DE102018212006B3 - Thioethers as modulators of Kv7.2 / Kv7.3 channels - Google Patents

Abstract

The invention relates to a compound of general formula (I) comprising a thioether structure, a process for their preparation and compounds obtained or obtainable by this process. Furthermore, the invention relates to a compound of general formula (I), or a compound of general formula (I) obtained or obtainable by the preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for use as a medicament, in particular for use as Analgesics and / or for use in tinnitus treatment or prevention and / or for use in the treatment or prevention of convulsive conditions and / or for use in the treatment or prevention of a disease selected from the group consisting of incontinence, anxiety, dependence, mania, bipolar disorder, cognitive disorder and dyskinesia. The invention further relates to a pharmaceutical preparation comprising a compound of the general formula (I) or a compound of the general formula (I) obtained or obtainable by the preparation process or a pharmaceutically acceptable salt of a compound of the general formula (I).

Description

The invention relates to a compound of general formula (I) comprising a thioether structure, a process for their preparation and compounds obtained or obtainable by this process.

Neural hyper excitability plays a crucial role in chronic pain, epilepsy or migraine. In this context, the hyperpolarization of the resting membrane potential of certain neurons by opening potassium channels and the resulting reduced generation of action potentials represents a therapeutic approach that is already being exploited clinically by the drugs flupirtine and retigabine. However, both drugs are associated with rare but serious adverse drug reactions (vide infra) and safer follow-up substances of these drugs are needed to obtain an important therapeutic alternative for many indications.

A voltage-dependent, slowly activating and nonactivating potassium ion current that occurs in the central and peripheral nervous systems is involved in the control of the firing behavior and thus in the regulation of the excitability of a large number of neuronal cells. [1] The molecular basis of M current is formed by Kv7 potassium channels. In most neurons, Kv7.2 and Kv7.3 subunits predominate. [2] In addition, Kv7 potassium channels also occur in the primary sensory cells of the inner ear, in skeletal muscle cells, and in smooth muscle cells. [3-5] Similar to other voltage-gated potassium channels, four subunits each form one channel. Both homo- and heterotetramers can be formed. [6,7] The effect of the drugs flupirtine and retigabine on Kv7 channels depends on which subunits they are constructed. [8] In particular, flupirtine and retigabine act at a molecular level to stabilize the open conformation of the heterotetrameric Kv7.2 / Kv7.3 channels, so far the exact binding site could only be estimated by computation but not determined by crystal structure analysis. [7-10]

Flupirtin was developed by Chemiewerk Hornburg (later ASTA Medica). It belongs chemically to the class of triaminopyridines and has been used in Europe since 1984 as an analgesic with muscle relaxant properties. [7] It is approved by the EMA for the treatment of pain and as a maleate salt in the form of capsules, tablets or injectable solutions. The drug reaches its analgesic effect on spinal and supraspinal levels. [11] Opiod receptors play just as little role as influencing prostaglandin synthesis, alpha ₁ or alpha ₂ adreno or 5-HT receptors. [12-14] Flupirtine is a positive modulator of Kv7 potassium channels and opens Kv7.2 /7.3 channels with an EC ₅₀ of about 5 μM. [15] At low micromolar plasma concentrations (2-6 μM), which are necessary for an analgesic effect, this is considered to be the main mode of action of flupirtine. [16]

Furthermore, there is evidence that flupirtine enhances the effect of GABA on the GABA _A receptor. [17] Flupirtin is approved for the treatment of acute and chronic pain, such as painful tension, degenerative joint disease, tension headache, cancer pain, menstrual pain or postoperative pain. Based on the mechanism of action of flupirtine, further indications are theoretically possible. These include multiple sclerosis, tinnitus prophylaxis, overactive bladder, neuropathic pain, peripheral neuropathy and fibromyalgia. [13] Furthermore, flupirtine has neuroprotective properties that are potentially therapeutically useful. [18] Studies in patients with postoperative or cancer pain have shown that flupirtine has approximately the same analgesic potency as weak opioids. [19-20] For a long time, flupirtine was generally considered to be well tolerated. Insomnia, drowsiness, dizziness, dry mouth, vomiting and discomfort in the gastrointestinal area are the most common side effects. In the context of pharmacovigilance, however, a serious problem became apparent. By 2013, 800 adverse drug reactions had been reported. More than a third of these reports describe liver or biliary disorders. Of the total number of case reports, 24 were fatal and 17 of these were again reported to have liver toxic reactions. Following a reassessment of benefit-risk, flupirtine-containing drugs are limited in their therapeutic target group and have a two-week treatment limit. [12] Meanwhile, the Pharmacavigilance Committee (PRAC) has recommended that the European Medicines Agency (EMA) withdraw authorizations for all medicinal products containing flupirtine (Katadolon® and others). [22]

Flupirtin also has anticonvulsant properties in higher doses. Structural optimization led to the development of the much more potent, highly analogous drug retigabine, which was subsequently approved as an antiepileptic drug. [23] Flupirtine and retigabine are shown below:

The postulated mechanism of action of retigabine is comparable to that of flupirtine. This retigabine has a completely different point of attack than any other currently available antiepileptic drugs. Retigabine is active in almost every epilepsy animal model with an overall profile that is different from any other antiepileptic drug. [23] This makes it an indispensable drug in drug-resistant epilepsies. [24, 25]

The most common adverse drug reactions during retigabine (Trobalt ®) treatment are drowsiness, fatigue, headache, confusion, and weakness. In addition, renal and urological disorders were observed in 12% of those treated. Retigabine causes urinary retention, which can be explained by the activation of Kv7 channels in the smooth muscle of the bladder. [26] However, unlike some other antiepileptic drugs, retigabine is not associated with cardiac, hematological or hepatic toxicity and is neither teratogenic nor toxic to reproduction. Furthermore, retigabine has little potential for drug interactions because it does not induce or inhibit liver enzymes. [6] These properties generally make retigabine a well tolerated antiepileptic. However, reports from long-term clinical studies on pigmentation (discoloration) of ocular tissues including the retina and blue-gray discoloration of the skin, lips and nails have limited the use. Retigabine is now approved as adjunctive therapy for pharmacoresistant seizures with or without secondary generalization in adult patients with epilepsy if other appropriate drugs are inadequate or intolerable. [27] In June 2017, the worldwide market withdrawal of Trobalt® by GlaxoSmithKline finally took place. One possible explanation for the observed toxicity is the formation of toxic metabolites. [28] In Greifswald, a clinical study was therefore conducted in which evidence for resulting oxidation products (quinonediimines) were found, which formed dimers or trimers in subsequent reactions and could be converted with glutathione into corresponding conjugates. [29] These glutathione conjugates are likely to be metabolized to cysteine and mercapturic acid conjugates in subsequent reactions. [30] It was deduced from these findings that the chinondiimine intermediates of flupirtine are responsible for hepatotoxicity. [31] This is known, for example, for paracetamol and diclofenac, which also form quinoneimine intermediates. [32,33]

In another in vitro reaction, the carbamate group of flupirtine could be cleaved by porcine liver esterases. The resulting primary aromatic amino group was in turn acetylated by human N-acetyltransferases to the metabolite D13223. This was found to be less reactive to peroxidases compared to flupirtine. The corresponding quinonediimines could be detected in vitro, i. they are relatively stable. [29] This may also explain that the metabolite D13223 of flupirtin is less hepatotoxic than flupirtine itself. [31] In addition, the N-acetylated metabolite of flupirtine still accounts for about one quarter of the analgesic effect of the parent compound. [34] The metabolism of retigabine is somewhat different from that of flupirtine. What is common is that retigabine is also metabolised by acetylation and glucuronidation. Both metabolites were detected in human urine after single oral administration of 600 mg retigabine. [35] As with the metabolism of flupirtine, no involvement of cytochrome P450 monooxygenases could be demonstrated. [36] Oxidative processes seem to play a lesser role in retigabine metabolism than in flupirtine. There are no reports of in vivo formation of dimers. Dousa et al. however, identified retigabine dimers as process-related impurities from retigabine synthesis. [37] Duggan et al. synthesized fluorinated retigabine analogues in order to arrive at analogues with better properties without, however, paying particular attention to metabolism. [38] At Grünenthal, the structure of flupirtine and retigabine, which has been proven to be safe and well-tolerated, has been completely eliminated, so that the problems associated with it are avoided, but no longer due to the unusually broad experience of use in the vast majority of patients Long-term compatibility of the proven structures can be referred. [39]

The object of the present invention was therefore to provide novel compounds which have a similarly good activity as, for example, flupirtine or retigabine and in which the structural toxicity is abolished.

wobei

R₁

ausgewählt ist aus verzweigter oder unverzweigter C1- bis C5-Alkylgruppe und -NR_1aR_1b-Gruppe, wobei R_1a und R_1b unabhängig voneinander ausgewählt sind aus Wasserstoffatom und verzweigter oder unverzweigter C1- bis C5- Alkylgruppe oder miteinander und mit dem Stickstoffatom der NR_1aR_1b-Gruppe eine C3- bis C9-Heterocycloalklygruppe bilden, wobei die C3- bis C9-Heterocycloalklygruppe jeweils mindestens einen Substituenten ausgewählt aus Wasserstoffatom und C1- bis C3-Alklygruppe aufweist und gegebenenfalls zusätzlich zum Stickstoffatom der NR_1aR_1b-Gruppe mindestens ein weiteres Heteroatom im Cyclus, bevorzugt ein Stickstoff- oder Sauerstoffatom, aufweist;

R₂

ausgewählt ist aus der Gruppe bestehend aus verzweigter oder unverzweigter C1- bis C10-Alkylgruppe, C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, wobei mindestens ein Heteroatom ausgewählt aus Stickstoff- und Sauerstoffatom umfasst ist, C6- bis C12-Arylgruppe; und C5- bis C11-Heteroarylgruppe, wobei die C1- bis C10-Alkylgruppe, C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, C6- bis C12-Arylgruppe, oder C5- bis C11-Heteroarylgruppe jeweils mindestens einen weiteren Substituenten R_2a aufweist, wobei R_2a ausgewählt ist aus der Gruppe bestehend aus Wasserstoffatom, Halogenatom (F, Cl, Br, I, bevorzugt F), verzweigter oder unverzweigter C1- bis C3-Alkylgruppe, verzweigter oder unverzweigter (per)halogenierter C1- bis C3-Alkylgruppe (F, Cl, Br, I, bevorzugt F) und verzweigter oder unverzweigter C1- bis C3-Alkoxygruppe, und wobei die C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, C6- bis C12-Arylgruppe oder C5- bis C11-Heteroarylgruppe ein oder mehrere Ringsysteme umfasst, welche kondensiert oder separiert sind;

R₃

ausgewählt ist aus der Gruppe bestehend aus verzweigter oder unverzweigter C1- bis C10-Alkoxygruppe; verzweigter oder unverzweigter C1-C10-Alkylgruppe und -(CH₂)_p-C6- bis C12-Arylgruppe, welche mindestens einen Substituenten ausgewählt aus Wasserstoffatom, Halogenatom (F, Cl, Br, I, bevorzugt F) und C1- bis C3-Alkoxygruppe aufweist und wobei die C6- bis C12-Arylgruppe einen oder mehrere Ringsysteme umfasst, welche kondensiert oder separiert sind;

R₄

ausgewählt ist aus Wasserstoff, Halogenatom (F, Cl, Br, I, bevorzugt Br) und verzweigter oder unverzweigter C1-C5-Alkylgruppe;

X

ein Stickstoffatom oder eine -CH- Gruppe ist;

n

Null oder eine ganze Zahl im Bereich von 1 bis 3 ist;

m

Null oder eine ganze Zahl im Bereich von 1 bis 5 ist; und

p

Null oder eine ganze Zahl im Bereich von 1 bis 5 ist.

The problem has been solved with compound of general formula (I)

in which

R ₁

is selected from branched or unbranched C1 to C5 alkyl group and -NR _1a R _1b group, wherein R _1a and R _{1b are} independently selected from hydrogen atom and branched or unbranched C1 to C5 alkyl group or with each other and with the nitrogen atom of NR _1a R _1b group form a C3 to C9 heterocycloalkly group, wherein the C3 to C9 heterocycloalkly group each has at least one substituent selected from hydrogen atom and C1 to C3 alkyl group and optionally in addition to the nitrogen atom of the NR _1a R _1b group at least one further heteroatom in the cycle, preferably a nitrogen or oxygen atom;

R ₂

is selected from the group consisting of branched or unbranched C1 to C10 alkyl group, C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, wherein at least one heteroatom selected from nitrogen and oxygen atom is included, C6 to C12 aryl group ; and C5 to C11 heteroaryl group, wherein the C1 to C10 alkyl group, C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group, or C5 to C11 heteroaryl group each has at least one further substituent R _2a , wherein R _{2a is} selected from the group consisting of hydrogen atom, halogen atom (F, Cl, Br, I, preferably F), branched or unbranched C1 to C3 alkyl group, branched or unbranched (per) halogenated C1 to C3 alkyl group (F, Cl, Br, I, preferably F) and branched or unbranched C1 to C3 alkoxy group, and wherein the C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group or C5 to C11 heteroaryl group comprises one or more ring systems which are fused or separated;

R ₃

is selected from the group consisting of branched or unbranched C1 to C10 alkoxy group; branched or unbranched C 1 -C 10 -alkyl group and - (CH ₂ ) _p -C 6 to C 12 -aryl group which has at least one substituent selected from hydrogen atom, halogen atom (F, Cl, Br, I, preferably F) and C 1 to C 3 Alkoxy group and wherein the C6 to C12 aryl group comprises one or more ring systems which are condensed or separated;

R ₄

is selected from hydrogen, halogen atom (F, Cl, Br, I, preferably Br) and branched or unbranched C1-C5 alkyl group;

X

is a nitrogen atom or a -CH group;

n

Is zero or an integer in the range of 1 to 3;

m

Is zero or an integer in the range of 1 to 5; and

p

Is zero or an integer in the range of 1 to 5.

It has been found that replacement of the N atom of the secondary amino group of flupirtine or retigabine by a sulfur atom alters the properties of the molecules obtained in such a way that oxidation no longer takes place at the triply substituted ring but at the thioether bridge. advantageously, The thioethers are oxidized by oxidants not to toxic quinone diimines, but to sulfoxides, which are chemically unreactive unlike the chinodiimines of flurpitins and can not react with endogenous nucleophiles, thus repealing the basis of toxicity at the molecular level. The formed sulfoxides, which are also the likely metabolites in vivo, are not per se toxic and it has been shown that by altering the substitution pattern, more potent thioethers can be obtained, whose sulfoxide metabolites are not hepatotoxic.

Surprisingly, the newly presented thioethers show in vitro an at least equivalent effect on Kv7 .2 / 7.3 channels such as flupirtine and retigabine, ie they are potential K _v 7.2 / 3 openers; A potassium channel assay was used to demonstrate the potassium channel activity of the substances based on EC ₅₀ and Emax. The half-maximal effect concentration (EC ₅₀ ) is the concentration of a substance at which the half-maximal effect of the potassium channel assay is observed when the logarithmic concentration of a substance is plotted against the associated corrected ΔF / F values, indicating the potency (potency ) of a compound is characterized. The intrinsic activity of a test compound is described by its E _max value. This is the maximum response, based on the assay used, which is possible by the corresponding test substance (plateau effect). When evaluating the assay used, Emax of the flupirtine used as the reference, which was used as maleate (flupirtine maleate), was set to 100% and the maximum responses of the respective substance of the general formula (I) were based on this value.

The lower the EC ₅₀ value, the higher the potency, the larger the E _max value, the more effective the compound. Many compounds of general formula (I) showed a low EC ₅₀ value of less than 0.9 micromolar (micromoles per liter). Emax was at least 60% for all active compounds of the general formula (I), from which a potassium flux at least approximately comparable to the flupirtine maleate is to be expected; many of the compounds of the general formula (I) have E _max values of more than 100%, indicating a greater potassium flux compared to flupirtine maleate.

To assess the hepatotoxic potential of a substance, the IC ₅₀ value was used. The IC ₅₀ was defined as the concentration of a compound at which the viability of the cells decreased to 50%. From the interaction of the results of Kv7.2 / 3-opening activity and the hepatotoxicity results in vitro, it can be seen that total compounds of the general formula (I) significantly exceed the effect of the reference substance flupirtine maleate.

In one embodiment of the compound of the general formula (I), n is zero. In one embodiment of the compound of the general formula (I), m is 1. In one embodiment of the compound, X is a nitrogen atom.

The radical R ₁ is preferably selected from the group consisting of branched or unbranched C1 to C3 alkyl group, and -NR _1a R _1b group, wherein R _1a and R _1b are independently selected from hydrogen and branched or unbranched C1 to C3 Alkyl group or with each other and with the nitrogen atom of NR _1a R _1b group form a C4 to C6, preferably C4, -Heterocycloalklygruppe, wherein the C4 to C6, preferably C4, heterocycloalkly group each having at least one substituent selected from hydrogen atom and C1 to C3 alkyl group and optionally in addition to the nitrogen atom of the NR _1a R _1b group has at least one further oxygen atom in the cycle; wherein R _{1 is} more preferably selected from the group consisting of NH ₂ group, methyl group, NH (CH ₃ ) group, pyrrolidine group and morpholine group, more preferably pyrrolidine group.

The radical R ₂ is preferably selected from the group consisting of mono- or difluorophenyl group; CF ₃ phenyl group; F ₅ S-phenyl group; C4 to C8 cycloalkyl group, preferably cyclohexyl group, cyclobutyl group or cyclopropyl group; branched or unbranched C 1 - to C 5 -alkyl group, preferably 2-propyl group; Biphenyl group; Pyridine group; Piperidine group; Tetrahydropyran group; Dioxolane group and phenyl group, wherein the phenyl group has at least one substituent R _2a selected from hydrogen atom and methoxy group; wherein R _{2 is} more preferably selected from the group consisting of C4 to C8 cycloalkyl group, preferably cyclohexyl group, cyclobutyl group or cyclopropyl group, more preferably cyclohexyl group; and branched or unbranched C1 to C5 alkyl group, preferably 2-propyl group; wherein R _{2 is} more preferably a cyclohexyl group or 2-propyl group.

The radical R ₃ is preferably selected from the group consisting of ethoxy group, propyl group, tert-butyl group, - (CH ₂ ) _p -phenyl group which has at least one substituent selected from hydrogen atom, halogen atom (F, Cl, Br , I, preferably F) and methoxy group and wherein p is zero or 1 or 2; wherein R _{3 is} more preferably a (CH ₂ ) _p -phenyl group having at least one substituent selected from hydrogen atom, halogen atom (F, Cl, Br, I, preferably F) and methoxy group, and p is 1 or 2.

The radical R ₄ is preferably a hydrogen atom, a bromine atom or a methyl group, more preferably a hydrogen atom.

In one embodiment of the compounds of general formula (I), n is zero; m is 1; X is a nitrogen atom, R ₁ is selected from the group consisting of NH ₂ group, methyl group, NH (CH ₃ ) group, pyrrolidine group and morpholine group, preferably pyrrolidine group; R ₂ is selected from the group consisting of cyclohexyl group and 2-propyl group; R ₃ is a - (CH ₂ ) _p -phenyl group which has at least one substituent selected from hydrogen atom, halogen atom (F, Cl, Br, I, preferably F) and methoxy group, wherein p is 1 or 2; and R ₄ is a hydrogen atom.

In a preferred embodiment of the compounds of the general formula (I), the compound has the formula (Ia):

In a further preferred embodiment of the compounds of the general formula (I), the compound has the formula (Ib):

In a further preferred embodiment of the compounds of the general formula (I), the compound has the formula (Ic):

In the compound of formula (Ic), which is the most potent substance described in the experimental section 11 Thus, in order to achieve the half-maximal effect on potassium channels, such low substance concentrations (0.0014 μmol per liter) are required that toxic effects are very unlikely in the resulting dosage. The determined toxicity data show that in the case of the solubility in the test medium limited concentrations of 63 .mu.mol per liter for compound 11 no toxic effects were observed and the IC ₅₀ can therefore be given as greater than 63 μmol per liter. Accordingly, the ratio of IC ₅₀ to EC _{50 is} at least 85 times greater in substance 11 as with flupirtine maleate. Liver damage during therapy should therefore be less likely. Also, the biological effect of the test substance should be greater (eg stronger analgesia), since Emax was over 100% and thus a stronger Kaliumefflux is expected than with flupirtine maleate.

wobei R₁, R₂, R₃, R₄, X, n, m und p die gleiche Bedeutung haben wie voranstehend zu den Verbindungen der allgemeinen Formel (I) definiert, umfassend:

1. (i) Bereitstellen einer Nitro-Verbindung der allgemeinen Formel (II)
   
   wobei R₁, R₂, R₄, m und n die gleiche Bedeutung haben wie voranstehend zu den Verbindungen der allgemeinen Formel (I) definiert;
2. (ii) Umsetzen der Nitro-Verbindung der allgemeinen Formel (II) gemäß (i) mit einer Verbindung H-R₃, wobei R₃ die gleiche Bedeutung hat wie voranstehend zu den Verbindungen der allgemeinen Formel (I) definiert, unter Erhalt einer Verbindung der allgemeinen Formel (I).

The invention further relates to a process for the preparation of a compound of general formula (I),

wherein R ₁ , R ₂ , R ₃ , R ₄ , X, n, m and p have the same meaning as defined above for the compounds of the general formula (I), comprising:

1. (i) providing a nitro compound of the general formula (II)
   
   wherein R ₁ , R ₂ , R ₄ , m and n have the same meaning as defined above for the compounds of general formula (I);
2. (ii) reacting the nitro compound of the general formula (II) according to (i) with a compound HR ₃ , wherein R _{3 has} the same meaning as defined above for the compounds of the general formula (I), to obtain a compound of the general Formula (I).

The invention further relates to compound of general formula (I), obtained or obtainable by a process as described above.

wobei

R₁

ausgewählt ist aus verzweigter oder unverzweigter C1- bis C5-Alkylgruppe und -NR_1aR_1b-Gruppe, wobei R_1a und R_1b unabhängig voneinander ausgewählt sind aus Wasserstoffatom und verzweigter oder unverzweigter C1- bis C5- Alkylgruppe oder miteinander und mit dem Stickstoffatom der NR_1aR_1b-Gruppe eine C3- bis C9-Heterocycloalklygruppe bilden, wobei die C3- bis C9-Heterocycloalklygruppe jeweils mindestens einen Substituenten ausgewählt aus Wasserstoffatom und C1- bis C3-Alklygruppe aufweist und gegebenenfalls zusätzlich zum Stickstoffatom der NR_1aR_1b-Gruppe mindestens ein weiteres Heteroatom im Cyclus, bevorzugt ein Stickstoff- oder Sauerstoffatom, aufweist;

R₂

ausgewählt ist aus der Gruppe bestehend aus verzweigter oder unverzweigter C1- bis C10-Alkylgruppe, C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, wobei mindestens ein Heteroatom ausgewählt aus Stickstoff- und Sauerstoffatom umfasst ist, C6- bis C12-Arylgruppe; und C5- bis C11-Heteroarylgruppe, wobei die C1- bis C10-Alkylgruppe, C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, C6- bis C12-Arylgruppe, oder C5- bis C11-Heteroarylgruppe jeweils mindestens einen weiteren Substituenten R_2a aufweist, wobei R_2a ausgewählt ist aus der Gruppe bestehend aus Wasserstoffatom, Halogenatom (F, Cl, Br, I, bevorzugt F), verzweigter oder unverzweigter C1- bis C3-Alkylgruppe, verzweigter oder unverzweigter (per)halogenierter C1- bis C3-Alkylgruppe (F, Cl, Br, I, bevorzugt F) und verzweigter oder unverzweigter C1- bis C3-Alkoxygruppe, und wobei die C3- bis C10-Cycloalkylgruppe, C3- bis C10-Heterocycloalkylgruppe, C6- bis C12-Arylgruppe oder C5- bis C11-Heteroarylgruppe ein oder mehrere Ringsysteme umfasst, welche kondensiert oder separiert sind;

R₃

ausgewählt ist aus der Gruppe bestehend aus verzweigter oder unverzweigter C1- bis C10-Alkoxygruppe; verzweigter oder unverzweigter C1-C10-Alkylgruppe und -(CH₂)_p-C6- bis C12-Arylgruppe, welche mindestens einen Substituenten ausgewählt aus Wasserstoffatom, Halogenatom (F, Cl, Br, I, bevorzugt F) und C1- bis C3-Alkoxygruppe aufweist und wobei die C6- bis C12-Arylgruppe einen oder mehrere Ringsysteme umfasst, welche kondensiert oder separiert sind;

R₄

ausgewählt ist aus Wasserstoff, Halogenatom (F, Cl, Br, I, bevorzugt Br) und verzweigter oder unverzweigter C1-C5-Alkylgruppe;

X

ein Stickstoffatom oder eine -CH- Gruppe ist;

n

Null oder eine ganze Zahl im Bereich von 1 bis 3 ist;

m

Null oder eine ganze Zahl im Bereich von 1 bis 5 ist; und

p Null oder eine ganze Zahl im Bereich von 1 bis 5 ist; oder eines pharmazeutisch akzeptablen Salzes einer Verbindung der allgemeinen Formel (I)
zur Verwendung als Arzneimittel, bevorzugt als Human- oder Veterinär-Arzneimittel, weiter bevorzugt als Human-, Kleintier- oder Heimtier-Arzneimittel.Furthermore, the invention relates to a compound of general formula (I)

in which

R ₁

is selected from branched or unbranched C1 to C5 alkyl group and -NR _1a R _1b group, wherein R _1a and R _{1b are} independently selected from hydrogen atom and branched or unbranched C1 to C5 alkyl group or with each other and with the nitrogen atom of NR _1a R _1b group form a C3 to C9 heterocycloalkly group, wherein the C3 to C9 heterocycloalkly group each has at least one substituent selected from hydrogen atom and C1 to C3 alkyl group and optionally in addition to the nitrogen atom of the NR _1a R _1b group at least one further heteroatom in the cycle, preferably a nitrogen or oxygen atom;

R ₂

is selected from the group consisting of branched or unbranched C1 to C10 alkyl group, C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, wherein at least one heteroatom selected from nitrogen and oxygen atom is included, C6 to C12 aryl group ; and C5 to C11 heteroaryl group, wherein the C1 to C10 alkyl group, C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group, or C5 to C11 heteroaryl group each has at least one further substituent R _2a , wherein R _{2a is} selected from the group consisting of hydrogen atom, halogen atom (F, Cl, Br, I, preferably F), branched or unbranched C1 to C3 alkyl group, branched or unbranched (per) halogenated C1 to C3 alkyl group (F, Cl, Br, I, preferably F) and branched or unbranched C1 to C3 alkoxy group, and wherein the C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group or C5 to C11 heteroaryl group comprises one or more ring systems which are fused or separated;

R ₃

is selected from the group consisting of branched or unbranched C1 to C10 alkoxy group; branched or unbranched C 1 -C 10 -alkyl group and - (CH ₂ ) _p -C 6 to C 12 -aryl group which has at least one substituent selected from hydrogen atom, halogen atom (F, Cl, Br, I, preferably F) and C 1 to C 3 Alkoxy group and wherein the C6 to C12 aryl group comprises one or more ring systems which are condensed or separated;

R ₄

is selected from hydrogen, halogen atom (F, Cl, Br, I, preferably Br) and branched or unbranched C1-C5 alkyl group;

X

is a nitrogen atom or a -CH group;

n

Is zero or an integer in the range of 1 to 3;

m

Is zero or an integer in the range of 1 to 5; and

p is zero or an integer in the range of 1 to 5; or a pharmaceutically acceptable salt of a compound of general formula (I)
for use as a medicament, preferably as a human or veterinary drug, more preferably as a human, small animal or pet drug.

Preferred embodiments of the compound of the general formula (I) for use as a medicament, preferably as a human or veterinary medicament, more preferably as a human, small animal or pet drug, are described in the introduction with regard to the compound of the general formula (I) even; the same applies to the corresponding pharmaceutically acceptable salts, ie for the pharmaceutically acceptable salts are the same compounds of general formula (I) as described above with respect to the compound of general formula (I) itself, preferably; The same applies to connections of the general formula (I) or obtainable by the production process described above. Particular preference is given to using a compound of the formula (Ia), or a compound of the formula (Ib), or a compound of the formula (Ic), which also comprises the use of a pharmaceutically acceptable salt; more preferably, a compound of formula (Ic) is used, which also includes the use of a pharmaceutically acceptable salt.

Pharmaceutically acceptable salts are known to those skilled in the art and, for example, in Berge et al. "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19 (1977) described. "Pharmaceutically acceptable salt" means a non-toxic, organic or inorganic acid addition salt of a compound of general formula (I) wherein the compound of general formula (I) is in cationic form in the acid addition salt. The pharmaceutically acceptable salt is neutrally charged overall.

A pharmaceutically acceptable salt may be prepared from a compound of general formula (I) with an organic or inorganic acid. The salt can be prepared in situ during isolation and purification of the compound of general formula (I) or by separate reaction of a purified compound of general formula (I) with a suitable organic or inorganic acid and subsequent isolation of the salt produced.

Preferably, the anion of the pharmaceutically acceptable salt is selected from the group consisting of sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, Formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexine-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, adipate, alginate, Mucate, aspartate, benzenesulfonate, butyrate, camphorate, camphorsulfonate, cyclopentaneproprionate, digluconate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, hexanoate, 2-hydroxyethanesulfone at, 2-naphthalenesulfonate, nicotinate, nitrate, palmitate, pectinate, persulfate, picrate, pivalate, salicylate, thiocyanate, tosylate, arginate and undecanoate.

In one embodiment, the compound of the general formula (I) is used as an acid addition salt in the form of the chloride, sulfate, mesylate, besylate, tosylate or the mono-, di- or tricarboxylic acid salt, wherein the mono-, di- or tricarboxylic acid is preferably selected from the group consisting of fumaric acid, malonic acid, malic acid, succinic acid, lactic acid, glycolic acid, maleic acid, citric acid, aspartic acid and mandelic acid.

The compound of the general formula (I) is used alone or as a mixture of two or more compounds of the general formula (I) or as a pharmaceutically acceptable salt of a compound of the general formula (I) or as a mixture of two or more pharmaceutically acceptable salts of Compounds of the general formula (I). Likewise, mixtures of one or more compound (s) of general formula (I) with one or more pharmaceutically acceptable salt (s) of compound (s) of general formula (I) may be employed.

In one embodiment, the invention relates to a compound of general formula (I) as described above or to a compound of general formula (I) obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for use as Painkiller.

In one embodiment, the invention relates to a compound of general formula (I) as described above or to a compound of general formula (I) obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for use in tinnitus treatment or prevention.

In one embodiment, the invention relates to a compound of general formula (I) as described above or to a compound of general formula (I) obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for use in the treatment or prevention of convulsions, preferably in the treatment or prevention of neonatal seizures.

In one embodiment, the invention relates to a compound of general formula (I) as described above or to a compound of general formula (I) obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for use in the treatment or prevention of a disease selected from the group consisting of incontinence, anxiety, dependence, mania, bipolar disorder, cognitive disorder and dyskinesia.

The invention furthermore relates to a pharmaceutical preparation comprising a compound of the general formula (I) as described above or obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of the general formula (I) as described above.

The invention further relates to a method for the prevention or treatment of pain conditions, for the prevention or treatment of tinnitus or for the prevention or treatment of convulsive conditions comprising the administration of a preventively or therapeutically effective amount of a compound of general formula (I) as described above or a compound of the general formula (I) obtained or obtainable by the above-described preparation method or a pharmaceutically acceptable salt of a compound of the general formula (I).

The invention also relates to a method for the treatment or prevention of a disease selected from the group consisting of incontinence, anxiety, dependence, mania, bipolar disorder, cognitive disorder and dyskinesia comprising administering a preventively or therapeutically effective amount of a compound of the general formula (I ) as described above, or a compound of the general formula (I) or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of the general formula (I).

The invention further relates to a method of treating or preventing a disease which is at least partially associated with alteration of Kv7.2 / Kv7.3 K ⁺ channels comprising administering to a subject (human or animal) receiving such treatment or prevention, a therapeutically or preventively active amount of a compound of general formula (I) as described above, or a compound of general formula (I) obtained or obtainable by the above-described preparation method or a pharmaceutically acceptable salt of a compound of general formula ( I).

The invention also relates to a use of a compound of general formula (I) as described above or a compound of general formula (I) obtained or obtainable by the above-described preparation process or a pharmaceutically acceptable salt of a compound of general formula (I) for the preparation a medicament, in particular a medicament for the prevention or treatment of pain, for the prevention or treatment of tinnitus or for the prevention or treatment of convulsive conditions or for the treatment or prevention of a disease selected from the group consisting of incontinence, anxiety, dependence, mania, bipolar disorder, cognitive disorder and dyskinesia.

The invention will be further explained by way of examples without, however, being limited thereto.

Examples

Preparation of compounds of general formula (I)

Preparation of nitro precursors

6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine

10 mmol of 6-amino-5-nitropyridine-2-thiol (1.71 g) were dissolved in 40 ml of DMF and 4.3 ml of 3 M NaOH added. To the dark brown solution was added 10 mmol of 4-fluorobenzylbromide and stirred for 3 h at room temperature. Subsequently, the product was precipitated by adding 100 ml of water. Finally, the product was washed with 200 ml of water and 200 ml of n-hexane. Yellow solid (mp. 139-141 ° C). Yield: 1.98 g (70.0%). R _f = 0.35 (n-hexane / ethyl acetate). ¹ H-NMR [ppm]: δ = 4.46 (s, 2H), 6.61 (d, 1H, J = 8.8 Hz), 7.13 (m, 2H), 7.53 (m, 2H), 8.15 (d, 1H, J = 8.8 Hz), 8.18 (bs, 2H). ¹³ C-NMR [ppm]: δ = 32.2, 110.3, 115.1 (d, 2C, ² J _{C, F} = 22 Hz), 123.5, 131.1 (d, 2C, ³ J _{C, F} = 8 Hz), 134.2 (d, ⁴ J _{C, F} = 3 Hz), 134.5, 153.1, 160.1 and 162.5 (d, ¹ J _{C, F} = 242 Hz ), 166.3. IR [cm ^-1 ]: ṽ = 1342, 1558, 3332, 3456.

additional nitro precursors

Additional thioether precursors were synthesized analogously according to the synthesis of 1.1.1. Table 1 shows the respective starting materials and the resulting nitro compound. Table 1 Overview of the synthesized thioether precursors and the educts used for the synthesis. reactants product

Preparation of compounds of general formula (I)

Ethyl {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} carbamate

In a 3-necked flask, 3.65 mmol of 6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine (1.02 g) with 18.6 mmol SnCl ₂ • 2H ₂ O (5, 1 eq, 4.2 g) and suspended in 40 ml of ethanol. The reaction batch was heated to 70 ° C. under argon atmosphere for 25 h. It was then cooled to 40 ° C and 37.2 mmol of Et ₃ N (10.2 eq, 5.16 ml) and 4.6 mmol of ethyl chloroformate (1.26 eq, 0.44 ml) were added. To The temperature was raised to 55 ° C for 30 minutes and held for one hour. After cooling to room temperature, the mixture was filtered through kieselguhr and the residue was washed with ethanol. The filtrate was concentrated on a rotary evaporator, taken up in DCM and washed with water. Evaporation of the organic phase and washing of the residue with n-hexane gave the clean product. Colorless solid (mp. 138-140 ° C). Yield: 0.75 g (47%). R _f = 0.83 (cyclohexane / ethanol / Et ₃ N 6: 2: 2). ¹ H-NMR [ppm]: δ = 1.22 (t, 3H, J = 7.1 Hz), 4.09 (q, 2H, J = 7.1 Hz), 4.29 (s, 2H) , 5.94 (s, 2H), 6.43 (d, 1H, J = 8.0 Hz), 7.07-7.12 (m, 2H), 7.40-7.45 (m, 3H), 8.57 (m, 1H). ¹³ C-NMR [ppm]: δ = 14.5, 32.6, 60.3, 109.3, 115.0 (d, 2C, ² J _{C, F} = 21 Hz), 115.2, 130, 7 (d, 2C, ³ J _{C, F} = 8 Hz), 131.8, 135.0 (d, ⁴ J _{C, F} = 3 Hz), 150.2, 152.8, 154.4, 159, 9 and 162.3 (d, ¹ J _{C, F} = 243 Hz). IR [cm ^-1]: v = 1454, 1678, 2984, 3145, 3260, 3403. HRMS: [C ₁₅ H ₁₆ N ₃ O ₂ FS + H] ⁺ calculated: 322.1020, Measured: 322.1027.

Ethyl [2-amino-6- (benzylthio) pyridin-3-yl] carbamate

2.31 mmol of 6- (benzylthio) -3-nitropyridin-2-amine (0.60 g) were suspended in a mixture of 15 ml of water and 25 ml of isopropanol. 6.93 mmol iron powder (3 eq, 0.39 g) and 10.4 mmol NH ₄ Cl (4.5 eq, 0.56 g) were added and the suspension heated to reflux for 6 h. The resulting dark brown mixture was filtered and the residue washed with 50 ml of isopropanol. Subsequently, 13.9 mmol Et ₃ N (6.0 eq, 1.91 ml) and 5.54 mmol ethyl chloroformate (2.4 eq, 0.53 ml) were added. To complete the reaction, 5.54 mmol of ethyl chloroformate were added after one and two hours, respectively. After an additional 5 hours, the solution was concentrated on a rotary evaporator and the residue was taken up in 100 ml of ethyl acetate. The organic phase was washed with 2 × 100 ml of water and 100 ml of saturated brine. After drying over Na ₂ SO ₄ , the crude product was purified by flash chromatography (mobile phase: n-hexane / ethyl acetate 7: 3) and the resulting brown solid washed with a little ice-cold diethyl ether. Light blue solid (mp. 100-101 ° C). Yield: 0.23 g (33.0%). R _f = 0.72 (cyclohexane / ethanol / Et ₃ N 6: 2: 2). ¹ H-NMR [ppm]: δ = 1.22 (t, 3H, J = 7.1 Hz), 4.09 (q, 2H, J = 7.1 Hz), 4.30 (s, 2H) , 5.93 (s, 2H), 6.44 (d, 1H, J = 8.0 Hz), 7.21 (t, 1H, J = 7.3 Hz), 7.28 (t, 2H, J = 7.3 Hz), 7.39-7.41 (m, 3H), 8.57 (s, 1H). ¹³ C-NMR [ppm]: δ = 14.5, 33.5, 60.3, 109.2, 115.2, 126.8, 128.3 (2C), 128.8 (2C), 131, 8, 138.6, 150.5, 152.7, 154.4. IR [cm ^-1]: v = 1457, 1681, 2975, 3140, 3281, 3405. HRMS: [C ₁₅ H ₁₇ N ₃ O ₂ S + H] ⁺ calculated: 304.1114, Measured: 304.1099.

Ethyl {2-amino-6 - [(3,4-difluorobenzyl) thio] pyridin-3-yl} carbamate

5 mmol of 6 - [(3,4-difluorobenzyl) thio] -3-nitropyridin-2-amine (1.49 g) was suspended in a mixture of 30 ml of water and 50 ml of isopropanol. 15 mmol iron powder (3.0 eq, 0.84 g) and 22.5 mmol NH ₄ Cl (4.5 eq, 1.2 g) were added and the mixture heated to reflux for 3.5 h. The resulting suspension was filtered and the residue washed with 100 ml of isopropanol. 15 mmol Et ₃ N (3.0 eq, 2.07 ml) and 6 mmol ethyl chloroformate (1.2 eq, 0.57 ml) were added to the solution and stirred for 2 h at 40 ° C. To complete the reaction, another 5.5 mmol of ethyl chloroformate and 5 mmol of Et ₃ N were added. After a further 5 h, the solution was concentrated on a rotary evaporator, the residue taken up in 100 ml of ethyl acetate and washed with 2 × 100 ml of water and 100 ml of saturated brine. The crude product was purified by flash chromatography (mobile phase: n-hexane / ethyl acetate 7: 3). Colorless solid (mp. 137-140 ° C).

Yield: 0.95 g (56.0%). R _f = 0.39 (n-hexane / ethyl acetate 7: 3). ¹ H-NMR [ppm]: δ = 1.22 (t, 3H, J = 7.1 Hz), 4.09 (q, 2H, J = 7.1 Hz), 4.29 (s, 2H) , 5.99 (s, 2H), 6.42 (d, 1H, J = 8.0 Hz), 7.24-7.36 (m, 2H), 7.40 (d, 1H, J = 6.3 Hz), 7.45-7.50 (m, 1H), 8.57 (s, 1H). ¹³ C-NMR [ppm]: δ = 14.5, 32.2, 60.4, 109.3, 115.3, 117.1 (d, ² J _{C, F} = 17 Hz), 117.8 ( d, ² J _{C, F} = 17 Hz), 125.7 (dd, ³ J _{C, F} = 6 Hz, ⁴ J _{C, F} = 3 Hz), 132.0, 137.0 (dd, ³ J _{C , F} = 6 Hz, ⁴ J _{C, F} = 4 Hz), 147.2 and 149.6 (dd, ¹ J _{C, F} = 245 Hz, ² J _{C, F} = 13 Hz). 147.8 and 150.2 (dd, ¹ J _{C, F} = 245 Hz, ² J _{C, F} = 13 Hz), 152.9, 154.4. IR [cm ^-1]: v = 1203, 1454, 1510, 1674, 2991, 3149, 3272, 3396. HRMS: [C ₁₅ H ₁₅ N ₃ O ₂ F ₂ S + H] ⁺ calculated: 340.0926 measured, : 340.0919.

Ethyl (2-amino-6 - {[4- (trifluoromethyl) benzyl] thio} pyridin-3-yl) carbamate

In a 3-neck flask, 1.52 mmol 3-nitro-6 - {[4- (trifluoromethyl) benzyl] thio} pyridin-2-amine (0.50 g) and 7.9 mmol SnCl ₂ • 2H ₂ O ( 5.2 eq, 1.79 g) and suspended in 20 ml of ethanol. The reaction mixture was heated to 70 ° C. under argon atmosphere for 23 h. The reaction was then cooled to 40 ° C and 15.4 mmol Et ₃ N (10.2 eq, 2.14 ml) and 1.9 mmol ethyl chloroformate (1.27 eq, 0.18 ml) were added. After 30 minutes, the temperature was raised to 55 ° C and stirred for an additional hour. After subsequent cooling to room temperature, the suspension was filtered through kieselguhr and the residue was washed with ethanol. The filtrate was concentrated on a rotary evaporator and the residue taken up in DCM. After washing the organic phase with water and then drying over Na ₂ SO ₄ , the solvent was removed in a partial vacuum and the residue was washed with n-hexane. Colorless solid (mp 166-168 ° C). Yield: 0.42 g (74.0%). R _f = 0.58 (n-hexane / ethyl acetate 7: 3). ¹ H-NMR [ppm]: δ = 1.22 (t, 3H, J = 7.1 Hz), 4.09 (q, 2H, J = 7.1 Hz), 4.39 (s, 2H) , 5.97 (s, 2H), 6.44 (d, 1H, J = 8.0 Hz), 7.41 (d, 1H, J = 5.6 Hz), 7.64 (s, 4H) , 8.57 (s, 1H). ¹³ C-NMR [ppm]: δ = 14.5, 32.8, 60.4, 109.3, 115.3, 122.9 and 125.6, 125.1 (q, 2C, ⁴ J _{C, F} = 4 Hz), 127.4 (q, ² J _{C, F} = 32 Hz), 129.6 (2C), 131.8, 144.1, 149.6, 152.7, 154.4. IR [cm ^-1]: v = 1113, 1460, 1685, 2980, 3174, 3307, 3423. HRMS: [C ₁₆ H ₁₆ N ₃ O ₂ F ₃ S + H] ⁺ calculated: 372.0988; found: 372 , 0970th

Ethyl (2-amino-6 - {[4- (pentafluoro-A ⁶ -sulfanyl) benzyl] thio} pyridin-3-yl) carbamate

2 mmol of 3-nitro-6 - ((4- (pentafluoro-λ ⁶ -sulfanyl) benzyl) thio) pyridin-2-amine (0.78 g) were suspended in a mixture of 12 ml of water and 20 ml of isopropanol. After addition of 6 mmol iron powder (3.0 eq, 0.34 g) and 9 mmol NH ₄ Cl (4.5 eq, 0.48 g) was heated at reflux for 2 h. The suspension was then filtered and the residue washed with 100 ml of isopropanol. To the filtrate was added 10 mmol Et ₃ N (5 eq, 1.38 ml) and 2.4 mmol ethyl chloroformate (1.2 eq, 0.23 ml) and the reaction heated to 40 ° C. After 1.5 h, 2.4 mmol of ethyl chloroformate were again added and stirred for a further 7 h. The solution was then concentrated on a rotary evaporator, the residue taken up in 50 ml of ethyl acetate and washed with 2x50 ml of water and 50 ml of saturated brine. The crude product was then purified by flash chromatography (mobile phase n-hexane / ethyl acetate 7: 3). Blue solid (mp. 157-158 ° C). Yield: 0.45 g (52.4%). ¹ H-NMR [ppm]: δ = 1.22 (t, 3H, J = 7.1 Hz), 4.09 (q, 2H, J = 7.1 Hz), 4.39 (s, 2H) , 5.97 (s, 2H), 6.44 (d, 1H, J = 8.0 Hz), 7.42 (d, 1H, J = 6.1 Hz), 7.64 (d, 2H, J = 8.5 Hz), 7.80 (d, 2H, J = 8.8 Hz), 8.57 (s, 1H). ¹³ C-NMR [ppm]: δ = 14.5, 32.2, 60.3, 109.3, 115.4, 125.7 (t, 2C, ³ J _{C, F} = 5 Hz), 129, 7 (2C), 131.9, 144.2, 149.4, 151.3 (q, ² J _{C, F} = 15 Hz), 152.8, 154.4. IR [cm ^-1]: V = 827, 1076, 1218, 1461, 1507, 1649, 1693, 2992, 3163, 3325, 3392nd HRMS: [C ₁₅ H ₁₆ N ₃ O ₂ F ₅ S ₂ + H] ⁺ calculated: 430.0677, measured: 430.0684.

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} butyric acid

1 mmol of 6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine (0.28 g) was suspended in 15 ml of isopropanol and a spatula tip of Raney nickel added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred vigorously for 5 h. To the reaction was added 1.4 mmol Et ₃ N (1.4 eq, 0.2 ml) followed by a solution of 1.2 mmol butyric acid chloride (1.2 eq, 0.12 ml) in 2 ml of ethyl acetate over 10 min added in pots. After 2 h, the catalyst was filtered off and the filtrate was concentrated on a rotary evaporator, dissolved in a little isopropanol and precipitated with water. The solid was filtered off, recrystallised from DCM and washed with a little n-hexane. Light yellow solid (mp. 163-164 ° C). Yield: 0.11 g (34.5%). ¹ H-NMR [ppm]: δ = 0.91 (t, 3H, J = 7.4 Hz), 1.60 (se, 2H, J = 7.4 Hz), 2.28 (t, 2H, J = 7.4 Hz), 4.30 (s, 2H), 5.90 (s, 2H), 6.44 (d, 1H, J = 8.0 Hz), 7.10 (m, 2H) , 7.44 (m, 2H), 7.44 (d, 1H, J = 8.0 Hz), 8.98 (s, 1H). ¹³ C-NMR [ppm]: δ = 13.6, 18.5, 32.6, 37.7, 109.2, 115.0 (d, 2C, ² J _{C, F} = 21 Hz), 115, 2, 130.7 (d, 2C, ³ J _CF = 8 Hz), 132.7, 135.0 (d, ⁴ J _{C, F} = 3 Hz), 150.6, 152.9, 159.9 and 162.3 (d, J _{C, F} = 243 Hz), 171.5. IR [cm ^-1]: V = 753, 1208, 1456, 1504, 1589, 1650, 2960, 3147, 3334. HRMS: [C ₁₆ H ₁₈ N ₃ SFO + H] ⁺ calculated: 320.1227; found: 320 , 1232nd

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} pivalinsäureamid

1 mmol of 6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine (0.28 g) was dissolved in 10 ml of THF and a spatula tip of Raney nickel was added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred vigorously for 4.5 h. To the reaction was added 1.4 mmol Et ₃ N (1.4 eq, 0.2 ml) and then a solution of 1.3 mmol pivaloyl chloride (1.3 eq, 0.16 ml) in 2 ml THF over 10 min added in pots. After 2 hours, the catalyst was filtered off and the product was precipitated by the addition of water. The solid was filtered off and recrystallized from toluene. Pale yellow platelets (mp. 165-166 ° C). Yield: 0.17 g (53.3%). ¹ H-NMR [ppm]: δ = 1.21 (s, 9H), 4.32 (s, 2H), 5.70 (s, 2H), 6.47 (d, 1H, J = 7.9 Hz), 7.08-7.12 (m, 2H), 7.20 (d, 1H, J = 7.9 Hz), 7.43-7.47 (m, 2H), 8.68 (s , 1H). ¹³ C-NMR [ppm]: δ = 27.3 (3C), 32.5, 109.5, 115.0 (d, 2C, ² J _{C, F} = 21 Hz), 115.2, 130.7 (d, 2C, ³ J _{C, F} = 8 Hz), 135.0 (d, ⁴ J _{C, F} = 4 Hz), 151.6, 154.4, 159.6 and 162.3 (d, ¹ J _{C, F} = 243 Hz), 177.0. IR [cm ^-1 ]: ṽ = 753, 1220, 1440, 1504, 1590, 1647, 2960, 3181, 3381. HRMS: [C ₁₇ H ₂₀ N ₃ OFS + H] ⁺ calcd: 334.1384, measured: 334 , 1388th

N- {2-amino-6 - [(cyclohexylmethyl) thio] pyridin-3-yl} butyric acid

2 mmol of 6 - [(cyclohexylmethyl) thio] -3-nitropyridine-2-amine (0.53 g) was dissolved in 20 ml of THF and a spatula tip of Raney nickel was added. The reaction batch was placed under a hydrogen atmosphere with the aid of a balloon and stirred vigorously for 1 h. The suspension was cooled to 0 ° C and 1.4 mmol Et ₃ N (2.8 eq, 0.39 ml) added. Subsequently, a solution of 2.4 mmol butyric acid chloride (1.2 eq, 0.25 ml) in 2 ml of THF was added dropwise over 10 min. After 3 h, the reaction was filtered and added to the filtrate 120 ml of water. The product was filtered off and recrystallized twice from toluene. Light yellow solid (mp. 107-109 ° C). Yield: 0.11 g (18.7%). ¹ H-NMR [ppm]: δ = 0.91 (t, 3H, J = 7.4 Hz), 0.97 (m, 2H), 1.10-1.23 (m, 3H), 1, 48 (m, 1H), 1.59 (se, 2H, J = 7.3 Hz), 1.65-1.68 (m, 3H), 1.81 (d, 2H, J = 11.4 Hz ), 2.28 (t, 2H, J = 7.4 Hz), 2.94 (d, 2H, J = 6.6 Hz), 5.79 (s, 2H), 6.43 (d, 1H , J = 8.0 Hz), 7.42 (d, 1H, J = 8.0 Hz), 9.00 (s, 1H). ¹³ C-NMR [ppm]: δ = 13.6, 18.5, 25.5 (2C), 25.9, 32.1 (2C), 36.4, 37.5, 37.7, 109, 2, 114.8, 132.7, 151.9, 152.9, 171.4. IR [cm ^-1]: v = 1227, 1454, 1504, 1588, 1644, 2849, 2925, 3157, 3319, 3395. HRMS: [C ₁₆ H ₂₅ N ₃ OS + H] ⁺ calculated: 308.1791, measured : 308.1797.

2- (3,5-Dimethoxyphenyl) -N- [6- (isobutylthio) -2- (pyrrolidin-1-yl) pyridin-3-yl] acetamide

1 mmol of 6- (isobutylthio) -3-nitro-2- (pyrrolidin-1-yl) pyridine (0.28 g) was dissolved in 10 ml of THF and a spatula tip of Raney nickel added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred vigorously for 0.5 h. Subsequently, the suspension was cooled to 0 ° C. 1.4 mmol Et ₃ N (1.4 eq, 0.2 ml) was added to the reaction followed by a solution of 1.2 mmol 2- (3,5-dimethoxyphenyl) acetyl chloride (0.26 g) in 1, 5 ml DCM was added dropwise over 15 min. After 3 h, 100 ml of water were added to the mixture and extracted with 100 ml of diethyl ether. The organic phase was washed with 2 × 100 ml of water and saturated brine, and dried over Na ₂ SO ₄ . The solution was concentrated on a rotary evaporator whereupon a green oil remained. This crystallized on storage in the refrigerator and could then be recrystallized from methanol. Slightly greenish needles (mp. 137-138 ° C). Yield: 0.09 g (19.8%). ¹ H-NMR [ppm]: δ = 0.97 (d, 6H, J = 6.7 Hz), 1.77 (t, 4H, J = 6.6 Hz), 1.87 (sep, 1H, J = 6.7 Hz), 2.99 (d, 2H, J = 6.7 Hz), 3.37 (t, 4H, J = 6.6 Hz), 3.51 (s, 2H), 3 , 73 (s, 6H), 6.39 (t, 1H, J = 2.3 Hz), 6.48 (d, 1H, J = 8.0 Hz), 6.50 (d, 2H, J = 2.4 Hz), 7.12 (d, 1H, J = 7.9 Hz), 9.39 (s, 1H). ¹³ C-NMR [ppm]: δ = 21.7 (2C), 24.9 (2C), 28.4, 37.5, 42.9, 48.1 (2C), 55.1 (2C), 98.4, 107.2 (2C), 109.4, 114.5, 137.8, 137.9, 152.8, 153.9, 160.3 (2C), 169.5. IR [cm ^-1]: v = 1054, 1148, 1203, 1439, 1515, 1594, 1649, 2863, 2959, 3256. HRMS: [C ₂₃ H ₃₁ N ₃ O ₃ S + H] ⁺ calculated: 430.2159 , measured: 430.2171.

N- {6 - [(Cyclohexylmethyl) thio] -2- (pyrrolidin-1-yl) pyridin-3-yl} -2- (3,5-dimethoxyphenyl) acetamide

1 mmol of 6 - [(cyclohexylmethyl) thio] -3-nitro-2- (pyrrolidin-1-yl) pyridine (0.32 g) was dissolved in 10 ml of THF and a spatula tip of Raney nickel was added. The reaction batch was placed under a hydrogen atmosphere with the aid of a balloon and stirred vigorously for 1 h. 1.4 mmol Et ₃ N (1.4 eq, 0.2 ml) was added to the reaction followed by a solution of 1.2 mmol 2- (3,5-dimethoxyphenyl) acetyl chloride (0.26 g) in 1, 5 ml DCM was added dropwise over 15 min. After 1 h, the suspension was filtered and the filtrate was concentrated on a rotary evaporator. The residue was taken up in diethyl ether and washed with water, saturated NaHCO ₃ solution and saturated brine. The organic phase was dried over Na ₂ SO ₄ and, after removal of the solvent, recrystallized from methanol. The product was then purified by preparative HPLC (gradient: 50-90% methanol / water, self-packed C18 column). This precipitated the product after the methanol was removed on a rotary evaporator. Colorless solid (mp. 142-144 ° C). Yield: 0.04 g (8.5%). ¹ H-NMR [ppm]: δ = 0.95-0.98 (m, 2H), 1.09-1.24 (m, 5H), 1.55-1.82 (m, 10H), 2 , 98 (d, 2H, J = 5.8 Hz), 3.37 (m, 4H), 3.51 (s, 2H), 3.73 (s, 6H), 6.39-6.50 ( m, 4H), 7.11 (d, 1H, J = 7.6 Hz), 9.39 (s, 1H). ¹³ C-NMR [ppm]: δ = 24.9 (2C), 25.6 (2C), 25.9, 32.1 (2C), 36.0, 37.7, 42.9, 48.1 (2C), 55.1 (2C), 98.4, 107.2 (2C), 109.4, 114.4, 137.8, 137.9, 152.9, 153.9, 160.3 ( 2C), 169.6. IR [cm ^-1]: v = 1064, 1142, 1200, 1446, 1517, 1589, 1650, 2848, 2919, 3243. HRMS: _[26 H ₃₅ N ₃ O ₃ S + H C] ⁺ calculated: 470.2472 , measured: 470.2485.

3- (3,5-difluorophenyl) -N- [6- (isobutylthio) -2- (pyrrolidin-1-yl) pyridin-3-yl] propionamide

1 mmol of 6- (isobutylthio) -3-nitro-2- (pyrrolidin-1-yl) pyridine (0.28 g) was dissolved in 10 ml of THF and a spatula tip of Raney nickel added. The reaction batch was placed under a hydrogen atmosphere with the aid of a balloon and stirred vigorously for 1 h. To the reaction was added 1.4 mmol of Et ₃ N (1.4 eq, 0.2 ml), followed by a solution of 1 mmol of 3- (3,5-difluorophenyl) propanoyl chloride (1.0 eq, 0.20 g). in 1.1 ml of DCM over 10 min. After 30 minutes, the catalyst was filtered off, the reaction mixture was added to 100 ml of water and the product was extracted with 100 ml of diethyl ether. The organic phase was washed with 100 ml of water, 100 ml of saturated NaHCO ₃ solution and 100 ml of saturated brine. After drying the ether phase over Na ₂ SO ₄ and evaporation of the solvent resulted in a green-colored solid which was recrystallized from an ethanol / water mixture (9: 1). Colorless solid (mp. 138-140 ° C). Yield: 0.17 g (40.6%). ¹ H-NMR [ppm]: δ = 0.97 (d, 6H, J = 6.7 Hz), 1.77 (t, 4H, J = 6.6 Hz), 1.87 (sep, 6, 7 Hz), 2.62 (t, 2H, J = 7.3 Hz), 2.92 (t, 2H, J = 7.3 Hz), 2.99 (d, 2H, J = 6.6 Hz ), 3.32 (t, 4H, J = 6.6 Hz, 6.48 (d, 1H, J = 7.8 Hz), 6.97-7.08 (m, 3H), 7.06 ( d, 1H, J = 7.8 Hz), 9.26 (s, 1H) ¹³ C-NMR [ppm]: δ = 21.7 (2C), 25.0 (2C), 28.4, 30 , 2, 36.2, 38.9, 48.1 (2C), 101.4 (t, ² J _{C, F} = 26 Hz), 109.4, 111.6 (dd, 2C, ² J _{C, F} = 18 Hz, ³ J _{C, F} = 6 Hz), 114.4, 137.8, 146.0 (t, ³ J _{C, F} = 9 Hz), 152.7, 154.0, 160.0 and 163.5 (dd, 2C ¹ J _{C, F} = 246 Hz, ³ J _{C, F} = 14 Hz), 170.7 IR [cm ^-1 ]: ṽ = 849, 1110, 1232, 1449, 1515, 1592, 1643, 2845, 2961, 3267th HRMS: [C ₂₂ H ₂₇ N ₃ OF ₂ S + H] ⁺ calculated: 420.1916, measured: 420.1904.

propionamide -3- (3,5-difluorophenyl) - N- {[(cyclopropylmethyl) thio] pyridin-3-yl 2-amino-6}

2 mmol of 6 - [(cyclopropylmethyl) thio] -3-nitropyridin-2-amine (0.45 g) was dissolved in 20 ml of THF and a spatula tip of Raney nickel was added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred vigorously for 3 h. To the reaction was added 2.8 mmol Et ₃ N (1.4 eq, 0.39 ml) followed by a solution of 2 mmol 3- (3,5-difluorophenyl) propanoyl chloride (1.0 eq, 0.40 g). in 2.2 ml of DCM over 10 min. After 1 h, the catalyst was filtered off and the solution was concentrated on a rotary evaporator. The resulting green solid was recrystallized twice from an ethanol / water (8: 2) mixture. Colorless solid (mp 122-125 ° C). Yield: 0.24 g (33.1%). ¹ H-NMR [ppm]: δ = 0.24-0.28 (m, 2H), 0.49-0.54 (m, 2H), 1.02-1.11 (m, 1H), 2 , 66 (t, 2H, J = 15.3), 2.94 (t, 2H J = 15.2 Hz), 2.99 (d, 2H, J = 7.0 Hz), 5.81 (s , 2H), 6.44 (d, 1H, J = 8.0 Hz), 7.00-7.07 (m, 3H), 7.37 (d, 1H, J = 8.0 Hz), 9 , 03. ¹³ C-NMR [ppm]: δ = 5.5 (2C), 10.8, 30.4, 34.9, 36.4, 101.4 (t, ² J _{C, F} = 26 Hz), 109 , 1, 111.4 (dd, 2C, ² J _{C, F} = 18 Hz, ³ J _{C, F} = 6 Hz), 114.5, 132.8, 145.9 (t, ³ J _{C, F} = 9 Hz), 152.2, 153.1, 161.1 and 163.5 (dd, 2C, ¹ J _{C, F} = 246 Hz, ³ J _{C, F} = 14 Hz), 170.4. IR [cm ^-1]: V = 771, 832, 968, 1114, 1463, 1523, 1592, 1639, 3091, 3154, 3283, 3465. HRMS: [C ₁₈ H ₁₉ N ₃ OF ₂ S + H] ⁺ calculated : 364.1290, measured: 364.1282.

Ethyl {2-amino-5-bromo-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} carbamate

0.5 mmol of ethyl {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} carbamate (0.17 g) was dissolved in a mixture of 5 ml of THF and 5 ml of ACN, O, Add 0.05 mmol of ammonium acetate (0.1 eq, 0.004 mg) and cool the reaction to 0 ° C. Portions of 0.5 mmol of N-bromosuccinimide (1 eq, 0.09 g) were then added. After 2 h, the suspension was concentrated on a rotary evaporator and the crude product was purified by MPLC (gradient: 0-100% ethyl acetate / n-hexane). The resulting product was then recrystallized from DCM. Colorless solid (mp 171-173 ° C). Yield: 0.06 g (28.5%). ¹ H-NMR [ppm]: δ = 1.23 (t, 3H, J = 7.1 Hz), 4.11 (q, 2H, J = 7.1 Hz), 4.33 (s, 2H) , 6.24 (s, 2H), 7.07-7.11 (m, 2H), 7.47-7.50 (m, 2H), 7.71 (s, 1H), 8.73 (s , 1H). ¹³ C-NMR [ppm]: δ = 14.4, 32.9, 60.6, 101.2, 114.9 (d, 2C, ² J _{C, F} = 21 Hz), 116.2, 131, 0 (d, 2C, ³ J _{C, F} = 8 Hz), 133.5, 135.1 (d, ⁴ J _{C, F} = 3 Hz), 151.4, 154.3, 159.9 and 162, 3 (d, ¹ J _{C, F} = 243 Hz). IR [cm ^-1]: V = 755, 837, 1054, 1217, 1265, 1434, 1505, 1608, 1664, 2985, 3244, 3362, 3470. HRMS: [C ₁₅ H ₁₅ N ₃ O ₂ FSBR + H] ⁺ calculated: 400.0125, measured: 400.0113.

Ethyl {2-amino-6 - [(4-fluorobenzyl) thio] -5-methylphenyl} carbamate

1.75 mmol of 6 - [(4-fluorobenzyl (thio] -5-methyl-3-nitropyridin-2-amine (0.51 g) was added along with 17.5 mmol iron powder (10 eq, 0.95 g) and 17.5 mmol of NH ₄ Cl (0.94 g) were suspended in a mixture of 12 ml of ethanol and 3 ml of water The reaction mixture was refluxed for 4 h and then filtered through kieselguhr The residue was washed with 50 ml of ethyl acetate and 50 ml of water were added to the filtrate, the phases were separated and the aqueous phase was extracted with 2 × 50 ml of ethyl acetate 17.5 mmol of Et ₃ N (10 eq, 2.4 ml) and 2.21 mmol of ethyl chloroformate (1 , 3 eq, 0.21 ml) was added and the solution was stirred overnight at 40 ° C. The resulting solution was concentrated on a rotary evaporator and purified by flash chromatography (mobile phase: ethyl acetate / n-hexane / 0.1% Et ₃ N). Colorless solid (mp: 114-115 ° C) Yield: 0.08 g (22.14%); ¹ H NMR [ppm]: δ = 8.56 (s, 1H), 7.45 (m, 2H), 7.31 (s, 1H), 7.10 (m, 2H), 5.70 (s , 2H), 4.36 (s, 2H), 4.12 (q, J = 7.04 Hz, 2H), 1.98 (s, 3H), 1.25 (t, J = 6.96 Hz , 2H); ¹³ C NMR [ppm]: δ = 162.2 (d, J = 243 Hz, 1C), 154.5, 135.6 (d, J = 3 Hz, 1C), 131.0 (d, J = 8 Hz, 2C), 117.0, 115.0 (d, J = 21 Hz, 2C), 60.3, 31.8, 16.6, 14.5; IR [cm ^-1]: v = 3446, 3355, 3287, 1679, 1509, 1442, 1259, 1219, 1083. HRMS: [C ₁₆ H ₁₈ N ₃ O ₂ FS + H] ⁺ calculated: 3336.1177 precisely measured, : 336.1171.

Ethyl {6 - [(4-fluorobenzyl) thio] -2-methyl-pyridin-3-yl} carbamate

1.8 mmol 6 - [(4-fluorobenzyl) thio] -2-methyl-3-nitropyridine (0.50 g) and 9 mmol SnCl ₂ • 2H ₂ O (5 eq, 2.03 g) were dissolved in 20 ml Ethanol and stirred under argon atmosphere at 70 ° C overnight. The temperature was then lowered to 40 ° C and 18.36 mmol Et ₃ N (10.2 eq, 2.56 ml) and 2.34 mmol of ethyl chloroformate (1.3 eq, 0.22 ml) were added. After 3 h, the suspension was filtered through kieselguhr and the residue was washed with 30 ml of ethanol. The filtrate was concentrated on a rotary evaporator and the product was precipitated by addition of water. Beige solid (mp. 102-103 ° C); Yield: 0.6 g (84%); ¹ H NMR [ppm]: δ = 8.99 (s, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.46 (m, 2H), 7.13 (m, 3H ), 4.36 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 2.40 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H ); ¹³ C NMR [ppm]: δ = 162.3 (d, J = 243 Hz, 1C), 154.4, 152.0, 134.7 (d, J = 3 Hz, 1C), 132.8, 130 , 8 (d, J = 8Hz, 2C), 129.5, 119.5, 115.1 (d, J = 21Hz, 2C), 60.43, 32.8, 20.8, 14.5 ; IR [cm ^-1 ]: ṽ = 3284, 2980, 1689, 1509, 1442, 1250, 1061; HRMS: [C ₁₆ H ₁₇ N ₂ O ₂ FS + H] ⁺ calculated: 321.1068, Measured: 321.1060.

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} -3,4-difluorobenzamide

5 mmol of 6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine (1.40 g) was added along with 50 mmol iron powder (10 eq, 2.80 g) and 50 mmol NH ₄ Cl (10 eq , 2.68 g) in a mixture of 12 ml of ethanol and 3 ml of water. The reaction mixture was heated to reflux for one hour and then filtered through kieselguhr. To the filtrate was added 50 ml of water and extracted with 2x50 ml of ethyl acetate. To the organic phase was added 7.5 mmol Et ₃ N (1.5 eq, 1.05 mL) and the solution cooled to 0 ° C. 5 mmol of 3,4-difluorobenzoyl chloride (1 eq, 0.63 ml) was added dropwise over 30 minutes and then the mixture was stirred for 1 h at room temperature. Concentration of the solution on a rotary evaporator and storage in the refrigerator precipitated the product and was finally recrystallized from DCM. Colorless solid (mp. 150-151 ° C). Yield: 0.68 g (35%); ¹ H NMR [ppm]: δ = 9.64 (s, 1H), 8.08 (m, 1H), 7.86 (d, J = 2.0 Hz, 1H), 7.63 (m, 1H ), 7.56 (m, 2H), 7.34 (d, 1H), 7.13 (m, 2H), 6.49 (d, J = 7.9 Hz, 1H), 6.09 (s , 2H), 4.34 (s, 2H); ¹³ C NMR [ppm]: δ = 167.3, 162.3 (d, J = 241 Hz, 1C), 154.6, 152.5, 150.3, 147.8, 135.2, 135.0 (d, J = 3Hz, 1C), 130.8 (d, J = 8Hz, 2C), 125.4, 117.5 (d, J = 9Hz, 1C), 117.3 (d, J = 9 Hz, 1C), 115.1 (d, J = 21 Hz, 2C), 114.1, 109.0, 32.5; IR [cm ^-1 ]: ṽ = 3396, 3298, 3150, 1629, 1504, 1462, 1226, 750; HRMS: [C ₁₉ H ₁₄ N ₃ OF ₃ S + H] ⁺ calculated: 388.0737, Measured: 388.0747.

Ethyl (6 - {[(1,1'-biphenyl) -4-ylmethyl] thio} -2-aminopyridine-3-yl) carbamate

2 mmol of 6 - {[(1,1'-biphenyl) -4-ylmethyl] thio} -3-nitropyridin-2-amine (0.68 g) was added along with 10 mmol SnCl ₂ • 2H ₂ O (2.26 g) suspended in 20 ml of ethanol and stirred under argon atmosphere at 70 ° C for 24 h. The temperature was then reduced to 40 ° C and 20.4 mmol Et ₃ N (10.2 eq, 2.85 ml) and 2.6 mmol of ethyl chloroformate (1.3 eq, 0.25 ml) were added. The reaction mixture was stirred overnight and the suspension was then filtered through kieselguhr. The filtrate was concentrated on a rotary evaporator and the product was precipitated by addition of water. Finally, it was recrystallized from DCM. Beige solid (mp 165-167 ° C); Yield: 0.3 g (38%); ¹ H NMR [ppm]: δ = 8.58 (s, 1H), 7.65 (m, 2H), 7.60 (m, 2H), 7.50 (m, 5H), 7.56 (m , 1H), 6.48 (d, J = 8.0 Hz, 1H), 5.96 (s, 2H), 4.35 (s, 2H), 4.12 (q, J = 7.1 Hz , 2H), 1.24 (t, J = 7.1 Hz, 3H); ¹³ C NMR [ppm]: δ = 154.9, 154.2, 152.0, 139.6, 139.5, 130.8 (2C), 129.9, 128.9 (2C), 127.5 , 126.5 (2C), 126.4 (2C), 120.1, 108.6, 60.6, 58.7, 14.4; IR [cm ^-1 ]: ṽ = 3484, 3290, 3161, 1703, 1622, 1460, 1219, 1065, 747; HRMS: [C ₂₁ H ₂₁ N ₃ O ₂ S - H] - Calculated: 378.1282; found: 378.1272.

Ethyl {2-amino-6 - [(3,5-dimethoxybenzyl) thio] pyridin-3-yl} carbamate

2.5 mmol 6 - [(3,5-dimethoxybenzyl) thio] -3-nitropyridin-2-amine (0.80 g) was added along with 12.5 mmol SnCl ₂ • 2H ₂ O (5 eq, 2.82 g g) suspended in 20 ml of ethanol and stirred under argon atmosphere at 70 ° C overnight. The temperature was then lowered to 40 ° C and 25.5 mmol of Et ₃ N (10.2 eq, 3.5 ml) and 3.25 mmol of ethyl chloroformate (1.3 eq, 0.31 ml) were added to the suspension. After 4 h, the reaction mixture was filtered through kieselguhr and the residue was washed with 60 ml of ethanol. The filtrate was concentrated on a rotary evaporator and the residue was purified by flash chromatography (mobile phase: ethyl acetate / n-hexane). The product was finally dissolved in a little ethanol and precipitated by addition of water. Colorless solid (mp. 84-85 ° C); Yield = 0.46 g (52%); ¹ H NMR [ppm]: δ = 8.57 (bs, 1H), 7.41 (d, J = 5.1 Hz, 1H), 6.57 (d, J = 2.1 Hz, 2H), 6.44 (d, J = 8.0Hz, 1H), 6.34 (t, 1H), 5.94 (s, 2H), 4.22 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.70 (s, 6H), 1.24 (t, J = 7.0 Hz, 3H); ¹³ C NMR [ppm]: δ = 160.2 (2C), 154.4, 152.8, 150.4, 140.9, 131.8, 115.2, 109.4, 106.7 (2C) , 98.7, 60.3, 55.1 (2C), 33.7, 14.5; IR: ṽ = 3475, 3285, 3163, 2935, 1685, 1598, 1520, 1460, 1202, 1148, 1057 cm ^-1 ; HRMS: [C ₁₇ H ₂₁ N ₃ O ₄ S + H] ⁺ calculated: 364.1326, Measured: 364.1317.

Ethyl {2-amino-6 - [(pyridin-2-ylmethyl) thio] pyridin-3-yl} carbamate

1 mmol of 3-nitro-6 - [(pyridin-2-ylmethyl) thio] pyridin-2-amine (0.26 g) was added together with 4.6 mmol SnCl ₂ • 2H ₂ O (4.6 eq, 1, 04 g) in 10 ml of ethanol and stirred under argon atmosphere at 70 ° C for 22 h. Subsequently, the temperature was lowered to 40 ° C and 10.2 mmol Et ₃ N (10.2 eq, 1.4 ml) and 1.3 mmol of ethyl chloroformate (0.12 ml) was added. The reaction mixture was stirred overnight and then filtered through kieselguhr. The filtrate was concentrated on a rotary evaporator, the residue was treated with water and extracted with DCM. The organic phase was evaporated and the residue was purified by flash chromatography. Brown solid (mp. 129-131 ° C); Yield: 0.09 g (30%); ¹ H NMR [ppm]: δ = 8.57 (s, 1H), 8.49 (d, J = 4.3 Hz, 1H), 7.73 (m, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 6.0 Hz, 1H), 7.25 (m, 1H), 6.49 (d, J = 8.0 Hz, 1H), 5.94 (s, 2H), 4.39 (s, 2H), 4.12 (q, J = 7.1Hz, 2H), 1.24 (t, J = 7.1Hz, 3H); ¹³ C NMR [ppm]: δ = 158.2, 154.5, 152.8, 150.3, 149.0, 136.7, 131.9, 130.1, 123.1, 122.1, 115 , 2, 109, 2, 60, 4, 45, 6, 35, 7, 14, 5; IR [cm ^-1 ]: ṽ = 3361, 3227, 2979, 2929, 1721, 1524, 1456, 1245, 1221, 1071; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S + H] ⁺ calculated: 305.1067, Measured: 305.1061.

Ethyl (2-amino-6 - {[2- (piperidin-1-yl) ethyl] thio} pyridin-3-yl) carbamate

1 mmol of 3-nitro-6 - {[2- (piperidin-1-yl) ethyl] thio} pyridin-2-amine (0.28 g) was added together with 4.6 mmol of SnCl ₂ • 2H ₂ O (4, 6 eq, 1.02 g) in 10 ml of ethanol and stirred under argon atmosphere at 70 ° C for 25 h. The temperature was then lowered to 40 ° C and 10.2 mmol Et ₃ N (10.2 eq, 1.4 ml) and 2.6 mmol of ethyl chloroformate (2.6 eq, 0.24 ml) was added. The suspension was stirred for a further 3 h, then filtered through kieselguhr and the residue was washed with 60 ml of ethanol. The filtrate was concentrated on a rotary evaporator, the residue was combined with 50 ml of water and extracted with DCM. The organic phase was finally evaporated. Brown solid (mp. 136-137 ° C); Yield: 0.1 g (32%); ¹ H NMR [ppm]: δ = 8.55 (s, 1H), 7.40 (d, J = 6.1 Hz, 1H), 6.44 (d, J = 8.0 Hz, 1H), 5, 82 (s, 2H), 4.12 (q, J = 7.1 Hz, 2H), 3.14 (q, J = 8.8 Hz, 2H), 2.50 (m, 2H), 2, 38 (m, 4H), 1.51 (m, 4H), 1.39 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H); ¹³ C NMR [ppm]: δ = 154.5, 152.8, 151.1, 131.9, 114.9, 109.3, 60.3, 58.2, 53.8 (2C), 26, 9, 25.5 (2C), 24.0, 14.5; IR [cm ^-1 ]: ṽ = 3341, 3212, 2934, 1713, 1530, 1452, 1214, 1065; HRMS ESI m / z [C ₁₅ H ₂₄ N ₄ O ₂ S + H] ⁺ calculated: 325.1693, Measured: 325.1698.

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} -2- (3,5-difluorpheny) acetamide

1.5 mmol of 6 - [(4-fluorobenzyl) thio] -3-nitropyridin-2-amine (0.42 g) was added along with 15 mmol iron powder (10 eq, 0.84 g) and 15 mmol NH ₄ Cl ( 10 eq, 0.81 g) in a mixture of 12 ml of ethanol and 3 ml of water. The reaction mixture was heated to reflux for one hour, then filtered through a paper filter and the residue washed with 75 ml of ethyl acetate. 1.5 mmol of Et ₃ N (1 eq, 0.21 ml) and 2 mmol of 2- (3,5-difluorophenyl) acetyl chloride were added to the filtrate and the mixture was stirred at 40 ° C. for 1 h. The solution was then concentrated on a rotary evaporator and the residue was purified by flash chromatography (mobile phase: ethyl acetate / n-hexane). Finally, it was recrystallized from ethanol. Colorless solid (mp: 166-167 ° C). Yield: 0.08 g (21.4%); ¹ H NMR [ppm]: δ = 9.29 (s, 1H), 7.46 (m, 2H), 7.40 (d, J = 7.96 Hz, 1H), 7.12 (m, 5H ), 6.44 (d, J = 7.96 Hz, 1H), 6.00 (s, 2H), 4.31 (s, 2H), 3.71 (s, 2H); ¹³ C NMR [ppm]: δ = 168.5, 163.4 (dd, J = 13 Hz, J = 240 Hz, 2C), 162.3, 159.9, 153.3, 151.4, 140, 4 (t, J = 10Hz, 1C), 135.0 (d, J = 3Hz, 1C), 133.2, 130.8 (d, J = 8Hz, 2C), 115.1 (d, J = 21 Hz, 2C), 114.6, 112.7 (dd, J = 7 Hz, J = 18 Hz, 2C), 109.1, 102.2 (t, J = 26 Hz, 1C), 41 , 8, 32.5; IR [cm ^-1 ]: ṽ = 3404, 3179, 1644, 1505, 1454, 1223, 1119, 839; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S + H] ⁺ calculated: 404.1039, Measured: 404.1038.

N- [6- (benzylthio) -2- (methylamino) pyridin-3-yl] -2- (3,5-difluorophenyl) acetamide

2.8 mmol of 6- (benzylthio) -N-methyl-3-nitropyridin-2-amine (0.77 g) was added along with 28 mmol iron powder (10 eq, 1.57 g) and 28 mmol NH ₄ Cl (10 eq, 1.50 g) in a mixture of 12 ml of ethanol and 3 ml of water. The reaction mixture was heated to reflux for 2 h, then the suspension was filtered through kieselguhr and the residue was washed with 2 × 50 ml of ethyl acetate. 50 ml of water were added and the phases separated. The aqueous phase was extracted with 2x50 ml of ethyl acetate and to the combined organic phases were added 5.6 mmol HATU (2 eq, 2.10 g) and 2.8 mmol 2,6-dichlorophenylacetic acid (1 eq, 0.60 g) and the mixture was stirred at 40 ° C overnight. The suspension was concentrated on a rotary evaporator and purified by flash chromatography (mobile phase: ethyl acetate / n-hexane). The crude product was dissolved in a little ethanol and precipitated by addition of water. Lavender solid (m.p. 201-202 ° C); Yield: 0.08 g (8%); ¹ H NMR [ppm]: δ = 9.25 (s, 1H), 7.40 (m, 2H), 7.30 (m, 4H), 7.13 (m, 3H), 6.41 (i.e. , J = 7.8 Hz, 1H), 6.23 (d, J = 4.6 Hz, 2H), 4.38 (s, 2H), 3.70 (s, 2H), 2.89 (i.e. , J = 4.6 Hz, 2H); ¹³ C NMR [ppm]: δ = 168.7, 163.4 (dd, J = 13 Hz, J = 244 Hz, 2C), 153.2, 151.6, 140.4 (t, J = 10 Hz , 1C), 138.9, 133.2, 128.6 (2C), 128.3 (2C), 126.8, 115.1, 112.7 (dd, J = 6 Hz, J = 17 Hz, 2C), 107.9, 102.2 (t, J = 26 Hz, 1C), 41.8, 33.3, 27.9; IR [cm ^-1 ]: ṽ = 3442, 3404, 3269, 1652, 1591, 1496, 1389, 1230, 1119, 991, 696; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S - H] - Calculated: 398.1144; found: 398.1157.

N- (2-amino-6 - {[(tetrahydro-2H-pyran-2-yl) methyl] thio} pyridin-3-yl) -2- (3,5-difluorophenyl) -acetamide

2- (3,5-difluorophenyl) acetyl chloride

10 mmol of 3,5-difluorophenylacetic acid (1.72 g) was dissolved in 15 ml of dry DCM and 11 mmol of oxalyl chloride (1.1 eq, 0.93 ml) was added under a nitrogen atmosphere. To the reaction mixture was carefully added 4 drops of dry DMF and stirred at room temperature overnight. The solution was used without further purification for further reactions.

N- (2-amino-6 - {[(tetrahydro-2H-pyran-2-yl) methyl] thio} pyridin-3-yl) -2- (3,5-difluorophenyl) acetamide

0.8 mmol of 3-nitro-6 - {[(tetrahydro-2H-pyran-2-yl) -methyl] -thio} -pyridine-2-amine (0.22 g) was added together with 8 mmol iron powder (10 eq, 0, 45 g) and NH ₄ Cl (10 eq, 0.43 g) suspended in a mixture of 12 ml of ethanol and 3 ml of water. The reaction mixture was heated to reflux for 3 h, then the suspension was filtered through a paper filter and the residue was washed with 50 ml of ethyl acetate. To the filtrate was added 1.5 mmol of Et ₃ N (1.9 eq, 0.17 ml) and 1.2 mmol of 2- (3,5-difluorophenyl) acetyl chloride from (a). The solution was heated to 40 ° C for 1.5 h and then concentrated in a rotary evaporator. The product was finally purified by flash chromatography (mobile phase: ethyl acetate / n-hexane). Colorless solid (mp. 136-137 ° C). Yield: 0.21 g (54%); ¹ H NMR [ppm]: δ = 9.27 (s, 1H), 7.38 (d, J = 7.96 Hz, 1H), 7.14 (m, 3H), 6.44 (d, J = 7.96 Hz, 1H), 5.91 (s, 2H), 3.87 (m, 1H), 3.70 (s, 2H), 3.40 (m, 3H), 3.21 (m , 2H), 1.78 (m, 7H); ¹³ C NMR [ppm]: δ = 168.5, 163.4 (dd, J = 13 Hz, J = 244 Hz, 2C), 153.3, 152.1, 140.4 (t, J = 10 Hz , 1C), 133.3, 114.3, 112.7 (dd, J = 7 Hz, J = 19 Hz, 2C), 109.1, 102.2 (t, J = 26 Hz, 1C), 76 , 4, 67.5, 41.8, 34.9, 30.6, 25.4, 22.7; IR [cm ^-1 ]: ṽ = 3305, 3180, 2940, 1645, 1463, 1114, 671; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S + H] ⁺ calculated: 394.1395, Measured: 394.1390.

N- (6 - {[(1,3-dioxolan-2-yl) methyl] thio} -2-morpholinopyridine-3-yl) -2- (3,5-difluorophenyl) acetamide

2 mmol of 4- (6 - {[(1,3-dioxolan-2-yl) methyl] thio} -3-nitropyridin-2-yl) morpholine (0.65 g) was suspended in 15 ml of THF and two Raney spatula tips Nickel added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred for 2 h at room temperature. Subsequently, 2 mmol of Et ₃ N (1 eq, 0.28 ml) and 3 mmol of 2- (3,5-difluorophenyl) acetyl chloride from 1.2.23 (a) were added and the mixture was stirred for a further 4 h. The suspension was then filtered through kieselguhr, the filtrate was concentrated on a rotary evaporator and the residue was purified by flash chromatography (mobile phase: ethyl acetate / n-hexane). Finally, the product was recrystallized from ethanol. Colorless solid (mp. 104-105 ° C). Yield: 0.24 g (27%); ¹ H NMR [ppm]: δ = 9.34 (s, 1H), 7.73 (d, J = 8.20 Hz, 1H), 7.17 (m, 3H), 6.92 (d, J = 8.2 Hz, 1H), 5.08 (t, J = 4.52 Hz, 1H), 3.94 (m, 2H), 3.84 (m, 2H), 3.75 (s, 2H ), 3.44 (m, 4H), 3.35 (m, 4H), 3.05 (m, 4H); ¹³ C NMR [ppm]: δ = 168.2, 163.5 (dd, J = 13 Hz, J = 243 Hz, 2C), 154.2, 150.5, 140.1 (t, J = 10 Hz , 1C), 134.5, 120.8, 115, 0, 112.5 (dd, J = 7Hz, J = 17Hz, 2C), 102.5, 102.3, 65.8 (2C), 64.6 (2C), 48.7 (2C), 42.2, 32.5; IR [cm ^-1 ]: ṽ = 3265, 1657, 1516, 1463, 1118, 958; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S + H] ⁺ calculated: 452.1450, Measured: 452.1452.

N- {2-amino-6 - [(cyclobutylmethyl) thio] pyridin-3-yl} -2- (3,5-difluorophenyl) acetamide

2 mmol of 6 - [(cyclobutylmethyl) thio] -3-nitropyridin-2-amine (0.48 g) were suspended in 15 ml of THF and 2 Raney nickel spatula tips added. The reaction batch was placed under a hydrogen atmosphere using a balloon and stirred for 2 h at room temperature. The suspension was then filtered through kieselguhr and the residue was washed with 75 ml of ethyl acetate. The filtrate was cooled to 0 ° C and 2 mL of Et ₃ N (1 eq, 0.28 mL) and 2 mmol of 2- (3,5-difluorophenyl) acetyl chloride added dropwise. After 2 hours, the solution was concentrated on a rotary evaporator, whereupon the product precipitated. Finally, it was recrystallized from DCM. Colorless solid (mp. 199-200 ° C). Yield: 0.47 g (65%); ¹ H NMR [ppm]: δ = 9.99 (s, 1H), 7.81 (d, J = 8.00 Hz, 1H), 7.14 (m, 3H), 6.71 (d, J = 8.12 Hz, 1H), 3.82 (s, 2H), 3.22 (d, J = 7.56 Hz, 2H), 2.07 (m, 2H), 1.85 (s, 4H ); ¹³ C NMR [ppm]: δ = 168.9, 163.4 (dd, J = 13 Hz, J = 245 Hz, 2C), 151.5, 140.1 (t, J = 10 Hz, 1C), 134.6, 112.8 (dd, J = 7 Hz, J = 17 Hz, 2C), 110.8, 102.3 (t, J = 26 Hz, 1C), 41.7, 37.3, 34 , 2, 27, 1 (2C), 17.4; IR [cm ^-1 ]: ṽ = 3071, 1650, 1524, 1251, 1115, 991, 838; HRMS: [C ₁₄ H ₁₆ N ₄ O ₂ S + H] ⁺ calculated: 364.1290, Measured: 364.1287.

In vitro investigations

used substances

Ethyl-2-amino-6-[(4-fluorbenzyl)-amino]-3-pyridincarbamat-Maleat (Flupirtin-Maleat) 1

Ethyl-{2-amino-6-[(4-fluorbenzyl)-thio]pyridin-3-yl}carbamat 2

Ethyl-(2-amino-6-{[4-(trifluormethyl)-benzyl]thio}pyridin-3-yl)carbamat 3

Ethyl-{2-amino-6-[(3,4-difluorbenzyl)thio]pyridin-3-yl}carbamat 4

Ethyl-{2-amino-5-brom-6-[(4-fluorbenzyl)thio]pyridin-3-yl}carbamat 5

Ethyl-(2-amino-6-{[4-(pentafluor-λ⁶-sulfanyl)benzyl]thio}pyridin-3-yl)carbamat 6

N-{2-Amino-6-[(4-fluorbenzyl)-thio]pyridin-3-yl}buttersäureamid 7

N-{2-Amino-6-[(4-fluorbenzyl)-thio]pyridin-3-yl}pivalinsäureamid 8

N-{2-Amino-6-[(cyclohexylmethyl)-thio]pyridin-3-yl}buttersäureamid 9

N-{6-[(Cyclohexylmethyl)thio]-2-(pyrrolidin-1-yl)pyridin-3-yl}-2-(3,5-dimethoxyphenyl)acetamid 10

2-(3,5-Dimethoxyphenyl)-N-(6-(isobutylthio)-2-[pyrrolidin-1-yl)pyridin-3-yl]acetamid 11

3-(3,5-Difluorphenyl)-N-(6-[isobutylthio)-2-(pyrrolidin-1-yl)pyridin-3-yl]propionsäureamid 12

N-{2-Amino-6-[(cyclopropylmethyl)-thio]pyridin-3-yl}-3-(3,5-difluorphenyl)propionsäureamid 13

Ethyl-{2-amino-6-[(pyridin-2-ylmethyl)thio]pyridin-3-yl}carbamat 14

Ethyl-(2-amino-6-{[2-(piperidin-1-yl)ethyl]thio}pyridin-3-yl)carbamat 15

Ethyl-[2-amino-6-(benzylthio)pyridin-3-yl]carbamat (6b) 16

Ethyl-{2-amino-6-[(3,5-dimethoxybenzyl)thio]pyridin-3-yl}carbamat 17

Ethyl-(6-{[(1 ,1'-biphenyl)-4-ylmethyl]thio}-2-aminopyridin-3-yl)carbamat 18

Ethyl-{6-[(4-fluorbenzyl)thio]-2-methylpyridin-3-yl)carbamat 19

N-{2-Amino-6-[(4-fluorbenzyl)-thio]pyridin-3-yl}-3,4-difluorbenzamid 20

N-[6-(Benzylthio)-2-(methylamino)pyridin-3-yl]-2-(3,5-difluorphenyl)acetamid 21

Ethyl-{2-amino-6-[(4-fluorbenzyl)thio]-5-methylphenyl}carbamat 22

N-{2-Amino-6-[(4-fluorbenzyl)-thio]pyridin-3-yl}-2-(3,5-difluorphenyl)acetam id 23

N-(2-Amino-6-{[(tetrahydro-2H-pyran-2-yl)methyl]thio}pyridin-3-yl)-2-(3,5-difluorphenyl)acetamid 24

N-(6-{[(1,3-Dioxolan-2-yl)methyl]thio}-2-morpholinopyridin-3-yl)-2-(3,5-difluorphenyl)acetamid 25

N-{2-Amino-6-[(cyclobutylmethyl)thio]pyridin-3-yl}-2-(3,5-difluorphenyl)acetam id The Substances used in the in vitro studies are shown in Table 2. Table 2 in the in vitro tests substances used number structure Surname Comparative Example 1

Ethyl 2-amino-6 - [(4-fluorobenzyl) amino] -3-pyridinecarbamate maleate (flupirtine maleate) 1

Ethyl {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} carbamate 2

Ethyl (2-amino-6 - {[4- (trifluoromethyl) benzyl] thio} pyridin-3-yl) carbamate 3

Ethyl {2-amino-6 - [(3,4-difluorobenzyl) thio] pyridin-3-yl} carbamate 4

Ethyl {2-amino-5-bromo-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} carbamate 5

Ethyl (2-amino-6 - {[4- (pentafluoro-λ ⁶ -sulfanyl) benzyl] thio} pyridin-3-yl) carbamate 6

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} butyric acid 7

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} pivalinsäureamid 8th

N- {2-amino-6 - [(cyclohexylmethyl) thio] pyridin-3-yl} butyric acid 9

N- {6 - [(Cyclohexylmethyl) thio] -2- (pyrrolidin-1-yl) pyridin-3-yl} -2- (3,5-dimethoxyphenyl) acetamide 10

2- (3,5-dimethoxyphenyl) -N- (6- (isobutylthio) -2- [pyrrolidin-1-yl) pyridin-3-yl] acetamide 11

3- (3,5-difluorophenyl) -N- (6- [isobutylthio) -2- (pyrrolidin-1-yl) pyridin-3-yl] propionamide 12

propionamide -3- (3,5-difluorophenyl) - N- {[(cyclopropylmethyl) thio] pyridin-3-yl 2-amino-6} 13

Ethyl {2-amino-6 - [(pyridin-2-ylmethyl) thio] pyridin-3-yl} carbamate 14

Ethyl (2-amino-6 - {[2- (piperidin-1-yl) ethyl] thio} pyridin-3-yl) carbamate 15

Ethyl [2-amino-6- (benzylthio) pyridin-3-yl] carbamate (6b) 16

Ethyl {2-amino-6 - [(3,5-dimethoxybenzyl) thio] pyridin-3-yl} carbamate 17

Ethyl (6 - {[(1,1'-biphenyl) -4-ylmethyl] thio} -2-aminopyridin-3-yl) carbamate 18

Ethyl {6 - carbamate [(4-fluorobenzyl) thio] -2-methyl-pyridin-3-yl) 19

N- {2-amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} -3,4-difluorobenzamide 20

N- [6- (benzylthio) -2- (methylamino) pyridin-3-yl] -2- (3,5-difluorophenyl) acetamide 21

Ethyl {2-amino-6 - [(4-fluorobenzyl) thio] -5-methylphenyl} carbamate 22

N- {2-Amino-6 - [(4-fluorobenzyl) thio] pyridin-3-yl} -2- (3,5-difluorophenyl) acetamide id 23

N- (2-amino-6 - {[(tetrahydro-2H-pyran-2-yl) methyl] thio} pyridin-3-yl) -2- (3,5-difluorophenyl) acetamide 24

N- (6 - {[(1,3-dioxolan-2-yl) methyl] thio} -2-morpholinopyridine-3-yl) -2- (3,5-difluorophenyl) acetamide 25

N- {2-Amino-6 - [(cyclobutylmethyl) thio] pyridin-3-yl} -2- (3,5-difluorophenyl) acetamide id

Potassium channel assay

Today, it is widely believed that the analgesic effect of flupirtine is due to its opening of the 7.2 / 3 potassium channel in the CNS. That's why an in vitro screening Method to evaluate the opening of this channel in the human potassium channel K _v 7.2 / 3 transfected cells by test substances. [40] The principle of the assay is based on the fact that HEK 293 cells expressing K _v 7.2 / 3 (voltage-dependent potassium channels 7.2 / 3) are loaded with a thallium-sensitive dye, which is cleaved by the cell-internal esterases to the active thallium-complexing agent. This forms fluorescent complexes with thallium ions, which flow through opened K _v 7.2 / 3. The intensity of the thallium signal is proportional to the number of open K _v 7.2 / 3 channels. Since the test substances are potential K _v 7.2 / 3 openers, the assay is suitable for determining the potassium channel activity of the substances.

To characterize this property, the EC ₅₀ and Emax of a test substance are determined. The half-maximal effect concentration (EC ₅₀ ) is the concentration of a substance at which the half-maximal effect of the assay described below is observed when the logarithmic concentration of a substance is plotted against the associated corrected ΔF / F values. Thus, the power of a connection is characterized. The intrinsic activity of a test compound is described by its E _max value. This is the maximum response, based on the present assay, the plateau effect, which is possible by the corresponding test substance. For this, Emax of the flupirtine maleate used as a reference was set to 100% and the maximum responses of the test substances relative to this value.

For the cell-based assay, HEK 293 cells (human embryonic kidney cells, SB-HEK-Kv7.2 / 7.3, SB Drug Discovery) expressing K _v 7.2 / 3 were used. These adherent cells were incubated in cell culture flasks (e.g., T75 TC standard, # 83.3911, Sarstedt) in incubator at 37 ° C, 5% CO ₂ and 95% humidity with MEM (Minimum Essential Medium, # 10370047, Thermo Fisher containing 10% FBS (fetal calf serum, F7524, Sigma Aldrich, heat-inactivated), 2 mM L-glutamine (G7513, Sigma Aldrich), 4 μg / ml blasticidin S HCl (# 203350-25, VWR), 1% penicillin. Streptomycin mix (# 15070063, Thermo Fisher) and 0.78 mg / ml G418 sulphate (CP11.2, Carl Roth) cultured. On the day before the experiment, cells of approximately 80% confluence were washed with phosphate buffered saline (eg L1825, Biochrom), detached from the cell culture flask bottom using TrypLE ™ Express (1x) (# 12604013, Thermo Fisher) (2 min 37 ° C), then suspended with MEM, then centrifuged for 2 min at 1000 rpm and resuspended in MEM after supernatant. The cell number of the suspension was determined with the automatic cell counter EVE ™ (NanoEnTEK). Then, 60,000 cells / well / 100 μL were seeded in 96 well Vision Plates ™ (96-well sterile black TC-treated 96-well microtiter plates, 4ti-0221, 4titude) and at 37 ° C, 5% CO _2, and 95 h for 24 h % Humidity incubated.

F_i(t) bzw. F_k(t) ist die Fluoreszenzintensität zu einem definierten Zeitpunkt (Erläuterung s.o.) einer Test-Substanzkonzentration bzw. der Negativkontrolle. <F_B> ist der Mittelwert der Fluoreszenzintensitäten, welche während der Hintergrundmessung generiert wurden. Mithilfe der GraphPad Prism Software (Version 6) wurde eine nichtlineare Regression der berechneten korrigierten ΔF/F-Werte und der zugehörigen logarithmierten Konzentrationswerte einer Verbindung durchgeführt, wobei als Passform der Kurve die vorgefertigte Gleichung „log(Agonist) gegen Effekt“ (variabler Anstieg, 4 Parameter) verwendet wurde, um unter anderem einen EC₅₀-Wert zu generieren. Beim E_max-Wert einer Substanz handelt es sich um die Differenz des höchsten und niedrigsten korr. ΔF/F-Werts (Angabe im Auswertedatenblatt der jeweiligen nichtlinearen Regressionsanalyse durch Software) der erhaltenden sigmoidalen Kurve prozentual bezogen auf den maximal erreichten Aktivität des als Referenzsubstanz verwendeten Flupirtinmaleats.The FLIPR ^® Potassium Assay Kit (R8222 Explorer Version, Molecular Devices) was used to measure the potassium channel activity of the substances. The assay was performed according to the manufacturer's instructions, which will be discussed in more detail below. To prepare the Loading Buffer, Component C was dissolved in 30 μL DMSO (# A994.2, Carl Roth) and transferred together with 10 ml of Component B into the vessel of Component A. Mixing produced a homogeneous solution. In addition, the Loading Buffer Probenecid (sc-202773A, Santa Cruz Biotechnology) was added at a concentration of 5 mM initially by this additive prepared a 500 mM stock solution in 1 N NaOH solution, which was subsequently 1: 1 (v / v ) was diluted with component B. The pH of the Loading Buffer was then adjusted to 7.4 with 1 M HCl by means of a pH electrode. To the cells was added 100 μL / well loading. The mixture was then incubated in the dark at room temperature for 1 h. Of the compounds to be tested, serial serial dilutions (at least five concentrations) were made in DMSO (# A994.2, Carl Roth) with concentrations 100 times the desired later in-well concentrations. The cells were incubated with the prepared substance concentrations, added in the ratio 1: 100 (v / v), for a further 30 min in the dark at room temperature. The negative control included only solvent (1% DMSO). For the fluorescence intensity measurements (485 nm excitation, 535 nm emission, measurement mode floor) the microtiter plate reader Infinite ^® F200 Pro (Tecan) was used. The plate to be measured was placed in the device. The fluorescence intensity of each well was determined over a period of 20 s, with all 2.5-3 s readings being taken by the instrument (background measurement). Thereafter, a rapid 1: 5 (v / v) stimulus buffer containing 7.5 mM Tl ₂ SO ₄ , 12.5 mM K ₂ SO ₄ and 1 x chloride-free buffer in distilled water was added to the individual wells , The fluorescence intensity of the respective wells was determined approximately every 2 min periodically every 2.5-3 s. The determination of the potassium channel opening activity of a compound based on the recorded data was carried out in several steps. First, for each dilution step of a substance, its fluorescence intensity change was calculated at a defined time relative to the averaged fluorescence intensities of the background measurement (ΔF / F). Furthermore, this determined result was adjusted by the analogously calculated value of the negative control (corr. ΔF / F). At the mentioned defined time (t) was the sum the respective corrected by the negative control fluorescence intensity changes the dilution series of a substance maximum. By visual inspection of an associated fluorescence intensity-time diagram (fluorescence intensity corrected for negative control, relative to respective background), the timing selection was controlled. The following equation summarizes the described calculation: $$kOrriGierte\frac{\Delta F}{F}=\frac{Fi\left(t\right)-<FiB>}{<FiB>}-\left(1-\frac{FK\left(t\right)}{<FKB>}\right)$$

F _i (t) or F _k (t) is the fluorescence intensity at a defined time (see above) of a test substance concentration or the negative control. <F _B > is the mean of the fluorescence intensities generated during the background measurement. Using the GraphPad Prism software (version 6), a nonlinear regression of the calculated corrected ΔF / F values and the associated logarithmic concentration values of a compound was performed using the prefixed equation "log (agonist) versus effect" (variable slope, 4 parameters) was used to generate, inter alia, an EC ₅₀ value. The E _max value of a substance is the difference between the highest and lowest corr. ΔF / F value (data in the evaluation data sheet of the respective nonlinear regression analysis by software) of the sigmoidal curve obtained, expressed as a percentage of the maximum activity of the flupirtine maleate used as the reference substance.

shows two representative dose-response curves for the opening of the K _v 7.2 / 3 potassium channel by Flupirtinmaleat or the most potent substance 11. From these curves both the potency as EC ₅₀ value and the intrinsic activity of the substance as Emax to mediate. The lower the EC ₅₀ value, the higher the potency (also called potency), while the larger the E _max value, the more effective the compound is.

Hepatocytenviabilitätsassy

The side effect of flupirtine, which was also responsible for its withdrawal from the market, is hepatotoxicity. For this reason, two hepatotoxicity cell culture models have been developed to test for the toxic effects of the new substances and to identify toxic candidates at an early stage. As a cell viability assay, the established MTT viability assay was performed. [41] The principle of the assay is based on the ability of viable cells to convert the yellow, water-soluble MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide) to the insoluble purple formazan product to reduce mitochondrial dehydrogenases (with consumption of NADH or NADPH), whose absorption can then be determined photometrically. The first cell-based assay was performed with the transforming growth factor-a transgenic mouse hepatocytes (TAMH) cell line, which has been used in previous studies with flupirtine [41] as well as other hepatotoxic agents and is useful as a predictor of hepatotoxicity. [42] Secondly, a human hepatocarcinoma cell line using human hepatocellular carcinoma (HEP-G2) as the second hepatotoxicity model [43] was used to compare the results from the TAMH cell line with a human cell line. Both cell lines grow as monolayers in culture, the TAHM cell lines in serum-free medium, the HEP-G2 line in culture medium with 10% fetal calf serum.

The two adherent cell lines were incubated in cell culture flasks (eg T75 TC standard, # 83.3911, Sarstedt) in the incubator at 37 ° C., 5% CO ₂ and 95% relative humidity, in the case of the TAMH cell line, DMEM / F12 (1: 1 mix) (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12, P04-41500, PAN Biotech) containing 5% Panexin NTA (P04-95750, PAN Biotech), 10mM Nicotinamide (N0636, Sigma Aldrich) and 0.1% (v / v) of 50 mg / ml gentamicin sulfate solution (in distilled water, P06-03001P, PAN Biotech), or concerning HEP-G2 cells, with RPMI 1640 (P04-16500, PAN Biotech ) containing 10% FBS (fetal calf serum, F7524, Sigma Aldrich, heat-inactivated) and 1% penicillin-streptomycin mix (P06-07100, PAN Biotech).

TAMH cells, which had a confluence of at least 80%, were washed with HBSS (Hanks balanced salt solution, P04-34500, PAN-Biotech) using trypsin-EDTA solution (T3924, Sigma Aldrich), which was used for about 10 sec on the cells, detached from the cell culture flask bottom (2 min at 37 ° C), then suspended with soybean trypsin inhibitor (T6522, Sigma Aldrich), then centrifuged at 400 g for 5 min and after supernatant with DMEM / F12 (1: 1 Mix ) resuspended. In turn, HEP-G2 cells, which had a confluency of approximately 70%, were washed with phosphate buffered saline (e.g., L1825, Biochrom), stripped from the cell culture flask bottom using trypsin-EDTA solution (at 37 ° C for approximately 5 minutes) ), then suspended with RPMI 1640, then centrifuged at 125 g for 5 min and after Supernatant removed resuspended with RPMI 1640. The cell count of both cell suspensions was determined with the EVE ™ Automated Cell Counter (NanoEnTEK). Of the murine cells, 20,000 cells / well / 200 μL were then seeded in 96 well TC plates, Cell +, F (# 83.3924.300, Sarstedt) and 2-3 days to complete confluency at 37 ° C, 5% CO ₂ and 95% humidity incubated. Of the human cells, 15,000 cells / well / 100 μL were seeded in 96 well TC plates, Standard, F (# 83.3924, Sarstedt) and incubated for 24 h at 37 ° C, 5% CO ₂ and 95% humidity. Of the compounds to be tested, serial serial dilutions (at least five concentrations) were made in DMSO (# A994.2, Carl Roth) with concentrations 100 times the desired later in-well concentrations. After incubation of both cell lines, the respective medium contained in the plates was discarded and the wells loaded according to a specific pattern with 200 μL / well each with the various solutions described below. The prepared dilution series of a substance was admixed to the appropriate medium in a ratio of 1: 100 (v / v) and added to the cells. Control wells contained the appropriate cells which were treated only with the respective medium and 1% DMSO. Wells containing no cells, but only the appropriate medium containing 1% DMSO were used to determine the background (blank). Each condition was determined at least as triplicate. The plates were incubated for 24 h at 37 ° C, 5% CO ₂ and 95% humidity. Subsequently, any liquid was removed from the plates and a mixture of the respective medium and a 2.5 mg / ml MTT solution (in Dulbecco's buffer, L11939.06, Alfa Aesar) in the ratio 1: 5 (v / v) to the Given cells. This was followed by an incubation of 1.5-4 h at 37 ° C, 5% CO ₂ and 95% humidity. Now the plates were completely freed from the added mixture and formed Formazankristalle using DMSO (# 7029.2, Carl Roth) brought into solution. The absorption of the individual wells was determined at λ = 570 nm using the SpectraMax 190 microplate reader incl. SoftMax Pro software (Molecular Devices). The viability of the cells treated with the substance to be tested was expressed in relation to the control. For this, the absorbance value of a single well, which included cells treated with a dilution step of the substance (T), was divided by the mean of the absorbance values of the control wells (<C>) and multiplied by 100. Both terms were previously corrected for the mean value of the measured absorbances of the wells for the blank determination (<Blank>). This results in the following formula: $$Viabilität\left[\%\right]=\frac{T-<blank>}{<G>-<Blank>}*100$$

To assess the hepatotoxic potential of a substance, the IC ₅₀ value was used. The IC ₅₀ was defined as the concentration of a compound at which the viability of the cells dropped to 50% ( ). To determine this, an analysis of the linear regression of the determined vivilities and the associated (logarithmic) concentrations of a substance was again carried out with the help of the MS Excel software (2013, Microsoft) and the IC _{50 was} determined by means of the resulting linear equation.

The results of the hepatotoxicity study are summarized in Table 3. In most cases, no IC ₅₀ value could be calculated because the upper solubility limit of the substances in cell culture medium was below the 50% toxic concentration; ie, the substances precipitated before an IC ₅₀ concentration could be achieved.

Results of biological testing

Table 3 summarizes almost all the biological test results of the synthesized substances and compares them to the reference substance flupritin maleate. From the interaction of the results of Kv7.2 / 3-opening activity and the hepatotoxicity results in vitro, it can be seen that total compounds of the general structural forms (I) significantly exceed the effect of the reference substance flupirtine maleate. In particular, compounds having a low EC ₅₀ value, preferably an EC ₅₀ value of less than 0.9 micromolar (micromoles per liter), more preferably less than 0.1, more preferably less than 0.01 micromolar, are especially suitable. For the most potent substance 11, such low substance concentrations (0.0014 μmol per liter) are needed to achieve the half-maximal effect on potassium channels that toxic effects in the resulting dosage are very unlikely. The determined toxicity data show that at concentrations of 63 μmol per liter for compound 11, which are limited by solubility in the test medium, no toxic effects were observed and the IC ₅₀ can therefore be given as greater than 63 μmol per liter. Accordingly, the ratio of IC ₅₀ to EC _{50 is} at least 85 times greater for substance 11 than for flupirtine maleate. Liver damage during therapy should therefore be less likely. Also, the biological effect of the test substance should be greater (for example, stronger analgesia), since Emax was over 100% and thus a stronger potassium effluent is to be expected than with flupirtine maleate. Table 3 Results of K _v 7.2I3 opening activity study and hepatotoxicity study No. Kv7.2 / 3 opening activity Hepatotoxicity in vitro TAMH HEP-G2 EC ₅₀ [μM] ± SD [μM] E _max [%] IC ₅₀ [μM] ± SD [μM] IC ₅₀ [μM] ± SD [μM] Comparative Example 1 0.918 ± 0.099 (n = 6) 100 487 ± 51 (n = 4) 547 ± 111 (n = 7) 1 1.279 ± 0.293 (n = 4) 93 ± 13 > 40 (n = 3) > 40 (n = 3) 2 0.237 ± 0.022 (n = 3) 115 ± 19 > 20 (n = 4) > 20 (n = 4) 3 0.771 ± 0.076 (n = 4) 96 ± 9 > 30 (n = 4) > 30 (n = 4) 4 > 10 (n = 2) - > 7.5 (n = 4) > 7.5 (n = 4) 5 0.240 ± 0.066 (n = 4) 78 ± 10 > 30 (n = 4) not determined 6 2.134 ± 0.457 (n = 3) 140 ± 11 > 63 (n = 4) > 63 (n = 5) 7 4,544 ± 1,457 (n = 3) 118 ± 28 > 63 (n = 4) > 63 (n = 5) 8th 1.320 ± 0.440 (n = 3) 120 ± 19 52 ± 16 (n = 4) > 250 (n = 5) 9 0.021 ± 0.006 (n = 3) 162 ± 9 > 125 (n = 3) > 125 (n = 4) 10 0.0245 ± 0.0069 (n = 3) 141 ± 11 > 125 (n = 3) > 125 (n = 4) 11 0.001378 ± 0.00006 (n = 3) 136 ± 13 > 63 (n = 3) > 63 (n = 4) 12 0.515 ± 0.035 (n = 3) 117 ± 22 > 250 (n = 3) > 250 (n = 4) 13 > 10 (n = 3) - > 1000 (n = 5) 831 ± 149 (n = 3) 14 > 10 (n = 4) - > 500 (n = 3) > 500 (n = 3) 15 1.746 ± 0.347 (n = 3) 110 ± 15 > 250 (n = 3) 221 ± 102 (n = 5) 16 > 10 (n = 2) - > 250 (n = 3) 134 ± 22 (n = 4) 17 0.253 ± 0.042 (n = 3) 69 ± 9 > 250 (n = 2) > 250 (n = 4) 18 0.269 ± 0.031 (n = 3) 129 ± 3 > 10 (n = 5) > 30 (n = 3) 19 0.260 ± 0.082 (n = 4) 100 ± 23 > 30 (n = 3) 8 ± 3 (n = 4) 20 0.015 ± 0.002 (n = 3) 147 ± 9 > 7.5 (n = 3) > 10 (n = 3) 21 0.477 ± 0.293 (n = 3) 72 ± 4 > 63 (n = 3) 58 ± 12 (n = 4) 22 0.066 ± 0.013 (n = 3) 114 ± 16 > 5 (n = 6) > 10 (n = 3) 23 1.348 ± 0.185 (n = 3) 106 ± 17 > 250 (n = 4) 203 ± 59 (n = 4) 24 0.122 ± 0.031 (n = 3) 112 ± 11 > 250 (n = 2) > 250 (n = 2) 25 0.021 ± 0.003 (n = 3) 107 ± 7 > 63 (n = 3) > 63 (n = 4)

list of figures

• , shows representative dose-response curves for the opening of the K _v 7.2 / 3 potassium channel by flupirtine maleate and substance 11;
• , shows representative dose-response curves for flupirtine maleate in TAMH and HEP-G2 cell lines.

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Claims (9)

Compound of the general formula (I)

wherein R _{1 is} selected from branched or unbranched C 1 to C 5 alkyl group and -NR _1a R _1b group, wherein R _1a and R _{1b are} independently selected from hydrogen and branched or unbranched C 1 to C 5 alkyl group or with each other and with the nitrogen atom of the NR _1a R _1b group form a C3 to C9 heterocycloalkyl group, the C3 to C9 heterocycloalkyl group each having at least one substituent selected from hydrogen atom and C1 to C3 alkyl group and optionally in addition to the nitrogen atom of the NR _1a R _1b group has at least one further heteroatom in the cycle; R _{2 is} selected from the group consisting of branched or unbranched C 1 to C 10 alkyl, C 3 to C 10 cycloalkyl, C 3 to C 10 heterocycloalkyl, wherein at least one heteroatom selected from nitrogen and oxygen is included, C 6 to C 12 aryl group; and C5 to C11 heteroaryl group, wherein the C1 to C10 alkyl group, C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group, or C5 to C11 heteroaryl group each have at least one further substituent R _2a , wherein R _{2a is} selected from the group consisting of hydrogen atom, halogen atom, branched or unbranched C1 to C3 alkyl group, branched or unbranched (per) halogenated C1 to C3 alkyl group and branched or unbranched C1 to C3 Alkoxy group, and wherein the C3 to C10 cycloalkyl group, C3 to C10 heterocycloalkyl group, C6 to C12 aryl group or C5 to C11 heteroaryl group includes one or more ring systems which are condensed or separated; R _{3 is} selected from the group consisting of branched or unbranched C 1 to C 10 alkoxy group; branched or unbranched C1-C10 alkyl group, and - _p (CH ₂₎ -C6- C12 aryl group having at least one substituent selected from hydrogen atom, halogen atom, and comprises C1 to C3 alkoxy group and wherein the C6 to C12 aryl group, a or more ring systems which are condensed or separated; R _{4 is} selected from hydrogen, halogen and branched or unbranched C 1 -C 5 alkyl group; X is a nitrogen atom or a -CH group; n is zero or an integer in the range of 1 to 3; m is zero or an integer in the range of 1 to 5; and p is zero or an integer in the range of 1 to 5. Compound of general formula (I) according to Claim 1 where n is zero and / or m is 1 and / or X is a nitrogen atom. Compound of general formula (I) according to Claim 1 or 2 in which R _{1 is} selected from the group consisting of branched or unbranched C 1 - to C 3 -alkyl group and -NR _1a R _1b group, where R _1a and R _1b are selected independently of one another from hydrogen atom and branched or unbranched C 1 - to C 3 -cycloalkyl Alkyl group or with each other and with the nitrogen atom of the NR _1a R _1b group form a C4 to C6 Heterocycloalklygruppe, wherein the C4 to C6 Heterocycloalklygruppe each having at least one substituent selected from hydrogen atom and C1 to C3 alkyl group and optionally in addition to Nitrogen atom of the NR _1a R _1b group has at least one further oxygen atom in the cycle; and / or R _{2 is} selected from the group consisting of mono- or difluorophenyl group; CF ₃ phenyl group; F ₅ S-phenyl group; C4 to C8 cycloalkyl group; branched or unbranched C1 to C5 alkyl group; Biphenyl group; Pyridine group; Piperidine group; Tetrahydropyran group; Dioxolane group and phenyl group, wherein the phenyl group has at least one substituent R _2a selected from hydrogen atom and methoxy group; and / or R _{3 is} selected from the group consisting of ethoxy group, propyl group, tert-butyl group, - (CH ₂ ) _p -phenyl group which has at least one substituent selected from hydrogen atom, halogen atom and methoxy group and where p is zero or 1 or 2; and / or R _{4 is} a hydrogen atom, a bromine atom or a methyl group. Compounds of the general formula (I) according to one of the Claims 1 to 3 in which the compound has the formula (Ia)

or (Ib)

or (Ic)

Process for the preparation of a compound of general formula (I),

wherein R ₁ , R ₂ , R ₃ , R ₄ , X, n, m and p have the same meaning as in one of Claims 1 to 7 comprising, (i) providing a nitro compound of the general formula (II)

wherein R ₁ , R ₂ , R ₄ , m and n have the same meaning as in one of Claims 1 to 7 Are defined; (ii) reacting the nitro compound of general formula (II) according to (i) with a compound HR ₃ , wherein R _{3 has} the same meaning as in one of Claims 1 to 4 has been defined to obtain a compound of general formula (I). A compound of the general formula (I) obtained or obtainable by a process according to Claim 5 , Compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 or pharmaceutically acceptable salt of a compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 for use as a medicine. Compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 or pharmaceutically acceptable salt of a compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 for use as a painkiller and / or for use in tinnitus treatment or prevention and / or for use in the treatment or prevention of convulsive conditions and / or for use in the treatment or prevention of a disease selected from Group consisting of incontinence, anxiety, dependence, mania, bipolar disorder, cognitive disorder and dyskinesia. Pharmaceutical preparation comprising a compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 or a pharmaceutically acceptable salt of a compound of the general formula (I) according to one of the Claims 1 to 4 or according to Claim 6 ,

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