FR2541271A1 - New synthesis of hpGRF in the liquid phase - Google Patents

Abstract

Process for the preparation of hpGRF via fragments, characterised in that there are coupled, one after the other and in order of sequence, the fragments in which: a. The side acid functional groups of aspartic and glutamic acids and the side amine functional group of lysine are protected by protecting groups which are stable under the conditions for the deprotection of the Boc group; b. The guanidine functional group of arginine is protected by protonation; and c. The N-terminal amino acid is protected on the amine by the Boc group, the Boc group being selectively removed from the N-terminal amine of the peptide in the elongation phase by hydrolysis with trifluoroacetic acid, the said coupling being carried out in an aprotic polar solvent and all the protecting groups are removed at the end of the sequence by hydrolysis using a 0.1 to 1M solution of methanesulphonic or trifluoromethanesulphonic acid in trifluoroacetic acid.

Description

New synthesis of hpGRF in liquid phase.

HpGRF (Human Pancreatic Growth Hormone Releasing
Factor) or Somatocrinine is a peptide consisting of the chain of 44 amino acids. His sequence is as follows
1 5 10
H - Tyr - Ala - Asp - Ala - Ile - Phe - Thr - Asn - Ser - Tyr
Arg - Lys - Val - Leu - Gly - Gln - Leu - Ser - Ala - Arg - Lily
25 30
Leu - Leu - Gln - Asp - Ile - Piet - Ser - Arg - Gln - Gln - Gly 35 40
Glu - Ser - Asn - Gln - Glu - Arg - Gly - Ala - Arg - Ala - Arg 44
Leu - NH2
It has recently been discovered by A. Guillemin et al.

 (Science, 218, 585-587 (1982)) from extracts of a human pancreatic tumor.

 This peptide is particularly active in stimulating the release of growth hormone (GH) both in vitro and in vitro, in particular its efficacy is shown at doses of. some fento moles / ml (ED50 = 15 fento moles / ml).

The therapeutic interest in human medicine of this substance will therefore be in the treatment of dwarfism and pediatric growth delays. Other applications are possible in cases of anabolic protein deficiency (stress ulcers, repair of fractures or cartilage damage, extensive burns (during the anabolic phase), skin repairs, osteoporosis ...).

In the veterinary field, this compound has an obvious interest in the weight growth of livestock (cattle, sheep, pigs, chickens, ...) and the increase in lactation (cattle, sheep, ...)
The industrial development of this polypeptide compound requires the synthesis of large quantities of this substance. Conventional methods of solid phase synthesis allow the preparation in short times of small amounts of this active ingredient (Science, 218, 585-587 (1982)) at very high costs and incompatible with large scale pharmaceutical development.

 bizous describe a liquid phase synthesis process that can be transposed on an industrial scale allowing access to the active ingredient with an excellent yield and purity ratio.

 This process is based on the principle of fragment synthesis.

 The example described uses 9 fragments whose cut points have been determined on the basis of chemical considerations (minimal risks of racemization during the coupling phases of the fragments). The division of these fragments could have been organized differently and the dzerite synthesis is only an example. An obvious advantage of a fragmented method over a stepwise strategy is that it is possible to synthesize these fragments in parallel, which results in a significant time saving.

At the level of complete elongation of the peptide from these 9 fragments, everything happens as if a nonapeptide were being synthesized.

The process of the present invention is further characterized in that one couples, one after the other and in the order of the sequence, the fragments in which a) the side acid functions of aspartic and glutamic acids
and the amine side function of lysine are protected by
stable protecting groups under the conditions of deprotection
of the Boc group, b) the guanidine function of arginine is protected by protonation, and c) the N-terminal amino acid is protected on the amine by the grouping
Boc, selectively removing the Boc group from the amine N-termi
of the peptide in the elongation phase by tri-acid hydrolysis
fluoroacétique, said coupling being carried out in a solvent
aprotic polar and is removed at the end of the sequence all protecting groups by hydrolysis using a 0.1 to 1 Vii solution of acid
methanesulfonic or trifluoromethanesulfonic acid in tri acid
-fluoro.

The process of the present invention can also be applied to the synthesis of hpGRF 1-40, a natural product also isolated by
R. GUILLEMIN, almost as active as hpGRF 1-44 and usable in the same therapeutic indications.

 The subject of the present invention is therefore the preparation of hpGRF, a liquid phase fragment synthesis method according to a strategy of the type schematized below with respect to hpGRF 1-44.

GRF - SYNTHESIS STRATEGY

 In which the α-amino acids are represented by the symbols recommended by the IUPAC-IUB nomenclature commission biochemistry section.

Ala: alanine
Arg: arginine
Asn: asparagine
asp: aspartic acid
Gln: glutamine
Glu: glutamic acid
Gly. wistaria
Island: isoleucine
Leu: leucine
Lily: lysine
Met: methionine
Phe: phenylalanine
Ser: serine
Thr: Threonine
Tyr: tyrosine
Val: valine
They all have the L configuration, each amino acid is assigned its sequence number in the GRF sequence.

The side chain protections are symbolized by
X1 = ester of the benzyl type (0 Bzl),
X2 = carbamate protection of the benzyloxyearbonyl (Z) type.

The vertical arrows delimit the fragments used in the described synthesis and each of them is designated by a capital letter
A: fragment 40 - 44
5: fragment 33 - 39
C: fragment 28 - 32
D: fragment 25 - 27
E: fragment 21 - 24
F: fragment 16 - 20
G: fragment 12 - 15
H: fragment 5 - 11
I: fragment 1 - 4
The GRF synthesis process described is further characterized by the following aspects - application of the principle of minimum protection at the level of
functionalized side chains; - Temporary protection of the lateral guanidine function of the ar
ginine by the nitro group. Arginine is introduced in
sequence in nitro granidine form.The nitro function is then
eliminated as soon as possible (see summary of fragments) by
catalytic hydrogenation using Pd / coal, or using
reading instead of hydrogen gas a hydrogen generator,
such as formic acid or ammonium formate. So, all
synthesized fragments with arginine in their sequence
(A, B, F, H) have at the end of synthesis the functions guanidine sim
protected by protonation with a strong acid (acid
hydrolytic, for example).

 X1 - The carboxylic acid functions of the side chains of glutamic acid (B) and aspartic acid (DI) are protected by cleavable groups by catalytic hydrogenation (H2 / Pd / charcoal) or in strong acidic medium, such as mixtures methanesulphonic acid (0.5 M) - trifluoroacetic acid, or trifluoromethanesulphonic acid (0.5 M) - trifluoroacetic acid. We recommend as protectors the (benzyl) or 2,6-dichlorobenzyl ester. These protective groups are stable under the conditions of intermittent deprotection of the amines at α (removal of the t-butyloxyearbonyl: Boc groups by trifluoroacetic acid).

 X2 - The amine functions of the lysine side chains are protected by cleavable groups under the same conditions as above. We recommend benzyloxycarbonyl (Z), 2-chloro or 2-bromobenzyloxyearbonyl (2-Cl or 2-3r Z) groups that are stable under the conditions of intermittent deprotection with trifluoroacetic acid.

 The hydroxyl functions present in threonine, serine and tyrosine are not protected.

 The elongation of the peptide from the synthesized fragments (A to I) is carried out using as coupling agent benzotriazolyloxyphosphonium hexafluorophosphate (B 0 P), or dicyclohexylcarbodiimide in the presence of hydro-1-benzotriazole, in a solvent. suitable as dimethylformamide or dimethylsulfoxide. The isolation of the product from the reaction medium is carried out by introducing a third insolubilizing solvent (ether, ethyl acetate, etc.) which precipitates the peptide.

 During the coupling steps, we limit ourselves to summary purification of the solid phase washing type using appropriate solvents in order to eliminate the slight excess of the last coupled fragment, as well as the impurities introduced by the coupling agents.

Each coupling operation of a fragment is followed by an intermittent deprotection phase of the protective group
Boc (tert-butyloxycarbonyl), at the amine on which will be carried out the following coupling. This deprotection is provided by trifluoroacetic acid in methylene chloride (50/50 by volume).

 Deprotections of the side chains at the end of the elongation of the peptide may be carried out by hydrogenation in the presence of a catalyst (such as Pd / C) using hydrogen gas under slight pressure (1 to 5 bar). or with a hydrogen generator, such as formic acid or ammonium formate. It is also possible to eliminate this type of protecting groups by a strong acid, such as mixtures of methanesulfonic acid in trifluoroacetic acid (0.5 M) or trifluoromethanesulfonic acid in trifluoroacetic acid (0.5 M).

 The product is purified using the counter-current distribution technique using an immiscible two-phase system, such as Partridge medium (n-butanol-water-acetic acid), and the purification is completed. by gel filtration of Sephadex G 50 in dilute acetic acid (10-20%).

Each of the fragments involved in the synthesis of
GRF described has been synthesized according to a strategy adapted to each case.

 The different strategies used for the synthesis of fragments A to I are reported in the following tables. They all belong to "Step-Wise" processes in the liquid phase.

 In the case where it is desired to synthesize hpGRF 1-40, fragment A is reduced to alanine amide (H-Al-NH 2).

TABLE I (fragment A)

<tb><SEP> Ala <SEP> Arg <SEP> a <SEP> ----- <SEP> A <SEP> ra <SEP> I --- Leu <SEP> NH2
<tb><SEP> s <SEP> I- <SEP> t <SEP> t
<tb><SEP>, * <SEP> N 2
<tb><SEP>'2
<tb><SEP>,<SEP> /
<tb><SEP> L <SEP> Boc <SEP> L <SEP> Boc <SEP>'t<SEP> OH <SEP> 2
<tb><SEP> t <SEP> t <SEP> t <SEP> t
<tb><SEP> t <SEP> I <SEP> t
<tb><SEP> t <SEP> t <SEP> NO
<tb><SEP> Soc <SEP> TFA <SEP> NH2
<tb><SEP> I <SEP> t
<tb><SEP> t <SEP> t <SEP> t <SEP> t
<tb><SEP> I <SEP> t <SEP> NO
<tb><SEP> I <SEP> I <SEP> t <SEP> t <SEP> 2 <SEP> t
<tb><SEP> t <SEP> t <SEP> t <SEP> I <SEP> I
<tb><SEP> 1 <SEP> I <SEP> Bar <SEP> - <SEP> -! <SEP> NH
<tb><SEP> I <SEP> 2
<tb><SEP>)<SEP> I <SEP> 1 <SEP> BOP <SEP> NO, <SEP> I
<tb><SEP> I <SEP> + <SEP>'<SEP> 2
<tb><SEP> t <SEP> I <SEP> v
<tb><SEP> OC <SEP> NH
<tb><SEP> t <SEP> BOc -----, <SEP> ---- <SEP> 2
<tb><SEP> t <SEP> I <SEP> I <SEP> I <SEP> I
<tb><SEP> t <SEP> TFA
<tb><SEP> NO2
<tb><SEP> So <SEP> BOC <SEP> H ~~~~~~~~~~~~~~~~~~~~~~~
<tb><SEP> I <SEP> I
<tb><SEP> NH
<tb><SEP> 2
<tb><SEP> BOP <SEP> I <SEP> BOP
<tb><SEP> I <SEP> t <SEP> I <SEP> t <SEP> I
<tb><SEP> Soc <SEP>, <SEP> 4 <SEP> wN02
<tb><SEP>,<SEP> Boc <SEP> m <SEP>'H2
<tb><SEP> 1NO2 <SEP>'NO
<tb><SEP> t <SEP>'2
<tb><SEP> H <SEP>: <SEP>, N02 <SEP> I <SEP> N02 <SEP> I
<tb><SEP> H
<tb> t <SEP> I <SEP> I <SEP> t
<tb> t <SEP> I <SEP> t <SEP> 2
<tb><SEP> i <SEP> NO2 <SEP> t <SEP> NO
<tb><SEP> 4, <SEP> 2 <SEP> Ni '
<tb> z1 <SEP> t <SEP> I <SEP> t <SEP> 2
<tb><SEP> t <SEP> SS <SEP> H <SEP> I
<tb><SEP> t <SEP> H, <SEP> t <SEP> H <SEP> +
<tb><SEP> I <SEP>'<SEP> + <SEP> 2 <SEP> H <SEP> t
<tb><SEP> I <SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> I
<tb> R <SEP> NH2,3HCl
<Tb>
TABLE II (fragment B)

<tb><SEP> Glu <SEP> Ser <SEP> - <SEP> AsDr -------- Gln <SEP> ---- <SEP> Glu <SEP> Arg <SEP> Arg <SEP> ~ "<SEP> Gly
<tb><SEP> I <SEP> I <SEP> t <SEP> I <SEP>
<tb><SEP> I <SEP> t <SEP> I <SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> I
<tb><SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> t <SEP> I <SEP> t <SEP> t
<tb><SEP> t <SEP> I <SEP> I <SEP> t <SEP>: 1
<tb><SEP> t <SEP> t <SEP> t <SEP> t <SEP> I <SEP>, O <SEP> t
<tb><SEP> t <SEP> t <SEP> I <SEP> t <SEP> 2
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> z <SEP> I <SEP> I <SEP> 1 <SEP> 2
<tb><SEP> I <SEP> LH
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> 5 <SEP> t <SEP> I
<tb><SEP> I <SEP> I <SEP> t <SEP> t <SEP> I <SEP> M02 <SEP> I
<tb><SEP> t <SEP> I <SEP> I <SEP> I <SEP> J <SEP> t <SEP> I
<tb><SEP> t <SEP> I <SEP> s <SEP> t <SEP> t <SEP> t
<tb><SEP> I <SEP> I <SEP> t <SEP> I <SEP> r <SEP> O3zi
<tb><SEP> t <SEP> I <SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> t <SEP> I <SEP> I <SEP> t <SEP> +
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> H <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> Bzl <SEP> t <SEP> 21 <SEP> There
<tb><SEP> t <SEP> I <SEP> t <SEP> :: 0 <SEP> I
<tb><SEP> I <SEP> I <SEP> t <SEP> s <SEP> I <SEP> I
<tb><SEP> t <SEP> I <SEP> I <SEP> Np <SEP> H <SEP> OH
<tb><SEP> I <SEP> t <SEP> t <SEP>Boc;<SEP> I
<tb><SEP> t <SEP> oB-l <SEP> I <SEP> I <SEP> OH
<tb><SEP> t <SEP> I <SEP> I <SEP> I <SEP> H:
<tb><SEP> I <SEP> I <SEP> t <SEP> .oBzlV
<tb><SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> I <SEP> t <SEP>'-ff<SEP> OH
<tb><SEP> I <SEP> t <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> 2, / <SEP>'<SEP> IHO
<tb><SEP> I <SEP> I <SEP> I <SEP> I <SEP> t
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> I <SEP> I <SEP> I <SEP> ItO
<tb><SEP> Boc <SEP> I <SEP> t
<tb><SEP> I <SEP> I <SEP> t <SEP> t <SEP> Bzl <SEP>
#####
<tb><SEP> I <SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> HNp <SEP> t
<tb><SEP> t <SEP>: <SEP> oBzl <SEP>:
<tb><SEP> I <SEP> t <SEP> I <SEP> t
<tb><SEP>''<SEP>,<SEP> oSzl <SEP> /
<tb><SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP>, <SEP>, '<SEP> Boc <SEP> t <SEP> I
<tb><SEP> I <SEP> 5 <SEP> There
<tb><SEP> I <SEP> I <SEP> r <SEP> ptH <SEP> 1 | TFA
<tb><SEP> I <SEP> t <SEP> I <SEP> TFA <SEP> I
<tb><SEP> t <SEP> I
<tb><SEP> t <SEP> t <SEP> t
<tb><SEP> I <SEP> t <SEP> t <SEP> t
<tb><SEP> I <SEP> t
<tb><SEP> t <SEP> I
<tb><SEP> I <SEP> H <SEP> I <SEP>;<SEP> OH
<tb><SEP> 50cW <SEP> e-rr - t-Ot
<tb><SEP> I <SEP> t <SEP> +
<tb><SEP> I <SEP> I <SEP> I <SEP> I <SEP> t <SEP> I
<tb><SEP> I <SEP> Bzl <SEP> I
<tb><SEP> t <SEP> t <SEP> oBzl <SEP> t <SEP> H <SEP>:
<tb><SEP> I <SEP> I <SEP> t
<tb><SEP> I <SEP> I <SEP> I <SEP> t
<tb><SEP>(<SEP>:<SEP> Boc. <SEP>: <SEP> t
<tb><SEP>:<SEP> Bzl <SEP> +
<tb><SEP>:<SEP>',<SEP>!,<SEP> oBzl <SEP>'<SEP>, TFA
<tb><SEP> I <SEP> t <SEP> I
<tb><SEP> Boc <SEP> BocONbH <SEP>: <SEP>. <SEP> OH
<tb><SEP> I <SEP> -I <SEP> t <SEP> + <SEP> I
<tb><SEP> I <SEP> r <SEP>, <SEP> I <SEP> t
<tb><SEP>'I<SEP> t <SEP> oBz <SEP> H <SEP> t
<tb><SEP> I <SEP> t <SEP> t <SEP> I
<tb><SEP> t <SEP> t <SEP> I <SEP> t <SEP> I
<tb><SEP> Boc <SEP> + <SEP> OH
<tb><SEP> t <SEP> I <SEP> OH
<tb><SEP> pBzl <SEP> I <SEP> t <SEP> + <SEP> TFA
<tb><SEP>'<SEP> 3zl <SEP> I <SEP> I <SEP> t <SEP> t <SEP> H
<tb><SEP>:<SEP> o <SEP> Bz0 <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> t <SEP> I
<tb><SEP> oc <SEP> - <SEP> I <SEP> t <SEP> I <SEP>: tOH
<tb><SEP> Ip <SEP> H-
<SEP> I <SEP> I <SEP> I <SEP> I <SEP> +
<tb><SEP> B <SEP> o3zl <SEP> 1 <SEP> t <SEP> I <SEP> t <SEP> tH
<tb><SEP> t <SEP> I <SEP> I <SEP> t <SEP> o <SEP> t
<tb><SEP> I <SEP> I
<tb><SEP> floc <SEP> t <SEP> I <SEP> t <SEP> I <SEP> t
<tb> r <SEP> OH
<Tb>
TABLE III (fragment C)

<tb><SEP><SEP> Ar5g <SEP> Gfn
<tb><SEP> I <SEP> s <SEP> s <SEP> s
<tb><SEP> s <SEP> s <SEP> J <SEP> s <SEP> J
<tb><SEP> s <SEP> J <SEP> s <SEP> s <SEP> t
<tb><SEP> s <SEP> J <SEP> I <SEP> s <SEP> I
<tb><SEP> s <SEP> s <SEP> s <SEP> s <SEP> s
<tb><SEP> I. <SEP> t <SEP> J
<tb><SEP> I <SEP> t
<tb><SEP> j <SEP>, <SEP>,
<tb><SEP>!<SEP>,<SEP> Z <SEP> ~~~~~~~ OH <SEP> H <SEP> 30p
<tb><SEP> I <SEP> s <SEP>'<SEP> I <SEP> Z!
<tb><SEP> GCEA
<tb><SEP> I <SEP> J <SEP> t <SEP> J
<tb><SEP> N 2 <SEP>'<SEP> s <SEP> s <SEP> s <SEP> 2 / pu <SEP> s
<tb><SEP> s <SEP> 2 <SEP> s <SEP> s
<tb><SEP> s <SEP> s <SEP> s <SEP> s
<tb><SEP> s <SEP> s
<tb><SEP> Oc, <SEP> H <SEP>: <SEP> I <SEP> CH3
<tb><SEP> I <SEP> s <SEP> s
<tb><SEP> N 2 <SEP> Bop <SEP> s <SEP>,
<tb><SEP>,'<SEP>, / <SEP> i
<tb><SEP> s <SEP> J <SEP> 2 <SEP> s <SEP> s
<tb><SEP> s <SEP> s <SEP> s <SEP> s
<tb><SEP> s <SEP> I
<tb><SEP> Boc <SEP> - <SEP>, <SEP> OCH3
<tb><SEP> CH
<tb><SEP> s <SEP>><SEP> s <SEP> s <SEP> J <SEP> s <SEP> 3
<tb><SEP> s <SEP> t <SEP> J <SEP> J <SEP> s
<tb><SEP> I <SEP> 2 <SEP> s
<tb><SEP> I <SEP> I
<tb><SEP> ONb <SEP><<SEP> s <SEP> oc3s
<tb> Boy <SEP> s <SEP> s <SEP> - <SEP> s <SEP> OCH3
<tb><SEP> I <SEP> I <SEP> s <SEP> s <SEP> I
<tb><SEP> NO2 <SEP> NO2 <SEP>, <SEP>,
<tb><SEP> I <SEP> s <SEP> s <SEP> s
<tb><SEP> J <SEP> s <SEP> s <SEP> J
<tb> floc <SEP>><SEP> -------, 1-rs <SEP> s <SEP> OCH
<tb><SEP> I <SEP>, <SEP> H + <SEP>, <SEP> d
<tb><SEP> * <SEP> I <SEP> | <SEP> H2 / Pd
<tb> Boc
<tb> Boc <SEP> IIPPs. <SEP> I <SEP> t <SEP> 0CR3
<tb><SEP> X <SEP> X <SEP> yz <SEP> 3
<tb><SEP> I <SEP> 9 <SEP> s <SEP> OH
<tb> I <SEP>: <SEP> x <SEP> OH
<tb><SEP> s <SEP>: <SEP> OH
<Tb>
TABLE IV (fragment D)

<tb>SEP> Asp <SEP> I1 <SEP> e <SEP> ~ <SEP> binds
<tb><SEP> I <SEP> I
<tb><SEP> t <SEP> I
<tb><SEP> t <SEP> t
<tb><SEP> I <SEP> I
<tb><SEP> Soc <SEP>'<SEP> OH <SEP> H <SEP>'<SEP> MH-NH-Troc
<tb><SEP>,
<tb><SEP>! 1A,
<tb><SEP> I <SEP> I <SEP> t
<tb><SEP> s <SEP> Boc <SEP> s <SEP> s
<tb><SEP> i <SEP> Soc <SEP> NH-NH-Troc
<tb><SEP> OBzl <SEP> j <SEP> TFA <SEP> | <SEP>,
<tb> Boc <SEP>. <SEP> -1 / <SEP> ONSu <SEP> ~ I ~ <SEP> t <SEP> B <SEP> I <SEP> NH-NH-Troc
<tb><SEP> I <SEP> 1
<tb><SEP>'Bzl<SEP> t
<tb><SEP> s <SEP> t <SEP> s
<tb> Soc <SEP>, <SEP> - <SEP> CH2 <SEP> NH-NH-Troc
<tb><SEP>.
<Tb>

<SEP> OSzl
<tb><SEP> Soc <SEP>: <SEP>?; H-NH2
<tb><SEP> t <SEP> s
<tb><SEP> OBzl <SEP> NO2Na
<tb><SEP> v <SEP> t <SEP> N3
<Tb>
TABLE V (fragment E)

<tb><SEP> Lys <SEP> Leu <SEP> Leu <SEP>. <SEP> The, u <SEP> Gln
<tb><SEP> I <SEP> t <SEP> I
<tb><SEP> I <SEP> 1 <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> t
<tb><SEP>,<SEP> Z <SEP>'<SEP> OH <SEP> H <SEP>'<SEP> -O <SEP> 3Ut
<tb><SEP> I <SEP> 1 <SEP> - <SEP> I
<tb><SEP> I <SEP> I <SEP> MA <SEP> I
<tb><SEP> I <SEP> t <SEP> j
<tb><SEP> t <SEP> I <SEP> I <SEP> It
<tb><SEP> t <SEP> Z <SEP> O <SEP>-'-<SEP> - <SEP> 3 <SEP> Sut
<tb><SEP> I <SEP> I
<tb><SEP> t <SEP> I
<tb><SEP> 1 <SEP> 1 <SEP> I <SEP> 1 <SEP> I
<tb><SEP> I <SEP> I <SEP> I
<tb><SEP> 8, <SEP> Bor <SEP>'<SEP> NH <SEP> H <SEP>'<SEP> O <SEP> 3ut.
<Tb>

<SEP> I <SEP> s <SEP> s <SEP> t
<tb><SEP> I <SEP> I <SEP> t <SEP> t
<tb><SEP> t <SEP> t <SEP> I
<tb><SEP> MA <SEP> MUA <SEP> g <SEP> I
<tb><SEP> t <SEP> J <SEP> s <SEP> I
<tb><SEP> Bor <SEP>'<SEP>.<SEP>.<SEP> - <SEP> O <SEP> Purpose
<tb><SEP> t <SEP> I <SEP> t <SEP> t
<tb><SEP> I <SEP> I <SEP>, <SEP> I
<tb><SEP> I <SEP> t, <SEP> T.FA
<tb> Boc <SEP>; OSSU <SEP> H <SEP> 4 <SEP> OH
<tb><SEP> I <SEP> t <SEP> I <SEP> I
<tb><SEP> I <SEP> t <SEP> I
<tb><SEP> 7
<tb><SEP> Soc <SEP> / <SEP> .... <SEP> I <SEP>. <SEP>, t <SEP> OH
<Tb>
TABLE VI (fragment F)

<tb><SEP> Gln <SEP> Leu <SEP> - <SEA> Ser <SEP> Ser <SEA> Al <SEP> a <SEP>. <SEP> Arg
<tb><SEP> I <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> I
<tb><SEP> s <SEP> i <SEP> t <SEP> t
<tb><SEP> I <SEP>'<SEP>,<SEP>',<SEP> Soc <SEP>'<SEP> OBZt
<tb><SEP> t <SEP> I <SEP> 1
<tb><SEP> s <SEP> I <SEP> Tt
<tb><SEP> t <SEP>T; A '<SEP> I <SEP> r5
<tb><SEP> I <SEP> 5 <SEP> I
<tb><SEP> I <SEP> 5 <SEP> t <SEP> s <SEP> s
<tb><SEP> t <SEP> s <SEP> s <SEP> I
<tb><SEP> t <SEP>',<SEP> I <SEP> Boc <SEP>'<SEP>.<SEP> OH <SEP> z
<tb><SEP> f <SEP> 1 <SEP> I
<tb><SEP> I <SEP> t <SEP> s <SEP> I
<tb><SEP> I <SEP> 80P <SEP> X <SEP>',
<tb><SEP> s <SEP> I <SEP> I <SEP> I
<tb><SEP>,,><SEP> s <SEP> I
<tb><SEP> I <SEP> s <SEP> -Nb <SEP> 60c <SEP> I
<tb><SEP> I <SEP>. <SEP> uNb <SEP> Boc
<tb><SEP> 1
<tb><SEP> t <SEP> I <SEP> I
<tb><SEP> I <SEP> I <SEP> t <SEP> NO2
<tb><SEP> I <SEP> t <SEP> I
<tb><SEP>,<SEP> I <SEP> I
<tb><SEP> I <SEP> t
<tb><SEP>(<SEP> SOC <SEP> t <SEP> I <SEP> OBZI
<tb><SEP> s <SEP> t
<tb><SEP> I <SEP> I <SEP>: <SEP> TFI \ <SEP> I
<tb><SEP> I <SEP> ,, <SEP> t <SEP> TFA <SEP> I <SEP> c
<tb><SEP> I <SEP> I <SEP> OH <SEP> I <SEP> t
<tb><SEP> 3ocOH <SEP> H <SEP> - <SEP>! <SEP> I <SEP> OBzl
<tb><SEP> s <SEP> f <SEP> BOP <SEP> 3 / <SEP> I <SEP> 1 <SEP> I
<tb><SEP>,<SEP> I <SEP> I <SEP> I <SEP> It <SEP> Z
<tb><SEP> s <SEP> s <SEP> I
<tb><SEP> I <SEP> i <SEP> I <SEP> I
<tb><SEP> I <SEP> S <SEP> I <SEP> I <SEP> OSzi
<tb><SEP>Boc;<SEP> =
<tb><SEP> I <SEP> I <SEP> 15 <SEP> s <SEP> I <SEP> 1
<tb><SEP> TFA <SEP>} <SEP> t <SEP>: <SEP> 12
<tb><SEP> s <SEP> i <SEP> s
<tb><SEP> I <SEP> I <SEP>'<SEP>,
<tb><SEP> IOBz
<tb> Boc-q <SEP> -ONp <SEP> H <SEP> z
<tb><SEP> I <SEP> t <SEP> t
<tb><SEP> s <SEP> s <SEP> 2
<tb><SEP> t <SEP> s <SEP> s
<tb><SEP> s <SEP> I
<tb> s <SEP> I <SEP> t
<tb> Boct <SEP>, <SEP> OBzi
<tb><SEP> s <SEP> I <SEP> t
<tb><SEP> H / Pd <SEP> 1 <SEP>: <SEP> H *
<tb><SEP> I <SEP> t <SEP> 2 <SEP>'y<SEP> I
<tb><SEP> I <SEP> I <SEP> I
<tb>(<SEP>'<SEP> t <SEP> t <SEP> t <SEP> OH
<Tb>
TABLE VII (fragment G)

<tb><SEP> Lys <SEP> I <SEP> Val <SEP> Leu <SEP>. <SEP> Gly
<tb><SEP> 5 <SEP> s
<tb><SEP> I <SEP> t <SEP> t
<tb><SEP> 5 <SEP> S
<tb><SEP> I <SEP> 5 <SEP> s <SEP> ~
<tb><SEP> z <SEP> 5 <SEP> ONp <SEP> H <SEP> ON <SEP> H <SEP> I <SEP> - <SEP> CH
<tb><SEP> 1 <SEP> l <SEP> s <SEP> I
<tb><SEP> I <SEP> 5
<tb><SEP> I <SEP> I <SEP> S
<tb><SEP> I <SEP> s <SEP> I
<tb><SEP> i <SEP> I <SEP> Z <SEP> ~~~~~~~~~~~~~~~~~~~~~~~~~~~ <SEP> CH
<tb><SEP> S <SEP> I <SEP> I <SEP> O
<tb><SEP> t
<tb><SEP> I <SEP> 5 <SEP> I
<tb><SEP> s <SEP> I <SEP> S <SEP> / Pc: <SEP> s
<tb><SEP> S <SEP> I
<tb><SEP> ZOH <SEP> H <SEP> W <SEP> OCH3
<tb><SEP> I <SEP> 1
<tb><SEP> MA <SEP> | <SEP>'<SEP> MA
<tb><SEP> I <SEP> S
<tb><SEP> I <SEP> I <SEP> st <SEP> I <SEP> S
<tb><SEP> s <SEP> I <SEP> S
<tb><SEP> z '<SEP> I <SEP> OCH
<tb><SEP> i <SEP> 5 <SEP> ~ <SEP> ~ <SEP> tt <SEP> OCH3 <SEP> I
<tb><SEP> I <SEP> I <SEP> s <SEP> I
<tb><SEP> I <SEP> S <SEP> I
<tb><SEP> S <SEP> / <SEP> s
<tb>Soc'ONp<SEP> S <SEP> s <SEP> S
<tb><SEP> H <SEP>: <SEP>'<SEP> OCH3
<tb><SEP> s <SEP> I
<tb><SEP>.<SEP> Z <SEP> I <SEP> S
<tb><SEP> S <SEP> I <SEP> I
<tb><SEP> I <SEP> 5 <SEP>,
<tb> Boc <SEP> - <SEP> g <SEP> t <SEP> ~ <SEP> t <SEP> | <SEP> OCH3
<tb><SEP> OH <SEP> t
<tb><SEP> I <SEP> 4 <SEP> OH
<tb> S <SEP> S
<tb> Soc <SEP>'I<SEP> I <SEP>'<SEP> OH
<Tb>
TABLE VIII (fragment H)

<tb><SEP> IlPhr <SEP> Thr -------- Asn <SEP> Ser-7yr <SEP> ---- <SEP> Arg
<tb><SEP><SEP> I <SEP> I <SEP> j
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> I
<tb><SEP> i <SEP> I <SEP> r <SEP> I <SEP> i <SEP> g <SEP> t
<tb><SEP> I <SEP> j <SEP> j <SEP> j <SEP> I <SEP> j <SEP> t
<tb><SEP> I <SEP> t <SEP> j <SEP> j <SEP>"I!<SEP> ti-2 <SEP> j
<tb><SEP> I <SEP> j <SEP> j
<tb><SEP><SEP> Sot <SEP> H <SEP> Boc
<tb><SEP> I <SEP> 1 <SEP>'BSP<SEP>:<SEP> NO2
<tb><SEP>(<SEP> F <SEP> F <SEP> I <SEP> 1 <SEP> Company <SEP> TrA <SEP> NO2
<tb><SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> ≈ <SEP> TFA <SEP> 1 <SEP> N02
<tb><SEP> I <SEP> j <SEP> j <SEP> I <SEP> j <SEP> j
<tb><SEP> ^ Bz
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> j <SEP> t <SEP> j
<tb><SEP> I <SEP> i <SEP> i <SEP> I <SEP> i <SEP> I <SEP> i <SEP> r ',
<tb><SEP>(<SEP> t <SEP> j <SEP> j <SEP> 3oc0Nb <SEP> ONb <SEP> z,
<tb><SEP> 1 <SEP> I <SEP> I <SEP> I
<tb><SEP> i <SEP> i <SEP> F <SEP> | <SEP> l <SEP> | <SEP> H <SEP>:, <SEP> N02
<tb><SEP>
<tb><SEP> j <SEP> I <SEP>, <SEP>, <SEP> s <SEP> s <SEP> j
<tb><SEP> I <SEP> 5 <SEP> 1-OEzi
<tb><SEP> I <SEP> I <SEP> i <SEP>'<SEP> 3oc <SEP> z
<tb><SEP> j <SEP> I <SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> j <SEP> 15 <SEP>: <SEP> j <SEP> j <SEP> TrA <SEP> NO2
<tb><SEP> I <SEP><SEP> t <SEP> I <SEP>: <SEP>
<tb><SEP> F <SEP> j <SEP> t <SEP> j <SEP> j <SEP> 5 <SEP> I
<tb><SEP> j <SEP> j <SEP> 1 <SEP> 1 <SEP> I <SEP> j <SEP> j
<tb><SEP> I <SEP> j <SEP> j <SEP> j <SEP> t <SEP> r
<tb><SEP>,<SEP> Boc <SEP>: <SEP> --ONb <SEP> I <SEP> 1 <SEP> t
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> t <SEP> j <SEP> I
<tb><SEP> j <SEP> j <SEP> I02
<tb><SEP> t <SEP> I <SEP> t <SEP> | <SEP> I <SEP> t <SEP> PrO <SEP> 2
<tb><SEP> I <SEP> j <SEP> j <SEP> j <SEP> j <SEP> j <SEP> t
<tb><SEP> I <SEP> t <SEP> k <SEP> y <SEP> j <SEP> j <SEP> j
<tb><SEP> I <SEP> t <SEP> j <SEP> I <SEP> j <SEP> Szl
<tb><SEP> t <SEP> I <SEP> s <SEP> Boc <SEP> j <SEP> L <SEP> Bzl
<tb><SEP> I <SEP> t <SEP> j <SEP> 5 <SEP> j
<tb><SEP> j <SEP> j <SEP> j <SEP> I <SEP> j
<tb><SEP> FA <SEP>, <SEP> I <SEP> I <SEP> j <SEP> I
<tb><SEP> I3Bzl
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> t
<tb><SEP> j <SEP> j <SEP> I
<tb><SEP> j <SEP> j
<tb><SEP>,<SEP> Boc <SEP><SEP> 3ocfr0NSu <SEP> R <SEP><<SEP> t
<tb><SEP> I <SEP> j <SEP> j <SEP> j <SEP> j <SEP> I <SEP> t
<tb><SEP> I <SEP> I <SEP> l <SEP> l <SEP> l
<tb><SEP> t <SEP> j <SEP> I <SEP> t <SEP> j <SEP> j <SEP>'' 2
<tb><SEP> I <SEP> t <SEP> j <SEP> j <SEP> I
<tb><SEP> I <SEP> t <SEP> j <SEP> j <SEP> t <SEP> j
<tb><SEP> t <SEP> Soc <SEP>: <SEP>('<SEP> ----- <SEP> Boc <SEP> (
<tb><SEP> j <SEP> j <SEP> j <SEP> t <SEP> j
<tb><SEP> I <SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP><SEP><SEP> I <SEP> t <SEP> 9 <SEP> 1 <SEP> NO2
<tb><SEP> 1 <SEP> H <SEP> j <SEP> j <SEP> OBzl
<tb><SEP> I <SEP> Boc <SEP> t
<tb><SEP> 111
<tb><SEP> t <SEP> t <SEP> t <SEP> t <SEP> tj <SEP> t <SEP> t <SEP> its <SEP> O
<tb><SEP> j <SEP> a <SEP>, <SEP> j <SEP> t <SEP> I <SEP> I
<tb><SEP> j <SEP> t <SEP> j <SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> j <SEP> j <SEP> I <SEP> t <SEP> t <SEP> r '
<tb><SEP> j <SEP> Boc
<tb><SEP> t <SEP> t <SEP> j <SEP> j <SEP> t <SEP> i
<tb><SEP> j <SEP> t <SEP> j <SEP> j <SEP>: <SEP>
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> t <SEP> t <SEP> tî.
<Tb>

<SEP> j <SEP> t <SEP> nBzl
<tb><SEP> j <SEP> I <SEP> j <SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> t <SEP> j <SEP> y <SEP> I <SEP> t <SEP> n
<tb> Bocf- <SEP> OH <SEP> j
<tb><SEP> j <SEP> flOP <SEP>: <SEP> NO
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> s <SEP> t <SEP> 2
<tb><SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> j <SEP> j <SEP> t <SEP> j <SEP> I <SEP> j
<tb><SEP> 5 <SEP> j <SEP> j <SEP> I <SEP> j
<tb> flos <SEP>: <SEP> tj <SEP> F <SEP>;<SEP> t <SEP> II <SEP> j
<tb><SEP> j <SEP> j <SEP> I
<tb><SEP> t <SEP> t <SEP> j <SEP> H
<tb><SEP> t <SEP> j <SEP> j <SEP> j <SEP> j <SEP>;
<tb><SEP> j <SEP> j <SEP> j <SEP> j <SEP> j <SEP> j <SEP> j
<tb><SEP> j <SEP> 5 <SEP> I <SEP> j <SEP> t <SEP> j
<tb> I <SEP> I <SEP><SEP>(<SEP>)<SEP>'<SEP>'<SEP>!,<SEP> Z <SEP> OH
<Tb>
TABLE IX (fragment I)

<tb> Tyr <SEP> Ala <SEP> Asp <SEP> Ala
<tb><SEP> t <SEP> j <SEP> t
<tb><SEP> Tire <SEP> ... <SEP> 5 <SEP> Al <SEP> s
<tb><SEP> s <SEP> 5 <SEP> s <SEP> OSz
<tb><SEP> 5 <SEP>,
<tb><SEP> I <SEP> I <SEP> I / <SEP> I
<tb><SEP> I <SEP> I <SEP> IC)
<tb><SEP> AT4NSii <SEP>, OSut
<tb><SEP> Soc <SEP> H
<tb><SEP> 5 <SEP> 5
<tb><SEP>: 0 <SEP> Szl
<tb><SEP> Soc <SEP> Y <SEP>. <SEP>',<SEP> .... <SEP> OBut
<tb><SEP> 5 <SEP> j
<tb><SEP> I <SEP> I <SEP> TFA
<tb><SEP> I <SEP> I <SEP> 1 / <SEP> I-- <SEP> I
<tb><SEP> 30c <SEP> w <SEP> OTcp <SEP> H <SEP>: ;; i <SEP>, OH
<tb><SEP> j <SEP>, '<SEP> 5 <SEP> OH
<tb><SEP>.<SEP> s
<tb><SEP> Soc <SEP> 5
<tb><SEP> Boc <SEP> Boc <SEP> 5
<tb><SEP> 5 <SEP> OBzl <SEP> TFA5
<tb><SEP> I <SEP> IG <SEP>'
<tb><SEP> ZrONp <SEP> H <SEP>, <SEP> / <SEP>'<SEP>''<SEP> OH
<tb><SEP>,<SEP> OB21
<tb><SEP>,<SEP>.
<Tb>

<SEP>, <SEP>
<tb><SEP> OH
<tb><SEP> 7 <SEP> 5 <SEP> j
<Tb>
In the present application, the abbreviations used have the following meanings: CC-thin-layer chromatography
ED 50 effective dose 50
OBzl benzyl ester

Z benzyloxycarbonylamino (carbamate)
#
Boc tertiobutyloxyearbonyl-amino
(Carbamate)

DMF dimethylformamide
NEM N-ethylmorpholine
TFA trifluoroacetic acid
MY method of coupling with anhydrides
mixed
ONSU ester activated with N-hydroxy
succinimide

ON ester activated with ortho
nitrophenol

o
Troe trichloroethoxycarbonylamino CCl3-CH2-OC-NH
(carbamate) 0but ester with tert-butanol

OTcp ester activated with 2,3,5-tri
chlorophenol
OHBT N-hydroxybenzotriazole
ONb ester activated with N-hydroxy
norbornene-5-dicarboximide-2,3

BOR hexafluorophosphate benzo
triazolyloxyphosphonium

The nonlimiting examples below illustrate the invention
tion
EXAMPLE I Synthesis of IL-Ala-Arg-Ala-Arg-Leu-NH2
(fragment A)
NO2 1. H-Arg-Leu-NH2
Dissolve at room temperature 130 g of leucine amide (H-Leu-NH 2) in 1.5 l of DMF. Add 333 g of Boc-Arg (NO2) -OH and 500 g of BOP. Adjust the pH to 7 on pH paper (on small samples diluted with water) and with N-ethylmorpholine (NEM).

The medium is stirred and the evolution of the reaction is monitored by C.C.M.

The reaction is complete after 4 hours. The medium is evaporated to dryness under vacuum at 25 ° C. The residue is taken up in 1 liter of water and a solid is obtained which is washed with water and then with an aqueous solution of 5% NaHCO 3 in water, with ethyl acetate and, finally, the solid is dried in the air. It is controlled by C.C.M.

 The above solid is introduced into 2 1 of a 50-50 by volume mixture of trifluoroacetic acid-methylene chloride.

The medium is stirred for 10 min at room temperature and evaporated to dryness under vacuum at room temperature. The evaporation residue is taken up in ether, drained, dried and monitored by C.C.M. and R.M.N.

Yield: 339 g (90%) of trifluoroacetate of a white solid.

2. H - Ala - Arg (NO2) Leu - NH2
443 g of H-Arg trifluoroacetate (NO2) Leu-NH2 are dissolved in 2 l of DMF. 200 g of Boc - Ala - OH and then 500 g of BOP are added. PH 7 is adjusted to pH paper (on small samples of the reaction medium) using NEM. The medium is stirred and the evolution of the reaction is monitored by CCM

The reaction is complete after 4 hours. The medium is evaporated to dryness under vacuum at 25 ° C. The residue taken up in 2 1 water and 2 1 of ethyl acetate. The organic phase is washed with an aqueous solution of
5% NaHCO3, with water: dried and evaporated. The tripeptide is recrystallized from ethyl acetate-ether and finally dried under vacuum.

It is controlled by C.C.M. and R.M.N.

 The previous dried product is treated with a 50-50 (volume) mixture of TFA-CH2Cl2 under the conditions of the preceding example (I-1). The isolenent is also carried out under the same conditions.

Yield: 412 g (80%) expressed as trifluoroacetate of a product
powdery white (checked by TLC and NMR).

3. Arg (NO2) - Ala - Arg (NO2) - Leu - NH2
From 515 g of H - Ala - Arg (NO2) - Leu - NH2 in 2.5 l of DbE and 333 g of Boc - Arg (NO2) - OH and 500 g of BOP are obtained using the operating conditions. described in Example II, after treatment with the TFA-CH 2 Cl 2 mixture, 60% (85%) of a white solid controlled by TLC and NMR

4. Z - Ala - Arg (NO2) - Ala - Arg - Leu - NH2
From 715 g of trifluoroacetate of H - Arg (NO2) Ala
Arg (NO2) - Leu - NH2 in solution in 4 l of DMF and 233 g of Z - Ala - OH and 500 g of BOP using the same technique as described in I - 2 (and without treatment in this case by TFA - CH2C12), after recrystallization from the DMF-ether mixture, 612 g of
Z - Ala - Arg (NO2) - Ala - Arg (NO2) Leu - NH2 (76%) in the form
of a powdery white solid, controlled by TLC and NMR

5. Ala - Arg - Ala - Arg - Leu - NH2, 3 HCl
200 g of Z - Ala - Arg (NO2) Ala - Arg (N2) - Leu - NH2 (0.25 moles) are suspended in 2 liters of methanol containing 0.8 moles of HCl. 40 g of Pd / C containing 10% of Pd are added, and the medium is stirred under an atmosphere of hydrogen under 1.2 bar of pressure for 24 hours. After this time, the end of the reaction is checked by CCX. The catalyst is removed by filtration and the solvent is evaporated under vacuum at room temperature.

 The solid residue is purified by chromatography on silica gel using butanol, pyridine, HO2CCH3, OH2 (50-122 by volume) as eluting medium.

 The fractions containing the pure product are combined, evaporated and lyophilized.

Yield: 119 g (69%) of a white powdery solid.

Controls: K.M.N., C.C.X.

Amino acid analysis: Leu 1.03 (1)
Arg 1.92 (2)
Ala 1.95 (2)
EXAMPLE II
Synthesis of Poc - Glu - Ser - Asn - Gln - Glu - Arg - Gly - OH
(fragment B) 1. Z - Arg (NO2) - Gly - O Bzl
353 g of glycyl benzyl ester tosylate (H-Gly-O-Bzl, tosylate) are dissolved in 2 l of DMF, 115 g of NEM and then 353 g of Z-Arg (NO 2) -OH are added and the mixture is heated. 500 g of BOP. The pH of the medium is adjusted to 7 using NEM and using pH indicator paper on samples of the reaction medium diluted with water.

The reaction medium is stirred and the progress of the reaction followed by C.C.M. After 4 hours, the reaction is complete and the medium evaporated to dryness under vacuum at 35 ° C. The evaporation residue is taken up in 2 l of ethyl acetate and 2 l of water. Under these conditions, a solid is obtained which is drained and washed successively in solid phase with a 5% aqueous solution of HKSO 4 T K 2 SO 4, with pure water, with a 5% aqueous solution of NaHCO 3 and finally with water.

The product is air dried and monitored by NMR and
TLC

Yield: 410 g (82%).

2. H - Glu (OBzl) - Arg - Gly - OH
500 g of the preceding product in solution in 3 l of HC1N in DMF and in the presence of 100 g of Pd / C at 10% Pd are hydrogenated under a pressure of 1.5 bar of hydrogen. After 24 hours, the reaction is complete (TLC control). The catalyst is filtered and the pH of the medium adjusted to 7 by addition of N-ethylmorpholine and using pH indicator paper on samples diluted with water. 458 g of Boc - Glu (O Bzol) - 0 Np in solution in 1 liter of DMP are introduced. The reaction medium is diluted by addition of 1 liter of water and 135 g of OHBT and
NMS in sufficient quantities to maintain the pH at 7.

 The medium is stirred for 4 h at room temperature. It is ensured that the reaction is complete and the solvent is evaporated under vacuum at a temperature of 350 ° C. The evaporation residue is taken up in ethyl acetate. Under these conditions, the product is organized in solid. It is drained and chromatographed on silica gel using as eluent chloroform-methanol (70-30 by volume).

Evaporation of the chromatography fractions containing the pure product resulted in 412 g (75%) of a white powdery compound after lyophilization. It is controlled by C.C.M. and N.N.N.

 The previous product is dissolved at room temperature in 3 L of a mixture (50/50) of TFA and CH2Cl2. The medium is kept at room temperature for 15 minutes and evaporated to dryness in vacuo, keeping the temperature at 200 ° C. The residue is taken up in ethyl ether and the solid obtained is drained and dried. The yield is 423 g (100%) as trifluoroacetate.

3. H - Gln - Glu (O Bzl) - Arg - Gly - OH
564 g of H-Glu trifluoroacetate (O Bzl) - Arg-Gly-OH are dissolved in 3 l of dimethylformamide. The pH is adjusted to 7 by addition of NEM on samples diluted with water and using pH indicator paper.

 367 g of Boc - Gln - ONp in solution in 1 liter of DMP are then introduced. The reaction medium is diluted by addition of 1 liter of water. 135 g of QHBT and the sufficient amount of NEM are gradually added so that the pH is maintained at 7.

 The mixture is stirred for 4 h at room temperature. The end of the reaction is monitored by C.C.M. and the solvent is evaporated under high vacuum to 35. The evaporation residue is triturated in ethyl acetate. Under these conditions, a hygroscopic solid is obtained which is used as it is for the treatment with trifluoroacetic acid - CH 2 Cl 2 (2 l of the 50-50 by volume mixture).

The medium is stored for 10 minutes at room temperature
and finally evaporated in vacuo at 200. The evaporation residue is triturated in ethyl ether to give 525 g (76%) of
H - Gln - Glu (O Bzl) - Arg - Gly - OH (controlled by NMR and TLC).

4. H - Asn - Gln - Glu (O Bzl) Arg - Gly - OH
By rigorously using the conditions of
Example II-2 and from H - Gln - Glu (O Bzl) - Arg Gly - OH,
trifluoroacetate (691 g) and 393 g of Boc - Asn - ONb, we obtain
685 g (85% of a white solid product controlled by NMR and CCM

H - Ser - Asn - Gln - Glu (O Bzl) - Arg - Gly - OH
Also under the conditions of Example II-2 and from 806 g of H - Asn - Gln - Glu (O Bzl) Arg - Gly - OH and 366 g of Boc - Ser - ONb, the controlled product is obtained. by NMR and CCN with a yield of 688 g (77%) and in trifluoroacetate form.

6. Boc - Glu (O Bzl) - Ser - Asn - Gln - Glu (O Szl) - Arg - Gly - OH (fragment B)
From 894 g of H - Ser - Asn - Gln - Glu (O Bzl)
Arg - Gly - OH trifluoroacetate and 458 g of Boc - Glu (O Bzl) - ONp give 824 g (80%) of the expected product after purification by crystallization in DMF - ether medium (50-50 by volume).

The product is monitored by NMR, TLC and amino acid analysis
Gln - Glu: 2.92 (3)
Ser: 0.97 (1)
Asn: 1.03 (1)
Arg: 0.97 (1)
Gly: 0.95 (1)
By following the strategies presented in Tables III, IV, V, VI, VII, VIII and IX and the techniques described in Examples I and II, the following compounds can be obtained Boc - Ser - Arg - Gln - Gln - Gly - OH (fragment C)
Boc - Asp (O Bzl) - Ile - Met - N3 (fragment D)
Boc - Lys (Z) - Leu - Leu - Gln - OH (fragment E) floc - Gln - Leu - Ser - Ala - Arg - OH (fragment F)
Boc - Lys (Z) - Val - Leu - Gly - OH (fragment G)
Boc - island - Phe - Thr - Asn - Ser - Tyr - Arg - OH (fragment H) Z - Tyr Ala - Asp - Ala - OH (fragments I)
EXAMPLE III
Synthesis of Boc - Glu (O Bzl) - Ser - Asn - Gin - Glu (O Bzl) - Arg
Gly - Ala - Arg - Ala - Arg - Leu - NH2 (fragment BA)
69.2 g of the fragment A hydrochloride are dissolved in 500 ml of DMF. Just pH 7 on samples diluted with water and using pH indicator paper and NEM. 117 g of fragment B, 55 g of BOP are then added, the fold is again adjusted to 7 with NEM and the medium is stirred for 14 hours at room temperature. After end-of-reaction control by TLC, 2 liters of ethyl ether are added to the reaction medium. A white powdery precipitate is deposited under these conditions. It is purified by recrystallization in DMP-ethyl acetate. The drained product is then washed with ethyl acetate in solid phase and dried.

149 g (81%) of a white product controlled by
TLC and NMR

Amino acid analysis:
Gln - Glu: 2.92 (3)
Ser: 1.02 (1)
Asn: 0.97 (1)
Arg: 2.89 (3)
Gly: 1.01 (1)
Ala: 1.98 (2)
Leu: 0.93 (1)
EXAMPLE IV
The product described in the preceding example (184 g) is introduced into a mixture consisting of 750 ml of TFA and 750 ml of CH 2 Cl 2 with stirring and at room temperature. After dissolution, the mixture is stored for 15 minutes at the same temperature and evaporated under vacuum while maintaining the evaporation medium at 250 ° C. The evaporation residue is triturated in ether and, under these conditions, Iton obtains a white powdery solid which is dried under vacuum and monitored by TLC and NMR.

 The following compounds which can be obtained according to the techniques of Examples III and IV and representing the different stages of the elongation of the peptide are claimed.

Fragment: C - B - A
Boc - Ser - Arg - Gln - Gln - Gly - Glu (O Bzl) - Ser - Asn - Gln - Glu - (H)
Arg - Gly - Ala - Arg - Ala - Arg - Leu - NH2
Fragment: D - C - B - A
OBzl
Boc - Asp - island - Met - Ser - Arg - Gin - Gln - Gly - Glu (O Bzl) - Ser - (H)
Asn - Gln - Glu (O Bzl) - Arg - Gly - Ala - Arg - Ala - Arg
Leu - NH2
In this particular case, the fragment D is introduced using azide activation (-C-N3) (common in peptide synthesis). O
As soon as the methionine is in sequence, a sweeper will be used during the various treatments with TFA-CH2Cl2 during the phases of selective deprotection (elimination of Boc).

We recommend thioanisole as an agent. methionine protection to oxidation in the proportion of 5% in the medium TFA - CH2C12.

Fragment: E - D - C - B - A
Boc - Lys (Z) - Leu - Leu - Gln - Asp (O Bzl) - Ile - Met - Ser - Arg - Gln (H) Gln - Gly - Glu (O Bzl) - Ser - Asn - Gln - Glu (O) Bzl) - Arg - Gly
Ala - Arg - Ala - Arg - Leu - NH2
Fragment: F - E - D - C - B - A
Boc - Gln - Leu - Ser - Ala - Arg - Lys (Z) - Leu - Leu - Gln - Asp (OBzl) (H)
Met - Ser - Arg - Gln - Gln - Gly - Glu (O Bzl) - Ser - Asn
Gin - Glu (O Bzl) - Arg - Gly - Ala - Arg - Ala - Arg - Leu - NH2
Fragment: F - F - E - D - C - B - A
Boc - Lys (Z) - Val - Leu - Gly - Gln - Leu - Ser - Ala - Arg
Lys (Z) - Leu - Leu - Ser - Asp (O Bzl) - Ile - Met - Ser - Arg Gln - Gln - Gly - Glu (O Bzl) - Ser - Asn - Gln - Glu (O Bzl) - Arg
Gly - Ala - Arg - Ala - Arg - Leu - NH2
Fragment: H - G - F - E - D - C - B - A
Boc - island - Phe - Thr - Asn - Ser - Tyr - Arg - Lily (Z) - Val - Leu - (H)
Gly - Gln - Leu - Ser - Ala - Arg - Lys (Z) - Leu - Leu - Gln
Asp - (O Bzl) - Ile - Met - Ser - Arg - Gln - Gln - Gly - Glu (O Bzl)
Ser - Asn - Gln - Glu (O Bzl) - Arg - Gly - Ala - Arg - Ala - Arg
Leu - NH2 hp GRF protected
Z - Tyr - Al. - Asp (O Bzl) - Ala - Ile - Phe - Thr - Asn - Ser
Tyr - Arg - Lys (Z) - Val - Leu - Gly - Gln - Leu - Ser - Ala - Arg
Lys (Z) - Leu - Leu - Gln - Asp (O Bzl) - Ile - Met - Ser - Arg Gln - Gln - Gly - Glu (O Bzl) - Ser - Asn - Gln - Glu (O Bzl) - Arg
Gly - Ala - Arg - Ala - Arg - Leu - NH2
EXAMPLE V
Deprotection of protected hp GRF side chains
60 g of protected GRF hP are dissolved at 0 ° C. in 1 liter of trifluoroacetic acid containing 0.2 mol / l of methanesulfonic acid and 50 ml of thioanisole. The medium is stirred for 10 min at 0 ° C. and then 60 min at 250 ° C.

 The medium is evaporated at 250 under vacuum and the evaporation residue is taken up in ether and extensively washed in ethyl acetate. Under these conditions, a powdery solid of ocher color is obtained.

Yield: 55 g
EXAMPLE VI
Purification of hp GRF
The reaction crude obtained in the preceding example is purified using the counter-flow distribution technique using the butanol-water-acetic acid solvent system (4-5-1, by volume). The fractions having the maximum purity are combined, evaporated and lyophilized.

 The previous lyophilizate is dissolved in 30% acetic acid and subjected to fine Sephadex G 50 gel permeation chromatography.

 The fractions of chromatography exhibiting the maximum purity (C.C.M. control) are combined, evaporated, and the residue redissolved in water and freeze-dried.

 The state of purity was considered sufficient based on C.C.M. controls, HPLC and amino acid analysis.

Analysis of aminoacids: Tyr: 1.91 (2)
Ala: 4.88 (5)
Asn, Asp: 3.86 (4)
Island: 1.96 (2)
Phe: 0.93 (1)
Thr: 1.02 (1)
Ser: 3.88 (4)
Arg: 5.89 (6)
Lily: 1.98 (2)
Val: 1,01 (1)
Leu: 5.10 (5)
Gly: 2.98 (3)
Gln, Glu: 6.82 (7)
Met: 0.91 (t)

Claims (4)

 1 - Process for the preparation of hp GRF by fragments, characterized in that one couples one after the other and in sequence of the sequence the fragments in which a) the lateral acid functions of the aspartic acid and gluta  and the amine side function of lysine are protected  by stable protective groups in the conditions of deprotec  (b) the guanidine function of arginine is protected by protonation,  and c) the N-terminal amino acid is protected on the amine by the Boc group, selectively removing the Boc group from the N-terminal amine of the peptide in the trifluoroacetic acid hydrolytic elongation phase, said coupling being carried out in an aprotic polar solvent and at the end of the sequence all the protecting groups are removed by hydrolysis using a 0.1 to 1 M solution of methanesulphonic or trifluoromethanesulphonic acid in trifluoroacetic acid.  2 - Process according to claim 1, characterized in that after each coupling the product obtained is isolated from the reaction medium by precipitation with an apolar solvent.  3 - Process according to claim 1, characterized in that the crude reaction product is purified by the countercurrent distribution and gel permeation chromatography.  4 - Method according to one of claims 1 to 3, characterized in that the first fragment introduced in sequence is llala nine amide, thus leading to hp GRF 1-40.