Ulllted States Patent [19]
`Villhauer
`
`[54] N-(SUBSTITUTED GLYCYL)-4-
`CYANOTHIAZOLIDINES,
`PHARMACEUTICAL COMPOSITIONS
`CONTAINING THEM AND THEIR USE IN
`INHIBITING DIPEPTIDYL PEPTIDASE-IV
`
`[75] Inventor: Edwin Bernard Villhauer, MorristoWn,
`N.J.
`
`[73] Assignee: NOVaI‘tiS AG, Basel, Switzerland
`
`[ * ]
`
`Notice:
`
`This patent issued on a continued pros-
`ecution application ?led under 37 CFR
`153((1), and is subject to the twenty year
`patent term provisions of 35 U.S.C.
`154(a)(2)_
`
`[21] Appl' No‘: 09/339 503
`’
`JllIl- 24, 1999
`
`[22] Filedi
`
`7
`
`............................................
`[2;]
`/
`,
`/
`. ........................................... ..
`.
`I.
`[
`]
`[58] Fleld 0f Search ............................ .. 548/200; 514/365
`_
`References C‘ted
`Us PATENT DOCUMENTS
`
`[56]
`
`5,939,560
`
`8/1999 Jenkins .................................. .. 548/535
`
`FOREIGN PATENT DOCUMENTS
`
`555 824 8/1993 European Pat. Off. .
`1581 09 12/1982 Germany -
`296 075 11/1991 Germany'
`90/12005 10/1990 WIPO '
`93/O8259
`4/1993 WIPO _
`
`91/16339 10/1991 WIPO .
`
`95/11689
`95/13069
`
`5/1995 WIPO .
`5/1995 WIPO .
`
`US006110949A
`[11] Patent Number:
`[45] Date of Patent:
`
`6,110,949
`*Aug. 29, 2000
`
`6/1995 WIPO.
`95/15309
`95/29190 11/1995 WIPO.
`95/29691 11/1995 WIPO
`95/34538 12/1995 WIPO -
`98/19998 5/1998 WIPO.
`99/38501
`8/1999 WIPO.
`
`OTHER PUBLICATIONS
`
`Archives of Biochemistry and Biophysics, vol. 323, No. 1,
`PP~ 148—154 (1995)
`Journal of Neurochemistry, vol. 66, pp. 2105—2112 (1996).
`Bulletin of the Chemical Society of Japan, vol. 50, No. 7, pp.
`18274830 (1977).
`Bulletin of the Chemical Society of Japan, vol. 51, No. 3, pp.
`878—883 (1978).
`DerWent Abstract 95: 302548.
`DerWent Abstract 84: 177689.
`DGI‘WGHI Abstract 962 116353.
`Biochimica et Biophysica, vol. 1293, pp. 147—153.
`Bioorganic and Medicinal Chemistry Letters, vol. 6, No. 10,
`pp. 1163—1166 (1996).
`J'Med'Chem', V01‘ 39, pp. 2087_2094 (1996).
`Diabetes, V01‘ 44, pp 1126_1131(Sep',96)'
`Bioorganic and Medicinal Chemistry Letters‘ Vol' 6, N0' 22,
`pp. 2745—2748 (1996).
`Eur. J. Med. Chem., V01. 32, pp. 301—309 (1997).
`Biochemistry, vol. 38, pp. 11597—11603 (1999).
`
`Primary EXamin@r—R0b9rt Gerstl
`Attorney, Agent, or Firm—Joseph J. Borovian
`[57]
`ABSTRACT
`
`The invention discloses certain N-(substituted glycyl)-4
`cyanothiaZolidines, pharmaceutical compositions contain
`ing said compounds as an active ingredient thereof, and the
`use of said compounds In Inhibiting dipeptidyl peptIdase-IV.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`.
`
`38 Claims, N0 Drawings
`
`AstraZeneca Exhibit 2158
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 11
`
`

`
`1
`N-(SUBSTITUTED GLYCYL)-4
`CYANOTHIAZOLIDINES,
`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)-4-cyanothiaZolidines, phar
`maceutical compositions containing said compounds, and
`the use of said compounds in inhibiting dipeptidyl
`peptidase-IV.
`
`BACKGROUND OF THE INVENTION
`
`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 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 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 signi?cant 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 bene?cial effects on glucose disposal, DPP-IV inhi
`bition appears to represent an attractive approach for treating
`non-insulin-dependent 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 suffer from the above-mentioned limita
`tions of knoWn DPP-IV inhibitors.
`
`DESCRIPTION OF THE PRIOR ART
`
`W0 95/ 15309 discloses certain peptide derivatives Which
`are inhibitors of DPP-IV and, therefore, are useful in treating
`a number of DPP-IV mediated processes.
`W0 95/ 13069 discloses certain cyclic amine compounds
`Which are useful 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. 323, 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—2112
`(1996) discloses certain Fmoc-aminoacylpyrrolidine-2
`nitriles Which are useful in inhibiting prolyl oligopeptidase.
`Bulletin of the Chemical Society of Japan, Vol. 50, No. 7,
`pgs. 1827—1830 (1977) discloses the synthesis of an
`aminohexapeptide, viZ., Z-Val-Val-lmPro-Gly-Phe-Phe
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`6,110,949
`
`2
`OMe, and its related aminopeptides. In addition, the anti
`microbial properties of said compounds Were examined.
`Bulletin of the Chemical Society of Japan, 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 al. Were erroneous.
`WO 90/12005 discloses certain amino acid compounds
`Which inhibit prolylendopeptidase activity and, therefore,
`are useful in treating dementia or amnesia.
`Derivent Abstract 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.
`Derivent Abstract 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.
`Derivent Abstract 96: 116353 discloses certain 3-amino
`2-mercapto-propyl-proline compounds Which are Ras
`farnesyl-transferase inhibitors useful in treating various car
`cinomas or myeloid leukemias.
`WO 95/34538 discloses certain pyrrolidides,
`phosphonates, aZetidines, peptides and aZaprolines Which
`inhibit DPP-IV and, therefore, are useful in treating condi
`tions mediated by DPP-IV inhibition.
`WO 95/29190 discloses certain compounds characteriZed
`by a plurality of KPR-type repeat patterns carried by a
`peptide matrix enabling their multiple presentation to, and
`having an affinity for, the enZyme DPP-IV, Which com
`pounds exhibit the ability to inhibit the entry of HIV into
`cells.
`WO 91/16339 discloses certain tetrapeptide boronic acids
`Which are DPP-IV inhibitors useful in treating autoimmune
`diseases and conditions mediated by IL-2 suppression.
`WO 93/0825 9 discloses certain polypeptide boronic acids
`Which are DPP-IV inhibitors useful in treating autoimmune
`diseases and conditions mediated by IL-2 suppression.
`WO 95/11689 discloses certain tetrapeptide boronic acids
`Which are DPP-IV inhibitors useful in blocking the entry of
`HIV into cells.
`East German Patent 158109 discloses certain N-protected
`peptidyl-hydroxamic acids and nitrobenZoyloxamides
`Which are useful as, inter alia, DPP-IV inhibitors.
`WO 95/29691 discloses, inter alia, certain dipeptide pro
`line phosphonates Which are DPP-IV inhibitors useful in the
`treatment of immune system disorders.
`East 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 in?uence of side chain modi?
`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. Chem., Vol. 39, pgs. 2087—2094 (1996) discloses
`certain prolineboronic acid-containing dipeptides Which are
`inhibitors of DPP-IV.
`Diabetes, Vol. 44, pgs. 1126—1131 (September 1996) is
`directed to a study Which demonstrates that GLP-I amide is
`
`Page 2 of 11
`
`

`
`6,110,949
`
`4
`or a pharmaceutically acceptable acid addition salt thereof.
`Preferred compounds are those of formula Ia:
`
`3
`rapidly degraded When administered by subcutaneous or
`intravenous routes to diabetic and non-diabetic subjects.
`Bioorganic and Medicinal Chemistry Letters, Vol. 6, No.
`22, pgs. 2745—2748 (1996) discloses certain
`4-cyanothiaZolidides Which are inhibitors of DPP-IV.
`Eur J. Med. Chem., Vol. 32, pgs. 301—309 (1997) dis
`closes certain homologues and 3-substituted analogues of
`pyrrolidides and thiaZolidides Which inhibit DPP-IV.
`
`SUMMARY OF THE INVENTION
`
`10
`
`The present invention provides neW DPP-IV inhibitors
`Which are effective in treating conditions mediated by DPP
`IV inhibition. More particularly, the present invention
`relates to certain N-(substituted glycyl)-4
`cyanothiaZolidines Which inhibit DPP-IV. In addition, the
`present invention provides pharmaceutical compositions
`useful in inhibiting DPP-IV comprising a therapeutically
`effective amount of a certain N-(substituted glycyl)-4
`cyano-thiaZolidine. Moreover, the present invention pro
`vides a method of inhibiting DPP-IV comprising adminis
`tering to a mammal in need of such treatment a
`therapeutically effective amount of a certain N-(substituted
`glycyl)-4-cyanothiaZolidine.
`
`15
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`The essence of the instant invention is the discovery that
`certain N-(substituted glycyl)-4-cyanothiaZolidines are use
`ful in inhibiting DPP-IV. In one embodiment, the present
`invention provides compounds of formula I:
`
`3O
`
`R1‘ is an unsubstituted pyridine ring; or a pyridine ring
`mono- or di- substituted by halo, tri?uoromethyl, cyano
`or nitro; and
`R4‘ is a (C2_4)alkoXy group;
`or a pharmaceutically acceptable acid addition salt thereof.
`More preferred compounds are those of formula Ib:
`
`Wherein
`R is C1_12alkyl; a group
`
`an unsubstituted (C3_7)cycloalkyl ring; a group
`—(—CH2—)—2R2; a group
`
`R3
`
`R1 is an unsubstituted pyridine ring; a pyridine ring
`mono- or di-substituted by halo, tri?uoromethyl, cyano
`or nitro; an unsubstituted pyrimidine ring; or a pyrimi
`dine ring monosubstituted by halo, tri?uoromethyl,
`cyano or nitro;
`R2 is an unsubstituted phenyl ring; or a phenyl ring
`mono-, di- or tri-substituted by halo or (C1_3)alkoXy;
`each R3, independently, is an unsubstituted phenyl ring; or
`a phenyl ring monosubstituted by halo or (C1_3)alkoXy;
`and
`R4 is a 2-oXopyrrolidine group or a (C2_4)alkoXy group;
`
`35
`
`Where
`R“ is C1_8alkyl; a group
`
`an unsubstituted (C4_6)—cycloalkyl ring; or a group
`—(—CH2—)—3R4‘;
`R1“ is a pyridine ring mono- or di-substituted by halo,
`tri?uoromethyl, cyano or nitro; and
`R4‘ is as de?ned above;
`or a pharmaceutically acceptable acid addition salt thereof.
`Even more preferred compounds are those of formula Ic:
`
`45
`
`Ic
`
`55
`
`Where
`R‘" is C1_6alkyl; a group
`
`an unsubstituted (C4_6)—cycloalkyl ring; or a group
`—<—CHZ—)—3R4:
`R‘" is a pyridine ring monosubstitued by halo,
`tri?uoromethyl, cyano or nitro; and
`
`Page 3 of 11
`
`

`
`6,110,949
`
`5
`
`R4‘ is as de?ned above;
`or a pharmaceutically acceptable acid addition salt thereof.
`In another embodiment, the instant invention provides
`pharmaceutical compositions useful in inhibiting DPP-IV
`comprising a pharmaceutically 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 Ib 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 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 pharma
`ceutically acceptable acid addition salt thereof, more pref
`erably a compound of formula Ib above, or a pharmaceuti
`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 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 the above de?nitions, it should be noted that the “alkyl”
`and “alkoxy” signi?cances are 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, sulfuric,
`phosphoric, citric, lactic and acetic acid may also be utiliZed.
`The compounds of formula I may be prepared by the
`folloWing three-step reaction:
`
`10
`
`15
`
`25
`
`35
`
`45
`
`STEP 1
`
`0
`
`O
`
`O
`
`NH2
`
`HN
`
`i "'1
`
`+
`
`Y O
`NQL
`R/
`OH
`III
`
`DIC
`HOBt
`
`55
`
`1
`OTJL O>/NH2
`R U S
`
`imidazole
`IV POC13
`
`S
`
`V
`
`STEP 3
`
`TFA
`V —> I
`
`Where R is as de?ned above.
`
`As to the individual steps, Step 1 involves the coupling of
`an N-t-boc protected N-substituted glycine compound of
`formula III With a slight molar eXcess of the amide com
`pound of formula II employing 1,3-diisopropylcarbodiimide
`as the coupling agent and 1-hydrobenZotriaZole hydrate as
`the activator therefor to obtain a t-boc protected amide
`compound of formula IV. The coupling reaction is con
`ducted in the presence of an inert, organic solvent, prefer
`ably a cyclic ether such as tetrahydrofuran, at a temperature
`of from 10° to 35° C. for a period of betWeen 8 and 36 hours.
`
`The second step concerns the dehydration of the com
`pound prepared in Step 1, i.e., a t-boc protected amide of
`formula IV, With betWeen 2.5 and 3 equivalents of phos
`phoryl chloride to obtain a t-boc protected nitrile compound
`of formula V. The dehydration is conducted in the presence
`of a miXture of pyridine and imidaZole, at a temperature of
`from —20° to —45° C. for a period of betWeen 30 minutes and
`2.5 hours.
`
`The third step involves the deprotection of the compound
`prepared in the second step, i.e., a t-boc protected nitrile
`compound of formula V, employing tri?uoroacetic acid as
`the deprotecting agent to obtain an N-substituted glycyl-4
`cyanothiaZolidine compound of formula I. The deprotection
`is carried out in the presence of an inert, organic solvent,
`preferably a cyclic ether such as tetrahydrofuran, at a
`temperature of from 10° to 35° C. for a period of betWeen
`2 and 6 hours.
`
`The amide compound of formula II may be prepared in
`accordance With the folloWing four-step reaction scheme:
`
`STEP A
`
`O
`
`k OH
`
`HN
`
`L +
`
`S
`VI
`
`.101.
`y K
`
`alkali metal
`base
`
`Page 4 of 11
`
`

`
`6,110,949
`
`7
`-continued
`
`S
`
`VII
`
`STEP B
`
`O
`
`HO
`VII + \ N
`
`E>
`
`O
`
`O
`
`O
`N
`\_ /
`O
`i $
`0
`
`LS
`
`VIII
`
`STEP C
`
`O
`
`VIII ammonia
`
`3
`
`O
`
`NHZ
`
`i s
`O
`N/>
`
`LS
`
`IX
`
`STEP D
`
`O
`
`:‘
`-'
`
`H\
`
`NHZ
`
`1) TFA
`2) ion-exchange resin
`
`—
`
`With regard to the individual steps, Step A involves the
`amino protection of the carboxylic acid compound of for
`mula VI employing di-t-butyl dicarbonate as the activating
`agent to obtain the t-boc protected carboxylic acid com
`pound of formula VII. The reaction is conducted in the
`presence of an alkali metal base, preferably an alkali metal
`hydroxide such as sodium hydroxide, and an aqueous mix
`ture comprising a cyclic either, e.g., a mixture of Water and
`dioxane. The reaction is conducted at a temperature of from
`10° to 35° C. for a period of betWeen 1 and 4 hours.
`Step B concerns the coupling of the compound prepared
`in Step A, i.e., the t-boc protected carboxylic acid of formula
`VII, With a slight molar excess of N-hydroxysuccinimide
`employing 1,3-diisopropylcarbodiimide as the coupling
`agent to obtain a mixture of the t-boc protected anhydride
`compound of formula VIII and 1,3-diisopropylurea. The
`coupling reaction is conducted in the presence of an inert,
`
`15
`
`3O
`
`45
`
`55
`
`65
`
`8
`organic solvent, preferably a cyclic ether such as
`tetrahydrofuran, at a temperature of from 10° to 35° C. for
`a period of betWeen 1 and 4 hours.
`Step C relates to the amidation of the compound prepared
`in Step B, i.e., the t-boc protected anhydride of formula VIII,
`employing ammonia to obtain a mixture of the t-boc pro
`tected amide compound of formula IX and 1,3
`diisopropylurea. The amidation is conducted in the presence
`of an inert, organic solvent, preferably an aliphatic haloge
`nated hydrocarbon such as methylene chloride, at a tem
`perature of from 10° to 35° C. for a period of betWeen 2 and
`6 hours.
`The ?rst part of Step D involves the acidic decarboxyla
`tion of the compound prepared in Step C, i.e., the t-boc
`protected amide compound of formula IX, employing trif
`luoroacetic acid to obtain a mixture of the tri?uoroacetic
`acid salt of the desired amide compound of formula II and
`1,3-diisopropylurea. The acidic decarboxylation is con
`ducted at a temperature of from 10° to 35° C. for a period
`of betWeen 1 and 4 hours.
`The second part of Step D involves subjecting the mixture
`obtained in the ?rst part to an ion-exchange resin, preferably
`Amberlite IRA 400(OH), to obtain the amide compound of
`formula II. The ion-exchange is conducted in the presence of
`an inert, organic solvent, preferably a cyclic ether such as
`tetrahydrofuran, at a temperature of from 10° to 35° C. for
`a period of betWeen 15 and 45 minutes.
`The N-t-boc protected N-substituted glycine compounds
`of formula III may be prepared by the folloWing three-step
`reaction:
`
`0
`
`Bryk
`
`O
`
`STEP 1A
`
`inorganic base
`+ R—NH2
`XI
`
`X
`
`O
`
`H
`\
`N
`/
`R
`
`o
`
`XII
`
`STEP 2A
`
`Xffm E
`X.
`
`XIII
`
`Page 5 of 11
`
`

`
`6,110,949
`
`9
`
`STEP 3A
`Pd/C
`XIII —> III
`2-4 atm.H
`
`Where R is as de?ned above
`As regards to the individual steps, Step 1A involves the
`coupling of the compound of formula X, viZ., benZyl
`2-bromoacetate, With at least 3 equivalents of a primary
`amine compound of formula XI in the presence of an
`inorganic base, preferably an alkali metal carbonate such as
`potassium carbonate, to obtain an amine compound of
`formula XII. The coupling is conducted in the presence of an
`inert, organic solvent, preferably an aliphatic halogenated
`hydrocarbon such as methylene chloride, initially at ice
`Water temperature for a period of betWeen 30 minutes and 5
`hours, and then at a temperature of from 10° to 35° C. for
`a period of betWeen 5 and 60 hours.
`Step 2A concerns the amino protection of the compound
`prepared in Step 1A, i.e., an amine compound of formula
`XII, employing di-t-butyl dicarbonate as the activating agent
`to obtain a t-boc protected amine compound of formula XIII.
`The reaction is conducted in the presence of an inert, organic
`solvent, preferably an aliphatic halogenated hydrocarbon
`such as methylene chloride, initially at ice-Water tempera
`ture for a period of betWeen 30 minutes and 4 hours, and
`then at a temperature of from 10° to 35° C. for a period of
`betWeen 6 and 24 hours.
`Step 3A involves the deesteri?cation via hydrogenation of
`the compound prepared in Step 2a, i.e., a t-boc protected
`amine compound of formula XII, employing a hydrogena
`tion catalyst such as 10% palladium on carbon in the
`presence of betWeen 2 and 4 atmospheres of hydrogen to
`obtain an N-t-boc protected N-substituted glycine com
`pound of formula III. The hydrogenation is typically carried
`out in the presence of an inert, organic solvent typically
`utiliZed for this purpose, e.g., ethyl acetate, at a temperature
`of betWeen 20° and 30° C. for a period of betWeen 12 and
`24 hours.
`The primary amine compounds of formula XI are knoWn
`and may be prepared by procedures Well documented in the
`literature. For example: a) 2-[(5-chloropyridin-2-yl)amino]
`ethylamine can be prepared by re?uxing a mixture of
`2,5-dichloropyridine With ethylenediamine in an oil bath for
`a period of betWeen 6 and 12 hours. (b) Similarly, 2-[(5
`tri?uoromethylpyridin-2-yl)amino]ethylamine can be pre
`pared by re?uxing a mixture of 2-chloro-5-tri?uoromethyl
`pyridine With ethylenediamine in an oil bath for a period of
`betWeen 6 and 12 hours. (c) 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. (d) 2-[(pyrimidin-2-yl)amino]ethylamine can
`be prepared by adding ethylenediamine 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.
`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,
`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.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`10
`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 American Type Culture Collection (AT CC
`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, 0.15 M NaCl, 0.04 t.i.u.
`aprotinin, 0.5% nonidet-P40, pH 8.0, Which is centrifuged at
`35,000 g for 30 min. at 4° C. to remove cell debris. The assay
`is conducted by adding 20 pg solubiliZed Caco-2 protein,
`diluted to a ?nal volume of 125 pl 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 pl 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 pl
`volume of 25% glacial acetic acid is added to stop the
`reaction. Test compounds are typically added as 30 pl
`additions and the assay buffer volume is reduced to 95 pl. A
`standard curve of free p-nitroaniline is generated using
`0—500 pM 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 ICSOs Were obtained:
`
`
`
`Compound Ex. 1
`
`
`
`Caco-2 DPP-IV 0.007
`
`Ex. 2
`Ex. 3
`
`0.007
`0.006
`
`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 modi?ed 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). Brie?y, ?ve pl of plasma are added to
`96-Well ?at-bottom mictotiter plates (Falcon), folloWed by
`the addition of 5 pl of 80 mM MgCl2 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 pl 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,
`?uorescence is measured using a CytoFluor 2350 ?uorim
`eter (Excitation 380 nm Emission 460 nm; sensitivity setting
`4). Test compounds are typically added as 2 pl additions and
`
`Page 6 of 11
`
`

`
`6,110,949
`
`11
`the assay buffer volume is reduced to 13 pl. A ?uorescence
`concentration curve of free AMC is generated using 0—50
`pM 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 ICSO, is calculated from
`8-point, dose-response curves using a 4 parameter logistic
`function.
`The folloWing ICSOs Were obtained:
`
`
`
`Compound EX. 1
`
`
`
`human plasma DPP-IV 0.01
`
`
`
`rat plasma DPP-IV 0.01
`
`EX. 2
`EX. 3
`
`0.005
`0.003
`
`0.001
`0.003
`
`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 ?ndings in the literature, it is expected that the com
`pounds disclosed herein are useful in the treatment of
`non-insulin-dependent diabetes mellitus, arthritis, obesity,
`allograft rejection in transplantation and calcitonin
`osteoporosis. More speci?cally, for example, the compounds
`of formula I, and their corresponding pharmaceutically
`acceptable acid addition salts, improve early insulin
`response to an oral glucose challenge and, therefore, are
`useful in treating non-insulin-dependent diabetes mellitus.
`The ability of the compounds of formula I, and their
`corresponding pharmaceutically acceptable acid addition
`salts, to improve early insulin response to an oral glucose
`challenge may be measured in insulin resistant rats accord
`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 pmol/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 speci?c
`anti-rat insulin antibody from Linco Research (St. Louis,
`M0). The RIA has a loWer limit of detection of 0.5 pU/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 ampli?ed 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 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
`
`12
`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
`is effective for treating conditions mediated by DPP-IV
`inhibition, such compositions in unit dosage form and such
`compositions comprising a pharmaceutically acceptable car
`rier.
`The compounds of formula I (including those of each of
`the subscopes thereof and each of the examples) may be
`administered in enantiomerically pure form (e.g., ee§98%,
`preferably 299%) or together With the S enantiomer, e.g., in
`racemic form. The above dosage ranges are based on the
`compounds of formula I (excluding the amount of the S
`enantiomer).
`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
`3-[(cyclohexyl)amino]acetyl-4-cyano-(R)-thiaZolidine
`monohydrochloride
`A) Preparation of N-t-boc protected 3-[(cyclohexyl)
`amino]acetyl-4-amide-(R)-thiaZolidine
`To a solution of 1.62 g (6.31 mmol) of N-t-boc protected
`N-cyclohexyl glycine in 18.9 ml of anhydrous tetrahydro
`fuiran is added, successively, 1.0 g (7.58 ml) of (R)-(—)-4
`amide thiaZolidine, 1.28 g (9.46 mmol) of
`1-hydroxybenZotriaZole hydrate and 1.97 ml of 1,3
`diisopropylc