RU2784866C1 - Optimized method for production of dimeric peptide-phospholipid conjugate - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a new effective method for the production of a KDR-targeted peptide-phospholipid conjugate, which can be used in therapeutic and diagnostic compositions and, in particular, in the production of echo-contrast preparations.

EFFECT: obtainment of a peptide-phospholipid conjugate.

10 cl, 2 dwg, 1 tbl, 5 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to the field of KDR-targeted peptide-phospholipid conjugates that are used in therapeutic and diagnostic compositions, and, in particular, to methods for their preparation.

BACKGROUND OF THE INVENTION

Angiogenesis is the formation of new blood vessels from an already existing vasculature and plays an important role not only in normal physiological processes, but also in the pathogenesis of diseases such as malignancy, rheumatoid arthritis and diabetic microangiopathy. For example, angiogenesis is involved in the transition of a tumor from hyperplastic to neoplastic growth. Thus, the suppression of associated pathological processes is very important in therapeutic and diagnostic studies of malignant tumors.

In the case where angiogenic growth factors are produced in excess of angiogenesis inhibitors, endothelial cells are stimulated to proliferate. Among the known and best characterized proangiogenic agents or growth factors, the vascular endothelial growth factor (VEGF) family, and in particular the KDR (kinase insert domain receptor, also known as VEGFR-2 or Flk-1), are proangiogenic agents or growth factors that are of greater interest because they show more robust endothelial cell expression and a dominant angiogenic response ¹ . KDR expression is significantly upregulated in angiogenic vessels, in particular tumors, causing a strong angiogenic response ² .

The in vivo VEGF binding activity of KDR is of great importance for angiogenesis, so the ability to detect its activation on endothelial cells or detect VEGF/KDR binding complexes would be extremely useful in detecting or monitoring angiogenesis.

It is known that for diagnostic and therapeutic purposes, for example for vascular and visceral imaging, it would be particularly advantageous to include in gas-filled echo contrast preparations any targeting vector composition that exhibits a high binding affinity to the desired target, such as KDR.

For example, KDR targeted peptide-phospholipid conjugates can be used to produce gas-targeted echocontrast preparations.

It is well known that gas-filled echo contrast agents are highly effective ultrasonic reflectors for echography. For example, the introduction into the bloodstream of living organisms of suspensions of gas-filled microbubbles in a carrier fluid will significantly enhance the visualization of echography, thereby facilitating the visualization of internal anatomical structures, such as blood vessels.

One targeting vector composition that exhibits high binding affinity to the target KDR, or VEGF/KDR complex, is, for example, the following compound (I), a targeting peptide-phospholipid (lipopeptide) conjugate, which was first described in patent application WO2007 /067979 A2 and showed a high ability to bind to KDR-expressing tissues.

The specified compound (I), described herein below and in more detail in figure 1, structurally consists of a heterodimeric peptide formed by two different monomeric peptide chains (both consisting of 23 amino acids), linked by a glutaryl linker and conjugated to a polyethylene glycol fragment (PEG), such as DSPE-PEG2000-NH2 via a second glutaryl linker.

A method for preparing a KDR-binding peptide-phospholipid conjugate (I) has been described in WO2007/067979 A2 (see Example 5 here, pages 52-54, and Figure 4 for details).

The conjugate is prepared by starting with automated solid phase synthesis of peptide monomers, followed by their combination and activation using succinimidyl glutarate (DSG) and subsequent conjugation of the resulting derivative with DSPE-PEG ₂₀₀₀ -NH ₂ via a glutaryl bond.

The last conjugation step is also illustrated in the following Scheme 1.

Scheme 1

It follows from the above description that the conjugation step may have some drawbacks in terms of yield and purity of the final product (see paragraph [0063]: "any free phospholipid may complicate the purification and isolation of the final product"). In fact, to prevent the formation of impurities and the need for laborious purifications, the phospholipid reagent DSPE-PEG ₂₀₀₀ -NH ₂ (III), which is difficult to separate from the final product (I) by known purification methods, is added by default to the succinimidyl dipeptide (II) (DSPE ratio -PEG ₂₀₀₀ -NH ₂ and succinimidyl dipeptide: 0.9 to 1 equivalent).

However, this approach, while limiting impurities in the final product, causes the loss of valuable heterodimer (II) and provides a yield at the conjugation stage of no more than 60% or even no more than 30%. In addition, the final compound after purification by preparative HPLC as described in example 5 in WO2007/067979 still contains almost 2% impurities with a purity profile that does not meet the requirements set by government authorities for a pharmaceutical product.

Another disadvantage of the known process is associated with the high content of trifluoroacetic acid (TFA), which is added during the synthesis for solubilization and in the mobile phases for purification by preparative HPLC. Furthermore, in addition to the fact that TFA is considered a pharmaceutically unacceptable salt, when the product is stored as a salt of TFA in the form of a lyophilisate at 5°C or in solution, degradation is observed, probably determined by TFA-activated acid hydrolysis of one of the esters. phospholipids and fatty acids in a dimeric conjugate to form a lyso compound as an unwanted impurity, as described in paragraphs [0065] - [0066] in WO2007/067979. Thus, additional cumbersome and time-consuming procedures must be carried out to "convert the TFA salts of the dimeric peptide-phospholipid conjugate" to another more stable salt.

Briefly, the main problems of the known method are represented by the unfavorable loss of the costly heterodimer (II) during the conjugation step with the pegylated phospholipid (III); a complex purification step that reduces the purity and yield of the final product; and instability of the final product obtained and stored as a TFA salt.

Therefore, the described approach, although highly effective, can be to a large extent onerous and still does not represent a valid and industrially applicable method. Until now, indicators of purity and production efficiency have not been observed.

At the same time, for the use of such a targeted peptide-phospholipid conjugate in vivo for vascular and visceral imaging, it would be particularly useful to have an efficient method for large-scale production of highly purified forms of the product.

SUMMARY OF THE INVENTION

The present invention provides a novel process for the preparation of a peptide-phospholipid compound (I) as defined above, characterized by optimized conditions for the conjugation and purification steps. This process is especially effective for the production of gas-filled echo contrast preparations.

In this context, an efficient analytical procedure has been found to significantly improve the separation of the final compound from by-products, thereby allowing an increase in the amount of starting material (III) during conjugation and obtaining compound (I) in higher yields, with a better purity profile and a suitable stability profile. The used to scale the entire process.

Accordingly, a first aspect of the present invention is a process for the preparation of compound (I) or pharmaceutically acceptable salts thereof,

including the stage

(i) combinations of the appropriate succinimidyl ester intermediate (II)

(II)

(II)

with phospholipid DSPE-PEG ₂₀₀₀ -NH ₂ (III)

in the presence of DIEA,

moreover, the specified phospholipid (III) is present in excess relative to the compound (II).

In a preferred embodiment, the combination is carried out with 1.1 or more equivalents of phospholipid (III) per equivalent of compound (II).

In a more preferred embodiment, the combination is carried out with two equivalents of phospholipid (III) per one equivalent of compound (II).

In another aspect, the present invention provides said method further comprising the steps of:

(ii) isolating the crude product (I) recovered from the reaction mixture in step (i);

(iii) optionally diluting the crude product obtained in step (ii) with water and adding a base to achieve a pH value in the range of 6 to 8; as well as

(iv) purification of the crude product from the solution of step (iii).

The addition of a base in step (iii) can help to completely solubilize the crude product in water by maintaining the pH of the solution in the range of 6 to 8. Preferably, an appropriate amount of 0.1 N NaOH is added to achieve a pH in the range of 6.5 to 7.5, more preferably to achieve a pH value of 7.3.

According to the invention, the purification in step (iv) can be carried out by reverse phase high performance liquid chromatography (RP-HPLC) or by ion exchange chromatography, or by both RP-HPLC and ion exchange chromatography.

In a preferred embodiment, the purification is carried out by RP-HPLC only. According to the invention, chromatographic separation by RP-HPLC is preferably achieved using eluents having a pH of 6 to 8 and containing easily removable ammonium carbonate. The optimal mobile phase can be represented, for example, by a combination of eluent A, consisting of 10 mm AcONH ₄ in water, and eluent B, consisting of 10 mm AcONH ₄ in a mixture of water/acetonitrile in a ratio of 1/9.

In another preferred embodiment, the purification is carried out by ion exchange chromatography. Preferably, such chromatography is carried out with an anion exchange resin (e.g., ANX Sepharose resin) and a buffer solution selected from those commonly used for ion chromatography, at a pH preferably in the range of 7 to 8, optionally with the addition of water-miscible solvents to improve solubilization. phospholipid fragment.

According to a preferred embodiment of the invention, purification is successfully carried out using Tris HCl/NaCl buffer as eluent. A suitable chromatographic separation is achieved, for example, using 0.05 M Tris HCl + 0.10 M NaCl (pH 7.5) + 35% iPrOH as fixation buffer and 0.05 M Tris HCl + 1.00 M NaCl ( pH 7.5) + 35% iPrOH as elution buffer.

In another aspect of the invention, the above process is provided wherein compound (II) is prepared by activating the terminal alkylamine moiety of the corresponding intermediate of formula (IV) with di(N-succinimidyl)glutarate (V) as shown in the following Scheme 2 (step i') ).

Scheme 2

Thus, the method of the invention is characterized by the use of an excess of the phospholipid reagent DSPE-PEG ₂₀₀ 0-NH ₂ (III) which reacts with all the activated heterodimer (II) present, for example to eliminate any loss of the last expensive intermediate. The yields of the coupling steps (i') and in particular (i) are now predominantly higher compared to the yields obtained by the previously known procedure.

In fact, the previous method only provides below 60% yield of the dimeric peptide-phospholipid conjugate that needs to be converted from the TFA salt to a more stable salt, further reducing the efficient recovery of the final product.

At the same time, the present method provides compound (I) with improved yields of at least 69%, and, most importantly, it provides more efficient methods for analytical separation and preparative purification of the final product from unwanted impurities and removal of excess reagents; thus, optimized purification conditions allow to obtain a final product with a purity of more than 99% after RP-HPLC.

Therefore, in accordance with several advantages provided by the present method, the new procedure for the synthesis of compound (I) eliminates the disadvantages of the previously described procedure and can be particularly effective for scale-up and industrial production.

Description of the invention

Brief description of the drawings

Figure 1 shows the structure of the compound (I) obtained in accordance with the invention.

Fig. 2 shows the chromatogram of UPLC-ELSD (sample light scattering detection) of compound (I) after final purification by HPLC.

Definitions

In the present description, unless otherwise indicated, the following terms have the following meanings.

The term "heterodimer" refers to a molecule consisting of two polypeptide chains that differ in composition, that is, in the order, number or type of their amino acid residues. In particular, referred to herein with reference to the compound of formula (I) or its predecessors (II) and (IV), as defined above.

The term "pegylated" refers to a molecule that is covalently attached to the polymer chain of polyethylene glycol (PEG). The PEGylated compound can be prepared by incubating the reactive PEG derivative, preferably after functionalization at one or both ends with the reactive moiety, with the target molecule.

The term “anion exchange solid” means a solid support capable of exchanging anions with a solution or suspension in contact with it. The specified contact can be carried out by elution through a column filled with an appropriate solid phase.

Detailed description of embodiments

The process described herein relates to the preparation of a compound of formula (I) as defined above and has the advantage of saving expensive starting material while providing a final product in high yields and optimum purity.

These results can be achieved, among others, by discovering two effective purification methods that can be used alone or in combination, effectively removing unreacted phospholipid (III) added in excess in the coupling step (i), together with any other unwanted by-product.

The preparation of compound (I) according to the synthesis method described in WO2007/067979 provides for the activation of the heterodimer (IV) as defined above, obtained, for example, by known methods of solid phase synthesis, with di(N-succinimidyl)glutarate (V) and subsequent conjugation of the activated heterodimer (II) with DSPE-PEG ₂₀₀₀ -NH ₂ (III). The last step, as described above, is carried out when the phospholipid is deficient, so the coupling is carried out only with unsatisfactory yield results.

On the other hand, the optimized process presented by the present invention provides a significant improvement in the conjugation step, as detailed below.

The heterodimer (IV) can be activated, for example, according to a similar procedure described in example 5 of WO2007/067979 (paragraph [00124]), i.e. by reacting the heterodimer precursor with an excess of di(N-succinimidyl)glutarate and a base such as DIEA , for example, with a 5-fold excess of both reagents, to eliminate the coupling of the heterodimer. After completion of the reaction, the mixture can be diluted with a suitable solvent, such as anhydrous ethyl acetate, to precipitate the mono-NHS ester of heterodimeric glutaric acid (II) monoamide, which is then isolated and washed to remove remaining trace reagents. Alternatively, the mixture may be concentrated to remove the solvent and the dry crude product may be washed with, for example, EtOAc and centrifuged to recover the solid from the vessel.

Combination with phospholipid

In accordance with the invention, and as described in more detail in the experimental part, the compound of formula (II) is incubated with an excess of DSPE-PEG ₂₀₀₀ -NH ₂ (III) dissolved in DMF and in the presence of a base such as DIEA. The ratio between the equivalents of the precursor heterodimer (II) and the equivalents of the phospholipid (III) is at least 1:1.1, but more preferably is from 1:1.1 to 1:5. Preferably 1:2.

Completion of the coupling reaction can be monitored by analytical HPLC.

Product isolation

The crude product can be collected after concentration of the reaction mixture. For example, some of the excess reactants can be removed by washing the dry crude oil with a suitable solvent and centrifuging the mixture. Alternatively, a solvent such as ethyl acetate may be added to accelerate the precipitation of the final product, which may then be isolated by filtration and dried.

Preferably, the mixture is purified by chromatography, as described below. Before the chromatography step, the reaction mixture can be concentrated under reduced pressure to recover the crude product, which is then dissolved in an aqueous medium such as water, optionally by adding a base such as 0.1 N NH ₄ OH, for example, to promote complete solubilization at pH value from 6 to 8, preferably at a pH value of about 7.3; the clear solution is preferably filtered on a 0.2 µm filter.

Chromatographic Purification

According to the invention, the crude product is purified by RP-HPLC, ion exchange chromatography, or both.

RP-HPLC separation is generally preferred because it is more efficient in separating pure product from excess reagents. However, in cases where residual trace amounts of phospholipid (III) remain in the final product (typically more than 1%), a fast and reliable ion exchange purification step can also be added or carried out.

Preparative HPLC purification according to the invention is preferably carried out on a preparative C4 reverse phase column eluting with a mobile phase containing an AcONH ₄ salt. In one embodiment, the mobile phases are aqueous solutions of 10 mM AcONH ₄ and 10 mM AcONH ₄ /acetonitrile 1/9 mixed in a gradient composition capable of effectively separating the product from the phospholipid.

The ion exchange purification of the invention is effectively carried out on an anion exchange resin, preferably a weak anion exchange resin with tertiary amine groups attached to the base matrix. Separation of the final product from excess reagents and other impurities is carried out by selecting suitable buffers for the fixation and elution phase. In accordance with the invention, optimal results have been obtained using a Tris-HCl/NaCl buffer solution at various salt concentrations and at a pH of about 7 to about 8, with the addition of a relative amount of a solvent such as, for example, iPrOH.

Changing the salt concentration in the buffer ensures that the product is fixed to the solid phase while eluting all by-products and then eluting and collecting the pure product.

This effective method for removing all trace amounts of phospholipid (III) allows the use of this reagent even in large excess amounts in the course of the conjugation reaction with the heterodimer (II).

Accordingly, as broadly described above, the present invention provides a more convenient and reliable method for producing compound (I) with high yields and purity, which can also be applied on an industrial scale.

In fact, the product obtained by this process is essentially free of by-products and meets the purity specifications required for its use in the manufacture of gas-filled echo contrast preparations.

Hereinafter, the present invention will be illustrated by examples, which are not intended to limit its scope.

experimental part

Materials and equipment

Solvents such as DMF and ethyl acetate have always been used neat and dried to minimize exposure time to ambient air.

Heterodimeric acetate (IV) was provided by Bachem (Bubendorf, Switzerland). Ammonium salt DSPE-PEG ₂₀₀₀ -NH ₂ was purchased from Avanti Polar Lipids Inc. (USA).

Reverse phase analytical HPLC was performed on a SHIMADZU UFLC system consisting of a UFLC binary pump, a UFLC controller (CBM-20A) and an UV/VIS HPLC detector (SPD-20A). Analyzes were performed using a linear gradient of phase A (10 mM AcONH ₄ in H ₂ O) and phase B (10 mM AcONH ₄ in ACN/H ₂ O 9/1) at 1.5 ml/min with UV detection at wavelength 214 nm. Injected 40 μl, the temperature of the column was 25°C.

Preparative RP-HPLC was performed on a Shimadzu preparative system consisting of a binary HPLC pump, an HPLC fraction collector (FRC-10A), an HPLC controller (SCL-10A), and an HPLC ultraviolet-visible detector (SPD-10AV). The system was equipped with a Kromasil C4 300Å (10×250 mm) column. Purification was performed by linear gradient elution of phase A (10 mM AcONH4 in H2O) and phase B (10 mM AcONH ₄ in ACN/H ₂ O 9/1) at 5 ml/min with UV detection at 214 nm. Introduced 3 ml, and the temperature of the column was 25°C.

The purity of the final product was determined using an Acquity ^TM Ultra Performance (Waters) HPLC system equipped with a TUV detector and an Acquity BEH Phenyl 1.7 µm (2.1×150 mm) column, or using an Agilent 1100 HPLC system equipped with a UV detector and an evaporative detector. by light scattering (ELSD Sedex 85) and column Zorbax 300SB 3.5 µm (3×150 mm).

Abbreviations for individual amino acid residues are generally accepted: for example, Asp or D correspond to aspartic acid, Gly or G correspond to glycine, Arg or R correspond to arginine. It is to be understood that the amino acids referred to herein have the configuration of the L-isomer, unless otherwise indicated.

List of abbreviations

AA, aa Amino acid ACN Acetonitrile AcOEt ethyl acetate Adoa 8-amino-3,6-dioxaoctanoic acid DIEA N,N-diisopropylethylamine DMF Dimethylformamide DSG Di(N-succinimidyl) glutarate DSPE-PEG ₂₀₀₀ - _NH2 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] ELSD Evaporative Light Scattering Detector Eq. Equivalent fmoc 9-fluorenylmethoxycarbonyl g Acceleration of gravity _H2O Water HPLC High Performance Liquid Chromatography iPrOH Isopropyl alcohol mg milligram(s) min minutes ml Milliliter(s) mm millimolar Rt retention time TFU Trifluoroacetic acid THF Tetrahydrofuran TUV Tunable UV UV UV About. Volume UHPLC Ultra high performance liquid chromatography

Example 1 Preparation of Intermediate (II)

Prior to conjugation to the PEGylated phospholipid, heterodimer (IV) was activated by coupling with a di(N-succinimidyl)glutarate moiety as a binding agent.

A solution of acetate heterodimer (49.08 mg) in 500 μl DMF was added in portions (7 x 70 μl) every 2 minutes to a solution of disuccinimidylglutarate with DIEA (130 μl). An excess of DSG (5 eq.) and DIEA (5 eq.) was used to prevent dimer coupling. After stirring at room temperature for 30 minutes after the last addition, the activated heterodimer was isolated and analyzed by HPLC to confirm the completion of the interaction. The following chromatographic conditions were used for these assays:

Column: Phenomenex Luna 5 µm C18 (250 x 4.6 mm)

Eluent A: 10 mM AcONH ₄ in H ₂ O

Eluent B: 10 mM AcONH ₄ in H ₂ O/ACN (1/9)

Flow rate: 1.5 ml/min

Detector: UV 214 nm

Gradient: 25% to 52% mobile phase A

Retention time: 12.69 min

Example 2 Isolation of Compound (II)

Compound (II) obtained in Example 1 was isolated to remove excess DSG from the reaction mixture. The suspension was concentrated under reduced pressure to remove DMF. The dry crude product was washed with 10 ml of EtOAc and then centrifuged for 3 min at 2500 g. The supernatant was poured into a 100 ml round bottom flask, the solid was washed twice with 15 ml EtOAc and dried under reduced pressure to give 47.02 mg of a white powder.

Example 3 Synthesis of Compound (I)

The synthesis of compound (I) was carried out according to the steps shown in Scheme 1. The progress of the reaction was monitored using reverse phase analytical HPLC or HPLC with a UV detector at 220 nm or an ELSD detector.

Steps i)-iii) Conjugation and isolation of product (I)

A sample of DSPE-PEG ₂₀₀₀ -NH ₂ ammonium salt (18 µmol, 50.23 mg, 2 eq) was dissolved in 300 µl anhydrous DMF and then DIEA (2 eq) was added to achieve a total volume of 315 µl.

Compound (II) was solubilized in 400 μl of DMF, then added in five portions to a mixture of DSPE-PEG ₂₀₀₀ -NH ₂ and DIEA and left overnight with stirring.

An aliquot was collected for analytical HPLC control and the profile showed a major elution peak at a retention time of about 12.5 minutes. The mixture was then concentrated under reduced pressure to give 105.5 mg of a crude product.

First, 5 ml of water was added to reach a pH value of 4.8; however, to achieve complete solubilization and obtain a clear solution, an additional 20 drops of 0.1 N NH ₄ OH were added to achieve a pH of 7.3. The solution was then filtered on a 0.2 µm filter and washed to a final volume of about 9 ml, ready for purification by preparative HPLC.

Example 4 Purification of Compound (I) by RP-HPLC

Purification of the final crude product (I) by preparative HPLC was carried out with the same stationary phase as for the analytical control of the coupling reaction. Accordingly, a Kromasil 10 µm 300Å C4 column (250×10 mm) was equilibrated with 10 mM AcONH ₄ in water:acetonitrile (1:9) before loading the sample divided into 3 aliquots (3×3 ml). The liquid phases and elution conditions used for purification are detailed below:

Column: Kromasil 10 µm 300Å C4 (250 x 10 mm)

Eluent A: 10 mM AcONH ₄ in H ₂ O

Eluent B: 10 mM AcONH ₄ in H ₂ O/ACN (1/9)

Flow rate: 5 ml/min.

Injection volume: 3.0 ml

Column temperature: 25°C

Detector: UV 214 nm

Gradient:

Time (meen) Eluent A % Eluent B % 0 100 0 5 100 0 45 0 100 55 0 100 60 100 0 65 100 0

The collection was carried out in fractions of 10 ml, which provided 30 ml of purified product for each cycle. Then 90 ml resulting from 3 cycles, concentrated under reduced pressure, removing most of the acetonitrile before lyophilization. The final product was isolated as a white solid (51.6 mg) with a yield of 69% of the starting heterodimer (IV).

UHPLC-UV analysis of the purified product (I) confirmed the purity to be 99%. No traces of DSPE-PEG ₂₀₀₀ -NH ₂ were detected (see FIG. 2).

Example 5 Purification of Compound (I) by Ion Exchange chromatography

The ion exchange chromatography method was optimized for further purification of the crude product (I) in the presence of trace amounts of DSPE-PEG ₂₀₀₀ -NH ₂ .

For this purpose, a column packed with ANX Sepharose 4 high flow resin (GE Healthcare) was used with the following buffers:

- for the fixation step: 0.05 M Tris HCl - 0.10 M NaCl - pH 7.5 + 35% iPrOH

- for the elution step: 0.05 M Tris HCl - 1.00 M NaCl - pH 7.5 + 35% iPrOH

- for the desalting step: 0.02 M Tris HCl pH 7.5

Two samples of compound (I) (solutions A and B containing 0.5 mg and 1.5 mg of compound (I) respectively) were loaded onto an ANX column. In addition, a similar experiment was carried out with DSPE-PEG ₂₀₀₀ -NH ₂ (solutions C and D, respectively, containing 0.5 mg and 1.5 mg DSPE-PEG ₂₀₀₀ -NH ₂ ).

Fraction analysis showed that DSPE-PEG ₂₀₀₀ -NH ₂ was completely eluted by the first two column volumes (CV) with fixing buffer. At the same time, compound (I) was not found in the first two fractions, which means that the separation could be effective, since one of them passes directly, while the other compound remains fixed on the resin.

Indeed, after a total of 4 CVs of the fixing buffer was passed, the elution buffer was introduced, ensuring the collection of compound (I) by fractions in two CVs (see table 1a, solutions A and B). In addition, in a second experiment, it was confirmed that DSPE-PEG ₂₀₀₀ -NH ₂ did not elute after the second fraction even with elution buffer (Table 1b, solutions C and D).

The pool of collected fractions was passed through a Sephadex G-25M desalting resin column prior to lyophilization.

Table 1a - Retention and elution of compound (I) (solutions A and B) on an ANX column

Column volume mg compound (I) extraction mg compound (I) extraction Solution A (0.5 mg) Solution B (1.5 mg) CV1 (fixation) < 0.010 < 2% 0 0% CV2 (fixation) 0 0% 0 0% CV 3+4 (fixation) 0 0% 0 0% CV1 (elution) 0.415 90% 1.659 108% CV2 (elution) 0.050 eleven% 0.014 one% CV3 (elution) 0 0% 0 0%

Table 1b - Retention and elution of DSPE ₂₀₀₀ -NH ₂ (solutions C and D) on an ANX column

Column volume mg DSPE ₂₀₀₀ -NH ₂ extraction mg DSPE ₂₀₀₀ -NH ₂ extraction Solution C (0.5 mg) Solution D (1.5 mg) CV1 (fixation) 0.477 95.4% 1.403 93.5% CV2 (fixation) 0.004 0.7% 0.014 0.9% CV3+4 (fixation) 0 0% 0 0% CV1 (elution) 0 0% 0 0% CV2 (elution) 0 0% 0 0% CV3 (elution) 0 0% 0 0%

All fractions were analyzed using a 1100 LC/MSD system (Agilent) and quantitated against a calibration standard using a UV detector for compound (I) or an ELSD detector for DSPE-PEG ₂₀₀₀ -NH ₂ (III).

Links

1. Ferrara N. et al., “Vascular Endothelial Growth Factor: Basic Science and Clinical Progress”, Endocrine Reviews, 2004, 25(4), 581-611.

2. Veikkola T. et al., “Regulation of Angiogenesis via Vascular Endothelial Growth Factor Receptors”, Cancer Res ., 2000, 60, 203-212.

Claims (22)

1. The method of obtaining the compound (I) or its pharmaceutically acceptable salts,

including stages (i) combinations of the appropriate succinimidyl ester intermediate (II)

with phospholipid DSPE-PEG ₂₀₀₀ -NH ₂ (III)

in the presence of DIEA, wherein said phospholipid (III) is present in an excess of 1.1 or more equivalents relative to compound (II); (ii) isolating the crude product (I) recovered from the reaction mixture of step (i); (iii) optionally diluting the crude product obtained in step (ii) with water and adding a base to achieve a pH of 6 to 8; and (iv) purification of the crude product from the solution of step (iii) by HPLC, ion exchange chromatography, or both. 2. The method according to p. 1, characterized in that the combination is carried out with 2 equivalents of phospholipid (III) per one equivalent of compound (II). 3. The method according to p. 1, characterized in that the pH of the solution obtained in stage (iii) is adjusted to a value of 7.0-7.5. 4. The method according to claim 1, characterized in that the purification in step (iv) is carried out by reverse phase high performance liquid chromatography (RP-HPLC). 5. The method according to p. 1, characterized in that the purification of stage (iv) is carried out by ion exchange chromatography. 6. The method according to claim 1, characterized in that the purification in step (iv) is carried out by both reverse phase high performance liquid chromatography (RP-HPLC) and ion exchange chromatography. 7. The method according to claim 4 or 6, characterized in that the purification by RP-HPLC is carried out using a mobile phase containing an AcONH ₄ salt. 8. The method according to claim 5 or 6, characterized in that the ion exchange chromatography is carried out using a weak anion exchange resin and a buffer solution at a pH value of from 7 to 8. 9. The method according to p. 8, characterized in that the elution solution is a buffer 0.05 M Tris HCl + 1.00 M NaCl + 35% iPrOH. 10. The method according to p. 1, characterized in that the compound (II) is obtained by activation of the corresponding intermediate compound of formula (IV) with di(N-succinimidyl)glutarate

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