DE112012004878T5 - Heterocyclic dihydro five-membered ketone derivatives as DHODH inhibitor and their use - Google Patents

Abstract

The present invention relates to the synthesis and use of heterocyclic dihydro five-membered ketone derivatives of the general formula (I) which are used as a DHODH inhibitor for Plasmodium falciparum, and the compound according to the invention can be used for the treatment and prevention of DHODH related diseases, including the treatment of parasitic diseases, such as malaria, caused by malaria parasites.

Description

Field of the invention

The present invention relates to the field of treatment of malaria, in particular to the synthesis and use of 3 (2H) thiophenone derivatives as a DHODH inhibitor.

Background of the invention

Malaria is an insect-borne infectious disease caused by plasmodium infection via mosquito bites, and is still one of the world's problems affecting human health. Currently, typical commercially available drugs for the treatment of malaria are chloroquine, artemisinin, pyrimethamine, etc. In recent decades, many agents that have previously been very useful, more and more cases of drug resistance, such as quinine, chloroquine, mel-quinoline and the like; After Plasmodium has been treated with chloroquine, its nucleus breaks open, blisters appear in the cytoplasm, and the malaria pigment contracts. Chloroquine can not kill the parasite directly, but it can interfere with its reproduction. It has a strong binding force to nucleoprotein, and the negatively charged 7-chloro atom of the quinoline ring comes close to the guanine 2-amino group in the Plasmodium DNA, inserting chloroquine into the two strands of the DNA double helix and forming a complex with DNA forms, thus preventing DNA replication and RNA transcription. Chloroquine can also prevent phosphate from being incorporated into the DNA and RNA of Plasmodium, thereby interfering with the proliferation of the parasite due to a decrease in nucleic acid synthesis. In recent years, however, it has been reported and confirmed that the efficacy of chloroquine and its derivatives as antimalarial drugs is rapidly decreasing, and in patients in some areas, even complete resistance to chloroquine occurs, and therefore longer treatment cycles for removal of the parasite necessary, and there are recurrences (Wu, T., S. Nagle, A., K. Chatterjee, A., Road Towards New Antimalarials - Overview of the Strategies and Their Chemical Progress, Current Medicinal Chemistry 2011, 18 (6) , 853-871).

Artemisinin is the only antimalarial drug that has not been reported to have widespread and severe drug resistance, but in recent years it has been reported that malaria mosquitoes that carry plasmodium and have artemisinin resistance in vivo are found in the border area between Thailand and Cambodia. Related aftercare outcomes have further increased people's fear of increasing resistance to artemisinin therapies (Eastman, RT, Artemisinin-based combination therapies: a vital tool in the effort to eliminate malaria, Nat Rev Micro 2009, 7 (12)). , 864-874).

In addition, early malaria drugs, such as pyrimethamine, may inhibit Plasmodium folate reductase, which in turn disturbs the normal folate metabolism of Plasmodium. The active ingredient is effective against the early phase of the Plasmodium falciparum cell cycle and can therefore be used as an etiologically preventive agent. In addition, the drug also inhibits the development of malaria parasites in mosquitoes so that transmission can be interrupted (McKie, JH, Douglas, KT, Chan, C., Roser, SA, Yates, R., Read, M., Hyde, JE ; Dascombe, MJ; Yuthavong, Y., Sirawaraporn, W., Rational Drug Design Approach for Overcoming Drug Resistance: Application to Pyrimethamine Resistance in Malaria, Journal of Medicinal Chemistry 1998, 41 (9), 1367-1370). The active substance can be used for the prevention of malaria and as antiretabolic therapy in the resting phase. However, after being used for many years, off-target effects due to enzyme mutations are constantly occurring, and the above drugs have more or less some side effects such as diarrhea, rash, hypertension, and liver enzyme system disorders. Therefore, it is of great academic value and practical value to search for a novel Plasmodium falciparum DHODH inhibitor with high selectivity, high efficiency, safety, and good target affinity.

Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent iron-containing mitochondrial enzyme (McConkey A, Fishwick CWG; Johnson AP; The first de novo designed inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase [J] Bioorganic & Medicinal Chemistry Letters, 2006, 16: 88- 92), which catalyzes the dehydrogenation of dihydroorotate to orotate, which is the fourth step in the de novo synthesis of pyrimidine. Therefore, DHODH is a key enzyme for the synthesis of pyrimidine in the nucleic acid, and it is also a unique enzyme within the parasite that catalyzes the formation of orotate from dihydroorotate and participates primarily in the catalysis of the fourth step of de novo biosynthesis of pyrimidine is. In humans and Plasmodium, DHODH is localized on the inner mitochondrial membrane, and the catalytic process requires other cofactors. Structurally, DHODH has two parts on, ie an α-helical structure region at the N-terminus and an α / β-barrel structure at the C-terminus. DHODH adheres to the unique structure of the N-terminus on the inner mitochondrial membrane, and the C-terminal region is the major catalysis center. There are binding sites on DHODH for the substrate and the cofactor flavin mononucleotide (FMN) and coenzyme Q (CoQ). The catalytic process occurs in two steps: First, dihydroorotate is oxidized to orotate, and FMN accepts the hydrogen released during the process simultaneously and is reduced to FMNH ₂ ; then FMNH _{2 is} again dehydrated under the action of CoQ and oxidized to FMN. Any compound that can competitively combine with the substrate or cofactor can block the action of DHODH and thereby also block the synthesis of DNA or RNA. Plasmodium falciparum DHODH inhibitors achieve therapeutic effects in malaria mainly by blocking the biological synthesis of pyrimidine within Plasmodium and inhibiting the proliferation and growth of Plasmodium.

In addition, pyrimidine base can be obtained in most organisms via de novo synthesis and salvage synthesis, but rapidly differentiating human cells, such as activated T lymphocytes, B lymphocytes, and tumor cells are also involved in the de novo synthesis of Pyrimidine instructed to meet their growth requirements. Therefore, a DHODH inhibitor can be used as an anti-cell proliferation agent for the treatment of tumors and for a particular immunosuppression. Because of the general mechanism of action of DHODH on DNA and RNA synthesis, inhibition of DHODH also affects many other downstream signaling pathways. It has been shown (Proceedings of the National Academy of Sciences 2010, 107 (29), 12828) that inhibition of mitochondrial DHODH induces a p53 stress response, which in turn can be used to treat tissue damage. Another reference (Annals of the Rheumatic Diseases 2006, 65 (6), 728-735) reports that inhibition of DHODH prevents TNF-induced phosphorylation of the transcription factor NF-κB, activating AP-1 and c-jun. N-terminal kinase inhibit and ultimately can inhibit TNF-induced apoptosis.

Research on the use of dihydroorotic acid analogs as inhibitors of DHODH has been reported (Biochemical pharmacology 1988, 37 (20), 3807-3816). Currently, research on DHODH inhibitors is directed primarily at the binding sites of CoQ, with Brequinar and leflunomide being used clinically. Brequinar is used as an antitumor agent and as a remedy for the organ transplant-induced immune response of the host. However, the therapeutic window of Brequinar is narrow and side effects such as thrombocytopenia and mucositis are easily caused when it is orally administered in combination with cisplatin or cyclosporin A, and therefore its broad clinical application is limited. Launched in 1998, leflunomide strongly inhibits a variety of autoimmune diseases as well as acute and chronic reactions and organograft-induced xenograft rejection, and is used clinically for the treatment of lupus and rheumatoid arthritis and can be used to prevent and prevent graft rejection to treat.

Other diseases that can be treated with a DHODH inhibitor and related literature include rheumatoid arthritis (Herrmann, ML, Schleyerbach, R. and Kirschbaum, BJ, Leflunomide: immunomodulatory drug for the treatment of rheumatoid arthritis and other autoimmune diseases. Immunopharmacology 2000, 47 (2-3), 273-289), colitis (Fitzpatrick, LR, DemI, L., Hofmann, C., Small, JS, Groeppel, M., Hamm, S., Lemstra, S., Leban, J. and Ammendola, A., 4SC-101, a novel immunosuppressive drug, inhibits IL-17 and attenuates colitis in two murine models of inflammatory bowel disease, Inflammatory bowel diseases 2010, 16 (10), 1763-1777), systemic lupus erythematosus (Kulkarni, OP, Sayyed, SG, Kantner, C., Ryu, M., Schnurr, M., Sardy, M., Leban, J., Jankowsky, R., Ammendola, A. and Doblhofer, R , 4SC-101, A Novel Small Molecule Dihydroorotate Dehydrogenase Inhibitor, Suppressive Systemic Lupus Erythematosus in MRL (Fas) Ipr Mice, Am J. P athol. 2010, 176 (6), 2840-2847), psoriatic arthritis (Kaltwasser, JP, Nash, P., Gladman, D., Rosen, CF, Behrens, F., Jones, P., Wollenhaupt, J., Falk , FG and Mease, P., Efficacy and safety of leflunomide in the treatment of psoriatic arthritis and psoriasis: A multinational, double-blind, randomized, placebo-controlled clinical trial, Arthritis & Rheumatism 2004, 50 (6), 1939-1950 ), Psoriasis (White, RM, Cech, J., Ratanasirintrawoot, S., Lin, CY, Rahl, PB, Burke, CJ, Langdon, E., Tomlinson, ML, Mosher, J. and Kaufman, C., DHODH transcriptional elongation in the neural crest and melanoma, Nature 2011, 471 (7339), 518-522), graft rejection (Makowka, L .; Sher, L .; Cramer, D. The development of Brequinar as an immunosuppressive drug for transplantation, Immunological reviews 1993, 136, 51), glomerular disease (Zeng Jianying, Zhang Jianlin, Clinical Observation of Leflunomide for Glomerular Disease, Practical Medicine, 2006 (15)); etc.

Brief description of the invention

wobei
X₁ aus O, S, NH und CH₂ ausgewählt ist;
X₂ aus O, S, NH, NOH und C₁-C₆-Imino ausgewählt ist;
R¹ aus H, C₁-C₁₀-Alkyl, ungesättigtem Kohlenwasserstoffrest, das nicht mehr als 10 Kohlenstoffatome enthält, gegebenenfalls substituiertem Aryl, Nitro, Amino, NR⁴R⁵ und Halogen ausgewählt ist;
R² aus H, C₁-C₆-Alkyl, ungesättigtem C₂-C₆-Kohlenwasserstoffrest, C₁-C₆-Alkylcarbonyl, gegebenenfalls substituiertem Benzoyl, Carboxy, Aminocarbonyl, C₁-C₆-Alkoxycarbonyl, C₁-C₆-Alkylaminocarbonyl, Hydroxy, C₁-C₆-Alkoxy, C₃-C₈-Cycloalkylaminocarbonyl, C₃-C₈-Cycloalkyl-C₁-C₆-alkylaminocarbonyl, C₃-C₈-Cycloalkyl-C₁-C₆-alkoxycarbonyl ausgewählt ist;
R³ aus H, C₁-C₆-Alkyl, gegebenenfalls substituiertem ungesättigten C₂-C₆-Kohlenwasserstoffrest, C₁-C₆-Alkylcarbonyl, gegebenenfalls substituiertem Benzoyl, Carboxy, Aminocarbonyl, C₁-C₆-Alkoxycarbonyl, C₁-C₆-Aminocarbonyl, Hydroxy, C₁-C₆-Alkoxy ausgewählt ist;
R⁴ und R⁵ unabhängig aus H, C₁-C₆-Alkyl, -C(O)NHR⁶, C₁-C₆-Alkoxycarbonyl, halogensubstituiertem Alkyl, ungesättigtem C₂-C₆-Kohlenwasserstoffrest, gegebenenfalls substituiertem Aryl, C₁-C₆-Alkylcarbonyl, gegebenenfalls substituiertem Benzoyl, einer gegebenenfalls substituierten heterocyclischen Gruppe, gegebenenfalls substituiertem Heterocyclocarbonyl und C₁-C₆-Alkoxycarbonyl ausgewählt sind;
R⁶ aus gegebenenfalls substituiertem Aryl and einer gegebenenfalls substituierten heterocyclischen Gruppe ausgewählt ist.A DHODH inhibitor binds to the coenzyme binding pocket located at the N-terminus of the enzyme DHODH, and based on the binding principle, the inhibitor would have a polar head and a hydrophobic tail, allowing the inhibitor to be effectively within the coenzyme Gujjar, R .; El Mazouni, F. Kaminsky, W. Malmquist, NA; Goldsmith, S. Rathod, PK; Phillips, MA, Structural plasticity of malaria dihydroorotate dehydrogenase [3] J. Biol. Chem., 2009, 284, 26999-27009). Accordingly, the present inventors have extensively utilized methods and techniques in the field of computational drug design, medicinal chemistry, and molecular biology, and have found a number of heterocyclic dihydro five-membered ketone derivatives that meet the above structural requirements. These derivatives are completely different from previously described high potency plasmodium falciparum DHODH inhibitors in terms of the framework. Some of the compounds have significant inhibitory effects against Plasmodium cell lines and immunological effects at the cellular level and are promising as drugs. Based on the leading compounds of this series, the inventors have designed and synthesized a series of dihydro five-membered heterocyclic ketone derivatives, and the structural formula is shown as follows:

in which
X _{1 is selected} from O, S, NH and CH ₂ ;
X _{2 is selected} from O, S, NH, NOH and C ₁ -C ₆ -imino;
R ^{1 is selected} from H, C ₁ -C ₁₀ alkyl, unsaturated hydrocarbon radical containing not more than 10 carbon atoms, optionally substituted aryl, nitro, amino, NR ⁴ R ⁵ and halogen;
R ^{2 is} H, C ₁ -C ₆ -alkyl, unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -alkylaminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy, C ₃ -C ₈ -cycloalkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkoxycarbonyl is selected;
R ^{3 is} H, C ₁ -C ₆ -alkyl, optionally substituted unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -aminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy;
R ⁴ and R ^{5 are} independently selected from H, C ₁ -C ₆ alkyl, -C (O) NHR ⁶ , C ₁ -C ₆ alkoxycarbonyl, halo-substituted alkyl, unsaturated C ₂ -C ₆ hydrocarbyl, optionally substituted aryl, C ₁ -C ₆ alkylcarbonyl, optionally substituted benzoyl, an optionally substituted heterocyclic group, optionally substituted heterocycliccarbonyl and C ₁ -C ₆ -alkoxycarbonyl radicals;
R ^{6 is selected} from optionally substituted aryl and an optionally substituted heterocyclic group.

wobei
X₁ aus O, S, NH und CH₂ ausgewählt ist;
R² aus H, C₁-C₆-Alkyl, ungesättigtem C₂-C₆-Kohlenwasserstoffrest, C₁-C₆-Alkylcarbonyl, gegebenenfalls substituiertem Benzoyl, Carboxy, Aminocarbonyl, C₁-C₆-Alkoxycarbonyl, C₁-C₆-Alkylaminocarbonyl, Hydroxy, C₁-C₆-Alkoxy, C₃-C₈-Cycloalkylaminocarbonyl, C₃-C₈-Cycloalkyl-C₁-C₆-alkylaminocarbonyl, C₃-C₈-Cycloalkyl-C₁-C₆-alkoxycarbonyl ausgewählt ist;
R⁷ aus gegebenenfalls substituiertem Aryl, gegebenenfalls substituiertem Arylcarbonyl, gegebenenfalls substituiertem stickstoffhaltigen Heterocyclocarbonyl, einer gegebenenfalls substituierten heterocyclischen Gruppe und gegebenenfalls substituiertem Aryloxy und C₁-C₃-Alkylcarbonyl ausgewählt ist.In one embodiment, the compounds are selected from compounds of formula II:

in which
X _{1 is selected} from O, S, NH and CH ₂ ;
R ^{2 is} H, C ₁ -C ₆ -alkyl, unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -alkylaminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy, C ₃ -C ₈ -cycloalkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkoxycarbonyl is selected;
R ^{7 is selected} from optionally substituted aryl, optionally substituted arylcarbonyl, optionally substituted nitrogen-containing heterocyclocarbonyl, an optionally substituted heterocyclic group and optionally substituted aryloxy and C ₁ -C ₃ -alkylcarbonyl.

As used herein, "alkyl" refers generally to a saturated straight or branched alkyl of 1 to 10 carbon atoms, preferably alkyl of 1 to 6 carbon atoms, more preferably alkyl of 1 to 4 or 1 to 3 carbon atoms. "Cycloalkyl" refers to cyclic alkyl, which generally has from 3 to 8 ring carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, cyclohexyl and the like.

"Unsaturated hydrocarbon radical" here includes alkenyl and alkynyl. "Alkenyl" means a straight or branched chain of 2 to 10 carbon atoms containing at least one double bond between two carbon atoms in the chain. Alkenyl is preferably an alkenyl group containing from 2 to 4 carbon atoms. Typically, alkenyl includes ethenyl, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl and 2-butenyl.

The term "alkynyl" as used herein means a straight or branched chain of 2 to 10 carbon atoms containing at least one triple bond between two carbon atoms in the chain. Alkynyl is preferably an alkynyl group containing from 2 to 4 carbon atoms. Typically, alkynyl includes ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl and 2-butynyl.

"Aryl" as used herein refers to a monocyclic, bicyclic or tricyclic aromatic group containing from 6 to 14 carbon atoms; these include phenyl, naphthyl, phenanthryl, anthryl, indenyl, fluorenyl, tetralin, indanyl and the like. Aryl may optionally be substituted with 1 to 5 (eg 1, 2, 3, 4 or 5) substituents selected from: halogen, C ₁ -C ₄ aldehyde group, straight or branched C ₁ -C ₅ - alkyl, cyano, nitro, amino, hydroxy, hydroxymethyl, halo-substituted alkyl (eg. trifluoromethyl), halo-alkoxy (eg. trifluoromethoxy), carboxy, C ₁ -C ₄ alkoxy, C ₁ -C ₄ -Methylthiol, -SF ₅ , morpholinyl, optionally substituted aryl (e.g., optionally substituted phenyl), optionally substituted aryloxy (e.g., optionally substituted phenoxy) and optionally substituted benzyloxy. For example, aryl may be substituted with 1 to 3 groups selected from: fluoro, chloro, bromo, C ₁ -C ₄ alkyl, trifluoromethyl, morpholinyl, methoxy, phenyl, methoxy-substituted phenyl, phenoxy, benzyloxy, halo-substituted benzyloxy, ethoxy , Nitro and the like.

The term "heterocyclic group" as used herein means a single or fused ring structure which may be aromatic or non-aromatic and preferably contains 3-20 ring atoms, more preferably 5-14 ring atoms, at least one and preferably up to four ring atoms being heteroatoms are selected from O, S and N. Examples of heterocyclic groups are furyl, thienyl, pyrrolyl, pyrrolidinyl, imidazolyl, triazolyl, thiazolyl, tetrazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzothiazolyl, benzoxazolyl, benzothienyl, Benzofuranyl, morpholinyl, carbazolyl, dibenzothiophene, coumarin and 1,2-methylenedioxyphenyl. The heterocyclic group may optionally be substituted by 1 to 3 substituents as described herein.

The term "heteroatom" as used herein includes O, S, and N. When the heteroatom is N, the N atom may be further substituted with a group, for example, hydrogen or C ₁ -C ₁₀ alkyl. When the heteroatom is S, the S atom may be further substituted with a group, for example, C ₁ -C ₁₀ alkyl.

The term "heteroaryl" or "aromatic heterocyclic group" as used herein means a heterocyclic group having aromatic properties as described above; these include, but are not limited to, furyl, thienyl, pyrrolyl, pyridyl, oxazolyl, pyrazinyl, pyridazinyl, pyrimidinyl and the like.

The term "halogen" as used herein includes fluoro, chloro, bromo and iodo.

Unless otherwise indicated, the term "optionally substituted" used herein means that the group modified by this term may optionally be substituted with 1 to 5 (generally 1, 2 or 3) substituents selected from: C ₁ - C ₄ alkyl, carboxy, halo, C ₁ -C ₄ alkoxy, cyano, nitro, amino, hydroxy, aldehyde group, C ₁ -C ₆ acyl, hydroxymethyl, halo-substituted C ₁ -C ₄ alkyl (e.g. Trifluoromethyl), C ₁ -C ₁₀ thioalkyl (e.g., pentafluorothiomethyl), C ₁ -C ₁₀ thioalkoxy (e.g., pentafluorothiomethoxy), halo-substituted C ₁ -C ₄ alkoxy (e.g., trifluoromethoxy), thiol - and C ₁ -C ₄ acyl.

In this case, amido (aminocarbonyl) itself or as part of another group refers to a "C ₁ -C ₆ -alkyl-CO-NH" group, "C ₃ -C ₈ -cycloalkyl-CO-NH-" or "C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkyl-CO-NH- ". Exemplary amido groups include formamido, acetamido, propionamido, butyramido, cyclopropylamido, cyclopropylmethylamido, and the like.

In this case, an acyl group itself or as part of another group can contain 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. Exemplary acyl groups include formyl, acetyl, and the like.

Preferred compounds of the invention are the compounds wherein X ₁ = S or O and X ₂ = 0.

Preferred compounds of the invention are the compounds wherein R ³ = H.

In some embodiments, compounds 14-21 are not among the compounds of the invention.

Preferred compounds of the invention are the compounds wherein R ² = C ₁ -C ₅ alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, cyclopropoxycarbonyl, cyclopropylmethoxycarbonyl, etc.), C ₁ -C ₅ -aminocarbonyl (e.g., formamido, Acetamido, propionamido, butyramido, cyclopropylamido, cyclopropylmethylamido, etc.).

Preferred compounds of the invention are the compounds in which R ¹ = -NH-optionally-substituted-phenyl, -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralin or NH-optionally substituted-quinolyl.

Preferred compounds of the invention are the compounds wherein the substituents on the optionally substituted phenyl or naphthyl in R ^{1 are} halo, halo-substituted C ₁ -C ₄ alkyl, C ₁ -C ₄ alkyl or C ₁ -C ₄ alkoxy.

In a preferred embodiment, the present invention comprises the compounds wherein X _{1 is} S or O, X _{2 is} O, R ^{3 is} H, R ^{2 is} C ₁ -C ₅ alkoxycarbonyl (eg methoxycarbonyl, Ethoxycarbonyl, cyclopropyloxycarbonyl, cyclopropylmethoxycarbonyl, etc.), C ₁ -C ₅ -aminocarbonyl (eg, formamido, acetamido, propionamido, butyramido, cyclopropylamido, cyclopropylmethylamido, etc.), R ¹ = -NH-optionally-substituted-phenyl, NH is optionally substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralinic or -NH-optionally-substituted-quinolyl. The substituents on the optionally substituted phenyl or naphthyl in R ¹ are particularly preferably halogen, halogen-substituted C ₁ -C ₄ -alkyl, C ₁ -C ₄ -alkyl or C ₁ -C ₄ -alkoxy.

In a particularly preferred embodiment, X ₁ = S or O, X ₂ is O, R ³ is H, R ² is C ₁ -C ₆ alkoxycarbonyl (eg, methoxycarbonyl, ethoxycarbonyl, etc.), R ¹ is -NH optionally substituted-phenyl, -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralinic or -NH-optionally-substituted-quinolyl; Phenyl is preferably unsubstituted or has 1 to 3 substituents selected from halogen, trifluoromethyl, methyl, nitro and methoxy. Preferably, the substituent is at the 3, 4 and / or 5 position. Preferably, compounds 14-21 are not included.

In a particularly preferred embodiment, X ₁ = S or O, X ₂ is O, R ³ is H, R ² is C ₂ -C ₄ alkoxycarbonyl (e.g., ethoxycarbonyl, cyclopropyloxycarbonyl, cyclopropylmethoxycarbonyl, etc.), R ¹ -NH-optionally-substituted-phenyl, -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralinic or -NH-optionally-substituted-quinolyl; Phenyl is preferably unsubstituted or has 2 to 3 substituents selected from halogen and C ₁ -C ₄ alkyl. Preferably, compounds 14-21 are not included.

In a particularly preferred embodiment, the compounds of the invention include the compounds wherein R ^{1 is} -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralinic or -NH-optionally-substituted -Chinolyl is.

Preferred compounds of the invention are those of the number 1-67, as shown in the following Table 1, and particularly preferred are those having an inhibition rate of more than 50%. In particular, the compounds of the invention comprise compounds 1, 2, 4, 6, 7, 10, 22-25, 35, 38, 39, 44, 61 and 62.

The compounds of the invention can be prepared by the following method:

In the above scheme, R ^{1 is} a substituent on phenyl in the final product, the definition of which is the same as that of the substituents on aryl, as defined above. In accordance with the actual requirements of the preparation, the person skilled in the art can prepare the compounds of the invention using various conventionally obtained compounds as starting material. For example, a compound of formula II of the present invention may be prepared based on the method described by Du Xiaohua et al. Peaches 2004, 43 (2), 78-79).

In the above scheme, R ^{1 is} a substituent on phenyl in the final product, the definition of which is the same as that of the substituents on aryl, as defined above. The definition of R ² is the same as that of the C ₁ -C ₆ -aminocarbonyl as defined above.

In the above scheme, R ^{1 is} a substituent on phenyl in the final product, the definition of which is the same as that of the substituents on aryl, as defined above. The definition of R ³ is the same as that of the C ₁ -C ₅ -aminocarbonyl as defined above.

In the above scheme, R ^{1 is} a substituent on phenyl in the final product, the definition of which is the same as that of the substituents on aryl, as defined above.

In the second aspect of the invention there is provided a pharmaceutical composition, the composition comprising a therapeutically effective amount of compounds of formula I or II of the Invention or a pharmaceutically acceptable salt thereof, as well as a pharmaceutically acceptable carrier or excipient.

Examples of the pharmaceutically acceptable salt of compounds of the invention include inorganic salts and organic salts, for example hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; and inorganic and organic salts formed with a base such as sodium hydroxide, tris (hydroxymethyl) aminomethane (IRIS, tromethamine) and N-methylglucamine.

One skilled in the art can determine the optimal dosage of each active ingredient in the pharmaceutical compositions of the invention, although the needs are individually different. In general, the compounds of the present invention or a pharmaceutically acceptable salt thereof are orally administered to a mammal at a daily dose of about 0.0025 to 50 mg / kg of body weight and are preferably orally administered at a daily dose of about 0.01 to 10 mg / kg of body weight , For example, an oral dosage unit may comprise about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of compounds of the invention. A unit dose may be administered one or more times a day or more, each tablet containing about 0.1 to 50 mg, preferably about 0.25 to 10 mg of the compounds of the invention or a solvate thereof.

The pharmaceutical composition of the invention can be suitably prepared into forms for various routes of administration; These include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical administration for the treatment of cancer and other diseases. The administration amount is the amount that effectively relieves or heals one or more diseases. For the treatment of a particular disease, the effective amount is that amount sufficient to alleviate or mitigate disease-related symptoms. Such an amount may be administered in a single dose or according to an effective therapeutic regimen. The amount of administration can cure diseases effectively, but usually the purpose is to alleviate symptoms of disease. In general, administration must be repeated to achieve relief of the symptoms. The dose may be determined according to the age, health status and weight of the patient, concurrent treatments, frequency of treatment, and desired therapeutic benefit.

The pharmaceutical preparations of the invention may be administered to any mammal as long as the compounds of the present invention are effective on them. Among the mammals, humans are the most important.

The compound or pharmaceutical composition of the invention can be used to treat and prevent various diseases mediated by DHODH, particularly as an inhibitor in diseases related to DHODH. Disorders mediated by DHODH include: parasitic diseases including malaria tropica, malaria tertiana, malaria ovale, malaria quartana, gram trypanosomiasis, dengue fever and other parasitic diseases, diseases caused by rapid proliferation of certain cells, such as cancer; Inflammation; and various autoimmune diseases. DHODH mediated diseases include rejection induced by allo- and xeno-organ transplantation in a host.

In particular, DHODH mediated diseases include, but are not limited to, Gram trypanosomiasis, malaria tropica, dengue fever, malaria tertiana, malaria ovale, malaria quartana, rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus (including systemic lupus erythematosus), glomerulopathy (including secondary glomerulopathy and primary glomerulopathy ), Rejection of organ transplants, melanomas, psoriatic arthritis and psoriasis.

The pharmaceutical preparations of the invention can be prepared in a known manner, for example by conventional mixing, granulation, tabletting, dissolving or lyophilization processes. When preparing an oral preparation, a solid excipient and an active compound may be combined together, and the mixture may optionally be ground. If necessary, suitable additives may be added and the mixture may be made into particles to obtain tablets or pastille seeds.

Suitable excipients, in particular fillers, include, for example, sugars, such as lactose or sucrose, mannitol or sorbitol, cellulose preparations or calcium phosphates, such as Tricalcium phosphate or calcium hydrogen phosphate; and a binder such as starch paste including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose or polyvinylpyrrolidone. If desired, a disintegrants such as the above-mentioned starches and carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate may be added. Adjuvants include in particular flow modifiers and lubricants such as silica, talc, stearates such as magnesium and calcium stearate, stearic acid or polyethylene glycol. If necessary, the pastille core may be suitably coated so that it is enteric-coated. For this purpose, concentrated saccharide solutions may be applied, which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. For the production of enteric coatings, suitable cellulose solutions may be used, such as cellulose acetate phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments may be added to the tablets or the coating of the pastille seeds, for example, to identify or characterize the dosage combinations of active ingredients.

Accordingly, the third aspect of the invention provides a method of treating or preventing DHODH-mediated diseases, which comprises administering compound I or compound II or the pharmaceutical composition of the invention to a patient in need of such administration.

Methods of administration include, but are not limited to, those methods known in the art which can be determined according to the condition of the patient; These include, but are not limited to, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical administration.

The invention also provides the use of Compound I or Compound II of the present invention for the manufacture of a medicament for the treatment or prevention of DHODH-mediated diseases.

The invention also provides the use of Compound I or Compound II of the present invention for the manufacture of a medicament for the treatment or prevention of Plasmodium DHODH-mediated diseases.

The invention also provides the use of Compound I or Compound II of the present invention for the manufacture of a medicament for inhibiting the activity of Plasmodium DHODH.

Preferably, compounds 1-4, 6, 7, 10, 12, 16-18, 20-25, 35, 38, 39, 44, 61 and 62 are used in the treatment and prevention methods and uses described above.

Detailed description of the invention

The following examples further illustrate the present invention. These examples are intended to illustrate but not limit the invention in any way.

Synthesis:

General principle for the synthesis of ethyl 2-anilino-4-carbonyl-4,5-dihydrothiophene-3-carboxylate:

Synthesis: 3 mmol of substituted aniline was dissolved in 3 ml of acetone, and then 9 mmol of triethylenediamine was added, and 15 ml of carbon disulfide was added dropwise thereto. The resulting mixture was stirred at room temperature for 24 hours. A yellow solid precipitated and the solid was filtered off and dried to give an intermediate. The intermediate was placed directly in a flask, and then 10 ml of chloroform was added to the flask to obtain a suspension. Into the suspension was added 10 ml of chloroform solution comprising 1 mmol of triphosgene and reacted overnight. The mixture was subjected to vacuum filtration to give a mother liquor. The Mother liquor was concentrated under reduced pressure, and by column chromatography, substituted phenylisothiocyanate was obtained in a yield of about 45%.

General Principle for the Synthesis of Ethyl 2-Anilino-4-Carbonyl-4,5-Dihydrothiophene-3-Carboxylate (Faull WA; Hull R; Some Reactions of Ethyl 2-Anilino-4-Oxo-4,5-Dihydrothiophene) 3-carboxylates [3], J. Chem. Society, Perkin 1981, 1078-1082):

Synthesis: To 0.55 mmol of freshly prepared solid sodium methoxide and 10 ml of dry DME was added 0.55 mmol of 4-chloroethyl acetoacetate and the resulting mixture was stirred for 1 hour at room temperature. In 10 ml of DME, 0.5 mmol of substituted phenyl isothiocyanate was dissolved, and the resulting solution was slowly added dropwise under argon. The reaction was carried out for 6 hours and then stopped. DME was removed under reduced pressure. The residue was neutralized with dilute hydrochloric acid, washed with saturated brine and extracted with ethyl acetate. The organic layer was concentrated and the product was purified by column chromatography. In the above scheme, R is a substituent on phenyl in the final product, the definition of which is the same as that of the substituents on aryl, as defined above.

Synthesis: In an ice bath, 3 mmol of ethyl 2- (4- (trifluoromethyl) anilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate in a solvent mixture of methanol / water = 5: 1 (total 18 ml) dissolved, and then lithium hydroxide monohydrate (5 eq.) was added to the resulting solution. The resulting mixture was stirred for half an hour and then the ice bath was removed. Then the reaction mixture was heated to 60 ° C and kept at this temperature overnight. Methanol was removed under reduced pressure. The residue was adjusted to pH = 2 by addition of a little water and 10% dilute hydrochloric acid, a large amount of white solid precipitated, the solid was filtered and dried to give the product.

Synthesis: To a round bottom flask was added the corresponding dried acid (1 mmol, 285 mg) and substituted amines (1.2 mmol) and then 5 mL dichloromethane, HOBt (1.2 mmol, 260 mg) and EDC (1, 2 mmol, 330 mg). The resulting mixture was stirred overnight at room temperature and TLC showed that the starting material was still present, but apparently a new spot appeared. The mixture was washed by successively using a saturated ammonium chloride solution, a saturated sodium carbonate solution and brine. The organic layer was concentrated, dried and purified by column chromatography (PE: EA = 5: 1, v / v) to give a pale yellow solid.

Synthesis: Dried acid (1 mmol), alcohol (1 mmol) and triphenylphosphine (1.1 mmol) were added in 5 mL of toluene, and then diethyl azodicarboxylate (1.2 mmol) was added dropwise under argon. The reaction was carried out for half an hour, and then the mixture was raised to 50 ° C heated and reacted overnight. The mixture was washed with brine and purified by column chromatography (PE: EA = 3: 1, v / v) to give a pale yellow solid.

Synthesis: To a 50 ml flask was added 3 mmol of 60% sodium hydroxide (about 240 mg) and 1.8 ml of anhydrous THF. In an ice bath, 1.8 ml of anhydrous THF solution of diethyl malonate (6 mmol) was added dropwise. After 10 minutes, 3 ml of chloroacetyl chloride solution (3 mmol) was added dropwise. The mixture was kept in an ice bath for one hour, and then the reaction was carried out at 40-45 ° C for 1 hour. Substituted arylamine was added dropwise at room temperature. The reaction was carried out overnight at room temperature and TLC showed that little starting material was left. After 2 hours of reflux, DC showed that there was still starting material. The reaction was stopped. After the pH was adjusted to 7 with dilute HCl, the mixture was extracted with ethyl acetate and washed twice with brine. The organic layer was concentrated, dried and purified by column chromatography (PE: EA = 2: 1, v / v) to give a pure product as a white powder, yield 35-40%.

Spectrum of the target compound

Ethyl 2-anilino-4-carbonyl-4,5-dihydrothiophene-3-carboxylate

Ethyl 2- (4-chloroanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 1)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.12 (s, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 4.25-4.19 (q, J = 7.2 Hz, 2H), 3.68 (s, 2H), 1.25 (t, J = 7.2 Hz , 3H); HRMS (ESI) calcd for C ₁₃ H ₁₂ ClNO ₃ S [M + H ⁺ ] 298.0305, found 298.0307;

Ethyl 2- (4-trifluoromethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 2)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.34 (s, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 4.26-4.21 (q, J = 7.2 Hz, 2H), 3.74 (s, 2H), 1.26 (t, J = 7.2 Hz , 3H); HRMS (ESI) calculated for C ₁₄ H ₁₂ F ₃ NO ₃ S [M + H ^+] 332.0568, found 332.0559;

Ethyl 2- (4-bromo-3-methylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 3)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.91 (s, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H), 4.24-4.19 (q, J = 6.8 Hz, 2H), 3.32 (s, 2H), 2.22 (s, 3H), 1.25 (t, J = 6.8 Hz, 3H); HRMS (ESI) calcd for C ₁₄ H ₁₄ BrNO ₃ S [M + H ⁺ ] 355.9956, found 355.9955;

Ethyl 2- (3,5-dichloroanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 4)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.12 (s, 1H), 7.66 (s, 1H), 7.61 (s, 2H), 4.25-4 , 20 (q, J = 7.2 Hz, 2H), 3.71 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H); HRMS (ESI) calcd for C ₁₃ H ₁₁ C ₁₂ NO ₃ S [M + H ⁺ ] 331.9915, found 331.9916;

Ethyl 2- (4-methyl-3-fluoroanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 5)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.10 (s, 1H), 7.39 (t, J = 8.4 Hz, 1H), 7.30 (d, J = 10.8 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 4.25-4.19 (q, J = 7.2 Hz, 2H), 3.68 (s , 2H), 2.26 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H); HRMS (ESI) calculated for C ₁₄ H ₁₄ FNO ₃ S [M + H ^+] 296.0757, found 296.0757;

Ethyl 2- (3,4-methylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 6)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.36 (s, 1H), 7.21 (d, J = 7.6 Hz, 1H), 7.13-7.10 (m, 2H), 4.42-4.37 (q, J = 7.2Hz, 2H), 3.63 (s, 2H), 2.31 (s, 6H), 1.42 (t, J = 7.2 Hz, 3H); HRMS (ESI) calculated for C ₁₅ H ₁₇ NO ₃ S [M + H ^+] 292.1007, found 292.1007;

Ethyl 2- (9-ethyl-9H-carbazolylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 7)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 11.42 (s, 1H), 8.10 (d, J = 7.6 Hz, 2H), 7.55 (t, J = 7 , 2Hz, 1H), 7.48-7.41 (m, 3H), 7.30 (d, J = 7.2Hz, 1H), 4.46-4.39 (m, 4H), 3 , 64 (s, 2H), 1.51-1.48 (m, 6H); HRMS (ESI) calculated for C ₂₁ H ₂₀ N ₂ O ₃ S [M + H ^+] 381.1273, found 381.1274;

Ethyl 2- (4-bromo-3-trifluoromethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 8)

• ¹ H-NMR (400 MHz, DMSO-d ₅ ) δ (ppm): 11.66 (s, 1H), 7.73 (s, 1H), 7.61 (d, J = 8.4 Hz, 1H ), 7.53 (t, J = 8.4 Hz, 1H), 4.43-4.37 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H); HRMS (ESI) calculated for C ₁₄ H ₁₁ BrF ₃ NO ₃ S [M + H ^+] 409.9673, found 409.9675;

Ethyl 2- (4-chloro-3-trifluoromethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 9)

• ¹ H-NMR (400 MHz, DMSO-d ₅ ) δ (ppm): 11.67 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H ), 7.45 (d, J = 8.4Hz, 1H), 4.43-4.36 (q, J = 6.8Hz, 2H), 3.71 (s, 2H), 1.42 (t, J = 6.8 Hz, 3H); HRMS (ESI) calculated for C ₁₄ H ₁₁ ClF ₃ NO ₃ S [M + H ^+] 366.0179, found 366.0177;

Ethyl 2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 10)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.34 (s, 1H), 8.05-7.96 (m, 4H), 7.60-7.54 (m, 3H), 4.28-4.22 (q, J = 7.2Hz, 2H), 3.70 (s, 2H), 1.27 (t, J = 7.2Hz, 3H); HRMS (ESI) calculated for C ₁₇ H ₁₅ NO ₃ S [M + H ^+] 314.0851, found 314.0849;

Ethyl 2- (β-anthrylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 11)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 8.46 (d, J = 12.8 Hz, 2H), 8.10 (d, J = 9.2 Hz, 1H), 8.04 (s, 2H), 8.02 (s, 1H), 7.53 (t, J = 4.8 Hz, 2H), 7.40 (d, J = 8.4 Hz, 1H), 4.44 (q, J = 7.2Hz, 2H), 3.76 (s, 2H), 1.45 (t, J = 7.2Hz, 3H). HRMS (ESI) calculated for C ₂₁ H ₁₈ NO ₃ S [M + H ^+] 364.1007, found 364.1005;

Ethyl 2- (anilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 12)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 7.47 (t, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.34 (s, 1H), 4.39 (q, J = 7.2Hz, 2H), 3.67 (s, 2H), 1.42 (t, J = 7.2Hz, 3H) HRMS (ESI) calculated for C ₁₃ H ₁₄ NO ₃ S [M + H ^+] 264.0694, found 264.0694;

Ethyl 2- (6-dibenzothiophenamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 13)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.27 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.42 (dd, J ₁ = 2.0 Hz, J ₂ = 1.6 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.08 (dd, J ₁ = 1.6 Hz, J ₂ = 2.4 Hz, 1H), 7.56 (m, 3H), 4.25 (q, J ₁ = 7.2 Hz, 2H), 3.67 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H) HRMS (ESI) calculated for C ₁₉ H ₁₆ NO ₃ S ₂ [M + H ^+] 370.0572, found 370.0572;

Ethyl 2- (2,3-dihydro-1H-indenylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 22)

¹ H-NMR (400 MHz, DMSO-d ₅ ): δ (ppm): 11.08 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 7.16 (d, J = 8.0 Hz, 1H), 4.22 (q, J = 7.2 Hz, 2H), 3.65 (s, 2H), 2.89 (t, J = 7.6Hz, 4H), 2.09-2.02 (m, 2H), 1.26 (t, J = 7.2Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₇ NO ₃ S [M + H ^+] 304.1007, found 304.1009.

Ethyl 2- (4-pentafluorothio) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 23)

• ¹ H NMR (400 MHz, DMSO-d ₆ ): δ 11.38 (s, 1H), 8.03 (d, J = 9.2 Hz, 2H), 7.69 (d, J = 8.4 Hz, 2H), 4.25 (q, J = 6.8 Hz, 2H), 3.76 (s, 2H), 1.26 (t, J = 6.8 Hz, 3H). HRMS (ESI) calculated for C ₁₃ H ₁₂ F ₅ NO ₃ S ₂ [M + H ⁺ ] 390.0262, found 390.0257.

Ethyl 2- (3-aminoquinoline) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 24)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.31 (s, 1H), 8.92 (d, J = 2.4 Hz, 1H), 8.52 (d, J = 2.4 Hz, 1H), 8.08 (t, J = 8.4 Hz, 2H), 7.87-7.82 (m, 1H), 8.08 (t, 3 = 7.6 Hz , 1H), 4.26 (q, J = 6.8Hz, 2H), 3.73, (s, 2H), 1.28 (t, J = 6.8Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₅ N ₂ O ₃ S [M + H ^+] 315.0803, found 315.0806.

Ethyl 2- (2- (5,6,7,8-tetrahydronaphthalenes) amino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 25)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.06 (s, 1H), 7.17-7.13 (m, 2H), 7.12 (s, 1H), 4 , 22 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 2.74 (t, J = 9.6 Hz, 4H), 1.75 (t, J = 3, 2 Hz, 4H), 1.26 (t, J = 7.0 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₉ NO ₃ S [M + H ^+] 318.1164, found 318.1168.

Ethyl 2- (4-tert-butylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 26)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 11.44 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 9 , 6Hz, 2H), 4.40 (q, J = 6.8Hz, 2H), 3.66 (s, 2H), 1.43 (t, J = 6.8Hz 3H), 1.36 (s, 9H). HRMS (ESI) calcd for C ₁₇ H ₂₁ NO ₃ S [M + H ⁺ ] 320.1320 found 320.1318.

2- (4-trifluoromethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropylmethyl) formamide (Compound 27)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.81 (s, 1H), 8.89 (t, J = 5.2 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 3.95 (s, 2H), 3.17 (t, J = 6.4 Hz, 2H), 1.08-0.98 (m, 1H), 0.46 (d, J = 6.8Hz, 2H), 0.23 (d, J = 4.8Hz, 2H). HRMS (ESI) calculated for C ₁₆ H ₁₅ F ₃ N ₂ O ₂ S [M + H ^+] 357.0885, found 357.0885

2- (4-trifluoromethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropyl) formamide (Compound 28)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.73 (s, 1H), 8.75 (t, J = 3.6 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 3.94 (s, 2H), 2.80-2.74 (m, 1H), 0.75 -0.73 (m, 2H), 0.50 (d, J = 1.6 Hz, 2H). HRMS (ESI) calculated for C ₁₅ H ₁₃ F ₃ N ₂ O ₂ S [MH ^+] 341.0572, found 341.0565

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropylmethyl) formamide (Compound 29)

• ¹ H-NMR (400 MHz, DMSO-d ₅ ) δ (ppm): 12.34 (s, 1H), 8.88 (t, J = 9.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J ₁ = 2.4 Hz, 32 = 8.0 Hz, 1H), 3.85 (s, 2H) , 3.15 (t, J = 6.0 Hz, 2H), 2.24 (d, J = 3.2 Hz, 6H), 1.02-0.97 (m, 1H), 0.48- 0.43 (m, 2H), 0.23-0.19 (m, 2H). HRMS (ESI) calculated for C ₁₇ H ₂₀ N ₂ O ₂ S [M + H ^+] 317.1316, found 317.1324.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropyl) formamide (Compound 30)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.27 (s, 1H), 8.75 (d, J = 7.6 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.15 (dd, J ₁ = 2.0 Hz, 3 ₂ = 8.0 Hz, 1H), 3.83 (s, 2H), 2.77-2.73 (m, 1H), 2.24 (d, J = 1.6Hz, 6H), 0.75-0.70 (m, 2H) , 0.50-0.48 (m, 2H). HRMS (ESI) calculated for C ₁₆ H ₁₈ N ₂ O ₂ S [M + H ^+] 303.1158, found 303.1167.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-formamide (compound 31)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.92 (s, 1H), 8.92 (s, 1H), 7.88 (s, 1H), 7.26 (i.e. , J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.06 (dd, J ₁ = 2.0 Hz, J ₂ = 8.0 Hz, 1H), 3.81 (s , 2H), 2.25 (s, 6H). HRMS (ESI) calculated for C ₁₃ H ₁₄ N ₂ O ₂ S [M + H ^+] 263.0853, found 263.0854.

2- (2,3-dihydro-1H-indenylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropylmethyl) formamide (Compound 32)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.33 (s, 1H), 8.88 (t, J = 5.2 Hz, 1H), 7.33-7.26 (m, 2H), 7.16 (d, J = 8.0Hz, 1H), 3.85 (s, 2H), 3.15 (t, J = 6.4Hz, 2H), 2.88 (q, J = 6.8Hz, 4H), 2.09-2.02 (m, 2H), 1.03-0.97 (m, 1H), 0.45 (d, J = 7.6 Hz, 2H), 0.22 (d, J = 4.8 Hz, 2H). HRMS (ESI) calculated for C ₁₈ H ₂₀ N ₂ O ₂ S [M + H ^+] 329.1342, found 329.1315.

2- (2,3-Dihydro-1H-indenylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropyl) -formamide (Compound 33)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.27 (s, 1H), 8.76 (s, 1H), 7.34-7.28 (m, 2H), 7 , 17 (d, J = 8.0 Hz, 1H), 3.83 (s, 2H), 2.89 (d, J = 5.6 Hz, 4H), 2.75 (d, J = 3, 6 Hz, 1H), 2.05 (t, J = 7.2 Hz, 1H), 2.05 (t, J = 7.2 Hz, 2H), 0.73 (d, J = 5.6 Hz , 2H), 0.49 (s, 2H). HRMS (ESI) calculated for C ₁₇ H ₁₈ N ₂ O ₂ S [M + H ^+] 315.1167, found 315.1174

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropylmethyl) formamide (Compound 34)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.92 (s, 1H), 8.89 (s, 1H), 7.91 to 7.82 (m, 4H), 7.56 -7.42 (m, 3H), 3.73 (s, 2H), 3.28 (t, J = 6.4Hz, 2H), 1.07 (m, 1H), 0.59-0, 54 (m, 2H), 0.31-0.27 (m, 2H). HRMS (ESI) calculated for C ₁₉ H ₁₈ N ₂ O ₂ S [M + Na ^+] 361.0987, found 361.0997

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclopropyl) formamide (Compound 35)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.92 (s, 1H), 8.89 (s, 1H), 7.92 to 7.83 (m, 4H), 7.54 -7.43 (m, 3H), 3.73 (s, 2H), 2.83 (s, 1H), 0.90-0.84 (m, 2H), 0.67-0.63 (m , 2H). HRMS (ESI) calculated for C ₁₈ H ₁₆ N ₂ O ₂ S [M + Na ^+] 347.0830, found 347.0834.

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-formamide (Compound 36)

• ¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 10.47 (s, 1H), 7.96 (m, 4H), 7.62 (dd, J ₁ = 2.0 Hz, J ₂ = 8.8 Hz, 1H), 7.54 (m, 2H), 4.75 (s, 2H), 4.25 (q, J = 6.8 Hz, 2H), 1.28 (t, J = 6.4 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₅ NO ₄ [M + Na ^+] 320.0899, found 320.0897.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-ethyl) formamide (Compound 37)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.38 (s, 1H), 8.77 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 8 , 0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J ₁ = 2.0 Hz, J ₂ = 7.6 Hz, 1H), 3.84 (s, 2H), 3.27 (q, J = 6.8Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.11 (t, J = 7.2Hz, 3H). HRMS (ESI) calculated for C ₁₅ H ₁₈ N ₂ O ₂ S [M + H ^+] 291.1167, found 291.1169.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-propyl) formamide (Compound 38)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.36 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J ₁ = 2.0 Hz, J ₂ = 7.6 Hz, 1H), 3.85 (s, 2H ), 3.23 (q, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.53-1.48 (m, 2H), 0 , 89 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₂₀ N ₂ O ₂ S [M + Na ^+] 327.1143, found 327.1134.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3- (N-butyl) formamide (Compound 39)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.36 (s, 1H), 8.80 (t, J = 5.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.15 (dd, J ₁ = 2.0 Hz, J ₂ = 7.6 Hz, 1H), 3.84 (s, 2H ), 3.27 (q, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.51-1.44 (m, 2H), 1 , 37-1.28 (m, 3H), 0.91 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₂₂ N ₂ O ₂ S [M + Na ^+] 341.1300, found 341.1305.

2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-ethyl) formamide (Compound 40)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 12.71 (s, 1H), 8.80 (d, J = 5.6 Hz, 1H), 8.05-7.95 (m, 4H), 7.60-7.53 (m, 3H), 3.90 (s, 2H), 3.31 (q, J = 7.2Hz, 2H), 1.12 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₆ N ₂ O ₂ S [M + H ^+] 313.1011, found 313.1022.

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-propyl) formamide (Compound 41)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.95 (s, 1H), 8.91 (s, 1H), 7.92 to 7.84 (m, 4H), 7.56 -7.29 (m, 3H), 3.75 (s, 2H), 1.66 (q, J = 6.8Hz, 2H), 1.36-1.28 (m, 2H), 1, 02 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₈ H ₁₈ N ₂ O ₂ S [M + H ^+] 327.1167, found 327.1165.

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-butyl) formamide (Compound 42)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.94 (s, 1H), 8.89 (s, 1H), 7.93 to 7.84 (m, 4H), 7.58 -7.44 (m, 3H), 3.75 (s, 2H), 1.66-1.59 (m, 2H), 1.45 (q, J = 7.2 Hz, 2H), 1, 36-1.28 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₉ H ₂₀ N ₂ O ₂ S [M + H ^+] 341.1324, found 341.1320.

2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylic acid (compound 43)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 7.26 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 4.02 (s, 2H), 2.26 (s, 6H). HRMS (ESI) calculated for C ₁₃ H ₁₃ NO ₃ S [M + H ^+] 264.0694, found 264.0690

2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylic acid (Compound 44)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.55 (s, 1H), 8.08-7.89 (m, 4H), 7.62-7.40 (m, 3H), 4.07 (s, 2H), HRMS (ESI) calculated for C ₁₅ H ₁₁ NO ₃ S [M + H ^+] 286.0538, found 286.0541

Methyl 2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 45)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.03 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H ), 7.15 (d, J = 8.0 Hz, 1H), 3.72 (s, 3H), 3.67 (s, 2H), 2.26 (s, 6H). HRMS (ESI) calculated for C ₁₅ H ₁₇ NO ₃ S [M + H ^+] 292.1007, found 292.1007.

Propyl 2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 46)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.07 (s, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H ), 7.15 (d, J = 8.0 Hz, 1H), 4.13 (t, J = 6.8 Hz, 2H), 3.66 (s, 2H), 2.26 (s, 6H ), 1.68-1.61 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₉ NO ₃ S [M + H ^+] 306.1164, found 306.1170

Butyl 2- (3,4-dimethylanilino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 47)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.06 (s, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H ), 7.15 (d, J = 8.0 Hz, 1H), 4.17 (t, J = 6.4 Hz, 2H), 3.65 (s, 2H), 2.25 (s, 6H ), 1.66-1.59 (m, 2H), 1.45-1.36 (m, 2H), 0.92 (t, J = 7.2Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₂₁ NO ₃ S [M + Na ^+] 342.1140, found 342.1123

Cyclopropylmethyl 2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 48)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 11.32 (s, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.13 (s, 1H), 7.10 (d, J = 8.8Hz, 1H), 4.17 (t, J = 6.8Hz, 2H), 3.64 (s, 2H), 1.30-1.27 (m , 1H), 0.61 (d, J = 6.8 Hz, 2H), 0.40 (d, J = 4.4 Hz, 2H). HRMS (ESI) calculated for C ₁₇ H ₁₉ NO ₃ S [M + H ^+] 318.1164, found 318.1168.

Methyl 2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 49)

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.30 (s, 1H), 8.06-7.97 (m, 4H), 7.66-7.54 (m, 3H), 3.76 (s, 3H), 3.71 (s, 2H). HRMS (ESI) calculated for C ₁₆ H ₁₃ NO ₃ S [M + H ^+] 300.0694, found 300.0699.

Propyl 2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 50)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 11.67 (s, 1H), 7.95 to 7.86 (m, 4H), 7.58 to 7.44 (m, 3H) , 4.13 (t, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.87-1.82 (m, 2H), 1.06 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₈ H ₁₇ NO ₃ S [M + Na ^+] 350.0827, found 350.0833.

Butyl 2- (2-naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 51)

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 11.66 (s, 1H), 7.95 to 7.86 (m, 4H), 7.57 to 7.44 (m, 3H) , 4.35 (t, J = 6.8 Hz, 2H), 3.69 (s, 2H), 1.85-1.77 (m, 2H), 1.55-1.45 (m, 2H ), 1.00 (t, J = 8.8 Hz, 3H). HRMS (ESI) calculated for C ₁₉ H ₁₉ NO ₃ S [M + Na ^+] 364.0983, found 364.0991.

Connection 52

• ¹ H-NMR (400 MHz, CDCl ₃ ) δ (ppm): 12.55 (s, 1H), 8.84 (s, 1H), 7.27 (d, J = 10.8 Hz, 1H), 7.22 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 3.70 (s, 2H), 3.42 (m, 2H), 2.94 (q, J = 7.2 Hz, 4H), 2.14 (t, J = 7.4 Hz, 2H), 1.64 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₈ N ₂ O ₂ S [M + Na] ⁺ 325.0987, found 325.0985.

Connection 53

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 12.55 (s, 1H), 8.89 (s, 1H), 7.27 (d, J = 10.8 Hz, 1H), 7.22 (s, 1H), 7.12 (d, J = 8.0 Hz, 1H), 3.71 (s, 2H), 3.35 (q, J = 7.2 Hz, 2H), 2.94 (q, J = 7.2Hz, 4H), 2.14 (m, 2H), 1.64 (m, 2H), 1.00 (t, J = 7.4Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₂₀ N ₂ O ₂ S [M + Na] ⁺ 339.1143, found 339.1137.

Connection 54

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.38 (s, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.49 (dd, J ₁ = 2.0 Hz, J ₂ = 8.4 Hz, 1H), 6.45 (s, 1H), 4.25 (q, J = 6.8 Hz, 2H), 3.76 (s, 2H), 2.47 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₅ NO ₅ S [M + H] ⁺ 346.0749, found 346.0746.

Connection 55

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.23 (s, 1H), 7.86 (s, 1H), 7.78-7.72 (m, 3H), 4 , 24 (q, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1.26 (t, J = 6.8 Hz, 3H). HRMS (ESI) calculated for C ₁₄ H ₁₂ F ₃ NO ₃ S [M + H ^+] 332.0568, found 332.0572.

Connection 56

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.14 (s, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.85 (d, J = 2.8 Hz, 1H), 7.67 (dd, J ₁ = 2.4 Hz, J ₂ = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6.59 (d, J = 9.2Hz, 1H), 4.23 (q, J = 7.2Hz, 2H), 3.69 (s, 2H), 1.26 (t, J = 7 , 2 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₃ NO ₅ S [M + H ^+] 332.0593, found 332.0603.

Connection 57

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.17 (s, 1H), 9.11 (s, 1H), 8.27 (s, 1H), 7.85 (i.e. , J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 3.84 (s, 2H) HRMS (EI) m / z calculated for C ₁₂ H ₉ F ₃ N ₂ O ₂ S [M ⁺ ] 302.0337, found 302.0338

Connection 58

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 7.88 (d, J = 8.0 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 4.04 (s, 2H). HRMS (EI) m / z calcd for C ₁₂ H ₈ F ₃ NO ₃ S [M ^+] 303.0177, found 303.0175

Connection 59

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 7.33 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.18 (d, J = 8.0 Hz, 1H), 4.00 (s, 2H), 2.87 (q, J = 7.2 Hz, 4H), 2.06-2.00 (m, 2H). HRMS (EI) m / z calcd for C ₁₄ H ₁₃ NO ₃ S [M ^+] 275.0616, found 275.0617

Connection 60

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 11.21 (s, 1H), 7.64 (s, 1H), 7.54 (d, J = 7.2 Hz, 2H ), 7.48 (t, J = 7.2 Hz, 2H), 7.43 (d, J = 7.2 Hz, 1H), 7.30-7.24 (m, 3H), 4.29 (q, J = 7.2Hz, 2H), 2.28 (s, 6H), 1.31 (t, J = 7.2Hz, 3H). HRMS (EI) m / z calcd for C ₂₂ H ₂₁ NO ₃ S [M ^+] 379.1242, found 379.1245

Connection 61

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 10.47 (s, 1H), 7.96 (m, 4H), 7.62 (dd, J ₁ = 2.0 Hz, 32 = 8.8 Hz, 1H), 7.54 (m, 2H), 4.75 (s, 2H), 4.25 (q, J = 6.8 Hz, 2H), 1.28 (t, J = 6.4 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₅ NO ₄ [M + Na ^+] 320.0899, found 320.0897.

Connection 62

• ¹ H-NMR (400 MHz, CDCl ₃₎ δ (ppm): 10.15 (s, 1H), 7.22 (s, 1H), 7.17 (s, 2H), 4.66 (s, 2H ), 4.22 (q, J = 7.1 Hz, 2H), 2.23 (s, 3H), 2.22 (s, 3H), 1.26 (t, J = 7.2 Hz, 3H ). HRMS (ESI) calculated for C ₁₅ H ₁₇ NO ₄ [M + Na ^+] 298.1055, found 298.1059.

Connection 63

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.62 (s, 1H), 8.99 (d, J = 2.4 Hz, 1H), 8.44 (d, J = 2.4 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.79-7.75 (m , 1H), 7.67-7.63 (m, 1H), 4.75 (s, 2H), 4.26 (q, J = 7.2Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₄ N ₂ O ₂ [M + Na ^+] 321.0843, found 321.0851.

Connection 64

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.16 (s, 1H), 7.15 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 1H), 4.68 (s, 2H), 4.22 (q, J = 6.8 Hz, 2H), 2.71 (s, 4H), 1.73 (s, 4H ), 1.26 (t, J = 6.8 Hz, 3H). HRMS (ESI) calculated for C ₁₇ H ₁₉ NO ₄ [M + Na ⁺ ] 324.1027, found 324.1212.

Connection 65

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.19 (s, 1H), 7.31 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H ), 7.18 (d, J = 8.0 Hz, 1H), 4.66 (s, 2H), 4.22 (q, J = 6.8 Hz, 2H), 2.86 (q, J = 7.2 Hz, 4H), 2.04 (t, J = 7.2 Hz, 2H), 1.26 (t, J = 6.8 Hz, 3H). HRMS (ESI) calculated for C ₁₆ H ₁₇ NO ₄ [M + Na ^+] 310.1056, found 310.1055.

Connection 66

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.52 (s, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.71 (d, J = 8.8 Hz, 2H), 4.76 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.26 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₃ H ₁₂ F ₅ NO ₄ S [M + H ^+] 374.0485, found 374.0490.

Connection 67

• ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ (ppm): 10.49 (s, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 4.75 (s, 2H), 4.24 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). HRMS (ESI) calculated for C ₁₄ H ₁₃ F ₃ NO ₄ [M + Na ^+] 338.0607, found 338.0616.

Activity test for DHODH

example 1

The inhibitory effects of the compounds of the present invention in vitro on dihydroorotate dehydrogenase (DHODH):
The catalytic region 159-565 of PfDHODH (Plasmodium falciparum dihydroorotate dehydrogenase) was cloned into the vector pET101 / D (The Journal of Biological Chemistry, 2008, 283, 35078-35085), the resulting vector was inserted into the expression strain E. coli BL21 introduced. 1.5 ml of pET101 / D-PfDHODH bacterial fluid was inoculated into 500 ml of 2x VT medium containing 250 μM ampicillin and cultured on a shaker table at 37 ° C for about 4 hours at 230 rpm. When the OD value of the cell suspension reached 0.81, IPTG was added to the medium so that the final IPTG concentration became 0.5 mM, and the induction was carried out at 25 ° C for 4-6 hours. At 4 ° C, the induced cells were recovered by centrifugation at 4000 rpm for 20 minutes and then resuspended in 15 ml deionized water. The sediment was recovered by centrifugation at 10,000 rpm for 30 minutes and stored overnight at -80 ° C. The cell cake was resuspended in 20 ml of lysis buffer, 50 mM HEPES (pH 7.5), 500 mM NaCl, 40 mM imidazole, 0.1% Triton X-100 and then sonicated to disrupt the cells. The protein was eluted with lysis fluid containing 300 mM imidazole, dialyzed overnight against 50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, and the subsequent determination of Enzyme activity and inhibitor screening.

The inhibitor screening can be carried out by means of DCIP colorimetry. DCIP was reduced in the reaction and is the final electron acceptor for DHO (dihydroorotate). Therefore, the degree of hydrolysis of the substrate DHO can be determined from the decrease rate of the absorbance of DCIP at 600 nm. The faster the absorbance decreases, the higher the activity of the enzyme, that is, the lower the inhibitory effect of compounds on the enzyme (DMSO in the same volume as negative control, A77 1726 in the same amount as positive control) (see literature, The Immunosuppressive Metabolite of Leflunomide Is a Potent Inhibitor of Human Dihydroorotate Dehydrogenase).

All reagents including ampicillin, VT medium, IPTG, imidazole, HEPES, Triton X-100, NaCl, DCIP, DHO were purchased from Sigma Corporation. Compounds 14-21 were purchased from the Dutch Association Library SPECS.

Example 2

The inhibitory effects of compounds of the present invention on two Plasmodium falciparum cell lines in vitro:
Chloroquine was purchased from Sigma Corporation. SYBR Green was purchased from Invitrogen Corporation. Chloroquine-sensitive Plasmodium falciparum 3D7 cell lines and chloroquine-resistant Plasmodium falciparum Dd2 cell lines were used to test for antimalarial activity in vitro. The cell lines were cultured by the culture method of Trager and Jensen except that 0.5% of Albumax II (Invitrogen Corporation) was used in place of human serum. Two insect strains of P. falciparum were purchased from MR4 (Malaria Research and Reference Reagent Resource Center), USA.

In the test, SYBR Green was used as a fluorescent marker and antimalarial activity was determined by measuring the fluorescence data. The culture procedure was shown as follows: the initial infection rate is 1%, the cell packing volume is 2%, and erythrocytes were cultured in the presence or absence of drug, the final volume being 100 μl. A dilution series of each drug was prepared and the concentration is 0.15525 μM to 20 μM. As a negative control 0.2% DMSO was used, various concentrations of chloroquine were used as a positive control, and 100 μl of uninfected erythrocytes with a cell packing volume of 2% was used as background control. The cells were incubated at 37 ° C for 72 hours. For each experimental setup, three parallel controls were provided. All of the above cultures were performed on 96-well cell culture plates. After 72 hours of culture, the culture fluid was spun down, the supernatant was discarded and to each well was added 100 μl of erythrocyte lysis fluid containing SYBR Green I (8.26 g / L NH ₄ Cl, 1 g / L KHCO ₃ , 0.037 g / l EDTA and 5x SYBR Green I). The Petri dishes were placed in the dark for one hour at room temperature and then all samples were transferred to black microtiter plates (Corning 3925). The fluorescence was detected with a Synergy MX multifunctional microplate detector from Biotek Cooperation and read at 485/520 nm. The experiment was repeated twice.

A data analysis was carried out after a modification of the method described by Michael et al. described method performed. The data analysis is briefly described below. Background fluorescence data generated in uninfected erythrocytes was subtracted from the negative control fluorescence data, and the resulting data represented the maximum amount of P. falciparum DNA normally cultured in the experiment, and was used as fluorescence data of the Wells recorded with the negative control. The fluorescence data from each test group and the positive control were treated as above. The inhibition rate at various concentrations was calculated according to the following formula: Inhibition Rate (%) = [1 - (mean fluorescence values in test wells / mean fluorescence values in wells with negative control)] × 100%.

The curve was adjusted using the Origin / 8.0 software Growth / Sigmoidal, the half maximal inhibition concentration (IC50 values) was calculated, and the mean and standard deviation were calculated using Microsoft Excel.

The results of Examples 1 and 2 are shown in the following table. Inhibition rate and IC50 value of thiophenone derivatives Connection no. pfDHODH 10 μM inhibition rate (%) pfDHODH IC ₅₀ (μM) Pf3D7 IC ₅₀ PfDd2 IC ₅₀ 1 74.9983 0.5999 ± 0.0078 2 63.9962 0.3677 ± 0.0060 0.946385 1.040115 3 38.2071 > 20 > 20 4 56.0507 1.1882 ± 0.1812 4.030645 5.99622 5 5.047945 3.729975 6 73.5205 0.2860 ± 0.0047 1.99155 1.31749 7 1.233025 1.51301 8th 5.614265 5.53863 9 2.19099 3.14673 10 80.1390 0.0034 ± 0.0006 0.077275 0.057365 12 69.27 1.34 16 70.6713 0.8547 ± 0.0458 17 61.0476 3.7177 ± 0.1358 18 66.1669 1.0713 ± 0.0077 20 32.047 21 19.306 22 73.33042336 0.851 ± 0.027 1.40117 1.572815 23 76.37532615 0.55898 ± 0.0135 0.306715 0.696995 24 84.58971104 0.35061 ± 0.00438 .2533 0.289575 25 86.90055674 0.092 ± 0.00016 0.16932 0.31484 27 5.615976422 > 20 > 20 28 12.47050528 12.83785 12.25155 29 49.298907 3.34247 3.499835 30 31.53426812 6.61833 5.53157 31 2.059884462 > 20 > 20 32 30.57561604 9.793915 9.97029 33 23.6284108 6,884 10.95979 34 11.96568749 12.402 > 20 35 58.74702609 13.20958 ± 0.1038 10.23289 7.94425 36 4.73749994 37 30.2244076 8.47004 6.62546 38 58.24222769 4.73106 ± 0.19 2.69638 2.77772 39 63.78877439 5.73275 ± 0.2855 3.17317 3.926755 40 40.99553838 11.14765 8.491925 43 44.62101375 44 59.45378691 1.23842 ± 0.011 1.86025 1.04883 52 20.56110617 17.14 15.31 53 26.62551656 54 6.74 4.87 55 36.46239003 14.35 13.59 56 37.76443461 > 20 > 20 57 5.043710956 > 20 > 20 58 9.195823097 > 20 > 20 59 29.6098525 13.82 10.42 61 91.59259371 0.00597 ± 5.8E-06 0.01567 0.01842 62 82.10318913 0.699764 ± 0.01335 0.372205 0.485605

Claims (11)

Compound of the formula I:

wherein X _{1 is selected} from O, S, NH and CH ₂ ; X _{2 is selected} from O, S, NH, NOH, NHR and C ₁ -C ₆ -imino; R ^{1 is selected} from H, C ₁ -C ₁₀ alkyl, unsaturated hydrocarbon radical containing not more than 10 carbon atoms, optionally substituted aryl, nitro, amino, NR ⁴ R ⁵ , halogen; R ^{2 is} H, C ₁ -C ₆ -alkyl, unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -alkylaminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy, C ₃ -C ₈ -cycloalkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkoxycarbonyl is selected; R ^{3 is} H, C ₁ -C ₆ -alkyl, optionally substituted unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -aminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy; R ⁴ and R ^{5 are} independently selected from H, C ₁ -C ₆ alkyl, -C (O) NHR ⁶ , C ₁ -C ₆ alkoxycarbonyl, halo-substituted alkyl, unsaturated C ₂ -C ₆ hydrocarbyl, optionally substituted aryl, C C ₁ -C ₆ alkylcarbonyl, optionally substituted benzoyl, optionally substituted heterocyclic group, optionally substituted heterocyclocarbonyl and C ₁ -C ₆ alkoxycarbonyl; R ^{6 is selected} from optionally substituted aryl and an optionally substituted heterocyclic group. A compound according to claim 1, wherein the compound is selected from compounds of formula II:

wherein X _{1 is selected} from O, S, NH and CH ₂ ; R ^{2 is} H, C ₁ -C ₆ -alkyl, unsaturated C ₂ -C ₆ -hydrocarbon radical, C ₁ -C ₆ -alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ -alkoxycarbonyl, C ₁ -C ₆ -alkylaminocarbonyl, hydroxy, C ₁ -C ₆ -alkoxy, C ₃ -C ₈ -cycloalkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkylaminocarbonyl, C ₃ -C ₈ -cycloalkyl-C ₁ -C ₆ -alkoxycarbonyl is selected; R ^{7 is selected} from optionally substituted aryl, an optionally substituted heterocyclic group, optionally substituted arylcarbonyl, optionally substituted nitrogen-containing heterocyclocarbonyl and optionally substituted aryloxy and C ₁ -C ₃ -alkylcarbonyl. A compound according to claim 2 wherein the aryl and heterocyclic group are optionally substituted with 1 to 5 substituents selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₃ -C ₈ cycloalkyl, C ₁ -C 4 alkoxy , optionally substituted phenyl, optionally substituted phenoxy, benzyloxy, CF ₃ , F, Cl, Br and I. A compound according to claim 2, wherein R ^{2 is} H, C ₁ -C ₆ alkyl, C ₂ -C ₆ unsaturated hydrocarbon radical, C ₁ -C ₃ alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C ₁ -C ₆ - Alkoxycarbonyl, C ₁ -C ₆ -aminocarbonyl, hydroxy and C ₁ -C ₆ -alkoxy. A compound according to claim 1, wherein the compound is selected from:

A pharmaceutical composition comprising compounds according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. Use of compounds according to any one of claims 1 to 5 for the manufacture of a medicament for the treatment or prevention of dihydroorotate dehydrogenase mediated diseases. Use according to claim 7, wherein the dihydroorotate dehydrogenase-mediated disease is selected from malaria tropica, malaria tertiana, malaria ovale, malaria quartana, Gram trypanosomiasis, dengue fever and other parasitic diseases. Use according to claim 7, wherein the dihydroorotate dehydrogenase-mediated disease is selected from rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus glomerulopathy and rejection of organ transplants. Use according to claim 7, wherein the dihydroorotate dehydrogenase-mediated disease is selected from a melanoma and by allo- and xeno-organ transplantation in a host-induced rejection. Use of compounds according to any one of claims 1 to 5 for the manufacture of a medicament for inhibiting the activity of dihydroorotate dehydrogenase.