Bicyclic heteroaromatic compounds

专利摘要:
The present invention relates to a bicyclic heteroaromatic derivative according to formula (I) or a pharmaceutically acceptable salt thereof. In addition, the present invention can be used in pregnancy control therapy. Formula I Where R 1 is (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14C) aryl or (4-13C) heteroaryl, all of which are optionally substituted with one or more substituents; R 2 is (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, or (6-14C) aryl or (4-13C) heteroaryl; R 3 is (1-8C) alkyl, (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14) aryl or (4-13C) heteroaryl; Y is CH or N; Z is NH 2 or OH; A is S, N (H), N (R 4 ), O or a bond; B is N (H), O or a bond; X 1 -X 2 is C═C, C (O) —NH, NH—C (O), C (O) —O, O—C (O), C═N, N═C, or S or O.
公开号:KR20030040483A
申请号:KR10-2003-7004132
申请日:2001-09-17
公开日:2003-05-22
发明作者:팀머스코르넬리스마리우스；카르스텐스빌렘프레데릭요한
申请人:악조 노벨 엔.브이.；
IPC主号:

专利说明:

Bicyclic heteroaromatic compound {BICYCLIC HETEROAROMATIC COMPOUNDS}
[2] Gonadotropins perform important functions in a variety of body functions, including metabolism, thermoregulation, and reproductive processes. The pituitary gonadotropin FSH, for example, plays a central role in stimulation of follicle development and maturation, while LH induces ovulation (Sharp, RM Clin Endocrinol. 33 : 787-807, 1990; Dorrington and Armstrong, Recent Prog.Horm . Res. 35 : 301-342, 1979). Currently, LH is clinically combined with FSH for ovarian stimulation, ie ovarian contusion for induction of in vitro fertilization (IVF) and ovulation in infertile ovulation women (Insler, V., Int. J. Fertility 33 : 85-97 , 1988, Navot and Rosenwaks, J. Vitro Fert.Embryo Transfer 5 : 3-13, 1988), as well as male sexual dysfunction and male infertility.
[3] Gonadotropin acts to initiate ovarian and testicular differentiation and steroid synthesis, depending on the specific gonad cell type. The action of these pituitary and placental hormones is mediated by specific plasma membrane receptors that are members of a large class of G-protein coupled receptors. They consist of a single polypeptide with seven transmembrane domains and can interact with the Gs protein, activating adenyl cyclase.
[4] Gonadal stimulating hormones designated for therapeutic purposes can be isolated from human urine sources and are of low purity (Morse et al, Amer. J. Reproduct. Immunol. And Microbiology 17 : 143, 1988). Alternatively, they can be prepared as recombinant gonadotropin.
[5] Subcutaneous or intramuscular injection of gonadotropin along with other therapeutic proteins is necessary. However, it would be advantageous to activate the receptor with small molecules that can be administered via the oral or transdermal route.
[1] The present invention relates to compounds having glycoprotein hormone agonist or antagonistic activity, specifically compounds having luteinizing hormone (LH) agonist activity. The present invention also relates to bicyclic heteroaromatic derivatives, pharmaceutical compositions containing them, as well as the use of such compounds in medical treatment, in particular as pregnancy control agents.
[6] The present invention seeks to describe a method for preparing such low molecular weight hormone analogs that selectively activate one of the gonadotropin receptors. This should be considered as one of the main advantages of the present invention.
[7] Accordingly, the present invention relates to bicyclic heteroaromatic derivatives according to formula (I) or a pharmaceutically acceptable salt thereof:
[8]
[9] Where
[10] R ¹ is (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14C) aryl or (4-13C) heteroaryl, preferably R ¹ is (6-14C) aryl or ( 4-13C) heteroaryl;
[11] R ² is (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, or (6-14C) aryl or (4-13C) heteroaryl;
[12] R ³ is (1-8C) alkyl, (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14) aryl or (4-13C) heteroaryl;
[13] Y is CH or N;
[14] Z is NH ₂ or OH;
[15] A is S, N (H), N (R ⁴ ), O or a bond;
[16] R ⁴ may be selected from the same groups as described for R ² ;
[17] B is N (H), O or a bond.
[18] R ¹ within the ring system is ^{R 5, NHR 5, N (} R 4) R 5, OR 5 and / or optionally may be substituted by one or more substituents selected from SR ^5, where R ⁵ is (6-14C) aryl , (4-13C) heteroaryl, (6-14C) arylcarbonyl, (2-7C) heterocycloalkyl, (3-8C) cycloalkyl, (6-14C) aryloxycarbonyl, (6-14) Arylsulfonyl, (6-14C) arylaminocarbonyl, (6-14C) aryloxycarbonyl, (6-14C) arylaminosulfonyl, (6-14C) aryloxysulfonyl, (2-8C) al Kenyl, (2-8C) alkynyl, (2-7C) heterocycloalkylcarbonyl, (2-8C) alkenylsulfonyl, (2-8C) alkeneoxycarbonyl or (1-8C) alkyl, (1- 8C) alkylcarbonyl, (1-8C) alkylsulfonyl, (1-8C) (di) alkylaminocarbonyl, (1-8C) alkoxycarbonyl, (1-8C) (di) alkylaminosulfonyl or (1-8C) alkoxysulfonyl, wherein the alkyl group is hydroxyl, (1-8C) alkoxy, (2-7C) heterocycloalkyl (1-8C) alkoxy, (3-8C) cycloalkyl (1-8C) Alkoxy, (6-14C) aryl (1-8C) alkoxy, (4-13C) Teraryl (1-8C) alkoxy, (2-7C) heterocycloalkyl, (3-8C) cycloalkyl, (6-14C) aryl, (4-13C) heteroaryl, (1-8C) alkoxycarbonyl, (6-14C) aryloxycarbonyl, (1-8C) alkylcarbonyloxy, (6-14C) arylcarbonyloxy, (1-8C) alkylcarbonyl, (6-14C) arylcarbonyl, amine, (1-8C) alkylaminocarbonyl, (6-14C) arylaminocarbonyl, (1-8C) alkylcarbonylamino, (6-14C) arylcarbonylamino, (6-14C) (di) arylamino And (di) [(1-3C) alkoxy (1-3C) alkyl] amino and / or (1-8C) (di) alkylamino. The substituent in R ¹ is preferably selected from NHR ⁵ or OR ⁵ . Any substituent in R ⁵ in R ¹ is (2-7C) heterocycloalkyl-carbonyl, (6-14C) arylcarbonyl or (1-8C) alkyl, (1-8C) alkylcarbonyl or (l- 8C) (di) alkylaminocarbonyl, the alkyl group being (2-7C) heterocycloalkyl, (4-13C) heteroaryl, (1-8C) alkoxycarbonyl, (1-8C) alkylamino Carbonyl, (1-8C) alkylcarbonylamino, (6-14C) arylcarbonylamino, amine and / or (1-8C) (di) alkylamino. Most preferred substituents on the alkyl group are (2-7C) heterocycloalkyl, (1-8C) (di) alkylamino, amine and (1-8C) (di) alkylaminocarbonyl. Most preferred in R ¹ is phenyl optionally substituted with one of the above substituents, preferably the meta position. In the compounds according to the invention, X 1 -X 2 is C═C, C (O) —NH, NH—C (O), C (O) —O, OC (O), C═N or N═C. R ⁵ is (1-8C) alkylsulfonyl, (6-14C) arylsulfonyl, (1-8C) (di) alkylaminocarbonyl, (6-14C) arylaminocarbonyl, (1-8C) alkoxy Carbonyl, (6-14C) aryloxycarbonyl, (1-8C) (di) alkylaminosulfonyl, (6-14C) arylaminosulfonyl, (1-8C) alkoxysulfonyl, (2-7C) When heterocycloalkylcarbonyl, (2-8C) alkenylsulfonyl, (2-8C) alkenoxycarbonyl or (6-14C) aryloxysulfonyl, X 1 -X 2 may further be S or O.
[19] Preferred compounds according to the invention are compounds of formula I, wherein B is N (H) or a bond and / or Z is NH ₂ . Among these preferred compounds, it is particularly preferable that B is N (H) or a bond and Z is NH ₂ . Most preferred compounds, in addition to the above definitions of B and Z, R ¹ is at least one substituent selected from N (R ⁴ ) R ⁵ , NHR ⁵ , R ⁵ , OR ⁵ and / or SR ⁵ , preferably NHR ⁵ or OR ⁵ To (6-14) aryl or (4-13C) heteroaryl optionally substituted by.
[20] N is preferred for Y in all of the above compounds, and N (H) or bond is preferred for B. When B is a bond, R ³ is preferably (2-7C) heterocycloalkyl.
[21] In all the above compounds, X 1 -X 2 is preferably C = C, C = N or N = C, and most preferably C = C. R ⁵ is (1-8C) alkylsulfonyl, (6-14C) arylsulfonyl, (1-8C) (di) alkylaminocarbonyl, (6-14) arylaminocarbonyl, (1-8C) alkoxy Carbonyl, (6-14C) aryloxycarbonyl, (1-8C) (di) alkylaminosulfonyl, (6-14C) arylaminosulfonyl, (1-8) alkoxysulfonyl, (2-7C) In the case of heterocycloalkylcarbonyl, (2-8C) alkenylsulfonyl, (2-8C) alkenoxycarbonyl or (6-14C) aryloxysulfonyl, S is preferred in addition to the above groups for X 1 -X 2.
[22] Most preferred compound is t-butyl 5-amino-2-methylthio-4- (3-((N, N-diethylamino) carbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6 -Carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- (methoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3-allyloxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio -4- (3- (ethoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- ( (Morpholin-4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- (1 , 2,3,6-tetrahydropyridinocarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-phenyl-4- (3- ((N, N-dimethylamino) carbonylamino) phenyl) thieno [2,3-d] pi The pyrimidine compound is selected from 6-carboxamide.
[23] Compound Ethyl 5-hydroxy-2-methyl-4- (piperidin-1-yl) pyrido [2,3-d] pyrimidine-6-carboxylate, ethyl 5-hydroxy-2-methyl- 4- (morpholin-4-yl) pyrido [2,3-d] pyrimidine-6-carboxylate and ethyl 5-hydroxy-2-methyl-4- (pyrrolidin-1-yl) pyrid Figure [2,3-d] pyrimidine-6-carboxylate is excluded from the present invention.
[24] Compounds not claimed in the above are those relating to the disclosure of Chem. Pharm. Bull. 18 (7), 1385-1393 (1970).
[25] The term (1-8C) alkyl used in the definition of formula (I) is a branched or unbranched alkyl group having 1 to 8 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, t- Butyl, hexyl and octyl. (1-6C) alkyl groups are preferred, and (1-3C) alkyl groups are most preferred.
[26] The term (2-8C) alkenyl refers to branched or unbranched alkenyl groups having 2 to 8 carbon atoms, such as ethenyl, 2-butenyl and the like. (1-6C) alkenyl groups are preferred, with (1-3C) alkenyl groups being most preferred.
[27] The term (2-8C) alkynyl refers to branched or unbranched alkynyl groups having 2 to 8 carbon atoms, such as ethynyl and propynyl. Most preferred is a (2-4C) alkynyl group.
[28] The term (6-14C) aryl means aromatic hydrocarbons having 6 to 14 carbon atoms, such as phenyl, naphthyl, tetrahydronaphthyl, indenyl, atrasyl, which are not intended to be limiting, but are not intended to be limiting, such as hydroxy Optionally substituted with halogen, nitro, trifluoromethyl, cyano, (1-8C) alkylcarbonylamino, (1-8C) alkylaminocarbonyl or (1-8C) (di) alkylamino, The alkyl moiety has the same meaning as defined above. The (6-10C) aryl group is more preferred. Most preferred aromatic hydrocarbon group is phenyl.
[29] The term (3-8C) cycloalkyl is a cycloalkyl group having 3 to 8 carbon atoms, ie cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl.
[30] The term (2-7C) heterocycloalkyl refers to a heterocycloalkyl group having from 2 to 7, preferably 2 to 5 carbon atoms and comprising at least one hetero atom selected from N, O or S. Preferred heteroatoms are N or O. The nitrogen containing heterocycloalkyl group can be linked via a carbon or nitrogen atom. Most preferred heterocycloalkyl groups are piperidine, morpholine and pyrrolidine.
[31] The term (2-7C) heterocycloalkylcarbonyl refers to a heterocycloalkyl group having from 2 to 7 carbon atoms as defined above, linked to a carbonyl group.
[32] The term (1-8C) alkoxy means an alkoxy group having from 1 to 8 carbon atoms in which the alkyl moiety has the same meaning as defined above. (1-6C) alkoxy is preferred, and (1-3C) alkoxy is most preferred.
[33] The term (1-8C) alkoxycarbonyl group means an alkoxycarbonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkoxycarbonyl group is preferred, and the (1-3C) alkoxycarbonyl group is most preferred.
[34] The term (2-8C) alkenoxycarbonyl means an alkenoxycarbonyl in which the alkenyl group contains 2 to 8 carbon atoms and has the same meaning as defined above. (2-6C) Alkenoxycarbonyl groups are preferred. And a (2-3C) alkeneoxycarbonyl group is most preferred.
[35] The term (1-8C) alkoxysulfonyl means an alkoxysulfonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkoxysulfonyl group is preferable, and the (1-3C) alkoxysulfonyl group is most preferable.
[36] The term (1-8C) (di) alkylamino refers to a (di) alkylamino group having 1 to 8 carbon atoms and the alkyl moiety has the same meaning as defined above. The (1-6C) (di) alkylamino group is more preferable.
[37] The term di [(1-3C) alkoxy (1-3C) alkyl] amino refers to a (di) [alkoxyalkyl] amino group having both the alkyl moiety and the alkoxy moiety having from 1 to 3 carbon atoms and having the same meaning as defined above it means.
[38] The term (6-14C) (di) arylamino means a (di) arylamino group having 6 to 14 carbon atoms, in which the aryl moiety has the same meaning as defined above. The (6-10C) (di) arylamino group is more preferred. Most preferred (di) arylamino groups are (di) phenylamino.
[39] The term (1-8C) alkylthio means an alkylthio group having from 1 to 8 carbon atoms in which the alkyl moiety has the same meaning as defined above. Most preferred are (1-4C) alkylthio groups.
[40] The term (6-14C) aryloxycarbonyl means an aryloxycarbonyl group containing 6 to 14, more preferably 6 to 10 carbon atoms, and having the same meaning as defined above. Most preferred are phenoxycarbonyl groups.
[41] The term (6-14C) aryloxysulfonyl means an aryloxycarbonyl group in which the aryl group contains 6 to 14, more preferably 6 to 10 carbon atoms, and has the same meaning as defined above. Most preferred are phenoxysulfonyl groups.
[42] The term (6-14C) aryl (1-8C) alkyl means an arylalkyl group having 7 to 22 carbon atoms, wherein as defined above, the alkyl group is a (1-8C) alkyl group and the aryl group is (6-14C) Aryl group. More preferred is a (6-10C) aryl (1-4C) alkyl group. Phenyl (1-4C) alkyl groups such as benzyl are the most preferred arylalkyl groups.
[43] The term (4-13C) heteroaryl refers to a substituted or unsubstituted aromatic group having 4 to 13 carbon atoms, preferably 4 to 9 carbon atoms, including one or more heteroatoms selected from N, O and / or S. Dazolyl, thienyl, benzthienyl, quinolyl, tetrahydroquinolyl, isoquinolyl, tetrahydroisoquinolyl, indolyl, acridinolyl, furyl or pyridyl. Substituents on the heteroaryl group may be selected from the group of substituents listed for the aryl group. Preferred heteroaryl groups are thienyl, furyl, pyridyl and pyrimidyl. The nitrogen containing heteroaryl group may be linked via a carbon atom or a nitrogen atom.
[44] The term halogen means fluorine, chlorine, bromine or iodine.
[45] The term (2-7C) heterocycloalkyl (1-8C) alkoxy contains 2 to 7 carbon atoms as defined above wherein the alkoxy moiety is bonded to a (1-8C) alkoxy group having the meaning as defined above. Means a heterocycloalkyl group. More preferred is a (2-5C) heterocycloalkyl (1-4C) alkoxy group.
[46] The term (3-8C) cycloalkyl (1-8C) alkoxy includes 3 to 8 carbon atoms as defined above wherein the alkoxy moiety is bonded to a (1-8C) alkoxy group having the meaning as defined above. It means a cycloalkyl group. More preferred is a (3-6C) cycloalkyl (1-4C) alkoxy group.
[47] The term (6-14C) aryl (1-8C) alkoxy is aryl containing 6 to 14 carbon atoms as defined above, wherein the alkoxy moiety is bonded to a (1-8C) alkoxy group having the meaning as defined above. Means a flag. The (6-10C) aryl (1-4C) alkoxy group is more preferred, and the phenyl (1-4C) alkoxy group is most preferred. (4-13C) heteroaryl (1-8C) alkoxy groups are analogs of (6-14C) aryl (1-8C) alkoxy groups containing one or more heteroatoms selected from N, O and S in the heteroaryl ring. More preferred is a (4-9C) heteroaryl (1-4C) alkoxy group.
[48] The term (1-8C) alkylcarbonyl means an alkylcarbonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkylcarbonyl group is more preferred, and the (1-4C) alkylcarbonyl group is most preferred.
[49] The term (6-14C) arylcarbonyl means an arylcarbonyl group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) arylcarbonyl group is more preferred, and the phenylcarbonyl group is most preferred.
[50] The term (1-8C) alkylsulfonyl means an alkylsulfonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkylsulfonyl group is more preferable, and the (1-4C) alkylsulfonyl group is most preferable.
[51] The term (2-8C) alkenylsulfonyl means an alkenylsulfonyl group in which the alkenyl group contains 2 to 8 carbon atoms and has the same meaning as defined above. The (2-6C) alkenylsulfonyl group is more preferable, and the (2-4C) alkenylsulfonyl group is most preferable.
[52] The term (6-14C) arylsulfonyl means an arylsulfonyl group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) arylsulfonyl group is more preferred, and the phenylsulfonyl group is most preferred.
[53] The term (1-8C) alkylcarbonyloxy means an alkylcarbonyloxy group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkylcarbonyloxy group is more preferable. Most preferred are (1-4C) alkylcarbonyloxy groups.
[54] The term (6-14C) arylcarbonyloxy means an arylcarbonyloxy group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) arylcarbonyloxy group is more preferred, and the phenylcarbonyloxy group is most preferred.
[55] The term (1-8C) (di) alkylaminocarbonyl means a (di) alkylaminocarbonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) (di) alkylaminocarbonyl group is more preferred, and the (1-4C) (di) alkylaminocarbonyl group is most preferred.
[56] The term (6-14C) (di) arylaminocarbonyl means a (di) arylaminocarbonyl group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) (di) arylaminocarbonyl group is more preferred, and the (di) phenylaminocarbonyl group is most preferred.
[57] The term (1-8C) (di) alkylaminosulfonyl means a (di) alkylaminosulfonyl group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) (di) alkylaminosulfonyl group is more preferable, and the (1-4C) (di) alkylaminosulfonyl group is most preferred.
[58] The term (6-14C) (di) arylaminosulfonyl means a (di) arylaminosulfonyl group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) (di) arylaminosulfonyl group is more preferable, and the (di) phenylaminosulfonyl group is most preferred.
[59] The term (1-8C) alkylcarbonylamino means an alkylcarbonylamino group in which the alkyl group contains 1 to 8 carbon atoms and has the same meaning as defined above. The (1-6C) alkylcarbonylamino group is more preferred, and the (1-4C) alkylcarbonylamino group is most preferred.
[60] The term (6-14C) arylcarbonylamino means an arylcarbonylamino group in which the aryl group contains 6 to 14 carbon atoms and has the same meaning as defined above. The (6-10C) arylcarbonylamino group is more preferred, and the phenylcarbonylamino group is most preferred.
[61] The term (2-7C) heterocycloalkyloxy means a heterocycloalkyl group containing 2 to 7 carbon atoms as defined above, bonded to an oxygen atom. Most preferred is a (2-5C) heterocycloalkyloxy group.
[62] The term (3-8C) cycloalkyloxy means a cycloalkyl group containing 3 to 8 carbon atoms as defined above, bonded to an oxygen atom.
[63] The term (6-14) aryloxy means an aryl group containing 6 to 14 carbon atoms as defined above, bonded to an oxygen atom. The (6-10C) aryloxy group is more preferred, and the phenoxy group is most preferred. (4-13C) heteroaryloxy groups are analogs of (6-14C) aryloxy groups containing one or more heteroatoms selected from N, O and S in the heteroaryl ring. More preferred is a (4-9C) heteroaryloxy group.
[64] The compounds of formula (I) are capable of binding to LH receptors and have been shown to exhibit functional LH activity.
[65] The present invention also relates to pharmaceutical compositions comprising bicyclic heteroaromatic derivatives of formula (I) or salts thereof.
[66] Thus, the compounds according to the invention can be used for treatment. Another aspect of the invention relates to the use of a bicyclic heteroaromatic compound of formula (I) for the manufacture of a medicament for controlling pregnancy. The compounds of the present invention are preferably used to activate the LH receptor.
[67] The bicyclic heteroaromatic derivatives of the present invention may have one or more chiral carbon atoms. Therefore, the compounds of the present invention can be obtained as chirally pure compounds or as mixtures of diastereomers and / or enantiomers. Methods of obtaining chirally pure compounds, such as crystallization or chromatography, are well known in the art.
[68] For therapeutic purposes, the salts of the compounds of formula (I) are those in which the counter ion is pharmaceutically acceptable. However, acid addition salts of bases according to formula (I) can also be used for use in the preparation or purification of pharmaceutically acceptable compounds, for example. All salts which are pharmaceutically acceptable or not acceptable are included within the scope of the present invention.
[69] Examples of acid addition salts are those derived from inorganic acids such as hydrochloric acid, phosphoric acid, sulfuric acid, preferably hydrochloric acid, and organic acids such as citric acid, tartaric acid, acetic acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid and the like.
[70] In addition, suitable routes of administration for the compounds of formula (I) or pharmaceutically acceptable salts thereof according to the invention as active ingredients are intramuscular injection, subcutaneous injection, intravenous injection or intraperitoneal injection, oral administration and intranasal administration. The compound of the present invention is preferably administered orally. The exact dosage and administration regimen of the active ingredient or pharmaceutical composition thereof will depend essentially on the therapeutic effect (infertility treatment; contraception) desired to be achieved, and the specific compound, route of administration, and age of the individual patient to which the drug is administered. It will vary depending on the condition.
[71] In general, parenteral administration requires a lower dosage than other methods of administration, which are more dependent on adsorption. However, the dosage for humans preferably contains 0.0001 to 25 mg per kg body weight. Certain doses may be given as a single dose, or as multiple sub-doses administered at appropriate intervals over the day, or as female doses at appropriate routine intervals throughout the menstrual cycle. In addition to the dosage form, the dosage regimen may differ between female and male recipients.
[72] For in vitro or ex vivo applications, for example in IVF applications, the compounds of the invention may be used in incubation media at concentrations of approximately 0.01-5 μg / ml.
[73] Accordingly, the present invention also relates to pharmaceutical compositions comprising a bicyclic heteroaromatic compound according to formula (I) and optionally other therapeutic agents in admixture with a pharmaceutically acceptable adjuvant. Adjuvants should be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the recipient. The pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (eg transdermal, buccal and sublingual), vaginal or parenteral (eg subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions of the present invention are those described in any method well known in the pharmaceutical art, such as in Gennaro et al., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing company, 1990, in particular in Part 8: Pharmaceutical Preparations and Their Manufacture. It can be prepared using the same method.
[74] Such methods include the step of bringing into association the active ingredient with any adjuvant. Auxiliary (s), also called auxiliaries, are those conventional in the art (Gennaro, supra) such as fillers, binders, diluents, disintegrants, lubricants, colorants, flavors and wetting agents.
[75] Pharmaceutical compositions suitable for oral administration may be presented as separate formulation units such as pills, tablets or capsules, or as powders or granules, or as solutions or suspensions. In addition, the active ingredient may be provided as a bullion or a paste. The compositions of the present invention may be further processed as suppositories or enemas for rectal administration.
[76] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injectables. The compositions of the present invention may be provided in unit dosage or multiple dosage containers, such as sealed vials and ampoules, and may be stored in lyophilized (freeze drying) conditions where only adding water prior to use is a sterile liquid carrier, such as. .
[77] Compositions or formulations suitable for nasal inhalation administration include fines or mists that may be generated by metered dose pressurized aerosols, nebulizers or blowers.
[78] The bicyclic heteroaromatic derivatives of the present invention may also be administered in the form of implantable pharmaceutical devices consisting of a core of active substance embedded in a release rate controlling membrane. Such implants will be applied subcutaneously or topically to release the active ingredient at approximately constant rate over a relatively long period of time, such as weeks to years. Methods of making such implantable pharmaceutical devices are known in the art, for example as described in European Patent No. 0,303,306 (Akzo V. V.).
[79] Thus, the compounds according to the invention can be used for the same clinical purposes as natural LH, and have the advantage that they exhibit altered stability properties and can be administered differently.
[80] In general, the compounds of the present invention represented by the following formula (I-a), wherein B is NH, can be prepared by condensation reactions known in the art of an acid of formula (II) and an amine of formula (III).
[81]
[82] The reaction is carried out at room temperature in a suitable solvent such as N, N-dimethylformamide (DMF) or an aprotic solvent such as dichloromethane, O- (benzotriazol-1-yl) -N, N, N ', N'. Coupling agents such as tetramethyluronium tetrafluoroborate (TBTU) or bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP) and tertiary amine bases such as N, N-diisopropylethylamine (DiPEA) Do it using
[83] Likewise, compounds of formula (I), wherein B is O, represented by formula (I-b), can be prepared in the same manner as described above for compounds of formula (I-a), starting with an acid of formula (II) and an alcohol of formula (IV).
[84]
[85] The compound of formula (I), wherein B is a bond represented by formula (Ic), can be obtained by reacting a suitable organometallic reagent with a derivative of formula (V) in an aprotic solvent such as THF. Related substitution reactions can be found in SV Frye, MC Johnson, NLValvano, J. Org. Chem. 56 : 3750, 1991. The Weinreb amide of Formula V can be synthesized from the acid of Formula II and N-methoxy-N-methyl amide using the conditions described for preparation in the amide of Formula Ia.
[86]
[87] A suitable method for the preparation of intermediate acid II is the base mediated saponification reaction known in the art of the ethyl ester of formula VI: The saponification reaction is carried out in aqueous dioxane at high temperature (40 ° C. to reflux temperature) in the presence of a base such as lithium hydroxide, potassium hydroxide or sodium hydroxide, followed by acidic work up.
[88]
[89] Bicyclic lactams of formula (VII) are useful starting materials for the preparation of the imides (VIII). In a typical experiment, lactam is converted to the corresponding chloroimine using phosphoryl chloride at high temperature (60 ° C. to reflux temperature) in a suitable solvent such as 1,4-dioxane. By treatment with a reducing agent such as hydrogen in the presence of a suitable catalyst in ethanol, certain imines of formula (VIII) can be isolated. Related reduction reactions have been reported in the literature (see, eg, E. Bisagni, C. Landras, S. Thirot and C. Huel, Tetrahedron 52 : 10427, 1996).
[90]
[91] Bicyclic lactams of formula (VII) can be prepared by the condensation reaction of an acid of type IX with diethyl aminomalonate under the operation of a coupling agent such as TBTU / DiPEA and by cyclization of the intermediate amide in ethanol under basic conditions. Aromatization together with the accompanying deethoxycarbonylation then gives a bicyclic type VII. In an alternative procedure, glycine ethyl ester can be used in place of diethyl aminomalonate. See, eg, M. Blanco, MG Lorenzo, I. Perillo, CB Schapira, J. Heterocycl. Chem. 33 : 361, 1996. In addition, cyclization of the intermediate amide can be carried out by tin chloride (IV). The use of tin (IV) chloride in the ring closure reaction to related systems has been reported (AC Veronese, R. Callegari, CF Morelli, Tetrahedron 51 : 12277, 1995).
[92]
[93] By treating the acid of formula IX with ethyl bromoacetate in an aprotic solvent under the influence of a non-nucleophilic base such as potassium carbonate or potassium t-butoxylate, an aromatic lactone of formula X can be obtained. Related structures are described in C. Bhakta, Indian J. Chem. Sect. B. 25 : 189, 1986.
[94]
[95] When the vinyl derivative of the formula (XI) is treated with diethyl malonate under basic conditions, a conjugate addition reaction occurs. Intermediate adducts can be cyclized to bicyclic formula (XII) by tin chloride (IV) mediated reactions and work up.
[96] Alternatively, t-butyl ethyl malonate can be used in this process instead of diethyl malonate.
[97]
[98] Treatment at high temperatures with a suitable oxidizing agent such as DDQ, or a palladium catalyst in a suitable solvent such as xylene, results in aromatization of the derivative of formula (XII) to quinazoline or quinoline of formula (XIII). See also, K. Kobayashi, T. Uneda, K. Takada, H. Tanaka, T. Kitamura, O. Morikawa, H. Konishi, J. Org. Chem. 62: 664, 1997.
[99]
[100] In another approach, methyl pyri (mi) dine of formula XIV is reacted with lithium hexamethyldisilazane (LiHMDS) or lithium diisopropylamide (LDA) in a suitable aprotic solvent such as THF at low temperature (-78 ° C). Deprotonation in the methyl group using a strong base such as. This anion is then reacted with ethyl 3-ethoxyacrylate. K. Kobayshi, K. Takada, H. Tanaka, T. Uneda, T. Kitamura, Chem. Lett . : 25 , 1996; K. Kobayashi, T. Uneda, K. Takada, H. Tanaka, T. Kitamura , O. Morikawa, H. Konishi, J. Org. Chem. 62 : 664, 1997), after the conjugate addition reaction, cyclization of quinoline or quinazoline of formula XIII occurs.
[101]
[102] Treatment of the chloride of general formula (XV) with (N-alkyl) beta-aniline ethyl ester of general formula (XVI) under basic conditions gives a bicyclic compound of general formula (XVII). Related variants are described in PY Boamah, N. Haider, G. Heinisch, Arch. Pharm. (Weinheim) 323 : 207, 1990.
[103]
[104] Compounds of formula (XVII) wherein R ⁶ is H can be oxidized to derivatives of formula (XVIII) using palladium on charcoal at high temperatures. See, eg, M. Onda, K. Kawakami, Chem. Pharm. Bull. 20 : 1484, 1972. Compounds of formula XVII wherein R ⁶ is H or alkyl are described in H. Ishii, Chem. Pharm. Bull. 26 : 864, 1978; JI DeGraw, PH Christie, WT Colwell, FM Sirotnak, J. Med. Chem. 35 : 320, 1992), DDQ can be used to convert to imines of formula XVIII.
[105]
[106] Alternatively, the imine of formula (XVIII) can be converted to the chloroimine in a similar manner (see above) as the positional isomer of formula (VIII), i.e. by converting to chloroimine using POCl ₃ and then dehalogenated using hydrogen and a suitable catalyst. It can be prepared from lactams.
[107]
[108] Lactam of formula (XIX) can be prepared by acylating the aminopyri (mi) dine of formula (XX) with chloroethyl malonate in the presence of a suitable base. Subsequent ring closure of the intermediate acyclic malonamide under the action of base (sodium ethoxylate in ethanol) or tin (IV) gives a bicyclic lactam of type XIX. Similar variations are described in, for example, AC Veronese, R. Callegari, CF Morelli, Tetrahedron 51 : 12277, 1995; W. Stadlbauer, S. Prattes, W. Fiala, J. Heterocycl. Chem. 35 : 627, 1998 Reported in
[109]
[110] According to the substitution pattern of the amino pyri (mi) dine of formula (XX), the lactam of formula (XIX) is tin chloride (IV) mediated coupling of diethylmalonate, followed by high temperature in a suitable solvent (eg, 240 ° C. in diphenyl ether). It can be prepared by cyclization.
[111] In a similar manner, lactones of the following type XXII can be prepared by O-acylating lactams of formula XXI with chloroethyl malonate followed by cyclization.
[112]
[113] Furopyri (mi) dine of formula (XXIII) can be obtained by selectively O-alkylating lactams of type XXI with potassium carbonate in acetone and then ring-closing under the influence of sodium ethoxylate in ethanol.
[114]
[115] Thienopyri (mi) dine of the general formula XXIV can be obtained by treating the chloride of formula XV with ethyl mercaptoacetate under the influence of a strong base. In a typical experiment, 1 equivalent of chloride of formula XV is reacted with 1.5 equivalents of ethyl mercaptoacetate and 2 equivalents of potassium t-butoxide in THF. Under these conditions, the acyclic sulfide undergoes spontaneous cyclization to form thienopyri (mi) dine of the following formula XXIV. When R ¹ is a (hetero) aryl substituted with an electron withdrawing group such as nitro, the cyclization involves the isolation of the intermediate thioether followed by treatment with a tertiary base such as DIPEA in toluene / EtOH at reflux temperature. Perform thiophene ring formation by following a two step procedure.
[116]
[117] The pyri (mi) dine carboxylates of formula (IX) are prepared using a strong base such as lithium hydroxide or potassium hydroxide at high temperature (40 ° C. to reflux temperature) in a mixture of water and an organic cosolvent such as 1,4-dioxane or methanol The alkoxycarbonyl pyri (mi) dine of the following general formula (XXV) can be obtained by saponification and then acid work up. When W is CO ₂ alkyl, R ⁷ is preferably benzyl capable of selective hydrogenolysis of benzyl ester functionality without affecting substituent W.
[118]
[119] The compounds of formula (XXV) can be prepared by palladium catalyzed alkoxycarbonylation of chlorides of formula (XV) in the presence of carbon monoxide and the appropriate alcohols (R ⁷ OH, XXVI). Similar variations are shown in the literature (see, eg, Y. Bessard, R. Crettaz, Heterocycles 51 : 2589, 1999).
[120]
[121] Palladium catalyzed reaction of chloride of formula (XV) with vinyl tributylstannan or tetravinylstanan yields vinyl pyri (mi) dine of formula (XI). Conversions of this type are described in, for example, LL Gundersen, AK Bakkestuen, AJ Aasen, H. Oeveras, F. Rise, Tetrahedron 50 : 9743, 1994; F. Guillier, F. Nivoliers, A. Godard, F. Marsais, G Queguiner, J. Heterocycl. Chem. 36 : 1157, 1999).
[122] According to the substitution pattern of the chloride of formula (XV), vinyl pyri (mi) dine of formula (XI) can also be prepared by substituting the chloride with methylene triphenyl phosphane and then reacting with (para) formaldehyde.
[123]
[124] Similarly, the chloride of formula XV is treated with trimethylalan in an aprotic solvent such as THF in the presence of a palladium catalyst to give methyl pyri (mi) dine of formula XIV. Related synthesis is described in Q. Lu, I. Mangalagiu, T. Benneche and K. Undheim, Acta Chem. Sc. 51 : 302, 1997.
[125]
[126] The compounds of formula (XXI), wherein Y is N, represented by formula (XXI-a) can be prepared by the methods described in several documents.
[127]
[128] For example, derivatives of formula XXI-a, wherein R ¹ is (6-14C) aryl or (4-13C) heteroaryl, may be substituted with an aldehyde of formula XXVIII with an aldehyde of formula XXVIII, By condensation reaction with a compound of formula XXIX, which may be urea (XXIX-a), isourea (XXIX-b), monosubstituted guanidine (XXIX-c), disubstituted guanidine (XXIX-d) or amidine (XXIX-e) Can be synthesized.
[129]
[130] In a typical experiment, the components of formulas XXVII, XXVIII and XXIXa-e are suspended in a suitable solvent such as ethanol, methanol, N, N-dimethylformamide, N-methylpyrrolidinone, tetrahydrofuran or pyridine and potassium carbonate , Base such as sodium acetate, sodium methoxide or sodium ethoxylate is added. The reaction takes place at high temperature (70 ° C. to reflux temperature). See, eg, S. Kambe, K. Saito and H. Kishi, Synthesis : 287 , 1979; AM Abd-Elfattah, SM Hussain and AM El-Reedy, Tetrahedron 39 : 3197, 1983; SM Hussain, AA El-Barbary and SA Mansour, J. Heterocycl. Chem. 22 : 169, 1985). When W is C (O) OEt, aromatization occurs upon addition of an oxidant such as DDQ or oxygen. Related cyclization can also be carried out on a solid support such as Merrifield resin using a suitable linker, see for example AL Mrzinzik and ER Felder, J. Org. Chem. 63 : 723, 1998; T. Masquelin, D. Sprenger, R. Baer, F. Gerber and Y. Mercadal, Helv. Chim. Acta 81 : 646, 1998).
[131] Compounds of formula (XX), wherein Y is N, represented by formula (XX-a), can be prepared by similar condensation methods using malonitrile.
[132]
[133] Alternatively, the compound of formula XX may be obtained by ammonia decomposition of the chloride of formula XV using a suitable organic cosolvent such as 1,4-dioxane of chlorides such as aqueous ammonia and 1,4-dioxane. This modification can also be accomplished with tertiary amine bases such as ammonium chloride and DiPEA in an aprotic solvent such as DMF.
[134]
[135] The chloride of formula XV can be synthesized by known reaction of lactam of formula XXI with POCl ₃ at high temperature (60 ° C. to reflux temperature) in a suitable solvent such as 1,4-dioxane.
[136]
[137] Derivatives of formula (XV) wherein Y is N and R ¹ is not (6-14C) aryl or (4-13C) heteroaryl can be prepared by monosubstituted Cl with various nucleophiles in derivatives of formula XXX: Related substitution reactions are described in, for example, S. Kohra, Y. Tominaga and A. Hosomi, J. Heterocycl. Chem. 25 : 959, 1988; AA Santilli, DH Kim and SV Wanser, J. Heterocycl.Chem. 8 : 445 , 1971; J. Clark, MS Shannet, D. Korakas and G. Varvounis, J. Heterocycl.Chem. 30 : 1065, 1993; S. Tumkevicius, Liebigs Ann.Org.Borgorg.Chem . 9 : 1703, 1995). You can find it.
[138]
[139] The pyridine of Formula XXI, wherein Y is CH, A is S, and W is CN, represented by Formula XXI-b, is described in the following formula XXXI, as described in P. Milart, Tetrahedron 54 : 15643-15656, 1998 Α, β-unsaturated dinitrile of can be obtained by continuous alkylation with carbon disulfide and alkyl iodide R ² -I to obtain a compound of formula XXXII. The compounds of formula (XXXII) are cyclized under acidic conditions as described in K. Peseke, Z. Chem. 29 : 442-443 (1989) to afford pyridine of formula XXI-b.
[140]
[141] Compounds of formula (XXI), wherein A is a bond, Y is CH, and W is as defined above, are represented by formula (XXI-c) in a suitable solvent such as dimethylsulfoxide in the presence of oxygen Unsaturated ketones of Formula (XXXIII) can be prepared by reacting W with an amide of Formula (XXXIV) as defined above. Related cyclization reactions are described in R. Jain, F. Roschangar, MA Ciufolini, Tetrahedron Lett. 36 : 3307, 1995.
[142]
[143] Methods for measuring receptor binding, as well as in vitro and in vivo assays for measuring the biological activity of gonadotropin, are well known. In general, the expressed receptor is contacted with the compound to be tested and the binding or stimulation or inhibition of the functional response is measured.
[144] To measure the functional response, isolated DNA encoding the LH receptor gene, preferably the human receptor, is expressed in a suitable host cell. Such cells may be Chinese hamster ovaries, but other cells are also suitable. The cells are preferably of mammalian origin (Jia et al., Mol. Endocrin. , 5 : 759-776, 1991).
[145] Methods of constructing recombinant LH expressing cell lines are well known in the art (Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, latest edition). Expression of the receptor is obtained by expression of DNA encoding a given protein. Techniques for site directed mutagenesis, ligation of additional sequences, PCR and construction of suitable expression systems are all well known in the art by now. Some or all of the DNA encoding a given protein may be synthetically constructed using standard solid phase techniques, preferably to include restriction sites for ease of ligation. Suitable control elements for transcription and translation of the included coding sequence can be provided to the DNA coding sequence. As is well known, expression systems are currently useful with a wide variety of hosts including prokaryotic hosts such as bacteria and eukaryotic hosts such as yeast, plant cells, insect cells, mammalian cells, algal cells and the like.
[146] Cells expressing the receptor are then contacted with a test compound to observe binding or stimulation or inhibition of a functional response.
[147] Alternatively, the binding of the compound is measured using an isolated cell membrane containing the expressed receptor.
[148] For the measurement of binding, radioactive or fluorescently labeled compounds can be used. Human recombinant LH can be used as a reference compound. Alternatively, competitive binding assays can also be performed.
[149] Another assay involves screening for LH receptor agonist compounds by measuring stimulation of receptor mediated cAMP accumulation. Thus, such methods involve expressing a receptor on a host cell surface and exposing the cell to a test compound. Then, the amount of cAMP is measured. The level of cAMP will decrease or increase as it inhibits or stimulates the effect of the test compound upon binding to the receptor.
[150] In addition to direct measurement of cAMP, for example in exposed cells, cell lines transfected with DNA encoding the receptor, as well as with a second DNA whose expression encodes a reporter gene that responds to cAMP receptors, can be used. . Such reporter genes may be cAMP inducible or may be constructed in a way that links them to novel cAMP reactive elements. In general, reporter gene expression can be regulated by any response element that acts to change cAMP levels. Examples of suitable reporter genes are LacZ, alkaline phosphatase, firefly luciferase and green fluorescent protein. The principles of such cross activation assays are well known in the art and are described, for example, in Stratowa, Ch, Himmler, A and Gzernilofsky, AP (1995) Curr. Opin. Biotechnol . 6 : 574.
[151] In order to select active compounds, testing at 10 ^-5 M must represent a case of using a material based on an LH, more than 20% activity of the maximum activity. Another criterion would be an EC _{50 which} should be less than 10 ⁻⁵ M, preferably less than 10 ⁻⁷ M.
[152] Those skilled in the art will recognize that the preferred EC ₅₀ value depends on the compound tested. For example, compounds having an EC ₅₀ of less than 10 ⁻⁵ M are generally considered candidates for drug selection. This value is preferably less than 10 ⁻⁷ M. However, compounds with high EC ₅₀ , which are selective for certain receptors, may be better candidates.
[153] In addition, screening for LH receptor agonist compounds can be performed by mouse Leydig cell bioassay (Van Damme, M., Robersen, D. and Diczfalusy, E. (1974). Acta Endocrinol. 77 : 655-671 Mannaerts, B., Kloosterboer, H. and Schuurs, A. (1987). Neuroendocrinology of reproduction.R. Rolland et al. Eds., Elsevier Science Publishers BV, 49-58). In this assay, the activity of LH receptor mediated testosterone production can be measured in Lydig cells isolated from male mice.
[154] To measure the in vivo activity of LH receptor agonist compounds, ovulation induction in immature mice can be studied. In this assay, immature female mice can be primed with urine FSH and treated with LH agonistic compounds after approximately 48 hours. This animal can be sacrificed after LH agonist treatment and the number of ovaries in the fallopian tubes can be measured under a microscope.
[155] The compounds of the present invention can be clinically applied in regimes where LH or hCG is currently used. These include intermediate cycle administration to induce LH substitutions, ovulation (induction of ovulation (OI) or controlled stimulation (COH) or corpus luteum) in patients with male or female sexual dysfunction.
[156] The following examples illustrate the invention and should not be construed as limiting the scope of the invention.
[157] Example 1
[158] t-butyl 5-amino-2-methylthio-4- (3- (methoxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[159] (a) 5-cyano-4- (3-methoxyphenyl) -2-methylthio-6-hydroxypyrimidine
[160] A mixture of S-methylisothiourea sulfate (139 mg), 3-methoxybenzaldehyde (243 μl), ethyl cyanoacetate (112 μl) and potassium carbonate (145 mg) in anhydrous ethanol (2 mL) was brought to 60 ° C. Stirred for 5 hours. The reaction mixture was cooled to 0 ° C. in an ice bath, filtered and the residue heated in water (H ₂ O) until a clear solution was obtained. The solution was acidified to pH 2 with 2N aqueous HCl and cooled to 0 ° C. in an ice bath. The resulting crystals were collected by filtration and dried in vacuo.
[161] Yield: 186 mg
[162] MS-ESI: [M + H] ⁺ = 274.2
[163] TLC: R _f = 0.50, silica gel, dichloromethane (CH ₂ Cl ₂ ) / methanol (CH ₃ OH) =
[164] 9/1 (v / v)
[165] (b) 6-chloro-5-cyano-4- (3-methoxyphenyl) -2-methylthiopyrimidine
[166] Phosphorous oxychloride (0.75 mL) was dissolved in 5-cyano-4- (3-methoxyphenyl) -2-methylthio-6-hydroxypyrimidine in 1,4-dioxane (1 mL) (Example 1a). 305 mg) was added to the stirred solution. N, N-dimethylaniline droplets were added. After 3 h at 80 ° C., the mixture was cooled to 0 ° C. in an ice bath and the crushed ice was added slowly. After stopping the exothermic reaction, H ₂ O (3 mL) was added. The solid was collected by filtration and dried in vacuo.
[167] Yield: 244 mg
[168] MS-ESI: [M + H] ⁺ = 292.2
[169] TLC: R _f = 0.86, silica gel, CH ₂ Cl ₂
[170] (c) ethyl 5-amino-4- (3-methoxyphenyl) -2-methylthiothieno [2,3-d] pyrimidine-6-carboxylate
[171] t-Butoxylated potassium (150 mg) was added ethyl 2-mercaptoacetate (92 μl) and 6-chloro-5-cyano-4- (3-methoxyphenyl) in anhydrous tetrahydrofuran (THF) (4 mL). ) To a stirred solution of 2-methylthiopyrimidine (Example 1b, 244 mg). After 1 h, the mixture was cooled to 0 ° C. in an ice bath and diluted with H ₂ O (10 mL). The solid was collected by filtration and dried in vacuo.
[172] Yield: 260 mg
[173] MS-ESI: [M + H] ⁺ = 376.2
[174] TLC: R _f = 0.44, silica gel, CH ₂ Cl ₂
[175] (d) 5-amino-4- (3-methoxyphenyl) -2-methylthiothieno [2,3-d] pyrimidine-6-carboxylic acid
[176] Ethyl 5-amino-4- (3-methoxyphenyl) -2-methylthiothieno [2,3-d] pyrimidine-6-carboxylate (Example 1c, 9.27 g) was diluted with 1,4-di. It was dissolved in a mixture of oxane (270 mL) and H ₂ O (30 mL). Lithium hydroxide (10 g) was added and the mixture was stirred at 80 ° C. for 48 hours. 1,4-dioxane was evaporated off the mixture and the residue was dissolved in H ₂ O. To the remaining solution was added aqueous 3N aqueous HCl and acidified to pH 2. The resulting precipitate was filtered off and washed with H ₂ O. Traces of water in the precipitate were removed by co-evaporation with 1,4-dioxane followed by diethyl ether and dried in vacuo at 50 ° C. overnight.
[177] Yield: 8.45 g
[178] MS-ESI: [M + H] ⁺ = 348.0
[179] TLC: R _f = 0.2, silica gel, CH ₂ Cl ₂ / CH ₃ OH = 9/1 (v / v)
[180] (e) t-butyl 5-amino-2-methylthio-4- (3-methoxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[181] 5-Amino-4- (3-methoxyphenyl) -2-methylthiothieno [2,3-d] pyrimidine-6-carboxylic acid (Example 1d, 7.0 g) was dissolved in anhydrous CH ₂ Cl ₂ ( 100 ml). Benzotriazol-1-yl-N, N, N, N'-tetramethyluroniumtetrafluoroborate (TBTU) (8.0 g), N, N-diisopropylethylamine (DIPEA) (6.6 mL) and t-butylamine (4.0 mL) was added and the mixture was stirred at rt for 5 h. The reaction mixture was washed with 5% aqueous NaHCO ₃ (2 × 100 mL) and 1 M aqueous HCl (2 × 100 mL) and concentrated in vacuo. The title compound was purified by chromatography on silica gel with heptane / ethyl acetate (EtOAc) = 1/0 to 3/2 as eluent.
[182] Yield: 6.5 g
[183] MS-ESI: [M + H] ⁺ = 403.0
[184] HPLC: R _t = 33.56 min, Column 3 μm Luna C-18 (2) 100 × 2.0 mm,
[185] Flow rate 0.25 ml / min, oven temperature 40 ° C., detection 210 nm + 254 nm,
[186] Eluent within 50 minutes H ₂ O / acetonitrile (CH ₃ CN) / CH ₃ OH =
[187] 0/95/5 (v / v) at 70 / 28.5 / 1.5
[188] (f) t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[189] t-butyl 5-amino-2-methylthio-4- (3-methoxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1e, 1.8 g) was dissolved in anhydrous CH ₂ Cl ₂ (30 mL) and the resulting solution was cooled to 0 ° C. A solution of boron tribromide in anhydrous CH ₂ Cl ₂ (30 mL) was added dropwise and the mixture was stirred overnight at room temperature. Saturated aqueous NaHCO ₃ was added dropwise to the reaction mixture until the exotherm ceased. CH ₂ Cl _{2 was} then evaporated off the mixture and a large amount of EtOAc was added. The organic layer was washed with saturated aqueous NaHCO ₃ , dried (MgSO ₄ ) and concentrated in vacuo.
[190] Yield: 1.3 g
[191] MS-ESI: [M + H] ⁺ = 389.2
[192] HPLC: R _t = 17.44 min, column Luna C-18 (see Example 1e), eluent
[193] Within 50 minutes H ₂ O / CH ₃ CN / CH ₃ OH = 0/95/5 (v / v) at 90 / 9.5 / 0.5
[194] (g) t-butyl 5-amino-2-methylthio-4- (3- (methoxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[195] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) was dissolved in anhydrous CH ₂ Cl Dissolved in ₂ (5 mL). DIPEA (500 μl) and methyl chloroformate (199 μl) were added and the reaction mixture was stirred at rt overnight. The reaction mixture was washed with H ₂ O. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[196] Yield: 93 mg
[197] MS-ESI: [M + H] ⁺ = 447.4
[198] HPLC: R _t = 17.56 min, column Luna C-18 (see Example 1e), eluent
[199] Within 25 minutes 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[200] Example 2
[201] t-butyl 5-amino-2-methylthio-4- (3- (allyloxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[202] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) and allyl chloroformate (274 μl) of the reaction was carried out according to the method described in Example 1g. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[203] Yield: 102 mg
[204] MS-ESI: [M + H] ⁺ = 473.4
[205] HPLC: R _t = 19.82 min, column Luna C-18 (see Example 1e), eluent
[206] Within 25 minutes 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[207] Example 3
[208] t-butyl 5-amino-2-methylthio-4- (3- (benzyloxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[209] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) and benzyl chloroformate (368 μl) was carried out according to the method described in Example 1g. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[210] Yield: 112 mg
[211] MS-ESI: [M + H] ⁺ = 523.2
[212] HPLC: R _t = 22.22 min, column Luna C-18 (see Example 1e), eluent
[213] Within 25 minutes 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[214] Example 4
[215] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrobenzyloxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[216] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) and p-nitrobenzyl The reaction of chloroformate (554 mg) was performed according to the method described in Example 1g. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[217] Yield: 47 mg
[218] MS-ESI: [M + H] ⁺ = 568.4
[219] HPLC: R _t = 21.45 min, column Luna C-18 (see Example 1e), eluent
[220] Within 25 minutes 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[221] Example 5
[222] t-butyl 5-amino-2-methylthio-4- (3- (phenoxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[223] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) with phenyl chloroformate (324 μl) of the reaction was carried out according to the method described in Example 1g. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[224] Yield: 89 mg
[225] MS-ESI: [M + H] ⁺ = 509.4
[226] HPLC: R _t = 21.12 min, column Luna C-18 (see Example 1e), eluent
[227] Within 25 minutes 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[228] Example 6
[229] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[230] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 400 mg) and p-nitrophenyl The reaction of chloroformate (207 mg) was performed according to the method described in Example 1g. The solvent was evaporated under reduced pressure to afford the crude title compound.
[231] Yield: 569 mg
[232] MS-ESI: [M + H] ⁺ = 554.6
[233] TLC: R _f = 0.5, silica gel, hept / EtOAc = 3/2 (v / v)
[234] Example 7
[235] t-butyl 5-amino-2-methylthio-4- (3-((N, N-diethylamino) carbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[236] t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 1f, 100 mg) was dissolved in anhydrous CH ₂ Cl ₂ (5 mL) and several drops of N, N-dimethylformamide (DMF) were added. Diethylcarbamoyl chloride (68 mg) and DIPEA (217 μl) were added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with H ₂ O. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / H ₂ O = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[237] Yield: 75 mg
[238] MS-ESI: [M + H] ⁺ = 488.4
[239] HPLC: R _t = 0.6 min, silica gel, hept / EtOAc = 1/1 (v / v)
[240] Example 8
[241] t-butyl 5-amino-2-methylthio-4- (3- (1,2,3,6-tetrahydropyridinocarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6- Carboxamide
[242] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 6, 142 mg) was dissolved in CH ₂ Cl ₂ . 1,2,3,6-tetrahydropyridine (117 μl) and DIPEA (224 μl) were added and the mixture was stirred overnight at room temperature. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer was concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 100/0 (v / v) at CH ₃ CN / H ₂ O = 20/80 within 45 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[243] Yield: 76 mg
[244] MS-ESI: [M + H] ⁺ = 498.2
[245] HPLC: R _t = 13.90 min, column 5 μm Luna C-18 (2) 150 × 4.60 mm,
[246] Flow rate 1 ml / min, detection 210 nm + 254 nm, eluent within 15 minutes
[247] 0/100 (v / v) at H ₂ O / CH ₃ CN = 40/60
[248] Example 9
[249] t-butyl 5-amino-2-methylthio-4- (3- (p-toluenesulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[250] (a) 5-cyano-4- (3-nitrophenyl) -2-methylthio-6-hydroxypyrimidine
[251] A mixture of S-methylisothiourea sulfate (69.0 g), 3-nitrobenzaldehyde (75.0 g), ethyl cyanoacetate (56.0 mL) and potassium carbonate (72.5 g) in anhydrous EtOH (1500 mL) at 60 ° C Stir for 16 hours. The reaction mixture was cooled to 0 ° C. in an ice bath. The resulting precipitate was filtered off, washed with anhydrous EtOH and dissolved in hot water (100 ° C.). The solution was cooled to room temperature, acidified to pH 2 with 2N HCl and washed with ice water. Water remaining in the precipitate was removed by co-evaporation with 1,4-dioxane.
[252] Yield: 54.0 mg
[253] MS-ESI: [M + H] ⁺ = 289.0
[254] TLC: R _f = 0.3, silica gel, DCM / MeOH = 9/1 (v / v)
[255] (b) 6-chloro-5-cyano-4- (3-nitrophenyl) -2-methylthiopyrimidine
[256] POCl ₃ (100 mL) was added to 5-cyano-4- (3-nitrophenyl) -2-methylthio-6-hydroxypyrimidine in 1,4-dioxane (300 mL) (Example 9 (a ), 25.0 g) was added to the stirred solution. After 3 h at 90 ° C., the mixture was cooled to rt and concentrated under reduced pressure. The residue was dissolved in 1,4-dioxane (100 mL) and the resulting solution was cooled to 0 ° C. Shaved water was carefully added. The resulting precipitate was filtered off and washed with water. Water remaining in the precipitate was removed by co-evaporation with 1,4-dioxane.
[257] Yield: 26.0 g
[258] MS-ESI: [M + H] ⁺ = 307.0
[259] TLC: R _f = 0.5, silica gel, heptane / EtOAc = 3/2 (v / v)
[260] (c) ethyl 5-cyano-4- (3-nitrophenyl) -2-methylthio-6- (ethoxycarbonylmethylthio) pyrimidine
[261] DIPEA (15.7 mL) was added ethyl 2-mercaptoacetate (9.3 mL) and 6-chloro-5-cyano-4- (3-nitrophenyl) -2 in a mixture of EtOH (250 mL) and DCM (250 mL). -To a stirred solution of methylthiopyrimidine (Example 9 (b), 26.0 g). After 1 hour at room temperature, 0.1 N aqueous HCl (500 mL) was added to the mixture, then extracted with DCM (3 × 500 mL), dried (MgSO ₄ ) and concentrated under reduced pressure.
[262] Yield: 28.0 g
[263] MS-ESI: [M + H] ⁺ = 390.4
[264] TLC: R _f = 0.5, silica gel, heptane / EtOAc = 3/2 (v / v)
[265] (d) ethyl 5-amino-4- (3-nitrophenyl) -2-methylthiothieno [2,3-d] pyrimidine-6-carboxylate
[266] Ethyl 5-cyano-4- (3-nitrophenyl) -2-methylthio-6- (ethoxycarbonylmethylthio) pyrimidine in a mixture of toluene (150 mL) and EtOH (150 mL) (Example 9 (c), 28.0 g) and DIPEA (30 mL) were stirred at reflux (100 ° C.) for 16 h. Then the mixture was cooled to room temperature and concentrated under reduced pressure. Residual DIPEA was removed by coevaporation with toluene.
[267] Yield: 28.0 g
[268] MS-ESI: [M + H] ⁺ = 391.2
[269] TLC: R _f = 0.6, silica gel, heptane / EtOAc = 3/2 (v / v)
[270] (e) t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[271] Ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) thieno [2,3-d] pyrimidine-6-carboxylate (Example 9d, 780 mg) was added 1,4-dioxane. (10 mL). Ethanol (10 mL) and tin (II) chloride (1.1 g) were added and the reaction mixture was stirred at 90 ° C. overnight. After the reaction mixture was concentrated in vacuo, the residue was redissolved in EtOAc (50 mL), washed with 4M aqueous NaOH (10 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The resulting derivative ethyl ester group in ethyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxylate (558 mg), Example 1d. The saponification with the acid (430 mg) was carried out using the method described in the following, followed by reaction with t-butylamine (200 μl) to form the corresponding t-butylamide (according to Example 1e). The title compound was purified by chromatography on silica gel with hept / EtOAc = 3/1 (v / v) as eluent.
[272] Yield: 391 mg
[273] MS-ESI: [M + H] ⁺ = 388.0
[274] TLC: R _f = 0.43, silica gel, hept / EtOAc = 3/2 (v / v)
[275] (f) t-butyl 5-amino-2-methylthio-4- (3- (p-toluenesulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[276] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) was dissolved in anhydrous pyridine (5 mL )). p-toluenesulfonyl chloride (70 mg) was added and the mixture was stirred at rt for 2 h. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with 0.1 M aqueous HCl. The organic layer was dried (MgSO 4) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent.
[277] Yield: 63 mg
[278] MS-ESI: [M + H] ⁺ = 542.4
[279] HPLC: R _t = 23.46 min, Column Luna C-18 (see Example 1e), eluent 50 min
[280] Phosphate buffer within 50 mM pH 2.1 / H ₂ O / CH ₃ CN / CH ₃ OH = 10/72/17/1
[281] 10/18/68/4 (v / v)
[282] Example 10
[283] t-butyl 5-amino-2-methylthio-4- (3- (vinylsulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[284] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 2.5 g) was added CH ₂ Cl ₂ ( 25 mL) and pyridine (25 mL). 2-bromoethanesulfonyl chloride was prepared as described by Bull. Chem. Soc. Jpn. 39, 1937-1941 (1966). A solution of 2-bromoethanesulfonyl chloride (2 g) in CH ₂ Cl ₂ (5 mL) was added dropwise and the reaction mixture was stirred at rt for 2 h. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with saturated aqueous NaHCO ₃ . The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent.
[285] Yield: 1.4 g
[286] MS-ESI: [M + H] ⁺ = 478.6
[287] TLC: R _f = 0.80, silica gel, hept / EtOAc = 3/2 (v / v)
[288] Example 11
[289] t-butyl 5-amino-2-methylthio-4- (3- (2-piperidinoethanesulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[290] t-butyl 5-amino-2-methylthio-4- (3- (vinylsulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 10, 87 mg) It was dissolved in anhydrous THF (5 mL). Piperidine (181 μl) was added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with saturated NaHCO ₃ . The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was first purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent and then by HPLC using a Luna C-18 column with the following gradient: 30 min Within CH ₃ CN / 0.1% aqueous trifluoroacetic acid (TFA) = 10/10 at 90/10 (v / v). The title compound was then lyophilized from a mixture of 1,4-dioxane and 0.1% aqueous TFA.
[291] Yield: 89 mg (TFA salt)
[292] MS-ESI: [M + H] ⁺ = 563.4
[293] HPLC: R _t = 18.4 min, column Luna C-18 (see Example 1e), eluent 25 min
[294] Within H ₂ O / CH ₃ CN = 60/40 to 0/100 (v / v)
[295] Example 12
[296] t-butyl 5-amino-2-methylthio-4- (3- (2- (thiomorpholin-4-yl) ethanesulfonamido) phenyl) thieno [2,3-d] pyrimidine-6- Carboxamide
[297] Thiomorpholine (184 μl) and t-butyl 5-amino-2-methylthio-4- (3- (vinylsulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide ( Example 10, 87 mg) was carried out according to the method described in Example 11. The title compound was first purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent and then by HPLC using a Luna C-18 column with the following gradient: 30 min Within CH ₃ CN / 0.1% aqueous TFA = 10/10 to 90/10 (v / v). The title compound was then lyophilized from a mixture of 1,4-dioxane and 0.1% aqueous TFA.
[298] Yield: 120 mg (TFA salt)
[299] MS-ESI: [M + H] ⁺ = 581.2
[300] HPLC: R _t = 17.2 min, column Luna C-18 (see Example 1e), eluent 25 min
[301] Within H ₂ O / CH ₃ CN = 60/40 to 0/100 (v / v)
[302] Example 13
[303] t-butyl 5-amino-2-methylthio-4- (3- (2- (bis- (2-methoxyethyl) amino) ethanesulfonamido) phenyl) thieno [2,3-d] pyrimidine -6-carboxyamide
[304] Bis (2-methoxyethyl) amine (244 mg) and t-butyl 5-amino-2-methylthio-4- (3- (vinylsulfonamido) phenyl) thieno [2,3-d] pyrimidine The reaction of -6-carboxyamide (Example 10, 87 mg) was performed according to the method described in Example 11. The title compound was first purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent and then by HPLC using a Luna C-18 column with the following gradient: 30 min Within CH ₃ CN / 0.1% aqueous TFA = 10/10 to 90/10 (v / v). The title compound was then lyophilized from a mixture of 1,4-dioxane and 0.1% aqueous TFA.
[305] Yield: 60 mg (TFA salt)
[306] MS-ESI: [M + H] ⁺ = 611.4
[307] HPLC: R _t = 17.9 min, column Luna C-18 (see Example 1e), eluent 25 min
[308] Within H ₂ O / CH ₃ CN = 60/40 to 0/100 (v / v)
[309] Example 14
[310] t-butyl 5-amino-2-methylthio-4- (3- (2- (N-methylpiperazino) ethanesulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxy amides
[311] N-methyl piperazine (184 μl) and t-butyl 5-amino-2-methylthio-4- (3- (vinylsulfonamido) phenyl) thieno [2,3-d] pyrimidine-6-carboxy The reaction of the amide (Example 10, 87 mg) was carried out according to the method described in Example 11. The title compound was first purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent and then by HPLC using a Luna C-18 column with the following gradient: 30 min Within CH ₃ CN / 0.1% aqueous TFA = 10/10 to 90/10 (v / v). The title compound was then lyophilized from a mixture of 1,4-dioxane and 0.1% aqueous TFA.
[312] Yield: 85 mg (TFA salt)
[313] MS-ESI: [M + H] ⁺ = 578.4
[314] HPLC: R _t = 16.1 min, column Luna C-18 (see Example 1e), eluent 25 min
[315] Within H ₂ O / CH ₃ CN = 60/40 to 0/100 (v / v)
[316] Example 15
[317] t-butyl 5-amino-2-methylthio-4- (3- (methoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[318] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) was dissolved in anhydrous CH ₂ Cl ₂ (5 ml). Methyl chloroformate (199 μl) and DIPEA (500 μl) were added and the reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with H ₂ O. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / 10% aqueous CH ₃ CN = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[319] Yield: 80 mg
[320] MS-ESI: [M + H] ⁺ = 446.2
[321] HPLC: R _t = 20.44 min, column Luna C-18 (see Example 1e), eluent 20 min
[322] Within phosphate buffer 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/72/18
[323] 10/18/72 (v / v)
[324] Example 16
[325] t-butyl 5-amino-2-methylthio-4- (3- (allyloxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[326] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) and allyl chloroformate ( 274 μl) was carried out using the method described in Example 15. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / 10% aqueous CH ₃ CN = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[327] Yield: 66 mg
[328] MS-ESI: [M + H] ⁺ = 472.2
[329] HPLC: R _t = 22.37 min, column Luna C-18 (see Example 1e), eluent 20 min
[330] Within phosphate buffer 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/72/18
[331] 10/18/72 (v / v)
[332] Example 17
[333] t-butyl 5-amino-2-methylthio-4- (3- (benzyloxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[334] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) and benzyl chloroformate ( 368 μl) was carried out using the method described in Example 15. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / 10% aqueous CH ₃ CN = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[335] Yield: 112 mg
[336] MS-ESI: [M + H] ⁺ = 522.4
[337] HPLC: R _t = 24.10 min, Column Luna C-18 (see Example 1e), eluent 20 min
[338] Within phosphate buffer 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/72/18
[339] 10/18/72 (v / v)
[340] Example 18
[341] t-butyl 5-amino-2-methylthio-4- (3- (ethoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[342] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) and ethyl chloroformate ( 247 μl) was carried out using the method described in Example 15. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / 10% aqueous CH ₃ CN = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[343] Yield: 74 mg
[344] MS-ESI: [M + H] ⁺ = 460.4
[345] HPLC: R _t = 21.17 min, column Luna C-18 (see Example 1e), eluent 20 min
[346] Within phosphate buffer 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/72/18
[347] 10/18/72 (v / v)
[348] Example 19
[349] t-butyl 5-amino-2-methylthio-4- (3- (phenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[350] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 100 mg) and phenyl chloroformate ( 324 μl) was carried out using the method described in Example 15. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 90/10 (v / v) at CH ₃ CN / 10% aqueous CH ₃ CN = 10/90 within 30 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[351] Yield: 47 mg
[352] MS-ESI: [M + H] ⁺ = 508.4
[353] HPLC: R _t = 23.25 min, column Luna C-18 (see Example 1e), eluent 20 min
[354] Within phosphate buffer 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/72/18
[355] 10/18/72 (v / v)
[356] Example 20
[357] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[358] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 9e, 1 g) was dissolved in anhydrous CH ₂ Cl ₂ (10 mL). Then a solution of p-nitrophenyl chloroformate (520 mg) in anhydrous CH ₂ Cl ₂ (10 mL) was added dropwise and the reaction mixture was stirred at room temperature. After 1 hour, the reaction mixture was washed with H ₂ O. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure.
[359] Yield: 1.42 g
[360] MS-ESI: [M + H] ⁺ = 553.6
[361] TLC: R _f = 0.7, silica gel, hept / EtOAc = 3/2 (v / v)
[362] Example 21
[363] t-butyl 5-amino-2-methylthio-4- (3-((morpholin-4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[364] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 20, 142 mg) was dissolved in anhydrous CH ₂ Cl ₂ (5 mL). Morpholine (112 μl) and DIPEA (225 μl) were added and the reaction mixture was stirred at rt overnight. The reaction mixture was diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer was concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[365] Yield: 22 mg
[366] MS-ESI: [M + H] ⁺ = 501.2
[367] HPLC: R _t = 8.62 min, Column Luna C-18 (see Example 8), eluent 15 min
[368] Within H ₂ O / CH ₃ CN = 40/60 to 0/100 (v / v)
[369] Example 22
[370] t-butyl 5-amino-2-methylthio-4- (3- (o-anisidinocarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[371] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 20, 142 mg) and orthoanisidine (159 mg) were performed using the method described in Example 21. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[372] Yield: 18 mg
[373] MS-ESI: [M + H] ⁺ = 537.2
[374] HPLC: R _t = 12.94 min, column Luna C-18 (see Example 8), eluent 15 min
[375] Within H ₂ O / CH ₃ CN = 40/60 to 0/100 (v / v)
[376] Example 23
[377] t-butyl 5-amino-2-methylthio-4- (3- (1,2,3,6-tetrahydropyridinocarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6- Carboxamide
[378] t-butyl 5-amino-2-methylthio-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 20, 142 mg) and 1,2,3,6-tetrahydropyridine (118 μl) urea coupling was performed using the method described in Example 21. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then freeze dried from a mixture of 1,4-dioxane and H ₂ O.
[379] Yield: 18 mg
[380] MS-ESI: [M + H] ⁺ = 497.2
[381] HPLC: R _t = 11.19 min, column Luna C-18 (see Example 8), eluent 15 min
[382] Within H ₂ O / CH ₃ CN = 40/60 to 0/100 (v / v)
[383] Example 24
[384] t-butyl 5-hydroxy-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxyamide
[385] (a) ethyl 2-methylthio-4- (3-methoxyphenyl) -6-methyl-1,4-dihydropyrimidine-5-carboxylate
[386] A mixture of S-methylisothiourea sulfate (13.9 g), 3-methoxybenzaldehyde (7.5 g), ethyl acetoacetate (6.5 g) and sodium hydrogencarbonate (21 g) in DMF (200 mL) was added at 70 ° C. Stir for 18 hours. The reaction mixture was cooled to rt, diluted with diethyl ether and washed with H ₂ O and saturated aqueous NaCl. The title compound was purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent.
[387] Yield: 7.3 g
[388] MS-ESI: [M + H] ⁺ = 321.0
[389] TLC: R _f = 0.2, silica gel, hept / EtOAc = 3/1 (v / v)
[390] (b) ethyl 2-methylthio-4- (3-methoxyphenyl) -6-methylpyrimidine-5-carboxylate
[391] Ethyl-2-methylthio-4- (3-methoxyphenyl) -6-methyl-1,4-dihydropyrimidine-5-carboxylate (Example 24a, 7.65 g) was dissolved in toluene (200 mL). Dissolved in a mixture of CH ₂ Cl ₂ (100 mL). 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (5.45 g) was added and the reaction mixture was stirred at room temperature for 15 minutes. 0.2 M aqueous NaOH (250 mL) was added. The organic layer was separated, washed with H ₂ O (2 × 250 mL) and saturated aqueous NaCl (2 × 250 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with hept / EtOAc = 4/1 (v / v) as eluent.
[392] Yield: 4.0 g
[393] MS-ESI: [M + H] ⁺ = 319.2
[394] TLC: R _f = 0.4, silica gel, hept / EtOAc = 3/1 (v / v)
[395] (c) ethyl 5-hydroxy-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxylate
[396] Cool a solution of ethyl 2-methylthio-4- (3-methoxyphenyl) -6-methylpyrimidine-5-carboxylate (Example 24b, 318 mg) in dry THF (2 mL) to -78 ° C. To freshly prepared solution of LDA in dry THF. The mixture was stirred at −78 ° C. for 30 minutes and 3-ethoxy acrylate (217 μl) was added. The mixture was then stirred for 3 h at -78 ° C to room temperature. 0.1 M aqueous HCl (20 mL) was added to the reaction mixture, which was then extracted with EtOAc (25 mL). The organic layer was washed with water (25 mL) and aqueous NaCl (25 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with hept / EtOAc = 3/1 (v / v) as eluent and recrystallized from CH ₃ OH.
[397] Yield: 38 mg
[398] MS-ESI: [M + H] ⁺ = 371.2
[399] TLC: R _f = 0.6, silica gel, hept / EtOAc = 2/3 (v / v)
[400] (d) t-butyl 5-hydroxy-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxyamide
[401] Ethyl 5-hydroxy-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxylate (Example 24c, 38 mg) was added 1,4-dioxane (4 mL) and 1 M. It was dissolved in a mixture of aqueous KOH (0.5 mL). The mixture was refluxed for 48 hours, then cooled to room temperature and acidified by addition of 0.1 M aqueous HCl (15 mL). The mixture was extracted with CH ₂ Cl ₂ (15 mL). The organic layer was dried (MgSO ₄ ) and evaporated under reduced pressure to give 5-hydroxy-4- (3-methoxyphenyl) -2-methylthioquinazolin-6 -Carboxylic acid was obtained. This was dissolved in DMF (2 mL). t-butylamine (53 μl) and TBTU (96 mg) were added and the mixture was stirred at room temperature for 3 hours. EtOAc (15 mL) was added and the organic layer was washed with saturated aqueous NaHCO ₃ (15 mL) and saturated aqueous NaCl (15 mL), dried (MgSO ₄ ) and evaporated under reduced pressure. The title compound was first purified by chromatography on silica gel with hept / EtOAc = 7/3 (v / v) as eluent. Purification was then by HPLC in the following gradient: 7/90/3 (v / v) at 10% aqueous CH ₃ CN / CH ₃ CN / 0.1% aqueous TFA = 57/40/3 within 30 minutes. The title compound was lyophilized from a mixture of water and 1,4-dioxane.
[402] Yield: 25 mg
[403] MS-ESI: [M + H] ⁺ = 398.2
[404] HPLC: R _t = 9.75 min, Column Luna C-18 (see Example 1e), eluent 15 min
[405] Phosphate buffer within 50 mM pH 2.1 / H ₂ O / CH ₃ CN / CH ₃ OH =
[406] 5/10/81/4 (v / v) to 5/35/57/3
[407] Example 25
[408] t-butyl 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxide
[409] (a) 5-cyano-4- (3-methoxyphenyl) -2-methylthio-6-vinylpyrimidine
[410] 6-Chloro-5-cyano-4- (3-methoxyphenyl) -2-methylthiopyrimidine (Example 1b, 1.46 g) was suspended in 1,4-dioxane (10 mL). Tetrakis (triphenylphosphine) palladium (0) (350 mg) was added and flushed with nitrogen atmosphere. Tetravinyltin (1.26 mL) was added and the mixture was refluxed for 5 hours. The reaction mixture was then poured into a mixture of EtOAc (100 mL) and H ₂ O (100 mL). The organic layer was washed with saturated aqueous NaCl, dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel at 1/3 (v / v) at hept / CH ₂ Cl ₂ = 1/0.
[411] Yield: 1.05 g
[412] MS-ESI: [M + H] ⁺ = 284.2
[413] TLC: R _f = 0.4, silica gel, hept / CH ₂ Cl ₂ = 1/2 (v / v)
[414] (b) 5-cyano-4- (3-methoxyphenyl) -2-methylthio-6- (1,1-bis (ethoxycarbonyl) prop-3-yl) pyrimidine
[415] 5-Cyano-4- (3-methoxyphenyl) -2-methylthio-6-vinylpyrimidine (Example 25a) was dissolved in a mixture of ethanol (2 mL) and toluene (2 mL). Potassium carbonate (690 mg) and diethylmalonate (272 μl) were added and the mixture was stirred at rt for 4 h. The reaction mixture was then poured into a mixture of 0.5 M aqueous HCl (25 mL) and EtOAc (50 mL). The organic layer was washed with H ₂ 0 (50 mL) and saturated aqueous NaCl (50 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel from toluene / EtOAc = 100/0 to 95/5 (v / v) as eluent.
[416] Yield: 344 mg
[417] MS-ESI: [M + H] ⁺ = 444.2
[418] TLC: R _f = 0.3, silica gel, toluene / EtOAc = 95/5 (v / v)
[419] (c) ethyl 5-amino-2-methylthio-4- (3-methoxyphenyl) -7,8-dihydroquinazolin-6-carboxylate
[420] 5-cyano-4- (3-methoxyphenyl) -2-methylthio-6- (1,1-bis (ethoxycarbonyl) prop-3-yl) pyrimidine (Example 25b, 81 mg ) Was dissolved in anhydrous CH ₂ Cl ₂ (1 mL). A 1 M solution of tin (IV) in CH ₂ Cl ₂ (1 mL) was added dropwise and the mixture was stirred at rt for 1 h. Then H ₂ 0 (10 mL) and EtOAc (10 mL) were added to the reaction mixture. The organic layer was washed with H ₂ 0 (10 mL) and saturated aqueous NaCl (10 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel from hept / EtOAc = 1/0 to 3/2 (v / v) as eluent.
[421] Yield: 24 mg
[422] MS-ESI: [M + H] ⁺ = 372.2
[423] TLC: R _f = 0.5, silica gel, hept / EtOAc = 3/1 (v / v)
[424] (d) ethyl 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxylate
[425] Ethyl 5-amino-2-methylthio-4- (3-methoxyphenyl) -7,8-dihydroquinazolin-6-carboxylate (Example 25c, 22 mg) was added CH ₂ Cl ₂ (1 mL). )). A 0.06 M solution of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone in CH ₂ Cl ₂ (1.2 mL) was added and the mixture was stirred at rt for 15 min. The reaction mixture was concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with toluene / EtOAc = 95/5 (v / v) as eluent.
[426] Yield: 20 mg
[427] MS-ESI: [M + H] ⁺ = 370.0
[428] TLC: R _f = 0.3, silica gel, hept / EtOAc = 1/3 (v / v)
[429] (e) 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxylic acid
[430] Ethyl 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxylate (Example 25d, 490 mg) was dissolved in 15 ml of 1,4-dioxane and 2M Aqueous KOH solution (3 mL) was added. The mixture was heated to reflux for 5 hours and heated to 70 ° C. for 76 hours. The mixture was cooled to room temperature and 0.5 M HCl solution in water was added. The mixture was extracted with CH ₂ Cl ₂ (2 × 50 mL) and the combined organic layers were dried (MgSO ₄ ) and concentrated under reduced pressure to afford crude title compound.
[431] Yield: 406 mg
[432] MS-ESI: [M + H] ⁺ = 342.2
[433] TLC: R _f = 0.0, silica gel, hept / EtOAc = 2/3 (v / v)
[434] (f) t-butyl 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxyamide
[435] To a 1.8 M solution of 5-amino-2-methylthio- (3-methoxyphenyl) quinazolin-6-carboxylic acid in DMF (1.8 mL) add TBTU (173 mg) and t-butylamine (95 mL). Was added. The reaction mixture was stirred for 2 hours and then poured into a mixture of EtOAc (25 mL) and saturated aqueous NaHCO ₃ (50 mL). The organic phase was separated, washed with 0.5 M aqueous HCl (50 mL) and brine (50 mL), then dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel using hept / EtOAc = 1/0 to 2/3 (v / v) as eluent. The title compound was lyophilized from a mixture of dioxane and water containing 1.5 equivalents of HCl.
[436] Yield: 44 mg
[437] MS-ESI: [M + H] ⁺ = 397.2
[438] HPLC: R _t = 21.52 min, Column Luna C-18 (see Example 1e), eluent 20 min
[439] Phosphate buffer at 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/70/20
[440] 10/10/80 (v / v)
[441] Example 26
[442] t-butyl 5-amino-2-methylthio-4- (3- (2- (pyrrolidin-1-yl) ethoxy) phenyl) quinazolin-6-carboxyamide
[443] (a) t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) quinazolin-6-carboxyamide
[444] A solution of t-butyl 5-amino-2-methylthio-4- (3-methoxyphenyl) quinazolin-6-carboxyamide (Example 25f, 1.5 g) in anhydrous CH ₂ Cl ₂ was cooled to 0 ° C. . A solution of BBr ₃ (1.1 mL) in CH ₂ Cl ₂ (25 mL) was added dropwise and after the addition was complete, the mixture was stirred at rt for 3 h. The mixture was diluted with CH ₂ Cl ₂ and saturated aqueous NaHCO ₃ (200 mL) was added carefully. The mixture was stirred vigorously for 1.5 hours until all solids dissolved. The aqueous layer was extracted twice with CH ₂ Cl ₂ . The first organic layer was washed with saturated aqueous NaHCO ₃ and brine. The combined organic layers were dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel at 1/1 (v / v) at hept / EtOAc = 1/0 as eluent.
[445] Yield: 930 mg
[446] MS-ESI: [M + H] ⁺ = 383.4
[447] TLC: R _f = 0.3, silica gel, hept / EtOAc = 2/3 (v / v)
[448] (b) t-butyl 5-amino-2-methylthio-4- (3- (2- (pyrrolidin-1-yl) ethoxy) phenyl) quinazolin-6-carboxyamide
[449] K ₂ CO ₃ (1.0 g) in acetone, 1- (2-chloroethyl) pyrrolidine hydrochloride (66 mg) and t-butyl 5-amino-2-methylthio-4- (3-hydroxyphenyl) quina Zoline-6-carboxyamide (Example 26a, 121 mg) was heated to reflux overnight. The mixture was cooled to rt, the solids were filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in EtOAc and washed with water and brine. The organic phase was dried (MgSO ₄ ) and concentrated in vacuo. The title compound was purified by HPLC using a Luna C-18 column in the following gradient: 27/70/3 at 10% aqueous CH ₃ CN / CH ₃ CN / 0.1% aqueous TFA = 72/25/3 within 30 minutes. (v / v / v). The title compound was lyophilized from a mixture of water, TFA and CH ₃ CN.
[450] Yield: 42 mg (TFA salt)
[451] MS-ESI: [M + H] ⁺ = 480.4
[452] HPLC: R _t = 12.93 min, column Luna C-18 (see Example 1e), eluent 20 min
[453] Phosphate buffer at 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/70/20
[454] 10/10/80 (v / v)
[455] Example 27
[456] t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxyamide
[457] (a) 5-cyano-2-methylthio-4- (3-nitrophenyl) -6-[(triphenylphosphaneylidene) methyl] pyrimidine
[458] To a suspension of anhydrous methyltriphenylphosphonium bromide (29.5 g) in dimethoxyethane (400 mL) was added a 1.6 M solution of n-butyllithium in hexane at -78 ° C. The mixture was stirred at -78 ° C for 1 hour and 6-chloro-5-cyano-4- (3-nitrophenyl) -2-methylthiopyrimidine in dimethoxyethane (100 mL) (Example 9b, 10.1 g) solution was added and the cooling bath was removed. After 1 hour, the reaction was terminated and water (15 mL) was added. After the solid was filtered off, the reaction mixture was concentrated to give a dark residue, which was stirred with ethyl acetate to give a suspension. The residue was filtered, washed with water and brine, the organic layer was dried over MgSO ₄ and concentrated in vacuo. The title compound was purified by chromatography on silica gel at 1/1 (v / v) at hept / EtOAc = 4/1 as eluent.
[459] Yield: 7.04 g
[460] MS-ESI: [M + H] ⁺ = 299.2
[461] TLC: R _f = 0.4, silica gel, hept / EtOAc = 3/2 (v / v)
[462] (b) 5-cyano-2-methylthio-4- (3-nitrophenyl) -6-vinylpyrimidine
[463] Solution of 5-cyano-2-methylthio-4- (3-nitrophenyl) -6-[(triphenylphosphaneylidene) methyl] pyrimidine (Example 27a, 7.04 g) in THF (64 mL) Was treated with aqueous formaldehyde (37% by weight, 3.55 mL) at 60 ° C for 1 hour. After the mixture was cooled to rt, it was diluted with EtOAc (100 mL), washed with water (2 × 50 mL), dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel using 3/2 (v / v) at hept / EtOAc = 9/1 as eluent.
[464] Yield: 1.42 g
[465] MS-ESI: [M + H] ⁺ = 547.2
[466] TLC: R _f = 0.6, silica gel, hept / EtOAc = 3/2 (v / v)
[467] (c) t-butyl ethyl (2- (5-cyano-2-methylthio-4- (3-nitrophenyl) pyrimidin-6-yl) ethyl) malonate
[468] Potassium carbonate (1.88 g) and t-butyl ethylmalonate are suspended in EtOH (82 mL) and 5-cyano-2-methylthio-4- (3-nitro in toluene / CH ₂ Cl ₂ (33 mL) A solution of phenyl) -6-vinylpyrimidine (Example 27b, 2.71 g) was added slowly (about 1.5 hours). After the addition was complete, the mixture was stirred for 40 more minutes. The mixture was diluted with EtOAc and washed with water (2 x) and brine. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel with hept / CH ₂ Cl ₂ = 1/1 (v / v) as eluent.
[469] Yield: 1.42 g
[470] MS-ESI: [M + H] ⁺ = 487.2
[471] TLC: R _f = 0.5, silica gel, hept / EtOAc = 3/2 (v / v)
[472] (d) ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) -7,8-dihydroquinazolin-6-carboxylate
[473] T-butyl ethyl {2- [5-cyano-2-methylthio-4- (3-nitrophenyl) pyrimidin-6-yl] ethyl} malonate in CH ₂ Cl ₂ (15 mL) (Example 27c , 1.40 g) was cooled to 0 ° C. A solution of SnCl ₄ (1 M in CH ₂ Cl ₂ , 11.5 mL) was added dropwise, the ice bath was removed and stirred for 30 minutes at room temperature. Water (64 mL) and EtOAc (64 mL) were added and the mixture was vigorously stirred until the solid dissolved. The organic phase was washed with water and brine, dried (MgSO ₄ ) and concentrated under reduced pressure to afford the title compound as crude product.
[474] Yield: 1.19 g (crude)
[475] MS-ESI: [M + H] ⁺ = 387.2
[476] TLC: R _f = 0.5, silica gel, hept / EtOAc = 3/2 (v / v)
[477] (e) ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) quinazolin-6-carboxylate
[478] Crude ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) -7,8-dihydroquinazolin-6-carboxylate in CH ₂ Cl ₂ (31 mL) (Example 27d, 1.19 The solution of g) was cooled to 0 ° C. A solution of DDQ (1.14 g) in toluene (31 mL) was added dropwise, the ice bath was removed and the solution stirred for 30 more minutes at room temperature. The mixture was diluted with CH ₂ Cl ₂ (about 100 mL) and washed with saturated aqueous solution of NaHCO ₃ (3 × 100 mL) and brine (2 × 50 mL). The combined aqueous layers were back extracted with CH ₂ Cl ₂ . The combined organic layers were dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel using 3/2 (v / v) at hept / EtOAc = 9/1 as eluent.
[479] Yield: 735 mg
[480] MS-ESI: [M + H] ⁺ = 385.2
[481] TLC: R _f = 0.5, silica gel, hept / EtOAc = 3/2 (v / v)
[482] (f) ethyl 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxylate
[483] 1, 4-dioxane (35 ㎖) of ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) quinazolin-6-carboxylate (example 27e, 1.54 g) and SnCl ₂ · 2H of _To a mixture of ₂ 0 (4.52 g) was added EtOH (35 mL) and concentrated aqueous HCl (690 mL). The reaction mixture was stirred at 90 ° C. for 5 hours. After cooling to room temperature and concentration under reduced pressure, the residue was suspended in EtOAc (35 mL). 2 M NaOH was added to the mixture to pH 10, and THF and brine were added. The resulting mixture was stirred for 40 minutes, then the organic layer was separated, dried (MgSO ₄ ) and concentrated under reduced pressure. The title compound was purified by chromatography on silica gel using hept / EtOAc = 9/1 to 3/2 as eluent.
[484] Yield: 818 mg
[485] MS-ESI: [M + H] ⁺ = 355.2
[486] TLC: R _f = 0.3, silica gel, hept / EtOAc = 3/2 (v / v)
[487] (g) 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxylic acid
[488] A solution of 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxylate (Example 27f, 658 mg) in 1,4-dioxane was KOH at 70 ° C. for 18 hours. Treated with an aqueous solution of (2 M, 4.2 mL). The reaction mixture was cooled to room temperature and then acidified to pH 1 with 4 N HCl. The mixture was extracted with CH ₂ Cl ₂ (3 ×). The combined organic layers were dried (MgSO ₄ ) and concentrated under reduced pressure to afford crude title compound.
[489] Yield: 215 mg
[490] MS-ESI: [M + H] ⁺ = 327.2
[491] TLC: R _f = 0, silica gel, hept / EtOAc = 3/2 (v / v)
[492] (h) t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxyamide
[493] 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxylic acid (Example 27g, 192 mg) was converted to the corresponding t-butyl amide according to the method described in Example 1e. . The title compound was purified by chromatography on silica gel using 3/2 (v / v) at hept / EtOAc = 9/1 as eluent.
[494] Yield: 243 mg
[495] MS-ESI: [M + H] ⁺ = 382.2
[496] HPLC: R _t = 6.57 min, column Luna C-18 (see Example 1e), eluent 20 min
[497] Within H ₂ O / CH ₃ CN = 45/55 to 0/100 (v / v)
[498] Example 28
[499] t-butyl 5-amino-2-methylthio-4- (3- (2- (morpholin-4-yl) acetamido) phenyl) quinazolin-6-carboxyamide
[500] (a) t-butyl 5-amino-2-methylthio-4- (3- (2-bromoacetamido) phenyl) quinazolin-6-carboxyamide
[501] DIPEA (1.08) in a suspension of t-butyl 5-amino-2-methylthio-4- (3-aminophenyl) quinazolin-6-carboxyamide (Example 27h, 791 mg) in CH ₂ Cl ₂ (60 mL). ML) was added and bromoacetyl chloride (242 mL) in CH ₂ Cl ₂ (20 mL) was added slowly. After 40 minutes at room temperature, the reaction mixture was washed with saturated aqueous NHCO ₃ (3 ×), then dried (MgSO ₄ ) and concentrated under reduced pressure. The crude title compound was used in the next step without further purification.
[502] Yield: 1.20 g (crude)
[503] MS-ESI: [M + H] ⁺ = 504.2
[504] TLC: R _f = 0.4, silica gel, hept / EtOAc = 3/2 (v / v)
[505] (b) t-butyl 5-amino-2-methylthio-4- (3- (2- (morpholin-4-yl) acetamido) phenyl) quinazolin-6-carboxyamide
[506] In a solution of t-butyl 5-amino-2-methylthio-4- (3- (2-bromoacetamido) phenyl) quinazolin-6-carboxyamide (Example 28a, 88 mg) in acetonitrile Pauline (148 mL) was added and the mixture was stirred for 18 hours. Then CH ₂ Cl ₂ (15 mL) was added and the mixture was washed with saturated aqueous NaHCO ₃ . The organic layer was dried (MgSO ₄ ) and concentrated in vacuo. The title compound was purified by chromatography on silica gel using CH ₂ Cl ₂ / MeOH = 1/0 at 9/1 (v / v) as eluent. The title compound was lyophilized from a mixture of acetonitrile and water containing 1.5 equivalents of HCl.
[507] Yield: 82 mg (HCl salt)
[508] MS-ESI: [M + H] ⁺ = 509.2
[509] HPLC: R _t = 13.00 min, Column Luna C-18 (see Example 1e), eluent 20 min
[510] Within H ₂ 0 / CH ₃ CN = 75/25 to 0/100 (v / v)
[511] Example 29
[512] t-butyl 5-amino-2-methylthio-4- (3-((N- (t-butyl) glycinyl) amino) phenyl) quinazolin-6-carboxyamide
[513] T-butylamine (275 μl) was added to t-butyl 5-amino-2-methylthio-4- (3- (2-bromoacetamido) phenyl) quinazolin-6- according to the method described in Example 28b. Reaction with carboxyamide (Example 28a, 130 mg). The title compound was first purified by chromatography on silica gel with hept / EtOAc = 3/2 (v / v) as eluent and then by HPLC using a Luna C-18 column with the following gradient: within 30 minutes 90/10 (v / v) at CH ₃ CN / 0.1% aqueous TFA = 10/90. The title compound was then lyophilized from a mixture of 1,4-dioxane and aqueous HCl.
[514] Yield: 28 mg (HCl salt)
[515] MS-ESI: [M + H] ⁺ = 495.4
[516] HPLC: R _t = 12.93 min, column Luna C-18 (see Example 1e), eluent 20 min
[517] Phosphate buffer at 50 mM pH 2.1 / H ₂ O / CH ₃ CN = 10/70/20
[518] 10/10/80 (v / v)
[519] Example 30
[520] Ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) -7-hydroxypyrido [2,3-d] pyrimidine-6-carboxylate
[521] (a) 6-amino-5-cyano-2-methylthio-4- (3-nitrophenyl) pyrimidine
[522] A solution of 6-chloro-5-cyano-4- (3-nitrophenyl) -2-methylthiopyrimidine (Example 9b, 10.0 g) was treated with ammonium hydroxide (28% NH _{3 in} water, 15 mL ), The mixture was stirred overnight. The crystals formed were collected by filtration and washed with water. The product was dried in vacuo at 50 ° C. to afford the title compound.
[523] Yield: 8.9 g
[524] MS-ESI: [M + H] ⁺ = 288.2
[525] TLC: R _f = 0.3, silica gel, hept / EtOAc = 3/2 (v / v)
[526] (b) diethyl 2- (amino [6-amino-2-methylthio-4- (3-nitrophenyl) pyrimidin-5-yl] methylene) malonate
[527] To a suspension of 6-amino-5-cyano-2-methylthio-4- (3-nitrophenyl) pyrimidine in 1,2-dichloropropane (200 mL) was added diethylmalonate (9.1 mL). The mixture was cooled to 0 ° C. and a solution of SnCl ₄ (14 mL) in 1,2-dichloropropane (50 mL) was added dropwise. After the addition was complete, the suspension was heated to reflux for 18 hours. The mixture was cooled to room temperature and after the solid had settled the 1,2-dichloropropane was carefully decanted. Stir with EtOAc (300 mL) and water (300 mL) until the resulting solid is completely dissolved. The organic layer was washed with water (500 mL) and brine (500 mL), dried (MgSO ₄ ) and concentrated in vacuo. The title compound was purified by chromatography on silica gel using hept / EtOAc = 1/0 to 3/2 (v / v) as eluent.
[528] Yield: 3.78 g
[529] MS-ESI: [M + H] ⁺ = 448.4
[530] TLC: R _f = 0.2, silica gel, hept / EtOAc = 3/2 (v / v)
[531] (c) ethyl 5-amino-2-methylthio-4- (3-nitrophenyl) -7-hydroxypyrido [2,3-d] pyrimidine-6-carboxylate
[532] Diethyl 2- (amino [6-amino-2-methylthio-4- (3-nitrophenyl) pyrimidin-5-yl] methylene) malonate in diphenyl ether (30 mL) (Example 30b, 1.34 g) ) Is heated to 240 ° C. for 2 hours under a stream of nitrogen. After the mixture was cooled to room temperature, heptane was added (200 mL) and the solids were collected by filtration. The title compound was purified by chromatography on silica gel using CH ₂ Cl ₂ / acetone = 1/0 to 4/1 (v / v) as eluent.
[533] Yield: 590 mg
[534] MS-ESI: [M + H] ⁺ = 402.4
[535] TLC: R _f = 0.3, silica gel, CH ₂ Cl ₂ / acetone = 9/1 (v / v)
[536] Example 31
[537] t-butyl 5-amino-2-phenyl-4- (3-((thiomorpholin-4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[538] (a) 5-cyano-4- (3-nitrophenyl) -2-phenyl-6-hydroxypyrimidine
[539] A mixture of benzamide hydrochloride (16.4 g), 3-nitrobenzaldehyde (15.1 g), ethyl cyanoacetate (11.2 mL) and potassium carbonate (16.6 g) in anhydrous EtOH (250 mL) was stirred at 60 ° C. for 8 hours. . The reaction mixture was cooled to 0 ° C. in an ice bath. The resulting precipitate was filtered off, washed with anhydrous EtOH and heated in water until a clear solution was obtained (100 ° C.). The solution was cooled to 50 ° C., acidified to pH 2 by addition of 2N aqueous HCl, and cooled to 0 ° C. in an ice bath. The resulting precipitate was filtered off and washed with ice water. The residual water was removed by coevaporation with 1,4-dioxane.
[540] Yield: 15.0 g
[541] MS-ESI: [M + H] ⁺ = 319.2
[542] TLC: R _f = 0.3, silica gel, DCM / MeOH = 9/1 (v / v)
[543] (b) 6-chloro-5-cyano-4- (3-nitrophenyl) -2-phenylpyrimidine
[544] POCl 3 (50 mL) was added 5-cyano-4- (3-nitrophenyl) -2-phenyl-6-hydroxypyrimidine in 1,4-dioxane pa (200 mL) (Example 31 (a)). , 15.0 g) and dimethylaniline (0.5 mL) were added to a stirred solution. After 3 h at 90 ° C., the warmed mixture was filtered off and the filtrate was concentrated under reduced pressure. The residue was dissolved in 1,4-dioxane and ice water was added. The resulting precipitate was filtered off and washed with water. The residual water was removed by coevaporation with 1,4-dioxane.
[545] Yield: 19.7 g
[546] MS-ESI: [M + H] ⁺ = 421.2
[547] TLC: R _f = 0.8, silica gel, heptane / EtOAc = 3/2 (v / v)
[548] (c) ethyl 5-cyano-4- (3-nitrophenyl) -2-phenyl-6- (ethoxycarbonylmethylthio) pyrimidine
[549] DIPEA (8.71 mL) was added ethyl 2-mercaptoacetate (5.15 mL) and 6-chloro-5-cyano-4- (3-nitrophenyl) in a mixture of EtOH (125 mL) and DCM (125 mL) under a nitrogen atmosphere. ) To a stirred solution of 2-phenylpyrimidine (Example 31 (b), 15.8 g). After 2 h at rt, the mixture was diluted with DCM, washed with 0.5 N aqueous HCl, dried (MgSO ₄ ) and concentrated under reduced pressure until dissolution was complete.
[550] Yield: 19.7 g
[551] MS-ESI: [M + H] ⁺ = 421.2
[552] TLC: R _f = 0.7, silica gel, heptane / EtOAc = 3/2 (v / v)
[553] (d) ethyl 5-amino-4- (3-nitrophenyl) -2-phenylthieno [2,3-d] pyrimidine-6-carboxylate
[554] DIPEA (20.0 mL) was added ethyl 5-cyano-4- (3-nitrophenyl) -2-phenyl-6- (ethoxycarbonylmethylthio) in a mixture of dry EtOH (100 mL) and toluene pa (100 mL). ) Was added to a stirred solution of pyrimidine (Example 31 (c), 19.7 g). After 48 h at 100 ° C, the mixture was cooled to 0 ° C. The resulting precipitate was filtered off, washed with cold EtOH and vacuum dried at 40 ° C.
[555] Yield: 17.0 g
[556] MS-ESI: [M + H] ⁺ = 421.2
[557] TLC: R _f = 0.5, silica gel, heptane / EtOAc = 3/2 (v / v)
[558] (e) ethyl 5-amino-4- (3-aminophenyl) -2-phenylthieno [2,3-d] pyrimidine-6-carboxylate
[559] A solution of tin (II) chloride (23.0 g) in anhydrous EtOH (250 mL) was added ethyl 5-amino-4- (3-nitrophenyl) -2-phenylthieno in 1,4-dioxane pa (250 mL). To a solution of [2,3-d] pyrimidine-6-carboxylate (Example 31 (d), 16.6 g) was added. 37% aqueous HCl (6.9 mL) was added and the mixture was heated to reflux (90 ° C.) for 16 h. The mixture was cooled to rt and concentrated under reduced pressure. The residue was suspended in EtOAc (500 mL). 4 N aqueous NaOH was added to pH 10-11. To the mixture was diluted by addition of saturated aqueous NaCl. The organic layer was separated, dried (MgSO ₄ ) and concentrated under reduced pressure.
[560] Yield: 17.0 g
[561] MS-ESI: [M + H] ⁺ = 421.2
[562] TLC: R _f = 0.5, silica gel, heptane / EtOAc = 3/2 (v / v)
[563] (f) 5-amino-4- (3-aminophenyl) -2-phenylthieno [2,3-d] pyrimidine-6-carboxylic acid
[564] Potassium hydroxide (20.0 g) was added with ethyl 5-amino-4- (3-aminophenyl) -2-phenylthieno [2,3- in a mixture of 1,4-dioxane (210 mg) and water (80 mL). d] to a solution of pyrimidine-6-carboxylate (Example 31 (e), 17.0 g). After 16 h at 90 ° C, the mixture was cooled to 0 ° C. The resulting precipitate was filtered off, suspended in water (300 mL) and cooled to 0 ° C. 2 N aqueous citric acid was added to the mixture and acidified to pH 3 and stirred at 0 ° C. to room temperature for 2 hours. The resulting precipitate was filtered off, washed with water and dried in vacuo at 40 ° C.
[565] Yield: 13.3 g
[566] MS-ESI: [M + H] ⁺ = 363.0
[567] TLC: R _f = 0.2, silica gel, DCM / MeOH = 95/5 (v / v)
[568] (g) t-butyl 5-amino-4- (3-aminophenyl) -2-phenylthieno [2,3-d] pyrimidine-6-carboxyamide
[569] DIPEA (15.3 mL), t-butylamine (9.3 mL) and TBTU (14.1 g) were added to a 5-amino-4- (3-aminophenyl) in a mixture of DCM (250 mL) and DMF (50 mL) under nitrogen atmosphere. To a mixture of -2-phenylthieno [2,3-d] pyrimidine-6-carboxylic acid. After 3 h at rt, the mixture was diluted with DCM and washed with saturated aqueous NaHCO ₃ , 0.1 N aqueous HCl and saturated aqueous NaCl. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel using heptane / EtOAc = 3/7 as 1/1 (v / v) as eluent.
[570] Yield: 14.7 g
[571] MS-ESI: [M + H] ⁺ = 418.4
[572] TLC: R _f = 0.4, silica gel, heptane / EtOAc = 3/2 (v / v)
[573] (h) t-butyl 5-amino-2-phenyl-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[574] t-butyl 5-amino-2-phenyl-4- (3-aminophenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 31 (g), 2.0 g) was dissolved in anhydrous CH ₂ Dissolved in Cl ₂ (20 mL). Then a solution of p-nitrophenyl chloroformate (520 mg) in anhydrous CH ₂ Cl ₂ (10 mL) was added dropwise and the reaction mixture was stirred at room temperature. After 3 hours, the reaction mixture was washed with H ₂ O. The organic layer was dried (MgSO ₄ ) and concentrated under reduced pressure.
[575] Yield: 2.9 g
[576] MS-ESI: [M + H] ⁺ = 583.2
[577] TLC: R _f = 0.6, silica gel, heptane / EtOAc = 1/1 (v / v)
[578] (i) t-butyl 5-amino-2-phenyl-4- (3-((thiomorpholin-4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxy amides
[579] Thiomorpholine (300 μl) was added t-butyl 5-amino-2-phenyl-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3- in dichloromethane (5 mL). d] To a solution of pyrimidine-6-carboxyamide (Example 31 (h), 150 mg) was added and the reaction mixture was stirred at rt overnight. The reaction mixture was then diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer was concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then lyophilized from a mixture of 1,4-dioxane and H ₂ O.
[580] Yield: 89 mg
[581] MS-ESI: [M + H] ⁺ = 547.2
[582] HPLC: R _t = 11.71 min, column Luna C-18 (2), 3 μm, 100 × 2.0 mm, detection
[583] UV = 210 nm, oven temperature = 40 ° C., flow rate = 0.25 ml / min, eluent
[584] Phosphate buffer at 50 mM pH 2.1 / water / ACN = 10/30/60
[585] 10/5/85 (v / v / v), Run time = 20 minutes
[586] Example 32
[587] t-butyl 5-amino-2-phenyl-4- (3-((N, N-dimethylamino) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[588] Dimethyl amine hydrochloride (150 mg) was diluted with N, N-diisopropylethylamine (DIPEA, 0.50 mL) in dichloromethane (5 mL) and t-butyl 5-amino-2-phenyl-4- (3- (p- To a solution of nitrophenoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide (Example 31 (h), 250 mg) was added and the reaction mixture was stirred at rt overnight. . The reaction mixture was then diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer was concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then lyophilized from a mixture of 1,4-dioxane and H ₂ O.
[589] Yield: 75 mg
[590] MS-ESI: [M + H] ⁺ = 489.2
[591] HPLC: R _t = 19.58 min, column Luna C-18 (2), 3 μm, 100 × 2.0 mm, detection
[592] UV = 210 nm, oven temperature = 40 ° C., flow rate = 0.25 ml / min, eluent
[593] Phosphate buffer at 50 mM pH 2.1 / water / ACN = 10/60/30
[594] 10/5/85 (v / v / v), Run time = 20 minutes
[595] Example 33
[596] t-butyl 5-amino-2-phenyl-4- (3-((morpholin-4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide
[597] Morpholine (250 mg) was dissolved in t-butyl 5-amino-2-phenyl-4- (3- (p-nitrophenoxycarbonylamino) phenyl) thieno [2,3-d in dichloromethane (5 mL). ] Was added to a solution of pyrimidine-6-carboxyamide (Example 31 (h), 150 mg) and the reaction mixture was stirred overnight at room temperature. The reaction mixture was then diluted with CH ₂ Cl ₂ and washed with H ₂ O. The organic layer was concentrated under reduced pressure. The title compound was purified by HPLC using a Luna C-18 column with the following gradient: 0/100 (v / v) at H ₂ O / CH ₃ CN = 80/20 within 45 minutes. The title compound was then lyophilized from a mixture of 1,4-dioxane and H ₂ O.
[598] Yield: 63 mg
[599] MS-ESI: [M + H] ⁺ = 489.2
[600] HPLC: R _t = 19.39 min, column Luna C-18 (2), 3 μm, 100 × 2.0 mm, detection
[601] UV = 210 nm, oven temperature = 40 ° C., flow rate = 0.25 ml / min, eluent
[602] Phosphate buffer at 50 mM pH 2.1 / water / ACN = 10/60/30
[603] 10/5/85 (v / v / v), Run time = 20 minutes
[604] Example 34
[605] CHO-LH and CHO-FSH in vitro viability
[606] The LH agonistic activity of the compounds was tested with Chinese hamster ovary (CHO) cells stably transfected with human LH receptors and cotransfected with cAMP reactive element (CRE) / promoter detecting expression of the firefly luciferase reporter gene. . Binding of the ligand to the Gs-coupled LH receptor will increase cAMP and in turn will result in increased cross activity of the luciferase reporter construct. Luciferase signals were quantified using an luminescence counter. For test compounds, EC ₅₀ values (concentrations of test compound causing half maximal (50%) stimulation) were calculated. For that purpose, a soft program GraphPad PRISM, version 3.0 (GraphPad Software Inc., San Diego, USA) was used. The results showed that the EC ₅₀ values for the compounds of Examples 9, 11, 12, 13, 14, 25 and 27 were 10 ⁻⁶ to 10 ⁻⁷ M. The compounds of Examples 1, 2, 3, 4, 5, 17, 22, 26, 28, 29, 31 and 33 have an EC ₅₀ value of 10 ⁻⁷ to 10 ⁻⁸ M whereas Examples 7, 15, 16 EC ₅₀ values of the compounds of 18, 21, 23 and 32 were less than 10 ⁻⁸ M.

权利要求:
Claims (15)
[1" claim-type="Currently amended] Bicyclic heteroaromatic compounds according to formula (I) or a pharmaceutically acceptable salt thereof:
Formula I

Where
R ¹ is (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14C) aryl or (4-13C) heteroaryl, all of which are N (R ⁴ ) R ⁵ , NHR ⁵ , Optionally substituted with one or more substituents selected from R ⁵ , OR ⁵ and / or SR ⁵ ;
R ² is (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, or (6-14C) aryl or (4-13C) heteroaryl;
R ³ is (1-8C) alkyl, (3-8C) cycloalkyl, (2-7C) heterocycloalkyl, (6-14) aryl or (4-13C) heteroaryl;
R ⁴ may be selected from the same groups as described for R ² ;
R ⁵ is (6-14C) aryl, (4-13C) heteroaryl, (6-14C) arylcarbonyl, (2-7C) heterocycloalkyl, (2-7C) heterocycloalkylcarbonyl, (2- 8C) alkenylsulfonyl, (2-8C) alkenoxycarbonyl, (3-8C) cycloalkyl, (6-14C) arylsulfonyl, (6-14C) arylaminocarbonyl, (6-14C) aryloxy Carbonyl, (6-14C) arylaminosulfonyl, (6-14C) aryloxysulfonyl, (2-8C) alkenyl, (2-8C) alkynyl or (1-8C) alkyl, (1-8C ) Alkylcarbonyl, (1-8C) alkylsulfonyl, (1-8C) (di) alkylaminocarbonyl, (1-8C) alkoxycarbonyl, (1-8C) (di) alkylaminosulfonyl or ( 1-8C) alkoxysulfonyl, the alkyl group being hydroxyl, (1-8C) alkoxy, (2-7C) heterocycloalkyl (1-8C) alkoxy, (3-8C) cycloalkyl (1-8C) alkoxy , (6-14C) aryl (1-8C) alkoxy, (4-13C) heteroaryl (1-8C) alkoxy, (2-7C) heterocycloalkyl, (3-8C) cycloalkyl, (6-14C) Aryl, (4-13C) heteroaryl, (1-8C) alkoxycarbonyl, (6-14C) aryloxycarbonyl, (1-8C ) Alkylcarbonyloxy, (6-14C) arylcarbonyloxy, (1-8C) alkylcarbonyl, (6-14C) arylcarbonyl, amine, (1-8C) alkylaminocarbonyl, (6-14C ) Arylaminocarbonyl, (1-8C) alkylcarbonylamino, (6-14C) arylcarbonylamino, (6-14C) (di) arylamino, (di) [(1-3C) alkoxy (1- 3C) alkyl] amino and / or (1-8C) (di) alkylamino optionally substituted with one or more substituents;
Y is CH or N;
Z is NH ₂ or OH;
A is S, N (H), N (R ⁴ ), O or a bond;
R ⁴ may be selected from the same groups as described for R ² ;
B is N (H), O or a bond;
X1-X2 is C = C, C (O) -NH, NH-C (O), C (O) -O, OC (O), C = N, N = C, or R ⁵ is (1-8C) ) Alkylsulfonyl, (6-14C) arylsulfonyl, (1-8C) (di) alkylaminocarbonyl, (6-14C) arylaminocarbonyl, (1-8C) alkoxycarbonyl, (6-14C ) Aryloxycarbonyl, (1-8C) (di) alkylaminosulfonyl, (6-14C) arylaminosulfonyl, (1-8C) alkoxysulfonyl, (6-14C) aryloxysulfonyl, (2 -7C) heterocycloalkylcarbonyl, (2-8C) alkenylsulfonyl or (2-8C) alkenylcarbonyl, further S or O;
Provided that the compound is ethyl 5-hydroxy-2-methyl-4- (piperidin-1-yl) pyrido [2,3-d] pyrimidine-6-carboxylate, ethyl 5-hydroxy- 2-methyl-4- (morpholin-4-yl) pyrido [2,3-d] pyrimidine-6-carboxylate or ethyl 5-hydroxy-2-methyl-4- (pyrrolidine-1 -Yl) pyrido [2,3-d] pyrimidine-6-carboxylate.
[2" claim-type="Currently amended] The compound of claim 1, wherein B is N (H) or a bond.
[3" claim-type="Currently amended] The compound of claim 1 or 2, wherein Z is NH ₂ .
[4" claim-type="Currently amended] 4. The compound of claim 1, wherein R ¹ is optionally substituted with one or more substituents selected from N (R ⁴ ) R ⁵ , NHR ⁵ , R ⁵ , OR ^5, or SR ⁵ . ) Aryl or (4-13C) heteroaryl.
[5" claim-type="Currently amended] The compound of claim 4, wherein R ⁵ is (6-14C) arylcarbonyl, (2-7C) heterocycloalkylcarbonyl or (1-8C) alkyl, (1-8C) alkylcarbonyl, (1-8C) (Di) alkylaminocarbonyl, wherein the alkyl group is (2-7C) heterocycloalkyl, (4-13C) heteroaryl, (1-8C) alkoxycarbonyl, (1-8C) alkylaminocarbonyl, (1 -8C) alkylcarbonylamino, (6-14C) arylcarbonylamino, amine and / or (1-8C) (di) alkylamino.
[6" claim-type="Currently amended] 6. The compound of claim 4, wherein R ¹ is phenyl optionally substituted with one or more substituents.
[7" claim-type="Currently amended] The compound of claim 6, wherein R ¹ is substituted at the meta position.
[8" claim-type="Currently amended] 8. The compound of claim 1, wherein Y is N. 9.
[9" claim-type="Currently amended] The compound of any one of claims 1-8, wherein B is N (H) or B is a bond and R ³ is (2-7C) heterocycloalkyl.
[10" claim-type="Currently amended] The compound of claim 1, wherein X 1 -X 2 is C═C, C═N, S or N═C.
[11" claim-type="Currently amended] The compound of claim 10, wherein X 1 -X 2 is C═C or S. 12.
[12" claim-type="Currently amended] t-butyl 5-amino-2-methylthio-4- (3-((N, N-diethylamino) carbonyloxy) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- (methoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino- 2-methylthio-4- (3- (allyloxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- (ethoxycarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3-((morpholine -4-yl) carbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide, t-butyl 5-amino-2-methylthio-4- (3- (1,2, 3,6-tetrahydropyridinocarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxyamide or t-butyl 5-amino-2-phenyl-4- (3-((N , N-dimethylaminocarbonylamino) phenyl) thieno [2,3-d] pyrimidine-6-carboxylic acid Compound is selected from the group consisting of draw.
[13" claim-type="Currently amended] The compound of any one of claims 1 to 12 for use in therapy.
[14" claim-type="Currently amended] A pharmaceutical composition comprising a bicyclic heteroaromatic compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt or solvate thereof in admixture with a pharmaceutically acceptable adjuvant.
[15" claim-type="Currently amended] Use of a bicyclic heteroaromatic derivative according to any one of claims 1 to 12 or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for controlling pregnancy.

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引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-09-22|Priority to EP00203287

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2000-09-22|Priority to EP00203287.8

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2001-09-17|Application filed by 악조 노벨 엔.브이.

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2001-09-17|Priority to PCT/EP2001/010743

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2003-05-22|Publication of KR20030040483A

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2006-12-11|First worldwide family litigation filed

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2008-09-25|Publication of KR100860040B1

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2008-09-25|Application granted

优先权:

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申请号 | 申请日 | 专利标题

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EP00203287|2000-09-22|

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EP00203287.8|2000-09-22|

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PCT/EP2001/010743|WO2002024703A1|2000-09-22|2001-09-17|Bicyclic heteroaromatic compounds|