CA2191612C - A process for preparing cardiodilatin fragments; highly purified cardiodilatin fragments and intermediate products for the preparation of same - Google Patents

Abstract

The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same.

Description

4012/oA/Wo A Process for Preparing Cardiodilatin Fraginents; Highly Pu-rified -cardiodilatin Fragments and Intermediate Products for the Preparation of Same The invention relates to a process for the prepara-tion of cardiodilatin fragments, to highly purified cardio-dilatin fragments, and to appropriate intermediates for'the preparation of said fragments.

The present invention is directed to a processfor the preparation of cardiodilatin fragments of formula I
R1-ANP(105-121)-R2 (1), having a chain length of 17 - 37 amino acids in to-tal, wherein ANP(105-121) represents the amino acid se- --quence [SEQ ID NO. 1], R1 represents -an amino acid chain of sequence ANP(90-104) [SEQ ID NO. 2] or fragments thereof having a chain length of 0 - 15 amino acids, and R2 represents an amino acid chain of sequence ANP(122--126) [SEQ ID NO. 3] or fragments thereof having a chain length of 0 - 5 amino acids, wherein synthesisis effected via condensation of at least three partial fragments, and condensation of the partial fragments to give the cardiodilatin fragments of formula I is carried out between the amino acid positions G1y108 and Arg109 and the amino acid positions G1y120 and Cys121.

Cardiodilatin is a peptide of the class of natriu-retic peptides. These peptides play an important role in regulating the balance of salts and water in the body. The prototype of natriuretic_hormones is cardiodilatin, also referred to in literature as atrial natriuretic peptide ~ 2191612 (CDD/ANP), The isolation of cardiodilatin andthe prepara-tion of biologically active fragments of cardiodilatin are known from US-PS 4,751,284 (cf., W.G. Forssmann et al., Klin. Wochenschr. 1986, 64 (Supp1. VI), 4-12). A review on isolation and characterization of cardiodilatin and frag-ments thereof, as well as their physiological properties has been published in Eur. J. Clin. Invest. 1986, 16; 439-451 (W.G. Forssmann). From EP 0,349,545, a specific cardio-dilatin fragment having a chain length of 32 amino acids is known. Meanwhile, this fragment is also referred to in 1it-erature as urodilatin (INN: ularitide). Furthermore, US
5,354,9b0 (Suntory) describes a biologically active frag-ment having a chain length of 28 amino acids, known as a-hANP.. Further biologically active cardiodilatin fragments or derivatives thereof have been described in EP 0,180,615.
Therein, in particular, cardiodilatin fragments are de-scribed which begin with the amino acid position Arg7-02 at the N-terminus and end with the amino acid position Arg125 or Arg126 at the C-terminus. Instead of the designation cardiodilatin, the literature frequently uses the designa- ----_--tion "atrial natriuretic peptide" (A.NP). In the numbering of the sequences of the cardiodilatin amino acids used in the following, reference is made to the nomenclature used-for theANF/CDD (1-126) peptide (=ANP) in EP 0,349,545.

A common structural feature of all hitherto known biologicaily active cardiodilatin fragments is the forma-tion of_a disulfide bridge between the amino acids Cys105 and Cys121, resulting in a stable ring of 17 amino acids.
It is believed that the formation of this ring is substan-tially responsible for the biological activity of the car-diodilatin derivatives. At position Cys105 the cardiodi-latin fragments are substituted by an amino acid chain R1 having a chain length of 0_- 15 amino acids, and at posi-tion Cys121 by a chain R2 having a chain length of 0 - 5-- -amino acids. In the [SEQ ID NO. 1], the central region ANP(105-121) is presented in linearized form.

~ 2191612 Rl-Cys Phe Gly Gly Arg Met -I
Asp Arg Ile Gly Ala Gln Ser Giy Leu Gly Cys-R2 115 120 -_-The cardiodilatin fragment ANP(95-126), with the INN designation ularitide, is a particularly stable' and biologically active human peptide, having diuretic activity and a relaxing effect on the smooth vascular muscles, which is formed of 32 amino acids and has the following sequence, wherein both the cysteine amino acids at positions 11 and 27 in the peptide are forming a disulfide bridge:

H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-I
Ile-G ly-Ala-Gln-Ser-GIy-Leu-Gly-Cy s-As n-Ser-Phe-Arg-Ty r-0H.

Urodilatin is found in human urine. EP 0,349,545 describes a process for recovering urodilatin from urine using alginic acid, wherein the peptides adsorbed to al-ginic acid are eiuted, the eluate is fractionated according to conventional purification methods, and the active frac-tion is recovered using a test based on the examination of the relaxing effect of urodilatin on the smooth muscles.

Furthermore, EP 0,349,545 describes a stepwise chemical synthesis of urodilatin using the Merrifield proc-ess (J. Am. Chem. Soc. 1963, 85; 2149-2156), at a solid phase according to the ABI standard program following the Boc strategy. In addition, this patent specification de--scribes, the preparation of urodilatin from the partial fragment ANP(99-126). This fragment is bound to a solid phase, and is reacted with a second partial fragment, the tetrapeptide Boc-Thr(But)-A1a-Pro-Arg(Tos). The peptide_ ANP(95-126) obtained from the condensation is removed from the support, subjected to cyclization after removal of the ~ 2191612 protecting groups and subsequently, is processed and puri-fied in a per se known manner.

Similarly, EP 0,180,615 describes the chemical syn-thesis using a solid support, wherein formation of the car-diodilatin fragments described therein is effected succes-sively, starting from the C-terminus in direction of the N-terminus. Here, condensation via partial fragments is not described. -However, the cardiodilatin fragments prepared ac-cording to the procedures described in literature did not have the purity necessary for clinical studies and for the authorization as medicinal product because, due to the syn-thesis, peptide impurities had been introduced into the fi-nal product which could not be removed even by subsequent purification processes. Due to their immunogenic proper-ties, the impurities may give rise to undesirable side-ef-fects when administered to the patient, so that therapeutic application involved risk. Moreover, the synthesis could be accomplished at only a small scale under reasonable techni-cal input and was not economically suitable for a larger production scale. Furthermore, another drawback of known processes for synthesis was the existing potential risk of racemization due to which the urodilatin was obtained with lower purity, lower biological activity and in insufficient yield. Racemization of the product which frequently occurs with existing syntheses often resulted in insufficient op-tical purity of the final product, and these impurities frequently cannot be removed or- only with exceedingly high technical input.

Thus, it is an object of the invention to develop an improved process for the chemical synthesis of cardiodi- -latin fragments which does not involve the above-mentioned drawbacks.

~ 2191612 The object of the invention is attained by perform-ing the synthesis of cardiodilatin fragments on the basis of the Merrifield proce-ss using a specific- selection of peptide fragments.

Surprisingly, the course of synthesis has be-en found to be optimal when the cardiodilatin fragments are formed using three partial fragments, with the condensation of the-partial fragments to give the cardiodilatin fragment of formula I being performed in such fashion that the for- .
mation is effected via condensation of partial fragments and bond formation between the amino acid positions Gly108 and Arg109 and the amino acid positions Gly120 and Cys121 This process is advantageous in that the cardiodilatin fragments of formula I can be obtained in higher yields and in higher purity as compared to the synthetic processes ---known from prior art. -The synthesis of the cardiodilatin fragments of formula I is effected in such way that initially, the three -partial. fragmerits having the sequences R1-ANP(105-108), ANP(109-120) and ANP(121)-R2 are prepared according to the Merrifield process. Then, preferably, condensation of the --three partial fragments to give the cardiodilatfn fragment of formula I is effected in two partial steps, whereby in a first step, condensation between the amino acid positions -- -G1y120 and Cys121 of the partial fragments ANP(109-120) and Cys7-21-R2 is effected, with the intermediate fragment ANP(109-121)-R2 being formed. Then, in a subsequent second step, condensation of the thus obtained fragment ANP(109-121)-R2 with the third partial fragment Rl-ANP(105-108) 3s effected, forming the desired cardiodilatin fragment of formula I. Using the process according to the invention, the yield of cardiodilatin fragments is between 15 and 20%, based on the amount of each cardiodilatin partial fragment -used as.starting material.
- - -The three partial fragments having the sequences R1-ANP(105-108), ANP(109-120) and ANP(121)-R2 are prepared according to the Merrifield process, wherein the amino ac-ids with functional groups (hydroxy, carboxy, amino, or mercapto groups) present in the sequence are substituted by appropriate protecting groups. For example, as suitable protecting groups the following groups are possible:
protecting groups for hydroxy groups: . Boc (t-butyloxycarbonyl), tBu (t-butyl ether);
protecting groups for amino functions: Fmoc (9-fluorenylmethoxycarbonyl), Pbf (2,2,4,6,7-pentamethyldi-hydrobenzofuran-5-sulfonyl), Pmc (2,2,5,7,8-pentamethyl-chroman-6-sulfonyl), Trt (trityl);
protecting groups for carboxy groups: OtBu (t-butyl ester);
protecting groups for mercapto - groups: Acm (acetamidomethyl) or Trt.
Here, the following protecting groups are preferred for thefollowing amino acids: tBu for the amino acids Thr, Asn, Tyr or Ser; Pbf or Pmc for the amino acid Arg; Acm for the amino acid Cys; OtBu for the amino acid Asp; Trt for the amino acids Gln, Asn or Cys.

Using the Fmoc strategy (B. Riniker et al., Tetra-hedron 1993, 49; 9307-9320), the protected partial frag-ments ANP(109-120), R1-ANP(105-108) and ANP(121)-R2 are formed on a solid support material. All the materials gen-erally used in the Merrifield synthesis may serve as solid support, materials. Preferred as support material is po1y--styrene functionalized as aminomethyl or benzhydrylamino compound. The superacid-sensitive bonding of the peptide fragments to the resin by means of the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid linker allows their removal without impeding the side-chain protection. The fragments are purified by digestion with various solvents. Thus, the three starting fragments ANP(109-120), R1-ANP(105-108) and ANP(121)-R2 are obtainedwith a C-terminal free carboxyl group and in good purity. When forming the peptides on the support resin, the yield in every single step of addition of one amino acid is nearly quantitative and is about 97-99%

The flow diagram in Figure 1 illustrates the prin-ciple of synthesis, with urodilatin ANP(95-126) as an exam-ple. Here, condensation of the fragment Boc-1-14-OH. (1) [this nomenclature corresponds to the general designation of fragment R1-ANP(105-108), wherein R1 = ANP(95-104)J with the fragment H-15-32-OtBu (5) [corresponding to an ANP no-menclature of ANP(109-121)-R2, wherein R2 =.ANP(122-126)]
is effected. This fragment (5) is synthesized from the fragments Fmoc-15-26-OH (2) [corresponding to an ANP nomen-clature: of ANP(109-120)] and H-27-32-OtBu (3c) [corresponding to an ANP nomenclature of ANP(121)-RZ].
'Figure 2 represents the fragments synthesized and modified with protecting groups.

In the next step, the carboxyl group of fragment (3a) is converted to the t-butyl ester (3b) (cf., Riniker et al., 22nd Europ. Peptide Symposium Interlaken, September 1992 (L7)). Subsequent removal of the Fmoc group from frag-ment (3b) leads tothe -product (3c) . This is fused with fragment (2), resulting in fragment (4). Removal of the Fmoc protecting group and condensation of the obtained fragment (5) with fragment (1) leads to the fully protected urodilatin (6). Removal of the protecting groups by treat-.--ment with trifluoroacetic acid and 1,3-propanedithiol as a scavenger provides the linear peptide (7) which is cyclized to crude urodilatin (8) by oxidation with iodine solution.
This is' desalted, purified and may be lyophilized subse-quently. The synthesis of other cardiodilatin fragments is --"
conducted in an analogous fashion.

The synthesis according to the invention, involving the described partial fragments ANP(109-120), R1-ANP(105-~ 2191612 108) and ANP(121)-R2 may be applied to all the cardiodi-latin fragments of formula I. In particular, cardiodilatin fragments are possible, wherein R1 has a chain length of 0-15 amino acids of the sequence ANP(90-104) or fragments thereof. Preferred for R1 are chain lengths of 1-15 or 3-10 amino acids, particularly the sequences ANP(95-104), ANP(99-104) and ANP(102-104). In particular, the group R2 represents a chain length of 1-5 amino acids of the se-quence ANP(122-126) or fragments -thereof_ Preferably, how-ever, the sequences ANP(122-126) and ANP(122-125) are pos-sible for R2.

Preferably, the cardiodilatin fragments ANP(95-126), ANP(99-126) and ANP(102-126) may be prepared accord-ing to, the process of the invention. The cardiodilatin fragments prepared by means of the process of the inven-tion, as well as the partial fragments required for conden-sation have high optical purity in the range of about 96.-99.91, particularly about 98-99%.

Similarly, the synthesis is suitable for all the other derivatives of cardiodilatin fragments wherein one or more amino acids in the sequenceof human ANP are replaced by other amino acids. In this meaning, replacement of amino acids includes corresponding substitutions, deletions or insertions of amino acids. For example, single or multiple amino acids may be replaced by the corresponding D-amino acids (cf., EP 0,180,615). Likewise, peptides of similar structure and with a corresponding cyclic basic structure of 15-20 amino acids may be prepared in this way. Examples of such peptides are BNP (brain natriuretic peptide) or CNP
(C-type natriuretic peptide). The structures of these pep- -tides a're described in J. Hypertension 1994, 12; 329-336 (N.C. Davidson and A.D. Struthers).

Likewise, the present invention is directed to novel partial fragments of ANP which are utilized for the ~ 2191612 preparation of cardiodilatin fragments of formula I accord-ing to the process of the invention.

More specifically, corresponding peptide fragments arethose of the type R1-ANP(105-108), wherein Rl repre-sents an amino acid chain of sequence ANP(90-104) or frag-ments thereof having a chain length of 0-15 amino acids, as well as their derivatives modified by protecting groups.
Here, in particular, R1 has the above-mentioned meanings.
Another novel peptide fragment is the fragment having the amino acid sequence ANP(109-120), as well as its deriva-tives modified by protecting groups, which is employed as a starting material in the condensation with the partial fragment ANP(121)-R2. Likewise, the corresponding ANP(121)-R2 type peptide fragments represent a novelty and a subject matter of the invention, wherein R2 represents ari amino acid chain of sequence ANP(122-126) or fragments thereof. having a chain length of 0-5 amino acids, as well as their derivatives modified by protecting groups. In par-ticular; R2 has the previously mentioned meaning. In addi-tion, the invention is directed to the intermediate ANP(109-121)-R2 which is formed from the condensation reac-tion of the partial fragments ANP(109-120) and ANP(121)-R2 effected in the first reaction step.

Furthermore, the present invention relates to_ a process' for preparing high-purity cardiodilatin fragmerits of formula I. Conventional synthetic processes and subse-quent purification procedures on cardiodilatin fragments suffered from the drawback that in many cases a peptide pu--rity in a range of merely 97-98% could be achieved.

EP 0,349,545 describes a purity level of about 98%
in the case of urodilatin; therein, the amount of urodilat-in prepared was merely on a smaller laboratory scale in the range of a few milligrams. The purification procedure de-scribed in Example 5 therein is based on a chromatography on a LH column (eluant: l". AcOll, 1~. 'I'FE'tOI1) and subsequent chromatogr.aphy ()n ,, '1'SK c:oluinn (F ra (,tf_) d (' l TM '1'SK-EIW ~ 0), wherein an aqueous ::,;o1 iit i on o f ] 0'illcOfi and 1, 'I'VEtOIi was used as the eluant. Ir1 a final puri_fication step, purifica-tion using preparative I{PLC is effected, without any fur-ther indications on the eluant beirlg made. Within the scope of later experiments on the preparation of larger amounts of urodilatin in the range of a few grams for performing clinical tests, it was determined, however, that in spite of multiple purification steps, the synthesized material could not be purified beyond a purity level of more than A comparable situation resulted in the case of car-diodilatin fragments described in EP 0,180,615. Therein, for example, the purification for fragment ANP(102-126) -in Example III.A.3 referred to as hANVP(127-151) - by chro-matography on a type G25F SephadexM column is described, where 0.5 M AcOH was used as the eluant. In a subsequent purification step by means of ion exchange chromatography on CM Sepharose'or CM Cellulose using a solvent gradient of 0.01 M NHqOAc/300 mM NHqOAc at pH 4.5, the peptide is ob-tained in a purity of about 97;',. Likewise, this purity achieved is not satisfactory for the requirements in drug manufacturing.

Surprisingly, it has been found that high-purity cardiodilatin fragments of formula I can be prepared if the crude product is purified using a reversed-phase HPLC col-umn, and the cardiodilatin fragment is eluted using a buffer system containing triethylammonium phosphate (TEAP) and acetonitrile in aqueous solution. Here, preferably, the pH value of the elution buffer is adjusted to a value of 2-5, more specifically, of 2-3. Preferably, a type C18 col-umn, for example, Biotage module type filled with YMC C18 is used as the reversed-phase HPLC column. This column is equilibrated with triethylammonium phosphate buffer prior to loading the cardiodilatin fragments to be purified. For example, a solution of 10-200 mM TEAP, preferably 50 mM
TEAP, is employed-as a suitable buffer solution.-The amount of buffer for column equilibration depends on the column size and this, in turn, on the amount of peptide to be pu-rified. According to experience, a column volume of 75 x 300 mm (diameter x length) is required to-purify an amount of peptide of 3-8 g of crude peptide. In this case, about 300 ml of a 50 mM TEAP buffer solution is required for equilibration. Subsequently, a solution of the concen-trated crude product of cardiodilatin fragment is applied.
As a solvent, for example, 10% acetic acid is suitable.
Thereafter, the peptide is eluted in a continuous gradient by continuous charging of eluant (mixture of an aqueous so- --lution of 10-200 mM TEAP and acetonitrile at a volume ratio of 2:3; pH 2-5). Elution of peptide is particularly advan-tageous if a continuous gradient of eluant is applied, where 22-28% of solvent gradient is used for a period of 90 minutes, followed by 28% of solvent gradient for 10 minutes and, eventually, 28-40% of gradient for 20 minutes. Prefer-ably, the flow rate is 100-200 ml/min, more specifically, about 140 ml/min. In the meaning of the purification proc-ess according to the invention, a buffer mixture of tri-ethylammonium phosphate in water and acetonitrile at a mix-ing ratio of from 1:3 to 2:1 (v/v), more specifically of about 2--3 (v/v) is used as elution buffer. The pH valueof the buffer solution is 2-5, preferably 2-3, and more spe-cifically about 2.25. TEAP may be used at a concentration -of 10-200 mM, preferably 20-100mM, and more specifically, of about 50 mM. According to the invention, optimum separa-tion is achieved in the reversed-phase HPLC by equilibrat-ing the column using 50 mM TEAP, pH 2.25, and eluting the peptide with a buffer consisting of 50 mM TEAP, pH 2.25, and acetonitrile at a ratio of 2:3.

Conventional purification procedures using aqueous 0.1o trifluoroacetic acid (TFA), for example, are not capa-ble of.further separating the polar impurities contained in the crude products, as are revealed in Fig. 5 in the exam-of urodilatin (Fig. 6). In contrast, in the case of the f ple eluants used according to the invention, there is signifi-cant separation of both impurities (see Fig. 5) Further-more, use of the eluant according to the invention is ad-vantageous in that the base line in the HPLC chromatogram takes an absolutely steady course, while in the case of -_:
TFA, a strong drift can be observed. In addition, use of TFA suffers from the drawback that a higher back pressure-builds up on the HPLC column, which is not the case for the -eluant according to the invention.

Using the process according to the invention, high-purity cardiodilatin fragments of formula I are obtained in a purity of at least 99% and preferably, of up to 99.9%.
Optionally, the cardiodilatin fragments may subsequently be converted to their physiologically acceptable salts, such as the acetate or citrate salts. The cardiodilatin frag-ments obtained are substantially free of peptide impurities so that not only the reversed-phase HPLC exhibits a single peak but also the much more sensitive method of capillary electrophoresis (CE) provides a single migration peak. In the case of urodilatin, the latter shows a mass of 3505.9 1 in the MS analysis, without byproducts being de-tected. It turned out that the use of capillary electropho-resis allows an excellent demonstration of the differences - -between cardiodilatin fragments obtained according to prior art and the cardiodilatin fragments according to the inven-S tion. Figure 3 illustrates the CE chromatogram of a urodi-latin production batch produced according to prior art.
Herein, it can be clearly seen that the product still con-tains impurities. In contrast, Figure 4 represents the CE chromatogram of a urodilatin production batch produced ac-cording to the process of the invention and purified corre-spondingly. =
It is clearly obvious that the product is sub-stantially free of other peptide impurities and exhibits a -single migration peak in the capillary electrophoresis.

Therefore, the invention is directed to high-purity cardiodilatin fragments of formula I which are remarkable in that they do not contain substantial peptide impurities detectable -by capillary electrophoresis and MS analysis, and that the purity analysis using capillary electrophore-sis exhibits a single migration peak.

Similarly, the purification procedure according to the invention is also suitable for the preparation of analogous high-purity peptide compounds such as, e.g., BNP
(brain natriur-etic peptide), CNP (C-type natriuretic pep-tide) or derivatives thereof. The cyclic structure of ANP
is based on the oxidation of two cysteine residues within the amino acid sequence, forming a cyclic ring of 17 amino acids. Other peptides which also form the characteristic -cyclic -structure of 15-20 amino acids, particularly 17 amino acids, such as, e.g., BNP or CNP, may be converted to the high-purity forms in the same fashion using the purifi-procedure according to the invention.
cation In the following embodiments, the invention will be illustrated using the selected representative cardiodilatin fragments ANP(95-126), ANP(99-126) and ANP(102-126).

Example 1 General Procedures of Solid-Phase Synthesis Accordinq to the Merrifield Process a) Solid-Phase synthesis on a support resin Starting from the C-terminus of the peptide to be =
synthesized, the first amino acid (AA) protected.by the Fmoc groiip at the N-terminal end, is bound to the support ~ 2191612 resin (FYnoc-AA-OHMPB-support resin) . With a standard batch of 6.66 mmoles, the Fmoc protecting group is subsequently removed by adding 100 ml of a solvent mixtureof piperidine and N-methylpyrrolidine (1:4 v/v) . Then, the resin suspen-sion is stirred for 10 minutes, subsequently filtrated, and again, 100 ml of the piperidine and NMP solvent mixture is added. -Then, the suspension is stirred for 10 minutes, fil-trated and subsequently washed with NMP an isopropanol,- and completeness -of the reaction is checked using the Kaiser test.

Thereafter, the next amino acid is coupled to the resin. Iriitially, 20 mmoles of a 0.5 M solution of diiso-propylethanylamine (DIPEA) in NMP is added to the resin, then 2.5 mmoles of a 0.5 M solution of 1-hydroxybenzotriazole (OHBT) in NMP, followed by 10 mmoles of the amino acid to be coupled in - 25 ml of NMP.
Thereafter, 11 mmoles of a 0.25 M solution of TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro- --borate) in NMP is added and stirred for 10 minutes.
Completeness of the reaction is checked using the Kaiser test. Subsequently, the resin is filtrated and washed with NMP. -This process is continued in the same way, until the peptide chain of desired chain length of amino acids is built up on the resin. When synthesis is complete, the resin is dried to constant weight at 40 C.

b) Removal of the protected peptides from the support resin Each of 10 suction flasks is charged with 75 ml of methanol and 3 ml of pyridine. 50 g of the support resin prepared according to step a) is stirred 10 times with 250 ml of 1% TFA in dry methylene chloride for one minute on the suction funnel, and is filtrated directly into the respective suction flask. These 10 filtrates are checked using thin layer chromatography. Fractions containing prod-uct are combined and evaporated_ to dryness. The residue is triturated with deionized water, and the crystalline resi-due is filtrated off and dried.

Example 2 Preparation of Fragment ANP(109-120) Following the general procedures of Example 1, and starting from 273 g of Fmoc-Gly-OHMPB-support resin (corresponding to 130 mmoles), 170.3 g of the fully pro-tected cardiodilatin fragment ANP(109-120) is obtained.
Example 3 Preparation of Fragment ANP(121-126) Following the general- procedures of Example 1, and starting from 264 g of Fmoc-Tyr-OHMPB-support resin (corresponding to 115 mmoles), 150.7 g of the fully pro-tected cardiodilatin fragment ANP(121-126) is obtained.
Here, the N-terminal end of the fragment is protected by the Fmoc group.

Subsequently, the terminal hydroxy group at the C-terminal end of the fragment is converted to the OtBu protecting group. For esterification, 149 g of the fully protected fragment is dissolved in 500 ml of triflubroetha-nol and 4.1 1 of chloroform. This is followed by addition of 141 ml of TBTA (t-butyl-2,2,2-trichloroacetimidate), and the solution is heated at reflux for one hour. After the reaction is completed, the solution is concentrated to give --a crystalline-oily residue, 6.8 1 of diisopropyl ether is added, and the suspension is stirred at room temperature for 14 hours. The product is filtrated off and dried to i, 2191612 constant weight. 136.7 g of fragment 3b indicated in Fig. 2 is obtained.

Subsequently, the Fmoc protecting group at the N-terminal ernd of the fragment is removed, and conversion to fragment 3c indicated in Fig. 2 is effected. To this end, a solution of fragment 3b (135.7 g) in 1.8 1 of DMF
and 74 ml of diethylamine is stirred at room temperature for 3 hours. The solution is evaporated to complete dryness in a vacuum. The residue is digested with 1.4 1 of deion-ized water and filtrated off. The wet product is taken up in 3 1 of MTBE (methyl t-butyl ether). The solution is ex-with a saturated NaCl solution (2 x 100 ml), and tracted the organic phase is dried with sodium sulfate. The solu-tion is concentrated to a volume of 500 ml. Following addi-tion of 1.5 1 of isopropyl ether, stirring for two hours is effected. The product is filtrated and dried. The yield is 104.6 g of fragment 3c indicated in Fig. 2.

Example 4 Preparation of Fragment ANP(121-125) In an analogous manner as described in Example 3, starting from 264 g of Fmoc-Arg(Pbf)-OHMPB-support resin and following the procedure described, 115.1 g of cardio-dilatin fragment ANP(121-125) is obtained.

Example ,5 Preparation of Fragment ANP(95-108) Following the general procedures of Example 1, and starting from 210 g of Fmoc-Gly-OHMPB-support resin, 151.5 g of the fully protected cardiodilatin fragment ANP(95-108) is obtained.

Example 6 Preparation of Fragment ANP(99-108) Following the general procedures of Example 1, and starting from 190 g of Fmoc-Gly-OHMPB-support resin, 145.1 cj of the fully protected cardiodilatin fragment ANP(99-108) is obtained. - -Example 7 Preparation of Fragment ANP(102-108) Following the general procedures of Example1, and starting from 220 g of Fmoc-Gly-OHMPB-support resin, 165.3 g of the fully protected cardiodilatin fragment ANP(102-108) is obtained.

Example 8 Condensation of the Partial Fragments to the Intermediate Product The fragment ANP(109-120) is converted to the in-termediate ANP(109-121)-R2 by condensation with the C-terminal fragment ANP(121)-R2 according to the following general.process:

The fragment ANP(109-120), the amino terminus of which is protected by the Fmoc group, is dissolved in N-methylpyrrolidone. Subsequently, TBTU (2-(1H-benzotri-azol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), 1-hydroxybenzotriazole and diisopropylethylamine are added to the solution at room temperature with stirring. There-after, the fragment ANP(121)-R2 provided with an appropri-ate protecting group at the C-terminal end and dissolved in N-methylpyrrolidone is added to the solution. In the fol-lowing, the reaction is monitored by thin layer chromato-graphy. After about 2 hours, the reaction is complete.
Then, the reaction mixture is dripped onto diisopropyl ether with stirring and subsequently stirred for about 30 minutes.. The precipitate is filtrated _on a porcelain suc-tion funriel over hard filter and washed twice with diiso-propyl ether. Thereafter, the residue is suspended in ace-tonitrile and digested at room temperature with stirring.
Subsequently, the product is filtrated on a porcelain suc- -tion funnel, rewashed with acetonitrile and dried to con-stant weight in a vacuum chamber at 40 C. The thus obtained crude product represents the cardiodilatin fragment Finoc-ANP(109-121)-R2 protected at the amino terminus by the Fmoc protecting group. Thereafter, the Fmoc group is removed ac-cording to known procedures to obtain the intermediate.
product H-ANP(109-121)-R2.

Example.9 Condensation of Fragments ANP(109-120) with ANP(121-126) to ANP(109-126) Following the general procedure described in Exam- - =
ple 8, 21.6 g of Fmoc-ANP(109-120) is dissolved in 650 ml of N-methylpyrrolidone. Subsequently, 3.2 g of TBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium tetrafluoro-borate), 1.5 g of 1-hydroxybenzotriazole and 3.5 ml of diisopropylethylamine are added to. the solution at room temperature with stirring. Thereafter, a solution of H-ANP(121-126)-OtBu, dissolved in 150 ml of N-methylpyr-rolidone, is added. In the following, the reaction is moni-tored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mixture is _ dripped onto 4 1 of diisopropyl ether with stirring and subsequently stirred for about 30 minutes. The precipitate is filtrated on a porcelain suction funnel over hard filter and washed twice with 500 ml of diisopropyl ether. There-after, the residue is_suspended in 600 m1 of acetonitrile and digested at roomtemperature with stirring. Subse-quently, the product is filtrated on a porcelain suction funnel, rewashed with 300 ml of ac tonitrile and dried to constant weight in a vacuum chamber at 40 C. Subsequently, the crude product Fmoc-ANP(109-126) thus obtained in an amount of 32.3 g is converted to the unprotected ANP(109- =
126) by addition of diethylamine. The yield is 30.2 g.

Example10 Condensation of Fragments ANP(109-120) with ANP(121-125) to ANP(109-125) Following the general procedure described in Exam-ple 8, 18.6 g of Fmoc-ANP(109-L20) is dissolved in 600 ml of N-methylpyrrolidone. Subsequently, 3.0 g of TBTU (2-(1H-benzotriazol-l-y1)-1,1,3,3-tetramethyluronium tetrafluoro-borate), 1.2 g of 1-hydroxybenzotriazole and -3.0 ml of diisopropylethylamine are added to the solution at room temperature with stirring. Thereafter, a solution of -H-ANP(121-125)-OtBu, dissolved in 150 ml of N-methylpyr-rolidone, is added. In the following, the reaction is moni-tored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mixture is -,--_-_ dripped onto 4 1 of diisopropyl ether with stirring and subsequently stirred for about 30 minutes. The precipitate is filtrated on a porcelain suction funnel over hard filter and washed twice with 450 ml of diisopropyl ether. There-after, the residue is suspended in 500 m1 of acetonitrrile and digested at room temperature with stirring. Subse-quently, the product is filtrated off on a porcelain suc-tion funnel, rewashed with 250 ml of acetonitrile and dried to constant weight in a vacuum chamber at 40 C. Subse-quently, the crude product Fmoc-ANP(109-125) thus obtained in an amount of 29.1 q is converted to the unprotected ANP(109-125) by addition of diethylamine. The yield is 28.2 g.

Example 11 Condensation of the Partial Fragments to the Final Product The intermediate ANP(109-121)-R2 is converted to the final product R1-ANP(105-121)-R2 by condensation with the amino-terminal fragment R1-ANP(105-108) according to the following general process:

The fragment R1-ANP(105-108), the amino terminus of which is protected by an appropriate protecting group, is dissolved in N-methylpyrrolidone. Subsequently, TBTU
(2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium tetra-fluoroborate), 1-hydroxybenzotriazole and diisopropylethyl-amine are added to the solution at room temperature with stirring. Thereafter, the fragment ANP(109-121)-R2 provided with an appropriate protecting group at the C-terminal end and dissolved in N-methylpyrrolidone is added to the solu-tion. In the following, the reaction is monitored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mixture is dripped onto diiso-propyl ether with stirring and subsequently stirred for about 30 minutes. The precipitate is filtrated on a porce-lain suction funnel over hard filter and washed twice with diisopropyl ether. Thereafter, the residue is suspended in acetonitrile and digested at room temperature with stir-ring. Subsequently, the product is filtrated off on a por-celain suction funnel, rewashed with acetonitrile and dried to constant weight in a vacuum chamber at 40 C. The thus ob-tained crude product represents the cardiodilatin fragment Rl-ANP(105-121)-R2 protected by appropriate protecting groups at the amino terminus and the C-terminus. There-after, the protecting group is removed according to known procedures to obtain the intermediate product H-R1-ANP(109-121)-R2. Following complete removal of the protecting groups, the obtained cardiodilatin fragment is converted to the cyclized derivative by oxidation and according to known procedures, for example, using iodine.

Example 12 Condensation of Fragments ANP(109-126) and ANP(95-108) to ANP(95-126) a) Preparation of ANP(95-126) Following the general procedure described in Exam-ple 11, 20.6 g of Boc-ANP(95-108) is dissolved in 404ml of N-methylpyrrolidone. Subsequently, 2.7 g of TBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium tetrafluoro-borate), 1.3 g of 1-hydroxybenzotriazole and 2.7 ml of diisopropylethylamine are added to the solution at room temperature with stirring. Thereafter, a solution of 29.4 g of H-ANP(109-126)-OtBu, dissolved in 400 ml of N-methylpyrrolidone, is added. In the following, the reac- _ tion is monitored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mix-ture is dripped onto 6.5 .1 of diisopropyl ether with stir-ring and subsequently stirred for about 30 minutes. The =
precipitate is filtrated on a porcelain suction funnel over hard filter and washed twice with 500 ml of diisopropyl ether. :Thereafter, the residue is suspended in 600 ml of acetonitrile and digested at room temperature with stir-ring.
Subsequently, the product is filtrated off on a por-celain suction funnel, rewashed with 500 ml of acetonitrile and dried to constant weight in a vacuum chamber at 40 C.
Subsequently, the crude product Boc-ANP(95-126)-OtBu thus obtained in an amount of 42.5 g is converted to the unpro-tected ANP(95-126) and dried. The yield is 27.5 g.

b) Cyclization of the deprotected linear ANP(95-126) 60 g of unprotected ANP(95-126) is dissolved in 16 1 of 5% acetiC acid in deionized water--(v/v) and oxi-dized by addition of 570 ml of a 0.02 M methanolic iodine solution. The reaction is complete after 5 minutes. Excess iodine is destroyed by addition of a 0.1 M sodium thiosul-fate solution. The cyclization solution obtained is__sub-jected directly to further processing.

Example 13 _ Condensation of Fragments ANP(109-126) and ANP(99-108) to ANP(99-126) Analogous to the procedure described in Example 12, 22.5 g of Boc-ANP(99-108) is dissolved in 400 m1 of N-methylpyrrolidone. Subsequently, 2.9 g of TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoro-.
borate), 1.4 g of 1-hydroxybenzotriazole and 2.8 ml of diisopropylethylamine are added to the solution at room temperature with stirring. Thereafter, a solution of 30.6 q of H-ANP(109-126)-OtBu, dissolved in 400 ml of N-methylpyrrolidone, is added. In the following, the reac-.
tion is monitored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mix-ture is dripped onto 6.5 1 of diisopropyl ether with stir-ring and subsequently stirred for about 30 minutes.. The precipitate is filtrated on a porcelain suction funnel over hard filter and washed twice with 500 ml of diisopropyl ether.-Thereafter, the residue is suspended in 600 ml of acetonitrile and digested at room temperature with stir-ring. Subsequently, the product is filtrated off on a por- ..
celain suction funnel, rewashed with 500 ml of acetonitrile and dried to constant weight in a vacuum chamber at 40 C.
Subsequently, the crude product Boc-ANP(99-126)-OtBu thus ~ 2191612 obtained in an amount of 44.7 g is converted to the unpro-=
tected.ANP(99-126) and dried. The yield is 28.1 g.

Example 14 Condensation of Fragments ANP(109-126) and ANP(102-108) to ANP(102-126) Analogous to the procedure described in Example 12, 20.4 g of Boc-ANP(102-108) is dissolved in 360 ml of N-methylpyrrolidone. Subsequently, 2.7 g of TBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium tetrafTuoro-borate), 1.4 g of 1-hydroxybenzotriazole and 2.6 ml of "
diisopropylethylamine are added to the solution at room temperature with stirring. Thereafter, a solution of 30.1 g of H-ANP(109-126)-OtBu, dissolved in 400 m1 of N-methylpyrrolidone, is added. In the following, the reac-tion is monitored by thin layer chromatography. After about 2 hours, the reaction is complete. Then, the reaction mix-ture is dripped onto 6.5 1 of diisopropyl ether with stir-ring and subsequently stirred for about 30 minutes. The precipitate is filtratedon a porcelain suction funnel over hard filter and washed twice with 500 ml of diisopropyl ether. Thereafter, the residue is suspended in 600 ml of acetonitrile and digested at room temperature with stir-ring. Subsequently, the product is filtrated off on a por-celain suction funnel, rewashed with 500 ml of acetonitrile 1and dried to constant weight in a vacuum chamber at 40 C.
Subsequently, the crude product Boc-ANP(102-126)-OtBu thus obtained in an amount of 41.2 g is converted to the unpro-tected ANP(102-126) and dried. The yield is 26.9 g.

Example 15 Purification of ANP(95-126) and Preparation of the Hiqh-Pu-rity Form a) Concentrating the cyclized urodilatin [ANP(95-126)]

The cyclization solution (about 17 liters of 5%
AcOH, in deionized water (v/v), contains about 60 g of cyclized urodilatin) is applied (flow rate 130 ml/min) on a glass column (diameter: 70 mm, length: 900 mm, filled with Vydac 218 TPB 2030) equilibrated with 1000 ml of buffer A3 (0.1% TFA (v/v) in deionized water).

Once application by pumping is finished, the peptide is eluted by continuous charging of buffer B3 (0.1% TFA in deionized water/ACN 2:3 v/v) in a continu-ous gradient (0% buffer B during 40 min; 15-35%
buffer B during 90 min; 35% buffer B during 10 min;
flow rate 130 ml/min).

urodilatin fractions showing a purity of more than 75% on monitoring bv analytical HPLC are combined.
These combined fractions are diluted with one volume equivalent of deionized water and applied (flow rate 140-m1/min) on a Biotage module (diameter: 75 mm, length: 300 mm, filled with YMC C181120 A, 10 pm) equilibrated with 300 ml of buffer A3.

Subsequently, the concentrated peptide is eluted by washing the column with 100% buffer B3, and the acetonitrile is evaporated. The remaining solution is lyophilized.

Between 17 and 20 g of urodilatin with a purity of more than 90% is obtained.

b) Purification of the concentrated urodilatin 4.5 g of the concentrated urodilatin is dis-solved in 250 ml of 10% AcOH in deionized water (v/v) and applied (flow rate 140 ml/min) on a Biotage module (diameter: 75 mm, length: 300 mm, filled with YMC C18, 120 A, 10 pm) equilibrated with 300 ml of buffer A4 (50 mM TEAP, pH 2.25, in deionized water).

The peptide is eluted by continuous charging of buffer B4 (50 mM TEAP, pH 2.25 in deionized water/ACN
2:3 v/v) in a continuous gradient (22-28% B during 90 min; 28% B during 10 min; 28-40% B during 20 min; flow rate 140 ml/min).

urodilatin fractions showing a purity of more than 99% and impurities of not more than 0.5% on moni-toring by analytical HPLC are combined. These combined fractions are diluted with one volume equivalent of deionized water and pumped onto the Biotage module pre-viously cleaned with 1000 ml of buffer B3 and subse-quently equilibrated with 300 ml of buffer A3. For de-salting, a washing with 1200 ml of buffer A3 is made.

The pure product is eluted by washing the col-umn with 1500 ml of buffer B3, and the acetonitrile is evaporated. The remaining solution is lyophilized.

The result is between 2.3 and 2.7 g of high-pu-rity urodilatin.

c) Resalting of urodilatin x TFA to urodilatin acetate ----2.5 g of high-purity urodilatin x TFA salt is 1 dissolved in 80 ml of 5% AcOH, in deionized water v/v, and applied to a chromatography column (diameter:

~ 2191612 20 mm, length: 300 mm, filled with 40 ml of- Merck ion exchanger III acetate form) washed with 5% AcOH. A
washing with 40 ml of 5% AcOH is made. The eluate, about 125 ml, is applied once more to the same ion ex-change column. A washing with 55 m1 of 5% AcOH is made.
The eluate, about 180 ml, is filtrated clear over a polysulfone membrane (diameter 47 mm, 0.2 pn). The so-lution is lyophilized.

The result is between 2.05 and 2.30 g of high-purity urodilatin acetate.

Example 16 -Purification of ANP(99-126) and Preparation of the High-Pu-rity Form a) Concentrating the cyclized cardiodilatin fragment ANP(99-126) Analogous to Example 15a), the cyclization so-lution (about 15 liters of 5% AcOH, in deionized water (v/v), with a peptide content of about 50 g) is applied (flow rate 130 ml/min) on a glass column equilibrated with 1000 ml of buffer A3 (0.1% TFA (v/v) in deionized water). Once application by pumping is finished, the peptide is eluted by continuous charging of buffer B3 (0.1% TFA in deionized water/ACN 2:3 v/v) in a continu-ous gradient (0% buffer B during 40 min; 15-35%
buffer B during 90 min; 35% buffer B during 10 min;
flow rate 130 m1/min). Peptide fractions showing a pu-rity of more than 75% on monitoring by analytical HPLC
are combined. These combined fractions are diluted with one volume equivalent of deionized water and applied (flow rate 140 ml/min) on a Biotage module equilibrated with 300 ml of buffer A3. Subsequently, the concen-trated peptide is eluted by washing the column with ~ 2191612 100$ buffer B3, and the acetonitrile is evaporated. The remaining solution is lyophilized. -The result is between 14 and 17 g of cardiodi-latin fragment ANP(99-126) with a purity of more than 90%.

b) Purification of the concentrated ANP(99-126) 3.5 g of the cardiodilatin fragment concen-trated according to Example 16a) is dissolved in 200-m1 of 10% AcOH in deionized water (v/v) and applied (flow rate 140 ml/min) on a Biotage module equilibrated with 300 ml of buffer A4 (50 mM TEAP, pH 2.25, in deionized water). The peptide-is eluted by continuous charging-of buffer B4 (50 mM TEAP, pH 2.25 in deionized water/ACN
2:3 v/v) in a continuous gradient (22-28% B during 90 min; 28% B during 10 min; 28-40% B during 20 min; flow rate 140 ml/min).

Peptide fractions showing a purity of more than 99% and impurities of not more than 0.5% on monitoring by analytical HPLC are combined. These combined frac- -tions are diluted with one volume equivalent of deion-ized water and pumped onto the Biotage module previ-ously .cleaned with 1000 mI of buffer B3 and subse-quently equilibrated with 300 ml of buffer A3. For de- _ salting, a washing with 1000 ml of buffer A3 is made.

The pure product is eluted by washing the col-umn with 1500 ml of bufferB3, and the acetonitrile is evaporated. The remaining solution is lyophilized.

The result is between 1.7 and 2.2 g of high-pu-rity cardiodilatin fragment ANP(99-126) . Analogous to -the procedure described in Example 14c), this fragment is converted to the corresponding acetate salt. The re-i( 2191612 sult is between 1.3 and 1.7 g of high-purity ANP(99-126) acetate.

Example 17 Purification of ANP(102-126) and Preparation of the High-Purity Form =
a) Concentrating the cyclized cardiodilatin fragment ANP(102-126) Analogous to Example 15a), the cyclization so-lution (about 18 liters of 5% AcOH, in deionized water (v/v), with a peptide content of about 65 g) is applied (flow rate 130 ml/min) on a glass column equilibrated with 1000 ml of buffer A3 (0.1% TFA (v/v) in deionized water). Once application by pumping is finished, the peptide is eluted by continuous charging of buffer B3 (0.1% TFA in deionized water/ACN 2:3 v/v) in a continu-ous gradient (0% buffer B during 40 min; 15-35%
buffer B during 90 min; 35% buffer B during 10 min;
flow rate 130 m1/min). Peptide fractions showing a pu-rity of more than 75% on monitoring by analytical HPLC
are:combined. These combined fractions are diluted with one volume equivalent of deionized water and applied (flow rate 140 ml/min) on a Biotage module equilibrated with 300 ml of buffer A3. Subsequently, the concen-trated peptide is eluted by washing the column with 100% buffer B3, and the acetonitrile is evaporated. The remaining solution is lyophilized.

The result is between 19 and 23 g of cardiodi-latin fragment ANP(102-126) with a purity of more than 90?.

b) Purification of the concentrated ANP(102-126) 4.8 g of the cardiodilatin fragment concen-trated according to Example 17a) is dissolved in 200 ml of 10% AcOH in deionized water (v/v) and applied (flow rate 140 ml/min) on a Biotage module equilibrated with 300 ml of buffer A4 (50 mM TEAP, pH 2.25, in deionized water). The peptide is eluted by continuous charging of buffer B4 (50 mM TEAP, pH 2.25 in deionized water/ACN
2:3 v/v) in a continuous gradient (22-28% B during 90 min; 28~ 3 during 10 min; 28-40% B during 20 min; flow rate_140 ml/min).

Peptide fractions showing a purity of more than 99% and impurities of not more than 0.5% on monitoring by analytical HPLC are combined. These combined frac-tions are diluted with one volume equivalent of deion-ized water and pumped onto the Biotage module previ-ously cleaned with 1000 ml of buffer B3 and subse-quently equilibrated with 300 ml of buffer A3. For de-salting, a washing with 1000 ml of buffer A3 is made.

The pure product is eluted by washing the col-umn with 1500 ml of buffer B3, and the acetonitrile is evaporated. The remaining solution is lyophilized.

The result is between 1.9 and 2.4 g of high-pu-rity cardiodilatin fragment ANP(102-126). Analogous to the procedure described in Example 14c), this fragment is converted to the corresponding acetate salt. The re-suit is between 1.5 and 1.9 g of high-purity ANP(99-126) acetate.

Example 18 Analytical HPLC Examinations UsinQ the ANP(95 ].26) Example a) Elution with TEAP buffer, pH 2.25 50 g of ANP(95-126) is injected onto an_ana-lytical HPLC column. A linear gradient of buffer B of 25-45% during 20 minutes (buffer A: 50 mM TEAP, pH 2.25; buffer B: mixture of A and acetonitrile at a volume ratio of 2:3) served as the eluant. The chroma-togram in Fig. 5 reveals that two polar impurities are contained which may be separated by the eluant em-ployed.

Legend to kig. 5:
25 - 45 %~ in 20 min.
Buffer A: 50mM TEAP pH 2,25 Buffer B: A:ACN (2:3) 215 nm 1,0 ml/'C-Nr. 4040465 C
M+N 250/1/4 /3 Nuc 300 AS u C18 Method:.50 g; TAG 243 CFi:l; Peak reject: 5000 File: 1; Calculation method: areas~; Table: 0; conc: area ~ 2191612 N2 RT Area 5 7,82 53358 0,311 BV

6 8,08 84196 0,491 W
7 9,07 386602 2,255 W
8 9,78 1265799 7,384 vV
9 10,56 4701290 27,430 VV
10,92 10557085 61,582 W
11 11,91 27613 0,161 TBB
12 12,82 8763 0,051 TBB
13 13,76 14346 0,084 BB
14 14,86 31959 0,186 BB
19,04 10892 0,064 BB
Total 17143003 100,00 b) Elution with 0.1% TFA (trifluoroacetic acid) Analogous to Example 18a), 50 g of ANP(95-126) of the same production batch is applied onto an ana-lytical HPLC column. A linear gradient of buffer B of 30-50g during 20 minutes (buffer A: 0.1g TFA in water;
buffer B: mixture of A and acetonitrile at a volume ra-tio of 2:3) served as the eluant. The chromatogram in Fig. 6 reveals that separation of the contained impuri-ties by means of this eluant is not effected- Compared to the chromatogram in Example a), the main peak is broader and the isolated product contains both of the polar impurities which can be recognized in the chroma- -togram ofFig. 5_ - - --Legend to FicL 6 : 30 - 50 g B in 20 min.

Buffer A: 0,1 % TFA in water =
Buffer B: A:ACN (2:3) 215 nm 1,0 ml/"C-Nr. 4011079 C
M+N 250/1/4 /3 Nuc 300 LA 5u C18 Method: 50 g; TAG 142; CH:1; Peak reject: 5000 File: 2; Calculation method: area*; Table: 0; conc: area No. B~ Area 2 3,64 5073 0,040 BV
4 5,10 6624 0,053 BB
5,92 8161 0,065 BB
6 7,36 6814 0,054 BB
7 9,11 252878 2,012 BB
9 11,73 87629 0,697 BB
12,60 258273 2,055 BB
11 13,09 4578590 36,428 V57 12 13,26 7175177 57,086 W
13 14,67 179155 1,425 TBB
14 17,48 10611 0,084 BB
Total 12568985 100,00 Example 19 Purity Check by Cauillarv ElectroDhoresis Lyophilized samples of the final products of car-diodilatin fragments from Examples 15 through 17 are dis-solved in water at a concentration of 1 mg/ml and analyzed immediately. Capillary electrophoresis was performed using the Beckmann P/ACE 2100 system under the following condi-tions:

Capillary: Fused Silica by Supelco~,N separation length 50 cm, internal diameter 75 m Detection wave length: 200 nm Injection period: 1 s Separation buffer: 100 mM sodium phosphate, pH 2.5; 0.020 hydroxypropylmethylcellulose Separation parameters: 25 C, 80 A, 30 m121 Figure 3 shows tlie chromatogram obtaineci for prior art urodilatin.
Figure 4 shows the chroinatogram for high-purity urodilatin obtained according to Example 15.

A comparison of both chromatograms reveals that the urodilatin according to the invention differs significantly from prior art urodilatin. The urodilatin according to the invention is free of peptide impurities.

= 2191612 INDEX OF ABBREVIATIONS

Amino acids Ala L-Alanine Asn L-Asparagine Asp L-Asparaginic acid Arg L-Arginine Cys L-Cysteine Gln L-Glutamine Gly Glycine Ile L-Isoleucine Leu L-Leucine Met L-Methionine Phe L-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine Tyr L-Tyrosine Protecting grouQs Boc t-Butyloxycarbonyl Fmoc :9-Fluorenylmethoxycarbonyl OtBu t-Butyl ester Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl tBu t-Butyl ether-Acm Acetamidomethyl Trt Trityl Reagents/Solvents ACN Acetonitrile TFA Trifluoroacetic:acid TEAP Triethylammonium phosphate SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Corange International Ltd.
(B) STREET: 36Dover Street (C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): WIX 3RB

(A) NAME: Forssmann, Wolf-Georg, Prof. Dr. med. Dr. h.c.

(B) STREET: Bluecherstr,- 5 (C) CITYc Hannover - - - - -(E) COUNTRY: Germany (F) POSTAL CODE (ZIP): 30175 (A) NAME: Adermann, Knut, Dr.
(B) STREET: Schleidenstr. 5 (C) CITY: Hannover (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): 30177 (A) NAME: Inuner, Hans-Ueli, Dr_ (B) STREET: Hasenweg 6 (C) CITY: Balsthal (E) COUNTRY: Switzerland (F) POSTAL CODE (ZIP): 4710 (A) NAME: Klessen, Christian, Dr.
(B) STREET: Hauptstr. 26 (C) CITY: Lauterecken (E) COUNTRY: Germany (F) POSTAL CODE (ZIP): 67742 ~ 2191612 (ii) TITLE OF INVENTION: A Process for Preparing Cardiodilatin Fragments; Highly Purified Cardiodilatin Fragments and Intermediate Products for the Preparation of Same (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: DE P 44 20 381_0 (B) FILING DATE: JUNE 02, 1994 - -(vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: DE 195 13 784_1 (B) FILING DATE: APRIL 10, 1994 ---- - (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No: 1: Cys Phe Gly Gly Arg Met Asp Arg Ile Gly Ala Gln Ser Gly Leu Gly Cys ~ 2191612 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Leu Arg Ala Leu Leu Thr Ala Pro Arg SerLeu Arg Arg Ser Ser -_-(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID No: 3: Asn Ser Phe Arg Tyr

Claims (6)

1. A process for preparing cardiodilatin fragments of formula I

R1-ANP (105-121) -R2 (I) having a total chain length of from 17 to 37 amino acids, wherein ANP(105-121) represents amino acid sequence [SEQ ID NO:1];

R1 represents an amino acid chain of sequence ANP (90-104)[SEQ ID NO: 2] or a fragment thereof having a chain length of from 0 to 15 amino acids; and R2 represents an amino acid chain of sequence ANP(122-126) [SEQ ID NO:3] or a fragment thereof having a chain length of from 0 to 5 amino acids;

characterized in that synthesis is effected through a condensation of at least three partial fragments, wherein the condensation of partial fragments to the cardiodilatin fragments of formula I is performed between amino acid position Gly108 and Arg109 and between amino acid positions Gly120 and Cys121 and is effected through partial fragment ANP(109-120).

2. The process according to claim 1, wherein (a) in a first step, the condensation of the partial fragments between amino acid positions Gly120 and Cys121 is performed from the partial fragments ANP (109-120) and Cys121-R2; and (b) in a second step, the condensation of the partial fragments between amino acid positions Gly108 and Arg109 is performed from the partial fragments ANP(109-121)-R2 obtained from step (a) and the partial fragment R1-ANP(105-108).

3. The process according to claim 1 or 2, characterized in that R1 represents an amino acid sequence selected from the group consisting of ANP(95-104), ANP(99-104) and ANP(102-104), and R2 represents an amino acid sequence selected from the group consisting of ANP(122-125) and ANP(122-126).

4. The process for preparing cardiodilatin fragments R1-ANP(105-121)-R2 having a total chain length of from 17 to 37 amino acids according to any one of claims 1 to 3, wherein R1 represents an amino acid chain of the sequence ANP(90-104) or fragments thereof having a chain length of from 0 to 15 amino acids, and R2 represents an amino acid chain of the sequence ANP(122-126) or fragments thereof having a chain length of from to 5 amino acids, characterized in that the purification of the raw product is effected by means of a reversed-phase HPLC column, and the cardiodilatin fragment is eluted with a buffer system containing triethylammonium phosphate and acetonitrile.

5. Protected fragments of ANP(95-126) selected from the group of fragments having the following structures:
Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser (tBu) -Ser (tBu) -Cys (Acm) -Phe-Gly-Gly-OH 1 Fmoc-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-OH 2 R1-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-O-R2 3a: R1 = Fmoc; R2 = OH
3b: R1 = Fmoc; R2 = tBu 3c: R1 = H; R2 = OtBu and R3-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr (tBu) -OtBu 4: R3 = Fmoc 5: R3 = H.

6. Use of peptide fragments selected from the group of amino acid sequences:

(a) R1-ANP (105-108) , wherein R1 represents an amino acid chain of the sequence ANP(90-104) or a fragment thereof having a chain length of from 0 to 15 amino acids;

(b) ANP(109-120);

(c) ANP (109-121) -R2, wherein R2 represents an amino acid chain of the sequence ANP(122-126) or a fragment thereof having a chain length of from 0 to 5 amino acids; and (d) Cys121-R2, wherein R2 represents an amino acid chain of the sequence ANP(122-126) or a fragment thereof having a chain length of from 3 to 5 amino acids;

and the derivatives modified by protective groups in a process for preparing cardiodilatin fragments according to any one of claims 1 to 3.