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`washed with 5% aqueousacetonitril (15 ml), and finally liberated from the cartridge by eluti-
`
`on with TFA (25 ml). The solvent was concentrated in vacuo, and the residue purified by co-
`
`lumn chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard
`
`acetonitri/TFA system. The column was heated to 65°C and the acetonitril gradient was 0-
`
`100% in 60 minutes. The title compound (2.1 mg, 16 %) was isolated, and the product was
`
`analysed by PDMS.
`
`Example 16
`Synthesis of Arg?***, Lys? (N*-(y-glutamyl(N*-hexadecanoyl))) GLP-1 (7-37)-OH.
`
`To a mixture of Arg***, Lys” GLP-1 (7-37)-OH (11.6 mg, 3.4 umol), EDPA (12.3
`
`mg, 94.9 pmol}, NMP (1.6 ml) and water (0.8 ml) was added a solution of Pal-Glu(ONSu)-
`
`OBut' (5.5 mg, 10.2 wmol) in NMP (137 yl). The reaction mixture was gently shaken for 5 min.
`
`at room temperature, and then allowed to stand for an additional 90 min. at room temperatu-
`
`re. The reaction was quenchedby the addition of a solution of glycine (5.6 mg, 74.6 pmol) in
`
`water (560 pl). A 0.5 % aqueous solution of ammonium acetate (34 ml) was added, and the
`
`resulting mixture eluted onto a Varian 5g C8 Mega Bond Elut®, the immobilised compound
`
`washed with 5% aqueous acetonitril (15 ml), andfinally liberated from the cartridge byeluti-
`
`on with TFA (25 ml). The solvent was concentrated in vacuo, and the residue purified by co-
`
`lumn chromatography using a cyanopropy!l column (Zorbax 300SB-CN) and a standard
`
`acetonitril/TFA system. The column was heated to 65°C and the acetonitril gradient was 0-
`
`100% in 60 minutes. The title compound (3.1 mg, 24 %) wasisolated, and the product was
`
`analysed by PDMS.
`
`Example 17
`Synthesis of Arg?***Lys'® (N°-(y-glutamyl(N*-hexadecanoyl))) GLP-1 (7-37)-OH
`To a mixture of Arg”, Lys'® GLP-1 (7-37)-OH (11.7 mg, 3.4 pmol), EDPA (12.2
`mg, 94.6 umol), NMP (1.6 ml) and water (0.8 mi) was added a solution of Pal-Glu(ONSu)-
`OBu! (5.5 mg, 10.2 wmol) in NMP (137 pl). The reaction mixture was gently shaken for 5 min.
`at room temperature, and then allowed to stand for an additional 90 min. at room temperatu-
`re. The reaction was quenched bythe addition of a solution of glycine (5.6 mg, 74.6 umol) in
`
`water (560 yl). A 0.5 % aqueous solution of ammonium acetate (34 ml) was added, and the
`
`resulting mixture eluted onto a Varian 5g C8 Mega Bond Elut®, the immobilised compound
`
`washed with 5% aqueousacetonitril (25 ml), and finally liberated from the cartridge by eluti-
`
`on with TFA (25 ml). The solvent was concentrated in vacuo, and the residue purified by co-
`
`20
`
`25
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`30
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`lumn chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard
`
`acetonitri/TFA system. The column was heated to 65°C and the acetonitril gradient was 0-
`
`100% in 60 minutes. Thetitle compound (1.9 mg, 15 %) was isolated, and the product was
`
`analysed by PDMS.
`
`Example 18
`Synthesis of Arg™,Lys”® (N*-(octanoyl)) GLP-1 (7-37)-OH.
`
`To a mixture of Arg* GLP-1 (7-37)-OH (41.1 mg, 12.2 umol), EDPA (44 mg, 341
`
`pmol), NMP (5.76 m!) and water (2.88 ml) was added a solution of Cap-ONSu (8.8 mg, 36.5
`
`pmol, prepared as described in example 10,
`
`in NMP (106 tl). The reaction mixture was
`
`gently shaken for 5 min. at room temperature, and then allowed to stand for an additional
`
`115 min. at room temperature. The reaction was quenched by the addition of a solution of
`
`glycine (20 mg, 268 umol) in water (200 ul). The solvent was concentrated in vacuo, and the
`
`residue purified by column chromatography using a cyanopropy! column (Zorbax 300SB-CN)
`
`and a standard acetonitril/TFA system. The column was heated to 65°C and the acetonitril
`
`gradient was 0-100% in 60 minutes. Thetitle compound (18.8 mg, 44 %) was isolated, and
`
`10
`
`15
`
`the product was analysed by PDMS.
`
`Example 19
`
`20
`
`Synthesis of Arg**,Lys° (N*-(dodecanoyl)) GLP-1 (7-37)-OH.
`
`To a mixture of Arg™ GLP-1 (7-37)-OH (41.1 mg, 12.2 umol), EDPA (44 mg, 341
`
`pmol), NMP (5.76 ml) and water (2.88 ml) was added a solution of Lau-ONSu (8.8 mg, 36.5
`
`pmol, prepared in a similar manner as described for Cap-ONSu in example 10), in NMP (271
`
`pl). The reaction mixture was gently shaken for 5 min. at room temperature, and then al-
`
`25
`
`lowed to stand for an additional 100 min. at room temperature. The reaction was quenched
`
`by the addition of a solution of glycine (20.1 mg, 268 pmol) in water
`
`(200 pl). The solvent
`
`was concentrated in vacuo, and the residue purified by column chromatography using a
`
`cyanopropyl column (Zorbax 300SB-CN) and a standard acetonitril/TFA system. The column
`
`was heated to 65°C and the acetonitril gradient was 0-100% in 60 minutes. Thetitle com-
`
`30
`
`pound (18 mg, 42 %) wasisolated, and the product was analysed by PDMS.
`
`Example 20
`
`Synthesis of Pal-GABA-ONSu.
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`A mixture of Pal-ONSu (3 g, 8.48 mmol), y-aminobutyric acid (0.87 g, 8.48 mmol) in
`
`DMF (200 ml) wasstirred at room temperature for 60 h. The reaction mixture wasfiltered
`
`and thefiltrate was added drop wise to 10% aqueouscitric acid (500 ml). The precipitated N-
`
`acylated intermediate wascollected and dried in vacuo. To a suspension of the dried inter-
`
`mediate in DMF (35 ml) was added a solution of DCC (1.45 g, 7.0 mmol) in dichloromethane
`
`(20 ml). The resulting mixture wasstirred at room temperature for 20h, and thenfiltered.
`
`The solvent was removedin vacuo to give a solid residue. The residue was recrystallised
`
`from a mixture of n-heptane (50 ml) and 2-propanol (2.5 ml) to give the title compound (2.5
`
`g, 75 %).
`
`Example 21
`Synthesis of Arg™,Lys” (N*-(y-aminobutyroyl(N'-hexadecanoyl))) GLP-1 (7-37)-OH.
`
`To a mixture of Arg™, Lys*° GLP-1 (7-37)-OH (41.1 mg, 12.2 umol), EDPA (44 mg,
`
`341 umol), NMP (5.76 mi) and water (2.88 ml) was added a solution of Pal-GABA-ONSu (16
`
`mg, 36.5 pmol, prepared as described in example 20) in NMP (400 ul). The reaction mixture
`
`was gently shaken for 5 min. at room temperature, and then allowed to stand for an additio-
`
`nal 100 min. at room temperature. The reaction was quenched by the addition of a solution
`
`of glycine (20 mg, 268 pmol) in water (200 ul). The solvent was concentrated in vacuo, and
`
`the residue purified by column chromatography using a cyanopropyl column (Zorbax 300SB-
`
`10
`
`16
`
`20
`
`CN) and a standard acetonitri/TFA system. The column was heated to 65°C and the aceto-
`
`nitril gradient was 0-100% in 60 minutes. Thetitle compound (15.8 mg, 35 %) wasisolated,
`
`and the product was analysed by PDMS.
`
`Example 22
`
`25
`
`Synthesis of N*-hexadecanoyl-D-glutamic acid a-t-butyl ester-y-2,5-dioxopyrrolidin-1-yl ester.
`
`A mixture of Pal-ONSu (6.64 g, 18.8 mmol), D-glutamic acid a-tert-buty! ester (4.5
`
`g, 18.8 mmol) and EDPA (4.85 g, 37.5 mmol) in DMF (538 ml) wasstirred at room tempe-
`
`rature for 60 h. The solvent was removed and the residue dissolved in ethyl acetate (175
`
`ml). The resulting solution was extracted with 10% aqueouscitric acid (2x125 ml), and the
`
`30
`
`organic phase concentrated in vacuo. The residue was dissolved in DMF (60 ml), and the
`
`resulting mixture slowly added to 10% aqueouscitric acid (500 ml). The precipitated com-
`
`pound wascollected and dried in vacuo, to give the crude N-acylated glutamic acid interme-
`
`diate. The crude intermediate was dissolved in DMF (35 ml), and a solution of DCC (3.5 g,
`
`17 mmol) in dichloromethane (70 ml) was added. The resulting mixture was stirred at room
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`temperature for 20 h, and thenfiltered. Thefiltrate was concentrated in vacuo, and the solid
`
`residue recrystallised from a mixture of n-heptane (75 ml) and 2-propanol (5 ml), to give the
`
`title compound (5.2 g, 50 %)
`
`Example 23
`Synthesis of Arg™,Lys”® (N*-(y-D-glutamyl(N°-hexadecanoyl))) GLP-1 (7-37)-OH.
`
`To a mixture of Arg™, Lys*° GLP-1 (7-37)-OH (41.1 mg, 12.2 umol), EDPA (44 mg,
`
`341 mol), NMP (5.76 ml) and water (2.88 ml) was added a solution of N*-hexadecanoy!-D-
`
`glutamic acid a-t-butyl ester-y-2,5-dioxopyrrolidin-1-yl ester (19.7 mg, 36.5 pmol) in NMP
`
`(491 ul). The reaction mixture was gently shaken for 5 min. at room temperature, and then
`
`allowed to stand for an additional 95 min. at room temperature. The reaction was quenched
`
`by the addition of a solution of glycine (20 mg, 268 mol) in water (2 ml). A 0.5 % aqueous
`
`solution of ammonium acetate (120 ml) was added, and the resulting mixture divided into to
`
`equal portions, and each portion eluted onto a Varian 5g C8 Mega Bond Elut®, the immobi-
`
`15
`
`lised compound washed with 5% aqueous acetonitril (25 ml), and finally liberated from the
`
`cartridge by elution with TFA (25 ml). The combined eluates were concentrated in vacuo,
`
`and the residue purified by column chromatography using a cyanopropyl column (Zorbax
`
`300SB-CN) and a standard acetonitril/TFA system. The column was heated to 65°C and the
`
`acetonitril gradient was 0-100% in 60 minutes. Thetitle compound (10.5 mg, 23 %) wasiso-
`
`20
`
`lated, and the product was analysed by PDMS.
`
`Example 24
`Synthesis of Lys* (N*-(y-glutamyl(N*-tetradecanoyl))) GLP-1 (7-37).
`
`To a mixture of GLP-1 (7-37)-OH (33.6 mg, 8.9 umol), EDPA (32.4 mg, 250 pmol), NMP (2.1
`
`25
`
`ml) and water (2.1 ml) was added a solution of Myr-Glu(ONSu)-OBu! (9.1 mg, 17.9 mol),
`
`prepared as described in PCT application no. PCT/DK97/00340, in NMP (228 il). The reac-
`
`tion mixture was gently shaken for 5 min., and then allowed to stand for an additional 80 min.
`
`at room temperature. The reaction was quenched by the addition of a solution of glycine
`
`(14.8 mg, 197 pmol) in water (1.47 ml). A 0.5% aqueous solution of ammonium acetate (100
`
`30
`
`ml) was added, and the resulting mixture divided into two equal portions, and each portion
`
`eluted onto a Varian 5g C8 Mega Bond Elut®, the immobilised compound washed with 5%
`
`aqueous acetonitril (2x25 ml), and finally liberated from the cartridge by elution with TFA
`
`(2x25 ml). The combined eluates were concentrated in vacuo, and the residue purified by
`
`column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a standard
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`acetonitril/TFA system. The column was heated to 65°C and the acetonitril gradient was 0-
`
`100% in 60 minutes. Thetitle compound (0.19 mg, 0.6%) was isolated, and the product was
`
`analysed by PDMS. The m/z value for the protonated molecular ion was found to be 3693 +
`
`3. The resulting molecular weight is thus 3692 + 3 amu (theoretical value 3695 amu).
`
`Example 25
`Synthesis of Arg”*** Lys®(N‘-(y-glutamyl(N*-hexadecanoyl))) GLP-1 (7-37).
`
`To a mixture of Arg?®**, Lys®-GLP-1 (7-37)-OH (10.3 mg, 3 pmol), EDPA (10.8 mg, 83 pmol),
`
`NMP (1.44 ml) and water (0.72 ml) was addeda solution of Pal-Glu(ONSu)-OBu!'(4.8 mg,
`
`8.9 nmol), prepared as described in PCT application no. PCT/DK97/00340, in NMP (120 ul).
`
`The reaction mixture was gently shaken for 5 min., and then allowed to stand for an additio-
`
`nal 70 min. at room temperature. The reaction was quenched by the addition of a solution of
`
`glycine (4.9 mg, 65.3 umol) in water (490 jl). A 0.5% aqueous solution of ammonium ace-
`
`tate (30 ml) was added, and the resulting mixture eluted onto a Varian 5g C8 Mega Bond
`
`15
`
`Elut®, the immobilised compound washed with 5% aqueous acetonitril (25 mi), and finally
`
`liberated from the cartridge by elution with TFA (25 ml). The eluate was concentrated in
`
`vacuo, and the residue purified by column chromatography using a cyanopropy! column
`
`(Zorbax 300SB-CN)and a standard acetonitril/TFA system. The column was heated to 65°C
`
`and the acetonitril gradient was 0-100% in 60 minutes. Thetitle compound (3.2 mg, 28%)
`
`20
`
`wasisolated, and the product was analysed by PDMS. The m/z value for the protonated
`
`molecular ion was found to be 3836 + 3. The resulting molecular weight is thus 3835 + 3
`
`AMU (theoretical value 3836 AMU).
`
`Example 26
`
`25
`
`Synthesis of Lau-Glu(ONSu)-OBu'.
`
`To a solution of H-Glu-OBu' (3 g, 15 mmol) in DMF (344 ml) was added EDPA(2.58 ml, 15
`
`mmol) and a solution of Lau-ONSu (4.5 g, 15 mmol), prepared in a similar manner as de-
`
`scribed for Cap-ONSu in example 10, in DMF (74 ml). The resulting mixture wasstirred at
`
`ambient temperature for 18 h, and the solvent removed in vacuo. The oily residue was parti-
`
`30
`
`tioned between ethyl acetate (150 ml) and 5% aqueouscitric acid (250 ml). The organic
`
`phase wasconcentrated in vacuo. The residue was dissolved in DMF (40 ml) and the soluti-
`
`on added drop by drop to a 10% aqueouscitric acid solution (350 ml). The precipitated pro-
`
`duct wascollected, washed with water and dried in vacuo for 18 h to give the intermediate
`
`free acid. To solution of the free acid intermediate in DMF (25 ml) was added N-
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`hydroxysuccinimide (1.7 g, 14.8 mmol) and a solution of N-(3-dimethylaminopropyl)-N'-
`
`ethylcarbodiimide (2.58 g, 13.5 mmol) in dichloromethane (52 ml). The resulting mixture was
`
`stirred at room temperature for 18 h, and the solvents removedin vacuo. The oily residue
`
`was partitioned between dichloromethane (80 ml) and water (80 ml). The organic phase was
`
`washedwith 5% aqueouscitric acid, dried (MgSO,), and concentrated in vacuoto a solid.
`
`The solid residue wascrystallised from a mixture of n-heptane (77 ml) and 2-propanol (50
`
`ml), andfinally recrystallised from n-heptane (76 ml) to give the title compound (2.96 g,
`
`46%).
`
`10
`
`15
`
`Example 27
`Synthesis of Arg™,Lys?°(N°-(y-glutamyl(N*-dodecanoyl))) GLP-1 (7-37).
`
`To a mixture of Arg**-GLP-1 (7-37)-OH (20.6 mg, 6.1 umol), EDPA (22 mg, 171 umol), NMP
`
`(2.88 ml) and water (1.44 ml) was added a solution Lau-Glu(ONSu)-OBu' (10.2 mg, 21.2
`
`pmol), prepared as described in example 26, in NMP (255 pl). The reaction mixture was
`
`gently shaken for 5 min., and then allowed to stand for an additional 75 min. at room tempe-
`
`rature. The reaction was quenchedbythe addition of a solution of glycine (10 mg, 134 wmol)
`
`in water (100 ul). A 0.5% aqueous solution of ammonium acetate (61 ml) was added, and
`
`the resulting mixture eluted onto a Varian 5g C8 Mega BondElut®, the immobilised com-
`
`pound washed with 5% aqueous acetonitril (25 ml), and finally liberated from the cartridge by
`
`20
`
`elution with TFA (25 ml). The eluate was concentrated in vacuo, and the residue purified by
`
`column chromatography using a cyanopropyl! column (Zorbax 300SB-CN) and a standard
`
`acetonitril/TFA system. The column was heated to 65°C and the acetonitril gradient was 0-
`
`100% in 60 minutes. Thetitle compound (8.2 mg, 36%) wasisolated, and the product was
`
`analysed by PDMS. The m/z value for the protonated molecular ion was found to be 3693 +
`
`25
`
`3. The resulting molecular weight is 3692 + 3 AMU (theoretical value 3693 AMU).
`
`Example 28
`
`Synthesis of Lau-B-Ala-ONSu.
`
`To a solution of Lau-ONSu (4.25 g, 14.3 mmol), prepared in a similar manner to in DMF
`
`30
`
`(400 ml) was added EDPA(1.84 g, 14.3 mmol) and B-alanine (1.27 g, 14.3 mmol). The re-
`
`sulting mixture wasstirred at ambient temperature for 18 h. Water (250 ml) and DMF (50 mi)
`
`were added andthe solution stirred for 1 h at room temperature. The solvents were removed
`
`in vacuoto give a solid. The solid residue was dissolved in DMF (50 ml) and the solution ad-
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`ded drop by drop to a 5% aqueoussolution ofcitric acid (200 ml). The precipitate collected,
`
`washed with water (50 ml) and dried in vacuoto give the title compound (3.6 g, 93%).
`
`Example 29
`
`Synthesis of Pal-B-Ala-ONSu.
`
`To a solution of Pal-ONSu (4.25 g, 14.3 mmol) in DMF (400 ml) was added EDPA (1.84 g,
`
`14.3 mmol) and B-alanine (1.27 g, 14.3 mmol). The resulting mixture wasstirred at ambient
`
`temperature for 18 h. Water (250 ml) and DMF (50 ml) were addedandthesolution stirred
`
`for 1 h at room temperature. The solvents were removed in vacuo to give a solid. The solid
`
`10
`
`residue was dissolved in DMF (50 ml) and the solution added drop by drop to a 5% aqueous
`
`solution of citric acid (200 ml). The precipitate collected, washed with water (50 ml) and dried
`
`in vacuoto give the title compound (3.6 g, 93%).
`
`Example 30
`
`15
`
`Synthesis of Myr-GABA-ONSu.
`
`To a solution of Myr-ONSu (4 g, 12.3 mmol) in DMF (350 ml) was added EDPA(1.58 g, 12.3
`
`mmol) and y-aminobutyric acid (1.26 g, 12.3 mmol). The resulting mixture was stirred at am-
`
`bient temperature for 18 h. Water (60 ml) was added andthe solution stirred for 1h at room
`
`temperature. The solvents were removed jn vacuo to give a solid. The solid residue wasdis-
`
`solved in DMF (75 ml) and the solution added drop by drop to a 5% aqueoussolution ofcitric
`
`acid (250 ml). The precipitate collected, washed with water (100 ml) and dried in vacuo to
`
`give the free acid intermediate (3.65 g, 95%). To a solution of the free acid intermediate (3 g,
`
`9.6 mmol), N-hydroxysuccinimide (1.65 g, 14.4 mmol) and N-(3-dimethylaminopropyl)-N'-
`
`ethylcarbodiimide hydrochloride (3.67 g, 19.1 mmol) in DMF (330 ml) wasstirred for 18 h at
`
`room temperature, and the solvent removed in vacuoto give a solid. The solid residue was
`
`dissolved in dichloromethane (100 ml) and washedwith brine (100 ml). The organic phase
`
`wasdried (MgSO,) and concentrated in vacuo to give a solid. The solid residue was recry-
`
`stallised from n-heptane (75 ml) to give thetitle compound (2.8 g, 71%).
`
`20
`
`25
`
`30
`
`Example 31
`
`Synthesis of Pal-B-Ala-ONSu.
`
`To a solution of Pal-ONSu (0.9 g, 2.8 mmol) in DMF (100 ml) were added N-
`
`hydroxysuccinimide (0.35 g, 3 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
`
`(0.79 g, 4.1 mmol). The resulting mixture was stirred at ambient temperature for 40h, and
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`the solvent removedin vacuo. The solid residue was partitioned between water (50 ml) and
`
`dichioromethane (50 ml). The organic phase was separated, dried (MgSO,) and the solvent
`
`removed in vacuoto give thetitle compound (1.1 g, 94%).
`
`Example 32
`
`Synthesis of Arg*Lys?*(N°-(B-alanyl(N*-hexadecanoy!))) GLP-1 (7-37).
`
`To a mixture of Arg*-GLP-1 (7-37)-OH (19.2 mg, 5.7 umol), EDPA (20.5 mg, 159 umol),
`
`NMP (2.7 ml) and water (1.35 ml) was added a solution Pal-B-Ala-ONSu (7.2 mg, 17 pmol),
`
`prepared as described in example 31, in NMP (181 pl). The reaction mixture was gently sha-
`
`10
`
`ken for 5 min., and then allowed to stand for an additional 90 min. at room temperature. The
`
`reaction was quenched by the addition of a solution of glycine (9.3 mg, 125 ymol) in water
`
`(93 ul). The reaction mixture was purified by column chromatography using a cyanopropy!
`
`column (Zorbax 300SB-CN) and a standard acetonitril/TFA system. The column was heated
`
`to 65°C and the acetonitrii gradient was 0-100% in 60 minutes. Thetitle compound (11.6 mg,
`
`15
`
`55%) wasisolated, and the product was analysed by PDMS. The m/z value for the protona-
`
`ted molecular ion was found to be 3694 + 3. The resulting molecular weight is thus 3693 + 3
`
`AMU (theoretical value 3693 AMU).
`
`Example 33
`
`20
`
`Synthesis of Pal-Glu(OBu')-ONSu.
`
`To a solution of H-Glu(OH)-OBu!(2.7 g, 11.3 mmol) and Pal-ONSu (3.98 g, 11.3 mmol)in
`
`DMF (300 ml) was added EDPA(3.2 g, 24.8 mmol). The resulting mixture wasstirred at am-
`
`bient temperature for 18h, and the solvent concentrated in vacuo to give an oil. The oily resi-
`
`due wasdissolved in DMF (60 ml) and the solution added drop by drop to a 10% aqueous
`
`25
`
`solution of citric acid (300 ml) whereby a precipitate was formed. The precipitate was collec-
`
`ted, washed with cold water (25 ml), and dried in vacuoto give free acid intermediate (4.44
`
`g, 89%). The free acid intermediate (4 g, 9.1 mmol) was dissolved in DMF (50 ml) and N-
`
`hydroxysuccinimide (1.15 g, 10 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide
`
`hydrochloride (2.6 g, 13.6 mmol) were added. The resulting mixture wasstirred at room
`
`30
`
`temperature for 60h, the solvent concentrated in vacuo to give the crudetitle compound (8.2
`
`9)
`
`Example 34
`Synthesis of Arg™,Lys”°(N°-(a.-glutamyl(N*-hexadecanoyl))) GLP-1 (7-37).
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`To a mixture of Arg**-GLP-1 (7-37)-OH (25.6 mg, 7.6 pmol), EDPA (27.4 mg, 212 umol),
`
`NMP (3.5 ml) and water (1.75 ml) was added a solution of Pal-Glu(OBu')-ONSu (12.2 mg,
`
`22.7 pmol), prepared as described in example 33, in NMP (305 ul). The reaction mixture was
`
`gently shaken for 5 min., and then allowed to stand for an additional 100 min. at room tem-
`
`perature. The reaction was quenchedby the addition of a solution of glycine (12.5 mg, 168
`
`nmol) in water (125 pl). A 0.5% aqueous solution of ammonium acetate (72.5 ml) was ad-
`
`ded, and the resulting mixture eluted onto a Varian 5g C8 Mega Bond Elut®, the immobilised
`
`compound washed with 5% aqueousacetonitril (25 ml), and finally liberated from the car-
`
`tridge by elution with TFA (30 ml). The eluate was concentrated in vacuo, and the residue
`
`10
`
`purified by column chromatography using a cyanopropyl column (Zorbax 300SB-CN) and a
`
`standard acetonitril/TFA system. The column was heated to 65°C and the acetonitril gradient
`
`was 0-100% in 60 minutes. Thetitle compound (6.1 mg, 22%) was isolated, and the product
`
`was analysed by PDMS. The m/z value for the protonated molecular ion was found to be
`
`3751 + 3. The resulting molecular weight is thus 3750 + 3 AMU (theoretical value 3751
`
`AMU).
`
`Example 35
`
`Synthesis of Ste-GABA-ONSu.
`
`To a solution of Ste-ONSu (3 g, 7.9 mmol) in DMF (270 ml) was added EDPA(1 g, 7.9
`
`20
`
`mmol) and a solution of y-aminobutyric acid (0.81 g, 7.9 mmol) in water (40 ml). The resul-
`
`ting suspension wasstirred at ambient temperature for 18 h, and then concentrated jn vacuo
`
`to a final volume of 50 ml. The resulting suspension was added to a 5% aqueoussolution of
`
`citric acid (500 ml) whereby a precipitate is formed. The precipitate was collected and
`
`washed with water (50 ml), and dried fn vacuo for 4h to give the free acid intermediate (2.8
`
`25
`
`g, 97%). To a mixture of the free acid intermediate (2.6 g, 7 mmol), N-hydroxysuccinimide
`
`(1.21 g, 10.5 mmol) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (2.69
`
`g, 14 mmol) in NMP (300 ml) wasstirred for 70 h, and the solvent removed in vacuo to give
`
`a solid. The solid residue was dissolved in dichloromethane (100 ml) and washedwith brine
`
`30
`
`(2x100 ml). The organic phase was dried (MgSO,) and concentrated in vacuoto give a solid.
`Thesolid residue was recrystallised from n-heptane (75 ml) to give the title compound (2.2 g,
`67%).
`
`Example 36
`
`Synthesis of Pal-lsonip-ONSu.
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`To a suspension of 1-hexadecanoylbenzotriazole (3 g, 8.4 mmol), prepared as described in
`
`the literature (Kreutzberger; van der Goot, Arch.Pharm., 307, 1974), in DMF (350 ml) were
`
`added EDPA(1.08 g, 8.4 mmol) and a solution of piperidine-4-carboxylic acid in water ( 20
`
`ml). The resulting suspension wasstirred at room temperature for 12d, and then concentra-
`
`ted in vacuo to an oil. The oily residue was added drop by drop to a 5% aqueoussolution of
`
`citric acid (300 ml) whereby a precipitate was formed. The precipitate was collected and
`
`washedwith water (50 ml), dried in vacuo for 2 h to give the free acid intermediate (3 g,
`
`97%). To a solution of the free acid intermediate (2.8 g, 7.6 mmol), N-hydroxysuccinimide
`
`(1.31 g, 11.4 mmol) in DMF (250 ml) was added N-(3-dimethylaminopropyi)-N'-
`
`ethylcarbodiimide hydrochloride (2.92 g, 15.2 mmol). The resulting mixture wasstirred at
`
`ambient temperature for 18h, and the solvent removed in vacuo to give anoil. Theoily resi-
`
`due wasdissolved in dichloromethane (100 ml), washed with brine (50 ml), dried (MgSO,)
`
`and concentrated in vacuoto give the crudetitle compound (4.1 g, quant.).
`
`Example 37
`Synthesis of Arg™,Lys”*(N*-(piperidinyl-4-carbonyl(N-hexadecanoyl))) GLP-1 (7-37).
`
`To a mixture of Arg™-GLP-1 (7-37)-OH (25 mg, 7.4 pmol), EDPA (26.7 mg, 207 umol), NMP
`
`(3.5 ml) and water (1.75 ml) was added a solution Pal-lsonip-ONSu (13.7 mg, 30 pmol), pre-
`
`pared as described in example 36 in NMP (343 ul). The reaction mixture was gently shaken
`
`for 5 min., and then allowed to stand for an additional 90 min. at room temperature. The re-
`
`action was quenchedbythe addition of a solution of glycine (12.2 mg, 163 pmol) in water
`
`(122 wl). The reaction mixture was purified by column chromatography using a cyanopropy!|
`
`column (Zorbax 300SB-CN)and a standard acetonitril/TFA system. The column was heated
`
`to 65°C and the acetonitril gradient was 0-100% in 60 minutes. Thetitle compound (12 mg,
`
`44%) wasisolated, and the product was analysed by PDMS. The m/z value for the protona-
`
`ted molecular ion was found to be 3734 + 3. The resulting molecular weight is thus 3733 + 3
`
`AMU (theoretical value 3733 AMU).
`
`Example 38
`
`Synthesis of Arg™,Lys?°(N*-(y-glutamyl(N*-decanoyl))) GLP-1 (7-37)
`To a mixture of Arg**-GLP-1 (7-37)-OH (25 mg, 7.4 pmol), EDPA (26.7 mg, 207 umol), NMP
`(3.5 ml) and water (1.75 ml) was added a solution of Cac-Glu(ONSu)-OBu! (10 mg, 22.1
`
`umol) in NMP (252 wl). The reaction mixture was gently shaken for 5 min., and then allowed
`
`to stand for an additional 140 min. at room temperature. The reaction was quenched by the
`
`20
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`25
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`30
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`addition of a solution of glycine (12.2 mg, 162 umol) in water (122 ul). A 0.5% aqueous solu-
`
`tion of ammonium acetate (73 ml) was added, and the resulting mixture eluted onto a Varian
`
`5g C8 Mega BondElut®, the immobilised compound washed with 5% aqueousacetonitril (25
`
`mil), andfinally liberated from the cartridge by elution with TFA (25 ml). The eluate was con-
`
`centrated in vacuo, and the residue purified by column chromatography using a cyanopropyl
`
`column (Zorbax 300SB-CN) and a standard acetonitril/TFA system. The column was heated
`
`to 65°C and the acetonitril gradient was 0-100% in 60 minutes. Thetitle compound (12.2 mg,
`
`45%) wasisolated, and the product was analysed by PDMS. The m/z value for the proto-
`
`nated molecular ion was found to be 3669.7 + 3. The resulting molecular weight is thus
`
`10
`
`3668.7 + 3 amu (theoretical value 3667 amu).
`
`BIOLOGICAL FINDINGS
`
`Protraction of GLP-1 derivatives after s.c. administration
`
`15
`
`20
`
`The protraction of a number GLP-1 derivatives of the invention was determined by
`
`monitoring the concentration thereof in plasma after sc administration to healthy pigs, using
`
`the method described below. For comparison also the concentration in plasma of GLP-1(7-
`
`37) after sc. administration was followed. The protraction of other GLP-1 derivatives of the
`
`invention can be determined in the same way.
`
`Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40 kg) were fasted
`
`from the beginning of the experiment. To each pig 0.5 nmol of test compound per kg body
`
`weight was administered in a 50 uM isotonic solution (6 mM phosphate, pH 7.4, 0.02% Twe-
`en®-20 (Merck), 45 mg/ml mannitol (pyrogen free, Novo Nordisk). Blood samples were
`
`drawn from a catheter in vena jugularis at the hours indicated in Table 1. 5 ml of the blood
`
`samples were pouredinto chilled glasses containing 175 ul of the following solution: 0.18 M
`
`25
`
`EDTA, 1500 KIE/m! aprotinin (Novo Nordisk) and 3% bacitracin (Sigma), pH 7.4. Within 30
`
`min, the samples were centrifuged for 10 min at 5-6000*g. Temperature was kept at 4°C.
`
`The supernatant waspipettedinto different glasses and kept at minus 20°C until use.
`
`The plasma concentrations of the peptides were determined by RIA using a mo-
`
`noclonal antibody specific for the N-terminal region of GLP-1(7-37). The cross reactivities
`
`30
`
`were less than 1% with GLP-1(1-37) and GLP-1(8-36)amide and < 0.1% with GLP-1(9-37),
`
`GLP-1(10-36)amide and GLP-1(11-36)amide. The entire procedure wascarried outat 4°C.
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`The assay was carried out as follows: 100 ul plasma was mixed with 271 pl 96% ethanol,
`
`mixed using a vortex mixer and centrifuged at 2600*g for 30 min. The supernatant was de-
`
`canted into Minisorp tubes and evaporated completely (Savant Speedvac AS290). The eva-
`
`poration
`
`residue was
`
`reconstituted
`
`in
`
`the
`
`assay buffer
`
`consisting
`
`of
`
`80 mM
`
`NaH,PO,/Na,HPO,, 0.1 % HSA (Orpha 20/21, Behring), 10 mM EDTA, 0.6 mM thiomersal
`
`(Sigma), pH 7.5. Samples were reconstituted in volumes suitable for their expected concen-
`
`trations, and were allowed to reconstitute for 30 min. To 300 pl sample, 100 yl antibody so-
`
`lution in dilution buffer containing 40 mM NaH,PO,/Na,HPO,, 0.1 % HSA, 0.6 mM thiomer-
`
`sal, pH 7.5, was added. A non-specific sample was prepared by mixing 300 ul buffer with
`
`100 ul dilution buffer. Individual standards were prepared from freeze dried stocks, dissolved
`
`in 300 ul assay buffer. All samples were pre-incubated in Minisorp tubes with antibody as
`
`described above for 72 h. 200 ul tracerin dilution buffer containing 6-7000 CPM was added,
`
`samples were mixed and incubated for 48 h. 1.5 ml of a suspension of 200 mi perlitre of he-
`
`parin-stabilised bovine plasma and 18 g per litre of activated carbon (Merck) in 40 mM
`
`NaH,PO,/Na,HPO,, 0.6 mM thiomersal, pH 7.5, was added to each tube. Before use, the
`
`suspension was mixed and aliowed to stand for 2 h at 4°C. All samples were incubatedfor 1
`
`h at 4°C and then centrifuged at 3400*g for 25 min. Immediately after the centrifugation, the
`
`supernatant was decanted and counted in a y-counter. The concentration in the samples
`
`wascalculated from individual standard curves.
`
`The findings show that the GLP-1 derivatives of the invention have a protracted
`
`profile of action relative to GLP-1(7-37) and are much morepersistent in plasma than GLP-
`
`1(7-37). The time at which the peak concentration in plasma is achieved varies within wide
`
`limits, depending on the particular GLP-1 derivative selected.
`
`10
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`15
`
`20
`
`25
`
`Stimulation of cAMP formation in a cell line expressing the cloned human GLP-1 re-
`
`ceptor
`
`In order to demonstrate efficacy of the GLP-1 derivatives, their ability to stimulate for-
`
`mation of cAMP in a cell line expressing the cloned human GLP-1 receptor was tested. An
`
`EC,, was calculated from the dose-response curve.
`
`30
`
`Baby hamster kidney (BHK) cells expressing the human pancreatic GLP-1 receptor
`
`were used (Knudsen and Pridal, 1996, Eur. J. Pharm. 318, 429-435). Plasma membranes we-
`
`re prepared (Adelhorst ef a/, 1994, J. Biol. Chem. 269, 6275) by homogenisation in buffer (10
`
`mmol/l Tris-HCI and 30 mmol/l NaCl pH 7.4, containing,
`
`in addition, 1 mmol/l dithiothreitol, 5
`
`mg/l leupeptin (Sigma, St. Louis, MO, USA), 5 mg/l pepstatin (Sigma, St. Louis, MO, USA),
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`100 mg/l bacitracin (Sigma, St. Louis, MO, USA), and 16 mg/| aprotinin (Novo Nordisk A/S,
`
`Bagsvaerd, Denmark)). The homogenate wascentrifuged on top of a layer of 41 w/v% sucro-
`
`se. The white band between the two layers was diluted in buffer and centrifuged. Plasma
`
`membranes were stored at -80°C until used.
`
`The assay was carried out in 96-well microtiter plates in a total volume of 140 ul. The
`
`buffer used was 50 mmol/l Tris-HCl, pH 7.4 with the addition of 1 mmol/l EGTA, 1.5 mmol/l
`
`MgSO,, 1.7 mmol/L ATP, 20 mM GTP, 2 mmol/l 3-isobutyl-1-methylxanthine, 0.01 % Tween-20
`
`and 0.1 % human serum albumin (Reinst, Behringwerke AG, Marburg, Germany). Compounds
`
`to be tested for agonist activity were dissolved and diluted in buffer, added to the membrane
`
`10
`
`preparation and the mixture was incubated for 2 h at 37°C. The reaction was stopped by the
`
`addition of 25 ul of 0.05 mol/f HCI. Samples werediluted 10 fold before analysis for cAMP by a
`
`scintillation proximity assay (RPA 538, Amersham, UK).
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`CLAIMS
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`212
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`PCT/DK99/00082
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`A derivative of GLP-1 analog of formula I:
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`#15
`
`#16
`
`«#417
`
`His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa-
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`2