Heterocyclic dihydro five-membered ring ketone derivatives as DHODH inhibitors and their uses.

专利摘要:
The present invention relates to the synthesis and use of heterocyclic dihydro-five-membered ketone derivatives of general formula (I) which are used as DHODH inhibitor for Plasmodium falciparum, and the compound according to the invention can be used for the treatment and prevention of with DHODH related diseases, including the treatment of parasitic diseases such as malaria caused by malarial parasites.
公开号:CH707498B1
申请号:CH00795/14
申请日:2012-11-14
公开日:2016-05-31
发明作者:Li Honglin；Zhao Zhenjiang；Huang Jin；Xu Yufang；Xu Minghao；Diao Yanyan；Zhou Hongchang；Jin Huangtao；Gao Rui；Zhu Junsheng
申请人:Univ East China Science & Tech；
IPC主号:

专利说明:

Field of 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 DHODH inhibitors.
Background of the invention
Malaria is an insect-borne infectious disease caused by Plasmodium infection via mosquito bites, and it is still one of the global problems affecting human health. At present, typical commercially available drugs for the treatment of malaria are chloroquine, artemisinin, pyrimethamine, etc. In recent decades, many drugs that used to be very useful, such as quinine, chloroquine, mel-quinoline and others, have become more and more drug resistant like; After treating Plasmodium with chloroquine, its nucleus ruptures, bubbles appear in the cytoplasm, and the malaria pigment agglomerates. Chloroquine cannot kill the parasite directly, but it can interfere with its reproduction. It has a strong binding force to nucleoprotein, and the negatively charged 7-chlorine atom of the quinoline ring comes close to the 2-amino group of guanine in Plasmodium DNA, inserting chloroquine into the two strands of the DNA double helix and forming a complex with Forms DNA, thus preventing DNA replication and RNA transcription. Chloroquine can also prevent phosphate from being incorporated into Plasmodium's DNA and RNA, thereby disrupting the parasite's reproduction due to a decrease in nucleic acid synthesis. In recent years, however, it has been reported and confirmed that the effectiveness of chloroquine and its derivatives as antimalarial agents is rapidly declining, and patients in some areas even develop complete resistance to chloroquine and therefore longer treatment cycles are required for removal of the parasite necessary, and relapses occur (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 agent that has not been reported to have widespread and severe drug resistance, but in recent years it has been reported that in the border area between Thailand and Cambodia, malaria mosquitoes carrying Plasmodium and having artemisinin resistance in vivo have appeared . The relevant follow-up results only increased people's fear of increasing resistance to artemisinin therapies (Eastman, RT; Fidock, DA, Artemisinin-based combination therapies: a vital tool in efforts to eliminate malaria, Nat Rev Micro 2009, 7 (12)) , 864-874).
In addition, early malaria agents, such as pyrimethamine, can inhibit Plasmodium's folate reductase, which in turn disrupts Plasmodium's normal folate metabolism. The active ingredient is effective against the early phase of the Plasmodium falciparum cell cycle and can therefore be used as an etiological preventive agent. The active ingredient 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 ingredient can be used for the prevention of malaria and as anti-recurrence 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 important academic and practical value to search for a new 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. Hence, DHODH is a key enzyme in the synthesis of pyrimidine in nucleic acid, and it is also a unique enzyme within the parasite that catalyzes the formation of orotate from dihydroorotate and is primarily involved in catalysis of the fourth step of de novo biosynthesis of pyrimidine is. In humans and in Plasmodium, DHODH is located on the inner mitochondrial membrane, and the catalytic process also requires other cofactors. Structurally, DHODH has two parts, i.e. an α-helix structure region at the N-terminus and an α / β-barrel structure at the C-terminus. DHODH adheres to the inner mitochondrial membrane through the unique structure of the N-terminus, and the C-terminal region is the main center for catalysis. There are binding sites on DHODH for the substrate and cofactor flavin mononucleotide (FMN) and coenzyme Q (CoQ). The catalytic process takes place in two steps: first, dihydroorotate is oxidized to orotate, and FMN accepts the hydrogen released during the process and is reduced to FMNH2; then FMNH2 is again dehydrated and oxidized to FMN under the action of CoQ. 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 primarily by blocking the biological synthesis of pyrimidine within Plasmodium and inhibiting the reproduction and growth of Plasmodium.
In addition, the 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 de novo -Synthesis of pyrimidine relied on to meet their growth requirements. Therefore, a DHODH inhibitor can be used as an anti-cellular agent for the treatment of tumors and for a certain 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 the inhibition of mitochondrial DHODH induces a p53 stress response, which in turn can be used to treat tissue damage. Another literature reference (Annals of the Rheumatic Diseases 2006, 65 (6), 728-735) reports that the inhibition of DHODH prevents the TNF-induced phosphorylation of the transcription factor NF-κB, the activation of AP-1 and c-jun- N-terminal kinase and ultimately can inhibit the TNF-induced apoptosis.
Research into 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 focused primarily on the binding sites of CoQ, with brequinar and leflunomide being used clinically. Brequinar is used as an anti-tumor agent and as an agent against the host's immune response induced by organ transplantation. However, the therapeutic window of Brequinar is narrow and side effects such as thrombocytopenia and mucositis are easily caused when it is administered orally in combination with cisplatin or cyclosporine A, and hence its wide clinical application is limited. Leflunomide, which was launched in 1998, is a potent inhibitor of a wide variety of autoimmune diseases, acute and chronic reactions and organ transplant-induced xenograft rejection and is used clinically to treat lupus and rheumatoid arthritis and can be used to prevent and control 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: an immunomodulatory drug for the treatment of rheumatoid arthritis and others autoimmune diseases, Immunopharmacology 2000, 47 (2-3), 273-289), colitis (Fitzpatrick, LR, Deml, 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, Suppresses Systemic Lupus Erythematosus in MRL- (Fas) Ipr Mice. Am. J. Pathol. 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, 3. and Kaufman, C., DHODH modulates transcriptional elongation in the neural crest and melanoma, Nature 2011, 471 (7339), 518-522), transplant 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
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 effective to bind within the coenzyme Q binding pocket (Deng, X .; Gujjar, R .; El Mazouni, F .; Kaminsky, W .; Malmquist, NA; Goldsmith, EJ; Rathod, PK; Phillips, MA, Structural plasticity of malaria dihydroorotate dehydrogenase allows selective binding of diverse chemical scaffolds. [J], J. Biol. Chem. 2009, 284, 26999-27009). Accordingly, the inventors extensively used methods and techniques in the fields of computational drug design, medicinal chemistry, and molecular biology in preparatory work, and found a series of dihydro five-membered ring heterocyclic ketone derivatives that meet the above structural requirements. These derivatives are completely different from the previously described highly effective Plasmodium falciparum DHODH inhibitors with regard to the structural framework. Some of the compounds have significant inhibitory effects against Plasmodium cell lines and immunological effects at the cell level and are promising drugs. Based on the lead compounds in this series, the inventors designed and synthesized a series of dihydro five-membered ring heterocyclic ketone derivatives, and the structural formula is shown as follows:
in whichX1 is selected from O, S, NH and CH2;X2 is selected from O, S, NH, NOH and C1-C6 imino;R <1> is selected from H, C1-C10-alkyl, unsaturated hydrocarbon radical containing no more than 10 carbon atoms, optionally substituted aryl, nitro, NR <4> R <5> and halogen;R <2> from H, C1 – C6-alkyl, unsaturated C2 – C6-hydrocarbon radical, C1 – C6-alkylcarbonyl, optionally substituted 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;R <3> from H, C1-C6-alkyl, optionally substituted unsaturated C2-C6-hydrocarbon radical, C1-C6-alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C1-C6-alkoxycarbonyl, C1-C6-aminocarbonyl, hydroxy, C1-C6 alkoxy is selected;R <4> and R <5> independently from H, C1-C6-alkyl, -C (O) NHR <6>, C1-C6-alkoxycarbonyl, halogen-substituted alkyl, unsaturated C2-C6-hydrocarbon radical, optionally substituted aryl, C1 -C6-alkylcarbonyl, optionally substituted benzoyl, an optionally substituted heterocyclic group, optionally substituted heterocyclocarbonyl and C1-C6-alkoxycarbonyl;R 6 is selected from optionally substituted aryl and an optionally substituted heterocyclic group.
In one embodiment, the compounds are selected from compounds of the formula II:
in whichX1 is selected from O, S, NH and CH2;R <2> from H, C1 – C6 alkyl, unsaturated C2 – C6 hydrocarbon radical, C1 – C6 alkylcarbonyl, optionally substituted 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;R 7 is selected from optionally substituted aryl, optionally substituted nitrogen-containing heterocyclocarbonyl, an optionally substituted heterocyclic group and C1-C3-alkylcarbonyl.
"Alkyl" here generally refers to a saturated straight-chain or branched alkyl having 1 to 10 carbon atoms, preferably alkyl having 1 to 6 carbon atoms, particularly preferably alkyl having 1 to 4 or 1 to 3 carbon atoms. "Cycloalkyl" refers to cyclic alkyl, which generally has 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 group of 2 to 10 carbon atoms that contains at least one double bond between two carbon atoms in the chain. Alkenyl is preferably an alkenyl group containing 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 group having 2 to 10 carbon atoms which contains at least one triple bond between two carbon atoms in the chain. Alkynyl is preferably an alkynyl group containing 2 to 4 carbon atoms. Typically, alkynyl includes ethynyl, 1-propynyl, 1-methyl-2-propynyl, 2-propynyl, 1-butynyl, and 2-butynyl.
As used herein, "aryl" refers to a monocyclic, bicyclic, or tricyclic aromatic group containing 6 to 14 carbon atoms; these include phenyl, naphthyl, phenanthryl, anthryl, indenyl, fluorenyl, tetralin group, indanyl and the like. Aryl can optionally be substituted with 1 to 5 (eg 1, 2, 3, 4 or 5) substituents selected from: halogen, C1-C4-aldehyde group, straight-chain or branched C1-C6-alkyl, cyano, nitro, amino , Hydroxy, hydroxymethyl, halogen-substituted alkyl (e.g. trifluoromethyl), halogen-substituted alkoxy (e.g. trifluoromethoxy), carboxy, C1-C4-alkoxy, C1-C4-methylthiol, -SF5, morpholinyl, optionally substituted aryl (e.g. optionally substituted phenyl), optionally substituted Aryloxy (for example optionally substituted phenoxy) and optionally substituted benzyloxy. For example, aryl can be substituted with 1 to 3 groups selected from: fluorine, chlorine, bromine, C1-C4-alkyl, trifluoromethyl, morpholinyl, methoxy, phenyl, methoxy-substituted phenyl, phenoxy, benzyloxy, halogen-substituted benzyloxy, ethoxy, nitro and the same.
The term "heterocyclic group" as used herein means a single or fused ring structure which can be aromatic or non-aromatic and preferably contains 3-20 ring atoms, particularly preferably 5-14 ring atoms, with at least one and preferably up to four ring atoms being heteroatoms 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, triazolyl, quinolinyl, benzothiazole, benzothiazole, Benzofuranyl, morpholinyl, carbazolyl, dibenzothiophene group, coumarin and 1,2-methylenedioxyphenyl. The heterocyclic group can optionally be substituted with 1 to 3 substituents, as described here.
The term "heteroatom" used here includes O, S and N. If the heteroatom is N, the N atom can be further substituted with a group, for example hydrogen or C1-C10-alkyl. If the hetero atom is S, the S atom can furthermore be substituted with a group, for example C1-C10-alkyl.
The term "heteroaryl" or "aromatic heterocyclic group" as used herein means a heterocyclic group having aromatic properties as described above; these include furyl, thienyl, pyrrolyl, pyridyl, oxazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, and the like.
The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.
Unless stated otherwise, the term "optionally substituted" as used herein means that the group modified by this term can optionally be substituted with 1 to 5 (generally 1, 2 or 3) substituents selected from: C1-C4-alkyl, carboxy, halogen, C1-C4-alkoxy, cyano, nitro, amino, hydroxy, aldehyde group, C1-C6-acyl, hydroxymethyl, halogen-substituted C1-C4-alkyl (e.g. trifluoromethyl), C1-C10-thioalkyl (e.g. pentafluorothiomethyl), C1-C10-thioalkoxy (e.g. pentafluorothiomethoxy), halogen-substituted C1-C4-alkoxy (e.g. trifluoromethoxy), thiol and C1-C4-acyl.
Amido (aminocarbonyl) itself or as part of another group refers to a “C1-C6-alkyl-CO-NH” group, “C3-C8-cycloalkyl-CO-NH-” or “C3-C8” -Cycloalkyl-C1-C6-alkyl-CO-NH- ». Exemplary amido groups include formamido, acetamido, propionamido, butyramido, cyclopropylamido, cyclopropylmethylamido and the like.
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 in which X1 = S or O and X2 = O.
Preferred compounds of the invention are the compounds in which R 3 = H.
In some embodiments, compounds 14-21 are not included in the compounds of the invention.
Preferred compounds of the invention are the compounds in which R 2 = C1-C6-alkoxycarbonyl (eg methoxycarbonyl, ethoxycarbonyl, cyclopropoxycarbonyl, cyclopropylmethoxycarbonyl, etc.), C1-C6-aminocarbonyl (eg 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 group or -NH-optionally-substituted-quinolyl.
Preferred compounds of the invention are the compounds in which the substituents on the optionally substituted phenyl or naphthyl in R <1> are halogen, halogen-substituted C1-C4-alkyl, C1-C4-alkyl or C1-C4-alkoxy.
In a preferred embodiment, the present invention comprises the compounds in which X1 = S or O, X2 = O, R 3 = H, R 2 = C1-C6-alkoxycarbonyl (eg methoxycarbonyl, Ethoxycarbonyl, cyclopropyloxycarbonyl, cyclopropylmethoxycarbonyl etc.), C1-C6-aminocarbonyl (e.g. formamido, acetamido, propionamido, butyramido, cyclopropylamido, cyclopropylmethylamido etc.) is, R 1 = -NH-optionally-substituted-phenyl, -NH -substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralin group or -NH-optionally-substituted-quinolyl. The substituents on the optionally substituted phenyl or naphthyl in R <1> are particularly preferred halogen, halogen-substituted C1-C4-alkyl, C1-C4-alkyl or C1-C4-alkoxy.
In a particularly preferred embodiment, X1 = S or O, X2 is O, R 3 is H, R 2 is C1-C6 alkoxycarbonyl (eg methoxycarbonyl, ethoxycarbonyl, etc.), R 1 is - NH-optionally-substituted-phenyl, -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralin group or -NH-optionally-substituted-quinolyl; Phenyl is preferably unsubstituted or has 1 to 3 substituents selected from halogen, trifluoromethyl, methyl, nitro and methoxy. The substituent is preferably located in the 3-, 4- and / or 5-position. Compounds 14-21 are preferably not included.
In a particularly preferred embodiment, X1 = S or O, X2 is O, R 3 is H, R 2 is C2-C4 alkoxycarbonyl (for example ethoxycarbonyl, cyclopropyloxycarbonyl, cyclopropylmethoxycarbonyl, etc.), R 1 is -NH-optionally-substituted-phenyl, -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralin group or -NH-optionally-substituted-quinolyl; Phenyl is preferably unsubstituted or has 2 to 3 substituents selected from halogen and C1-C4-alkyl. Compounds 14-21 are preferably not included.
In a particularly preferred embodiment, the compounds of the invention comprise the compounds in which R <1> -NH-optionally-substituted-naphthyl, -NH-optionally-substituted-indanyl, -NH-optionally-substituted-tetralin group or - Is NH-optionally-substituted-quinolyl.
Preferred compounds of the invention are those numbered 1-67 as shown in Table 1 below, and particularly preferred are those having a rate of inhibition greater than 50%. In particular, the compounds of the invention include compounds 1, 2, 4, 6, 7, 10, 22-25, 35, 38, 39, 44, 61 and 62.
The compounds of the invention can be prepared using the following procedure:
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. According to actual production requirements, those skilled in the art can produce the compounds of the invention using various conventionally obtained compounds as a starting material. For example, a compound of Formula II of the present invention can be prepared based on the method described by Du Xiaohua et al. (non-sulfur phosgene synthesis of phenyl isothiocyanate difficult to be synthesized. [J]. Pesticides 2004, 43 (2), 78-79).
[0034]
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 C1-C6 aminocarbonyl as defined above.
[0035]
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 C1-C6 aminocarbonyl as defined above.
[0036]
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, which composition comprises a therapeutically effective amount of compounds of formula I or II of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.
Examples of the pharmaceutically acceptable salt of compounds of the invention include inorganic salts and organic salts such as hydrochloride, hydrobromide, sulfate, citrate, lactate, tartrate, maleate, fumarate, mandelate and oxalate; as well as inorganic and organic salts formed with a base such as sodium hydroxide, tris (hydroxymethyl) aminomethane (TRIS, tromethamine) and N-methylglucamine.
The person skilled in the art can determine the optimal dosage of each active ingredient in the pharmaceutical compositions of the invention, even if the needs vary individually. In general, the compounds of the present invention or a pharmaceutically acceptable salt thereof are orally administered to a mammal in a daily dose of about 0.0025 to 50 mg / kg body weight, and preferably orally administered in a daily dose of about 0.01 to 10 mg / kg body weight . For example, a unit oral dose may comprise about 0.01 to 50 mg, preferably about 0.1 to 10 mg, of compounds of the invention. A unit dose can be administered one or more times, one or more tablets per day, 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 formulated into forms for various routes of administration; these include parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal or topical administration for the treatment of cancer and other diseases. The amount administered is that amount which is effective in ameliorating or curing one or more diseases. For the treatment of a particular disease, the effective amount is that amount which is sufficient to alleviate or alleviate disease-related symptoms. Such amount can be administered in a single dose or according to an effective therapeutic regimen. The administration amount can effectively cure diseases, but usually the purpose is to relieve symptoms of diseases. In general, the administration must be repeated to achieve symptom relief. The dose can be determined according to the age, health status and weight of the patient, concurrent treatments, the frequency of treatment and the desired therapeutic benefit.
The pharmaceutical preparations of the invention can be administered to any mammal so long as the compounds of the present invention are effective on them. Most important of all mammals is man.
The compound or pharmaceutical composition of the invention can be used to treat and prevent various diseases mediated by DHODH, particularly as an inhibitor of diseases associated with DHODH. The diseases mediated by DHODH include: parasitic diseases including malaria tropica, malaria tertiana, malaria ovale, malaria quartana, gram's trypanosomiasis, dengue fever and other parasitic diseases, diseases caused by the rapid proliferation of certain cells, such as cancer; Inflammation; and various autoimmune diseases. Diseases mediated by DHODH also include rejection induced by allo- and xeno-organ transplantation in a host.
In particular, diseases mediated by DHODH include, but are not limited to, Gram's trypanosomiasis, tropic malaria, dengue fever, tertian malaria, ovale malaria, quartana malaria, rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus (including systemic lupus erythematosus), glomeropathic secondary disease and primary glomerulopathy), organ transplant rejection, melanoma, psoriatic arthritis, and psoriasis.
The pharmaceutical preparations of the invention can be produced in a known manner, for example by conventional mixing, granulating, tableting, dissolving or lyophilizing processes. When preparing an oral preparation, a solid excipient and an active compound can be combined and the mixture can be milled if necessary. If necessary, suitable additives can be added and the mixture can be made into particles to obtain tablets or lozenge cores.
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, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose or polyvinylpyrrolidone. If desired, a disintegrant such as the above starches as well as carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar or alginic acid or a salt thereof such as sodium alginate can be added. Adjuvants include, in particular, flow modifiers and lubricants such as silicon oxide, talc, stearates such as magnesium and calcium stearate, stearic acid or polyethylene glycol. If necessary, the lozenge core can be coated in a suitable manner so that it is enteric-coated. For this purpose, concentrated saccharide solutions can be applied, which can contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Suitable cellulose solutions, such as cellulose acetate phthalate or hydroxypropylmethyl cellulose phthalate, can be used for the production of enteric coatings. Dyes or pigments can be added to the tablets or the coating of the lozenge cores, for example in order to identify or characterize the dosage combinations of active ingredients.
Accordingly, in the third aspect of the invention there is provided 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.
The methods of administration include, but are not limited to, methods known in the art that can be determined according to the patient's condition; these include, inter alia, 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 treating or preventing 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 only intended to illustrate the invention and are not intended to limit it 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. 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. 10 ml of chloroform solution containing 1 mmol of triphosgene were added to the suspension and reacted overnight. The mixture was subjected to vacuum filtration to obtain a mother liquor. The mother liquor was concentrated under reduced pressure, and substituted phenyl isothiocyanate was obtained by column chromatography 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-carboxylate [J] J. Chem. Society. Perkin. 1981, 1078-1082):
Synthesis: To 0.55 mmol of freshly prepared solid sodium methoxide and 10 ml of dry DME were added 0.55 mmol of 4-chloroethylacetoacetate, and the resulting mixture was stirred at room temperature for 1 hour. 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 sodium chloride solution 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. The reaction mixture was then heated to 60 ° C. and kept at this temperature overnight. Methanol was removed under reduced pressure. The residue was adjusted to pH = 2 by adding a little water and 10% strength dilute hydrochloric acid, a large amount of white solid precipitated, the solid was filtered and dried, the product being obtained.
Synthesis: In a round bottom flask the appropriate dried acid (1 mmol, 285 mg) and substituted amines (1.2 mmol) were added, and then 5 ml of dichloromethane, HOBt (1.2 mmol, 260 mg) and EDC (1, 2 mmol, 330 mg) added. The resulting mixture was stirred at room temperature overnight and TLC showed the starting material was still present, but a new stain appeared to appear. The mixture was washed using a saturated ammonium chloride solution, a saturated sodium carbonate solution, and brine in turn. 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 placed in 5 ml of toluene and then diethylazodicarboxylate (1.2 mmol) was added dropwise under argon. The reaction was carried out for half an hour, and then the mixture was heated to 50 ° C. and reacted overnight. The mixture was washed with saturated saline and purified by column chromatography (PE: EA = 3: 1, v / v) to give a pale yellow solid.
Synthesis: 3 mmol of 60% sodium hydroxide solution (about 240 mg) and 1.8 ml of anhydrous THF were placed in a 50 ml flask. In an ice bath, 1.8 ml of anhydrous THF solution of diethyl malonate (6 mmol) was added dropwise. After 10 min, 3 ml of chloroacetyl chloride solution (3 mmol) were 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 remained. After 2 hours of reflux, TLC showed that starting material was still present. 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%.
Target compound spectrum
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-d6) 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 C13H12CINO3S [M + H <+>] 298.0305, found 298.0307;
Ethyl 2- (4-trifluoromethylanilino) -4-carbonyI-4,5-dihydrothiophene-3-carboxylate (compound 2)
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C14H12F3NO3S [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-d6) 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 C14H14BrNO3S [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-d6) 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 C13H11CI2NO3S [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-d6) 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) calcd for C14H14FNO3S [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-d6) 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 = 7.2 Hz, 2H), 3.63 (s, 2H), 2.31 (s, 6H), 1.42 (t , J = 7.2 Hz, 3H); HRMS (ESI) calcd for C15H17NO3S [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, CDCl3) δ (ppm): 11.42 (s, 1H), 8.10 (d, 3 = 7.6 Hz, 2H), 7.55 (t, J = 7.2 Hz, 1H), 7.48-7.41 (m, 3H), 7.30 (d, J = 7.2 Hz, 1H), 4.46-4.39 (m, 4H), 3.64 (s, 2H), 1.51-1.48 (m, 6H); HRMS (ESI) calcd for C21H20N2O3S [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-d6) 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) calcd for C14H11BrF3NO3S [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-d6) δ (ppm): 11.67 (s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.73 (s, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.43-4.36 (q, J = 6.8 Hz, 2H), 3.71 (s, 2H), 1, 42 (t, J = 6.8 Hz, 3H); HRMS (ESI) calcd for C14H11CIF3NO3S [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-d6) δ (ppm): 11.34 (s, 1H), 8.05-7.96 (m, 4H), 7.60-7.54 (m , 3H), 4.28-4.22 (q, J = 7.2 Hz, 2H), 3.70 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H); HRMS (ESI) calcd for C17H15NO3S [M + H <+>] 314.0851, found 314.0849;
Ethyl 2- (β-anthrylamino) -4-carbonyI-4,5-dihydrothiophene-3-carboxylate (Compound 11)
<1> H-NMR (400 MHz, DMSO-d6) 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 = 7.2 Hz, 2H), 3.76 (s, 2H), 1.45 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C21H18NO3S [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-d6) 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 = 7.2 Hz, 2H), 3.67 (s, 2H), 1.42 (t, J = 7.2 Hz, 3H) HRMS (ESI) calcd for C13H14NO3S [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-d6) δ (ppm): 11.27 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.42 (dd, J1 = 2.0 Hz, J2 = 1.6 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H), 8.08 (dd, J1 = 1.6 Hz, J2 = 2, 4 Hz, 1H), 7.56 (m, 3H), 4.25 (q, J1 = 7.2 Hz, 2H), 3.67 (s, 2H), 1.28 (t, J = 7, 2 Hz, 3H) HRMS (ESI) calcd for C19H16NO3S2 [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-d6): δ (ppm): 11.08 (s, 1H), 7.32 (d, 3 = 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 = 7.6 Hz, 4H), 2.09-2.02 (m, 2H), 1.26 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C16H17NO3S [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-d6): 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) calcd for C13H12F5NO3S2 [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-d6) δ (ppm): 11.31 (s, 1H), 8.92 (d, 3 = 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, J = 7.6 Hz, 1H), 4.26 (q, J = 6.8 Hz, 2H), 3.73, (s, 2H), 1.28 (t, J = 6.8 Hz, 3H). HRMS (ESI) calcd for C16H15N2O3S [M + H <+>] 315.0803, found 315.0806.
Ethyl 2- (2- (5,6,7,8-tetrahydronaphthalene) amino) -4-carbonyl-4,5-dihydrothiophene-3-carboxylate (Compound 25)
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C17H19NO3S [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, CDCl3) δ (ppm): 11.44 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 9.6 Hz, 2H), 4.40 (q, J = 6.8 Hz, 2H), 3.66 (s, 2H), 1.43 (t, J = 6.8 Hz 3H), 1, 36 (s, 9H). HRMS (ESI) calcd for C17H21NO3S [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-d6) 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 = 6.8 Hz, 2H), 0.23 (d, J = 4.8 Hz, 2H). HRMS (ESI) calcd for C16H15F3N2O2S [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-d6) 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) calcd for C15H13F3N2O2S [M-H <+>] 341.0572, found 341.0565
2- (3,4-Dimethylanilino) -4-carbonyi-4,5-dihydrothiophene-3- (N-cyclopropylmethyl) formamide (compound 29)
<1> H-NMR (400 MHz, DMSO-d6) 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, J1 = 2.4 Hz, J2 = 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) calcd for C17H20N2O2S [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-d6) 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, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 3 , 83 (s, 2H), 2.77-2.73 (m, 1H), 2.24 (d, J = 1.6 Hz, 6H), 0.75-0.70 (m, 2H), 0.50-0.48 (m, 2H). HRMS (ESI) calcd for C16H18N2O2S [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-d6) δ (ppm): 10.92 (s, 1H), 8.92 (s, 1H), 7.88 (s, 1H), 7.26 ( d, J = 8.0 Hz, 1H), 7.13 (s, 1H), 7.06 (dd, J1 = 2.0 Hz, J2 = 8.0 Hz, 1H), 3.81 (s, 2H), 2.25 (s, 6H). HRMS (ESI) calcd for C13H14N2O2S [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-d6) δ (ppm): 12.33 (s, 1H), 8.88 (t, J = 5.2 Hz, 1H), 7.33 -7.26 (m, 2H), 7.16 (d, J = 8.0 Hz, 1H), 3.85 (s, 2H), 3.15 (t, J = 6.4 Hz, 2H) , 2.88 (q, J = 6.8 Hz, 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) calcd for C18H20N2O2S [M + H +] 329.1342, found 329.1315.
2- (2,3-Dihydro-1H-indenylamino) -4-carbonyl-4,5-dihydrothiophene-3- (N-cyclo-propyl) formamide (compound 33)
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C17H18N2O2S [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, CDCl3) δ (ppm): 12.92 (s, 1H), 8.89 (s, 1H), 7.91-7.82 (m, 4H), 7, 56-7.42 (m, 3H), 3.73 (s, 2H), 3.28 (t, J = 6.4 Hz, 2H), 1.07 (m, 1H), 0.59-0 , 54 (m, 2H), 0.31-0.27 (m, 2H). HRMS (ESI) calcd for C19H18N2O2S [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, CDCl3) δ (ppm): 12.92 (s, 1H), 8.89 (s, 1H), 7.92-7.83 (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) calcd for C18H16N2O2S [M + Na <+>] 347.0830, found 347.0834.
2- (2-Naphthylamino) -4-carbonyl-4,5-dihydrothiophene-3-formamide (Compound 36)
<1> H-NMR (400 MHz, CDCl3) δ (ppm): 10.47 (s, 1H), 7.96 (m, 4H), 7.62 (dd, J1 = 2.0 Hz, J2 = 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) calcd for C17H15NO4 [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, CDCl3) 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, J1 = 2.0 Hz, J2 = 7.6 Hz, 1H), 3.84 (s, 2H), 3 .27 (q, J = 6.8 Hz, 2H), 2.25 (s, 3H), 2.24 (s, 3H), 1.11 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C15H18N2O2S [M + H <+>] 291.1167, found 291.1169.
2- (3,4-Dimethylanilino) -4-carbonyI-4,5-dihydrothiophene-3- (N-propyI) formamide (compound 38)
<1> H-NMR (400 MHz, DMSO-d6) 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, J1 = 2.0 Hz, J2 = 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) calcd for C16H20N2O2S [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-d6) 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, J1 = 2.0 Hz, J2 = 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) calcd for C17H22N2O2S [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-d6) 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 = 7.2 Hz, 2H), 1.12 (t , J = 7.2 Hz, 3H). HRMS (ESI) calcd for C17H16N2O2S [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, CDCl3) δ (ppm): 12.95 (s, 1H), 8.91 (s, 1H), 7.92-7.84 (m, 4H), 7, 56-7.29 (m, 3H), 3.75 (s, 2H), 1.66 (q, J = 6.8 Hz, 2H), 1.36-1.28 (m, 2H), 1 .02 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C18H18N2O2S [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, CDCl3) δ (ppm): 12.94 (s, 1H), 8.89 (s, 1H), 7.93-7.84 (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) calcd for C19H20N2O2S [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-d6) 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) calcd for C13H13NO3S [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-d6) δ (ppm): 10.55 (s, 1H), 8.08-7.89 (m, 4H), 7.62-7.40 (m , 3H), 4.07 (s, 2H), HRMS (ESI) calcd for C15H11NO3S [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-d6) 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) calcd for C15H17NO3S [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-d6) 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) calcd for C16H19NO3S [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-d6) 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 = 7.2 Hz, 3H). HRMS (ESI) calcd for C17H21NO3S [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, CDCl3) δ (ppm): 11.32 (s, 1H), 7.20 (d, J = 8.8 Hz, 1H), 7.13 (s, 1H) , 7.10 (d, J = 8.8 Hz, 1H), 4.17 (t, J = 6.8 Hz, 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) calcd for C17H19NO3S [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-d6) 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) calcd for C16H13NO3S [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, CDCl3) δ (ppm): 11.67 (s, 1H), 7.95-7.86 (m, 4H), 7.58-7.44 (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) calcd for C18H17NO3S [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, CDCl3) δ (ppm): 11.66 (s, 1H), 7.95-7.86 (m, 4H), 7.57-7.44 (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) calcd for C19H19NO3S [M + Na <+>] 364.0983, found 364.0991.
Compound 52
<1> H-NMR (400 MHz, CDCl3) 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) calcd for C16H18N2O2S [M + Na <+>] 325.0987, found 325.0985.
Compound 53
<1> H-NMR (400 MHz, CDCl3) 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 = 7.2 Hz, 4H), 2.14 (m, 2H), 1.64 (m, 2H), 1.00 (t, J = 7.4 Hz, 3H) . HRMS (ESI) calcd for C17H20N2O2S [M + Na <+>] 339.1143, found 339.1137.
Compound 54
<1> H-NMR (400 MHz, DMSO-d6) 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, J1 = 2.0 Hz, J2 = 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) calcd for C17H15NO5S [M + H <+>] 346.0749, found 346.0746.
Compound 55
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C14H12F3NO3S [M + H <+>] 332.0568, found 332.0572.
Compound 56
<1> H-NMR (400 MHz, DMSO-d6) 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, J1 = 2.4 Hz, J2 = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 6 .59 (d, J = 9.2 Hz, 1H), 4.23 (q, J = 7.2 Hz, 2H), 3.69 (s, 2H), 1.26 (t, J = 7, 2 Hz, 3H). HRMS (ESI) calcd for C16H13NO5S [M + H <+>] 332.0593, found 332.0603.
Compound 57
<1> H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.17 (s, 1H), 9.11 (s, 1H), 8.27 (s, 1H), 7.85 ( d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.0 Hz, 2H), 3.84 (s, 2H) HRMS (EI) m / z calculated for C12H9F3N2O2S [M <+ >] 302.0337, found 302.0338
Compound 58
<1> H-NMR (400 MHz, DMSO-d6) 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 C12H8F3NO3S [M <+>] 303.0177, found 303.0175
Compound 59
<1> H-NMR (400 MHz, DMSO-d6) 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 C14H13NO3S [M <+>] 275.0616, found 275.0617
Compound 60
<1> H-NMR (400 MHz, DMSO-d6) 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 = 7.2 Hz, 2H), 2.28 (s, 6H), 1.31 (t, J = 7.2 Hz, 3H). HRMS (EI) m / z calcd for C22H21NO3S [M <+>] 379.1242, found 379.1245
Compound 61
<1> H-NMR (400 MHz, CDCl3) δ (ppm): 10.47 (s, 1H), 7.96 (m, 4H), 7.62 (dd, J1 = 2.0 Hz, J2 = 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) calcd for C17H15NO4 [M + Na <+>] 320.0899, found 320.0897.
Compound 62
<1> H-NMR (400 MHz, CDCl3) 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) calcd for C15H17NO4 [M + Na <+>] 298.1055, found 298.1059.
Compound 63
<1> H-NMR (400 MHz, DMSO-d6) 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 = 7.2 Hz, 2H), 1.28 (t, J = 7.2 Hz, 3H). HRMS (ESI) calcd for C16H14N2O2 [M + Na <+>] 321.0843, found 321.0851.
Compound 64
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C17H19NO4 [M + Na <+>] 324.1027, found 324.1212.
Compound 65
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C16H17NO4 [M + Na <+>] 310.1056, found 310.1055.
Compound 66
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C13H12F5NO4S [M + H <+>] 374.0485, found 374.0490.
Compound 67
<1> H-NMR (400 MHz, DMSO-d6) 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) calcd for C14H13F3NO4 [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):
[0116] 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, 35 078-35 085), the resulting vector became introduced into the E. coli BL21 expression strain. 1.5 ml of pET101 / D-PfDHODH bacterial fluid were inoculated into 500 ml of 2 × YT medium with 250 μM ampicillin, and cultivation was carried out on a shaker table at 37 ° C. at 230 rpm for about 4 hours. When the OD value of the cell suspension reached 0.8-1, IPTG was added to the medium so that the final IPTG concentration was 0.5 mM, and induction was carried out at 25 ° C for 4-6 hours. At 4 ° C., the induced cells were obtained by centrifugation at 4000 rpm for 20 min and then resuspended in 15 ml of deionized water. The sediment was collected by centrifugation at 10,000 rpm for 30 minutes and stored at -80 ° C overnight. 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 treated with ultrasound 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 in the subsequent determination of the Enzyme activity and inhibitor screening.
The inhibitor screening can be carried out with the aid 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 rate of decrease in the absorbance of DCIP at 600 nm. The faster the extinction decreases, the higher the activity of the enzyme, i.e. the lower the inhibitory effect of compounds on the enzyme (DMSO in the same volume as negative control, A771726 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, YT medium, IPTG, imidazole, HEPES, Triton X-100, NaCl, DCIP, DHO were purchased from Sigma Corporation. Compounds 14-21 were obtained from the Dutch compound 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 obtained from Invitrogen Corporation. Chloroquine-sensitive Plasmodium falciparum 3D7 cell lines and chloroquine-resistant Plasmodium falciparum Dd2 cell lines were used to test antimalarial activity in vitro. The cell lines were cultured using the Trager and Jensen culture method, except that 0.5% Albumax II (Invitrogen Corporation) was used in place of human serum. Two insect strains of P. falciparum were obtained from MR4 (Malaria Research and Reference Reagent Resource Center), USA.
In the test, SYBR Green was used as a fluorescent marker, and the antimalarial activity was determined by taking the fluorescence data. The culture process 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 active ingredient, the final volume being 100 μl. A dilution series was made for each active ingredient and the concentration is 0.15625 µM to 20 µM. 0.2% DMSO was used as a negative control, various concentrations of chloroquine were used as a positive control, and 100 µl of uninfected erythrocytes with a cell packing volume of 2% were used as a background control. The cells were incubated at 37 ° C for 72 hours. Three parallel controls were provided for each experimental setup. All of the above cultures were performed on 96-well cell culture plates. After 72 hours of cultivation, the culture fluid was centrifuged off, the supernatant was discarded, and 100 μl of erythrocyte lysis fluid, the SYBR Green I (8.26 g / l NH4CI, 1 g / l KHCO3, 0.037 g / l EDTA) were added to each well and 5 x 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 following a modification of the method described by Michael et al. described procedure carried out. The data analysis is briefly described below. Background fluorescence data generated in uninfected erythrocytes was subtracted from the fluorescence data of the negative control, and the resulting data represented the maximum amount of DNA from P. falciparum normally cultured in the experiment and was taken as fluorescence data from Wells recorded with the negative control. The fluorescence data from each test group and the positive control were treated as above. The rate of inhibition at different 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 fitted with the Growth / Sigmoidal program of the Origin 8.0 software, the concentration of half maximum inhibition (IC50 values) was calculated, and the mean value and the standard deviation were calculated with the aid of Microsoft Excel.
The results of Examples 1 and 2 are shown in the following table.
Inhibition rate and IC50 value of thiophenone derivatives
[0126] 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 8 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 0.2533 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 0.69764 ± 0.01335 0.372205 0.485605

权利要求:
Claims (11)
[1]
1. Compound of formula I:
in whichX1 is selected from O, S, NH and CH2;X2 is selected from O, S, NH, NOH and C1-C6 imino;R <1> is selected from H, C1-C10-alkyl, unsaturated hydrocarbon radical which contains no more than 10 carbon atoms, optionally substituted aryl, nitro, NR <4> R <5>, halogen;R <2> from H, C1 – C6 alkyl, unsaturated C2 – C6 hydrocarbon radical, C1 – C6 alkylcarbonyl, optionally substituted 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;R <3> from H, C1-C6-alkyl, optionally substituted unsaturated C2-C6-hydrocarbon radical, C1-C6-alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C1-C6-alkoxycarbonyl, C1-C6-aminocarbonyl, hydroxy, C1-C6 alkoxy is selected;R <4> and R <5> independently from H, C1-C6-alkyl, -C (O) NHR <6>, C1-C6-alkoxycarbonyl, halogen-substituted alkyl, unsaturated C2-C6-hydrocarbon radical, optionally substituted aryl, C1 -C6-alkylcarbonyl, optionally substituted benzoyl, an optionally substituted heterocyclic group, optionally substituted heterocyclocarbonyl and C1-C6-alkoxycarbonyl;R 6 is selected from optionally substituted aryl and an optionally substituted heterocyclic group.
[2]
2. A compound according to claim 1, wherein the compound is selected from compounds of the formula II:
in whichX1 is selected from O, S, NH and CH2;R <2> from H, C1 – C6 alkyl, unsaturated C2 – C6 hydrocarbon radical, C1 – C6 alkylcarbonyl, optionally substituted 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;R 7 is selected from optionally substituted aryl, an optionally substituted heterocyclic group, optionally substituted nitrogen-containing heterocyclocarbonyl and C1-C3-alkylcarbonyl.
[3]
3. A compound according to claim 2, wherein the aryl and the heterocyclic group are optionally substituted by 1 to 5 substituents selected from the following group: C1-C10-alkyl, C3-C8-cycloalkyl, C1-C4-alkoxy, optionally substituted phenyl, optionally substituted phenoxy, benzyloxy, CF3, F, Cl, Br and I.
[4]
4. A compound according to claim 2, wherein R 2 is selected from H, C1-C6-alkyl, unsaturated C2-C6-hydrocarbon radical, C1-C3-alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C1-C6-alkoxycarbonyl, C1-C6 aminocarbonyl, hydroxy and C1-C6 alkoxy.
[5]
5. A compound according to claim 1, wherein the compound is selected from:
[6]
6. A pharmaceutical composition which comprises compounds according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.
[7]
7. Use of compounds according to any one of claims 1 to 5 for the production of a medicament for the treatment or prevention of diseases mediated by dihydroorotate dehydrogenase.
[8]
8. Use according to claim 7, wherein the disease mediated by dihydroorotate dehydrogenase is selected from malaria tropica, malaria tertiana, malaria ovale, malaria quartana, gram trypanosomiasis, dengue fever and other parasitic diseases.
[9]
9. Use according to claim 7, wherein the dihydroorotate dehydrogenase mediated disease is selected from rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus glomerulopathy and organ transplant rejection.
[10]
10. Use according to claim 7, wherein the disease mediated by dihydroorotate dehydrogenase is selected from melanoma and rejection induced by allo- and xeno-organ transplantation in a host.
[11]
11. Use of compounds according to any one of claims 1 to 5 for the production of a medicament for inhibiting the activity of dihydroorotate dehydrogenase.

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公开号 | 公开日

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DE112012004878T8|2014-10-23|

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CN103842350B|2015-11-25|

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DE112012004878T5|2014-08-14|

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CN103842350A|2014-06-04|

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CN103127095A|2013-06-05|

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WO2013075596A1|2013-05-30|

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法律状态:

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CN2011103766682A|CN103127095A|2011-11-23|2011-11-23|Synthesis and application of dihydrothiophenone derivative as pfDHODH inhibitor|

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