ZA200006601B - Thienopyridine compounds, their production and use. - Google Patents

Description

DESCRIPTION

Thienopyridine Compounds, Their Production and Use

TECHNICAL FIELD

The present invention relates to thieno[2,3-blpyridine derivatives exhibiting gonadotropin releasing hormone (GnRH) antagonizing activity, their production and use.

BACKGROUND ART

The secretion of hypophysial anterior lobe hormone is regulated by the peripheral hormone secreted by each target organ and the secretion-promoting or secretion-suppressing hormone secreted by the hypothalamus, which is the center superior to the hypophysial anterior lobe, and this group of hormones hereinafter generically referred to as hypothalamic hormone in this specification. To date, nine hypothalamic hormones have been identified, for example, thyroid- stimulating hormone-releasing hormone (TRH), and gonadotropin releasing hormone [GnRH, also known as luteinizing hormone releasing hormone (LH-RH)], etc. It is conjectured that these hypothalamic hormones exhibit their hormone actions etc. via receptors assumed to be present in the hypophysial anterior lobe, and analyses of receptor genes specific to these hormones, including humans, are ongoing.

Antagonists or agonists that act specifically and selectively on these receptors would therefore regulate the action of hypothalamic hormones and hence regulate the secretion of hypophysial anterior lobe hormone. As a result, such antagonists or agonists are expected to prevent or treat diseases depending on these hypophysial anterior lobe hormone.

Known compounds possessing GnRH-antagonizing activity include GnRH-derived linear peptides (USP 5,140,009 and USP 5,171,835), a cyclic hexapeptide derivative (JP-A-61- 191698), a bicyclic peptide derivative [Journal of Medicinal

Co WO 00/00493 PCTAP99/03379

A 2

Chemistry, Vol. 36, pp. 3265-3273 (1993)], and so forth.

Non-peptide compounds possessing GnRH-antagonizing activity include compounds described in WO 95/28405, WO 97/14697, WO 97/14682, WO 97/41126 and so forth.

Peptide compounds pose a large number of problems to be resolved with respect to oral absorbability, dosage form, dose volume, drug stability, sustained action, metabolic stability etc. There is strong demand for an oral GnRH antagonist, especially one based on a non-peptide compound, that has excellent therapeutic effect on hormone-dependent

Co cancers, e.g., prostatic cancer, endometriosis, precocious puberty etc., and that does not show transient } hypophysial-gonadotropic action (acute action).

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a diagrammatic representation of the ameliorating effect of suppression of plasma LH concentrations in castrated monkeys after oral administration of the compound of the following Example 1-2, in which '-'B* represents control (1) (vehicle), codes represents control (2) (vehicle), SNE represents control (3) (vehicle), — A — represents compound (1) [the test animal administered the compound of Ex. No. 1-2] and —A— represents compound (2) [the test animal administered the compound of Ex. No. 1-2]. .

DISCLOSURE OF INVENTION

The present inventors produced various thienopyridine derivatives, investigated their actions, and found that some compounds possess excellent GnRH-antagonizing activity.

The inventors have conducted further investigation based on this finding, and developed the present invention.

Accordingly, the present invention relates to:

[1] a compound of the formula:

i 3 cH;-N 0 0]

N | (1)

R— S N F

ES

E wherein rl represents a Cy_7 alkyl group which may be substituted, a C3_9 cycloalkyl group which may be substituted, a Cj_g alkoxyamino group which may be substituted or a hydroxyamino group which may be substituted; and

RZ represents a Cy _v alkyl group which may be substituted or a phenyl group which may be substituted; when R! is an unsubstituted C;_5 alkyl group, then R?2 is a substituted Cy_9 alkyl group or a substituted phenyl group, or a salt thereof [hereinafter sometimes referred to briefly as compound (I)1];

[2] a compound of the above (1) or a salt thereof, wherein RY is (1) a C;.7 alkyl group which may be substituted by 1 to 5 substituents selected from the group consisting of (i) hydroxy, (ii) C;_5 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy _g alkoxy-carbonyl, benzyloxycarbonyl, Cj _3 acyl, Cy_3 alkylsulfonyl and Cq_3 alkyl, (iv) Ci.10 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_7 cycloalkyloxy-carbonyl and Ci-3 alkoxy and (v) Cj_g alkoxy-carbonyl, (2) aCz_9 cvcloalkyl group which may be substituted by 1 to

R © 4 3 substituents selected from the group consisting of (i) hydroxy, (ii) Cy. acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy_g alkoxy-carbonyl, benzyloxycarbonyl, Ci-3 acyl, C;.; alkylsulfonyl and C;_3 alkyl, (iv) Cj_jg alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_y cycloalkyloxy-carbonyl and Cy _3 alkoxy and (v) Cj_¢ alkoxy-carbonyl, (3) a Cy_¢ alkoxyamino group which may be substituted by 1 to 5 substituents selected from the group consisting of (i) hydroxy. (ii) Cy. acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cj _g alkoxy-carbonyl, benzyloxycarbonyl, Cj_3 : acyl, Ci_3 alkylsulfonyl and C;_3 alkyl, (iv) Cj_jo alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_» cycloalkyloxy-carbonyl and C;_3 alkoxy and (v) Cj_¢ alkoxy-carbonyl, or (4) a hydroxyamino group which may be substituted by 1 or 2 substituents selected from the group consisting of i) Cy. acyloxy, (ii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cqi_¢ alkoxy-carbonyl, benzyloxycarbonyl, Cqy_3 acyl, Cy.3 alkylsulfonyl and Cy_3 alkyl, (iii) Cy_30 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_< cycloalkyloxy-carbonyl and Cy _3 alkoxy and (iv) Cq.g alkyl; and

Co RZ is (1) a C;.7 alkyl group which may be substituted by 1 to 5 substituents selected from the group consisting of (1). hydroxy, (ii) Cy_7 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy _g alkoxy-car penzyloxycarbonyl, Cj _3

B WO 00/0493 PCT/IP99/03379 acyl, Cy_3 alkylsulfonyl and Cj_3 alkyl, (iv) Cj.jp alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_7 cycloalkyloxy-carbonyl and Cy.3 alkoxy and (v) Cj_¢ alkoxy-carbonyl, or (2) a phenyl group which may be substituted by 1 to 5 substituents selected from the group consisting of halogen,

Ci-3 alkyl and Cj_3 alkoxy:

[3] a compound of the above (1) or a salt thereof, wherein Rl is a Ci1-7 alkyl group which may be substituted or a Cy. 9 cycloalkyl group which may be substituted;

[4] a compound of the above (1) or a salt thereof, wherein rR! is a substituted branched Ci.7 alkyl group or a substituted C3_7 cycloalkyl group:

[5] a compound of the above (1) or a salt thereof, wherein Rl is a Ci-7 alkyl group substituted by hydroxy or a C3_y cycloalkyl group substituted by hydroxy:

[6] a compound of the above (1) or a salt thereof, wherein Rl! is a substituted C3_- cycloalkyl group;

[7] a compound of the above (1) or a salt thereof, wherein rR! is a cyclopropyl group which may be substituted by hydroxy:

[8] a compound of the above (1) or a salt thereof, wherein R? is a branched C3_97 alkyl group which may be substituted;

[9] a compound of the above (1) or a salt thereof, wherein R? is a phenyl group which may be substituted;

[10] a compound of the above (1) or a salt thereof, wherein R? is a phenyl group:

[11] a compound of the above (1) or a salt thereof, wherein rR! is a Cs3.7 cycloalkyl group and RZ is a Ci-6 alkyl group;

[12] a compound of the above (1) or a salt thereof, wherein R! is (1) a Cy.4 alkyl group substituted by 1 or 2 hydroxy, (2) a C3. cycloalkyl group substituted by hydroxy. or (3) a Cj_3 alkoxyamino group; and

R?2 is an isopropyl group Or a phenyl group;

[13] a compound of the above (1) or a salt thereof, wherein R! is (1) a C;.7 alkyl group which may be substituted by 1 or 2 substituents selected from the group consisting of hydroxy. Ci_3 alkyl-carbonyloxy, amino, benzyloxycarbonylamino, Cy; _3 alkoxy, Cq_3alkoxy-Cj_3 alkoxy and Cq_3 alkoxy-carbonyl, (2) a C3_7 cycloalkyl group which no may be substituted by a hydroxy or a Ci1-3 alkyl-carbonyloxy, or (3) a Cj.3 alkoxyamino group; and

RZ is (1) an isopropyl group which may be substituted by a hydroxy or (2) a phenyl group;

[14] 3- (N-benzyl-N-methylaminomethyl)-4, 7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2- (4- cyclopropanecarbonylaminophenyl) -4-oxothieno[2,3- : blpyridine or a salt thereof; a

[15] 3-(N-benzyl-N-methylaminomethyl)-4, 7-dihydro-5- jsobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2- methylpropionylamino)phenyl]-4 -oxothieno[2,3-blpyridine or a salt thereof;

[16] 3- (N-benzyl-N-methylaminomethyl)-4, 7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1- hydroxycyclopropyl)carbonylamino Jphenyl]-4-oxothieno [2,3- blpyridine or a salt thereof; [17) 3- (N-benzyl-N-methylaminomethyl) -5-benzoyl-7-

Tr (2,6-difluorobenzyl)-4,7-dihydro-2-{4-0(1-_ hydroxycyclopropyl)carbonylamino phenyl] -4- oxothieno [2,3- blpyridine or a salt thereof;

[18] a process for producing a compound of the above [1] or a salt thereof, which comprises reacting a compound of the oo WO 00/00493 PCT/IP99/03379 formula: cH; N Oo O 2 wa vsen i (II)

STONT OF

Xo wherein RZ represents a Cj_7 alkyl group which may be substituted or a phenyl group which may be substituted, or a salt thereof [hereinafter sometimes referred to briefly as compound (II)], with i) a compound of the formula: rR1iacoon wherein rla represents a C;_9 alkyl group which may be substituted or a C3_y cycloalkyl group which may be substituted, or a salt thereof or a reactive derivative thereof; or ii) carbonyldiimidazole, phosgene or a chloroformate, followed by reacting with a compound of the formula:

R1PH wherein r1b represents a Cy _g alkoxyamino group which may be substituted or a hydroxyamino group which may be substituted, or a salt thereof;

[19] a process for producing a compound of the above [3] or a salt thereof, which comprises reacting compound (II) with a compound of the formula: r12cooH wherein rla represents a Cj_4 alkyl group which may be substituted or a C3_7y cycloalkyl group which may be

BN WO 00/00493 PCT/JP99/03379 substituted, or a salt thereof or a reactive derivative thereof; :

[20] a pharmaceutical composition which comprises a compound of the above (1) or a salt thereof; .

[21] a pharmaceutical composition of the above (20) which is for antagonizing gonadotropin-releasing hormone;

[22] a pharmaceutical composition of the above (21) which is for preventing or treating a sex hormone dependent disease;

[23] a method for antagonizing gonadotropin-releasing hormone in a mammal in need thereof which comprises administering to said mammal an effective amount of a compound ov of the above (1) or a salt thereof with a pharmaceutically - acceptable excipient, carrier or diluent;

[24] use of a compound of the above (1) or a salt thereof = for manufacturing a pharmaceutical composition for antagonizing gonadotropin-releasing hormone, and so forth.

Each symbol in the above formulae is hereinafter described in more detail.

The "C,_5 alkyl group" of the "Cj;_y alkyl group which may be substituted" for Rl includes, for example, a straight-chain Cy_4 alkyl group such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, etc.; a branched C3_7 alkyl group such as isopropyl, isobutyl, sec-butyl, tert- butyl, isopentyl, neopentyl, etc., and so forth. Among others, preferred is a branched C3_7 alkyl group. More preferred is isopropyl.

The "substituents" of the "C;_5 alkyl group which may — - 30 be substituted" for R! include, for example, (i) hydroxy, (ii)

Ci-7 acyloxy (e.g., Ci_¢ alkyl-carbonyloxy such as acetoxy, propionyloxy, etc.; benzoyloxy etc.), (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cj_g alkoxy-carbonyl (e.g., methoxycarbonyl. ethoxycarbonyl, tert-butoxycarbonyl, etc. ), benzyloxycarbonyl, Cy_3 acyl (e.g., Ci.» alkyl-carbonyl such as acetyl, propionyl, etc.), Ci.3 alkylsulfonyl (e.g., methanesulfonyl etc.) and Cy_3 alkyl (e.g., methyl, ethyl, etc.), etc. [e.g.; amino, methoxycarbonylamino, ethoxycarbonylamino, tert- but oxycarbonylbenzyloxycarbonylamino , acetylamino, methanesulfonylamino, methylamino, dimethylamino, etc.), (iv) Cq1_-10 (preferably C;.4) alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of

C3.7 cycloalkyloxycarbonyl (e.g... cyclohexyloxycarbonyloxy, etc.) and Cy_3 alkoxy (e.g... methoxy, ethoxy, etc.) [e.g.; methoxy, ethoxy, Propoxy. tert-butoXy, cyclohexyloxycarbonyloxy- l-ethoxy. methoxymethoxy, ethoxymethoxy, etc.], (v) Ci-6 alkoxy- carbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, etc.), and so forth. Among others, preferred is hydroxy.

The "Cy _7 alkyl group" may have 1 to 5, preferably 1 to 3 substituents as mentioned above at possible positions and, when the number of substituents is two or more, those substituents may be the same as or different from one another.

The "C3_5 cycloalkyl group" of the "C3.7 cycloalkyl group which may be substituted” for R! includes, for example, cyclopropyl. cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc. Among others, preferred is cyclopropyl.

The "substituents" of the "C3_4y cycloalkyl group which may be substituted" for Rl are the same as those mentioned above for the "substituents" of the "Ci.7 alkyl group which may be substituted" for Rl. The number of substituents is 1 to 3. When the number of substituents is two or more, those i 10 substituents may be the same as or different from one another.

The "Cj.g alkoxyamino group" of the "Cq_g alkoxyamino group which may be substituted" for Rl includes, for example, mono- Or di-Ci.¢ alkoxyamino (e.g., methoxyamino, ethoxyamino, dimethoxyamino, diethoxyamino, ethoxymethoxyamino, etc. ), etc. Among others, preferred is mono-Cy_3 alkoxyamino (e.g., methoxyamino, etc.) .

The “substituents” of the "Cj _g alkoxyamino group which may be substituted” for rR! and their number are the same as those mentioned above for the “substituents” of the "Ci.7 alkyl group which may be substituted” for Rl. When the number . of substituents is two or more, those substituents may be the . same as or different from one another. The "Ci-6 alkoxy" or the "nitrogen atom of an amino group” of the Cj_¢ alkoxyamino group may be substituted by the above "gsubstituents”.

Such "Cy _g alkoxyamino group which may be substituted” is exemplified by methoxyamino, N-methyl-N-methoxyamino,

N-ethyl-N-methoxyamino, ethoxyamino, dimethoxyamino, diethoxyamino, ethoxymethoxyamino, etc. Preferred;is Cq_ : 20 3 alkoxyamino, N-C;.3 alkyl-N-Cj_3 alkoxyamino, etc.

The "substituents” of the "hydroxyamino group which may be substituted” for ri may be located on the "hydroxy group" of the hydroxyamino group or the "nitrogen atom of an amino group" of the hydroxyamino group. Such “substituents” on the "hydroxy group” include, for example, (i) Cj_7 acyloxy (e.g., Ci_¢ alkyl-carbonyloxy such as acetoxy, propionyloxy, — etc.; benzoyloxy etc.), (ii) amino which may be substituted by 1 cr 2 substituents selected from. the group consisting of

C;.g alkoxy-carbonyl (e.g., methoxycarbonyl, Te ethoxycarbonyl, tert-butoxycarbonyl, etc. ) benzyloxycarbonyl, Cq_3 acyl (e.g., oo ) alkyl-carbonyl such as acetyl, propionyl, etc.), Cy_3 alkylsulfonyl (e.g.. methanesulfonyl etc.) and Ci-3 alkyl (e.g., methyl, ethyl. etc.), etc. [e.g.; amino, methoxycarbonylamino, ethoxycarbonylamino, tert- butoxycarbonylbenzyloxycarbonylamino , acetylamino, methanesulfonylamino, methylamino, dimethylamino, etc. 1. (iii) Cy.1o (preferably Cy.4) alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of

C3. cycloalkyloxycarbonyl (e.g... cyclohexyloxycarbonyloxy, etc.) and Cy_3 alkoxy (e.g.. methoxy, ethoxy, etc.) [e.g.; methoxy, ethoxy, PIOpPOXY, tert-butoxy, cyclohexyloxycarbonyloXy- l1-ethoxy, methoxymethoxy, ethoxymethoxy, etc.], and so forth. The number of substituents is 1 to 5, preferably 1 to 3. When the number of substituents is two or more, those substituents may be the same as or different from one another. Such "substituents" on the “nitrogen atom of the amino group" include, for example, (1) each group as described in the above (1) to (iii) and (2) C3_¢ alkyl (e.g., methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, etc. The number of substituents is 1 to 5, preferably 1 to 3. When the number of substituents is two or more, those substituents may be the same as or different from one another.

Preferable examples of the "hydroxyamino group which may be substituted" include N-Cj_g alkyl-N-hydroxyamino (e.qg.,

N-methyl-N-hydroxyamino, N-ethyl-N-hydroxyamino, etc.) and so forth. More preferred is N-Cq.3 alkyl-N-hydroxyamino, etc.

The "Cqi_7 alkyl group" of the "Ci1-9 alkyl group which may be substituted" for R2 includes, for example, a straight-chain or branched Cj_7 alkyl group such as methyl,

. PCT/JP99/03379 ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, etc. Among others, preferred is Cj_3 alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc. More preferred is isopropyl.

Trie “substituents” of the "Cj_5 alkyl group which may be substituted” for RZ and their number are the same as those mentioned above for the "substituents" of the "Cq.7 alkyl group which may be substituted” for Rl. When the number of substituents is two or more, those substituents may be the same as or different from one another.

The “substituents” of the "phenyl group which may be substituted" for RZ includes, for example, halogen (e.g.. fluoro, chloro, bromo, iodo, etc.), Cy_3 alkyl (e.g., methyl, ethyl, propyl, isopropyl. etc. ). Cq.3 alkoxy (e.g., methoxy, ethoxy, DPropoxy, 1SOpPIrOpoOXYy, etc.), etc. Among others, preferred is halogen, more preferred is fluoro.

The "phenyl group" may have 1 to 5, preferably 1 to 3 substituents as mentioned above at possible positions and, when the number of substituents is two or more, those substituents may be the same as or different from one another. rl is preferably a substituted branched C3 _~ alkyl group or a substituted C3_~ cycloalkyl group, more preferablyaC;_5 alkyl group substituted by hydroxy or a C3_9 cycloalkyl group substituted by hydroxy. Among others, preferred is a substituted C3_7 cycloalkyl group. Also, a Ci-3 alkyl group which may be substituted by hydroxy, a C3_7 cycloalkyl group oo which may be substituted by a hydroxy, mono-C; _3 alkoxyamino,

N-Cq_3 alkyl-N-hydroxyamino, hydroxyamino, "is preferred. - --

Especially preferably rR! is (i) cyclopropyl which may be substituted by a hydroxy or (ii) methoxyamino, etc. Most preferred is cyclopropyl which may be substituted by a hydroxy group.

RZ is preferably a Cj_v alkyl group which may be substituted. More preferred is a Ci1-3 alkyl group which may be substituted by a hydroxy group. Especially preferred is isopropyl. Phenyl is also preferred. preferable examples of compound (I) include a compound wherein, rR! is a Cq.3 alkyl group which may be substituted by a hydroxy group, a C3.7 cycloalkyl group which may be substituted by a hydroxy group or a mono-Cjy _3 alkoxyamino group; and

RZ is a C;.3 alkyl group, or a salt thereof.

More preferred is a compound wherein Rl is (1) a Cq- 4 alkyl group substituted by 1 or 2 hydroxy. (2) a C3_7 cycloalkyl group substituted by hydroxy, OT (3) a Cy_3 alkoxyamino group; and

RZ is an isopropyl group or a phenyl group, Or a salt theteof.

A compound wherein rR! is (1) a Ci-7 alkyl group which may be substituted by 1 or 2 substituents selected from the group consisting of hydroxy, Cy_3 alkyl-carbonyloxy, amino, benzyloxycarbonylamino, Cj _3 alkoxy, Cj.3 alkoxy-Cy.gz alkoxy and C;_3 alkoxy-carbonyl, (2) aCz_9q cycloalkyl group which may be substituted by a hydroxy group or a Cyi-3 alkyl- i carbonyloxy, or (3) a Cj_3 alkoxyamino group; and

RZ is (1) an isopropyl group which may be substituted by a hydroxy group or (2) a phenyl group, OT salt thereof is also preferred.

As compound (I), concretely mentioned are 3- (N-benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryi-7-(2,6-difluorobenzyl)-2-(4-

cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3- bipyridine or a salt thereof,

5-benzoyl-3- (N-benzyl-N-methylaminomethyl)-7-(2,6- difluorobenzyl)-4,7-dihydro-4-oxo-2-[4-(3-hydroxy:2-

nethylpropionylamino)phenyllthienol2,3-blpyridine,

5-(4-fluorobenzoyl)-3-(N-benzyl-N- methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-4- oxo-2- (4-cyclopropanecarbonylaminophenyl)thienol2,3- blpyridine,

3- (N-benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2- methylpropionylamino)phenyl]-4-oxothieno[2,3-blpyridine,

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5- ) jsobutyryl-7-(2,6-difluorobenzyl)-2-(4-N'- h methoxyureidophenyl)-4-oxothieno[2,3-b]pyridine,

3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-14-[(1- hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3- blpyridine,

(R)-4,7-dihydro-2-[4-(3-hydroxy-2- IEE methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno{2,3- bipyridine,

4,7-dihydro-2-[4-(2-hydroxy-2- :

methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl)=-5-isobutyryl-4-oxothieno{2,3- blpyridine,

4,7-dihydro-2-[4-(3-hydroxy-3- methylbutyrylamino)phenyl]}-7-(2,6-difluorobenzyl)-3-(N-

benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2.3-

— blpyridine,

(R)-4,7-dihydro-2-[4-(2,3- EE dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3- (N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-

oxothieno{2,3-blpyridine,

3- (N-benzyl-N-methylaminomethyl) -5-benzoyl-7-(2,6- difluorobenzyl)-4,7-dihydro-2-14-[(1- hydroxycyclopropyl )carbonylamino jphenyl]-4- oxothienol2,3- b]pyridine, and salts thereof. compound (I) can be produced in any per se known manner, for example, according to the methods disclosed in WO 95/28405 or analogous methods thereto. Concretely mentioned are the following Production method 1 and Production method 2.

Production method 1: compound (I) is produced by reacting compound (II) with a compound of the formula: rlacooH wherein R12 represents a

Ci-7 alkyl group which may be substituted ora C3_7 cycloalkyl group which may be substituted, or a salt thereof or a reactive derivative thereof [hereinafter sometimes referred to briefly as compound (III)].

The "Cy_7 alkyl group which may be substituted” and the "Ci_9 cycloalkyl group which may be substituted" for R12 are the same as those mentioned above for the "Ci-7 alkyl group which may be substituted” and the "C3_7 cycloalkyl group which may be substituted" for Rl, respectively.

The "reactive derivative" of the "compound of the formula: Rl8COOH, or a salt thereof or a reactive derivative thereof" includes, for example, a compound of the formula: rl2coy wherein Y represents halogen and rl2 is same as defined above.

The amount of compound (III) to be reacted is about 1 to 3 mol, relative to one mol of compound (II).

This reaction is also carried out in the presence of a base. The "base" is exemplified by inorganic bases such as sodium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, sodium hydroxide,

oo PCT/JP99/03379 potassium hydroxide and thallium hydroxide, and organic bases such as triethylamine and pyridine, etc.

The amount of the "base" is about 1 to 10 mol, preferably about 2 to 5 mol, relative to one mol of compound (IT) .

In the case where a compound of the formula: RY2COOH or a salt thereof is used, a condensing reagent which is generally used in peptide chemistry such as benzotroazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (pyBop) may be employed to form an amide bond.

This reaction is advantageously carried out in a solvent inert to the reaction. Examples of the solvent include ethers (e.g., ethyl ether, dioxane, dimethoxyethane, 7 4 tetrahydrofuran, etc.), aromatic hydrocarbons (e.g.., benzene, toluene, etc.), amides (e.g... dimethylformamide, dimethylacetamide, etc.), halogenated hydrocarbons (e.g... chloroform, dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150 °C, preferably room temperature (about 15 to 25 °c). The reaction time is usually about 1 to 12 hours. B compound (II) can be produced in any per se known manner, for example, by the methods disclosed in WO 95/28405 or analogous methods thereto. compound (1) wherein rl isaCy_¢ alkoxyamino group which may be substituted or a hydroxyamino group which may be substituted can also be produced in according to the method of the following Production method 2.

Production method 2: —— 30 compound (I) is produced by reacting compound (II) with carbonyldiimidazole (N,N'-carbonyldiimidazole: —eDI)i- phosgene (monomer, dimer or trimer) or a chloroformate, followed by reacting with a compound of the formula: R1bH wherein plD represents a Cj _g alkoxyamino group which may be

: PCT/IP99/03379 substituted or a hydroxyamino group which may be substituted, or a salt thereof [hereinafter sometimes referred to briefly as compound (IV)].

The "chloroformate" includes, for example, a compound of the formula: C1-COOY' wherein Y' represents a Ci-6 alkyl group, such as chloroformate ethyl, etc.

The "C;.¢ alkoxyamino group which may be substituted” and the "hydroxyamino group which may be substituted" for r1P are the same as those mentioned above for the "Ci1-6 alkoxyamino group which may be substituted” and the "hydroxyamino group which may be substituted" for rl, respectively.

In the reaction of compound (II) with carbonyldiimidazole, phosgene oI chloroformates, carbonyldiimidazole, phosgene or chloroformates is used in amount of about 1 to 3 mol, relative to one mol of compound (II).

This reaction is advantageously carried out in a solvent inert to the reaction. Examples of the solvent include ethers (e.g., ethyl ether, dioxane, dimethoxyethane, tetrahydrofuran, etc.) , aromatic hydrocarbons (e.g... benzene, toluene, etc.), amides (e.g.. dimethylformamide, dimethylacetamide, etc. ), halogenated hydrocarbons (e.g., chloroform, dichloromethane, etc.), and so forth.

The reaction temperature is usually about 0 to 150 °C, preferably room temperature (about 15 to 25 °C). The reaction time is usually about 1 to 36 hours.

This reaction is also carried out in the presence of a pase. The "base" is exemplified by inorganic bases such as codium carbonate, sodium hydrogen carbonate, potassium carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium nydroxide and thallium hydroxide, and organic bases such as triethylamine and pyridine, etc.

The amount of the "base" is about 2 to 20 mol, preferably about 5 to 12 mol, relative to one mol of compound (II).

The following reaction with compound (IV) can be carried out in the same condition of. the above reaction of compound (II) with carbonyldiimidazole, phosgene or a chloroformate.

The amount of compound (IV) is about 2 to 20 mol, preferably ~ about 5 to 10 mol, relative to one mol of compound (II).

The reaction temperature 1is usually about 0 to 150 °C, preferably room temperature (about 15 to 25°C). The reaction time is usually about 1 to 6 hours.

Compound (I) of the present invention may be isolated . and purified by ordinary means of separation such as 5 recrystallization, distillation and chromatography, etc. :

When the compound of the formula (I) is obtained in free form, : it can be converted to a salt by per sé known methods or analogous thereto. When compound (I) is obtained in salt form, it can be converted to the free form or another salt by per se known methods Or analogous thereto.

Salts of compound (I) are preferably physiologically acceptable acid addition salts. Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid. hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid) and physiologically acceptable acid addition salts with organic acids (e.g., formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p- toluenesulfonic acid). When compound (I) has an acidic group, it may form a physiologically acceptable salt with an —- inorganic base (e.g., alkali metals such as sodium, potassium, calcium and magnesium, alkaline earth metals, ammonia) cr an organic base (e.g., trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-

dibenzylethylenediamine).

Compound (I) may be a hydrate or a non-hydrate. The hydrate is exemplified by monohydrate, sesquihydrate and dihydrate. when compound (I) is obtained as a mixture (racemate) of optically active configurations, it can be resolved into the (R)- and (S)-forms by the conventional optical resolution techniques.

Compound (I) of the present invention (hereinafter also referred to as "compound of the present invention") possesses excellent GnRH-antagonizing activity and low toxicity. In addition, it is excellent in oral absorbability, action sustainability, stability and pharmacokinetics.

Furthermore, it can be easily produced. The compound of the present invention can therefore be safely used in a mammal (e.g., human, monkey, bovine, horse, dog, cat, rabbit, rat, mouse) for the preventing and/or treating diseases depending on male or female hormones, diseases due to excess of these hormone, etc., by suppressing gonadotropin secretion by its

GnRH receptor-antagonizing action to control blood sex hormone concentrations.

For example, the compound of the present invention is useful for preventing and/or treating sex hormone-dependent cancers (e.g., prostatic cancer, uterine cancer, breast cancer, pituitary tumor, etc.), prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty, amenorrhea, premenstrual syndrome, multilocular ovary syndrome, pimples etc. The compound of the present invention is also useful for the regulation of reproduction in males and females (e.g., pregnancy regulators, menstruation cycle regulators, etc.). The compound of the present invention also be used as a male or female contraceptive, or as a female ovulation inducer. Based on its rebound effect after withdrawal, the compound of the present invention can be used to treat infertility.

PCT/JP99/03379

In addition, the compound of the present invention is useful for regulation of animal estrous, improvement of meat quality and promotion of animal growth in the field of animal husbandry. The compound of the present invention 1s also useful as a fish spawning promoter. x

Although the compound of the present invention can be used alone, it is effective to use in combination with a steroidal or non-steroidal anti-androgen agent or anti- estrogen agent. The compound of the present invention can also be used to suppress the transient rise in blood testosterone concentration (flare phenomenon) observed in administration of a super-agonist such as Leuprorelin . acetate. The compound of the present invention can ‘be used . in combination with a super-agonist such as leuprorelin acetate, gonadrelin, buserelin, triptorelin, goserelin, nafarelin, histrelin, deslorelin, meterelin, lecirelin, and so forth. Among others, preferred is leuprorelin acetate.

The compound of the present invention also may be used with a chemotherapeutic agent for cancer. A preferred example of such combination is the compound of the present invention in combination with chemotherapeutic agents such as ifosfamide,

UTF, adriamycin, peplomycin and cisplatin for prostatic cancer. For breast cancer, the compound of the present invention can be used with chemotherapeutic agents such as cyclophosphamide, 5-FU, UFT, methotrexate, adriamycin, mitomycin C and mitoxantrone.

When the compound of the present invention is used for preventing and/or treating (as a prophylactic and/or therapeutic agent for) the above-mentioned diseases OI used in the filed of animal husbandry or fishery, it can be — administered orally or non-orally, as formulated with a pharmaceutically acceptable carrier, normally in the form of solid preparations such as tablets, capsules, granules and powders for oral administration, or in the form of intravenous, subcutaneous, intramuscular or other injections, suppositories or sublingual tablets for non-oral administration. It may also be sublingually. subcutaneously, intramuscularly or otherwise administered in the form of sustained-release preparations of sublingual tablets, microcapsules etc. Depending on symptom severity: subject age, sex, weight and sensitivity: duration and intervals of administration; property, dispensing and kind of pharmaceutical preparation; kind of active ingredient etc., daily dose is not subject to limitation. For use in the treatment of the above-described sex hormone -dependent cancers (e.g., prostatic cancer, uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy, hysteromyomz, endometriosis, precocious puberty etc., daily dose is normally about 0.01 to 30 mg, preferably about 0.02 to 10 mg, and more preferably 0.1 to 10 mg, per kg weight of mammal, normally in 1 to 4 divided dosages.

The above doses are applicable to the use of the compound of the present invention in the filed of animal husbandry or fishery. Daily dose is about 0.01 to 30 mg, preferably about 0.1 to 10 mg, per kg weight of subject organism, normally in 1 to 3 divided dosages.

In the pharmaceutical composition of the present invention, the amount of compound (I) is 0.01 to 100 % by weight or so of the total weight of the composition.

Pharmaceutically acceptable carriers are various organic or inorganic carrier substances in common use as pharmaceutical materials, including excipients, lubricants, binders ané disintegrants for solid preparations, and solvents, dissolution aids, suspending agents, isotonizing agents, bufiers and soothing agents for liquid preparations.

Other pharmaceutical additives such as preservatives, antioxidants, coloring agents and sweetening agents may be used as necessary. preferable excipients include, for example, lactose,

sucrose, D-mannitol, starch, crystalline cellulose and light silicic anhydride. Preferable lubricants include, for example, magnesium stearate, calcium stearate, talc and colloidal silica. Preferable binders include, for example, crystalline cellulose, sucrose, D-mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose and polyvinylpyrrolidone. preferable disintegrants include, for example, starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, crosslinked carmellose sodium and carboxymethyl starch sodium. Preferable solvents include, for example, water for injection, alcohol, propylene glycol, macrogol, sesame oil and corn oil. preferable dissolution aids include, for example, polyethylene glycol, propylene - glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and sodium citrate. Preferable suspending agents include, for example, surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and monostearic glycerol; and hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose. Preferable isotonizing agents include, for example, sodium chloride, glycerol and D-mannitol. Preferable buffers include, for example, buffer solutions of phosphates, acetates, carbonates, citrates etc. Preferable soothing agents include, for example, benzyl alcohol. Preferable preservatives include, for example, paraoxybenzoic acid — esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid and sorbic acid. preferable antioxidants include, for example, sulfites and ascorbic acid.

By adding suspending agents, dissolution aids, stabilizers, isotonizing agents, preservatives, and so

R <0 4 forth, the compound of the present invention can be prepared as an intravenous, subcutaneous OI intramuscular injection by a commonly known method. In such cases, the compound of the present invention can be freeze-dried as necessary by a commonly known method. In administration to humans, for example, the compound of the present invention can be safely administered orally or non-orally as such or as a pharmaceutical composition prepared by mixing it with a pharmacologically acceptable carrier, excipient and diluent selected as appropriate.

Such pharmaceutical compositions include oral preparations (e.g., powders, granules, capsules, tablets), injections, drip infusions, external preparations (e.q., nasal preparations, transdermal preparations) and suppositories (e.g., rectal suppositories, vaginal suppositories).

These preparations can be produced by commonly known methods in common use for pharmaceutical making processes.

An injection can be produced by, for example, preparing the compound of the present invention as an agueous injection along with a dispersing agent (e.g., Tween 80, produced by

Atlas Powder Company, USA, HCO 60, produced by Nikko Chemicals

Co., Ltd.., polyethylene glycol, carboxymethyl cellulose, sodium alginate), a preservative (e.g., methyl paraben, propyl paraben, benzyl alcohol), an isotonizing agent (e.g., sodium chloride, mannitol, sorbitol, glucose) and other additives, or as an oily injection in solution, suspension or emulsion in a vegetable oil such as olive oil, sesame oil, cottonseed oil or corn oil, propylene glycol or the like.

An oral preparation can pe produced by shaping the compound of the present invention by a commonly known method after addition of an excipient (e.g... lactose, sucrose, starch), a disintegrant (e.g., starch, calcium carbonate), a binder (e.g., starch, gum arabic, carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose), a lubricant

(e.g., talc, magnesium stearate, polyethylene glycol 6000) and other additives, and, where necessary, coating the shaped product for the purpose of taste masking, enteric dissolution or sustained release by a commonly known method. Coating agents for this purpose include, for example, hydroxypropylmethyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethylene glycol, Tween 80, Prulonic F68, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, hydroxymethyl cellulose acetate succinate, Eudragit (produced by Rohm Company, Germany; methacrylic acid/acrylic acid copolymer) and dyes (e.g., iron oxide, titanium So dioxide). For an enteric preparation, an intermediate phase ry may be provided between the enteric phase and the drug- containing phase for the purpose of separation of the two phases by a commonly known method.

An external preparation can be produced by compounding the compound of the present invention as a solid, semi-solid or liquid composition by a commonly known method. Such a solid composition is produced by, for example, powdering the compound of the present invention as such or in mixture with an excipient (e.g.. glycol, mannitol, starch, microcrystalline cellulose), a thickening agent (e.g., natural rubber, cellulose derivative, acrylic acid polymer) and other additives. Such a liquid composition 1is produced by preparing the compound of the present invention as an oily or agueous suspension in almost the same manner as with the injection. The semi-solid composition is preferably an aqueous or oily gel, or an ointment. All these compositions may contain pH regulators (e.g., carbonic acid, phosphoric — : acid, citric acid, ‘hydrochloric acid, sodium hydroxide), preservatives (e.g., paraoxybenzoic acid esters, ~~ chlorobutanol, benzalkonium chloride) and other additives.

A suppository is produced by preparing the compound of the present invention as an oily or aqueous solid, semi-solid or liquid composition by a commonly known method. Useful oily bases for such compositions include glycerides of higher fatty acids (e.g., cacao fat, uitepsols, produced by Dynamite

Nobel Company. Germany) . moderate fatty acids (e.g... MIGLYOL, produced by Dynamite Nobel Company, Germany), and vegetable oils (e.g., sesame oil, soybean oil, cottonseed oil).

Aqueous bases include, for example, polyethylene glycols and propylene glycol. Bases for aqueous gels include, for example, natural rubbers, cellulose derivatives, vinyl polymers and acrylic acid polymers.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is hereinafter described in more detail by means of, put is not limited to, the following reference examples, examples, preparation examples and experimental examples. 1H-NMR spectra are determined with tetramethylsilane as the internal standard, using the GEMINI 200 (200 MHz) spectrometer (produced by varian, Ltd.). the LAMBDA 300 (300

MHz) spectrometer (produced by JEOL, Ltd.) or the Bruker AM500 (500 MHz) spectrometer (produced by Bruker); all 0 values are shown in ppm. Unless otherwise specifically indicated, "%" is by weight. Yield indicates mol/mol %.

The symbols used herein have the following definitions: s: singlet d: doublet t: triplet dt: double triplet m: multiplet br: broad

The term "at room temperature” indicates the range from about 15 To 25 °C, but is not to be construed as strictly limitative.

’ PCT/JP99/03379

Examples

Reference Example 1 : production of 2-amino-5-phenylthiophen-3-carboxylic acid ethyl ester Lo

To a mixture of ethyl cyanoacetate (6.1 g, 50 mmol), sulfur (1.61 g, 50 mmol), triethylamine (3.5 ml, 25 mmol), and dimethylformamide (10 ml). phenylacetaldehyde (50% solution in diethyl phthalate; 12.05 g, 50 mmol) was added dropwise with stirring at 45 °c over 20 minutes. After i0 stirring at 45 °c for 9 hours, the reaction mixture was concentrated and the obtained residue was extracted with ethyl acetate, washed with saline and dried (MgSO4). the : solvent was distilled off under reduced pressure. The residue was chromatographed on silica gel and recrystallized : 15 from ether-hexane to yield pale yellow tabular crystals (5.55 g, 45%). mp 124.5-125.5 °C (1it.; 123-124 °C).

Elemental analysis for Cq13H13NO,S

C (%) H (%) N(¥) E

Calculated : 63.13 ; 5.30 ; 5.66

Found . 62.99 ; 5.05 ; 5.63 14-NMR (300MHz, CDCly) 6: 1.37 (3H, t, J=7.1Hz), 4.30 (2H, d, J=7.1Hz), 5.97 (2d, br), 7.17-7.46 (6H, m).

IR (KBr): 3448, 3320, 1667, 1590, 1549 cm 1. :

Reference Example 2

Production of 2-amino-4-methyl-5-(4- methoxyphenyl)thiophen-3-carboxylic acid ethyl ester

A mixture of 4-methoxyphenylacetone (16.5g, 0.10 mol), oo 30 ethyl cyanoacetate (12.29, 0.10 mol), ammonium acetate (1.55 } g, 20 mmol), acetic acid (4.6 ml, 80 mmol), and benzene (20 ml) was heated and refluxed for 24 hours, while the water being produced was removed using a Dean-Stark apparatus. After cooling, the reaction mixture was concentrated under reduced

PCT/JP99/03379 pressure and the residue was partitioned between dichloromethane and aqueous sodium bicarbonate. After the organic layer was washed with saline and dried (MgSO0y4) the solvent was distilled off under reduced pressure. To a solution in ethanol (30 ml) of the obtained residue, were added sulfur (3.21 g, 0.10 mol) and diethylamine (10.4 ml, 0.10 mol), followed by stirring at 50 to 60 °C for 2 hours. successively the reaction mixture was concentrated. The obtained residue was extracted with ethyl acetate, washed with saline and dried (MgSOy). the solvent was distilled off under reduced pressure. The residue was chromatographed on silica gel and recrystallized from ether-hexane to yield light yellow tabular crystals (11.5 9. 40%). mp 79-80 °C.

Elemental analysis for C,gHy7NO3S

Cc (¢) H (%) N (2) S (%) calculated : 61.83 ; 5.88 ; 4.81 : 11.01

Found ~~: 61.81 ; 5.75 ; 4.74 ; 10.82 1H-NMR (200MHz, CDCl3) 8: 1.37 (3H, t, J=7.1Hz), 2.28(3H, s), 3.83 (3H, s). 4.31 (2H, q, J=7.1Hz), 6.05 (2H, brs), 6.91 (2H, d, J=8.8Hz), 7.27 (2H, 4, J=8.8Hz).

IR (KBr): 3426, 3328, 1651, 1586, 1550, 1505, 1485 cm 1.

FAB-MS m/z: 291 (M').

Reference Example 3

Using some acetone derivatives in place of 4- methoxyphenylacetone, the following compound was obtained in the same manner as in Reference Example 2.

H,C COOCH,CH,

. 0 00/00493 PCT/JP99/03379

Yield: 40% mp 64-65 °C.

Reference Example 4 :

Production of [ 3-ethoxycarbonyl-5-(4-methoxyphenyl)- 4-methylthiophen-2-yl)aminomethylenemalonic acid diethyl ester

To the compound obtained in Reference Example 2 (10 g, 34.3 mmol), ethoxymethylenemalonic acid diethyl ester (7.45 g, 34.5 mmol) was added, followed by stirring at 120 °C for B 2 hours. After the reaction mixture was cooled, the crystal precipitated was collected by filtration via the addition of ether, again washed with ether, and dried over phosphorus . pentaoxide under reduced pressure to yield yellow crystals (14.2 g, 90%). mp 122-123 °C. 1H-NMR (200MHz, CDCl) &: 1.32 (3H, t, J=7.1Hz), 1.38 (3H, t, J=7.2Hz), 1.41 (3H, t, J=7.2Hz), 2.34 (3H, s), 3.85 (3H, s), 4.25 (2H, q, J=7.1Hz), 4.38 (2H, Qq. J=7.2Hz), 4.45 (2H, q, J=7.2Hz), 6.95 (2H, d, J=8.8Hz), 7.31 (2H, 4, J=8.8Hz), 8.22 (1H, 4d, J=13.4Hz), 12.74 (1H, 4d, J=13.1Hz).

IR (KBr): 2984, 1720, 1707, 1688, 1653, 1599, 1518, 1499 em™1.

Reference Example 5

Using the compound obtained in Reference Example 3 as a starting material, the following compound was obtained in the same manner as in Reference Example 4.

EE : H,C ,COOCH,CH, oo

S NH

C000,

COOCH,CH,

. x

Yield: 92% mp 108-109 °C.

Reference Example 6 : ‘Production of 3-carboxy-5-[ (4-methoxyphenyl)-4- methylthiophen-2-yllaminomethylenemalonic acid diethyl ester

To a solution in dioxane (20 ml) of the compound obtained in Reference Example 4 (7.0 g, 15.2 mmol) was added, a solution of potassium hydroxide (5.0 g, 75.7 mmol) in ethanol (30 ml) with stirring at 60 to 70 °C. After stirring at this temperature for 1 hour, the reaction mixture was kept standing at room temperature for 1 hour. To the mixture was added 2

N hydrochloric acid (40 ml, 80 mmol) with ice cooling, the reaction mixture was concentrated under reduced pressure.

The yellow precipitate was collected by filtration, washed with cold water-ethanol, and dried over phosphorus pentaoxide under reduced pressure to yield yellow powders (6. 1g, 93%). mp 184-187 °C. 1H-NMR (200MHZz, DMSO-dg) 6: 1.24 (3H, t, J=7.1Hz), 1.28 (3H, ¢ 3=7.2Hz), 2.30 (3H, s), 3.80 (3H, 5), 4.15 (2H, q. J=7.1Hz), 4.24 (2H, q, J=7.2Hz), 7.03 (2H, 4, J=8.7Hz), 7.37 (2H, 4,

J=8.7Hz), 8.08 (1H, d, J=13.6Hz), 12.41 (1H, 4, J=13.6Hz).

IR (KBr): 3422, 2980, 1719, 1653, 1607, 1551, 1512 em™1.

Reference Example 7

Using the compound obtained in Reference Example 5 as a starting material, the following compound was obtained in the same manner as in Reference Example 6.

PCT/JP99/03379

H,C COOH

S NH

\ coooHoH,

COOCH,CH,

Yield: 98% mp 187-190 °C.

Reference Example 8

Production of 4-hydroxy-2-(4-methoxyphenyl)-3- methylthieno[2,3-blpyridine-5-carboxylic acid ethyl ester

With stirring at 120 °C, small portions of the compound obtained in Reference Example 6 (6.0 g, 13.8 mmol) was added ot to polyphoshoric acid ester (PPE) (90 ml). After stirring a at the same temperature for 30 minutes, the reaction mixture was poured into ice water and extracted with ethyl acetate.

The combined extracts were washed with saline, and dried (MgsS0,4). the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel toyield yellow powders (3.659, 77%). For a sample for elemental analysis, the powders obtained were recrystallized from ethanol to yield yellow crystals. mp 162-163 °C.

Elemental analysis for CqgHj7NO4S

Cc (%) H (%) N (%) S (%)

Calculated : 62.96 ; 4.99 ; 4.08 ; 9.34

Found . 62.89 ; 5.04 ; 4.01 ; 9.34 l4-NMR (200MHz, CDCl) 8: 1.47 (3H, t, J=7.1Hz). 2.63 (3H, s), 4.87 (3H, s), 4.49 (2H, q, J=7.1Hz), 6.99 (2H, 4, J=8.8Hz), 7.44 (2H, 4, J=8.8Hz), 8.84 (1H,s), 1251 (1H, 8s)

IR (KBr): 3434, 2992, 1692, 1601, 1582, 1535, 1504 cm.

FAB-MS m/z: 344 (MH).

; PCT/JP99/03379

Reference Example 9

Using the compound obtained in Reference Example 7 as a starting material, the following compound was obtained in the same manner as in Reference Example 8.

H,GC OH

OY

S Nd

Yield: 60% mp 155-157 °C.

Reference Example 10

Production of 4-hydroxy-2-(4-nitrophenyl)-3- methylthieno[2,3-blpyridine-5-carboxylic acid ethyl ester

To a solution in concentrated sulfuric acid (10 ml) of the compound obtained in Reference Example 9 (3.76 g, 12.0 mmol) was added dropwise a solution of sodium nitrate (1.27 g, 15.0 mmol) in concentrated sulfuric acid (5 ml) with ice cooling. After stirring at this temperature for 30 minutes, the reaction mixture was poured into ice water and extracted with chloroform. The combined extracts were washed with line, and dried (MgSO), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield yellow powders, which was recrystallized from ethanol to yield yellow crystals (1.75 g, 41%). mp 260-261 °C.

Elemental analysis for C17H14N2058

Cc (3) H (3) N (¥)

Calculated : 56.98 ; 3.94 ; 7.82

Found . 56.66 ; 3.91 ; 7.86 lH-NMR (200MHz, CDCly) 8: 1.49(3H, t. J=7.1Hz), 2.70(3H, s), 4.51 (2H, g, J=7.1Hz), 7.70(2H, d, Jj=8.8Hz), B.34(2H, 4,

j=8.8Hz), 8.89(1H,s), 12.27(1H, s).

IR (KBr): 3002, 1692, 1605, 1514, 1350, 1290 cm L.

FAB-MS m/z: 358 (MH').

Reference Example 11

Production of 4,7-dihydro-7-(2-methoxybenzyl)-2-(4- methoxyphenyl) - 3-methyl-4-oxothieno[2, 3-blpyridine-5- carboxylic acid ethyl ester

To a suspension of sodium hydride (60% dispersion inoil; 123 mg, 3.08 mmol) in dimethylformamide (3 ml) was added a solution of the compound obtained in Reference Example 8 (1.0 g, 2.91 mol) in dimethylformamide (20 ml) under nitrogen stream with ice cooling. After stirring at this temperature for 30 minutes, to the mixture was added dropwise a solution of 2-methoxybenzyl chloride (0.92 g, 5.87 mmol) in dimethylformamide (3 ml). After stirring at room temperature for 23 hours and at 70 °C for 2 hours, the reaction mixture was concentrated, and then the obtained residue was partitioned between ethyl acetate and an agueous solution of ammonium chloride. The water layer was extracted with ethyl acetate. The combined extracts were washed with saline, and dried (MgSOy4) , the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield light yellow amorphous powders (0.95 g, 70%). For a sample for elemental analysis, the obtained powders were recrystallized from dichloromethane-ether to yield yellow prismatic crystals. mp 165-167 °C.

Elemental analysis for C,gH,5NOgS 0.5H,0 er 30 : Cc (%) H (2) N (%)

Calculated : 66.08 ; 5.55 ; 2.96

Found . 66.33 ; 5.44 ; 2.74 14-NMR (200MHz, CDClg) 8: 1.41(3H, t, J=7.1Hz), 2.65(3H, s). 3.85(3H, s:, 3.86(3H, s), 4.39(2H, q, J=7.1Hz), 5.16(2H, s).

6.92-7.00(4H, m), 7.21-7.41(4H, m), 8.41(1H, s).

IR (KBr): 2980, 1727, 1684, 1609, 1590, 1497, 1464 cm-1.

Reference Example 12

The following compound was produced by the same method as described in Reference Example 11, using benzyl 2,6- difluorochloride in place of 2-methoxybenzyl chloride with the compound obtained in Reference Example 10 as the starting material.

H,C 0

COOCH,CH, $7 NT OF be

Yield: 81% mp 215-217 °C.

Reference Example 13

Production of 3-bromomethyl-4 ,7-dihydro-7-(2- methoxybenzyl)-2-(4 -methoxyphenyl)-4-oxothieno [2,3- blpyridine-5-carboxylic acid ethyl ester

A mixture of the compound obtained in Reference Exam ple 11 (0.35 g, 0.755 mmol), N-bromosuccinimide (0.135 g, 0.758 mmol), a,c’ -azobisisobutyronitrile (13 mg, 0.079 mmol), and carbon tetrachloride (5 ml) was heated and refluxed for 2 hours. After cooling, the insoluble substances were filtered off, and then the filtrate was diluted with chloroform. After the organic layer was washed with saline and dried (MgSO), the solvent was distilled off under reduced pressure. The obtained residue was recrystallized from ethyl acetate to yield colorless needles (0.272 g, 66%).

: PCT/JP99/03379 mp 200-201 °C.

Elemental analysis for C,H 4NOg SBI

C (%) H (%) N (3)

Calculated : 57.57 : 4.46 ; 2.58

Found . 57.75 ; 4.31 ; 2.31 14-NMR (200MHz, CDClg) 8: 1.40(3H, t, J=7.1Hz), 3.86(6H, s), 4.40(2H, gq, J=7.1Hz), 5.05(2H, s), 5.16(2H, s}, 6.92-7.04(4H, m), 7.23-7.28(1H, m), 7.34-7.43(1H, m), 7.57(2H, 4, J=8.9Hz), 8.46(1H, s).

IR (KBr): 2980, 1725, 1607, 1588, 1497 cm™1. .

Reference Example 14

Using the compounds obtained in Reference Examples 12, 22 and 27 as a starting material, respectively, the following compounds (Reference Examples 14-1 to 14-3) were obtained in the same manner as in Reference Example 13.

Reference Example 14-1: gen, 9%

OCH,CH

ON 74 | PARLEY

ST ONT OF

F

Yield: 81% mp 200-202 °C.

Reference Example 14-2:

: PCT/JP99/03379 gel, $9

ASSO As §T ONT OF yield: BO%

Amorphous.

Reference Example 14-3:

BrCH, o 0

CH, au"

STN F °° 0

Yield: 79% mp 189-192 °C.

Reference Example 15

Production of 3-benzylaminomethyl-4, 7-dihydro-7-(2- methoxybenzyl)-2-( 4-methoxyphenyl)-4 -oxothieno(2,3- blpyridine-5-carboxylic acid ethyl ester hydrochloride

To a solution of the compound obtained in Reference

Example 13 (0.245 g, 0.452 mmol) in dimethylformamide (5 ml) were added triethylamine (0.10 ml, 0.717 mmol) and benzylamine (80 ul, 0.732 mmol) with ice cooling. After stirring at room temperature for 1.5 hours, the reaction mixture was concentrated, and then the obtained residue was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The water layer was extracted with ethyl acetate. The combined organic extracts were dried (MgS0Oy,)., the solvent was distilled off under reduced pressure. The

WQ 00/00493 PCT/IP99/03379 obtained residue was chromatographed on silica gel to yield a colorless oil (0.135 g, 53%). To a solution of this oil in ethanol (4 ml) was added 10 N ethanolic hydrochloric acid (0.2 ml) with ice cooling, followed by stirring at this temperature for 10 minutes. The reaction mixture was concentrated, and then the obtained residue was crystallized from ethyl acetate-ether to yield the hydrochloride (0.113 g) as white crystals. mp [Hydrochloride] 118-119 °C.

Elemental analysis for C33H39N055 HCl 0.9H,0

C (8) H (8) N (3) | co

Calculated : 63.79 ; 5.64 ; 4.51 vos .

Found . 64.03 ; 5.44 ; 4.51 | Lo 14 NMR (300MHz, CDCly) [Free amine] &: 1.40(3H, t, J=7.1Hz), Lu 2.05(1H, br),3.81(3H, s), 3.86(3H, s), 3.87(2H, s), 3.94(2H, } s), 4.40(2H, gq. J=7.1Hz), 5.18(2H, s), 6.80(2H, d, J=8.8Hz), 6.91-6.99(2H, m), 7.20-7.42(9H, m), 8.45(1H, s).

IR (KBr) [Hydrochloride]: 3422, 2938, 1719, 1605, 1560, 1545, 1502, 1460 cml. SE

Co

Reference Example 16

Production of 4-methyl-2-[ (4-methyl-3-oxo-1l-penten- 1-yl)amino}-5-phenylthiophen-3-carboxylic acid ethyl ester

The compound obtained in Reference Example 3 (10 g), 1 -methoxy-4-methyl-1l-penten-3-one of 85% content (6.9 g), p-toluenesulfonic acid monohydrate (0.219 g), and toluene (100 ml) were mixed and stirred at room temperature for 2.5 hours. After the reaction mixture was diluted with ethyl acetate and washed with a saturated aqueous solution of sodium hydrogen carbonate, the water layer was extracted with ethyl oo acetate. The combined extracts were washed with saturated saline, the organic layers were dried over anhydrous magnesium sulfate. The residue was crystallized via the addition cf a seed crystal, after which it was triturated with hexane, collected by filtration, and washed, to yield the title compound (12.64 g, 92%). mp 104-108 °C.

Reference Example 17

Production of 4. 7-dihydro-7-(2-methoxybenzyl)-2-(4- me thoxyphenyl) - 3-methyl-4-oxothienol2,3 -bl}pyridine-N- benzylpiperazinyl-5- carboxamide

To 1-benzylpiperazine (0.77 gd, 4.37 mmol) was added dropwise a solution of diisobutyl aluminum hydride (DIBAL) in toluene (1.5N, 2.9ml, 4 .37 mmol) with ice cooling. After the addition was completed, the solution was allowed to warm to room temperature and stirred for further 0.5 hours. To this solution was added a solution of the compound obtained in Reference Example 11 (0.50 g, 1.08 mmol) in toluene (5 ml) at room temperature. After the mixture was further stirred at room temperature for 15 hours, methylene chloride (30 ml) was added. After water washing, the solution was dried over sodium sulfate, and then the solvent was concentrated under reduced pressure to yield a solid (1.03 g), which was recrystallized from methylene chloride-n-hexane to yield the title compound (0.48 g, 78%). mp 233-235 °C.

Elemental analysis for Ca5H35N3045 1/2H,0

C (%) H (%) N (%) S (%)

Calculated : 69.75 ; 6.02 ; 6.97 ; 5.32.

Found . 69.88 ; 6.06 ; 6.98 ; 5.39. 14_NMR (300MHz, CDCl,) 8: 2.45-2.55(4H, m), 2.63(3H, s), 3.43-3.49(2H, m), 3.55(2H, s), 3.73-3.82(2H, m), 3.84(6H, s), 5.11(2H, s), 6.89-6.98(4H, m), 7.21-7.40(9H, m), 7.79(1H, s).

Reference Example 18

Using the compound obtained in Reference Example 12 as a startinc material and N,O-dimethylhydroxy in place of 1-benzylciperazine, the following compound was obtained in the same manner as in Reference Example 17. he OQ nC wd TY

ST ONT OF 3 x

Yield: 80% mp 223-224 °C.

Reference Example 19

Production of 4-hydroxy-5-isobutyryl-3-methyl-2- phenylthieno[2,3-b] pyridine

The compound obtained in Reference Example 16 (50 g) and diphenyl ether (500 ml) were mixed, and then this mixture was heated and refluxed for 4 hours, while the ethanol being produced with the progress of the reaction was distilled off.

After cooling, the diphenyl ether was distilled off under reduced pressure, and then the crude crystal precipitated was washed with n-hexane to yield the title compound (35.1 g, 81%). mp 114-117 °C.

Reference Example 20

Using the compound obtained in Reference Example 14- 1 as starting material and N-methylbenzylamine in place of benzylamine, the following compounds (Reference Examples 20-1 to 20-3) were obtained in the same manner as in Reference

Example 15.

Reference Example 20-1:

PCT/JP99/03379 _N 0

H,C

COOCH,CH,

ON 49

STON” OF

Yield: 83% mp 140-144 °C.

Reference Example 20-2:

Pi Oo

H,C N

COOCH,CH,

ON 4%

STNT F

Yield: 91% mp 145-147 °C.

Reference Example 20-3:

N 0

H,C

COOCH,CH,

ON / A\ CI — ST NT Cl

PCT/JP99/03379

Yield: 78% mp 175-177 °C.

Reference Example 21

Using the compounds obtained in Reference Example 20 as starting materials, the following compounds (Reference

Examples 21-1 to 21-3) were obtained in the same manner as in Reference Example 28.

Reference Example 21-1:

H N 0 3 COOCH,CH,

HN 7]

STONT OF

Yield: 79% mp 158-160 °C.

Reference Example 21-2:

H MN 0 3 COOCH,CH,;

HN 28

STONT OF

Yield: 96% mp 195-196 °C.

. PCT/IP99/03379

Reference Example 21-3:

NN Oo

HC COOCH,CH, i 9 I

ST NT Cl x0

Yield: 71% mp 144-146 °C.

Reference Example 22

Production of 7-(2, 6-difluorobenzyl)-4,7 -dihydro-5- isobutyryl-3-methyl- 4-o0x0-2-phenyl- thieno[2,3-b] pyridine

The compound obtained in Reference Example 19 (35 ag). potassium carbonate (18.6 g), and N,N-dimethylformamide (280 ml) were mixed, and then 2,6-difluorobenzyl bromide (27.9 9) was added, followed by stirring at room temperature for 4 i5 hours. To the reaction mixture was added dropwise water (560 ml), followed by stirring for 30 minutes, and then the mixture was stirred for 1 hour with ice cooling. The crude crystal was collected by filtration, washed with water, and dried, successively suspended in a 1:1 mixed solvent (250 ml) of ethyl acetate and diisopropyl ether and stirred at 25 to 40 °c for 1 hour, then stirred with ice cooling for 1 hour, the residual crystal was collected by filtration and washed with the above mixed solvent (125 ml) to yield the title compound (44.6 g, 92%). mp 205-207 °C.

Reference Example 23

PCT/JP99/03379 : B 42

Using the compounds obtained in Reference Example 14-2 and Reference Example 24 as starting materials and N- methylbenzylamine in place of benzylamine, the following compounds (Reference Examples 23-1 to 23-2) were obtained in the same manner as in Reference Example 15.

Reference Example 23-1:

N oO 0

H,C on IY

SH NE ~

Yield: 83% mp 197-199 °C.

Reference Example 23-2: _N 0 0)

HC CH,

ON au.

STNT F 7

F

Yield: 66% mp 1S51-1E82 °C.

Reference Example 24

Production of 3-promomethyl-7-(2,6-difluorobenzyl)- 4 7-qihydro-5-1isobutyryl-2-(4-nitrophenyl)-4-

- i 43 oxothieno[2,3-blpyridine

After the compound obtained in Reference Example 14- 3 (1 g) was dissolved in methanesulfonic acid (5 ml) with cooling at 10 to 12 °c, a solution of sodium nitrate (0.165 g) in methanesulfonic acid (2.5ml) was added dropwise to this mixture. After being stirred as was for 2 hours, the mixture was poured into cold water, and then the crystal precipitated was collected by filtration, washed with water and diisopropyl ether, and dried to yield a crude product (1.04 i0 g), which was then triturated with ethyl acetate (15ml), then cooled with ice, filtered, and washed with cold ethyl acetate to yield the title compound (0.647 g, 59.5%). mp 202 to 204 °c (recrystallized from methanol).

Reference Example 25

Production of 4.7 -dihydro-7-(2,6 -difluorobenzyl)-2- (4 -nitrophenyl)-3- (N-benzyl-N-methylaminomethyl) -4- oxothieno[2,3-blpyridine- 5- (N-isopropyl) carboxamide

To a solution of isopropylamine (0.296 g, 5 mmol) in anhydrous methylene chloride (5 ml) was added dropwise a solution of trimethylaluminum in hexane (15%, 2.41 ml, 5.0 mmol) at 0 °C. After the addition was completed, the solution was allowed to warm to room temperature and further stirred for 1 hour. To this solution was added a solution of the compound obtained in Reference Example 20-2 (0.12 g, 0.25 mmol) in anhydrous methylene chloride (3 ml) with ice cooling (0 °C) over 30 minutes. After the mixture was stirred at room temperature for further 1 hour, chloroform (50 m1) was added, followed by washing with water. The combined organic layers were dried with sodium sulfate, and then the solvent was concentrated under reduced pressure to yield a solid, which was recrystallized from chloroformethyl acetate-ethyl ether to yield colorless crystals (0.096 g. 70%). mp 200-202 °C.

PCT/JP99/03379 14-NMR (500MHz, CDCly) [Free amine] 8: 1.30(6H, d, J=6.7Hz), 2.15(3H, s), 3.66(2H, s). 4.18(2H, s), 4.18-4.31(1H, m), 5.32(2H, s), 7.00(2H, t, J=7.26Hz), 7.13-7.25(5H, m), 7.42(1H, t, J=7.3Hz), 8.02(2H, 4, J=8.9Hz), 8.26(2H, 4d,

J=8.9Hz), 8.73(1H, s), 10.02(1H, 4d, J=9.1Hz).

IR (KBr) : 2974, 1661, 1597, 1547, 1497, 1346, 1212, 1035 em™1

FAB-Mass m/z 617 (MH)”

Reference Example 26

Using the compound obtained in Reference Example 20- 2 as a starting material and N,O-dimethylhydroxyamine, the following compound was obtained in the same manner as in

Reference Example 25.

N 0 0)

HyC OCH;

O,N 71 1

CH

TNT OF T° be

Yield: 96% mp 100-102 °C.

Reference Example 27

Production of 4,7-dihydro-7-(2, 6-difluorobenzyl)-2- (4-nitrophenyl)-5-benzoyl- 3-methyl-4-oxothieno[2,3- b]pyridine

The compound obtained in Reference Example 18 (3.93 g, 7.87 mmol) was dissolved in anhydrous tetrahydrofuran (THF) (300 ml) with slightly warming. While this solution was kept at 0 °C, a solution of phenylmagnesium bromide in THF (1 M, 15.7 ml, 15.7 mmol) was added dropwise to the mixture over 10 minutes. After the addition was completed, the solution i - 45 was stirred for further 1 hour. The reaction mixture was partitioned between ethyl acetate (300 ml) and water (50ml)., and then the water layer was again extracted with ethyl acetate. The combined organic layers were dried with magnesium sulfate, the solvent was concentrated under reduced pressure to yield a residue, which was chromatographed on silica gel to yield yellow crystals (3.00 g, 74%), which was then recrystallized from ethyl acetate-hexane to give yellow crystals. mp 114-116 °C.

Elemental analysis for C,gH1gN204SF) 0.7H50

C (3%) H (3%) N (%)

Calculated : 63.56 ; 3.70 ; 5.29

Found : 63.83 ; 3.95 ; 5.08

H-NMR (500MHz, CDCl3) §: 2.68(3H, s), 5.30(2H, s), 7.02(2H, t, J=8.1Hz), 7.43(3H, t, J=7.2Hz), 7.52-7.63(3H, m), 7.86(2H, da, J=7.5Hz), 7.99(1H, s), 8.30(2H, 4, J=8.7Hz).

IR (KBr): 3422, 3068, 1665, 1615, 1491, 1473, 1346, 853 cml.

FAB-Mass m/z 517(MH)*

Reference Example 28

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-7-(2.,6 -difluorobenzyl)-2-(4- aminophenyl)-5- benzoyl-4-oxothienol 2,3-blpyridine

To a mixture of the compound obtained in Reference

Example 23-1 (0.30 g, 0.47 mmol) in ethyl alcohol (6 ml) were added water (2 ml), and then one drop of concentrated hydrochloric acid was added to yield a uniform solution. To this solution were added iron powder (0.105 g, 2.0 mmol) and concentrated hydrochloric acid (0.39ml, 4.7 mmol) carefully.

After the addition was completed, the mixture was stirred at room temperature for 5 hours and filtered through Celite. A small amount of aqueous ammonia was added, and then the reaction filtrate was concentrated under reduced pressure.

Co PCT/JP99/03379

The obtained residue was poured into ice water, neutralized with sodium bicarbonate, and extracted with ethyl acetate.

After the combined organic layers were washed with saline, and dried (MgSOy4), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from isopropyl ether to yield yellow needles (0.24 g, 84%). mp 126-128 °C.

Elemental analysis for CagHpgN30,5F 1/2H,0 }

C (3) H (3%) N (%) calculated : 68.93 ; 5.04 ; 6.70

Found : 68.71 ; 5.18 ; 6.62 1H-NMR (300MHz, CDCl1l3) 8: 2.13 (3H, s),3.65 (2H, s), 3.87 (2H, br s), 4.14(2H, s). 5.28(2H, s), 6.74(2H, da, J=8.7Hz), 7.00(2H, t, J=7.8Hz), 7.16-7.24(5H, m), 7.36-7.46(3H, m), 7.53(1H, t., J=7.2Hz), 7.62(2H, d, J=8.4Hz), 7.89(2H, 4,

J=7.2Hz), 7.94(1H, s).

IR (KBr) : 3358, 1607, 1495, 1473, 1035 cm-l.

FAB-Mass m/z 606(MH)™

Reference Example 29

Production of 2-(4-aminophenyl)-7-(2,6- difluorobenzyl)-4,7-dihydro- 5-isobutyryl-3-(N-benzyl-N- methylaminomethyl)-4 -oxothieno[2,3-bjpyridine

HCN oO O

CH,

NF 8

Xo

B wo 00/00493

PCT/JP99/03379 i 47

To a solution of the compound obtained in Reference

Example 23-2 (0.259, 0.415 mmol) in methanol (5 ml) were added dropwise iron powder (0.093 4g, 1.66 mmol) and concentrated hydrochloric acid (0.8 ml) with ice cooling. After the addition was completed, the mixture was stirred at room temperature for 1 hour and filtered through Celite. A small amount of a saturated aqueous solution of sodium hydrogen carbonate (10 ml) was added, followed bY extraction with methylene chloride (30 ml x 3). The combined extracts were washed with saline, and dried (MgSOy4) the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield light yellow amorphous powders (0.203 g. 86%) . 14.NMR(300MHz,CDC13)d: 1.18(6H, 4), 2.11(3H, s). 3.65(2H, s). 3.85(2H, br s), 4.17(2H, s), 4.18(1H, m), S5.25(2H, s), 6.73(2H, 4d). 6.95(2H, t). 7.10-7.26(5H, m), 7.42(1H, m), 7.58(2H, 4), 8.27(1H, s).

Reference Example 30

Production of 5-pbenzoyl-7-(2, ¢-difluorobenzyl) -4,7- dihyrdro-3-( N-benzyl-N-methylaminomethyl) -2-(4- propionylaminophenyl )-4-oxothieno(2, 3-blpyridine

The compound obtained in Reference Example 28 (0.14 g, 0.23 mmol) was dissolved in anhydrous methylene chloride (2 ml), and then to the mixture was added triethylamine (0.038 ml) with ice cooling (0 °C). After this solution was stirred for a while, to the mixture was added propionyl chloride (0.021 ml, 0.243 mmol). After the addition was completed, the solution was further stirred with ice cooling (0 °C) for 40 minutes. The reaction mixture was partitioned between methylene chloride (25 ml) and very thinly overlaid water (10 ml). The water layer was again extracted with methylene chloride (25 ml). The combined organic layers were washed with saline, and dried (MgSOy), the solvent was distilled off under reduced pressure. The solid obtained was recrystallized from ethyl acetate-isopropyl ether to yield yellow needles (0.10 g, 65%). mp 226-228 °C. g

Elemental analysis for C3qH33N303SF, 0.7H,0

C (%) H (%) N (3)

Calculated : 69.46 ; 5.14 ; 6.23

Found : 69.60 ; 5.18 ; 6.04

Thus obtained compound was dissolved in ethyl acetate, and then a saturated solution of hydrogen chloride (HCl) in ether was added in equal to slightly excess amount, and then the crystal precipitated was recrystallized from isopropyl ether to yield light yellow needles (0.095 g, 61%). mp 218-220 °C.

Elemental analysis for C3gH33N303SF,; HCl 3.5H,0

C (8) H (2) N (%)

Calculated : 61.53 ; 5.43 ; 5.52

Found : 61.83 ; 5.33 ; 5.30 1y-NMR (300MHz, DMSO-dg) §: 1.11(3H, t, J=7.2Hz),1.93(3H, s),2.35(2H, q,J=7.5Hz), 3.44(2H, s), 4.00(2H, s), 5.62(2H, s), 7.11-7.25(6H, m), 7.43-7.72(10H, m), 7.79(2H, 4,

J=7.5Hz), 8.40(1H, s), 10.03(1H, s).

IR (KBr) : 3422, 3068, 1603, 1502, 1473, 1035 cm™ 1,

FAB-Mass m/z 662(MH)™

Reference Example 31

The following compounds (Reference Examples 31-1 through 31-10) were produced by the same method as described in Reference Example 30, using various acid chlorides, isocyanates, and chlorocarbonic acid esters in place of propionyl chloride, and also using the amines shown in Example 16 and carbonyldiimidazole, with the compounds obtained in

Reference Examples 28 and 29 as the starting materials. The following compounds (Reference Examples 31-11 through 31- 16) can also be produced in the same manner.

Reference Example 31-1-1: _N 0 0)

H,C

H

HOTT OC

N— STONT OF

H,C 0]

F

Yield: 68% mp 238-240 °C.

Reference Example 31-1-2:

CO Hel

N 0 0

H,C

H

OTC

N= STONT OF

H,C 0]

F mp 230-232 °C.

Reference Example 31-2-1:

. ° 50

N 0 0)

H.C CH,

H 2

HG N Ld, —( STON OF TO 0 be}

F

Yield: 64% mp 201-204 °C.

Reference Example 31-2-2: aN HCI

N 0] 0 ’ H.C CH,

HCN aes L, — STONT FT

F mp 207-214 °C.

Reference Example 31-3-1:

N 0 0

H,C CH,

H / hoN Lb

N— STONT FTF

H,C 0)

F

Yield: 55%

mp 207-210 °C.

Reference Example 31-3-2: oN HC wre 0 0]

CH,

CH

N— S N F 3

H,C 0)

F mp 222-226 °C.

Reference Example 31-4:

CO HCl

N 0] 0

H,C

H

CH,CH,SO,N ¢ am ®

STON” F

F

Yield: 49% mp 185-187 °C.

Reference Example 31-5:

N WO 00/00493 PCT/JP99/03379 aS HCI

N 0 0

H,C

H wo HOLY C

H,C 0)

F

Yield: 79% mp 216-218 °C.

Reference Example 31-6:

Oy wo

N 0) 0

H,C

H wo HOLT TC

H.C (0)

F

Yield: 73% mp 180-183 °C.

Reference Example 31-7:

N 0) 0

CH H.C

SH wo LAITY TC

N— S N F

F

Yield: 65%

mp 245-247 °C.

Reference Example 31-8: .N 0] 0]

H.C

H

OT TC

Ov STON” OF

F

Yield: 65%

Reference Example 31-9: = Hel

N oO 0)

H.C

H

LTC

CF,CH,0—{ STN" OF

F

Yield: 70% mp 232-234 °C.

Reference Example 31-10:

C3 HCI

H c 0 O

H Y CH, — S N F 3

H,C 0)

F

Yield: 73% mp 192-197 °C.

Reference Example 31-11:

N oO oO

H,C CH,

HiC § 4 Sh

Pond Ss N F 3

H,C lo}

F

Reference Example 31-12:

SN HCI

N oOo 0

H.C CH,

HG, N 2 Ly

Jn ER

H,C 0]

F

Reference Example 31-13:

N oO 0

HC CH,

H

H,C— N 4 | | by

Jn STONT FT

H,C 0

F

Reference Example 31-14: { HCI _N 0 0

H,C cH,

H

HC— ~~ N 7 | | hn

Jo STONT oF

H,C lo}

F

Reference Example 31-15:

N o ©0

H.C CH,

H

LA ams

R— Ss N FE 3 ho— 0

F

Reference Example 31-16:

- PCT/JP99/03379 a HCI

N oO 0

HC CH,

LN Sms

Pa Hp Ch

H,C o

F

Reference Example 32

Production of 3-(N-benzyl-N-methylaminomethyl)-4,7- dihyrdro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-5- benzoyl-4-oxothieno[2,3-blpyridine

The compound obtained in Reference Example 26 (1.91 g, 3.09 mmol) was dissolved in anhydrous tetrahydrofuran (THF) (30 ml) with warming, and then to the mixture was added dropwise a solution of phenylmagnesium bromide in THF (1 M, 6.18 ml, 6.2 mmol) with ice cooling (0 °C) over 10 minutes.

After stirring under ice cooling conditions for 1 hour, the reaction mixture was partitioned between ethyl acetate (100 ml) and aqueous hydrochloric acid (0.5 N, 100 ml), and then the organic layer was again washed with saturated saline (100 ml). After the organic layer was dried (MgSO,), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield yellow crystals (1.00 g, 51%), which was then recrystallized from isopropyl ether to yield yellow needles. mp 197-199 °C.

Elemental analysis for C3gH,7N3045F 0.7H,0

C (%¥) H (%) N (%)

Calculated : 66.70 ; 4.42 ; 6.48

Found : 66.59 ; 4.48 ; 6.42 14-NMR (300MHz, CDCl) &: 2.17(3H, s), 3.61(2H. s), 4.16(2H, s), 5.30(2%, s), 7.03(2H, t, J=8.1Hz). 7.19-7.25(5H, m),

© PCT/JP99/03379 7.40-7.47(3H, m), 7.56(1H, t, J=7.5Hz), 7.88(2H, da, J=6.9Hz), 7.96(1H, s), 8.10(2H, 4, J=8.7Hz), 8.28(2H, a, J=8.7Hz).

IR (KBr) : 3430,1663, 1611, 1518, 1473, 1348, 853 cml.

FAB-Mass m/z 636(MH)

Reference Example 33

Production of 3. (N-benzyl-N-methylaminomethyl)-4.7- dihyrdro-7-(2.,6 -difluorobenzyl)-2-(4- isobutyrylaminophenyl )-5-(2 -bromoisobutyryl)-4- oxothieno[2,3-b] pyridine

The compound obtained in Reference Example 31-10 (0.48 g, 0.75 mmol) was dissolved in dichloromethane (15 m1). To the solution was added dropwise 47% hydrobromic acid (0.35 ml, 3.0 mmol) at room temperature for 1 minute, followed by further stirring for 10 minutes. To this reaction mixture was added dropwise bromine (39 pl, 0.75 mmol), followed by further stirring at room temperature for 24 hours. The reaction mixture was partitioned between dichloromethane (30 ml) and aqueous sodium bicarbonate (saturated, 60 ml), and then the organic layer was again washed with saturated saline (100 ml). After the organic layer was dried (MgSOy). the solvent was distilled off under reduced pressure to yield brown amorphous crystals (0.53 g).

Reference Example 34

Production of 3-(N-benzyl-N-methylaminomethyl) -4,7- dihyrdro-7-(2,6 -difluorobenzyl)-2-(4- isobutyrylaminophenyl )-5-methacryloyl- 4-oxothieno[2,3- blpyridine

The compound obtained in Reference Example 33 (0.52 g, 0.72 mmol) was dissolved in dimethylformamide (DMF) (30 ml), and then to this solution was added potassium acetate (2.0 g, 20 mmol) at room temperature, followed by stirring at 100 °c for 20 hours. The reaction mixture was partitioned between ethyl acetate (30 ml) and water (30 ml), and then the water layer was again washed with ethyl acetate (10 ml). The combined organic layers were again washed with saturated saline (30 ml). After the organic layer was dried (MgSO,). the solvent was distilled off under reduced pressure to yield a brown oil (0.56 g).

Example 1

Example 1-1:

Production of 3- (N-benzyl-N-methylaminomethyl)-4,7- dihydro-5-isobutyryl-7- (2,6-difluorobenzyl)-2- (4- cyclopropanecarbonylaminophenyl) -4-oxothienof 2, 3- blpyridine ‘

H,c-N 0 O

OLY

>= sow” FO

BE.

F

To a solution of the compound obtained in Reference

Example 29 (2.57 g, 4.5 mmol) in dichloromethane (50 ml) were added cyclopropanecarboxylic acid (861 mg, 10 mmol) and diisopropylethylamine (2.59 g. 20 mmol), and then benzotriazol-1-yl-oxy tripyrrolidino phosphonium hexafluorophosphoric acid (PyBop) (5.409, 10 mmol) was added to it in a small portion with ice cooling. After stirring at room temperature for 1 day, the reaction mixture was poured into a 0.1 N aqueous solution of potassium hydroxide and extracted with chloroform. The combined extracts were washed with saline, and dried (Na,SO4) , the solvent was distilled cff under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from a. 00493 PCT/JP99/03379 ethyl acetate to yield white powdery crystals (2.15 g, 79%). 1H-NMR (300MHz, CDCl) 8: 0.85-0.90(2H.m), 1.10-1.15(2H,m), 1.18(6H, 4), 1.53-1.59(1H, m), 2.09(3H, s), 4.12-4.20(1H, m), 4.16(2H, s), 5.25(2H, s). 6.99(2H, t), 7.10-7.25(6H, m), 7.35-7.45(1H, m), 7.61(2H, d), 7.67(1H, br s), 7.76(2H, 4), 8.27(1H, s).

FAB-Mass m/z 608(MH)¥

Example 1-2:

Production of 3-(N-benzyl-N-methylaminomethyl) -4,7- dihydro-5 -isobutyryl-7-(2, 6-aifluorobenzyl)-2-(4- cyclopropanecarbonylaminophenyl )-4-oxothieno([2,3- blpyridine hydrochloride

Oy

HCN 0 0]

CH

H / 3

N EY

F

The white powdery crystals obtained in Example 1-1 were dissolved in ethanol, and then a solution of hydrogen chloride in ethyl ether was added to it in a small excess amount, followed by recrystallization from ethanol-ethyl ether, to yield white powdery crystals (2.20 g, 72%). mp 254-256 °C.

Elemental analysis for C37H35N3035F HCl

C (%) H (8) N (8)

Calculated : 65.72 : 5.37 ; 6.21

Found . 65.61 ; 5.38 ; 6.25

IR (KBr) : 2950, 1673, 1595, 1502, 1473, 1408, 1313, 1183 cm 1.

: PCT/¥P99/03379

Example 2

Production of 4,7-dihydro-2-[4-(3-acetoxy-2- methylpropionylamino )phenyl]}-7-(2,6 -difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl)-5-isobutyryl-4- oxothieno[2,3- blpyridine

HeN— Ff

CH

H 3

NA 5 N - CH,

F .

To a solution of the compound obtained in Reference

Example 29 (1.14 g, 2.0 mmol) in dichloromethane (30 ml) were added dropwise triethylamine (500 mg, 5 mmol) and 3- acetoxyisobutyryl chloride (0.5 g, 3 mmol) with ice cooling over 1 minute, followed by further stirring at room temperature for 1 hour. The reaction mixture was partitioned between a saturated aqueous solution of sodium hydrogen carbonate (30 ml) and dichloromethane (30 ml), and then the water layer was again extracted with dichloromethane (10 ml).

The combined extracts were washed with saline, and dried (Na,SO4), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield brown crystals (1.4 g). 1H NMR (300MHz, CDClgy) 5: 1.18(6H,d), 1.30(3H,4), 2.08(3H,s), 2.12(3H,s), 2.73-2.84(1H,m), 3.65(2H,s), 4.11-4.36(3H,m), 4.17(2H,s), 5.27(2H,s), 7.00(2H,t), 7.13-7.22(5H,m), 7.36-7.46(1H,m), 7.52(1H,br s). 7.62- 7.82(4H,q), 8.29(1H, s).

: PCT/YP99/03379

Example 3

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-5-isobutyryl-7 -(2, 6-difluorobenzyl)-2-[4- (3- hydroxy- 2-methylpropionylamino )phenyl]l-4- oxothieno[2,3- blpyridine

H,c~N 0 0

CH

H 3

HO N 77 7 1

Jy < \ . H,

H,C 0

F

The compound obtained in Example 2 (1.40 g) was dissolved in methanol (100 ml), and then to the solution was added potassium carbonate (2.0 g, 15 mmol), followed by stirring at room temperature for 15 minutes. The reaction mixture was partitioned between ethyl acetate (200 ml) and distilled water (300 ml), and then the water layer was extracted with ethyl acetate (100 ml). The combined extracts were washed with saline, and dried (Na5,S0y) , the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from ether to yield white crystals (0.95 g, 72%). mp 161-162 °C.

Flemental analysis for C39H37N3045F2 0.5H,0

Cc (%) H (%) N (%)

Calculated : 66.65 ; 5.74 ; 6.30

Found . 66.70 ; 5.52 ; 6.40 1, NMR (300MHz, CDClz) 8: 1.17(6H,d), 1.18(3H.d), 2.06(3H,s)., 2.67-2.78(1H,m), 3.61(2H,s)., 3.57-3.84(2H,m),

PCT/JP99/03379 - = 62 . 3.99-4.08(1H,m), 4.15(2H,8), 5.52(2H,8), 7.08(2H,t). 7.15(5H,s), 7.44-7.54(1H,m). 7.58(2H,4), 7.69(2H,4), 8.46 (1H, s).

FAB-Mass m/z 658 (MH) *

Example 4

Using the compound obtained in Reference Example 29, the following compounds (Examples 4-1 to 4-3) were obtained in the same manner as in Example 1.

Example 4-1: no 0 0

CH CH,

Cc 3 H " Jo N / L

H,C — s—~y~ F Ob

F

Yield: 67% mp 201-203 °C.

Example 4-2:

H _N 0 0 0 © CH,

H H 7

NN 0 L, 0 sT>NT F T°

F

Yield: 58% mp 135-137 °C.

0/00493 PCT/JP99/03379 - 63

Example 4-3:

N Oo O

H,C 0] CH,

H 4 r N N LI en iS STNT F T°

F

Yield: 75% mp 205-207 °C.

Example 5

Example 5-1:

Production of 3- (N-benzyl-N-methylaminomethyl)-4,7- dihydro-7-(2,6-difluorobenzyl)-2-[4-(2- hydroxyacetylamino)phenyll-5-isobutyryl-4-oxothieno[2,3- blpyridine

H,c-N Oo O

H 4 CH,

N | EY 4 STON” FTE

HO Oo

F

The compound obtained in Example 4-1 (0.137 g) was dissolved in dichloromethane (1.0 ml), and then to the solution was added dropwise trifluoroacetic acid (1.0 ml) with ice cooling. After stirring under ice cooling conditions for 1 hour, the reaction mixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between chloroform (50 ml) and saturated aqueous sodium bicarbonate (50 ml), and then the water layer was again extracted with chloroform (10 ml). The combined organic layers were washed with saturated saline (30 ml). After the organic layer was dried (MgsSO,), the solvent was distilled off under reduced pressure, and then the obtained residue was chromatographed on silica gel and recrystallized from ethanol-ethyl acetate-ethyl ether to vield white crystalline powders (0.12g, 95%).

Example 5-2:

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-7-(2,6-difluorobenzyl)-2-[4-(2- hydroxyacetylamino)phenyl ]-5-isobutyryl-4-oxothienol[2,3- blpyridine hydrochloride oN HCI

H,C-N oO O

ONT

— sn" fF Os

HO 0

F

The white crystalline powders obtained in Example 5- 1 were dissolved in dichloromethane (1.0 ml), and then to this solution was added an ethanolic solution containing 10 N hydrogen chloride (1.0 ml) with ice cooling. The solvent was distilled off under reduced pressure, and then the residue was recrystallized from ethanol-ethyl acetate to yield white crystalline powders (0.12 g, 84%). mp 197-199 °C.

Elemental analysis for C3gH33N30,4SF) HCl 2.5H,0

: PCT/JP99/03379

Cc (8) H (%) N (3)

Calculated : 59.11 : 5.53 ; 5.91

Found . 59.06 ; 5.25 ; 5.93 14-NMR (300MHz, CDCl) 8: 1.18(6H, d), 2.10(3H. s). 3.63(2H,s), 3.72(1H,br), 4.09-4.17(1H,m), 4.16(2H,s), 4.24(2H,s), 5.24(2H,s), 6.99(2H,t), 7.18-7.23(5H,m), 7.35-7.45(1H,m), 7.63(2H,d4), 7.73(2H,4), 8.25(1H,s), 8.59(1H,s).

Example 6

Using the compounds obtained in Example 4-2 and Example 4-3, the following compounds (Examples 6-1 to 6-2) were obtained in the same manner as in Example 5.

Example 6-1: as HCI me O 0 3 CH,

H /

HNN [7 I, ~— S N F 3

F

Yield: 76% mp 192-194 °C.

Example 6-2:

to PCT/JP99/03379

H MN 0) 0 3 CH,

H /

HN N

Ny sp Cr

F

Yield: 81% mp 193-195 °C.

Example 7

Production of 3- (N-benzyl-N-methylaminomethyl)-4,7- dihydro-7-(2,6-difluorobenzyl)-2-(4- i sobutyrylaminophenyl)-5-(2-hydroxy-2-methylpropionyl)-4- oxothieno[2,3-blpyridine

HCN Oo O

H CH, 4

HC N || OH — sn Fe

H,C 0)

F

The brown amorphous crystal (0.13 g) obtained by re- peating the method described in Reference Example 33 using the compound obtained in Reference Example 31-10 was hy- drolyzed with potassium acetate by the same method as de- scribed in Reference Example 34. The solid obtained was chromatographed on silica gel to remove the compound obtained in Reference Example 34, and the byproduct was recrystallized from methanol to yield colorless crystalline powders (0.03 g).

PCT/JP99/03379 mp 170-172 °C.

Elemental analysis for C37H37N3045F) 1/6H,0

Cc (¢) H (2) N (%)

Calculated : 67.26 ; 5.69 ; 6.36

Found . 67.18 ; 5.74 ; 6.46 ly_NMR (300MHz, CDCl3) 5: 1.28(6H,4), 1.49(6H,s). 2.06(3H,s), 2.51-2.60(1H,m)}, 3.61(2H,s)., 4.13(2H,s). 5.31(2H,s), 7.01(2H,t), 7.12-7.21(5H,m), 7.37-7.47(2H,m). 7.64(2H,4). 7.74(2H,4), 8.04(1H,s), 8.35(1H,s).

IR (KBr) : 3466,1669, 1499, 1313, 1075 cm 1.

Example 8

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-7-(2, 6-difluorobenzyl)-2- (4- isobutyrylaminophenyl )-5-(3-hydroxy-2 -methylpropionyl )-4- oxothieno[2,3-b] pyridine

HCN oO ©O

H

HG N aemn OH — STON FT

H,C 0)

F

The compound obtained in Reference Example 34 (0.56 g) was dissolved in trifluoroacetic acid (10 ml) and stirred at room temperature for 1 hour. The reaction mixture was concentrated to dryness under reduced pressure, and the obtained residue was partitioned between dichloromethane (30 ml) and saturated aqueous sodium bicarbonate (60 ml), and then the water layer was again extracted with dichloromethane (10 ml). The combined organic layers were washed with saturated saline (30 ml). After the organic layer was dried (MgSOy). the solvent was distilled off under reduced pressure to yield brown amorphous crystals (0.42 g), which was chromatographed : on silica gel to yield yellow crystals (0.07g, overall recovery rate as calculated from the compound obtained in

Reference Example 31-10 12%). 1H-NMR (300MHz, CDClj) &: 1.15(3H.d), 1.26(6H, 4), 2.07(3H, s), 2.53-2.62(1H, m), 3.58(2H, 4d), 3.66(2H, 4d), 3.82(1H, 4d), 3.93(1H, dd), 4.03(1H, 4d), 4.22(1H, 4), 4.27-4.37(1H, m), 5.26(2H, s), 6.99(2H, t), 7.13-7.21(5H, m}, 7.36-7.46(1H, mm), 7.63(2H, 4), 7.67(2H, da), 7.79(1H, s), 8.30(1H, s).

Example 9

Production of (R)-4,7-dihydro-2-{4-(3-t-butoxy-2- methylpropionylamino)phenyl ]-7-(2,6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl) -5-isobutyryl-4-oxothieno(2,3- blpyridine

H,Cc-N oO O

HC NITY), CH,

Pa STONT Fe 0 0) ae 0 3 F

To a solution of the compound obtained in Reference

Example 29 (1.14 g, 2.0 mmol) in dichloromethane (20 ml) were added diisopropylethylamine (520 mg, 4 mmol) and (2R)-3- t-butoxy-2-methylpropanoic acid (0.43 g, 3 mmol), followed by stirring with ice cooling. To this solution was added penzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent) (1.33 g, 3 mmol). After stirring with ice cooling for 1 hour, the solution was further stirred at room temperature for 4 hours. The reactionmixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between water (50 ml) and chloroform (50 ml). The water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgSO4). the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield yellow powders (1.1149, 78%). 14-NMR (300MHz, CDClz) &: 1.18(6H,d), 1.25(3H.d). 1.30(9H,s), 2.62(3H,s), 2.63-2.71(1H,m), 3.51(1H,t like), 3.61(1H,d44), 3.64(2H,s)., 4.18(2H,s), 4.18(1H,qguint), 5.27(2H,s), 7.00(2H,t), 7.14-7.22(5H,m), 7.36-7.46(1H,m), 7.60(2H,d), 7.76(2H,d), 8.28(1H, s), 9.00(1H,s).

Example 10

Example 10-1:

Production of (R)-4, 7-dihydro-2-[4-( 3-hydroxy-2- methylpropionylamino yphenyll-7-(2,6 -difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl )-5-isobutyryl-4- oxothienol[2,3- bipyridine

H,C-N oO ©O

H CH,

Daa STNT F 78

HO 0

F

The compound obtained in Example 9 (1.11 g) was dissolved in trifluoroacetic acid (5 ml) under ice cooling conditions, and then after stirring for 1 hour, the solution was allowed to warm to room temperature and stirred for further 12 hours.

oo PCT/JP99/03379

The reaction mixture was concentrated to dryness under reduced pressure, and then the residue was dissolved in methanol (20 ml) and again cooled with ice, and then a 5 N solution of sodium hydroxide was added to reach pH 9.0. The reaction mixture was stirred for 30 minutes and further stirred at room temperature for 30 minutes. The reaction mixture was concentrated to dryness under reduced pressure at low temperature, and then the obtained residue was chromatographed on silica gel. The light yellow amorphous ie crystals obtained (0.83 g, 82%) were recrystallized from ether to yield light yellow powdery crystals (0.69 a). mp 161-162 °C.

Elemental analysis for C37H37N3045F 0.5H,0

C (2) H (%) N (%)

Calculated : 66.65 ;: 5.74 ; 6.30

Found . 66.47 ; 5.73 ; 6.10 14.NMR (300MHz, CDCl) 8: 1.18(6H,d), 1.28(3H.d). 2.11(3H,s). 2.71(1H,m), 3.65(2H,s), 3.83-3.85(2H,m), 4.16(1H,quint), 4.18(2H,s), 5.27(2H,s8), 7.00(2H,t), 7.14- 7.23(5H,m), 7.36-7.44(1H,m), 7.64(2H,4)., 7.77(2H,4), 8.09(1H, s), 8.28(1H,s).

FAB-Mass m/z 658(MH)™

Example 10-2:

Production of (R)-4,7-dihydro-2-[4-(3-hydroxy-2- nethylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N- penzyl-N-methylaminomethyl) -5-isobutyryl-4-oxothienol2,3- blpyridine hydrochloride a HCI

H,C-N OO O

CH,

Pa S N F 3

HO 0 0

F

The title compound was obtained in the same manner as in Example 1. mp 214-216 °C.

Example 11

Example 11-1:

Production of 4, 7-dihydro-2-[4-(2-hydroxy-2- methylpropionylamino)phenyl] -7-(2,6 -difluorobenzyl)-3- (N- benzyl-N-methylaminomethyl )-5-isobutyryl-4-oxothieno [2,3- blpyridine

H,C-N oO O

He Vase

HO—>— sn F Os

H.C 0

F

To a solution of the compound obtained in Reference

Example 29 (1.14 g, 2.0 mmol) in dichloromethane (30 ml) were added diisopropylethylamine (1.04 g, 8 mmol) and 2- hydroxyisobutanoic acid (0.416 g, 4 mmol), followed by stirring under ice cooling conditions. To this solution was added benzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After

: PCT/JP99/03379 stirring under ice cooling conditions for 1 hour, the solution was further stirred at room temperature for 4 days. The reaction mixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between water (50 ml) and chloroform (50 ml). The water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgSO4), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from ether to yield yellow powdery crystald (0.56 g, 43%). mp 178-180 °C.

Elemental analysis for C37H37N30485F) 0.5H,0

C (%) H (3) N (%)

Calculated : 66.65 ; 5.74 ; 6.30

Found . 66.54 ; 5.49 ; 6.36 14-NMR (300MHz, CDCly) 8: 1.18(6H,d), 1.58(6H.s). 2.10(3H,s)., 3.05(1H,brs), 3.64(2H,s), 4.16(2H,s), 4.12- 4.22(1H,m), 5.23(2H,s), 6.99(2H,t), 7.05-7.25(5H,m), 7.34-7.44(1H,m), 7.64(2H,d), 7.75(2H,4), 8.25(1H, s), 8.86(1H, s).

Example 11-2:

Production of 4,7-dihydro-2-[4-(2-hydroxy-2- methylpropionylamino )phenyl]-7-(2, 6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl )-5-isobutyryl-4 -oxothieno[2,3- blpyridine hydrochloride

PCT/JP99/03379 i 73 { HCl

HCN 0 O

CH,

H /

HO~>— S N~ F 3

HC O 0

F

The title compound was obtained in the same manner as in Example 1. mp 213-215 °C.

Example 12

Example 12-1:

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-5-isobutyryl-7- (2, 6-difluorobenzyl)-2- [4-1(1- nydroxycyclopropyl ycarbonylamino jphenyll-4- oxothieno[2,3- b)pyridine

H,C-N Oo O

H ) CH,

N LY a

S N F 3

HO O

F

To a solution of the compound obtained in Reference

Example 29 (0.57g, 1.0 mmol) in dichloromethane (10 ml) were added diisopropylethylamine (0.52 g, 4 mmol) and 2- nydroxycyclopropanecarboxylic acid (0.204 g, 2 mmol), followed by stirring under ice cooling conditions. To this solution was added benzotriazol-1l- yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP

} PCT/JP99/03379 : i 74 reagent) (1.76 9. 4 mmol). After stirring under ice cooling conditions for 1 hour, the solution was further stirred at room temperature for 4 days. The reaction mixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between water (50 ml) and chloroform (50 ml). The water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgS0Q4) , the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from ether to yield yellow powdery crystals (0.27 9. 41%). 1H-NMR (300MHz, CDCl3) §: 1.16-1.20(2H,m), 1.18(6H,4), 1.48-1.51(2H,m), 2.09(3H,s), 3.64(2H,s), 3.95(1H,br s). 4.14(2H,s8). 4.12-4.19(1H,m), 5.20(2H,s), 6.99(2H,t), 7.10-7.25(5H,m), 7.34-7.46(1H,m), 7.57(2H,4), 7.70(2H,4), 8.21(1H, s). 8.82(1H, s)-.

Example 12-2: production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dinydro- 5-isobutyryl-7-(2, 6-difluorobenzyl)-2-[4- [(1- hydroxycyclopropyl ycarbonylamino]) phenyll-4- oxothienol2,3- blpyridine hydrochloride

OO HCI

H,C-N oO O

CH a STON FR

HO ©O L

F

The title compound, white powdery crystals (0.12g) was obtained in the same manner as in Example 1.

Solvent for recrystallization: ethanol-ether mp 220-222 °C.

Elemental analysis for C37H35N3045F HCl 0.5H,0

C (8) H (%) N (%) calculated : 63.37 ; 5.32 ; 5.99

Found . 63.35 ; 5.24 ; 5.82

Example 13

Example 13-1:

Production of 4,7-dihydro-2-[4-(3-hydroxy-3- methylbutyrylamino)phenyl])-7-(2,6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothienol2,3- blpyridine

HO CH HoT i CH

BCHOLY

S N F 3 0] OO

F

To a solution of the compound obtained in Reference

Example 29 (1.14 g, 2.0 mmol) in dichloromethane (10 ml) were added diisopropylethylamine (1.04 g, 8 mmol) and 3- hydroxy-3-methylbutanoic acid (0.47 g, 4 mmol), followed by stirring under ice cooling conditions. To this solution was added penzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After stirring under ice cooling conditions for 1 hour, the solution was further stirred at room temperature for 4 days. The reaction mixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between water (50 ml) and chloroform (50 ml). The water layer was again extracted with chloroform (10 m1). The combined

: PCT/JP99/03379 extracts were washed with saline, and dried (MgSO). the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel and recrystallized from ether to yield yellow powdery crystals (0.50 g, 37%). 14 NMR (300MHz, CDCly) 8: 1.17(6H,d), 1.39(6H,s). 2.11(3H,s). 2.58(2H,s)., 3.12(1H, br s), 3.65(2H,s), 4.12- 4.19(1H,m), 4.18(2H,s), 5.27(2H,s)., 7.00(2H,t), 7.10- 7.23(5H,m), 7.32-7.44(1H,m), 7.61(2H,4). 7.79(2H,4d), 8.24(1H, s). 8.28(1H, s).

Example 13-2:

Production of 4, 7-dihydro-2-[4-(3-hydroxy-3- methylbutyrylamino)phenyll-7-(2,6-difluorobenzyl)-3-(N- benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothienof2.3- blpyridine hydrochloride 8 \ HCI

HCN oO ©O

HO, CH, 1 CH,

H,C N CTT Ly

STN" F 73

F .

The title compound, white powdery crystals (0.429) were obtained in the same manner as in Example 1.

Solvent for recrystallization: ethanol mp 216-218 °C.

Elemental analysis for C3gH3zgN3045F HC1 HO

C (%) H (%) N (%)

Calculated : 62.84 ; 5.83; 5.79

Found . 62.70 ; 5.75 ; 5.82

R co -

PCT/JP99/03379 - - 77

Example 14

The following compounds (Examples 14-1 through 14-10) were produced by the same method as described in Examples 9, 11, 12, and 13, using the compound obtained in Reference

Example 29, and also using various carboxylic acid compounds in place of, for example, (2R)-3- t-butoxy-2-methylpropanolc acid, which was used in Example 9,

Example 14-1:

H,C-N Oo O

CH

H 3

H,C, ONT TL

H,C_ © 0]

H.C~X

CH,

F

Yield: 46% amorphous. 1, NMR (300MHz, CDClz) 6: 1.18(6H,d), 1.25(3H.d). 1.30(9H,s). 2.11(3H,s). 2.60(1H,br), 3.51(1H, t like), 3.59-3.65(1H,m), 3.65(2H,s), 4.17(1H.q). 4.18(2H,s), 5.27(2H.,s). 7.00(2H,t), 7.12-7.22(5H,m), 7.38-7.44(1H,m), 7.60(2H,d)., 7.75(2H,4), 8.28(1H, s), 9.00(1H, s).

Example 14-2: -N oO ©O

H.C CH, H.C ° X H CH,

HC o— N am

Q Re son FFs

YN © o H o

Yield: 47%

PCT/JP99/03379 mp 145-150 °C.

Example 14-3:

HCN 0 9 ! ) CH,

H,C, N IT o—~ STONT FC

F

Yield: 99% mp 96-98 °C.

Example 14-4: on (O 9) HCN o 0 > H / cs o N LI én

I= be

F

Yield: 57% mp 92-94 °C.

Example 14-5:

HCN 2 9 eo 0

H 3

CH,cH,0—4 N SW"

STN F °°

F

PCT/JP99/03379

Yield: 71% mp 57-62 °C.

Example 14-6:

HCN QQ

CH

H 3

CH, N SE

H,C— a S N F 3

HA be a F

Yield: 92% mp 79-81 °C.

Example 14-7: h,c-N o 9

CH i ahs

HC, Wa STN" F 7%

TC hb " yield: 97% mp 91-96 °C.

Example 14-8:

HCN Q @ a CH,

He HN 1 } aa Wa or © be i oo 3 PCT/JP99/03379

Yield: 91% mp 75-80 °C.

Example 14-9:

H,C-N Oo O =0 N 0 Ly o_ /— STON F T°

Oo 7X

A

Yield: 87% mp 83-88 °C.

Example 14-10: £ HCN oO O i.

H.C H 3 © To CHN 28m = STN FO

H.C 0

F

Yield: 50% mp 130-135 °C.

Example 15

Using the compound obtained in Example 14, the following compounds (Examples 15-1 to 15-6) in the same manner as in

Example 1C.

Example 15-1:

BN WO 00/00493 PCT/JP99/03379

HCN o 9 y > CH,

HC ~~ N LJ ey

F

Yield: 69% mp 161-162 °C.

Example 15-2:

OO =

HCN Q 0 ’ ) CH,

HC STN I

HO 0]

F

Yield: 94% mp 153-155 °C.

Example 15-3:

HCN o 0

CH

H Yi ®

HC, N Lb da — SS

F

Yield: 62% mp 224-226 °C.

N PCT/JP99/03379

Example 15-4:

Ow

HCN o 0

CH,

H Vi

H,C N IJ ew ha sow gO

F

Yield: 56% mp 230-232 °C.

Example 15-5:

HCN o 9

HO N

J — STON F Ca

F

Yield: 90% mp 92-94 °C.

Example 15-6:

H,C-N o Q y y CH,

HO N LU ay ha ST EC

F

Yield: 34% mp 107-112 °C.

PCT/JP99/03379 : . B3

Example 16

Example 16-1:

Production of 3- (N-benzyl-N-methylaminomethyl) -4,7- dihydro-5 -isobutyryl-7-(2, 6-difluorobenzyl)-2- (4-N'- methoxyureidophenyl) -4 -oxothienof2,3 -blpyridine

H,CN Oo ©O y TTS CH,

N— sw FO

H,C-O 0) \

F

To a solution of the compound obtained in Reference

Example 29 (0.40 g, 0.7 mmol) in dichloromethane (15 ml) were added triethylamine (0.20 ml, 1.43 mmol) and N,N’'- carbonyldiimidazole (0.228 g, 1.41 mmol), followed by stirring under ice cooling conditions. After the solution was allowed to warm to room temperature and further stirred for 2.5 days, to the mixture were added O-methylhydroxylamine hydrochloride (0.59 g, 7.06 mmol) and triethylamine (0.98 ml, 7.03 mmol) with ice cooling. While the solution was allowed to return to room temperature, it was stirred for 1.5 hours.

After dilution with water (50 ml), the reaction mixture was extracted with chloroform (50 ml). The water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgS0y4) . the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield yellow amorphous powders (0.349 g, 77%). 1H-NMR (300MHz, CDCl3) 8: 1.19(6H,d). 2.17(3H,s), 3.72(2H,brs), 3.83(3H,s), 4.10-4.20(1H,m), 4.22(2H,brs), 5.28(2H,s), 7.00(2H,t). 7.15-7.24 (5H, m) , 7.37-7.46(2H,m),

PCT/JP99/03379 7.59(2H,4), 7.75(2H,d4), 7.83(1H, br). 8.31(1H,s).

Example 16-2:

Production of 4,7-dihydro-2-(4-N’"- methoxyureidophenyl)-7- (2, 6-difluorobenzyl)-3-(N-benzyl-

N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3- blpyridine hydrochloride

H,C-N OO O . NTL

N— SEA

H,C-O Oo

F

The title compound, white amorphous powders (0.335 g) were obtained in the same manner as in Example 1.

Solvent for recrystallization: ethyl acetate-ether mp 225-230 °C.

Elemental analysis for C3gH34N4045F 7 HCl 0.5H;0

Cc (%) H (3%) N (%) calculated : 60.91 : 5.26 ; 8.12

Found . 60.80 ; 5.07 ; 8.17

Example 17

Example 17-1:

Production of (R)-4,7-dihydro-2-[4-(2,3- 4inydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3- (N-benzyl-N-methylaminomethyl)-5-isobutyryl-4- oxothieno{2,3-blpyridine

H,C-N 0 O i. = SE

HO © 0

F

To a 2 N aqueous solution of potassium hydroxide was added (R)-(+)-2, 2-dimethyl-1,3 -dioxolan-4-carboxylic acid methyl ester (1.00 g, 6.24 mmol), followed by stirring under ice cooling conditions for 3 hours. After neutralization with hydrochloric acid, the solution was salted out and extracted with ethyl acetate (50 ml) to yield (R)Y-(+)- 2.2-dimethyl-1,3-dioxolan-4-carboxylic acid as colorless amorphous powders (0.72 g). To a solution of the compound obtained in Reference Example 29 (0.572 g. 1.0 mmol) in dichloromethane (5 ml) were added diisopropylethylamine (1.04 g, 8 mmol) and the (R)-(+)-2,2-dimethyl-1,3-dioxo- lane-4-carboxylic acid obtained (0.292 g, 2 mmol), followed by stirring under ice cooling conditions. To this solution was added penzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate (BOP reagent) (0.885 g, 2 mmol). After stirring under ice cooling conditions for 1 hour, the solution was further stirred at room temperature for 4 days. The reaction mixture was concentrated to dryness under reduced pressure, and then the obtained residue was partitioned between water (50ml) and chloroform (50 ml). The water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgSO4). the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield colorless amorphous powders (0.5 g), which was then dissolved in trifluoroacetic acid (10 ml) and stirred at room

PCT/JP99/03379 temperature for 16 hours. The reaction mixture was concentrated to dryness under reduced pressure and treated with alkali, after which it was partitioned between chloroform (50 ml) and water (50 ml), and then the water layer was again extracted with chloroform (10 ml). The combined extracts were washed with saline, and dried (MgS0O4), the solvent was distilled off under reduced pressure. The obtained residue was chromatographed on silica gel to yield 0.5 g of colorless amorphous powders (76%). , “H-NMR (300MHz, DMSC-dg) 8: 1.09(6H.d), 1.95(3H,s). 3.51(2H,br), 3.55-3.72(2H,m), 4.01-4.08(4H,m), 4.84(1H,t). 5.63(2H,brs), 5.80(1H,d4), 7.15-7.24(7H,m), 7.49- 7.57(1H,m), 7.66(2H,d), 7.84(2H,4d), 8.53(1H,s), 9.82(1H,s).

Example 17-2:

Production of (R)-4,7-dihydro-2-[4-(2,3- dihydroxypropionylamino yphenyll-7-(2, 6-difluorobenzyl)-3- (N-benzyl-N-methylaminomethyl) -5-isobutyryl-4- oxothieno[2,3-blpyridine hydrochloride 3 HCI

HCN oO O

H ) CH,

HO N |. y ha soy” FO

F

The title compound, white powdery crystals (0.43 g) were obtained in the same manner as in Example 1.

Solvent for recrystallization: ether-ethanol mp 146-148 °C.

Example 18

Production of 4, 7-dihydro-2-(4-N'-hydroxy-N'- nethylureidophenyl)-7- (2, 6-difluorobenzyl) -3- (N-benzyl-N- methylaminomethyl)-5-isobutyryl-4-oxothienof2,3- blpyridine _N oO O

HC J : N _ on,

HO © eS

F

To a solution of the compound obtained in Reference

Example 29 (1. 0g, 1.75 mmol) in dichloromethane (40 ml) were added and triethylamine (0.49 m1, 3.50 mmol) and N,N'- carbonyldiimidazole (0.567 mg, 3.50 mmol), followed by stirring under ice cooling conditions. After the solution was allowed to warm to room temperature and further stirred for 17 hours, to the mixture was added N- methylhydroxylammonium chloride (730 mg, 8.75 mmol) and triehylamine (1.23 ml, 8.75 mmol) with ice cooling. While the solution was allowed to warm to room temperature, it was stirred for 5 hours. The reaction mixture was partitioned between chloroform (300 ml) and aqueous sodium bicarbonate (saturated, 200 ml), and then the organic layer was again washed with saline (200 ml). After the organic layer was dried (MgSO), the solvent was distilled off under reduced pressure. The residue obtained was chromatographed on silica gel to yield pale yellow amorphous powder (0.38 g, 34%). 1H-NMR (300MHz, CDCl,) 6: 1.18( 6H, d), 2.15( 3H, s), 3.26(3H, s), 3.71(2%, s), 4.19(2H, s), 5.25(2H, s), 7.00(2H, t),

7.22-7.26( 5H, m), 7.41( 1H, m), 7.49( 2H,d), 7.60( 2H.4), 8.06( 1H, s), 8.26( 1H, s).

Elemental analysis for C3sH34N404SF, 0.8H20

C (3) H (3) N (%)

Calculated : 63.78 ; 5.44 ; 8.50

Found . 63.73 ; 5.24 ; 8.41

Example 19

Production of 2-[4-[(1- i0 acetoxycyclopropyl) carbonylamino]lphenyl]-3- (N-benzyl-N- methylaminomethyl)- 5-benzoyl-7-(2,6 -difluorobenzyl)-4,7- dihydro-4-oxothieno[2, 3-blpyridine

HCN oO O g FOL 0

AE

Hc 0

F

To a solution of the compound obtained in Reference : Example 28 (0.42 g, 0.7 mmol) and triethylamine (0.08 g, 0.8 mmol) in dichloromethane (10 ml) was added 1- acetoxycyclopropanecarbonyl chloride (0.13 g, 0.8 mmol) with ice-cooling. The mixture was stirred at 0 °C for 30 minutes and at room temperature for 30 minutes. The reaction mixture was diluted with chloroform (20 ml) and the solution was washed with aqueous sodium bicarbonate (saturated, 20 ml), and brine (20ml). After the organic layer was dried (Na;SO;), the solvent was distilled off under reduced pressure. The residue obtained was chromatographed on silica gel to yield pale yellow amorphous powders (0.34 g, 66%). 14-NMR (300MHz, CDCls) &: 1.22( 2H, ad), 1.68( 2H, dd),

: PCT/JP99/03379 2.12(3H, s), 2.21(3H, s), 3.64(2H, s), 4.13(2H, Ss). 5.28(2H, s), 7.01( 2H, t), 7.17-7.23( 5H, m), 7.37-7.44( 3H, m), 7.51-7.56( 1H, m), 7.60( 2H, a), 7.81( 2H, d), 7.87-7.90(3H, m), 7.94(1H,S).

FAB-Mass m/z 732 (MH)”

Example 20

Using the compound obtained in Reference Example 28, the following compound Examples 20-1 was obtained in the same manner as in Example 19

Example 20-1:

N 0] 0]

HC”

H ofr fi OL CC

S$” °N” F x, °

HC ©

F

Yield : 80% 14_NMR (300MHz, CDC13) 8: 1.77(6H, s), 2.12(3H, s), 2.17(3H, s), 3.63(2H, s), 4.13(2H, s), 5.29(2H, s), 7.01(2H, t), 7.15-7.23(5H, m), 7.38-7.45(3H, m), 7.51-7.56(1H, m), 7.64(2H, 4), 7.85(2H, d), 7.88-7.90(3H, m), 7.94(1H, s).

FAB-Mass m/z 734(MH)"

Using the compound obtained in Reference Example 28, the following compound Examples 20-2 was obtained in the same manner as in Example 19

Example 20-2:

- PCT/JP99/03379 oO oO

H we HOLT TC a STON OF

H,C 0 0

HEX

CH, E

Yield: 84% : 14-NMR (300MHZz, CDCl1l,) &: 1.25(3H, d), 1.30(9H, s), 2.10(3H, s), 2.65-2.67(1H, br m), 3.51(2H, t)., 3.60(2H, s). 4.12(2H, s), 5.28(2H, s), 7.00(2H, t). 7.16-7.23(5H, m), 7.37-7.42 (3H, m), 7.50-7.53(1H, m), 7.61(2H, d), 7.80(2H, 4). 7.89(2H, d), 7.94(1H, s), 9.01(1H, s).

FAB-Mass m/z 748(MH)’

Example 21

Production of 3- (N-benzyl-N-methylaminomethyl)-5- penzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1- hydroxycyclopropyl )carbonylamino] phenyl] -4 -oxothieno[2,3- bipyridine

N

H,C~ 0) 0

H

OC

ST” NT F

HO 0

F

A mixture of the compound obtained in Example 19 (0.24 g, 0.33 mmol) and 5N sodium hydroxide (0.07 ml) in ethanol (8 ml) was stirred at room temperature for 1 hour. After the

Co PCT/JP99/03379 solvent was distilled off under reduced pressure, the residue obtained was dissolved in chloroform (30 ml) and the solution was washed with agueous sodium bicarbonate (saturated, 20 ml) and brine (20ml). After the organic layer was dried (Na;SOa). the solvent was distilled off under reduced pressure and the residue obtained was recrystallized from chloroform-ether to yield pale yellow powders (0.17 4g. 75%). mp 186-188 °C. 14-NMR (300MHz, CDCly) 8: 1.15( 2H, ad). 1.44( 2H, 4d), 2.05(3H, s). 3.56(2H, s), 4.01(2H, s). 4.56(1H, br Ss). 5.24(2H, s), 7.00( 2H, t). 7.14-7.22( 5H, m), 7.41( 3H, t)., 7.55( 2H, 4), 7.55(1H, 8s), 7.70( 2H, 4), 7.88(2H, 4d), 7.90(1H, s), 8.88(1H, 5s).

Elemental analysis for C40H33N3048F2 1.0H.0

Cc (2) H (%) N (%)

Calculated: 67.88 ; 4.98 ; 5.94

Found . 67.75 ; 4.70 ; 5.90

FAB-Mass m/z 690 (MH)’

Example 22

Production of 3- (N-benzyl-N-methylaminomethyl) -5- benzoyl-7-(2, 6-difluorobenzyl)-4. 7-dihydro-2-1{4- (2- hydroxy-2 -methylpropionylamino )phenyl]}-4- oxothieno[2,3- blpyridine

HCN o © gf TC 3 = S N F

HO 0

F

Using the compound obtained in Example 20-1, the title

Co PCT/JP99/03379 : i 92 compound was obtained in the same manner as in Example 21.

Solvent for recrystallization: chloroform-ether mp 222-223 °C. 1H-NMR (300MHz, CDCls) 6: 1.57( 6H, s), 2.09(3H, s), 2.50(1H, s), 3.59(2H, s), 4.10(2H, s), 5.28(2H, s), 7.01(2H, t), 7.16-7.25(5E, m), 7.38-7.44(3H, m), 7.51-7.56(1H, m), 7.67(2H, 4), 7.82(2H, da), 7.89(2H, 4), 7.93(1H, s), 8.82(1H, s).

FAB-Mass m/z 692 (MH)"

Example 23

Production of (R)-3- (N-benzyl-N-methylaminomethyl) - 5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihyaro-2-[4-[{3- nydroxy-2-methylbutyrylamino phenyl] -4- oxothienol[2,3- blpyridine

HCN oO ©O

HG TY

Dan SN” F

HO 0)

F

Using the compound obtained in Example 20-2, the title compound was obtained in the same manner as in Example 10.

Solvent for recrystallization: chloroform-ether mp 145-146 °C. 1y-NMR (300MHz, CDCls) 8: 1.25(3H, 4). 2.07(3H, s), 2.63- 2.70(1H, m}, 2.84(1H, br s), 3.57(2H, s)., 3.78-3.81(2H, m), 4.08(2H, s), 5.27(2H, s), 7.00(2H, t), 7.17-7.20(5H, m), 7.37-7.43(3H, m), 7.51-7.56(1H, m), 7.61(2H, 4). 7.77(2H, 4), 7.89(2H, a). 7.94(1H, s), 8.31(1H, br s).

o PCT/JP99/03379

FAB-Mass m/z 692 (MH)

Preparation Example 1

Using 100 mg of the compound produced in Example 1-2, 165 mg of lactose, 25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesium stearate, tablets are produced by a conventional method.

Preparation Example 2

The compound produced in Example 1-2 (5 g) is dissolved in distilled water for injection to make a total volume of 100 ml. This solution is aseptically filtered through a 0.22 um membrane filter (produced by Sumitomo Electric Industries,

Ltd. or Sartorius) and dispensed at 2 ml per washed sterile vial, followed by freeze-drying by a conventional method, to yield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 3

Using 100 mg of the compound produced in Example 3, 165 mg of lactose, 25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg of magnesium stearate, tablets are produced by a conventional method.

Preparation Example 4

The compound produced in Example 3 (5 g) is dissolved in distilled water for injection to make a total volume of 100 ml. This solution is aseptically filtered through a 0.22 um membrane filter (produced by Sumitomo Electric Industries,

Ltd. or Sartorius) and dispensed at 2 ml per washed sterile vial, followed by freeze-drying by a conventional method, to yield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 5 (1) Compound produced in Example 1-2 5g (2) Lactose/crystalline cellulose (particles) 330 g

(3) D-mannitol 29 g (4) Low-substitutional hydroxypropyl cellulose 20 g (5) Talc 25 g (6) Hydroxypropyl cellulose 50 g (7) Aspartame 3 g (8) Dipotassium glycyrrhizinate 3g (9) Hydroxypropylmethyl cellulose 2910 30 g (10)Titanium oxide 3.5 g (11)Yellow iron sesquioxide 0.5 g (12)Light silicic anhydride lg components (1), (3), (4), (5), (6), (7) and (8) are suspended or dissolved in purified water and coated on the core particles (2) to yield base fine subtilae, which are then further coated with components (9) through (11) to yield coated fine subtilae, which are then mixed with component (12) to yield 500 g of 1% fine subtilae of the compound. These subtilae are divided to 500 mg folded subtilae.

Experimental Example 1

Preparation of 1251_31euprorelin

To a tube containing 10 pl of a 3x 10-4 M aqueous solution of leuprorelin and 10 ul of 0.01 mg/ml lactoperoxidase, 10 ul (37MBqg) of a solution of Nal291 was added. After stirring, 10 pl of 0.001% H,0, was added, and a reaction was carried out at room temperature for 20 minutes. By adding 700 ul of a0.05% TFA (trifluoroacetic acid) solution, the reaction was stopped. followed by purification by reversed-phase HPLC.

The HPLC conditions used are shown below. 1257 _jeuprorelin was eluted at a retention time of 26 to 27 minutes.

Column: TSKgel ops-80T™ (TM indicates a registered trademark; the same applies below) CTR (4.6 mm x 10 cm)

Eluents: Solvent A (0.05% TFA)

Solvent B (40% CH3CN-0.05% TFA) 0 minute (100% Solvent A) - 3 minutes (100% Solvent

A) - 7 minutes (50% Solvent A + 50% Solvent B) - 40 minutes (100% Solvent B)

Eluting temperature: Room temperature

Elution rate: 1 ml/min

Experimental Example 2

Preparation of a rat pituitary anterior lobe membrane fraction containing GnRH receptors

Anterior lobes of the pituitary glands were isolated from forty Wistar rats (8 weeks old, male), and washed with ice-cooled homogenate buffer [25 mM Tris (tris (hydroxymethyl)aminomethane)-HC1, 0.3 M sucrose, 1 mM

EGTA (glycol-etherdiamine-N,N,N' N'-tetraacetic acid), 0.25 mM PMSF (phenylmethylsulfonyl fluoride), 10 U/ml aprotinin, 1 mg/ml pepstatin, 20 mg/ml leupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH 7.5]. The pituitary tissue was floated in 2 ml of the homogenate puffer and homogenized using a Polytron homogenizer. The homogenate was centrifuged at 700 x g for 15 minutes. The supernatant was taken in an ultracentrifuge tube and centrifuged at 100,000 x g for 1 hour to provide a membrane fraction pellet.

This pellet was suspended in 2 ml of assay buffer [25 mM

Tris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 0.1%

BSA (bovine serum albumin), 0.25 mM PMSF, 1 mg/ml pepstatin, 20 mg/ml leupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH 7.5) and the suspension was centrifuged at 100,000 x g for 1 hour. The membrane fraction recovered as a pellet was resuspended in 10 ml of assay buffer, divided into portions, preserved at _80 °C and thawed when needed.

Experimental Example 3

Preparation of CHO (Chinese hamster ovarian) cell membrane fraction containing human GnRH receptor

PCT/JP99/03379 oo : 96

Human GnRH receptor-expressing CHO cells (EP-A-678577) (10° cells) were suspended in phosphate-buffered saline supplemented with 5 mM EDTA (ethylenediaminetetraacetic acid) (PBS-EDTA) and centrifuged at 100 x g for 5 minutes.

To the cell pellet, 10 ml of a cell homogenate buffer (10 mM

NaHCO3, 5 mM EDTA, pH 7.5) was added, followed by homogenization using the Polytron homogenizer. After centrifugation at 400 x g for 15 minutes. the supernatant was transferred to an ultracentrifugation tube and centrifuged i0 at 100,000 x g for 1 hour to yield a membrane fraction precipitate. This precipitate was suspended in 2 ml of an assay buffer and centrifuged at 100,000 x g for 1 hour. The membrane fraction recovered as a precipitate was again suspended in 20 ml of the assay buffer, dispensed, and stored at -80 °C before use upon thawing.

Experimental Example 4

Determination of 1251 _1euprorelin binding inhibition rate

The rat and human membrane’ fractions prepared in

Experimental Examples 2 and 3 were diluted with the assay puffer to yield a 200 mg/ml dilution, which was then dispensed at 188 ml per tube. Where the rat pituitary anterior lobe membrane fraction was used, to each tube, 2 ml of a solution of 0.1 mM compound in 60% DMSO (dimethyl sulfoxide) and 10 ul of 38 nM 1251 jeuprorelin were added simultaneously.

Where the cell membrane fraction of the CHO with human GnRH receptors expressed, to each tube, 2 ml of a solution of 2 mM compound in 60% DMSO and 10 ul of 38 nM 1257.1euprorelin © were added simultaneously. To determine maximum binding quantity, a reaction mixture of 2 ul of 60% DMSO and 10 pl of 38 nM 1257_31euprorelin was prepared. To determine non-specific binding amount, a reaction mixture of 2 ul of

Bn PCT/JP99/03379 100 pM leuprorelin in solution in 60% DMSO and 10 pl of 38 nM 1251-leuprorelin was prepared.

Where the rat or povine pituitary anterior lobe membrane fraction was used, the reaction was conducted at 4 °c for 90 minutes. Where the membrane fraction of the CHO with human

GnRH receptors expressed was used, the reaction was carried out at 25 °C for 60 minutes. After each reaction, the reaction mixture was aspirated and filtered through a polyethyleneimine-treated Whatman glass filter (GF-F).

After this filtration, the radioactivity of 12571_jeuprorelin remaining on the filter paper was measured with a y-counter.

The expression (TB-SB)/(TB-NSB) x 100 (where SB = radioactivity with the compound added, TB = maximum bound radioactivity, NSB = nonspecifically bound radioactivity) was calculated to find the binding inhibition rate (%) of each test compound. Furthermore, the inhibition rate was determined by varying the concentration of the test substance and the 50% inhibitory concentration (ICsg value) of the compound was calculated from Hill plot. The results are shown in below.

IC. value binding inhibition ICgq value

Test Compound rate (%) (pM)

Rat (1 uM) Human (20 uM) Rat Human compd. of Ex.1-2 96 102 0.06 0.0001 compd. of Ex.3 62 NT 0.6 0.0002

NT: not measured

Experimental Example 5

Suppression of blood LH in castrated monkeys

The compound produced in Example 1-2 was orally administered to castrated male cynomolgus monkeys (Macaca fascicularis)., and blood LH was guantified. The male cynomolgus monkeys, used at 3 years 8 months to 7 years 7 months

PCT/JP99/03379 of age at time of experimentation, had been castrated more than half a year prior to the examination. Test animals [n = 2; compound (1) and compound (2)] were given 30 mg/kg (3 ml/kg) of the compound suspended in 0.5% methyl cellulose at a final concentration of 1% by oral administration, and control animals [n = 3; control (1), control (2) and control (3)] were given 3 ml/kg of the 0.5% methyl cellulose dispersant alone by oral administration. At 24 hours and immediately before administration and at 2, 4, 6, 8, 24, and 48 hours after administration, blood was collected for heparinized plasma samples via the femoral vein and immediately stored under freezing conditions. plasma LH concentrations were determined by a bioassay oe using mouse testicular cells. The testicular cells were ~ collected frommale BALB/cmice (8 to 9 weeks of age) and washed three times with 1 ml of Dulbecco's modified Eagle medium (DMEM-H) containing 20 mM HEPES and 0.2% BSA per testis.

After incubation at 37 °c for 1 hour, the cells were passed through anylon mesh filter (70 pm) and dispensed to test tubes at 8 x 10° cells/tube. After the cells were washed twice with 0.4 ml of DMEM-H, 0.4 ml of a DMEM-H solution containing either equine LH (Sigma company), as the standard LH, or monkey plasma, previously diluted up to 1,000 fold, as the test sample, was added, followed by a reaction at 37 °c for 2 hours.

The testosterone concentration in the culture supernatant was determined by a radioimmunoassay (CIs Diagnostics Company) , and the LH concentration in the test monkey plasma was calculated from the standard curve for the standard equine

LH.

The results are given together in Figure 1.

The compound indicated is the compound obtained in

Example 1-2.

For the control (1), the control (2), and the control (3), changes over time in the LH concentrations in each animal are expressed as percent ratios to the respective reference values, which comprise the LH concentrations immediately before administration in each control animal (cynomolgus monkey). Similarly, for the compound (1) and the compound (2), changes over time in the LH concentrations in each animal (cynomolgus monkey) receiving the compound of Example 1-2 are expressed as percent ratios to the respective reference values, which comprise the LH concentrations immediately before administration of the compound. The time of administration was defined as zero (0), and hours before and after administration shown by minus and plus, respectively.

INDUSTRIAL APPLICABILITY

The compound of the present invention possesses excellent gonadotropin-releasing hormone antagonizing activity. Itisalsogoodin oral absorbability and excellent in stability and pharmacokinetics. with low toxicity, it is also excellent in safety.

The compound of the present invention can therefore be used as a prophylactic or therapeutic agent for hoxrmone- dependent diseases etc. Specifically, it is effective as a prophylactic or therapeutic agent for seX hormone -dependent cancers (e.g.., prostatic cancer, uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy. hysteromyoma, endometriosis, precocious puberty, amenorrhea syndrome, multilocular ovary syndrome, pimples etc, or as a pregnancy regulator (e.g., contraceptive), infertility remedy or menstruation regulator. It is also effective as an animal estrous regulator, food meat guality improving agent or animal growth regulator in the field of animal husbandry, and as a fish spawning promoter in the field of fishery.

Claims (24)

B WO 00/00493 PCT/IP99/03379 CLAIMS

1. A compound of the formula: CH; N oO O R—( STN” F TX F wherein Rl represents a Cj_5 alkyl group which may be substituted, a C3_5 cycloalkyl group which may be substituted, a C,_g alkoxyamino group which may be substituted or a hydroxyamino group which may be substituted; and RZ represents a Cy _9 alkyl group which may be substituted or a phenyl group which may be substituted; when Rl is an unsubstituted Ci-7 alkyl group, then R? is a substituted C;_4 alkyl group or a substituted phenyl group, or a salt thereof.

2. A compound of Claim 1 or a salt thereof, wherein Rl is (1) a Cq_7 alkyl group which may be substituted by 1 to 5 substituents selected from the group consisting of (i) hydroxy, (ii) C;_9 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy _g alkoxy-carbonyl, benzyloxycarbonyl, Cj _3 acyl, Ci_3 alkylsulfonyl and C,_3 alkyl, (iv) Cj._;0 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_4 cycloalkyloxy-carbonyl and C;_4 alkoxy and (v) Ci_¢ alkoxy-carbonyl, (2) aC3_9¢ cycloalkyl group which may be substituted by 1 to 3 substituents selected from the group consisting of (1) hydroxy, (ii) C;_9 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy _g alkoxy-carbonyl, benzyloxycarbonyl, Ci-3 acyl, Cy_3 alkylsulfonyl and Cj _3 alkyl, (iv) Ci_1j0 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_~ cycloalkyloxy-carbonyl and C1.3 alkoxy and (v) Cj_¢g alkoxy-carbonyl, (3) a Ci_¢ alkoxyamino group which may be substituted by 1 to 5 substituents selected from the group consisting of (1) hydroxy, (ii) Cj.-7 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cy _g alkoxy-carbonyl, benzyloxycarbonyl, Cj_.3 acyl, C;_3 alkylsulfonyl and C3._3 alkyl, (iv) Cj_10 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_5 cycloalkyloxy-carbonyl and Cy_3 alkoxy and (v) Cj_¢ alkoxy-carbonyl, or (4) a hydroxyamino group which may be substituted by 1 or 2 substituents selected from the group consisting of (i) Cj_97 acyloxy, (ii) amino which may be substituted by l or 2 substituents selected from the group consisting of Cy1_¢ alkoxy-carbonyl, benzyloxycarbonyl, Cj_3 acyl, Ci_3 alkylsulfonyl and Cy_3 alkyl, (iii) Cy_30 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_9 cycloalkyloxy-carbonyl and Cy _3 alkoxy and (iv) Cy_¢ alkyl; and RZ is (1) a Ci1-7 alkyl group which may be substituted by 1 to 5 substituents selected from the group consisting of (i) hydroxy, (ii) C;_7 acyloxy, (iii) amino which may be substituted by 1 or 2 substituents selected from the group consisting of Cj _g alkoxy-carbonyl, benzyloxycarbonyl, Cy _3 acyl, Ci_3 alkylsulfonyl and Ci-3 alkyl, (iv) C;_q0 alkoxy which may be substituted by 1 to 3 substituents selected from the group consisting of C3_+ cycloalkyloxy-carbonyl and Cj_3 alkoxy and (v) Cj_g alkoxy-carbonyl, or (2) a phenyl group which may be substituted by 1 to 5 substituents selected from the group consisting of halogen, Ci-3 alkyl and Ci-3 alkoxy.

3. A compound of Claim 1 or a salt thereof, wherein Rl is a C;._7 alkyl group which may be substituted or a C3_4 cycloalkyl group which may be substituted.

4. A compound of Claim 1 or a salt thereof, wherein Rl is a substituted branched C3_5 alkyl group or a substituted C3_7 cycloalkyl group.

5. A compound of Claim 1 or a salt thereof, wherein rR! is a C,.7 alkyl group substituted by hydroxy or a C3_7 cycloalkyl group substituted by hydroxy.

6. A compound of Claim 1 or a salt thereof, wherein Rl is a substituted Cz_7 cycloalkyl group.

7. A compound of Claim 1 or a salt thereof, wherein Rl isa cyclopropyl group which may be substituted by hydroxy.

8. A compound of Claim 1 or a salt thereof, wherein RZ? is a branched C3_7 alkyl group which may be substituted.

9. A compound of Claim 1 or a salt thereof, wherein R? is a phenyl group which may be substituted.

10. A compound of Claim 1 or a salt thereof, wherein R? is a phenyl group.

11. A compound of Claim 1 or a salt thereof, wherein Rl is a C3_7 cycloalkyl group and R? is a C,.g alkyl group.

12. A compound of Claim 1 or a salt thereof, wherein

Co WO 00/00493 PCT/JP99/03379 Rl is (1) a Cy_4 alkyl group substituted by 1 or 2 hydroxy. (2) a C3_7 cycloalkyl group substituted by hydroxy, or (3) a Cy_3 alkoxyamino group: and R2 is an isopropyl group or a phenyl group.

13. A compound of Claim 1 or a salt thereof, wherein Rl is (1) a Cq.7 alkyl group which may be substituted by 1 or 2 substituents selected from the group consisting of hydroxy. Cqi_3 alkyl-carbonyloxy, amino, benzyloxycarbonylamino, C;_3 alkoxy, C;.3 alkoxy-C;_3 alkoxy and Cy_3 alkoxy-carbonyl, (2) a C3_7 cycloalkyl group which may be substituted by a hydroxy or a Cy_3 alkyl-carbonyloxy. or (3) a C;_3 alkoxyamino group; and R2 is (1) an isopropyl group which may be substituted by a hydroxy or (2) a phenyl group.

14. 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-(4- cyclopropanecarbonylaminophenyl) -4-oxothieno[2,3~ b]pyridine or a salt thereof.

15. 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2- methylpropionylamino)phenyl]-4-oxothieno[2,3-blpyridine or a salt thereof.

16. 3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5- isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1- nydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3- blpyridine or a salt thereof.

17. 3-(N-Benzyl-N-methylaminomethyl)-5-benzoyl-7- (2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1- hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3- blpyridine or a salt thereof.

18. A process for producing a compound of Claim 1 or a salt thereof, which comprises reacting a compound of the formula:

¢ \ oo WO 00/00493 PCT/JP99/03379 cH; N Oo O 2 STON OF be wherein RZ represents a C;_y alkyl group which may be substituted or a phenyl group which may be substituted, or . a salt thereof, with i) a compound of the formula: r1acooH wherein R12 represents a C;_5 alkyl group which may be substituted or a C3_g cycloalkyl group which may be substituted, or a salt thereof or a reactive derivative thereof; or ii) carbonyldiimidazole, phosgene or a chloroformate, followed by reacting with a compound of the formula: r1bg wherein r1b represents a Cj _g alkoxyamino group which may be substituted or a hydroxyamino group which may be substituted, or a salt thereof.

19. A process for producing a compound of Claim 3 or a salt thereof, which comprises reacting a compound of the formula:

cH N Oo , STN” F xX wherein R2 represents a Cy_7 alkyl group which may be substituted or a phenyl group which may be substituted, or a salt thereof, with a compound of the formula: r12cooH wherein R12 represents a Cj_7 alkyl group which may be substituted or a C3_7y cycloalkyl group which may be substituted, or a salt thereof or a reactive derivative thereof. :

20. A pharmaceutical composition which comprises a compound of Claim 1 or a salt thereof.

21. A pharmaceutical composition of Claim 20 which is for antagonizing gonadotropin-releasing hormone.

22. A pharmaceutical composition of claim 21 which is for preventing OI treating a sex hormone dependent disease.

23. A compound of Claim 1 or a salt thereof with a pharmaceutically acceptable excipient, carrier or diluent for use in a method for antagonizing gonadotropin-releasing hormone in a mammal in need thereof.

24. Use of a compound of Claim 1 or a salt thereof for manufacturing a pharmaceutical composition for antagonizing gonadotropin-releasing hormone. AMENDED SHEET 13.11.2001