RU2802514C1 - New platinum iv complexes with essentially increased antitumor efficiency - Google Patents

Abstract

FIELD: new complexes of platinum (IV).

SUBSTANCE: invention relates to a complex of platinum (IV) with a geometric configuration of cis-(ammine,1-adamantylamine)-trans-bis(1-adamantylcarboxylate)-cis-(chloride, X) Pt (IV ) of general formula (III), where X is a conjugated anion of a strong mineral or organic acid and where X is selected from the group consisting of nitrate, monochloroacetate and trifluoroacetate. A method is also proposed for producing these new platinum (IV) complexes, a pharmaceutical composition and a method for treating a tumour disease.

EFFECT: proposed platinum (IV) complexes have improved antitumor efficacy and can be used to treat, preferably, difficult cancer, most preferably pancreatic cancer.

15 cl, 3 dwg, 6 tbl, 5 ex

(III)

Description

Field of technology

The invention relates to new platinum(IV) complexes with substantially increased antitumor efficacy. A method for obtaining these new platinum(IV) complexes is described. A pharmaceutical composition and final dosage form for oral administration of novel platinum(IV) complexes are described. Described is the use of a novel platinum(IV) complex in a method for treating a tumor disease, preferably a difficult to treat cancer, most preferably pancreatic cancer, preferably by oral administration.

State of the art

Cancer is a serious disease with a continuous increase in incidence and prevalence. Despite significant advances in immunotherapy, cell therapy and biological therapy, there is still a constant need for small molecule anticancer drugs with increased antitumor efficacy, reduced toxicity, good availability and affordable price.

Platinum(II) complexes are anticancer drugs that have been used for many years. They have great antitumor efficacy, synergistic effects in combination therapy, good availability and affordable price. Platinum(II) complexes used in medicine have a planar structure with a central Pt ²⁺ ion surrounded by two cis -neutral non-leaving amino ligands and two cis -anionic leaving ligands X ₁ , X ₂ , as shown in figure (I):

(I)

where R ₁ , R ₂ are hydrogen or together 1R,2R -1,2-cyclohexyl and X ₁ , X ₂ are chloride or together dicarboxylate. The high antitumor efficacy of platinum (II) complexes is based on a unique mechanism of action. The leaving ligands of platinum(II) complexes are replaced by water in the intracellular space (a process of "hydration") to form highly reactive water complexes, which then react with DNA, causing its deformation. Extensive damage to the DNA structure induces a cascade of intracellular mechanisms leading to cell death (apoptosis). In parallel, the cancer cell fights such platinum (II) xenobiotics by converting them into inactive Pt complexes, for example, with the help of metallothionein, methionine, glutathione, etc. The inactivated Pt complexes are then removed from the cell. The relationship between the structure of platinum (II) complexes and their antitumor activity was studied in the articles [1] - Cleare MJ, Hoeschele JD: Platinum Metals Rev., Vol.17, 2-13, 1973 and [2] - Ridgway HJ, Speer RJ et al.: Journal of Clinical Hematology and Oncology, Vol.7, No.1, 220-230, 1976. The rate of “hydration” and the associated cytotoxicity and antitumor efficacy depend mainly on the strength of the bond between the leaving anionic ligand and the central platinum ion (II). The weaker this bond, the faster the formation of reactive aqueous complexes and the higher the associated toxicity and activity. The stronger this bond, the slower the formation of reactive aqueous complexes and the less associated toxicity and activity. Loosely bound conjugate bases of strong acids, such as nitrates or sulfates, were very reactive, but they also caused severe toxicity (eg, seizures) and their testing had to be stopped. In contrast, strongly bound conjugated bases of weak acids, such as SCN ^- , NO ₂ ^- , CNO ^- , were unreactive, exhibiting only marginal toxicity and activity. Toxicity and antitumor efficacy in Pt(II) complexes are mutually inseparable, both due to the same mechanism of action and due to the low selectivity of action on tumor cells. Moderately bound anionic ligands as chlorides (cisplatin) or dicarboxylates (carboplatin, oxaliplatin) have been developed for therapeutic use as a compromise between the toxicity and antitumor efficacy of platinum(II) complexes. Their unwanted toxicity was then partially eliminated by premedicating patients; however, the problem of their serious toxicity remains.

Platinum(IV) complexes are prodrugs of platinum(II) complexes. Their axial positions in the structure are occupied by two other anionic ligands, usually carboxylates. The axial ligands prevent water from accessing the central Pt ion, so the necessary "hydration" process can only occur after their reductive removal. Platinum(IV) complexes provide all the benefits of platinum(II) complexes while improving their stability and toxicity to healthy cells. An overview of the current state of platinum (IV)-based drugs is given in reviews [3] - Johnston TC, et al.: “The next Generation of Platinum Drugs: Targeted Pt (II) Agents, Nanoparticle Delivery and Pt (IV) Prodrugs”, Chem. Rev. 2016, 116, 3436-3486] and [3] - Kenny RG, et al.: “Platinum (IV) Prodrugs-A Step Closer to Ehrlich's Vision?”, Eur. J. Inorg. Chem. 2017, 1596-1612. The first promising lipophilic platinum(IV) complex for oral administration was discovered by Johnson Matthey, [4]-EP0328274, Johnson Matthey, priority 02 February 1988. It has the configuration cis- (ammin, cyclohexylamine) -trans -bis-acetate -cis- Pt(IV) dichloride, and shortly thereafter it entered clinical trials under the name satraplatin. Although great efforts have been made over the past 30 years to introduce Pt(IV) complexes into medical practice, this has not yet been achieved.

The platinum(IV) complex TU31 with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (dichloride)Pt(IV) of formula (II) is a very promising platinum(IV) complex for cancer therapy in the prior art:

(II)

This platinum(IV) complex TU31 was discovered by VUAB Pharma, [5]-EP3189064, VUAB Pharma a.s., filed September 03, 2014. This platinum(IV) complex represents a combination of the unique properties of 1-adamantyl ligands with the unique antitumor mechanisms of platinum(II) complexes ). The introduction of three 1-adamantantyl ligands with organic diamond properties imparts superior stability, symmetry, compactness, and permeability to the TU31 complex, resulting in improved stability, cellular uptake, and antitumor efficacy. Reductive cleavage of axial bulky and symmetrical 1-adamantyl carboxylate ligands requires increased levels of cell-protective reducing agents (e.g., metallothionein, methionine, or glutathione) that are more specific to tumor cells as opposed to healthy cells. This makes it possible to suppress toxicity in healthy cells, as opposed to tumor cells. This was tested and confirmed in the preclinical stage of TU31 complex, where toxic symptoms were not achieved even with a single oral administration of TU31 complex in both rats and dogs.

However, the same lipophilicity, compactness and inertness lead to poor solubility of the TU31 complex in water, which limits its bioavailability for the same reasons. This disadvantage has been overcome by reducing the particle size and selecting a suitable carrier, but the limited bioavailability of the TU31 complex remains. This calls for the development of new TU31 derivatives with further enhancement of antitumor efficacy while maintaining the advantages of the “mother” platinum(IV) complex of TU31, mainly low toxicity and broad antitumor efficacy.

The essence of the invention

According to a first aspect of the invention, there is provided a platinum(IV) complex with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride,X)Pt(IV) of general formula (III):

(III)

where X is the conjugate anion of a strong mineral or organic acid.

In a preferred embodiment, ligand X is selected from the group consisting of nitrate, mesylate, monochloroacetate and trifluoroacetate.

In a preferred embodiment, ligand X is a nitrate.

In a preferred embodiment, ligand X is a mesylate.

In a preferred embodiment, ligand X is monochloroacetate.

In a preferred embodiment, ligand X is trifluoroacetate.

Without being bound by theory, the introduction of a weakly bound conjugated strong acid anion X into platinum(II) complexes is not possible due to the high reactivity of the Pt-X bond and the great corresponding toxicity of the subsequently formed water-platinum(II) species. However, the same introduction of weakly bound ligand X into the platinum(IV) complex of TU31 may have a beneficial effect, since the reactive Pt-X bond is spatially shielded by bulky axial 1-adamantylcarboxylate ligands, which are more difficult to remove in healthy cells than in cancer cells. This leads to suppression of the toxicity of this platinum(IV) complex in healthy cells, despite the fact that it contains a highly reactive Pt-X bond. Conversely, removal of shielding axial ligands in such a platinum(IV) complex in cancer cells is possible due to increased concentrations of shielding reducing agents (e.g., metallothionein, glutathione) specific to cancer cells. This leads to the rapid formation of toxic aqueous platinum(II) complexes in cancer cells with a corresponding antitumor effect, in contrast to healthy cells.

In a second aspect of the present invention there is provided a method for the preparation of a platinum (IV) complex of formula (III), where X is a conjugated anion of a strong mineral or organic acid, comprising the following preparation steps:

a) Reaction of the starting platinum(IV) complex with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, iodide) Pt(IV) (TU31-monoiodide) with a stoichiometric excess silver salt of a strong acid, in a mixture of a chlorinated hydrocarbon solvent, preferably chloroform, and a corresponding strong acid at the boiling point of the solvent for the reaction time necessary to convert to obtain the mixture.

b) Removing solids from the mixture by filtration to obtain a crude filtrate.

c) Washing the crude filtrate with water and drying the separated organic phase.

d) Removing the solvent from the dried organic phase by evaporation to obtain a platinum(IV) complex of formula (III), where X is the conjugated anion of a strong mineral or organic acid.

In a preferred embodiment of the invention, the method for preparing a platinum (IV) complex of general formula (III), where X is a conjugated anion of a strong mineral or organic acid, further comprises the step

e) Dissolving the platinum(IV) complex in acetone, adding activated carbon, ultrafiltrating the resulting suspension and precipitating the resulting ultrafiltered solution of pure platinum(IV) complex in a large amount of water to obtain a pure platinum(IV) complex of general formula (III), where X is conjugated anion of a strong mineral or organic acid in the form of nanoparticles.

The preferred preparation method of the invention is to introduce the weakly bound ligand anion X directly into the TU31 complex prepared according to Figure 1.

It is preferable to introduce one iodide ligand instead of one chloride ligand into the parent TU31 complex in step a) to promote the conversion of the parent TU31 complex into a new platinum(IV) complex. The original TU31-monoiodide complex with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, iodide) Pt(IV), which corresponds to the platinum(IV) complex of formula (III) , where X is substituted with iodide, can be prepared according to a three-step reaction procedure already known from the prior art. For example, the chloride ligand can be replaced by an iodide ligand in the platinum(II) complex intermediate TU9 (TU9 - see Figure 1) by reacting the platinum(II) complex TU9 with a stoichiometric amount of KI in an aqueous suspension at about 50°C for 24 h to obtain the platinum(II) complex TU9-monoiodide. The platinum(II) complex TU9-monoiodide is then oxidized to the corresponding platinum(IV) complex according to a known procedure, described for example in EP0328274, Johnson Mathey. Briefly, the Pt(II) TU9-monoiodide complex is oxidized with a 30-50 stoichiometric excess of hydrogen peroxide at approximately 60℃ for 6-8 hours to produce the platinum(IV) TU11-monoiodide complex (TU11 - see Scheme 1). Finally, the platinum(IV) TU11-monoiodide complex is esterified with an excess of 1-adamantylcarbonyl chloride to obtain the starting TU31-monoiodide complex according to a known procedure, described, for example, in EP 3189064, VUAB Pharma. Briefly, one stoichiometric equivalent of the TU11-monoiodide complex is reacted with 5 stoichiometric equivalents of 1-adamantylcarbonyl chloride with an appropriate amount of pyridine in 1,4-dioxane to obtain the crude TU31-moniodide complex. The crude TU31-monoiodide complex is purified by repeated leaching in 1,4-dioxane and water. The resulting product is then dissolved in acetone, activated carbon is added, the suspension is ultrafiltered, and the resulting ultrafiltered solution is precipitated in a large amount of water to obtain the pure starting TU31-monoiodide complex.

In a preferred embodiment, the yields of the new platinum(IV) complexes obtained in accordance with the invention in steps a) - e) are 62-78% relative to the original TU31-moniodide complex. The purity of the resulting platinum(IV) complex is typically 96-98.5% (HPLC-UV).

In a preferred embodiment, the particle size of the resulting platinum(IV) complexes is in the range of 100-300 nm, measured by DLS (dynamic light scattering).

The molecular weight of the resulting platinum(IV) complexes can be further confirmed by HPLC-MS. The preparation of platinum (IV) complexes of general formula (III) is explained in more detail in examples 1, 2 and 3 .

According to a third aspect of the invention, there is provided a pharmaceutical composition comprising a platinum(IV) complex according to formula (III) with a pharmaceutically acceptable carrier.

In a preferred embodiment, the platinum (IV) complex pharmaceutical composition according to formula (III) can be administered orally.

In a preferred embodiment, the pharmaceutically acceptable carrier is a self-emulsifying carrier, preferably stearoyl macrogol glycerides, most preferably Gelucire 50/13.

In a preferred embodiment, the pharmaceutical composition contains a platinum (IV) complex in accordance with formula (III) in the form of nanoparticles.

In a preferred embodiment, a final dosage form for use in the treatment of tumor diseases is provided, preferably for oral administration, containing a pharmaceutical composition of platinum (IV) complexes according to the invention, preferably in the form of nanoparticles, preferably with a self-emulsifying carrier, enclosed in acid-resistant capsules. The final dosage form of the invention addresses both the poor solubility with corresponding poor bioavailability of the new platinum(IV) complexes and the instability of this complex in the stomach and intestines, where the concentration of reducing agents and other biodegrading agents is also elevated, as in tumor cells. Conversion of microparticles of platinum(IV) complexes of formula (III) into nanoparticles, preferably less than 250 nm, improves the solubility and bioavailability of the new platinum(IV) complexes, preferably after oral administration.

The preparation of nanoparticles of new platinum (IV) complexes is described in examples of the preparation of such platinum (IV) complexes.

In a preferred embodiment, the use in the composition of a self-emulsifying carrier, preferably stearoyl macrogol glycerides, most preferably Gelucire 50/13, supports both the protection of the new platinum(IV) complexes from premature activation in the intestine and the bioavailability of this complex in the body by encapsulating it into vesicles. Gelucire 50/13 has a dew point of approximately 50℃ and low melt viscosity, which makes it easy to prepare the hot melt composition of the present invention at temperatures above 50℃. This carrier is also relatively simple, cheap, non-toxic and certified for pharmaceutical use.

In a preferred embodiment, the mass fraction of Gelucire 50/13 is at least 80 wt%. composition is recommended to ensure maximum supportive and protective effect of this carrier.

The use of acid-resistant capsules in accordance with a preferred embodiment protects the novel platinum(IV) complexes from premature activation in the stomach after oral administration. The preferred acid-resistant capsules are hydroxypropyl methylcellulose phthalate (HPMCP) or hydroxypropyl methylcellulose acetate succinate (HPMCAS) capsules, which dissolve quickly in the upper intestine, unlike the commonly used enteric-coated capsules. The platinum(IV) complex of the invention should be protected as much as possible in the gastrointestinal tract to avoid the risk of unwanted toxicity. This dual protection of the novel platinum(IV) complex, through both an acid-resistant capsule and a self-emulsifying carrier, minimizes the risk of premature reconstitution/activation with attendant toxicity until the complex reaches its target cancer cells.

According to a fourth aspect of the invention, there is provided the use of novel platinum(IV) complexes of general formula (III) for use in a method of treating neoplastic diseases, preferably difficult to treat cancers, most preferably pancreatic cancer, by oral administration.

The present invention covers any of the following aspects:

1) Platinum (IV) complex with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride,X)Pt (IV) of general formula (III):

(III)

where X is the conjugated anion of a strong mineral or organic acid.

2) The platinum (IV) complex according to aspect 1), where X is selected from the group consisting of nitrate, mesylate, monochloroacetate and trifluoroacetate.

3) A method for producing a platinum (IV) complex according to aspects 1) or 2), including the following stages of production:

a) Interaction of the initial platinum (IV) complex with the geometric configuration of cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, iodide) Pt (IV) with a stoichiometric excess of the silver salt of a strong acid, in a mixture of a chlorinated hydrocarbon solvent, preferably chloroform, and a suitable strong acid at the boiling point of the solvent for the reaction time necessary to convert to obtain the mixture.

b) Removing solids from the mixture by filtration to obtain a crude filtrate.

c) Washing the crude filtrate with water and drying the separated organic phase.

d) Removing the solvent from the dried organic phase by evaporation to obtain the platinum(IV) complex according to aspect 1) or 2).

4) The method according to aspect 3), including the additional step of:

f) Dissolving the platinum (IV) complex in acetone, adding activated carbon, ultrafiltration of the resulting suspension and precipitation of the resulting ultrafiltered solution of pure platinum (IV) complex in a large amount of water to obtain pure platinum (IV) complex according to aspect 1) or 2) in the form nanoparticles

5) The pharmaceutical composition of a platinum (IV) complex according to aspect 1) or 2), preferably for oral administration, containing a Pt (IV) complex and a pharmaceutically acceptable carrier.

6) The pharmaceutical composition according to aspect 5), wherein the pharmaceutically acceptable carrier is a self-emulsifying carrier, preferably stearoyl macrogol glycerides, most preferably Gelucire 50/13.

7) Pharmaceutical composition according to aspect 5) or 6), where the platinum (IV) complex is present in the form of nanoparticles.

8) Pharmaceutical composition according to aspect 6) or 7), where the mass fraction of the self-emulsifying carrier is more than 80% of the mass.

9) The pharmaceutical composition according to any one of aspects 5)-8), wherein the pharmaceutical composition is placed in acid-resistant capsules, preferably hydroxypropyl methylcellulose phthalate or hydroxyperopyl methylcellulose acetate succinate capsules.

10) A platinum (IV) complex according to aspect 1) or 2) or a pharmaceutical composition according to any of aspects 5) - 9) for use in a method for treating a tumor disease, preferably a difficult-to-treat cancer, most preferably pancreatic cancer, preferably by oral administration.

11) A method for treating a tumor disease, preferably a difficult-to-treat cancer, most preferably pancreatic cancer, in a subject in need thereof, comprising administering, preferably orally, a platinum (IV) complex according to aspect 1) or 2) or a pharmaceutical composition according to any of aspects 5) - 9).

More detailed description of the invention

The authors unexpectedly discovered that platinum (IV) complexes of general formula (III) have significantly improved antitumor efficacy compared to the prior art. This has been demonstrated in both in vitro and in vivo experiments. The XTT method was used in in vitro experiments. The original platinum (IV) complex TU31 (where X=Cl ^- ), three new platinum (IV) complexes according to the invention were tested, where X=ClCH ₂ COO ^- (TU105 complex), F ₃ CCOO ^- (TU106 complex) and NO ₃ ^- (TU113 complex), reference platinum(II) complex oxaliplatin and reference platinum(IV) complex satraplatin. Two cancer cell lines of difficult-to-treat tumors, Capan-2 (pancreatic adenocarcinoma) and PC-3 (prostate carcinoma), were used in the XTT tests. The results obtained are summarized as IC ₅₀ values in the following Table 1:

Table 1: IC ₅₀ values measured for the new platinum(IV) complexes and the reference platinum(II) complex Oxaliplatin

Tested Platinum Complex IC ₅₀ µM Capan-2 cancer cell line PC-3 cancer cell line Oxaliplatin (Pt(II) reference complex) >100 55.7 Satraplatin (Pt(IV) reference complex) >30 10.2 TU31 (Pt(IV) mother complex) 0.60 0.91 TU105 (new Pt(IV) complex) 0.04 0.06 TU106 (new Pt(IV) complex) 0.02 0.07 TU113 (new Pt(IV) complex) 0.11 0.23

Capan-2=pancreatic adenocarcinoma, DSMZ, ACC-245

PC-3=prostate carcinoma, ATCC® CRL-1435™

We have unexpectedly discovered that platinum(IV) complexes of formula (III) have significantly increased antitumor efficacy in vitro compared to the prior art. The parent TU31 complex has approximately 50-100 times better in vitro antitumor efficacy than reference oxaliplatin and 11-55 times better in vitro antitumor efficacy than reference satraplatin. The excellent antitumor effects of the TU31 complex compared to the reference oxaliplatin and satraplatin, combined with its very low toxicity and the possibility of oral administration, are good prerequisites for the success of the TU31 complex in clinical trials. However, the new platinum(IV) complexes TU105, TU106 and TU113 of general formula (III) according to the invention have IC ₅₀ values even more than 1000 times better than the reference oxaliplatin, more than 100 times better than the reference satraplatin and more than 15 times better than the parent TU31 complex for both Capan-2 and PC-3 cancer cell lines. This further improvement appears to be significant compared to the prior art and creates good preconditions for the further development of new platinum(IV) complexes according to the invention, in particular for the treatment of tumors that are difficult to treat, such as pancreatic tumors.

More detailed information on the effectiveness and test results of XTT is given in Example 4.

Two new platinum(IV) complexes TU105 and TU106 from the XTT assays were further studied in vivo in a nude mouse model bearing two pancreatic tumors. The platinum(IV) complex satraplatin and the parent complex TU31 were again used as reference compounds. Two difficult to treat pancreatic tumors, Capan-2 and PaTu8902, were cultured and then subcutaneously injected into mice at a dose of 1 x 10 ⁷ cells/mouse. After the subcutaneous tumors had expanded to a volume of approximately 0.2 cm ³ , two doses of the appropriate complexes of 2 x 20 mg/kg were administered orally every 1 week. The control group of mice did not receive the drug. Tumor volume was continuously measured throughout the experiment. Three weeks after the second dose, mice were sacrificed, tumors were removed, weighed, and the data obtained were scored as % tumor volume inhibition (TVI%) and % tumor weight inhibition (TWI%), where:

TVI%= 100 - [(TVexp/TVcont) x 100]

TVexp=average volume of treated tumors in cm ³

TVcont=average volume of control tumors in ^cm3

TWI%= 100 - [(TWexp/TWcont) x 100]

TWexp=average weight of treated tumors in city.

TWcont=average weight of control tumors in g.

The results obtained are summarized in Table 2 and Table 3.

Table 2. Results of antitumor efficacy of platinum (IV) complexes TU31, TU105, TU106 in %TWI, single oral dose of 20 mg/kg, experimental duration 28 days (28 days)

Mouse: athymic nude -FOXN-1 ⁿ , 6 mice per group (3 males + 3 females).

Tumor: Capan-2, pancreatic adenocarcinoma (DSMZ, ACC-245)

Pt(IV) complex Average tumor volume during the experiment in days (g), in cm ³ TVI% Average tumor weight in g
28 d TWI% 1d 4d 8d 11d 15d 18d 22d 25d 29d Control 0.20 0.27 0.35 0.44 0.53 0.63 0.73 0.85 1.00 - 0.793 - Satraplatin 0.21 0.23 0.30 0.39 0.47 0.56 0.66 0.77 0.90 10.0 0.711 10.3 TU31 0.21 0.23 0.27 0.31 0.37 0.43 0.49 0.56 0.63 37.0 0.371 53.2 TU105 0.20 0.21 0.22 0.23 0.27 0.30 0.34 0.37 0.40 60.0 0.213 73.1 TU106 0.21 0.21 0.23 0.24 0.25 0.26 0.28 0.29 0.31 69.0 0.166 79.1

Table 3. Results of antitumor efficacy of platinum (IV) complexes TU31, TU105, TU106 in %TWI, single oral dose of 20 mg/kg, experimental duration 28 days (28 days)

Mouse: Athymic hairless -FOXN-1 ^nu , 6 mice per group (3 males + 3 females)

Tumor: PaTu8902, pancreatic adenocarcinoma, metastatic, stage II (DSMZ, ACC-179)

Pt(IV) complex Average tumor volume during the experiment in days (g), in cm ³ TVI% Average tumor weight in g
28 d TWI% 1d 4d 8d 11d 15d 18d 22d 25d 29d Control 0.21 0.58 1.15 2.20 3.59 5.44 7.79 10.59 13.65 - 8,186 - Satraplatin 0.21 0.50 0.99 1.94 3.10 4.80 6.80 9.35 11.90 12.8 7,245 11.5 TU31 0.21 0.28 0.54 0.86 1.44 2.27 3.35 4.62 5.96 56.3 4,017 50.9 TU105 0.21 0.21 0.40 0.62 1.06 1.65 2.46 3.40 4.35 68.1 2,522 69.2 TU106 0.21 0.21 0.37 0.60 1.01 1.60 2.35 3.27 4.20 69.2 2,317 71.7

From the results obtained, it follows that the parent TU31 complex has very good antitumor efficacy in both pancreatic tumors, having a TWI of approximately 50%, in contrast to the reference satraplatin complex. However, the new platinum(IV) complexes TU105 and TU106 of the invention demonstrated significantly improved antitumor efficacy, expressed in TWI%, of approximately 70-80%, which is approximately 7 times higher than the reference platinum(IV) complex satraplatin and significantly greater than that of the reference platinum(IV) complex satraplatin. than for the original TU31 complex, also for both pancreatic tumors studied. More detailed information on the effectiveness and results of in vivo testing in the nude mouse model is given in Example 5 .

The results obtained in both in vitro and in vivo tests confirm the significantly improved antitumor efficacy of the new platinum(IV) complexes compared to the prior art. The new platinum(IV) complexes of general formula (III) according to the invention offer very promising and affordable agents for oral therapy of cancer, preferably difficult to treat cancer, more preferably pancreatic cancer. The broad antitumor efficacy of the new platinum (IV) complexes according to the invention is based on the known and unique mechanism of action of platinum (II) metabolites, which is little dependent on the variability of signaling pathways and tumor markers in cells. In this case, the problem of drug targeting of toxic complexes is solved by selective activation of the platinum (IV) complex in tumor cells according to the invention.

The invention is further explained and illustrated, but is not limited to, by the following examples and drawings.

The drawings show

Figure 1: scheme for obtaining platinum (IV) complex TU31,

Figure 2: Capan-2 tumor growth curve, and

Figure 3: PaTu8902 tumor growth curve.

Examples

EXAMPLE 1.

Receipt cis- (ammine, 1-adamantylamine) - trance -bis(1-adamantylcarboxylate)- cis -(chloride, monochloroacetate) Pt (IV), new Pt (IV) complex TU105, molecular weight 850.77 g/mol.

The preparation is carried out in a fume hood in the absence of light. The starting Pt(IV) TU31-monoiodide complex is prepared in accordance with the prior art.

70 mg of silver oxide (0.3 mmol) and 7.9 g of monochloroacetic acid (83.6 mmol) are stirred in 20 ml of chloroform for 1 hour at room temperature. Then 360 mg of the original Pt(IV)-monoiodide complex (0.4 mmol) was added and the mixture was stirred for 6 hours at 63℃ (low reflux). The resulting suspension is cooled to room temperature and the solid phase is removed by filtration. The resulting organic solution is extracted three times with 20 ml of water to remove water-soluble compounds from the organic solution. The washed organic solution is dried using molecular sieves and then the chloroform is removed from the clear dried solution by vacuum distillation to obtain the crude TU105 complex. The crude TU105 complex is dissolved in 33 ml of acetone and 20 mg of activated carbon is added to obtain a suspension, which is stirred for 0.5 hours at room temperature. The suspension is then ultrafiltered through a 0.22 µm porosity filter to obtain an ultrafiltered product solution, which is then precipitated in 100 ml of cold water to obtain 270 mg of pure TU105 complex, 78% theoretical yield, 98.1% purity (HPLC method) and particle size 200-400 nm (DSR method). MS spectrometry confirms the molecular weight.

EXAMPLE 2.

Receipt cis- (ammine, 1-adamantylamine) - trance -bis(1-adamantylcarboxylate)- cis -(chloride, trifluoroacetate) Pt (IV), new Pt (IV) complex TU106, molecular weight 870.30 g/mol.

The preparation is carried out in a fume hood in the absence of light. The starting Pt(IV) TU31-monoiodide complex is prepared in accordance with the prior art.

A mixture of 360 mg of the starting Pt(IV)-monoiodide complex (0.4 mmol), 135 mg of trifluoroacetic acid silver salt (0.6 mmol), 9.6 g of trifluoroacetic acid (84.2 mmol) and 20 ml of chloroform is stirred for 6 hours at 63℃ (gentle reflux). The resulting suspension is cooled to room temperature and the solid phase is removed by filtration. The resulting organic solution is extracted three times with 20 ml of water to remove water-soluble compounds from the organic solution. The washed organic solution is dried using molecular sieves and then the chloroform is removed from the clear dried solution by vacuum distillation to obtain the crude TU106 complex. The crude TU106 complex is dissolved in 33 ml of acetone and 20 mg of activated carbon is added to obtain a suspension, which is stirred for 0.5 hours at room temperature. The suspension is then ultrafiltered through a 0.22 µm porosity filter to obtain an ultrafiltered product solution, which is then precipitated in 100 ml of cold water to obtain 287 mg of pure TU106 complex, 81% theoretical yield, 98.5% purity (HPLC method) and particle size 200-400 nm (DSR method). MS spectrometry confirms the molecular weight.

EXAMPLE 3.

Receipt cis- (ammine, 1-adamantylamine) - trance -bis(1-adamantylcarboxylate)- cis -(chloride, nitrate) Pt (IV), new Pt (IV) complex TU113, molecular weight 819.29 g/mol.

The preparation is carried out in a fume hood in the absence of light. The starting Pt(IV) TU31-monoiodide complex is prepared in accordance with the prior art.

A mixture of 360 mg of the original Pt(IV) monoiodide complex (0.4 mmol), 100 mg of AgNO ₃ (0.6 mmol), 6 ml (8.46 g) 65% nitric acid (87.3 mmol) and 20 ml chloroform is stirred for 10 hours at 63℃ (gentle reflux). The resulting suspension is cooled to room temperature and the solid phase is removed by filtration. From the filtrate, the organic phase is separated from the aqueous phase with HNO ₃ . The resulting organic phase is extracted three times with 20 ml of water to remove water-soluble compounds from the organic solution. The washed organic solution is dried using molecular sieves and then the chloroform is removed from the clear dried solution by vacuum distillation to obtain the crude TU113 complex. The crude TU113 complex is dissolved in 33 ml of acetone and 20 mg of activated carbon is added to obtain a suspension, which is stirred for 0.5 hours at room temperature. The suspension is then ultrafiltered through a 0.22 µm porosity filter to obtain an ultrafiltered product solution, which is then precipitated in 100 ml of cold water to obtain 227 mg of pure TU113 complex, 69% theoretical yield, 96.2% purity (HPLC method) and particle size 200-350 nm (DSR method). MS spectrometry confirms the molecular weight.

EXAMPLE 4.

Study of antitumor efficacy in vitro new Pt(IV) complexes according to the present invention, severe for the treatment of pancreatic and prostate tumors, XTT test.

The antitumor efficacy of the new Pt(IV) complexes according to the invention is being studied on difficult-to-treat pancreatic and prostate cancer lines. The XTT method is used. The results are assessed by IC ₅₀ values and compared with the reference Pt(II) complex oxaliplatin and the reference Pt(IV) complex satraplatin.

Tested connections:

1. Reference complex Pt(II) oxaliplatin, ( 1R,2R -1,2-cyclohexanediamine-N,N´) (ethanedioato-O,O´) platinum(II), CAS No: 61825-94-3, purity: 99.7% (HPLC), source VUAB Pharma as

2. Reference Pt(IV) complex satraplatin, cis- (ammine, cyclohexylamine) -trans -bis-acetate -cis- Pt(IV) dichloride, CAS No.: 129580-63-8, > 98.8% (HPLC), source WATSON Inc., Ltd, CN.

3. Pt(IV) starting complex TU31, cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate)- cis- (dichloride)Pt(IV), CAS No.: NCE, purity 99.5% ( HPLC), source VUAB Pharma as

4. New Pt(IV) complex TU105, cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, monochloroacetate) Pt(IV), CAS No.: NCE, purity 98.1 % (HPLC), source VUAB Pharma as

5. New Pt(IV) complex TU106, cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate)- cis- (chloride, trifluoroacetate) Pt(IV), CAS No.: NCE, purity 98.5 % (HPLC), source VUAB Pharma as

6. New Pt(IV) complex TU113, cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, nitrate) Pt(IV), CAS No.: NCE, purity 96.2 % (HPLC), source VUAB Pharma as

Cancer cell lines:

Fresh tumor cell lines are purchased from a Cell Bank to ensure their quality and activity.

a) Capan-2, pancreatic duct adenocarcinoma, DSMZ, ACC-245

b) PC-3, prostate carcinoma, ATCC® CRL-1435™

Conditions for culturing tumor cell lines:

Reducing the connections used:

DMSO: dimethyl sulfoxide

PBS: phosphate buffered saline

XTT: 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt

PMS: N-methyl-dibenzopyrazine methyl sulfate

FBS: fetal bovine serum

NEAA: Essential Amino Acids

L-glu: L-glutamine

DMEM: Dulbecco's modified Eagle's medium (SigmaAldrich)

FMS: phenazine methosulfate

Cultivation conditions: 37°C, 5% CO ₂

Growth media: DMEM, 10% FBS, 2 mM L-glut., NEAA 100x

XTT Method:

Test compounds are dissolved in DMSO and diluted with PBS to the test concentration range immediately before adding to the cell lines in the wells. PBS was used as a positive control, and DMSO at a final concentration of 20% was used as a negative control. All concentrations of compounds were determined in triplicate. Each determination is carried out twice and is blinded to the experimenter.

Testing is carried out on a 96-well plate. The dosage of tumor cells is approximately 2.5 x 10 ⁴ cells per well, the dosage of culture medium is 100 μl per well. After 24 hours, the nutrient medium is aspirated and 80 μl of fresh nutrient medium and 20 μl of a solution with different concentrations of the test substance are added to the wells. After 72 hours, the medium is aspirated and 100 μl of Optimem reagent solution containing XTT and PMS is added to the wells. After another 4 hours, the optical density is measured at 450 nm (the standard is at 630 nm).

The results in the form of IC ₅₀ are assessed using a graph of normalized cell viability plotted against the logarithm of the substance concentration.

Results:

The results are shown in Table 1:

Table 1: Measured IC ₅₀ values for the new Pt(IV) complexes and the reference Pt(II) complex of oxaliplatin

Tested Platinum Complex IC ₅₀ µM Capan-2 cancer cell line PC-3 cancer cell line Oxaliplatin (Pt(II) reference complex) >100 55.7 Satraplatin (Pt(IV) reference complex) >30 10.2 TU31 (Pt(IV) mother complex) 0.60 0.91 TU105 (new Pt(IV) complex) 0.04 0.06 TU106 (new Pt(IV) complex) 0.02 0.07 TU113 (new Pt(IV) complex) 0.11 0.23

Conclusion on the antitumor efficacy of new Pt(IV) complexes in vitro:

The parent TU31 complex has approximately 50-100 times better in vitro antitumor efficacy compared to the reference oxaliplatin, and 11-55 times better in vitro antitumor efficacy compared to the reference satraplatin. The excellent antitumor effects of the TU31 complex compared to the reference oxaliplatin and satraplatin, combined with its very low toxicity and the possibility of oral administration, are good prerequisites for the success of the TU31 complex in clinical trials.

However, the new platinum(IV) complexes TU105, TU106 and TU113 of the invention have IC ₅₀ values even more than 1000 times better than the reference oxaliplatin, more than 100 times better than the reference satraplatin and more than 15 times better than the parent TU31 complex for both Capan-2 and PC-3 cancer cell lines. This improvement of the new Pt(IV) complexes according to the invention appears to be significant compared to the prior art.

EXAMPLE 5.

Study of antitumor efficacy in vivo new Pt(IV) complexes of the present invention on two difficult to treat pancreatic tumors, athymic mouse model.

In vivo model: Athymic nude -FOXN-1 ^nu , 6 mice per group (3 males + 3 females).

Cancer cell lines used:

a) Capan-2, pancreatic adenocarcinoma, ductal (DSMZ, ACC-245)

b) PaTu9802, pancreatic adenocarcinoma, ductal, highly metastatic, grade II (DSMZ, ACC-179)

Pt(IV) complexes used:

a) reference complex Pt(IV) satraplatin, see example 4 for detailed specification.

b) Pt(IV) TU31 starting complex, see Example 4 for detailed specification.

c) new Pt(IV) complex TU105, detailed specification see example 4.

d) new Pt(IV) complex TU106, detailed specification see example 4.

Oral doses used:

A homogeneous bulk formulation of each tested Pt(IV) complex (20 wt%) with Gelucire 50/13 (80 wt%) was converted into an aqueous emulsion prior to oral administration to mice.

Conditions for culturing cancer cell lines: 37°C, 5% CO₂, DMEM (Dulbecco's modified Eagle's medium, Sigma Aldrich), 10% FBS (fetal bovine serum), 2 mM L-glutamine, nonessential amino acids.

Cancer cell transplantation. Each experimental mouse is subcutaneously transplanted with a dose of 1 x 10 ⁷ cells, then the mice are divided into separate experimental groups. Each experimental group has a control group that does not receive the drug.

Running the test:

After subcutaneous tumors have enlarged to a volume of approximately 0.2 cm ³ , two doses of the appropriate complexes of 2 × 20 mg/kg are administered orally every 1 week. The control group of mice does not receive the drug. The volume of tumors is continuously measured throughout the experiment. Three weeks after the second dose, mice are sacrificed, tumors are removed, weighed, and the resulting data are scored as % tumor volume inhibition (TVI%) and % tumor weight inhibition (TWI%), where:

TVI%= 100 - [(TVexp/TVcont) x 100]

TVexp=average volume of treated tumors in cm ³

TVcont=average volume of control tumors in ^cm3

TWI%= 100 - [(TWexp/TWcont) x 100]

TWexp=average weight of treated tumors in city.

TWcont=average weight of control tumors in g.

Results:

The results obtained are summarized in Table 2 and Table 3.

Table 2. Results of antitumor efficacy of platinum (IV) complexes TU31, TU105, TU106 in %TWI, single oral dose of 20 mg/kg, experimental duration 28 days (28 days)

Tumor: Capan-2, pancreatic adenocarcinoma (DSMZ, ACC-245)

Pt(IV) complex Average tumor volume during the experiment in days (g), in cm ³ TVI% Average tumor weight in g
28 d TWI% 1d 4d 8d 11d 15d 18d 22d 25d 29d Control 0.20 0.27 0.35 0.44 0.53 0.63 0.73 0.85 1.00 - 0.793 - Satraplatin 0.21 0.23 0.30 0.39 0.47 0.56 0.66 0.77 0.90 10.0 0.711 10.3 TU31 0.21 0.23 0.27 0.31 0.37 0.43 0.49 0.56 0.63 37.0 0.371 53.2 TU105 0.20 0.21 0.22 0.23 0.27 0.30 0.34 0.37 0.40 60.0 0.213 73.1 TU106 0.21 0.21 0.23 0.24 0.25 0.26 0.28 0.29 0.31 69.0 0.166 79.1

Table 3. Results of antitumor efficacy of platinum (IV) complexes TU31, TU105, TU106 in %TWI, single oral dose of 20 mg/kg, experimental duration 28 days (28 days)

Tumor: PaTu8902, pancreatic adenocarcinoma, metastatic, stage II (DSMZ, ACC-179)

Pt(IV) complex Average tumor volume during the experiment in days (g), in cm ³ TVI% Average tumor weight in g
28 d TWI% 1d 4d 8d 11d 15d 18d 22d 25d 29d Control 0.21 0.58 1.15 2.20 3.59 5.44 7.79 10.59 13.65 - 8,186 - Satraplatin 0.21 0.50 0.99 1.94 3.10 4.80 6.80 9.35 11.90 12.8 7,245 11.5 TU31 0.21 0.28 0.54 0.86 1.44 2.27 3.35 4.62 5.96 56.3 4,017 50.9 TU105 0.21 0.21 0.40 0.62 1.06 1.65 2.46 3.40 4.35 68.1 2,522 69.2 TU106 0.21 0.21 0.37 0.60 1.01 1.60 2.35 3.27 4.20 69.2 2,317 71.7

The achieved results in Tables 2 and 3 are further illustrated by the tumor growth curves for both pancreatic tumors in Figures 2 and 3.

Conclusion on antitumor efficacy in vivo new Pt(IV) complexes according to the invention, athymic mouse model:

The new Pt(IV) complexes TU105 and TU106 of the invention demonstrated significantly improved antitumor efficacy, expressed in both TVI% and TWI%, equal to approximately 70-80%, which is approximately 7 times higher than that of the reference Pt(IV) complex ) satraplatin and significantly more than for the original TU31 complex, also for both of the difficult-to-treat pancreatic tumors studied. The results obtained confirm the significantly improved antitumor efficacy of the new Pt(IV) complexes according to the invention compared to the prior art.

The new Pt(IV) complexes of general formula (III) according to the invention offer a very promising and affordable agent for oral therapy of cancer, preferably difficult to treat cancer, more preferably pancreatic cancer.

Claims (24)

1. Platinum (IV) complex with the geometric configuration cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride,X) Pt (IV) of general formula (III): (III) where X is the conjugated anion of a strong mineral or organic acid and where X is selected from the group consisting of nitrate, monochloroacetate and trifluoroacetate. 2. A method for producing a platinum (IV) complex according to claim 1, including the following stages of production: a) interaction of the initial platinum (IV) complex with the geometric configuration of cis- (ammine,1-adamantylamine) -trans -bis(1-adamantylcarboxylate) -cis- (chloride, iodide) Pt (IV) with a stoichiometric excess of the silver salt of a strong acid, in a mixture of a chlorinated hydrocarbon solvent, and a suitable strong acid at the boiling point of the solvent for the reaction time necessary to convert to obtain the mixture; b) removing solids from the mixture by filtration to obtain a crude filtrate; c) washing the crude filtrate with water and drying the separated organic phase; d) removing the solvent from the dried organic phase by evaporation to obtain the platinum (IV) complex according to claim 1. 3. The method according to claim 2, wherein in step a) the chlorinated hydrocarbon solvent is chloroform. 4. Method according to claim 2 or 3, including an additional stage: f) dissolving the platinum (IV) complex in acetone, adding activated carbon, ultrafiltration of the resulting suspension and precipitation of the resulting ultrafiltered solution of pure platinum (IV) complex in a large amount of water to obtain pure platinum (IV) complex according to claim 1 in the form of nanoparticles. 5. Platinum complex (IV) according to claim 1 for use in a method of treating a tumor disease, preferably difficult to treat cancer, most preferably pancreatic cancer, preferably by oral administration. 6. Pharmaceutical composition for the treatment of tumor diseases, containing the Pt (IV) complex according to claim 1 and a pharmaceutically acceptable carrier. 7. The pharmaceutical composition according to claim 6, characterized in that the pharmaceutically acceptable carrier is a self-emulsifying carrier, preferably stearoyl macrogol glycerides, most preferably Gelucire 50/13. 8. Pharmaceutical composition according to claim 6 or 7, characterized in that the platinum (IV) complex is present in the form of nanoparticles. 9. Pharmaceutical composition according to claim 7 or 8, characterized in that the mass fraction of the self-emulsifying carrier is more than 80% wt. 10. Pharmaceutical composition according to any one of paragraphs. 6-9, characterized in that the pharmaceutical composition is placed in acid-resistant capsules, preferably capsules of hydroxypropyl methylcellulose phthalate or hydroxyperopyl methylcellulose acetate succinate. 11. Pharmaceutical composition according to any one of paragraphs. 6-10 for use in a method of treating a tumor disease, preferably a difficult to treat cancer, most preferably pancreatic cancer, preferably by oral administration. 12. A method for treating tumor diseases in a subject in need, comprising administering a platinum complex (IV) according to claim 1 or a pharmaceutical composition according to any one of paragraphs. 6-10. 13. The method of treatment according to claim 12, including oral administration of the complex. 14. The method of treatment according to claim 12 or 13, where the disease is a difficult-to-treat cancer. 15. Method of treatment according to any one of paragraphs. 12-14, where the disease is pancreatic cancer.

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