Heteroaryl amide derivatives as sodium channel inhibitors

Abstract

The present invention relates generally to compounds of Formula (I) that inhibit sodium channel (NaV) activity; in particular, the present invention relates to compounds that are heteroaryl amide derivatives, including zwitterionic forms and pharmaceutically acceptable salts thereof, methods of making the compounds, and therapeutic uses thereof. The compounds of the present invention can be used, for example, to treat a number of disorders associated with NaV, such as respiratory diseases. (I).

Description

[0001] Invention Field

[0002] This invention generally relates to compounds that inhibit the activity of voltage-gated sodium channels (NaV) (hereinafter referred to as NaV inhibitors); the invention relates to compounds that are heteroarylamide derivatives, including pharmaceutically acceptable salts thereof, methods for preparing said compounds, and their therapeutic uses.

[0003] The compounds of this invention can be used, for example, to treat many disorders related to sodium channel receptor mechanisms, such as respiratory diseases. Background of the Invention

[0005] Sensory afferent nerves carry impulses originating from different stimuli (heat, cold, thermal, mechanical, and chemical) and mediate the cough reflex.

[0006] Coughing is a defensive reflex designed to protect the airways from foreign objects and help remove debris from the airways. Chronic cough is defined as a cough lasting more than 8 weeks. Hypersensitive and pica cough states are often chronic, lasting more than three months and can manifest as many respiratory disease 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. Furthermore, an inappropriate cough reflex can manifest acutely and persistently following a viral infection. Finally, chronic cough can be idiopathic with an unknown etiology (Dicpinigaitis, PV et al, Pharmacol Rev, 2014, 66, 468-512; Patil, MJ et al, Pulmonary Pharmacology & Therapeutics, 2019, 56, 15-19).

[0007] Due to the failure of many cough suppression treatment strategies in clinical practice, increased attention in recent years has focused on potential inhibitors of voltage-gated sodium channels (NaVs), which are absolutely necessary for conducting action potentials originating in nerve endings in response to a variety of stimuli, including many inflammatory mediators. At least nine members of the voltage-gated sodium channel (NaV) α-subunit family are currently known. The NaV family has been phylogenetically divided into two subfamilies: NaV1.x (all except SCN6A) and NaV 2.x (SCN6A). The NaV 1.x subfamily can be further functionally divided into two groups: those sensitive to tetrodotoxin blockade (TTX-sensitive or TTX-s) and those resistant to tetrodotoxin blockade (TTX-resistant or TTX-r). Cough is a complex process triggered and regulated by two independent neural pathways that can interfere with each other: mechanosensitive vagal nodular Aδ fibers and chemosensitive vagal jugular C-fibers. Of the various known NaV1.x subunits, only a few are expressed in the C-fibers of the vagus nerve and jugular vein in the respiratory tract, involving TTX-sensitive NaV l.7 and TTX-resistant NaV l.8 and NaV l.9 (Kollarik, M., et al, J Physiol, 2018, 596, 1419-1432). NaV 1.7 (PN1, SCN9A) is sensitive to blockade by tetrodotoxin and is preferentially expressed in peripheral sympathetic and sensory neurons.

[0008] The cough reflex can be suppressed by effective sodium channel blockers, such as lidocaine. Lidocaine blocks all sensory input (as in local anesthesia) and mechanoreceptors and motor neurons, resulting in numbness and paralysis. Lidocaine spray is routinely used to suppress the cough reflex before bronchoscopy. The relief of intractable cough by lidocaine inhalation was first reported in 1977 (C Fletcher et al, Br Med J, 1977 1(6077), 1645-8), and then recently confirmed in clinical trials as a throat spray for patients with intractable chronic cough (RCC) (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, and has off-target effects (burning sensation upon injection), and its use (as a cough suppressant) is limited by its inferior risk / benefit profile (blocking the cardiac NaV 1.5 channel).

[0009] Sensory neurons express several channels with macropores (TRPV1, TRPA1, ASICS, P2X3, etc.), which, upon activation, allow charged molecules that normally cannot permeate the neuronal membrane to pass through. This is different from neutral molecules such as lidocaine, which can penetrate the lipid membrane surrounding the channel to reach the Nav binding site in the channel pore (Frazier et al, JPET, 2015; 171,1.; Brenneis et al, J Neuroscience, 2013, 33(1):315-26).

[0010] In 1999, Nortran Pharmaceutical filed a patent application (WO99 / 63985) relating to a pharmaceutical composition having antitussive activity and a method of treating warm-blooded animals affected by cough or bronchial stenosis by administering an effective amount of the inventive pharmaceutical composition. An example active antitussive agent reported therein is a charged quaternary ammonium compound, namely N,N-di(phenylcarbamoylmethyl)dimethylammonium chloride (i.e., carbaryl).

[0011] Carcainium was first disclosed by Astra in 1962 for the treatment of arrhythmias (US 3255207). Its antitussive effects have been studied in various animal models: in guinea pig models, carcainium showed high antitussive efficacy and a mechanism of action somewhat different from lidocaine (JJ Adcock, British Journal of Pharmacology, 2003, 138, 407-416). In 2013, Verona Pharma filed a patent application (WO2013 / 03490) concerning carcainium in a salt (anionic form of a pharmaceutically acceptable acid) for the treatment and / or suppression of cough, cough attack, or cough flare-up in patients. The compound was found to be effective in preliminary studies of patients with chronic cough and idiopathic interstitial pneumonia (Lavorini et al, Pulmonary Pharmacology & Therapeutics, 2016, 40, 91-94), but it was never commercialized because it failed to reduce the frequency and severity of cough in larger follow-up studies (Satia et al, Thorax, 2015;70 (Suppl 3): A1-A254).

[0012] Currently available cough medications often have limited efficacy. Cough can severely impact a patient's quality of life, with physical (including fatigue, wheezing, sleep disturbances, and incontinence), psychological (including anger, frustration, depression, and anxiety), and social consequences. Therefore, continuous efforts are needed to find new and promising therapies and targets for cough treatment, and chronic cough remains an unmet medical need.

[0013] The prior art does not describe or suggest the general formula (I) amide derivative compounds of the present invention, which may represent a solution to the unmet medical needs mentioned above.

[0014] Invention Summary

[0015] In a first aspect, the present invention relates to compounds of formula (I)

[0016]

[0017] (I)

[0018] It exists in zwitterionic or pharmaceutically acceptable salt form.

[0019] At least one of Y and Z is S, and the other is CR4;

[0020] R1 and R2 are independently -(C1-C6)alkyl or fused to form -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl may optionally be substituted by one or more groups selected from halogens, -(C1-C6)alkyl and -(C1-C6)alkyl-OR8;

[0021] R3 and R4 are independently H or -(C1-C6)alkyl, or, in the case that Y is CR4, R3 and R4 are fused to form an aryl group;

[0022] R5 and R6 are independently H or selected from -(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)heterocyclicalkyl and -(C3-C7)heterocyclicalkyl, or fused to form -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C6)aminoalkyl or -(C1-C6)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C6)alkyl, -C(O)OR8 and -SO2R7;

[0023] R7 and R8 are independently H or selected from -(C1-C6)alkyl, -(C3-C6)cycloalkyl and -(C1-C6)haloalkyl, or fused to form -(C3-C7)heteroalkyl;

[0024] L1 is a bond or is selected from -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-;

[0025] A is a ring selected from aryl and heteroaryl groups, wherein any aryl or heteroaryl group may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; provided that any aryl group is not substituted by -(C1-C6)alkyl when L1 is a bond.

[0026] In a second aspect, the present invention relates to pharmaceutical compositions comprising a compound of formula (I) and one or more pharmaceutically acceptable carriers or excipients mixed therewith.

[0027] In a third aspect, the present invention relates to the use of compounds of formula (I) as pharmaceuticals.

[0028] In another aspect, the present invention relates to compounds of formula (I) for the treatment of diseases, disorders or conditions associated with voltage-gated sodium channels (NaV) dysregulation.

[0029] In another aspect, the present invention relates to compounds of formula (I) for the prevention and / or treatment of respiratory diseases selected from the following: cough, subacute or chronic cough, treatment of resistant cough, intractable 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. Invention Details

[0031] Unless otherwise specified, the term "formula (I)" means its stereoisomers, tautomers, pharmaceutically acceptable salts, zwitterionic forms, or solvates.

[0032] The term "stereoisomer" refers to isomers with identical constitutions but different atomic spatial arrangements. Enantiomers and diastereomers are examples of stereoisomers.

[0033] The term "enantiomer" refers to one of a pair of molecules that are mirror images of each other and cannot be superimposed.

[0034] The term "diamera" refers to a stereoisomer that is not a mirror image.

[0035] The term "racemate" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts, wherein the composition is optically inactive.

[0036] The symbols "R" and "S" represent the configuration of the substituents surrounding (one or more) chiral carbon atoms and are expected to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry, 68:2193-2222(1996)).

[0037] The term "tautomer" refers to each of two or more isomers of a compound, which exist together in equilibrium and are readily interconvertible by the migration of atoms or groups within the molecule.

[0038] Compound (I) is a quaternary ammonium compound. The term "pharmaceutically acceptable salt" as used herein refers to a compound of formula (I), wherein the compound is provided as an addition salt of any acid conventionally expected to be pharmaceutically acceptable. Suitable examples of such salts may thus include inorganic or organic acid addition salts of 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.

[0039] Alternatively, the compound of formula (I) according to the invention can be provided as an "amphoteric form", also known as an internal salt or dipole ion, which is a form in which both positive and negative charges are present.

[0040] The term "solvent" refers to the physical combination of the compound of the present invention with one or more solvent molecules (whether organic or inorganic). This physical combination includes the formation of hydrogen bonds. In some cases, the solvate may be separated by crystallization, for example, in the case where one or more solvent molecules are incorporated into a crystalline solid lattice. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules.

[0041] The terms "halogen" or "halogen atom" or "halogenated" as used herein include fluorine, chlorine, bromine, and iodine atoms.

[0042] The term "-(C)" x -C y "alkyl" where x and y are integers refers to a straight-chain or branched alkyl group having x to y carbon atoms. Thus, for example, in the case where x is 1 and y is 6, the term includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, and n-hexyl.

[0043] The term "-(C)" x -C y )alkylene-" where x and y are integers, referring to (C) alkylene groups having a total of two unsatisfied valences. x -C y )alkyl residues. The "-(C x -C y Examples of alkylene groups may include, for example, divalent methylene residues and -CH(CH3)-.

[0044] The term "-(C)" x -C y ) Haloalkyl, where x and y are integers, refers to the above-defined "(C x -Cy)alkyl" group, wherein one or more hydrogen atoms are replaced by one or more halogen atoms that can be the same or different. x -C y Examples of "halogenated alkyl" groups can thus include halogenated, polyhalogenated and fully halogenated alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as trifluoromethyl.

[0045] The term "-(C)" x -C y )aminoalkyl" where x and y are integers, referring to "(C" as defined above) x -C y An alkyl group, wherein one or more hydrogen atoms are replaced by one or more amino groups that can be the same or different. x -C y Examples of "aminoalkyl" include, for example, aminomethyl.

[0046] The term "-(C)" x -C y )hydroxyalkyl" where x and y are integers, referring to "-(C" as defined above. x -C y The "-(C)" group, wherein one or more hydrogen atoms are replaced by one or more hydroxyl (OH) groups. x -C y Examples of "hydroxyalkyl" include, for example, hydroxymethyl.

[0047] The term "partially unsaturated" refers to a ring portion that includes at least one double or triple bond. The term "partially unsaturated" is intended to cover rings having multiple unsaturated sites but not necessarily to include aryl or heteroaryl portions as defined herein.

[0048] The term "-(C)" x -C y"Cycloalkyl" where x and y are integers refers to a monocyclic or polycyclic hydrocarbon group containing the stated number of cyclic carbon atoms, either saturated or partially unsaturated. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0049] Term "(C x -C y "(Cycloalkylene)" where x and y are integers, refers to a mono- or polycyclic alkyl group containing the specified number of cyclic carbon atoms, either saturated or partially unsaturated. x -C y Cycloalkyl groups have two unsatisfied valences on the same carbon atom. x -C y Examples of "-(C1-C4)cycloalkyl-" include "-(C1-C6)cycloalkyl-", such as cyclopropyl.

[0050] The term "-(C)" x -C y Heterocyclic alkyl groups, where x and y are integers, refer to saturated or partially unsaturated monocyclic, polycyclic, or polycyclic spirocyclic groups containing the indicated number of ring carbon atoms, wherein at least one ring carbon atom is replaced by at least one heteroatom or heterogroup (e.g., N, NH, S, or O) or carries an oxo (=O) substituent. The heterocyclic alkyl group (i.e., a heterocyclic residue or group) may be optionally substituted by one or more specific groups at possible positions in the ring, i.e., on a carbon atom, or on a possible substituted heteroatom or heterogroup. In the presence of more than one substituent, they may involve the same or adjacent positions in the ring, for example, "(C3-C..." 10 Heterocyclic alkyl groups can be 1,1-, 1,2-, or 1,3-disubstituted. Substitution at the carbon atom includes spirodisubstituted groups, wherein an additional spirofused 3- to 6-membered ring is formed. The spirodisubstituted groups are included as polycyclic spiro groups in (C3-C4) 10 The meaning of "heterocyclic alkyl" is as follows. (C x -C y Representative non-limiting examples of "heterocyclic alkyl" are pyrrolidinyl, piperazinyl, piperidinyl, morpholinyl, thiomorpholinyl, dihydro- or tetrahydro-pyridinyl, oxocyclic butyl, isoindolyl, tetrahydropyranyl, pyranyl, dihydro- or tetrahydrofuranyl.

[0051] The term "-(C)" x -C y ) Heterocyclic alkyl-" where x and y are integers, referring to a mono- or polycyclic alkyl group containing the specified number of cyclic carbon atoms, either saturated or partially unsaturated. x -C y Heterocyclic alkyl groups have two unsatisfied valences on the same carbon atom.

[0052] The group may be optionally substituted, wherein the term "optionally substituted" means substituted or unsubstituted. In the case where the term "one or more" refers to any atom or group acting as a substituent for a group of a compound of formula (I), it is desirable that there are 1 to 3, preferably 1 to 2, more preferably 1 substituent that may replace the hydrogen on the variable.

[0053] The term "aryl" refers to a monocyclic or bicyclic unsaturated or partially unsaturated carbocyclic system in which at least one ring is aromatic. Examples of suitable aryl ring systems include, for example, phenyl, dihydro-1H-indenyl, or naphthyl.

[0054] The term "heteroaryl" refers to a monocyclic, bicyclic, or tricyclic system having 5 to 20, preferably 5 to 15, ring atoms, wherein at least one ring is aromatic and at least one ring atom is a heteroatom (e.g., N, NH, S, or O). Examples of "heteroaryl" include thiophene, benzothiophene, pyrrole, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and triazinyl.

[0055] Keys pointing to waveforms or wavy lines, such as those used in the structural formulas of this article. This describes a bond that is the attachment point of the part or substituent to the core or skeletal structure.

[0056] The term "bond" used to define a substituent refers to a situation where the substituent is connected to two functional groups that are directly linked to each other without any additional atoms in between.

[0057] A dash ("-") not between two letters or symbols is intended to represent the attachment point of a substituent.

[0058] The terms “sodium channel receptor” or “voltage-gated sodium channel receptor” or “NaV receptor” used to refer to the inhibitory activity of the compounds of the present invention are intended to include members of the voltage-gated sodium channel (NaV) α subunit family expressed in the C-fibers of the jugular vein of the vagus nerve of the respiratory tract, namely NaV 1.7, NaV 1.8 and NaV 1.9.

[0059] The term "NaV inhibitor" means that the compounds of the present invention are inhibitors of NaV receptors, particularly but not limited to NaV 1.7 receptors.

[0060] The term "IC" 50 "The half-maximal inhibitory concentration" refers to the concentration at half maximum, which serves as a measure of a substance's effectiveness in inhibiting specific biological or biochemical functions.

[0061] The term "pIC" 50 "refers to IC50 expressed as molar concentration" 50 The negative logarithm of the value.

[0062] Whenever other basic amino or quaternary ammonium groups are present in a compound of formula (I), pharmaceutically acceptable anions selected from chloride, bromide, iodide, trifluoroacetate, formate, sulfate, phosphate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate, oxalate, succinate, benzoate, p-toluenesulfonate, emipamoate, sine, and naphthalenedisulfonate may be present. Similarly, in the presence of acidic groups such as COOH groups, corresponding pharmaceutically acceptable cations may be present, including alkali metal or alkaline earth metal ions, such as sodium, potassium, and calcium.

[0063] As noted above, the present invention relates to a series of compounds represented by general formula (I) having inhibitory activity against voltage-gated sodium channel receptors, as described in detail below.

[0064] The inhibitory activity of compound (I) was tested in an automated patch-clamp assay on CHO cells stably expressing the human NaV 1.7 channel, as described in the experimental section.

[0065] For some compounds of this invention, pIC was calculated. 50 Values ​​show the intracellular inhibition of the tested compounds on the NaV 1.7 receptor (see Qian, Binbin et al, Current protocols in pharmacology, 2020, 89, 1).

[0066] Indeed, the compounds of formula (I) of the present invention can act as inhibitors of NaV 1.7 in a substantial and effective manner. In particular, as noted in the experimental section, the compounds of formula (I) of the present invention exhibit intracellular inhibition of NaV 1.7, as reported in Table 2 for representative compounds.

[0067] Compound (I) is a heteroaryl quaternary ammonium amide derivative in salt or zwitterionic form, and it exhibits particularly good inhibitory activity against NaV 1.7.

[0068] As noted in the experimental section comparing the compound segments, particularly in Table 4, the presence of an amide-substituted tiophene or benzothiophene ring in the compounds of the present invention unexpectedly and significantly determines the associated increase in intracellular inhibitory activity against the NaV 1.7 receptor, unlike carbaryl, which is considered a comparative compound C1.

[0069] Furthermore, as noted in the same experimental section, the reported data demonstrate that, in contrast to the comparative compound C2, which is characterized by a different heteroaryl ring, the presence of an amide-substituted thiophene or benzothiophene ring in the compounds of the present invention unexpectedly and notably determines the associated increase in inhibitory activity against the NaV receptor.

[0070] Compounds of formula (I) in the form of pharmaceutically acceptable salts or zwitterions, hereinafter alternatively referred to as compounds of the present invention, may be used as pharmaceuticals. Accordingly, the present invention also provides the use of compounds of formula (I) in the form of pharmaceutically acceptable salts or zwitterions as pharmaceuticals.

[0071] The compounds of formula (I) of the present invention can act as inhibitors of the NaV1.7 receptor in a substantial and effective manner, which is particularly recognized by those skilled in the art (in the search for compounds that may treat respiratory diseases such as cough, asthma, IPF and COPD, and especially chronic cough).

[0072] Accordingly, compounds of formula (I) can be used to treat respiratory diseases selected from cough, asthma, IPF and COPD, and especially chronic cough, provided that NaV receptors are involved.

[0073] As can be recognized, the compounds of the present invention shown in Table 2 exhibit relevant potency in their intracellular inhibitory activity against the NaV 1.7 receptor, expressed as a pIC value equal to or greater than 5, preferably 5 to 5.5, more preferably equal to or greater than 5.5. 50 value.

[0074] More advantageously, compounds represented by general formula (I) are able to inhibit one or more voltage-gated ion channels when exposed to or administered to cells, but show little or no inhibition when administered extracellularly.

[0075] A similar protocol was used for extracellular inhibition, i.e., after exchanging the external solution for a solution containing the test compound, compound (I) was also tested in an automated patch-clamp assay. The experimental values ​​obtained were used to calculate the pIC representing extracellular inhibition of the NaV1.7 receptor. 50 value.

[0076] As indicated in the experimental section, the compound of formula (I) of this invention exhibits lower extracellular inhibition of NaV 1.7 than intracellular inhibition, as shown in Table 2.

[0077] Accordingly, the difference between intracellular and extracellular efficacy of the compounds of the present invention in inhibiting NaV 1.7, as shown in Table 2, is defined as DELTA, which is equal to or greater than 0.5, preferably between 0.5 and 1, and more preferably equal to or greater than 1.

[0078] This difference between intracellular and extracellular inhibition can lead to greater safety of the compound for sensory neurons.

[0079] The compounds of this invention have also been tested in safety pharmacology studies, particularly in in vitro testing of the human ether-a-go-go related gene (hERG) channel. Safety pharmacology studies are non-clinical studies that test the safety of new drugs in humans from a pharmacological perspective. Guidelines for safety pharmacology studies aimed at examining the safety of investigational substances in humans and predicting their adverse effects have been established at 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 these guidelines, as part of safety pharmacology studies, it is necessary to examine the arrhythmic effects of the investigational substance, particularly the presence or absence of QT interval prolongation on an electrocardiogram. The QT interval is an electrocardiogram measurement used to evaluate certain electrical properties of the heart and approximates the time taken from the onset of ventricular contraction to the end of diastole. To protect patients from ventricular tachycardia, torsades de pointes, and fatal arrhythmias associated with drug-induced QT interval prolongation, it is important in drug development to detect QT interval prolongation effects that can induce these serious adverse effects.

[0080] To date, many drugs known to prolong the QT interval have been found to inhibit delayed rectifier potassium channels in cardiomyocytes. hERG channels are believed to act as a major component protein in delayed rectifier potassium channels. Therefore, in the draft guidelines for non-clinical evaluation of the ability of human drugs to delay ventricular repolarization (QT interval prolongation), the use of hERG channel-transfer cell ion channel assays as a non-clinical study is recommended.

[0081] In addition to its significant efficacy in inhibiting NaV receptors, the in vitro inhibition of hERG channels by the compound of formula (I) of this invention was also tested and showed low toxicity to hERG, thereby reducing the likelihood of adverse events being observed during clinical studies.

[0082] Therefore, in one aspect, the present invention relates to compounds of general formula (I).

[0083]

[0084] (I)

[0085] It exists in zwitterionic or pharmaceutically acceptable salt form.

[0086] At least one of Y and Z is S, and the other is CR4;

[0087] R1 and R2 are independently -(C1-C6)alkyl or fused together as -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl may optionally be substituted by one or more groups selected from halogens, -(C1-C6)alkyl and -(C1-C6)alkyl-OR8;

[0088] R3 and R4 are independently H or -(C1-C6)alkyl, or, in the case that Y is CR4, R3 and R4 are fused to form an aryl group;

[0089] R5 and R6 are independently H or selected from -(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)heterocyclicalkyl and -(C3-C7)heterocyclicalkyl, or fused to form -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C6)aminoalkyl or -(C1-C6)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C6)alkyl, -C(O)OR8 and -SO2R7;

[0090] R7 and R8 are independently H or selected from -(C1-C6)alkyl, -(C3-C6)cycloalkyl and -(C1-C6)haloalkyl, or fused to form -(C3-C7)heteroalkyl;

[0091] L1 is a bond or is selected from -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-;

[0092] A is a ring selected from aryl and heteroaryl groups, wherein any aryl or heteroaryl group may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; provided that any aryl group is not substituted by -(C1-C6)alkyl when L1 is a bond.

[0093] All listed groups in the variable portions Y, Z, R1, R2, R3, R4, R5, R6, R7, R8, L1, and A of the compounds of this invention must be considered as alternatives and can be combined with each other to form embodiments included within the scope of this invention.

[0094] The 5-membered ring containing Y and Z is an aromatic ring, also known as a thiophene ring, which is substituted by the -C(O)NR5R6 group.

[0095] Preferred halogens, alone and in -(Cx -C y The alkyl halide substituents are fluorine and bromine, with fluorine being more preferred.

[0096] In a preferred embodiment, Z is S and Y is CR4. In a more preferred embodiment, Z is S and Y is CH.

[0097] In yet another embodiment, Y is S and Z is CR4, wherein R4 is preferably hydrogen.

[0098] In one embodiment, R1 and R2 are independently -(C1-C6)alkyl or fused to form -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl may optionally be substituted with one or more groups selected from halogens, -(C1-C6)alkyl, and -(C1-C6)alkyl-OR8. In a preferred embodiment, R1 and R2 are independently -(C1-C6)alkyl. In a more preferred embodiment, R1 and R2 are independently -(C1-C4)alkyl. In an even more preferred embodiment, R1 and R2 are methyl. In yet another preferred embodiment, R1 and R2 are fused to form a -(C3-C7)heteroalkyl, optionally substituted with one or more groups selected from halogens, -(C1-C6)alkyl, and -(C1-C6)alkyl-OR8. In a more preferred embodiment, R1 and R2 are fused to form a -(C3-C7)heteroalkyl group, optionally substituted with one or more groups selected from halogens, methyl groups, and -(C1-C4)alkyl-OR8. In the most preferred embodiment, R1 and R2 are fused to optionally substituted piperidinyl or azirheptanyl groups.

[0099] In one embodiment, R3 and R4 are independently H or -(C1-C6)alkyl, or, if Y is CR4, R3 and R4 are fused to form an aryl group. In yet another embodiment, R3 and R4 are H. In a more preferred embodiment, R3 and R4 are independently H or -(C1-C4)alkyl; even more preferred, R3 and R4 are independently H or methyl, and most preferably R3 is methyl and R4 is H. In the most preferred embodiment, Z is S and Y is CH, and R3 is H or methyl.

[0100] In yet another preferred embodiment, R3 and R4 are fused to form an optionally substituted phenyl ring, which is fused with a 5-membered ring to form an optionally substituted benzothiophene ring system.

[0101] In one embodiment, R5 and R6 are independently H or selected from -(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)heterocyclic alkyl and -(C3-C7)heterocyclic alkyl, or fused to form -(C3-C7)heterocyclic alkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclic alkyl, -(C3-C7)heterocyclic alkyl, -(C1-C6)aminoalkyl or -(C1-C6)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C6)alkyl, -C(O)OR8 and -SO2R7. In another embodiment, R5 and R6 are independently H or selected from -(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)heterocyclic alkyl, and -(C3-C7)heterocyclic alkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclic alkyl, -(C3-C7)heterocyclic alkyl, -(C1-C6)aminoalkyl, or -(C1-C6)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C6)alkyl, -C(O)OR8, and -SO2R7. In a preferred embodiment, R5 and R6 are independently H or -(C1-C6)alkyl. In a more preferred embodiment, R5 and R6 are independently H or -(C1-C4)alkyl. In a more preferred embodiment, R5 and R6 are independently H or Me. In yet another preferred embodiment, R5 and R6 are independently selected from H, -(C1-C4)aminoalkyl, -(C1-C4)alkyl-OR8, -(C1-C4)alkyl-NR7R8, -(C1-C4)alkyl-NR7-C(O)OR8, -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, and -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C4)aminoalkyl, or -(C1-C4)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C4)alkyl, -C(O)OR8, and -SO2R7. In another embodiment, R5 and R6 are fused together to form a -(C3-C7) heterocyclic alkyl group, wherein the -(C3-C7) heterocyclic alkyl group may optionally be substituted by one or more groups selected from -(C1-C6) alkyl, -C(O)OR8 and -SO2R7.In a preferred embodiment, R5 and R6 are fused into a -(C3-C7) heterocyclic alkyl group, wherein the -(C3-C7) heterocyclic alkyl group may optionally be substituted with one or more groups selected from -(C1-C4) alkyl groups. In a more preferred embodiment, R5 and R6 are fused into a -(C4-C7) heterocyclic alkyl group selected from pyrrolidine, piperazine, and morpholine, wherein the -(C3-C7) heterocyclic alkyl group may optionally be substituted with one or more groups selected from -(C1-C4) alkyl groups.

[0102] In one embodiment, R7 and R8 are independently H or selected from -(C1-C6)alkyl, -(C3-C6)cycloalkyl, and -(C1-C6)haloalkyl, or fused to form -(C3-C7)heteroalkyl. In a preferred embodiment, R7 and R8 are independently H or -(C1-C4)alkyl. In yet another preferred embodiment, R7 and R8 are independently H or methyl. In still a preferred embodiment, R7 and R8 are H or R7 and R8 are methyl.

[0103] In one embodiment, L1 is a bond or selected from -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-. In a preferred embodiment, L1 is a bond or selected from -(C1-C4)alkylene- and -(C1-C4)cycloalkylene-. In yet another preferred embodiment, L1 is a bond. In yet another more preferred embodiment, L1 is selected from methylene, cyclopropylene, and -CH(CH3)-.

[0104] In one embodiment, A is a ring selected from aryl and heteroaryl groups, wherein any aryl or heteroaryl group may optionally be substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8, and -(C1-C6)alkyl; provided that any aryl group is not substituted with -(C1-C6)alkyl when L1 is a bond. In a preferred embodiment, A is a heteroaryl group, optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8, and -(C1-C6)alkyl. In yet another preferred embodiment, A is a heteroaryl group selected from optionally substituted thiophene, benzothiophene, pyrrole, pyrazol, imidazolyl, isoxazol, oxazol, isothiazol, thiazolyl, pyridinyl, pyrimidinyl, pyrazinyl, and triazinyl. In yet another preferred embodiment, A is an aryl group, optionally substituted with one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8, and -(C1-C6)alkyl, provided that the aryl group is not substituted with -(C1-C6)alkyl when L1 is a bond. In a preferred embodiment, A is an aryl group, wherein any aryl group may optionally be substituted with one or more groups selected from -OR8, -C(O)OR8, and -C(O)NR7R8. In a more preferred embodiment, A is selected from phenyl, tiophenyl, and isoxazolyl, optionally substituted with one or more groups selected from -OR8, -C(O)OR8, and -C(O)NR7R8. In other preferred embodiments, A is phenyl or selected from unsubstituted isoxazolyl or isoxazolyl substituted with one or more methyl groups.

[0105] All preferred groups of the variable portions Y, Z, R1, R2, R3, R4, R5, R6, R7, R8, L1 and A of the compounds of the present invention listed above can be combined with each other to form embodiments included within the scope of the present invention.

[0106] In yet another preferred embodiment, the present invention relates to compounds of formula (I) as defined above, wherein

[0107] Z, Y, R1, R2, R3, R4, R5, R6, R7, R8 are as defined above;

[0108] L1 is selected from -(C1-C4)alkylene- and -(C1-C4)cycloalkylene-; and

[0109] A is a ring selected from aryl and heteroaryl, wherein any of the aryl or heteroaryl groups may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C4)alkyl.

[0110] In a preferred embodiment, L1 is selected from -(C1-C4)alkylene and -(C1-C4)cycloalkylene. In an even more preferred embodiment, L1 is selected from methylene, cyclopropylene, and -CH(CH3)-.

[0111] In another preferred embodiment, A is a ring selected from aryl and heteroaryl groups, wherein any of the aryl and heteroaryl groups may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, and -C(O)NR7R8. In a more preferred embodiment, A is a heteroaryl group, wherein any of the heteroaryl group may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, and -C(O)NR7R8. In yet another more preferred embodiment, A is an aryl group, wherein any of the aryl group may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, and -C(O)NR7R8.

[0112] In yet another embodiment, the present invention relates to compounds of formula (I), wherein L1 is a bond, represented by formula (Ia):

[0113]

[0114] (Ia)

[0115] It exists in zwitterionic or pharmaceutically acceptable salt form.

[0116] in

[0117] Z, Y, R1, R2, R3, R4, R5, R6, R7, and R8 are as defined above; and

[0118] A is a ring selected from aryl and heteroaryl groups, wherein any of the aryl groups may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8 and -C(O)NR7R8, and wherein any of the heteroaryl groups may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl groups.

[0119] In a more preferred embodiment, A is a heteroaryl group, wherein any of the heteroaryl group may optionally be substituted with one or more -OR8, -C(O)OR8, -C(O)NR7R8, and -(C1-C6)alkyl groups. In an even more preferred embodiment, A is a heteroaryl group, wherein any of the heteroaryl group is selected from thienyl, pyrroloyl, pyrazolyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, pyridyl, pyrimidinyl, pyrazinyl, and triazinyl, and any of the heteroaryl group may optionally be substituted with one or more methyl, ethyl, isopropyl, or tert-butyl groups.

[0120] In yet another preferred embodiment, the present invention relates to compounds of formula (I) as defined above.

[0121] in

[0122] At least one of Y and Z is S, and the other is CR4;

[0123] R1 and R2 are independently -(C1-C4)alkyl or fused to -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl may optionally be substituted by one or more groups selected from halogens, -(C1-C4)alkyl and -(C1-C4)alkyl-OR8;

[0124] R3 and R4 are independently H or -(C1-C6) alkyl, or wherein Y is CR4, and R3 and R4 are fused to form an aryl group, preferably optionally a substituted aryl group;

[0125] R5 and R6 are independently H or selected from -(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)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, or fused to form -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C4)aminoalkyl or -(C1-C4)alkyl-NR7R8 may optionally be substituted by one or more groups selected from -(C1-C4)alkyl, -C(O)OR8 and -SO2R7;

[0126] R7 and R8 are independently H or selected from -(C1-C4)alkyl, -(C3-C6)cycloalkyl and -(C1-C4)haloalkyl, or fused to form -(C6-C7)heteroalkyl;

[0127] L1 is a bond or is selected from -(C1-C4)alkylene and -(C1-C4)cycloalkylene;

[0128] A is a ring selected from aryl and heteroaryl groups, wherein any aryl or heteroaryl group may optionally be substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C4)alkyl; provided that any aryl group is not substituted by -(C1-C4)alkyl when L1 is a bond.

[0129] In a preferred embodiment, R1 and R2 are fused together to form a -(C6-C7) heterocyclic alkyl group, wherein the -(C6-C7) heterocyclic alkyl group is optionally substituted by one or more groups selected from -OR8, -(C1-C6) alkyl and -(C3-C7) heterocyclic alkyl groups.

[0130] In yet another preferred embodiment, the present invention relates to compounds of formula (I) as defined above.

[0131] Where Z is S and Y is CH or Z is CH and Y is S; R3 is H or methyl, and R1, R2, R5, R6, R7, R8, L1, A are as defined above.

[0132] In a preferred embodiment, the present invention relates to at least one of the compounds listed in Table 1 below, in zwitterionic form or a pharmaceutically acceptable salt thereof.

[0133] Table 1 - List of Preferred Compounds

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] It should be understood that all individual enantiomers, diastereomers and mixtures thereof in any proportion of the compounds of formula (I) of the present invention are covered within the scope of the present invention.

[0150] In a preferred embodiment, the present invention relates to compounds of formula (I) as NaV inhibitors. In this regard, compounds of formula (I) of the present invention have been found to have a pIC (p-IC value) representing a NaV 1.7 receptor. 50 The efficacy of an inhibitory drug is equal to or greater than 5.

[0151] Preferably, the compounds of the present invention have a pIC ratio of 5 to 5.5 to NaV 1.7. 50 More preferably, the compounds of the present invention have a pIC value of 5.5 or higher relative to NaV 1.7. 50 Even more preferred are compounds of the present invention having a pIC of 5.5 to 6 against NaV 1.7. 50 .

[0152] The present invention also relates to pharmaceutical compositions comprising a compound of formula (I) in the form of a zwitterionic or pharmaceutically acceptable salt, and mixed therewith at least one or more pharmaceutically acceptable carriers and / or excipients.

[0153] As used in this application, "effective amount" of a compound of formula (I) (in zwitterionic or pharmaceutically acceptable salt form) or other pharmaceutically active agent means an amount of compound sufficient to treat a patient's condition but low enough to avoid serious side effects, and which can still be routinely determined by those skilled in the art.

[0154] Compounds of formula (I) may be administered once or according to a dosage regimen in which multiple doses are administered at varying time intervals for a given duration. Typical daily doses may vary depending on the chosen route of administration.

[0155] In one embodiment, the present invention relates to pharmaceutical compositions of a compound of formula (I) mixed with one or more pharmaceutically acceptable carriers or excipients (e.g., those described in Remington's Pharmaceutical Sciences Handbook, XVII Ed., Mack Pub., NY, USA).

[0156] In therapeutic applications, compounds of formula (I) may be administered via any convenient, suitable, or effective route. Administration of the compounds of the present invention and their pharmaceutical compositions may be achieved as needed by the patient, for example, orally, nasally, parenterally (subcutaneously, intravenously, intramuscularly, intrasternalally, and by infusion), and by inhalation.

[0157] Preferably, the compounds of the present invention are administered orally or by inhalation.

[0158] In a more preferred embodiment, the compound of the present invention or a pharmaceutical composition thereof is administered by inhalation.

[0159] In yet another preferred embodiment, the compound of the present invention or a pharmaceutical composition thereof is administered orally.

[0160] In a preferred embodiment, the pharmaceutical composition comprising a compound of formula (I) in the form of a zwitterion or a pharmaceutically acceptable salt is a solid oral dosage form such as tablets, gel caps, capsules, tablets, granules, sugar tablets, and packaged powders.

[0161] The compounds of the present invention can be applied alone or in combination with various pharmaceutically acceptable carriers, diluents (e.g., sucrose, mannitol, lactose, starch) and known excipients, including suspending agents, solubilizers, buffers, binders, disintegrants, preservatives, colorants, flavoring agents, lubricants, etc.

[0162] In yet another embodiment, the pharmaceutical composition comprising the compound of formula (I) is a liquid oral dosage form, such as a solution of water and non-aqueous substances, emulsion, suspension, or syrup. Such liquid dosage forms may also comprise a suitable known inert diluent (e.g., water) and suitable known excipients (e.g., preservatives, wetting agents, sweeteners, flavoring agents, etc.), as well as reagents for emulsifying and / or suspending the compounds of the present invention.

[0163] In another embodiment, the pharmaceutical composition comprising the compound of formula (I) is an inhalable formulation such as an inhalable powder, a metered-dose aerosol containing propellant, or an inhalable formulation without propellant. The inhalable formulation can be administered via a suitable inhalation device, selected from dry powder inhalers, pressurized metered-dose inhalers, or nebulizers.

[0164] In yet another embodiment, the invention also relates to an apparatus comprising a pharmaceutical composition comprising a compound of formula (I) according to the invention (obtained according to the invention as described above) in the form of a zwitterionic or pharmaceutically acceptable salt, in the form of a single-dose or multi-dose dry powder inhaler or metered-dose inhaler.

[0165] For administration as a dry powder, single-dose or multi-dose inhalers known in the art can be used. In this case, the powder can be filled into gelatin, plastic, or other capsules, cartridges, or blister packs or stored in a reservoir.

[0166] A chemically inert diluent or carrier, such as lactose, or any other additive suitable for improving absorbable fractions, can be added to the powdered compounds of the present invention.

[0167] Inhalation aerosols containing propellant gases such as hydrofluorocarbons may contain the compounds of this invention in solution or dispersion form. Propellant-driven formulations may also contain other components, such as cosolvents, stabilizers, and optional other excipients.

[0168] Propellant-free inhalable formulations containing the compounds of the present invention may be in the form of solutions or suspensions in water, alcohol, or water / alcohol media, and they may be delivered by jet atomizers or ultrasonic atomizers known from the prior art or by soft atomizers.

[0169] The compounds of this invention can be administered as the sole active agent or in combination with other pharmaceutical active ingredients.

[0170] The dosage of the compounds of this invention depends on various factors, including, in particular, the specific disease to be treated, the severity of symptoms, the route of administration, etc.

[0171] In another aspect, the present invention relates to the use of compounds of formula (I) in the preparation of pharmaceutical products. In yet another aspect, the present invention relates to the use of compounds of formula (I) as pharmaceutical products.

[0172] Therefore, the present invention relates to the use of compounds of formula (I) for the preparation of pharmaceuticals, preferably for the treatment of disorders related to voltage-gated sodium channel (NaV) receptor mechanisms.

[0173] In yet another embodiment, the present invention relates to compounds of formula (I) for the prevention and / or treatment of diseases, disorders or conditions associated with voltage-gated sodium channel (NaV) dysregulation.

[0174] In one aspect, the invention also relates to a method for preventing and / or treating disorders related to the NaV receptor mechanism, the method comprising administering a therapeutically effective amount of a compound of formula (I) to a patient requiring treatment.

[0175] Preferably, the compounds of the present invention are used to treat and / or prevent respiratory diseases.

[0176] 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).

[0177] In another aspect, the present invention relates to pharmaceutical compositions comprising a compound of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients for the prevention and / or treatment of respiratory diseases.

[0178] In yet another embodiment, the present invention provides a method for preventing and / or treating respiratory diseases, the method comprising administering a pharmaceutical composition containing a compound of formula (I).

[0179] On the other hand, the respiratory diseases mentioned above are selected from cough, subacute or chronic cough, treatment-resistant cough, idiopathic chronic cough, intractable chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD, and cough associated with respiratory diseases such as COPD, asthma, and bronchospasm.

[0180] In one embodiment, the present invention relates to the use of a compound of formula (I) or a pharmaceutical composition thereof for the preparation of a medicament for the treatment and / or prevention of cough, subacute or chronic cough, treatment of resistant cough, idiopathic chronic cough, intractable chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

[0181] In another embodiment, the present invention relates to a compound or pharmaceutical composition of formula (I) for the prevention and / or treatment of respiratory diseases, wherein the respiratory diseases are selected from: cough, subacute or chronic cough, treatment-resistant cough, idiopathic chronic cough, intractable chronic cough, post-viral cough, iatrogenic cough, asthma, IPF, COPD and cough associated with respiratory diseases such as COPD, asthma and bronchospasm.

[0182] In another embodiment, the present invention relates to a method for treating and / or preventing respiratory diseases selected from cough, subacute or chronic cough, treatment-resistant cough, idiopathic chronic cough, intractable 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 a pharmaceutical composition of a compound of formula (I) or a compound of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients.

[0183] In a preferred embodiment, the respiratory disease mentioned above is chronic cough.

[0184] In yet another preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutical composition thereof for the prevention and / or treatment of chronic cough.

[0185] In yet another preferred embodiment, the present invention relates to the use of a compound of formula (I) or a pharmaceutical composition thereof for the preparation of a medicament for the treatment and / or prevention of chronic cough.

[0186] In yet another preferred embodiment, the present invention relates to a method for treating and / or preventing chronic cough, the method comprising a pharmaceutical composition of a compound of formula (I) or a compound of formula (I) and one or more pharmaceutically acceptable carriers and / or excipients.

[0187] Compounds of formula (I) can be administered to mammals, particularly humans, using any suitable route of administration.

[0188] The dosage of the compound of formula (I) for prevention or treatment will, of course, vary depending on the severity of the condition being treated and the route of administration, and will generally be determined through clinical trials required in the pharmaceutical field. It will also vary based on the individual patient's age, weight, and response.

[0189] All preferred groups or embodiments described above for compounds of formula (I) can also be combined with each other and applied with necessary modifications.

[0190] The compounds of this invention can be prepared in a variety of ways known to those skilled in the art of organic synthesis. It should be understood that the functionalities present on the molecule should correspond to the transformations mentioned. Sometimes this will require modification of the order of synthetic steps to obtain the desired compound of this invention. Although optimal reaction conditions can vary depending on the specific reactants or solvents used, these conditions can be readily determined by those skilled in the art through conventional optimization methods. Therefore, the processes described below should not be considered as limiting the scope of synthetic methods that can be used to prepare the compounds of this invention. In some cases, in accordance with general chemical principles, generally known protecting groups (PGs) can be used when necessary to mask or protect sensitive or reactive moieties (Protective group in organic syntheses, 3rd ed. TW Greene, PGM Wuts).

[0191] Compounds of formula (I), including all the compounds listed above, can generally be prepared according to the procedures described in schemes 1 to 2 below, wherein at least one non-limiting synthetic route is provided for the preparation of the illustrative compounds (i.e., examples).

[0192] Option 1

[0193]

[0194] In one embodiment of the present invention, compound (I) can be prepared from compounds (VI) and (VIII) according to scheme 1.

[0195] Compound (VI) can be prepared from compound (II). Compound (V) can be obtained from compound (II) via a two-step procedure: step a) involves acylation with a suitable haloacetyl halide (III), wherein X1 and X2 are halogens, preferably and independently chlorides or bromides. Step b) involves alkylation with a suitable amine NHR1R2 (IV). Compound (VI) can then be prepared from compound (V) via a two-step procedure: step c) involves ester hydrolysis mediated by a base such as NaOH or KOH or an acid such as HCl or H2SO4. The subsequent step d) involves amide formation with a suitable amine NHR5R6 (IX) in the presence of a suitable coupling agent such as chloro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (TCFH). Compound (VIII) can be prepared from compound (VII) by acylation with a suitable haloacetyl halide (III), wherein X1 and X2 are halogens, preferably and independently chlorides or bromides. Compound (I) can then be obtained from compounds (VI) and (VIII) by a nitrogen quaternization reaction (Menshutkin reaction).

[0196] Option 2

[0197]

[0198] In another embodiment of the present invention, the compound of formula (I) can be prepared according to scheme 2.

[0199] Compound (X) can be prepared from compound (V) via a two-step procedure: step a) involves an ester hydrolysis mediated by a base such as NaOH or KOH or an acid such as HCl or H₂SO₄, and step b) involves the introduction of a suitable carboxylic acid ester protecting group (=PG), such as tert-butyl. Compound (XI) can be prepared by reacting compound (X) and compound (VIII) via a nitrogen quaternization reaction (Menshutkin reaction). Compound (I) can then be obtained from compound (XI) via a two-step procedure: step c) involves a carboxylic acid protecting group removal reaction, and step d) involves an amide formation reaction with a suitable amine NHR5R6 (IX) in the presence of a suitable coupling agent such as TCFH.

[0200] Option 3

[0201]

[0202] In another embodiment of the invention, the compound of formula (I) can be prepared according to scheme 3. Compound (XIV) can be prepared from compound (XII): by an amide formation reaction with a suitable amine NHR5R6 (IX) in the presence of a suitable coupling agent such as TCFH. Compound (XVI) can be prepared from compound (XIV): by an acylation reaction with a suitable haloacetyl halide (III), wherein X1 and X2 are halogens, preferably and independently chlorides or bromides. Compound (XV) can be prepared from compound (VIII): by an alkylation reaction with a suitable amine NHR1R2 (IV). Finally, the compound of formula (I) can be prepared from compounds (XV) and (XVI) by an alkylation reaction.

[0203] Accordingly, the present invention provides intermediate compounds (V), (VI), (VIII), (X), (XI), (XIV), (XV) and (XVI) as defined above, and their use in preparing compounds of formula (I).

[0204] In another aspect, the present invention provides the use of intermediate compounds selected from compounds (V), (VIII), (X), (XI), (XV) and (XVI) as defined above (especially in schemes 1, 2 and 3) for the preparation of compounds of formula (I) as defined above.

[0205] In cases where compounds of formula (I) are separated by precipitation followed by grinding or crystallization from the reaction mixture, they are generally obtained as salts, wherein the anion is either a chloride anion or a bromide anion, depending on the meaning of X2 in compounds (III), (VIII), and (XVI) used to form the quaternary ammonium salt (see schemes 1, 2, and 3). Compounds of formula (I) in salt form can be obtained as zwitterions by subsequent treatment under basic conditions (e.g., purification by silica gel chromatography with amino-functionalized silica gel). The zwitterions of compounds of formula (I) can be treated with acids such as hydrochloric acid to obtain the corresponding salts of compounds of formula (I).

[0206] The preparation methods are illustrated in the experimental section below.

[0207] Preparation of intermediates and examples

[0208] The chemical names of compounds are generated using the PerkinElmer ChemDraw® Professional Edition application (v. 20.0.0.41.) Structure-to-Name Tool or generic chemical names. All reagents not described in the experimental section are commercially available or are known compounds or can be formed from known compounds by methods known to those skilled in the art.

[0209] In the following method, some of the raw materials are identified by the numbers indicated in the "Intermediates" or "Examples" steps. This is provided solely to assist chemists in the art.

[0210] When references are made to the use of "similar" or "analogous" procedures, those skilled in the art will recognize that the above procedures may involve minor variations that will be apparent to them, such as reaction temperature, reagent / solvent volume, reaction time, post-treatment conditions, or chromatographic purification conditions. All final compounds are obtained as pharmaceutically acceptable salts unless otherwise stated.

[0211] All reported intermediates and embodiments were obtained via LC-MS and / or as described herein. 1 H-NMR analysis characterization reveals that the optimal proton frequency and solvent conditions can be varied and easily determined by those skilled in the art through conventional optimization procedures.

[0212] abbreviation

[0213] ACN = Acetonitrile; AMU = Atomic mass unit; CDCl3 = Deuterated chloroform; CyHex = Cyclohexane; DCM = Dichloromethane; DMF = Dimethylformamide; DMSO = Dimethyl sulfoxide; DMSO-d6 = Deuterated dimethyl sulfoxide; EGTA = Ethylene glycol-di(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; Et2O = Diethyl ether; EtOAc = Ethyl acetate; eq. = Equivalent; FCC = Rapid column chromatography; h = Hour; HATU = 1-[Di(dimethylamino)methylene]-1H-1,2,3-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 = Methanol; min = Minute; NMR = Nuclear magnetic resonance; RT / rt = Room temperature; t R =Retention time; ss =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-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II)methanesulfonate.

[0214] Analytical methods

[0215] NMR characterization:

[0216] 1H-NMR spectra were obtained as follows: on a 400 Agilent INOVA, 500 Agilent VNMRS, or 600 Agilent INOVA spectrometer, at 400, 500, and 600 MHz (proton frequency), or on a Bruker Avance III 400 spectrometer.

[0217] Chemical shifts are reported as δ values ​​in ppm relative to the internal standard tetramethylsilane (TMS). Coupling constants (J values) are provided in Hertz (Hz) and multiplicity is reported using the following abbreviations (s = singlet, d = doublet, t = triplet, quin = quintet, m = multiplet, br = broad).

[0218] Information about the zwitterionic or salt form of compound (I) is obtained through 1 ¹H-NMR spectroscopy reveals that the zwitterionic form generally shows a missing amide NH signal. HBr or HCl salts generally show the full amide NH signal. Formate (i.e., formic acid) is obtained through the reaction of formic acid... 1 H-NMR signal examination and quantification.

[0219] LC-MS:

[0220] LCMS can record under the following conditions: Waters LC / PDA / MS Acquity TM System coupling MicromassZQ TM Alternatively, on a Waters SQD single quadrupole mass spectrometer, operating in positive and / or negative electron spray (ES) ionization mode, diode array (DAD) chromatograms, mass spectrometry chromatograms, and mass spectra can be acquired.

[0221] LC / UV / MS characterization:

[0222] It is estimated that the LC / MS retention time (t) R The time is affected by experimental error by ±0.5 minutes.

[0223] LC-MS Method 1: Acquity CSH C18 2.1 x 50 mm 1.7 μm column, column temperature 40 °C; mobile phase solvent A is milliQ water + 0.1% HCOOH, mobile phase solvent B is ACN + 0.1% HCOOH. Flow rate is 1 mL / min.

[0224] The gradient table is 97% A 3% B at t=0 min, 0.1% A 99.9% B at t=1.5 min, 0.1% A 99.9% B at t=1.9 min, and 97% A 3% B at t=2 min. The UV detection range is 210-350 nm, while the ES+ / ES- range is 100 to 1000 AMU.

[0225] LC-MS Method 2: Column: Kinetex EVO C18 2.1 x 50 mm 1.7 μm, column temperature: 40 °C; mobile phase solvent A: 10 mM NH4HCO3 aqueous solution (adjusted to pH=10 with ammonia); mobile phase solvent B: ACN. Flow rate: 1 mL / min. Gradient table: 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. UV detection range: 210–350 nm; ES+ / ES- range: 100–1200 AMU.

[0226] Purification methods

[0227] Purification of some compounds was achieved by semi-preparative HPLC: using an MDAP Waters instrument with mass spectrometry detection (MS: ZQ2000) and DAD detection 220, equipped with a CSH C18 (30 x 100 mm, 5 µm) column and a 1 mL injection loop. The gradient was ACN / H2O + 0.1% HCOOH from 50% to 70% over 10 min (flow rate = 40.00 mL / min.), used as the mobile phase.

[0228] Other compounds were purified by FCC using Biotage® columns and the conditions specified in each example.

[0229] General synthesis procedure

[0230] Intermediate 1: Methyl 3-(2-bromoacetamido)-4-methylthiophene-2-carboxylic acid ester

[0231]

[0232] 2-Bromoacetyl bromide (6.11 mL, 71.0 mmol) was added dropwise to a suspension of methyl 3-amino-4-methylthiophene-2-carboxylic acid (10.0 g, 58.4 mmol) in water (235 mL) at 0 °C, and a creamy white solid was observed to form. The reaction was allowed to warm to RT and stirred overnight. The solid was filtered, washed with water, and dried under high vacuum to give the title compound (intermediate 1: 16.0 g, 55.0 mmol, 94% yield) as a creamy white powder.

[0233] LC-MS Method 1: t R = 0.81 minutes, MS (ESI) m / z = 292.0 / 294.0 [M+H]+

[0234] Intermediate 2: 2-Bromo-N-(isoxazo-3-yl)acetamide

[0235]

[0236] 2-Bromoacetyl bromide (1.82 mL, 20.9 mmol) was added dropwise to a DCM suspension of potassium carbonate (6.58 g, 47.6 mmol) and isoxazol-3-amine (1.44 mL, 19.0 mmol) (73 mL). After 3 hours, the reaction mixture was washed with water (2x) and then with a saturated aqueous solution of NaHCO3. The organic phase was separated, dried and concentrated under reduced pressure on a phase separator to provide a crude product, which was purified by FCC (KP Sfär silica, 10-80% EtOAc / cyclohexane) to provide the title compound (intermediate 2: 1.60 g, 7.80 mmol, 41% yield) as an orange solid.

[0237] LC-MS Method 1: t R = 0.53 minutes, MS (ESI) m / z = 205.0 / 207.0 [M+H]+

[0238] Intermediate 3: (R)-2-bromo-N-(1-phenylethyl)acetamide

[0239]

[0240] Similar to intermediate 1, intermediate 3 is prepared as follows: starting from a suitable amine:

[0241] (R)-1-Phenylacetyl-1-amine: 1.00 g, 8.25 mmol (1.0 equivalent)

[0242] Yield / Quantity: 1.69 g, 7.00 mmol, 84% yield.

[0243] LC-MS Method 2: t R = 0.83 minutes, MS (ESI) m / z = 242.0 / 244.0 [M+H]+

[0244] Example 1: 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethyl-1-ammonium (zwitterion)

[0245] Step 1 - Methyl 3-(2-chloroacetamido)-4-methylthiophene-2-carboxylic acid ester (Intermediate 4)

[0246]

[0247] 2-Chloroacetyl chloride (9.30 mL, 116 mmol) was added dropwise to an aqueous solution of methyl 3-amino-4-methylthiophene-2-carboxylate (5.00 g, 29.0 mmol) at 0 °C (117 mL). The reaction was allowed to warm to RT overnight, and a white precipitate was observed to form. The precipitate was filtered and washed with water (3x) and pentane (2x) to provide the title compound (intermediate 4: 7.40 g, quantitative yield) as a creamy white powder.

[0248] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 248.0 [M+H]+

[0249] Step 2 - Methyl 3-(2-(dimethylamino)acetamido)-4-methylthiophene-2-carboxylic acid (Intermediate 5)

[0250]

[0251] Dimethylamine (2.0 M, in THF) (2.42 mL, 4.84 mmol) was added to a solution of methyl 3-(2-chloroacetamido)-4-methylthiophene-2-carboxylic acid intermediate 4 (1.00 g, 4.0 mmol) and sodium bicarbonate (0.68 g, 8.1 mmol) in a vessel (10 mL) under stirring at RT. The mixture was stirred overnight at RT. Water was added, and the reaction was extracted with EtOAc (3x). The collected organic layer was dried on Na2SO4, filtered, and concentrated to provide the title compound (intermediate 5: 0.91 g, 3.6 mmol, 88% yield), which is a yellow oil.

[0252] LC-MS Method 1: t R = 0.38 minutes, MS (ESI) m / z = 257.1 [M+H]+

[0253] Step 3 - 2-(dimethylamino)-N-(4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)acetamide (intermediate 6)

[0254]

[0255] Four balls were loaded into a 10 mL ball mill reactor with pyrrolidine (64 mL, 0.78 mmol), potassium tert-butoxide (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 sealed and shaken at 13 Hz for 4 hours. The reactor was then opened and water and EtOAc were added, the reactor was sealed, and shaken. The organic layers were separated, and the aqueous layer was extracted with EtOAc (2x). The collected organic layers were dried on Na2SO4, filtered, and concentrated to provide the title compound (intermediate 6: 104 mg, 0.35 mmol, 90% yield).

[0256] LC-MS Method 1: t R = 0.43 minutes, MS (ESI) m / z = 296.1 [M+H]+

[0257] Step 4 - 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethyl-1-ammonium (zwitterion) (Example 1)

[0258] Potassium iodide (3.8 mg, 0.020 mmol) was added to a stirred solution of N-benzyl-2-chloroacetamide (46 mg, 0.25 mmol) and 2-(dimethylamino)-N-(4-methyl-2-(pyrrolidine-1-carbonyl)thiophene-3-yl)acetamide (intermediate 6, 70 mg, 0.23 mmol) in ACN (0.8 mL). The reaction was stirred overnight at 80 °C. After this time, the reaction was cooled to RT and concentrated under high vacuum. The residue was purified by FCC (Sfäramino, 0-5% MeOH / DCM) to provide the title compound (Example 1: 35 mg, 0.079 mmol, 32% yield) as a pale yellow powder.

[0259] LC-MS Method 2: t R = 0.98 minutes, MS (ESI) m / z = 443.2 [M]+

[0260] 1H NMR (400 MHz, CDCl3)δppm 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, 4H), 2.13 (s, 3 H), 1.85 (br s, 4 H).

[0261] Example 2: 2-(benzylamino)-N-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium carbamate

[0262] Step 1 - Sodium chloride of 3-(2-(dimethylamino)acetamido)-4-methylthiophene-2-carboxylic acid (intermediate 7)

[0263]

[0264] To a solution of intermediate 5 (180 mg, 0.700 mmol) in THF (2 mL), water (0.4 mL) and a 2 M aqueous solution of sodium hydroxide (0.42 mL, 0.84 mmol) were added, and the reaction was heated at 50 °C for 16 h. The reaction was then cooled to 0 °C and neutralized by adding a 2 M aqueous solution of HCl until pH = 7. The volatiles were removed under reduced pressure, providing the title compound (intermediate 7: 230 mg, 92% wt, quantitative yield) as an orange solid.

[0265] LC-MS Method 1: t R = 0.32 minutes, MS (ESI) m / z = 243.1 [M+H]+

[0266] Step 2 - 3-(2-(dimethylamino)acetamido)-N,N,4-trimethylthiophene-2-carboxamide (Intermediate 8)

[0267]

[0268] HATU (315 mg, 0.83 mmol) was added to a DMF (2 mL) solution of intermediate 7 (230 mg, 92% wt, 0.70 mmol). After stirring for 5 min, a 2 M THF solution of dimethylamine (0.69 mL, 1.38 mmol) was added. The reaction was stirred overnight at RT. An aqueous solution of NaOH at pH = 9 was added, and the reaction was extracted with EtOAc (3x). The collected organic layer was dried on Na2SO4, filtered, and concentrated to provide a crude product, which was purified by FCC (Sfär amino, 1% MeOH / DCM) to provide the title compound (intermediate 8: 176 mg, 0.65 mmol, 93% yield).

[0269] LC-MS Method 1: t R = 0.36 minutes, MS (ESI) m / z = 270.1 [M+H]+

[0270] Step 3 - 2-(benzylamino)-N-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium carbamate (Example 2)

[0271] Similar to Example 1, Example 2 was prepared as follows: starting from a suitable amine and chloride.

[0272] Intermediate 8: 176 mg, 0.65 mmol (1.0 equivalent)

[0273] N-Benzyl-2-chloroacetamide: 120 mg, 0.65 mmol (1.0 equivalent)

[0274] Purified by FCC (Sfär-C18, 10-30% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0275] Dosage / Yield: 9.9 mg, 0.021 mmol, 3% yield.

[0276] LC-MS Method 2: t R = 0.87 minutes, MS (ESI) m / z = 417.5 [M]+

[0277] 1H NMR (600 MHz, CDCl3) δppm 11.92 (br s, 1 H), 9.13 (s, 1 H), 8.66 (br s, 1H), 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).

[0278] Example 3: 2-(benzylamino)-N-(2-((2-(tert-butylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium carbamate

[0279] Step 1 - N-(tert-butyl)-3-(2-(dimethylamino)acetamido)-4-methylthiophene-2-carboxamide (Intermediate 9)

[0280]

[0281] Similar to intermediate 8, intermediate 9 is prepared as follows: starting from suitable carboxylic acids and amines:

[0282] Intermediate 7: 150 mg, 0.62 mmol (1.0 equivalent)

[0283] 2-Methyl-2-propylamine: 0.33 mL, 3.1 mmol (5 equivalents)

[0284] Dosage / Yield: 140 mg, 0.47 mmol, 76% yield.

[0285] LC-MS Method 1: t R = 0.45 minutes, MS (ESI) m / z = 298.1 [M+H]+

[0286] Step 2 - 2-(benzylamino)-N-(2-((2-(tert-butylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium carbamate (Example 3)

[0287] N-benzyl-2-bromoacetamide (118 mg, 0.52 mmol) was added to a 1.7 mL solution of intermediate 9 (140 mg, 0.47 mmol) in ACN. The reaction was stirred overnight at 80 °C. The residue was purified by a semi-preparative HPLC method to provide the title compound (Example 3: 10 mg, 0.021 mmol, 4% yield).

[0288] LC-MS Method 2: t R = 0.66 minutes, MS (ESI) m / z = 445.2 [M]+

[0289] 1 H NMR (400 MHz, CDCl3) δppm 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, 2H), 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).

[0290] Example 4: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0291] Step 1 - Methyl 3-(2-(azacycloheptane-1-yl)acetamido)-4-methylthiophene-2-carboxylate (Intermediate 11)

[0292]

[0293] To a stirred solution of intermediate 1 (2.0 g, 6.78 mmol) in ACN (27 mL), perpiperidine (0.99 mL, 8.81 mmol) and potassium carbonate (1.21 g, 8.81 mmol) were added, and the mixture was stirred at RT for 16 h. The reaction was filtered to remove salts and concentrate the organic phase. The residue was dissolved in EtOAc and water. The phases were mixed in an extractor, the organic layer was separated, and the aqueous layer was extracted with EtOAc (2x). The collected organic layer was dried over Na2SO4, filtered, and concentrated to provide the title compound (intermediate 11: 1.77 g, 5.69 mmol, 84% yield), an orange oil.

[0294] LC-MS Method 1: t R= 0.48 minutes, MS (ESI) m / z = 311.2 [M+H]+

[0295] Step 2 - 2-(azacycloheptan-1-yl)-N-(4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)acetamide (intermediate 12)

[0296]

[0297] Similar to intermediate 6, intermediate 12 is prepared as follows: starting from suitable amines and esters:

[0298] Intermediate 11: 200 mg, 0.64 mmol (1.0 equivalent)

[0299] Morpholine: 0.11 mL, 1.29 mmol (2.0 equivalent)

[0300] Potassium tert-butoxide: 65 mg, 0.58 mmol (0.9 equivalents)

[0301] Dosage / Yield: 182 mg, 0.5 mmol, 77% yield.

[0302] LC-MS Method 1: t R = 0.36 minutes, MS (ESI) m / z = 366.2 [M+H]+

[0303] Step 3 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 4)

[0304] To a stirred solution of N-benzyl-2-bromoacetamide (41 mg, 0.18 mmol) in 0.9 mL of ACN, 2-(azacycloheptan-1-yl)-N-(4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)acetamide (intermediate 12, 60 mg, 0.16 mmol) was added. The reaction was stirred overnight at 80 °C. The reaction was cooled and concentrated under high vacuum. The residue was purified by FCC (Sfäramino, 1-5% MeOH / DCM) to provide the title compound (Example 4: 42 mg, 0.082 mmol, 45% yield).

[0305] LC-MS Method 1: t R = 0.63 minutes, MS (ESI) m / z = 513.3 [M]+

[0306] 1H NMR (500 MHz, DMSO-d6) δppm 9.13 - 10.77 (m, 1 H), 7.19 - 7.38 (m, 5H), 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, 7H), 1.62 (br s, 4 H).

[0307] Example 5: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0308] Step 1 - Sodium chloride of 3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid (intermediate 13)

[0309]

[0310] Similar to intermediate 7, intermediate 13 is prepared as follows: starting from the corresponding ester:

[0311] Intermediate 11: 5.0 g, 16.1 mmol (1.0 equivalent)

[0312] Sodium hydroxide (2 M, in water): 9.7 mL, 19.3 mmol (1.2 equivalents)

[0313] Yield / Quantity: 6.3 g, 90% wt, quantitative yield.

[0314] LC-MS Method 1: t R = 0.43 minutes, MS (ESI) m / z = 297.2 [M+H]+

[0315] Step 2 - 3-(2-(azacycloheptane-1-yl)acetamido)-N,4-dimethylthiophene-2-carboxamide (Intermediate 14)

[0316]

[0317] Similar to intermediate 8, intermediate 14 is prepared as follows: starting from the corresponding acid and amine.

[0318] Intermediate 13: 284 mg, 0.80 mmol (1.0 equivalent)

[0319] Methylamine 2 M, in THF: 0.80 mL, 1.60 mmol (2.0 equivalents)

[0320] Purified by FCC (Sfär amino, 20% EtOAc / DCM)

[0321] Yield / Quantity: 212 mg, 0.69 mmol, 86% yield

[0322] LC-MS Method 1: t R = 0.38 minutes, MS (ESI) m / z = 310.2 [M+H]+

[0323] Step 3 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 5)

[0324] Similar to Example 4, Example 5 was prepared as follows: starting from the corresponding bromide and amine.

[0325] Intermediate 14: 70 mg, 0.23 mmol (1.0 equivalent)

[0326] N-Benzyl-2-bromoacetamide: 52 mg, 0.23 mmol (1.0 equivalent)

[0327] Purification via FCC (Sfär amino, 10-20% EtOAc / DCM)

[0328] Dosage / Yield: 34 mg, 0.074 mmol, 32% yield

[0329] LC-MS Method 1: t R = 0.59 minutes, MS (ESI) m / z = 457.3 [M]+

[0330] 1 H NMR (400 MHz, DMSO-d6) δppm 9.19 (t, J=5.7 Hz, 1 H), 8.99 (q, J=4.4 Hz, 1H), 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).

[0331] Example 6: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0332] Step 1 - 2-(azacycloheptan-1-yl)-N-(4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)acetamide (Intermediate 15)

[0333]

[0334] Similar to intermediate 6, intermediate 15 is prepared as follows: starting from the corresponding amine and ester.

[0335] Intermediate 11: 200 mg, 0.63 mmol (1.0 equivalent)

[0336] Pyrrolidine: 0.10 mL, 1.26 mmol (2.0 equivalent)

[0337] Potassium tert-butoxide: 64 mg, 0.57 mmol (0.9 equivalents)

[0338] Yield / Quantity: 134 mg, 0.38 mmol, 61% yield

[0339] LC-MS Method 1: t R = 0.51 minutes, MS (ESI) m / z = 350.2 [M+H]+

[0340] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 6)

[0341] Similar to Example 4, Example 6 was prepared as follows: starting from the corresponding bromide and amine.

[0342] Intermediate 15: 70 mg, 0.20 mmol (1.0 equivalent)

[0343] N-Benzyl-2-bromoacetamide: 58 mg, 0.22 mmol (1.0 equivalent)

[0344] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0345] Dosage / Yield: 60 mg, 0.10 mmol, 52% yield

[0346] LC-MS Method 1: t R = 0.70 minutes, MS (ESI) m / z = 497.2 [M]+

[0347] 1 H NMR (400 MHz, DMSO-d6) δppm 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).

[0348] Example 7: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0349] Similar to Example 4, Example 7 was prepared as follows: starting from the corresponding bromide and amine.

[0350] Intermediate 15: 70 mg, 0.20 mmol (1.0 equivalent)

[0351] Intermediate 2: 58 mg, 0.26 mmol (1.3 equivalents)

[0352] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0353] Dosage / Yield: 33 mg, 0.069 mmol, 35% yield.

[0354] LC-MS Method 1: t R = 0.58 minutes, MS (ESI) m / z = 474.2 [M]+

[0355] 1H NMR (400 MHz, CDCl3) δppm 8.20 (d, J=1.4 Hz, 1 H), 6.96 (s, 1 H), 6.89 (d, J=1.4 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).

[0356] Example 8: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0357] Similar to Example 4, Example 8 was prepared as follows: starting from the corresponding bromide and amine.

[0358] Intermediate 12: 60 mg, 0.16 mmol (1.0 equivalent)

[0359] Intermediate 2: 44 mg, 0.21 mmol (1.3 equivalents)

[0360] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0361] Dosage / Yield: 11 mg, 0.022 mmol, 14% yield.

[0362] LC-MS Method 1: t R = 0.51 minutes, MS (ESI) m / z = 490.2 [M]+

[0363] 1 H NMR (400 MHz, CDCl3)δppm 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).

[0364] Example 9: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate

[0365] Similar to Example 4, Example 9 was prepared as follows: starting from the corresponding bromide and amine.

[0366] Intermediate 14: 70 mg, 0.23 mmol (1.0 equivalent)

[0367] Intermediate 2: 60 mg, 0.29 mmol (1.3 equivalents)

[0368] Purification via FCC (Sfär-C18, 10%-25% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0369] Dosage / Yield: 14 mg, 0.029 mmol, 13% yield.

[0370] LC-MS Method 1: t R = 0.46 minutes, MS (ESI) m / z = 434.2 [M]+

[0371] 1 H NMR (500 MHz, CDCl3) δppm 8.65 (s, 1 H), 8.25 (d, J=1.1 Hz, 1 H), 7.96 (brs, 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).

[0372] Example 10: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0373] Step 1 - 3-(2-(azacycloheptane-1-yl)acetamido)-N,N,4-trimethylthiophene-2-carboxamide (Intermediate 16)

[0374]

[0375] Similar to intermediate 8, intermediate 16 is prepared as follows: starting from the corresponding acid and amine.

[0376] Intermediate 13: 250 mg, 0.70 mmol (1.0 equivalent)

[0377] N-methylmethylamine 2 M, in THF: 0.70 mL, 1.41 mmol (2.0 equivalent)

[0378] Purification via FCC (Sfäramino, 20-40% EtOAc / cyclohexane)

[0379] Dosage / Yield: 157 mg, 0.49 mmol, 69% yield.

[0380] LC-MS Method 1: t R = 0.44 minutes, MS (ESI) m / z = 324.2 [M+H]+

[0381] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 10)

[0382] Similar to Example 4, Example 10 was prepared as follows: starting from the corresponding bromide and amine.

[0383] Intermediate 16: 77 mg, 0.24 mmol (1.0 equivalent)

[0384] N-Benzyl-2-bromoacetamide: 54 mg, 0.24 mmol (1.0 equivalent)

[0385] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0386] Dosage / Yield: 65 mg, 0.14 mmol, 58% yield

[0387] LC-MS Method 1: t R = 0.64 minutes, MS (ESI) m / z = 471.2 [M]+

[0388] 1H NMR (400 MHz, CDCl3) δppm 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).

[0389] Example 11: 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0390] Similar to Example 4, Example 11 was prepared as follows: starting from the corresponding bromide and amine.

[0391] Intermediate 16: 77 mg, 0.24 mmol (1.0 equivalent)

[0392] Intermediate 2: 59 mg, 0.29 mmol (1.2 equivalents)

[0393] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0394] Dosage / Yield: 22 mg, 0.049 mmol, 20% yield.

[0395] LC-MS Method 1: t R = 0.50 minutes, MS (ESI) m / z = 448.2 [M]+

[0396] 1 H NMR (500 MHz, CDCl3)δppm 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=1.9 Hz, 4 H), 1.77 (br s, 4 H).

[0397] Example 13: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onthionyl carboxylate

[0398] Step 1 - Methyl 4-methyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxylic acid (Intermediate 17)

[0399]

[0400] Similar to intermediate 11, intermediate 17 is prepared as follows: starting from the corresponding bromide and amine.

[0401] Intermediate 1: 2.0 g, 6.85 mmol (1.0 equivalent)

[0402] Piperidine: 0.88 mL, 8.9 mmol (1.3 equivalents)

[0403] Potassium carbonate: 1.23 g, 8.9 mmol (1.3 equivalents)

[0404] Yield / Quantity: 1.3 g, 4.5 mmol, 65% yield

[0405] LC-MS Method 1: t R = 0.45 minutes, MS (ESI) m / z = 297.1 [M+H]+

[0406] Step 2 - N-(2-methoxyethyl)-4-methyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxamide (Intermediate 18)

[0407]

[0408] Similar to intermediate 6, intermediate 18 is prepared as follows: starting from a suitable amine and ester.

[0409] Intermediate 17: 300 mg, 0.93 mmol (1.0 equivalent)

[0410] 2-Methoxyethylamine: 0.28 ml, 2.1 mmol (2.20 equivalents)

[0411] Potassium tert-butoxide: 102 mg, 0.91 mmol (0.98 equivalents)

[0412] Dosage / Yield: 110 mg, 0.32 mmol, 35% yield.

[0413] LC-MS Method 1: t R= 0.39 minutes, MS (ESI) m / z = 340.2 [M+H]+

[0414] Step 3 - 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onthionyl carbamate (Example 13)

[0415] Similar to Example 4, Example 13 was prepared as follows: starting from the corresponding bromide and amine.

[0416] Intermediate 18: 55 mg, 0.16 mmol (1.0 equivalent)

[0417] Intermediate 2: 110 mg, 0.16 mmol (1.0 equivalent)

[0418] Purification via FCC (Sfär-C18, 5-25% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0419] Dosage / Yield: 6.2 mg, 0.08 mmol, 7.5% yield

[0420] LC-MS Method 1: t R = 0.47 minutes, MS (ESI) m / z = 464.2 [M]+

[0421] 1 H NMR (500 MHz, CDCl3) δppm 8.72 (s, 1H), 8.27 (d, J=1.6 Hz, 1 H), 7.35 (brs, 1 H), 6.94 (s, 1 H), 6.86 (d, J=1.6 Hz, 1 H), 4.88 (br s, 4 H), 3.90 - 4.08 (m, 4H), 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).

[0422] Example 14: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion)

[0423] Step 1 - Sodium chloride of 4-methyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxylic acid (intermediate 19)

[0424]

[0425] Similar to intermediate 7, intermediate 19 is prepared as follows: starting from the corresponding ester.

[0426] Intermediate 17: 0.60 g, 2.0 mmol (1.0 equivalent)

[0427] Sodium hydroxide (2 M, in water): 1.2 mL, 2.4 mmol

[0428] Yield / Quantity: 0.62 g, 1.83 mmol, 90% yield

[0429] LC-MS Method 1: t R = 0.39 minutes, MS (ESI) m / z = 283.0 [M+H]+

[0430] Step 2 - N,N,4-Trimethyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxamide (Intermediate 20)

[0431]

[0432] Similar to intermediate 8, intermediate 20 is prepared as follows: starting from the corresponding acid and amine.

[0433] Intermediate 19: 400 mg, 1.42 mmol (1.0 equivalent)

[0434] N-methylmethylamine 2 M, in THF: 1.4 mL, 2.84 mmol (2.0 equivalent)

[0435] Purification via FCC (Sfäramino, 20-40% EtOAc / cyclohexane)

[0436] Dosage / Yield: 100 mg, 0.32 mmol, 23% yield

[0437] LC-MS Method 1: t R = 0.41 minutes, MS (ESI) m / z = 310.1 [M+H]+

[0438] Step 3 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion) (Example 14)

[0439] Similar to Example 4, Example 14 was prepared as follows: starting from the corresponding bromide and amine.

[0440] Intermediate 20: 60 mg, 0.19 mmol (1.0 equivalent)

[0441] N-Benzyl-2-bromoacetamide: 51 mg, 0.19 mmol (1.0 equivalent)

[0442] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0443] Dosage / Yield: 34 mg, 0.074 mmol, 39% yield

[0444] LC-MS Method 1: t R = 0.61 minutes, MS (ESI) m / z = 457.2 [M]+

[0445] 1 H NMR (400 MHz, CDCl3) δppm 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, 2H), 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, 6H).

[0446] Example 15: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate

[0447] Step 1 - (2-(3-(2-(azacycloheptane-1-yl)acetamido)-4-methylthiophene-2-carbamate)ethyl)(methyl)carbamate tert-butyl ester (intermediate 10)

[0448]

[0449] Similar to intermediate 6, intermediate 10 is prepared as follows: starting from a suitable amine and ester.

[0450] Intermediate 11: 600 mg, 1.72 mmol (1.0 equivalent)

[0451] N-(2-aminoethyl)-N-methylcarbamate tert-butyl ester: 600 mg (3.44 mmol) (2.0 equivalent)

[0452] Potassium tert-butoxide: 193 mg, 1.72 mmol (1.0 equivalent)

[0453] Purification via FCC (Sfäramino, 10-30% EtOAc / cyclohexane)

[0454] Dosage / Yield: 110 mg, 0.32 mmol, 15% yield.

[0455] LC-MS Method 1: t R = 0.63 minutes, MS (ESI) m / z = 453.2 [M+H]+

[0456] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carbamate (Example 15)

[0457] Similar to Example 4, Example 15 was prepared as follows: starting from the corresponding bromide and amine.

[0458] Intermediate 10: 95 mg, 0.21 mmol (1.0 equivalent)

[0459] N-Benzyl-2-bromoacetamide: 61 mg, 0.23 mmol (1.0 equivalent)

[0460] Purified by FCC (Sfär-C18, 5-40% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH). Yield / Quantity: 29 mg, 0.045 mmol, 21% yield.

[0461] LC-MS Method 1: t R = 0.77 minutes, MS (ESI) m / z = 600.2 [M]+

[0462] 1H NMR (500 MHz, CDCl3)δppm 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, 3H), 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).

[0463] Example 16: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0464] Step 1 - 3-(2-(azacycloheptan-1-yl)acetamido)-N-(2-methoxyethyl)-4-methylthiophene-2-carboxamide (Intermediate 21)

[0465]

[0466] Similar to intermediate 8, intermediate 21 is prepared as follows: starting from the corresponding acid and amine.

[0467] Intermediate 13: 350 mg, 0.99 mmol (1.0 equivalent)

[0468] 2-Methoxyethylamine: 133 mg, 1.78 mmol (1.8 equivalents)

[0469] Purification via FCC (Sfäramino, 10-40% EtOAc / cyclohexane)

[0470] Dosage / Yield: 200 mg, 0.57 mmol, 57% yield

[0471] LC-MS Method 1: t R = 0.43 minutes, MS (ESI) m / z = 354.2 [M+H]+

[0472] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 16)

[0473] Similar to Example 4, Example 16 was prepared as follows: starting from the corresponding bromide and amine.

[0474] Intermediate 21: 65 mg, 0.18 mmol (1.0 equivalent)

[0475] N-Benzyl-2-bromoacetamide: 45 mg, 0.20 mmol (1.1 equivalents)

[0476] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0477] Dosage / Yield: 65 mg, 0.13 mmol, 72% yield

[0478] LC-MS Method 1: t R = 0.61 minutes, MS (ESI) m / z = 501.2 [M]+

[0479] 1 H NMR (400 MHz, DMSO-d6) δppm 9.32 (br s, 1 H), 9.16 (br t, J=5.5 Hz, 1H), 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).

[0480] Example 17: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion)

[0481] Similar to Example 4, Example 17 was prepared as follows: starting from the corresponding bromide and amine.

[0482] Intermediate 18: 55 mg, 0.16 mmol (1.0 equivalent)

[0483] N-Benzyl-2-bromoacetamide: 43 mg, 0.16 mmol (1.0 equivalent)

[0484] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0485] Dosage / Yield: 14 mg, 0.028 mmol, 18% yield

[0486] LC-MS Method 1: t R = 0.61 minutes, MS (ESI) m / z = 487.2 [M]+

[0487] 1 H NMR (400 MHz, CDCl3)δppm 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).

[0488] Example 18: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0489] Step 1 - 3-(2-(azacycloheptan-1-yl)acetamido)-N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-methylthiophene-2-carboxamide (Intermediate 22)

[0490]

[0491] Similar to intermediate 8, intermediate 22 is prepared as follows: starting from the corresponding acid and amine.

[0492] Intermediate 13: 513 mg, 1.45 mmol (1.0 equivalent)

[0493] 2-Aminoethoxy(tert-butyl)dimethylsilane: 507 mg, 2.89 mmol (2.0 equivalents)

[0494] Purification via FCC (Sfäramino, 10-30% EtOAc / cyclohexane)

[0495] Yield / Quantity: 409 mg, 0.90 mmol, 62% yield

[0496] LC-MS Method 1: t R = 0.80 minutes, MS (ESI) m / z = 454.3 [M+H]+

[0497] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butyldimethylsilyl)oxy)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Intermediate 23)

[0498]

[0499] Similar to intermediate 4, intermediate 23 is prepared as follows: starting from the corresponding bromide and amine.

[0500] Intermediate 22: 136 mg, 0.30 mmol (1.0 equivalent)

[0501] N-Benzyl-2-bromoacetamide: 75 mg, 0.33 mmol (1.1 equivalents)

[0502] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0503] Dosage / Yield: 71 mg, 0.12 mmol, 39% yield.

[0504] LC-MS Method 1: t R = 0.94 minutes, MS (ESI) m / z = 601.3 [M]+

[0505] Step 3 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 18)

[0506] To a solution of 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butyldimethylsilyl)oxy)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide intermediate 23 (70 mg, 0.10 mmol) in DCM (2 mL), 0.5 mL of 2 M Et2O solution of hydrogen chloride was added dropwise. After 3 hours, the evaporation was evaporated, and the residue was purified by column chromatography (Sfäramino, 0-10% MeOH / DCM) to provide the title compound (Example 18: 10 mg, 0.020 mmol, 19% yield) as a white powder.

[0507] LC-MS Method 1: t R = 0.56 minutes, MS (ESI) m / z = 487.2 [M]+

[0508] 1 H NMR (400 MHz, DMSO-d6) δppm 9.39 (br t, J=5.5 Hz, 1 H), 9.16 (t, J=5.6Hz, 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).

[0509] Example 19: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride hydrochloride

[0510] Step 1 - 1-(2-((2-((2-((tert-Butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate (Intermediate 24)

[0511]

[0512] Similar to intermediate 4, intermediate 24 is prepared as follows: starting from a suitable bromide and amine.

[0513] Intermediate 10: 95 mg, 0.21 mmol (1.0 equivalent)

[0514] Intermediate 2: 67 mg, 0.23 mmol (1.1 equivalents)

[0515] Purification was performed via FCC (Sfär amino, 1-5% MeOH / DCM) followed by FCC (Sfär-C18, 5-30% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0516] Dosage / Yield: 63 mg, 0.10 mmol, 49% yield.

[0517] LC-MS Method 1: t R = 0.66 minutes, MS (ESI) m / z = 577.3 [M]+

[0518] Step 2 - 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride hydrochloride (Example 19)

[0519] Similar to Example 18, Example 19 was prepared as follows: starting from a suitable Boc-protected amine. After removing volatiles, a pure product was obtained without FCC.

[0520] Intermediate 24: 61 mg, 0.10 mmol (1.0 equivalent)

[0521] Dosage / Yield: 47 mg, 0.090 mmol, 86% yield

[0522] LC-MS Method 2: t R = 0.67 minutes, MS (ESI) m / z = 239.1 [(M+H) / 2]+

[0523] 1H NMR (500 MHz, DMSO-d6) δppm 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).

[0524] Example 20: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate

[0525] Similar to Example 4, Example 20 was prepared as follows: starting from the corresponding bromide and amine.

[0526] Intermediate 21: 65 mg, 0.18 mmol (1.0 equivalent)

[0527] Intermediate 2: 59.24 mg, 0.200 mmol (1.1 equivalents)

[0528] Purification via FCC (Sfär-C18, 5-30% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0529] Dosage / Yield: 4.4 mg, 0.0090 mmol, 5% yield

[0530] LC-MS Method 1: t R = 0.49 minutes, MS (ESI) m / z = 478.2 [M]+

[0531] 1H NMR (400 MHz, CDCl3) δppm 8.70 (s, 1H), 8.29 (d, J=1.3 Hz, 1 H), 7.39 (brs, 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, 4H).

[0532] Example 21: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxecyclobutane-3-ylcarbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0533] Step 1 - 3-[[2-(azacycloheptan-1-yl)acetyl]amino]-4-methyl-N-(oxecyclobutan-3-yl)thiophene-2-carboxamide (Intermediate 25)

[0534]

[0535] Similar to intermediate 8, intermediate 25 is prepared as follows: starting from a suitable acid and amine.

[0536] Intermediate 13: 400 mg, 1.13 mmol (1.0 equivalent)

[0537] 3-Oxycyclic butylamine: 165 mg, 2.25 mmol (2.0 equivalent)

[0538] Purification via FCC (Sfär amino, 5-20% EtOAc / DCM)

[0539] Yield / Quantity: 256 mg, 0.73 mmol, 65% yield

[0540] LC-MS Method 1: t R = 0.80 minutes, MS (ESI) m / z = 352.1 [M+H]+

[0541] Step 2 - 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetane-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 21) Similar to Example 4, Example 21 was prepared as follows: starting from a suitable bromide and amine.

[0542] Intermediate 25: 80 mg, 0.23 mmol (1.0 equivalent)

[0543] Intermediate 2: 64 mg, 0.27 mmol (1.2 equivalents)

[0544] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0545] Dosage / Yield: 10 mg, 0.021 mmol, 9% yield

[0546] LC-MS Method 1: t R = 0.48 minutes, MS (ESI) m / z = 476.2 [M]+

[0547] 1 H NMR (500 MHz, DMSO-d6) δppm 11.91 (br s, 1 H), 9.59 (br d, J=5.9 Hz, 1H), 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).

[0548] Example 22: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate

[0549] Step 1 - N-[2-[[3-[[2-[1-[2-(benzylamino)-2-oxo-ethyl]azacycloheptane-1-onthiol-1-yl]acetyl]amino]-4-methyl-thiophene-2-carbonyl]amino]ethyl]-N-methyl-carbamate tert-butyl ester (zwitterion) (intermediate 26)

[0550]

[0551] Similar to intermediate 4, intermediate 26 is prepared as follows: starting from a suitable bromide and amine.

[0552] Intermediate 10: 247 mg, 0.55 mmol (1.0 equivalent)

[0553] N-Benzyl-2-bromoacetamide: 125 mg, 0.55 mmol (1.0 equivalent)

[0554] Purification via FCC (Sfär amino, 0-10% MeOH / DCM)

[0555] Dosage / Yield: 100 mg, 0.17 mmol, 30% yield.

[0556] LC-MS Method 1: t R = 0.75 minutes, MS (ESI) m / z = 600.5 [M]+.

[0557] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate (Example 22)

[0558] Similar to Example 18, Example 22 was prepared as follows: starting from a suitable Boc-protected amine.

[0559] Intermediate 26: 100 mg, 0.17 mmol (1.0 equivalent)

[0560] Dosage / Yield: 66 mg, 0.12 mmol, 71% yield.

[0561] Purification via FCC (Sfär-C18, 5-50% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0562] LC-MS Method 2: t R = 0.98 minutes, MS (ESI) m / z = 500.4 [M]+

[0563] 1H NMR (400 MHz, DMSO-d6) δppm 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).

[0564] Example 23: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxecyclobutane-3-ylcarbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0565] Similar to Example 4, Example 23 was prepared as follows: starting from a suitable bromide and amine.

[0566] Intermediate 25: 80 mg, 0.22 mmol (1.0 equivalent)

[0567] N-Benzyl-2-bromoacetamide: 55 mg, 0.24 mmol (1.1 equivalents)

[0568] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0569] Dosage / Yield: 29 mg, 0.058 mmol, 26% yield

[0570] LC-MS Method 1: t R = 0.62 minutes, MS (ESI) m / z = 499.2 [M]+

[0571] 1H NMR (500 MHz, DMSO-d6) δppm 9.85 (br d, J=6.7 Hz, 1 H), 9.09 (t, J=5.7Hz, 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).

[0572] Example 24: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onthionyl carboxylate

[0573] Step 1 - N-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-methyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxamide (Intermediate 27)

[0574]

[0575] Similar to intermediate 8, intermediate 27 is prepared as follows: starting from a suitable carboxylic acid and amine.

[0576] Intermediate 19: 365 mg, 1.30 mmol (1.0 equivalent)

[0577] (2-Aminoethoxy)(tert-butyl)dimethylsilane: 0.53 mL, 2.6 mmol (2.0 equivalent)

[0578] Purification via FCC (Sfäramino, 0-40% EtOAc / cyclohexane)

[0579] Dosage / Yield: 260 mg, 0.59 mmol, 46% yield

[0580] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 440.2 [M+H]+

[0581] Step 2 -1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onthionyl carboxylate (Example 24)

[0582] Similar to Example 4, Example 24 was prepared as follows: starting from a suitable bromide and amine.

[0583] Intermediate 27: 90 mg, 0.21 mmol (1.0 equivalent)

[0584] N-Benzyl-2-bromoacetamide: 48 mg, 0.21 mmol (1.0 equivalent)

[0585] Purification via FCC (Sfär-C18, 5-28% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0586] Dosage / Yield: 12 mg, 0.022 mmol, 11% yield

[0587] LC-MS Method 1: t R = 0.55 minutes, MS (ESI) m / z = 473.2 [M]+

[0588] 1 H NMR (400 MHz, CDCl3)δppm 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).

[0589] Example 26: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium chloride hydrochloride

[0590] Step 1 - Methyl(2-(4-methyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carbamate)ethyl)tert-butyl carbamate (Intermediate 28)

[0591]

[0592] Similar to intermediate 8, intermediate 28 is prepared as follows: starting from the corresponding acid and amine.

[0593] Intermediate 19: 384 mg, 1.3 mmol (1.0 equivalent)

[0594] N-(2-aminoethyl)-N-methylcarbamate tert-butyl ester: 0.46 mL, 2.6 mmol (2.0 equivalent)

[0595] Purification via FCC (Sfäramino, 0-40% EtOAc / cyclohexane)

[0596] Dosage / Yield: 250 mg, 0.57 mmol, 44% yield.

[0597] LC-MS Method 1: t R = 0.60 minutes, MS (ESI) m / z = 439.2 [M+H]+

[0598] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion) (intermediate 29)

[0599]

[0600] Similar to intermediate 4, intermediate 29 is prepared as follows: starting from a suitable bromide and amine.

[0601] Intermediate 28: 100 mg, 0.23 mmol (1.0 equivalent)

[0602] N-Benzyl-2-bromoacetamide: 61 mg, 0.23 mmol (1.0 equivalent)

[0603] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0604] Dosage / Yield: 73 mg, 0.11 mmol, 48% yield

[0605] LC-MS Method 1: t R = 0.78 minutes, MS (ESI) m / z = 586.2 [M]+

[0606] Step 3 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium chloride hydrochloride (Example 26)

[0607] Similar to Example 18, Example 26 was prepared as follows: starting from a suitable Boc-protected amine.

[0608] Intermediate 29: 73 mg, 0.11 mmol (1.0 equivalent)

[0609] Amount / Yield: 43 mg, 0 mm. 19 mmol, 71% yield.

[0610] LC-MS Method 1: t R = 0.41 minutes, MS (ESI) m / z = 486.3 [M]+

[0611] 1 H NMR (500 MHz, DMSO-d6) δppm 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, 2H), 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).

[0612] Example 25: 1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0613] Similar to Example 4, Example 25 was prepared as follows: starting from a suitable bromide and amine.

[0614] Intermediate 22: 136 mg, 0.30 mmol (1.0 equivalent)

[0615] Intermediate 2: 85 mg, 0.36 mmol (1.2 equivalents)

[0616] Purification was achieved via FCC (Sfär amino, 1-10% MeOH / DCM) followed by grinding with DCM.

[0617] Dosage / Yield: 14 mg, 0.030 mmol, 10% yield

[0618] LC-MS Method 1: t R = 0.44 minutes, MS (ESI) m / z = 464.2 [M]+

[0619] 1 H NMR (500 MHz, DMSO-d6) δppm 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, 1H), 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, 2H), 1.93 (s, 7 H), 1.63 (br s, 4 H).

[0620] Example 28: 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion)

[0621] Step 1 - 2-Bromo-N-(4-methylisoxazol-3-yl)acetamide (Intermediate 30)

[0622]

[0623] 2-Bromoacetyl bromide (0.78 mL, 8.55 mmol) was added dropwise 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). After 3 h, water was added, and the reaction was extracted with EtOAc (3x). The collected organic layer was dried on Na2SO4, filtered, and concentrated to provide a crude product, which was purified by FCC (KP Sfär silica, 0-60% EtOAc / cyclohexane) to provide the title compound (intermediate 30: 1.23 g, 94% wt).

[0624] LC-MS Method 1: t R = 0.54 minutes, MS (ESI) m / z = 219.0 / 221.0 [M+H]+

[0625] Step 2 - 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion) (Example 28)

[0626] Similar to Example 4, Example 28 was prepared as follows: starting from a suitable bromide and amine.

[0627] Intermediate 20: 26 mg, 0.084 mmol (1.0 equivalent)

[0628] Intermediate 30: 18.4 mg, 0.084 mmol (1.0 equivalent)

[0629] Purified by FCC (Sfär amino, 0-5% MeOH / DCM).

[0630] Dosage / Yield: 4 mg, 0.009 mmol, 11% yield

[0631] LC-MS Method 1: t R = 0.51 minutes, MS (ESI) m / z = 448.2 [M]+

[0632] 1 H NMR (400 MHz, CDCl3)δppm 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, 3H), 2.05 (s, 3H), 1.92 - 2.04 (m, 4H), 1.61 - 1.90 (m, 2H).

[0633] Example 29: 1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium carboxylate

[0634] Similar to Example 4, Example 29 was prepared as follows: starting from a suitable bromide and amine.

[0635] Intermediate 21: 65 mg, 0.18 mmol (1.0 equivalent)

[0636] Intermediate 30: 47 mg, 0.20 mmol (1.2 equivalents)

[0637] Purified by a semi-preparative HPLC method.

[0638] Dosage / Yield: 31 mg, 0.054 mmol, 29% yield.

[0639] LC-MS Method 1: t R = 0.51 minutes, MS (ESI) m / z = 492.3 [M]+

[0640] 1 H NMR (500 MHz, CDCl3) δppm 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 (brs, 4 H).

[0641] Example 12: 1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0642] Similar to Example 4, Example 12 was prepared as follows: starting from a suitable bromide and amine.

[0643] Intermediate 14: 70 mg, 0.23 mmol (1.0 equivalent)

[0644] Intermediate 30: 50 mg, 0.23 mmol (1.0 equivalent)

[0645] Purification was performed via FCC (Sfär amino, 1-5% MeOH / DCM) followed by FCC (Sfär-C18, 10-25% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0646] Dosage / Yield: 2.7 mg, 0.006 mmol, 3% yield.

[0647] LC-MS Method 1: t R = 0.48 minutes, MS (ESI) m / z = 448.1 [M]+

[0648] 1 H NMR (500 MHz, CDCl3)δppm 11.37 (br s, 1 H), 8.11 (s, 1 H), 7.23 (br s, 1H), 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).

[0649] Example 39: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-(morpholin-4-carbonyl)benzo[b]thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide

[0650] Step 1 - Methyl 3-(2-bromoacetamido)benzo[b]thiophene-2-carboxylate (Intermediate 31)

[0651]

[0652] Similar to intermediate 1, intermediate 31 is prepared as follows: starting from a suitable amine.

[0653] 3-Aminobenzo[b]thiophene-2-carboxylic acid methyl ester: 2.50 g, 12.0 mmol (1.0 equivalent)

[0654] Yield / Quantity: 1.80 g, 5.50 mmol, 45% yield

[0655] LC-MS Method 1: t R = 1.00 minutes, MS (ESI) m / z = 328.0 / 330.1 [M+H]+

[0656] Step 2 - 3-(2-(azacycloheptane-1-yl)acetamido)benzo[b]thiophene-2-carboxylic acid methyl ester (intermediate 32)

[0657]

[0658] Similar to intermediate 11, intermediate 32 is prepared as follows: starting from a suitable bromide.

[0659] Intermediate 31: 1.30 g, 3.96 mmol (1.0 equivalent)

[0660] Perpiperidine: 0.47 mL, 3.96 mmol (1.0 equivalent)

[0661] Yield / Quantity: 1.37 g, 3.95 mmol, 99% yield.

[0662] LC-MS Method 1: t R = 0.61 minutes, MS (ESI) m / z = 347.1 [M+H]+

[0663] Step 3 - 3-(2-(azacycloheptane-1-yl)acetamido)benzo[b]thiophene-2-carboxylic acid (intermediate 33)

[0664]

[0665] Lithium hydroxide (86 mg, 3.59 mmol) was added to a suspension of methyl 3-(2-(azacycloheptan-1-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), and the reaction was stirred at 45 °C for 6 h. The organic phase was evaporated under reduced pressure, and 2 M HCl aqueous solution was added to the remaining aqueous phase until pH = 7. The aqueous phase was then extracted with DCM and DCM / MeOH (9 / 1). The collected organic layer was evaporated under reduced pressure, providing the title compound (33: 790 mg, 2.38 mmol, 99% yield).

[0666] LC-MS Method 1: t R = 0.57 minutes, MS (ESI) m / z = 333.3 [M+H]+

[0667] Step 4 - 2-(azacycloheptan-1-yl)-N-(2-(morpholin-4-carbonyl)benzo[b]thiophen-3-yl)acetamide (intermediate 34)

[0668]

[0669] Sulfoxide (320 mL, 4.51 mmol) was added to a DCM (6 mL) suspension of 3-(2-(azacycloheptan-1-yl)acetamido)benzo[b]thiophene-2-carboxylic acid (intermediate 33, 750 mg, 2.26 mmol) 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 product was purified by FCC (KP Sfär silica, 0-10% MeOH / DCM) to provide the title compound (intermediate 34: 95 mg, 0.24 mmol, 10% yield) as an orange solid.

[0670] LC-MS Method 1: t R = 0.57 minutes, MS (ESI) m / z = 402.3 [M+H]+

[0671] Step 5 - 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-(morpholin-4-carbonyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide (Example 39)

[0672] To a solution of intermediate 34 (30 mg, 0.070 mmol) in 0.5 mL of ACN, 2-bromo-N-(isoxazo-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 Pasteurized pipette, and the solid was ground with cyclohexane / EtOAc (7 / 3 v / v) and then with cold ACN to give the title compound (Example 39: 13 mg, 0.021 mmol, 28% yield).

[0673] LC-MS Method 1: t R = 0.63 minutes, MS (ESI) m / z = 526.38 [M]+

[0674] 1H NMR (400 MHz, DMSO-d6) δppm 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, 1H), 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).

[0675] Example 43: 1,1-Di(2-((2-(dimethylcarbamoyl)benzo[b]thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0676] Step 1 - 3-Aminobenzo[b]thiophene-2-carboxylic acid (Intermediate 35)

[0677]

[0678] Lithium hydroxide (115 mg, 4.83 mmol) was added to a suspension of methyl 3-aminobenzo[b]thiophene-2-carboxylate (500 mg, 2.41 mmol) in isopropanol (20 mL) / water (4 mL), and the reaction mixture was stirred under reflux for 1.5 h. The mixture was then cooled to RT, diluted with water, and the volatile organic compounds were evaporated under reduced pressure. The remaining aqueous phase was neutralized with 3 M HCl until pH = 6, at which point a white precipitate was observed to form. The solid was filtered, washed with cold water (2x), and dried to give the title compound (intermediate 35: 460 mg, 2.38 mmol, 99% yield) as a creamy white solid.

[0679] LC-MS Method 1: t R = 0.83 minutes, MS (ESI) m / z = 194.2 [M+H]+

[0680] Step 2 - 3-Amino-N,N-dimethylbenzo[b]thiophene-2-carboxamide (Intermediate 36)

[0681]

[0682] To a solution of 3-aminobenzo[b]thiophene-2-carboxylic acid (intermediate 35, 175 mg, 0.91 mmol), HATU (482 mg, 1.27 mmol), and 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 overnight at RT. Water was added, and the aqueous phase was extracted with EtOAc (3x). The collected organic layer was washed with a saturated aqueous solution of NaHCO3, a 2 M aqueous solution of potassium carbonate, and then brine. The organic phase was dried on a phase separator and evaporated under reduced pressure to give the title compound (intermediate 36, 170 mg, 0.77 mmol, 85% yield).

[0683] LC-MS Method 1: t R = 0.93 minutes, MS (ESI) m / z = 221.0 [M+H]+

[0684] Step 3 - 3-(2-bromoacetamido)-N,N-dimethylbenzo[b]thiophene-2-carboxamide (Intermediate 37)

[0685]

[0686] Similar to intermediate 1, intermediate 37 is prepared as follows: starting from a suitable amine.

[0687] Intermediate 36: 170 mg, 0.77 mmol (1.0 equivalent)

[0688] Dosage / Yield: 260 mg, 0.76 mmol, 99% yield.

[0689] LC-MS Method 1: t R = 0.79 minutes, MS (ESI) m / z = 341.1 / 343.2 [M+H]+

[0690] Step 4 - 3-(2-(azacycloheptane-1-yl)acetamido)-N,N-dimethylbenzo[b]thiophene-2-carboxamide (intermediate 38)

[0691]

[0692] A solution of 3-(2-bromoacetamido)-N,N-dimethylbenzo[b]thiophene-2-carboxamide (intermediate 37, 100 mg, 0.53 mmol) in ACN (4.2 mL) was added to azircycloheptane (0.13 mL, 1.11 mmol), and the reaction mixture was stirred at RT for 1.5 h. The volatiles and crude product were removed under reduced pressure and purified by FCC (KP Sfär silica, 0-50% EtOAc / cyclohexane) to provide the title compound (intermediate 38: 150 mg, 0.42 mmol, 79% yield).

[0693] LC-MS Method 1: t R = 0.55 minutes, MS (ESI) m / z = 360.3 [M+H]+

[0694] Step 5 - 1,1-Di(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 43)

[0695] Similar to Example 4, Example 43 was prepared as follows: starting from a suitable amine and bromide.

[0696] Intermediate 38: 35 mg, 0.10 mmol (1.0 equivalent)

[0697] Intermediate 37: 3-(2-bromoacetamido)-N,N-dimethylbenzo[b]thiophene-2-carboxamide: 33 mg, 0.10 mmol (1.0 equivalent)

[0698] Dosage / Yield: 3.8 mg, 0.006 mmol, 6% yield.

[0699] Purified by FCC (Sfär amino, 0-5% MeOH / DCM).

[0700] LC-MS Method 1: t R = 0.80 minutes, MS (ESI) m / z = 620.2 [M]+

[0701] 1H NMR (500 MHz, DMSO-d6) δppm 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 (brs, 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).

[0702] 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)azacycloheptane-1-onium (zwitterion)

[0703] Similar to Example 4, Example 68 was prepared as follows: starting from a suitable bromide and amine.

[0704] Intermediate 14: 81 mg, 0.26 mmol (1.0 equivalent)

[0705] Intermediate 1: 76 mg, 0.26 mmol (1.0 equivalent)

[0706] Purification via FCC (Sfär amino, 1-5% MeOH / DCM)

[0707] Dosage / Yield: 69 mg, 0.13 mmol, 50% yield.

[0708] LC-MS Method 1: t R = 0.61 minutes, MS (ESI) m / z = 521.2 [M]+

[0709] 1 H NMR (500 MHz, DMSO-d6) δppm 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)

[0710] Example 69: 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion)

[0711] Step 1 - 3-Amino-N,4-Dimethylthiophene-2-carboxamide (Intermediate 55)

[0712]

[0713] Similar to intermediate 36, intermediate 55 is prepared as follows: starting from a suitable acid and amine.

[0714] 3-Amino-4-methylthiophene-2-carboxylic acid: 640 mg, 4.07 mmol (1.0 equivalent)

[0715] Methylamine hydrochloride: 550 mg, 8.14 mmol (2.0 equivalents)

[0716] Purification via FCC (Sfäramino, 20% EtOAc / cyclohexane)

[0717] Dosage / Yield: 259 mg, 1.5 mmol, 37% yield

[0718] LC-MS Method 1: t R = 0.57 minutes, MS (ESI) m / z = 171.3 [M+H]+

[0719] Step 2 - 3-(2-Chloroacetamido)-N,4-Dimethylthiophene-2-carboxamide (Intermediate 56)

[0720]

[0721] 2-Chloroacetyl chloride (0.097 mL, 1.22 mmol) was slowly added to a suspension of intermediate 55 (190 mg, 1.1 mmol) and TEA (0.155 mL, 0.89 mmol) in ACN (9 mL) at 0 °C. The reaction mixture was stirred at RT overnight. The reaction mixture was then washed with a saturated aqueous solution of NaHCO3 and water. The organic phase was dried on a phase separator and concentrated under reduced pressure to provide a crude product, which was purified by FCC (Sfär silica, 20-50% EtOAc / cyclohexane) to provide the title compound (intermediate 56: 200 mg, 0.81 mmol, 74% yield).

[0722] LC-MS Method 1: t R= 0.58 minutes, MS (ESI) m / z = 247.4 [M+H]+

[0723] Step 3 - N,4-Dimethyl-3-(2-(piperidin-1-yl)acetamido)thiophene-2-carboxamide (Intermediate 57)

[0724]

[0725] 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 at RT for 3 h. The reaction was quenched with a saturated aqueous solution of NaHCO3, the organic layer was separated, and the aqueous layer was extracted with DCM (3x). The collected organic layer was filtered through a phase separator and concentrated to provide the title compound (intermediate 57: 62 mg, 0.21 mmol, 75% yield).

[0726] LC-MS Method 2: t R = 0.73 minutes, MS (ESI) m / z = 296.1 [M+H]+

[0727] Step 4 - 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (zwitterion) (Example 69)

[0728] Similar to Example 4, Example 69 was prepared as follows: starting from a suitable bromide and amine.

[0729] Intermediate 57: 62 mg, 0.21 mmol (1.0 equivalent)

[0730] Intermediate 1: 61.3 mg, 0.21 mmol (1.0 equivalent)

[0731] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[0732] Dosage / Yield: 41 mg, 0.081 mmol, 38% yield.

[0733] LC-MS Method 1: t R = 0.59 minutes, MS (ESI) m / z = 507.2 [M]+

[0734] 1H NMR (500 MHz, DMSO-d6) δppm 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)

[0735] Example 73: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)piperidine-1-onthionyl carboxylate

[0736] Step 1 - N-benzyl-2-(4,4-di(hydroxymethyl)piperidin-1-yl)acetamide (intermediate 58)

[0737]

[0738] Similar to intermediate 11, intermediate 58 is prepared as follows: starting from a suitable bromide and amine.

[0739] N-Benzyl-2-bromoacetamide: 150 mg, 0.66 mmol (1.0 equivalent)

[0740] Piperidine-4,4-Ideoxydimethylhydrochloride: 120 mg, 0.66 mmol (1.0 equivalent)

[0741] Potassium carbonate: 227 mg, 1.6 mmol (2.5 equivalents)

[0742] Yield / Quantity: 190 mg, 0.64 mmol, 98% yield

[0743] LC-MS Method 2: t R = 0.52 minutes, MS (ESI) m / z = 293.2 [M+H]+

[0744] Step 2 - 3-Amino-N,N,4-Trimethylthiophene-2-carboxamide (Intermediate 59)

[0745]

[0746] Similar to intermediate 36, intermediate 59 is prepared as follows: starting from a suitable acid and amine.

[0747] 3-Amino-4-methylthiophene-2-carboxylic acid: 900 mg, 5.72 mmol (1.0 equivalent)

[0748] Dimethylamine hydrochloride: 934 mg, 11.45 mmol (2.0 equivalent)

[0749] Purified by FCC (Sfär silica, 70% EtOAc / cyclohexane)

[0750] Yield / Quantity: 410 mg, 1.5 mmol, 39% yield

[0751] LC-MS Method 1: t R = 0.68 minutes, MS (ESI) m / z = 185.0 [M+H]+

[0752] Step 3 - 3-(2-bromoacetamido)-N,N,4-trimethylthiophene-2-carboxamide (Intermediate 60)

[0753]

[0754] Similar to intermediate 30, intermediate 60 is prepared as follows: starting from a suitable amine, at 0°C:

[0755] Intermediate 59: 190 mg, 1.03 mmol (1.0 equivalent)

[0756] Purified by FCC (Sfär silica, 90% EtOAc / cyclohexane)

[0757] Amount / Yield: 187 mg, 0.61 mmol, 59% yield.

[0758] LC-MS Method 1: t R = 0.64 minutes, MS (ESI) m / z = 305.0 / 307.0 [M+H]+

[0759] Step 4 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)piperidine-1-onthionyl carboxylate (Example 73)

[0760] Similar to Example 4, Example 73 was prepared as follows: starting from a suitable bromide and amine.

[0761] Intermediate 58: 180 mg, 0.61 mmol (1.0 equivalent)

[0762] Intermediate 60: 187 mg, 0.61 mmol (1.0 equivalent)

[0763] Purification via FCC (Sfär-C18, 10-30% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[0764] Crystallization via vapor diffusion from Et2O into iPrOH

[0765] Dosage / Yield: 131 mg, 0.23 mmol, 38% yield.

[0766] LC-MS Method 1: t R = 0.59 minutes, MS (ESI) m / z = 517.2 [M]+

[0767] 1 H NMR (400 MHz, DMSO-d6)δppm 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).

[0768] Example 74: (2-(4,4-dimethyl-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-onthiol-1-yl)acetyl)(isoxazo-3-yl)amidocarbamate

[0769] Step 1 - 2-Chloro-N-(isoxazo-3-yl)acetamide (Intermediate 53)

[0770]

[0771] 2-Chloroacetyl chloride (0.40 mL, 5.0 mmol) was added 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) at 0 °C. After 10 min, the reaction mixture was warmed to RT and stirred for 2 h. Water was then added, the organic phase was separated, and the aqueous phase was extracted with DCM (2x). The collected organic phase was dried on a phase separator and concentrated under reduced pressure. The crude product was purified by FCC (Sfär amino, 5-50% cyclohexane / EtOAc) to give the title compound (intermediate 53: 610 mg, 3.80 mmol, 80% yield) as a white powder.

[0772] LC-MS Method 2: t R = 0.50 minutes, MS (ESI) m / z = 161.2 [M+H]+

[0773] Step 2 - 2-(4,4-dimethylpiperidin-1-yl)-N-(isoxazo-3-yl)acetamide (intermediate 61)

[0774]

[0775] Similar to intermediate 57, intermediate 61 is prepared as follows: starting from a suitable amine and chloride.

[0776] Intermediate 53: 2.3 g, 12.9 mmol (1.0 equivalent)

[0777] 4,4-Dimethylpiperidine hydrochloride: 1.92 g, 12.9 mmol (1.0 equivalent)

[0778] Potassium carbonate: 4.46 g, 32.2 mmol (2.5 equivalents)

[0779] Purification via FCC (Sfär amino, 10% EtOAc / cyclohexane)

[0780] Yield / Quantity: 2.88 g, 12.1 mmol, 94% yield.

[0781] LC-MS Method 1: t R = 0.39 minutes, MS (ESI) m / z = 238.2 [M+H]+

[0782] Step 3 - 3-(2-bromoacetamido)-N,4-dimethylthiophene-2-carboxamide (Intermediate 62)

[0783]

[0784] Similar to intermediate 30, intermediate 62 is prepared as follows: starting from a suitable amine, at 0°C:

[0785] Intermediate 55: 265 mg, 1.55 mmol (1.0 equivalent)

[0786] Purified by FCC (Sfär silica, 50% EtOAc / cyclohexane)

[0787] Dosage / Yield: 340 mg, 1.34 mmol, 86% yield.

[0788] LC-MS Method 1: t R = 0.65 minutes, MS (ESI) m / z = 290.9 / 292.9 [M+H]+

[0789] Step 4 - (2-(4,4-dimethyl-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-onthiol-1-yl)acetyl)(isoxazol-3-yl)amidocarbamate (Example 74)

[0790] Similar to Example 4, Example 74 was prepared as follows: starting from a suitable bromide and amine.

[0791] Intermediate 62: 100 mg, 0.34 mmol (1.0 equivalent)

[0792] Intermediate 61: 81 mg, 0.34 mmol (1.0 equivalent)

[0793] Purification by HPLC semi-preparative method

[0794] Dosage / Yield: 26 mg, 0.053 mmol, 15% yield.

[0795] LC-MS Method 1: t R = 0.60 minutes, MS (ESI) m / z = 448.2 [M]+

[0796] 1H NMR (500 MHz, DMSO-d6)δppm 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 = 6.9Hz, 6H)

[0797] Example 70: 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetane-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0798] Similar to Example 4, Example 70 was prepared as follows: starting from a suitable bromide and amine.

[0799] Intermediate 25: 32 mg, 0.09 mmol (1.0 equivalent)

[0800] Intermediate 1: 27 mg, 0.09 mmol (1.0 equivalent)

[0801] Purification via FCC (Sfär amino, 0-2% MeOH / DCM)

[0802] Dosage / Yield: 13 mg, 0.02 mmol, 25% yield.

[0803] LC-MS Method 1: t R = 0.66 minutes, MS (ESI) m / z = 563.3 [M]+

[0804] 1 H NMR (400 MHz, DMSO-d6) δppm 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)

[0805] Example 55: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride

[0806] Step 1 - 3-(2-(azacycloheptane-1-yl)acetamido)-N-(2-hydroxyethyl)-N,4-dimethylthiophene-2-carboxamide (Intermediate 63)

[0807]

[0808] Similar to intermediate 36, intermediate 63 is prepared as follows: starting from a suitable acid and amine.

[0809] Intermediate 13: 700 mg, 1.97 mmol (1.0 equivalent)

[0810] 2-(Methylamino)ethanol-1-ol: 222 mg, 2.96 mmol (1.5 equivalents)

[0811] Purification was performed via FCC (Sfär silica, 0-5% MeOH / DCM) followed by FCC (Sfär-C18, 5-40% ACN + 0.1% (33% aq NH4OH) / H2O + 0.1% (33% aq NH4OH)

[0812] Dosage / Yield: 336 mg, 0.95 mmol, 48% yield

[0813] LC-MS Method 1: t R = 0.40 minutes, MS (ESI) m / z = 354.2 [M+H]+

[0814] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride (Example 55)

[0815] N-benzyl-2-bromoacetamide (114 mg, 0.48 mmol) was added to a solution of intermediate 63 (168 mg, 0.48 mmol) in ACN (4.7 mL) under stirring. The mixture was stirred in a sealed vial at 80 °C for 18 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by FCC (Sfäramino, 0-5% MeOH / DCM) to provide the desired product, which is zwitterionic. It was dissolved in DCM (2.0 mL) and HCl 2M Et2O solution (4 mL) was added under stirring at RT. After 5 minutes, the volatiles were removed under reduced pressure to provide the title compound (Example 55: 101 mg, 0.19 mmol, 40% yield).

[0816] LC-MS Method 1: t R = 0.60 minutes, MS (ESI) m / z = 501.3 [M]+

[0817] 1 H NMR (500 MHz, DMSO-d6) δppm10.44 (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 = 5.6Hz, 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)

[0818] Example 67: 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)piperidine-1-onthionyl carboxylate

[0819] Step 1 - 3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-N,4-dimethylthiophene-2-carboxamide (Intermediate 64)

[0820]

[0821] Similar to intermediate 57, intermediate 64 is prepared as follows: starting from a suitable amine and chloride.

[0822] Intermediate 56: 69 mg, 0.28 mmol (1.0 equivalent)

[0823] 4,4-Dimethylpiperidine hydrochloride: 42 mg, 0.28 mmol (1.0 equivalent)

[0824] Dosage / Yield: 86 mg, 0.27 mmol, 95% yield.

[0825] LC-MS Method 2: t R = 0.88 minutes, MS (ESI) m / z = 324.1 [M+H]+

[0826] Step 2 - 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)piperidine-1-onthionyl carboxylate (Example 67)

[0827] Similar to Example 4, Example 67 was prepared as follows: starting from a suitable bromide and amine.

[0828] Intermediate 64: 86 mg, 0.27 mmol (1.0 equivalent)

[0829] Intermediate 1: 78 mg, 0.27 mmol (1.0 equivalent)

[0830] Purified by FCC (Sfär-C18, 5-20% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0831] Dosage / Yield: 71 mg, 0.12 mmol, 45% yield.

[0832] LC-MS Method 1: t R = 0.63 minutes, MS (ESI) m / z = 535.6 [M]+

[0833] 1H NMR (500 MHz, DMSO-d6) δppm 11.05 (br s, 1 H), 8.41 (s, 1 H), 8.20 (brs, 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)

[0834] Example 75: 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)-1-(2-oxo-2-((1-phenylcyclopropyl)amino)ethyl)piperidine-1-onthionyl carboxylate

[0835] Step 1 - 2-Bromo-N-(1-Phenylocyclopropyl)acetamide (Intermediate 65)

[0836]

[0837] 2-Bromoacetyl bromide (0.13 mL, 1.47 mmol) was added to a stirred solution of 1-phenylcyclopropyl-1-amine hydrochloride (250 mg, 1.47 mmol) and TEA (0.41 mL, 2.94 mmol) in 15 mL of DCM at 0 °C. After stirring at 0 °C for 1 h, a saturated aqueous solution of NH4Cl was added to separate the organic phase, and the aqueous phase was extracted with DCM (2x). The collected organic phase was dried on a phase separator and concentrated under reduced pressure. The crude product was purified by FCC (Sfär silica, 40% EtOAc / cyclohexane) to provide the title compound (intermediate 65: 279 mg, 1.09 mmol, 74% yield) as a white solid.

[0838] LC-MS Method 2: t R = 0.64 minutes, MS (ESI) m / z = 253.1 / 255.1 [M+H]+

[0839] Step 2 - 3-(2-(4,4-di(hydroxymethyl)piperidin-1-yl)acetamido)-N,N,4-trimethylthiophene-2-carboxamide (Intermediate 66)

[0840]

[0841] To a stirred solution of intermediate 60 (50 mg, 0.16 mmol) in ACN (4 mL), piperidine-4,4-imidediethanol hydrochloride (30 mg, 0.16 mmol) and potassium carbonate (56 mg, 0.41 mmol) were added, and the mixture was stirred at RT for 16 hours. The reaction was filtered to remove the salt, and the organic phase was concentrated under reduced pressure to provide the title compound (intermediate 66: 55 mg, 0.15 mmol, 90% yield) as a white solid.

[0842] LC-MS Method 2: t R = 0.50 minutes, MS (ESI) m / z = 370.2 [M+H]+

[0843] Step 3 - 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)-1-(2-oxo-2-((1-phenylcyclopropyl)amino)ethyl)piperidine-1-onthionyl carboxylate (Example 75)

[0844] Similar to Example 4, Example 75 was prepared as follows: starting from a suitable bromide and amine.

[0845] Intermediate 66: 55 mg, 0.15 mmol (1.0 equivalent)

[0846] Intermediate 65: 38 mg, 0.15 mmol (1.0 equivalent)

[0847] Purified by FCC (Sfär-C18, 5-35% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0848] Dosage / Yield: 39 mg, 0.066 mmol, 44% yield.

[0849] LC-MS Method 1: t R = 0.60 minutes, MS (ESI) m / z = 543.5 [M]+

[0850] 1H NMR (400 MHz, DMSO-d6)δppm 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)

[0851] Example 66: 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0852] Similar to Example 4, Example 66 was prepared as follows: starting from a suitable bromide and amine.

[0853] Intermediate 11: 90 mg, 0.26 mmol (1.0 equivalent)

[0854] Intermediate 60: 55 mg, 0.26 mmol (1.0 equivalent)

[0855] Purification via FCC (Sfär amino, 0-2% MeOH / DCM)

[0856] Dosage / Yield: 55 mg, 0.10 mmol, 39% yield.

[0857] LC-MS Method 1: t R = 0.65 minutes, MS (ESI) m / z = 535.2 [M]+

[0858] 1H NMR (400 MHz, DMSO-d6) δppm 13.99 (br s, 1 H), 7.29 (s, 1 H), 7.21 (s, 1H), 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)

[0859] Example 64: 1-(2-((2-(di(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carboxylate

[0860] Step 1 - 3-(2-(azacycloheptane-1-yl)acetamido)-N,N-bis(2-hydroxyethyl)-4-methylthiophene-2-carboxamide (Intermediate 67)

[0861]

[0862] Add 2,2'-azaalkylidene di(ethylene-1-ol) (0.41 mL, 0.42 mmol) to a DMF (1.4 mL) suspension of sodium chloride (intermediate 13, 100 mg, 0.28 mmol), HATU (129 mg, 0.34 mmol), and DIPEA (147 mL, 0.85 mmol), and stir the reaction mixture overnight at 80 °C. Dilute the mixture with EtOAc. Separate the two phases; wash the organic layer with a saturated aqueous solution of NaHCO3 followed by brine. Back-extract the aqueous phase with a DCM:iPrOH 2:1 solution (2x). Dry the collected organic phase on Na2SO4, filter, and concentrate under reduced pressure. The residue was purified by FCC (Sfär-C18, 0-80% ACN / H2O) to give the title compound (intermediate 67: 35 mg, 0.09 mmol, 32% yield).

[0863] LC-MS Method 1: t R = 0.37 minutes, MS (ESI) m / z = 384.2 [M+H]+

[0864] Step 2 - 1-(2-((2-(di(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onthionyl carbamate (Example 64)

[0865] Similar to Example 4, Example 64 was prepared as follows: starting from a suitable bromide and amine.

[0866] Intermediate 67: 35 mg, 0.09 mmol (1.0 equivalent)

[0867] Intermediate 1: 27 mg, 0.09 mmol (1.0 equivalent)

[0868] Purified by FCC (Sfär-C18, 5-30% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0869] Dosage / Yield: 14.1 mg, 0.022 mmol, 24% yield.

[0870] LC-MS Method 1: t R = 0.59 minutes, MS (ESI) m / z = 595.2 [M]+

[0871] 1 H NMR (500 MHz, DMSO-d6) δppm 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, 6H), 1.92 - 2.09 (m, 4 H), 1.67 (br s, 4 H).

[0872] Example 42: 1-(2-((2-(dimethylcarbamoyl)benzo[b]thiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium bromide

[0873] Similar to Example 39, Example 42 was prepared as follows: starting from suitable amines and bromides.

[0874] Intermediate 38: 50 mg, 0.14 mmol (1.0 equivalent)

[0875] Intermediate 2: 29 mg, 0.14 mmol (1.0 equivalent)

[0876] Precipitate from EtOAc and then grind with DCM:MeOH (9 / 1 v / v).

[0877] Dosage / Yield: 31 mg, 0.054 mmol, 39% yield.

[0878] LC-MS Method 1: t R = 0.62 minutes, MS (ESI) m / z = 484.2 [M]+

[0879] 1 H NMR (500 MHz, DMSO-d6) δppm 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).

[0880] Intermediate 40: 3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid tert-butyl ester

[0881]

[0882] The stirred suspension of 3-(2-(azacycloheptan-1-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, 1,1-di-tert-butoxy-N,N-dimethylmethylamine (7.66 mL, 32.0 mmol) was added dropwise. The reaction was stirred under reflux for 24 h. The solvent was evaporated to produce a crude mixture, which was purified by FCC (Sfäramino, 0-20% DCM / EtOAc (1 / 1 v / v), in cyclohexane) to provide the title compound (intermediate 40: 3.15 g, 8.93 mmol, 56% yield).

[0883] LC-MS Method 1: t R = 0.65 minutes, MS (ESI) m / z = 353.2 [M+H]+

[0884] Intermediate 39: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(tert-butoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0885]

[0886] Intermediate 40 (2.10 g, 5.84 mmol) was added to a stirred solution of N-benzyl-2-bromoacetamide (1.33 g, 5.84 mmol) in ACN (30 mL). The reaction was stirred at 80 °C for 36 h. The reaction was cooled and concentrated under high vacuum. The residue was purified by FCC (Sfäramino, 0–5% MeOH / DCM) to give the title compound (Intermediate 39: 2.54 g, 5.08 mmol, 87% yield).

[0887] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 500.3 [M]+

[0888] Intermediate 41: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-carboxy-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride

[0889]

[0890] Complete dissolution was observed upon dropwise addition of a 2 M Et₂O solution (30 mL, 60 mmol) of HCl to a suspension of intermediate 39 (2.50 g, 5.00 mmol) in THF (20 mL) and DCM (30 mL). After 48 hours, the evaporation was evaporated under reduced pressure, yielding a solid. This solid was then ground with Et₂O to yield the title compound (intermediate 41: 1.92 g, 4.00 mmol, 80% yield), a white solid.

[0891] LC-MS Method 1: t R = 0.64 minutes, MS (ESI) m / z = 445.2 [M]+

[0892] Intermediate 42: 1-(2-((2-(tert-butoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0893]

[0894] Intermediate 40 (1.40 g, 3.85 mmol) was added to a 20 mL solution of ACN containing intermediate 2 (789 mg, 3.85 mmol) under stirring. The reaction was stirred overnight at 80 °C. The reaction was cooled and concentrated under high vacuum. The residue was purified by FCC (Sfär amino, 0–5% MeOH / DCM) to provide the title compound (intermediate 42: 1.42 g, 2.98 mmol, 77% yield).

[0895] LC-MS Method 1: t R = 0.74 minutes, MS (ESI) m / z = 477.2 [M]+

[0896] Intermediate 43 - 1-(2-((2-carboxy-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium chloride

[0897]

[0898] At RT, a solution of 2 M Et2O in HCl (37 mL) was added dropwise to a solution of 3-[[2-[1-[2-(isoxazol-3-ylamino)-2-oxo-ethyl]azacycloheptane-1-onthiol-1-yl]acetyl]amino]-4-methyl-thiophene-2-carboxylic acid tert-butyl ester; bromide (intermediate 42, 1.42 g, 2.98 mmol) in DCM (37 mL). The reaction was stirred at RT for 16 h, and then the evaporation was evaporated under reduced pressure to provide the title compound (intermediate 43: 1.15 g, 2.52 mmol, 86% yield).

[0899] LC-MS Method 1: t R = 0.51 minutes, MS (ESI) m / z = 421.2 [M]+

[0900] Example 27: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride hydrochloride

[0901] 1-Methylimidazole (205 mg, 2.50 mmol) was added to a suspension of intermediate 41 (400 mg, 0.83 mmol) in ACN (2.6 ml), followed by the addition of TCFH (257 mg, 0.92 mmol) at 0 °C. After 1 hour, 1-methylpiperazine (125 mg, 1.25 mmol) was added, and the mixture was stirred at 70 °C for 14 hours. The evaporated volatiles and residues were purified by FCC (Sfär amino, 0-5% MeOH / DCM) followed by FCC (Sfär-C18, 10-30% ACN / H2O + 0.05% HCl 37%) to provide the title compound (Example 27: 32 mg, 0.053 mmol, 6% yield).

[0902] LC-MS Method 2: t R = 1.16 minutes, MS (ESI) m / z = 526.5 [M]+

[0903] 1 H NMR (400 MHz, DMSO-d6) δppm 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 (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, 4H)

[0904] Similar to the description of Example 27, the following examples were prepared by starting with a suitable intermediate and a corresponding commercially available amine.

[0905]

[0906]

[0907]

[0908]

[0909] Intermediate 44: 1-(2-((2-((2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0910]

[0911] Similar to Example 27, intermediate 44 was prepared as follows: starting from intermediate 43.

[0912] Intermediate 43: 100 mg, 0.22 mmol (1.0 equivalent)

[0913] 4-(2-Aminoethyl)piperazine-1-carboxylic acid tert-butyl ester: 50 mg, 0.22 mmol (1.0 equivalent)

[0914] Purified by FCC (Sfär amino, 0-5% MeOH / DCM).

[0915] Dosage / Yield: 75 mg, 0.12 mmol, 54% yield.

[0916] LC-MS Method 1: t R = 0.46 minutes, MS (ESI) m / z = 632.3 [M]+

[0917] Intermediate 45: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-(4-(tert-butoxycarbonyl)piperazin-1-yl)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0918]

[0919] Similar to Example 27, intermediate 45 was prepared as follows: starting from a suitable carboxylic acid and amine.

[0920] Intermediate 41: 125 mg, 0.23 mmol (1.0 equivalent)

[0921] 4-(2-Aminoethyl)piperazine-1-carboxylic acid tert-butyl ester: 50 mg, 0.22 mmol (1.0 equivalent)

[0922] Purified by FCC (Sfär amino, 0-5% MeOH / DCM).

[0923] Yield / Dose: 137 mg, 0.21 mmol, 95% yield

[0924] LC-MS Method 1: t R = 0.63 minutes, MS (ESI) m / z = 655.5 [M]+

[0925] Intermediate 68: Methyl 3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid ester

[0926]

[0927] 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 at RT for 2 h. The reaction mixture was diluted with water and EtOAc, the organic layers were separated, and the aqueous layer was extracted with EtOAc (3x). The collected organic layers were dried over Na2SO4, filtered, concentrated, and yielded methyl 3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid (intermediate 68: 2.2 g, 6.78 mmol, 99% yield).

[0928] LC-MS Method 1: t R = 0.58 minutes, MS (ESI) m / z = 325.2 [M+H]+

[0929] Intermediate 69: Sodium chloride of 3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid

[0930]

[0931] Similar to intermediate 7, intermediate 69 is prepared as follows: starting from the corresponding ester:

[0932] Intermediate 68: 2.2 g, 6.3 mmol (1.0 equivalent)

[0933] Sodium hydroxide (2 M, in water): 3.8 mL, 7.6 mmol (1.2 equivalents)

[0934] Yield / Quantity: 2.4 g, quantitative yield.

[0935] LC-MS Method 1: t R = 0.52 minutes, MS (ESI) m / z = 311.2 [M+H]+

[0936] Intermediate 70: (2-(3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carbamate)ethyl)tert-butyl carbamate

[0937]

[0938] Similar to intermediate 36, intermediate 70 is prepared as follows: starting from a suitable acid and amine.

[0939] Intermediate 69: 0.60 g, 1.63 mmol (1.0 equivalent)

[0940] (2-Aminoethyl) tert-butyl carbamate: 0.43 g, 2.44 mmol (1.5 equivalents)

[0941] Purification via FCC (Sfäramino, 50% EtOAc / cyclohexane)

[0942] Yield / Quantity: 1.10 g, quantitative yield (impure compounds were used as is in subsequent steps).

[0943] LC-MS Method 1: t R = 0.67 minutes, MS (ESI) m / z = 467.3 [M+H]+

[0944] Intermediate 71: 1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethylpiperidine-1-onthionyl carbamate

[0945]

[0946] Similar to Example 4, intermediate 71 was prepared as follows: starting from the corresponding bromide and amine.

[0947] Intermediate 70: 130 mg, 0.28 mmol (1.0 equivalent)

[0948] Intermediate 1: 81 mg, 0.28 mmol (1.0 equivalent)

[0949] Purification was performed via FCC (Sfär amino, 0-5% MeOH / DCM) followed by FCC (Sfär-C18, 5%-40% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0950] Dosage / Yield: 34 mg, 0.050 mmol, 18% yield

[0951] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 678.5 [M]+

[0952] Intermediate 72: 1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-((methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium carboxylate

[0953]

[0954] Similar to Example 4, intermediate 72 was prepared as follows: starting from the corresponding bromide and amine.

[0955] Intermediate 10: 108 mg, 0.24 mmol (1.0 equivalent)

[0956] Intermediate 1: 88 mg, 0.29 mmol (1.2 equivalents)

[0957] Purification was performed via FCC (Sfär amino, 0-2% MeOH / DCM) followed by FCC (Sfär-C18, 5%-50% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[0958] Dosage / Yield: 80 mg, 0.113 mmol, 47% yield

[0959] LC-MS Method 1: t R = 0.78 minutes, MS (ESI) m / z = 664.4 [M]+

[0960] Intermediate 73: (2-(3-amino-4-methylthiophene-2-carbamoyl)ethyl)(cyclopropyl)carbamate tert-butyl ester

[0961]

[0962] Similar to intermediate 36, intermediate 73 is prepared as follows: starting from a suitable acid and amine.

[0963] (2-Aminoethyl)(cyclopropyl)carbamate tert-butyl ester: 0.51 mL, 2.67 mmol (1.0 equivalent)

[0964] 3-Amino-4-methylthiophene-2-carboxylic acid: 600 mg, 3.80 mmol (1.4 equivalents)

[0965] Purified by FCC (Sfär silica, 40% EtOAc / cyclohexane)

[0966] Dosage / Yield: 490 mg, 1.44 mmol, 54% yield

[0967] LC-MS Method 1: t R = 1.02 minutes, MS (ESI) m / z = 340.5 [M+H]+

[0968] Intermediate 74: (2-(3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carbamate)ethyl)(cyclopropyl)tert-butyl carbamate

[0969]

[0970] 2-Bromoacetyl bromide (0.10 mL, 1.22 mmol) was added dropwise to a suspension of potassium carbonate (340 mg, 2.45 mmol) and (2-(3-amino-4-methylthiophene-2-carbamoyl)ethyl)(cyclopropyl)carbamate tert-butyl ester (intermediate 73, 490 mg, 1.22 mmol) in ACN (12 mL) at 0 °C. After stirring at RT for 1 h, azircycloheptane (0.41 mL, 3.66 mmol) was added, and the reaction was stirred at RT for 4 h. The solid and filtrate were filtered off and concentrated under reduced pressure. The crude product was purified by FCC (KP Sfär silica, 5-40% EtOAc / cyclohexane) to provide the title compound (intermediate 74: 550 mg, 94% wt, quantitative yield).

[0971] LC-MS Method 1: t R = 0.68 minutes, MS (ESI) m / z = 479.3 [M+H]+

[0972] Intermediate 75: 1-(2-((2-((2-((tert-butoxycarbonyl)(cyclopropyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0973]

[0974] Similar to Example 4, intermediate 75 was prepared as follows: starting from the corresponding bromide and amine.

[0975] Intermediate 74: 150 mg, 0.31 mmol (1.0 equivalent)

[0976] Intermediate 1: 110 mg, 0.29 mmol (1.0 equivalent)

[0977] Purification via FCC (Sfär amino, 0-10% MeOH / DCM)

[0978] Dosage / Yield: 70 mg, 0.091 mmol, 29% yield

[0979] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 694.4 [M]+

[0980] Intermediate 102: 4-((3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carboxamido)methyl)piperidine-1-carboxylic acid tert-butyl ester

[0981]

[0982] Similar to intermediate 36, intermediate 102 is prepared as follows: starting from a suitable acid and amine.

[0983] Intermediate 13: 500 mg, 1.41 mmol (1.0 equivalent)

[0984] 4-(aminomethyl)piperidine-1-carboxylic acid tert-butyl ester: 0.450 mL, 2.11 mmol (1.5 equivalents)

[0985] Purification via FCC (Sfäramino, 20% EtOAc / cyclohexane)

[0986] Dosage / Yield: 140 mg, 0.28 mmol, 20% yield

[0987] LC-MS Method 1: t R = 0.79 minutes, MS (ESI) m / z = 493.3 [M+H]+

[0988] Intermediate 76: 1-(2-((2-(((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-((methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[0989]

[0990] Similar to Example 4, intermediate 76 was prepared as follows: starting from the corresponding bromide and amine.

[0991] Intermediate 102: 90 mg, 0.180 mmol (1.0 equivalent)

[0992] Intermediate 1: 53 mg, 0.180 mmol (1.0 equivalent)

[0993] Purification via FCC (Sfär amino, 0-2.5% MeOH / DCM)

[0994] Dosage / Yield: 60 mg, 0.085 mmol, 47% yield

[0995] LC-MS Method 1: t R = 0.78 minutes, MS (ESI) m / z = 704.8 [M]+

[0996] Intermediate 77: 4-(3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carbonyl)piperazine-1-carboxylic acid tert-butyl ester

[0997]

[0998] Similar to intermediate 36, intermediate 77 is prepared as follows: starting from the corresponding acid and amine. The reaction is carried out at 60°C.

[0999] Intermediate 13: 560 mg, 1.68 mmol (1.0 equivalent)

[1000] Piperazine-1-carboxylic acid tert-butyl ester: 470 mg, 2.52 mmol (1.5 equivalents)

[1001] Purification was performed via FCC (Sfär silica, 30% EtOAc / cyclohexane) followed by FCC (Sfär-C18, 10-30% ACN / H2O).

[1002] Dosage / Yield: 84 mg, 0.18 mmol, 10% yield

[1003] LC-MS Method 1: t R = 0.67 minutes, MS (ESI) m / z = 465.4 [M+H]+

[1004] Intermediate 78: 1-(2-((2-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1005]

[1006] Similar to Example 4, intermediate 78 was prepared as follows: starting from the corresponding bromide and amine.

[1007] Intermediate 77: 760 mg, 1.64 mmol (1.0 equivalent)

[1008] Intermediate 1: 493 mg, 1.64 mmol (1.0 equivalent)

[1009] Purified by FCC (Sfär amino, 0-10% MeOH / DCM) followed by Sfär-C18 (5-100% ACN / H2O).

[1010] Dosage / Yield: 107 mg, 0.14 mmol, 9% yield

[1011] LC-MS Method 1: t R = 0.82 minutes, MS (ESI) m / z = 676.3 [M]+

[1012] Intermediate 79: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1013]

[1014] Similar to Example 27, intermediate 79 was prepared as follows: starting from the corresponding acid and amine.

[1015] Intermediate 41: 200 mg, 0.42 mmol (1.0 equivalent)

[1016] tert-butyl methyl (2-(methylamino)ethyl)carbamate: 118 mg, 0.63 mmol (1.5 equivalents)

[1017] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[1018] Yield / Quantity: 77 mg, 0.12 mmol, 28% yield

[1019] LC-MS Method 1: t R = 0.81 minutes, MS (ESI) m / z = 614.4 [M]+

[1020] Intermediate 80: N2-(3-amino-4-methylthiophene-2-carbonyl)-N 6-(tert-Butoxycarbonyl)-D-Lysine methyl ester

[1021]

[1022] Similar to intermediate 36, intermediate 80 is prepared as follows: starting from a suitable acid and amine.

[1023] N 6 -(tert-Butyloxycarbonyl)-D-Lysine methyl ester hydrochloride: 1.96 g, 6.62 mmol (1.3 equivalents)

[1024] 3-Amino-4-methylthiophene-2-carboxylic acid: 800 mg, 5.09 mmol (1.0 equivalent)

[1025] Purified by FCC (Silica, 20% (EtOAc / EtOH) / cyclohexane)

[1026] Yield / Quantity: 0.75 g, 1.88 mmol, 37% yield

[1027] LC-MS Method 1: t R = 0.99 minutes, MS (ESI) m / z = 400.2 [M+H]+

[1028] Intermediate 81: N 2 -(3-(2-bromoacetamido)-4-methylthiophene-2-carbonyl)-N 6 -(tert-Butoxycarbonyl)-D-Lysine methyl ester

[1029]

[1030] 2-Bromoacetyl bromide (0.164 mL, 1.88 mmol) was added dropwise to a suspension of intermediate 80 (0.75 g, 1.88 mmol) in ACN (17 mL) at 0 °C. A creamy-white solid was observed to form. The reaction was stirred at RT for 1 h. The solid and the liquid phase were then filtered off and evaporated under reduced pressure. The crude product was purified by FCC (Sfär silica, 10% EtOAc / cyclohexane) to provide the title compound (intermediate 81: 0.48 g, 0.92 mmol, 49% yield), a colorless oil.

[1031] LC-MS Method 1: t R = 1.00 minutes, MS (ESI) m / z = 520.2 / 522.2 [M+H]+

[1032] Intermediate 101: N 2-(3-(2-(azacycloheptane-1-yl)acetamido)-4-methylthiophene-2-carbonyl)-N 6 -(tert-Butoxycarbonyl)-D-Lysine methyl ester

[1033]

[1034] Similar to intermediate 66, intermediate 101 is prepared as follows: starting from a suitable bromide and amine.

[1035] Intermediate 81: 700 mg, 0.77 mmol (1.0 equivalent)

[1036] Azacycloheptadecane: 0.17 mL, 1.53 mmol (2.0 equivalent)

[1037] Dosage / Yield: 170 mg, 0.32 mmol, 41% yield.

[1038] LC-MS Method 1: t R = 0.65 minutes, MS (ESI) m / z = 540.4 [M+H]+

[1039] Intermediate 82: (R)-1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((6-((tert-butoxycarbonyl)amino)-1-methoxy-1-oxohexane-2-yl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1040]

[1041] Similar to Example 4, intermediate 82 was prepared as follows: starting from the corresponding bromide and amine.

[1042] Intermediate 101: 170 mg, 0.32 mmol (1.0 equivalent)

[1043] N-Benzyl-2-bromoacetamide: 72 mg, 0.32 mmol (1.0 equivalent)

[1044] Purification via FCC (Sfär amino, 0-10% MeOH / DCM)

[1045] Dosage / Yield: 85 mg, 0.11 mmol, 35% yield

[1046] LC-MS Method 1: t R = 0.78 minutes, MS (ESI) m / z = 686.8 [M]+

[1047] Intermediate 83: methyl 3-(2-(4-fluoro-4-methylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid

[1048]

[1049] Add 4-fluoro-4-methylpiperidine hydrochloride (300 mg, 1.95 mmol) and potassium carbonate (674 mg, 4.88 mmol) to a stirred solution of intermediate 1 (570 mg, 1.95 mmol) in DCM (13 mL), and stir the mixture at RT for 16 h. Filter the reaction to remove the salt and concentrate the organic phase. Dissolve the residue in EtOAc and water. Mix the phases in an extractor, separate the organic layer, and extract the aqueous layer with EtOAc (2x). Dry the collected organic layer on Na2SO4, filter, and concentrate to provide the title compound (intermediate 83: 600 mg, 1.83 mmol, 94% yield), as a yellow solid.

[1050] LC-MS Method 1: t R = 0.47 minutes, MS (ESI) m / z = 329.2 [M+H]+

[1051] Intermediate 84: 3-(2-(4-fluoro-4-methylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carboxylic acid

[1052]

[1053] Similar to intermediate 7, intermediate 84 is prepared as follows: starting from the corresponding ester:

[1054] Intermediate 83: 600 mg, 1.83 mmol (1.0 equivalent)

[1055] Sodium hydroxide (2 M, in water): 1.1 mL, 2.19 mmol (1.2 equivalents)

[1056] Purification via FCC (Sfär-C18, 0-10% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH)

[1057] Amount / Yield: 150 mg, 0.48, 26% yield.

[1058] LC-MS Method 1: t R = 0.43 minutes, MS (ESI) m / z = 315.1 [M+H]+

[1059] Intermediate 85: (2-(3-(2-(4-fluoro-4-methylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carbamate)ethyl)(methyl)carbamate tert-butyl ester

[1060]

[1061] Similar to Example 27, intermediate 85 was prepared as follows: starting from a suitable carboxylic acid and amine:

[1062] Intermediate 84: 150 mg, 0.48 mmol (1.0 equivalent)

[1063] (2-Aminoethyl)(methyl)carbamate tert-butyl ester: 83 mg, 0.48 mmol (1.0 equivalent)

[1064] Purification via FCC (Sfäramino, 0-60% EtOAc / cyclohexane)

[1065] Dosage / Yield: 100 mg, 0.21 mmol, 45% yield

[1066] LC-MS Method 1: t R = 0.62 minutes, MS (ESI) m / z = 471.4 [M+H]+

[1067] Intermediate 86: 1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4-fluoro-1-(2-((2-((methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4-methylpiperidin-1-onium (zwitterion) (a mixture of diastereomers)

[1068]

[1069] Similar to Example 4, intermediate 86 was prepared as follows: starting from the corresponding bromide and amine.

[1070] Intermediate 85: 100 mg, 0.21 mmol (1.0 equivalent)

[1071] Intermediate 1: 62 mg, 0.21 mmol (1.0 equivalent)

[1072] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[1073] Dosage / Yield: 47 mg, 0.062 mmol, 29% yield

[1074] LC-MS Method 1: tR = 0.79 minutes, MS (ESI) m / z = 682.4 [M]+

[1075] Intermediate 87: N 2 -(3-amino-4-methylthiophene-2-carbonyl)-N 6 -(tert-Butoxycarbonyl)-L-L-lysine methyl ester

[1076]

[1077] Similar to intermediate 36, intermediate 87 is prepared as follows: starting from a suitable acid and amine. The reaction is carried out in ACN.

[1078] N 6 -(tert-Butyloxycarbonyl)-L-lysine methyl ester hydrochloride: 1.88 g, 6.36 mmol (1.0 equivalent)

[1079] 3-Amino-4-methylthiophene-2-carboxylic acid: 1.00 g, 6.36 mmol (1.0 equivalent)

[1080] Purified by FCC (Sfär silica, 10% ACN / DCM)

[1081] Dosage / Yield: 910 mg, 2.28 mmol, 36% yield

[1082] LC-MS Method 1: t R = 1.00 minutes, MS (ESI) m / z = 300.2 [M+H]+

[1083] Intermediate 88: N 2 -(3-(2-(azacycloheptane-1-yl)acetamido)-4-methylthiophene-2-carbonyl)-N 6 -(tert-Butoxycarbonyl)-L-L-lysine methyl ester

[1084]

[1085] Similar to intermediate 74, intermediate 88 is prepared as follows: starting from a suitable amine.

[1086] Intermediate 87: 910 mg, 2.28 mmol (1.0 equivalent)

[1087] Purified by FCC (Silica, 80% (EtOAc:EtOH 9:1) / cyclohexane)

[1088] Dosage / Yield: 500 mg, 0.93 mmol, 40% yield

[1089] LC-MS Method 1: t R = 0.69 minutes, MS (ESI) m / z = 539.5 [M+H]+

[1090] Intermediate 89: (S)-1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((6-((tert-butoxycarbonyl)amino)-1-methoxy-1-oxohexane-2-yl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1091]

[1092] Similar to Example 4, intermediate 89 was prepared as follows: starting from the corresponding bromide and amine.

[1093] Intermediate 88: 250 mg, 0.27 mmol (1.0 equivalent)

[1094] N-Benzyl-2-bromoacetamide: 73 mg, 0.27 mmol (1.0 equivalent)

[1095] Purified by FCC (Sfär amino, 0-100% ACN / DCM)

[1096] Dosage / Yield: 40 mg, 0.058 mmol, 22% yield

[1097] LC-MS Method 1: t R = 0.80 minutes, MS (ESI) m / z = 686.3 [M]+

[1098] Intermediate 90: (3-(3-(2-(4,4-dimethylpiperidin-1-yl)acetamido)-4-methylthiophene-2-carbamate)propyl)(methyl)carbamate tert-butyl ester

[1099]

[1100] Similar to intermediate 36, intermediate 90 is prepared as follows: starting from a suitable acid and amine.

[1101] Intermediate 69: 1.00 g, 2.71 mmol (1.0 equivalent)

[1102] (3-Aminopropyl)(Methyl)carbamate tert-butyl ester: 766 mg, 4.07 mmol (1.5 equivalents)

[1103] Purification via FCC (Sfäramino, 50% EtOAc / cyclohexane)

[1104] Yield / Quantity: 1.60 g, quantified yield (82% wt)

[1105] LC-MS Method 1: t R = 0.71 minutes, MS (ESI) m / z = 484.1 [M+H]+

[1106] Intermediate 91: Methyl 3-((2-bromoacetamido)methyl)benzoate

[1107]

[1108] Similar to intermediate 65, intermediate 91 is prepared as follows: starting from a suitable amine.

[1109] 3-(aminomethyl)benzoate hydrochloride: 300 mg, 1.49 mmol (1.0 equivalent)

[1110] Purified by FCC (Silica, 0-10% MeOH / DCM)

[1111] Yield / Dose: 334 mg, 1.17 mmol, 79% yield

[1112] LC-MS Method 1: t R = 0.76 minutes, MS (ESI) m / z = 284.4 / 286.4[M+H]+

[1113] Intermediate 92: 1-(2-((2-((3-((tert-butoxycarbonyl)(methyl)amino)propyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((3-(methoxycarbonyl)benzyl)amino)-2-oxoethyl)-4,4-dimethylpiperidin-1-onium (zwitterion)

[1114]

[1115] Similar to Example 4, intermediate 92 was prepared as follows: starting from the corresponding bromide and amine.

[1116] Intermediate 90: 152 mg, 0.31 mmol (1.0 equivalent)

[1117] Intermediate 91: 90 mg, 0.31 mmol (1.0 equivalent)

[1118] Purification via FCC (Sfär amino, 0-2% MeOH / DCM)

[1119] Dosage / Yield: 56 mg, 0.08 mmol, 26% yield

[1120] LC-MS Method 1: t R = 0.81 minutes, MS (ESI) m / z = 686.9 [M]+

[1121] Intermediate 93: Ethyl 3-(2-bromoacetamido)-4-methylthiophene-2-carboxylic acid ester

[1122]

[1123] Similar to intermediate 1, intermediate 93 is prepared as follows: starting from a suitable amine:

[1124] Ethyl 3-amino-4-methylthiophene-2-carboxylate: 820 mg, 4.41 mmol (1.0 equivalent)

[1125] Yield / Quantity: 1.36 g, quantitative yield.

[1126] LC-MS Method 1: t R = 0.92 minutes, MS (ESI) m / z = 306.1 / 308.1 [M+H]+

[1127] Intermediate 94: 1-(2-((2-((3-((tert-butoxycarbonyl)(methyl)amino)propyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(ethoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethylpiperidin-1-onium (zwitterion)

[1128]

[1129] Similar to Example 4, intermediate 94 was prepared as follows: starting from the corresponding bromide and amine.

[1130] Intermediate 90: 120 mg, 0.21 mmol (1.0 equivalent)

[1131] Intermediate 93: 77 mg, 0.21 mmol (1.0 equivalent)

[1132] Purification via FCC (Sfär amino, 0-2% MeOH / DCM)

[1133] Dosage / Yield: 43 mg, 0.06 mmol, 29% yield

[1134] LC-MS Method 1: t R = 0.93 minutes, MS (ESI) m / z = 706.3 [M]+

[1135] Intermediate 95: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(((1-(tert-butoxycarbonyl)piperidin-4-yl)methyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1136]

[1137] Similar to Example 4, intermediate 95 was prepared as follows: starting from the corresponding bromide and amine.

[1138] Intermediate 102: 130 mg, 0.26 mmol (1.0 equivalent)

[1139] N-Benzyl-2-bromoacetamide: 60 mg, 0.26 mmol (1.0 equivalent)

[1140] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[1141] Yield / Dose: 76 mg, 0.11 mmol, 40% yield

[1142] LC-MS Method 1: t R = 0.85 minutes, MS (ESI) m / z = 640.4 [M]+

[1143] Intermediate 96: 4-(3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carboxamido)piperidine-1-carboxylic acid tert-butyl ester

[1144]

[1145] Similar to intermediate 36, intermediate 96 is prepared as follows: starting from a suitable acid and amine.

[1146] Intermediate 13: 500 mg, 1.41 mmol (1.0 equivalent)

[1147] 4-Aminopiperidine-1-carboxylic acid tert-butyl ester: 423 mg, 2.11 mmol (1.3 equivalents)

[1148] Purified by FCC (Sfär silica, 30% EtOAc / cyclohexane)

[1149] Yield / Quantity: 400 mg, 0.84 mmol, 59% yield

[1150] LC-MS Method 1: t R = 0.67 minutes, MS (ESI) m / z = 479.3 [M+H]+

[1151] Intermediate 97: 1-(2-((2-((1-(tert-butoxycarbonyl)piperidin-4-yl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-((methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1152]

[1153] Similar to Example 4, intermediate 97 was prepared as follows: starting from the corresponding bromide and amine.

[1154] Intermediate 1: 128 mg, 0.41 mmol (1.0 equivalent)

[1155] Intermediate 96: 199 mg, 0.41 mmol (1.0 equivalent)

[1156] Purification via FCC (Sfär amino, 0-3% MeOH / DCM)

[1157] Dosage / Yield: 110 mg, 0.16 mmol, 38% yield

[1158] LC-MS Method 1: t R = 1.02 minutes, MS (ESI) m / z = 690.7 [M]+

[1159] Intermediate 98: (2-(3-(2-(azacycloheptan-1-yl)acetamido)-4-methylthiophene-2-carbamate)ethyl)(methyl)carbamate tert-butyl ester

[1160]

[1161] Similar to intermediate 36, intermediate 98 is prepared as follows: starting from a suitable amine and carboxylic acid.

[1162] Intermediate 13: 2.0 g, 6.75 mmol (1.0 equivalent)

[1163] (2-Aminoethyl)(methyl)carbamate tert-butyl ester: 1.76 g, 10.0 mmol (1.5 equivalents)

[1164] Purified via FCC (Sfäramino, 65% (EtOAc:EtOH 9:1) / DCM)

[1165] Dosage / Yield: 700 mg, 1.54 mmol, 23% yield.

[1166] LC-MS Method 1: t R = 0.60 minutes, MS (ESI) m / z = 453.7 [M]+

[1167] Intermediate 99: 2-Bromo-N-(2-(trifluoromethoxy)benzyl)acetamide

[1168]

[1169] 2-Bromoacetyl bromide (0.78 mL, 8.55 mmol) was added dropwise to a suspension of potassium carbonate (1.34 g, 9.69 mmol) and (2-(trifluoromethoxy)phenyl)methylamine (0.56 g, 5.70 mmol) in ACN (20 mL) at 0 °C. After stirring at RT for 2 h, the solid and the fraction filtered off by vacuum evaporation were collected. The crude product was purified by FCC (KP Sfär amino, 3% MeOH / DCM) to provide the title compound (intermediate 99: 737 mg, 2.36 mmol, 45% yield).

[1170] LC-MS Method 1: t R = 0.92 minutes, MS (ESI) m / z = 312.0 / 314.0 [M+H]+

[1171] Intermediate 100: 1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-oxo-2-((2-(trifluoromethoxy)benzyl)amino)ethyl)azacycloheptane-1-onium (zwitterion)

[1172]

[1173] Similar to Example 4, intermediate 100 was prepared as follows: starting from the corresponding bromide and amine.

[1174] Intermediate 98: 200 mg, 0.42 mmol (1.0 equivalent)

[1175] Intermediate 99: 170 mg, 0.42 mmol (1.0 equivalent)

[1176] Purification via FCC (Sfär amino, 10% MeOH / DCM)

[1177] Dosage / Yield: 68 mg, 0.099 mmol, 24% yield

[1178] LC-MS Method 1: t R= 0.84 minutes, MS (ESI) m / z = 684.3 [M]+

[1179] Example 33: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride hydrochloride

[1180] At RT, a 2 M Et2O solution (0.5 mL, 1 mmol) of HCl was added to a DCM (1 mL) solution as a chloride salt of Example 34 (17 mg, 0.030 mmol). After 16 minutes, the evaporation was evaporated and the solid was washed with Et2O to provide the title compound (Example 33: 12 mg, 0.021 mmol, 79% yield).

[1181] LC-MS Method 2: t R = 0.66 minutes, MS (ESI) m / z = 489.2 [M]+

[1182] 1 H NMR (500 MHz, DMSO-d6) δppm 11.82 (br s, 1 H), 10.84 (s, 1 H), 9.16 (brs, 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).

[1183] Similar to the description of Example 33, the following examples are prepared by starting from suitable intermediates or examples.

[1184]

[1185]

[1186]

[1187]

[1188] Example 49: 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1189] Step 1 - 1-(2-((2-(tert-butoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((2-((methoxycarbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide (Intermediate 46)

[1190]

[1191] Similar to intermediate 42, intermediate 46 is prepared as follows: starting from a suitable amine and bromide:

[1192] Intermediate 40: 200 mg, 0.57 mmol (1.0 equivalent)

[1193] Intermediate 1: 167 mg, 0.57 mmol (1.0 equivalent)

[1194] Dosage / Yield: 226 mg, 0.35 mmol, 62% yield.

[1195] LC-MS Method 1: t R = 0.83 minutes, MS (ESI) m / z = 564.3 [M]+

[1196] Step 2 - 1-(2-((2-carboxy-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium chloride (Intermediate 47)

[1197]

[1198] Similar to intermediate 43, intermediate 47 is prepared as follows: starting from a suitable ester.

[1199] Intermediate 46: 223 mg, 0.35 mmol (1.0 equivalent)

[1200] Yield / quantity: 226 mg, 83% wt, quantitative yield.

[1201] Method 1: t R = 0.65 minutes, MS (ESI) m / z = 508.2 [M]+

[1202] Step 3 - 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 49)

[1203] Similar to Example 27, Example 49 was prepared as follows: starting from suitable amines and carboxylic acids.

[1204] Intermediate 47: 115 mg, 0.21 mmol (1.0 equivalent)

[1205] 1-Methylpiperazine: 42 mg, 0.42 mmol (2.0 equivalent)

[1206] Purified by FCC (Sfär amino, 0-5% MeOH / DCM).

[1207] Dosage / Yield: 12 mg, 0.020 mmol, 9% yield.

[1208] LC-MS Method 1: t R = 1.04 minutes, MS (ESI) m / z = 590.4 [M]+

[1209] 1 H NMR (500 MHz, DMSO-d6)δppm 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, 4H), 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)

[1210] Example 54: 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1211] Similar to Example 27, Example 54 was prepared as follows: starting from suitable amines and carboxylic acids.

[1212] Intermediate 47: 115 mg, 0.21 mmol (1.0 equivalent)

[1213] Morpholine: 0.027 mL, 0.32 mmol (1.5 equivalents)

[1214] Purification via FCC (Sfär amino, 0-5% MeOH / DCM)

[1215] Dosage / Yield: 38 mg, 0.066 mmol, 29% yield.

[1216] LC-MS Method 1: t R = 0.66 minutes, MS (ESI) m / z = 577.3 [M]+

[1217] 1 H NMR (500 MHz, DMSO-d6) δppm 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)

[1218] Example 37: 1-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1219] Step 1 - Methyl 3-iodo-4-methylthiophene-2-carboxylate (Intermediate 48)

[1220]

[1221] Under a nitrogen atmosphere, a solution of methyl 3-amino-4-methylthiophene-2-carboxylic acid (3.00 g, 17.5 mmol) in ACN (44 mL) was added to diiodomethane (5.65 mL, 70.1 mmol), and the reaction mixture was stirred at 70 °C for 20 min. After this 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 complete, the reaction mixture was stirred at 60 °C for 4 h. The mixture was then slowly poured into a 0.5 M aqueous solution of HCl (30 mL) and extracted with EtOAc (3x). The collected organic layer was dried over MgSO4 and concentrated under reduced pressure to give a crude product, which was purified by FCC (KP Sfär silica, 0-10% EtOAc / cyclohexane) to provide the title compound (intermediate 48: 2.90 g, 10.3 mmol, 59% yield).

[1222] LC-MS Method 1: t R = 1.15 minutes, MS (ESI) m / z = 283.0 [M+H]+

[1223] Step 2 - Methyl 3-cyano-4-methylthiophene-2-carboxylate (Intermediate 51)

[1224]

[1225] A round-bottom 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 purged and backfilled with nitrogen (3x), and then potassium hexacyanoferrate(II) 0.1 N standard solution (60.0 mL, 6.00 mmol) and 1,4-dioxane (52 mL) were added under a nitrogen atmosphere. Three vacuum-nitrogen cycles were performed, and the reaction mixture was heated at 100 °C with stirring for 4 hours. XPhos Pd G3 (0.16 mg, 0.19 mmol) and potassium hexacyanoferrate(II) 0.1 N standard solution (60.0 mL, 6.00 mmol, degassed before addition) were then added at RT. The reaction mixture was heated again at 100 °C with stirring for 5 hours. It was then cooled to RT and extracted with EtOAc. The collected organic layer was concentrated under reduced pressure to provide a crude product, which was purified by FCC (KP Sfär silica, 0-10% EtOAc / cyclohexane) to give the title compound (intermediate 51: 0.78 g, 4.30 mmol, 42% yield).

[1226] LC-MS Method 1: t R = 0.89 minutes, MS (ESI) m / z = 181.9 [M+H]+

[1227] Step 3 - 3-(aminomethyl)-4-methylthiophene-2-carboxamide (intermediate 49)

[1228]

[1229] In a hydrogenation flask under a nitrogen atmosphere, a solution of methyl 3-cyano-4-methylthiophene-2-carboxylate (intermediate 51, 680 mg, 3.75 mmol) in MeOH (100 mL) was added to Raney®-nickel (70.0 mg, 50% aqueous slurry), followed by a 7 M MeOH solution of ammonia (15.0 mL, 185 mmol). The flask was emptied, backfilled with hydrogen (1 atm), and stirred for 24 hours. The solid was then allowed to settle, and the organic phase was removed under a nitrogen atmosphere. The remaining solid was washed with MeOH and DCM. The collected organic layer was concentrated under reduced pressure to provide a crude product, which was purified by FCC (Sfär amino, 0-100% EtOAc / MeOH (9 / 1 v / v), in cyclohexane) to provide the title compound (intermediate 49: 173 mg, 1.01 mmol, 27% yield).

[1230] LC-MS Method 2: t R = 0.46 minutes, MS (ESI) m / z = 171.1 [M+H]+

[1231] Step 4 - 3-((2-(azacycloheptan-1-yl)acetamido)methyl)-4-methylthiophene-2-carboxamide (Intermediate 50)

[1232]

[1233] 2-Chloroacetyl chloride (70 mL, 0.89 mmol) was slowly added 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). The reaction was stirred at RT for 4 h. Then, azircycloheptane (600 mL, 5.36 mmol) was added to the reaction mixture at RT. After stirring for 3 h, the volatiles were removed under reduced pressure, and the crude product was purified by FCC (Sfäramino, 0-100% EtOAc / MeOH (9 / 1 v / v), in cyclohexane) to provide the title compound (intermediate 50: 140 mg, 0.452 mmol, 51% yield).

[1234] LC-MS Method 2: t R = 0.82 minutes, MS (ESI) m / z = 310.2 [M+H]+

[1235] Step 5 - 1-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Example 37)

[1236] Similar to Example 39, Example 37 was prepared as follows: starting from a suitable amine and bromide.

[1237] Intermediate 50: 40 mg, 0.13 mmol (1.0 equivalent)

[1238] Intermediate 2: 30 mg, 0.15 mmol (1.1 equivalents)

[1239] Purification was performed via FCC (Sfäramino, 0-100% EtOAc / MeOH (9 / 1 v / v), in cyclohexane).

[1240] Dosage / Yield: 10 mg, 0.023 mmol, 18% yield.

[1241] LC-MS Method 2: t R = 0.59 minutes, MS (ESI) m / z = 434.3 [M]+

[1242] 1H NMR (500 MHz, DMSO-d6) δppm 9.23 (br t, J=5.0 Hz, 1 H), 8.33 (d, J=1.5Hz, 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=1.6Hz, 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).

[1243] Example 40: (R)-1-(2-(((2-carbamoyl-4-methylthiophene-3-yl)methyl)amino)-2-oxoethyl)-1-(2-oxo-2-((1-phenylethyl)amino)ethyl)azacycloheptane-1-onium bromide

[1244] Similar to Example 39, Example 40 was prepared as follows: starting from suitable amines and bromides.

[1245] Intermediate 50: 50 mg, 0.16 mmol (1.0 equivalent)

[1246] Intermediate 3: 44 mg, 0.18 mmol (1.1 equivalents)

[1247] Purified by FCC (Sfär-C18, 0-50% ACN + 0.1% HCOOH / H2O + 0.1% HCOOH).

[1248] Dosage / Yield: 7.5 mg, 0.014 mmol, 8% yield.

[1249] LC-MS Method 1: t R = 0.65 minutes, MS (ESI) m / z = 471.32 [M]+

[1250] 1H NMR (500 MHz, DMSO-d6) δppm 9.11 (d, J=7.7 Hz, 1 H), 8.76 (t, J=5.4 Hz, 1H), 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).

[1251] The conversion of the zwitterionic form of the compounds of the present invention to obtain the corresponding salt form can be carried out as described in the following examples (chloride salts).

[1252] Example 1, as a chloride salt: 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethyl-1-ammonium chloride

[1253] Example 1 (zwitterion) (33 mg, 0.070 mmol) was dissolved in DCM (1.0 mL) and HCl was added dropwise in a 2 M Et2O solution (50 µL, 0.11 mmol) at RT. After 5 minutes, the volatiles were removed under reduced pressure, yielding the title compound (Example 1, as a chloride salt: 7 mg, 0.015 mmol, 21% yield), as a white powder.

[1254] LC-MS Method 1: t R = 0.63 minutes, m / z = 443.4 [M]+

[1255] 1 H NMR (400 MHz, DMSO-d6) δppm 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)

[1256] Similar to the procedure described in Example 1 as a chloride salt, starting from a suitable example as a zwitterion, the following examples have been prepared.

[1257]

[1258]

[1259] Example 78: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide

[1260] Step 1 - Methyl 4-(2-bromoacetamido)-5-methylthiophene-3-carboxylic acid ester (Intermediate 103)

[1261]

[1262] Similar to intermediate 1, intermediate 103 is prepared as follows: starting from a suitable amine:

[1263] 4-Amino-5-methylthiophene-3-carboxylic acid methyl ester hydrochloride: 1.50 g, 7.22 mmol (1.0 equivalent)

[1264] Yield / Quantity: 1.57 g, 5.36 mmol, 74% yield.

[1265] UPLC Method 1: rt 0.79 minutes, MS (ESI) m / z = 292.0 / 294.0 [M+H]+

[1266] Step 2 - Methyl 4-(2-(azacycloheptan-1-yl)acetamido)-5-methylthiophene-3-carboxylic acid (Intermediate 104)

[1267]

[1268] Similar to intermediate 11, intermediate 104 is prepared as follows: starting from a suitable bromide:

[1269] Intermediate 103: 1.57 g, 5.36 mmol (1.0 equivalent)

[1270] Yield / Quantity: 1.52 g, 4.90 mmol, 91% yield.

[1271] UPLC Method 1: rt 0.46 minutes, MS (ESI) m / z = 311.1 [M+H]+

[1272] Step 3 - Sodium chloride of 4-(2-(azacycloheptan-1-yl)acetamido)-5-methylthiophene-3-carboxylic acid (intermediate 105)

[1273]

[1274] Similar to intermediate 7, intermediate 105 is prepared as follows: starting from a suitable ester:

[1275] Intermediate 104: 1.52 g, 4.90 mmol (1.0 equivalent)

[1276] Yield / quantity: 1.97 g, 88% wt, quantitative yield.

[1277] UPLC Method 1: rt 0.41 minutes, MS (ESI) m / z = 297.1 [M+H]+

[1278] Step 4 - 2-(azacycloheptan-1-yl)-N-(2-methyl-4-(morpholin-4-carbonyl)thiophen-3-yl)acetamide (intermediate 106)

[1279]

[1280] Similar to intermediate 8, intermediate 106 is prepared as follows: starting from a suitable acid and amine:

[1281] Intermediate 105: 350 mg, 0.97 mmol (1.0 equivalent)

[1282] Morpholine: 0.17 mL, 1.97 mmol (2.0 equivalent)

[1283] Amount / Yield: 211 mg, 0.57 mmol, 59% yield.

[1284] UPLC Method 1: rt 0.44 minutes, MS (ESI) m / z = 366.2 [M+H]+

[1285] Step 5 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide (Example 78)

[1286] Similar to Example 3, Example N1568-18-1 CHD-074186 was prepared as follows: starting from suitable amines and bromides:

[1287] Intermediate 106: 110 mg, 0.30 mmol (1.0 equivalent)

[1288] N-Benzyl-2-bromoacetamide: 76 mg, 0.33 mmol (1.1 equivalents)

[1289] Dosage / Yield: 121 mg, 0.20 mmol, 68% yield.

[1290] UPLC Method 1: rt 0.64 minutes, MS (ESI) m / z = 513.2 [M]+

[1291] 1 H NMR (500 MHz, DMSO-d6) δ= 10.55 - 10.16 (m, 1H), 9.05 (br s, 1H), 7.56 -7.06 (m, 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 - 3.39 (m, 4H), 2.23 (br s, 3H), 2.01 - 1.86 (m, 4H), 1.72 -1.57 (m, 4H)

[1292] Example 79: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide

[1293] Step 1 - 2-(azacycloheptan-1-yl)-N-(2-methyl-4-(pyrrolidine-1-carbonyl)thiophen-3-yl)acetamide (intermediate 107)

[1294]

[1295] Similar to intermediate 8, intermediate 107 is prepared as follows: starting from a suitable acid and amine:

[1296] Intermediate 104: 350 mg, 0.97 mmol (1.0 equivalent)

[1297] Pyrrolidine: 0.16 mL, 1.97 mmol (2.0 equivalent)

[1298] Amount / Yield: 204 mg, 0.58 mmol, 59% yield.

[1299] UPLC Method 1: rt 0.50 minutes, MS (ESI) m / z = 350.2 [M+H]+

[1300] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide (Example 79)

[1301] Similar to Example 3, Example 79 was prepared as follows: starting from suitable amines and bromides:

[1302] Intermediate 107: 110 mg, 0.32 mmol (1.0 equivalent)

[1303] N-Benzyl-2-bromoacetamide: 79 mg, 0.35 mmol (1.1 equivalents)

[1304] Dosage / Yield: 96 mg, 0.17 mmol, 53% yield.

[1305] UPLC Method 1: rt 0.68 minutes, MS (ESI) m / z = 497.3 [M]+

[1306] 1 H NMR (500 MHz, DMSO-d6)δ= 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 (brt, 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)

[1307] Example 80: 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-((2-methoxyethyl)(methyl)carbamoyl)-2-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide

[1308] Step 1 - 4-(2-(azacycloheptan-1-yl)acetamido)-N-(2-methoxyethyl)-N,5-dimethylthiophene-3-carboxamide (intermediate N1568-17-1)

[1309]

[1310] Similar to intermediate 8, intermediate N1568-17-1 is prepared as follows: starting from a suitable acid and amine:

[1311] Intermediate 101: 350 mg, 0.97 mmol (1.0 equivalent)

[1312] 2-Methoxy-N-methylethyl-1-amine: 0.20 mL, 1.97 mmol (2.0 equivalents)

[1313] Dosage / Yield: 200 mg, 0.54 mmol, 55% yield.

[1314] UPLC Method 1: rt 0.47 minutes, MS (ESI) m / z = 368.2 [M+H]+

[1315] Step 2 - 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-((2-methoxyethyl)(methyl)carbamoyl)-2-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium bromide (Example 80)

[1316] Similar to Example 3, Example 80 was prepared as follows: starting from suitable amines and bromides:

[1317] Intermediate N1568-17-1: 110 mg, 0.30 mmol (1.0 equivalent)

[1318] N-Benzyl-2-bromoacetamide: 75 mg, 0.33 mmol (1.1 equivalents)

[1319] Dosage / Yield: 97 mg, 0.16 mmol, 54% yield.

[1320] UPLC Method 1: rt 0.66 minutes, MS (ESI) m / z = 515.3 [M]+

[1321] 1 H NMR (500 MHz, DMSO-d6) δ= 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 - 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 - 1.57 (m,4H)

[1322] Compounds C1 and C2 are prepared as described below.

[1323] C1 is characterized by having a phenyl ring instead of an amide-substituted tiophene or benzothiophene ring in the compound of the present invention.

[1324] C2 is characterized by having a different heteroaryl ring instead of the amide-substituted tiophene or benzothiophene ring in the compound of the present invention.

[1325] Compound C1: N,N-bis(phenylcarbamoylmethyl)dimethylammonium chloride (i.e., carbochloride) was prepared according to the description on page 6 of WO99 / 63985.

[1326] Compound C2: 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((5-(methoxycarbonyl)-3-methylisoxazo-4-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion)

[1327] Step 1 - Methyl 4-(2-bromoacetamido)-3-methylisoxazole-5-carboxylic acid ester (Intermediate 52)

[1328]

[1329] 2-Bromoacetyl bromide (0.31 mL, 3.52 mmol) was added dropwise to a DCM suspension of potassium carbonate (1.10 g, 8.01 mmol) and methyl 4-amino-3-methylisoxazole-5-carboxylic acid (500 mg, 3.20 mmol). After 3 h, water was added and the organic phase was separated, dried on a phase separator, and concentrated under reduced pressure. The residue was purified by FCC (KP Sfär silica, 0-40% cyclohexane / EtOAc) to provide the title compound (intermediate 52,800 mg, 2.89 mmol, 90% yield) as a creamy white powder.

[1330] LC-MS Method 1: t R = 0.66 minutes, MS (ESI) m / z = 277.1 / 279.0 [M+H]+

[1331] Step 2 - 2-(azacycloheptan-1-yl)-N-(isoxazo-3-yl)acetamide (intermediate 54)

[1332]

[1333] A solution of intermediate 53 (200 mg, 1.25 mmol) in ACN (5 mL) was added with azircycloheptane (0.28 mL, 2.49 mmol) and potassium carbonate (361 mg, 2.62 mmol), and the mixture was stirred at RT for 2 h. The evaporation was distilled off, and the residue was partitioned between water and EtOAc. The organic phase was separated, and the aqueous phase was extracted with EtOAc. The collected organic layer was washed with brine and concentrated under reduced pressure to provide the title compound (intermediate 54: 270 mg, 1.21 mmol, 97% yield), an orange oil.

[1334] LC-MS Method 2: t R = 0.78 minutes, MS (ESI) m / z = 224.4 [M]+

[1335] Step 3 - 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((5-(methoxycarbonyl)-3-methylisoxazo-4-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (zwitterion) (Compound C2)

[1336] Intermediate 52 (50 mg, 0.180 mmol) was added to a 1.2 mL solution of ACN containing intermediate 54 (40 mg, 0.180 mmol). The mixture was stirred at RT for 24 h. The mixture was then concentrated under reduced pressure, and the residue was purified by FCC (Sfär amino, 0–5% MeOH / DCM) to provide the title compound (compound C2: 57 mg, 0.114 mmol, 75% yield).

[1337] LC-MS Method 2: t R = 0.52 minutes, MS (ESI) m / z = 420.1 [M]+

[1338] 1 H NMR (500 MHz, DMSO-d6) δppm 12.25 - 14.42 (m, 1 H), 8.76 (d, J=1.6 Hz, 1H), 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)

[1339] Pharmacological activity of the compounds of this invention

[1340] hNaV 1.7 cell assay protocol

[1341] The intracellular and extracellular inhibition of NaV 1.7 was tested in the automated patch-clamp test described herein using representative embodiments of the invention.

[1342] Cell culture

[1343] CHO cells stably expressing the 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 µg / ml hygromycin B (Invitrogen, Waltham MA). Cells were grown and maintained at 37°C in a humidified environment with 5% CO2 / air. Cells were removed from culture flasks for passage and harvesting using TrypLE Express (Sigma-Aldrich, St. Louis, MO). Experimental flasks were prepared with antibiotic-free 2–4 day-old cells and used at ~80% confluence. Prior to experiments, cells were treated with Ca-free... 2+ and Mg 2+ Cells were washed with PBS and desorbed with pre-warmed TrypLE Express. After a brief centrifugation step, the cells were resuspended in CHO-S-serum-free medium II (Life Technologies, Carlsbad CA). Cells were counted, with a final concentration set at 3-4 million cells per mL.

[1344] Patch clamp solution & drugs

[1345] The intracellular solution (or internal solution) contains the following: 140 mM CsF, 10 mM NaCl, 1 mM EGTA, and 10 mM HEPES, adjusted to pH 7.2 with CsOH, and the osmolality is adjusted to 325 with sucrose. The extracellular solution (or external solution) contains the following: 40 mM NMDG, 100 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose, adjusted to pH 7.4 with HCl, and the osmolality is adjusted to 310 with sucrose. All chemicals were sourced from Sigma-Aldrich, St. Louis, MO.

[1346] For both intracellular and extracellular applications of the test compound, the spotted compound was dissolved in intracellular and extracellular solutions, respectively, and tested up to 60 μM, resulting in a pIC value of not less than 4.2.50 value.

[1347] Automated Patch Clamp Testing Solution

[1348] The automated patch-clamp procedure was performed as follows: The temperature was set to 22°C on a Qube 384 equipped with multi-well QChips (Sophion Bioscience A / S, Ballerup, Denmark). A whole-cell configuration was formed using the default Qube seal and break-in parameters. The membrane potential was maintained at -100 mV before the voltage program began. The voltage program consisted of 20 pulses, with 30 scans for intracellular applications and 55 scans for extracellular applications, both before and after compound administration. Cells were held at -100 mV, and a depolarization pulse to -20 mV was applied for 10 ms. With the holding potential at -100 mV, the scan interval was set to 9 s.

[1349] Internal blocking effect (intracellular inhibition) of the test compound

[1350] For intracellular application of the compound, remove the QChip from the recording chamber. Replace the intracellular solution with a solution containing the test compound. After the intracellular solution exchange, return the QChip to the recording chamber and apply a voltage pulse.

[1351] Data filtering was performed using Sophion analyzer software. Only wells with a minimum sealing resistance of 40 MΩ and a pre-compound application current of 1 nA were used for further analysis using Aplus software. The average peak current amplitude of the 20th pulse of the last three programs applied in both the pre-compound application and post-compound application groups was calculated. For each well, the post-compound application / pre-compound application ratio was calculated.

[1352] The results were then standardized as follows: 0.3% DMSO control was considered 0%, and complete blockade (100 µM N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium chloride, i.e., QX314, for internal administration) was considered 100% inhibition. %activity values ​​were plotted, and concentration-response curves were fitted using a 4-parameter logarithmic Hill formula. The IC50 was estimated from the fitted curves. 50 Values ​​and calculation of intracellular pIC 50 value.

[1353] External blocking effect (extracellular inhibition) of the test compound

[1354] For extracellular administration of the compound, the extracellular solution is replaced with a solution containing the test compound. A voltage pulse is applied after the extracellular solution exchange.

[1355] Data filtering was performed using Sophion analyzer software. Only wells with a minimum sealing resistance of 50 MΩ and a pre-compound application current of 1 nA were used for further analysis using Aplus software. The average peak current amplitude of the 20th pulse of the last three programs applied in both the pre-compound application and post-compound application groups was calculated. For each well, the post-compound application / pre-compound application ratio was calculated.

[1356] The results were then standardized as follows: 0.3% DMSO control was considered 0%, and complete blockade (300 µM tetracaine, for external application) was considered 100% inhibition. %activity values ​​were plotted, and concentration-response curves were fitted using a 4-parameter logarithmic Hill formula. The IC50 was estimated from the fitted curves. 50 Values ​​and calculations of extracellular pIC 50 value.

[1357] A pIC value ≤4.2 was obtained at the maximum compound concentration of 60 μM. 50 At this point, the inhibition rate is less than or equal to 50%.

[1358] Intracellular and extracellular inhibition reports for representative embodiments are shown in Table 2.

[1359] Table 2 - Intracellular and extracellular inhibition of representative examples

[1360]

[1361]

[1362] As can be recognized, the compounds in Table 2 (i.e., the compounds according to the invention) exhibit high intracellular inhibitory activity against the NaV 1.7 receptor. Conversely, the compounds in Table 2 exhibit lower extracellular inhibitory activity against the NaV 1.7 receptor.

[1363] Table 3 shows the inhibition data of the selected embodiments, tested in both zwitterionic and salt (chloride) forms. As can be appreciated, both forms exhibit high intracellular inhibitory activity against the NaV 1.7 receptor. Negligible variations were observed in some cases between the respective zwitterionic and salt forms, which can be considered within the range of experimental error.

[1364] Table 3 - Comparison of intracellular and extracellular inhibition in representative examples presented in zwitterionic or salt form, hNav 1.7

[1365]

[1366] Accordingly, the compounds of the present invention, in both zwitterionic and salt forms, can be used to treat respiratory diseases such as cough, asthma, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD), and especially chronic cough.

[1367] Comparison of compounds

[1368] Compounds C1 and C2 were tested in the same binding assay described above, and their intracellular and extracellular pIC values ​​were examined. 50 The report is in Table 4.

[1369] Table 4 - Comparison of intracellular and extracellular inhibition of compounds

[1370]

[1371] As shown in Tables 2, 3, and 4 (Example 7), the compounds of formula (I) of the present invention exhibit intracellular inhibitory activity against NaV 1.7, expressed as pIC values ​​higher than 5, 5 to 5.5, higher than 5.5, or 5.5 to 6. 50 value.

[1372] These data show that, unlike comparative compound C1, the presence of an amide-substituted thiophene or benzothiophene ring in the compounds of the present invention leads to a surprisingly and notably significant increase in intracellular inhibitory activity against NaV 1.7 and increases the difference in intracellular and extracellular inhibition of NaV 1.7 (reported as DELTA in Table 4).

[1373] Furthermore, as noted in the same section of the Experimental section, the reported data show that, in contrast to the comparative compound C2, the presence of an amide-substituted thiophene ring instead of a different heteroaryl ring in the compounds of the present invention, particularly in Example 7, unexpectedly and notably determined a significant increase in intracellular inhibitory activity against the NaV 1.7 receptor and increased the difference in intracellular and extracellular inhibition of NaV 1.7 (DELTA).

[1374] hERG testing solution

[1375] The effects of test substances on the tail current of human ether-à-go-go-related genes (hERG) were evaluated using a QPatch device (Sophion Bioscience A / S, Ballerup, Denmark) in an automated patch-clamp protocol.

[1376] Cell culture

[1377] hERG was expressed in HEK293 cells under tetracycline induction.

[1378] Cells were cultured in the following minimum essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% non-essential amino acids, 1% sodium pyruvate, 2 mM L-glutamine, 1% penicillin-streptomycin, 15 µg / ml isoprothiolane, and 100 µg / ml hygromycin. hERG cells were induced with 10 µg / ml tetracycline for 24 to 72 hours prior to recording.

[1379] Prior to the experiment, cells were washed with calcium- and magnesium-free Dulbecco phosphate-buffered saline (DPBS), desorbed with TrypLE, and then resuspended and cell aggregates broken in serum-free medium containing 25 mM Hepes and soybean trypsin inhibitor. Cells were counted and the final concentration was set at 4–6 million cells per mL. All chemicals used for cell culture were from Life Technologies Italia Srl, Monza, Italy.

[1380] Patch clamp solution & drugs

[1381] The intracellular solution contained the following (mM): KCl 130, MgCl2 1, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 5, MgATP 5, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) 10, adjusted to pH 7.2 with 1 M KOH.

[1382] The external solution contains the following (mM): NaCl 137; KCl 4; CaCl2 1.8; MgCl2 1; d-glucose 10; HEPES 10; and is adjusted to pH 7.4 with NaOH.

[1383] All chemicals used in the patch clamp solution are sourced from Merck Life Science Srl, Milano, Italy.

[1384] To evaluate the degree of external blockade, the compound was first dissolved in DMSO and then diluted in an extracellular buffer to achieve final experimental concentrations of 0.6 μM, 6 μM, and 60 μM in 0.3% DMSO.

[1385] Voltage program

[1386] A single-well QPlate chip (Sophion Bioscience, Denmark) was used for voltage-clamp experiments. The membrane potential was held at -80 mV before the voltage program began. The voltage program consisted of the following steps in sequence: -50 mV for 200 ms, +20 mV for 4.8 s, -50 mV for 5 s, and then back to the holding potential. The hERG tail current was measured during the repolarization step from +20 mV to -50 mV. Each voltage program scan lasted 15 s and was continuously repeated at a frequency of 0.066 Hz during the experiment.

[1387] Liquid program

[1388] The external solution and then the medium solution (equivalent to the external solution containing 0.3% DMSO) were each applied for 3 minutes and 45 seconds to achieve a stable current recording. Then, three test compounds at increased concentrations (0.6, 6, and 60 μM, in 0.3% DMSO) were each applied for 5 minutes.

[1389] Data Analysis

[1390] Using Sophion Analyzer software (Sophion Bioscience, Denmark), for each concentration in each cell, the mean tail current was measured from the last four voltage scans during each compound administration procedure and used to calculate the % inhibition by comparison with the pretreated group. IC50 was estimated using the Hill fitting formula in the Sophion Analyzer software. 50 value.

Claims

1. Compound of formula (I) (I) At least one of Y and Z is S, and the other is CR4; R1 and R2 are independently -(C1-C6)alkyl or fused to form -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl is optionally substituted by one or more groups selected from halogens, -(C1-C6)alkyl and -(C1-C6)alkyl-OR8; R3 and R4 are independently H or -(C1-C6)alkyl, or, in the case that Y is CR4, R3 and R4 are fused to form an aryl group; R5 and R6 are independently H or selected from -(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)heterocyclicalkyl and -(C3-C7)heterocyclicalkyl, or fused to form -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C6)aminoalkyl or -(C1-C6)alkyl-NR7R8 is optionally substituted by one or more groups selected from -(C1-C6)alkyl, -C(O)OR8 and -SO2R7; R7 and R8 are independently H or selected from -(C1-C6)alkyl, -(C3-C6)cycloalkyl and -(C1-C6)haloalkyl, or fused to form -(C3-C7)heteroalkyl; L1 is a bond or selected from -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-; and A is a ring selected from aryl and heteroaryl, wherein any aryl or heteroaryl is optionally substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; provided that any aryl is not substituted by -(C1-C6)alkyl when L1 is a bond. It exists in zwitterionic or pharmaceutically acceptable salt form.

2. The compound of formula (I) according to claim 1, wherein Z, Y, R1, R2, R3, R4, R5, R6, R7 and R8 are as defined in claim 1; L1 is selected from -(C1-C6)alkylene- and -(C1-C6)cycloalkylene-; and A is a ring selected from aryl and heteroaryl, wherein any of the aryl or heteroaryl groups are optionally substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C4)alkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

3. The compound of formula (I) according to claim 1, wherein L1 is a bond, represented by formula (Ia). (Ia) in Z, Y, R1, R2, R3, R4, R5, R6, R7, and R8 as defined in claim 1; and A is a ring selected from aryl and heteroaryl, wherein any of the aryl groups are optionally substituted by one or more groups selected from -OR8, -C(O)OR8 and -C(O)NR7R8, and wherein any of the heteroaryl groups are optionally substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

4. The compound according to any one of the preceding claims, wherein R1 and R2 are independently -(C1-C4)alkyl or fused to -(C3-C7)heteroalkyl, wherein the -(C3-C7)heteroalkyl is optionally substituted by one or more groups selected from halogens, -(C1-C4)alkyl and -(C1-C4)alkyl-OR8; R3 and R4 are independently H or -(C1-C6)alkyl, or, in the case that Y is CR4, R3 and R4 are fused to form an aryl group; R5 and R6 are independently H or selected from -(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)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, or fused to form -(C3-C7)heterocyclicalkyl, wherein the -(C1-C4)alkyl-(C3-C7)heterocyclicalkyl, -(C3-C7)heterocyclicalkyl, -(C1-C4)aminoalkyl or -(C1-C4)alkyl-NR7R8 is optionally substituted by one or more groups selected from -(C1-C4)alkyl, -C(O)OR8 and -SO2R7; and R7 and R8 are independently H or selected from -(C1-C4)alkyl, -(C3-C6)cycloalkyl and -(C1-C4)haloalkyl, or fused to form -(C6-C7)heteroalkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

5. The compound according to any one of the preceding claims, wherein R1 and R2 are fused together to form a -(C6-C7)heterocyclic alkyl group, wherein the -(C6-C7)heterocyclic alkyl group is optionally substituted by one or more groups selected from -OR8, -(C1-C4)alkyl and -(C3-C7)heterocyclic alkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

6. The compound according to any one of the preceding claims, wherein R3 and R4 are independently H or -(C1-C4) alkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

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; It exists in zwitterionic or pharmaceutically acceptable salt form.

8. The compound according to any one of the preceding claims, wherein A is a heteroaryl group, optionally substituted by one or more groups selected from -OR8, -C(O)OR8, -C(O)NR7R8 and -(C1-C6)alkyl; It exists in zwitterionic or pharmaceutically acceptable salt form.

9. The compound according to any claim 1, selected from: 2-(benzylamino)-N,N-dimethyl-N-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)-2-oxoethyl-1-ammonium (Example 1); 2-(benzylamino)-N-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium (Example 2); 2-(benzylamino)-N-(2-((2-(tert-butylcarbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-N,N-dimethyl-2-oxoethyl-1-ammonium (Example 3); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 4); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 5); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 6); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 7); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 8); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium, (Example 9); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 10); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 11); 1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 12); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 13); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 14); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-((tert-butoxycarbonyl)(methyl)amino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 15); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 16); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 17); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 18); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 19); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 20); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetane-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 21); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 22); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxecyclobutane-3-ylcarbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 23); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 24); 1-(2-((2-((2-hydroxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 25); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 26); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 27); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 28); 1-(2-((2-((2-methoxyethyl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-((4-methylisoxazol-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 29); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-morpholinoethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 30); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 31); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-(methanesulfonyl)piperazine-1-carbonyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 32); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 33); 1-(2-((2-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 34); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 35); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-(methanesulfonyl)piperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 36); 1-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 37); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 38); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-(morpholin-4-carbonyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 39); (R)-1-(2-(((2-carbamoyl-4-methylthiophen-3-yl)methyl)amino)-2-oxoethyl)-1-(2-oxo-2-((1-phenylethyl)amino)ethyl)azacycloheptane-1-onium (Example 40); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(trimethylammonium)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 41); 1-(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)-1-(2-(isoxazo-3-ylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 42); 1,1-Di(2-((2-(dimethylcarbamoyl)benzo[b]thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 43); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 44); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 45); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(trimethylammonyl)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 46); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 47); 1-(2-(isoxazo-3-ylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(piperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 48); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(4-methylpiperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 49); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-methoxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 50); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(piperazin-1-yl)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 51); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((2-(4-methylpiperazin-1-yl)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 52); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 53); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 54); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-((2-hydroxyethyl)(methyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 55); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-(methyl(2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 56); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(piperazin-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 57); (S)-1-(2-((2-((6-amino-1-methoxy-1-oxohexane-2-yl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(benzylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 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)azacycloheptane-1-onium (Example 59); 4-Fluoro-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4-methyl-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 60); 1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophene-3-yl)amino)-2-oxoethyl)-1-(2-oxo-2-((2-(trifluoromethoxy)benzyl)amino)ethyl)azacycloheptane-1-onium (Example 61); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-methyl-2-((piperidin-4-ylmethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 62); 1-(2-((2-((2-(cyclopropylamino)ethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 63); 1-(2-((2-(di(2-hydroxyethyl)carbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 64); 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)azacycloheptane-1-onium (Example 65); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (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)piperidine-1-onium (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)azacycloheptane-1-onium (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)piperidine-1-onium (Example 69); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-1-(2-((4-methyl-2-(oxetane-3-ylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 70); 1-(2-((2-(methoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethyl-1-(2-((4-methyl-2-((2-(methylamino)ethyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 71); 1-(2-((2-(ethoxycarbonyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-dimethyl-1-(2-((4-methyl-2-((3-(methylamino)propyl)carbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidine-1-onium (Example 72); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)piperidine-1-onium (Example 73); (2-(4,4-dimethyl-1-(2-((4-methyl-2-(methylcarbamoyl)thiophen-3-yl)amino)-2-oxoethyl)piperidin-1-onthiol-1-yl)acetyl)(isoxazo-3-yl)amide (Example 74); 1-(2-((2-(dimethylcarbamoyl)-4-methylthiophen-3-yl)amino)-2-oxoethyl)-4,4-di(hydroxymethyl)-1-(2-oxo-2-((1-phenylcyclopropyl)amino)ethyl)piperidine-1-onium (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)azacycloheptane-1-onium (Example 76); and (R)-1-(2-((2-((6-amino-1-methoxy-1-oxohexane-2-yl)carbamoyl)-4-methylthiophene-3-yl)amino)-2-oxoethyl)-1-(2-(benzylamino)-2-oxoethyl)azacycloheptane-1-onium (Example 77); 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(morpholin-4-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 78) 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((2-methyl-4-(pyrrolidine-1-carbonyl)thiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 79) 1-(2-(benzylamino)-2-oxoethyl)-1-(2-((4-((2-methoxyethyl)(methyl)carbamoyl)-2-methylthiophen-3-yl)amino)-2-oxoethyl)azacycloheptane-1-onium (Example 80) It exists in zwitterionic or pharmaceutically acceptable salt form.

10. Use of an intermediate compound selected from compounds (V), (VIII), (X), (XI), (XV), and (XVI) for the preparation of a compound of formula (I) according to any one of claims 1 to 9. Wherein R1, R2, R3, R4, R5, L1 and A are as defined in claims 1 to 9, X2 is a halogen, preferably chlorine or bromine, and PG is a -(C1-C6) alkyl group.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9 and one or more pharmaceutically acceptable carriers or excipients mixed therewith.

12. The pharmaceutical composition of claim 11, formulated for administration by inhalation.

13. The compound of formula (I) according to any one of claims 1-9 or the pharmaceutical composition according to claim 11 or 12, used as a medicine.

14. The compound or pharmaceutical composition of formula (I) according to claim 13, for the prevention and / or treatment of diseases, disorders or conditions related to sodium channel receptor mechanisms.

15. The compound or pharmaceutical composition of formula (I) according to claim 13 or 14, for the prevention and / or treatment of respiratory diseases selected from the following: cough, subacute or chronic cough, treatment-resistant cough, intractable 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. The compound or pharmaceutical composition of formula (I) according to claim 15, for the prevention and / or treatment of chronic cough.

Images