CN112759559B - Sulfonamide compounds as sodium channel blockers and uses thereof - Google Patents

Abstract

The invention provides a sulfonamide compound serving as a sodium channel blocker and application thereof, wherein the sulfonamide compound simultaneously has double inhibitory activities on Nav1.7 and Nav1.3 and can be used as a medicine for treating extensive pain.

Description

Sulfonamide compounds as sodium channel blockers and uses thereof

Technical Field

The invention relates to sulfonamide compounds with double inhibitory activity on sodium ion channels, in particular to Nav1.3 and Nav1.7 sodium ion channels and application thereof.

Background

Voltage-gated sodium channels (VGSCs) are microporous transmembrane glycoproteins widely distributed on excitable cell membranes such as neurons and are mainly responsible for Na ⁺ Is the most important ion channel required by neurons to produce excitability and exert normal electrophysiological functions. Voltage-gated sodium channels are composed of an alpha subunit and a plurality of beta subunits, wherein the alpha subunit is the main functional unit and is now available9 subtypes (Nav1.1. about. Nav1.9) were found. It is surrounded by 4 highly similar homeodomains, each with 6 alpha helical transmembrane segments (S1-S6), forming a central pore of the ion channel, with the amino acid sequence of S4 being highly conserved and considered as baroreceptors for voltage-gated sodium channels. The beta subunit has 4 subtypes (beta 1-beta 4), plays an auxiliary role in the positioning and stability of the alpha subunit on the membrane, and is involved in regulating the voltage sensitivity and inactivation process of the alpha subunit. There is extensive homology, but also great differences, of voltage-gated sodium channels in different animals. Sodium ion channels are classified into TTX-sensitive (TTX-S) and TTX-insensitive (TTX-R) according to their sensitivity to blockade by tetrodotoxin (TTX). TTX-R type sodium ion channels include Nav1.5, Nav1.8 and Nav1.9, the rest is TTX-S type sodium ion channels.

Physiological and pharmacological studies (Catterall, WA. et al. Pharmacol. Rev.2005,57, 397-) 409) have demonstrated the expression profile and pharmacological profile of 9 sodium channels in mammals as shown in Table 1:

TABLE 1

Among them, Nav1.1, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 in normal adult animal DRG neurons expression, and Nav1.3 in immature neurons higher expression. Nociceptors on the dorsal root ganglion convert various nociceptive thermal, mechanical and chemical signals into electrical impulses which are transmitted to the central nervous system, and Nav determines the generation and propagation of action potentials, and functional changes thereof have important effects on excitability and pain conductance of neurons. The subtypes currently known to be closely associated with pain include: nav1.3, Nav1.7, Nav1.8, Nav1.9.

Nav1.7 is a transmembrane protein encoded by SCN9A, specifically expressed in peripheral sensory nerve endings and sympathetic ganglion neurons, and mainly expressed on large diameter DRG neurons and unmyelinated small diameter DRG, i.e., on 85% of nociceptors, suggesting that Nav1.7 has a very critical role in pain conductance. Animal model studies have shown that Nav1.7 protein or mRNA expression is increased in carrageenan and Complete Freund's Adjuvant (CFA) induced inflammatory pain models, and these changes occur primarily in DRG neurons or afferent neurons. An increase in TTX-S current in DRG neurons was also simultaneously detected in the carrageenan inflammatory pain model. In rat diabetic neuralgia model, Nav1.7 protein expression is increased. Knockdown of Nav1.7 in DRG can relieve thermal pain sensitivity, mechanical pain sensitivity and cold pain hypersensitivity of animals. Recent studies have also found that Nav1.7 in the DRG and sympathetic ganglia, respectively, are involved in different types of pain information transmission. After conditional knock-out of nav1.7 in mouse DRG, the basal mechanical and thermal pain valves were significantly elevated, while the inflammatory pain caused by 4% formaldehyde was also alleviated. In human disease research, when the mutation of the SCN9A gene causes Nav1.7 to be difficult to inactivate after opening, the function of the Nav1.7 is enhanced, and the mutation belongs to function-enhanced mutation, so that Primary Erythromelalgia (PE) is caused. When the SCN9A gene has null mutation, Nav1.7 completely loses function, i.e., loss of function type mutation, resulting in congenital painless syndrome (CIP), the patient completely loses the ability to sense pain. Intervention in Nav1.7 expression or administration of Nav1.7 inhibitors can produce analgesia. Meanwhile, research also shows that the medicine taking Nav1.7 as the target point has small toxic and side effects. The results show that Nav1.7 has a core effect on pain signal conduction and maintenance, and has become a very important target point for analgesic drug development. Currently, several pharmaceutical companies and scientific institutions are conducting selective Nav1.7 blocker studies (Sun, S.Y.et al.Bioorg.Med.chem.Lett.2014,24, 4397-4401; Dimauro, E.F.et al.J.Med.chem.2016,59, 7818-7839; Wu, Y.J.et al.J.Med.chem.2017,60, 2513-2525; Jennifer, M.F.J.Med.chem.2016,59, 3373-3391; Marx, I.E.et al.ACS.Med.Lett.201712017120184658, WO2017082688, WO2017058821, WO 20171067172802, WO 201201602826).

Nav1.3 is a rapid restart voltage-gated sodium channel, normal state only in the development of sensory neurons and adult central nervous system expression, in the adult peripheral nervous system low level expression. However, peripheral nerve injury, such as spinal nerve cutting, ligation or chronic compressive injury, can lead to a large upregulation of Nav1.3 expression in dorsal root ganglion neurons. This phenomenon indicates that Nav1.3 is involved in the development and maintenance of peripheral neuropathic pain. A series of aryl ether selective Nav1.3 inhibitors have been reported by Pryde D.C. et al to be useful in the treatment of pain (Pryde D.C. et al. MedChemcomm,2017,8, 1255-1267). WO2006122014A2 discloses a class of bicyclic Nav1.3 blockers useful for treating chronic pain, acute pain, inflammatory pain, and anxiety and depression. Samad et al demonstrated the potential of nav1.3 knockout for pain treatment in a rat model and suggested gene therapy as a potential treatment option (Samad, o.a. et al. mol. ther.2013,21, 49-56). An increasing number of researchers believe that Nav1.3 is also a very potential target for the development of analgesic drugs.

Nav1.8 and Nav1.9 belong to TTX-R class of sodium ion channels, in adult DRG expression, is thought to be involved in pain, but its exact mechanism is not completely clear. Nav1.1 and Nav1.2 are highly expressed in the brain and are critical for normal brain function (Ray, C.K.el.J.biol.chem.2004, 279, 46234-46241). Studies have shown that some loss of function caused by the Nav1.1 mutation can cause epilepsy (Yu, F.H.et al. Nat. neuroscience,2006,9, 1142-1149). Therefore, although Nav1.1 is also expressed in the peripheral nervous system and inhibition of Nav1.1 is beneficial for pain relief, it may also cause side effects such as anxiety or hyperexcitability. Nav1.4 is mainly expressed in skeletal muscle, and Nav1.4 inhibition may cause myotonia, even paralysis adverse effect. Nav1.5 is mainly expressed in cardiomyocytes, including atria, ventricles, sinoatrial node, atrioventricular node, etc. Inhibition of Nav1.5 may cause arrhythmia syndromes, including QT prolongation, Brugada syndrome, sudden nocturnal sudden death syndrome, sudden infant death syndrome, and the like (Liu, H.et al. am.J. pharmacogenomics,2003,3, 173-. While Nav1.6 may be associated with dyskinesias.

In summary, Nav1.7 is a very potential target for the treatment of pain or pain-related diseases, and is the most sodium channel currently studied, but some highly selective Nav1.7 blockers are not clinically ideal for analgesic effect, probably due to the compensatory mediation of pain signal in the peripheral nervous system by the expression of Nav1.3. WO2012125973A reports on the use of polypeptides with potent and selective activity against Nav1.3 and Nav1.7 for the prevention and treatment of pain. However, the wide application of protein and polypeptide drugs is often limited due to the limitations of stability, single administration mode and the like. Therefore, research on novel, more potent and less side effects of Nav1.3 and Nav1.7 small molecule dual inhibitors is an important research direction.

Disclosure of Invention

The invention aims to provide sulfonamide compounds with double inhibitory activity on sodium ion channels, particularly Nav1.3 and Nav1.7 sodium ion channels, and application thereof.

To achieve the above objects, the present invention provides a pharmaceutical composition consisting of formula I, a solvate, tautomer or pharmaceutically acceptable salt thereof:

wherein R is ₁ Selected from 5-10 membered aromatic heterocycles containing up to 3 heteroatoms of N, O, S, optionally 5-10 membered aromatic heterocycle substituted with halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano;

R ₂ 、R ₃ Independently selected from hydrogen, halogen, alkyl or cycloalkyl, and R ₂ 、R ₃ At least 1 is halogen;

x is-CONH-, - (CH) ₂ ) _m O-or- (CH) ₂ ) _m NH-, wherein m is an integer of 1 to 2;

R ₄ selected from hydrogen, halogen, C _1-4 Alkyl radical, C _1-4 Alkoxy radical, C _1-4 Haloalkyl, C _1-4 Haloalkoxy, cyano;

a is an N-linked 3-to 10-membered monocyclic saturated non-aromatic ring, which in addition to the linking N may contain 1 to 2 heteroatoms selected from nitrogen, oxygen and sulfur, optionally the 3-to 10-membered monocyclic saturated non-aromatic ring is substituted with halogen, alkyl, alkoxy, haloalkyl, haloalkoxy, amino.

In another example, R ₁ Selected from 5-6 membered monocyclic aromatic heterocycles containing up to 3 heteroatoms of N, O, S, optionally, the 5-6 membered monocyclic aromatic heterocycle is substituted by halogen, C _1-6 Alkyl, -OC _1-6 Alkyl radical, C _1-6 Haloalkyl, -OC _1-6 Haloalkyl, cyano. In another example, R ₁ Is selected from a 5-6 membered monocyclic aromatic heterocycle containing up to 3 heteroatoms of N, O, S, optionally the 5-6 membered monocyclic aromatic heterocycle is substituted with a halogen.

In another example, R ₁ Selected from 5-membered aromatic heterocycles comprising up to 3 heteroatoms of N, O, S, optionally the 5-membered aromatic heterocycle being substituted with halogen.

In another example, R ₁ Is selected from

In another example, R ₂ 、R ₃ Independently selected from hydrogen or halogen, and R ₂ 、R ₃ At least 1 item is halogen.

In another example, X is selected from-CH ₂ O-or-CH ₂ NH-。

In another example, R ₄ Selected from hydrogen, halogen, C _1-4 Alkyl radical, C _1-4 Alkoxy radical, C _1-4 Haloalkyl, C _1-4 A haloalkoxy group.

In another example, R ₄ Selected from hydrogen or halogen.

In another example, A is

Wherein n is an integer from 1 to 5, optionally, A is substituted with amino.

In another example, A is

In another example, the compound of formula I is specifically selected from the following:

a second object of the present invention is to provide a pharmaceutical composition comprising a compound according to the first object of the present invention, a solvate, tautomer or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

A third object of the present invention is to provide the use of a compound of the first object of the present invention, a solvate, a tautomer or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of pain.

In another embodiment, the pain is selected from: neuropathic pain, inflammatory pain, visceral pain, cancer pain, chemotherapy pain, trauma pain, surgical pain, labor pain, neurogenic bladder pain, ulcerative colitis pain, chronic pain, persistent pain, peripherally mediated pain, centrally mediated pain, migraine, sinus headache, tension headache, phantom limb pain, dental pain, peripheral nerve injury pain, or a combination thereof; or the pain is associated with a disease or condition selected from the group consisting of: HIV, HIV therapy-induced neuropathy, trigeminal neuralgia, post-herpetic neuralgia, acute pain, thermal sensitivity, sarcoidosis, irritable bowel syndrome, Crohn's disease, Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), diabetic neuropathy, peripheral neuropathy, arthritis, rheumatoid arthritis, atherosclerosis, paroxysmal dystonia, myasthenia syndrome, myotonia, malignant hyperthermia, cystic fibrosis, pseudoaldosteronism, rhabdomyolysis, hypothyroidism, bipolar depression, anxiety, schizophrenia, sodium channel toxin-related diseases, familial erythromelalgia, primary erythromelalgia, familial rectal pain, cancer, epilepsy, partial and systemic megaly attacks of pain, restless leg syndrome, arrhythmia, fibromyalgia, neuroprotection in ischemic conditions resulting from stroke or nerve trauma, neuro trauma, post-traumatic injury, post-herpetic neuralgia, post-herpetic syndrome, post-herpetic neuralgia, acute pain, heat sensitivity, sarcoidosis, irritable bowel syndrome, Crohn's disease, myotonia, myasthenia syndrome, myotonia, a, malignant hyperthermia, cystic hyperkeratosis, cystic fibrosis, a, myasthenia syndrome, cystic fibrosis, a myasthenia gravis, a myasthenia syndrome, a myasthenia, Atrial fibrillation and ventricular fibrillation.

In another embodiment, the pain is neuropathic pain or inflammatory pain.

Definition of

As used herein, the term "alkyl" by itself or as part of another substituent means (unless otherwise specified) a straight or branched chain hydrocarbon group (i.e., C) having the specified number of carbon atoms _1-8 Meaning one to eight carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

As used herein, the term "heteroaromatic ring" refers to a monocyclic heteroaromatic ring having 5 to 10 ring atoms, preferably 5 or 6 membered or a bicyclic heteroaromatic ring having 8 to 10 membered ring atoms; and a group having 1 to 3 hetero atoms in addition to carbon atoms. "heteroatom" means nitrogen, oxygen or sulfur.

As used herein, "5 to 6 membered monocyclic aromatic heterocycle" refers to a mono heteroaryl ring containing 5 to 6 ring atoms, including for example (but not limited to): thiophene ring, N-alkylpyrrole ring, furan ring, thiazole ring, imidazole ring, oxazole ring, pyrrole ring, pyrazole ring, triazole ring, isoxazole ring, oxadiazole ring, thiadiazole ring, pyridine ring, pyridazine ring, pyrimidine ring, pyrazine ring, etc.

As used herein, "8 to 10 membered bicyclic aromatic heterocycle" refers to a bis heteroaryl ring containing 8 to 10 ring atoms, including for example (but not limited to): a benzofuran ring, a benzothiophene ring, an indole ring, an isoindole ring, a quinoline ring, an isoquinoline ring, an indazole ring, a benzothiazole ring, a benzimidazole ring, a quinazoline ring, a quinoxaline ring, a cinnoline ring, a phthalazine ring.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is replaced with a substituent, and may include variations of deuterium and hydrogen, so long as the valence of the particular atom is normal and the substituted compound is stable. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.

As used herein, the term "halogen" by itself or as part of another substituent means (unless otherwise specified) fluorineChlorine, bromine or iodine atoms. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C _1-4 Haloalkyl "is intended to include trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, difluoromethyl, and the like.

As used herein, "alkoxy" represents the above alkyl group having the specified number of carbon atoms attached through an oxygen bridge, unless otherwise specified, C _1-6 Alkoxy radicals comprising C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ And C ₆ Alkoxy group of (2). Examples of alkoxy groups include, but are not limited to: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy.

As used herein, "N-linked 3-10 membered monocyclic saturated non-aromatic ring" represents a 3-10 membered monocyclic saturated alkyl group having a nitrogen heteroatom thereon such that the monocyclic saturated alkyl group participates in the linkage through the nitrogen heteroatom.

As used herein, a wavy line intersecting a bond in a chemical structure indicates the point of attachment of the bond, which wavy bond in the chemical structure intersects the rest of the molecule.

Unless otherwise specified, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational) forms of the structure; for example, the R and S configurations of each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Thus, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of these compounds are within the scope of the invention. Unless otherwise specified, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise specified, the structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, except that hydrogen is replaced by deuterium or tritium or carbon is replaced by ¹³ C-or ¹⁴ C-enriched carbon instead of compounds having the structure of the present invention are within the scope of the present invention.

As herein describedAs used herein, "solvate" refers to a physical association of a compound of the present application with one or more solvent molecules; this physical association involves various degrees of ionic and covalent bonding, including hydrogen bonding; in certain cases, such as when one or more solvent molecules are introduced into the crystal lattice of a crystalline solid, the solvate will be able to be isolated; "solvate" encompasses both solution phase and isolatable solvates; non-limiting examples of suitable solvents include, but are not limited to, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine, and the like; "hydrate" is where the solvent molecule is H ₂ A solvate of O.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. The phrase "pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

As used herein, the term "pharmaceutically acceptable salt" is intended to include salts of the active compounds prepared with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese salts, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines (including substituted amines, cyclic amines, naturally occurring amines, and the like), such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, histidine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as from relatively nontoxic organic acids such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginine and the like, and organic acids such as glucuronic or galacturonic acids.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of an active agent of the present invention, without interfering with the biological activity of the active agent, and without toxic side effects to the host or patient, and representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, and the like. These include suspending agents, viscosity enhancers, skin penetration enhancers, and the like. Their preparation is known to those skilled in the cosmetic or topical pharmaceutical field.

Compositions comprising compounds of formula I are generally formulated in accordance with standard pharmaceutical practice as pharmaceutical compositions. Typical formulations are prepared by mixing a compound of the invention with a diluent, carrier or excipient. The formulations may also include one or more of buffering agents, stabilizing agents, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, fragrances, flavoring agents, diluents, and other known additives.

The compounds of the present invention may be administered in any convenient form of use, such as tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches and the like. Such compositions may contain ingredients conventional in pharmaceutical formulations, such as diluents, carriers, pH adjusting agents, sweeteners, fillers and additional active agents.

The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, intracerebral, intraocular, intralesional or subcutaneous administration.

The compounds were named manually or by Chemdraw software and the commercially available compounds were given the supplier catalog name.

Compared with the prior art, the invention has the main advantages that:

provides a series of sulfonamide derivatives with novel structures, which simultaneously have double inhibitory activities on Nav1.7 and Nav1.3 and can be used as medicaments for treating extensive pains.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. The chemical reactions described in the examples (preparations) can be readily modified to prepare a number of other compounds of the invention, and alternative methods for preparing the compounds of the invention are considered to be within the scope of the invention. Unless otherwise defined, terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention.

Example 1

Preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Step a): preparation of 4-chloro-2- (pyrrolidin-1-yl) benzonitrile

Mixing pyrrolidine(2.51g,35.356mmol), 4-chloro-2-fluoro-benzonitrile (5.00g,32.142mmol), potassium carbonate (8.88g,64.284mmol) and DMSO (75mL) are added into a reaction flask, the temperature is raised to 60 ℃, stirring is carried out for reaction for 12 hours, cooling is carried out to room temperature, water (150mL) is added for dilution, precipitated solid is filtered, filter cake is washed by water (75mL), then petroleum ether (75mL) is used for leaching, 4-chloro-2- (pyrrolidine-1-yl) benzonitrile is obtained by decompression and vacuum drying, the yield is 97.10%, ¹ H NMR(400MHz,DMSO-d ₆ )δ:7.50(d,J＝8.4Hz,1H),6.77(d,J＝2.0Hz,1H),6.71(dd,J ₁ ＝8.4Hz,J ₂ ＝2.0Hz,1H),3.53-3.50(m,4H),1.95-1.92(m,4H),ESI-MS(m/z)：207.0[M+H] ⁺ 。

step b): preparation of 4-chloro-2-pyrrolidin-1-yl-benzylamine

B is to be ₂ H ₆ Adding tetrahydrofuran solution (110mL,1M) into tetrahydrofuran solution (100mL) of 4-chloro-2- (pyrrolidine-1-yl) benzonitrile (2.40g,11.613mmol), heating to reflux for 6 hours, slowly and carefully quenching the reaction with methanol (50mL), continuing reflux for 0.5 hour, cooling to room temperature, concentrating under reduced pressure, purifying the obtained crude product by a silica gel chromatographic column (eluent: dichloromethane/methanol ═ 10:1v/v) to obtain 4-chloro-2-pyrrolidine-1-yl-benzylamine with yield of 33.6%, ¹ H NMR(400MHz,CDCl ₃ )δ:7.18(d,J＝8.0Hz,1H),6.89(d,J＝2.0Hz,1H),6.85(dd,J ₁ ＝8.0Hz,J ₂ ＝2.0Hz,1H),3.88(s,2H),3.23-3.12(m,4H),2.01-1.89(m,4H),ESI-MS(m/z):194.1[M-16] ⁺ 。

step c): preparation of N- (2, 4-dimethoxybenzyl) -1,2, 4-thiadiazole-5-amine

1,2, 4-thiadiazole-5-amine (5.0g, 49.441mmol), 2, 4-dimethoxy-benzaldehyde (8.6g, 51.913mmol) and dichloromethane (250mL) are added into a reaction flask, stirred and dissolved, tetraisopropyl titanate (15.5g, 54.385mmol) is added into a reaction solution at 0 ℃, after the addition is finished, the reaction solution is heated to room temperature for 6 hours, sodium triacetoxyborohydride (18.9g, 88.994mmol) is added in portions, and the reaction solution is stirred at room temperature for 15 hours. After the reaction, saturated ammonium chloride aqueous solution (12mL) was slowly added to the reaction solution, the mixture was stirred for 10min, 100-mesh 200-mesh silica gel (25g) was added and mixed, the mixture was filtered through a sand core funnel, and the filter cake was mixed with an appropriate amount of dichloromethane/methanol (100: 1) mixtureWashing, combining filtrates, concentrating under reduced pressure, purifying the obtained crude product with silica gel chromatography column (eluent: petroleum ether/ethyl acetate ═ 1:1v/v) to obtain N- (2, 4-dimethoxybenzyl) -1,2, 4-thiadiazole-5-amine, yield is 68.0%, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.69(s,1H),7.90(s,1H),7.17(d,J＝8.4Hz,1H),6.58(d,J＝2.4Hz,1H),6.49(dd,J ₁ ＝8.4Hz,J ₂ ＝2.4Hz,1H),4.37(d,J＝5.6Hz,2H),3.80(s,3H),3.75(s,3H),ESI-MS(m/z):252.1[M+H] ⁺ 。

step d): preparation of 5-chloro-N- (2, 4-dimethoxy-benzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazol-5-yl) -benzenesulfonamide

Slowly adding a tetrahydrofuran solution (30mL) of N- (2, 4-dimethoxybenzyl) -1,2, 4-thiadiazole-5-amine (900mg,3.581mmol) into a tetrahydrofuran suspension of LiHMDS (1M,3.96mL) at-60 ℃, stirring for reaction for 1 hour, slowly dropwise adding a tetrahydrofuran (4mL) solution of 5-chloro-2, 4-difluoro-benzenesulfonyl chloride (885mg,3.581mmol) into the reaction solution, stirring for reaction for 3 hours, after the reaction is finished, slowly pouring the reaction solution into a saturated ammonium chloride solution at 0 ℃, extracting with ethyl acetate (30 mL. times.3), combining organic layers, washing with a saturated sodium chloride solution (50 mL. times.2), drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, purifying the obtained crude product by silica gel column chromatography (eluent: petroleum ether/ethyl acetate:. times.2: 1v/v), to obtain 5-chloro-N- (2, 4-dimethoxy-benzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazole-5-yl) -benzenesulfonamide with the yield of 80.5 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.50(s,1H),7.79(m,2H),7.12(d,J＝8.4Hz,1H),6.42(dd,J ₁ ＝8.4Hz,J ₂ ＝2.4Hz,1H),6.26(d,J＝2.4Hz,1H),5.28(s,2H),3.71(s,3H),3.61(s,3H)，ESI-MS(m/z)：462.1[M+H] ⁺ 。

Step e): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxy-benzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

4-chloro-2-pyrrolidin-1-yl-benzylamine (200mg,0.949mmol), 5-chloro-N- (2, 4-dimethoxy-benzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazol-5-yl) -benzenesulfonamide (438mg,0.949mmol), potassium carbonate (262mg,1.898mmol) and dry DMSO (12mL) were reacted under nitrogen at 25 ℃ for 15 hours to complete the reactionAfter that, saturated aqueous ammonium chloride (60mL) was added to dilute, dichloromethane (40mL × 2) was used for extraction, the organic layers were combined, washed with water (20mL × 3), saturated sodium chloride solution (50mL) in that order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product which was dispersed in 10mL of petroleum ether: filtering the mixed solution of ethyl acetate and 5:1 to obtain 5-chloro-4- ((4-chloro-2- (pyrrolidine-1-yl) benzyl) amino) -N- (2, 4-dimethoxy-benzyl) -2-fluoro-N- (1,2, 4-thiadiazole-5-yl) benzenesulfonamide, wherein the yield is 61.9%, and ESI-MS (m/z): 652.1[ M + H] ⁺ 。

Step f): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Adding 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxy-benzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (374mg,0.574mmol) into a reaction bottle, adding about 2N ethyl acetate hydrochloride solution (10mL) for dissolving, stirring at room temperature for reaction for 20 hours, performing suction filtration after the reaction is finished, washing a filter cake with ethyl acetate (5mL), performing vacuum drying under reduced pressure, dissolving the obtained product in 2mL of ethanol and 10mL of pure water, and performing freeze drying to obtain 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -2-fluoro-N- (1), 2, 4-thiadiazole-5-yl) benzenesulfonamide, the yield is 85.4%, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.48(s,1H),7.59(d,J＝7.2,1H),7.25-7.00(m,4H),6.44(d,J＝12.8Hz,1H),4.48(s,2H),3.28(s,4H),1.95(s,4H),ESI-MS(m/z)：502.1[M+H] ⁺ 。

Example 2

Preparation of 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in example 1 except that 2,4, 5-trifluorophenyl-1-sulfonyl chloride in step d is replaced with 2,4, 5-trifluorophenyl-1-sulfonyl chloride with 2, 4-difluoroben-sulfonyl chloride to give 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide in a yield of 65.3%, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.51(s,1H),7.53(d,J＝7.2,1H),7.25-7.00(m,4H),6.85-6.84(m,1H)6.45(d,J＝12.8Hz,1H),4.38(s,2H),3.40(s,4H),1.93(s,4H)，ESI-MS(m/z):468.1[M+H] ⁺ 。

example 3

Preparation of 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (5-chlorothiazol-2-yl) -2, 5-difluoro-benzenesulfonamide

The procedure is as in example 1 except that 1,2, 4-thiadiazol-5-amine in step c is replaced with 5-chloro-thiazol-2-amine and 5-chloro-2, 4-difluoro-benzenesulfonyl chloride in step d is replaced with 2,4, 5-trifluorophenyl-1-sulfonyl chloride, respectively, to give 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (5-chlorothiazol-2-yl) -2, 5-difluoro-benzenesulfonamide, ¹ H NMR(400MHz,DMSO-d ₆ )δ8.94(s,1H),7.51(s,1H),7.39(dd,J＝11.0,6.5Hz,1H),7.21(s,1H),7.08(d,J＝8.0Hz,1H),6.69(d,J＝1.8Hz,1H),6.63(dd,J＝8.0,1.8Hz,1H),6.32(dd,J ₁ ＝12.4,J ₂ ＝6.8Hz 1H),4.48(s,2H),3.28(s,4H),1.91(s,4H),ESI-MS(m/z):519.1[M+H] ⁺ 。

example 4

Preparation of 5-chloro-N- (5-chlorothiazol-2-yl) -2-fluoro-4- ((2- (pyrrolidin-1-yl) -4- (trifluoromethyl) benzyl) amino) benzenesulfonamide

The procedure is as in example 1, except that 4-chloro-2-fluoro-benzonitrile in step a is replaced with 2-fluoro-4- (trifluoromethyl) benzonitrile and 1,2, 4-thiadiazol-5-amine in step c is replaced with 5-chloro-thiazol-2-amine, respectively, to give 5-chloro-N- (5-chlorothiazol-2-yl) -2-fluoro-4- ((2- (pyrrolidin-1-yl) -4- (trifluoromethyl) benzyl) amino) benzenesulfonamide, ESI-MS (M/z):569.0[ M + H ] benzenesulfonamide ] ⁺ 。

Example 5

Preparation of 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-2-fluoro-N- (thiazol-2-yl) benzenesulfonamide

Step a): preparation of N- (2, 4-dimethoxybenzyl) thiazol-2-amine

The procedure is as in step c of example 1, except that 1,2, 4-thiadiazol-5-amine is replaced by thiazol-2-amine to give N- (2, 4-dimethoxybenzyl) thiazol-2-amine.

Step b): preparation of 5-bromo-N- (2, 4-dimethoxybenzyl) -2, 4-difluoro-N- (thiazol-2-yl) benzenesulfonamide

The procedure is as in example 1, except that N- (2, 4-dimethoxybenzyl) -1,2, 4-thiadiazole-5-amine is replaced with N- (2, 4-dimethoxybenzyl) thiazol-2-amine and 5-chloro-2, 4-difluoro-benzenesulfonyl chloride is replaced with 5-bromo-2, 4-difluoro-benzenesulfonyl chloride, respectively, to give 5-bromo-N- (2, 4-dimethoxybenzyl) -2, 4-difluoro-N- (thiazol-2-yl) benzenesulfonamide.

Step c): preparation of 5-bromo-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide

The procedure is as in example 1, except that 5-chloro-N- (2, 4-dimethoxy-benzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazol-5-yl) -benzenesulfonamide is replaced with 5-bromo-N- (2, 4-dimethoxybenzyl) -2, 4-difluoro-N- (thiazol-2-yl) benzenesulfonamide to give 5-bromo-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide.

Step d): preparation of 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide

5-bromo-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide (400mg, 0.574mmol) and dioxane (8mL) were added to a reaction flask, followed by cyclopropylboronic acid (148mg, 1.722mmol), palladium acetate (26mg, 0.115mmol), potassium phosphate (487mg, 2.296mmol) and tricyclohexylphosphineTetrafluoroborate (85mg, 0.230mmol), degassing the reaction system by introducing argon flow for 15min, heating to 100 ℃ for reaction for 5H, cooling to room temperature after the reaction is finished, suction-filtering the reaction solution with diatomite, washing the filter cake with a proper amount of ethyl acetate, combining the filtrates, concentrating under reduced pressure, purifying the obtained crude product by a preparation thin layer (developing solvent: dichloromethane/methanol ═ 20:1v/v) to obtain 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide, wherein the yield is 35.5%, and ESI-MS (M/z):657.2[ M + H ] benzenesulfonamide] ⁺ 。

Step e): preparation of 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-2-fluoro-N- (thiazol-2-yl) benzenesulfonamide

The procedure is as in example 1, except that 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxy-benzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide is replaced with 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (thiazol-2-yl) benzenesulfonamide to give 4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -5-cyclopropyl-2-fluoro- N- (thiazol-2-yl) benzenesulfonamide, yield 86.2%, ¹ H NMR(400MHz,DMSO-d ₆ )δ:7.83(s,1H),7.24(d,J＝3.6Hz,1H),7.04(d,J＝8.0Hz,1H),6.98(s,1H)，6.89(d,J＝3.6Hz,1H),6.81(s,1H),6.66(s,1H),6.61(d,J＝8.0Hz,1H),4.48(s,2H),3.22(s,4H),1.90(s,4H),1.85-1.79(m,1H),0.89-0.78(m,2H),0.58-0.54(m,2H)，ESI-MS(m/z):507.1[M+H] ⁺ 。

example 6

Preparation of 5-chloro-4- ((4-chloro-2- (piperidin-1-yl) benzyl) amino) -N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide

The procedure is as in example 1 except that in step a, the pyrrolidine is replaced with piperidine and in step c, the 1,2, 4-thiadiazol-5-amine is replaced with 5-chloro-thiazol-2-amine, respectively, to give 5-chloro-4- ((4-chloro-2- (piperidin-1-yl) benzyl) amino) -N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide in 68.9% yield, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.56(s,1H),7.84(s,1H),7.28-7.25(m,1H),7.07(d,J＝8.0Hz,1H),6.76(s,1H),6.72-6.60(m,2H),6.32-6.25(m,1H),4.17(s,2H),3.25(s,4H),1.89-1.85(m,6H),ESI-MS(m/z):549.1[M+H] ⁺ 。

example 7

Preparation of 4- ((2- (homopiperidin-1-yl) -4-chlorobenzyl) amino) -5-chloro-N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide

The procedure is as in example 1, except that in step a the pyrrolidine is replaced with homopiperidine and in step c the 1,2, 4-thiadiazol-5-amine is replaced with 5-chloro-thiazol-2-amine, respectively, to give 4- ((2- (homopiperidin-1-yl) -4-chlorobenzyl) amino) -5-chloro-N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide, ESI-MS (M/z):563.1[ M + H ] M ] ⁺ 。

Example 8

Preparation of 5-chloro-2-fluoro-4- ((4-fluoro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (pyrimidin-4-yl) benzenesulfonamide

The procedure is as in example 1, except that in step a, 4-chloro-2-fluoro-benzonitrile is replaced with 2, 4-difluorobenzonitrile and in step c, 1,2, 4-thiadiazol-5-amine is replaced with pyrimidin-4-amine, respectively, to give 5-chloro-2-fluoro-4- ((4-fluoro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (pyrimidin-4-yl) benzenesulfonamide, ESI-MS (M/z):480.1[ M + H ] 480.1] ⁺ 。

Example 9

Preparation of 4- ((2- (homopiperidin-1-yl) -4-chlorobenzyl) amino) -5-chloro-2-fluoro-N- (pyrazin-2-yl) benzenesulfonamide

The procedure was as in example 1 except thatRespectively replacing pyrrolidine in the step a with homopiperidine, and replacing 1,2, 4-thiadiazole-5-amine in the step c with pyrazine-2-amine to obtain 4- ((2- (homopiperidin-1-yl) -4-chlorobenzyl) amino) -5-chloro-2-fluoro-N- (pyrazine-2-yl) benzene sulfonamide, ESI-MS (M/z):524.2[ M + H] ⁺ 。

Example 10

Preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyloxy) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Step a): preparation of methyl 4-chloro-2- (pyrrolidin-1-yl) benzoate

Pyrrolidine (905mg,12.727mmol), 4-chloro-2-fluoro-benzoic acid methyl ester (2.00g,10.606mmol), potassium carbonate (2.9g,21.212mmol) and DMF (20mL) were added to a reaction flask, warmed to 60 ℃ under argon atmosphere, stirred for reaction for 2 hours, cooled to room temperature, diluted with water (150mL), extracted with ethyl acetate (75mL × 3), the organic layers combined, washed with saturated aqueous sodium chloride solution (100mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give a pale yellow solid which was used in the next reaction without purification. ESI-MS (m/z): 240.1[ M + H ] ⁺ 。

Step b): preparation of 4-chloro-2- (pyrrolidin-1-yl) benzyl alcohol

4-chloro-2- (pyrrolidin-1-yl) benzoic acid methyl ester (500mg,2.086mmol) and THF (20mL) were added to a reaction flask and stirred at 0 ℃ for 0.5h, lithium aluminum hydride (118mg,3.129mmol) was added, the reaction was continued to stir at 0 ℃ for 0.5h, after the reaction was completed, it was quenched at 15 ℃ with saturated aqueous ammonium chloride (200mL), extracted with ethyl acetate (200 mL. times.3), the organic layers were combined, washed with saturated aqueous sodium chloride (100 mL. times.2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a pale yellow oil which was used in the next reaction without purification ESI-MS (m/z): 212.1[ M + H] ⁺ 。

Step c): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyloxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

4-chloro-2- (pyrrolidin-1-yl) benzyl alcohol (200mg,0.945mmol), 5-chloro-N- (2, 4-dimethoxy-benzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazol-5-yl) -benzenesulfonamide (436mg,0.945mmol), potassium carbonate (261mg,1.890mmol) and dry DMF (12mL) were reacted at 25 ℃ under nitrogen for 15 hours, after completion of the reaction, a saturated aqueous ammonium chloride solution (60mL) was added and diluted, extracted with ethyl acetate (40mL x 2), the organic layers were combined, washed with water (20mL x 3) and a saturated sodium chloride solution (50mL) in this order, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure, and the resulting crude product was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 10:1v/v)), to obtain 5-chloro-4- ((4-chloro-2- (pyrrolidine-1-yl) benzyloxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazole-5-yl) benzenesulfonamide with the yield of 69.9%, ESI-MS (M/z): 653.1[ M + H ] ⁺ 。

Step d): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyloxy) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyloxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (200mg,0.306mmol) and ethyl acetate hydrochloride solution (4M, 10mL) were added to a reaction flask, reacted at 20 ℃ for 1 hour, after the reaction is finished, the reaction solution is concentrated under reduced pressure, the obtained crude product is purified by preparative HPLC to obtain 5-chloro-4- ((4-chloro-2- (pyrrolidine-1-yl) benzyloxy) -2-fluoro-N- (1,2, 4-thiadiazole-5-yl) benzene sulfonamide with the yield of 34.8 percent, ¹ H NMR(400MHz,DMSO-d ₆ )δ:8.43(s,1H),7.61(d,J＝7.2,1H),7.28-7.02(m,4H),6.44(d,J＝12.8Hz,1H),5.18(s,2H),3.35(s,4H),1.98(s,4H)，ESI-MS(m/z)：503.1[M+H] ⁺ 。

example 11

Preparation of 5-chloro-4- ((4-chloro-2-morpholinylbenzyl) amino) -N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide

The procedure is as in example 1, except that in step a, the pyrrolidine is replaced by morpholine and in step c, the 1,2, 4-thiadiazole-5-amine is replaced by morpholine5-chloro-thiazol-2-amine substitution to give 5-chloro-4- ((4-chloro-2-morpholinobenzyl) amino) -N- (5-chlorothiazol-2-yl) -2-fluoro-benzenesulfonamide, ESI-MS (M/z):551.0[ M + H] ⁺ 。

Example 12

Preparation of 5-chloro-4- ((4-chloro-2- (piperazin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiazol-5-yl) benzenesulfonamide

The procedure is as in example 1, except that the pyrrolidine in step a is replaced with 1-tert-butoxypiperazine, respectively, to give 5-chloro-4- ((4-chloro-2- (piperazin-1-yl) benzyl) amino) -2-fluoro-N- (1,2, 4-thiazol-5-yl) benzenesulfonamide, ESI-MS (M/z):517.1[ M + H ] ⁺ 。

Example 13

Preparation of 4- ((2- (3-aminopyrrolin-1-yl) -4-chlorobenzyl) amino) -5-chloro-2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in example 1, except that the pyrrolidine in step a is replaced with tert-butyl pyrrolidin-3-carbamate, respectively, to give 4- ((2- (3-aminopyrrolin-1-yl) -4-chlorobenzyl) amino) -5-chloro-2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide, ESI-MS (M/z):517.1[ M + H] ⁺ 。

Example 14

Preparation of N- (4- (N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -2-chloro-5-fluorophenyl) -4-chloro-2- (pyrrolidin-1-yl) benzamide

Step a): preparation of benzyl (5-chloro-2-fluoro-4-nitrophenyl) sulfide

1-chloro-4, 5-difluoro-2-nitrobenzene (5.0g, 25.836mmol), anhydrous K ₂ CO ₃ (5.4g, 38.754mmol) and dry DMF (40mL) were added to the flask, and benzyl mercaptan (3.2g, 25.836mmol) was slowly added dropwise to the reaction mixture at 0 ℃ and after addition, the temperature was slowly raised to room temperature for 2 hours. After the reaction is finished, pouring the reaction into ice water, filtering and collecting precipitated yellow solid, washing with a proper amount of water, and drying to obtain crude benzyl (5-chloro-2-fluoro-4-nitrobenzene) thioether, wherein the crude benzyl (5-chloro-2-fluoro-4-nitrobenzene) thioether is directly used for the next reaction without further purification.

Step b): preparation of 5-chloro-2-fluoro-4-nitro-benzenesulfonyl chloride

Benzyl (5-chloro-2-fluoro-4-nitrophenyl) sulfide (5.0g, 16.793mmol) and methylene chloride (150mL) were added to a reaction flask, HCl (4M, 125.00mL) and NaClO (4.38g, 58.776mmol) were slowly added dropwise with stirring at 0 deg.C, reacted at 0 deg.C for 1 hour, and warmed to 10 deg.C for 12 hours. After the reaction is finished, the organic phase is separated and concentrated, the obtained solid is pulped by petroleum ether (20mL) at the temperature of minus 30 ℃ to obtain the 5-chloro-2-fluoro-4-nitro-benzenesulfonyl chloride with the yield of 45.6 percent, and ESI-MS (M/z):273.9[ M + H ])] ⁺ 。

Step c): preparation of 5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-4-nitro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in example 1, except that 5-chloro-2, 4-difluoro-benzenesulfonyl chloride is reacted with 5-chloro-2-fluoro-4-nitro-benzenesulfonyl chloride to give 5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-4-nitro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide.

Step d): preparation of 4-amino-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Adding 5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-4-nitro-N- (1,2, 4-thiadiazole-5-yl) benzenesulfonamide (1.0g, 2.045mmol) and ethanol (30mL) into a reaction flask, stirring for dissolving, adding saturated ammonium chloride solution (10mL), adding zinc powder (669mg, 10.225mmol), heating to 50 deg.C, stirring for reacting for 12 hours, cooling to room temperature, filtering through a silica gel pad to remove insoluble solids, washing filter cakes with ethyl acetate (50mL), combining filtrates, extracting with ethyl acetate (50mL) and water (100mL), washing organic layer with saturated sodium chloride solution (100mL), drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, and passing the obtained crude product through silica gel column layer Purification by chromatography (eluent: petroleum ether/ethyl acetate 1: 1v/v) gave 4-amino-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide in 73.5% yield, ESI-MS (m/z): 459.1[ M + H] ⁺ . Step e): preparation of 4-chloro-N- (2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluorophenyl) -2- (pyrrolidin-1-yl) benzamide

Adding 4-amino-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (100mg, 0.218mmol), 4-chloro-2- (pyrrolidin-1-yl) benzoic acid (54mg, 0.240mmol), benzotriazol-1-yloxy-tris (tetrahydropyrrolyl) phosphonium hexafluorophosphate (PyBOP, 170mg, 0.327mmol), DIPEA (85mg, 0.654mmol) and dry DMSO (5mL) into a reaction flask, stirring at room temperature for reaction for 20 hours, pouring the reaction solution into water after the reaction is finished, extracting with ethyl acetate (20mL x 3), combining organic layers, sequentially adding saturated ammonium chloride aqueous solution (20mL x 3) and water (20mL), Saturated aqueous sodium chloride (10mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product purified by preparative thin layer (developing solvent: dichloromethane/methanol ═ 10: 1v/v) to give 4-chloro-N- (2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -5-fluorophenyl) -2- (pyrrolidin-1-yl) benzamide in 41.1% yield, ESI-MS (M/z):666.1[ M + H ] sulfonamide ] ⁺ 。

Step f): preparation of N- (4- (N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -2-chloro-5-fluorophenyl) -4-chloro-2- (pyrrolidin-1-yl) benzamide

Adding 4-chloro-N- (2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazole-5-yl) sulfamide) -5-fluorophenyl) -2- (pyrrolidine-1-yl) benzamide (80mg,0.120mmol) into a reaction bottle, adding 2N ethyl acetate hydrochloride solution (10mL) for dissolving, stirring at room temperature for 1 hour, after the reaction is finished, concentrating under reduced pressure, purifying the obtained crude product by preparative HPLC to obtain N- (4- (N- (1,2, 4-thiadiazole-5-yl) sulfamide) -2-chloro-5-fluorophenyl) -4-chloro-2- (pyrrolidine-1-yl) benzamide, yield 35.4%, ESI-MS (m/z): 516.1[ M + H] ⁺ 。

Example 15

Preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) phenyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Step a): preparation of 1- (5-chloro-2-nitrophenyl) pyrrolidine

The procedure is as in step a of example 1, except that 4-chloro-2-fluoro-benzonitrile is replaced with 4-chloro-2-fluoro-1-nitrobenzene to give 1- (5-chloro-2-nitrophenyl) pyrrolidine, ESI-MS (M/z):227.1[ M + H] ⁺ 。

Step b): preparation of 4-chloro-2- (pyrrolidin-1-yl) aniline

The procedure is as in step d of example 13, except that 5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-4-nitro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide is replaced with 1- (5-chloro-2-nitrophenyl) pyrrolidine to give 4-chloro-2- (pyrrolidin-1-yl) aniline, ESI-MS (M/z):197.1[ M + H ] aniline ] ⁺ 。

Step c-d): preparation of 4-bromo-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in example 1, steps c-d, except that 5-chloro-2, 4-difluoro-benzenesulfonyl chloride is replaced with 4-bromo-5-chloro-2-fluoro-benzenesulfonyl chloride to give 4-bromo-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide, ESI-MS (M/z):521.9[ M + H] ⁺ 。

Step e): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

A mixture of 4-chloro-2- (pyrrolidin-1-yl) aniline (300mg, 1.525mmol), 4-bromo-5-chloro-N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (957mg, 1.830mmol), Pd (OAc) ₂ (34mg, 0.153mmol), BINAP (190mg, 0.306mmol) and Cs ₂ CO ₃ (1.49g, 4.575mmol) is dispersed in dry dioxane (10mL), the reaction is refluxed for 12 hours under the protection of nitrogen, after the reaction is finished, the reaction product is cooled to room temperature, filtered, a filter cake is washed by ethyl acetate (50mL), then ethyl acetate (50mL) and water (100mL) are added for extraction,the organic layer was washed with saturated sodium chloride solution (100mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: dichloromethane/methanol ═ 20:1v/v) to give 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide in 52.5% yield, ESI-MS (m/z): 638.1[ M + H ] ⁺ 。

Step f): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) phenyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in step d of the example, except that 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyloxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide is replaced with 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide to give 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) phenyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide - (1,2, 4-thiadiazol-5-yl) benzenesulfonamide, yield 50.0%, ESI-MS (m/z): 488.1[ M + H] ⁺ 。

Example 16

Preparation of 5-chloro-4- (4-chloro-2- (pyrrolidin-1-yl) phenoxy) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Step a): preparation of 4-chloro-2- (pyrrolidin-1-yl) phenol

Adding 4-chloro-2- (pyrrolidine-1-yl) aniline (200mg, 1.017mmol) into a mixed solvent of acetic acid (2mL) and water (2mL) at 0 ℃, uniformly stirring, adding sodium nitrite (842mg, 12.204mmol), stirring at room temperature for reaction for 1 hour, pouring a reaction solution into a mixture of ethyl acetate and water (30mL/20mL), separating an organic layer, sequentially washing with a saturated aqueous solution of sodium bicarbonate (10mL) and a saturated aqueous solution of sodium chloride (10mL), drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, dissolving a crude product with methanol (4mL), adding water (1mL) and potassium carbonate (281mg, 2.034mmol), heating to 60 ℃ for reaction for 20 hours, after the reaction is finished, evaporating under reduced pressure to remove the solvent, and removing the residual solvent Dissolving the residue with ethyl acetate, washing with water and saturated sodium chloride, drying with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, purifying the obtained crude product by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 1: 1v/v) to obtain 4-chloro-2- (pyrrolidin-1-yl) phenol with yield of 46.2%, ESI-MS (M/z):198.1[ M + H] ⁺ 。

Step b): preparation of 5-chloro-4- (4-chloro-2- (pyrrolidin-1-yl) phenoxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

Adding 4-chloro-2- (pyrrolidin-1-yl) phenol (50mg, 0.253mmol), 5-chloro-N- (2, 4-dimethoxybenzyl) -2, 4-difluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (117mg, 0.253mmol), potassium carbonate (70mg, 0.506mmol) and dry DMSO (2mL) into a reaction flask, reacting at 50 ℃ for 15 hours under nitrogen protection, after the reaction is finished, adding water (30mL) to dilute, extracting with ethyl acetate (20mL x 3), combining organic layers, washing with water (10mL x 3) and saturated sodium chloride solution (30mL), drying over anhydrous sodium sulfate, filtering, concentrating under reduced pressure, purifying the obtained crude product by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 3:1v/v), to obtain 5-chloro-4- (4-chloro-2- (pyrrolidine-1-yl) phenoxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazole-5-yl) benzenesulfonamide with yield of 74.5%, ESI-MS (m/z): 639.1[ M + H ] ⁺ 。

Step c): preparation of 5-chloro-4- (4-chloro-2- (pyrrolidin-1-yl) phenoxy) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

5-chloro-4- (4-chloro-2- (pyrrolidin-1-yl) phenoxy) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide (80mg) was added to a reaction flask, a mixture of trifluoroacetic acid and dichloromethane (4mL, v/v ═ 1:6) was added, the reaction was stirred at room temperature for 1 hour, after completion of the reaction, excess trifluoroacetic acid and dichloromethane were distilled off under reduced pressure, and the residue was saturated NaHCO ₃ The solution (10mL) was extracted with ethyl acetate (20mL), the aqueous layer was extracted with ethyl acetate (10mL x 3), the organic layers were combined, washed with saturated sodium chloride solution (40mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the resulting crude product was purified by preparative thin layer (developing solvent: dichloromethane/methanol ═ 10: 1) to give 5-chloro-4- (4-chloro-2- (pyrrolidin-1-yl) benzeneOxy) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide, ESI-MS (m/z): 489.0[ M + H] ⁺ 。

Example 17

Preparation of 4- (N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) benzyl) -5-fluoro-benzamide

Step a): preparation of 4- (benzylsulfanyl) -2-chloro-5-fluoro-benzoic acid

2-chloro-4, 5-fluorobenzoic acid (3.0g, 15.580mmol) and DMF (30mL) were added to a reaction flask, and Cs was added ₂ CO ₃ (10.2g, 31.160mmol) and phenylmethanethiol (1.94g, 15.580mmol), heating to 70 ℃ for 6 hours, cooling to room temperature after the reaction is finished, pouring into water (500mL), extracting with ethyl acetate (100mL x 3), adjusting the pH of the water layer to 1 with 4nhci (aq), filtering and drying the precipitated solid to obtain 4- (benzylthio) -2-chloro-5-fluoro-benzoic acid, yield 79.0%, ESI-MS (m/z): 297.1[ M + H] ⁺ 。

Step b): preparation of methyl 4- (benzylsulfanyl) -2-chloro-5-fluorobenzoate

4- (benzylthio) -2-chloro-5-fluoro-benzoic acid (3.0g, 10.110mmol) and methanol (30mL) were added to a reaction flask and concentrated H was added slowly dropwise ₂ SO ₄ (198mg, 2.022mmol), after addition, the reaction was warmed to reflux for 48 h, concentrated under reduced pressure, diluted with ethyl acetate (100mL), and then sequentially diluted with water (50mL) and saturated NaHCO ₃ Aqueous solution (50mL), saturated aqueous sodium chloride solution (50mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give methyl 4- (benzylsulfanyl) -2-chloro-5-fluorobenzoate in 85.0% yield, ESI-MS (m/z): 311.1[ M + H] ⁺ 。

Step c): preparation of methyl 2-chloro-4- (chlorosulfonyl) -5-benzoate

4- (benzylthio) -2-chloro-5-fluorobenzoic acid methyl ester (2.0g, 6.436mmol) and dichloromethane (60mL) were added to a reaction flask, HCl (4N, 50.0mL) and NaClO (1.68g, 22.526mmol) were slowly added dropwise with stirring at 0 deg.C, reacted at 0 deg.C for 1 hour, then heated to 10 deg.C and reacted for 12 hours After the reaction is finished, the organic layer is separated, decompressed and concentrated, the obtained crude product is ultrasonically beaten by petroleum ether (5mL) at the temperature of minus 30 ℃ to obtain the 2-chloro-4- (chlorosulfonyl) -5-methyl benzoate with the yield of 45.2 percent and ESI-MS (M/z):287.0[ M + H ]/]] ⁺ 。

Step d): preparation of methyl 2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluoro-benzoate

The procedure is as in step d of example 1, except that 5-chloro-2, 4-difluoro-benzenesulfonyl chloride is replaced with methyl 2-chloro-4- (chlorosulfonyl) -5-benzoate to give methyl 2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonamide) -5-fluoro-benzoate.

Step e): preparation of 2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluoro-benzoic acid

2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluoro-benzoic acid methyl ester (500mg, 0.996mmol) and LiOH ₂ O (63mg, 1.494mmol) was added to THF (10mL) and water (50mL) and reacted at 10 ℃ for 1 hour, after the reaction was completed, a mixed solution of 10% methanol and dichloromethane (100mL × 3) was extracted, the organic layers were combined, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give 2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -5-fluoro-benzoic acid in 86.2% yield, ESI-MS (m/z): 488.1[ M + H ] ⁺ 。

Step f): preparation of 2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) phenyl) -4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -5-fluoro-benzamide

Adding 2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazole-5-yl) sulfonamide) -5-fluoro-benzoic acid (100mg,0.205mmol), 4-chloro-2- (pyrrolidin-1-yl) aniline (40mg,0.205mmol), benzotriazol-1-yloxy-tris (tetrahydropyrrolyl) phosphonium hexafluorophosphate (PyBOP, 213mg, 0.410mmol), DIPEA (79mg, 0.615mmol) and dry DMSO (5mL) into a reaction flask, stirring at room temperature for reaction for 24 hours, after the reaction is finished, pouring the reaction solution into water, extracting with ethyl acetate (20mL x 3), combining the organic layers, and sequentially using saturated ammonium chloride solution (10mL x 3)mL × 3), water (10mL), saturated sodium chloride solution (10mL), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the resulting crude product purified by preparative thin layer (developing solvent: dichloromethane/methanol 20: 1v/v) to obtain 2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) phenyl) -4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -5-fluoro-benzamide, with a yield of 36.8%, ESI-MS (M/z):666.1[ M + H] ⁺ 。

Step f): preparation of 4- (N- (1,2, 4-thiadiazol-5-yl) sulfonamido) -2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) benzyl) -5-fluoro-benzamide

The procedure is as in example 14, except that 4-chloro-N- (2-chloro-4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluorophenyl) -2- (pyrrolidin-1-yl) benzamide is replaced with 2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) phenyl) -4- (N- (2, 4-dimethoxybenzyl) -N- (1,2, 4-thiadiazol-5-yl) sulfonylamino) -5-fluoro-benzamide to give 4- (N- (1,2, 4-Thiadiazol-5-yl) sulfonylamino) -2-chloro-N- (4-chloro-2- (pyrrolidin-1-yl) benzyl) -5-fluoro-benzamide in 32.8% yield and ESI-MS (M/z):516.1[ M + H] ⁺ 。

Example 18

Preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidine-1-methylene) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonyl

Step a): preparation of 4-chloro-2- (pyrrolidine-1-methylene) benzonitrile

The procedure is as in example 1, except that 4-chloro-2-fluoro-benzonitrile is replaced with 2- (bromomethyl) -4-chlorobenzonitrile to give 4-chloro-2- (pyrrolidin-1-methylene) benzonitrile ESI-MS (M/z):221.1[ M + H ] benzonitrile] ⁺ 。。

Step b): preparation of (4-chloro-2- (pyrrolidine-1-methylene) benzylamine

The procedure was the same as in step b of example 1 except that 4-chloro-2- (pyrrolidin-1-yl) benzonitrile was replaced with 4-chloro-2- (pyrrolidin-1-methylene) benzonitrile to give (4-chloro-2- (pyrrolidin-1-yl) benzonitrile Methylene) benzylamine, ESI-MS (M/z):225.1[ M + H] ⁺ 。

Step c): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-methylene) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide

The procedure is as in example 1, except that 4-chloro-2-pyrrolidin-1-yl-benzylamine is replaced by (4-chloro-2- (pyrrolidin-1-methylene) benzylamine to give 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-methylene) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide, ESI-MS (M/z):666.1[ M + H-5-yl) benzenesulfonamide] ⁺ 。

Step d): preparation of 5-chloro-4- ((4-chloro-2- (pyrrolidine-1-methylene) benzyl) amino) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonyl

The procedure is as in example 1, except that 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-yl) benzyl) amino) -N- (2, 4-dimethoxy-benzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide is replaced with 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-methylene) benzyl) amino) -N- (2, 4-dimethoxybenzyl) -2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonamide to give 5-chloro-4- ((4-chloro-2- (pyrrolidin-1-methylene) benzyl) amino) - 2-fluoro-N- (1,2, 4-thiadiazol-5-yl) benzenesulfonyl, ESI-MS (M/z):516.1[ M + H] ⁺ 。

Electrophysiological assay

Patch voltage clamp electrophysiology allows direct measurement and quantification of the blockade of the sodium channel of the voltage gate (NaV), as well as determination of the time and voltage dependence of the blockade, which has been explained in conjunction with the differences in the resting, open and inactive states of the sodium channel (hill, b.et al, Journal of General Physiology (1977),69: 497-.

Representative compounds of the invention the effect of compounds on ion channel current was determined by a manual patch clamp assay using a stable HEK293 cell line (purchased from synergetics cell bank) transfected with specific ion channels.

The manual patch clamp experimental protocol was as follows:

1) cell culture

Nav1.7

hNav1.7 sodium channel Stable expressionThe HEK293 cell line is cultured in a DMEM medium containing 10% fetal calf serum and 0.2mg/mL Hygromycin B at 37 ℃ and CO ₂ The concentration is 5%; when the cells grow to a certain stage, carrying out cell passage by removing the old culture medium, washing the cells with PBS once, adding 1mL of Trypsin-EDTA solution, incubating for one minute at 37 ℃, adding 5mL of complete culture medium preheated at 37 ℃, slightly blowing and beating the cell suspension by using a suction pipe to separate the aggregated cells, transferring the cell suspension into a sterile centrifuge tube, and centrifuging for 5 minutes at 1000rmp to collect the cells; for expansion or maintenance culture, cells are seeded in 6cm cell culture dishes, each cell culture dish being seeded with a cell amount of 2.5 x 10 ⁵ cells; 3 x 10 were separated by Trypsin-EDTA separation prior to patch clamp experiments ³ The cells were plated on a cover glass, cultured in a 24-well plate (final volume: 500. mu.L), and after 18 hours, the experimental examination was carried out.

Nav1.5

The CHO cell line stably expressed by the hNav1.5 sodium channel is cultured in an F12 culture medium containing 10% fetal calf serum, the culture temperature is 37 ℃, and the concentration of CO2 is 5%; when the cells grow to a certain stage, carrying out cell passage, wherein the method comprises the steps of removing an old culture medium, washing the old culture medium with PBS once, adding 1mL of de 0.25% -Trypsin-EDTA solution, incubating for one minute at 37 ℃, adding 5mL of complete culture medium preheated at 37 ℃ when the cells are separated from the bottom of a dish, slightly blowing and beating the cell suspension by using a suction pipe to separate the aggregated cells, transferring the cell suspension into a sterile centrifuge tube, and centrifuging for 5 minutes at 1000rmp to collect the cells; for expansion or maintenance culture, cells were seeded in 6cm cell culture dishes, each cell culture dish being seeded with a cell mass of 2.5 × 105 cells; 3 x 10 were separated by Trypsin-EDTA separation prior to patch clamp experiments ³ The cells were plated on a cover glass, cultured in a 24-well plate (final volume: 500. mu.L), and after 18 hours, the experimental examination was carried out.

Nav1.3

The hNav1.3 sodium channel stably expressed HEK293 cell line is cultured in a DMEM medium containing 10% fetal bovine serum and 1.2mg/mL G418 at the temperature of 37 ℃ and CO ₂ The concentration is 5%; when the cells have grown to a certain stage, they are passaged, which isRemoving an old culture medium, washing the old culture medium with PBS once, adding 1mL of Trypsin-EDTA solution, incubating for one minute at 37 ℃, adding 5mL of complete culture medium preheated at 37 ℃ when cells are separated from the bottom of a dish, gently blowing and beating a cell suspension with a suction pipe to separate aggregated cells, transferring the cell suspension into a sterile centrifuge tube, and centrifuging for 5 minutes at 1000rmp to collect the cells; for expansion or maintenance culture, cells are seeded in 6cm cell culture dishes, each cell culture dish being seeded with a cell amount of 2.5 x 10 ⁵ cells; 3 x 10 were separated by Trypsin-EDTA separation prior to patch clamp experiments ³ The cells were plated on a cover glass, cultured in a 24-well plate (final volume: 500. mu.L), and after 18 hours, the experimental examination was carried out.

2) Preparation of Compound samples

The compounds prepared in the examples of the present invention were dissolved in dimethyl sulfoxide (DMSO) and prepared into DMSO stock solution at a concentration of 10mM for experiments. Extracellular fluid for 10mM DMSO stock solution (140mM NaCl, 4mM KCl, 1mM MgCl) ₂ ，2mM CaCl ₂ 5mM D-Glucose monohydrate, 10mM HEPES, pH 7.4 adjusted with NaOH) to various concentrations, and the final concentration of DMSO in each compound was 0.1% or less.

3) Membrane clamp measurement of sodium ion channel blocking effect

Whole cell patch clamp voltage stimulation protocols for recording hNav1.7, hNav1.5 and hNav1.3 sodium channel currents were as follows: after forming a whole cell seal, the cell voltage is clamped at-120 mV, the clamping voltage is depolarized to 0mV and maintained for 20 milliseconds, then the voltage is restored to-75 mV and maintained for 8 seconds, then the cell membrane potential is restored to-120 mV, the maintaining time is 20 milliseconds, then the voltage is depolarized to 0mV and maintained for 20 milliseconds, finally the clamping voltage is restored to-120 mV and maintained for 30 milliseconds, data acquisition is repeated every 20 seconds, and the effect of the drug on the current peak values of the hNav1.7, hNav1.5 and hNav1.3 sodium channels is observed.

A capillary glass tube (BF150-86-10, Sutter Instruments) was drawn into a recording electrode using a microelectrode drawing machine (P97, Sutter Instruments). The recording electrode was brought into contact with the cell under an inverted microscope (AE31E, Motic) by manipulating a microelectrode manipulator (86PW420600, MCI Instruments) and applying negative pressure suction to form a G.OMEGA.seal. And carrying out rapid capacitance compensation after forming the G omega seal, then continuously applying negative pressure to the G omega seal to suck and break cell membranes to form a whole cell recording mode, and then carrying out slow capacitance compensation and recording membrane capacitance and series resistance.

Dosing was initiated when whole cell recorded currents of hNav1.7, hNav1.5, and hNav1.3 stabilized, and the next concentration was measured after each drug concentration had been applied for 5 minutes (or current to stabilization), with multiple concentrations being measured for each test compound. All electrophysiological experiments were performed at room temperature. Specifically, eight concentrations were set for each compound, percent inhibition of sodium channels by compounds was determined by calculating the percent inhibition of peak current generated after treatment of cells with each concentration of compound relative to peak current generated before treatment with compound, and IC was calculated using IGOR software ₅₀ The value is obtained. And the inhibition IC of the compounds on hNav1.7, hNav1.5 and hNav1.3 was assessed as follows ₅₀ ：

hNav1.7：IC ₅₀ ：+(>1μM)、++(100nM～1μM)、+++(50～100nM)、++++(<50nM)

hNav1.5：IC ₅₀ ：+(>1μM)、++(100nM～1μM)、+++(50～100nM)、++++(<50nM)

hNav1.3：IC ₅₀ ：+(>1μM)、++(100nM～1μM)、+++(50～100nM)、++++(<50nM)

The results show that all compounds in the examples have IC of inhibitory activity on hNav1.5 sodium channel associated with cardiac dysfunction ₅₀ Are all larger than 10 mu M. While the inhibitory activity IC on hNav1.7 and hNav1.3 ₅₀ The calculation results are shown in table 2 below.

TABLE 2

ExamplesNumbering	IC 50 (hNav1.7)	IC 50 (hNav1.3)	Example numbering	IC 50 (hNav1.7)	IC 50 (hNav1.3)
						Example 1	++++	+++	Example 10	++++	++++
Example 2	+++	++++	Example 11	+++	+++
						Example 3	++++	++++	Example 12	+++	+++
Example 4	++++	++++	Example 13	++++	+++
						Practice ofExample 5	++++	+++	Example 14	+++	++++
Example 6	++++	+++	Example 15	++++	+
						Example 7	++++	++++	Example 16	++	+++
Example 8	++++	++++	Example 17	++++	+
						Example 9	+++	++++	Example 18	++++	+

Claims (10)

1. A compound of formula I:

wherein R is ₁ Is selected from a 5-6 membered monocyclic aromatic heterocycle containing up to 3 heteroatoms of N, O, S, optionally the 5-6 membered monocyclic aromatic heterocycle is substituted with halogen;

R ₂ 、R ₃ independently selected from hydrogen, halogen, cyclopropyl, and R ₂ 、R ₃ At least 1 is halogen;

x is-CONH-, -CH ₂ O-or-CH ₂ NH-；

R ₄ Selected from hydrogen, halogen, C _1-4 A haloalkyl group;

a is

Wherein n is an integer from 2 to 4;

or A is

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R is ₁ Selected from 5-membered aromatic heterocycles comprising up to 3 heteroatoms of N, O, S, optionally the 5-membered aromatic heterocycle being substituted with halogen.

3. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is ₁ Is selected from

4. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is ₂ 、R ₃ Independently selected from hydrogen or halogen, and R ₂ 、R ₃ At least 1 item is halogen.

5. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein X is selected from-CH ₂ O-or-CH ₂ NH-。

6. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R is ₄ Selected from hydrogen or halogen.

7. Compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that said compound is selected from:

8. a pharmaceutical composition comprising a compound of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

9. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of pain.

10. Use according to claim 9, characterized in that the pain is neuropathic pain or inflammatory pain.