WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
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`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`
`(51) International Patent Classification 5 :
`
`C07D 207/00, 401/00, C07K 5/00
`
`
`
`
`
`
`(11) International Publication Number: WO 98/19998
`14 May 1998 (l4.05.98)
`
`(43) International Publication Date:
`
`PCT/EP97/06125
`
`_
`
`US
`
`(21) International Application Number:
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, Fl, GB, GE,
`
`GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK,
`(22) International Filing Date:
`5 November 1997 (05.1 1.97)
`
`LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO,
`NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR,
`T1‘, UA, UG, US, UZ, VN, YU, ZW, ARIPO Patent (GH,
`KE, LS, MW, _SD, SZ, UG, ZW), Eurasian patent (AM, AZ,
`BY, KG, KZ, MD, RU,_TJ, TM), European patent (AT, BE,
`"CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF,'CG, CI, CM, GA, GN,
`ML, MR, NE,‘ SN, TD, TG)._
`'
`"
`
`
`
`'
`
`Published
`Without international search report and to be republished
`upon receipt of that report.
`
`
`
`
`
`
`(30) Priority Datai .
`,
`,
`-
`,
`08/746,295
`' 7 November 1996 (07.11.96)
`
`
`
`
`(71) Applicant (for all designated States except’ US): NOVARTIS
`AG [CH/CH]; Schwarzwaldallee 215, CH—4058 Basel (CH);
`
`’
`’
`'
`'
`-
`(72) Inventor; and
`(for US only): VILLHAUER, Edwin,
`(75) InventorlApplicant
`Bernard [US/US]; 20 Dorothy Drive, Morristown, NJ
`07960 (US).
`
`(74) Agent: ROTH, Bernhard, M.; Novartis AG, Patent— und
`Markenabteilung, Lichtstrasse 35, CH-4002 Basel (CH).
`
`(54) Title: N—SUBSTITUTED 2—CYANOPYRROLIDINES
`
`
`
`(57) Abstract
`
`the
`N~(N’~substituted glycy1)—2—cyanopyrrolidines, e.g.
`compounds of formula (I) wherein R has various significances, are
`novel. They inhibit DPP—lV (dipeptidyl-peptidase—lV) activity.
`They are therefore indicated for use as pharmaceuticals in inhibiting
`DPP—lV and in the treatment of conditions mediated by DPP—IV,
`such as non—insulin—dependen’t diabetes mellitus, arthritis, obesity,
`osteoporosis and further conditions of impaired glucose tolerance.
`
`H
`
`\
`
`/
`R
`
`N
`
`\\\‘
`‘
`
`AURO — EXHIBIT 1008
`
`

`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`Albania
`Annenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d’Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`[E
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`N0
`NZ
`PL
`PT
`R0
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`SZ
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`US
`UZ
`VN
`YU
`ZW
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`

`
`W0 98/ 19998
`
`PCT/EP97/06125 _
`
`N -SUBSTITUTED 2-CYANOPYRROLIDINES
`
`Field
`
`The present invention relates to N-substituted 2-cyanopyrrolidines. More particularly,
`
`it provides novel N-glycyl—2—cyanopyrrolidine derivatives.
`
`Background
`
`Dipeptidyl peptidase-IV (DPP-IV) is a serine protease which cleaves N—terminal
`
`dipeptides from a peptide chain containing, preferably, a proline residue in the penultimate
`
`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. DPP-IV is responsible for inactivating glucagon-like peptide-1 (GLP-1).
`
`More particularly, DPP-IV cleaves the amino-terrninal 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 inhibition appears to represent an
`
`attractive approach for treating non-insulin-dependent diabetes mellitus (NIDDM).
`
`Although a number of DPP-IV inhibitors have been described, all have limitations
`
`relating to potency, stability or toxicity. Accordingly, a great need exists for novel DPP-IV
`
`inhibitors which are useful in treating conditions mediated by DPP-IV inhibition and which do
`
`not suffer from the above-mentioned limitations.
`
`

`
`W0 98/ 19998
`
`PCT/EP97/06125 _
`
`Summa;v_ of the invention
`
`The invention provides novel N—(N’—substituted glycyl)-2—cyanopyrrolidines which are
`
`effective as DPP-IV inhibitors in treating conditions mediated by DPP-IV. It also concerns
`
`corresponding pharmaceutical compositions, a process for their preparation, a method of
`
`inhibiting DPP-IV comprising administering to a patient in need of such treatment a
`
`therapeutically effective amount thereof, the compounds for use as a pharmaceutical, and their
`
`use in a process for the preparation of a medicament for treating a condition mediated
`
`by DPP-IV.
`
`Detailed descrigtion
`
`The invention concerns N-(N’-substituted glycyl)—2-cyanopyrrolidines, hereinafter
`
`briefly named "the compounds of the invention"; more particularly, it concerns compounds
`
`of formula I:
`
`wherein R is:
`
`a) R1R1.N(CH2).,,- wherein
`
`R, is a pyridinyl or pyrimidinyl moiety optionally mono- or independently
`
`disubstituted with (C1-4)alkyl, (C1-4)alkoxy, halogen, trifluoromethyl,
`
`cyano or nitro; or phenyl optionally mono- or independently disubstituted
`
`with (CM)a1kyl, (C,-4)alkoxy or halogen;
`
`R1, is hydrogen or (C1-g)alkyl; and
`
`m is 2 or 3;
`
`b) (C3.12)cycloalkyl optionally monosubstituted in the 1-position with (C1.3)hydroxyalkyl;
`
`c) R2(CH2)..- wherein either
`
`R2 is phenyl optionally mono- or independently di- or independently trisubstituted
`
`with (CM)alkyl, (CM)alkoxy, halogen or phenylthio optionally monosubstituted
`
`in the phenyl ring with hydroxymethyl; or is (C1-g)alkyl; a [3.l.1]bicyclic
`
`carbocyclic moiety optionally mono- or plurisubstituted with (C1.g)alkyl;
`
`

`
`WO 98/19998
`
`PCT/EP97/06125 ,
`
`-3-
`
`a pyridinyl or naphthyl moiety optionally mono- or independently disubstituted
`
`with (CM)alkyl, (C14)alkoxy or halogen; cyclohexene; or adamantyl; and
`
`n is 1 to 3; or
`
`R2 is phenoxy optionally mono- or independently disubstituted with (CM)a1kyl,
`
`(C14)alkoxy or halogen; and
`
`n is 2 or 3;
`
`d) (R3);CH(CH2)2- wherein each R3 independently is phenyl optionally mono- or
`
`independently disubstituted with (CM)alkyl,
`
`(CM)alkoxy or halogen;
`
`e) R4(CH2),,- wherein R4 is 2-oxopyrrolidinyl or (C2.4)a1koxy and
`
`p is 2 to 4;
`
`f) isopropyl optionally monosubstituted in 1-position with (C,_3)hydroxyalkyl;
`
`g) R5 wherein R5 is:
`
`indanyl; a pyrrolidinyl or piperidinyl moiety optionally substituted with
`
`benzyl; a [2.2. l]- or [3.l.1]bicyclic carbocyclic moiety optionally mono- or
`
`plurisubstituted with (C,-g)alkyl; adamantyl; or (C1-g)alkyl optionally mono- or
`
`independently plurisubstituted with hydroxy, hydroxymethyl or phenyl optionally mono-
`
`or independently disubstituted with (C1-4)alkyl, (CM)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 acetic 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. R1,, preferably is hydrogen. R2 preferably is
`
`phenyl optionally substituted as defined above. R3 preferably is unsubstituted phenyl.
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`

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`W0 98/ 19998
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`PCT/EP97/06125 _
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`-4-
`
`R4 preferably is alkoxy as defined above. R5 preferably is optionally substituted alkyl as defined
`
`above. in 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. Pyrirnidinyl preferably is pyrimidin-2-yl. It preferably is unsubstituted
`
`or monosubstituted, preferably in 4—position. Preferred as substitutents for pyridinyl and
`
`pyrirnidinyl are halogen, cyano and nitro, especially chlorine.
`
`When it is substituted, phenyl preferably is monosubstituted; it preferably is substituted
`
`with halogen, preferably 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 1 or 2 carbon
`
`atoms, it especially is methoxy.
`
`(C2-4)all<oxy 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-g)alkyl preferably is of 1 to 6, preferably 1 to 4 or 3 to 5,
`
`especially of 2 or 3 carbon atoms, or methyl. (C14) alkyl preferably is methyl or ethyl,
`
`especially methyl. (C1_3)hydroxyalkyl preferably is hydroxymethyl.
`
`A [3. l .l]bicyclic carbocyclic moiety optionally substituted as defined above preferably
`
`is bicyclo[3.l.l]hept-2-yl optionally disubstituted in 6-position with methyl, or
`
`bicyclo[3.1.l]hept-3-yl optionally trisubstituted with one methyl in 2-position and two methyl
`groups in 6-position. A [2.2.l]bicyclic carbocyclic moiety optionally substituted as defined
`
`above preferably is bicyclo[2.2. l]hept-2-yl.
`
`Naphthyl preferably is 1-naphthyl. Cyclohexene preferably is cyclohex-1-en—1—yl.
`
`Adamantyl preferably is l- or 2-adamantyl.
`
`A pyrrolidinyl or piperidinyl moiety optionally substituted as defined above preferably is
`
`pyrrolidin-3-yl or piperidin-4-yl. When it is substituted it preferably is N-substituted.
`
`

`
`WO 98/19998
`
`PCT/EP97/06125 -
`
`-5-
`
`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 R, ’ is pyridinyl optionally mono- or independently disubstituted with
`
`halogen, trifluoromethyl, cyano or nitro; or unsubstituted
`
`pyrirnidinyl;
`
`-
`
`(C3.7)cyc1oa1ky1 optionally monosubstituted in 1-position with (C,_3)hydroxyalkyl;
`
`— R4’(CH;;_)3— wherein R4’ is (C24)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 Ib), whereby R" is:
`
`- R1"NH(CH2);;_- wherein R1" is pyridinyl mono- or independently disubstituted with halogen,
`
`trifluoromethyl, cyano or nitro;
`
`-
`
`(C4_5)cycloa1kyl 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 (C 1.g)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(CH2)2— wherein R," is as defined above;
`
`-
`
`(C4_5)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.
`
`

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`W0 98/19998
`
`PCT/EP97/06125 _
`
`-5-
`
`A further group of compounds of the invention is compounds Ip, wherein R is R”,
`
`which is:
`
`a) R1"NH(CH;»_);;_- wherein R1” is a pyridinyl or pyrimidinyl moiety optionally mono- or
`
`independently disubstituted with halogen, trifluoromethyl,
`
`cyano or nitro;
`
`b) (C3.7)cycloalkyl optionally monosubstituted in 1-position with (C1-3)hydroxyalkyl;
`
`c) R2"(CH2)2- wherein R2” is phenyl optionally mono- or independently di- or independently
`
`d)
`
`(R3")2CH(CH2)2- wherein each R3” independently is phenyl optionally monosubstituted with
`
`trisubstituted with halogen or (C1.3)alkoxy;
`
`halogen or (C1_3)a1koxy;
`
`e) R4(CH2)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 compounds Is, wherein R is R‘,
`
`which is:
`
`a) R1sR,a‘(CH2)ms- wherein R1‘ is pyridinyl optionally mono- or independently disubstituted
`
`with chlorine, trifluoromethyl, cyano or nitro; pyrimidinyl
`
`optionally monosubstituted with chlorine or trifluoromethyl;
`
`or phenyl;
`
`R1: is hydrogen or methyl; and
`
`ms is 2 or 3;
`
`b)
`
`(C3_12)cycloalkyl optionally monosubstituted in 1-position with hydroxymethyl;
`
`c) R2‘(CH2),,s- wherein either
`
`R25 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-e)alkyl; 6,6-dimethylbicyclo[3. 1 . 1]hept-2—yl; pyridinyl;
`
`naphthyl; cyclohexene; or adamantyl; and ns is 1 to 3; or
`
`R25 is phenoxy; and ns is 2;
`
`d) (3,3-dipheny1)propyl;
`
`

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`WO 98/19998
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`PCT/EP97/06125 ,
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`-7-
`
`e) R4‘(CH2),,s wherein R4‘ is 2—oxopyrrolidin—1-yl or isopropoxy and
`
`ps is 2 or 3;
`
`f) isopropyl optionally monosubstituted in 1~position with hydroxymethyl;
`
`g) R55 wherein R5‘ is:
`
`indanyl; a pyrrolidinyl or piperidinyl moiety optionally N-substituted
`
`with benzyl; bicyclo[2.2.l]hept-2-yl; 2,6,6-trimethylbicyc1o-
`
`[3.1.l]hept-3-yl; adamantyl; or (C1-g)alky1 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 process which comprises
`
`coupling a reactive (2-cyanopyrrolidino)carbonylmethylene compound with an appropriate
`
`substituted amine; more particularly, for the preparation of the compounds of formulal it
`
`comprises reacting a compound of formula H
`
`CN
`
`11
`
`0 I
`
`I
`
`X\/C\N:
`
`wherein X is a reactive group,
`
`with a compound of formula III
`
`wherein R is as defined above,
`
`NHZR
`
`III
`
`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 conventional 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.
`
`

`
`WO 98/19998
`
`PCT/EP97/06125 ,
`
`-3-
`
`The starting materials may also be prepared in conventional manner.
`
`The compounds of formula Il may e.g. be prepared by the following two—step reaction
`
`scheme:
`
`STEP 1
`
`STEP 2
`
`.~
`
`J
`
`H-N
`
`NH2
`
`X
`
`C
`\/ \ X
`Et3N’ DMAP
`
`0
`
`\/C\N
`
`IV
`
`V
`
`TFAA
`(at least 2 eq.)
`
`H
`
`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 triethylamine
`
`and a catalytic amount of dimethylarninopyridine (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 preferably is
`
`conducted in the presence of an inert, organic solvent such as tetrahydrofuran or 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.
`
`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.
`
`

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`WO 98/19998
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`PCT/EP97/06125 _
`
`The following Examples illustrate the invention. All temperatures are in degrees
`
`Celsius.
`
`-9-
`
` Example 1: 1- 2-
`
`(S 2-pyrrolidine
`
`To a 500 ml flask is added 16.6 g of 2-[(5-chloropyridin-2-yl)amino]ethylamine and
`
`100 ml of tetrahydrofuran and the mixture is cooled in an ice bath. To the cooled mixture is
`
`added 7.0 g of (2-cyanopyrrolidino)carb0nylmethylene-(S)-bromide dissolved in 30 ml of
`
`tetrahydrofuran. The resultant mixture is stirred for 2 hours at 0°, 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 aqueous layer is then washed twice with ethyl
`
`acetate. The combined organic layers are washed successively with water and brine, dried over
`
`sodium sulfate and concentrated to obtain the desired free base compound in crude form. The
`
`crude form is then purified on silica gel employing a mixture of 5% methanol in methylene
`
`chloride as the eluent to yield the title compound in free base form as a light brown oil.
`
`After dissolving the free base in 30 ml of dry tetrahydrofuran, hydrogen chloride gas
`
`is bubbled into the solution for five seconds. The off-white precipitate that forms is filtered,
`
`washed with dry tetrahydrofuran and the solvent is removed by high vacuum pumping to
`
`obtain the title compound in dihydrochloride acid addition salt form (off-white solid;
`
`m.p. 265°—267°; NMR: see * at bottom of Table hereunder).
`
`The starting material is obtained as follows:
`
`a) 22.37 g of (S)-2-carbamoylpyrrolidine, 30.1 ml of triethylamine and 30.0 mg of
`
`dimethylaminopyridine (DMAP) are dissolved in 200 ml of methylene chloride and the solution
`
`is then added, dropwise, to an ice-cold solution of 18.8 ml of bromoacetylbromide in 192 ml
`of methylene chloride, over a period of 60 minutes under a calcium sulfate drying tube. The
`
`resultant solution is stirred for 2 hours at ice-water temperature under a calcium sulfate drying
`
`tube, then poured into 3.5 liters of ethyl acetate. The resultant precipitate is filtered, washed
`
`with ethyl acetate, and the filtrate is concentrated to obtain (2-carbamoylpyrrolidino)-
`
`carbonylmethylene-(S)-bromide (hard yellow taffy).
`
`

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`W0 98/19998
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`PCT/EP97/06125 ,
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`-10-
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`b) 50.0 g of the bromide compound prepared in a) above is dissolved in 300 ml of methylene
`
`chloride and the solution is cooled in an ice water bath under a calcium sulfate drying tube.
`
`The cooled solution is then poured into 60.2 ml of trifluoroacetic anhydride over a 2 minute
`
`period, the resultant solution is stirred at ice-water temperature under a calcium sulfate drying
`
`tube for 4 hours, and partitioned between methylene chloride and saturated aqueous sodium
`
`bicarbonate. The product is extracted into the methylene chloride layer and the aqueous layer
`
`is washed twice with methylene chloride. The combined organic
`
`layers are washed successively with water and brine and then dried over sodium sulfate. The
`
`solution is filtered and the solvent is removed by rotovaping and high vacuum pumping to
`
`obtain (2-cyanopyrrolidino)carbonylmethylene-(S)-bromide (dark yellow solid).
`
`

`
`WO 98/19998
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`PCT/EP97/06125
`
`-11
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`
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`
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`

`
`WO 98/19998
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`W0 98/ 19998
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`PCT/EP97/06125 :
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`
`WO 98/19998
`
`PCT/EP97/06125 0
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`

`
`WO 98/19998
`
`PCT/EP97/06125
`
`_15_
`
`EB5%Ea:n
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`W0 98/19998
`
`PCT/EP97/06125
`
`-15-
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`
`

`
`WO 98/19998
`
`PCT/EP97/06125 _
`
`-17-
`
`The compounds of the invention in free form or in pharmaceutically acceptable acid
`
`addition salt form, hereinafter briefly named "the agents of the invention", in particular, the
`
`compounds of formula I in free form or in pharmaceutically acceptable acid addition salt form,
`
`possess pharmacological activity. They are therefore indicated for use as pharmaceuticals.
`
`In particular, they inhibit DPP—IV. This activity 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 can be obtained from the American Type Culture Collection (ATCC HTB 37).
`
`Differentiation of the cells to induce DPP-IV expression is accomplished as described by
`
`Reisher et al. in Proc.Natl.Acad.Sci.USA E (1993) 5757-5761. Cell extract is prepared from
`
`cells solubilized in 10 mM Tris-HC1, 0.15 M NaCl, 0.04 t.i.u. (trypsin inhibitor unit)
`
`aprotinin, 0.5% non-ionic detergent 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 mg solubilized Caco-2
`
`protein, diluted to a final volume of 125 ml in assay buffer (25 mM Tris-HC1 pH 7.4, 140 mM
`
`NaCl, 10 mM KC1, 1% bovine serum albumin) to microtiter plate wells. The reaction is
`
`initiated by adding 25ml 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 ml volume of
`
`25% glacial acetic acid is added to stop the reaction. Test compounds are typically added as
`
`30 ml additions and the assay buffer volume is reduced to 95 ml. A standard curve of free
`p-nitroaniline is generated using 0-500 mM 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 /rnin). 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 IC5o, is calculated from 8-point, dose-response
`
`curves using a 4-parameter logistic function.
`
`In the above test, IC5o values of from about 10 nM to about 900 nM are obtained with
`
`the agents of the invention, e.g. 22 nM for the agent of Example 3.
`
`The DPP-IV inhibition 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 Clin.Exp.Immunol. §_9_ (1992) 192-197. Briefly, five ml
`
`of plasma are added to 96-well flat-bottom microtiter plates (Falcon), followed by the addition
`
`

`
`WO 98/19998
`
`PCT/EP97/06125
`
`-18-
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`of 5 ml 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
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`initiated by the addition of 10 ml of incubation buffer containing 0.1 mM substrate (H-Glycine-
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`Proline-AMC; AMC is 7-amino-4-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 fluorimeter (Excitation 380 nm
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`Emission 460 nm; sensitivity setting 4). Test compounds are typically added as 2 ml additions
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`and the assay buffer volume is reduced to 13 ml. A fluorescence-concentration curve of free
`
`AMC is generated using 0-50 mM solutions of AMC in assay buffer. The curve generated is
`
`linear and is used for interpolation of substrate consumption (catalytic activity in nmoles
`
`substrate cleaved/rnin). As with the previous assay, the potency of the test compounds as
`
`DPP—IV inhibitors, expressed as IC5o, is calculated from 8-point, dose-response curves using a
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`4 parameter logistic function.
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`In the above assay, IC5o values of from about 7 nM to about 2000 nM are obtained in
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`human plasma, and of from about 3 nM to about 400 nM in rat plasma, e.g., for the agent of
`
`Example 3, of 7 nM in human and 6 nM in rat plasma, respectively.
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`In view of their ability to inhibit DPP-IV, the agents of the inventi