United States Patent (19)
`Villhauer
`
`54 USE OF N-(SUBSTITUTED GLYCYL)-2-
`CYANOPYRROLDINES IN INHIBITING
`DIPEPTIDYL PEPTIDASE-IV
`
`75 Inventor: Edwin Bernard Villhauer, Morristown,
`N.J.
`
`73 Assignee: Novartis AG, Basel, Switzerland
`
`Appl. No.: 09/428,224
`21
`22 Filed:
`Oct. 20, 1999
`Related U.S. Application Data
`62 Division of application No. 08/962,168, Oct. 31, 1997, Pat.
`No. 6,011,155.
`60 Provisional application No. 60/030,570, Nov. 7, 1996.
`51) Int. Cl." ........................ A61K 31/505; A61K 31/44;
`A61K 31/415; A61K 31/40
`52 U.S. Cl. .......................... 514/272; 514/343; 514/406;
`514/412; 514/423: 514/333
`58 Field of Search ..................................... 514/272,343,
`514/333, 406, 412,423
`
`56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,229,465. 10/1980 Ohkuma et al. ...................... 424/27 X
`4,849,435
`7/1989 Wallweber et al.......
`... 514/343
`4,923,883 5/1990 Wallweber et al.......
`... 514/343
`4,977,182 12/1990 Wallweber et al...................... 514/343
`FOREIGN PATENT DOCUMENTS
`
`
`
`USOO6124305A
`Patent Number:
`11
`(45) Date of Patent:
`
`6,124,305
`Sep. 26, 2000
`
`OTHER PUBLICATIONS
`Liet 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. 878–833 (1978).
`Derwent Abstract 95-302548.
`Derwent Abstract 84–177689.
`Derwent Abstract 96-116353.
`Kaspari et al., Biochimica et Biophysica, Vol. 1293, pp.
`147-153.
`Ashworth et al., Bioorganic and Medicinal Chemistry Let
`ters, vol. 6, No. 10, pp. 1163-1166 (1996).
`Coutts et al., J. Med. Chem., vol.39, pp. 2087-2094 (1996).
`Deacon et al., Diabetes, vol. 44, pp. 126–1131 (Sep. 96).
`Ashworth et al., Bioorganic and Medicinal Chemistry Let
`ters, vol. 6, No. 22, pp. 2745-2748 (1996).
`Augustyns et al., Eur. J. Med. Chem., vol. 32, pp. 301-309
`(1997).
`Primary Examiner Floyd D. Higel
`Attorney, Agent, or Firm Joseph J. Borovian
`57
`ABSTRACT
`N-(N-substituted glycyl)-2-cyanopyrrolidines of formula I
`
`O
`
`CN
`
`I
`
`646454. 11/1991
`339422 11/1989
`555 824 A1 8/1993
`1581 09 12/1982
`296 O75 A5 U 11/1991
`WO90/12005 10/1990
`WO91/16339 10/1991
`WO93/08259 4/1993
`WO95/11689 5/1995
`WO95/13069 5/1995
`WO95/15309 6/1995
`WO95/29 190 11/1995
`WO95/29691. 11/1995
`WO95/34538 12/1995
`
`Australia.
`European Pat. Off..
`European Pat. Off..
`Germany.
`Germany.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`WIPO.
`
`wherein R is as defined herein. Compounds of formula I
`inhibit DPP-IV (dipeptidyl-peptidase-IV) activity. They are
`therefore indicated for use as pharmaceuticals in inhibiting
`DPP-IV and in the treatment of conditions mediated by
`DPP-IV, such as non-insulin-dependent diabetes mellitus,
`arthritis, obesity, osteoporosis and further conditions of
`impaired glucose tolerance.
`
`10 Claims, No Drawings
`
`Merck Exhibit 2191, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`1
`USE OF N-(SUBSTITUTED GLYCYL)-2-
`CYANOPYRROLIDINES IN INHIBITING
`DIPEPTIDYL PEPTIDASE-IV
`
`6,124,305
`
`2
`R is hydrogen or (Cs)alkyl, and
`m is 2 or 3;
`b) (C2)cycloalkyl optionally monosubstituted in the
`1-position with (C.)hydroxyalkyl,
`c) R-(CH2),
`wherein either
`R is phenyl optionally mono- or independently di- or
`independently trisubstituted with (Cl)alkyl, (C)
`alkoxy, halogen or phenylthio optionally monoSub
`Stituted in the phenyl ring with hydroxymethyl, or is
`(Cs)alkyl, a 3.1.1 bicyclic carbocyclic moiety
`optionally mono- or plurisubstituted with (Cs)
`alkyl, a pyridinyl or naphthyl moiety optionally
`mono- or independently disubstituted with (C)
`alkyl, (C)alkoxy or halogen; cyclohexene; or ada
`mantyl; and
`n is 1 to 3; or
`R is phenoxy optionally mono- or independently dis
`ubstituted with (Cl)alkyl, (C)alkoxy or halogen;
`and
`n is 2 or 3;
`d) (R)-CH(CH-)-- wherein each R- independently is
`phenyl optionally mono- or independently disubstituted
`with (Cl)alkyl, (C)alkoxy or halogen;
`e) R(CH2) - wherein R is 2-oxopyrrolidinyl or (C)
`alkoxy and p is 2 to 4,
`f) isopropyl optionally monoSubstituted in 1-position with
`(C1-)hydroxyalkyl,
`g) Rs wherein Rs 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 (Cs)alkyl, adamantyl; or (Cs)
`alkyl optionally mono- or independently plurisubsti
`tuted with hydroxy, hydroxymethyl or phenyl option
`ally mono- or independently disubstituted with (C)
`alkyl, (C)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
`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. R preferably
`is a pyridinyl or pyrimidinyl moiety optionally Substituted as
`defined above. R preferably is hydrogen. R. preferably is
`phenyl optionally Substituted as defined above. R prefer
`ably is unsubstituted phenyl. R. preferably is alkoxy as
`defined above. Rs 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 substituents for pyridinyl and pyrimidinyl are
`halogen, cyano and nitro, especially chlorine.
`When it is substituted, phenyl preferably is monosubsti
`tuted; it preferably is substituted with halogen, preferably
`
`5
`
`1O
`
`15
`
`This is a divisional of U.S. application Ser. No. 08/962,
`168, filed Oct. 31, 1997, now issued as U.S. Pat. No.
`6,011,155, which application claims the benefit of U.S.
`Provisional Application No. 60/030,570, filed Nov. 7, 1996.
`FIELD OF THE INVENTION
`The present invention relates to the area of dipeptidyl
`peptidase-IV (DPP-IV) inhibition. DPP-IV is a serine pro
`tease which cleaves N-terminal dipeptides from a peptide
`chain containing, preferably, a proline residue in the penul
`timate position. Although the biological role of DPP-IV in
`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
`Sible for inactivating glucagon-like peptide-1 (GLP-1).
`More particularly, DPP-IV cleaves the amino-terminal His
`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
`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
`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
`maceutical compositions useful in inhibiting DPP-IV com
`40
`prising a therapeutically effective amount of an
`N-(Substituted glycyl)-2-cyanopyrrolidine disclosed herein.
`Moreover, the present invention provides a method of inhib
`iting DPP-IV comprising administering to a mammal in
`need of Such treatment a therapeutically effective amount of
`a N-(Substituted glycyl)-2-cyanopyrrolidine.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The instant invention relates to novel N-(substituted
`glycyl)-2-cyanopyrrolidines of formula I:
`
`25
`
`45
`
`50
`
`N C
`
`55
`
`wherein R is:
`a) RRN(CH2)- wherein
`R is a pyridinyl or pyrimidinyl moiety optionally
`mono- or independently disubstituted with (C)
`alkyl, (C)alkoxy, halogen, trifluoromethyl, cyano
`or nitro, or phenyl optionally mono- or indepen
`dently disubstituted with (Cl)alkyl, (C)alkoxy or
`halogen;
`
`60
`
`65
`
`Merck Exhibit 2191, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`3
`chlorine, or methoxy. It preferably is substituted in the 2-, 4
`and/or 5-position, especially in the 4-position.
`(C2)cycloalkyl preferably is cyclopentyl or cyclohexyl.
`When it is substituted, it preferably is substituted with
`hydroxymethyl. (Cl)alkoxy preferably is of 1 or 2 carbon
`atoms, it especially is methoxy. (Cl)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. (Cs)alkyl prefer
`ably is of 1 to 6, preferably 1 to 4 or 3 to 5, especially of 2
`or 3 carbon atoms, or methyl. (Cl)alkyl preferably is
`methyl or ethyl, especially methyl. (Cl-)hydroxyalkyl pref
`erably is hydroxymethyl.
`A 3.1.1 bicyclic carbocyclic moiety optionally Substi
`tuted as defined above preferably is bicyclo3.1.1 hept-2-yl
`optionally disubstituted in the 6-position with methyl, or
`bicyclo3.1.1 hept-3-yl optionally trisubstituted with one
`methyl in the 2-position and two methyl groups in the
`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
`ably is cyclohex-1-en-1-yl. Adamantyl preferably is 1- or
`2-adamantyl.
`A pyrrolidinyl or piperidinyl moiety optionally Substi
`tuted as defined above preferably is pyrrolidin-3-yl or
`piperidin-4-yl. When it is substituted it preferably is
`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:
`R'NH(CH)- wherein R is pyridinyl optionally
`mono- or independently disubstituted with halogen,
`trifluoromethyl, cyano or nitro, or unsubstituted pyri
`midinyl;
`(C-7)cycloalkyl optionally monosubstituted in the
`1-position with (C1-)hydroxyalkyl,
`R"(CH-)-- wherein R is (Cl)alkoxy; or
`Rs, wherein Rs 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 Ib),
`whereby R" is:
`R"NH(CH) - wherein R." is pyridinyl mono- or inde
`pendently disubstituted with halogen, trifluoromethyl,
`cyano or nitro,
`(Ce)cycloalkyl monosubstituted in the 1-position with
`(C1-)hydroxyalkyl,
`R(CH) - wherein R is as defined above; or
`Rs' wherein Rs' is a 2.2.1- or 3.1.1 bicyclic carbocyclic
`moiety optionally mono- or plurisubstituted with
`(Cs)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:
`R"NH(CH) - wherein R." is as defined above;
`(Ce)cycloalkyl monosubstituted in the 1-position with
`hydroxymethyl;
`R(CH) - wherein R is as defined above; or
`Rs" wherein Rs" is adamantyl;
`in free form or in acid addition Salt form.
`A further group of compounds of the invention is com
`pounds Ip, wherein R is R, which is:
`a) R'NH(CH) - wherein R is a pyridinyl or pyrim
`idinyl moiety optionally mono- or independently dis
`ubstituted with halogen, trifluoromethyl, cyano or
`nitro,
`
`4
`b) (C7)cycloalkyl optionally monosubstituted in the
`1-position with (C.)hydroxyalkyl,
`c) R(CH-)- wherein R is phenyl optionally mono
`or independently di- or independently trisubstituted
`with halogen or (C1-)alkoxy;
`d) (R)-CH(CH-)- wherein each Rindependently is
`phenyl optionally monoSubstituted with halogen or
`(C1-)alkoxy;
`e) R(CH-)-- wherein R is as defined above; or
`f) isopropyl optionally monosubstituted in the 1-position
`with (Cl-)hydroxyalkyl,
`in free form or in pharmaceutically acceptable acid addition
`Salt form.
`A further group of compounds of the invention is com
`pounds Is, wherein R is R, which is:
`a) RR (CH)- 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 is hydrogen or methyl, and
`ms is 2 or 3;
`b) (C)cycloalkyl optionally monosubstituted in the
`1-position with hydroxymethyl;
`c) R-(CH2)- wherein either
`R 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, (C)alkyl,
`6,6-dimethylbicyclo3.1.1 hept-2-yl; pyridinyl;
`naphthyl, cyclohexene; or adamantyl; and nS is 1 to
`3; or
`R is phenoxy; and nS is 2,
`d) (3,3-diphenyl)propyl;
`e) R. (CH), wherein R is 2-oxopyrrolidin-1-yl or
`isopropoxy and pS is 2 or 3;
`f) isopropyl optionally monosubstituted in the 1-position
`with hydroxymethyl;
`g) Rs wherein Rs is: indanyl; a pyrrolidinyl or piperidi
`nyl moiety optionally N-substituted with benzyl;
`bicyclo2.2.1]hept-2-yl; 2,6,6-trimethylbicyclo-3.1.1
`hept-3-yl; adamantyl; or (Cs)alkyl optionally mono
`or independently disubstituted with hydroxy,
`hydroxymethyl or phenyl;
`in free form or in acid addition salt form.
`The compounds of the invention may be prepared by a
`proce SS which comprise S 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
`
`N
`
`C
`
`wherein X is a reactive group,
`with a compound of formula III
`
`NHR
`
`wherein R is as defined above,
`
`II
`
`III
`
`6,124,305
`
`5
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
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`65
`
`Merck Exhibit 2191, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`S
`and recovering the resultant compound of formula I in free
`form or in acid addition salt form.
`X preferably is a halogen Such as bromine, chlorine or
`iodine.
`The process of the invention may be effected in conven
`tional manner.
`The compound of formula II is preferably reacted with at
`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
`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
`tional manner.
`The compounds of formula II may e.g. be prepared by the
`following two-step reaction Scheme:
`
`STEP 1
`
`
`
`C N1 Nx NH2 X
`Hess
`EtN, DMAP
`
`H-N
`
`IV
`
`His II
`-
`(at least 2 eq.)
`
`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
`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
`ably is conducted in the presence of an inert, organic Solvent
`Such as tetrahydrofuran or a chlorinated, aliphatic hydrocar
`bon 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.
`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
`mented in the literature. More particularly,: a)
`
`6,124,305
`
`6
`1-hydroxymethylcyclopentylamine can be prepared by the
`reduction of 1-amino-1-cyclopentane carboxylic acid with
`lithium aluminum hydride as set forth below:
`
`OH
`
`HN
`
`COOH
`
`NH2
`
`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)aminoethylamine can be prepared by
`refluxing a mixture of 2,5-dichloropyridine with ethylene
`diamine in an oil bath for a period of between 6 and 12
`hours. (c) Similarly, 2-(5-trifluoromethylpyridin-2-yl)
`aminoethylamine 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)
`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)aminoethylamine can be prepared by adding ethylene
`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. (f) 1-amino-1-
`cyclohexanemethanol can be prepared by the reduction of
`1-amino-1-cyclohexane carboxylic acid with lithium alumi
`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) 203
`aminopropylamino)-5-cyanopyridine can be prepared by
`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
`positions useful in inhibiting DPP-IV comprising a pharma
`ceutically acceptable carrier or diluent and a therapeutically
`effective amount of a compound of formula I, or a pharma
`ceutically acceptable acid addition Salt thereof.
`In Still another embodiment, the instant invention pro
`vides a method of inhibiting DPP-IV comprising adminis
`tering to a mammal in need of Such treatment a therapeuti
`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
`method of treating conditions mediated by DPP-IV inhibi
`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 I,
`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
`maceutically acceptable acid addition salts, to inhibit DPP
`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 AmericanType Culture Collection (ATCC
`
`15
`
`25
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`35
`
`40
`
`45
`
`50
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`Merck Exhibit 2191, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6,124,305
`
`7
`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
`cell line Caco-2” in Proc. Natl. Acad. Sci., Vol. 90, pgs.
`5757-5761 (1993). Cell extract is prepared from cells solu
`bilized in 10 mM Tris-HCl, 0.15M 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
`is conducted by adding 20 lig Solubilized Caco-2 protein,
`diluted to a final volume of 125ul in assay buffer (25 mM
`1O
`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 ul of 1 mM Substrate (H-Alanine
`Proline-pNA; pNA is p-nitroaniline). The reaction is run at
`room temperature for 10 minutes after which time a 19 ul
`Volume of 25% glacial acetic acid is added to Stop the
`reaction. Test compounds are typically added as 30 ul
`additions and the assay buffer volume is reduced to 95 ul. A
`Standard curve of free p-nitroaniline is generated using
`0-500 uM 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 microtiter plate
`reader. The potency of the test compounds as DPP-IV
`inhibitors, expressed as ICso, is calculated from 8-point,
`dose-response curves using a 4-parameter logistic function.
`The following ICs were obtained:
`
`15
`
`25
`
`8
`
`-continued
`
`Caco-2 DPP-IV (nM)
`50
`38O
`240
`140
`240
`850
`5
`700
`150
`1O
`35
`12
`23
`250
`2O
`860
`240
`270
`350
`470
`50
`390
`6OO
`310
`270
`46
`22O
`8O
`60
`
`Compound
`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. SO
`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
`
`Compound
`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. 7D
`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
`
`Caco-2 DPP-IV (nM)
`36
`176
`22
`140
`26
`50
`165
`8
`175
`990
`290
`295
`54
`215
`382
`388
`279
`227
`1O
`50
`3O
`60
`OO
`2O
`90
`390
`50
`50
`70
`40
`70
`310
`90
`3O
`650
`500
`50
`1O
`37
`3O
`60
`22O
`
`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
`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 ul of plasma are added to
`96-well flat-bottom microtiter plates (Falcon), followed by
`the addition of 5ul of 80 mM MgClin 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 ul of incubation buffer
`containing 0.1 mM substrate (H-Glycine-Proline-AMC;
`AMC is 7-amino-4-methylcoumarin). The plates are cov
`ered 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 fluorim
`eter (Excitation 380 nm Emission 460 nm, sensitivity setting
`4). Test compounds are typically added as 2 ul additions and
`the assay buffer Volume is reduced to 13 ul. A fluorescence
`concentration curve of free AMC is generated using 0-50
`luM solutions of AMC in assay buffer. The curve generated
`is linear and is used for interpolation of Substrate consump
`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 ICs, is calculated from
`8-point, dose-response curves using a 4 parameter logistic
`function.
`The following ICsos were obtained:
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`Compound
`
`human plasma DPP-IV (nM) rat plasma DPP-IV (nM)
`
`65
`
`Ex. 1
`Ex. 3
`
`27
`7
`
`22
`6
`
`Merck Exhibit 2191, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`6,124,305
`
`9
`
`-continued
`
`Compound
`
`human plasma DPP-IV (nM) rat plasma DPP-IV (nM)
`
`Ex. 4
`Ex. 5
`Ex. 6
`Ex. 8
`Ex. 10
`Ex. 12
`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. SO
`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
`
`40
`37
`22
`12
`51
`95
`95
`70
`70
`250
`60
`8O
`8O
`210
`70
`40
`32
`1O
`240
`50
`8O
`28
`8O
`8O
`60
`2O
`277
`1090
`70
`OO
`65
`22O
`340
`OO
`40
`240
`1O
`2130
`28O
`11
`60
`8
`60
`18O
`2O
`490
`90
`140
`140
`42O
`2O
`28O
`250
`260
`190
`60
`150
`90
`130
`
`23
`18
`32
`11
`19
`38
`24
`40
`60
`2O
`70
`50
`50
`1O
`60
`40
`19
`40
`70
`60
`60
`9
`90
`OO
`3O
`1O
`61
`340
`8O
`50
`23
`2OO
`370
`50
`18O
`12O
`1O
`390
`60
`5
`3O
`3
`40
`150
`1O
`400
`60
`90
`1OO
`150
`1OO
`130
`110
`8O
`1OO
`3O
`60
`40
`40
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`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
`tions mediated by DPP-IV inhibition. Based on the above
`and findings in the literature, it is expected that the com
`pounds disclosed herein are useful in the treatment of
`conditions Such as non-insulin-dependent diabetes mellitus,
`arthritis, obesity, allograft transplantation, and calcitonin
`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
`
`55
`
`60
`
`65
`
`10
`corresponding pharmaceutically acceptable acid addition
`Salts, to improve early insulin response to an oral glucose
`challenge may be measured in insulin resistant rats accord
`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
`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 1 g/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
`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 Linco 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
`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:
`
`Compound
`
`Ex. 1
`Ex. 3
`Ex. 5
`Ex. 8
`Ex. 12
`
`Increase of Insulin Response
`at 10 umol/kg
`
`61%
`66%
`1.08%
`14.4%
`59%
`
`The precise dosage of the compounds of formula I, and
`their corresponding pharmaceutically acceptable acid addi
`tion Salts, to be employed for treating conditions mediated
`by DPP-IV inhibition depends upon several factors, includ
`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
`ated by DPP-IV inhibition are effectively treated when a
`compound of formula I, or a corresponding pharmaceuti
`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
`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
`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
`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
`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
`positions containing an amount of the active Substance that
`
`Merck Exhibit 2191, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`11
`is effective for treating conditions mediated by DPP-IV
`inhibition, Such compositions in unit dosage form and Such
`compositions comprising a pharmaceutically acceptable car
`C.
`
`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., eee.98%,
`preferably 2.99%) or together with the Renantiomer, e.g., in
`racemic form. The above dosage ranges are based on the
`compounds of formula I (excluding the amount of the R
`enantiomer).
`Prior U.S. Provisional Application No. 60/030,570, filed
`on Nov. 7, 1996 is incorporated by reference herein, in its
`entirety.
`The following examples show representative compounds
`encompassed by this invention and their Synthesis.
`However, it should be clearly understood that they are for
`purposes of illustration only.
`EXAMPLE 1.
`1-2-(5-chloropyridin-2-yl)aminoethylamino
`acetyl-2-cyano-(S)-pyrrolidine dihydrochloride
`A. Prep a ration of 2-carbamoyl pyrrollidine
`carbonylmethylene-(S)-bromide.
`22.37 g (196 mmol) of (S)-2-carbamoylpyrrolidine, 30.1
`ml (216 mmol) of triethylamine and 30.0 mg of dimethy
`laminopyridine (DMAP) are dissolved in 200 ml of meth
`ylene chloride and the Solution is then added, dropwise, to
`an ice-cold solution of 18.8 ml (216 mmol) of bromoacetyl
`bromide in 192 ml of methylene chloride, over a period of
`60 minutes under a calcium Sulfate drying tube. The result
`ant Solution is then Stirred for 2 hours at ice-water tempera
`ture under a calcium Sulfate drying tube, after which time it
`is poured into 3.5 liters of ethyl acetate. The resultant
`precipitate is filtered, washed with ethyl acetate, and the
`filtrate is concentrated to obtain the desired compound as a
`hard yellow taffy.
`B. Preparation of 2-cyanopyrrolidino-carbonylmethylene
`(S)bromide.
`50.0 g (213 mmol) of the bromide