KR101149095B1 - 4-Methylhexanoic kahalalide F compound - Google Patents

Abstract

The present invention is directed to new kahalalide antitumoral compounds, in particular to analogues of kahalalide F, useful as antitumoral, antiviral, antifungal agents and in the treatment of psoriasis.

Description

 4-Methylhexanoic kahalalide F compound

The present invention provides novel kahalarid antitumor compounds, more particularly homologues of kahaladide F in which fatty 5-methylhexanoic acid is substituted by 4-methylhexanoic acid, pharmaceutical compositions comprising the same and antitumor agents thereof , Antiviral and antifungal agents, and therapeutic use of psoriasis.

Kahalarid compounds are peptides isolated from Elysia rufescens , a Hawaiian herbivore marine species, and Elysia rufescens , and its prey, Bryopsis sp .

Kahalarid AG is published in Hamann, M. et al., J. Org. Chem, 1996, 61, 6594-6600: "Kahalalides: bioactive peptides from a marine mollusk Elysia rufescens and its algal diet Bryopsis sp." It is explained.

Kahalarids H and J are described in Schuer P.J. et al., Nat. Prod. 1997, 60, 562-567: "Two acyclic kahalalides from the sacoglossan mollusk Elysia rufescens".

Kahalarid O is described in Schuer P.J. et al., Nat. Prod. 2000, 63 (1) 152-4: A new depsipeptide from the sacoglossan mollusk Elysia ornata and the green alga Bryopsis species ".

Kahalarid K is described in Kan, Y. et al., J. Nat. Prod. 1999 62 (8) 1169-72: "Kahalalide K: A new cyclic depsipeptide from the hawaiian green alga bryopsis species". have.

As a related article, Goets et al., Tetrahedron, 1999, 55; 7739-7746: "The absolute stereochemistry of Kahalalide F"; Albericio, F. et al., Tetrahedron Letters, 2000, 41, 9765-9769: "Kahalalide B. Synthesis of a natural cyclodepsipeptide"; Becerro et al., J. Chem. Ecol. 2001, 27 (11), 2287-99: "Chemical defenses of the sarcoglossan mollusk Elysia refescens and its host Alga bryopsis sp."

Of these kahalarid compounds, kahalarid F is the most promising because of its antitumor activity. Its structure is complex, including six amino acids that are cyclic moieties and seven amino acids that have terminal fatty acid groups that are cyclic outer chains. Its inhibitory activity against in vitro culture of human lung carcinoma A-549 and human colon carcinoma HT-29 is disclosed in European Patent EP 610 078. Kahalarid F is also reported to have antiviral and antifungal properties.

In preclinical in vivo studies, the maximum tolerated dose (MTD) of kahalad following single bolus intravenous injection in female mice was 280 μg / kg. The MTD intravenous injection is extremely toxic, even if the animal showing signs of neurotoxicity eventually dies, 280 μg / kg of Kahalarid F can be administered repeatedly on five schedules once daily No clear evidence of acute toxicity was found in this case. In this regard, F. et al., Proceedings of the 1999 AACR NCI EORTC International Conference, abstract 315: "Preclinical pharmacology studies with the marine natural product Kahalalide F".

WO 02 36145 discloses a pharmaceutical composition containing kahalarid F and a novel use of the compound for the treatment of cancer, the entire contents of which are hereby incorporated by reference. It is done.

International Publication No. WO 03 33012, the basis of the priority claimed herein, discloses the clinical use of kahalarid compounds in oncology, the entire contents of which are hereby incorporated by reference. It is included.

Further, British Patent Application No. GB 0304367, the underlying application of the claims claimed herein, describes the use of kahalarid compounds in the treatment of psoriasis and related diseases, the entire contents of which are hereby incorporated by reference. It is included in the present specification.

Synthesis and cytotoxic activity of natural and synthetic kahalarid compounds are disclosed in WO 01 58934, the entire contents of which are hereby incorporated by reference. WO 01 58934 discloses the synthesis of compounds having a similar structure in which the kahalarid F and the terminal fatty acid chain are substituted with other fatty acids.

There is a need to provide yet another anti-tumor compound, especially another kahalarid compound with improved properties.

Summary of the Invention

We unexpectedly found that one of the kahalarid homologue compounds exhibited significant activity and improved antitumor efficacy in an in vivo model.

The present invention relates to a compound of formula 1 and pharmaceutically acceptable salts, produrg, tautomers, and solvates thereof.

Equation 1

The compound corresponds to kahalarid F having a 4-methylhexano terminal fatty acid chain, hereinafter referred to as 4-methylhexano KF.

In a preferred embodiment, the present invention relates to a compound containing (4S) -methylhexanoic acid and a pharmaceutically acceptable salt, prodrug, tautomer, and solvate thereof, having the formula (2) below. This compound is hereinafter referred to as (4S) -methylhexano KF.

Equation 2

The invention also relates to a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier, excipient or diluent.

The present invention also provides a method of treating a mammal, preferably a human, having cancer or psoriasis comprising administering a therapeutically effective amount of the compound to a subject having cancer or psoriasis.

The present invention can be used particularly in the treatment of patients with refractory cancer that is not alleviated by other therapies. More specifically, the composition according to the present invention can be used after other chemotherapy has been tried and not treated by it.

The present invention relates in particular to the treatment of patients with prostate cancer, breast cancer, hepatocellular carcinoma, melanoma, colon cancer, kidney cancer, ovarian cancer, NSCL cancer, epithelial cancer, pancreatic cancer and tumors overexpressing the Her2 / neu oncogene.

Another aspect of the invention relates to the use of said compound in the manufacture of a medicament. In a preferred embodiment, the medicament is for the treatment of cancer, psoriasis, viral infection or fungal infection.

The present invention also relates to a kit comprising a pharmaceutical composition comprising said compound and a separate container containing a regenerant. A method of regeneration thereof is also provided.

The invention also relates to a process for the preparation of said compounds. Preferably the method uses 4-methylhexanoic acid as starting material. In a preferred embodiment, the 4-methylhexanoic acid is (4S) -methylhexanoic acid. In the most preferred embodiment the method is solid phase synthesis.

Detailed description of the invention

We have identified kahalarid F homologs that show significantly improved activity against kahalarid F. As shown in the comparative examples, 4-methylhexano KF showed unexpectedly improved efficacy in cancer models in vivo. In view of the minor structural differences between 4-methylhexano KF and 5-methylhexano KF, this marked change in efficacy was surprising.

The compound of the present invention is kahalarid F having a 4-methylhexano fatty acid chain instead of 5-methylhexano, and has a structure of the following formula (1).

Equation 1

In particular, the compound preferably has a stereochemistry of formula 2.

Equation 2

Nevertheless, the compounds of the present invention have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. The present invention relates to the use of all optical isomers and stereoisomers of the compounds of the present invention, and also to all pharmaceutical compositions and methods of treatment that may utilize or include the same.

For convenience, the compounds of the present invention, preferably compounds 1 and 2, will be referred to as 4-methylhexyl kahalarid F compounds or 4-MeHex KF compounds. Preferably the 4-MeHex KF compound of the present invention is almost free of, substantially free of, or completely free of other kahalarid compounds. For example, it is preferable that the 4-MeHex KF of the present invention does not include kahalarid F having a 5-methylhexyl side chain. Specifically, the 4-MeHex KF of the present invention contains at least 25%, 10%, 5%, 2%, 1% or 0.5%, or 0.5% of any other kahalarid, preferably kahalarid F. It is desirable to. In a related aspect, 4-MeHex KF compounds of the present invention are provided in substantially pure form. Particularly suitable for the pharmaceutical compositions and methods of treatment of the present invention are 4-MeHex KF compounds which do not contain any such degree of other kahalarides.

The present invention includes compounds of the present invention and their pharmaceutically acceptable salts in which one or more hydrogen, carbon or other atoms are substituted by their isotopes. The compounds can be used as research and diagnostic tools in metabolic pharmacokinetic studies and binding assays.

As used herein, a compound of the present invention comprising a compound of formulas 1 and 2 is defined to include pharmaceutically acceptable derivatives or prodrugs thereof. Said “pharmaceutically acceptable derivative or prodrug” is any pharmaceutically acceptable compound of the present invention that can provide (directly or indirectly) a compound of the invention or a metabolite or residue thereof when administered to a recipient. Acceptable salts, esters, salts of esters or other derivatives. Particularly preferred derivatives and prodrugs increase the bioavailability of the compounds of the invention when the compounds are administered to a patient (eg, by making the orally administered compounds easier to absorb into the blood), or To enhance delivery to the biological compartment, to increase solubility that permits administration by injection, to alter metabolism, or to alter the rate of secretion.

Salts of the compounds of the present invention may include acid addition salts derived from nitrogen in the compound of formula 1 or 2. Although it is desirable to be pharmaceutically acceptable to the patient for therapeutic and prophylactic purposes, the therapeutically active moiety of the site derived from the compound of the invention as defined herein and the same site of the other compound are less important. Pharmaceutically acceptable acid addition salts include mineral acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid and tartaric acid, acetic acid, trifluoroacetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, glutamic acid Organic acids such as choline acid, succinic acid and methanesulfonic acid and arylsulfonic acid, such as those derived from p-toluenesulfonic acid. Preferred salts are trifluoroacetic salts.

The compounds of the present invention are suitable for the synthesis process disclosed in WO 01 58934, such as (S) or (R) 4-methylhexa, instead of 5-methylhexanno in Example 3 of WO 01 58934. It can be prepared by adding an acid. Thus, the present invention also encompasses a process for the preparation of the compounds according to formula 1 or 2. Preferably the method uses 4-methylhexanoic acid as starting material. Most preferably the starting material is (4S) -methylhexanoic acid. Preferably the synthesis is a solid phase synthesis method. A more detailed description of the synthesis is given in the Examples.

The process of the invention can be carried out in an enantiomeric or stereoisomer-controlled and rapid method from the starting material using a solid phase synthesis method characterized by the attachment of molecules in preparation to insoluble supports during all synthetic operations.

Pharmaceutical formulations of the compounds of the present invention can be adjusted to be administered by any suitable route. Such suitable routes include, for example, the oral (including buccal or sublingual), rectal, nose, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes. Such formulations may be prepared by any method known in the art of pharmacy, such as by mixing the active ingredient with a carrier or excipient.

Preferably the pharmaceutical composition of the compound of the present invention comprises a liquid (solution, suspension or emulsion) together with a suitable composition for intravenous administration and is in the form of a pure compound or mixed with any carrier or other pharmacologically active compound Can be. Further guidance regarding such pharmaceutical compositions is given in WO 02 36145, the entire contents of which are incorporated herein by reference.

Therefore, it is appropriate to use mixtures of nonionic surfactants and organic acids with bulking agents that provide a pressure-freeze-dried form of the compounds of the present invention suitable for regeneration. Preferably the regeneration is achieved with a mixture of emulsifying solubilizers, alkanols and water.

Preferably the reduced pressure lyophilized composition comprises primarily a bulking agent, such as at least 90% or at least 95% bulking agent. Examples of bulking agents are well known and include sucrose and mannitol. Other bulking agents than the above may be used.

The nonionic surfactant in the vacuum-dried composition is preferably a sorbitan ester, more preferably a polyethylene sorbitan ester such as polyoxyethylene sorbitan alkanoate, in particular polyoxyethylene sorbitan mono-oleate such as polysorb Bait 80. Generally, such nonionic surfactants comprise several percent of the composition, such as two to three or four percent of the composition, such as 0 to 5 percent of the composition.

The organic acid in the vacuum-dried composition is generally a fatty acid, preferably a hydrocarbon acid and more preferably a hydroxypolycarboxylic acid, most preferably citric acid. Generally, the organic acid comprises several percent of the composition, such as two to three or four percent of the composition, such as 0 to 5 percent of the composition.

The amount of the compound of the present invention in the reduced pressure lyophilized composition is generally less than 1% or often less than 0.1% of the mixture. Suitable amounts range from 50 to 200 μg, preferably 100 μg, per 100 mg of the composition.

Suitable emulsifying solubilizers for the regenerant are polyethylene glycol ethers, preferably esters of fatty acids, more preferably PEG oleates such as PEG-35 oleate. The emulsifying solubilizer is suitably 0 to 10%, generally about 3 to 7%, preferably 5% of the regenerant. The alkanol is generally ethanol and suitably 0 to 10%, generally about 3 to 7%, preferably 5% of the regenerant. The residue of the regenerant is water, producing a regenerated solution suitable for intravenous injection.

Further dilution of the regeneration solution with 0.9% saline may be appropriate for the infusion of kahalarid compound. Suitable injection equipment preferably includes a glass container rather than a polyethylene container. The material of the tube is preferably silicon.

Next, the preferred regenerant is 2 to 7%, preferably 5% of an emulsifying dissolving agent; 2-7%, preferably 5% alcohol; And the remaining water.

The formulations may be provided in the form of containers in unit-dose or multi-dose form, such as sealed ampoules and vials, and may require only the addition of sterile liquid carriers, such as water for injection, immediately prior to use. Under reduced pressure freeze drying).

Accordingly, the present invention also provides a kit comprising a separate container containing the pressure-freeze-dried composition and a regenerant. A method of regeneration thereof is also provided.

Administration of the compounds or compositions of the invention is by intravenous infusion. Injection times up to 72 hours, more preferably 1 to 24 hours, can be used, with about 1 hour or about 3 hours being most preferred. Particular preference is given to short infusion times which allow the treatment to be carried out without hospitalization for more than one night. However, the infusion may be within 24 hours or more if necessary.

Administration is preferably carried out periodically with a preferred method of application. That is, the compound of the present invention is injected intravenously into the patient in the first week of each cycle and the patients recover during the rest of each cycle. The preferred duration of each cycle is 1, 3 or 4 weeks; Multiple cycles can be used if necessary. In an alternative dosing protocol, the compounds of the present invention are administered for about 1 hour daily for five consecutive days every three weeks. Other protocols can be designed in their variant ways.

Dosage delays and / or dose reductions and schedule adjustments can be performed if necessary, depending on the patient's treatment resistance, in particular dose reductions above normal serum levels of liver transaminase or alkaline phosphatase. Recommended for patients with levels.

In one aspect, the present invention relates to administering a compound of the present invention to a human patient with cancer at 1200 mcg / m 2 / day or less, preferably at most 930 mcg / m 2 / day and more preferably at most 800 mcg / m 2 / day Provided is a method of treating a human patient with cancer comprising. The dose is suitably at least 320 mcg / m 2 / day. Preferably the dose is 400-900 mcg / m 2 / day, more preferably 500-800 mcg / m 2 / day, even more preferably 600-750 mcg / m 2 / day. Especially preferably, the dose is about 650-700 mcg / m 2 / day.

In another aspect, the present invention provides a human patient with cancer having a resting period for 1 to 4 weeks, wherein the compound of the present invention is administered up to 930 mcg / m 2 / day or less daily for 5 days, followed by no administration of the kahalarid compound. It provides a method of treating a human patient with cancer comprising the. Preferably the dose is 650-750 mcg / m 2 / day, more preferably about 700 mcg / m 2 / day. The injection time is preferably 1 to 14 hours, more preferably 1 to 3 hours. The resting period is preferably 2-3 weeks, more preferably about 2 weeks.

The present invention also provides a method of treating a human patient with cancer comprising administering a compound of the present invention to a human patient with cancer at a dose of 800 mcg / m 2 / day or less once a week. Preferably the dose is 600-700 mcg / m 2 / day, more preferably about 650 mcg / m 2 / day. The injection time is preferably 1 to 24 hours, more preferably 1 to 3 hours. Particularly preferably, the injection time is about 1 hour.

Although the instructions for dosage are described above, the exact dosage of a compound of the invention will vary depending on the individual agent, dosage form, particular site, host, and tumor to be treated. Other factors such as age, weight, sex, diet, time of administration, rate of secretion, host conditions, drug mix, sensitivity of reaction and severity of disease will be considered. Administration can be carried out continuously or periodically within the range of the maximum tolerated dose.

The present invention relates in particular to the treatment of patients with prostate cancer, breast cancer, hepatocellular carcinoma, melanoma, colon cancer, kidney cancer, ovarian cancer, NSCL cancer, epithelial cancer, pancreatic cancer and tumors overexpressing the Her2 / neu oncogene. Most preferably the present invention relates to the treatment of hepatocellular carcinoma, melanoma, breast cancer and prostate cancer.

The present invention also relates to a method for treating a skin disease associated with hyperproliferation of dermal cells in a mammal comprising administering an effective and nontoxic amount of a compound of the invention to the mammal. The skin disease is preferably psoriasis. Preferably the present invention relates to the treatment of human patients with psoriasis, in particular severe psoriasis.

The compounds and compositions of the present invention can be used in combination with other agents to provide combination therapy. The other medicament may be provided as a separate composition for forming part of the same composition or for administration at the same time or at different times. Although combinations with other chemotherapeutic, hormonal or antibody agents are possible, the type of other drugs is not particularly limited. The amount of the compound of the invention and the relative timing of other pharmaceutically active agents or agents and administration will be selected to achieve the desired combined therapeutic effect.

Example 1: Preparation of (4S) -methylhexano KF

General processes and initial steps of the production process are disclosed in WO 01 58934.

(4S) -MeHex-D-Val-Thr ( t Bu) -Val-D-Val-D-Pro-Orn (Boc) -D- allo -Ile-D- allo -Thr (Val-Alloc) -D- allo- Ile-D-Val-O-TrtCl-resin:

Remove the Fmoc group, remove Fmoc-Val-OH (678 mg, 2 mmol, 4 equiv), DIPCDI (233 μl, for 1.5 mmol and 3 equiv; 310 μl, for 2 mmol and 4 equiv; and 388 μl, for 2.5 mmol and 5 equiv) and HOBt (230 mg, for 1.5 mmol and 3 equiv; 307 mg, for 2 mmol and 4 equiv; and 395 mg, for 2.5 mmol and 5 equiv) for 90 minutes using the peptidyl-resin In Example 3, Fmoc-Thr (tBu) -OH (992 mg, 2.5 mmol, 5 equiv), Fmoc-D-Val-OH (678 mg, 2 mmol, 4 equiv), and (4S) -MeHex- OH (195 mg, 1.5 mmol, 3 equiv) was added sequentially. In all cases, 90 minutes after binding, the ninhydrin test was negative. Removal and washing of the Fmoc group was performed as described in General Procedures.

(4S) -MeHex-D-Val-Thr ( t Bu) -Val-D-Val-D-Pro-Orn (Boc) -D- allo -Ile-D- allo -Thr (Val-Z-Dhb-Phe -Alloc) -D- allo -Ile-D-Val-O-TrtCl-resin:

Remove Alloc group using Pd (PPh ₃ ) ₄ (58 mg, 0.05 mmol, 0.1 equiv) in argon atmosphere and in the presence of PhSiH ₃ (617 μl, 5 mmol, 10 equiv), Alloc-Phe-Z-Dhb -OH (666 mg, 2 mmol, 4 equiv) and HOAt (273 mg, 2 mmol, 4 equiv) were dissolved in DMF (1.25 mL) and added to peptidyl-resin, followed by DIPCDI (310 μL, 2 mmol, 4 equiv) was added and the mixture was stirred for 5 hours and the ninhydrin test was negative. After washing with DMF and CH ₂ Cl ₂ , one drop of peptidyl-resin was treated with TFA-H ₂ O (1:99) for 1 minute and the product was measured by MALDI-TOF-MS, C Calcd for ₈₈ H ₁₄₆ N ₁₄ 0 ₂₁ , 1,736.18. Found: m / z 1,758.67 [M + Na] ⁺ , 1,774.62 1,618.2 [M + K] ⁺ .

(4S) -MeHex-D-Val-Thr ( t Bu) -Val-D-Val-D-Pro-Orn (Boc) -D- allo -Ile-D- allo -Thr (Val-Z-Dhb-Phe -H) -D- allo -Ile-D-Val-OH:

After washing with DMF and CH ₂ Cl ₂ , using Pd (PPh ₃ ) ₄ (58 mg, 0.05 mmol, 0.1 equiv) under argon atmosphere and in the presence of PhSiH ₃ (617 μl, 5 mmol, 10 equiv) To remove the Alloc group. The protected peptide was separated from the resin by TFA-CH ₂ Cl ₂ (1:99) (5 × 30 seconds). The filtrate was collected on H ₂ O (4 mL) and partial removal of H ₂ O in rotavapor. ACN was then added to dissolve the solids indicated during H ₂ O removal, and the solution was freeze-dried to give 639 mg (387 μmol, 77% yield) of the title compound, followed by HPLC (Condition A, t _R 10.5 min). Its purity, measured by, was at least 95%.

(4S) -MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D- allo- Ile-cyclo (D-allo-Thr-D-allo-Ile-D-Val-Phe- Z-Dhb-Val) = (4S) methylhexano KF

The protected peptide (Example 6) (639 mg, 387 μmol) was added to CH ₂ Cl ₂ (390 mL, 1 mM) and HOBt (237 mg, 1.55 mmol) dissolved in the minimum volume of DMF to dissolve HOBt. Dissolve and add DIEA (203 μl, 1.16 mmol, 3 equiv) and DIPCDI (240 μl, 1.55 mmol, 4 equiv). The mixture was stirred for 1 hour, then the course of the cyclization step was checked by HPLC. The solvent is removed by evaporation under reduced pressure. The protected cyclic peptide was dissolved in TFA-H ₂ O (19: 1, 85 mL) and the mixture was stirred for 1 hour. The solvent was removed by evaporation under reduced pressure, dioxane was added (30 mL) and the solvent was removed by evaporation under reduced pressure (the process was repeated three times), followed by the addition of H ₂ O (40 mL) and freezing under reduced pressure. Dried. The crude product was purified by detection at HPLC (Kromasil C8 5 μm, 205 × 50 mm), equivalent 44% acetonitrile (+ 0.05% TFA), 55 mL / h, 220 nm in water (+ 0.05% TFA). The title product (192 mg, 0.13 mmol, 26% yield, 92.3%) was obtained. MALDI-TOF-MS, calcd for C ₇₅ H ₁₂₄ N ₁₄ 0 ₁₆ , 1,477.9. Found: m / z 1,500.12 [M + Na] ⁺ , 1,515.97 [M + K] ⁺ . ¹ H-NMR (2.5 mM; 500 MHz, H ₂ OD ₂ O (9: 1) The spectrum of the compound is shown in Table 1).

TABLE 1

¹ H-NMR spectroscopy [1H, NOESY, TOCSY at (278K)] was performed on Varian Unity Plus (500 MHz). Chemical shifts (d) are expressed in ppm downfield from TMS. Coupling constants are expressed in hertz.

Example 2: Preparation of (4R) -Methylhexano KF

(4R) -Methylhexano KF was prepared according to the experimental procedure described in Example 1 starting with 1 g of resin except that (4S) -MexHex was replaced with (4R) -MexHex. It was. The product (220 mg, 0.15 mmol, 30%, 92,3% purity) was measured by ES-MS. C ₇₅ H ₁₂₄ N ₁₄ O ₁₆ , Found: m / z 1,499.07 [M + Na] ⁺ , 1,514.94 [M + K] ⁺ .

Example 3: In vitro Cytotoxic Activity

The finality of this assay is to stop the growth of "in vitro" tumor cell culture by continuously exposing the cells to the sample to be tested.

Cell line

Designation N ° ATCC Species Tissue Characteristics

p-388 CCL-46 mouse ascites lymphocyte neoplasia

K-562 CCL-243 Human leukemia Eritros leukemia (breast effusion)

A-549 CCL-185 Human Lung Lung Carcinoma "NSCL"

SK-MEL-28 HTB-72 Human Melanoma Malignant Melanoma

HT-29 HTB-38 Human Colon Adenocarcinoma

LoVo CCL-229 Human Colon Adenocarcinoma

LoVo-Dox Human Colon Adenocarcinoma (MDR)

SW620 CCL-228 human colon adenocarcinoma (lymph node metastasis)

DU-145 HTB-81 Human Prostate Prostate Carcinoma, Androgen Receptor

                                          no

LNCaP CRL-1740 Human Prostate Prostate Adenocarcinoma, Androgen Receptor

                                          sieve

SK-BR-3 HTB-30 Human Breast Breast Adenocarcinoma, Her2 / neu +, (Chest

                                          Membrane exudation)

MCF-7 HTB-22 Human Breast Breast Adenocarcinoma, (Thymus Exudation)

MDA-MB-231 HTB-26 Human Breast Breast Adenocarcinoma, Her2 / neu +, (Chest

                                          Membrane exudation)

IGROV-1 Human Ovarian Ovarian Adenocarcinoma

IGROV-ET Human Ovarian Ovarian Adenocarcinoma, ET-743 Resistant Cells

                                          This feature

SK-OV-3 HTB-77 human ovarian ovarian adenocarcinoma (malignant ascites)

OVCAR-3 HTB-161 Human Ovarian Ovarian Adenocarcinoma

HeLa CCL-2 Human Cervical Cervical Epithelial Carcinoma

HeLa-APL CCL-3 Human Cervical Cervical Epithelial Carcinoma, In Apridine

                                          Sex cells are characterized

A-498 HTB-44 Human Kidney Kidney Carcinoma

PANC-1 CRL-1469 Human and Interest Epithelial Carcinoma

HMEC1 human endothelial

Colorimetric assays using sulfohodamine B (SRB) reactions were used to quantify cell growth and viability [Philip Skehan et al. (1990), New colorimetric cytotoxicity assay for anticancer drug screening, J. Natl. Following the techniques described in Cancer Inst., 82: 1107-1112].

This type of assay uses a 9 mm diameter 96 well cell culture microplate (Faircloth, 1988; Mosmann, 1983). Most cell lines derived from different human cancer types can be obtained from the American Type Culture Collection (ATCC).

Cells were maintained in RPMI 1640 10% FBS supplemented with 0.1 g / L penicillin and 0.1 g / L streptomycin sulfate and then incubated at 37 ° C., 5% CO ₂ and 98% humidity. For experiments, cells were harvested from subconfluent cultures with trypsin and suspended in fresh medium before plating.

Cells were inoculated in 96 well microtiter plates at 5 × 10 ³ cells / well in 195 μl of medium and allowed to adhere to the plate surface by growing for 18 hours in drug free medium. The sample was then added to 5 μl of water in the range of 10 to 10 ⁻⁸ μg / ml dissolved in DMSO / EtOH / PBS (0.5: 0.5: 99). After 48 hours exposure, antitumor effect is measured by SRB method: Cells are fixed by adding 50 μl of 50% (weight / volume) of cold trichloroacetic acid (TCA) and incubating at 4 ° C. for 60 minutes. The plates are washed with deionized water and dried. 100 μl of SRB solution (0.4% in 1% acetic acid by weight / volume) is added to each microtiter well and incubated for 10 minutes at room temperature. Unbound SRB is removed by washing with 1% acetic acid. The plate is air dried and the bound stain is dissolved in Tris buffer. Optical density is read on an automated spectrophotometry plate reader at a single wavelength of 490 nm.

Values for mean +/- SD of data from three wells were calculated. Some parameters for the cellular response can be calculated: GI = growth inhibition, TGI = total growth inhibition (cell proliferation inhibitory effect) and LC = cell death (cytotoxic effect).

The results are shown in Table 2. There was no significant difference between the compounds.

Table 2 Activity Data (Mole)

Example 4: In Vitro Toxicity

To assess the cytotoxicity of the drug against normal cells, normal cell lines stored according to the guidelines of ATCC: plated at a density of 5000 cells / well with AML-12, normal mouse liver cells and NRK-52E, normal rat kidney cells 96 well plates were used. The cells in each plate were allowed to stand overnight before the test drug was added. To each well (100 μl medium) 10 μl of drug in medium was added at different concentrations (1 × 10 ⁻¹⁰ −0.01 mg / ml final concentration) and further incubated overnight at 37 ° C., 5% CO ₂ . All experiments were repeated at least three times and analyzed twice. After 24 hours an MTS assay (CellTiter 96 aqueous) was performed (for all cell types) according to the manufacturer's (Promega) instructions. Cell viability (mitochondrial activity) is determined through enzymatic conversion of the formazan substrate.

As can be seen from the results in Table 3, there was no significant difference between compounds 5 methylhexano KF and (4S) -methylhexano KF.

<Table 3>

ALM
IC ₅₀ ([mu] M) NRK height
IC ₅₀ ([mu] M) 5-methylhexano KF 3.4 2.7 (4S) -methylhexano KF 5.4 2.7

Example 5: In vivo MTD in CD-1 Mice and Thymus Free Animals

The maximum tolerated dose for each drug after one bolus dose and five daily doses was determined in CD-1 and thymus-free mice (positive, respectively). The results are shown in Table 4 and there was no significant difference between compounds 5 methylhexano KF and (4S) -methylhexano KF.

TABLE 4

5-methylhexano KF (4S) -methylhexano KF Male CD-1 Mouse MTD Bolus 300 300 Female CD-1 Mouse MTD Bolus 200 200 Male CD-1 Mouse MTD 5DD 175-350 175-350 Female CD-1 Mouse MTD 5DD 175-350 175-350 Male thymus-free mouse MTD bolus 350 350 Female thymus-free mouse MTD bolus 325 325 Male thymus-free mouse MTD 5DD 350 350 Female thymus-free mouse MTD 5DD 325 325

Example 6 In vivo Efficacy in Breast Xenografts (5DD)

Maximum tolerated dose (MTD) level of 3/4 in breast xenografts according to the dosing schedule of 5 consecutive days (ie QDx5) intravenous (iv) bolus injection (0-4 days) in female thymus-free mice The efficacy of each of 5-methylhexano KF and (4S) -methylhexano KF in was analyzed. The compounds were provided as a vial solution in a freshly prepared carrier [Cremophore-EL / ethanol / water (5: 5: 90)]. Each daily dose (QDx5 schedule) was administered intravenously in an injection volume of 0.2 ml per 20 g of animal.

Evaluation of net growth in tumors of the corresponding treated group relative to the carrier control (ie% ΔT / ΔC) showed that the lowest (optimal) value appeared at 3 days after the start of drug treatment for all groups. In addition, pair-wise statistical analysis (using Mann-Whitney, a non-parametric method) has surprisingly different efficacy differences between the two compounds, given their very similar structure and in view of the previous examples. Showed that

Based on the efficacy studies disclosed herein and previously conducted toxicity studies (Example 4), a therapeutic index of at least 1.33 (1 × MTD / 0.75 × MTD, the dose at which the drug is toxic / dose at which the drug is efficacious) is ( 4S) -methylhexano KF. In addition, biologically relevant observations showed that the antitumor effect of (4S) -methylhexano KF in the breast at the same relevant MTD dose lasted longer than in prostate xenografts (Example 7). In summary, it appears clear that (4S) -methylhexano KF is a more potent isomer in breast xenografts and suggests that the duration of its biological activity has a long lasting effect on this type of tumor.

TABLE 5

Volume
(Μg / kg) Net growth of tumor
mm ³ , n = 9 % ΔT / ΔC Control group 236 100 5-methylhexano KF 262 126 53 (4S) -methylhexano KF 262 62 26

Example 7 In vivo Efficacy in Prostate Xenografts (5DD)

5-Methylhexano at each dose for prostate xenografts according to the dosing schedule of 5 consecutive days (ie QDx5) intravenous (iv) bolus injection (0-4 days) in male thymus-free mice The efficacy of KF and (4S) -methylhexano KF was analyzed. The compounds were provided as a vial solution in a freshly prepared carrier [Cremophore-EL / ethanol / water (5: 5: 90)]. Each daily dose (QDx5 schedule) was administered intravenously in an injection volume of 0.2 ml per 20 g of animal.

Evaluation of net growth in tumors of the corresponding treated group relative to the carrier control (ie% ΔT / ΔC) showed that the lowest (optimal) value appeared at 3 days after the start of drug treatment for all groups. In addition, it was confirmed by pair-wise statistical analysis (Mann-Whitney, using non-parametric methods) that more significant efficacy was achieved when (4S) -methylhexano KF was administered at a dose of 262 μg / kg / day. It was. Based on the efficacy studies disclosed herein and previously conducted toxicity studies (Example 4), a therapeutic index of at least 1.33 (1 × MTD / 0.75 × MTD, the dose at which the drug is toxic / dose at which the drug is efficacious) is ( 4S) -methylhexano KF. The results are shown in Figure 6 below.

<Table 6>

Volume
(Μg / kg) Net growth of tumor
mm ³ , n = 10 % ΔT / ΔC Control group 167 100 5-methylhexano KF 245 108 65 (4S) -methylhexano KF 245 74 44

Example 8: Antitumor Activity of Kahalarid F Homologs in Hollow Fibers Using Panels of Human Tumor Cell Lines

The antitumor activity of the Kahalarid F homologues described above is expressed in human tumor cell lines, namely SK-Hep-1 (liver cancer), HepG2 (hepatocellular carcinoma), Panc-1 (Izacar) and Mel-28 (melanoma). It was tested in a hollow fiber (HF) system using a panel of. The human tumor cells are encapsulated in in vitro HFs and subsequently transplanted into mice without thymus in vivo.

Doses of 5-methylhexano KF and (4S) -methylhexano KF were selected based on MTD experiments performed in thymus-free mice above, with the value being 325 μg / kg / day (see Example 5). ). Five consecutive daily doses were administered intraperitoneally (ip) at an injection volume of 0.2 ml per 20 g of animal.

Overall, KF-4 (S) -Met showed statistically significant antitumor activity against liver cancer (sc), hepatocellular carcinoma (both ip and sc), and cancer (ip). In the sc section, there was a remarkable tendency (ie, P = 0.059). In contrast, KF-5-Met had activity only in some tumor types, ie only in immature cancer (both ip and sc) and melanoma (sc), but not in any form of liver cancer tested. . The results are summarized in the table below. The table below clearly shows the difference between the compounds.

drug Tumor Type / HF Location (Random Cell Number / HF) SK-Hep-1 HepG2 Panc-1 Mel-28 ip sc ip sc ip sc ip sc carrier 0.575 0.872 0.576 0.509 0.200 0.392 2.078 1.77 KF-4 (S) -Met 0.485 0.525 ^* 0.335 ^* 0.319 ^* 0.129 ^* 0.237 ^§ 1.906 1.51 KF-5-Met 0.693 0.686 0.475 0.361 0.149 ^* 0.192 ^* 1.771 1.56

^* Statistically significant, P <0.05.

^{§ A} prominent trend, ie P = 0.059.

Claims (41)

A compound of formula 1 or a pharmaceutically acceptable salts, tautomers, or solvates thereof.

Equation 1 The compound of claim 1, wherein the compound is (4S) -MeHex-D-Val-L-Thr-L-Val-D-Val-D-Pro-L-Orn-D- allo -Ile- cyclo (D- allo -Thr-D- allo -Ile-D-Val-L-Phe-Z-Dhb-L-Val), or a pharmaceutically acceptable salt, tautomer, or solvate thereof. The compound of claim 1, wherein the compound is (4S) -MeHex-D-Val-L-Thr-L-Val-D-Val-D-Pro-L-Orn-D- allo -Ile- cyclo (D- allo -Thr-D- allo -Ile-D-Val-L-Phe-Z-Dhb-L-Val), or a pharmaceutically acceptable salt thereof. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising up to 25% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising up to 10% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising up to 5% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising up to 2% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising less than 1% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising less than 0.5% of kahalad F having a 5-methylhexyl side chain. A composition for the treatment of cancer, said composition comprising as an active ingredient the compound of any one of claims 1 to 3 and comprising less than 0.5% of kahalad F having a 5-methylhexyl side chain. The compound of claim 3, wherein the compound is pure. delete delete delete delete A composition for the treatment of cancer comprising the compound of any one of claims 1 to 3 as an active ingredient. delete The composition of claim 16, wherein the composition is for treating a mammal having cancer by administering a therapeutically effective amount of the active ingredient to the mammal having cancer. delete The composition of claim 18, wherein the mammal is a human. delete The composition of claim 20, wherein the human is suffering from refractory cancer that is not alleviated by other treatments. delete 21. The method of claim 20, wherein the cancer is selected from tumors that overexpress prostate cancer, breast cancer, hepatocellular carcinoma, melanoma, colon cancer, kidney cancer, ovarian cancer, NSCL cancer, epithelial cancer, pancreatic cancer and Her2 / neu oncogenes. Phosphorus composition. delete A cancer treatment kit comprising a pharmaceutical composition for treating cancer comprising the compound of any one of claims 1 to 3 as an active ingredient and an individual container containing a reconstituting agent. delete A method for producing the compound of any one of claims 1 to 3. delete 29. The method of claim 28 wherein 4-methylhexanoic acid is used as starting material. delete 31. The method of claim 30, wherein (4S) -methylhexanoic acid is used as starting material. delete A method of treating any mammalian subject, except human, with cancer, comprising administering the compound of any one of claims 1 to 3 to a subject with cancer. delete 35. The method of claim 34, wherein said treatment is for any mammal, except humans, having cancer, comprising administering a therapeutically effective amount of said compound to a subject with cancer. delete The method of claim 36, wherein the subject has refractory cancer that is not alleviated by other treatments. delete 37. The method of claim 36, wherein the cancer is selected from prostate cancer, breast cancer, hepatocellular carcinoma, melanoma, colorectal cancer, kidney cancer, ovarian cancer, NSCL cancer, epithelial cancer, pancreatic cancer and tumors overexpressing the Her2 / neu oncogene. How to be. delete