United States Patent [19J
`Villhauer
`
`[54] N-(SUBSTITUTED GLYCYL)-2-
`CYANOPYRROLIDINES,
`PHARMACEUTICAL COMPOSITIONS
`CONTAINING THEM AND THEIR USE IN
`INHIBITING DIPEPTIDYL PEPTIDASE-IV
`
`[75]
`
`Inventor: Edwin Bernard Villhauer, Morristown,
`N.J.
`
`[73] Assignee: Novartis AG, Basle, Switzerland
`
`[21] Appl. No.: 08/962,168
`
`[22] Filed:
`
`Oct. 31, 1997
`
`[51]
`
`Related U.S. Application Data
`[ 60] Provisional application No. 60/030,570, Nov. 7, 1996.
`Int. Cl? ...................... C07D 207/34; C07D 207/42;
`C07D 401!06; C07D 405/10; C07D 409/06
`[52] U.S. Cl. .......................... 544/333; 544/326; 544/330;
`546/208; 546/276.4; 546/279.1; 548/530;
`548/540
`[58] Field of Search ..................................... 548/530, 540,
`548/517; 546/276.4, 279.1, 208; 544/516,
`330, 328, 333
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,229,465
`4,849,435
`4,923,883
`4,977,182
`
`10/1980 Ohkuma et a!. ...................... 424/27 X
`7/1989 Wollweber, eta!. ................... 514/343
`5/1990 Wollweber, eta!. ................... 514/343
`12/1990 Wollweber, eta!. ................... 514/423
`
`FOREIGN PATENT DOCUMENTS
`
`0646454
`0339422
`555 824 A1
`1581 09
`296 075 A5
`W090/12005
`W09116339
`W09308259
`W095/13069
`W09511689
`W095/15309
`W09529190
`W09529691
`
`11/1991
`11/1989
`8/1993
`12/1982
`11/1991
`10/1990
`10/1991
`4/1993
`5/1995
`5/1995
`6/1995
`11/1995
`11/1995
`
`Australia ............................... 548/540
`European Pat. Off. ............... 548/540
`European Pat. Off ..
`Germany.
`Germany.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`
`111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US006011155A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,011,155
`Jan.4,2000
`
`W09534538 12/1995 WIPO .
`
`OTHER PUBLICATIONS
`
`Li et al, Archives of Biochemistry and Biophysics vol. 323,
`No. 1, pp. 148-154 (1995).
`Li et al, Journal of Neurochemistry, vol. 66, pp. 2105-2112
`(1996).
`Yamada et al, Bulletin of the Chemical Society of Japan, vol.
`50, No. 7, pp. 1827-1830 (1977).
`Yamada et al., Bulletin of the Chemical Society of Japan,
`vol. 51, No. 3, pp. 878-883 (1978).
`Chemical Abstracts 95: 302548. (1995).
`Chemical Abstracts 84: 177689. (1984).
`Chemical Abstracts 96: 116353 (1996).
`Kaspari et al., Biochimica et Biophysica, vol. 1293, pp.
`147-153. (1996).
`Ashworth et al., Bioorganic and Medicinal Chemistry Let(cid:173)
`ters, vol. 6, No. 10, pp. 1163-1166 (1996).
`Coutts et al., J. Med. Chern., vol. 39, pp. 2087-2094 (1996).
`Deacon et al., Diabetes, vol. 44, pp. 1126-1131 (Sep. '96).
`Ashworth et al., Bioorganic and Medicinal Chemistry Let(cid:173)
`ters, vol. 6, No. 22, pp. 2745-2748 (1996).
`Augustyns et al., Eur. J. Med. Chern., vol. 32, pp. 301-309
`(1997).
`
`Primary Examiner-Floyd D. Rigel
`Attorney, Agent, or Firm-Joseph J. Borovian
`ABSTRACT
`[57]
`
`N-(N'-substituted glycyl)-2-cyanopyrrolidines of formula I
`
`Compounds of formula I inhibit DPP-IV (dipeptidyl(cid:173)
`peptidase-IV) activity. They are therefore indicated for use
`as pharmaceuticals in inhibiting DPP-IV and in the treat(cid:173)
`ment of conditions mediated by DPP-IV, such as non(cid:173)
`insulin-dependent diabetes mellitus, arthritis, obesity,
`osteoporosis and further conditions of impaired glucose
`tolerance.
`
`11 Claims, No Drawings
`
`SAXA-DEF-00130
`
`Page 1 of 20
`
`AstraZeneca Exhibit 2023
` Mylan v. AstraZeneca
` IPR2015-01340
`
`

`
`6,011,155
`
`1
`N-(SUBSTITUTED GLYCYL)-2-
`CYANOPYRROLIDINES,
`PHARMACEUTICAL COMPOSITIONS
`CONTAINING THEM AND THEIR USE IN
`INHIBITING DIPEPTIDYL PEPTIDASE-IV
`
`This application claims the benefit of Provisional Appli(cid:173)
`cation number 60/030,570 filed on Nov. 7, 1996.
`
`5
`
`2
`R 1 a is hydrogen or (C1 _8)alkyl; and
`m is2or3;
`b) (C3 _12)cycloalkyl optionally monosubstituted m the
`1-position with (C1 _3)hydroxyalkyl;
`c) RiCH2)n- wherein either
`R2 is phenyl optionally mono- or independently di- or
`independently trisubstituted with (C1 _4)alkyl, (C1 _4 )
`alkoxy, halogen or phenylthio optionally monosub(cid:173)
`stituted in the phenyl ring with hydroxymethyl; or is
`(C1 _8)alkyl; a [3.1.1]bicyclic carbocyclic moiety
`optionally mono- or plurisubstituted with (C1 _8 )
`alkyl; a pyridinyl or naphthyl moiety optionally
`mono- or independently disubstituted with (C1 _4 )
`alkyl, (C1 _4)alkoxy or halogen; cyclohexene; or ada(cid:173)
`mantyl; and
`n is 1 to 3; or
`R2 is phenoxy optionally mono- or independently dis(cid:173)
`ubstituted with (C1 _4)alkyl, (C1 _4)alkoxy or halogen;
`and
`n is 2 or 3;
`d) (R3 ) 2 CH(CH2 ) 2 - wherein each R3 independently is
`phenyl optionally mono- or independently disubstituted
`with (C1 _4)alkyl, (C1 _4)alkoxy or halogen;
`e) RiCH2)P- wherein R4 is 2-oxopyrrolidinyl or (C2 _4 )
`alkoxy and p is2to4;
`f) isopropyl optionally monosubstituted in 1-position with
`(C1 _3)hydroxyalkyl;
`g) R5 wherein R5 is: indanyl; a pyrrolidinyl or piperidinyl
`moiety optionally substituted with benzyl; a [2.2.1]- or
`[3.1.1]bicyclic carbocyclic moiety optionally mono- or
`plurisubstituted with (C1 _8)alkyl; adamantyl; or (C1 _8 )
`alkyl optionally mono- or independently plurisubsti(cid:173)
`tuted with hydroxy, hydroxymethyl or phenyl option(cid:173)
`ally mono- or independently disubstituted with (C1 _4 )
`alkyl, (C1 _4)alkoxy or halogen;
`in free form or in acid addition salt form.
`The compounds of formula I can exist in free form or in
`acid addition salt form. Salt forms may be recovered from
`the free form in known manner and vice-versa. Acid addition
`salts may e.g. be those of pharmaceutically acceptable
`organic or inorganic acids. Although the preferred acid
`addition salts are the hydrochlorides, salts of
`methanesulfonic, sulfuric, phosphoric, citric, lactic and ace(cid:173)
`tic acid may also be utilized.
`The compounds of the invention may exist in the form of
`optically active isomers or diastereoisomers and can be
`separated and recovered by conventional techniques, such as
`chromatography.
`"Alkyl" and "alkoxy" are either straight or branched
`chain, of which examples of the latter are isopropyl and
`tert-butyl.
`R preferably is a), b) or e) as defined above. R1 preferably
`is a pyridinyl or pyrimidinyl moiety optionally substituted as
`defined above. R 1 a preferably is hydrogen. R2 preferably is
`55 phenyl optionally substituted as defined above. R3 prefer(cid:173)
`ably is unsubstituted phenyl. R4 preferably is alkoxy as
`defined above. R5 preferably is optionally substituted alkyl
`as defined above. m preferably is 2. n preferably is 1 or 2,
`especially 2. p preferably is 2 or 3, especially 3.
`Pyridinyl preferably is pyridin-2-yl; it preferably is
`unsubstituted or monosubstituted, preferably in 5-position.
`Pyrimidinyl preferably is pyrimidin-2-yl. It preferably is
`unsubstituted or monosubstituted, preferably in 4-position.
`Preferred as substitutents for pyridinyl and pyrimidinyl are
`65 halogen, cyano and nitro, especially chlorine.
`When it is substituted, phenyl preferably is monosubsti(cid:173)
`tuted; it preferably is substituted with halogen, preferably
`
`10
`
`FIELD OF THE INVENTION
`The present invention relates to the area of dipeptidyl
`peptidase-TV (DPP-IV) inhibition. DPP-IV is a serine pro(cid:173)
`tease which cleaves N-terminal dipeptides from a peptide
`chain containing, preferably, a proline residue in the penul(cid:173)
`timate position. Although the biological role of DPP-IV in 15
`mammalian systems has not been completely established, it
`is believed to play an important role in neuropeptide
`metabolism, T-cell activation, attachment of cancer cells to
`the endothelium and the entry of HIV into lymphoid cells.
`More recently, it was discovered that DPP-IV is respon- 20
`sible for inactivating glucagon-like peptide-1 (GLP-1).
`More particularly, DPP-IV cleaves the amino-terminal His(cid:173)
`Ala dipeptide of GLP-1, generating a GLP-1 receptor
`antagonist, and thereby shortens the physiological response
`to GLP-1. Since the half-life for DPP-IV cleavage is much 25
`shorter than the half-life for removal of GLP-1 from
`circulation, a significant increase in GLP-1 bioactivity (5- to
`10-fold) is anticipated from DPP-IV inhibition. Since GLP-1
`is a major stimulator of pancreatic insulin secretion and has
`direct beneficial effects on glucose disposal, DPP-IV inhi- 30
`bition appears to represent an attractive approach for treating
`non-insulin-dependent diabetes mellitus (NIDDM).
`
`SUMMARY OF THE INVENTION
`The present invention provides new DPP-IV inhibitors 35
`which are effective in treating conditions mediated by DPP-
`IV. More particularly, the present invention relates to certain
`N-(substituted glycyl)-2-cyanopyrrolidines which inhibit
`DPP-IV. In addition, the present invention provides phar(cid:173)
`maceutical compositions useful in inhibiting DPP-IV com- 40
`prising a therapeutically effective amount of a N-(substituted
`glycyl)-2-cyanopyrrolidine disclosed herein. Moreover, the
`present invention provides a method of inhibiting DPP-IV
`comprising administering to a mammal in need of such
`treatment a therapeutically effective amount of a 45
`N -(substituted gl ycy 1)-2-cyanopyrrolidine.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The instant invention relates to novel N-(substituted
`glycyl)-2-cyanopyrrolidines of formula I:
`
`50
`
`wherein R is:
`a) R1R1aN(CH2)m- wherein
`R1 is a pyridinyl or pyrimidinyl moiety optionally
`mono- or independently disubstituted with (C1 _4 )
`alkyl, (C1 _4)alkoxy, halogen, trifiuoromethyl, cyano
`or nitro; or phenyl optionally mono- or indepen(cid:173)
`dently disubstituted with (C1 _4)alkyl, (C1 _4)alkoxy or
`halogen;
`
`60
`
`SAXA-DEF-00131
`
`Page 2 of 20
`
`

`
`6,011,155
`
`10
`
`35
`
`4
`b) (C3_7 )cycloalkyl optionally monosubstituted in
`1-position with (C1 _3)hydroxyalkyl;
`c) R2P(CH2 ) 2 - wherein R2P is phenyl optionally mono- or
`independently di- or independently trisubstituted with
`halogen or (C1 _3)alkoxy;
`d) (R3P) 2 CH(CH2 ) 2 - wherein each R/ independently is
`phenyl optionally monosubstituted with halogen or
`(C1 _3)alkoxy;
`e) RiCH2 ) 3- wherein R4 is as defined above; or
`f) isopropyl optionally monosubstituted in 1-position with
`(C1 _3)hydroxyalkyl;
`in free form or in pharmaceutically acceptable acid addition
`salt form.
`A further group of compounds of the invention is com(cid:173)
`pounds Is, wherein R is Rs, which is:
`a) R/R1as(CH2)ms- wherein R/ is pyridinyl optionally
`mono- or independently disubstituted with chlorine,
`trifluoromethyl, cyano or nitro; pyrimidinyl optionally
`monosubstituted with chlorine or trifluoromethyl; or
`phenyl; R 1 as is hydrogen or methyl; and ms is 2or3;
`b) (C3_12)cycloalkyl optionally monosubstituted in
`1-position with hydroxymethyl;
`c) R2s(CH2)ms- wherein either
`R2s is phenyl optionally mono- or independently di- or
`independently trisubstituted with halogen, alkoxy of
`1 or 2 carbon atoms or phenylthio monosubstituted
`in the phenyl ring with hydroxymethyl; (C1 _6)alkyl;
`6,6-dimethylbicyclo[3.1.1 ]hept-2-yl; pyridinyl;
`naphthyl; cyclohexene; or adamantyl; and
`ns is 1 to 3; or
`R2s is phenoxy; and ns is 2;
`d) (3,3-diphenyl)propyl;
`e) R4s(CH2)pswherein R4s is 2-oxopyrrolidin-1 -yl or
`isopropoxy and ps is2or3;
`f) isopropyl optionally monosubstituted in 1-position with
`hydroxymethyl;
`g) R/ wherein R/ is: indanyl; a pyrrolidinyl or piperidi(cid:173)
`nyl moiety optionally N-substituted with benzyl;
`bicyclo[2.2.1 ]hept-2-yl; 2,6,6trimethylbicyclo-[3.1.1]
`hept-3-yl; adamantyl; or (C1 _8)alkyll optionally mono(cid:173)
`or independently disubstituted with hydroxy,
`hydroxymethyl or phenyl;
`45 in free form or in acid addition salt form.
`The compounds of the invention may be prepared by a
`process which comprises coupling a reactive
`(2-cyanopyrrolidino )carbonylmethylene compound with an
`appropriate substituted amine; more particularly, for the
`preparation of the compounds of formula I it comprises
`reacting a compound of formula II
`
`50
`
`55
`
`3
`chlorine, or methoxy. It preferably is substituted in 2-, 4-
`and/or 5-position, especially in 4-position.
`(C3_12)cycloalkyl preferably is cyclopentyl or cyclohexyl.
`When it is substituted, it preferably is substituted with
`hydroxymethyl. (C1 _4)alkoxy preferably is of I or 2 carbon 5
`atoms, it especially is methoxy. (C2 _4)alkoxy preferably is of
`3 carbon atoms, it especially is isopropoxy. Halogen is
`fluorine, chlorine, bromine or iodine, preferably fluorine,
`chlorine or bromine, especially chlorine. (C1 _8)alkyl prefer(cid:173)
`ably is of 1 to 6, preferably I to 4 or 3 to 5, especially of 2
`or 3 carbon atoms, or methyl. (C1 _4 ) alkyl preferably is
`methyl or ethyl, especially methyl. (C1 _3)hydroxyalkyl pref(cid:173)
`erably is hydroxymethyl.
`A [3.1.1]bicyclic carbocyclic moiety optionally substi(cid:173)
`tuted as defined above preferably is bicyclo[3.1.1 ]hept-2-yl
`optionally disubstituted in 6-position with methyl, or bicyclo 15
`[3.1.1]hept-3-yl optionally trisubstituted with one methyl in
`2-position and two methyl groups in 6-position. A [2.2.1]
`bicyclic carbocyclic moiety optionally substituted as defined
`above preferably is bicyclo[2.2.1 ]hept-2-yl.
`Naphthyl preferably is 1-naphthyl. Cyclohexene prefer- 20
`ably is cyclohex-1-en-1-yl. Adamantyl preferably is 1- or
`2-adamantyl.
`A pyrrolidinyl or piperidinyl moiety optionally substi(cid:173)
`tuted as defined above preferably is pyrrolidin-3-yl or
`piperidin-4yl. When it is substituted it preferably is 25
`N -substituted.
`A preferred group of compounds of the invention is the
`compounds of formula I wherein R is R' (compounds Ia),
`whereby R' is:
`R1 'NH(CH2 ) 2 - wherein R1 ' is pyridinyl optionally mono- 30
`or independently disubstituted with halogen,
`trifluoromethyl, cyano or nitro; or unsubstituted pyri(cid:173)
`midinyl;
`(C3_7)cycloalkyl optionally monosubstituted in 1-position
`with (C1 _3)hydroxyalkyl;
`R4 '(CH2 ) 3- wherein R4 ' is (C2 _4)alkoxy; or
`R5 , wherein R5 is as defined above;
`in free form or in acid addition salt form.
`More preferred compounds of the invention are those
`compounds of formula I wherein R is R" (compounds lb ), 40
`whereby R" is:
`R1 "NH(CH2) 2 - wherein R1 " is pyridinyl mono- or inde(cid:173)
`pendently disubstituted with halogen, trifluoromethyl,
`cyano or nitro;
`(C4 _6)cycloalkyl monosubstituted in 1-position with (C1 _
`3)hydroxyalkyl;
`R4 '(CH2 ) 3- wherein R4 ' is as defined above; or
`R5 ' wherein R5 ' is a [2.2.1]- or [3.1.1]bicyclic carbocyclic
`moiety optionally mono- or plurisubstituted with (C1 _
`s)alkyl; or adamantyl;
`in free form or in acid addition salt form.
`Even more preferred compounds of the invention are the
`compounds of formula I wherein R is R"' (compounds Ic),
`whereby R"' is:
`R1 "NH(CH2) 2 - wherein R1 " is as defined above;
`(C4 _6)cycloalkyl monosubstituted in 1-position with
`hydroxymethyl;
`R4 '(CH2 ) 3- wherein R4 ' is as defined above; or
`R5 " wherein R5 " is adamantyl;
`in free form or in acid addition salt form.
`A further group of compounds of the invention is com(cid:173)
`pounds Ip, wherein R is RP, which is:
`a) R/NH(CH2 ) 2 - wherein R/ is a pyridinyl or pyrim(cid:173)
`idinyl moiety optionally mono- or independently dis(cid:173)
`ubstituted with halogen, trifluoromethyl, cyano or
`nitro;
`
`II
`
`III
`
`60 wherein X is a reactive group,
`with a compound of formula III
`
`65 wherein R is as defined above,
`and recovering the resultant compound of formula I in free
`form or in acid addition salt form.
`
`SAXA-DEF-00132
`
`Page 3 of 20
`
`

`
`6,011,155
`
`6
`
`5
`X preferably is a halogen such as bromine, chlorine or
`iodine.
`The process of the invention may be effected in conven(cid:173)
`tional manner.
`The compound of formula II is preferably reacted with at 5
`least 3 equivalents of a primary amine of formula III. The
`reaction is conveniently conducted in the presence of an
`inert, organic solvent, preferably a cyclic ether such as
`tetrahydrofuran. The temperature preferably is of from about
`0° to about 35° C., preferably between about 0° and about 10
`25° c.
`The compounds of the invention may be isolated from the
`reaction mixture and purified in conventional manner, e.g.
`by chromatography.
`The starting materials may also be prepared in conven(cid:173)
`tional manner.
`The compounds of formula II may e.g. be prepared by the
`following two-step reaction scheme:
`
`STEP 1
`
`IV
`
`STEP2
`
`TFAA
`(at least 2 eq.)
`
`II
`
`v
`
`The reduction is conducted in the presence of an inert,
`organic solvent, preferably a cyclic ether such as
`tetrahydrofuran, at the reflux temperature of the solvent for
`a period of between 14 and 24 hours. (b) 2-[(5-
`chloropyridin-2-yl)amino ]ethylamine can be prepared by
`refluxing a mixture of 2,5-dichloropyridine with ethylene-
`15 diamine in an oil bath for a period of between 6 and 12
`hours. (c) Similarly, 2-[(5-trifluoromethylpyridin-2-yl)
`amino ]ethylamine can be prepared by refluxing a mixture of
`2-chloro-5-trifluoromethyl pyridine with ethylenediamine in
`an oil bath for a period of between 6 and 12 hours. (d)
`20 2-[(5-cyanopyridin-2-yl)amino ]-ethylamine can be prepared
`by stirring a mixture of 2-chloropyridine-5-carbonitrile and
`ethylenediamine at a temperature between 20° and 30° C.,
`for a period of between 4 and 6 hours. (e) 2-[(pyrimidin-2-
`yl)amino ]ethylamine can be prepared by adding ethylene-
`25 diamine to ice-bath cooled 2-chloropyrimidine and allowing
`the mixture to react at a temperature between 20° and 30° C.,
`for a period of between 12 and 20 hours. (t) 1-amino-1-
`cyclohexanemethanol can be prepared by the reduction of
`1-amino-1-cyclohexane carboxylic acid with lithium alumi-
`30 num hydride. The reduction is conducted in the presence of
`an inert, organic solvent, preferably a cyclic ether such as
`tetrahydrofuran, at the reflux temperature of the solvent for
`a period of between 14 and 24 hours. (g) 2(3-
`aminopropylamino )-5-cyanopyridine can be prepared by
`35 refluxing a mixture of 2,5-dichloropyridine with 1,3 propyl
`diamine in an oil bath for a period of between 6 and 12
`hours. Alternatively, the above examples (a) through (g) may
`be carried out at room temperature.
`The instant invention also includes pharmaceutical com-
`40 positions useful in inhibiting DPP-IV comprising a pharma(cid:173)
`ceutically acceptable carrier or diluent and a therapeutically
`effective amount of a compound of formula 1, or a phar(cid:173)
`maceutically acceptable acid addition salt thereof.
`In still another embodiment, the instant invention pro-
`45 vides a method of inhibiting DPP-IV comprising adminis(cid:173)
`tering to a mammal in need of such treatment a therapeuti(cid:173)
`cally effective amount of a compound of formula I, or a
`pharmaceutically acceptable acid addition salt thereof.
`In a further embodiment, the instant invention provides a
`50 method of treating conditions mediated by DPP-IV inhibi(cid:173)
`tion comprising administering to a mammal in need of such
`treatment a therapeutically effective amount of a compound
`of formula I above, or a pharmaceutically acceptable acid
`addition salt thereof.
`As indicated above, all of the compounds of formula 1,
`and their corresponding pharmaceutically acceptable acid
`addition salts, are useful in inhibiting DPP-IV. The ability of
`the compounds of formula I, and their corresponding phar(cid:173)
`maceutically acceptable acid addition salts, to inhibit DPP-
`60 IV may be demonstrated employing the Caco-2 DPP-IV
`Assay which measures the ability of test compounds to
`inhibit DPP-IV activity from human colonic carcinoma cell
`extracts. The human colonic carcinoma cell line Caco-2 was
`obtained from the American Type Culture Collection (ATCC
`65 HTB 37). Differentiation of the cells to induce DPP-IV
`expression was accomplished as described by Reisher, et al.
`in an article entitled "Increased expression of ... intestinal
`
`Step 1 involves the reaction of the pyrrolidine of formula
`IV with a slight molar excess of a haloacetylhalide such as
`bromoacetylbromide or chloroacetylchloride and triethy(cid:173)
`lamine and a catalytic amount of dimethylaminopyridine
`(DMAP). The reaction conveniently is conducted in the
`presence of an inert, organic solvent, preferably a
`chlorinated, aliphatic hydrocarbon such as methylene
`chloride, at a temperature of from about 0° to about 25° C.,
`preferably at a temperature between about 0° and about 15°
`c.
`Step 2 concerns the dehydration of the compound of
`formula V, prepared in Step 1, with at least 2 equivalents of
`trifluoroacetic anhydride (TFAA). The dehydration prefer(cid:173)
`ably is conducted in the presence of an inert, organic solvent
`such as tetrahydrofuran or a chlorinated, aliphatic hydrocar(cid:173)
`bon such as methylene chloride, at a temperature of from
`about 0° to about 25° C., preferably at a temperature 55
`between about 0° and about 15° C.
`Insofar as its preparation is not particularly described
`herein, a compound used as starting material is known or
`may be prepared from known compounds in known manner
`or analogously to known methods or analogously to methods
`described in the Examples.
`For example, the primary amine compounds of formula
`III are known and may be prepared by procedures docu(cid:173)
`mented in the literature. More particularly,: a)
`1-hydroxymethylcyclopentylamine can be prepared by the
`reduction of 1-amino-1-cyclopentane carboxylic acid with
`lithium aluminum hydride as set forth below:
`
`SAXA-DEF-00133
`
`Page 4 of 20
`
`

`
`6,011,155
`
`7
`cell line Caco-2" in Proc. Natl. Acad. Sci., Vol. 90, pgs.
`5757-5761 (1993). Cell extract is prepared from cells solu(cid:173)
`bilized in 10 mM Tris-HCl, 0.15 M NaCl, 0.04 t.i.u.
`aprotinin, 0.5% nonidet-P40, pH 8.0, which is centrifuged at
`35,000 g for 30 min. at 4° C. to remove cell debris. The assay 5
`is conducted by adding 20 jAg solubilized Caco-2 protein,
`diluted to a final volume of 125 pi in assay buffer (25 mM
`Tris-HCl pH 7.4, 140 mM NaCl, 10 mM KCl, 1% bovine
`serum albumin) to microtiter plate wells. The reaction is
`initiated by adding 25 tAl of 1 mM substrate (H-Alanine- 10
`Proline-pNA; pNA is p-nitroaniline). The reaction is run at
`room temperature for 10 minutes after which time a 19 tAl
`volume of 25% glacial acetic acid is added to stop the
`reaction. Test compounds are typically added as 30 fAl
`additions and the assay buffer volume is reduced to 95 tAL A
`standard curve of free p-nitroaniline is generated using
`0-500 jAM solutions of free pNA in assay buffer. The curve
`generated is linear and is used for interpolation of substrate
`consumption (catalytic activity in nmoles substrate cleaved/
`min). The endpoint is determined by measuring absorbance
`at 405 nm in a Molecular Devices UV Max microfiter plate
`reader. The potency of the test compounds as DPP-IV
`inhibitors, expressed as IC-50, is calculated from 8-point,
`dose-response curves using a 4-parameter logistic function.
`The following IC50s were obtained:
`
`15
`
`20
`
`25
`
`8
`-continued
`
`Compound
`
`Caco-2 DPP-IV (nM)
`
`Ex. 42
`Ex. 43
`Ex. 44
`Ex. 45
`Ex. 46
`Ex. 47
`Ex. 48
`Ex. 49
`Ex. 50
`Ex. 51
`Ex. 52
`Ex. 53
`Ex. 54
`Ex. 55
`Ex. 56
`Ex. 57
`Ex. 58
`Ex. 59
`Ex. 60
`Ex. 61
`Ex. 62
`Ex. 63
`Ex. 64
`Ex. 65
`Ex. 66
`
`240
`850
`5
`700
`150
`10
`35
`12
`23
`250
`20
`860
`240
`270
`350
`470
`50
`390
`600
`310
`270
`46
`220
`80
`60
`
`Compound
`
`Caco-2 DPP-IV (nM)
`
`Ex. 1
`Ex. 2
`Ex. 3
`Ex. 4
`Ex. 5
`Ex. 6
`Ex. 7A
`Ex. 8
`Ex. 7B
`Ex. 9A
`Ex. 7C
`Ex. 9C
`Ex. 10
`Ex. 11
`Ex. 70
`Ex. 7E
`Ex. 12
`Ex. 13
`Ex. 14
`Ex. 15
`Ex. 16
`Ex. 17
`Ex. 18
`Ex. 19
`Ex. 20
`Ex. 21
`Ex. 22
`Ex. 23
`Ex. 24
`Ex. 25
`Ex. 26
`Ex. 27
`Ex. 28
`Ex. 29
`Ex. 30
`Ex. 31
`Ex. 32
`Ex. 33
`Ex. 34
`Ex. 35
`Ex. 36
`Ex. 37
`Ex. 38
`Ex. 39
`Ex. 40
`Ex. 41
`
`36
`176
`22
`140
`26
`50
`165
`8
`175
`990
`290
`295
`54
`215
`382
`388
`279
`227
`110
`150
`130
`60
`100
`120
`90
`390
`150
`50
`70
`140
`170
`310
`90
`130
`650
`500
`150
`10
`37
`130
`160
`220
`50
`380
`240
`140
`
`The ability of the compounds of formula I, and their
`corresponding pharmaceutically acceptable acid addition
`salts, to inhibit DPP-IV may also be demonstrated by
`measuring the effects of test compounds on DPP-IV activity
`30 in human and rat plasma employing a modified version of
`the assay described by Kubota, et al. in an article entitled
`"Involvement of dipeptidylpeptidase IV in an in vivo
`immune response" in Clin. Exp. Immunol., Vol. 89, pgs.
`192-197 (1992). Briefly, five tAl of plasma are added to
`35 96-well fiat-bottom mictotiter plates (Falcon), followed by
`the addition of 5 tAl of 80 mM MgC12 in incubation buffer (25
`mM HEPES, 140 mM NaCl, 1% RIA-grade BSA, pH 7.8).
`After a 5 min. incubation at room temperature, the reaction
`is initiated by the addition of 10 tAl of incubation buffer
`40 containing 0.1 mM substrate (H-Glycine-Proline-AMC;
`AMC is 7-amino4-methylcoumarin). The plates are covered
`with aluminum foil (or kept in the dark) and incubated at
`room temperature for 20 min. After the 20 min. reaction,
`fluorescence is measured using a CytoFluor 2350 fiuorim-
`45 eter (Excitation 380 nm Emission 460 nm; sensitivity setting
`4). Test compounds are typically added as 2 tAl additions and
`the assay buffer volume is reduced to 13 tAL A fluorescence(cid:173)
`concentration curve of free AMC is generated using 0-50
`jAM solutions of AMC in assay buffer. The curve generated
`50 is linear and is used for interpolation of substrate consump(cid:173)
`tion (catalytic activity in nmoles substrate cleaved/min). As
`with the previous assay, the potency of the test compounds
`as DPP-IV inhibitors, expressed as IC50 , is calculated from
`8-point, dose-response curves using a 4 parameter logistic
`55 function.
`The following IC50s were obtained:
`
`Compound
`60 Ex. 1
`Ex. 3
`Ex. 4
`Ex. 5
`Ex. 6
`Ex. 8
`65 Ex. 10
`Ex. 12
`
`human plasma DPP-IV (nM)
`
`rat plasma DPP-IV (nM)
`
`27
`7
`40
`37
`22
`12
`51
`95
`
`22
`
`23
`18
`32
`11
`19
`38
`
`SAXA-DEF-00134
`
`Page 5 of 20
`
`

`
`6,011,155
`
`9
`-continued
`
`Compound
`
`human plasma DPP-IV (nM)
`
`rat plasma DPP-IV (nM)
`
`Ex. 14
`Ex. 15
`Ex. 16
`Ex. 17
`Ex. 18
`Ex. 19
`Ex. 20
`Ex. 21
`Ex. 22
`Ex. 23
`Ex. 24
`Ex. 25
`Ex. 26
`Ex. 27
`Ex. 28
`Ex. 29
`Ex. 30
`Ex. 31
`Ex. 32
`Ex. 33
`Ex. 34
`Ex. 35
`Ex. 36
`Ex. 37
`Ex. 38
`Ex. 39
`Ex. 40
`Ex. 41
`Ex. 42
`Ex. 43
`Ex. 44
`Ex. 45
`Ex. 46
`Ex. 47
`Ex. 48
`Ex. 49
`Ex. 50
`Ex. 51
`Ex. 52
`Ex. 53
`Ex. 54
`Ex. 55
`Ex. 56
`Ex. 57
`Ex. 58
`Ex. 59
`Ex. 60
`Ex. 61
`Ex. 62
`Ex. 63
`Ex. 64
`Ex. 65
`Ex. 66
`
`95
`70
`170
`250
`160
`180
`180
`210
`170
`40
`32
`110
`240
`150
`180
`28
`80
`80
`160
`20
`277
`1090
`170
`100
`65
`220
`340
`100
`140
`240
`10
`2130
`280
`11
`60
`8
`60
`180
`20
`490
`90
`140
`140
`420
`20
`280
`250
`260
`190
`60
`150
`90
`130
`
`24
`40
`60
`120
`70
`50
`150
`110
`60
`40
`19
`140
`70
`160
`60
`9
`90
`100
`130
`10
`161
`340
`80
`150
`23
`200
`370
`50
`180
`120
`10
`390
`60
`5
`30
`3
`40
`150
`10
`400
`60
`90
`100
`150
`100
`130
`110
`80
`100
`30
`60
`40
`40
`
`In view of their ability to inhibit DPP-IV, the compounds
`of formula I, and their corresponding pharmaceutically
`acceptable acid addition salts, are useful in treating condi(cid:173)
`tions mediated by DPP-IV inhibition. Based on the above
`and findings in the literature, it is expected that the com(cid:173)
`pounds disclosed herein are useful in the treatment of
`conditions such as non-insulin-dependent diabetes mellitus,
`arthritis, obesity, allograft transplantation, and calcitonin(cid:173)
`osteoporosis. More specifically, for example, the compounds
`of formula I, and their corresponding pharmaceutically
`acceptable acid addition salts, improve early insulin
`response to an oral glucose challenge and, therefore, are
`useful in treating non-insulin-dependent diabetes mellitus.
`The ability of the compounds of formula I, and their
`corresponding pharmaceutically acceptable acid addition
`salts, to improve early insulin response to an oral glucose
`challenge may be measured in insulin resistant rats accord(cid:173)
`ing to the following method:
`Male Sprague-Dawley rats that had been fed a high fat
`diet (saturated fat=57% calories) for 2-3 weeks were fasted
`
`5
`
`10
`for approximately 2 hours on the day of testing, divided into
`groups of 8-10, and dosed orally with 10 ,umol/kg of the test
`compounds in CMC. An oral glucose bolus of lg!kg was
`administered 30 minutes after the test compound directly
`into the stomach of the test animals. Blood samples,
`obtained at various timepoints from chronic jugular vein
`catheters were analyzed for plasma glucose and immunore(cid:173)
`active insulin (IRI) concentrations, and plasma DPP-IV
`activity. Plasma insulin levels were assayed by a double
`antibody radioimmunoassay (RIA) method using a specific
`anti-rat insulin antibody from Linea Research (St. Louis,
`Mo.). The RIA has a lower limit of detection of 0.5 ,uU/ml
`with intra- and inter-assay variations of less than 5%. Data
`are expressed as % increase of the mean of the control
`animals. Upon oral administration, each of the compounds
`15 tested amplified the early insulin response which led to an
`improvement in glucose tolerance in the insulin resistant test
`animals. The following results were obtained:
`
`10
`
`20
`
`25
`
`Compound
`
`Ex. 1
`Ex. 3
`Ex. 5
`Ex. 8
`Ex. 12
`
`Increase of Insulin Response
`at 10 .umol/k:g
`
`61%
`66%
`108%
`144%
`59%
`
`The precise dosage of the compounds of formula I, and
`their corresponding pharmaceutically acceptable acid addi-
`30 tion salts, to be employed for treating conditions mediated
`by DPP-IV inhibition depends upon several factors, includ(cid:173)
`ing the host, the nature and the severity of the condition
`being treated, the mode of administration and the particular
`compound employed. However, in general, conditions medi-
`35 ated by DPP-IV inhibition are effectively treated when a
`compound of formula I, or a corresponding pharmaceuti(cid:173)
`cally acceptable acid addition salt, is administered enterally,
`e.g., orally, or parenterally, e.g., intravenously, preferably
`orally, at a daily dosage of 0.002-5, preferably 0.02-2.5
`40 mg/kg body weight or, for most larger primates, a daily
`dosage of 0.1-250, preferably 1-100 mg. A typical oral
`dosage unit is 0.01-0.75 mg!kg, one to three times a day.
`Usually, a small dose is administered initially and the
`dosage is gradually increased until the optimal dosage for
`45 the host under treatment is determined. The upper limit of
`dosage is that imposed by side effects and can be determined
`by trial for the host being treated.
`The compounds of formula I, and their corresponding
`pharmaceutically acceptable acid addition salts, may be
`50 combined with one or more pharmaceutically acceptable
`carriers and, optionally, one or more other conventional
`pharmaceutical adjuvants and administered enterally, e.g.,
`orally, in the form of tablets, capsules, caplets, etc. or
`parenterally, e.g., intravenously, in the form of sterile inject-
`55 able solutions or suspensions. The enteral and parenteral
`compositions may be prepared by conventional means.
`The compounds of formula I, and their corresponding
`pharmaceutically acceptable acid addition salts, may be
`formulated into enteral and parenteral pharmaceutical com-
`60 positions containing an amount of the active substance that
`is effective for treating conditions mediated by DPP-IV
`inhibition, such compositions in unit dosage form and such
`compositions comprising a pharmaceutically acceptable ear(cid:173)
`ner.
`The compounds of formula I (including those of each of
`the subscopes thereof and each of the examples) may be
`administered in enantiomerically pure form (e.g., ee~98%,
`
`65
`
`SAXA-DEF-00135
`
`Page 6 of 20
`
`

`
`6,011,155
`
`12
`gas is bubbled into the solution for five seconds. The
`off-white precipitate that forms is then filtered, washed with
`dry tetrahydrofuran and the solvent is removed by high
`vacuum pumping to obtain the title compound as an off(cid:173)
`white solid, m.p. 265°-267° C.
`
`EXAMPLE 2
`
`1-[2-[ (5-trifluoromethylpryrdin-2-yl)amino]
`ethylamino ]acetyl-2-cyano-(S)-pyrrolidine
`
`To a 25 ml. flask is added 1.15 g (5.61 mmol) of
`2-[(5-trifluoromethylpyrdin-2-yl)-amino ]ethylamine and 10
`ml of tetrahydrofuran and the mixture is cooled in an ice
`bath. To the cooled mixture is added 0.404 g (1.87 mmol) of
`15 the bromide compound of Example 1b) dissolved in 5 ml of
`tetrahydrofuran. The resultant mixture is stirred for 2 hours
`at 0° C., the solvent is removed by rotovaping and the
`mixture is partitioned between ethyl acetate and water. The
`product is then extracted into the ethyl acetate layer and the
`20 aqueous layer is then washed twice with ethyl acetate. The
`combined organic layers are then washed successively with
`water and br