CA1040623A - Derivatives of retro-enantio-somatostatin, intermediates therefor and process therefor - Google Patents
Derivatives of retro-enantio-somatostatin, intermediates therefor and process thereforInfo
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- CA1040623A CA1040623A CA234,424A CA234424A CA1040623A CA 1040623 A CA1040623 A CA 1040623A CA 234424 A CA234424 A CA 234424A CA 1040623 A CA1040623 A CA 1040623A
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Abstract
DERIVATIVES OF RETRO-ENANTIO-SOMATOSTATlN.
INTERMEDIATES THEREFOR,AND PROCESS THEREFOR
Abstract of the Disclosure Compounds of the formula I or Ia (I.) (Ia.) in which R is hydrogen or CONHCH2CONHCH2CH3 are disclosed. The compounds are obtained by a process which comprises the following step: preparing peptide fragments II, III, V and VII see below, by a series of condensations involving the reaction of an appropriately protected peptide having an activated ester group with an appropriately protected peptide having a free amino group; condensing (II) by means of the azide method with (III) followed by hydrogenolysis of the reaction product to obtain (IV), condensing the latter by means of the azide method with (V) followed by treating the resulting compound with hydrazine hydrate to obtain (VI), condensing the latter by means of the azide method with HNH?HCH2STrt (VII) in which R is hydrogen or CONHCH2CONHCH2CH3 to obtain the linear protected peptide
INTERMEDIATES THEREFOR,AND PROCESS THEREFOR
Abstract of the Disclosure Compounds of the formula I or Ia (I.) (Ia.) in which R is hydrogen or CONHCH2CONHCH2CH3 are disclosed. The compounds are obtained by a process which comprises the following step: preparing peptide fragments II, III, V and VII see below, by a series of condensations involving the reaction of an appropriately protected peptide having an activated ester group with an appropriately protected peptide having a free amino group; condensing (II) by means of the azide method with (III) followed by hydrogenolysis of the reaction product to obtain (IV), condensing the latter by means of the azide method with (V) followed by treating the resulting compound with hydrazine hydrate to obtain (VI), condensing the latter by means of the azide method with HNH?HCH2STrt (VII) in which R is hydrogen or CONHCH2CONHCH2CH3 to obtain the linear protected peptide
Description
104~623 - Backqround of this Invention a. Field of Invention This invention relates to deriva~ives of the te1radecapeptide - somatostatin. More particularly, this invention concerns peptide derivatives of retro-enantio-somatostatin and salts thereof, a process for preparing the peptide derivatives and salts, intermediates used in the process and methods for using the peptide derivatives and their ~ , salts.
; b. Prior Art The name "somatos+atin" has been proposed for the factor found in hypothalamic extracts which inhibits the secretion of growth ~ ,. . .
hormone (somatotropin). The structure~of this factor has been -~
elucidated by P. Brazeau, et al., Science, 179, 77 (1973) and reported to be the following tetradecapeptide structure: -H-Ala-Gly-Cys-Lys-Asn-phe-phe-Trp-Lys-Thr-phe-Thr-ser-cys-oH.
:1 . I I '':
The abbreviations used herein for the various amino acids are Ala, alanine; Asn, asparagine; Cys, cysteine; Gly, glycine; Lys, Iysine; Phe, phenylalanine; Ser, serine; Thr, threon7ne; and Trp, tryptophane.
' The constitution of the tetradecapeptide somatostatin has been confirmed by synthesis; for example, see D. Sarantakis and W. A.
McKinley, Biochem. Biophys. Res. Comm., 54, 234 (1973), J. Rivier, et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H.U. Immer et al., Helv. Chim. Acta, 57, 730 (1974).
The important physiological activity of this tetradecapeptide established it as a compound of significance for clinical pharmacology relating to the treatment of acromegaly and the management of diabetes;
for example, see K. Lundbaek, et al., Lancet, 2, 131 (1970) and R. Guillemin in "Chemistry and Biology of Peptides" J. Meienhofer, Ed., 3rd American Peptide Symposium Boston 1972, Ann Arbor Science Publications, Ann Arbor, Mich., 1972, pp 585 - 600.
3 ~i ~
1046)6Z3 The linear form of somatostatin, having two sulfhydryl groups instead of a disulfide bridge, has been prepared recently by J.W.F. Rivier, J. Amer. Chem. Soc., 96, 2986 (1974). He reports that the linear form is equipotent to somatostatin based on the ability of 1he two compounds 1o inhibit the rate of secretion of growth hormone by rat pituitary cells in monolayer tissue cultures.
Only recently have there been reported polypeptides, other than the natural hormone and its linear form, having somatostatin-like activity. D. Sarantakis, et al., Biochem. Biophys. Res. Comm., 55, 538 t1973) reportèd~`the synthesis of the somatostatin analog, ~AIa ]-somatostatin, by solid phase methods. This analog ¦ : -exhibited a very small amount of activity~ about 0.01% of somato-statin s potency. P. Brazeau, et al., Biochem. Biophys. Res. Comm., 60, 1202 (1974) recently reported the synthesis of a number of acylated des[Ala -Gly ]-somatostatin compounds, by solid phase methods.
The prcsent invention discloses new analogs of somatostatin based on the principle of the retro-enantio system. This system is achieved by construction of a reversed sequence of amino acids havtng opposlle configuration, i.e., D Tnstead of L, to give the ¦
retro-enantio tsomer of the natural peptide. It is surprising that the retro-enantio derivatives of somatostatin of formulae I or la have been found to retain the activity of the natural hormone somatostatin notwithstanding the fact that other hormones of the retro-enantio system have shown a range of retffntil)n of full activity to complete loss-Df activity, as reported in the review by J. Rudinger, The Design of Peptide Hormone Analogs, pp 368 - 369 in I
Drug Design, Vol. II, Ed. E.J. Ariëns, Academic Press, New York and :
London, 1971.
. ' ':':
4 . ~:
, ::
. , ~,~ , .. .. . ,, . : ..... . . .
. . .
.
'' 104~623 The present inven~ion discloses retro-enantio peptlde ~ ¦
derivatives which retain the activity of the natural hormone somatostatin. The derivatives are prepared readily by a convenient process, which includes the following advantages: the process starts ., from readily available materials, avoids noxious reagents, is executed .
facilely and utilizes easily removable protecting groups.
The foregoing advantages and attributes render the peptides of this invention useful for the management of diabetes and ¦' the treatmenl of acromegaly.
SummarY of the Invention'.
,, . ~
The pept,id,es ot this invention are represented by formulae I
and la; formula I representing the cyclic peptides of this invention and formula la representing the linear reduced form S-CH CH C0-D-Ser-; 2 3 4 5 6 7 7 9 1O ~ 2 2 1, ~
'. D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHICHCH2~ !1, .' R 1:
'' ' (I) ,~ H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .:
.I D-Lys-NHlCHCH2SH
.I R
', 20 . - . (la) .
'I In-which R is hydrogen or CONHCH2ÇONHCH2CH3.
, When R is CONHCH2CONHCH2CH3,the terminal group NHCIHCH25 ~ R
1 may be written alternatively as D-Cys-Gly-NHEt, and formulae I and la 1 25 may be written as ;D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .
D-Lys-D-Cys-Gly-NHEt (I) ~
or HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-D-Cys-Gly-NHEt (la) , ~N
. I~ .
~ . ' :~
., . ~, ' ' ., . "
' ' . , ' . ' . . . ' . .
~ .' AHP-o550 104~6Z3 The pharmaceutically acceptable salts of the compounds of formulae I and la are also included within the scope of this ir,vention.
The peptides of this invention are prepared by a process comprising:
Preparing peptide fragments 11, III,IV, V, Vl and Vll, see below, by a series of condensations involving the reactlon of an appropriately protected peptide having an activated ester group with an appropriately protected peptide having a free amino group.
The tetrapeptide of formula Z-D-TIhr-D Llys-D-Trp-D-Phe-NHNH2 Bu 8 c ~, ~
tll) containing the amino!and residues 4 - 7 is condensed by means ~ -of the azide me,t"hod with a tripeptide (8 - 10) of formula ,' H-'D-Phe-D-Asn-D-Lys-OMe (111) to yield the heptapeptide (4 ~ 10) of '' ,,, - Boc formula'Z-D-TLr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lrs-OMe. The latter is . ;;
hydrogenoltzed by means of hydrogen and a noble metal catalyst to yield the heptapeptide (4 - 10) of formula H-D-TIlt-D-Lrs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV). .
Bu Boc Boc Sald last-named compound (IV? is condensed by means of the azide method with the tripeptide (I - 3) of formula 2CH2CO D Sltr D Thltr D-P~e-NHNH2 (V) to yield the decapeptide ~
~, ~ of formula n Trt-s-cH2cH2co-D-ser-D-T7r-D-phe-D-T~r-D-Lys-D-Trp-D-phe-D Phe D As ' Blt Bu Bu Boc D-Lys-NHNH2 (Vl) containing the amino acid residues I - 10. ;~
, BbC ''~, ~
Said last-named compound (Vl) is condensed by means of the ;
azide method with HNH~HCH25 Trt (Vll) in which R is hydrogen ~VIIa) or :
11' .: ---`-- _ :' , ~ . , ~...... .. .. . ..
A~IP-G550 104~1623 CONHCH2CONHCH2CH3 (Vllb) to yield the linear protected peptide of formula tVIII) '' ' r S CH2CH2C-D sTt, D T~tr-D-phe-D-Tsr-D-LJs-D-Trp-D-phe-D-phe Lys-N ~ HCH2S Trt tV111);'1n which R is as defined herein followed by B c R
oxidizing said linear protected peptide tVIII) with iodine or thio-cyanogen to obtain the corresponding cyclic disulfide of formula tlX)
; b. Prior Art The name "somatos+atin" has been proposed for the factor found in hypothalamic extracts which inhibits the secretion of growth ~ ,. . .
hormone (somatotropin). The structure~of this factor has been -~
elucidated by P. Brazeau, et al., Science, 179, 77 (1973) and reported to be the following tetradecapeptide structure: -H-Ala-Gly-Cys-Lys-Asn-phe-phe-Trp-Lys-Thr-phe-Thr-ser-cys-oH.
:1 . I I '':
The abbreviations used herein for the various amino acids are Ala, alanine; Asn, asparagine; Cys, cysteine; Gly, glycine; Lys, Iysine; Phe, phenylalanine; Ser, serine; Thr, threon7ne; and Trp, tryptophane.
' The constitution of the tetradecapeptide somatostatin has been confirmed by synthesis; for example, see D. Sarantakis and W. A.
McKinley, Biochem. Biophys. Res. Comm., 54, 234 (1973), J. Rivier, et al., Compt. Rend. Ser. D, 276, 2737 (1973) and H.U. Immer et al., Helv. Chim. Acta, 57, 730 (1974).
The important physiological activity of this tetradecapeptide established it as a compound of significance for clinical pharmacology relating to the treatment of acromegaly and the management of diabetes;
for example, see K. Lundbaek, et al., Lancet, 2, 131 (1970) and R. Guillemin in "Chemistry and Biology of Peptides" J. Meienhofer, Ed., 3rd American Peptide Symposium Boston 1972, Ann Arbor Science Publications, Ann Arbor, Mich., 1972, pp 585 - 600.
3 ~i ~
1046)6Z3 The linear form of somatostatin, having two sulfhydryl groups instead of a disulfide bridge, has been prepared recently by J.W.F. Rivier, J. Amer. Chem. Soc., 96, 2986 (1974). He reports that the linear form is equipotent to somatostatin based on the ability of 1he two compounds 1o inhibit the rate of secretion of growth hormone by rat pituitary cells in monolayer tissue cultures.
Only recently have there been reported polypeptides, other than the natural hormone and its linear form, having somatostatin-like activity. D. Sarantakis, et al., Biochem. Biophys. Res. Comm., 55, 538 t1973) reportèd~`the synthesis of the somatostatin analog, ~AIa ]-somatostatin, by solid phase methods. This analog ¦ : -exhibited a very small amount of activity~ about 0.01% of somato-statin s potency. P. Brazeau, et al., Biochem. Biophys. Res. Comm., 60, 1202 (1974) recently reported the synthesis of a number of acylated des[Ala -Gly ]-somatostatin compounds, by solid phase methods.
The prcsent invention discloses new analogs of somatostatin based on the principle of the retro-enantio system. This system is achieved by construction of a reversed sequence of amino acids havtng opposlle configuration, i.e., D Tnstead of L, to give the ¦
retro-enantio tsomer of the natural peptide. It is surprising that the retro-enantio derivatives of somatostatin of formulae I or la have been found to retain the activity of the natural hormone somatostatin notwithstanding the fact that other hormones of the retro-enantio system have shown a range of retffntil)n of full activity to complete loss-Df activity, as reported in the review by J. Rudinger, The Design of Peptide Hormone Analogs, pp 368 - 369 in I
Drug Design, Vol. II, Ed. E.J. Ariëns, Academic Press, New York and :
London, 1971.
. ' ':':
4 . ~:
, ::
. , ~,~ , .. .. . ,, . : ..... . . .
. . .
.
'' 104~623 The present inven~ion discloses retro-enantio peptlde ~ ¦
derivatives which retain the activity of the natural hormone somatostatin. The derivatives are prepared readily by a convenient process, which includes the following advantages: the process starts ., from readily available materials, avoids noxious reagents, is executed .
facilely and utilizes easily removable protecting groups.
The foregoing advantages and attributes render the peptides of this invention useful for the management of diabetes and ¦' the treatmenl of acromegaly.
SummarY of the Invention'.
,, . ~
The pept,id,es ot this invention are represented by formulae I
and la; formula I representing the cyclic peptides of this invention and formula la representing the linear reduced form S-CH CH C0-D-Ser-; 2 3 4 5 6 7 7 9 1O ~ 2 2 1, ~
'. D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHICHCH2~ !1, .' R 1:
'' ' (I) ,~ H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .:
.I D-Lys-NHlCHCH2SH
.I R
', 20 . - . (la) .
'I In-which R is hydrogen or CONHCH2ÇONHCH2CH3.
, When R is CONHCH2CONHCH2CH3,the terminal group NHCIHCH25 ~ R
1 may be written alternatively as D-Cys-Gly-NHEt, and formulae I and la 1 25 may be written as ;D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn- .
D-Lys-D-Cys-Gly-NHEt (I) ~
or HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-D-Cys-Gly-NHEt (la) , ~N
. I~ .
~ . ' :~
., . ~, ' ' ., . "
' ' . , ' . ' . . . ' . .
~ .' AHP-o550 104~6Z3 The pharmaceutically acceptable salts of the compounds of formulae I and la are also included within the scope of this ir,vention.
The peptides of this invention are prepared by a process comprising:
Preparing peptide fragments 11, III,IV, V, Vl and Vll, see below, by a series of condensations involving the reactlon of an appropriately protected peptide having an activated ester group with an appropriately protected peptide having a free amino group.
The tetrapeptide of formula Z-D-TIhr-D Llys-D-Trp-D-Phe-NHNH2 Bu 8 c ~, ~
tll) containing the amino!and residues 4 - 7 is condensed by means ~ -of the azide me,t"hod with a tripeptide (8 - 10) of formula ,' H-'D-Phe-D-Asn-D-Lys-OMe (111) to yield the heptapeptide (4 ~ 10) of '' ,,, - Boc formula'Z-D-TLr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lrs-OMe. The latter is . ;;
hydrogenoltzed by means of hydrogen and a noble metal catalyst to yield the heptapeptide (4 - 10) of formula H-D-TIlt-D-Lrs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV). .
Bu Boc Boc Sald last-named compound (IV? is condensed by means of the azide method with the tripeptide (I - 3) of formula 2CH2CO D Sltr D Thltr D-P~e-NHNH2 (V) to yield the decapeptide ~
~, ~ of formula n Trt-s-cH2cH2co-D-ser-D-T7r-D-phe-D-T~r-D-Lys-D-Trp-D-phe-D Phe D As ' Blt Bu Bu Boc D-Lys-NHNH2 (Vl) containing the amino acid residues I - 10. ;~
, BbC ''~, ~
Said last-named compound (Vl) is condensed by means of the ;
azide method with HNH~HCH25 Trt (Vll) in which R is hydrogen ~VIIa) or :
11' .: ---`-- _ :' , ~ . , ~...... .. .. . ..
A~IP-G550 104~1623 CONHCH2CONHCH2CH3 (Vllb) to yield the linear protected peptide of formula tVIII) '' ' r S CH2CH2C-D sTt, D T~tr-D-phe-D-Tsr-D-LJs-D-Trp-D-phe-D-phe Lys-N ~ HCH2S Trt tV111);'1n which R is as defined herein followed by B c R
oxidizing said linear protected peptide tVIII) with iodine or thio-cyanogen to obtain the corresponding cyclic disulfide of formula tlX)
2 H2C0 D S~t D Thl~tr-D-Phe-D-T~r-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-I I B Bu B~ Boc Lys-NH~HCH2S 7n whTch R is as defined herein and subsequently B c ,; ~
removing all remaining protecting groups under moderately acidic conditions to obtain the corresponding peptide of formula l; or followed by subjecting saTd ITnear peptTde of formula tVlll)to ; 15 treatment with eTther mercuric.acetate, mercuric chloride, silver , acetate or sl'lver n1trate to remove selectively the sulfhydryl pro-~ tecting groups to obtaTn the mercuric or dTsTIver salt, respectTvely, of the correcpondlng dlsulfhydryl derTvatlve; converttng the latter salt to its correspondTng free dTsulfhydryl derivatTve by treatment wlth`hydrogen sulfide, oxTdizTng said last-named derivative by treatment wTth oxygen, 1,2-diiodoethane, sodium or potassium ferrTcy- ' anide or TodTne to obtain the correspondTng cyclic d'isulfide der-. .
7vative, and removing the remaining protectTng groups under moderately acidic conditions to obtain the desired peptide of formula 1.
'~ 25 Alternatively, saTd cyclTc disulfTde derivatlve Ts reduced to saTd ~ correspondTng free dTsulfhydryl derivative by agents known to be :~ effective for reducTng known cyclTc disulfTdes to their corresponding dTsulfhydryl derTvatTves.
:
.
~ ~, _ AHP-6550 ~104V623 A further aspect of this invention comprises the removal of all the protec1ing groups from the aforementioned linear protected peptide of formula (Vlll) or the aforementioned disulfhydryl der- j ivatives under moderately acidic conditions to obtain the linear reduced form of the peptide of this invention of formula la, H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH~HCH~SH in which R is as defined herein.
R
! The latter compound is also obtained by direct reduction of the cyclic peptide of .formula I by agents known to be effective for reducing known cyclic disulfides to their corresponding disulfhydryl i derivatives. If désired said reduced form of the cyclic peptide y is converted to the corresponding derivative of formula I by one of ;1 the above oxidizing agents.
Details of the Invention In general the abbreviations used herein for designating the ¦;
j amino acids ana the protective groups are based on recommendations ¦ of the IUPAC-IUB Commission on Biochemicàl Nomenclature, see ¦ Btochernlstry, Il, 1726-1732 (1972). For instance, Gly, Cys~ Lys, Asn, Phe, Trp, Thr, and Ser represent the "residues"of glycine, cysteine, ~ ;
Iyslne, asparagine, phenylalanine, tryptophane, threonine and serine, respectivsly. By the residue is meant a radical derived from the ¦
corresponding D-amino acid by eliminating the OH portion of the ¦
carboxyl group and the H portion of the amino group. All the amino acids have the unnatural D-configuration.
~i A number of procedures or techniques for the preparation of l ~ I
peptid~s have hitherto been well established. For instance, the I
I functional groups which are not involved in the peptide bond ~ -; ; formation reaction are optionally protected by a protecting group or groups prior to the condensation reaction. For example, pro- -tecting groups which may be chGsen for an amino function of a '. .' ~: : : 8 - :-` , ~. ..
.,~.................................. . .
~ ' .
~ .. . :
:'~ .~ I : .~, ~ ~ ., ' , . , ' , ~'' 1~40623 :
peptide or amino acid not involved in the peptide bond formation are the alkoxycarbonyls which include benzyloxycarbonyl (represented - by Z), t-butyloxycarbonyl (represented by Boc), ~Ja-dimethyl-3~5 j dimethoxybenzyloxycarbonyl (represented by Ddz), 2-(P-biphenyl)-isopropyloxycarbonyl (represented by Bpoc), i~-chlorobenzyloxy-carbonyl, D~methoxybenzyloxycarbonyl, isopropyloxycarbonyl, or ethoxycarbonyl; the acyl type protecting groups which include ~ormyl, trifluoroacetyl, phthalyl, acetyl, or toluenesulfonyl; the alkyl type protecting groups which include triphenylmethyl or trityl (represented by Trt) or benzyl, The preferred protecting groups are benzyloxyca~bonyl, t-butyloxycarbo~yl, triphenyImethyl and dimethyl-3~5-dimethoxybenzyloxycarbonyl~ The protecting groups ~ ~ .
for the hydroxyl of serine and tyrosine are represented by acetyl, tosyl, benzoyl, tert-butyl trepresented by But), trityl, and benzyl.
The preferred protecting group is tert-butyl. The protecting group on the sulfur of cysteine or modified cysteine is illustrated by ~enzyl, triphenylmethyl or trltyl trepresented by Trt), benzyloxy-carbonyl, or acetamldomethyl trepresented by Acm), the preferred protecting groups are trltyl and acetamidomethyl. The carboxylic acid function of a peptide or amino acid can be considered protected by lower alkyl or lower aralkyl esters which include methyl (represented by OME), ethyl ~represented by OEt), or benzyl , (represented by OBzl), and also by substituted hydrazides which -include t-butyloxycarbonyl hydrazide (represented bV NHNH Boc), ~; 25 benzyloxycarbonyl hydrazide (represented by NHNH Z),or a,~-dimethyl-
removing all remaining protecting groups under moderately acidic conditions to obtain the corresponding peptide of formula l; or followed by subjecting saTd ITnear peptTde of formula tVlll)to ; 15 treatment with eTther mercuric.acetate, mercuric chloride, silver , acetate or sl'lver n1trate to remove selectively the sulfhydryl pro-~ tecting groups to obtaTn the mercuric or dTsTIver salt, respectTvely, of the correcpondlng dlsulfhydryl derTvatlve; converttng the latter salt to its correspondTng free dTsulfhydryl derivatTve by treatment wlth`hydrogen sulfide, oxTdizTng said last-named derivative by treatment wTth oxygen, 1,2-diiodoethane, sodium or potassium ferrTcy- ' anide or TodTne to obtain the correspondTng cyclic d'isulfide der-. .
7vative, and removing the remaining protectTng groups under moderately acidic conditions to obtain the desired peptide of formula 1.
'~ 25 Alternatively, saTd cyclTc disulfTde derivatlve Ts reduced to saTd ~ correspondTng free dTsulfhydryl derivative by agents known to be :~ effective for reducTng known cyclTc disulfTdes to their corresponding dTsulfhydryl derTvatTves.
:
.
~ ~, _ AHP-6550 ~104V623 A further aspect of this invention comprises the removal of all the protec1ing groups from the aforementioned linear protected peptide of formula (Vlll) or the aforementioned disulfhydryl der- j ivatives under moderately acidic conditions to obtain the linear reduced form of the peptide of this invention of formula la, H-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH~HCH~SH in which R is as defined herein.
R
! The latter compound is also obtained by direct reduction of the cyclic peptide of .formula I by agents known to be effective for reducing known cyclic disulfides to their corresponding disulfhydryl i derivatives. If désired said reduced form of the cyclic peptide y is converted to the corresponding derivative of formula I by one of ;1 the above oxidizing agents.
Details of the Invention In general the abbreviations used herein for designating the ¦;
j amino acids ana the protective groups are based on recommendations ¦ of the IUPAC-IUB Commission on Biochemicàl Nomenclature, see ¦ Btochernlstry, Il, 1726-1732 (1972). For instance, Gly, Cys~ Lys, Asn, Phe, Trp, Thr, and Ser represent the "residues"of glycine, cysteine, ~ ;
Iyslne, asparagine, phenylalanine, tryptophane, threonine and serine, respectivsly. By the residue is meant a radical derived from the ¦
corresponding D-amino acid by eliminating the OH portion of the ¦
carboxyl group and the H portion of the amino group. All the amino acids have the unnatural D-configuration.
~i A number of procedures or techniques for the preparation of l ~ I
peptid~s have hitherto been well established. For instance, the I
I functional groups which are not involved in the peptide bond ~ -; ; formation reaction are optionally protected by a protecting group or groups prior to the condensation reaction. For example, pro- -tecting groups which may be chGsen for an amino function of a '. .' ~: : : 8 - :-` , ~. ..
.,~.................................. . .
~ ' .
~ .. . :
:'~ .~ I : .~, ~ ~ ., ' , . , ' , ~'' 1~40623 :
peptide or amino acid not involved in the peptide bond formation are the alkoxycarbonyls which include benzyloxycarbonyl (represented - by Z), t-butyloxycarbonyl (represented by Boc), ~Ja-dimethyl-3~5 j dimethoxybenzyloxycarbonyl (represented by Ddz), 2-(P-biphenyl)-isopropyloxycarbonyl (represented by Bpoc), i~-chlorobenzyloxy-carbonyl, D~methoxybenzyloxycarbonyl, isopropyloxycarbonyl, or ethoxycarbonyl; the acyl type protecting groups which include ~ormyl, trifluoroacetyl, phthalyl, acetyl, or toluenesulfonyl; the alkyl type protecting groups which include triphenylmethyl or trityl (represented by Trt) or benzyl, The preferred protecting groups are benzyloxyca~bonyl, t-butyloxycarbo~yl, triphenyImethyl and dimethyl-3~5-dimethoxybenzyloxycarbonyl~ The protecting groups ~ ~ .
for the hydroxyl of serine and tyrosine are represented by acetyl, tosyl, benzoyl, tert-butyl trepresented by But), trityl, and benzyl.
The preferred protecting group is tert-butyl. The protecting group on the sulfur of cysteine or modified cysteine is illustrated by ~enzyl, triphenylmethyl or trltyl trepresented by Trt), benzyloxy-carbonyl, or acetamldomethyl trepresented by Acm), the preferred protecting groups are trltyl and acetamidomethyl. The carboxylic acid function of a peptide or amino acid can be considered protected by lower alkyl or lower aralkyl esters which include methyl (represented by OME), ethyl ~represented by OEt), or benzyl , (represented by OBzl), and also by substituted hydrazides which -include t-butyloxycarbonyl hydrazide (represented bV NHNH Boc), ~; 25 benzyloxycarbonyl hydrazide (represented by NHNH Z),or a,~-dimethyl-
3,5-dimethoxybenzyloxycarbonyl hydrazide (represented by NHNH Ddz).
'1 ' 1~ ~ 9 1 r ~ ~i .,~ .. i1~ -~1~
.. :~
104~623 -To promote facile condensation of the peptide carboxyl group with a free amino group of another peptide to form a new peptide bond, the terminal carboxyl group mus~ be activa1ed.
Descriptions of such carboxyl-activating groups are found in general textbooks of peptide chemistry; for example K.D. Kopple, "Peptides and Amino Acids", W.A. Benjamin, Inc., New York, 1966, pp. 45 - 51 and E. Schroder and K. LUbke, "The Peptides"; Vol. 1, Academic Press, New York, 1965, pp. 77 - 128. Examples of the activated form of the ; terminal carboxyl are acid chloride, anhydride, azide, activated ester, or _-acyl urea of a dialkylcarbodiimide. The following activated esters have proved to be particularly su~table in the process of this invention: 2,4,5-trichlorophenyl (represented by OTcp), ~ -pentachlorophenyl trepresented by OPcp), D-nitrophenyl (represented by ONp), or l-benzotriazolyl. The succlnim7do group is also useful for activating a carboxyl. -The term "azide method" as used herein refers to th~ method of coupling two pepttde fragmenTs wh7ch comprlses the reactlon of a pept7de hydrazide wtth a reagent which furnishes nitrous acid In ~ ;
sltu. Suitable reagents for this purpose include organic n7trites (e.g. _-butyl nitrite, isoamyl n7tr7te) or an alkal7 metal n7trite salt ~e.g. sodium nitrite, potassium nitrite) in the presence of a strong acid such as hydrogen chloride or sulfur7c or phosphoric ac7d. The ;; corresponding peptide azide thus obtained is then reacted with a peptide having a free amino group to obtain the desired peptide.
Preferred condit70ns for the az7de method of coupl7ng cornprises reacting :!: . . -.
~ , . '. ' ' , ,:
~ ' :~ . .. .
~--. . . : : ' : ,; , . : ' AHP~6~50 1~ 623 . ' . .
the peptide hydrazide with nitrous acid, generated In situ frorn an organic nitrite in the presence of a mineral acid, preferably hydrogen chloride, (pH ranging usually from 0.1 to 2), in an anhydrous inert organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichlorids, tetrahydrofuran, dioxane, and the like at -30 to 20C, preferably at about -15C, for 10 to 30 minutes to obta1n the corresponding azide. The peptide azide can be isolated and crystallized or is prèferably allowed to remain in the reaction mixture, and thereafJer reacting the azid~e in the said mixture with the peptide unit having the free amino group at temperatures rangtng ;
from -30 to 20C for about one to two hours and then at 0 to 30C for 10 to 30 hours. An acid acceptor, preferably an organic base, for ~¦
Zl example N-ethyldiisopropylamine, N-ethylmorpholine or triethyl-amine, is present in the reaction medium in order to make the reaction medium sllghtly alkaline, preferably pH 7.0 to 7.5. See also the above clted textbooks of Kopple or Schr~der and LUbke for add7tional descriptions of thls method.
The terms "peptide, polypeptide, tripeptide, hexapeptide, and the like" as used herein are not limited to refer to the respective parent peptides but also are used in reference to modified peptides ; havlng functionalized or protecting groups. The term "peptide" as ;~
iZ~ used herein is used in reference to a peptide with two to twelve amTno ac7d residues. In addition the residue "NH~HCH2S" as - R
defTned herein is written as H-D-Cys-Gly-NHEt when R is CONHCH2CONHCH2CH3, ~ and is written as a modified residue of cysteine when R 7s H, viz., 2-"'t~ ~ thioethylamine.
~: : ' ,,~,, ' :
.~ , ~: ''' ~ lZ
. .. ,~
:- . ~ .. ... . :. :
:: - ~ . : : . . .. .
:: . .
1~4~)623 The abbreviation Me represents a methyl group and NHN~12 represents a hydrazide group. In addition, the following abbreviations are used: dimethylformamide (DMF), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), methanol (MeOH), ethyl acetate (EtOAc), methylene dichlortde (CH2C12), N,N'-dicyclohexylcarbodiimide (DCC), sodium chloride (NaCI), sodium bicarbonate (NaHC03), sodium sulfate (Na2S04) magnesium sulfate (MgS04), and 5~ palladium on charcoal (5% Pd/C).
The term l'lower alkyll' as used herein contemplates hydro- ~-carbon radicals having one to three carbon atoms and includes methyl, slO ethyl and propyl.
The term 'Imineral acidll as used herein contemplates the strong inorganic acids and includes hydrochloric, hydrobromic, suJfuric, phosphoric and the like. When the term is used in con-1~ junction wtth an anhydrous system, hydrogen chloride is the preferred jl5 mlneral acid.
! The term llmiIdly acidtc conditionsll as used herein contem- ~ ' plates conditlons in which a dilute aqueous solutlon of an organic ~ i aclc, for example 30 - 80% or mixi~ures thereof, is a prlncipal çr-component of the reaction medlum. ~ -~o The term "moderately acidlc conditions" as used herein contemplates conditions in which concentrated organic acids or ~ ~ -f aqueous solutions of the mineral acids are used as a principal -~
component of the reaction medium,at temperatures ranging from ' ~ about -30 to 30C. Examples of preferred conditions in this j case include the use of 50 to 100% trifluoroacetic acid at 0 ... . . . .
~i~ to 30C or 0.1 - 12N hydrochloric acid in aqueous or anhydrous organic ' - ~ -solvents at -20 to 10C.
:1 ' .. , he term 'lorganic nitrite'l includes the commercially available alkyl nitrites~ for instance, t-butyl nitrite or isoamyl nitrite.
:
f ~' ., ' .
-.f Al-IP-6550 104~623 :' The term organic base as used herein includes triethyl-amine, N-ethylmorpholine, or N-ethyldiisopropylamine.
~ The term strong base as used herein contemplates both - organic bases, as described above, and strong inorganic bases including the hydroxldes and carbonates of sodium and potassium.
The peptldes of thls invention, includlng the cycllc and the llnear reduced forms, are obtained in the form of the free base or a~ an acid additlon salt thereof elther directly from the process of thls inventlon or by reacting the peptide with one or more equivalenTs of the appropriate acTd. Examples of preferred salts are those wlth pharmaceuttcally acceptable organic acids, e.g. acetic, -~
lactlc, succlnic, benzolc, salicyllc, methanesulfonlc or toluene- ;
sulfonlc acid, as well as polymeric acids such as tannic acld or carboxymethyl cellulose, and salts wlth inorganic acids such as -` 15 hydrohallc aclds, e.g. hydrochlorlc acid, or sulfurlc acld, or phos-, phorlc acld. It should be noted that the peptldes have two baslc ! nltrogens givlng rlse to addltlon salts wlth one to posslbly two i equlvalenTs of acid. If deslred a partlcular acid addition salt J, is converted into another acid addition salt, e.g., a salt with a ~ -non-toxic, pharmaceutically acceptable acld, by treatment with the ,~ - appropriate ion exchange resln In the manner descrlbed by R. A.
-~ ; Boissonas, et al., Helv. Chlm. Acta, 43, 134g (1960). Suitable ion exchange reslns are cellulose based cation exchangers, for example carboxymethylcellulose or chemically modlfled, cross-llnked dextran cation exchangers, for example, those of the Sephadex*C type, and strongly basic anion exchange resins, for example those listed in J.P. Greenste7n and M. Wtnitz Chemistry of the Amlno Acids , John J Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456.
t * Sephadex is a trade mark ~, 13 ., , ., .
~ .. }
~ - , 1041)623 The peptides of this invention of formulae I and la give complex sal-l-s wi-rh heavy metal ions. An example of a pharmaceutically acceptable heavy metal complex is a complex formed with zinc or with zinc protamine.
The peptides of formulae I or la, as well as their ;
corresponding pharmaceutically acceptable salts, are useful because they possess the pharmacological activity of the natural hormone somatostatin. Their activity is demonstrated ~;
readily in pharmacological tests such as a modification [A.V. Schally, et al., B~i`ochem. Biophys. Res. Commun., 52, 1314 (Ig73~; J. RivierJ et al., C.R. Acad. Sci. Paris, Ser. D, 276, ~ ~
2737 (1973)] of the n vitro method of M. Saffran and A.V. Schally, ~-Can. J. Biochem. Physiol., 33, 405 (1955).
The activity of the peptides of formulae I or la is , 15 demonstrated also In vivo 7n a modification of the pentobarbital- ;
! 7nduced increase in plasma growth hromone level in the rat as described by Brazeau, et al., cited above. In this test the peptldes of this Inventlon show a level of actlvity which is of the same order as that of somatostatin.
The peptides of formulae I or la and their salts are useful for the treatmert of acromegaly and other hypersecretory endocrine states and in the management of diabetes in mammals; see for example, -P. Brazeau, et al., cited above. When a peptide of formula t or la or a salt thereof is employed for such treatment or management, it 1~ 25 is administered systemically, preferably parenterally " n combination ¦~ - with a pharmaceutically acceptable liquid or solid carrier. The proportion of the peptide or salt thereof is determined by its ,~ solubility in the given carrier, by the given carrier, or by the `~ chosen route of administration, and by standard biological practice.
For parenteral administration to animals the peptide or a salt : - ~
thereof is used in a sterile aqueous solution which may also contain : : 14 i ... :.. , .. ... .. . ,.. , . ,.. , ,. ....... . , ,. :.. .. .. . .... .. . . ...
: . ,: . .... . . . . . . . . .. . ,. , ~ . .
Al-IP-6550 ~ .
1()40~Z3 other solutes such as buffers or preservatives, as well as sufficient pharmaceutically acceptable salts or glucose to make the solutlon isotonic. The dosage will vary with the form of administration and with the particular species of animal to be treated and Ts preferably kept at alevel of from 5 mcg to 300 mcg per kilogram -body weight. However, a dosage level in the range of from about 10 mcg to about 50 mcg per kilogram body weight is most desirably employed in order to achieve effecttve results.
The peptides or salts thereof may also be administered in one of the long acting,~slow-release or depot dosage for~s descrtbed below~ preferably by intramuscular injection or by Implantation. Such dosage forms are designed to release from about 0.5 mcg to about 50 mcg per kilogram body weight per day.
It is often desirable to administer a peptide of formula I
or ~ continuously over prolonged periods of time in long-acting, slow-release, or depot dosage forms. Such dosage forms may elther contatn a pharmaceutically acceptable salt of the peptide having ¦ a low degree of solubillty In body flulds, for example one of those salts descrlbed below, or they may contain the peptlde in the form of a w3ter-soluble salt together with a protective carrier whtch preve~ts ¦~ rapld release. In the latter case, for example, the peptide may beformulated wtth a non-antigenic par~ially hydrolyzed gelatin ~,~ in the form of a viscous liquld; or the peptide may be absorbed on~1 a pharmaceutically acceptable solid carrier, for example ztnc hydroxide, and may be administered in suspension in a pharmaceutlcally acceptable liquid vehTcle; or the peptide may be formulated in gels or I: :
suspensions with a protective non-antigenic hydrocolloid, for example ,~ sodium carboxymethylcellulose, polyvtnylpyrrolidone, sodium alg7nate, gelatine, polygalacturonic acids, for example, pectin, or certaln mucopolysaccharTdes, together with aqueous or non-aqueous pharmaceuti-cally acceptable liquid vehtcles, preservatives, or surfactants.
. .
1 :
104~623 : ~
Examples of such formulations are found in standard pharmaceutical - texts, e.g. in Remington's Pharmaceutical Sciences, 14th Ed., Mack y;~ ~ :
Publishing Co., Easton; Pennsylvania, 1970. Long-acting, slow-release preparations of the peptide of formulae I or la may also be obtained by microencapsulation in a pharmaceutically acceptable coating, for exampie gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coat7ng mater7als and of the processes used for m7croencapsulat70n are descr7bed by J.A. Herb7g 7n "Encycloped7a of Chemical Technology", Vol. 13, 2nd Ed., Wiley, New York 1967, pp 436 - 456. Such formulat70ns, as well as suspens70ns of salts ¦;
of the agent wh7,ch are only sparingly soluble in body fluidsJ for ' example salts with pamoic acid or tannic acid, are designed to j-release from about 5.0 mcg to about 100 mcg of the active compound per k7logram body weight per day, and are preferably administered by ~! : I
i 15 7ntramuscular 7nject70n. Alternat7vely, some of the solid dosage forms 17sted above, for example certa7n spar7ngly water-soluble salts or ~' dlspers70ns 7n or adsorbates on solld carr7ers of salts of the agent, . :.., ,.,, . ,-: ... .
~ for example d7spers70ns 7n a neutral hydrogel of a polymer of ethylene -¦ glycol methacrylate or s7m71ar monomers cross-17nked as descr7bed inU.S. Patent 3,551,556 may also be formulated 7n the form of pellets 1 1 releas7ng about the same amounts as shown above and may be 7mplanted subcutaneously or 7ntramuscularly. i Process i For conven;ence and clar7ty 7n the following dtscussion the indlv7dual pept7de un7t (i.e., amino acid) is designated sometlmes by a number which refers to the pos7tion in which the part7cular am7no ac7d appears 7n the sequence of the amino acids as illustrated in the formula 1.
.:~, . ... .:
, : , ., :' ' Ij,; :' I ...~
:
~:
1046)623 t:
Thef process of this invention is carrier out in the following manner. .' : With reference to the tripeptide fragment 1-3, the ff~ `
tripeptide is prepared by reacting a protected activated ester of ~ -D-threonine, preferably Z-D-T3r-OH with a lower alkyl ester of '~ ,, D-phenylalanine, preferably H-D-Phe-OMe, to obtain the corresponding ~ ;:
lower alkyl ester of the dipeptide Z~D-Thr-D-Phe-OH, which after removal B~t : .
the terminal protecting grou~ (Z) using hydrogen in the presence of ~ -a noble metal catf~kys~t yields the corresponding lower alkyl ester of :. H-D-T~r-D-Phe-OH, preferably H-D-T~r-D-Phe-OMe. In turn the latter ~ .
Blt BLt' , , ., :,- ,-,, compound is reacted with a protected activated ester of D-serine, :: -preferably the benzotriozolyl ester to obtain the corresponding lower ¦ r,~
alkyl ester of Z-D-Selr-D-T3r-D-Phe-OH, preferably Z-D-Sftr-D-T~r-D-Plle-OMe.
:,~ ' . .
j Subsequent removal of the terminal amino protecting group of the latter .
f compound usTng hydrogen in the presence of a noble met31 catalyst yields 20 the corresponding lower alkyl ester of H-D-Ser-D-Thr-D-Phe-OH, pre-~; ferably H-D-SIer-D-Thlr-D-Phe OMe. Condensation of said last-named compound with a protected activated esler of thiopropionic acid, : preferably the benzotriazolyl ester, gives the corresponding protected ;
f 25 lower :Ikyl ester of Trt-S-CH~CH2CO-D-Ser-D-T~r-D-Phe-OH, preferably : ;
Trt S H2CH2CO D Sltr D r3tr-D-Phe-OMe. The latter compound is treated ~
with hydrazine hydrate to obtain the hydrazide of the tripeptide ~ :
ragment 1 3 of formula Trt S CH2CH2CO-D-SIt-D-T~r-D-Phe-NHNH2 (V). ;
~ 7 .. ~`' ': . ~ :
``` ~.
Al-IP-6550 104~623 ~
In a preferred embodiment of the preparation of the above tripeptide fragment 1-3, a mixture of substantially equimolar amounts of Z-D-TIhr-OH, prepared from D-threonine in the same manner B t 1 5 as described for the corresponding L-isomer by E. Schr~der, Justus Liebigs Ann. Chem.,- 670, 127 (1963) and H-D-Phe-OMe HCI, prepared from D-phenylalanine in the same manner as described for the L-isomer by F. Bergel, J.M. Jchnson, and R. Wade, J. Chem. Soc., 3802 (1962), in an Inert organic solvent, preferably DMF or THF, at -20 to 10C, ¦~
preferably at 0C, is tre~ated with a molar excess, preferably with 1.1 ~i to 1.3 molar equivalents of a strong organic base, preferably N-ethyl-morpholine, to p~ 7-8. A molar excess~preferably 1.1 to 1.3 molar equivalents, of l-hydroxybenzotriazole is added followed by the dropwise aodition of a substantTally molar equivalent of DCC (1.0 to I
1.3 molar equivalents) in an inert organic solvent, preferably DMF or THF. The mixture is kept at -20 to 10C, preferably at 0C from ~ 30 rninutes to 2 hours and then at 20 to 30C for ar, additional hour, ;7 filtered, and the filtrate evaporated. The residue is taken up in a , ] substantlally water-lmmtsclble organic solven~, preferably dlethyl ~
etller, washed, drled, and evaporated. The res1due is taken up in a ¦ ;
mixture of a lower alkyl ester of a lower alkanoic acid, preferably ¦
ethyl acetate, and a hydrocarbon, preferably hexane, and is purified by chromatography on silica gel to yield the ~ipeptide of formula Z-D-T~r-D-Phe-OMe. Said last-named compound is then subjected to : -i~ 25 hydrogenation in the presence of a noble metal catalyst, preferably 5%
palladium on charcoal (5% Pd/C), and of an e~iimolar amount of pyridine ;~
hydrochloride or of an excess of acetic acid. Methanol, ethanol, acetic acid, or mixtures thereof are convenie~t solvents for this hydrogenation. In this manner the terminal a~ano protecting group (Z) , 18 I :
,~,:
... .
: -- --` . ' !
. . .` - , . . .
of the above dipeptide is removed to give the corresponding dipeptide of formula H-D-T~r-D-Phe-OMe as its acetic acid or hydrochloric acid ,~ , Blt , addition salt. Said last-named compound and a substantially equimolar amount of Z-D-S~r-OH, prepared as described for the L-isomer by , Bbt E. Schr~der, Justus Liebigs Ann. Chem., 670, 127 (1963), in an inert organtc solvent, preferably DMF or THF, at -20 to 10C, preferably at 0C, is treated with a molar excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine, to ?r pH 7 - 8. A substantially-equimolar amount of l-hydroxybenzotriazole is added followed by the addition of a substantially molar equivalent -of DCC (1.0 to 1.3 molar equivalents) in an inert organic solvent, preferably DMF or THF, and the mixture is cooled to -20 to 10C, pre-~I ferably to 0C. The mixture is kept at -20 to 10C, preferably at 0C~ from 30 mTnutes to two hours and then at 20 to 30C for an additionalj Hour, fiItered, and the fiItrate is evaporated. The residue is taken up tn a substantially water-immiscible organic solvent, preferably dlethyl ether, washed, dried, and evaporated. The resldue is taken up In a mixture of a lower alkyl ester of a lower alkanoic acid, pre-ferably ethyl acetate, and a hydrocarbon, preferably hexane, and is puriiied by chromatography on silica gel to yield Z-D-Selr-D-T~r-D-Phe-OMe.
~1~j Said last-named compound is dissolved in a lower alkanol or a lower alkanoic acid or a mixture thereof, preferably in acetic, a noble metal catalyst, preferably 5% Pd/C, is added and ~he mixture is agitated in an atmosphere of hydrogen at room temperature for 10-30 hours, preferably for about 20 hour, until substantially one molar equivalent of hydrogen :
has been taken up. FiItration of the catalyst and evaporation of the filtrate yields the tripeptide of formula H-D-Ser-D-Thr-D-Phe-OMe .,~ , ., ~9 . ':
., ' ,:':
::
` ~ .
Al iP-6550 1~623 as the acetic acid addition salt. Said last-named compound is dissolved in an inert organic solvent, preferably DMF or THF, ~-at -20~ to 10C, preferably at 0C, and 1-reated with a molar - excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine ,to pH 7 - 8. A
substantially equimolar amount of 3-tritylthiopropionic acid, pre-pared as described by E. Biliman and N.V. Due, Bull. Soc. Chim.
Fr., 35, 384 (1924), in an inert organic solvent, preferably DMF
or THF, is added followed by the addition of a substantially molar equivalent of l-hydro,xybenzotriazole. A molar excess of DCC (1.1 to 1.3~mp~ar equivalents) in an inert organic solvent, 1 prefeably DMF or THF, is added and the mixture is kept at -20 :11 to .10C, preferably at 0C from 30 minutes to 2 hours and then -I at 20 to 30C for an additional hour, fiItered, and the fiItrate eva?orated. The residue Ts taken up in a substantially water-Immisctble organic solvent, preferably diethyl ether, the pre-.
, cTpttate is removed by fiItration, washed, dried, and evarporated.
J The residue 7s taken up In a mlxture of a lower alkyl ester of a j lower alkanolc acid, preferably ethyl acetate, an aromatic hydro-¦ 20 carbon, preferably benzene, and a strong organic base, preferably ~ :
, trlethylamine, and is purified by chromatography on silica gel.
:~ Crystallization of the purified matRrial yields H2C0 D S~tr D T~tr-D-Phe-OMe. Said last-named compound is dtssolved in an inert organic solvent, for example methanol, ethanol, 1; DMF, preferably methanol, and the solutlon i5 treated with an excess `~ of hydrazine hydrate, for example with 20 to 50 molar equivalents. ;
m The reaction mixture is kept at -20 to 10C, preferably at 0C, from :
1 30 minutes to 2 hours and then at 20 to 30C for 15 to 30 hours, 1 ~ ~
3~ ::
. f ~ O
, ' .
' .~', ', - :
104~)6Z3 ; preferably for 24 hours. Water is added, the resulting precipitate is collected by fil1ration and dried to yield the tripeptide fragment 1-3 of formula Trt-S~OH2CH2CO-D-S~r-D-T~r-D-Phe-NHNH2 (V).
~ B Bu r~-~With reference to the tetrapeptide fragment 4-7, the tetrapeptide is prepared by reacting an amino protected D-tryptophane, preferably Z-D-Trp-OH, with a lower alkyl ester of D-phenylalanine, preferably H-D-Phe-OMe, to obtain an amino protected lower alkyl ester of the dipeptide H-D-Trp-D-Phe-OH, preferably Z-D-Trp-D~Phe-OMe, I which after removal of thè terminal protecting group (Z) using hyarogen in the presence,o~ a noble metal catalyst yields the corresponding ! lower alkyl ester of H-D-Trp-D-Phe-OH, preferably H-D-Trp-D-Phe-OMe.
In turn, the latter compound is reacted with an activated ester of Z-D-Lys-OH to give the corresponding lower alkyl ester of Boc Z-D-Lys-D-Trp-D-Phe-OH, preferably Z-D-Lys-D-Trp-D-Phe-CMe. Subsequent Boc Boc removal of the terminal amlno protecting group of the latter compound ¦
(Z) using hydrogen in the presence of a noble metal catalyst gtves ¦ the corresponding lower alkyl ester of H-D-Lys-D-Trp-D-Phe-OH, pre-¦~ 20 Boc ferably H-D-Lrs-D-Trp-D-Phe-OMe. Condensatlon of the last-named ompound with an activated ester of Z-D-TIr-OH yields the corresponding 1¦
~ Z5 ~ But Bo ! ~ Z-D-T~r~D-Lrs-D-Trp-D-Phe-OMe. The latter compound Is treated w7th hydrazine hydrate to obtain the tetrapeptida fragment 4-7 of formula Z~D-TIlr-D-L~s-D Trp-D-Phe NHNH2 --~ 30 ~ Bu Boc 21 ~ ¦
.':: :, .: ~ .
~ ~. . `1.
.: : ` - .,.. , .
"".. ~ , ,, ;,...... ., . : ~ : ; : . ~ , ' : . , In a preferred embodiment of the preparation of'the above tetrapeptide fragment 4-7, substantially equimolar amounts of '' Z-D-Trp-OH, prepared as described for the L-isomer hy E. Klieger, -~
E. Schr~der, and H. Gibian, Justus Liebigs' Ann. Chem., 640, 157 (1961), and ~ Phe-OMe-HCI (see F. Bergel et al, cited above) with an excess, preferably 1.5 to 2.5 molar equivalents, of l-hydroxybenzo-triazole, in an inert organic solvent, prefeably DMF, at -20 to 10C, ; preferably 0C, is treated with an excess, preferably 1.1 to 1.3 molar ; equivalents, of an organic base, preferably N-ethylmorpholine, to pH
7 - 8. A substantially equ~imolar amount of DCC in an inert organic solvent, preferab,ly~DMF, at -10 to 10C, is added dropwise. The .. .. . .
-1 mixture is kept at -20 to 10C for an additional hour, cooled to -10 .i..................................... . .
'~; to 10C, filtered, and the filtrate evaporated. The residue is taken ' up in a subslantially water-imm7scible solvent, preferably ethyl ';~-1 ! ,: .. .
acetate, washed, dried and evaporated. The residue is taken up in "' -' a mixture of a halogenated hydrocarbon solvent, preferably chloroform, -and a lower alkanol, preferably methanol. The solution is passed through a column of sil7ca gel. Evaporatton of the eluate and crystalllzation of the residue ylelds the dipeptlde fragment 6-7 of formula Z-D-Trp-D-Phe-OMe. Said last-named compound is then subjected ~' ' to hydrogenation in the presence of a noble metal catalyst, preferably ' 5~ Pd/C. Methanol, ethanol, acetic acid, or mixtures thereof are conven7ent solvents for this hydrogenation. When acetic acid is used ¦
, the product will be isolated as the acetic acid add7tion salt. ¦
~' 25 Filtration of the catalyst, and evaporation of the filtrate yields the dipeptide fragment 6-7 of formula H-D-Trp-D-Phe-OMe.
i '' :i : ~ :. -..... . ... : . , . : , .
~\HP-6~C~O
1041)623 Said last-named compound in an inert organic solv~nt, preferably DMF, at -20 to 10C, preferably 0C, is treated with an excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine, to pH 7 - 8,the mixture is then treated with a su~stantially molar equivalent of a protected activated ester of D-lysine, preferably Z-D-Lys-ONp prepared in the same manner from Boc D-lysin~ as described for the L-isomer by E. Sandrin and R. A.
Boissonnas, Helv. Chim. Acta., 46, 1637 (1963). The solution is stlrred at about 0C for 30`minutes to two hours, at 20 - 30C for two to four days, and e,vaporated. The residue is taken up in a ~ ~
substantially water-immiscible solvent, preferably ethyl acetate, ~ -washed, dried, and evaporated. The residue is taken up in a halcgenated hydrocarbon solvent, preferably chloroform, a lower alkanol, preferably methanol, and a strong organic base, pre-ferably pyr;dine. The solution is passed through silica gel. , -After evaporation of the eluate the residue is crystallized to ` ¦
yield the tripeptide fragment 5-7 of formula Z-D-L~s-D-Trp-D-Phe-OMe.
Boc Said last-named compound is then subjected to hydrogenation in the presence of a noble metal catalyst, preferably 5~ Pd/C. Methanol, ethanol, acetic acid, or mixtures thereof are convenient solvents ~1 for thts hydrogenation,when acetic acid is used the product will be ~`
isolated as the acetic acid addition salt. FiItration of the catalyst, , .
and evaporation of the fiItrate yields the tripeptide fragment 5-7 of formula H-D-Lys-D-Trp-D-phe-oMe. Said last-named compound, sub-Bocstantially equimolar amount of a protected D-threonine, preferably .
- :
: :~ 23 ~
: - .
.
: . . .` .... . . ... .
~ . .. . , . , . , . :
104~)6Z3 Z-D-TIhr-OH (see E. Schr~der cited above), and about one to two ~ -Bu molar equivalents of l-hydroxybenzotriazole in an inert organic solvent, preferably DMF, at -20 to 10C, preferably 0C, is treated with an excess, preferably 1.1 to 1.3 moiar equivalents, of a strong organic base, preferably N-ethylmorpholine,to pH 7 - 8.
A substantially equimolar amount of DCC in an inert solvent, pre-ferably DMF, at about 0C is slowly added dropwlse. The mixture is stirred at about 0C for 30 minutes to two nours, at 20 - 30C
for one to two hours, fiItered, and evaporated. The residue is .~ 1O J"" ~
taken up in a halogen3ted hydrocarbon sJolvent, preferably chloroform, and a lower alkanol, preferably methanol. The solution is passed .~ . - -:
~` through a column of silica gel. Evaporation of the eluate and crystallization of the residue yields the tetrapeptide fragment 4-7 ~ of formula Z-D-T~r-D-Lyls-D-Trp-D-Phe-OMe. Said last-named compound ,~ ,, is dissolved in an inert organic solvent, for example methanol, ethanol, or DMF, preferably DMF. The solution is treated with an excess of hydrazine hydrate, for example 20 to 50 molar equivalents, . .
and is kept at -20 to 10C, preferably at 0C, for one and a half to three hours, preferably two hours. Water is added; the pre-cipitate is collected by f71tration, dried, and crystallized to 1~ :
, yield the tetrapeptide fragment 4-7 of formula Z-D-T~r-D-Lys-D-Trp-D-Phe-NHNH2 ( I I ,.
~ , With reference to the tripeptTde fragment 8-10, the ~¦ tripeptide is prepared by reacting a protected activated ester of 1~ D-asparagine, preferably Z-D-Asn-OTcp, with a lower alkyl ester of - a protected D-lysine, preferably H-D-Lys-OMe, to obtain the corresponding 1~ Boc 2 ~ .
:~ ................................. .
.. :
..
.. . . .. . . ~. . : ;, .. = .: ` , . ., . . , ~ ~ ..
.. .,, . . ~ . . ., .. ., :
; . ` ; . . . .. . .
` . . .. . .. . . . . . . . .
protected lower alkyl ester of the dipeptide D-asparaginyl-D-lysine, preferably Z-D-Asn-D-Lyls-OMe, which after removal of the terminal amino Boc protecting group (Z) using hydrogen in the presence of a noble metal catalyst gives the corresponding lower alkyl ester of H-D-Asn-D-L~s-OH, ~ Boc preferably Z-D-Phe-D-Asn~D-Lys-OMe. Subsequent removal of the terminal Boc amlno protecting group of the latter compound (Z) using hydrogen in the presence of a noble metal catalyst gives the corresponding lower alkyl ester of H-D-Phe-D-Asn-D-Lyls-OH, preferably the tripeptide fragment -, B c - -8-10 of formuia~H,D~Phe-D-Asn-D-Lrs-OMe (111).
Boc ', ' :
In a preferred embodiment of the preparat70n of the tri-peptide fragment 8-10, substantially equtmolar ambunts of Z-D-Asn-OTcp, ,;
¦ prepared from D-asparagine in the same manner as described for the ~` corresponding L-isomer by J. Beacham, G. Dupuis, F.M. Finn, H.T. Storey, C. Yanaihara, N. Yanaihara, and K. Hofmann, J. Amer. Chem., Soc., 93, 5526 ~1971), an~ H-D-Lys-OMe, prepared from D-lysine tn the same B c monner as described for the corresponding L-isomer by R. Schwyzer and W. Rittel, Helv. Chim. Acta, 44, 159 (1961), in an inert organic I solvent, preferably DMF, at -20 to 10C, preferably 0C, is treated with~a substantiDlly molar equivalent of an organic base,-preferably i : . ~ 1 3~ 25 N-ethylmorpholine and stirred at -20 to 10C, preferably 0C, for ; two to four hours, and then at 20 to 30C for 15 to 30 hours. The solutlon is evaporated, the residue is triturated with an alkyl ether, .1 ,- .
preferably diethyl ether and dried to yield the dTpeptide fragment 9-10 ~`
of formula Z-D-Asn-D-L,ys-OMe. Said last-named compound is then subjected oc 2 ~
r : .
, , - ,, , . , .. , ., .. , ., ,, , . ~ .
1~)4~6Z3 to hydrogenation in the presence of a noble metal catalyst, pre-ferably 5% Pd/C. Methanol, ethanol, acetic acid or mixtures ~ thereof are convenient solvents for this hydrogenation. The catalyst is removed by fiItration, the fiItrate is treated with ~ . .
a substantially equimolar amount of a mineral acid, prefera~ly hydrochloric acid, an a evaporated to yield the dipeptide fragment 9-10 of formula H-D-Asn-D-Lys-OMe as the hydrochloric acid addition --60c salt. Said last-named compound and a substantially equimolar ; amount of Z-D-Phe-OTcp, prepared from D-phenylalanine in the same manner described for the corresponding L-isomer by J. Pless -~-and R. A. Boissonnas, Helv. Chim. Acta.~ 46 IGO9 (1963), in an inert organic solvent, preferably DMF or THF, at -20 to 10C, preferably 0C, is treated with an excess, prefer~bly 1.1 to 1.3 ^1 15 molar equivalents, of a organic base, preferably N-ethylmorpholine. ;~
j The solutlon is stirred at about 0C for 20 to 30 hours. The precipitate is collected by f71tratTon and crystallized to obtain the trtpepttde fragment 8-10 of formula Z-D-Phe-D-Asn-D-Lys-OMe.
80c The latter compound is subjected to hydrogenatton in the presence of a noble metal catalyst, preferably 5% Pd/C. Methanol, ethanol, acet!c actd, or mixtures thereof are convenient solvents for this hydrogenation, when acettc actd ts ùsed the product wtll be tsolated as the acettc acid addttion salt. The catalyst is removed by ~tltrat70n, the filtrate evaporated, the residue taken up in an aromatic hydrocarbon, preferably benzene, and evaporated to obtain the tri-pepttde fragment 8-10 of formula H-D-Pho-D-Asn-D-L~s-OMe ~111). ~
j~ Boc ,~ -1 , .
i. ~ ' .
;
. . , .. ,~ . . ..
.- , . . . . .. . . .
,. . . i . , ~ . . ..
..
` 104~623 j With reference to the fragment HNHCIHCH25 Trt (Vll) in which f:: :
R is CONHCH2CONHCH2CH3, i.e. 1-he fragmant VlIb alternatively written as I H-D-C~s-Gly-NHEt, said fragment is prepared by reacting a protected Trt lower alkyl ester of the dipeptide D-cysteinyl-glycine, preferably !~ -Trt-D-Cys-Gly-oMe~ with ethylamine, to obtain the correspondingly 1 Trt protected ethylamide of the dipeptide D-cysteinyl-glycine, preferably , Trt-D-Cys-Gly-NHEt. Removal of the termTnal amino protecting group ~Trt) using miIdly acidic conditions, preferably a mixture of water and acetic acid or formic acid yields the corresponding addition salt of the fragment H NHC~HCH2S Trt ~VII~ in which R is CONHCH2CONHCH2CH3, ~' . . . .
i.e. H-D-C~s-Gly-NHEt (Vllb).
Trt In a preferred embodiment of the preparation ot the fragment (Vll) in which R Is CONHCH2CONHCH2CH3,the dipeptide Trt-D-Crs-Gly-OMe, prepared from D-cysteine in the same manner I as described for the corresponding L-isomer by G. Amlard, Bull.
;1 Soc. Chim. ~Fr.), 1956, 698, Is treated with a molar excess, preferably 50 - 200 molar equivalents, of ethylamine at -15 to 15C, preferably 5C, for 20 to 30 ~hours. The solution is evaporated, and the residue is dissolved in a solution of a - lower alkyl ester of a lower alkanoic acid, preferably ethyl acetate, and an aromatic hydrocarbon, preferably ben7ene, and purified by chromatography on silica gel to obtain the dipeptide of '~ ~ formul a Trt- D-C~s-Gly-NHEt. Said last-named compound, is subjected ~j ' to miIdly actdic conditions, preferably 70 to 90% acetic acid at ¦~
30 to 50C, preferably 45C, for ten to 20 minutes, preferably ~-- 30 15 minutes. Water is added, the mixture is filtered, the filtrate , ;` 2 7 1 ~
: ~ ,~.
,;., ` ii , - - - - - `= _ ~.
~ ...... .. , . . . , . . . . . . . ......... .... .... .
',: '. ''' - 104~6Z3 is treated with a substantially molar equivalent of a mineral acid, ;~ preferably hydrochloric acid, to obtain the fragment HNHIHCH2STrt (Vll) in which R is CONHCHzCONHCH2CH3 alternatively written as H-D-Cys-Gly-NHEt (Vllb).
The heptapeptide fragment 4-10 is conveniently prepared by coupling the fragment 4-7 and th~ fragment 8-10 according to the azide coupl7ng method in the following manner. A solution of the -tetrapeptide fragment 4 7~of formula Z-D-T~r-D-L~s-D-Trp-D-Phe-NHNH2 (Il), obtatned as desc~SIbéd above, in an inert anhydrous organic solvent, preferably DMF, is cooled to a temperature of from about -30C to ~
about -10C and mixed with a solution of about two to five molar -~ -jJ equivalents, preferably three molar equivalents, of a mineral acid, I
J 15 preferably hydrogen chloride, in an inert anhydrous organic solvent, preferably ethyl acetate. An organic nitrite, preferably t-butyl nitrltTe or isoamyl nitrite in a substantially equimolar amount is added with stirring. The solutTon Is stlrred for 10-30 minUTes, preferably for about 15 mlnutes, at a temperature of from about -20C to about -10C. Keeplng the solution at a temperature of from about -30C to about -10C, a solution of a substant7ally ¦
equimolar amount of the tripept7de fragment 8-10 of formula ¦
H-D-Phe-D-Asn-D-Lyls-OMe (lil), preferably as the acetic acid add7t70n salt obtalned as descr7bed above, and of about three to f7ve molar t~ ~ 25 equivalents, preferably abo_T 3.5 molar equ7valents, of an organ7c , base, preferably N-ethyldi7sopropylam7ne, 7n an 7nert anhydrous ;
organ7c solvent, prefe mbly DMF, 7s added slowly with stirr7ng. -~
,:
' :', - 2~ ~1 .,~ ~ .... j~ .
,~, ~.. ,......... . ~ ~ - .- .. . . ... . .
~he mixture is stirred for 30 - 60 rninules at about -20C to about -10C, then at about 2~ to 30C for 20 to ~0 hours. The solution is evaporated and the resid~Je is triturated with cold aqueous citric acid (0.5 to 2N), water and dried. The residue is taken up in a mixture of a halogenated hydrocarbon, preferably ch!oroform, and a lower alkanol, preferably methanol, and purifie~ by chromatography on silica gel. The solvent is evaporated and the residue crystallized to yleld the heptapeptide fragment 4-10 of formula Z-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-oMe.
Bl Boc Boc Said last-named ~ompound is subjected to hydrogenation in the presence of~a;n~ble metal catalyst, preferably 5% Pd/C.
Methanol, ethanol, acetic acid, or mixtures thereof are suitable solvents for this hydrogenation, and when acetic acid is used the '3 15 product is isolated as the acetic acid addition sait. The catalyst , 1s removed by filtration; the filtrate evaporated, the residue taken up In an aromatic hydrocarbon, preferably benzene, evaporated, and 1 drled over strong alkali, preferably potassium hydroxide or sodium hydroxlde, to obtain the heptapeptide frag~ent 4 10 of formula H-D-T~rD-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV).
The decapeptide fragment~~l-10 is conveniently prepared by coupling the fragment 1-3 and th~ fragment 4-10 according to -- the azide coupling method in the following manner. A solution of ~j 25 the trTpeptide fragment 1-3 of formula ~- -S CH2CH2CO D Sltr D Tltr-D-Phe-NHNH2 (V), obtained as described . . .
~~ above, in an inert anhydrous solvent, preferably DMF, is cooled tc ~, -.:: - ~. :a temperature of from about -30C to about -10C and mixed with a `~
solution of about two to five molar equivalents, preferably three molar equivalents, of a strong mineral acid, preferably hydrogen hloride, in an anhydrous organic solvent, preferably ethyl acetate.
2~ ~:
. 1 ' .
- . i .- -. . , ,. .. . .. .. ., ., ,. ., : .
. .~; ~. ~ . , .. . ` ,, . :. . .
-: . : .. . , . , : ` - . . .
AHP-~550 An organic nitrite, preferably t-butyl nitri1e or isoamyl n7trite, in a substantially equimolar amount, is added with stirring. The solution is stirred for lO - 30 minutes~ preferably for about 15 minu~es, at a temperature of from about -20C to about -10C.
Keeping the solution at a temperature of from about -30C to about -10C, a solution of a substantially equimolar amount of the heptapeptide fragment 4-10 of formula H-D-Thr-D-Lys-D-Trp-D-Phe-D-Bl Boc Phe-D-Asn-G-Lys-OMe ~IV) obtained as described above, and of about Boc :; 10 ~`
about three to five molar e!quivalents, preferably about 3.5 molar equivalents, of ~a~ organic base, preferably N-ethyldiisopropylamine, in an inert anhydrous organic solvent, preferably DMF, is slowly added with stirring. Stirring of the resulting mixture is continued for 30 - 60 minutes at about -20C to about -10C, then at about 2~ to 30C for 20 to 30 hours, The solution is evaporated, the residue is trlturated with cold aqueous citric acid (0.5 to 2N), water and dried. The residue is taken up in a mixture of a halogenated ' hydrocarbon, preferably chloroform, and a lower alkanol, preferably methanol, purified by~chromatography on silica gel and crystallized i20 to yieid the decapeptide fragment 1-lO of formula Trt-S-CH2CH2CO-D-Sir-D T~tr D Phe D-Thit-D-L~5-D-Trp-D-phe-D Phe_D_A5n-D-Lys-oMe.
Bu Bu B c Boc Said last-named compound is taken up in an inert organic solvent, for example methanol, ethanol, or DMF, preferably DMF, and ` treated with an excess of hydrazine hydrate, for example 20 to 50 molar equivalents. The mixture is kept at -20 to 10C, preferably .`~ . ' . ' , .
, .
.
.
: :
'~' ' "
; 0C, for 15 to 45 minutes and at 20 to 30C, for 20 to 30 hours. , Water is added, the precipitate is collected by fiItration, washed - with water, and dried to yield the decapeptide fragment 1-10 of formula Trt-S-CH2CH2CO-D-Ser-D-Th,r-D-Phe D-T~r-D-Lys-D-Trp-D-Phe~-Phe- '' Bl B~t Blt Boc ~' D-Asn-D-Ly,s-NHNH2 (V I ) . . .
Boc `-The linear peptide Vlll is convenient;y prepared by coupling the fragment 1- ! (Vl) and the fragment ~11) according to the azide ', coupling method in the fo~'lowing manner. A solùtion of the decapeptide fragment 1-10 (Vl)~ obtained as described above, is taken up in an inert anhydrous organ?c solvent, preferably a mixture of DMF and DMSO, cooled to a temperature of from about -30C to about -10C and mixed i with a solution of about two to five molar equivalents, preferably three molar equivalents of a mineral acid, preferably hydrogen chloride, in an anhydrous organic solvent, preferably efhyl acetate. -A sùbstantially equimolar amount of an organTc nitrite, preferably t-butyl nitrite or tsoamyl nitrite,is added, and the solution is stlrred for 10-30 mlnutes, preferably for about 15 minutes, at a ; temperature of from about -20C to about -10C. Keeping the stirred '-!; solution at a temperature of from'ai30ut -30C to about -10C, a soiution of a substantially equimolar amount of the fragmen~ ; ~' HNHCHeH2STrt (Vll) in which R is CONHCH2CONHCH2CH3(Vllb) obtained as des- ~i~
cribed above or the fragment HNH~HCH2STrt (Vll) in which R is hydrogen (Vlla) - :~ 25 ~ ~ R
described by F. 1. Carroll et al., J. Org. Chem. 30, 36 (1965)], and of about three to five molar equivalents, preferably about 3.5 molar equivalents, of an organic base,~preferably N-ethyldiisopropylamine, ' ~' ~
. , : ': :.
: ~ : :~ :.-, :.
,~
3 1 ::
~-' -, ` . ,., : .::
' :.
, .
104~)623 in an inert anhydrous organic solvent, preferably DMF, is added slowly. Stirring of the resulting mixture is continued for 45 - 75 minutes at about -20C to aboul -10C, then at about 2Q to 30C
. for 20 to 30 hours. Evaporation of the solution, trituration of , 5 the residue with cold aqueous citric acid (0.5 to 2N), water, methanol, and drying yields the corresponding linear protected peptlde of formula (Vlll) Trt-S-CH CH C0-D-S~r-D-T~r-D-Phe-D-Thr-D-Ly,s-D-Trp-D-Phe~D-Phe~Asn~-L~s-NH~HCH25 Trt (Vlll) 7n which R is as "4; Boc Boc R
o " ~
defined herein. J
The conversion of the above linear protected peptide, 1 obtained as described above, to the compound of formula I is ~ ;~
3' accomplished conveniently and efficiently by first subjecting the linear protected peptide to the action of iodine, preterably in the presence of a lower alkanol or a lower alkanoic acid whereby slmultaneous removal of the sulfhydryl protecting groups, i,e. Trt, ~ and formatlon of tne disulflde brldge occurs to glve the corresponding I cycli~c disulfide of formula IX
S-CH2CH2C0-D-Sfr-D-TIr-D-Phe-D-T~r~-D-Llys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-Bu Bu Bu Boc B c ~; N ~HCH2~ (IX) in which R is as defined hercin, subsequent treafment of j~ ~ of the latter compound under moderately acidic conditions removes the 3- ~ ~ remaining protecting groups (i.e. Boc and Bu ) to gTve the correspondlng compound of formula 1.
- J :: ~
,~, .
., - . :
, . i . ~ , .
,.' : ~ ' :
,~ -32-j,,, : . ~ . .
: 104~623 In a preferred embodiment of the above transformation, the Iinear protected peptide (Vll!) is dissolved in a lower alkanol or a lower alkanoic acid, preferably acetic acid, and added to an 1;
excess of iodine (5 to 25 molar equivalents, preferably 10 mclar equivalents) dissolved in a lower-alkanol or a lower alkanoic acid, preferably methanol, at a concentration of about 2 - 5~ iodine.
The time and temperature of this reaction is not critical; however, it is desirable to keep the reaction between 0 and 30C by regulat7ng the addition to the iodine solution or by cooling of the reactton mixture, or by a combination of both. Under these ¦-~ conditions the addition usually takes 30 to 60 minutes. After the ,J' addit7On the mixturé is stirred at 20 to 30C for 30 to 120 minutesJ
1 preferably 60 minutes. Thereafter the mixture is cooled to about 0C -i and an excess of a miId reducing agent, preferably sodium thiosulfate in ~
aqueous solution is added. The mixture is concentrated and the residue Is suspended in water. Collection of the solid material affords the deslred corresponding cyclic disulfide of formula IX.
7 Alternatively, the linear protected peptide (Vll) is converted to the aforementioned corresponding cyclic disulfide by the method of ¦ 20 R.G. Hlskey and R. L. Smith, J. Amer. Chem. Soc., 90, 2677 (1968) using thlocyanogen. ;
Again alternatively, the cyclic disulfide (1X) is also obtained by selectively removing the sulfhydryl protecting groups of t,le above - ¦
linear protected peptide (Vlll)by the action of a mercuric or silver salt, !
for example, mercuric acetate, mercuric chloride, silver acetate or silver nitrate, in an inert organ7c solvent, for example DMF or acetic acid, according to known methods; for example, see B. Kamber, and N. Rlttel, Heiv. Chem. Acta, 52, 1074 tl964), L. Zervas, et al., J. Amer. Chem.
- Soc.,87, 4922 (1965) and R. G. Denkewalter et al., ~. Amer. Chem. ~
.: ~ - . . : :' Soc., 91, 502 (1969). -The mercuric or disilver salt thus obtained is ~ . :
., , - :.
then converted to the corresponding free disulfhydryl derivative (X) 2 2 Itr D T~tr D Phe D-TIhr-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHCHCH2SH (X)in which R is as defined herein B c by treatment with hydrogen sulfide, see L. Zervas, et al., cited above.
The latter derivative is then converted to the aforementioned cyclic disulfide ~IX) by a mild oxidizing agent selected from the group consisting of iodine according to the method described hereinbefore, oxygen according to the~me~thod of J. Rivier, et al., C.R. Acad. Sci.
Ser. D, 276, 2737 (1973), 1,2-diiodoethane according to the method of , , ~,:
F. Weygand and G. Zumach, Z. Naturforsch~ 17b, 807 (1962), and sodium or potassium ferricyanide according to the method of D. Jarvis, et al., J. Amer. Chem.-Soc., 83, 4780 (1961).
,, Finally, the aforementioned cyclic disulfide of formula IX is transformed into the corresponding cyclic peptide of formula 1, by subJecting the former to moderately acidTc conditions whereby the re~aining protecting groups of tne cyclic disulfide (IX) are removed.
~, Generally this step is carried out by treating the cyclic disulflde (IX) wlth 50 - 100~ trifluoroacetic acid or with an aqueous solution con-I tain'ng a mineral acid at 0 to 20C for 10 to about 60 minutes~
.j . . ~ . ~:Examples of such mineral acids are 10 to 20% aqueous sulfuric ac7d, -~ 10% phosphoric acid, 10 - 30% hydrobromic acid and 10 to 36% hydrochloric ; acid. An extremely useful acid is concentrated hydrochloric acid.
i Preferred conditions for the present step include dissolving ~he cyclic di 5U Ifide in a minimum of concentrated hydrochloric acid cooled to O~C and stirring the mixture at 0C for five to ten minutes under a nitrogen atmosphere. Thereafter glacial acetic acid ,..
,, - , ,~
,,, , .
.
.
..
-3~-, .
. . . . ~ .
;: . . .
; ' ' ' '. , ' ' ,'~; : ~
._ 104~623 (10 X vols.) is added, the solution is cooled to about -70C and Iyophilized to give the corresponding cyclic peptide of formula The latter product is purified further by ion exchange chromatography using a carboxymethylcellulose cation exchanger and ammontum acetate as the eluant. In the latter case the product Ts obtained in the form of its acid addition salt with acetic acid. Alternatively, the product is purified by partltlon chromatography on a chemically modified cross-llnked dextran, for example Sephadex*LH 20 or Sephadex G-25, using methanol or acetlc acld, respectively, as the eluting solvent. In the case where Sephadex*LH-20 and methanol as the eluting solvent is employed, the product Ts obtalned In the form of its hydrochloric acid addition salt. In the case where - -Sephadex G-25~,and acet7c acid is employed, the product is obtained in the form of its acetic ac1d addltion salt. Repeated IyophllizatTon .. .
from water of the product In the form of its acetic acid additlon salt ! ylelds the substantlally pure cycllc peptlde of formula I in which R Is as deflned hereln, In the form of the free base.
The llnear reduced form of the peptlde of formula la -is obtained by removal of the protectlng groups from the aforem,entioned llnear protected peptide of formula (Vl~
~, Trt-S-CH2CH2CO-D-Selr-D-T~r-D-Phe-D-T~,r-D-Lys-D-Trp-D-phe-D-phe-D-Asn 81 BL Bl Boc D-Lys-NHCHCH2S Trt (Vll~) !~ 80c R ~ ~
in whlch R is as defined hereln from the aforementloned dlsulfhydryl ~;j derivatlve of formula (X) t 8~t D T~t~ D-L~s-D-Trp-D-Phe-D-Phe-D-Asn D-Lys-NH~HCH2SH (X) -' -Boc R
-, * Sephadex is a trade mark ~35~
AI~P-6550 ` 1041)623 in which R is as defined herein under moderately acidic conditions.
Preferred conditions for this deprotection step comprises dissolving the linear protected peptide (Vlll) or the disulfhydryl derivative (X) in concentrated hydrochloric acid at 0 to 5C in an inert atmosphere, for example, nitrogen or argon. The mixture is kept at this temperature for five to ten minutes. Subsequent isolation of the -linear reduced form is accomplished in the same manner as described previously for the isolation of the cyclic peptide of formula 1.
Also, the linear reduced form (la) is obtained directly lo by reduction of the cycltc peptide of formula 1. Reduction with dithiothreitol according to the method~of W.W. Cleland, Biochem. _, , 480 (1964) is preferred, although other agents known to be effective j for the reduction of cyclic disulfides to the correspondTng disulf-hydryl derivative are applicable, for example, sodium bisulfite ~ -followed by hydrolysis of the intermediate dithiosulfate derivat.ive.
j Finally it will be apparent to those skilled in the art J fhat: equ7valent amino, hydroxy or thtol protecting groups, ¦ ~ equlvalent methods of coupling peptide fragments, and equlvalent ¦ metnods for removal of amlno, hydroxy or thiol protecting groups, j 20 other than those disclosed herein could be used in the embodimentsof this tnvention without departing from the scope and spirit of the tnventTon. Such apparent alterations are intended to be included withtn the scope of this invention. ~ -The following flow diagrams and examples illustrate further this invention.
.
::~
.
. . .
!
. ' .. , . ... ,- . .. ... ... . . .
, . . .. .. . .. . . .. .
:
.: .
'1 ' 1~ ~ 9 1 r ~ ~i .,~ .. i1~ -~1~
.. :~
104~623 -To promote facile condensation of the peptide carboxyl group with a free amino group of another peptide to form a new peptide bond, the terminal carboxyl group mus~ be activa1ed.
Descriptions of such carboxyl-activating groups are found in general textbooks of peptide chemistry; for example K.D. Kopple, "Peptides and Amino Acids", W.A. Benjamin, Inc., New York, 1966, pp. 45 - 51 and E. Schroder and K. LUbke, "The Peptides"; Vol. 1, Academic Press, New York, 1965, pp. 77 - 128. Examples of the activated form of the ; terminal carboxyl are acid chloride, anhydride, azide, activated ester, or _-acyl urea of a dialkylcarbodiimide. The following activated esters have proved to be particularly su~table in the process of this invention: 2,4,5-trichlorophenyl (represented by OTcp), ~ -pentachlorophenyl trepresented by OPcp), D-nitrophenyl (represented by ONp), or l-benzotriazolyl. The succlnim7do group is also useful for activating a carboxyl. -The term "azide method" as used herein refers to th~ method of coupling two pepttde fragmenTs wh7ch comprlses the reactlon of a pept7de hydrazide wtth a reagent which furnishes nitrous acid In ~ ;
sltu. Suitable reagents for this purpose include organic n7trites (e.g. _-butyl nitrite, isoamyl n7tr7te) or an alkal7 metal n7trite salt ~e.g. sodium nitrite, potassium nitrite) in the presence of a strong acid such as hydrogen chloride or sulfur7c or phosphoric ac7d. The ;; corresponding peptide azide thus obtained is then reacted with a peptide having a free amino group to obtain the desired peptide.
Preferred condit70ns for the az7de method of coupl7ng cornprises reacting :!: . . -.
~ , . '. ' ' , ,:
~ ' :~ . .. .
~--. . . : : ' : ,; , . : ' AHP~6~50 1~ 623 . ' . .
the peptide hydrazide with nitrous acid, generated In situ frorn an organic nitrite in the presence of a mineral acid, preferably hydrogen chloride, (pH ranging usually from 0.1 to 2), in an anhydrous inert organic solvent, for example, dimethylformamide, dimethyl sulfoxide, ethyl acetate, methylene dichlorids, tetrahydrofuran, dioxane, and the like at -30 to 20C, preferably at about -15C, for 10 to 30 minutes to obta1n the corresponding azide. The peptide azide can be isolated and crystallized or is prèferably allowed to remain in the reaction mixture, and thereafJer reacting the azid~e in the said mixture with the peptide unit having the free amino group at temperatures rangtng ;
from -30 to 20C for about one to two hours and then at 0 to 30C for 10 to 30 hours. An acid acceptor, preferably an organic base, for ~¦
Zl example N-ethyldiisopropylamine, N-ethylmorpholine or triethyl-amine, is present in the reaction medium in order to make the reaction medium sllghtly alkaline, preferably pH 7.0 to 7.5. See also the above clted textbooks of Kopple or Schr~der and LUbke for add7tional descriptions of thls method.
The terms "peptide, polypeptide, tripeptide, hexapeptide, and the like" as used herein are not limited to refer to the respective parent peptides but also are used in reference to modified peptides ; havlng functionalized or protecting groups. The term "peptide" as ;~
iZ~ used herein is used in reference to a peptide with two to twelve amTno ac7d residues. In addition the residue "NH~HCH2S" as - R
defTned herein is written as H-D-Cys-Gly-NHEt when R is CONHCH2CONHCH2CH3, ~ and is written as a modified residue of cysteine when R 7s H, viz., 2-"'t~ ~ thioethylamine.
~: : ' ,,~,, ' :
.~ , ~: ''' ~ lZ
. .. ,~
:- . ~ .. ... . :. :
:: - ~ . : : . . .. .
:: . .
1~4~)623 The abbreviation Me represents a methyl group and NHN~12 represents a hydrazide group. In addition, the following abbreviations are used: dimethylformamide (DMF), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), methanol (MeOH), ethyl acetate (EtOAc), methylene dichlortde (CH2C12), N,N'-dicyclohexylcarbodiimide (DCC), sodium chloride (NaCI), sodium bicarbonate (NaHC03), sodium sulfate (Na2S04) magnesium sulfate (MgS04), and 5~ palladium on charcoal (5% Pd/C).
The term l'lower alkyll' as used herein contemplates hydro- ~-carbon radicals having one to three carbon atoms and includes methyl, slO ethyl and propyl.
The term 'Imineral acidll as used herein contemplates the strong inorganic acids and includes hydrochloric, hydrobromic, suJfuric, phosphoric and the like. When the term is used in con-1~ junction wtth an anhydrous system, hydrogen chloride is the preferred jl5 mlneral acid.
! The term llmiIdly acidtc conditionsll as used herein contem- ~ ' plates conditlons in which a dilute aqueous solutlon of an organic ~ i aclc, for example 30 - 80% or mixi~ures thereof, is a prlncipal çr-component of the reaction medlum. ~ -~o The term "moderately acidlc conditions" as used herein contemplates conditions in which concentrated organic acids or ~ ~ -f aqueous solutions of the mineral acids are used as a principal -~
component of the reaction medium,at temperatures ranging from ' ~ about -30 to 30C. Examples of preferred conditions in this j case include the use of 50 to 100% trifluoroacetic acid at 0 ... . . . .
~i~ to 30C or 0.1 - 12N hydrochloric acid in aqueous or anhydrous organic ' - ~ -solvents at -20 to 10C.
:1 ' .. , he term 'lorganic nitrite'l includes the commercially available alkyl nitrites~ for instance, t-butyl nitrite or isoamyl nitrite.
:
f ~' ., ' .
-.f Al-IP-6550 104~623 :' The term organic base as used herein includes triethyl-amine, N-ethylmorpholine, or N-ethyldiisopropylamine.
~ The term strong base as used herein contemplates both - organic bases, as described above, and strong inorganic bases including the hydroxldes and carbonates of sodium and potassium.
The peptldes of thls invention, includlng the cycllc and the llnear reduced forms, are obtained in the form of the free base or a~ an acid additlon salt thereof elther directly from the process of thls inventlon or by reacting the peptide with one or more equivalenTs of the appropriate acTd. Examples of preferred salts are those wlth pharmaceuttcally acceptable organic acids, e.g. acetic, -~
lactlc, succlnic, benzolc, salicyllc, methanesulfonlc or toluene- ;
sulfonlc acid, as well as polymeric acids such as tannic acld or carboxymethyl cellulose, and salts wlth inorganic acids such as -` 15 hydrohallc aclds, e.g. hydrochlorlc acid, or sulfurlc acld, or phos-, phorlc acld. It should be noted that the peptldes have two baslc ! nltrogens givlng rlse to addltlon salts wlth one to posslbly two i equlvalenTs of acid. If deslred a partlcular acid addition salt J, is converted into another acid addition salt, e.g., a salt with a ~ -non-toxic, pharmaceutically acceptable acld, by treatment with the ,~ - appropriate ion exchange resln In the manner descrlbed by R. A.
-~ ; Boissonas, et al., Helv. Chlm. Acta, 43, 134g (1960). Suitable ion exchange reslns are cellulose based cation exchangers, for example carboxymethylcellulose or chemically modlfled, cross-llnked dextran cation exchangers, for example, those of the Sephadex*C type, and strongly basic anion exchange resins, for example those listed in J.P. Greenste7n and M. Wtnitz Chemistry of the Amlno Acids , John J Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p. 1456.
t * Sephadex is a trade mark ~, 13 ., , ., .
~ .. }
~ - , 1041)623 The peptides of this invention of formulae I and la give complex sal-l-s wi-rh heavy metal ions. An example of a pharmaceutically acceptable heavy metal complex is a complex formed with zinc or with zinc protamine.
The peptides of formulae I or la, as well as their ;
corresponding pharmaceutically acceptable salts, are useful because they possess the pharmacological activity of the natural hormone somatostatin. Their activity is demonstrated ~;
readily in pharmacological tests such as a modification [A.V. Schally, et al., B~i`ochem. Biophys. Res. Commun., 52, 1314 (Ig73~; J. RivierJ et al., C.R. Acad. Sci. Paris, Ser. D, 276, ~ ~
2737 (1973)] of the n vitro method of M. Saffran and A.V. Schally, ~-Can. J. Biochem. Physiol., 33, 405 (1955).
The activity of the peptides of formulae I or la is , 15 demonstrated also In vivo 7n a modification of the pentobarbital- ;
! 7nduced increase in plasma growth hromone level in the rat as described by Brazeau, et al., cited above. In this test the peptldes of this Inventlon show a level of actlvity which is of the same order as that of somatostatin.
The peptides of formulae I or la and their salts are useful for the treatmert of acromegaly and other hypersecretory endocrine states and in the management of diabetes in mammals; see for example, -P. Brazeau, et al., cited above. When a peptide of formula t or la or a salt thereof is employed for such treatment or management, it 1~ 25 is administered systemically, preferably parenterally " n combination ¦~ - with a pharmaceutically acceptable liquid or solid carrier. The proportion of the peptide or salt thereof is determined by its ,~ solubility in the given carrier, by the given carrier, or by the `~ chosen route of administration, and by standard biological practice.
For parenteral administration to animals the peptide or a salt : - ~
thereof is used in a sterile aqueous solution which may also contain : : 14 i ... :.. , .. ... .. . ,.. , . ,.. , ,. ....... . , ,. :.. .. .. . .... .. . . ...
: . ,: . .... . . . . . . . . .. . ,. , ~ . .
Al-IP-6550 ~ .
1()40~Z3 other solutes such as buffers or preservatives, as well as sufficient pharmaceutically acceptable salts or glucose to make the solutlon isotonic. The dosage will vary with the form of administration and with the particular species of animal to be treated and Ts preferably kept at alevel of from 5 mcg to 300 mcg per kilogram -body weight. However, a dosage level in the range of from about 10 mcg to about 50 mcg per kilogram body weight is most desirably employed in order to achieve effecttve results.
The peptides or salts thereof may also be administered in one of the long acting,~slow-release or depot dosage for~s descrtbed below~ preferably by intramuscular injection or by Implantation. Such dosage forms are designed to release from about 0.5 mcg to about 50 mcg per kilogram body weight per day.
It is often desirable to administer a peptide of formula I
or ~ continuously over prolonged periods of time in long-acting, slow-release, or depot dosage forms. Such dosage forms may elther contatn a pharmaceutically acceptable salt of the peptide having ¦ a low degree of solubillty In body flulds, for example one of those salts descrlbed below, or they may contain the peptlde in the form of a w3ter-soluble salt together with a protective carrier whtch preve~ts ¦~ rapld release. In the latter case, for example, the peptide may beformulated wtth a non-antigenic par~ially hydrolyzed gelatin ~,~ in the form of a viscous liquld; or the peptide may be absorbed on~1 a pharmaceutically acceptable solid carrier, for example ztnc hydroxide, and may be administered in suspension in a pharmaceutlcally acceptable liquid vehTcle; or the peptide may be formulated in gels or I: :
suspensions with a protective non-antigenic hydrocolloid, for example ,~ sodium carboxymethylcellulose, polyvtnylpyrrolidone, sodium alg7nate, gelatine, polygalacturonic acids, for example, pectin, or certaln mucopolysaccharTdes, together with aqueous or non-aqueous pharmaceuti-cally acceptable liquid vehtcles, preservatives, or surfactants.
. .
1 :
104~623 : ~
Examples of such formulations are found in standard pharmaceutical - texts, e.g. in Remington's Pharmaceutical Sciences, 14th Ed., Mack y;~ ~ :
Publishing Co., Easton; Pennsylvania, 1970. Long-acting, slow-release preparations of the peptide of formulae I or la may also be obtained by microencapsulation in a pharmaceutically acceptable coating, for exampie gelatine, polyvinyl alcohol or ethyl cellulose.
Further examples of coat7ng mater7als and of the processes used for m7croencapsulat70n are descr7bed by J.A. Herb7g 7n "Encycloped7a of Chemical Technology", Vol. 13, 2nd Ed., Wiley, New York 1967, pp 436 - 456. Such formulat70ns, as well as suspens70ns of salts ¦;
of the agent wh7,ch are only sparingly soluble in body fluidsJ for ' example salts with pamoic acid or tannic acid, are designed to j-release from about 5.0 mcg to about 100 mcg of the active compound per k7logram body weight per day, and are preferably administered by ~! : I
i 15 7ntramuscular 7nject70n. Alternat7vely, some of the solid dosage forms 17sted above, for example certa7n spar7ngly water-soluble salts or ~' dlspers70ns 7n or adsorbates on solld carr7ers of salts of the agent, . :.., ,.,, . ,-: ... .
~ for example d7spers70ns 7n a neutral hydrogel of a polymer of ethylene -¦ glycol methacrylate or s7m71ar monomers cross-17nked as descr7bed inU.S. Patent 3,551,556 may also be formulated 7n the form of pellets 1 1 releas7ng about the same amounts as shown above and may be 7mplanted subcutaneously or 7ntramuscularly. i Process i For conven;ence and clar7ty 7n the following dtscussion the indlv7dual pept7de un7t (i.e., amino acid) is designated sometlmes by a number which refers to the pos7tion in which the part7cular am7no ac7d appears 7n the sequence of the amino acids as illustrated in the formula 1.
.:~, . ... .:
, : , ., :' ' Ij,; :' I ...~
:
~:
1046)623 t:
Thef process of this invention is carrier out in the following manner. .' : With reference to the tripeptide fragment 1-3, the ff~ `
tripeptide is prepared by reacting a protected activated ester of ~ -D-threonine, preferably Z-D-T3r-OH with a lower alkyl ester of '~ ,, D-phenylalanine, preferably H-D-Phe-OMe, to obtain the corresponding ~ ;:
lower alkyl ester of the dipeptide Z~D-Thr-D-Phe-OH, which after removal B~t : .
the terminal protecting grou~ (Z) using hydrogen in the presence of ~ -a noble metal catf~kys~t yields the corresponding lower alkyl ester of :. H-D-T~r-D-Phe-OH, preferably H-D-T~r-D-Phe-OMe. In turn the latter ~ .
Blt BLt' , , ., :,- ,-,, compound is reacted with a protected activated ester of D-serine, :: -preferably the benzotriozolyl ester to obtain the corresponding lower ¦ r,~
alkyl ester of Z-D-Selr-D-T3r-D-Phe-OH, preferably Z-D-Sftr-D-T~r-D-Plle-OMe.
:,~ ' . .
j Subsequent removal of the terminal amino protecting group of the latter .
f compound usTng hydrogen in the presence of a noble met31 catalyst yields 20 the corresponding lower alkyl ester of H-D-Ser-D-Thr-D-Phe-OH, pre-~; ferably H-D-SIer-D-Thlr-D-Phe OMe. Condensation of said last-named compound with a protected activated esler of thiopropionic acid, : preferably the benzotriazolyl ester, gives the corresponding protected ;
f 25 lower :Ikyl ester of Trt-S-CH~CH2CO-D-Ser-D-T~r-D-Phe-OH, preferably : ;
Trt S H2CH2CO D Sltr D r3tr-D-Phe-OMe. The latter compound is treated ~
with hydrazine hydrate to obtain the hydrazide of the tripeptide ~ :
ragment 1 3 of formula Trt S CH2CH2CO-D-SIt-D-T~r-D-Phe-NHNH2 (V). ;
~ 7 .. ~`' ': . ~ :
``` ~.
Al-IP-6550 104~623 ~
In a preferred embodiment of the preparation of the above tripeptide fragment 1-3, a mixture of substantially equimolar amounts of Z-D-TIhr-OH, prepared from D-threonine in the same manner B t 1 5 as described for the corresponding L-isomer by E. Schr~der, Justus Liebigs Ann. Chem.,- 670, 127 (1963) and H-D-Phe-OMe HCI, prepared from D-phenylalanine in the same manner as described for the L-isomer by F. Bergel, J.M. Jchnson, and R. Wade, J. Chem. Soc., 3802 (1962), in an Inert organic solvent, preferably DMF or THF, at -20 to 10C, ¦~
preferably at 0C, is tre~ated with a molar excess, preferably with 1.1 ~i to 1.3 molar equivalents of a strong organic base, preferably N-ethyl-morpholine, to p~ 7-8. A molar excess~preferably 1.1 to 1.3 molar equivalents, of l-hydroxybenzotriazole is added followed by the dropwise aodition of a substantTally molar equivalent of DCC (1.0 to I
1.3 molar equivalents) in an inert organic solvent, preferably DMF or THF. The mixture is kept at -20 to 10C, preferably at 0C from ~ 30 rninutes to 2 hours and then at 20 to 30C for ar, additional hour, ;7 filtered, and the filtrate evaporated. The residue is taken up in a , ] substantlally water-lmmtsclble organic solven~, preferably dlethyl ~
etller, washed, drled, and evaporated. The res1due is taken up in a ¦ ;
mixture of a lower alkyl ester of a lower alkanoic acid, preferably ¦
ethyl acetate, and a hydrocarbon, preferably hexane, and is purified by chromatography on silica gel to yield the ~ipeptide of formula Z-D-T~r-D-Phe-OMe. Said last-named compound is then subjected to : -i~ 25 hydrogenation in the presence of a noble metal catalyst, preferably 5%
palladium on charcoal (5% Pd/C), and of an e~iimolar amount of pyridine ;~
hydrochloride or of an excess of acetic acid. Methanol, ethanol, acetic acid, or mixtures thereof are convenie~t solvents for this hydrogenation. In this manner the terminal a~ano protecting group (Z) , 18 I :
,~,:
... .
: -- --` . ' !
. . .` - , . . .
of the above dipeptide is removed to give the corresponding dipeptide of formula H-D-T~r-D-Phe-OMe as its acetic acid or hydrochloric acid ,~ , Blt , addition salt. Said last-named compound and a substantially equimolar amount of Z-D-S~r-OH, prepared as described for the L-isomer by , Bbt E. Schr~der, Justus Liebigs Ann. Chem., 670, 127 (1963), in an inert organtc solvent, preferably DMF or THF, at -20 to 10C, preferably at 0C, is treated with a molar excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine, to ?r pH 7 - 8. A substantially-equimolar amount of l-hydroxybenzotriazole is added followed by the addition of a substantially molar equivalent -of DCC (1.0 to 1.3 molar equivalents) in an inert organic solvent, preferably DMF or THF, and the mixture is cooled to -20 to 10C, pre-~I ferably to 0C. The mixture is kept at -20 to 10C, preferably at 0C~ from 30 mTnutes to two hours and then at 20 to 30C for an additionalj Hour, fiItered, and the fiItrate is evaporated. The residue is taken up tn a substantially water-immiscible organic solvent, preferably dlethyl ether, washed, dried, and evaporated. The resldue is taken up In a mixture of a lower alkyl ester of a lower alkanoic acid, pre-ferably ethyl acetate, and a hydrocarbon, preferably hexane, and is puriiied by chromatography on silica gel to yield Z-D-Selr-D-T~r-D-Phe-OMe.
~1~j Said last-named compound is dissolved in a lower alkanol or a lower alkanoic acid or a mixture thereof, preferably in acetic, a noble metal catalyst, preferably 5% Pd/C, is added and ~he mixture is agitated in an atmosphere of hydrogen at room temperature for 10-30 hours, preferably for about 20 hour, until substantially one molar equivalent of hydrogen :
has been taken up. FiItration of the catalyst and evaporation of the filtrate yields the tripeptide of formula H-D-Ser-D-Thr-D-Phe-OMe .,~ , ., ~9 . ':
., ' ,:':
::
` ~ .
Al iP-6550 1~623 as the acetic acid addition salt. Said last-named compound is dissolved in an inert organic solvent, preferably DMF or THF, ~-at -20~ to 10C, preferably at 0C, and 1-reated with a molar - excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine ,to pH 7 - 8. A
substantially equimolar amount of 3-tritylthiopropionic acid, pre-pared as described by E. Biliman and N.V. Due, Bull. Soc. Chim.
Fr., 35, 384 (1924), in an inert organic solvent, preferably DMF
or THF, is added followed by the addition of a substantially molar equivalent of l-hydro,xybenzotriazole. A molar excess of DCC (1.1 to 1.3~mp~ar equivalents) in an inert organic solvent, 1 prefeably DMF or THF, is added and the mixture is kept at -20 :11 to .10C, preferably at 0C from 30 minutes to 2 hours and then -I at 20 to 30C for an additional hour, fiItered, and the fiItrate eva?orated. The residue Ts taken up in a substantially water-Immisctble organic solvent, preferably diethyl ether, the pre-.
, cTpttate is removed by fiItration, washed, dried, and evarporated.
J The residue 7s taken up In a mlxture of a lower alkyl ester of a j lower alkanolc acid, preferably ethyl acetate, an aromatic hydro-¦ 20 carbon, preferably benzene, and a strong organic base, preferably ~ :
, trlethylamine, and is purified by chromatography on silica gel.
:~ Crystallization of the purified matRrial yields H2C0 D S~tr D T~tr-D-Phe-OMe. Said last-named compound is dtssolved in an inert organic solvent, for example methanol, ethanol, 1; DMF, preferably methanol, and the solutlon i5 treated with an excess `~ of hydrazine hydrate, for example with 20 to 50 molar equivalents. ;
m The reaction mixture is kept at -20 to 10C, preferably at 0C, from :
1 30 minutes to 2 hours and then at 20 to 30C for 15 to 30 hours, 1 ~ ~
3~ ::
. f ~ O
, ' .
' .~', ', - :
104~)6Z3 ; preferably for 24 hours. Water is added, the resulting precipitate is collected by fil1ration and dried to yield the tripeptide fragment 1-3 of formula Trt-S~OH2CH2CO-D-S~r-D-T~r-D-Phe-NHNH2 (V).
~ B Bu r~-~With reference to the tetrapeptide fragment 4-7, the tetrapeptide is prepared by reacting an amino protected D-tryptophane, preferably Z-D-Trp-OH, with a lower alkyl ester of D-phenylalanine, preferably H-D-Phe-OMe, to obtain an amino protected lower alkyl ester of the dipeptide H-D-Trp-D-Phe-OH, preferably Z-D-Trp-D~Phe-OMe, I which after removal of thè terminal protecting group (Z) using hyarogen in the presence,o~ a noble metal catalyst yields the corresponding ! lower alkyl ester of H-D-Trp-D-Phe-OH, preferably H-D-Trp-D-Phe-OMe.
In turn, the latter compound is reacted with an activated ester of Z-D-Lys-OH to give the corresponding lower alkyl ester of Boc Z-D-Lys-D-Trp-D-Phe-OH, preferably Z-D-Lys-D-Trp-D-Phe-CMe. Subsequent Boc Boc removal of the terminal amlno protecting group of the latter compound ¦
(Z) using hydrogen in the presence of a noble metal catalyst gtves ¦ the corresponding lower alkyl ester of H-D-Lys-D-Trp-D-Phe-OH, pre-¦~ 20 Boc ferably H-D-Lrs-D-Trp-D-Phe-OMe. Condensatlon of the last-named ompound with an activated ester of Z-D-TIr-OH yields the corresponding 1¦
~ Z5 ~ But Bo ! ~ Z-D-T~r~D-Lrs-D-Trp-D-Phe-OMe. The latter compound Is treated w7th hydrazine hydrate to obtain the tetrapeptida fragment 4-7 of formula Z~D-TIlr-D-L~s-D Trp-D-Phe NHNH2 --~ 30 ~ Bu Boc 21 ~ ¦
.':: :, .: ~ .
~ ~. . `1.
.: : ` - .,.. , .
"".. ~ , ,, ;,...... ., . : ~ : ; : . ~ , ' : . , In a preferred embodiment of the preparation of'the above tetrapeptide fragment 4-7, substantially equimolar amounts of '' Z-D-Trp-OH, prepared as described for the L-isomer hy E. Klieger, -~
E. Schr~der, and H. Gibian, Justus Liebigs' Ann. Chem., 640, 157 (1961), and ~ Phe-OMe-HCI (see F. Bergel et al, cited above) with an excess, preferably 1.5 to 2.5 molar equivalents, of l-hydroxybenzo-triazole, in an inert organic solvent, prefeably DMF, at -20 to 10C, ; preferably 0C, is treated with an excess, preferably 1.1 to 1.3 molar ; equivalents, of an organic base, preferably N-ethylmorpholine, to pH
7 - 8. A substantially equ~imolar amount of DCC in an inert organic solvent, preferab,ly~DMF, at -10 to 10C, is added dropwise. The .. .. . .
-1 mixture is kept at -20 to 10C for an additional hour, cooled to -10 .i..................................... . .
'~; to 10C, filtered, and the filtrate evaporated. The residue is taken ' up in a subslantially water-imm7scible solvent, preferably ethyl ';~-1 ! ,: .. .
acetate, washed, dried and evaporated. The residue is taken up in "' -' a mixture of a halogenated hydrocarbon solvent, preferably chloroform, -and a lower alkanol, preferably methanol. The solution is passed through a column of sil7ca gel. Evaporatton of the eluate and crystalllzation of the residue ylelds the dipeptlde fragment 6-7 of formula Z-D-Trp-D-Phe-OMe. Said last-named compound is then subjected ~' ' to hydrogenation in the presence of a noble metal catalyst, preferably ' 5~ Pd/C. Methanol, ethanol, acetic acid, or mixtures thereof are conven7ent solvents for this hydrogenation. When acetic acid is used ¦
, the product will be isolated as the acetic acid add7tion salt. ¦
~' 25 Filtration of the catalyst, and evaporation of the filtrate yields the dipeptide fragment 6-7 of formula H-D-Trp-D-Phe-OMe.
i '' :i : ~ :. -..... . ... : . , . : , .
~\HP-6~C~O
1041)623 Said last-named compound in an inert organic solv~nt, preferably DMF, at -20 to 10C, preferably 0C, is treated with an excess, preferably 1.1 to 1.3 molar equivalents, of a strong organic base, preferably N-ethylmorpholine, to pH 7 - 8,the mixture is then treated with a su~stantially molar equivalent of a protected activated ester of D-lysine, preferably Z-D-Lys-ONp prepared in the same manner from Boc D-lysin~ as described for the L-isomer by E. Sandrin and R. A.
Boissonnas, Helv. Chim. Acta., 46, 1637 (1963). The solution is stlrred at about 0C for 30`minutes to two hours, at 20 - 30C for two to four days, and e,vaporated. The residue is taken up in a ~ ~
substantially water-immiscible solvent, preferably ethyl acetate, ~ -washed, dried, and evaporated. The residue is taken up in a halcgenated hydrocarbon solvent, preferably chloroform, a lower alkanol, preferably methanol, and a strong organic base, pre-ferably pyr;dine. The solution is passed through silica gel. , -After evaporation of the eluate the residue is crystallized to ` ¦
yield the tripeptide fragment 5-7 of formula Z-D-L~s-D-Trp-D-Phe-OMe.
Boc Said last-named compound is then subjected to hydrogenation in the presence of a noble metal catalyst, preferably 5~ Pd/C. Methanol, ethanol, acetic acid, or mixtures thereof are convenient solvents ~1 for thts hydrogenation,when acetic acid is used the product will be ~`
isolated as the acetic acid addition salt. FiItration of the catalyst, , .
and evaporation of the fiItrate yields the tripeptide fragment 5-7 of formula H-D-Lys-D-Trp-D-phe-oMe. Said last-named compound, sub-Bocstantially equimolar amount of a protected D-threonine, preferably .
- :
: :~ 23 ~
: - .
.
: . . .` .... . . ... .
~ . .. . , . , . , . :
104~)6Z3 Z-D-TIhr-OH (see E. Schr~der cited above), and about one to two ~ -Bu molar equivalents of l-hydroxybenzotriazole in an inert organic solvent, preferably DMF, at -20 to 10C, preferably 0C, is treated with an excess, preferably 1.1 to 1.3 moiar equivalents, of a strong organic base, preferably N-ethylmorpholine,to pH 7 - 8.
A substantially equimolar amount of DCC in an inert solvent, pre-ferably DMF, at about 0C is slowly added dropwlse. The mixture is stirred at about 0C for 30 minutes to two nours, at 20 - 30C
for one to two hours, fiItered, and evaporated. The residue is .~ 1O J"" ~
taken up in a halogen3ted hydrocarbon sJolvent, preferably chloroform, and a lower alkanol, preferably methanol. The solution is passed .~ . - -:
~` through a column of silica gel. Evaporation of the eluate and crystallization of the residue yields the tetrapeptide fragment 4-7 ~ of formula Z-D-T~r-D-Lyls-D-Trp-D-Phe-OMe. Said last-named compound ,~ ,, is dissolved in an inert organic solvent, for example methanol, ethanol, or DMF, preferably DMF. The solution is treated with an excess of hydrazine hydrate, for example 20 to 50 molar equivalents, . .
and is kept at -20 to 10C, preferably at 0C, for one and a half to three hours, preferably two hours. Water is added; the pre-cipitate is collected by f71tration, dried, and crystallized to 1~ :
, yield the tetrapeptide fragment 4-7 of formula Z-D-T~r-D-Lys-D-Trp-D-Phe-NHNH2 ( I I ,.
~ , With reference to the tripeptTde fragment 8-10, the ~¦ tripeptide is prepared by reacting a protected activated ester of 1~ D-asparagine, preferably Z-D-Asn-OTcp, with a lower alkyl ester of - a protected D-lysine, preferably H-D-Lys-OMe, to obtain the corresponding 1~ Boc 2 ~ .
:~ ................................. .
.. :
..
.. . . .. . . ~. . : ;, .. = .: ` , . ., . . , ~ ~ ..
.. .,, . . ~ . . ., .. ., :
; . ` ; . . . .. . .
` . . .. . .. . . . . . . . .
protected lower alkyl ester of the dipeptide D-asparaginyl-D-lysine, preferably Z-D-Asn-D-Lyls-OMe, which after removal of the terminal amino Boc protecting group (Z) using hydrogen in the presence of a noble metal catalyst gives the corresponding lower alkyl ester of H-D-Asn-D-L~s-OH, ~ Boc preferably Z-D-Phe-D-Asn~D-Lys-OMe. Subsequent removal of the terminal Boc amlno protecting group of the latter compound (Z) using hydrogen in the presence of a noble metal catalyst gives the corresponding lower alkyl ester of H-D-Phe-D-Asn-D-Lyls-OH, preferably the tripeptide fragment -, B c - -8-10 of formuia~H,D~Phe-D-Asn-D-Lrs-OMe (111).
Boc ', ' :
In a preferred embodiment of the preparat70n of the tri-peptide fragment 8-10, substantially equtmolar ambunts of Z-D-Asn-OTcp, ,;
¦ prepared from D-asparagine in the same manner as described for the ~` corresponding L-isomer by J. Beacham, G. Dupuis, F.M. Finn, H.T. Storey, C. Yanaihara, N. Yanaihara, and K. Hofmann, J. Amer. Chem., Soc., 93, 5526 ~1971), an~ H-D-Lys-OMe, prepared from D-lysine tn the same B c monner as described for the corresponding L-isomer by R. Schwyzer and W. Rittel, Helv. Chim. Acta, 44, 159 (1961), in an inert organic I solvent, preferably DMF, at -20 to 10C, preferably 0C, is treated with~a substantiDlly molar equivalent of an organic base,-preferably i : . ~ 1 3~ 25 N-ethylmorpholine and stirred at -20 to 10C, preferably 0C, for ; two to four hours, and then at 20 to 30C for 15 to 30 hours. The solutlon is evaporated, the residue is triturated with an alkyl ether, .1 ,- .
preferably diethyl ether and dried to yield the dTpeptide fragment 9-10 ~`
of formula Z-D-Asn-D-L,ys-OMe. Said last-named compound is then subjected oc 2 ~
r : .
, , - ,, , . , .. , ., .. , ., ,, , . ~ .
1~)4~6Z3 to hydrogenation in the presence of a noble metal catalyst, pre-ferably 5% Pd/C. Methanol, ethanol, acetic acid or mixtures ~ thereof are convenient solvents for this hydrogenation. The catalyst is removed by fiItration, the fiItrate is treated with ~ . .
a substantially equimolar amount of a mineral acid, prefera~ly hydrochloric acid, an a evaporated to yield the dipeptide fragment 9-10 of formula H-D-Asn-D-Lys-OMe as the hydrochloric acid addition --60c salt. Said last-named compound and a substantially equimolar ; amount of Z-D-Phe-OTcp, prepared from D-phenylalanine in the same manner described for the corresponding L-isomer by J. Pless -~-and R. A. Boissonnas, Helv. Chim. Acta.~ 46 IGO9 (1963), in an inert organic solvent, preferably DMF or THF, at -20 to 10C, preferably 0C, is treated with an excess, prefer~bly 1.1 to 1.3 ^1 15 molar equivalents, of a organic base, preferably N-ethylmorpholine. ;~
j The solutlon is stirred at about 0C for 20 to 30 hours. The precipitate is collected by f71tratTon and crystallized to obtain the trtpepttde fragment 8-10 of formula Z-D-Phe-D-Asn-D-Lys-OMe.
80c The latter compound is subjected to hydrogenatton in the presence of a noble metal catalyst, preferably 5% Pd/C. Methanol, ethanol, acet!c actd, or mixtures thereof are convenient solvents for this hydrogenation, when acettc actd ts ùsed the product wtll be tsolated as the acettc acid addttion salt. The catalyst is removed by ~tltrat70n, the filtrate evaporated, the residue taken up in an aromatic hydrocarbon, preferably benzene, and evaporated to obtain the tri-pepttde fragment 8-10 of formula H-D-Pho-D-Asn-D-L~s-OMe ~111). ~
j~ Boc ,~ -1 , .
i. ~ ' .
;
. . , .. ,~ . . ..
.- , . . . . .. . . .
,. . . i . , ~ . . ..
..
` 104~623 j With reference to the fragment HNHCIHCH25 Trt (Vll) in which f:: :
R is CONHCH2CONHCH2CH3, i.e. 1-he fragmant VlIb alternatively written as I H-D-C~s-Gly-NHEt, said fragment is prepared by reacting a protected Trt lower alkyl ester of the dipeptide D-cysteinyl-glycine, preferably !~ -Trt-D-Cys-Gly-oMe~ with ethylamine, to obtain the correspondingly 1 Trt protected ethylamide of the dipeptide D-cysteinyl-glycine, preferably , Trt-D-Cys-Gly-NHEt. Removal of the termTnal amino protecting group ~Trt) using miIdly acidic conditions, preferably a mixture of water and acetic acid or formic acid yields the corresponding addition salt of the fragment H NHC~HCH2S Trt ~VII~ in which R is CONHCH2CONHCH2CH3, ~' . . . .
i.e. H-D-C~s-Gly-NHEt (Vllb).
Trt In a preferred embodiment of the preparation ot the fragment (Vll) in which R Is CONHCH2CONHCH2CH3,the dipeptide Trt-D-Crs-Gly-OMe, prepared from D-cysteine in the same manner I as described for the corresponding L-isomer by G. Amlard, Bull.
;1 Soc. Chim. ~Fr.), 1956, 698, Is treated with a molar excess, preferably 50 - 200 molar equivalents, of ethylamine at -15 to 15C, preferably 5C, for 20 to 30 ~hours. The solution is evaporated, and the residue is dissolved in a solution of a - lower alkyl ester of a lower alkanoic acid, preferably ethyl acetate, and an aromatic hydrocarbon, preferably ben7ene, and purified by chromatography on silica gel to obtain the dipeptide of '~ ~ formul a Trt- D-C~s-Gly-NHEt. Said last-named compound, is subjected ~j ' to miIdly actdic conditions, preferably 70 to 90% acetic acid at ¦~
30 to 50C, preferably 45C, for ten to 20 minutes, preferably ~-- 30 15 minutes. Water is added, the mixture is filtered, the filtrate , ;` 2 7 1 ~
: ~ ,~.
,;., ` ii , - - - - - `= _ ~.
~ ...... .. , . . . , . . . . . . . ......... .... .... .
',: '. ''' - 104~6Z3 is treated with a substantially molar equivalent of a mineral acid, ;~ preferably hydrochloric acid, to obtain the fragment HNHIHCH2STrt (Vll) in which R is CONHCHzCONHCH2CH3 alternatively written as H-D-Cys-Gly-NHEt (Vllb).
The heptapeptide fragment 4-10 is conveniently prepared by coupling the fragment 4-7 and th~ fragment 8-10 according to the azide coupl7ng method in the following manner. A solution of the -tetrapeptide fragment 4 7~of formula Z-D-T~r-D-L~s-D-Trp-D-Phe-NHNH2 (Il), obtatned as desc~SIbéd above, in an inert anhydrous organic solvent, preferably DMF, is cooled to a temperature of from about -30C to ~
about -10C and mixed with a solution of about two to five molar -~ -jJ equivalents, preferably three molar equivalents, of a mineral acid, I
J 15 preferably hydrogen chloride, in an inert anhydrous organic solvent, preferably ethyl acetate. An organic nitrite, preferably t-butyl nitrltTe or isoamyl nitrite in a substantially equimolar amount is added with stirring. The solutTon Is stlrred for 10-30 minUTes, preferably for about 15 mlnutes, at a temperature of from about -20C to about -10C. Keeplng the solution at a temperature of from about -30C to about -10C, a solution of a substant7ally ¦
equimolar amount of the tripept7de fragment 8-10 of formula ¦
H-D-Phe-D-Asn-D-Lyls-OMe (lil), preferably as the acetic acid add7t70n salt obtalned as descr7bed above, and of about three to f7ve molar t~ ~ 25 equivalents, preferably abo_T 3.5 molar equ7valents, of an organ7c , base, preferably N-ethyldi7sopropylam7ne, 7n an 7nert anhydrous ;
organ7c solvent, prefe mbly DMF, 7s added slowly with stirr7ng. -~
,:
' :', - 2~ ~1 .,~ ~ .... j~ .
,~, ~.. ,......... . ~ ~ - .- .. . . ... . .
~he mixture is stirred for 30 - 60 rninules at about -20C to about -10C, then at about 2~ to 30C for 20 to ~0 hours. The solution is evaporated and the resid~Je is triturated with cold aqueous citric acid (0.5 to 2N), water and dried. The residue is taken up in a mixture of a halogenated hydrocarbon, preferably ch!oroform, and a lower alkanol, preferably methanol, and purifie~ by chromatography on silica gel. The solvent is evaporated and the residue crystallized to yleld the heptapeptide fragment 4-10 of formula Z-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-oMe.
Bl Boc Boc Said last-named ~ompound is subjected to hydrogenation in the presence of~a;n~ble metal catalyst, preferably 5% Pd/C.
Methanol, ethanol, acetic acid, or mixtures thereof are suitable solvents for this hydrogenation, and when acetic acid is used the '3 15 product is isolated as the acetic acid addition sait. The catalyst , 1s removed by filtration; the filtrate evaporated, the residue taken up In an aromatic hydrocarbon, preferably benzene, evaporated, and 1 drled over strong alkali, preferably potassium hydroxide or sodium hydroxlde, to obtain the heptapeptide frag~ent 4 10 of formula H-D-T~rD-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe (IV).
The decapeptide fragment~~l-10 is conveniently prepared by coupling the fragment 1-3 and th~ fragment 4-10 according to -- the azide coupling method in the following manner. A solution of ~j 25 the trTpeptide fragment 1-3 of formula ~- -S CH2CH2CO D Sltr D Tltr-D-Phe-NHNH2 (V), obtained as described . . .
~~ above, in an inert anhydrous solvent, preferably DMF, is cooled tc ~, -.:: - ~. :a temperature of from about -30C to about -10C and mixed with a `~
solution of about two to five molar equivalents, preferably three molar equivalents, of a strong mineral acid, preferably hydrogen hloride, in an anhydrous organic solvent, preferably ethyl acetate.
2~ ~:
. 1 ' .
- . i .- -. . , ,. .. . .. .. ., ., ,. ., : .
. .~; ~. ~ . , .. . ` ,, . :. . .
-: . : .. . , . , : ` - . . .
AHP-~550 An organic nitrite, preferably t-butyl nitri1e or isoamyl n7trite, in a substantially equimolar amount, is added with stirring. The solution is stirred for lO - 30 minutes~ preferably for about 15 minu~es, at a temperature of from about -20C to about -10C.
Keeping the solution at a temperature of from about -30C to about -10C, a solution of a substantially equimolar amount of the heptapeptide fragment 4-10 of formula H-D-Thr-D-Lys-D-Trp-D-Phe-D-Bl Boc Phe-D-Asn-G-Lys-OMe ~IV) obtained as described above, and of about Boc :; 10 ~`
about three to five molar e!quivalents, preferably about 3.5 molar equivalents, of ~a~ organic base, preferably N-ethyldiisopropylamine, in an inert anhydrous organic solvent, preferably DMF, is slowly added with stirring. Stirring of the resulting mixture is continued for 30 - 60 minutes at about -20C to about -10C, then at about 2~ to 30C for 20 to 30 hours, The solution is evaporated, the residue is trlturated with cold aqueous citric acid (0.5 to 2N), water and dried. The residue is taken up in a mixture of a halogenated ' hydrocarbon, preferably chloroform, and a lower alkanol, preferably methanol, purified by~chromatography on silica gel and crystallized i20 to yieid the decapeptide fragment 1-lO of formula Trt-S-CH2CH2CO-D-Sir-D T~tr D Phe D-Thit-D-L~5-D-Trp-D-phe-D Phe_D_A5n-D-Lys-oMe.
Bu Bu B c Boc Said last-named compound is taken up in an inert organic solvent, for example methanol, ethanol, or DMF, preferably DMF, and ` treated with an excess of hydrazine hydrate, for example 20 to 50 molar equivalents. The mixture is kept at -20 to 10C, preferably .`~ . ' . ' , .
, .
.
.
: :
'~' ' "
; 0C, for 15 to 45 minutes and at 20 to 30C, for 20 to 30 hours. , Water is added, the precipitate is collected by fiItration, washed - with water, and dried to yield the decapeptide fragment 1-10 of formula Trt-S-CH2CH2CO-D-Ser-D-Th,r-D-Phe D-T~r-D-Lys-D-Trp-D-Phe~-Phe- '' Bl B~t Blt Boc ~' D-Asn-D-Ly,s-NHNH2 (V I ) . . .
Boc `-The linear peptide Vlll is convenient;y prepared by coupling the fragment 1- ! (Vl) and the fragment ~11) according to the azide ', coupling method in the fo~'lowing manner. A solùtion of the decapeptide fragment 1-10 (Vl)~ obtained as described above, is taken up in an inert anhydrous organ?c solvent, preferably a mixture of DMF and DMSO, cooled to a temperature of from about -30C to about -10C and mixed i with a solution of about two to five molar equivalents, preferably three molar equivalents of a mineral acid, preferably hydrogen chloride, in an anhydrous organic solvent, preferably efhyl acetate. -A sùbstantially equimolar amount of an organTc nitrite, preferably t-butyl nitrite or tsoamyl nitrite,is added, and the solution is stlrred for 10-30 mlnutes, preferably for about 15 minutes, at a ; temperature of from about -20C to about -10C. Keeping the stirred '-!; solution at a temperature of from'ai30ut -30C to about -10C, a soiution of a substantially equimolar amount of the fragmen~ ; ~' HNHCHeH2STrt (Vll) in which R is CONHCH2CONHCH2CH3(Vllb) obtained as des- ~i~
cribed above or the fragment HNH~HCH2STrt (Vll) in which R is hydrogen (Vlla) - :~ 25 ~ ~ R
described by F. 1. Carroll et al., J. Org. Chem. 30, 36 (1965)], and of about three to five molar equivalents, preferably about 3.5 molar equivalents, of an organic base,~preferably N-ethyldiisopropylamine, ' ~' ~
. , : ': :.
: ~ : :~ :.-, :.
,~
3 1 ::
~-' -, ` . ,., : .::
' :.
, .
104~)623 in an inert anhydrous organic solvent, preferably DMF, is added slowly. Stirring of the resulting mixture is continued for 45 - 75 minutes at about -20C to aboul -10C, then at about 2Q to 30C
. for 20 to 30 hours. Evaporation of the solution, trituration of , 5 the residue with cold aqueous citric acid (0.5 to 2N), water, methanol, and drying yields the corresponding linear protected peptlde of formula (Vlll) Trt-S-CH CH C0-D-S~r-D-T~r-D-Phe-D-Thr-D-Ly,s-D-Trp-D-Phe~D-Phe~Asn~-L~s-NH~HCH25 Trt (Vlll) 7n which R is as "4; Boc Boc R
o " ~
defined herein. J
The conversion of the above linear protected peptide, 1 obtained as described above, to the compound of formula I is ~ ;~
3' accomplished conveniently and efficiently by first subjecting the linear protected peptide to the action of iodine, preterably in the presence of a lower alkanol or a lower alkanoic acid whereby slmultaneous removal of the sulfhydryl protecting groups, i,e. Trt, ~ and formatlon of tne disulflde brldge occurs to glve the corresponding I cycli~c disulfide of formula IX
S-CH2CH2C0-D-Sfr-D-TIr-D-Phe-D-T~r~-D-Llys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-Bu Bu Bu Boc B c ~; N ~HCH2~ (IX) in which R is as defined hercin, subsequent treafment of j~ ~ of the latter compound under moderately acidic conditions removes the 3- ~ ~ remaining protecting groups (i.e. Boc and Bu ) to gTve the correspondlng compound of formula 1.
- J :: ~
,~, .
., - . :
, . i . ~ , .
,.' : ~ ' :
,~ -32-j,,, : . ~ . .
: 104~623 In a preferred embodiment of the above transformation, the Iinear protected peptide (Vll!) is dissolved in a lower alkanol or a lower alkanoic acid, preferably acetic acid, and added to an 1;
excess of iodine (5 to 25 molar equivalents, preferably 10 mclar equivalents) dissolved in a lower-alkanol or a lower alkanoic acid, preferably methanol, at a concentration of about 2 - 5~ iodine.
The time and temperature of this reaction is not critical; however, it is desirable to keep the reaction between 0 and 30C by regulat7ng the addition to the iodine solution or by cooling of the reactton mixture, or by a combination of both. Under these ¦-~ conditions the addition usually takes 30 to 60 minutes. After the ,J' addit7On the mixturé is stirred at 20 to 30C for 30 to 120 minutesJ
1 preferably 60 minutes. Thereafter the mixture is cooled to about 0C -i and an excess of a miId reducing agent, preferably sodium thiosulfate in ~
aqueous solution is added. The mixture is concentrated and the residue Is suspended in water. Collection of the solid material affords the deslred corresponding cyclic disulfide of formula IX.
7 Alternatively, the linear protected peptide (Vll) is converted to the aforementioned corresponding cyclic disulfide by the method of ¦ 20 R.G. Hlskey and R. L. Smith, J. Amer. Chem. Soc., 90, 2677 (1968) using thlocyanogen. ;
Again alternatively, the cyclic disulfide (1X) is also obtained by selectively removing the sulfhydryl protecting groups of t,le above - ¦
linear protected peptide (Vlll)by the action of a mercuric or silver salt, !
for example, mercuric acetate, mercuric chloride, silver acetate or silver nitrate, in an inert organ7c solvent, for example DMF or acetic acid, according to known methods; for example, see B. Kamber, and N. Rlttel, Heiv. Chem. Acta, 52, 1074 tl964), L. Zervas, et al., J. Amer. Chem.
- Soc.,87, 4922 (1965) and R. G. Denkewalter et al., ~. Amer. Chem. ~
.: ~ - . . : :' Soc., 91, 502 (1969). -The mercuric or disilver salt thus obtained is ~ . :
., , - :.
then converted to the corresponding free disulfhydryl derivative (X) 2 2 Itr D T~tr D Phe D-TIhr-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHCHCH2SH (X)in which R is as defined herein B c by treatment with hydrogen sulfide, see L. Zervas, et al., cited above.
The latter derivative is then converted to the aforementioned cyclic disulfide ~IX) by a mild oxidizing agent selected from the group consisting of iodine according to the method described hereinbefore, oxygen according to the~me~thod of J. Rivier, et al., C.R. Acad. Sci.
Ser. D, 276, 2737 (1973), 1,2-diiodoethane according to the method of , , ~,:
F. Weygand and G. Zumach, Z. Naturforsch~ 17b, 807 (1962), and sodium or potassium ferricyanide according to the method of D. Jarvis, et al., J. Amer. Chem.-Soc., 83, 4780 (1961).
,, Finally, the aforementioned cyclic disulfide of formula IX is transformed into the corresponding cyclic peptide of formula 1, by subJecting the former to moderately acidTc conditions whereby the re~aining protecting groups of tne cyclic disulfide (IX) are removed.
~, Generally this step is carried out by treating the cyclic disulflde (IX) wlth 50 - 100~ trifluoroacetic acid or with an aqueous solution con-I tain'ng a mineral acid at 0 to 20C for 10 to about 60 minutes~
.j . . ~ . ~:Examples of such mineral acids are 10 to 20% aqueous sulfuric ac7d, -~ 10% phosphoric acid, 10 - 30% hydrobromic acid and 10 to 36% hydrochloric ; acid. An extremely useful acid is concentrated hydrochloric acid.
i Preferred conditions for the present step include dissolving ~he cyclic di 5U Ifide in a minimum of concentrated hydrochloric acid cooled to O~C and stirring the mixture at 0C for five to ten minutes under a nitrogen atmosphere. Thereafter glacial acetic acid ,..
,, - , ,~
,,, , .
.
.
..
-3~-, .
. . . . ~ .
;: . . .
; ' ' ' '. , ' ' ,'~; : ~
._ 104~623 (10 X vols.) is added, the solution is cooled to about -70C and Iyophilized to give the corresponding cyclic peptide of formula The latter product is purified further by ion exchange chromatography using a carboxymethylcellulose cation exchanger and ammontum acetate as the eluant. In the latter case the product Ts obtained in the form of its acid addition salt with acetic acid. Alternatively, the product is purified by partltlon chromatography on a chemically modified cross-llnked dextran, for example Sephadex*LH 20 or Sephadex G-25, using methanol or acetlc acld, respectively, as the eluting solvent. In the case where Sephadex*LH-20 and methanol as the eluting solvent is employed, the product Ts obtalned In the form of its hydrochloric acid addition salt. In the case where - -Sephadex G-25~,and acet7c acid is employed, the product is obtained in the form of its acetic ac1d addltion salt. Repeated IyophllizatTon .. .
from water of the product In the form of its acetic acid additlon salt ! ylelds the substantlally pure cycllc peptlde of formula I in which R Is as deflned hereln, In the form of the free base.
The llnear reduced form of the peptlde of formula la -is obtained by removal of the protectlng groups from the aforem,entioned llnear protected peptide of formula (Vl~
~, Trt-S-CH2CH2CO-D-Selr-D-T~r-D-Phe-D-T~,r-D-Lys-D-Trp-D-phe-D-phe-D-Asn 81 BL Bl Boc D-Lys-NHCHCH2S Trt (Vll~) !~ 80c R ~ ~
in whlch R is as defined hereln from the aforementloned dlsulfhydryl ~;j derivatlve of formula (X) t 8~t D T~t~ D-L~s-D-Trp-D-Phe-D-Phe-D-Asn D-Lys-NH~HCH2SH (X) -' -Boc R
-, * Sephadex is a trade mark ~35~
AI~P-6550 ` 1041)623 in which R is as defined herein under moderately acidic conditions.
Preferred conditions for this deprotection step comprises dissolving the linear protected peptide (Vlll) or the disulfhydryl derivative (X) in concentrated hydrochloric acid at 0 to 5C in an inert atmosphere, for example, nitrogen or argon. The mixture is kept at this temperature for five to ten minutes. Subsequent isolation of the -linear reduced form is accomplished in the same manner as described previously for the isolation of the cyclic peptide of formula 1.
Also, the linear reduced form (la) is obtained directly lo by reduction of the cycltc peptide of formula 1. Reduction with dithiothreitol according to the method~of W.W. Cleland, Biochem. _, , 480 (1964) is preferred, although other agents known to be effective j for the reduction of cyclic disulfides to the correspondTng disulf-hydryl derivative are applicable, for example, sodium bisulfite ~ -followed by hydrolysis of the intermediate dithiosulfate derivat.ive.
j Finally it will be apparent to those skilled in the art J fhat: equ7valent amino, hydroxy or thtol protecting groups, ¦ ~ equlvalent methods of coupling peptide fragments, and equlvalent ¦ metnods for removal of amlno, hydroxy or thiol protecting groups, j 20 other than those disclosed herein could be used in the embodimentsof this tnvention without departing from the scope and spirit of the tnventTon. Such apparent alterations are intended to be included withtn the scope of this invention. ~ -The following flow diagrams and examples illustrate further this invention.
.
::~
.
. . .
!
. ' .. , . ... ,- . .. ... ... . . .
, . . .. .. . .. . . .. .
:
.: .
4 5 6 7104~623 8 9 10 Z-D-T~r-D-L~s-D-Trp-D-Phe-NHNH + H-D-Phe-D-Asn-D Lys-OMe Bu Boc Boc .
4 5 6 7 ~ 9 1 o : ~ -~: H-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe Blt Boc . Boc ; ~ :
'. ( l V ) ~
::
.1 1 2 3 . ~ Trt-S-CH2CH2CO-D-Ser-D-T~r-D-Phe-NHNH :
~ ~S'" ~) , J ~---- ~
< ,~
i 1 2 3 4 S 6 7 8 9 IO
r-D-Tlr-D-phe-D-T~r-D-Lyls-D-Trp-D-phe-D-phe-D-Asn-D-Lys-oMe Bu B Bu Boc Boc Corresponding hydrazide (Vl) 1 ~ + HNHCH2CH2STrt or + HNH-IH-CH2S-Tr~
¦ ~ ~Vllaj ONHCH2CON~H2CH3 -alternatively written as \ H D-C~s-Gly-NHEt b Linea~r protected peptid~e (V~ L~ :
(R = ~ \ (R = CONHCH2CONHCH2CH3' idation \ I) Ag or Hg / \ 2) H2S
dation ~ ~ . .
. Cycl7c disul ide derivative (IX) Disulfhydryl derivative (X) : Reduction /
Depr tection Depl otection / . ~ :
Reduction~
- Cyclic peptide (I) Linear reduced for~ of peptide (la) ~ ~ ~ Oxidation i:~, . : : . :
' . : .. -~: : ' ,"' :'' -.
'I ~ . , . ' . , ,, ' , , . , ' , ~ ' " " " ~ "
~04~)623 ,,~ ~.
t -t Z ~ / ~ _ ~
~0 ~m~m ;i~ t~ i~m ~,m ~ m ~m~ ~
_ _ . _ _ _ O ~ ~ ~ s I ~¦ --I ~¦
=1 ~ ~ lo~
~ I ¦ ¦~ ¦ 1~ ¦~
L ~ ~m ~ ~ ~m '~ ~ O ~ O O Z , ~ C~ .
T/m 1~ l~o /m /m ~ /a~ ~ ~ _ I , N
-:
3û-1040623 ~..
- EXAMPLE-I - -.
BenzYloxvcarbonYI~ t-butYl)-D-threonYl-l3-phenylalan7ne MethYI Ester A' . (Z-D-Tt)r-D-Phe-OMe j Blt . . - I
A mixture of Z-D-Ttlr-OH (3.1 9, 10 mmoles) and h-D-phe-oMe-H
Bu ;;~
(2.16 9, 10 mmoles) in THF (15 ml) is cooled to 0C and N-ethylmorpholine (1.8 ml) added to attain a pH of 7 - 8. I-Hydroxybenzotriazole (1.4 g, 10.3 mmoles) is added followed by dropwise addition of DCC t2.1 9, 10 1O mmoles) in THF (20 ml). The mixture is stirred at 0C for 45 min and at T room temperature for I hr. After filtration, the filtrate is con-centrated under reduced pressure and the~residue dissolved in ether~
1 After fiItration of the precipitate, the fiItrate is washed with t saturated NaHC03 solution,saturated NaCI solution, 5~ aqueous citric, 15 acid solution, and saturated NaCI solution. The ether solution is dried .
over Na2S04 and evaporated under reduced pressure. The residue is subjected to chromatography on silica gel (200 9) using 25% EtOAc in !~ -hexane. Evaporation of tne solvent under reduced pressure g7ves the tl~le compound as an oil, EOl]D = -25.1 tc = 1, DMF~, nmr 20 tCDC13) ~ 1.08 (d,J = 6.5Hz, 3H), 1.15 (S, 9H), 3.70 (s, 3H), 5.18 ;~ (s~ 2H), 7.35 tm, IOH).
;, - . .. `~.` "
.~ . :
~ ~ '''' ~1 : ' : '' t ~
1 . :
'. .: '-. . l . ~
.,, . , - .. ~ : . . : . . .. . .
Benzyloxvcarbonvl-(O-t-butYI)-D-serYl-(O-t-butYl)-D-threon~l-D~phenvl-- alanine MethYI Ester (Z-D-Se,r-D-Thr-D-Phe-OMe) ., Blt B¦t , ..
Z-D-T~r-D-Phe-OMe (7.5 9, 15.9 mmoles, described in Example 1) Bu - dissolved in MeOH (90 ml) containing pyridine hydrochloride (1.83 9, 15.9 ' mmoles) is hydrogenated with 5% Pd/C as a catalyst. The mixture is flItered and the fiItrate taken to dryness under reduced pressure to givè H-D-Thr-O-Phe-OMe-HCI. The above product and Z-D-Ser-OH
~ 10 t4.67 9, 15.9 mmoles) are d7ssolved in dry THF (45 ml), cooled to OqC
'' and 2.7 ml N-ethylmorphol7ne'is added. I-Hydroxybenzotriazole (2.16 9, . . .
15.9 mmoles) is addéd followed by a cold ~0C) solution of DCC (3.27 9, 15.9 mmoles) in THF (30 ml). The mixture is stirred for 45 min at 0C
and then I hr at room temperature. After filtratlon, the THF is removed under reduced pressure, the residue taken up in ether and filtered.
I The ftItrate Is washed with saturated NaHC03 solution, saturated NaCI
¦ solution, ice-cold 5% citric acid solution, saturated NaCI solution,satu'rated NaHC03 solution and saturated NaCI solutlon. The resldue (9.2 9) obtained after drylng the ether layer wlth Na2S04 and evaporating under reduced pressure Is subjected to chromatography on a column of slllca gel (200 9) using 30% EtOAc !n hexane. The solvent is evaporated `~ under reduced pressure to glve the title compound as an oil, nmr (CDC13)j 1.05 ~d, J = 6.5 Hz, 3H), 1.18 (s, 18H), 3.75 (s, 3H), 5.18 (s, 2H),'3~ '25 7.4 (m, IOH).
~. -~40- ` ' , 104~)623 (O-t-butyl)-D-seryl-tO-t-butYI)-D-threonyl-D-phenYlalanine MethYI Ester Acetate (H D-S~t D Tlht 3 2 ;
Z-D-Ser-D-Thr-D-Phe-OMe (8.4 9, 13.6 mmoles, described in Blt B¦t . , Example 2), dissoived in acetic acid (84 ml), is hydrogenated with 5%
Pd/C as a catalyst for 20 hr. The mixture is filtered and the fiitrate - taken to dryness under reduced pressure to gtve the title compound as an oil: nmr (CDC13) ~ 1.05 (d, J = 6.5 Hz, 3H), 1.19 (s, 18H), 2.06 (s, 3H), 3.76 (s, 3H).
., ~
1 . ' '' ''~'~ ' .~
.
':
.' ., . ~
,- . ~
:
' ' :
104~623 N-rl-oxo-3-(tritvl)thiolpropvl-(O-t-h~ltYl)-D-seryl-(o-t-buty!---D-threon D-phenylalanine Mel-hYl Ester (Trt-S-CH2 H ~ Tr-D-T~r-D-Phe-~Me) A solutior, of H-D-S~r-D-TIhr-D-Phe-OMe-CH3CO2H (1'.6 mmoles described in Example 3) in dry THF (25 ml) is cooled to 0C
and N~ethylmorphollne (1.7 ml) added until pH 7 is attained. A
solution of 3-tritylthiopropionic acid (4.7 g, 13.6 mmoles) in THF
(25 ml) is added followed ~by l-hydroxybenzotriazole (1.~ g, 13.6 mmoles) , and DCC (2.8 9, 13.6 mmoles~ in THF (25 ml). The mixture is stirred at 0C for 45 min "and at room temperatur0 for 2 hr. The precipitate is removed by filtration, and the filtrate is concentrated under reduced pressure. The residue is dissolved in ether and the precipitate ` 15 removed by fiItra~ion. The fiItrate is washed with saturated NaHC03 ¦ solution, saturated NaCI solution, cold 5% citric acid solution, saturated ¦ NaCI solution, saturated NaHC03 solution and saturated NaC! solution.
The ether solutton Is dried over Na2S04 and evaporated under reduced pressure. The residue is subjected to chromatography or a column of silica gel (220 g) using 30% EtOAc-in benzene containing 0.2% tri~thyl-amine. After evaporation of the eluates, under reduced pressure the residue j~5 crystallized from ether to give the title compound, mp 117 -125C [~]D = 18.6 ~c = 1, DMF)~
, ~ ''' ,~- ' - , j - .. : .
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, . . .
~: . : .' . ~ ' N-rl-oxo-3-(trityl)thiolpropyl-lo-t-bu1-yl)-D-seryl-(o-t-butyl)-D--hreon D-phenvlalanine Hydra~ide (V, Trt-~-CH2__ ~ elr-D-TIr~D~phe--NHNH2) H2 2 D Sltr D T~r-D-Phe-OMe (2.025 g, described in Example 4) in MeOH (70 ml) and hydrazine hydrate (2 ml) is stirred at 0C for I hr and at room temperature for 24 hr. The precipitate which is obtained upon addttion of water is collected on a sTntered glass flIterand dried over phosphorus pentoxide to give the title compound;
nmr (CDC13) ~ 0.98 (s, 3H),I1.16 (s, 18H), 7.4 (m, 20H). ~ -. ' " J ,'~ ~ -, ' ~ ' ' '. ' ' '' 4, 1 '. ':
i j, 1'.'"
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: . `, : - :, . , ` , . . . .. :.:i 104~623 EXAMPLF=_ Benzyloxycarbonvl-D-tryptophyl-D-phenylalanine Methvl Ester (Z-D-lrp-D-Phe-OMe) A solution of Z-D-Trp-OH (3.62 9, 10.7 mmoles), H-D-Phe-OMe-HCI
(2.3 9, 10.7 mmoles) and l-hydroxybenzotriazole (2.89 9, 21.4 mmoles) in dry DMF (25 ml) is cooled to 0C and N-ethylmorpholine (1.37 ml, 10.7 mmoles) is added. A cold (0C) solution of DCC (2.27 9, 11 mmoles) In DMF (6 ml) is added dropwise and the reaction mixture is stirred for I hr at 0C and I hr at room temperature. The reaction mixture is then cooled to 0C, fiItered, the fiItrate evaporated under reduced pressure and taken up in EtOA~. The EtOAc solution is washed with saturated NaHC03 soii~tion, water, cold citric acid (2N), water, saturated NaHC03 solution and saturated NaCI solution, dried with MgS04, and evaporated under reduced pressure to afford the crude title product I
whtch is subjected to chromatography on a co;lumn of silica gel '1 . : .
~500 9) with CHC13 containing MeOH (1%) as eluent. Tha eluates are evaporaled under reduced pressure and the residue is crystallized ;~ from EtOAc-petroleum ether to give the t;tle compound; mp 130 - 131C, [~X]D5 = +34.1 (c = I, DMF).
: .
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~ 550 .
104()~
BenzyloxYcarbon~l-N~-t-butvloxycal-bonyl-D- Iysvl-D-trY~tor)hy!-D-phenylalanine Methyl Ester (Z-D-Lys-D-Trp-D-Phe-O~le) Boc A mixture of Z-D-Trp-D-Phe-OMe (2.00 g, 4.0 mmoles, described -I in Example 6) and 5% Pd/C (0.25 g) in acetic acid is stirred rapidly under an atmosphere of hydrogen. After completion of hydrogen uptake a stream of nitrogen is passed tllrough the mixture for 15 m7n, the catalyst is removed by filtration, and the acetic IO acid is rcmoved under reduced pressure. The residue is taken ; 7n~o benzene, evaporate~d under reduced pressure (twice), and dried under reduced pressure over KOH pellets to give H-D-Trp-D-Phe-~Mé CH3C02H. The above product is dissolved in ! dry DMF (20 ml) and cooled to 0C. N-ethylmorpholine (0.512 ml, 4 mmoles) is added, followed by Z-D-L~s-ONp (2.00 g) 4 mmoles). -`~
Boc .i, : .
The solutTon is stirred at 0C for 30 min and for 3 days at room temperature. The solvent is evaporated under reduced pressure, the res7due i~ taken Tnto EtOAc and washed with cold sa1urated NaHC03 solutlon, water, cold citric actd (2N), water, saturated NaHC03 solution and saturated NaCI solution. The EtOAc solution is dried with MgS04 and evaporated under reduced pressure to ~;~
afford the crude title product. After chromatography on a column of silica gel ~300 9) with CHCI3 containing MeOH (5%) and pyridine 2 (0-1%) as eluent, the chromatographically pure product is triturated ~ I
with ether, filtered, dried under reduced pressure, and crystallized from MeOH-CH2CI2-isopropyl ether to give the title compound; mp 172 -174C, [~]D = +18.9 (c = I, DMF).
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' :' ~ .: ` :.' ' .' ., ' ' B~IP-6550 104~6Z3 N~-t-ButyloxYcarbonv!-D-lYsYI-D-tryptophyl-D--p-enylalanine Methyl-Ester Acetate (H-D-L~vs-D-Trp-D-Phe-OMe CH3CO
` Boc - --A mixture of Z-D-L~s-D-Trp-D-Phe-OMe (2.218 9, 3.04 mmoles, :~
B c ~: described in Example 7) and 5% Pd/C (0.25 g) in acetic acid is ~ rapidly stirred under an atmosphere of hydrogen for 20 hr. The '~, catalyst is removed by filtration and the filtrate evaporated under reduced pressure to give the title compound as an oil~
nmr (CDC13) ~ 1.4 (s, 9H), 2.07 (s, 3H), 3.6 (s, 3H). -", , .
, . ' ., - ~''-'.
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~ 1 : ~ . . . . :, :.- . , ~ -~ 1040623 BenzvloxYcarbonY!-(O-t-butYI)-D-threonyl-N~-t-butYlox~arb D-lYs~l-D-t~y~tophvl-D-phenvlalanine Methvl Ester (Z-D-Thr-D-Lys-D-Trp-D-phe--oMe) ~ BLt B~c
4 5 6 7 ~ 9 1 o : ~ -~: H-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe Blt Boc . Boc ; ~ :
'. ( l V ) ~
::
.1 1 2 3 . ~ Trt-S-CH2CH2CO-D-Ser-D-T~r-D-Phe-NHNH :
~ ~S'" ~) , J ~---- ~
< ,~
i 1 2 3 4 S 6 7 8 9 IO
r-D-Tlr-D-phe-D-T~r-D-Lyls-D-Trp-D-phe-D-phe-D-Asn-D-Lys-oMe Bu B Bu Boc Boc Corresponding hydrazide (Vl) 1 ~ + HNHCH2CH2STrt or + HNH-IH-CH2S-Tr~
¦ ~ ~Vllaj ONHCH2CON~H2CH3 -alternatively written as \ H D-C~s-Gly-NHEt b Linea~r protected peptid~e (V~ L~ :
(R = ~ \ (R = CONHCH2CONHCH2CH3' idation \ I) Ag or Hg / \ 2) H2S
dation ~ ~ . .
. Cycl7c disul ide derivative (IX) Disulfhydryl derivative (X) : Reduction /
Depr tection Depl otection / . ~ :
Reduction~
- Cyclic peptide (I) Linear reduced for~ of peptide (la) ~ ~ ~ Oxidation i:~, . : : . :
' . : .. -~: : ' ,"' :'' -.
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~04~)623 ,,~ ~.
t -t Z ~ / ~ _ ~
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_ _ . _ _ _ O ~ ~ ~ s I ~¦ --I ~¦
=1 ~ ~ lo~
~ I ¦ ¦~ ¦ 1~ ¦~
L ~ ~m ~ ~ ~m '~ ~ O ~ O O Z , ~ C~ .
T/m 1~ l~o /m /m ~ /a~ ~ ~ _ I , N
-:
3û-1040623 ~..
- EXAMPLE-I - -.
BenzYloxvcarbonYI~ t-butYl)-D-threonYl-l3-phenylalan7ne MethYI Ester A' . (Z-D-Tt)r-D-Phe-OMe j Blt . . - I
A mixture of Z-D-Ttlr-OH (3.1 9, 10 mmoles) and h-D-phe-oMe-H
Bu ;;~
(2.16 9, 10 mmoles) in THF (15 ml) is cooled to 0C and N-ethylmorpholine (1.8 ml) added to attain a pH of 7 - 8. I-Hydroxybenzotriazole (1.4 g, 10.3 mmoles) is added followed by dropwise addition of DCC t2.1 9, 10 1O mmoles) in THF (20 ml). The mixture is stirred at 0C for 45 min and at T room temperature for I hr. After filtration, the filtrate is con-centrated under reduced pressure and the~residue dissolved in ether~
1 After fiItration of the precipitate, the fiItrate is washed with t saturated NaHC03 solution,saturated NaCI solution, 5~ aqueous citric, 15 acid solution, and saturated NaCI solution. The ether solution is dried .
over Na2S04 and evaporated under reduced pressure. The residue is subjected to chromatography on silica gel (200 9) using 25% EtOAc in !~ -hexane. Evaporation of tne solvent under reduced pressure g7ves the tl~le compound as an oil, EOl]D = -25.1 tc = 1, DMF~, nmr 20 tCDC13) ~ 1.08 (d,J = 6.5Hz, 3H), 1.15 (S, 9H), 3.70 (s, 3H), 5.18 ;~ (s~ 2H), 7.35 tm, IOH).
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Benzyloxvcarbonvl-(O-t-butYI)-D-serYl-(O-t-butYl)-D-threon~l-D~phenvl-- alanine MethYI Ester (Z-D-Se,r-D-Thr-D-Phe-OMe) ., Blt B¦t , ..
Z-D-T~r-D-Phe-OMe (7.5 9, 15.9 mmoles, described in Example 1) Bu - dissolved in MeOH (90 ml) containing pyridine hydrochloride (1.83 9, 15.9 ' mmoles) is hydrogenated with 5% Pd/C as a catalyst. The mixture is flItered and the fiItrate taken to dryness under reduced pressure to givè H-D-Thr-O-Phe-OMe-HCI. The above product and Z-D-Ser-OH
~ 10 t4.67 9, 15.9 mmoles) are d7ssolved in dry THF (45 ml), cooled to OqC
'' and 2.7 ml N-ethylmorphol7ne'is added. I-Hydroxybenzotriazole (2.16 9, . . .
15.9 mmoles) is addéd followed by a cold ~0C) solution of DCC (3.27 9, 15.9 mmoles) in THF (30 ml). The mixture is stirred for 45 min at 0C
and then I hr at room temperature. After filtratlon, the THF is removed under reduced pressure, the residue taken up in ether and filtered.
I The ftItrate Is washed with saturated NaHC03 solution, saturated NaCI
¦ solution, ice-cold 5% citric acid solution, saturated NaCI solution,satu'rated NaHC03 solution and saturated NaCI solutlon. The resldue (9.2 9) obtained after drylng the ether layer wlth Na2S04 and evaporating under reduced pressure Is subjected to chromatography on a column of slllca gel (200 9) using 30% EtOAc !n hexane. The solvent is evaporated `~ under reduced pressure to glve the title compound as an oil, nmr (CDC13)j 1.05 ~d, J = 6.5 Hz, 3H), 1.18 (s, 18H), 3.75 (s, 3H), 5.18 (s, 2H),'3~ '25 7.4 (m, IOH).
~. -~40- ` ' , 104~)623 (O-t-butyl)-D-seryl-tO-t-butYI)-D-threonyl-D-phenYlalanine MethYI Ester Acetate (H D-S~t D Tlht 3 2 ;
Z-D-Ser-D-Thr-D-Phe-OMe (8.4 9, 13.6 mmoles, described in Blt B¦t . , Example 2), dissoived in acetic acid (84 ml), is hydrogenated with 5%
Pd/C as a catalyst for 20 hr. The mixture is filtered and the fiitrate - taken to dryness under reduced pressure to gtve the title compound as an oil: nmr (CDC13) ~ 1.05 (d, J = 6.5 Hz, 3H), 1.19 (s, 18H), 2.06 (s, 3H), 3.76 (s, 3H).
., ~
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104~623 N-rl-oxo-3-(tritvl)thiolpropvl-(O-t-h~ltYl)-D-seryl-(o-t-buty!---D-threon D-phenylalanine Mel-hYl Ester (Trt-S-CH2 H ~ Tr-D-T~r-D-Phe-~Me) A solutior, of H-D-S~r-D-TIhr-D-Phe-OMe-CH3CO2H (1'.6 mmoles described in Example 3) in dry THF (25 ml) is cooled to 0C
and N~ethylmorphollne (1.7 ml) added until pH 7 is attained. A
solution of 3-tritylthiopropionic acid (4.7 g, 13.6 mmoles) in THF
(25 ml) is added followed ~by l-hydroxybenzotriazole (1.~ g, 13.6 mmoles) , and DCC (2.8 9, 13.6 mmoles~ in THF (25 ml). The mixture is stirred at 0C for 45 min "and at room temperatur0 for 2 hr. The precipitate is removed by filtration, and the filtrate is concentrated under reduced pressure. The residue is dissolved in ether and the precipitate ` 15 removed by fiItra~ion. The fiItrate is washed with saturated NaHC03 ¦ solution, saturated NaCI solution, cold 5% citric acid solution, saturated ¦ NaCI solution, saturated NaHC03 solution and saturated NaC! solution.
The ether solutton Is dried over Na2S04 and evaporated under reduced pressure. The residue is subjected to chromatography or a column of silica gel (220 g) using 30% EtOAc-in benzene containing 0.2% tri~thyl-amine. After evaporation of the eluates, under reduced pressure the residue j~5 crystallized from ether to give the title compound, mp 117 -125C [~]D = 18.6 ~c = 1, DMF)~
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, . . .
~: . : .' . ~ ' N-rl-oxo-3-(trityl)thiolpropyl-lo-t-bu1-yl)-D-seryl-(o-t-butyl)-D--hreon D-phenvlalanine Hydra~ide (V, Trt-~-CH2__ ~ elr-D-TIr~D~phe--NHNH2) H2 2 D Sltr D T~r-D-Phe-OMe (2.025 g, described in Example 4) in MeOH (70 ml) and hydrazine hydrate (2 ml) is stirred at 0C for I hr and at room temperature for 24 hr. The precipitate which is obtained upon addttion of water is collected on a sTntered glass flIterand dried over phosphorus pentoxide to give the title compound;
nmr (CDC13) ~ 0.98 (s, 3H),I1.16 (s, 18H), 7.4 (m, 20H). ~ -. ' " J ,'~ ~ -, ' ~ ' ' '. ' ' '' 4, 1 '. ':
i j, 1'.'"
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: . `, : - :, . , ` , . . . .. :.:i 104~623 EXAMPLF=_ Benzyloxycarbonvl-D-tryptophyl-D-phenylalanine Methvl Ester (Z-D-lrp-D-Phe-OMe) A solution of Z-D-Trp-OH (3.62 9, 10.7 mmoles), H-D-Phe-OMe-HCI
(2.3 9, 10.7 mmoles) and l-hydroxybenzotriazole (2.89 9, 21.4 mmoles) in dry DMF (25 ml) is cooled to 0C and N-ethylmorpholine (1.37 ml, 10.7 mmoles) is added. A cold (0C) solution of DCC (2.27 9, 11 mmoles) In DMF (6 ml) is added dropwise and the reaction mixture is stirred for I hr at 0C and I hr at room temperature. The reaction mixture is then cooled to 0C, fiItered, the fiItrate evaporated under reduced pressure and taken up in EtOA~. The EtOAc solution is washed with saturated NaHC03 soii~tion, water, cold citric acid (2N), water, saturated NaHC03 solution and saturated NaCI solution, dried with MgS04, and evaporated under reduced pressure to afford the crude title product I
whtch is subjected to chromatography on a co;lumn of silica gel '1 . : .
~500 9) with CHC13 containing MeOH (1%) as eluent. Tha eluates are evaporaled under reduced pressure and the residue is crystallized ;~ from EtOAc-petroleum ether to give the t;tle compound; mp 130 - 131C, [~X]D5 = +34.1 (c = I, DMF).
: .
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:.
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::.,.-: : ~ -. . :: :,.. : .
~ 550 .
104()~
BenzyloxYcarbon~l-N~-t-butvloxycal-bonyl-D- Iysvl-D-trY~tor)hy!-D-phenylalanine Methyl Ester (Z-D-Lys-D-Trp-D-Phe-O~le) Boc A mixture of Z-D-Trp-D-Phe-OMe (2.00 g, 4.0 mmoles, described -I in Example 6) and 5% Pd/C (0.25 g) in acetic acid is stirred rapidly under an atmosphere of hydrogen. After completion of hydrogen uptake a stream of nitrogen is passed tllrough the mixture for 15 m7n, the catalyst is removed by filtration, and the acetic IO acid is rcmoved under reduced pressure. The residue is taken ; 7n~o benzene, evaporate~d under reduced pressure (twice), and dried under reduced pressure over KOH pellets to give H-D-Trp-D-Phe-~Mé CH3C02H. The above product is dissolved in ! dry DMF (20 ml) and cooled to 0C. N-ethylmorpholine (0.512 ml, 4 mmoles) is added, followed by Z-D-L~s-ONp (2.00 g) 4 mmoles). -`~
Boc .i, : .
The solutTon is stirred at 0C for 30 min and for 3 days at room temperature. The solvent is evaporated under reduced pressure, the res7due i~ taken Tnto EtOAc and washed with cold sa1urated NaHC03 solutlon, water, cold citric actd (2N), water, saturated NaHC03 solution and saturated NaCI solution. The EtOAc solution is dried with MgS04 and evaporated under reduced pressure to ~;~
afford the crude title product. After chromatography on a column of silica gel ~300 9) with CHCI3 containing MeOH (5%) and pyridine 2 (0-1%) as eluent, the chromatographically pure product is triturated ~ I
with ether, filtered, dried under reduced pressure, and crystallized from MeOH-CH2CI2-isopropyl ether to give the title compound; mp 172 -174C, [~]D = +18.9 (c = I, DMF).
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' :' ~ .: ` :.' ' .' ., ' ' B~IP-6550 104~6Z3 N~-t-ButyloxYcarbonv!-D-lYsYI-D-tryptophyl-D--p-enylalanine Methyl-Ester Acetate (H-D-L~vs-D-Trp-D-Phe-OMe CH3CO
` Boc - --A mixture of Z-D-L~s-D-Trp-D-Phe-OMe (2.218 9, 3.04 mmoles, :~
B c ~: described in Example 7) and 5% Pd/C (0.25 g) in acetic acid is ~ rapidly stirred under an atmosphere of hydrogen for 20 hr. The '~, catalyst is removed by filtration and the filtrate evaporated under reduced pressure to give the title compound as an oil~
nmr (CDC13) ~ 1.4 (s, 9H), 2.07 (s, 3H), 3.6 (s, 3H). -", , .
, . ' ., - ~''-'.
j ' ' . . '~, '' ` 3 ~
~ ' .' ~ ,.
- , i . ' :~ ``-- 46- - ; :.
~ 1 : ~ . . . . :, :.- . , ~ -~ 1040623 BenzvloxYcarbonY!-(O-t-butYI)-D-threonyl-N~-t-butYlox~arb D-lYs~l-D-t~y~tophvl-D-phenvlalanine Methvl Ester (Z-D-Thr-D-Lys-D-Trp-D-phe--oMe) ~ BLt B~c
5- A solut;on of H-D-Lys-D-Trp-D-Phe-OMe-CH3C02H (3.04 mmoles, - - B c - , ,~ . -. . ' : .
,,1, described in Example 8), Z-T~$-OH (0.940 9, 3.0A mmoles) and 1- ;' hydroxybenzotriazole (0.820 9, 6.08 mmoles) in dry DMF 15 ml), is cooled to 0C and N-ethylmorpholine (0.39 ml, 3.04 mmoles) is ,, I added. A cold ~0C')'~solution of DCC (0.626 9, 3.04 mmoles) in DMF ~4 ml) is added dropwise and the reaction mixture is stirred '-I hr at 0C and I hr at room temperature. After filtration the DMF
is removed under reduced pressure. The oily residue obtained after drying is subjected to chromatography on a column of silica gel (270 9) ,' wtth 2% MeOH in CHC13 as eluent. The chromatographically pure product , Is'crystalltzed from MeOH/isopropyl ether to give the ' tltle compound;,mp 124 - 126C, [a]D25 = +3.9o (c = 1, DMF).
.
I: ' ~,!
.
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-47- ~
~04~)6Z3 Ben~yloxycarbonyl-~0-t-b~y1~__threonv!-N~-t-bulyloxycarbonyl-D-IYsYl-D-l-rvptop-hyl-D-~henvl~laninc HYdrazide ( ! ! . Z-D-T~r-D-Lys-D-Trp-D-Phe-NHNH2) But Boc A solution of Z-D-T~$-D-Lys-D-Trp-D-phe-oMe (1.307 9, 1.475 : :
Bb Bdc mmoles, described in Example 9) and hydrazine hydrate (2.74 ml) in : :
:~ DMF (8 ml) is stirred at 0C for 2 hr. Water is added, and the precipitate is collected by filtration and dried (1.267 g) over phosphorus ; pentoxide. The dried residue is crystallized from : 10 MeOH-CH2C12-isopropyt~.ther to give the l-itle compound; mp 203 -204C, [a]D5 - +11.6~ ~c=l, DMF).
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` EXAMPLE II
BenzyloxYcarbonyl-~-asparaqinyl-N ~ butyloxvcarbonyl-D-lysine Methyl Ester ~Z-D-Asn-D-L~s-OMe) ~
Boc - ;
A solution of Z-D-Asn-OTcp (1.48 9, 6.0 mmole) and H-D-Lys-nMe I
Boc (2.67 g, 6.0 mmole) in DMF (40 ml) and .'~-ethylmorpholine (0.5 ml) is stirred at 0C for 3 hr and at 25C for 20 hr. The soivent is removed under reduced pressure and the residue triturated with ~,' 1Oether. The residu~ i~s dried under reduced pressure to give the title compound; mp 147 - 149C, nmr (MeOH-d4): ~ 1.4 ~, 9H~, 3,7 Is, 3H), 5.15 (s, 2H),~7.37 (s, 3H).
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-49- , I .
, ~ ~ ..' ' ' ' , ' . : . .. ' EXAMPLE_12 Benzyloxycarbonyl-D-phenylalanine-D-asparaginyl-N~-t-butyloxy-carbonyl-D-lysine Methyl-Ester ~Z-D-Phe-D-Asn-D-Lys~t3Me~
130c - 5 A mixture of Z-D-Asn-D-Lys-OMe (2.32 9, 4.67 mmoles, ~' Boc ~
described in Example 11) and 5% Pd/C tO.30 9) in acetic acid (30 ml) Is rapidly stirred under an atmosphere of hydrogen for 1.5 hours. The mixture is ftItered and IN hydrochloric acid ; 1O (4.67 ml, 4.67 mmoles) is added to the filtrate. The filtrate ¦ is concentrated under'reduced pressure, MeOH (50 ml) is added, and the-solve~t i,s removed under reduced pressure to give H-D-Asn-D-Lyls-OMe-HCI.
Boc 1, A solution of the above compound (0.88 9, 2.14 mmoles) 'I ' Z-D-Phe-~Tcp (1.023 9, 2.14 mmoles), and N-ethylmorpholine (I ml) 1 - 1n dry THF (30 ml) is stirred at 0C for 24 hr. The precipitate Is collected by filtration and crystallized from MeOH-isopropyl ether to gTve the t7tle compound; mp 173 - 175C, ~D5 = ~13-4 (c - 1, DMF~. -., . : , ~ ~ ~ .
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....... l Al-IP-6~50 ¦ .
. ~ . ,' ' : 104U623 ~
.EXAMPLE 13 ..
D-Pheny!alanYI-D-asp ~ uty!oxycarbonyl~D-l~L~ne Me~hY!
Ester hcetate~ H-D-Phe~D-Asn=D~L~s~~Me C!-I ~ ~ ;~
~ ^ B c - .. ~
A mixture of Z~D-Phe-D-Asn-D-L~s-OMe (0.641 9, 0.994 mmole, . ~ :
. Boc ~ . .
described in Example 12) and 5%.Pd/C (0.065 9) in acetic acid (9 ml) is rapidly stirred under an atmosphere of hydrogen for 20 hr.
: The mixture is fiItered and the fiItrate is evaporated under :.
.' 1O reduced pressure. The residue is dissolved in benzen~ and : :
, evaporated under reduced pressure (twice~ and dried -~ under reduced;préssure over KOH pell,ets to give the title compound .
.1 as an oil: nmr (CDC13) 6 1.38 (s, 9H), 2.02 (s, 3H), 3.7 (s, 3H), ;-: ;
1~ 7.4 (m, 5H).
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_, Benzyloxycarbonyl-~O-t-butyl)-D-threonyl-N~-t-butvloxycarbonyl-D-l~y !-D-tryptop~ l-D-phenylalanyl-D-phenvlalanyl-D-asparaainyl-N~=t-but ~ -carbonyl-D-lysine Methvl Ester-(Z-D-T;r-D-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-- 5 l~s-OMe) - But Boc B c - -Z-D-T3r-D-Lys-D-Trp-D-Phe-NHNH2 (0.88 g, 0.994 mmole, described B B c In Example 10) is dissolved in dry DMF ~20 ml) with slight heating and the clear solution is cooled to -20C. Hydrochloric acid in EtOAc ~2N: 1.25 ml) is added followed by t-butyl nitrite (0.137 ml, 1.2 mmole).
The mixture is stirr~d for 15 min at -15Cv The mixture is stirred for 15 mln at -15C. A soluflon ot H-D-Phe-D-Asn-D-Lys-OMe CH3CO~H -(0.994 mmole, described in Example 13) in DMF (6 ml) containing N-ethyl-d7isopropylamine (0.60 ml, 3.5 mmoles) is cooled to -15C and added dropwise to the above mixture. Stirring is continued at -15C for I hr 1 and at room temperature overnight. The reaction mixture is evaporated ¦~
¦ under reduced pressure at 35C, the resTdue triturated with ice cold citrlc acld (IN), the m1xture is filtered and the precipitate is washed , with wate~ and dried over phosphorus pentoxide (1.27 g). The solid ¦ residue is subjected to chromatography on a column of silica gel (127 9) with 5~ MeOH in CHC13 as eluent. The chromatographically pure product ~ is evaporated under reduced pressure and the residue is crystallized 1~ from MeOH-CH2C12~isopropyl ether to give the title compound; mp 213 - ~ ~-25 215C, [a]D = ~13.4 (c = 1, DMF). `
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~ AHP-6550 ~04~)623 '- EXAMPLE-15 ' '' (O-t-Butyl)-D-threonyl-N -t-butYloxYcarbonYI-D-lYsyl-D-tryptophyl-D-phenylalanyl-D-pheny!a anYl-D-asParaqinY!-N~-t-butyloxycarbonyl-D-lysine ~ ' ', Methyl Ester Acetate-(IV~ H-D-T~r-D-Lys-D-l'rP-D-Phe-D-Phe-D-Asn-D-~- 5 L s-OMe-CH CO H) Bl Bo~
-r 3- ~
, Boc A mixture of Z-D-T3r-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-LIys-OMe B Boc Boc (0.71 9, 0.515 mmole, described in Example 14) and 5~ Pd/C (0.065 9) in acetic acid is rapidly stiired under an atmosphere of hydrogen for 20 hr.
The mixture is filtered and the filtrate evaporated under reduced pressure.
,~ The residue is dissolved in benzene, evaporated under reduced pressure . , , (~twice),and dried under reduced pressure over KOH pellets to give the title compound as an oil: nmr (CDC13) ~ 0.95 (s, 9H), 1.50 ~, 15 ~5/ 9H), 2.04 (s, 3H), 3.81 (:, 3H).
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, -53-N-rl-Oxo-3-(tritv!)thiolpr~a~!L ~ butyl)-D-seryl-(O-t-butyl)-D-~threonyl-D-phenYlalanYI-(O-t-but~l)-D-threony__N -t-butyloxycarbonvl-D-lvsYI-D-try~Dto~l-D--æhen-ylalanyl-D-~henylalanyl-D-asDaraqin~-Ne-t-but~
carbonyl-D-lvsine MethYI Ester (Trt-S-CH2 H~CO-D-S~rr-D-T~r-D-Phe-D-T~r-D-L~s-D-~u~ _ Phe-D-Phe-D-A n-D-L ~ Bbt Bbt Bbt B c Boc ; The tripeptlde hydrazide (Y) Trt-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-NHNH2 (0.418 9, 0.515 mmole, described In Example 5) is dissolved in dry DMF (6 ml) and cooled to -20C. Hydrogen chloride in EtOAc (2N, 0.645 ml) is added;followed by t-butyl n7trite (0.0706 ml, 0.619 mmole). The mixture is stirred for 15 min at -15C. A solution of H-D-T~r-D-Ly,s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe-CH3C02H (0.515 mmole, Bu Boc Boc : , described in Example 15) in DMF (10 ml) containing N-ethyldiisopropylamine (0.310 mi, 1.8 mmole) is cooled to -15C and added dropwise to the ~ ~ -;1 above mixture. Stirrlng Is continued at -15C for I hr and at room temperature overn19ht. The reactlon mtxture Ts evaporated under reduced pressure at 35C, the resldue Is trlturated wlth Ice cold cltrlc acld ;1~ iltered, washed With Water and dried over phosphorus pentoxlde.
~ The ~sorld resldue is subJected to chromatography on a column of silica -I gel~(100 g) wlth 5% MeOH in CHC13 as eluent. The chromatographically purë product is crystallized from MeOH/CH2C12/isopropyl ether to glve the title compound mp 205 - 207C [a]D5 = ~4.3 (c = 1, DMF).
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1040623 ~
EXAMPL~. 17 l~'-rl-Oxo-3-(trityl)thiol?ropvl-(0-t-butyl)-D-se_~-(0-t-butYI)-D-threonyl-D-phenylalanyl-(0-t-butyl)-D-threonyl-N~-;-butyloxycarbonyl-D- IYSVI_D ~ .
~: trvptophy!-D~phenYlalanyl-D-phenylalanyl-D-asparaqlnyl-N~-t-butyl_xy-carbonyl-D-Iysine Hydra~ide (VI, Trt-S-CH2 II~CO-D-Selr-D-Thlr-D-Phe-D-TIhr-D-lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHNH2) But But Bu Boc Boc .
A solution of Trt-S-CH2CH2C0-D-SIr-D-T~r-D-Phe-D-T~r-D-Llys D-B B B Boc :, Trp-D-Phe-D-Phe-D-Asn-D-Lyls-OMe (0.504 9, 0.249 mmole, described in Boc .l .
Example 16) in pMl~t~ ml) and hydrazine fhydrate (0.464 ml) is stirred ,. at 0C for 30 min and at room temperature for 23 hr. The product is :
precipitated by addition of cold water and filtered. The precipitate is washed several times with water and dried under reduced pressure over phosphorus pentoxide to give the title compound:.amino acid ~
analysis: Lys, 1.91, Thr, 1.62,Ser, 0.76, Asp, 1.00, Phe, 2.90. ~ :
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~ 104~6Z3 N;S-Ditrityl-D-cysteinylqlvcine-ethylarr.?ide tTrt-D-C~s-Gly-NHEt) Trt A solution of Trt-D-C~Is-Gly-OEt (10 9, prepared as described Trt by G. Amiard, Bull. Soc. Chim. (Fr.), 1956, 698 for the L-isomer of cysteine) and ethylamine (15 mlj is allowed to stand at 5C for 24 hr.
The solution is evaporated under reduced pressure and the residue is subjected to chromatography on a column ofstlicagelt200 9) uslng 15%to 30% EtOAc In benzene as eluent. The eluants are evaporated under reduced pressure to give the titlë c,ompound; nmr (CDC13) a o.s (t, J = 7 Hz, 3H)"~7.35 (m, 30H). --.~, ~ ,.
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S-Tritvl-D-cysteinvlqIycine-ethylamide ~Yd-roc-h!or-ide ~ _S Trt, R = C0NHCH~CONHCH2CH3LVIIb) A solution of Tr1--D-Clys-Gly-NHEt (1.03 9, 1.52 mmoles, Trt ~ described in Example 18) in acetic acid t8 ml) and water (1.9 ml) -~
-~ is stirred at 45C for 15 min. Water (8 ml) is added an~ the precipitate is removed by fiItration. Hydrochloric acid (IN, 1.5 ml) is added to the flItrate and the soluTTon Iyophilized. The dry product is washed with ether to give the title compound; nmr (DMS0-d6) ,1 ~ 1.02 (t J=7 Hz, 3H), 3.20 (m, 2H), 7.40 (s, 15H).
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` 10406Z3 cXAMPLE 20 N-rl-Oxo-3-(tritYl)thiolpropyl-(o-t-butyl)-D-seryl-(o-t-butyl)-D-threonyl- . .:
D-phenYlalanyl-(o-~-butyl)-D-threonYl-N -t-butYloxycarbonYl-D-lysYl-D
tryptophyl-D~phenylalanyl-~-phenylalanyl-D-asparaginyl-N~-t-butvloxy - 5 carbonyl-D-lysvl-(S-trityl)-~-cvsteinylqlycine-ethylamide.
t Bl't ~y s-D-Trp-D-Phe-D- Phe-D-Asn-D-Lys-Nl~CH~S Trt, ~ ~ -~
Boc R
Trt-S-CH2CH2CO-p-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-phe-D-phe ¦t 1 Blt Boc D-Asn-D-L~s-NHNH2 (~.467 9, 0.231 mmole, ~escribed in Example 17) is Boc dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and cooled to -20C. Hydrogen chloride in EtOAc (1.4 N, 0.412 ml, 0.577 mmole) is added followed by t-butyl nitrite (0.0313 ml, 0~276 mmole?.
The mixture is stirred for 15 min at -15C. A solution of H-D-Cys-Gly-Trt j NHEt-HCI (0.112 9, 0.231 mmole, described in Example 19) in DMF
(2.5 ml) containlng N-ethyldiisopropylamine (0.138 ml), 0.807 mmole) is cooled to -15C and added dropwise to the above reaction mixture.
Stirring is continued at -15C for l~hr and at room temperature over- j~
night. The reaction mixture is evaporated under reduced pressure, the residue is triturated with ice cold citric acid (IN), filtered and washed with water. The solid residue is washed three times with MeOH
and dried under reduced pressure to give the title compound; amino ~ .., . , ..: , .
~ acid analysis: ratio Asp/Gly = 1.08:1.
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4~6Z3 EXAMPL E 2 !, Cvclic Disulfide of-N~ oxo-3-thio)propyl-(0-t-butyl)-D-servl-(O-t-butyl)-D-threonYI-D-phenYtalanYl-~O-t-butvl)-D-threonYl-N3-t-butyloxycarbonyl-D
lYsYl-D-trvPtophYl-D-phenyialanyl-D-~henvlalanyl-D-asparaqinyl-N -t-butvl-oxvcarbonyi-~-lysYI-D-cvsteinYlt~lYcine-ethvlamids (S-CH~CH~CO-D-SIer-D-T
Phe-D-T ~ ys-D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NH~HCH~S,R~ CONHCH2CONHCHC~,IX) B Boc B c -. . _ _ _ _ _ _ _D- .
A solution of Trt-S-CH2CH2CO-D-S~tr-D-TIh~r D Phe D T~tr D Lyjs 10 Trp-D-Phe-D-Phe-D-Asn-D-L~-D-Cys-Gly-NHEt (0.32 9, 0.132 mmole, B Tr~ -,~ I
described in Exam,p;le,20) in acetic acid is slowly added to a stirred solution of iodine (0.336 9, 1.32 mmole) in MeOH (66 ml) at room ; temperature. After completion of addition the solution is stirred at :
¦ 15 room temperature for I hr. The sctiution is coo~ed to 0C and a solution of sodium thiosulfate in water (IN) iS slowly added to destroy the excess of iodine until a colorless solution is obtained. The solvent Ts evaporated under reduced pressure almost to dryness; the residue is dissolved in MeOH and added to 7ce cold water. The precipitate is collected by filtration, washed with water and dried under reduced pressure over phosphorus pentoxide to give the title compound; amino 1~ 1 acid analysis: Lys, 1.92; Ser, 0.75; Asp, !.05; Gly, 1.00; Thr, 1.78; ¦
Phe, 3.02; Cysteic acid, 0.93.
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Afl-lp-r~j5o 1040623 ~
EXAMj~LE 22 CYCI;C Disulfide of N-(l-oxo-3-thio)DroDvl-D-serYl-D-thrffonyl-D-Dllcnyl-alanYl-D-threonYI-D-lvsYl-D-tryDtoDnyl-D-phenylalanyl-D-Dhenylalanyl-D
asDaraqinYl-D-IYsvl-D-cvsteinYlalvcine-ethylamide ; 5 ~ ~ ~C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D-TrD-D-Phe-D-Phe-D-Asn-D-LYs-Nllt~H2Ch2 . 1. R = CONHCH2CONHCH2fCH
A solution of the cyclic disulfide of ~CH2CH2C0-D-Ser-D-TIhr-D-Phe-D-TIhr-D-Livs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lyls-D-C s-Gly-NHEt (0.250 9, But Bdc Boc j 0.132 mmole, described in Example 21) is vigorously stirred at 0C under an atmosphere of nTtrogen for 10 min in conc. hydrochloriG acid (11 ml).
Acetic acid (150 ml) is added and the solution is Iyophilized. The residue is taken in water, filtered and again Iyophilized. The residue (0.20 9) is dissolved in 0.01 M ammonium acetate (10 ml), the solution obtained is app~ied to a column (2.1 cm x 30 cm) of carboxymethyl ~ ~-ii cellulose (Whatman CM-23) and eluted first with O.OIM ammonlum acetate (200 ml), to remove the impurlties. The pure compound is eluted with 0.05 M
I and 0.06 M ammonium acetate sfnd Iyophllized to give the tltle i compound as a whlte solld in the form of its acetic acid ,~ 20 addition salt; vMmeaXoH 289 (6310), 283 nm (6 6310~. Repeated Iyophilization i of the latter product from water gives the title compound as the free base;amino acid analysis: Lys, 1.88; Asp, 1.00;
Thr, 1.89; Ser, 0.92; Gly, 0.71; Cys, 0.43; Phe, 2.93. Lyophilization ¦ 25 of the latter product from IN hydrochlor!c acid gives the title compound in the form of its hydrochloric acid additlon salt; amino acid ~ analysls: Lys, 1.92; Asp, 1.00; Thr, 1.90; Ser, 0.90; Gly, 0.78; Cys, 1 0.52; Phe, 2.90.
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N-(!-Oxo-3-thio)propyl-D-seryl--D-threonyl-D-~-henyla!a-nyl-D-th~l--D-lysYI-D-trYptophyl-D-phenyla_lanyl-D-phenylalanYl-D-asparaainvl-D-lysyl-D-cysteinvlqlycine-ethylamide tH-S-CH2__C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D- ~ -he-D-phe-D-Asn-D-Lvs-NHci-2 _~SH. Ia. R = CONHCH ~ ~C_ ~
By following the procedure of Example 22 but replacing the title cyclic disulfide of Exa~nple 21 with an equivalent amount of the tTtle compound of Example 20 Trt-S-CH2CH2C0-D-Selr-D-Thr-D-Phe-D-Thr-D-But Bbt B~t Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-D-cys-Gly-NHEt the title compound B c ~ ~ Boc T~t of this example is obtained; amino acid analysis: Lys, 2.02; Asp, 1.00;
Thr, 1.95; Ser, 0.~8; Gly, 0.82; Cys, 0.49; Phe, 2.97.
In the same manner, by replacing the title compound of Example ' 20 with the corresponding disulfhydryl derivative of formula X in which ! R is CONHCH2CONHEt HS-CH2CH2CO-D-Se,r-D-T~t-D-Phe-D-TIht D LYis D Trp D
Bu B Bu B c Phe-D-Phe-D-Asn-D-Lys-D-C~s-Gly-NHEt and proceding as above the title J B~c Trt compound Is also obtained.
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104~623 AHP-6550 EXAMPLE ?~4 N-rl-Oxo-3-ttritYl)thiolpropYI-~O-t-butyl)-D-serYl-(o-t-butyl)-D-threon D-PhenYlalanYI-(O-t-butyl)-D-threonyl-N~-t-butvloxycarbonvl-D-lYsyl-D
tryptophyl-D-phenYlalanYl-D-Dhenylalanyl-D-asparaqinYI-N -t-butvloxy-carbonyl-D-lysine 2-(tritYlthio)ethYlamide tTrt-S-CH2CH_CO-D-Sle~
Phe-D-Thr-D-Lyjs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHCH2_~S Trt. Vlll. R - H) B~ Boc B~c Trt-S-CH2CH CO-D-Sçr-D-T~r-D-Phe-D-T~r-D-Lys-D-Trp-D-Phe-D-Phe-2 B~t B~t B1t Boc D-Asn-D-Lys-NHNH2 ~0.458 g, 0.227 mmole, described in Example 17) is B c ' dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and't . cooled to -20C. Hydrogen chloride in EtOAc (2.0 N; 0.286 ml, 0.567 ~ mmole) is added f~oliowed by t-butyl nitrite (0.0314 ml, 0.276 mmole). '-.. , :' The mixture i5 stirred for 15 min at -15C. A solution of 2-tritylthio-ethylamine [0.080 9, 0.250 mmole, prepared as described by F.l Carroll ,j et al., J. Ora. Chem~, 30, 36 (1965)] in DMF (3 ml) containing N-ethyl-diisopropylamine (0.055 mlj 0.348 mmole)'is cooled to 15C and added ' dropwise to the above reaction mixture. Stirring is continued at -15C
for l hr and at room temperature for 3 days. The reaction mixture is ~ evaporated under reduced pressure, the resldue Is trlturated wlth Ice 'i 20 "¦ cold cltric acid (IN) and filtered. The precipitate is washed with ! -water, drled and crystallized from chloroform-methanol to give the title compound,mp 234 - 237C. anal: calcd for C130 H165 N15 019 S2 : C, 67.71; H, 7.21; N, 9.11. Found: C, 67.64; H, 7.28; N, 9.24. ¦
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-., 1~)40623 Cyclic DisulfidQ of N-(l-Oxo-3-thio)propvl-(0-t-butYI)-D-serYl-(O-t-butYi)-, D-threonvi-D-phenvialanYI-(O-t-butYl)-D-threonyl-N~-t-but~loxycarbonvl-D-lysvl-D-trYDtophyl-D-phenYIalanyl-D-phenylalanyl-D-asparaqinyl-N -t-butvl-oxycarbonyl-D-Ivsine_2-thioethylamide (5 ~ 2C l--e~3t -~- -D-L~s D-Tr~-D-Phe-D-Phe-D-Asn-D-L ~ 2__2S. IX, R = H
B c B c B~t B~t B~t B~c B~" 2 2 9, 0.096 mmole, described in Example ,24) in acetic acid (40 ml) is slowly added to a stirred solution of iodine (0.956 mmole) in acetic acid (50 ml) at room ~-temperature. After completion of addition, the solution is stirred at . , , room temperature for I hr. The solution is cooled to 0C and a solution of sodium thiosulfate in water (IN) is slowly added to destroy , the excess of iodine until a colorless solution is obtained. The solvent Is evaporated under reduced pressure almost to dryness and the ~ restdue Is triturated wi ih water. The preclp1tate is collected by 3~ 20 filtration and dried under reduced pressure over phosphorus pentoxlde ;
~t ~o give the title compound; amino acid analysis: Lys, q.89; Ser, 0.71;
" ~
Y Asp, ~.06; Thr, 1.75; Phe 3.12. -~-'' ' ' ., :
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- 1~4~6Z3 AHP-6550 :: EXAMPLE 26 CYCIjC Disulfide of N-(!-Oxo-3-thio)propyl-D--ser~l-D-threony!--D-phen alanYI-D-threonvl-D-lysy!-D-tryptophyl-D-phenylalanyl-D-pheny!a!anyl-D-asparaainyl-D-lysine 2-thioethylamide (Sl-CH2___C0-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH_~ 1. R = H) A solution of the cyclic disulfide of S ~ C0 D Sltr-D-T~r-D Phe D T~r D LYIS-D-TrP-D-Phe-D-Phe-D-ASn-D-~YIS-NHC~2CH2 (0.096 mmole, BU BOC B C 1.
described in Example 25) is vigorously stirred at 0C under an :' atmosphere of nitrogen for~10 min in conc. hydrochloric acid (10 ml). -Acetic acid (100 ml) is added and the solution is Iyophilized. The , " ~ ~ .
residue is taken up in 5% acetic acid in water and again Iyophilized The resldue is dissolved in the upper phase of n-butanol-acetic acid-water (4:1:5) and fiItered. The fiItrate is subjected to partition chromatography on a column of a chemically modified cross- ~;
linked dextran ("Sephadex G-25 M", 3 x 50 cm, equilibrated in the lower phase of n-butanol-acetic acid-water (4:1:5) and then equilibrated In the upper phase) using the upper phase to desorb the cyclic peptide .
The fractions containing the pure cycllc peptide are combined and ;yophilTzed to give the title compound in the form of its acetic acid addition salt; ~MmaxH 290 (~ 5,415)-, 282 (~ 6,000) and 274 nm (~ 5,660). ,1 Repeated Iyophilization of the latter compound from water gives the '¦
tTtle compound in the form of its free base; amino acid analysis: ~
Lys, 2.10; Asp, 0.93; Thr, 1.83; Ser, 0.93; Phe, 3.00. !
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- A~IP-G550 11;)4~623 N~ Oxo-3-thio)propyl-D-seryl-D-threonVI-D-phenYlalanYI-D-threonYl-D-lysYl-D-tryptophvl-D-phenylalanvl-D-phenylalanyl-D-asr)araqinyl-D-lyslne 2-thioetnv!amide (H-S-CH2CH~CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lvs-D-TrD-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH~SH. Ia, R = H) By following the procedure of Example 26 but replacing the title cyclic disulfide of Exampl e 25 with an equivalent amount of the title compound of Example 24 Trt-S CH2cH2co-D-ser-D-Thlr-D-phe-D-Ter-D-L
D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NHCH2CH2S Trt the title compound of thls B c example is obtain~ amino acid analysis Lys, 1.95; Asp, 1.13; Thr, 1.91;
Ser, 0186; Phe, 3.00.
; In the same manner, but replacing the title compound of Example 24 with the correspondingdisulfhydryl derivative of formula X in which R is hydrogen HS-CH2CH2CO-D-S~r-D-T3r-D-Phe-D-Tt~r-D-LYjs-D-TrP-D-. B B But Boc j Phe-D-Phe-D-Asn-D-Lys-NHCH2CH25H and proceeding as above the title compound Is also obtained. ;
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- ,: . .
,,1, described in Example 8), Z-T~$-OH (0.940 9, 3.0A mmoles) and 1- ;' hydroxybenzotriazole (0.820 9, 6.08 mmoles) in dry DMF 15 ml), is cooled to 0C and N-ethylmorpholine (0.39 ml, 3.04 mmoles) is ,, I added. A cold ~0C')'~solution of DCC (0.626 9, 3.04 mmoles) in DMF ~4 ml) is added dropwise and the reaction mixture is stirred '-I hr at 0C and I hr at room temperature. After filtration the DMF
is removed under reduced pressure. The oily residue obtained after drying is subjected to chromatography on a column of silica gel (270 9) ,' wtth 2% MeOH in CHC13 as eluent. The chromatographically pure product , Is'crystalltzed from MeOH/isopropyl ether to give the ' tltle compound;,mp 124 - 126C, [a]D25 = +3.9o (c = 1, DMF).
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~04~)6Z3 Ben~yloxycarbonyl-~0-t-b~y1~__threonv!-N~-t-bulyloxycarbonyl-D-IYsYl-D-l-rvptop-hyl-D-~henvl~laninc HYdrazide ( ! ! . Z-D-T~r-D-Lys-D-Trp-D-Phe-NHNH2) But Boc A solution of Z-D-T~$-D-Lys-D-Trp-D-phe-oMe (1.307 9, 1.475 : :
Bb Bdc mmoles, described in Example 9) and hydrazine hydrate (2.74 ml) in : :
:~ DMF (8 ml) is stirred at 0C for 2 hr. Water is added, and the precipitate is collected by filtration and dried (1.267 g) over phosphorus ; pentoxide. The dried residue is crystallized from : 10 MeOH-CH2C12-isopropyt~.ther to give the l-itle compound; mp 203 -204C, [a]D5 - +11.6~ ~c=l, DMF).
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` EXAMPLE II
BenzyloxYcarbonyl-~-asparaqinyl-N ~ butyloxvcarbonyl-D-lysine Methyl Ester ~Z-D-Asn-D-L~s-OMe) ~
Boc - ;
A solution of Z-D-Asn-OTcp (1.48 9, 6.0 mmole) and H-D-Lys-nMe I
Boc (2.67 g, 6.0 mmole) in DMF (40 ml) and .'~-ethylmorpholine (0.5 ml) is stirred at 0C for 3 hr and at 25C for 20 hr. The soivent is removed under reduced pressure and the residue triturated with ~,' 1Oether. The residu~ i~s dried under reduced pressure to give the title compound; mp 147 - 149C, nmr (MeOH-d4): ~ 1.4 ~, 9H~, 3,7 Is, 3H), 5.15 (s, 2H),~7.37 (s, 3H).
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, ~ ~ ..' ' ' ' , ' . : . .. ' EXAMPLE_12 Benzyloxycarbonyl-D-phenylalanine-D-asparaginyl-N~-t-butyloxy-carbonyl-D-lysine Methyl-Ester ~Z-D-Phe-D-Asn-D-Lys~t3Me~
130c - 5 A mixture of Z-D-Asn-D-Lys-OMe (2.32 9, 4.67 mmoles, ~' Boc ~
described in Example 11) and 5% Pd/C tO.30 9) in acetic acid (30 ml) Is rapidly stirred under an atmosphere of hydrogen for 1.5 hours. The mixture is ftItered and IN hydrochloric acid ; 1O (4.67 ml, 4.67 mmoles) is added to the filtrate. The filtrate ¦ is concentrated under'reduced pressure, MeOH (50 ml) is added, and the-solve~t i,s removed under reduced pressure to give H-D-Asn-D-Lyls-OMe-HCI.
Boc 1, A solution of the above compound (0.88 9, 2.14 mmoles) 'I ' Z-D-Phe-~Tcp (1.023 9, 2.14 mmoles), and N-ethylmorpholine (I ml) 1 - 1n dry THF (30 ml) is stirred at 0C for 24 hr. The precipitate Is collected by filtration and crystallized from MeOH-isopropyl ether to gTve the t7tle compound; mp 173 - 175C, ~D5 = ~13-4 (c - 1, DMF~. -., . : , ~ ~ ~ .
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.EXAMPLE 13 ..
D-Pheny!alanYI-D-asp ~ uty!oxycarbonyl~D-l~L~ne Me~hY!
Ester hcetate~ H-D-Phe~D-Asn=D~L~s~~Me C!-I ~ ~ ;~
~ ^ B c - .. ~
A mixture of Z~D-Phe-D-Asn-D-L~s-OMe (0.641 9, 0.994 mmole, . ~ :
. Boc ~ . .
described in Example 12) and 5%.Pd/C (0.065 9) in acetic acid (9 ml) is rapidly stirred under an atmosphere of hydrogen for 20 hr.
: The mixture is fiItered and the fiItrate is evaporated under :.
.' 1O reduced pressure. The residue is dissolved in benzen~ and : :
, evaporated under reduced pressure (twice~ and dried -~ under reduced;préssure over KOH pell,ets to give the title compound .
.1 as an oil: nmr (CDC13) 6 1.38 (s, 9H), 2.02 (s, 3H), 3.7 (s, 3H), ;-: ;
1~ 7.4 (m, 5H).
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_, Benzyloxycarbonyl-~O-t-butyl)-D-threonyl-N~-t-butvloxycarbonyl-D-l~y !-D-tryptop~ l-D-phenylalanyl-D-phenvlalanyl-D-asparaainyl-N~=t-but ~ -carbonyl-D-lysine Methvl Ester-(Z-D-T;r-D-L~s-D-Trp-D-Phe-D-Phe-D-Asn-D-- 5 l~s-OMe) - But Boc B c - -Z-D-T3r-D-Lys-D-Trp-D-Phe-NHNH2 (0.88 g, 0.994 mmole, described B B c In Example 10) is dissolved in dry DMF ~20 ml) with slight heating and the clear solution is cooled to -20C. Hydrochloric acid in EtOAc ~2N: 1.25 ml) is added followed by t-butyl nitrite (0.137 ml, 1.2 mmole).
The mixture is stirr~d for 15 min at -15Cv The mixture is stirred for 15 mln at -15C. A soluflon ot H-D-Phe-D-Asn-D-Lys-OMe CH3CO~H -(0.994 mmole, described in Example 13) in DMF (6 ml) containing N-ethyl-d7isopropylamine (0.60 ml, 3.5 mmoles) is cooled to -15C and added dropwise to the above mixture. Stirring is continued at -15C for I hr 1 and at room temperature overnight. The reaction mixture is evaporated ¦~
¦ under reduced pressure at 35C, the resTdue triturated with ice cold citrlc acld (IN), the m1xture is filtered and the precipitate is washed , with wate~ and dried over phosphorus pentoxide (1.27 g). The solid ¦ residue is subjected to chromatography on a column of silica gel (127 9) with 5~ MeOH in CHC13 as eluent. The chromatographically pure product ~ is evaporated under reduced pressure and the residue is crystallized 1~ from MeOH-CH2C12~isopropyl ether to give the title compound; mp 213 - ~ ~-25 215C, [a]D = ~13.4 (c = 1, DMF). `
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~ AHP-6550 ~04~)623 '- EXAMPLE-15 ' '' (O-t-Butyl)-D-threonyl-N -t-butYloxYcarbonYI-D-lYsyl-D-tryptophyl-D-phenylalanyl-D-pheny!a anYl-D-asParaqinY!-N~-t-butyloxycarbonyl-D-lysine ~ ' ', Methyl Ester Acetate-(IV~ H-D-T~r-D-Lys-D-l'rP-D-Phe-D-Phe-D-Asn-D-~- 5 L s-OMe-CH CO H) Bl Bo~
-r 3- ~
, Boc A mixture of Z-D-T3r-D-Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-LIys-OMe B Boc Boc (0.71 9, 0.515 mmole, described in Example 14) and 5~ Pd/C (0.065 9) in acetic acid is rapidly stiired under an atmosphere of hydrogen for 20 hr.
The mixture is filtered and the filtrate evaporated under reduced pressure.
,~ The residue is dissolved in benzene, evaporated under reduced pressure . , , (~twice),and dried under reduced pressure over KOH pellets to give the title compound as an oil: nmr (CDC13) ~ 0.95 (s, 9H), 1.50 ~, 15 ~5/ 9H), 2.04 (s, 3H), 3.81 (:, 3H).
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, -53-N-rl-Oxo-3-(tritv!)thiolpr~a~!L ~ butyl)-D-seryl-(O-t-butyl)-D-~threonyl-D-phenYlalanYI-(O-t-but~l)-D-threony__N -t-butyloxycarbonvl-D-lvsYI-D-try~Dto~l-D--æhen-ylalanyl-D-~henylalanyl-D-asDaraqin~-Ne-t-but~
carbonyl-D-lvsine MethYI Ester (Trt-S-CH2 H~CO-D-S~rr-D-T~r-D-Phe-D-T~r-D-L~s-D-~u~ _ Phe-D-Phe-D-A n-D-L ~ Bbt Bbt Bbt B c Boc ; The tripeptlde hydrazide (Y) Trt-S-CH2CH2CO-D-Ser-D-Thr-D-Phe-NHNH2 (0.418 9, 0.515 mmole, described In Example 5) is dissolved in dry DMF (6 ml) and cooled to -20C. Hydrogen chloride in EtOAc (2N, 0.645 ml) is added;followed by t-butyl n7trite (0.0706 ml, 0.619 mmole). The mixture is stirred for 15 min at -15C. A solution of H-D-T~r-D-Ly,s-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-OMe-CH3C02H (0.515 mmole, Bu Boc Boc : , described in Example 15) in DMF (10 ml) containing N-ethyldiisopropylamine (0.310 mi, 1.8 mmole) is cooled to -15C and added dropwise to the ~ ~ -;1 above mixture. Stirrlng Is continued at -15C for I hr and at room temperature overn19ht. The reactlon mtxture Ts evaporated under reduced pressure at 35C, the resldue Is trlturated wlth Ice cold cltrlc acld ;1~ iltered, washed With Water and dried over phosphorus pentoxlde.
~ The ~sorld resldue is subJected to chromatography on a column of silica -I gel~(100 g) wlth 5% MeOH in CHC13 as eluent. The chromatographically purë product is crystallized from MeOH/CH2C12/isopropyl ether to glve the title compound mp 205 - 207C [a]D5 = ~4.3 (c = 1, DMF).
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1040623 ~
EXAMPL~. 17 l~'-rl-Oxo-3-(trityl)thiol?ropvl-(0-t-butyl)-D-se_~-(0-t-butYI)-D-threonyl-D-phenylalanyl-(0-t-butyl)-D-threonyl-N~-;-butyloxycarbonyl-D- IYSVI_D ~ .
~: trvptophy!-D~phenYlalanyl-D-phenylalanyl-D-asparaqlnyl-N~-t-butyl_xy-carbonyl-D-Iysine Hydra~ide (VI, Trt-S-CH2 II~CO-D-Selr-D-Thlr-D-Phe-D-TIhr-D-lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHNH2) But But Bu Boc Boc .
A solution of Trt-S-CH2CH2C0-D-SIr-D-T~r-D-Phe-D-T~r-D-Llys D-B B B Boc :, Trp-D-Phe-D-Phe-D-Asn-D-Lyls-OMe (0.504 9, 0.249 mmole, described in Boc .l .
Example 16) in pMl~t~ ml) and hydrazine fhydrate (0.464 ml) is stirred ,. at 0C for 30 min and at room temperature for 23 hr. The product is :
precipitated by addition of cold water and filtered. The precipitate is washed several times with water and dried under reduced pressure over phosphorus pentoxide to give the title compound:.amino acid ~
analysis: Lys, 1.91, Thr, 1.62,Ser, 0.76, Asp, 1.00, Phe, 2.90. ~ :
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~ 104~6Z3 N;S-Ditrityl-D-cysteinylqlvcine-ethylarr.?ide tTrt-D-C~s-Gly-NHEt) Trt A solution of Trt-D-C~Is-Gly-OEt (10 9, prepared as described Trt by G. Amiard, Bull. Soc. Chim. (Fr.), 1956, 698 for the L-isomer of cysteine) and ethylamine (15 mlj is allowed to stand at 5C for 24 hr.
The solution is evaporated under reduced pressure and the residue is subjected to chromatography on a column ofstlicagelt200 9) uslng 15%to 30% EtOAc In benzene as eluent. The eluants are evaporated under reduced pressure to give the titlë c,ompound; nmr (CDC13) a o.s (t, J = 7 Hz, 3H)"~7.35 (m, 30H). --.~, ~ ,.
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S-Tritvl-D-cysteinvlqIycine-ethylamide ~Yd-roc-h!or-ide ~ _S Trt, R = C0NHCH~CONHCH2CH3LVIIb) A solution of Tr1--D-Clys-Gly-NHEt (1.03 9, 1.52 mmoles, Trt ~ described in Example 18) in acetic acid t8 ml) and water (1.9 ml) -~
-~ is stirred at 45C for 15 min. Water (8 ml) is added an~ the precipitate is removed by fiItration. Hydrochloric acid (IN, 1.5 ml) is added to the flItrate and the soluTTon Iyophilized. The dry product is washed with ether to give the title compound; nmr (DMS0-d6) ,1 ~ 1.02 (t J=7 Hz, 3H), 3.20 (m, 2H), 7.40 (s, 15H).
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` 10406Z3 cXAMPLE 20 N-rl-Oxo-3-(tritYl)thiolpropyl-(o-t-butyl)-D-seryl-(o-t-butyl)-D-threonyl- . .:
D-phenYlalanyl-(o-~-butyl)-D-threonYl-N -t-butYloxycarbonYl-D-lysYl-D
tryptophyl-D~phenylalanyl-~-phenylalanyl-D-asparaginyl-N~-t-butvloxy - 5 carbonyl-D-lysvl-(S-trityl)-~-cvsteinylqlycine-ethylamide.
t Bl't ~y s-D-Trp-D-Phe-D- Phe-D-Asn-D-Lys-Nl~CH~S Trt, ~ ~ -~
Boc R
Trt-S-CH2CH2CO-p-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-phe-D-phe ¦t 1 Blt Boc D-Asn-D-L~s-NHNH2 (~.467 9, 0.231 mmole, ~escribed in Example 17) is Boc dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and cooled to -20C. Hydrogen chloride in EtOAc (1.4 N, 0.412 ml, 0.577 mmole) is added followed by t-butyl nitrite (0.0313 ml, 0~276 mmole?.
The mixture is stirred for 15 min at -15C. A solution of H-D-Cys-Gly-Trt j NHEt-HCI (0.112 9, 0.231 mmole, described in Example 19) in DMF
(2.5 ml) containlng N-ethyldiisopropylamine (0.138 ml), 0.807 mmole) is cooled to -15C and added dropwise to the above reaction mixture.
Stirring is continued at -15C for l~hr and at room temperature over- j~
night. The reaction mixture is evaporated under reduced pressure, the residue is triturated with ice cold citric acid (IN), filtered and washed with water. The solid residue is washed three times with MeOH
and dried under reduced pressure to give the title compound; amino ~ .., . , ..: , .
~ acid analysis: ratio Asp/Gly = 1.08:1.
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4~6Z3 EXAMPL E 2 !, Cvclic Disulfide of-N~ oxo-3-thio)propyl-(0-t-butyl)-D-servl-(O-t-butyl)-D-threonYI-D-phenYtalanYl-~O-t-butvl)-D-threonYl-N3-t-butyloxycarbonyl-D
lYsYl-D-trvPtophYl-D-phenyialanyl-D-~henvlalanyl-D-asparaqinyl-N -t-butvl-oxvcarbonyi-~-lysYI-D-cvsteinYlt~lYcine-ethvlamids (S-CH~CH~CO-D-SIer-D-T
Phe-D-T ~ ys-D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NH~HCH~S,R~ CONHCH2CONHCHC~,IX) B Boc B c -. . _ _ _ _ _ _ _D- .
A solution of Trt-S-CH2CH2CO-D-S~tr-D-TIh~r D Phe D T~tr D Lyjs 10 Trp-D-Phe-D-Phe-D-Asn-D-L~-D-Cys-Gly-NHEt (0.32 9, 0.132 mmole, B Tr~ -,~ I
described in Exam,p;le,20) in acetic acid is slowly added to a stirred solution of iodine (0.336 9, 1.32 mmole) in MeOH (66 ml) at room ; temperature. After completion of addition the solution is stirred at :
¦ 15 room temperature for I hr. The sctiution is coo~ed to 0C and a solution of sodium thiosulfate in water (IN) iS slowly added to destroy the excess of iodine until a colorless solution is obtained. The solvent Ts evaporated under reduced pressure almost to dryness; the residue is dissolved in MeOH and added to 7ce cold water. The precipitate is collected by filtration, washed with water and dried under reduced pressure over phosphorus pentoxide to give the title compound; amino 1~ 1 acid analysis: Lys, 1.92; Ser, 0.75; Asp, !.05; Gly, 1.00; Thr, 1.78; ¦
Phe, 3.02; Cysteic acid, 0.93.
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EXAMj~LE 22 CYCI;C Disulfide of N-(l-oxo-3-thio)DroDvl-D-serYl-D-thrffonyl-D-Dllcnyl-alanYl-D-threonYI-D-lvsYl-D-tryDtoDnyl-D-phenylalanyl-D-Dhenylalanyl-D
asDaraqinYl-D-IYsvl-D-cvsteinYlalvcine-ethylamide ; 5 ~ ~ ~C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D-TrD-D-Phe-D-Phe-D-Asn-D-LYs-Nllt~H2Ch2 . 1. R = CONHCH2CONHCH2fCH
A solution of the cyclic disulfide of ~CH2CH2C0-D-Ser-D-TIhr-D-Phe-D-TIhr-D-Livs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lyls-D-C s-Gly-NHEt (0.250 9, But Bdc Boc j 0.132 mmole, described in Example 21) is vigorously stirred at 0C under an atmosphere of nTtrogen for 10 min in conc. hydrochloriG acid (11 ml).
Acetic acid (150 ml) is added and the solution is Iyophilized. The residue is taken in water, filtered and again Iyophilized. The residue (0.20 9) is dissolved in 0.01 M ammonium acetate (10 ml), the solution obtained is app~ied to a column (2.1 cm x 30 cm) of carboxymethyl ~ ~-ii cellulose (Whatman CM-23) and eluted first with O.OIM ammonlum acetate (200 ml), to remove the impurlties. The pure compound is eluted with 0.05 M
I and 0.06 M ammonium acetate sfnd Iyophllized to give the tltle i compound as a whlte solld in the form of its acetic acid ,~ 20 addition salt; vMmeaXoH 289 (6310), 283 nm (6 6310~. Repeated Iyophilization i of the latter product from water gives the title compound as the free base;amino acid analysis: Lys, 1.88; Asp, 1.00;
Thr, 1.89; Ser, 0.92; Gly, 0.71; Cys, 0.43; Phe, 2.93. Lyophilization ¦ 25 of the latter product from IN hydrochlor!c acid gives the title compound in the form of its hydrochloric acid additlon salt; amino acid ~ analysls: Lys, 1.92; Asp, 1.00; Thr, 1.90; Ser, 0.90; Gly, 0.78; Cys, 1 0.52; Phe, 2.90.
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N-(!-Oxo-3-thio)propyl-D-seryl--D-threonyl-D-~-henyla!a-nyl-D-th~l--D-lysYI-D-trYptophyl-D-phenyla_lanyl-D-phenylalanYl-D-asparaainvl-D-lysyl-D-cysteinvlqlycine-ethylamide tH-S-CH2__C0-D-Ser-D-Thr-D-Phe-D-Thr-D-LYs-D- ~ -he-D-phe-D-Asn-D-Lvs-NHci-2 _~SH. Ia. R = CONHCH ~ ~C_ ~
By following the procedure of Example 22 but replacing the title cyclic disulfide of Exa~nple 21 with an equivalent amount of the tTtle compound of Example 20 Trt-S-CH2CH2C0-D-Selr-D-Thr-D-Phe-D-Thr-D-But Bbt B~t Lyls-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-D-cys-Gly-NHEt the title compound B c ~ ~ Boc T~t of this example is obtained; amino acid analysis: Lys, 2.02; Asp, 1.00;
Thr, 1.95; Ser, 0.~8; Gly, 0.82; Cys, 0.49; Phe, 2.97.
In the same manner, by replacing the title compound of Example ' 20 with the corresponding disulfhydryl derivative of formula X in which ! R is CONHCH2CONHEt HS-CH2CH2CO-D-Se,r-D-T~t-D-Phe-D-TIht D LYis D Trp D
Bu B Bu B c Phe-D-Phe-D-Asn-D-Lys-D-C~s-Gly-NHEt and proceding as above the title J B~c Trt compound Is also obtained.
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104~623 AHP-6550 EXAMPLE ?~4 N-rl-Oxo-3-ttritYl)thiolpropYI-~O-t-butyl)-D-serYl-(o-t-butyl)-D-threon D-PhenYlalanYI-(O-t-butyl)-D-threonyl-N~-t-butvloxycarbonvl-D-lYsyl-D
tryptophyl-D-phenYlalanYl-D-Dhenylalanyl-D-asparaqinYI-N -t-butvloxy-carbonyl-D-lysine 2-(tritYlthio)ethYlamide tTrt-S-CH2CH_CO-D-Sle~
Phe-D-Thr-D-Lyjs-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NHCH2_~S Trt. Vlll. R - H) B~ Boc B~c Trt-S-CH2CH CO-D-Sçr-D-T~r-D-Phe-D-T~r-D-Lys-D-Trp-D-Phe-D-Phe-2 B~t B~t B1t Boc D-Asn-D-Lys-NHNH2 ~0.458 g, 0.227 mmole, described in Example 17) is B c ' dissolved in a mixture of dry distilled DMF (8 ml) and DMSO (2 ml) and't . cooled to -20C. Hydrogen chloride in EtOAc (2.0 N; 0.286 ml, 0.567 ~ mmole) is added f~oliowed by t-butyl nitrite (0.0314 ml, 0.276 mmole). '-.. , :' The mixture i5 stirred for 15 min at -15C. A solution of 2-tritylthio-ethylamine [0.080 9, 0.250 mmole, prepared as described by F.l Carroll ,j et al., J. Ora. Chem~, 30, 36 (1965)] in DMF (3 ml) containing N-ethyl-diisopropylamine (0.055 mlj 0.348 mmole)'is cooled to 15C and added ' dropwise to the above reaction mixture. Stirring is continued at -15C
for l hr and at room temperature for 3 days. The reaction mixture is ~ evaporated under reduced pressure, the resldue Is trlturated wlth Ice 'i 20 "¦ cold cltric acid (IN) and filtered. The precipitate is washed with ! -water, drled and crystallized from chloroform-methanol to give the title compound,mp 234 - 237C. anal: calcd for C130 H165 N15 019 S2 : C, 67.71; H, 7.21; N, 9.11. Found: C, 67.64; H, 7.28; N, 9.24. ¦
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.
:, .
-., 1~)40623 Cyclic DisulfidQ of N-(l-Oxo-3-thio)propvl-(0-t-butYI)-D-serYl-(O-t-butYi)-, D-threonvi-D-phenvialanYI-(O-t-butYl)-D-threonyl-N~-t-but~loxycarbonvl-D-lysvl-D-trYDtophyl-D-phenYIalanyl-D-phenylalanyl-D-asparaqinyl-N -t-butvl-oxycarbonyl-D-Ivsine_2-thioethylamide (5 ~ 2C l--e~3t -~- -D-L~s D-Tr~-D-Phe-D-Phe-D-Asn-D-L ~ 2__2S. IX, R = H
B c B c B~t B~t B~t B~c B~" 2 2 9, 0.096 mmole, described in Example ,24) in acetic acid (40 ml) is slowly added to a stirred solution of iodine (0.956 mmole) in acetic acid (50 ml) at room ~-temperature. After completion of addition, the solution is stirred at . , , room temperature for I hr. The solution is cooled to 0C and a solution of sodium thiosulfate in water (IN) is slowly added to destroy , the excess of iodine until a colorless solution is obtained. The solvent Is evaporated under reduced pressure almost to dryness and the ~ restdue Is triturated wi ih water. The preclp1tate is collected by 3~ 20 filtration and dried under reduced pressure over phosphorus pentoxlde ;
~t ~o give the title compound; amino acid analysis: Lys, q.89; Ser, 0.71;
" ~
Y Asp, ~.06; Thr, 1.75; Phe 3.12. -~-'' ' ' ., :
'~
,., ~: ' ': ', ~ ' ij :
~ 1 ~
-63~
'~ !
- 1~4~6Z3 AHP-6550 :: EXAMPLE 26 CYCIjC Disulfide of N-(!-Oxo-3-thio)propyl-D--ser~l-D-threony!--D-phen alanYI-D-threonvl-D-lysy!-D-tryptophyl-D-phenylalanyl-D-pheny!a!anyl-D-asparaainyl-D-lysine 2-thioethylamide (Sl-CH2___C0-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH_~ 1. R = H) A solution of the cyclic disulfide of S ~ C0 D Sltr-D-T~r-D Phe D T~r D LYIS-D-TrP-D-Phe-D-Phe-D-ASn-D-~YIS-NHC~2CH2 (0.096 mmole, BU BOC B C 1.
described in Example 25) is vigorously stirred at 0C under an :' atmosphere of nitrogen for~10 min in conc. hydrochloric acid (10 ml). -Acetic acid (100 ml) is added and the solution is Iyophilized. The , " ~ ~ .
residue is taken up in 5% acetic acid in water and again Iyophilized The resldue is dissolved in the upper phase of n-butanol-acetic acid-water (4:1:5) and fiItered. The fiItrate is subjected to partition chromatography on a column of a chemically modified cross- ~;
linked dextran ("Sephadex G-25 M", 3 x 50 cm, equilibrated in the lower phase of n-butanol-acetic acid-water (4:1:5) and then equilibrated In the upper phase) using the upper phase to desorb the cyclic peptide .
The fractions containing the pure cycllc peptide are combined and ;yophilTzed to give the title compound in the form of its acetic acid addition salt; ~MmaxH 290 (~ 5,415)-, 282 (~ 6,000) and 274 nm (~ 5,660). ,1 Repeated Iyophilization of the latter compound from water gives the '¦
tTtle compound in the form of its free base; amino acid analysis: ~
Lys, 2.10; Asp, 0.93; Thr, 1.83; Ser, 0.93; Phe, 3.00. !
. `' ' . l :' ': . ; , ~ ~ .
, : : .
,: :
:~:
-, ` . :
:
i- '~
: : :
'- . : :.
.,, ~, -` . . . . . . . : .
.. . : , , , . ~ . . .
- A~IP-G550 11;)4~623 N~ Oxo-3-thio)propyl-D-seryl-D-threonVI-D-phenYlalanYI-D-threonYl-D-lysYl-D-tryptophvl-D-phenylalanvl-D-phenylalanyl-D-asr)araqinyl-D-lyslne 2-thioetnv!amide (H-S-CH2CH~CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lvs-D-TrD-D-Phe-D-Phe-D-Asn-D-LYs-NHCH2CH~SH. Ia, R = H) By following the procedure of Example 26 but replacing the title cyclic disulfide of Exampl e 25 with an equivalent amount of the title compound of Example 24 Trt-S CH2cH2co-D-ser-D-Thlr-D-phe-D-Ter-D-L
D-Trp-D-Phe-D-Phe-D-Asn-D-L~s-NHCH2CH2S Trt the title compound of thls B c example is obtain~ amino acid analysis Lys, 1.95; Asp, 1.13; Thr, 1.91;
Ser, 0186; Phe, 3.00.
; In the same manner, but replacing the title compound of Example 24 with the correspondingdisulfhydryl derivative of formula X in which R is hydrogen HS-CH2CH2CO-D-S~r-D-T3r-D-Phe-D-Tt~r-D-LYjs-D-TrP-D-. B B But Boc j Phe-D-Phe-D-Asn-D-Lys-NHCH2CH25H and proceeding as above the title compound Is also obtained. ;
~ ~ -.
, ::
.
, .
.~
.. . ~ .
.. . .
- ,: . .
Claims (29)
1. A process for preparing a peptide of formula I or Ia (1) (Ia) in which R is hydrogen or CONHCH2CONHCH2CH3, or a pharmaceutically acceptable salt thereof, which comprises selecting a process from the group of processes consisting of:
(a) when a compound of formula I is required, reacting a decapeptide of formula VI
with a reagent that furnishes nitrous acid in situ in the presence of a strong acid to convert said decapeptide to the corresponding azide and reacting said azide with a compound of formula VII, HNH?HCH2STrt, in which R is as defined herein to obtain the corresponding linear pro-tected peptide of formula VIII
in which R is as defined herein followed by oxidizing said linear protected peptide with iodine or thiocyanogen to obtain the corresponding cyclic disulfide derivative of formula IX
in which R is as defined herein and subsequently removing all remaining protecting groups under moderately acidic condition to obtain the corresponding peptide of formula I; or subjecting said linear peptide to treatment with either mercuric acetate, mercuric chloride, silver acetate or silver nitrate to remove selectively the sulfhydryl protecting groups to obtain the mercuric or disilver salt, respectively, of the corresponding di-sulfhydryl derivative; converting the latter salt to its corresponding free disulfhydryl derivative of formula X by treatment with hydrogen sulfide, oxidizing said last-named derivative by treatment with oxygen, 1,2-dilodoethane, sodium or potassium ferricyanide or iodine to obtain the corresponding cyclic disulfide and removing the remaining protecting groups under moderately acidic conditions to obtain the desired peptide of formula I;
(b) when a compound of formula Ia is required, removing the protecting groups under moderately acidic conditions from said linear protected peptide of formula VIII to obtain said corresponding peptide of formula Ia; and (c) when a pharmaceutically acceptable salt of said compound of formula I or Ia is required, reacting said compound of formula I or Ia with a pharmaceutically acceptable acid to obtain the corresponding pharmaceutically acceptable salt of the corresponding compound of formula I or Ia.
(a) when a compound of formula I is required, reacting a decapeptide of formula VI
with a reagent that furnishes nitrous acid in situ in the presence of a strong acid to convert said decapeptide to the corresponding azide and reacting said azide with a compound of formula VII, HNH?HCH2STrt, in which R is as defined herein to obtain the corresponding linear pro-tected peptide of formula VIII
in which R is as defined herein followed by oxidizing said linear protected peptide with iodine or thiocyanogen to obtain the corresponding cyclic disulfide derivative of formula IX
in which R is as defined herein and subsequently removing all remaining protecting groups under moderately acidic condition to obtain the corresponding peptide of formula I; or subjecting said linear peptide to treatment with either mercuric acetate, mercuric chloride, silver acetate or silver nitrate to remove selectively the sulfhydryl protecting groups to obtain the mercuric or disilver salt, respectively, of the corresponding di-sulfhydryl derivative; converting the latter salt to its corresponding free disulfhydryl derivative of formula X by treatment with hydrogen sulfide, oxidizing said last-named derivative by treatment with oxygen, 1,2-dilodoethane, sodium or potassium ferricyanide or iodine to obtain the corresponding cyclic disulfide and removing the remaining protecting groups under moderately acidic conditions to obtain the desired peptide of formula I;
(b) when a compound of formula Ia is required, removing the protecting groups under moderately acidic conditions from said linear protected peptide of formula VIII to obtain said corresponding peptide of formula Ia; and (c) when a pharmaceutically acceptable salt of said compound of formula I or Ia is required, reacting said compound of formula I or Ia with a pharmaceutically acceptable acid to obtain the corresponding pharmaceutically acceptable salt of the corresponding compound of formula I or Ia.
2. A process as claimed In Claim I in which the decapeptide of formula VI is prepared by reacting a tripeptide of formula V, , with a reagent that furnishes nitrous acid in situ in the presence of a strong acid to convert said tripeptide to the corresponding azide and reacting said azide with a hepta-peptide of formula IV, , to obtain the decapeptide of formula , followed by reacting said last-named compound with hydrazine hydrate and isolating said decapeptide of formula VI.
3. A process as claimed in Claim I in which the compound of for-mula VII, HNH?HCH2STrt, in which R is CONHCH2CONHCH2CH3 (VIIb) is prepared by reacting the dipeptide of formula with ethylamine to obtain the corresponding dipeptide of formula and removing the terminal amino protecting group (Trt) of said last-named compound under mildly acidic conditions to obtain said compound of formula HNH?HCH2STrt in which R-is CONHCH2CONHCH2CH3.
4. A process as claimed in Claim 2 in which the tripeptide of formula V, , is prepared by reacting a tripeptide of formula witn an activated ester of Trt-S-CH2CH2CO2H to obtain , followed by reacting the last-named compound with hydrazine hydrate and isolating said tripeptide of formula V.
5. A process as claimed in Claim 4 in which the tripeptide of formula is prepared by reacting a dipeptide of formula with an activated ester of to obtain and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation, in the presence of a noble metal catalyst to obtain said tripeptide.
6. A process as claimed in Claim 5 in which the dipeptide of formula is prepared by reacting H-D-Phe-OMe witn an activated ester of to obtain and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation in the presence of a noble metal catalyst to obtain said dipeptide.
7. A process as claimed in Claim 2 in which the heptapeptide of formula IV, , is prepared by reacting the tetrapeptide of formula II, , with a reagent that furnishes nitrous acid in situ in the presence of a strong acid to convert said tetrapeptide to the corresponding azide and reacting said azide with a tripeptide of formula III, , to obtain a heptapeptide of formula and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation in the presence of a noble metal catalyst and isolating said heptapeptide.
8. A process as claimed in Claim 7 in which the tetrapeptide of formula II, , is prepared by reacting a tripeptide of formula with an activated ester of to obtain a tetrapeptide of formula , followed by reacting said last-named compound with hydrazine hydrate and isolating said tetrapeptide.
9. A process as claimed in Claim 8 in which the tripeptide of formula , is prepared by reacting a dipeptide of formula H-D-Trp-D-Phe-OMe with an activated ester or to obtain a tripeptide of formula and removing the terminal protecting group (Z) of said last-named compound by hydrogenation in the presence of a noble metal catalyst to obtain said tripeptide.
10. A process as claimed in Claim 9 in which the dipeptide of formula H-D-Trp-D-Phe-OMe is prepared by reacting H-D-Phe-OMe with an activated ester of Z-D-Trp-OH to obtain a dipeptide of formula Z-D-Trp-D-Phe-OMe and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation in the presence of a noble metal catalyst to obtain said dipeptide.
11. A process as claimed in Claim 7 in which the tripeptide of formula III, , is prepared by reacting a dipeptide of formula with an activated ester of Z-D-Phs-OH to obtain a tripeptide of formula and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation in the presence of a noble metal catalyst to obtain said tripeptide.
12. A process as claimed in Claim 11 in which the dipeptide of formula is prepared by reacting with an activated ester of Z-D-Asn-OH to obtain a dipeptide of formula and removing the terminal amino protecting group (Z) of said last-named compound by hydrogenation in tne presence of a noble metal catalyst to obtain said dipepttde.
13. A process as claimed in Claim 1 in which the linear protected peptide of formula VIII as defined therein is subjected to moderately acidic conditions to obtain the corresponding compound of formula Ia HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH in which R is as defined therein.
14. A process as claimed in Claim 1 in which the corresponding disulfhydryl derivative as defined therein is subjected to moderately acidic conditions to obtain the corresponding compound of formula Ia HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-Asn-D-Lys-NH?HCH2SH in which R is as defined therein.
15. The process as claimed in Claim 1 wherein said linear pro-tected peptide of formula VIII is subjected to treatment with iodine in the presence of a lower alkanol or acetic acid to obtain the corresponding cyclic disulfide derivative of formula IX.
16. The process as claimed in Claim 1 wherein said linear protected peptide of formula VIII is subjected to treatment with iodine at from about 0° to 30°C for about 30 to 180 minutes in a lower alkanol or acetic acid to obtain the corresponding cyclic disulfide derivative of formula IX.
17. The process according to step (a) of Claim 1 for preparing the compound of formula I, as defined in Claim 1, in which R is hydrogen.
18. The process according to step (a) of Claim 1 for preparing the compound of formula I, as defined in Claim 1, in which R is CONHCH2CONHCH2CH3.
19. The process according to step (b) of Claim 1 for preparing the compound of formula Ia, as defined in Claim 1, in which R is hydrogen.
20. The process according to step (b) of Claim 1 for preparing the compound of formula Ia, as defined in Claim 1, in which R is CONHCH2CONHCH2CH3.
21. The process according to step (c) of Claim 1 wherein a compound of formula I or Ia is reacted with acetic acid to obtain the acetic acid addition salt of the corresponding compound of formula I or Ia.
22. The process according to step (c) of Claim 1 wherein a compound of formula I or Ia is reacted with hydrochloric acid to obtain the hydrochloric acid addition salt of the corresponding compound of formula I or Ia.
23. A compound of formula I or Ia (I) HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
(Ia) in which R is hydrogen or CONHCH2CONHCH2CH3 or a pharmaceutically acceptable salt thereof, when prepared by the process of Claim 1, or an obvious chemical equivalent thereof.
(Ia) in which R is hydrogen or CONHCH2CONHCH2CH3 or a pharmaceutically acceptable salt thereof, when prepared by the process of Claim 1, or an obvious chemical equivalent thereof.
24. The compound of formula I
in which R is hydrogen, when prepared by the process of Claim 17, or an obvious chemical equivalent thereof.
in which R is hydrogen, when prepared by the process of Claim 17, or an obvious chemical equivalent thereof.
25. The compound of formula I
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 18 or an obvious chemical equivalent thereof.
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 18 or an obvious chemical equivalent thereof.
26. The compound of formula Ia HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
in which R is hydrogen, when prepared by the process of Claim 19, or an obvious chemical equivalent thereof.
in which R is hydrogen, when prepared by the process of Claim 19, or an obvious chemical equivalent thereof.
27. The compound of formula Ia HS-CH2CH2CO-D-Ser-D-Thr-D-Phe-D-Thr-D-Lys-D-Trp-D-Phe-D-Phe-D-Asn-D-Lys-NH?HCH2SH
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 20, or an obvious chemical equivalent thereof.
in which R is CONHCH2CONHCH2CH3, when prepared by the process of Claim 20, or an obvious chemical equivalent thereof.
28. The acetic acid addition salt of a compound of formula I or Ia of Claim 23, when prepared by the process of Claim 21, or an obvious chemical equivalent thereof.
29. The hydrochloric acid addition salt of a compound of formula I or Ia of Claim 23, when prepared by the process of Claim 22, or an obvious chemical equivalent thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA234,424A CA1040623A (en) | 1975-08-28 | 1975-08-28 | Derivatives of retro-enantio-somatostatin, intermediates therefor and process therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA234,424A CA1040623A (en) | 1975-08-28 | 1975-08-28 | Derivatives of retro-enantio-somatostatin, intermediates therefor and process therefor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1040623A true CA1040623A (en) | 1978-10-17 |
Family
ID=4103927
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA234,424A Expired CA1040623A (en) | 1975-08-28 | 1975-08-28 | Derivatives of retro-enantio-somatostatin, intermediates therefor and process therefor |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1040623A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994005311A1 (en) * | 1992-08-27 | 1994-03-17 | Deakin Research Limited | Retro-, inverso-, and retro-inverso synthetic peptide analogues |
-
1975
- 1975-08-28 CA CA234,424A patent/CA1040623A/en not_active Expired
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994005311A1 (en) * | 1992-08-27 | 1994-03-17 | Deakin Research Limited | Retro-, inverso-, and retro-inverso synthetic peptide analogues |
| US6261569B1 (en) | 1992-08-27 | 2001-07-17 | Deakin Research Limited | Retro-, inverso- and retro-inverso synthetic peptide analogues |
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