4, Example 16 was prepared by starting from the corresponding bromide and amine.

[0398] Intermediate 21 : 65 mg, 0.18 mmol (1.0 eq)

[0399] N-benzyl-2-bromo-acetamide: 45 mg, 0.20 mmol (1.1 eq)

[0400] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0401] Amount / yield: 65 mg, 0.13 mmol, 72% yield

[0402] LC-MS Method 1: ta = 0.61 min, MS (ESI) m / z = 501.2 [M]+ 'H NMR (400 MHz, DMSO-d6) 5 ppm 9.32 (br s, 1 H), 9.16 (br t, J=5.5 Hz, 1 H), 7.19 - 7.39 (m, 5 H), 6.99 (s, 1 H), 4.65 (s, 2 H), 4.33 (d, J=5.7 Hz, 2 H), 4.22 (s, 2 H), 3.78 (br dd, J=6.0, 3.2 Hz, 4 H), 3.34 - 3.43 (m, 4 H), 3.27 (s, 3 H), 1.93 (s, 3 H), 1.91 (br s, 4 H), 1.63 (br s, 4 H).

[0403] Example 17: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-methoxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion)

[0404] Analogously to Example 4, Example 17 was prepared by starting from the corresponding bromide and amine.

[0405] Intermediate 18: 55 mg, 0.16 mmol (1.0 eq)

[0406] N-benzyl-2-bromo-acetamide: 43 mg, 0.16 mmol (1.0 eq)

[0407] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0408] Amount / yield: 14 mg, 0.028 mmol, 18% yield

[0409] LC-MS Method 1 : tz? = 0.61 min, MS (ESI) m / z = 487.2 [M]+

[0410] 'H NMR (400 MHz, CDCh) 5 ppm 9.86 (br d, J=0.8 Hz, 1 H), 8.87 (br s, 1 H), 7.21 - 7.34 (m, 5H), 6.96 (s, 1 H), 4.38 (s, 2 H), 4.34 (br s, 2 H), 4.00 (s, 2 H), 3.60 - 3.73 (m, 2 H), 3.43 - 3.59 (m, 6 H), 3.34 (s, 3 H), 1.99 (s, 3 H), 1.91 - 2.02 (m, 4 H), 1.75 - 1.87 (m, 1 H), 1.65 - 1.73 (m, 1 H).

[0411] Example 18: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-hydroxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0412] Step 1 - 3-(2-(azepan-l-yl)acetamido)-7V-(2-((tert-butyldimethylsilyl) oxy)ethyl)-4- methylthiophene-2-carboxamide (Intermediate 22)

[0413] Analogously to Intermediate 8, Intermediate 22 was prepared by starting from the corresponding acid and amine.

[0414] Intermediate 13: 513 mg, 1.45 mmol (1.0 eq)

[0415] 2-aminoethoxy(te / 7-butyl)dimethylsilane: 507 mg, 2.89 mmol (2.0 eq)

[0416] Purified by FCC (Sfar amino, 10-30% EtOAc in cyclohexane)

[0417] Amount / yield: 409 mg, 0.90 mmol, 62% yield

[0418] LC-MS Method 1 : ty? = 0.80 min, MS (ESI) m / z = 454.3 [M+H]+

[0419] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert- butyldimethylsilyl)oxy)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan- 1-ium (zwitterion) (Intermediate 23)

[0420] Analogously to Intermediate 4, Intermediate 23 was prepared by starting from the corresponding bromide and amine.

[0421] Intermediate 22: 136 mg, 0.30 mmol (1.0 eq)

[0422] N-benzyl-2-bromo-acetamide: 75 mg, 0.33 mmol (1.1 eq)

[0423] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0424] Amount / yield: 71 mg, 0.12 mmol, 39% yield.

[0425] LC-MS Method 1 : tz? = 0.94 min, MS (ESI) m / z = 601.3 [M]+

[0426] Step 3 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-hydroxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 18)

[0427] To a solution of l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert- butyldimethylsilyl)oxy)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l- ium bromide Intermediate 23 (70 mg, 0.10 mmol) in DCM (2 mL), hydrogen chloride 2 M in Et20 (0.5 mL, 1.0 mmol) was added dropwise at rt. After 3 h volatiles were evaporated and the residue was purified by column chromatography (Sfar amino, 0-10% MeOH in DCM) to afford title compound (Example 18: 10 mg, 0.020 mmol, 19% yield) as white powder.

[0428] LC-MS Method 1 : tz? = 0.56 min, MS (ESI) m / z = 487.2 [M]+

[0429] 'H NMR (400 MHz, DMSO-d6) 5 ppm 9.39 (br t, J=5.5 Hz, 1 H), 9.16 (t, J=5.6 Hz, 1 H), 7.20 - 7.36 (m, 5 H), 6.98 (d, J=0.9 Hz, 1 H), 4.82 (t, J=5.0 Hz, 1 H), 4.66 (s, 2 H), 4.34 (d, J=5.7 Hz, 2 H), 4.17 - 4.28 (m, 2 H), 3.76 (br s, 4 H), 3.48 (q, J=5.5 Hz, 2 H), 3.23 - 3.34 (m, 2 H), 1.78 - 1.98 (m, 7 H), 1.63 (br d, J=3.3 Hz, 4 H).

[0430] Example 19: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-

[0431] (methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride hydrochloride

[0432] Step 1 - l-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l- ium formate (Intermediate 24)

[0433] Analogously to Intermediate 4, Intermediate 24 was prepared by starting from the suitable bromide and amine.

[0434] Intermediate 10: 95 mg, 0.21 mmol (1.0 eq)

[0435] Intermediate 2: 67 mg, 0.23 mmol (1.1 eq)

[0436] Purified by FCC (Sfar amino, 1-5% MeOH in DCM), then by FCC (Sfar-C18, 5-30% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0437] Amount / yield: 63 mg, 0.10 mmol, 49% yield.

[0438] LC-MS Method 1: ta = 0.66 min, MS (ESI) m / z = 577.3 [M]+

[0439] Step 2 - l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-

[0440] (methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride hydrochloride (Example 19)

[0441] Analogously to Example 18, Example 19 was prepared by starting from the suitable Boc- protected amine. Pure product was obtained after removal of volatiles without FCC.

[0442] Intermediate 24: 61 mg, 0.10 mmol (1.0 eq)

[0443] Amount / yield: 47 mg, 0.090 mmol, 86% yield

[0444] LC-MS Method 2: ta = 0.67 min, MS (ESI) m / z = 239.1 [(M+H) / 2]+

[0445] ‘H NMR (500 MHz, DMSO-d6) 5 ppm 11.82 (br s, 1 H), 10.99 (br s, 1 H), 8.89 (d, J=1.6 Hz, 1 H), 8.76 (br s, 2 H), 8.31 (t, J=5.6 Hz, 1 H), 7.41 (d, J=0.8 Hz, 1 H), 6.90 (d, J=1.6 Hz, 1 H), 4.80 (s, 2 H), 4.67 - 4.74 (m, 2 H), 3.77 - 4.03 (m, 4 H), 3.51 (q, J=5.8 Hz, 2 H), 3.01 - 3.14 (m, 2 H), 2.57 (br s, 3 H), 2.02 (s, 3 H), 1.89 - 2.15 (m, 4 H), 1.66 (br s, 4 H).

[0446] Example 20: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((2-((2- methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium formate

[0447] Analogously to Example 4, Example 20 was prepared by starting from the corresponding bromide and amine.

[0448] Intermediate 21 : 65 mg, 0.18 mmol (1.0 eq)

[0449] Intermediate 2: 59.24 mg, 0.200 mmol (1.1 eq) Purified by FCC (Sfar-C18, 5-30% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0450] Amount / yield: 4.4 mg, 0.0090 mmol, 5% yield)

[0451] LC-MS Method 1 : tz? = 0.49 min, MS (ESI) m / z = 478.2 [M]+

[0452] 'H NMR (400 MHz, CDCh) 5 ppm 8.70 (s, 1H), 8.29 (d, J=1.3 Hz, 1 H), 7.39 (br s, 1 H), 6.99 (s, 1 H), 6.86 (s, 1 H), 4.80 - 4.99 (m, 4 H), 3.87 - 4.06 (m, 4 H), 3.43 - 3.58 (m, 4 H), 3.30 (s, 3 H), 2.07 (s, 3 H), 1.91 - 2.15 (m, 4 H), 1.68 - 1.86 (m, 4 H).

[0453] Example 21 : l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(oxetan-3- ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0454] Step 1 - 3-[[2-(azepan-l-yl)acetyl]amino]-4-methyl-7V-(oxetan-3-yl)thiophene-2- carboxamide (Intermediate 25)

[0455] Analogously to Intermediate 8, Intermediate 25 was prepared by starting from the suitable acid and amine.

[0456] Intermediate 13: 400 mg, 1.13 mmol (1.0 eq)

[0457] 3-oxetanamine: 165 mg, 2.25 mmol (2.0 eq)

[0458] Purified by FCC (Sfar amino, 5-20% EtOAc in DCM)

[0459] Amount / yield: 256 mg, 0.73 mmol, 65% yield

[0460] LC-MS Method 1 : tz? = 0.80 min, MS (ESI) m / z = 352.1 [M+H]+

[0461] Step 2 - l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(oxetan-3- ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 21)

[0462] Analogously to Example 4, Example 21 was prepared by starting from the suitable bromide and amine.

[0463] Intermediate 25: 80 mg, 0.23 mmol (1.0 eq)

[0464] Intermediate 2: 64 mg, 0.27 mmol (1.2 eq)

[0465] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0466] Amount / yield: 10 mg, 0.021 mmol, 9% yield

[0467] LC-MS Method 1 : t« = 0.48 min, MS (ESI) m / z = 476.2 [M]+

[0468] XHNMR (500 MHz, DMSO-d6) 5 ppm 11.91 (br s, 1 H), 9.59 (br d, J=5.9 Hz, 1 H), 8.76 (d, J=1.5 Hz, 1 H), 7.09 (s, 1 H), 6.90 (s, 1 H), 4.83 - 4.94 (m, 1 H), 4.74 (t, J=6.9 Hz, 2 H), 4.69 (s, 2 H), 4.48 (t, J=6.4 Hz, 2 H), 4.38 (s, 2 H), 3.76 - 3.90 (m, 4 H), 1.96 (s, 3 H), 1.87 - 2.03 (m, 4 H), 1.64 (br s, 4 H). Example 22: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-

[0469] (methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl) azepan-l-ium formate

[0470] Step 1 - tert-butyl N-[2-[[3-[[2-[l-[2-(benzylamino)-2-oxo-ethyl]azepan-l-ium-l- yl] acetyl] amino]-4-methyl-thiophene-2-carbonyl] amino] ethyl]-7V-methyl-carbamate (zwitterion) (Intermediate 26)

[0471] Analogously to Intermediate 4, Intermediate 26 was prepared by starting from the suitable bromide and amine.

[0472] Intermediate 10: 247 mg, 0.55 mmol (1.0 eq)

[0473] N-benzyl-2-bromo-acetamide: 125 mg, 0.55 mmol (1.0 eq)

[0474] Purified by FCC (Sfar amino, 0-10% MeOH in DCM)

[0475] Amount / yield: 100 mg, 0.17 mmol, 30% yield.

[0476] LC-MS Method 1: tR = 0.75 min, MS (ESI) m / z = 600.5 [M]+.

[0477] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-

[0478] (methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl) azepan-l-ium formate (Example 22)

[0479] Analogously to Example 18, Example 22 was prepared by starting from the suitable Boc- protected amine.

[0480] Intermediate 26: 100 mg, 0.17 mmol (1.0 eq)

[0481] Amount / yield: 66 mg, 0.12 mmol, 71% yield.

[0482] Purified by FCC (Sfar-C18, 5-50% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0483] LC-MS Method 2: tR = 0.98 min, MS (ESI) m / z = 500.4 [M]+

[0484] 'H NMR (400 MHz, DMSO-d6) 5 ppm 9.26 (br s, 1 H), 8.46 - 8.64 (m, 1 H), 8.42 (s, 1 H), 7.24 - 7.36 (m, 5 H), 7.22 (br s, 1 H), 4.48 - 4.63 (m, 4 H), 4.29 - 4.40 (m, 2 H), 3.72 - 3.90 (m, 4 H), 3.23 - 3.35 (m, 2 H), 2.63 (t, J=6.2 Hz, 2 H), 2.30 (s, 3 H), 1.98 (s, 3 H), 1.85 - 1.97 (m, 4 H), 1.53 - 1.73 (m, 4 H).

[0485] Example 23: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(oxetan-3- ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0486] Analogously to Example 4, Example 23 was prepared by starting from the suitable bromide and amine.

[0487] Intermediate 25: 80 mg, 0.22 mmol (1.0 eq)

[0488] A-benzyl-2-bromo-acetamide: 55 mg, 0.24 mmol (1.1 eq) Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0489] Amount / yield: 29 mg, 0.058 mmol, 26% yield

[0490] LC-MS Method 1 : ta = 0.62 min, MS (ESI) m / z = 499.2 [M]+

[0491] 'HNMR (500 MHz, DMSO-d6) 5 ppm 9.85 (br d, J=6.7 Hz, 1 H), 9.09 (t, J=5.7 Hz, 1 H), 7.18 - 7.36 (m, 5 H), 7.03 (d, J=0.8 Hz, 1 H), 4.90 (sxt, J=6.8 Hz, 1 H), 4.73 (t, J=6.9 Hz, 2 H), 4.64 (s, 2 H), 4.47 (t, J=6.4 Hz, 2 H), 4.28 - 4.37 (m, 4 H), 3.72 - 3.92 (m, 4 H), 1.94 (s, 3 H), 1.80 - 2.03 (m, 4 H), 1.62 (br s, 4 H).

[0492] Example 24: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-hydroxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium formate

[0493] Step 1 - N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-3-(2-(piperidin-l- yl)acetamido)thiophene-2-carboxamide (Intermediate 27)

[0494] Analogously to Intermediate 8, Intermediate 27 was prepared by starting from the suitable carboxylic acid and amine.

[0495] Intermediate 19: 365 mg, 1.30 mmol (1.0 eq)

[0496] (2-aminoethoxy)(tert-butyl)dimethylsilane: 0.53 mL, 2.6 mmol (2.0 eq)

[0497] Purified by FCC (Sfar amino, 0-40% EtOAc in cyclohexane)

[0498] Amount / yield: 260 mg, 0.59 mmol, 46% yield

[0499] LC-MS Method 1 : ty? = 0.82 min, MS (ESI) m / z = 440.2 [M+H]+

[0500] Step 2 -l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-hydroxyethyl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium formate (Example 24)

[0501] Analogously to Example 4, Example 24 was prepared by starting from the suitable bromide and amine.

[0502] Intermediate 27: 90 mg, 0.21 mmol (1.0 eq)

[0503] N-benzyl-2-bromo-acetamide: 48 mg, 0.21 mmol (1.0 eq)

[0504] Purified by FCC (Sfar-C18, 5-28% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0505] Amount / yield: 12 mg, 0.022 mmol, 11% yield

[0506] LC-MS Method 1 : tz? = 0.55 min, MS (ESI) m / z = 473.2 [M]+XH NMR (400 MHz, CDCh) 5 ppm 8.97 - 9.13 (m, 1 H), 8.68 ppm (s, 1H), 7.60 (br s, 1 H), 7.27 - 7.44 (m, 5 H), 7.05 (s, 1 H), 4.63 (s, 3 H), 4.37 - 4.49 (m, 4 H), 3.82 - 4.23 (m, 4 H), 3.55 - 3.65 (m, 2 H), 3.32 - 3.43 (m, 2 H), 2.05 (s, 3 H), 1.52 - 2.03 (m, 6 H).

[0507] Example 26: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-

[0508] (methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium chloride hydrochloride

[0509] Step 1 - tert-butyl methyl(2-(4-methyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2- carboxamido)ethyl)carbamate (Intermediate 28)

[0510] Analogously to Intermediate 8, Intermediate 28 was prepared by starting from the corresponding acid and amine.

[0511] Intermediate 19: 384 mg, 1.3 mmol (1.0 eq)

[0512] A-(2-aminoethyl)-A-methyl carbamic acid tert-butyl ester: 0.46 mL, 2.6 mmol (2.0 eq)

[0513] Purified by FCC (Sfar amino, 0-40% EtOAc in cyclohexane)

[0514] Amount / yield: 250 mg, 0.57 mmol, 44% yield.

[0515] LC-MS Method 1 : tz? = 0.60 min, MS (ESI) m / z = 439.2 [M+H]+

[0516] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert- butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)piperidin-l-ium (zwitterion) (Intermediate 29)

[0517] Analogously to Intermediate 4, Intermediate 29 was prepared by starting from the suitable bromide and amine.

[0518] Intermediate 28: 100 mg, 0.23 mmol (1.0 eq) A-benzyl-2-bromo-acetamide: 61 mg, 0.23 mmol (1.0 eq)

[0519] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0520] Amount / yield: 73 mg, 0.11 mmol, 48% yield

[0521] LC-MS Method 1 : tz? = 0.78 min, MS (ESI) m / z = 586.2 [M]+

[0522] Step 3 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-((2-(methylamino) ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium chloride hydrochloride (Example 26)

[0523] Analogously to Example 18, Example 26 was prepared by starting from the suitable Boc- protected amine.

[0524] Intermediate 29: 73 mg, 0.11 mmol (1.0 eq)

[0525] Amount / yield: 43 mg, 0 mm.19 mmol, 71% yield.

[0526] LC-MS Method 1: ta = 0.41 min, MS (ESI) m / z = 486.3 [M]+

[0527] 'H NMR (500 MHz, DMSO-d6) 5 ppm 11.03 (s, 1 H), 9.35 (t, J=5.8 Hz, 1 H), 8.82 - 9.04 (m, 2 H), 8.22 (t, J=5.7 Hz, 1 H), 7.43 (d, J=0.8 Hz, 1 H), 7.31 - 7.36 (m, 2 H), 7.24 - 7.31 (m, 3 H), 4.90 (s, 2 H), 4.58 (s, 2 H), 4.36 (d, J=5.6 Hz, 2 H), 3.64 - 3.91 (m, 4 H), 3.51 (q, J=5.6 Hz, 2 H), 2.99 - 3.17 (m, 2 H), 2.55 (t, J=5.4 Hz, 3 H), 2.02 (s, 3 H), 1.81 - 2.09 (m, 4 H), 1.52 - 1.70 (m, 2 H).

[0528] Example 25: l-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)-l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0529] Analogously to Example 4, Example 25 was prepared by starting from the suitable bromide and amine.

[0530] Intermediate 22: 136 mg, 0.30 mmol (1.0 eq)

[0531] Intermediate 2: 85 mg, 0.36 mmol (1.2 eq)

[0532] Purified by FCC (Sfar amino, 1-10% MeOH in DCM), then by trituration with DCM Amount / yield: 14 mg, 0.030 mmol, 10% yield

[0533] LC-MS Method 1 : ta = 0.44 min, MS (ESI) m / z = 464.2 [M]+

[0534] ‘HNMR (500 MHz, DMSO-d6) 5 ppm 11.88 (br s, 1 H), 9.05 (br s, 1 H), 8.76 (d, J=1.4 Hz, 1 H), 7.05 (s, 1 H), 6.90 (d, J=0.8 Hz, 1 H), 4.70 (s, 1 H), 4.82 (br s, 1 H), 4.32 (s, 2 H), 3.72 - 3.86 (m, 4 H), 3.47 (t, J=5.8 Hz, 2 H), 3.26 (q, J=5.6 Hz, 2 H), 1.93 (s, 7 H), 1.63 (br s, 4 H).

[0535] Example 28: l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- l-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion)

[0536] Step 1 - 2-bromo-7V-(4-methylisoxazol-3-yl)acetamide (Intermediate 30) To a suspension of potassium carbonate (1.34 g, 9.69 mmol) and 4-methylisoxazol-3-amine (0.56 g, 5.70 mmol) in ACN (20 mL), was added 2-bromoacetyl bromide (0.78 mL, 8.55 mmol) dropwise. After 3h water was added, and the reaction extracted with EtOAc (3x). The collected organic layers were dried overNa2SO4, filtered and concentrated to afford a crude that was purified by FCC (KP Sfar silica, 0-60% EtOAc in cyclohexane) to give title compound (Intermediate 30: 1.23 g, 94% wt).

[0537] LC-MS Method 1 : tz? = 0.54 min, MS (ESI) m / z = 219.0 / 221.0 [M+H]+

[0538] Step 2 - l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2- ((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion) (Example 28)

[0539] Analogously to Example 4, Example 28 was prepared by starting from the suitable bromide and amine.

[0540] Intermediate 20: 26 mg, 0.084 mmol (1.0 eq)

[0541] Intermediate 30: 18.4 mg, 0.084 mmol (1.0 eq)

[0542] Purified by FCC (Sfar amino, 0-5% MeOH in DCM).

[0543] Amount / yield: 4 mg, 0.009 mmol, 11% yield

[0544] LC-MS Method 1 : tz? = 0.51 min, MS (ESI) m / z = 448.2 [M]+

[0545] 'HNMR (400 MHz, CDCh) 5 ppm 8.69 (br s, 1 H), 8.10 (s, 1 H), 6.97 (s, 1 H), 5.07 (br s, 2 H), 4.75 (s, 2 H), 3.86 - 4.28 (m, 4 H), 3.08 (br s, 6 H), 2.19 (s, 3 H), 2.05 (s, 3 H), 1.92 - 2.04 (m, 4 H), 1.61 - 1.90 (m, 2 H).

[0546] Example 29: l-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)-l-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azepan-l-ium formate

[0547] Analogously to Example 4, Example 29 was prepared by starting from the suitable bromide and amine.

[0548] Intermediate 21 : 65 mg, 0.18 mmol (1.0 eq)

[0549] Intermediate 30: 47 mg, 0.20 mmol (1.2 eq)

[0550] Purified by HPLC Semi-prep, method.

[0551] Amount / yield: 31 mg, 0.054 mmol, 29% yield.

[0552] LC-MS Method 1: ta = 0.51 min, MS (ESI) m / z = 492.3 [M]+

[0553] 'H NMR (500 MHz, CDCh) 5 ppm 8.72 (s, 1 H), 8.10 (s, 1 H), 7.08 (br s, 1 H), 7.01 (s, 1 H), 5.01 (br s, 2 H), 4.77 (br s, 2 H), 3.88 - 4.21 (m, 4 H), 3.45 - 3.58 (m, 4 H), 3.33 (s, 3 H), 2.09 (s, 3 H), 2.06 (s, 3 H), 1.96 - 2.07 (m, 4 H), 1.78 (br s, 4 H).

[0554] Example 12: l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)-l- (2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0555] Analogously to Example 4, Example 12 was prepared by starting from the suitable bromide and amine. Intermediate 14: 70 mg, 0.23 mmol (1.0 eq)

[0556] Intermediate 30: 50 mg, 0.23 mmol (1.0 eq)

[0557] Purified by FCC (Sfar amino, 1-5% MeOH in DCM), then by FCC (Sfar-C18, 10-25% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0558] Amount / yield: 2.7 mg, 0.006 mmol, 3% yield.

[0559] LC-MS Method 1 : ta = 0.48 min, MS (ESI) m / z = 448.1 [M]+

[0560] ‘H NMR (500 MHz, CDCh) 5 ppm 11.37 (br s, 1 H), 8.11 (s, 1 H), 7.23 (br s, 1 H), 7.02 (s, 1 H), 5.04 (br s, 2 H), 4.82 (br s, 2 H), 3.86 - 4.06 (m, 4 H), 2.89 (d, J=4.7 Hz, 3 H), 2.09 (s, 6 H), 2.05 (br s, 4 H), 1.78 (br s, 4 H).

[0561] Example 39: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((2-(morpholine-4- carbonyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide

[0562] Step 1 - methyl 3-(2-bromoacetamido)benzo[b]thiophene-2-carboxylate (Intermediate

[0563] 31)

[0564] Analogously to Intermediate 1, Intermediate 31 was prepared by starting from the suitable amine.

[0565] Methyl 3-aminobenzo[b]thiophene-2-carboxylate: 2.50 g, 12.0 mmol (1.0 eq)

[0566] Amount / yield: 1.80 g, 5.50 mmol, 45% yield

[0567] LC-MS Method 1: ta = LOO min, MS (ESI) m / z = 328.0 / 330.1 [M+H]+

[0568] Step 2 - methyl 3-(2-(azepan-l-yl)acetamido)benzo[b]thiophene-2-carboxylate (Intermediate 32)

[0569] Analogously to Intermediate 11, Intermediate 32 was prepared by starting from the suitable bromide.

[0570] Intermediate 31 : 1.30 g, 3.96 mmol (1.0 eq)

[0571] Homopiperidine: 0.47 mL, 3.96 mmol (1.0 eq)

[0572] Amount / yield: 1.37 g, 3.95 mmol, 99% yield.

[0573] LC-MS Method 1: ta = 0.61 min, MS (ESI) m / z = 347.1 [M+H]+ Step 3 - 3-(2-(azepan-l-yl)acetamido)benzo[b]thiophene-2-carboxylic acid

[0574] (Intermediate 33)

[0575] To a suspension of methyl 3-(2-(azepan-l-yl)acetamido)benzo[b]thiophene-2-carboxylate (Intermediate 32, 829 mg, 2.39 mmol) in water (2.7 mL) / MeOH (2.7 mL) / THF (2.7 mL), Lithium hydroxide (86 mg, 3.59 mmol) was added and the reaction was stirred at 45°C for 6h. The organic phase was evaporated under reduced pressure and HC1 2M in water was added to the remaining aqueous phase until pH = 7. Then the aqueous phase was extracted with DCM and DCM / MeOH (9 / 1). The collected organic layers were evaporated under reduced pressure to give the title compound (33: 790 mg, 2.38 mmol, 99 % yield).

[0576] LC-MS Method 1: ta = 0.57 min, MS (ESI) m / z = 333.3 [M+H]+

[0577] Step 4 - 2-(azepan-l-yl)-7V-(2-(morpholine-4-carbonyl)benzo[b]thiophen-3- yl)acetamide (Intermediate 34)

[0578] To a suspension of 3-(2-(azepan-l-yl)acetamido)benzo[b]thiophene-2-carboxylic acid (Intermediate 33, 750 mg, 2.26 mmol) in DCM (6 mL), thionyl dichloride (320 mL, 4.51 mmol) was added at 0 °C and the reaction mixture was stirred at RT for 2 h. Morpholine (0.78 mL, 9.00 mmol) was added and the reaction mixture was stirred at 35°C for 4 h. Volatiles were removed under reduced pressure. The crude was purified by FCC (KP Sfar silica, 0-10% MeOH in DCM) to give the title compound (Intermediate 34: 95 mg, 0.24 mmol, 10% yield) as an orange solid.

[0579] LC-MS Method 1: ta = 0.57 min, MS (ESI) m / z = 402.3 [M+H]+

[0580] Step 5 - l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((2-(morpholine-4- carbonyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide (Example 39)

[0581] To a solution of Intermediate 34 (30 mg, 0.070 mmol) in ACN (0.5 mL), 2-bromo-A- (isoxazol-3-yl)acetamide Intermediate 2 (16 mg, 0.070 mmol) was added and the reaction mixture was stirred overnight at 70°C. The reaction was cooled to RT and the solid precipitate was allowed to settle. The liquid phase was removed with a Pasteur pipette and the solid was triturated with cyclohexane / EtOAc (7 / 3 v / v), then with cold ACN to obtain the title compound (Example 39: 13 mg, 0.021 mmol, 28% yield).

[0582] LC-MS Method 1 : ty? = 0.63 min, MS (ESI) m / z = 526.38 [M]+

[0583] ‘H NMR (400 MHz, DMSO-d6) 5 ppm 11.73 (br s, 1 H), 10.99 (br s, 1 H), 8.90 (d, J=1.5 Hz, 1 H), 8.03 (d, J=8.1 Hz, 1 H), 7.82 (d, J=7.8 Hz, 1 H), 7.47 - 7.54 (m, 1 H), 7.41 - 7.46 (m, 1 H), 6.94 (d, J=1.0 Hz, 1 H), 4.77 (s, 2 H), 4.67 (s, 2 H), 3.78 - 4.03 (m, 4 H), 3.42 - 3.73 (m, 8 H), 1.85 - 2.11 (m, 4 H), 1.55 - 1.80 (m, 4 H).

[0584] Example 43: l,l-bis(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium (zwitterion)

[0585] Step 1 - 3-aminobenzo[b]thiophene-2-carboxylic acid (Intermediate 35)

[0586] To a suspension of methyl 3-aminobenzo[b]thiophene-2-carboxylate (500 mg, 2.41 mmol) in isopropanol (20 mL) / water (4 mL), lithium hydroxide (115 mg, 4.83 mmol) was added, and the reaction mixture was stirred at reflux for 1.5 h. Then it was cooled to RT, diluted with water and the organic volatiles were evaporated under reduced pressure. The remained aqueous phase was neutralized with HC1 3 M in water until pH = 6 observing the formation of a white precipitate. The solid was filtered, washed with cold water (2x) and dried to obtain the title compound (Intermediate 35: 460 mg, 2.38 mmol, 99% yield) as an off-white solid.

[0587] LC-MS Method 1 : tz? = 0.83 min, MS (ESI) m / z = 194.2 [M+H]+

[0588] Step 2 - 3-amino- / V,7V-dimethylbenzo[b]thiophene-2-carbo amide (Intermediate 36)

[0589] To a solution of 3-aminobenzo[b]thiophene-2-carboxylic acid (Intermediate 35, 175 mg, 0.91 mmol), HATU (482 mg, 1.27 mmol), diisopropylethylamine (160 mL, 0.91 mmol) in DMF (9 mL), dimethylamine hydrochloride (155 mg, 1.90 mmol) was added, and the reaction mixture was stirred at RT overnight. Water was added and the aqueous phase was extracted with EtOAc (3x). The collected organic layers were washed with NaHCCh aq. sat. sol., potassium carbonate 2 M in water, and then brine. The organic phase was dried over a phase separator and evaporated under reduced pressure to obtain the title compound (Intermediate 36, 170 mg, 0.77 mmol, 85% yield).

[0590] LC-MS Method 1 : tz? = 0.93 min, MS (ESI) m / z = 221.0 [M+H]+

[0591] Step 3 - 3-(2-bromoacetamido)- / V,7V-dimethylbenzo[b]thiophene-2-carboxamide (Intermediate 37)

[0592] Analogously to Intermediate 1, Intermediate 37 was prepared by starting from the suitable amine.

[0593] Intermediate 36: 170 mg, 0.77 mmol (1.0 eq)

[0594] Amount / yield: 260 mg, 0.76 mmol, 99% yield.

[0595] LC-MS Method 1 : ty? = 0.79 min, MS (ESI) m / z = 341.1 / 343.2 [M+H]+

[0596] Step 4 - 3-(2-(azepan-l-yl)acetamido)- / V,7V-dimethylbenzo[b]thiophene-2-carboxamide (Intermediate 38)

[0597] To a solution of 3-(2-bromoacetamido)-A,A-dimethylbenzo[b]thiophene-2-carboxamide (Intermediate 37, 100 mg, 0.53 mmol) in ACN (4.2 mL), azepane (0.13 mL, 1.11 mmol) was added, the reaction mixture was stirred at RT for 1.5 h. Volatiles were removed under reduced pressure and the crude was purified by FCC (KP Sfar silica, 0-50% EtOAc in cyclohexane), to afford the title compound (Intermediate 38: 150 mg, 0.42 mmol, 79% yield).

[0598] LC-MS Method 1: ta = 0.55 min, MS (ESI) m / z = 360.3 [M+H]+

[0599] Step 5 - l,l-bis(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium (zwitterion) (Example 43)

[0600] Analogously to Example 4, Example 43 was prepared by starting from the suitable amine and bromide.

[0601] Intermediate 38: 35 mg, 0.10 mmol (1.0 eq)

[0602] Intermediate 37: 3-(2-bromoacetamido)-A,A-dimethylbenzo[b]thiophene-2-carboxamide: 33 mg, 0.10 mmol (1.0 eq)

[0603] Amount / yield: 3.8 mg, 0.006 mmol, 6% yield. Purified by FCC (Sfar amino, 0-5% MeOH in DCM).

[0604] LC-MS Method 1 : ty? = 0.80 min, MS (ESI) m / z = 620.2 [M]+

[0605] ‘H NMR (500 MHz, DMSO-d6) 5 ppm 13.87 (br s, 1 H), 7.88 (d, J=8.1 Hz, 2 H), 7.67 (br d, J=8.1 Hz, 2 H), 7.35 (t, J=7.3 Hz, 2 H), 7.20 (t, J=7.5 Hz, 2 H), 4.40 (br s, 4 H), 3.78 (br s, 4 H), 2.97 (br s, 12 H), 1.98 (br s, 4 H), 1.67 (br s, 4 H).

[0606] Example 68: l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0607] Analogously to Example 4, Example 68 was prepared starting from the suitable bromide and amine.

[0608] Intermediate 14: 81 mg, 0.26 mmol (1.0 eq)

[0609] Intermediate 1 : 76 mg, 0.26 mmol (1.0 eq)

[0610] Purified by FCC (Sfar amino, 1-5% MeOH in DCM)

[0611] Amount / yield: 69 mg, 0.13 mmol, 50% yield.

[0612] LC-MS Method 1 : tz? = 0.61 min, MS (ESI) m / z = 521.2 [M]+

[0613] 'HNMR (500 MHz, DMSO-d6) 5 ppm 11.24 (br s, 1H), 8.73 (br s, 1H), 7.53 (s, 1H), 7.04 (s, 1H), 4.68 (br s, 2H), 4.39 (s, 2H), 3.85 - 3.77 (m, 2H), 3.73 (s, 3H), 3.77 - 3.70 (m, 2H), 2.62 (d, J = 4.7 Hz, 3H), 2.01 (s, 3H), 1.96 (s, 3H), 2.00 - 1.90 (m, 4H), 1.69 - 1.58 (m, 2H)

[0614] Example 69: l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion)

[0615] Step 1 - 3-amino-N,4-dimethylthiophene-2-carboxamide (Intermediate 55)

[0616] Analogously to Intermediate 36, Intermediate 55 was prepared by starting from the suitable acid and amine.

[0617] 3-amino-4-methylthiophene-2-carboxylic acid: 640 mg, 4.07 mmol (1.0 eq)

[0618] Methanamine hydrochloride: 550 mg, 8.14 mmol (2.0 eq)

[0619] Purified by FCC (Sfar amino, 20% EtOAc in cyclohexane)

[0620] Amount / yield: 259 mg, 1.5 mmol, 37% yield

[0621] LC-MS Method 1: ta = 0.57 min, MS (ESI) m / z = 171.3 [M+H]+

[0622] Step 2 - 3-(2-chloroacetamido)-N,4-dimethylthiophene-2-carboxamide (Intermediate

[0623] 56)

[0624] To a suspension of Intermediate 55 (190 mg, 1.1 mmol) and TEA (0.155 mL, 0.89 mmol) in ACN (9 mL), 2-chloroacetyl chloride (0.097 mL, 1.22 mmol) was slowly added at 0°C. The reaction was allowed to stir at RT overnight. Then the reaction mixture was washed with NaHCCh aq. sat. sol. and water. The organic phase was dried over a phase separator and concentrated under reduced pressure to afford a crude that was purified by FCC (Sfar silica, 20-50% EtOAc in cyclohexane) to afford the title compound (Intermediate 56: 200 mg, 0.81 mmol, 74 % yield).

[0625] LC-MS Method 1 : tz? = 0.58 min, MS (ESI) m / z = 247.4 [M+H]+

[0626] Step 3 - \.4-dimethyl-3-(2-(piperidin-l-yl)acetamido)thiophene-2-carbox:imide (Intermediate 57)

[0627] To a stirred solution of Intermediate 56 (69 mg, 0.28 mmol) in ACN (1 mL), piperidine (0.028 mL, 0.28 mmol) and potassium carbonate (58 mg, 0.42 mmol) were added, and the mixture was stirred for 3 h at RT. The reaction was quenched NaHCCh aq. sat. sol, the organic layer was separated and the aqueous one extracted with DCM (3x). The collected organic layers were filtered through a phase separator and concentrated to afford title compound (Intermediate 57: 62 mg, 0.21 mmol, 75% yield).

[0628] LC-MS Method 2: ty? = 0.73 min, MS (ESI) m / z = 296.1 [M+H]+

[0629] Step 4 - l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2- ((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-l-ium (zwitterion) (Example 69)

[0630] Analogously to Example 4, Example 69 was prepared by starting from the suitable bromide and amine.

[0631] Intermediate 57: 62 mg, 0.21 mmol (1.0 eq)

[0632] Intermediate 1 : 61.3 mg, 0.21 mmol (1.0 eq) Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0633] Amount / yield: 41 mg, 0.081 mmol, 38% yield.

[0634] LC-MS Method 1 : tz? = 0.59 min, MS (ESI) m / z = 507.2 [M]+

[0635] 'HNMR (500 MHz, DMSO-d6) 5 ppm 11.24 (br s, 1H), 8.73 (br s, 1H), 7.53 (s, 1H), 7.04 (s, 1H), 4.68 (br s, 2H), 4.39 (s, 2H), 3.85 - 3.77 (m, 2H), 3.73 (s, 3H), 3.77 - 3.70 (m, 2H), 2.62 (d, J = 4.7 Hz, 3H), 2.01 (s, 3H), 1.96 (s, 3H), 2.00 - 1.90 (m, 4H), 1.69 - 1.58 (m, 2H)

[0636] Example 73: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-bis(hydroxymethyl)piperidin-l-ium formate

[0637] Step 1 - N-benzyl-2-(4,4-bis(hydroxymethyl)piperidin-l-yl)acetamide (Intermediate 58)

[0638] Analogously to Intermediate 11, Intermediate 58 was prepared by starting from the suitable bromide and amine.

[0639] N-benzyl-2-bromoacetamide: 150 mg, 0.66 mmol (1.0 eq)

[0640] Piperidine-4,4-diyldimethanol hydrochloride: 120 mg, 0.66 mmol (1.0 eq)

[0641] Potassium carbonate: 227 mg, 1.6 mmol (2.5 eq)

[0642] Amount / yield: 190 mg, 0.64 mmol, 98% yield

[0643] LC-MS Method 2: tz? = 0.52 min, MS (ESI) m / z = 293.2 [M+H]+

[0644] Step 2 - 3-amino-N,N,4-trimethylthiophene-2-carboxamide (Intermediate 59)

[0645] Analogously to Intermediate 36, Intermediate 59 was prepared starting from the suitable acid and amine.

[0646] 3-amino-4-methylthiophene-2-carboxylic acid: 900 mg, 5.72 mmol (1.0 eq) dimethylamine hydrochloride: 934 mg, 11.45 mmol (2.0 eq)

[0647] Purified by FCC (Sfar silica, 70% EtOAc in cyclohexane) Amount / yield: 410 mg, 1.5 mmol, 39% yield LC-MS Method 1: ta = 0.68 min, MS (ESI) m / z = 185.0 [M+H]+

[0648] Step 3 - 3-(2-bromoacetamido)-N,N,4-trimethylthiophene-2-carboxamide

[0649] (Intermediate 60)

[0650] Analogously to Intermediate 30, Intermediate 60 was prepared starting from the suitable amine at 0°C:

[0651] Intermediate 59: 190 mg, 1.03 mmol (1.0 eq)

[0652] Purified by FCC (Sfar silica, 90% EtOAc in cyclohexane)

[0653] Amount / yield: 187 mg, 0.61 mmol, 59 % yield.

[0654] LC-MS Method 1 : tz? = 0.64 min, MS (ESI) m / z = 305.0 / 307.0 [M+H]+

[0655] Step 4 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(dimethylcarbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-bis(hydroxymethyl)piperidin-l-ium formate (Example 73)

[0656] Analogously to Example 4, Example 73 was prepared starting from the suitable bromide and amine.

[0657] Intermediate 58: 180 mg, 0.61 mmol (1.0 eq)

[0658] Intermediate 60: 187 mg, 0.61 mmol (1.0 eq)

[0659] Purified by FCC (Sfar-C18, 10-30% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH)

[0660] Crystallization by vapour diffusion of Et20 in iPrOH

[0661] Amount / yield: 131 mg, 0.23 mmol, 38% yield.

[0662] LC-MS Method 1: ta = 0.59 min, MS (ESI) m / z = 517.2 [M]+

[0663] 'HNMR (400 MHz, DMSO-d6) 5 ppm 10.98 (br s, 1H), 9.65 - 9.22 (m, 1H), 8.51 (s, 1H), 7.41 - 7.21 (m, 6H), 5.25 - 4.56 (m, 4H), 4.54 (s, 2H), 4.35 (d, J = 5.7 Hz, 2H), 3.89 - 3.64 (m, 4H), 3.46 - 3.33 (m, 4H), 2.89 (s, 6H), 2.06 (s, 3H), 1.80 - 1.63 (m, 4H).

[0664] Example 74: (2-(4,4-dimethyl-l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3- yl)amino)-2-oxoethyl)piperidin-l-ium-l-yl)acetyl)(isoxazol-3-yl)amide formate

[0665] Step 1 - 2-chloro-N-(isoxazol-3-yl)acetamide (Intermediate 53)

[0666] To a stirred solution of isoxazol-3-amine (400 mg, 4.76 mmol) and triethylamine (0.73 mL, 5.23 mmol) in DCM (34 mL), 2-chloroacetyl chloride (0.40 mL, 5.0 mmol) was added at 0°C. After 10 min, the reaction mixture was warmed to RT and stirred for 2h. Then water was added, the organic phase was separated and the aqueous phase extracted with DCM (2x). The collected organic phases were dried on a phase separator and concentrated under reduced pressure. The crude was purified by FCC (Sfar amino, 5-50% cyclohexane in EtOAc) to title compound (Intermediate 53: 610 mg, 3.80 mmol, 80% yield) as white powder.

[0667] LC-MS Method 2: tz? = 0.50 min, MS (ESI) m / z = 161.2 [M+H]+

[0668] Step 2 - 2-(4,4-dimethylpiperidin-l-yl)-N-(isoxazol-3-yl)acetamide (Intermediate 61)

[0669] Analogously to Intermediate 57, Intermediate 61 was prepared by starting from the suitable amine and chloride.

[0670] Intermediate 53: 2.3 g, 12.9 mmol (1.0 eq)

[0671] 4,4-Dimethylpiperidine hydrochloride: 1.92 g, 12.9 mmol (1.0 eq)

[0672] Potassium carbonate: 4.46 g, 32.2 mmol (2.5 eq)

[0673] Purified by FCC (Sfar amino, 10% EtOAc in cyclohexane)

[0674] Amount / yield: 2.88 g, 12.1 mmol, 94% yield.

[0675] LC-MS Method 1 : tz? = 0.39 min, MS (ESI) m / z = 238.2 [M+H]+

[0676] Step 3 - 3-(2-bromoacetamido)-N,4-dimethylthiophene-2-carboxamide (Intermediate 62)

[0677] Analogously to Intermediate 30, Intermediate 62 was prepared by starting from the suitable amine at 0°C:

[0678] Intermediate 55: 265 mg, 1.55 mmol (1.0 eq)

[0679] Purified by FCC (Sfar silica, 50% EtOAc in cyclohexane)

[0680] Amount / yield: 340 mg, 1.34 mmol, 86 % yield. LC-MS Method 1: ta = 0.65 min, MS (ESI) m / z = 290.9 / 292.9 [M+H]+

[0681] Step 4 - (2-(4,4-dimethyl-l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)- 2-oxoethyl)piperidin-l-ium-l-yl)acetyl)(isoxazol-3-yl)amide formate (Example 74)

[0682] Analogously to Example 4, Example 74 was prepared by starting from the suitable bromide and amine.

[0683] Intermediate 62: 100 mg, 0.34 mmol (1.0 eq)

[0684] Intermediate 61 : 81 mg, 0.34 mmol (1.0 eq)

[0685] Purified by HPLC Semi-prep.

[0686] Amount / yield: 26 mg, 0.053 mmol, 15% yield.

[0687] LC-MS Method 1 : tz? = 0.60 min, MS (ESI) m / z = 448.2 [M]+

[0688] 'HNMR (500 MHz, DMSO-d6) 5 ppm 8.84 (d, J = 1.5 Hz, 1H), 8.35 (s, 1H), 8.17 (br q, J = 4.0 Hz, 1H), 7.29 (s, 1H), 6.91 (s, 1H), 4.85 - 4.64 (m, 4H), 3.89 - 3.73 (m, 4H), 2.67 (d, J = 4.5 Hz, 3H), 1.98 (s, 3H), 1.87 - 1.62 (m, 4H), 1.07 (d, J = 6.9 Hz, 6H)

[0689] Example 70: l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l- (2-((4-methyl-2-(oxetan-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0690] Analogously to Example 4, Example 70 was prepared by starting from the suitable bromide and amine.

[0691] Intermediate 25: 32 mg, 0.09 mmol (1.0 eq)

[0692] Intermediate 1 : 27 mg, 0.09 mmol (1.0 eq)

[0693] Purified by FCC (Sfar amino, 0-2% MeOH in DCM)

[0694] Amount / yield: 13 mg, 0.02 mmol, 25% yield.

[0695] LC-MS Method 1: ta = 0.66 min, MS (ESI) m / z = 563.3 [M]+

[0696] 'HNMR (400 MHz, DMSO-d6) 5 ppm 11.26 (s, 1H), 9.58 (s, 1H), 7.54-7.07 (s, 2H), 4.80 (m, 9H), 4.70 - 4.41 (m, 4H), 4.64 - 4.41 (m, 4H), 3.85 (s, 4H), 3.72 (s, 3H), 2.00 - 1.97 (m, 10H), 1.67 (s, 4H)

[0697] Example 55: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2- hydroxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride

[0698] Step 1 - 3-(2-(azepan-l-yl)acetamido)-N-(2-hydroxyethyl)-N,4-dimethylthiophene-2- carboxamide (Intermediate 63)

[0699] Analogously to Intermediate 36, Intermediate 63 was prepared by starting from the suitable acid and amine.

[0700] Intermediate 13: 700 mg, 1.97 mmol (1.0 eq)

[0701] 2-(methylamino)ethan-l-ol: 222 mg, 2.96 mmol (1.5 eq)

[0702] Purified by FCC (Sfar silica, 0-5% MeOH in DCM), then by FCC (Sfar-C18, 5-40% ACN + 0.1% (33% aq NH4OH) in H2O + 0.1% (33% aq NH4OH)

[0703] Amount / yield: 336 mg, 0.95 mmol, 48% yield

[0704] LC-MS Method 1 : ty? = 0.40 min, MS (ESI) m / z = 354.2 [M+H]+

[0705] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-hydroxyethyl)(methyl) carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride (Example 55)

[0706] To a stirred solution of Intermediate 63 (168 mg, 0.48 mmol) in ACN (4.7 mL), N-benzyl- 2 -bromoacetamide (114 mg, 0.48 mmol) was added. The mixture was stirred at 80 °C for 18 h in a sealed vial. The reaction mixture was concentrated under reduced pressure and the residue was purified by FCC (Sfar amino, 0-5% MeOH in DCM) affording the desired product as zwitterion. This was dissolved in DCM (2.0 mL) and HC1 2 M in Et20 (4 mL) was added at RT under stirring. After 5 min, volatiles were removed under reduced pressure to afford title compound (Example 55: 101 mg, 0.19 mmol, 40% yield).

[0707] LC-MS Method 1 : tz? = 0.60 min, MS (ESI) m / z = 501.3 [M]+

[0708] 'H NMR (500 MHz, DMSO-d6) 5 ppml0.44 (br s, 1H), 9.12 (br t, J = 5.7 Hz, 1H), 7.38 - 7.23 (m, 6H), 4.80 (br s, 1H), 4.65 (s, 2H), 4.45 (s, 2H), 4.35 (d, J = 5.6 Hz, 2H), 3.86 - 3.71 (m, 4H), 3.53 (br s, 2H), 3.38 (br s, 2H), 2.94 (br s, 3H), 2.07 (s, 3H), 1.95 (br d, J = 4.3 Hz, 4H), 1.64 (br s, 4H)

[0709] Example 67 : l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- 4,4-dimethyl-l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2- oxoethyl)piperidin-l-ium formate

[0710] Step 1 - 3-(2-(4,4-dimethylpiperidin-l-yl)acetamido)-N,4-dimethylthiophene-2- carboxamide (Intermediate 64)

[0711] Analogously to Intermediate 57, Intermediate 64 was prepared by starting from the suitable amine and chloride.

[0712] Intermediate 56: 69 mg, 0.28 mmol (1.0 eq)

[0713] 4,4-Dimethylpiperidine hydrochloride: 42 mg, 0.28 mmol (1.0 eq)

[0714] Amount / yield: 86 mg, 0.27 mmol, 95% yield.

[0715] LC-MS Method 2: ty? = 0.88 min, MS (ESI) m / z = 324.1 [M+H]+

[0716] Step 2 - l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4- dimethyl-l-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin- 1-ium formate (Example 67)

[0717] Analogously to Example 4, Example 67 was prepared by starting from the suitable bromide and amine.

[0718] Intermediate 64: 86 mg, 0.27 mmol (1.0 eq)

[0719] Intermediate 1 : 78 mg, 0.27 mmol (1.0 eq)

[0720] Purified by FCC (Sfar-C18, 5-20% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH).

[0721] Amount / yield: 71 mg, 0.12 mmol, 45% yield.

[0722] LC-MS Method 1 : tz? = 0.63 min, MS (ESI) m / z = 535.6 [M]+

[0723] 'HNMR (500 MHz, DMSO-d6) 5 ppm 11.05 (br s, 1 H), 8.41 (s, 1 H), 8.20 (br s, 1 H), 7.58 (s, 1 H), 7.26 (br s, 1 H), 4.80 (br s, 2 H), 4.69 (br s, 2 H), 3.77 - 3.90 (m, 4 H), 3.74 (s, 3 H), 2.62 (d, J=4.5 Hz, 3 H), 2.05 (s, 3 H), 2.00 (s, 3 H), 1.78 (br s, 4 H), 1.09 (s, 6 H)

[0724] Example 75: l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)- 4,4-bis(hydroxymethyl)-l-(2-oxo-2-((l-phenylcyclopropyl)amino) ethyl)piperidin-l-ium formate

[0725] Step 1 - 2-bromo-N-(l-phenylcyclopropyl)acetamide (Intermediate 65)

[0726] To a stirred solution of 1-phenylcyclopropan-l -amine hydrochloride (250 mg, 1.47 mmol) and TEA (0.41 mL, 2.94 mmol) in DCM (15 mL), 2-bromoacetyl bromide (0.13 mL, 1.47 mmol) was added at 0°C. After 1 h of stirring at 0°C, NH4Q aq. sat. sol. was added, the organic phase was separated, and the aqueous phase extracted with DCM (2x). The collected organic phases were dried on a phase separator and concentrated under reduced pressure. The crude was purified by FCC (Sfar silica, 40% EtOAc in cyclohexane) to afford title compound (Intermediate 65: 279 mg, 1.09 mmol, 74% yield) as white solid.

[0727] LC-MS Method 2: tz? = 0.64 min, MS (ESI) m / z = 253.1 / 255.1 [M+H]+

[0728] Step 2 - 3-(2-(4,4-bis(hydroxymethyl)piperidin-l-yl)acetamido)-N,N,4- trimethylthiophene-2-carboxamide (Intermediate 66)

[0729] To a stirred solution of Intermediate 60 (50 mg, 0.16 mmol) in ACN (4 mL), piperidine-4,4- diyldimethanol hydrochloride (30 mg, 0.16 mmol) and potassium carbonate (56 mg, 0.41 mmol) were added, and the mixture was stirred for 16 h at RT. The reaction was filtered to remove the salts and the organic phase was concentrated under reduced pressure to afford title compound (Intermediate 66: 55 mg, 0.15 mmol, 90% yield) as a white solid.

[0730] LC-MS Method 2: tz? = 0.50 min, MS (ESI) m / z = 370.2 [M+H]+

[0731] Step 3 - l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4- bis(hydroxymethyl)-l-(2-oxo-2-((l-phenylcyclopropyl)amino)ethyl)piperidin-l-ium formate (Example 75)

[0732] Analogously to Example 4, Example 75 was prepared by starting from the suitable bromide and amine.

[0733] Intermediate 66: 55 mg, 0.15 mmol (1.0 eq)

[0734] Intermediate 65: 38 mg, 0.15 mmol (1.0 eq)

[0735] Purified by FCC (Sfar-C18, 5-35% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH).

[0736] Amount / yield: 39 mg, 0.066 mmol, 44% yield.

[0737] LC-MS Method 1 : tz? = 0.60 min, MS (ESI) m / z = 543.5 [M]+

[0738] 'H NMR (400 MHz, DMSO-d6 ) 5 ppm 11.00 (br s, 1H), 9.68 (br s, 1H), 8.51 (s, 1H), 7.32 (s, 1H), 7.28 - 7.21 (m, 2H), 7.20 - 7.11 (m, 3H), 4.70 (br s, 2H), 4.52 (s, 2H), 4.78 (br s, 2H), 3.73 (br t, J = 4.9 Hz, 4H), 3.45 - 3.36 (m, 4H), 2.90 (s, 6H), 2.06 (s, 3H), 1.82 - 1.61 (m, 4H), 1.30 - 1.09 (m, 4H) Example 66: l-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-

[0739] 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0740] Analogously to Example 4, Example 66 was prepared by starting from the suitable bromide and amine.

[0741] Intermediate 11 : 90 mg, 0.26 mmol (1.0 eq)

[0742] Intermediate 60: 55 mg, 0.26 mmol (1.0 eq)

[0743] Purified by FCC (Sfar amino, 0-2% MeOH in DCM)

[0744] Amount / yield: 55 mg, 0.10 mmol, 39% yield.

[0745] LC-MS Method 1 : ty? = 0.65 min, MS (ESI) m / z = 535.2 [M]+

[0746] 'H NMR (400 MHz, DMSO-d6) 5 ppm 13.99 (br s, 1 H), 7.29 (s, 1 H), 7.21 (s, 1 H), 4.61 (br s, 2 H), 4.10 (s, 2 H), 3.73 - 3.89 (m, 2 H), 3.65 (s, 3 H), 3.57 - 3.69 (m, 2 H), 2.91 (br s, 6 H), 1.94 (br s, 4 H), 1.87 - 1.90 (m, 6 H), 1.64 (br s, 4 H)

[0747] Example 64: l-(2-((2-(bis(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-

[0748] 2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan- 1 -in in formate

[0749] Step 1 - 3-(2-(azepan-l-yl)acetamido)-N,N-bis(2-hydroxyethyl)-4-methylthiophene-2- carboxamide (Intermediate 67)

[0750] To a suspension of 3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2-carboxylic acid sodium chloride (Intermediate 13, 100 mg, 0.28 mmol), HATU (129 mg, 0.34 mmol), DIPEA (147 mL, 0.85 mmol) in DMF (1.4 mL), 2,2'-azanediylbis(ethan-l-ol) (0.41 mL, 0.42 mmol) was added, and the reaction mixture was stirred at 80°C overnight. The mixture was diluted with and EtOAc. The two phases were separated, and the organic layer was washed with NaHCCh aq sat sol and then brine. The water phase was back extracted with a solution of DCM:iPrOH 2: 1 (2x). The collected organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by FCC (Sfar-C18, 0-80% ACN in H2O) to afford title compound (Intermediate 67: 35 mg, 0.09 mmol, 32% yield).

[0751] LC-MS Method 1 : tz? = 0.37 min, MS (ESI) m / z = 384.2 [M+H]+ Step 2 - l-(2-((2-(bis(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l- ium formate (Example 64)

[0752] Analogously to Example 4, Example 64 was prepared by starting from the suitable bromide and amine.

[0753] Intermediate 67: 35 mg, 0.09 mmol (1.0 eq)

[0754] Intermediate 1 : 27 mg, 0.09 mmol (1.0 eq)

[0755] Purified by FCC (Sfar-C18, 5-30% ACN + 0.1 % HCOOH in H2O + 0.1 % HCOOH).

[0756] Amount / yield: 14.1 mg, 0.022 mmol, 24% yield.

[0757] LC-MS Method 1 : tz? = 0.59 min, MS (ESI) m / z = 595.2 [M]+

[0758] ‘HNMR (500 MHz, DMSO-d6 ) 5 ppm 9.79 - 11.60 (m, 2 H), 8.45 (s, 1H), 7.55 (br s, 1 H), 7.28 (s, 1 H), 4.44 - 4.81 (m, 4 H), 4.08 - 5.93 (m, 2 H), 3.77 - 3.89 (m, 4 H), 3.74 (s, 3 H), 3.48 - 3.62 (m, 4 H), 3.39 - 3.48 (m, 4 H), 2.00 - 2.07 (m, 6 H), 1.92 - 2.09 (m, 4 H), 1.67 (br s, 4 H).

[0759] Example 42: l-(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)- l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium bromide

[0760] Analogously to Example 39, Example 42 was prepared by starting from the suitable amine and bromide.

[0761] Intermediate 38: 50 mg, 0.14 mmol (1.0 eq)

[0762] Intermediate 2: 29 mg, 0.14 mmol (1.0 eq)

[0763] Precipitation from EtOAc, then trituration with DCM:MeOH (9 / 1 v / v).

[0764] Amount / yield: 31 mg, 0.054 mmol, 39% yield.

[0765] LC-MS Method 1 : tz? = 0.62 min, MS (ESI) m / z =484.2 [M]+

[0766] 'HNMR (500 MHz, DMSO-d6) 5 ppm 11.77 (s, 1 H), 10.86 (s, 1 H), 8.91 (d, J=1.6 Hz, 1 H), 8.02 (d, J=8.1 Hz, 1 H), 7.78 (d, J=8.0 Hz, 1 H), 7.46 - 7.54 (m, 1 H), 7.38 - 7.45 (m, 1 H), 6.95 (d, J=1.5 Hz, 1 H), 4.77 (s, 2 H), 4.67 (s, 2 H), 3.79 - 3.97 (m, 4 H), 2.85 - 3.03 (m, 6 H), 1.99 (br d, J=3.4 Hz, 4 H), 1.67 (br s, 4 H).

[0767] Intermediate 40: tert-butyl 3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxylate

[0768] A stirred suspension of 3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2-carboxylic acid sodium chloride (Intermediate 13, 6.30 g, 16.0 mmol) in Toluene (113 mL), was refluxed for 15 min. To the resulting solution, l , l -di- / c / 7-butoxy-AA-dimethylmethanamine (7.66 mL, 32.0 mmol) was added dropwise. The reaction was stirred at reflux for 24 h. The solvent was evaporated to yield a crude mixture that was purified by FCC (Sfar amino, 0-20% DCMZEtOAc (1 / 1 v / v) in cyclohexane) to afford the title compound (Intermediate 40: 3.15 g, 8.93 mmol, 56% yield).

[0769] LC-MS Method 1 : tz? = 0.65 min, MS (ESI) m / z = 353.2 [M+H]+

[0770] Intermediate 39: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(tert-butoxycarbonyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0771] To a stirred solution of N-benzyl-2 -bromoacetamide (1.33 g, 5.84 mmol) in ACN (30 mL),

[0772] Intermediate 40 (2.10 g, 5.84 mmol) was added. The reaction was stirred at 80°C for 36 h. The reaction was cooled down and concentrated under high vacuum. The residue was purified by FCC

[0773] (Sfar amino, 0-5% MeOH in DCM), affording the title compound (Intermediate 39: 2.54 g, 5.08 mmol, 87% yield).

[0774] LC-MS Method 1 : ty? = 0.82 min, MS (ESI) m / z = 500.3 [M]+

[0775] Intermediate 41 : l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-carboxy-4-methylthiophen-

[0776] 3-yl)amino)-2-oxoethyl)azepan-l-ium chloride

[0777] To a suspension of Intermediate 39 (2.50 g, 5.00 mmol) in THF (20 mL) and DCM (30 mL), HC1 2 M in Et2O (30 mL, 60 mmol) was added dropwise at RT observing full dissolution. After 48 h volatiles were evaporated under reduced pressure to afford a solid that was triturated with Et2O to afford the title compound (Intermediate 41 : 1.92 g, 4.00 mmol, 80 % yield) as a white solid.

[0778] LC-MS Method 1 : tz? = 0.64 min, MS (ESI) m / z = 445.2 [M]+ Intermediate 42: l-(2-((2-(tertebutoxycarbonyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)-l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0779] To a stirred solution of Intermediate 2 (789 mg, 3.85 mmol) in ACN (20 mL), Intermediate 40 (1.40 g, 3.85 mmol) was added. The reaction was stirred at 80°C overnight. The reaction was cooled down and concentrated under high vacuum. The residue was purified by FCC (Sfar amino, 0-5% MeOH in DCM) affording the title compound (Intermediate 42: 1.42 g, 2.98 mmol, 77% yield).

[0780] LC-MS Method 1 : ty? = 0.74 min, MS (ESI) m / z = 477.2 [M]+

[0781] Intermediate 43 - l-(2-((2-carboxy-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2- (isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium chloride

[0782] To a solution of tert-butyl 3-[[2-[l-[2-(isoxazol-3-ylamino)-2-oxo-ethyl]azepan-l-ium-l- yl]acetyl]amino]-4-methyl-thiophene-2-carboxylate;bromide (Intermediate 42, 1.42 g, 2.98 mmol) in DCM (37 mL), HC1 in 2 M Et2O (37 mL, 74 mmol) was added dropwise at RT. The reaction was stirred at RT for 16 h, then volatiles were evaporated under reduced pressure, to afford the title compound (Intermediate 43: 1.15 g, 2.52 mmol, 86% yield).

[0783] LC-MS Method 1 : tz? = 0.51 min, MS (ESI) m / z = 421.2 [M]+

[0784] Example 27: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(4-methylpiperazine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride hydrochloride

[0785] To a suspension of Intermediate 41 (400 mg, 0.83 mmol) in ACN (2.6 ml), 1- methylimidazole (205 mg, 2.50 mmol) was added, followed by TCFH (257 mg, 0.92 mmol), that was added at 0°C. After 1 h, 1 -methylpiperazine (125 mg, 1.25 mmol) was added and the mixture was stirred at 70°C for 14 h. Volatiles were evaporated and the residue was purified by FCC (Sfar amino, 0-5% MeOH in DCM), then by FCC (Sfar-C18, 10-30% ACN in H2O + 0.05% of HC1 37%), to afford the title compound (Example 27: 32 mg, 0.053 mmol, 6% yield).

[0786] LC-MS Method 2: ta = 1.16 min, MS (ESI) m / z = 526.5 [M]+

[0787] 'HNMR (400 MHz, DMSO-d6) 5 ppm 11.03 (br s, 1 H), 10.82 (s, 1 H), 9.21 (t, J=5.8 Hz, 1 H), 7.41 (s, 1 H), 7.23 - 7.37 (m, 5 H), 4.74 (s, 2 H), 4.46 (s, 2 H), 4.36 (d, J=5.7 Hz, 2 H), 4.13

[0788] (br s, 2 H), 3.68 - 3.93 (m, 4 H), 3.21 - 3.51 (m, 4 H), 2.93 - 3.10 (m, 2 H), 2.74 (br s, 3 H), 2.13 (s, 3 H), 1.85 - 2.09 (m, 4 H), 1.64 (br s, 4 H)

[0789] The following Examples were prepared analogously to what described for Example 27, by starting from the suitable Intermediate and corresponding commercially available amine.

[0790] Intermediate 44: l-(2-((2-((2-(4-(tert-butoxycarbonyl)piperazin-l-yl)ethyl)carbamoyl)- 4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l- ium (zwitterion)

[0791] Analogously to Example 27, Intermediate 44 was prepared by starting from the Intermediate

[0792] 43.

[0793] Intermediate 43: 100 mg, 0.22 mmol (1.0 eq) tert-butyl 4-(2-aminoethyl)piperazine-l -carboxylate: 50 mg, 0.22 mmol (1.0 eq)

[0794] Purified by FCC (Sfar amino, 0-5% MeOH in DCM).

[0795] Amount / yield: 75 mg, 0.12 mmol, 54% yield.

[0796] LC-MS Method 1: ta = 0.46 min, MS (ESI) m / z = 632.3 [M]+

[0797] Intermediate 45: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-(4-(tert- butoxycarbonyl)piperazin-l-yl)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium (zwitterion) Analogously to Example 27, Intermediate 45 was prepared by starting from the suitable carboxylic acid and amine.

[0798] Intermediate 41 : 125 mg, 0.23 mmol (1.0 eq) tert-butyl 4-(2-aminoethyl)piperazine-l -carboxylate: 50 mg, 0.22 mmol (1.0 eq)

[0799] Purified by FCC (Sfar amino, 0-5% MeOH in DCM).

[0800] Amount / yield: 137 mg, 0.21 mmol, 95% yield

[0801] LC-MS Method 1 : tz? = 0.63 min, MS (ESI) m / z = 655.5 [M]+

[0802] Intermediate 68: methyl 3-(2-(4,4-dimethylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxylate

[0803] To a stirred solution of Intermediate 1 (2 g, 6.85 mmol) in ACN (38 mL), 4,4- Dimethylpiperidine hydrochloride (1.00 g, 8.9 mmol) and potassium carbonate (2.46 g, 17.8 mmol) were added, and the mixture was stirred for 2 h at RT. The reaction was diluted with water and EtOAc, the organic layer was separated and the aqueous one extracted with EtOAc (3x). The collected organic layers were dried over Na2SO4, filtered, and concentrated to afford methyl 3-(2- (4,4-dimethylpiperidin-l-yl)acetamido)-4-methylthiophene-2-carboxylate (Intermediate 68: 2.2 g, 6.78 mmol, 99% yield).

[0804] LC-MS Method 1 : ty? = 0.58 min, MS (ESI) m / z =325.2 [M+H]+

[0805] Intermediate 69: 3-(2-(4,4-dimethylpiperidin-l-yl)acetamido)-4-methylthiophene-2- carboxylic acid sodium chloride

[0806] Analogously to Intermediate 7, Intermediate 69 was prepared by starting from the corresponding ester:

[0807] Intermediate 68: 2.2 g, 6.3 mmol (1.0 eq)

[0808] Sodium hydroxide (2 M in water): 3.8 mL, 7.6 mmol (1.2 eq)

[0809] Amount / yield: 2.4 g, quantitative yield.

[0810] LC-MS Method 1 : ta = 0.52 min, MS (ESI) m / z =311.2 [M+H]+

[0811] Intermediate 70: tert-butyl (2-(3-(2-(4,4-dimethylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxamido)ethyl)carbamate

[0812] Analogously to Intermediate 36, Intermediate 70 was prepared by starting from the suitable acid and amine.

[0813] Intermediate 69: 0.60 g, 1.63 mmol (1.0 eq) tert-butyl (2-aminoethyl)carbamate: 0.43 g, 2.44 mmol (1.5 eq)

[0814] Purified by FCC (Sfar amino, 50% EtOAc in cyclohexane)

[0815] Amount / yield: 1.10 g, quantitative yield (impure compound used as such in next step)

[0816] LC-MS Method 1 : ty? = 0.67 min, MS (ESI) m / z = 467.3 [M+H]+

[0817] Intermediate 71: l-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino) ethyl)carbamoyl)- 4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)-4,4-dimethylpiperidin-l-ium formate

[0818] Analogously to Example 4, Intermediate 71 was prepared by starting from the corresponding bromide and amine.

[0819] Intermediate 70: 130 mg, 0.28 mmol (1.0 eq)

[0820] Intermediate 1 : 81 mg, 0.28 mmol (1.0 eq)

[0821] Purified by FCC (Sfar amino, 0-5% MeOH in DCM), then by FCC (Sfar-C18, 5%-40% ACN + 0.1 % HCOOH in H2O+ 0.1 % HCOOH)

[0822] Amount / yield: 34 mg, 0.050 mmol, 18% yield

[0823] LC-MS Method 1 : tz? = 0.82 min, MS (ESI) m / z = 678.5 [M]+

[0824] Intermediate 72: l-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino) ethyl)carbamoyl)- 4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium formate

[0825] Analogously to Example 4, Intermediate 72 was prepared by starting from the corresponding bromide and amine.

[0826] Intermediate 10: 108 mg, 0.24 mmol (1.0 eq)

[0827] Intermediate 1 : 88 mg, 0.29 mmol (1.2 eq)

[0828] Purified by FCC (Sfar amino, 0-2% MeOH in DCM), then by FCC (Sfar-C18, 5%-50% ACN

[0829] + 0.1 % HCOOH in H2O + 0.1 % HCOOH)

[0830] Amount / yield: 80 mg, 0.113 mmol, 47% yield

[0831] LC-MS Method 1 : ty? = 0.78 min, MS (ESI) m / z = 664.4 [M]+

[0832] Intermediate 73: tert-butyl (2-(3-amino-4-methylthiophene-2- carboxamido)ethyl)(cyclopropyl)carbamate

[0833] Analogously to Intermediate 36, Intermediate 73 was prepared by starting from the suitable acid and amine. tert-butyl (2-aminoethyl)(cyclopropyl)carbamate: 0.51 mL, 2.67 mmol (1.0 eq) 3-amino-4-methylthiophene-2-carboxylic acid: 600 mg, 3.80 mmol (1.4 eq) Purified by FCC (Sfar silica, 40% EtOAc in cyclohexane)

[0834] Amount / yield: 490 mg, 1.44 mmol, 54% yield

[0835] LC-MS Method 1 : tz? = 1.02 min, MS (ESI) m / z = 340.5 [M+H]+

[0836] Intermediate 74: tert-butyl (2-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxamido)ethyl)(cyclopropyl)carbamate To a suspension of potassium carbonate (340 mg, 2.45 mmol) and tert-butyl (2-(3-amino- 4-methylthiophene-2-carboxamido)ethyl)(cyclopropyl)carbamate (Intermediate 73, 490 mg, 1.22 mmol) in ACN (12 mL) at 0°C, 2-bromoacetyl bromide (0.10 mL, 1.22 mmol) was added dropwise. After 1 h of stirring at RT, azepane (0.41 mL, 3.66 mmol) was added, and the reaction was stirred at RT for 4 h. The solid was filtered off and the filtrate was concentrated under reduced pressure. Crude was purified by FCC (KP Sfar silica, 5-40% EtOAc in cyclohexane) to give title compound (Intermediate 74: 550 mg, 94% wt, quantitative yield).

[0837] LC-MS Method 1 : ty? = 0.68 min, MS (ESI) m / z = 479.3 [M+H]+

[0838] Intermediate 75: l-(2-((2-((2-((tert-butoxycarbonyl)

[0839] (cyclopropyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2- (methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0840] Analogously to Example 4, Intermediate 75 was prepared by starting from the corresponding bromide and amine.

[0841] Intermediate 74: 150 mg, 0.31 mmol (1.0 eq)

[0842] Intermediate 1 : 110 mg, 0.29 mmol (1.0 eq)

[0843] Purified by FCC (Sfar amino, 0-10% MeOH in DCM)

[0844] Amount / yield: 70 mg, 0.091 mmol, 29% yield

[0845] LC-MS Method 1 : tz? = 0.82 min, MS (ESI) m / z = 694.4 [M]+

[0846] Intermediate 102: tert-butyl 4-((3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxamido)methyl)piperidine-l-carboxylate Analogously to Intermediate 36, Intermediate 102 was prepared by starting from the suitable acid and amine.

[0847] Intermediate 13: 500 mg, 1.41 mmol (1.0 eq) tert-butyl 4-(aminomethyl)piperidine-l -carboxylate: 0.450 mL, 2.11 mmol (1.5 eq)

[0848] Purified by FCC (Sfar amino, 20% EtOAc in cyclohexane)

[0849] Amount / yield: 140 mg, 0.28 mmol, 20% yield

[0850] LC-MS Method 1 : tz? = 0.79 min, MS (ESI) m / z = 493.3 [M+H]+

[0851] Intermediate 76: l-(2-((2-(((l-(tert-butoxycarbonyl)piperidin-4-yl)methyl)carbamoyl)- 4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0852] Analogously to Example 4, Intermediate 76 was prepared by starting from the corresponding bromide and amine.

[0853] Intermediate 102: 90 mg, 0.180 mmol (1.0 eq)

[0854] Intermediate 1 : 53 mg, 0.180 mmol (1.0 eq)

[0855] Purified by FCC (Sfar amino, 0-2.5% MeOH in DCM)

[0856] Amount / yield: 60 mg, 0.085 mmol, 47% yield

[0857] LC-MS Method 1 : ty? = 0.78 min, MS (ESI) m / z = 704.8 [M]+

[0858] Intermediate 77: tert-butyl 4-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carbonyl)piperazine-l-carboxylate

[0859] Analogously to Intermediate 36, Intermediate 77 was prepared by starting from the corresponding acid and amine. Reaction was carried out at 60°C.

[0860] Intermediate 13: 560 mg, 1.68 mmol (1.0 eq) tert-butyl piperazine-l-carboxylate: 470 mg, 2.52 mmol (1.5 eq) Purified by FCC (Sfar silica, 30% EtOAc in cyclohexane), then by FCC (Sfar-C18, 10-30% ACN in H2O)

[0861] Amount / yield: 84 mg, 0.18 mmol, 10% yield

[0862] LC-MS Method 1 : ty? = 0.67 min, MS (ESI) m / z = 465.4 [M+H]+

[0863] Intermediate 78: l-(2-((2-(4-(tert-butoxycarbonyl)piperazine-l-carbonyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0864] Analogously to Example 4, Intermediate 78 was prepared by starting from the corresponding bromide and amine.

[0865] Intermediate 77: 760 mg, 1.64 mmol (1.0 eq)

[0866] Intermediate 1 : 493 mg, 1.64 mmol (1.0 eq)

[0867] Purified by FCC (Sfar amino, 0-10% MeOH in DCM), then by Sfar-C18, 5-100% ACN in H2O).

[0868] Amount / yield: 107 mg, 0.14 mmol, 9% yield

[0869] LC-MS Method 1 : tz? = 0.82 min, MS (ESI) m / z = 676.3 [M]+

[0870] Intermediate 79: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((2-((tert- butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium (zwitterion)

[0871] Analogously to Example 27, Intermediate 79 was prepared by starting from the corresponding acid and amine.

[0872] Intermediate 41 : 200 mg, 0.42 mmol (1.0 eq) tert-butyl methyl(2-(methylamino)ethyl)carbamate: 118 mg, 0.63 mmol (1.5 eq) Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0873] Amount / yield: 77 mg, 0.12 mmol, 28% yield

[0874] LC-MS Method 1 : ty? = 0.81 min, MS (ESI) m / z = 614.4 [M]+

[0875] Intermediate 80: methyl N2-(3-amino-4-methylthiophene-2-carbonyl)-N6-(tert-

[0876] Analogously to Intermediate 36, Intermediate 80 was prepared by starting from the suitable acid and amine. methyl N6-(tert-butoxycarbonyl)-D-lysinate hydrochloride: 1.96 g, 6.62 mmol (1.3 eq)

[0877] 3-amino-4-methylthiophene-2-carboxylic acid: 800 mg, 5.09 mmol (1.0 eq)

[0878] Purified by FCC (Sfar silica, 20% (EtOAc / EtOH) in cyclohexane)

[0879] Amount / yield: 0.75 g, 1.88 mmol, 37% yield

[0880] LC-MS Method 1 : tz? = 0.99 min, MS (ESI) m / z = 400.2 [M+H]+

[0881] Intermediate 81: methyl N2-(3-(2-bromoacetamido)-4-methylthiophene-2-carbonyl)- N6-(tert-butoxycarbonyl)-D-lysinate

[0882] To a suspension of Intermediate 80 (0.75 g, 1.88 mmol) in ACN (17 mL), 2-bromoacetyl bromide (0.164 mL, 1.88 mmol) was added dropwise at 0°C. observing the formation of an off- white solid. The reaction was stirred at RT for 1 h. Then the solid was filtered off and the filtrated liquid phase was evaporated under reduced pressure. Crude was purified by FCC (Sfar silica, 10% EtOAc in cyclohexane), to afford title compound (Intermediate 81 : 0.48 g, 0.92 mmol, 49% yield) as a colourless oil.

[0883] LC-MS Method 1 : tz? = 1.00 min, MS (ESI) m / z = 520.2 / 522.2 [M+H]+

[0884] Intermediate 101: methyl N2-(3-(2-(azepan-l-yl)acetamido)-4-methyl thiophene-2- carbonyl)-N6-(tert-butoxycarbonyl)-D-lysinate

[0885] Analogously to Intermediate 66, Intermediate 101 was prepared starting from the suitable bromide and amine.

[0886] Intermediate 81 : 700 mg, 0.77 mmol (1.0 eq)

[0887] Azepane: 0.17 mL, 1.53 mmol (2.0 eq)

[0888] Amount / yield: 170 mg, 0.32 mmol, 41% yield.

[0889] LC-MS Method 1 : tz? = 0.65 min, MS (ESI) m / z = 540.4 [M+H]+

[0890] Intermediate 82: (R)-l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((6-((tert- butoxycarbonyl)amino)-l-methoxy-l-oxohexan-2-yl)carbamoyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0891] Analogously to Example 4, Intermediate 82 was prepared by starting from the corresponding bromide and amine.

[0892] Intermediate 101 : 170 mg, 0.32 mmol (1.0 eq)

[0893] N-benzyl-2-bromoacetamide: 72 mg, 0.32 mmol (1.0 eq)

[0894] Purified by FCC (Sfar amino, 0-10% MeOH in DCM)

[0895] Amount / yield: 85 mg, 0.11 mmol, 35% yield

[0896] LC-MS Method 1: ta = 0.78 min, MS (ESI) m / z = 686.8 [M]+

[0897] Intermediate 83: methyl 3-(2-(4-fluoro-4-methylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxylate To a stirred solution of Intermediate 1 (570 mg, 1.95 mmol) in DCM (13 mL), 4-Fluoro-4- methylpiperidine hydrochloride (300 mg, 1.95 mmol) and potassium carbonate (674 mg, 4.88 mmol) were added, and the mixture was stirred for 16 h at RT. The reaction was filtered to remove the salts and the organic phase was concentrated. The residue was dissolved in EtOAc and water. The phases were mixed in an extractor, the organic layer was separated and the aqueous one extracted with EtOAc (2x). The collected organic layers were dried over Na2SO4, filtered and concentrated to afford the title compound (Intermediate 83: 600 mg, 1.83 mmol, 94% yield) as yellow solid.

[0898] LC-MS Method 1 : ty? = 0.47 min, MS (ESI) m / z = 329.2 [M+H]+

[0899] Intermediate 84: 3-(2-(4-fluoro-4-methylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxylic acid

[0900] Analogously to Intermediate 7, Intermediate 84 was prepared by starting from the corresponding ester:

[0901] Intermediate 83: 600 mg, 1.83 mmol (1.0 eq)

[0902] Sodium hydroxide (2 M in water): 1.1 mL, 2.19 mmol (1.2 eq)

[0903] Purified by FCC (Sfar-C18, 0-10% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH)

[0904] Amount / yield: 150 mg, 0.48, 26% yield.

[0905] LC-MS Method 1 : tz? = 0.43 min, MS (ESI) m / z = 315.1 [M+H]+

[0906] Intermediate 85: tert-butyl (2-(3-(2-(4-fluoro-4-methylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxamido)ethyl)(methyl)carbamate

[0907] Analogously to Example 27, Intermediate 85 was prepared by starting from the suitable carboxylic acid and amine:

[0908] Intermediate 84: 150 mg, 0.48 mmol (1.0 eq) tert-butyl (2-aminoethyl)(methyl)carbamate: 83 mg, 0.48 mmol (1.0 eq)

[0909] Purified by FCC (Sfar amino, 0-60% EtOAc in cyclohexane)

[0910] Amount / yield: 100 mg, 0.21 mmol, 45% yield LC-MS Method 1 : tz? = 0.62 min, MS (ESI) m / z = 471.4 [M+H]+

[0911] Intermediate 86: l-(2-((2-((2-((tert-butoxycarbonyl) (methyl)amino)ethyl)carbamoyl)- 4-methylthiophen-3-yl)amino)-2-oxoethyl)-4-fluoro-l-(2-((2-(methoxycarbonyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-4-methylpiperidin-l-ium (zwitterion) (mixture of diastereoisomers)

[0912] Analogously to Example 4, Intermediate 86 was prepared by starting from the corresponding bromide and amine.

[0913] Intermediate 85: 100 mg, 0.21 mmol (1.0 eq)

[0914] Intermediate 1 : 62 mg, 0.21 mmol (1.0 eq)

[0915] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0916] Amount / yield: 47 mg, 0.062 mmol, 29% yield

[0917] LC-MS Method 1 : ty? = 0.79 min, MS (ESI) m / z = 682.4 [M]+

[0918] Intermediate 87: methyl N2-(3-amino-4-methylthiophene-2-carbonyl)-N6-(tert- butoxycarbonyl)-L-lysinate

[0919] Analogously to Intermediate 36, Intermediate 87 was prepared by starting from the suitable acid and amine. The reaction was carried out in ACN. methyl N6-(tert-butoxycarbonyl)-L-lysinate hydrochloride: 1.88 g. 6.36 mmol (1.0 eq)

[0920] 3-amino-4-methylthiophene-2-carboxylic acid: 1.00 g, 6.36 mmol (1.0 eq)

[0921] Purified by FCC (Sfar silica, 10% ACN in DCM)

[0922] Amount / yield: 910 mg, 2.28 mmol, 36% yield

[0923] LC-MS Method 1 : tz? = 1.00 min, MS (ESI) m / z = 300.2 [M+H]+

[0924] Intermediate 88: methyl N2-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carbonyl)-N6-(tert-butoxycarbonyl)-L-lysinate

[0925] Analogously to Intermediate 74, Intermediate 88 was prepared by starting from the suitable amine.

[0926] Intermediate 87: 910 mg, 2.28 mmol (1.0 eq)

[0927] Purified by FCC (Sfar silica, 80% (EtOAc:EtOH 9: 1) in cyclohexane)

[0928] Amount / yield: 500 mg, 0.93 mmol, 40% yield

[0929] LC-MS Method 1 : ty? = 0.69 min, MS (ESI) m / z = 539.5 [M+H]+

[0930] Intermediate 89: (S)-l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-((6-((tert- butoxycarbonyl)amino)-l-methoxy-l-oxohexan-2-yl)carbamoyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0931] Analogously to Example 4, Intermediate 89 was prepared by starting from the corresponding bromide and amine.

[0932] Intermediate 88: 250 mg, 0.27 mmol (1.0 eq)

[0933] N-benzyl-2-bromo-acetamide: 73 mg, 0.27 mmol (1.0 eq)

[0934] Purified by FCC (Sfar amino, 0-100% ACN in DCM)

[0935] Amount / yield: 40 mg, 0.058 mmol, 22% yield

[0936] LC-MS Method 1 : tz? = 0.80 min, MS (ESI) m / z = 686.3 [M]+

[0937] Intermediate 90: tert-butyl (3-(3-(2-(4,4-dimethylpiperidin-l-yl)acetamido)-4- methylthiophene-2-carboxamido)propyl)(methyl)carbamate

[0938] Analogously to Intermediate 36, Intermediate 90 was prepared by starting from the suitable acid and amine.

[0939] Intermediate 69: 1.00 g, 2.71 mmol (1.0 eq) tert-butyl (3-aminopropyl)(methyl)carbamate: 766 mg, 4.07 mmol (1.5 eq)

[0940] Purified by FCC (Sfar amino, 50% EtOAc in cyclohexane)

[0941] Amount / yield: 1.60 g, quantitative yield (82 % wt)

[0942] LC-MS Method 1 : ta = 0.71 min, MS (ESI) m / z = 484.1 [M+H]+

[0943] Intermediate 91: methyl 3-((2-bromoacetamido)methyl)benzoate

[0944] Analogously to Intermediate 65, Intermediate 91 was prepared by starting from the suitable amine.

[0945] Methyl 3-(aminomethyl)benzoate hydrochloride: 300 mg, 1.49 mmol (1.0 eq)

[0946] Purified by FCC (Sfar silica, 0-10% MeOH in DCM)

[0947] Amount / yield: 334 mg, 1.17 mmol, 79% yield

[0948] LC-MS Method 1 : tz? = 0.76 min, MS (ESI) m / z = 284.4 / 286.4[M+H]+

[0949] Intermediate 92: l-(2-((2-((3-((tert-butoxycarbonyl)(methyl)amino) propyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((3- (methoxycarbonyl)benzyl)amino)-2-oxoethyl)-4,4-dimethylpiperidin-l-ium (zwitterion)

[0950] Analogously to Example 4, Intermediate 92 was prepared by starting from the corresponding bromide and amine. Intermediate 90: 152 mg, 0.31 mmol (1.0 eq)

[0951] Intermediate 91 : 90 mg, 0.31 mmol (1.0 eq)

[0952] Purified by FCC (Sfar amino, 0-2% MeOH in DCM)

[0953] Amount / yield: 56 mg, 0.08 mmol, 26% yield

[0954] LC-MS Method 1 : tz? = 0.81 min, MS (ESI) m / z = 686.9 [M]+

[0955] Intermediate 93: ethyl 3-(2-bromoacetamido)-4-methylthiophene-2-carboxylate

[0956] Analogously to Intermediate 1, Intermediate 93 was prepared by starting from the suitable amine: ethyl 3-amino-4-methylthiophene-2-carboxylate: 820 mg, 4.41 mmol (1.0 eq)

[0957] Amount / yield: 1.36 g, quantitative yield.

[0958] LC-MS Method 1 : ty? = 0.92 min, MS (ESI) m / z = 306.1 / 308.1 [M+H]+

[0959] Intermediate 94: l-(2-((2-((3-((tert-butoxycarbonyl)(methyl)amino) propyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(ethoxycarbonyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethylpiperidin-l-ium (zwitterion)

[0960] Analogously to Example 4, Intermediate 94 was prepared by starting from the corresponding bromide and amine.

[0961] Intermediate 90: 120 mg, 0.21 mmol (1.0 eq)

[0962] Intermediate 93: 77 mg, 0.21 mmol (1.0 eq)

[0963] Purified by FCC (Sfar amino, 0-2% MeOH in DCM)

[0964] Amount / yield: 43 mg, 0.06 mmol, 29% yield

[0965] LC-MS Method 1: ta = 0.93 min, MS (ESI) m / z = 706.3 [M]+

[0966] Intermediate 95: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-(((l-(tert- butoxycarbonyl)piperidin-4-yl)methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2- oxoethyl)azepan-l-ium (zwitterion)

[0967] Analogously to Example 4, Intermediate 95 was prepared by starting from the corresponding bromide and amine.

[0968] Intermediate 102: 130 mg, 0.26 mmol (1.0 eq)

[0969] N-benzyl-2-bromo-acetamide: 60 mg, 0.26 mmol (1.0 eq)

[0970] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[0971] Amount / yield: 76 mg, 0.11 mmol, 40% yield

[0972] LC-MS Method 1 : tz? = 0.85 min, MS (ESI) m / z = 640.4 [M]+

[0973] Intermediate 96: tert-butyl 4-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxamido)piperidine-l-carboxylate

[0974] Analogously to Intermediate 36, Intermediate 96 was prepared by starting from the suitable acid and amine.

[0975] Intermediate 13: 500 mg, 1.41 mmol (1.0 eq) tert-butyl 4-aminopiperidine-l -carboxylate: 423 mg, 2.11 mmol (1.3 eq)

[0976] Purified by FCC (Sfar silica, 30% EtOAc in cyclohexane)

[0977] Amount / yield: 400 mg, 0.84 mmol, 59% yield

[0978] LC-MS Method 1 : tz? = 0.67 min, MS (ESI) m / z = 479.3 [M+H]+

[0979] Intermediate 97 : l-(2-((2-((l-(tert-butoxycarbonyl)piperidin-4-yl)carbamoyl)-4- methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3- yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[0980] Analogously to Example 4, Intermediate 97 was prepared by starting from the corresponding bromide and amine.

[0981] Intermediate 1 : 128 mg, 0.41 mmol (1.0 eq)

[0982] Intermediate 96: 199 mg, 0.41 mmol (1.0 eq)

[0983] Purified by FCC (Sfar amino, 0-3% MeOH in DCM)

[0984] Amount / yield: 110 mg, 0.16 mmol, 38% yield

[0985] LC-MS Method 1: ta = 1.02 min, MS (ESI) m / z = 690.7 [M]+

[0986] Intermediate 98: tert-butyl (2-(3-(2-(azepan-l-yl)acetamido)-4-methylthiophene-2- carboxamido)ethyl)(methyl)carbamate

[0987] Analogously to Intermediate 36, Intermediate 98 was prepared by starting from the suitable amine and carboxylic acid.

[0988] Intermediate 13: 2.0 g, 6.75 mmol (1.0 eq) tert-butyl (2-aminoethyl)(methyl)carbamate: 1.76 g, 10.0 mmol (1.5 eq)

[0989] Purified by FCC (Sfar amino, 65% (EtOAc:EtOH 9: 1) in DCM)

[0990] Amount / yield: 700 mg, 1.54 mmol, 23% yield.

[0991] LC-MS Method 1 : tz? = 0.60 min, MS (ESI) m / z = 453.7 [M]+

[0992] Intermediate 99: 2-bromo-N-(2-(trifluoromethoxy)benzyl)acetamide To a suspension of potassium carbonate (1.34 g, 9.69 mmol) and (2- (trifluoromethoxy)phenyl)methanamine (0.56 g, 5.70 mmol) in ACN (20 mL) at 0°C, 2- bromoacetyl bromide (0.78 mL, 8.55 mmol) was added dropwise. After 2 h of stirring at RT the solids were filtered off and the filtrated was evaporated under reduced pressure. The crude was purified by FCC (KP Sfar amino, 3% MeOH in DCM) to give title compound (Intermediate 99: 737 mg, 2.36 mmol, 45 % yield).

[0993] LC-MS Method 1 : ty? = 0.92 min, MS (ESI) m / z = 312.0 / 314.0 [M+H]+

[0994] Intermediate 100: l-(2-((2-((2-((tert-butoxycarbonyl)

[0995] (methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-oxo-2-((2- (trifluoromethoxy)benzyl)amino)ethyl)azepan-l-ium (zwitterion)

[0996] Analogously to Example 4, Intermediate 100 was prepared by starting from the corresponding bromide and amine.

[0997] Intermediate 98: 200 mg, 0.42 mmol (1.0 eq)

[0998] Intermediate 99: 170 mg, 0.42 mmol (1.0 eq)

[0999] Purified by FCC (Sfar amino, 10% MeOH in DCM)

[1000] Amount / yield: 68 mg, 0.099 mmol, 24% yield

[1001] LC-MS Method 1 : tz? = 0.84 min, MS (ESI) m / z = 684.3 [M]+

[1002] Example 33: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((4-methyl-2-(piperazine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride hydrochloride

[1003] To a solution of Example 34 as chloride salt (17 mg, 0.030 mmol) in DCM (1 mL), HC1 2 M in Et2O (0.5 mL, 1 mmol) was added at RT. After 16 volatiles were evaporated and the solid washed with Et2O, to afford the title compound (Example 33: 12 mg, 0.021 mmol, 79% yield).

[1004] LC-MS Method 2: tz? = 0.66 min, MS (ESI) m / z = 489.2 [M]+

[1005] 'H NMR (500 MHz, DMSO-d6) 5 ppm 11.82 (br s, 1 H), 10.84 (s, 1 H), 9.16 (br s, 2 H), 8.89 (d, J=1.8 Hz, 1 H), 7.40 (d, J=1.0 Hz, 1 H), 6.92 (d, J=1.6 Hz, 1 H), 4.71 (s, 2 H), 4.66 (s, 2 H), 3.77 - 4.03 (m, 4 H), 3.67 - 3.75 (m, 4 H), 3.13 (br s, 4 H), 2.11 (s, 3 H), 1.92 - 2.09 (m, 4 H), 1.66 (br s, 4 H). The following Examples were prepared analogously to what described for Example 33, by starting from the suitable Intermediate or Example.

[1006] Example 49: l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l- (2-((4-methyl-2-(4-methylpiperazine-l-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan- 1-ium (zwitterion) Step 1 - l-(2-((2-(tert-butoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-

[1007] (2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide (Intermediate 46)

[1008] Analogously to Intermediate 42, Intermediate 46 was prepared by starting from suitable amine and bromide:

[1009] Intermediate 40: 200 mg, 0.57 mmol (1.0 eq)

[1010] Intermediate 1 : 167 mg, 0.57 mmol (1.0 eq)

[1011] Amount / yield: 226 mg, 0.35 mmol, 62% yield.

[1012] LC-MS Method 1 : ty? = 0.83 min, MS (ESI) m / z = 564.3 [M]+

[1013] Step 2 - l-(2-((2-carboxy-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2-((2-

[1014] (methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium chloride (Intermediate 47)

[1015] Analogously to Intermediate 43, Intermediate 47 was prepared by starting from the suitable ester.

[1016] Intermediate 46: 223 mg, 0.35 mmol (1.0 eq)

[1017] Amount / yield: 226 mg, 83% wt, quantitative yield.

[1018] Method 1 : tz? = 0.65 min, MS (ESI) m / z = 508.2 [M]+

[1019] Step 3 - l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l-(2- ((4-methyl-2-(4-methylpiperazine-l-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l- ium (zwitterion) (Example 49)

[1020] Analogously to Example 27, Example 49 was prepared by starting from the suitable amine and carboxylic acid. Intermediate 47: 115 mg, 0.21 mmol (1.0 eq)

[1021] 1 -methylpiperazine: 42 mg, 0.42 mmol (2.0 eq)

[1022] Purified by FCC (Sfar amino, 0-5% MeOH in DCM).

[1023] Amount / yield: 12 mg, 0.020 mmol, 9% yield.

[1024] LC-MS Method 1 : tz? = 1.04 min, MS (ESI) m / z = 590.4 [M]+

[1025] 'HNMR (500 MHz, DMSO-d6) 5 ppm 7.36 (br s, 1 H), 7.25 (s, 1 H), 4.61 (br s, 2 H), 4.05 - 4.43 (m, 2 H), 3.67 - 3.87 (m, 4 H), 3.68 (s, 3 H), 3.40 - 3.50 (m, 4 H), 2.27 (br t, J=4.7 Hz, 4 H), 2.15 (s, 3 H), 1.93 - 2.04 (m, 4 H), 1.93 (br s, 6 H), 1.58 - 1.73 (m, 4 H)

[1026] Example 54: l-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-l- (2-((4-methyl-2-(morpholine-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[1027] Analogously to Example 27, Example 54 was prepared starting from the suitable amine and carboxylic acid.

[1028] Intermediate 47: 115 mg, 0.21 mmol (1.0 eq)

[1029] Morpholine: 0.027 mL, 0.32 mmol (1.5 eq)

[1030] Purified by FCC (Sfar amino, 0-5% MeOH in DCM)

[1031] Amount / yield: 38 mg, 0.066 mmol, 29% yield.

[1032] LC-MS Method 1: ta = 0.66 min, MS (ESI) m / z = 577.3 [M]+

[1033] 'HNMR (500 MHz, DMSO-d6) 5 ppm 14.14 (br s, 1 H), 7.33 (br s, 1 H), 7.24 (s, 1 H), 4.60 (br s, 2 H), 4.15 (br s, 2 H), 3.66 (s, 3 H), 3.61 - 3.86 (m, 4 H), 3.55 - 3.62 (m, 4 H), 3.45 - 3.52 (m, 4 H), 1.91 - 2.04 (m, 4 H), 1.84 - 1.92 (m, 6 H), 1.65 (br s, 4 H)

[1034] Example 37 : l-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-l- (2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium (zwitterion)

[1035] Step 1 - methyl 3-iodo-4-methylthiophene-2-carboxylate (Intermediate 48)

[1036] To a solution of methyl 3-amino-4-methylthiophene-2-carboxylate (3.00 g, 17.5 mmol) in ACN (44 mL) under nitrogen atmosphere, diiodomethane (5.65 mL, 70.1 mmol) was added and the reaction mixture was stirred at 70°C for 20 min. After that time, the reaction mixture was allowed to cool to RT and tert-butyl nitrite (3.80 mL, 35.0 mmol) was added dropwise over 10 min. After the addition was completed, the reaction mixture was stirred at 60°C for 4 h. Then the mixture was slowly poured into an aqueous solution of HC1 0.5 M (30 mL) and extracted with EtOAc (3x). The collected organic layers were dried over MgSCh and concentrated under reduced pressure to obtain a crude that was purified by FCC (KP Sfar silica, 0-10% EtOAc in cyclohexane) to afford the title compound (Intermediate 48: 2.90 g, 10.3 mmol, 59% yield).

[1037] LC-MS Method 1: ta = 1.15 min, MS (ESI) m / z = 283.0 [M+H]+

[1038] Step 2 - methyl 3-cyano-4-methylthiophene-2-carboxylate (Intermediate 51)

[1039] A round bottomed flask was loaded with methyl 3-iodo-4-methylthiophene-2-carboxylate (Intermediate 48, 2.90 g, 10.3 mmol), potassium acetate (1.41 g, 14.4 mmol), XPhos (0.49 g, 1.03 mmol) and XPhos Pd G3 (0.87 g, 1.03 mmol). The flask was evacuated and backfilled with nitrogen (3x), then potassium hexacyanoferrate (II) 0.1 N standardized solution (60.0 mL, 6.00 mmol) and 1,4-di oxane (52 mL) were added under nitrogen atmosphere. Three further vacuum - nitrogen cycles were performed and the reaction mixture was heated at 100 °C under stirring for 4 h. Then XPhos Pd G3 (0.16 mg, 0.19 mmol) and potassium hexacyanoferrate (II) 0.1 N standardized solution (60.0 mL, 6.00 mmol, degassed prior addition) were added at RT. The reaction mixture was heated at 100°C under stirring for further 5 h. Then it was cooled to RT and extracted with EtOAc. The collected organic layers were concentrated under reduced pressure affording a crude that was purified by FCC (KP Sfar silica, 0-10% EtOAc in cyclohexane) to obtain the title compound (Intermediate 51 : 0.78 g, 4.30 mmol, 42 % yield).

[1040] LC-MS Method 1 : tz? = 0.89 min, MS (ESI) m / z = 181.9 [M+H]+

[1041] Step 3 - 3-(aminomethyl)-4-methylthiophene-2-carboxamide (Intermediate 49)

[1042] To a solution of methyl 3-cyano-4-methylthiophene-2-carboxylate (Intermediate 51, 680 mg, 3.75 mmol) in MeOH (100 mL) in a hydrogenation flask under nitrogen atmosphere, Raney®- Nickel (70.0 mg, 50% slurry in water) was added followed by ammonia 7 M in MeOH (15.0 mL, 185 mmol). The flask was evacuated, backfilled with hydrogen (1 atm) and stirred for 24 h. Then the solid was allowed to settle and the organic phase was removed under nitrogen atmosphere. The remaining solid was washed with MeOH and DCM. The collected organic layers were concentrated under reduced pressure to afford a crude that was purified by FCC (Sfar amino, 0- 100% EtOAc / MeOH (9 / 1 v / v) in cyclohexane) to give the title compound (Intermediate 49: 173 mg, 1.01 mmol, 27% yield).

[1043] LC-MS Method 2: tz? = 0.46 min, MS (ESI) m / z = 171.1 [M+H]+

[1044] Step 4 - 3-((2-(azepan-l-yl)acetamido)methyl)-4-methylthiophene-2-carboxamide (Intermediate 50)

[1045] To a solution of 3-(aminomethyl)-4-methylthiophene-2-carboxamide (Intermediate 49, 152 mg, 0.89 mmol) and triethylamine (0.12 mL, 0.89 mmol) in ACN (9 mL), 2-chloroacetyl chloride (70 mL, 0.89 mmol) was slowly added at 0°C. The reaction was allowed to stir at RT for 4 h. Then azepane (600 mL, 5.36 mmol) was added at RT to the reaction mixture. After stirring for 3 h, volatiles were removed under reduced pressure and the crude was purified by FCC (Sfar amino, 0-100% EtOAc / MeOH (9 / 1 v / v) in cyclohexane) to give the title compound (Intermediate 50: 140 mg, 0.452 mmol, 51 % yield).

[1046] LC-MS Method 2: tz? = 0.82 min, MS (ESI) m / z = 310.2 [M+H]+

[1047] Step 5 - l-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-l-(2- (isoxazol-3-ylamino)-2-oxoethyl)azepan-l-ium (zwitterion) (Example 37)

[1048] Analogously to Example 39, Example 37 was prepared by starting from the suitable amine and bromide.

[1049] Intermediate 50: 40 mg, 0.13 mmol (1.0 eq)

[1050] Intermediate 2: 30 mg, 0.15 mmol (1.1 eq)

[1051] Purified by FCC (Sfar amino, 0-100% EtOAc / MeOH (9 / 1 v / v) in cyclohexane) Amount / yield: 10 mg, 0.023 mmol, 18% yield.

[1052] LC-MS Method 2: tz? = 0.59 min, MS (ESI) m / z = 434.3 [M]+

[1053] 'HNMR (500 MHz, DMSO-d6) 5 ppm 9.23 (br t, J=5.0 Hz, 1 H), 8.33 (d, J=1.5 Hz, 1 H), 7.90 (br s, 1 H), 7.48 (br s, 1 H), 7.26 (d, J=0.7 Hz, 1 H), 6.75 (d, J=1.6 Hz, 1 H), 4.57 (s, 2 H), 4.50 (d, J=5.1 Hz, 2 H), 4.04 (s, 2 H), 3.61 - 3.79 (m, 4 H), 2.16 (s, 3 H), 1.85 (br s, 4 H), 1.58 (br s, 4 H).

[1054] Example 40: l? -l-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2- oxoethyl)-l-(2-oxo-2-((l-phenylethyl)amino)ethyl)azepan-l-ium bromide

[1055] Analogously to Example 39, Example 40 was prepared by starting from the suitable amine and bromide.

[1056] Intermediate 50: 50 mg, 0.16 mmol (1.0 eq) Intermediate 3: 44 mg, 0.18 mmol (1.1 eq)

[1057] Purified by FCC (Sfar-C18, 0-50% ACN + 0.1% HCOOH in H2O + 0.1% HCOOH).

[1058] Amount / yield: 7.5 mg, 0.014 mmol, 8% yield.

[1059] LC-MS Method 1 : ty? = 0.65 min, MS (ESI) m / z = 471.32 [M]+

[1060] 'HNMR (500 MHz, DMSO-d6) 5 ppm 9.11 (d, J=7.7 Hz, 1 H), 8.76 (t, J=5.4 Hz, 1 H), 7.39 - 7.95 (m, 2 H), 7.30 - 7.37 (m, 4 H), 7.29 (s, 1 H), 7.22 - 7.27 (m, 1 H), 4.92 (quin, J=7.1 Hz, 1 H), 4.46 - 4.57 (m, 2 H), 4.44 (s, 2 H), 4.37 (s, 2 H), 3.55 - 3.78 (m, 4 H), 2.15 (s, 3 H), 1.86 (br s, 4 H), 1.58 (br s, 4 H), 1.37 (d, J=7.1 Hz, 3 H).

[1061] Conversion of the zwitterionic forms of the compounds of the invention to obtain the corresponding salt forms may be performed as described for following examples (chloride salts).

[1062] Example 1 as chloride salt: 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2- (pyrrolidine-l-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethan-l-aminium chloride

[1063] Example 1 (zwitterion) (33 mg, 0.070 mmol) was dissolved in DCM (1.0 mL) and HC1 2 M in Et2O (50 pL, 0.11 mmol) was added dropwise at RT. After 5 min volatiles were removed under reduced pressure affording the title compound (Example 1 as chloride salt: 7 mg, 0.015 mmol, 21% yield) as a white powder.

[1064] LC-MS Method 1 : tz? = 0.63 min, m / z = 443.4 [M]+

[1065] 'H NMR (400 MHz, DMSO-d6) 5 ppm 10.37 (s, 1H), 9.06 (t, J = 5.8 Hz, 1H), 7.40 - 7.21 (m, 6H), 4.56 (s, 2H), 4.40 (s, 2H), 4.36 (d, J = 5.8 Hz, 2H), 3.40 (s, 6H), 3.48 - 3.35 (m, 4H), 2.07 (d, J = 1.0 Hz, 3H), 1.81 (br s, 4H)

[1066] The following examples have been prepared analogously to the procedure described for Example 1 as chloride salt, starting from the suitable Examples as zwitterion.

[1067] Example 78: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-methyl-4-(morpholine-4- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide

[1068] Step 1 - methyl 4-(2-bromoacetamido)-5-methylthiophene-3-carboxylate

[1069] (Intermediate 103)

[1070] Analogously to Intermediate 1, Intermediate 103 was prepared by starting from the suitable amine: methyl 4-amino-5-methylthiophene-3-carboxylate hydrochloride: 1.50 g, 7.22 mmol (1.0 eq)

[1071] Amount / yield: 1.57 g, 5.36 mmol, 74% yield.

[1072] UPLC Method 1 : r.t. 0.79 min, MS (ESI) m / z = 292.0 / 294.0 [M+H]+

[1073] Step 2 - methyl 4-(2-(azepan-l-yl)acetamido)-5-methylthiophene-3-carboxylate

[1074] (Intermediate 104)

[1075] Analogously to Intermediate 11, Intermediate 104 was prepared by starting from the suitable bromide:

[1076] Intermediate 103: 1.57 g, 5.36 mmol (1.0 eq)

[1077] Amount / yield: 1.52 g, 4.90 mmol, 91% yield.

[1078] UPLC Method 1 : r.t. 0.46 min, MS (ESI) m / z = 311.1 [M+H]+

[1079] Step 3 - 4-(2-(azepan-l-yl)acetamido)-5-methylthiophene-3-carboxylic acid sodium chloride (Intermediate 105)

[1080] Analogously to Intermediate 7, Intermediate 105 was prepared by starting from the suitable ester:

[1081] Intermediate 104: 1.52 g, 4.90 mmol (1.0 eq)

[1082] Amount / yield: 1.97 g, 88% wt, quantitative yield.

[1083] UPLC Method 1 : r.t. 0.41 min, MS (ESI) m / z = 297.1 [M+H]+

[1084] Step 4 - 2-(azepan-l-yl)-N-(2-methyl-4-(morpholine-4-carbonyl)thiophen-3- yl)acetamide (Intermediate 106)

[1085] Analogously to Intermediate 8, Intermediate 106 was prepared by starting from the suitable acid and amine:

[1086] Intermediate 105: 350 mg, 0.97 mmol (1.0 eq) Morpholine: 0.17 mL, 1.97 mmol (2.0 eq)

[1087] Amount / yield: 211 mg, 0.57 mmol, 59% yield.

[1088] UPLC Method 1 : r.t. 0.44 min, MS (ESI) m / z = 366.2 [M+H]+

[1089] Step 5 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-methyl-4-(morpholine-4- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide (Example 78)

[1090] Analogously to Example 3, Example N1568-18-1 CUD-074186 was prepared by starting from the suitable amine and bromide:

[1091] Intermediate 106: 110 mg, 0.30 mmol (1.0 eq)

[1092] N-benzyl-2-bromoacetamide: 76 mg, 0.33 mmol (1.1 eq)

[1093] Amount / yield: 121 mg, 0.20 mmol, 68% yield.

[1094] UPLC Method 1 : r.t. 0.64 min, MS (ESI) m / z = 513.2 [M]+

[1095] 'HNMR (500 MHz, DMSO-d6) 5 = 10.55 - 10.16 (m, 1H), 9.05 (br s, 1H), 7.56 - 7.06 (m,

[1096] 6H), 4.36 (d, J = 5.1 Hz, 2H), 4.61 - 4.21 (m, 4H), 3.89 - 3.63 (m, 4H), 3.61 - 3.55 (m, 4H), 3.53 -

[1097] 3.39 (m, 4H), 2.23 (br s, 3H), 2.01 - 1.86 (m, 4H), 1.72 - 1.57 (m, 4H)

[1098] Example 79: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-methyl-4-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide

[1099] Step 1 - 2-(azepan-l-yl)-N-(2-methyl-4-(pyrrolidine-l-carbonyl)thiophen-3- yl)acetamide (Intermediate 107)

[1100] Analogously to Intermediate 8, Intermediate 107 was prepared by starting from the suitable acid and amine:

[1101] Intermediate 104: 350 mg, 0.97 mmol (1.0 eq)

[1102] Pyrrolidine: 0.16 mL, 1.97 mmol (2.0 eq)

[1103] Amount / yield: 204 mg, 0.58 mmol, 59% yield.

[1104] UPLC Method 1 : r.t. 0.50 min, MS (ESI) m / z = 350.2 [M+H]+

[1105] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((2-methyl-4-(pyrrolidine-l- carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide (Example 79)

[1106] Analogously to Example 3, Example 79 was prepared by starting from the suitable amine and bromide:

[1107] Intermediate 107: 110 mg, 0.32 mmol (1.0 eq)

[1108] N-benzyl-2-bromoacetamide: 79 mg, 0.35 mmol (1.1 eq) Amount / yield: 96 mg, 0.17 mmol, 53% yield. UPLC Method 1 : r.t. 0.68 min, MS (ESI) m / z = 497.3 [M]+

[1109] 'H NMR (500 MHz, DMSO-d6) 5 = 10.28 (br s, 1H), 9.78 - 8.64 (m, 1H), 7.64 - 7.40 (m, 1H), 7.38 - 7.18 (m, 5H), 4.64 - 4.20 (m, 6H), 3.90 - 3.66 (m, 4H), 3.43 (br t, J = 6.3 Hz, 2H), 3.33 - 3.25 (m, 2H), 2.24 (br s, 3H), 2.00 - 1.87 (m, 4H), 1.85 - 1.71 (m, 4H), 1.69 - 1.59 (m, 4H)

[1110] Example 80: l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-((2- methoxyethyl)(methyl)carbamoyl)-2-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide

[1111] Step 1 - 4-(2-(azepan-l-yl)acetamido)-N-(2-methoxyethyl)-N,5-dimethylthiophene-3- carboxamide (Intermediate N1568-17-1)

[1112] Analogously to Intermediate 8, Intermediate N1568- 17-1 was prepared by starting from the suitable acid and amine:

[1113] Intermediate 101 : 350 mg, 0.97 mmol (1.0 eq)

[1114] 2-methoxy-N-methylethan-l -amine: 0.20 mL, 1.97 mmol (2.0 eq)

[1115] Amount / yield: 200 mg, 0.54 mmol, 55% yield.

[1116] UPLC Method 1 : r.t. 0.47 min, MS (ESI) m / z = 368.2 [M+H]+

[1117] Step 2 - l-(2-(benzylamino)-2-oxoethyl)-l-(2-((4-((2- methoxyethyl)(methyl)carbamoyl)-2-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-l-ium bromide (Example 80)

[1118] Analogously to Example 3, Example 80 was prepared by starting from the suitable amine and bromide:

[1119] Intermediate N1568- 17-1 : 110 mg, 0.30 mmol (1.0 eq)

[1120] N-benzyl-2-bromoacetamide: 75 mg, 0.33 mmol (1.1 eq)

[1121] Amount / yield: 97 mg, 0.16 mmol, 54% yield.

[1122] UPLC Method 1 : r.t. 0.66 min, MS (ESI) m / z = 515.3 [M]+

[1123] 'H NMR (500 MHz, DMSO-d6) 5 = 10.22 (br s, 1H), 9.48 - 8.63 (m, 1H), 7.49 - 7.37 (m, 1H), 7.37 - 7.22 (m, 5H), 4.65 - 4.39 (m, 4H), 4.35 (br d, J = 5.1 Hz, 2H), 3.89 - 3.65 (m, 4H), 3.55

[1124] - 3.38 (m, 4H), 3.27 - 3.18 (m, 3H), 3.06 - 2.83 (m, 3H), 2.24 (br s, 3H), 1.99 - 1.85 (m, 4H), 1.73

[1125] - 1.57 (m, 4H)

[1126] Comparative compounds Ci and C2 were prepared as described below.

[1127] Ci is characterized by having a phenyl ring replacing the ami de- substituted tiophene or benzothiophene ring of the compounds of the invention. Ci is characterized by having a different heteroaryl ring replacing the ami de- substituted tiophene or benzothiophene ring of the compounds of the invention.

[1128] Compound Ci: N,N-bis(phenylcarbamoylmethyl)dimethylammonium chloride (aka Carcainium chloride) was prepared as described in WO99 / 63985, pag. 6.

[1129] Compound C2: l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((5-(methoxycarbonyl)-3- methylisoxazol-4-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion)

[1130] Step 1 - methyl 4-(2-bromoacetamido)-3-methylisoxazole-5-carboxylate (Intermediate 52)

[1131] To a suspension of potassium carbonate (1.10 g, 8.01 mmol) and methyl 4-amino-3- methylisoxazole-5-carboxylate (500 mg, 3.20 mmol) in DCM (20 mL), was added 2-bromoacetyl bromide (0.31 mL, 3.52 mmol) dropwise. After 3h, water was added and the organic phase was separated, dried over a phase separator and concentrated under reduced pressure. The residue was purified by FCC (KP Sfar silica, 0-40% cyclohexane in EtOAc) to afford title compound (Intermediate 52, 800 mg, 2.89 mmol, 90% yield) as an off-white powder.

[1132] LC-MS Method 1: ta = 0.66 min, MS (ESI) m / z = 277.1 / 279.0 [M+H]+

[1133] Step 2 - 2-(azepan-l-yl)-N-(isoxazol-3-yl)acetamide (Intermediate 54)

[1134] To a stirred solution of Intermediate 53 (200 mg, 1.25 mmol) in ACN (5 mL), azepane (0.28 mL, 2.49 mmol) and potassium carbonate (361 mg, 2.62 mmol) were added and the mixture was stirred for 2h at RT. Volatiles were evaporated and the residue partitioned between water and EtOAc. The organic phase was separated and the aqueous phase extracted with EtOAc. The collected organic layers were washed with brine and concentrated under reduced pressure to afford title compound (Intermediate 54: 270 mg, 1.21 mmol, 97% yield) as an orange oil.

[1135] LC-MS Method 2: ta = 0.78 min, MS (ESI) m / z = 224.4 [M]+

[1136] Step 3 - l-(2-(isoxazol-3-ylamino)-2-oxoethyl)-l-(2-((5-(methoxycarbonyl)-3- methylisoxazol-4-yl)amino)-2-oxoethyl)azepan-l-ium (zwitterion) (Compound C2) To a stirred solution of Intermediate 54 (40 mg, 0.180 mmol) in ACN (1.2 mL), Intermediate 52 (50 mg, 0.180 mmol) was added. The mixture was stirred at RT for 24h. Then the mixture was concentrated under reduced pressure and the residue was purified by FCC (Sfar amino, 0-5% MeOH in DCM) affording the title compound (Compound C2: 57 mg, 0.114 mmol, 75% yield).

[1137] LC-MS Method 2: ty? = 0.52 min, MS (ESI) m / z = 420.1 [M]+

[1138] 'HNMR (500 MHz, DMSO-d6) 5 ppm 12.25 - 14.42 (m, 1 H), 8.76 (d, J=1.6 Hz, 1 H), 6.92 (d, J=1.6 Hz, 1 H), 4.61 (s, 2 H), 4.14 (s, 2 H), 3.74 (s, 3 H), 3.68 - 3.85 (m, 4 H), 2.11 (s, 3 H), 1.93 (br s, 4 H), 1.63 (br s, 4 H)

[1139] PHARMACOLOGICAL ACTIVITY OF THE COMPOUNDS OF THE INVENTION hNaV 1.7 cellular assay Protocol

[1140] Representative examples of the invention were tested for intracellular and extracellular inhibition of NaV 1.7 in the automated patch clamp assay herein described.

[1141] Cell Culture

[1142] CHO cells stably expressing human NaV 1.7 channel were obtained from B’SYS GmbH, Switzerland. Cells were cultured in F12 Nutmix (Gibco, Carlsbad CA) supplemented with 10% Fetal Bovine Serum (Invitrogen, Waltham MA), 1% Penicillin-Streptomycin (Gibco, Carlsbad CA) and 200 pg / ml Hygromycin B (Invitrogen, Waltham MA). Cells were grown and maintained at 37 °C in a humidified environment containing 5% CO2 in air. Cells were detached from the culture flask for passage and harvested using TrypLE Express (Sigma-Aldrich, St. Louis, MO). Experiment flasks were prepared with 2-4 days cells without antibiotics and used with -80% confluency. Before experiments, cells were washed with Ca2+and Mg2+free PBS and detached with pre-warmed TrypLE Express. After a brief centrifugation step, cells were resuspended in CHO-S-Serum Free Media II (Life Technologies, Carlsbad CA). Cells were counted and the final concentration was set at 3-4 million cells per mL.

[1143] Patch Clamp Solutions & Drugs

[1144] The intracellular solution (or internal solution) contained the following: 140 mM CsF, 10 mM NaCl, 1 mM EGTA and 10 mM HEPES, adjusted to pH 7.2 with CsOH, and osmolarity to 325 with sucrose. The extracellular solution (or external solution) contained the following: 40 mM NMDG, 100 mM NaCl, 4 mM KC1, 1 mM MgCh, 2 mM CaCh, 10 mM HEPES, 10 mM Glucose, adjusted to pH 7.4 with HC1 and osmolality to 310 with sucrose. All chemicals were from Sigma-Aldrich, St. Louis, MO.

[1145] For both intracellular and extracellular application of test compounds, the spotted compound was dissolved in the intracellular and extracellular solution respectively and tested up to 60 pM, which results in a pICso not lower than 4.2 value. Automated Patch Clamp Assay Protocol

[1146] Automated Patch Clamp was performed on Qube 384 (Sophion Bioscience A / S, Ballerup, Denmark) with multihole QChips at a temperature setting of 22 °C. The whole cell configuration was formed with default Qube seal and break-in parameter. The membrane potential was held at - 100 mV before the voltage protocol began. The voltage protocol consisted of 20 pulse protocols with 30 sweeps for intracellular and 55 sweeps for extracellular applications for both pre- and postcompound applications. Cells were held at -100 mV with a depolarizing pulse to -20 mV for 10 ms. The interval between sweeps was set at 9 s when the holding potential was -100 mV.

[1147] Internal Block by Test Compounds (intracellular inhibition)

[1148] For intracellular application of the compounds, the QChip was removed from the recording chamber. The intracellular solution was replaced with the solution containing the test compound. After the intracellular solution exchange, QChip was placed back in the recording chamber and the voltage pulses were applied.

[1149] Data filtering was performed by Sophion Analyzer software. Only wells with minimum 40 MQ seal resistance and 1 nA pre-compound current were used for further analysis using Aplus software. The average of the peak current amplitude of the 20th pulse of the last three trains applied in pre-compound and post-compound were calculated. For each well, post- / pre-compound ratios were calculated.

[1150] The results were then normalized to 0.3% DMSO control, which was considered as 0%, and to full block (100 pM N-(2,6-dimethylphenylcarbamoylmethyl) tri ethylammonium chloride, aka QX314, for internal application), considered as 100% inhibition. The values of % activity were plotted and concentration response curves were fitted using the 4-parameters logistic Hill equation. From the fitted curves, ICso values were estimated and intracellular pICso values calculated.

[1151] External Block by Test Compounds (extracellular inhibition)

[1152] For extracellular application of the compounds, the extracellular solution was replaced with the solution containing the test compound. After the extracellular solution exchange, voltage pulses were applied.

[1153] Data filtering was performed by Sophion Analyzer software. Only wells with minimum 50 MQ seal resistance and 1 nA pre-compound current were used for further analysis using Aplus software. The average of the peak current amplitude of the 20th pulse of the last three trains applied in pre-compound and post-compound were calculated. For each well, post- / pre-compound ratios were calculated.

[1154] The results were then normalized to 0.3% DMSO control, which was considered as 0%, and to full block (300 pM tetracaine for external application), considered as 100% inhibition. The values of % activity were plotted and concentration response curves were fitted using the 4- parameters logistic Hill equation. From the fitted curves, IC50 values were estimated and extracellular pICso values calculated.

[1155] A pICso of < 4.2 was obtained when the inhibition was minor or equal to 50% at the maximum compound concentration tested of 60 pM. Intracellular and Extracellular inhibition for representative Examples are reported in Table

[1156] 2.

[1157] Table 2 - Intracellular and Extracellular inhibition for representative Examples

[1158] As it can be appreciated, the compounds of Table 2, i.e. compounds according to the invention, show a high intracellular inhibitory activity on NaV 1.7 receptor. Conversely, the compounds of Table 2 show a lower extracellular inhibitory activity on NaV 1.7 receptor.

[1159] Table 3 shows inhibition data for selected Examples that were tested both in their zwitterionic and salt (chloride) forms. As it can be appreciated, both forms possess high intracellular inhibitory activity on NaV 1.7 receptor. Negligible variations were observed between corresponding zwitterionic and salt forms in some instances, that can be considered within the experimental error range. Table 3 - Comparison of Intracellular and Extracellular inhibition for representative

[1160] Examples in zwitterionic or salt form, hNav 1.7

[1161] Accordingly, the compounds of the invention, in both zwitterionic and salt forms, may be used for treating respiratory diseases such as, for example, cough, asthma, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), and in particular chronic cough.

[1162] Comparative Compounds

[1163] Compounds Ci and C2 were tested in the same binding assay described above and their intracellular and extracellular pICso are reported in Table 4.

[1164] Table 4 - Intracellular and Extracellular inhibition for Comparative Compounds As shown in Table 2, Table 3 and Table 4 (Example 7), the compounds of formula (I) of the present invention show an intracellular inhibitory activity on NaV 1.7 expressed as pICso value higher than 5, between 5 and 5.5, higher than 5.5, or between 5.5 and 6.

[1165] These data show that, unlike the comparative Compound Ci, the presence of an amidesubstituted thiophene or benzothiophene ring in the compounds of the present invention causes unexpectedly and notably a significant increase in the intracellular inhibitory activity on NaV 1.7 and increases the difference between NaV 1.7 intracellular and extracellular inhibition, reported in the Table 4 as DELTA.

[1166] Furthermore, as indicated in the same section of the experimental part, the reported data show that, contrary to the comparative Compound C2, the presence of the ami de- substituted thiophene ring, in place of a different heteroaryl ring, in the compounds of the present invention, in particular in Example 7, unexpectedly and noticeably determines a significant increase in the intracellular inhibitory activity on the NaV 1.7 receptors and increases the difference between NaV 1.7 intracellular and extracellular inhibition (DELTA). hERG assay Protocol

[1167] The effect of test substances on the human Ether-a-go-go-Related Gene (hERG) tail current was assessed in automated patch clamp protocol by QPatch instrument (Sophion Bioscience A / S, Ballerup, Denmark).

[1168] Cell Culture hERG was expressed in HEK293 cells upon induction with tetracycline.

[1169] Cells were cultured in minimum essential medium (MEM) supplemented with 10% heat inactivated Fetal Bovine Serum, 1% Non-essential Amino Acids, 1% Sodium Pyruvate, 2mM L- Glutamine, 1% Penicillin-Streptomycin, 15pg / ml Blasticidin and lOOpg / ml Hygromycin. hERG cells were induced with 10 pg / ml Tetracycline for 24 to 72 h before recording.

[1170] Before experiments, cells were washed with Dulbecco's Phosphate Buffered Saline (DPBS), without calcium and without magnesium, detached with TrypLE and then triturated in serum-free media containing 25 mM Hepes and Soybean Trypsin inhibitor to resuspend the cells and to break apart cell aggregates. Cells were counted and the final concentration was set at 4-6 million cells per mL. All chemicals for cell culture were from Life Technologies Italia Sri, Monza, Italy.

[1171] Patch Clamp Solutions & Drugs

[1172] The intracellular solution contained the following (in mM): KC1 130, MgCh 1, ethyleneglycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 5, MgATP 5, N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) 10, pH 7.2 with 1 M KOH.

[1173] The external solution contained the following (in mM): NaCl 137; KC1 4; CaCh 1.8; MgCh 1; d-glucose 10; HEPES 10; pH 7.4 with NaOH. All chemicals for patch clamp solutions were from Merck Life Science Sri, Milano, Italy.

[1174] To evaluate the degree of external block, compounds were first dissolved in DMSO and then diluted in extracellular buffer to achieve final test concentrations of 0.6 pM, 6 pM, 60 pM, in 0.3% DMSO.

[1175] Voltage Protocol

[1176] Single hole QPlate chips (Sophion Bioscience, Denmark) were used for voltage clamp experiments. The membrane potential was held at -80 mV before the voltage protocol began. The voltage protocol included the following sequential steps: -50 mV for 200 ms, +20 mV for 4.8 s, -50 mV for 5 s, then back to holding potential. hERG tail current was measured during the repolarisation step to -50 mV from +20 mV. Every voltage protocol sweep lasted 15 s and was continuously repeated at a frequency of 0.066 Hz during the experiment.

[1177] Liquid Protocol

[1178] External solution then vehicle solution, corresponding to 0.3% DMSO in external solution, were applied for 3 min 45 s each, to reach a stable current recording. Then, three increasing concentrations (0.6, 6 and 60 pM in 0.3% DMSO) of the test compound were applied for 5 min each.

[1179] Data Analysis

[1180] Average tail current was measured from last four voltage sweeps during each compound application protocol and used to calculate the % inhibition, compared to vehicle pre-treatment, for every tested concentration in every cell, using Sophion Analyser software (Sophion Bioscience, Denmark). ICso values were estimated using Hill Fit equation in Sophion Analyser software.

Claims

1. Compound of formula (I) , where at least one of Y and Z is S and the other is CR4; R1 and R2 independently represent -(C1-C6)alkyl or are fused together to form -(C3-C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl is optionally substituted with one or more groups selected from the group consisting of halogen, -(C1-C6)alkyl and -(C1-C6)alkyl-OR8; R3 and R4 are independently H or -(C1-C6)alkyl, or if Y is CR4, R3 and R4 are fused together to form aryl; R5 and R6 are independently H or selected from the group consisting of -(C1-C6)alkyl, -(C1-C6)aminoalkyl, -(C1-C6)alkyl-OR8, -(C1-C6)alkyl-NR7R8, -(C1-C4)alkyl-NR7-C(O)OR8, -(C1-C4)alkyl-(C3-C7)heterocycloalkyl and -(C3-C7)heterocycloalkyl, or fused together to form -(C3-C7)heterocycloalkyl, wherein said -(C1-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, -(C1-C6)aminoalkyl or -(C1-C6)alkyl-NR7R8 are optionally substituted with one or more groups selected from -(C1-C6)alkyl, -C(O)OR8 and -SO2R7; R7 and R8 are independently H or selected from the group consisting of -(C1-C6)alkyl, -(C3-C6)cycloalkyl and -(C1-C6)haloalkyl, or fused together to form -(C3-C7)heterocycloalkyl; L1 is a bond or is selected from the group consisting of -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-; A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl is optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8, and -(C1-C6)alkyl; provided that if L1 is a bond, any of such aryl is not substituted with -(C1-C6)alkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

2. A compound of formula (I) according to claim 1, wherein Z, Y, R1, R2, R3, R 4, R 5, R6, R7 and R8 are as defined in paragraph 1; L1 is selected from the group consisting of -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-; and A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl or heteroaryl is optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C4)alkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

3. A compound of formula (I) according to claim 1, wherein L1 is a bond represented by formula (Ia) where Z, Y, R1, R2, R3, R 4, R 5, R 6, R7 and R8 are as defined in paragraph 1; and A is a ring selected from the group consisting of aryl and heteroaryl, wherein any of such aryl is optionally substituted with one or more groups selected from -OR8, -C(O)OR8 and -C(O)NR7R8, and wherein any of such heteroaryl is optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

4. A connection according to any of the preceding paragraphs, where R1 and R2 independently represent -(C1-C4)alkyl or are fused together to -(C3-C7)heterocycloalkyl, wherein said -(C3-C7)heterocycloalkyl is optionally substituted with one or more groups selected from the group consisting of halogen, -(C1-C4)alkyl and -(C1-C4)alkyl-OR8; R3 and R4 are independently H or -(C1-C6)alkyl, or if Y is CR4, R3 and R4 are fused together to form aryl; R5 and R6 are independently H or selected from the group consisting of -(C1-C4)alkyl, -(C1-C4)aminoalkyl, -(C1-C4)alkyl-OR8, -(C1-C4)alkyl-NR7R8, -(C1-C4)alkyl-NR7-C(O)OR8, -(C1-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, or fused together to form -(C3-C7)heterocycloalkyl, wherein said -(C1-C4)alkyl-(C3-C7)heterocycloalkyl, -(C3-C7)heterocycloalkyl, -(C1-C4)aminoalkyl or -(C1-C4)alkyl-NR7R8 are optionally substituted with one or more groups selected from -(C1-C4)alkyl, -C(O)OR8 and -SO2R7; R7 and R8 are independently H or selected from the group consisting of -(C1-C4)alkyl, -(C3-C6)cycloalkyl and -(C1-C4)haloalkyl, or fused together to form -(C6-C7)heterocycloalkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

5. A connection according to any of the preceding paragraphs, where R1 and R2 are fused together to form -(C6-C7)heterocycloalkyl, wherein said -(C6-C7)heterocycloalkyl is optionally substituted with one or more groups selected from -OR8, -(C1-C4)alkyl and -(C3-C7)heterocycloalkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

6. A connection according to any of the preceding paragraphs, where R3 and R4 independently represent H or -(C1-C4)alkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

7. A compound according to any one of the preceding claims, wherein Z is S and Y is CH, and R3 is H or methyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

8. A connection according to any of the preceding paragraphs, where A is heteroaryl optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; in the form of a zwitterion or a pharmaceutically acceptable salt.

9. A compound according to any one of claim 1, selected from the group consisting of: 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethane-1-aminium (Example 1); 2-(benzylamino)-N-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethane-1-aminium (Example 2); 2-(benzylamino)-N-(2-((2-(tert-butylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethane-1-aminium (Example 3); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholine-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 4); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 5); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 6); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 7); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholine-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 8); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium, (Example 9); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 10); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-1-ium (Example 11); 1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 12); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ia (Example 13); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ia (Example 14); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 15); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 16); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ium (Example 17); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 18); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 19); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 20); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetan-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 21); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 22); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetan-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 23); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidin-1-ium (Example 24); 1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazole-3-ylamino)-2-oxoethyl)azepan-1-ia (Example 25); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ium (Example 26); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 27); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidin-1-ia (Example 28); 1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 29); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-morpholinoethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 30); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 31); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-(methylsulfonyl)piperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 32); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 33); 1-(2-((2-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazole-3-ylamino)-2-oxoethyl)azepan-1-ium (Example 34); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 35); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-(methylsulfonyl)piperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 36); 1-(2-(((2-carbamoyl-4-methylthiophene-3-yl)methyl)amino)-2-oxoethyl)-1-(2-(isoxazole-3-ylamino)-2-oxoethyl)azepan-1-ia (Example 37); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 38); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((2-(morpholin-4-carbonyl)benzo[b]thiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 39); (R)-1-(2-(((2-carbamoyl-4-methylthiophene-3-yl)methyl)amino)-2-oxoethyl)-1-(2-oxo-2-((1-phenylethyl)amino)ethyl)azepan-1-ia (Example 40); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(trimethylammonio)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 41); 1-(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazol-3-ylamino)-2-oxoethyl)azepan-1-ium (Example 42); 1,1-bis(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 43); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 44); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 45); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(trimethylammonio)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 46); 1-(2-(isoxazol-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 47); 1-(2-(isoxazole-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(piperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 48); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Primary 49); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 50); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(piperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 51); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 52); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 53); 1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Primary 54); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)(methyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 55); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methyl(2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 56); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Primary 57); (S)-1-(2-((2-((6-amino-1-methoxy-1-oxohexan-2-yl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(benzylamino)-2-oxoethyl)azepan-1-ium (Primary 58); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-yl (Primer 59); 4-fluoro-1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-4-methyl-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-pyrimethyl-1-p 60); 1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)-1-(2-oxo-2-((2-(trifluoromethoxy)benzyl)amino)ethyl)azepan-1-ia (Example 61); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((piperidin-4-ylmethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azepan-1-ia (Example 62); 1-(2-((2-((2-(cyclopropylamino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)aze (Primary-1-3)); 1-(2-((2-(bis(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azepan-1-6 (Primary 4); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperidin-4-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 65); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 66); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethyl-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ium (Example 67); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 68); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ium (Example 69); 1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetane-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ia (Primary 70); 1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-4,4-dimethyl-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-pyoxycarbonyl (1-periethyl) 71); 1-(2-((2-(ethoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-4,4-dimethyl-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophene-3-yl)amino)-2-pyridine-co-pyrime 72); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-4,4-bis(hydroxymethyl)piperidin-1-ium (Example 73); (2-(4,4-dimethyl-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-ia-1-yl)acetyl)(isoxazol-3-yl)amide (Example 74); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-bis(hydroxymethyl)-1-(2-oxo-2-((1-phenylcyclopropyl)amino)ethyl)piperidin-1-ium (Example 75); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-((piperidin-4-ylmethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 76); and (R)-1-(2-((2-((6-amino-1-methoxy-1-oxohexan-2-yl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(benzylamino)-2-oxoethyl)azepan-1-ium (Example 77); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(morpholine-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 78) 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 79) 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-((2-methoxyethyl)(methyl)carbamoyl)-2-methylthiophen-3-yl)amino)-2-oxoethyl)azepan-1-ium (Example 80) in the form of a zwitterion or a pharmaceutically acceptable salt.

10. Using an intermediate compound selected from the group consisting of compound (V), (VIII), (X), (XI), (XV) and (XVI) to obtain a compound of formula (I) according to any one of claims 1 to 9 where R1, R2, R3, R4, R 5, L1 and A are as defined in paragraphs 1-9, X2 is halogen, preferably chlorine or bromine, and PG is -(C1-C6)alkyl.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9, in admixture with one or more pharmaceutically acceptable carriers or excipients.

12. A pharmaceutical composition according to claim 11, formulated for administration by inhalation.

13. A compound of formula (I) according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 11 or 12 for use as a medicine.

14. A compound of formula (I) or a pharmaceutical composition for use according to claim 13 for the prevention and / or treatment of diseases, disorders or conditions associated with sodium channel receptor mechanisms.

15. A compound of formula (I) or a pharmaceutical composition for use according to claim 13 or 14 for the prevention and / or treatment of diseases of the respiratory system selected from cough, subacute or chronic cough, treatment-resistant cough, refractory chronic cough, idiopathic chronic cough, post-viral cough, iatrogenic cough, asthma, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

16. A compound of formula (I) or a pharmaceutical composition for use according to claim 15 for the prevention and / or treatment of chronic cough.

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