GB1590668A - Process for the preparation of thymosin a1 and an analogue - Google Patents

Process for the preparation of thymosin a1 and an analogue Download PDF

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GB1590668A
GB1590668A GB1574/78A GB1587478A GB1590668A GB 1590668 A GB1590668 A GB 1590668A GB 1574/78 A GB1574/78 A GB 1574/78A GB 1587478 A GB1587478 A GB 1587478A GB 1590668 A GB1590668 A GB 1590668A
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bzl
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    • C07K14/57581—Thymosin; Related peptides
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Abstract

Peptide derivatives, which are intermediates in the preparation of thymosin alpha 1 and [Asn<2>]-thymosin alpha 1, of the general formula H3C-CO-Ser(R<1>)-X-Ala-Ala-Val-Asp(OR<2>)-Thr(R<3>)-Ser(R<1>)-Ser(R<1> )-Glu(OR <4>)-I le-Thr(R<3>)-Thr(R<3>)-Lys(R<5>)-Asp(OR<2>)-Leu-Lys(R<5>)-Glu(OR<4>)-L ys(R <5 )-Lys(R<5>) -Glu(OR<4>)-Val-Val-Glu(OR<4>)-Glu(OR<4>)-Ala-Glu(OR<4>)-Asn-OR<6)<I> in which X represents Asn or Asp(OR<2>); R<1> represents a group which can be removed with formation of the hydroxyl group of the seryl radical; R<2>, R<4> and R<6> represent a group which can be removed with formation of a carboxyl group; R<3> represents a group which can be removed with formation of the hydroxyl group of the threonyl radical and R<5> represents a group which can be removed with formation of the omega -amino group of the lysyl radical.

Description

(54) A PROCESS FOR THE PREPARATION OF THYMOSIN a AND AN ANALOGUE (71) We, F. HOFFMANN-LA ROCHE & CO., AKTIENGESELLSCHAFT, a Swiss Company of 124-184 Grenzacherstrasse, Basle, Switzerland, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- Thymosin a, is a heat stable, acidic polypeptide compound of 28 amino acid residues of the following sequence: 5 10 H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu- 15 20 Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu- 25 Val-Val-Glu-Glu-Ala-Glu-Asn-OH.
This peptide which is a potent immunopotentiating agent was isolated from thymosin fraction 5 by Goldstein et al. by a combination of ion-exchange chromatography and gel filtration (Proc. Natl. Acad. Sci. USA 74, 725-729 (1977)).
The present invention relates to a synthesis of thymosin a1 or its [Asn2]analogue and the pharmaceutically acceptable salts thereof by removing the protecting groups from a protected octacosapeptide of the sequence: H3C-CO-Ser(Rl)-X-Ala-Ala-Val-Asp(OR2)-Thr(R3)-Ser(R' )- Ser(R' )-Glu(OR4)-Ile-Thr(R3)-Thr(R3)-Lys(R5)- Asp(OR2)-Leu-Lys(R5)-Glu(OR4)-Lys(R5)-Lys(R5)- Glu(OR4)-Val-Val-Glu(OR4)-Glu(OR4)-Ala-Glu(OR4)-Asn-ORs (I) wherein X is Asn or Asp(OR2); R' is a protecting group for the hydroxyl group of the serine radical; R2, R4 and Re are protecting groups for the carboxyl groups of the aspartic acid radical, the glutamic acid radical and the asparagine radical respectively; R3 is a protecting group for the hydroxyl group of the threonine radical and R5 is a protecting group, for the E-amino group of the lysine radical, and, if desired, converting the compound obtained into a pharmaceutically acceptable salt.
Examples of R1 are benzyl, acetyl, benzoyl, tert.-butyl, trityl, 4-bromobenzyl, 2,6-dichlorobenzyl and benzyloxycarbonyl with benzyl being preferred. Examples of R2, R4 and R6 are aryl groups particularly phenyl or phenyl substituted with lower (C,~6) alkyl, halo, nitro, mercapto or substituted mercapto such as methylthio; aralkyl groups such as benzyl or benzyl substituted with methoxy,halo or nitro; lower (C,~6) alkyl groups such as methyl, ethyl, tert.-butyl and tert.-amyl; substituted lower (C,~6) alkyl groups such as 2-haloethyl, p,p-dimethylaminoethyl and cyanomethyl; benzhydryl and phenacyl groups with benzyl being preferred.
Examples of R3 are benzyl, acetyl, benzoyl, tert.-butyl, trityl, 2,6-dichlorobenzyl, 4bromobenzyl and benzyloxycarbonyl with benzyl being preferred. Examples of R5 are benzyloxycarbonyl which may be optionally substituted in the aromatic ring such as by 4-chloro, 2-bromo, 4-bromo, 2,4-dichloro, 4-nitro, 4-methoxy, 3,5dimethoxy, 4-methyl, 2,4,6-trimethyl, 4-phenylazo, 4-(4-methoxyphenylazo), 2 (N,N-dimethylcarbamido) and 2-nitro-4,5-dimethoxy, urethane type protecting groups such as 4-toluenesulfonylethyloxycarbonyl and 9fluorenylmethyloxycarbonyl, 5-benzisoxazolylmethyleneoxycarbonyl, methylthioand methylsulfonylethyloxycarbonyl, isonicotinyloxycarbonyl, haloethyloxycarbonyl, diisopropylmethyloxycarbonyl, benzhydryloxycarbonyl, isobornyloxycarbonyl, dinitrodiphenylmethyloxycarbonyl, tert.-butyloxycarbonyl, tert.-amyloxycarbonyl, adamantyloxycarbonyl, cyclopentyloxycarbonyl, methylcyclobutyloxycarbonyl, methylcyclohexyloxycarbonyl, 2arylisopropyloxycarbonyl groups such as 2-(p-biphenylyl)-isopropyloxycarbonyl and 2-(4-pyridyl)-isopropyloxycarbonyl; acyl groups such as formyl, trifluoroacetyl, phthaloyl, benzenesulfonyl, acetoacetyl, chloroacetyl, 2-nitrobenzoyl, 4toluenesulfonyl, sulfenyl groups such as benzenesulfenyl and 0-nitrophenylsulfenyl, and aryl-lower alkyl groups such as diphenylmethyl and triphenylmethyl, with the benzyloxycarbonyl group being preferred.
The invention also includes protected octacosapeptides of sequence I hereinabove.
In one embodiment of the invention the protecting groups are removed from a protected octacosapeptide of the sequence H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)- Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(Bzl)-Thr(Bzl)-Lys(Z)- Asp(OBZI)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)- Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (lea) In another embodiment of the invention the protecting groups are removed from a protected octacosapeptide of the sequence H3C-CO-Ser(Bzl)-Asn.Ala.Ala-Val-Asp(Obzl)-Thr(Bzl)- Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(Bzl)-Thr(Bzl)- Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z) Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (Ib) Removal of the protecting groups from the protected octacosapeptide of sequence I is readily accomplished by procedures known per se, for example by treatment with anhydrous acid such as hydrogen fluoride preferably in the presence of anisole.
In accordance with the present invention the protected octacosapeptide of sequence I can be synthesized by condensation of a protected tetradecapeptide of the sequence H3C-CO-Ser(Rl)-X-Ala-Ala-Val-Asp(OR2)-Thr(R3)-Ser(Rl)- Ser(Rl)-Glu(OR4)-Ile-Thr(R3)-Thr(R3)-Lys(R5)-OR7 II with a protected tetradecapeptide of the sequence H-Asp(OR2)-Leu-Lys(R5)-Glu(OR4)-Lys(Rs)-Lysf Rub).
Glu(OR4)-Val-Val-Glu(OR4)-Glu(OR4)-Ala-Glu(OR4).Asn.OR6 III wherein X, R', R2, R3, R4, Rub and Rub are defined as above and R7 is hydrogen or an activated ester group, derived for example from p-nitrophenol, N hydroxysucciniimide or l-Hydroxybenzotriazole.
The aforesaid coupling reaction can be carried out utilizing procedures well known in solution phase peptide synthesis. Thus, for example, the amino terminal tetradecapeptide can be reacted with l-hydroxybenzotriazole (HOBT) and dicyclohexylcarbodiimide (DCC) to yield the activated ester which is then reacted with the carboxyl terminal tetradecapeptide to yield the desired octacosapeptide in protected form.
Thus the protected octacosapeptide of sequence Ia may be obtained by condensation of the activated protected tetradecapeptide of the sequence H3C-CO-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl)- Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-lle-Thr(B zl)-Thr(B zl)-Lys(Z)-OBT (lia) with the trifluoroacetic acid salt of the protected tetradecapeptide of the sequence H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)- Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)Asn-OBzl (lIla) wherein BT is benzotriazolyl.
Similarly, the protected octacosapeptide of sequence Ib may be obtained by condensation of the activated protected tetradecapeptide of the sequence H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z).OBT (lIb) with the trifluoroacetic acid salt of the protected tetradecapeptide of the sequence H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl) Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (ill) wherein BT is benzotriazolyl.
The protected tetradecapeptides can be prepared in accordance with methods well-known in the art.
The strategy employed in the chemical synthesis of the protected carboxyl terminal tetradecapeptide III [X=Asp(OBzl)] was as follows: H-Glu(OBzl)-OH was first coupled to Boc-Ala-OSu to give the protected dipeptide fragment Boc-Ala-Glu(OBzl)-OH which was then condensed with HCl. H-Asn-OBzl via the DCC/HOSu procedure of Wunsch and Drees, Chem.
Ber. 99, 110(1966). The hydrochloride salt of asparagine benzyl ester was prepared from Boc-Asn-OBzl which in turn was synthesized from commercially available Boc-Asn-OH and benzyl bromide using the cesium salt of the amino acid. The Bocprotecting group was removed by a 30 minutes treatment with 4M HCI in dry THF.
Reaction between H - Glu(OBzl) - OH and Boc - Glu(OBzl) - OSu produced Boc - Glu(OBzl) - Glu(OBzl) - OH as a colorless clear oil. It was subsequently utilized in the synthesis of the protected pentapeptide Boc - Glu(OBzl) Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl in a DCC/HOSu mediated fragment condensation using HCI - H - Ala - Glu(OBzl) - Asn - OBzl that was derived from Boc - Ala - Glu(OBzl) - Asn - OBzl upon 4M HCVTHF treatment. The aforesaid protected pentapeptide was obtained in good yield as a crystalline pure material.
For the preparation of the protected octapeptide Boc - Glu(OBzl) - Val Val - Glu(OBzl)- Glu(OBzl)- Ala - Glu(OBzl)- Asn - OBzl, the required protected tripeptide Boc - Glu(OBzl) - Val - Val - OH was first prepared. Boc Val - OSu was allowed to react with free valine to provide Boc - Val - Val - OH which on deblocking with 4M HCI in THF followed by reaction with Boc Glu(OBzl)-OSu yielded the desired tripeptide which was crystallized as cyclohexylamine salt Boc - Glu(OBzl) - Val - Val - OH CHA. The cyclohexylamine salt was converted to the free acid and was then coupled by DCC in the presence of HOSu to HCI - H - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) Asn - OBzl that was derived from Boc - Glu(OBzl)- Glu(OBzl)- Ala Glu(OBzl) - Asn - OBzl on treatment with HCI in THF.
For the synthesis of the protected undecapeptide Boc - Glu(OBzl) - Lys(Z) Lys(Z)- Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) Asn - OBzl, the required tripeptide fragment was synthesized starting from Boc Lys(Z) - OSu and H - Lys(Z) - OH. The dipeptide Boc - Lys(Z) - Lys(Z) - OH thus obtained was treated with 4M HCI in THF and the ensuing salt HCl - H Lys(Z) - Lys(Z) - OH was then allowed to react with Boc - Glu(OBzl) - OSu to provide the desired tripeptide Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - OH. The tripeptide was then activated with DCC and HOSu according to the procedure of Weygand et al., Z. Naturforsch. 21b, 426 (1966), and the solution of the active tripeptide ester Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - OSu generated in situ was combined with the trifluoroacetate salt of H - Glu(OBzl) - Val - Val Glu(OBzl)- Glu(OBzl)- Ala - Glu(OBzl)- Asn - OBzl derived from the corresponding blocked octapeptide by a 30 minutes treatment with TFA. Upon addition of a small amount of a base the desired protected undecapeptide Boc Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) Ala - Glu(OBzl) - Asn - Obzl was thus obtained.
The synthesis of the protected tetradecapeptide followed a similar pattern.
Boc - Leu - Osu was coupled to H - Lys(Z) - OH to provide Boc - Leu - Lys(Z) OH. After removal of the N" - Boc - group with 4M HCI in THF and reaction with Boc - Asp(OBzl)- OSu the protected tripeptide Boc - Asp(OBzl) - Leu Lys(Z) - OH was obtained as a crystalline pure solid. It was converted into the active ester Boc - Asp(OBzl)- Leu - Lys(Z) - OSu and condensed with the trifluoroacetate salt of H - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val Glu(OBzl)- Glu(OBzl) - Ala - Glu(OBzl)- Asn - OBzl obtained from TFA treatment of the corresponding blocked undecapeptide. The desired product Boc Asp - (OBzl) - Leu - Lys(Z) - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl was obtained in the good yield. Thin layer chromatography indicated that the product was homogeneous.
In a similar way the protected amino terminal tetradecapeptide H3C - CO Ser(Bzl) - Asp(OBzl) - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Hzl) - Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH was assembled from an acetyl tetrapeptide fragment, a hexapeptide, and another tetrapeptide using procedures well known in the peptide synthesis art. For the synthesis of the amino terminal acetyl tetrapeptide, H3C - CO - Ser(Bzl) - OBzl was prepared from H3C - CO - Ser(Bzl) - OH and benzyl bromide via the cesium salt of the amino acid. Hydrazinolysis of H3C - CO - Ser(Bzl) - OBzl gave H3C - CO Ser(Bzl) - HNNH2 as a pure crystalline solid in good yield. Deblocking of Boc Ala - Ala - OH afforded the dipeptide hydrochloride salt HCI H - Ala - Ala OH. Coupling of this dipeptide with Boc - Asp(OBzl) - OSu provided the protected tripeptide Boc - Asp(OBzl) - Ala - Ala - OH which was isolated as the dicyclohexylamine salt. Removal of the amino protecting group and condensation with H3C - CO - Ser(Bzl) - HNNH2 via the azide procedure of Honzyl and Rudinger, Collection Czech. Chem. Commun. 26, 2333 (1961), gave the partially protected tetrapeptide H3C - Ser(Bzl) - Asp(OBzl) - Ala - Ala - OH which on reaction with equivalent amounts of hydrazine mediated by DCC in the presence of HOBT produced the desired intermediate H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala - Ala - HNNH2.
For the synthesis of the protected hexapeptide Boc - Val - Asp(OBzl) Thr(Bzl) - Ser(Bzl) - Ser(Bzl)- Glu(OBzl)- HNNH2, a process involving the stepwise elongation of the peptide chain from the C-terminal end was adopted.
Thus, H - Glu(OBzl) - OH was coupled with Boc - Ser(Bzl) - OSu to form the protected dipeptide Boc - Ser(Bzl) - Glu(OBzl) - OH which on removal, of the Boc - group and further reaction with Boc - Ser(Bzl) - OSu gave rise to the tripeptide Boc - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH. Deprotection of the amino group followed by condensation of the resultant tripeptide salt HCI H - Ser(Bzl) Ser(Bzl) - Glu(OBzl) - OH with Boc - Thr(Bzl) - OSu yielded the protected tetrapeptide Boc - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - G[u(OBzl) - OH which on treatment with HCI in THF removed the Boc-group and further reaction with Boc - Asp(OBzl) - OSu resulted in the formation of the protected pentapeptide Boc - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH. The Bocgroup of this compound was removed by HCI treamment and the ensuing product HCl. H - Asp(OBzl)- Thr(Bzl)- Ser(Bzl) - Ser(Bzl) - Glu(OBzl)- OH was subsequently coupled to Boc - Val - OSu to give Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH as a crystalline pure material.
The a-carboxyl group on the terminal glutamic acid residue of the aforesaid hexapeptide was then specifically converted into the hydrazide function by the reaction with an equivalent amount of hydrazine using DCC as coupling agent in the presence of HOBT. The desired protected hexapeptide hydrazide Boc - Val Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - HNNH2 was isolated as pure crystalline solid in a reasonable yield.
For the synthesis of the protected tetrapeptide Boc - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z)- OH, a similar stepwise procedure using the Nhydroxysuccinimide active ester procedure of Anderson et al., J. Amer. Chem.
Soc. 86, 1839 (1964), was utilized. Reaction between Boc - Thr(Bzl) - OSu and H Lys(Z) - OH gave the dipeptide Boc - Thr(Bzl) - Lys(Z) - OH as an oil which was deprotected at the a-amino end and allowed to react with Boc - Thr(Bzl) - OSu to provide the tripeptide Boc-Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH as a crystalline solid.
Removal of the Boc-group and reaction of the resultant material HCl. H Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH with Boc - Ile - OSu yielded the desired protected tetrapeptide Boc - Ile - Thr(Bzl)- Thr(Bzl) - Lys(Z)- OH as a crystalline pure compound after chromatography on a silica gel column. This tetrapeptide fragment was then deprotected at the amino terminal and condensed with the protected hexapeptide Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) Ser(Bzl) - Glu(OBzl) - HNNH2 by the azide method to produce the protected decapeptide Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH in good yield.
Removal of the Boc-group from the decapeptide compound with TFA and subsequent coupling with the N-terminal tetrapeptide Ac - Ser(Bzl) - Asp(OBzl) Ala - Ala - HNNH2 through the azide procedure resulted in formation of the required protected tetradecapeptide H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH. For the final coupling, this acetyl tetradecapeptide was activated with DCC and HOBT and the ensuing active ester was then allowed to react with TFA H - Asp(OBzl) - Leu - Lys(Z) - Glu(OBzl) Lys(Z)- Lys(Z)- Glu(OBzl)- Val - Val - Glu(OBzl)- Glu - OBzl) - Ala Glu(OBzl) - Asn - OBzl which was derived from the corresponding blocked compound on treatment with TFA to give the protected acetyl octacospeptide H3 CO - Ser(Bzl) - Asp(OBzl), - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) Ser(Bzl) - Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - Asp(OBzl) - Leu Lys(Z)- Glu(OBzl) - Lys(Z)- Lys(Z)- Glu(OBzl) - Val - Val - Glu(OBzl) Glu(OBzl) - Ala - Glu(OBzl)- Asn - OBzl. Treatment with anhydrous HF removed all the protecting groups and purification on ion-exchange chromatography yielded thymosin a1.
The synthesis of [Asn2] - thymosin a, followed the same pattern as the synthesis of thymosin a1. All of the intermediates used were the same except the Nterminal acetyl tetrapeptide hydrazide H3C - CO - Ser(Bzl) - Asn - Ala - Ala HNNH2. For the synthesis of this compound, Boc - Ala - Ala - OH was first converted into Boc - Ala - Ala - OBzl which on selective removal of the Bocgroup with HCI gave the dipeptide ester salt HCl. H - Ala - Ala - OBzl. The dipeptide was then coupled with Boc - Asn - OH using the DCC/HOBT procedure of Knig and Geiger, Chem. Ber. 103, 788 (1970), to give the protected tripeptide ester Boc - Asn - Ala - Ala - OBzl which was treated with HCI in THF to remove the Boc-group. The resultant product HCI H - Asn - Ala - Ala - OBzl was then condensed (DCC,HOBT procedure) with H3C - CO - Ser(Bzl) - OH DCHA to afford the desired protected tetrapeptide H3C - CO - Ser(Bzl) - Asn - Ala - Ala OBzl.
The corresponding hydrazide H3C - CO - Ser(Bzl) - Asn - Ala - Ala HNNH2 was obtained in good yield on hydrazinolysis of this compound. Fragment condensation between the hydrazide and the decapeptide TFA H - Val Asp(OBzl)- Thr(Bzl)- Ser(Bzl)- Ser(Bzl)- Glu(OBzl)- Ile - Thr(Bzl) Thr(Bzl) - Lys(Z) - OH produced the protected tetradecapeptide H3C - CO Ser(Bzl) - Asn - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH. This compound was then coupled to the deblocked C-terminal tetradecapeptide TFA salt discussed above to give the corresponding protected [Asn2] - thymosin a,. Removal of all the protecting groups by treatment with anhydrous HF followed by column ionexchange chromatographic purification gave the desired [Asn2] - thymosin (z,.
Several of the intermediate peptides used in the above synthesis of thymosin a, and [Asn2] - thymosin a1 have activity in the regulation, differentiation and function of T-cells, namely H-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH; H-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH; H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH; H3C-CO-Ser-Asn-Ala-Ala.Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH; and H3C-CO-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-OH .
The first three named above are the subject of our Divisional Application No.
7937369 (Serial No. 1590688).
Thymosin cr, and [Asn2] - thymosin a, and the pharmaceutically acceptable salts thereof may be administered to warm blooded mammals by parenteral application either intravenously, subcutaneously or intramuscularly. These compounds are potent immunopotentiating agents with a daily dosage in the range of 1 to 100 mgjkg of body weight per day for intravenous administration. Obviously the required dosage will vary with the particular condition being treated, the severity of the condition and the duration of treatment. A suitable dosage form for pharmaceutical use is I mg of lyophilized thymosin , or [Asn2] - thymosin al, to be reconstituted prior to use by the addition of sterile water or saline.
Also included within the scope of the present invention are the pharmaceutically acceptable salts of thymosin a1 and [Asn2] - thymosin a1.
Suitable salts include the sodium and potassium salts or salts with a strong organic base such as guanidine. In addition, the counter ions of these cations such as the chloride, bromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate and ascorbate may be included in the preparation.
Abbreviations used herein have the following meaning: Boc=tbutyloxycarbonyl; Bzl=benzyl; DCC=dicyclohexylcarbodiimide; DMF=dimethylformamide; THF=tetrahydrofuran; HOSu=N- hydroxysuccinimide; Triton B (trade mark)=40% by weight methanolic solution of trimethylbenzylammonium hydroxide; NMM=N-methylmorpholine; CHA=cyclohexylamine; DCHA=dicyclohexylamine; Z=benzyloxycarbonyl; DMSO=dimethyl sulfoxide; TFA-trifluoroacetic acid; TLC=thin layer chromatography; Et3N=triethylamine; HOBT=l-hydroxybenzotriazole.
The following Examples describe in detail the synthesis of thymosin ct, and [Asn2] - thymosin a1.
While specific protecting groups have been employed in describing the synthesis of thymosin a1 and [Asn2] - thymosin at it is within the skill of the art to utilize equivalent protecting groups in such synthesis.
In the Examples reagent percentages are given as weight percent; yield percentages as mole percent. "Ether" is diethyl ether.
Example 1 A.a) Boc - Asn - OH (11.0 g, 47.5 mmol) was dissolved in 200 ml of MeOH and 20 ml of water was added. The solution was titrated to pH 7.0 with a 20 /" aq.
solution of Cs2Co3 (ca. 55 ml). The mixture was evaporated to dryness and the residue reevaporated twice from DMF (120 ml each, 45"C). The white solid obtained was then stirred with 8.9 g of benzyl bromide (52 mmol) in 120 ml DMF for 6 hours. On evaporation to dryness and treatment with a large volume of water, the product solidified immediately. It was collected by filtration, dissolved in ethyl acetate, washed with water, dried over Na2SO4, evaporated to a solid mass and crystallized from ethyl acetate with petroleum ether. Yield 13.8 g (90.3%) of Boc Asn - OBzl; m.p. 120--1220C; [al25=l7.290 (c=l, DMF).
Boc - Asn - OBzl (13.7 g, 42.4 mmol) was dissolved in 80 ml of THF and treated with 500 ml of 4M HCI in THF. The mixture was left standing for 45 minutes during which time some product began to precipitate. On treatment with 1000 ml of ether, a white solid material formed immediately. The product was filtered washed with ether and dried over NaOH pellets in vacuo. Yield: 10.3 g (94%) of HCI H - Asn OBzl; m.p. 122-1260C; [a]D5=+6.82 .
b) H - Glu(OBzl) - OH (7.0 g, 29.5 mmol) was finely ground in a mortar and pestle and then stirred with 8.88 g (32.3 mmol) of Boc - Ala - OSu for 48 hours in 250 ml DMF in the presence of 6 ml NMM. Some more NMM was added to maintain the reaction slightly basic during the reaction. The solvent was evaporated and the residue partitioned between 300 ml ethyl acetate and 500 ml H2O containing 2 ml of 10% H2SO4. The organic layer was then washed three times with water, dried over Na2SO4 and evaporated to dryness. The product was taken up in a small volume of ether and treated with a large volume of petroleum ether. A white amorphous solid was obtained which was homogeneous on TLC. Yield: 11.0 g (91.5%) of Boc Ala - Glu(OBzl) - OH; m.p. 84--880C; [al2B=8.08O (c=l, DMF).
Boc - Ala - Glu(OBzl) - OH (10.4 g, 25.4 mmol), HCI H - Asn - OBzl (6.56 g, 25.4 mmol) and HOSu (5.9 g, 50.8 mmol) were dissolved in DMF (250 ml, 0 C).
DCC (5.7 g, 27.6 mmol) was added followed immediately by Et3N (3.5 ml). The mixture was stirred at OOC for 2 hours and then at 250C for 40 hours during which period some more Et3N was added from time to time to maintain the reaction slightly basic. The insoluble by-products formed were filtered off and the filtrate evaporated to dryness. The residual oily material solidified on treatment with water. The crude product was taken up in CHCl3, washed with water (3x), dried over Na2SO4 and evaporated to a smaller volume. Some solid formed at this stage was filtered off (heavily contaminated with dicyclohexylurea) and the filtrate treated with petroleum ether. A crystalline product was obtained. Yield: 8.0 g (51.4% of Boc - Ala - Glu(OBzl) - Asn - OBzl; m.p. 102-1050C; [aj2Bl2.5O (c=l, DMF).
c) H - Glu(OBzl) - OH (4.74 g, 20 mmol) was ground in a mortar and pestle and stirred with Boc - Glu(OBzl) - OSu (0.7 g, 20 mmol) in DMF for 36 hours in the presence of 3.6 ml NMM. The ensuing solution was evaporated to a syrup and treated with water. The oily precipitate was taken up in ethyl acetate, washed successively with 5% HOAc and water (3x), dried over Na2SO4 and evaporated to dryness yielding 14.03 g of a clear oil. It was left standing submerged under petroleum ether. The residual oily Boc - Glu(OBzl) - Glu(OBzl) - OH weighed 10.2 g (90.0%). TLC indicated that the product was homogeneous. [aln2B=7.590 (c=l, DMF).
d) Boc - Ala - Glu(OBzl) - Asn - OBzl (28.2 g; 46 mmol) was treated with 1.1 liter of 4M HCl in THF for 1 hour. Evaporation of the solvent and excess acid left an oil which was evaporated twice more with fresh THF. The residual oil turned into a solid when treated with a large volume of ether. The solid HCI - H - Ala Glu(OBzl) - Asn - OBzl was stirred with Boc - Glu(OBzl) - Glu(OBzl) - OH(25.6 g, 46 mmol), HOSu (10.6 g, 92 mmol) and DCC (10.9 g, 53 mmol) in DMF (540 ml) at 0 C for I hour and then at 250C for 48 hours. Et3N was added to maintain the reaction slightly basic over the entire period of time (ca.16 ml Et3N total). The insoluble by-products formed were filtered off and the filtrate evaporated to dryness. The crude product was dissolved in CHCl3, washed with water (3x), dried over Na2SO4 and evaporated to dryness. The product solidified when treated with petroleum ether. Recrystallization from isopropanol yielded 28.9 g (59.8%) of Boc - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl; m.p. 169-175 C; [a]D5=11.78 (c=l, DMF).
Boc - Glu(OBzl)-Gu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl (3.9 g, 3.48 mmol) was treated with 15 ml 4M HCI in THF for 30 minutes. Some crystalline product started to form. Ether (210 ml) was added and the precipitated solid was collected and washed with ether. The crude material was crystallized from MeOH and ether. Yield: 2.58 g (75.1%) of HCI-H - Glu(OBzl) - Glu(OBzl) - Ala Glu(OBzl) - Asn - OBzl; m.p. 148-1510C; [&alpha;]D25=-3.65 (c=l, DMF).
e) Boc - Val - OSu (12.6 g, 40 mmol) and H - Val - OH (4.68 g, 40 mmol) were condensed in DMF (250 ml) for 96 hours in the presence of 2 ml Et3N. More Et3N was added when needed to maintain the reaction slightly basic. The remaining insoluble material was filtered off and the filtrate evaporated to dryness (45 C).
The residue was partitioned between ether and dil (45"C). The remaining oil residue solidified when treated with water. The crude solid was dissolved in DMF (50 ml) and precipitated with MeOH (300 ml). Yield: 2.25 g (58.7%) of Boc - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala Glu(OBzl) - Asn - OBzl; m.p. 277-2800C; [a]5=-12.430 (c=l, DMF).
This product (0.72 g, 0.49 mmol) was hydrogenated over 5% Pd/BaSO4 (0.5 g) for 3 hours at 3.4 atm in a mixture of 40 ml DMF/30 ml MeOH/2 ml H2O. The mixture was then filtered and the filtrate evaporated to dryness.
f) Boc - Lys(Z) - OH (15 g, 39.5 mmol) was stirred with HOSu (5.8 g, 50.5 mmol) and DCC (8.66 g, 42 mmol) in THF (250 ml) for 3 hours. An insoluble byproduct was filtered off and the filtrate evaporated to dryness. The residual syrup (24.2 g) was treated with iso-propanol (150 ml) and petroleum ether (150 ml) to yield an oily product (21 g) which failed to crystallize. The crude active ester Boc Lys(Z) - OSu was thus used for condensation with H - Lys(Z) - OH (10.6 g, 38 mmol) in DMF (250 ml) for 72 hours in the presence of 5.5 ml Et3N. More Et3N was added occasionally in order to maintain the stirred reaction mixture slightly basic.
Some small quantity of undissolved material was then filtered off and the filtrate evaporated to dryness (45"C). The remaining oily residue was treated with I liter of 5% HOAc. The product precipitated was extracted into.ethyl acetate and the organic phase washed with water, dried over Na2SO4 and evaporated to an oil. It was crystallized from ethyl acetate (300 ml) containing DCHA (10 ml) as a salt.
Recrystallization from MeOH and ether yielded 22.7 g (72.5%) of Boc - Lys(Z) Lys(Z) - OH DCHA; m.p. 161620C; [(u]5=-2.21" (c=l, MeOH).
Boc - Lys(Z) - Lys(Z) - OH DCHA (10 g, 12.14 mmol) was partitioned between EtOAc (1 liter) and 0.1 N H2SO4-(l liter). The organic layer was then washed with water (3x), dried over Na2SO4 and evaporated to dryness (7.9 g). The free acid, Boc - Lys(Z) - Lys(Z) - OH, thus obtained was treated with freshly prepared 4M HCI in THF for 30 minutes. The solvent and the excess acid was evaporated (30"C) and the residue re-evaporated twice with THF. The remaining residue solidified when treated with ether. The salt HCI H - Lys(Z) - Lys(Z) - OH was collected by filtration and washed several times with ether to yield 6.7 g of white powder. It was dissolved in DMF (70 ml), chilled in an ice-bath and treated with Et3N (1.63 ml) followed by Boc - Glu(OBzl) - OSu (5.54 g, 12.76 mmol). The mixture was stirred at OOC for 1 hour and then at 250C for 24 hours. More Et3N was added during this time to maintain the reaction at approximately pH 7.5. A few ml of acetic acid was added to make the reaction acidic (pH 3.5) and the solvent removed by evaporation. The ensuing residue was taken up in EtOAc, washed with water (3x), dried over Na2SO4 and evaporated to dryness when the product began to solidify. It was triturated in ether and recrystallized from ethyl acetate. Yield 7.26 g (69.5%) of Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - OH; m.p. 1531550C; [a]D5=2.71 (c=l, THF).
g) Boc - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) Asn - OBzl (1.7 g, 1.16 mmol) was treated with TFA (24 ml) for 30 minutes. After evaporation of the excess acid (300 C) the residue was triturated with ether. The powder obtained was washed thoroughly with ether and petroleum ether and dried over NaOH in vacuo to give the trifluoroacetate salt of H - Glu(OBzl) - Val Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl (1.71 g). The active ester Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - OSu was then generated in situ by stirring Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - OH (0.998 g, 1.16 mmol), HOSu (0.16 g, 1.4 mmol) and DCC (0.274 g, 1.33 mmol) in 15 ml DMF at 0 C for 3 hours. To this solution containing the tripeptide active ester, the octapeptide salt CF3COOH H - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala Glu(OBzl) - Asn - OBzl (1.71 g) was added together with 0.2 ml of Et3N. A few more drops of Et3N and DMF (15 ml) were added and the mixture was stirred for 3 days at 250C. A gelatinous semi-solid formed. It was acidified with acetic acid and treated with water. The white solid precipitate was collected and washed (H2O, MeOH, ether) to yield 2.25 g of crude product melting at 310--3130C. It was dissolved in DMF and precipitated with MeOH. Yield: 1.75 g (68.3%) of Boc Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) Ala - Glu(OBzl) - Asn - OBzl; m.p. 314--316"C; [a]5=13.68" (c=l, DMSO); homogeneous on TLC.
h) Boc - Leu - OSu (4.0 g, 12.2 mmol) and H - Lys(Z) - OH (3.42 g, 12.2 mmol) were condensed in DMF (75 ml) during 48 hours in the presence of Et3N (1.7 ml). The reaction pH was maintained at 7.5 by addition of Et3N periodically as usual. The remaining insoluble material was filtered off and the filtrate evaporated to dryness. The ensuing foamy glass was dissolved in ether (200 ml) and the mixture treated with 3 ml of DCHA to produce crystalline material which was collected washed with ether and recrystallized from MeOH and ether. Yield: 5.7 g (69.5%) of Boc - Leu - Lys(Z) - OH DCHA; m.p. 140--1420C; [a]5=-7.20" (c=l, MeOH).
Boc - Leu - Lys(Z) - OH DCHA (2.97 g, 4.4 mmol was converted into the free acid (partitioned between EtOAc and 0.1 N H2SO4) and the colorless oil obtained (2.2 g) was treated with 4M HCI in THF (40 ml) for 30 minutes. The excess acid and the solvent were evaporated (300 C) and the residue treated with ether. The remaining oil was dissolved in ether and evaporated twice more with fresh ether. The residue was then stirred with Boc - Asp(OBzl) - OSu (1.85 g, 4.4 mmol) in the presence of ET3N (1.85 ml) overnight. The reaction mixture was then evaporated to dryness giving an oily residue which was taken up in ethyl acetate, washed with water (3x), dried over Na2SO4 and evaporated to dryness again. The crude product thus obtained was crystallized from ethyl acetate and petroleum ether and yielded 1.52 g (49.6%) of pure Boc - Asp(OBzl) - Leu - Lys(Z) - OH; m.p. 109-1110C; [a!]D5=16.14 (c=l, DMF).
Boc - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl (1.2 g, 0.545 mmol) was treated with 35 ml TFA for 30 minutes. The excess acid was quickly evaporated and the residue triturated with ether several times to give 1.2 g of H - Glu(OBzl) - Lys(Z) Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl)- Ala - Glu(OBzl) Asn - OBzl TFA-salt as a white powder. It was dissolved in a mixture of DMF (5 ml) and DMSO (2 ml) and treated with Boc - Asp(OBzl) - Leu - Lys(Z) - OSu generated in situ by stirring Boc - Asp(OBzl) - Leu - Lys(Z) - OH (0.381 g, 0.545 mmol) with HOSu (0.126 g, 1.1 mmol) and DCC (0.124 g, 0.599 mmol) in 3 ml DMF at OOC for 3 hours. The mixture containing the tripeptide active ester and undecapeptide was stirred at OOC for 2 hours and then at 250C for 3 days, during which period Et3N was added from time to time- in order to maintain the pH slightly basic. A gelatineous substance formed. It was triturated with 5% HOAc and the resulting white solid was filtered and washed with water, MeOH and ether to give 1.28 g of crude material melting at 325-3260C. reprecipitation from DMF/DMSO (10 mV5 ml) and MeOH (230 ml) yielded 1.22 g (80.2%) of pure Boc Asp(OBzl) - Leu - Lys(Z) - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl; m.p. 326--3270C; [a]025=-l5.710 (c=l, DMF/DMSO).
B.a) H - Ser(Bzl) - OH (18.6 g, 95.4 mmol) was dissolved in 45 ml Triton B, evaporated to dryness, and the residue re-evaporated twice with DMF (100 ml each). The residue was then stirred with AcOSu (16.9 g, 95.4 mmol) in 150 ml DMF for 20 hours. N-Methylmorpholine was added from time to time in order to maintain the reaction slightly basic. The solvent was removed and extraction of the product into EtOAc was followed by washing with small volumes of 10% HOAc, and H2O (the product is water soluble, use a small volume of H2O). Drying over Na2SO4 and evaporation.to dryness again gave a clear oil (14.5 g, Ac - Ser(Bzl) - OH). The compound failed to crystallize. It was thus dissolved in a mixture of 300 ml of MeOH and 300 ml of H2O, titrated to pH 7.0 with 20% Cs2CO3 and evaporated to a solid mass. The salt was reevaporated twice more with DMF and stirred with benzyl bromide (15.4 g, 91 mmol) in 250 ml DMF for 18 hours. On evaporation of the solvent, the residue was taken up in H2O (600 ml) and the oily product formed was extracted into EtOAc. It was washed with H2O, dried over Na2SO4 and evaporated to a - syrup which on seeding crystallized immediately. It was recrystallized from EtOAc and petroleum ether to yield 10.42 g (32.2% overall) of H3C - CO - Ser(Bzl) - OBzl; m.p. 89-910C- H3C - CO - Ser(Bzl) - OBz1(2.2 g, 6.73 mmol) was dissolved in 75 ml EtOH and stirred gently with 5 ml of H2NNH2 overnight. Some insoluble matter precipitated was filtered off and the filtrate was evaporated to an oil which solidified when treated with ether. The product was recrystallized from a small volume of EtOH and ether to yield 1.40 g (82.8%) of H3CO - Ser(Bzl) - HNNH2; m.p. 128-1300C; [a]D5=+5.80O (c=l, MeOH).
b) L-Alanine (3.57 g, 40 mmol) was dissolved in 18.8 ml of Triton B (40 mmol), evaporated to dryness, and the oily residue re-evaporated twice with DMF (30 ml each). The salt obtained was stirred with 11.45 g of Boc - Ala - OSu (40 mmol) in 40 ml DMF, with 4 ml of NMM added, for 20 hours. The solvent was removed and the residue taken up in 10% HOAc (100 ml). The product was extracted into EtOAc (4x 100 ml), washed twice with a small volume of H2O, dried over Na2SO4, evaporated to a small volume, and treated with petroleum ether until cloudiness developed. A crystal-line product formed on storage in the refrigerator overnight.
Yield: 8.2 g (76.3%) of Boc - Ala - Ala - OH; m.p. 115-1180C.
Boc - Ala - Ala - OH (36.2 g, 139 mmol) was treated with 3 1 of 4 M HCI in THF for 30 minutes. Evaporation and work-up as usual gave an oily mass which solidified when treated with ether. The product was recrystallized from methanol with ether to yield 9.1 g (33.3%) of HCI H - Ala - Ala - OH; m.p. 209--211"C.
HCI H - Ala - Ala - OH (2.36 g, 12 mmol) was dissolved in 20 ml of DMF, chilled in an ice-bath, and treated with 1.68 ml of Et3N (12 mmol) followed by Boc - Asp(OBzl) - OSu (12 mmol). The mixture was stirred gently at 0OC for 2 hours and then at 25"C overnight during which time one more equivalent of Et3N (12 mmol) was added, in small proportions, maintaining the reaction pH near 8.0. A few ml of HOAc was added and the acidified mixture evaporated to dryness. The product formed was extracted into EtOAc, washed with H2O (3 times), dried over Na2SO4, and evaporated to an oily residue (6 g). It was dissolved in EtOAc and titrated to pH 8.0 with DCHA. The crystalline salt precipitated and was recrystallized from iso-propanol and petroleum ether to yield 5.1 g (65.7) of Boc - Asp(OBzl) - Ala - Ala - OH.DCHA; m.p. 138-1410C; la]n2B=l3.33o (c=l, MeOH).
Boc - Asp(OBzl) - Ala - Ala - OH DCHA (3.5 g, 5.4 mmol) was partitioned between 500 ml EtOAc and 350 ml H2O containing 10 ml of 10% H2SO4. The aqueous layer was extracted once more with EtOAc (250 ml) and the combined EtOAc layer washed twice with H2O, dried over Na2SO4, and evaporated to dryness, leaving a glassy solid of Boc - Asp(OBzl) - Ala - Ala - OH (2.5 g). This material was treated with 200 ml of freshly prepared 4 M HCI in TH-F for 30 minutes, evaporated at 320C to a syrup, and re-evaporated twice more with THF.
The oily residue solidified when treated with ether. This HCl H - Asp(OBzl) Ala - Ala - OH (1.93 g, 4.83 mmol) was then used in the next reaction involving azide coupling with H3C - CO - Ser(Bzl) - N3 that was prepared from 1.24 g H3C CO - Ser - (Bzl) - HNNH2 (4.9 mmol) in 25 ml DMF (-25"C) with 7.42 ml of 3.3 N HCI in THF (24.5 mmol) and 0.99 ml of i-amylnitrite (7.35 mmol) stirred at -300C for 30 minutes. The azide solution prepared was cooled down to -350C, mixed with 4.1 ml of Et3N and then treated with the white powder of HCI H - Asp(OBzl) Ala - Ala - OH (1.93 g) prepared above. The mixture was stirred at -200C for 30 minutes and then at 40C for 2 days. Some more .Et3N was added to keep the reaction slightly basic. Work-up as usual gave a crystalline mass which was recrystallized from THF and petroleum ether to yield 1.85 g (65.6%) of H3C - CO Ser(Bzl) - Asp(OBzl) - Ala - Ala - OH; m.p. 167--1700C; [Ct]D5=18.91 (c=l, DMSO).
H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala - Ala - OH (0.825 g, 1.41 mmol) was dissolved in 4 ml DMF and chilled to OOC in an ice-bath. To the solution, H2NNH2 (54.3 mg; 1.69 mmol) was added followed by HOBT H2O (0.475 g, 3.10 mmol) and DCC (0.32 g, 1.55 mmol). The mixture was adjusted to pH 7.5 with NMM and stirred at OOC for 2 hours followed by 17 hours at 250C. The reaction became a gel during this time. It was diluted with MeOH and the solid material remaining was collected on a suction filter and washed thoroughly with MeOH, ether and petroleum ether to give a material melting at 229--2320C. The product was then precipitated from DMF and MeOH to yield 0.51 g (61.0%) of H3C - CO Ser(Bzl) - Asp(OBzl) - Ala - Ala - HNNH2; m.p. 23232"C; [CE]DS=17.94 (c=l, DMSO).
c) H - Glu(OBzl) - OH (39.4 g, 166 mmol) was stirred with Boc - Ser(Bzl) OSu (65.0 g, 166 mmol) in 900 ml DMF overnight in the presence of Et3N (2.3 ml, 165 mmol). More Et3N was added during this time in order to maintain the reaction slightly basic. The clear solution was evaporated to dryness and the oily residue partitioned between EtOAc (1.5 liters) and 5% HOAc (2 liters). The organic layer was washed with H2O (2 times), dried over Na2SO4, and concentrated to a clear oil (90.0 g) which was taken up in 3 liters of ether and treated with 25 ml of cyclohexylamine. The solid formed was recrystallized from MeOH and ether.
Yield: 76.2 g (74.8%) of Boc - Ser(Bzl) - Glu(OBzl) - OH CHA; m.p. 154- 156.5"C; [al2B=+6.320 (c=l, MeOH).
Boc - Ser(Bzl) - Glu(OBzl) - OH CHA (76.2 g 124 mmol) was suspended in a mixture of 1.5 liters each of H2O and EtOAc. To this mixture, 10% H2SO4 was added until it became acidic (pH ca. 2.5) and the solid dissolved. The organic layer containing the Boc - Ser(Bzl) - Glu(OBzl) - OH was washed with H2O (2 times), dried, and evaporated to dryness leaving a clear oil (68.5 g). It was treated with 3 liters of freshly prepared 4.1 M HCI in THF for 45 minutes and evaporated to an oily residue which was re-evaporated twice more with THF. The residue (HCl H - Ser(Bzl) - Glu(OBzl) - OH) was dissolved in DMF (500 ml), chilled to OOC, and treated with Boc - Ser(Bzl) - OSu (48.66 g, 124 mmol), followed immediately by 27 ml of Et3N. The mixture was stirred overnight at 250C during which time more Et3N was added occasionally in order to maintain the reaction slightly basic some small quantities of insoluble matters were removed by filtration and the filtrate was evaporated to an oil which was taken up in EtOAc, washed with 5% HOAc and H2O, dried over Na2SO4 and evaporated again to dryness. The product was crystallized from EtOAc with petroleum ether. Yield: 71.8 g (83.7%) of Boc Ser(Bzl)- Ser(Bzl)- Glu(OBzl)- OH; m.p. 112--1130C; [a15=+17.91" (c=l, THF).
Boc - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH (71.6 g, 104 mmol) was treated with 2.7 liters of freshly prepared 3.9 M HCI in THF for 45 minutes. The mixture was evaporated to dryness and the residue evaporated twice more with THF to give a solid mass (59.3 g; m.p. 161-1650C). It was collected and washed with ether and stirred in 500 ml of DMF with Boc - Thr(Bzl) - OSu (38.2 g, 94 mmol) in the presence of Et3N (25 ml) at OOC for 1 hour and then at 250C for 15 hours. More Et3N (14.5 ml) was added in several portions during this time to maintain the reaction slightly basic. Some insoluble matter formed was filtered off and the filtrate evaporated to an oil which was dissolved in EtOAc (1.5 liters), washed with 5% HOAc, H2O (2x), dried over Na2SO4 and evaporated to a solid mass. The product was recrystallized from EtOAc and petroleum ether to yield 64.8 g (78.1%) of Boc - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH; m.p. 115--1180C; [a]D5=+11.64 (c=l, DMSO).
Boc - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH (54.5 g, 61.7 mmol) was treated with HCI in THF (1.5 liters; 4.1 M) and worked up as usual to give HCI H - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - QH (46.3 g, 56.6 mmol) as a white powder. It was then stirred in DMF (500 ml) with Boc - Asp(OBzl) - OSu (23.7 g 56 mmol) at OOC for 2 hours in the presence of Et3N (16 ml). The mixture was further stirred at 250C for 15 hours during which time additional 7.4 ml of Et3N was added. The product was worked up as usual and crystallized from CH2CI2 and petroleum ether. Yield: 50.35 g (82.7%) of Boc - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) Ser(Bzl) - Glu(OBzl) - OH; m.p. 111--1130C; [a]5=+7.210 (c=l, DMSO).
Boc - Asp(OBzl) - - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH (50.0 g, 46 mmol) was deprotected with HCl (4.15 M) in THF and worked up as usual to give 45.4 g of white solid. It was dissolved in THF (1.5 liters) and treated with ether (7 liters). On standing at OOC overnight a white solid powder was obtained (44.0 g, m.p. 179-1840C). Part of this material, HCI H - Asp(OBzl) - Thr(Bzl) Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH (43.7 g, 42.7 mmol), was then dissolved in 500 ml of DMF, cooled to OOC, and treated with Boc - Val - OSu (15.4 g, 49 mmol) and Et3N (10 ml). The mixtuie was stirred for 15 hours during which time more Et N (7.9 ml) was added in several portions maintaining the reaction slightly basic.
insoluble matter was removed by filtration and the filtrate evaporated to dryness.
The oily residue was dissolved in CH2CI2, washed with 5% HOAc, H2O, dried over Na2SO4, and evaporated to a smaller volume (0.5 liter) when treated with petroleum ether. The product crystallized slowly during overnight standing. It was recrystallized from THF and iso-propanol to yield 26.3 g (51.4%) of Boc - Val Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH; m.p. 174--1770C; [a]5=+0.84" (c=l, THF).
Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - OH (13.0 g; 10.94 mmol) was dissolved in DMF (50 ml), cooled to OOC, and treated with H2NNH2 (0.421g; 13.14 mmol), HOBT (3.688 g; 24.1 mmol), and DCC (2.48 g; 12.04 mmol). NMM was then added until the reaction showed pH 7.5. The mixture was stirred for 18 hours and filtered to remove the insoluble by-products. The filtrate was evaporated to dryness and the residue treated with H2O. The solid formed was collected and crystallized from DMF and iso-propanol to yield 8.7 g (66.4%) of Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) HNNH2; m.p. 215-2180C; I]DS=+7.62O (c=l, DMSO).
d) Boc - Thr(Bzl) - Lys(Z) - OH (14.0 g, 24.5 mmol) was treated with 500 ml of 4.0 M HCI in THF for 30 minutes, evaporated to dryness, and re-evaporated twice with fresh THF. The oily residue solidified when treated with ether. The dried powder (11.4 g, 21.6 mmol) of the dipeptide hydrochloride salt was then dissolved in 140 ml DMF, cooled to OOC, and treated with Boc - Thr(Bzl) - OSu (8.8 g, 21.6 mmol) followed by 3.5 ml of Et3N. A few drops of Et3N was added to maintain a slightly basic condition while the mixture was stirred for an additional 24 hours at 250 C. It was acidified with 5 ml of HOAc and then diluted with a large volume of water. The solid crude product precipitated was collected, dissolved in EtOAc, washed with H2O, dried over Na2SO4, and evaporated to dryness, leaving a glassy solid mass. Crystallization from EtOAc and petroleum ether yielded 13.8 g (83.7) of Boc - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH; m.p. 110--1129C; [a] D2B=+ 19450 (c=l, EtOAc).
Boc - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (41.5 g, 54 mmol) was treated with 500 ml of freshly prepared 3.55 M HCI in THF for 25 minutes and evaporated to a syrup which was re-evaporated twice with fresh THF. The oily residue solidified when treated with ether. It was collected and washed with ether to give 37.4 g of crude hydrochloride salt of H - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH dissolved in 500 ml of DMF, chilled to OOC, and treated with Boc - Ile - OSu (17.4 g, 53 mmol), followed by 16 ml of Et3N. The mixture was stirred at 250C overnight during which time more Et3N was added in small portions (6.2 ml total) to maintain a slightly basic condition. The resultant mixture was filtered and the filtrate evaporated to an oil which was extracted into EtOAc, washed with 5% HOAc, H2O, dried over Na2SO4, and evaporated to give a yellowish oil. It was crystallized from EtOAc and petroleium ether. The crude solid thus obtained (39.6 g, mp 14-1420C) was found to be contaminated with several minor impurities. The material was then chromatographed on a silica gel column (4.7x67 cm) using CHCl3/MeOH (95:5, v/v as eluent. The fractions containing the desired product (monitored by TCL) were pooled and evaporated to give an oily product which was crystallized from CHCI3 and petroleum ether. Yield: 19.1 g (41.2 /n) of Boc - Ile - Thr(Bzl) - Thr(Bzl) Lys(Z) - OH; m.p. 144--1460C; [a]D5=+2.40 (c=l, CHCl3.
Boc - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (0.439 g, 0.496 mmol) was treated with 4 M HCI in THF for 30 minutes and worked up as usual to give 0.39 g of HCl. H - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH. Boc - Val - Asp(OBzl) Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(Obzl) - HNNH2 (0.59 g, 0.492 mmol) was then dissolved in 6 ml of DMF, cooled to -250C, and treated with 0.57 ml of 4.3 M.HCI in THF (2.46 mmol) followed immediately by 0.1 ml .of i-amylnitrite (0.74 mmol) Aft r stirring at --200 to -250C for 30 minutes, the temperature was lowered to -3 when 0.42 ml of Et3N and HCI H - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (0.39 g, prepared above). was added. The mixture was stirred at -200C for 30 minutes and then at 4"C for 48 hours during which time Et3N was added from time to time in order to keep the pH at about 7.5. The mixture was then diluted with 250 ml of 5% HOAc and the solid product formed was collected and washed with H2O, MeOH, ether, and dried over NaOH pellets in vacuo to give 0.82 g of crude material (m.p. 244--2540C). It was dissolved in DMSO and precipitated by addition of MeOH. Yield: 0.698 g (81.7%)' of Boc - Val - Asp(OBzl) - Thr(Bzl) Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH; m.p.
268-271 0C.
H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala - Ala - HNNH2 (0.408 g, 0.68 mmol) suspended in 10 ml DMF was cooled to -200C and treated with freshly prepared 5.43 M HCI in THF (0.627 ml, 3.4 mmol) followed by 10% i-amylnitrite in DMF (1.39 ml, 1.03 mmol). After stirring for 30 minutes, it was cooled down to -300C when Et3N (0.476 ml, 3.4 mmol) was added followed by the TFA salt of the decapeptide H - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (1.334 g, 0.68 mmol). The mixture was stirred at -200C for 30 minutes and then at 40C for 5 days during which time more Et3N and DMSO were added in order to maintain the reaction at a slightly basic condition and to keep the gel from forming. The reaction mixture was poured into 5% HOAc (300 ml) and the solid precipitate formed was collected, washed with H2O, MeOH, ether, and dried to give 1.49 g of material melting at 296--2990C. The product was reprecipitated from DMSO with MeOH. Yield: 1.40 g (85.37%) of H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) Ser(Bzl)- Ser(Bzl)- Glu(OBzl) - Ile - Thr(Bzl)- Thr(Bzl) - Lys(Z) - OH; []D5=+6.37O (c=l, DMSO).
The tetradecapeptide obtained (1.35 g, 0.558 mmol) was stirred with HOBT H2O (0.188 g, 1.23 mmol) fdr a few minutes in a mixture of 15 ml each of DMF and DMSO. The mixture was then cooled in an ice-bath when DCC (0.126 g, 0.614 mmol) was added and stirring continued for 24 hours at the same temperature.
In a separate flask Boc - Asp(Obzl)- Leu - Lys(Z)- Glu(OBzl)- Lys(Z) Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) Asn - OBzl (4.0 g, 1.43 mmol) was treated with 40 ml of TFA for 25 minutes and the TFA salt ot the ensuing H - Asp(OBzl) - Leu - Lys(Z) - Glu(OBzl) - Lys(Z) Lys(Z) - Glu(OBzl) - Va1 - Va1 - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) Asn - OBzl precipitated quickly with addition of a large volume of ether. The solid formed was collected and washed thoroughly with ether to give 3.74 g of white powder (TFA salt of C-terminal H - Asp(OBzl) - Leu - Lys(Z) - Glu(OBzl) Lys(Z)- Lys(Z)- Glu(OBzl)- Val - Val - Glu(OBzl) - Glu(OBzl)- Ala Glu(OBzl) - Asn - OBzl). Part of this material (1.567 g, 0.5583 mmol) was added to the active ester derived from the amino terminal tetradecapeptide as prepared above in a DMF - DMSO mixture. A few drops of NMM was added to bring the pH of the reaction to 7.5-8.0, and the stirring was continued for I hour at OOC and then 5 days at 250C. The reaction mixture was then poured into 1.5 1 of 5% acetic acid. The precipitated product was washed thoroughly with H2O, MeOH, DMF, MeOH and ether to give 2.21 g of the desired product, H3C - CO - Ser(Bzl) Asp(OBzl)- Ala - Ala - Val - Asp(OBzl) - Thr(Bzl)- Ser(Bzl)- Ser(Bzl) Thr(Bzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - Asp(OBzl) - Leu - Lys(Z) Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) Ala - Glu(OBzl) - Asn - OBzl, melting above 300"C.
The protected octacosapeptide (2.21 g, 0.435 mmol) was dissolved in 8 ml of TFA, mixed with 4 ml of anisol 4.0 M HCI in THF for 30 minutes. The excess acid and solvent were evaporated off and the residue re-evaporated twice with fresh THF. The oily product solidified when treated with ether. It was triturated with more fresh ether and collected to give a white powder (1.85 g). The hydrochloride salt thus obtained was dissolved in 40 ml DMF and chilled to OOC when H3C - CO - Ser - Ser(Bzl) - OH. DCHA (1.97 g, 4.75 mmol) was added. Afler stirring at OOC for 30 minutes, precipitation of DCHA HCI was observed and HOBT (1.19 g) was added followed by DCC (1.08 g, 5.23 mmol). The reaction was then adjusted to pH 7.5 with a few drops of NMM and stirred at 0 C for 2 hours and then at 250C overnight. The insoluble by-product was removed by filtration and the filtrate evaporated to a slightly colored solid mass. It was washed thoroughly with H2O and EtOAc to give a buff colored powder which was crystallized from DMF (40 ml) and iso-propanol (500 ml). Yield: 1.57 g (56.9%) of H3C - CO - Ser(Bzl) - Asn - Ala - Ala - OBzl; m.p. 213-2150C; [a]D5=23.11 (c=l, DMSO).
H3C - CO - Ser(Bzl) - Asn - Ala - Ala - OBzl (1.57 g, 2.69 mmol) was dissolved in 20 ml DMF and stirred with 2 ml of H2NNH2 for 18 hours. The solid product formed was collected and washed thoroughly with DMF, EtOH, and ether to yield 1.22 g (89.6%) of H3C - CO - Ser(Bzl) - Asn - Ala - Ala - HNNH2; m.p.
262--264"C; [a]D5=26.7 (c=l, DMSO).
Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - glu(OBzl) - Ile Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (0.698 g, 0.358 mmol) was treated with 10 ml of TFA for 30 minutes and the peptide salt was precipitated with ether. It was collected on a suction filter, washed with ether and dried to give 0.652 g of material (0.333 mmol as TFA salt). In a separate flask, H3C - CO - Ser(Bzl) - Asn - Ala Ala - HNNH2 (0.17 g, 0.335 mmol) was suspended in 7 ml DMF and treated with 0.27 ml of 6.18 N HCI in THF at -20 C. To the mixture, 0.68 ml of 10% iamylnitrite in DMF was added and the solution stirred at the same temperature for 30 minutes. The temperature was lowered to -300C when 0.234 ml of Et3N (1.67 mmol) was added followed by the TFA salt of decapeptide (0.652 g) prepared above. The mixture was diluted with 3 ml of DMSO at -200C and adjusted to a slightly basic condition (pH 7.5) with a few drops of Et3N. It was stirred at -200C for 30 minutes and then at 40C for 5 days. More DMSO (5 ml) and Et3N were added during this period of time to maintain the slightly basic conditions and to keep the reaction from becoming a gel. The entire solution was then poured into 5% HOAc (250 ml) to give a white precipitate which was collected, washed with H2O, MeOH, ether, and dried. The crude product (0.702 g; m.p. 292910C) was reprecipitated from DMSO with MeOH. Yield: 0.348 g (42.0% of H3C - CO - Ser(Bzl) - Asn Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH; m.p. 296-2980C (dec.); [a]D5=+3 77 (c=l, DMSO).
The product obtained (0.866 g, 0.373 mmol) was stirred with HOBT. H2O (0.126 g, 0.82 mmol) in a mixture of DMSO (8 ml) and DMF (6 ml) and chilled in an ice-bath. The mixture was treated with DCC (0.085 g, 0.411 mmol) and then stirred at 0 for 24 hours. It was then mixed with the TFA salt of the tetradecapeptide prepared as in Example 1B.d) (1.05 g, 0.373 mmol) and 2 ml more of DMSO. A few drops of NMM were added to bring the pH to 7.5-8.0 and the stirring was continued at OOC for 1 hour and then at 250C for 5 days. Work up as described in Example 1B.d) gave 1.5775 g of the fully protected octacosapeptide H3C - CO Ser(Bzl) - Asn - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - (Ser(Bzl) - Ser(Bzl) Glu(OBzl) - Ile - Thr(Bzl)- Thr(Bzl)- Lys(Z) - Asp(OBzl)- Leu - Lys(Z) Glu(OBzl) - Lys(Z) - Lys Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) Ala - Glu(OBzl) - Asn - OBzl.
The fully protected octacosapeptide (1.577 g, 0.3 mmol) was dissolved in 10 ml of TFA which contained 3 ml of anisole. The mixture was stirred with 45 ml of HF at 0 C for 30 minutes and then worked up as described in Example 1B.d).
Purification on Sephadex G-10 and DEAE - Sephadex columns as described above gave 0.283 g of H3C - CO - Ser - Asn - Ala - Ala - Val - Asp - Thr - Ser - Ser Glu - Ile - Thr - Thr - Lys - Asp - Leu - Lys - Glu - Lys - Lys - Glu - Val - Val - Glu - Glu - Ala - Glu - Asn - OH ((Asn2) - thymosin a1] as white amorphous product. The compound migrated at the position slightly less acidic than natural thymosin a1 on acrylamide gel isoelectric focusing in agreement with difference in the structure.
WHAT WE CLAIM IS: 1. A process for the preparation of thymosin a1 or its [Asn2] - analogue (as
**WARNING** end of DESC field may overlap start of CLMS **.

Claims (1)

  1. **WARNING** start of CLMS field may overlap end of DESC **.
    4.0 M HCI in THF for 30 minutes. The excess acid and solvent were evaporated off and the residue re-evaporated twice with fresh THF. The oily product solidified when treated with ether. It was triturated with more fresh ether and collected to give a white powder (1.85 g). The hydrochloride salt thus obtained was dissolved in 40 ml DMF and chilled to OOC when H3C - CO - Ser - Ser(Bzl) - OH. DCHA (1.97 g, 4.75 mmol) was added. Afler stirring at OOC for 30 minutes, precipitation of DCHA HCI was observed and HOBT (1.19 g) was added followed by DCC (1.08 g, 5.23 mmol). The reaction was then adjusted to pH 7.5 with a few drops of NMM and stirred at 0 C for 2 hours and then at 250C overnight. The insoluble by-product was removed by filtration and the filtrate evaporated to a slightly colored solid mass. It was washed thoroughly with H2O and EtOAc to give a buff colored powder which was crystallized from DMF (40 ml) and iso-propanol (500 ml). Yield: 1.57 g (56.9%) of H3C - CO - Ser(Bzl) - Asn - Ala - Ala - OBzl; m.p. 213-2150C; [a]D5=23.11 (c=l, DMSO).
    H3C - CO - Ser(Bzl) - Asn - Ala - Ala - OBzl (1.57 g, 2.69 mmol) was dissolved in 20 ml DMF and stirred with 2 ml of H2NNH2 for 18 hours. The solid product formed was collected and washed thoroughly with DMF, EtOH, and ether to yield 1.22 g (89.6%) of H3C - CO - Ser(Bzl) - Asn - Ala - Ala - HNNH2; m.p.
    262--264"C; [a]D5=26.7 (c=l, DMSO).
    Boc - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - glu(OBzl) - Ile Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH (0.698 g, 0.358 mmol) was treated with 10 ml of TFA for 30 minutes and the peptide salt was precipitated with ether. It was collected on a suction filter, washed with ether and dried to give 0.652 g of material (0.333 mmol as TFA salt). In a separate flask, H3C - CO - Ser(Bzl) - Asn - Ala Ala - HNNH2 (0.17 g, 0.335 mmol) was suspended in 7 ml DMF and treated with 0.27 ml of 6.18 N HCI in THF at -20 C. To the mixture, 0.68 ml of 10% iamylnitrite in DMF was added and the solution stirred at the same temperature for 30 minutes. The temperature was lowered to -300C when 0.234 ml of Et3N (1.67 mmol) was added followed by the TFA salt of decapeptide (0.652 g) prepared above. The mixture was diluted with 3 ml of DMSO at -200C and adjusted to a slightly basic condition (pH 7.5) with a few drops of Et3N. It was stirred at -200C for 30 minutes and then at 40C for 5 days. More DMSO (5 ml) and Et3N were added during this period of time to maintain the slightly basic conditions and to keep the reaction from becoming a gel. The entire solution was then poured into 5% HOAc (250 ml) to give a white precipitate which was collected, washed with H2O, MeOH, ether, and dried. The crude product (0.702 g; m.p. 292910C) was reprecipitated from DMSO with MeOH. Yield: 0.348 g (42.0% of H3C - CO - Ser(Bzl) - Asn Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile Thr(Bzl) - Thr(Bzl) - Lys(Z) - OH; m.p. 296-2980C (dec.); [a]D5=+3 77 (c=l, DMSO).
    The product obtained (0.866 g, 0.373 mmol) was stirred with HOBT. H2O (0.126 g, 0.82 mmol) in a mixture of DMSO (8 ml) and DMF (6 ml) and chilled in an ice-bath. The mixture was treated with DCC (0.085 g, 0.411 mmol) and then stirred at 0 for 24 hours. It was then mixed with the TFA salt of the tetradecapeptide prepared as in Example 1B.d) (1.05 g, 0.373 mmol) and 2 ml more of DMSO. A few drops of NMM were added to bring the pH to 7.5-8.0 and the stirring was continued at OOC for 1 hour and then at 250C for 5 days. Work up as described in Example 1B.d) gave 1.5775 g of the fully protected octacosapeptide H3C - CO Ser(Bzl) - Asn - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) - (Ser(Bzl) - Ser(Bzl) Glu(OBzl) - Ile - Thr(Bzl)- Thr(Bzl)- Lys(Z) - Asp(OBzl)- Leu - Lys(Z) Glu(OBzl) - Lys(Z) - Lys Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) Ala - Glu(OBzl) - Asn - OBzl.
    The fully protected octacosapeptide (1.577 g, 0.3 mmol) was dissolved in 10 ml of TFA which contained 3 ml of anisole. The mixture was stirred with 45 ml of HF at 0 C for 30 minutes and then worked up as described in Example 1B.d).
    Purification on Sephadex G-10 and DEAE - Sephadex columns as described above gave 0.283 g of H3C - CO - Ser - Asn - Ala - Ala - Val - Asp - Thr - Ser - Ser Glu - Ile - Thr - Thr - Lys - Asp - Leu - Lys - Glu - Lys - Lys - Glu - Val - Val - Glu - Glu - Ala - Glu - Asn - OH ((Asn2) - thymosin a1] as white amorphous product. The compound migrated at the position slightly less acidic than natural thymosin a1 on acrylamide gel isoelectric focusing in agreement with difference in the structure.
    WHAT WE CLAIM IS:
    1. A process for the preparation of thymosin a1 or its [Asn2] - analogue (as
    hereinbefore defined) and the pharmaceutically acceptable salts thereof which process comprises removing the protecting groups from a protected octascosapeptide of the sequence H3C-CO-Ser( R1 )-X-Ala-Ala-Val-Asp(OR2)-Thr( R3)-Ser( R1 )- Serf R')-Glu(OR4)-Ile-Thr(R3)-Thr(R3)-Lys(R5)-Asp(OR2)- Leu-Lys(R5)-Glu(OR4)-Lys(R5)-Lys(Rs)-Glu(OR4)-Val-Val- Glu(OR4)-Glu(OR4)-Ala-Glu(OR4)-Asn-ORs (I) wherein X is Asn or Asp(OR2) R' is a protecting group for the hydroxyl group of the serine radical; R2, R4 and RB are protecting groups for the carboxyl groups of the aspartic acid radical, the glutamic acid radical and the asparagine radical, respectively; R3 is a protecting group for the hydroxyl group of the threonine radical and R5 is a protecting group for the amino group of the lysine radical and, if desired, converting the compound obtained into a pharmaceutically acceptable salt.
    2. A process for the preparation of thymosin a, which process comprises removing the protecting groups from a protected octacosapeptide of the sequence H3C-CO-Ser(Bzl)-Asp(OBzl)Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z) Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl) Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OBzl (lea)
    3. A process for the preparation of [Asn2]thymosin ct, which process comprises removing the protecting groups from a protected octacosapeptide of the sequence H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)- Lys(Z)-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)- Glu(OBzl)-Val-Val-Glu(OBzl)-Glu(OBzl-Ala-Glu(OBzl)-Asn-OBzl (Ib) 4. A process for the preparation of thymosin a, or its [Asn2] - analogue according to claim 2 or claim 3 characterized in that removal of the protecting groups is achieved with anhydrous hydrogen fluoride.
    5. A process for the preparation of thymosin a, or its [Asn2] - analogue according to claim I which process is characterized in that the protected octacosapeptide of the sequence I is prepared by condensation of a protected tetra decapeptide of the sequence H3C-CO-Ser(R' )-X-Ala-Ala-Val-Asp(OR2)-Thr(R3)- Ser(R')-Ser(R')-Glu(OR4)-Ile-Thr(R3)-Thr(R3)-Lys(Rs)-OR7 (Il) with a protected tetradecapeptide of the sequence H-Asp(OR2)-Leu-Lys(R5)-Glu(O R4)-Lys( RB).Lys( R5)-Glu(O R4)- Val-Val-Glu(OR4)-Glu(OR4)-Ala-Glu(OR4)-Asn-OR8 (III) wherein X, R', R2, R3, R4, RB and Rub are defined as in claim 1 and R7 is hydrogen or an activated ester group.
    6. A process for the preparation of thymosin cr, according to claim 2 or claim 4 characterized in that the protected octacosapeptide of the sequence Ia as defined in claim 2 is obtained by condensation of the activated protected tetradecapeptide of the sequence H3C-Co-Ser(Bzl)-Asp(OBzl)-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bz1)-Lys(Z)-OBT (IIa) with the trifluoroacetic acid salt of the protected tetradecapeptide of the sequence H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)- Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OBzl)-Asn-OB zl (IIIa) wherein BT is benzotriazolyl.
    7. A process for the preparation of [Asn2] - thymosin cr, according to claim 3 or claim 4 characterized in that the protected octacosapeptide of the sequence IB as defined in claim 3 is obtained by condensation of the activated protected tetradecapeptide of the sequence H3C-CO-Ser(Bzl)-Asn-Ala-Ala-Val-Asp(OBzl)-Thr(Bzl) Ser(Bzl)-Ser(Bzl)-Glu(OBzl)-Ile-Thr(Bzl)-Thr(Bzl)-Lys(Z)-OBT (IIb) with the trifluoroacetic acid salt of the protected tetradecapeptide of the sequence H-Asp(OBzl)-Leu-Lys(Z)-Glu(OBzl)-Lys(Z)-Lys(Z)-Glu(OBzl)- Val-Val-Glu(OBzl)-Glu(OBzl)-Ala-Glu(OB zl)-Asn-OBzl) (IIIa) wherein BT is benzotriazolyl.
    8. A process for the preparation of thymosin a, or its [Asn2] - analogue and their pharmaceutically acceptable salts as hereinbefore particularly described with reference to the foregoing Examples I and 2.
    9. Thymosin &alpha;1, whenever prepared according to a process claimed in any one of claims 1, 2, 4--6 and 8 or by an obvious chemical equivalent thereof.
    10. [Asn2] - Thymosin a, whenever prepared according to a process claimed in any one of claims 1, 3-5, 7 and 8 or by an obvious chemical equivalent thereof.
    11. [Asn2] - Thymosin a, (as hereinbefore defined) and pharmaceutically acceptable salts thereof.
    12. H3C - CO - Ser(R') - Asn - Ala - Ala - Val - Asp(OR2) - Thr(R3) Ser(Rl) - Ser(R') - Glu(OR4) - Ile - Thr(R3) - Thr(R3) - Lys(R5) - Asp(OR2) Leu - Lys(Rs) - Glu(OR4) - Lys(R5) - Lys(R5) - Glu(OR4) - Val - Val - Glu(OR4) Glu(OR4) - Ala - Glu(OR4) - Asn - OR6 wherein R1, R2, R3, R4, R5 and RB are as defined in claim 1.
    13. H3C - CO - Ser(Bzl) - Asn - Ala - Ala - Val - Asp(OBzl) - Thr(Bzl) Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) - Asp(OBzl) Leu - Lys(Z) - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl.
    14. H3C - CO - Ser(Rl) - Asp(OR2) - Ala - Ala - Val - Asp(OR2) - Thr(R3) Ser(Rl) - Ser(Rl) - Glu(OR4) - Ile - Thr(R3) - Thr(R3) - Lys(R5) - Asp(OR2) Leu - Lys(R5) - Glu(OR4)- Lys(RS)- Lys(RS)- Glu(OR4)- Val - Val Glu(OR4) - Glu(OR4) - Ala - Glu(OR4) - Asn - OR6.
    wherein R', R2, R3, R4, Rub and RB are as defined in claim 1.
    15. H3C - CO - Ser(Bzl) - Asp(OBzl) - Ala - Ala - Val - Asp(OBzl) Thr(Bzl) - Ser(Bzl) - Ser(Bzl) - Glu(OBzl) - Ile - Thr(Bzl) - Thr(Bzl) - Lys(Z) Asp(OBzl) - Leu - Lys(Z) - Glu(OBzl) - Lys(Z) - Lys(Z) - Glu(OBzl) - Val - Val - Glu(OBzl) - Glu(OBzl) - Ala - Glu(OBzl) - Asn - OBzl.
GB1574/78A 1977-04-22 1978-04-21 Process for the preparation of thymosin a1 and an analogue Expired GB1590668A (en)

Applications Claiming Priority (2)

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US78989877A 1977-04-22 1977-04-22
US05/871,563 US4116951A (en) 1977-04-22 1978-01-23 [Asn2 ]-thymosin α1 and analogs thereof

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CA (1) CA1113088A (en)
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DK (1) DK147918C (en)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0741746B1 (en) * 1994-01-28 2006-01-11 SciClone Pharmaceuticals, Inc. Analogs of thymosin alpha 1

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4116951A (en) * 1977-04-22 1978-09-26 Hoffmann-La Roche Inc. [Asn2 ]-thymosin α1 and analogs thereof
DE2919592A1 (en) * 1979-05-15 1981-01-15 Max Planck Gesellschaft METHOD FOR PRODUCING THYMOSINE ALPHA 1 AND DERIVATIVES THEREOF
EP0033384B1 (en) * 1980-01-18 1984-02-15 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Medicaments containing fragments of thymosin-alpha-1 with immunostimulating activity, and thymosin-alpha-1 fragments
US4339427A (en) * 1980-04-14 1982-07-13 Hoffmann-La Roche Inc. Radioimmunoassay of thymosinα
EP0056594B1 (en) * 1981-01-14 1984-09-12 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Thymosin-alpha-1 fragments and pharmaceutical compositions with immunoregulating action containing them
CN1058500C (en) * 1993-02-03 2000-11-15 施塞克龙药品公司 Thymosin alpha-1 derivatives

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0741746B1 (en) * 1994-01-28 2006-01-11 SciClone Pharmaceuticals, Inc. Analogs of thymosin alpha 1

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JPS53137914A (en) 1978-12-01
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DE2817082A1 (en) 1978-11-02
PT67937A (en) 1978-05-01
NO821608L (en) 1978-10-24
DK169478A (en) 1978-10-23
FR2401134A1 (en) 1979-03-23
CH640218A5 (en) 1983-12-30
ATA286178A (en) 1981-03-15
CH641153A5 (en) 1984-02-15
SE7804612L (en) 1978-12-20
PT67937B (en) 1980-04-07
FI781241A (en) 1978-10-23
DK147918C (en) 1985-08-19
LU79488A1 (en) 1979-05-25
IT1113134B (en) 1986-01-20
IT7822629A0 (en) 1978-04-21
CH641152A5 (en) 1984-02-15
NL7804364A (en) 1978-10-24
GR71886B (en) 1983-08-04
FR2401134B1 (en) 1983-09-30
DK147918B (en) 1985-01-07
NO148924B (en) 1983-10-03
FR2405926B1 (en) 1983-09-09
SE447262B (en) 1986-11-03
FR2405926A1 (en) 1979-05-11
CA1113088A (en) 1981-11-24
AT364470B (en) 1981-10-27
HU180783B (en) 1983-04-29
NO781404L (en) 1978-10-24

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