Ulllted States Patent
`
`[19]
`
`[11] Patent Number:
`
`6,107,317
`
`Villhaucr
`
`[45] Date of Patent:
`
`*Aug. 22, 2000
`
`US(JiJ6lU7317A
`
`[54] N-{SUBSTITUTED GI.YCYl.)-
`THlAZ0LlDlNl55s PHARMACEUTICAL
`COMPOSITIONS CONTAINING THEM AND
`THEIR USE IN INHIBITING DIPEPTIDYI.
`PEPTIDASEIW
`
`[75]
`
`Inventor: Edwin Bernard Villlmucr, Morrislown.
`NJ.
`
`........................................... .. 514,665; 5482194
`[52] U..‘. Cl.
`[58]
`1-'"i1:|d of S1.-z1n:|1 ............................ .. 548,! 194; 514.1365
`
`[56]
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`
`9()x'39384
`
`12119915 WIPO .................................. .. 5431194
`()'l'II]_".R PUBI_I(_'Afl'I()NS
`
`173]
`
`‘°“°*‘“3"°°‘ N""“”i5 AG’ B“3°1~S“’i‘Z°”a""
`
`Schon Biol. Chem. [I()ppe—Sey]t:r 372(5) 305-11, 1991.
`
`I *] Nolicc:
`
`This palcnl issued on a conlinucd prose
`cculion applicalion lilcd under 37 CIR
`l.53(d}, and is suhjccl lo lhc lwenly year
`palcnl
`lcrm provisions of 35 U.S.C.
`l54(a)(2)-
`
`P;-.f_m.m-_3; Exmm',I1er—R0[)cr[ Gel-s1[
`Armrricy, Agem, or Fi1"m—.Ioseph J. Borovian
`_, 1
`V 1
`ABSIRALI
`[57]
`Tho invention discloses ccrtain N—{subslilulcd g]ycy])—
`lhiazolitlinics, pharmaceutical compositions containing said
`c1n11pL1unds as an a1:1i\1c -ingredient ihcrctll‘, and [he use of
`said compounds In lnhlblllng dlpeplldyl pcplldasutz-IV.
`
`A9111. NU-l 0933395502
`Filcdj
`Jun. 24’ 1999
`
`[31]
`[22]
`
`[51]
`
`Int. Cl.7 ..................... .. C07D 277.I'04; AOIK 311425
`
`20 Claims, N0 Drawings
`
`Page 1 of 9
`
`Astraleneca Exh1b1t 2157
`Mylan v. Astraleneca
`lI’R2015-01340
`
`AstraZeneca Exhibit 2157
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 9
`
`

`
`6,107,317
`
`2
`
`1
`N-(SUBSTITUTED GI.YCYL_}-
`THIAZOLIDINES, PHARMACEUTICAL
`COMPOSITIONS CONTAINING THEM AND
`THEIR USE IN INHIBITING DIPEPTIDYL
`PEPTIDASE-IV
`
`FIELD OF THE INVENTION
`
`The present invention relates to the area of dipeptidyl
`peptidase—IV inhibition and, more particularly, relates to
`certain N{substituted glycyl)—thiazolidines, pharmaceutical
`compositions containing said compounds, and the use of
`said compounds in inhibiting dipeptidyl peptidase-IV.
`
`IIACKGROUNI) (J15 'l"IIl-E. INVEN'I'l()N
`
`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 DI-’I-’-IV in mam-
`malian 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 o1'IllV into lymphoid cells.
`More recently, it was discovered that l)PP-IV is respon-
`sible for inactivating glucagon—Iike peptide—l
`(GLP—1).
`More particularly, DPP—IV cleaves the amino—ter1nir1al His-
`Ala dipeptide of (iI.P-1, generating a GI.P-1 receptor
`antagonist, and thereby shortens the physiological response
`to (.}I.l-’-l. Since the hall’-life for DPI-"-IV cleavage is much
`shorter than the half—life for removal of GLP—l
`from
`
`circulation, a significant increase in GLP—l bioactivity {5— to
`10-fold) is anticipated from DPP-IV inhibition. Since (.iI.I-’-1
`is a major stimulator ol‘ pancreatic insulin secretion and has
`direct beneficial effects on glucose disposal, DPP—IV inhi-
`bition appears to represent an attractive approach for treating
`non—ir1su Iinxlependent diabetes mellitus (NIDDM).
`Although a number of DPP—IV inhibitors have been
`described in the literature, all have limitations relating to
`potency, stability or toxicity. Accordingly, it is clear that a
`great need exists for novel DPP—IV inhibitors which are
`useful in treating conditions mediated by DPP—IV inhibition
`and which do not suller from the above-mentioned limita-
`tions of known DPP-IV inhibitors.
`
`‘I0
`
`15
`
`10
`
`30
`
`40
`
`DESCRIPTION OF THE PRIOR ART
`
`Bulletin of the Chemical Society oflapan, Vol.51, No. 3,
`pgs. 878-883 (1978) discloses the synthesis of two known
`peptide antibiotics, viz., Bottromycins B1 and B2 according
`to the structures proposed by Nakamura, et al. However,
`since the resultant compounds were devoid of antimicrobial
`properties, it was concluded that the structures proposed by
`Nakamura, et at. were erroneous.
`W0 90i‘12005 discloses certain amino acid compounds
`which inhibit prolylen—dopeptidase activity and, therefore,
`are useful in treating dementia or amnesia.
`Chemical Abstracts 95: 302548 discloses certain N-(aryl
`(alkyl)carbonyl) substituted heterocyclic compounds which
`are cholinesterase activators with enhanced peripheral selec-
`tivity useful in treating conditions due to the lowering of
`cholinesterase activity.
`Chemical /\bstracLs 84: 177689 discloses certain 1-acyl-
`pyrrolidine-2-carbonitrile compounds which are useful as
`intermediates for proline compounds exhibiting angiotensin
`converting enzyme (ACE) inhibiting activity.
`ChemicalAbstracts 96: 116353 discloses certain 3—amino—
`
`2—mercapto—propyl—proline compounds which are Ras
`lamsyl-translierase inhibitors useful in treating various car-
`cinomas or myeloid leukemias.
`WC) 95334538 discloses certain pyrrolidides,
`phosphonates, azetidines, peptides and azaprolines which
`inhibit DPP—[V and, therefore, are useful in treating condi-
`tions mediated by DPP-IV inhibition.
`W0 95;"29l9U discloses certain compounds characterized
`by a plurality of Kl-‘R-type repeat patterns carried by a
`peptide matrix enabling their multiple presentation to, and
`having an aflinity for,
`the enzyme DPP—IV, which com-
`pounds exhibit the ability to inhibit the entry of HIV into
`cells.
`
`WO 91,3] 6339 discloses certain tetrapeptide boronic acids
`which are D1-‘I’-IV inhibitors useful in treating autoimmune
`diseases and conditions mediated by IL-2 suppression.
`WO 93108259 discloses certain polypeptide boronic acids
`which are DPP—IV inhibitors useful in treating autoimmune
`diseases and conditions mediated by IL-2 suppression.
`WC) 95;’11689 discloses certain tetrapeptide boronic acids
`which are l)PP-IV inhibitors useful in blocking the entry of
`HIV into cells.
`
`German Patent 158109 discloses certain N—protected
`peptidyl—hydroxamic acids and nitrobenzoyloxamides
`which are useful as, inter alia, DPP-IV inhibitors.
`
`‘W0 9.3329691 discloses, inter alia, certain dipeptide pro-
`line phosphonates which are DPP-IV inhibitors useful in the
`treatment of tmmu ne system disorders.
`German Patent 296075 discloses certain amino acid
`amides which inhibit DPP—IV.
`
`Biochimica et Biophysica Acta, Vol. 1293, pgs. 147-153
`discloses the preparation of certain di- and tri-peptide
`p-nitroanilides to study the inlluence of side chain modifi-
`cations on their DPP—IV and PEP—catalyzed hydrolysis.
`Bioorganic and Medicinal Chemistry Letters, Vol. 6, No.
`10, pgs. 1163-1166 (1996) discloses certain
`2-cyanopyrrolidines which are inhibitors of DPP-IV.
`J. Med. (_'hen‘t., Vol. 39, pgs. 2U8'i'—2(l'94 (1996) discloses
`certain prolineboronic acid-containing dipeptides which are
`inhibitors of DPP—IV.
`
`60
`
`Diabetes, Vol. 44, pgs. 1126-1131 (September ’96) is
`directed to a study which demonstrates that GLP—I amide is
`rapidly degraded when administered by subcutaneous or
`intravenous routes to diabetic and non-diabetic subjects.
`
`W0 95! 15309 discloses certain peptide derivatives which
`are inhibitors of DPP—IV and, therefore, are useful in treating
`a number of I)PI’—IV mediated processes.
`W0 95.113069 discloses certain cyclic amine compounds
`which are useliul
`in stimulating the release of natural or
`endogenous growth hormone.
`European Patent 555,824 discloses certain benzimida—
`zolyl compounds which prolong thrombin time and inhibit
`thrombin and serine-related proteases.
`Archives of Biochemistry and Biophysics, Vol. 313, No.
`1, pgs. 148-154 (1995) discloses certain
`aminoacylpyrrolidine—2—nitriles which are useful as DPP—IV
`inhibitors.
`
`Journal of Neurochemistry, Vol. 66, pgs. 2105-2] 1?.
`(1996) discloses certain Fmoc-aminoacylpyrrolidine-2-
`nitrilcs which are useful in inhibiting prolyl oligopeptidase.
`Bulletin of the Chemical Society of Japan, Vol. 50, No. '2',
`pgs. 1827-1830 (197?) discloses the synthesis of an
`aminohexapeptide, viz., Z—Val—Val—lmPro—Gly—Phe—Phe—
`()Me, and its related aminopeptides. In addition, the anti-
`microbial properties of said compounds were examined.
`
`Page 2 of 9
`
`Page 2 of 9
`
`

`
`6,107,317
`
`3
`Bioorganic and Medicinal Chemistry Letters, Vol. 6, No.
`22, pgs. 2745-2748 (1996) discloses certain
`4—cyanothiazolidides which are inhibitors of DPP—IV.
`
`SUMMARY OI“ TIIE lNVl3NTI()N
`
`The present invention provides new DPP-[V inhibitors
`which are elTective in treating conditions mediated by DH’-
`IV inhibition. More particularly,
`the present
`invention
`relates to certain N—{substiruted glycyl)—thiazolidines which
`inhibit DPP—IV. In addition, the present invention provides
`pharmaceutical compositions useful in inhibiting DPP-[V
`comprising a therapeutically effective amount of a certain
`N—(substituted glycyl)—thiazotidine. Moreover,
`the present
`invention provides a method of inhibiting DPP—IV compris-
`ing administering to a mammal in need of such treatment a
`therapeutically effective amount of a certain N-(substituted
`glycyl)-thiazolidine.
`
`DETAILED DESCRIPTION OF THE
`lNVEN'l'I()N
`
`The essence of the instant invention is the discovery that
`certain N-(substituted glycyl)-thiazolidines are useful
`in
`inhibiting DPP-IV. In one embodiment, the present inven-
`tion provides com pounds of fonnula I:
`
`4
`
`R4 is a 2-oxopyrrolidine group or a ((_':_,,}alkoxy group;
`or a pharmaceutically acceptable acid addition salt thereof.
`Preferred compounds are those of formula Ia:
`
`‘Ill
`
`15
`
`10
`
`30
`
`where
`
`R’ is a group
`
`H
`—(—c:H;—;q—N—R‘.;
`
`(C3_—,)
`a
`ring;
`an unsubstituted (C3_—,.)cycloalkyl
`cycloalkyl
`ring substituted in the 1-position by a
`hydroxy((T,_3)al.kyl group; a group —(—(TI'I2—);R'_.; an
`isopropyl group; or an isopropyl group substituted in
`the t—position by a hydroxy (C1_3)a]kyl group;
`R'1, is an unsubstituted pyridine ring; or a pyridine ring
`n'Iono- or di- substituted by halo, trifluoromethyl or
`cyano; and
`R',, is a ((_'2_,,)alkoxy group;
`or a phannaceutically acceptable acid addition salt thereof
`More preferred cornpounds are those of fonnula lb:
`
`wherein
`
`R is a group
`
`H
`—(-(TH:-}rl\‘:R 1:
`
`40
`
`where
`
`R“ is a group
`
`ring; a (C34)
`an unsubstituted (C3_,)—cycloalkyl
`cycloalkyl
`ring substituted in the 1-position by a
`hydroxy((_',_3)alkyl group; a group —(-CIl2—}5R:;
`a group
`
`It
`[I
`—(—CII3—ig—N—R 1:
`
`ring; a (C_,_,,,)
`an unsubstituted (C,,_6} cycloalkyl
`cycloalkyl
`ring substituted in the 1-position by a
`hydroxy(C1_3)a]kyl group;
`a group —{CH3—)~_;R‘4; an
`isopropyl group; or an isopropyl group substituted in
`the 1-position by a hydroxy((_‘1_3)alkyl group;
`R", is an unsubstituted pyridine ring; or a pyridine ring
`monosubstituted by halo, trifluoromethyl or cyano; and
`R',, is as defined above;
`or a pharmaceutically acceptable acid addition salt thereof.
`Even more preferred oompou nds are those of fonnula Ie:
`
`[c
`
`R,
`
`a group —(CH2—)3R4; an isopropyl group; or an isopro-
`pyl group substituted in the t—position by a hydroxy
`{C1_3)alkyl group;
`is an unsubstituted pyridine ring; a pyridine ring
`mor1o- or di-substituted by halo,
`trifluoromethyl or
`cyano; an unsubstituted pyrimidine ring; or a pyrimi-
`dine ring monosubstiruted by halo, trifluoromethyl or
`cyano;
`
`60
`
`R3 is an unsubstituted phenyl ring; or a phenyl ring
`rnono-, di- or tri-substituted by halo or (C1_3)al.koxy;
`
`each
`
`R3, independently, is an unsubstituted phenyl ring; or a
`phenyl ring monosubstituted by halo or {C,_3)a1koxy;
`and
`
`where
`
`Page 3 of 9
`
`Page 3 of 9
`
`

`
`R"' is a group
`
`H
`—(-CHr_:'}'_!,—N
`
`R''.:
`
`6,107,317
`
`6
`-continued
`Step 2
`
`a (C44,-)
`ring;
`an unsubstituted (C44,) cycloalkyl
`cycloalkyl
`ring substituted in the 1-position by a
`hydroxymethyl group; a group +CIl2——}3R',,; an isopro-
`pyl group; or an isopropyl group substituted in the
`1—position by a hydroxymnethyl group; and
`R‘, and R‘, are as defined above;
`or a pharrnaceutically acceptable acid addition salt thereof.
`In another embodiment, the instant
`invention provides
`pharmaceutical compositions useful in inhibiting DPP-[V
`comprising a phanrlaceulically acceptable carrier or diluent
`and a therapeutically effective amount of a compound of
`formula I above, or a pharmaceutically acceptable acid
`addition salt thereof, preferably a compound of formula Ia
`above, or a pharmaceutically acceptable acid addition salt
`thereof, more preferably a compound of formula lb above,
`or a pharmaceutically acceptable acid addition salt thereof,
`and even more preferably a compound of formula Ic above,
`or a pharmaceutically 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 elfective amount of a compound of formula I above, or
`a pharmaceutically acceptable acid addition salt
`thereof,
`preferably a compound of formula Ia above, or a pharma-
`ceutically acceptable acid addition salt thereof, more pref-
`erably a compound of formula lb above, or a pham1aceuti-
`cally acceptable acid addition salt thereof, and even more
`preferably a compound of formula Ic above, or a pharma-
`ceutically 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 elfective amount of a compound
`of formula I above, or a pharmaceutically acceptable acid
`addition salt thereof, preferably a compound of formula Ia
`above, or a pharmaceutically acceptable acid addition salt
`thereof, more preferably a compound of formula lb above,
`or a pharmaceutically acceptable acid addition salt thereof,
`and even more preferably a compound of formula Ic above,
`or a phanrtaceutically acceptable acid addition salt thereof.
`In the above definitions,
`it should be noted that
`the
`"alkoxy” significance is either straight or branched chain, of
`which examples of the latter are isopropyl and t—butyl.
`The acid addition salts of the compounds of formula I may
`be those of pharmaceutically acceptable organic or inorganic
`acids. Although the preferred acid addition salts are the
`hydrochlorides, salts of methanesulfonic, sulturic,
`phosphoric, citric, lactic and acetic acid may also be utilized.
`The compounds of formula I may be prepared by the
`following two—step reaction:
`
`Step ‘I
`
`if
`Cl
`C‘
`xg ‘KCI
`
`inorganic base
`
`H—-\,
`l a—-\|
`E;
`
`10
`
`‘I5
`
`10
`
`30
`
`40
`
`<.- (D
`
`60
`
`NH;R —- I
`(at least 3 cq.)
`
`III
`
`IV
`
`As to the individual steps, Step 1 involves the reaction of
`thiazolidine of formula II with at
`least
`1 equivalent of
`chloroacetylchloride and an inorganic base, eg, an alkali
`metal carbonate such as sodium carbonate to obtain the
`chloride compound of formula III. The reaction is conducted
`in the presence of an inert, organic solvent, preferably a
`cyclic ether such as tetrahydrofuran, at a temperature of
`from 0° to 25° (.‘., preferably at a temperature between 0°
`and 15° C., for a period of between 2 and 6 hours, preferably
`between 2 and 4 hours.
`Step 2 concerns the reaction of the compound prepared in
`Step 1, i.e., the chloride compound of fonrtula III, with at
`least 3 equivalents of a primary amine compound of fonnula
`IV to obtain an N—(substituted glycyl)—thiazolidine com-
`pound of formula I. The reaction is conducted in the
`presence of an inert, organic solvent, preferably a cyclic
`ether such as tetrahydrofuran, at a temperature of from 0° to
`15° C., for a period of between 2 and 14 hours, and then at
`a temperature of from 20° to 30° C. for a period of between
`12 and 20 hours.
`The primary amine compounds of formula IV are known
`and may be prepared by procedures well documented in the
`literature.
`For
`example:
`(a)
`l-hydroxymethylcyclopentylamine can be prepared by the
`reduction of 1-amino-l-cyclopentane carboxylic acid with
`lithium aluminum hydride as set forth below:
`
`OH
`
`HEN
`
`coon
`
`NH3
`
`mun,
`
`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—
`ehloropyridin—2—yl)amino]ethylamine 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)
`amino]ethylam ine 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-cyanopyriclin-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-
`diamine to ice-bath cooled Z-chloropyrimidine and allowing
`the mixture to react at a temperature between 20° and 30° C.,
`for a period of between 12 and 20 hours.
`As indicated above,
`the compounds of formula I form
`pharmaceutically acceptable acid addition salts. For
`example, the free base of a compound of formula I can be
`reacted with hydrochloric acid in gaseous form to form the
`corresponding mono— and di—hydrochloride salt
`forms,
`
`Page 4 of 9
`
`Page 4 of 9
`
`

`
`6,107,317
`
`7
`whereas reacting the free base with methanesulfonic acid
`forms the corresponding mesylate salt form. All pharmaceu-
`tically acceptable acid addition salt forms of the compounds
`of formula I are intended to be embraced by the scope of this
`invention.
`
`As indicated above, all of the compounds of formula I,
`and their corresponding pharmaceutically acceptable acid
`addition salts, are useful in inhibiting DPP—lV. The ability of
`the compounds of fonnula I, and their corresponding phar-
`maceutically acceptable acid addition salts, to inhibit [)PP-
`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
`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). (Tell extract is prepared from cells solu-
`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° (3. to remove cell debris. The assay
`is conducted by adding 20 gig solubilized (.‘aco-2 protein,
`diluted to a final volume of 125 _ul 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,trl of 1 mM substrate (['I-/\lanine-
`Proline-pN/\; pNA is p-nitroaniline). The reaction is run at
`room temperature for 10 minutes after which time a 1.0 _ul
`volume of 25% glacial acetic acid is added to stop the
`reaction. Test compounds are typically added as 30 girl
`additions and the assay buffer volume is reduced to 95 git. A
`standard curve of free p-nitroaniline is generated using
`0-500 ,ttM 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 IC50, is calculated from 8-point,
`dose-response curves using a 4-parameter logistic function.
`The following l(I5Us were obtained:
`
`(TcrI1p-nund
`Ex. 1
`line. 2
`["»X. 3
`Ex. 4
`
`(faco-1 |)l’P-l\«" (UM)
`10.3
`25.3
`16.2
`31.1
`
`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 DI-’l’-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,
`live rd of plasma are added to
`96-well flat-bottorn miclotiter plates (Falcon), followed by
`the addition of 5 pl of 80 mM MgCl: 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 Jul of incubation buffer
`containing 0.1 ITIM substrate {Il-Glycine-Proline-AMCT;
`
`8
`AMC is 7-arnino-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 rim; sensitivity setting
`4). Test com pounds are typically added as 2 ,nl additions and
`the assay buffer volume is reduced to 13 gtl. A fluorescence-
`concentration curve of free AMC is generated using 0-50
`,ttM solutions of /\M(T in assay buffer. The curve generated
`is linear and is used for interpolation of substrate consump-
`tion (catalytic activity in nmoles substrate cleavedfmin}. As
`with the previous assay, the potency of the test compounds
`as DPP—lV inhibitors, expressed as [C50, is calculated from
`8-point, dose-response curves using a 4 parameter logistic
`function.
`
`The following lC50S were obtained:
`
`Compound
`Ex. 1
`lix. 3
`Ex. 3
`EX. 4
`
`hurnan plasma DPl"—W [uh-I)
`6.3
`5.7
`13.9
`21.0
`
`rat plasma DP[’—[\r' (ulvl)
`10.5
`3.3
`3.6
`7.]
`
`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 DPI’-IV inhibition. For example,
`the
`compounds of formula I, and their corresponding pharma-
`ceutically acceptable acid addition salts, improve early insu-
`lin 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-
`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 ].r.rI'I'I0l;'l(g of the test
`compounds in CMC. An oral glucose bolus of 1 gfkg was
`administered 30 minutes after the test compound directly
`into the stomach of the test animals. Blood samples,
`obtained at various timcpoints from chronic jugular vein
`catheters were analyzed for plasma glucose and immuno1‘e-
`active insulin (IRI) concentrations, and plasma DPP-[V
`activity. Plasma insulin levels were assayed by a double
`antibody radioinmmunoassay (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 _itU;’ml
`with intra- and inter-assay variations of less than 5%. Data
`are expressed as ‘Z:
`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 precise dosage of the compounds of formula 1, 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 phan'naceuti-
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`6,107,317
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`9
`cally acceptable acid addition salt, is administered enterally,
`e.g., orally, or parenterally, e.g., intravenously, preferably
`orally, at a daily dosage of 010-100, preferably l—75 mgfkg
`body weight or, for most larger primates, a daily dosage of
`5—'r',(l0[l, preferably 25—5,0[)0, more preferably 5{)—2,5U0
`mg. Atypical oral dosage unit is 0.5—l0 mgfkg, 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 elfects 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
`is effective for treating conditions mediated by DPP—IV
`inhibition, such compositions in unit dosage form and such
`compositions comprising a pharmaceutically acceptable car-
`I’16]’.
`
`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-[( pyridin -2- yl)amino]ethylamino]
`acetylthia‘/iolidine dihydrochloride
`
`a) Preparation of I-chloroacetylthiazolidine
`Asolution of 2.5 g (22.0 mmol) of chloroacetylchloride in
`25 ml of tetrahydrofuran is added, dropwise over a period of
`20 tninutes, into an ice—water cold solution containing 2.0 g
`(22.0 mmol) of thiazotidine, 12.4 g (90.0 mmol) of potas-
`sium carbonate and 50 ml of tetrahydrofuran. The reaction
`mixture is then stirred at ice-water temperature under a
`calcium sulfate drying tube for 2 hours. The potassium salts
`are then removed via filtration to yield the desired com-
`pound in a clear, colorless solution.
`b) Preparation of the title compound in free base form
`In a 500 ml flask is dissolved 9.2 g (67.0 mmol) of
`2-[(pyridin-2-yl)amino]ethylar11ino in 50 ml of tetrahydro-
`furan and the mixture is cooled in an ice bath. To the cooled
`mixture is added, dropwise over 30 minutes, the solution
`prepared in a) above containing 3.7 g (22.0 mmol) of
`1-chloroacetylthiaviolidine ((based on the yield had the reac-
`tion in a) above been completed). The resultant mixture is
`then stirred at ice-water temperature for 2 hours under a
`calcium sulfate drying tube and then allowed to stir at room
`temperature for 18 hours. The solvent is then removed by
`rotovaping and the resulting oily paste is partitioned
`between methylene chloride and water. The product is then
`extracted into the methylene chloride layer and the aqueous
`layer is then washed twice with methylene chloride. The
`combined organic layers are then washed successively with
`water and brine, dried over sodium sulfate, and concentrated
`to obtain the desired compound in cr1.tde form as an orange
`
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`oil. The crude f0]'l1'I is then purified on silica gel employing
`a mixture of 5% methanol in methylene chloride as the
`eluent to yield the free base of the title compound as an
`olI—white solid.
`
`c) Preparation of the title compound
`After dissolving the free base compound prepared in b)
`above in 100 ml of dry tetrahydrofuran, hydrogen chloride
`gas is bubbled into the solution for approximately 10 sec-
`onds. The 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 a
`white solid, m.p. 214°—2l6° C.
`
`EXAMPLE 2
`
`l-[(1-hydroxymethylcyclopent-l-yl)amino]
`acetylthiazotidine hydrochloride
`
`a} Preparation of the title compound in free base form
`In a 500 ml flask is dissolved 4.26 g (3? mmol) of
`(1—hydroxymethyl}cyclopentylamine in 44 ml of tetrahydro-
`furan and the mixture is cooled in an ice bath. To the cooled
`mixture is added, dropwise, a solution containing 3.63 g (22
`mmol of the compound of Example la) in 75 ml of tetrahy-
`drofuran. The resultant mixture is allowed to warm to room
`
`temperature and then stirred at room temperature for 18
`hours. The solvent is then removed by rotovaping and the
`resultant product is partitioned between methylene chloride
`and water. The product is then extracted into the methylene
`chloride layer and the aqueous layer is then washed twice
`with methylene chloride. The combined organic layers are
`then washed successively with water and brine, dried over
`StlCliLll'|'I sulfate, and concentrated to obtain the desired com-
`pound in cr1.tde form. The crude form is then purified on
`silica gel employing initially a mixture of 2% methanol in
`methylene chloride and then a mixture of 5% methanol in
`methylene chloride as the eluent to yield the free base of the
`title Compound as a very light yellow liquid.
`b) Preparation of the title compound
`Following essentially the procedure of Lixample 1c), and
`using in place of the free base therein, the free base obtained
`in a) above, the title compound is obtained as a white fiulfy
`Solid, m.p. 72°—75° C. (with foaming).
`
`EXAMPLE 3
`
`l -[2-[( 5-cya nop yridin-2-yl)amino]eth ylamino]
`acetylthia’/iolidirte dihydrochloride
`
`a} Preparation of 1-chloroacetylthiamlidine
`A solution of {L95 g (8 mmol} of chloroacetylchloride in
`16.8 ml of dimethylforrnamide is added, dropwise, into an
`ice—water cold solution containing 0.7.0 g (8.4 mmol) of
`thiazolidine, 4.65 g (33 mmol) of potassium carbonate, and
`16.8 ml of dimethylforrnaide. The reaction mixture is then
`stirred at
`ice water temperature under a calcium sulfate
`drying tube for 2.5 hours. The potassium salts are then
`removed via filtration to yield the desired compound in a
`clear, colorless solution.
`b) Preparation of the title compound in free base form
`In a 500 ml flask is dissolved 4.089 g (25 mmol) of
`2-[(5-cyanopyridin-2-yl)amino]ethylamine in 16.8 ml of
`dimethytforniamide and the mixture is cooled in an ice bath.
`To the cooled mixture is added, dropwise,
`the solution
`prepared in a} above containing 1.38 g (8.4 mmol) of
`1-chloroacetylthiaxolidine {(based on the yield had the reac-
`tion in a) above been completed). The resultant mixture is
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`6,107,317
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`allowed to wanu to room temperature and then stirred at
`room temperature for 18 hours. The solvent is then removed
`by rotovaping and the resultant product
`is partitioned
`between methylene chloride and water. The product is then
`extracted into the methylene chloride layer and the aqueous
`layer is then washed twice with methylene chloride. The
`combined organic layers are then washed successively with
`water and brine, dried over sodium sulfate, and concentrated
`to obtain the desired compound in cmde form as a dark
`brown oil. The crude form is then purified on silica gel
`employing initially a mixture of 2% methanol in methylene
`chloride and then a mixture of 5% methanol in methylene
`chloride as the eluent
`to yield the free base of the title
`compound as a light brown Ilulfy solid.
`c) Preparation of the title compound
`Following essentially the procedure of Example 1c}, and
`using in place ofthe free base therein, the free base obtained
`in b) above, the title is obtained as a light brown solid, mp.
`218°—22U° C.
`
`EX/-\MP[.lj 4
`
`1—[2-[(5-chlorop yridi n-2-yl)ami no]ethylan1ino]
`acetylthiamlidine
`Frlllowing essentially the procedure of Example lb), and
`using in place of the amine therein an equivalent amount of
`2—[(5—chloropyridin—2—yl)amino]ethylai:nine, and using in
`place of the eluent therein a mixture of 2% methanol in
`methylene chloride initially and then a mixture of 4%
`methanol
`in methylene chloride as the eluent,
`the title
`compound is obtained as a dark yellow solid, m.p. 88°—90°
`C
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`iWhat is claimed is:
`l. A compound of formula I:
`
`ring
`R: is an unsubstituted phenyl ring; or a phenyl
`mono-, di— or tri—substituted by halo or (C,_3)alkoxy;
`
`each
`
`R3, independently, is an unsubstituted phenyl ring; or a
`phenyl ring monosubstituted by halo or (C,_3)alkoxy;
`and
`
`R4 is a 2-oxopy1Tolidine group or a (C'2_;,)alkoxy group; or
`a pharmaceutical